Four interleukin-2 surface binding proteins detected in rat spleen cells.

Four specific interleukin-2 (IL-2) surface binding proteins can be detected by covalent cross-linking of [125I]IL-2 to rat spleen cells that have been activated with various stimuli including concanavalin A (Con A), phytohaemagglutinin (PHA), calcium ionophore, and phorbol dibutyrate (PDB) with or without calcium ionophore. These four cross-linked proteins could not be demonstrated in either unstimulated T cells or in activated T cells when binding was performed in the presence of a 20-100-fold excess of unlabelled IL-2. The molecular weights of the four cross-linked proteins, after subtraction of the molecular weight contribution of IL-2 are: 53,000, 70,000, 90,000 and 118,000. The 53,000 MW protein was identified as the rat IL-2 receptor (IL-2R) alpha-chain by immune precipitation. Additionally, results suggest that the rat IL-2R alpha-chain is tightly complexed to both the 118,000 and 90,000 MW IL-2 binding proteins. Purification of surface labelled proteins from activated cells using IL-2 affinity chromatography yields four proteins with similar molecular weight to those identified by cross-linking plus an additional non-ligand cross-linked protein of 46,000 MW. The 46,000 MW band may be a non-binding associated protein since it was not seen following [125I]IL-2 binding cross-linking. Tryptic digests and two-dimensional separation of the affinity-isolated proteins indicate that unique peptide maps are generated for the 46,000, 53,000 and 70,000 MW proteins and excludes the possibility that the bands identified by cross-linking represents cross-linking of multiple ligands to the 53,000 MW subunit. However, the 90,000 and 118,000 MW bands yield peptide maps that closely resemble each other suggesting that these binding proteins may be related. These results suggest that at least four IL-2 surface binding proteins may constitute the rat IL-2R system.


Hodgkin's disease cell lines: a model for interleukin-1-independent accessory cell function.

The haemopoietic origins of the Hodgkin's disease (HD)-derived cell lines L428, KM-H2 and HDLM-2 remain controversial. Analysis of T-cell receptor (TcR) and Ig rearrangements cannot resolve this, and lineage promiscuity limits the interpretation of isolated surface antigen expression. Nonetheless the cell marker profile of L428 has similarities with human dendritic cells (DC), and L428 strongly stimulates in the mixed leucocyte reaction (MLR). We therefore undertook an extended immunophenotypic comparison of the HD lines with that recently defined for DC, prior to examining their ability to stimulate allogenic T lymphocytes, and comparing the molecular interactions involved with those of primary MLR stimulatory cells. The immunophenotype of the HD lines failed to establish either a lymphoid or monocytoid derivation. The profile of L428 appeared similar to the human DC. All three lines were potent stimulators in the primary MLR, and each expressed relevant adhesion and signal-transducing molecules important for co-stimulating T lymphocytes. Inhibition studies using monoclonal antibodies indicated similar contributions within HD line-T cell MLR to that documented in human tonsil DC-T cell MLR. The HD lines produced no detectable interleukin-1 (IL-1) by biological or immunological analysis. Moreover they stimulated allogeneic T lymphocytes in the presence of anti-IL-1 antibodies. Thus although IL-1 mRNA can be detected in both HDLM-2 and KM-H2 by polymerase chain reaction, these lines, and L428, share with DC the ability to stimulate allogeneic T lymphocytes in an IL-1-independent manner [corrected]. HD lines, particularly L428, may provide a standardized, reproducible, IL-1-independent model for dissection of the co-stimulatory requirements of the human primary MLR.


Intravenous injection of interferon-gamma inhibits the proliferation of Listeria monocytogenes in the liver but not in the spleen and peritoneal cavity.

In the present study the effects of intravenous administration of recombinant interferon-gamma (IFN-gamma) on both the proliferation of Listeria monocytogenes in the liver and spleen of mice and the listericidal activity of their peritoneal macrophages were investigated. A single intravenous injection of 1 x 10(6) U or three injections of 2 x 10(5) U recombinant IFN-gamma (rIFN-gamma) induced optimal activation of resident and exudate peritoneal macrophages, as judged by their ability to inhibit the intracellular proliferation of Toxoplasma gondii and their enhanced release of H2O2 and NO2-. The rate of intracellular killing of L. monocytogenes by the rIFN-gamma-activated resident and exudate macrophages was not higher than that by resident macrophages. Addition of 10 ng lipopolysaccharides (LPS) to the rIFN-gamma also did not enhance the bactericidal activity of the activated peritoneal macrophages. The decrease in the number of L. monocytogenes in the peritoneal cavity of mice that had received an i.p. injection of 1 x 10(4) U rIFN-gamma was similar to that in control mice. Intravenous administration of 1 x 10(5) rIFN-gamma activated cells in the liver, as indicated by the increased expression of Ia antigen, and reduced the rate of proliferation of L. monocytogenes in the liver relative to that in control mice when 0.1 LD50 or 1 LD50 L. monocytogenes were injected. However, when 10 LD50 L. monocytogenes were administered there was no effect on their proliferation. The number of L. monocytogenes found initially in the spleen of rIFN-gamma-treated mice was 20-30% of that in the spleen of control mice, but the rate of proliferation of L. monocytogenes was not reduced. These divergent results for the proliferation of L. monocytogenes in the liver, spleen and peritoneal cavity indicate that cells other than macrophages and/or as yet unknown local factors play an important role in the listericidal activity.


Haematopoietic cell lines capable of colonizing the thymus following in vivo transfer expressed T-cell receptor gamma-gene immature mRNA.

To clarify the mechanism by which progenitor T (pro-T) cells recognize and enter the thymus, an attempt was made to produce haematopoietic cell lines by the fusion of BALB/c nude mouse bone marrow or foetal liver cells (gestation 14 and 15 days) with AKR thymoma BW5147, thereby immortalizing cells with potency to colonize the thymus, a characteristic of pro-T cells rarely found in adult bone marrow or foetal liver. The hybridomas thus produced were classified according to the phenotype of surface markers, T-cell receptor (TcR) gene configuration and expression. All hybridomas were negative in the surface expression of T-cell markers such as TcR alpha beta, TcR gamma delta, CD3, CD4 and CD8. They had TcR beta-, gamma- and delta-genes, each with a different status with respect to configuration and transcription. Some possessed partially rearranged TcR genes and others expressed immature TcR mRNA. The cell lines were examined for their capacity to colonize the thymus following intravenous injection into recipient mice. It was found that the cells with capacity of colonizing the thymus expressed immature TcR delta mRNA, while the cell lines lacking TcR delta-genes did not home to the thymus. These findings imply that the potency for migrating to thymus is closely associated with the particular stage of prethymic cell differentiation which could be estimated by the analysis of TcR genes, and that some cell lines with the expression of TcR delta-gene mRNA and the ability to colonize the thymus are derived from pro-T cells.


Class II major histocompatibility complex (Ia) antigen-bearing dendritic cells within the iris and ciliary body of the rat eye: distribution, phenotype and relation to retinal microglia.

The density, distribution and surface phenotype of dendritic cells (DC) and macrophage populations within the ciliary body and iris of Wistar Furth rats were studied by a combination of flat mounting, tangential sectioning, pre-embedding fixation, with a single and double immunohistochemical techniques. Monoclonal antibodies included anti-Ia (OX6) and other dendritic cell/macrophage (ED1 and ED8) or mature tissue macrophage markers (ED2). Single and double staining revealed a network (approximately 400 cells/mm2) of Ia+ cells within the epithelium of the ciliary processes with the morphological and surface phenotypic characteristics of DC populations in other tissues. A minor proportion of DC co-expressed ED1 and ED8, but not ED2. In contrast the immunopositive cells in the lamina propria displayed a more generalized phenotype, including ED2 expression, and pleiomorphic morphology suggesting a preponderance of cells of macrophage lineage. Flat mounts of iris revealed a remarkably regular network of Ia+ DC at a density of 450 cells/mm2. The network of DC in the ciliary epithelium terminated at the cilioretinal junction where they formed a continuous syncytium with retinal microglia which did not display Ia staining. The demonstration of networks of cells with relevant morphological and phenotypical properties of professional antigen-presenting cells at strategic locations within the eye has important implications in relation to ocular immune regulation and on the theories of the mechanism of anterior chamber-associated immune deviation (ACAID). Namely, until now it has been assumed that 'immune privilege' in the anterior chamber of the eye is partly a consequence of there being a paucity of class II+ cells in the surrounding tissues. Dendritic cells in the eye may function as antigen-presenting cells, sampling endogenous and exogenous intraocular antigens and possibly migrating from the eye to draining lymphoid organs (the spleen) where they may generate systemic immune responses. Equally dendritic cells could potentially regulate local immune responses for example in various forms of autoimmune uveoretinal inflammatory disease.


The effect of ultraviolet B irradiation and urocanic acid isomers on dendritic cell migration.

Irradiation with ultraviolet-B light (UV-B) suppresses some cell-mediated immune responses to a variety of antigens, including contact sensitizers. Following UV irradiation there is modulation of Langerhans' cells' markers and keratinocytes are induced to synthesize and secrete tumour necrosis factor-alpha (TNF-alpha). Cis-urocanic acid (cis-UCA) has been suggested as a photoreceptor for UV and has been demonstrated to suppress immune responses in several experimental systems. UCA is found naturally in the stratum corneum as the trans-isomer and converts to the cis-isomer on irradiation. In the present study the migration of dendritic cells (DC) to lymph nodes following UV-B irradiation or epicutaneous application of UCA isomers was examined in unsensitized mice and mice sensitized with fluorescein isothiocyanate (FITC). It was found that UV-B irradiation alone induced DC migration to draining lymph nodes (DLN) and that UV-B irradiation prior to skin sensitization at the same site enhanced DC migration. A maximum number of DC was present in DLN 48 hr following irradiation. In sensitized mice, the percentage of DC bearing FITC and the quantity of FITC per DC was unaltered by prior UV exposure. In contrast, neither isomer of UCA had any significant effect on DC numbers in sensitized or unsensitized mice. It was concluded that UV-B irradiation induced the migration of DC from the epidermis to draining lymph nodes, an effect possibly mediated by TNF-alpha release, while UCA may act by a different mechanism, perhaps via histamine-like receptors in the epidermis.


Role of mast cells in plasma permeation due to immune injury of the skin basement membrane.

Immune injury of the basement membrane occurs in various human diseases. In the present study, an antibody specific for the basement membrane of mouse skin was injected i.d. into mast cell-deficient WBB6F1-W/Wv mice and their congenic controls, WBB6F1-(+/+). Vascular permeability changes, oedema and fibrin deposition were assessed. Plasma permeation, evaluated by dye exudation, was time and dose dependent in both groups of animals, but significantly less in WBB6F1-W/Wv than in normal mice. At 30 min, the time of maximum in congenic controls, extravasation of the dye was 60% less in mast cell-deficient than in WBB6F1-(+/+) mice. Pyrilamine decreased exudation by 40% in normal but not in WBB6F1-W/Wv mice, indicating that the mast cell mediator histamine contributes to the increase in vascular permeability. Mast cell deficiency also markedly reduced fibrin deposition as assessed by direct immunostaining. Oedema, measured as skin thickness, was 60% less in WBB6F1-W/Wv mice than in their congenic controls. A 5-lipoxygenase blocker inhibited plasma exudation and oedema in normal but not in WBB6F1-W/Wv mice. This indicates that leukotrienes are involved in these processes and that mast cells are important for their production. Local mast cell reconstitution restored dye extravasation and oedema to normal levels as well as the effect of the 5-lipoxygenase inhibitor. These findings show that mast cells and their mediators participate in these inflammatory processes which were initiated by the deposition of IgG on the skin basement membrane.


Assessment of cholinergic influences on a primary humoral immune response.

Cholinergic ligands can affect some lymphocyte functions, and binding of labelled cholinergic ligands to lymphocytes has been reported. However, the role of endogenous cholinergic stimulation in immunomodulation in vivo is unclear. It has been suggested that suppression of primary humoral immune responses in vivo by administration of organophosphorus compounds is caused by excessive cholinergic stimulation. If this is correct, it would demonstrate cholinergic immunomodulation in vivo and might serve as a useful model for the characterization of this phenomenon. In the present study, the organophosphorus insecticide parathion and its major metabolite, paraoxon, suppressed the primary IgM response to sheep red blood cells (SRBC) in vitro in Mishell-Dutton cultures at concentrations similar to those probably reached in vivo. In contrast, cholinergic agonists did not suppress the in vitro response, but tended to enhance it. However, antagonists also tended to enhance the response and the effects of agonists were not blocked by antagonists. Binding studies with a radiolabelled cholinergic antagonist ([3H-]QNB) did not indicate the presence of specific, saturable cholinergic receptors on lymphocytes. A membrane preparation from brain was used as a positive control, and specific, saturable binding was observed. These results suggest that suppression of primary immune responses in vivo by parathion is mediated at least in part by direct action of parathion and/or its major metabolite, paraoxon, on the immune system. The data provide no evidence that direct interaction of cholinergic ligands with the immune system contributes to parathion-induced immunosuppression. In fact, the absence of expected agonist-antagonist relationships in Mishell-Dutton cultures, the absence of saturable [3H]QNB binding, and puzzling inconsistencies in the literature on this subject cast doubt on the conclusion that lymphocytes express specific cholinergic receptors.


Antibody-secreting cell responses in the mouse liver.

To elucidate the origins of biliary IgA antibodies, we investigated the isotype and specificity of antibody-secreting cells (ASC) in the liver in comparison with the spleen and intestinal lamina propria of mice immunized by peroral or parenteral routes. The profile of specific IgM, IgG1, IgG2a, and IgA ASC in the liver resembled that of the spleen rather than the lamina propria, regardless of the route of immunization. Peroral immunization increased the proportion of specific IgA ASC in all three organs. However, liver mononuclear cells (MNC) contained a higher proportion of total IgA-secreting cells than spleen cells. After immunization, the number and proportion of B220+ B cells were increased in the liver but not in the spleen. Although the predominant isotype of Ig and specific antibody in bile in response to immunization by either route was IgA, IgM and IgG were clearly detectable. However, specific activities of biliary antibodies relative to total Ig isotype were generally higher than in serum. The predominance of IgA-secreting cells in the liver and the large amount of IgA secreted in the bile resemble the situation at other secretory sites of the mucosal immune system. However, specific antibody-secreting cells appear to accumulate in the liver promptly after immunization, regardless of isotype, and contribute locally produced antibodies to the bile.


Hyperprolactinaemia in hypophysectomized or intact male rats and the development of adjuvant arthritis.

In rats, complete Freund's adjuvant (CFA), injected at the base of the tail, induced a hyperactivation of cellular immune functions and triggered the development of adjuvant arthritis (AA). Before onset of arthritis (Days 9-10 upon CFA), the positive control rats showed significant increases of pituitary prolactin (Prl) mRNA accumulation (Days 3-5). On the other hand, production of pituitary growth hormone (GH) mRNA was significantly reduced from Day 3 onwards. During this early latent period, plasma Prl levels were transiently increased (at least on Day 4), while GH levels were reduced within 8 days (and onwards). Pituitary proopiomelanocortin (POMC) mRNA content progressively decreased with a nadir between Days 6 and 8, accompanied by a loss of the adrenocortical ornithine decarboxylase (ODC) circadian rhythm of activity and a transient reduction of plasma corticosterone (CS) levels (Days 3-6, obvious during the dark phase). At onset of arthritis, the POMC mRNA accumulation and adrenocortical ODC activity increased over their respective baselines. Elevation of plasma CS levels (obvious during the light phase) and important CS-induced thymolysis occurred. Further, hypophysectomized rats did not develop AA. However, hypophysectomized male rats carrying pituitary grafts under the kidney capsule had mild hyperprolactinaemia and developed a worsened arthritic response to CFA, compared to sham-operated controls. On the other hand, intact hyperprolactinaemic male rats showed a delay in the onset and a reduction in the severity of AA. This difference might be due to stimulation of the adrenal cortex in intact pituitary-grafted rats. Such rats showed increased baselines of pituitary POMC mRNA production, adrenocortical ODC activity and plasma CS levels. In addition, during the latent period after CFA, POMC mRNA accumulation, adrenocortical ODC activity and plasma CS levels were only partially suppressed, less than in sham-operated rats. Extensive thymolysis occurred after CFA in these animals--as in the sham-operated rats--but not in the hypophysectomized pituitary-implanted rats. This suggested that in the presence of adrenocortical deficiency, Prl released by the pituitary graft can freely act on the immune system, without being counter-regulated by CS.


HLA-B27/microbial mimicry: an in vivo analysis.

The association between three major spondyloarthritic diseases, ankylosing spondylitis, Reiter's syndrome, and reactive arthritis, and the major histocompatibility complex (MHC) class 1 antigen HLA-B27 is well documented. The hypothesis of cross-reactivity between HLA-B27 and the antecedent infection-causing Gram-negative pathogens such as Salmonella, Shigella and Yersinia has been suggested by in vitro studies employing monoclonal antibodies. We have examined the possibility of such cross-reactivity in vivo using various rabbit immune sera and patient sera as the source of cross-reacting antibody. Mouse L cells were transfected with HLA-A3 or HLA-B27 and used as a source of antigen. Western blot analysis employing denatured antigen, FACS analysis employing native antigen and immunoprecipitation studies were undertaken to detect cross-reacting antibodies generated in vivo to HLA-B27 antigen. Antibodies generated in vivo by infection in patients or immunization in animals against arthritogenic bacteria did not demonstrate any cross-reactivity with HLA-B27 by any of the methods used. As defined by the humoral immune response, molecular mimicry appears unlikely to explain the role of B27 in the pathogenesis of reactive arthritis.


Specific inhibition of IgE antibody production by an antisense oligodeoxynucleotide oligomer (Oligostick).

We have investigated the ability of an antisense oligonucleotide (ASE-1) to specifically inhibit IgE synthesis by a human myeloma cell line, U266. ASE-1 inhibited IgE production in a concentration-dependent manner, as assessed by isotype-specific ELISA measurement of immunoglobulin in myeloma cell supernatants. Inhibition of IgE production was specific and not due to cytotoxicity since IgG1 and IgM production by human myeloma cell lines ARH-77 and RPMI-1788 respectively, was not significantly affected by up to 20 microM ASE-1 whereas IgE production was inhibited by approximately 70% at this concentration. These results indicate that antisense oligonucleotides represent a potential therapeutic approach to the treatment of IgE-mediated allergic diseases.


CD3-gamma, -delta, -epsilon, -zeta, T-cell receptor-alpha and -beta transcripts are independently regulated during thymocyte ontogeny and T-cell activation.

CD3 proteins transduce signals delivered from the T-cell receptor (TcR) for antigen. The genes that encode the individual CD3 subunits are asynchronously regulated in tumour cell lines in vitro. In this report, we examined the expression of individual CD3 and TcR genes during normal murine thymocyte ontogeny in vivo. We show that CD3-gamma, -delta, -epsilon, and zeta transcripts are all expressed on Day 14 post-coitum (p.c.), along with IL-2R alpha and Thy-1 mRNA, and prior to the expression of functional TcR-alpha, -beta, CD4 and CD8 transcripts. Individual CD3 subunits display unique patterns of increased gene expression as ontogeny proceeds. Unique regulation of these T-cell transcripts is also observed in splenic cells activated with the T-cell mitogen concanavalin A (Con A). CD3-delta, -zeta, TcR-alpha and -beta mRNA expression increases, whilst CD3-gamma and -epsilon mRNA levels decrease after mitogenic activation. The potential implications of this regulation on the composition and expression of the TcR/CD3 complex during T-cell ontogeny and activation is discussed.


Lipopolysaccharide synergizes with tumour necrosis factor-alpha in cytotoxicity assays.

Lipopolysaccharide (LPS) from Escherichia coli was found to synergize with human recombinant tumour necrosis factor-alpha (TNF-alpha) in the lysis of L929 and WEHI 164 (clone 13) murine fibroblasts, two cell lines classically used in TNF-alpha bioassays. The effect was noted with TNF-alpha at low (sublytic or lightly lytic) concentrations and was significant for LPS concentrations in the ng range. The LPS effect could be inhibited by polymyxin B, and was not observed when the TNF-alpha assay was performed in the absence of actinomycin D. Enhancement of TNF-alpha lysis by LPS occurred in several assays for determining TNF-alpha, including MTT cleavage, crystal violet staining and lactate dehydrogenase release. Synergism was obtained only when LPS and TNF-alpha were added to cells simultaneously, but not when applied in sequence. The reported synergism may be relevant for TNF-alpha determinations by bioassay, and for the understanding of pathophysiology of Gram-negative sepsis.


Antibodies to 65 kDa and 70 kDa heat shock proteins in rheumatoid arthritis and systemic lupus erythematosus.

To test the potential role of autoimmunity to the highly conserved heat shock proteins (HSP) in immune arthritides, the sera from 99 patients with rheumatoid arthritis (RA), 48 patients with systemic lupus erythematosus (SLE) and 65 normal controls were examined by ELISA for IgG and IgM antibodies to the 65 kDa and 70 kDa heat shock proteins from Mycobacterium bovis (Bacille Calmette-Guerin; BCG). In RA sera there are significant numbers of individuals with increased IgM anti-65 kDa and anti-BCG reactivity as well as IgG anti-70 kDa when compared with controls. In SLE both IgM and IgG anti-BCG, together with IgM anti-65 kDa, differed significantly from controls. The results were compared with previous reports in similar groups of patients, and it is clear that no consistent pattern of reactivity emerges. While further work may be justified looking carefully at the disease duration and other subsets of both RA and SLE, it is difficult at this stage to conclude that antibodies to autologous HSP that cross-react with mycobacterial HSP play a major role in disease pathogenesis.


Local immunity in the male reproductive tract.

In previous studies we have demonstrated that the male urinary tract forms part of the common mucosal immune system, and that the gut contributes to local defences at this site. The question arises as to the extent to which the reproductive tract also forms part of the common mucosal immune system. Rats were immunized by a variety of routes designed to stimulate a local response in the intestine and/or the reproductive tract. Rats immunized only by the intratesticular (i.t.) route yielded no antibody-containing (ACC) response in any of the tissues examined. Intestinal immunization using intraperitoneal priming followed by intraduodenal challenge (i.p./i.d.) yielded a substantial IgA-specific ACC response in the jejunum, but no ACC were detectable in any of the reproductive tract tissues. However, when intestinal and testicular immunizations were combined, large numbers of IgA-specific antibodies were detected in all tissues examined. Chronic drainage of the thoracic lymphatic duct throughout the post-challenge period abrogated the ACC response in all tissues of the reproductive tract, indicating that the ACC appearing at these sites after immunization were of gut origin. The IgA-specific anti-OVA antibody detectable in serum, saliva and testis homogenate reflected the ACC counts in histological sections. The studies reported here confirm that the male reproductive tract does form part of a common mucosal immune system and that gut-associated lymphoid tissues may contribute cellular precursors for ACC, particularly those of IgA specificity, appearing in the tract after local challenge.(ABSTRACT TRUNCATED AT 250 WORDS)


In vitro metabolic inhibition of the human lymphocyte: influence on the expression of interleukin-2 receptors.

We have investigated the effects of energy depletion in human lymphocytes on the expression of the membrane-bound interleukin-2 receptor (IL-2R) and the release of soluble IL-2R. Concanavalin-A (Con-A) and IL-2-transformed lymphocytes were incubated with sodium fluoride, inhibiting the Embden-Meyerhof pathway, rotenone and 2,4-dinitrophenol (2,4-DNP), which are known to disturb mitochondrial high energy production. Rotenone was found to be the most potent inhibitor, with a half maximal effect at 10 nmol/L. 2,4-DNP and sodium fluoride, although not as potent as rotenone, showed marked inhibitory effects in higher concentrations, with half maximal effects at 50 mumol/L and 5 mmol/L respectively. No difference in the inhibition pattern for membrane-bound IL-2R compared with sIL-2R was observed. It is concluded that intracellular synthesis, transport and subsequent membrane insertion or release of receptors important for immunoregulation require high energy phosphate compounds and are sensitive to disturbances in intracellular energy levels.


Proliferation of the BCL1 B cell lymphoma induced by IL-4 and IL-5 is dependent on IL-6 and GM-CSF.

Lymphokine requirements for the in vitro proliferation of the spleen-dependent B cell lymphoma BCL1 have been analysed. Cells were found to respond by proliferation to added recombinant (r) interleukin-4 (IL-4), r-IL-5 and recombinant granulocyte-macrophage colony-stimulating factor (r-GM-CSF). Inhibition by antibodies specific for each of these lymphokines has confirmed growth factor-dependent growth. Anti-GM-CSF has, however, been found to inhibit the proliferation of BCL1 cells induced by r-IL-4 and r-IL-5, as well as r-GM-CSF, suggesting that BCL1 cells may express receptors for GM-CSF and that GM-CSF may be able to act synergistically with IL-4 and IL-5 in promoting cell proliferation. Anti-IL-6 antibody was also found to be a very effective inhibitor of BCL1 proliferation induced by either IL-4 or IL-5 but not by GM-CSF. Added IL-6 did not stimulate BCL1 proliferation, suggesting that endogenous IL-6 may regulate the autocrine growth of BCL1 cells. BCL1 cell proliferation in vitro appears to be regulated by interactions between multiple growth factors.


Intradermal and percutaneous transudation of IgG1 and transferrin in sheep.

The leakage of [125I]-IgG1 into skin sites following injection of mediators of enhanced vascular permeability and during induction of transudates on the skin surface under negative pressure was examined to determine whether IgG is selectively transported into cutaneous transudates. 111In-transferrin was employed as a marker of plasma leakage unaided by selective transport. The leakage of IgG1 into interstitial spaces of untreated skin, into inflammatory transudates and into transudate fluid drawn to the skin surface under vacuum occurred at a lower rate than did leakage of transferrin. No evidence was found in favour of a selective transport mechanism to aid transport of IgG1 into extravascular skin compartments.


T cell memory: a role for MHC class II molecules on T cells?

A proportion of human T cells express MHC class II molecules. In this paper, the hypothesis is advanced that these MHC class II molecules present antigenic peptides acquired from interaction with other antigen-presenting cells (e.g. during the primary response) and can therefore interact in a cognitive manner with T cells with the same specificity. Memory is maintained in the physical form as clusters of T cells expressing T cell receptors and the cognate peptide-MHC class II complex; by virtue of their cognate interaction with each other these cells turn over gradually and return to the cluster when they recirculate. When fresh antigen is introduced, it is presented to some of these T cells by non-T antigen-presenting cells that provide a second signal leading to vigorous activation of the T cell. The proximity and cognate interaction of other T cells of the same specificity allows a chain reaction to occur, leading to rapid development of the secondary response. The hypothesis, which is testable, is proposed because of doubts concerning the current concept of memory as applied to T cells, and a need to understand the consequences of the expression of MHC class II molecules by a subset of T cells.


An hypothesis to explain why cell-mediated immunity alone can contain infections by certain intracellular parasites and how immune class regulation of the response against such parasites can be subverted.

Cells with a low density of parasite-specific antigens on their surface are postulated to be susceptible to a cell-mediated attack but not to effector mechanisms normally activated following the binding of specific antibody to the infected cell. It is further postulated that such infected cells normally induce a cell-mediated response, and that cells infected with slow-growing intracellular parasites have a low density of parasite-specific antigens on their surface. Despite these general postulates, cell-mediated immunity is not invariably induced following natural infection by certain slow-growing parasites, such as those responsible for leprosy, tuberculosis, and the leishmaniases, and antibody can be induced that is exclusive of a strong, cell-mediated response. It is proposed that certain events in such cases subvert the normal regulatory processes that control the class of immunity induced. In these cases, the parasite-infected cells, bearing a low representation of parasite antigens, induce antibody even though they are not susceptible to antibody-dependent effector mechanisms, and so they are not eliminated. In this case, chronic infection and uncontrolled growth of the parasite occurs, often with fatal consequences.


Dietary supplementation with fish oil enhances in vivo synthesis of tumor necrosis factor.

Studies reported here investigate the influence of dietary fat types on cytokine production in response to endotoxin (LPS) challenge. Tumor necrosis factor (TNF) serum levels were markedly higher (by 10-fold) in mice fed chronically a diet rich in fish oil rather than either a diet rich in corn or coconut oil or a low fat diet. This in vivo hyper-responsiveness in LPS-induced TNF production following fish oil consumption concorded with similar exaggerated in vitro TNF release from macrophages exposed to LPS. These data suggest that high consumption of fish oils, by virtue of their high content of omega-3 polyunsaturated fatty acids, can lead to an exaggerated production of mediators of inflammation with potentially adverse consequences on the outcome and severity of infectious diseases.


Mycoplasma fermentans (incognitus strain) induces TNF alpha and IL-1 production by human monocytes and murine macrophages.

We have demonstrated that Mycoplasma fermentans (incognitus strain), as well as M. fermentans KL4, PG 18 and IM 1 strains have the ability to activate human peripheral blood monocytes and murine macrophages of two inbred strains to secrete a high level of tumor necrosis factor alpha (TNF alpha) in a dose-dependent manner. Secretion of interleukin-1 (IL-1) was also stimulated following the incubation of human monocytes with the organism. We suggest that cytokine secretion following infection with M. fermentans (incognitus strain) that was detected in AIDS patients may contribute to the pathological manifestations, including cachexia, in this disease.


Novel monoclonal antibodies to double stranded DNA that require Ca2+ or Mg2+ for their binding.

Murine monoclonal antibody (MoAb) 2B5 (IgG2aK) was obtained by its binding on a solid phase to double-stranded (ds) DNA from one of the mutant CBA/K1(CBA/K1Jms-1prcg/1prcg) mice which were recently found, in our institute, to develop lymphadenopathy associated with the production of anti-double-stranded (ds) antibodies. MoAb 2B5 was highly specific for dsDNA, as shown by enzyme-linked immunosorbent assay (ELISA). The dsDNA binding of 2B5 was decreased dose-dependently by the chelating agent EDTA, being lost completely with 2.5-5.0 mM EDTA, whereas dsDNA on the solid phase remained intact after incubation with EDTA. Addition of Ca2+ or Mg2+ to antibody in culture supernatant that had lost dsDNA binding activity by dialysis against Ca2+ and Mg(2+)-free buffer restored its binding with dsDNA to the original level, indicating that MoAb 2B5 requires Ca2+ or Mg2+ for its binding with dsDNA. It is unknown whether MoAb 2B5 recognizes new conformational epitopes created in the presence of Ca2+ or Mg2+, but this MoAb should be useful in studies on the modes of interaction of DNA with antibodies and DNA-binding proteins.


1,25-Dihydroxyvitamin D3-mediated suppression of T lymphocyte functions and failure of T cell-activating cytokines to restore proliferation.

1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) has been shown to be a potent inhibitor of lymphocyte proliferation in vitro. The present study was undertaken to determine if this is caused by a direct effect on the lymphocytes, and to evaluate to what degree this suppression may be restored by the addition of cytokines. 1,25-(OH)2D3, > or = 10(-10) M, significantly inhibited the proliferation of pokeweed mitogen (PWM)-driven human peripheral blood mononuclear cells (MNC). Depletion of monocytes did not alter the response to 1,25-(OH)2D3. The antiproliferative effect was preceded by decreased production of interleukin (IL)-1 alpha and lymphotoxin (LT), both of which are crucially involved in T cell activation. However, the suppressive effect of 1,25-(OH)2D3, seen in MNC cultures stimulated with PWM alone, was of the same magnitude as the effect seen in MNC cultures stimulated with a combination of PWM and recombinant (r)IL-1 alpha, rIL-6, recombinant tumour necrosis factor (rTNF) alpha, rIL-2 or rLT, as well as PWM plus conditioned medium. Although pretreatment of monocytes for 2 h with 1,25-(OH)2D3 caused significant reduction in the release of IL-1 alpha and TNF alpha, reconstitution of monocyte-depleted cultures with similarly treated monocytes had no inhibitory effect on the proliferative response. In conclusion, even though it cannot be excluded that a low but critical number of monocytes are essential for the suppressive effect of 1,25-(OH)2D3-mediated inhibition of MNC proliferation, the inhibition is most likely the result of a direct effect on the lymphocytes and independent of monocytes and exogenously added cytokines.(ABSTRACT TRUNCATED AT 250 WORDS)


Molecular mimicry by major histocompatibility complex molecules and peptides accounts for some alloresponses.

One explanation offered for the uniquely high precursor frequencies of T cells which recognize allogeneic major histocompatibility complex (MHC) molecules, and their lack of self-MHC restriction, is that the alloreactive cells are polyclonal populations the primary specificity of which is self-MHC plus peptide X1, X2, ... Xn. These are postulated to cross-react with allo-MHC plus peptides Y1, Y2, ... Yn. It has been further suggested that the structural basis for the crossreactivity between different MHC alleles is the similarity in amino acid sequence of that part of the molecule predicted to make contact with the T cell receptor (TcR). In order to test this concept, T cells were obtained with dual specificity for influenza haemagglutinin (HA), restricted by HLA-DR1Dw1, and for DR4Dw4/Dw14 expressed on allogeneic human B cell lines, and the specificity of one clone was studied in detail. The exposed, TcR-contacting surfaces of these two DR molecules are predicted to be identical. Although the HA-specific response was stimulated by DR1-expressing mouse DAP.3 transfectants, DAP.3 cells expressing the alloantigen DR4Dw4 were unable to stimulate, possibly because of a failure to present the necessary human peptide for anti-DR4 allorecognition. Therefore, the effects of pulsing the DR4Dw4-expressing DAP.3 cells with the HA peptide were examined. This peptide is known to bind to both DR1 and DR4. Addition of the HA peptide restored the anti-DR4Dw4 response. These data support the concept that allorecognition in some responder/stimulator combinations can be explained by cross-reactivity at the level of the MHC molecule and the peptide.


Effect of tuftsin and its retro-inverso analogue on the release of interferon (IFN-gamma) and tumor necrosis factor (TNF-alpha) by human leucocytes.

The aim of this work was to demonstrate whether natural tuftsin or a retro-inverso (r.i.) analogue may induce interferon (IFN) and tumor necrosis factor (TNF) in peripheral-blood-mononuclear-cells (PBMC). For this purpose tuftsin or its analogue were added at different molar concentrations to PBMC and the supernatants were tested for IFN and TNF activity. Both cytokines were released after 12 hours incubation with r.i. tuftsin at an optimum concentration of 10(-10) M. Under the same conditions no activity was observed in the presence of natural tuftsin. In comparison to natural tuftsin the stimulatory activity of this tuftsin analogue is likely to be due to its high stability.


T-cell immunodeficiency induced by T-cell mitogens combined with cyclophosphamide injection.

In the murine system in vivo administration of several T-cell mitogens (LcA, Con A, anti-Thy 1.2 mAb) followed by cyclophosphamide (CP) inhibited the functional activity of T-cell subsets (Th1, Th2, Ts) but not that of B cells. T-cell counts in the spleen of treated mice proved to be significantly decreased. Conversely, T mitogens or CP alone produced a negligible effect if any. Adoptively transferred thymocytes from intact donors restored T-dependent splenocyte responses in experimental mice. In addition, it did not suppress the normal response to sheep red blood cells (SRBC). Our results indicated that the acquired immune deficiency under study is caused by polyclonal elimination (deletion) of mitogen-stimulated T cells, and could be regarded as a model of CP-induced tolerance.


Immunosenescence of human NK cells: effects on tumor target recognition, lethal hit and interferon sensitivity.

Previous results from this laboratory have shown the preservation of non-MHC-restricted, constitutive oncolytic activity of human peripheral blood NK cells in the elderly as assessed by the chromium release assay which quantitates the lytic endpoint at the cell population level. We have now addressed this senescence-related change at single-cell level using 101 blood samples. Both the efficiency of the initial tumor target binding step i.e., recognition of K562, the NK-sensitive erythroleukemia cell line, as well as the ability of NK cells to deliver lethal hit are highly conserved during healthy aging. In fact, the elderly exhibit a statistically significant, moderately higher frequency of active killers among circulating lymphocytes. Analyzed in another way, a majority of "high NK responders" were found to be older donors, while none in the "low NK responders" group were > 70 years old. Gamma interferon, a gene product as well as an autocrine activator of NK cells, is effective in converting non-lytic "pre-NK" cells to active killers at single-cell level. This in vitro cytokine sensitivity of NK cells is unaltered during immune senescence. The intactness of the NK cell's capacity to be modulated may be vital in both tumor resistance and host viral defenses of aged humans.


Differential cell behaviour on polylysine and gelatin in primary culture of mouse mammary adenocarcinoma.

Cells dissociated from spontaneous and transplanted tumours of C3HJax mammary gland have been cultured on polylysine and gelatin substrates. The isolated cells proliferated to form monolayers with high degree of organoid structure as indicated by formation of alveolar cavities. Differences were observed in the cell attachment, growth pattern, number and size of alveolar cavities, cells which lined the cavity and cell morphology on polylysine and gelatin substrates as compared to conventional cell culture plastic surface. On polylysine more than 90% cells attached rapidly, within 15-45 min after plating, with or without serum and formed confluent monolayers marked by presence of large and small alveolar cavities. Multiple interacting cell types took part in organization of the cavity. Cells lining the cavity constantly proliferated and rearranged to expand it. On gelatin, 60-70% cells attached over a period of 6-24 hr in presence of serum and formed confluent monolayers dominated by small alveolar cavities. Cells forming the cavities were epithelial in nature and cavities once formed did not increase in size. Upon subculture, the cell morphology on these substrates was strikingly different. On polylysine, the predominant cell type had numerous irregular microvilli whereas on gelatin, cells had smoother boundaries with a few stunted cytoplasmic extensions. The cell attachment on conventional surface was low, 40-50%. When seeded at high cell density, formation of alveolar cavities was suppressed and at low cell density, cultures were marked by contact inhibition of cells and failure to attain confluence. These results suggest differential behaviour and interaction of mammary tumour epithelium with the substrates used.


Sex determination in sheep and goats using bovine Y-chromosome specific primers via polymerase chain reaction: potential for embryo sexing.

A simple and novel method, using polymerase chain reaction (PCR) has been standardized for accurate sex determination in sheep and goats. The assay utilizes a pair of bovine Y-chromosome specific primers and the genomic DNA isolated from blood samples of adult male and female sheep and goats. The primers recognize and amplify the Y-chromosome specific sequences in male goats and sheep. The assay is accurate, reliable and rapid.


Regulation of estrous cycle in hysterectomized albino rats by activation of follicular development.

Wistar strain albino rats were hysterectomized and the estrous cycle was compared with sham operated controls. Duration of estrous cycle in hysterectomized rats increased markedly with significant delay in the luteal phase and this was correlated to the inhibited follicular development of ovary. When these rats were treated with PGF2 alpha and PMSG and subjected to physical exercises, the estrous cycle was synchronised and the ovaries of such animals had active follicular development. Thus the deranged operation of sexual cycle in hysterectomized rats was regulated through physical exercises.


Effect of unilateral ovariectomy and subsequent ovum implantation in mice.

Unilateral ovariectomy (ULO) was done on any stage of the cycle and the animals were mated within day 1 to day 21 to observe the acute and long term effect of ULO on ovum implantation. Implantation reduced in proportion to single ovary if the animals were mated within 24 hr of ULO. Increase in ovarian weight along with an increase in implantation number continued in mated mice and reached at peak on day 19-21 of ULO (sacrificed after 6 days i.e., 25-27 days of ULO). After ULO the remaining ovary compensated within day 5-6 of ULO even during pregnancy. Ovarian histology showed stimulation of small antral follicles in mice mated on day 3 of ULO (sacrificed after 6 days i.e., day 9 of ULO) along with a decrease of large antral follicles and pre-antral follicles. Preantral follicles were at peak on day 12-14. Large antral follicles attained a peak on day 4 which slowly decreased. The occurrence of implantation in such ULO conditions are discussed.


Protection to glycolysis by a combination of 5-hydroxy-L-tryptophan and 2-aminoethylisothiuronium bromide hydrobromide in lethally irradiated rats.

Rate of glycolysis in vivo at different time intervals following 8 Gy [LD100(30)] whole body gamma radiation (WBGR) was evaluated by estimating liver glycogen, blood sugar, serum lactic dehydrogenase (LDH) and blood lactic acid concentration in adult male Sprague Dawley rats. Within 1 hr of radiation exposure, a significant fall in liver glycogen was observed in rats fed food and water ad libitum. The glycogen content increased after 24 hr and had returned to control level on 7th day after radiation exposure. Blood sugar, serum LDH and blood lactate levels increased significantly as compared to non irradiated controls. Pretreatment with 5-hydroxy-L-tryptophan (5-HTP; 100 mg/kg) + 2-aminoethylisothiuronium bromide hydrobromide (AET; 20 mg/kg) ip 30 min before 8 Gy WBGR, modified these values and restored them to normal level on 7th day post-irradiation.


Analgesic and hypnosis potentiation effect of some 1-(2-benzothiazolyl)-1-aryl-3-phenyl-4-aryl guanidines.

1-(2-benzothiazolyl)-1-aryl-3-phenyl-4-arylguanidines (I-X) were prepared by oxidation of 1,3-diarylthioureas. The compounds were screened for their analgesic and hypnotic activities in rats. Of these, p-methyl group substituted compound of the series was the most potent analgesic as compared to other compounds of the series. In hypnotic test all the compounds potentiated pentobarbitone-induced hypnosis.


Pathogenesis of age dependent paralysis by a temperature sensitive mutant (tsl) of Moloney murine leukemia virus-TB.

The tsl mutant of Moloney murine leukemia virus-TB produces neurological disease leading to fatal hind limb paralysis when inoculated in newborn BALB/c mice. The present study was under taken to assess the role of T and B lymphocytes in age dependent resistance to tsl induced paralysis in BALB/c mice. The adoptive transfer of non-immune splenic unseparated lymphoid cells, T cells and B cells and tsl immune B cells and T cells to newborn BALB/c mice infected with tsl did not prevent the development of paralysis. However, adoptive transfer of immune splenic unseparated lymphoid cells and immune T cells delayed the onset of paralysis by 5 to 10 days as compared to the mice which did not receive the immune lymphocytes. Athymic BALB/c nude mice inoculated with tsl at days 1 and 10 after birth failed to develop the paralytic disease. Transfer of tsl neutralising antibody also delayed the onset of paralysis. Mice (10 days old) treated with cyclophosphamide, cyclosporine A, cortisone acetate and anti-T cell serum when inoculated with tsl also did not develop neurological disease. The results suggest that age related resistance to neurological disease may not be associated with B cell mediated immunity.


Delayed effects of acute oral and chronic inhalational exposure to methylparathion on learning and memory in rats.

Impairment of acquisition phase of the learning process was observed in rats even at 3 weeks after single oral exposure to the near-lethal dose of commercial grade methylparathion (MP) followed by atropine resuscitation. Though there was a trend towards memory impairment in this group of animals, memory retention was not significantly affected. Chronic inhalational exposure to MP (one exposure/day for 3 weeks) did not significantly alter learning or memory. No significant alteration in red blood cell or brain acetylcholinesterase levels were observed in either of the two groups. It appears that behavioural effects can persist even 3 weeks after exposure to acute near-lethal doses of the pesticide, as occurs in the clinical situation of suicidal attempts; while repeated exposure to no-observed-effect-level doses as occurs in farm and factory workers, may not be associated with behavioural changes.


Gas chromatographic detection of anaerobic bacteria from environment.

A rapid method of detection of anaerobic bacteria in environment using gas chromatograph is described. Metabolically produced volatile and non-volatile fatty acid by the anaerobic bacteria are detected gas-chromatographically. Using this technique anaerobic bacteria are detected from soil, air, laboratory and operation theatre environments and drinking water samples. In the polluted drinking water apart from drug resistant E. coli, Clostridium difficile is isolated indicating faecal pollution of drinking water from cases of antibiotic associated pseudomembraneous colitis. The method has great significance in detection of anaerobic bacteria in environment especially in the management of war wounds.


Remote sensing system to study rodent behaviour.

The instrumental system assembled consists of video monitors which projects signals from remotely placed video camera in the rat pen with pan and tilt arrangement and all operations are done through control-room. Signals obtained from the camera will pass through the monitor and then fed into VCR system which records the behavioural events. Studies carried out with the above system has revealed that all the characteristic behaviour of gerbils (Meriones hurricane Jerdon) which is significantly superior (P < 0.001) over the direct observation method. The integrated sensing system is an ideal instrumental set up for viewing and recording the behaviour of rodents as well as other animals in the experimental pen throughout the year under varying weather and light conditions.


Insecticide interaction with carrier and neuroproteins.

Binding of alpha-, beta-, gamma-hexachlorocyclohexane, p,p'-DDT and p,p'-DDE with bovine serum albumin (BSA) and locust brain homogenate was studied. Binding affinities of pesticides were higher for the locust brain homogenates than for BSA. Results of uptake by isolated locust brain revealed higher uptake of gamma-HCH than alpha-HCH. gamma-HCH uptake was also higher from locust haemolymph than either from BSA or from buffer.


Acetylhomocysteine thiolactone protection against phosphamidon-induced alteration of regional superoxide dismutase activity in the central nervous system and its correlation with altered lipid peroxidation.

Phosphamidon intoxication (2 mg/kg body wt./day for 7 days) inhibited SOD activity, but enhanced the lipid peroxidation in various CNS regions. Administration of cithiolone (8 mg/kg body wt./day, ip for 7 days), however, elevated SOD activity and depleted lipid peroxidation. Interestingly, no significant change was observed either in SOD activity or in lipid peroxidation following simultaneous administration of phosphamidon and cithiolone.


HIV-2 antibodies in serum samples from Maharashtra state.

Two hundred serum samples were tested to detect the presence of HIV-2 infection in Maharashtra state. The serum samples were derived from various groups including those with high risk behaviour for HIV infection. All samples were tested by three combined HIV-1 and HIV-2 ELISA kits. The reactivity was confirmed by LiaTek HIV 1+2 immunoblot. The study confirmed that HIV-2 infection exists in Maharashtra, as 14 samples showed antibodies to HIV-2 and 14 showed antibodies to both HIV-1 and HIV-2. Antibodies to HIV-2 or HIV-1 and HIV-2 were detected mainly in persons with high risk behaviour.


Characterisation of non-01 Vibrio cholera isolated from stools of diarrhoeal patients.

A characterisation of the biochemical and physiological properties of 30 strains of non 01 V. cholerae isolated from faecal samples, was undertaken. The strains consistently expressed a large number of characters that define them to be closer to the El Tor biotype than their classical counterparts. They did not show much heterogeneity in their cultural and biochemical properties. The strains were strongly haemolysing (100%), haemagglutinating (96%) and also salt-tolerant, thereby acquiring a unique taxonomic status.


Diagnostic accuracy of IgM antibody detection in typhoid by a modification in Widal test.

Specific IgM antibodies were detected indirectly in sera of patients of typhoid by observing fourfold or more fall in the agglutinin titres after treatment of the serum with 2-mercaptoethanol (2-ME) for removal of IgM antibodies. Significant fall in titres was observed in 94 per cent patients of bacteriologically confirmed typhoid and 67 per cent patients in whom a diagnosis of typhoid was based only on a significant Widal test result. Patients of non-typhoid febrile illnesses showed either no fall or an insignificant fall in their titres. The detection of specific IgM antibodies by this simple modification of Widal test thus seems to enhance the sensitivity and specificity of the test.


Auxotyping of Neisseria gonorrhoeae as an additional epidemiological marker.

Auxotypes and penicillin sensitivity of 102 strains of N. gonorrhoeae were studied. Ten distinct auxotypes on the basis of growth requirement to seven amino acids were observed. The commonest pattern seen was zero auxotype (33.3%), followed by auxotype requiring proline (18.6%). MIC of 72 (71.5%) strains ranged from < 0.003 IU/ml to 0.062 IU/ml of benzyl penicillin. MIC of the other 30 (29.5%) strains was found to be > or = 0.125 IU/ml, indicating penicillin resistance. None of the strains were penicillinase producers. A case of reinfection was also detected on the basis of change in auxotype pattern.


Occurrence of multiple antimicrobial resistance among Haemophilus influenzae type b causing meningitis.

Among the 157 children with culture proven pyogenic meningitis who were admitted during 1987-1991, in the Department of Child Health at the Christian Medical College and Hospital, Vellore, 40 had H. influenzae type b (HIB) meningitis; 17 of these had multiantimicrobial resistant HIB (MRHIB) and 23 others had usual susceptibility HIB (USHIB) meningitis. The two groups were compared. Children with MRHIB meningitis who received optimal treatment with cefotaxime had excellent recovery. When cefotaxime therapy was delayed, mortality was noted in all except one child who survived with severe neurological sequelae. MRHIB meningitis treated with drugs other than cefotaxime had 100 per cent mortality. Cefotaxime is therefore recommended in children below 3 yr of age presenting with purulent meningitis, as MRHIB is a possible causative agent in this group.


Role of bacterial flora in the pathogenesis & management of atopic dermatitis.

The carriage state of Staphylococcus aureus and its role in the pathogenesis and management of atopic dermatitis were evaluated in 50 patients, aged 3 months to 12 yr. An equal number of age and sex matched controls were also studied. The positivity of Staph.aureus in patients with atopic dermatitis was 50 per cent from eczematous skin, 34 per cent from anterior nares and 26 per cent from normal skin. In controls, the comparative figures were 14 per cent from anterior nares and 10 per cent from normal skin. After institution of oral erythromycin or cloxacillin therapy (according to sensitivity), the colony counts dropped to 18 per cent from eczematous skin, 14 per cent from anterior nares and 8 per cent from normal skin after one week and to zero after 3 wk. This was associated with significant clinical improvement. The results of this study suggest that Staph. aureus aggravates the eczematous process in patients with atopic dermatitis and antibiotics decrease the severity and are useful in long term prognosis of the disease.


Anopheline fauna of parts of Tirap district, Arunachal Pradesh with reference to malaria transmission.

In a survey on the anopheline fauna in highly malaria endemic areas of the Tirap district of Arunachal Pradesh, 7476 anophelines belonging to 17 species were collected, including seven species of anophelines which are recognized malaria vectors in India. Anopheles tessellatus and A. jamesii were recorded for the first time in this area. The parasitological survey revealed that the area was endemic for malaria particularly P. falciparum, the slide positivity rate and slide falciparum rate being 25.63 and 19.21 per cent respectively. On dissection of 10 anopheles species, malarial infection was detected in two viz., A. minimus and A. dirus.


Estradiol promotes cell shape changes and glial fibrillary acidic protein redistribution in hypothalamic astrocytes in vitro: a neuronal-mediated effect.

We have previously shown that in hypothalamic mixed neuronal-glial cultures both astrocytic shape and distribution of glial fibrillary acidic protein (GFAP) are modified by estradiol. In the present study, we have investigated whether or not the presence of neurons is necessary for these hormonal effects. In mixed neuronal-glial hypothalamic cultures the proportion of process-bearing GFAP-immunoreactive cells was significantly increased after treatment for 30 min with 10(-12) M 17 beta estradiol. This effect was present for at least 1 day and was reverted by incubating the cells in estradiol-free medium. Estradiol incubation resulted in a progressive differentiation of GFAP-immunoreactive cells from a flattened epithelioid morphology to bipolar, radial, and stellate shapes. This effect was not observed in pure hypothalamic glial cultures. Furthermore, incubation of hypothalamic glial cells with medium conditioned by estradiol-treated mixed hypothalamic cultures did not affect the shape of GFAP-immunoreactive astrocytes. In contrast, addition of hypothalamic neurons, but not cerebellar neurons or fibroblasts, to established hypothalamic glial cultures affected the development of estradiol sensitivity in astrocytes. These results indicate that estradiol induction of shape changes in hypothalamic astrocytes is not only dependent on the presence of hypothalamic neurons, but that physical contact between astrocytes and neurons is necessary for the manifestation of the effect of this hormone.


Interleukin-1 beta regulates proenkephalin gene expression in astrocytes cultured from rat cortex.

Glial cells execute essential functions in central nervous system (CNS) development and are also believed to play important roles during gliosis in response to trauma or disease. These developmental and pathological states have also been associated with elevated expression of opioid genes. Because levels of the cytokine interleukin-1 beta (IL-1 beta) increase following CNS lesions, we examined the possible influence of IL-1 beta on the expression of opioid genes in astrocytes cultured from rat cortex. Proenkephalin mRNA expression was stimulated by IL-1 beta in a time- and concentration-dependent manner, being maximal with 5 U/ml IL-1 beta at 4 h. Although the beta-adrenergic agonist isoproterenol was also active, interferon, glutamate, and carbachol were not. Unlike isoproterenol, the actions of IL-1 beta were not associated with a cyclic adenosine monophosphate (AMP)-dependent pathway. Interleukin-1 beta also regulated a proenkephalin-chloramphenicol acetyltransferase fusion gene transiently transfected into astrocytes, with a dose-response similar to that active in proenkephalin mRNA. These effects of IL-1 beta were region-specific, not being observed with either cerebellar or hippocampal astrocytes; however, isoproterenol was active in the latter cell populations. Proenkephalin mRNA in cortical astrocytes was stimulated following a temperature stress. These results suggest that enhanced proenkephalin gene expression in astrocytes by IL-1 beta may be important in neuroimmune interactions and in trauma-induced CNS injury or stress.


The glial framework of central white matter tracts: segmented rows of contiguous interfascicular oligodendrocytes and solitary astrocytes give rise to a continuous meshwork of transverse and longitudinal processes in the adult rat fimbria.

The cellular skeleton of the adult rat fimbria consists of regularly spaced interfascicular glial rows of considerable length, running in the longitudinal (axonal) axis of the tract. Each row consists of a series of repeated segments made up of a stretch of interfascicular oligodendrocytes lying in direct contact with each other, and separated from the adjacent segments by usually solitary interfascicular astrocytes. A typical segment would be around 60 microns long, and have an axial core of about eight contiguous oligodendrocytes surrounded by a shell of about 1,200 axons, 70% of which are myelinated. In the transverse plane of the tract, adjacent segments are stacked together with a core-to-core distance of around 15 microns. The interfascicular oligodendrocytes have radial stem processes (in a plane transverse to the axonal axis) which give rise to the longitudinal myelinating (internodal) processes. Both transverse and longitudinal oligodendrocytic processes are longer than the dimensions of the segment (in which their cell bodies lie) and its axonal shell. They thus cooperate in myelinating axons of adjacent segments in both planes. The interfascicular astrocytes have three distinct types of processes: radial, longitudinal, and vascular (bearing end feet). The radial astrocytic processes are thick and tapering, and the processes of individual astrocytes extend transversely (in the plane of the original embryonic radial glial processes) for a total of at least 100 microns. The considerably more numerous longitudinal astrocytic processes arise from all parts of the cell bodies and radial processes. They are up to at least 30 microns long, thin, untapering, and largely unbranched, and are interdigitated among the fimbrial axons. In the radial plane, the astrocytic radial processes spread out through a wide swathe of adjacent segments, so that the integrated meshwork of interpenetrating longitudinal processes arising from overlapping radial processes of astrocytes from many different interfascicular rows provides a continuous longitudinal substrate for the fimbrial axons.


Purification and characterization of an astrocyte GABA-carrier inducing protein (GABA-CIP) released from cerebellar granule cells in culture.

A glycoprotein that induces gamma-aminobutyric acid (GABA) carriers in cultured cerebellar astrocytes was isolated and purified from conditioned media from cultured cerebellar granule cells by anion exchange chromatography, affinity chromatography, and gel filtration. Following gel filtration three fractions corresponding to M(r) 30,000, 60,000, and 240,000 exhibited GABA carrier inducing activity. SDS-PAGE of the M(r) 30,000 fraction revealed under non-reducing conditions three bands corresponding to M(r) 30,000, 60,000, and 120,000. Under reducing conditions only the band corresponding to an M(r) of 30,000 was visible. An identical N-terminal amino acid sequence and amino acid composition was found in the M(r) 30,000 and the M(r) 60,000 fraction from the gel filtration. These results suggest that the protein polymerizes into di- and tetramers. Computer base analysis of the N-terminal amino acid sequence revealed no obvious homology with previously reported N-terminal amino acid sequences. Application of the glycoprotein to cerebellar astrocytes led time and dose dependently to an increased GABA uptake. The effect became maximal after 24 h exposure of the cells. Kinetic analysis of the GABA uptake showed that exposure of the astrocytes to the glycoprotein led to an increase in Vmax for GABA uptake without affecting Km, suggesting an increase in the number of GABA carrier molecules. Addition of actinomycin D together with the glycoprotein abolished this effect suggesting that the glycoprotein acts by stimulating de novo synthesis of GABA carriers. Hence, the newly purified protein secreted from neurons is named GABA-carrier inducing protein (GABA-CIP).


Gastrointestinal motility in pregnancy.

The gallbladder and gut should be viewed as hormonally responsive organs the normal physiology of which may be altered by the hormones of pregnancy. The gallbladder enlarges and empties sluggishly in response to meals during pregnancy. Small bowel transit is slowed, and the resting pressure of the lower esophageal sphincter is reduced. All these effects are reversed by delivery; motility reverts toward normal in the postpartum period. The rapid return of normal motility suggests that the effects of pregnancy are hormonally related. Most studies have demonstrated that progesterone, not estrogen, may be the hormone responsible. Although incompletely defined, one mechanism of the effects of pregnancy on motility may be progesterone-induced inhibition of the mobilization of intracellular calcium within smooth muscle cells.


Gastroesophageal reflux disease in pregnancy.

Heartburn during pregnancy occurs in approximately two thirds of all pregnancies. The origin is multifactorial, but the predominant factor is a decrease in LES pressure resulting from female sex hormones, especially progesterone. Mechanical factors play a small role. Serious reflux complications during pregnancy are rare outside the obstetric setting. Therapy involves lifestyle modifications and nonsystemic medications as the initial choices. H2 blockers are probably safe in severe and refractory cases.


Diagnosis and management of irritable bowel syndrome, constipation, and diarrhea in pregnancy.

Irritable bowel syndrome, constipation, and diarrhea may complicate a pregnancy. Complaints of IBS and constipation may be managed by nonpharmaceutical methods. A careful history should be conducted to determine whether these complaints are of an acute or a long-standing nature. Conservative treatment of IBS is recommended and may include stool-bulking agents, a high-fiber diet, elimination of offensive foods, and the behavioral treatment of passive muscle relaxation, biofeedback or supportive psychotherapy. Constipation is generally self-limiting. It also may be treated conservatively with stool-bulking agents, increases in dietary fiber, and the addition of pelvic muscle exercises, preferably using electromyographic biofeedback. Laxatives should be used judiciously (Table 1). Diarrhea is caused most often by infectious agents in pregnancy but may also be from food poisoning or a viral disease. Infectious diarrhea may be treated by mild antidiarrheal agents and safe antibiotics. Fluid replacement is the mainstay of treatment, and care should be taken, remembering that the treatment involves two patients. These complaints can generally be managed conservatively, but persistent cases should be investigated as in a nonpregnant patient.


Gallstone disease and pancreatitis in pregnancy.

Controversy exists over whether pregnancy is a risk factor for gallstone formation; however, changes in hepatobiliary function do occur during pregnancy to create a lithogenic environment; these changes include gallbladder stasis and secretion of bile with increased amounts of cholesterol and decreased amounts of chenodeoxycholic acid. In women with existing gallstones, pregnancy may bring out symptoms, including pain and even acute cholecystitis. This may be more common during the postpartum period than during pregnancy itself; however, the overall occurrence of symptomatic biliary disease in association with pregnancy is low. The effects of pregnancy, if any, on pancreatic exocrine function are undefined. Acute pancreatitis can occur during pregnancy but does not appear to do so with either increased or, alternatively, decreased frequency. The concept of pancreatitis caused by pregnancy per se is not valid, although in susceptible women with lipid disorders, hypertriglyceridemia can occur and serve as an etiologic factor. Gallstones are a common cause of pancreatitis, but in contrast to nonpregnant women, alcohol is unusual as a cause. Although the presentation of both acute cholecystitis and acute pancreatitis may be similar to that in the nonpregnant state, the differential diagnosis of both these disorders is expanded because of unique pregnancy-related conditions and the shift of abdominal viscera by the enlarging uterus. The diagnosis is clinical and supported with conventional laboratory studies and ultrasound; management is supportive and in most patients successful. Cholecystectomy is seldom necessary during pregnancy, either for acute cholecystitis or gallstone pancreatitis, but can be safely performed if necessary after the first trimester. Endoscopic papillotomy and stone removal for choledocholithiasis are possible during pregnancy and may be the treatment of choice for this unusual condition. Specific enteral or parenteral nutrition may be necessary in women with pancreatitis associated with hypertriglyceridemia.


Peptic ulcer disease in pregnancy.

In general, peptic ulcer disease during pregnancy is relatively rare. Certainly, gastroesophageal reflux symptomatology and hyperemesis gravidarum are the primary pregnancy-associated upper gastrointestinal tract illnesses. The symptoms of dyspepsia accompanies all three diagnoses and makes it difficult to determine whether peptic ulcer is playing a role in the patient's symptomatology. Patients with a previous history of complicated peptic ulcer diatheses should be suspected of having recurrent ulcer disease and treated accordingly. Endoscopy is not to be feared if needed to confirm a diagnosis of peptic ulcer disease or to aid in the diagnosis of the patient with upper gastrointestinal tract hemorrhage. There is thought to be some improvement in peptic ulcer disease with pregnancy, which may be secondary to lower gastric acid output and increased protective mucus production associated with elevated progesterone levels. This may afford some level of protection against this disease process in pregnant women. Patients who are smokers and have a previous history of peptic ulcer disease are at highest risk for ulcer disease during pregnancy. Multiple agents have been found to be relatively safe and effective for ulcer healing, with H2 antagonists the mainstay of therapy during pregnancy.


Inflammatory bowel disease in pregnancy.

This article provides the current state of knowledge on the interrelations between ulcerative colitis and Crohn's disease and pregnancy and its multiple concerns, including inheriting inflammatory bowel disease, fertility, influence of inflammatory bowel disease activity on pregnancy and vice versa, the safety of drug therapy, and risk factors. The author provides some conclusions and his recommendations.


Hyperemesis gravidarum.

Hyperemesis gravidarum is a poorly understood disorder and one for which many physicians have little sympathy, perhaps because we find it difficult to understand its pathogenesis and to treat. It may be associated with dysfunction in a variety of organs, including the thyroid and liver. Many theories, prominently including psychological or behavioral abnormalities, have been proposed to explicate this syndrome. On examination, however, no single theory seems to provide an adequate explanation for hyperemesis gravidarum. Treatment for hyperemesis gravidarum includes fluid and electrolyte supplementation, nutritional support, and total parenteral nutrition, as well as psychological and behavioral therapies and, in selected cases, pharmacotherapy.


The syndrome of preeclampsia.

Preeclampsia has traditionally been viewed as one of several forms of hypertension complicating pregnancy. More recently, the multisystem nature of this unique gestational disorder has been emphasized. Pathophysiologic events, including abnormal placentation and heightened vascular reactivity, may occur weeks or months prior to clinical recognition of the disease. Although most frequently presenting as hypertension and proteinuria, hepatic (abdominal pain and elevation of transaminases) and hematologic (intravascular hemolysis and thrombocytopenia) involvement may be important features of the disease. Current theories suggest that multiorgan dysfunction may be caused by widespread vascular endothelial dysfunction, vasospasm, and variable activation of coagulation mechanisms. Pending delivery, which is the only definitive therapy for preeclampsia, maternal complications of intracerebral hemorrhage and eclampsia may be prevented with judicious use of antihypertensive medication (e.g., hydralazine) and magnesium sulfate, respectively. Finally, data from a number of small trials suggest that low-dose aspirin (60-100 mg/d) may reduce the incidence of preeclampsia in patients at high risk without adversely affecting the fetus or newborn; however, it is recommended that aspirin not be used as a routine prophylactic intervention until publication of results of several ongoing large multicenter trials, which will help to more fully clarify the benefits and risks of this approach.


Viral hepatitis, A though E, complicating pregnancy.

Jaundice during pregnancy may be the result either of disease uniquely associated with the pregnant state or disease totally unrelated to the pregnancy. In the United States, the most common cause of jaundice in pregnant women is viral hepatitis. With the exception of hepatitis E virus infection, the clinical course and histologic findings do not differ between pregnant and nonpregnant patients.


Pregnancy in patients with chronic liver disease.

The pregnant patient with established chronic liver disease presents a unique situation in medicine. Although neither the pregnancy nor the liver disease is likely to specifically worsen the other, the combination can result in fatal complications for mother and infant. Fertility is decreased in patients with advanced liver disease and may provide a degree of protection for many patients who would be at increased risk should they become pregnant; however, pregnancy may occur even with advanced liver disease, and it is necessary to anticipate and plan for possible complications of the specific hepatic disease encountered. Counseling prior to pregnancy is the best policy, with consideration to transplantation prior to childbearing or to sterilization if it is more appropriate. Most problems associated with advanced liver disease are managed as in the nonpregnant patient; however, variceal bleeding may be a particularly difficult problem, and management here may necessitate portacaval shunt surgery.


The spectrum of liver and gastrointestinal disease seen in cholestasis of pregnancy.

A mild form of intrahepatic cholestasis is an infrequent complication of pregnancy, with a spontaneous cure almost immediately after delivery and that often recurs in future pregnancies. Pruritus alters maternal well-being, and a subclinical steatorrhea may impair the patient's nutritional status; otherwise, it is a mild disease in the mother, and no maternal mortality has been attributed to it. In contrast, cholestasis of pregnancy is often identified as a risk of increased perinatal morbidity and mortality. The cause of cholestasis of pregnancy is unknown. A hereditary predisposition seems to induce in the maternal liver an abnormal reaction to female sex hormones, but some still unidentified environmental (possibly dietary) factor could also be involved in the pathogenesis of the disease. Pruritus, but not the biochemical alterations, can be alleviated by the use of cholestyramine, silymarin, or epomediol. Ursodeoxycholic acid has been beneficial in pruritus and in liver function tests; an improvement in fetal prognosis should be evaluated in future controlled studies.


Obstetric management of gastroenterologic complications of pregnancy.

Key elements in the management of gastroenterologic complications during pregnancy are knowledge of the natural history of the disease process, understanding the effects of pregnancy on the disease and of the disease on pregnancy, concern about fetal effects of medications, careful antepartum fetal assessment, and appropriate timing of the delivery. Vaginal delivery is preferred in most pregnancies complicated by gastrointestinal disease.


Care of the pregnancy complicated by HELLP syndrome.

Pregnancies complicated by HELLP syndrome require a well-formulated management plan. The development of this syndrome after 34 weeks' gestation or with documentation of fetal lung maturity is an indication for delivery. Vaginal delivery can be accomplished in most cases; however, if cesarean section is required, the use of general anesthesia, subfascial drains, and preoperative platelet transfusion for platelet counts less than 50,000/mm3 can reduce the incidence of complications. It is advisable that patients with complications of HELLP syndrome such as pulmonary edema, acute renal failure, liver rupture, or extreme prematurity be referred to a tertiary care center where maternal and neonatal intensive care facilities are available.


Experiences of developed management in the Cardiovascular Service at Royal Adelaide Hospital.

Devolved management structures are on trial at Royal Adelaide Hospital with assistance from the Clinical Devolution Project. The trial is to assess the suitability of devolved management structures to support clinical decision making, and to evaluate financial management reporting systems and information support systems. A nurse at Assistant Director of Nursing level and a doctor head each of the three trial Clinical services. Both have equal responsibilities and delegations of authority for day to day operations utilising an allocated budget. The impact of devolution of responsibility to clinical units is considerable. Such reorganisation presents opportunities and difficulties. Nurse/doctor collaboration is essential to manage high patient throughout effectively and efficiently as well as to prepare for the impact of casemix and output based funding. Clinical department size, budget allocation, roles and organisational infrastructure affect the success or failure of devolved management structures, even if a service works within an allocated budget and achieves high patent throughput and decreasing length of patient stay.


Hormonal status in postmenopausal androgenetic alopecia.

The development of androgenetic alopecia is thought to be caused by increased androgen action on hair follicles with menopause. Testosterone, estradiol and sex hormone binding globulin (SHBG) serum levels were determined in ten postmenopausal women with androgenetic alopecia and in ten sex and age matched healthy controls. No statistically significant differences were found in the hormone levels between the patients and the controls. These findings suggest that a genetically determined functional alteration of androgen receptors and/or a metabolic disturbance may exist in the hair follicle keratinocytes in androgenetic alopecia.


Cutaneous alterations in patients with chronic renal failure.

The purpose of this study was to evaluate the prevalence of dermatologic problems among patients with chronic renal failure (CRF) undergoing hemodialysis or peritoneal dialysis. One-hundred and two patients with CRF were examined for the presence of cutaneous alterations. All patients examined had at least one cutaneous lesion. The most prevalent findings were alterations in the cutaneous pigmentation. Of particular interest was an increased prevalence of hyperpigmented macules on the palms and soles. Other manifestations, seen particularly in the hemodialysis group, included the half-and-half nail, pruritus, and keratotic pits of the palms and soles. Infectious processes were more prevalent in the group undergoing peritoneal dialysis. This study showed that all patients with CRF have some type of cutaneous alteration and that the type of dialysis could have some influence upon the incidence of these changes.


Pelgeroid-like anomalous cells in the diagnosis of neutrophilic dermatosis associated with myelodysplastic syndrome.

Neutrophilic dermatoses (ND), with or without accompanying myelodysplastic syndrome (MDS), were examined in terms of nuclear abnormality like pelgeroid anomaly of infiltrating cells into skin lesions. Six ND accompanying MDS showed 1.0 to 13.5% of such anomalous cells among infiltrating cells. In contrast, ND without accompanying myeloproliferative disorders rarely had such anomalous cells. Our findings suggest that pelgeroid-like anomalous cells infiltrating into ND are probably a good marker of underlying MDS.


Curative effect of dexamethasone-cyclophosphamide pulse therapy for the treatment of pemphigus vulgaris.

In 1982, five patients having pemphigus vulgaris, four men and one woman, ranging in age between 16 and 48 years, were treated with an arbitrary regimen designed by us. The regimen consisted of giving 100 mg dexamethasone in 500 mL of 5% glucose by a slow intravenous infusion on three consecutive days, along with 500 mg cyclophosphamide on one day only. Such dexamethasone-cyclophosphamide pulses (DCP) were meant to be given once a month, but most patients were not regular in this treatment. In between these pulses the patients received only 50 mg cyclophosphamide orally per day. Oral corticosteroids were given only when necessary. After having received a total of 14 to 48 DCPS, further treatment for pemphigus was withdrawn. All the patients are in continuous clinical remission for the last 4 to 9 years and without any treatment for 2 to 7 years. Further studies on more pemphigus patients have yielded similar results suggesting that it may be possible to cure pemphigus.


Electrotrichogenesis: further evidence of efficacy and safety on extended use.

These data represent a subset of data from the original 36-week study conducted by Maddin et al., which was in itself a preliminary study of a pulsed electrical stimulation device in male subjects alone. The extension phase of this study, which is summarized here, was undertaken to gather data on longer-term efficacy and safety and to study clinical effects in control subjects who were then switched to active treatment. Thirteen subjects had active treatment for 70 weeks, and 14 subjects were included in the crossover group, which had sham treatment for 36 weeks followed by active treatment for 30 additional weeks. On average, terminal hair counts increased from 82 to 276 in the active treatment group. Among those in the crossover group, a mild increase, from 124 to 160, was observed during the sham treatment period and a more notable increase, from 160 to 249, occurred during the subsequent active treatment period. The results presented here provide evidence of the efficacy and safety of this device during extended use; however, the generalizability of these findings is limited by the small subset of subjects for whom complete data are available.


Design, synthesis and structural characterization of model heterodimeric coiled-coil proteins.

We report the design and synthesis of model heterodimeric coiled-coil proteins and the packing contribution of interchain hetero-hydrophobic side-chains to coiled-coil stability. The heterodimeric coiled-coils are obtained by oxidizing two 35-residue polypeptide chains, each containing a cysteine residue at position 2 and differing in amino acid sequences in the hydrophobic positions ("a" and "d") responsible for the formation and stabilization of the coiled-coil. In each peptide, a single Ala residue was substituted for Leu at position "a" or "d". The formation and stability of heterodimeric coiled-coils were investigated by circular dichroism studies in the presence and absence of guanidine hydrochloride and compared to the corresponding homodimeric coiled-coils. The coiled-coil proteins with an Ala substitution at position "a" were less stable than those with an Ala substitution at position "d" in both the homodimeric (Ala-Ala interchain interactions) and heterodimeric (Leu-Ala interchain interactions ) coiled-coils. The 70-residue disulfide bridged peptides (homo- and heterodimeric coiled-coils) can be readily separated by reversed-phase chromatography (RPC) even though they have identical amino acid compositions as well as in the hydrophobic "a" and "d" positions. The elution of the 70-residue peptides prior to their corresponding 35-residue monomers suggests that these proteins are retaining a large portion of their coiled-coil structure during RPC at pH2 and their retention behavior correlates with protein stability.


In situ neutralization in Boc-chemistry solid phase peptide synthesis. Rapid, high yield assembly of difficult sequences.

Simple, effective protocols have been developed for manual and machine-assisted Boc-chemistry solid phase peptide synthesis on polystyrene resins. These use in situ neutralization [i.e. neutralization simultaneous with coupling], high concentrations (> 0.2 M) of Boc-amino acid-OBt esters plus base for rapid coupling, 100% TFA for rapid Boc group removal, and a single short (30 s) DMF flow wash between deprotection/coupling and between coupling/deprotection. Single 10 min coupling times were used throughout. Overall cycle times were 15 min for manual and 19 min for machine-assisted synthesis (75 residues per day). No racemization was detected in the base-catalyzed coupling step. Several side reactions were studied, and eliminated. These included: pyrrolidonecarboxylic acid formation from Gln in hot TFA-DMF; chain-termination by reaction with excess HBTU; and, chain termination by acetylation (from HOAc in commercial Boc-amino acids). The in situ neutralization protocols gave a significant increase in the efficiency of chain assembly, especially for "difficult" sequences arising from sequence-dependent peptide chain aggregation in standard (neutralization prior to coupling) Boc-chemistry SPPS protocols or in Fmoc-chemistry SPPS. Reported syntheses include HIV-1 protease(1-50,Cys.amide), HIV-1 protease(53-99), and the full length HIV-1 protease(1-99).


Effects of resin swelling and substitution on solid phase synthesis.

Resins sold by several companies were examined for swelling, uniformity of beads, and substitution in the case of resins sold with the first amino acid attached. Effects of resin swelling, uniformity, and substitution on the solid phase synthesis of long, structured and/or branched peptides were evaluated.


A rational design of synthetic peptide vaccine with a built-in adjuvant. A modular approach for unambiguity.

We describe a peptide vaccine model containing a built-in adjuvant. This model used a multiple antigen peptide system (MAPS) to amplify peptide antigens and a lipoamino acid, tripalmitoyl glyceryl cysteine (P3C), as a built-in adjuvant. An 18-residue peptide antigen (B2) derived from the third variable domain (amino acid 312-329) of the glycoprotein gp120 of type I human immunodeficiency virus (HIV-1) was used in this model. This peptide antigen is a suitable target since it consists of neutralizing, T-helper, and T-cytotoxic epitopes. The peptide antigen in a tetravalent MAPS format (B2M-P3C) with a lipophilic attachment was synthesized by two routes for comparison: a direct stepwise approach and an indirect modular approach. In the stepwise approach, each residue was sequentially added to the peptide resin to give B2M-P3C and the P3C was incorporated to the side chain of a carboxyl terminal lysine as Fmoc-Lys(P3C). In the modular approach, a module containing a chloroacetylated core matrix of MAPS (M-P3C) with a carboxyl tetrapeptide bearing Lys(P3C) and a second module containing the peptide antigen B2 with a cysteine at its terminus were synthesized and purified separately, and then coupled to each other to form B2M-P3C. In the modular approach, the molecular ion of B2M-P3C was unambiguously identified by ion-spray mass spectrometry. B2M-P3C, administered in liposomes without any adjuvant such as Freund's complete adjuvant, was used to immunize mice and found to induce gp120-specific antibodies in vitro, and prime cytotoxic T lymphocytes in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)


Synthesis and biological activity of new conformationally restricted analogues of pepstatin.

A new statine derivative, 3-hydroxy-4-amino-5-mercaptopentanoic acid; cysteinylstatine (CySta), was synthesized and used to prepare a series of conformationally restricted analogues of pepstatin (Iva-Val-Val-Sta-Ala-Sta) in which the conformational constraint was introduced via a bis-sulfide connecting the appropriately substituted residues in the P1 and the P3 inhibitor side chains. The precursor peptide, Iva-Cys-Val-CySta-Ala-Iaa, was synthesized and alkylated with a series of dibromoalkanes and alkenes to produce the cyclic structures. This strategy permitted the carbon atom spacing between the P1 and the P3 inhibitor side chains to be systematically varied so as to produce inhibitors with 15-, 16-, and 17-membered ring systems. Additional non-cyclic analogues were synthesized as controls by alkylating the bisthiol intermediates with methyl iodide. The inhibitory potency of the analogues were determined against porcine pepsin and penicillopepsin by using standard enzyme kinetic assays. The cyclic inhibitor were found to be potent inhibitors of both aspartic proteases; inhibitor that contained a trans-2-butene link between the two sulfur atoms was found to be the most potent inhibitor with a Ki less than 1 nM against pepsin and 3.94 nM against penicillopepsin. This series of compounds illustrates a new type of conformational restriction that can be used to probe for the bioactive conformation of peptides.


Influence of glycine residues on peptide conformation in membrane environments.

Transmembrane (TM) segments of integral membrane proteins are putatively alpha-helical in conformation, yet their primary sequences are rich in residues known in globular proteins as helix-breakers (Gly) and beta-sheet promoters (Ile, Val, Thr). To examine the specific 2 degrees structure propensities of such residues in membrane environments, we have now designed and synthesized a series of model 20-residue peptides with "guest" hydrophobia segments embedded in "host" N- and C-terminal hydrophilic matrices. Molecular design was based on the prototypical sequence NH2-(Ser-Lys)2-Ala5-Leu6-x7-Ala8-Leu9-y10-Trp 11-Ala12-Leu13-z14-(Lys-Ser)3-OH. The 10-residue hydrophobic mid-segment 5-14 is expected to act as ca. three turns of an alpha-helix. In the present work, we compare the 20-residue peptide having three "helix-forming" Ala residues [x = y = z = Ala (peptide 3A)] to the corresponding peptide 3G (x = y = z = Gly) which contains three "helix-breaking" Gly residues. Trp was inserted to provide a measure of aromatic character typical of TM segments; Ser and Lys enhanced solubility in aqueous media. Circular dichroism studies in water, in a membrane-mimetic [sodium dodecylsulfate (SDS)], medium, and in methanol solutions, demonstrated the exquisite sensitivity of the conformations of these peptides to environment, and proved that despite its backbone flexibility, Gly can be accommodated as readily as Ala into a hydrophobic alpha-helix in a membrane. Nevertheless, the relative stability of Ala- vs. Gly-containing helices emerged in methanol solvent titration and temperature dependence experiments in SDS.(ABSTRACT TRUNCATED AT 250 WORDS)


Molecular dynamics simulations of opioid peptide analogs containing multiple conformational restrictions.

Molecular dynamics simulations were performed on the potent and slightly mu-receptor selective cyclic dermorphin analog H-Tyr-D-Orn-Phe-Glu-NH2 as well as on analogs containing a conformationally restricted phenylalanine derivative in place of Phe in the 3 position of the peptide sequence. Peptides studied included the potent and highly mu-selective analogs H-Tyr-D-Orn-Aic-Glu-NH2 (Aic = 2-aminoindan-2-carboxylic acid), H-Tyr-D-Orn-Atc-Glu-NH2 (Atc = 2-aminotetralin-2-carboxylic acid) and H-Tyr-D-Orn-D-Atc-Glu-NH2, and the weakly active analog H-Tyr-D-Orn-Tic-Glu-NH2 (Tic = tetrahydroisoquinoline-3-carboxylic acid). Four different starting conformations were chosen for each peptide, and after equilibration each simulation was allowed to proceed for 100 picoseconds at 600 degrees K. The 14-membered ring structures in the Phe-, Aic-, L- and D-Atc-containing analogs showed moderate structural flexibility, while the peptide ring in the Tic-containing analog was more rigid. As theoretically predicted, the phi 3 and psi 3 angles of the Aic-, L- and D-Atc-containing analogs were limited to values of either about +50 degrees or -50 degrees during almost the entire period of the simulations. In the Tic-containing analog the phi 3 and psi 3 angles were 0 degrees and 90 degrees, respectively, and did not change for the entire duration of the simulation. The side chains of the constrained amino acids showed limited movement, but transitions between the allowed conformations did occur on the time scale of the simulations.(ABSTRACT TRUNCATED AT 250 WORDS)


Semisynthesis of human growth hormone-releasing factor by trypsin catalyzed coupling of leucine amide to a C-terminal acid precursor.

Human growth hormone-releasing factor, GRF(1-44)-NH2, was synthesized by trypsin catalyzed coupling of Leu-NH2 to Arg43 of the precursor, GRF(1-43)-OH, prepared by solid phase peptide synthesis. The semisynthetic GRF(1-44)-NH2 was fully characterized and showed full potency in the rat pituitary in vitro bioassay. Conversion to GRF(1-44)-NH2 was limited to 60-70% in both 75% v:v N,N'-dimethylacetamide and 95% v:v 1,4-butanediol due to competing transpeptidations at Arg41 and Arg38 generating [Leu42]-GRF(1-42)-NH2 and [Leu39]-GRF(1-39)-NH2 side-products, respectively. The rates of formation and yields of GRF(1-44)-NH2 versus pH, Leu-NH2 concentration, and solvent composition were also studied.


HIV protease (HIV PR) inhibitor structure-activity-selectivity, and active site molecular modeling of high affinity Leu [CH(OH)CH2]Val modified viral and nonviral substrate analogs.

This report details the structure-activity relationships of the HIV gag substrate analog Val-Ser-Gln-Asn-Leu psi[CH(OH)CH2]Val-Ile-Val (U-85548E), an inhibitor exhibiting subnanomolar affinity towards HIV type-1 aspartic proteinase (HIV-1 PR). Our data show that the P1-P2' tripeptidyl sequence provides the minimal chemical determinant for HIV-1 PR binding. We describe the structure-activity properties of Leu psi[CH(OH)CH2]Val substitution in other peptidyl ligands of nonviral substrate origin (e.g., angiotensinogen, insulin and pepstatin). Furthermore, the aspartic proteinase selectivities of a few key compounds are summarized relative to evaluation against human renin, human pepsin, and the fungal enzyme, rhizopuspepsin. These studies have led to the rational design of nanomolar potent inhibitors of both HIV-1 and HIV-2 PR. Finally, a 2.5 A resolution X-ray crystallographic structure of U-85548E complexed to synthetic HIV-1 PR dimer (Jaskolski et al., Biochemistry 30, 1600 [1991]) provided a 3-D picture of the inhibitor bound to the enzyme active site, and we performed computer-assisted molecular modeling studies to explore the possible binding modes of the above series of Leu psi[CH(OH)CH2]Val substituted HIV-1 PR inhibitors.


Novel approach to the design of synthetic radioiodinated linear V1A receptor antagonists of vasopressin.

We report the solid phase synthesis of six analogs of the potent and selective linear AVP vasopressor (V1a receptor) antagonist: Phaa1-D-Tyr(Et)2-Phe3-Gln4-Asn5-Lys6-Pro7-Arg-NH(8)2(A) (where Phaa = phenylacetyl) in which the Phaa1 residue is replaced by hydroxyphenylacetyl (HO-Phaa), hydroxyphenylpropionyl (HO-Phpa) and phenylpropionyl (Phpa) and the D-Tyr(Et)2 and Lys6 residues by D-Tyr(Me)2 and Arg6 substituents. The phenolic-containing peptides were synthesized to test the feasibility of using this approach for the design of high affinity selective ligands for AVP V1a receptors. The following analogs of A were synthesized: 11 [(HO)Phaa1]; 2. [(HO)Phaa1,D-Tyr(Me)2]; 3. [(HO)Phaa1,D-Tyr(Me)2, Arg6]; 4. [(HO)Phaa1,Arg6]; 5. [Phpa1]; 6. [(HO)Phpa1]. All six peptides were examined for agonistic and antagonistic potencies in vasopressor (V1a-receptor) and antidiuretic (V2-receptor) and in vitro oxytocic assays in rats. The affinities of the phenolic-containing peptides for hepatic V1a and uterine receptors were also determined. The phenolic-containing peptides all exhibit potent V1a antagonism. Their anti-V1a pA2 values range from 8.23 to 8.63 (the anti-V1a pA2 value of A = 8.69). Their inhibition constants (Ki in nM) range 0.4 to 1.0. They are weak antidiuretic agonists with activities ranging from 0.022 U/mg to 0.13 U/mg (A = 0.033 U/mg). They all exhibit OT antagonism in vitro. Their anti-OT pA2 values range from 7.28 to 7.71 (A = 7.62). All five phenolic compounds were iodinated using iodine chloride and tested in the same in vivo and in vitro assay system.(ABSTRACT TRUNCATED AT 250 WORDS)