Expression of Wnt receptors in adult spiral ganglion neurons: frizzled 9 localization at growth cones of regenerating neurites.

Little is known about signaling pathways, besides those of neurotrophic factors, that are operational in adult spiral ganglion neurons. In patients with sensorineural hearing loss, such pathways could eventually be targeted to stimulate and guide neurite outgrowth from the remnants of the spiral ganglion towards a cochlear implant, thereby improving the fidelity of sound transmission. To systematically identify neuronal receptors for guidance cues in the adult cochlea, we conducted a genome-wide cDNA microarray screen with 2-month-old CBA/CaJ mice. A meta-analysis of our data and those from older mice in two other studies revealed the presence of neuronal transmembrane receptors that represent all four established guidance pathways--ephrin, netrin, semaphorin, and slit--in the mature cochlea as late as 15 months. In addition, we observed the expression of all known receptors for the "wingless-related MMTV integration site" (Wnt) morphogens, whose neuronal guidance function has only recently been recognized. In situ hybridizations located the mRNAs of the Wnt receptors frizzled 1, 4, 6, 9, and 10 specifically in adult spiral ganglion neurons. Finally, frizzled 9 protein was found in the growth cones of adult spiral ganglion neurons that were regenerating neurites in culture. We conclude from our results that adult spiral ganglion neurons are poised to respond to neurite damage, owing to the constitutive expression of a large and diverse collection of guidance receptors. Wnt signaling, in particular, emerges as a candidate pathway for guiding neurite outgrowth towards a cochlear implant after sensorineural hearing loss.

Physiological (antioxidant) responses of estuarine fishes to variability in dissolved oxygen.

Cycles of dissolved oxygen (DO) in estuaries can range from anoxia to various levels of supersaturation (200-300%) over short time periods. Aerobic metabolism causes formation of damaging reactive oxygen species (ROS), a process exacerbated by high or low DO. Fish can generate physiological defenses (e.g. antioxidant enzymes) against ROS, however, there are little data tying this to environmental conditions. We investigated physiological defenses generated by estuarine fishes in response to high DO and various DO cycles. We hypothesized that chemical defenses and/or oxidative damage are related to patterns of DO supersaturation. Specific activities of antioxidants in fish tissues should be positively correlated with increasing levels of DO, if high DO levels are physiologically stressful. We caged common benthic fishes (longjaw mudsucker, Gillichthys mirabilis, and staghorn sculpin, Leptocottus armatus, in CA and spot, Leiostomus xanthurus and pinfish, Lagodon rhomboides, in NC) during summer 1998 in two estuarine sites in southern North Carolina and two in central California. At each site a water quality meter measured bottom DO, salinity, temperature, depth, pH and turbidity at 30 min intervals throughout the study. These sites exhibited a wide variety of dissolved oxygen patterns. After 2 weeks in the cages, fish gills and livers were analyzed for antioxidant enzymes (glutathione peroxidase, catalase and superoxide dismutase) and the metabolite glutathione. All fish exhibited antioxidant enzyme activity. There was a significant site-dependent effect on all enzyme activities at the NC sites, with the most activity at the site with the highest DO cycling and the most DO supersaturation. There was a trend towards higher enzyme activities under high DO levels at the CA sites.

Immunomagnetic purging of Ewing's sarcoma from blood and bone marrow: quantitation by real-time polymerase chain reaction.

PURPOSE: A propensity for hematogenous spread with resulting contamination of autologous cell products complicates cellular therapies for Ewing's sarcoma. We used a new approach to purge artificially contaminated cellular specimens of Ewing's sarcoma and show the capacity for real-time polymerase chain reaction (PCR) to quantify the contamination level of Ewing's sarcoma in such specimens. PATIENTS AND METHODS: Binding of monoclonal antibody (MoAb) 8H9 to Ewing's sarcoma cell lines and normal hematopoietic cells was studied using flow cytometry. Using real-time PCR--based amplification of t(11;22), levels of Ewing's contamination of experimental and clinical cellular products were monitored. Purging was accomplished using immunomagnetic-based depletion. Monitoring of the function of residual hematopoietic progenitors and T cells was performed using functional assays. RESULTS: MoAb 8H9 shows binding to Ewing's sarcoma but spares normal hematopoietic tissues. Nested real-time PCR is capable of detecting contaminating Ewing's sarcoma cells with a sensitivity of one cell in 10(6) normal cells. After 8H9-based purging, a 2- to 3-log reduction in contaminating Ewing's sarcoma was shown by real-time PCR, with purging to PCR negativity at levels of contamination of 1:10(6). Levels of contamination in clinical samples ranged from 1:10(5) to 10(6). Therefore, 8H9-based purging of clinical samples is predicted to reduce tumor cell contamination to a level below the limit of detection of PCR. CONCLUSION: These results demonstrate a new approach for purging contaminated cellular products of Ewing's sarcoma and demonstrate the capacity of real-time PCR to provide accurate quantitative estimates of circulating tumor burden in this disease.

Interleukin-7 restores immunity in athymic T-cell-depleted hosts.

Thymic-deficient hosts rely primarily on antigen-driven expansion to restore the peripheral T-cell compartment following T-cell depletion (TCD). The degree to which this thymic-independent pathway can restore immune competence remains poorly understood but has important implications for a number of clinical conditions including stem cell transplantation and human immunodeficiency virus (HIV) infection. A model of HY-mediated skin graft rejection by athymic, TCD mice was used to show that restoration of naive and recall responses via peripheral expansion requires transfer of only 25 x 10(6) lymph node (LN) cells representing approximately 10% of the T-cell repertoire. Constitutive expression of bcl-2 in the expanding inocula restored recall responses to HY at a substantially lower LN cell dose (1 x 10(6)), which is normally insufficient to induce HY-mediated graft rejection in athymic hosts. Interestingly, bcl-2 had no effect on primary responses. Interleukin-7 (IL-7) potently enhanced thymic-independent peripheral expansion and led to HY graft rejection using an LN cell dose of 1 x 10(6) in both primary and recall models. The restoration of immune competence by IL-7 appeared to be mediated through a combination of programmed cell death inhibition, improved costimulation, and modulation of antigen-presenting cell (APC) function. These results show that immune competence for even stringent antigens such as HY can be restored in the absence of thymic function and identify IL-7 as a potent modulator of thymic-independent T-cell regeneration.

Mutational analysis of patients with p47-phox-deficient chronic granulomatous disease: The significance of recombination events between the p47-phox gene (NCF1) and its highly homologous pseudogenes.

OBJECTIVE: The aim of this study was to determine the molecular basis of p47-phox-deficient chronic granulomatous disease (CGD), the most common autosomal recessive form of the disease. CGD is an inherited condition characterized by defective oxygen radical production due to defects in the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Mutational analysis of p47-phox-deficient CGD patients previously demonstrated that the majority of patients have a GT dinucleotide (Delta GT) deletion at the start of exon 2, a signature sequence also observed in the highly homologous pseudogenes of NCF1. MATERIALS AND METHODS: We performed genetic analysis of NCF1 and its pseudogenes using genomic DNA in 29 p47-phox-deficient CGD patients from 22 separate families. First-strand cDNA analysis was performed in 17 of the 29 patients. RESULTS: We confirmed the significance of the Delta GT mutation; in 27 of 29 patients, only the Delta GT sequence was detectable. All but one of the 27 had at least one additional signature sequence, specific to the pseudogene, in either intron 1 and/or intron 2. We extended our analysis to look at signature sequence differences in exons 6 and 9 and detected both the wild-type and pseudogene sequences in all patients tested. CONCLUSIONS: Although detection of only Delta GT sequence accounts for over 85% of affected patients, the molecular basis is most likely due to partial cross-over events between the wild-type and pseudogene(s) of p47-phox at different recombination sites. Our results suggest that complete gene conversion or deletion of the p47-phox gene (NCF1) occurs rarely, if it all.

Genomic structure of the human p47-phox (NCF1) gene.

The cytosolic factor p47-phox, encoded by the NCF1 gene, is an essential component of the phagocyte NADPH-oxidase system. Upon activation of this multicomponent system, p47-phox translocates to the membrane and participates in the electron transfer from NADPH to molecular oxygen. A deficiency or absence of p47-phox is the most common autosomal form of chronic granulomatous disease (CGD). We have cloned and characterized the NCF1 gene from four bacteriophage clones, a P1 clone and genomic DNA from normal individuals. The gene is 15,236 base pairs long and includes 11 exons. It is 98.6% homologous in sequence to at least one pseudogene that maps to the same region of chromosome 7q11.23. Slightly more than half (50.37%) of the wild-type NCF1 gene consists of repetitive elements. In particular, the density of Alu sequences is high (1.4 Alu/kb); there are 21 Alu repeats interspersed through 10 introns. These findings are consistent with the observation that recombination events between the wild-type gene and its highly homologous pseudogenes account for the majority of potentially lethal mutations in p47-phox-deficient chronic granulomatous disease. Analysis of 1.96 kb of sequence 5' of the start of translation revealed a high homology (99.6%) between wild-type and pseudogene clones. Characterization of NCF1 establishes a foundation for detailed molecular analysis of p47-phox-deficient CGD patients as well as for the study of the regulation of the NCF1 gene and pseudogenes, both of which are present as full-length transcripts in normal individuals.

Isolation of a complex of respiratory burst oxidase components from resting neutrophil cytosol.

The respiratory burst oxidase of neutrophils is a multicomponent enzyme, dormant in resting cells, that catalyzes the reduction of oxygen to O2- at the expense of NADPH. In the resting neutrophil, some of the components of the oxidase, including proteins p47phox and p67phox, are in the cytosol, while the rest are in a fraction that usually copurifies with plasma membrane. Recent evidence has suggested that at least some of the cytosolic oxidase components exist as a complex. We have now purified such a complex from the cytoplasm of resting neutrophils using an affinity column prepared with an antibody that recognizes the C-terminal decapeptide of p47phox. Immunoblotting showed that the complex contained both p47phox and p67phox. When supplemented with recombinant p67phox, the complex displayed considerable activity in a cell-free oxidase-activating system, and even without added p67phox, the complex could more than double O2- production in an oxidase-activating system supplemented with suboptimal amounts of cytosol. Isolation of the complex was blocked by preincubating the affinity column with CFSTKRKLASAV, the peptide against which the antibody was raised. On gel filtration, the complex migrated with a molecular weight of 240-300K, similar to that observed with whole neutrophil cytosol. The p47phox/p67phox ratio in the gel-filtered complex was approximately 1 to 1. These results indicate that in resting neutrophil cytosol, p47phox and p67phox exist as a complex.

Clinical manifestations, disability and use of folk medicine in dracunculus infection in Nigeria.

A cross-sectional survey of households was carried out in a dracunculiasis endemic village in Oyo state Nigeria. Data were collected on history of dracunculiasis, occupational and domestic sources of drinking water, clinical manifestations, disability, use of folk medicine, and incorporation of previous dracunculiasis control programmes. The findings indicated that dracunculiasis patients were usually unaware of their infection 3-5 days before the appearance of a bleb; that religious affiliation appeared to be positively related to increasing morbidity; and that ulcers were predominantly in the ankles and feet, particularly among young children. Severe disability was related to age, site and number of ulcers, and the form of selected treatment. Indigenous remedy was the treatment of choice, although traditional healers in the community indicated no knowledge of any efficacious remedy. Mortality from secondary tetanus infection was associated with outbreak of dracunculiasis. The impact of dracunculiasis on agricultural, economic and recreational activities was considerable, with the infected farmers being unable to attend to their farms at the critical farming period. Sixty-one per cent of the residents were opposed on religious and aesthetic grounds to the treatment of the local surface water which contained cyclops species. Sixty-three per cent regarded the boiling and filtration of portions of their domestic water as an additional burden, cumbersome and impracticable.(ABSTRACT TRUNCATED AT 250 WORDS)

Source of drinking water supply and transmission of guinea worm disease in Nigeria.

During 1982, ecological factors associated with freshwater pollution were investigated in Idere, a rural Nigerian community with endemic guinea worm infection. Data were collected on the quality of all pond water sources, and on rainfall patterns and potable piped water available in the community. Pond water provided 76% of the total water used in Idere. This source of drinking water provided the classical ecological environment for the transmission of Dracunculus medinensis, other helminth parasites and bacterial enteric infections. The bacteriological analysis of drinking water from the ponds reflects the absence of sanitary arrangements for human waste disposal in the community, as the ponds are collectors of storm run-offs. Okina, the spring-fed pond which was nearest to the households, was the most reliable year-round source of water to the community; however, Okina also contained the highest density of infective Thermocyclops and the highest faecal coliform (FC) to faecal Streptococcus (FS) ratio (FC/FS), thus providing a central reservoir for guinea worm and bacterial infections. The transmission season of guinea worm infection corresponded with the period of greatest water scarcity in Idere. The amount of portable water available to Idere residents in 1981 was 3.6 litres per person per day. Frequent mechanical breakdowns, electric power failures, lack of fuel to run the water pumping engines and the direct link of the water pipeline supplying water to Idere with a water pipeline serving another major city in the same district were some of the reasons for potable water shortage in the community.

Age and sex differences in new and recurrent cases of guinea worm disease in Nigeria.

In this population-based cross-sectional survey of the prevalence and incidence of guinea worm disease in Idere, a rural agricultural community of Oyo state in Nigeria, epidemiological data were collected by household interview of all 501 households (6527 persons, 3594 females and 2933 males). 86% of the households had at least one case. The prevalence was 32.4% in the overall population, but varied markedly by age and sex. About 10% of prevalent cases were first-time infections or new cases. Males over 19 years of age had a higher prevalence rate than adult females; however, females had a higher prevalence rate at younger ages. The prevalence in females peaked (47%) at 35 to 44 years; for males the proportion was highest (57%) at ages 45 to 54 years.

Uptake of dehydroascorbic acid and ascorbic acid to isolated nerve terminals and secretory granules from ox neurohypophyses.

When uptake of L-[14C]ascorbic acid ([14C]AA) to various organs in guinea-pigs was studied after intracardiac injection, the adenohypophysis, pars intermedia, and the neurohypophysis had an uptake per milligramme protein which was about half of the uptake to the adrenals. Adrenal uptake was 20 +/- 2.8 pmol mg-1 protein microCi-1 injected. The uptake to the different parts of the hypophysis was considerably higher than the uptake to pancreas, liver, kidney, spleen and other organs. When isolated nerve endings (neurosecretosomes) from ox neurohypophyses were incubated with a medium containing labelled dehydroascorbic acid ([14C]DHA), the uptake was much slower than when the medium contained labelled ascorbic acid. The uptake of [14C]DHA showed a linear dependence on concentration, and was not influenced by addition of Mg2+ and ATP. Addition of Mg2+ + ATP, omission of Ca2+ and Mg2+ or exchange of Na+ in the medium with K+ had no effect on the uptake of ascorbic acid. When isolated secretory granules from ox neurohypophyses were incubated with a medium containing [14C]DHA, uptake was considerably faster than the uptake when they were incubated in a medium containing [14C]AA. The uptake of dehydroascorbic acid was linear with the concentration in the medium and was not changed by addition of Mg2+ ATP. Addition of 10 mM NH4Cl or exchange of 120 mM K+ in the incubation medium with Na+ did not change the uptake of dehydroascorbic acid. The contents of copper, zinc, iron and cobalt were determined in isolated nerve endings (A) and membranes (B) as well as in lysate (C) from isolated neurosecretory granules. The results (in nmol mg-1 protein) were for Cu: (A): 0.25 +/- 0.01 (SEM), (B): 0.67 +/- 0.16, (C): 0.22 +/- 0.06; for Zn: (A): 0.53 +/- 0.13, (B): 6.97 +/- 0.75, (C): 1.8 +/- 0.53; and for Fe: (A): 15.6 +/- 1.9, (B): 6.92 +/- 0.32, (C): 3.15 +/- 0.43. In all preparations the cobalt content was below the detection limit (less than 5 pmol mg-1 protein).

Impact of guinea worm disease on children in Nigeria.

School attendance records of all primary schools in a guinea worm-endemic village in southwestern Nigeria were examined to determine the cause of missed school days and school drop-outs. At the time of the survey, 1,495 pupils (768 boys and 727 girls were registered in the 4 primary schools in the village, of which 21% of the pupils were infected with guinea worm disease (GWD). Female pupils had a higher infection rate than their male counterparts. Guinea worm-infected pupils missed up to 25% of school year days compared to a non-guinea worm-infected absence of 2.5%. At the height of guinea worm season in the study area, guinea worm-related absences contributed virtually all of the absenteeism recorded in the schools. Implications of the findings within the context of educational attainment of the pupils are discussed.

Total parenteral nutrition-related cholestasis in infants.

An epidemiological study was conducted to estimate the proportion of infants on total parenteral nutrition (TPN) who developed cholestasis and to identify risk factors associated with the development of this disease. Data were abstracted from medical records of 624 infants less than or equal to 30 days of age who were treated with TPN. A case of TPN-related cholestasis was defined as an infant whose serum level of direct bilirubin was greater than or equal to 1.5 mg/dl subsequent to initiation of TPN. Risk factors were assessed using multiple logistic regression analysis. Forty-six of 624 infants in the cohort (7.4%) were classified as having TPN-related cholestasis. The multivariable analysis indicated that cholestasis was associated with intracranial hemorrhage, patent ductus arteriosus, sepsis and gastrointestinal conditions that require surgery. Two distinct processes appear to have occurred: in infants who experienced patent ductus arteriosus or intracranial hemorrhage (conditions associated with hypoxia) where TPN may be the necessary trigger for the development of cholestasis, and in infants with gastrointestinal conditions requiring surgery or sepsis, where cholestasis may develop with or without parenteral infusions.

Wegener's granulomatosis diagnosed by open lung biopsy.

A 21 year old man was diagnosed as having Wegener's granulomatosis at thoracotomy. Treatment with prednisolone and cyclophosphamide was initiated but the patient died 4 months later. Wegener's granulomatosis is a rare disease which should be considered in the differential diagnosis of pulmonary malignancies, tuberculosis and lung abscess. Early diagnosis is mandatory for a favourable outcome. Open lung biopsy is recommended, if other examinations have failed, as large representative biopsies can be obtained.

Ascorbic acid uptake to isolated nerve terminals and secretory granules from ox neurohypophyses.

Isolated nerve terminals (neurosecretosomes) from cow neurohypophyses accumulated radioactivity when they were incubated with L[14C]-ascorbic acid in an ionic medium dominated by NaCl. Uptake of radioactivity was saturable with ascorbic acid concentration. Replacement of Na+ with Li+ in the incubation medium or presence of ouabain inhibited the accumulation. Isolated, purified cow neurosecretory granules contained 14 +/- 2 nmol ascorbate (n = 10) per mg of protein. When such granules were incubated with L[14C]-ascorbic acid in a KCl dominated medium, they took up radioactivity slowly. The accumulation was not saturable with ascorbic acid concentration and was not influenced by the presence of Mg2+ATP.

Blood pressure, fatness, and fat patterning among USA adolescents from two ethnic groups.

Many studies have explored the relationship between blood pressure and body size and composition in adults and preadults, but none has inquired into the relationship of blood pressure and the anatomical distribution of subcutaneous fat (fat patterning). Fat patterning has an association with chronic diseases (diabetes and cardiovascular disease) in adults. We sought the relationship between fatness, fat patterning, weight, and height among adolescents (12 to 17 years of age) of two ethnic groups (black and white) from the Health Examination Survey. Systolic blood pressure adjusted for age was related to body build variables in all sex/ethnic groups in decreasing order of importance as follows: body weight (independent of height), fatness (as assessed by a two skinfold index), and an excess of fat on trunk relative to fat on the leg (pattern index). Neither fatness nor fat patterning was significantly related to blood pressure after weight entered the regression equation. Relative fat patterning may be less important in predicting cardiovascular risk factors in adolescents than it is in adults. The fact that body weight was more important than fatness suggests that the weight/blood pressure association is due to components of body mass other than body fat.

Antepartum hematocrit, maternal smoking and birth weight.

Infants born to women with hematocrit values below 31% showed an increased frequency of low birth weight. However, when the women were stratified by smoking history, differences in birth weight became apparent at the higher hematocrit levels. The data suggested an increased risk of low birth weight among women who smoke and have hematocrit values in the 41--47 range. Since smoking alters plasma volume, care should be taken when evaluating hematocrit levels measured late in pregnancy.