Neuropeptide Y-immunoreactive cells in the caudal medulla project to the median preoptic nucleus

The region surrounding the anteroventral third ventricle, particularly the median preoptic nucleus, has been implicated in the control of fluid balance and blood pressure. Previous studies indicate that catecholaminergic inputs from the caudal medulla are important in these controls. In this study we report that neuropeptide Y-immunoreactive cells in the caudal medulla project to the median preoptic nucleus in the basal forebrain. Notably these cells are found in the caudal ventral lateral medulla and the nucleus of the solitary tract. Since catecholaminergic projections to the median preoptic nucleus also arise from these regions the possibility of interactions between NPY and catecholamines exists, particularly in the control of fluid balance and blood pressure.     

Epstein-Barr virus-transformed human precursor B cell lines: altered growth phenotype of lines with germ-line or rearranged but nonexpressed heavy chain genes

A series of lymphoblastoid cell lines (LCLs) have been established by in vitro infection of fetal bone marrow and fetal liver cells with Epstein-Barr virus (EBV). While most lines showed the usual mature B cell phenotype, a small proportion were cytoplasmic and surface immunoglobulin (Ig) heavy and light chain negative. Analysis of gene rearrangements indicated that the Ig- lines were either germ-line or nonproductively rearranged when probed for JH and were in germ-line configuration for C chi; no mu or chi mRNA could be detected in such cells. Precursor B cell lines were indistinguishable from their normal Ig+ counterparts in their expression of a wide variety of cell surface markers including "activation" antigens usually associated with the lymphoblastoid state; even the single LCL showing germ-line heavy and light chain genes expressed B lineage-specific cell surface antigens. However, the Ig- lines were distinct from their Ig+ counterparts in three important respects: (a) they grew much more slowly and achieved lower saturation densities, (b) they showed unusually high proportions (8-16%) of cells in EBV-productive cycle, and (c) they contained unusually high proportions (up to 40%) of cells expressing free joining (J) chain. These results suggest that precursor B cells differ in their response to the growth-transforming effects of EBV such that the virus-cell interaction in precursor B cell lines is inherently less stable than in conventional LCL. In particular there may be a greater movement of cells out of cycle and along the B cell maturation pathway. It is possible that such movement leads in individual cells either to virus replication or to a "sterile" plasmacytoid differentiation with J chain expression in the absence of Ig synthesis.     

Mechanisms by which Candida albicans induces endothelial cell prostaglandin synthesis.

One strategy for improving resistance to opportunistic pathogens is to determine host cellular responses during the invasion process and upregulate those responses that are relevant to host defense mechanisms. Within this context, we have shown previously that invasion of endothelial cells by Candida albicans in vitro causes increased production of prostaglandins. As a prerequisite for modulating endothelial cell prostaglandin production, we now characterize the mechanisms through which this process occurs. Endothelial cell invasion by C. albicans appeared to stimulate the conversion of arachidonic acid into prostaglandins by upregulating the synthesis of endothelial cell cyclooxygenase and increasing the activity of the endothelial cell phospholipase. The enhanced activities of these two enzymes were independent of calphostin C-sensitive protein kinase C and resulted in the increased production and extracellular secretion of prostaglandin I2 (PGI2), PGF2 alpha, and PGE2. The secretion of these prostaglandins had no effect on the amount of endothelial cell injury induced by C. albicans. The role of the increased prostaglandin secretion by endothelial cells is likely related to modulation of the leukocyte response at the candida-leukocyte-endothelial cell interface.     

Increased inflammatory reactivity in newly formed lining tissue

The air pouch has been shown to provide a convenient model for studying the behaviour of synovial lining tissue. Air pouches of different ages were used to study the reactivity of newly developing lining tissue towards irritants known to cause inflammation. Pouches of 1 day in age were relatively inert in their reactivity as judged by the number of cells and volume of the exudate accumulating in the pouch. In contrast, 3-day-old pouches responded to a much greater extent, and 6-day-old pouches were highly responsive with a further increase in cell numbers and fluid volume. The different responses of 1-, 3- and 6-day-old pouches could be explained by (a) developing vascularity of the pouch, (b) formation of an organised lining of phagocytic cells, or (c) an increasingly organised mechanical barrier that retains the irritant and products of the inflammatory response. These studies of air pouch lining development permit a dissection of those components necessary for inflammatory reactivity of a lining tissue and may help explain the sensitivity of synovium to chronic inflammation.     

Influence of preimmunization antibody levels on the specificity of the immune response to related polysaccharide antigens

We studied the influence of preimmunization antibody level on the immune response of adults to one of two structurally related yet immunologically distinct type-specific polysaccharides from Type III Group B streptococcus and Type 14 pneumonococcus. Four weeks after immunization with multivalent pneumococcal vaccine, 20 subjects with low levels of antibody to Type III Group B streptococcus antigen had no significant increase in antibody to this antigen (P greater than 0.05), but all volunteers with moderate to high preimmunization antibody levels who were immunized with Pneumovax had significant increases (P less than 0.01). However, the streptococcal antibody response to pneumococcal Type 14 antigen was weaker and briefer than that in 10 adults given Type III Group B streptococcus vaccine(P less than 0.05). Preimmunization antibody levels influenced the immune response to a structurally similar polysaccharide antigen, but specific Type III polysaccharide antigen appeared necessary to induce a primary antibody response in "nonimmune" adults. We conclude that immunization of mothers with pneumococcal vaccine is not likely to prevent neonatal Type III Group B streptococcal infection, despite immunologic similarities between the two organisms.     

The response to lipopolysaccharide of mouse spleen lymphocytes fractionated on the basis of surface immunoglobulin and complement receptor using fluorescence-activated cell sorting and rosetting techniques.

Mouse spleen lymphocytes were stained with rabbit antisera specific for either μ chain or δ chain, followed by fluorescein-conjugated goat anti-rabbit immunoglobulin. The cells were analysed and fractionated using a fluorescence-activated cell sorter. Fifty-five per cent of the lymphocytes stained with a polyspecific anti-Ig reagent or with a combination of anti-μ and anti-δ reagents, while about 40% of the lymphocytes were stained when either the anti-μ reagent or the anti-δ reagent was used alone. Three per cent of the lymphocytes stained with the anti-μ reagent, but not with the anti-δ reagent, and eight per cent stained only with the anti-δ reagent. Unfractionated spleen cells and populations depleted of μ- or δ-bearing cells were cultured in the presence of lipopolysaccharide. All three populations responded by incorporating [³H]-thymidine and secreting IgM and IgG.Spleen cells were fractionated by a rosetting technique into complement receptor-positive and negative populations. Both populations were able to respond to lipopolysaccharide and to synthesize Ig of both the IgM and IgG classes. Unfractionated cells and complement receptor-negative populations were stained for surface μ or δ chain and analysed on the fluorescence-activated cell sorter. The distribution of staining intensity suggested that the complement receptor-bearing population was enriched in cells which stain weakly for μ and cells which stain with a low to intermediate intensity for δ chain.It is concluded that the precursors of IgM- and IgG-secreting cells are not limited to any one of the three populations of cells defined on the basis of surface immunoglobulin or to either of the populations defined on the basis of the complement receptor.     

Adherence to and damage of endothelial cells by Cryptococcus neoformans in vitro: role of the capsule.

Escape from the intravascular compartment is likely a critical step in the development of hematogenously disseminated cryptococcal infections, such as meningitis. The capsule of Cryptococcus neoformans is considered to be a virulence factor because of its antiphagocytic properties. To further investigate the role of the capsule in escape from the intravascular compartment, we used isogenic strain pairs, an acapsular mutant, and an encapsulated clinical isolate to determine the effects of the capsule of C. neoformans on adherence to, phagocytosis by, and damage of endothelial cells in vitro. Acapsular C. neoformans adhered significantly more to endothelial cells and caused greater endothelial cell injury than did encapsulated organisms. Coating of an acapsular strain with cryptococcal glucuronoxylomannan decreased both adherence to and damage of endothelial cells by 61.7% +/- 9.1% and 76.6% +/- 10.2%, respectively. Transmission electron microscopy demonstrated internalization of acapsular, but not encapsulated, organisms by endothelial cells. Internalization of an acapsular strain occurred through endothelial cell phagocytosis and was inhibited by cytochalasin D. Phagocytosis required a heat-labile serum factor, probably complement. These results suggest that acapsular or poorly encapsulated C. neoformans may be the form(s) that escapes from the vasculature during initiation of hematogenously disseminated disease.     

Enzyme-linked immunosorbent assay for streptococcal M protein antibodies.

The enzyme-linked immunosorbent assay (ELISA) described by Engvall and Perlmann, which uses antigen-coated tubes and enzyme-labeled anti-immunoglobulin, has been used for the detection of antibodies against streptococcal M protein. The antigen used in the assay was obtained by guanidine extraction of type M-12 streptococcal cell walls followed by hydroxyapatite chromatography. This antigen has the capacity to elicit bactericidal antibodies in rabbits. The results show that the ELISA is specific and highly sensitive for the detection of antibodies in rabbit and human antisera. Preliminary results suggest that, when M-12 antigen is used, the antibodies detected by ELISA are the same antibodies detected in the bactericidal test. The assay has been performed with human and rabbit sera. There was a 96% agreement between bactericidal and ELISA results with rabbit sera and 97.5% agreement with human sera. All bactericidal antibody-positive sera tested thus far yielded positive ELISA results.     

Resistance to platelet microbicidal protein results in increased severity of experimental Candida albicans endocarditis.

Thrombin-induced platelet microbicidal protein (tPMP) exerts potent in vitro microbicidal activity against pathogens commonly found in the bloodstream, including Staphylococcus aureus, Staphylococcus epidermidis, and Candida albicans. Localized platelet release of tPMP may be important in defense against infections involving the vascular endothelium caused by tPMP-susceptible organisms. In contrast, pathogens capable of surviving in the presence of tPMP could then exploit the platelet as an adhesive surface for attachment to damaged endothelium. To examine these hypotheses, we derived a tPMP-resistant (tPMP(r)) C. albicans strain from its tPMP-sensitive (tPMP(s)) parental strains were equivalent in vitro as assessed by genotyping (electrophoretic karyotype and restriction endonuclease analysis of genomic DNA), biotyping, germination, platelet aggregation, adherence to vascular endothelial cells, and growth characteristics. In addition, the tPMP(r) phenotype was stable following multiple in vitro and in vivo passages. We then investigated the in vivo relevance of tPMP susceptibility on endovascular infection using a rabbit model of endocarditis and hematogenous dissemination. Rabbits with transaortic catheters (n = 15 in each group) were challenged with either the tPMP(s) or tPMP(r) C. albicans strain. All rabbits developed C. albicans-induced endocarditis, as determined by the presence of infected vegetations. In rabbits challenged with tPMP(s) strain (P < 0.001). These results were seen in the absence of differences in either initial adherence of strains to cardiac valves or vegetation weights. Furthermore, although these C. albicans strains induced equivalent rates and extent of hematogenous renal infection, only the tPMP(r) strain disseminated hematogenously to the spleen (15 of 15 rabbits) versus 0 of 15 [tpmp(s) strain]; P < 0.0001). Thus, tPMP(r) C. albicans caused more-severe endocarditis and produced greater metastatic sequelae than the tPMP(s) counterpart.     

Processing of radical prostatectomy specimens for correlation of data from histopathological, molecular biological, and radiological studies: a new whole organ technique

AIMS: To develop a method of processing non-formalin fixed prostate specimens removed at radical prostatectomy to obtain fresh tissue for research and for correlating diagnostic and molecular results with preoperative imaging. METHODS/RESULTS: The method involves a prostate slicing apparatus comprising a tissue slicer with a series of juxtaposed planar stainless steel blades linked to a support, and a cradle adapted to grip the tissue sample and receive the blades. The fresh prostate gland is held in the cradle and the blades are moved through the cradle slits to produce multiple 4 mm slices of the gland in a plane perpendicular to its posterior surface. One of the resulting slices is preserved in RNAlater. The areas comprising tumour and normal glands within this preserved slice can be identified by matching it to the haematoxylin and eosin stained sections of the adjacent slices that are formalin fixed and paraffin wax embedded. Intact RNA can be extracted from the identified tumour and normal glands within the RNAlater preserved slice. Preoperative imaging studies are acquired with the angulation of axial images chosen to be similar to the slicing axis, such that stained sections from the formalin fixed, paraffin wax embedded slices match their counterparts on imaging. CONCLUSIONS: A novel method of sampling fresh prostate removed at radical prostatectomy that allows tissue samples to be used both for diagnosis and molecular analysis is described. This method also allows the integration of preoperative imaging data with histopathological and molecular data obtained from the prostate tissue slices.