Age-related changes in the cerebrospinal fluid outflow glycosaminoglycans

The glycosaminoglycan distribution patterns of the cerebrospinal fluid (CSF) outflow pathway, dura mater and cerebral cortex of young New Zealand red rabbits and 1-, 3- and 12-week-old C-57 mice were identified by analyses of the glycosaminoglycan moieties and by the use of zone electrophoresis. The glycosaminoglycans were identified by specific degradation procedures, i.e., hyaluronate lyase, chondroitin ABC lyase, endo-gb-D-galactosidase and nitrous acid treatment. The CSF outflow pathway and dura mater glycosaminoglycan components were primarily hyaluronic acid and chondroitin sulfatedermatan sulfate, whereas the cerebral cortex glycosaminoglycan components were hyaluronic acid, chondroitin sulfatedermatan sulfate, keratan sulfate and heparan sulfate. The glycosaminoglycan components of the dura mater and cerebral cortex decreased and those of the CSF outflow pathway increased as a function of age. These results demonstrate the feasibility of analyses of the CSF outflow pathway glycosaminoglycan components and suggest that topographical changes in the glycosaminoglycan distribution profiles may contribute to the pattern of cerebrospinal fluid outflow.     

Comparison of the sensitivities of the version 1.5 and version 1.0 ultrasensitive Roche AMPLICOR HIV-1 MONITOR kits at low concentrations of human immunodeficiency virus RNA

The sensitivities of the version 1.5 and 1.0 Roche UltraSensitive AMPLICOR HIV-1 MONITOR tests were compared using panels of coded samples of subtype B human immunodeficiency virus type 1 spiked into plasma at predetermined concentrations. Results indicate that the version 1.5 kit is more sensitive than the version 1.0 kit.     

Clonal differences in response to T cell replacing factor (TRF) for IgM secretion and TRF receptors in a human B lymphoblast cell line

A number of human B lymphoblast cell lines were tested to find a suitable model for induction of immunoglobulin-secreting cells (ISC) by T cell replacing factor (TRF). Partially purified TRF was size fractionated from the conditioned medium of irradiated (1000 rds) human spleen cells stimulated with pokeweed mitogen. IgM line SKW 6 showed high levels of TRF-stimulated immunoglobulin secretion and was chosen for cloning experiments. Clone 11 had a very low level of ISC with or without TRF (less than 0.1% ISC). Clone 4 had low background numbers of ISC (0.2%) and showed the highest degree of stimulation by TRF (to 6% IFC). High secreting clone 8-2 (6%) was not stimulated significantly by TRF. These clones had the same HLA-DR antigens, and their levels of ISC and sensitivity to TRF were relatively stable over three months of observation. The TRF preparation also had strong helper activity for normal peripheral blood B cell differentiation. TRF activity for both normal B cells and clone 4 cell line was absorbed by all 3 clones, but not by a pre-B line. This suggests that the effector molecules for normal B cell and cell line differentiation were the same. The three typical clones corresponding to nonresponding, responding, and high rate-secreting B cells provide basic models for analyzing B cell receptors for TRF and the biochemical effects of TRF during B cell differ entiation.     

Activated Pulmonary Macrophages Are Insufficient for Resistance against Pneumocystis carinii

CD4⁺ T cells are pivotal for elimination of Pneumocystis carinii from infected lungs, and alveolar macrophages are considered the main effector cells clearing the infected host of P. carinii organisms. To investigate this issue, several mutant mouse strains were used in a previously established experimental setup which facilitates natural acquisition of disease through inhalation of airborne fungal organisms. Mutant mice deficient in major histocompatibility complex class II molecules (Aβ^−/−), T-cell receptor αβ cells (TCRβ^−/−), or all mature T and B lymphocytes (RAG-1^−/−) were naturally susceptible to P. carinii, whereas mouse mutants lacking the gamma interferon (IFN-γ) receptor (IFN-γ-R^−/−) or tumor necrosis factor alpha (TNF-α) type I receptor (p55) (TNF-α-RI^−/−) resisted disease acquisition. Analysis of pulmonary cytokine patterns and free radical expression revealed the presence of superoxide, nitric oxide, and interleukin-1 (IL-1) mRNA and elevated levels of IFN-γ, TNF-α, and IL-12 in diseased TCRβ^−/− and RAG-1^−/− mice. Pulmonary macrophages of all diseased mouse mutants expressed scavenger and mannose receptors. Morbid Aβ^−/− mutants displayed significant NO levels and IL-1 mRNA only, whereas heterozygous controls did not exhibit any signs of disease. Interestingly, neither IFN-γ nor TNF-α appeared to be essential for resisting natural infection with P. carinii, nor were these cytokines sufficient for mediating resistance during established disease in the absence of CD4⁺ T lymphocytes. Taken together, the results indicated that an activated phagocyte system, as evidenced by cytokine and NO secretion, in diseased mutants was apparently operative but did not suffice for parasite clearance in the absence of CD4⁺ TCRαβ cells. Therefore, additional pathways, possibly involving interactions of inflammatory cytokines with CD4⁺ T lymphocytes, must contribute to successful resistance against P. carinii.Immunocompromised patients, especially those suffering from AIDS, are at elevated risk of acquiring Pneumocystis carinii pneumonia (PCP), a major cause of premature mortality among AIDS patients (8, 35, 53). Various studies have emphasized that CD4⁺ T lymphocytes play a pivotal role in the orchestration of resistance to P. carinii (22, 43, 45), an opportunistic fungus, but the mechanisms underlying protection remain a conundrum. Pulmonary macrophages are considered the main effector cells in clearing the immunocompetent host from invading P. carinii organisms (25). It seems conceivable, therefore, that macrophage-activating functions mediated by CD4⁺ T cells are central to resistance. Impaired gamma interferon (IFN-γ) production by T cells from AIDS patients is thought to enhance susceptibility to P. carinii (34, 41). This notion is supported by reports that application of exogenous IFN-γ ameliorates disease in experimental animal models (2, 45). In contrast, in vivo neutralization of IFN-γ in spleen cell-reconstituted severe combined immunodeficiency (SCID) mice by a specific monoclonal antibody (MAb) does not affect parasite clearance (5). Further studies point to a critical role of tumor necrosis factor alpha (TNF-α) (5) and interleukin-1 (IL-1) (6) in maintaining an immunocompetent state. Both cytokines are mainly produced by macrophages and induce inflammatory responses (4, 10, 26). Overall, these findings support involvement of macrophage-derived cytokines in successful host resistance against P. carinii.To analyze in more depth the role of inflammatory and Th1/Th2-related pulmonary defense mechanisms in control of aerogenically acquired PCP, we took advantage of naturally susceptible gene disruption mutant mice lacking major histocompatibility complex (MHC) class II molecules (and therefore conventional CD4⁺ T cells) (Aβ^−/−), T-cell receptor (TCR) αβ cells (TCRβ^−/−), or all mature T and B lymphocytes (RAG-1^−/−) (19). We further exploited mice deficient in the IFN-γ receptor (IFN-γ-R^−/−) or the TNF-α type I receptor (p55) (TNF-α-RI^−/−) to analyze their capacity to cope with aerogenic P. carinii organisms.Bronchoalveolar lavage (BAL) cells of healthy and diseased mice were investigated for expression of the proinflammatory cytokines IL-1, TNF-α, IFN-γ, and IL-12, as well as IL-4, IL-5, and IL-10. The latter three cytokines counteract IFN-γ- and IL-12-mediated responses but participate in protection against certain extracellular pathogens (9). Moreover, production of superoxide (SO) and nitric oxide (NO), putative effector molecules of antimicrobial defense, was taken as a further indicator of macrophage activation. Contact with foreign material induces a rapid respiratory burst in professional phagocytes which results in SO production as a first line of defense. SO has been implicated in destruction of P. carinii (31), whereas NO produced by IFN-γ-stimulated macrophages encountering pathogens (4, 18, 30) does not appear to participate in control of P. carinii infection (47). Of further interest was the role of macrophage-expressed mannose receptors (MR) and scavenger receptors (SR). MR were previously found crucial for mediating P. carinii internalization (11, 37). The relevance of SR with respect to PCP has not been evaluated, but they are mainly expressed by tissue macrophages (36) and nonspecifically bind a large array of molecules, including surface molecules of microorganisms (39). Receptors with such broad pattern reactivity may be involved in direct differentiation of self from non-self, and recent data suggest that not only MR but also SR aid pattern recognition by macrophages and subsequent internalization of invading pathogens (27).We found that BAL cells from P. carinii-diseased RAG-1^−/− and TCRβ^−/− mutants secreted elevated IFN-γ, TNF-α, IL-12, NO, and SO levels and expressed IL-1 mRNA. In contrast, cells from morbid Aβ^−/− mice produced IL-1 mRNA and high levels of NO only, whereas all other parameters were low to absent in these mutants. SR were expressed on pulmonary macrophages of all diseased RAG-1^−/−, TCRβ^−/−, and Aβ^−/− mutants, whereas MR were also expressed by macrophages of healthy animals. Yet, the apparently activated phagocyte system in the lung, most pronounced in morbid TCRβ^−/− and RAG-1^−/− mutant mice, was insufficient for protection against natural P. carinii infection. Elevated levels of IFN-γ and TNF-α in morbid mutants (not in Aβ^−/− mice) and the naturally resistant status of IFN-γ-R^−/− and TNF-α-RI^−/− mice further argue not only for independence from IFN-γ and TNF-α. Our findings indicate that CD4⁺ αβ T lymphocytes prevent and clear infection with P. carinii by mechanisms distinct from, or in addition to, pulmonary macrophage activation.(This study is part of the Ph.D. thesis of R. Hanano.)     

Water intake during the development of renal hypertension (2K-1C) in mice

For both practical and methodological reasons, mice have been the most widely employed species for development of transgenic and gene knockin and knockout animals. However, basic behavioral and physiology control and regulatory mechanisms in mice are not well characterized. To broaden our understanding of the processes maintaining body fluid and blood pressure homeostasis in the mouse, the objectives of this study were to evaluate voluntary water, and sodium intakes during the development of renal hypertension and to examine the relationship between hypertension and the quantities of water and salt ingested. In male, C57BL/6J mice, two-kidney, one-clip renal hypertension (2K-1C) was induced, and water and 1.8% NaCl intakes were monitored for 2 weeks. At the end of this period, all animals received arterial catheters for direct recording of blood pressure. The mice that received renal artery clips were sorted into hypertensive (152+/-4 mm Hg) and normotensive (122+/-2 mm Hg) groups and were compared to control (117+/-4 mm Hg) animals that underwent a sham renal clipping procedure. Hypertensive 2K-1C animals had significantly elevated water intake compared to control animals. On most of the postsurgical days, the normotensive 2K-1C animals did not display increased water intake in comparison to the control group. No significant effect was detected for 1.8% saline intake between any of the pairs of groups. In summary, the reduction of blood flow to a single kidney in the 2K-1C model of renal hypertension induces high blood pressure accompanied by sustained hyperdipsia in the mouse.     

ZK 91296, a partial agonist at benzodiazepine receptors

ZK 91296 (ethyl 5-benzyloxy-4-methoxymethyl--carboline-3-carboxylate) is a potent and selective ligand for benzodiazepine (BZ) receptors. Biochemical investigations indicate that ZK 91296 may be a partial agonist at BZ receptors. Such partial agonism may explain to some extent why ZK 91296 needs higher BZ receptor occupancy than diazepam for the same effect against chemical convulsants and for behavioural effects. The lack of sedatiye effects, and the very potent inhibition of reflex epilepsy, spontaneous epilepsy and DMCM-induced seizures suggest, furthermore, that ZK 91296 may possess pharmacological selectivity for a particular type of BZ receptor interaction, perhaps including topographic as well as receptor subtype differentiation.