Enhanced lymphokine production and lymphokine receptor expression in multiple antibody-stimulated human CD4+ peripheral blood lymphocytes. Treatment of T lymphocytes with antibodies directed against the T cell receptor CD3 complex results in cellular activation that can be augmented by costimulation through other cell surface receptors. The activities of anti-CD3-stimulated human CD4+ PBL were compared to anti-CD3 plus anti-CD2-, anti-CD4-, or anti-CD11a (LFA-1)-stimulated cells. [3H]thymidine incorporation, lymphokine receptor expression, expansion of cell numbers, and lymphokine mRNA and protein were measured. Forty-eight hours after activation, costimulated CD4+ cells demonstrated increased numbers of cells positive for surface IL-2R alpha-chain, IL-2R beta-chain, IFN-gamma receptors, and TNF-alpha receptors. By day 6, costimulated cells exhibited a sevenfold greater expansion in cell numbers compared to cells stimulated with anti-CD3 alone. Anti-CD3 plus anti-CD11a stimulation consistently induced the highest secretion of IL-2 and IFN-gamma, whereas variation in secretion of TNF-alpha and IL-4 between different donors was noted. Analysis of lymphokine receptor mRNA demonstrated increased levels of mRNA for IL-2R alpha-chain and IFN-gamma receptor that preceded the phenotypic changes on the cell surface. In contrast, levels of IL-2R beta-chain and the TNF-alpha receptor mRNA decreased after stimulation. Amounts of IL-2, IL-4, and TNF-alpha, but not IFN-gamma, secreted also correlated with the levels of mRNA measured in the cells. Although costimulation through CD2, CD4, or LFA-1 appeared equally effective for the induction of the lymphokine receptors, these additional stimuli had a different impact on lymphokine secretion. These results indicate that specific control of lymphokine secretion and receptor induction can be another function of the CD2, CD4, and CD11a cell surface receptors. This control is evident at both transcriptional and post-transcriptional levels.


Intercellular adhesion molecule-1 (ICAM-1)-dependent and ICAM-1-independent adhesive interactions between polymorphonuclear leukocytes and human airway epithelial cells infected with parainfluenza virus type 2. Acute respiratory virus infections are often associated with an early influx of neutrophils (PMN) into the airways. Maximal cytoxic injury by PMN depends on tight cell-cell adhesion. Infection of some cell types by respiratory and other viruses has been shown to increase PMN adhesion to these cells by undefined mechanisms. We studied adhesion by human PMN to monolayers of primary (1 degree) human tracheal epithelial cells (TEC) or an immortalized cell line derived from human TEC, 9HTEo-, that had been infected with parainfluenza virus type 2 (PiV2). PMN adhesion to uninfected 1 degree TEC was very low (< 5%), but PMN adhesion to PiV2-infected 1 degree TEC was greatly increased (89 +/- 7%). PMN adhesion to 9HTEo- cells was 47 +/- 6%, but increased, 87 +/- 8%, for PiV2-infected 9HTEo- cells. Surface intercellular adhesion molecule-1 (ICAM-1) expression on 1 degree TEC, as determined by immunofluorescence flow cytometry, was relatively low (23 fluorescence units) but doubled by 24 h after PiV2 infection and tripled by 48 h. The 9HTEo- cells constitutively expressed higher levels of surface ICAM-1 (120 units) which did not increase with PiV2 infection. Treatment of non-PiV2-infected 9HTEo- cells with mAb (R6.5) to ICAM-1 reduced PMN adhesion to these cells from 47 +/- 8 to 23 +/- 5%. Identical mAb treatment of either 1 degree TEC or 9HTEo- cells infected with PiV2 had no significant effect on PMN adhesion. Treatment of the PMN with mAb against CD11a, CD11b, or CD18 markedly reduced PMN adhesion to PiV2-infected 1 degree TEC and 9HTEo- cells. We conclude that PiV2 infection of human TEC causes a marked increase in their adhesive interactions with PMN by inducing increased surface expression of both ICAM-1 and one or more, as yet uncharacterized, non-ICAM-1 adhesion molecules that function as counter-receptors for CD11/CD18 on PMN. These mechanisms of adhesion may play a role in epithelial damage during acute respiratory virus infections.


Inhibition of lymphocyte endothelial adhesion and in vivo lymphocyte migration to cutaneous inflammation by TA-3, a new monoclonal antibody to rat LFA-1. Lymphocyte function-associated Ag-1 (LFA-1) or CD11a/CD18 mediates lymphocyte adhesion to cultured vascular endothelial cells (EC). Thus, LFA-1 likely plays a major role in lymphocyte migration out of the blood, but there is little information on this in vivo. Small peritoneal exudate lymphocytes (sPEL) and lymph node (LN) lymphoblasts adhere to cytokine-activated EC and preferentially migrate to cutaneous inflammatory sites. The role of LFA-1 in the adherence and in vivo migration of these T cells was determined. Because of a lack of anti-rat LFA-1, mAb were prepared to rat T cells. One mAb, TA-3, inhibited homotypic aggregation; T cell proliferation to Ag, alloantigens, and mitogens; stained all leukocytes; and immunoprecipitated 170- and 95-kDa polypeptides from lymphocytes and neutrophils. TA-3 binding to lymphocytes also required Ca2+, but not Mg2+. Thus, TA-3 appears to react with rat LFA-1. TA-3 inhibited spleen T cell adhesion to unstimulated EC by 30% and to IFN-gamma, TNF-alpha, IL-1 alpha, and LPS stimulated EC by 50 to 60% but inhibited sPEL EC adhesion by only 10%. TA-3 also strongly inhibited anti-CD3-stimulated LN T cell adherence. The migration of spleen T cells to delayed-type hypersensitivity and skin sites injected with LPS, poly I:C, IFN-gamma, IFN-alpha/beta, and TNF was inhibited by 72 to 88% by TA-3, and was decreased by 50% to peripheral LN. TA-3 caused less but still 50 to 60% inhibition of sPEL migration to inflamed skin. Lymphoblast migration to skin was inhibited 40 to 80% and to PLN by 30%. Migration of lymphocytes from all sources to mesenteric LN was inhibited by 32 to 60%. In conclusion, LFA-1 mediates much of the adherence of spleen T cells and lymphoblasts to EC in vitro, most of the migration of these cells to dermal inflammation and about 50% of the homing of LN and spleen T cells to peripheral and mesenteric LN. sPEL are less dependent on LFA-1 for adhesion to EC in vitro and for migration to inflamed skin and LN in vivo.


IL-4 synthesis by in vivo primed keyhole limpet hemocyanin-specific CD4+ T cells. I. Influence of antigen concentration and antigen-presenting cell type. The development of IL-4 synthesis is a critical step in the regulation of immune responses. Our studies focused on the production of IL-4 by CD4+ T cells taken from mice primed with the Ag keyhole limpet hemocyanin (KLH). In vitro stimulation of such CD4+ T cells with KLH resulted in little or no IL-4 production in the first 24 h of stimulation, indicating that little IL-4 synthesis persists in vivo after immunization. However, IL-4 was generated later at 24 to 96 h of in vitro stimulation, indicating that the potential to produce IL-4 was retained by the KLH-primed CD4+ T cells, but that in vitro maturation of the T cells was required before initiation of IL-4 production. The amount of IL-4 produced in vitro by KLH-primed T cells from BALB/c mice was influenced by several factors. First, stimulation of KLH-primed CD4+ T cells with higher in vitro concentrations of KLH resulted in more IL-4 synthesis, but this was accompanied by more IFN-gamma as well. Second, primed CD4+ T cells from lymph nodes (axillary and popliteal) produced significantly more IL-4 than primed splenic T cells. Third, when primed B cells were utilized to present low concentrations of KLH to the T cells, IL-4 but not IFN-gamma was produced. In contrast, use of splenic adherent cells resulted in IFN-gamma but not IL-4 synthesis. These restricted patterns of lymphokine synthesis, however, were observed only with low concentrations of KLH. Fourth, the amount of IL-4 produced and its regulation by the presence of IFN-gamma differed among mouse strains, in that BALB/c T cells produced much more IL-4 than H-2 identical DBA/2 T cells. Our results characterizing the APC and Ag dose requirements for IL-4 synthesis in KLH-primed T cells from different strains of mice are consistent with previous observations that distinct strains of mice differ in the type of immune response generated against different pathogens, and with the concept that low Ag concentrations preferentially result in high levels of IgE synthesis, which is absolutely dependent on IL-4 production.


Natural killer (NK) cell stimulatory factor or IL-12 has differential effects on the proliferation of TCR-alpha beta+, TCR-gamma delta+ T lymphocytes, and NK cells. We have analyzed the effects of NK cell stimulatory factor/IL-12, on proliferation of PBL and their subsets. IL-12 synergizes with lectins and phorbol diesters to induce proliferation of CD4+ and CD8+ peripheral blood T lymphocytes. In the case of phorbol-diester-induced proliferation, the effect of IL-12 is in part mediated by induced IL-2 production, as suggested by the observation that IL-12 enhances IL-2 production in these cultures and that anti-IL-2 antibodies inhibit proliferation. IL-12 synergizes also with anti-CD3 antibodies and with allogeneic stimulation in MLC in inducing T cell proliferation. IL-12 alone is mitogenic for preactivated T and NK lymphoblasts. This mitogenic effect is observed with similar doses of IL-12 on NK lymphoblasts as well as on CD4+ and CD8+ TCR-alpha beta+ and on TCR-gamma delta+ lymphoblasts. On TCR-alpha beta+ T lymphocytes the effect of IL-12 is always additive to that of IL-2 over a wide dose range. The same effect is observed on highly activated, actively proliferating NK cells. However, on NK and TCR-gamma delta+ lymphoblasts reverting to a resting state after stimulation and on a TCR-gamma delta+ acute leukemia-derived T cell line, IL-12 inhibits significantly the proliferation induced by moderate to high doses (10 to 100 U/ml) of IL-2. This inhibitory effect is, at least in part, indirect, and depends on IL-12-induced production of TNF. Neutralizing anti-TNF antibodies, but not anti-IFN-gamma and anti-transforming growth factor antibodies, restore by more than 70% the inhibition of proliferation induced by IL-12 in these cultures. However, TNF alone cannot mimic the inhibitory effect of IL-12 on the IL-2-induced proliferation of NK and TCR-gamma delta+ lymphoblasts, suggesting the involvement of additional mechanisms. The relevance of these findings for the biology of lymphocyte subsets mediating MHC nonrestricted cytotoxicity is discussed.


Granuloma T lymphocytes in murine schistosomiasis mansoni have somatostatin receptors and respond to somatostatin with decreased IFN-gamma secretion. The granulomas of mice infected with Schistosoma mansoni for 8 wk have macrophages that secrete somatostatin 1-14 (SOM). Within the granuloma, SOM has no known function. To uncover the possible significance of SOM produced within this granulomatous inflammation, we sought SOM receptors on distinct cellular components of the granuloma to identify cells targeted for SOM action. [125I]SOM 1-14 bound to dispersed granuloma inflammatory cells specifically and reversibly. Scatchard analysis suggested one receptor type (kDa 4.28 x 10(-9) M). Octreotide, a stable SOM derivative, displaced radioligand (kDa 1.01 x 10(-10) M), but SOM 1-28, substance P, and vasoactive intestinal peptide did not. The SOM receptor localized exclusively to a subset of granuloma CD4+ T lymphocytes. Using IL-5 and IFN-gamma ELISA, it was shown that granuloma T cells can secrete appreciable IL-5 and IFN-gamma when stimulated with Ag or mitogen. Both SOM and octreotide at concentrations as low as 10(-10) M substantially decreased IFN-gamma secretion from Ag or mitogen-stimulated T cells, but at concentrations as high as 10(-6) did not affect IL-5 production. Octreotide administered to animals in vivo decreased the intensity of the granulomatous response. Thus, some granuloma T cells have SOM 1-14 receptors. SOM 1-14, a product of granuloma macrophages, may participate in regulation of the granulomatous response by modulating the secretion of some lymphokines. Octreotide, a clinically useful SOM analog, mimics the action of SOM on the immune system.


Role of neutrophils and CD4+ T lymphocytes in the primary and memory response to nonimmunogenic murine mammary adenocarcinoma made immunogenic by IL-2 gene. TS/A is a spontaneous adenocarcinoma, apparently not immunogenic in BALB/cnAnCr mice. TS/A cells are unable to stimulate a syngeneic antitumor response either in vitro or in vivo. To evaluate the immunogenic potential of IL-2-releasing neoplastic cells, we used an expression vector to introduce the cDNA coding for murine IL-2 into TS/A cells. Six clones releasing between 30 and 6800 U of IL-2/10(5) cells/ml/48 h have been isolated. Both low (30 U, B1.30) and high (6000 U, B4.6000) IL-2-releasing clone are capable of stimulating a proliferative and cytotoxic response in syngeneic cultures. While the B1.30 clone grows in 60% of syngeneic mice with a delayed pattern, the five clones that release higher levels of IL-2 are promptly rejected. Rejection is associated with neutrophil infiltration, the intensity of which is directly proportional to the amount of IL-2 released. NK cells and CD4+ lymphocytes are uninfluential, whereas CD8+ lymphocytes play only a minor role. This neutrophil-dominated rejection leaves a long-lasting, tumor-specific, T lymphocyte-mediated immune memory. For its induction, CD4+ lymphocytes are required. Their specific activation appears to depend on both the amount of IL-2 released and the granulocyte-mediated reaction that may lead to a more efficient presentation of tumor-associated Ag. These data support the notion that, after transduction of IL-2 gene, cancer cells may elicit an immune antitumor response, and stress the potential use of IL-2 as a component of new tumor vaccines.


Comparison of airway and blood eosinophil function after in vivo antigen challenge. Eosinophils (EOS) are important effector cells in allergic diseases and asthma. However, functional characteristics of the EOS have been derived primarily from studies of blood cells, and it is unlikely that such assessments reflect events occurring in tissues or airways. To establish more precisely the function of airway EOS, segmental Ag challenge was used to elicit and isolate large numbers of these cells. Airway, as well as blood, EOS were isolated from allergic patients 48 h after segmental Ag challenge. Both blood and bronchoalveolar lavage (BAL) EOS were fractionated over Percoll density gradients; by using this protocol, three density-distinct populations of pure (>90%) EOS were obtained from BAL fluid (1.100, 1.095, and 1.090 g/ml) and one from blood (1.100 g/ml). The functions of these various populations were compared by measuring superoxide generation, adherence to collagen and endothelial cell monolayers, cell surface receptors, and in vitro survival. BAL EOS of all three densities had greater superoxide generation and adherence with FMLP activation than did corresponding blood EOS. In contrast, blood and airway EOS responded similarly to PMA. BAL EOS also had increased expression of CD11b/CD18 and HLA-DR. The intracellular calcium concentration ([Ca2+]i) was measured with the fluorescent marker indo-1/acetoxymethyl ester. FMLP caused a greater and more sustained increase in [Ca2+]i with BAL than blood EOS. EGTA blocked the sustained component of the [Ca2+]i response to FMLP. Our findings indicate that BAL EOS have an enhanced [Ca2+]i response to activation that may contribute to their functional up-regulation.


Eradication of hantavirus infection among laboratory rats by application of caesarian section and a foster mother technique. Hantavirus antibodies were demonstrated by the indirect immunofluorescent antibody assay, in the serum of inbred strains of laboratory rats, during the period 1973-1982, at the Unit of Experimental Immunology in the Catholic University of Louvain, Brussels, Belgium. LOU rats, as well as immunocytomas, which were requested by laboratories in the U.K. and The Netherlands, were supplied at a time when the infection was unknown and unsuspected in Europe. Hantavirus-infected laboratory rats were rendered free of virus through re-derivation by caesarian section and suckling by virus-free foster mothers. Immunocytomas were tested for the presence of hantaviruses by implantation into seronegative laboratory rats. The strain of hantavirus causing the laboratory infection was clearly different from the one circulating in free-living bankvoles in Belgium. The exchange of laboratory rats and rat tumours in relation to the potential risk of laboratory-acquired hantavirus infection, is discussed.


Genetic linkage between the collagen VII (COL7A1) gene and the autosomal dominant form of dystrophic epidermolysis bullosa in two Dutch kindreds. Epidermolysis bullosa is a heterogeneous group of heritable blistering skin diseases affecting epidermis and the dermal-epidermal junction zone. Recently, genetic linkage to the type VII collagen gene (Z = 8.77; theta = 0.00) localized on chromosome 3p21 was shown in three Finnish families with the autosomal dominant form of dystrophic epidermolysis bullosa. Two Dutch kindreds with intrafamilial characteristics of both the Cockayne-Touraine type and Bart's syndrome of autosomal dominant dystrophic epidermolysis bullosa have been studied. Two-point linkage analysis in these two families with the COL7A1 marker revealed a combined lod score of Z = 6.08 at theta = 0.00. These data strongly suggest that the type VII collagen gene is the candidate gene in these Dutch pedigrees. At least two (Cockayne-Touraine and Bart) of the three subtypes of dominant dystrophic epidermolysis bullosa seem to represent different forms of expression of the same gene defect.


Human epidermal keratinocyte expression of sialyl-Lewis X. Cell-surface oligosaccharides can function as ligands for intercellular adhesion receptors, matrix proteins, and growth factors. We report that human neonatal and adult epidermal keratinocytes (KC) express sialyl Lewis X [s-Le(x); SA alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3R], a ligand for endothelial and platelet selectins. Freshly isolated or cultured KC bind FH6 monoclonal antibody (MoAb), which is specific for s-Le(x)-containing oligosaccharides. The relevant epitope is bona fide s-Le(x), because sialidase treatment of KC suspensions abrogates FH6 binding while generating de novo KC reactivity with anti-Le(x). KC stained in ice-cold suspension display a knobby membrane distribution of s-Le(x) detectable by immunofluorescence microscopy. As others have reported, FH6 appeared not to bind KC in perpendicular skin sections. However, basal KC in intact epidermal sheets exhibited obvious "honeycomb" reactivity with FH6 when stained and viewed en face, suggesting that s-Le(x) in intact epidermis may occur in bands that parallel the major tissue axis. FH6 specifically immunoprecipitated proteins of Mr 34 kd, 44 kd, and 56 kd from [35S]-labeled KC, and anti-Le(x) precipitated similar proteins from sialidase-treated KC. The enzymatic basis for KC s-Le(x) expression was studied by analyzing acceptor specificities and other properties of KC fucosyltransferases. Results indicate that KC express both Lewis- and myeloid-type alpha 1-3fucosyltransferases. KC s-Le(x) could be an important element of the epithelial milieu, because both epithelial cells and immune cells that home to epithelia express s-Le(x) and related structures, and because KC s-Le(x) is well positioned for selectin-mediated platelet binding after trans-cutaneous wounding. The apparent distributions of s-Le(x) in epidermis and on isolated KC are compatible with a functional role for s-Le(x) in these intercellular interactions.


Co-stimulatory functions of antigen-presenting cells. Antigen-presenting cells, in addition to presenting processed antigen, provide co-stimulatory signals that are necessary for stimulating maximal lymphokine production by CD4+ T cells. For interleukin 2 (IL-2)-producing CD4+ T cells, the B7 molecule provides an important co-stimulatory signal through interaction with its ligand on the T-cell surface, CD28. Populations of antigen-presenting cells that express high levels of B7 (e.g., dendritic cells) are much more potent stimulators of T-cell activation than cells that fail to express B7 (e.g., resting B cells). An increase in B7 expression could therefore explain the increased accessory function gained by Langerhans cells as they leave the skin and migrate to the draining lymph node.


Doses of ultraviolet radiation that modulate accessory cell activity and ICAM-1 expression are ultimately cytotoxic for murine epidermal Langerhans cells. Mechanisms that underlie immunomodulatory properties of ultraviolet (UV) radiation remain incompletely characterized. Recently, we have studied effects of UV on the functional activity of epidermal Langerhans cells (LC) and have attempted to relate inhibitory effects of UV on LC function to modulatory effects of UV on adhesion molecule expression by LC. Exposure of LC in vitro to amounts of UVB, UVC, or psoralen+UVA (PUVA) radiation that inhibited LC function also prevented increased expression of intercellular adhesion molecule-1 by LC in vitro. Subsequent studies revealed that amounts of UV radiation that inhibited LC function and modulated ICAM-1 expression also decreased LC survival in vitro, although UV-induced LC cytotoxicity did not become apparent until 48-72 h after UV exposure. Our results are consistent with those of previous studies that suggested that low doses of UV radiation were cytotoxic for LC in situ. The potential cytotoxicity of UV radiation for LC should be considered when studies of effects of UV radiation on immune responses in skin are interpreted.


Regulation of the skin immune system by retinoids during carcinogenesis. One of the immunosuppressive effects of both ultraviolet (UV) light and chemical carcinogens is to deplete Langerhans cells (LC) from the epidermis, suggesting that these cells play an important role in inducing immune responses to developing tumors during the early phases of carcinogenesis. Retinoids such as all-trans-retinoic acid (RA) are natural or synthetic derivatives of vitamin A; RA binds to nuclear receptors in the skin, effecting transcription of a wide range of genes. Topical application of RA prevents the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) from depleting the density of LC in murine epidermis. In contrast, topical RA did not itself alter the normal LC density. RA also inhibited the development of TPA-induced immunosuppression to a locally applied contact sensitizer. Topical RA also prevented UV light from reducing the density of both LC and Thy-1+ dendritic epidermal cells (Thy-1+ dEC). However, the RA treatment did not prevent local immunosuppression to the contact sensitizer from developing in response to UV irradiation. The reasons for this are unclear, however, it is possible that RA does not inhibit some other immunosuppressive effect of UV light. Temarotene, a recently developed synthetic retinoid also inhibited UV light from reducing the LC and Thy-1+ dEC density from murine epidermis. Thus part of the anti-carcinogenic activity of retinoids may be due to their ability to protect LC during the early stages of carcinogenesis.


Epidermal compromise in American cutaneous leishmaniasis. In American cutaneous leishmaniasis (ACL), Leishmania parasites enter the epidermis of the host via the bite of infected sandflies. Immune responses against the parasite vary from "effective" in localized (LCL) to a state of "selective anergy" in diffuse (DCL) cutaneous leishmaniasis, whereas the intermediate muco-cutaneous form (MCL) is characterized by an exacerbated cell-mediated immunity. We have shown that in LCL epidermis, Langerhans cells (LC) are increased, HLA-DR is universally expressed and intercellular adhesion molecule-1 (ICAM-1) immunoreactivity is distributed in patches. In addition, mRNA for IL-1 beta, IL-8, TNF alpha, TNF beta, and INF gamma may be detected in epidermal sheets by reverse transcriptase followed by polymerase chain reaction (RT-PCR). In contrast, DCL epidermis shows fewer LC than LCL epidermis, and expression of ICAM-1, HLA-DR, and IL-1 beta mRNA cannot be detected. MCL lesions show a mucosal epithelium lacking LC, but ICAM-1 is universally expressed. The clinical manifestations of ACL can be reproduced experimentally in different strains of inbred mice. In healthy mice, we have shown a positive correlation between LC and dendritic epidermal T cells (DETC) numbers. This correlation was not, however, observed in L. mexicana-infected mice, suggesting that infection alters the balance between the two cell types. In addition, agents that modulate LC and DETC cell densities change the development of experimental leishmaniasis. These results suggest that the epidermis is essential in determining the type of immune response that is developed against the Leishmania parasites.


Association of antibody to human immunodeficiency virus type 1 core protein (p24), CD4+ lymphocyte number, and AIDS-free time. Serum antibody to p24 (anti-p24) and p24 antigen, alone and in combination with CD4+ lymphocyte number, were evaluated as predictors of progression of human immunodeficiency virus type 1 (HIV-1) infection. Two hundred six HIV-1-prevalent seropositive men in the Multi-center AIDS Cohort Study since 1984-1985 were studied cross-sectionally and 84 seroconverters were evaluated longitudinally. Cross-sectional analyses revealed significant associations among titer of anti-p24, CD4+ cell count, disease status (Centers for Disease Control class), and progression to AIDS. A high titer of anti-p24 was associated with lack of p24 antigenemia. Longitudinal studies of seroconverters demonstrated that a low titer of anti-p24, low CD4+ cell count, and detection of HIV-1 p24 antigen are individually strong predictors of AIDS, but only low CD4+ cell count retains its independent predictive value in multivariate analysis of the three markers during the period immediately after infection with HIV-1.


Incidence and natural history of cytomegalovirus disease in patients with advanced human immunodeficiency virus disease treated with zidovudine. The Zidovudine Epidemiology Study Group. Data were analyzed from a multicenter observational cohort study of 1002 persons with AIDS or AIDS-related complex (ARC) and total CD4 cell count < 0.25 x 10(9)/L treated with zidovudine between April 1987 and April 1988. Cytomegalovirus (CMV) disease developed in 109 patients (10.9%), with a 2-year actuarial risk of 15%. Manifestations included retinitis (93 patients), esophagitis (10), colitis (8), gastritis (1), hepatitis (1), and encephalitis (1). The probability of CMV disease at 2 years for patients with initial counts < 0.1 x 10(9)/L was 21.4%, compared with 10.3% for patients with initial counts > or = 0.1 x 10(9)/L (P < .001). By proportional hazards analysis, baseline CD4 cell count < 0.1 x 10(9)/L, enrollment diagnosis of AIDS, and homosexuality were significantly associated with subsequently developing CMV disease. Median survival after diagnosis of CMV disease was 173 days, and CMV was an independent predictor of death. CMV contributes to AIDS-related morbidity and mortality. As new anti-CMV drugs become available, prophylaxis should be targeted at individuals with CD4 cell counts < 0.1 x 10(9)/L.


Localized, aggregative, and diffuse adherence to HeLa cells, plastic, and human small intestines by Escherichia coli isolated from patients with diarrhea. Adherence of diarrhea-associated Escherichia coli was studied by scanning electron microscopy. Enteropathogenic E. coli (EPEC) adherence factor-positive (EAF+) E. coli of EPEC serotypes (class I EPEC) adhered to plastic and human jejunal and ileal mucosa, similar to case and HeLa cells. Localized adherence, elongation of cell microvilli, and "locking" of the bacterial aggregates by the elongated microvilli were evident after incubation for 20 min. EAF+ E. coli adhered strikingly to mucus but rarely to M cells in Peyer's patch-associated epithelium. Most enteroaggregative E. coli (EAggEC) strains adhered to plastic, similar to HeLa cells. Some diffuse-adhering E. coli (DAEC) strains displayed no adherence to plastic but formed "dimples" on HeLa cells. Both EAggEC and DAEC adhered at lower levels to human small intestines (except M cells) than did EAF+ E. coli. In all cases of EAF+ E. coli, EAggEC, and DAEC, strains were found with atypical characteristics. The data demonstrate the unique adherence characteristics of EAF+ E. coli, EAggEC, and DAEC.


Course of primary candidiasis in T cell-depleted mice infected with attenuated variant cells. Anti-CD4, anti-CD8, or anti-interferon-gamma (IFN-gamma) antibodies or combinations of them were administered in the early stages of chronic infection of mice with a Candida albicans live vaccine strain, and the animals were monitored for course of primary infection, development of delayed-type hypersensitivity, resistance to reinfection, production of interleukin 2 (IL-2) and IFN-gamma in vitro by splenic lymphocytes, and levels of IL-2 and IFN-gamma transcripts in these cells. CD4+ cell and IFN-gamma depletion resulted in the development of fatal candidiasis by the attenuated yeast vaccine. In contrast, either treatment alone modified the course but not the outcome of primary infection, though each prevented the development of resistance to reinfection. Our data thus indicate that both IFN-gamma and CD4+ cells participate in resistance to primary infection with attenuated yeast cells and are critical in the induction of persistent systemic anticandidal immunity.


Construction and expression of an enzymatically active human-mouse chimeric double-stranded RNA-dependent protein kinase. The interferon (IFN)-inducible double-stranded (ds) RNA-activated protein kinase (p68 kinase) is a physiologically important enzyme that regulates the rate of cellular and viral protein synthesis by phosphorylating and thereby inactivating the peptide chain initiation factor 2. We have generated a partial cDNA clone, which probably represents the murine p68 kinase, by reverse transcription-polymerase chain reaction (RT-PCR) using sequence information of the human p68 kinase. The 725-bp cDNA clone encoded the carboxyl-terminal 238 amino acid residues of the mouse kinase. It has 67% overall identity with the corresponding region of the human kinase. All the protein kinase catalytic domains are conserved in the mouse protein. Moreover, there are additional stretches of residues that are totally conserved between the two proteins. The functional equivalence of the two proteins was tested by constructing a chimeric cDNA that encoded a protein whose amino-terminal 364 residues were of human origin and carboxyl-terminal 187 residues were of mouse origin. The chimeric protein was as efficient as the human p68 kinase in binding to the dsRNA, autophosphorylating and phosphorylating exogenous substrate.


Macrophage cytoskeleton association with CR3 and CR4 regulates receptor mobility and phagocytosis of iC3b-opsonized erythrocytes. Alveolar macrophages (AM phi) were examined for CR1 (C3b receptor, CD35), CR3 (iC3b receptor; CD11b/CD18), and CR4 (iC3b receptor; CD11c/CD18) by assays for binding of C3-opsonized sheep erythrocytes (EC3b or EC3bi) and uptake of specific monoclonal antibodies (mAbs). In AM phi isolates from nine normal volunteers, 49% of cells bound EC3b and 71% bound EC3bi. Quantitation of receptors per cell with [125I]mAbs showed 8.5 x 10(4) CR4, 5.1 x 10(4) CR3, and 2.6 x 10(4) CR1. With most AM phi preparations, CR3 was the major receptor mediating attachment of EC3bi, despite the predominance of CR4 antigens. Anti-CR3 inhibited EC3bi rosettes by > or = 50%, whereas anti-CR4 blocked rosettes by < or = 18%. U937 cells differentiated with phorbol myristate acetate resembled AM phi in receptor expression but exhibited almost no CR4-dependent rosetting. Despite the relative inability of CR4 to mediate EC3bi attachment, AM phi ingestion of [51Cr]EC3bi was blocked by either anti-CR3 or anti-CR4. Two lines of evidence indicated that CR3 were more mobile within the membrane than were CR4. Immunofluorescence staining demonstrated patching and occasional capping of CR3, whereas CR4 remained uniformly distributed. This patching and capping of CR3 required the actin cytoskeleton, as it was inhibited by cytochalasin D. Modulation experiments using surfaces coated with anti-CR3 or anti-CR4 also showed that CR3 was more mobile than was CR4. However, there was some variation among AM phi isolates from different donors. In seven isolates, no CR4 modulation was produced with anti-CR4, whereas in six other isolates, CR4 was modulated by 66%. Incubation of cells in cytochalasin D increased modulation of both CR3 and CR4 on mAb-coated surfaces. Cells exhibiting increased mobility of CR4 showed an increased ability to form CR4-dependent EC3bi rosettes. The data are consistent with the hypothesis that CR3 and CR4 exhibit a variable association with the cytoskeleton that regulates their mobility and function. A relatively mobile subset of CR3 and/or CR4 mediates EC3bi attachment, whereas a relatively immobile subset of CR3 and/or CR4 fails to mediate EC3bi attachment but functions to promote ingestion of EC3bi.


Role of CD4+ T lymphocytes and interleukin-5 in antigen-induced eosinophil recruitment into the site of cutaneous late-phase reaction in mice. Previous studies suggested that the eosinophil recruitment into the site of cutaneous late-phase reaction (LPR) was dependent on IgE antibody and mast cells. In this study, we determined the role of CD4+ T cells and CD8+ T cells in causing antigen-induced eosinophil recruitment of LPR in mouse skin. Eosinophil infiltration into the subcutaneous tissue of ovalbumin (OVA)-sensitized BALB/c mice was biphasic, reaching the first peak at 6 h after the subcutaneous challenge with OVA and the second peak at 24 to 48 h. The in vivo depletion of CD4+ T cells by pretreatment with anti-L3T4 monoclonal antibody (mAb) significantly decreased the second peak (at 24 h and 48 h), but not the first peak (at 6 h), of OVA-induced eosinophil infiltration into the skin of OVA-sensitized mice. However, the depletion of CD8+ T cells by pretreatment with anti-Lyt-2 mAb had no significant effect on either the first peak or second peak of OVA-induced cutaneous eosinophilia. Pretreatment with anti-murine interleukin-5 (IL-5) mAb also decreased the second peak, but not the first peak, of OVA-induced cutaneous eosinophilia. In contrast to the inhibitory effects of depletion of CD4+ T cells and of anti-IL-5 mAb on the second peak of antigen-induced cutaneous eosinophilia, disodium cromoglycate and a selective antagonist for platelet activating factor (PAF) CV-6209 decreased the first peak of OVA-induced cutaneous eosinophilia in the mouse. These results indicate that CD4+ T cells, but not CD8+ T cells, cause the second peak of antigen-induced eosinophil recruitment of cutaneous LPR and that IL-5 mediates this eosinophil recruitment. In contrast, the first peak of antigen-induced eosinophil recruitment of cutaneous LPR is mediated by mast cells and PAF.


A rapid and reliable method for direct genotyping of codon 360 in the human apolipoprotein A-IV gene. Human apolipoprotein A-IV exhibits a polymorphism, first investigated at the protein level, that is caused by a single amino acid substitution of glutamine to histidine at codon 360. Detection of this polymorphism requires polymerase chain reaction (PCR) and direct sequencing of the amplified products, radiolabeled allele-specific oligonucleotides (ASOs) technique, or restriction enzyme analysis of the amplified products. However, these techniques involve the use of radioactivity and/or are not well suited to the rapid processing of a large number of samples. In this paper, we propose a new technique, a bispecific-allele primer amplification, in which a simple electrophoresis of PCR products is used for typing the variation at codon 360. The 3' primer of PCR hybridizes with one or other homologous sequence in the apoA-IV gene, depending on the presence or the absence of the mutation. This differential hybridization of the primer is used for typing the variation. In order to demonstrate the validity of this system, 120 individuals phenotyped by two-dimensional electrophoresis and genotyped by ASO were analyzed by this new technique. The results obtained by the latter method are in agreement with those found by the other techniques. However, this method is simple, more reliable, and will facilitate population studies without using radioactive materials.


Detection of three separate DNA polymorphisms in the human lipoprotein lipase gene by gene amplification and restriction endonuclease digestion. A rapid detection method was developed for DNA polymorphisms in the human lipoprotein lipase (LPL) gene. The examined polymorphisms include an A-C transversion in the 5'-region of intron 3, a T-G transversion that occurs within a Hind III site of intron 8, and the previously described C-T transition that causes a Pvu II polymorphism in intron 6. Gene fragments encompassing each polymorphic site were amplified by the polymerase chain reaction (PCR) and digested with an appropriate restriction enzyme whose recognition site was either naturally affected by the polymorphism or artificially created with a mismatched PCR-primer. According to the digestion profiles, genotypes were unambiguously distinguished. With this method, respective allelic frequencies were determined for 50 or 70 normal subjects. The procedure will facilitate LPL genotyping in the large population.


Differential regulation of the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase, synthase, and low density lipoprotein receptor genes. The ability of mitogenic stimulation of human T lymphocytes to alter the expression of genes involved in sterol metabolism was examined. Messenger RNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, HMG-CoA synthase, and low density lipoprotein (LDL) receptor were quantified in resting and mitogen-stimulated T lymphocytes by nuclease protection assay. Mitogenic stimulation increased HMG-CoA synthase mRNA levels by 5-fold and LDL receptor by 4-fold when cells were cultured in lipoprotein-depleted medium whereas HMG-CoA reductase gene expression was not significantly increased. When cultures were supplemented with concentrations of low density lipoprotein sufficient to saturate LDL receptors, expression of all three genes was inhibited in resting lymphocytes, as effectively as was noted with fibroblasts. Similarly, LDL down-regulated gene expression in mitogen-activated lymphocytes so that mitogenic stimulation did not increase either HMG-CoA reductase or synthase mRNA levels, although LDL receptor gene expression was enhanced. These results indicate that expression of three of the genes involved in sterol metabolism is differentially regulated by LDL and mitogenic stimulation. Moreover, the increase in rates of endogenous sterol synthesis and the activity of HMG-CoA reductase in mitogen-stimulated T lymphocytes cannot be accounted for by increases in HMG-CoA reductase mRNA levels.


Microfilaments, cell shape changes, and the formation of primary mesenchyme in sea urchin embryos. Primary mesenchyme formation in sea urchin embryos occurs when a subset of epithelial cells of the blastula move from the epithelial layer into the blastocoel. The role of microfilaments in producing the cell shape changes that characterize this process, referred to as ingression, was investigated in this study. f-Actin was localized by confocal microscopy using labeled phalloidin. The distribution of f-actin was observed before, during, and after ingression and was correlated with cellular movements. Prior to the onset of ingression, staining became intense in the apical region of putative primary mesenchyme and disappeared following the completion of mesenchyme formation. The apical end of these cells constricted coincidentally with the appearance of the intensified staining, indicating that f-actin may be involved in this constriction. In addition, papaverine, a smooth muscle cell relaxant that interferes with microfilament-based contraction, and that was shown in this study to inhibit cytokinesis, diminished apical constriction and delayed ingression. Despite this interference with apical constriction, the basal surface of ingressing cells protruded into the blastocoel. It is suggested that apical constriction, while not necessary for ingression, does contribute to the efficient production of mesenchyme and that protrusion of the basal surface results from changes that occur independent of apical constriction.


Dominant mutation of the murine Hox-2.2 gene results in developmental abnormalities. Genes carrying the homeobox were originally identified in Drosophila, in which they are now known to play key roles in establishing segmentation patterns and in determining segment identities. A number of genes with striking homology to the Drosophila homeobox genes have now been found in the mouse genome, and mutational analysis is beginning to shed light on their function in mammalian development. To understand better the developmental significance of the murine Hox-2.2 gene, we have generated gain of function mutants by using the chicken beta-actin promoter to drive ubiquitous expression in transgenic mice. The resulting Hox-2.2 misexpression produces early postnatal lethality as well as craniofacial and axial skeletal perturbations that include open eyes at birth, cleft palate, micrognathia, microtia, skull bone deficiencies, and structural and positional alterations in the vertebral column. We repeatedly observe complete or partial absence of the supraoccipital bone and malformations of the exoccipital and the basioccipital bones. These results suggests a role for the Hox-2.2 gene in specifying positional identity along the anterior-posterior axis.


Dangerous marine life.

Dangerous marine life. All physicians must be educated in treating injuries incurred when a diver comes into contact with any dangerous marine life. Stinging invertebrates are the most commonly encountered dangerous marine animals. Venomous vertebrate marine animals are less common than stinging invertebrates and easier to recognize. However, they may be much more deadly. Sharks pose the greatest danger to divers. However, bites from other marine animals can be painful, become infected and require extensive medical treatment.


Receptive field properties of rod-driven horizontal cells in the skate retina. The large receptive fields of retinal horizontal cells result primarily from extensive intercellular coupling via gap (electrical) junctions; thus, the extent of the receptive field provides an index of the degree to which the cells are electrically coupled. For rod-driven horizontal cells in the dark-adapted skate retina, a space constant of 1.18 +/- 0.15 mm (SD) was obtained from measurements with a moving slit stimulus, and a comparable value (1.43 +/- 0.55 mm) was obtained with variation in spot diameter. These values, and the extensive spread of a fluorescent dye (Lucifer Yellow) from the site of injection to neighboring cells, indicate that the horizontal cells of the all-rod retina of skate are well coupled electrically. Neither the receptive field properties nor the gap-junctional features of skate horizontal cells were influenced by the adaptive state of the retina: (a) the receptive field organization was unaffected by light adaptation, (b) similar dye coupling was seen in both dark- and light-adapted retinae, and (c) no significant differences were found in the gap-junctional particle densities measured in dark- and light-adapted retinas, i.e., 3,184 +/- 286/microns 2 (n = 8) and 3,073 +/- 494/microns 2 (n = 11), respectively. Moreover, the receptive fields of skate horizontal cells were not altered by either dopamine, glycine, GABA, or the GABAA receptor antagonists bicuculline and picrotoxin. We conclude that the rod-driven horizontal cells of the skate retina are tightly coupled to one another, and that the coupling is not affected by photic and pharmacological conditions that are known to modulate intercellular coupling between cone-driven horizontal cells in other species.


Evidence of genomic variations between infectious pancreatic necrosis virus strains determined by restriction fragment profiles. Infectious pancreatic necrosis virus (IPNV) is the aetiological agent of an important disease in hatchery-reared salmonid fish in North America, Europe and Japan. It belongs to the family Birnaviridae and shows a high degree of antigenic heterogeneity. However, genomic variations between the 10 identified serotypes have not yet been studied. In order to correlate genomic heterogeneity with the different serotypes, oligonucleotides were synthesized according to the published sequence of the Jasper strain (serotype A9). They were used as primers for the amplification of a 359 bp cDNA fragment of the viral genome using the polymerase chain reaction. Fragments amplified from 37 strains were digested with five different restriction enzymes. Restriction fragment profiles obtained an agarose gels showed heterogeneity not only between strains of different serotypes, but also among those belonging to serotype A1. A cluster analysis of the restriction patterns showed that IPNV strains can be divided into three major groups, corresponding approximately to serotypes A1, A2 and A3, and 10 subgroups which do not correlate with the serotyping of the strains.


Demonstration of scrapie strain diversity in infected PC12 cells. Scrapie strain replication in the nerve growth factor-induced, differentiated PC12 cell culture system was examined. Differences in replication between mouse-derived agents were demonstrated, with the 139A scrapie strain yielding 100- to 1000-fold higher levels of infectivity than the ME7 scrapie strain. Replication was not detected in PC12 cells infected with either the hamster-derived 263K or rat-derived 139R scrapie strains. Studies on the neurotransmitters in infected PC12 cells demonstrated that the adrenergic pathway was unchanged but the cholinergic pathway was altered. Furthermore, the degree of alteration correlated with the level of scrapie strain replication. Comparison of infectivity titres and enzymatic changes in ME7-infected PC12 cells with those in Chandler agent-infected mouse neuroblastoma cells suggests that the significant changes in neurotransmitter levels in cultures exhibiting low titres of infectivity involve factors in addition to strain replication. The variation in the range of scrapie strain replication in PC12 cells is discussed in relationship to species barrier, cell targeting, genetic susceptibility and species strain specificity. These studies further emphasize the value of the PC12 cell model system in examining the scrapie strain-host cell interaction and in addition support the concept of variation among scrapie strains.


Longitudinal outcome and medication noncompliance among manic patients with and without mood-incongruent psychotic features. The prognostic utility of mood-incongruent psychotic features was examined in a sample of 23 hospitalized manic patients. Patients were initially subdivided according to whether they met Research Diagnostic Criteria (RDC) for schizoaffective, mainly affective (mood-incongruent) manic disorder (SAM; N = 11) or RDC primary manic (mood-congruent or nonpsychotic) manic disorder (PM; N = 12). Patients were then followed over a 9-month posthospitalization period and rated every 3 months for relapse status, symptom severity, social adjustment, and medication noncompliance. Patients with SAM and PM did not differ at follow-up on rates or timing of manic or depressive relapses or on cycling of symptoms of mood disorder. However, at follow-up, SAM patients had more severe positive and negative psychotic symptoms and poorer social adjustment, and were less medically compliant than PM patients. Results are consistent with the view that mania with mood-incongruent psychotic features is a poor-prognosis subtype of bipolar disorder.


Structure-activity relationship of the neurotransmitter alpha-bag cell peptide on Aplysia LUQ neurons: implications regarding its inactivation in the extracellular space. Alpha-bag cell peptide [alpha-BCP (Ala-Pro-Arg-Leu-Arg-Phe-Tyr-Ser-Leu)] is a neurotransmitter that mediates bag cell-induced inhibition of left-upper-quadrant (LUQ) neurons L2, L3, L4, and L6 in the abdominal ganglion of Aplysia. Our recent biochemical studies have shown that alpha-BCP[1-9] is cleaved into alpha-BCP[1-2], [3-9], [1-5], [6-9], and [7-9] by a combination of three distinct peptidase activities located within the extracellular spaces of the CNS: A diaminopeptidase-IV (DAP-IV)-like enzyme cleaves alpha-BCP[1-9] at the 2-3 peptide bond; a neutral metalloendopeptidase (NEP)-like enzyme cleaves either alpha-BCP[1-9] or alpha-BCP[3-9] at the 5-6 bond; an aminopeptidase M-II (APM-II)-like enzyme cleaves alpha-BCP[6-9] at the 6-7 bond, but cleaves neither alpha-BCP[1-9], nor the other ganglionic peptidase products. To further understand the manner in which alpha-BCP is inactivated after release, that is loses its electrophysiological activity, we studied its structure-activity relationship by recording intracellularly from LUQ neurons in isolated abdominal ganglia that were arterially perfused with peptides dissolved in artificial sea water. The effects of alpha-BCP[1-9] and 15 of its fragments ([1-8], [1-7], [1-6], [1-5], [2-9], [3-9], [3-8], [6-9], [7-9], [8-9], [6-7], [6-8], [1-2], Phe, Tyr) indicated that the sequence Phe6-Tyr7 was both necessary and sufficient to produce LUQ inhibitory activity.(ABSTRACT TRUNCATED AT 250 WORDS)


Transient expression of somatostatin peptide is a widespread feature of developing sensory and sympathetic neurons in the embryonic rat. Previous studies from this and other laboratories demonstrated that many embryonic sensory ganglion cells in the rat transiently express the catecholamine synthesizing enzyme tyrosine hydroxylase (TH), a trait not expressed by most mature sensory neurons. We, therefore, sought to determine whether transient expression was uniquely associated with catecholaminergic traits, or, alternatively, whether embryonic ganglion cells transiently expressed peptidergic properties as well. Of the four peptides examined (somatostatin [somatotropin release inhibiting factor] (SRIF), galanin (Gal), calcitonin gene-related peptide (CGRP), and substance P (SP)), only SRIF was found to be transiently expressed during early stages of sensory gangliogenesis. Surprisingly, SRIF immunoreactivity was observed in virtually all cranial and spinal sensory ganglion cells on embryonic day (E) 12.5. In addition to perikaryal labeling, intense SRIF immunoreactivity was also observed in the central and peripheral processes of E12.5 sensory neurons, suggesting the peptide may be released from nerve endings. The time course of SRIF appearance in cranial ganglion cells paralleled that previously described for TH, and double-labeling studies revealed extensive co-localization of these two phenotypes. By E16.5, however, the number of neurons expressing SRIF had diminished markedly, indicating that SRIF is only transiently expressed by most sensory neurons during early stages of ganglion development. An unexpected finding was that transient expression of SRIF is also a prominent feature of sympathetic ganglion cells; however, the temporal pattern of staining in the sympathetic and sensory ganglia differed substantially. Whereas virtually no SRIF staining was observed in E12.5 sympathetics, the vast majority of cells in the E16.5 superior cervical ganglion (SCG) were labeled. This contrasted sharply with the adult SCG, in which only low levels of SRIF expression were found. These findings demonstrate that SRIF peptide is transiently expressed at high levels in peripheral sensory and sympathetic neurons during embryogenesis. The time course and widespread distribution of SRIF expression indicates that the peptide may play a role in early stages of ganglion cell growth and development. Moreover, these data, in conjunction with previous studies demonstrating SRIF immunoreactivity in developing central neurons, suggest that transient expression of this peptide is a common property of diverse neuronal cell types.


Establishment of mouse-immortalized hybrid clones expressing characteristics of differentiated neurons derived from the cerebellar and brain stem regions. Two clonal immortalized neurons designated CL8c4.7 and CL8a5.2 were established by somatic cell fusion between a hypoxanthine phosphoribosyltransferase-(HPRT-) deficient neuroblastoma N18TG2 and newborn mouse cerebellar/brain stem neurons. In the serum-containing medium without extra differentiating agents, both clones exhibited a morphology of differentiated neurons. They contained high levels of glutamate but no gamma-aminobutyric acid (GABA). The CL8a5.2 clone synthesized choline acetyltransferase and serotonin. In immunocytochemical studies, both clones expressed 200 kD neurofilament protein, neuron-specific enolase, microtubule-associated protein 2 (MAP2), tau protein, neuronal cell adhesion molecule (N-CAM), HNK-1, Thy-1.2, saxitoxin-binding sodium channel protein, and glutamate. Synaptophysin immunoreactivity was identified in the neuritic terminals of CL8c4.7 cells. Most of these antigens were barely detectable on N18TG2 cells. Electrophysiologically, both clones generated action potentials in response to electrical stimuli. The hybrid clones that express characteristics of differentiated neurons derived from the cerebellar and brain stem regions might be invaluable for the study of the molecular basis of neuronal differentiation and degeneration in these regions.


Toxicity and residual action of the photoactivated compound, cyano-alpha-terthienyl, and its efficacy for reducing pre-imaginal populations of mosquitoes. The photoactivated compound, cyano-alpha-terthienyl (cyano-alpha-T), was highly toxic to pre-imagines of the mosquitoes Culex restuans, Cx. tarsalis and Culiseta inornata when synergized with piperonyl butoxide (PBO). Lethal concentrations for 50% mortality, determined during an outdoor trial using caged fourth-instar Culex spp. larvae, were 19.4, 15.4 and 12.9 g/ha at 24, 48 and 72 h after treatment, respectively. No residual activity of cyano-alpha-T was observed beyond 24 h following treatment. In artificial pool tests, greatest population reductions were achieved using dosages of 20 and 40 g/ha; statistically significant reductions were not observed following applications of 5 g/ha. Cyano-alpha-T plus PBO was more effective for reducing mosquito populations than alpha-terthienyl (alpha-T) plus PBO at comparable dosages, although it exhibited slightly lower insecticidal activity at a dosage of 20 g/ha than a formulation of Bacillus thuringiensis var. israelensis (Vectobac 12 AS, 0.12 ml/m2). Greatest effectiveness of cyano-alpha-T plus PBO was observed in pools with low organic content relative to pools high in organic content.


New state record for Culiseta impatiens in Maryland. Culiseta impatiens is reported for the first time from Maryland. Collections from the southwestern border of Fort George G. Meade extend the range of this species over 400 km farther south than previous records. Multiple collections from 4 separate traps during 2 years indicate a population of Cs. impatiens is probably established in the collection area.


Occurrence of Psorophora howardii in Suffolk County, Long Island, New York. Psorophora howardii has been confirmed in New York State for the first time, based on larvae from a rain pool and an adult female from a CDC trap catch. Both collections were reported from Suffolk County, Long Island, NY.


Regulation of expression of dopamine beta-hydroxylase in PC12 cells by glucocorticoids and cyclic AMP analogues. Regulation of catecholamine biosynthesis is crucial in the adaptation to various physiological conditions, such as stress, and in several disorders, including hypertension and depression. In this study we have found that in PC12 cells, the mRNA levels of dopamine beta-hydroxylase (DBH), the enzyme that catalyzes the formation of norepinephrine from dopamine, can be regulated by glucocorticoids and cyclic AMP (cAMP) analogues. Treatment with dexamethasone increased DBH mRNA levels by 6 h. with maximal elevation (four- to fivefold) obtained after 1 day of exposure, and these levels were maintained for up to 4 days. DBH mRNA levels were also elevated on treatment of PC12 cells with 8-bromo cAMP for 8 h to 1 day. The response to 8-bromo cAMP, however, was bimodal, because DBH mRNA levels declined below control values on treatment for > 1 day. In combined treatments with 8-bromo cAMP and dexamethasone, the cAMP effect was dominant. To begin to characterize the regulation of DBH mRNA, genomic clones for rat DBH were isolated, and 1 kb of the 5' flanking region was sequenced. Several putative regulatory elements, which may be involved in cAMP and glucocorticoid regulation, were identified, including two adjacent cAMP response elements, another element that can also bind members of the ATF/CREB family of transcription factors, a NF-kappa B-like sequence, several AP-2 sites, and three core glucocorticoid receptor binding sequences.


Tetrahydrobiopterin turnover in cultured rat sympathetic neurons: developmental profile, pharmacologic sensitivity, and relationship to norepinephrine synthesis. We have examined the turnover of 5,6,7,8-tetrahydrobiopterin (BH4) and the effect of decreasing BH4 levels on in situ tyrosine hydroxylase (TH) activity and norepinephrine (NE) content in a homogeneous population of NE-containing neurons derived from the superior cervical ganglion (SCG) of the neonatal rat and maintained in tissue culture. Initial studies indicated that the level of BH4 within SCG cultures increased fourfold between 5 and 37 days in vitro (DIV). This increase in BH4 levels was determined to result from an increase in the rate of BH4 biosynthesis without a change in the rate of degradation. Regardless of culture age, the BH4 content of SCG neurons was observed to turn over with a half-life of approximately 2.5 h. BH4 synthesis by SCG neurons was found to be five times more sensitive to inhibition by 2,4-diamino-6-hydroxypyrimidine (DAHP) and 25 times less sensitive to inhibition by N-acetylserotonin than was previously reported for CNS neurons in culture. Under basal conditions, the rates of in situ TH activity and BH4 biosynthesis were similar. In response to inhibition of BH4 biosynthesis by DAHP and a 90-95% decrease in BH4 levels, in situ TH activity declined by 75%. NE levels declined by 30% following a 24-h period of inhibition of BH4 synthesis. After 2 days of BH4 synthesis inhibition, the level of NE was decreased by 47%. On treatment days 3 and 4, the decline in NE content plateaued at 24% of control levels.(ABSTRACT TRUNCATED AT 250 WORDS)


Stimulation of cultured cerebellar granule cells via glutamate receptors induces TRE- and CRE-binding activities mediated by common DNA-binding complexes. By use of nuclear mini-extracts prepared from cultured cerebellar granule cells in a gel-mobility assay, exogenous N-methyl-D-aspartate (NMDA) or kainate was shown to increase both 12-O-tetradecanoylphorbol 13-acetate-responsive element (TRE)- and cyclic AMP-responsive element (CRE)-binding activity. These increases were specifically prevented by the NMDA receptor antagonist D,L-2-amino-5-phosphonovalerate and the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, respectively. The increase of TRE-binding activity was dependent on de novo protein synthesis, and its inductions by both NMDA and kainate required extracellular Ca2+. TRE-binding activity was competitively inhibited by the CRE, and vice versa, showing higher DNA-binding affinity to the CRE than to the TRE. A proteolytic clipping bandshift assay demonstrated that the increase in CRE-binding activity could be mediated by the TRE-binding activity. Thus, the TRE-binding activity cross-binding to the CRE could be activated by NMDA or kainate stimulation. The involvement of c-Fos or Fos-related proteins in the TRE- and CRE-binding complexes was shown by a supershift gel-mobility assay using anti-c-Fos antiserum.


Biphasic 24-hour variations in cyclic GMP accumulation in the rat pineal gland are due to corresponding changes in the activity of cytosolic and particulate guanylate cyclase. Various parameters of the rat pineal gland display a 24-h rhythm. However, nothing is known about possible 24-h variations in cyclic GMP (cGMP) metabolism. In the present study, 24-h variations in pineal gland cGMP accumulation were investigated by determining the increase in cGMP level with and without inhibitors of phosphodiesterase at different time points over a light/dark cycle (12/12 h). Furthermore, the activity of guanylate cyclase (GC) was determined under substrate-saturated conditions regarding the cytosolic and particulate forms of the enzyme. It has been found that cGMP accumulation and GC activity display biphasic 24-h variations with two peaks--one approximately 7 h after lights "on" and the other approximately 7 h after lights "off." The activity of cytosolic GC remains unchanged in the presence of the nitric oxide (NO) synthesis inhibitor N-monomethyl-L-arginine, indicating that 24-h variations in the activity do not reflect changes in the synthesis of the GC stimulator NO.


Differential effects of tetanus toxin on inhibitory and excitatory neurotransmitter release from mammalian spinal cord cells in culture. The effect of tetanus toxin on depolarization-evoked and spontaneous synaptic release of inhibitory and excitatory neurotransmitters was examined in murine spinal cord cell cultures. Toxin action on the release of radiolabeled glycine and glutamate was followed over time intervals corresponding to the early phase of convulsant activity through the later phase of electrical quiescence. Tetanus toxin inhibited potassium-evoked release of [3H]glycine and [3H]glutamate in a time- and dose-dependent manner. Ninety minutes after the application of toxin (6 x 10(-10) M), the stimulated release of [3H]glycine was blocked completely, whereas stimulated release of [3H]glutamate was not blocked completely until 150-210 min after toxin application. Fragment C, the binding portion of the tetanus toxin molecule, had no effect on stimulated release of either transmitter. The spontaneous synaptic release of [3H]glycine was blocked totally within 90 min of toxin exposure. In contrast, the spontaneous release of [3H]glutamate, in toxin-exposed cultures, was elevated to nearly twice that of control cultures at this time. Thus, toxin-induced convulsant activity is characterized by a reduction in the spontaneous synaptic release of inhibitory neurotransmitter with a concomitant increase in the release of excitatory neurotransmitter, as well as the more rapid onset of blockade of depolarization-evoked release of inhibitory versus excitatory neurotransmitter.


Neomycin is an agonist at a polyamine site on the N-methyl-D-aspartate receptor. Neomycin appears as a full agonist and spermidine as a partial agonist at the site where polyamines enhance 1-[1-(2-thienyl)cyclohexyl][3H]piperidine ([3H]TCP) binding on the N-methyl-D-aspartate (NMDA) receptor. Other aminoglycosides also enhance [3H]TCP binding with efficacies roughly proportional to the number of primary amine groups. The polyamine antagonists ifenprodil and arcaine inhibit enhancement of [3H]TCP binding by spermidine or neomycin. The inhibition of [3H]TCP binding by arcaine is apparently competitively reduced by neomycin and spermidine, supporting a common site. Diethylenetriamine (previously described as a polyamine antagonist) may be a partial agonist. Enhancement by neomycin or spermidine is not additive to that of Mg2+, consistent with competition of Mg2+ and spermidine or neomycin at the site where these compounds enhance [3H]TCP binding. Polyamines also enhance the binding of the competitive antagonist 2-(2-carboxypiperazin-4-yl)[3H]propyl-1-phosphonic acid ([3H]CPP). Neomycin, which does not enhance [3H]CPP binding, inhibits the enhancement by spermidine. That this site is distinct from the site where spermidine and neomycin increase [3H]TCP binding is supported by different pharmacology. Arcaine and diethylenetriamine do not inhibit spermidine enhancement of [3H]CPP binding. Mg2+ also does not compete with the spermidine enhancement of [3H]CPP binding. Ifenprodil inhibits the spermidine enhancement of [3H]CPP binding. The data suggest two or more polyamine sites, with arcaine selective for the site that enhances [3H]TCP binding. Neomycin is an agonist at one polyamine site and antagonist to the second.


Neuropeptide Y inhibits beta-adrenergic agonist- and vasoactive intestinal peptide-induced cyclic AMP accumulation in rat pinealocytes through pertussis toxin-sensitive G protein. The effects of neuropeptide Y (NPY) on pineal gland cyclic AMP (cAMP) accumulation were investigated using dispersed pinealocytes from rats. NPY inhibited the intracellular cAMP accumulation stimulated by isoproterenol and norepinephrine in a dose-dependent manner during a 10-min incubation of pinealocytes. NPY (1 x 10(-7) M) also inhibited vasoactive intestinal peptide (VIP)- and cholera toxin-induced cAMP accumulation. The inhibitory effect of NPY on isoproterenol-induced cAMP accumulation was completely abolished by a 5-h pretreatment of pinealocytes with 1 microgram/ml of pertussis toxin (PT). These results suggest that NPY participates in modulation of cAMP production in the rat pineal gland through PT-sensitive G protein. Yohimbine, an alpha 2-adrenergic antagonist, blocked NPY inhibition of isoproterenol-stimulated cAMP accumulation. On the other hand, the alpha 2-adrenergic agonist clonidine by itself did not affect cAMP accumulation stimulated by isoproterenol but significantly potentiated NPY action. The present study demonstrates that NPY inhibits beta-adrenergic or VIPergic stimulation of the pineal gland cAMP accumulation. The inhibitory effect of NPY is mediated through PT-sensitive G protein. Our results also suggest that NPY exerts its action to affect alpha 2-adrenoceptor function.


Differing neurochemical and morphological sequelae of global ischemia: comparison of single- and multiple-insult paradigms. The purpose of this investigation was to investigate pathomechanisms responsible for the deleterious effects of repeated episodes of brief forebrain ischemia. Halothane-anesthetized male Wistar rats were subjected to either (a) a single 15-min period or (b) three 5-min periods (separated by 1 h) of global forebrain ischemia by bilateral carotid artery occlusions plus hypotension (50 mm Hg), followed by various periods of recirculation. Brain temperature was normothermic throughout. In one series of rats, extracellular levels of glutamate, glycine, and gamma-aminobutyric acid (GABA) were measured in the dorsolateral striatum (n = 6-8 per group) and lateral thalamus (n = 4-6 per group) by microdialysis and HPLC before and during ischemia and during 3-5 h of recirculation. In a parallel series of rats (n = 6 per group), ischemic cell change was quantified at 2 (dark neurons), 24, or 72 h following either single or multiple ischemic insults. A single 15-min ischemic period led to massive glutamate release (13-fold increase; p = 0.001), which returned to normal by 20-30 min of recirculation and remained normal thereafter. By contrast, in rats with three 5-min periods of ischemia, the glutamate level rise with each repeated insult (four- to 4.5-fold; p < or = 0.02) was smaller than that observed during the single 15-min insult, but a late sustained rise (five- to six-fold; p < 0.05) occurred at 2-3 h of recirculation. Brief ischemia-induced elevations of glycine and GABA levels were detected in both the single- and multiple-insult groups, with normalization during recirculation. In contrast, the excitotoxic index, a composite measure of neurotransmitter release ([glutamate] x [glycine]/[GABA]), differed markedly following single versus multiple insults (p = 0.002 by repeated-measures analysis of variance) and increased by seven- to 12-fold (p < 0.05) at 1-3 h following the third insult. The total amount of glutamate released was 3.3-fold higher in the multiple-insult than in the single-insult group (p < 0.02). At 2 h of recirculation, histopathological analysis of dorsolateral striatum showed a significantly greater frequency of dark neurons in the multiple- than in the single-insult group (p < 0.05 by analysis of variance). In the thalamus, a higher frequency of ischemic neurons was seen in the multiple-than in the single-insult group at all intervals studied. Thus, in rats with multiple ischemic insults, accelerated ischemic damage was found in the striatum, and severe ischemic injury was documented in the thalamus.(ABSTRACT TRUNCATED AT 400 WORDS)


Nicotinic cholinergic regulation of tyrosine hydroxylase gene expression and catecholamine synthesis in isolated bovine adrenal chromaffin cells. Isolated bovine adrenal chromaffin cells were used to study the nicotinic regulation of tyrosine hydroxylase (TH) gene expression. Continuous exposure of the cells to carbachol or the nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) produces a time- and concentration-dependent increase in TH enzyme activity, whereas muscarine has no effect. DMPP at 1 microM (EC50 = 0.3 microM) elicits a two- to threefold elevation of both TH activity and TH immunoreactive protein level after 3-5 days in the presence of 2.5 mM calcium; the increase in enzyme levels is significantly less at lower extracellular calcium levels. The rate of hydroxylation of tyrosine to dopamine (DA) in intact cells, an index of endogenous TH activity, increases in parallel with the rise in TH levels. The TH mRNA level is elevated before the increase in protein levels. As determined by nuclear run-on assays, TH gene transcription is stimulated two- to threefold within 30 min of addition of 1 microM DMPP to the cells; transcription returns to basal levels by 2 h. Nitrendipine (20 microM) blocks the stimulation of transcription by DMPP. Pretreatment of the cells with cycloheximide (5 microM) does not prevent the DMPP stimulation of transcription. Forskolin (10 microM) also increases TH transcription (fourfold in 15 min) by a mechanism that is not blocked by cycloheximide. These results show that nicotinic receptor stimulation increases TH mRNA synthesis, TH protein levels, and TH activity in a calcium-dependent manner. Furthermore, the nicotinic influence on TH gene expression does not appear to require the synthesis of a protein factor for its effects. That in situ DA synthesis rates are elevated consequent to the rise in TH levels demonstrates that TH induction serves as a mechanism for enhancing the catecholamine-synthesizing capacity of the chromaffin cell on a long-term basis.


Taxol: quantitative internuclear proton-proton distances in CDCl3 solution from nOe data: 2D nmr ROESY buildup rates at 500 MHz. Quantitative nmr internuclear proton-proton distance measurements obtained by observation of the initial buildup rates of nOe's in 2D ROESY spectra of taxol [1] in CDCl3 are reported. A comparison to the X-ray crystal structure of taxotere [2] is made, and the results are discussed in terms of previous studies of structure-activity relationships.


An immunogenetic heterogeneity in multiple sclerosis. Two clinical forms of multiple sclerosis (MS), primarily chronic progressive MS (PCP MS) and relapsing/remitting MS (R/R MS) have been shown to differ in several respects. The results of genomic HLA class II typing with restriction fragment length polymorphism analysis of 62 MS patients from Western Norway, 42 with R/R MS and 20 PCP MS, are reported on here. As in previous studies of Swedish patients, the haplotype DRw17(3), DQw2 was found to be five times more common in R/R MS than in PCP MS. This finding supports the hypothesis that R/R and PCP MS are immunogenetically separate entities. In contrast with a previous investigation of Norwegian MS patients, no association of MS with glutamine at position 34 of the HLA-DQ alpha chain or with defined sequences of the HLA-DQB1 gene was found.


Determinants of total high density lipoprotein cholesterol and high density lipoprotein subfraction levels among Hispanic and non-Hispanic white persons with normal glucose tolerance: the San Luis Valley Diabetes Study. Determinants of total high-density lipoprotein cholesterol (HDL-C) and HDL subfractions were assessed in Hispanic and non-Hispanic white persons (n = 932), aged 20-74 years, in the San Luis Valley, Colorado. Using multiple regression, BMI was negatively associated with HDL-C, HDL2-C, and HDL3-C in men and HDL-C and HDL3-C in women. Among females, current smokers had lower HDL-C and subfractions. Women on beta-blockers had lower HDL3-C levels. For both sexes, a positive association was observed between age and HDL-C and subfractions and physical activity with HDL-C and HDL3-C. Drinking alcohol (> or = 50 g/week) was associated with higher HDL-C and HDL3-C in both sexes and HDL2-C in women. The positive association of age and negative associations of the subscapular/triceps ratio and fasting insulin had consistent relationships with HDL-C, HDL2-C, and HDL3-C in men and women. Ethnicity was not significantly associated with HDL-C or subfractions after controlling for body fat distribution or fasting insulin.


The action of the putative neurotransmitters N-acetylaspartylglutamate and L-homocysteate in cat dorsal lateral geniculate nucleus. 1. We have examined the actions and pharmacology of two putative optic nerve transmitters, N-acetylaspartylglutamate (NAAG) and L-homocysteic acid (L-HCA), in the feline dorsal lateral geniculate nucleus (dLGN). We compared the responses obtained to iontophoretic application of these substances with those elicited by visual stimulation and application of specific N-methyl-D-aspartate (NMDA) and non-NMDA receptor agonists. The relative effects of the selective NMDA antagonist 3-[(+/-)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP) and the selective non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were tested on these responses. 2. There was a pronounced contrast between the influence of iontophoretically applied NAAG and L-HCA on dLGN cells. Iontophoretic application of NAAG [ejection current range 75-200 nA (mean 125 nA)] evoked either no effect (17/37), or very weak and sluggish excitatory (16/37) or inhibitory (4/37) effects. Conversely, L-HCA application [current range 25-136 nA (mean 67 nA)] elicited brisk and powerful excitatory responses (32/32) that were comparable with those produced by visual stimulation and iontophoresis of NMDA, kainate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). 3. Responses to L-HCA were selectively antagonized by application of the NMDA receptor antagonist CPP but were generally much less affected by the non-NMDA receptor antagonist CNQX. The weak and inconsistent responses to NAAG were not compatible with an evaluation of antagonist effects. 4. CPP application at dose levels selective for NMDA with respect to kainate and AMPA did not exert equal effects on L-HCA and NMDA. Whereas the mean responses to L-HCA were reduced to 32% of control for Y cells and 21% for X cells, those to NMDA were 11 and 11%, respectively. However, the level of reduction of the visual response for X and Y cells was very similar to that of L-HCA, visual responses being reduced to 35 and 22% of control for Y and X cells. 5. CNQX application reduced the visual response level of Y cells to 64% of control and that of X cells to 65%. The mean level for the L-HCA response of Y cells was 106% of control; the mean for X cells, 79%, was substantially below control. The responses to kainate and AMPA were reduced to a much greater extent. 6. The data suggest that it is unlikely that NAAG is the optic nerve transmitter.(ABSTRACT TRUNCATED AT 400 WORDS)


Dopamine and glutamate agonists stimulate neuron-specific expression of Fos-like protein in the striatum. 1. The monoamine dopamine and the amino acid glutamate are major neurotransmitters in the basal ganglia implicated in the normal functions of the striatum and in extrapyramidal disease states. To study the effects of these neurotransmitters on gene transcription in striatal neurons, we treated rats with dopamine (monoamine) agonists and with glutamate agonists and monitored the induction of Fos-like protein in striatal neurons. We administered the indirect monoamine agonists cocaine and amphetamine intraperitoneally and gave the glutamate agonist quinolinic acid by direct intrastriatal injection. We identified the phenotypes of the responsive neurons by immunohistochemistry and by enzyme histochemistry in double staining protocols. 2. Both the indirect monoamine agonists and the glutamate receptor agonist stimulated rapid nuclear expression of Fos-like protein in specific classes of striatal neurons. The induction by cocaine and amphetamine was blocked by pretreatment with the dopamine D1-like receptor antagonist SCH23390, and the induction by quinolinic acid was blocked by pretreatment with MK-801, a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) glutamate receptor. 3. The monoamine and glutamate agonists both induced Fos-like protein exclusively in striatal neurons that constitutively expressed the protein phosphatase inhibitor DARPP-32 (dopamine and cAMP-regulated phosphoprotein). 4. The dopamine agonists failed to induce detectable Fos-like protein in striatal neurons expressing enkephalin, even though many such neurons expressed DARPP-32. By contrast, many enkephalinergic neurons did express Fos-like protein in response to glutamatergic stimulation. 5. Glutamate agonist stimulation, but not dopamine agonist stimulation, induced Fos-like protein in a subpopulation of striatal interneurons, namely, a group of neurons exhibiting NADPH-diaphorase activity. 6. These findings suggest that stimulation of dopamine D1-like receptors (or related monoamine receptors) and glutamate NMDA receptors activates neuron-specific programs of immediate-early gene expression in the striatum. Our findings further suggest that monoamine and glutamate may act cooperatively at the transcriptional level on a functionally defined subset of striatal neurons.


Blockade of excitation reveals inhibition of dentate spiny hilar neurons recorded in rat hippocampal slices. 1. Extracellular and intracellular recordings in rat hippocampal slices were used to compare the synaptic responses to perforant path stimulation of granule cells of the dentate gyrus, spiny "mossy" cells of the hilus, and area CA3c pyramidal cells of hippocampus. Specifically, we asked whether aspects of the local circuitry could explain the relative vulnerability of spiny hilar neurons to various insults to the hippocampus. 2. Spiny hilar cells demonstrated a surprising lack of inhibition after perforant path activation, despite robust paired-pulse inhibition and inhibitory postsynaptic potentials (IPSPs) in adjacent granule cells and area CA3c pyramidal cells in response to the same stimulus in the same slice. However, when the slice was perfused with excitatory amino acid antagonists [6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), or CNQX with 2-amino-5-phosphonovaleric acid (APV)], IPSPs could be observed in spiny hilar cells in response to perforant path stimulation. 3. The IPSPs evoked in spiny hilar cells in the presence of CNQX were similar in their reversal potentials and bicuculline sensitivity to IPSPs recorded in dentate granule cells or hippocampal pyramidal cells in the absence of CNQX. 4. These results demonstrate that, at least in slices, perforant path stimulation of spiny hilar cells is primarily excitatory and, when excitation is blocked, underlying inhibition can be revealed. This contrasts to the situation for dentate and hippocampal principal cells, which are ordinarily dominated by inhibition, and only when inhibition is compromised can the full extent of excitation be appreciated.(ABSTRACT TRUNCATED AT 250 WORDS)


Mu opioid receptor-mediated modulation of synaptic currents in dentate granule cells of rat hippocampus. 1. The effect of a selective mu opioid agonist, [N-MePhe3-D-Pro4]morphiceptin (PL017), on synaptic transmission in the dentate gyrus was examined in hippocampal slices. Synaptic currents were evoked by stimulation of the outer molecular layer and recorded from granule cells using whole-cell voltage-clamp techniques. 2. Monosynaptic inhibitory postsynaptic currents (IPSCs) were evoked in the presence of D(-)-2-amino-5-phosphonovaleric acid (D-APV), and N-methyl-D-aspartate (NMDA) receptor antagonist, and 6,7-dinitroquinoxaline-2,3-dione (DNQX), a non-NMDA type of glutamate receptor antagonist. The IPSCs consisted of a gamma-aminobutyric acid (GABA)A receptor-mediated early component and a GABAB receptor-mediated late component. 3. Bath application of PL017 (0.3-3 microM) induced a dose-dependent reduction in the amplitude of both early IPSCs (21-56%) and late IPSCs (43-81%). These effects could be reversed by the opiate antagonist naloxone (1 microM) or prevented by the selective mu antagonist beta-funaltrexamine hydrochloride (10 microM). 4. NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) were revealed in the presence of DNQX and the GABAA antagonist bicuculline methiodide. PL017 (3 microM) caused a 35% reduction in the amplitude of NMDA EPSCs. NMDA receptor-mediated population EPSPs recorded extracellularly were also inhibited by 3 microM PL017 to a similar degree. 5. Non-NMDA receptor-mediated EPSCs were demonstrated in the presence of D-APV and bicuculline methiodide. The amplitude of non-NMDA EPSCs was not affected by PL017.(ABSTRACT TRUNCATED AT 250 WORDS)


Calcium transients evoked by climbing fiber and parallel fiber synaptic inputs in guinea pig cerebellar Purkinje neurons. 1. Calcium transients related to climbing fiber (CF) and parallel fiber (PF) synaptic potentials were recorded from Purkinje cells in guinea pig cerebellar slices. Transients were measured using either absorbance changes of arsenazo III or fluorescence changes of fura-2, which were injected into individual cells in the slice. 2. All-or-none somatically recorded CF potentials elicited by white matter stimulation had all-or-none Ca transients. These signals began with a delay of > or = 2 ms from the start of the electrically recorded synaptic potential. The recovery time of CF-induced arsenazo III absorbance transients was < 50 ms in the fine dendrites in conditions that minimized the effects of dye buffering. 3. Ca2+ entry through voltage-gated Ca channels opened by Ca action potentials was the dominant source of the rise in [Ca2+]i after CF activation. There was no significant change in [Ca2+]i corresponding to the plateau potential that followed the large CF response. 4. The appearance and amplitude of distal CF-evoked Ca signals was more variable than proximal signals, suggesting that CF potentials do not reliably spread to the fine distal dendrites. The distal transient could be enhanced by intrasomatic depolarizing pulses, suggesting that it was a property of the postsynaptic membrane and not the presynaptic side of the CF synapse that was responsible for this variability. 5. Parallel fiber responses were evoked by electrical stimulation near the pial surface. Graded synaptic potentials and related Ca transients were reversibly blocked by 2 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Small synaptic potentials induced small, localized Ca transients. With increasing stimulus intensity, the PF electrical response developed a regenerative component. Larger dendritic Ca transients were detected corresponding to this component. Ca transients evoked by the regenerative responses had the same rapid rise times and fall times as those related to somatically stimulated Ca action potentials, suggesting that they also were due to Ca2+ entry through voltage-sensitive channels. 6. During trains of PF responses, we observed an increase in the spatial extent of related Ca transients. This effect could be modulated by changes in the resting potential, suggesting that the same intrinsic mechanism was affecting the spread of both CF and PF signals.


Membrane properties of mesopontine cholinergic neurons studied with the whole-cell patch-clamp technique: implications for behavioral state control. 1. The whole-cell patch-clamp technique was used to study the membrane properties of identified cholinergic and noncholinergic laterodorsal tegmental neurons in slices of rat brain maintained in vitro. 2. On the basis of their expression of the transient outward potassium current IA and the transient inward calcium current IT, three classes of neurons were observed: type I neurons exhibited a large IT; type II neurons exhibited a prominent IA; and type III neurons exhibited both IA and IT. 3. Combining intracellular deposition of biocytin with NADPH diaphorase histochemistry revealed that the vast majority of type III neurons were cholinergic, whereas only a minority of type I and type II neurons were cholinergic. Thus mesopontine cholinergic neurons possess intrinsic ionic currents capable of inducing burst firing. 4. Delineation of the intrinsic membrane properties of identified mesopontine cholinergic neurons, in concert with recent results regarding the responses of these neurons to neurotransmitter agents, has led us to present a unifying and mechanistic hypothesis of brain stem cholinergic function in the control of behavioral states.


Fibroblast growth factor differentially modulates the neurotransmitter phenotype of cultured sympathetic neurons. Fibroblast growth factors (FGFs) are multifunctional growth factors that increase the proliferation of mesoderm- and neuroectoderm-derived cells and promote neuronal survival and neurite outgrowth in various regions of the brain, yet the physiological role(s) they may play in nervous system function and/or development is unclear. The present report demonstrates, using a well-characterized system, avian sympathetic neurons in vitro, that acidic and basic FGFs increase ChAT but decrease tyrosine hydroxylase (TH) activity in these cells, without affecting neuronal growth and survival. Heparin, which binds to FGFs with a high affinity, potentiates the activity of FGF on ChAT, but not TH. The time course of FGF action on the neurotransmitter phenotype is slow since effects start to appear after 1-2 d only. FGFs may thus modulate the activities of ChAT and TH by differentially regulating the expression of the genes coding for these enzymes. In conclusion, this report provides evidence supporting the hypothesis that FGFs may play a role in regulating neurotransmitter expression in sympathetic neurons during development independently of any effect on neuronal survival.


Calcium concentration dynamics produced by synaptic activation of CA1 hippocampal pyramidal cells. The spatial and temporal dynamics of many electrophysiological and biochemical processes in nerve cells are in turn dependent on the concentration dynamics of the second messenger calcium. We have used microfluorimetry of the calcium indicator fura-2 (Grynkiewicz et al., 1985) to measure and characterize synaptically activated calcium changes in individual CA1 pyramidal cells contained within guinea pig hippocampal slices. One component of the calcium changes was largely produced by influx through voltage-dependent Ca2+ channels (VDCCs). It consisted of large transient accumulations in the proximal-apical and basal dendrites; the amplitude was smaller in the distal-apical dendrites and the soma. This spatial profile was insensitive to the method of cell activation: stimulation of inputs located at different positions on the dendritic tree as well as antidromic stimulation produced only slight modifications. This component was not blocked by the NMDA antagonist 5-amino-4-phosphonovalerate (AP5) (Collingridge et al., 1983), was greatly reduced by Cd2+, partially reduced by nifedipine, and was increased by Bay-K 8644, providing the evidence that it was largely produced by influx through VDCCs. Blocking postsynaptic Na+ channels with QX-314 greatly reduced the accumulation amplitude, and spatial differences between proximal-dendritic and distal-dendritic regions were less pronounced, suggesting that active sodium conductances contribute significantly to the spatial activation of calcium conductances. Residual spatial differences that persist in QX-314 experiments are consistent with the idea that VDCCs have decreased density on distal-apical dendrites. A second component of accumulation was induced by ionic currents through NMDA receptor channels. It was blocked by AP5, unaffected by QX-314, attenuated and slowed down by elevated calcium buffering, and spatially localized to regions receiving activated synaptic inputs. The magnitude of this component was strongly dependent on the frequency and amplitude of synaptic activation. At high frequency, it was generally very large, often saturating the fura-2 (> 2 microM). Measurements made with the indicator furaptra also showed large localized AP5-sensitive fluorescence changes. Our results suggest that in dendritic regions near activated input fibers calcium levels may reach 2-10 microM. In general, our measurements of calcium dynamics provide an experimental basis for evaluating the spatial distribution of calcium conductances, the spatial distribution of calcium-activated electrophysiological and biochemical processes, and the spatial uniformity of calcium buffering and removal systems in CA1 hippocampal pyramidal cells. The time course and amplitude of Ca2+ transients we measured suggest that activation of Ca(2+)-dependent conductances [e.g., IK(Ca)] will be markedly different for different cellular regions.(ABSTRACT TRUNCATED AT 400 WORDS)


Long-term synaptic depression in the striatum: physiological and pharmacological characterization. The effect of tetanic activation of corticostriatal glutamatergic fibers was studied in striatal slices by utilizing extracellular and intracellular recording techniques. Tetanic stimulation produced a long-term synaptic depression (LTD) (> 2 h) of both extracellularly recorded field potentials and intracellularly recorded EPSPs. LTD was not coupled with changes of intrinsic membrane properties of the recorded neurons. In some neurons, repetitive cortical activation produced a short-term posttetanic potentiation (1-3 min). Subthreshold tetanic stimulation, which under control condition did not cause LTD, induced LTD when associated with membrane depolarization. Moreover, LTD was not expressed in cells in which the conditioning tetanus was coupled with hyperpolarization of the membrane. Bath application of aminophosphonovalerate (30-50 microM), an antagonist of NMDA receptors, did not affect the amplitude of the synaptic potentials and the expression of LTD. Striatal LTD was significantly reduced by the pretreatment of the slices with 30 microM 2-amino-3-phosphonopropionic acid, an antagonist of glutamate metabotropic receptors. LTD was not blocked by bicuculline (30 microM), a GABA(A) receptor antagonist. Scopolamine (3 microM), an antagonist of muscarinic receptors, induced a slight, but significant, increase of the amplitude of LTD. Both SCH 23390 (3 microM), an antagonist of D1 dopamine (DA) receptors, and I-sulpiride (1 microM), an antagonist of D2 DA receptors, blocked LTD. LTD was also absent in slices obtained from rats in which the nigrostriatal DA system was lesioned by unilateral nigral injection of 6-hydroxydopamine. In DA-depleted slices, LTD could be restored by applying exogenous DA (30 microM) before the conditioning tetanus. In DA-depleted slices, LTD could also be restored by coadministration of SKF 38393 (3-10 microM), a D1 receptor agonist, and of LY 171555 (1-3 microM), a D2 receptor agonist. Application of a single class of DA receptor agonists failed to restore LTD. These data show that striatal LTD requires three main physiological and pharmacological conditions: (1) membrane depolarization and action potential discharge of the postsynaptic cell during the conditioning tetanus, (2) activation of glutamate metabotropic receptors, and (3) coactivation of D1 and D2 DA receptors. Striatal LTD may alter the output signals from the striatum to the other structures of the basal ganglia. This form of synaptic plasticity can influence the striatal control of motor activity.


Energy requirements of glutamatergic pathways in rabbit retina. In vitro rabbit retina was used as an example of CNS tissue in experiments designed to measure the energy requirements associated with the activation of different types of glutamate receptors. Retinas were exposed to glutamate and to four analogs: kainate, 2-amino-4-phosphonobutyric acid (APB), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and 2-amino-5-phosphonovaleric acid (APV). The changes in O2 consumption and lactate production were determined using a recently developed experimental system that permitted simultaneous measurements of the rates at which O2 was removed from the medium and acid was added. The glutamatergic agents had relatively little effect on oxidative metabolism, but they caused large changes in glycolysis. Kainate increased retinal lactate production by 50%, whereas APB, CNQX, and APV reduced it by 23%, 19%, and 35%, respectively. Glutamate increased lactate production by 16% when administered after APB, but decreased it by 12% when administered after CNQX. The changes in energy metabolism coincided with changes in electrophysiological function. Since the energy metabolism of many retinal cells was presumably not much affected by the glutamatergic agents, the changes measured as a percent of total retinal glycolysis must have reflected considerably larger fractional changes in the cells most affected. From the response to inhibitors, it seems probable that even under resting conditions in darkness, activity in glutamatergic pathways is responsible for more than 50% of the glycolytically derived energy used by the cells involved. It also seems probable (particularly from the response to kainate) that under some circumstances the cells' energy metabolism and/or transport capability cannot meet the requirements imposed by glutamate-induced increases in function.(ABSTRACT TRUNCATED AT 250 WORDS)


Identified postnatal mesolimbic dopamine neurons in culture: morphology and electrophysiology. To examine the intrinsic properties of postnatal mesolimbic dopamine (DA) neurons, we dissociated the ventral tegmental area (VTA) from postnatal rats, enriched for DA neurons by microdissection or gradient purification, and grew the cells in culture. In these cultures, up to 50% of neurons were dopaminergic. DA neurons resembled their in vivo counterparts in soma shapes, and in showing two levels of tyrosine hydroxylase (TH) expression, axodendritic differentiation, two sizes of synaptic vesicles, nest-like synaptic arrangements with non-DA cells, and synaptic specializations. Electrophysiologically, however, they could not be distinguished from non-DA cells, which could be consistent with heterogeneity in cell properties. To examine a functional subset of VTA DA neurons, we retrogradely labeled VTA neurons projecting to the nucleus accumbens. These mesoaccumbens neurons were 86% TH positive, 56% cholecystokinin positive, and 0% neurotensin positive; they also displayed the soma shapes characteristic of DA neurons more generally and two levels of TH expression. Like their in vivo counterparts, mesoaccumbens cells generally fired single broad spikes that were triggered by slow depolarizations and had robust spike afterhyperpolarizations, low- and high-threshold Ca2+ spikes, rapid accommodation of firing, time-dependent anomalous rectification, and hyperpolarizing autoreceptor responses. Strikingly, the expression of these active properties did not change with time in culture. Mesoaccumbens DA cells could be identified by a distinctive subset of properties that made up an electrophysiological signature; however, unlike their in vivo counterparts, they were less often spontaneously active and never fired in bursts. These results suggest that most DA cell properties are intrinsic to the cells, including a significant heterogeneity that is maintained in postnatal culture; their level and mode of activity, however, appear to require afferent input. Culturing identified postnatal VTA DA neurons now makes possible examination of the impact of their individual properties on synaptic function.


The regional vulnerability to hypoglycemia-induced neurotoxicity in organotypic hippocampal culture: protection by early tetrodotoxin or delayed MK-801. Profound hypoglycemia selectively damages CA1 and the dentate gyrus of the hippocampus. We have examined the time course of hippocampal neuronal injury in organotypic cultures following in vitro "hypoglycemia," using the fluorescent vital dye propidium iodide to observe directly the regional distribution of early neuronal membrane injury in living cultures. The in vivo hippocampal pattern of hypoglycemic injury was reproduced by a 2 hr exposure to glucose-free media, which resulted in simultaneous, selective propidium staining of CA1 and the dentate gyrus starting by 4 hr after exposure. After 24 hr of recovery, CA3 remained spared. A similar pattern of propidium staining was produced by incubation of cultures for briefer periods in glucose-free medium containing 5 mM 2-deoxyglucose (2-DG) to inhibit glycolysis. This "hypoglycemic" pattern and time course of neuronal injury was mimicked by 300 microM aspartate but not by glutamate. The NMDA receptor antagonists MK-801 and CPP, but not the relatively selective non-NMDA receptor antagonist 6-cyano-7-dinitroquinoxaline-2,3-dione, prevented the development of propidium staining. MK-801 protected against injury even if added to the recovery media 30 min after the insult, while TTX (10 microM) protected only if added by the end of the exposure. The appearance of propidium staining after 4-6 hr of recovery was well correlated with histological observation of pyknotic neuronal nuclei in the injured regions. The characteristic hippocampal regional vulnerability of CA1 and the dentate gyrus to injury following profound hypoglycemia can be reproduced in organotypic hippocampal culture and appears to be mediated both by an early TTX-sensitive component and by a more prolonged period of toxic NMDA receptor activation, extending for at least 30 min into the recovery period.