Repair of long tracheal defects with cryopreserved cartilaginous allografts.

Tracheoplasties with various autografts (cartilage, periosteum, pericardium) have been used in the treatment of long-segment tracheal stenosis. Previous studies have shown that cartilage allografts survive transplantation on a long-term basis in various sites of the body. In this study we set out to determine if cryopreserved cartilage and cryopreserved tracheal allografts would survive when used to cover tracheal defects in animals. A rectangular defect (2.8 +/- 0.3 cm long and incorporating 50% of tracheal circumference) was created in the thoracic trachea of 18 piglets. The defect was covered with the excised tracheal segment in 6 (group A, control group), with a cryopreserved tracheal allograft in 6 (group B), and with a cryopreserved cartilage allograft harvested from the scapula in 6 (group C). The allografts were cryopreserved, by a standard slow-freezing technique, at -80 degrees C for more than 21 days. All animals survived the grafting procedure and were killed after 2 months. None had signs of airway obstruction. Using the trachea above the defect as the standard, the mean sagittal narrowing of the airway in the repaired trachea was 0.4 mm in group A, 0.7 mm in group B, and 0.6 mm in group C; the coronal diameter in normal and grafted trachea was similar. The lumen of all grafts was lined by regenerating respiratory epithelium, and cilia were seen in many. Some cartilage was reabsorbed in group A and B but cartilage islands were present in all. In group A, reabsorption of cartilage was minimal. These findings suggest that segments of trachea or cartilage allografts can be cryopreserved, stored, and, subsequently, used when necessary for tracheoplasty.     

Pharmacokinetics of tacrolimus (FK 506) in children and adolescents with renal transplants

Background: Only few data exist on pharmacokinetics of tacrolimus in children. Patients: In 1995 and 1996, 14 children (mean age 13 years, range 5-23 years) received tacrolimus after renal transplantation; 10 of these after biopsy-proven steroid-resistant rejection (2 with vascular rejection), two for cyclosporin A (CsA)-induced severe nephrotoxicity, one for untreatable gingival hyperplasia on CsA, and one child was treated primarily after transplantation because of severe liver involvement in nephronophthisis. Pharmacokinetic investigations were performed after establishing a stable maintenance dose with trough levels in the desired window of 5-12 ng/ml. Results: Mean follow-up time was 6 months (range 3-25 months). Eleven patients were still on tacrolimus. Two were discontinued because of severe aggravation of chronic persistent hepatitis C (one of them also developed diabetes mellitus),and one patient was subsequently switched to conventional immunosuppression because of tacrolimus-associated nephrotoxicity. All tacrolimus levels were measured by a modified assay (MEIA, Tacrolimus, Abbott) with improved sensitivity. At the time of switch, median serum creatinine was 234±82 7mgr;mol;l and 6 months after switch 201±99 &mgr;mol/l. All grafts are still functioning. Mean FK-506 dose was 0.16 mg/kg body weight/day (range 0.036-0.30 mg/kg). Mean trough level was 7.1±2.6 ng/ml in the morning and 6.5±2.0 ng/ml in the evening. Median time of maximum concentration (tmax) was 120 min after application, and the mean maximum concentration (Cmax) was 15.2±6.7 ng/ml. Mean area under the curve (AUC) was 104±33 ng * h/ml, with a range from 65 to 169 ng * h/ml. No patient had unsatisfactorily low trough levels during the study. There was only a weak but significant (P<0.05) correlation between dose per kg body weight and AUC and, as expected, an excellent correlation (r²=0.73, P<0.001) between AUC and trough level. Conclusion: Because of interindividual variation between patients, therapeutic drug monitoring of tacrolimus is mandatory. In this study, a daily dose of 0.15 mg/kg was sufficient in most patients. We recommend the performance of at least one pharmacokinetic study after establishing stable FK 506 trough levels to ascertain a safe profile.     

Mechanisms by which Candida albicans induces endothelial cell prostaglandin synthesis.

One strategy for improving resistance to opportunistic pathogens is to determine host cellular responses during the invasion process and upregulate those responses that are relevant to host defense mechanisms. Within this context, we have shown previously that invasion of endothelial cells by Candida albicans in vitro causes increased production of prostaglandins. As a prerequisite for modulating endothelial cell prostaglandin production, we now characterize the mechanisms through which this process occurs. Endothelial cell invasion by C. albicans appeared to stimulate the conversion of arachidonic acid into prostaglandins by upregulating the synthesis of endothelial cell cyclooxygenase and increasing the activity of the endothelial cell phospholipase. The enhanced activities of these two enzymes were independent of calphostin C-sensitive protein kinase C and resulted in the increased production and extracellular secretion of prostaglandin I2 (PGI2), PGF2 alpha, and PGE2. The secretion of these prostaglandins had no effect on the amount of endothelial cell injury induced by C. albicans. The role of the increased prostaglandin secretion by endothelial cells is likely related to modulation of the leukocyte response at the candida-leukocyte-endothelial cell interface.     

Adherence to and damage of endothelial cells by Cryptococcus neoformans in vitro: role of the capsule.

Escape from the intravascular compartment is likely a critical step in the development of hematogenously disseminated cryptococcal infections, such as meningitis. The capsule of Cryptococcus neoformans is considered to be a virulence factor because of its antiphagocytic properties. To further investigate the role of the capsule in escape from the intravascular compartment, we used isogenic strain pairs, an acapsular mutant, and an encapsulated clinical isolate to determine the effects of the capsule of C. neoformans on adherence to, phagocytosis by, and damage of endothelial cells in vitro. Acapsular C. neoformans adhered significantly more to endothelial cells and caused greater endothelial cell injury than did encapsulated organisms. Coating of an acapsular strain with cryptococcal glucuronoxylomannan decreased both adherence to and damage of endothelial cells by 61.7% +/- 9.1% and 76.6% +/- 10.2%, respectively. Transmission electron microscopy demonstrated internalization of acapsular, but not encapsulated, organisms by endothelial cells. Internalization of an acapsular strain occurred through endothelial cell phagocytosis and was inhibited by cytochalasin D. Phagocytosis required a heat-labile serum factor, probably complement. These results suggest that acapsular or poorly encapsulated C. neoformans may be the form(s) that escapes from the vasculature during initiation of hematogenously disseminated disease.     

Resistance to platelet microbicidal protein results in increased severity of experimental Candida albicans endocarditis.

Thrombin-induced platelet microbicidal protein (tPMP) exerts potent in vitro microbicidal activity against pathogens commonly found in the bloodstream, including Staphylococcus aureus, Staphylococcus epidermidis, and Candida albicans. Localized platelet release of tPMP may be important in defense against infections involving the vascular endothelium caused by tPMP-susceptible organisms. In contrast, pathogens capable of surviving in the presence of tPMP could then exploit the platelet as an adhesive surface for attachment to damaged endothelium. To examine these hypotheses, we derived a tPMP-resistant (tPMP(r)) C. albicans strain from its tPMP-sensitive (tPMP(s)) parental strains were equivalent in vitro as assessed by genotyping (electrophoretic karyotype and restriction endonuclease analysis of genomic DNA), biotyping, germination, platelet aggregation, adherence to vascular endothelial cells, and growth characteristics. In addition, the tPMP(r) phenotype was stable following multiple in vitro and in vivo passages. We then investigated the in vivo relevance of tPMP susceptibility on endovascular infection using a rabbit model of endocarditis and hematogenous dissemination. Rabbits with transaortic catheters (n = 15 in each group) were challenged with either the tPMP(s) or tPMP(r) C. albicans strain. All rabbits developed C. albicans-induced endocarditis, as determined by the presence of infected vegetations. In rabbits challenged with tPMP(s) strain (P < 0.001). These results were seen in the absence of differences in either initial adherence of strains to cardiac valves or vegetation weights. Furthermore, although these C. albicans strains induced equivalent rates and extent of hematogenous renal infection, only the tPMP(r) strain disseminated hematogenously to the spleen (15 of 15 rabbits) versus 0 of 15 [tpmp(s) strain]; P < 0.0001). Thus, tPMP(r) C. albicans caused more-severe endocarditis and produced greater metastatic sequelae than the tPMP(s) counterpart.