Rejection of male skin grafts by splenectomized female mice.

Female mice of the C3H strain normally do not reject skin grafts from males of the same strain; however, 40 percent of splenectomized C3H female mice completely rejected C3H male skin grafts applied 2 weeks later. All splenectomized females showed at least transitory signs of graft rejection.

Frequency of trisomy 20 in nonmalignant bronchial epithelium from lung cancer patients and cancer-free former uranium miners and smokers.

Lung cancer is the leading cause of cancer-related deaths. The development of sensitive screening methods to identify at-risk individuals before emergence of clinical disease would permit early intervention that could decrease this mortality. Our previous studies have shown that cells with trisomy 7 can be detected in bronchial epithelium from cancer-free smokers and former uranium miners. However, the use of more than one molecular marker could increase the chance of identifying at-risk individuals. Trisomy 20, which is found in 43-57% of non-small cell lung cancers, is a candidate marker. The purpose of the current investigation was to determine the percentage of cells with trisomy 20 in persons with a high risk for lung cancer. Bronchial epithelial cells that had been assayed for trisomy 7 were assayed for trisomy 20 by fluorescence in situ hybridization. Trisomy 20 was detected in bronchial epithelial cells from lung cancer patients and from smokers and ex-uranium miners without lung cancer. In some cases, patients who were negative for trisomy 7 exhibited trisomy 20. Consequently, more people with field cancerization were identified using both markers. However, the two markers combined did not appear to stratify the risk for lung cancer.

Alpha-difluoromethylornithine resistance in Leishmania donovani is associated with increased ornithine decarboxylase activity.

The promastigote form of Leishmania donovani is sensitive to growth inhibition by DL-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), the first enzyme of the polyamine biosynthetic pathway, with an EC50 value of approximately 30 microM. Exposure of a wild type (DI700) cell population to gradually increasing concentrations of DFMO resulted in the selection of a strain of Leishmania, DFMO-10, which was capable of proliferating in 10 mM DFMO. DFMO-10 cells possessed an EC50 value for DFMO greater than 4 mM, and were cross-resistant to alpha-methylornithine, alpha-monofluoromethyl-3,4-dehydroornithine methyl ester, and delta-methyl-acetylenic putrescine, three other inhibitors of ODC activity. DI700 and DFMO-10 cells accumulated and/or transported [3H]DFMO and a spectrum of basic, neutral, and acidic amino acids at comparative rates. However, the DFMO-resistant Leishmania, if suspended in culture medium in the absence of DFMO for several days, expressed up to 15-fold greater levels of ODC activity than did wild-type cells. The overexpressed ODC in mutant cells appeared kinetically normal, since the ODC activities from DI700 and DFMO-10 cells possessed similar apparent Km values for ornithine and were equally sensitive to inactivation by DFMO. Incubation of extracts of DFMO-10 cells, but not of wild-type parental cells, with [3H]DFMO for 1 h resulted in the labeling of a polypeptide, presumably ODC, which migrated with a molecular weight of 76,000 +/- 4000 on SDS-gel electrophoretograms. As a consequence of the elevated ODC activities, the levels of putrescine in mutant cells released from DFMO exposure were also elevated by about 15-fold over those of wild-type cells, although spermidine levels in DI700 and DFMO-10 cells were similar. In the absence of prolonged selective pressure, the resistance to DFMO, the ODC activity, and the putrescine levels of DFMO-10 cells all returned to those of wild type cells, indicating that the mutant phenotype of DFMO-selected L. donovani was unstable.

Purification and characterization of the adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase activities from Leishmania donovani.

The adenine phosphoribosyltransferase (APRTase) and hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) activities from promastigotes of Leishmania donovani have been purified to homogeneity using ammonium sulfate precipitation, DEAE-cellulose exclusion, and either AMP-agarose (APRTase) or GTP-agarose (HGPRTase) affinity chromatography. The specific activities of the affinity-purified APRTase and HGPRTase fractions were 326-fold and 1341-fold greater than those in the 40-80% ammonium sulfate precipitate, respectively. The purified APRTase migrated as a single band on sodium dodecyl sulfate (SDS) polyacrylamide gels with a size of 29 kDa, while HGPRTase was also determined to be homogeneous by SDS gel electrophoresis with a size of 24 kDa. In addition, a mutant cell line, APPB2, partially deficient in APRTase activity, still contained quantities of purifiable APRTase protein, while a clonal secondary derivative of the APPB2 cell line that is completely deficient in APRTase activity, APPB2-640A3, failed to express purifiable APRTase protein. The homogeneous enzymes possessed apparent Km values for their nucleobase substrates between 2.0 and 5.0 microM, and both enzymes were inhibited by their immediate or ultimate reaction endproducts, APRTase by AMP and PPi and HGPRTase by GMP, GTP, and PPi. The generation of homogeneous preparations of APRTase and HGPRTase protein will serve as a prerequisite for the generation of immunological and molecular biological probes to analyze the leishmanial phosphoribosyltransferases.

Developments in management of the newborn foal in respiratory distress 1: Evaluation.

Developments in evaluation of newborn foals with respiratory distress are discussed. Major causes of respiratory distress are outlined and discussed in terms of the similar respiratory signs exhibited by foals with this clinical syndrome. History, physical examination, clinical pathology, chest radiography and blood gas analyses are discussed as important elements of the evaluation of the condition of these foals. Foals with respiratory disease are grouped into three major categories on the basis of clinical signs and arterial blood gas profiles. The evaluation of foals with respiratory distress is designed not only to reach an accurate diagnosis of the aetiology but also to define the foal's need for respiratory support.

Developments in management of the newborn foal in respiratory distress 2: Treatment.

New developments in therapy for foals in respiratory distress are discussed. Therapy is based on preservation of the foal's life by maintenance of a patent airway, resuscitation with fluids and warmth, provision of humidified oxygen to raise the fractional concentration of inspired oxygen sufficient to avoid hypoxia and provision of ventilatory support when hypercapnia becomes critical. Ventilatory support described includes assisted and controlled ventilation, positive end expiratory pressure, continuous positive airway pressure and intermittent mandatory ventilation. The aims of these techniques are discussed together with their associated indications, disadvantages and complications. Secondary therapy includes coupage, airway hygiene, drug therapy and stress management. Knowledge of equine neonatology is limited in comparison with human neonatology. More information in basic physiology and pharmacology relating to equine neonatology is needed and the efficacy of various modes of therapy must be evaluated.

Validation of probe EFD52 (D17S26) for forensic DNA analysis.

Validation studies that meet TWGDAM (The Working Group on DNA Analysis Methods) and CAC (California Association of Criminalists) guidelines for RFLP (restriction fragment length polymorphism) analysis were performed with the DNA probe EFD52 (D17S26). These studies demonstrate that the probe EFD52 is suitable for forensic casework. No unexpected DNA banding patterns were obtained from controlled studies examining various tissues, sample consistency over many gels, mixtures of body fluids, various substrates, various contaminants and non-human DNA sources. Of all the animal DNAs tested, only one higher primate yielded a single band to EFD52 hybridization. The sensitivity of EFD52 was shown to be comparable to that of other forensic probes. Population frequency distribution tables were prepared from over 4000 alleles and two-locus studies were conducted on nine forensically useful probes. Black, White, Hispanic and Lumbee Indian populations were found to be in Hardy-Weinberg and linkage equilibrium. Comparisons between victim blood standards and epithelial fractions of mixed strains from sexual assault cases were used to demonstrate the robustness of the EFD52 probe in forensic casework.

An illustrated guide to RFLP troubleshooting.

A large number of reagents and steps are required for restriction fragment length polymorphism (RFLP) analysis, which at times make determining the cause of any observed anomaly difficult. Troubleshooting problems in RFLP analysis is difficult and often the exact cause of a problem cannot be determined. In this paper a collection of controlled experiments detail the consequences of a number of human or materials problems. Although the focus is on forensic applications, this troubleshooting guide will be helpful to anyone employing Southern analysis.

Monoclonal antibodies: the promise and the reality.

Monoclonal antibodies, or "MoAbs," have revolutionized clinical approaches to diagnostic imaging and therapy of many diseases. The use of MoAbs for diagnosing and treating cancer has been especially promising. However, the full potential of these "magic bullets" has yet to be realized. This article examines the current and potential uses of MoAbs, describes problems with the technology and looks at potential solutions. Along with descriptions of how MoAbs are made and prepared for use in the clinic, the article provides examples of the ways in which MoAbs can be used to complement and expand the information obtained from standard diagnostic imaging modalities. Specific examples of the use of monoclonal antibodies for treating cancer and other diseases also are provided.

Teleradiology: the practice of radiology enters Cyberspace.

Teleradiology refers to the use of computers and electronic communication networks to transmit diagnostic images acquired at one location to another location for review and interpretation. The use of teleradiology has grown tremendously during the past few years. Many of the early problems with teleradiology are being resolved by the rapid advances taking place in telecommunications and computer technologies, while changes in health care economics are driving the need to establish cost-effective and efficient communications links between rural and urban health care providers and tertiary care specialists. Effective arguments can be made both for and against the role of teleradiology in improving patient care. These arguments, as well as an overview of the impact of teleradiology on the the practice of radiology, are discussed in this article.

MRI's role in assessing and managing breast disease.

As medical imaging techniques become more sensitive and treatment regimes become focused on "cure" for Stage I and Stage II breast cancers, it is essential that screening and diagnostic techniques become more specific. Mammography is the primary imaging modality used to detect early, clinically occult breast cancer. However, limitations in the sensitivity and specificity of mammograms have stimulated exploration into alternative or adjunctive imaging methods. This article discusses magnetic resonance imaging as a technology for assessing and managing breast disease.

Leishmania donovani: isolation and characterization of sodium stibogluconate (Pentostam)-resistant cell lines.

Leishmania donovani promastigotes were generated by virtue of their resistance to incrementing concentrations of sodium stibogluconate (Pentostam) under completely defined growth conditions. The PENT0400 and PENT03200 cell lines were isolated after prolonged exposure to 0.4 mg/ml and 3.2 mg/ml Pentostam (Sb concentration), respectively. Whereas the effective concentration of Pentostam which inhibited the growth of wild type cells by 50% (EC50 value) was 0.1-0.15 mg/ml, the EC50 values for the PENT0400 and PENT03200 cells were approximately 1 and 4 mg/ml, respectively. The decreased sensitivities of both PENT0400 and PENT03200 cells to Pentostam were maintained after 6 months of continuous culture in the absence of selective pressure, indicating that the Pentostam resistance in the mutant organisms was a stable genetic trait. Interestingly, wild type and PENT03200 cells were equally sensitive to growth inhibition and cytotoxicity caused by SbCl5 and SbCl3, as well as to a variety of other cations such as Cd, Zn, and As. Wild type and PENT03200 cells also displayed equivalent growth sensitivities to a spectrum of other antiprotozoal agents, including antimony potassium tartrate, melarsoprol, pyrimethamine, pentamidine, formycin B, and difluoromethylornithine. These results illustrate a potentially useful model system to study Pentostam resistance in Leishmania and suggest that Pentostam resistance in vitro may be independent of antimony toxicity.

Methotrexate-resistant Leishmania donovani genetically deficient in the folate-methotrexate transporter.

From a mutagenized population of wild type Leishmania donovani promastigotes, a clone was isolated in a single step by virtue of its resistance to 1 mM methotrexate, a potent inhibitor of dihydrofolate reductase. This methotrexate-selected cell line, MTXA5, was cross-resistant to aminopterin but just as sensitive to growth inhibition caused by pyrimethamine, trimethoprim, and cytotoxic purine and pyrimidine analogs. Unlike previously characterized methotrexate-resistant Leishmania (Coderre, J. A., Beverley, S. M., Schimke, R., and Santi, D. V. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2132-2136), resistance to the antimetabolite was not due to gene amplification or increased dihydrofolate reductase activity. The genetic defect in MTXA5 cells appeared to be in the methotrexate-folate transport system. The rate of uptake and transport of [3H]methotrexate and [3H]folate into MTXA5 cells was less than 1% of that of wild type parental cells. Neither wild type nor MTXA5 cells could multiply in folate-deficient medium, and thymine and thymidine at concentrations which circumvented methotrexate toxicity, did not restore the ability of Leishmania to grow. The concentration of exogenous folate that restored growth of wild type and mutant cells, however, was virtually identical, although MTXA5 cells, unlike parental cells, could not proliferate in folate-deficient medium supplemented with 10 microM biopterin. Interestingly, methotrexate and aminopterin could stimulate the growth of both leishmanial strains in folate-deficient medium, suggesting that these antifolate analogs were serving as a pteridine source for the parasite. These somatic cell genetic studies of folate transport in Leishmania provide genetic evidence for a specific folate permease in L. donovani promastigotes and have important implications concerning the mechanisms by which these parasites utilize exogenous pteridines and folates and by which they might become resistant to parasite-directed chemotherapeutic regimens.

Genetic analysis of 2',3'-dideoxycytidine incorporation into cultured human T lymphoblasts.

In order to analyze the cellular determinants that mediate the action of 2',3'-dideoxycytidine, the growth inhibitory and cytotoxic effects and the metabolism of the dideoxynucleoside were examined in wild type human CEM T lymphoblasts and in mutant populations of CEM cells that were genetically deficient in either nucleoside transport or deoxycytidine kinase activity. Whereas 2',3'-dideoxycytidine at a concentration of 5 microM inhibited growth of the wild type CEM parental strain by 50%, two nucleoside transport-deficient clones were 4-fold resistant to the pyrimidine analog. The deoxycytidine kinase-deficient cell line was virtually completely resistant to growth inhibition by the dideoxynucleoside at a concentration of 1024 microM. An 80% diminished rate of 2',3'-[5,6-3H]dideoxycytidine influx into the two nucleoside transport-deficient lines could account for their resistance to the dideoxynucleoside, while the resistance of the deoxycytidine kinase-deficient cells to 2',3'-dideoxycytidine toxicity could be explained by a virtually complete failure to incorporate 2',3'-[5,6-3H]dideoxycytidine in situ. Two potent inhibitors of mammalian nucleoside transport, 4-nitrobenzylthioinosine and dipyridamole, mimicked the effects of a genetic deficiency in nucleoside transport with respect to 2',3'-dideoxycytidine toxicity and incorporation. These data indicate that the intracellular metabolism of 2',3'-dideoxycytidine in CEM cells is initiated by the nucleoside transport system and the cellular deoxycytidine kinase activity.

Gene enrichment in an American Indian population: an excess of severe combined immunodeficiency disease.

A subgroup of Athabascan Indians in Arizona and New Mexico was found to have an unusually high incidence of severe combined immunodeficiency, probably due to founder effect. Closed genetic pressures have limited their histocompatibility-antigen heterogeneity, enabling 3 patients to be grafted, 2 of them across HLA-B locus barriers.

Is radiation treatment volume a predictor for acute or late effect on pulmonary function? A prospective study of patients treated with breast-conserving surgery and postoperative irradiation.

BACKGROUND: When tangential radiation beams are used in patients with breast cancer after breast-conserving surgery, the amount of lung included in the radiation field varies because of patient anatomy and treatment technique. The question of how much lung tissue can be irradiated incidentally without acute or late complications requires quantitative study. METHODS: Thirty-four women were enrolled in a prospective study of pulmonary function after breast-conserving surgery and radiotherapy for early stage breast cancer. The percentage of lung volume irradiated was estimated from computed tomography scans. Pulmonary function tests including spirometrics, lung volume, and diffusing capacity of carbon monoxide (DLCO) were performed before, during, and at regular intervals after radiotherapy. Both acute and long term changes in pulmonary function were analyzed in 29 eligible patients. RESULTS: Acutely, DLCO values dropped, but they returned to normal levels by 24 months. At 5 years, pulmonary function did not vary significantly according to the percentage of lung irradiated, the use of regional lymphatic irradiation, or the addition of chemotherapy. Symptomatic pneumonitis occurred only in two women with baseline deficits in DLCO (P = 0.016), who had more than 10% of the total lung volume irradiated. Patients with a smoking history had a clinically significant baseline deficit of 32% in DLCO values (P = 0.0011) but showed a 21% improvement (P = 0.11), which probably correlated with quitting smoking. CONCLUSION: Within the range evaluated in this study, the volume of lung irradiated did not predict a late decrease in pulmonary function, although pneumonitis was observed only when more than 10% of the lung was irradiated.

Women uranium workers: a case study of perceived hazardous exposures and health effects.

An exploratory study of seventy-four women uranium workers employed in the western United States (miners, millers, truck haulers, and office workers) was conducted. These uranium industry workers were employed primarily during the 1970s and 1980s. It was found that approximately 60 percent perceived overall moderate to high levels of dust during their employment, and about 50 percent reported the likelihood of having past, present, or future health problems related to their uranium work. Two of the most-often-identified health problems were respiratory symptoms or illnesses and cancer. Issues regarding public policy, research, and worker rights are discussed.