Growth models for Salmonella, E. coli O157:H7 and L. monocytogenes give different predictions for pathogen growth in cut leafy greens transportation, but are consistent in identifying higher risk conditions.

Leafy greens are frequently implicated in foodborne disease outbreaks and cut-leafy greens are a food that requires time and temperature control for safety. Predictive microbiology uses mathematical models to predict the growth of bacteria based on environmental conditions. The objective of our study was to compare published square root growth models for Salmonella (n = 6), pathogenic E. coli (n = 6) and Listeria monocytogenes (n = 4) using real world transport temperature data. Data from trucks transporting fresh-cut leafy greens during cross-country shipments were used as temperature inputs to the models. Bacterial growth was computed using the temperatures from each probe in every truck over the duration of transit, which resulted in 12-18 growth predictions per truck for each model. Each model generally gave significantly different predictions than other models for the same organism. The exception was for the two Salmonella models predicting the least growth and the two Salmonella models predicting the most growth which gave predictions that were not significantly different. Although different models tended to give different predictions, their ability to rank risk by truck was generally consistent across models. While absolute risk might be dependent upon choice of model, relative risk is independent of model choice.

Cohort antler size signals environmental stress in a moderate climate.

Research in northern latitudes confirms that climate teleconnections exert important influences on ungulate fitness, but studies from regions with milder climates are lacking. We explored the influence of the Pacific Decadal Oscillation (PDO), Northern Atlantic Oscillation (NAO), and El Niño-Southern Oscillation (ENSO) on male, 2.5-year-old white-tailed deer (Odocoileus virginianus) antler and body mass in Mississippi, USA, a region with mild winters and warm, humid summers. Explanatory variables were seasonal averages of each climate index extending back to 3 years prior to account for possible maternal and lag effects. Seasonal climate indices from the period of gestation and the first year of life were correlated with deer morphometrics. Reduced antler mass was largely correlated (R2 = 0.52) with PDO values indicating dry conditions during parturition and neonatal development and NAO values indicating warmer than normal winters during gestation and the first year of life. Body mass was less correlated (R2 = 0.16) to climate indices, responding negatively to warmer winter weather during the first winter of life. Climate may promote variable fitness among cohorts through long-term effects on male competition for dominance and breeding access. Because broad-scale climate indices simplify complex weather systems, they may benefit management at larger scales. Although this study compared climate with morphological variables, it is likely that demographic characteristics can likewise be modeled using climate indices. As climate change in this region is projected to include greater variability in summer precipitation, we may see concomitantly greater variability in fitness among cohorts of white-tailed deer.

Marginal zone B cells are critical to factor VIII inhibitor formation in mice with hemophilia A.

Although factor VIII (FVIII) replacement therapy can be lifesaving for patients with hemophilia A, neutralizing alloantibodies to FVIII, known as inhibitors, develop in a significant number of patients and actively block FVIII activity, making bleeding difficult to control and prevent. Although a variety of downstream immune factors likely regulate inhibitor formation, the identification and subsequent targeting of key initiators in inhibitor development may provide an attractive approach to prevent inhibitor formation before amplification of the FVIII immune response occurs. As the initial steps in FVIII inhibitor development remain incompletely understood, we sought to define early regulators of FVIII inhibitor formation. Our results demonstrate that FVIII localizes in the marginal sinus of the spleen of FVIII-deficient mice shortly after injection, with significant colocalization with marginal zone (MZ) B cells. FVIII not only colocalizes with MZ B cells, but specific removal of MZ B cells also completely prevented inhibitor development following FVIII infusion. Subsequent rechallenge with FVIII following MZ B-cell reconstitution resulted in a primary antibody response, demonstrating that MZ B-cell depletion did not result in FVIII tolerance. Although recipient exposure to the viral-like adjuvant polyinosinic:polycytidylic acid enhanced anti-FVIII antibody formation, MZ B-cell depletion continued to display similar effectiveness in preventing inhibitor formation following FVIII infusion in this inflammatory setting. These data strongly suggest that MZ B cells play a critical role in initiating FVIII inhibitor formation and suggest a potential strategy to prevent anti-FVIII alloantibody formation in patients with hemophilia A.

A subset of high-titer anti-factor VIII A2 domain antibodies is responsive to treatment with factor VIII.

The primary B-cell epitopes of factor VIII (fVIII) are in the A2 and C2 domains. Within the C2 domain, antibody epitope and kinetics are more important than inhibitor titer in predicting pathogenicity in a murine bleeding model. To investigate this within the A2 domain, the pathogenicity of a diverse panel of antihuman fVIII A2 domain monoclonal antibodies (MAbs) was tested in the murine model. MAbs were injected into hemophilia A mice, followed by injection of human B domain-deleted fVIII. Blood loss after a 4-mm tail snip was measured. The following anti-A2 MAbs were tested: high-titer type 1 inhibitors 4A4, 2-76, and 1D4; 2-54, a high-titer type 2 inhibitor; B94, a type 2 inhibitor; and noninhibitory MAbs GMA-012, 4C7, and B25. All high-titer type 1 MAbs produced blood loss that was significantly greater than control mice, whereas all non-inhibitory MAbs produced blood loss that was similar to control. The type 2 MAbs were not pathogenic despite 2-54 having an inhibitor titer of 34 000 BU/mg immunoglobulin G. In addition, a patient with a high-titer type 2 anti-A2 inhibitor who is responsive to fVIII is reported. The discrepancy between inhibitor titer and bleeding phenotype combined with similar findings in the C2 domain stress the importance of inhibitor properties not detected in the standard Bethesda assay in predicting response to fVIII therapy.

High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors.

Inhibitor formation in hemophilia A is the most feared treatment-related complication of factor VIII (fVIII) therapy. Most inhibitor patients with hemophilia A develop antibodies against the fVIII A2 and C2 domains. Recent evidence demonstrates that the C1 domain contributes to the inhibitor response. Inhibitory anti-C1 monoclonal antibodies (mAbs) have been identified that bind to putative phospholipid and von Willebrand factor (VWF) binding epitopes and block endocytosis of fVIII by antigen presenting cells. We now demonstrate by competitive enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry that 7 of 9 anti-human C1 mAbs tested recognize an epitope distinct from the C1 phospholipid binding site. These mAbs, designated group A, display high binding affinities for fVIII, weakly inhibit fVIII procoagulant activity, poorly inhibit fVIII binding to phospholipid, and exhibit heterogeneity with respect to blocking fVIII binding to VWF. Another mAb, designated group B, inhibits fVIII procoagulant activity, fVIII binding to VWF and phospholipid, fVIIIa incorporation into the intrinsic Xase complex, thrombin generation in plasma, and fVIII uptake by dendritic cells. Group A and B epitopes are distinct from the epitope recognized by the canonical, human-derived inhibitory anti-C1 mAb, KM33, whose epitope overlaps both groups A and B. Antibodies recognizing group A and B epitopes are present in inhibitor plasmas from patients with hemophilia A. Additionally, group A and B mAbs increase fVIII clearance and are pathogenic in a hemophilia A mouse tail snip bleeding model. Group A anti-C1 mAbs represent the first identification of pathogenic, weakly inhibitory antibodies that increase fVIII clearance.

Complement Dependence of Murine Costimulatory Blockade-Resistant Cellular Cardiac Allograft Rejection.

Building on studies showing that ischemia-reperfusion-(I/R)-injury is complement dependent, we tested links among complement activation, transplantation-associated I/R injury, and murine cardiac allograft rejection. We transplanted BALB/c hearts subjected to 8-h cold ischemic storage (CIS) into cytotoxic T-lymphocyte associated protein 4 (CTLA4)Ig-treated wild-type (WT) or c3-/- B6 recipients. Whereas allografts subjected to 8-h CIS rejected in WT recipients with a median survival time (MST) of 37 days, identically treated hearts survived >60 days in c3-/- mice (p < 0.05, n = 4-6/group). Mechanistic studies showed recipient C3 deficiency prevented induction of intragraft and serum chemokines/cytokines and blunted the priming, expansion, and graft infiltration of interferon-γ-producing, donor-reactive T cells. MST of hearts subjected to 8-h CIS was >60 days in mannose binding lectin (mbl1-/- mbl2-/- ) recipients and 42 days in factor B (cfb-/- ) recipients (n = 4-6/group, p < 0.05, mbl1-/- mbl2-/- vs. cfb-/- ), implicating the MBL (not alternative) pathway. To pharmacologically target MBL-initiated complement activation, we transplanted BALB/c hearts subjected to 8-h CIS into CTLA4Ig-treated WT B6 recipients with or without C1 inhibitor (C1-INH). Remarkably, peritransplantation administration of C1-INH prolonged graft survival (MST >60 days, p < 0.05 vs. controls, n = 6) and prevented CI-induced increases in donor-reactive, IFNγ-producing spleen cells (p < 0.05). These new findings link donor I/R injury to T cell-mediated rejection through MBL-initiated, complement activation and support testing C1-INH administration to prevent CTLA4Ig-resistant rejection of deceased donor allografts in human transplant patients.

Comparing Normothermic Machine Perfusion Preservation With Different Perfusates on Porcine Livers From Donors After Circulatory Death.

The utilization of normothermic machine perfusion (NMP) may be an effective strategy to resuscitate livers from donation after circulatory death (DCD). There is no consensus regarding the efficacy of different perfusates on graft and bile duct viability. The aim of this study was to compare, in an NMP porcine DCD model, the preservation potential of three different perfusates. Twenty porcine livers with 60 min of warm ischemia were separated into four preservation groups: cold storage (CS), NMP with Steen solution (Steen; XVIVO Perfusion Inc., Denver, CO), Steen plus red blood cells (RBCs), or whole blood (WB). All livers were preserved for 10 h and reperfused to simulate transplantation for 24 h. During preservation, the NMP with Steen group presented the highest hepatocellular injury. At reperfusion, the CS group had the lowest bile production and the worst hepatocellular injury compared with all other groups, followed by NMP with Steen; the Steen plus RBC and WB groups presented the best functional and hepatocellular injury outcomes, with WB livers showing lower aspartate aminotransferase release and a trend toward better results for most parameters. Based on our results, a perfusate that contains an oxygen carrier is most effective in a model of NMP porcine DCD livers compared with Steen solution. Specifically, WB-perfused livers showed a trend toward better outcomes compared with Steen plus RBCs.

B cell activating factor (BAFF) and a proliferation inducing ligand (APRIL) mediate CD40-independent help by memory CD4 T cells.

Donor-reactive memory T cells undermine organ transplant survival and are poorly controlled by immunosuppression or costimulatory blockade. Memory CD4 T cells provide CD40-independent help for the generation of donor-reactive effector CD8 T cells and alloantibodies (alloAbs) that rapidly mediate allograft rejection. The goal of this study was to investigate the role of B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) in alloresponses driven by memory CD4 T cells. The short-term neutralization of BAFF alone or BAFF plus APRIL synergized with anti-CD154 mAb to prolong heart allograft survival in recipients containing donor-reactive memory CD4 T cells. The prolongation was associated with reduction in antidonor alloAb responses and with inhibited reactivation and helper functions of memory CD4 T cells. Additional depletion of CD8 T cells did not enhance the prolonged allograft survival suggesting that donor-reactive alloAbs mediate late graft rejection in these recipients. This is the first report that targeting the BAFF cytokine network inhibits both humoral and cellular immune responses induced by memory CD4 T cells. Our results suggest that reagents neutralizing BAFF and APRIL may be used to enhance the efficacy of CD40/CD154 costimulatory blockade and improve allograft survival in T cell-sensitized recipients.

Acute and chronic rejection: compartmentalization and kinetics of counterbalancing signals in cardiac transplants.

Acute and chronic rejection impact distinct compartments of cardiac allografts. Intramyocardial mononuclear cell infiltrates define acute rejection, whereas chronic rejection affects large arteries. Hearts transplanted from male to female C57BL/6 mice undergo acute rejection with interstitial infiltrates at 2 weeks that resolve by 6 weeks when large arteries develop arteriopathy. These processes are dependent on T cells because no infiltrates developed in T cell-deficient mice and transfer of CD4 T cells restored T cell as well as macrophage infiltrates and ultimately neointima formation. Markers of inflammatory macrophages were up-regulated in the interstitium acutely and decreased as markers of wound healing macrophages increased chronically. Programmed cell death protein, a negative costimulator, and its ligand PDL1 were up-regulated in the interstitium during resolution of acute rejection. Blocking PDL1:PD1 interactions in the acute phase increased interstitial T cell infiltrates. Toll-like receptor (TLR) 4 and its endogenous ligand hyaluronan were increased in arteries with neointimal expansion. Injection of hyaluronan fragments increased intragraft production of chemokines. Our data indicate that negative costimulatory pathways are critical for the resolution of acute interstitial infiltrates. In the arterial compartment recognition of endogenous ligands including hyaluronan by the innate TLRs may support the progression of arteriopathy.

Anti-huCD20 antibody therapy for antibody-mediated rejection of renal allografts in a mouse model.

We have reported that B6.CCR5(-/-) mice reject renal allografts with high serum donor-specific antibody (DSA) titers and marked C4d deposition in grafts, features consistent with antibody-mediated rejection (AMR). B6.huCD20/CCR5(-/-) mice, where human CD20 expression is restricted to B cells, rejected A/J renal allografts by day 26 posttransplant with DSA first detected in serum on day 5 posttransplant and increased thereafter. Recipient treatment with anti-huCD20 mAb prior to the transplant and weekly up to 7 weeks posttransplant promoted long-term allograft survival (>100 days) with low DSA titers. To investigate the effect of B cell depletion at the time serum DSA was first detected, recipients were treated with anti-huCD20 mAb on days 5, 8, and 12 posttransplant. This regimen significantly reduced DSA titers and graft inflammation on day 15 posttransplant and prolonged allograft survival >60 days. However, DSA returned to the titers observed in control treated recipients by day 30 posttransplant and histological analyses on day 60 posttransplant indicated severe interstitial fibrosis. These results indicate that anti-huCD20 mAb had the greatest effect as a prophylactic treatment and that the distinct kinetics of DSA responses accounts for acute renal allograft failure versus the development of fibrosis.

Graft-derived CCL2 increases graft injury during antibody-mediated rejection of cardiac allografts.

The pathogenic role of macrophages in antibody-mediated rejection (AMR) remains unclear. Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a potent chemotactic factor for monocytes and macrophages. The current studies used a murine model of AMR to investigate the role of graft-derived CCL2 in AMR and how macrophages may participate in antibody-mediated allograft injury. B6.CCR5−/−/CD8−/− recipients rejected MHC-mismatched WT A/J allografts with high donor-reactive antibody titers and diffuse C4d deposition in the large vessels and myocardial capillaries, features consistent with AMR. In contrast, A/J.CCL2−/− allografts induced low donor-reactive antibody titers and C4d deposition at Day 7 posttransplant. Decreased donor-reactive CD4 T cells producing interferon gamma were induced in response to A/J.CCL2−/− versus WT allografts. Consequently, A/J.CCL2−/− allograft survival was modestly but significantly longer than A/J allografts. Macrophages purified from WT allografts expressed high levels of IL-1ß and IL-12p40 and this expression and the numbers of classically activated macrophages were markedly reduced in CCL2-deficient allografts on Day 7. The results indicate that allograft-derived CCL2 plays an important role in directing classically activated macrophages into allografts during AMR and that macrophages are important contributors to the inflammatory environment mediating graft tissue injury in this pathology, suggesting CCL2 as a therapeutic target for AMR.

IL-15 induces alloreactive CD28(-) memory CD8 T cell proliferation and CTLA4-Ig resistant memory CD8 T cell activation.

The presence of CD28(-) memory CD8 T cells in the peripheral blood of renal transplant patients is a risk factor for graft rejection and resistance to CTLA-4Ig induction therapy. In vitro analyses have indicated poor alloantigen-induced CD28(-) memory CD8 T cell proliferation, raising questions about mechanisms mediating their clonal expansion in kidney grafts to mediate injury. Candidate proliferative cytokines were tested for synergy with alloantigen in stimulating CD28(-) memory CD8 T cell proliferation. Addition of IL-15, but not IL-2 or IL-7, to co-cultures of CD28(-) or CD28(+) memory CD8 T cells and allogeneic B cells rescued proliferation of the CD28(-) and enhanced CD28(+) memory T cell proliferation. Proliferating CD28(-) memory CD8 T cells produced high amounts of interferon gamma and tumor necrosis factor alpha and expressed higher levels of the cytolytic marker CD107a than CD28(+) memory CD8 T cells. CTLA-4Ig inhibited alloantigen-induced proliferation of CD28(+) memory CD8 T cell proliferation but had no effect on alloantigen plus IL-15-induced proliferation of either CD28(-) or CD28(+) memory CD8 T cells. These results indicate the ability of IL-15, a cytokine produced by renal epithelial during inflammation, to provoke CD28(-) memory CD8 T cell proliferation and to confer memory CD8 T cell resistance to CTLA-4Ig-mediated costimulation blockade.

Endogenous memory CD8 T cells are activated within cardiac allografts without mediating rejection.

Endogenous memory CD8 T cells infiltrate MHC-mismatched cardiac allografts within 12-24 h posttransplant in mice and are activated to proliferate and produce IFN-γ. To more accurately assess the graft injury directly imposed by these endogenous memory CD8 T cells, we took advantage of the ability of anti-LFA-1 mAb given to allograft recipients on days 3 and 4 posttransplant to inhibit the generation of primary effector T cells. When compared to grafts from IgG-treated recipients on day 7 posttransplant, allografts from anti-LFA-1 mAb-treated recipients had increased numbers of CD8 T cells but these grafts had marked decreases in expression levels of mRNA encoding effector mediators associated with graft injury and decreases in donor-reactive CD8 T cells producing IFN-γ. Despite this decreased activity within the allograft, CD8 T cells in allografts from recipients treated with anti-LFA-1 mAb continued to proliferate up to day 7 posttransplant and did not upregulate expression of the exhaustion marker LAG-3 but did have decreased expression of ICOS. These results indicate that endogenous memory CD8 T cells infiltrate and proliferate in cardiac allografts in mice but do not express sufficient levels of functions to mediate overt graft injury and acute rejection.

Creating labial bone for immediate implant placement: a minimally invasive approach by using orthodontic therapy in the esthetic zone.

Orthodontic extrusion of nonrestorable teeth has been used for almost 20 years as an alternative to bone grafting in preparation for implant placement. Although this technique predictably creates bone and soft tissue, and improves the socket diameter and depth, most of the bone apposition occurs in the marginal alveolar and periapical areas of the extruded tooth. To create more labial bone, the standard orthodontic extrusion technique was modified to apply pressure on the hopeless tooth both coronally and palatally, which allowed bone at the site to develop apically and labially. Gingival thickness on the labial aspect was also increased, and the tissue biotype was improved. A clinical treatment is presented that illustrates the use of this technique.

Antibody-mediated rejection of single class I MHC-disparate cardiac allografts.

Murine CCR5(-/-) recipients produce high titers of antibody to complete MHC-mismatched heart and renal allografts. To study mechanisms of class I MHC antibody-mediated allograft injury, we tested the rejection of heart allografts transgenically expressing a single class I MHC disparity in wild-type C57BL/6 (H-2(b)) and B6.CCR5(-/-) recipients. Donor-specific antibody titers in CCR5(-/-) recipients were 30-fold higher than in wild-type recipients. B6.K(d) allografts survived longer than 60 days in wild-type recipients whereas CCR5(-/-) recipients rejected all allografts within 14 days. Rejection was accompanied by infiltration of CD8 T cells, neutrophils and macrophages, and C4d deposition in the graft capillaries. B6.K(d) allografts were rejected by CD8(-/-)/CCR5(-/-), but not µMT(-/-)/CCR5(-/-), recipients indicating the need for antibody but not CD8 T cells. Grafts recovered at day 10 from CCR5(-/-) and CD8(-/-)/CCR5(-/-) recipients and from RAG-1(-/-) allograft recipients injected with anti-K(d) antibodies expressed high levels of perforin, myeloperoxidase and CCL5 mRNA. These studies indicate that the continual production of antidonor class I MHC antibody can mediate allograft rejection, that donor-reactive CD8 T cells synergize with the antibody to contribute to rejection, and that expression of three biomarkers during rejection can occur in the absence of this CD8 T cell activity.

The spleen is the major source of antidonor antibody-secreting cells in murine heart allograft recipients.

Antibody-mediated allograft rejection is an increasingly recognized problem in clinical transplantation. However, the primary location of donor-specific alloantibody (DSA)-producing cells after transplantation have not been identified. The purpose of this study was to test the contribution of allospecific antibody-secreting cells (ASCs) from different anatomical compartments in a mouse transplantation model. Fully MHC-mismatched heart allografts were transplanted into three groups of recipients: nonsensitized wild type, alloantigen-sensitized wild-type and CCR5(-/-) mice that have exaggerated alloantibody responses. We found that previous sensitization to donor alloantigens resulted in the development of antidonor alloantibody (alloAb) with accelerated kinetics. Nevertheless, the numbers of alloantibody-secreting cells and the serum titers of antidonor IgG alloantibody were equivalent in sensitized and nonsensitized recipients 6 weeks after transplantation. Regardless of recipient sensitization status, the spleen contained higher numbers of donor-reactive ASCs than bone marrow at days 7-21 after transplantation. Furthermore, individual spleen ASCs produced more antidonor IgG alloantibody than bone marrow ASCs. Taken together, our results indicate that the spleen rather than bone marrow is the major source of donor-reactive alloAb early after transplantation in both sensitized and nonsensitized recipients.

Antibody-mediated rejection--an ounce of prevention is worth a pound of cure.

The presence of preformed, donor-specific alloantibodies inpatients undergoing renal transplantation is associated with a high risk of hyperacute and acute antibody-mediated rejection (ABMR), and often limits potential recipients' access to organs from living and deceased donors. Over the last decade, understanding of ABMR has improved markedly and given rise to numerous, diverse strategies for the transplantation of allosensitized recipients. Antibody desensitization programs have been developed to allow renal transplant recipients with a willing but antibody-incompatible living donor to undergo successful transplantation, whereas kidney paired exchange schemes circumvent the antibody incompatibility altogether by finding suitable pairs to donors and recipients. Recognizing the complexity of ABMR and the recent developments that have occurred in this important clinical research field, the Roche Organ Transplantation Research Foundation (ROTRF) organized a symposium during the XXIII Congress of The Transplantation Society in Vancouver, Canada, to discuss current understanding in ABMR and ways to prevent it. This Meeting Report summarizes the presentations of the symposium, which addressed key areas that included the interactions between alloantibodies and the complement system in mediating graft injury, technological advancements for assessing antibody-mediated immune responses to HLA antigens, and the potential benefits and challenges of desensitization and kidney paired donation schemes.