Cerebral Blood Flow in Polytrauma: Transcranial Doppler Analysis in a Nonhuman Primate Shock Model.

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.

Nonhuman Primate (Rhesus Macaque) Models of Severe Pressure-Targeted Hemorrhagic and Polytraumatic Hemorrhagic Shock.

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) ±â€Šadditional 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 ±â€ŠSEM; 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% ±â€Š2, 59% ±â€Š3, 52% ±â€Š3, 49% ±â€Š2, and 54% ±â€Š2 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.

Generation of complement molecular complex C5b-9 (C5b-9) in response to poly-traumatic hemorrhagic shock and evaluation of C5 cleavage inhibitors in non-human primates.

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.

Cryopreservation of human whole blood allows immunophenotyping by flow cytometry up to 30days after cell isolation.

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.

Development of a Nonhuman Primate (Rhesus Macaque) Model of Uncontrolled Traumatic Liver Hemorrhage.

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.

Inflammatory cytokine and chemokine expression is associated with heterotopic ossification in high-energy penetrating war injuries.

OBJECTIVE: Heterotopic ossification (HO) develops frequently after modern high-energy penetrating war injuries. The purpose of this prospective study was to identify and characterize the unique cytokine and chemokine profile associated with the development of HO as it pertained to the systemic inflammatory response after penetrating combat-related trauma. METHODS: Patients with high-energy penetrating extremity wounds were prospectively enrolled. Surgical debridement along with the use of a pulse lavage and vacuum-assisted-closure device was performed every 48-72 hours until definitive wound closure. Wound bed tissue biopsy, wound effluent, and serum were collected before each debridement. Effluent and serum were analyzed for 22 relevant cytokines and chemokines. Tissue was analyzed quantitatively for bacterial colonization. Correlations between specific wound and patient characteristics were also analyzed. The primary clinical outcome measure was the formation of HO as confirmed by radiographs at a minimum of 2 months of follow-up. RESULTS: Thirty-six penetrating extremity war wounds in 24 patients were investigated. The observed rate of HO in the study population was 38%. Of the 36 wounds, 13 (36%) demonstrated HO at a minimum follow-up of 2 months. An elevated injury severity score was associated with the development of HO (P = 0.006). Wound characteristics that correlated with the development of HO included impaired healing (P = 0.005) and bacterial colonization (P < 0.001). Both serum (interleukin-6, interleukin-10, and MCP-1) and wound effluent (IP-10 and MIP-1α) cytokine and chemokine bioprofiles were individually associated and suggestive of the development of HO (P < 0.05). CONCLUSIONS: A severe systemic and wound-specific inflammatory state as evident by elevated levels of inflammatory cytokines, elevated injury severity score, and bacterial wound colonization is associated with the development of HO. These findings suggest that the development of HO in traumatic combat-related wounds is associated with a hyper-inflammatory systemic response to injury. LEVEL OF EVIDENCE: Prognostic Level II. See Instructions for Authors for a complete description of levels of evidence.

Inflammatory response is associated with critical colonization in combat wounds.

BACKGROUND: Modern combat- or blast-related injuries are characterized by devastatingly massive zones of injury that violate soft tissue, bone, and neurovascular structures. In our translational research program, we have determined that healing of traumatic combat wounds is dependent on the immune response. Although the majority of combat wounds are not critically colonized with bacteria, there exists a correlation between critical colonization and the concentration of inflammatory cytokines and chemokines measured in wound effluent or patient serum. METHODS: Patients with penetrating extremity wounds sustained during combat operations were studied prospectively, being followed for 30 days after definitive wound closure. Surgical debridement was repeated every 48-72 h until wound closure at the discretion of the attending surgeon. Serum, wound effluent, and wound bed tissue biopsy were collected at each debridement. Serum and wound effluent were analyzed with a multiplex assay for cytokines, chemokines, and inflammatory proteases, whereas wound tissue was assessed for microbial colonization via quantitative cultures. Correlations between serum and effluent cytokines and chemokines and the degree of tissue colonization were evaluated. RESULTS: Samples from 154 debridements in 38 wounds from 25 male patients were investigated. Many of the patients sustained multi-system trauma (mean Injury Severity Score 21±12 points) and were critically ill (mean Acute Physiology and Chronic Health Evaluation II score 7±5 points). Healing failure occurred in 23.7% of wounds. A marked inflammatory profile, including increased serum and wound effluent cytokines and chemokines, was associated with the extent of critical colonization. CONCLUSIONS: The correlation between systemic and local inflammatory cytokines and quantitative culture suggests that the interplay between the systemic response to injury and the local wound environment is a determinant of outcome. This relationship remains ill defined and requires further investigation in both clinical and pre-clinical studies. A biomarker panel reflective of colonization may provide clinically useful, objective criteria indicating when wound closure is appropriate for successful healing.

Incidence of pulmonary embolus in combat casualties with extremity amputations and fractures.

BACKGROUND: The objective of this retrospective study was to determine the incidence of pulmonary embolism (PE) in casualties of wartime extremity wounds and specifically in casualties with a trauma-associated amputation. METHODS: Records of all combat-wounded evacuated and admitted between March 1, 2003, and December 31, 2007, were retrospectively reviewed. Continuous and categorical variables were studied with the Student's t test, Fisher's exact test or χ² test; multivariate analysis was performed using a stepwise regression logistic model. RESULTS: A total of 1,213 records were reviewed; 263 casualties met the inclusion criteria. One hundred three (41.5%) had amputations and 145 (58.5%) had long-bone fractures not requiring amputation. The observed rate of PE in these 263 casualties was 5.7%. More casualties with amputations, 10 (3.7%), developed PE than those with long-bone fractures in the absence of amputation, 5 (1.9%) (p = 0.045). Casualties with bilateral lower extremity trauma-associated amputations had a significantly higher incidence of PE compared with those sustaining a single amputation (p = 0.023), and the presence of bilateral lower extremity amputations was an independent risk factor for development of a PE (p = 0.007, odds ratio 5.9) (univariate and multivariate analysis, respectively). CONCLUSION: The cumulative incidence of PE was 5.7%. The incidence of PE is significantly higher with trauma-associated amputation than with extremity long-bone fracture without amputation. Bilateral amputations, multiple long-bone fractures, and pelvic fractures are independent risk factors for the development of PE. The use of aggressive prophylaxis, deep venous thrombosis screening with ultrasound, and use of prophylactic inferior vena cava filters should be considered in this patient population.

Comparative analysis of angiogenic gene expression in normal and impaired wound healing in diabetic mice: effects of extracorporeal shock wave therapy.

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.

Probabilistic (Bayesian) modeling of gene expression in transplant glomerulopathy.

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.

Metalloproteinase expression is associated with traumatic wound failure.

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.

Development of a Bayesian model to estimate health care outcomes in the severely wounded.

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.

Assessment of cadaveric organ viability during pulsatile perfusion using infrared imaging.

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.

Extracorporeal shock wave therapy suppresses the early proinflammatory immune response to a severe cutaneous burn injury.

Following severe burn injury, persistent inflammation perpetuated by surface eschar, bacterial colonisation and neutrophil proteolytic activity can impede normal healing and result in further tissue damage. Extracorporeal shock wave treatment (ESWT) has been shown in the clinical setting to promote the healing of burn and difficult-to-heal wounds; however, the mechanism is unclear. We investigated the role of ESWT on the early proinflammatory response using a severe, full-thickness and highly inflammatory cutaneous burn wound in a murine model. Various wound-healing parameters were measured and leukocyte infiltration quantitated. A panel of 188 candidate genes known to be involved in acute inflammation and wound healing was screened. We show that ESWT of burn wounds 1 hour postwounding significantly blunts polymorphonuclear neutrophil and macrophage infiltration into the wound. ESWT treatment potently attenuates both CC- and CXC-chemokine expression, acute proinflammatory cytokine expression and extracellular matrix proteolytic activity at the wound margin. Given these findings and the clinical success of ESWT, we speculate that ESWT may be a potential therapeutic modality to treat severe wounds wherein excessive inflammatory responses involving increased levels of inflammatory cells, proinflammatory cytokines and proteases may become self-resolving allowing wound healing to progresses by way of normal physiological repair processes.

New directions for induction immunosuppression strategy in solid organ transplantation.

BACKGROUND: Solid organ transplant centers are increasingly using induction immunosuppression strategies. Induction immunosuppression involves the use of intense therapy at the time of transplantation with the goal of preventing acute rejection and ultimately inducing a tolerogenic state. The objective of this review is to examine specialized induction agents currently in clinical use and highlight novel therapeutics on the horizon for induction immunosuppression. METHODS: A literature search using the PubMed and MEDLINE databases identified salient basic science and clinical research articles on induction immunosuppression for solid organ transplantation. CONCLUSIONS: While current induction immunosuppression agents have reduced the incidence of acute rejection, the goal of transplant tolerance has not been realized. Furthermore, the long-term allograft survival rate is not clearly influenced by the practice of induction immunosuppression. New approaches to tolerance induction, such as costimulatory-based therapy, mixed chimerism, and adoptive cellular transfer, hold promise for more effective induction immunosuppression in solid organ transplantation.

Angiogenic response to extracorporeal shock wave treatment in murine skin isografts.

Skin grafts are commonly utilized and proven effective methods of open wound coverage. Revascularization through neoangiogenesis is a pivotal mechanism for skin graft integration and durability. Extracorporeal shock-wave treatment (ESWT) has been demonstrated to accelerate wound repair; however, its mechanism-of-action is unclear. We investigated the role of ESWT in early revascularization of full-thickness skin isografts in a murine model. Cohorts of mice were euthanized and skin grafts were harvested 6 h, 2, 4, and 7 days post grafting +/- ESWT. Various aspects of graft neovascularization were measured including gross morphology, quantitative microscopy (vessel number, density), immunohistochemistry (CD31), cDNA SuperArrays for 84 angiogenesis-specific genes, and custom-designed 'Wound Repair' TaqMan Low Density Array (TLDA) cards to assess expression of 188 wound repair genes. We demonstrate that a single administration of ESWT immediately following skin grafting significantly enhances recipient graft revascularization (increased vessel number, size, and density). An augmented early pro-angiogenic and suppressed delayed pro-inflammatory response to ESWT was accompanied by significantly increased expression of both skin graft CD31 and angiogenesis pathway-specific genes, including ELR-CXC chemokines (CXCL1, CXCL2, CXCL5), CC chemokines (CCL2, CCL3, CCL4), cytokines (IL-1 beta, IL-6, G-CSF, VEGF-A), matrix metalloproteinases (MMP3, MMP9, MMP13), hypoxia-inducible factors (HIF-1 alpha), and vascular remodeling kinase (Mst1), as early as 6 h and up to 7 days post grafting and treatment. These findings suggest that early pro-angiogenic and anti-inflammatory effects of ESWT promote tissue revascularization and wound healing by augmenting angiogenesis and dampening inflammation.

Human brain endothelial cells (HUBEC) promote SCID repopulating cell expansion through direct contact.

The objective of this study was to re-evaluate the previously published hematopoietic stem cell (HSC) expansion work using human brain endothelial cells (HUBEC). The expansion effect of contact and non-contact conditions was reported to be equivalent by others. However, we report here different results that the expansion can be achieved only with direct contact. We co-cultured human CD34+ cells with and without HUBEC contact for seven days with cytokines and the readouts were CD34+ / CD38 - phenotype and SCID repopulating cell (SRC) frequency. Also tested was the inhibitory effect of Wnt receptor inhibitor Dkk-1 on HUBEC contact ex vivo expansion; whether an increased expression of Wnt3 occurs on the HUBEC surface; and detection of an increased nuclear localization of beta-catenin in CD34+ / CD38- cells in HUBEC contact culture condition. We conclude that the successful expansion by HUBEC contact culture is a candidate explanation based on the Wnt family protein, possibly Wnt3, expression on HUBEC.

Differential cutaneous wound healing in thermally injured MRL/MPJ mice.

Adult wound repair occurs with an initial inflammatory response, reepithelialization, and the formation of a permanent scar. MRL/MpJ mice following ear-hole punch biopsies display accelerated healing and tissue regeneration. In this study, we characterized the healing responses in both MRL/MpJ and BALB/c mice following a 15% total body surface area full-thickness cutaneous burn injury. Macroscopic and histological observations show that delayed wound closure in MRL/MpJ mice is accompanied by an increase in edema, reduced neutrophil infiltration, and more prominent eschar. In vivo bromodeoxyuridine labeling showed no defect in keratinocyte proliferation and migration (reepithelialization). In comparison with BALB/c mice, MRL/MpJ wounds had greater collagen deposition, less granulation tissue formation, and contained fewer alpha-smooth muscle actin-positive myofibroblasts. An observed reduction in dermal neutrophil infiltration and myofibroblast development correlated with enhanced angiogenesis. Overall, BALB/c wounds contracted sooner and to a larger degree, resulting in a significant decrease in scar formation. Interestingly, MRL/MpJ mice showed overt abnormalities in hair follicle proliferation, morphogenesis, and subsequent hair regrowth postburn injury. No substantial evidence of tissue regeneration was observed in either BALB/c or MRL/MpJ wounds. Our results convincingly demonstrate that MRL/MpJ skin burn wounds heal with scar formation with delays in two critical wound healing events: wound closure, and myofibroblast development.

The role of NO in macrophage dysfunction at early stage after burn injury.

AIM: To explore the role of nitric oxide (NO) in macrophage dysfunction at early stage after burn injury. METHOD: Peritoneal macrophages were isolated and cultured from early stage burnt mice. NO production and inducible NO synthase (iNOS) expression in the macrophages were checked by the Greiss method and real-time PCR (TaqMan), respectively. l-Arginine, the substrate of NO producing, or N-monomethyl-l-arginine (l-NMMA), a competing blocker of NOS was administered to the culture, the changes of NO, TNF-alpha and PGE2 productions were measured, additionally the changes of the iNOS, TNF-alpha and COX-2 expression were assayed by real-time PCR. After that, the effects of l-arginine and l-NMMA were determined on burnt macrophage influencing the proliferation of normal splenic lymphocytes. RESULT: A large amount of NO was produced by macrophages from post burn hour 6 (6PBH) with a high level of iNOS expression. l-Arginine could increase NO production in a dosage-dependent manner, while l-NMMA attenuated NO production, but neither could affect iNOS expression. Moreover, l-arginine enhanced productions of both the latter produced TNF-alpha and PGE2 from burnt macrophages, and the expressions of TNF-alpha and COX-2 were improved significantly, while l-NMMA did reverse ways. It was found that macrophages from post burn hour 24 mice could inhibit Con A-stimulated normal splenic lymphocytes dramatically, l-NMMA could decrease this function significantly, but l-arginine could not influence the suppression. CONCLUSION: Our experiment indicated NO derived from burnt macrophage played a vital role in macrophage producing excessive TNF-alpha and PGE2, and suppressing lymphocyte function at early stage after burn injury.

Effects of combined treatment with CD25- and CD154-specific monoclonal antibodies in non-human primate allotransplantation.

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.