Significance of Tim-3 and Its Ligand Galectin-9 in Th1/Th2 Imbalance in Patients with Multiple Myeloma / 中国实验血液学杂志

OBJECTIVE@#To investigate the significance of Tim-3 and Galectin-9 in Th1/Th2 imbalance in patients with multiple myeloma (MM).@*METHODS@#55 newly diagnosed MM patients and 20 healthy controls were included. Flow cytometry was used to detect the expression of Tim-3 on CD4+T cells, the proportion of Th1, Th2, Tim-3+Th1 and Tim-3+Th2 cells in peripheral blood. ELISA was used to detect the levels of cytokines IFN-γ and IL-4 in serum, and PCR was used to detect the level of Galectin-9 mRNA. Then the correlations between Galectin-9 mRNA expression and Th-cell subsets and related cytokine levels, as well as the relationship between Tim-3+Th1/Tim-3+Th2 ratio and corresponding clinical features were analyzed.@*RESULTS@#Compared with the control group, the expression of Tim-3 on CD4+T cells in peripheral blood of MM patients was significantly increased (P<0.05), the proportions of Tim-3+Th1 cells, Tim-3+Th2 cells and Tim-3+Th1/Tim-3+Th2 ratio in MM patients were also increased (P<0.05), while the proportion of Th1 cells and Th1/Th2 ratio in MM patients were significantly decreased (P<0.05). The level of cytokine IFN-γ and IFN-γ/IL-4 ratio in MM patients were significantly decreased (P<0.05), while the level of cytokine IL-4 was increased (P<0.05). The mRNA levels of Galectin-9 in MM patients were significantly increased (P<0.05). The levels of Galectin-9 mRNA were positively correlated with Tim-3+CD4+T cells (r=0.663), Tim-3+Th2 cells (r=0.492) and IL-4 (r=0.470), while negatively correlated with IFN-γ (r=-0.593). The ratios of Tim-3+Th1/Tim-3+Th2 in MM patients were positively correlated with ISS stage (r=0.511), osteolytic damage (r=0.556) and chromosome abnormality (r=0.632).@*CONCLUSION@#These results suggest that Tim-3 and Galectin-9 are involved in Th1/Th2 imbalance in MM patients, and the high ratio of Tim-3+Th1/Tim-3+Th2 is associated with poor clinical prognosis.

Upregulation of IL-18 expression in blood CD4+ Th2 cells of patients with allergic rhinitis / 细胞与分子免疫学杂志

Objective To investigate the expressions of IL-18, IL-18 binding protein isoform a (IL-18BPa) and IL-18 receptor α (IL-18Rα) in blood CD4+ Th2 cells of patients with allergic rhinitis (AR) and the effects of allergens on their expressions. Methods Blood samples of AR patients and healthy control subjects (HCs) were collected. Peripheral blood mononuclear cells (PBMCs) and CD4+ T cells sorted by immunomagnetic beads were stimulated by crude extract of Artemisia sieversiana wild allergen (ASWE), Platanus pollen (PPE) and house dust mite extract (HDME). Flow cytometry was used to detect the expression of IL-18, IL-18BPa and IL-18Rα in CD4+ Th2 cells, and BioPlex was used to detect the level of plasma IL-4 and analyze its correlation with the proportion of IL-18+ Th2 cells. Results Compared with HCs, the proportion of IL-18+ cells was increased in Th2 cells of AR patients; MFI of IL-18 was increased, while that of IL-18Rα was decreased. Moreover, allergens induced IL-18 and IL-18Rα expression in sorted CD4+ Th2 cells of HCs and induced IL-18Rα in that of AR patients. Additionally, elevated plasma IL-4 level was found in AR patients, which was moderately correlated with the percentage of IL-18+ Th2 cells. Conclusion Allergens may be involved in the pathogenesis of AR by inducing expression of IL-18 in peripheral blood CD4+ Th2 cells.

The number of TIGIT+CD8+ T cells increases but their cytokine secretion decreases in the lungs of Plasmodium yoelii infected mice / 细胞与分子免疫学杂志

Objective To investigate the effect of T cell immunoreceptor with Ig and ITIM domains (TIGIT) on the function of CD8+ T cells in the lungs of Plasmodium infected mice. Methods The lungs of the mice infected with Plasmodium yoelii were isolated, weighed and photographed after 12 days' infection. After dissolution, lung lymphocytes were isolated, counted and stained, and then the contents of CD8+ and TIGIT+CD8+ T cells were detected by flow cytometry. The expressions of L selectin (CD62L), CD69, programmed death 1 (PD-1), CD25, and C-X3-C motif chemokine receptor 1 (CX3CR1) on TIGIT+CD8+ T cells were detected by flow cytometry. After stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, the ability of TIGIT+CD8+T cells to secrete interferon γ(IFN-γ), interleukin 21 (IL-21), IL-4, IL-17, and IL-10 was detected. Results The body mass of mice with Plasmodium infection was reduced. The lungs became darker, and the ratio of the lung mass to body mass was significantly increased. Compared with the normal mice, the percentages and absolute quantity of CD8+ and TIGIT+CD8+ T cells in the lungs of the infected mice were significantly increased. The percentage of TIGIT+CD8+ T cells expressing CD62L in the infected group was significantly lower, while the percentage of the CD69, PD-1, and CX3CR1 cells were significantly higher than that of TIGIT+CD8+ T cells from the normal mice. The percentages of TIGIT+CD8+ T cells secreting IL-21, IL-4, IL-17 and IL-10 cells in the infected group were significantly lower. Conclusion The lung lesions from mice with Plasmodium infection are obvious, the numbers of TIGIT+CD8+ T cells increase, and these cells express a variety of activation-related molecules, but the ability to secrete cytokines is reduced.

Improvement effect of Shegan Mahuang Decoction on rats with cold-induced asthma based on TRPV1/NRF-1/mtTFA pathway / 中国中药杂志

This study investigated the therapeutic effect of Shegan Mahuang Decoction(SGMHD) on cold-induced asthma in rats and explored its underlying mechanism. Seventy-two healthy male SD rats of specific pathogen free(SPF) grade were randomly divided into a blank group, a model group, a positive control group(dexamethasone, 0.4 mg·kg~(-1)), and low-, medium-, and high-dose SGMHD groups(3.2, 6.4, and 12.8 g·kg~(-1)). The blank group received saline, while the other groups were sensitized by intraperitoneal injection of ovalbumin(OVA) solution. Subsequently, the rats were placed in a cold chamber adjustable to 0-2 ℃, and OVA solution was ultrasonically nebulized to induce cold-induced asthma in rats. After three weeks of treatment, the general behaviors of rats were observed. Hematoxylin-eosin(HE) staining was used to evaluate pathological changes in lung tissues, periodic acid-Schiff(PAS) staining assessed mucin changes, and Masson staining was performed to examine collagen deposition. Enzyme-linked immunosorbent assay(ELISA) was used to measure the levels of the inflammatory factors interleukin-4(IL-4) and vascular endothelial growth factor(VEGF) in serum and bronchoalveolar lavage fluid(BALF). Real-time quantitative polymerase chain reaction(RT-PCR) was employed to assess the mRNA expression levels of transient receptor potential vanilloid subfamily member 1(TRPV1), nuclear respiratory factor 1(NRF-1), and mitochondrial transcription factor A(mtTFA) in lung tissues. Western blot was used to measure the protein expression levels of TRPV1, NRF-1, and mtTFA in lung tissues. Compared with the blank group, the model group exhibited signs of rapid respiration, increased frequency of defecation with looser stools, and disheveled and dull fur. Pathological results showed significant infiltration of inflammatory cells in lung tissues, narrowing of bronchial lumens, increased mucin secretion, and enhanced collagen deposition in the model group. Additionally, the levels of IL-4 and VEGF in serum and BALF were significantly elevated, and the mRNA and protein expression levels of TRPV1, NRF-1, and mtTFA in lung tissues were significantly increased. Compared with the model group, SGMHD improved the behaviors of rats, alleviated pathological changes in lung tissues, mucin production, and collagen deposition, significantly decreased the levels of IL-4 and VEGF in serum and BALF, and reduced the mRNA expression levels of TRPV1, NRF-1, and mtTFA in lung tissues, with the medium-dose SGMHD group showing the most significant effect. Moreover, the protein expression levels of TRPV1, NRF-1, and mtTFA in lung tissues were also reduced, with the medium-dose SGMHD group exhibiting the most significant effect. In conclusion, this study demonstrates that SGMHD can alleviate airway inflammation and inhibit airway remodeling in cold-induced asthma rats. These effects may be associated with the modulation of the TRPV1/NRF-1/mtTFA signaling pathway.

Bombyx Batryticatus extract promotes microglia polarization to improve neuron injury and behaviors of cerebral ischemia/reperfusion rats / 中国中药杂志

This study aims to investigate the effect of Bombyx Batryticatus extract(BBE) on behaviors of rats with global cerebral ischemia reperfusion(I/R) and the underlying mechanism. The automatic coagulometer was used to detect the four indices of human plasma coagulation after BBE intervention for quality control of the extract. Sixty 4-week-old male SD rats were randomized into sham operation group(equivalent volume of normal saline, ip), model group(equivalent volume of normal saline, ip), positive drug group(900 IU·kg~(-1) heparin, ip), and low-, medium-, and high-dose BBE groups(0.45, 0.9, and 1.8 mg·g~(-1)·d~(-1) BBE, ip). Except the sham operation group, rats were subjected to bilateral common carotid artery occlusion followed by reperfusion(BCCAO/R) to induce I/R. The administration lasted 7 days for all the groups. The behaviors of rats were examined by beam balance test(BBT). Morphological changes of brain tissue were observed based on hematoxylin-eosin(HE) staining. Immunofluorescence method was used to detect common leukocyte antigen(CD45), leukocyte differentiation antigen(CD11b), and arginase-1(Arg-1) in cerebral cortex(CC). The protein expression of interleukin-1β(IL-1β), interleukin-4(IL-4), interleukin-6(IL-6), and interleukin-10(IL-10) was detected by enzyme-linked immunosorbent assay(ELISA). The non-targeted metabonomics was employed to detect the levels of metabolites in plasma and CC of rats after BBE intervention. The results of quality control showed that the BBE prolonged the activated partial thromboplastin time(APTT), prothrombin time(PT), and thrombin time(TT) of human plasma, which was similar to the anticoagulation effect of BBE obtained previously. The results of behavioral test showed that the BBT score of the model group increased compared with that of the sham operation group. Compared with the model group, BBE reduced the BBT score. As for the histomorphological examination, compared with the sham operation group, the model group showed morphological changes of a lot of nerve cells in CC. The nerve cells with abnormal morphology in CC decreased after the intervention of BBE compared with those in the model group. Compared with the sham operation group, the model group had high average fluorescence intensity of CD45 and CD11b in the CC. The average fluorescence intensity of CD11b decreased and the average fluorescence intensity of Arg-1 increased in CC in the low-dose BBE group compared with those in the model group. The average fluorescence intensity of CD45 and CD11b decreased and the average fluorescence intensity of Arg-1 increased in medium-and high-dose BBE groups compared with those in the model group. The expression of IL-1β and IL-6 was higher and the expression of IL-4 and IL-10 was lower in the model group than in the sham operation group. The expression of IL-1β and IL-6 was lower and the expression of IL-4 and IL-10 was higher in the low-dose, medium-dose, and high-dose BBE groups than in the model group. The results of non-targeted metabonomics showed that 809 metabolites of BBE were identified, and 57 new metabolites in rat plasma and 45 new metabolites in rat CC were found. BBE with anticoagulant effect can improve the behaviors of I/R rats, and the mechanism is that it promotes the polarization of microglia to M2 type, enhances its anti-inflammatory and phagocytic functions, and thus alleviates the damage of nerve cells in CC.

Protective effects of three kinds of borneol on different brain regions in acute cerebral ischemia/reperfusion model rats / 中国中药杂志

This study compared the ameliorating effects of L-borneol, natural borneol, and synthetic borneol on the injury of different brain regions in the rat model of acute phase of cerebral ischemia/reperfusion(I/R) for the first time, which provides a reference for guiding the rational application of borneol in the early treatment of ischemic stroke and has important academic and application values. Healthy specific pathogen-free(SPF)-grade SD male rats were randomly assigned into 13 groups: a sham-operation group, a model group, a Tween model group, a positive drug(nimodipine) group, and high-, medium-, and low-dose(0.2, 0.1, and 0.05 g·kg~(-1), respectively) groups of L-borneol, natural borneol, and synthetic borneol according to body weight. After 3 days of pre-administration, the rat model of I/R was established by suture-occluded method and confirmed by laser speckle imaging. The corresponding agents in different groups were then administered for 1 day. The body temperature was monitored regularly before pre-administration, days 1, 2, and 3 of pre-administration, 2 h after model awakening, and 1 d after model establishment. Neurological function was evaluated based on Zea-Longa score and modified neurological severity score(mNSS) 2 h and next day after awakening. The rats were anesthetized 30 min after the last administration, and blood was collected from the abdominal aorta. Enzyme-linked immunoassay assay(ELISA) was employed to determine the serum levels of tumor necrosis factor-alpha(TNF-α), interleukin-6(IL-6), IL-4, and transforming growth factor-beta1(TGF-β1). The brain tissues were stained with triphenyltetrazolium chloride(TTC) for the calculation of cerebral infarction rate, and hematoxylin-eosin(HE) staining was used for observing and semi-quantitatively evaluating the pathological damage in different brain regions. Immunohistochemistry was employed to detect the expression of ionized calcium binding adapter molecule 1(IBA1) in microglia. q-PCR was carried out to determine the mRNA levels of iNOS and arginase 1(Arg1), markers of polarization phenotype M1 and M2 in microglia. Compared with the sham-operation group, the model group and the Tween model group showed significantly elevated body temperature, Zea-Longa score, mNSS, and cerebral infarction rate, severely damaged cortex, hippocampus, and striatum, increased serum levels of IL-6 and TNF-α, and decreased serum levels of IL-4 and TGF-β1. The three borneol products had a tendency to reduce the body temperature of rats 1 day after modeling. Synthetic borneol at the doses of 0.2 and 0.05 g·kg~(-1), as well as L-borneol of 0.1 g·kg~(-1), significantly reduced Zea-Longa score and mNSS. The three borneol products at the dose of 0.2 g·kg~(-1) significantly reduced the cerebral infarction rate. L-borneol at the doses of 0.2 and 0.1 g·kg~(-1) and natural borneol at the dose of 0.1 g·kg~(-1) significantly reduced the pathological damage of the cortex. L-borneol and natural borneol at the dose of 0.1 g·kg~(-1) attenuated the pathological damage of hippocampus, and 0.2 g·kg~(-1) L-borneol attenuated the damage of striatum. The 0.2 g·kg~(-1) L-borneol and the three doses of natural borneol and synthetic borneol significantly reduced the serum level of TNF-α, and the 0.1 g·kg~(-1) synthetic borneol reduced the level of IL-6. L-borneol and synthetic borneol at the dose of 0.2 g·kg~(-1) significantly inhibited the activation of cortical microglia, and 0.2 g·kg~(-1) L-borneol up-regulated the expression of Arg1 and down-regulated the expression level of iNOS. In conclusion, the three borneol products may alleviate inflammation to ameliorate the pathological damage of brain regions of rats in the acute phase of I/R by inhibiting the activation of microglia and promoting the polarization of microglia from M1 type to M2 type. The protective effect on brain followed a trend of L-borneol > synthetic borneol > natural borneol. We suggest L-borneol the first choice for the treatment of I/R in the acute phase.

Regulating the immune response to carbon tetrachloride-induced liver fibrosis in mice by blocking inducible co-stimulatory molecules and interleukin-33 / 中华肝脏病杂志

Objective: To investigate the effects of combined blockade of interleukin-33 (IL-33) and inducible co-stimulatory molecule (ICOS) on carbon tetrachloride-induced chronic liver fibrosis and imbalance of T helper lymphocyte subsets in mice. Methods: There were 40 BALB/c mice in each model and control group. Flow cytometry was used to determine the proportion of Th1/Th2/Th17 cells in the splenic lymphocyte suspension of mice, the expression levels of interferon γ, IL-4, and IL-17 in the splenic lymphocyte suspension of liver fibrosis mice after combined blockade of IL-33 and ICOS, and the pathological changes of liver histopathology in mice with liver fibrosis. Two independent sample t-test was used to compare data between groups. Results: Compared with the non-blocking group, the proportion of Th2 and Th17 cells in the IL-33/ICOS blocking group was significantly down-regulated (Th2: 65.96% ± 6.04% vs. 49.09% ± 7.03%; Th17: 19.17% ± 4.03% vs. 9.56% ± 2.03%), while the proportion of Th1 cells and Th1/Th2 ratio were up-regulated (Th1: 17.14% ± 3.02% vs. 31.93% ± 5.02%; Th1/Th2: 0.28 ± 0.06 vs. 0.62 ± 0.23), and the difference was statistically significant (t = 5.15, 6.03, 7.14, 4.28, respectively, with P < 0.05). After entering the chronic inflammation stage of liver fibrosis in mice (10 weeks), compared with the non-blocking group, the expression levels of IL-4 and IL-17 in the blockade group were significantly down-regulated [IL-4: (84.75 ± 14.35) pg/ ml vs. (77.88 ± 19.61) pg/ml; IL-17: (72.38 ± 15.13) pg/ml vs. (36.38 ± 8.65) pg/ml], while the expression of interferon γ was up-regulated [(37.25 ± 11.51) pg/ml vs. (77.88 ± 19.61) pg/ml], and the difference was statistically significant (t: IL-4: 4.71; IL-17: 5.84; interferon γ: 5.05, respectively, with P < 0.05). Liver histopathological results showed that hepatic necrosis, hepatic lobular structural disorder, and fibrous tissue hyperplasia were significantly lower in the blockade group than those in the non-blocking group at 13 weeks of liver fibrosis. Conclusion: Combined blockade of the ICOS signaling pathway and IL-33 can regulate Th2 and Th17 polarization, down-regulate the inflammatory response, and inhibit or prevent the occurrence and progression of fibrosis.

Total Saponin Fraction of Dioscorea Nipponica Makino Improves Gouty Arthritis Symptoms in Rats via M1/M2 Polarization of Monocytes and Macrophages Mediated by Arachidonic Acid Signaling / 中国结合医学杂志

OBJECTIVE@#To explore the mechanism of effects of total saponin fraction from Dioscorea Nipponica Makino (TSDN) on M1/M2 polarization of monocytes/macrophages and arachidonic acid (AA) pathway in rats with gouty arthritis (GA).@*METHODS@#Seventy-two Sprague Dawley rats were randomly divided into 4 groups (n=18 in each): normal, model, TSDN at 160 mg/kg, and celecoxib at 43.3 mg/kg. Monosodium urate crystal (MSU) was injected into the rats' ankle joints to induce an experimental GA model. Blood and tissue samples were collected on the 3rd, 5th, and 8th days of drug administration. Histopathological changes in the synovium of joints were observed via hematoxylin and eosin (HE) staining. The expression levels of arachidonic acid (AA) signaling pathway were assessed via real-time polymerase chain reaction (qPCR) and Western blot. Flow cytometry was used to determine the proportion of M1 and M2 macrophages in the peripheral blood. An enzyme-linked immunosorbent assay (ELISA) was used to detect interleukine (IL)-1 β, tumor necrosis factor-alpha (TNF-α), IL-4, IL-10, prostaglandin E2 (PGE2), and leukotriene B4 (LTB4).@*RESULTS@#HE staining showed that TSDN improved the synovial tissue. qPCR and Western blot showed that on the 3rd, 5th and 8th days of drug administration, TSDN reduced the mRNA and protein expressions of cyclooxygenase (COX)2, microsomal prostaglandin E synthase-1 derived eicosanoids (mPGES-1), 5-lipoxygenase (5-LOX), recombinant human mothers against decapentaplegic homolog 3 (Smad3), nucleotide-binding oligomerization domain-like receptor protein 3 (NALP3), and inducible nitric oxide synthase (iNOS) in rats' ankle synovial tissues (P<0.01). TSDN decreased COX1 mRNA and protein expression on 3rd and 5th day of drug administration and raised it on the 8th day (both P<0.01). It lowered CD68 protein expression on days 3 (P<0.01), as well as mRNA and protein expression on days 5 and 8 (P<0.01). On the 3rd, 5th, and 8th days of drug administration, TSDN elevated the mRNA and protein expression of Arg1 and CD163 (P<0.01). Flow cytometry results showed that TSDN decreased the percentage of M1 macrophages while increasing the percentage of M2 in peripheral blood (P<0.05 or P<0.01). ELISA results showed that on the 3rd, 5th, and 8th days of drug administration, TSDN decreased serum levels of IL-1 β, TNF-α, and LTB4 (P<0.01), as well as PGE2 levels on days 3rd and 8th days (P<0.05 or P<0.01); on day 8 of administration, TSDN increased IL-4 serum levels and enhanced IL-10 contents on days 5 and 8 (P<0.05 or P<0.01).@*CONCLUSION@#The anti-inflammatory effect of TSDN on rats with GA may be achieved by influencing M1/M2 polarization through AA signaling pathway.

Triptolide inhibits inflammatory response and migration of fibroblast like synovial cells in rheumatoid arthritis through the circRNA 0003353/JAK2/STAT3 signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effect of triptolide (TPL) on inflammatory response and migration of fibroblast like synovial cells (FLS) in rheumatoid arthritis (RA-FLS) and the mechanism of circular noncoding RNA (circRNA) 0003353 for mediating this effect.@*METHODS@#We collected peripheral blood mononuclear cells (PBMCs) and serum samples from 50 hospitalized RA patients and 30 healthy individuals for detecting the expression of circRNA 0003353, immune and inflammatory indexes (ESR, CRP, RF, anti-CCP, IgA, IgG, IgM, C3, and C4) and DAS28 score. Cultured RA-FLS was treated with 10 ng/mL TPL and transfected with a circRNA 0003353 overexpression plasmid, and cell counting kit-8 (CCK-8) assay and Transwell assay were used to detect the changes in the viability and migration of the cells. Enzyme-linked immunosorbent assay (ELISA) was used to examine the cytokines IL-4, IL-6, and IL-17, and real-time fluorescence quantitative PCR (RT-qPCR) was performed to detect the expression of circRNA 003353; Western blotting was used to detect the expressions of p-JAK2, pSTAT3, JAK2 and STAT3 proteins in the treated cells.@*RESULTS@#The expression of circRNA 0003353 was significantly increased in PBMCs from RA patients and showed a good performance in assisting the diagnosis of RA (AUC=90.5%, P < 0.001, 95% CI: 0.83-0.98). CircRNA 0003353 expression was positively correlated with ESR, RF and DAS28 (P < 0.05). Treatment with TPL significantly decreased the expression of circRNA 0003353, suppressed the viability and migration ability, decreased the expressions of IL-6 and IL-17, and increased the expression IL-4 in cultured RA-FLS in a time-dependent manner (P < 0.01). TNF-α stimulation of RA-FLS significantly increased the ratios of p-JAK2/JAK2 and p-STAT3/STAT3, which were obviously lowered by TPL treatment (P < 0.01). TPL-treated RA-FLS overexpressing circRNA 0003353 showed significantly increased cell viability and migration ability with decreased IL-4 expression and increased IL-6 and IL-17 expressions and ratios of p-JAK2/ JAK2 and p-STAT3/STAT3 (P < 0.01).@*CONCLUSION@#The expression of circRNA 0003353 is increased in PBMCs in RA patients and in RA-FLS. TPL treatment can regulate JAK2/STAT3 signal pathway and inhibit the inflammatory response and migration of RA-FLS through circRNA 0003353.

STAT6 Expression and IL-13 Production in Association with Goblet Cell Hyperplasia and Worm Expulsion of Gymnophalloides seoi from C57BL/6 Mice

In intestinal helminth infections, Th2 immune respones are generally associated with mucin secretion for worm expulsion from the host intestine. In particular, IL-4 and IL-13 are the important cytokines related with intestinal mucus production via STAT6 signalling in nematode infections. However, this perspective has never been studied in Gymnophalloides seoi infection. The present study aimed to observe the STAT6 signalling and cytokine responses in C57BL/6 mice, a mouse strain resistant to infection with this trematode. The results showed that worm expulsion occurred actively during days 1-2 post-infection (PI), when goblet cells began to proliferate in the small intestine. The STAT6 gene expression in the mouse spleen became remarkable from day 2 PI. Moreover, G. seoi infection induced a significant increase of IL-13 from day 4 PI in the spleen of infected mice. Our results suggested that goblet cell hyperplasia and worm expulsion in G. seoi-infected mice should be induced by STAT6 signalling, in which IL-13 may be involved as a dominant triggering cytokine.

Sclareol reduces CD4+ CD25+ FoxP3+ t-reg cells in a breast cancer model in vivo

Sclareol is a phytochemical used in people's diet in Southeast Asia. To investigate the immunotherapeutic effectiveness of Sclareol against breast cancer by direct intraperitoneal injection. Sclareol was isolated and purified from Salvia sclarea. Effect of Sclareol on cell growth inhibition was evaluated by MTT assay. Intraperitoneally injected Sclareol effects on reducing the tumor volume and shifting the cytokine profile were investigated. We also assessed if intraperitoneally injected Sclareol could improve the outcome of cancer therapy through suppressing the regulatory T cells. The results confirmed a significant decrease in the tumor size. Furthermore, a significant decrease in the level of IL-4 and an increase in the level of IFN-gamma were noticed in the intraperitoneally injected Sclareol group [p<0.05]. It was also observed that the splenocytes of treated animals significantly increase in cell proliferation assay. Moreover, measurements of splenic T regulatory cell indicated that intraperitoneally injected Sclareol significantly decreased the number of splenic T regulatory cell. Our results suggest that Sclareol, by reducing T-reg cells frequency and also tumor size can enhance the effect of cancer therapy as an immunostimulant

Protease-Activated Receptor 2 Is Involved in Th2 Responses against Trichinella spiralis Infection

In order to get a better understanding of the role of protease-activated receptor 2 (PAR2) in type 2 helper T (Th2) cell responses against Trichinella spiralis infection, we analyzed Th2 responses in T. spiralis-infected PAR2 knockout (KO) mice. The levels of the Th2 cell-secreted cytokines, IL-4, IL-5, and IL-13 were markedly reduced in the PAR2 KO mice as compared to the wild type mice following infection with T. spiralis. The serum levels of parasite-specific IgE increased significantly in the wild type mice as the result of T. spiralis infection, but this level was not significantly increased in PAR2 KO mice. The expression level of thymic stromal lymphopoietin, IL-25, and eotaxin gene (the genes were recently known as Th2 response initiators) of mouse intestinal epithelial cells were increased as the result of treatment with T. spiralis excretory-secretory proteins. However, the expression of these chemokine genes was inhibited by protease inhibitor treatments. In conclusion, PAR2 might involve in Th2 responses against T. spiralis infection.

The Soluble Tumor Necrosis Factor-Alpha Receptor Suppresses Airway Inflammation in a Murine Model of Acute Asthma

PURPOSE: Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that has been implicated in many aspects of the airway pathology in asthma. TNF-alpha blocking strategies are now being tried in asthma patients. This study investigated whether TNF-alpha blocking therapy inhibits airway inflammation and airway hyperresponsiveness (AHR) in a mouse model of asthma. We also evaluated the effect of TNF-alpha blocking therapy on cytokine production and adhesion molecule expression. MATERIALS AND METHODS: Ovalbumin (OVA) sensitized BALB/c female mice were exposed to intranasal OVA administration on days 31, 33, 35, and 37. Mice were treated intraperitoneally with soluble TNF-alpha receptor (sTNFR) during the OVA challenge. RESULTS: There were statistically significant decreases in the numbers of total cell and eosinophil in bronchoalveolar lavage fluid (BALF) in the sTNFR treated group compared with the OVA group. However, sTNFR-treatment did not significantly decrease AHR. Anti-inflammatory effect of sTNFR was accompanied with reduction of T helper 2 cytokine levels including interleukin (IL)-4, IL-5 and IL-13 in BALF and vascular cell adhesion molecule 1 expression in lung tissue. CONCLUSION: These results suggest that sTNFR treatment can suppress the airway inflammation via regulation of Th2 cytokine production and adhesion molecule expression in bronchial asthma.

The Role of Cyclosporine and Mycophenolate in an Orthotopic Porcine-to-Rat Corneal Xenotransplantation

We performed this study to investigate the feature of rejection in porcine-to-rat corneal orthotopic transplantation and to evaluate the effect of cyclosporine and mycophenolate on the xeno-rejection. Orthotopic corneal transplantation was done at 91 Sprague-Dawley rats, and they were divided into 10 groups based on the combination of immunosuppressants including dexamethasone, cyclosporine, and mycophenolate mofetil. Graft survival was analyzed and grafted eyes were examined with Hematoxylin & Eosin and CD4 or CD8 staining. Enzyme-linked immunosorbent assays were done for interleukin-2 (IL-2), IL-4, IL-5, IL-10, and interferon (IFN)-gamma in cornea, lacrimal gland, and cervical lymph nodes. The longest median survival of the immune suppressant group was 11.00+/-1.96 days, which showed no statistical differences compared with that of control (8.00+/-1.52 days). The neutrophils were prominent in the early phase but soon gave way to the monocytes. The number of CD8+ cells was higher than that of CD4+ cells. IL-2 and IFN-gamma markedly increased at 10 to13 days in cornea, lacrimal glands, and cervical lymph nodes, which showed a decrease with immunosuppressants except in the cornea. In conclusion, cyclosporine and mycophenolate could not prevent the rejection in porcine to rat orthotopic corneal xenograft associated with infiltraton of CD8+ and innate immune cells.

The Effect of Interleukin-4 and Amphiregulin on the Proliferation of Human Airway Smooth Muscle Cells and Cytokine Release

Airway smooth muscle (ASM) hyperplasia and angiogenesis are important features associated with airway remodeling. We investigated the effect of IL-4 and amphiregulin, an epidermal growth factor family member, on the proliferation of human ASM cells and on the release of vascular endothelial growth factor (VEGF) and monocyte chemotactic protein (MCP)-1 from human ASM cells. Human ASM cells were growth-arrested for 48 hr and incubated with platelet-derived growth factor (PDGF)- BB, interleukin (IL)-4, amphiregulin, and VEGF to evaluate cell proliferation. The cells were treated with PDGF, IL-4 and amphiregulin to evaluate the release of VEGF, MCP-1. IL-4 suppressed unstimulated and PDGF-stimulated ASM cell proliferation. Amphiregulin stimulated ASM cell proliferation in a dose-dependent manner. VEGF did not have any influence on ASM cell proliferation. IL-4 stimulated VEGF secretion by the ASM cells in a dose-dependent manner and showed added stimulatory effects when co-incubated with PDGF. Amphiregulin did not promote VEGF secretion. IL-4 and amphiregulin showed no stimulatory effects on MCP-1 secretion. The results of this study showed that IL-4 had bifunctional effects on airway remodeling, one was the suppression of the proliferation of the ASM cells and the other was the promotion of VEGF release by the ASM cells, and amphiregulin can promote human ASM cell proliferation.

The effect of vitamin A on secretion of IFN-gamma and IL-4 in A549 cells induced by Mycoplasma pneumoniae / 华中科技大学学报（医学）（英德文版）

In order to investigate the effect of vitamin A (VA) on the secretion of IFN-gamma and IL-4 in Mycoplasma Pneumoniae (MP)-induced A549 cells, A549 cells were co-cultured with MP for different time lengths and then the levels of IFN-gamma and IL-4 in the cell culture supernatants were detected before and after treatment with different concentrations of VA by using the enzyme-linked immunosorbent assay (ELISA). The results showed that the level of IFN-gamma and IL-4 in the supernatants of MP-induced A549 cells was much higher than that in non-induced cells (P<0.01). After application of VA, IL-4 level was not increased until the concentration of VA was up to 0.5x10(-5) mol/L (P<0.01). However, with concentration of VA increased up to 1x10(-4) mol/L, IL-4 was significantly suppressed (P<0.01). It was concluded that MP could induce the secretion of IFN-gamma and IL-4 in A549 cells. VA could inhibit the secretion of IFN-gamma and increase the IL-4 level in MP-induced A549 cells. However, high concentration of VA had an inhibitory effect on the secretion of IL-4 as well as on the IFN-gamma. These data provided a theoretical basis for the application of VA in MP pneumonia in the clinical practice.

Differential T helper cell response in tuberculous pleuritis.

PURPOSE: This study was conducted to understand the in vivo and in vitro immune responses and to find whether there exists any difference in the systemic and localized immune responses in tuberculous pleuritis. METHODS: The in vivo levels of IFN-gamma and IL-4 were compared in plasma (BL) and pleural fluid (PF) of 47 tuberculous (TB) and 31 nontuberculous pleuritis (Non-TB) patients. In vitro cytokine response to various mycobacterial antigens was studied in 19 TB patients by ELISA. Both ex vivo and in vitro cytokine responses were further ascertained by intracellular cytokine staining on purified CD4+ T cells from pleural fluid mononuclear cells (PFMC) of 10 TB patients. RESULTS: The ex vivo results showed a significant increase in IFN-gamma levels and higher IFN-gamma + T cells in PF. On the other hand, in vitro results showed simultaneous increase in both IFN-gamma and IL-4 levels in the supernatants of antigen stimulated PFMC. Similarly antigen specific increase was observed in both IFN-gamma + and IL-4+ T cells in all cultured conditions. However, the percentile increase was more in IL-4 secreting T cells, significant for PPD stimulation (P < 0.05), indicating that in vitro cellular response was dominated by Th2 type. CONCLUSIONS: These results showed a differential T-helper response in TB pleuritis suggestive of predominant Th1 in vivo and mixed response (Th1 and Th2) under in vitro conditions.

Pre- & post-treatment evaluation of immunological features in Indian visceral leishmaniasis (VL) patients with HIV co-infection.

BACKGROUND & OBJECTIVE: The risk of human immunodeficiency virus (HIV) co-infection in patients with visceral leishmaniasis (VL) or kala-azar in endemic areas has posed a major challenge in control programmes. We undertook this study to identify the high risk groups vulnerable to Leishmania-HIV co-infection in VL endemic State of Bihar, India. Further, immunological responses were also evaluated in these patients before and after treatment for VL to see the immune impairment associated with CD4 T cell count. METHODS: A total of 1511 subjects attending Voluntary Counselling and Testing Centre (VCTC) at Patna, Bihar were included in this study. VL was confirmed by splenic or bone marrow aspirates testing for parasite. HIV states was confirmed by two kits. Immunological parameters (CD4, CD8, IFN-gamma, IL-4) were studied in co-infection patients. RESULTS: Of the 280 (18.53%) HIV-positive individuals, eight were diagnosed serologically and pathologically as VL patients co-infected with HIV. The humoral and cellular immune responses were evaluated in 18 Indian VL patients with (n = 8) or without HIV (n = 10) and 10 HIV seropositive subjects. Among the eight confirmed cases of VL, false negative direct agglutination test (DAT) result was observed in two who had HIV co-infection (sensitivity 80%), while none in 10 other VL cases who were HIV negative (sensitivity 100%). A very low CD4 cell count was observed in VL cases that had HIV co-infection compared to HIV negative VL or controls. All VL cases with or without HIV infection had lower Th1/Th2 ratio compared to controls. VL patients with or without HIV infection responded well to anti-leishmanial/anti-retroviral therapy with considerable degree of immunological reconstitution. INTERPRETATION & CONCLUSION: A different immune response was noticed in patients with co-infection of HIV and Leishmania. Anti-leishmanial drug treatment led to improvement in immunological response in co-infected patients. Further studies need to be done to see the effect of combined therapy for VL and HIV on immunological parameters in these patients.

Identification of 38kDa Brugia malayi microfilarial protease as a vaccine candidate for lymphatic filariasis.

A FPLC purified 38kDa protease (Bm mf S-7) isolated from B. malayi microfilarial soluble antigen was identified. It showed pronounced reactivity with sera collected from 'putatively immune' asymptomatic and amicrofilaraemic individuals residing in an endemic area for bancroftian filariasis. Further the immune protective activity of Bm mf S-7 antigen was evaluated in susceptible hosts, jirds (Meriones unguiculatus) against B. malayi filarial infection. The antigen showed 89% cytotoxicity against mf and 87-89% against infective (L3) larvae in in vitro antibody dependent cellular cytotoxicity Assay (ADCC) and in situ micropore chamber methods. Bm mf S-7 immunized jirds after challenge infection showed 81.5% reduction in the adult worm burden. The present study has shown that, the 38kDa microfilarial proteases (Bm mf S-7) could stimulate a strong protective immune response against microfilariae and infective larvae in jird model to block the transmission of filariasis. Analysis of IgG subclasses against Bm mf S-7 revealed a significant increase in IgG2 and IgG3 antibodies in endemic normals. Lymphocyte proliferation to Bm mf S-7 was significantly high in endemic normal group as compared to that in clinical and microfilarial carriers. Significantly enhanced levels of IFN-gamma in the culture supernatant of PBMC of endemic normals followed by stimulation with Bm mf S-7 suggest that the cellular response in this group is skewed towards Th 1 type.

Increased circulating CCR3+ type 2 helper T cells in house dust mite-sensitive Chinese patients with allergic diseases.

Chemokine receptor expression has been shown to be associated with the differentiation of T helper cells. The CCR3, CXCR4 and CCR5 expression on circulating T cells were studied in 30 house dust mite sensitive-patients with allergic diseases and in another 30 healthy controls. The expression was analyzed in CD4, CD8 and double negative (DN) T cells by triple fluorescence staining. In addition, intracellular cytokine staining was performed in the CCR3+ CD4+ T cells. Increased circulating portions of CCR3+ CD4+ T cells and CCR3+ DN T cells were found in these patients (p < 0.01). There was no statistically significant difference in the expression of CXCR4 and CCR5 on T cells. The follow-up data of the patients did not show a statistically significant change in the CCR3 expression. IL-4 was expressed within CCR3+ CD4+ T cells upon activation. The IL-4 secreting CCR3+ type 2 T helper cells may play a pathogenetic role in immune responses of house dust mite-sensitive Chinese patients with allergic diseases.