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Background: In combat-related trauma, resuscitation goals are to attenuate tissue hypoxia and maintain circulation. During hemorrhagic shock, compensatory and autoregulatory mechanisms are activated to preserve cerebral blood flow. Transcranial Doppler (TCD) ultrasonography may be an ideal noninvasive modality to monitor cerebral hemodynamics. Using a nonhuman primate (NHP) model, we attempted to characterize cerebral hemodynamics during polytraumatic hemorrhagic shock using TCD ultrasonography., Materials and Methods: The ophthalmic artery was insonated at multiple time points during varying stages of shock. Hemorrhage was controlled and pressure targeted to 20 mmHg to initiate and maintain the shock period. Mean flow velocity (MFV), peak systolic velocity (PSV), end diastolic velocity (EDV), pulsatility index (PI), and resistance index (RI) were recorded. Results represent mean ± standard deviation; statistical significance is P < 0.05; n = 12., Results: Compared to baseline, MFV, PSV, EDV, and RI show significant changes after 60 min of hemorrhagic shock, (9.81 ± 3.60 cm/s; P < 0.01), (21.15 ± 8.59 cm/s; P < 0.01), (5.15 ± 0.21 cm/s; P < 0.01), (0.70 ± 0.11; P < 0.05), respectively. PI did not change during hemorrhagic shock. At end of prehospital care (T30), cerebral flow recovers for MFV, PSV, and RI while EDV remained decreased at T30 (6.15 ± 1.13 cm/s; P < 0.01) and 1 h of simulated transport (T90) (5.87 ± 0.62 cm/s; P < 0.01). Changes in PI at T30 and T90 were not significant. MFV diminished (16.45 ± 3.85 cm/s; P < 0.05) at T90., Conclusions: This study establishes baseline and hemorrhagic shock values for NHP cerebral blood flow velocities and cerebrovascular indices. TCD ultrasonography may represent an important area of research for targeted resuscitation investigations using a hemorrhagic shock model in NHPs., (Published by Elsevier Inc.)

Background: We endeavored to develop clinically translatable nonhuman primate (NHP) models of severe polytraumatic hemorrhagic shock., Methods: NHPs were randomized into five severe pressure-targeted hemorrhagic shock (PTHS) ± additional injuries scenarios: 30-min PTHS (PTHS-30), 60-min PTHS (PTHS-60), PTHS-60 + soft tissue injury (PTHS-60+ST), PTHS-60+ST + femur fracture (PTHS-60+ST+FF), and decompensated PTHS+ST+FF (PTHS-D). Physiologic parameters were recorded and blood samples collected at five time points with animal observation through T = 24 h. Results presented as mean ± SEM; statistics: log transformation followed by two-way ANOVA with Bonferroni multiple comparisons, Wilcoxon nonparametric test for comparisons, and the Friedmans' one-way ANOVA; significance: P < 0.05., Results: Percent blood loss was 40% ± 2, 59% ± 3, 52% ± 3, 49% ± 2, and 54% ± 2 for PTHS-30, PTHS-60, PTHS-60+ST, PTHS-60+ST+FF, and PTHS-D, respectively. All animals survived to T = 24 h except one in each of the PTHS-60 and PTHS-60+ST+FF groups and seven in the PTHS-D group. Physiologic, coagulation, and inflammatory parameters demonstrated increasing derangements with increasing model severity., Conclusion: NHPs exhibit a high degree of resilience to hemorrhagic shock and polytrauma as evidenced by moderate perturbations in metabolic, coagulation, and immunologic outcomes with up to 60 min of profound hypotension regardless of injury pattern. Extending the duration of PTHS to the point of decompensation in combination with polytraumatic injury, evoked derangements consistent with those observed in severely injured trauma patients which would require ICU care. Thus, we have successfully established a clinically translatable NHP trauma model for use in testing therapeutic interventions to trauma.

Immunophenotyping of whole blood (WB) by flow cytometry (FC) is used clinically to assess a patient's immune status and also in biomedical research. Current protocols recommend storage of immunolabeled samples at 4°C with FC analysis to be completed within seven days. This data acquisition window can be extended to up to one year post-labeling, but this requires cryopreservation of the samples at ultra-low temperatures (≤-80°C or in liquid nitrogen). In this study we optimized a standardized cryopreservation protocol to enable preservation of immunolabeled, human WB samples at -20°C for FC and tested its effectiveness after 0, 5, 15 or 30days. Analysis of stored samples shows that this protocol effectively preserves immunolabeled WB samples and that the duration of storage has no effect on morphology, viability or frequency of WB cell subpopulations, and that the intensity of fluorescent signal from labeled extracellular markers is fully preserved for at least 15days, and up to 30days for some markers. We demonstrate that using this protocol, we are able to differentiate resting versus activated WB cells as demonstrated by detection of significantly increased expression of CD11b by myeloid cells in WB samples pretreated with LPS (100μg/mL for 12h). Finally, we show that this method allows for labeling and detection of the intracellular cytokine (IL-8) up to 30days following cryopreservation from myeloid cells, in previously labeled and cryopreserved WB samples., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Severe trauma initiates a systemic inflammatory cascade and that involves early activation of complement and cleavage of C5 into C5a (anaphylatoxin) and C5b (C5b-9 membrane attack complex). We examined activation of C5 in non-human primate (NHP) models of hemorrhagic shock. Blood plasma concentrations of C5b-9 were significantly increased in NHPs in response to hemorrhage alone and were further increased with the addition of tissue trauma. The onset of increased C5 cleavage was accelerated in NHPs that experienced decompensated poly-traumatic hemorrhagic shock. Next, to identify an effective inhibitor of NHP C5 cleavage in vitro, as a first step in the development of a potential therapy, three inhibitors of human C5 cleavage and hemolysis were tested in vitro. NHP C5 cleavage and complement-mediated hemolysis were successfully inhibited by pre-treatment of serum samples with a small, inhibitory peptide RA101348. Commercially-available C5 inhibitory antibodies were found to exhibit species-specific efficacy in vitro. Quidel's A217 antibody demonstrated dose-dependent inhibition of C5 cleavage and hemolysis in NHP samples, whereas LGM-Eculizumab only inhibited complement-mediated hemolysis in human samples. This study shows that complement activation in NHPs following experimental poly-traumatic hemorrhagic shock is consistent with clinical reports, and that cleavage of C5 and complement-mediated hemolysis can be effectively inhibited in vitro using a small peptide inhibitor. Taken together, these findings offer a clinically-relevant vehicle and a potential strategy for treatment of hemorrhagic shock with poly-traumatic injury., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Hemorrhage is the leading cause of potentially survivable trauma mortality, necessitating the development of improved therapeutic interventions. The objective of this study was to develop and characterize a reproducible clinically translatable nonhuman primate model of uncontrolled severe hemorrhage. Such a model is required to facilitate the development and meaningful evaluation of human-derived therapeutics. In Rhesus macaques, a laparoscopic left-lobe hepatectomy of 25% (n = 2), 50% (n = 4), or 60% (n = 6) was performed at T = 0 min, with no attempt at hemorrhage control until T = 120 min. A constant-rate infusion of normal saline was administered between T = 15 and 120 min to a total volume of 20 mL/kg. At T = 120 min, a laparotomy was performed to gain surgical hemostasis and quantify blood loss. Physiological parameters were recorded, and blood samples were collected at defined intervals until termination of the study at T = 480 min. Statistical analyses used Student t tests, with P < 0.05 considered statistically significant. Results are reported as mean ± SEM. The calculated percent blood loss for the 25% hepatectomy group was negligible (2.3% ± 0.2%), whereas the 50% and 60% hepatectomy groups exhibited 26.6% ± 7.1% and 24.9% ± 3.8% blood loss, respectively. At T = 5 min, blood pressure for the 25%, 50%, and 60% hepatectomy groups was reduced by 13.8%, 60.8%, and 63.2% from the respective baseline values (P < 0.05). In the 60% hepatectomy group, alterations in thromboelastometry parameters and systemic inflammatory markers were observed. The development of a translatable nonhuman primate model of uncontrolled hemorrhage is an ongoing process. This study demonstrates that 60% hepatectomy offers a significant reproducible injury applicable for the evaluation of human-derived therapeutics.

Background: Hemorrhagic shock results in systemic activation of the immune system and leads to ischemia-reperfusion injury. Lymphocytes have been identified as critical mediators of the early innate immune response to ischemia-reperfusion injury, and immunomodulation of lymphocytes may prevent secondary immunologic injury in surgical and trauma patients., Methods: Yorkshire swine were anesthetized and underwent a grade III liver injury with uncontrolled hemorrhage to induce hemorrhagic shock. Experimental groups were treated with a lymphocyte depletional agent, porcine polyclonal anti-thymocyte globulin (PATG) (n = 8) and compared to a vehicle control group (n = 9). Animals were observed over a 3 day survival period. Circulating lymphocytes were examined with FACS analysis for CD3/CD4/CD8, and central lymphocytes with mesenteric lymph node and spleen staining for CD3. Circulating and lung tissue16 infiltrating neutrophils were measured. Circulating CD3 lymphocytes in the blood and in central lymphoid organs (spleen/lymph node) were stained and evaluated using FACS analysis. Immune-related gene expression from liver tissue was quantified using RT-PCR., Results: The overall survival was 22% (2/9) in the control and 75% (6/8) in the PATG groups, p = 0.09; during the reperfusion period (following hemorrhage) survival was 25% (2/8) in the control and 100% (6/6) in the PATG groups, p = 0.008. Mean blood loss and hemodynamic profiles were not significantly different between the experimental and control groups. Circulating CD3+CD4+ lymphocytes were significantly depleted in the PATG group compared to control. Lymphocyte depletion in the setting of hemorrhagic shock also significantly decreased circulating and lung tissue infiltrating neutrophils, and decreased expression of liver ischemia gene expression., Conclusions: Lymphocyte manipulation with a depletional (PATG) strategy improves reperfusion survival in experimental hemorrhagic shock using a porcine liver injury model. This proof of principle study paves the way for further development of immunomodulation approaches to ameliorate secondary immune injury following hemorrhagic shock.

The inflammatory response to severe traumatic injury results in significant morbidity and mortality. Lymphocytes have recently been identified as critical mediators of the early innate immune response to ischemia-reperfusion injury. Experimental manipulation of lymphocytes following hemorrhagic shock may prevent secondary immunologic injury in surgical and trauma patients. The objective of this study is to evaluate the lymphocyte sequestration agent FTY720 as an immunomodulator following experimental hemorrhagic shock in a swine liver injury model. Yorkshire swine were anesthetized and underwent a grade III liver injury with uncontrolled hemorrhage to induce hemorrhagic shock. Experimental groups were treated with a lymphocyte sequestration agent, FTY720, (n = 9) and compared to a vehicle control group (n = 9). Animals were observed over a 3 day survival period after hemorrhage. Circulating total leukocyte and neutrophil counts were measured. Central lymphocytes were evaluated with mesenteric lymph node and spleen immunohistochemistry (IHC) staining for CD3. Lung tissue infiltrating neutrophils were analyzed with myeloperoxidase (MPO) IHC staining. Relevant immune-related gene expression from liver tissue was quantified using RT-PCR. The overall survival was 22.2% in the vehicle control and 66.7% in the FTY720 groups (p = 0.081), and reperfusion survival (period after hemorrhage) was 25% in the vehicle control and 75% in the FTY720 groups (p = 0.047). CD3(+) lymphocytes were significantly increased in mesenteric lymph nodes and spleen in the FTY720 group compared to vehicle control, indicating central lymphocyte sequestration. Lymphocyte disruption significantly decreased circulating and lung tissue infiltrating neutrophils, and decreased expression of liver immune-related gene expression in the FTY720 treated group. There were no observed infectious or wound healing complications. Lymphocyte sequestration with FTY720 improves survival in experimental hemorrhagic shock using a porcine liver injury model. These results support a novel and clinically relevant lymphocyte immunomodulation strategy to ameliorate secondary immune injury in hemorrhagic shock.

Impaired wound healing is a persistent clinical problem which has been treated with mixed results. Studies aimed at elucidating the mechanism of impaired wound healing have focused on small cohorts of genes which leave an incomplete picture of the wound healing process. We aimed to investigate impaired wound healing via a comprehensive panel of angiogenic/inflammation-related genes and wound closure kinetics with and without the application of extracorporeal shock wave therapy (ESWT), which has been demonstrated to improve wound healing. Full-thickness skin from the dorsal surface of "normal" (BALB/c) and "impaired" (db (+)/db (+)) mice was excised, and wound margin tissue was harvested 2, 7, and 10 days post injury. A separate, but identical wound model was established over 40 days in order to measure wound closure kinetics. Over time, the normal non-ESWT treated wounds exhibited varying patterns of elevated expression of 25-30 genes, whereas wounds with impaired healing displayed prolonged elevated expression of only a few genes (CXCL2, CXCL5, CSF3, MMP9, TGF-α). In response to ESWT, gene expression was augmented in both types of wounds, especially in the expression of PECAM-1; however, ESWT had no effect on wound closure in either model. In addition, multiple doses of ESWT exacerbated the delayed wound healing, and actually caused the wounds to initially increase in size. These data provide a more complete picture of impaired wound healing, and a way to evaluate various promising treatments.

Transplant glomerulopathy (TG) is associated with rapid decline in glomerular filtration rate and poor outcome. We used low-density arrays with a novel probabilistic analysis to characterize relationships between gene transcripts and the development of TG in allograft recipients. Retrospective review identified TG in 10.8% of 963 core biopsies from 166 patients; patients with stable function were studied for comparison. The biopsies were analyzed for expression of 87 genes related to immune function and fibrosis by using real-time PCR, and a Bayesian model was generated and validated to predict histopathology based on gene expression. A total of 57 individual genes were increased in TG compared with stable function biopsies (P < 0.05). The Bayesian analysis identified critical relationships between ICAM-1, IL-10, CCL3, CD86, VCAM-1, MMP-9, MMP-7, and LAMC2 and allograft pathology. Moreover, Bayesian models predicted TG when derived from either immune function (area under the curve [95% confidence interval] of 0.875 [0.675 to 0.999], P = 0.004) or fibrosis (area under the curve [95% confidence interval] of 0.859 [0.754 to 0.963], P < 0.001) gene networks. Critical pathways in the Bayesian models were also analyzed by using the Fisher exact test and had P values <0.005. This study demonstrates that evaluating quantitative gene expression profiles with Bayesian modeling can identify significant transcriptional associations that have the potential to support the diagnostic capability of allograft histology. This integrated approach has broad implications in the field of transplant diagnostics.

Background: Graphical probabilistic models have the ability to provide insights as to how clinical factors are conditionally related. These models can be used to help us understand factors influencing health care outcomes and resource utilization, and to estimate morbidity and clinical outcomes in trauma patient populations., Study Design: Thirty-two combat casualties with severe extremity injuries enrolled in a prospective observational study were analyzed using step-wise machine-learned Bayesian belief network (BBN) and step-wise logistic regression (LR). Models were evaluated using 10-fold cross-validation to calculate area-under-the-curve (AUC) from receiver operating characteristics (ROC) curves., Results: Our BBN showed important associations between various factors in our data set that could not be developed using standard regression methods. Cross-validated ROC curve analysis showed that our BBN model was a robust representation of our data domain and that LR models trained on these findings were also robust: hospital-acquired infection (AUC: LR, 0.81; BBN, 0.79), intensive care unit length of stay (AUC: LR, 0.97; BBN, 0.81), and wound healing (AUC: LR, 0.91; BBN, 0.72) showed strong AUC., Conclusions: A BBN model can effectively represent clinical outcomes and biomarkers in patients hospitalized after severe wounding, and is confirmed by 10-fold cross-validation and further confirmed through logistic regression modeling. The method warrants further development and independent validation in other, more diverse patient populations.

Soldiers wounded in modern warfare present with extensive and complicated acute wounds, confounded by an overwhelming inflammatory response. The pathophysiology of acute wounds is unknown and timing of wound closure remains subjective. Collagen gene expression profiles are presented for 24 patients. Impaired healing wounds showed a twofold decrease in the up-regulation of COL1A1 and COL3A1 genes in the beginning of the wound healing process, compared with normal healing wounds. By the final debridement, however, collagen gene expression profiles for normal and impaired healing wounds were similar for COL1A1 and COL3A1. In addition, Raman spectroscopic maps were collected of biopsy tissue sections, from the first and last debridements of 10 wounds collected from nine patients. Tissue components obtained for the debridement biopsies were compared to elucidate whether or not a wound healed normally. Raman spectroscopy showed a loss of collagen in five patients, indicated by a negative percent difference in the 1,665/1,445 cm(-1) band area ratios. Four healed patients showed an increased or unchanged collagen content. Here, we demonstrate the potential of Raman spectroscopic analysis of wound biopsies for classification of wounds as normal or impaired healing. Raman spectroscopy has the potential to noninvasively monitor collagen deposition in the wound bed, during surgical wound debridements, to help determine the optimal time for wound closure.

The Combat Wound Initiative (CWI) program is a collaborative, multidisciplinary, and interservice public-private partnership that provides personalized, state-of-the-art, and complex wound care via targeted clinical and translational research. The CWI uses a bench-to-bedside approach to translational research, including the rapid development of a human extracorporeal shock wave therapy (ESWT) study in complex wounds after establishing the potential efficacy, biologic mechanisms, and safety of this treatment modality in a murine model. Additional clinical trials include the prospective use of clinical data, serum and wound biomarkers, and wound gene expression profiles to predict wound healing/failure and additional clinical patient outcomes following combat-related trauma. These clinical research data are analyzed using machine-based learning algorithms to develop predictive treatment models to guide clinical decision-making. Future CWI directions include additional clinical trials and study centers and the refinement and deployment of our genetically driven, personalized medicine initiative to provide patient-specific care across multiple medical disciplines, with an emphasis on combat casualty care.

Background: Matrix metalloproteinases (MMPs) are crucial in the inflammatory and remodeling phases of wound healing. We previously reported the correlation between pro-inflammatory cytokines and timing of successful combat-wound closure. We now extend our studies to investigate the correlation between wound-remodeling MMP expression and wound healing., Methods: Thirty-eight wounds in 25 patients with traumatic extremity combat wounds were prospectively studied. Surgical debridement with vacuum-assisted closure (VAC) device application was repeated every 48 to 72h until surgical wound closure. Wound effluent and patient serum were collected at each wound debridement and analyzed for five matrix metalloproteinases using the Luminex multiplex system; Millipore Corp, Billerica, MA. The primary outcome was wound healing within 30 d of definitive wound closure. Impairment was defined as delayed wound closure (>21 d from injury) or wound dehiscence. MMP expression was compared between impaired and normal healing wounds., Results: Elevated levels of serum MMP-2 and MMP-7 and reduced levels of effluent MMP3 were seen in impaired wounds (n = 9) compared with wounds that healed (n = 29; P<0.001). Receiver operating characteristic (ROC) curve analysis yielded area-under-the-curve (AUC) of 0.744, 0.783, and 0.805, respectively., Conclusions: Impaired wound healing is characterized by pro-inflammatory MMP-2 and MMP-7. Serum and effluent concentrations of MMP-2, MMP-3, and MMP-7 can effectively predict the outcome of traumatic war wounds and can potentially provide decision-supportive, objective evidence for the timing of wound closure.

Background: Modern war ballistics and blast injuries inflict devastating extremity injuries, violating soft tissue, bone, and neurovascular structures. Despite advances in complex wound management, appropriate timing of war wound closure remains subjective. In addition, the pathophysiology of acute wound failure is poorly defined., Methods: Patients with penetrating extremity wounds sustained during combat were prospectively studied and followed for 30 days after definitive wound closure. The primary outcome was wound healing. Wound dehiscence was defined as spontaneous partial or complete wound disruption after closure. Serum, wound effluent, and wound bed tissue biopsy were collected at each surgical wound debridement. Serum and wound effluent were analyzed with a multiplex array of 22 cytokines and chemokines, and wound tissue for corresponding gene transcript expression., Results: Fifty-two penetrating extremity war wounds in 33 male patients were investigated. Nine (17%) wounds dehisced. Concomitant vascular injury, increased wound size, and higher injury severity score correlated with wound dehiscence. Both serum and wound effluent cytokine and chemokine protein profiles were statistically associated with healing outcome at various time points. Wound biopsy gene transcript expression demonstrated increased tissue inflammation associated with wound failure. Multiple protein and gene transcript biomarkers predictive of wound healing were identified., Conclusions: The cytokine and chemokine protein and gene transcript expression patterns demonstrate a condition of inflammatory dysregulation associated with war wound failure. A molecular biomarker panel may predict combat wound healing outcome and warrants prospective validation.

This pilot study compared the use of the Lifor Organ Preservation Medium (RTLF) at room temperature with hypothermic Belzer machine preservation solution (CMPS) and room in vitro temperature Belzer machine preservation solution (RTMPS) in a porcine model of uncontrolled donation after cardiac death (DCD). In this study, 5 porcine kidneys for each perfusate group were recovered under a DCD protocol. The kidneys were recovered, flushed, and placed onto a renal preservation system following standard perfusion procedures. The average flow rate for CMPS was 36.2 +/- 7.2549 mL/min, RTMPS was 90.2 +/- 9.7159 mL/min, and RTLF was 103.1 +/- 5.1108 mL/min. The average intrarenal resistance for CMPS was 1.33 +/- 0.1709 mm Hg/mL per minute, RTMPS was 0.84 +/- 0.3586 and RTLF was 0.39 +/- 0.04. All perfusion parameters were statistically significant (P < .05) at all time points for the CMPS when compared with both RTMPS and RTLF. All perfusion parameters for RTMPS and RTLF were equivalent for the first 12 hours; thereafter, RTLF became significantly better than RTMPS at 18 and 24 hours. It appears that both RTMPS and RTLF have equivalent perfusion characteristic for the initial 12 hours of perfusion, but LF continues to maintain a low resistance and high flow up to 24 hours. The results of this pilot study indicate that RTLF may represent a better alternative to pulsatile perfusion with CMPS and requires validation in an in vivo large animal transplant model.

Introduction: Human lyophilized platelets hold promise as a novel hemostatic infusion agent for the control of traumatic hemorrhage. Rehydrated, lyophilized platelets (Stasix) were investigated as an infusible hemostatic agent in experimental non-compressible hemorrhage, using a porcine liver injury model., Methods: Yorkshire swine underwent a grade III liver injury and uncontrolled bleeding. After 15 min, animals were infused with Stasix (n = 10) or normal saline vehicle (n = 10). At 2 h, the liver was repaired, and the animals were monitored for another4 h. Resuscitation, including blood transfusion, was administered during the hospital phase. Laboratory data, including arterial blood gas, complete blood count, thromboelastography (TEG), and coagulation parameters, were collected. All animals underwent necropsy with complete histopathologic examination., Results: Overall survival in the Stasix group [8/10 (80%)] was significantly higher than in the control group [2/10 (20%)] (P = 0.023). Mean total blood loss index (g kg(-1)) was lower in Stasix-treated animals (22.2 +/- 3.5) than in control animals (34.7 +/- 3.4) (P = 0.019). Hemodynamic parameters were improved in the Stasix group, and a trend towards higher hemoglobin and lower lactate was observed. Coagulation and TEG parameters were not different between the groups. One surviving animal in the Stasix group had evidence of thrombi on necropsy., Conclusions: This is the first reported study to evaluate rehydrated, lyophilized platelets as an infusible hemostatic agent for non-compressible hemorrhage. Stasix improved survival and reduced blood loss in a liver injury porcine model. However, evidence of thrombotic complications warrants further investigation prior to human use in the setting of traumatic hemorrhage.

Assessment of pulsatile perfusion (PP) is limited to measurements of flow (V) and resistance (R). We investigated infrared (IR) imaging during PP as a means for precise organ assessment. IR was used to monitor 10 porcine kidneys during 18 hr of PP in an uncontrolled Donation after Cardiac Death model. An IR camera (Lockheed Martin) was focused on the anterior surfaces of the kidneys. The degree of temperature homogeneity was compared with standard measurements of V and R. IR thermal images correlated with V and R (R=0.92, P<0.001). IR detected an increase in homogeneity during PP by comparing standard deviation differences before and after PP (P=0.002), which was not evident by standard measurements of V and R. Finally, IR assessment allowed for measurement of dynamic changes in perfusion.

The CD154-specific monoclonal antibody (Mab) hu5c8 greatly prolongs allograft survival in primates. The CD25-specific Mab daclizumab has not, to date, been paired with hu5c8. We evaluated the effects of hu5c8 in vitro, alone and in combination with daclizumab on rhesus-mixed lymphocyte reactions (MLRs). We then evaluated therapy with hu5c8 and daclizumab in four monkey renal allograft recipients compared with monkeys untreated or contemporaneously treated with hu5c8 alone. Lymphocyte proliferation in MLR was reduced by both daclizumab and hu5c8, and their combined effects were additive. Rejection-free allograft survival in monkeys treated with both hu5c8 and daclizumab (74-479 days) was not significantly better than animals treated with hu5c8 alone (257-587 days), and one combined therapy animal rejected while still on hu5c8 therapy, a condition not typically seen with hu5c8 monotherapy. Although daclizumab and hu5c8 are additively effective in MLR, they do not appear to be synergistic in vivo in rhesus monkeys.

Background: Profound T-cell depletion before allotransplantation with gradual posttransplant T-cell repopulation induces a state of donor-specific immune hyporesponsiveness or tolerance in some animal models. Alemtuzumab (Campath-1H, Millennium Pharmaceuticals, Cambridge, MA) is a humanized CD52-specific monoclonal antibody that produces profound T-cell depletion in humans and reduces the need for maintenance immunosuppression after renal transplantation. We therefore performed a study to determine if pretransplant T-cell depletion with alemtuzumab would induce tolerance in human renal allografts and to evaluate the nature of the alloimmune response in the setting of T-cell depletion., Methods: Seven nonsensitized recipients of living-donor kidneys were treated perioperatively with alemtuzumab and followed postoperatively without maintenance immunosuppression. Patients were evaluated clinically by peripheral flow cytometry, protocol biopsies evaluated immunohistochemically, and real-time polymerase chain reaction-based transcriptional analysis., Results: Lymphocyte depletion was profound in the periphery and secondary lymphoid tissues. All patients developed reversible rejection episodes within the first month that were characterized by predominantly monocytic (not lymphocytic) infiltrates with only rare T cells in the peripheral blood or allograft. These episodes were responsive to treatment with steroids or sirolimus or both. After therapy, patients remained rejection-free on reduced immunosuppression, generally monotherapy sirolimus, despite the recovery of lymphocytes to normal levels., Conclusions: T-cell depletion alone does not induce tolerance in humans. These data underscore a prominent role for early responding monocytes in human allograft rejection.

Previous studies has shown that human anti-pig reactivity in mixed lymphocyte cultures require the indirect presentation of antigens by human antigen presenting cells (APC). Xenoreactivity was inhibited by blockade of human costimulatory molecules. We investigated the role of porcine costimulatory molecules in their ability to activate human T cells directly. Porcine CD80 was cloned from lipopolysaccharide (LPS)-activated porcine lymphocytes. Sequence analysis showed a high degree of conservation in residues involved in CD28/CTLA4. COS cells transfected with porcine CD80 was able to activate human T cells in a cyclosporine independent manner, demonstrating that porcine CD80 can costimulate human T cells. Tumor necrosis factor-alpha (TNF-alpha) activated porcine splenocytes have been shown to up-regulate B7s. In order to test the effect of costimulation blockade in a xeno system, activated splenocytes were cultured with purified CD4+ T cells. The results demonstrated that these cells were capable of activating human T cells and this activation can be blocked by using an antihuman CD80 antibody that demonstrated cross-reactivity to porcine CD80. Non-cross reactive antibodies had no effect, again suggesting direct activation of the human T cells. These data suggest that a reagent that can block both the direct and indirect activation is necessary for a discordant xenotransplant.

Anti-CD154 variably prolongs allograft survival in nonhuman primates. Rodent studies suggest that adding pretransplant donor-specific transfusion (DST) and/or rapamycin to anti-CD154 improves survival. The CD154-specific Ab IDEC-131 was tested alone and in combination with rapamycin for its ability to inhibit rhesus MLRs. The ability of the Ab to block endothelial activation was also assessed. IDEC-131 was then tested alone and in combination with DST and/or rapamycin for its ability to prevent rejection of full-thickness, MHC-mismatched rhesus skin allografts. Animals were monitored for donor-specific hyporesponsiveness by MLR and alloantibody determination. IDEC-131 modestly inhibited rhesus MLRs and inhibited CD154-dependent endothelial cell activation. Rapamycin combined with IDEC-131 additively inhibited MLRs. IDEC-131 modestly prolonged allograft survival when compared with no treatment, rapamycin alone, or DST plus rapamycin. Adding DST to IDEC-131 did not prolong survival beyond IDEC-131 alone. IDEC-131 plus rapamycin was effective in prolonging graft survival, although animals had episodes of acute rejection before graft demise. Therapy with IDEC-131, rapamycin, and DST induced long-term allograft survival without intermittent acute rejection. However, no evidence for MLR inhibition was seen, and most animals eventually developed alloantibody. All animals ultimately rejected their grafts after drug withdrawal. IDEC-131 modestly prolongs rhesus skin allograft survival. Rapamycin and rapamycin plus DST improves the efficacy of IDEC-131 in prolonging allograft survival. IDEC-131, rapamycin, and DST are a promising combination for clinical evaluation in allotransplantation.

Background: Antibodies and fusion proteins specific for CD80, CD86, and CD154 have shown promise as agents capable of inducing donor-specific tolerance in rodents. These agents have also been shown to be synergistic with one another in many settings of counter-adaptive immunity. In the nonhuman primate, monoclonal antibodies specific for CD80 and CD86 have prolonged the time to rejection of renal allografts but have not resulted in tolerance. A monoclonal antibody specific for CD154 has resulted in markedly prolonged survival of kidney, islet, cardiac, and skin allografts, but again most animals have eventually developed rejection after prolonged periods of rejection-free survival off therapy., Methods: A combination of monoclonal antibodies specific for CD80, CD86, and CD154 were used in a mismatched nonhuman primate renal-allograft model. Doses used were based on optimized treatment protocols for each agent individually., Results: Treatment of four rhesus macaques with this combination yielded a mean rejection-free survival of 565 days (311-911 days), significantly greater than untreated controls (mean survival=7.0 days, P=0.001) and animals treated with only a combination of anti-CD80 and CD86 (mean survival=191 days, P=0.01). The survival of animals treated with this combination of monoclonal antibodies was not significantly greater than those treated with anti-CD154 alone, but the production of alloantibody was delayed compared with monotherapy anti-CD154., Conclusion: These data suggest that a synergy exists between these agents, particularly with regard to T-dependent B-cell responses, but that they fail to induce durable tolerance in nonhuman primates.

Pneumocystis carinii pneumonia (PCP) is a frequent and serious opportunistic infection in immunocompromized patients. Although the pathogenesis of PCP-mediated lung injury is poorly understood, a central involvement of host inflammatory responses has been implicated. We have found that while the loss of specific T cell costimulatory signals increases susceptibility to the spontaneous pneumocystis infection, PCP-induced pulmonary injury (and subsequent morbidity and mortality) involves other intact costimulatory pathways. Mice that are genetically deficient for the costimulatory receptor CD154 (CD154 knockout (ko) mice) spontaneously developed PCP, consistent with the increased susceptibility of X-linked hyper IgM syndrome patients (caused by CD154 gene mutations) to P. carinii infection. In these mice PCP was manifested by progressive weight loss, dyspnea and death. In contrast, CD154 ko mice also genetically lacking ICAM1 (CD154 koxICAM1 ko) or CD28 (CD154 koxCD28 ko) costimulatory receptors had later onset of weight loss and significantly prolonged survival. Although onset of infection and age-matched P. carinii organism burden were equivalent, the CD154 single knockout mice had evidence of greater pulmonary inflammation vs. the double ko's. These findings suggest that costimulation-dependent T cell-mediated inflammation plays an important role in both susceptibility to and pathogenesis of PCP, and may identify potential molecular targets for novel immunomodulatory treatment approaches.

The anti-CD154 antibody hu5C8 prevents acute allograft rejection and prolongs allograft survival after withdrawal of therapy in nonhuman primates. This study describes the use of hu5C8 as a rescue agent for rejection developing after the withdrawal of hu5C8. Twelve rhesus monkeys that had received renal allografts under hu5C8 induction and subsequently rejected were studied. Rescue with hu5C8 was analyzed based on the histological character of the rejection (acute versus chronic) and whether conventional therapy was received at the time of rescue or induction. The diagnosis of rejection and response to therapy was based on allograft function and histology. Four monkeys that had acute rejection associated with conventional immunosuppression and hu5C8 were not reversed by hu5C8 rescue. Four animals with isolated chronic rejection following prolonged rejection-free survival after the withdrawal of hu5C8 did not respond to hu5C8 rescue therapy. Hu5C8 rescue therapy effectively reversed acute rejection occurring in two monkeys after hu5C8 withdrawal. One of two animals with combined acute on chronic rejection responded to hu5C8 rescue therapy. Hu5C8 effectively reverses acute but not chronic allograft rejection and appears to have no synergistic effect with conventional rescue agents.

A regimen combining sirolimus, tacrolimus, and daclizumab has recently been shown to provide adequate immunosuppression for allogeneic islet transplantation in humans, but remains unproven for primarily vascularized allografts. We evaluated this regimen for renal allograft transplantation in mismatched nonhuman primates. Dosages of sirolimus and tacrolimus were adjusted for trough levels of 10-15 ng/mL and 4-6 ng/mL, respectively. Treated monkeys (n = 5) had significantly prolonged allograft survival, with a mean survival of 36 days vs. 7 days in untreated controls (n = 6, p = 0.008). Four of five treated animals, but none of the controls, developed fibrinoid vascular necrosis of the small intestine. A review of gut histology from animals on other immunosuppressive protocols performed by our laboratory suggested that these lesions were a result of sirolimus exposure. In summary, this regimen prolongs the survival of vascularized renal allografts, but is limited by profound GI toxicity in rhesus macaques.

Background: Delayed xenograft rejection is associated with endothelial cell activation, platelet sequestration, and subsequent thrombosis. We evaluated whether human platelets could directly activate porcine endothelium (PEC), and if so, whether this was mediated by an interaction between platelet-bound CD154 and PEC CD40., Methods: Platelet activation was achieved by thrombin exposure and confirmed by evaluation of up-regulated CD62P and CD154. Co-incubation of platelets or D1.1 cells with PEC was performed, and PEC activation was evaluated by up-regulation of CD62E., Results: Co-incubation of resting platelets that lacked significant expression of CD62P and were void of CD154 did not activate PEC. In contrast, thrombin-activated human platelets expressing considerable amounts of both CD62P and CD154 induced PEC activation. This activation could be completely inhibited by coincubation with a humanized monoclonal antibody directed at human CD154 (hu5c8). Similarly, human D1.1 cells expressing CD154 were shown to activate PEC in a CD154-dependent manner., Conclusion: Human CD154 expressed on activated human platelets or on T cells interacts with CD40 expressed on PEC leading to PEC activation. This interaction can be inhibited by a monoclonal antibody directed against CD154, suggesting that an interaction between human CD154 and PEC CD40 is at least in part responsible for PEC activation seen in delayed xenograft rejection. These data strengthen the rationale for the use of CD154-directed therapy in discordant xenotransplantation.

Background: Allogeneic skin transplantation remains a rigorous test of any immune intervention designed to prevent allograft rejection. To date, no single, clinically available immunosuppressant has been reported to induce long-term primary skin allograft survival in primates. We have previously shown that treatment with the humanized CD154-specific monoclonal antibody, humanized 5C8 (hu5C8), induces long-term renal allograft survival in nonhuman primates. In this study, we evaluated the efficacy of hu5C8 in preventing primary skin allograft rejection in rhesus monkeys., Methods: Ten rhesus monkeys were transplanted with full-thickness skin allografts mismatched at both class I and class II major histocompatibility loci. Of these, two were given no treatment, five were treated with hu5C8 alone, and three received hu5C8 combined with whole blood donor-specific transfusion (DST). All recipients also received skin autografts for comparison. Animals were followed by inspection, serial biopsy, mixed lymphocyte culture, and alloantibody determination., Results: Treatment with hu5C8 alone or hu5C8 plus DST greatly prolonged allograft survival. Rejection occurred in the untreated group within 7 days. Mean allograft survival in the monotherapy hu5C8 group was >236 days and in the DST group was >202 days; these differences were not significant. Rejection eventually occurred in most animals. Allograft survival was not correlated with the development of T cell hyporesponsiveness in mixed lymphocyte culture. Rejection was not predicted by the development of donor-specific alloantibody., Conclusion: These results show that treatment with the CD154-specific monoclonal antibody, hu5C8, greatly delays the onset of acute skin allograft rejection.

CD80 and CD86 (also known as B7-1 and B7-2, respectively) are both ligands for the T cell costimulatory receptors CD28 and CD152. Both CD80 and CD86 mediate T cell costimulation, and as such, have been studied for their role in promoting allograft rejection. In this study we demonstrate that administering monoclonal antibodies specific for these B7 ligands can delay the onset of acute renal allograft rejection in rhesus monkeys. The most durable effect results from simultaneous administration of both anti-B7 antibodies. The mechanism of action does not involve global depletion of T or B cells. Despite in vitro and in vivo evidence demonstrating the effectiveness of the anti-B7 antibodies in suppressing T cell responsiveness to alloantigen, their use does not result in durable tolerance. Prolonged therapy with murine anti-B7 antibodies is limited by the development of neutralizing antibodies, but that problem was avoided when humanized anti-B7 reagents are used. Most animals develop rejection and an alloantibody response although still on antibody therapy and before the development of a neutralizing antibody response. Anti-B7 antibody therapy may have use as an adjunctive agent for clinical allotransplantation, but using the dosing regimens we used, is not a tolerizing therapy in this non-human primate model.

CD154 plays a critical role in determining the outcome of a transplanted organ. This simple statement is amply supported by experimental evidence demonstrating that anti-CD154 antibodies are potent inhibitors of allograft rejection in many rigorous transplant models. Unfortunately, despite intensive investigation over the past ten years, the precise mechanisms by which antibodies against CD154 exert their anti-rejection effects have remained less obvious. Though originally classified with reference to B-cell function, CD154-CD40 interactions have also been shown to be important in T cell-antigen-presenting cell interactions. Accordingly, CD154 has been classified as a T-cell co-stimulatory molecule. However, mounting data suggest that treatment with anti-CD154 antibodies does not simply block costimulatory signals, but rather that the antibodies appear to induce signalling in receptor-bearing T cells. Other data suggest that anti-CD154 effects may be mediated by endothelial cells and possibly even platelets. In fact, the current literature suggests that CD154 can either stimulate or attenuate an immune response, depending upon the model system under study. CD154 has secured a fundamental place in transplant biology and general immunology that will no doubt be the source of considerable investigation and therapeutic manipulation in the coming decade.

Background: T-cell costimulatory blocking agents inhibit allospecific T-cell responses in vitro and prevent allograft rejection in vivo. Costimulatory requirements for discordant xenospecific cellular responses remain undefined. We have evaluated costimulatory molecule expression by porcine endothelial cells (PEC) after interaction with human cells and tested agents known to inhibit allospecific responses for their ability to inhibit xenospecific responses in vitro., Methods: Human-specific agents were screened for their ability to bind porcine costimulatory molecules by FACS. Up-regulation of B7 molecules on PEC was evaluated by FACS after exposure to human cells or supernatants. The effect of human and/or porcine costimulatory blockade was tested in xeno-mixed lymphocyte reactions (XMLRs) and in natural killer (NK) cell cytotoxicity assays., Results: B7 expression was induced on PEC after exposure to human T and NK cells or T cell-conditioned medium. The human XMLR was attenuated by human CTLA4-Ig and anti-human CD154 (hu5C8), and the combination was synergistic. Anti-human CD80 and CD86 antibodies alone had minor effects in the XMLR, but in combination with hu5C8 were as effective as human CTLA4-Ig plus hu5C8. Anti-hCD80 and hCD86 antibodies that did not cross-react with porcine CD80 or CD86 were as effective in blocking the MLR as those that did cross-react, indicating that the predominant costimulation in vitro was derived from the responding cells. None of the agents affected the xeno-NK response., Conclusions: We conclude that the costimulation-modulating agents block human anti-porcine T-cell responses in vitro predominantly through interruption of costimulation derived from responding cells. They have no effect on NK cell-mediated cytotoxicity.

Signals generated through CD28-B7 and CD40 ligand (CD40L)-CD40 interactions have been shown to be crucial for the induction of long-term allograft survivability. We have recently demonstrated that humanized anti-CD40L (hu5C8) prevents rejection of mismatched renal allografts in primates. To investigate potential mechanisms of CD40L-induced allograft acceptance, we coimmobilized hu5C8 with suboptimal amounts of anti-CD3 to stimulate CD4(+) T cells. We now report that anti-CD3/CD40L costimulation results in CD28-independent activation and subsequent deletion of resting T cells. Coligation of CD3 and CD40L increased expression of CD69, CD25, and CD54 on CD4(+) T cells. We also found that costimulation with anti-CD3/CD40L resulted in enhanced production of interleukin (IL)-10, interferon gamma, and tumor necrosis factor alpha but not IL-2 or IL-6. Interestingly, after several days, anti-CD3/CD40L-mediated activation was followed by apoptosis in a significant population of cells. Consistent with that observation, anti-CD3/CD40L did not enhance the antiapoptotic proteins Bcl-2 and Bcl-xL. Further, the addition of CD28 at 24 h failed to rescue those cells induced to die after costimulation with anti-CD3/CD40L. Together, these data suggest that the graft-sparing effect of hu5C8 in vivo may result in part from early and direct effects on CD4(+) T cells, including a vigorous induction of immunomodulatory cytokines and/or apoptosis of allograft-specific T cells.

CD154 is the ligand for the receptor CD40. This ligand-receptor pair mediates endothelial and antigen-presenting cell activation, and facilitates the interaction of these cells with T cells and platelets. We demonstrate here that administration of a CD154-specific monoclonal antibody (hu5C8) allows for renal allotransplantation in outbred, MHC-mismatched rhesus monkeys without acute rejection. The effect persisted for more than 10 months after therapy termination, and no additional drug was required to achieve extended graft survival. Indeed, the use of tacrolimus or chronic steroids seemed to antagonize the anti-rejection effect. Monkeys treated with antibody against CD154 remained healthy during and after therapy. The mechanism of action does not require global depletion of T or B cells. Long-term survivors lost their mixed lymphocyte reactivity in a donor-specific manner, but still formed donor-specific antibody and generated T cells that infiltrated the grafted organ without any obvious effect on graft function. Thus, therapy with antibody against CD154 is a promising agent for clinical use in human allotransplantation.

The biological importance of Leu15 of epidermal growth factor (EGF) is suggested by its conservation through evolution, its critical location in the domain-domain interface of EGF and its close proximity to Arg41, a residue that is crucial for receptor binding and activation. Mutagenesis of Leu15 of human EGF (hEGF) was employed to examine the role of this residue in the ligand-receptor interaction. The relative receptor affinities of the hEGF variants, as determined by radioreceptor competition assays, varied depending on the amino acid substitution. The L15F, L15W and L15V hEGF analogues had receptor affinities 45, 26 and 18% respectively of wild type hEGF. The L15A and L15R analogues displayed receptor affinities of only 2.4 and 1.6% relative to wild type hEGF. No binding of the L15E analogue was detected. The relative agonist activities, as measured by receptor tyrosine kinase stimulation assays, generally followed a similar trend. The L15F, L15W and L15V analogues stimulated the receptor kinase to a level (Vmax) similar to that for wild type hEGF. A striking difference was observed between the L15A and L15R variants; although having similar binding affinities, the L15A mutant activated the receptor to only approximately 5% of the wild type Vmax in contrast to 53% for the L15R mutant. 1H-NMR analysis of the L15R and L15A mutants showed only minor structural alterations that were not sufficient to account for the dramatic losses in binding and agonist activities. The results indicate that both the size and hydrophobicity of the gamma-branched aliphatic side chain of Leu15 of hEGF are important in the formation of a catalytically active ligand-receptor complex.

Using members of the epidermal growth factor (EGF) family as well as site-directed recombinant human EGF mutants, we investigated how ligand binding properties influence endosomal sorting. Mouse EGF (mEGF), human EGF (hEGF), and transforming growth factor alpha (TGF alpha) bind to the human EGF receptor (EGFR) with similar affinities at pH 7.4. However, the binding properties of these ligands have substantially different pH sensitivities resulting in varying degrees of dissociation from the receptors at lower pH levels characteristic of endosomes. We employed a steady-state sorting assay to determine the fraction of ligand sorted to recycling versus degradation as a function of the number of intracellular ligand molecules in mouse B82 fibroblasts. mEGF, hEGF, and TGF alpha display significantly different steady-state endosomal sorting patterns which correspond to the extent of their dissociation at endosomal pH. Moreover, several recombinant hEGF mutants with differing affinities exhibit altered endosomal sorting compared to hEGF, demonstrating a similar direct relationship between ligand binding properties and endosomal sorting outcomes. Intracellular trafficking of the EGF ligands was also monitored by measuring the observed degradation rate constants. These likewise show marked differences that correlate with the differing pH sensitivities of the ligands' binding properties.

The tyrosine at position 13 of epidermal growth factor (EGF) has been implicated as playing a role in receptor binding due to its close proximity to the critical arginine 41 residue as well as its high degree of conservation in EGF and EGF-like proteins that can bind to the EGF receptor. Site-directed mutagenesis of tyrosine 13 in human EGF (hEGF) was employed to examine the role of this residue in ligand-receptor interaction. The removal of the hydroxyl moiety of the tyrosine by substitution with phenylalanine had little effect on the binding, indicating that it is not involved in any crucial hydrogen bonds with either the receptor or with other regions of the EGF molecule. The substitution of the aromatic tyrosine side-chain with the nonpolar leucine side-chain caused the receptor affinity to decrease only slightly, indicating that aromaticity of the amino acid at this site is also not critical. Substitutions with other hydrophobic residues, isoleucine, valine, and alanine, resulted in a significant decrease in receptor affinity as a function of decreasing hydrophobicity. Substitution of tyrosine 13 with the polar residues histidine and arginine markedly decreased receptor binding affinity, and complete removal of the side-chain by substitution with glycine dramatically lowered the binding affinity to 0.3% as compared to wild type. Analysis of three hEGF mutants, Tyr13-->Leu, Tyr13-->Arg, and Tyr13-->Gly, by circular dichroism showed that the major structural features of hEGF were not significantly altered. The results demonstrate that the decreased receptor affinities of these hEGF mutants are due to disruption of the functional contribution(s) of the tyrosine 13 residue rather than alteration(s) in the overall structural integrity. Overall, the results suggest that the tyrosine 13 side-chain plays a critical role in receptor binding by contributing to hydrophobic receptor-ligand interactions.

Four residues in the carboxy-terminal domain of human epidermal growth factor (hEGF), glutamate 40, glutamine 43, arginine 45, and aspartate 46 were targeted for site-directed mutagenesis to evaluate their potential role in epidermal growth factor (EGF) receptor-ligand interaction. One or more mutations were generated at each of these sites and the altered recombinant hEGF gene products were purified and evaluated by radioreceptor competition binding assay. Charge-conservative replacement of glutamate 40 with aspartate resulted in a decrease in receptor binding affinity to 30% relative to wild-type hEGF. On the other hand, removal of the electrostatic charge by substitution of glutamate 40 with glutamine or alanine resulted in only a slightly greater decrease in receptor binding to 25% relative receptor affinity. The introduction of a positive charge upon substitution of glutamine 43 with lysine had no effect on receptor binding. The substitution of arginine 45 with lysine also showed no effect on receptor binding, unlike the absolute requirement observed for the arginine side-chain at position 41 [Engler DA, Campion SR, Hauser MR, Cook JS, Niyogi, SK: J Biol Chem 267:2274-2281, 1992]. Subsequent elimination of the positive charge of lysine 45 by reaction with potassium cyanate showed that the electrostatic property of the residue at this site, as well as that at lysine 28 and lysine 48, was not required for receptor-ligand association.(ABSTRACT TRUNCATED AT 250 WORDS)