Th2-type Cytokine Responses Research Articles

Impaired IL-17 and cytotolytic responses to influenza vaccine antigens in aging nonhuman primates

Abstract Despite widespread flu vaccination, influenza remains a major cause of morbidity and mortality in the elderly. The early immune response, including CD4+ helper T cells, and CD8+ CTLs, plays a critical role in efficient control of virus and initiation of adaptive immunity. However, the correlates of protection in the elderly are not well understood. Here, we sought to investigate early cellular responses to re-exposure with seasonal flu vaccine antigens in a cohort of pre-vaccinated young and old rhesus macaques (human age equivalent of 30–45 yrs, and 55–85 yrs). In vitro T cell responses were examined following overnight stimulation of PBMCs with flu vaccine antigens by flow cytometry for Th1-type and Th17-type cytokine responses, and cytolytic functions. Notably, older macaques showed significantly impaired IL-17 production and CD107a upregulation in response to vaccine antigens, along with greater HLA-DR +/PD-1 +CD8 T cells. Further, they produced significantly higher TNF-α than young macaques. These results suggest that circulating flu vaccine-specific T cells in older macaques are deficient in mucosal barrier-protective and cytotolytic functions and possess a dominant proinflammatory function. These findings suggest that re-exposure to flu vaccine antigens in older individuals likely induces a proinflammatory response that is impaired in cytolytic capacity and the ability to repair lung mucosal damage during influenza infection, thereby resulting in more severe disease outcomes. Further studies on in vivo modulation of proinflammatory T cells while enhancing IL-17 functions during vaccination could support the development of more effective influenza vaccine strategies for older adults.

Magnitude and breadth of antibody cross-reactivity induced by recombinant influenza hemagglutinin trimer vaccine is enhanced by combination adjuvants

The effects of adjuvants for increasing the immunogenicity of influenza vaccines are well known. However, the effect of adjuvants on increasing the breadth of cross-reactivity is less well understood. In this study we have performed a systematic screen of different toll-like receptor (TLR) agonists, with and without a squalene-in-water emulsion on the immunogenicity of a recombinant trimerized hemagglutinin (HA) vaccine in mice after single-dose administration. Antibody (Ab) cross-reactivity for other variants within and outside the immunizing subtype (homosubtypic and heterosubtypic cross-reactivity, respectively) was assessed using a protein microarray approach. Most adjuvants induced broad IgG profiles, although the response to a combination of CpG, MPLA and AddaVax (termed ‘IVAX-1’) appeared more quickly and reached a greater magnitude than the other formulations tested. Antigen-specific plasma cell labeling experiments show the components of IVAX-1 are synergistic. This adjuvant preferentially stimulates CD4 T cells to produce Th1>Th2 type (IgG2c>IgG1) antibodies and cytokine responses. Moreover, IVAX-1 induces identical homo- and heterosubtypic IgG and IgA cross-reactivity profiles when administered intranasally. Consistent with these observations, a single-cell transcriptomics analysis demonstrated significant increases in expression of IgG1, IgG2b and IgG2c genes of B cells in H5/IVAX-1 immunized mice relative to naïve mice, as well as significant increases in expression of the IFNγ gene of both CD4 and CD8 T cells. These data support the use of adjuvants for enhancing the breath and durability of antibody responses of influenza virus vaccines.

A Herbal Concoction of Cinnamomum cassia and Artemisa annua Extracts Ameliorates Allergic Rhinitis in OVA-Induced Balb/C Mice by Inhibiting Th2 Signaling

Allergic rhinitis (AR) is an inflammatory airway disease (IAD) that is characterized by itching, nasal obstruction, and sneezing. AR is induced by Th-2 inflammatory responses such as those mediated by IgE and IL-4. This study aims to investigate the therapeutic effects of an herbal concoction, which is a combination of Cinnamomum cassia and Artemisa annua extracts (CIAR) against ovalbumin (OVA)-induced allergic rhinitis in a Balb/C mouse model. The effect of CIAR on the Th-2 mediated inflammatory response in the AR mouse model was studied by analyzing blood or nasal fluid samples. Experimental results revealed that OVA inhalation increased IgE, IL-4, IL-33, and TSLP levels, leading to Th2-type cytokine response. CIAR was found to significantly reduce the Th-2 response and levels of cytokines, including IL-4, IL-33, and thymic stromal lymphopoietin (TSLP). CIAR also down-regulated eosinophil (EOS) and basophil (BASO) levels in the blood. Histological analyses demonstrated decreased OVA-induced thickness of the respiratory epithelium in the CIAR-treated group. Collectively, our results suggest that the herbal concoction CIAR can effectively ameliorate the development of allergic rhinitis through the inhibition of Th-2 mediated responses.

Fermented Red Ginseng Alleviates Ovalbumin-Induced Inflammation in Mice by Suppressing Interleukin-4 and Immunoglobulin E Expression.

Ginseng (the root of Panax ginseng Meyer) has been reported to have many biologic therapeutic effects, including anti-inflammatory properties, and ginsenosides are considered as one of the factors responsible for these therapeutic effects. To improve their therapeutic action, probiotic bacteria are used to ferment and chemically transform ginsenosides in red ginseng (RG). In this study, we aimed to investigate the beneficial effects of RG fermented by probiotic bacteria (FRG) against ovalbumin (OVA)-induced allergic rhinitis in a mouse model. We induced the mouse model via OVA inhalation; experimental results revealed increased immunoglobulin E (IgE) and interleukin (IL)-4 levels, leading to Th2-type cytokine response. The mice with induced allergy were then orally administered RG and FRG over 2 weeks, as a result of which, IL-4 and IgE levels in bronchoalveolar lavage fluid, nasal fluid, and serum were found to be ameliorated more effectively by FRG than by RG, suggesting that FRG has better immune regulatory effects than RG. FRG also downregulated immune cell levels, such as those of eosinophils and basophils, and significantly decreased the thickness of OVA-induced respiratory epithelium compared to RG. Collectively, the results showed that FRG treatment alleviates inflammation, thereby extending a protective effect to mice with OVA-induced inflammatory allergic rhinitis.

Chronic Stress Physically Spares But Functionally Impairs Innate-Like Invariant T Cells

Long-term stress disrupts the neuroimmune interface, often resulting in immunosuppression. The deleterious effects of stress on mainstream T cells are well-documented. However, innate-like invariant T cells have been overlooked. Chief among these are invariant natural killer T (iNKT) and mucosa-associated invariant T (MAIT) cells, which fulfill important functions in anticancer and antimicrobial immunity. We found stress to surprisingly abrogate both TH1- and TH2-type responses mediated by iNKT cells. This was not due to cell death since iNKT cells were unusually refractory to stress-inflicted apoptosis. Activated iNKT cells in stressed mice triggered an abnormal response characterized by a ‘split’ inflammatory signature along with a sudden and robust induction of IL-10, IL-23 and IL-27. Pharmacological inhibitors, conditional knockout systems and gene expression analyses revealed that dysregulated iNKT cell responses were driven by iNKT cell-intrinsic glucocorticoid receptor signaling. Accordingly, the inhibitory effects of chronic stress on iNKT cell functions were lost upon habituation to predictable stressors. Importantly, iNKT cells in stressed mice failed to potentiate cytotoxicity against lymphoma targets or promote immune surveillance against metastatic melanoma. Using a congenic mouse model in which MAIT cells are abundant, these cells remained resistant to stress-induced death but demonstrated hindered TH1- and TH2-type cytokine responses, reminiscent of their iNKT cell counterparts. This was corroborated in culture systems in which we interrogated human peripheral blood and hepatic iNKT and MAIT cells. Our findings uncover a previously unappreciated mechanism of stress-induced immunosuppression and implicate the stress response as a potential barrier to iNKT cell- and MAIT cell-based immunotherapies.

1537P Phase I/II study with CXCL12 inhibitor NOX-A12 and pembrolizumab in patients with microsatellite-stable, metastatic colorectal or pancreatic cancer

NOX-A12 is a CXCL12 inhibitor that prevents activation of receptors CXCR4 and CXCR7 and blocks formation of CXCL12 concentration gradients. The Opera study is a phase I/II study to investigate pharmacodynamics of NOX-A12 monotherapy and safety and efficacy of a combination with pembrolizumab in metastatic MSS colorectal (CRC) and pancreatic (PaC) cancer where PD-1 inhibition alone has no clinical benefit. Patients received NOX-A12 twice weekly for 2 weeks. Biopsies were taken from liver metastases before and after treatment to analyze immune cells and cytokines. Subsequently, patients received repeated 21-day cycles of NOX-A12 and pembrolizumab. 11 patients had CRC and 9 PaC. All patients were heavily pretreated with 5 (CRC) and 3 mean lines (PaC) of prior treatment. Best response to last prior treatment was progressive disease in 95% of cases. The AE profile was comparable with the pembrolizumab profile and typical for the underlying diseases. 2 weeks of NOX-A12 monotherapy induced changes in the tumor microenvironment: an upregulated cassette of Th1 cytokines in approx. 50% of patients, being associated with better clinical stabilization. Biopsy analysis before and after NOX-A12 monotherapy showed agglomeration of T cells and migration of T cells towards cancer cells in patients where NOX-A12 induced a Th1-type response. These trends suggest that CXCL12 inhibition increased effector immune cell infiltration of the tumor, resulting in a more effective immune response. 25% of patients achieved stable disease (SD) and long term disease control. Remarkably, 7 patients showed prolonged time on treatment vs. prior therapy. Median PFS was 1.87 months, OS was 42% at 6 and 22% at 12 months. Agglomeration of T cells within tumors was seen in ∼half of the patients where NOX-A12 had induced a Th1-type cytokine response. This was accompanied by reduced distances between T and cancer cells, i.e. more engaged T cells. Increased infiltration of effector immune cells into tumor was associated with SD in 25% of patients and prolonged time on treatment vs. prior therapy for 35% of pts. Safety profile of the combination was consistent with that of pembrolizumab in advanced cancer patients.

Abstract CT117: Phase 1/2 study with CXCL12 inhibitor NOX-A12 and pembrolizumab in patients with microsatellite-stable, metastatic colorectal or pancreatic cancer

Abstract Background: NOX-A12 is an inhibitor of the chemokine CXCL12 which prevents engagement with its receptors CXCR4 and CXCR7 and blocks the formation of chemotactic CXCL12 concentration gradients in tissue. The Opera study (NCT03168139) is a Phase 1/2 study to evaluate pharmacodynamic effects and safety of monotherapy with NOX-A12 as well as safety and efficacy of a combination with pembrolizumab in metastatic microsatellite-stable colorectal and pancreatic cancer where PD-1 inhibition alone has not shown clinical benefit. Methods: Patients received 300 mg NOX-A12 twice weekly during a 2-week monotherapy phase. Biopsies were taken from liver metastases before and after NOX-A12 monotherapy for analysis of immune cell infiltration and cytokine signature. In a combination phase, patients received repeated 21-day cycles of 300 mg NOX-A12 and 200 mg pembrolizumab until progression or intolerable toxicity. Results: 11 of the patients had CRC and 9 PaC. All patients were heavily pretreated with 5 (colorectal cancer) and 3 median lines (pancreatic cancer) of prior treatment. Best responses to last prior treatment was progressive disease for 95% of patients. The AE profile was comparable with the pembrolizumab profile or typical for the underlying diseases. Two weeks of NOX-A12 monotherapy induced Th1 cytokines (IFNγ, IL-2, IL-16) in approx. half of the patients. Comparison of tumor biopsies from before and after NOX-A12 monotherapy shows trends towards (i) agglomeration of T cells and (ii) migration of T cells towards cancer cells in the patient sub-group where NOX-A12 induced a Th1-type cytokine response. These trends are consistent with preclinical data (Feig 2013, PNAS 110:20212; Zboralski 2017, Cancer Immunol Res 5:950), suggesting that neutralization of CXCL12 signaling - either through one or both receptors - increased infiltration of effector immune cells into tumor tissue and resulted in a more effective immune response. Although there were no objective responses, 25% of patients achieved stable disease; 7 patients showed prolonged time on treatment vs. the prior line of therapy. Median progression-free survival was 1.87 months, overall survival was 42% at 6 months and 22% at 12 months. Conclusions: A trend towards agglomeration of T cells within tumors was observed in about half of the patients where NOX-A12 had induced a Th1-type cytokine response. This agglomeration was accompanied by reduced distances between T cells and cancer cells, suggesting increased infiltration of effector immune cells into tumor tissue. Treatment with NOX-A12 plus pembrolizumab in the combination therapy part of the study resulted in stable disease in 25% of patients, and prolonged time on treatment vs. prior therapy for 35% of patients. The safety profile of the combination therapy was consistent with that of pembrolizumab in advanced cancer patients. Citation Format: Niels Halama, Anja Williams, Ulrike Prüfer, Anna Frömming, Diana Beyer, Dirk Eulberg, Jarl Ulf Jungnelius, Aram Mangasarian. Phase 1/2 study with CXCL12 inhibitor NOX-A12 and pembrolizumab in patients with microsatellite-stable, metastatic colorectal or pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT117.

AST-008: A novel approach to TLR9 agonism with PD-1 blockade for anti-PD-1 refractory Merkel cell carcinoma (MCC) and cutaneous squamous cell carcinoma (CSCC).

TPS3164 Background: AST-008 is a potent spherical nucleic acid configuration of a toll-like receptor (TLR) 9 agonist oligonucleotide being developed for the treatment of MCC and CSCC in patients (pts) progressing on immune checkpoint inhibitor (CPI) monotherapy. Previously presented data suggests AST-008 elicits a Th1-type cytokine response and immune cell activation. This parallel-arm phase II design will permit assessment of efficacy in two advanced skin cancers with high unmet medical need. Methods: The study has an open label, multicenter phase Ib/II dose escalation/expansion design. The phase 1b dose escalation used a 3+3 design with increasing doses of intratumoral (IT) AST-008 plus standard flat dose pembrolizumab. The phase II dose expansion is using the recommended regimen (RP2D) of IT AST-008 plus flat dose pembrolizumab or cemiplimab to treat two cohorts of pts with advanced/metastatic MCC or CSCC, respectively. The key objective of the phase II expansion is to provide an estimate of preliminary efficacy of IT AST-008 with pembrolizumab or cemiplimab in pts whose disease progressed on a single-agent CPI therapy. Endpoints also include safety, pharmacokinetic and pharmacodynamic assessments. Pts must have at least two evaluable lesions by RECIST v1.1. One or more lesions may be injected with AST-008, but one lesion (the “witness” lesion) must remain un-injected throughout the study to differentially assess effects of AST-008 on the injected and witness lesions. The RP2D of AST-008 was derived from the dose escalation phase together with pembrolizumab. That RP2D is being used not only in the MCC cohort where AST-008 is combined with pembrolizumab, but also in the CSCC cohort where AST-008 is combined with cemiplimab. To ensure safety of the AST-008/cemiplimab combination, the first 6 pts in the CSCC cohort will be monitored for dose limiting toxicities through the first 5 weeks of dosing, and then the data review committee will assess safety before approving additional patient enrollment to that cohort. Each expansion cohort will enroll up to 29 pts with a Simon two-stage optimal design. In the first stage, 10 pts will be accrued. If there are 0 responses in these 10 pts, enrollment in that arm will be stopped. Otherwise, 19 additional pts will be accrued for a total of 29. The null hypothesis will be rejected if 4 or more responses are observed in 29 pts. This design yields a type I error rate of 0.05 and power of 80% when the true response rate is 20%. The planned phase II enrollment is 58 pts across about 15 US-based sites. Clinical trial information: NCT03684785 .

ESX5 associated PE proteins of Mycobacterium tuberculosis drives Th1 type polarization: an immunomodulatory paradigm

Abstract Mycobacterium tuberculosis (M.tb) harbors various effector molecules that affect human immune system. There exists a varied difference between the slow growing pathogenic mycobacteria compared to the fast growing non-pathogenic strains. One marked difference is the presence of ESX5, a type VII secretion system that is found to be most recently evolved and has been acquired by the pathogenic M.tb. ESX5 is involved in the secretion of an enigmatic PE/PPE family of proteins that are exclusive to the genus mycobacteriacea. PE/PPE are the effector virulent molecules that play vivid role in modulating host immune system. ESX5 locus has 2 pe genes and 3 ppe genes. Of the translated products, two PE proteins are paralogs, but contrastingly found to have different PPE binding partners as these proteins function as heterodimers, homodimers and individual proteins. THP1 macrophages (differentiated human monocytes) when treated with different micro-concentrations of PE 1X and PE 1Y induce differential Th1 type cytokine responses. PE 1X and PE 1Y induced stronger TNF-α response at 24hrs of protein exposure that increased markedly at 48hrs. IL-12 p70 was found to decrease from 24hrs to 48hrs during PE 1X treatment whereas PE 1Y exposure led to the secretion of IL-12 p70 which was constantly maintained from 24hrs to 48hrs of exposure time. PE 1X specifically mediated TLR2 dependent activation of macrophages that was responsible for the secretion of pro-inflammatory cytokines. On the contrary PE 1Y activated macrophages through TLR4 dependent cascade. These results therefore, conclude that PE 1X and PE 1Y contribute profoundly towards Th1 polarization of the immune response.

Proof of concept in utilizing in-trans surface display system of Lactobacillus plantarum as mucosal tuberculosis vaccine via oral administration in mice

BackgroundTuberculosis is one of the most common and deadliest infectious diseases worldwide affecting almost a third of the world’s population. Although this disease is being prevented and controlled by the Bacille Calmette Guérin (BCG) vaccine, the protective efficacy is highly variable and substandard (0–80%) in adults. Therefore, novel and effective tuberculosis vaccine that can overcome the limitations from BCG vaccine need to be developed.ResultsA novel approach of utilizing an in-trans protein surface display system of Lactobacillus plantarum carrying and displaying combination of Mycobacterium tuberculosis subunit epitope antigens (Ag85B, CFP-10, ESAT-6, Rv0475 and Rv2031c) fused with LysM anchor motif designated as ACERL was constructed, cloned and expressed in Esherichia coli Rossetta expression host. Subsequently the binding capability of ACERL to the cell wall of L. plantarum was examined via the immunofluorescence microscopy and whole cell ELISA where successful attachment and consistent stability of cell wall binding up to 4 days was determined. The immunization of the developed vaccine of L. plantarum surface displaying ACERL (Lp ACERL) via the oral route was studied in mice for its immunogenicity effects. Lp ACERL immunization was able to invoke significant immune responses that favor the Th1 type cytokine response of IFN-γ, IL-12 and IL-2 as indicated by the outcome from the cytokine profiling of spleen, lung, gastrointestinal tract (GIT), and the re-stimulation of the splenocytes from the immunized mice. Co-administration of an adjuvant consisting of Lactococcus lactis secreting mouse IL-12 (LcIL-12) with Lp ACERL was also investigated. It was shown that the addition of LcIL-12 was able to further generate significant Th1 type cytokines immune responses, similar or better than that of Lp ACERL alone which can be observed from the cytokine profiling of the immunized mice’s spleen, lung and GIT.ConclusionsThis study represents a proof of concept in the development of L. plantarum as a carrier for a non-genetically modified organism (GMO) tuberculosis vaccine, which may be the strategy in the future for tuberculosis vaccine development.

Clinical characteristics and cytokine profiles of children with acute lower respiratory tract infections caused by human rhinovirus.

The clinical profile of human rhinovirus (HRV) with regard to lower respiratory infections remains unclear. We analyzed the clinical features and cytokine responses of HRV isolates in children with respiratory infections. Quantitative analysis and genotyping of the HRV-positive samples from 601 nasopharyngeal aspirates (NPAs) were performed using VP4/VP2 sequencing. To compare T-helper1 (Th1) type (IFN-γ, TNF-α) and Th2 type (IL-4, IL-10) cytokine responses between HRV-A, B and C, the levels of the four cytokines were measured. The HRV-positive children had shorter fever duration (P = 0.018), and higher frequencies of chest retraction (P = 0.002) and wheezing (P = 0.022) than did the HRV-negative group. HRV-A was identified in 55 cases (58.5%), HRV-B in 8 (8.5%), and HRV-C in 31 (33.0%). There were no significant differences in the clinical data or NPA cytokines levels between patients with HRV-A and HRV-C infections. HRV is an important pathogen of the lower respiratory tract in young children. HRV-A and HRV-C are the dominant species that cause respiratory difficulty in young children.

Enhanced Anti-Mycobacterium tuberculosis Immunity over Time with Combined Drug and Immunotherapy Treatment

It is estimated that one third of the world’s population is infected with Mycobacterium tuberculosis (Mtb). This astounding statistic, in combination with costly and lengthy treatment regimens make the development of therapeutic vaccines paramount for controlling the global burden of tuberculosis. Unlike prophylactic vaccination, therapeutic immunization relies on the natural pulmonary infection with Mtb as the mucosal prime that directs boost responses back to the lung. The purpose of this work was to determine the protection and safety profile over time following therapeutic administration of our lead Mtb vaccine candidate, ID93 with a synthetic TLR4 agonist (glucopyranosyl lipid adjuvant in a stable emulsion (GLA-SE)), in combination with rifampicin, isoniazid, and pyrazinamide (RHZ) drug treatment. We assessed the host inflammatory immune responses and lung pathology 7–22 weeks post infection, and determined the therapeutic efficacy of combined treatment by enumeration of the bacterial load and survival in the SWR/J mouse model. We show that drug treatment alone, or with immunotherapy, tempered the inflammatory responses measured in brochoalveolar lavage fluid and plasma compared to untreated cohorts. RHZ combined with therapeutic immunizations significantly enhanced TH1-type cytokine responses in the lung over time, corresponding to decreased pulmonary pathology evidenced by a significant decrease in the percentage of lung lesions and destructive lung inflammation. These data suggest that bacterial burden assessment alone may miss important correlates of lung architecture that directly contribute to therapeutic vaccine efficacy in the preclinical mouse model. We also confirmed our previous finding that in combination with antibiotics therapeutic immunizations provide an additive survival advantage. Moreover, therapeutic immunizations with ID93/GLA-SE induced differential T cell immune responses over the course of infection that correlated with periods of enhanced bacterial control over that of drug treatment alone. Here we advance the immunotherapy model and investigate reliable correlates of protection and Mtb control.

Induction of Broad-Spectrum Protective Immunity against Disparate Cryptococcus Serotypes.

Cryptococcosis is a fungal disease caused by multiple Cryptococcus serotypes; particularly C. neoformans (serotypes A and D) and C. gattii (serotypes B and C). To date, there is no clinically available vaccine to prevent cryptococcosis. Mice given an experimental pulmonary vaccination with a C. neoformans serotype A strain engineered to produce interferon-γ, denoted H99γ, are protected against a subsequent otherwise lethal experimental infection with C. neoformans serotype A. Thus, we determined the efficacy of immunization with C. neoformans strain H99γ to elicit broad-spectrum protection in BALB/c mice against multiple disparate Cryptococcus serotypes. We observed significantly increased survival rates and significantly decreased pulmonary fungal burden in H99γ immunized mice challenged with Cryptococcus serotypes A, B, or D compared to heat-killed H99γ (HKH99γ) immunized mice. Results indicated that prolonged protection against Cryptococcus serotypes B or D in H99γ immunized mice was CD4+ T cell dependent and associated with the induction of predominantly Th1-type cytokine responses. Interestingly, immunization with H99γ did not elicit greater protection against challenge with the Cryptococcus serotype C tested either due to low overall virulence of this strain or enhanced capacity of this strain to evade host immunity. Altogether, these studies provide “proof-of-concept” for the development of a cryptococcal vaccine that provides cross-protection against multiple disparate serotypes of Cryptococcus.

Superoxide dismutase 3 attenuates experimental Th2-driven allergic conjunctivitis

Allergic conjunctivitis is an inflammatory eye disease mediated by Th2 type immune response. The role of extracellular superoxide dismutase 3 (SOD3) in immune response and allergic conjunctival inflammation was examined in a murine model for experimental allergic conjunctivitis (EAC). Allergic conjunctivitis was induced in mice by allergen challenge with ovalbumin in alum via the conjunctival sac. SOD3 was topically applied and allergy indicators were compared. Clinical signs associated with conjunctivitis, such as OVA-specific IgE production, IgG1/G2a ratio and eosinophil infiltration, were drastically reduced in mice treated with SOD3. They also had less dendritic cells and CD4+ T cells in conjunctiva than controls. Attenuated allergic inflammation was accredited to reduced Th2 type cytokine responses and increased Treg cytokine in draining lymph node. The characteristics of EAC were attributed to the absence of SOD3. Our findings suggest that SOD3 might be considered as a potential target for Th2-driven allergic conjunctival inflammation.

Transcription levels of Cyp450s, H99, Hsps and Dmp53 in Drosophila melanogaster larvae treated with zearalenona

Cryptococcus neoformans is an opportunistic fungal pathogen that threatens individuals with impaired cell-mediated immunity (CMI). Presently, there are no standardized vaccines available to prevent cryptococcal infections and conventional anti-fungal drug therapy does not induce host immune reactivity and thus cannot efficiently resolve C. neoformans infections in immunocompromised individuals. The present study was designed to characterize pulmonary immune responses following infection with an avirulent temperature-sensitive (ts) mutant, calcineurin A1 (cna1) compared to the pathogenic C. neoformans strain H99 and its potential to induce protective anti-cryptococcal immunity. Host CMI responses in cna1-inoculated mice were observed to be dose-dependent, and comprise increases in pulmonary macrophages and CD4+ T lymphocytes. However, cytokine analysis demonstrated a mixed pulmonary cytokine response (increases in IL-4, and MCP-1) with no induction of IFN-γ. Also, pre-immunization with the ts cna1 mutant did not result in protection from a subsequent secondary pulmonary infection with the pathogenic C. neoformans strain H99. Taken together, these results suggest that host pulmonary CMI responses to the ts cna1 mutant that is eventually eliminated from the host without the induction of IFN-γ appear to be dose-dependent, diverse, and require further stimulation to induce C. neoformans-specific Th1-type cytokine responses to resolve subsequent experimental pulmonary cryptococcal infections.

Subset-specific alterations in frequencies and functional signatures of γδ T cells in systemic sclerosis patients.

Here, we evaluated the distribution and functional profile of circulating CD27+ and CD27- γδ T-cell subsets in systemic sclerosis (SSc) patients to assess their potential role in this disorder. Peripheral blood from 39 SSc patients and 20 healthy individuals was used in this study. The TCR-γδ repertoire, cytokine production and cytotoxic signatures of circulating γδ T-cell subsets were assessed by flow cytometry. Gene expression of EOMES, NKG2D and GZMA was evaluated by quantitative RT-PCR in both purified γδ T-cell subsets. Absolute numbers of γδ T-cell subsets were significantly decreased in SSc groups, likely reflecting their mobilization to the inflamed skin. Both γδ T-cell subsets preserved their relative proportions and Th1-type cytokine responses. However, cytotoxic properties showed significant disease-associated and subset-specific changes. SSc patients exhibited increased percentages of CD27+ γδ T cells expressing granzyme B or perforin and upregulated GZMA expression in diffuse cutaneous SSc. Conversely, EOMES and NKG2D were downregulated in both SSc γδ T-cell subsets vs. normal controls. Interestingly, patients with pulmonary fibrosis showed a biased TCR repertoire, with a selected expansion of effector Vγ9+ γδ T cells associated with increased frequency of cells expressing granzyme B, but decreased IFN-γ production. Significant alterations on circulating γδ T-cell subsets suggest a deregulated (increased) cytotoxic activity and thus enhanced pathogenic potential of CD27+ γδ T cells in SSc.

Construction and Immunogenicity of Recombinant Swinepox Virus Expressing Outer Membrane Protein L of Salmonella.

Salmonella spp. are gram-negative flagellated bacteria that cause a variety of diseases in humans and animals, ranging from mild gastroenteritis to severe systemic infection. To explore development of a potent vaccine against Salmonella infections, the gene encoding outer membrane protein L (ompL) was inserted into the swinepox virus (SPV) genome by homologous recombination. PCR, western blot, and immunofluorescence assays were used to verify the recombinant swinepox virus rSPV-OmpL. The immune responses and protection efficacy of rSPV-OmpL were assessed in a mouse model. Forty mice were assigned to four groups, which were immunized with rSPV-OmpL, inactive Salmonella (positive control), wildtype SPV (wtSPV; negative control), or PBS (challenge control), respectively. The OmpLspecific antibody in the rSPV-OmpL-immunized group increased dramatically and continuously over time post-vaccination, and was present at a significantly higher level than in the positive control group (p < 0.05). The concentrations of IFN-γ and IL-4, which represent Th1-type and Th2-type cytokine responses, were significantly higher (p < 0.05) in the rSPVOmpL- vaccinated group than in the other three groups. After intraperitoneal challenge with a lethal dose of Salmonella typhimurium CVCC542, eight out of ten mice in the rSPV-OmpLvaccinated group were protected, whereas all the mice in the negative control and challenge control groups died within 3 days. Passive immune protection assays showed that hyperimmune sera against OmpL could provide mice with effective protection against challenge from S. typhimurium. The recombinant swinepox virus rSPV-OmpL might serve as a promising vaccine against Salmonella infection.

Synthesis of α-C-Galactosylceramide via Diastereoselective Aziridination: The New Immunostimulant 4'-epi-C-Glycoside of KRN7000.

A new immunostimulant, the 4'-epimer of α-C-GalCer, was synthesized from a C2-symmetric dienediol and α-C-allyl galactoside. The intramolecular aziridination and the following reductive ring opening provided the core of the aliphatic amino alcohol with excellent regio- and stereocontrol. The new immunostimulants 3d and 3e gave a better polarized Th1-type cytokine response in murine NKT cells than the benchmarked α-C-GalCer.

Study of TH1/TH2 Cytokine Profiles in HIV/AIDS Patients in a Tertiary Care Hospital in India

Background and Objectives: Switch of cytokines from a T helper 1 (Th1) to a Th2 is an important factor in the progression of HIV infection to AIDS. Hence, our objectives were to analyze the levels of Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-10) cytokines and their correlation with clinical and immunological profiles in HIV/AIDS patients. Methods: We studied 234 symptomatic HIV positive patients (case group) attending OPDs, ART clinic and medical wards of the Hospital. CD4+T-cell count was determined by flow cytometry using Fluorescent Activated Cell Sorter Count system. Quantitative determination of cytokines (Th1 subtype: IL-2, IFN-γ and Th2 subtype: IL-4, IL-10) was done by ELISA. Results: Patients (08-68 years) had CD4+T-cell counts ranged from 16-1033 cells/μl. The median CD4+T cell count was 204.50 cells/μl and the mean was 265.48 cells/μl. The concentration of IL-2 and IFN-γ were significantly lower in case group compared to asymptomatic HIV patients (P<0.001) while the IL-4 in symptomatic HIV negative was higher than healthy controls and case group compared to asymptomatic HIV patients. Concentration of IL-10 was also higher in case group compared to asymptomatic HIV patients. A positive correlation was found between IL-4, IFN-γ, IL-2 with CD4+T-cells and a negative correlation between IL-10 and CD4+T-cells among the case group. Conclusions: Hence a decline in type-1 cytokines (IFN-γ, IL-2) and rise in type-2 cytokines (IL-4, IL-10) was observed in symptomatic patients with HIV infection in comparison to asymptomatic HIV patients suggesting a shift from Th1 to Th2 type cytokine response.

A novel mannoside-glycocluster adjuvant: Compared in vitro to CpG ODN and MPL and tested in vivo in mouse asthma model

BackgroundAllergen-specific immunotherapy balances the Th2-biased immunity towards Th1 and Treg responses. Adjuvants are used in allergen preparations to intensify the immune responses. The increased prevalence of allergies in developed societies has been associated with decreased microbial load during childhood. This has initiated a search for microbial structures to be used as adjuvants. Our study has shown that a synthetic triacedimannose (TADM) may suppress the Th2-type allergic inflammatory response. The aim of this study was to compare the properties of TADM with capacities of other adjuvants, CpG ODN and MPL, to modulate cytokine production in PBMC and regulate sensitisation in an OVA-sensitised mouse asthma model. MethodsThe effects of TADM were studied in vitro on birch stimulated PBMC cultures of birch allergic rhinitis patients with other known adjuvants. Cytokines in supernatants were measured by Luminex. Effects of TADM were analysed in vivo in a mouse model of OVA-induced allergic asthma by analysing BAL, cytokine mRNA and serum antibodies. ResultsTADM was the only adjuvant that significantly suppressed the production of all birch induced Th2-type cytokines. In a murine model, TADM significantly suppressed the specific IgE production and enhanced IFN-γ production. ConclusionsTADM suppresses the birch allergen induced Th2-type cytokine responses in allergic subjects more efficiently than the two other adjuvants, MPL and CpG ODN. TADM is immunomodulatory also in vivo and decreases the IgE levels and increases the IFN-γ responses in a murine model. These results suggest that TADM may be a promising candidate for novel adjuvants in immunotherapy.