Use of modified nucleotides and uracil-DNA glycosylase (UNG) for the control of contamination in the PCR-based amplification of RNA.

The inadvertent carryover of amplified fragments of nucleic acids (amplicons) is a potential source of contamination in the polymerase chain reaction. Recently, a method has been developed to generate amplicons with deoxyuracil triphosphate (dUTP) and to specifically hydrolyze these amplicons with uracil-DNA glycosylase (UNG) following the completion of the assay. We evaluated this system for the specific amplification of RNA from coxsackievirus A3 and B3. We found that RNA from both viruses could be amplified with dUTP, although the use of this triphosphate in place of TTP resulted in some loss of assay sensitivity. We also found that the dUTP-containing amplicons could be efficiently hydrolyzed by UNG, resulting in a 10,000,000-fold reduction in amplicon concentration with little effect on the native nucleic acid. The dUTP-UNG method has a great deal of potential for reducing amplicon contamination during the routine performance of nucleic acid amplification reactions.     

Persistent diarrhea and fecal shedding of retroviral nucleic acids in children infected with human immunodeficiency virus

Gastrointestinal dysfunction is a serious problem in many children infected with human immunodeficiency virus (HIV), the etiology of which has not been clearly defined. Quantitative nucleic acid amplification was used to study the correlation between shedding of HIV nucleic acids and gastrointestinal symptoms in HIV-infected infants and children. Many with HIV infection and persistent diarrheal disease shed HIV nucleic acids in their feces, as did an HIV-infected patient without apparent diarrheal disease. HIV nucleic acids were not found in feces of non-HIV-infected individuals. Intestinal infection with HIV appears to be important in the pathophysiology of gastrointestinal dysfunction in infants and children with HIV infection. Furthermore, the fecal shedding of HIV may play a role in HIV transmission in environments prone to high levels of fecal-oral contamination.     

Allergenicity of orally administered immunoglobulin preparations in food-allergic children

Passive immunization by the oral administration of immunoglobulin preparations derived from bovine milk, chicken egg, and human sera has been proposed as a method for the prevention and treatment of enteric diseases. However, the allergenic potential of these proteins may be a factor limiting their widespread use for disease prevention. An in vitro study with sera from milk- and egg-allergic children was performed to determine whether these immunoglobulin preparations have allergenic potential. Protein extracts of milk, bovine immunoglobulin, egg white, human immune globulin, and five egg yolk antiviral immunoglobulin preparations were bound to nitrocellulose paper. These preparations were probed for specific IgE binding with sera from milk- and egg-allergic patients. Of 22 milk-hypersensitive patients, 16 had specific IgE binding against the bovine immunoglobulin preparation. Of 28 egg-allergic patients 15 had specific IgE binding against one or more of the egg yolk-derived antiviral chicken immunoglobulins. Control sera were negative against the milk and egg preparations. Western blot analysis confirmed that milk- and egg-allergic patients had IgE-specific antibodies for bovine and chicken immunoglobulin molecules. Therefore, the removal of contaminating proteins from milk and egg antibody preparations would be unlikely to eliminate their allergenic potential. In contrast, sera from milk- and egg-allergic patients displayed no detectable IgE binding to human immunoglobulin preparations. These data indicate that the administration of antibody preparations derived from bovine and chicken sources may lead to severe allergic reactions in milk- or egg-sensitized patients and to sensitization in some nonallergic individuals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzyme Immunoassays for Measurement of Cytomegalovirus Immunoglobulin M Antibody

The diagnosis of congenital cytomegalovirus (CMV) infection is often accomplished by the detection of circulating antibody directed against CMV. We devised a method for measuring CMV-specific immunoglobulin M (IgM) based on the isolation of IgM antibody by reaction with a solid phase coated with antihuman IgM. The determination of IgM antibody specific for CMV was accomplished by the subsequent addition of CMV or control antigen and enzyme-labeled CMV antibody (solid phase-IgM method). We compared the sensitivity and specificity of this method with those of a conventional form of solid-phase enzyme immunoassay in which CMV antigen is bound to the solid phase (solid phase-antigen method). Both assay systems were capable of detecting CMV-specific IgM antibody in the sera of 10 babies with documented CMV infection and in those of the mothers of 4 of these babies. The solid phase-IgM method yielded negative results in all 66 sera available from babies who did not have congenital CMV infection. On the other hand, the solid phase-antigen system yielded false-positive results in 12 (18%) of these sera. In addition, the solid phase-antigen system yielded false-positive results in 8 of 12 sera obtained from patients with demonstrable rheumatoid factor. However, the solid phase-IgM system yielded negative results for the rheumatoid sera, provided that appropriate control reactions were performed. The solid phase-IgM system is thus a specific and sensitive method for the determination of CMV IgM antibody.     

Whole body protein turnover in children with human immunodeficiency virus (HIV) infection

The purpose of this study was to determine the rate of whole body protein turnover (WBPT) in human immunodeficiency virus (HIV)-infected children, and to determine the relationship between WBPT and growth. The rate of WBPT was calculated from the cumulative excretion of labeled urinary ammonia after a single intravenous dose of 15N-glycine in three groups of children: 1) HIV+ with growth retardation (HIV+ Gr); 2) HIV+ with normal growth (HIV+); and 3) HIV-uninfected with normal growth (HIV-). Twenty-six children between 2 and 11 y of age were studied (10 HIV+ Gr, 12 HIV+, 4 HIV-). All children were afebrile and free of acute infection during the study. Rates of WBPT (mean +/- SD) for the study groups were: HIV+ Gr, 12.2 +/- 4.8; HIV+, 10.7 +/- 5.1; and HIV-, 8.6 +/- 2.1 g.protein.kg-1.d-1 (NS, P > 0.05). Although not statistically significant, mean WBPT was 42% greater in HIV+ Gr, and 24% greater in HIV+ compared to HIV-. Statistically significant correlations were found between WBPT and Z scores for height (r = -0.39, P = 0.05) and weight-for-age (r = -0.51, P = 0.01) and dietary intake of protein (r = 0.39, P = 0.05), and between protein balance (synthesis-catabolism) and intakes of energy (r = 0.47, P = 0.02) and protein (r = 0.40, P = 0.04). There was no statistically significant correlation between WBPT and resting energy expenditure (r = 0.27, P = 0.19), or CD4 cell number (r = 0.05, P = 0.82). These data suggest an association between increased rates of protein turnover and low weight and height-for-age Z scores, and that it may be possible to achieve positive protein balance given an adequate intake of nutrients.     

Disease-specific alterations in frontal cortex brain proteins in schizophrenia, bipolar disorder, and major depressive disorder. The Stanley Neuropathology Consortium

Severe psychiatric disorders such as schizophrenia, bipolar disorder and major depressive disorder are brain diseases of unknown origin. No biological marker has been documented at the pathological, cellular, or molecular level, suggesting that a number of complex but subtle changes underlie these illnesses. We have used proteomic technology to survey postmortem tissue to identify changes linked to the various diseases. Proteomics uses two-dimensional gel electrophoresis and mass spectrometric sequencing of proteins to allow the comparison of subsets of expressed proteins among a large number of samples. This form of analysis was combined with a multivariate statistical model to study changes in protein levels in 89 frontal cortices obtained postmortem from individuals with schizophrenia, bipolar disorder, major depressive disorder, and non-psychiatric controls. We identified eight protein species that display disease-specific alterations in level in the frontal cortex. Six show decreases compared with the non-psychiatric controls for one or more diseases. Four of these are forms of glial fibrillary acidic protein (GFAP), one is dihydropyrimidinase-related protein 2, and the sixth is ubiquinone cytochrome c reductase core protein 1. Two spots, carbonic anhydrase 1 and fructose biphosphate aldolase C, show increase in one or more diseases compared to controls. Proteomic analysis may identify novel pathogenic mechanisms of human neuropsychiatric diseases.     

Randomised controlled trial of zinc supplementation in malnourished Bangladeshi children with acute diarrhoea

OBJECTIVE: To evaluate the impact of zinc supplementation on the clinical course, stool weight, duration of diarrhoea, changes in serum zinc, and body weight gain of children with acute diarrhoea. DESIGN: Randomised double blind controlled trial. Children were assigned to receive zinc (20 mg elemental zinc per day) containing multivitamins or control group (zinc-free multivitamins) daily in three divided doses for two weeks. SETTING: A diarrhoeal disease hospital in Dhaka, Bangladesh. PATIENTS: 111 children, 3 to 24 months old, below 76% median weight for age of the National Center for Health Statistics standard with acute diarrhoea. Children with severe infection and/or oedema were excluded. MAIN OUTCOME MEASURES: Total diarrhoeal stool output, duration of diarrhoea, rate of weight gain, and changes in serum zinc levels after supplementation. RESULTS: Stool output was 28% less and duration 14% shorter in the zinc supplemented group than placebo (p = 0.06). There were reductions in median total diarrhoeal stool output among zinc supplemented subjects who were shorter (less than 95% height for age), 239 v 326 g/kg (p < 0.04), and who had a lower initial serum zinc (< 14 mmol/l), 279 v 329 g/kg (p < 0.05); a shortening of mean time to recovery occurred (4.7 v 6.2 days, p < 0.04) in those with lower serum zinc. There was an increase in mean serum zinc in the zinc supplemented group (+2.4 v -0.3 mumol/l, p < 0.001) during two weeks of supplementation, and better mean weight gain (120 v 30 g, p < 0.03) at the time of discharge from hospital. CONCLUSIONS: Zinc supplementation is a simple, acceptable, and affordable strategy which should be considered in the management of acute diarrhoea and in prevention of growth faltering in children specially those who are malnourished.     

Platelet kinetics in patients with idiopathic thrombocytopenic purpura and moderate thrombocytopenia

We studied ten normal subjects and 20 patients with stable, untreated idiopathic thrombocytopenic purpura (ITP) and platelet counts in the range of 35,000 to 110,000/microL. The diagnosis was made by clinical criteria. Platelet-associated IgG was increased in all nine of the nine patients studied. Autologous platelets were labeled with chromium 51 and reinfused for measurement of mean cell life and platelet production rate. Mean cell life was calculated by two methods, weighted mean and multiple hit, with excellent agreement between the two. As expected, mean cell life was significantly reduced in ITP patients as compared to the normal subjects (2.9 days v. 8.0 days, P less than .001). However, mean platelet production rates in ITP patients and normal subjects, 3.5 and 3.8 X 10(9) platelets/k/d respectively, were not significantly different. Platelet production rate was above and below the normal range (2 to 5.6 X 10(9) platelets/k/d) in two and four patients, respectively. We conclude that the rate of platelet production is not increased in most patients with ITP who have platelet counts greater than 35,000/microL. We did find that platelet size was increased in eight of the 12 patients in whom it was measured, including two of the patients with low platelet production.     

High-dose cytosine arabinoside and daunorubicin as consolidation therapy for acute nonlymphocytic leukemia in first remission: a pilot study

High-dose (HD) cytosine arabinoside (ARA-C) is more effective treatment than conventional-dose ARA-C regimens for patients with relapsed acute nonlymphocytic leukemia (ANLL). We report here that HD ARA-C given during the first remission of ANLL has resulted in long remission durations and a high proportion of patients who survive more than three years free of disease. From August 1979 to September 1983, 36 adult patients with ANLL in first remission received one to three courses of HD ARA-C (3 g/m2 by one-hour infusion every 12 hours for 12 doses on days 1 through 6) alone or with daunorubicin (30 mg/m2 for two or three doses on days 7 through 9). Three patients died of sepsis or hemorrhage during consolidation, and 14 patients have relapsed from five to 48 months after diagnosis. The remaining 19 patients are in continued complete remission (CCR) from 11 to 62 months. Denoting all deaths in remission as relapse, the actuarial probability of CCR is 42% at 62 months, with an apparent plateau in the survival curve. Of the first 22 patients treated, ten remain in CCR from 37 to 62 months with no therapy for at least three years. Due to its heightened anti-leukemic activity, HD ARA-C allows brief but effective consolidation of ANLL in first remission, with long-term disease-free survival comparable to other approaches.