Molecular evidence of organic ion transporters in the rat adrenal cortex with adrenocorticotropin-regulated zonal expression.

Experimental evidence suggested that secretion of steroid hormones from adrenocortical cells involves carrier-mediated transport: Cortisol release from, and uptake of p-[3H]aminohippurate into, bovine adrenocortical cells showed properties of the renal p-[3H]aminohippurate/anion exchanger OAT1. Other poly-specific transporters such as organic anion-transporting polypeptides (oatps) and organic cation transporters (OCTs) could also be involved in steroid hormone release. A homology-cloning procedure was established to detect these transporters in rat adrenal gland cDNA. PCR revealed the presence of OAT1, oatp1, oatp2, and oatp3. In situ hybridization localized OAT1 in the outer zona fasciculata, oatp3 in the zona glomerulosa, and oatp1 and oatp2 in the inner zona fasciculata and outer zona reticularis. An OCT2-specific probe produced signals in the zona glomerulosa and outer zona fasciculata. Pretreatment of rats with ACTH increased the expression of OAT1 mRNA that spread to all zones, and hypophysectomy strongly decreased it. A less pronounced regulation was detected for OCT2 and oatp3. Specific antibodies confirmed the localization of OAT1 in the outer zona fasciculata, supporting a possible role of OAT1 in cortisol release. The zonated distribution of transporters furthermore suggest that oatp1-3 and OCT2 may be important for the endocrine function of rat adrenocortical cells.

Interaction of the metal chelator 2,3-dimercapto-1-propanesulfonate with the rabbit multispecific organic anion transporter 1 (rbOAT1).

The metal chelator DMPS (2,3-dimercapto-1-propanesulfonate) is used to treat heavy metal intoxication because it increases renal excretion of these toxins, which are accumulated in proximal tubule cells. To evaluate the involvement of the organic anion transporter 1 (OAT1) in the renal flux of DMPS, we examined the effect of DMPS on transport mediated by the rabbit ortholog of OAT1 and compared these characteristics with those observed in intact isolated rabbit proximal tubules. The rabbit OAT1 (rbOAT1) cDNA consisted of 2124 base pairs encoding a protein of 551 amino acids. Heterologous expression in COS-7 cells revealed rbOAT1-mediated transport of p-aminohippurate (PAH; K(t) = 16 microM). A 1 mM concentration of unlabeled PAH, alpha-ketoglutarate, urate, or probenecid inhibited [(3)H]PAH uptake by 70 to 90%. cis-Inhibition and trans-stimulation experiments using several Krebs cycle intermediates implicated alpha-ketoglutarate as the main intracellular exchange anion. Reduced DMPS inhibited rbOAT1-mediated fluorescein transport with an apparent K(i) of 102 microM. These characteristics paralleled those observed in isolated rabbit proximal tubules. PAH was transported into nonperfused single proximal tubule S(2) segments with a K(t) of 76 microM. DMPS inhibited FL uptake into single tubule segments with a K(i-app) of 71 microM. Fluorescein efflux from preloaded tubules was trans-stimulated by 1 mM PAH and 1 mM DMPS, consistent with DMPS entry into tubule cells by rbOAT1. In summary, rbOAT1 mediates basolateral uptake of DMPS into proximal tubule cells, implicating this process in the detoxification process of heavy metals in the kidneys.

Molecular physiology of renal p-aminohippurate secretion.

Renal proximal tubules secrete various organic anions, including drugs and p-aminohippurate (PAH). Uptake of PAH from blood into tubule cells occurs by exchange with intracellular alpha-ketoglutarate and is mediated by the organic anion transporter 1. PAH exit into tubule lumen is species specific and may involve ATP-independent and -dependent transporters.

Genomic structure and in vivo expression of the human organic anion transporter 1 (hOAT1) gene.

The human organic anion transporter 1 (hOAT1) plays a key role in the secretion of an array of potentially toxic organic anions including many clinically important drugs. Here we report on the genomic cloning of hOAT1. A human genomic library was used for screening of a PAC (P1 artificial chromosome) clone applying PCR techniques. Sequencing of several restriction subclones and of a PCR-generated clone revealed that the hOAT1 gene spans 8.2 kb and is composed of 10 exons divided by 9 introns. RT-PCR studies in a human kidney specimen led to the detection of two new splice variants, hOAT1-3 and hOAT1-4, showing a 132-bp in-frame deletion. Using fluorescence in situ hybridization (FISH) we mapped the hOAT1 gene as a single signal to chromosome 11q13.1-q13.2. Additionally, 600 bp of the 5' flanking region was analyzed, illustrating the probable transcription start site at nt -280, a NF-kappaB-site at nt -397 and several putative transcription factor binding sites.

Detection of different viral strains of hepatitis B virus in chronically infected children after seroconversion from HBsAg to anti-HBs indicating viral persistence.

BACKGROUND/AIMS: Seroconversion to anti-HBs or the loss of HBsAg is usually associated with complete elimination of the replicative hepatitis B virus. Usually in these patients hepatitis B virus DNA (HBV DNA) becomes undetectable. Routine controls of patients who underwent anti-HBs seroconversion by more sensitive tests showed that in some cases the virus persisted in the patient. Therefore the aim of our study was to evaluate if virus persistence could also be found in children with chronic hepatitis B after anti-HBs seroconversion. The virus pool should be characterized before and after seroconversion. METHODS: Viral DNA was extracted from nine HBsAg negative or anti-HBs positive sera of children, previously diagnosed as chronic HBsAg carriers. HBV DNA was amplified by polymerase chain reaction. Subsequently the nucleotide sequences of the polymerase chain reaction product in the a-determinant region (aa 121-161) were analyzed on an automatic fluorescent sequencer. RESULTS: In the sera of seven children, HBV DNA was detected in the HBsAg negative phase of the HBV infection. Mutations in codons 122, 125, 127, 131, 134, 143, 159 and 161 of the S gene could be documented, resulting in amino acid changes. In three patients the sequence analysis revealed changes in the HBV genotype from genotype A (serotype adw) to genotype D (serotype ayw) during seroconversion to anti-HBs. CONCLUSIONS: These data demonstrate that persistence of the hepatitis B virus can also occur in HBsAg negative and anti-HBs positive children. After loss of HBsAg, no specific HBV variant was identified. Although a conclusive explanation for the selection process cannot be provided, it remains a fact that the 'surviving' viral strain was mostly represented by genotype D.

Selection of a precore mutant after vertical transmission of different hepatitis B virus variants is correlated with fulminant hepatitis in infants.

The incidence of perinatal transmission of hepatitis B virus (HBV) depends on the HBeAg/anti-HBe status of the mother. While children of HBeAg-positive mothers have a 90% probability of acquiring a chronic hepatitis B virus carrier state, babies of anti-HBe-positive mothers are more likely to develop fulminant hepatitis within the first 3 to 4 months of life. There is evidence that precore (pre-C) mutations of the HBV can be associated with fulminant hepatitis. The pre-C region was therefore examined in sera from nine infants with fulminant hepatitis after vertical transmission, one HBeAg-positive and seven anti-HBe-positive mothers by polymerase chain reaction (PCR) and direct sequence analysis. In five mother/infant pairs the virus populations were characterized in addition by analysing clones of the amplified products. All mothers were infected with two or four variants of HBV with mutations at different positions of the preC genome including position 1896, which results in a stop codon. While the precore stop codon was detected in a portion of the virus populations of the HBeAg-positive and of four anti-HBe-positive mothers the dominating viral strain was represented by the wild type virus in three. In contrast, the virus populations of all babies showed the 1896 precore variant as the prevalent virus strain during the phase of active disease. In the surviving baby only wild type sequences were detected after recovery. Subtype ayw was found in all mothers and infants and adw2 was present in three mothers and in the surviving child. The findings suggest that all mothers carried a wild type HBV population with a certain number of different HBV variants. After transmission of the mixed virus population a selection process was started in the baby. The association of subtype ayw with the precore mutations and with the fatal outcome of the hepatitis B might be the result of a directed selection of this variant with a particular advantage in the viral life cycle.

Redistribution of Lyt-bearing T cells in acute murine experimental allergic encephalomyelitis: selective migration of Lyt-1 cells to the central nervous system is associated with a transient depletion of Lyt-1 cells in peripheral blood.

Experimental allergic encephalomyelitis (EAE) was induced in SJL/J mice by using two injections of spinal cord homogenate in incomplete Freund's adjuvant supplemented with mycobacteria. Analysis of circulating Lyt-bearing subsets by indirect immunofluorescence during the course of acute EAE revealed the following: 1) during the pre-clinical phase of EAE (1 to 2 days before the onset of paralysis), there was a decrease in the percentage of Lyt-1- but not of Lyt-2-bearing cells in peripheral blood, and of both Lyt-1- and Lyt-2-bearing cells in spleen; 2) with the onset of clinically evident EAE, there was a decrease in both Lyt-1 and Lyt-2 cells in peripheral blood and an increase in the percentage of Lyt-1-bearing cells in pooled inguinal and axillary lymph node; and 3) after these early changes, there was a rapid reconstitution of the percentages of total Lyt-bearing cells and of both Lyt-1- and Lyt-2-bearing cells in peripheral blood. Immunohistochemical analysis of the central nervous system infiltrate revealed that the earliest lesions consisted predominantly of Lyt-1 T lymphocytes, with few Lyt-2 cells present. These results demonstrate that the influx of cells of the Lyt-1 inducer subset to the central nervous system in acute EAE is accompanied by a transient decrease in Lyt-1 cells in peripheral blood.

Experimental endocarditis induced by dental manipulation and oral streptococci.

Circumstantial evidence has strongly implicated dental manipulation as an etiologic factor in the development of infective endocarditis. The introduction of human oral streptococci via the oral cavity in rabbits with vegetative cardiac lesions yields a 94 per cent incidence of infective endocarditis, if the number of inoculated organisms is above a threshold level of 10(7).

Immunization with dextransucrases, levansucrases, and glycosidic hydrolases from oral streptococci. II. Immunization with glucosyltransferases, fructosyltransferases, and glycosidic hydrolases from oral streptococci in monkeys.

The feasibility of immunizing monkeys with enzymes from oral streptococci in an attempt to reduce dental caries was investigated. Forty rhesus monkeys, Macaca mulatta, were used. Cariogenic streptococci, S mutans, were implanted into all the monkeys' mouths. There was no pathological effect resulting from immunization. Of the 40 animals, 30 retained the implanted flora throughout the experiment; the remaining 10 were reimplanted until the streptococci remained. In six months, gross carious lesions were evident with plaque. Inhibitiors present in the monkey sera after immunization inhibited glucosyltransferase, fructosyltransferase, and neuraminidase activities. It was presumed the inhibitors were antibodies. There was a reduction of 68.6% in the total carious lesions in the animals immunized intraorally with glucosyltransferase, 62.4% reduction in those injected with fructosyltransferase, and 57.4% reduction in total lesions in those immunized with glycosidic hydrolases after 19 months, as compared to the control group. There were no gross lesions apparent in the group immunized with glycosidic hydrolases. It appears that immunization with enzymes significantly reduces carries and is feasible in a primate model.

Ethical issues in investigation of screening strategies.

Screening programs can be designed to be useful for data collection to study effectiveness of screening. High-risk populations may be identified, and screening techniques applied to a randomly selected subgroup with another subgroup serving as controls. This raises the ethical question of not applying helpful surveillance to individuals known to be at high-risk. Attention should be paid to management of controls, for example, to screen them less frequently, or at least to inform them of their increased risk and advise periodic exams. Against such recommendations is that differences between test and control groups would be minimized and the study less conclusive. Another approach is to use adaptive design, selecting the study group for a large population which is being subject to medical surveillance for some other reason. As in other clinical trails, studies of screening programs must include appropriate surveillance for controls to properly safeguard their rights and medical needs.

Population cancer screening.

Controlled trials to evaluate mass screening programs for cancer detection have been singularly lacking. High cost, lack of medical manpower, and low yield have contributed to this problem. A new program in cancer detection (CANSCREEN) has been developed jointly by The Fox Chase Cancer Center in Philadelphia and the Preventive Medicine Institute in New York City. This program attempts to provide a quality cancer-detection examination with increased cost effectiveness. Features include: 1) a self-administered questionnaire on medical history, symptoms, and risk factors; 2) nonphysican examiners; 3) risk-facotr analysis with a predetermined decision logic to determine type and periodicity of examination; and 4) primary intervention (health education). This collaborative program between two institutions in two cities demonstrates the feasibility of introducing similar programs elsewhere. A data base shared by cooperating centers permits information on all patients to be used for evaluation of new techniques, end results, etc. A randomized controlled trial has been designed to evaluate the effectiveness and efficiency of the questionnaire alone and of the questionnaire and examination.

Inhibition of glucan and levan synthesis and neuraminidase activity of oral streptococci by monkey antiserums.

A demonstration that antiserums from monkeys immunized with fructosyltransferase and glucosyltransferase reduced the activities of the enzymes in producing glucans and levans was done. The nature of the inhibiting factor has not yet been identified. The local immunization of monkeys with glycosidic hydrolases produced a sufficient amount of inhibiting principle, presumably antibody, to inhibit neuraminidase one month after the first immunization and this degree of immunization, which reached degrees of 50%, remained constant throughout 11 months of immunization of these monkeys. The immunizing principle has not yet been found in saliva. A criterion for the feasibility for immunization has been met. A significant reduction in streptoccal glucosyltransferase, fructosyltransferase, and neuraminidase activity was noted after immunization with enzymes.