Optimal primer design using the novel primer design program: MSPprimer provides accurate methylation analysis of the ATM promoter.

Methylation-specific polymerase chain reaction (PCR) (MSP) is frequently used to study gene silencing by promoter hypermethylation. However, non-specific primer design can lead to false-positive detection of methylation. We present a novel, web-based algorithm for the design of primers for bisulfite-PCRs (MSP, sequencing, COBRA and multiplex-MSP), allowing the determination of a specificity score, which is based on the thermodynamic characteristics of the primer 3'-end. PCR amplification with primers not reaching a high specificity score can result in false-positive findings. We used MSPprimer to design MSP primers for analysis of the ATM promoter. In 37 non-small cell lung cancer (NSCLC) samples and 43 breast cancer samples no promoter methylation was detected. Conversely, published MSP primers not reaching the required specificity score led to non-specific amplification of DNA not converted by bisulfite. The result was a false-positive incidence of ATM promoter methylation of 24% in NSCLC and 48% in breast cancers, similar to published studies. This highlights the critical need for specific primer design for MSP. MSPprimer is a convenient tool to achieve this goal, which is available free of charge to the scientific community.

Modulation of CD28 expression: distinct regulatory pathways during activation and replicative senescence.

The costimulatory molecule CD28 has a restricted tissue distribution and is expressed on T cells and some plasmacytoma cells. Although CD28 is constitutively expressed, its expression is transiently down-regulated following T cell activation and declines progressively with in vitro senescence. In vivo, CD8+ T cells and, less frequently, CD4+ T cells may completely lose CD28 surface expression during chronic infections and with aging. This correlates with changes of nuclear protein-binding activities to two motifs, site alpha and beta, within the CD28 minimal promoter. Both alpha- and beta-bound complexes are found only in lymphoid tissues, in CD28+ T cells, and in some transformed B cells. These complexes are coordinately expressed except during replicative senescence, which is characterized by the down-modulation of site beta- but not site alpha-binding activities. In contrast, T cell activation induces a parallel decline in both site alpha- and beta-binding activities. CD4+ and CD8+ T cells differ in their beta-binding profiles, which may explain the more pronounced down-regulation of CD28 in senescent CD8+ T cells. In vivo expanded CD4+CD28null and CD8+CD28null T cells uniformly lack alpha- and beta-bound complexes, resembling the pattern seen in chronically activated cells and not of senescent cells.

Functional properties of CD4+ CD28- T cells in the aging immune system.

The aging immune system is characterized by a progressive decline in the responsiveness to exogenous antigens and tumors in combination with a paradoxical increase in autoimmunity. From a clinical viewpoint, deficiencies in antibody responses to exogenous antigens, such as vaccines, have a major impact and may reflect intrinsic B cell defects or altered performance of helper T cells. Here we describe that aging is associated with the emergence of an unusual CD4 T cell subset characterized by the loss of CD28 expression. CD28 is the major costimulatory molecule required to complement signaling through the antigen receptor for complete T cell activation. CD4+ CD28- T cells are long-lived, typically undergo clonal expansion in vivo, and react to autoantigens in vitro. Despite the deficiency of CD28, these unusual T cells remain functionally active and produce high concentrations of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2). The loss of CD28 expression is correlated with a lack of CD40 ligand expression rendering these CD4 T cells incapable of promoting B cell differentiation and immunoglobulin secretion. Aging-related accumulation of CD4+ CD28- T cells should result in an immune compartment skewed towards autoreactive responses and away from the generation of high-affinity B cell responses against exogenous antigens. We propose that the emergence of CD28-deficient CD4 T cells in the elderly can partially explain age-specific aberrations in immune responsiveness.

Optimization of dialysate flow and mass transfer during automated peritoneal dialysis.

The purpose of this study was to evaluate the effect of patient position on the mass transfer area coefficient (KoA) and to characterize drain/fill profiles in an effort to enhance efficiency of automated peritoneal dialysis. Over 100 exchanges were performed in 38 stable peritoneal dialysis patients to either determine the small solute KoA in the supine versus upright position or to characterize fill/drain profiles. The KoA for all solutes tested was significantly greater in the supine position compared with the upright position (P < 0.05). Fill profiles revealed the fill rate to be a function of fill height (P < 0.001) and patient position (supine > upright [P < 0.001]). Analysis of drain flow rate versus time revealed an initial segment of high outflow (350 +/- 89 mL/min) followed by an abrupt transition to a segment characterized by slow drainage (36 +/- 21 mL/min). The first segment of drain only took 5.6 +/- 2.3 minutes (42% of the total drain time); in that time, 83% +/- 10% of the dialysate was drained. The transition volume (volume of dialysate remaining at the time the transition occurs, excluding residual volume) correlated with body surface area (R = 0.52, P < 0.01). In conclusion, automated peritoneal dialysis treatment (including intermittent peritoneal dialysis, which may be done in the upright position) should be done in the supine position to optimize the KoA, and shortening drain time to include only the initial segment of high outflow will improve the efficiency and convenience of therapy.

Relationship between body size, fill volume, and mass transfer area coefficient in peritoneal dialysis.

A peritoneal dialysate fill volume of 2 L has become the standard of clinical practice, but the relationships between body size, fill volume, and mass transfer area coefficient (KoA) have not been well established. These relationships were studied in 10 stable peritoneal dialysis patients who underwent six peritoneal equilibration studies (2 h each) at fill volumes of 0.5, 1, 1.5, 2, 2.5, and 3 L. The concentration-time profiles for urea, creatinine, and glucose were measured at each fill volume, and residual volumes were calculated from the preceding dwell period. A modified Henderson equation was used to calculate the KoA for the three solutes as a function of fill volume. By normalizing the KoA for each solute to the value at 2 L, the data for all three solutes collapsed onto the same trend line when plotting the normalized KoA versus dialysate volume. Between 0.5- and 2-L fill volumes, the average normalized KoA increases in an almost linear fashion, its value almost doubling over this range. Between 2- and 3-L fill volumes, there is less than a 10% change in the normalized KoA. However, fill volumes for peak urea KoA were found to increase with increasing body surface area (R = 0.76), being around 2.5 L for an average-sized patient and increasing to between 3 and 3.5 L for body surface areas > 2 m2. To maximize solute transport, these relationships between body size, volume, and KoA should be considered when choosing fill volumes for continuous ambulatory peritoneal dialysis and automated peritoneal dialysis and when deciding reserve and tidal volumes for tidal peritoneal dialysis.

Hepatitis C is a poor prognostic indicator in black kidney transplant recipients.

BACKGROUND: The significance of hepatitis C in kidney transplant recipients is unclear. The prevalence of antibodies to hepatitis C among candidates for transplantation is up to 50% in some centers. METHODS: We screened 640 frozen serum samples obtained pretransplantation from all kidney recipients at the Medical College of Wisconsin between January 1979 and March 1990 for antibody to hepatitis C using the second generation immunoassay. Charts were reviewed from all hepatitis C antibody-positive (anti-HCV+) patients and 256 randomly chosen hepatitis C antibody negative (anti-HCV-) controls. Actuarial patient and graft survival in these two groups were determined. RESULTS: The prevalence of anti-HCV was 8.3%. Blacks and i.v. drug users were disproportionately represented in the anti-HCV+ group. Of the anti-HCV+ patients, 18.9% developed chronic hepatitis independent of race. Black anti-HCV+ patients had a 5-year graft survival of 28 +/- 11% compared to 67 +/- 7% in black anti-HCV- patients (P = 0.003). Black anti-HCV-, white anti-HCV-, and white anti-HCV+ patients all had similar graft survival. Anti-HCV was not a poor prognostic indicator for overall patient survival or the development of aplastic anemia and malignancies including hepatocellular carcinoma. CONCLUSIONS: Anti-HCV is a significant risk factor for reduced kidney graft survival in blacks apart from i.v. drug abuse. Black anti-HCV- patients had graft survival similar to white transplant recipients, indicating that anti-HCV may be one marker for the poorer graft survival in blacks that has been observed in most transplant programs. Anti-HCV in kidney transplant recipients increases the risk for the development of chronic hepatitis post-transplant.

Clinical outcome of continuous ambulatory peritoneal dialysis predicted by urea and creatinine kinetics.

The effectiveness of urea kinetics (Kt/V, where K is urea clearance, t is treatment time, and V is the volume of distribution for urea) to assess the adequacy of continuous ambulatory peritoneal dialysis (CAPD) and clinical outcome has not been established prospectively, and cross-sectional clinical studies have been inconclusive. A minimum weekly creatinine clearance of 40 to 50 L is recommended, but the adequacy of this dose is unproven. We introduced a simpler approach to creatinine kinetics in the form of an efficacy number (EN) calculated from data obtained in a standardized 4-h dwell exchange. To determine the most effective model for predicting CAPD adequacy, residual renal function, weekly Kt/V urea, weekly creatinine clearance standardized to body surface area, and EN (liters per gram of creatinine per day) were measured in 18 stable CAPD patients followed prospectively for at least 12 months. Patients were divided into three groups, good (G), intermediate (I), and poor (P), on the basis of uremic symptoms, mortality, hospital days, biochemical indices, and the need for transfer to hemodialysis. When comparing groups G (N = 6) and P (N = 8), weekly Kt/V were 2.3 +/- 0.2 versus 1.5 +/- 0.1 (P less than 0.005), weekly creatinine clearances were 71.5 +/- 8.6 versus 35.1 +/- 1.3 L (P less than 0.001), and EN were 7.4 +/- 0.8 versus 3.6 +/- 0.2 L/g of creatinine/day (P less than 0.005). Creatinine kinetics (weekly clearance and EN) but not urea kinetics could differentiate group I (N = 4) from groups G or P. Both urea and creatinine kinetics predict clinical outcome in CAPD.(ABSTRACT TRUNCATED AT 250 WORDS)

Obstructive acute renal failure by a gravid uterus: a case report and review.

Acute renal failure caused from an obstruction by the gravid uterus is a rare complication of pregnancy. Only 13 cases have been reported. We report a case of obstructive acute renal failure in a patient at 34 weeks' gestation with a twin pregnancy complicated by polyhydramnios. The serum creatinine peaked at 1,078 mumol/L (12.2 mg/dL), higher than in the previously reported patients. The striking feature of her clinical course was the immediate resolution of anuria following amniotomy, thus avoiding the need for dialysis, ureteral stenting, or immediate surgical delivery. This patient illustrates the potential importance of increased uterine pressure on the ureters as a cause of significant obstructive renal failure during pregnancy.

A method to assess efficacy of CAPD: preliminary results.

Adequacy of dialysis is a primary concern when caring for patients undergoing continuous peritoneal dialysis (CAPD). To determine objectively the efficacy of CAPD, the use of an 'efficacy number' (EN) calculated from the data obtained in a peritoneal equilibration test (PET) for creatinine (cr) is proposed: EN = [cr(D/P) x V24] divided by ACPPD3 Where, cr(D/P) is PET-derived dialysate/plasma ratio for creatinine at 4 hrs; V24 is the volume of exchanges (L) prescribed for 24 hrs; ACPPD is adjusted creatinine production based on daily dialysate creatinine appearance. PET were performed and the EN calculated in two groups of CAPD patients observed over a 10 month period. One group (n = 8) had a poor clinical outcome in terms of uremic parameters. The EN in this group was 3.85 +/- 0.45 (+/- 1 SD) L/g creatinine/day. The other group (n = 4) was considered well dialyzed and had a good clinical outcome over 10 months. The EN in this group was 6.07 +/- 0.40 L/g creatine/day, p less than 0.001. There was no statistically significant difference between the two groups in regard to sex, age, length of time on dialysis, underlying kidney disease, baseline creatinine, or D/P ratios of creatinine and BUN. The 'efficacy numbers' appears to be more useful than the D/P ratio alone in determining the adequacy of CAPD. A simple to use nomogram is presented which provides guidelines for the clinician to alter the dialysis prescription.

Pregnancy in lymphocytic hypophysitis: case report and review.

Lymphocytic hypophysitis has reported in 22 cases, all but one in women. More than 80% of the cases in women occurred during pregnancy or in the postpartum period. We describe a girl with partial hypopituitarism secondary to lymphocytic hypophysitis who, five years after the diagnosis, successfully delivered a healthy infant at term. Of the previously reported cases, most patients remain amenorrheic after the diagnosis. From the present case report and review of the literature, it would appear that a diagnosis of lymphocytic hypophysitis does not necessarily preclude the patient's ability to conceive and successfully carry the pregnancy. Pituitary hormone deficiency in this disease, therefore, does not always include the gonadotropins.

Pyelonephritis complicating relapsing acute pancreatitis.

A case of right pyelonephritis with hydronephrosis complicating relapsing acute pancreatitis and right pararenal phlegmon formation is presented. Hydronephrosis is a reportedly rare complication of extrapancreatic inflammation; the only 6 previous cases involving the right side are reviewed. The present case report, to our knowledge, is the first to describe clinical and laboratory evidence of pyelonephritis secondary to partial obstruction of the right upper renal tract by an extrapancreatic phlegmon. The clinician caring for patients with acute pancreatitis should be aware of this important complication, since the presentation of pyelonephritis-flank pain and fever--could erroneously be attributable solely to the pancreatitis.