Investigation of IL-35 and IL-39, New Members of the IL-12 Family, in Different Clinical Presentations of Brucellosis.

Brucellosis is significantly influenced by the interactions between the causative Brucella bacteria and host immunity. Recently identified cytokines have been described for their immunomodulatory effects in numerous inflammatory, autoimmune and infectious diseases. Some of them are new members of cytokine superfamilies, including several members of the IL-12 superfamily (IL-35, IL-39). The major purpose of the present study was to investigate the role of these new immunomodulatory cytokines in Brucella infections. The levels of IL-35 and IL-39 in the serum of 40 acute and 40 chronic brucellosis patients and 40 healthy controls were measured by ELISA. The mRNA levels of IL-35 and IL-39 in PBMCs were detected by RT-qPCR. Both IL-35 and IL-39 serum concentrations were significantly higher in healthy control subjects than in brucellosis patients, and IL-35 and IL-39 serum levels of chronic brucellosis patients were higher than those of acute cases. It was also found that the expression of Ebi3/IL-12A (IL-35 genes) and Ebi3/IL-23A (IL-39 genes) was upregulated in chronic brucellosis patients compared to healthy controls. Moreover, the expression of the Ebi3/IL-12A and Ebi3/IL-23A genes was lower in patients with acute brucellosis than in patients with chronic brucellosis. Overall, this study showed that IL-35 and IL-39 are positively correlated in brucellosis and significantly decreased during the disease. Significantly lower levels of IL-35 and IL-39 in acute brucellosis than in chronic brucellosis and healthy controls suggest that these cytokines may play a key role in suppressing the immune response to brucellosis and its progression to chronicity.

IL-35 and IL-39, new members of the IL-12 cytokine family, are immunomodulatory cytokines characterized as anti-inflammatory and pro-inflammatory, respectively.In acute and chronic brucellosis, serum IL-35 and IL-39 are significantly decreased.In acute brucellosis, serum IL-35 are significantly lower than in chronic brucellosis, suggesting that this cytokine may play a role in chronification.A positive correlation was found between IL-35 and IL-39 in acute and chronic brucellosis, suggesting that the common protein subunit Ebi may be suppressed.According to the results of this study, IL-35 and IL-39 may play a role in the pathogenesis of brucellosis.

Assessment of CD39 expression in regulatory T-cell subsets by disease severity in adult and juvenile COVID-19 cases.

COVID-19 is a disease characterized by acute respiratory failure and is a major health problem worldwide. Here, we aimed to investigate the role of CD39 expression in Treg cell subsets in COVID-19 immunopathogenesis and its relationship to disease severity. One hundred and ninety COVID-19 patients (juveniles, adults) and 43 volunteers as healthy controls were enrolled in our study. Flow cytometric analysis was performed using a 10-color monoclonal antibody panel from peripheral blood samples. In adult patients, CD39+ Tregs increased with disease severity. In contrast, CD39+ Tregs were decreased in juvenile patients in an age-dependent manner. Overall, our study reveals an interesting profile of CD39-expressing Tregs in adult and juvenile cases of COVID-19. Our results provide a better understanding of the possible role of Tregs in the mechanism of immune response in COVID-19 cases.

Effects of DNA Methylation on Progression to Interstitial Fibrosis and Tubular Atrophy in Renal Allograft Biopsies: A Multi-Omics Approach.

Progressive fibrosis of the interstitium is the dominant final pathway in renal destruction in native and transplanted kidneys. Over time, the continuum of molecular events following immunological and nonimmunological insults lead to interstitial fibrosis and tubular atrophy and culminate in kidney failure. We hypothesize that these insults trigger changes in DNA methylation (DNAm) patterns, which in turn could exacerbate injury and slow down the regeneration processes, leading to fibrosis development and graft dysfunction. Herein, we analyzed biopsy samples from kidney allografts collected 24 months posttransplantation and used an integrative multi-omics approach to understand the underlying molecular mechanisms. The role of DNAm and microRNAs on the graft gene expression was evaluated. Enrichment analyses of differentially methylated CpG sites were performed using GenomeRunner. CpGs were strongly enriched in regions that were variably methylated among tissues, implying high tissue specificity in their regulatory impact. Corresponding to this methylation pattern, gene expression data were related to immune response (activated state) and nephrogenesis (inhibited state). Preimplantation biopsies showed similar DNAm patterns to normal allograft biopsies at 2 years posttransplantation. Our findings demonstrate for the first time a relationship among epigenetic modifications and development of interstitial fibrosis, graft function, and inter-individual variation on long-term outcomes.

Real Time Central Assessment of Kidney Transplant Indication Biopsies by Microarrays: The INTERCOMEX Study.

The authors conducted a prospective trial to assess the feasibility of real time central molecular assessment of kidney transplant biopsy samples from 10 North American or European centers. Biopsy samples taken 1 day to 34 years posttransplantation were stabilized in RNAlater, sent via courier overnight at ambient temperature to the central laboratory, and processed (29 h workflow) using microarrays to assess T cell- and antibody-mediated rejection (TCMR and ABMR, respectively). Of 538 biopsy samples submitted, 519 (96%) were sufficient for microarray analysis (average length, 3 mm). Automated reports were generated without knowledge of histology and HLA antibody, with diagnoses assigned based on Molecular Microscope Diagnostic System (MMDx) classifier algorithms and signed out by one observer. Agreement between MMDx and histology (balanced accuracy) was 77% for TCMR, 77% for ABMR, and 76% for no rejection. A classification tree derived to provide automated sign-outs predicted the observer sign-outs with >90% accuracy. In 451 biopsy samples where feedback was obtained, clinicians indicated that MMDx more frequently agreed with clinical judgment (87%) than did histology (80%) (p = 0.0042). In 81% of feedback forms, clinicians reported that MMDx increased confidence in management compared with conventional assessment alone. The authors conclude that real time central molecular assessment is feasible and offers a useful new dimension in biopsy interpretation. ClinicalTrials.gov NCT#01299168.

The Banff 2015 Kidney Meeting Report: Current Challenges in Rejection Classification and Prospects for Adopting Molecular Pathology.

The XIII Banff meeting, held in conjunction the Canadian Society of Transplantation in Vancouver, Canada, reviewed the clinical impact of updates of C4d-negative antibody-mediated rejection (ABMR) from the 2013 meeting, reports from active Banff Working Groups, the relationships of donor-specific antibody tests (anti-HLA and non-HLA) with transplant histopathology, and questions of molecular transplant diagnostics. The use of transcriptome gene sets, their resultant diagnostic classifiers, or common key genes to supplement the diagnosis and classification of rejection requires further consensus agreement and validation in biopsies. Newly introduced concepts include the i-IFTA score, comprising inflammation within areas of fibrosis and atrophy and acceptance of transplant arteriolopathy within the descriptions of chronic active T cell-mediated rejection (TCMR) or chronic ABMR. The pattern of mixed TCMR and ABMR was increasingly recognized. This report also includes improved definitions of TCMR and ABMR in pancreas transplants with specification of vascular lesions and prospects for defining a vascularized composite allograft rejection classification. The goal of the Banff process is ongoing integration of advances in histologic, serologic, and molecular diagnostic techniques to produce a consensus-based reporting system that offers precise composite scores, accurate routine diagnostics, and applicability to next-generation clinical trials.

Learning curves of robotic technology in an orthopedic teaching hospital.

BACKGROUND: In recent years there has been an increasing implementation of robotic technology in arthroplasty. Due to the unclear data situation the aim of this study was to analyze the learning curve for robotic technology in residency training. METHODS: After its introduction, the first 351 consecutive robotic knee replacements were prospectively included in the study. Surgical times, preoperative and postoperative radiographs, intraoperatively recorded alignment data and complications were analyzed. Satisfaction, revision, and referral rates were determined in a 90-day follow-up survey. Data from the last 350 navigated total knee arthroplasties were analyzed as a historical control group. RESULTS: A learning curve of between 3 and 53 procedures was identified, depending on the surgeon, with further reductions in time measured even after 1 year of use. The operative times of the navigated technique were achieved by all surgeons. With respect to precision (alignment outliers) and patient satisfaction rate, no learning curve was evident. Comparison between tutorial and non-tutorial surgery showed a 16-min increase in operating time, but no significant differences in precision, complications, and patient satisfaction rate. CONCLUSION: The study showed that there was a learning curve in terms of duration of surgery but not in terms of precision, complications, and patient satisfaction. Robotic tutorial surgery requires more time but provides the same outcome compared to experienced surgeons. Thus, the robotic surgical technique appears to be an excellent training tool in knee arthroplasty.

Risk factors for developing upper limb cellulitis after breast cancer treatment.

Cellulitis is one of the most important troubling complications of breast cancer treatment. Therefore, elucidating the risk factors for cellulitis in patients that have undergone breast cancer treatment is crucial. This is a retrospective medical record study among 523 patients who had received breast cancer treatment and were referred to the Lymphedema Clinic. Data on age, height, weight, BMI (body mass index), education level, arm dominance, history of previous surgery, axillary lymph node dissection, radiotherapy, and chemotherapy were noted. The time between operation and onset of lymphedema, duration of lymphedema, history of cellulitis, and number of cellulitis attacks were recorded. Circumference measurements were taken at four points on the upper limb. Univariate analysis showed that longer duration of lymphedema, larger circumference of the unaffected arm and larger circumference of the arm with lymphedema were associated with higher risk of cellulitis (p=0.008, p=0.007, p< 0.001, respectively). The incidence of cellulitis was higher in patients with lymphedema than patients who had no lymphedema (p< 0.001). Moreover, the frequency of cellulitis was higher in patients with lower education level (p=0.015). It was deter-mined that patients with cellulitis needed more compression garments (p< 0.001) and multi-layered bandage therapy (p< 0.001) than those without. Regression analysis revealed that presence of lymphedema (p=0.036), duration of lymphedema (p=0.048), radiotherapy (p=0.01) and educational level (0.019) are significantly associated with developing upper extremity cellulitis. It is important to consider these risk factors for the prevention and management of cellulitis in patients who undergo treatment for breast cancer. Early detection and treatment of lymphedema also remains essential for these patients.

The Age-Dependent Role of Th22, Tc22, and Tc17 Cells in the Severity of Pneumonia in COVID-19 Immunopathogenesis.

Coronavirus disease 2019 (COVID-19) has clinical manifestations ranging from mild symptoms to respiratory failure, septic shock, and multi-organ failure. Lymphocytes are divided into different subtypes based on their cytokine production pattern. In this study, we investigated the role of cytokine expressions of CD4+ T (T helper [Th]1, Th2, Th17, Th22) and CD8+ T cell subtypes (T cytotoxic [Tc]1, Tc2, Tc17, Tc22) in the pathogenesis of COVID-19. Peripheral blood mononuclear cells (PBMCs) were extracted with Ficoll by density gradient centrifugation from blood samples of 180 COVID-19 patients (children and adults) and 30 healthy controls. PBMCs were stimulated with PMA and Ionomycin and treated with Brefeldin A in the fourth hour, and a 10-colored monoclonal antibody panel was evaluated at the end of the sixth hour using flow cytometry. According to our findings, the numbers of Th22 (CD3+, CD4+, and interleukin [IL]-22+) and Tc22 (CD3+, CD8+, IL-22+) cells increased in adult patients regardless of the level of pneumonia (mild, severe, or symptom-free) as compared with healthy controls (p < 0.05). In addition, the number of Tc17 (CD3+, CD8+, and IL-17A+) cells increased in low pneumonia and severe pneumonia groups compared with the healthy controls (p < 0.05). Both IL-22 and IL-17A production decreased during a follow-up within 6 weeks of discharge. Our findings suggest that the increase in only IL-22 expressed Tc22 cells in the 0-12 age group with a general symptom-free course and higher levels of Th22 and Tc22 in uncomplicated adult cases may indicate the protective effect of IL-22. On the contrary, the association between the severity of pneumonia and the elevation of Tc17 cells in adults may reveal the damaging effect of IL-22 when it is co-expressed with IL-17.

CD28-B7 blockade after alloantigenic challenge in vivo inhibits Th1 cytokines but spares Th2.

Blocking the CD28-B7 T cell costimulatory pathway with the fusion protein CTLA4Ig inhibits alloimmune responses in vitro and in vivo and induces tolerance to cardiac allografts in mice and rats, but the mechanisms mediating the tolerant state in vivo are unknown. Here, we report the effects and potential mechanisms of CTLA4Ig in the rat renal allograft model. LEW rats were nephrectomized and received renal allografts from major histocompatibility complex-incompatible WF rats. While all untreated and control immunoglobulin (Ig)-treated animals acutely rejected their allografts and died, 86% of rats that received a single injection of CTLA4Ig on day 2 after transplantation had prolonged survival (> 60-100 days) with preserved renal function. By contrast, only 29% of animals that received CTLA4Ig on the day of engraftment had prolonged survival. Long-term survivors (> 100 days) exhibited donor-specific tolerance, accepting donor-matched WF but acutely rejecting third-party BN cardiac allografts. Immunohistological analysis of grafts sampled at 1 week after transplantation showed that both control and CTLA4Ig-treated animals had mononuclear cell infiltrates, with a higher percentage of CD4+ cells in the CTLA4Ig-treated group. However, while this was associated with vasculitis and tubulitis in control grafts, there was no evidence of tissue injury in CTLA4Ig-treated animals. The immune response leading to graft rejection in control animals was characterized by expression of the T helper (Th) type 1 cytokines interleukin (IL)-2 and interferon-gamma. In contrast, the persistent CD4+ infiltrate without graft rejection in CTLA4Ig-treated animals was associated with increased staining for the Th2-related cytokines IL-4 and IL-10. Furthermore, grafts from CTLA4Ig-treated animals had marked upregulation of intragraft staining for IgG1, but not IgG2a or IgG2b. Administration of rIL-2 to CTLA4Ig-treated animals restored allograft rejection in 50% of animals tested. These results confirm that blockade of the CD28-B7 pathway after alloantigenic challenge induces donor-specific acceptance of vascularized organ allografts, and indicates in this model that CTLA4Ig inhibits Th1 but spares Th2 cytokines in vivo.

Factors associated with failure to list HIV-positive kidney transplant candidates.

With improved survival in the antiretroviral era, data from ongoing studies suggest that HIV patients can be safely transplanted. The disproportionate burden of HIV-related end-stage renal disease in minority populations may impose additional obstacles to successful completion of the transplant evaluation. We retrospectively reviewed 309 potentially eligible HIV patients evaluated for kidney transplant at our institution since 2000. Only 20% of HIV patients have been listed, compared to 73% of HIV-negative patients evaluated over the same period (p < 0.00001). Failure to provide documentation of CD4 and viral load (36% of candidates) was the most common reason for failure to progress beyond initial evaluation. Other factors independently associated with failure to complete the evaluation included CD4 < 200 at initial evaluation (OR 15.17; 95% CI 1.94-118.83), black race (OR 2.33; 95% CI 1.07-5.06), and history of drug use (OR 2.56; 95% CI 1.22-5.37). More efficient medical record sharing and an awareness of factors associated with failure to list HIV-positive transplant candidates may enable transplant centers to more effectively advocate for these patients.

The burden of chronic kidney disease in long-term liver transplant recipients.

BACKGROUND: Chronic kidney disease (CKD) is an important risk factor for morbidity and mortality post-liver transplantation (OLT). This study focused on investigating the incidence and risk factors associated with the development CKD after OLT. METHODS: We performed a retrospective cohort study of recipients followed at least 5 years at our institution. CKD was diagnosed and classified according to National Kidney Foundation and the Kidney Disease Outcomes Quality Initiative guidelines. RESULTS: There were 231 patients, 64% men, 67% Caucasian, 16% African-American, and 17% others, with a mean age of 56 +/- 13 years. The mean glomerular filtration rate (GFR) of the population was 56 +/- 28 mL/min/1.73 m2. CKD was defined as GFR less than 60 mL/min; 144 patients (61%) were identified as having CKD. When these patients were compared to the non-CKD group, the former were significantly older (62 +/- 9 vs 52 +/- 12 years, P = .03), more likely to be hypertensive (59% vs 38%, P = .003), and required more antihypertensive medications (0.83 +/- 0.81 vs 0.52 +/- 0.77, P = .02); 26% of all patients had diabetes. However, the incidence of diabetes (43.3% vs 19.3%, P = .02) as well as the incidence of insulin dependency (21.6% vs 12.5%, P = .001) was significantly higher in the CKD population. Mean uric acid levels were higher in CKD patients compared to non-CKD patients (8.00 +/- 2.00 mg/dL vs 6.70 +/- 1.99 mg/dL respectively, P = .001); patients with uric acid more than 6.0 had a 1.7 risk of having CKD. CONCLUSIONS: CKD defined as GFR < 60 mL/min is highly prevalent in long-term OLT survivors. Older age, elevated systolic blood pressure, insulin-dependent diabetes mellitus, and elevated uric acid levels are independently associated with CKD.

Chronic cardiac rejection in the LEW to F344 rat model. Blockade of CD28-B7 costimulation by CTLA4Ig modulates T cell and macrophage activation and attenuates arteriosclerosis.

CTLA4Ig, a fusion protein that blocks CD28-B7 costimulation, was studied in a LEW to F344 rat model of chronic cardiac rejection. In rats treated with a single dose of CTLA4Ig (0.5 mg intraperitoneally) 2 d after transplantation, allografts survived significantly longer ( > 70 d in 64%) than in untreated controls or rats treated with control Ig (all rejected within 25 d). Only 25% of grafts from rats treated with a single, high dose of cyclosporine A (25 mg/kg, 2 d after transplantation) survived longer than 70 d. Reverse transcriptase PCR and immunostaining analyses of tissue from 75-d, CTLA4Ig-treated allografts showed reduced expression of the T cell factor IFN-gamma and macrophage activation factors monocyte chemoattractant protein-1, inducible nitric oxide synthase, and galactose/N-acetylgalactosamine macrophage lectin, as well as TGF-beta. Grafts from longterm survivors ( > 120 d) treated with CTLA4Ig showed significant reductions in the frequency and severity of arteriosclerosis in comparison with cyclosporine A-treated rats. Thus, T cell activation is a proximal event in the cascade that culminates in the arteriosclerosis of chronic rejection. Strategies for blocking T cell costimulation may help prevent chronic rejection in clinical transplantation.

Gene polymorphisms and transplantation.

The influence of gene polymorphisms in key immunoregulatory molecules on the clinical course post-transplant has become an area of active research, since it offers a possible explanation for the heterogeneity in outcomes between individuals. Several groups have now investigated the association of polymorphisms in molecules--including cytokines, cytokine receptors, adhesion molecules and costimulatory molecules--that participate in the immune response to an allograft. Several interesting observations have been made that would suggest that genetic variability influencing allograft survival reaches beyond that of the MHC molecules.

MicroRNA Expression Patterns of CD8+ T Cells in Acute and Chronic Brucellosis.

Although our knowledge about Brucella virulence factors and the host response increase rapidly, the mechanisms of immune evasion by the pathogen and causes of chronic disease are still unknown. Here, we aimed to investigate the immunological factors which belong to CD8+ T cells and their roles in the transition of brucellosis from acute to chronic infection. Using miRNA microarray, more than 2000 miRNAs were screened in CD8+ T cells of patients with acute or chronic brucellosis and healthy controls that were sorted from peripheral blood with flow cytometry and validated through qRT-PCR. Findings were evaluated using GeneSpring GX (Agilent) 13.0 software and KEGG pathway analysis. Expression of two miRNAs were determined to display a significant fold change in chronic group when compared with acute or control groups. Both miRNAs (miR-126-5p and miR-4753-3p) were decreased (p <0.05 or fold change > 2). These miRNAs have the potential to be the regulators of CD8+ T cell-related marker genes for chronic brucellosis infections. The differentially expressed miRNAs and their predicted target genes are involved in MAPK signaling pathway, cytokine-cytokine receptor interactions, endocytosis, regulation of actin cytoskeleton, and focal adhesion indicating their potential roles in chronic brucellosis and its progression. It is the first study of miRNA expression analysis of human CD8+ T cells to clarify the mechanism of inveteracy in brucellosis.

Emphysematous cystitis and pyelitis in a diabetic renal transplant recipient.

Emphysematous cystitis is a rare complication of urinary tract infection. Patients with diabetes mellitus, neurogenic bladder, bladder outlet obstruction, and recurrent urinary tract infection are at increased risk for the disease. We present a case of emphysematous cystitis and pyelitis in a diabetic renal transplant recipient. He was treated with antibiotics alone with complete clinical and radiologic resolution. The clinical course was benign, as described in most patients. The prognosis of emphysematous cystitis is good after early diagnosis and prompt treatment with appropriate antibiotics, blood glucose control, and adequate urinary drainage.

The indirect pathway of allorecognition. The occurrence of self-restricted T cell recognition of allo-MHC peptides early in acute renal allograft rejection and its inhibition by conventional immunosuppression.

There is evidence that T cells can "directly" recognize intact allo-MHC molecules on the surface of allogeneic stimulator or target cells, and/or "indirectly" recognize processed allo-MHC peptides presented by self antigen-presenting cells (APCs). We and others have recently demonstrated that in vivo-primed rat CD4+ T cells recognize and proliferate to specific polymorphic amino acid sequences when presented as MHC allopeptides by self APCs. Studies on the mechanisms of indirect T cell recognition of alloantigen are now reported. First, we studied the immunogenicity of 4 synthetic polymorphic class II MHC allopeptides representing full-length sequences of the hypervariable domains of RT1.Du beta (DR or I-E-like) in several responder strains: LEW (RT1(l)), ACI (RT1a), BUF (RT1b), BN (RT1n), and control syngeneic WF (RT1u) strains. Immunogenicity of the individual 25mer allopeptides varied in the different responder strains, indicating that self-restricted T cell recognition of allo-MHC peptides is determined not only by polymorphisms, but also by the responder MHC genotype. Self-restricted CD4+ T cell recognition of processed allo-MHC peptides has been shown to occur during acute skin and cardiac allograft rejection, and there is evidence that this pathway may play an important role in initiating and amplifying the immune response to allografts. T cells from LEW animals primed in vivo by WF (RT1u) vascularized renal allografts were capable of proliferating to the RT1.Du beta peptides as early as 3 days postengraftment, when presented by self APCs. We then tested the effects of various immunosuppressive drugs on self-restricted primed T cell proliferative response to an immunogenic MHC allopeptide in vitro. Methylprednisolone, cyclosporine, and FK506 inhibited the proliferative response of RT1.Du beta 2-primed LEW T cells in a dose-dependent fashion. In addition, a single injection of cyclosporine (25 mg/kg i.m.) to LEW recipients of WF renal allografts on the day of transplantation completely abolished the proliferative response of in vivo-primed T cells to RT1.Du beta 2, indicating the susceptibility of the indirect pathway of allorecognition to conventional immunosuppressive drugs.

CD28-B7 T cell costimulatory blockade by CTLA4Ig in the rat renal allograft model: inhibition of cell-mediated and humoral immune responses in vivo.

Blocking CD28-B7 T-cell costimulation by CTLA4Ig induces tolerance to rat renal allografts and inhibits Th1, but spares Th2, cytokines. We now report on the mechanisms of CD28-B7 blockade in this model. Lymphocytes from CTLA4Ig-treated animals showed significant reduction of mixed lymphocyte response, as well as antidonor cytotoxic T-cell effector function, as compared with rejecting controls. Flow cytometry studies on sera of renal allograft recipients showed complete inhibition of antidonor humoral responses by CTLA4Ig. Analysis by reverse transcriptase-polymerase chain reaction and immunohistology showed that intragraft macrophage products, monocyte chemoattractant protein-1 and inducible nitric oxide synthase, were reduced by CTLA4Ig therapy. Immunohistologic studies also showed reduced intragraft macrophage infiltration and decreased staining for the fibrogenic and mitogenic growth factor, transforming growth factor-beta. These results indicate that CD28-B7 blockade inhibits cell-mediated and humoral immune responses, and suggest that strategies targeting T-cell costimulation may provide a novel approach to prevent chronic allograft rejection.

Intrathymic tolerance in the Lewis-to-F344 chronic cardiac allograft rejection model.

Successful induction of donor-specific unresponsiveness by intrathymic inoculation of alloantigen in several experimental acute rejection models has led us to hypothesize that similar immune manipulations can prevent chronic rejection and development of graft arteriosclerosis in the Lewis-to-F344 rat chronic cardiac allograft rejection model. Recipient F344 rats were treated with donor (Lewis) splenocytes by intrathymic injection (i.t.) alone (10 x 10(6) cells/lobe); with donor splenocytes i.t. plus a one-time dose of ALS (1 mg) by intraperitoneal injection (i.p.); or with ALS i.p. (1 mg) alone 2 and 6 weeks prior to heterotopic Lewis heart transplantation. Control F344 recipients received saline i.t. Allografts were monitored by daily palpation, and long-term surviving grafts were harvested on day 90 for histopathologic analysis. Control allografts had 28.6% long-term survival (> 90 days) with mean graft survival of 46.7 +/- 12.2 days. At day 90 the surviving control allografts were enlarged and fibrotic with barely palpable heartbeat (mean heartbeat grade 0.29 +/- 0.18), and histologically showed diffuse moderate mononuclear cell infiltrates and advanced graft arteriosclerosis (mean vessel score 3.57 +/- 0.10 and 89 +/- 1% vessels diseased). Recipient treatment with intrathymic donor splenocytes alone significantly prolonged graft survival (89% long-term survival; mean 83.8 +/- 6.2 days, P < 0.04), but did not significantly inhibit the development of graft arteriosclerosis (score 2.98 +/- 0.53 and 79 +/- 8% diseased, P = NS). By contrast, treatment with i.t. donor splenocytes plus ALS 2 weeks prior to transplantation prolonged graft survival (100% long-term; mean 90.0 +/- 0.0 days, P < 0.04), and markedly inhibited graft arteriosclerosis (score 0.80 +/- 0.14, P < 0.05; 27 +/- 4% diseased, P < 0.05). ALS alone given two weeks prior to transplantation also prolonged graft survival (100% long-term; mean 90.0 +/- 0.0 days, P < 0.04), and inhibited graft arteriosclerosis (score 0.89 +/- 0.31, P < 0.05; 25 +/- 7% diseased, P < 0.05). However, when ALS was given 6 weeks prior to heart transplantation the beneficial effect of ALS alone was abolished, suggesting that lymphocyte depletion may have been responsible for the observed effects when ALS was administered at 2 weeks. Interestingly, intrathymic donor splenocytes plus ALS 6 weeks prior to transplantation, on the other hand, showed significant prolongation of allograft survival (100% long-term, mean 90.0 +/- 0.0 days, P < 0.04), and inhibited graft arteriosclerosis (score 0.41 +/- 0.02, P < 0.05; 16 +/- 2% diseased, P < 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)

Binding of activated protein C to a specific receptor on human mononuclear phagocytes inhibits intracellular calcium signaling and monocyte-dependent proliferative responses.

Upon activation, mononuclear phagocytes (Mphi) play key roles in the development of septic shock and multiple host immune responses, but details of the regulation of Mphi activation are little understood. We recently showed that the physiologic anticoagulant molecule, activated protein C (APC), blocks responses of human blood Mphi, alveolar Mphi, or THP-1 cells induced by LPS, IFN-gamma, or PMA, including TNF-alpha production and down-regulation of several LPS binding-related proteins. We now report a possible mechanism of action through inhibition of the rapid intracellular calcium signaling that occurs at the onset of Mphi activation, and characterization of a specific Mphi receptor for APC. Flow cytometry studies using Fluo-3 showed that Mph activation by Fc-receptor cross-linking or rIFN-gamma caused a rapid increase in free intracellular calcium, a primary event in multiple signal transduction pathways, which was blocked by pretreatment with APC. Consistent with this, addition of APC inhibited PHA-induced T cell proliferation in a dose- and time-dependent manner. Peak suppression (> 70%) required addition of APC within the first hour of 72 hr cocultures of Mphi and lymphocytes, and proliferative responses were not restored by addition of IL-2 or TNF-alpha. Biochemical studies showed that 125I-labeled APC bound specifically to M phi in a time-dependent and saturable manner. Scatchard analysis indicated there were 180,690 binding sites for APC per cell, which were of high affinity (Kd value of 12.9 mM). Binding of 125I-APC was doubled by activation of Mphi with LPS, and bound APC was not displaced by the zymogen, protein C (PC), or by enzymatically inactive (diisopropyl fluorophosphate- or PPACK-treated) APC, indicating an absolute requirement for the active site of APC in its binding to Mphi. APC binding was blocked by a polyclonal Ab to human PC/APC, but not by protein S, factor Va or Xa, or a polyclonal antithrombomodulin antibody. When 125I-APC was crosslinked to its receptor, immunoprecipitated and analyzed by SDS-PAGE under reducing conditions, a covalent complex (110-115 kD) of 125I-APC (62 kD) and its receptor was seen. In addition, a Mphi membrane protein of 50-55 kD, as determined by SDS-PAGE, was affinity-purified using an APC-Affigel column, and confirmed by ligand binding. Taken together, our findings document the presence of a M phi surface receptor for APC, which appears distinct from a recently described endothelial receptor for PC and APC, and which may be involved in the inhibitory effects of APC on activation of human Mphi, including Mphi-dependent T cell proliferation.

Gene expression analysis in human renal allograft biopsy samples using high-density oligoarray technology.

BACKGROUND: High-density oligoarray technology is a novel method for screening the expression of thousands of genes in a small tissue sample. Oligoarray analysis of genes expressed during human renal allograft rejection has not been reported previously. METHODS: Seven human renal allograft biopsies with histologic evidence of acute cellular rejection and three renal allograft biopsies without evidence of rejection (control) were analyzed for the expression of 6800 human genes using high-density oligoarrays (GeneChip, Affymetrix, Santa Clara, CA). Quantitative expression of gene transcripts was determined and a comparison analysis between acute rejection and control biopsy samples was performed. Up-regulation of a specific gene transcript during acute rejection was considered to be significant if transcript abundance increased fourfold or more relative to control biopsy samples. RESULTS: Comparison analysis revealed that between 32 and 219 gene transcripts are up-regulated (>fourfold) during acute rejection. Of these transcripts, only four (human monokine induced by interferon-gamma, T-cell receptor active beta-chain protein, interleukin-2 stimulated phosphoprotein, and RING4 (a transporter involved in antigen presentation)) were consistently up-regulated in each acute rejection sample relative to at least two of three control biopsy samples. Six other genes were up-regulated in six of seven acute rejection samples. These were interferon-stimulated growth factor-3, complement factor 3, nicotinamide N-methyltransferase, macrophage inflammatory protein-3beta, myeloid differentiation protein, and CD18. Only two gene transcripts were down-regulated in five of seven acute rejection samples. Significant up-regulation of cytotoxic T-cell effector molecules, previously reported as markers of acute renal rejection in humans, was not detected. CONCLUSIONS: High-density oligoarray technology is useful for screening gene expression in transplanted tissues undergoing acute rejection. Because this method does not rely on a priori knowledge of which genes are involved in acute rejection, it is likely to yield novel insights into the mechanisms and diagnosis of rejection.