Australian scientists develop a nasal spray that could stop SARS-CoV-2 infection

Australian scientists develop a nasal spray that could stop SARS-CoV-2 infection

  • September 30, 2020

As the coronavirus disease (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to wreak havoc globally, scientists race to develop an effective medicine or vaccine to fight the infection. With more than 33.5 million people infected since the pandemic started in December 2019, finding a treatment for those infected is crucial to stemming its spread.

Now, a biotech company in Australia, Ena Respiratory, said that a nasal spray it is developing might help boost the human immune system to fight flu and common colds, significantly reduced the growth of the coronavirus in a recent study in animals.

The potential nasal spray may not only help treat COVID-19 but also prevent it.

The nasal spray

The novel product, called INNA-051, is being developed by Ena Respiratory, and laboratory experiments have shown that it reduced viral replication by as much as 96 percent in the animal study. Spearheaded by Public Health England’s (PHE) Deputy Director, Professor Miles Carroll, the new study described the potential treatment and has been published in the open-source preprint server bioRxiv*.

Used as a nasal spray, it aims to boost the natural immune system of the body to fight common colds and flu. It works by triggering the innate immune system, which is the body’s first line of defense against infection from a pathogen. When the drug has enhanced the immune system, it also prevented the infection and replication of SARS-CoV-2 in the laboratory.

Clinical observations. (a) Schematic of experimental design. Ferrets received INNA-051 and PBS treatments 4 days and 1 day prior to challenge with 5.0 x 106 pfu/ml SARS-CoV-2. Nasal wash and throat swabs were collected at days 1, 3, 5, 7, 10 & 12 post challenge (p.c.) for all treatment groups and control group. Scheduled culls were performed for 6/24 ferrets on day 3 p.c. and 18/24 ferrets on days 12-14 p.c. (b) Temperatures were measured twice daily (approximately 8 hours apart), using implanted temperature/ID chips. Mean temperatures +/- standard error of the mean (SEM) are displayed. Temperature dip post SARS-CoV-2 challenge (*) was attributed to sedation. (b) Weight was recorded daily and percentage change from the ferret weight prior to treatment plotted. Mean percentage weight change +/- SEM are displayed.

Clinical observations. (a) Schematic of experimental design. Ferrets received INNA-051 and PBS treatments 4 days and 1 day prior to challenge with 5.0 x 106 pfu/ml SARS-CoV-2. Nasal wash and throat swabs were collected at days 1, 3, 5, 7, 10 & 12 post challenge (p.c.) for all treatment groups and control group. Scheduled culls were performed for 6/24 ferrets on day 3 p.c. and 18/24 ferrets on days 12-14 p.c. (b) Temperatures were measured twice daily (approximately 8 hours apart), using implanted temperature/ID chips. Mean temperatures +/- standard error of the mean (SEM) are displayed. Temperature dip post SARS-CoV-2 challenge (*) was attributed to sedation. (b) Weight was recorded daily and percentage change from the ferret weight prior to treatment plotted. Mean percentage weight change +/- SEM are displayed.

The study

Respiratory tract diseases, including those that cause flu, common colds, and the coronavirus infection, represent major ongoing global health threats. These viruses have caused outbreaks to pandemics, endangering the lives of those who are at higher risk, such as children, older adults, and those who are immunocompromised.

The SARS-CoV-2 infection is actively spreading worldwide, and it spreads quickly from one person to another through close contact and respiratory droplets. One of the biggest threats of the current pandemic is that several people infected with the virus are asymptomatic, which means that they do not know that they carry the virus. As a result, they are called silent spreaders.

The research team from the National Infection Service, Public Health England (PHE) wanted to develop the nasal spray to prevent the replication of the virus in the nasal area, the most common point of entry of the virus.

To test the nasal spray, the team obtained nasal wash and throat swab samples four days before the viral challenge. Upon analyzing the viral RNA in nasal wash samples, the team confirmed infection in all treatment groups, with lower viral RNA levels seen in the INNA-051 treatment.

The team has found that prophylactic intra-nasal administration of INNA-051 in the SARS-CoV-2 ferret infection model has reduced levels of viral RNA in the nose and threat.

“The results of our study support clinical development of a therapy based on prophylactic TLR2/6 innate immune activation in the URT to reduce SARS-CoV-2 transmission and provide protection against COVID-19,” the team wrote in the paper.

Further, the research team added that the prophylactic approach is important to people at a high risk of community transmission or development of the severe disease from COVID-19, such as older adults, people with comorbidities, and those who are immunocompromised.

“We’ve been amazed by just how effective our treatment has been. By boosting the natural immune response of the ferrets with our treatment, we’ve seen a rapid eradication of the virus,” Christophe Demaison, the Ena Respiratory Managing Director, said.

“If humans respond similarly, the benefits of treatment are two-fold. Individuals exposed to the virus would most likely rapidly eliminate it, with the treatment ensuring that the disease does not progress beyond mild symptoms. This is particularly relevant to vulnerable members of the community. In addition, the rapidity of this response means that the infected individuals are unlikely to pass it on, meaning a swift halt to community transmission,” he added.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.


Journal reference:

An immune cocktail therapy to realize multiple boosting of the cancer-immunity cycle by combination of drug/gene delivery nanoparticles

An immune cocktail therapy to realize multiple boosting of the cancer-immunity cycle by combination of drug/gene delivery nanoparticles

  • September 30, 2020


Immune checkpoint blockade therapy (ICT) has shown potential in the treatment of multiple tumors, but suffers poor response rate in clinic. We found that even combining ICT with chemotherapy, which was wildly used in clinical trials, failed to achieve satisfactory tumor inhibition in the B16F10 model. Thus, we further constructed a previously unexplored immune cocktail therapy and realized multiple boosting of the cancer-immunity cycle. Cocktail therapy consisted of two kinds of tumor microenvironment-responsive drug and gene delivery nanoparticles to achieve specific delivery of doxorubicin and codelivery of plasmids expressed small hairpin RNA of PD-L1 (pshPD-L1) and hyaluronidase (pSpam1) in the tumor area. Experimental evidences proved that any component in the cocktail therapy was indispensable, and the cocktail therapy exhibited excellent antitumor effects against different types of tumors. The cocktail therapy presented here offers a searching strategy for more synergistic units with ICT and is meaningful for developing more efficient antitumor immunotherapy.


Immune checkpoint blockade therapy (ICT), which interferes with the interactions of immune checkpoints and their receptors, has achieved inspiring therapeutic effects in many tumor types (1). However, the poor response rate of ICT has largely restricted its further development (2). Previous findings have shown that inducing tumor cell immunogenic cell death (ICD) could largely increase the therapeutic effect of ICT (3). Specific chemotherapeutic agents, such as doxorubicin (DOX) and oxaliplatin, have been proven to effectively induce tumor ICD (4). Through exposing calreticulin (CRT) on the cell surface, ICD promotes the tumor antigen uptake by dendritic cells (DCs). Meanwhile, the release of danger signal molecules, including high-mobility group box 1 protein (HMGB1) and adenosine triphosphate (ATP), facilitates DC recruitment and activation (5). ICD is associated with the subsequent activation of tumor-specific T cells, thus exerting a synergistic effect with immune checkpoint antibodies (the most widely used ICT agent in the clinic).

However, the intravenous injection of DOX and checkpoint antibodies induces undesirable adverse effects (6, 7). Using nanodelivery systems could partially solve this problem (8). Compared with normal tissues, tumor areas usually have a lower pH value, ranging from 6.5 to 7.2 (9). Acid-responsive nanodelivery systems could increase the accumulation of nanoparticles via the enhanced permeability and retention (EPR) effect and release more cargo when stimulated by the tumor-specific acid environment (10). Furthermore, using small interfering RNA or small hairpin RNA to silence checkpoints locally in the tumor area would induce negligible immune-related adverse effects (irAEs) compared to the systemic administration of checkpoint antibodies (11).

On the basis of these observations, we developed two kinds of acid-responsive drug and gene delivery systems to achieve combined therapy with immunogenic chemotherapy and ICT (binary therapy). The as-prepared drug delivery system consisted of DOX-loaded poly(l-glutamic acid)-g-methoxy poly(ethylene glycol) (abbreviated DOX NPs), and the as-prepared gene delivery system consisted of pshPD-L1–loaded aldehyde-modified polyethylene glycol (OHC-PEG-CHO), poly(l-glutamic acid) (PLG), and polyethylenimine (PEI) (abbreviated shPD-L1 NPs). Unexpectedly, this combined therapy of immunogenic chemotherapy and ICT was insufficient to control the tumor growth in a highly malignant B16F10 mouse model. It was speculated that the dense extracellular matrix (ECM) in the tumor area restricted the therapeutic effect in two ways. First, the immunosuppressive tumor microenvironment (TME) induced by the ECM continuously instigated T cells into a dysfunctional state that was weakly responsive to ICT. Second, ECM acted as a physical barrier, preventing the infiltration of functionally normal peripheral T cells. Therefore, eliminating the tumor ECM would have the potential to adjust the immunosuppressive TME and promote peripheral T cell infiltration simultaneously.

In this work, an immune cocktail therapy was proposed to improve the above binary therapy by introducing an ECM destroyer as a third component. A sperm adhesion molecule 1 plasmid (pSpam1), expressing hyaluronidase (HAase), was chosen to perform the role of ECM elimination. pSpam1 can effectively degrade hyaluronic acid (HA), which is a critical component of the tumor ECM and overexpressed in 25 to 30% of the tumor types (12). Cocktail therapy was accomplished by exploiting two kinds of nanoparticles, DOX NPs and (shPD-L1 + Spam1) dual-gene codelivery NPs, both of which exhibited acid-responsive behavior (Fig. 1). The cocktail therapy has the following superiorities: (i) facilitating T cell priming via inducing tumor ICD, (ii) polarizing an immunosuppressive TME to an immune-active phenotype, (iii) notably increasing the amount of peripheral CD8+ T cell infiltration, and (iv) inducing strong immune memory effects and effectively preventing tumor metastasis. Benefiting from these advantages, outstanding immunotherapeutic effects were achieved in different murine tumor types.

Fig. 1 Schematic diagram of the immune cocktail therapy to realize multiple boosting of the cancer-immunity cycle by the combination of drug/gene delivery nanoparticles.

We constructed a cocktail therapy that consisted of an acid-responsive drug delivery system (DOX NPs) and an acid-responsive dual gene delivery system [(shPD-L1 + Spam1) NPs]. DOX NPs could induce tumor cell ICD and promoted tumor antigens uptake of DCs. (shPD-L1 + Spam1) NPs could generate shPD-L1 and HAase in situ at tumor areas, worked as checkpoint blockade and ECM elimination, respectively. The immune cocktail therapy held the following superiorities: (I) facilitating T cell activation, (II) reprogramming the immunosuppressive TME into an immune active TME, (III) increasing the infiltration of peripheral CD8+ T cells into the tumor, and (IV) inducing strong immune memory effect.

In summary, we reported an innovative immune cocktail therapy for antitumor treatment by combining immunogenic chemotherapy, immune checkpoint blockade, and ECM elimination. In addition, a set of drug/gene nanodelivery systems matching the cocktail therapy was developed. This work presents a promising comprehensive immunotherapy strategy that integrates multiple aspects in regulating the cancer-immunity cycle. We expect this could be an instructive study for other cancer immunotherapies.


Synthesis and characterization of acid-responsive DOX NPs

Although DOX is a widely used drug for chemotherapy in clinical trials, it still has some side effects, especially cardiotoxicity, which makes many patients suffer from unexpected pain (6). To solve this problem, nano-scaled drug delivery systems were developed in the last few decades (13). In this work, the biocompatible poly(l-glutamic acid)-g-methoxy poly(ethylene glycol) (PLG-g-mPEG) was used to encapsulate DOX (fig. S1, A and C). The drug-loading efficiency (DLE) and the drug-loading content (DLC) of the DOX NPs was 80.3 and 28.6%, respectively, calculated by the ultraviolet-visible (UV-Vis) absorbance of the DOX-encapsulated nanoparticles (fig. S1B). The DOX NPs exhibited a uniform particle size around 95 nm and zeta potential of −23.2 mV, which could enhance tumor accumulation via the EPR effect and achieve longer blood circulation (fig. S1, D and E). A pH-responsive drug-releasing characteristic of DOX NPs was observed in our experiments (fig. S1F). Lower pH could reduce interactions between PLG-g-mPEG and DOX via changing the hydrophobicity and charges of PLG, causing faster DOX release in the tumor area (14). The DOX NPs exhibited comparable cytotoxicity with free DOX in the B16F10 cells (fig. S1G).

DOX NPs induce ICD in vitro and in vivo

Effective antigen presentation is one of the most important steps in T cell activation. Immunologic cell death can help DCs maturation and enhance their antigen presentation ability (15). CRT, ATP, and HMGB1 are three representative markers of ICD (16). To provide the evidence that DOX NPs worked as immunogenic chemotherapy, we marked the CRT on the B16F10 cell surface after treated with different materials. The expression of CRT increased obviously in the DOX or DOX NPs treatment group, accompanied by a higher ATP value in cellular supernatant (fig. S1, H and I). We also observed an HMGB1 release from nuclei in the DOX NPs treatment group (fig. S1J). To verify whether DOX NPs can induce ICD of B16F10 cell in vivo via intravenous injection, we designed an animal experiment using C57BL/6 mice (fig. S1K). DOX NPs were given twice via intravenous injection. Tumors were harvested 2 days after the last injection. The immunohistochemistry results showed that DOX NPs up-regulated CRT expression and HMGB1 release in the tumor area, induced ICD successfully in the B16F10 tumor model in vivo (fig. S1L).

Synthesis and characterization of an acid-responsive nanocarrier for gene delivery

For more efficient and precise gene delivery to the tumor site, we chose OHC-PEG-CHO/PEI/PLG as the gene delivery system. OHC-PEG-CHO/[(PLG/PEI)/pDNA] nanoparticles (P[(GP)D] NPs) were prepared (fig. S2, A to C). The amino groups of PEI could quickly form Schiff bases with the aldehyde groups of OHC-PEG-CHO in neutral aqueous solution, whereas the Schiff bases were labile at pH 6.8 (fig. S2D). The P[(GP)D] NPs exhibited pH-triggered size/charge dual-rebound characteristics (fig. S2E) and higher cellular uptake efficiency at pH 6.8 than at pH 7.4 in our experiment (fig. S2F). Last, an optimized mass ratio of PEG:PLG:PEI:DNA = 5:1:2.5:1 was chosen for the P[(GP)D] NPs according to an in vitro transfection experiment to achieve relatively higher transfection efficiency in the tumor and avoid undesirable gene expression in the normal tissues (fig. S2G).

Combinational therapy by using DOX NPs and shPD-L1 NPs

For the first-stage antitumor treatment, we combined DOX NPs with shPD-L1 NPs for treating a B16F10 model in C57BL/6 mice (Fig. 2A). The results showed that immunogenic chemotherapy could generate synergistic effects with ICT when we compared the DOX NPs + shPD-L1 NPs group with the shPD-L1 NPs group (Fig. 2B), which could be attributed to the increased numbers of CD8+ T cells (Fig. 2, C and D). Unexpectedly, the number of intratumoral CD8+ T cells was not significantly different between the DOX NPs + shPD-L1 NPs group and the DOX NPs group, which induced a similar therapeutic effect. As a result, the DOX NPs + shPD-L1 NPs only achieved 60.6% tumor inhibition (Fig. 2B). In a normal situation, combining immunogenic chemotherapy with ICT should achieve much higher CD8+ T cell numbers compared with chemotherapy (17). There might be some special immunosuppressive factors in the B16F10 tumor that restricted further increases in CD8+ T cell numbers in the DOX NPs + shPD-L1 NPs treatment.

Fig. 2 Combination of immunogenic chemotherapy and ICT on the subcutaneous B16F10 model.

(A) Combinational treatment scheme of DOX NPs and shPD-L1 NPs. Mice received shPD-L1 NPs on days 12 and 16 via intravenous injection. Mice received DOX NPs on days 14 and 18 via intravenous injection. (B) Tumor growth curves in phosphate-buffered saline (PBS), DOX NPs, shPD-L1 NPs, and DOX NPs + shPD-L1 NPs treated groups. (C) CD45+CD3+CD8+ T cell ratios in the tumor of different treatment groups on day 21. (D) Immunofluorescence staining in tumor tissues for CD8 of different treatment groups. Sections were stained with anti-CD8 antibody (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. Significant differences in (B) and (C) were assessed using t test (ns represented not significant, **P < 0.01, ***P < 0.001). Results were expressed as means ± SD (n = 4).

Recent reports have shown that most preexisting CD8+ T cells in the immunosuppressive TME were dysfunctional T cells, which could only partially improve their function after ICB treatment but had nearly no proliferative capacity (18). Recruiting functional normal T cells from the periphery would be a wise strategy for improving the antitumor efficiency of the current DOX NPs + shPD-L1 NPs treatment. In addition, reversing the immunosuppressive TME is also important because it would continuously exhaust the intratumoral T cells, whether they were preexisting or newly infiltrated (19). Considering that B16F10 is a high-grade malignant tumor type, dense ECM during the growing process might generate an immunosuppressive TME and largely hinder the attraction of peripheral CD8+ T cells (20). We supposed that introducing extra therapy targeting the ECM would increase the therapeutic effect of immunogenic chemotherapy and ICT.

Immune cocktail therapy for antitumor treatment

HA is an important component of the tumor ECM and performs multiple immunosuppressive functions (21). In this work, pSpam1 was chosen to generate mouse HAase, codelivered with pshPD-L1 via our gene delivery system. We first designed an experiment to observe the antitumor effect of immune cocktail therapy, which consisted of DOX NPs and (shPD-L1 + Spam1) NPs (Fig. 3A). As we expected, the cocktail therapy group exhibited the most effective tumor inhibition, demonstrating 97.3% tumor shrinkage (Fig. 3B). This was a marked improvement in the therapeutic effect when we compared to 60.3% tumor shrinkage in the DOX NPs + shPD-L1 NPs group (Fig. 3B) and looked back to the 60.6% tumor shrinkage for the DOX NPs + shPD-L1 NPs group in the previous experiment (Fig. 2B). Further analyses of the tumor inhibition showed that any other formulations exhibited poorer antitumor efficiency compared to the cocktail therapy, indicating the indispensability of any component in the cocktail therapy. The tumor weight in the different treatment groups also exhibited the same tendencies (Fig. 3C). Furthermore, in the cocktail therapy group, five of six mice had a partial response (PR), indicating that cocktail therapy could increase the overall response rate and present a potential benefit to more patients (Fig. 3D). The photos and hematoxylin and eosin (H&E) staining of the excised tumors further proved the superior treatment effect of the cocktail therapy (Fig. 3E and fig. S3A). The weight of the mice did not change obviously during the therapeutic process (fig. S3B). The H&E results of other major organs indicated that all treatments had only negligible side effects (fig. S3C). In addition, we evaluated the systemic cytotoxicity of different treatment groups by measuring uric acid, blood urea nitrogen, creatinine, alkaline phosphatase, glutamate pyruvic transaminase, and aspartate aminotransferase in the serum (fig. S3D). No abnormal indicator was observed in our cocktail therapy when compared with the phosphate-buffered saline (PBS) group.

Fig. 3 Immune cocktail therapy on the subcutaneous B16F10 model.

(A) Treatment scheme of different combinational formulations. Mice received gene therapies on days 10 and 14 via intravenous injection. Mice received DOX NPs on days 12 and 16 via intravenous injection. (B) Tumor growth curves of different treatment groups. (C) Tumor weight of different treatment groups on day 19. (D) Individual tumor growth curves of each mouse after treated with different groups. PR represented tumors’ longer diameter was reduced by more than 30% compared with their initial longer diameter. (E) Tumor photos and H&E of different treatment groups. Group 1, PBS; group 2, DOX NPs; group 3, shPD-L1 NPs; group 4, DOX NPs + shPD-L1 NPs; group 5, Spam1 NPs; group 6, DOX NPs + Spam1 NPs; group 7, (shPD-L1 + Spam1) NPs; group 8, DOX NPs + (shPD-L1 + Spam1) NPs. Scale bar in tumor photos represented 5 mm. Scale bar in H&E represented 50 μm. Significant differences in (B) and (C) were assessed using t test (***P < 0.001). Results were expressed as means ± SD (n = 6). Photo credit (E): Jiayan Wu, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences.

Cocktail therapy reprograms an immunosuppressive TME to an active immune phenotype

To further interrogate the mechanisms of how the cocktail therapy worked, immunohistochemistry was used to evaluate related biological markers in the tumor sections. As clearly shown in fig. S4A, shPD-L1 NPs significantly reduced PD-L1 expression, whether in monotherapy or in combinational therapies. At the same time, we monitored the level of FoxP3 in the tumor sections, which could reflect the amounts of regulatory T (Treg) cells. Excess Treg cells could inhibit the activity of CD8+ T cells (22). In the DOX NPs + shPD-L1 NPs group, the amount of FoxP3 was increased compared to the PBS group, probably because the immune system tried to achieve immune homeostasis to balance the active immune state. After combined therapy with pSpam1, the immunostained area of FoxP3 was reduced in the cocktail therapy group because degrading HA could deactivate the Treg cells (23). Furthermore, HAase generated by pSpam1 remarkably reduced the immunostained area of hypoxia-inducible factor 1-α, which could help more immune cells function better (24).

For a comprehensive understanding of the changes in the immune cell populations, flow cytometry analyses were carried out. DOX-induced tumor ICD successfully increased the percentage of CD80+ major histocompatibility complex (MHC) II+ mature DCs in the tumor-draining lymph nodes (TDLNs) (Fig. 4A and fig. S4B). The cocktail therapy group showed a marked increase in intratumoral T cells (Fig. 4B). The intratumoral number of CD8+ T cells in the cocktail therapy group was 7.9-fold higher than that in the PBS group and 2.4-fold higher than that in the DOX NPs + shPD-L1 NPs group (Fig. 4C). The cocktail therapy group also showed an increase in the number of CD4+ T cells compared to the PBS group (Fig. 4D). Further calculations showed that the cocktail therapy group exhibited high CD8+T/CD4+ T cell ratios (Fig. 4E and fig. S4C), indicating an active immune status in the tumor. Besides, cocktail therapy significantly reduced the percentage of M2 macrophages compared to the PBS group or DOX NPs + shPD-L1 NPs group, which would help CD8+ T cells function normally (Fig. 4F) (25).

Fig. 4 The immune cocktail therapy induced an immune-active TME.

Flow cytometry analyses of (A) CD80+MHC II+ matured DCs of the CD11c+ DCs ratios in TDLNs. (B) CD45+CD3+ T cell ratios in the tumor. (C) CD45+CD3+CD8+ T cell ratios in the tumor. (D) CD45+CD3+CD4+ T cell ratios in the tumor. (E) CD8+ T/CD4+ T cell ratios in the tumor. (F) CD206+F4/80+ M2 macrophages (M2φ) of the CD11b+ macrophages ratios in tumor. ELISA results reflected the relative fold change of (G) PD-L1, (H) HAase, (I) IFN-γ, (J) granzyme B, (K) IL-6, and (L) TNF-α in tumor tissues compared with the PBS group. (M) Immunofluorescence staining in tumor tissues for CD8 (green) and granzyme B (purple) of different treatment groups. Scale bars, 50 μm. (N) The heatmap of multiple gene expression levels according to RT-qPCR results measured from tumor tissues in different treatment groups. Each square indicated one individual test. Group 1, PBS; group 2, DOX NPs; group 3, shPD-L1 NPs; group 4, DOX NPs + shPD-L1 NPs; group 5, Spam1 NPs; group 6, DOX NPs + Spam1 NPs; group 7, (shPD-L1 + Spam1) NPs; group 8, DOX NPs + (shPD-L1 + Spam1) NPs. Significant differences in (A) to (L) were assessed using t test (ns represented not significant, *P < 0.05, **P < 0.01, ***P < 0.001). Results were expressed as means ± SD (n = 5).

Next, the immune status of the TME was further investigated by enzyme-linked immunosorbent assay (ELISA) and reverse transcription and quantitative real-time polymerase chain reaction (RT-qPCR) analysis of the protein and gene levels. The ELISA results showed that the cocktail therapy decreased PD-L1 expression (Fig. 4G) and increased the amount of HAase (Fig. 4H) in the tumors, indicating that the therapeutic gene-loaded NPs could function as intended. Interferon-γ (IFN-γ) and granzyme B are two key elements in antitumor efficiency (26). The cocktail therapy generated 1.9-fold more intratumoral IFN-γ than the PBS group and 1.3-fold more than the DOX NPs + shPD-L1 NP group (Fig. 4I). Furthermore, the amount of granzyme B in the cocktail therapy group was 3.8-fold higher than in the PBS group and 1.6-fold higher than the DOX NPs + shPD-L1 NPs group (Fig. 4J). The higher levels of CD8 and granzyme B in the cocktail therapy group was further confirmed by immunofluorescence (Fig. 4M and fig. S5A). These results proved that the cocktail therapy exerted much stronger killing ability against the tumors, indicated by the up-regulation of IFN-γ and granzyme B. In addition, interleukin-6 (IL-6) and tumor necrosis factor–α (TNF-α) are two typical proinflammatory factors that can facilitate immune cell recruitment, maturation, or activation (27). The cocktail therapy further increased these two cytokines compared to the PBS group and the DOX NPs + shPD-L1 NPs group (Fig. 4, K and L). Furthermore, the heatmap of related genes showed that the cocktail therapy could down-regulate the expression of immunosuppressive genes (Pd-l1 and Tgf-β) and up-regulate the expression of chemokines (Cxcl10, Cxcl9, Ccl5, and Ccl3), adhesion molecules (Icam1 and Vcam1), proinflammatory factors (Il-1α, Il-1β, Il-6, and Tnf-α), and nuclear transcription factor κB compared to the PBS group (Fig. 4N). Together, with pSpam1 assistance, cocktail therapy demonstrated a powerful capacity for TME regulation at the protein and gene levels.

Cocktail therapy increases peripheral T cell infiltration

In the previous experiment, we speculated that most of the T cells in the DOX NPs + shPD-L1 NPs group were dysfunctional T cells and were weakly responsive to ICT. Peripheral functionally normal CD8+ T cells could have a better synergistic effect with ICT, continuing proliferation and activation. To study whether pSpam1 could promote peripheral CD8+ T cell infiltration in the tumors, FTY720, a widely used peripheral T cell depletion agent (28), was used to establish a peripheral CD8+ T cell depletion model (Fig. 5A). The tumor growth curves were recorded and the CD8+ T cells in the peripheral blood and tumors were analyzed (Fig. 5, B to D).

Fig. 5 Cocktail therapy increased peripheral T cell infiltration.

(A) Treatment scheme for peripheral T cells depletion experiment. (B) Left: Tumor growth curves in different treatment groups. Right: Amplified region of tumor growth curves. (C) Relative fold change of peripheral CD45+CD3+CD8+ T cells in blood compared with the PBS group. (D) CD45+CD3+CD8+ T cells in tumor compared with the PBS group. (E) Immunofluorescence staining in tumor tissues for CD8 (green) and granzyme B (purple) of different treatment groups. Scale bars, 50 μm. (F) Immunofluorescence staining in tumor tissues for α-SMA (green) and CD31 (purple). Scale bars, 100 μm. (G) RT-qPCR results of Cxcl9, Cxcl10, Icam1, and Vcam1 mRNA expression level in tumor tissues. (H) Quantitative measurement of ICAM1 and VCAM1 in tumor tissues. (I) Immunofluorescence staining in tumor tissues for HA (green). Scale bars, 100 μm. (J) The schematic diagram for suggested mechanisms that could affect peripheral T cell infiltration. We concluded four crucial steps: vessel normalization, chemokines level, adhesion molecules level, and ECM elimination. The score table for the comprehensive contributions to peripheral T cell infiltration was also shown in the figure. We supposed that the cocktail therapy group gained 3.5 “points,” whereas the DOX NPs + shPD-L1 NPs group gained 0.5 points in the score table. Significant differences in (B) to (D), (G), and (H) were assessed using t test (ns represented not significant, *P < 0.05, **P < 0.01, ***P < 0.001). Results were expressed as means ± SD (n = 5 or 3).

First, the CD8+ T cells in peripheral blood clearly showed that FTY720 decreased the numbers of peripheral CD8+ T cells by 80 to 90% in the related groups, indicating that this model was successfully established (Fig. 5C). The tumor growth curves showed that the PBS + FTY720 group was not obviously different from the PBS group (Fig. 5B). This could be attributed to the poor infiltration of the peripheral CD8+ T cells into the tumor area (Fig. 5D). In the B16F10 tumor–bearing mice, it was difficult for the tumor-specific peripheral CD8+ T cells to infiltrate the tumor because of the high ECM density. Thus, the peripheral CD8+ T cells contributed little to the tumor inhibition in the PBS group. It was understandable that the depletion of peripheral CD8+ T cells by FTY720 in the PBS group would not cause an obvious difference in the tumor growth curves. In contrast, an extra FTY720 injection significantly increased the tumor growth rate in mice treated with Spam1 NPs (Fig. 5B), indicating that peripheral CD8+ T cells played an important role in the pSpam1 NP group. pSpam1 NPs could effectively destroy the ECM by the in situ expression of HAase in the tumors, thus increasing the infiltration of peripheral CD8+ T cells into the tumors. The tumor inhibition ability of the pSpam1 group largely relied on the infiltration of peripheral CD8+ T cells. Therefore, it was obvious that FTY720 injections would significantly eliminate the possibility of peripheral CD8+ T cell infiltration into the tumors from the source (Fig. 5D), greatly decreasing the tumor-inhibiting ability of the pSpam1 NPs. Similarly, FTY720 injections could badly damage the tumor-inhibiting ability of the (shPD-L1 + Spam1) NPs (Fig. 5B). Originally, the (shPD-L1 + Spam1) NPs demonstrated the best therapeutic effect among all the treatment groups, which was attributed to the following aspects. First, the infiltration of peripheral CD8+ T cells into the tumors was improved. The second was the activation and proliferation of CD8+ T cells, which was boosted by PD-L1 silencing. Therefore, the depletion of CD8+ T cells by FTY720 injection would inevitably decrease the number of CD8+ T cells (Fig. 5, D and E) and the amount of granzyme B (Fig. 5E) in the tumors in the (shPD-L1 + Spam1) NPs group, further decreasing the antitumor efficiency. In conclusion, the ICT for the tumors with dense ECM was heavily dependent on the infiltration of peripheral CD8+ T cells into the tumors, which could be boosted by HA degradation. This was the critical reason why we included a functional HA degradation Spam1 gene in our cocktail therapy.

After confirming that pSpam1 could promote peripheral CD8+ T cell infiltration, we further explored the mechanism that could cause this phenomenon. Several aspects that could influence the T cell infiltration capacity were assessed, respectively. First, the degree of vessel normalization was evaluated by immunofluorescence (Fig. 5F). The colocalization of alpha-smooth muscle actin (α-SMA) and CD31 was seen as a biomarker of a normalized vessel (29). Second, Fig. 5G showed the gene expression levels of chemokines (Cxcl9 and Cxcl10) and adhesion molecules (Icam1 and Vcam1), which were measured by RT-qPCR. ELISA was further carried out to confirm the amount of ICAM1 and VCAM1 protein in the tumors (Fig. 5H and fig. S5, B and C). Last, HA degradation in the tumor was visualized by immunofluorescence (Fig. 5I). On the basis of the above results, we summarized the contribution of different factors that affected peripheral T cell infiltration and compared the tumors with dense HA to tumors after HA degradation (Fig. 5J). Although the gene expression level of the chemokines in the cocktail group (with HA degradation) was slightly lower than that in the DOX NPs + shPD-L1 NPs group (without HA degradation), the cocktail group gained more points in vessel normalization, adhesion molecule expression, and ECM physical barrier destruction in the TME. Therefore, introducing pSpam1 in the cocktail group could induce effective HA degradation in the tumor and act as a trigger for a series of cascading effects, and lastly, greatly improving the infiltration of peripheral CD8+ T cells. Besides promoting immune cell infiltration, HA degradation has also been proven to increase nanoparticle accumulation in tumors (30). The fluorescence imaging experiment result showed that Spam1 NPs induced a higher DOX NPs accumulation in the B16F10 tumor at different times (fig. S6, A to C). In addition, the photoacoustic (PA) imaging experiment also showed that Spam1 NPs could increase the average signal of contrast agent in the 4T1 tumor when compared with PBS-treated mice (fig. S6, D and E). We proved that Spam1 NPs increased the accumulation of nanoparticles in different tumor types, which were mainly attributed to the destruction of tumor ECM. This would also increase the antitumor efficiency.

Cocktail therapy induces strong immune memory effects

It was reported that effector T cells newly infiltrating into the tumor site, not preexisting dysfunctional T cells in the tumor site, might be transformed into memory T cells (31, 32). Therefore, the cocktail therapy was also expected to enhance the immune memory effect. Because the spleen is an important immune organ that provides a place for immune cell homing and differentiation (33), T cells in the spleen were carefully analyzed after the treatments. The cocktail therapy group showed increased numbers of CD8+ T and CD4+ T cells compared to the PBS group but no significant differences compared to the DOX NPs + shPD-L1 NP group (Fig. 6, A and B). However, as shown in Fig. 6C, the cocktail therapy group exhibited significantly increased numbers of CD3+CD8+CD44+CD62L effector memory T (TEM) cells in the spleen compared to the DOX NPs + shPD-L1 NP group (1.6-fold) and the PBS group (2.5-fold). Considering that a higher number of TEM cells indicate a profound and durable antitumor efficiency, the cocktail therapy group might prolong the survival and enhance the resistance to lung metastasis. As shown in Fig. 6 (D and E), the median survival in the cocktail therapy group was markedly increased to 38.5 days, compared with the PBS group (17 days) and the DOX NPs + shPD-L1 NP group (21.5 days). Next, a lung metastasis model was established to verify whether the cocktail therapy could protect mice from tumor metastasis (Fig. 6F). The H&E staining results clearly showed that the B16F10 tumors developed rapidly in the lungs of naive mice, but there was no metastasis in the cocktail therapy group (Fig. 6G). Another wound-healing experiment also proved that pSpam1 could impede tumor migration in vitro (fig. S7).

Fig. 6 Cocktail therapy largely increased immune memory cells in the spleen, prolonged medium survival, and could inhibit lung metastasis.

Flow cytometry results for (A) CD3+CD4+ T cell ratios, (B) CD3+CD8+ T cell ratios, and (C) CD3+CD8+ CD44+CD62 effective memory T cell ratios of different treatment groups in the spleen (n = 5). (D) Treatment scheme of survival experiment. (E) Mouse survival curves of different treatment groups (n = 6). (F) Treatment scheme of lung metastasis experiment. (G) H&E results for lungs of naive mouse group (control) and cocktail therapy group. Scale bars, 500 μm. Significant differences in (A) to (C) were assessed using t test (ns represents not significant, **P < 0.01, ***P < 0.001). Significant differences in (E) were assessed using log-rank (Mantel-Cox) test (*P < 0.05, ***P < 0.001).

Cocktail therapy shows enhanced therapeutic effect in other tumor types

Considering that the cocktail therapy has achieved superior antitumor efficiency in the B16F10 tumor, we further explored whether this strategy could be applied to other tumor types. Recently published studies have shown that combining chemotherapy with ICB could greatly increase the therapeutic effect in both breast cancer or colorectal cancer when compared with single ICB therapy (3, 17). More effective antitumor results were expected to be achieved in these kinds of tumors by the cocktail therapy. We then established subcutaneous 4T1 and CT26 models on BALB/c mice and performed the treatments according to fig. S8A. The results showed that the cocktail therapy group exhibited slowest 4T1 tumor growth rate and minimized tumor weight when compared with other treatment groups, including DOX NPs + shPD-L1 NPs group (fig. S8, B and C). Tumor photos of different treatment groups also showed that the cocktail therapy had the best antitumor efficiency (fig. S8F). The intratumoral immune cell analyses provided the evidence that the cocktail therapy could further elevate the CD3+ and CD8+ T cell amounts when compared with DOX NPs + shPD-L1 NPs group (fig. S8, D and E). Similar tendencies of therapeutic outcomes and immune cell analyses were also observed in the CT26 model (fig. S8, G to K).

In summary, the cocktail therapy could effectively inhibit the growth of several subcutaneously tumor models and could promote a strong immune memory effect with prolonged median survival and protection against tumor metastasis in the B16F10 model. It is worth mentioning that the cocktail therapy could further increase the intratumoral CD8+ T cells infiltration in multiple tumor types when compared with traditional combination therapy that consists of immunogenic chemotherapy and ICB. This could be a pivotal factor to produce a better therapeutic effect. We supposed that the cocktail therapy has the potential to be used in a variety of tumor types.


Although ICT has achieved great success in treating a variety of tumors, the effectiveness of ICT depends on patient characteristics (34). Even in melanoma, one of the most sensitive tumor types to ICT, nivolumab (a U.S. Food and Drug Administration–approved PD-1 antibody) could only achieve an objective response rate of 20.3 to 43.6% (35). Recent reports found that immunogenic chemotherapy could help increase the therapeutic effects of ICT by promoting immune activation. For example, short-term DOX treatment remarkably increased the PD-1 blockade response rate in patients with triple-negative breast cancer (3).

However, systemic administration of DOX and checkpoint antibodies could induce undesirable side effects. Cardiotoxicity is the main side effect of DOX treatment, using nanotechnology to develop a tumor-specific drug delivery system could minimize the damage to normal tissues. TMEs have a lower pH value ranged from 6.5 to 7.2 compared with normal tissues. Developing acid-responsive nanodelivery systems with good biocompatibility could release cargos specifically in the TME and improve the therapeutic effect. For checkpoint antibodies, irAEs such as fatigue, skin disorders, and hematologic adverse effects are a major concern. Recently, reports have shown that using gene technology to hinder the generation of checkpoint molecules locally at tumor area may induce lower irAEs compared with the systemic administration of checkpoint antibodies (11, 36). Thus, in a preliminary study, we synthesized acid-responsive DOX NPs and shPD-L1 NPs, mirroring the common combinational therapy of immunogenic chemotherapy and ICT in clinical use and expected that the nanodelivery systems would have fewer side effects.

Unexpectedly, the shPD-L1 + DOX NP group only achieved 60.6% tumor inhibition in the mouse B16F10 model, which was insufficient for effective antitumor therapy (Fig. 2B). Dense tumor ECM facilitated the formation of an immunosuppressive TME, which continuously exhausted the T cells inside the tumor, making them insensitive to ICT. In addition, the ECM could also prevent peripherally functionally normal T cells from infiltrating the tumor. As a result, most T cells in the TME were dysfunctional T cells that were weakly responsive to ICT, eventually failing to inhibit tumor growth.

To further improve the therapeutic effect, an immune cocktail therapy that consisted of DOX NPs and (shPD-L1 + Spam1) NPs was created. The cocktail therapy induced 97.3% tumor shrinkage compared to the PBS group, and five of six mice in this group achieved PRs (Fig. 3, B and D). The mice that received cocktail therapy achieved longer median survival and better inhibition of lung metastasis. Cocktail therapy had the following superiorities: (i) promoting T cell priming via inducing tumor ICD, (ii) reprogramming the immunosuppressive TME into an active immune phenotype, (iii) facilitating peripheral T cell infiltration into the tumor, and (iv) inducing a vigorous immune memory effect. In addition, we proved that the cocktail was also suitable for other tumor types, such as the 4T1 model and CT26 model (fig. S8).

Previously reported immunotherapies related to ECM clearance usually observed an increase of CD8+ T cells into the tumor after treatment, but few of them studied the source of these T cells (4). Whether increased T cells came from the proliferation of originally resident T cells or the infiltration and proliferation of peripheral T cells remained unknown. In this study, we established a peripheral T cell depletion model and successfully demonstrated that peripheral T cell infiltration was an important factor in increasing the number of CD8+ T cells in the tumor (Fig. 5, B and D). pSpam1, the key component of cocktail therapy, could promote peripheral T cell infiltration via vascular normalization, chemokine up-regulation, adhesion molecule up-regulation, and physical barrier (ECM) degradation (Fig. 5J). It was worth noting that the antitumor effect obtained with pSpam1 treatment alone was limited. Combining pSpam1 with DOX and shPD-L1 was necessary because the combination could effectively increase T cell priming and avoid T cell exhaustion.

There were some potential safety concerns in this work. One was whether the acid-responsive carriers could reduce the toxicity to normal organs and minimize irAEs. Throughout the treatments, there were no obvious mouse weight changes or irAEs in any treatment group (fig. S4B). The H&E results of the major organs and measurements for liver and kidney functions proved that our cocktail therapy had little damage to normal organs (fig. S4, C and D). However, ECM elimination might be a double-edged sword in immunotherapy. Some reports pointed out that endogenous HAase could promote tumor growth and cause faster tumor metastasis (37). Actually, HAase as a tumor promoter or suppressor depends on its intratumoral concentration (38). In this study, no identifiable metastasis could be observed in any treatment group according to the H&E results in the major organs (fig. S4C). There might be another problem regarding whether the normalization of blood vessels caused by ECM elimination would increase the number of some immunosuppressive cells in the tumor. Our immunohistochemistry and flow cytometry results showed that both the ratio of Treg cells and M2 macrophages were decreased in the cocktail therapy group compared to the PBS group (Fig. 4F and fig. S5A). Together, our cocktail therapy was a safe and effective antitumor strategy.

We believe that in a sturdy cancer-immunity cycle, some steps are crucial (39). These steps include but not limited to (1) tumor antigen release, (2) DC maturation and antigen uptake, (3) antigen presentation and T cell priming, (4) T cells trafficking from the periphery to the tumor, (5) successful recognition of the tumor cells by tumor-specific T cells, (6) killing of tumor cells, and (7) the generation of immune memory T cells. ICT could be helpful for restoring the cytotoxic effect of tumor-specific T cells but could only achieve a limited promotion of the abovementioned steps. In contrast, our cocktail therapy expanded the range of regulation, and at least, affected steps (1), (2), (4), (6), and (7). In view of the excellent antitumor efficiency of our cocktail therapy, any strategy that could promote a positive shift in the cancer-immunity cycle would be a possible cornerstone for new cocktail therapy. We look forward to the further innovation of “super cocktail therapies” with pleiotropic regulations of the cancer-immunity cycle, which would help people fight tumors more effectively.



Branched PEI (molecular weight 25,000 Da) and 4′,6-diamidino-2-phenylindole (DAPI) and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). DOX hydrochloride (DOX·HCl) was purchased from Meilun Biotechnology Corporation (Dalian, China). Fingolimod hydrochloride (FTY720) was purchased from Sunsurechem Biotechnology Corporation (Nanjing, China). DNA labeled with Cyanine 5 (Cy5-DNA) was purchased from RiboBio (Guangzhou, China). A luciferase reporter gene assay kit was purchased from Promega (Mannheim, Germany). SYBR Green qPCR Mix for qPCR was purchased from TIANGEN Corporation (Beijing, China). The plasmid expressed shPD-L1, and all the primers for qPCR were synthesized by Sangon (Shanghai, China). The plasmid DNA-expressed mouse Spam1 was synthesized by GenePharma (Shanghai, China). An ATP assay kit was purchased from Beyotime Biotechnology Corporation (Shanghai, China). The flow cytometry antibodies were purchased from Thermo Fisher Scientific or Abcam. Immunohistochemistry or immunofluorescence antibodies were purchased from Thermo Fisher Scientific, Abcam, R&D Systems, or Servicebio Biotechnology Corporation (Wuhan, China). A PV-6000 two-step immunohistochemistry kit was purchased from Zhongshan Goldbridge Biotechnology (Beijing, China). ELISA kits were purchased from Thermo Fisher Scientific or Anoric Bio-technology Corporation (Tianjin, China). The other reagents were purchased from Sinopharm Chemical Reagent Co. Ltd., China. Antibodies used in this study were listed in table S1. Primers sequences used in this study were listed in table S2.

Synthesis and characterization of PLG

PLG was prepared according to the previously reported method (39). Bruker AV-300 nuclear magnetic resonance (NMR) spectrometer (Bruker, Ettlingen, Germany) was used to characterize the final product. D2O was used as the solvent.

Synthesis and characterizations of PLG-g-mPEG

PLG-g-mPEG was prepared according to the previously reported method (40). The final product was characterized by 1H NMR spectra through a Bruker AV-300 NMR spectrometer (Bruker, Ettlingen, Germany) in D2O.

Synthesis and characterizations of aldehyde group modified PEG (OHC-PEG-CHO)

OHC-PEG-CHO was prepared according to the previously reported method (10). Bruker AV-300 NMR spectrometer (Bruker, Ettlingen, Germany) was used to characterize the final product. CDCl3 was used as the solvent. To verify the pH sensitivity of the reaction between PEI and OHC-PEG-CHO, they were dissolved in D2O, adjusted pH of the solution by DCl and NaOD and reacted for 10 min. Then, the 1H NMR spectra were detected.

Preparation of the pH-responsive NPs for gene delivery system

For the preparation of pH-responsive NPs for in vivo gene delivery, the method was described in the following steps. First, PEI 25k aqueous solution (0.25 mg/ml) was mixed with PLG aqueous solution (0.1 mg/ml) in equal volume. After 15-s vortex and 10-min incubation at room temperature, DNA aqueous solution (0.1 mg/ml) was added into the previous solution for 15-s vortex and 10-min incubation. Last, different concentration of OHC-PEG-CHO aqueous solution was added into the above prepared solution and kept at room temperature for 5 min. PEG[(PLG/PEI)/DNA] (P[(GP)D]) complexes with mass ratio of (2.5 ~ 10):1:2.5:1 for PEG[(PLG/PEI)/DNA] were obtained.

Preparation of the pH-responsive NPs for drug delivery system

For the preparation of pH-responsive NPs for immunogenic chemotherapy, the method was described in the following steps. First, PLG-g-mPEG (100 mg) was dissolved in Milli-Q water (0.5 ml). After completely dissolved, dimethyl sulfoxide (DMSO) (4.5 ml) was added into the solution. The final concentration of PLG-g-mPEG was 20 mg/ml. DOX·HCl (50 mg) was dissolved in DMSO (5.0 ml) to form 10 mg/ml solution. Next, PLG-g-mPEG solution was mixed with DOX·HCl solution in equal volume. After 15-s vortex, the mixture solution was added into distilled water drop by drop and stirred for 0.5 hours in the dark to form stable micelle NPs. The excess drug was removed by dialysis [molecular weight cut off (MWCO 2000)] against deionized water for 48 hours. For determination of DLE and DLC, the solution after dialysis was diluted with 50 volumes of DMSO and measured by UV-Vis spectrometer at 480 nm. DLE and DLC were calculated according to the following equation

DLE (wt%)=(weight of loaded drug/weight of feeding drug)×100%

DLC (wt%)=(weight of loaded drug/weight of NPs)×100%

Zeta potential and particle size

The zeta potential and particle size of PEG[(PLG/PEI)/DNA] NPs and PLG-g-mPEG/DOX NPs (DOX NPs) were measured at room temperature by using a zeta potential/BI-90Plus particle size analyzer (Brookhaven, USA). Data were shown as means ± SD based on triplicate independent experiments. The morphological characteristic of the PLG-g-mPEG/DOX NPs was observed by field emission scanning electron microscope (Zeiss Merlin FE-SEM).

pH-responsive drug release behavior of DOX NPs

The pH-responsive drug release behavior of DOX NPs was evaluated in PBS of pH 7.4 or pH 6.8 or pH 5.5 at 37°C by a dialysis method. Briefly, DOX NPs were added in dialysis bag at a concentration of 2 mg/ml (4 ml) and set in a 50-ml beaker, 36 ml of PBS at different pH was added into the beaker. Then, put the beaker into a 37°C constant temperature shaking box, take out the liquid in 1 ml of the beaker at intervals and measure the UV absorption at 480 nm, and replenish the lost liquid in the beaker.

Cell lines and animals

Murine B16F10 melanoma cancer cells, murine 4T1 breast cancer cells and murine CT26 colorectal cancer cells were obtained from the cell bank of Chinese Academy of Sciences (Shanghai, China). All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) contains 10% fetal bovine serum. Cells were incubated at 37°C under a humidified atmosphere containing 5% CO2.

C57BL/6 mice, BALB/c mice, and BALB/c nude mice were ordered from Changsheng Experimental Animal Center (Liaoning, China). All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals, and all procedures were approved by the Animal Care and Use Committee of Jilin University.

Cell uptake

Cell uptake experiment was tested by flow cytometry. Briefly, B16F10 cells were seeded in six-well plates at a density of 1.5 × 105 cells per well. After 24 hours of incubation in 37°C, PEG[(PLG/PEI)/DNA] NPs (Cy5-DNA was used for NPs preparation) or DOX NPs were added to each well, the cells were incubated for another 4 hours. After incubation, the cells were washed twice with cold PBS and digested with trypsin for 30 s. DMEM was added and the cells were collected after washed twice with cold PBS. The cells were tested with a Guava EasyCyte flow cytometer (Guava Technologies).

In vitro DNA transfection

The DNA transfection experiment of the PEG[(PLG/PEI)/DNA] NPs was carried out in a murine B16F10 melanoma cancer cells. The luciferase plasmid DNA was used as the reporter gene. B16F10 cells were seeded in 96-well plates at a density of 1.0 × 104 cells per well and then cultured at 37°C for 12 hours. The culture medium in the plates was replaced with new DMEM with different pH values (7.4 and 6.8) before transfection, the PEG[(PLG/PEI)/DNA] NPs at various mass ratios were prepared and added into the plates and incubated for 2 hours. After that, the culture medium was replaced by fresh DMEM and then incubated for another 46 hours. The luciferase level in each well was measured according to the manufacturer’s instruction.

Cytotoxicity assay

The cytotoxicity of the NPs was measured by MTT assay. Briefly, B16F10 cells were seeded in 96-well plates at a density of 8000 cells per well and then cultured at 37°C for 12 hours. Different concentration of DOX and DOX NPs were added to each well, and the plates were returned back to the incubator for 48 hours. MTT (20 μl, 5 mg/ml) was then added to each well. After 4 hours of incubation, the medium was removed and 150-μl DMSO was added to each well for dissolving the formazan crystals. The samples were measured by using a TECAN Infinite F50 at 492 nm.

Confocal laser scanning microscopy

B16F10 cells were seeded in six-well plates at a density of 1.0 × 105 cells per well. After 24 hours of incubation in 37°C, DOX NPs were added to each well. After 2 hours of incubation, the cells were washed twice with PBS and fixed with 4.0% paraformaldehyde for 18 min at room temperature. After washed four times with PBS, the cell nuclei were stained by DAPI for 10 min. Last, the coverslips were carefully taken out and placed on the slides, enclosed with glycerol. The samples were observed by confocal laser scanning microscopy (CLSM) (ZEISS LSM780, Germany).

Induction of ICD with the DOX NPs in vitro and in vivo

To determine chemotherapy-induced ICD of the B16F10 tumor cells in vitro, surface expression of CRT, extracellular release of HMGB1, and ATP secretion were examined via different methods. For flow cytometry detection of CRT expression, B16F10 cells were seeded in six-well plates at a density of 3.0 × 105 cells per well. After 12 hours of incubation in 37°C, DOX and DOX NPs were added into each well at the identical DOX concentration of 10 μM and incubated for 6 hours. The cells were then washed twice with PBS and incubated with anti-CRT Alexa Fluor 647 for 45 min. The cells were washed twice with PBS and tested with a Guava easyCyte flow cytometer.

Intracellular HMGB1 release was visualized using immunofluorescence analysis. B16F10 cells were seeded in six-well plates at a density of 1.5 × 105 cells per well. After 12 hours of incubation in 37°C, DOX NPs were added into each well at the identical DOX concentration of 2 μM and incubated for 24 hours. The cells were then washed, fixed with 4% paraformaldehyde for 18 min, and permeabilized with 0.1% Triton X-100 for 10 min. After blocking with goat serum for 30 min, the cells were incubated with the anti-HMGB1 antibody for 1 hour and then incubated with Cy5-conjugated secondary antibody for 30 min. The cells were then stained with DAPI for 20 min and observed using CLSM.

Extracellular release of ATP was tested using an ATP assay kit. B16F10 cells were seeded in six-well plates at a density of 1.5 × 105 cells per well. After 12 hours of incubation in 37°C, DOX and DOX NPs were added into each well at the identical DOX concentration of 2 μM and incubated for 24 hours. Next day, the supernatants were collected and measured with an ATP assay kit according to the manufacturer’s instruction.

To demonstrate the DOX NPs could induce ICD in vivo via intravenous injection, C57BL/6 mice were injected by 1.0 × 106 B16F10 cells at the right flank. When the tumor volume reached 200 mm3, mice were intravenously injected with DOX NPs at a DOX dose of 5 mg/kg. Treatment was given twice every 4 days. Twenty-four hours after the last treatment, mice were euthanized and the tumors were harvested and fixed to make paraffin section. CRT and HMGB1 in tumor tissues were visualized via a typical immunohistochemistry (IHC) procedure. Anti-CRT antibody and anti-HMGB1 antibody were used as primary antibodies, respectively.

Establishment of animal models for in vivo tumor inhibition experiments

Subcutaneous B16F10 melanoma model was established by injection of 1.0 × 106 B16F10 cells in 100-μl PBS into the right dorsal flank of female C57BL/6 mice. Subcutaneous 4T1 or CT26 models for tumor inhibition were established by injection of 1.5 × 106 4T1 or CT26 cells into the right dorsal flank of female BALB/c mice. Subcutaneous 4T1 or CT26 models for PA imaging were stablished by injection of 2.0 × 106 4T1 or CT26 cells into the right dorsal flank of female BALB/c nude mice.

B16F10 tumor-bearing C57BL/6 mice, 4T1 tumor-bearing BALB/c mice, or CT26 tumor-bearing BALB/c mice were randomized distribute into different treatment groups, and the investigator was blinded to each group during the whole experiments. When the tumor volume reached 80 to 120 mm3, mice in different treatment groups were intravenously injected with PBS, DOX NPs, shPD-L1 NPs, DOX NPs + shPD-L1 NPs, Spam1 NPs, DOX NPs + Spam1 NPs, (shPD-L1 + Spam1) NPs, and DOX NPs + (shPD-L1 + Spam1) NPs (cocktail therapy group). We used PBS solution during injection to ensure a pH range around 7.4. The dosage for immunogenic chemotherapy was 5 mg/kg body weight on a DOX basis. The dosage for gene therapy was 1.2 mg/kg body weight on a total pDNA basis. The tumor volume and body weight were monitored every other day. Tumor volume was calculated by the formula: L × S2/2, where L referred to the longer diameter and S referred to the shorter diameter. PR represented tumors’ longer diameter was reduced by more than 30% compared with their initial longer diameter. After treatment, the animals were euthanized and the tumors and major organs were collected for further analysis. Tumors were divided equably into several pieces for flow cytometry assay, ELISA, RT-qPCR assay, and immunohistochemistry assay. In the survival experiment, mice were euthanized when their tumor size was larger than 2000 mm3. For the establishment of peripheral T cell depletion model, mice received FTY720 every other day via intraperitoneal injection at a dosage of 1 mg/kg in glucose solution. For the establishment of lung metastasis model, naive mice and mice treated by the cocktail therapy received 3.0 × 105 B16F10 cells via intravenous injection.

PA imaging experiment

To investigate whether Spam1 NPs could increase the accumulation and permeation of nanoparticles in tumor, PA imaging was carried out. Our previously reported FeT NPs, which composed of PLG-g-mPEG, ferric iron, and tannic acid, were used as contrast agent (41). For the in vivo PA imaging, FeT NPs (100 μl, 250 μg ml−1) were administered into 4T1 or CT26 bearing BALB/c nude mice via intravenous injection. Mice received PBS or Spam1 NPs via intravenous injection 2 days before FeT NPs injection. The PA signals of tumors were evaluated by the MSOT InVision 128 system (iThera Medical, Germany) at different time points after injection.

Flow cytometry assay

After treatment, tumor tissues, TDLNs, and spleen were made into single-cell suspensions. Lymphocytes were quantitatively analyzed by flow cytometry (BD Canto II) after staining. Briefly, tissues were harvested and grinded with 1-ml PBS in 12-well plates. The suspension was filtered through a 200-mesh sieve and then washed with PBS. Cells were collected and dispersed with 100 μl of PBS and stained by the addition of fluorescence-conjugated antibodies. Markers are as follows: CD3+ T cells (CD45+CD3+), CD8+ T cells (CD45+CD3+CD4CD8+), and CD4+ T cells (CD45+CD3+CD4+CD8). Another tube was stained to investigate M2 macrophages (CD11b+CD206+F4/80+) in tumor tissues. Maturation DCs were marked as (CD11c+CD80+MHC II+) in TDLNs. Cells in spleen were stained to investigate T lymphocyte subsets including CD4+ T cells, CD8+ T cells, and the TEM cells (CD3+CD8+CD44+CD62L). Antibodies used in flow cytometry were listed in table S1.

Immunohistochemistry or immunofluorescence analyses

To estimate the safety and therapeutic effect of NPs, heart, liver, spleen, lung, kidney, and tumors were stored in 4% paraformaldehyde and paraffin sections were made. H&E were used as dye for pathological analysis. For the IHC or immunofluorescence experiment, the already prepared paraffin sections were put in a 65°C drying oven for 1 hour, followed by deparaffinization and rehydration in xylene and graded alcohols (100, 95, and 75%). Citrate was used to retrieve antigen epitopes via high-pressure thermal repair. Primary antibodies used in IHC or IF were listed in table S1.

RT-qPCR assay

Tumor tissues were grinded in liquid nitrogen treated mortar, and total RNA was extracted by TRIzol according to the manufacturer’s instruction. The RNA was reversely transcribed to cDNA. RT-qPCR experiment was performed using SYBR Green qPCR Mix according to the manufacturer’s instruction on Roche LightCycler 96 instrument. Primers sequences used in this study were listed in table S2.

Enzyme-linked immunosorbent assay

Different kinds of proteins in tumor tissues were measured by mouse ELISA kits for tumor tissue homogenate. Tumor tissues were chipped and homogenized in cold PBS with ice bath. After centrifugation, the supernatants were collected and measured according to the manufacturer’s instruction.

Statistics and data analysis

GraphPad Prism 8.0 software was used for constructing graphs and analyzing statistical significance. FlowJo v10 software was used for flow cytometry analyses. Student’s t test and log-rank (Mantel-Cox) test were used to calculate statistical significance.

Acknowledgments: We would like to thank all the laboratory members for cooperation in this study. Funding: This work is supported by the National Natural Science Foundation of China (51925305, 51873208, 51520105004, 51833010, 51973217, and 51803210), National Science and Technology Major Projects for Major New Drugs Innovation and Development (2018ZX09711003-012), and Jilin Province Science and Technology Development Program (20180414027GH and 20200201075JC). Patent: We are applying for a Chinese invention patent. Application number: 202010530812.2. Author contributions: J.W. and H.T. designed the experiments. J.W. performed the experiments. J.C., Y.F., S.Z., L.L., P.S., and C.X. contributed to the bioassays. Z.G. instructed the synthesis. J.W., J.C., H.T., and X.C. analyzed the data and wrote the manuscript. H.T. supervised the study. Competing interests: X.C., H.T., J.W., J.C., L.L., Z.G., P.S., and C.X. are inventors on a pending patent related to this work filed by State Intellectual Property Office of China (SIPO) [no. 202010530812.2, filed (12 June 2020)]. The other authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

‘Provocative results’ boost hopes of antibody treatment for COVID-19 | Science

‘Provocative results’ boost hopes of antibody treatment for COVID-19 | Science

  • September 30, 2020

Companies are developing COVID-19 treatments using monoclonal antibodies, Y-shaped immune proteins that target the pandemic coronavirus.

KTSDESIGN/Science Source

Sciences COVID-19 reporting is supported by the Pulitzer Center and the Heising-Simons Foundation.

A second company has now produced strong hints that monoclonal antibodies, synthetically produced versions of proteins made by the immune system, can work as treatments in people who are infected with the pandemic coronavirus but are not yet seriously ill.

The biotech Regeneron Pharmaceuticals has developed a cocktail of two monoclonal antibodies that attach to the surface protein of that coronavirus, SARS-CoV-2, and attempt to block it from infecting cells. Yesterday at an investor and media webcast, the firm revealed early results.

The company showed slides with detailed data from 275 infected people in a placebo-controlled trial that ultimately plans to enroll 2100 individuals who are asymptomatic or, at worst, moderately ill. The analysis divides patients into two groups: those who had detectable antibodies against SARS-CoV-2 at the trial’s start and those who did not, a so-called seronegative group. The monoclonal cocktail showed little effect on people who already had antibodies against the virus. But it appeared to help the seronegative patients, powerfully reducing the amount of virus found in nasopharyngeal swabs and alleviating symptoms more quickly. “These are provocative results,” says Myron Cohen of the University of North Carolina, Chapel Hill, who was not involved with the study but is helping Regeneron test its monoclonal cocktail as a preventive.

Cohen notes that Regeneron’s data look similar to those in a press release from Eli Lilly 2 weeks ago about early results from a trial of its single monoclonal antibody against SARS-CoV-2. “Both of these reports go in the same direction,” Cohen says. But he cautions that neither has been published, both trials are ongoing, and more data are needed to understand how—or whether—these experimental medicines can best help patients. Lilly, oddly, did not see an impact at the highest dose of antibody tested, and Regeneron saw no difference between its low- and high-dose preparations used in the study. 

James Crowe, a viroimmunologist at Vanderbilt University who is working with AstraZeneca to develop COVID-19 monoclonal antibodies, welcomed Regeneron’s detailed preliminary results. “I applaud Regeneron for releasing so much information,” Crowe says. “They’re contributing to public health by releasing this as soon as possible.” But he notes even people who did well on the monoclonal cocktail still had low levels of virus detectable after their treatment, which in theory could cause problems. “I was surprised that there was any virus at all given that these are such potent antibodies,” he says, adding that the residual virus detected in the swab tests may not be capable of copying itself.

The monoclonal antibodies from the two companies are clones of potent SARS-CoV-2 antibodies that can “neutralize” the virus in test tube studies. Researchers plucked the genes for these antibodies from humans who recovered from COVID-19 or from mice artificially infected with the virus. The companies then put the genes in Chinese hamster ovary cells to bulk manufacture the antibodies, which were given to the COVID-19 patients as infusions.

At the webcast that announced Regeneron’s results, George Yancopoulos, president and co-founder of the company, emphasized how the “target population” for the monoclonal cocktail are SARS-CoV-2 infected people who have “not yet mounted their own immune response” and have exceedingly high levels of the virus. “What we really want to do is turn them into patients who have already started to effectively fight the virus,” Yancopoulos said.

In the Regeneron data, the most dramatic drops in SARS-CoV-2 were seen in seronegative patients who had the highest levels of virus at the trial’s start. In comparison with patients who received the placebo, the results were clearly statistically significant.

Daniel Skovronsky, Lilly’s chief scientific officer, says the Regeneron data are “quite confirmatory” of their own. “I don’t expect there to be large differences between good neutralizing antibodies,” Skovronsky says. “Antibodies will work best in people who can’t clear the virus on their own.” One key difference between the two studies, he says, is that Lilly enrolled fewer seronegative people and still found an impact—although the company, in contrast to Regeneron, is withholding details until it publishes results. Lilly also stressed that people receiving its antibody were shown to have fewer hospitalizations or emergency room visits: five out of 302 (1.7%) treated patients versus nine out of 150 (6%) in the placebo group. “Yes, these are small numbers by some measures,” Skovronsky says, “but by other measures, there are significant differences in hospitalization.”

Regeneron hasn’t yet accumulated enough data to show the same protection. Its trial had only 12 patients who had “COVID-19related medically attended visits.” Although there was a trend toward more of these in the placebo group than treatment arms, only one was hospitalized.

Regeneron’s data raise difficult questions about when to use its cocktail. People who test positive for SARS-CoV-2 aren’t routinely screened for antibodies to it or for levels of the virus. “If the decision is going to be made to deploy such a therapeutic solution in the patients who might benefit the most and need it most, we’re going to have to solve the problem of using the right point-of-care diagnostic tools, either for serology or high viral load,” Yancopoulos said, noting that their partners—including Roche—are developing these types of assays.

Skovronsky says Lilly has a simpler plan: Offer monoclonals to people who test positive for the virus if they are in high-risk groups for developing severe disease, which include the elderly and people with underlying diseases such as diabetes or who are overweight. Running extra tests before treating people, as Regeneron suggests, “is just not going to meet the needs of the population,” he says

Both Lilly and Regeneron say they are discussing their data with regulators to see whether their monoclonal antibodies might warrant moving to widespread use more quickly through mechanisms like the U.S. Food and Drug Administration’s emergency use authorization process. Additional studies of their monoclonal treatments are underway in hospitalized COVID-19 patients and, separately, as preventives in uninfected people.

Monoclonal antibodies are more difficult to make than many drugs and often are extremely expensive, which means that supply could outstrip demand and many countries might not be able to afford them. The U.S. government’s Operation Warp Speed has invested $450 million in Regeneron to produce up to 300,000 “doses” of its cocktail by the end of the year, which would be distributed to Americans free of charge. “A substantial fraction of those are already available,” Yancopoulos said—although it’s not yet clear what constitutes a single dose of the company’s cocktail. Nonetheless, Regeneron, which is partnering with Roche to increase production capability, says it hopes to ramp up to produce 250,000 doses per month.

Skovronsky says if the lowest dose Lilly is testing works, it could have up to 1 million doses by the end of the year. Lilly is partnering with Amgen to scale up production to “several million doses” next year. “We’re rooting for Regeneron’s success, just as Regeneron is rooting for Lilly’s success,” he says. “None of us can make enough antibodies to meet the need.”

“Immune Boosting” in the time of COVID: selling immunity on Instagram | Allergy, Asthma & Clinical Immunology

“Immune Boosting” in the time of COVID: selling immunity on Instagram | Allergy, Asthma & Clinical Immunology

  • September 30, 2020

Our daily collection of top posts over a week amounted to 28 samples (n = 28) from 26 unique accounts, following the removal of duplicate content appearing on multiple days. The average number of days for a post to remain in the top 10 was 2.42 (see Table 1). These 28 top posts included 55 tags on images, more than 17,000 likes, 539 hashtags (413 unique; average of 19.25 hashtags per post), 1 URL, and 17 unique @s directed to other Instagram accounts. Collectively, the 26 account holders of these top posts tally more than 500,000 followers, demonstrating considerable influence among some Instagram users.

Table 1 Complete coding analysis of the unique top Instagram posts using #immunebooster over one week in May 2020 (n = 28)

We found 17 of the 28 (61%) posts feature “immune boosting” as a central idea and 11 posts as peripheral. All the posts portray “immune boosting” as beneficial. Most posts portray or intimate a general benefit to “immune boosting.” Specific benefits are associated with improved mood, anti-inflammation, increased metabolism, disease prevention, personal protection, gut health, better cognition, and skin care. None of the posts critique or question the value or validity of “immune boosting” in any way. Seven posts refer or appeal to scientific or medical authorities, including dietitians, nutritionists, doctors, and experts. One post mentions scientific research or evidence, which, in this instance, was a single unsubstantiated reference to “clinical studies.” 8 posts refer to COVID-19 through such phrases and hashtags as “Now more than ever it’s important for us to boost our immunity,” “#lockdowncooking,” and “#quarantineandchill.”

Of the 26 unique accounts sampled, 25 were commercial accounts, which we defined as any account that sells or advertises commercial products or services. This definition includes “influencer” accounts that advertise other people’s or companies’ products or services. Three posts are listed as paid promotions. We identified a total of 62 different companies tagged or mentioned in these posts, 53 of which are distinct from the accountholder. 75% of the posts tagged one or more companies. In order of decreasing frequency, the focus of these companies are related to food (ingredients and cooking); general health and wellness; clothing, fashion, and accessories; exercise advice and products; beauty products and advice; nutrition supplements; essential oils; home décor and furnishings; self-help and self-improvement; travel; technology; brand management; and a humanitarian non-government organization.

About 30% of the posts appeal to some form of medical or scientific authority and several have text suggesting the rhetoric of scientific evidence, which is “text that draws on scientific sounding language in order to create a veneer of legitimacy” [20]. For instance, post 13 recommends a dinner recipe that “helps boost your Microbiome and immunity.” That post was made by a self-identified general practitioner and health journalist who sells a series of diet and recipe books. Post 5 featured a “registered nutritionist, author, media regular, and mom” offering some tips “to boost your #immunity,” which entailed taking their brand of supplement and probiotic products. Another example using scientific rhetoric is Post 15, from the account of a personal trainer and “Creative Brand Consultant” who sells protein bars. The advertised product in Post 15 is a supplement, which is described as “a unique formula made with clinically-studied microbiome strains” and containing “organic ashwagandha- a plant known for its adoptogenic properties.” Post 9 includes a softer appeal to scientific knowledge from an influencer’s account, which advertises a variety of healthy food and supplement products with dedicated discount codes. This post has an image of a labelled jar of apple cider vinegar gummies held above a “loaded oatmeal bowl.” The theme is about healthy living and the caption connects the product to the COVID-19 pandemic: “Apple cider vinegar is known to improve digestion, detoxes the body, and provides immunity boosting which I think we could all use a little extra right now!”

Three accountholders included medical warnings concerning their immune-boosting products or services. The caption for post 21 has the following bolded disclaimer: “The Content is not intended to be a substitute for professional medical advice, diagnosis, or treatment. Always seek the advice of your physician or other qualified health provider with any questions you may have regarding a medical condition. Never disregard professional medical advice or delay in seeking it.” This emphatic deference to medical expertise contrasts with another post’s warning. Post 6 lists numerous contraindications and cautions for an “immunity booster pose.” The caption suggests the yoga pose “powers our immunity by stimulating the digestive system and stimulate the thymus, an organ located behind the chest bone that is responsible for the growth of T-cells.” Taken with the medical jargon, the warning in post 6 serves to legitimize the claim that the pose offers real and substantial immunity benefit.

Most posts expressly or implicitly advertised products or services as “immune boosting.” The image in post 2, for example, has a woman holding a jar of juice and is captioned as an “ORGANIC IMMUNE BOOSTING TONIC” comprised of “Naturally FRESH homemade immune booster.” The accompanying description claims this “vitamin powered” tonic is anti-inflammatory, antioxidant, antibacterial, and anti-fungal and can fight infections, prevent asthma, boost metabolism, aid “health cell growth,” and counter viral infections. The post also promotes the accountholder’s IGTV channel dedicated to selling the “immune boosting” tonic. Many commercial posts associated “immune boosting” with other popular health and lifestyle topics, including gut health, anti-inflammation, veganism, and “natural” foods and products. Post 21 comes from an account selling a line of essential-oil health and beauty products. The post has an infographic for “FOODS TO BOOST THE BODY IMMUNE SYSTEM,” which graphic is reminiscent of a nutrition guide. This post also includes the bolded disclaimer discussed above.

Some posts mentioned the COVID-19 pandemic in connection to their advertised products and services. Post 4 offered “a FREE 30-day #GetImmunityUpgraded challenge.” The caption promised that the advertised challenge, which includes workout routines, food recipes, and expert advice, will “protect you, your family, your friends and our world,” and ultimately “help flatten the curve.”

9 Health Benefits of Limes: Are Limes Good For You?

9 Health Benefits of Limes: Are Limes Good For You?

  • September 30, 2020

Lime, this precious little green fruit. Some might recognize it from the gin cocktails and vodka drinks they are often put in, some of you might have tried its taste in salads or even cake. We believe we can all agree that not only does lime look good, it’s also tasty. However, there is much more to limes besides their beauty and taste. Science has proven that limes have their fair share of health benefits.

Do you want to know what those benefits are? Then, take a look at the list we compiled of nine benefits of limes.

1. They Can Give Immunity a Boost

Limes are quite rich in vitamin C. We’ve all heard of vitamin C’s importance for foods that boost immunity. In numerous studies that have been done on the subject of immunity, vitamin C seems to be the nutrient that impacts the growth of the white blood cells — the cells that fight against viruses and infections. Even if the white cells couldn’t overcome the viruses, vitamin C shortens the duration and severity of the cold.

Besides vitamin C, limes are abundant in antioxidants. Antioxidants join the vitamin C in the fight to strengthen the immune system.

2. They May Enhance the Heart Function

We mentioned that limes brim with antioxidants, among many other healthy components. These antioxidants have a significant impact on the cardiovascular system. They help keep the arteries and the circulatory system healthy and functional. The arteries’ good functionality and flexibility mean that the blood pumped by the heart will be transported without any problem.

Vitamin C can also help lower blood pressure — one of the leading causes of heart diseases worldwide. The said vitamin can also help keep the arteries from forming plaques, fighting atherosclerosis disease.

While a human study on this subject hasn’t been done yet, study results on rabbits suggest that lime juice and peels could slow down the progression of atherosclerosis.

3. They Can Improve Skin Health

Antioxidants and vitamin C, two elements that limes are known for, are two of the ingredients that many skin products have. Vitamin C levels of collagen production, a protein that keeps your skin strong, elastic, and hydrated. The cosmetic industry has been using this ingredient in its products and for years now, and you can even find it in protein powders.

What if we tell you that you could get the needed vitamins and antioxidants to keep skin healthy through a glass of water and a hefty squeeze of lime?

The impact of vitamin C on skin health has been studied for a while. In one of the studies, in which 4,000 women participated, those who consumed vitamin C experienced a lower risk of developing wrinkles and dry skin as they aged.

4. They May Reduce the Risk of Developing Cancer

Cancer is characterized by the abnormal growth of cells and their spread around different organs and tissues. While there isn’t one specific leading cause of this disease, our lifestyle can greatly impact the development of cancerous cells. Just as there are types of foods that can cause cancer, there are foods that fight it. One of them happens to be lime. Being a citrus fruit and having its properties, lime is inevitably linked to a lower risk of cancer.

Another study in citrus fruits such as lime showcases their ability to subdue various cancerous cells’ growth and their spread in different parts of the human body.

5. They Can Improve Digestion

As we all know, we have a digestive system whose function is to process the food we eat. Some types of food take longer to digest, and some take less time. However, one can aid the stomach in this process by consuming food known for its digestive properties. Lime is one of the fruits that, thanks to its acidity, may help the digestive system by breaking down food. Also, drinking lime water first thing in the morning can help wake up the digestive system.

Lime water can help even those that suffer from acid reflux, commonly known as heartburn. Drinking warm lime water 30 minutes before a meal might help prevent reflux symptoms and discourage overeating.

6. They May Prevent the Formation of Kidney Stones

Kidney stones are a common condition that occurs in people of both genders. While we still don’t know what exactly causes them, there is a list of possible factors that significantly impact their development. Luckily, there are options that one can choose to prevent kidney stone formation.

Being careful about the diet we choose to consume daily can be one of these options. Making lime part of the daily diet can help prevent kidney stones from forming in the first place. How can that happen, you might be wondering? Lime has high citric acid levels that can hinder kidney stones by raising levels of citrate and stone-forming minerals in the urine.

7. They May Increase the Nutrient Absorption

Iron is one of the minerals that the body needs to function properly. Said more precisely, iron is a crucial element in the production of blood and hemoglobin. Low levels of this mineral can cause a host of issues. Vitamin C, meanwhile, has an essential role in the body’s absorption of iron. It seizes non-heme iron and stores it in a form that is easier for the body to absorb. A study conducted on the effect of vitamin C on iron absorption showed that taking 100mg of this vitamin with a meal increases iron absorption by 13%.

8. They Can Help Boost Your Metabolism

Losing weight has to be one of countless people’s most sought-after goals. Everybody wants to achieve and maintain a healthy weight. However, when it comes to losing extra weight, most of the time, it is easier said than done.

Don’t lose hope entirely, as you can still achieve your goal weight. You just have to commit to a lifestyle that will make it easier for you to accomplish what you want. You can start by adding lime to your diet. This healthy fruit filled with citric acid can kick-start your metabolism and, in turn, help you burn more calories and store less fat.

However, one thing to keep in mind is that you shouldn’t rely entirely on the effects of the lime in the metabolism. You have to keep your end of the bargain as well. Try to dedicate some time to physical activity like running or workouts. That, combined with the lime properties, will surely bring the results you’re looking for.

9. They Can Reduce Inflammation

Inflammation is the leading cause of many joint conditions. As such, one is always on the lookout for medical and natural treatments for these diseases. Luckily, there is a natural remedy for those who suffer from joint inflammation. Lime water can alleviate some of the symptoms these conditions have. Thanks to the minerals lime water has, especially Vitamin C, one can get rid of the joint pain. Studies show that those who have a lower intake of Vitamin C experience an increased risk of developing arthritis.

Limes seem to be the all-in-one-package fruit. They are pretty to look at, taste great, and have many benefits to the human body. What more can one ask from a fruit? We’re sure that from now on, limes will be even more present in your life. Not only will they make your life more colorful, but also healthier.

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Virtual Immunity Champions of India Awards 2020 held to honour COVID-19 Fighters

  • September 30, 2020

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In times when a strong immunity is top priority, companies fighting on the frontlines for immunity boosting were honoured for their noble services

While the importance of having a strong immune system was emphasised on since time immemorial, the COVID-19 pandemic threw much more light on the sensitive issue. In such days, when hectic and stressful lifestyles are the new normal, dangers of having a weak immune system loom overhead. To help us realise this, several pharma companies and health organisations have been consistent in their efforts to help us boost our immune systems. Thus, to honour their diligent efforts and to offer them a token of appreciation for their invaluable services, the virtual edition of Immunity Champions of India Awards was organised by HEAL Foundation. The ceremony lauded efforts on both organisational as well as individual levels.

On the Organisational level, there were five categories of awards – Food and Technology Award, Immunity Booster of the Year, Immunity Startup Award, Corporate Social Responsibility (CSR) Initiative of the Year Award, Innovation in Food Supplementation Award and Nutrition Campaign of the Year Award. Appreciating the efforts of each and every company part of the Immunity Booster initiative, Dr Swadeep Srivastava, Founder, HEAL Foundation, said, “In such times of hardship and stress, there is no award or trophy big enough to congratulate our awardees for the noble work that they are doing. Day and night, they have been wracking their brains and coming up with solutions and ideas which can help enhance public immunity and eventually drive out the deadly virus from our beloved country. HEAL Foundation hopes that this small token of appreciation on our behalf can serve as a constant reminder to these brave warriors to keep on working for their cause, and always remember that they have the faith of the nation in them.”

Immunity Champions of India Awards 2020 have been conferred upon the following organisations for their respective categories:

S.No. Award Category Recipient Award Given for Exemplary Work
01. FOOD AND TECHNOLOGY AWARD ASPARTIKA BIOTECH Used indigenous technology to come up with ‘Daily Drops’ and ‘Beam Roti’, two immunity booster products which were supplied to migrant workers as ready to bake food during lockdown.
02. IMMUNITY BOOSTER OF THE YEAR MAHARISHI AYURVEDA Their product, Maharishi Amrit Kalash, has already been sold in 50+ countries for the last 35 years. They have created a global awareness of immunity-boosting measures through Ayurveda, provided tangible solutions and also organised a global webinar with the AYUSH Ministry and 20 countries worldwide.
03. IMMUNITY BOOSTER OF THE YEAR CONVERGE BIOTECH One of the first strong promoters of high dose Vitamin C in the treatment of COVID-19, Converge Biotech also organised highly informative webinars and scientific sessions with renowned doctors from the early stages of the pandemic itself. They also significantly contributed to increasing COVID-19 recoveries during a very crucial phase and made the treatment for COVID-19 induced ARDS extremely cost-effective.
04. IMMUNITY BOOSTER OF THE YEAR KEPLER HEALTHCARE (MAKERS OF MOKTEL IMMUNE) They launched ‘Moktel Immune’, a multi-herb formula for enhancing doctors’ immunity during the global pandemic. Over 1 lakh chemists and doctors have been benefitted by Moktel Immune and been consuming it to protect themselves from the virus,
05. IMMUNITY STARTUP AWARD MALLIPATHRA NUTRACEUTICALS The company has developed novel process and product for immunity boosting comprising Cordyceps in the form of Capsules, Fruiting body, Infusions and fruiting body powder with the support of BIRAC-GoI, CCAMP, Sir MVIT, BBC and KITS-GoK.
06. CORPORATE SOCIAL RESPONSIBILITY (CSR) INITIATIVE OF THE YEAR STEADFAST MEDISHIELD The company took the noble initiative of committing INR 15 Lakh for setting up COVID-19 Testing Labs. They also supported the Institute of Kidney Diseases and Research (IKDRC), Ahmedabad
07. INNOVATION IN FOOD SUPPLEMENTATION STEADFAST MEDSHIELD They launched ‘HerbFizz’, a blend of nine neo-ancient herbs to boost immunity, improve digestion and detox toxins from the body.
08. NUTRITION CAMPAIGN OF THE YEAR BAXTER INDIA Right Lipid Right Nutrition: The parent company which launched ‘Oliclionomel’, a third-generation olive oil- based emulsion. Oliclinomel is indicated in Parental nutrition for adult and children more than 2 years when oral and enteral nutrition is impossible, insufficient or contraindicated.

The HEAL Foundation also took up the initiative to honour individuals who made a special mark through their brilliant efforts during the COVID-19 pandemic. The winners of India Immunity E-Summit 2020 (Individual Categories) have been mentioned below:

S.No. Award Category Recipient Award Given for Exemplary Work
01. NUTRITION CHAMPION OF THE YEAR APARNA TANDON JAIN She has worked upon two new products with more protein and antioxidants as the head of New Product Development. She has also educated her audience well through several informative blogs on nutrition.
02. NUTRITION CHAMPION OF THE YEAR MEHAK SETHI She took up the noble initiative of providing free nutrition counselling to 15-18 individuals on a daily basis, resulting in the improvement of health of nearly 500 people free of cost.
03. INNOVATION IN IMMUNITY AND DIET MODULE JAYA MOHAN She created an innovative diet to help a patient with hypothyroid conceive. With 800+ clients all over the country, she has played a keen role in promoting awareness surrounding physical and mental health.
04. INNOVATION IN IMMUNITY AND DIET MODULE GAURI ANAND She promoted iodine consumption and integrated healthy diet planning and consumption to help out clients free of cost. She created innovative diets to aid people in such testing times with no charge.
05. INNOVATION IN IMMUNITY AND DIET MODULE SAGOLSEM REEDYNA She counselled 50+ patients daily, donning on PPE kits to physically visit them and ensure that they are keeping healthy. She also emphasised on the consumption of locally-available natural food products and their importance in a healthy lifestyle.

Talking about the role of these essential health workers during the COVID-19 pandemic, Dr C S Pandav, one of the esteemed Jury Members, said, “We need such inspirational, young leaders to take our country in the right direction. I reached this realisation when I came across the exemplary work that they have done and the things they have achieved in such a short span- the right people should be brought to the forefront of our battle against COVID-19.” Soni Sangwan, another member on the panel, added, “Eating intelligently is an art. After coming across the wonderful speakers in this event, I have realised that it is as much a science. My heartiest congratulations to all the winners, whose small steps have made such a major contribution to changing lifestyles.”

Scientists propose immune cocktail therapy to boost cancer-immunity cycle in multiple aspects

Scientists propose immune cocktail therapy to boost cancer-immunity cycle in multiple aspects

  • September 30, 2020
Scientists propose immune cocktail therapy to boost cancer-immunity cycle in multiple aspects
Schematic diagram of immune cocktail therapy. Credit: CIAC

Immune checkpoint blockade therapy (ICT) has shown potential in the treatment of multiple tumors, but the poor response rate has restricted its further application.

Although scientists have developed some combination treatments to enhance the efficacy of ICT, satisfactory tumor inhibition in a variety of tumor models has not been achieved.

Recently, however, a research team led by Prof. Tian Huayu from the Changchun Institute of Applied Chemistry (CIAC) of the Chinese Academy of Sciences proposed an innovative immune that combined ICT along with other therapeutic approaches. The cocktail therapy achieved multiple boosting of the cancer-immunity cycle by utilizing a nano-delivery system. The study was published in Science Advances on Sept. 30.

The proposed cocktail therapy achieved anti-tumor treatment by combining immunogenic chemotherapy, immune checkpoint blockade, and extracellular matrix elimination. It consists of two kinds of tumor microenvironment (TME) responsive drug and gene delivery nanoparticles. The smart nano delivery systems can achieve specific delivery of doxorubicin and co-delivery of plasmids expressed small hairpin RNA of PD-L1 and hyaluronidase in the tumor area, leading to improved .

The cocktail therapy can also facilitate T cell priming by inducing tumor immunogenic cell death and polarizing an immunosuppressive TME to an immune-active phenotype.

Benefiting from these advantages, outstanding immunotherapeutic effects were achieved in multiple tumor types. For example, the cocktail therapy induced dramatic tumor shrinkage in B16F10, CT26 and 4T1 tumor models, making it more efficient than a traditional combination of chemotherapy and ICT.

“These excellent outcomes are mainly attributed to the increasing amount of peripheral CD8+ T cell infiltration in tumors, which can also induce strong immune memory effects and effectively prevent tumor metastasis,” said Prof. Tian.

This work presents a promising comprehensive immunotherapy strategy that integrates multiple aspects of regulating the cancer-immunity cycle, such as tumor antigen release, T cell trafficking from the periphery to the tumor, effective killing of cells, and the generation of immune memory T .

The cocktail therapy strategy provides a new technique for combining treatment approaches synergistic with ICT. It is a way to develop more efficient antitumor immunotherapy in the clinic.

Nanomaterials based strategies for treatment of hypoxic tumor

More information:
“An immune cocktail therapy to realize multiple boosting of the cancer-immunity cycle by combination of drug/gene delivery nanoparticles” Science Advances (2020). DOI: 10.1126/sciadv.abc7828

Scientists propose immune cocktail therapy to boost cancer-immunity cycle in multiple aspects (2020, September 30)
retrieved 30 September 2020

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These 11 Foods Are Exactly What to Reach for to Fight Stress

These 11 Foods Are Exactly What to Reach for to Fight Stress

  • September 30, 2020

We’ve all experienced it, short-term, long-term, physically, or mentally: Stress. It’s an innate reaction to life experiences, good and bad. It’s something we can’t completely control, but we can stay mindful by meditating, running, or listening to music, reading, taking a walk, phoning a friend, or whatever else helps you personally to alleviate tension. One of the most important ways to mitigate stress and anxiety starts with your diet, since certain foods contain vitamins, minerals, and amino acids that boost your brain’s “feel good” hormones serotonin, dopamine, and reduce cortisol, often called the “stress hormone.”

People deal with stress mechanisms differently, but many of us eat for comfort. If you run to your kitchen to cope with anxiety, tension, worry, or self-doubt, there are foods that you can reach for that will benefit your mood. The Beet complied a list of the top 11 foods to eat that will help you fight stress, boost productivity, and make you feel calmer and more joyful in no-time. Here’s what to eat to fight stress and find calm.

1. Tahini Contains an Amino Acid That Treats Anxiety and Stress

Tahini is a sesame spread, the core ingredient in hummus, and prominently used in the Mediterranean diet. You probably eat this regularly without even knowing. This creamy and flavorful spread is an incredible source of the amino acid L-tryptophan, a serotonin precursor that helps reduce stress and anxiety. In a review study, researchers say that L-tryptophan is “verifying a nutritional approach to the treatment of anxiety.” A deficiency in tryptophan may lower your serotonin system and cognition function according to a different study. The best to load up on tahini is to drizzle the spread onto your greek salad or dip your favorite veggies in an oil-free hummus!

2. Matcha Powder Contains a Natural Amino Acid That Helps You Relax

This green tea powder is used as a natural caffeine replacement since it doesn’t give you the same jittery kick that coffee does, and lasts longer to avoid the crash you’re probably familiar with coffee. Matcha gained its popularity over the past few years since the vibrant green color makes for a likable photo opportunity, but has been around for centuries and can be traced back to the Tang Dynasty in China.

Pretty aesthetic aside, matcha delivers some powerful health benefits and is loaded with antioxidants that help reduce stress. In a study, researchers analyzed the relationship between the intake of matcha components and the suppression of stress in a mouse model. Results concluded that a “significant stress-reducing effect was observed.” Matcha has a balanced combination of caffeine and L-theanine, which is a natural amino acid found in plants that helps with relaxation without the drowsiness. In a study, researchers note, “L-theanine alone improved self-reported relaxation, tension, and calmness starting at 200 mg. Generally, one scoop of matcha, 1 gram, equals the same caffeine in an espresso shot, or 35 mg of caffeine.

3. Swiss Chard Contains High Levels of Magnesium Reducing the Activation of Stress

This leafy green vegetable is loaded with stress-reducing minerals and nutrients like magnesium. In a review study, researchers examined the “link between magnesium deficiency and stress, focusing on the association between magnesium and various stress pathologies, magnesium’s potential interaction with stress pathways, and magnesium’s effects on the brain.” The results concluded that an increase in magnesium may help reduce stress and if you have low levels on this mineral, then you are more likely to activate stressors. In a different study, “findings suggest oral magnesium supplementation alleviated stress in healthy adults with low magnesemia.”

One cup of cooked Swiss chard, (175 grams), contains 36% of the recommended intake for magnesium, so this leafy green vegetable is a healthy way to consume this essential mineral.

4. Acerola Cherries Are a Good Source of Vitamin C Which Gets Depleted by Stress

The National Library of Medicine says acerola cherry powder is an untapped functional superfruit. This fruit powder is a rich source of antioxidants like ascorbic acid also known as vitamin C that “appear to be a promising candidate in combating various diseases associated with the oxidative stress,” according to the study. When you stress, vitamin C disappears from the bloodstream at an extremely rapid rate, according to Dr. James L. Wilson, author of Adrenal Fatigue: The 21st Century Stress Syndrome. Consumption of vitamin C during stressful times since it’s lost in your body. In that case, acerola cherries are your best friend since they are one of the most concentrated sources of vitamin C you will find in plants. One fruit contains 80.5 mg of vitamin C, that’s almost more than half the vitamin C in oranges and almost triple the vitamin C in lemons.

5. Kimchi is a Fermented Food that Contains Probiotics That Reduce Social Anxiety

If you love fermented food, say hello to your new favorite snack, kimchi. This cabbage medley is loaded with health benefits, packed with beneficial bacterias, probiotics, and essential vitamins and minerals. In a study, researchers “determined whether the consumption of fermented foods likely to contain probiotics interacts with neuroticism to predict social anxiety symptoms.” Results found that the consumption of fermented foods that contain probiotics may reduce social anxiety and stress levels. In a different review study, researchers said, “higher frequency of fermented food consumption was associated with fewer symptoms of social anxiety.” In another study, “findings suggest that kimchi is a promising functional food with an antioxidative effect and fermentation of kimchi led to the elevation of antioxidative activity.”

6. Artichokes Contain Prebiotics That May Uplift Your Mood

It’s no joke that artichokes are extremely high in fiber and promote gut health since they contain prebiotics that may help reduce stress levels. Artichokes are rich in a prebiotic called fructooligosaccharides, often known as FOSs. In a study, researchers tested “whether chronic prebiotic treatment modifies behavior across domains relevant to anxiety, depression, cognition, stress response, and social behavior,” results found that the prebiotic FOSs is beneficial for stress-related behaviors and may reduce the stress that builds up in your gut. Depending on your stressful situation, if it’s short-term you will experience the stressful pain in your gut which causes constipation, loss of appetite, and slowed digestion. If the stress is long-term you may experience gastrointestinal (GI) issues and should contact your doctor. Another study review suggests, “high-quality diets, prebiotics, and probiotics may beneficially affect mood.”

7. Garlic is Often Used as a Therapeutic Food that Protects Cells From Stress

You might add this vegetable to your pasta and bread because garlic’s distinctive taste is the perfect addition to any savory dish. But, others may add garlic to food since it’s classified as a “therapeutic food,” because it contains CYP2E1 inhibitors and CYP3A enzymes that are used to treat diseases like alcoholism, therapeutically. Garlic also contains sulfur compounds increasing the antioxidant called glutathione. “Glutathione provides a critical defense system for the protection of cells from many forms of stress,” according to a study.

8. Parsley Reduces Stress and Supports the Cellular Antioxidant Defense System

Parsley is arguably the best ingredient in a roasted veggie dish since it brings out a bitter yet flavorful flavor, but more importantly, this is an essential herb that may reduce stress levels and make you feel a bit more joyful. In a study, researchers examined “parsley-rich diets in terms of eliminating stress-induced oxidative gastric injury were evaluated.” The study says “oxidative stress has been shown to play a principal role in the pathogenesis of stress-induced gastric injury.” The results concluded, “oral administration of parsley is effective in reducing stress-induced gastric injury by supporting the cellular antioxidant defense system.”

9. Broccoli is a Cruciferous Vegetable That Boosts Positive Mental Health

Broccoli is a renowned cruciferous vegetable known for its many health benefits. Several studies claim diets rich in cruciferous vegetables may reduce your risk of cancer, heart disease, and mental health disorders. In one study, researchers investigated different foods that promoted recovery from depressive disorders. The results found that the highest scoring foods were all plant-based and included cruciferous vegetables. Like garlic, broccoli contains the compound sulfur, increasing levels of glutathione, an antioxidant that protects against many forms of stress, according to a study. Preferably, it’s healthier to eat broccoli raw since the sulforaphane gets activated when the vegetable is cut, chopped, and chewed, instead of being cooked. To learn more about Broccoli and health benefits, click here.

10. Chamomile Tea Produces Calming Effects and Reduces Anxiety

Traditionally, chamomile tea was used to treat insomnia and induce sedation, calming effects since the herb contains flavonoid, apigenin that binds to benzodiazepine receptors in the brain, medical tranquilizers that relaxes you, according to a study. In a sleep study, researchers concluded that the “inhalation of the vapor of chamomile oil reduced a stress-induced increase in plasma adrenocorticotropic hormone (ACTH) levels.” ACTH is a hormone that stimulates the production of cortisol and helps boost the immune system. In a study, researches evaluated the association between cortisol and stress in the body with chamomile therapy among subjects with generalized anxiety disorders. The results found that the 45 participants who consumed chamomile extract over the 8-week period, decreased cortisol levels, and improved anxiety symptoms.

11. Blueberries Contain Polyphenols, Helping Boost Your Immune System and Mood

If you love blueberries, eat more! These small and delicious berries are high in flavonoids, an antioxidant that contains mood-boosting properties according to a study. “Blueberries are rich in polyphenols that may be beneficial to cognitive performance and mood,” another study suggests. Polyphenols are micronutrients found in many plant-based foods, especially blueberries, and protect your immune system and help to fight off cardiovascular diseases. The Beet reported on 5 Foods For Better Brain Health, Focus, and Mood, and blueberries were recommended by Dr. Naidoo, a Harvard trained psychiatrist, and professional chef. He said, “Berries are my personal favorite fruit, they’re lower on the glycemic index and a great option when adding fruit to your daily meal plan. The bright colors of berries represent the polyphenols, which provide that antioxidant boost.”  Cheers.

8 foods that boost your immune system and can help keep you healthy

8 foods that boost your immune system and can help keep you healthy

  • September 30, 2020
  • Foods that can help boost your immune system include citrus fruits, leafy green vegetables, red bell peppers, yogurt, and green tea.
  • Many of these foods contain vitamin C and vitamin A, which are known to help support immune function. 
  • You can also add ginger, garlic, and turmeric to your food to gain additional immune benefits. 
  • This article was reviewed by Jason R. McKnight, MD, MS, a family medicine physician and clinical assistant professor at Texas A&M College of Medicine
  • Visit Insider’s Health Reference library for more advice.

The foods you eat can help support your immune system and keep you healthy. 

“What we eat is really central to our overall health, and that includes the immune system,” says Janine Souffront, RDN, a registered dietitian and supervisor of Health Education for L.A. Care Health Plan. “Eating healthy, nutrient-rich foods, can help your body fight off illness.”

Of course, you won’t be able to rely on these foods or nutrients as a miracle cure for sickness. But on top of preventative behaviors — like washing your hands often and getting a flu shot — these healthy foods may help reduce your risk of contracting illness. 

“While there is not one food or supplement that can straight-on prevent illness, you can support your immune system by including foods with nutrients that play a role in tissue health and integrity,” Souffront says. 

The following foods can contribute to a strong immune system: 

1. Citrus fruits

Citrus fruits like oranges or grapefruits are packed with vitamin C, which is well-known for immune support. Adult women should get 75 mg of vitamin C per day, while men should get 90 mg.

Here’s how much vitamin C you can get from common citrus fruits:

  • One medium orange provides about 70 mg
  • One grapefruit provides about 80 mg
  • One lemon provides about 30 mg

To support the immune system, vitamin C helps the body repair tissue and keeps skin and blood vessels healthy. In addition, vitamin C is an important antioxidant, a substance that prevents cell deterioration and improves immune function. 

A 2017 review published in Biomed Research International found that taking 1 gram (1,000 mg) of vitamin C per day reduced the duration of a cold by 8% in adults and 18% in children. And while it can’t necessarily prevent you from getting a cold, it may help your ability to fight it. 

“Contrary to popular belief, it is unlikely that vitamin C will keep you from getting a cold but there is some limited evidence that high doses of vitamin C may decrease the length of cold symptoms,” Souffront says.

2. Leafy green vegetables

Leafy greens like spinach, kale, and collard greens are high in vitamin A, which is important for immune function. Men need a recommended 900 micrograms (mcg) of vitamin A each day, while women need 700 mcg. Here’s how to get the recommended daily dose:

  • One cup of raw spinach contains 469 mcg
  • One cup of cooked kale contains 190 mcg 
  • One cup of collard greens contains 90 mcg

According to a 2018 study published in the Journal of Clinical Medicine, scientists know that vitamin A is important for the immune system, but they don’t understand exactly why. They believe that vitamin A affects bone marrow production. Bone marrow produces immune cells like lymphocytes, a type of white blood cell that can help fight off infections. 

A 2019 study published in Nutrition Reviews found that leafy greens are also rich in dietary nitrate, an organic compound that has anti-inflammatory properties and can help regulate the immune system.

3. Red bell peppers 

Red bell peppers are especially beneficial for immune health. The European Journal of Clinical Nutrition published a paper in April 2020 recommending red bell peppers as part of a healthy quarantine diet because of their content of vitamin A and C. 

“Red bell peppers are good for the immune system because they have both vitamin A and C, and beta carotene,” Souffront says. “Vitamin C has important healing benefits and antioxidants. Beta carotene helps the body make Vitamin A, which protects against infections.”

4. Yogurt 

Yogurt is a great source of protein, Souffront says, which can help keep your bones and skin healthy. “Healthy tissues are the first barrier against infections,” she says. When your skin is healthy, it keeps out harmful bacteria or viruses, for example. 

In addition to providing protein, most yogurts contain live culture, which are bacteria that improve the health of your gut microbiome. A 2015 study published in the Annual Review of Immunology concluded that the health of the gut microbiome impacts immune function and contributes to a person’s ability to ward off infection.

“Yogurt and kefir are good sources of probiotics, which can also have a positive effect in the immune system by keeping up the ‘good’ bacteria and down the ‘bad’ bacteria in the intestinal tract,” Souffront says. 

5. Green tea 

“Green tea is very high in catechins and polyphenols, antioxidants that help prevent cell damage,” Souffront says. “Healthier cells in general will allow the body to have a better immune response.”

For example, a 2016 study published in the Journal of Agricultural and Food Chemistry found that green tea catechins can improve the response of t-cells, which are cells that attack viruses. Increased t-cells are associated with an improved immune response. A 2018 study published in the Journal of Immunology Research also found that polyphenols help the body signal when an immune response is needed. 

6. Ginger 

Ginger has antioxidant and anti-inflammatory properties. A 2013 study published in the International Journal of Preventive Medicine found that ginger supports the immune system and may even be effective at preventing cancer. 

A 2019 study published in the Journal of Nutrition & Intermediary Metabolism looked at the effect of 8.3 grams of ginger extract on smokers and non-smokers. Both groups had immune system improvements, though from different causes. The smokers experienced more lymphocytes. The non-smokers had increased immunoglobulin M, a type of antibody. 

However, Souffront recommends sticking with fresh ginger. “The whole food source ginger is more effective than supplements,” she says. “For that reason, using fresh ginger in cooking or teas is best.”

7. Garlic 

“For centuries, people have touted the health benefits of garlic, with suggestions that it can help with heart disease, high cholesterol, and colds and flu,” Souffront says. 

The health benefits of garlic are rooted in allicin, a compound that is released when garlic is chopped or crushed. “Allicin and the antioxidants inside garlic help fight off infection and support the immune system,” Souffront says. 

A 2018 study published in Clinical Nutrition found that garlic extract boosted the immune system for obese adults. In the randomized, double-blind study, a group of 51 adults were given 3.6 grams of aged garlic extract or a placebo for 6 weeks. At the end of the six weeks, the garlic group had lower inflammation and better distribution of immune system cells. 

In addition, a 2016 study published in the Journal of Nutrition examined a group of 120 healthy participants that were given 2.56 grams of aged garlic extract or a placebo for 90 days. The garlic and placebo groups reported the same amount of illnesses, but the garlic group reported fewer symptoms, reduced illness severity, and fewer days of school or work missed.  

8. Turmeric

Turmeric’s immune benefits are linked to curcumin, the component that gives it its deep yellow color, Souffront says. 

“Curcumin seems to have the ability to modulate the immune system by activating some of the immunity related cells and dampening the effect of some pro-inflammatory compounds,” Souffront says. 

An August 2020 report published in Food and Science reports concluded that turmeric could be a helpful immune booster. It can be easily added to teas or incorporated into recipes like curry. 


No dietary approach can ensure that you won’t get sick. However, eating a well-rounded diet that incorporates the foods and nutrients mentioned above can help you stay healthy. Just remember not to each too much of these foods — for example, it is possible to have too much vitamin A.

Plus, it’s important to take other steps to boost your immunity, Souffront says. 

“Diet alone cannot boost your immune system,” she said. “You can also support your immune system by supporting your body through an overall healthy lifestyle that includes physical activity, plenty of sleep, and managing stress, along with balanced nutrition.”

Why Doing Good Boosts Health And Well-Being

Why Doing Good Boosts Health And Well-Being

  • September 30, 2020

Previous studies have suggested that people who engage in helping others are happier and have better mental and physical health than those who don’t spend as much time helping others. During hard times such as the pandemic, a body of research suggests that helping gestures assuage worry and concern. Often during emergencies and crises, people start performing acts of kindness at random. Helping others through a crisis by performing good deeds can make you feel in control—even give you bursts of euphoria called “the helper’s high.”

The obvious benefit when you reach out to help someone else is that you get a break from your own worries for a while. Contributing, giving, volunteering, donating and performing kind acts, no matter how small or brief, connect you to other people (and animals) in a deeply meaningful, humane way. But that’s just for starters.

The bursts of euphoria—known as “the helper’s high”—come from dopamine and endorphin squirts released in the brain. Medical studies show that the saliva of compassionate people contains more immunoglobulin A, which is an antibody that fights off infection. In addition to boosting the immune system, brain scans of benevolent people show that generosity gave them a calmer disposition, less stress, better emotional health and higher self-worth.

According to a new study published in the journal Psychological Bulletin, performing acts of kindness and helping other people can be good for your health and well-being. But not all goodhearted behavior is equally beneficial to the giver. The strength of the link depends on many factors, including the type of kindness, the definition of well-being and the giver’s age, gender and other demographic factors.

“Prosocial behavior — altruism, cooperation, trust and compassion — are all necessary ingredients of a harmonious and well-functioning society,” said lead author Bryant P.H. Hui, PhD, a research assistant professor at the University of Hong Kong. “It is part of the shared culture of humankind, and our analysis shows that it also contributes to mental and physical health.”

To better understand what drives that variation, Hui and his colleagues performed a meta-analysis of 201 independent studies, comprising 198,213 total participants, that looked at the connection between prosocial behavior and well-being. Overall, they found that there was a modest link between the two. Although the effect size was small, it is still meaningful, according to Hui, given how many people perform acts of kindness every day.

Digging deeper into the research, Hui and his colleagues found that random acts of kindness, such as helping an older neighbor carry groceries, were more strongly associated with overall well-being than formal prosocial behavior, such as scheduled volunteering for a charity. That may be because informal helping is more casual and spontaneous and may more easily lead to forming social connections, according to Hui. Informal giving is also more varied and less likely to become stale or monotonous, he said.

The effects varied by age, according to Hui, who began this research at the University of Cambridge. Younger givers reported higher levels of overall well-being and psychological functioning, while older givers reported higher levels of physical health. Also, women showed stronger relationships between prosociality and several measures of well-being compared with men — perhaps because women are stereotypically expected to be more caring and giving, and thus derive a stronger sense of good feelings for acting in accordance with those social norms, according to the study.


Other studies show that a priority of what American workers want and need is more compassion and empathy from businesses. Now that research shows increases in well-being and psychological functioning which can only foster job satisfaction and enhance engagement, performance and the company’s bottom line.


Bryant P. H. et al. (2020). Rewards of kindness? A meta-analysis of the link between prosociality and well-being.. Psychological Bulletin. DOI: 10.1037/bul0000298