A rat model of syngeneic bone marrow transplantation during breast cancer therapy.

The purpose of this study was to develop a breast cancer model in rats, in which myeloablative chemotherapy and syngeneic bone marrow transplantation (SBMT) could be evaluated systematically for therapeutic effect. The Wistar-Furth (WF) DMBA-4 breast cancer cell line transplanted into naive WF rats produced rapidly growing tumors that were lethal within 2 months. SBMT was performed following preparation with a regimen (Bu-Cy), consisting of busulfan 16 mg/kg by gastric gavage on days -3 and -2 followed by 250 mg/kg of cyclophosphamide i.p. on day -1. Marrow was prepared from the femurs of donors and infused i.v. into the recipient on day 0. In all, 15 rats treated with Bu-Cy without marrow died, while 22 of 25 transplanted rats survived. In total, 16 rats with measurable tumors showed tumor responses following transplantation, but tumors recurred and survival was minimally prolonged. Of nine rats transplanted before clinical tumors were detected, five became long-term survivors that resisted further tumor challenge. It was concluded that the DMBA-4 breast cancer in WF rats could serve to evaluate SBMT following myeloablative doses of chemotherapy at various tumor loads. At large tumor loads therapy was not curative, but at low tumor burdens cures were possible and resistance to subsequent tumor challenge was demonstrated. The model may be useful for further studies of stem cell infusion in rodent tumor systems.

A flow cytometric protocol for titering recombinant adenoviral vectors containing the green fluorescent protein.

As the use of adenoviral vectors in gene therapy protocols increases, there is a corresponding need for rapid, accurate, and reproducible titer methods. Multiple methods currently exist for determining titers of recombinant adenoviral vector, including optical absorbance, electron microscopy, fluorescent focus assay, and the "gold standard" plaque assay. This paper introduces a novel flow cytometric method for direct titer determination that relies on the expression of the green fluorescent protein (GFP), a tracking marker incorporated into several adenoviral vectors. This approach was compared to the plaque assay using 10(-4)- to 10(-6)-fold dilutions of a cesium-chloride-purified, GFP expressing adenovirus (AdEasy + GFP + GAL). The two approaches yielded similar titers: 3.25 +/- 1.85 x 10(9) PFU/mL versus 3.46 +/- 0.76 x 10(9) green fluorescent units/(gfu/mL). The flow cytometric method is complete within 24 h in contrast to the 7 x 10 days required by the plaque assay. These results indicate that the GFU/mL is an alternative functional titer method for fluorescent-tagged adenoviral vectors.

Successful management of myocardial infarction during pregnancy in a renal transplant recipient.

Myocardial infarction (MI) complicating pregnancy in a renal transplant recipient is described. Management challenges of MI in pregnancy and the possible predisposing roles of renal transplantation and erythropoietin (EPO) use are discussed.

Coronary artery fistula after cardiac transplantation.

A cardiac transplant recipient with multiple coronary artery fistulae draining into the right ventricle is described. These fistulae presumably resulted from repeated endomyocardial biopsies. The diagnosis of coronary artery fistulae was made at the annual coronary arteriography. The magnitude of the shunt remained small over eight years of follow-up.

Development of circulatory and metabolic shock following transient portal triad occlusion.

Liver ischemia is purposefully induced by portal triad occlusion (PTO) in several clinical situations including liver surgery for trauma, tumor, and transplantation. Despite significant morbidity from PTO, the hemodynamic and metabolic effects of PTO have not been evaluated relative to duration of ischemia. We investigated this using a total hepatic ischemia model. Rats received isoflurane anesthesia, carotid artery and jugular vein cannulation, and serial measurements of cardiac output (CO), mean arterial pressure (MAP), heart rate (HR), central venous pressure (CVP), stroke volume (SV), systemic vascular resistance (SVR), superior mesenteric artery blood flow (SMAF), intestinal vascular resistance (IVR), pH, pCO2, pO2, lactate, glucose, hematocrit (HCT), white blood cell count (WBC), and total neutrophils. Each group received 0, 15, 30, 45, or 60 min of PTO followed by 2 hr of reperfusion. All sham ischemia animals remained hemodynamically stable throughout the study. However, in the ischemic groups, there were significant time-dependent decreases in MAP, HR, CO, CVP, SV, SMAF, and pH, and increases in SVR, IVR, HCT, and lactate, while pCO2, pO2, glucose, and WBC remained stable. All of the ischemic animals survived except those that received 60 min of PTO. In this group, all of the animals survived the ischemic period; however, only one animal survived beyond 60 min of reperfusion. These data demonstrate a time-dependent circulatory and metabolic shock following PTO heralded by intestinal venous pooling and loss of intravascular fluid, and culminating in death. Careful hemodynamic monitoring and restoration of blood volume in the trauma patient may reduce morbidity and mortality.

Apophysomyces elegans infection in a renal transplant recipient.

A 50-year-old cadaveric renal transplant recipient on immunosuppressive therapy is described with post-traumatic cutaneous infection caused by Apophysomyces elegans. He showed no evidence of hematogenous dissemination and recovered fully after therapy with extensive local debridement and amphotericin B lipid complex. An apparent drug-drug interaction between amphotericin B lipid complex and cyclosporine was encountered. The course of A elegans infection in transplant recipients may be similar to that described in immunocompetent hosts. A elegans infection should be considered in evaluation of post-traumatic cutaneous infection not readily responsive to antibacterial therapy.

A new model for inducing total hepatic ischemia while preventing circulatory collapse secondary to splanchnic vascular congestion.

Animal models used to study liver ischemia are limited by the lethal effect of splanchnic venous engorgement from portal triad occlusion (PTO). We compared a passive porto-systemic shunt (PSS) to a pump-driven PSS. The passive and pumped PSS groups (n = 6) received 60 min of PTO followed by 2 h of reperfusion. A control group received all interventions, but no PTO, and remained stable throughout. During PTO, severe circulatory shock with intestinal ischemia occurred in the passive group, while the pumped group remained stable. During reperfusion, both shunted groups experienced varying degrees of metabolic acidosis with decreases in cardiac index, stroke volume, superior mesenteric artery flow, and increases in systemic and intestinal vascular resistance. The mortality rate for the passive group was 83% vs. 0% for the pumped group. These results suggest that pumped PSS prevents splanchnic engorgement and allows for reproducible, isolated total hepatic ischemia in vivo.

Clinical and medicoeconomic impact of the cyclosporine-diltiazem interaction in renal transplant recipients.

The effect of the diltiazem-cyclosporine interaction on cyclosporine pharmacokinetics, pharmacodynamics, and pharmacoeconomics was studied in 10 recipients of renal allografts. Each subject was studied while receiving diltiazem 60 mg twice/day and while not taking the drug. After achieving steady-state conditions, cyclosporine and metabolite concentrations were determined in whole blood from samples drawn after the morning cyclosporine dose. After pharmacokinetic analysis, all patients were followed for 6 months during treatment with cyclosporine plus diltiazem or cyclosporine alone. Cyclosporine blood clearance decreased significantly after treatment with diltiazem (18.0-11.0 ml/min.kg; p = 0.008). The apparent volume of cyclosporine distribution also decreased significantly (4.26-2.62 L/kg; p < 0.05). After 6 months, diltiazem had no effect on renal function indexes, and no apparent effect on immunosuppression. Alterations in cyclosporine clearance and apparent volume of distribution secondary to diltiazem result in dosage reduction and potential cost savings in transplant pharmacotherapy. The mean decrease in cyclosporine dosage requirements would produce a cost saving of $1520 or 28% per patient per year.

Evidence that the large loss of glutathione observed in ischemia/reperfusion of the small intestine is not due to oxidation to glutathione disulfide.

Reperfusion injury following ischemia is thought to be the consequence of reactive oxygen species possibly generated either by xanthine oxidase activity or by processes associated with neutrophil activation in the affected organ or tissue. The conversion of xanthine dehydrogenase to the oxidase as well as the interactions between endothelium and neutrophils in the margination and activation of the latter are all considered to be results of conditions resulting from the ischemic episode. Determination of the redox status of glutathione in an ischemic/reperfused organ is frequently employed as an indicator of oxidative stress created by the production of oxygen free radicals during the reperfusion period. In this procedure, the ratio of oxidized glutathione (GSSG) to total glutathione (GSH + GSSG) is utilized to demonstrate the proportion of glutathione oxidized during reperfusion. We determined this ratio in the rat small intestine during ischemia and reperfusion and found that while the ratio of GSSG/(GSH + GSSG) does increase, this increase was the result of GSH disappearance rather than an increase in GSSG, and that essentially all of this loss occurred during the ischemic episode. We demonstrated that no oxidation of GSH occurred that was attributable to reperfusion per se; nor was there an increase of GSSG during this reoxygenation period.

Blockade of protein C activation reduces microvascular surgical blood loss.

Protein C is a natural anticoagulant glycoprotein which prevents intravascular clot formation. Protein C functions as an anticoagulant when converted to an active serine protease (activated protein C). Activated protein C is formed at the site of the endothelial injury in response to blood clotting and helps limit the size of blood clots. We tested the hypothesis that by temporarily blocking the activation of intrinsic protein C, we could reduce subsequent surgical blood loss from a microvascular surgical wound. The formation of activated protein C was blocked systemically by intravenous administration of a monoclonal antibody (HPC4) which binds to circulating protein C and prevents its conversion to activated protein C. Domestic pigs were blindly pretreated with intravenous HPC4 or saline then underwent partial-thickness skin graft harvesting to create a reproducible microvascular wound. Blood loss was measured from each wound and the hemostatic effect of protein C blockade was compared to intravenous saline alone as well as to topical thrombin or thromboplastin. We found that blocking the activation of protein C significantly (P = 0.005) reduces surgical blood loss in this model by 27% compared to saline control animals. Intravenous HPC4 performed equally as well as topical thrombin or tissue thromboplastin. In addition, topical thrombin acted synergistically with HPC4 to reduce blood loss an additional 44% (P = 0.01) as compared to intravenous HPC4 or topical thromboplastin alone. Autopsies performed 1 week after HPC4 treatment showed no evidence of systemic thrombosis resulting from the protein C blockade.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone marrow transplantation in miniature swine. III. Graft-versus-host disease and the effect of T cell depletion of marrow.

Graft-versus-host disease (GVHD) has been evaluated in partially inbred miniature swine in order to study this complication of allogeneic bone marrow transplantation (BMT) in a major histocompatibility complex (MHC) genetically defined large animal model. Bone marrow from MHC homozygous ("parental") swine was injected into irradiated (900 rads total-body irradiation) MHC heterozygous ("F1") swine that shared one haplotype with the donor. All 18 animals successfully engrafted with donor bone marrow, and 17 of these developed skin rash of varying intensity depending on the extent of T cell depletion of infused marrow. Of 18 animals, 8 received undepleted bone marrow from exsanguinated donors and 2 also received additional peripheral blood lymphocytes (PBL) as a source of mature T cells. All 8 showed a moderate-to-severe rash, and the 2 pigs that received additional donor PBL developed the most severe rash. The cutaneous eruption seen in this model clinically, histologically, and immunologically resembled human GVHD. Two protocols of T cell depletion of donor bone marrow by antiporcine T cell monoclonal antibodies plus complement were tested for their effect on development of GVHD. The combination of two monoclonal antibodies, 74-12-4 (PT4) and 76-2-11 (PT8), had a marginal effect on the subsequent development of cutaneous manifestations of GVHD. However, treatment of the donor marrow by a combination of three monoclonal antibodies--PT4, PT8, and MSA4 (PT11)--effectively decreased the severity of the GVHD skin rash. These results indicate that (1) the GVHD associated with allogeneic bone marrow transplantation in swine is dependent on T cells in the marrow; (2) effective T cell depletion of donor marrow by monoclonal antibodies and complement does not prevent engraftment; and (3) this swine GVHD model, which allows study with F1 and homozygous parental combinations in an MHC genetically defined large animal, is particularly useful for the understanding of GVHD pathogenesis, prevention, and treatment.

Bone marrow transplantation in miniature swine. I. Development of the model.

Procedures for successful autologous and MHC-matched allogeneic bone marrow transplantation in partially inbred, MHC-defined miniature swine have been established. All marrow recipients were conditioned with single-dose total-body irradiation at the upper level of tolerance, and supported with antibiotics and irradiated blood products during aplasia. Surgical harvest of autologous and allogeneic marrow yielded sufficient numbers of cells to successfully reconstitute recipients. Radiation control animals that received no marrow failed to show recovery of marrow function. Pigs transplanted with autologous marrow at doses greater than 10(8) cells/kg routinely engrafted and recovered normal marrow function. The major clinical complications were acute and chronic infections and hemorrhage. T cell-depleted autologous marrow also engrafted, and there was no observed increase in clinical complications. In bone marrow transplantation across non-MHC allogeneic differences, engraftment and survival were similar to that observed for autologous transplants. The T cell depletion of marrow in such MHC-matched allogeneic recipients was associated in one animal, however, with early reconstitution by cells of autologous origin.

Bone marrow transplantation in miniature swine. II. Effect of selective genetic differences on marrow engraftment and recipient survival.

In order to study the effect of defined genetic differences on bone marrow transplantation in miniature swine, five different combinations of major histocompatibility complex (MHC)-matched and mismatched bone marrow transplants were performed. Eight of nine fully MHC-mismatched allogeneic bone marrow transplants failed to reconstitute, and one animal reconstituted briefly but then died quickly thereafter. Five of six class I-matched/class II-mismatched (g----c) bone marrow transplants engrafted, showed a skin rash typical of graft-versus-host (GVH) reaction, and died 3 weeks after the marrow transplantation. None of five class II-matched/class I-mismatched (g----d) transplants engrafted. Parental marrow transplants into F1 hosts engrafted and caused GVH skin rash, with survivals from 1 to 9 months (n = 5). Serologic typing of the F1 recipients of parental marrow showed only donor-type peripheral blood lymphocytes (PBL), suggesting complete marrow replacement. Conversely, F1 into parental marrow transplants showed no engraftment (n = 6). These results indicate that resistance to MHC-mismatched allogeneic bone marrow engraftment in swine represents a host response recognizing donor class I MHC differences. This response appears to interfere with engraftment of donor bone marrow cells despite host preparation with 900-1100 rads total-body irradiation. In the absence of donor MHC class I differences, engraftment was seen despite the existence of multiple non-MHC differences, and even in the presence of class II differences. Such engraftment also led to GVH, varying in intensity according to the strength of genetic disparity (i.e., worst in parent----F1 combination). These results suggest that miniature swine should provide an effective model for study of both GVH elimination (in the parent----F1 combination) and problems of engraftment (in the F1----parent combination), the two most important obstacles to clinical allogeneic transplantation.

Technique for intraoperative localization of urinary leakage.

Urinary leakage is an uncommon complication of renal transplantation with a near equal division occurring between ureteral and bladder origins. The diagnosis is usually entertained from a characteristic clinical course and confirmed by preoperative contrast cystography or radionuclide scanning. A technique for precise intraoperative localization of the site of urine extravasation using intracystic instillation of Intralipid has been described. It allows easy recognition without damaging or staining surrounding tissues. In addition, this technique may be applied in other situations to confirm anastomotic or hollow viscus closures.

Intestinal necrosis due to sodium polystyrene (Kayexalate) in sorbitol enemas: clinical and experimental support for the hypothesis.

Five patients who suffered catastrophic colonic necrosis are presented. All patients were uremic and received sodium polystyrene (Kayexalate) in sorbitol enemas for the treatment of hyperkalemia shortly before the development of signs and symptoms of colonic necrosis. In all specimens extensive ischemic necrosis was present, and Kayexalate crystals were noted in the intestinal lumen. Four of the five patients eventually died. To further investigate the occurrence of colonic necrosis after the administration of Kayexalate in sorbitol enemas, a series of experiments were performed in rats. Two groups of Sprague-Dawley rats were studied. One group was made uremic by performance of bilateral nephrectomy. The other group underwent sham operation. Enemas of saline, Kayexalate alone, sorbitol alone, or Kayexalate in sorbitol were administered. In nonuremic rats, transmural necrosis was noted in seven of 10 rats receiving sorbitol enemas and in six of 10 rats receiving Kayexalate in sorbitol enemas. No significant pathologic changes were noted in the rats receiving other enemas. In uremic rats, extensive transmural necrosis was noted in all rats receiving enemas of sorbitol or Kayexalate in sorbitol. All of these 19 rats died within the period of observation compared with no deaths in 18 rats that received enemas without sorbitol (p less than 0.001).

Reversal of progressive renal allograft dysfunction with OKT3.

The safety and efficacy of treatment with Orthoclone OKT3 was evaluated in 14 renal transplant patients with allograft dysfunction unresponsive to conventional therapy. Eleven of the 14 initially improved on OKT3, and 9 have retained their kidneys after a median 13-month follow-up. The mean serum creatinine among successes is 2.4 mg/dl. It is concluded that OKT3 is a safe and effective drug for treatment of rejection, and may be used during temporary discontinuation of ciclosporin in cases in which nephrotoxicity is questioned.

Mucormycosis in renal transplant patients--a report of two cases and review of the literature.

Mucormycosis is a rare complication of diabetic ketoacidosis and immunosuppressed states, such as those occurring after renal transplantation. We describe two cases of mucormycosis in renal allograft recipients and present a review of similar cases previously reported. An analysis of these data suggests that renal transplant patients with concomitant diabetes mellitus are most susceptible to developing this complication.

Immunologic characterization of MHC recombinant swine. Production of SLA class specific antisera and detection of Ia antigens on both B and non-B PBL.

Two intra-MHC recombinant haplotypes have been examined to document the nature of the recombination and to generate MHC-specific alloantisera. Cells from progeny of recombinant pigs have been compared by mixed lymphocyte reaction, by complement-dependent cytotoxicity with standard alloantisera, and by sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) analysis of immune precipitates of radio-labeled extracts. The results demonstrate that both recombinant haplotypes, f and g, have inherited the SLA-A,B loci of the c haplotype and the SLA-D loci of the d haplotype, and that no differences between the two recombinant chromosomes are detectable. Class specific anti-SLA-A,B and anti-SLA-DR sera were produced in or against the g haplotype. In terms of cytotoxicity and SDS-PAGE these sera exhibited the expected reactivities, except that the high-titered anti-SLA-DR sera gave an unexpectedly high percentage of lysis of swine peripheral blood lymphocytes. That cells other than pig B cells were being lysed by the anti-SLA-DR sera was confirmed by analyses of subpopulations of peripheral blood lymphocytes. Approximately 50% of nylon nonadherent T cells were specifically lysed by allo-anti-Ia sera. Similar lysis of T cells was found with crossreactive mouse anti-Ia sera. Thus, unlike other species in which Ia antigens are expressed on T cells at low levels and are difficult to detect, the SLA-D region products are readily detectable on swine peripheral blood T lymphocytes.

Transplantation in miniature swine. X. Evidence for non-SLA-linked immune response gene(s) controlling rejection of SLA-matched kidney allografts.

The survival of renal allografts between SLA-matched swine has been found to be subject to non-SLA-linked Ir gene control. Using three herds of miniature swine, each homozygous for a different SLA haplotype (designated aa, cc, and dd, respectively), we initially observed that all animals of the c haplotype rejected SLA-matched renal allografts. In contrast, the subset of dd animals which were the product of continuous homozygous matings since establishment of the dd herd (ddR) accepted SLA-matched grafts indefinitely without any immunosuppression. A formal backcross study was therefore performed in which offspring of (cd x ddR) matings (designated cdbc and ddbc) were bilaterally nephrectomized and transplanted with SLA-matched renal allografts. Acceptors and rejectors were found among both backcross types, with a total of 6 of 17 (35%) of the animals dying of renal failure secondary to rejection. When ddbc animals were used as donors for ddR recipients, all grafts were accepted, ruling out the possibility that rejection was attributable to strong non-SLA antigens segregating within the cc herd. These results are consistent with a model in which rejection of SLA-matched renal allografts is controlled by either one or two non-SLA-linked immune response genes. These findings raise the possibility that the observed 5 to 15% frequency of rejection of HLA-identical living related donor renal allografts in man could involve similar immune response gene control.

Transplantation in miniature swine. VIII. Recombination within the major histocompatibility complex of miniature swine.

Offspring of heterozygous parents derived from three herds of miniature swine, each of which is homozygous at the major histocompatibility complex (MHC), were screened for recombination within the MHC. The swine were typed serologically at weaning and later typed by mixed lymphocyte reaction (MLR). Two intra-MHC recombinants were discovered, both of which involved the exchange of D region specificities without apparent dissociation of ABC region specificities, confirming the localization of the SLA-D region outside of the SLA-ABC regions. The first recombinant was the offspring of an SLAc/d (cd) by dd mating and typed serologically as cd but typed by MLR as dd. The second recombinant was the offspring of a cd by cd mating. It typed serologically as cc but stimulated cc in one-way MLR and retained its reactivity to dd, thus suggesting a possible recombination within the D region. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of 3H-leucine-labeled lymphocyte surface antigens demonstrated that corresponding Ia antigens were also exchanged during these recombinant events supporting the hypothesis that genes coding for Ia antigens are identical or closely linked to D region genes encoding the MLR specificities.