Clinical, immunologic, and genetic spectrum of 696 patients with combined immunodeficiency.

BACKGROUND: Combined immunodeficiencies (CIDs) are diseases of defective adaptive immunity with diverse clinical phenotypes. Although CIDs are more prevalent in the Middle East than Western countries, the resources for genetic diagnosis are limited. OBJECTIVES: This study aims to characterize the categories of patients with CIDs in Iran clinically and genetically. METHODS: Clinical and laboratory data were obtained from 696 patients with CIDs. Patients were subdivided into those with syndromic (344 patients) and nonsyndromic (352 patients) CIDs. Targeted DNA sequencing was performed on 243 (34.9%) patients. RESULTS: The overall diagnostic yield of the 243 sequenced patients was 77.8% (189 patients). The clinical diagnosis of hyper-IgE syndrome (P < .001), onset of disease at greater than 5 years (P = .02), and absence of multiple affected family members (P = .04) were significantly more frequent in the patients without a genetic diagnosis. An autosomal recessive disease was found in 62.9% of patients, reflecting the high rate of consanguinity in this cohort. Mutations impairing VDJ recombination and DNA repair were the most common underlying causes of CIDs. However, in patients with syndromic CIDs, autosomal recessive mutations in ataxia-telangiectasia mutated (ATM), autosomal dominant mutations in signal transducer and activator of transcription 3 (STAT3), and microdeletions in 22q11.21 were the most commonly affected genomic loci. Patients with syndromic CIDs had a significantly lower 5-year survival rate rather than those with nonsyndromic CIDs. CONCLUSIONS: This study provides proof of principle for the application of targeted next-generation sequencing panels in countries with limited diagnostic resources. The effect of genetic diagnosis on clinical care requires continued improvements in therapeutic resources for these patients.

Gene expression pattern of CCL2, CCL3, and CXCL8 in patients with bipolar disorder.

BACKGROUND: Bipolar disorder (BD) is one of the most important psychiatric disorders in the world. There is evidence suggesting the role of inflammatory mediators such as chemokines in the etiology of BD. The objective of the current study was to evaluate the gene expression of CCL2, CCL3, and CXCL8 in patients with BD and compare them to healthy controls. MATERIALS AND METHODS: A total of 48 patients with confirmed BD and 48 healthy controls enrolled in this study. All patients were recruited from April to August 2016 at Ibn-Sina Psychiatric Hospital, Mashhad University of Medical Sciences, Mashhad, Iran. RNA was extracted from the whole blood samples and then cDNA was synthesized. Gene expression of CCL2, CCL3, and CXCL8 was measured using SYBR® Green real-time polymerase chain reaction. The difference of delta-CT values between patients and healthy controls was compared with the independent samples t-tests. RESULTS: CCL2 and CXCL8 genes expressed at higher levels in patients with BD as compared to healthy controls, but not significant. On the contrary, we found lower expression levels for CCL3 gene in our patients compared to healthy controls, but the difference was not statistically significant. CONCLUSION: Our findings do not show an association between the gene expression of CCL2, CCL3 and CXCL8 and BD. Increasing the sample size and evaluation on the gene expression of other chemokines in depression and mania phases of BD might be helpful to get a better conclusion.

Fourth Update on the Iranian National Registry of Primary Immunodeficiencies: Integration of Molecular Diagnosis.

BACKGROUND: The number of inherited diseases and the spectrum of clinical manifestations of primary immunodeficiency disorders (PIDs) are ever-expanding. Molecular diagnosis using genomic approaches should be performed for all PID patients since it provides a resource to improve the management and to estimate the prognosis of patients with these rare immune disorders. METHOD: The current update of Iranian PID registry (IPIDR) contains the clinical phenotype of newly registered patients during last 5 years (2013-2018) and the result of molecular diagnosis in patients enrolled for targeted and next-generation sequencing. RESULTS: Considering the newly diagnosed patients (n = 1395), the total number of registered PID patients reached 3056 (1852 male and 1204 female) from 31 medical centers. The predominantly antibody deficiency was the most common subcategory of PID (29.5%). The putative causative genetic defect was identified in 1014 patients (33.1%) and an autosomal recessive pattern was found in 79.3% of these patients. Among the genetically different categories of PID patients, the diagnostic rate was highest in defects in immune dysregulation and lowest in predominantly antibody deficiencies and mutations in the MEFV gene were the most frequent genetic disorder in our cohort. CONCLUSIONS: During a 20-year registration of Iranian PID patients, significant changes have been observed by increasing the awareness of the medical community, national PID network establishment, improving therapeutic facilities, and recently by inclusion of the molecular diagnosis. The current collective study of PID phenotypes and genotypes provides a major source for ethnic surveillance, newborn screening, and genetic consultation for prenatal and preimplantation genetic diagnosis.

Exploring Common Symptoms in Patients with Respiratory Allergies Using K-Means Algorithm in the North-East of Iran in 2012-2015.

Background: As a common disease among people of almost any age, allergic rhinitis has many adverse effects such as lowering the quality of life and efficiency at work or school. Considering these conditions and the collection of large amounts of data, the present research was conducted on allergic rhinitis and asthma patients' data to extract the common symptoms of these diseases using cluster analysis and the k-means algorithm. Materials and Methods: The present cross-sectional research was conducted in Mashhad city. The inclusion criteria were affliction with one or two respiratory allergy diseases diagnosed by an allergy specialist through clinical history taking and physical examination. A researcher-made checklist was used in the present study for data collection. Then, the K-means algorithm's cluster analysis model was conducted to extract clusters (WEKA software (3, 6, 9)). Results: Overall, 1,231 patients met the inclusion criteria. The result of the Cluster analysis consisted of Cluster 1 in allergic rhinitis consisted of 702 patients, and cluster 2 consisted of 382 patients.46 asthma patients were assigned to cluster 1 and 23 to cluster 2.Also, 60 asthma and allergic rhinitis patients were assigned to cluster 1 and 19 to cluster 2. The most common symptoms in all patients were rhinorrhea, sneezing, nasal congestion, and itchy nose. Conclusion: Overall, Salsola kali was the most common allergen in allergic rhinitis and asthma patients. Also, the most common symptoms in patients are rhinorrhea, sneezing, itchy nose, and nasal congestion. This study can help physicians diagnose allergic rhinitis and asthma in geographical areas with a high prevalence of Salsola kali.

Mesenchymal Stem/Stromal Cells Overexpressing CXCR4R334X Revealed Enhanced Migration: A Lesson Learned from the Pathogenesis of WHIM Syndrome.

C-X-C chemokine receptor type 4 (CXCR4), initially recognized as a co-receptor for HIV, contributes to several disorders, including the WHIM (Warts, Hypogammaglobulinemia, Infections, and Myelokathexis) syndrome. CXCR4 binds to its ligand SDF-1 to make an axis involved in the homing property of stem cells. This study aimed to employ WHIM syndrome pathogenesis as an inspirational approach to reinforce cell therapies. Wild type and WHIM-type variants of the CXCR4 gene were chemically synthesized and cloned in the pCDH-513B-1 lentiviral vector. Molecular cloning of the synthetic genes was confirmed by DNA sequencing, and expression of both types of CXCR4 at the protein level was confirmed by western blotting in HEK293T cells. Human adipose-derived mesenchymal stem cells (Ad-MSCs) were isolated, characterized, and subjected to lentiviral transduction with Wild type and WHIM-type variants of CXCR4. The presence of copGFP-positive MSCs confirmed the high efficiency of transduction. The migration ability of both groups of transduced cells was then assessed by transwell migration assay in the presence or absence of a CXCR4-blocking agent. Our qRT-PCR results showed overexpression of CXCR4 at mRNA level in both groups of transduced MSCs, and expression of WHIM-type CXCR4 was significantly higher than Wild type CXCR4 (P<0.05). Our results indicated that the migration of genetically modified MSCs expressing WHIM-type CXCR4 had significantly enhanced towards SDF1 in comparison with Wild type CXCR4 (P<0.05), while it was reduced after treatment with CXCR4 antagonist. These data suggest that overexpression of WHIM-type CXCR4 could lead to enhanced and sustained expression of CXCR4 on human MSCs, which would increase their homing capability; hence it might be an appropriate strategy to improve the efficiency of cell-based therapies.

High proviral load of human T cell lymphotropic virus type-1 facilitates coronary artery diseases.

OBJECTIVES: Coronary artery disease (CAD) is known as a life threatening disease, worldwide. In this study the role of HTLV-1 infection was evaluated on cardiac involvement in an endemic region of northeastern Iran. MATERIALS AND METHODS: Serologic and molecular tests for HTLV-1 infection were carried out in subjects who had coronary angiography. A real-time PCR, TaqMan method, to quantify HTLV-1 proviral load (PVL), and routine hematological and biochemical tests were performed for study subjects. RESULTS: Twenty nine patients were HTLV-1+CAD+ and 13 cases were HTLV-1+CAD-. Although, there were no significant differences for risk factors like FBS, HDL, triglyceride, systolic and diastolic blood pressure (Cbp, Dbp), waist circumference (WC), hip circumference (WL), cholesterol (P=0.003), and LDL (P=0.007) levels, and monocyte count (P=0.05) had meaningful differences. The mean HTLV-1 PVL in HTLV-1+CAD+ subjects was 992.62±120 which was higher compared with HTLV-1+CAD- group (406.54±302 copies/104 PBMCs). Moreover, HTLV-1 PVL in males (833±108) was lower compared with females (1218±141 copies/104 PBMCs) (P=0.05). Patients with HTLV-1-PVL of more than 500 copies/104 had more diffused atherosclerosis plaque than patients with less than 500 (OR=6.87, 95% CI=1.34-35.05; P=0.016). Furthermore, patients with diffused coronary atherosclerosis had significantly higher levels of HTLV-1 PVL than patients with middle, proximal, and normal location of coronary sclerotic lesions (P<0.05). CONCLUSION: Taken together, in endemic area, HTLV-1 infection, more likely is a facilitating factor for heart complications and the high HTLV-1 PVL might affect CAD manifestations.

Comparison of Common Monogenic Defects in a Large Predominantly Antibody Deficiency Cohort.

BACKGROUND: Predominantly antibody deficiencies (PADs) are the most common primary immunodeficiencies, characterized by hypogammaglobulinemia and inability to generate effective antibody responses. OBJECTIVE: We intended to report most common monogenic PADs and to investigate how patients with PAD who were primarily diagnosed as suffering from agammaglobulinemia, hyper-IgM (HIgM) syndrome, and common variable immunodeficiency (CVID) have different clinical and immunological findings. METHODS: Stepwise next-generation sequencing and Sanger sequencing were performed for confirmation of the mutations in the patients clinically diagnosed as suffering from agammaglobulinemia, HIgM syndrome, and CVID. RESULTS: Among 550 registered patients, the predominant genetic defects associated with agammaglobulinemia (48 Bruton's tyrosine kinase [BTK] and 6 µ heavy chain deficiencies), HIgM syndrome (21 CD40 ligand and 7 activation-induced cytidine deaminase deficiencies), and CVID (17 lipopolysaccharides-responsive beige-like anchor deficiency and 12 atypical Immunodeficiency, Centromeric instability, and Facial dysmorphism syndromes) were identified. Clinical disease severity was significantly higher in patients with µ heavy chain and CD40 ligand mutations compared with patients with BTK (P = .003) and activation-induced cytidine deaminase (P = .009) mutations. Paralysis following live polio vaccination was considerably higher in patients with µ heavy chain deficiency compared with BTK deficiency (P < .001). We found a genotype-phenotype correlation among patients with BTK mutations regarding clinical manifestation of meningitis and chronic diarrhea. Surprisingly, we noticed that first presentations in most patients with Immunodeficiency, Centromeric instability, and Facial dysmorphism were respiratory complications (P = .008), whereas first presentations in patients with lipopolysaccharides-responsive beige-like anchor deficiency were nonrespiratory complications (P = .008). CONCLUSIONS: This study highlights similarities and differences in the clinical and genetic spectrum of the most common PAD-associated gene defects. This comprehensive comparison will facilitate clinical decision making, and improve prognosis and targeted treatment.

Immunotherapy in Allergic Rhinitis: It's Effect on the Immune System and Clinical Symptoms.

BACKGROUND: Allergic rhinitis is one of the most common allergic diseases and characterised by sneezing, rhinorrhea, nasal congestion and nasopharyngeal itching. Subcutaneous immunotherapy (SCIT) for specific allergens is an effective treatment and induces the inhibitory effect of T regulatory lymphocytes and decreases clinical symptoms in allergic rhinitis. AIM: In this study effect of subcutaneous immunotherapy with specific allergens on clinical symptoms and T regulatory and T Helper cells cytokines, in patients with allergic rhinitis are evaluated. METHODS: In this study, 30 patients with moderate to severe allergic rhinitis according to clinical criteria and positive skin prick test for aeroallergens were selected and treated by SCIT. Clinical symptoms and T cells cytokines IL4, IL17, IFN gamma, TGF beta, GITR, FOXP3 and IL-10 (by RT-PCR) were evaluated before and one year after initiation of treatment. RESULTS: Thirty (30) patients with allergic rhinitis at age range 15-45 years old were treated by SCIT, and 23 (14 female, 9 male) patients continued the study, and 7 patients did not continue treatment. After immunotherapy, clinical symptoms decreased significantly. The specific cytokines TGF beta and IL10 levels increased and changes were statistically significant. (Respectively P = 0.013 and P = 0.05) The IL17 level was also increased, but not statistically significant. (P = 0.8) IFN gamma, IL4, GITR, FOXP3, all decreased, but the changes were not statistically significant (P > 0.05). CONCLUSION: Subcutaneous Immunotherapy for specific allergens decreases clinical symptoms in patients with allergic rhinitis and induces tolerance in T lymphocytes, especially by increasing T regulatory cells cytokines, TGF beta and IL10.

GATA3 Gene Polymorphisms Associated with Allergic Rhinitis in an Iranian Population.

BACKGROUND: The development of allergic rhinitis (AR) is caused by the interaction between genetic predisposition and environmental factors. In this study, the association between GATA3 single nucleotide polymorphisms and AR in an Iranian population was identified. METHODS: This case-control study was performed on 86 patients with AR and 86 healthy subjects. This study aimed to evaluate a potential association between two GATA3 SNPs, rs1269486 and rs2229360, and AR. Blood samples were collected and DNA was extracted for the evaluation of these SNPs by RFLP-PCR. RESULTS: A statistically-significant association was found between rs1269486 and AR (P<0.001). The frequencies of the A and GA genotypes were less in patients than in controls. The frequencies of the G allele and the GG genotype were greater in patients than in controls (P < 0.001). CONCLUSIONS: SNP rs1269486 of GATA3 was associated with AR and sensitivity to aeroallergens in our population. Because of the significance of this gene in AR, studying the association between GATA3 polymorphisms and AR is recommended for other populations.

Multiple Right and Left Pulmonary Arteries and Subdivisions of Inferior Mesenteric Artery Aneurysms in Behcet's Disease Case: A Rare Clinical Presentation.

Behcet's disease is a multi-systemic inflammatory disorder with cutaneous acneiform eruptions, orogenital aphthae, uveitis, arthritis and systemic vascular inflammation. One of the rare vascular manifestations is thoraco-abdominal aortic and pulmonary aneurysm that is associated with high risk of morbidity and mortality. We report a 36-year-old man with chronic cough, hemoptysis, significant weight loss, and orogenital ulcers from one year before referral. Initial assessments revealed multiple parahillar nodules in chest X-ray, chronic inflammatory anemia, erythrocyte sedimentation rate more than 100, and positive Human Leukocyte Antigen B5 and B51. Evaluation for infection and malignancies was unremarkable. Open exploratory lung study showed multiple pulsatile nodules in both lungs. AMIGO computed tomogram confirmed multiple right and left pulmonary artery aneurysms and impending to rupture aneurysm at subdivision of inferior mesenteric artery. After beginning of three methylprednisolone and cyclophosphamide pulse doses, the clinical aspect of the patient dramatically improved. Although pulmonary aneurysm is a rare manifestation of Behcet's disease and it is more infrequent in the distal branches, it can be seen in patients presenting with inflammatory disease and respiratory manifestations and with Behcet's disease diagnosis. Corticosteroid pulse-therapy could be considered as the first line of medical treatment in these patients.

Association of ADAM33 gene polymorphisms with allergic asthma.

OBJECTIVES: Asthma results from the interaction between genetic and environmental factors. ADAM33 gene on chromosome 20p13 is associated with asthma and airway hyperresponsiveness. MATERIALS AND METHODS: This is a case-control study, where four SNPs S1 (rs3918396), T1 (rs2280091), T2 (rs2280090), V4 (rs2787094) of ADAM33 gene have been assessed in patients with allergic asthma and normal controls (95 patients and 86 normal). Blood samples of these participants have been genotyped by PCR and the RFLP method. RESULTS: There was no association between asthmatic patients and polymorphisms of alleles, genotypes and haplotypes of the ADAM33 gene. When categorizing the asthmatic patients in severe, moderate and mild groups, associations in the subcategories of asthmatic patients were found. There were associations between polymorphisms of C allele of T1 SNP with severe asthmatic patients and G allele of V4 SNP with moderate asthmatics respectively (P=0.006, P=0.01). There was a significant association between sensitivity to mite and polymorphism of C allele of T1 SNP (P=0.02). Besides, there was a significant association between sensitivity to weeds and genotype GG of V4 SNP (P=0.05). CONCLUSION: Polymorphisms of ADAM33 gene might be associated with severe asthma and sensitivity to aeroallergens in northeast of Iran, but further studies are needed to determine the polymorphisms in this area and other regions of our country.

Long-term evaluation of a historical cohort of Iranian common variable immunodeficiency patients.

OBJECTIVES: Common variable immune deficiency (CVID) is the most frequent form of symptomatic primary immunodeficiency disease, characterized by hypogammaglobulinemia, recurrent infections and increased predisposition to autoimmunity and malignancies. The aim of this study was to reconsider important points of previously performed studies on Iranian CVID patients diagnosed and followed from 1984 to 2013. METHODS: Diagnosis was made using approved criteria including reductions of serum levels of immunoglobulins and exclusion of well-known single gene defects in individuals with an age >4 years and evidence of specific antibody deficiency. RESULTS: Detailed information on demographic data, survival rates, clinical phenotypes, immunologic and genetic data and treatment of 173 patients are provided. The early onset presentation (74.5%) and rate of consanguineous marriage (61.2%) were considerably higher in our cohort. Our study revealed clinically related correlations regarding consanguinity, the population of naïve CD4(+) T cells and switched-memory B cells, cytokine levels and special genetic factors (including HLA and AID genes). CONCLUSION: Despite current efforts, more comprehensive studies are needed, especially for classification and investigation of the genetic background and prognostic factors for patients with CVID in order to better managment and followup of patinets.

Is there any Association Between Human Lymphotropic Virus Type I (HTLV-I) Infection and Systemic Lupus Erythematosus? An Original Research and Literature Review.

OBJECTIVE(S): Systemic lupus erythematosus (SLE) is an autoimmune disease with unknown etiology. Some environmental factors can induce SLE in genetically susceptible individuals; for example, sun exposure and some viral infections may emerge the disease manifestations. Human T lymphotropic virus type 1 (HTLV-I) can dysregulate the human immune system, and the role of this virus in the pathogenesis of autoimmune diseases is under investigation. There are conflicting data about the role of HTLV-I in the pathogenesis of several autoimmune diseases such as SLE. In this study, we have focused on the correlation between HTLV-I infection and SLE in the northeast of Iran, an endemic area for the virus. MATERIALS AND METHODS: One hundred and thirty women with SLE and 915 healthy controls were screened for HTLV-I by enzyme linked immunosorbent assay (ELISA). Western blot method was used for confirmation of the positive results done by ELISA in the patients and the control group. RESULTS: Two (1.5%) of the patients and 23 (2.5%) of the healthy controls were HTLV-I seropositive. There was not a statistical difference between patients and controls in the number of HTLV-I seropositive samples (P=0.49). CONCLUSION: This cross-sectional case-control study did not find any association between HTLV-I and SLE. With regard to the previous studies, these controversies may stem from differences in ethnic background. Geographical and environmental factors should also be taken into account.

Oral administration of the purple passion fruit peel extract reduces wheeze and cough and improves shortness of breath in adults with asthma.

Asthma, affecting as many as 400 million individuals worldwide, is one of the most prevalent chronic health condition in the United States. With an increasing number of patients with asthma and the frequent inability of conventional lifestyle modification and therapy to effectively control the problem, nutritional and dietary therapies are being sought. This study was undertaken to investigate the efficacy of the purple passion fruit peel (PFP) extract, a novel mixture of bioflavonoids, on asthma symptoms. Patients with asthma were studied in a 4-week randomized, placebo-controlled, double-blind trial with oral administration of PFP extract (150 mg/d) or placebo pills. The effects of PFP extract were evaluated by assessing the clinical symptoms of asthma and spirometry tests. Most clinical symptoms of asthma of the PFP extract-treated group were moderated significantly compared to the baseline. The prevalence of wheeze, cough, as well as shortness of breath was reduced significantly in group treated with PFP extract (P < .05), whereas the placebo caused no significant improvement. Purple passion fruit peel extract supplementation resulted in a marked increase in forced vital capacity (P < .05) as placebo showed no effect. However, no significant improvement was observed in the forced expiratory volume at 1 second of those supplemented with PFP extract. No adverse effect was reported by any of study participants. The PFP extract may be safely offered to asthmatic subjects as an alternative treatment option to reduce clinical symptoms.

Association between HLA-DRB1*01 and HLA-Cw*08 and outcome following HTLV-I infection.

BACKGROUND: Human T cell lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory disease which occurs in less than 2% of HTLV-I -infected individuals. High proviral load, high HTLV-I-specific CD8+ cytotoxic T lymphocyte frequency (CTL) and host genetic factors such as HLA all appear to be associated with HTLV-I infection. Previous studies have shown that HLA-DRB1*01 increases the risk of HAM/TSP in Japanese HTLV-I infected individuals. OBJECTIVE: To investigate the association between HLA class II DRB1 alleles and HLA class I alleles (HLA-Cw*08, B54, A*02 and A-30) in HTLV-I infected individuals in Mashhad. METHODS: Here we determined the frequency of HLA class II DRB1, using INNO-LIPA reverse hybridization line probe assay, and HLA class I alleles (HLA-Cw*08,B54, A*02 and A-30) by PCR-SSCP method in healthy controls, HAM/TSP patients and HTLV-I infected individuals born and resident in Mashhad. RESULTS: The frequency of HLA-DRB1*01 alleles in this population was different from other areas of Iran. The frequency of HLA-DRB1*01 was significantly increased in HAM/TSP patients compared with carriers (p 0.028; OR=9.4). The frequency of HLA-Cw*08 was also significantly increased in HAM/TSP patients compared with controls (p=0.03; OR=13.5). CONCLUSION: Our results may suggest that possession of HLA-DRB1*01 increases the risk of HAM/TSP in HTLV-I-infected individuals and HLA-Cw*08 correlates with low CTL immune response in HAM/TSP patients.

Serum Levels of TNF-, IL-6, and Selenium in Patients with Acute and Chronic Coronary Artery Disease.

BACKGROUND: Selenium (Se) is part of the glutathione peroxidase enzyme complex (GSH-PX) that plays an important role in antioxidant mechanisms in body, also it has been demonstrated that populations with low Se intake have 2-3 times greater risk of ischemic heart disease. OBJECTIVE: To determine the circulating levels of IL- 6, TNF-alpha, Cu, Zn, and Se in patients with chronic coronary artery disease (CCAD), acute myocardial infarction (AMI), and normal individuals. METHODS: Patients were divided into two groups: 25 subjects with CCAD and 25 patients with AMI. The control group included 50 normal individuals who did not have any history of ischemic heart disease, and were sex and age matched with the patients. Blood samples were collected during the first hours after the onset of chest pain in AMI group. Serum concentration of Se, Cu, and Zn were determined by atomic absorption spectrometry and TNF-alpha and IL-6 levels were measured using ELISA method. RESULTS: In both groups of patients there was a significant reduction in serum Se levels (82.36 + 11.31 g/L in CCAD, 74.08+11.31g/L in AMI, and 105+32.52g/L in the control group, p=0.03). TNF-alpha titers were increased in AMI patients compared with CCAD and control group. Mean TNF-alpha levels were 37.44 pg/ml in CCAD, 914.32 pg/ml in AMI, and 4.80 pg/ml in the control group (p=0.01). Serum levels of IL-6 in CCAD and AMI patients were 3.28 15.55 pg/ml and 472207.88 pg/ml, respectively, and 1.28 pg/ml in the control group (p=0.001). CONCLUSION: These findings confirm previous studies and demonstrate that patients suffering from AMI exhibit lower plasma concentrations of Se and higher concentrations of pro-inflammatory cytokines of TNF-alpha and IL-6.