Portal venous donor-specific transfusion in conjunction with sirolimus prolongs renal allograft survival in nonhuman primates.

Pretransplant exposure to donor antigen is known to modulate recipient alloimmunity, and frequently results in sensitization. However, donor-specific transfusion (DST) can have a protolerant effect that is dependent on route, dose and coadministered immunosuppression. Rodent studies have shown in some strain combinations that portal venous (PV) DST alone can induce tolerance, and uncontrolled clinical use of PVDST has been reported. In order to determine if pretransplant PVDST has a clinically relevant salutary effect, we studied it and the influence of concomitant immunosuppression in rhesus monkeys undergoing renal allotransplantation. Animals received PVDST with unfractionated bone marrow and/or tacrolimus or sirolimus 1 week prior to transplantation. Graft survival was assessed without any posttransplant immunosuppression. PVDST alone or in combination with tacrolimus was ineffective. However, PVDST in combination with sirolimus significantly prolonged renal allograft survival to a mean of 24 days. Preoperative sirolimus alone had no effect, and peripheral DST with sirolimus prolonged graft survival in 2/4 animals, but resulted in accelerated rejection in 2/4 animals. These data demonstrate that PVDST in combination with sirolimus delays rejection in a modest but measurable way in a rigorous model. It may thus be a preferable method for donor antigen administration.

Successful renal transplantation in patients with chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a genetic disease caused by structural mutations in the enzyme NADPH oxidase that results in severe immunodeficiency. End-stage renal disease occurs in this patient population, and is often attributed to the necessary use of nephrotoxic anti-infectives. In this report, we present the experiences of two centers in transplantation of three patients with CGD: one transplanted with CGD, one cured of his CGD with bone marrow transplantation who subsequently underwent kidney transplantation and one that received a kidney transplant prior to being cured of CGD via a sequential peripheral blood stem cell transplant (SCT). All three recipients have enjoyed excellent outcomes. Their courses demonstrate the absolute requirements for a multidisciplinary and compulsive approach before, during and after transplantation. These case reports also highlight the unexpectedly benign effects of immunosuppressive therapy in this patient population.

The Art and Science of Immunosuppression: the Fifth Annual American Society of Transplant Surgeon's State-of-the-Art Winter Symposium.

The 2005 American Society of Transplant Surgeons (ASTS) Winter Symposium entitled 'The Art and Science of Immunosuppression' explored ways to maximize existing immunosuppressive protocols and to develop new strategies incorporating novel agents and emerging diagnostic technologies to customize immunosuppression and reduce side effects. Several presentations evaluated steroid withdrawal or avoidance protocols reflecting the significant difficulties of bone loss, glucose control and growth retardation in children associated with long-term steroid use. Calcineurin-inhibitor related renal dysfunction of both native and transplanted kidneys was identified as significant, but no consensus was reached concerning effective prevention. Similarly, recurrence of Hepatitis C following liver transplantation was identified as problematic without identifying a preferred immunosuppressive regimen in this setting. Control of T-cell mediated rejection was found to be excellent, but recognition and treatment of non-T cell causes of allograft damage (i.e. B- or NK-cell mediated) was identified as an area of current interest. Immunosuppressive agents under development, such as those blocking co-stimulation or cytokine signals, and JAK-3 inhibitors were discussed. Finally, the available technologies for molecular and genetic diagnostics and the clinical correlation in the post-transplant setting were discussed.

Molecular evaluation of BK polyomavirus nephropathy.

Understanding at a molecular level, the immunologic response of polyomavirus nephropathy (PVN), a critical cause of kidney graft loss, could lead to new targets for treatment and diagnosis. We undertook a transcriptional evaluation of kidney allograft biopsies from recipients with PVN or acute rejection (AR), as well as from recipients with stable allograft function (SF). In both the PVN and AR groups, Banff histologic scores and immunohistochemical analysis of inflammatory infiltrates were similar. Despite their different etiologies, the transcriptional profiles of PVN and AR were remarkably similar. However, transcription of genes previously linked to AR including CD8 (65.9 +/- 18.8) and related molecules IFN-gamma(55.1 +/- 17.0), CXCR3 (49.9 +/- 12.8) and perforin (153.8 +/- 50.4) were significantly higher in PVN compared to AR (30.9 +/- 2.0, 14.0 +/- 7.3, 12.1 +/- 7.3 and 15.6 +/- 3.8-fold, respectively; p < 0.01). Importantly, transcription of molecules associated with graft fibrosis including matrix collagens, TGFbeta, MMP2 and 9, as well as markers of epithelial-mesenchymal transformation (EMT) were significantly higher in PVN than AR. Thus, renal allografts with PVN transcribe proinflammatory genes equal in character and larger in magnitude to that seen during acute cellular rejection. BK infection creates a transcriptional microenvironment that promotes graft fibrosis. These findings provide new insights into the intrarenal inflammation of BK infection that promotes graft loss.

Preclinical evaluation of tolerance induction protocols and islet transplantation in non-human primates.

Non-human primate studies of tolerance induction strategies in solid organ transplantation represent a critical bridge between studies in rodents and humans. Our work demonstrates that strategies involving the blockade of co-stimulatory molecules, especially the CD40-CD154 pathway, have great potential for clinical adaptation. While the combination of anti-CD154 antibody with blockade of the CD28 pathway reduced donor antibody production, graft survival was not significantly improved over that achieved with anti-CD154 antibody alone. Moreover, although long courses of steroids seem to interfere with this approach, it may be possible to combine blockade of the CD40-CD154 pathway with other conventional immunosuppressants without sacrificing efficacy. This is a key issue for reducing the risk associated with eventual clinical trials. Work in the non-human primate islet transplant model demonstrates that viable islets can be recovered, isolated and infused in a reliable fashion. It also confirms the efficacy of a steroid sparing approach to immunosuppression for islet transplantation. These data have been expanded to the kidney allograft model, setting the stage for kidney islet transplantation studies. Overall, tolerance induction and islet transplant studies in non-human primates permit the preclinical screening of promising immunomodulatory approaches developed in rodents and reduce the inherent uncertainties associated with adapting new regimens to the clinic.

Establishment of stable multilineage hematopoietic chimerism and donor-specific tolerance without irradiation.

BACKGROUND: Induction of tolerance to organ transplants will increase graft survival and decrease patient mortality and morbidity. Radiation-induced cytoreduction/ablation followed by donor hematopoietic cell reconstitution has been the most consistently successful approach to experimental tolerance induction. However, reluctance of clinicians to expose recipients to radiation has hampered its clinical application. METHODS: In the studies described, administration of polyclonal antilymphocyte serum (ALS), donor-specific bone marrow (DSBM) (150x10(6) cells), and sirolimus (24 mg/kg) in a completely mismatched murine model (B10.A donor, C57B/10 recipient) produced 100% indefinite (>250 days) skin graft survival. The level and character of donor-specific chimerism was evaluated with flow cytometry. RESULTS: Specific tolerance was confirmed by continued acceptance of primary and secondary donor-specific skin allografts and rejection of third-party grafts. The level and duration of chimerism induced was directly related to the dose of DSBM administered. Mice given 150x10(6) DSBM cells showed levels of 8-10% donor peripheral blood mononuclear cell chimerism by 30 days, and these levels persisted indefinitely (>250 days) in association with permanent tolerance of donor grafts. Eighty percent of donor chimeric cells were B lymphocytes (MHC class I and II positive, Fc receptor positive, CD45/B220 positive but negative for CD4, CD8 and Thy 1.2) and 20% were sorted in the macrophage monocyte population. CONCLUSIONS: These studies demonstrate for the first time that cytoreduction/ablation with ALS combined with sirolimus and reconstitution with donor bone marrow induces tolerance and chimerism in a completely mismatched murine combination. The use of ALS and sirolimus, currently employed therapies in clinical transplantation, and the lack of requirement for radiation make this tolerance protocol attractive for clinical application.

Determination of an improved sirolimus (rapamycin)-based regimen for induction of allograft tolerance in mice treated with antilymphocyte serum and donor-specific bone marrow.

BACKGROUND: Posttransplant donor-specific bone marrow (BM) infusion in mice treated with antilymphocyte serum (ALS) induces specific unresponsiveness (tolerance) to skin allografts, which can be augmented by the adjuvant administration of chemotherapeutic immunosuppressive agents. The purpose of this study was to determine the optimal dose and timing of administration of sirolimus (rapamycin) to induce maximal skin allograft survival in ALS-treated, BM-infused recipients. METHODS: DBA/2 donor skin grafts were placed on B6AF1 recipients (class I- and II-disparate). Groups of recipient mice (n=10 each) received combinations of the following treatment protocols: ALS, 0.5 ml on days -1 and 2; BM, 25x10(6) donor-specific cells on day 7; sirolimus, 6, 12, 18, or 24 mg/kg at times indicated; and cyclosporine, 50 mg/kg at times indicated. The immune status of putatively tolerant animals was examined with mixed lymphocyte cultures, cell-mediated lympholysis assays (CML), and limiting dilution analyses. RESULTS: When administered in conjunction with ALS/BM, a single dose of sirolimus (6 mg/kg) on days 21, 18, 14, 10, or 7 resulted in median skin graft survival times of 35, 26, 40, 46, and 103 days, respectively, versus a median survival of 27 days in mice given ALS and BM alone. The addition of cyclosporine to sirolimus (6 mg/kg) given on day 7 or days 7 and 10 did not significantly increase graft survival over that achieved with sirolimus alone. A single dose (18 or 24 mg/kg) of sirolimus administered on day 7 to ALS/BM-treated recipients resulted in 100% 200-day skin graft acceptance. Tolerant mice demonstrated nonspecific suppression of the mixed lymphocyte culture assays at 90 and 200 days and a nonspecific reduction of the CML assay at 50 days. By 200 days, the third-party CML response was restored, whereas donor-specific cell-mediated cytotoxicity remained suppressed. There was a donor-specific reduction in the number of alloreactive cytotoxic T lymphocyte clones by limiting dilution assay at 120 days. In vivo specificity of immunosuppression induced with this protocol was demonstrated by indefinite survival of second donor-specific skin grafts placed on putatively tolerant mice at day 90, whereas third-party skin grafts were rejected in 14 days. CONCLUSION: A single dose of sirolimus (18-24 mg/kg) administered on day 7, within the context of an ALS/BM immunosuppressive regimen, reliably induces permanent skin allograft acceptance in this model. In vitro measures of immunocompetence demonstrated an early nonspecific suppression of the recipients immune status and later recovery of third-party immunoreactivity. In vivo testing indicates an operationally tolerant state that is donor-specific 90 days after treatment.

Use of CTLA4-Ig in combination with conventional immunosuppressive agents to prolong allograft survival.

BACKGROUND: The objective of our study was to determine the effectiveness of CTLA4-Ig, a novel immunosuppressive agent, in augmenting allograft survival when combined with either cyclosporine, sirolimus, donor-specific bone marrow alone (BM), or bone marrow in conjunction with antilymphocyte serum (ALS). METHODS: Full-thickness skin allografts were used in C3H to B6AF1 (class I mismatch) and AKR to C57BL/6 (complete mismatch) models. Groups of mice (n=6-14) were treated with various combinations of the following treatment protocols: murine CTLA4-Ig, L-6 control Ig, sirolimus, cyclosporine, ALS, or ALS/BM. RESULTS: In the class I mismatch model, L-6 control Ig had no effect whereas use of CTLA4-Ig alone resulted in a doubling of the median graft survival compared with controls. The addition of either sirolimus or cyclosporine to CTLA4-Ig increased graft survival over that achieved with CTLA4-Ig alone. CTLA4-Ig demonstrated no efficacy when used in combination with BM, ALS, or ALS/BM. CTLA4-Ig was clearly less effective in the complete mismatch model. CONCLUSION: These data suggest that CTLA4-Ig may be effective clinically in combination with cyclosporine or sirolimus but offers no additional effectiveness in combination with antilymphocyte serum with or without donor-specific bone marrow.

Appendectomy: a contemporary appraisal.

OBJECTIVE: The authors present an accurate and comprehensive snapshot of appendicitis and the practice of appendectomy in the 1990s. METHODS: Appendectomies were performed on 4950 patients in 147 Department of Defense hospitals worldwide over a 12-month period ending January 31, 1993. RESULTS: The median age was 23 years (range, 6 months to 82 years) with 64% males and 36% females. The patients were assigned a diagnosis of normal appendix in 632 (13%) cases, acute appendicitis in 3286 (66%) cases, and perforated appendicitis in 1032 (21%) cases. There were no differences in perforation and normal appendix rates between those operations performed in teaching hospitals versus community hospitals or between high-volume hospitals (> or = 100 appendectomies/year) versus low-volume hospitals. Both a preoperative temperature > or = 100.5 and a preoperative leukocyte count > or = 10,000 were incapable of discriminating between patients with appendicitis and those with a normal appendix. Multivariate analysis showed a significantly increased risk of perforation associated with age younger than or equal to 8 years (38% vs. 18%) and age older than or equal to 45 years (49% vs. 18%). Females had a significantly higher rate of normal appendices (19% vs. 9%) and a lower rate of perforation (18% vs. 23%). The complication rates to include reoperation and intraabdominal sepsis were markedly increased in those patients with perforation. There were four deaths in this series (0.08%). CONCLUSIONS: Despite a marked decline in associated mortality over the past 50 years, rates of perforation and negative appendectomy remain unchanged because they are influenced strongly by factors untouched by the intervening technologic advances.

Superiority of sirolimus (rapamycin) over cyclosporine in augmenting allograft and xenograft survival in mice treated with antilymphocyte serum and donor-specific bone marrow.

BACKGROUND: Sirolimus is a potent immunosuppressive agent with great therapeutic potential. The objective of our study was to evaluate the efficacy of sirolimus versus cyclosporine in augmenting the unresponsiveness induced by an antilymphocyte serum (ALS)/donor-specific bone marrow (BM)-based regimen across three levels of histoincompatibility: class I and II disparate (DBA/2 to B6AF1), complete mismatch (AKR to C57BL/6), and xenograft (ACI rat to B6AF1). METHODS: Full-thickness skin grafts were taken from donors and placed on recipients in standard fashion. Seven groups of recipient mice (n=10-28) received various combinations of the following treatment protocols: sirolimus, 1.5 mg/kg (3.0 mg/kg for xenografts) every other day from day 0 to day 12; cyclosporine, 50 mg/kg every other day from day 10 through 22; ALS, 0.5 ml on days -1 and 2 for allografts and days -1, 2, and 4 for xenografts; and BM, 25 million donor-specific cells IV on day 7. RESULTS: The administration of ALS or ALS/BM resulted in modest but significant prolongation of skin graft survival in all combinations tested. Cyclosporine combined with ALS or ALS/BM significantly extended allograft survival compared with ALS or ALS/BM alone (P<0.05) but had no effect on xenograft survival. In contrast, the combination of sirolimus with ALS or ALS/BM resulted in a two- to threefold increase in allograft survival and over a fourfold increase in xenograft survival when compared with the comparable cyclosporine-based regimen. Additionally, lymphocytes isolated from class I and II incompatible mice with skin grafts surviving >100 days demonstrated markedly reduced interleukin 2 and interferon-gamma secretion in response to irradiated donor-specific lymphocytes in culture. CONCLUSIONS: In the regimens tested, sirolimus was superior to cyclosporine in augmenting donor BM-induced skin graft prolongation in ALS-treated mice across all levels of histoincompatibility.

Appendectomy. Improving care through quality improvement.

OBJECTIVE: To evaluate the practice of appendectomy in Department of Defense hospitals worldwide in a large-scale quality improvement initiative. DESIGN: Case series study. POPULATION AND SETTING: A total of 4950 consecutive nonincidental appendectomies performed in 147 Department of Defense hospitals worldwide during a 12-month period ending January 31, 1993. RESULTS: The mean age was 25.5 years, with 64% males and 36% females. The patients were assigned a diagnosis of normal appendix (negative appendectomy) in 632 cases (12.8%), acute appendicitis in 3286 cases (66.4%), and perforative appendicitis in 1032 cases (20.9%). The influence of inpatient and outpatient delays on perforation and negative appendectomy rates were studied. In at least 52% of all patients ultimately assigned a diagnosis of perforative appendicitis, the perforation occurred before the first outpatient contact with the health care system, and in at least 68% of all patients ultimately assigned the diagnosis of perforative appendicitis, the perforation occurred before surgical evaluation and admission. Neither outpatient delay in diagnosis nor inpatient delay in diagnosis and treatment was associated with a significant change in the rate of negative appendectomy. CONCLUSIONS: Perforation rates are determined predominantly by patient- and primary care-related factors over which surgeons have little control. Negative appendectomies are predominantly related to the wide overlap in presenting signs and symptoms between appendicitis and the diseases that most often mimic it but do not require operative intervention. Whereas studies of this type are useful for identifying potential problems at the health care system level, the relatively small number of appendectomies performed by each surgeon precludes analysis at the practitioner level.

Reversibility of deoxycholate-induced cellular hypercalcemia in rabbit gastric mucosal cells.

BACKGROUND: Bile acid exposure produces cellular hypercalcemia in gastric and hepatic cells. It is not known, however, whether this event contributes to cell injury or if it results from passive equilibration of calcium ion concentrations across the membranes of irreversibly damaged cells. This study was performed to determine whether the cellular hypercalcemia produced by bile acid exposure in gastric cells is reversible and to determine whether the source of this hypercalcemia is from intracellular stores of calcium, extracellular sources, or both. METHODS: Cytosolic free calcium concentrations ([Ca]i) were measured in rabbit gastric mucosal cells that had been loaded with the intracellular probe FURA-2. Measurements were performed in suspensions of dispersed cells by using standard spectrofluorometry and in primarily cultured cells by using fluorescence videomicroscopy. Measurements were made before and after exposure to 0.2, 0.5, and 1.0 mmol/L deoxycholic acid (DC). These measurements were made in the presence of 1 mmol/L extracellular calcium and in the absence of any extracellular calcium (0.5 mmol/L EGTA). RESULTS: In experiments with dispersed cells and spectrofluorometry, [Ca]i increased from a pretreatment level of 194 +/- 8 nmol/L to 396 +/- 21 nmol/L within 3 minutes of exposure to 0.2 mmol/L DC. When these cells were washed and resuspended in DC-free medium, [Ca]i] decreased to 180 +/- 5 nmol/L. In experiments with cultured cells and fluorescence videomicroscopy, rapid, reversible hypercalcemia was observed after exposure to 0.5 and 1.0 mmol/L DC. Removal of extracellular calcium from the incubating medium reduced both the magnitude and duration of the observed hypercalcemia. CONCLUSIONS: These data show that the cellular hypercalcemia that accompanies DC-induced injury in gastric cells is a reversible event. The initial increase in [Ca]i appears to come from both intracellular and extracellular sources, although sustained hypercalcemia requires a source of extracellular calcium. As a reversible event, cellular hypercalcemia may be an important pathophysiologic feature of bile acid induced injury of the upper gastrointestinal tract.

Long-term impact of previous breast biopsy on breast cancer screening modalities.

Approximately 80% of breast biopsies are performed for what proves to be a benign process. The patients who undergo these procedures should continue screening with breast physical examination and mammography. The long-term impact of breast biopsy on these screening modalities has not been well studied. We performed a prospective, follow-up evaluation in 63 patients who underwent needle localization biopsy with benign histology at our institution between 6 and 7 years ago. This evaluation consisted of a directed history, breast physical examination, and follow-up mammogram. Two patients (3%) had undergone mastectomy for an interval breast cancer; 17 others (28%) had undergone subsequent biopsies. No patient had changes on physical examination of the biopsy site. All mammograms were evaluated as normal or as having benign abnormalities. Excisional breast biopsy does not generally produce long-term changes affecting the interpretation of breast physical examination or mammography.

Pediatric appendectomy.

PURPOSE: To define patterns of care and outcome for pediatric appendectomy. METHODS: A study was designed to evaluate all pediatric appendectomies performed in the 147 Department of Defense hospitals worldwide. Cases of nonincidental appendectomy were identified through discharge diagnoses and operative logs, and 98.6% of the charts were retrieved for review. All charts were abstracted, and data were entered into a 127-field database for analysis. RESULTS: Over a 12-month period, ending January 1993, appendectomy was performed on 1,366 pediatric patients in the Department of Defense hospital system. The patients' median age was 12 years (range, 6 months to 18 years); 59% were male. The diagnosis was normal appendix for 157 patients (12%), acute nonperforated appendicitis for 930 (68%), and perforated appendicitis for 279 (20%). Age < or = 8 years was predictive (P < .001) of a higher rate of perforated appendicitis (33% v 18%) but was not predictive of normal pathology (13% v 11%). Female gender was associated with a significantly higher rate of normal pathology (17% v 8%; P < .001) but not of perforation (18% v 22%). Temperature elevation and right lower quadrant pain and tenderness did not clinically distinguish between diagnostic groups. Sixty-two percent of patients with a normal appendix had a white blood cell count of more than 10,000/mm3, as did 91% of patients with acute or perforated appendicitis. Those with perforated appendicitis received pre- and postoperative antibiotics, primarily ampicillin/gentamicin/clindamycin or Flagyl (41%), cefoxitin (34%), or Unasyn (15%). In 77% of this subgroup, intraoperative cultures were positive, with isolates for Escherichia coli (76%) Enterococcus (30%), Bacteroides (24%), and Pseudomonas (20%) predominating. There were no deaths. Major complications occurred in 1.2% of patients with acute appendicitis and in 6.4% of those with perforated appendicitis; there were no major complications in the group with normal appendectomies. The hospitalization period was more than 7 days for 1.6%, 40%, and 3.8%, respectively. CONCLUSION: This large series, from a large number of hospitals, with multiple practitioners, can serve as a community standard for pediatric appendectomy in the 1990s.

Selective digital enhancement of conventional film mammography.

Screening mammography continues to play a key role in the early diagnosis of non-palpable breast cancer. Approximately 5% of patients will have lesions on their mammograms that appear neither clearly benign nor malignant. The proper management of these patients is not well defined. In this study, the mammograms of 64 women who underwent breast biopsy were reviewed. After the initial review, the mammograms were reevaluated using a personal computer driven digital conversion and enhancement system. The majority of indeterminate lesions were assigned a determinate diagnosis by enhancing the areas of abnormality through enlargement, contrast adjustment, and brightness adjustment. The radiologists were able to maintain baseline sensitivity and specificity throughout this process. In contrast, enhancement of lesions initially assigned a diagnosis of characteristically benign or malignant demonstrated no advantage over plain film mammography. These findings suggest a role for the selective use of digital mammography and enhancement techniques.

Complete colonic obstruction caused by a sigmoid colon cancer incarcerated in an inguinal hernia sac.

An 85-year-old man with a clinical and x-ray picture consistent with a large bowel obstruction had a large left inguinal hernia with an incarcerated loop of sigmoid colon containing a small annular constricting colon carcinoma. Colonic obstruction due to inguinal hernia alone is so rare that a thorough evaluation should be done preoperatively in any patient believed to have colonic obstruction due to inguinal hernia.

Small intestinal transplantation in nonhuman primates.

Small intestinal transplantation represents a potentially therapeutic procedure for individuals with short gut syndrome. The purpose of this study was to develop a model for small intestinal transplantation in primates that is: technically feasible without microsurgery; consistent in the prevention of allograft rejection; functional in terms of nutrient absorption; and compatible with harvest for multiple organ procurement. First, autotransplantations on four rhesus monkeys were performed in order to study a variety of harvesting techniques and vascular anastomoses. Then, a study was performed with 14 heterotopic allotransplants in 4 baboons and 10 rhesus primates. The successful donor model consisted of division of the pancreas, harvesting the small bowel with a superior mesenteric artery and portal vein pedicle. The allograft vascular pedicle was anastomosed to the recipient's common iliac vessels in end-to-side fashion. The graft was transplanted as an out-of-continuity loop, both ends being exteriorized as stomas providing access for absorption studies and biopsy. Three immunosuppressive regimens were tested: (1) cyclosporine A (CyA) 20 mg/kg/d, solumedrol (SML) 2 mg/kg/d, and graft irradiation (150 rad) (n = 4); (2) CyA 20 mg/kg/d and SML 2 mg/kg/d (n = 3); and (3) CyA 40 mg/kg/d, SML 2 mg/kg/d, and azathioprine 5 mg/kg/d (n = 3). There were 4 deaths due to technical error in the first 24 hours. Weekly graft biopsy, serum CyA levels, complete blood count, and automated 24-channel serum analysis were performed. Grafts surviving greater than 14 days underwent absorption study via luminal perfusion with sucrose, maltose, dextrose, Pregestimil, xylose, and cyclosporine.(ABSTRACT TRUNCATED AT 250 WORDS)