Platelet lysate and tendon healing: comparative analysis of autologous frozen-thawed PRP and ketorolac tromethamine in the treatment of patients with rotator cuff tendinopathy.

Platelet-rich blood derivatives are being nowadays increasingly used in the treatment of tendon-related pathologies as a rich source of growth factors. We sought to ascertain if local application of platelet lysate (PL) to augment rotator cuff repair ameliorates patient outcomes compared to ketorolac tromethamine treated group. A total of forty patients, with clinical diagnosis of Rotator Cuff Tendinopathy were randomized to receive sub acromial injections of PL every week for a total of 3 injections and two injection of ketorolac tromethamine once every two weeks. Subjective assessments included VAS, SPADI and shoulder range of motion were assessed at baseline and at 1 and 6 months after injection. Taking both control and PL groups, it was vividly seen that the outcomes were identical at the initial state, as well as the short-term one; whereas, when considering the 6-month period, there is a seemingly remarkable superiority in PL group in all parameters.

Expression level of immune checkpoint inhibitory factors in preeclampsia.

Preeclampsia (PE) is a severe complication in pregnancy, and its symptoms (proteinuria and hypertension) manifest after 20 weeks of gestation, affecting up to 8 % of pregnancies. The pregnant women's immune system uses different tolerance mechanisms to deal with a semi-allogeneic fetus. The T-cell subsets including CD8+, CD4+, and Treg play a critical role in maintaining pregnancies. The expression of immune checkpoint molecules in T-cells can ensure pregnancy at the feto-maternal interface by controlling immune responses. This research aims to evaluate the expression level of immune checkpoint factors, including PD-1, LAG-3, CTLA-4, and TIM-3 in normal pregnant women and PE patients. Decidual tissue was collected from 50 participants (25 PE and 25 control). For evaluating the genes expression, real-time PCR was employed. The western blot was used to assess the proteins level. The results of real-time PCR indicated significantly decreased expression level of these immune checkpoints in PE patients. In parallel to gene expression results, the protein level of PD-1, LAG-3, CTLA-4, and TIM-3 in the PE group was also reduced. We revealed that the profile of proteins and genes expression of immune checkpoints in the decidua of PE mothers are different from normal pregnancy and these results indicate aberrant expression of immune checkpoints such as PD-1, LAG-3, CTLA-4, and TIM-3 may cause maladaptation immune response which results in PE manifestation.

Intrauterine administration of autologous hCG- activated peripheral blood mononuclear cells improves pregnancy outcomes in patients with recurrent implantation failure; A double-blind, randomized control trial study.

We aimed to investigate the effect of intrauterine administration of autologous hCG-activated PBMCs in RIF women with low Th-17/Treg cell ratio. 248 women with a history of implantation failure volunteered to receive PBMC-therapy. After immunologic consultation and doing flow cytometry analysis, 100 women with at least three IVF/ET failure who had low Th-17/Treg ratio in comparison with healthy control were enrolled in this study. These 100 patients were randomly divided into two groups as PBMC receiving (n = 50) and controls (n = 50). Then PBMCs were obtained from patients and treated with hCG for 48 h. Afterward, PBMCs were administered into the uterine cavity of the patient in the study group, two days before ET. The concentration of inflammatory cytokines was examined in the supernatant of cultured PBMCs after 2, 24, and 48 h of incubation using the ELISA method. The frequency of Th-17, Treg, and the Th-17/Treg ratio was significantly lower in RIF women than the healthy controls (P < 0.0001). The secretion of inflammatory cytokines was significantly higher after 48 h compared to 2 and 24 h (P < 0.0001). The pregnancy and live birth rate were significantly increased in women undergoing the PBMC-therapy compared to control (PBS-injecting) group (P = 0.032 and P = 0.047, respectively). The miscarriage rate was considerably lower in PBMC-therapy group (P = 0.029). Our findings suggest that intrauterine administration of autologous in vitro hCG-activated PBMCs improves pregnancy outcomes in patients with at least three IVF/ET failures.

The effects of nanocurcumin on Treg cell responses and treatment of ankylosing spondylitis patients: A randomized, double-blind, placebo-controlled clinical trial.

AIM: Ankylosing spondylitis (AS) is an inflammatory rheumatic disease with increased bone mass in the main sites of inflammation. Regulatory T (Treg) cells have been reported to involve in pathology of AS. This study designed at investigating the effects of nanocurcumin on Treg cell responses in peripheral blood (PB) of AS patients. METHODS: Test group including 12 AS patients received nanocurcumin daily for 4 months and control group including 12 patients received placebo. The frequency of Treg was measured by flow cytometry. The expression levels of FoxP3 and several associated microRNAs (miRNAs; miR-27, miR-17, and miR-146a) and cytokines including Interleukin-10 (IL-10), TGF-ß, and IL-6 were assessed by real-time polymerase chain reaction. Furthermore, enzyme-linked immunosorbent assay was done to determine the secretion levels of cytokines. RESULTS: After treatment with nanocurcumin the frequency of Treg cells in AS patients increased significantly. The RT-PCR data indicated that the expression of miR-17 and miR-27 were significantly decreased following nanocurcumin treatment while miR-146a and FoxP3 were significantly increased. Moreover, nanocurcumin-treated group had high levels of IL-10 and TGF-ß and low levels of IL-6 production than control group. CONCLUSION: The findings suggested that dysregulation of Treg cells in PB influences the AS development and nanocurcumin therapy could regulate the Treg cells, and so could be useful in the treatment of AS and may be other autoimmune diseases. This study is registered with IRCT.ir, number IRCT2017052927520N7.

The role of IL-10-producing B cells in repeated implantation failure patients with cellular immune abnormalities.

There are a few data of the role of B cells in RIF pathogenesis. Accordingly, the objective of the current study was to determine the role of IL-10-producing B cells in RIF. Twenty-three RIF women with cellular immune abnormalities and 25 normal controls were enrolled in this experiment. Isolated naïve B cells from peripheral blood of the subjects were cultured in vitro, divided into two parts and activated by CpG ODN and imiquimod as TLR agonists. Afterwards, the number of CD19+ IL-10+ B cells was evaluated by flow cytometry and their related IL-10 cytokine level was assessed by ELISA. The mRNA expression levels of related genes in just CPG stimulated B cell population were also analyzed using real-time PCR. RIF patients exhibited a decreased level of the cells (P = 0.014, P = 0.023, respectively) and IL-10 cytokine (P = 0.009, P = 0.045, respectively) in both CPG and imiquimod stimulated B cell groups. IL-10 serum level was also lower in these patients (P = 0.0014). Additionally, we found a negative relationship between the frequency of these cells with the number of failed ET and total IgG titers in RIF patients. The mRNA levels of IL-10-producing B cells related genes (IL-10 and PD-L1) was also significantly lower in RIF women, whereas the expression of plasma cells-associated transcriptional factors (BLIMP1, IRF4, and XBP1) was higher. Summing up the obtained results, we concluded that peripheral blood IL-10-producing B cells down-regulation might result in RIF pathogenesis. It is further suggested that these cells can suppress autoantibody generation and contribute to a successful implantation.

Cytokine profile, Treg/Th17 cell frequency changes during different posttransplantational time points in patients undergoing renal transplantation.

Renal transplantation is the best therapeutic approach for end stage renal dysfunction patients. There are direct relationships between proportions of Treg cells, Treg/Th17 cells ratio and secreted immunosuppressive cytokines with increased survival rate of the transplanted organ. The aim of this study was the measurement of Treg and Th17 cells frequency and their secreted cytokines. Ninety renal-transplanted patients were divided in three groups based on times after transplantation (1-6 month, 6-36 month, and more than 3 years). Treg and Th17 cells frequency, expression level of their transcription factors and cytokines, and their secreted cytokines level were measured in these groups. Higher expression level of Interleukin (IL)-10 and FoxP3 mRNA were observed in patients who had longer posttransplantation time. In contrast, lower mRNA expressions of IL-6, IL-17, IL-23, and TGF-ß were observed in this group. Receptor γt showed no significant changes in studied groups. In addition IL-10 level was increased and IL-6, IL-17, IL-23, and TGF-ß level were decreased in patients who had longer posttransplantation time. Treg cells frequency was raised in mentioned group whereas no remarkable changes were observed in Th17 cell frequency. The present study declared that in stable renal transplantation, over time, the percentage of Treg cells and Treg/Th17 ration is increased. This increase in ratio induces a change in cytokine profile, resulting in an increased immunosuppressive cytokines such as IL-10 leading to increase in the survival rate of the transplanted organ.

Cyclosporine A improves pregnancy outcomes in women with recurrent pregnancy loss and elevated Th1/Th2 ratio.

Recurrent pregnancy loss (RPL) is a one of the most common obstetrical complications. Since, the successful pregnancy occurs in T helper 2 (Th2)-dominant situation and since, Th1 type immunity is related to pregnancy failure, we investigated the effects of cyclosporine on Th1 and Th2 cells in RPL women. Totally, 76 RPL patients (38 women as treated group and 38 as control group) were included in this study. Flow cytometry was utilized to analyze the frequency of Th1 and Th2 in blood samples. Also, real-time polymerase chain reaction was carried out to assess the messenger RNA (mRNA) expression of transcription factors and enzyme-linked immunosorbent assay was used to evaluate Th1 and Th2 related cytokines. Significant decrease in Th1 frequency (p = 0.0004), Th1/Th2 ratio (p < 0.0001), T-bet mRNA expression (p < 0.0001), interferon-γ (p = 0.0007), and tumor necrosis factor α (p = 0.0002) secretion level were observed in cyclosporine group. Moreover, significant increase in Th2 frequency (p < 0.0001), mRNA expression of GATA binding protein 3 (p = 0.0001), and interleukin 10 secretion level (p = 0.0027) was also evident in treated group. At the end of the investigation, 31 (81.5%) patients in cyclosporine-treated group had successful childbirth when compared with 16 (42.1%) women in control group (p = 0.0001). Given this, cyclosporine treatment for RPL patients with elevated Th1/Th2 ratio can result in improved pregnancy outcome.

Nanocurcumin: A novel strategy in treating ankylosing spondylitis by modulating Th17 cells frequency and function.

Ankylosing spondylitis (AS) is a chronic rheumatic disease which mainly affects the axial skeleton and sacroiliac joints. T-helper 17 (Th17) cells have been reportedly involved in AS pathogenesis. Nanocurcumin is considered to be beneficial, as an anti-inflammatory compound, in AS patients treatment. In this study, Th17-related immunological parameters were evaluated in AS patients. Transcription factors messenger RNA (mRNA) expression level, cytokines, and related microRNAs (miRNAs) were measured by real-time polymerase chain reaction. Additionally, Th17 frequency and cytokines secretion were evaluated by flow cytometry and enzyme-linked immunosorbent assay tests, respectively. The frequency of Th17 was higher in AS patients. Gained data from nanocurcumin group also demonstrated that retinoic acid-related orphan receptor γ (RORγt) and interleukin-17 (IL-17) mRNA expression levels were significantly decreased (P = 0.0001 and 0.0006, respectively), as the decrease also happened in Th17-associated miRNAs including miR-141, miR-155, and miR-200 ( P = 0.04, P = 0.02, and P < 0.0001, respectively). Posttreatment data of miR-155 and miR-200 in the nanocurcumin and placebo groups also showed a higher expression level in the placebo group compared with nanocurcumin-treated patients. Some clinical symptoms of AS patients were also improved at the end of the treatment process. The results of this study showed the potential ability of nanocurcumin to regulate Th17 cells activity in AS patients. This study provided further evidence on the function and underlying mechanism of nanocurcumin helping better treatment of AS.

Metabolic syndrome mediates proinflammatory responses of inflammatory cells in preeclampsia.

PROBLEM: To investigate whether metabolic syndrome (MetS) is associated with exacerbation of inflammatory responses in preeclamptic (PE) patients, the dynamic changes of Th17 and Treg cells, monocytes, cytokines, and transcription pattern of inflammasome-related genes were analyzed in 35 women with PE suffering from MetS in comparison to 38 PE women without MetS and healthy pregnant women. METHOD OF STUDY: Expression of inflammasome-related genes, cytokines, and also TLR4 was measured using real-time PCR. Serum and medium supernatant cytokines levels of PBMCs and serum levels of HMGB1 and Caspase-1 were also evaluated by ELISA. Monocytes, Th17, and Treg cells frequency were also determined by flow cytometry. RESULT: PE women with MetS exhibited increased percentage of non-classical and intermediate monocytes and Th17 cells (P = 0.025). Furthermore, decreased Treg cells frequency was also observed in PE women with MetS compared to PE women (P = 0.019). The mRNA expression of inflammasome-related genes (Caspase-1, NLRP3, HMGB1), TLR4, IL-1ß, IL-6, IL-17, IL-18, and TNF-α was significantly higher in PE patients with MetS than that of the healthy pregnant individuals (P < 0.0001) and PE patients (P < 0.0001). Serum levels of TGF-ß and TNF-α in PE patients with MetS were increased compared to other two groups, while IL-10 levels were significantly reduced. A significant sFlt (P = 0.016), Caspase-1 (P = 0.012), HMGB1 (P = 0.016) upregulation, and VEGF (P = 0.023) downregulation were also observed in the serum of PE women having MetS compared to PE women. CONCLUSION: MetS is closely related to the exacerbation of inflammatory reactions in PE. This study indicates that, in order to diminish the systemic features of PE, prior to conceive and start a pregnancy, MetS should be severely considered and managed.

Toll-like receptors signaling network in pre-eclampsia: An updated review.

Toll-like receptors (TLRs) are innate immune cells receptors. They are expressed on leukocytes, epithelial cells, and more particularly on placental immune cells and chorion trophoblast. Upregulation of innate immune response occurs during normal pregnancy, but its excessive activity is involved in the pathology of pregnancy complications including pregnancy-induced hypertension and pre-eclampsia (PE). The recent studies about the overmuch inflammatory responses and aberrant placentation are associated with increased expression of TLRs in PE patients. This review has tried to focus on the relationship between some activities of TLRs and the risk of preeclampsia development.

MicroRNAs and signaling networks involved in epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a phenomenon in which epithelial cells lose their cell-to-cell connection and are detached from the base membrane. EMT is fundamental for many biological processes such as embryonic development and neurogenesis. It also plays a significant role in cancer progression and metastasis. EMT regulation occurs through a sophisticated network of transcription regulations that include many signaling pathways. The exact mechanism of cancer gene regulation has not been understood yet. However, it is interesting to study the role of microRNAs and epigenetics mechanism in the cancer development. In this review, the transcription regulation of EMT and the analysis of possible overlap between microRNAs and their targets which are involved in the cancer development are scrutinized.

T cell Subsets in Peripheral Blood of Women with Recurrent Implantation Failure.

The objective of this study was to determine whether there are any differences in the T cell composition and the expression of specific factors (i.e., IRF4, TBX21, GATA3, and GITR) of T cells between women with Repeated Implantation Failure (RIF) and fertile women. We observed a decrease in circulating Tregs and exhausted CD8 + T cells in RIF patients when compared to the controls whereas exhausted Treg and Th17 cells were more frequent. Using real-time PCR, we determined that the expression of IRF-4 and TBX21 was significantly elevated in the cases. In contrast, mRNAs encoding GATA3 and GITR were reduced. Furthermore, the expression of some miRNAs involved in T cell differentiation and their target gene candidates were examined in T cells from women with RIF and fertile control women. The patients showed significant up-regulation of miR-25, miR-93, and miR-326. miR-155 and miR-146a demonstrated significant down-regulation in RIF patients. The results revealed that the expression pattern of target genes was in line with data for miRNAs expression from purified Treg and Th17 cells. The findings of real-time PCR analysis provided insights into the genetic pathways underlying this aberration in the proportions of T cell subsets. Our data suggest that a combination of higher pro-inflammatory Th17 and exhausted Treg cells, and lower Treg and exhausted CD8 + T cells may co-exist in the peripheral blood of women with RIF. Moreover, the expression level of transcription factors and miRNAs controlling T cell differentiation may differ in women with RIF influencing pregnancy outcomes in these women.

The imbalance of Th17/Treg axis involved in the pathogenesis of preeclampsia.

PROBLEM: Inappropriate activation of the immune system, particularly the imbalance of T-helper type 17 (Th17)/regulatory T (Treg) cells is thought to play considerable roles in preeclampsia (PE). To investigate the probable effects of the adaptive immune system in the pathophysiology of PE, we analyzed the dynamic changes of Th17/Treg cells, cytokines profile, and transcription pattern of Th17/Treg-related genes and microRNAs (miRNAs) in 50 women suffering from PE in comparison with 50 healthy pregnant women. METHODS: Expressions of cytokines, specific transcription factors, and related miRNAs were measured by real-time polymerase chain reaction (PCR). Enzyme-linked immunosorbent assay (ELISA) was used to test the interleukin (IL)-17, IL-23, IL-6, and IL-10 and transforming growth factor ß in serum and supernatant of peripheral blood mononuclear cells (PBMCs). The frequency of Th17 and Treg cells were determined by flow cytometry. RESULTS: PE patients exhibited a decreased number of Treg cells (p = 0.006), while Th17 cells were increased ( p = 0.004). Forkhead box P3 and IL-10 mRNA expressions were reduced ( p = 0.0001 and 0.0028, respectively), while expressions of retinoic acid receptor-related orphan nuclear receptor γt, IL-17, IL-23, and IL-6 were enhanced ( p < 0.0001, 0.0018, 0.0014, and 0.027, respectively). ELISA results also showed increased levels of IL-6, IL-17, and IL-23 ( p = 0.022, 0.0005, 0.0081, respectively), and decreased levels of IL-10 in the supernatant of PBMCs of PE patients compared with control group ( p = 0.0011). There was significant upregulation of miR-106b and miR-326 ( p = 0.0048 and 0.028, respectively) in PE patients in comparison with the control group. CONCLUSIONS: These findings suggest that imbalance of Th17/Treg cells, regulated possibly via microRNAs, may be involved in the pathogenesis of PE, emphasizing on the importance of these cells in feto-maternal immune cross-talk.

Disturbed Th17/Treg balance, cytokines, and miRNAs in peripheral blood of patients with Behcet's disease.

Impaired inflammatory immune cells have been implicated in the pathogenesis of Behcet's disease (BD). In the current study, we aimed to evaluate the frequency of T helper (Th) 17 and regulatory T (Treg) cells, cytokine secretion, the expression of transcription factors related to Th17 and Treg cells, and microRNAs (miRNAs) targeting these transcription factors in BD patients. Blood samples from 47 BD patients and 58 healthy subjects were drawn, and the peripheral blood mononuclear cells (PBMCs) were separated and isolated. The frequency of Th17 and Treg cells was assessed using flow cytometry. Transcription factors related to these cells and miRNAs targeting these transcription factors were quantified using real-time polymerase chain reaction. Finally, the levels of associated cytokines were measured using enzyme-linked immunosorbent assay. A significant reduction in the percentage of Treg cell frequency and the levels of interleukin (IL)-10 and forkhead box P3 messenger RNA (mRNA) expressions were observed. The proportion of Th17 cells was notably increased, which was accompanied by a increased levels of IL-17, IL-23, and retinoic acid-related orphan receptor ɣ (RORɣt) mRNA expressions in BD patients. The level of Th17-associated cytokines in the supernatant was found to be elevated in BD patients. T-cell-associated miRNA expression levels, miR-25, miR-106b, miR-326, and miR-93 were significantly upregulated, while miR-146a and miR-155 levels were lower in PBMCs of patients with BD when compared with the controls. The increase in the proportion of Th17 cells alongside the decrease in Treg cells are possibly the involving factors in the pathogenesis of BD. Therefore, the evaluation of immune cells and related miRNA profile may serve as both prognostic biomarker and therapeutic approach in treating patients with BD.

Intravenous immunoglobulin G treatment increases live birth rate in women with recurrent miscarriage and modulates regulatory and exhausted regulatory T cells frequency and function.

Exhausted T cells and regulatory T (Treg) cells have been recently proposed to be new risk factors for recurrent miscarriage (RM). Intravenous immunoglobulin G (IVIG) treatment reported to modulate various immune cells. In this study, the effects of IVIG on the frequency and function of exhausted T cells, exhausted Tregs, and Treg cells, as well as pregnancy outcome in women with unexplained RM (URM), were investigated. Ninety-four pregnant women with RM were enrolled. At the time of positive pregnancy, blood samples were drawn. Forty-four patients with URM were included as IVIG receiving treated group and received 400 mg/kg of IVIG and the rest fifty patients were considered as a control group and received no IVIG administration. IVIG was given intravenously every 4 weeks during 32 weeks of gestation. Blood samples of patients were collected after the latest administration. Exhausted T cells, exhausted Tregs, and Treg cells were evaluated pre- and posttreatment in both groups. IVIG induced a significant decrease in the frequency of exhausted Tregs population and function as well as a significant increase in Treg cells population, however, IVIG failed to affect population and the function of exhausted T cells. Pregnancy outcome was successful in IVIG treated women (86.3%) and were significantly different (P = 0.0006) in compared with the untreated URM subjects (42%). Therefore, employing of IVIG increases Treg cells and diminishes exhausted Tregs responses in RM patients with cellular immune anomalies throughout the pregnancy. Immunemodulatory effects of IVIG are probably associated with successful pregnancy outcome.

Intravenous immunoglobulin (IVIG) treatment modulates peripheral blood Th17 and regulatory T cells in recurrent miscarriage patients: Non randomized, open-label clinical trial.

BACKGROUND: Th17 cells and Treg cells have been proposed as new risk factors for recurrent miscarriage (RM). In this study, we investigated the effect of Intravenous immunoglobulin G (IVIG) on the levels and function of Th17 and Treg cells and pregnancy outcome in women with RM. MATERIALS AND METHODS: 94 pregnant women with RM were enrolled in this study. Blood was drawn at the time of positive pregnancy. On the same day, IVIG 400mg/kg was administered intravenously for 44 patients. 50 other RM patients were included as no IVIG interfering control group. Following the first administration, IVIG was given every 4 weeks through 32 weeks of gestation. Peripheral blood was drawn after the last administration (32 weeks after pregnancy). RESULTS: IVIG down-regulated Th17 cells population and function and up-regulated Treg cells population and function were significant in the treated group. Pregnancy outcome in IVIG treated subjects was successful in 38 out of 44 RM women (86.3%). However, pregnancy outcome was successful in 21 out of 50 untreated RM women (42%). CONCLUSION: Administration of IVIG in RM women with cellular immune cells abnormalities during pregnancy influences Th17/Treg ratio in peripheral blood and enhances Treg and decreases Th17 responses.