Heteroduplex formation and S1 digestion for mapping alternative splicing sites.

The identification of alternatively spliced transcripts has contributed to a better comprehension of developmental mechanisms, tissue-specific physiological processes and human diseases. Polymerase chain reaction amplification of alternatively spliced variants commonly leads to the formation of heteroduplexes as a result of base pairing involving exons common between the two variants. S1 nuclease cleaves single-stranded loops of heteroduplexes and also nicks the opposite DNA strand. In order to establish a strategy for mapping alternative splice-prone sites in the whole transcriptome, we developed a method combining the formation of heteroduplexes between 2 distinct splicing variants and S1 nuclease digestion. For 20 consensuses identified here using this methodology, 5 revealed a conserved splice site after inspection of the cDNA alignment against the human genome (exact splice sites). For 8 other consensuses, conserved splice sites were mapped at 2 to 30 bp from the border, called proximal splice sites; for the other 7 consensuses, conserved splice sites were mapped at 40 to 800 bp, called distal splice sites. These latter cases showed a nonspecific activity of S1 nuclease in digesting double-strand DNA. From the 20 consensuses identified here, 5 were selected for reverse transcription-polymerase chain reaction validation, confirming the splice sites. These data showed the potential of the strategy in mapping splice sites. However, the lack of specificity of the S1 nuclease enzyme is a significant obstacle that impedes the use of this strategy in large-scale studies.

Vaccine recommendations for patients on chronic dialysis. The Advisory Committee on Immunization Practices and the American Academy of Pediatrics.

Pediatric patients on dialysis should receive all the vaccines currently recommended by the ACIP and the AAP for healthy children, except the oral polio vaccine (34, 35). Adult patients should receive the hepatitis B vaccine series, pneumococcal vaccine, yearly influenza vaccinations, tetanus-diphtheria toxoids, and varicella vaccine, if they are susceptible (33, 48, 69). Vaccines are well tolerated by these patients (33), but higher doses and/or additional boosters may be required periodically to adequately protect dialysis patients from vaccine-preventable diseases (33, 36, 37, 82, 83). Following vaccination, antibody concentrations for hepatitis B vaccine should be measured annually and booster doses administered when antibody concentrations fall below protective levels (33, 38). Although both children and adults on dialysis may show an impaired and/or delayed immunologic response to certain antigens, particularly hepatitis B virus and S. pneumoniae, appropriate immunizations can significantly reduce the risk of serious complications from vaccine-preventable diseases (11, 84). Because the protection these vaccines provide may be incomplete or transient, infection control strategies at hospitals and other health care facilities should be implemented simultaneously. Health care providers are encouraged to assess each patients need for vaccinations individually and formulate immunization strategies early in the course of progressive renal disease, ideally before the patient requires dialysis.

Rubella outbreaks among Hispanics in North Carolina: lessons learned from a field investigation.

OBJECTIVE: To describe the epidemiology and the lessons learned from two simultaneous, but unrelated, outbreaks of rubella in North Carolina affecting mostly Hispanic immigrants of Mexican origin. METHODS: A case and contact investigation was conducted at industrial work sites and Hispanic communities between March 26 and June 15, 1996, using both structured and informal interviews. Active surveillance was conducted at hospitals, clinical laboratories, primary care physicians' offices, local health departments, and migrant health centers to identify additional cases. Rubella specific IgM testing was performed by the North Carolina State Laboratory to confirm cases. Vaccination clinics were conducted in communities and at work sites with a large Hispanic population in affected counties to reduce the number of susceptible persons. RESULTS: Eighty-three confirmed cases of rubella were reported: 75 cases from the first outbreak and 8 from the second. The mean age of cases from both outbreaks was 24 and 20 years, respectively. Only three cases occurred among children under five years of age, two in the first outbreak and one in the second. Seventy-one (95%) cases in the first outbreak and all 8 cases in the second outbreak were Hispanics; 21 (28%) cases from the first and 3 (37%) from the second outbreak were females, and a total of 65 (78%) cases from both outbreaks were industrial workers. Six women with confirmed cases in the first outbreak were pregnant at the time of exposure. No females cases were pregnant in the second outbreak. CONCLUSIONS: The outbreaks in North Carolina confirmed the persistent susceptibility to rubella in Hispanics and persons migrating from countries where the rubella vaccine is not used for routine childhood vaccination. The ultimate goal of rubella vaccination programs is to prevent fetal infection and congenital rubella syndrome (CRS). Thus, to eliminate rubella from the United States, efforts should focus on understanding new emerging patterns of disease transmission and vaccinating susceptible adults in settings where they congregate.

Relapse on endocrine treatment in patients with stage D2 prostate cancer. Does second-line hormonal therapy affect survival?

Patients with Stage D2 prostate cancer relapsing on endocrine treatment have a grim prognosis. The role of a second-line hormonal treatment is debatable. We analyzed retrospectively, response and survival among 119 men (57 black, 62 white) progressing on a variety of first-line hormonal therapy. Sixty-one received a second-line hormonal treatment. Fifty-eight received only supportive therapy after first line therapy was discontinued. Age and race were not a factor in survival. Based on this retrospective study the optimal management for progressive metastatic prostate cancer cannot be delineated. However, the best results were in patients treated with diethylstilbestrol (DES) as a single treatment or when employed as either first or second treatment in patients receiving two therapies. The other modalities, e.g., bilateral orchiectomy, LH-RH analogues, and anti-androgens resulted in comparable outcomes when used either as single treatment or in combinations. Further clarification of the trends shown in this report require randomized controlled studies.

In vitro study of the effect of hyperthermia on normal bladder cell line and on five different transitional cell carcinoma cell lines.

Intraluminal hyperthermia is potentially useful in the management of superficial bladder cancer. The potential inhibitory effect of hyperthermia on various human bladder cancer cell lines, normal human bladder cells and the murine MBT-2 bladder cancer cell line has been studied in vitro. These cell lines were exposed for one hour to 43 +/- 0.5C and compared to controls. Cell survival was assessed comparing the cell growth curve and colony formation. The human transitional cell carcinoma (TCC) cell lines vary in their sensitivity to heat. MGH-U1 was the most heat sensitive cell line. The human A-1698, CUB-2, UM-UC-3 and the murine MBT-2 lines were heat insensitive. We conclude that the cytocidal effect of hyperthermia in bladder transitional cell carcinoma is variable. Further experiments using the combination of hyperthermia and intravesical anticancer agents are in progress.

Efficacy of terazosin in patients with benign prostatic hyperplasia.

Terazosin, a selective, long-acting alpha 1-adrenergic blocker, was evaluated in 44 men with benign prostatic hyperplasia. The dose was titrated from 2 to 20 mg nightly depending on improvement in symptoms and flow rate. All men completed at least 3 months of therapy, 26 had 6 months and 19 received 9-12 months of terazosin. There was an average increase of 2 ml/s in the peak urinary flow rate compared to baseline. This was statistically significant at the 3-month level. Residual urine decreased under treatment at each 3-month time interval. Prior to initiation of terazosin the mean was 165 ml, and it was 62, 100, and 41 ml at 3, 6 and 9 months respectively. There was a statistically significant improvement in both the obstructive and irritative symptom scores. Side effects were minimal; only 1 patient discontinued terazosin due to a hypotensive episode. Terazosin was found to be safe and effective in the dose range of 2-20 mg taken at bedtime in men with symptoms related to benign prostatic hyperplasia. The present study did not identify any baseline parameters such as initial prostate volume, peak flow rates, or obstructive or irritative symptom scores that correlated with clinical outcome.

Influence of nephrectomy on the growth of a murine Wilms tumor: a study using parabiotic rats.

It has been reported that unilateral nephrectomy causes acceleration of the growth of a Wistar/Furth rat Wilms tumor. We studied this phenomenon in parabiotic rats by measuring tumor growth after either sham nephrectomy, or excision of 1, 2 or 3 kidneys. We observed no stimulation of tumor growth in the experimental groups. Renal function was significantly decreased after removal of 2 or 3 kidneys. Serum creatinine levels were significantly different between right and left members of parabiotic pairs in these 2 groups. The effect of unilateral and bilateral nephrectomy on tumor growth in single rats also was examined. In these rats progressive increases in tumor growth were observed after unilateral and bilateral nephrectomy. Our inability to demonstrate a tumor-stimulating factor in the parabiotic model may be due partly to incomplete sharing of humoral factors between parabionts. Serum transfer studies in vitro may prove more fruitful in demonstrating such a factor.

Animal models of renal cell carcinoma.

Animal models are extremely valuable in studying disease processes and evaluating new therapeutic modalities. RCC in men is resistant to any type of chemo- or hormonal therapeutic regimens. Developing appropriate models is therefore important to test new therapeutic modalities or to understand basic mechanisms of this malignancy. From our review renal cell tumors appear spontaneously with a certain frequency in humans, rats, mice, and hamsters, but are extremely rare in rabbits and have not been described in guinea pigs. Some tumors have been used as renal carcinoma models, such as the VX-2 in the rabbit or a mouse tumor described by Myers, which on further evaluation do not represent true RCC. The tumors described here are either chemically induced or spontaneous tumors in a variety of species and will serve investigators as a valuable resource regarding the origin and the biologic characteristics to be applied to specific experiments.