Lymphocyte Balance Research Articles

Change of Th17 Lymphocytes and Treg/Th17 in Typical and Atypical Optic Neuritis.

BackgroundTypical and atypical optic neuritis (ON) are two clinical types of autoimmune inflammatory diseases of the optic nerve that causes acute vision loss, and are difficult to distinguish in their early stages. The disturbance in the balance of Th17 and Treg lymphocytes is thought to play an essential role in these autoimmune inflammatory diseases.ObjectivesTo detect the clinical relevance of Th17 and Treg in peripheral blood and the ratio of Treg/Th17 in patients with typical and atypical ON. To determine whether analysis of Th17 and Treg lymphocytes will provides insights into the different disease phenotypes of typical and atypical ON.MethodsWe studied a consecutive series of patients aged 14–70 years who presented to our neurological department with typical ON (n = 30) or atypical ON (n = 33) within 4 weeks of their acute attacks. Routine clinical tests and ophthalmological examination were performed in all patients. Blood samples were collected from untreated patients and from gender- and age-matched healthy controls (n = 30). The proportion of peripheral blood Th17 cells and Treg cells was determined by flow cytometry.ResultsPatients with atypical ON had a higher proportion of Th17 cells than patients with typical ON (3.61±1.56 vs 2.55±1.74, P<0.01) or controls (1.45±0.86, P<0.01). The proportion of Th17 cells in patients with typical ON was also markedly higher than in controls (P<0.01). The mean percentage of Treg cells in atypical ON (6.31±2.11) and typical ON (6.80±2.00) were significantly lower when compared to controls (8.29±2.32, both P<0.01). No significant difference in Treg frequency was observed between typical ON and atypical ON (p>0.05).ConclusionsThe frequency of Th17 cells is higher in atypical ON than typical ON, and patients with atypical ON have a greater imbalance of pro-inflammatory and regulatory cells than patients with typical ON when compared with controls. These changes are indicative of distinct pathological mechanisms and may provide useful information to distinguish typical and atypical ON.

Assessment of bone marrow lymphocytic status during tyrosine kinase inhibitor therapy and its relation to therapy response in chronic myeloid leukaemia.

Tyrosine kinase inhibitors (TKIs) used in the treatment of chronic myeloid leukaemia have been reported to induce immunomodulatory effects. We aimed to assess peripheral blood (PB) and bone marrow (BM) lymphocyte status at the diagnosis and during different TKI therapies and correlate it with treatment responses. BM and PB samples were acquired from 105 first-line TKI-treated patients. Relative number of BM lymphocytes was evaluated from MGG-stained BM aspirates, and immunophenotypic analyses were performed with multicolour flow cytometry. Early 3-month expansion of BM lymphocytes was found during all different TKIs (imatinib n = 71, 20 %; dasatinib n = 25, 21 %; nilotinib n = 9, 22 %; healthy controls n = 14, 12 %, p < 0.0001). Increased PB lymphocyte count was only observed during dasatinib therapy. The BM lymphocyte expansion was associated with early molecular response; patients with 3-month BCR-ABL1 <10 % showed higher lymphocyte counts than patients with BCR-ABL1 >10 % (23 vs. 17 %, p < 0.05). Detailed phenotypic analysis showed that BM lymphocyte expansion consisted of various lymphocyte subclasses, but especially the proportion of CD19+ B cells and CD3negCD16/56+ NK cells increased from diagnostic values. During dasatinib treatment, the lymphocyte balance in both BM and PB was shifted more to cytotoxic direction (increased CD8+CD57+ and CD8+HLA-DR+ cells, and low T regulatory cells), whereas no major immunophenotypic differences were observed between imatinib and nilotinib patients. Early BM lymphocytosis occurs with all current first-line TKIs and is associated with better treatment responses. PB and BM immunoprofile during dasatinib treatment markedly differs from both imatinib- and nilotinib-treated patients.

Bone Marrow Lymphocytic Status during Tyrosine Kinase Inhibitor Therapy and Its Relation to Therapy Response in Chronic Phase Chronic Myeloid Leukemia Patients

IntroductionThe introduction of tyrosine kinase inhibitors (TKIs) has revolutionized the treatment of chronic myeloid leukemia (CML). In addition to blocking their main target kinase, the BCR-ABL oncoprotein, several studies have reported that TKIs could also have secondary effects on the immune system and lymphocyte behavior. The aim of this study was to assess the bone marrow (BM) lymphocyte status at diagnosis and during different first-line TKI therapies and correlate it with treatment responses.MethodsAltogether 105 first-line TKI treated patients were included in the study (imatinib n=71, dasatinib n=25 and nilotinib n=9) and samples from 14 healthy bone marrow donors served as controls. BM aspirate samples were taken from patients at the diagnosis and at 3, 6, 12 and 18 months after the TKI therapy start, and MGG-stained BM aspirate slides were examined for cellularity and individual cellular proportions. Treatment responses were evaluated with standard karyotyping and real-time quantitative PCR. Patients were divided in different groups according to ELN criteria based on their therapy response at 12 months. In addition, multi-color flow cytometry was done from both BM and peripheral blood (PB) samples using 5 different antibody panels including markers for T, B, NK and regulatory T cells.ResultsWe found an early (3 months) expansion of BM lymphocytes during all different TKI therapies (imatinib median lymphocyte count 20%; dasatinib 21%; nilotinib 22%; healthy controls 12%, p<0.0001). Increased PB lymphocyte count was only observed during dasatinib therapy, possibly due to mobilization phenomenon observed only in dasatinib, and not in imatinib or nilotinib treated patients. The BM lymphocytic expansion was associated with better early molecular response; patients with 3 months BCR-ABL<10% showed higher lymphocyte counts than patients with BCR-ABL>10% (median 23% and 17% respectively; p<0.05). Interestingly, patients with suboptimal (warning) response (no major molecular remission at 12 months) showed significantly higher lymphocyte counts at 3 months than patients with optimal response or failure (28%, 19.5%, and 18.5% respectively, p<0.0001). During the treatment at 12 and 18 months when patients were already in complete cytogenetic and molecular remission, BM lymphocytosis seemed to persist (median lymphocyte counts 19% and 18%), thus suggesting that the lymphocyte increase is TKI therapy induced and not only related to myeloablation and eradication of leukemia cells.Detailed phenotypic analysis showed that lymphocyte expansion consisted of various lymphocyte subclasses, but especially the proportion of CD19+ B cells and CD3negCD16/56+ NK cells increased from diagnostic values (3 months NK cells: 19.4% vs. 16.8 % at diagnosis, p<0.05; B cells 10.7% vs. 8.6%, p<0.05). Among different TKIs studied, the proportion of CD3+CD16/56+ NKT-like cells was higher in dasatinib treated patients, but no differences were observed in the amount of B, NK or T cells. However, within the T cell population dasatinib group had significantly more BM CD8+ T cells (48% and 51% at 3 and 6 months) when compared to imatinib (41% and 39%) and nilotinib treated patients (43% and 33%). The percentage of CD57+ expressing T cells was also clearly increased in dasatinib patients (40% vs. 21% and 25% in the imatinib and nilotinib groups, p=0.004) as well as the percentage of HLA-DR expressing cells (HLA-DR+ from CD4+ T cells 18% vs. 7% and 7% in imatinib and nilotinib groups, p<0.05 and from CD8+ T cells 41% vs. 22% vs. 15%, respectively, p<0.05). The proportion of regulatory T cells in the BM was significantly lower in dasatinib-treated patients (3.2%) than in the imatinib (5.7%, p<0.05) or nilotinib group (5%).ConclusionBM lymphocytosis occurs during all current first-line TKI treatments, and it is associated with early molecular response. On the contrary, PB lymphocytosis occurs only during dasatinib treatment, and the overall lymphocyte balance in dasatinib group is shifted more to cytotoxic direction (increased CD8+CD57+ and CD8+HLA-DR+ cells, and low T regulatory cells). In accordance with shared kinase inhibition spectrum, no marked changes in the immunoprofile are observed between imatinib- and nilotinib-treated patients. DisclosuresBjerrum:Bristoll Myers Squibb, Novartis and Pfizer: Other: educational activities. Porkka:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Hjorth-Hansen:Novartis: Honoraria; Ariad: Honoraria; Bristol-Myers Squibb: Research Funding; Pfizer: Honoraria, Research Funding. Mustjoki:Novartis: Honoraria, Research Funding; Academy of Finland: Research Funding; the Finnish Cancer Societies: Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Sigrid Juselius Foundation: Research Funding; Finnish Cancer Institute: Research Funding; Signe and Ane Gyllenberg Foundation: Research Funding.

Immunomodulatory activity of Nerium indicum through inhibition of nitric oxide and cyclooxygenase activity and modulation of TH1/TH2 cytokine balance in murine splenic lymphocytes.

Nerium indicum is a medicinal plant which is used in the treatment of wide range of illness in different ethnopharmacological practices. We have studied the immunomodulatory activity of the 70% hydro-methanolic extract of N. indicum leaves (NILE) by studying plaque forming cell assay, haemagglutination titre, cell proliferation assay, inhibition of nitric oxide expression and cyclooxygenase activity, estimation of IL-2, IFN-γ, IL-4, IL-10, TNF-α and IgM levels. Furthermore, we have characterized NILE with Fourier Transform Infra-red (FTIR) spectroscopy. The results displayed that NILE possessed potent immunomodulatory activity by up-regulating IL-2, IFN-γ, IL-10 expression and down-regulating IL-4, TNF-α expression in vitro. NILE also stimulated the in vivo expression of immunoglobulin level in mouse. FTIR analysis revealed phytocompounds of diverse chemical nature present in NILE. Thus, the potent immunomodulatory activity of N. indicum may have serious implications in the treatment of inflammatory diseases in the future.

Perforin is a novel immune regulator of obesity-related insulin resistance.

Obesity-related insulin resistance is associated with an influx of pathogenic T cells into visceral adipose tissue (VAT), but the mechanisms regulating lymphocyte balance in such tissues are unknown. Here we describe an important role for the immune cytotoxic effector molecule perforin in regulating this process. Perforin-deficient mice (Prf1(null)) show early increased body weight and adiposity, glucose intolerance, and insulin resistance when placed on high-fat diet (HFD). Regulatory effects of perforin on glucose tolerance are mechanistically linked to the control of T-cell proliferation and cytokine production in inflamed VAT. HFD-fed Prf1(null) mice have increased accumulation of proinflammatory IFN-γ-producing CD4(+) and CD8(+) T cells and M1-polarized macrophages in VAT. CD8(+) T cells from the VAT of Prf1(null) mice have increased proliferation and impaired early apoptosis, suggesting a role for perforin in the regulation of T-cell turnover during HFD feeding. Transfer of CD8(+) T cells from Prf1(null) mice into CD8-deficient mice (CD8(null)) resulted in worsening of metabolic parameters compared with wild-type donors. Improved metabolic parameters in HFD natural killer (NK) cell-deficient mice (NK(null)) ruled out a role for NK cells as a single source of perforin in regulating glucose homeostasis. The findings support the importance of T-cell function in insulin resistance and suggest that modulation of lymphocyte homeostasis in inflamed VAT is one possible avenue for therapeutic intervention.

Oxidative Balance in Lymphocytes From Patients With Nonalcoholic Steatohepatitis

Oxidative stress is linked to several human diseases, including nonalcoholic steatohepatitis (NASH). In this study, lymphocytes were used as a model to study this disease. These cells offer several advantages for cellular and molecular studies such as easy accessibility, and they are easily accessible and constitute a "time-persistent” system capable of reflecting the condition of the whole organism. Lymphocytes from patients with NASH display oxidative stress features. Among the possible causes for the overproduction of reactive oxygen species in NASH lymphocytes, there might be alterations of enzymatic pathways, autooxidation of glucose and mitochondrial superoxide production, which, in turn, would lead to protein oxidative damage. increased oxidative stress in lymphocytes from patients with NASH may result in a pro-oxidative environment, which, in turn, could modify the pathway of the enzymatic activities. The data confirm that an imbalance between pro-oxidant and antioxidant defense mechanisms may be an important factor in NASH.

Non-myeloablative autologous haematopoietic stem cell transplantation expands regulatory cells and depletes IL-17 producing mucosal-associated invariant T cells in multiple sclerosis.

Autologous haematopoietic stem cell transplantation has been tried as one experimental strategy for the treatment of patients with aggressive multiple sclerosis refractory to other immunotherapies. The procedure is aimed at ablating and repopulating the immune repertoire by sequentially mobilizing and harvesting haematopoietic stem cells, administering an immunosuppressive conditioning regimen, and re-infusing the autologous haematopoietic cell product. 'Non-myeloablative' conditioning regimens to achieve lymphocytic ablation without marrow suppression have been proposed to improve safety and tolerability. One trial with non-myeloablative autologous haematopoietic stem cell transplantation reported clinical improvement and inflammatory stabilization in treated patients with highly active multiple sclerosis. The aim of the present study was to understand the changes in the reconstituted immune repertoire bearing potential relevance to its mode of action. Peripheral blood was obtained from 12 patients with multiple sclerosis participating in the aforementioned trial and longitudinally followed for 2 years. We examined the phenotype and function of peripheral blood lymphocytes by cell surface or intracellular staining and multi-colour fluorescence activated cell sorting alone or in combination with proliferation assays. During immune reconstitution post-transplantation we observed significant though transient increases in the proportion of CD4+ FoxP3+ T cells and CD56(high) natural killer cell subsets, which are cell subsets associated with immunoregulatory function. CD8+ CD57+ cytotoxic T cells were persistently increased after therapy and were able to suppress CD4+ T cell proliferation with variable potency. In contrast, a CD161(high) proinflammatory CD8+ T cell subset was depleted at all time-points post-transplantation. Phenotypic characterization revealed that the CD161(high)CD8+ T cells were mucosal-associated invariant T cells, a novel cell population originating in the gut mucosa but expressing the central nervous system-homing receptor CCR6. Detection of mucosal-associated invariant T cells in post-mortem multiple sclerosis brain white matter active lesions confirmed their involvement in the disease pathology. Intracellular cytokine staining demonstrated interferon γ and interleukin 17 production and lack of interleukin 10 production, a pro-inflammatory profile. Mucosal-associated invariant T cell frequency did not change in patients treated with interferon β; and was more depleted after autologous haematopoietic stem cell transplantation than in patients who had received high-dose cyclophosphamide (n = 7) or alemtuzumab (n = 21) treatment alone, suggesting an additive or synergistic effect of the conditioning regime components. We propose that a favourably modified balance of regulatory and pro-inflammatory lymphocytes underlies the suppression of central nervous system inflammation in patients with multiple sclerosis following non-myeloablative autologous haematopoietic stem cell transplantation with a conditioning regimen consisting of cyclophosphamide and alemtuzumab.

The relationship of plasma aluminum to oxidant–antioxidant and inflammation status in asthma patients

Aluminum (Al) is a non-essential mineral which human beings are exposed to on day-to-day life. The purpose of this study was to assess the concentrations of plasma Al and the relationship of those levels with risk factors for asthma. In total, 27 allergic asthmatics and 30 healthy volunteers were enrolled. Plasma Al and selected blood parameters were measured, and a pulmonary function test was performed. Higher Al concentrations were found in the asthmatics than the healthy controls. Increased immunoglobulin E, high-sensitivity C-reactive protein, lipid peroxidation products, and pro-inflammatory cytokines (tumor necrosis factor-α and interleukin [IL]-4) were observed, but IL-10 and overall antioxidant and enzyme activities were lower. Associations between oxidative-antioxidant status and inflammatory markers with plasma Al levels in asthmatics were noted. Al status was also linked to cytokine concentrations and pulmonary function. In conclusion, abnormal Al distribution may further precipitate oxidative stress and inflammation, alter Th1/Th2 lymphocyte balance, and therefore contribute to the development of asthma.

A Novel Role of IGFBP7 in Mouse Uterus: Regulating Uterine Receptivity through Th1/Th2 Lymphocyte Balance and Decidualization

Previously we have screened out Insulin-like Growth Factor Binding Protein 7 (IGFBP7) as a differentially expressed gene in post-implantation uterus versus pre-implantation uterus by suppressive subtractive hybridation. However its function in uterus was not clearly identified. In this research, the expression and function of IGFBP7 during post-implantation were studied. We found that IGFBP7 was mainly located in the glandular epithelium and the stroma, and was upregulated after embryo implantation. The vector pCR3.1-IGFBP7-t expressing partial IGFBP7 was constructed. Inhibition of IGFBP7 by specific DNA immunization induced significant reduction of implanted embryos and pregnancy rate. The number of implanted embryos (5.68±0.46) was significantly reduced after immunization with pCR3.1-IGFBP7-t, as compared with that of the mice immunized with the control vector (12.29±0.36) or saline (14.58±0.40) (p<0.01). After specific inhibition of IGFBP7, the T helper type 1 (Th1) cytokine IFNγ, was significantly elevated (p<0.05) and the Th2 cytokines IL-4 and IL-10, were reduced in uteri (p<0.05). The increase of Tbet and the decrease of Gata3 were found in mice peripheral lymphocytes by flow cytometry. The expression of decidualization marker IGFBP1 and angiogenesis regulator VEGF were declined in uteri (p<0.05). The expression of apoptosis-associated proteins, caspase3 and Bcl-2, were also declined (p<0.05). These results showed that inhibition of IGFBP7 induced pregnancy failure by shifting uterine cytokines to Th1 type dominance and repressing uterine decidualization.

English

This study aimed to investigate the effect and mechanism of tranilast (TR) on bleomycin (BLM) induced pulmonary fibrosis in a rat model, pulmonary fibrosis was induced in rats by using intratracheal bleomycin and the serum and lungs were collected 28 days later. The serum levels of IL-4 and IFN-γ were determined by ELISA and IL-4/IFN-γ was calculated. Reverse transcription polymerase chain reaction (RT-PCR) was employed to measure the mRNA expression of TGF-β1. Immunohistochemistry was employed to detect the protein expression of TGF-β1 followed by evaluation of alveolitis and fibrosis. When compared with BLM group and glucocorticoid (GC) group, the INF-γ level was markedly increased and IL-4 level dramatically decreased in the TR group with a significant decrease of IL-4/INF-γ ratio. The mean optical density and mRNA expression of TGF-β1 were markedly decreased accompanied by low scores of alveolitis and fibrosis. Significant differences were observed between TR groups (medium and high dose) and BLM group as well as control group (P < 0.05) in the aforementioned parameters. Moreover, marked difference was also observed among TR groups (P < 0.05) in the aforementioned parameters. Both TR and GC can affect the Th1/Th2 lymphocyte balance and inhibit the TGF-β1 expression improving the BLM induced pulmonary fibrosis. Key words: Th1/Th2, tranilast, pulmonary fibrosis, interferon-γ, transforming growth factor-β1.

Allergic Bronchopulmonary Aspergillosis After Infliximab Therapy for Sarcoidosis: A Potential Mechanism Related to T-Helper Cytokine Balance

We report a case of allergic bronchopulmonary aspergillosis (ABPA) that occurred in a man receiving infliximab for systemic sarcoidosis. His symptoms associated with ABPA were temporally related to his infliximab infusions. We suspect that infliximab disrupted the T-helper (Th) type 1-Th2 lymphocyte balance such that Th2 cytokines were left relatively unopposed, promoting the development of ABPA.

Evaluation of TH1/TH2 Lymphocyte Balance and Lypopolysaccharide Receptor Expression in Asthma Patients

Introduction: An increase in the number of asthma patients which has recently been observed depends on their place of residence and their occupation. This suggests that both external factors and genetic predispositions affect the development of the disease. The contact with bacterial lypopolysaccharide (LPS) may suppress the development of asthma among rural inhabitants. The mechanism of LPS effect most probably consists in the activation of macrophages and granulocytes by TLR4 and CD14 receptors for the production of cytokines, which affect Th1/Th2 balance. The objective of the study was the evaluation of CD14 and TLR4 expression on mononuclear cells and the analysis of Th1/Th2 balance in peripheral blood among asthma patients. Material and Methods: The study group covered 22 patients with bronchial asthma (mean age 45 ± 15), and was conducted by the method of flow cytometry with the use of fluorochrome-labelled monoclonal antibodies. CD14 and TLR expression was assessed in peripheral blood monocytes. Th1/Th2 balance was determined by the measurement of intracellular IL-2, IFN-γ, IL-4 and IL-10 expression in T-helper cells after culture with the stimulation of cytokine production. Results: A negative correlation was noted between TLR4 expression and the percentage of Th2 lymphocytes, while a positive correlation was observed between expression of TLR4 and percentage of Th1 cells. No relationship was found between CD14 expression on monocytes and the percentage of Th1 and Th2 lymphocytes. Conclusions: An increased percentage of lymphocytes with TLR4 expression is associated with the change in Th1/Th2 balance in favour of Th1 lymphocytes in asthma patients.

Effect of Dietary Fatty Acid Composition on Th1/Th2 Polarization in Lymphocytes

It has become increasingly clear that polyunsaturated fatty acids (PUFAs) have immunomodulatory effects. However, the intake of these fatty acids used in animal studies often greatly exceeds dietary human intake. Whether differences in the composition of fatty acids that are consumed in amounts consistent with normal dietary intake can influence immune function remains uncertain. We manufactured 3 types of liquid diet, related to modified fatty acid composition (omega-6/omega-3 = 0.25, 2.27 and 42.9), but excluding eicosapentaenoic acid and docosahexaenoic acid, based upon a liquid diet used clinically in humans. We assessed CD3-stimulated cytokine production of splenocytes in female BALB/c mice (n = 4 per group) fed 1 of 3 liquid diets for 4 weeks. We also measured the cytokine production of peripheral blood mononuclear cells stimulated with phorbol myristate acetate and ionomycin in humans at the end of a 4-week period of consumption of 2 different liquid diets (omega-6/omega-3 = 3 and 44). We found that the ratio of interfero omega-gamma (IFN-gamma) / interleukin-4 (IL-4) was significantly higher in mice fed the omega-3 rich diet than in others. In humans, IFN-gamma / IL-4 was significantly higher after the omega-3 versus the omega-6 enhanced diet. Differences in the composition of omega-3 and omega-6 PUFAs induces a shift in the Th1/Th2 balance in both mouse and human lymphocytes, even when ingested in normal dietary amounts. An omega-3 rich diet containing alpha-linolenic acid modulates immune function.

Differential Th1/Th2 balance in peripheral blood lymphocytes from patients suffering from flea bite-induced papular urticaria

BackgroundThe Th1/Th2 balance has not been characterized in patients suffering from flea bite-induced papular urticaria (FBPU). Our aim was to improve understanding of the immunopathogenesis of CD4+and CD8+T-cells in humans suffering from flea bite-induced papular urticaria. MethodsPeripheral blood mononuclear cells were obtained from 18 pediatric patients and 10 age-matched healthy controls. Cellular phenotypes, intracellular production of interferon gamma (IFNγ) and interleukin-4 (IL-4) in T-cells stimulated with polyclonal stimuli was determined by flow cytometry following short-term in vitro stimulation. ResultsThe results revealed lower frequencies of IFNγ-secreting (p=0.02) and higher frequencies of IL-4-secreting (p=0.03) CD4+T-cells in patient lymphocyte cultures compared to healthy control cultures in the presence of polyclonal stimuli. This is the first description of differential cytokine patterns in papular urticaria patients. ConclusionPatients suffering from papular urticaria have an atopic status compared to healthy children.

Flow Cytometric Analysis of Lymphocyte Subpopulations and Th1/Th2 Balance in Patients with Polymyositis and Dermatomyositis

Polymyositis (PM) and dermatomyositis (DM) are idiopathic inflammatory myopathies; autoimmune mechanisms are thought to play an important role in their pathogenesis. We investigated the immunocytochemical characteristics and Th1/Th2 balance of peripheral blood lymphocytes in PM and DM using flow cytometry. Eight patients with PM and 13 with DM were enrolled in this study. Of these, 8 patients with DM were examined before and after clinical remission. No patients were receiving any treatment for PM or DM at enrollment. Ten healthy subjects were used as controls. Patients with PM showed significant increases in CD3+CD4+HLA-DR+ (p<0.01) and CD19+CD23+ cells (p<0.05), and significant decreases in CD3+CD4+ (p<0.005) and CD4+CD45RO+ cells (p<0.05) compared with controls. Patients with DM showed significant increases in CD19+ (p<0.05) and CD19+CD23+ cells (p<0.05), and significant decreases in CD4+CD45RO+ cells (p<0.005) and the CD4+CD45RO+/CD4+CD45RA+ ratio (p<0.005) compared with controls. CD4+interferon (IFN)-gamma+ cells and the intracellular IFN-gamma/interleukin (IL)-4 ratio in CD4+ cells were significantly lower in patients with DM than in those with PM (p<0.05) or controls (p<0.0005 and p<0.001, respectively). The intracellular IFN-gamma/IL-4 ratio in CD4+ cells was significantly increased in DM after clinical remission compared with before (p<0.05). Both B and helper T cells are activated in peripheral blood of active PM. Th2 cells predominate in peripheral blood of active DM, and the intracellular IFN-gamma/IL-4 ratio in CD4+ cells may be useful as a clinical marker indicating disease activity.

Changing survival, memory cell compartment, and T-helper balance of lymphocytes between severe and mild asthma

BackgroundAsthma is a complicated network of inflammatory reactions. It is classified into mild, moderate, and severe persistent asthma. The success of asthma therapy relies much on understanding the underlying mechanisms of inflammation at each stage of asthma severity. The aim of this study was to explore the differences in apoptotic potential, CD4/CD8 ratio, memory compartment, and T- helper (Th) 1 and 2 profile of peripheral blood lymphocytes (PBL) in patients with mild intermittent asthma and severe persistent asthma during exacerbation periods.ResultsFour research lines were investigated and compared among mild asthmatics, severe asthmatics, and healthy groups by applying immunocytochemical staining of PBL. Antiapoptotic and proapoptotic proteins with Bcl-2/Bax ratio, CD4, CD8 markers with CD4+/CD8+ ratio, CD45RO+, CD45RA+ markers with memory/naïve ratio (CD45RO+/CD45RA+). Th2/Th1 cytokines balance represented by IL-4/IFN-γ ratio was measured by enzyme-linked immunosorbent assay (ELISA) for in vitro PBL cytokine synthesis. It was found that Bcl-2/Bax ratio was higher in severe than in mild asthmatics which in turn was higher than in healthy group. And memory/naïve ratio of PBL was higher in severe than in mild asthmatics. Moreover, memory cells, CD45RO+ and CD45RO+/CD45RA+ ratio were correlated directly with Bcl-2/Bax, in severe and mild asthma patients. In contrast, CD4+/CD8+ ratio was not changed significantly among healthy group, mild and severe asthmatics. However, CD8+ cells were correlated directly with memory cells, CD45RO+, in severe asthmatics only. Interestingly, the dominant profile of cytokines appeared to change from T helper 2 (Th2) in mild asthmatics to T helper 1 (Th1) in severe asthmatics where the lowest in vitro IL-4/IFN-γ ratio and highest IFN-γ were found.ConclusionIt was concluded that the underlying mechanisms of inflammation might vary greatly with asthma stage of severity. Mild intermittent asthma is mainly Th2 allergen-oriented reaction during exacerbations with good level of apoptosis making the inflammation as self-limiting, while in severe persistent asthma, the inflammatory reaction mediated mainly by Th1 cytokines with progressive loss of apoptosis leading to longer exacerbations, largely expanded memory cells, CD45RO+, leading to persistent baseline inflammation.

Th1/Th2 balance in canine peripheral blood lymphocytes—A flow cytometric study

The canine immune system undergoes continuous remodeling with advancing age. We measured the Th1/Th2 balance in peripheral blood lymphocytes (PBLs) obtained from 23 Beagles ranging in age from 0.5 to 11.8 years by flow cytometric analysis using intracellular cytokine staining. The percentage of CD4 cells producing interferon-gamma (Th1) increased with age. The percentage of CD4 cells producing interleukin-4 (Th2) increased but much less so. In conclusion, we demonstrated that the Th1/Th2 balance in canine peripheral blood could be measured by this flow cytometry technique and the Th1/Th2 balance inclined to dominance of the Th1 subpopulation in PBLs as the dog matured.

T-helper type 1 cytokine release is enhanced by in vitro zinc supplementation due to increased natural killer cells

Objective We examined the influence of zinc on T-helper type 1 (Th1)/T-helper type 2 (Th2) balance in human lymphocytes. Methods Human peripheral blood mononuclear cells or diluted whole blood were cultured for 8 d in the presence of zinc (30 or 60 μM) or 1 μM of N, N, N′, N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) (a zinc-specific chelator). Phytohemagglutinin-induced cytokine release was measured by enzyme-linked immunosorbent assay, and expression of CD56/CD69, CCR4/CD3, and CCR5/CD3 and intracellular labile zinc were detected by flow cytometry. Results We found that our in vitro supplementation resulted in an increase of intracellular labile zinc comparable to that of a 7-wk administration of 10 mg of zinc per day in vivo. Zinc triggered interferon-γ release and impaired interleukin-10 release. Phenotypically, a Th2/Th1 shift could not be confirmed after detecting the Th1-specific chemokine receptor CCR5 or CCR4 for Th2 cells. Surprisingly, we detected a larger amount of CD56 + cells after zinc stimulation, leading us to the conclusion that the amount of interferon-γ release after zinc supplementation might be attributed to the upregulation of natural killer cells after in vitro zinc supplementation rather than to a Th2/Th1 shift. Conclusion We suggest that a nutritional intake of 10 mg of zinc increases the quantity of interferon-γ–producing natural killer cells and strengthens the immune system against neoplasms and viral infections.

Herbal medicine Gamgungtang down-regulates autoimmunity through induction of TH2 cytokine production by lymphocytes in experimental thyroiditis model

The crude herbal formulation, Gamgungtang (GGT), has been shown to protect animals against a wide range of spontaneously developing or induced autoimmune diseases. We have previously reported that GGT shows marked down-regulation of several experimental autoimmune diseases. Although very effective at preventing thyroid infiltrates in mice immunized with mouse deglycosylated thyroglobulin and complete Freund's adjuvant and in spontaneous models of thyroiditis, it completely failed to modify experimental autoimmune thyroiditis (EAT) induced in mice immunized with mouse thyroglobulin and lipopolysaccharide. In this study, in an effort to elucidate the mechanisms by which GGT suppresses EAT, and autoimmunity in general, we investigated the in vivo effects of this drug on the Th1/Th2 lymphocyte balance, which is important for the induction or inhibition of autoreactivity. Naive SJL/J mice were treated orally for 5 days with GGT (80 mg/(kg day)). Spleen cells were obtained at various time points during the treatment period and were stimulated in vitro with concanavalin A. Interleukins IL-4, IL-10 and IL-12, transforming growth factor-β (TGF-β) and interferon-γ (IFN-γ) cytokine production was evaluated at the protein levels of the cytokines in the medium and mRNA expressions. A significant upregulation of IL-4, IL-10 and TGF-β was observed following treatment with GGT, which peaked at day 5 (IL-10) or day 10 (IL-4). On the other hand, IL-12 and IFN-γ production were either unchanged or decreased. It seems therefore that GGT induces in vivo a shift towards Th2 lymphocytes which may be one of the mechanisms of down-regulation of the autoimmune reactivity in EAT. Our observations indicate that down-regulation of TH1 cytokines (especially IL-12) and enhancement of Th2 cytokine production may play an important role in the control of T-cell-mediated autoimmunity. These data may contribute to the design of new immunomodulating treatments for a group of autoimmune diseases.

Phase I clinical trial of hybrid cell vaccination in patients with gastrointestinal cancer

2579 Background: Vaccination with hybrid cells of dendritic cells (DCs) and tumor cells can induce specific ant-tumor immunity against multiple tumor antigens including those that are unknown. We report the results of phase I study of hybrid cell vaccines comprised of autologous DC and autologous tumor cells in patients with Stage IV or recurrent gastrointestinal cancer. Method: Thirteen patients with Stage IV or recurrent gastrointestinal cancer were enrolled in this study after informed consent was obtained. Tumor cells were cryopreserved following acquisition from the primary or a metastatic site. DCs were induced from mononuclear cells of patients by culture with cytokines (6 days culture with GM-CSF and IL-4, and a further 1 day culture with TNF-α, IL-1 β, IL-6 and PGE2). Fusion of irradiated tumor cells and DC was created with a combination of PEG and electrofusion (two-step fusion method). At 2 week interval, patients received 4 freshly prepared fusion vaccines, with a constant dose range of 1 × 107 to 4 × 107 cells. The endpoints of this study were assessments of safety, anti-tumor immune response and clinical response. Results: Serious adverse events were not observed in any of the enrolled patients and all Common Toxicity Criteria were grade I. Clinical responses are 1 with partial response (PR), 5 with stable disease (SD) and 7 with progressive disease (PD). Delayed skin tests changed to positive in 5 patients. In patients with PR and SD, a shift to Th1 in Th1/Th2 balance of lymphocytes (IFN γ/IL10, IFN γ/IL4) was demonstrated by ELISPOT and cytokine flow cytometry analysis. After vaccination, decrease of immunosuppressive factors (IAP, TGF-β) in serum were shown in patients with PR and SD. There was no significant differences of CD4+CD25+ cells by vaccination. Conclusion: These results suggests that a strategy of hybrid cell vaccine employing auto-DCs and auto-tumor cells is feasible in patients with gastrointestinal cancer and may show evidence of anti-tumor immune response. No significant financial relationships to disclose.