Serum leptin correlates with fat mass but not dietary energy intake in continuous ambulatory peritoneal dialysis patients.

OBJECTIVES: In view of previous studies demonstrating hyperleptinemia in uremic and hemodialysis patients, the aims of the present study were to determine whether serum leptin levels are elevated in peritoneal dialysis (PD) patients, to establish whether leptin is significantly removed by PD, and to elucidate the relationship of plasma leptin to body composition, dietary intake, nutritional indices, and dialysis adequacy. DESIGN: Cross-sectional analysis of PD patients and matched healthy controls. SETTING: Tertiary-care institutional dialysis center. PARTICIPANTS: The study included 49 PD patients [35 women and 14 men; median age 63 years, interquartile range (IQR) 49.5-68.5 yr; body mass index (BMI) 25.5 +/- 0.8] and 27 controls (11 men and 16 women; median age 42 years, IQR 34.8-51; BMI 27.2 +/- 0.9). For evaluation of leptin clearance, 8 patients receiving nocturnal intermittent PD were also evaluated. MAIN OUTCOME MEASURES: The primary outcome measure was plasma leptin concentration. Dialysate leptin concentration was also measured in 7 patients. RESULTS: Serum leptin levels were significantly higher (p < 0.01) in patients (males: median 11 ng/mL, IQR 9-19 ng/mL; females: 53 ng/mL, 19.5-128 ng/mL) compared with controls (males: 5.5 ng/mL, 4-9.5 ng/mL; females: 12 ng/mL, 9.8-17.3 ng/mL). Leptin levels in both groups correlated positively with BMI (r = 0.64 and 0.60, respectively; p < 0.0001) and with percentage body fat determined by dual-energy x-ray absorptiometry (r = 0.86 and 0.82, respectively; p < 0.01). Dialysis patients exhibited a greater increase in serum leptin for any given increase in BMI. No significant correlation was observed between leptin concentration and residual renal function, dialysis adequacy (Kt/V), dietary protein or caloric intake, or serum levels of albumin, prealbumin, C-reactive protein, glucose, and insulin-like growth factor-I. Although leptin was detectable in peritoneal dialysate after a 6-hour dwell (median 4.2 ng/mL, IQR 1.1-8.5 ng/mL, n = 8), serum leptin levels were not appreciably lowered following intermittent PD via an automated cycler (63.9 +/- 19.3 ng/mL vs 57.6 +/- 20.5 ng/mL, p = NS, n = 8). CONCLUSIONS: Serum leptin levels are elevated in PD patients and are not appreciably cleared by PD. Although hyperleptinemia correlates poorly with dialysis adequacy and protein intake, a strong and significant relationship was maintained between serum leptin and fat mass. Serum leptin could therefore serve as a useful clinical marker of body fat content in PD patients.

The cytokine and histological response in islet xenograft rejection is dependent upon species combination.

BACKGROUND: Islet xenografts have clinical potential, may avoid hyperacute rejection, and therefore are a good place to examine the cellular xenograft immune response. The aim of this study was to examine the cellular, humoral, and cytokine response in islet xenograft rejection and to determine the difference in the immune response with a different donor species. METHODS: Two islet xenograft models (DA rat islets to B6AF1 mouse and canine islets to B6AF1 mouse) and a mouse syngeneic control model were examined histologically and by a semiquantitative polymerase chain reaction method. RESULTS: There was significant up-regulation of all intragraft cytokines tested (interleukin [IL]-2, IL-4, IL-5, IL-10, and interferon-gamma) in both xenograft models compared with the controls. However, the dog islet grafts had higher levels of IL-4 and IL-5 gene expression than the rat islet grafts, which, conversely, had higher levels of interferon-gamma gene expression. These differences correlated with the histological and anti-donor antibody production differences between the two models. The dog to mouse model had an intense eosinophilic infiltrate and an early up-regulation of anti-donor antibody, whereas there was little eosinophilic infiltrate and a delayed anti-donor antibody up-regulation in the rat to mouse model. CONCLUSIONS: The mouse used different mechanisms to reject the rat and canine islets, suggesting that the immune response in islet xenograft rejection may be dependent on the species combination. It may not be possible to characterize the cellular xenograft rejection response in a bipolar manner as has been the case with humoral rejection response. Caution therefore needs to be taken before extrapolating the cellular immune responses seen in animal models to the clinical setting.

Experimental hyperacute rejection in pancreas allotransplants.

A model of sensitization by intraperitoneal lymph node inoculation was developed to test the hypothesis that hyperacute rejection (HAR) could occur in sensitized recipients of vascularized pancreas allografts. Ten pairs of outbred mongrel dogs that were lymphocytotoxic cross-match assay negative were inoculated with homogenized lymph nodes on either three or four occasions at fortnightly intervals before renal transplantation. A renal allograft from the same donor was used to test the HAR response and to further enhance sensitization by rejection of a vascularized organ. Pancreas transplants were performed 2 weeks later, with biopsies of the graft and blood samples taken at 0, 10, 20, and 30 min and then at 30-min intervals until the grafts were no longer viable. All renal and pancreas grafts were rejected in a classical hyperacute pattern. Within 4 min of revascularization of the pancreas, central lobular hemorrhage and vascular congestion appeared, followed by general edema. Histology demonstrated parallel changes of edema, vascular congestion, necrosis, hemorrhage, and leukocytic infiltrate, which all preceded graft infarction. A sharp decline in both arterial and venous white blood cell count and platelets occurred within 10 min of revascularization with initial sequestration and subsequent release of platelets from the graft (P=0.02). In summary, HAR of the allografted pancreas can be observed by the surgeon within minutes of revascularization, with predictable macroscopic and microscopic changes. This study supports the use of routine lymphocytotoxic cross-match tests for all recipients of pancreas transplants and implies that particular care is warranted in regraft pancreas allograft recipients.

HLA-DQA1 and -DQB1 genotyping by PCR-RFLP, heteroduplex and homoduplex analysis.

PCR-RFLP typing methods for DQA1 and DQB1 in conjunction with the analysis of heteroduplex and homoduplex patterns have allowed a simple method for typing all of the major DQA1 and DQB1 alleles. This method has advantages over PCR amplification with sequence-specific primers (PCR-SSP), PCR hybridization with sequence-specific oligonucleotide probes (PCR-SSO) and other PCR-RFLP strategies for typing DQ alleles. The analysis of heteroduplex and homoduplex patterns can be used in conjunction with other PCR typing systems such as PCR-SSP as a confirmatory step with little additional work. In addition, a PCR-RFLP strategy was designed for resolving the DQB1*0602 and DQB1*0603 alleles, which involved the use of a primer containing a base mutation, creating a new restriction site which distinguished the two alleles. These techniques have enabled resolution of the major homozygous and heterozygous combinations of these DQA1 and DQB1 alleles. The PCR-RFLP technique does not require the large number of oligonucleotides that are necessary for both the PCR-SSP and PCR-SSO techniques and is thus both time and cost effective for infrequent or small numbers of samples.

Diagnostic utility of whole blood cyclosporine measurements in renal transplantation using triple therapy.

Whole blood CsA concentrations measured by specific monoclonal RIA (CYCLO-Trac SP whole blood RIA, IncSTAR) were compared with episodes of renal dysfunction (n = 138) and protocol biopsies (n = 52) that occurred within the first 100 days in consecutive renal allograft recipients receiving triple therapy (n = 92). Histological confirmation of events was available in 98% episodes of acute rejection (n = 60/61), 59% of episodes of CsA nephrotoxicity (22/38), and 100% of the diagnoses of acute tubular necrosis (35/35). Mean, minimum, and maximum CsA levels were elevated in CsA nephrotoxicity compared with all other groups (P < 0.001). Interestingly, CsA levels achieved relative to administered dose also increased at the time of CsA nephrotoxicity compared with other groups (P < 0.01). In the context of acute dysfunction, the sensitivity and specificity of mean CsA levels above 400 ng/ml to predict CsA nephrotoxicity were 32% and 89%, respectively. The negative predictive value of a high CsA level to exclude acute rejection was 88% (at 400 ng/ml), 92% (450 ng/ml), and 95% (500 ng/ml). As a marker of effective immunosuppression, CsA levels were not correlated with in vitro proliferation of PHA-stimulated PBL and did not reduce the severity and degree of cellular infiltration in needle core biopsies during rejection. The sensitivity and specificity of a low CsA level (150 ng/ml) in the diagnosis of acute rejection were 31% and 91%, respectively. The majority of episodes of acute dysfunction, including 63% of CsA nephrotoxicity and 59% of acute rejections, occurred with CsA levels between 150 and 400 ng/ml. In summary, when using low dose triple therapy regimens, CsA levels within the range of 150-400 ng/ml were of little diagnostic value in acute allograft dysfunction. In contrast, levels outside this range were useful in the clinical diagnosis of CsA nephrotoxicity and acute allograft rejection.

Inhibition of PHA induced mononuclear cell proliferation by FK506 in combination with cyclosporine, methylprednisolone, 6-mercaptopurine and mycophenolic acid.

In vitro inhibition of human peripheral blood mononuclear cell (PBMC) proliferation by immunosuppressive drugs has been shown to correlate with clinical outcomes in kidney transplant patients. The aim of our study was to analyse the degree of variability of in vitro interactions of FK506 (FK) with combinations of cyclosporin A (CyA), methylprednisolone (MP), 6-mercaptopurine (6ME), and mycophenolic acid (MPA) using phytohaemagglutinin (PHA) stimulated PBMCs. Our hypotheses were: that a wide range of interindividual variation would be detected; and that certain combinations of drugs would have a synergistic inhibitory effect on PHA stimulated PBMCs. Cells were cultured in supplemented RPMI 1640 for 65 hours at 37 degrees C in humidified 5% CO2 - 95% air and proliferative responses measured by 3H-thymidine incorporation over a further 24 hours. Inhibition of PBMC proliferation was assessed over 10 FK concentrations ranging from 1 x 10(-8) to 10 micrograms/ml using both volunteer controls (n = 51) and dialysis patients (n = 23). A wide range of interindividual variability of FK inhibition occurred throughout the range of FK concentrations tested, becoming less variable at the higher concentrations. The interactions of FK with CyA, MP, 6ME and MPA were assessed using PBMCs from 12 volunteer controls. For FK at 1 x 10(-8) micrograms/ml; CyA, MP, 6ME and MPA were used at 0.01 microgram/ml. For FK at 1 x 10(-7) micrograms/ml; CyA, MP, 6ME and MPA were used at 0.1 microgram/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Histoplasma capsulatum. Isolation from an Australian cave environment and from a patient.

The fungus Histoplasma capsulatum, although commonly found in bat-frequented caves in many countries, has not previously been isolated from that environment in Australia. This report describes the isolation of H. capsulatum from several sources, including cave soil, the organs of mice exposed to the cave environment, and the sputum of a patient clinically diagnosed as having acute pulmonary histoplasmosis after exposure to the cave environment.