Ankit Bharat

Early posttransplant inflammation promotes the development of alloimmunity and chronic human lung allograft rejection

Background. Chronic human lung allograft rejection, represented by bronchiolitis obliterans syndrome (BOS), is the single most important factor that limits the long-term survival following lung transplantation (LT). However, the pathogenesis of BOS remains unclear. We hypothesized that the early posttransplant inflammation would promote the development of donor anti–human leukocyte antigen (HLA) alloimmunity and predispose to BOS. Methods. Serum levels of interleukin (IL)-1&bgr;, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, Eotaxin, IP-10, MIG, MCP-1, MIP-1&agr;, MIP-1&bgr;, RANTES, tumor necrosis factor (TNF)-&agr;, interferon (IFN)-&agr;, IFN-&ggr;, granulocyte-macrophage colony-stimulating factor, IL-1R&agr;, and IL-2R were serially analyzed in 31 BOS+ and matched 31 BOS− patients using quantitative multiplex bead immunoassays. Donor-specific HLA class II cellular immunity was analyzed using enzyme-linked immunospot (ELISPOT) by testing recipient peripheral blood mononuclear cells against mismatched donor HLA-DR peptides. Anti-HLA class II antibodies were monitored using flow panel reactive antibodies. Results. There was early posttransplant elevation in basal serum levels of proinflammatory chemokines IP-10 and MCP-1 and Th1-cytokines IL-1&bgr;, IL-2, IL-12, and IL-15 in BOS+ patients, compared to BOS− and normal subjects. In addition, a threefold decline in IL-10 levels was found during BOS development. BOS+ patients revealed increased development of HLA class II alloantibodies and Th1-predominant donor-specific cellular immunity with high frequency of IFN-&ggr; and low IL-5 producing T-cells. Conclusion. Early posttransplant elevation of proinflammatory mediators is associated with alloimmunity and chronic human lung allograft rejection.

Immunological Link Between Primary Graft Dysfunction and Chronic Lung Allograft Rejection

BACKGROUND Primary graft dysfunction (PGD) in the immediate post-lung transplant period strongly increases the risk of chronic rejection (broncholitis obliterans syndrome). Here, we hypothesized that PGD-induced inflammation augments alloimmunity, thereby predisposing to broncholitis obliterans syndrome. METHODS Primary graft dysfunction and broncholitis obliterans syndrome were diagnosed according to the established International Society for Heart and Lung Transplantation criteria. Anti-human leukocyte antigen (HLA) alloantibodies were analyzed using Flow-PRA. Donor HLA class II-specific T cells were analyzed using interferon (IFN)-gamma ELISPOT. Serum levels of 25 cytokines and chemokines were measured using LUMINEX. RESULTS Of the 127 subjects, 29 (22.8%) had no PGD (grade 0), 42 (33.2%) had PGD-1, 36 (28.3%) had PGD-2, and 20 (15.7%) had PGD-3. Patients with PGD grades 1 to 3 (PGD(1-3)) had elevated proinflammatory mediators MCP-1, IP-10, interleukin (IL)-1 beta, IL-2, IFN-gamma, and IL-12 in the sera during the early posttransplant period compared with patients with PGD grade 0 (PGD(0)). On serial analysis, PGD(1-3) patients revealed increased development of de novo anti-HLA-II (5 years: 52.2% versus PGD(0) 13.5%, p = 0.008). However, no difference was found in anti-HLA-I alloantibody development (PGD(1-3) patients 48% versus PGD(0) 39.6%, p = 0.6). Furthermore, PGD(1-3) patients had increased frequency of donor HLA class II-specific CD4(+) T cells [(91.4 +/- 19.37) x 10(-6) versus (23.6 +/- 15.93) x 10(-6), p = 0.003]. CONCLUSIONS Primary graft dysfunction induces proinflammatory cytokines that can upregulate donor HLA-II antigens on the allograft. Increased donor HLA-II expression along with PGD-induced allograft inflammation promotes the development of donor specific alloimmunity. This provides an important mechanistic link between early posttransplant lung allograft injury and reported association with broncholitis obliterans syndrome.

CD4+25+ Regulatory T Cells Limit Th1-Autoimmunity by Inducing IL-10 Producing T Cells Following Human Lung Transplantation

Chronic human lung allograft rejection is manifested by bronchiolitis obliterans syndrome (BOS). BOS has a multifactorial etiology. Previous studies have indicated that both cellular and humoral alloimmunity play a significant role in the pathogenesis of BOS. Recently, autoimmunity has also been demonstrated to contribute to lung allograft rejection in animal models. However, the significance of autoimmunity in BOS remains unknown. In this report, we investigated the role of naturally occurring CD4+CD25+ regulatory T cells (T‐regs) in modulating cellular autoimmunity to collagen type V (col‐V), a ‘sequestered’ yet immunogenic self‐protein present in the lung tissue, following lung transplantation (LT). We demonstrated that col‐V reactive CD4+ T cells could be detected in the peripheral blood of lung transplant recipients. There was a predominance of IL‐10 producing T cells (TIL‐10) reactive to col‐V with significantly lower levels of IFN‐γ and IL‐2 producing T cells (Th1 cells). The col‐V specific TIL‐10 cells suppressed the proliferation and expansion of col‐V specific Th1 cells by IL‐10‐dependent and contact‐independent pathways. The TIL‐10 cells were distinct but their development was dependent on the presence of T‐regs. Furthermore, during chronic lung allograft rejection there was a significant decline of TIL‐10 cells with concomitant expansion of col‐V‐specific IFN‐γproducing Th1 cells.

Antibodies to Self-Antigens Predispose to Primary Lung Allograft Dysfunction and Chronic Rejection

BACKGROUND Primary graft dysfunction (PGD) is a known risk factor for bronchiolitis obliterans syndrome (BOS) after lung transplantation. Here, we report that preformed antibodies to self-antigens increase PGD risk and promote BOS. METHODS Adult lung transplant recipients (n = 142) were included in the study. Primary graft dysfunction and BOS were diagnosed based on International Society for Heart and Lung Transplantation guidelines. Antibodies to self-antigens k-alpha-1 tubulin, collagen type V, and collagen I were quantitated using standardized enzyme-linked immunosorbent assays, and cytokines were analyzed using Luminex immunoassays (Biosource International, Camirillo, CA). Human leukocyte antigen (HLA) antibodies were measured using Flow-PRA (One Lambda, Canoga Park, CA). RESULTS Lung transplant recipients with pretransplant antibodies to self-antigens had increased risk of PGD (odds ratio 3.09, 95% confidence interval: 1.2 to 8.1, p = 0.02) compared with recipients without. Conversely, in patients with PGD, 34.7% were positive for pretransplant antibodies whereas in the PGD negative group, only 14.6% had antibodies (p = 0.03). Antibody positive patients demonstrated high levels of proinflammatory cytokines interleukin (IL)-1β (2.1-fold increase), IL-2 (3.0), IL-12 (2.5), IL-15 (3.0), and chemokines interferon-inducible protein-10 (3.9) and monocyte chemotactic protein-1 (3.1; p < 0.01 for all). On 5-year follow-up, patients without antibodies showed greater freedom from development of HLA antibodies compared with patients who had antibodies (class I: 67% versus 38%, p = 0.001; class II: 71% versus 41%, p < 0.001). Patients with pretransplant antibodies were found to have an independent relative risk of 2.3 (95% confidence interval: 1.7 to 4.5, p = 0.009) for developing BOS. CONCLUSIONS Presence of antibodies to self-antigens pretransplant increases the risk of PGD immediately after transplant period and BOS on long-term follow-up. Primary graft dysfunction is associated with an inflammatory cascade that augments the alloimmune (anti-HLA) response that predisposes to BOS.

Monocyte-derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span.

Little is known about the relative importance of monocyte and tissue-resident macrophages in the development of lung fibrosis. We show that specific genetic deletion of monocyte-derived alveolar macrophages after their recruitment to the lung ameliorated lung fibrosis, whereas tissue-resident alveolar macrophages did not contribute to fibrosis. Using transcriptomic profiling of flow-sorted cells, we found that monocyte to alveolar macrophage differentiation unfolds continuously over the course of fibrosis and its resolution. During the fibrotic phase, monocyte-derived alveolar macrophages differ significantly from tissue-resident alveolar macrophages in their expression of profibrotic genes. A population of monocyte-derived alveolar macrophages persisted in the lung for one year after the resolution of fibrosis, where they became increasingly similar to tissue-resident alveolar macrophages. Human homologues of profibrotic genes expressed by mouse monocyte-derived alveolar macrophages during fibrosis were up-regulated in human alveolar macrophages from fibrotic compared with normal lungs. Our findings suggest that selectively targeting alveolar macrophage differentiation within the lung may ameliorate fibrosis without the adverse consequences associated with global monocyte or tissue-resident alveolar macrophage depletion.

Patient and tumor characteristics associated with increased mortality in young women (≤40 years) with breast cancer

The goal of the current study is to identify predictors responsible for mortality disparities between young (≤40 years) and older (>40 years) women with breast cancer.

A novel technique of vascular anastomosis to prevent juxta-anastomotic stenosis following arteriovenous fistula creation

OBJECTIVES Juxta-anastomotic stenosis (JAS) is one of the predominant causes of arteriovenous fistula (AVF) failure, with the reported incidence as high as 65%. We hypothesized that technical modification to alter the outflow vein configuration using the novel piggyback Straight Line Onlay Technique (pSLOT) would prevent JAS and improve AVF maturation. METHODS Intention-to-treat analysis of the outcomes of consecutive distal radiocephalic (RC) fistulas performed by a single operator with three different anastomotic techniques using a prospectively maintained database. Traditional end-to-side technique (ETS), side-to-side straight-line onlay technique (SLOT, STS) and pSLOT in RC AVF created in 125 consecutive patients between 1/2004 and 12/2007 were compared. AVF maturation was evaluated by ultrasonography at 4 to 6 weeks and use for dialysis. RESULTS The mean age of the study group was 53.1 ± 20.7 years, the male-to-female ratio was 61:64, and the races studied were African American (66; 52.8%) and Caucasian (54; 43.2%). The primary disease for renal failure was hypertension (54; 43.2%) and diabetes (51; 40.8%). Brachial artery flow at maturation was 1103 ± 531 mL/min. Incidence of early JAS was 9.8% and late 14.6%. The clinico-demographic variables between ETS (n = 57), STS (n = 12), and pSLOT (n = 54) were similar. The median follow-up between three groups: ETS (19 months), STS (12 months), and pSLOT (19 months; P = .1), was similar. There was a significant decrease in JAS development in pSLOT patients (P = .04). pSLOT patients also revealed decreased overall fistula failure (ETS 40.3%, STS 33.3%, pSLOT 16.7%; P = .01). CONCLUSIONS There was significant reduction in JAS and improvement in AVF maturation with pSLOT. This study provides evidence highlighting the role of outflow vein configuration in AVF maturation. Minimal alteration of vein wall configuration and avoidance torsion using pSLOT technique improves AVF maturation.

Induction of IL-10 Suppressors in Lung Transplant Patients by CD4 + 25 + Regulatory T Cells through CTLA-4 Signaling

T cell-mediated autoimmunity to collagen V (col-V), a sequestered yet immunogenic self-protein, can induce chronic lung allograft rejection in rodent models. In this study we characterized the role of CD4+CD25+ regulatory T cells (Tregs) in regulating col-V autoimmunity in human lung transplant (LT) recipients. LT recipients revealed a high frequency of col-V-reactive, IL-10-producing CD4+ T cells (TIL-10 cells) with low IL-2-, IFN-γ-, IL-5-, and no IL-4-producing T cells. These TIL-10 cells were distinct from Tregs because they lacked constitutive expression of both CD25 and Foxp3. Expansion of TIL-10 cells during col-V stimulation in vitro involved CTLA-4 on Tregs, because both depleting and blocking Tregs with anti-CTLA4 F(ab′)2 mAbs resulted in loss of TIL-10 cells with a concomitant increase in IFN-γ producing Th1 cells (TIFN-γ cells). A Transwell culture of col-V-specific TIL-10 cells with Th1 cells (those generated in absence of Tregs) from the same patient resulted in marked inhibition of IFN-γ and proliferation of TIFN-γ cells, which was reversed by neutralizing IL-10. Furthermore, the TIL-10 cells were HLA class II restricted because blocking HLA class II on APCs resulted in the loss of IL-10 production. Chronic lung allograft rejection was associated with the loss of Tregs with a concomitant decrease in TIL-10 cells and an increase in TIFN-γ cells. We conclude that LT patients have col-V-specific T cells that can be detected in the peripheral blood. The predominant col-V-specific T cells produce IL-10 that suppresses autoreactive Th1 cells independently of direct cellular contact. Tregs are pivotal for the induction of these “suppressor” TIL-10 cells.

Disseminated Ureaplasma infection as a cause of fatal hyperammonemia in humans.

Disseminated infection with Ureaplasma species causes fatal hyperammonemia syndrome in lung transplant recipients, likely by disrupting ammonia metabolism. The killer within Hyperammonemia, or abnormal buildup of ammonia, is an uncommon but generally fatal condition that affects immunosuppressed patients, particularly those who receive a lung transplant. Although there are treatments that can lower the blood concentration of ammonia, their effects are short-lived, and they are typically ineffective for this condition. Now, Bharat et al. identified Ureaplasma bacteria as the cause of this condition and demonstrated that it can be successfully treated with antibiotics. These findings suggest that patients with hyperammonemia should be screened for Ureaplasma species and treated with the appropriate antibiotics for this infection. Moreover, the authors found evidence of Ureaplasma species in one donor’s lung fluid sample, indicating that donors may need to be screened for it as well. Hyperammonemia syndrome is a fatal complication affecting immunosuppressed patients. Frequently refractory to treatment, it is characterized by progressive elevations in serum ammonia of unknown etiology, ultimately leading to cerebral edema and death. In mammals, ammonia produced during amino acid metabolism is primarily cleared through the hepatic production of urea, which is eliminated in the kidney. Ureaplasma species, commensals of the urogenital tract, are Mollicutes dependent on urea hydrolysis to ammonia and carbon dioxide for energy production. We hypothesized that systemic infection with Ureaplasma species might pose a unique challenge to human ammonia metabolism by liberating free ammonia resulting in the hyperammonemia syndrome. We used polymerase chain reaction, specialized culture, and molecular resistance profiling to identify systemic Ureaplasma infection in lung transplant recipients with hyperammonemia syndrome, but did not detect it in any lung transplant recipients with normal ammonia concentrations. Administration of Ureaplasma-directed antimicrobials to patients with hyperammonemia syndrome resulted in biochemical and clinical resolution of the disorder. Relapse in one patient was accompanied by recurrent Ureaplasma bacteremia with antimicrobial resistance. Our results provide evidence supporting a causal relationship between Ureaplasma infection and hyperammonemia, suggesting a need to test for this organism and provide empiric antimicrobial treatment while awaiting microbiological confirmation.

Flow Cytometry Reveals Similarities Between Lung Macrophages in Humans and Mice.

Findings in murine models implicate subpopulations of alveolar macrophages in the pathogenesis of lung injury and fibrosis, however, the relevance of these findings for humans with chronic lung disease is unknown in part due to a lack of proper tools to identify macrophage heterogeneity in the human lung. Here we report a flow cytometry protocol that allows unambiguous identification of alveolar macrophages, interstitial macrophages and monocytes in the human lung and in bronchoalveolar lavage fluid. We validated this panel using normal lung tissue and tissue from patients with COPD and lung fibrosis. We found evidence of heterogeneity within human alveolar macrophage populations, which suggest parallels between murine and human macrophage development and differentiation. Abstract word count: 113.