Biologically active pituitary hormones in the rat brain amygdaloid nucleus.

While an attempt was being made to identify the source of the growth hormone releasing factor present in cerebral spinal fluid of man, it was discovered that cells of the rat amygdaloid nucleus, grown in tissue culture, produce a material that is immunologically and chromatographically identical to growth hormone found in the pituitary. Immunoperoxidase staining revealed dense accumulation of the peroxidase-antibody to growth hormone complex in amygdala cells. Significant amounts of growth hormone and adrenocorticotropin could be extracted from this limbic structure. Extracts containing immunoequivalent amounts of growth hormone were measured by bioassay in hypophysectomized rats. Stimulation of the growth of epiphyseal cartilage by extracts of the amygdala was comparable to the stimulation by extracts of anterior pituitary glands. The stimulatory effect of amygdala extracts on adrenal and gonadal size and weight and on growth of thyroid follicular epithelium was also comparable to that of pituitary extracts.

Salivary gland hyperglycemic factor: an extrapancreatic source of glucagon-like material.

Extracts of homogenates of rat, mouse, rabbit, and human submaxillary salivary glands contain a significant quantity of a material with glucagon-like immunoreactivity. Fractionation of this material on columns of Sephadex G-100 reveals a single peak immediately following a gamma globulin marker but in advance of a rat growth hormone marker, crystalline amylase, and isotopically labeled porcine insulin and glucagon. This material, which is urea stable, shows identical immunoassay dilution curves when measured with the highly specific K-30 glucagon antiserum. Study of paired glands in vitro shows that low concentrations of glucose stimulate and high concentrations of glucose suppress release of this material. Arginine promotes brisk release in vitro. Somatostatin does not influence arginine-stimulated secretion and insignificantly suppresses basal release in vitro. These findings lend support to previous speculations that the salivary glands may possess endocrine as well as exocrine functions. Salivary gland glucagon may also be the source of circulating glucagon recently reported in pancreatectomized and eviscerated rats.

Brain thyroid-stimulating hormone: effects of endocrine manipulations.

We have previously described the presence of and the immunologic, chromatographic and biologic characteristics of a thyroid-stimulating hormone (TSH)-like peptide, widely distributed in the rat and monkey central nervous system (CNS). In order to test the hypothesis that brain TSH, specifically hypothalamic TSH, participates in the turnover of pituitary TSH, we have assessed the effect on hypothalamic TSH of two endocrine manipulations, castration and adrenalectomy, known to significantly decrease pituitary and/or serum TSH in the rat. Orchidectomy led to a significant decline of pituitary and serum TSH while a significant increase in hypothalamic TSH concentrations was seen. Adrenalectomy also led to decreased pituitary concentration of TSH linked with a significant increase in hypothalamic TSH levels. Neither manipulation led to changes in TSH in the extrahypothalamic brain. The inverse relationship wherein serum and/or pituitary TSH decrease is accompanied by an increase in hypothalamic TSH is compatible with a role for hypothalamic TSH in pituitary TSH regulation. The possible significance of these findings in terms of TSH triiodothyronine interactions in the CNS is discussed.

Growth hormone (GH) immunoreactivity in the rodent and primate CNS: distribution, characterization and presence posthypophysectomy.

Using a specific sensitive radioimmunoassay, the distribution of growth hormone (GH) immunoreactivity in the rodent and primate central nervous system (CNS) was determined. Highest levels of extractable growth hormone-like materials were obtained from the rat amygdaloid nucleus, although other areas including cortex, hippocampus and the thalamus, contained immunoreactive material. Primate hypothalamus showed the highest levels of growth hormone immunoreactivity but it was also detectable in all regions examined. Forty-eight days posthypophysectomy, levels of GH immunoreactivity did not change in most rodent CNS areas studied. Moreover, levels in the amygdaloid nucleus and hypothalamus, although demonstrating an initial fall, actually rose above control levels several weeks following hypophysectomy. Dispersed CNS cells from both intact and hypophysectomized rats continuously released a GH-like material into the growth medium during a 20-day period of tissue culture. This phenomenon was suppressed with the addition of somatostatin to the growth medium. Characterization of this readily extractable GH-like material using column chromatography, parallel displacement curves, and biologic assay in the hypophysectomized rat showed a similarity between the CNS growth hormone-like material and its pituitary counterpart. The blood-brain barrier was found to be most likely intact to circulating pituitary growth hormone lending further support to the CNS origin of this biologically active and immunoreactive GH-like material in the brain.

Immunoreactive thyroid stimulating hormone (TSH)(: association with synaptosomally-rich fractions in the rat hypothalamus.

The subcellular compartmentalization of brain thyroid stimulating hormone (TSH) in the hypothalamus of the rat was investigated using differential and discontinuous sucrose density gradient centrifugation. When the mitochondrial fraction (P2) was layered on a discontinuous sucrose density gradient (0.32-1.4 M) and centrifuged for 60 min at 72,000 g, TSH recovered from the gradient was found, by double antibody radioimmunoassay, to be associated preferentially with the synaptosomally-rich layers. The separation was monitored by electron microscopic examination of all fractions obtained throughout the procedure. The addition of a large excess of either [125I]-labeled or unlabeled pituitary TSH at the time of homogenization did not influence the amount of immunoreactive TSH associated with the synaptosome-rich fractions, and both the unlabeled and labeled hormone were recoverable in the final supernatant indicating that the simple addition of peptide to hypothalamic homogenates did not result in any preferential association to any particular subcellular fraction. The apparent association of this brain-based pituitary peptide was not, therefore, an artifact of the homogenization process. It is concluded that an association exists between immunoassayable TSH and brain-based synaptosomes in homogenates of the rodent hypothalamus.

Growth hormone (GH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH)-like peptides in the rodent brain: non-parallel ontogenetic development with pituitary counterparts.

Brain and anterior pituitary growth hormone (GH), thyroid-stimulating hormone (TSH) and luteinizing hormone (LH) were measured during fetal, neonatal, and pubertal life and into adulthood. Immunoassayable GH and TSH could be found in the fetal whole brain before their detection in the fetal pituitary. Developmental patterns of pituitary and brain hormones differed in that pituitary hormones showed a gradual rise in levels from birth to puberty at approximately 20 days of age. Biochemically similar, brain-based peptides demonstrated a remarkable preparturitional surge in concentrations that was limited to a few days immediately preceding birth. Twenty-four hours after birth, brain GH, TSH, and LH had dropped to levels equal to or less than concentrations in the neonatal pituitary and subsequently rose to adult levels around the time of puberty. In these studies it could be shown that both the placental-fetal barrier and the neonatal blood-brain barrier were intact. These observations indicate the presence of two biochemically and immunologically similar but topographically distinct pools of peptides present in the developing brain and in the anterior pituitary gland.

Human immunodeficiency virus type I. Infection among dentists.

The largest collection yet assembled of year-to-year data on the seroprevalence of antibody to HIV in practicing dentists confirms that dentists--along with other health care workers--remain at low risk for occupationally acquired HIV infection.

Stimulation of prostaglandin production in rabbit ileal mucosa by bradykinin.

When rabbit ileal mucosa was incubated with exogenous [3H]arachidonic acid (AA), its major metabolites, identified by comigration with known standards on thin-layer chromatography, were prostaglandin (PG) E2, 6-keto-PGF1 alpha and to a lesser extent PGF2 alpha and PGD2. The rate of prostanoid release from the serosal surface of the mucosa only was increased after incubation th either bradykinin, lys-bradykinin, melittin or the calcium ionophore A 23187, in a rapid and dose-dependent fashion. Peptide concentrations as low as 10(-9) M were effective. Kinin-induced release of AA or its metabolites required the presence of Ca++ in the incubation medium. Stimulation of prostanoid release by lys-bradykinin was completely blocked by indomethacin. The combined lipoxygenase/cyclooxygenase inhibitors BW 755 and eicosa-5,8,11,14-tetraynoic acid and the lipoxygenase inhibitor nordihydroguaiaretic acid also blocked the stimulation of PG synthesis by lys-bradykinin. These inhibitors caused an increase in levels of AA released from the tissue by lys-bradykinin. The phospholipase inhibitors, mepacrine and U- 10029, inhibited the lys-bradykinin-stimulated release of both prostanoids and AA. At higher concentrations, U- 10029 inhibited the stimulation of transepithelial potential difference and short-circuit current across rabbit ileal mucosa produced by lys-bradykinin. These results support the hypothesis that bradykinin-stimulated intestinal secretion may be mediated by PGs.

TSH in the rat and monkey brain. Distribution, characterization and effect of hypophysectomy.

Characterization of the molecular, immunological and biological properties of a thyrotropin-stimulating hormone (TSH)-like peptide in rodent brain tissue showed similarities to its pituitary counterpart. The distribution of immunoreactivity in rodent brain was determined by radioimmunoassay in both intact and hypophysectomized animals. Easily detectable but smaller quantities of immunoassayable TSH were present in formalin fixed, acetone-stored brains from Macaca mullata. No change in the level of this TSH-like peptide was observed in most rat brain parts, although the hypothalamus did show a significant drop in hormone level after removal of the pituitary. Extracts of brain containing TSH-like material restored thyroid histology to normal when administered to hypophysectomized rats. Tissue cultured neural cells from both intact and hypophysectomized rats released a TSH-like material into the medium over a 30-day time period. Sodium l-thyroxine suppressed TSH release from pituitary monolayers but did not alter TSH release from brain derived cells that were similarly cultured. Surgical thyroidectomy resulted in a striking rise in serum TSH concentrations with no alteration in the content of brain TSH.

Impact of endocrine manipulations on brain-based rat growth hormone.

Our laboratory has previously described the widespread distribution of an immunoreactive and bioactive rat growth hormone (rGH)-like protein in rat brain. It has also been demonstrated that regulation of pituitary rGH secretion is at least partly mediated by a short-loop negative feedback system. In such a system, increased levels of rGH, acting at a suprapituitary locus, would decrease pituitary GH secretion. Thus, the present study has dealt with attempts to further investigate the hypothesis that one function of brain-based rGH might be as a mediator of the short-loop negative feedback system controlling pituitary rGH release. If brain-based rGH were to function as a mediator of such a system, then in situations where serum rGH levels are decreased, brain rGH concentrations should increase, indicating activation of a negative feedback loop. In the present communication we report that significantly decreased serum GH levels in oophorectomized and in thyroidectomized rats were coupled with a significant increase in rGH concentrations in the hypothalamus and in the amygdala. By contrast, adrenalectomy, which was not associated with any changes in levels of GH in serum caused no perturbations in levels of rGH in the brain. These discordant changes in serum and brain-based rGH are findings compatible with the hypothesis that one function of brain-based rGH is as a mediator of the short-loop negative feedback system regulating the release of pituitary GH.

Testicular dysfunction in experimental chronic renal insufficiency: a deficiency of nocturnal pineal N-acetyltransferase activity.

Biochemical correlates of neuroendocrine/gonadal function and nocturnal levels of serotonin N-acetyltransferase (NAT) activity were determined in partially nephrectomized (PNx), male, Long Evans rats following a 5-week period of chronic renal insufficiency (CRI). PNx animals demonstrated two to four-fold elevations in urea nitrogen and three to four-fold reductions (P less than 0.02) in plasma total testosterone concentrations as compared to sham-operated controls. The pituitary LH contents of PNx rats were decreased to approximately 60% of the control value (P less than 0.05). There were no differences in plasma prolactin levels between the control and PNx groups either at mid-day or in the middle of the night. Nocturnal pineal NAT activity in PNx rats was markedly reduced to approximately 20% of the control value (P less than 0.001). Similar evidence of gonadal dysfunction (reduced plasma total testosterone and testes testosterone content) and a significant decrease in night-time levels of pineal NAT activity were also observed after 13 weeks of CRI in PNx rats of the Sprague-Dawley strain that were housed under a different photoperiod. These results suggest that pineal gland dysfunction is a feature of CRI in the PNx model. Such an abnormality might contribute to the pathogenesis of gonadal dysfunction in CRI.

The effect of alcohol on quantitative and qualitative changes in luteinizing hormone (LH) in the female rat.

In order to study the impact of short-term ethanol feeding on pituitary luteinizing hormone (LH) levels, LH release and hormone microheterogeneity, 28 female oophorectomized rats were fed either a diet containing 36% ethanol or an isocaloric diet without ethanol for 16 days. On the 14th day of the experiment, all rats were given 50 micrograms of estradiol subcutaneously to provide a uniform steroid milieu and to induce an LH surge, and sacrificed 48 hours later. Total levels of pituitary and serum immunoreactive LH were identical between the ethanol and control groups. However, when pituitary LH was analyzed by isoelectric focusing on agarose gel a distinct difference was found. Four distinct immunoreactive peaks at pI's 3.50-4.55, 5.20-6.50, 7.00-7.50, 8.15-9.30 were noted. Ethanol-treated animals exhibited an 8-fold increase in the acidic LH subspecies in the pI range 3.50-4.55 and similarly more LH was determined at pI 7.00-7.50 compared to controls (P less than 0.01). More LH was found at the basic pI's from control pituitaries, compared to LH from the ethanol-exposed animals (P less than 0.01). In summary, short-term ethanol feeding in the female rats does not cause quantitative changes in pituitary LH levels or in the estrogen-induced LH surge. However, qualitative changes are present in isoelectric focusing patterns. A significant shift to the more acidic species of the pituitary LH isohormone was noted for ethanol-fed animals as compared to controls. These qualitative changes in the LH molecule, seen before quantitative changes could be appreciated, may help to explain why excessive exposure to alcohol results in reproductive impairments both in rats and in humans.

Comparison of two second-generation anti-hepatitis C virus ELISA on 21431 US blood donor samples.

We have compared two different second-generation (2.0) enzyme-linked immunosorbent assays (ELISA) for the presence of antibodies to hepatitis C virus (anti-HCV) in blood from volunteer, unpaid donors. At two separate blood centres, a total of 21,431 donor samples were tested with Abbott Anti-HCV 2.0 ELISA and Ortho Anti-HCV 2.0 ELISA. Samples found to be repeatedly reactive were tested by supplemental/investigational assays. MATRIX HCV (Abbott) and anti-HCV RIBA II (Ortho/Chiron), to 'confirm' the presence of anti-HCV. Discordant ELISA samples were additionally tested by the polymerase chain reaction (PCR) for the presence of HCV RNA. The Abbott anti-HCV assay had a repeatedly reactive rate of 0.59% (127/21,431) and the Ortho anti-HCV assay 0.51% (110/21,431). Overall agreement between assays was 99.76%, 72/127 (56.7%) of Abbott repeatedly reactive samples confirmed on MATRIX and 61/127 (48.0%) on RIBAII; 70/110 (63.6%) of Ortho repeatedly reactivate samples confirmed on MATRIX and 61/110 (55.5%) on RIBA II. Discordant ELISA samples tested by PCR yielded negative results. Hence the two ELISA had equal sensitivity, as defined by detection of true positive samples; the slightly lower specificity of the Abbott Anti-HCV 2.0 ELISA may be owing to culling of donors with a false positive test by Ortho's Anti-HCV 1.0 and 2.0 ELISA tests (the routine tests in place at each blood centre). A sample found to be repeatedly reactive by two different ELISA tests for anti-HCV is likely to be a true positive and may not require further 'confirmatory' testing.

Kinetics of antibody response to hepatitis B virus determinants and to recombinant vaccines in Italy.

The kinetics of antibody response to the group determinant a and subdeterminants d and y of hepatitis B virus were studied after infection and following immunisation with two recombinant DNA yeast-derived hepatitis B vaccines. The initial antibody response was to the subdeterminant epitopes, whereas anti-a antibody, which provides protection against different subtypes of the virus, was not detected for some weeks or months. The delay in the development of anti-a antibody after active immunisation raises important issues of early protection against infection.

Differential diagnosis of acute viral hepatitis using rapid, fully automated immunoassays.

We report the development of three rapid, fully automated immunoassays allowing the differential diagnosis of acute viral hepatitis. These assays detect HBsAg, IgM antibody to hepatitis B core antigen (IgM anti-HBc) and IgM antibody to hepatitis A virus (IgM anti-HAV) using the IMx instrument system. All IMx assays were run in less than 45 minutes and all steps were fully automated including specimen dilution steps. Specimens from blood donors, diagnostic and hospital patients, and individuals with a variety of infectious and immune diseases were tested for IgM anti-HAV (n = 1473) or for IgM anti-HBc (n = 1606) or for HBsAg (n = 9700) by the IMx and commercially available EIA and RIA. Each IMx assay showed 99.8% agreement with current EIA. Reproducibility in all hepatitis IMx assays was significantly better than that observed with manual or semiautomated assays; within-run and between-run % CV ranged from 2.2 to 4.8 and 3.5 to 10.3 respectively. In 29 acute hepatitis B patients studied, HBsAg and IgM anti-HBc were detected in the first available patient bleed collected from 0 to 4 week from the onset of symptoms. IgM anti-HBc persisted at reactive levels in the IMx assay for 1 to 24 weeks (mean 12.1 +/- 5.3 weeks) after the patient presented with symptoms. In individuals exposed to hepatitis A, IgM anti-HAV was detectable by IMx by 40 days post exposure (average 33.5 days) and IgM had declined to unreactive levels in IMx for all patients by from 3 to 6 months post exposure. These data demonstrate the use of these rapid IMx assays for differentiation of acute hepatitis A and B.

Prevalence of hepatitis C in tropical communities: the importance of confirmatory assays.

The prevalence of antibody to hepatitis C virus (HCV) was estimated in 3 tropical populations using 2 screening ELISAs to detect antibody to the c100-3 antigen and 2 supplementary assays designed to test the specificity of these tests. Two hundred and eighty-six of 385 (74.2%) sera from Kiribati, 17 of 138 (12.3%) sera from Vanuatu, and 39 of 173 (22.5%) sera from Zaire were reactive in the initial screening assay. The proportion of reactive sera which were also reactive in the second screening ELISA varied between populations (55.1% in Kiribati, 85.1% in Vanuatu, and 39.2% from Zaire). Reactive sera were selected at random for confirmatory testing. Only 3 of 49 (6.12%) of sera from Kiribati and 1 of 14 (4.76%) of sera from Vanuatu positive in the initial ELISA were reactive in the confirmatory assays. The proportion of confirmed positive sera from Zaire was higher 8 of 28 (28.5%). Based on the results of these supplementary assays the estimated prevalence of anti-HCV in these populations is 4.8% in Kiribati, less than 1% in Vanuatu, and 6.4% in Zaire. Reliance on a single screening ELISA to estimate the prevalence of anti-HCV in stored sera from tropical communities may lead to a gross over-estimate of the true prevalence in these populations.

Detection of HIV antigen and specific antibodies to HIV core and envelope proteins in sera of patients with HIV infection.

The sera of well-characterized populations were examined for three markers of human immunodeficiency virus (HIV) infection; HIV antigen (HIV Ag), and antibodies to HIV envelope (gp41) and core (p24) proteins. Of 563 serum samples tested, 251 were from HIV-infected patients diagnosed as having AIDS manifested by opportunistic infections (AIDS-OI), AIDS-associated Kaposi's sarcoma (AIDS-KS), or AIDS-related complex (ARC). One hundred seventy-six specimens tested were from asymptomatic high-risk individuals, and 136 were from heterosexual control subjects or patients with non-AIDS-related disease. None of the 136 control individuals tested had HIV Ag or HIV antibodies to either p24 or gp41. Of the 427 HIV-seropositive individuals, 99% to 100% were positive for gp41 antibodies to HIV. In contrast, the seroprevalence of p24 antibodies to HIV varied from 23% to 83% and appeared to be inversely associated with the severity of the patients' clinical symptoms. When specimens were analyzed for the presence of HIV Ag, in seropositive individuals the prevalence rate for this marker was lowest (1.4%) in asymptomatic individuals and highest (50%) in the AIDS-OI diagnosed group. Also, 240 cases with AIDS-KS, AIDS-OI, and ARC and the group of asymptomatic high-risk individuals were analyzed for T helper/T lymphocytes (T4) cell number and T4/T8 ratio; only one (2.0%) HIV Ag-positive case showed a T4 cell number greater than 400 and a normal T4/T8 ratio. These studies appear to demonstrate a direct correlation between the presence of HIV Ag and the severity of clinical complications of HIV infection.

Reliable detection of individuals seropositive for the human immunodeficiency virus (HIV) by competitive immunoassays using Escherichia coli-expressed HIV structural proteins.

We molecularly cloned the gag and env genes of the human immunodeficiency virus (HIV) and expressed fragments of these genes in Escherichia coli. Using the recombinant core and envelope proteins, we developed two competitive immunoassays (CIAs). Samples that recognized either the envelope or core proteins were considered positive for antibodies to HIV. This test system was comparable with western blot in detecting antibodies in patients with AIDS or AIDS-related complex that were repeatably reactive in the HIV screening test. All 360 individuals who were positive by western blot were positive by the CIA. A total of 844 samples repeatably reactive by an ELISA screening test were negative both by western blot and by the CIA; 48 samples positive by ELISA, but negative or indeterminate by western blot, were positive by the CIA. Alternate research procedures verified the positivity of these individuals. These data indicate that the CIA described here may be useful as an adjunct or alternative to the western blot.