A higher frequency of CD4+ CXCR5+ T follicular helper cells in patients with newly diagnosed Henoch–Schönlein purpura nephritis

T follicular helper (TFH) cells play an important role in the humoral immune responses. The aim of this study was to examine the frequency of different subsets of CD4⁺ CXCR5⁺ TFH cells and B cells in patients with new-onset Henoch–Schönlein purpura nephritis (HSPN). The numbers of different subsets of CD4⁺ CXCR5⁺ TFH cells, B cells and the constituents of serum cytokines were detected in a total of 25 patients with newly diagnosed HSPN before and after treatment, and in 14 healthy controls (HC). The potential connection of these cells with the clinical characteristics in HSPN patients was analyzed. The numbers of circulating CD4⁺ CXCR5⁺, CD4⁺ CXCR5⁺ ICOS⁺ and CD4⁺ CXCR5⁺ PD-1⁺ TFH cells, CD86⁺ CD19⁺, CD38⁺ CD19⁺ B cells and serum IL-2, IL-4, IL-17A, IL-21 and IFN-γ were significantly higher in HSPN patients (p < 0.05) than in HC. Before and after treatment the numbers of CD4⁺ CXCR5⁺ TFH cells were negatively correlated with the values of eGFR (r = − 0.7162, p < 0.05; r = − 0.732, p < 0.05, respectively). Similarly the numbers of CD4⁺ CXCR5⁺ PD-1⁺ TFH cells were negatively correlated with 24-h urinary proteins (r = − 0.4013, p < 0.05; r = − 0.7857, p < 0.05, respectively), and the numbers of CD4⁺ CXCR5⁺ ICOS⁺ TFH cells were positively correlated with the levels of serum IL-21 (r = 0.5186, p < 0.05; r = 0.8503, p < 0.05, respectively) and 24-h urinary protein (r = 0.6045, p < 0.05; r = 0.833, p < 0.05, respectively) in these patients, regardless of treatment. Following treatment the numbers of CD4⁺ CXCR5⁺, CD4⁺ CXCR5⁺ PD-1⁺, and CD4⁺ CXCR5⁺ ICOS⁺ TFH cells, as well as serum levels of IL-21 were significantly reduced, however IL-4 levels were noticeably increased (p < 0.05). A higher frequency of circulating CD4⁺ CXCR5⁺ TFH cells existed in patients with HSPN and may be a viable therapeutic target.     

CXCR5+ CD8+ T cells could induce the death of tumor cells in HBV-related hepatocellular carcinoma

The follicular CXCR5⁺ CD8⁺ T cells have recently emerged as a critical cell type in mediating peripheral tolerance as well as antiviral immune responses during chronic infections. In this study, we investigated the function of CXCR5⁺ CD8⁺ T cells in HBV-related hepatocellular carcinoma patients. Compared to CXCR5⁻ CD8⁺ T cells, CXCR5⁺ CD8⁺ T cells presented elevated PD-1 expression but reduced Tim-3 and CTLA-4 expression. Upon anti-CD3/CD28 stimulation, CXCR5⁺ CD8⁺ T cells demonstrated higher proliferation potency than CXCR5⁻ CD8⁺ T cells, especially after PD-1 blockade. CXCR5⁺ CD8⁺ T cells also demonstrated significantly higher granzyme B synthesis and release, as well as higher level of degranulation. Tumor cells were more readily eliminated by CXCR5⁺ CD8⁺ T cells than by CXCR5⁻ CD8⁺ T cells. Interestingly, we found that B cells were more resistant to CXCR5⁺ CD8⁺ T cell-mediated killing than tumor cells, possibly through IL-10-mediated protection. In addition, the CXCR5⁺ CD8⁺ T cell-mediated cytotoxic effects on tumor cells could be significantly enhanced by PD-L1 blockade. Together, we presented that in patients with in HBV-related hepatocellular carcinoma, CXCR5⁺ CD8⁺ T cells could mediate tumor cell death more potently than the CXCR5⁻ CD8⁺ T cells in vitro while the autologous B cells were protected.     

Gabapentin-induced changes of plasma cortisol level and immune status in hysterectomized women

AimWe have examined the effects of gabapentin (GBP) on stress-related changes of cortisol and catecholamines in patients who underwent hysterectomy because of uterine fibrinoids. Additionally, we have observed the effect of GBP on the immune status in the acute stress response to surgery.MethodsSixty patients scheduled for an abdominal hysterectomy were randomly assigned to the GBP administration 1 h before surgery (n = 30 pts), or to the placebo group (n = 30 pts). Blood samples were collected before and 24 h after the surgery. The intensity of pain was assessed by a visual analogue scale (VAS) every 8 h at rest. Immunomodulatory effects of GBP were determined by flow cytometry. We followed the total proportion of CD3⁺ lymphocytes, CD3⁺CD4⁺, CD3⁺CD8⁺, CD19⁺ B lymphocytes, CD16⁺CD56⁺CD3⁻NK cells and CD16⁺CD56⁺CD3⁺ NKT cells before and 24 h after hysterectomy. The plasma cortisol and catecholamines concentration was used to estimate the level of the stress response.ResultsVAS pain score at rest was significantly lower in the GBP group than in the placebo group (P = 0.003). Application of GBP significantly decreased the plasma cortisol level 24 h after the operation in comparison to the placebo group (P < 0,001). We found significant positive correlation between the VAS pain score and concentration of cortisol in all patients (P = 0.025). GBP reduced the concentration of catecholamines (p < 0.05). The proportion of CD3⁺ (P = 0.027) and CD3⁺CD4⁺cells (P = 0.006) was significantly lower in the GBP group 24 h after operation, while the contribution of CD19⁺ (P = 0.033) was significantly higher.ConclusionPreoperative administration of GBP reduced the pain scores at rest in patients at 0, 16 and 24 h after abdominal hysterectomy. Additionally, GBP reduced the stress response and changed immune parameters in the reaction to surgery.     

Association of the MDR1 (ABCB1) gene 3435C> T polymorphism with male infertility

Infertility is a common problem affecting one in six couples, and in 30% of infertile couples, the male factor is a major cause due to defective sperm quality. P-glycoprotein (P-gp), a product of the MDR1 (ABCB1) gene, may be a link between genetic and environmental factors contributing to the development of male infertility because pesticides (P-gp substrates) are well established factors of male infertility. The aim of the present study was to examine the effect of the MDR1 gene 3435C>T polymorphism on male infertility. In total, 162 male patients undergoing semen analysis due to initial infertility workup were included in the study. The control group consisted of 191 healthy males with proven fertility. MDR1 3435C>T genotyping was performed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Assessment of MDR1 genotypes among the infertile men showed that 17.9% of subjects were carriers of the CC genotype, 58.0% were CT and 24.1% were TT. Among fertile men, 30.4% of subjects were characterised by the CC genotype, 49.7% were CT and 19.9% were TT. In addition, the frequency of carriers of at least one T allele (i.e., CT and TT genotypes) among infertile and fertile subjects was 82.1% and 69.6%, respectively. The risk of infertility was significantly elevated by two-fold in individuals carrying at least one T allele (CT and TT genotypes: p = 0.009, OR = 2.00, 95% CI: 1.20–3.32). Furthermore, this elevated risk was still found when considering each of the CT and TT genotypes alone (TT genotype: p = 0.027, OR = 2.05, 95% CI: 1.09–3.86; CT genotype: p = 0.013, OR = 1.98, 95% CI: 1.16–3.36). This preliminary report suggests that P-gp may play some role in male infertility, mediating detrimental effects of environmental factors.     

Polybacterial immunomodulator Respivax restores the inductive function of innate immunity in patients with recurrent respiratory infections

Respivax (BulBio-NCIPD Ltd.) is an oral polybacterial immunomodulator intended for treatment and prevention of non-specific respiratory tract infections. We studied for the first time its effects on the inductive mechanisms of innate immunity, in the course of 3-month immunoprophylaxis of 25 patients with recurrent and chronic respiratory infections. The expression of pattern-recognition receptors on peripheral blood (PB) monocytes and plymorphonuclear cells (PMNs), the antigen-presenting and co-stimulatory potential of peripheral blood monocytes and dendritic cells; and the stimulated Th1/Th2 cytokine production were determined by flow cytometry. As compared to healthy controls, patients were characterized with down-regulation of TLR2 and TLR4/CD14 complex on PB monocytes (p < 0.01), decreased share of CD14+CD16+ DCs precursors (p < 0.01), decreased CD86 expression on PB DCs (p < 0.05) and a Th2 shift of cytokine profile. Respivax modulated differentially the surface expression of pattern-recognition receptors on PB monocytes, increasing TLR2 and CD14 without affecting TLR4 expression. Further on, Respivax enhanced the differentiation of mature CD86^high dendritic cells (DCs). Importantly, Respivax promoted a Th1 shift of cytokine profile and restored the Th1/Th2 cytokine balance without pro-inflammatory effects. Noteworthy, Th1/Th2 ratios in the patient's group correlated positively with the levels of TLR2 (R = 0.5, p < 0.001) and CD14 expression (R = 0.4, p < 0.05). We conclude that Respivax treatment restores the inductive function of innate immunity at three key levels: antigen recognition and presentation, co-stimulation of naïve T cells, and Th1/Th2 balance. This results, at least in part, from a differential modulation effect on the expression of pathogen-recognition receptors.     

Circulating CD4+ CXCR5+ T cells contribute to proinflammatory responses in multiple ways in coronary artery disease

Coronary artery disease (CAD) is a common subtype of cardiovascular disease. The major contributing event is atherosclerosis, which is a progressive inflammatory condition resulting in the thickening of the arterial wall and the formation of atheromatous plaques. Recent evidence suggests that circulating CD4⁺ CXCR5⁺ T cells can contribute to inflammatory reactions. In this study, the frequency, phenotype, and function of circulating CD4⁺ CXCR5⁺ T cells in CAD patients were examined. Data showed that circulating CD4⁺ CXCR5⁺ T cells in CAD patients were enriched with a PD-1⁺ CCR7⁻ subset, which was previously identified as the most potent in B cell help. The CD4⁺ CXCR5⁺ T cells in CAD patients also secreted significantly higher levels of IFN-γ, IL-17A, and IL-21 than those from healthy controls. Depleting the PD-1⁺ population significantly reduced the cytokine secretion. Interestingly, the CD4⁺ CXCR5⁺ PD-1⁻ T cells significantly upregulated PD-1 following anti-CD3/CD28 or SEB stimulation. CD4⁺ CXCR5⁺ T cells from CAD patients also demonstrated more potent capacity to stimulate B cell inflammation than those from healthy individuals. The phosphorylation of STAT1 and STAT3 were significantly higher in B cells incubated with CD4⁺ CXCR5⁺ T cells from CAD than controls. The IL-6 and IFN-γ expression were also significantly higher in B cells incubated with CD4⁺ CXCR5⁺ T cells from CAD. Together, this study demonstrated that CAD patients presented a highly activated CD4⁺ CXCR5⁺ T cell subset that could contribute to proinflammatory responses in multiple ways. The possibility of using CD4⁺ CXCR5⁺ T cells as a therapeutic target should therefore be examined in CAD patients.     

CXCR5+ CD8+ T cells present elevated capacity in mediating cytotoxicity toward autologous tumor cells through interleukin 10 in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is a common and aggressive subtype of non-Hodgkin's lymphomas, with limited treatment options in refractory and relapsed patients. Growing evidence supports the notion that CD8⁺ T cell immunity could be utilized to eliminate B cell lymphomas. CXCR5⁺ CD8⁺ T cell is a novel cell subtype and share CXCR5 expression with CD19⁺ tumor cells. In this study, we investigated the frequency and function of existing CXCR5⁺ CD8⁺ T cells in DLBCL patients. We found that DLBCL patients as a group demonstrated significantly higher level of CXCR5⁺ CD8⁺ T cells than healthy individuals, with huge variability in each patient. Using anti-CD3/CD28-stimulated CD8⁺ T cells as effector (E) cells and autologous CD19⁺ tumor cells as target (T) cells, at high E:T ratio, no difference between the intensities of CXCR5⁺ CD8⁺ T cell- and CXCR5⁻ CD8⁺ T cell-mediated cytotoxicity were observed. However, at intermediate and low E:T ratios, the CXCR5⁺ CD8⁺ T cells presented stronger cytotoxicity than CXCR5⁻ CD8⁺ T cells. The expressions of granzyme A, granzyme B, and perforin were significantly higher in CXCR5⁺ CD8⁺ T cells than in CXCR5⁻ CD8⁺ T cells, with no significant difference in the level of degranulation. Tumor cells in DLBCL were known to secrete high level of interleukin 10 (IL-10). We therefore blocked the IL-10/IL-10R pathway, and found that the expressions of granzyme A, granzyme B, and perforin by CXCR5⁺ CD8⁺ T cells were significantly elevated. Together, these results suggest that CXCR5⁺ CD8⁺ T cells are potential candidates of CD8⁺ T cell-based immunotherapies, could mediate elimination of autologous tumor cells in DLBCL patients, but are also susceptible to IL-10-mediated suppression.     

Mesenchymal stem cells upregulate Treg cells via sHLA-G in SLE patients

BackgroundSoluble human leukocyte antigen-G (sHLA-G) is a non-classical HLA class I molecule, exhibiting strong immunosuppressive properties by inducing the differentiation of T regulatory cells (Treg). Mesenchymal stem cells (MSCs) transplantation alleviates disease progression in systemic lupus erythematosus (SLE) patients. However, the underlying mechanisms are largely unknown.ObjectivesTo explore whether sHLA-G is involved in upregulating effects of MSCs on Treg, which contributes to therapeutic effects of MSCs transplantation in SLE.MethodsThe serum sHLA-G levels of SLE patients and healthy controls were detected by ELISA. The percentages of peripheral blood CD4 + ILT2 +, CD8 + ILT2 +, CD19 + ILT2 + cells and Treg cells were examined by flow cytometry. Ten patients with active SLE, refractory to conventional therapies, were infused with umbilical cord derived MSCs (UC-MSCs) and serum sHLA-G was measured 24 h and 1 month after infusion. The mice were divided into three groups: C57BL/6 mice, B6.MRL-Fas^lpr mice infused with phosphate buffer saline (PBS), and B6.MRL-Fas^lpr mice infused with bone marrow MSCs (BM-MSCs). Then, the concentrations of serum Qa-2 were detected. Peripheral blood mononuclear cells (PBMCs) were isolated from SLE patients and co-cultured with UC-MSCs for 3 days at different ratios (50:1, 10:1, and 2:1) with or without HLA-G antibody, and the frequencies of CD4 + CD25 + Foxp3 + T cells were then determined by flow cytometry.ResultsThe concentrations of serum sHLA-G were comparable between SLE patients and healthy controls. However, there was a negative correlation between sHLA-G levels and SLE disease activity index (SLEDAI) scores in active SLE patients (SLEDAI > 4). We found that serum sHLA-G levels were negatively correlated with blood urea nitrogen, serum creatinine and 24-hour urine protein in SLE patients. The sHLA-G levels were significantly lower in SLE patients with renal involvement than those without renal involvement. The expression of ILT2 on CD4 + T cells from SLE patients decreased significantly compared to that of healthy controls. A positive correlation between the frequencies of Treg and CD4 + ILT2 + T cells was found in SLE patients. The levels of sHLA-G increased 24 h post UC-MSCs transplantation. The concentrations of Qa-2 in BM-MSCs transplanted mice were significantly higher than those of control group. In vitro studies showed that MSCs increased the frequency of Treg cells in SLE patients in a dose-dependent manner, which was partly abrogated by the anti-HLA-G antibody.ConclusionsOur results suggested that MSCs may alleviate SLE through upregulating Treg cells, which was partly dependent on sHLA-G.     

Circulating follicular helper T cells presented distinctively different responses toward bacterial antigens in primary biliary cholangitis

Primary biliary cholangitis (PBC) is a chronic and progressive cholestatic liver disease with unknown causes. The initiation of PBC is associated with bacterial infections and abnormal immune correlates, such as the presence of self-reactive anti-mitochondrial antibodies and shifted balance of T cell subsets. In particular, the CD4⁺ CXCR5⁺ follicular helper T (Tfh) cells are highly activated in PBC patients and are significantly associated with PBC severity, but the underlying reasons are unknown. In this study, we found that the circulating CD4⁺ CXCR5⁺ T cells were enriched with the interferon (IFN)-γ-secreting Th1-subtype and the interleukin (IL)-17-secreting Th17-subtype, but not the IL-4-secreting Th2 subtype. We further demonstrated that a host of microbial motifs, including Pam3CSK4, poly(I:C), LPS, imiquimod, and CpG, could significantly stimulate IFN-γ, IL-17, and/or IL-21 from circulating CD4⁺ CXCR5⁺ T cells in PBC patients, especially in the presence of monocytes and B cells. Whole bacterial cells of Escherichia coli, Novosphingobium aromaticivorans, and Mycobacterium gordonae, could also potently stimulate IFN-γ, IL-17, and/or IL-21 production from circulating CD4⁺ CXCR5⁺ T cells. But interestingly, while the whole cell could potently stimulate circulating CD4⁺ CXCR5⁺ T cells from both healthy controls and PBC patients, the cell protein lysate could only potently stimulate circulating CD4⁺ CXCR5⁺ T cells from PBC patients, but not those from healthy controls, suggesting that circulating CD4⁺ CXCR5⁺ T cells in PBC patients had distinctive antigen-specificity from those in healthy individuals. Together, these data demonstrated that bacterial antigen stimulation is a potential source of aberrant Tfh cell activation in PBC patients.     

MicroRNA-106b regulates pro-allergic properties of dendritic cells and Th2 polarisation by targeting early growth response-2 in vitro

Recent evidence has suggested that miRNA is implicated in the immune response of allergic and inflammatory diseases. However, little is known about its role in the mechanism that underlies the establishment of pro-allergic DCs in allergic rhinitis is not fully understood. This study assessed whether and how microRNA (miR)-106b regulates the pro-allergic properties of DCs upon allergen stimulation in vitro. Bone marrow-derived dendritic cells (BMDCs) were generated and stimulated with ovalbumin (OVA) to identify the miRNA expression profile. After transfection with miR-106b mimics and inhibitors OVA-activated BMDCs were further evaluated for surface marker expression using flow cytometry, cytokine production using ELISA and subsequent effects on Th2 cell polarisation using flow cytometry. Moreover, the upstream controllers and potential target proteins of miR-106b were examined in a western blot analysis. Results showed that MiR-106b expression was significantly inhibited in activated BMDCs upon OVA stimulation (p < 0.05). Surface marker expression (e.g., MHC class II, CD80 and CD86) was significantly upregulated after the transfection of an miR-106b inhibitor (p < 0.05), and the proportion of GATA-3⁺ T cells was significantly increased among CD4⁺ T cells that were cocultured with miR-106b inhibitor-pretreated BMDCs (p < 0.05). Conversely, IL-12 production from OVA-activated BMDCs and the proportion of T-bet⁺ T cells increased significantly in a coculture of CD4⁺ T cells and miR-106b mimics-transfected BMDCs (p < 0.05). The early growth response (Egr)-2 was identified via luciferase reporter assays as a target gene of miR-106b, and significant Egr-2 upregulation was observed in OVA-activated BMDCs following transfection with a miR-106b inhibitor (p < 0.05). In conclusion, our results suggest that miR-106b negatively regulates the pro-allergic properties of BMDCs and subsequent Th2 polarisation upon OVA stimulation and might represent a promising therapeutic target for allergic inflammation.     

Inosine Acedoben Dimepranol promotes an early and sustained increase in the natural killer cell component of circulating lymphocytes: A clinical trial supporting anti-viral indications

Inosine Acedoben Dimepranol (IAD), licensed for the treatment of cell-mediated immune deficiencies associated with viral infections, has been reported to impact a variety of immune parameters both in vitro and in vivo. Here we report the results from a clinical trial where multiple lymphocyte subsets – CD19 + B cells, CD3 + T cells, CD4 + T-helper cells, FoxP3^hi/CD25^hi/CD127^lo regulatory T cells (Tregs), CD3 −/CD56 + NK cells, and CD3 +/CD56 + NKT cells – were, together with serum immunoglobulins and IgG subclasses, followed during 14 days of IAD administration to ten healthy volunteers; these selected from 27 individuals pre-screened in vitro for their capacity to respond to IAD as gauged by increases in the percentage of Treg and/or NKT cells arising in PHA-stimulated cultures. While a transient spike and dip in Treg and T-helper fractions, respectively, was noted, the outstanding consequence of IAD administration (1 g po, qds) was an early and durable rise in NK cells. For half the cohort, NK cells increased as a percentage of total peripheral blood lymphocytes within 1.5 h of receiving drug. By Day 5, all but one of the volunteers displayed higher NK cell percentages, such elevation – effectively a doubling or greater – being maintained at termination of study. The IAD-induced populations were as replete in Granzyme A and Perforin as basal NK cells. The novel finding of IAD boosting phenotypically competent NK numbers in healthy individuals supports the drug's indicated benefit in conditions associated with viral infection and reinforces the potential for uplift where immune performance may be compromised.     

l-arginine and docetaxel synergistically enhance anti-tumor immunity by modifying the immune status of tumor-bearing mice

l-arginine (l-Arg) supplementation has been reported to enhance the function of immune cells, including dendritic cells (DCs) and T lymphocytes, in cancer models thereby countering the suppressive effects of myeloid-derived suppressor cells (MDSCs). The balance of the active immune cells is one factor that determines the progression of cancers in vivo. Docetaxel (DTX), an immunomodulatory chemotherapeutic agent, is now widely used in several types of malignancies including breast cancer. We hypothesized that the combination of DTX and l-Arg would elicit a more robust antitumor response than either molecule alone. To test this hypothesis we utilized BALB/c mice inoculated with 4T1 mammary carcinoma cells. DTX and l-Arg synergistically limited tumor growth in vivo and moderately increased the life span of tumor bearing mice. The anti-tumor effects were associated with the proliferation of splenic CD8⁺ CTL and CD4⁺ Th1 effector cells, as well as increased serum levels of interferon gamma. More importantly, DTX + l-Arg effectively increased anti-tumor immunity within the tumor microenvironment. Furthermore, the combined therapy increased the number of myeloid (mDCs) and plasmacytoid (pDCs) dendritic cells, potent activators of the T cell response, and enhanced expression of the maturation markers CD86 and MHC II (required for antigen presentation). The combination therapy also reduced the proliferation of MDSCs. These data suggest that DTX + l-Arg may be a novel therapeutic strategy for breast cancer patients.     

APL-1, an altered peptide ligand derived from human heat-shock protein 60, selectively induces apoptosis in activated CD4 + CD25 + T cells from peripheral blood of rheumatoid arthritis patients

Rheumatoid arthritis (RA) is a chronic T-cell mediated autoimmune disease that affects primarily the joints. The induction of immune tolerance through antigen-specific therapies for the blockade of pathogenic CD4 + T cells constitutes a current focus of research. In this focus it is attempted to simultaneously activate multiple regulatory mechanisms, such as: apoptosis and regulatory T cells (Tregs). APL-1 is an altered peptide ligand derived from a novel CD4 + T-cell epitope of human heat-shock protein of 60 kDa, an autoantigen involved in the pathogenesis of RA. Previously, we have reported that APL-1 induces CD4 + CD25^highFoxp3 + Tregs in several systems. Here, we investigated the ability of APL-1 in inducing apoptosis in PBMCs from RA patients, who were classified as active or inactive according to their DAS28 score. APL-1 decreased the viability of PBMCs from active but not from inactive patients. DNA fragmentation assays and typical morphological features clearly demonstrated that APL-1 induced apoptosis in these cells. Activated CD4 + CD25 + T cells but not resting CD4 + CD25 − T cells were identified as targets of APL-1. Furthermore, CD4 + T-cell responses to APL-1 were found to be dependent on antigen presentation via the HLA-DR molecule. Thus, APL-1 is a regulatory CD4 + T cell epitope which might modulate inflammatory immune responses in PBMCs from RA patients by inducing CD4 + CD25^highFoxp3 + Tregs and apoptosis in activated CD4 + T cells. These results support further investigation of this candidate drug for the treatment of RA.     

Influence of anticancer agents on cell survival,proliferation, and CD4+CD25+Foxp3+ regulatory T cell-frequency in human peripheral-blood mononuclear cells activated by T cell-mitogen

Many anticancer agents currently used are considered to be cytotoxic not only to cancer cells but also to functional immune cells. To learn more about the immunosuppressive adverse influence of chemotherapeutic drugs in cancer chemotherapy, we examined the effects of arsenic trioxide, dacarbazine, 5-fluorouracil, and methotrexate on the survival, proliferation, cytokine production, and CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cell-frequency in human peripheral blood mononuclear cells (PBMCs) activated by T cell mitogen in vitro. Arsenic trioxide, dacarbazine, and 5-fluorouracil increased trypan-blue stained (dead) cell rates and suppressed the mitogen-activated proliferation of PBMCs significantly at 1–100 μM (p < 0.05). Methotrexate also significantly increased the percentages of dead cells and suppressed the mitogen-activated PBMC-proliferation at concentrations of more than 0.05 μM (p < 0.01). Arsenic trioxide significantly inhibited the production of interferon γ, interleukin (IL)-4, -6, and -10 from the activated PBMCs at 5 μM (p < 0.05). In contrast, the anticancer agents significantly increased Treg cell-frequency in the activated PBMCs at concentrations of more than 0.1 μM for methotrexate, 5 μM for arsenic trioxide and 5-fluorouracil, and 50 μM for dacarbazine, respectively (p < 0.05). These agents did not significantly influence the production of transforming growth factor (TGF) β from the activated PBMCs at a concentration range of 0.05–50 μM. Our data suggest that the anticancer agents: arsenic trioxide, dacarbazine, 5-fluorouracil, and methotrexate attenuate T cell mediated immunity by not only inhibiting the proliferative response of T cells but by also increasing the frequency of Treg cells, which may result in the suppression of the effector T cell function.     

In vitro induced CD4+CD25+Foxp3+ Tregs attenuate hepatic ischemia–reperfusion injury

Reperfusion injury causes liver dysfunction after warm or cold ischemia. Emerging data suggest a role of T cells as mediators in this ischemia/reperfusion (I/R) injury. In the T cells, a part of CD4⁺CD25⁺FoxP3⁺ T regulatory cells (Tregs) were reported to facilitate recovery from I/R injury. These Tregs can be induced by TGF-β in vitro. Interestingly, rapamycin was reported to selectively expand these Tregs in vitro. In the present study, addition of rapamycin to cultures containing TGF-β further increased the frequency and absolute number of functional CD4⁺ Tregs. Using a partial (70%) hepatic warm ischemia model, we investigated the effects of liver function recovery under the treatment of Tregs induced by rapamycin and TGF-β. The treatment of Tregs significantly reduced serum alanine aminotransferase and aspartate aminotransferase compared to I/R control animals at 24 h after reperfusion (P < 0.05). They also significantly attenuated the up-regulation of IFN-γ and IL-17 compared to the I/R control animals (P < 0.05). In conclusion, Tregs ameliorate the biochemical of hepatic I/R injury by preventing proinflammatory cytokines following a warm I/R insult. These data may pave the way to use Tregs as cell therapy to prevent hepatic I/R injury.     

Test of association between GABRA2 (SNP rs279871) and adolescent conduct/alcohol use disorders utilizing a sample of clinic referred youth with serious substance and conduct problems,controls and available first degree relatives

Recent findings have linked the GABRA2 gene with antisocial personality disorder and alcohol dependence (AD) in adults and conduct disorder (CD), but not AD symptoms, in children and adolescents. We sought to replicate previous findings and test for an association between a single nucleotide polymorphism (SNP) in the GABRA2 gene (rs279871) and CD among adolescents.MethodsAdolescent patients (n = 371), 13–18 years old, were recruited from a university substance abuse treatment program. Patient siblings (n = 245), parents of patients (n = 355), adolescent controls (n = 185), siblings of controls (n = 163) and parents of controls (n = 263) were included in these analyses (total sample n = 1582). Case-control (using only Caucasian and Hispanic probands) and family-based association tests were completed to test for association between rs279871 and several a priori CD and AD phenotypes.ResultsFor case-control association tests, rs279871 was significantly associated with CD (p = 0.02) but not AD phenotypes; the result did not survive strict correction for multiple testing. All family-based association tests were non-significant (CD p = 0.48; CD symptom count age corrected within sex p = 0.91; AD p = 0.84; alcohol use disorder p = 0.52).ConclusionsConsistent with previous findings, the results do not support the association between GABRA2 SNP rs279871 and AD in adolescents. Our results also do not support an association between rs279871 and CD; the study limitations are reviewed.     

Chicken egg yolk antibodies (IgY) modulate the intestinal mucosal immune response in a mouse model of Salmonella typhimurium infection

This study determined the effects of chicken egg yolk antibodies (IgY) on immune responses in the intestinal mucosal of mice infected with Salmonella typhimurium. Sixty, 28-day-old mice were divided into 4 groups and treated with streptomycin or sterile water for 2 days followed by 1 day without treatment. The control group was unchallenged whereas the mice in the other three groups were treated twice with 10⁹ CFU mL^− 1 S. typhimurium. For the next 3 days, control mice continued to receive no treatment whereas the mice in the remaining three groups were orally administered with 20 mg mL^− 1 of specific IgY, 20 mg mL^− 1 of nonspecific IgY or PBS. S. typhimurium activated gut-associated lymphoid tissue, increasing the release of IFN-γ and TNF-α in the mucosa and increased the number of activated T-lymphocytes and cytotoxic T-γδ. Specific IgY attenuated the increase in IFN-γ and TNF-α and the decrease in IL-10. S. typhimurium induced mobilization of CD8⁺ and CD8⁺ TCRγδ T cells in the epithelium and CD4⁺ and CD8⁺ T cells in the lamina propria reflecting an inflammatory process that was attenuated by IgY. These results suggest that specific IgY modulates intestinal mucosal immune responses during a S. typhimurium infection.     

CD4+ B220+ TCRγδ+ T cells produce IL-17 in lupus-prone MRL/lpr mice

Systemic lupus erythematosus is an autoimmune disease with comprehensive immune cell disorders. Recent studies suggested that pro-inflammatory cytokine IL-17 plays important role in lupus, leaving the cellular sources and their pathogenic and physiologic characters largely unknown. In the current study, by using lupus-prone MRL/lpr mice, we demonstrated that Th17 response prevails in lupus disease regarding significantly accumulated serum IL-17, increased IL-17-producing splenocytes, and elevated phospho-STAT3 in CD4⁺ T cells. Intracellular staining revealed that unusual CD4⁺ B220⁺ T cells are major IL-17-producing cells, whereas conventional CD4⁺ B220⁻ T cells are major IFN-γ-producing cells. Subsequent studies showed that CD4⁺ B220⁺ cells contains both αβ and γδ T cells in the spleen and thymus of MRL/lpr mice. Further study showed that around 60% of γδ T cells in MRL/lpr mice co-express both B220 and CD4 on their surface, and are the major RORγt⁺ cells in MRL/lpr mice. Finally, CD4⁺ B220⁺ T cells alone do not proliferate, but could enhance the proliferation and IFN-γ-production of conventional CD4⁺ B220⁻ T cells. Our findings suggest the pathogenic role of unusual CD4⁺ B220⁺ T cells in lupus disease in MRL/lpr mice according to their IL-17-producing ability and stimulatory function for conventional CD4⁺ B220⁻ T cells.     

Comparative evaluation of two dye probes in the rat everted gut sac model for unambiguous classification of P-gp substrate and inhibitor

IntroductionP-glycoprotein (P-gp) plays a crucial role in beta-amyloid efflux from the blood–brain barrier thus becoming a promising pharmacological target in the treatment of Alzheimer's disease (AD). The increase of P-glycoprotein expression and activity by a P-gp inducer could be an effective pharmacological strategy in slowing or halting the progression of AD. Commonly used in vitro methods to classify a P-gp interacting molecule as substrate, inhibitor, modulator or inducer are not always confirmed by in vivo experiments. Here we validate the new dye-probe beta-amyloid (1–40) HiLyte Fluor? TR-labeled (Ab-HiLyte) (Anaspec) P-gp mediated transport in the ex vivo rat everted gut sac assay by using MC18 or MC266, a fully characterized P-gp inhibitor and substrate, respectively, and compare it with the commonly used dye rhodamine.MethodsMale Wistar rats' everted intestines were divided into sacs, each sac was filled with 10 μM Ab-HiLyte with or without 50 μM of MC18 or MC266. Ab-HiLyte concentrations in mucosal fluid were measured spectrophotometrically at 594 nm at each appropriate time.ResultsThe Ab-HiLyte P-gp mediated efflux had a K = 1.00 × 10^? 2 min^? 1 and t_1/2 = 68.74 min, while in the presence of MC18, the Ab-HiLyte efflux turned out to be reduced by an order of magnitude (K = 1.65 × 10^? 3 min^? 1) and the half life is extremely increased (t_1/2 = 419 min). A P-gp substrate, like MC266, determines no change in the efflux of Ab: the kinetic constant and the half life turned out to be unmodified (K = 1.81 × 10^? 2 min^? 1 and t_1/2 = 38.28 min).DiscussionThe results demonstrate that the new dye probe, Ab-HiLyte, could be a probe of choice to unequivocally distinguish between a P-gp substrate and an inhibitor. This is particularly important as different groups obtain a controversial classification of the same compound.     

Vitamin D Binding Protein rs7041 polymorphism and high-residual platelet reactivity in patients receiving dual antiplatelet therapy with clopidogrel or ticagrelor

BackgroundVitamin D deficiency represents a major health problem in general population, especially for its association with cardiovascular disorders and thrombotic risk, even in patients on dual antiplatelet therapy (DAPT). Vitamin D Binding Protein (VDBP) is the main transporter of vitamin D in the bloodstream and genetic polymorphisms of this protein have been shown to account for a significant variability of vitamin D levels and its systemic effects. Contrasting data have linked the rs7041 T → G substitution with cardiovascular disease. However, no study has so far addressed the role of rs7041 polymorphism on platelet reactivity in patients on DAPT, that was the aim of the present study.MethodsPatients treated with DAPT (ASA and clopidogrel or ticagrelor) for an ACS or elective PCI were scheduled for platelet function assessment at 30–90 days post-discharge. Platelet function was assessed by Multiplate® (Roche Diagnostics AG), and VDBP genetic status by polymerase chain reaction and restriction fragment length polymorphism technique. Fasting samples were obtained for main chemistry parameters and vitamin D levels assessment.ResultsWe included 400 patients, 187 (46.8%) receiving clopidogrel and 213 (53.2%) ticagrelor. The genetic polymorphism rs7041 (T → G) was observed in 318 patients, (79.5%), in 38.7% of them in homozygosis. Main clinical and chemistry features did not significantly differ according to genetic status, but for a higher rate of ACE-inhibitors and beta-blockers use among the carriers of the G allele (p = 0.04 and p = 0.01, respectively).VDBP genetic status did not affect the rate of HRPR with ADP-antagonists (25.6% vs 24.6% vs 28.5%, p = 0.59; adjusted OR[95%CI] = 0.94[0.52–1.7], p = 0.83 for T/G patients; adjusted OR[95%CI] = 1.14[0.6–2.2], p = 0.67 for G homozygotes).However, the rate of HRPR with ADP-antagonists was influenced by severe hypovitaminosis D (< 10 ng/ml) only in patients carrying the G allele, especially in homozygosis (T/T: 25.9% vs 26.1%, p = 0.99; G carriers: 22.1% vs 35.3%, p = 0.02, p_interaction = 0.019; adjusted OR[95%CI] = 1.93[1.11–3.34], p = 0.02 for G carriers).ConclusionThe present study shows that rs7041 polymorphism of Vitamin D Binding Protein does not affect platelet reactivity or the rate of HRPR among patients receiving DAPT. However the carriage of the G allele could condition the impact of hypovitaminosis D on the response to antiplatelet agents, increasing the occurrence of HRPR especially in homozygotes, thus suggesting a more significant role of vitamin D deficiency among these patients.