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In spite of the advances in the knowledge of adipose-derived stem cells (ASCs), in situ location of ASCs and the niche component of adipose tissue (AT) remain controversial due to the lack of an appropriate culture system. Here we describe a fibrin matrix-supported three-dimensional (3D) organ culture system for AT which sustains the ASC niche and allows for in situ mobilization and expansion of ASCs in vitro. AT fragments were completely encapsulated within the fibrin matrix and cultured under dynamic condition. The use of organ culture of AT resulted in a robust outgrowth and proliferation in the fibrin matrix. The outgrown cells were successfully recovered from fibrin by urokinase treatment. These outgrown cells fulfilled the criteria of mesenchymal stem cells, adherence to plastic, multilineage differentiation, and cell surface molecule expression. In vitro label retaining assay revealed that newly divided cells during the culture resided in interstitium between adipocytes and capillary endothelial cells. These interstitial stromal cells proliferated and outgrew into the fibrin matrix. Both in situ mobilized and outgrown cells expressed CD146 and alpha-smooth muscle actin (SMA), but no endothelial cell markers (CD31 and CD34). The structural integrity and spatial approximation of CD31(-)/CD34(-)/CD146(+)/SMA(+) interstitial stromal cells, adipocytes, and capillary endothelial cells were well preserved during in vitro culture. Our results suggest that ASCs are natively associated with the capillary wall and more specifically, belong to a subset of pericytes. Furthermore, organ culture of AT within a fibrin matrix-supported 3D environment can recapitulate the ASC niche in vitro.

We describe synthetic extracellular matrix (sECM) hydrogel films composed of co-crosslinked thiolated derivatives of chondroitin 6-sulfate (CS) and heparin (HP) for controlled-release delivery of basic fibroblast growth factor (bFGF) to full-thickness wounds in genetically diabetic (db/db) mice. In this model for chronic wound repair, full-thickness wounds were treated with CS, CS-bFGF, or CS-HP-bFGF films. At 2 and 4 weeks postinjury, wound closure and formation of the new epidermis and dermis were determined. Both CS and CS-HP hydrogel films accelerated wound repair, even without bFGF. Addition of bFGF to CS films showed partial dose-dependent acceleration of wound repair. Importantly, addition of bFGF to co-crosslinked CS-HP sECM films showed a dramatic bFGF dose-dependent acceleration of wound healing, as well as improved dermis formation and vascularization. Compared with 27% wound closure in 2 weeks in the controls, 89% wound closure was observed for mice treated with the CS-HP-bFGF films. The synthetic CS-HP sECM films mimic the chemistry and biology of heparan sulfate proteoglycans, and may have clinical potential for topical delivery of growth factors to patients with compromised wound healing.

Chemically crosslinked glycosaminoglycan (GAG) hydrogel films were evaluated as biointeractive dressings in a porcine model for donor-site autograft wounds. Multiple 5 × 5 × 0.03 cm3 wounds were created on the dorsum of pigs. Half of the wounds were treated with a GAG film plus an occlusive dressing (Tegaderm™), whereas the other half were treated with Tegaderm™ alone. At 3, 5, or 7 days after surgery, the partially healed wounds were excised and evaluated histologically for three animals at each time point. By day 3, epithelial cells had proliferated and migrated from wound edges and from epithelial islands associated with residual hair follicles to begin to cover the wound bed. A statistically significant increase in coverage was observed for GAG + Tegaderm™-dressed wounds than for those with Tegaderm™ alone at day 3 and day 5 post-surgery. By day 7, all treatment groups were completely healed. Thus, GAG hydrogels accelerated wound healing by enhancing re-epithelialization.

Chemically-crosslinked glycosaminoglycan (GAG) hydrogel films were prepared and evaluated as bio-interactive wound dressings. Hyaluronan (HA) and chondroitin sulfate (CS) were first converted to the adipic dihydrazide derivatives and then crosslinked with poly(ethylene glycol) propiondialdehyde to give a polymer network. The crosslinking occurred at neutral pH in minutes at room temperature to give clear, soft hydrogels. After gelation, a solvent-casting method was used to obtain a GAG hydrogel film. A mouse model was used to evaluate the efficacy of these GAG films in facilitating wound healing. Full-thickness wounds were created on the dorsal side of Balb/c mice and were dressed with a GAG film plus Tegaderm' or TegadermT' alone. A significant increase in re-epithelialization was observed on day 5 (p < 0.001) and day 7 (p < 0.05) for wounds treated with a GAG film plus Tegaderm versus those treated with Tegaderm alone. While no significant differences in wound contraction or inflammatory response were found, wounds treated with either HA or CS films showed more fibro-vascular tissue by day 10. The GAG hydrogel films provide a highly hydrated, peri-cellular environment in which assembly of other matrix components. presentation of growth and differentiation factors, and cell migration can readily occur.

Calcium signaling patterns are important for the specific regulation of activation and effector function in lymphocytes. Studies of [Ca2+]i regulation in lymphocytes, including the involvement of ryanodine receptors (RyR) and the importance of caffeine-sensitive pools, have been carried out mainly in lymphocyte cell lines and the presence and functional importance of these pools in primary lymphocytes has not been addressed. Here we show by confocal microscopy that caffeine caused a prompt but transitory increase of [Ca2+]i in primary lymphocytes, an effect that was inhibited by pre-treatment with ryanodine. Furthermore, the increase of [Ca2+]i in CD4+ and CD8+ MLR T lymphocytes stimulated by 5 microg/ml concanavalin A was significantly inhibited by pretreatment with caffeine. In functional studies, caffeine decreased cytotoxicity against myocyte target cells which is probably related to an altered calcium signaling in CD8+ MLR lymphocytes. Caffeine also terminated spontaneous Ca2+ oscillations and induced a rise in [Ca2+]i in CD4- and CD8- MLR lymphocytes probably of B cell origin. These results demonstrate that caffeine alters Ca2+ signaling in primary lymphocytes, and suggest that RyR, probably the skeletal muscle receptor (RyR-1) and brain receptor (RyR-3), are involved in mediating this effect. It is also possible that blocking of inositol-1,4,5-triphosphate (IP3) receptors is involved in the effects of caffeine on lymphocyte activation.

Interleukin-18 (IL-18) and IL-12 have been shown to play an important role in the induction of interferon-gamma (IFN-gamma). IFN-gamma induces the proliferation of T cells and natural killer (NK) cells and augments the Th1 immune cascade. The role of IL-18 and IL-12 in the induction of IFN-gamma following allogeneic heart transplantation has not been described. We sought to characterize the IL-12 and IL-18 response to murine allogeneic heart transplantation, particularly with respect to IFN-gamma production and histologic transplant rejection. Forty-eight heterotopic heart transplants were performed in two groups of mice: syngeneic C3H/HeN to C3H/HeN mice and allogeneic BALB/C to C3H/HeN mice. Transplants were followed out to 2, 6, 10, and 14 days. Six transplants were performed in each group. Serum and splenic samples were used to evaluate the cytokine response by ELISA. Explanted heart tissue was processed for evidence of histologic rejection, and RT-PCR was performed to evaluate the IL-12, IL-18, and IFN-gamma signal qualitatively. Analysis of variance (ANOVA), Fisher's projected least significant difference (PLSD) was used for statistical analysis. Transplant rejection occurred in the allogeneic group histologically by day 6 and clinically by day 10. Serum IFN-gamma levels rose significantly by day 6 in the allogeneic group and then continued to rise in the splenocyte cultures. Serum IL-18 also rose significantly in the allogeneic group at day 6 compared with syngeneic group. RT-PCR revealed that the allogeneic tissue contained an increased signal for IL-12, IL-18, and IFN-gamma beginning at day 6 and peaking at day 10 after transplant. Beginning 6 days after transplantation, IL-12 and IL-18 appear to play a significant role in the induction of IFN-gamma in allogeneic heart transplants.

M. Ritter, Z. Su, S. Xu, J. Shelby and W. H. Barry. Cardiac Unloading Alters Contractility and Calcium Homeostasis in Ventricular Myocytes. Journal of Molecular and Cellular Cardiology (2000) 32, 577–584. Altered cardiac workload has an important effect on myocyte structure and function. Cardiac hypertrophy resulting from an increase in load has been studied extensively in the past. However, the effects of unloading and atrophy have recently become of more interest since devices for mechanical left ventricular unloading have been introduced into clinical practice for the treatment of patients with terminal heart failure, and a resulting improved cardiac and myocyte contractility have been reported. We used the heterotopic abdominal mouse heart transplant model in order to study the effects of 5 days of unloading on cell size (confocal microscopy), contractility (fractional shortening: video motion), calcium homeostasis ([Ca 2+ ] i transients, SR Ca 2+ content); and L-type Ca 2+ and sodium/calcium exchanger currents (whole cell patch clamp technique). We found unloading caused decreased cell volume consistent with atrophy. An increased fractional shortening and [Ca 2+ ] i transient were observed in myocytes from unloaded hearts as compared with controls. Transsarcolemmal I Ca,L and I Na/Ca densities, and SR Ca 2+ content were unaltered, as was membrane capacitance. A reduction in cell volume with mainteinance of internal and surface membrane areas, and/or a decrease in concentration of cellular protein Ca 2+ buffers, may contribute to the increase in the [Ca 2+ ] i transient in this model.

Background. Previous in vitro studies have suggested that both cytotoxic T lymphocyte (CTL)-mediated and non-CTL-mediated myocyte lysis occur during murine cardiac heterotopic allograft rejection, but the relative importance of these injury mechanisms on myocardial function is not established. We therefore compared the in vivo effects of depletion of CTL and inhibition of nitric oxide synthase (NOS) on contractility of the rejecting heart. Methods. Syngeneic (BALB/c into BALB/c) and allogeneic (BALB/c into C57/B16) heterotopic abdominal cardiac transplants were performed. In some of the allogeneic transplants, CD8 + lymphocytes were depleted by intraperitoneal injection of anti-CD8 monoclonal antibody. NOS inhibition was accomplished by continuous infusion of N G -monomethyl-L-arginine via a subcutaneous osmotic pump. Five days after transplantation, the abdominal cavity was opened and the transplanted heart exposed. Base to apex developed force was measured during spontaneous beating at a diastolic stretch of 4 g by placing a suture through the apex of the heart and attaching it to a strain gauge. Effects of interventions on graft survival were determined by recording the days required for loss of palpable graft contractions. Results. Allogeneic hearts showed a significant reduction in systolic force compared to non-rejecting syngeneic hearts. Depletion of CD8 + cells improved contractility significantly relative to non-depleted allogeneic hearts, but contractility remained significantly reduced relative to syngeneic hearts. Developed force in allogeneic hearts was also improved by NOS inhibition (P

Resuscitation from shock based on oxygen transport criteria has been widely used in trauma and surgical patients, but has not been examined in thermally injured patients. To study the possible efficacy of this type of resuscitation, the oxygen transport characteristics of burn resuscitation were studied in nine adults, of whom six had inhalation injuries, with a mean burn size of 45% total body surface area and a mean age of 33.4 years, who were resuscitated based on oxygen transport criteria. Pulmonary artery balloon flotation catheters were placed and hemodynamic and oxygen transport parameters (Fick method) were measured hourly for 6 hours. Patients received fluid boluses in addition to resuscitation calculated by the Parkland formula, until the pulmonary artery wedge pressure reached 15 mm Hg, after which dobutamine infusions (5 micrograms/kg/min) were initiated. Cardiac index increased from 2.51 to 6.57 L/min/m2 (p < 0.05), whereas systemic vascular resistance fell from 1534 to 584 dyne sec/cm5 (p < 0.05). Oxygen delivery (DO2I) and oxygen consumption (VO2I) indexes increased significantly during the study period (573 +/- 47 to 1028 +/- 57, and 132 +/- 8 to 172 +/- 16 ml/min/m2, respectively; p < 0.05). VO2I appeared dependent on DO2I at levels of DO2I less than 800 ml/min/m2. In this study, depressed cardiovascular function in patients with burn injuries responded to volume loading and inotropic support much as it does in patients with shock of other etiologies. Whether oxygen transport-based resuscitation is effective in improving survival or the incidence of multiple organ failure is unknown and will need to be evaluated in randomized trials.

Graft survival is prolonged by pretransplant transfusion of the graft recipient. It has been postulated that graft rejection is associated with Th1-like cytokines. We tested whether transfusion shifts cytokine production from a Th1-type (gamma-IFN production) to a Th2-type (IL-4 production). Transfusion prolonged cardiac allograft (C3H/HeN donor to a C57BL/6 recipient) survival (10.4+/-0.5 versus 7.2+/-0.2 days for controls, P

Rehabilitation of patients with burn injuries is receiving renewed interest because survival has improved, and health reform has mandated outcomes assessment. To determine factors affecting return to work, a survey was conducted among 234 employed patients treated from 1986 through 1993. The mean burn size was 13.3% total body surface area. Patients returned to work in a mean of 14.3 weeks; a number initially returned to light-duty or part-time jobs as a bridge to full-time employment. Length of hospitalization, number of surgeries, total and full-thickness burn size, and subjective assessments by patients of their functional ability correlated with time off work. Patients with health insurance were more likely to resume work than was expected, whereas those covered by Medicaid and those involved in injury-related lawsuits were less likely to return to work. It is hoped that this information can be used to design interventions aimed at improving this outcome of burn treatment. (J Burn Care Rehabil 1996;17:353-61)

Allogeneic blood transfusion (Allo/BT) and burn injury modify the cellular immune response in patients under a variety of circumstances. We designed this study to investigate the influence of Allo/BT, burn injury, and the combination of the two on in vivo natural killer (NK) cell activity in a murine model. This study demonstrated significant enhancement of in vivo NK cell activity in noninjured BALB/c mice receiving Allo/BT from C3H mice when compared to both the control and syngeneic blood transfusion group at posttransfusion day 5. When burn-injured mice were compared to sham-stressed mice, the burn-injured mice showed significant suppression of in vivo NK cell activity. Furthermore, in this strain combination model, Allo/BT modulated the suppressive effect of burn injury on in vivo NK cell activity at posttransfusion day 5 and postburn day 7.

Conventional systems for isolating adipose-derived stem cells (ASC) require enzymatic digestion of adipose tissue (AT), followed by monolayer culture to the enrich the stem cell population. However, these systems are hindered by low cell yields and a lack of reproducibility. The present study was aimed at developing a unique strategy for isolating ASC based on fibrin matrix-supported three-dimensional (3-D) organ culture of native AT. Furthermore, we tried to optimize the fibrin composition by adjusting the fibrinogen and thrombin concentrations to allow rapid outgrowth and proliferation of ASC in the 3-D fibrin matrix. Human cutaneous AT fragments were encapsulated within the fibrin matrix to construct a 3-D environment and cultured under dynamic conditions. During in vitro culture the fibrin matrix provided physical support for the AT and also allowed selective outgrowth of ASC from embedded AT fragments. In situ expanded outgrown cells were recovered from the fibrin matrix by selective fibrinolysis and propagated under monolayer culture conditions. The cultured cells fulfilled the following criteria for ASC: adhesion to culture plastic, multipotent differentiation, correct immunophenotypic profile. Fibrin matrix-supported 3-D organ culture produced ASC that with high competency in terms of growth and differentiation capabilities, and resulted in a larger and more consistent cell yield than obtained with conventional culture systems. The fibrinogen and thrombin concentrations inversely affected spreading, migration, and ASC outgrowth from native AT. Our results indicate that this 3-D organ culture system for AT can be used as an efficient and reproducible method for ASC isolation.

Mycoplasma arthritidis produces a potent superantigen (MAM) that activates specific murine and human T lymphocytes to proliferate and secrete lymphokines. We show here that MAM also influences both T- and B-cell functions in vivo. Lymphocytes from mice injected with MAM exhibit a suppression of proliferative responses to MAM in vitro but only a partial suppression of responses to other mitogens. This T-cell anergy not only decreased contact sensitivity to dinitrofluorobenzene but also prolonged survival of skin transplants. In contrast, B-cell reactivity is increased following in vivo injection of MAM, as evidenced by enhanced antibody responses to sheep red blood cells and ovalbumin. Also, there is a marked decrease in the ability of splenocytes from MAM-injected mice to produce interleukin-2 (IL-2) but a marked increase in their ability to produce IL-4 and IL-6. The combined results suggest that MAM induces a lymphokine profile that favors activation of B-cell functions, with a resulting potential for triggering of autoimmune disease.

Following activation of cytotoxic T cells and NK cells several genes encoding proteins putatively involved in cell-mediated cytotoxicity become expressed. The expression of genes encoding the cytotoxic T cell associated serine protease granzyme A and perforin was analyzed in cellular infiltrates of MHC mismatched (H-2d-->H-2k) heterotopic heart transplants both in immunosuppressed recipients treated with cyclosporine and in untreated recipients. Heart transplants were completely rejected by untreated animals on day 10 post-transplantation, whereas CsA treatment generally prolonged survival of the transplants beyond 30 days. In untreated recipients the number of granzyme A- and perforin-expressing cells in heart transplants increased from approximately 10 granzyme A-positive cells/mm2 and 1 perforin-positive cell/mm2 on day 2 posttransplantation to over 80 positive cells for both genes on day 5 posttransplantation. In contrast, these values remained always below 15 positive cells/mm2 for both genes between day 5 and day 30 posttransplantation in CsA-treated recipients. Comparison of the frequency of CD8+ T cells in the infiltrates showed that lower numbers of perforin and granzyme A-positive cells were mainly due to the immunosuppressive action of CsA rather than to reduced infiltration of transplants. The present study shows that expression of granzyme A and perforin gene can be used to discriminate between quiescent and activated cytotoxic cells also in immunosuppressed animals and further confirms that these can be used as sensitive markers for monitoring the fate of a transplant.

Modified limiting dilution analysis (LDA) techniques were used to evaluate the mobilization of antigen-stimulated helper T lymphocytes (HTL) and cytotoxic T lymphocytes (CTL) following allogeneic heterotopic cardiac transplantation. These modified LDA techniques allow a quantitative comparison of T cells that have been stimulated by antigen in vivo versus unstimulated precursor T cells of the same antigen specificity. Endothelial changes associated with mononuclear cell infiltration of the transplant were studied using endothelia-specific monoclonal antibodies and immunohistochemistry. Early (day 3) infiltration of cardiac allografts was characterized by a prevalence of donor alloantigen-specific HTL over CTL. Immunohistology revealed that the day-3 infiltrate was associated with areas of differentiated vascular endothelium, located primarily in the subepicardial region. Though donor-specific precursor HTL and CTL were present in the peripheral lymphoid tissues and blood, very few of them had been stimulated at this early time. During the latter phases of the response (days 6-9), antigen-stimulated HTL and CTL were present in the rejecting heart with CTL dominating the response. Accumulation of large numbers of donor-specific CTL in the allograft correlated with extensive inflammatory endothelial development, myocyte destruction, and loss of graft function by day 9. Stimulated HTL and CTL were detectable in peripheral lymphoid tissues at days 6 and 9. In addition, a marked increase in the number of donor-specific precursor CTL, but not precursor HTL, was observed in the lymphoid tissues at the peak of the response. Depletion of class II MHC-restricted T cells by in vivo treatment with anti-CD4 mAb eliminated HTL activity in all lymphoid compartments assessed and markedly reduced the number of CTL infiltrating the allograft. In addition, no stimulated CTL were detectable in lymphoid tissues, and the number of precursor CTL was not increased. In anti-CD4-treated recipients, cardiac allografts remained functional with minimal histological evidence of rejection for at least 21 days. Though graft-associated inflammatory endothelia were absent in anti-CD4-treated recipients at day 6, endothelial differentiation was observed in day 21 allografts in anti-CD4-treated recipients. These observations indicate that inflammatory endothelial development may precede T cell infiltration and subsequent loss of the cardiac allograft function. Thus, CD4-positive HTL are required for (1) graft-associated inflammatory endothelial development; (2) CTL activation in peripheral lymphoid tissues; (3) CTL accumulation in allografted tissues; and (4) acute cardiac allograft rejection.

The purpose of this study was to investigate two promising immunosuppressive agents, didemnin B (DB) and FK506 (FK), during pregnancy to assess potential adverse maternal or fetal effects. Pregnant C3H mice were randomized into control and high- and low-dose treatment groups for each drug. Animals received daily injections from day 1 to day 16, and on day 17 of gestation the maternal condition, litter size, fetal resorption rates, and fetal/placental unit weights were determined. Immunoglobulin (IgG) levels were obtained for DB treatment groups. Delayed type hypersensitivity was assessed in virgin females. Both DB and FK had dose-dependent immunosuppressive activity in the DTH assay, and DB caused elevated IgG concentrations. High doses of DB caused diarrhea and maternal wasting with no fetal survival; with low-dose DB, maternal weight gain was depressed, but pregnancy outcome was not different from control animals. High-dose FK had no obvious detrimental effects on maternal health but caused resorption of all fetuses; administration of low-dose FK resulted in a higher number of resorptions, but fetuses that survived did not appear different from controls. We conclude that these immunosuppressive drugs can have adverse effects on pregnancy, but the maternal and fetal toxicity are dose-dependent.

Intradermal or skin-graft sensitization always results in accelerated rejection of subsequent MHC-disparate mouse heart transplants; intravenous or intrasplenic sensitization almost always results in prolonged survival. The survival time after intraperitoneal sensitization is unpredictable: hyperacute or accelerated rejection, as well as normal or prolonged survival, occurs suggestive of a delicate balance between suppressive and rejecting immune responses. We have observed a positive correlation between transplant survival and the distribution of 51Cr-labeled antigenic cells early after i.v., i.d., i.p., and intrasplenic injection. Label recovery from the viscera was already high 2 hr after i.v. injection, while after i.d. injection virtually all label was still confined to the carcass, even at the end of a 24-hr period. After i.p. injection label recovery varied significantly from mouse to mouse, from very low to a level approximating that seen after i.v. injection. This close correlation suggests that the fate of a subsequent transplant is decided within a few hours after antigen deposition, well before placement of the transplant itself.

BACKGROUND The mechanisms by which ventricular function is altered during cardiac transplant rejection are not well understood. Therefore, an in vitro model system has been developed to facilitate investigation of lymphocyte-mediated myocyte injury. METHODS AND RESULTS Splenic lymphoid cells were obtained from mice 8-10 days after placement of a vascularized abdominal cardiac allograft and were restimulated in vitro with irradiated donor-type splenocytes for 5 days. Cytotoxic effects of these allogenically stimulated lymphocytes on syngeneic and donor strain fetal cultured myocytes were determined by a 51Cr release assay at different lymphocyte to myocyte ratios. 51Cr release from donor strain myocytes was detectable within 1 hour of exposure, was maximal by 3-5 hours of coincubation with sensitized lymphocytes, and was allospecific. Cell injury manifest by 51Cr release was calcium dependent and was inhibited by pretreatment of lymphocytes with phorbol ester to deplete protein kinase C. Myocyte injury was also prevented by pretreatment of sensitized lymphocytes with anti-Thy 1.2 or anti-CD8 antibody plus complement but not by treatment with anti-CD4 antibody, indicating that CD8+ cytotoxic T cells are involved. Altered myocyte contractile motion preceded myocyte lysis (51Cr release), was characterized by an initial reversible decrease in amplitude of contraction, and was followed by rapid and irregular beating with eventual complete cessation of contraction. Contractile alterations induced by sensitized lymphocytes were inhibited by elimination of CD8+ cells. CONCLUSIONS Myocyte injury can be produced by sensitized cytotoxic T lymphocytes in vitro and is calcium and protein kinase C dependent. The contractile abnormalities produced appear to be similar to those observed in cardiac transplant patients undergoing rejection, and thus this model system promises to allow investigation of the mechanisms involved.

Thermally injured mice that were given intraperitoneal injections of murine cytomegalovirus (MCMV) appeared to be clinically septic and to have increased mortality rates. To evaluate the possible role of MCMV infection in promoting bacterial translocation in burned mice, mesenteric lymph nodes were cultured from two strains of mice (BALB/c and CBA) that were given thermal injuries alone, MCMV alone, or both. BALB/c mice injected with 5 X 10(5) plaque-forming units MCMV following a 15% to 16% total body surface area scald injury had increased incidence of positive mesenteric lymph node cultures compared with other groups. No intestinal mucosal histologies, mucosal dry weights, or wet-to-dry weight ratios in any animals were abnormal. Differences in cecal bacterial concentrations were not observed. Murine cytomegalovirus infection appears to enhance bacterial translocation in this model.

Crosslinked hyaluronic acid (HA) hydrogels were evaluated for their ability to elicit new microvessel growth in vivo when preloaded with one of two cytokines, vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF). HA film samples were surgically implanted in the ear pinnas of mice, and the ears retrieved 7 or 14 days post implantation. Histologic analysis showed that all groups receiving an implant demonstrated significantly more microvessel density than control ears undergoing surgery but receiving no implant (p0.01). Moreover, aqueous administration of either growth factor produced substantially more vessel growth than an HA implant with no cytokine. However, the most striking result obtained was a dramatic synergistic interaction between HA and VEGF. Presentation of VEGF in crosslinked HA generated vessel density of NI = 6.7 at day 14, where NI is a neovascularization index defined below, more than twice the effect of the sum of HA alone (NI = 1.8) plus VEGF alone (NI=1.3). This was twice the vessel density generated by co-addition of HA and bFGF (NI=3.4, p0.001). New therapeutic approaches for numerous pathologies could be notably enhanced by the localized, synergistic angiogenic response produced by release of VEGF from crosslinked HA films.

As a first step to developing cytokine-impregnated hydrogels capable of promoting desired physiologic responses, we have tested in vivo angiogenesis in a mouse model. Hyaluronic acid (HA) hydrogel samples pre-loaded with vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) were surgically implanted in an ear pocket in the mouse. The implants were then retrieved at 7 and 14 days post surgery, and the number of ingrowing microvessels evaluated by direct counting. As compared to contralateral controls, we show that released degrading hyaluronan alone is itself an effective stimulus for new vessel growth. Pre-loading of the gel with VEGF, however, dramatically augments the rate of new microvessel growth, resulting in a 2.3-fold increase in neovascularization over the sum of the rates produced by HA and VEGF individually. In contrast, although bFGF alone greatly augments vessel growth, no such potentiation effect was observed between bFGF and HA. We present physiologic data demonstrating vessel growth and correlate those data with gene expression changes measured by microarray analysis. The results show that HA-cytokine combinations can be designed to optimize angiogenesis, and suggest potential mechanisms by which angiogenic vessel proliferation may occur.

A mild chemical modification of hyaluronic acid (HA) provides functionalized derivatives for fabrication of targeted drug delivery systems, wound dressings, tissue engineering scaffolds, and probes for cellular binding and transport of HA. First, we describe the use of covalent HA-anti-cancer agents for use as potential therapeutics. Data from cell culture, flow cytometry, and in vivo mouse models support this targcted anti-tumor strategy. Second, we describe new flexible hydrogel films composed of crosslinked chondroitin sulfate (CS) and HA, which have potential as wound dressings capable of biointegration and drug release. Lyophilization and rehydration of these flexible films also provide porous materials for cell growth and tissue engineering. Third, we describe progress on the elucidation of the structure determination of the HA-binding domain (HABD) of RHAMM and the use of this domain to identify peptides that mimic HA.

Long-term growth retardation occurs in children and osteopenias occur among children and adults who have been burned or suffer other injuries that result in a systemic inflammatory response. The purpose of this study was to define some of the growth, and to determine cancellous and cortical bone changes that occur following thermal injury in several contrasting strains of mice. Male C3H/HeN and Balb/c mice were given about a 20% total body thermal injury and 10 days later skeletal tissues were collected. The bone ash weights of the burned animals from both strains were less than their sham controls. In both strains, cancellous bone volumes were less in the burned animals than in their respective sham or baseline control groups. The loss of bone was particularly evident in the secondary spongiosa regions and also included a decrease in trabecular thickness and connectivity and an increase in trabecular separation. Longitudinal (endochondral) growth was suppressed in the burn animals. In the burned animals, indices of cancellous bone formation were substantially reduced whereas those in cortical bone were essentially nonexistent. The numbers of osteoclasts were increased in cancellous bone, and endocortical eroded surface was increased in the burn animals. These data show that there are rapid and profound changes in skeletal growth and metabolism in an experimental model of thermal injury. In general, a greater relative response was observed in the Balb/c vs. the C3H/HeN strain. Thermal injury resulted in a rapid and dramatic suppression of bone formation and endochondral growth with increased bone resorption in both cancellous and cortical bone.

Allogeneic blood transfusion is known to be immunosuppressive in the settings of cancer and transplantation, but the contribution of blood transfusion to immunomodulation after hemorrhage is unknown. Our purpose was to determine the influence of allogeneic blood transfusion upon cytokine profiles following hemorrhagic shock, using a model which approximates the clinical setting. Methods.Male C3H/HeN mice were hemorrhaged via femoral arterial catheters to a mean arterial pressure (MAP) of 35 ± 5 mm Hg, which was maintained for 1 h. Mice were resuscitated with autologous blood (auto BT) or allogeneic blood (allo BT) from Balb/c mice (both equivalent to volume of shed blood), and crystalloid (2X the volume of shed blood)-infused at 0.05 ml/min. Animals were sacrificed at 1, 2, and 5 days postshock, and splenocytes were cultured for 24 h with anti-CD3 antibody. Supernatants were assayed for IL-2, IL-6, IL-10, and γ-IFN by ELISA. Results.Regardless of transfusion status, hemorrhagic shock resulted in increased IL-6 and γ-IFN by Day 2 postshock. Distinct cytokine alterations after allogeneic transfusion were as follows. IL-2: transient elevation of splenocyte IL-2 production in the shock + allo BT group (P< 0.005 vs. shock + auto BT) at Postshock Day 2. IL-6: suppression in IL-6 production in the shock + allo BT group by Postshock Day 5 (P< 0.05 vs. shock + auto BT). IL-10: persistently elevated IL-10 production following shock + allo BT (Day 1,P< 0.001 vs. shock + auto BT; Day 5;P< 0.05 vs. shock + auto BT). γ-IFN: elevation in γ-IFN production by Day 5 in the shock + allo BT group (P< 0.0005 vs. shock + auto BT). Conclusions.Allogeneic blood transfusion results in distinct alterations in splenocyte production of IL-2, IL-6, IL-10, and γ-IFN after hemorrhagic shock. This model reflects the clinical usage of blood products and demonstrates some of the immune alterations after transfusion.

Biosynthesis of nitric oxide (NO) by mammalian cells has recently been demonstrated (Hibbs et al., 1987a; Iyengar et al., 1987; Hibbs et al., 1988; Marietta et al., 1988). NO synthesis is induced during a cell-mediated immune response to infection or tumor development. NO is biologically produced by the enzyme immune/inflammatory NO synthase (iNOS), which utilizes molecular oxygen to oxidatively deiminate a guanidino nitrogen atom of L-arginine with formation of L-citrulline and NO as the products (Hibbs et al., 1988; Marietta et al., 1988, Tayeh et al., 1989). iNOS is competitively inhibited by N-substituted L-arginine derivatives, including NG -monomethyl-L-arginine (MLA) (Hibbs et al., 1987a, 1987b; Granger et al., 1990, 1991). Nitric oxide reacts with O2 in a termolecular reaction to produce nitrate (NO3 −) and nitrite (NO2 −- NO2 − entering the vascular system is further oxidized by hemoglobin to NO3 − (Granger et al., 1991) and is excreted in the urine. When exogenous sources of nitrate are eliminated from the diet, urine nitrate can be used as a measure of NO biosynthesis (Granger et al., 1991).

Bone growth and remodeling are inhibited by severe burns in adult and pediatric patients, resulting in alterations in linear growth, bone mass, osteoporosis, and increased risk for pathologic fractures. This study of a mouse model of burn injury showed skeletal changes similar to those reported in patients with burn injuries. Baseline, control, sham, and burned mice were injected with fluorescent markers calcein and tetracycline for histomorphometric analysis. Total femur dry and ash weights and total calcium content were significantly lower 10 days after burn injury compared with sham and control animals. There also were decreases in the percentage of fluorochrome-labeled bone surfaces and bone formation rates in the burn-injured mice compared with control and sham mice; however, there were no differences in the mineral apposition rates. This model now provides an opportunity to examine cellular and molecular mechanisms contributing to skeletal pathology in a well-defined burn injury model.

Melatonin and 21-aminosteroids (lazaroids) are potent antioxidants and may attenuate the increased membrane permeability associated with profound shock. Our purpose was to test the effect of melatonin and a lazaroid (U74389-G) on cytokine production and fluid requirements after shock. Methods: Male C3H/HeN mice, 20–25 g, were hemorrhaged via a femoral artery catheter to a mean arterial pressure of 35 ± 5 mm Hg, which was maintained for 1 hr, and then resuscitated with shed blood and crystalloid (2× vol of shed blood). Experimental mice received melatonin at 10 or 50 mg/kg, U74389-G at 3 mg/kg, or vehicle iv upon resuscitation, and blood was returned at 0.1 cc/min and crystalloid at 0.05 cc/min. The percentage of total crystalloid required to reach stabilization (mean arterial pressure remaining within 2 mm Hg for 5 min) was recorded. Animals were sacrificed at 1 hr postshock. Serum and anti-CD3-stimulated splenocyte culture supernatants were assayed for interleukin-6 (IL-6) and γ-IFN by ELISA. Results: Mice receiving lazaroid or melatonin (50 mg/kg) required significantly less fluid to reach stabilization, with lazaroid-treated animals requiring 24 ± 1% and melatonin-treated animals requiring 28 ± 2% of total crystalloid compared to 40 ± 3% for untreated animals. Melatonin-treated mice (50 mg/kg) had lower serum IL-6 levels (368 ± 154 vs 1078 ± 146 pg/ml) and lazaroid-treated mice had lower γ-IFN levels (7 ± 6 vs 52 ± 15 pg/ml) compared to those of the untreated group (P< 0.05). There were no differences in splenocyte cytokine production. Conclusions: Treatment with lazaroid and melatonin both reduced postshock fluid requirements. Melatonin reduced serum IL-6 levels, while lazaroid reduced serum γ-IFN levels, suggesting different mechanisms of action.

Objectives . This study was performed to determine the mechanisms by which allosensitized lymphocytes cause contractile dysfunction in cultured ventricular myocytes and to compare the effects on isolated myocytes with those observed in an intact heart preparation during allograft rejection. Background . Allograft rejection may be associated with reversible abnormalities of both systolic and diastolic function. The immunologic mechanisms that cause ventricular dysfunction are poorly understood. Methods . Vascularized heterotopic abdominal heart transplantation was performed in mice. Contractile function of excised allografts undergoing rejection was assessed using a Langendorff perfusion apparatus and a strain gauge. Spontaneously beating monolayers of cultured ventricular myocytes from donor strain fetal mice were exposed to the allosensitizcd cytotoxic T lymphocytes, and the effects on myocyte motion, intracellular calcium transients, relaxation half-time, membrane potential and myocyte lysis (chromium-51 release) were measured. Results . In intact hearts, histologically mild rejection without myocyte necrosis was associated with decreased systolic function without slowing of relaxation. In cultured fetal myocytes, sensitized lymphocytes induced a progressive decrease in the amplitudes of myocyte motion and calcium transients, with cessation of beating within 40 min. Also, the diastolic membrane potential and amplitude of the action potential decreased. Relaxation half-time, as estimated by measurement of cell motion, was unchanged. The effect was allospecific and was reversible with early removal of lymphocytes from the myocyte monolayer. Pretreatment of lymphocytes with the degranulation inhibitor 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene blocked both the negative inotropic effect and myocyte lysis. Conclusions . We conclude that impaired relaxation is not a prominent feature of contractile dysfunction caused directly in myocytes by alloimmune injury from cytotoxic lymphocytes. Allosensitized lymphocytes can cause reversible systolic dysfunction in myocytes by means of a direct cell-cell interaction. This effect may be in part responsible for the reversible systolic dysfunction associated with allograft rejection.

Initial treatment of heterotopic cardiac transplant recipients with anti-CD4 mAb promotes long-term (60 days) allograft survival. We have used modified limiting dilution analysis to quantitate donor alloantigen-reactive helper T lymphocytes (HTL) and CTL in mice bearing long-term cardiac allografts. Despite repopulation of lymphoid tissues with CD4+ T cells, donor alloantigen-reactive IL-2 producing and IL-4-producing HTL were rare or not detectable in lymphoid tissues or in the graft. While donor-reactive precursor CTL were present in lymphoid tissues, modified limiting dilution analysis revealed that stimulated ("antigen-conditioned") CTL were not detectable, and few CTL were present in the graft. In addition, antibodies reactive with donor alloantigens were not detectable in the sera of mice bearing long-term cardiac allografts. To determine whether additional in vivo stimulation with donor alloantigens would elicit an immune response, sponge allografts were implanted into mice bearing long-term cardiac allografts. Previous reports from this laboratory have demonstrated that T cell infiltration of sponge allografts is dependent upon antigen-driven cytokine production. While third-party sponge allografts were readily infiltrated by third-party-reactive HTL and CTL, sponge allografts of the same strain as the cardiac allograft were not infiltrated by T cells. However, donor strain sponge allografts induced an IgM (but not IgG) alloantibody response. These data indicate that IgM alloantibody could be induced in the absence of a cellular response to the sponge allograft. Kinetic studies revealed that a transient IgM (but not IgG) response was induced by the initial cardiac transplantation in the absence of CD4+ cells. These IgM alloantibodies disappeared by day 21 despite the persistence of the allograft. These observations indicate that transient depletion of CD4+ T cells induces allograft-specific T cell tolerance, but does not eliminate the ability to mount an allograft-specific IgM response. To our knowledge, this is the first report of a transient humoral response to alloantigens that occurs in the absence of CD4+ T cells, and can be reinduced in "tolerant" animals that fail to mount a cellular immune response. Potential mechanisms involved in the development and maintenance of anti-CD4 mAb-induced tolerance are discussed.

Endogenous nitric oxide biosynthesis in mice receiving allogeneic heterotopic heart transplants was monitored as a function of time post-transplant. Nitric oxide production was measured by daily urine nitrate levels and by formation of paramagnetic heme-nitrosyl complexes in the cardiac tissue. Exogenous sources of urine nitrate and EPR signal were minimized by maintaining the animals on a low nitrite/nitrate diet. Urine nitrate peaked on postoperative day 7. A heme-nitrosyl EPR signal also appeared in the cardiac tissue on postoperative day 7 and remained unchanged in size until rejection on postoperative day 9 at which time the peak height of the signal nearly tripled. Some of the animals in the study were treated with the nitric oxide synthase inhibitor, N omega-monomethyl-L-arginine which caused marked inhibition of urinary nitrate excretion and prevented heme-nitrosyl complex formation in beating hearts. However, administration of the inhibitor did not increase graft survival time. Low intensity heme-nitrosyl signals were identified in inhibitor-treated allogeneic hearts after rejection. Syngeneic heart transplants did not induce urinary nitrate excretion nor EPR signal formation. These results show that cytokine induced high output nitric oxide synthesis from L-arginine is a prominent biochemical component of the cell-mediated immune response to cardiac allografts in mice. However, nitric oxide production was not essential for rejection of cardiac allografts mismatched at the major histocompatibility locus.

Clinical studies have revealed a correlation between cytomegalovirus (CMV) infection and acute allograft rejection. Likewise, for a murine model we observed that C3H (H-2k) recipients infected with murine CMV (MCMV) rejected BALB/c (H-2d) cardiac allografts earlier than uninfected recipients (6.9 +/- 0.1 d compared with 8.1 +/- 0.6 d; P < 0.001). It has been hypothesized that MCMV epitopes crossreact with alloantigens and in this manner induce rejection. However, we also demonstrated that MCMV caused accelerated rejection in the reverse combination (C3H heart engrafted to BALB/c recipient; 7.2 +/- 0.3 and 9.4 +/- 0.4 d for infected and control animals, respectively; P < 0.001) and accelerated rejection of grafts of a third, unrelated haplotype (C57Bl/6; H-2b; 8.0 +/- 0.7 d compared with 10.1 +/- 0.6 for infected and control C3H recipients, respectively; P < 0.03). Ultraviolet (UV) inactivation of MCMV before administration to the graft recipient abrogated the ability to induce rapid rejection. Activated T lymphocytes were present in grafts from infected recipients 2 d before control recipients (P < 0.02) and, at the time of graft rejection, were almost exclusively CD8+ for both infected and control recipients. Thus, MCMV accelerated rejection appears not to result from crossreaction between MCMV epitopes and MHC products. The failure of UV-inactivated virus to accelerate rejection and the high proportion of CD8+ T cells recovered from all rejected grafts suggest that the class I pathway of antigen presentation may be important in the induction of early rejection.

• Burned individuals display a reduced ability to elicit cellular and humoral immune responses and a depression in the in vitro production of certain T-cell lymphokines. Treatment of burned mice with 100 μg of dehydroepiandrosterone within 1 hour after injury resulted in preserving a completely normal capacity to produce T-cell—derived lymphokines and to generate cellular immune responses. In addition, dehydroepiandrosterone-treated thermally injured mice demonstrated an above-normal ability to resist an induced infection with the intracellular pathogen, Listeria monocytogenes. Dehydroepiandrosterone-treated animals also did not exhibit the sustained plasma levels of interleukin 6 that normally accompany thermal injury and infection. Because of its antiglucocorticoid effects and positive immunoregulatory influences, we believe dehydroepiandrosterone to be a beneficial form of therapy for thermally injured individuals. ( Arch Surg. 1993;128:318-325)

Phosphorus-31 nuclear magnetic resonance techniques using shallow penetrating coils have been used to noninvasively monitor severity and metabolic changes over time in skin wounds in rats. Ratios of phosphocreatine (PCr) to inorganic phosphate (Pi) indicate energy status in both thermal wounds and surgical flaps. In partial and full-thickness scald wounds, reductions in PCr/Pi ratios correlated with burn depth and improved over time postinjury, suggesting wound revascularization. No decrease in intracellular pH was noted in these wounds; the phosphate shifts may be primarily the result of tissue degradation followed by restoration of the microvasculature. Distal regions of caudally based dorsal 3 x 10 cm full-thickness skin flaps reveal progressively lower PCr/Pi ratios to 3-6 hours after elevation as well as drops in pH up to 0.5 units, presumably as a result of anaerobic glycolysis in these tissues. After 24 hours, the intracellular pH returned to normal (7.1-7.2) and the PCr/Pi ratios approached 70%-90% of the well-perfused proximal regions within 3-7 days. These results indicate the establishment of a microvasculature from the underlying bed as the distal regions survive as free grafts. The data demonstrate the potential usefulness of the technique in noninvasive measurement of the biochemical response to injury and wound healing in living organisms.

The crucial role of close relative care givers in the rehabilitation of the patient with burns indicates that the psychologic adjustment of noninjured relatives is of concern. This study examined the stress profiles of 14 spouses and parents of patients with burns of greater than 20% total body surface area. Four standardized measures of depression, anxiety, and cell-mediated immunity were used. Tests were given at two time intervals: less than 72 hours after admission and 2 to 5 weeks later. Depression and anxiety were high at the first test period; there was a significant drop in depression (p less than 0.05) but not in anxiety at the second testing. Immune function was suppressed at the first test but improved at the second test (p less than 0.05). There were significant negative correlations between immune response and psychologic distress, indicating that immune function declined as depressive symptoms increased. These results support an interaction between psychologic distress and immunity, and provide further evidence of the stressful nature of severe burn injury on close noninjured relatives.

Cytomegalovirus is seen frequently in patients with burns and may enhance morbidity and mortality rates. Burned BALB/c mice that were given murine cytomegalovirus by intraperitoneal injection demonstrate increased bacterial translocation, as evidenced by positive mesenteric lymph node culture at 5 days after burn injury. The present experiment was conducted to investigate the time course and mechanism of this effect. A significant delay in the resolution of positive mesenteric lymph node cultures was observed with thermal injury alone. This was further reduced by the addition of murine cytomegalovirus infection to thermal injury. No such delay was seen with murine cytomegalovirus infection alone or in controls.

Phenotypic characterization of the inflammatory infiltrate often provides very little information about the fate of a transplant. Therefore we decided to look for functional markers, characteristic for activated effector cells involved in the rejection of primarily vascularized MHC-mismatched heart transplants in mice. Infiltrating cells in the interstitium of eventually rejected allogeneic heart transplants were found to express the gene for the serine esterase HF (granzyme A) in high numbers already on day 2 following transplantation, whereas HF mRNA positive cells were absent, or only rarely found, in syngeneic nonrejecting transplants throughout the entire observation period of 10 days after transplantation. These findings could also become of importance for the prediction of the outcome of organ transplants in humans.

An elongated, narrow, slotted crossover surface coil provides surface localization capable of resolving in vivo 31P NMR spectra from skin tissues. The shallow B1 field penetration achieves localization objectives while the probe length maintains signal-to-noise requirements. Dielectric and inductive losses are minimized via the crossover design (see T. L. Nagel et al., Magn. Reson. Med. 13, xxx (1990). In vivo spectra with millimeter depth resolution were acquired in 5 min at 2 T without pulse localization sequences. Preliminary 31P NMR spectra of normal and thermally injured rat skin were completed using a 25 X 3-mm slotted probe with a 3 X 2-cm surface region of excitation. Normal rat skin tissue PCr/Pi ratios ranged from 3.8 to 4.7 for 5-, 10-, and 30-mus pulse widths, while partial- and full-thickness scald injured tissues ranged from 0 to 2.8. Evaluation of a single minor partial thickness injury 1 to 5 h postburn shows evidence of a localized hypermetabolic response associated with hyperemia. Determination of burn depth and tissue viability appears feasible using: (1) PCr/Pi ratios and (2) observation of localized hypermetabolism.

REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION ANALYSIS OF MYOCARDIAL GROWTH FACTOR MRNA IN MURINE HETEROTOPIC HEART TRANSPLANTS. • 206