Infection of P388D1 macrophages and respiratory epithelial cells by Histoplasma capsulatum: selection of avirulent variants and their potential role in persistent histoplasmosis.

We evaluated P388D1 macrophagelike cells as model host cells for studying the intracellular survival and strain-specific virulence of Histoplasma capsulatum. Previously characterized strains which were virulent for mice destroyed monolayers of these cells within a few days. In contrast, related avirulent "smooth" variants failed to do so even after 20 days, although they persisted within P388D1 cells for at least 7 days. On the basis of this observation, we developed a quantitative radiolabel assay to use as an initial screen for virulence. Another cell type lining the respiratory tract was then examined as a potential host for H. capsulatum. Hamster trachea epithelial (HTE) cells readily internalized a variety of strains lacking alpha-(1,3)-glucan in their cell walls; however, the tracheal cells were only rarely infected by organisms possessing this polysaccharide. We subsequently inoculated HTE cells with alpha-(1,3)-glucan-positive strains and enriched for the few yeasts infecting these cells. The progeny resembled smooth variants in terms of colony morphology, the absence of alpha-(1,3)-glucan in their cell walls, and their inability to kill macrophages. Did the HTE cells select for these variant yeasts from the parent inoculum or instigate a change from the parental phenotype? Following a 3-h uptake period, only 2% of the ingested yeasts lacked alpha-(1,3)-glucan. One day later, nearly half of the intracellular organisms lacked this polymer. This rapid conversion of a large proportion of the inoculum suggests some type of environmentally triggered change, perhaps analogous to phase variation seen in many other pathogens. Infection of epithelial cells or some other nonprofessional phagocyte during natural histoplasmosis might give rise to similar variants, thus establishing a reservoir of organisms capable of causing chronic or latent infections.     

Developmental changes in pre-adult behavior in confined colonies of golden hamsters.

Eight litters of hamsters living in a large enclosure were observed from birth to 120 days. For the first 65 days each litter was confined to a separate compartment. Basic motor coordination was reached by the 3rd week. Marking was seen during the 6th week when males began to mount their mothers sexually, but lordosis was not seen in female pups before the 7th week. Fighting appeared at this time with the females being the more aggressive. At 65 days the litters were combined into 2 groups of 4.A gradual shift followed in the relative aggressiveness of the sexes. Females, which had previously initiated over 90% of attacks, became less aggressive, whereas attacks by males increased to the former female level. This unusual behavior for hamsters, where females are normally dominant, may have been due to crowding. Also, severe disruption of maternal behavior occurred with marked infanticidal tendencies.     

Concentration and turnover of intraperitoneal hyaluronan during inflammation

Aseptic peritonitis was induced in rabbits by intraperitoneal injection of irritating agents, mainly starch suspensions. The inflammatory response was followed in the peritoneal lavage fluid by cell counts (average increase about 800-fold the first day) and hyaluronan concentration (average increase about 200-fold on the second and third days). The turnover rate of hyaluronan was studied by injecting tritium-labeled hyaluronan intraperitoneally and by following the appearance of tritiated water in serum. In control animals given trace amounts of hyaluronan, half-lives of 1–14 h were recorded. When the labeled polysaccharide had been mixed with 10 mg/ml of unlabeled hyaluronan, the half-life was approximately one day. Rabbits with ongoing peritonitis exhibited half-lives between 1 and 16 h. It was concluded that there was a large individual variation in uptake kinetics, that the removal process could be receptor mediated, and that the increase in intraperitoneal hyaluronan in peritonitis mainly was due to an increased production of the polysaccharide rather than a decreased rate of removal.     

T helper type 2-like cells and therapeutic effects of interferon-γ in combined immunodeficiency with hypereosinophilia (Omenn's syndrome)

We characterized the defects of CD4+ cells in a 17-month-old girl suffering from combined immunodeficiency with hypereosinophilia (Omenn's syndrome). Because the vast majority of peripheral blood CD4⁺ cells expressed the CD45R0 isoform, we purified circulating CD4⁺ CD45R0⁺ cells from the patient and healthy individuals in order to compare their production of cytokines. The patient's CD4⁺ CD45R0⁺ cells spontaneously produced high levels of interleukin-5 (IL-5) in vitro (1600 pg/ml after 24 h of culture) and this was associated with the presence of IL-5 in serum (323 pg/ml). After stimulation with phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187, they produced higher levels of IL-4 (306 vs. 55 ± 4 pg/ml) and IL-5 (2900 vs. 213 ± 72 pg/ml) and lower levels of IL-2 (17 vs. 63 ± 17 IU/ml) and interferon-γ (IFN-γ) (16 vs. 299 ± 70 IU/ml) than controls CD4⁺ CD45R0⁺ cells. This T helper type 2 (Th2) pattern was confirmed by the detection using reverse polymerase chain reaction of IL-4, IL-5 and IL-10 mRNA within peripheral blood mononuclear cells. During a therapeutic trial with human IFN-γ (40 μg/day) which ameliorated the clinical status of the patient, we observed a down-regulation of the in vivo expression of IL-5 and IL-10, a normalization of the eosinophil count and an improvement of the Tcell response to phytohemagglutinin. This observation indicates for the first time that Th2-like cells might be involved in certain forms of congenital immunodeficiency and that IFN-γ might down-regulate their activities in vivo.     

Impaired responses to toll-like receptor 4 and toll-like receptor 3 ligands in human cord blood

Toll-like receptor (TLR)-4 signaling pathway plays an essential role in host defense against gram-negative bacteria while TLR-3-mediated signaling is critically involved in anti-viral immunity. To gain insight into the defects responsible for impaired Th1 responses in human newborns, we investigated the responses of human cord blood cells to lipopolysaccharide, LPS, and to polyinosinic-polycytidylic acid, Poly (I:C), ligands of TLR-4 and TLR-3, respectively. Measurement of cytokine levels revealed a profound defect in IL-12 (p70) synthesis and an increased release of IL-10 in cord blood exposed to LPS or Poly (I:C), as compared to adult blood. Moreover, Poly (I:C)-induced IFN-alpha production was found to be significantly impaired in cord blood. Phenotypic maturation of myeloid DC in response to LPS or Poly (I:C) was next compared in cord and adult blood. We observed that neonatal myeloid DC displayed decreased upregulation of CD40, CD80 whereas CD86 and HLA-DR upregulation did not differ significantly between adults and neonates. Taken together, these findings might be relevant to the increased vulnerability of human newborns to intracellular pathogens and to their inability to develop efficient Th1-type responses.     

Utilization review of the use of BACTEC PLUS high-volume blood culture bottles.

The BACTEC PLUS 26 (NR26) (Becton Dickinson, Towson, Md.) high-volume blood culture bottle replaced the less expensive smaller-volume NR6A bottle in our hospital. An audit carried out several months after their introduction revealed that only 17.5% of the NR26 bottles received the required blood volume. Several audits and educational programs were required in order to achieve a compliance rate of > 60%.     

Elevated glucocorticoid receptor levels in lymphocytes of children with the fetal hydantoin syndrome (FHS)

Our recent studies of the teratogenic mechanisms of phenytoin (DPH) and glucocorticoids in mice have indicated that DPH utilizes the anti-inflammatory pathway of glucocorticoids in producing congenital defects, such as cleft palate. This pathway is influenced by H-2 and H-3 histocompatibility-linked genes in the mouse, such that congenic strains have H-2 or H-3 alleles that confer susceptibility to DPH-induced congenital defects, and susceptible H-2 congenic strains have high glucocorticoid receptor levels. However, other H-2 or H-3 alleles confer resistance to these defects in their otherwise genetically identical congenic partner strains, and "resistant" H-2 alleles are associated with low levels of these receptors. To determine whether this animal work is applicable to the human, we have sought to investigate whether the level of glucocorticoid receptors in circulating lymphocytes of children with the fetal hydantoin syndrome (FHS) is as it is in the animals. We found that children with FHS had glucocorticoid receptor levels significantly elevated above those of unaffected children with similar DPH exposure in control families. The receptor level of affected children was also significantly elevated above that of fathers of children with the FHS and of fathers and mothers of control children. These findings are consistent with those documented in the animal models and suggest that an elevated level of glucocorticoid receptors in lymphocytes may be a marker for susceptibility to the FHS syndrome.     

Factors associated with the use of psychiatric consultations in short-term general hospitals

A significant number of patients in general hospitals suffer from psychiatric distress or illness, but not all receive psychiatric consultation. This study examined several potential predictors of such consultation. These included patient characteristics (age, sex, race, insurance, disease stage, and number of medical diagnoses), characteristics of hospital stay (number of procedures, other consultations, length of stay, discharge destination), hospital characteristics (size, percent occupancy, teaching status, presence of a psychiatric unit, type of control), and community characteristics (region, urban/rural setting). Data were derived from a national sample of 327 hospitals. Parallel analyses using stepwise logistic regression were carried out across four samples: patients determined at discharge to have been hospitalized for diabetes, hip fracture, chronic obstructive pulmonary disease, and coronary artery bypass surgery. Results show a wide variation in the use of psychiatric consultations across different subgroups. Longer lengths of stay, urban setting. Northeast region, younger patient age, and other consultations were the most consistent predictors of the probability of psychiatric consultation. These variations may reflect differences in the need for consultation, differential recognition of these needs by providers, or differential availability of psychiatric consultation services. Moreover, they may have implications for equity and/or quality of hospital care.