B-cells absence in patients diagnosed as inborn errors of immunity: a registry-based study.

Hypogammaglobulinemia without B-cells is a subgroup of inborn errors of immunity (IEI) which is characterized by a significant decline in all serum immunoglobulin isotypes, coupled with a pronounced reduction or absence of B-cells. Approximately 80 to 90% of individuals exhibit genetic variations in Bruton's agammaglobulinemia tyrosine kinase (BTK), whereas a minority of cases, around 5-10%, are autosomal recessive agammaglobulinemia (ARA). Very few cases are grouped into distinct subcategories. We evaluated phenotypically and genetically 27 patients from 13 distinct families with hypogammaglobinemia and no B-cells. Genetic analysis was performed via whole-exome and Sanger sequencing. The most prevalent genetic cause was mutations in BTK. Three novel mutations in the BTK gene include c.115 T > C (p. Tyr39His), c.685-686insTTAC (p.Asn229llefs5), and c.163delT (p.Ser55GlnfsTer2). Our three ARA patients include a novel homozygous stop-gain mutation in the immunoglobulin heavy constant Mu chain (IGHM) gene, a novel frameshift mutation of the B-cell antigen receptor complex-associated protein (CD79A) gene, a novel bi-allelic stop-gain mutation in the transcription factor 3 (TCF3) gene. Three patients with agammaglobulinemia have an autosomal dominant inheritance pattern, which includes a missense variant in PIK3CD, a novel missense variant in PIK3R1 and a homozygous silent mutation in the phosphoinositide-3-kinase regulatory subunit (RASGRP1) gene. This study broadens the genetic spectrum of hypogammaglobulinemia without B-cells and presented a few novel variants within the Iranian community, which may also have implications in other Middle Eastern populations. Notably, disease control was better in the second affected family member in families with multiple cases.

TFPI2 and NDRG4 gene promoter methylation analysis in peripheral blood mononuclear cells are novel epigenetic noninvasive biomarkers for colorectal cancer diagnosis.

BACKGROUND: As a result of the growing prevalence of colorectal cancer (CRC), new screening and early detection methods are required. Among the novel biomarkers, DNA methylation has emerged as a high-potential diagnosis/screening molecular marker. The present study aimed to assess non-invasive early diagnosis of CRC by examining promoter methylation of TFPI2 and NDRG4 genes in peripheral blood mononuclear cells (PBMCs). METHODS: Fifty CRC patients and 50 normal controls were recruited to the present study. Quantitative methylation of the promoter region of the TFPI2 and NDRG4 genes was analyzed in DNA extracted from PBMCs of all cases and control subjects using a methylation-quantification endonuclease-resistant DNA (MethyQESD) method. RESULTS: The sensitivity and specificity of the TFPI2 gene for the diagnosis of CRC was 88% and 92%, respectively, and, for the NDRG4 gene, it was 86% and 92%, respectively. The methylation range for the TFPI2 gene was 43.93% and 11.56% in patients and controls, respectively, and, for the NDRG4 gene, it was 38.8% in CRC patients and 12.23% in healthy controls (p < 0.001). In addition, we observed that a higher percentage of methylation was correlated with the higher stage of CRC. CONCLUSIONS: The results of the present study reveal that PBMCs are reliable sources of methylation analysis for CRC screening. Furthermore, the TFPI2 and NDRG4 genes provide sufficiently high sensitivity and specificity to be nominated for use in a novel noninvasive CRC screening method in PBMCs.

The interleukin 28B gene polymorphism, rs8099917, in patients with chronic hepatitis C and response to the treatment with pegylated interferon and ribavirin.

BACKGROUND: The present study aimed to determine the frequency of the IL28B polymorphism rs8099917 in patients with genotype 1 hepatitis C virus (HCV) infection treated with pegylated-interferon-α2b (PEG-IFN-α2b) and ribavirin (RBV) and its treatment outcome. MATERIALS AND METHODS: The IL28B rs8099917 genotypes were determined among 100 HCV-infected patients and the viral load was also estimated. PEG-IFN-α2b and RBV combination were administrated to the patients for 48 weeks and the treatment outcome was defined. RESULTS: Sixty-seven (67%), 27 (27%), and 6 (6%) of 100 patients were determined as TT, GT, and GG genotype, respectively. The response rate to treatment was significantly higher in patients with TT genotype. CONCLUSION: According to the results of the present study, patients with IL28B rs8099917 TT genotype achieve higher sustained virological response than the GT and GG genotypes. Thus, when there are no alternatives, treatment with PEG-IFN-α2b and RBV combination can be suggested in patients with IL28B TT genotype.

Promising effect of rapamycin on multiple sclerosis.

The routine therapies for relapsing-remitting multiple sclerosis (RRMS) are common disease-modifying medications, yet are not effective in all patients. The aim of the present clinical trial was to evaluate the therapeutic effects of rapamycin on the clinical and radiological aspects, regulatory T cells proliferation and FOXP3 and GARP gene expression in the patients with RRMS. In this study, eight patients with RRMS were chosen and included in the trial. Patients received rapamycin (Rapacan, Biocon, India) for six months. Magnetic resonance imaging (MRI) of the patients' brain was taken before and after the therapy. Patients' expanded disability status scale (EDSS), and FoxP3 and GARP gene expression, and Treg cell proliferation were also been evaluated. All the patients had some degrees of significant reduction in mean plaque area size (P = 0.012, Z = -2.520), and minimum and maximum size of the plaques (P = 0.012, Z = -2.521). EDSS of 50% of patients was decreased after the treatment, yet it was not significant (P = 0.059, Z = -1.89). The expression rate of FOXP3 (P = 0.003) and GARP genes in Tregs increased after the therapy. We found a promising response to rapamycin among our cases with minor side effects and it may be considered as a therapeutic option of this disease.

Impaired IL-12- and IL-23-Mediated Immunity Due to IL-12Rß1 Deficiency in Iranian Patients with Mendelian Susceptibility to Mycobacterial Disease.

PURPOSE: Inborn errors of IFN-γ-mediated immunity underlie Mendelian Susceptibility to Mycobacterial Disease (MSMD), which is characterized by an increased susceptibility to severe and recurrent infections caused by weakly virulent mycobacteria, such as Bacillus Calmette-Guérin (BCG) vaccines and environmental, nontuberculous mycobacteria (NTM). METHODS: In this study, we investigated four patients from four unrelated consanguineous families from Isfahan, Iran, with disseminated BCG disease. We evaluated the patients' whole blood cell response to IL-12 and IFN-γ, IL-12Rß1 expression on T cell blasts, and sequenced candidate genes. RESULTS: We report four patients from Isfahan, Iran, ranging from 3 months to 26 years old, with impaired IL-12 signaling. All patients suffered from BCG disease. One of them presented mycobacterial osteomyelitis. By Sanger sequencing, we identified three different types of homozygous mutations in IL12RB1. Expression of IL-12Rß1 was completely abolished in the four patients with IL12RB1 mutations. CONCLUSIONS: IL-12Rß1 deficiency was found in the four MSMD Iranian families tested. It is the first report of an Iranian case with S321* mutant IL-12Rß1 protein. Mycobacterial osteomyelitis is another type of location of BCG infection in an IL-12Rß1-deficient patient, notified for the first time in this study.

Establishment and Development of the First Biobank of Inflammatory Bowel Disease, Suspected to Primary Immunodeficiency Diseases in Iran.

BACKGROUND: Inflammatory bowel disease (IBD) might be an immunodeficiency rather than an excessive inflammatory reaction. IBD, suspected to primary immunodeficiency diseases biobank (IBDSPIDB) as a resource for researches can help improve the prevention, diagnosis, and illness treatment and the health promotion throughout the society. Therefore, we launched the biobank of IBDSPID for the first time in Iran. MATERIALS AND METHODS: This study was designed to provide the IBDSPIDB to have a high-quality DNA, RNA, and cDNA. Among of 365 patients, 39 have inclusion criteria that were as below: (1) IBD diagnosis before 5 years of age. (2) Resistance to conventional therapy of IBD. (3) Severe IBD. (4) Signs of SPID (including ear infections or pneumonia or recurrent sinus within the 1-year period; failure to thrive; poor response to the prolonged use of antibiotics; persistent thrush or skin abscesses; or a family history of PID). RESULTS: Out of 39 patients, 51.3% were males. The mean age was 32.92 ± 15.90 years old. Ulcerative colitis (79.5%) was more than Crohn's disease. The majority of patients (50.0%) had severe IBDSPID. Resistance to drugs and consanguinity was 12.9% and 47.4%, respectively. Age at onset in 65.8% of patients was after 17 years old. Patients with autoimmune, allergy, and immunodeficiency disease history were 33.3%, 33.3%, and 10.36%, respectively. RNA and cDNA yields large quantities of high-quality DNA obtained and stored. CONCLUSION: Our biobank would be valuable for future genetic and molecular study to be more about the relation between IBD and PID.

Prevalence of intestinal parasites in Isfahan city, central Iran, 2014.

Intestinal parasites are important enteric pathogens. Poverty, low quality of food and water supply and poor sanitation systems are the important factors associated with intestinal parasitic infections. These kinds of infections can be a good index for hygienic and sanitation status of the society. This study aimed to determine the prevalence of intestinal parasitic infections among humans referred to Dr. Sharifi Clinical Laboratory, Isfahan, Iran, 2014. In this cross sectional study, 652 fecal samples (286 males and 366 females) from humans who had stool examination test from January to August 2014 were chosen. Microscopic examination for parasitic infections has been carried out using wet mount method. Indistinguishable samples underwent trichrome staining method for accurate identification of protozoa. Intestinal parasitic infections were observed in 68 (10.42 %) out of 652 studied humans. Forty eight Blastocystis hominis (7.36 %), thirteen Endolimax nana (1.99 %), nine Giardia lamblia (1.38 %), five Entamoeba coli (0.76 %), four Chilomastix mesnili (0.61 %) and two Iodamoeba butschlii (0.15 %) were the observed protozoa in the studied population. B. hominis, E. nana and C. mesnili were found to be significantly more prevalent in people with loose stool specimen. Considering the helminthic infections, only one case (0.15 %) that was excreted Taenia saginata proglottids has been documented among 652 studied humans. Based on the findings of the present study intestinal parasitic infections in Isfahan city has been dramatically decreased over the past years and shows a good hygienic and sanitation status of the city.

Th1, Th2 and Th17 Cytokine Profile in Patients with Multiple Sclerosis Following Treatment with Rapamycin.

BACKGROUND: Management of multiple sclerosis (MS) is based on the usage of immunosuppressive and immune-modulating medications. Cytokines play an important role in the pathogenesis of MS. OBJECTIVE: To evaluate the effects of rapamycin on the concentrations of Th1/Th2/Th17 serum cytokines in patients with MS. METHODS: Six patients with relapsing remitting MS as a case group and 6 healthy individuals as a control group were enrolled. The patients have been receiving 2 mg rapamycin daily for 6 months. The individuals in control group received nothing during 6 months of the experiment. Enzyme linked immunosorbent assay (Simultaneous Multi-Analyte ELISA) technique was used for determination of serum concentrations of IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17, IFN-γ, TNF-α, G-CSF and TGF-ß before and after therapy with rapamycin. RESULTS: The mean absorbance of 10 out of the 12 studied cytokines showed reduction after the therapy with rapamycin including IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17, IFN-γ and TNF-α. The only statistically significant reduction was observed in the absorbance of IFN-γ (p=0.028). Two cytokines illustrated increase in the patients sera after the therapy, including G-CSF and TGF-ß, but only increase in TGF-ß was statistically significant (p=0.046). None of the studied cytokines in the control group varied significantly after 6 months. CONCLUSION: Based on the findings of this study, rapamycin has some immunosuppressive effects, such as decreasing IFN -γ, which can improve the quality of life of the patients with multiple sclerosis. Also the increased level of TGF-ß may also have benefits on the disease, which needs further clinical studies.

Interleukin-28B (rs12979860) gene variation and treatment outcome after peginterferon plus ribavirin therapy in patients with genotype 1 of hepatitis C virus.

BACKGROUND: The success of treatment of chronic hepatitis C (CHC) with pegylated interferon-α (PEG-IFN-α) and ribavirin (RBV) is affected by several host, viral, and treatment factors. This study was designed to describe the association of interleukin (IL) 28B genotypes for rs12979860 with sustained virologic response (SVR) in patients with genotype 1 CHC infection treated with PEG-IFN α-2 and RBV. MATERIALS AND METHODS: Interleukin-28B genotype in 100 studied patients was detected by tagman real-time polymerase chain reaction. Before treatment blood samples were obtained, then patients were treated for 48-week with a combination therapy using of the PEG-IFN α-2 and RBV. SVR evaluated 6 months after stopping therapy, and was defined as undetectable plasma hepatitis C virus-RNA. RESULTS: Among studied patients, 65% were IL-28B CT, 27% CC, and 8% TT. In all studied patients, SVR was 58.3%, relapse 15.6%, and null virological response 26.1%. SVR rates were 76.9% in IL-28B-CC, 56.4% in IL-28B-CT, and 12.5% in IL-28B-TT patients. Relapse rates were 7.7% in IL-28B-CC, 12.9% in IL-28B-CT, and 62.5% in IL-28B-TT patients. There was a significant difference between response to treatment in patients IL-28B-CC, CT, and TT (P = 0.003). IL-28B genotype CC, (odds ratio = 0.053, 95% confidence interval; 0.005-0.54, P = 0.03), was the independent predicting factor. CONCLUSION: Interleukin-28B was an important predictor of CHC treatment outcome with Peg-IFN-α and RBV. IL-28B-CC seems to be more important than IL-28B-CT/TT in predicting positive treatment response.

Immunosuppressive effect of silymarin on mitogen-activated protein kinase signalling pathway: the impact on T cell proliferation and cytokine production.

Silymarin, a polyphenolic flavonoid derived from milk thistle (Silybum marianum), is known to have anti-inflammatory, hepatoprotective and anticarcinogenic effects. In this study, the in vitro immunomodulatory effect of silymarin was investigated using human CD4+ T cells. Peripheral blood mononuclear cells (PBMC) from healthy individuals were activated with anti-CD3 (5 µg/ml) plus anti-CD28 (2 µg/ml) and treated with 10, 50 and 100 µM silymarin. Cells were incubated 72 hr for proliferation assay using MTT and for viability analysis using PI staining and flow cytometry. Naive CD4+ T cell was also isolated from PBMC, activated with PHA/anti-CD28 and treated with 100 µM silymarin for 72 hr. MAPKs' activity of cell lysate from activated naive CD4+ T cells was assessed using an ELISA-based MAPKinase activity kit, and Th1/Th2/Th17-related cytokine expression was determined by Multi-analyte ELISA array kit. Results indicated a significant inhibition in proliferation of activated PBMC after 48-hr incubation with 100 µM silymarin without causing cell death. Moreover, MAPKs' activity (ERK1/2 and P38) and Th1-related cytokines (IL-2, TNF-α, IFN-γ) were significantly reduced in silymarin-treated cells compared with control after 72 hr. This study shows that silymarin has the ability to inhibit T cell proliferation and pro-inflammatory cytokine secretion in vitro. Furthermore, silymarin is able to inhibit ERK1/2 and P38 pathway activation in T cells stimulated through TCR engagement, a property that is likely associated with its ability to inhibit T cell proliferation and cytokine secretion. Therefore, silymarin, as an immune-response modifier, might be a valuable drug in therapeutic situations in which immunosuppression is required.

Cell cycle analysis of the CD133(+) and CD133(-) cells isolated from human colorectal cancer.

AIM: The CD133 antigen has been identified as a putative stem cell marker in colorectal cancer tissues. The aim of this study was to investigate the cell cycle state of CD133(+) and CD133(-) cells, isolated from primary human colorectal tumors. MATERIALS AND METHODS: After mechanical and enzymatic dissociation of the tumor samples, CD133(+) and CD133(-) subsets were identified and separated by magnetic cell sorting. Flow cytometric analysis was performed to compare the cell cycle of both CD133(+) and CD133(-) cells isolated from primary and liver metastatic cancer cells. RESULTS: The results indicated that CD133(+) cells isolated from both primary and liver metastatic colorectal cancers were found in higher percentage in the G0/G1 phases. However, the CD133(-) cells isolated from primary colorectal cancers were predominantly found in the S and G2/M phases. Surprisingly, the CD133(-) cells isolated from liver metastatic colorectal cancers were mostly found in the G0/G1 phase. CONCLUSION: The present study provides evidence that CD133(+) cells are in a quiescent state in colorectal cancer, representing a mechanism that would at least partially explain chemotherapy resistance and tumor recurrence in post-therapy patients.

The influence of iron loading and iron chelation on the proliferation and telomerase activity of human peripheral blood mononuclear cells.

BACKGROUND: Iron is an essential trace element in cell proliferation. Several investigations demonstrate that iron deprivation inhibits cell proliferation. However, the impact of iron on telomerase activity of activated lymphocytes remains unexplained to date. OBJECTIVE: In this study, the effect of iron on the proliferation and telomerase activity of lymphocytes stimulated by phytohemagglutinin (PHA) were investigated. METHODS: Iron loading was performed by incubating peripheral blood mononuclear cells in 500microM FeSO4.7H2O for 24 h and iron chelation was done by exposing cells to desferrioxamine, a potent iron chelator. The effects of silymarin, a flavonoid with both antioxidant and iron chelating activities, on the proliferation and telomerase activity of PHA-activated lymphocytes were also compared with desferrioxamine. Proliferation and telomerase activity were assessed using BrdU incorporation assay and Telomeric Repeat Amplification Protocol (TRAP), respectively. RESULTS: The proliferations of lymphocytes were significantly inhibited by 10 and 20 microg/ml desferrioxamine in a dose dependent manner, while iron loading recovered suppressed cell proliferation to the normal level. Silymarin at 20 microg/ml significantly increased the proliferation of lymphocytes in both normal and iron-treated conditions. Telomerase activity of lymphocytes was markedly increased by iron treatment and suppressed by desferrioxamine. Conversely, iron treatment had no effect on the telomerase activity of lymphocytes incubated with silymarin. CONCLUSION: Iron plays a significant role in the proliferation and telomerase activity of lymphocytes. The effects of silymarin on the proliferation and telomerase activity of lymphocytes were completely different from those of desferrioxamine, suggesting that the immunomodulatory effect of silymarin is probably not associated with its iron chelating activity.

Premature senescence of T lymphocytes from patients with beta-thalassemia major.

Several researches have demonstrated a suppressed cell mediated immunity in patients with beta-thalassemia major. To know whether the premature aging of T cells is involved in abnormalities of cell mediated immunity, the biomarkers of immunosenescence including telomerase activity, apoptosis, and the expression of CD28 and CD95 were evaluated in T lymphocytes from beta-thalassemia major patients. The ex vivo spontaneous apoptosis in CD4(+) or CD8(+) T cells from patients and healthy subjects was assessed by an in situ TdT mediated dUTP-biotin nick end labelling (TUNEL) assay after 24h incubation in medium. Flow cytometric data revealed that lymphocytes from beta-thalassemia patients were resistant to spontaneous apoptosis compared to the normal lymphocytes. Moreover, the percentages of TUNEL(+)CD4(+) or TUNEL(+)CD8(+) T cells from patients were significantly lower than those control cells. Quantitative determination of telomerase activity in resting and activated T cells was performed using the Telomeric Repeat Amplification Protocol (TRAP). The results showed a decreased telomerase activity of activated T cells in patients with thalassemia major compared to that in healthy controls. However, the percentages of CD8(+)CD28(-) and CD3(+)CD95(+) T lymphocytes were significantly higher in thalassemia patients, indicating the phenotypes associated with senescent T lymphocytes. These data provide evidences for the occurrence of accelerated aging of T cells in beta-thalassemia major; possibly result in abnormal T cell function leading to suppressed cell mediated immunity.

Effects of silymarin on the proliferation and glutathione levels of peripheral blood mononuclear cells from beta-thalassemia major patients.

Iron toxicity in beta-thalassemia major is the main cause of oxidative stress and cell mediated immune deficiencies. Despite indicative signs of severe oxidative deficiencies associated with beta-thalassemia major, such as decreased level of plasma antioxidants and depletion of erythrocyte glutathione, little is known about intracellular redox status of immune cells. Since glutathione is a primary intracellular antioxidant and plays an essential role in several functions in T cells, in this study intracellular glutathione (GSH) levels as well as proliferation of PHA-activated peripheral blood mononuclear cells (PBMC) were investigated in 28 beta-thalassemia major patients and 28 healthy age-matched individuals. Considering the potential benefits of flavonoids in the therapy of oxidative stress, the effects of silymarin on the GSH levels and proliferation of PBMC from normal and thalassemia individuals were further examined. Quantitative determination of intracellular GSH and proliferative response of PBMC to PHA were performed before and after 72 h incubation of PBMC with various concentrations of silymarin (0, 5, 10, or 20 mug/ml). Results demonstrated a significant reduction of GSH and proliferation in beta-thalassemia major cells; however treatment with silymarin led to restoration of both GSH levels and PBMC proliferation in thalassemia patients. Considerably low levels of GSH and depressed proliferative response of PBMC in beta-thalassemia major may be responsible for the cell mediated immune abnormalities in iron overload conditions. Moreover, the GSH restoration and improvement of PBMC growth by silymarin is a possible explanation for its recently reported antioxidant and immunostimulatory activities. These data suggest the benefit of using flavonoids to normalize immune dysfunction in beta-thalassemia major. The immunomodulatory effects of silymarin in beta-thalassemia major are currently under further investigation in a double blind clinical trial.