Ethanol-induced conditioned place preference is expressed through a ventral tegmental area dependent mechanism

The authors examined the role of the ventral tegmental area (VTA) and nucleus accumbens (NAc) in the expression of ethanol-induced conditioned place preference (CPP). After cannulas were implanted, male DBA/2J mice underwent an unbiased Pavlovian-conditioning procedure for ethanol-induced CPP. Before preference testing, the mice were injected intra-VTA (Experiments 1 and 3) or intra-NAc (Experiment 2) with the nonselective opioid antagonist methylnaloxonium (0-ng, 375-ng, or 750-ng total infusion; Experiments 1 and 2) or the gamma aminobutyric acid (GABA(B)) agonist baclofen (0-ng, 25-ng, or 50-ng total infusion; Experiment 3). Intra-VTA methylnaloxonium or baclofen decreased ethanol-induced CPP, whereas intra-NAc methylnaloxonium had no effect. These findings indicate that the conditioned rewarding effect of ethanol is expressed through a VTA-dependent mechanism that involves both opioid and GABA(B) receptors.     

Ability of bacteria associated with chronic inflammatory disease to stimulate E-selectin expression and promote neutrophil adhesion.

Porphyromonas gingivalis, Pseudomonas aeruginosa, and Helicobacter pylori have been shown to be associated with adult periodontal disease, chronic lung infections, and peptic ulcers, respectively. The ability of these bacteria to stimulate E-selectin expression and promote neutrophil adhesion, two components necessary for the recruitment of leukocytes in response to infection, was investigated. Little or no stimulation of E-selectin expression was observed with either P. gingivalis or H. pylori when whole cells, lipopolysaccharide (LPS), or cell wall preparations added to human umbilical cord vein endothelial cells were examined. P. aeruginosa was able to induce E-selectin to near-maximal levels; however, it required approximately 100 to 1,000 times more whole cells or LPS than that required by E. coli. Neutrophil-binding assays revealed that LPS and cell wall preparations obtained from these bacteria did not promote endothelial cell adhesiveness by E-selectin-independent mechanisms. In addition, P. gingivalis LPS blocked E-selectin expression by LPS obtained from other bacteria. We propose that lack of E-selectin stimulation and the inability to promote endothelial cell adhesiveness are two additional indications of low biologically reactive LPS. We suggest that this property of LPS may contribute to host tissue colonization. In addition, the ability of P. gingivalis to inhibit E-selectin expression may represent a new virulence factor for this organism.     

Immunoblot analysis of anti-Ureaplasma urealyticum antibody in pregnant women and newborn infants.

The purpose of the present study was to determine the frequency in which antibody reactive to Ureaplasma urealyticum could be detected in a population of pregnant women and newborn infants. Serum samples from a prospective cohort of 80 healthy, U. urealyticum culture-positive and culture-negative pregnant women and a retrospective cohort of 522 infants born at between 25 and 42 weeks of gestation were studied by immunoblot analysis. Cultures of specimens from the lower genital tract were positive for U. urealyticum for 83% of the pregnant women, and serum immunoglobulin G (IgG) antibody which reacted to U. urealyticum was detectable in 93% of the pregnant women. Samples from five women (8%) had increases in the number of anti-U. urealyticum IgG bands over the course of the pregnancy. Samples from four of these five women had corresponding increases in the number of antibody bands present in IgA immunoblots. Six of the 522 samples from newborns or cord blood (1.1%) were positive for anti-U. urealyticum IgA; 5 of these 6 samples were also positive for IgM. The six anti-U. urealyticum IgA-positive infants were distributed as follows; 3 of 67 (4.5%) infants were delivered at 25 to 30 weeks of gestation, 3 of 176 (1.7%) infants were delivered at 31 to 34 weeks of gestation, and 0 of 279 infants were delivered at > or = 35 weeks of gestation. An antibody response to U. urealyticum can be detected in pregnant women and preterm infants and may serve as a marker of infection.     

Microcarrier culture of hepatocytes in whole plasma for use in liver support bioreactors.

Contact activation of plasma clotting may limit the use of some microcarrier types for hepatocyte attachment in a liver assist device. Activation by seven microcarrier types was studied in plasma containing 2-500 units heparin/mL. Clotting was activated by dextran (Cytodex 1 and 2) and collagen-coated (Cytodex 3) microcarriers at 2-25 units/mL (Cytodex 1) and 2-100 units/mL (Cytodex 2 and 3). There was no activation by polystyrene, gelatin, glass or fibronectin-coated polystyrene microcarriers. Compared with culture medium, incubation of HepG2 cells in plasma did not affect cell viability but increased cell number (56.4 versus 65.1 x 10(4) cells; P less than 0.05) and incorporation of [3H]-amino acids into protein (204913 versus 279624 dpm; P less than 0.05). Polystyrene-attached cells demonstrated time-linear protein synthesis, glucose and 7-ethoxycoumarin metabolism. We conclude that polystyrene-attached hepatocytes maintain viability and metabolic activity in plasma and are of potential use in a liver support bioreactor.     

Use of Leu M1 and antiepithelial membrane antigen monoclonal antibodies for diagnosing Hodgkin's disease

Biopsies of 82 patients diagnosed as having Hodgkin's disease were reviewed. Seventeen were reclassified histologically as non-Hodgkin's lymphoma or reactive lymphoid hyperplasia. A substantial number of cases of Hodgkin's disease were negative when stained with Leu M1. Staining for Leu M1 was not found in the cases of non-Hodgkin's lymphoma or reactive lymphoid hyperplasia. With the exception of the lymphocyte predominant nodular subtype of Hodgkin's disease, epithelial membrane antigen staining was seen in a few cases of Hodgkin's disease and non-Hodgkin's lymphomas. This was not a useful discriminating feature.     

Resting whole blood viscosity of elite rowers is related to performance

This study investigated the relationships between resting whole blood viscosity (WBV), haemoglobin concentration (HGB), haematocrit (HCT), and performance in 25 highly-trained national squad rowers (11 women and 14 men). The WBV and HGB were measured at rest prior to a 2500 m simulated race on a Concept rowing ergometer when performance (P) was measured by average velocity. A group of 12 rowers were measured on just one occasion, another 11 were measured twice with an intervening 5 weeks of continued training and 2 were measured three times, the third test after another 4 weeks. Regression analyses making simultaneous use of both intra- and interindividual data indicated a significant inverse relationship between P and WBV (at both high and low shear rates), a relationship which was strengthened after statistically controlling for the effects of HGB, this effect being slightly more significant than HCT. A significant positive regression also emerged between P and HGB, but only after statistically controlling for the influence of WBV at high shear rate. Overall, stronger relationships were demonstrated in the male rowers compared with the female. These data, in the light of previous evidence that fitter people tend to have lower WBV, would indicate that blood rheology unrelated to HGB (or HCT) is related to performance in relatively homogeneous and already highly-trained athletes.     

Immunological Relationship between the Class I Epitope of Streptococcal M Protein and Myosin

The class I epitope of streptococcal M protein is an epidemiological marker for acute rheumatic fever (ARF)-associated serotypes of group A streptococci and is recognized by anti-M protein monoclonal antibody (MAb) 10B6. Using MAb 10B6, we determined the relationship between the class I epitope of M protein and the α-helical coiled-coil protein myosin. MAb 10B6 reacted by enzyme-linked immunosorbent assay and Western blotting with human cardiac myosin and rabbit skeletal myosin and its heavy meromyosin (HMM) subfragment. Overlapping synthetic peptides of M5 protein were used to identify the region of M5 protein recognized by MAb 10B6. Two C repeat peptides (C2A and C3) containing the amino acid sequence KGLRRDLDASREAK reacted with MAb 10B6. Partial sequence identity, RRDL, was found in the HMM fragment of myosin, which reacted with MAb 10B6. However, not all peptides of M5 protein and myosin containing the RRDL sequence reacted with MAb 10B6. ARF sera and sera from uncomplicated pharyngitis (UNC) reacted with C repeat region peptides of M protein, while acute glomerulonephritis sera were not as reactive. Affinity-purified human antibody to peptide C3 reacted with myosin. The data demonstrate that the class I epitope of M protein is immunologically cross-reactive with myosin and the HMM subfragment, and antibodies to peptide C3 and myosin were present in ARF and UNC sera.Acute rheumatic fever (ARF) is an inflammatory disease that can follow group A streptococcal pharyngitis. The most serious clinical manifestation is rheumatic carditis; however, arthritis, chorea, erythema marginatum, or subcutaneous nodules may be present (40, 41). The pathogenesis of ARF is thought to be mediated by autoimmune mechanisms activated during a streptococcal infection (40). The autoimmune hypothesis is supported by a number of previous observations, including a time interval of at least 3 weeks between the initial streptococcal throat infection and the onset of ARF (40, 41), the identification of heart-reactive immunoglobulin (Ig) and complement deposits in the myocardium of patients with fatal rheumatic carditis (25–27, 30), and the elevation of heart-reactive antibodies in the sera of patients with ARF (46). Cardiac myosin has been identified as one of the cardiac antigens recognized by these heart-reactive antistreptococcal autoantibodies (13, 29).Streptococcal M protein, an α-helical coiled-coil protein, structurally and immunologically mimics host tissue antigens, particularly the rod region of myosin (12, 14, 15, 17, 34, 35). Sequence analysis has revealed that streptococcal M proteins contain blocks of internally repeated amino acid sequences referred to as A, B, and C repeat regions (19). The NH2-terminal nonrepeat and A repeat regions contain determinants of type specificity, while epitopes found in the B and more highly conserved C repeat regions may be common to different M serotypes (19). While there are nearly 100 different serological types of group A streptococcal M protein, epidemiological studies indicate that only a limited number of M protein serotypes are associated with ARF outbreaks (6). This finding suggests that certain M protein serotypes may be more rheumatogenic than others. In a previous attempt to classify streptococcal serotypes according to their rheumatogenic capacity, Widdowson identified human antisera directed to a non-type-specific protein moiety of M protein known as M-associated protein (44, 45). However, a more recent classification scheme has been proposed by Bessen and colleagues, in which streptococcal serotypes were grouped based on the expression of a conserved surface-exposed M protein epitope (4). It was demonstrated that the M serotypes associated with the majority of ARF outbreaks possessed an epitope (class I) defined by monoclonal antibody (MAb) probes 10B6 and 10F5. The sequence of the 10B6 and 10F5 epitope was localized to a 15-amino-acid fragment within the C repeat region of the type 6 M protein (23). The remaining serotypes (class II) lack this epitope or the determinant is structurally inaccessible in those strains. There was a close parallel between serotypes designated class I and those serotypes previously classified as M-associated protein I by Widdowson (44, 45). The fact that only certain serotypes within class I streptococci are rheumatogenic implies that these organisms are of a phenotype that is capable of inducing ARF (4). This implication is supported in part by a recent publication in which it was shown that sera of ARF patients contained high levels of antibodies to the class I epitope, suggesting that their disease was the result of an infection by a class I streptococcus (5).Elevated titers of antibodies to many streptococcal antigens (2), including M protein and the self-antigen myosin (12–15, 17, 29), are associated with ARF. While antibodies to M protein are crucial for the opsonization of streptococci, they have also been implicated in the immunological cross-reactions between streptococci and host tissue antigens such as cardiac myosin (12–15, 17, 29). In earlier studies, many of these cross-reactive epitopes have been localized to the N-terminal, hypervariable A and B repeat regions of the M molecule (12, 15, 17). Myosin-reactive antibodies, found to be elevated in almost all cases of ARF (13), have been shown to bind to human heart tissue and to cross-react with streptococcal M protein (12). Previous studies have demonstrated that immunization of animals with the cell walls of certain strains of group A streptococci resulted in the production of heart-reactive antibodies which could be adsorbed with streptococcal extracts containing streptococcal M protein (16, 24, 28). Human MAbs or myosin affinity-purified antibodies produced from patients with ARF cross-reacted with streptococcal M protein and human cardiac myosin and contributed to the presence of heart-cross-reactive antistreptococcal antibodies in ARF (12, 13, 39). More recent studies have identified cytotoxic antistreptococcal/antimyosin MAbs from rheumatic carditis patients (1). Antimyosin antibody has been shown to deposit in the heart tissues of susceptible mice (31), and a cytotoxic mouse antistreptococcal/antimyosin antibody which binds to the surface of heart cells and to the α-helical coiled coil molecule laminin has been described (10).Identification of myosin cross-reactive epitopes of M protein recognized in ARF has been reported for the amino-terminal half of the molecule (12, 15, 17), and a study by Vashishtha and Fischetti demonstrated antimyosin antibody responses to the C repeat region. However, the reactivity was directed only to denatured myosin (43). More recently, studies of the C repeat or carboxy-terminal region of M protein have shown T-cell cross-reactions with myosin (38). The goal of the present study was to investigate the possibility that the class I epitope in the C repeat region of M protein cross-reacts immunologically with myosin. In this study we show that MAb 10B6, which recognizes the class I epitope of M protein, reacts with cardiac and skeletal myosin. This study also demonstrates that ARF and UNC sera react with a site in the conserved C repeat region of M protein within the class I epitope of rheumatogenic M protein serotypes. The new data show that in addition to previously described N-terminal epitopes, the class I epitope of streptococcal M protein is immunologically cross-reactive with myosin.(Portions of this work were presented at the XIII International Lancefield Society Meeting on Streptococci and Streptococcal Diseases at the Pasteur Institute in Paris, France, in September 1996.)     

Aberrant actin cytoskeleton leads to accelerated proliferation of corneal epithelial cells in mice deficient for destrin (actin depolymerizing factor)

Corneal disease is the most common cause of bilateral blindness in the world. Visual loss in this condition is often due to changes in morphology and function of the corneal epithelial surface. Corneal disease-1 (corn1) and corn1(2J) are spontaneous mouse mutants that develop irregular thickening of the corneal epithelium, similar to that observed in human corneal surface disease. These autosomal-recessive mutations cause an increase in the rate of proliferation of the corneal epithelial cells. Here, we report that the phenotypes in both mutants are caused by mutations within the destrin gene (also known as actin-depolymerizing factor). By positional cloning, we identified a deletion encompassing the entire coding sequence of the destrin gene in corn1 mice, and a point mutation (Pro106Ser) in the coding sequence of destrin in corn1(2J) mice. In situ analysis showed that destrin is highly expressed in the corneal epithelium. Consistent with the cellular roles for destrin, an essential regulator of actin filament turnover that acts by severing and enhancing depolymerization of actin filament, we observed that the corn1 mutations increased the content of filamentous actin in corneal epithelial cells. Our results suggest an in vivo connection between remodeling of the actin cytoskeleton and the control of cell proliferation, and a new pathway through which an aberrant actin cytoskeleton can cause epithelial hyperproliferation.     

Care of vaginal symptoms among HIV-infected women

OBJECTIVE: Gynecologic disease is common in HIV-infected women. We examine the sociodemographic, clinical, and provider factors associated with the care of women with vaginal symptoms. METHODS: Women enrolled in the HIV Cost and Services Utilization Study (HCSUS), a nationally representative probability sample of HIV-infected adults, were interviewed between January 1996 and April 1997. Women with vaginal symptoms who sought medical attention were asked, "Did your health care provider examine your vaginal area?" Women were also asked if they received medication for their symptoms. RESULTS: Among 154 women with vaginal symptoms, 127 sought care for their symptoms. Of those who sought care, 48% saw a gynecologist and 52% sought care from nongynecologists, most often their usual HIV care provider. Women who saw a gynecologist for their symptoms were more likely to have received a pelvic examination (92% versus 76%; p =.06) and vaginal fluid collection (98% versus 88%; p =.06) than those who saw their regular HIV provider. Fifteen percent of women received medication for their symptoms without having a pelvic examination; gynecologists were less likely to prescribe without an examination (8% versus 21%; p =.12). CONCLUSION: Gynecologists are more likely to provide adequate care of vaginal symptoms among HIV-infected women than nongynecologists who were HIV care providers. This specialty difference is consistent with quality of care studies for other medical conditions, but the potential gynecologic complications of inadequate evaluation and treatment warrants further investigation.     

Mutations in the Ca(2+)-sensing receptor gene cause autosomal dominant and sporadic hypoparathyroidism

Parathyroid hormone secretion is negatively regulated by a 7- transmembrane domain, G-protein coupled Ca(2+)-sensing receptor. We hypothesized that activating mutations in this receptor might cause autosomal dominant hypoparathyroidism (ADHP). Consistent with this hypothesis, we identified, in two families with ADHP, heterozygous missense mutations in the Ca(2+)-sensing receptor gene that cosegregated with the disorder. None of 50 normal controls had either mutation. We also identified a de novo, missense Ca(2+)-sensing receptor mutation in a child with severe sporadic hypoparathyroidism. The amino acid substitution in one ADHP family affected the N-terminal, extracellular domain of the receptor. The other mutations involved the transmembrane region. Unlike patients with acquired hypoparathyroidism, patients with these mutations had hypercalciuria even at low serum calcium concentrations. Their greater hypercalciuria presumably reflected activation of Ca(2+)-sensing receptors in kidney cells, where the receptor negatively regulates calcium reabsorption. This augmented hypercalciuria increases the risk of renal complications and thus has implications for the choice of therapy.