Modeling the interface between islet amyloid polypeptide and insulin-based aggregation inhibitors: correlation to aggregation kinetics and membrane damage.

Human islet amyloid polypeptide (hIAPP) forms cytotoxic fibrils in type-2 diabetes and insulin is known to inhibit formation of these aggregates. In this study, a series of insulin-based inhibitors were synthesized and assessed for their ability to slow aggregation and impact hIAPP-induced membrane damage. Computational studies were employed to examine the underlying mechanism of inhibition. Overall, all compounds were able to slow aggregation at sufficiently high concentrations (10× molar excess); however, only two peptides showed any inhibitory capability at the 1:1 molar ratio (EALYLV and VEALYLV). The results of density functional calculations suggest this is due to the strength of a salt bridge formed with the Arg11 side chain of hIAPP and the inhibitors' ability to span from the Arg11 to past the Phe15 residue of hIAPP, blocking one of the principal amyloidogenic regions of the molecule. Unexpectedly, slowing fibrillogenesis actually increased damage to lipid membranes, suggesting that the aggregation process itself, rather than the fibrilized peptide, may be the cause of cytotoxicity in vivo.

Sequence analysis or rat integrin alphaE1 and alphaE2 subunits: tissue expression reveals phenotypic similarities between intraepithelial lymphocytes and dendritic cells in lymph.

The integrin alphaOX-62 subunit is defined by the OX-62 monoclonal antibody that was raised against rat dendritic cells in lymph (veiled cells) and shows properties similar to those of human alphaE that is predominantly expressed on intraepithelial lymphocytes. To clone alphaOX-62, rat probes generated using primers specific for the human alphaE sequence were used to screen rat T cell cDNA libraries. cDNA clones encoding two similar but not identical alpha subunits that are closely related but distinct from human alphaE were isolated. alphaE1 is predicted to be the rat homolog of mouse alphaM290 and alphaE2 corresponds to rat alphaOX-62. Immunofluorescence analysis revealed that mouse alphaE1 and rat alphaE2 are expressed in dendritic epidermal T cells in the skin, intraepithelial lymphocytes in the small intestine and in cells with a dendritic morphology present at sites where gammadelta T cells occur in lymphoid organs. Unexpectedly, in veiled cells alphaE2 is co-expressed with intracellular CD3-delta and a 33-kDa CD3 chain but not the T cell receptor. These findings suggest that veiled cells may be derived from a lymphoid precursor. Furthermore, veiled cells show phenotypic similarities to intraepithelial lymphocytes.

An optimized set of human telomere clones for studying telomere integrity and architecture.

Telomere-specific clones are a valuable resource for the characterization of chromosomal rearrangements. We previously reported a first-generation set of human telomere probes consisting of 34 genomic clones, which were a known distance from the end of the chromosome ( approximately 300 kb), and 7 clones corresponding to the most distal markers on the integrated genetic/physical map (1p, 5p, 6p, 9p, 12p, 15q, and 20q). Subsequently, this resource has been optimized and completed: the size of the genomic clones has been expanded to a target size of 100-200 kb, which is optimal for use in genome-scanning methodologies, and additional probes for the remaining seven telomeres have been identified. For each clone we give an associated mapped sequence-tagged site and provide distances from the telomere estimated using a combination of fiberFISH, interphase FISH, sequence analysis, and radiation-hybrid mapping. This updated set of telomeric clones is an invaluable resource for clinical diagnosis and represents an important contribution to genetic and physical mapping efforts aimed at telomeric regions.

Examination of the X chromosome by STS-PCR screening for the presence of submicroscopic deletions.

Cytogenetically undetectable deletions are suspected to be an important cause of mental retardation and developmental delay, as suggested by the observation that about 7% of children with undiagnosed mental retardation have rearrangements affecting the chromosome ends. Screening the whole genome for regions of aneuploidy smaller than 5 Mb is not feasible, but the availability of a high resolution map of the X chromosome means that it is possible to look for deletions in males by PCR. We have screened 96 affected males and their 96 unaffected fathers with 110 markers distributed across the X chromosome. No deletions were found in either group. Our results show that the prevalence of deletions greater than 1 Mb in children with mental retardation is less than 3.9% (95% confidence interval). We conclude that X chromosome deletions in the size range 1-5 Mb are a rare cause of mental retardation in males.

Sequence analysis of rat integrin alpha E1 and alpha E2 subunits: tissue expression reveals phenotypic similarities between intraepithelial lymphocytes and dendritic cells in lymph.

The integrin alpha OX-62 subunit is defined by the OX-62 monoclonal antibody that was raised against rat dendritic cells in lymph (veiled cells) and shows properties similar to those of human alpha E2 that is predominantly expressed on intraepithelial lymphocytes. To clone alpha OX-62, rat probes generated using primers specific for the human alpha E sequence were used to screen rat T cell cDNA libraries. cDNA clones encoding two similar but not identical alpha subunits that are closely related to but distinct from human alpha E were isolated. alpha E1 is predicted to be the rat homolog of mouse alpha M290 and alpha E2 corresponds to rat alpha OX-62. Immunofluorescence analysis revealed that mouse alpha E1 and rat alpha E2 are expressed in dendritic epidermal T cells in the skin, intraepithelial lymphocytes in the small intestine and in cells with a dendritic morphology present at sites where gamma delta T cells occur in lymphoid organs. Unexpectedly, alpha E2 is co-expressed with intracellular CD3-delta and a 33-kDa CD3 chain but not the T cell receptor in veiled cells. These findings suggest that veiled cells may be derived from a lymphoid precursor. Furthermore, veiled cells show phenotypic similarities to intraepithelial lymphocytes.

Development of the airway intraepithelial dendritic cell network in the rat from class II major histocompatibility (Ia)-negative precursors: differential regulation of Ia expression at different levels of the respiratory tract.

The relative inefficiency of respiratory mucosal immune function during infancy is generally attributed to the immaturity of the neonatal T cell system. However, immune competence in the adult lung has recently been shown to be closely linked to the functional capacity of local networks of intraepithelial dendritic cells (DC). This study examines the density and distribution of these DC throughout the neonatal respiratory tract in rats, focusing particularly on microenvironmental regulation of their class II major histocompatibility complex (MHC) (Ia) expression. In animals housed under dust-controlled conditions, airway epithelial and alveolar Ia+ DC detectable by immunostaining with the monoclonal antibody (mAb) Ox6 are usually not seen until day 2-3 after birth, and adult-equivalent staining patterns are not observed until after weaning. In contrast, the mAb Ox62 detects large numbers of DC in fetal, infant, and adult rat airway epithelium. Costaining of these Ox62+ DC with Ox6 is rare in the neonate and increases progressively throughout infancy, and by weaning Ia+ DC comprised, on average, 65% of the overall intraepithelial DC population. In infant rats, Ia+ DC are observed first at the base of the nasal turbinates, sites of maximum exposure to inhaled particulates, suggesting that their maturation is driven in part by inflammatory stimuli. Consistent with this suggestion, densitometric analysis of Ia staining intensity of individual DC demonstrates that by 2-3 d after birth, Ia expression by nasal epithelial DC was comparable with that of Iahigh epidermal Langerhans cells in adjacent facial skin, at a time when expression by tracheal epithelial DC was 7-10-fold lower. Additionally, the rate of postnatal appearance of Iahigh DC in the airway epithelium was increased by administration of interferon gamma, and decreased by exposure of infant rats to aerosolized steroid. These findings collectively suggest that Ia expression by neonatal respiratory tract DC is locally controlled and can be upregulated by mediators that are produced within the lung and airway epithelium in response to inhalation of proinflammatory stimuli. It was also noted that Ialow neonatal airway DC expressed adult equivalent levels of class I MHC, which suggests differences in capacity to prime for CD8(+)-dependent versus CD4(+)-dependent immunity to inhaled pathogens, during the early postnatal period.

The MRC OX-62 antigen: a useful marker in the purification of rat veiled cells with the biochemical properties of an integrin.

A mouse immunoglobulin G1 monoclonal antibody (mAb), MRC OX-62 (OX-62), was raised against density gradient-enriched rat veiled (dendritic) cells obtained from lymph. In suspensions of lymphoid cells, the OX-62 mAb only labeled cells with the characteristics of veiled cells. The OX-62 mAb was used with a magnetic cell sorter to enrich or deplete veiled cells, and the enriched veiled cells were potent stimulators in the primary allogeneic mixed leukocyte reaction. Immunohistochemical staining of tissue sections showed that the OX-62 mAb did not label all classical dendritic cells and was not restricted to this cell type. In lymphoid tissues, the labeling correlated with dendritic cells, but in skin, major histocompatibility complex class II+ cells were OX-62-, while another CD3+ cell with dendritic morphology was strongly OX-62+. It seems that the OX-62 mAb may be restricted to dendritic cells and probably to gamma/delta T cells. The OX-62 mAb will be of use in delineating minor subsets of cells with dendritic morphology in various tissues. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of veiled cell-enriched populations immunoprecipitated with the OX-62 mAb gave bands with the biochemical characteristics of an integrin. The OX-62 mAb recognized the alpha-like subunit.

Deficient responsiveness of ovine popliteal peripheral lymphocytes.

Following lymphadenectomy, popliteal peripheral leucocytes present in afferent lymphatics draining the skin can be obtained relatively free of contaminating cells derived from blood and central lymph in contrast to prescapular and hepatic peripheral lymph which contain contaminating cells. Popliteal peripheral lymphocytes comprise a population of long-lived dividing cells that are deficient responders in mixed lymphocyte cultures (MLC) but elicit strong responses following stimulation with phorbol myristate acetate plus ionomycin or concanavalin A. Deficient responsiveness persisted when popliteal peripheral lymphocytes were cultured for 3 weeks prior to setting up MLC. The in vitro results correlate with in vivo results from normal lymphocyte transfer reactions and indicate that peripheral lymphocytes may comprise a population deficient in T-cell receptors mediating allogeneic stimulation.

Indoxyl-tetranitro blue tetrazolium method for detection of alkaline phosphatase in immunohistochemistry.

A sensitive method for detection of alkaline phosphatase in immunohistochemistry, using lymphoid cells, has been optimized. The conditions for staining are 0.23 mM 5-bromo-4-chloro-indoxyl phosphate, 0.55 mM tetranitro blue tetrazolium, 2.0 mM levamisole, 5.0 mM sodium azide, 10.0 mM magnesium chloride, and 0.15 mM 1-methoxyphenazine methosulfate dissolved in 100 mM Tris-HCl buffer, pH 9.5.

Automated fluorometric assay for T cell cytotoxicity.

A fluorometric assay avoiding the use of radioactivity has been developed for detecting cytotoxic T lymphocytes (Tc cells). The method involves labelling targets with Hoechst dye no. 33342 (H33342) which becomes brightly fluorescent on binding to DNA. Lysis of target cells by Tc cells is quantified by measuring the release of fluorescent H33342 into the supernatant of culture wells. The fluorescence is measured using an automated Microfluor reader which allows results to be obtained rapidly. The assay has been used to detect alloreactive Tc cells and H-2 restricted Tc cells against influenza virus in a short-term 6 h assay using P815 and L929 as targets with comparable results to those obtained with 51Cr labelling. In contrast, lymphocyte blasts were found to be less sensitive in 6 h fluorometric assays when compared with the 51Cr assay. In long-term overnight assays (possible because of the low spontaneous release of H33342 from targets) lymphocyte blasts gave high specific lysis and some anti-self reactivity. The cause of the anti-self reactivity may reflect fundamental differences between the H33342 and 51Cr release assays.

Analysis of the genetic control of lymphocyte positioning.

A possible role for the major histocompatibility complex (MHC) in the localization of lymphocytes in different lymphoid organs was investigated using inbred mouse strains. Lymphocytes labelled with the intracellular fluorochrome Hoechst 33342 (H33342) were transfused intravenously (IV) into unimmunized mice and the distribution of these labelled lymphocytes examined. In some combinations (e.g. C57BL/6----CBA) 2 h after injection allogeneic lymphocytes accumulated in the region between the marginal zones and outer aspects of the white pulp of the spleen. In contrast, in syngeneic controls (e.g. CBA----CBA) the lymphocytes migrated normally into the while pulp. Similar results were obtained in Peyer's patches. Mapping studies in the spleen indicated that the failure to migrate normally is predominantly controlled by the MHC complex, although some non-MHC genes may play a role. In the case of the MHC the most definitive combination was BALB/c-H-2dm2 (H-2L deletion mutant) lymphocytes transfused into BALB/c recipients, the mutant lymphocytes failing to migrate normally and, therefore, implicating the H-2L region in the phenomenon. No differences in the viability of labelled lymphocytes at 6 and 24 h after injection into either syngeneic or allogeneic recipients suggests that the inability of cells to passage through lymphoid organs may represent inappropriate receptors rather than elimination of the allogeneic lymphocytes by natural killer cells (NK) as previously proposed.

A role for early cytotoxic T cells in resistance to ectromelia virus infection in mice.

Ectromelia virus-specific cytotoxic T (Tc) cell precursors were present in the draining popliteal lymph node of all strains of mice tested at 2 to 3 days after footpad inoculation of a high dose (10(5) p.f.u.) of the virulent Moscow strain of ectromelia virus. To detect this response it was necessary to culture lymph node cells from infected mice in the presence of T cell growth factors and to use the more sensitive neutral red assay for measuring cytotoxicity. Cells with lytic activity were virus-specific, major histocompatibility complex-restricted TC cells. C57BL/6J resistant mice, which express a single dominant gene conferring innate resistance had virus-specific TC cell precursors 1 to 2 days sooner than did susceptible BALB/b mice. This TC cell-mediated immune response early after infection could account for the barrier to virus dissemination known to operate 1 to 2 days after infection to slow virus passage into the lymphoreticular system.

Modification of lymphocyte migration by sulfated polysaccharides.

The role of sulfated polysaccharides in lymphocyte migration has been analyzed in vivo using lymphocytes labeled with an intracellular DNA-binding fluorochrome Hoechst 33342. The influence of a panel of sulfated polysaccharides on entry (by injecting the sulfated polysaccharide prior to the labeled cells) and displacement from lymphoid organs (by injecting the sulfated polysaccharide after the labeled cells have localized) indicated that different sulfated polysaccharides have selective effects on entry and displacement, and furthermore positioning of subpopulations within organs. Additional experiments suggested that receptors for sulfated polysaccharides on high endothelial venules may interact with complementary structures on lymphocytes. The data supporting this conclusion were: (a) the normal localization behavior of lymphocytes preincubated with sulfated polysaccharides; (b) an inverse relationship between the expression of lymphocyte surface receptors for sulfated polysaccharides and the ability of the lymphocytes to enter lymphoid organs and (c) the selective binding of sulfated polysaccharide-coupled fluoresceinated beads to high endothelial venules. In this case only the beads coupled with the sulfated polysaccharides that inhibited entry bound to the high endothelial venules. These findings are discussed in terms of a fundamental cellular recognition system utilizing sulfated polysaccharides.

Topographical studies of lymphocyte localization using an intracellular fluorochrome.

A procedure for analysing the topographical localization in tissue sections or whole-organ mounts of lymphocytes labelled with an intracellular DNA-binding fluorochrome, Hoechst dye No. 33342, is described. The localization of intravenously injected lymphocytes in spleen, popliteal lymph nodes, and Peyer's patches was followed up to 7 days. In the case of spleen, both B and T lymphocytes initially localised in the marginal zone. Subsequently, B cells appeared to exit via the red pulp, while T cells aggregated around vessels in the white pulp. In Peyer's patches, B and T lymphocytes localized to different lymphoid areas. The advantages and potential applications of this technique are discussed.

Intracellular fluorescent labelling of cells for analysis of lymphocyte migration.

A procedure for analysing in vivo migration of lymphocytes labelled in vitro using intracellular fluorochromes is described. Comparison of carboxyfluorescein diacetate, a cytoplasmic label, with Hoechst dye No. 33342 (H33342), a DNA-binding fluorochrome, indicated that H33342 is superior. The concentration of H33342 used for labelling does not significantly affect viability or lymphocyte migration and permits long-term visualization. H33342 allows quantitation of in vivo migration in cell suspension and histological localization in frozen sections. Fluorescence is retained in fixed frozen sections for at least 3 months. This method can be used for analyses of lymphocyte migration and maturation.

Influence of one virus infection on a second concurrent primary in vivo antiviral cytotoxic T-cell response.

The influence of one virus on the in vivo cytotoxic T-cell response to a different concurrent viral infection was analyzed. Lymphocytic choriomeningitis and Newcastle disease viruses, known to induce high interferon titers, and the synthetic interferon inducer polyriboinosinic acid-polyribocytidylic acid inhibited the cytotoxic T-cell response against the second virus. In contrast, vaccinia and vesicular stomatitis viruses failed to induce inhibition. Inhibition directly correlated with the interferon titers; similarly, the interferon titers directly correlated with macrophage and natural killer cell activation. The involvement in vivo of interferon in macrophage and natural killer cell activation and the possible mechanisms of inhibition of the cytotoxic responses are shown by the inhibition of the effect by antibodies against interferon.

Neonatal tolerance of major histocompatibility complex antigens alters Ir gene control of the cytotoxic T cell response to vaccinia virus.

The K region of H-2 controls the Tc cell response to vaccinia-Db. The Kb, Kd, and Kq alleles allow good Tc cell responses against vaccinia-Db. In contrast, the presence of Kk in H-2 recombinants 2R (Kk,Db) and 4R (Kk,Db) or in F1 hybrids greatly reduces the anti-vaccinia-Db response. The defect does not lie in antigen presentation, as infected 4R cells can stimulate anti-vaccinia-Db Tc cells in vitro. Furthermore, nonresponder animals possess Tc cell precursors for vaccinia-Db, as transfer of F1 nonresponder spleen cells into infected, lethally irradiated responder recipients allowed generation of anti-vaccinia-Db effector Tc cells. Secondary responses to vaccinia-Db can also be obtained in vitro from T cells of 4R animals. Feedback inhibition was excluded in experiments with mixed chimeras in which Kk and Db were expressed on separate cell populations. Neonatal tolerance of B10 animals to Kk suppressed the anti-vaccinia-Db response but did not affect anti-vaccinia-Kb, anti-lymphocytic choriomeningitis virus, or anti-H-2d responses. In cold target competition experiments, H-2k competitors inhibited vaccinia-Db-specific target cell lysis by Tc cells, which suggests that anti-vaccinia-Db and anti-H-2Kk Tc cells may cross-react. Therefore, we propose that the suppressive influence of Kk on anti-vaccinia-Db Tc cell responses is a consequence of self-tolerance and that suppression of anti-Kk Tc cells results in cross-reactive suppression of anti-vaccinia-Db Tc cells.