ASSOCIATION OF FOXP3 GENE VARIANTS WITH RISK OF HASHIMOTO'S THYROIDITIS AND CORRELATION WITH ANTI-TPO ANTIBODY LEVELS.

CONTEXT: Regulatory T cells (Tregs) have critical roles in preventing autoimmune diseases such as Hashimoto's thyroiditis (HT). Forkhead box P3 (Foxp3), the master transcription factor of Tregs, plays a pivotal role in Treg function. OBJECTIVE: Herein, we investigated the association of two single nucleotide polymorphisms (SNPs) of the Foxp3 gene with HT development. METHODS AND STUDY DESIGN: A total of 129 HT patients and 127 healthy subjects were genotyped for rs3761548 (-3279 A/C) and rs3761549 (-2383 C/T) in the Foxp3 gene, using polymerase chain reaction-restriction fragment length polymorphism. RESULTS: Genotypic and allelic distribution of rs3761548 SNP showed a significant association with HT. The CC genotype was observed in 37.2% of patients versus 22.1% of the controls [P<0.008, odds ratio (OR): 2.1; 95% confidence interval (CI): 1.2-3.6] and the AC genotype in 41.1% of patients compared to 54.3% of the controls (P<0.025, OR: 2.1; CI: 1.2-3.6). In addition, higher frequency of C allele in patients compared to controls (P=0.05, OR: 1.4; 95% CI: 0.9-2) suggested that patients with the CC genotype and C allele had increased susceptibility to HT. There were significantly higher serum levels of anti-thyroid peroxidase (ATPO) antibody in patients with the rs3761548 CC genotype (1156±163 IU/mL) compared to the other genotypes (≈582-656 IU/mL; P<0.004). We observed a greater frequency of the AC genotype in patients who had decreased ATPO antibody levels (P=0.02). CONCLUSIONS: The association of the rs3761548 SNP with risk of HT and its influence on ATPO antibody levels suggested an important role for Foxp3 in the biology and pathogenesis of HT.

In Vitro Effects of Sodium Benzoate on Th1/Th2 Deviation in Patients with Multiple Sclerosis.

Interleukin 4 (IL-4) can improve the clinical manifestations in experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). Sodium benzoate (NaB) deviates the cytokine profile to Th2 (or IL-4 producing) cells in EAE and thus might be effective in the treatment of MS. Therefore, in this study the effect of different concentrations of NaB on the percentage and mRNA levels of IL-4 and interferon gamma (IFN-γ)-producing peripheral blood mononuclear cells (PBMCs) of 20 Relapsing-remitting multiple sclerosis (RR-MS) patients and eight healthy controls was evaluated in the presence of mitogen (phytohemagglutinin, PHA) or specific antigen (myelin basic protein, MBP). Our results showed that in the patient's group the percentage of CD4(+)IL-4(+) cells was significantly increased in the presence of all concentrations of NaB when PBMCs were stimulated by MBP (p = 0.001) or PHA (p < 0.03). The same results were obtained for normal donors in the highest concentration of NaB, 1000 µg/ml (p = 0.02). Moreover, in the patient's group the percentage of CD4(+)IFN-γ(+) cells was decreased significantly when the PBMCs were stimulated by PHA and NaB (p < 0.004) or by MBP and 1000 µg/ml of NaB (p < 0.03). The effect of NaB on IL-4 and IFN-γ production was also documented at the mRNA levels. In conclusion, our data suggest that NaB is able to induce IL-4 production by human PBMCs and therefore might be a useful candidate for conjunctive therapy in RR-MS.

Expression Profile of Human Fc Receptor-Like 1, 2, and 4 Molecules in Peripheral Blood Mononuclear Cells of Patients with Hashimoto's Thyroiditis and Graves' Disease.

Recently identified Fc receptor-like (FCRL) molecules are new members of the immunoglobulin superfamily dominantly expressed by B cells. Although FCRL expression patterns have been studied in normal and malignant cells, their biological functions and roles remain to be clearly identified in humans. Research has particularly focused on FCRL gene polymorphisms in autoimmune diseases, however, their involvement in the pathogenesis of autoimmune diseases is an interesting field for investigation. In the present study, we have investigated the gene expression profiles of FCRL1, 2, and 4 in 2 common thyroid diseases, Hashimoto's thyroiditis (HT) and Graves' disease (GD). FCRL1, 2, and 4 expressions were determined in peripheral blood samples of 55 HT patients, 40 GD patients and equal numbers of normal subjects by quantitative real-time PCR. Our results showed downregulation of FCRL1 and upregulation of FCRL2 transcripts in both HT and GD groups compared to healthy counterparts. Overexpression of FCRL4 was observed only in GD patients compared to controls. A significant correlation was observed between all FCRL gene expression levels in HT patients. Only FCRL2 and 4 had a correlation in GD patients. In addition, FCRL1, 2, and 4 gene expressions showed no correlations with the level of anti-thyroid peroxidase antibody (anti-TPO) or anti-thyroglobulin (anti-Tg) antibody from patients' sera. In conclusion, expressions of activating or inhibitory FCRL1, 2, and 4 showed significant alterations in HT and GD patients compared to healthy subjects.

Immunotherapy with tumor cell lysate-pulsed CD8α+ dendritic cells modulates intra-tumor and spleen lymphocyte subpopulations.

Using cellular adjuvants including dendritic cells (DCs) has provided a promising approach in immunotherapy of cancer. Our previous study showed that mice immunization with tumor cell lysate-pulsed DCs (TL-CD8α+DCs) could significantly suppress the tumor growth and increase mice survival. The aim of the present study was to investigate the impact of TL-CD8α+DC vaccine on intra-tumor and spleen lymphocyte subpopulations in tumor-bearing mice. ABalb/c mouse model of fibrosarcoma was used and changes in various lymphocyte subpopulations including CD4+, CD8+ and CD4+CD25+Foxp3+ T cells in mice immunized with TL-CD8α+ DCs were studied. The cytotoxic activity of the lymphocytes and tumor growth inhibitory rate were also measured. Immunotherapy with TL-CD8α+ DCs significantly enhanced both CD4+ and CD8+ lymphocytes, whereas decreased CD4+CD25+ Foxp3+ regulatory T cells as well as the tumor growth rate. There was also a decrease in the ratio of regulatory T cells to CD4+ and to CD8+ lymphocytes in both the tumor and spleen tissues as compared to that in the non-immunized control mice. Immunization with TL-CD8α+ DCs as well as CD8α+ DCs significantly increased the splenocytes cytotoxic activity by 45.1% and 18.2% of control, respectively. In conclusion, the current study indicated that TL-CD8α+ DCs can enhance tumor immunity against the fibrosarcoma by enhancing both the CD4+ and CD8+ lymphocytes and reducing regulatory T cells. This finding suggests the usefulness of TL-CD8α+DCs vaccine for cancer treatment.

Intrathecal Autologous Bone Marrow-Derived Hematopoietic Stem Cell Therapy in Neurological Diseases.

BACKGROUND: Cellular transplantation is a promising treatment strategy for neurological diseases. OBJECTIVE: To report the results of intrathecal hematopoietic stem cell therapy in different neurological diseases in the past 6 years in a single center. METHODS: From October 2011 to September 2018, 220 patients with various neurological diseases were transplanted intrathecally by their bone marrow stem cells. To have a longer follow up, we only reported the first 80 patients, transplanted up to July 2015-10 patients had spinal cord injuries and paralysis, 12 had advanced Parkinson's disease, 28 had cerebral palsy, 7 had hypoxic brain damage, 2 had autism, 4 had multiple sclerosis, 5 had progressive cerebellar atrophy, and 12 had other neurological diseases. The patients were admitted to the Bone Marrow Transplant Unit. On the first day, 50-200 (median 100) mL bone marrow was aspirated from the patients' posterior iliac crests, mixed with 120 mL culture media (RPMI), and 12 mL heparin. The samples were then transferred to immunology lab in cold box. Mononuclear cells (MNCs) were separated by a Ficoll-Hypaque gradient, washed, and suspended in ringers. Cell viability was assessed with trypan blue viability test. Transplantation was performed 3-4 hours after bone marrow collection. 5-10 mL of the cerebrospinal fluids were aspirated and about 20 mL MNCs (containing stem cells) in ringers were injected intrathecally (IT). The patients were laid down on their back for 4-5 hours. The median number of MNCs was 4×107 (range 1-450×107). The median viability of the cells was 90% (range 60%-98%). The patients received intravenous ceftriaxone every 12 hours and were discharged from the hospital few days after autologous stem cell therapy. RESULTS: We noted clinical improvements in 9 of 12 patients with Parkinson's disease, 20 of 28 patients with cerebral palsy, 6 of 7 patients with hypoxic brain damage, 2 of 4 patients with multiple sclerosis, and 4 of 5 patients with cerebellar atrophy. The improvements were noted after 2-4 weeks of cell therapy. There were no improvements in patients with spinal cord injury and complete paralysis and those with autism. There were variable improvements in other patients treated. CONCLUSION: Most patients with advanced Parkinson's disease, cerebral palsy, hypoxic brain damage, progressive cerebellar atrophy, and kernicterus neuropathy reported clinical effects of this safe intervention resulting in better functioning and an increased quality of life.

Anticancer effects of various Iranian native medicinal plants on human tumor cell lines.

In this study the antineoplastic activity of methanolic extracts of six medicinal plants that are native to Iran, including Galium mite, Ferulago angulata, Stachys obtusicrena, Cirsium bracteosum and Echinophora cinerea was investigated. Different tumor cell lines were exposed to the extracts and cytotoxic analysis using MTT colorimetric assay was performed. Quantification of percentage of cells undergoing apoptotic changes by flow cytometry, and DNA fragmentation analysis on sensitive cell lines was then carried out. Results obtained indicated that almost all of the extracts more or less had the capacity to decrease the proliferation of tumor cells. Among the plants, the highest activity against K562 leukemia cell line was found for E. cinerea and C. bracteosum with IC50 less than 20 microg/ml followed by G. mite with IC50 of 39.8 microg/ml. F. angulata and E. cinerea, mostly inhibited Jurkat cells proliferation (IC50 less than 8 microg/ml). Fifty percent inhibition of Fen bladder cell carcinoma due to exposure to F. angulata and E. cinerea was found at concentrations of nearly 180 microg/ml. A549, a lung carcinoma cell, was mostly affected by S. obtusicrena (IC50 more than 200 microg/ml). In flow cytometry analysis, C. bracteosum, E. cinerea, F. angulata and S. obtusicrena extracts demonstrated no remarkable effects on the cell cycle profile of K562 and Jurkat cells. Moreover, in DNA fragmentation analysis of treated cells, no ladder formation was detected. In contrary, G. mite caused more than 40% apoptosis in the K562 and Jurkat cells. In DNA fragmentation analysis G. mite extract produced ladder formation in these cells. In conclusion, these results indicated that the extracts used in this study have anti tumor activity particularly against the leukemia cell lines and that apoptosis is the possible cause of cell death observed due to the extract of G. mite.

Bcl-2 expression in acute myelogenous leukemia: the relation to myeloid antigen expression and response to therapy in Iranian patients.

Product of Bcl-2 gene prolongs survival of hematopoietic cells by inhibition of programmed cell death. The aim of this study is to examine the expression of the bcl-2 protein in a group of patients with AML and its relation to clinical features and response to therapy. Slides from the bone marrow or peripheral blood of 70 patients with AML were assessed for the expression of bcl-2 by immunocytochemistry. The expression of myeloid and non-lineage associated markers was detected by indirect immunofluorescence method. Correlation between bcl-2 and markers expression and patients characteristics was determined. More than 20% positivity for bcl-2 was found in 22 (31.4%) patients. Bcl-2 expression showed an association with M4 and M5 subtypes (p<0.01) and was correlated with clinical parameters including WBC and platelet count, extramedullary disease and Hb level. Bcl-2 expressing cells were significantly higher in CD15(+) and CD13(+) patients and lower in CD11b(+) and CD33(+) cases (p<0.001). Complete remission (CR) rate was significantly lower in cases with 20% or more bcl-2 positivity than others (24.4% v 75.6%). A shorter CR duration was observed in bcl-2+ patients when compared with bcl-2- ones (571+/-50 versus 850+/-17 days)(p=0.0001). The expression of bcl-2 was also associated with shorter survival (p=0.0001). Survival time for bcl-2+ patients was 831+/-44 days versus 1119+/-17 days for bcl-2- ones. CD11b and CD33 positivity was associated with longer survival whereas CD13 and CD15 positivity was correlated with lower survival (p<0.007). In multivariate analysis bcl-2 positivity was associated with poor survival. Bcl-2 expression showed a prognostic value in our patients indicating that even despite of some differences in treatment regimen, immunocytochemical analysis of this marker is still a simple and inexpensive method for evaluation of prognosis in AML patients. Bcl-2 expression may be related to the expression of differentiation associated markers.

Primary T-cell lymphoma of the breast with lymphoepithelial lesion. A case report.

Fine-needle aspiration, histomorphologic and immunologic findings in a case of primary T-cell lymphoma of the breast are reported. The tumor cells were diagnosed as large cell lymphoma with lymphoepithelial lesion and no nuclear multilobulation. No serologic evidence of HTLV-1 infection was detected. After simple mastectomy, lymph node dissection and chemotherapy, the patient is now doing well, having been followed for 30 months. To our knowledge, this is the first case report of primary T-cell lymphoma of the breast with prominent lymphoepithelial lesion, no nuclear lobularity, and an indolent clinical course.

Soluble HLA class I molecules in malignant pleural and peritoneal effusions and its possible role on NK and LAK cytotoxicity.

PURPOSE: To examine the amount of sHLA-I in malignant pleural and peritoneal effusions and its possible role in natural immune defense. METHODS: Three groups of patients (75 patients with malignancy, 21 with infection, and 27 with other diseases) were studied for sHLA-I value using an ELISA method. Cytolytic activity of freshly isolated pleural and peritoneal effusion-associated lymphoid (EAL) cells from 14 of cases with malignancy were examined and compared to that of ten non-cancerous patients. EAL cells were co-cultured with the autologous cell-free effusions immediately after collection and 3 days after incubation with IL-2. RESULTS: The mean value of sHLA-I in effusions was 1.01+/-1.36 micro g/ml, 0.97+/-1.20 micro g/ml, and 0.49+/-0.45 micro g/ml, respectively. Despite higher mean sHLA-I levels in malignant and infected patients, no significant difference between these groups was observed ( P >0.05). Generally, the amount of sHLA-I in peritoneal effusions was higher than that for pleural effusions, but the difference was not significant. There were also no statistical differences in the sHLA-I levels between sub-groups of patients with malignancy. EAL cells' killing activity in malignant and infected effusions was 68.15+/-11.73 and 78.28+/-14.41, respectively ( P=0.08). No correlation between sHLA-I level and NK activity of EAL cells from the patients was found. Almost all malignant cases after exposure to cell-free effusions displayed an increase in NK activity (from 68.66+/-11.13 to 74.2+/-12.39, P=0.042) and a decrease in LAK activity (74.5+/-18.30 vs 67.72+/-16.46, P=0.040). Whereas, the same experiment performed for non-malignant effusions showed a decrease in both NK activity and LAK activity. Changes in NK and LAK activity were not correlated with the amount of sHLA-I in the effusions. CONCLUSION: The presence of sHLA-I, particularly in malignant effusions, suggests a role for these molecules in tumor immunity in the peritoneal or plural environment; however, at least with these group of patients, sHLA-I appears not to be a unique determining factor on EAL cells' killing activity.

Profile of epidermal growth factor receptor (EGFr) expression in human malignancies: effects of exposure to EGF and its biological influence on established human tumour cell lines.

The aim of this study was to compare the profile of EGFr expression in transitional cell carcinoma of the bladder (TCC) and in oral squamous cell carcinoma (OSCC). In addition, to study the influence of EGF stimulation on the expression of major histocompatibility complex class I antigens, placental alkaline phosphatase (PLAP) as well as changes in tumour cell sensitivity to cisplatin using immunocytochemical staining, a colorimetric assay and SDS-gel electrophoresis. The results showed that: a) strong EGFr expression could be seen in 22/88 (27%) cases of TCCs. In oral tumours the values for non-invasive ameloblastoma and invasive OSCC were 4/25 (16%) and 30/41 (73%) respectively. b) EGFr expression in tumour cell lines paralleled that of tumour biopsies. The number of lines expressing high and low EGFr expression amongst TCCs were 4 and 4 and in OSCCs were 3 and 1 respectively. c) Exposure of tumour cell lines to EGF led to: i) an increase in EGFr expression (stimulatory indices SI, ranged from 1.06 to 2.58) for TCCs but a decrease in the case of OSCCs (SI ranged from 0.01 to 0.85). The corresponding SI values for class I antigens were 0.95-1.16 and 0.10-0.84. ii) A significant reduction in expression of PLAP by OSCC cell lines. iii) An increased susceptibility of OSCC cell lines to cisplatin by as much as 14% (p<0.001). These data demonstrated the overexpression of EGFr in a significant proportion of TCCs. As for oral tumours it depended on whether they were of an invasive or non-invasive type. In the invasive cases the majority overexpressed EGFr. The exposure of OSCC but not TCC tumour cells to EGF resulted in down regulation of EGFr and class I antigens. The expression of PLAP was also significantly reduced. Exposure of OSCC cells to EGF resulted in their increased susceptibility to cisplatin. The data supports the notion that the mitogenic activation of some tumour cells by EGF resulted in a reduction of their immune visibility, differentiation status and an increase in chemosensitivity.

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.

Cross-linking of CD38 molecules induced expression of interleukin-2 receptor (IL-2R) and HLA-DR on human thymocytes.

CD38 is a 45 KD glycoprotein which is mainly expressed on early lymphoid progenitors and plasma cells. Role and function of CD38 on thymocytes and plasma cells has not yet been elucidated. By cross-linking of CD38 molecules using a specific monoclonal antibody on thymocytes and a neoplastic T cell line (Jurkat), it was shown that percentage of HLA-DR and IL-2R molecules is significantly upregulated. Moreover, CD11a and CD18 were among two adhesion molecules which showed a sharp increase in expression, whereas no changes on expression of CD11b, CD44 and CD54 were detected. Spontaneous proliferation of Jurkat cell line after addition of different concentrations of CD38 monoclonal antibody was unchanged.

Antiphospholipid antibodies and hepatitis C virus infection in Iranian thalassemia major patients.

Although the precise nature of Antiphospholipid antibodies is still not clearly defined, they are known to have association with thromboembolic events and have been found in hepatitis C virus (HCV) infection. Moreover, high prevalence of HCV infection and thrombotic risk is described in thalassemia. We aimed at investigating the prevalence of anticardiolipin antibodies (aCLAbs), lupus anticoagulant (LA), and their relation with HCV infection in Iranian thalassemic patients. Presence of anti-HCV antibody, serum HCV-RNA, aCLAbs, and LA activity was determined in 131 patients with thalassemia major (male/female: 63/68 aged 3-29 years) registered at thalassemia unit, Dastgheib Hospital, Shiraz, Iran. Sixty-one healthy controls were also included. Anti-HCV antibody was positive in 24 (18.3%), IgG aCLAbs in 56 (42.7%), and LA activity in 9 (6.9%) patients. 87.5% of patients positive for aCLAbs had a low titer of aCLAbs. Although none of the participants had a previous history of thrombosis, higher prevalence of aCLAbs was detected in thalassemic patients compared with controls. No significant difference in the prevalence of aCLAbs was found between HCV-infected and noninfected patients. A high prevalence of aCLAbs, the majority in low titers, was detected in Iranian thalassemic patients irrespective of previous history of thrombosis and presence of HCV infection.