Production and characterization of monoclonal antibodies directed against Bordetella pertussis lipopolysaccharide.

Hybrid cell lines producing monoclonal antibodies against Bordetella pertussis lipopolysaccharide (LPS) were established. The specificity of the antibodies was ascertained by enzyme-linked immunosorbent assay (ELISA) and ELISA-inhibition experiments with LPS and delipidated polysaccharide fragments (PS-1 and PS-2) prepared from B. pertussis LPS. Monoclonal antibody 9-1-H5 reacted with B. pertussis LPS only, whereas monoclonal antibodies 6-4-H6 and 9-2-A8 reacted with PS-1 and PS-2 as well as B. pertussis LPS. The antibodies did not react with LPS prepared from B. parapertussis and B. bronchiseptica in an LPS-specific ELISA. A monoclonal antibody-based sandwich ELISA was developed for detection of B. pertussis LPS. This assay had a detection limit of B. pertussis LPS in concentrations ranging from 0.16 to 0.32 microgram/ml. The assay was also shown to be specific for the detection of whole B. pertussis bacteria. No cross-reactions were observed with strains of Branhamella catarrhalis, Neisseria meningitidis, Streptococcus miteor, Haemophilus influenzae, or Legionella pneumophila. The monoclonal antibodies might be useful for the detection of soluble antigens and whole bacteria in clinical samples and for studies of the immunochemical structure of B. pertussis LPS.     

Evolution of the neuropeptide Y receptor family: gene and chromosome duplications deduced from the cloning and mapping of the five receptor subtype genes in pig

Neuropeptide Y (NPY) receptors mediate a variety of physiological responses including feeding and vasoconstriction. To investigate the evolutionary events that have generated this receptor family, we have sequenced and determined the chromosomal localizations of all five presently known mammalian NPY receptor subtype genes in the domestic pig, Sus scrofa (SSC). The orthologs of the Y(1) and Y(2) subtypes display high amino acid sequence identities between pig, human, and mouse (92%-94%), whereas the Y(4), Y(5), and y(6) subtypes display lower identities (76%-87%). The lower identity of Y(5) is due to high sequence divergence in the large third intracellular loop. The NPY1R, NPY2R, and NPY5R receptor genes were localized to SSC8, the NPY4R to SSC14, and NPY6R to SSC2. Our comparisons strongly suggest that the tight cluster of NPY1R, NPY2R, and NPY5R on human chromosome 4 (HSA4) represents the ancestral configuration, whereas the porcine cluster has been split by two inversions on SSC8. These 3 genes, along with adjacent genes from 14 other gene families, form a cluster on HSA4 with extensive similarities to a cluster on HSA5, where NPY6R and >13 other paralogs reside, as well as another large cluster on HSA10 that includes NPY4R. Thus, these gene families have expanded through large-scale duplications. The sequence comparisons show that the NPY receptor triplet NPY1R-NPY2R-NPY5R existed before these large-scale duplications.     

Longitudinal study of viruses associated with canine infectious respiratory disease

In this investigation a population of dogs at a rehoming center was monitored over a period of 2 years. Despite regular vaccination of incoming dogs against distemper, canine adenovirus type 2 (CAV-2), and canine parainfluenza virus (CPIV), respiratory disease was endemic. Tissue samples from the respiratory tract as well as paired serum samples were collected for analysis. The development of PCR assays for the detection of CPIV, canine adenovirus types 1 and 2, and canine herpesvirus (CHV) is described. Surprisingly, canine adenovirus was not detected in samples from this population, whereas 19.4% of tracheal and 10.4% of lung samples were positive for CPIV and 12.8% of tracheal and 9.6% of lung samples were positive for CHV. As reported previously, a novel canine respiratory coronavirus (CRCoV) was detected in this population (K. Erles, C. Toomey, H. W. Brooks, and J. Brownlie, Virology 310:216-223, 2003). Infections with CRCoV occurred mostly during the first week of a dog's stay at the kennel, whereas CPIV and CHV were detected at later time points. Furthermore, the evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to CPIV and an immunofluorescence assay for detection of antibodies to CHV is described. This study shows that CPIV is present at kennels despite vaccination. In addition, other agents such as CHV and CRCoV may play a role in the pathogenesis of canine respiratory disease, whereas CAV-2 and canine distemper virus were not present in this population, indicating that their prevalence in the United Kingdom is low due to widespread vaccination of dogs.     

Endotoxins prevent murine IgE production,T(H)2 immune responses,and development of airway eosinophilia but not airway hyperreactivity

BACKGROUND: Contact with immunomodulatory factors, such as LPS, in early infancy is associated with decreased allergen sensitization. OBJECTIVE: We sought to study the effects of systemic or airway exposure with LPS on the development of allergen sensitization, eosinophilic airway inflammation, and increased in vivo airway reactivity (AR) in a mouse model. METHODS: BALB/c mice were systemically sensitized with ovalbumin (OVA) plus adjuvant on days 1 and 14 and challenged through the airways with allergen on days 34 to 36. We performed measurement of OVA-specific IgE serum levels, in vitro T(H)2 cytokine production, differential cell counts in bronchoalveolar lavage fluids, and assessment of in vivo AR to inhaled methacholine by means of barometric whole-body plethysmography. RESULTS: Systemic LPS administration before OVA sensitization reduced OVA-specific IgE serum levels (426 +/- 76 vs 880 +/- 104 U/mL, P <.01), T(H)2 cytokine production by splenic mononuclear cells (IL-4: 0.08 +/- 0.01 vs 0.17 +/- 0.01 ng/mL; IL-5: 1.98 +/- 0.52 vs 4.11 +/- 0.54 ng/mL; P <.01), and extent of airway eosinophilia (total cell counts: 93 vs 376 x 10(3)/mL; eosinophils: 23% vs 51%; P <.01) compared with that in OVA-sensitized mice. Local LPS administration to sensitized mice before airway allergen challenges particularly induced IFN-gamma production by peribronchial lymph node cells in vitro (1718 +/- 315 vs 483 +/- 103 ng/mL, P <.01) associated with reduced airway eosinophilia compared with that seen in OVA-sensitized mice. Development of increased AR was not affected by systemic or local LPS exposure. Inhibitory effects of LPS on allergen sensitization and eosinophilic airway inflammation were inhibited by administration of anti-IL-12 antibodies before LPS exposure. CONCLUSION: These data indicate that local and systemic application of LPS modulates systemic and local T(H)1/T(H)2 immune responses in a distinct but similarly IL-12-dependent mode.     

Yersinia pseudotuberculosis inhibits Fc receptor-mediated phagocytosis in J774 cells.

Nonopsonized as well as immunoglobulin-G (IgG)-opsonized Yersinia pseudotuberculosis resists phagocytic uptake by the macrophage-like cell line J774 by a mechanism involving the plasmid-encoded proteins Yops. The tyrosine phosphatase YopH was of great importance for the antiphagocytic effect of the bacteria. YopH-negative mutants did not induce antiphagocytosis; instead, they were readily ingested, almost to the same extent as that of the translocation mutants YopB and YopD and the plasmid-cured strain. The bacterial determinant invasin was demonstrated to mediate phagocytosis of nonopsonized bacteria by these cells. In addition to inhibiting uptake of itself, Y. pseudotuberculosis also interfered with the phagocytic uptake of other types of prey: J774 cells that had been exposed to virulent Y. pseudotuberculosis exhibited a reduced capacity to ingest IgG-opsonized yeast particles. This effect was impaired when the bacterium-phagocyte interaction occurred in the presence of gentamicin, indicating a requirement for in situ bacterial protein synthesis. The Yersinia-mediated antiphagocytic effect on J774 cells was reversible: after 18 h in the presence of gentamicin, the phagocytic capacity of Yersinia-exposed J774 cells was completely restored. Inhibition of the uptake of IgG-opsonized yeast particles was dependent on the Yops in a manner similar to that seen for blockage of Yersinia phagocytosis. This similarity suggests that the pathogen affected a general phagocytic mechanism. Despite a marked reduction in the capacity to ingest IgG-opsonized yeast particles, no effect was observed on the binding of the prey. Taken together, these results demonstrate that Yop-mediated antiphagocytosis by Y. pseudotuberculosis affects regulatory functions downstream of the phagocytic receptor and thereby extends to other types of phagocytosis.     

Effects of prednisolone treatment on cytokine expression in patients with leprosy type 1 reactions

Leprosy type 1 reactions (T1R) are due to increased cell-mediated immunity and result in localized tissue damage. The anti-inflammatory drug prednisolone is used for treatment, but there is little good in vivo data on the molecular actions of prednisolone. We investigated the effect of prednisolone treatment on tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-10, and transforming growth factor beta1 (TGF-beta1) mRNA and protein expression in blood and skin biopsies from 30 patients with T1R in India. After 1 month of prednisolone treatment the sizes of the skin granulomas were reduced, as were the grades of cells positive for TNF-alpha and IL-10 in skin lesions. Increased production of TGF-beta1 was seen in skin lesions after 6 months of prednisolone treatment. Expression of mRNA for TNF-alpha, IL-1beta, and TGF-beta1 was reduced, whereas no change in IL-10 mRNA expression was detected during treatment. The circulating cytokine profiles were similar in patients with and without T1R, and prednisolone treatment had no detectable effects on cytokine expression in the blood. The data emphasize the compartmentalization of pathology in T1R and the importance of the immune response in the skin. Clinical improvement and cytokine expression were compared. Surprisingly, patients with improved skin and nerve function and patients with nonimproved skin and nerve function had similar cytokine profiles, suggesting that clinical improvement is not directly mediated by the cytokines studied here. This in vivo well-controlled study of the immunosuppressive effects of prednisolone showed that the drug does not switch off cytokine responses effectively.     

Advances in the pharmacological control of the bladder

To effectively control bladder activity, and to treat urinary incontinence caused by bladder overactivity, identification of suitable targets for pharmacological intervention is necessary. Such targets may be found in the central nervous system (CNS) or peripherally. The causes of bladder overactivity are not known, but theoretically increased afferent activity, decreased inhibitory control in the CNS and/or peripheral ganglia, and increased sensitivity of the detrusor to efferent stimulation may be involved. Several CNS transmitters may modulate voiding, but few drugs with a defined CNS site of action have been developed for treatment of voiding disorders. Potentially, drugs affecting GABA, opioid, 5-HT, noradrenaline, dopamine, or glutamatergic receptors and mechanisms can be developed, but a selective action on the lower urinary tract may be difficult to obtain. Traditionally, drugs used for treatment of bladder overactivity have had a peripheral site of action, mainly the efferent neurotransmission or the detrusor muscle itself. Antimuscarinic drugs, beta-adrenoceptor agonists, alpha-adrenoceptor antagonists, drugs affecting membrane channels, prostaglandin synthetase inhibitors and several other agents have been used. However, none of them has been developed specifically for treatment of bladder disorders, and their efficacy, as judged from controlled clinical trials (where performed), is often limited. Recent information on the alpha-adrenoceptor, beta-adrenoceptor (beta 3), and muscarinic receptor subtypes of the human detrusor and outflow region can be the basis for the development of compounds with effect on bladder overactivity and with improved tolerance. New ways of decreasing acetylcholine release may represent a promising way of controlling bladder contraction. Potassium channel (KATP) openers are theoretically attractive, but the drugs available so far have targeted vascular rather than bladder smooth muscle, which has limited their clinical use. However, new drugs belonging to these groups with an interesting profile of action have been developed. Drugs decreasing afferent activity represent an attractive therapeutic approach and transmitters of afferent nerves and their receptors are possible targets for pharmacological interventions. Tachykinins, such as substance P, neurokinins A and B, and other neuropeptides have been demonstrated in nerves of the lower urinary tract and have been shown to influence bladder function. Agents affecting these nerves by causing release of tachykinins, such as capsaicin and resiniferatoxin, given intravesically can be effective in some cases of bladder overactivity, and agents antagonizing tachykinin receptors may also be of therapeutic interest. New drugs specifically directed for control of bladder activity are under development and will hopefully lead to improved treatment of urinary incontinence.     

PCR for Detection and Identification of Abiotrophia spp.

Members of the genus Abiotrophia, formerly known as nutritionally variant streptococci, are important pathogens causing septicemia and endocarditis. Cultivation and biochemical differentiation of Abiotrophia spp. are often difficult. Based on 16S rRNA sequences, two PCR assays for detection and identification of Abiotrophia spp. were developed. The first PCR assay was positive for all Abiotrophia spp. Subsequently performed restriction fragment length polymorphism analysis allowed the verification of the PCR amplicons and the differentiation of the three species. The second PCR assay was positive only for A. elegans, the most fastidious species of Abiotrophia. Both PCR assays were shown to be specific and sensitive and should facilitate the identification of Abiotrophia spp.     

Mouse macrophage development in the absence of the common γ chain: defining receptor complexes responsible for IL-4 and IL-13 signaling

The common γ chain (γ_c) forms a critical component of the receptors for interleukins (IL)-2, IL-4, IL-7, IL-9, and IL-15. We analyzed γ_c-deficient mice to define a role for γ_c signaling in the development and function of the macrophage lineage. No major differences in absolute cell numbers, cell surface phenotype, or in vitro function of γ^?_c compared to γ⁺_c macrophages were observed. We therefore conclude that signaling through the γ_c chain is not essential for the differentiation of mouse macrophages. Although B and T cells require γ_c for IL-4 responses, IL-4 up-regulated major histocompatibility class II molecules and inhibited nitric oxide production from γ^?_c macrophages following stimulation with lipopolysaccharide and interferon-γ. γ^?_c macrophages could also respond to IL-13, consistent with the model of a type II IL-4 receptor α/IL-13R which can function in the absence of γ_c. Both IL-4 and IL-13 responses could be completely inhibited with the mouse IL-4 antagonist QY, suggesting that all of the observed IL-13 responses pass through the type II receptor, making it the primary signaling receptor complex for IL-13 in mouse macrophages.     

Molecular characterization of human immunodeficiency virus (HIV)-1 and -2 in individuals from guinea-bissau with single or dual infections: predominance of a distinct HIV-1 subtype A/G recombinant in West Africa.

Guinea-Bissau in West Africa has the highest prevalence of human immunodeficiency virus (HIV)-2 infection in the world, but recently the HIV-1 prevalence increased rapidly with the subsequent appearance of HIV-1 and HIV-2 dual infections. Information about the genetic subtypes of HIV in the region is limited. Therefore, we characterized the env V3 region of HIV-1 and HIV-2 variants through direct DNA sequencing of peripheral blood mononuclear cell samples from 18 individuals with HIV-1 only and 9 individuals with dual infection. Phylogenetic analyses of these new sequences and database sequences from other West African countries showed that all HIV-1 and HIV-2 sequences from singly as well as dually infected individuals, except one, clustered among HIV-1 subtype A and HIV-2 subtype A, respectively. Importantly, a majority of the HIV-1 sequences from Guinea-Bissau and neighbouring countries were closely related with the isolates IbNG, DJ263, and DJ264, which share a common subtype A/G recombination pattern. Analysis of pol gene sequences from selected HIV-1 variants showed that "IbNG-like" viruses in Guinea-Bissau are also recombinant, indicating that the HIV-1 epidemic in Guinea-Bissau and neighbouring countries is dominated by an epidemic spread of a distinct subtype A/G recombinant, which is strikingly similar to the epidemic spread of a subtype A/E recombinant in Southeast Asia. Furthermore, the HIV-1 and HIV-2 variants carried by individuals with dual infection were intermixed with variants from singly infected individuals, indicating that variants involved in dual and single infections have common epidemiological histories.