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The ability to induce tolerance would be a major advance in the field of solid organ transplantation. Here, we investigated whether autologous (congenic) hematopoietic stem cell transplantation (HSCT) could promote tolerance to heart allografts in mice. In an acute rejection model, fully MHC-mismatched BALB/c hearts were heterotopically transplanted into C57BL/6 (CD45.2) mice. One week later, recipient mice were lethally irradiated and reconstituted with congenic B6 CD45.1 Lin−Sca1+ckit+ cells. Recipient mice received a 14-day course of rapamycin both to prevent rejection and to expand regulatory T cells (Tregs). Heart allografts in both untreated and rapamycin-treated recipients that did not undergo HSCT were rejected within 33 days (median survival time = 8 days for untreated recipients, median survival time = 32 days for rapamycin-treated recipients), whereas allografts in HSCT-treated recipients had a median survival time of 55 days (P < 0.001 vs. both untreated and rapamycin-treated recipients). Enhanced allograft survival following HSCT was associated with increased intragraft Foxp3+ Tregs, reduced intragraft B cells, and reduced serum donor-specific antibodies. In a chronic rejection model, Bm12 hearts were transplanted into C57BL/6 (CD45.2) mice, and congenic HSCT was performed two weeks following heart transplantation. HSCT led to enhanced survival of allografts (median survival time = 70 days vs. median survival time = 28 days in untreated recipients, P < 0.01). Increased allograft survival post-HSCT was associated with prevention of autoantibody development and absence of vasculopathy. These data support the concept that autologous HSCT can promote immune tolerance in the setting of allotransplantation. Further studies to optimize HSCT protocols should be performed before this procedure is adopted clinically.

Background Despite more than 2 years having elapsed since the onset of SARS-CoV-2 pandemic, a level of hesitation around increased SARS-CoV-2 vaccine toxicity in cancer patients receiving immunotherapy (IO) remains. Here, we explore serological responses to SARS-CoV-2 vaccination in patients treated with IO and we describe blood cytokines, autoantibody levels and immune-related adverse events (irAEs) post vaccination. Methods Serum anti-SARS-CoV-2 spike (S) protein receptor binding domain (RBD) antibodies, surrogate viral neutralization test (sVNT), Th1/Th2 cytokines and antibodies against self-antigens were quantified at baseline, between 1st and 2nd vaccine doses, at 1 week (1W), 1 month (1M), 4–6 months and 10–12 months after the 2nd dose. Grade 2 or higher (≥ gr2+) irAEs were captured prospectively. Results Fifty-one evaluable patients were enrolled in this longitudinal study, 35 on immunotherapy (IO) and 16 on non-immunotherapy (non-IO) treatment. Absolute levels and neutralization potential of anti-SARS-CoV-2 antibodies were not significantly different in the IO group compared to non-IO. Chemotherapy adversely affects seroconversion when compared to IO and/or targeted treatment with antibody levels of 67.6 U/mL vs 1441 U/mL (p = 0.006) and sVNT of 70.9% vs 94.5% (p = 0.009), at 1M after 2nd vaccine dose. Following vaccination, the prevalence of grade ≥ 2 irAEs in patients treated with IO was not higher than the usual reported IO toxicity. We report, for the first time, that post-vaccination, IgM autoantibodies against beta 2 glycoprotein (p = 0.02), myeloperoxidase (p = 0.03), nucleosome (p = 0.041), SPLUNC2 (p

BACKGROUND: Antibody-mediated rejection (AMR) accounts for >50% of kidney allograft loss. Donor-specific antibodies (DSA) against HLA and non-HLA antigens in the glomeruli and the tubulointerstitium cause AMR while inflammatory cytokines such as TNFα trigger graft injury. The mechanisms governing cell-specific injury in AMR remain unclear. METHODS: Unbiased proteomic analysis of laser-captured and microdissected glomeruli and tubulointerstitium was performed on 30 for-cause kidney biopsy specimens with early AMR, acute cellular rejection (ACR), or acute tubular necrosis (ATN). RESULTS: A total of 107 of 2026 glomerular and 112 of 2399 tubulointerstitial proteins was significantly differentially expressed in AMR versus ACR; 112 of 2026 glomerular and 181 of 2399 tubulointerstitial proteins were significantly dysregulated in AMR versus ATN (P

Left ventricular assist devices (LVADs) can serve as a bridge to transplant or destination therapy for patients with advanced heart failure. Implantation of LVADs is known to be associated with increases in anti-HLA antibodies, but less is known about how autoantibody levels change with the use of these devices.Autoantibody levels were quantified with the use of customized antigen microarrays in 22 patients both before and after LVAD. We observed an increase (1.5- to 2-fold) in 14 IgG autoantibodies in the serum of patients after LVAD, including autoantibodies against cardiac proteins (myosin, troponin I, tropomyosin), DNA, and structural proteins (collagen, laminin). There was also a small but significant rise in total serum IgG after LVAD. Increases in autoantibodies after LVAD were positively associated with increases in calculated panel-reactive antibody class II (P = .05) and negatively correlated with age (r = -0.45; P.05). Cytokines were evaluated to gain insights into the mechanism of antibody generation, and we observed a positive correlation between total IgG levels after LVAD and the level of monocyte chemoattractant protein 1 (r = 0.60; P.05).LVAD implantation is associated with increases in IgG autoantibodies, anti-HLA antibodies, and total IgG. Increases in IgG after LVAD implantation may relate to an inflammatory response triggered by these devices.

Objective We investigated the autoantibody (autoAb) profiles in ANA+ individuals lacking systemic autoimmune rheumatic disease (SARD) and early SARD patients to determine the key differences between these groups and identify factors that are associated with an increased risk of symptomatic progression within the next 2 years in ANA+ individuals. Methods Using custom antigen (Ag) microarrays, 144 IgM and IgG autoAbs were surveyed in 84 asymptomatic and 123 symptomatic (48 UCTD and 75 SARD patients) ANA+ individuals. AutoAbs were compared in ANA+ individuals lacking a SARD diagnosis with ≥2 years follow-up (n = 52), including all those who demonstrated progression (n = 14) during this period, with changes over time assessed in a representative subset. Results We show that ANA+ individuals have autoAb to many self-Ags that are not being captured by current screening techniques and very high levels of these autoAbs are predominantly restricted to early SARD patients, with SLE patients displaying reactivity to many more autoAgs than the other groups. In general, the symptoms that developed in progressors mirrored those seen in SARD patients with similar patterns of autoAbs. Only anti-Ro52 Abs were found to predict progression (positive predictive value 46%, negative predictive value 89%). Surprisingly, over 2 years of follow-up the levels of autoAbs remained remarkably stable regardless of whether individuals progressed or not. Conclusion Our findings strongly argue that development of assays with an expanded set of auto-Ags and enhanced dynamic range would improve the diagnostic and prognostic ability of autoAb testing.

Supplemental Digital Content is available in the text., Background. Antibody-mediated rejection (AMR) causes more than 50% of late kidney graft losses. In addition to anti-human leukocyte antigen (HLA) donor-specific antibodies, antibodies against non-HLA antigens are also linked to AMR. Identifying key non-HLA antibodies will improve our understanding of AMR. Methods. We analyzed non-HLA antibodies in sera from 80 kidney transplant patients with AMR, mixed rejection, acute cellular rejection (ACR), or acute tubular necrosis. IgM and IgG antibodies against 134 non-HLA antigens were measured in serum samples collected pretransplant or at the time of diagnosis. Results. Fifteen non-HLA antibodies were significantly increased (P < 0.05) in AMR and mixed rejection compared with ACR or acute tubular necrosis pretransplant, and 7 at diagnosis. AMR and mixed cases showed significantly increased pretransplant levels of IgG anti-Ro/Sjögren syndrome-antigen A (SS-A) and anti-major centromere autoantigen (CENP)-B, compared with ACR. Together with IgM anti-CENP-B and anti-La/SS-B, these antibodies were significantly increased in AMR/mixed rejection at diagnosis. Increased IgG anti-Ro/SS-A, IgG anti-CENP-B, and IgM anti-La/SS-B were associated with the presence of microvascular lesions and class-II donor-specific antibodies (P < 0.05). Significant increases in IgG anti-Ro/SS-A and IgM anti-CENP-B antibodies in AMR/mixed rejection compared with ACR were reproduced in an external cohort of 60 kidney transplant patients. Conclusions. This is the first study implicating autoantibodies anti-Ro/SS-A and anti-CENP-B in AMR. These antibodies may participate in the crosstalk between autoimmunity and alloimmunity in kidney AMR.

Background Primary sclerosing cholangitis (PSC) is an indication for liver transplantation, but recurrence after liver transplantation is associated with poor outcomes often requiring repeat transplantation. We investigated whether autologous hematopoietic stem cell transplantation (aHSCT) could be used to stop progression of recurrent PSC and promote operational tolerance. Methods Twelve patients with recurrent PSC were fully evaluated and 5 were selected for aHSCT. Autologous hematopoietic stem cells were collected, purified by CD34 immunomagnetic selection and cryopreserved. Immunoablation using busulfan, cyclophosphamide and rabbit anti-thymocyte globulin was followed by aHSCT. The primary endpoint of the study was the establishment of operational tolerance defined as lack of biochemical, histologic and clinical evidence of rejection while off immunosuppression at 2 years post-aHSCT. Results Two of the 5 patients achieved operational tolerance with no clinical or histological evidence of PSC progression or allo-rejection. A third patient developed sinusoidal obstruction syndrome following aHSCT requiring repeat liver transplantation but has no evidence of PSC recurrence while on sirolimus monotherapy now more than 3 years after aHSCT. A fourth patient was weaned off immunosuppression but died 212 days after aHSCT from pericardial constriction. A fifth patient died from multiorgan failure. Immunosuppression-free allograft acceptance was associated with deletion of T cell clones, loss of autoantibodies and increases in regulatory T cells, transitional B cells, and programmed cell death protein-1 expressing CD8+ T cells in the 2 long-term survivors. Conclusions Although operational tolerance occurred following aHSCT, the high morbidity and mortality observed renders this specific protocol unsuitable for clinical adoption.

Health care workers (HCWs) are at higher risk for SARS-CoV-2 infection and may play a role in transmitting the infection to vulnerable patients and members of the community. This is particularly worrisome in the context of asymptomatic infection. We performed a cross-sectional study looking at asymptomatic SARS-CoV-2 infection in HCWs. We screened asymptomatic HCWs for SARS-CoV-2 via PCR. Complementary viral genome sequencing was performed on positive swab specimens. A seroprevalence analysis was also performed using multiple assays. Asymptomatic health care worker cohorts had a combined swab positivity rate of 29/5776 (0.50%, 95%CI 0.32–0.75) relative to a comparative cohort of symptomatic HCWs, where 54/1597 (3.4%) tested positive for SARS-CoV-2 (ratio of symptomatic to asymptomatic 6.8:1). SARS-CoV-2 seroprevalence among 996 asymptomatic HCWs with no prior known exposure to SARS-CoV-2 was 1.4–3.4%, depending on assay. A novel in-house Coronavirus protein microarray showed differing SARS-CoV-2 protein reactivities and helped define likely true positives vs. suspected false positives. Our study demonstrates the utility of routine screening of asymptomatic HCWs, which may help to identify a significant proportion of infections.

Antibody-mediated rejection (AMR) causes >50% of late kidney graft losses. Although donor-specific antibodies (DSA) against HLA cause AMR, antibodies against non-HLA antigens are also linked to rejection. Identifying key non-HLA antibodies will improve our understanding of antibody-mediated injury.We analyzed non-HLA antibodies using protein microarrays in sera from 91 kidney transplant patients with AMR, mixed rejection, acute cellular rejection (ACR), or acute tubular necrosis (ATN). IgM and IgG antibodies against 134 non-HLA antigens were measured pre-transplant, at the time of biopsy-proven diagnosis, and post-diagnosis. Findings were validated in 60 kidney transplant patients from an independent cohort.Seventeen non-HLA antibodies were significantly increased (pThis is the first study that implicates autoantibodies against Ro/SS-A and CENP-B in AMR. These non-HLA antibodies may participate in the crosstalk between autoimmunity and alloimmunity in kidney AMR.SIGNIFICANCE STATEMENTAntibody-mediated rejection (AMR) causes >50% of kidney graft losses. Although donor-specific antibodies against HLA cause AMR, antibodies against non-HLA antigens are also linked to rejection. Serum samples of 91 kidney transplant patients were analyzed using protein arrays against 134 non-HLA antigens. AMR and mixed rejection cases showed significantly increased pre-transplant levels of IgG anti-Ro/SS-A and anti-CENP-B, compared to acute cellular rejection. Together with IgM anti-CENP-B and anti-La/SS-B, these antibodies were significantly increased in AMR/mixed rejection at diagnosis and were validated in a second, independent cohort. Increased IgG anti-Ro/SS-A, IgG anti-CENP-B and IgM anti-La/SS-B were associated with the presence of microvascular lesions and anti-HLA class-II antibodies. This is the first study to implicate anti-Ro/SS-A, anti-La/SS-B and anti-CENP-B autoantibodies in AMR.

We screened three separate cohorts of healthcare workers for SARS-CoV-2 via nasopharyngeal swab PCR. A seroprevalence analysis using multiple assays was performed in a subgroup. The asymptomatic health care worker cohorts had a combined swap positivity rate of 29/5776 (0.50%, 95%CI 0.32-0.75) compared to the symptomatic cohort rate of 54/1597 (3.4%) (ratio of symptomatic to asymptomatic 6.8:1). Sequencing demonstrated several variants. The seroprevalence (n=996) was 1.4-3.4% depending on assay. Protein microarray analysis showed differing SARS-CoV-2 protein reactivities and helped define likely true positives vs. suspected false positives. Routine screening of asymptomatic health care workers helps identify a significant proportion of infections.

Antibody-mediated rejection (AMR) accounts for >50% of kidney allograft losses. AMR is caused by donor-specific antibodies (DSA) against HLA and non-HLA antigens in the glomeruli and the tubulointerstitium, which together with inflammatory cytokines such as tumor necrosis factor alpha (TNFα) and interferon gamma (IFNɣ), trigger graft injury. Unfortunately, the mechanisms governing cell-specific injury in AMR remain unclear. We studied 30 for-cause kidney biopsies with early AMR, acute cellular rejection or acute tubular necrosis (‘non-AMR’). We laser-captured and microdissected glomeruli and tubulointerstitium, and subjected them to unbiased proteome analysis. 120/2026 glomerular and 180/2399 tubulointerstitial proteins were significantly differentially expressed in AMR vs. non-AMR biopsies (PSIGNIFICANCE STATEMENTAntibody-mediated rejection (AMR) accounts for >50% of kidney allograft loss, and is caused by donor-specific antibodies against HLA antigens, which induce maladaptive responses in the kidney glomeruli and tubulointerstitium. This is the first unbiased proteomics analysis of laser-captured/microdissected glomeruli and tubulointerstitium from 30 indication kidney biopsies with early AMR, acute cellular rejection or acute tubular necrosis. >2,000 proteins were quantified in each compartment. We discovered that basement membrane and extracellular matrix (ECM) proteins were significantly decreased in both AMR compartments. Two ECM-modifying proteins, LGALS1 and GSTO1, were significantly increased in glomeruli and tubulointerstitium, respectively. LGALS1 and GSTO1 were upregulated by anti-HLA antibodies or AMR-related cytokines in primary kidney cells, and may represent therapeutic targets to ameliorate ECM-remodeling in AMR.

Chronic lung allograft dysfunction (CLAD) limits long-term survival after lung transplant (LT). Ischemia-reperfusion injury (IRI) promotes chronic rejection (CR) and CLAD, but the underlying mechanisms are not well understood. To examine mechanisms linking IRI to CR, a mouse orthotopic LT model using a minor alloantigen strain mismatch (C57BL/10 [B10, H-2b ] → C57BL/6 [B6, H-2b ]) and isograft controls (B6→B6) was used with antecedent minimal or prolonged graft storage. The latter resulted in IRI with subsequent airway and parenchymal fibrosis in prolonged storage allografts but not isografts. This pattern of CR after IRI was associated with the formation of B cell-rich tertiary lymphoid organs within the grafts and circulating autoantibodies. These processes were attenuated by B cell depletion, despite preservation of allograft T cell content. Our observations suggest that IRI may promote B cell recruitment that drives CR after LT. These observations have implications for the mechanisms leading to CLAD after LT.

This chapter describes the factors involved in the disease prognosis, parameters of outcome evaluations, principles and techniques for progression prevention. In last section, the future perspectives in both basic and clinical investigations towards unmet medical needs in AECHB and HBV ACLF are discussed. 1. Factors affecting the prognosis of patients with severe hepatitis B include those related to the virus (including viral load, HBeAg expression, and gene mutation), patient age, co-morbidity, TBil, INR, serum Cr, and the host genetic background. Indicators associated with patient prognosis include TBil, total cholesterol, albumin and prealbumin, hepatic encephalopathy, kidney damage, alpha-fetoprotein and vitamin D binding protein, blood sodium level, virus HBeAg expression and genotype, and blood glucose. 2. In addition to TBil, INR, hepatic encephalopathy, Cr level and AFP as indicators for prognosis of severe hepatitis, some other parameters such as clinical signs, symptoms, serum levels of total cholesterol and albumin and natrium, and coagulation factors are all valuable in assessment. The roles of cell apoptosis, liver regeneration and immunological parameters in assessing patient prognosis are under study. Prognostic evaluating systems include MELD score, MELD-Na score, iMELD score, KCI and CTP score. 3. Prevention of severe hepatitis B should be started in asymptomatic patients. Close observation, sufficient rest, adequate nutrition, meticulous nursing and psychological care, preventing and removing exacerbating factors, treating concomitant diseases, reasonable antiviral and comprehensive therapies are helpful to prevent CHB patients from developing to severe hepatitis. For patients who already have severe hepatitis B, the prevention and management of complications is important for lowering mortality rate. 4. New research directions in acute-on-chronic liver failure include: (1) Additional well controlled studies using present or new liver systems are warranted. Other approaches include the use of granulocyte colony stimulating factor to treat infections as well as the potential of use of stem cells to restore immune integrity and enhance liver regeneration. (2) Using new cell lines and animal models to understand the molecular biology of HBV, the immune response and to develop novel therapies. (3) Development of new anti-HBV strategies, e.g. silencing or remove cccDNA, enhancing immunologic clearance of HBV infection, inhibiting virus entry or HBc expression and using CRISP to disrupt cccDNA.

Purpose Non-human leukocyte antigen (HLA) antibodies have been associated with chronic lung allograft dysfunction (CLAD). Whether they predate CLAD is unclear. We hypothesized that patients destined to develop CLAD might develop non-HLA antibodies prior to CLAD onset. Methods In a single centre retrospective study, we examined 100 consecutive crossmatch negative recipients of a bilateral lung transplant between 1 January 2013 and 31 December 2014. Using a two-colour fluorescent antigen microarray with 152 unique antigens, we measured IgG and IgM autoantibodies in pre-transplant and six months post-transplant serum samples. Log2-transformed mean fluorescent intensities (MFIs) were determined for each antigen. CLAD was determined using published definitions. Significance analysis of microarrays (SAM) was used to analyze differences in antigen reactivity with a false discovery rate of 0.05. Paired and unpaired analyses were used to examine differences within and between recipients who developed CLAD within 5 years and recipients who remained CLAD free at 5 years post-transplant. Results Serum samples were available for 96 recipients - 35 (36.5%) developed CLAD and 61 remained CLAD-free within 5 years. Total IgM and IgG reactivity decreased in most patients post-transplant (Fig 1A), although IgM levels were stable in patients developing CLAD. Three IgM autoantibodies - including anti-club cell secretory protein (CCSP, Fig 1B) - increased in most patients post-transplant; this increase was of greater magnitude in patients later developing CLAD. Patients destined to develop CLAD also had an increase in IgG fluorescence directed at human core histones (Fig 1C). Conclusion Our results suggest that there may be important differences in non-HLA antibody reactivity that appear before the onset of CLAD. These observations, which require validation in further studies, may also indicate a specific role for non-HLA antibodies directed at club cell secretory protein and nuclear antigens in CLAD development.

Fibrinogen-like protein 2 (FGL2) is an immunomodulatory protein that is expressed by regulatory T cells (Tregs). The objective of this study was to determine if recombinant FGL2 (rFGL2) treatment or constitutive FGL2 overexpression could promote transplant tolerance in mice. Although rFGL2 treatment prevented rejection of fully mismatched cardiac allografts, all grafts were rejected after stopping treatment. Next, we generated FGL2 transgenic mice (fgl2(Tg) ) that ubiquitously overexpressed FGL2. These mice developed normally and had no evidence of the autoimmune glomerulonephritis seen in fgl2(-/-) mice. Immune characterization showed fgl2(Tg) T cells were hypoproliferative to stimulation with alloantigens or anti-CD3 and anti-CD28 stimulation, and fgl2(Tg) Tregs had increased immunosuppressive activity compared with fgl2(+/+) Tregs. To determine if FGL2 overexpression can promote tolerance, we transplanted fully mismatched cardiac allografts into fgl2(Tg) recipients. Fifty percent of cardiac grafts were accepted indefinitely in fgl2(Tg) recipients without any immunosuppression. Tolerant fgl2(Tg) grafts had increased numbers and proportions of Tregs and tolerant fgl2(Tg) mice had reduced proliferation to donor but not third party antigens. These data show that tolerance in fgl2(Tg) recipients involves changes in Treg and T cell activity that contribute to a higher intragraft Treg-to-T cell ratio and acceptance of fully mismatched allografts.

Persistent viruses evade immune detection by interfering with virus‐specific innate and adaptive antiviral immune responses. Fibrinogen‐like protein‐2 (FGL2) is a potent effector molecule of CD4(+) CD25(+) FoxP3(+) regulatory T cells and exerts its immunosuppressive activity following ligation to its cognate receptor, Fcγ RIIB/RIII. The role of FGL2 in the pathogenesis of chronic viral infection caused by lymphocytic choriomeningitis virus clone‐13 (LCMV cl‐13) was assessed in this study. Chronically infected fgl2 (+/+) mice had increased plasma levels of FGL2, with reduced expression of the maturation markers, CD80, CD86 and MHC‐II on macrophages and dendritic cells and impaired production of neutralizing antibody. In contrast, fgl2 (−/−) mice or fgl2 (+/+) mice that had been pre‐treated with antibodies to FGL2 and Fcγ RIIB/RIII and then infected with LCMV cl‐13 developed a robust CD4(+) and CD8(+) antiviral T‐cell response, produced high titred neutralizing antibody to LCMV and cleared LCMV. Treatment of mice with established chronic infection with antibodies to FGL2 and Fcγ RIIB/RIII was shown to rescue the number and functionality of virus‐specific CD4(+) and CD8(+) T cells with reduced total and virus‐specific T‐cell expression of programmed cell death protein 1 leading to viral clearance. These results demonstrate an important role for FGL2 in viral immune evasion and provide a rationale to target FGL2 to treat patients with chronic viral infection.

Summary Therapies that promote tolerance in solid organ transplantation will improve patient outcomes by eliminating the need for long-term immunosuppression. To investigate mechanisms of rapamycin-induced tolerance, C3H/HeJ mice were heterotopically transplanted with MHC-mismatched hearts from BALB/cJ mice and were monitored for rejection after a short course of rapamycin treatment. Mice that had received rapamycin developed tolerance with indefinite graft survival, whereas untreated mice all rejected their grafts within 9 days. In vitro, splenic mononuclear cells from tolerant mice maintained primary CD4+ and CD8+ immune responses to donor antigens consistent with a mechanism that involves active suppression of immune responses. Furthermore, infection with lymphocytic choriomeningitis virus strain WE led to loss of tolerance suggesting that tolerance could be overcome by infection. Rapamycin-induced, donor-specific tolerance was associated with an expansion of regulatory T (Treg) cells in both the spleen and allograft and elevated plasma levels of fibrinogen-like protein 2 (FGL2). Depletion of Treg cells with anti-CD25 (PC61) and treatment with anti-FGL2 antibody both prevented tolerance induction. Tolerant allografts were populated with Treg cells that co-expressed FGL2 and FoxP3, whereas rejecting allografts and syngeneic grafts were nearly devoid of dual-staining cells. We examined the utility of an immunoregulatory gene panel to discriminate between tolerance and rejection. We observed that Treg-associated genes (foxp3, lag3, tgf-β and fgl2) had increased expression and pro-inflammatory genes (ifn-γ and gzmb) had decreased expression in tolerant compared with rejecting allografts. Taken together, these data strongly suggest that Treg cells expressing FGL2 mediate rapamycin-induced tolerance. Furthermore, a gene biomarker panel that includes fgl2 can distinguish between rejecting and tolerant grafts.

Although the newer continuous-flow left ventricular assist devices (CF-LVADs) provide clinical advantages over the pulsatile pumps, the effects of low pulsatility on inflammation are incompletely understood. The objective of our study was to examine the levels of inflammatory mediators in CF-LVAD recipients compared with both healthy control subjects and heart failure patients who were candidates for CF-LVAD support. Plasma levels of chemokines, cytokines, and inflammatory markers were measured in 18 CF-LVAD recipients and compared with those of 14 healthy control subjects and 14 heart failure patients who were candidates for CF-LVADs. The levels of granulocyte macrophage-colony stimulating factor, macrophage inflammatory proteins-1β, and macrophage-derived chemokine were significantly higher in the CF-LVAD group compared with both the heart failure and the healthy control groups, whereas no significant differences were observed between the healthy control subjects and the heart failure groups. Compared with the healthy controls, C-reactive protein, interferon gamma-induced protein-10, monocyte chemotactic protein-1, and interleukin-8 levels were significantly higher in both the CF-LVAD and heart failure groups, but no significant differences were observed between the CF-LVAD recipients and the heart failure patients. Inflammatory markers were elevated in CF-LVAD recipients compared with healthy control subjects and the heart failure patients. Further studies should investigate the clinical implications of elevated levels of inflammation in CF-LVAD recipients.

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels of autoantibodies to specific antigens are typically detected with the enzyme-linked immunosorbent assay (ELISA) technique. However, screening for multiple autoantibodies with ELISA can be time-consuming and requires a large quantity of patient sample. The antigen microarray technique is an alternative method that can be used to screen for autoantibodies in a multiplex fashion. In this technique, antigens are arrayed onto specially coated microscope slides with a robotic microarrayer. The slides are probed with patient serum samples and subsequently fluorescent-labeled secondary antibodies are added to detect binding of serum autoantibodies to the antigens. The autoantibody reactivities are revealed and quantified by scanning the slides with a scanner that can detect fluorescent signals. Here we describe methods to generate custom antigen microarrays. Our current arrays are printed with 9 solid pins and can include up to 162 antigens spotted in duplicate. The arrays can be easily customized by changing the antigens in the source plate that is used by the microarrayer. We have developed a two-color secondary antibody detection scheme that can distinguish IgG and IgM reactivities on the same slide surface. The detection system has been optimized to study binding of human and murine autoantibodies.

The T cell antigen receptor (TCR)-CD3 complex is unique in having ten cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs). The physiological importance of this high TCR ITAM number is unclear. Here we generated 25 groups of mice expressing various combinations of wild-type and mutant ITAMs in TCR-CD3 complexes. Mice with fewer than seven wild-type CD3 ITAMs developed a lethal, multiorgan autoimmune disease caused by a breakdown in central rather than peripheral tolerance. Although there was a linear correlation between the number of wild-type CD3 ITAMs and T cell proliferation, cytokine production was unaffected by ITAM number. Thus, high ITAM number provides scalable signaling that can modulate proliferation yet ensure effective negative selection and prevention of autoimmunity.

Autoantibodies directed against endogenous proteins including contractile proteins and endothelial antigens are frequently detected in patients with heart failure and after heart transplantation. There is evidence that these autoantibodies contribute to cardiac dysfunction and correlate with clinical outcomes. Currently, autoantibodies are detected in patient sera using individual ELISA assays (one for each antigen). Thus, screening for many individual autoantibodies is laborious and consumes a large amount of patient sample. To better capture the broad-scale antibody reactivities that occur in heart failure and post-transplant, we developed a custom antigen microarray technique that can simultaneously measure IgM and IgG reactivities against 64 unique antigens using just five microliters of patient serum. We first demonstrated that our antigen microarray technique displayed enhanced sensitivity to detect autoantibodies compared to the traditional ELISA method. We then piloted this technique using two sets of samples that were obtained at our institution. In the first retrospective study, we profiled pre-transplant sera from 24 heart failure patients who subsequently received heart transplants. We identified 8 antibody reactivities that were higher in patients who developed cellular rejection (2 or more episodes of grade 2R rejection in first year after transplant as defined by revised criteria from the International Society for Heart and Lung Transplantation) compared with those who did have not have rejection episodes. In a second retrospective study with 31 patients, we identified 7 IgM reactivities that were higher in heart transplant recipients who developed antibody-mediated rejection (AMR) compared with control recipients, and in time course studies, these reactivities appeared prior to overt graft dysfunction. In conclusion, we demonstrated that the autoantibody microarray technique outperforms traditional ELISAs as it uses less patient sample, has increased sensitivity, and can detect autoantibodies in a multiplex fashion. Furthermore, our results suggest that this autoantibody array technology may help to identify patients at risk of rejection following heart transplantation and identify heart transplant recipients with AMR.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) are critical to the maintenance of immune tolerance. Treg are known to utilize a number of molecular pathways to control immune responses and maintain immune homeostasis. Fibrinogen-like protein 2 (FGL2) has been identified by a number of investigators as an important immunosuppressive effector of Treg, which exerts its immunoregulatory activity by binding to inhibitory FcγRIIB receptors expressed on antigen-presenting cells including dendritic cells, endothelial cells, and B cells. More recently, it has been suggested that FGL2 accounts for the immunosuppressive activity of a highly suppressive subset of Treg that express T cell immunoreceptor with Ig and ITIM domains (TIGIT). Here we discuss the important role of Treg and FGL2 in preventing alloimmune and autoimmune disease. The FGL2-FcγRIIB pathway is also known to be utilized by viruses and tumor cells to evade immune surveillance. Moving forward, therapies based on modulation of the FGL2-FcγRIIB pathway hold promise for the treatment of a wide variety of conditions ranging from autoimmunity to cancer.

The activation state of beta-adrenergic receptors (beta-ARs) in vivo is an important determinant of hemodynamic status, cardiac performance, and metabolic rate. In order to achieve homeostasis in vivo, the cellular signals generated by beta-AR activation are integrated with signals from a number of other distinct receptors and signaling pathways. We have utilized genetic knockout models to test directly the role of beta1- and/or beta2-AR expression on these homeostatic control mechanisms. Despite total absence of beta1- and beta2-ARs, the predominant cardiovascular beta-adrenergic subtypes, basal heart rate, blood pressure, and metabolic rate do not differ from wild type controls. However, stimulation of beta-AR function by beta-AR agonists or exercise reveals significant impairments in chronotropic range, vascular reactivity, and metabolic rate. Surprisingly, the blunted chronotropic and metabolic response to exercise seen in beta1/beta2-AR double knockouts fails to impact maximal exercise capacity. Integrating the results from single beta1- and beta2-AR knockouts as well as the beta1-/beta2-AR double knock-out suggest that in the mouse, beta-AR stimulation of cardiac inotropy and chronotropy is mediated almost exclusively by the beta1-AR, whereas vascular relaxation and metabolic rate are controlled by all three beta-ARs (beta1-, beta2-, and beta3-AR). Compensatory alterations in cardiac muscarinic receptor density and vascular beta3-AR responsiveness are also observed in beta1-/beta2-AR double knockouts. In addition to its ability to define beta-AR subtype-specific functions, this genetic approach is also useful in identifying adaptive alterations that serve to maintain critical physiological setpoints such as heart rate, blood pressure, and metabolic rate when cellular signaling mechanisms are perturbed.

Women develop certain autoimmune diseases more often than men. It has been hypothesized that this may relate to the development of more robust T-helper (Th)1 responses in women. To test whether women exhibit a Th1 bias, we isolated naïve cluster of differentiation (CD)4 + T cells from peripheral blood of healthy women and men and measured the proliferation and cytokine production by these cells in response to submaximal amounts of anti-CD3 and anti-CD28. We observed that CD4 + T cells from women produced higher levels of IFNγ as well as tended to proliferate more than male CD4 + T cells. Intriguingly, male CD4 + T cells instead had a predilection toward IL-17A production. This sex dichotomy in Th cytokine production was found to be even more striking in the Swiss/Jackson Laboratory (SJL) mouse. Studies in mice and humans indicated that the sexual dimorphism in Th1 and Th17 cytokine production was dependent on the androgen status and the T-cell expression of peroxisome proliferator activated receptor (PPAR)α and PPARγ. Androgens increased PPARα and decreased PPARγ expression by human CD4 + T cells. PPARα siRNA-mediated knockdown had the effect of increasing IFNγ by male CD4 + T cells, while transfection of CD4 + T cells with PPARγ siRNAs increased IL-17A production uniquely by female T cells. Together, our observations indicate that human T cells exhibit a sex difference in the production of IFNγ and IL-17A that may be driven by expressions of PPARα and PPARγ.

Antibody-mediated rejection (AMR) is gaining increasing recognition as a major complication after heart transplantation, posing a significant risk for allograft failure, cardiac allograft vasculopathy, and poor survival. AMR results from activation of the humoral immune arm and the production of donor-specific antibodies (DSA) that bind to the cardiac allograft causing myocardial injury predominantly through complement activation. The diagnosis of AMR has evolved from a clinical diagnosis involving allograft dysfunction and the presence of DSA to a primarily pathologic diagnosis based on histopathology and immunopathology. Treatment for AMR is multifaceted, targeting inhibition of the humoral immune system at different levels with emerging agents including proteasome and complement inhibitors showing particular promise. While there have been significant advances in our current understanding of the pathogenesis, diagnosis, and treatment of AMR, further research is required to determine optimal diagnostic tools, therapeutic agents, and timing of treatment.

Objective: Broadly neutralizing antibodies (bNt Abs) against HIV-1 are rarely produced during natural infection, and efforts to induce such Abs by vaccination have been unsuccessful. Thus, elucidating the nature and cellular origins of bNt Abs is a high priority for vaccine research. As the bNt monoclonal Abs (MAbs) 2F5, 4E10 and 2G12 have been reported to bind select autoantigens, we investigated whether these MAbs display a broader range of autoreactivity and how their autoreactivity compares with that of pathogenic autoAbs. Methods: An autoantigen microarray comprising 106 connective tissue disease-related autoantigens and control antigens was developed and used, in combination with ELISAs, to compare the reactivity profiles of MAbs 4E10, 2F5 and 2G12 to those of four pathogenic autoAbs derived from patients with antiphospholipid-syndrome (APS), and to serum from a patient with systemic lupus erythematosus (SLE). Results: The APS MAbs and SLE serum reacted strongly with multiple autoantigens on the microarray, whereas anti-HIV-1 MAb reactivity was limited mainly to HIV-1-related antigens. The APS autoAbs reacted strongly with CL, yet only 4E10 bound CL at high concentrations; both 2F5 and 4E10 bound their HIV-1 epitopes with a 2―3-log higher apparent affinity than CL. Moreover, the polyreactivity of 4E10, but not CL15, could be blocked with dried milk. Conclusion: The reactivity profiles of bNt anti-HIV-1 MAbs are fundamentally distinct from those of pathogenic autoAbs that arise from dysregulated tolerance mechanisms. This suggests that the limited polyreactivity observed for the bNt MAbs, and for HIV-1-Nt Abs in general, may arise through alternative mechanisms, such as extensive somatic mutation due to persistent antigen selection during chronic infection.

Three subtypes of alpha 2 adrenergic receptors have been identified in the human and rat. The subtype located on human chromosome 2 (alpha 2-C2) is unique in that it is expressed mainly in the peripheral tissues and lacks sites for N-linked glycosylation. We isolated the gene encoding the mouse homolog of the human alpha 2-C2 adrenergic receptor (M alpha 2-2H). The deduced amino acid sequence of the M alpha 2-2H shows 82% and 96% identity to the human alpha 2-C2 and the rat RNG alpha 2 adrenergic receptors, respectively. Southern blot analysis demonstrated that the M alpha 2-2H was encoded by a single copy gene and was distinct from the mouse homologs of the alpha 2-C4 and alpha 2-C10 adrenergic receptors. When expressed in COS-7 cells, the M alpha 2-2H exhibited a pharmacological profile similar to the human alpha 2-C2 and rat RNG alpha 2 receptors.

Pathological angiogenesis contributes to tobacco-related diseases such as malignancy, atherosclerosis and age-related macular degeneration. Nicotine acts on endothelial nicotinic acetylcholine receptors (nAChRs) to activate endothelial cells and to augment pathological angiogenesis. In the current study, we studied nAChR subunits involved in these actions. We detected mRNA for all mammalian nAChR subunits except alpha(2), alpha(4), gamma, and delta in four different types of ECs. Using siRNA methodology, we found that the alpha(7) nAChR plays a dominant role in nicotine-induced cell signaling (assessed by intracellular calcium and NO imaging, and studies of protein expression and phosphorylation), as well as nicotine-activated EC functions (proliferation, survival, migration, and tube formation). The alpha(9) and alpha(7) nAChRs have opposing effects on nicotine-induced cell proliferation and survival. Our studies reveal a critical role for the alpha(7) nAChR in mediating the effects of nicotine on the endothelium. Other subunits play a modulatory role. These findings may have therapeutic implications for diseases characterized by pathological angiogenesis.

Purpose Previous studies have documented B-cell activation and increases in anti-HLA antibodies after LVAD support. We sought to determine if autoantibodies (non-HLA) antibodies as measured by antigen microarrays also increase after LVAD support. Methods and Materials We measured autoantibodies before and after LVAD support with antigen microarrays. Antigen microarrays were constructed by spotting 61 antigens onto nitrocellulose slides using a microarrayer. After blocking the slides, they were probed with diluted serum. They were then probed with fluorescently labeled secondary antibodies to differentially detect IgG and IgM reactivities. Fluorescent reactivities were detected with a scanner and significance analysis of microarrays (SAM) was used to detect significant changes in antigen reactivities. Total IgG levels were also measured by ELISA. Results We measured autoantibodies before and after implantation of continuous flow LVADs in 22 patients. Mean duration of support was 102 days. SAM analysis revealed 14 autoantibodies that were increased after LVAD. Figure 1 shows a heatmap of pre and post reactivities. Thirteen of these reactivities were IgG and one was IgM. The antibodies that increased were against heart proteins such as myosin, troponin I, and tropomyosin as well as extracellular proteins such as collagen and laminin. Although not statistically significant, there was a trend for increases in autoantibodies to correlate with increases in PRA. Total IgG levels also increased after LVAD support from 15.9 to 17.8 mg/ml. Conclusions We identified 14 autoantibodies against heart and extracellular proteins that increased after LVAD support. Increases in these reactivities and total IgG may reflect immune activation after LVAD.

alpha2-Adrenergic receptors (alpha2ARs) are essential components of the neural circuitry regulating cardiovascular function. The role of specific alpha2AR subtypes (alpha2a, alpha2b, and alpha2c) was characterized with hemodynamic measurements obtained from strains of genetically engineered mice deficient in either alpha2b or alpha2c receptors. Stimulation of alpha2b receptors in vascular smooth muscle produced hypertension and counteracted the clinically beneficial hypotensive effect of stimulating alpha2a receptors in the central nervous system. There were no hemodynamic effects produced by disruption of the alpha2c subtype. These results provide evidence for the clinical efficacy of more subtype-selective alpha2AR drugs.

beta-Adrenergic receptors (beta-AR) are essential regulators of cardiovascular homeostasis. In addition to their prominent function in the heart, beta-AR are located on vascular smooth muscle cells, where they mediate vasodilating effects of endogenous catecholamines. In this study, we have investigated in an isometric myograph different types of blood vessels from mice lacking beta(1)- and/or beta(2)-adrenergic receptor subtypes (beta(1)-KO, beta(2)-KO, beta(1)beta(2)-KO). In wild-type mice, isoproterenol induced relaxation of segments from thoracic aorta, carotid, femoral and pulmonary arteries, and portal vein. The relaxant effect of beta-receptor stimulation was absent in femoral and pulmonary arteries from beta(1)-KO mice. In aortic and carotid arteries and in portal veins, the vasodilating effect of isoproterenol was reduced in mice lacking beta(1)- or beta(2)-receptors. However, in these vessels the vasodilating effect was only abolished in double KO mice lacking both beta(1)- and beta(2)-receptors. Vessel relaxation induced by forskolin did not differ between wild-type and KO mice. Similar contributions of beta(1)- and beta(2)-receptors to isoproterenol-induced vasorelaxation were found when vessels from KO mice were compared with wild-type arteries in the presence of subtype-selective beta-receptor antagonists. These studies demonstrate that beta(1)-adrenergic receptors play a dominant role in the murine vascular system to mediate vasodilation. Surprisingly, beta(2)-receptors contribute to adrenergic vasodilation only in a few major blood vessels, suggesting that differential distribution of beta-adrenergic receptor subtypes may play an important role in redirection of tissue perfusion.

MAP kinase (ERK) translates cell surface signals into alterations in transcription. We have found that ERK also regulates hippocampal neuronal excitability during 5 Hz stimulation and thereby regulates forms of long-term potentiation (LTP) that do not require macromolecular synthesis. Moreover, ERK-mediated changes in excitability are selectively required for some forms of LTP but not others. ERK is required for the early phase of LTP elicited by brief 5 Hz stimulation, as well as for LTP elicited by more prolonged 5 Hz stimulation when paired with β1-adrenergic receptor activation. By contrast, ERK plays no role in LTP elicited by a single 1 s 100 Hz train. Consistent with these results, we find that ERK is activated by β-adrenergic receptors in CA1 pyramidal cell somas and dendrites.

beta-Adrenergic receptors (beta-ARs) are members of the superfamily of G-protein-coupled receptors that mediate the effects of catecholamines in the sympathetic nervous system. Three distinct beta-AR subtypes have been identified (beta1-AR, beta2-AR, and beta3-AR). In order to define further the role of the different beta-AR subtypes, we have used gene targeting to inactivate selectively the beta2-AR gene in mice. Based on intercrosses of heterozygous knockout (beta2-AR +/-) mice, there is no prenatal lethality associated with this mutation. Adult knockout mice (beta2-AR -/-) appear grossly normal and are fertile. Their resting heart rate and blood pressure are normal, and they have a normal chronotropic response to the beta-AR agonist isoproterenol. The hypotensive response to isoproterenol, however, is significantly blunted compared with wild type mice. Despite this defect in vasodilation, beta2-AR -/- mice can still exercise normally and actually have a greater total exercise capacity than wild type mice. At comparable workloads, beta2-AR -/- mice had a lower respiratory exchange ratio than wild type mice suggesting a difference in energy metabolism. beta2-AR -/- mice become hypertensive during exercise and exhibit a greater hypertensive response to epinephrine compared with wild type mice. In summary, the primary physiologic consequences of the beta2-AR gene disruption are observed only during the stress of exercise and are the result of alterations in both vascular tone and energy metabolism.

Publisher Summary The role that individual β AR subtypes play in specific physiological processes has been traditionally defined using subtype-specific ligands. Unfortunately, many of these ligands either possess poor selectivity or are used at concentrations in vivo that can lead to occupation of nonspecific subtypes. The advent of gene disruption techniques in the mouse now enables to selectively delete or alter cloned genes as a means of identifying the specific function(s) of their gene products. To better understand β AR subtype-specific functions in the context of either the whole animal or isolated organs and cells, the genes encoding both β 1 - and β 2 ARs have been disrupted. Gene targeting vectors containing β 1 AR or β 2 AR gene sequences interrupted or partially replaced by a bacterial neomycin resistance gene cassette were flanked by a viral thymidine kinase gene cassette. A standard positive-negative selection strategy (G418 + gancyclovir) has been used to isolate R1 embryonic stem (ES) cells, having undergone homologous recombination at β 1 AR or β 2 AR loci. The pharmacological profile of β 1 AR and β 2 AR knockouts is consistent with the loss of these specific receptor subtypes. When the nonspecific β AR antagonist [ 125 I]cyanopindolol is used in competition binding studies, excess unlabeled CGP 20712A ( β 1 AR-specific antagonist) reveals a selective loss of specific high-affinity sites in β 1 AR knockout heart or lung membranes while excess unlabeled ICI 118,551 ( β 2 AR-specific antagonist) reveals a selective loss of specific high-affinity sites in β 2 AR knockout lung membranes. These results provide further evidence that the β 1 AR and β 2 AR gene disruptions have abolished β AR protein expression. The physiological impact of knocking out either β 1 - or β 2 ARs has been studied in instrumented animals and in isolated tissues.

β-adrenergic receptors (β-ARs) are model G-protein coupled receptors that mediate signal transduction in the sympathetic nervous system. Despite the widespread clinical use of agents that target β-ARs, the signaling pathways that operate downstream of β-AR stimulation have not yet been completely elucidated. Here, we utilized a lysate microarray approach to obtain a broad-scale perspective of phosphoprotein signaling downstream of β-AR. We monitored the time course of phosphorylation states of 54 proteins after β-AR activation mouse embryonic fibroblast (MEF) cells. In response to stimulation with the non-selective β-AR agonist isoproterenol, we observed previously described phosphorylation events such as ERK1/2(T202/Y204) and CREB(S133), but also novel phosphorylation events such as Cdc2(Y15) and Pyk2(Y402). All of these events were mediated through cAMP and PKA as they were reproduced by stimulation with the adenylyl cyclase activator forskolin and were blocked by treatment with H89, a PKA inhibitor. In addition, we also observed a number of novel isoproterenol-induced protein dephosphorylation events in target substrates of the PI3K/AKT pathway: GSK3β(S9), 4E-BP1(S65), and p70s6k(T389). These dephosphorylations were dependent on cAMP, but were independent of PKA and correlated with reduced PI3K/AKT activity. Isoproterenol stimulation also led to a cAMP-dependent dephosphorylation of PP1α(T320), a modification known to correlate with enhanced activity of this phosphatase. Dephosphorylation of PP1α coincided with the secondary decline in phosphorylation of some PKA-phosphorylated substrates, suggesting that PP1α may act in a feedback loop to return these phosphorylations to baseline. In summary, lysate microarrays are a powerful tool to profile phosphoprotein signaling and have provided a broad-scale perspective of how β-AR signaling can regulate key pathways involved in cell growth and metabolism.

antibody levels decreased in both WT and GTKO recipients after implantation. WT recipients, however, retained elevated levels of anti-Gal IgG for at least the first year post implant. Conversely anti-Gal IgG levels in all GTKO recipients fell within one month. The area under the curve showed a significant increase of anti-Gal IgG in the WT BHV group compared to GTKO BHV recipients (p 0.01). Conclusions: Persistently and significantly (p 0.01) elevated levels of anti-Gal IgG were observed in WT but not GTKO BHV recipients. This indicates a BHV-specific continuing immune stimulation to the Gal antigen which is known to be present in current commercially available BHVs, and other bioprosthetic materials of animal origin. These data support the hypothesis that clinical use of current xenogeneic bioprosthetic materials can induce an anti-Gal antibody response which increasing evidence suggests promotes subsequent calcification. BHVs made from GTKO pigs would have reduced antigenicity, may have greater durability and be potentially used in younger patients with more active immune systems.

The mouse beta 1-adrenergic receptor was isolated from a genomic library and cloned into pBluescript SK-. Characterization of the clone revealed an open reading frame which encodes a predicted protein of 466 amino acids. The mouse beta 1 receptor is 92.7% identical to the human sequence, 98.5% identical to the rat sequence, and contains a consensus site for N-linked glycosylation at Asn-15 and a cAMP-dependent protein kinase phosphorylation site at Ser-301.