Biological Activity of a Mouse-Human Chimeric Immunoglobulin G2 Antibody to Cryptococcus neoformans Polysaccharide

The variable regions of the heavy and light chains of the protective murine monoclonal antibody (MAb) 2H1 (m2H1) were expressed with the human constant region genes for immunoglobulin G2 (IgG2) and kappa, respectively, to construct a chimeric antibody (ch2H1). ch2H1 retains the specificity of the parent MAb, exhibits biological activity, and lacks the toxicity of the parent murine IgG1 in chronically infected mice.     

The intra-cortical trajectory of the coeruleo-cortical projection in the rat: A tangentially organized cortical afferent

Cortical and sub-cortical lesions in the rat were used to analyze the intracortical trajectory of the noradrenergic axons, which were visualized by aldehyde-induced catecholamine histofluorescence and by immunohistochemistry using an antibody directed against rat dopamine-β-hydroxylase. Following subcortical lesions there is a slowly progressive reduction in the density of cortical noradrenergic axons, indicating that they undergo asynchronous anterograde degeneration. By 2 weeks after transection of the dorsal noradrenergic bundle, no dopamine β-hydroxylase-immunoreactive fibers are detectable in the ipsilateral cortex. Neither transection of the cingulum bundle, nor parasagittal incisions through the dorsal cortex lateral to the cingulum, diminished the noradrenergic innervation of medial or dorso-lateral cortex. A cortical lesion medial to the cingulum bundle markedly reduced the density of noradrenergic fibers in cingulate cortex caudal to the lesion, but did not affect the innervation of dorso-lateral cortex. In contrast, dorso-lateral frontal incisions and decortication (frontal lobotomy) produced a marked ipsilateral decrease in the noradrenergic fiber density throughout the remaining dorso-lateral cortex, while sparing the innervation of cingulate and infra-rhinal cortex.These results demonstrate that the dorso-lateral cortex is innervated by noradrenergic fibers in the medial forebrain bundle that reach the frontal pole, turn dorsally over the anterior portion of the forceps minor and continue caudally within the deep layers of frontal and dorso-lateral cortex, supplying the noradrenergic innervation throughout their trajectory. The medial cortex is innervated by a separate group of noradrenergic fibers that ascend through the septum, curve over the genu of the corpus callosum, and run caudally in the supracallosal stria.The present results show that the cingulum bundle is not a major intra-cortical noradrenergic pathway and does not provide branches that contribute significantly to the innervation of dorsal or lateral cortex. Thus the medial and lateral cortex can be selectively and differentially denervated of noradrenergic fibers and a coarse topographic order exists in the noradrenergic innervation of cortex. Since noradrenergic fibers travel long distances within the cortical grey matter, a small lesion of frontal cortex can have far-reaching effects on the innervation of distant, more caudal regions of cortex. The coeruleocortical projection has properties that differ from those of the best characterized cortical afferents and may be a useful model for the study of other ascending monoamine systems. The tangential, intracortical trajectory of the noradrenergic fibers would confer upon the coeruleo-cortical system the capacity to modulate neuronal activity simultaneously through a vast expanse of neocortex. A formulation of cortical organization is presented which integrates the tangential organization of the coeruleo-cortical projection with the concept of columnar organization of cortex.     

Identification and characterization of clinical isolates of members of the Staphylococcus sciuri group

A total of 28 staphylococcal isolates from human clinical specimens belonging to the Staphylococcus sciuri group were identified and characterized. The API Staph and ID32 STAPH correctly identified S. sciuri and S. lentus but not S. vitulinus strains. Identification to the subspecies level was possible only by a PCR-based method.     

Monoclonal antibodies define genus-specific, species-specific, and cross-reactive epitopes of the chlamydial 60-kilodalton heat shock protein (hsp60): specific immunodetection and purification of chlamydial hsp60.

Ocular and urogenital tract infections with Chlamydia trachomatis can progress to chronic inflammatory diseases that produce blindness and tubal infertility. The pathophysiology of these chronic disease conditions is thought to be immunologically mediated, and the chlamydial 60-kDa heat shock protein (hsp60) has been implicated as a major target antigen that stimulates the immunopathological response. The lack of chlamydial hsp60 antibodies and purified hsp60 has severely restricted studies to define more thoroughly the role of this protein in the immunopathogenesis of chlamydial disease. We produced a panel of antichlamydial hsp60 monoclonal antibodies (MAbs) and defined their specificities by immunoblotting against lysates of C. trachomatis, C. psittaci, and six other genera of bacteria. Three patterns of anti-hsp60 immunoreactivity were observed: chlamydial species specific, chlamydial genus specific, and cross-reactive. The epitopes recognized by these MAbs were localized within the primary amino acid sequence of hsp60 by immunoblotting against recombinant amino-terminal truncated hsp60 fusion polypeptides and then precisely mapped by use of overlapping synthetic peptides. The majority of the MAbs mapped to either the amino or the carboxyl termini of hsp60. Epitopes defining all three MAb reactivities mapped within amino-terminal residues 6 to 16. Genus-specific hsp60 MAbs mapped to epitopes located within this region and to residues 17 to 28 and 177 to 189. Antichlamydial hsp60 MAbs stained inclusions as effectively as MAbs specific for the major outer membrane protein. Homogeneous preparations of full-length recombinant chlamydial hsp60 and amino-terminal truncated recombinant hsp60 polypeptides were obtained by immunoabsorption chromatography with an hsp60 MAb reactive to the carboxyl terminus of the protein. Thus, the antichlamydial MAbs described here should be extremely useful for the specific immunodetection of hsp60 in tissues from individuals having different disease manifestations and for the purification of hsp60 or truncated hsp60 polypeptides for use in serologic and lymphocyte proliferation assays. The availability of these MAbs will facilitate studies to define more precisely the role of hsp60 in the immunopathogenesis of chlamydial disease.     

In situ analysis of the evolution of the primary immune response in murine Chlamydia trachomatis genital tract infection

Adaptive immune responses contribute to the resolution of Chlamydia trachomatis genital tract infection and protect against reinfection, but our understanding of the mechanisms of those protective responses is incomplete. In this study, we analyzed by in situ immunohistochemistry the progression of the inflammatory and cytokine responses in the genital tracts of mice vaginally infected with C. trachomatis strain mouse pneumonitis. The cellular inflammatory response was characterized by an initial elevation in myeloid cells in the vagina (day 3) and uterine horns (day 7), followed by a marked rise in the number of T cells, predominantly CD4(+) cells. CD8(+) T cells and CD45R(+) B cells were also detected but were much less numerous. Perivascular clusters of CD4(+) T cells, which resembled clusters of T cells seen in delayed-type hypersensitivity responses, were evident by 2 weeks postinfection. Following the resolution of infection, few CD8(+) T cells and CD45R(+) B cells remained, whereas numerous CD4(+) T cells and perivascular clusters of CD4(+) T cells persisted in genital tract tissues. Interleukin-12 (IL-12)- and tumor necrosis factor alpha (TNF-alpha)-producing cells were observed in vaginal tissue by day 3 of infection and in uterine tissues by day 7. Cells producing IL-4 or IL-10 were absent from vaginal tissues at day 3 of infection but were present in uterine tissues by day 7 and were consistently more numerous than IL-12- and TNF-alpha-producing cells. Thus, the evolution of the local inflammatory response was characterized by the accumulation of CD4(+) T cells into perivascular clusters and the presence of cells secreting both Th1- and Th2-type cytokines. The persistence of CD4(+)-T-cell clusters long after infection had resolved (day 70) may provide for a readily mobilizable T-cell response by which previously infected animals can quickly respond to and control a secondary infectious challenge.     

Mechanical Characterization of an In Vitro Device Designed to Quantitatively Injure Living Brain Tissue

Due to the nonlinear, viscoelastic material properties of brain, its mechanical response is dependent upon its total strain history. Therefore, a low strain rate, large strain will likely produce a tissue injury unique from that due to a high strain rate, moderate strain. Due to a lack of current understanding of specific in vivo physiological injury mechanisms, a priori assumptions cannot be made that a low strain rate injury induced by currently employed in vitro injury devices is representative of clinical, nonimpact, inertial head injuries. In the present study, an in vitro system capable of mechanically injuring cultured tissue at high strain rates was designed and characterized. The design of the device was based upon existing systems in which a clamped membrane, on which cells have been cultured, is deformed. However, the present system incorporates three substantial improvements: (1) noncontact measurement of the membrane deflection during injury; (2) precise and independent control over several characteristics of the deflection; and (3) generation of mechanical insults over a wide range of strains (up to 0.65) and strain rates (up to 15s^–1). Such a system will be valuable in the elucidation of the mechanisms of mechanical trauma and determination of injury tolerance criteria on a cellular level utilizing appropriate mechanical injury parameters.     

Development of hatching blastocysts from immature human oocytes following in-vitro maturation and fertilization using a co-culture system

Recently, in-vitro maturation (IVM) of immature human oocytes recovered from non-stimulated follicles has been applied in the treatment of infertility. However, in previous reports, very few embryos cultured in conventional medium have reached the expanded blastocyst stage following in-vitro maturation and fertilization (IVM/IVF). The objective of this study was to investigate whether the developmental competence of human embryos following IVM/IVF could be enhanced by the use of a human ampullary cell co-culture system. Immature human oocytes were aspirated from small follicles at Caesarean section and then cultured in medium containing human menopausal gonadotrophin for 36 to 48 h, followed by insemination. Zygotes were randomly cultured either in conventional culture medium alone or in the co-culture system. Of 48 embryos cultured in conventional medium alone, all arrested at the 2-16- cell stage on day 3 after insemination. Of 46 embryos cultured in the co-culture system, 26 embryos (56.5%) arrested at the 2-16-cell stage. Six embryos (13%) developed to the morula stage. Fourteen embryos (30.4%) developed to expanded blastocysts and two blastocysts were hatching on day 7 after insemination. We conclude that co-culture significantly enhances the development of blastocysts in embryos resulting from IVM/IVF.      

A mouse model of spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) is an adult-onset motor neuron disease, caused by the expansion of a trinucleotide repeat (TNR) in exon 1 of the androgen receptor (AR) gene. This disorder is characterized by degeneration of motor and sensory neurons, proximal muscular atrophy, and endocrine abnormalities, such as gynecomastia and reduced fertility. We describe the development of a transgenic model of SBMA expressing a full-length human AR (hAR) cDNA carrying 65 (AR(65)) or 120 CAG repeats (AR(120)), with widespread expression driven by the cytomegalovirus promoter. Mice carrying the AR(120) transgene displayed behavioral and motor dysfunction, while mice carrying 65 CAG repeats showed a mild phenotype. Progressive muscle weakness and atrophy was observed in AR(120) mice and was associated with the loss of alpha-motor neurons in the spinal cord. There was no evidence of neurodegeneration in other brain structures. Motor dysfunction was observed in both male and female animals, showing that in SBMA the polyglutamine repeat expansion causes a dominant gain-of-function mutation in the AR. The male mice displayed a progressive reduction in sperm production consistent with testis defects reported in human patients. These mice represent the first model to reproduce the key features of SBMA, making them a useful resource for characterizing disease progression, and for testing therapeutic strategies for both polyglutamine and motor neuron diseases.     

Expression of class II MHC gene products by macrophages in human uteroplacental tissue.

The expression of class II MHC determinants by fetal and maternal macrophages in human uteroplacental tissues was examined with monoclonal antibodies directed against HLA-DR, DP and DQ antigens. Maternal macrophages in early and full-term pregnancy decidua were HLA-DR positive, and a substantial proportion also expressed DP and DQ antigens. Fetal macrophages in chorionic villous stroma in first-trimester pregnancy were rarely HLA-DR positive, and DQ and DP antigens were never expressed. In term placental tissues, groups of villous stromal macrophages were HLA-DR positive, as were fetal macrophages within amniotic mesenchyme. In contrast, DP and DQ antigens were detected on a small minority of fetal macrophages in term placental tissues. DP and DQ antigens were not detected in the absence of DR antigens.