From caution to urgency: the evolution of HIV testing and counselling in Africa.

OBJECTIVE: To describe recent changes in policy on provider-initiated testing and counselling (PITC) for human immunodeficiency virus (HIV) infection in African countries and to investigate patients' experiences of and views about PITC. METHODS: A review of the published literature and of national HIV testing policies, strategic frameworks, plans and other relevant documents was carried out. FINDINGS: Of the African countries reviewed, 42 (79.2%) had adopted a PITC policy. Of the 42, all recommended PITC for the prevention of mother-to-child HIV transmission, 66.7% recommended it for tuberculosis clinics and patients, and 45.2% for sexually transmitted infection clinics. Moreover, 43.6% adopted PITC in 2005 or 2006. The literature search identified 11 studies on patients' experiences of and views about PITC in clinical settings in Africa. The clear majority regarded PITC as acceptable. However, women in antenatal clinics were not always aware that they had the right to decline an HIV test. CONCLUSION: Policy and practice on HIV testing and counselling in Africa has shifted from a cautious approach that emphasizes confidentiality to greater acceptance of the routine offer of HIV testing. The introduction of PITC in clinical settings has contributed to increased HIV testing in several of these settings. Most patients regard PITC as acceptable. However, other approaches are needed to reach people who do not consult health-care services.

Elevation of susceptibility to ozone-induced acute tracheobronchial injury in transgenic mice deficient in Clara cell secretory protein.

Increases in Clara cell abundance or cellular expression of Clara cell secretory protein (CCSP) may cause increased tolerance of the lung to acute oxidant injury by repeated exposure to ozone (O3). This study defines how disruption of the gene for CCSP synthesis affects the susceptibility of tracheobronchial epithelium to acute oxidant injury. Mice homozygous for a null allele of the CCSP gene (CCSP-/-) and wild type (CCSP+/+) littermates were exposed to ozone (0.2 ppm, 8 h; 1 ppm, 8 h) or filtered air. Injury was evaluated by light and scanning electron microscopy, and the abundance of necrotic, ciliated, and nonciliated cells was estimated by morphometry. Proximal and midlevel intrapulmonary airways and terminal bronchioles were evaluated. There was no difference in airway epithelial composition between CCSP+/+ and CCSP-/- mice exposed to filtered air, and exposure to 0.2 ppm ozone caused little injury to the epithelium of both CCSP+/+ and CCSP-/- mice. After exposure to 1.0 ppm ozone, CCSP-/- mice suffered from a greater degree of epithelial injury throughout the airways compared to CCSP+/+ mice. CCSP-/- mice had both ciliated and nonciliated cell injury. Furthermore, lack of CCSP was associated with a shift in airway injury to include proximal airway generations. Therefore, we conclude that CCSP modulates the susceptibility of the epithelium to oxidant-induced injury. Whether this is due to the presence of CCSP on the acellular lining layer surface and/or its intracellular distribution in the secretory cell population needs to be defined.

Colonic spirochetosis of colony-raised rhesus macaques associated with Brachyspira and Helicobacter.

Colonic spirochetosis is an inflammatory bowel disease that affects a broad range of hosts, including human and non-human primates. The disease in humans and non-human primates is characterized by intimate attachment of the anaerobic spirochetes Brachyspira aalborgi and B. pilosicoli, and some unclassified flagellated microbes along the apical membrane of colonic enterocytes. Although the presence of spiral-shaped bacteria with single polar flagella and blunted ends in colonic spirochetosis is well established, the identities of many of these organisms is still unknown. Recently, Helicobacter species with a morphology similar to the flagellated bacteria present in colonic spirochetosis have been cultured from intestinal specimens obtained from rhesus macaques, some with idiopathic colitis. The purpose of the present study was to determine whether or not the flagellated bacteria seen in the colons of rhesus macaques with colonic spirochetosis are Helicobacter. The presence of flagellated bacteria alone (n=2) or together with spirochetes (n=1) in formalin-fixed and paraffin-embedded colons of three rhesus macaques with the naturally occurring disease was demonstrated by immunohistochemical staining and ultrastructural examination. Total DNA extracted from affected and control intestinal specimens was amplified by polymerase chain reaction (PCR) using Helicobacter 16S rRNA gene-specific primers. Comparative nucleotide sequence analysis of PCR products cloned from positive reactions indicated that two distinct Helicobacter genomospecies were present either alone or in combination with Brachyspira in the colons of rhesus macaques with microscopic lesions indicative of colonic spirochetosis.

Allergic asthma induced in rhesus monkeys by house dust mite (Dermatophagoides farinae).

To establish whether allergic asthma could be induced experimentally in a nonhuman primate using a common human allergen, three female rhesus monkeys (Macaca mulatta) were sensitized with house dust mite (Dermatophagoides farinae) allergen (HDMA) by subcutaneous injection, followed by four intranasal sensitizations, and exposure to allergen aerosol 3 hours per day, 3 days per week for up to 13 weeks. Before aerosol challenge, all three monkeys skin-tested positive for HDMA. During aerosol challenge with HDMA, sensitized monkeys exhibited cough and rapid shallow breathing and increased airway resistance, which was reversed by albuterol aerosol treatment. Compared to nonsensitized monkeys, there was a fourfold reduction in the dose of histamine aerosol necessary to produce a 150% increase in airway resistance in sensitized monkeys. After aerosol challenge, serum levels of histamine were elevated in sensitized monkeys. Sensitized monkeys exhibited increased levels of HDMA-specific IgE in serum, numbers of eosinophils and exfoliated cells within lavage, and elevated CD25 expression on circulating CD4(+) lymphocytes. Intrapulmonary bronchi of sensitized monkeys had focal mucus cell hyperplasia, interstitial infiltrates of eosinophils, and thickening of the basement membrane zone. We conclude that a model of allergic asthma can be induced in rhesus monkeys using a protocol consisting of subcutaneous injection, intranasal instillation, and aerosol challenge with HDMA.

Three-dimensional mapping of ozone-induced acute cytotoxicity in tracheobronchial airways of isolated perfused rat lung.

Acute lung injury induced by reactive oxygen gases such as ozone (O(3)) is focal and site-selective. To define patterns of acute epithelial injury along intrapulmonary airways, we developed a new analytic approach incorporating labeling of permeable cells, airway microdissection, and laser scanning confocal microscopy, and applied it to isolated perfused rat lungs where ventilation and breathing pattern could be controlled. After exposure to O(3) (0, 0.25, 0.5, or 1.0 ppm), lungs were lavaged to assess lactate dehydrogenase (LDH) and protein, or infused with the permeability marker ethidium homodimer-1 (EthD-1) via tracheal cannula, gently lavaged, and fixed by airway infusion. The airway tree of the right middle lobe was exposed by microdissection of the axial pathway down to the terminal bronchioles; the dissection was incubated with a second nuclear dye, YOPRO-1, to label all nuclei; and whole mounts were examined by confocal microscopy. Abundance of EthD-1-positive (injured) cells was estimated as the number per epithelial volume using stereology on Z-series of projected images. For ozone concentrations of 1.0 ppm, lavage fluid LDH and total protein did not increase over controls. Exposure produced a concentration- dependent but nonhomogeneous increase in the abundance of EthD-1-labeled cells in proximal and distal conducting airways both in the main pathway, including terminal bronchioles, and in side branches. Overall, the highest EthD-1 labeling occurred in the side branches of the most proximal part of the airway tree at 1 ppm with the adjacent axial pathway airway having approximately one-third the labeling density. Density of EthD-1-labeled cells was lowest in terminal bronchioles at all O(3) doses. For the model we used, identification of injured epithelial cells by differential permeability and laser confocal microscopy appeared to be highly sensitive and permitted mapping of acute cytotoxicity throughout the airway tree and quantitative comparisons of sites with different branching histories and potential dosimetry rates.

Preimplantation embryo morphology following early luteal phase anti-nidatory treatment with mifepristone (RU486) in the rhesus monkey.

The ultrastructural characteristics of peri-implantation stage embryos recovered on day 6 after ovulation from rhesus monkeys with or without mifepristone (RU486) treatment during the early luteal phase were examined in the present study. Monkeys were randomly allocated to two groups; group 1 animals were injected s.c. with 2 ml vehicle (1:4, benzyl benzoate: olive oil, v/v, n = 21) and group 2 animals received a single dose of mifepristone (2 mg/kg body weight, w/v, n = 30) in the same volume of vehicle on day 2 after ovulation in mated cycles. On day 6 after ovulation, female monkeys of both groups were laparotomized and their reproductive tracts were flushed to retrieve preimplantation stage embryos. Embryos that showed frank degeneration or desynchrony on gross microscopical examination were not included in the present study. Preimplantation embryo growth on day 6 after ovulation was significantly (P < 0.05) affected in the morula-blastocyst transition stage in mifepristone-treated monkeys compared with that in the control group of monkeys. Ultrastructurally, administration of mifepristone on day 2 after ovulation depressed preimplantation stage embryo development, characterized by loss of cell polarity, lack of mitochondrial maturity, and lack of differentiation in trophoblast cells. Furthermore, preimplantation embryos from mifepristone-treated animals displayed a higher occurrence of inter-blastomere space, intra-cytoplasmic vacuoles, myelinoid bodies, accumulation of lipid droplets, lysosomes, lipofuscins, autophagosomes and multivesicular bodies. Collectively, it appears that the developmental potential of preimplantation embryos was significantly compromised in mifepristone-treated cycles.

Cross-linked features of mouse pelage hair resistant to detergent extraction.

Cross-linking defects in hair cuticle have been observed in certain rare human disorders (trichothiodystrophy, transglutaminase-deficient lamellar ichthyosis). The hypothesis being investigated is that defective cross-linking in the cuticle or other parts of the fiber is a feature of some mouse mutants in which the hair is sparse or appears structurally unsound. Pelage hair samples from 13 mouse mutants displaying defective hair were extracted with sodium dodecyl sulfate and dithiothreitol at neutral pH and examined by transmission electron microscopy. All samples were indistinguishable after extraction from normal hair fibers in appearance of the medulla and cortex. In the cortex, keratins were completely extractable, but material remaining at the cell boundaries was clearly evident. Cells of the medulla were largely unextracted, containing distinct nuclei and amorphous material in the cytoplasm. In two samples (from mice with the matted/flaky tail and naked mutations) cells of the cuticle, which readily detached from the fiber when incubated at 100 degrees C, were more extensively extracted than normal. Defective cross-linking is thus observable in a minority of mouse hair mutants. The observed perturbation of cross-linking in the cuticle was not accompanied by visible perturbation in the cortex or medulla, indicating that different proteins participate in cross-linking in the different cell types.

Cuticle cell defects in lamellar ichthyosis hair and anomalous hair shaft syndromes visualized after detergent extraction.

BACKGROUND: The biochemical bases for fragility in most of the rare brittle hair shaft syndromes are unknown. The hypothesis being investigated in several syndromes is that the hair cuticle cells show defects in cross-linked protein features. Since transglutaminases stabilize protein structures by cross-linking them, hair from autosomal recessive lamellar ichthyosis patients lacking keratinocyte transglutaminase was examined to find whether this enzyme participates in hair shaft stabilization. METHODS: Hair shaft samples from patients afflicted with lamellar ichthyosis or several brittle hair syndromes were examined ultrastructurally by transmission electron microscopy after vigorous extraction with detergent and reducing agent to reveal cross-linked protein features. RESULTS: In hair cuticle cells from three patients with lamellar ichthyosis the marginal band (A layer) was present but nonuniform and subject to breakage, while in a fourth sample it was missing altogether. The exocuticle appeared less dense than in normal hair, consistent with extensive protein loss during detergent extraction. In cuticle cells from trichothiodystrophy hair, the exocuticle layer was essentially fully extractable in one sample, while in two others (from siblings) the exocuticle appeared less dense and the A layer was absent or greatly reduced in thickness. A sample of proximal trichorrhexis nodosa also displayed defects in cuticle cells, in which the endocuticle layer appeared subject to rupture. The outer cuticle cells in monilethrix hair displayed a thinning of the A layer and less dense exocuticle, while the cortex exhibited regions lacking remnant cell borders. Pili annulati hair displayed large gaps in the cortex, presumably reflecting the air-filled cavities characteristic of this syndrome, and wavy borders of some cuticle cells. CONCLUSIONS: Observations with autosomal recessive lamellar ichthyosis hair indicate that keratinocyte transglutaminase has a major role in maturation of the cuticle but appears unnecessary for stabilization of cell borders in the cortex. Defective cross-linking was also evident in cuticle cells of trichothiodystrophy and monilethrix.

Ultrastructural visualization of cross-linked protein features in epidermal appendages.

Upon vigorous extraction with ionic detergent under reducing conditions, the macroscopic structures of mammalian hair, bird feather and horny teeth of the hagfish become swollen and flexible but were substantially preserved. In each case, removal in this way of solubilizable constituents, such as disulfide-bonded keratins and associated matrix proteins, left a residue of epsilon-(gamma-glutamyl)lysine cross-linked protein. Residual features in hair included cell envelopes in the cortex, cell envelopes and intracellular deposits in the medulla, and nearly the entire cuticle cells. In feather, extraction left largely intact the macroscopic barb structures but caused collapse of the rachis. In both rachis and barbs, considerable material resembling cell envelopes remained. In extracted hagfish teeth the cellular organization was clearly visible microscopically, including cell borders and remnant nuclei. Unlike the cornified envelopes of mature epidermal keratinocytes, which appear as doublets, reflecting their formation immediately beneath each plasma membrane of apposing cells, the borders of cells of hair cortex and hagfish teeth appeared single and continuous from one cell to the next. Thus the observed cross-linked features comprised four types: (i) condensation immediately beneath the plasma membrane (feather, hair medulla and cuticle) similar to cornified envelopes of epidermal keratinocytes; (ii) deposition between cells (hair cortex, horny teeth); (iii) cytoplasmic deposits (hair cuticle and medulla); and (iv) nuclear condensation (hair medulla, horny teeth). The results emphasize the importance of transglutaminases and their substrate proteins for the function of epidermal appendages and may provide a useful diagnostic test for perturbation of their normal structures.

Differentiated bronchiolar epithelium in alveolar ducts of rats exposed to ozone for 20 months.

The effects of exposure to 1.0 ppm of ozone for twenty months were studied in male Fischer 344 rats. Light microscopic, morphometric, and immunohistological approaches were used to determine the distribution and degree of differentiation of ciliated and nonciliated bronchiolar epithelial (Clara) cells lining alveolar ducts of the central acinus, a primary target of ozone-induced lung injury. Alveolar duct pathways extending beyond the level of the most proximal alveolar outpocketing of terminal bronchioles were isolated in longitudinal profile. The distance that ciliated and nonciliated bronchiolar epithelial (Clara) cells projected down each alveolar duct pathway was determined by placing concentric arcs radiating outward from a single reference point at the level of the first alveolar outpocketing. A high degree of heterogeneity in the magnitude of bronchiolar epithelial cell extension into alveolar ducts was noted for each isolation and animal. Age-matched control animals also demonstrated variation in the degree of bronchiolar epithelial cell extension down alveolar ducts. In animals exposed to ozone, a striking similarity was noted by scanning electron microscopy in the surface characteristics of cells lining both terminal bronchioles and alveolar ducts. The presence of Clara cell secretory protein in cells of bronchioles and alveolar ducts was also detected immunohistochemically and visualized using confocal laser scanning microscopy in the reflectance mode. Well-differentiated ciliated and nonciliated bronchiolar epithelial cells were found lining alveolar septal tips and alveoli up to a depth of 1,000 mu into the pulmonary acinus after 20 months of exposure to ozone. No evidence of inflammation was present in alveolar ducts, suggesting that epithelial cell transformations in alveolar ducts is a natural consequence of lifetime exposures to oxidant gases.

Nonolfactory surface epithelium of the nasal cavity of the bonnet monkey: a morphologic and morphometric study of the transitional and respiratory epithelium.

The purpose of the present study was to characterize ultrastructurally the nonolfactory nasal epithelium of a nonhuman primate, the bonnet monkey. Nasal cavities from eight subadult bonnet monkeys were processed for light microscopy, and scanning and transmission electron microscopy. Nonolfactory epithelium covered the majority of the nasal cavity and consisted of squamous (SE), transitional (TE), and respiratory epithelium (RE). Stratified SE covered septal and lateral walls of the nasal vestibule, while ciliated pseudostratified RE covered most of the remaining nasal cavity. Stratified, nonciliated TE was present between SE and RE in the anterior nasal cavity. This epithelium was distinct from the other epithelial populations in abundance and types of cells present. TE was composed of lumenal nonciliated cuboidal cells, goblet cells, small mucous granule (SMG) cells, and basal cells, while RE contained ciliated cells, goblet cells, SMG cells, basal cells, and cells with intracytoplasmic lumina lined by cilia and microvilli. TE and RE contained similar numbers of total epithelial cells and basal cells per millimeter of basal lamina. TE was composed of more SMG cells but fewer goblet cells compared to RE. We conclude that nonolfactory nasal epithelium in the bonnet monkey is complex with distinct regional epithelial populations which must be recognized before pathologic changes within this tissue can be assessed adequately.