Aberrant NKG2D expression with IL-17 production of CD4+ T subsets in patients with type 2 diabetes

Type 2 diabetes (T2D) is a systemic inflammatory disease. Although the natural killer group 2, member D (NKG2D) receptor, was not expressed normally on CD4+ T cells, the aberrant expression was found in pathological conditions such as in auto-immune diseases. However, the involvement of NKG2D in pathogenesis of T2D is unclear. We hypothesize that there is an inflammatory CD4+ T cell subpopulation expressing NKG2D and producing interleukin (IL)-17 in T2D. NKG2D expression on CD4+ T cells and their subsets were analyzed by multi-color staining using flow cytometry. Lymphocytes were activated by phorbol-12-myristate-13-acetate (PMA) and ionomycin, and were stained for intracellular IL-17. To investigate the mechanism of IL-17 production, patients’ lymphocytes were stimulated using specific anti-T cell receptor (TCR) alone, anti-NKG2D alone or a combination of the two antibodies. CD4+ T cells and particularly, CD4 + CD28^null T subset of T2D patients were highly expressed NKG2D and more prevalent compared to non-diabetic individuals (ND) (P = 0.039 and P = 0.022, respectively). Significantly higher percentages of CD4 + CD28^nullNKG2D + T cells of patients produced IL-17 when compared to those of ND (P = 0.024) and were positively correlated with the level of glycated hemoglobin A1c (HbA1c) (R² = 0.386, P = 0.041). Additionally, this cell population could be stimulated by specific monoclonal anti-NKG2D to produce IL-17. In conclusion, CD4 + CD28^nullNKG2D+ T cells were expanded in T2D, especially in patients with poor glycemic control. NKG2D may be one of the surrogate co-stimulatory receptors leading to irregular inflammatory function producing IL-17. An IL-17 producing CD4 + CD28^nullNKG2D + T cells may potentially be involved in pathogenesis and drive severity of the disease with the glycemic dependence. This particular cell type could be targeted for prognostic or therapeutic purposes.     

Effectiveness and safety of two 18-gauge needle types on native and allograft renal biopsies

Percutaneous biopsy is a key diagnostic tool for both native and allograft kidney diseases. Adequacy criteria vary, but at a minimum, a biopsy should allow the pathologist to reach a diagnosis and provide prognostic information such as the degree of interstitial fibrosis and tubular atrophy (IF/TA) and percentage of glomerulosclerosis. Whereas most studies use glomerular counts as a surrogate for biopsy adequacy, the amount and preservation of tubulointerstitium is equally important, considering IF/TA is a major prognostic parameter for most medical renal diseases. Many studies have compared the diagnostic adequacy of different gauge needles; however few have investigated performance differences between same gauge needles. In this study, we retrospectively analyzed 235 renal biopsies performed at a single center in Canada over 2 years to compare the utilization, safety, diagnostic and prognostic performance of two 18-gauge needles in native and allograft kidney biopsies. We found no significant difference in needle utilization between native and allograft kidneys, or between trainees and staff radiologists. The total tissue yielded area, glomerular counts, percentage of inadequate biopsies and number of passes were similar; however the number of cases in which IF/TA evaluation was deemed not possible was higher for biopsies using disposable instrument needles (4.3% vs. 0%; p = 0.01). These also showed greater number of tissue fragments (median 4 for reusable vs 3 for disposable; p = 0.04). We postulate that the increased tissue fragmentation might have impaired the pathologists ability to accurately assess interstitial fibrosis and tubular atrophy in biopsies obtained with the disposable instrument needles.     

Natural killer cell subsets and receptor expression in peripheral blood mononuclear cells of a healthy Korean population: Reference range,influence of age and sex,and correlation between NK cell receptors and cytotoxicity

Background

The purpose of this study was to identify CD56^bright and CD56^dim natural killer (NK) cell subsets and analyze their receptors expression in a healthy Korean population, and to determine whether receptor expression correlates with age, sex, and cytotoxicity.

Relevance of HLA-G,HLA-E and IL-10 expression in lip carcinogenesis

HLA-G, HLA-E and IL-10 are molecules which can provide tumor immunosuppression as well as the capacity of evasion to the immune system host. This study set out to evaluate HLA-G, HLA-E and IL-10 expression in lip squamous cell carcinoma (LSCC) and in a potentially malignant disorder (actinic cheilitis – AC), correlating the expression of these proteins with the degree of epithelial dysplasia. Immunohistochemistry was undertaken to identify HLA-G, HLA-E and IL-10 in samples from patients with LSCC (n = 20), AC (n = 30) and healthy lip mucosa (control) (n = 10). A semiquantitative scoring system was used for analysis. Differences between the groups were evaluated using the Pearson Chi-Squared test. The percentage of LSCC samples showing high immunoreactivity (IRS > 2) for HLA-G, HLA-E and IL-10 (neoplastic/epithelial cells) and HLA-E (stroma/connective tissue) was significantly higher that of the control (P < 0.05). A tendency for a progressive increase in the proteins analyzed was observed from the control to AC and to LSCC. The degree of dysplasia in the AC samples was not significantly associated with the proteins evaluated (P > 0.05). The high expression of HLA-G, HLA-E and IL-10 in AC and LSCC reflects the capacity that these pathologies have for evasion and progression.     

Patterns of systemic lupus erythematosus expression in Europe

ObjectivesTo analyse the differences in disease expression of European SLE patients based on gender, age at diagnosis, and ethnicity.MethodsA two-year, retrospective, multicentre, observational study was carried out in five countries (France, Germany, Italy, Spain and the UK). Patients' clinical manifestations including disease activity, organ involvement, organ damage and flares were analysed.ResultsThirty-one centres enrolled 412 consecutive eligible patients (90.5% of women), with active disease, stratified by disease severity (half severe and half non-severe).Baseline characteristics included; mean (SD) age: 43.3 (13.6) years, SLE duration: 10.7 (8.0) years and age at disease diagnosis: 32.6 (13.0) years old. The mean (SD) SELENA-SLEDAI and SLICC/ACR scores were: 8.1 (6.7) and 0.82 (1.36), respectively. Over half of patients experienced flares (54.9%). The average number of annual flares was 1.01 (0.71) flares/year.In males compared to females, the renal system was more frequently active (53.8% vs 30.0%, p = 0.002), the mean SLICC/ACR score was higher (1.15 vs 0.79, p = 0.039) and the pulmonary system was more likely to be damaged (12.8% vs 3.8%, p = 0.010). Furthermore, patients diagnosed at younger age displayed more renal system activity (young: 56.3% vs adult: 33.4% vs elder: 8.9%, p < 0.001) and renal damage (25.0% vs 6.9% vs 2.2%, p = 0.018) compared to the others. The annual number of flares (1.13 vs 1.05 vs 0.81 flares/year, p < 0.0001), including the occurrence of severe flares (0.58 vs 0.51 vs 0.20, p < 0.0001), was also higher in these patients.Conversely, greater organ damage was observed in patients diagnosed at an older age compared to the others. The mean SLICC/ACR score was higher (1.31 vs young: 0.88 and adult: 0.78, p < 0.001) in patients diagnosed in the older age groups. The pulmonary (13.3% vs younger: 0% vs adult: 3.7%, p = 0.030) and cardiovascular (17.8% vs younger: 0% vs adult: 2.9%, p < 0.001) systems were more frequently damaged in these patients.Black African descents showed greater disease activity compared to Caucasian patients. They flared more often (77.1% vs 48.6%, p = 0.001) and experienced a greater number of annual flares (1.57 vs 0.89 flares/year, p < 0.0001), mainly more severe flares (0.89 vs 0.38/year, p < 0.0001). They also were more likely to experience renal system damage.ConclusionThe study showed clearly two patient subsets. The disease was the most active in Black African descents, and this phenomenon has never been described before in continental Europe. The disease was also more active in patients diagnosed at a younger or adult. Greater disease damage was observed in males and in patients diagnosed at an older age.     

Nonclassical human leukocyte antigen (HLA-G,HLA-E,and HLA-F) in coronary artery disease

AimsSeveral evidences suggest the association between the evolution of coronary artery disease (CAD) and the development of coronary syndrome that is often associated with disrupted plaque and partial or complete thrombosis of the related artery. Because of the inflammatory nature of CAD, we investigated the human leukocyte antigen (HLA)-G, HLA-E, and HLA-F genetic polymorphisms within CAD patients and evaluated their potential association with this disease in Tunisian population.MethodsDifferent polymorphisms in HLA-G (14-bp Insertion/Deletion, +3142C/G), HLA-E (HLA-E*01:01/01:03 A/G), HLA-F (HLA-F*01:02 T/C, 01:03 C/T, 01:04 A/C) genes were typed using different laboratory techniques in a cohort of 89 CAD patients and 84 controls.ResultsA significant association was reported between the HLA-G +3142 G allele (OR = 1.64, 95% CI = 1.05–2.56, p = 0.02) and increased risk of CAD. No association was found for the other studied polymorphisms. When we considered the haplotypes, we found TDELCA and TDELGG haplotypes associated to CAD with p = 0.008 and p = 0.030, respectively, suggesting the potential interaction between HLA-G and HLA-E genes.ConclusionsOur findings indicated that the HLA-G +3142C/G polymorphism and TDELCA and TDELGG haplotypes can harbour a reliable diagnosis value for the risk of CAD development suggesting that HLA-G, -E and -F molecules might be involved in the pathogenesis of the disease. However, further studies are necessary to confirm our results.     

Long-term clinical outcomes following the use of synthetic hydroxyapatite and bone graft in impaction in revision hip arthroplasty

Impaction grafting using morsellised allograft bone restores bone stock, but carries the potential for transmission of infection. Synthetic bone graft substitutes can eliminate this risk but may, however, influence outcome. In this study we tested the hypothesis that a 50/50 mix of hydroxyapatite and allograft does not affect long-term function, survival or radiological outcome. Sixty-five patients had revision hip arthroplasty using impaction grafting with either pure allograft (42 patients) or a 50/50 mixture of allograft and solid particulate hydroxyapatite. Harris hip scores were assessed pre-operatively and annual intervals thereafter. Function was analyzed using multilevel modeling, the Kaplan–Meier method used for survival analysis and graft incorporation was assessed radiologically. The hip score improved in both groups but showed a small annual decline (average 1.2/year, p < 0.01). This decline was higher for females (average 3.4, p = 0.025) and significantly related to pre-op scores (p < 0.001). After adjusting for these, allograft patients had marginally higher scores (difference = 3.1, p = 0.3). The majority of revisions were for aseptic loosening. At 13 years survival in the allograft group was 84%, and 82% in the mixture group (p = 0.96, log rank test). Radiologically the graft incorporation was similar in both groups (p = 0.62). We conclude that long-term prosthesis survival and function following revision arthroplasty with a 50/50 mixture of allograft and hydroxyapatite are comparable to allograft alone.     

HLA-E-bound peptides influence recognition by inhibitory and triggering CD94/NKG2 receptors: preferential response to an HLA-G-derived nonamer

The HLA-E class Ib molecule constitutes a major ligand for the lectin-like CD94/NKG2 natural killer (NK) cell receptors. Specific HLA class I leader sequence-derived nonapeptides bind to endogenous HLA-E molecules in the HLA-defective cell line 721.221, inducing HLA-E surface expression, and promote CD94/NKG2A-mediated recognition. We compared the ability of NK clones which expressed either inhibitory or activating CD94/NKG2 receptors to recognize HLA-E molecules on the surface of 721.221 cells loaded with a panel of synthetic nonamers derived from the leader sequences of most HLA class I molecules. Our results support the notion that the primary structure of the HLA-E-bound peptides influences CD94/NKG2-mediated recognition, beyond their ability to stabilize surface HLA-E. Further, CD94/NKG2A⁺ NK clones appeared more sensitive to the interaction with most HLA-E-peptide complexes than did effector cells expressing the activating CD94/NKG2C receptor. However, a significant exception to this pattern was HLA-E loaded with the HLA-G-derived nonamer. This complex triggered cytotoxicity very efficiently over a wide range of peptide concentrations, suggesting that the HLA-E/G-nonamer complex interacts with the CD94/NKG2 triggering receptor with a significantly higher affinity. These results raise the possibility that CD94/NKG2-mediated recognition of HLA-E expressed on extravillous cytotrophoblasts plays an important role in maternal-fetal cellular interactions.     

Current outcomes of chronic active antibody mediated rejection – A large single center retrospective review using the updated BANFF 2013 criteria

BackgroundThe updated BANFF 2013 criteria has enabled a more standardized and complete serologic and histopathologic diagnosis of chronic active antibody mediated rejection (cAMR). Little data exists on the outcomes of cAMR since the initiation of this updated criteria.Methods123 consecutive patients with biopsy proven cAMR (BANFF 2013) between 2006 and 2012 were identified.ResultsPatients identified with cAMR were followed for a median of 9.5 (2.7–20.3) years after transplant and 4.3 (0–8.8) years after cAMR. Ninety-four (76%) recipients lost their grafts with a median survival of 1.9 years after diagnosis with cAMR. Mean C4d and allograft glomerulopathy scores were 2.6 ± 0.7 and 2.2 ± 0.8, respectively. 53.2% had class II DSA, 32.2% had both class I and II, and 14.5% had class I DSA only. Chronicity score >8 (HR 2.9, 95% CI 1–8.4, p = 0.05), DSA >2500 MFI (HR 2.8, 95% CI 1.1–6.8, p = 0.03), Scr >3 mg/dL (HR 3.2, 95% CI 1.6–6.3, p = 0.001) and UPC >1 g/g (HR 2.5, 95% CI 1.4–4.5, p = 0.003) were associated with a higher risk of graft loss.ConclusionscAMR was associated with poor graft survival after diagnosis. Improved therapies and earlier detection strategies are likely needed to improve outcomes of cAMR in kidney transplant recipients.     

Correlated analysis of semi-quantitative immunohistochemical features of E-cadherin,VEGF and CD105 in assessing malignant potentiality of oral submucous fibrosis

Oral submucous fibrosis, a potentially premalignant condition for oral squamous cell carcinoma, manifests both non-dysplastic and dysplastic grades. Early and specific identification of its malignant potentiality suffers from diagnostic limitations that may be addressed by correlated molecular pathology attributes having histopathological backdrop. Present study correlates expressional alteration in prime epithelial marker E-cadherin, with neo-angiogenic molecules viz. VEGF and CD105 for elucidation of malignant potentiality in different stages of oral submucous fibrosis. Sixty-eight incision biopsies from normal oral mucosa (n = 10), non-dysplastic (n = 18) and different dysplastic grades (n = 40) of oral submucous fibrosis were semi-quantitatively analyzed for immunohistochemical expressions of E-cadherin (membranous and cytoplasmic), VEGF and CD105 which were further statistically correlated. The loss of membranous E-cadherin with increase in cytoplasmic accumulation in differentiative layers of epithelium through the progression of dysplasia was noted along with up-regulation in VEGF expressions. The number of CD105⁺ blood vessels and their major axis also showed significant increase from non-dysplasia toward higher grades of dysplasia. The positive correlation between deregulated expression of epithelial cell–cell adhesion molecule and increase in neo-angiogenic attributes of oral submucous fibrosis with increase in dysplastic grades indicated elucidatory potential of molecular expression features in assessment of malignant potentiality in oral submucous fibrosis.     

Stronger Toll-like receptor 1/2, 4, and 7/8 but less 9 responses in peripheral blood mononuclear cells in non-infectious exacerbated asthmatic children

Toll-like receptors (TLR) initiate innate and often affect adaptive immune response. This study aimed to determine if TLR response and T regulatory cell (Treg) function in peripheral blood mononuclear cells (PBMC) correlate with clinical severity in non-infectious asthma. TLR1–9 expression and representative response cytokine TNF-α, IL-6, and IFN-β secretions were analyzed after stimulation by TLR1–9 ligands from 17 non-infectious asthmatic children. TNF-α production was higher in TLR1/2 (median 385.4 vs. 250.3 pg/ml in 1 μg/ml Pam3CSK4, p = 0.0078), TLR4 (2392.4 vs. 1355.9 in 1 μg/ml LPS; p = 0.0005), and TLR7/8 (10,776.2 vs. 4237.0 pg/ml in 1 μg/ml R848, p = 0.0079) of patients in exacerbation than those in convalescence and healthy controls despite equal TLR expression. TNF-α production stimulated by TLR9 agonist was significantly lower in exacerbation (17.7 vs. 34.9 pg/ml in 1 μg/ml ODN2216, p = 0.0175), while IL-6 production had similar patterns but was significantly lower in TLR3 signaling (119.7 vs. 245.0 pg/ml in 0.1 μg/ml poly(I:C), p = 0.0033). IFN-β production by TLR3 agonist also decreased in exacerbation but not statistically significant. Six older children showed decreased FOXP3 percentage in CD4 + CD25^high and decreased suppression capability in exacerbation but restored in stabilization (82.8% vs. 90.0%, p = 0.0061 and 60.9% vs. 81.7%, p = 0.0071; respectively). In conclusion, normalizing imbalanced TLR signaling and enhancing Treg cell capability may guide possible therapeutic strategies for non-infectious asthma in exacerbation.     

ABO incompatible renal transplants and decreased likelihood for developing immune responses to HLA and kidney self-antigens

Immune responses to HLA and tissue-restricted self-antigens (SAgs) have been proposed to play a role in the pathogenesis of renal allograft (KTx) rejection. However, ABO incompatible (ABOi) KTx recipients (KTxR) following depletion of antibodies (Abs) to blood group antigens had fewer rejections. To determine the mechanisms, pre- and post-transplant sera from ABOi (n = 18) and ABO-compatible (ABOc) (n = 45) KTxR were analyzed for Abs against HLA class I and II by LABScreen single antigen assay. The development of Abs to SAgs was measured by ELISA. Immunity to Collagen IV (Col-IV) and cytokines induced were measured by ELISPOT. While 8/45 (18%) ABOc KTxR developed new donor specific antibodies to HLA (DSA) following transplantation, 0/18 ABOi KTxR developed DSA. ABOi KTxR failed to develop Abs to kidney SAgs (Col-IV and fibronectin (FN)). In contrast, 7 ABOc KTxR developed Abs to both Col-IV and FN. Col-IV stimulation of lymphocytes from ABOc KTxR demonstrated increased IFNγ, IL-17 and decreased IL-10. In contrast ABOi recipients following stimulation with antigens resulted in more IL10 and reduced IFN-γ and IL17 production. At one year, the GFR in ABOi KTxR were significantly better (p < 0.04) than ABOc KTxR. De novo DSA and immune responses to SAgs are reduced or absent in ABOi KTxR which we propose leads to less acute rejection and better long term function following ABOi KTx.     

Stem cell markers OCT4 and nestin in laryngeal squamous cell carcinoma and their relation to survivin expression

We explored the expression of the stem cell markers OCT4 and nestin in laryngeal squamous cell carcinoma (LSCC) and investigated their relationship to survivin expression.Eighty-five LSCC, and 62 non-neoplastic laryngeal tissues were analyzed immunohistochemically for the presence of OCT4, nestin and survivin. Marker expression was correlated to clinicopathological parameters. The positive detection rates of OCT4 (42.35%) and nestin (51.76%) in LSCC were higher than those of non-neoplastic mucosa (p < 0.05). OCT4 expression was positively associated with nestin expression (p = 0.0001). High expression of both OCT4 and nestin was associated with higher tumor grade (p = 0.0001). Also, high OCT4 expression was related to higher T stage (p = 0.0001). Co-expression of OCT4 and nestin was more significantly associated with glottic location, higher T stage and nodal metastasis than high expression of either marker (p = 0.015, 0.006 and 0.008, respectively). Survivin expression was not significantly related to expression of OCT4 or nestin (p = 0.094 and 0.266, respectively).OCT4 and nestin are overexpressed in LSCC and may contribute to laryngeal carcinogenesis. Their co-expression may help to predict the lymph node metastatic potential of LSCC. No relationship was detected between expression of survivin and OCT4 or nestin.     

OR18 Identification of anti-endothelial non–HLA antibodies in kidney allograft rejection

Aim

The endothelium is the initial point of contact between the donor allograft and the transplant recipient’s immune system and therefore is a likely source for the identification of novel non-HLA Ab associated with rejection. The goal of this study was to identify novel anti-endothelial non-HLA Ab associated with kidney allograft rejection.

An evidence-based recommendation for the inclusion of specific local intrinsic factors in the study of knee osteoarthritis

ObjectiveAdequate characterization of the mechanical environment of the knee with osteoarthritis (OA) is important. These local intrinsic factors are difficult to measure and there is little evidence to guide their selection. This study makes an evidence-based recommendation for the inclusion of specific factors in the future study of knee OA.MethodForty-six subjects with knee OA were examined. Observed function was measured by the Timed Chair Rise (TCR). Self-reported function was measured by the WOMAC Function Scale and pain was measured by the WOMAC Pain Scale. Local intrinsic factors measured included varus/valgus alignment, anterior/posterior (A/P) laxity, proprioception, isometric knee extension (KE) strength, isometric knee flexion (KF) strength, and knee range of motion (ROM).ResultsFactors were recommended for inclusion in future research if they were significantly correlated with at least one measure of function or pain and if the factor made a significant unique contribution to a regression model when more than one local intrinsic factor was correlated with the same measure of function or pain. Alignment was correlated with pain (r = 0.48, p = 0.001) and WOMAC function (r = 0.38, p = 0.009). A/P laxity was correlated with pain (r = 0.30, p = 0.04) and WOMAC function (r = 0.37, p = 0.01). Knee ROM was correlated to WOMAC function (r = ? 0.35, p = 0.02). KE strength was correlated with TCR (r = 0.32, p = 0.03). Alignment made a significant contribution to prediction of pain (p = 0.003). A/P laxity (p = 0.004) and ROM (p = 0.008) made a significant contribution to WOMAC function.ConclusionWe recommend future knee OA studies include the variables varus/valgus alignment, A/P laxity, ROM, and KE strength.Level of EvidenceIII (correlational study).     

HLA-E regulates NKG2C+ natural killer cell function through presentation of a restricted peptide repertoire

NK cells interact with the HLA-E molecule via the inhibitory receptor NKG2A and the activating receptor NKG2C. Hence, HLA-E can have a dual role in the immune response. In the present study, we aim to investigate the functional consequences of HLA-E for NKG2A and NKG2C expressing NK cell subsets by using a panel of HLA-E binding peptides derived from CMV, Hsp60 and HLA class I. PBMC derived from healthy subjects were used as targets for isolated NK cells and NK cell activation was examined by analysis of the expression of the degranulation marker CD107a. Peptide induced HLA-E expression inhibited degranulation of NKG2A+ NK cell subsets with almost all peptides, whereas NKG2A− NKG2C+ NK cell responses were enhanced only after incubation with four peptides; 1.3-fold with CMV(I), A80 and B13 and 3.2-fold with HLA-G derived peptide. In addition, the HLA-E:G peptide complex triggered NKG2C receptor internalization, as evidenced by reduction in the percentage of NKG2C+ NK cells when incubated with the peptide, which could be restored by addition of Bafilomycin. In conclusion: in contrast to NKG2A, NKG2C is regulated by HLA-E only when HLA-E is in complex with a restricted peptide repertoire, especially in combination with the HLA-G leader peptide.     

Angiotensin II type-1 receptor antibody (AT1Rab) associated humoral rejection and the effect of peri operative plasma exchange and candesartan

Angiotensin II type 1 antibodies (AT1Rab) can mediate antibody mediated rejection (AMR). Pre transplant AT1Rab levels, and risk of rejection were assessed in Kidney Transplant Recipients (KTR) transplanted in our centre from 2013 to 2014 (n = 145). 14/145 (9.7%) KTR experienced antibody mediated rejection (AMR). The Hazard Ratio for AMR = 3.7 [95% CI 2–26] (p = 0.009) for KTR with AT1Rab levels >17.5 U/ml. 6/11 of KTR with levels >25 U/ml experienced AMR.In 2015 (n = 80) KTR were transplanted and 6/80 KTR experienced rejection (2 AMR and 4 TCMR with vascular lesions). 7/80 of KTR had AT1Rab 17.5–25 U/ml and none experienced rejection and were induced with ATG and candesartan. 7/80 had AT1Rab 25–40 U/ml and received pre and post-operative plasma exchange, ATG and candesartan and 1/7 experienced TCMR with a vascular lesion.This perioperative regimen may alter the risk of rejection in patients with high levels of AT1Ab and further studies are needed.     

Human T cell receptor-mediated recognition of HLA-E

The HLA-E class Ib molecule presents hydrophobic peptides derived from the leader sequences of other class I molecules, constituting the ligands for CD94/NKG2 lectin-like receptors. Along the course of our studies on human CD94+ T cells, we characterized an alpha beta CD8+CD94/NKG2C+ CTL clone (K14). In cytolytic assays against the murine TAP-deficient RMA-S cells transfected with human beta2 microglobulin and HLA-E (RMA-S/HLA-E), loaded with different synthetic peptides, K14 displayed a pattern of specific recognition distinct to that observed in CD94/NKG2C+ NK clones tested in parallel. RMA-S/HLA-E cells loaded with some but not all HLA class I leader sequence peptides were efficiently recognized by K14 but not by CD94/NKG2C clones, andvice versa. Remarkably, K14 also reacted with HLA-E loaded with a peptide derived from the BZLF-1 Epstein-Barr virus protein. Anti-CD94 mAb did not prevent K14 cytotoxicity against RMA-S/HLA-E cells, whereas incubation with anti-clonotypic mAb specific for the K14 TCR markedly inhibited lysis. Soluble HLA-E tetramers refolded with different peptides (i.e. VMAPRTVLL, VMAPRTLIL, VMAPRTLFL) specifically stained K14 cells. HLA-E tetramer binding was minimally reduced by pretreatment with anti-CD94 mAb alone, but was completely prevented in combination with anti-clonotypic mAb. Altogether, the data unequivocally imply the generation of human T cells potentially recognizing through the alpha beta TCR HLA-E molecules that bind to class I- and virus-derived peptides.     

Effect of lentivirus-mediated gene silencing,targeting toll-like receptor 2, on corneal allograft transplantation in rats

AimThe present work aims to assess the effectiveness of lentiviral vector (LV) mediated Toll-like receptor 2 (TLR2) gene silencing in the survival of transplanted corneal allografts, against immune rejection, in rats.MethodsLV mediated TLR2 small interference RNA (SiRNA) with enhanced green fluorescent protein (eGFP) [LV-TLR2-siRNA-eGFP] was realised and transfected to both rat corneal epithelial (EC) and stromal cells (SC). Multiplicity of infection (MOI) was optimized for transfection efficiency using flow cytometric (FCM) analysis. Viability of transfected cells and the success rate of TLR2 gene silencing were respectively determined by CCK-8 assay and western blot assay. The in-vivo experiments were subjected to a penetrating keratoplasty (PK) performed between host Sprague Dawley (SD) and donor Wistar/SD rats, randomly dividing them into 4 groups including the allograft, isograft, allograft treated with LV-eGFP (LV blank control) and allograft treated with LV-TLR2-siRNA-eGFP (LV treated group). The rejection index (RI) was then recorded under a slit lamp, every day following surgery. Expression of the TLR2 and Myeloid differentiation primary response gene 88 (MyD88) were detected by using immunohistochemistry on day 9 post-surgery, whereas grafts’s TLR2 and MyD88 mRNA were determined on day 5, 9, and 14 post-surgery performing RT-PCR and, normal rat corneas were included as additional controls.ResultsTransfected cells showed the strongest eGFP expression when MOI was 200 with an efficiency of 77.5% for EC and 76.3% for SC. CCK-8 assay, as measured at 72 and 96 h post transfection, showed no significant changes in the cell viability (both EC and SC) between the transfected and the control group (p > 0.05, p > 0.05). Western blot results demonstrated a successful down regulation of TLR2 expression by LV-TLR2-SiRNA-eGFP, in both EC and SC. In vivo, compared to allograft group, LV treated group demonstrated less edema, opacity and neovascularization. Immunohistochemical analysis revealed a lower expression of TLR2 and MyD88 in isograft and LV treated group as compared to allograft group. TLR2 and MyD88 mRNA were detected in all grafts, and increased over time. With its highest expression in allograft group (peak on day 9), the mRNA expression for TLR2 and MyD88 showed a significant difference amongst the groups, on both day 9 and 14 (p < 0.001).ConclusionsLV mediated TLR2 siRNA could effectively down regulate the TLR2 expression via RNA interference and prolong the survival of corneal grafts, although not necessarily able to prevent the rejection.     

In-stent restenosis is associated with neointimal angiogenesis and macrophage infiltrates

Restenosis after stenting occurs secondary to the neointima formation. Neovessels have been found in the neointima within stents. However, there are few studies correlating neointimal angiogenesis and in-stent restenosis in humans. We analyzed 65 post-mortem stented arteries from 33 patients with duration >3 months. Cause of death was determined incidental to the coronary findings in every case. Stented segments were embedded in paraffin and stained immunohistochemically for CD68 (macrophages), and endothelial marker PECAM-1 (CD31). Computerized morphometry was performed to quantitate neovessel density for CD31, macrophage infiltrates, as well as plaque and neointimal area. In-stent restenosis was defined as luminal narrowing ≥75% cross-section of the stented area. Underlying plaque morphology was classified as fibrous or atheromatous. Neovessels were present in the neointima of 57 stented segments (88%). Mean neovessel density was greater in restenotic vs. non-restenotic neointimas (p = 0.009) and macrophage density was also greater (p = 0.006). Neointimal area correlated positively with density of neointimal vessels (p = 0.002), as well as neointimal macrophage density (p = 0.006), but not type of stent, underlying plaque type, or underlying plaque macrophage score. We conclude that in-stent restenosis is associated with neointimal angiogenesis which is accompanied by macrophage inflammation. The relevance of these findings to treatment and prevention of in-stent restenosis needs to be further explored.