乳酸脱氢酶实验检测细胞新陈代谢的实验研究

目的 探索LDH实验检测细胞活力的可行性。方法 原代培养骨髓细胞和软骨细胞，用LDH实验测定上述两组细胞的活力，并与镜下活体观察到细胞的生长状况相比较。与目前比较成熟的测定细胞活力的MTS实验的测得的值相比较。结果 LDH实验对上述两组细胞的活力的测定结果与镜下活体观察到的结果相符合。与MTS实验的测得的结果经统计学处理无显著差异。结论 LDH实验可用于细胞活力的直接测定，而对活细胞的生存、繁殖无影响。     

Regulation of histamine H1 receptor-mediated phosphoinositide hydrolysis by histamine and phorbol esters in DDT1 MF-2 cells

The regulation of histamine-stimulated phosphoinositide turnover by histamine and phorbol esters was examined in intact DDT1 MF-2 cells grown in suspension culture. Histamine increased the incorporation of 32P into phosphatidylinositol (PI) in these cells, and this stimulation was inhibited by the H1 antagonist diphenhydramine but not by the H2 antagonist cimetidine. Pretreatment of cells with histamine or with phorbol 12-myristate 13-acetate (PMA) or other activators of protein kinase C induced a marked decrease in the subsequent stimulation by histamine. PMA, but not histamine, also decreased the ability of epinephrine to stimulate PI labelling through alpha 1-adrenoceptors. Thus, histamine appears to induce homologous desensitization of histamine H1 receptor-mediated PI turnover, whereas direct activation of protein kinase C in the absence of receptor occupancy by agonist induces nonspecific heterologous desensitization of both histamine H1- and alpha 1-adrenoceptor-mediated responses.     

Acid-base-electrolyte balance responses of Bufo marinus to aminoglutethimide, corticosterone, and aldosterone during hypercapnia.

Experiments were conducted to test the hypothesis that one or more interrenal steroids are active in regulatory responses to respiratory acidosis in the toad, Bufo marinus. Toads were divided into four experimental groups. The first group received sham injections. The second group received 1-3 mg of aminoglutethimide (AG) every 8 hr. AG inhibits the conversion of cholesterol to pregnenolone, thus inhibiting all steroid hormone synthesis. The third group received AG + 5 micrograms of aldosterone on the same schedule. The fourth group received AG + 25 micrograms of corticosterone on the same schedule as the other groups. All four groups were subjected to hypercapnia using 5% CO2 to induce a respiratory acidosis. The sham-operated animals displayed the normal compensatory pattern of producing a metabolic alkalosis (elevated plasma HCO3-) after 24 hr. AG-treated toads failed to elevate plasma HCO3-. Administration of interrenal steroids produced compensation in varying degrees. Aldosterone produced a small compensation while corticosterone produced a compensation similar to that seen in sham-operated animals. Analysis of steroid titers in toad plasma during hypercapnia showed that Bufo marinus does not elevate aldosterone during respiratory acidosis, but that corticosterone is elevated. AG blocked the corticosterone elevation, however. AG also produced a hyponatremia that was corrected with aldosterone or corticosterone. Normocapnic controls showed that AG does not produce deleterious effects on pH or blood gases in toads in the absence of a respiratory acidosis. We conclude that corticosterone is important in acid-base regulatory responses to respiratory acidosis in this amphibian.     

Mutations in the Ca(2+)-sensing receptor gene cause autosomal dominant and sporadic hypoparathyroidism

Parathyroid hormone secretion is negatively regulated by a 7- transmembrane domain, G-protein coupled Ca(2+)-sensing receptor. We hypothesized that activating mutations in this receptor might cause autosomal dominant hypoparathyroidism (ADHP). Consistent with this hypothesis, we identified, in two families with ADHP, heterozygous missense mutations in the Ca(2+)-sensing receptor gene that cosegregated with the disorder. None of 50 normal controls had either mutation. We also identified a de novo, missense Ca(2+)-sensing receptor mutation in a child with severe sporadic hypoparathyroidism. The amino acid substitution in one ADHP family affected the N-terminal, extracellular domain of the receptor. The other mutations involved the transmembrane region. Unlike patients with acquired hypoparathyroidism, patients with these mutations had hypercalciuria even at low serum calcium concentrations. Their greater hypercalciuria presumably reflected activation of Ca(2+)-sensing receptors in kidney cells, where the receptor negatively regulates calcium reabsorption. This augmented hypercalciuria increases the risk of renal complications and thus has implications for the choice of therapy.      

Prostaglandin E2 synthesis and suppression of fibroblast proliferation by alveolar epithelial cells is cyclooxygenase-2-dependent

Alveolar epithelial cells (AECs) may influence neighboring fibroblasts by the elaboration of prostaglandin E(2) (PGE(2)). This prostanoid can be synthesized via "constitutive" cyclooxygenase (COX)-1 and "inducible" COX-2 enzyme isoforms. We compared AECs isolated from wild-type (WT), COX-1 knockout (KO), and COX-2 KO mice to determine the contribution of COX isoforms to AEC PGE(2) synthesis and capacity for suppression of fibroblast proliferation in co-cultures. WT AECs constitutively expressed both COX-1 and COX-2 isoforms by immunoblot analysis. COX-1 KO cells and WT cells comparably augmented PGE(2) synthesis following incubation with lipopolysaccharide or interleukin-1, whereas COX-2 KO cells were unable to do so. Surprisingly, however, constitutive generation of PGE(2) was also dramatically reduced only in COX-2 KO cells. When co-cultured with WT murine lung fibroblasts, AECs from WT and COX-1 KO animals suppressed serum-induced fibroblast proliferation, whereas COX-2-deficient AECs caused a modest enhancement in fibroblast proliferation. These results indicate that PGE(2) synthetic capacity in AECs is predominantly COX-2-dependent under both basal and stimulated conditions. They also demonstrate conclusively that AECs can modulate fibroblast function by the elaboration of suppressive prostanoids. These alterations in AEC phenotype likely contribute to the propensity for pulmonary fibrosis observed in COX-2-deficient mice.     

The gamma interferon receptor is required for the protective pulmonary inflammatory response to Cryptococcus neoformans

Mice with a null deletion mutation in the gamma interferon (IFN-gamma) receptor gene were used to study the role of IFN-gamma responsiveness during experimental pulmonary cryptococcosis. Cryptococcus neoformans was inoculated intratracheally into mice lacking the IFN-gamma receptor gene (IFN-gammaR-/-) and into control mice (IFN-gammaR+/+). The numbers of CFU in lung, spleen, and brain were determined to assess clearance; cytokines produced by lung leukocytes were measured, and survival curves were generated. In the present study, we demonstrate the following points. (i) IFN-gammaR-/- mice are markedly more susceptible to C. neoformans infection than IFN-gammaR+/+ mice. (ii) In the absence of IFN-gamma signaling, pulmonary CFU continue to increase over the course of infection, and the infection disseminates to the brain. (iii) In the absence of IFN-gamma receptor, recruitment of inflammatory cells in response to pulmonary cryptococcal infection is not impaired. (iv) At week 5 postinfection, IFN-gammaR-/- mice have recruited greater numbers of leukocytes into their lungs, with neutrophils, eosinophils, and lymphocytes accounting for this cellular increase. (v) IFN-gamma signaling is required for the development of a T1 over a T2 immune response in the lung following cryptococcal infection. These results indicate that in the absence of IFN- gamma responsiveness, even though the recruitment of pulmonary inflammatory cells is not impaired and the secretion of IFN-gamma is not affected, IFN-gammaR-/- mice do not have the ability to resolve the cryptococcal infection. In conclusion, our data suggest that proper functional IFN-gamma signaling, possibly through a mechanism which inhibits the potentially disease-promoting T2 response, is required for mice to confine the cryptococcal infection.     

Induction of interleukin-12 and gamma interferon requires tumor necrosis factor alpha for protective T1-cell-mediated immunity to pulmonary Cryptococcus neoformans infection

The development of T1-cell-mediated immunity is required to clear a pulmonary Cryptococcus neoformans infection. The objective of these studies was to determine the mechanism by which tumor necrosis factor alpha (TNF-alpha) augments the development of pulmonary T1 immunity to C. neoformans infection. TNF-alpha expression was detected in lavage sample cells at days 2, 3, and 7 following C. neoformans infection. The numbers of CFU in the lung were not different between control and anti-TNF-alpha-treated mice at any time point examined during the afferent phase of the response (days 0 to 7). However, neutralization of TNF-alpha prevented the initiation of pulmonary clearance during the efferent phase of the response (day 14). Administration of anti-TNF-alpha monoclonal antibody (day 0) diminished the lung levels of TNF-alpha, interleukin-12 (IL-12), and gamma interferon (IFN-gamma) induced by C. neoformans at day 7 postinfection. Neutralization of TNF-alpha (day 0) also altered the IFN-gamma/IL-4 ratio in the lung-associated lymph nodes at day 7 following C. neoformans infection. Anti-TNF-alpha-treated mice developed a pulmonary eosinophilia at day 14 postinfection. Consistent with the pulmonary eosinophilia, anti-TNF-alpha-treated mice exhibited elevated serum immunoglobulin E and inhibition of the anticryptococcal delayed-type hypersensitivity response, indicating a shift toward a T2 response. Neutralization of IL-12 also prevented lung leukocyte production of IFN-gamma in response to the infection. These findings demonstrate that afferent-phase TNF-alpha production is essential for the induction of IL-12 and IFN-gamma and neutralization of early TNF-alpha results in a T2 shift of the T1/T2 balance of antifungal immunity.     

Transient neutralization of tumor necrosis factor alpha can produce a chronic fungal infection in an immunocompetent host: potential role of immature dendritic cells

The mechanisms underlying induction of immune dysregulation and chronic fungal infection by a transient tumor necrosis factor alpha (TNF-alpha) deficiency remain to be defined. The objective of our studies was to determine the potential contribution of neutropenia and immature dendritic cells to the immune deviation. Administration of an anti-TNF-alpha monoclonal antibody at day 0 neutralized TNF-alpha only during the first week of a pulmonary Cryptococcus neoformans infection. Transient neutralization of TNF-alpha resulted in transient depression of interleukin-12 (IL-12), monocyte chemotactic protein 1 (MCP-1), and gamma interferon (IFN-gamma) production but permanently impaired long-term clearance of the infection from the lungs even after the levels of these cytokines increased and a vigorous inflammatory response developed. Early neutrophil recruitment was defective in the absence of TNF-alpha. However, as demonstrated by neutrophil depletion studies, this did not account for the decrease in IL-12 and IFN-gamma levels and did not play a role in establishing chronic pulmonary cryptococcal infection. Transient TNF-alpha neutralization also produced a deficiency in CD11c(+) MHC II(+) cells and IL-12 in the lymph nodes, potentially implicating a defect in mature dendritic cell trafficking. Transfer of cryptococcal antigen-pulsed immature dendritic cells into naive mice prior to intratracheal challenge resulted in the development of a nonprotective immune response to C. neoformans that was similar to that observed in anti-TNF-alpha-treated mice (increased IL-4, IL-5, and IL-10 levels, pulmonary eosinophilia, and decreased clearance). Thus, stimulation of an antifungal response by immature dendritic cells can result in an immune deviation similar to that produced by transient TNF-alpha deficiency, identifying a new mechanism by which a chronic fungal infection can occur in an immunocompetent host.