Salmonella mutagenicity of three complex mixtures assayed with the microsuspension technique. A WHO/IPCS/CSCM study. In a collaborative study on complex mixtures, three complex mixtures and two pure compounds were assayed with the Salmonella microsuspension technique. The two pure compounds were benzo[a]pyrene (BaP) and 1-nitropyrene (1-NP). The three complex mixtures were standard reference materials (SRMs) from the U.S. National Institute of Standards and Technology, SRM 1649, SRM 1650 and SRM 1597. The two samples SRM 1649, an urban dust particulate matter, and SRM 1650, a diesel particulate matter, were sonicated with dichloromethane. Sample SRM 1597 was an extract of a coal tar sample with a complex mixture of polycyclic aromatic hydrocarbons. The microsuspension assay was performed with Salmonella strains TA98 and TA100 according to Kado et al. (1983) with minor modifications (Löfroth et al., 1988). The results showed that the microsuspension technique is a more sensitive assay than the plate incorporation method. Depending on sample, strain and metabolic condition the mutagenic responses were 3-37 times higher in the microsuspension assay than in the conventional plate incorporation assay. The microsuspension method is thus useful for environmental samples which are often available in only small amounts.


Studies on the genotoxicity of monocrotophos in somatic and germ-like cells of Drosophila. Parryfos, a farm-grade formulation of monocrotophos, was tested for genotoxicity in the wing primordial cells and the male germ-line cells of Drosophila melanogaster. Larvae of the 2nd or 3rd instar, heterozygous for the wing-cell marker mutations mwh and flr3, were exposed to different concentrations of the insecticide in the food. The wings of the hatched flies were screened for the presence of mutant mosaic spots exhibiting the marker phenotypes. The frequency of induction of sex-linked recessive lethal mutations was used to assess genotoxic effects in male germ-line cells. The tested compound was genotoxic in both the somatic and the germ-line cells of Drosophila.


Enhancement by butylated hydroxytoluene of the in vitro activation of 3,3'-dichlorobenzidine. Mutagenicity of Salmonella TA98 and covalent binding to DNA of 3,3'-dichlorobenzidine (DCB) were used to assess the influence of di-tert.-butylated hydroxytoluene (BHT) on the in vitro activation of the arylamine by rat hepatic S9 metabolic systems. BHT at a concentration of 4 or 20 microM enhanced the mutagenicity of DCB by 32 or 21%, respectively, and the covalent binding of DCB to added DNA by 76 or 328%, respectively. The antioxidant altered the HPLC profile of isolable DCB metabolites, causing a decrease in the formation of three metabolites, an increase in the formation of one metabolite, and the formation of an entirely new metabolite. BHT inhibited the mutagenicity of the promutagen 2-acetylaminofluorene (2-AAF) but had no effect on that of the direct-acting mutagen 2,4- dinitrophenylhydrazine (DNPH). The results show that BHT enhances the mutagenicity of and DNA binding by DCB, in contrast with the predominantly inhibitory effect of the antioxidant on the mutagenicity of other chemicals that require bioactivation.


Results of a comparative study on the Salmonella pre-incubation and plate incorporation assays using test samples from the IPCS collaborative study. Three complex mixtures (air particles, diesel particles and a coal tar fraction) and two pure compounds (benzo[a]pyrene and 1-nitropyrene) were tested in both the pre-incubation and the plate incorporation assay employing Salmonella typhimurium TA98 and TA100. Each experiment was conducted independently 2 or 4 times in duplicate in the presence and absence of metabolic activation. The mutagenic activities were calculated by least squares linear regression from the slope of the linear portion of each dose-response curve. Although slightly higher mutagenic activity was observed in the pre-incubation assay for the two pure compounds and with the plate incorporation assay for the diesel particulate sample, the overall data from both assays gave similar values and good correlations in TA100 and TA98. The results indicate that the pre-incubation assay could be used for these samples instead of the plate incorporation assay.


Germinal and somatic mutation induction in Drosophila after treatment of larvae with tritiated water. The present study was carried out to evaluate the mutagenicity of tritium, administered as tritiated water, in Drosophila melanogaster. Larvae were fed on tritium-treated medium during their development. Germinal and somatic mutation induction was detected by means of the sex-linked recessive lethal and the wing spot tests, respectively. Our results show that beta-radiation from tritium is able to induce significant increases in the frequency of both germinal and somatic mutations.


Evaluation of the co-mutagenicity of ethanol and delta 9-tetrahydrocannabinol with Trenimon. The mutagenic potential of chronic treatments of male CF-1 mice with ethanol and delta 9-tetrahydrocannibinol (THC), and their comutagenic potential with a known mutagenic agent, Trenimon, were examined. This was accomplished by measuring the frequency of dominant lethal mutations arising from mating of treated males with nontreated females. Adult male mice were treated with 5% (v/v) ethanol as part of a liquid diet (28% ethanol-derived calories) for five weeks; 10 mg/kg body weight (p.o.) THC every two days for five weeks; a single injection of Trenimon (0.125 mg/kg, i.p.) on day 28 of diet treatment; and all combinations of treatments. The control group was pair-fed a liquid diet in which isocaloric sucrose replaced ethanol; these males were also given sesame oil (vehicle for THC) and saline (vehicle for Trenimon) on the same schedule as that for the treated males. Neither body weights nor hematocrits were adversely affected by any treatment. Both ethanol and Trenimon treatments resulted in a small (8-9%; p less than 0.05) decrease in testicular weight. The effect of combined treatment with ethanol and Trenimon was roughly additive. Treatment with THC had no effect on testicular weight. Seminal vesicle weights were not affected by any treatment. Treatments were without significant effect on fertility, as measured by the frequency of males producing pregnancies. Ethanol and Trenimon treatments produced approximately 3- and 7-fold increases, respectively in the frequencies of preimplantational loss over that seen for the control group (7.3%), resulting in significant ethanol and Trenimon effects (p less than 0.001). No interactive effects of ethanol and Trenimon treatments were noted. Frequencies of dead fetuses per pregnancy in the ethanol- and Trenimon-treated groups were increased approximately 2.5- and 4-fold, respectively, over the control value of approximately 16%. However, the effect of combined treatments was not greater than that due to Trenimon alone, resulting in Trenimon and ethanol effects (p less than 0.001) and ethanol-Trenimon interaction (p less than 0.001). The calculated mutation index resulting from each treatment yielded significant (p less than 0.001) ethanol- and Trenimon-induced effects. In contrast to effects of ethanol and Trenimon treatments, THC, given alone, or in combination with ethanol and/or Trenimon, had no effect on either preimplantational loss, fetal mortality or the resulting mutation index. The data suggest that chronic ethanol treatment, at levels resulting in minimal fertility impairment, increases the frequency of dominant lethal mutations. In contrast, chronic treatment with THC, as administered in the present study, appears to be without effect.(ABSTRACT TRUNCATED AT 400 WORDS)


In vivo cytogenetic activity of diphenylhydantoin in mice. Diphenylhydantoin was tested in vivo in mice using a variety of cytogenetic endpoints to evaluate its genotoxicity. Injected doses of 125, 250 and 500 mg/kg failed to increase the number of chromosome aberrations in marrow cells at 17 h post-treatment, and 37.5, 75 and 150 mg/kg doses were likewise ineffective at 36 h. SCEs were significantly increased by doses of 125 mg/kg (but not 250 mg) after 23 h and modestly, in relation to dose, at 42 h. No increase in the number of micronuclei among marrow PCEs was seen following single i.v. injections ranging from 0.1 to 20 mg/kg. Three daily i.p. injections of doses up to 70 mg/kg also failed to increase the number of micronuclei in either marrow or peripheral blood PCEs. Some cytotoxic effect was evident following relatively high doses.


Clastogenicity of lead chromate particles in hamster and human cells. Several insoluble compounds of chromium, such as lead chromate, are respiratory carcinogens in experimental animals and suspected to be so in humans. Lead chromate induces neoplastic transformation in cultured cells but the mechanism of genotoxicity is unknown. We examined the effect of lead chromate on the integrity of chromosomes of Chinese hamster ovary (CHO) and human foreskin fibroblasts (HFF) after a 24-h exposure. At 0.4 microgram/cm2, 0.8 microgram/cm2, 2 microgram/cm2 and 8 microgram/cm2 lead chromate particles reduced survival of CHO cells to 86%, 62%, 2% and less than 1% respectively. These concentrations induced a dose-dependent 4-19-fold increase in the percent metaphases with damage. The HFF cells exhibited higher sensitivity in both cytotoxicity and clastogenicity. The spectrum of damage observed for both cell types was primarily achromatic lesions affecting one or both chromatids. To test for particle dissolution effects, CHO cells were treated for 24 h with either clarified medium that had been incubated for 24 h with lead chromate particles, or clarified medium that had been pre-conditioned by CHO cells treated with lead chromate particles for 24 h. No damage was detected in these cells, indicating that extracellular dissolution into ionic lead and chromate did not contribute to the genotoxicity. This is consistent with a previous study in which scanning electron micrographs illustrated internalization of the particles. These results suggest that clastogenesis may be a mechanism for lead chromate induced carcinogenesis.


Improved light-microscopical detection of microsporidia spores in stool and duodenal aspirates. The Enteric Opportunistic Infections Working Group. The diagnosis of infection with Enterocytozoon bieneusi, a microsporidian organism that causes chronic diarrhea in patients infected with the human immunodeficiency virus (HIV), has depended on invasive procedures. We have developed a new method to detect microsporidia spores in feces and duodenal aspirates. Stool was obtained from four HIV-infected patients with biopsy-confirmed intestinal microsporidiosis. Slides prepared from unconcentrated, formalin-fixed stool specimens were stained with a new chromotrope-based technique and examined by light microscopy. Methods of stool concentration were also compared. The technique was then evaluated by examining 215 specimens from 134 HIV-infected persons with or without diarrhea. In addition, duodenal aspirates from 10 patients with unexplained chronic diarrhea were examined by light microscopy after staining according to the new and the traditional techniques. E. bieneusi spores were found in all unconcentrated stool specimens from the four patients with microsporidiosis. The use of various methods of stool concentration did not improve the detection of microsporidia spores. In the prospective study, microsporidiosis was detected in samples from 6 of 27 patients with chronic diarrhea, but in none of those from 42 patients with acute diarrhea or 65 patients without diarrhea. The presence of microsporidia spores in stool specimens and duodenal aspirates allowed the successful prediction of the presence of microsporidia in small-bowel biopsy specimens from all four patients who subsequently underwent endoscopy. E. bieneusi is an important cause of chronic diarrhea in HIV-infected persons. This new diagnostic technique serves as a practical, noninvasive means to detect microsporidia spores in stool specimens and is also applicable to the examination of duodenal aspirates.


Vulvar vestibulitis: significant clinical variables and treatment outcome. The subject of vulvar vestibulitis was reviewed in regard to clinical variables that may be associated with this problem as well as the success of available treatment modalities. Questionnaires were returned by 71 patients diagnosed as having vulvar vestibulitis. Identical information was obtained from a comparison group of individuals with no clinical or physical findings suggesting this diagnosis. A history of recurrent candidiasis and previous condyloma acuminatum were the only variables noted more frequently in patients with vestibulitis. Among the patients treated by perineoplasty, 66% reported complete or significant alleviation of vulvar pain; 78% of the women noted a significant decrease in dyspareunia. Of the patients treated with intralesional interferon, six (50%) reported significant improvement in dyspareunia. Vulvar vestibulitis is a puzzling clinical entity whose etiology is not well understood. Perineoplasty still appears to be the treatment of choice in properly selected individuals.


Elevated maternal serum alpha-fetoprotein: association with placental sonolucencies, fetomaternal hemorrhage, vaginal bleeding, and pregnancy outcome in the absence of fetal anomalies. Elevated maternal serum alpha-fetoprotein (MSAFP) levels have been associated with an increased incidence of both placental sonolucencies and pregnancy complications. We designed a prospective study to test the hypothesis that the presence of these sonolucencies or a positive maternal Kleihauer-Betke stain would be associated with an elevated risk of obstetric complications. We enrolled 95 women with singleton pregnancies, elevated MSAFP, and no evidence of fetal anomalies on second-trimester ultrasound evaluation. Placental sonolucencies were documented at the time of ultrasound examination, and a maternal Kleihauer-Betke stain for fetal cells was obtained on the same day. Complications of pregnancy included fetal growth retardation, preterm delivery, late vaginal bleeding (at or after the 20th week of gestation), and fetal death. Women with elevated MSAFP had an increased incidence of placental sonolucencies, positive maternal Kleihauer-Betke stains, first-trimester vaginal bleeding, late vaginal bleeding, preterm delivery, fetal growth retardation, and fetal death compared with controls. Thirty-nine of 95 women with elevated MSAFP (41.1%) had at least one complication. In women with elevated levels, neither the presence of placental sonolucencies nor a positive Kleihauer-Betke stain correlated with first-trimester vaginal bleeding, the MSAFP level, or an increased risk of pregnancy complications. First-trimester vaginal bleeding was associated with an increased risk of preterm delivery in subjects with elevated MSAFP.


Marek's disease virus-mediated enhancement of avian leukosis virus gene expression and virus production. Direct interaction between two viruses in coinfected cells may promote replication and pathogenesis of one or both virus types. Synergism between herpesviruses and retroviruses is an important factor in diagnosis, treatment, and prevention of animal and human diseases. In birds, Marek's disease virus (MDV) may be an important cofactor in avian leukosis virus induced disease. Infection of susceptible cells with non-oncogenic serotype 2 MDV, an avian herpesvirus, and Rous-associated virus type 2 (RAV-2 ALV), a leukemogenic avian retrovirus, results in enhanced (greater than 3-fold) transcription of retroviral genes, relative to infection with ALV alone. A direct relationship between concentrations of retroviral gene expression and amount of input MDV suggests that MDV-encoded or -induced factors are responsible for enhanced ALV gene expression, ultimately leading to increased accumulation of ALV-specific RNA (greater than 5-fold) and protein (greater than 10-fold). At lower doses of input MDV, ALV virus production increased over 3-fold, relative to cells infected with ALV alone. Interactive laser cytometry was used to detect accumulation of both MDV and ALV antigens within single cells from coinfected cultures. These results suggest a direct role for MDV-encoded or -induced factors in enhancement of ALV gene expression and demonstrate the importance of herpesviruses as cofactors in retrovirus replication and pathogenesis in coinfected cells.


Mx genes show weaker primary response to virus than other interferon-regulated genes. Some interferon (IFN)-regulated genes are induced as a primary response to virus as well as secondarily through virus-induced IFN. Here we investigated whether this dual control mechanism would also regulate the activity of the human and mouse Mx genes that encode proteins with intrinsic antiviral potentials. To distinguish between a primary response to virus and a secondary response to virus-induced IFN, we studied virus-induced Mx gene expression in cell lines that lack a functional IFN system and in cells with blocked protein synthesis. In contrast to the two IFN-regulated human genes ISG56 and ISG15, the human MxA gene showed almost no primary response to Newcastle disease virus (NDV) or influenza virus. Similarly, direct activation of the mouse Mx1 gene by NDV or influenza virus was not significant in mouse embryo cells or explanted peritoneal macrophages. A moderate primary Mx1 response to NDV was observed in the permanent cell line L1210. Lack of a strong IFN-independent Mx response to virus indicates that this mode of gene regulation does not play a significant role in Mx-mediated resistance to viral disease.


Comoviruses and enteroviruses share a T cell epitope. An in vitro murine T cell proliferation assay was used to determine whether an antigenic epitope(s) recognized by enterovirus-immune T cells is held in common between plant comoviruses and human enteroviruses. Splenocytes isolated from C3H/HeJ mice infected with coxsackievirus B3 (CVB3) proliferated in vitro not only against a variety of enterovirus (CVB2, CVB3, CVB6, CVA16, PV1) antigens, but against comovirus (CPMV, BPMV) antigens as well. Splenocytes from mice inoculated with bean pod mottle virus (BPMV) also proliferated in response to comoviral and enteroviral antigens in vitro. However, if the viral inocula were highly purified prior to inoculation, then the splenocyte response was generated only against the group used to inoculate, suggesting that the epitope shared between the comoviruses and the enteroviruses resided in the nonstructural region. B (nonstructural) and M (structural) genomic segments of CPMV were translated in rabbit reticulocyte lysates and used as in vitro antigens. Splenocytes from mice inoculated with live CVB3 proliferated in response to the B-RNA-encoded but not the M-RNA-encoded polypeptides, confirming the nonstructural coding region location of the common epitope. Comparison of predicted amino acid sequences in the nonstructural coding regions of the comoviruses and picornaviruses suggested a potentially immunogenic linear epitope in protein 2C. The consensus peptide LEEKGI was synthezized and shown to be immunogenic for both BPMV- and CVB3-immune splenocytes.


Mechanism of HIV spread from lymphocytes to epithelia. Contact of human immunodeficiency virus (HIV)-infected MOLT-4 lymphocytes with epithelial cells derived from small intestine (I407; Intestine 407) resulted in a rapid polar budding of viral particles into an enclosed space formed by interdigitating microvilli of the contacting cells. Electron microscopy showed that released HIV was taken up into the mucosal cell via three independent mechanisms: (1) phagocytosis, (2) coated pits, and (3) direct fusion. Morphological evidence suggests that internalized HIV may escape into the cytoplasm of the target cell by uncoating at the endosomal membrane. Based on CD4 antibody binding and CD4 antibody blocking experiments, HIV entry does not appear to be mediated by a viral CD4 receptor. Productivity of I407 infection was confirmed by virus isolation from cocultured MT-4 lymphocytic cells, reverse transcriptase assay, p24 antigen ELISA, in situ HIV mRNA hybridization, and Southern dot blot analysis. Contrary to infection with free virus, the cell-to-cell infection was not blocked by anti-gp120 or antiviral serum from HIV-positive individuals. It appears that HIV transmission within the confined space between contacting cells enables HIV to evade immune protection provided by neutralizing antibodies. Our results reveal a mechanism of HIV infection of epithelial cells which is triggered by cell-cell contact. Furthermore, these observations offer an insight into the cellular sequence of events which may take place during sexual transmission of HIV across an intact epithelial barrier.


HIV-1 gene rev responsible for accumulated heterodisperse RNAs seen in infected T-cells. To analyze which gene of HIV-1 was responsible for the high accumulation of "heterodisperse RNAs" in virally infected T-cells, Northern blot analysis was performed. Total RNA from inducible cell lines expressing the envelope protein of HIV-1, or the regulatory gene rev, were blotted and hybridized with an Alu probe that detects heterodisperse RNAs. Results show that the rev gene is responsible for the accumulation of cellular heterodisperse RNAs seen earlier in HIV-1-infected T-cells.


Epitopic regions recognized by monoclonal antibodies against rat brain hexokinase: association with catalytic and regulatory function. Using direct and competitive epitope mapping methods, 23 monoclonal antibodies (Mabs) against rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) were divided into nine groups, each recognizing epitopes within defined surface regions of the N- or C-terminal domains; the latter have been associated with regulatory or catalytic functions, respectively. Reactivity of Mabs with the isolated domains was also studied. Based on the effect of various ligands on immunoreactivity, specific regions involved in ligand-induced conformational changes were identified. Adjacent epitopic regions, designated Regions F and G and located in the N- and C-terminal domains, respectively, were selectively affected by inhibitory hexose 6-phosphates (or analogs), marking these regions as being involved in transmission of the conformational signal from the regulatory N-terminal domain to the catalytic C-terminal domain. Consistent with this, the Ki for inhibition of the enzyme by the glucose 6-phosphate analog, 1,5-anhydroglucitol-6-phosphate, was markedly increased by Mabs binding in these regions, but unaffected by Mabs binding elsewhere in the molecule. Reactivity with Mabs recognizing conformationally sensitive epitopes in Region H of the C-terminal domain was greatly decreased by binding of substrate hexoses that induce closure of a cleft in the catalytic domain; selective recognition of the "open cleft" conformation, thereby preventing closure of the cleft required for progression of the catalytic cycle, can account for the marked decrease in Vmax that results from binding of these Mabs. Reactivity with Mabs binding to Region H was also decreased in the presence of inhibitory hexose 6-phosphates, implying that cleft closure was also induced by the latter; this is consistent with the suggestion that limitation of access to the C-terminal ATP binding site, resulting from cleft closure, is a factor in inhibition of the enzyme.


Domains of herpes simplex virus I glycoprotein B that function in virus penetration, cell-to-cell spread, and cell fusion. Herpes simplex virus 1 glycoprotein B (gB) is one of 10 glycoproteins in the virion envelope and in the membranes of infected cells. It is required for infection of cells in culture and functions in penetration of the cell by fusing the virion envelope with the plasma membrane. In studies to map the functional domains on HSV-1 gB, we reported that epitopes of potent neutralizing antibodies cluster in three major antigenic domains, D1, D2, and D5a. D1 contains continuous epitopes in the very amino terminus of gB. D2 comprises discontinuous epitopes that are assembled on gB derivatives 457 amino acids in length. D5a contains discontinuous epitopes that map between amino acids 600 and 690. We have now analyzed the function of these domains in virion infectivity by a detailed examination of the effects of 16 neutralizing antibodies on virion adsorption, penetration, plaque development, and cell fusion. Our results are as follows. (i) Ten antibodies with complement-independent neutralizing activity blocked penetration of virions into cells but not their adsorption to the cell surface. Treating cell-bound, neutralized virus with the fusogenic agent polyethylene glycol promoted their entry into cells. (ii) Ten antibodies with complement-dependent and -independent neutralizing activity interfered with plaque development by preventing spread of virus from infected to neighboring uninfected cells. (iii) Nine neutralizing antibodies, all complement-independent, prevented cell fusion induced by strain HFEM syn. We conclude that domains mapping in three regions of gB function in penetration of virions into cells, and that most neutralizing antibodies to these domains also block cell-to-cell spread of virus and cell fusion. The findings that three complement-independent neutralizing antibodies that blocked penetration did not inhibit plaque development, and that only one of these blocked cell fusion, indicate that the cell-to-cell spread of virus and cell fusion are related processes, but not identical to the penetration function.


Betaine:homocysteine methyltransferase from rat liver: purification and inhibition by a boronic acid substrate analog. Betaine:homocysteine methyltransferase (BHMT) from rat liver has been highly purified by an efficient procedure requiring only two chromatographic steps: Sephadex G-100 chromatography and fast protein liquid chromatography chromatofocusing. A 170-fold purification and 7.5% overall yield were achieved. Chromatofocusing yielded three active forms of BHMT with pI values near 8.0, 7.6, and 7.0. The subunit molecular weight of each active form is 45,000 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the native enzyme has a molecular weight of 270,000 as determined by exclusion chromatography. The stability of the purified enzyme was found to be potentiated by the presence of 1 mM dimethylglycine and 1 mM homocysteine. Boronate analogs of betaine (pinanyl N,N,N-trimethylaminomethaneboronate) (4) and dimethylglycine (pinanyl N,N-dimethylaminomethaneboronate) were synthesized from pinanyl iodomethaneboronate (3) and trimethylamine or dimethylamine, respectively. The free acid of the betaine analog (5) was reversibly generated from (4). The inhibition of BHMT by (5) appears competitive with a Ki = 45 microM. Since the Km for betaine measured with the purified enzyme is near 0.1 mM, the boronic acid analog of betaine appears to function effectively as a substrate analog inhibitor of BHMT. The analog does not appear to act as a methyl donor to homocysteine when (5) is substituted for betaine in the enzyme reaction. In addition, an enzyme assay based upon C3-cyano reverse phase HPLC detection of the o-phthalaldehyde derivative of methionine was developed as an alternative to the standard radiochemical assay. Betaine:homocysteine methyltransferase in the picomole range can be quantitated using this assay as indicated by a linear response of enzyme activity to protein concentration.


Multivariate analysis of serum tumor markers for diagnosis of skeletal metastases. Serum tumor markers, including carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), carbohydrate antigen 15-3 (CA 15-3), carbohydrate antigen 19-9 (CA 19-9), cancer antigen 125 (CA 125), and tissue polypeptide antigen (TPA), were measured in 26 patients with skeletal metastases and 11 patients with primary malignant bone tumors. TPA, which was elevated in 16 patients (61.5%), was the most sensitive marker for detection of skeletal metastases. Combined measurement of these markers was useful in detecting skeletal metastases from primary lesions, although tumor markers had little organ specificity. In addition, skeletal metastases could be completely differentiated from primary lesions by the use of multivariate discriminant analysis of markers. The most and least powerful discriminating factors were AFP and CA 19-9, respectively. On multidimensional scaling, the distance between AFP and CEA was longest, with the other markers scattered between them. Expression of individual markers can not be linked to that of other markers.


Malignant pilomatricoma. An immunohistochemical study with antihair keratin antibody. A case is reported of malignant pilomatricoma confirmed by immunohistochemistry using anti-human hair keratin (anti-HHK) antibody prepared by the authors. The tumor occurred in the soft tissue of the inguinal region of an 88-year-old woman, with later invasion of the epidermis. No other possible primary lesion was found at autopsy. Histologically, the tumor was squamous cell carcinoma with nests of tumor cells and shadow cell-like necrotic cells showing central keratinization and focal calcification. Immunohistochemically, the hair keratin was positive in this tumor and in benign pilomatricomas exclusively. All other skin lesions and various squamous cell carcinomas examined were negative for this antigen. The staining patterns of commercial antiepidermal keratin and antiinvolucrin antibodies were significantly different from that of anti-HHK in normal skin and in these lesions. To the authors' knowledge, this is the first case of malignant pilomatricoma tested with anti-HHK staining. Malignant pilomatricoma is generally a low-grade malignant tumor, but it can metastasize and be fatal as it was in this case.


Tumor and host response to arginine and branched chain amino acid-enriched total parenteral nutrition. A study involving Walker 256 carcinosarcoma-bearing rats. The metabolic effects of total parenteral nutrition (TPN) solutions containing different profiles of individual amino acids were investigated in the rats bearing Walker 256 carcinosarcoma. The rats were subcutaneously inoculated with 10(7) tumor cells and 6 days later were continuously infused intravenously for 8 days with three different TPN solutions. The experimental and control solutions were composed of high concentrations of branched chain amino acids (BCAA) and arginine (high Arg + BCAA TPN), high concentrations of BCAA (high BCAA TPN) and regular amino acids (regular TPN), respectively, and were isonitrogenous, isocaloric, and isovolemic. Rats receiving subcutaneous injection of saline rather than tumor cells received high Arg + BCAA TPN as pair-fed controls. The flooding dose method of 14C-leucine was used for the analysis of protein synthesis. With the feeding of high Arg + BCAA TPN, the tumor-bearing rats revealed a smaller increase of tumor volume and lower tumor fractional rates of growth (Kg) and synthesis (Ks) as well as protein synthesis (PS), compared with the high BCAA TPN and regular TPN in tumor-bearing rats. There were no significant differences of Ks and PS in liver and muscle between TPN groups, whereas tumor-bearing rats infused with high Arg + BCAA TPN displayed higher levels of whole-body Ks and PS than other TPN groups in tumor-bearing rats and pair-fed nontumorous rats. Except for liver RNA content which showed a lower level in tumor-bearing rats with high Arg + BCAA TPN, no other differences of DNA and RNA contents were found in tumor, liver, and muscle of the different TPN groups. The current results indicate that individual amino acids can influence tumor growth and protein metabolism, and that arginine, in combination with BCAA, may reduce tumor growth through a reduction in protein synthesis.


Kinetics of restitution of left ventricular relaxation. Although the kinetics of cardiac systolic force restitution have been well described, the restitution kinetics of left ventricular relaxation have not been examined. To define relaxation restitution behavior, we studied seven dogs chronically instrumented with left ventricular high-fidelity micromanometers and piezoelectric dimension crystals. After a priming period at a basic cycle length of 375 msec, test extrastimuli were introduced after a range of extrasystolic intervals (ESIs). Relaxation behavior of control and extrasystolic beats was characterized by the time constant of isovolumic relaxation, tau. Relaxation restitution can be described by two concatenated monoexponential curves, an early phase described by a rapid time constant and a late phase described by a slower time constant (TC1, 36.21 +/- 7.90 msec; TC2, 75.94 +/- 10.65 msec; p less than 0.05). The first phase of relaxation restitution parallels systolic force restitution over the same range and displays faster recovery (TCs, 58.93 +/- 10.01 msec, p less than 0.05). Postextrasystolic restitution of test pulses after beats at fixed ESIs depends on the initial ESI. Relaxation recovery of postextrasystolic beats proceeds faster with smaller initial ESIs (TC1 for ESI of 300 msec, 13.27 +/- 4.05 msec; TC1 for ESI of 450 msec, 72.85 +/- 21.72 msec; p less than 0.0001). The monoexponential pattern of restitution was seen with model-independent descriptors of relaxation as well as with tau.


Long-term EEG-video-audio monitoring: computer detection of focal EEG seizure patterns. Twelve individuals with medically refractory partial seizures had undergone EEG-video-audio (EVA) monitoring over 1-15 (mean 10.5) days. We selectively reexamined available 15-channel EEGs (video-cassettes) totaling 461 h and containing 253 EEG focal seizures. Computer analysis (CA) of these bipolar records was performed using a mimetic method of seizure detection at 6 successive computer settings. We determined the computer parameters at which this method correctly detected a reasonably large percentage of seizures (81.42%) while generating an acceptable rate of false positive results (5.38/h). These parameters were adopted as the default setting for identifying focal EEG seizure patterns in all subsequent long-term bipolar scalp and sphenoidal recordings. Factors hindering or facilitating automatic seizure identification are discussed. It is concluded that on-line computer detection of focal EEG seizure patterns by this method offers a satisfactory alternative to and represents a distinct improvement over the extremely time consuming and fatiguing off-line fast visual review (FVR). Combining CA with seizure signaling (SS) by the patients and other observers increased the correct detections to 85.38% CA is best used in conjunction with SS.


EEG coherence as a predictor of spike propagation. The relationship between resting EEG coherence and the propagation of spike activity from an experimental cortical focus was investigated in 6 rats. EEG was collected from an array of 6 epicortical electrodes positioned over the posterior hind limb sensorimotor (HL) and frontal (Fr1, Fr2) cortices. Coherence decreased non-linearly for all frequency bands with increasing interelectrode distance. The greatest decrement in coherence occurred in the region corresponding to the junction between hind limb (HL) and frontal (Fr1) cortices. The decrease in coherence was greatest for the highest frequency band for all interelectrode pairs. A spike focus was induced in all 6 animals following the application of bicuculline at the most posterior electrode of the cortical array. In 5 of the 6 animals spike propagation was delayed at the junction of HL and Fr1 cortices, where the greatest decrease in coherence was observed. In one animal spike activity never advanced across this region. These results demonstrate an association between intracortical coherence and the spatial propagation of spike activity. The function of short and long cortico-cortical pathways as mediators of both EEG coherence and cortical spike propagation are discussed.


Partial status epilepticus: short-term prediction of seizure outcome from on-line EEG analysis. Several combinations of ictal and interictal EEG abnormalities have previously been identified in partial status epilepticus (PSE). Some are associated with a consistently higher seizure index than others. On-line analysis of the initial segments of the EEG monitoring and familiarization with seizure indices corresponding to each combination may provide useful clues to rapidly foretell short-term seizure recurrence in patients with PSE. Referring to matrices reconstructed from an analysis of the initial min of the monitoring in 64 patients with recorded PSE can help to identify quickly those patterns which are most often associated with the highest probability of relapsing seizures. Awareness of the probability range of recording additional seizures observed with each of these various patterns may provide guidelines for judicious patient enrollment and for meaningful assessment of results in prospective studies of treatment efficacy in PSE.


Comparison and correlation of surface and sphenoidal electrodes with simultaneous intracranial recording: an interictal study. We prospectively compared and correlated interictal spikes recorded with simultaneous surface, sphenoidal, depth and subdural electrodes in 21 patients. Although the amplitude of sphenoidal spikes was often larger than that of surface spikes in patients with mesial basal temporal ictal and interictal foci, only 1 patient had exclusively sphenoidal spikes. Spikes with maximal amplitude at the sphenoidal electrode arose from mesial temporal, temporal neocortical and orbital frontal foci. An inferior vertical temporal dipole (hippocampal positive and inferior temporal neocortex negative) was associated with surface and sphenoidal spikes.


Detection of neonatal seizures through computerized EEG analysis. Neonatal seizures are a symptom of central nervous system disturbances. Neonatal seizures may be identified by direct clinical observation by the majority of electrographic seizures are clinically silent or subtle. Electrographic seizures in the newborn consist of periodic or rhythmic discharges that are distinctively different from normal background cerebral activity. Utilizing these differences, we have developed a technique to identify electrographic seizure activity. In this study, autocorrelation analysis was used to distinguish seizures from background electrocerebral activity. Autocorrelation data were scored to quantify the periodicity using a newly developed scoring system. This method, Scored Autocorrelation Moment (SAM) analysis, successfully distinguished epochs of EEGs with seizures from those without (N = 117 epochs, 58 with seizure and 59 without). SAM analysis showed a sensitivity of 84% and a specificity of 98%. SAM analysis of EEG may provide a method for monitoring electrographic seizures in high-risk newborns.


Localization of the sources of EEG delta, theta, alpha and beta frequency bands using the FFT dipole approximation. FFT dipole approximation and 3-dimensional dipole modelling were used to determine the locations of the equivalent dipole model sources of the delta, theta, alpha, beta-1 and beta-2 frequency bands in 13 normal subjects during resting. From each subject, 2 successive data sets were analysed, each consisting of 10 epochs of 2 sec randomly collected during 30 min. ANOVAs showed that over subjects, the source locations of EEG frequency bands differed significantly in the vertical and antero-posterior dimensions. Results of data set 2 confirmed those of data set 1. The source of delta was deepest and most anterior, theta more posterior and less deep, alpha most posterior and highest on the vertical dimension, beta-1 deeper and slightly more anterior than alpha, and beta-2 again more anterior and deeper than beta-1. Thus, the depth of source location was not linearly related to temporal frequency. The sources of all 5 bands were oriented in the sagittal direction; delta mean fields had steeper gradients anteriorly, alpha and beta-1 posteriorly. The power map for any frequency was well described by a single phase angle. The results indicate that the different EEG frequency bands during a given EEG epoch are generated by neural populations in different brain locations.


Decreased alpha bandwidth responsiveness to photic driving in Alzheimer disease. The power spectra of the photically activated occipital EEGs of 9 mildly to moderately demented probable Alzheimer disease (AD) patients (according to NINCDS-ADRDA criteria), 9 normal age-matched control and 27 normal subjects of different ages were compared. In normal subjects, photic stimulation with rhythmic flashes ranging between 2 and 20 Hz elicited a characteristic response in each EEG bandwidth (delta, theta, alpha, beta1 and beta2). The magnitude of each bandwidth response was a function of the frequency of the photic stimulus. In AD patients the alpha bandwidth response curve was significantly smaller than that of age-matched controls (MANOVA main effect of group, P = 0.018); all the other bandwidth response curves were normal. Therefore, in AD there is a selective abnormality in the alpha bandwidth responsiveness to photic stimulation, probably due to AD pathology in the neuronal generator of the alpha rhythm.


Macular dysfunction in multiple sclerosis revealed by steady-state flicker and pattern ERGs. Recent evidence indicates that the 2nd harmonics of steady-state (8 Hz) electroretinograms to either sinusoidal flicker (FERG) or to counterphased gratings (PERG) presented in the macular region (9 degrees) represent different subsets of generators in the inner retina. We evaluated the steady-state macular FERG and PERG 2nd harmonics (2F and 2P, respectively) in 19 normal subjects (19 eyes) and in 23 multiple sclerosis patients (44 eyes; 25 eyes with a history of clinical optic neuritis, and 19 eyes with no history of optic neuritis, subclinical eyes). The mean 2F and 2P amplitudes were significantly reduced, as compared to controls, in both subclinical and optic neuritis eyes. The 2P phase was significantly delayed, as compared to controls, in subclinical eyes, whereas 2F phase was delayed in eyes with optic neuritis. 2F was outside the 95% confidence limits (in amplitude or phase) in 11/19 subclinical eyes and in 25/25 optic neuritis eyes. 2P was outside the normal range in 12/19 subclinical eyes and in 24/25 optic neuritis eyes. These results show that FERG and PERG 2nd harmonics are significantly altered in multiple sclerosis eyes with or without a clinical history of optic neuritis. This finding suggests a dysfunction of inner macular layers which may result from direct retinal involvement or retrograde degeneration.


Electrophysiological evidence of different abilities to form cross-modal associations in children with autistic behavior. This study evaluates the amplitudes of auditory evoked responses and the variability of evoked responses, using a signal-to-noise ratio (SNR) method applied to individual evoked potential in a cross-modal (sound and light) association paradigm in 17 children with autistic behavior matched for sex and chronological age with normal children. Auditory evoked responses were smaller in children with autistic behavior than in normal children. The modifications of amplitudes and of SNR during cross-modal associations allowed the separation of children with autistic behavior into 3 subgroups who presented different patterns of ability to form cross-modal associations. These 3 subgroups of children presented different clinical profiles demonstrating that the differences observed in the ability to form cross-modal associations can be related to differences in the main psychophysiological functions such as attention, intention, motility, association, contact and communication.


On the possibility of independent activation of bilateral mismatch negativity (MMN) generators. Event-related potential (ERP) studies on auditory selective attention have suggested that the neuronal traces underlying the mismatch negativity (MMN) develop separately for left and right ear inputs. We investigated whether this feature of the MMN could be exploited to activate the MMN generators in each hemisphere independently. Given the location of the MMN sources, this would allow separate assessment of left and right hemisphere auditory cortical structures. The MMN was elicited in a dichotic oddball paradigm using stimuli and conditions of stimulation that should optimize transmission of left and right ear stimuli to the contralateral auditory cortices. The MMN demonstrated no interhemispheric asymmetries, indicating that the bilateral MMN generators were symmetrically involved. The results of a supplementary experiment provide evidence for the simultaneous existence of multiple traces for MMN generation.


Measurement of interhemispheric time differences in generalised spike-and-wave. We have compared interhemispheric time differences (ITD) calculated by coherence/phase and linear and non-linear correlation analyses of generalised spike-wave episodes in 30 patients (15 with primary generalised epilepsy (PGE), 5 with secondary generalised epilepsy (SGE) and 10 with a lateralised epileptogenic area). Most cases were recorded during routine departmental EEGs. No significant difference was found between measures of interhemispheric synchrony (IS) (proportion of ITDs that are synchronous) calculated using the linear and the non-linear correlation techniques, although the latter gave slightly more (average 7%) valid ITD estimates. This suggests that the non-linear correlation technique does not provide significantly more time difference information than its linear counterpart. Similarly, no significant differences in IS values were identified between the coherence/phase and the correlation techniques. For all 3 techniques, IS values greater than 50% were derived for most patients with PGE and SGE; the proportion of patients with a lateralised epileptogenic area showing this was smaller. However, broad overlap in the distribution of IS values between the 3 groups suggests that individual patients cannot reliably be distinguished using these methods.


Stimulation of lipolysis in cultured fat cells by tumor necrosis factor, interleukin-1, and the interferons is blocked by inhibition of prostaglandin synthesis. Multiple cytokines induce a number of alterations in lipid metabolism which can produce hyperlipidemia. Recent studies have demonstrated that tumor necrosis factor (TNF) increases lipolysis, resulting in an increase in circulating FFA levels, which stimulates hepatic triglyceride production, thereby contributing to the hyperlipidemia induced by TNF. In the present investigation we have determined the effects of a variety of cytokines on lipolysis in cultured 3T3-F442A adipocytes. TNF increased lipolysis approximately 3-fold with a maximal effect at 100 ng/ml and a half-maximal increase at 5-10 ng/ml. This increase was first observed 8 h after incubation with TNF. Interleukin-1 (IL-1) and interferon-alpha (IFN), -beta, and -gamma also stimulated lipolysis in cultured adipocytes. The half-maximal increase in lipolysis occurred at approximately 10 ng/ml IL-1, 5 ng/ml IFN alpha, 10 ng/ml IFN beta, and 8 ng/ml of IFN gamma. Maximal lipolysis was observed at approximately 100 ng/ml for each of these cytokines, with the exception of IFN beta, for which maximal stimulation was observed at 1000 ng/ml. Neither platelet-activating factor nor IL-6 stimulated lipolysis; therefore, it is unlikely that these compounds mediate the increase in lipolysis induced by cytokines. However, indomethacin, a well known inhibitor of prostaglandin synthesis, prevented the increase in lipolysis induced by TNF, IL-1, IFN alpha, IFN beta, or IFN gamma. Indomethacin did not affect basal lipolysis or the acute stimulation of lipolysis induced by epinephrine. These results demonstrate that multiple cytokines can increase lipolysis and that this increase is mediated by cytokine-induced stimulation of prostaglandin synthesis.


Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines. New insulin-secreting cell lines (INS-1 and INS-2) were established from cells isolated from an x-ray-induced rat transplantable insulinoma. The continuous growth of these cells was found to be dependent on the reducing agent 2-mercaptoethanol. Removal of this thiol compound caused a 15-fold drop in total cellular glutathione levels. These cells proliferated slowly (population doubling time about 100 h) and, in general, showed morphological characteristics typical of native beta-cells. Most cells stained positive for insulin and did not react with antibodies against the other islet hormones. The content of immunoreactive insulin was about 8 micrograms/10(6) cells, corresponding to 20% of the native beta-cell content. These cells synthesized both proinsulin I and II and displayed conversion rates of the two precursor hormones similar to those observed in rat islets. However, glucose failed to stimulate the rate of proinsulin biosynthesis. In static incubations, glucose stimulated insulin secretion from floating cell clusters or from attached cells. Under perifusion conditions, 10 mM but not 1 mM glucose enhanced secretion 2.2-fold. In the presence of forskolin and 3-isobutyl-1-methylxanthine, increase of glucose concentration from 2.8-20 mM caused a 4-fold enhancement of the rate of secretion. Glucose also depolarized INS-1 cells and raised the concentration of cytosolic Ca2+. This suggests that glucose is still capable of eliciting part of the ionic events at the plasma membrane, which leads to insulin secretion. The structural and functional characteristics of INS-1 cells remained unchanged over a period of 2 yr (about 80 passages). Although INS-2 cells have not been fully characterized, their insulin content was similar to that of INS-1 cells and they also remain partially sensitive to glucose as a secretagogue. INS-1 cells retain beta-cell surface antigens, as revealed by reactivity with the antigangloside monoclonal antibodies R2D6 and A2B5. These findings indicate that INS-1 cells have remained stable and retain a high degree of differentiation which should make them a suitable model for studying various aspects of beta-cell function.


Effects of continuous infusion of insulin-like growth factor I and II, alone and in combination with thyroxine or growth hormone, on the neonatal hypophysectomized rat. In this study, we examined the effects of systemically administered insulin-like growth factor (IGF)-I and -II on growth of the hypophysectomized (Hx) neonatal rat. Neonatal Wistar rats were Hx or sham Hx on postnatal day (PND) 6 and implanted sc with Alzet pumps on PND 10. Recombinant human IGF-I or -II were infused between PND 10 and 18 at an average dose of 1.9 micrograms/g body weight (BW) per day. In addition, some groups received daily sc injections of recombinant human GH or thyroxine (T4) at 2.5 micrograms and 25 ng/g BW per day, respectively. Pups were sacrificed on PND 18 and serum IGF levels determined. Despite restoration of serum IGF-I levels to sham control values in the Hx pups infused with IGF-I, no significant increase in BW occurred, although some increase in individual organ growth was observed (spleen, kidney, lung). Similarly, administration of IGF-II proved ineffective as a growth promoter in the neonatal Hx rat. In contrast, GH alone stimulated BW gain (P less than 0.001). T4 proved most potent in increasing skeletal growth (50% increase over Hx controls, P less than 0.001), without increasing serum IGF-I or -II levels. IGF-I and GH were equally effective in promoting a small yet statistically significant (17% over Hx controls, P less than 0.05) increase in skeletal growth. A synergistic effect on BW was observed with combined administration of T4 plus IGF-I to the Hx pups (P less than 0.05). The effects of hormonal therapy on serum IGF binding proteins (IGFBPs) was assessed by Western ligand blots. Administration of IGF-I, but not GH, resulted in increased levels of IGFBP-3, the predominant IGFBP of the adult rat. We conclude that systemically administered IGFs in doses that result in normalization of serum levels are ineffective promoters of somatic growth in neonatal rats. While normalization of serum IGF-I levels does result in modest skeletal growth, selective organ growth and increased serum IGFBP-3, growth stimulation does not equal that seen with GH (body weight) or thyroid hormone (skeletal growth). Differences in IGFBP profiles fail to account for the increased potency of GH as a promoter of BW gain. Thus, our data do not support a major endocrine role for IGF-I or -II in neonatal growth, but are consistent with an autocrine/paracrine action of IGF in the mediation of neonatal mammalian growth.


Expression and secretion of galanin during pregnancy in the rat. The expression of galanin messenger RNA (mRNA) in the pituitary and conceptus of pregnant rats has been studied at various stages of gestation. Using Northern blot analysis and in situ hybridization we have found that high levels of mRNA coding for galanin were detected in the conceptus during early pregnancy. The level of expression in conceptuses increased until day 11-12 after which the levels decreased rapidly. In contrast, in the pituitary galanin mRNA continued to increase throughout pregnancy, especially in the latter half of pregnancy as serum estradiol levels has been reported to be increased. The size of the galanin transcript was the same in the conceptus and pituitary (0.9 kilobase). Expression of this mRNA was confined to the decidua and was first seen at day 5 of pregnancy at a time when implantation swellings were first observed. Galanin antisense probe hybridized strongly to the decidual cells that surround the implantation site. At day 11 of pregnancy the galanin mRNA was found in both the antimesometrial and the mesometrial tissue with the highest concentration in the region lateral to the antimesometrial cells and continued toward the mesometrial cells. Serum galanin levels measured by an RIA using synthetic rat galanin as standard and antisera raised against porcine galanin, exhibited a temporal pattern similar to the pattern of mRNA expression in decidua, with a 7.1-fold increase at day 12 of pregnancy followed by a decline. In summary, decidual cells may differentiate into an endocrine cell during pregnancy. The galanin secreted by these cells, in addition to acting locally in a paracrine/autocrine fashion, may function as a placental hormone with systemic effects on distal target tissues.


Tissue distribution and regulation of insulin-like growth factor (IGF)-binding protein-3 messenger ribonucleic acid (mRNA) in the rat: comparison with IGF-I mRNA expression. Insulin-like growth factor-binding protein-3 (IGFBP-3) is the most abundant IGFBP in rat and human sera. The present study demonstrates the expression of the rat IGFBP-3 gene in a large number of tissues and coexpression, but not necessarily equal expression, with IGF-I mRNA. Tissues with a major abundance of IGFBP-3 were kidney, antrum of stomach, placenta, uterus, and liver. Changes in hepatic and renal levels of IGFBP-3 mRNA were analyzed after hypophysectomy (with and without GH treatment) and in the developing postnatal rat. These results were compared to changes in IGF-I mRNA levels under the same physiological conditions. Using S1 nuclease analysis, IGFBP-3 mRNA was present in the kidney and liver of 1-day-old rats and rose significantly in both organs by week 1. Thereafter, levels remained relatively constant, particularly in the liver. This is in marked contrast to the hepatic IGF-I pattern, which showed a continual rise up to 8 weeks. Hepatic IGFBP-3 gene expression was partially GH dependent, with IGFBP-3 mRNA levels falling (approximately 50%) after hypophysectomy and rising slightly after GH treatment. These changes were much less dramatic than those in IGF-I mRNA. In contrast, the renal levels of IGFBP-3 mRNA increased after hypophysectomy, (approximately 100%), but did not decrease with GH treatment. These data suggest that IGFBP-3 mRNA abundance is regulated differently in different tissues, and in at least some tissues is less sensitive to regulation than is IGF-I mRNA.


Expression of GLUT1 and GLUT2 glucose transporter isoforms in rat islets of Langerhans and their regulation by glucose. Previous studies revealed that rat islets express the GLUT2-liver facilitative glucose transporter isoform, a glucose carrier with a low affinity for glucose but a high capacity for glucose transport. These studies indicated the presence of a second glucose transporter in rat islets; however, they did not indicate to which of the five known facilitative glucose transporters it corresponded. In this study, we isolated RNA from rat islets of Langerhans and confirmed the presence of GLUT2 mRNA. In addition, we present data indicating that the second isoform expressed in islets is the GLUT1-erythrocyte isoform. The effect of culturing islets in 5.5, 8.3, or 11.1 mM glucose on the levels of GLUT1 and GLUT2 mRNA also was examined. The levels of GLUT1 and GLUT2 mRNA were two- and threefold higher, respectively, in islets cultured for 24 h in 11.1 mM glucose compared with those incubated in the presence of 5.5 mM glucose. Therefore, the previously observed increase in GLUT2 mRNA levels in the islets of rats made hyperglycemic by chronic infusion of glucose can be mimicked in vitro, implying that glucose regulates GLUT2 mRNA expression.


Sequence of human galanin and its inhibition of glucose-stimulated insulin secretion from RIN cells. Human progalanin cDNA was cloned with polymerase chain reaction techniques. The cDNA sequence predicts that the human form of galanin has a substitution of the glycine residue found at position 30 in other species and thus is likely to retain this residue during posttranslational processing and not be amidated at the COOH terminus. Furthermore, the cDNA sequence predicts three additional amino acid substitutions compared with known galanins. Human galanin was synthesized, and its bioactivity was compared with porcine and rat galanin based on inhibition of insulin release from a glucose-responsive rat insulinoma (RIN) cell line. Human galanin inhibited glucose-stimulated insulin secretion in a dose-dependent manner in RIN cells. Human, porcine, and rat galanin exhibited similar activity with ED50 less than 1 nM.


Intrahepatic up-regulated expression of extracellular matrix protein receptors in chronic active hepatitis type B. The aim of the present study was to investigate the differences in expression of beta 1 integrins between normal liver and the inflamed livers of patients with chronic active hepatitis B. Immunohistochemical staining with monoclonal antibodies that specifically recognize the common beta 1 chain and five different alpha subunits has been performed in frozen liver biopsy sections from 10 patients with chronic active hepatitis B and from 4 patients with normal livers. The major findings of our study were de novo expression in liver with chronic active hepatitis B of alpha 2 and alpha 3 subunits on both periportal hepatocytes and on lobular hepatocytes in close proximity to lymphocyte infiltrates. These results indicate the existence of an up-regulatory process in the expression of beta 1 integrins, especially the alpha 2 and alpha 3 subunits, in the inflamed liver tissue from patients with chronic active hepatitis B, suggesting that these integrins could play an important role in the development of liver fibrosis and in regulating intrahepatic lymphocyte migration.


Immunohistochemical demonstration of galaninlike immunoreactive nerves in the human pancreas. Galanin, the newly discovered 29 amino acid-residue peptide, has been shown to suppress glucose-induced insulin secretion in experimental animals, but its presence and physiological role in the human pancreas have not been established. In this study, the occurrence and distribution of galanin immunoreactivity in the human pancreas was investigated by immunohistochemistry. In addition, the possible coexistence of galanin and vasoactive intestinal peptide immunoreactivity in neural elements of the pancreas was examined. In the human pancreas, galanin immunoreactivity was localized in numerous nerve fibers around glandular acini, ductules and blood vessels, and in a few nerve fibers within islets. Nerve cells with galanin immunoreactivity were frequently noticed. Immunostainings for galanin and for vasoactive intestinal peptide on serial adjacent sections of intrapancreatic ganglia showed the coexistence of the two immunoreactivities in a large proportion (73.3%) of nerve cells. These observations may provide a morphological basis for the possible neurotransmitter or neuromodulator role of galanin in the human pancreas.


Ultrastructural localization of somatostatin- and substance P-immunoreactive nerve fibers in the feline liver. The distribution and the possible source of somatostatin- and substance P-immunoreactive nerve fibers were studied in the liver of the cat by immunocytochemical techniques. Abundant substance P-immunoreactive and a moderate number of somatostatin-immunoreactive nerve fibers were found running around the blood vessels in the perilobular connective tissue. Some somatostatin-immunoreactive nerve cell bodies were also observed in the liver. A moderate number of somatostatin-immunoreactive nerve profiles were found inside the hepatic lobules along the sinusoid endothelial cells and the hepatocytes. The interspace between the axon and hepatocyte and endothelial cells membranes was about 20 nm. Cutting the extrinsic hepatic nerves resulted in marked reduction of substance P-immunoreactive nerves but only a slight reduction of somatostatin-immunoreactive nerves in the liver. These findings provide a morphological basis for the possibility that somatostatin and substance P may act as transmitters or neuromodulators on the neighboring smooth muscle cells of vessels and may regulate the function of the hepatocytes. It is also possible that some of these fibers serve sensory function along the blood vessels.


Tachykinin antagonists inhibit nerve-mediated contractions in the circular muscle of the human ileum. Involvement of neurokinin-2 receptors. The effects of some newly developed tachykinin antagonists that are selective for the neurokinin (NK)-1 (L 668,169) or the NK-2 (MEN 10,207, L 659,877 and R 396) tachykinin receptor on the cholinergic and noncholinergic contraction and on the nonadrenergic noncholinergic relaxation produced by electrical field stimulation (50 Hz) were investigated in mucosa-free circular strips of the human ileum. The strips were contracted by substance P and neurokinin A as well as by selective NK-2-receptor ligands, [beta Ala8]neurokinin A(4-10), and MDL 28,564, the latter peptide being capable of discriminating between NK-2-receptor subtypes. The selectivity of the antagonists for NK-1 or NK-2 receptors was confirmed in pharmacological experiments using substance P, neurokinin A, and [beta Ala8]neurokinin A(4-10) as stimulants. Among the NK-2-selective antagonists, MEN 10,207 displayed the highest affinity, followed by L 659,877 and R 396. The antagonists MEN 10,207 and L 659,877 inhibited the noncholinergic contraction to electrical stimulation in a concentration-dependent manner; L 668,169 and R 396 were poorly effective. Thus the potency of antagonists toward the noncholinergic response closely paralleled their rank order of potency at NK-2 receptors. The cholinergic contraction and nonadrenergic noncholinergic relaxation were not inhibited by the antagonists. Both substance P- and neurokinin A-like immunoreactivities were detected in extracts of the human ileum, and the identity of the corresponding peptides was confirmed by reverse-phase high-performance liquid chromatography. It was concluded that in addition to NK-1 receptors, the circular muscle of the human ileum also contains NK-2 receptors. Activation of the latter is chiefly responsible for the noncholinergic contraction to nerve stimulation.


Detection of hepatitis C virus RNA in serum of patients with chronic hepatitis C treated with interferon-alpha. We tested serial serum samples for hepatitis C virus RNA from patients undergoing treatment for chronic hepatitis C with interferon-alpha using an assay that combined reverse transcription and polymerase chain reaction. The subjects studied were 20 patients with chronic hepatitis who had serum antibody to hepatitis C virus (anti-C100-3). Before therapy, hepatitis C virus RNA was detected in 18 (90%) and 20 (100%) patients using primer sets derived from the NS3 region or the 5'-noncoding region of hepatitis C virus, respectively. Hepatitis C virus RNA became undetectable in all patients whose ALT level fell into the normal range during therapy. However, hepatitis C virus RNA reappeared in all patients whose ALT levels rose again after therapy, usually before the relapse. In patients whose ALT levels did not become normal, hepatitis C virus RNA did not disappear during therapy. Thus therapy with interferon-alpha appears to be beneficial in chronic hepatitis C because of its suppressive effects on hepatitis C virus replication. Detection of hepatitis C virus RNA in serum is useful for evaluating the antiviral effect of interferon.


D-penicillamine prevents the development of hepatitis in Long-Evans Cinnamon rats with abnormal copper metabolism. The Long-Evans Cinnamon rat is a mutant strain that contracts hereditary hepatitis and, eventually, spontaneous hepatocellular carcinoma. Because we found a corresponding gross copper accumulation in the liver of the rats, we examined whether the development of hepatitis in our rat system could be prevented by administration of D-penicillamine. D-Penicillamine is a copper-chelating agent and one of the drugs effective for human Wilson's disease, in which abnormal copper metabolism is also observed. The results show that D-penicillamine treatment inhibited the elevation of serum transaminases, suppressed abnormal histological changes in the liver and completely prevented the onset of hepatitis in the Long-Evans Cinnamon rats. We further found that the copper concentration in the liver and serum copper and ceruloplasmin levels were decreased, whereas the urinary copper level was increased in the D-penicillamine-treated Long-Evans Cinnamon rats. These findings demonstrate that the pathogenesis of hereditary hepatitis in Long-Evans Cinnamon rats is due to abnormal copper accumulation in the liver.


Insulin-like growth factor-binding protein-3 is functionally normal in pregnancy serum. Insulin-like growth factor-binding protein-3 (IGFBP-3) carries most of the serum IGFs as a 150K ternary complex which increases during pregnancy. However, recent studies have demonstrated that IGFBP-3 is absent in pregnancy serum when measured by ligand blotting after electrophoresis. In the present study we demonstrate that, by several criteria, IGFBP-3 appears functionally intact in pregnancy serum. Serum samples were collected from pregnant women between 2-40 weeks gestation. Serum immunoreactive IGFBP-3 and acid-labile (alpha) subunit increased linearly throughout pregnancy. Ligand blotting confirmed diminution of IGFBP-3 at 2 weeks gestation and complete absence after 4 weeks gestation or when nonpregnancy serum was preincubated with amniotic fluid. When less harsh methods (neutral chromatography or transient acidification) were used, IGFBP-3 appeared functionally normal in pregnancy serum. Fractionation of pregnancy serum by Superose-12 gel permeation chromatography showed similar elution profiles for both IGFBP-3 and alpha-subunit compared to those for nonpregnancy serum. Preincubation of nonpregnancy serum with pregnancy serum or amniotic fluid had no effect on IGFBP-3 recovery. After acidification and neutralization of serum to destroy endogenous alpha-subunit, ternary complex formation, measured by radiolabeled alpha-subunit binding, was essentially identical in all nonpregnancy and pregnancy serum, except for a slight loss of activity in first trimester serum. Since the 150K complex cannot form unless IGFBP-3 binds IGFs and alpha-subunit normally, these results suggest that IGFBP-3 in native pregnancy serum is functionally normal.


The 24/25-kDa serum insulin-like growth factor-binding protein is increased in elderly women with hip and spine fractures. Fractures of the hip in elderly women represent a clinical syndrome (age-related osteoporosis) often marked by decreased calcium absorption and secondary hyperparathyroidism. We studied 13 elderly women with fractures of the hip and spine and 18 healthy similarly aged control women to determine whether serum insulin-like growth factor-I (IGF-I) or its carrier binding proteins (IGFBPs) were altered in this syndrome. Serum IGF-I concentrations were not different in the two groups (P = 0.50), but immunoreactive PTH was significantly higher in the fracture group (58.50 +/- 8.20 vs. 13.50 +/- 2.70 ng/L; P less than 0.003). Binding of [125I]IGF-I to IGFBP-3, IGFBP-2, and IGFBP-1, measured by ligand blotting, was not statistically different in the 2 groups, but binding intensities for the serum 24/25-kDa IGFBP were approximately 2.5 times greater in fracture women than control women (P less than 0.0005). In data pooled from both groups, PTH correlated strongly (r = 0.70; P less than 0.0001) with the relative binding intensities for the 24/25-kDa IGFBP. Based on previous work, we speculate that production of the 24/25-kDa IGFBP, which in vitro is known to inhibit IGF-I- and IGF-II-mediated osteoblast function, may be stimulated by PTH in patients with the syndrome of age-related osteoporosis.


The insulin-like growth factor (IGF)-binding proteins and IGF bioactivity in Laron-type dwarfism. Laron-type dwarfism (LTD) is caused by a variable defect in the GH receptor gene and is, therefore, an ideal model to study the physiology of the insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) in the complete absence of GH action. In this study we examined the overnight variation of the IGFs, IGFBPs, and IGF bioactivity in two prepubertal subjects with LTD. Subject 1 was a 14-yr-old female, 103 cm tall (-8.3 SD), and subject 2 was a 11.5-yr-old male, 103.6 cm tall (-5.9 SD). Both had serum IGF-I levels below 0.07 U/mL and low constant serum IGF-II levels overnight (185 +/- 10 and 232 +/- 8 micrograms/L), despite high serum GH levels [mean GH, 65 (32.5 micrograms/L) and 53 mU/L (26.5 micrograms/L)]. Serum IGFBP-1 levels increased overnight (from 24 and 22 micrograms/L at 2000 h to 83 and 110 micrograms/L at 0800 h) as serum insulin levels fell [from 19 (136 pmol/L) and 17 mU/L (122 pmol/L) at 2000 h to less than 2 (less than 14 pmol/L) and 5 mU/L (36 pmol/L) at 0800 h] in subjects 1 and 2, respectively. Serum IGFBP-2 levels remained constant overnight, as assessed on Western Ligand blotting and, despite the changes in IGFBP-1, remained the most prominent IGFBP throughout. On size separation, most of the IGF-II (greater than 60%) eluted with IGFBP-2 and the other low mol wt IGFBPs. Serum IGFBP-3 levels were reduced, and IGFBP-3 was not the major IGF carrier in LTD serum, in contrast to normal serum. An IGFBP-3-specific protease that was heat sensitive and cation dependent was identified as the cause of an apparent overnight rise of serum IGFBP-3 levels. No IGFBP-3 variation and no proteolytic activity was seen in normal serum or rapidly separated LTD plasma. Serum IGF bioactivity, measured in a porcine cartilage bioassay, was 0.18 and 0.55 U/mL in subjects 1 and 2; differences in bioactivity between subjects did not relate to serum IGF-II levels, but, rather, to differences in IGFBP-3 levels. Serum IGF bioactivity was not constant overnight and varied in a similar fashion in both subjects 1 and 2, with reduction in bioactivity between 0600-0800 h by 55% and 32%, suggesting the presence of inhibitory factors in the LTD serum; this decrease coincided with the rise in serum IGFBP-1 levels.(ABSTRACT TRUNCATED AT 400 WORDS)


Differential expression of aminopeptidase-N on human ovarian granulosa and theca cells. The expression of aminopeptidase-N and neutral endopeptidase in human ovarian tissue was examined using specific monoclonal antibodies for each of these peptidases and histochemical staining for enzyme activity. Aminopeptidase-N is a membrane-bound metalloprotease catalyzing the removal of N-terminal amino acids from peptides and was detected by immunofluorescence staining on theca interna cells in secondary follicles and on luteinized thecal cells in preovulatory follicles and corpora lutea. However, aminopeptidase-N was not detected on granulosa cells. Peptidase activity was also detected by histochemical staining on theca interna cells and luteinized thecal cells. Luteinized granulosa cells showed peptidase activity, despite the lack of aminopeptidase-N. Neutral endopeptidase was not detected in ovarian granulosa and thecal cells. These observations indicate that aminopeptidase-N can be a useful surface marker for thecal cells.


Regulation of human basophil activation. II. Histamine release is potentiated by K+ efflux and inhibited by Na+ influx. Na+ and K+ are the major extra- and intracellular cations, respectively. We have thus studied the role of these ions on human basophil histamine release by modifying their transmembrane gradients or by increasing membrane ion fluxes using ionophores. 1) When external Na+ (reduced to 4 mM) was replaced by the nonpermeating Na+ substitute N-methyl-D-glucamine, the release of histamine was enhanced in 2 mM Ca2+ (from 37.5 +/- 8.0% in 140 mM Na+ to 68.5 +/- 9.1% in low Na+) and became possible in the presence of low Ca2+ (at 1 microM Ca2+: from 0.6 +/- 0.7% in 140 mM Na+ to 36.2 +/- 8.0% in low Na+); moreover, in low Na+, the release of histamine became partly independent on Ca2+ influx. 2) Increasing the Na+ influx with the cation channel-forming gramicidin D inhibited the release of histamine by 33.2 +/- 13.6% (n = 6) in an external Na(+)-dependent manner. 3) Decreasing K+ efflux using K+ channel blockers (4-aminopyridine, quinine, sparteine) inhibited histamine release in a dose-response manner. 4) The K+ ionophore valinomycin, which increases K+ efflux, slightly enhanced IgE-mediated histamine release when used alone, whereas it potentiated the release of histamine from leukocytes previously treated with 4-aminopyridine by 57.0 +/- 18.6% (n = 7). 5) Decreasing K+ efflux by increasing external K+ inhibited IgE-mediated release in a similar manner as Na+ did. The inhibitory effects of Na+ and high K+ were not additive, thus suggesting that both cations inhibited the release by a common mechanism. In conclusion 1) our data evidence that histamine release from human basophils is inhibited by Na+ influx and potentiated by K+ efflux; 2) they suggest that K+ channels are present on the basophil membrane and that Na+ and K+ fluxes act on histamine release most probably via modulation of membrane potential.


Expression of variable exon A-, B-, and C-specific CD45 determinants on peripheral and thymic T cell populations. A mAb (I/24) has been generated that is specific for a determinant on mouse CD45 molecules. Reactivity of this mAb with a panel of CD45 transfected cell lines demonstrated that the determinant recognized is dependent upon expression of one or more CD45 variable exons and that exon C is sufficient for its expression. The exon C-specific epitope detected by I/24 is expressed at high density on essentially all B lymphocytes and at an intermediate density on the vast majority of CD8+ splenic T cells. Two distinct subpopulations of CD4+ splenic T cells were detected, a minor subpopulation that expresses this exon determinant at high density and a major subpopulation that expresses it at a much lower density. This first identification of a CD45RC-specific reagent allowed a comparison of the expression of exon A-, exon B-, and exon C-specific determinants on peripheral and thymic lymphoid populations. When splenic lymphocytes were analyzed for expression of CD45RA (reactive with mAb 14.8), CD45RB (reactive with mAb 23G2 or mAb 16.A), and CD45RC (reactive with mAb I/24) determinants, it was found that each of these CD45 determinants had a distinct pattern of expression on CD4+ and CD8+ T cells and B cells. CD45RB and RC epitopes were also detected at high density on a small proportion (0.7 to 4.1%) of thymocytes. Both CD45RB and RC epitopes were found predominantly on CD4-CD8- and CD4-CD8+ thymocytes but were also found on small numbers of CD4+CD8+ and CD4+CD8- cells. The population of thymocytes that expressed CD45RB and CD45RC determinants displayed a novel TCR CD3 phenotype characterized by a level of expression that was intermediate between that seen in the larger CD3 bright and CD3 dull populations of thymocytes.


Stimulation of human T lymphocytes by Leishmania lipophosphoglycan-associated proteins. Lipophosphoglycan (LPG) is a glycoconjugate present on the surface of Leishmania promastigotes that has been reported to promote intracellular survival of these parasites, to protect mice against leishmaniasis, and to elicit T cell responses in infected mice and humans. We investigated whether LPG and its components could elicit proliferative responses and cytokine secretion from leishmaniasis patient PBMC. LPG prepared by standard methods (LPG-1) stimulated patients T cells to proliferate and secrete IFN-gamma. LPG was fractionated into several components. An LPG-1-specific T cell line was shown to respond to the core region but not to the repeating saccharide units. LPG-1 was fractionated to yield an LPG-free- associated protein complex and an LPG-2 fraction that was more than 95% depleted of associated protein. The ability of LPG-2 to stimulate T cells was significantly decreased over that of LPG-1. In contrast, LPG-AP stimulated T cell proliferation and IFN-gamma production. Therefore, proteins associated with LPG were effective in eliciting patient T cell responses, whereas the glycolipid enriched moiety was weakly effective or ineffective at stimulating these responses.


Epitopes on the outer surface protein A of Borrelia burgdorferi recognized by antibodies and T cells of patients with Lyme disease. We have characterized immunogenic epitopes of the 31-kDa outer surface protein A (OspA) protein of Borrelia burgdorferi, which is a major surface Ag of the spirochete causing Lyme disease. Full length and truncated forms of rOspA proteins were expressed in Escherichia coli, and their reactivities with antibodies and human T cell clones isolated from patients with Lyme disease were determined. The epitopes recognized by three of four OspA-reactive T cell clones are contained within the 60 COOH-terminal amino acids. Each of the four OspA-reactive T cell clones has a different HLA class II molecule involved in Ag recognition and recognizes a distinct epitope. One T cell clone promiscuously recognized an epitope in the context of different HLA-DQ molecules. In addition, the binding of a murine monoclonal anti-OspA antibody, as well as antibodies in sera of three of five patients with Lyme disease, was dependent upon the amino acids in the carboxy-terminal protion of this protein. Taken together, our results indicate that the 60 COOH-terminal amino acids of OspA contain epitopes recognized by human antibodies and T cells.


Molecular cloning of a cDNA encoding CD34, a sialomucin of human hematopoietic stem cells. CD34 is a 115-kDa transmembrane glycoprotein of unknown function that is expressed on human hematopoietic progenitor cells and the small vessel endothelium of a variety of tissues. We have isolated a CD34 cDNA clone from a KG-1 cell library following three rounds of transient expression in COS cells and enrichment by panning with the anti-CD34 mAb MY10 and BI-3C5. The 5' and 3' ends of the full-length cDNA were subsequently amplified by polymerase chain reaction from KG-1 RNA; the final cDNA clone contained 2615 bp and ended in a poly(A) tail. COS cells transfected with the cDNA clone expressed a surface protein of approximately 110 kDa that was immunoprecipitated by MY10. Southern blot analysis suggested that CD34 is a single copy gene. A 2.7-kb CD34 transcript was observed in the hematopoietic cell lines KG-1, KMT-2, AML-1, RPMI 8402, and MOLT 13 and the endothelial cells BAE and EAhy926, but not in monocytes, resting T cells, or the cell lines Laz 509, HL-60, U937, K562, and HeLa. The cDNA sequence predicts a 40-kDa type I integral membrane protein with nine potential N-linked and numerous potential O-linked glycosylation sites in its extracellular domain. There are two consensus protein kinase C phosphorylation sites and one potential tyrosine kinase phosphorylation site in the cytoplasmic portion of CD34. CD34 has no significant sequence homology to any known protein but has some structural similarities to the heavily glycosylated leukocyte surface molecule CD43.


Neutrophil migration across monolayers of resting or cytokine-activated endothelial cells. Role of intracellular calcium changes and fusion of specific granules with the plasma membrane. Chemoattractants, used at concentrations to invoke optimal neutrophil chemotaxis, induce rapid changes in neutrophils such as a transient increase in intracellular Ca2+ ([Ca2+]i). We have previously observed that neutrophils adhering to cytokine-activated endothelial cells (EC) also respond with a rapid rise in [Ca2+]i caused by an endothelial membrane-bound form of platelet-activating factor. After preloading with the intracellular Ca(2+)-chelator bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM), neutrophils were no longer able to respond with a rapid rise in [Ca2+]i toward the chemoattractant FMLP or to rIL-1 beta-pretreated EC. These neutrophils were still able to adhere and migrate under the conditions tested. The only difference was that the BAPTA/AM-treated neutrophils migrated a little slower than untreated control neutrophils. This discrepancy was not observed at later time points. The BAPTA/AM-preloaded neutrophils did not differ from unloaded neutrophils in actin polymerization responses. Whereas untreated neutrophils demonstrated an up-regulation of the specific granule markers CD11b, CD45, and CD67 during migration (without any release from the azurophil granules), the BAPTA/AM pretreatment completely prevented this process. The BAPTA/AM-preloaded neutrophils did not release vitamin B12-binding protein from the specific granules upon treatment with FMLP. The down-modulation of the selectin member LAM-1, as seen upon neutrophil activation, was not affected by BAPTA/AM pretreatment of the neutrophils. Thus, neither the rapid rise in [Ca2+]i nor specific granule fusion with the plasma membrane constitute a prerequisite for neutrophil migration across resting or cytokine-activated EC.


Human dermal microvascular endothelial but not human umbilical vein endothelial cells express CD36 in vivo and in vitro. CD36 is an 88-kDa glycoprotein that has been identified on platelets, monocytes, and some endothelial cells. Experimental evidence suggests that CD36 mediates the binding of Plasmodium falciparum-infected RBC to a variety of cells, and therefore may play a role in the vascular complications associated with malaria. Additionally, CD36 may also bind the extracellular matrix proteins thrombospondin and collagen. Human umbilical vein endothelial cells have been used in in vitro models examining the binding of P. falciparum RBC to endothelial cells, but they do not consistently express cell surface CD36. Inasmuch as human dermal microvascular endothelial cells (HDMEC) differ in a variety of ways from large vessel endothelial cells, we have examined HDMEC for cell surface expression of CD36 in vivo and in vitro. Direct immunofluorescence of skin showed bright staining of HDMEC with antibody recognizing CD36 and flow cytometric analysis of cultured HDMEC revealed cell surface expression. In contrast, large vessel endothelial cells were not stained with antibody recognizing CD36 in vivo and cultured cells derived from umbilical vein failed to express cell surface CD36 in vitro. Western immunoblots of lysates of HDMEC but not human umbilical vein endothelial cells demonstrated an 88-kDa protein that comigrated with CD36 from platelets. Functional studies demonstrated that adherence of PRBC to HDMEC was inhibited up to 66% by mAb recognizing CD36. Furthermore, the expression of CD36 on HDMEC was increased in a dose- and time-dependent manner by IFN-gamma, and was decreased by protein kinase C agonists. These data demonstrate that HDMEC express functionally active CD36 and this expression can be positively and negatively regulated by soluble factors. This study demonstrates that HDMEC are useful in the study of CD36-mediated binding of PRBC to endothelial cells in vitro and provides further evidence of distinct phenotypic differences between HDMEC and large vessel endothelial cells.


Ordered appearance of zidovudine resistance mutations during treatment of 18 human immunodeficiency virus-positive subjects. The order of appearance in the reverse transcriptase gene of four mutations implicated in the development of resistance to zidovudine was investigated by selective polymerase chain reaction. Serial human immunodeficiency virus isolates were studied from 18 initially asymptomatic individuals who had been treated with zidovudine for 2 years. Most subjects had similar patterns. The first mutation occurred transiently at codon 70; its disappearance was paralleled by the appearance of a mutation at codon 215. Subsequently, in some individuals, the mutation at codon 70 reappeared. During the 2 years of treatment, no mutations developed at codon 219 and only one at codon 67, suggesting that most individuals developed only partly resistant virus. This was confirmed by plaque-reduction assay. Six subjects progressed to AIDS within the 2-year study period, confirming that the development of highly resistant isolates is not required for progression in treated individuals. No clear temporal relationship was found between the development of partial resistance and progression.


A comparison of the variable antigens expressed by clone IV-1 and subgroup III of Neisseria meningitidis serogroup A. Serogroup A Neisseria meningitidis of subgroup III has caused two pandemics of meningococcal meningitis since 1966 and recently spread to East Africa. The last epidemics in West Africa in the early 1980s were caused by clone IV-1. Surface antigens of clone IV-1 strains from West Africa and subgroup III strains from both pandemic waves were analyzed. Lipopolysaccharide was stable within clone IV-1 but variable in subgroup III. Pili from clone IV-1 possessed class I epitopes, while those from subgroup III also possessed class IIa epitopes. Certain class 5 protein variants were expressed by both bacterial clones, possibly reflecting either inheritance of primeval genes or horizontal transmission. Exposure of Gambians to clone IV-1 bacteria stimulated production of bactericidal antibodies cross-reactive with subgroup III bacteria in some individuals but of type-specific antibodies in others. Gambians without bactericidal antibodies usually became healthy carriers rather than developing meningococcal disease on exposure to virulent meningococci.


Chemotherapy for nonmetastatic osteogenic sarcoma: the Memorial Sloan-Kettering experience. Adjuvant chemotherapy improves disease-free survival (DFS) for patients with osteogenic sarcoma (OS). We reviewed our experience with OS to determine prognostic factors, the role of preoperative chemotherapy and subsequent histologic response, and the role of salvage chemotherapy after poor initial response. From 1975 to 1984, we saw 279 patients with previously untreated OS without metastasis. All patients received intensive chemotherapy and underwent surgical resection of primary tumor. Chemotherapy included high-dose methotrexate; Adriamycin (doxorubicin; Adria Laboratories, Columbus, OH); and bleomycin, cyclophosphamide, and dactinomycin (BCD). Selected patients also received cisplatin. DFS was not affected by use of preoperative chemotherapy versus immediate surgery, by use of limb-sparing surgery versus amputation, age, sex, or dose intensity of chemotherapy. DFS did correlate with serum lactate dehydrogenase (LDH), alkaline phosphatase, primary tumor site, race, and histologic response to preoperative chemotherapy. There was no difference in DFS for patients with a poor histologic response who did or did not receive cisplatin, although patients who did receive cisplatin had a longer time to relapse. The 5-year DFS was 76% for patients aged less than or equal to 21 years who had extremity primary tumor and were treated with the T10 protocol. Intensive chemotherapy can achieve DFS for a high proportion of patients with OS. Although it is a powerful predictor of DFS, histologic response to preoperative chemotherapy cannot be assessed at diagnosis. We have not shown an ability to salvage patients with an unfavorable response. We need to increase the proportion of patients with a favorable response, identify the patients who will have an unfavorable response, and develop novel treatments to salvage poor responders.


Effects of free and bound insulin-like growth factors on proteoglycan metabolism in articular cartilage explants. This article describes the effects of bound forms of insulin-like growth factors (IGFs) on proteoglycan metabolism by bovine articular cartilage in explant culture. When these growth factors were added to articular cartilage explants complexed with their native serum binding proteins (BPs), both IGF-I-BP complex and IGF-II-BP complex stimulated proteoglycan synthesis to different degrees over a 3-day period. When added to the medium of cultures of articular cartilage over 5 days, IGF-II-BP complex induced high rates of synthesis and low rates of catabolism of proteoglycans, giving rise to tissue levels of proteoglycan similar to those observed in fresh tissue. When articular cartilage was maintained in culture with the same concentration of IGF-I-BP complex, tissue levels of proteoglycans fell over the culture period because of lower rates of proteoglycan synthesis. Analysis of the proteoglycans synthesized by articular cartilage in the presence of free or bound IGF-I or IGF-II showed that these growth factors stimulated the rate of synthesis of the large proteoglycan species present in cartilage but did not affect the synthesis of the small proteoglycans.


S-100 protein immunostaining identifies cells expressing a chondrocytic phenotype during articular cartilage repair. The healing of articular surface defects has been studied with conventional histology, which relies on the staining of the extracellular matrix to identify the phenotype of the cells present. A chondrospecific cellular marker would be useful. S-100 protein has been found in all chondroid tissues studied, and we evaluated its usefulness in the study of articular cartilage repair. Full-thickness rabbit femoral condylar defects were made, and the specimens were studied at serial time intervals. S-100 protein staining positively showed chondroid cells in the 7- and 14-day specimens, which were not identifiable by conventional techniques. At 30 and 60 days, an S-100 positive band of cells separated a deep safranin-O positive hypertrophic layer from a fibrocellular surface layer. At 120 days, the presence of S-100 protein identified cells with chondrogenic potential, and the lack of S-100 protein in other cells embedded in conventionally stained matrix suggested that these cells were no longer of a chondroid phenotype. The presence of S-100 protein-identified chondroid cells early in the repair process when the cells had not begun to synthesize conventionally stainable matrix and the lack of S-100 protein in cells late in the repair positively identified a phenotypic change earlier than conventional histology.


Rabbit knee immobilization: bone remodeling precedes cartilage degradation. This study analyzed processes underlying osteoporosis and osteoarthrosis after short-term immobilization of the right hind limb of postadolescent (2.8 kg) and mature (4.0 kg) rabbits. After 3 weeks, the lateral posterior aspect of the lateral tibial plateau and the lateral femoral condyle of the immobilized limb exhibited prominent subchondral vascular eruptions. Femoral metaphyseal bone density decreased 27 and 18% in the immobilized limbs of postadolescent and mature rabbits, respectively. Calcein green fluorescence increased 1.9-fold (p less than 0.001) in the metaphyseal trabeculae of immobilized femurs. With immobilization, sulfate incorporation into femoral cartilage glycosaminoglycan increased, although total cartilage glycosaminoglycan and hydroxyproline levels were unchanged. Thymidine incorporation into DNA increased four- to fivefold in tibial and femoral cartilage of the immobilized limb. In this study, bone loss and remodeling preceded erosive cartilage degradation.


The Denver II: a major revision and restandardization of the Denver Developmental Screening Test. Since the Denver Developmental Screening Test was first published 23 years ago, it has been utilized worldwide and restandardized in more than a dozen countries. Concerns raised through the years by test users about specific items and features of the Denver Developmental Screening Test, coupled with a need for more current norms, have prompted a major revision and restandardization of the test. For the revision, 336 potential items were administered to more than 2000 children. The average number of times each item was administered was 540. Using regression analysis, composite norms for the total sample and norms for subgroups (based on gender, ethnicity, maternal education, and place of residence), were used to determine new age norms. The final selection of the 125 Denver II items was based on the following criteria: ease of administration and scoring, item appeal to child and examiner, item test-retest and inter-rater reliability, minimal "refusal" scores, minimal "no opportunity" scores, minimal subgroup differences, and a smooth step-like progression of ages at which 90% of children could perform the tasks. The major differences between the Denver II and the Denver Developmental Screening Test are: 1) an 86% increase in language items; 2) two articulation items; 3) a new age scale; 4) a new category of item interpretation to identify milder delays; 6) a behavior rating scale; and 7) new training materials.


Participation by pediatricians in early intervention: impetus from Public Law 99-457. Part H of the Individuals with Disabilities Education Act (originally enacted as Public Law 99-457) requires that participating states phase in a system of early intervention services by 1993. By recognizing the importance of good health in the development of infants and toddlers, Congress acknowledged the key role of medical care providers in a comprehensive program for young children with or at risk for developmental delay or dysfunction. National and state surveys of pediatricians suggest limited but growing awareness of this legislation and uncertainty about how they might participate effectively. A chief concern relates to mechanisms of payment for developmental screening and assessment as well as time-demands for participation in interdisciplinary team activities. The American Academy of Pediatrics and its state chapters are responding to requests for information with educational seminars and print materials. Pediatricians can enhance the quality of community support services for children with special needs by participating in planning efforts and by coordinating health care with other aspects of early intervention. Other professionals and parents are looking to pediatricians for leadership and willing participation in the implementation of PL 99-457.


Differential effects of dichlorodiphenyltrichloroethane analogs, chlordecone, and 2,3,7,8-tetrachlorodibenzo-p-dioxin on establishment of pregnancy in the hypophysectomized rat. Many of the organochlorine pesticides have been shown to elicit estrogenic responses in laboratory animals. Two estrogenic actions, initiation of implantation and maintenance of pregnancy, were examined in progesterone-primed, delayed-implanting, hypophysectomized rats exposed to several polychlorinated hydrocarbons. The insecticide P,P'-dichlorodiphenyltrichloroethane (DDT) was nearly devoid of estrogenic activity for initiating implantation, as was a dichloro analog, 1,1-dichloro-2-[p-chlorophenyl],2-[o-chlorophenyl]ethane (O,P'-DDD), but another such analog, 1,1-dichloro-2-(p-chlorophenyl),2-(o-chlorophenyl)ethylene (O,P'-DDE), was nearly as estrogenic as the O,P'-DDT isomer of DDT and the methoxylated analog methoxychlor. The latter three compounds not only initiated implantation, but maintained pregnancy when given in large (200 mg/kg) and repeated doses. Another insecticide, chlordecone (Kepone) was more estrogenic than any of the DDT analogs and maintained pregnancy with a single dose of 50 mg/kg. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a toxic contaminant of herbicide production, did not induce implantation at a dose of 125 micrograms/kg, but inhibited the implantation initiated by estrone in 35% of the animals. The mechanism of this antiestrogenicity is unknown but most probably does not involve direct action via the classical estrogen receptor. The possible interference with the normal blastocyst-uterine interactions of these polychlorinated xenobiotics may be an important factor in their being considered reproductive toxins.


Effectiveness of India ink as a long-term colonic mucosal marker. We prospectively studied the use of India ink as a long-term or "permanent" mucosal marker as part of a study investigating the natural history of diminutive distal colorectal polyps. Twenty-six patients had 32 India ink tatoos implanted. The tatoo sites of the 19 patients who were followed at least 6 months continued to display intensely stained mucosa at the original sites. No side effects or complications were encountered. India ink appears to be a safe and effective long-term marker for colonic mucosal lesions.