Stimulation of murine lymphocyte blastogenesis by mitogens in heat-killed Histoplasma capsulatum yeast cells.

In vitro blastogenesis by normal murine splenocytes from several mouse strains has been detected after exposure to heat-killed Histoplasma capsulatum yeast cells. Maximal lymphocyte stimulation induced by 10(4) heat-killed cells resulted in 20- to 45-fold increases in [3H]thymidine uptake by splenocytes when compared with responses by normal unstimulated lymphocytes. The kinetics for this response to heat-killed H. capsulatum cells has shown peak mitogenesis 3 days after culture. Examination of the mitogenic potential of soluble antigen preparations from H. capsulatum has revealed stimulation of lymphocyte blastogenesis with yeast cell sonicates and autolysates but not substances from autoclaved yeast cells. The levels of lymphocyte blastogenesis induced by sonicates or autolysates were comparable to mitogen responses stimulated by heat-killed cells. Preliminary biochemical characterization of the mitogenic factor(s) associated with yeast cell sonicates show two peaks of activity, at 178,000 and less than 12,000 Mr, which have a protein or glycoprotein nature. Finally, analysis of lymphocyte blastogenesis in cultures enriched for selected lymphocyte subpopulations has shown that T lymphocytes are preferentially stimulated by yeast cell mitogens.     

Mitogenic response of marmoset lymphocytes: cytokinetics and identification of responsive cells

Isotope (tritiated thymidine, [³H]Tdr) incorporation into lymphocytes from two marmoset species, New World primates which are haemopoietic chimeras, was studied using peripheral blood lymphocyte (PBL) cultures incubated in vitro for 1–5 days with different concentrations of Concanavalin A (Con A), leucoagglutinin (LA), bacterial lipopolysaccharide (LPS) from gram-negative bacteria, rabbit anti-marmoset immunoglobulin (anti-Ig) serum, thymosin or irradiated histoincompatible (xenogeneic) lymphocytes. Only the plant lectins and xenogeneic lymphoid cells stimulated the uptake of [³H]Tdr. Lymphocyte enrichment experiments demonstrated that cells responsive to plant lectins and xenoantigens were primarily thymus-derived (T) lymphocytes. Bacterial endotoxin (LPS), however, enhanced the mitogenic response of PBL to Con A when LPS and plant lectin were added together at culture initiation. Thymosin caused either enhancement or suppression of the response to Con A depending on its time of addition relative to lectin stimulation. Addition of thymosin to lymphocyte cultures with Con A or 24 h later caused a decrease in isotope incorporation, while addition of thymosin 48 h later caused a 12–100% increase in [³H]Tdr uptake. Lymphocyte chimerism did not influence the mitogenic response since single-born, nonchimeric marmosets responded to plant lectins in a manner similar to marmosets with varying proportions of chimeric blood elements. Cytological analysis of stimulated lymphocytes from heterosexual marmosets revealed the percentage chimerism in the polyclonal mitogenic response and the clonal mixed lymphocyte reaction to be similar.     

Oligonucleotide Ligation Assay for Detecting Mutations in the Human Immunodeficiency Virus Type 1 pol Gene That Are Associated with Resistance to Zidovudine, Didanosine, and Lamivudine

This report describes the detection of mutations in the pol gene of human immunodeficiency virus type 1 associated with resistance to zidovudine, didanosine, and lamivudine by genotyping by an oligonucleotide ligation assay specific codons in the pol gene amplified by PCR. Our studies demonstrate the sensitivity, simplicity, and specificity of this genotyping system.     

Sequence diversity and large-scale typing of SNPs in the human apolipoprotein E gene

A common strategy for genotyping large samples begins with the characterization of human single nucleotide polymorphisms (SNPs) by sequencing candidate regions in a small sample for SNP discovery. This is usually followed by typing in a large sample those sites observed to vary in a smaller sample. We present results from a systematic investigation of variation at the human apolipoprotein E locus (APOE), as well as the evaluation of the two-tiered sampling strategy based on these data. We sequenced 5.5 kb spanning the entire APOE genomic region in a core sample of 72 individuals, including 24 each of African-Americans from Jackson, Mississippi; European-Americans from Rochester, Minnesota; and Europeans from North Karelia, Finland. This sequence survey detected 21 SNPs and 1 multiallelic indel, 14 of which had not been previously reported. Alleles varied in relative frequency among the populations, and 10 sites were polymorphic in only a single population sample. Oligonucleotide ligation assays (OLA) were developed for 20 of these sites (omitting the indel and a closely-linked SNP). These were then scored in 2179 individuals sampled from the same three populations (n = 843, 884, and 452, respectively). Relative allele frequencies were generally consistent with estimates from the core sample, although variation was found in some populations in the larger sample at SNPs that were monomorphic in the corresponding smaller core sample. Site variation in the larger samples showed no systematic deviation from Hardy-Weinberg expectation. The large OLA sample clearly showed that variation in many, but not all, of OLA-typed SNPs is significantly correlated with the classical protein-coding variants, implying that there may be important substructure within the classical epsilon 2, epsilon 3, and epsilon 4 alleles. Comparison of the levels and patterns of polymorphism in the core samples with those estimated for the OLA-typed samples shows how nucleotide diversity is underestimated when only a subset of sites are typed and underscores the importance of adequate population sampling at the polymorphism discovery stage. [The sequence data described in this paper have been submitted to the GenBank data library under accession no. AF261279.]     

Causes and outcomes of the acute chest syndrome in sickle cell disease. National Acute Chest Syndrome Study Group

BACKGROUND: The acute chest syndrome is the leading cause of death among patients with sickle cell disease. Since its cause is largely unknown, therapy is supportive. Pilot studies with improved diagnostic techniques suggest that infection and fat embolism are underdiagnosed in patients with the syndrome. METHODS: In a 30-center study, we analyzed 671 episodes of the acute chest syndrome in 538 patients with sickle cell disease to determine the cause, outcome, and response to therapy. We evaluated a treatment protocol that included matched transfusions, bronchodilators, and bronchoscopy. Samples of blood and respiratory tract secretions were sent to central laboratories for antibody testing, culture, DNA testing, and histopathological analyses. RESULTS: Nearly half the patients were initially admitted for another reason, mainly pain. When the acute chest syndrome was diagnosed, patients had hypoxia, decreasing hemoglobin values, and progressive multilobar pneumonia. The mean length of hospitalization was 10.5 days. Thirteen percent of patients required mechanical ventilation, and 3 percent died. Patients who were 20 or more years of age had a more severe course than those who were younger. Neurologic events occurred in 11 percent of patients, among whom 46 percent had respiratory failure. Treatment with phenotypically matched transfusions improved oxygenation, with a 1 percent rate of alloimmunization. One fifth of the patients who were treated with bronchodilators had clinical improvement. Eighty-one percent of patients who required mechanical ventilation recovered. A specific cause of the acute chest syndrome was identified in 38 percent of all episodes and 70 percent of episodes with complete data. Among the specific causes were pulmonary fat embolism and 27 different infectious pathogens. Eighteen patients died, and the most common causes of death were pulmonary emboli and infectious bronchopneumonia. Infection was a contributing factor in 56 percent of the deaths. CONCLUSIONS: Among patients with sickle cell disease, the acute chest syndrome is commonly precipitated by fat embolism and infection, especially community-acquired pneumonia. Among older patients and those with neurologic symptoms, the syndrome often progresses to respiratory failure. Treatment with transfusions and bronchodilators improves oxygenation, and with aggressive treatment, most patients who have respiratory failure recover.     

Mining SNPs from EST databases

There is considerable interest in the discovery and characterization of single nucleotide polymorphisms (SNPs) to enable the analysis of the potential relationships between human genotype and phenotype. Here we present a strategy that permits the rapid discovery of SNPs from publicly available expressed sequence tag (EST) databases. From a set of ESTs derived from 19 different cDNA libraries, we assembled 300,000 distinct sequences and identified 850 mismatches from contiguous EST data sets (candidate SNP sites), without de novo sequencing. Through a polymerase-mediated, single-base, primer extension technique, Genetic Bit Analysis (GBA), we confirmed the presence of a subset of these candidate SNP sites and have estimated the allele frequencies in three human populations with different ethnic origins. Altogether, our approach provides a basis for rapid and efficient regional and genome-wide SNP discovery using data assembled from sequences from different libraries of cDNAs.     

Effects of ACTH on membranous whorls in the adrenal gland of the Mongolian gerbil

The ultrastructure of whorls of rough endoplasmic reticulum in the adrenal gland of the Mongolian gerbil (Meriones unguiculatus) was examined during and after treatment with ACTH. Whorls were loosely wound after treatment for one day and consisted of only a few paired membranes. Focal areas of increased tubular smooth endoplasmic reticulum were seen throughout the cytoplasm of the cells. Whorls disappeared altogether after three days of ACTH treatment. The structures reappeared one day after stopping the ACTH injections and seemed to enlarge progressively by addition of membranes to the periphery of the structures. Numerous vesicles and sometimes parallel cisternae of rough endoplasmic reticulum were observed in the cytoplasm of adrenocortical cells containing newly forming whorls. The whorls apparently serve as a readily available reserve of rough endoplasmic reticulum, which can transform into smooth reticulum upon stimulation with ACTH.     

Measuring contraceptive values: An alternative approach

Previous assessments of individuals' values for various contraceptive consequences have employed one of four methodologies: free elicitation, direct ratings, multiple regression, or factor analysis. All four methodologies are flawed because they produce group rather than individual values, rely on rating scales, and fail to incorporate information regarding consequence trade-offs. Axiomatic conjoint measurement is proposed as an alternative methodology and used to determine individuals' values for a selected set of contraceptive consequences at two stages of the family-planning career.Preparation of this paper was supported in part by Grants HD-10802 and HD-14403 from the National Institute of Child Health and Human Development. Appreciation is due the Statistical Computing Facility of the University of California at Berkeley. Requests for reprints should be sent to the Publications Librarian, Center for Research on Judgment and Policy, Muenzinger Psychology Building, University of Colorado, Boulder, Colorado 80309-0344.     

Validity and utility of urinary CXCL10/Cr immune monitoring in pediatric kidney transplant recipients

Individualized posttransplant immunosuppression is hampered by suboptimal monitoring strategies. To validate the utility of urinary CXCL10/Cr immune monitoring in children, we conducted a multicenter prospective observational study in children <21 years with serial and biopsy-associated urine samples (n = 97). Biopsies (n = 240) were categorized as normal (NOR), rejection (>i1t1; REJ), indeterminate (IND), BKV infection, and leukocyturia (LEU). An independent pediatric cohort of 180 urines was used for external validation. Ninety-seven patients aged 11.4 ± 5.5 years showed elevated urinary CXCL10/Cr in REJ (3.1, IQR 1.1, 16.4; P < .001) and BKV nephropathy (median = 5.6, IQR 1.3, 26.9; P < .001) vs. NOR (0.8, IQR 0.4, 1.5). The AUC for REJ vs. NOR was 0.76 (95% CI 0.66–0.86). Low (0.63) and high (4.08) CXCL10/Cr levels defined high sensitivity and specificity thresholds, respectively; validated against an independent sample set (AUC = 0.76, 95% CI 0.66–0.86). Serial urines anticipated REJ up to 4 weeks prior to biopsy and declined within 1 month following treatment. Elevated mean CXCL10/Cr was correlated with first-year eGFR decline (ρ = −0.37, P ≤ .001), particularly when persistently exceeding ≥4.08 (ratio = 0.81; P < .04). Useful thresholds for urinary CXCL10/Cr levels reproducibly define the risk of rejection, immune quiescence, and decline in allograft function for use in real-time clinical monitoring in children.     

The status of women at one academic medical center. Breaking through the glass ceiling

Despite recent gains in admission to medical school and in obtaining junior faculty positions, women remain underrepresented at senior academic ranks and in leadership positions in medicine. This discrepancy has been interpreted as evidence of a "glass ceiling" that prevents all but a few exceptional women from gaining access to leadership positions. We analyzed data from Columbia University College of Physicians & Surgeons, New York, NY, for all faculty hired from 1969 through 1988 and found that the likelihood of promotion on the tenure track was 0.40 for women and 0.48 for men (ratio, 0.82; 95% confidence interval, 0.56 to 1.20); on the clinical track the likelihood of promotion was 0.75 for women and 0.72 for men (ratio, 1.04; 95% confidence interval, 0.56 to 1.94). Additional analysis of current faculty showed that in the academic year 1988-1989 the proportion of women at each tenure track rank at the College of Physicians & Surgeons equaled or exceeded the national proportion of women graduating from medical school, once allowance was made for the average time lag necessary to attain each rank. On the clinical track women were somewhat overrepresented, particularly at the junior rank. National data that describe medical school faculty, which combine tenure and clinical tracks, showed that in 1988 women were proportionately represented at each rank once the lead time from graduation was considered. We conclude that objective evidence shows that women can succeed and are succeeding in gaining promotions in academic medicine.