Jane Shelby

Release of basic fibroblast growth factor from a crosslinked glycosaminoglycan hydrogel promotes wound healing

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.

Mobilization of T lymphocytes following cardiac transplantation. Evidence that CD4-positive cells are required for cytotoxic T lymphocyte activation, inflammatory endothelial development, graft infiltration, and acute allograft rejection.

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.

Resuscitation of thermally injured patients with oxygen transport criteria as goals of therapy

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.

Nω-monomethyl-l-arginine inhibits nitric oxide production in murine cardiac allografts but does not affect graft rejection

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.

Glycosaminoglycan Hydrogels as Supplemental Wound Dressings for Donor Sites

Chemically crosslinked glycosaminoglycan (GAG) hydrogel films were evaluated as biointeractive dressings in a porcine model for donor-site autograft wounds. Multiple 5 x 5 x 0.03 cm 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.

Prostaglandin production and suppressor cell induction in transfusion-induced immune suppression.

Two models were used to examine the role of prostaglandin (PGE) in the inductive phase of transfusion-induced suppression. First, it was observed that postoperative allogeneic third-party blood transfusion resulted in prolonged major histocompatibility complex (MHC)-compatible rat heart allograft survival. Additionally, pretransplant antigen-specific allogeneic transfusion decreased graft-versus-host (GVH) reactivity in mice. An inhibitor of PGE synthesis, indometha-cin, blocked transfusion-induced suppression when it was administered to either transfused rat heart recipients, or graft-versus-host responder mice. Neutralization of endogenous PGE by anti-PGE antibody also blocked allogeneic blood induced suppression. Alloge-neic-blood-induced splenic suppressor cells down-regulated normal GVH responsiveness, and appeared to be generated via a prostaglandin-dependent pathway.

The effect of two new immunosuppressive agents, FK506 and didemnin B, in murine pregnancy.

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.

Interleukin-18 Production Following Murine Cardiac Transplantation: Correlation with Histologic Rejection and the Induction of IFN-γ

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), Fishers 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.

Severe burn injury : effects on psychologic and immunologic function in noninjured close relatives

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.

Human skin storage techniques: a study utilizing a nude mouse recipient.

The nude mouse model is an excellent technique for studying the viability of stored human skin. In this study Roswell Park Memorial Institute (RPMI) tissue culture medium provided reliable storage of viable human skin for 20 days. Antibiotic concentration plays a role in stored skin viability with low concentrations of penicillin and streptomycin providing adequate bacterial control and improved viability when compared to identical media with high concentrations of the same antibiotics. Addition of fetal calf serum to the storage medium did not improve human skin viability in this experiment.