mTORC1 inhibition may improve T lymphocytes affected by aging.

BACKGROUND: Due to the increase of the elderly's population and related social and economic problems, it is very important to provide strategies on health. In this regard, induction of T lymphocytes responses, the most important cells of the immune system, may be a good approach. Among different agents considered as antiaging factors, mTORC1 pathway inhibitors are significant. So, the purpose of this study was to evaluate the effect of two mTORC1 inhibitors, Everolimus and Metformin, on age-related features of activated T cells. MATERIALS AND METHODS: Optimum doses of drugs was determined with evaluating the effect of treatments on IL-2 gene expression. T cells isolated from old and young mice were treated with drugs and PHA. IL-2 production was evaluated by ELISA. Also, the expression of CD28, PD-1, and KLRG-1, proliferation, and intracellular oxidative stress were assessed by flow cytometry-based assays, phenotyping, CFSE, and DCF-DA assay respectively. RESULTS: Both drugs increased IL-2 production in the T cells of old mice. Also, using drugs especially Metformin could improve age-related phenotypical markers and increase the proliferation of T cells of old mice significantly. In addition, Metformin and Everolimus reduced intracellular oxidative stress in aged cells. However, the effect of both drugs on the T cells of young mice wasn't significant or was in opposite to the results of old mice T cells. DISCUSSION: In line with studies noting mTOR inhibitors as antiaging drugs, Metformin and Everolimus may improve T cells affected from aging in vitro, and a decrease in intracellular oxidative stress may be one of their mechanism of function.

Association between Interleukin-21, 23 and 27 Expression and Protein Level with Cytomegalovirus Infection in Liver Transplant Recipients.

BACKGROUND: Cytokines have regulatory crosstalk with CMV infection leading to manage of post-liver transplantation virus-related outcomes. OBJECTIVE: To investigate the link between IL-21, IL-23 and IL-27 mRNA and protein level with active CMV infection, which was evaluated in reactivated and non-reactivated liver transplant recipients. METHODS: Two groups of liver transplant recipients were enrolled in this study-54 without and 15 with active CMV infection. 3 EDTA-treated blood samples were taken on day 1, 4, and 7 post-liver transplantation. Plasma and buffy coats of all samples were separated. All samples were analyzed for CMV reactivation using antigenemia technique. The separated plasma of positive samples was used for viral DNA extraction and protein evaluation. For evaluating the mRNA expression level by real-time PCR, RNA extraction and cDNA synthesis were done for all samples. Also, the protein level of studied genes was estimated by ELISA. RESULTS: The expression level of IL-21, IL-23A and IL-27A cytokine genes was increased in CMV reactivated liver transplant recipients in comparison with CMV non-reactivated ones; IL-27A expression pattern was significant (p=0.001) at all sampling times. IL-21 significantly increased on the 2nd and 3rd (p=0.028 and 0.01, respectively) sampling days in CMV reactivated compared with non-reactivated patients. The expression level of IL-23A cytokine significantly increased on the 3rd (p=0.017) sampling day in CMV reactivated compared with non-reactivated liver transplant recipients. CONCLUSION: Elevation in the expression level of IL-21, IL-23A and IL-27A mRNA and protein level in CMV reactivated patients emphasized on the antiviral role of these cytokines in CMV reactivated liver transplant recipients.

T cell responses to an HLA-A2-restricted adipophilin peptide correlate with BMI in patients with atherosclerosis.

INTRODUCTION: Atherosclerosis is an inflammatory disease causing a vast array of cardiovascular diseases. Adipophilin has been reported to be highly expressed in atherosclerotic lesions. This study investigated the possible existence of auto-reactive T cells against an HLA-A02-restricted adipophilin-derived peptide as well as peptides from Epstein-barr virus (EBV), Cytomegalovirus (CMV) and influenza (Flu) virus in patients with atherosclerosis. METHODS: HLA-A02 expression on peripheral blood mononuclear cells (PBMCs) was examined by flow cytometry. PBMCs from HLA-A02 individuals were stimulated with adipophilin, CMV, EBV, and Flu peptides at a concentration of 10 µM. Interferon (IFN)-γ production was evaluated in the culture supernatant using a commercial ELISA test. RESULTS: The levels of IFN-γ production against an HLA-A02-restricted adipophilin peptide and peptides from CMV, EBV, and Flu revealed no statistically significant differences between patients and healthy controls. However, we found a positive correlation between IFN-γ production against adipophilin and Body mass index (BMI) of patients (R = 0.8, P = 0.003), whereas no significant correlation was found in healthy controls (R = -0.267, P = 0.378). No correlation between BMI and IFN-γ production against CMV, EBV, or Flu peptides was found. DISCUSSION: Atherosclerotic patients with higher BMIs might have greater numbers of T cells against adipophilin that is highly expressed in atherosclerotic plaques. Therefore, autoimmune reactions may have a greater role in the development of atherosclerosis in individuals with higher BMI.

Association of IL-12 and TNF-α Polymorphisms with Graft-Versus-Host Disease in Allogeneic Hematopoietic Stem Cell Transplant Recipients.

BACKGROUND: Cytokines are important factors determining the outcome of transplantation. The host ability in cytokine production may be affected by cytokine genes polymorphisms. OBJECTIVE: To investigate the effect of IL-12 and TNF-α gene polymorphisms on outcome of hematopoietic stem cell transplantation. METHODS: 90 bone marrow transplant recipients were included in this study. 30 (33%) of 90 recipients experienced graft-versus-host disease (GVHD). IL-12 and TNF-α gene polymorphisms were evaluated by PCR-RFLP and ARMS-PCR method, respectively. RESULTS: No significant difference in the distribution of IL-12 (rs3212227 +1188 A/C) and TNF-α (rs 1800629 -308 G/A) genotypes and alleles was observed between those with and without GVHD. There was no significant association between the distribution of genotypes and the recipient sex. CONCLUSION: IL-12 (rs3212227 +1188 A/C) and TNF-α (rs 1800629-308 G/A) genotypes and alleles were not risk factors for development of GVHD.

Blood Product Transfusion in Liver Transplantation and its Impact on Short-term Survival.

BACKGROUND: Estimation of the amount of blood products required during liver transplantation can help provision of adequate blood supply, minimize transfusion-associated complications, and plan for preventive measures in high risk patients. OBJECTIVE: To investigate independent predictors of peri-operative blood product transfusion and its impact on short-term survival of liver transplant recipients. METHODS: In a cross-sectional study, old charts of patients who underwent liver transplantation between March 2003 and March 2013 at Namazi Hospital, Shiraz, Iran, were reviewed. The mean amount of blood product utilized during surgery and hospital stay and the related factors, including demographic characteristics, pre-transplant laboratory data, pre-transplant clinical data, operation data, and post-transplantation data were recorded. RESULTS: We studied 1198 patients who underwent liver transplantation. The mean±SD amounts of red blood cells, fresh frozen plasma, and platelet transfusion during surgery and hospital stay were 2.67±3.5, 2.06±3.8, and 1.6±3.8 units, respectively. The mortality rate was significantly higher in patients who received high amounts of blood products (p<0.001). The mean amount of blood products' utilized during operation was significantly (p<0.001) decreased from 2003 to 2013.The mean amount of packed cell usage during operation and hospital stay was significantly (p<0.001) correlated with age, technique of surgery, serum albumin level, cirrhosis, blood urea nitrogen, length of operation, and prothrombin time. CONCLUSION: Pre-operative factors may predict blood transfusion requirements in patients undergoing liver transplantation. Therefore, evaluation of patients before operation should be considered to provide adequate blood supply and minimize transfusion-associated complications. Understanding pre-operative factors associated with rate of transfusion may help us to best utilize the limited available blood resources.

mRNA Expression of Interferon Regulatory Factors during Acute Rejection of Liver Transplants in Patients with Autoimmune Hepatitis.

BACKGROUND: Interferon regulatory factors (IRFs) can play a critical role in the regulation of many facets of innate and adaptive immune responses through transcriptional activation of type I interferons, other proinflammatory cytokines, and chemokines. However, their roles in transplantation immunity still remain to be elucidated. OBJECTIVE: To evaluate the time course of mRNA expression of all 9 members of IRFs family of transcription factors during liver allograft acute rejection. METHODS: Blood samples of 19 patients with autoimmune hepatitis receiving liver transplants were collected on days 1, 3, 5, and 7 post-transplantation. The patients were followed for 6 months after transplantation and divided into two groups of acute rejection (AR) (n=4) and non-acute rejection (non-AR) (n=15). RESULTS: All of the studied transcription factors were down-regulated in AR-group on days 3, 5, and 7 post-transplantation compared to non-AR group. The mean±SEM IRF5 on day 7 post-transplantation was significantly (p=0.005) lower in AR-group than in non-AR group (0.7±0.21 vs. 1.91±0.27, respectively); expression of other IRFs family members was not significantly different between the two groups on days 3, 5, and 7 post-transplantation. CONCLUSION: IRF5 may have an important role during the acute rejection of liver transplants.

Association between IRF1 Gene Expression and Liver Enzymes in HBV-infected Liver Transplant Recipients with and without Experience of Rejection.

BACKGROUND: Liver function indices and anti-viral immune regulatory markers can both improve graft outcomes, which lead to better post-transplantation management and increase the possibility of surveillance in liver transplant recipients with chronic hepatitis B virus (HBV) infection. OBJECTIVE: To determine the association between the interferon regulatory factor 1 (IRF1) mRNA levels and liver enzymes in HBV-infected liver transplant recipients with and without experience of rejection. METHODS: A total of 46 chronic HBV-infected patients who had undergone liver transplant surgery was divided into 2 groups of recipients "with rejection" and "without rejection.". Blood samples were collected form each patient on days 1, 4, and 7 post-transplantation. A SYBER GREEN real-time PCR was used to evaluate the expression level of IRF1 in liver recipients. Liver enzyme activities were also measured in all patients. RESULTS: The expression of IRF1 in the patients with rejection was up-regulated at all 3 follow-up days compared with those without rejection. The serum levels of ALT and AST were more than normal levels at 3 follow-up times in both study groups. Significant differences were found in IRF1 gene expression levels and also serum ALT levels between those with and without rejection after 7 days post-transplantation. CONCLUSION: The IRF1 expression and serum ALT levels were increased significantly in patient with rejection compared to those without rejection. IRF1, an inflammatory factor, may also intensify induction of inflammatory pathways in engrafted liver and promote liver inflammation and injuries leading to liver enzymes elevation in patients with graft rejection.

The Immunomodulatory Effect of Bone-Marrow Mesenchymal Stem Cells on Expression of TLR3 and TLR9 in Mice Dendritic Cells.

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent cells with immunomodulatory effect on immune cells including dendritic cells (DCs). DCs are the most potent antigen-presenting cells (APC). MSCs have been found to modulate both differentiation and function of DCs. DCs express a broad range of Toll-like receptors (TLR), which play a critical role in DCs maturation and function. OBJECTIVE: To evaluate expression level of TLR3 and TLR9 transcripts in DCs following treatment with MSCs supernatant. METHODS: MSCs and DCs were derived from adult BALB/c mice bone marrow and spleen, respectively. MSCs supernatant was harvested following 24, 48, and 72 hours. Isolated DCs were treated with MSCs supernatant and incubated for 24 and 48 hours. TLR3 and TLR9 transcript levels were evaluated using real-time PCR. RESULTS: The results showed that 48 and 72 hours MSCs supernatant significantly decreased the expression of TLR3 in DCs following 24 and 48 hours incubation in comparison with untreated cells (p<0.01). Moreover, 48 hours of treatment with 24, 48 and 72 hours MSCs supernatant significantly decreased TLR9 transcript level (p<0.05). CONCLUSION: TLR3 and TLR9 mRNA expression decreases in DCs after incubation with MSCs culture supernatant. This confirms the immunomodulatory role of MSCs in cell-base therapy.

Association of GSTO2 (N142D) Genetic Polymorphism and Acute Rejection of Liver.

BACKGROUND: Acute rejection is the main problem in liver transplantation that occurs in the first days or months of transplantation. It includes histological and cellular rejection. Acute histological rejection is confirmed by biopsy. Glutathione S-transferase family is the most important genes in phase II detoxification working in xenobiotic and drug metabolism. GSTO2 is one of the members of this family. GSTO2 (N142D) polymorphism may influence metabolism of immunosuppressive drugs. OBJECTIVE: To determine if GSTO2 polymorphism has association with acute liver rejection. METHODS: The present study included 120 patients with histological-proven acute liver rejection and 182 patients without acute rejection. Both groups were matched for sex and age. To determine variants of GSTO2, we used polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: There was a significant association between the GSTO2 genotype and acute liver rejection (NN: OR: 3.642, 95% CI: 1.179-5.444) and (ND: OR: 2.533, 95% CI: 1.672-8.149) compared to those with DD geneotype. CONCLUSION: Recipients with either NN or ND genotype for GSTO2 are more likely to develop acute liver rejection compared to those with DD genotype.

Tolerance Induction in Liver.

Liver is an exclusive anatomical and immunological organ that displays a considerable tolerance effect. Liver allograft acceptance is shown to occur spontaneously within different species. Although in human transplant patients tolerance is rarely seen, the severity level and cellular mechanisms of transplant rejection vary. Non-paranchymal liver cells, including Kupffer cells, liver sinusoidal endothelial cells, hepatic stellate cells, and resident dendritic cells may participate in liver tolerogenicity. The mentioned cells secret anti-inflammatory cytokines such as TGF-ß and IL-10 and express negative co-stimulatory molecules like PD-L1 to mediate immunosuppression. Other mechanisms such as microchimerism, soluble major histocompatibility complex and regulatory T cells may take part in tolerance induction. Understanding the mechanisms involved in liver transplant rejection/tolerance helps us to improve therapeutic options to induce hepatic tolerance.

Generation of Islet-like Cell Aggregates from Human Adipose Tissue-derived Stem Cells by Lentiviral Overexpression of PDX-1.

BACKGROUND: Pancreatic duodenal homeobox1 (PDX-1) is a transcription factor that is important in regulating pancreas development and maintaining ß-cell function. ß-cell replacement is an effective approach for the treatment of type 1 diabetes. Human adipose-mesenchymal stem cells (hAMSCs) are the ideal population cells for differentiating into insulin-producing cells. OBJECTIVE: To determine if islet-like cell aggregates production could be generated from hAMSCs by lentiviral overexpression of PDX-1. METHODS: After isolation of hAMSCs, characteristics of these cells were identified by flow-cytometic analysis and multilineage differentiation studies. PDX-1 gene delivered into hAMSCs through lentiviral vector for differentiating hAMSCs into insulin-producing cells (IPCs) at the utilized protocol for 14 days. Characteristics of IPCs were evaluated by immunocytofluorescence, dithizone staining, and quantitative reverse transcription PCR. In response to high glucose medium, insulin release was detected by chemiluminescence enzyme immunoassay. RESULTS: The islet-like cell aggregates appeared about 10 days after introduction of PDX-1 into hAMSCs. PDX-1 induced its own expression (auto-induction), a number of islet-related genes such as Ngn3, Nkx2-2, and insulin. The insulin-positive cells were detected in the PDX-1 transduced cells. In response to glucose challenge test, secretion of insulin hormone in the medium with high glucose concentration significantly increased in the PDX-1-transduced cells related to medium with low glucose concentration. CONCLUSION: Introduction of lentiviral PDX-1 significantly induces hAMSCs to differentiate into islet-like cell aggregates, which may provide a source of adipose stem cells-derived insulin-producing cells for cell replacement therapy in type 1 diabetes.

Polymorphism of the IL-18 and CD40 genes and Liver Transplant Outcome in Iranian Patients.

BACKGROUND: Cytokines and co-stimulatory molecules are important factors determining the outcome of transplantation. OBJECTIVE: To investigate the effect of IL-18 and CD40 gene polymorphisms on the outcome of liver transplantation. METHODS: 150 liver transplant recipients were included in this study. Alleles and genotypes frequencies for IL-18 (rs1946519) and CD40 (rs1883832) were determined in 28 acutely rejected (AR group) and 122 non-acutely rejected (non-AR group) liver transplant recipients. IL-18 and CD40 gene polymorphisms were evaluated by PCR-RFLP methods. RESULTS: There were no significant associations between IL-18 and CD40 polymorphism with acute rejection in liver transplant patients. IL-18TT and TG genotypes had a significant association with rejection in women compared to men. After grouping the liver recipients according to living vs cadaver donors, a significant association was found between CC genotype of CD40 and rejection in male living donor recipients. IL-18 TG genotype had a significant association with rejection in female cadaver donor recipients. CONCLUSION: There is no correlation between all genotype and alleles of IL-80 and CD40 polymorphism and the outcome of liver transplantation. However, gender and type of donor affect the correlation between all genotype and alleles of IL-18 and CD40, and the outcome of liver transplantation.

Tolerogenic dendritic cells produced by lentiviral-mediated CD40- and interleukin-23p19-specific shRNA can ameliorate experimental autoimmune encephalomyelitis by suppressing T helper type 17 cells.

Down-regulation of soluble or membrane-bound co-stimulatory molecules by RNAi in dendritic cells can prevent the activation of immune responses. Therefore, this study was designed to evaluate the therapeutic efficacy of bone marrow-derived DCs (BMDCs) transduced with lentiviral vectors to permanently expressed shRNA specific for CD40 (CD40LV-DCs) and/or p19 subunit of interleukin (IL)-23 (p19LV-DCs) mRNAs in experimental autoimmune encephalomyelitis (EAE). In-vitro studies showed that double-transduced BMDCs (CD40(+) p19LV-DCs) resemble tolerogenic DCs due to profound down-regulation of CD40, lower expression of proinflammatory cytokines (IL-6 and IL-12), increased IL-10 production and stronger stimulation of myelin oligodendrocyte glycoprotein (MOG)35-55 -specific T cells for production of IL-10 compared with CD40LV-DCs, p19LV-DCs and BMDCs transduced with control lentiviral vector (CoLV-DCs). Moreover, injection of transduced CD40(+) p19LV- BMDCs in EAE mice resulted in more reduction in clinical score, significant reduction in IL-17 or increased production of IL-10 by mononuclear cells derived from the lymph nodes or spinal cord compared with CoLV-DCs-treated EAE mice. In conclusion, simultaneous knock-down of CD40 and IL-23 production by BMDCs may represent a promising therapeutic tool for the treatment of IL-17-dependent autoimmune diseases, including multiple sclerosis.

Glutathione S-transferase Omega 2 Genetic Polymorphism and Risk of Hepatic Failure that Lead to Liver Transplantation in Iranian Population.

BACKGROUND: Liver transplantation is the treatment of choice for both acute and chronic hepatic failure. GSTs family is one of the most important genes in phase II detoxification interfering with the xenobiotics and free radical metabolism. GSTO2 (N142D) is a member of this family the polymorphism of which may influence the metabolism of active components and free radicals and may contribute to hepatic failure. OBJECTIVE: To investigate the association between GSTO2 genetic polymorphism and the susceptibility of hepatic failure that would lead to liver transplantation. METHODS: This case-control study included 330 healthy people and 302 patients with liver transplantation as a result of hepatic failure. To determine the variants of GSTO2, we used polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: There was a significant association between D allele and hepatic failure, thus, people with DD genotype are more susceptible to develop heaptic failure leading to liver transplantation (OR=1.8, 95% CI: 1.10-2.95, p=0.02). We also observed that male sex increases the chance of hepatic failure (OR=2.69, 95% CI: 1.95-3.71, p=0.001). CONCLUSION: D allele may reduce the detoxification ability of liver so people with mutant D allele are more prone to develop hepatic failure.

Hematologic and biochemical indices and viral hepatitis in liver transplant patients.

BACKGROUND AND PURPOSE OF STUDY: The pathogenic role of important hepatotropic viral agents to induce hepatic dysfunction and failure may lead to the need for liver transplantation. We focused on the use of hematologic and biochemical laboratory diagnostic indexes to follow the clinical impact of hepatitis B virus (HBV); hepatitis C virus (HCV); and hepatitis G virus-related liver complications in transplant patients. MATERIALS AND METHODS: We collected 141 EDTA-treated blood samples pre- and post-liver transplantation for 2 years among 67 transplant patients. We evaluated the statistical relationships between hematologic and biochemical indices with HBV, HCV, and HGV infections among transplant recipient samples using version 15 of SPSS software. RESULTS: HBV polymerase chain reaction (PCR) positivity significantly correlated with partial thromboplastin (P=.011) pretransplant, with creatinine (P=.026) and Na (P=.034) levels at 1-week posttransplant, and also with alkaline phosphatase (P=.027) and mean corpuscular hemoglobin concentration (P=.050) at 2 weeks posttransplantation. Significant correlations were detected between HCV-reverse transcriptase (RT)-PCR-positive results and blood urea nitrogen (P=.008) and Na (P=.021) levels in the first aspartate aminotransferase and with (P=.025) in the second week after liver transplantation. Also, significant relationships were noted between HGV-RT-PCR-positive results and alkaline phosphatase (P=.05) and creatinine (P=.002) levels in the first and second weeks after liver transplant, respectively. CONCLUSION: Detection of significant correlations between HBV, HCV, and HGV infections with laboratory indices suggested that monitoring hematologic and biochemical liver function-related criteria aid the management of clinical complications of viral hepatitis in liver transplant patients.

The molecular and antigenic tissue impact of viral infections on liver transplant patients with neonatal hepatitis.

BACKGROUND: Pathogenesis of neonatal hepatitis relates to various underlying causes including viral infections. Both hepatotropic and non-hepatotropic viruses may induce liver failures in infants before birth, during delivery, or shortly after birth. OBJECTIVES: The tissue impact of HCMV, HSV, HBV, HCV, and rotavirus and adenovirus infections was evaluated in studied infants with neonatal hepatitis. METHODS: The history of viral infections was analyzed in paraffin-embedded biopsy and autopsy tissues of 22 infants with neonatal hepatitis between years 1996 and 2007, retrospectively. The tissue molecular presentation of HBV, HCV, HCMV, HSV, adenovirus, and rotavirus was evaluated by different qualitative simple and nested PCR and RT-PCR protocols. Immunohistochemistry (IHC) method was used for studying the antigenic prevalence of HSV-1, 2; HBV, HCMV and adenovirus infections. Also the laboratory liver indices of all patients with neonatal hepatitis were analyzed. RESULTS: The HBV and HSV genomes were detected in 3 (14%) of 22 infants. The rotavirus and HCV-RNA and also the HCMV-DNA were detected separately in 1 (4%) of 26 paraffin-embedded autopsy and biopsy tissues. The HBV and HSV-1 specific antigens were separately diagnosed in 1 (4%) of 26 neonatal samples by IHC protocols. Also the HSV-2 antigen was seen in 5 (23%) of 22 liver autopsy and biopsy specimens. Co-infections with HCMV, HSV, HBV, HCV, and rotavirus were detected in these infants with hepatitis. CONCLUSION: Diagnosis of single and mixed molecular and antigenic traces of HCMV, HSV, HBV, HCV and rotavirus underlines the etiologic role of these viruses in clinical pathogenesis of neonatal hepatitis.

Association of the Co-stimulatory Molecules Polymorphisms with CMV Infection in Liver Transplant Recipients.

BACKGROUND: Co-stimulatory molecules play a critical role in regulating T-cell function during CMV infection after liver transplantation. OBJECTIVE: To investigate the relationship between the polymorphisms of the co-stimulatory genes and the susceptibility to CMV infection after liver transplantation. METHODS: Single nucleotide polymorphisms (SNPs) in PD-1 gene (PD1.1 A/G, PD1.3 A/G, PD1.9 C/T) ICOS (-693 A/G, 1720 C/T), CTLA-4 gene (318 C/T, 1722 T/C, 1661 A/G, 49 A/G) and CD28 (+17 C/T) were analyzed by PCR-RFLP in 70 liver transplant patients. CMV infection was determined in these patients by antigenemia test. RESULTS: CTLA-4 49G showed significant association with CMV infection (p=0.03, OR=3.82, 95% CI: 0-3.5; p=0.01, OR=004, 95% CI: 0-1.3). G and T alleles in CTLA-4 gene (318 C/T and 1661 A/G) (p=0.03, OR=0, 95% CI: 0-3.5; p=0.01, OR=0.04, 95% CI: 0-1.3) were significantly higher in CMV-infected rejector group. CONCLUSION: CTLA-4 have significant role in CMV pathogenesis and rejection among CMV-positive liver transplant patients.

Evaluation of the immunomodulatory effects of five herbal plants.

A group of medicinal plants including, Silybum marianum, Matricaria chamomilla, Calendula officinalis, Cichorium intybus and Dracocephalum kotschyi which grow in Iran, were extracted with ethanol 70% and the mitogenic activity was examined both on human peripheral blood lymphocytes and thymocytes. Effect of these extracts on proliferative responsiveness of human lymphocytes to phytohemagglutinin (PHA) and on the mixed lymphocyte reaction (MLR) was also investigated. The results obtained indicated that none of the extracts had a direct mitogenic effect on human lymphocytes or thymocytes (stimulation index, SI<0.07). Among the plants studied, C. intybus and C. officinalis showed a complete inhibitory effect on the proliferation of lymphocytes in the presence of PHA (SI range 0.01-0.49). A dose dependent inhibitory effect was obtained in the case of D. kotschyi. Extract of M. chamomilla showed almost no stimulatory effect. A significant decrease in proliferation assay due to 0.1-10 microg/ml of S. marianum was observed (SI<0.46, P<0.05). In MLR, a markedly stimulatory effect with some lower concentrations of all the extracts except Dracocephalum was detected. The highest stimulatory effect was due to 100 microg/ml of S. marianum (SI 2.82). Treatment of mixed lymphocytes with 0.1-10 microg/ml of C. officinalis (SI range 1.34-1.80) and 10 microg/ml of M. chamomilla and C. intybus (SI 2.18 and 1.70, respectively) strongly increased the cell proliferation. In conclusion, this in vitro study revealed the capacity of all the extracts except Dracocephalum to enhance the proliferation of lymphocytes after stimulation with the allogenic cells.