Serum Levels Of Th1 Cytokines Research Articles

Chlorophyll a and novel synthetic derivatives alleviate atopic dermatitis by suppressing Th2 cell differentiation via IL-4 receptor modulation

Atopic dermatitis (AD) treatment has largely relied on non-specific broad immunosuppressants despite their long-term toxicities until the approval of dupilumab, which blocks IL-4 signaling to target Th2 cell responses. Here, we report the discovery of compound 4aa, a novel compound derived from the structure of chlorophyll a, and the efficacy of chlorophyll a to alleviate AD symptoms by oral administration in human AD patients. 4aa downregulated GATA3 and IL-4 in differentiating Th2 cells by potently blocking IL-4 receptor dimerization. In the murine model, oral administration of 4aa reduced the clinical severity of symptoms and scratching behavior by 76% and 72%, respectively. Notably, the elevated serum levels of Th2 cytokines reduced to levels similar to those in the normal group after oral administration of 4aa. Additionally, the toxicological studies showed favorable safety profiles and good tolerance. In conclusion, 4aa may be applied for novel therapeutic developments for patients with AD.

Milk fat globule membrane supplementation protects against β-lactoglobulin-induced food allergy in mice via upregulation of regulatory T cells and enhancement of intestinal barrier in a microbiota-derived short-chain fatty acids manner

Milk fat globule membrane (MFGM), which contains abundant glycoproteins and phospholipids, exerts beneficial effects on intestinal health and immunomodulation. The aim of this study was to evaluate the protective effects and possible underlying mechanisms of MFGM on cow’s milk allergy (CMA) in a β-lactoglobulin (BLG)-induced allergic mice model. MFGM was supplemented to allergic mice induced by BLG at a dose of 400 mg/kg body weight. Results demonstrated that MFGM alleviated food allergy symptoms, decreased serum levels of lipopolysaccharide, pro-inflammatory cytokines, immunoglobulin (Ig) E, IgG1, and Th2 cytokines including interleukin (IL)-4, while increased serum levels of Th1 cytokines including interferon-γ and regulatory T cells (Tregs) cytokines including IL-10 and transforming growth factor-β. MFGM modulated gut microbiota and enhanced intestinal barrier of BLG-allergic mice, as evidenced by decreased relative abundance of Desulfobacterota, Rikenellaceae, Lachnospiraceae, and Desulfovibrionaceae, while increased relative abundance of Bacteroidetes, Lactobacillaceae and Muribaculaceae, and enhanced expressions of tight junction proteins including Occludin, Claudin-1 and zonula occludens-1. Furthermore, MFGM increased fecal short-chain fatty acids (SCFAs) levels, which elevated G protein-coupled receptor (GPR) 43 and GPR109A expressions. The increased expressions of GPR43 and GPR109A induced CD103+ dendritic cells accumulation and promoted Tregs differentiation in mesenteric lymph node to a certain extent. In summary, MFGM alleviated CMA in a BLG-induced allergic mice model through enhancing intestinal barrier and promoting Tregs differentiation, which may be correlated with SCFAs-mediated activation of GPRs. These findings suggest that MFGM may be useful as a promising functional ingredient against CMA.

Type 2 cytokines and scleroderma interstitial lung disease.

Interstitial lung disease (ILD) is a life-threatening complication of systemic sclerosis (SSc). Type 2 (Th2) cytokines play a pivotal role in airway disease. Study aim was to evaluate serum level of Th2 interleukin (IL) and chemokine in SSc-ILD. Serum levels of IL-4, IL-5, IL-11, IL-13, IL-21, IL-31 and CXCL-13 were measured by Bio-Plex Multiplex Immunoassays in 60 SSc patients and 20 healthy controls (HC). Pulmonary function tests with diffusion lung capacity for carbon monoxide (DLco) and high resolution computed tomography (HRCT) were performed in SSc patients. ILD is defined as fibrotic changes (ground glass, reticular and honeycombing), assessed by Computer-Aided Lung Informatics for Pathology Evaluation and Ratings (CALIPER) software, affecting at least 10% of the lungs. Serum levels of Th2 cytokines were higher in SSc patients than HC. A linear correlation was observed between ground glass and IL-13 (r = 0.342, p < 0.01), IL-21 (r = 0.345, p < 0.01), IL-31 (r = 0.473, p < 0.001), IL-4 (r = 0.863, p < 0.001), IL-5 (r = 0.249, p < 0.05) and peripheral blood eosinophils (r = 0.463, p < 0.001). We found a negative correlation between DLco and IL-4 (r = - 0.511, p < 0.001) and peripheral blood eosinophils (r = - 0.446, p < 0.001). In the logistic regression analysis, IL-4 is associated with DLco ≤ 60% of the predicted [OR 1.039 (CI 95%: 1.015-1.064), p < 0.001], whilstmRSS [OR 1.138 (CI 95%: 1.023-1.266), p < 0.05] and IL-4 [OR 1.017 (CI 95%: 1-1.034), p < 0.05] were associated with ILD. Th2 inflammation could play a key role in early phase of SSc-ILD.

Identification of mite-specific eosinophils in the colon of patients with ulcerative colitis

The pathogenesis of ulcerative colitis (UC) is unclear. House dust mite (HDM) is associated with immune inflammation in the body. This study is designed to identify the association between HDM and UC clinical symptoms. UC patients (n = 86) and non-UC control (NC) subjects (n = 64) were recruited. Colon lavage fluids (CLF) were collected from HDM skin prick test positive patients during colonoscopy, and analyzed by immunological approaches. HDM was detected in fecal samples, which was positively correlated with UC clinical symptoms. HDM-specific eosinophils and Th2 cells were detected in CLF, which could be specifically activated by exposing to HDM in the culture. Direct exposure to HDM induced eosinophil activation in the colon of UC patients. UC patients displayed elevated levels of Th2 cytokines in the serum. UC clinical symptom scores were positively correlated with serum levels of Th2 cytokines. HDM was detected in UC patients’ stools, which was positively correlated with UC clinical symptoms. Direct exposure to HDM could trigger eosinophilic activation of the colon.

Th1/Th2 cytokines profile in overweight/obese young adults and their correlation with airways inflammation

ObjectivesThis study aims to compare the Th1/Th2 cytokines of subjects with general/abdominal obesity and non-obese individuals, and to correlate them with the biomarker of airways inflammation and different body compositions. MethodsEighty subjects were divided into 37 normal weight (BMI >25) and 43 overweight/obese groups (BMI ≥25). All participants were further categorised by waist circumference (WC) into an abdominal obesity group (n = 32) and a group without abdominal obesity (n = 48). Serum levels of Th1 cytokines (INF-γ, TNF-α, IL-2,) and Th2 cytokines (IL-4, IL-5, IL-13) were measured using a multiplex ELISA technique. The fractional exhaled nitric oxide (FeNO) was used as a biomarker for airways inflammation. Different body compositions were assessed using a bioelectrical body composition analyser. ResultsSerum IL-5 and TNF-α were significantly increased in groups with general or abdominal obesity compared to control groups. IL-5 showed a significant positive correlation with FeNO. BMI and total fat percentage were positively correlated to IL-5 and TNF-α, whereas WC and visceral fat percentage were correlated with the levels of IL-5 and IL-4. ConclusionThis study confirms the elevation of certain Th1 and Th2 cytokines in subjects with general and abdominal obesity. IL-5 was positively correlated with FeNO, which may link obesity to airways inflammation.

Eosinophil count, serum CCL17/18/26 and immunoglobulin E levels in atopic dermatitis: upadacitinib phase 2 study analysis

To assess upadacitinib (UPA), a selective JAK1 inhibitor, treatment on absolute eosinophil count (AEC), serum Type 2 biomarkers, total and allergen-specific immunoglobulin E (IgE) levels in patients with atopic dermatitis (AD). Adults with moderate-to-severe AD were randomized to daily placebo (PBO) or UPA 7.5, 15, or 30mg in a 16-wk, phase 2 study. Wk 16 AEC, serum biomarkers, total and allergen-specific IgE (ImmunoCap) levels, changes in Eczema Area and Severity Index (EASI) and pruritus numeric rating scale (NRS, weekly average of daily patient assessments) were analyzed. Statistical analysis was done using analysis of variance and Spearman correlation. Mean percentage improvements from baseline to wk 16 in EASI (39.4%/61.7%/74.4% vs 23.0%; P<0.05/<0.001/<0.001) and pruritus NRS (39.6%/48.0%/68.9% vs 9.7%; P<0.01/<0.001/<0.001) were significantly greater with UPA 7.5/15/30mg vs PBO. AEC was significantly lower with 30mg UPA vs PBO (P=0.01) at wk 16; significant differences were observed as early as wk 2 in UPA 15mg (P=0.003) and 30mg groups (P<0.0001) vs PBO. Serum levels of the Th2 cytokines, CCL18 and CCL26, and the Th22 cytokine, IL-22, were significantly lower with 15mg and 30mg UPA vs PBO as early as wk 2 (P<0.01); these significant decreases were sustained at wk 16 in UPA 15 and 30mg groups (P<0.01). CCL17 (TARC) showed similar trends (significant at wks 2 and 8 vs baseline) with UPA 30mg dose. There were no clear trends in Th17 cytokine levels (IL-17A, IL-17F, or CCL20). Baseline levels of AEC significantly correlated with baseline EASI (r=0.39, P<0.0001); in addition, percentage change from baseline to wk 16 in AEC correlated with percentage change at wk 16 in EASI (r=0.62, P<0.0001) and pruritus NRS (r=0.66, P<0.0001). Baseline serum levels of Th2 and Th22 cytokines significantly correlated with baseline EASI scores (CCL17/18/26: r=0.44/0.43/0.41, P<0.0001; IL-22: r=0.43, P<0.001). Changes from baseline to wk 16 in Th2 and Th22 cytokines correlated with percentage changes in EASI (CCL18/26: r=0.58/0.57, P<0.0001; IL-22: r=0.48, P<0.0001) and pruritus NRS (CCL18/26: r=0.37/0.43, P<0.01; IL-22: r=0.38, P<0.001). While baseline levels of serum total IgE correlated with baseline EASI (r=0.35, P<0.001), no trends in allergen-specific or total IgE levels as a function of study duration were observed. Eosinophil counts and serum levels of Th2 (CCL18/26) and Th22 (IL-22)-attracting chemokines were significantly reduced with UPA treatment (15/30mg) as early as wk 2, suggesting UPA may have early and robust effects on Th2, Th22, and eosinophil axes. No significant changes in total and specific IgE levels were observed, suggesting that UPA clinical efficacy is independent of IgE levels, and this observation coupled with the lack of clinical efficacy for anti-IgE strategies in patients with AD argues against the primary role of IgE in AD pathogenesis.

IL-33/ST2 axis is involved in disease progression in the spleen during Leishmania donovani infection

BackgroundDuring infection with Leishmania donovani, parasite control is linked to the systemic Th1 immune response, but in infected organs (liver, spleen and bone marrow), the response differs according to the micro-environment. The pleiomorphic cytokine interleukin-33 (IL-33) exerts various roles during infection, either protective or detrimental. In this study, we explored the role of IL-33 in the outcome of Leishmania infection in the spleen.MethodsWe used several mouse models, on BALB/c and C57BL/6 (B6) backgrounds, infected with L. donovani and sacrificed at 15, 30 or 60 days after infection and characterized mRNA expression of immune markers, immune cell populations, histological response, and parasite loads.ResultsDuring infection IL-33 and ST2 mRNA increased in parallel in the spleen of wild type (wt) animals and paralleled the immunodetection of ST2+ and IL-33+ cells; their expression was twice as high in BALB/c, compared to B6 mice. Mice treated with twice-weekly injections of rIL-33 had higher splenic parasite burdens on D15 (BALB/c) or on D60 (B6). In BALB/c, IL-33 treatment led to immune exhaustion with abolition of Th1 cytokine expression (IFN-γ and IL-12) in the spleen and higher serum levels of Th2 cytokines (IL-4, IL-5 and IL-13). In B6, IL-33 treatment induced the Treg cell pathway with a dramatic increase of FoxP3 mRNA induction and expression on tissue sections. IL-33-KO mice had lower parasite loads and a higher Th1 response than their wt counterparts.ConclusionsIL-33 appears as a factor of aggravation of the disease in the spleen tissue of mice infected with L. donovani.

Fibrocytes in Chronic Periaortitis: A Novel Mechanism Linking Inflammation and Fibrosis.

Chronic periaortitis (CP) is a rare disease characterized by periaortic and periiliac fibroinflammatory tissue. The pathogenic mechanisms leading to tissue accumulation and activation of fibroblasts are unclear. This study was undertaken to explore the role of fibrocytes, circulating precursors of tissue fibroblasts, in patients with CP. We studied 44 patients with newly diagnosed CP and 30 healthy controls. Circulating fibrocytes were identified as Col1+CD45+ cells using flow cytometry. Retroperitoneal tissue biopsy samples from 9 CP patients were stained with anti-type I procollagen, anti-CXCR4, and anti-CD45 antibodies and analyzed by confocal microscopy to detect tissue-infiltrating fibrocytes. Circulating levels and tissue expression of CXCL12, a CXCR4 ligand that promotes fibrocyte homing, were investigated using enzyme-linked immunosorbent assay and immunohistochemistry, respectively. We also characterized T helper polarization in biopsy samples from CP patients and measured serum levels of a panel of cytokines that are hallmarks of T helper responses and capable of influencing fibrocyte differentiation. The frequency of circulating Col1+CD45+ fibrocytes was higher in patients than in controls (P = 0.0371). CD45+proCol1+ and CXCR4+proCol1+ cells were detected in all examined biopsy samples from CP patients. Serum levels of CXCL12 were also higher in CP patients than controls (P = 0.0056), and tissue-infiltrating inflammatory cells intensely expressed CXCL12. Increased serum levels of Th2 cytokines (e.g., interleukin-13 [IL-13] and IL-10) were found in patients, and immunohistochemistry revealed a dominant infiltration of GATA-3+ cells, also indicating Th2 polarization; Th2-skewed responses are known to promote fibrocyte differentiation. Our findings indicate that fibrocytes are enriched in the peripheral blood of CP patients and infiltrate target lesions. The accumulation of fibrocytes in the pathologic tissue might be driven by CXCL12, and Th2-skewed immune responses are likely to facilitate their differentiation.

Effects of magnesium isoglycyrrhizinate on AST, ALT, and serum levels of Th1 cytokines in patients with allo-HSCT

This study aimed to investigate the protective effects of magnesium isoglycyrrhizinate (MGL) on aspartate aminotransferase (AST), alanine aminotransferase (ALT), and serum levels of T helper 1 (Th1) cytokines in patients with allogeneic hematopoietic stem cell transplantation (allo-HSCT). The study included 42 patients prepared for allo-HSCT, who were divided equally into MGL and reduced glutathione groups. The ALT and AST levels were detected 1day before pretreatment and transplantation, and 7, 14, and 21days after transplantation. The total days and times of fever, treatment time of patients in the laminar flow room, white blood cell (WBC) count, platelet (PTL) implantation time, and success rate of transplantation were recorded. The serum levels of Th1/Th2 cytokines were detected. MGL had a significant protective effect on AST 1day before transplantation and 7, 14, and 21days after transplantation, while ALT had a statistical difference only 7days after transplantation. MGL could shorten the duration of fever during transplantation and advance the WBC and PTL implantation time. Significant differences in Th1-like cytokines (P<0.05) and higher levels of Th2-like cytokines but with no statistical significance (P>0.05) were found in the MGL group compared with the control group. MGL had significant protective effects on AST after transplantation. MGL could reduce the duration of fever during transplantation, help the reconstruction and recovery of WBCs and PTLs, and regulate Th1 cytokines, revealing its protective effects on hepatic transaminases and graft versus host disease in allo-HSCT patients.

Transfer of Immunity Using Vaccinate and Boost Strategy with Bispecific Antibody Armed T Cells (BATs) in Metastatic Breast Cancer Patients

Background. In our recent phase I immunotherapy (IT) trial in 23 women with metastatic breast cancer (MBC), 8 infusions of activated T cells (ATC) armed with anti-CD3 x anti-HER2 bispecific antibody (HER2Bi) given in combination with interleukin-2 (IL-2) and granulocyte-macrophage colony stimulating factor (GM-CSF) induced specific anti-breast cancer immunity. This study investigated whether specific cellular and humoral anti-breast cancer immunity induced by infusions of HER2 bispecific antibody armed T cells (BATs) could be transferred after high dose chemotherapy (HDC) and stem cell transplant (SCT) in MBC patients. Methods. T cell were activated with OKT3 and expanded in IL-2. ATC were harvested, armed with HER2Bi, and cryopreserved in 8 doses for twice weekly infusions for 4 weeks along with IL-2 and GM-CSF. Seven to 14 days after the last infusion of BATs, the patient was leukapheresed again to obtain and expand immune T cells. Multiple infusions of immune ATC after the HDC and SCT were given to boost the transferred cellular and humoral immune responses which were monitored up to 24 months. Results. Six of 8 MBC patients enrolled were evaluable in the protocol, no dose-limiting toxicity, delays in engraftment, or life-threatening infections were observed. Five of 6 evaluable patients exhibited increased anti- breast cancer cytotoxicity and IFN-γ Elispots after vaccination with BATs and up to 18 months post SCT. Serum and culture supernatants from in vitro antibody synthesis assay showed gradual increases in anti-SK-BR-3 IgG levels after SCT. Serum cytokine profile showed increases in IL-12 and Th1 cytokines. One out of 6 evaluable patients who rapidly progressed showed poor immune response, had high serum levels of Th2 cytokines and no evidence of transfer of immunity. Flow cytometry analysis of Vβ repertoire pattern in PBMC collected post IT and post SCT indicate transfer of the major Vβ clones post SCT that produced IFN-γ upon stimulation with breast cancer cells (Fig. 1). A significant correlation (r=1.0; p<0.002) between immune ATC cytotoxicity directed at breast cancer cells and time to progression (TTP) suggests that more robust vaccinations with a Th1 shift in cytokine profiles can lead to clinical benefit (Fig. 2). Conclusions. Our pilot study suggests that cellular and humoral immunity was transferred and boosted using immune T cells after SCT. There was robust reconstitution of T and B cell functions early after SCT as evidenced by CTL and NK activity, IFN-γ EliSpots, in vivo/in vitro antibody synthesis, and Th1 cytokine responses.BATs induced endogenous anti- breast cancer cellular, humoral and innate immunity that could be detected after SCT and may have provided clinically meaningful anti-tumor immunity. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Adaptive immune response in symptomatic and asymptomatic enteric protozoal infection: evidence for a determining role of parasite genetic heterogeneity in host immunity to human giardiasis

The genetic basis of the ultimate clinical outcomes of human giardiasis has been the subject of numerous investigations. We previously demonstrated roles for both host and parasite factors in determining the outcome of enteric infection in a murine model of Giardia duodenalis infection. In the current study, fecal and serum specimens from healthy controls and human subjects infected with the intestinal parasite G. duodenalis were assessed. Using a semi-nested PCR method, clinical isolates were genetically characterized based on the gdh and tpi loci, and the phylogenetic trees were constructed. Using a sandwich ELISA method, the serum levels of representative TH1 and TH2 cytokines were measured in infected human subjects and healthy controls. Here we showed that symptomatic human giardiasis was characterized by significantly elevated serum levels of the TH1 cytokine IFN-γ compared to healthy controls, whereas asymptomatic human subjects and healthy controls had comparable levels of serum IFN-γ. Further analyses showed that human subjects infected with G. duodenalis genotype AI had significantly elevated levels of serum IFN-γ and IL-10, but not IL-5, whereas human subjects infected with AII had similar levels of those cytokines compared to healthy controls. These data demonstrate roles for both host and parasite factors in the determination of the outcome of enteric infections and may further broaden our understanding of host-parasite interaction during enteric protozoal infections.

80 Nicotine increases eclampsia-like seizure threshold and attenuates microglial activity in rat hippocampus through the A7 nicotinic acetylcholine receptor

Introduction A considerable number of studies have demonstrated that nicotine, a α 7-nicotinic acetylcholine receptor ( α 7-nAChR) agonist, can dampen immune response through the cholinergic anti-inflammatory pathway. Evidence suggests that inflammation plays a critical role in eclampsia, which contributes to maternal and fetal morbidity and mortality. Objectives In the present study, possible anti-inflammation and neuro-protective effects of nicotine via α 7-nAChRs have been investigated after inducing eclampsia-like seizures in rats. Methods Rat eclampsia-like models were established by administering lipopolysaccharide (LPS) plus pentylenetetrazol (PTZ) in pregnant rats. Rats were given nicotine from gestation day (GD) 14–19. Then, clinical symptoms were detected. Seizure severity was recorded by behavioral tests, serum levels of inflammatory cytokines were measured by Luminex assays, microglia and astrocyte expressions were detected by immunofluorescence, and changes in neuronal number in the hippocampal CA1 region among different groups were detected by Nissl staining. Results Our results revealed that nicotine effectively improved fetal outcomes. Furthermore, it significantly decreased systolic blood pressure, and maternal serum levels of Th1 cytokines (TNF- α , IL-1 β , IL-6 and IL-12P70) and an IL-17 cytokine (IL-17A), dramatically increased Th1 cytokine (IL-4) and eclampsia-like seizure threshold. Moreover, this attenuated neuronal loss and decreased the expression of microglial activation markers of the hippocampal CA1 region in the eclampsia-like group. Additionally, pretreatment with α -bungarotoxin, a selective α 7-nAChR antagonist could prevent the protective effects of nicotine in eclampsia-like model rats. Conclusion Our findings indicate that the administration of nicotine may increase eclampsia-like seizure threshold and attenuate microglial activity in rat hippocampus through the α 7 nicotinic receptor.

Nicotine increases eclampsia-like seizure threshold and attenuates microglial activity in rat hippocampus through the α7 nicotinic acetylcholine receptor

ObjectiveA considerable number of studies have demonstrated that nicotine, a α7-nicotinic acetylcholine receptor (α7-nAChR) agonist, can dampen immune response through the cholinergic anti-inflammatory pathway. Evidence suggests that inflammation plays a critical role in eclampsia, which contributes to maternal and fetal morbidity and mortality. In the present study, possible anti-inflammation and neuro-protective effects of nicotine via α7-nAChRs have been investigated after inducing eclampsia-like seizures in rats. MethodsRat eclampsia-like models were established by administering lipopolysaccharide (LPS) plus pentylenetetrazol (PTZ) in pregnant rats. Rats were given nicotine from gestation day (GD) 14–19. Then, clinical symptoms were detected. Seizure severity was recorded by behavioral tests, serum levels of inflammatory cytokines were measured by Luminex assays, microglia and astrocyte expressions were detected by immunofluorescence, and changes in neuronal number in the hippocampal CA1 region among different groups were detected by Nissl staining. ResultsOur results revealed that nicotine effectively improved fetal outcomes. Furthermore, it significantly decreased systolic blood pressure, and maternal serum levels of Th1 cytokines (TNF-α, IL-1β, IL-6 and IL-12P70) and an IL-17 cytokine (IL-17A), and dramatically increased eclampsia-like seizure threshold. Moreover, this attenuated neuronal loss and decreased the expression of microglial activation markers of the hippocampal CA1 region in the eclampsia-like group. Additionally, pretreatment with α-bungarotoxin, a selective α7-nAChR antagonist could prevent the protective effects of nicotine in eclampsia-like model rats. ConclusionOur findings indicate that the administration of nicotine may attenuate microglial activity and increase eclampsia-like seizure threshold in rat hippocampus through the α7 nicotinic receptor.

Trypanosoma cruzi infection and benznidazole therapy independently stimulate oxidative status and structural pathological remodeling of the liver tissue in mice.

This study used a murine model of Chagas disease to investigate the isolated and combined impact of Trypanosoma cruzi infection and benznidazole (BZ) therapy on liver structure and function. Male C57BL/6 mice were challenged with T. cruzi and BZ for 15 days. Serum levels of cytokines and hepatic enzymes, liver oxidative stress, morphology, collagen, and glycogen content were monitored. Separately, T. cruzi infection and BZ treatment resulted in a pro-oxidant status and hepatic reactive damage. Concurrently, both T. cruzi infection and BZ treatment induced upregulation of antioxidant enzymes and pathological reorganization of the liver parenchyma and stroma. T. cruzi infection increased serum levels of Th1 cytokines, which were reduced by BZ in both infected and non-infected animals. BZ also induced functional organ damage, increasing serum levels of liver enzymes. When combined, T. cruzi infection and BZ therapy elicited intense hepatic reactive damage that was not compensated by antioxidant enzymatic reaction, subsequently culminating in more severe morphofunctional hepatic injury. Taken together, these findings indicate that during specific treatment of Chagas disease, hepatic pathology may be a result of an interaction between BZ metabolism and specific mechanisms activated during the natural course of T. cruzi infection, rather than an isolated toxic effect of BZ on liver structure and function.

Multiplex cytokine analysis of Werner syndrome.

We reported a minor inflammation-driven ageing (inflammageing) assessed by highly sensitive CRP (hsCRP) in normal individuals and patients with Werner syndrome (WS), followed by an ageing associated Th2-biased cytokine change in normal ageing in the previous papers. To further study the association of hsCRP and 26 cytokines/chemokines in 35 WS patients, a multiple cytokine array system was used in the same serum samples as were examined for hsCRP. The serum levels of Th2 cytokines (IL-4, IL-6, IL-10, and GM-CSF), Th1 products (IL-2, TNFα, IL-12, and IFNγ) and monocyte/macrophage products (MCP-1, basic FGF and G-CSF) in WS were significantly elevated compared with normal ageing. Elevated hsCRP level in WS was significantly correlated with IL-6, IL-12 and VEGF levels, if age and sex were taken into account. A pro-inflammatory cytokine/chemokine circuit-stimulated immunological shift to Th2 in WS was similar to normal ageing. These cytokine/chemokine changes may induce a systemic chronic inflammation monitored by hsCRP, though these immunological changes in WS were more complicated than normal ageing, possibly due to the WS-specific chronic inflammation such as skin ulcer, diabetes mellitus and central obesity with visceral fat deposition. Further study may warrant the pathophysiology of Th2 shift and Th2-biased inflammageing in normal ageing and WS.

Th2-type inflammation under conditions of pre-existing chronic disease is associated with liver damage in patients with avian influenza H7N9 virus

Most patients infected with avian H7N9 influenza virus develop severe disease, including respiratory failure, acute respiratory distress syndrome (ARDS), and multi-organ failure. The pathogenesis of H7N9 infection is not fully understood. This study revealed that H7N9-infected patients who had fatal outcomes or critical illness all had pre-existing chronic diseases. The patients did not have obvious systemic inflammation compared to the healthy controls. However, their fatal outcomes and critically severe illness were significantly associated with high serum levels of tumor necrosis factor (TNF)-α. Interestingly, the degree of liver damage in these patients significantly correlated with their serum levels of Th2 cytokines, interleukin (IL)-4, and IL-9. Taken together, our results suggest that Th2-type inflammation in H7N9-infected patients with pre-existing chronic diseases likely contributes to the pathogenesis of H7N9 infection and is linked to poor clinical outcomes.

Cytokine Profiles and Hepatic Injury in Occult Hepatitis C Versus Chronic Hepatitis C Virus Infection

Occult hepatitis C virus (HCV) infection is a new entity that should be considered when diagnosing patients with abnormal liver functions of unknown origin. This work was carried out to evaluate T-helper 1/T-helper 2 (Th1/Th2) cytokine profiles in patients with occult HCV infection versus chronic hepatitis C (CHC) infection, also to investigate any association between theses cytokines and liver histological features in both groups. Serum levels of Th1 cytokines (IL-2, IFN-gamma) and Th2 (IL-4 and IL-10) were measured in 35 patients with occult HCV infection compared to 50 patients with chronic hepatitis C infection and 30 healthy controls. We have found that Th1 cytokines were significantly increased in patients with CHC infection than in both occult HCV infection and control groups (p less than 0.001). On the other hand, serum IL-4 levels were higher in occult HCV infection than in CHC and control groups (p less than 0.001). Furthermore, serum IL-10 levels were higher in both patient groups vs control group (pless than 0.001), with no significant difference between CHC and occult HCV groups. Finally, only serum IL-10 levels were significantly higher among patients with high activity (A2-A3) than those with low activity (A0-A1) in both CHC and occult HCV groups (p=0.038, p=0.025, respectively). Patients with occult HCV infection exhibited a distinct immunoregulatory cytokine pattern that is shifted towards the Th2 arm.

Changes of Th1/Th2 cytokines in immunocompetent patients with pulmonary cryptococcosis

To study the changes of Th1/Th2 cytokines in immunocompetent patients with pulmonary cryptococcosis (PC). Twenty immunocompetent patients with PC were identified by histopathological examination and enrolled along with the age- and gender-matched healthy controls. The serum concentrations of interferon-γ (IFN-γ), interleukin-4 (IL-4) and interleukin-12 (IL-12) were measured by enzyme linked immunosorbent assay (ELISA). Peripheral blood mononuclear cells (PBMC) in both groups were isolated and incubated with or without recombinant human IL-12 (rhIL-12) for 48 hours, and the concentrations of IFN-γ and IL-4 in the supernatant were measured by ELISA. (1) Serum IFN-γ levels were significantly decreased in the patients compared with the control group [(14.5 ± 2.7) vs (81.8 ± 9.8) ng/L (t = 6.590, P < 0.01)], while no significant difference was observed in serum IL-12 and IL-4 levels [(2.5 ± 0.5) vs (2.52 ± 0.6) ng/L and (6.9 ± 1.3) vs (7.3 ± 1.5) ng/L, (t = 0.0035 and 0.2136, P > 0.05) ]. (2) The concentrations of IFN-γ and IL-4 in the supernatant of PBMC without rhIL-12 stimulation showed no differences between the 2 groups [(55.7 ± 13.6) vs (51.1 ± 17.5) ng/L and (5.1 ± 0.7) vs (5.0 ± 0.6) ng/L (t = 0.2979 and 0.0325, P > 0.05) ]. (3) Treatment with rhIL-12 stimulated the release of IFN-γ, but the increase in the patients [(4.3 ± 0.5) folds] was less compared with that in the controls [(7.9 ± 1.1) folds] (t = 3.01, P < 0.01) , while IL-4 concentration in the supernatant of PBMC was not increased in both groups[ (0.9 ± 0.4) vs (1.3 ± 0.4) folds (t = 0.7240, P > 0.05) ]. Serum Th1 cytokine (IFN-γ) levels may be dampened in immunocompetent patients with PC, without significant change in serum levels of Th2 cytokines (IL-4). Deficiency in the response to IL-12 stimulation of Th1 cells may be one of the underlying mechanisms for the decline in serum IFN-γ levels.

Human mesenchymal stem cells creating an immunosuppressive environment and promote breast cancer in mice

Human mesenchymal stem cells (hMSC) can home to tumor sites and promote tumor growth. The effects of hMSC on tumor growth are controversial and involvement of hMSC in tumor immunology has not been adequately addressed. Therefore, we investigated whether injection of hMSC affects tumor appearance, growth and metastasis, and anti-tumor immunity in an experimental animal model of metastatic breast cancer. Injection of hMSC in BALB/c mice bearing mammary carcinoma promoted tumor growth and metastasis, which was accompanied by lower cytotoxic activity of splenocytes, NK cells and CD8+ T cells in vitro. Tumor-bearing mice that received hMSC had significantly lower percentages of CD3+NKp46+ NKT-like, higher percentages of CD4+Foxp3+ T cells, increased serum levels of Th2 and decreased serum levels of Th1 cytokines, and significantly higher number of CD4+ cells expressing IL-10. These results demonstrate that immunosuppressive environment created by hMSC promoted breast tumor growth and metastasis in mice.

Transfer of Cellular and Humoral Anti-Tumor Immunity with Immune T Cells After Stem Cell Transplant (SCT) for Metastatic Breast Cancer Following “Vaccination” with Anti-CD3 x Anti-Her2/Neu Bispecific Antibody (Her2Bi) Armed Activated T Cells

Abstract 1914Novel therapeutic approaches are needed for women with metastatic breast cancer (BrCa). In our phase I clinical trial, infusions of anti-CD3 activated T cells (ATC) armed with anti-CD3 x anti-Her2/neu bispecific antibody (Her2Bi) induced specific cytotoxicity (SC) directed at SK-BR-3 breast cancer cells by fresh peripheral blood lymphocytes (PBL) and induced elevated serum levels of Th1 cytokines. In this study, we took advantage of armed ATC induced anti-tumor immune responses by infusing “immune” T cells collected by leukopheresis. We expanded “immune T cells” with anti-CD3 and IL-2, followed by a cryopreservation for multiple re-infusions of ATC after high dose chemotherapy (HDC) and autologous stem cell transplant (SCT). We asked whether this approach would transfer anti-tumor responses back into patients after HDC and SCT to accelerate the development of cellular and humoral anti-tumor responses. This strategy of “prime” with armed ATC and “boost” with ATC was performed in 5 evaluable patients. The “priming” portion involved 8 infusions of (2 infusions/week for 4 weeks) armed ATC given with daily low dose IL-2 (300,000 IU/m2/day) and twice weekly GM-CSF (250 μg/m2). Approximately 3 weeks after the armed ATC infusions, patients underwent a second leukopheresis for the collection and expansion of ATC. The expanded ATC from 6 patients at an effector:target ratio (E/T) of 25:1 exhibited specific cytotoxicity (SC) ranging from 3.7–25.8 (mean = 13.6%) directed at the SK-BR-3. Phenotyping of the ATC showed a mean of 50.2 % (25–74) CD4+ cells, 30.4 % (16.3-51.3) CD8+ cells, 11.6% (4.5-24.3) CD56+CD16+ cells, and 29.5% (10.4–41) CD4+CD25+ cells. A separate leukopheresis after G-CSF stimulation was performed to obtain CD34+ cells for the SCT. After HDC and PBSCT, 5 evaluable patients received multiple infusions with a mean total of 54×109 ATC (16–110 × 109) beginning day +1 after SCT. No G-CSF was given to accelerate engraftment. There were no dose-limiting side effects or delays in engraftment. One patient developed sepsis, multiple organ failure and recovered fully with supportive care and antibiotics. Phenotyping at 2 weeks after SCT showed the mean proportions of CD4+ and CD8+ cells to be 55.5 and 17.7%, respectively. Specific cytotoxicity (SC) directed at SK-BR-3 targets ranged from 4.7 to 70% at E/T of 25:1 up to 18 months post SCT but not against a negative control cell line-Daudi. Mean serum anti-SK-BR-3 antibody levels were 800 ng/ml preSCT and 1500, 1080 and 1360 ng/ml at 1, 2, and 3 months post SCT, respectively. In vitro anti-SK-BR-3 antibody synthesis was assessed using a new assay (Thakur et al, Cancer Immunol Immunother EPub, 2011) showed easily detectable levels of in vitro anti-SK-BR-3 antibody synthesis. The mean anti-SK-BR-3 antibody synthesis in the presence of CpG in pre-immunotherapy (Pre-IT), mid-IT, 1 months post-IT, pre-SCT and 1, 2, and 3 months post-SCT is summarized in the table. PBL produced anti-SK-BR-3 antibody pre-SCT and there was clear recovery of anti-SK-BR-3 antibody synthesis by PBL at 2 and 3 months after SCT. These data show infusions of immune ATC transferred cytotoxic T lymphocytes and humoral antibody activity directed at tumor antigens. These novel findings suggest that adaptive immunity was transferred into patients by ATC infusions and the stem cell product after myeloablation and SCT leading to rapid reconstitution of anti-tumor immunity.Time PointsCulture ConditionsMean±SD (n=3)Pre-ITCpG23±27Mid-ITCpG66±671M Post-ITCpG41±67Pre-SCTCpG92±101M Post-SCTCpG9±182M Post-SCTCpG45±273M Post-SCTCpG82±55 Disclosures:Lum:Transtarget Inc: Equity Ownership, Founder of Transtarget.