Altered airway responsiveness in CD38-deficient mice

Cyclic ADP-ribose (cADPR) mobilizes calcium from intracellular stores and contributes to agonist-induced intracellular calcium elevation in airway smooth muscle (ASM). In this study we determined the functional role of CD38/cADPR signaling in the regulation of airway tone using CD38 deficient (cd38(-/-)) mice. The responsiveness to different doses of methacholine, as determined by changes in lung resistance and dynamic compliance, was significantly (P < or = 0.05) lower in cd38(-/-) mice compared with wild-type controls. To determine the mechanism responsible for the reduced responsiveness, we measured the intracellular calcium responses to contractile agonists in ASM cells. In ASM cells isolated from cd38(-/-) mice, the intracellular calcium responses to acetylcholine and endothelin-1 were significantly lower than in controls. Pretreatment of ASM cells with a cADPR antagonist resulted in attenuated intracellular calcium responses to endothelin-1 in cells isolated from wild-type mice, but not in those isolated from the cd38(-/-) mice. Very low cADPR levels and no detectable ADP-ribosyl cyclase activity were observed in lung tissue from cd38(-/-) mice, suggesting that CD38 is a critical source for cADPR synthesis. The results of the present study demonstrate that CD38/cADPR contributes to airway smooth muscle tone and responsiveness through its effects on agonist-induced elevation of intracellular calcium in ASM cells.     

Expression of human pim family genes is selectively up-regulated by cytokines promoting T helper type 1, but not T helper type 2, cell differentiation

Cytokines are the most important inducers of T helper (Th) cell differentiation. Interleukin-12 (IL-12) and interferon-alpha (IFN-alpha) are responsible for human Th1-cell differentiation, while IL-4 is the critical cytokine promoting Th2-cell development. These two subsets of cells co-ordinate immunological responses to pathogens as well as autoimmune or allergic reactions. The pim family of proto-oncogenes encodes serine/threonine-specific kinases involved in cytokine-mediated signalling pathways in haematopoietic cells. Here we demonstrate that expression of pim-1 and pim-2 mRNAs is selectively up- or down-regulated in human cord-blood-derived CD4+ cells freshly induced to polarize towards Th1 or Th2 cells, respectively, whereas their expression is inhibited in both cell types by the immunosuppressive transforming growth factor beta (TGF-beta). Moreover, the Th1-specific cytokines IL-12 and IFN-alpha, but not the Th2-specific cytokine IL-4, transiently up-regulate pim-1 and pim-2 mRNA expression in human peripheral blood T cells and natural killer cells. In addition, the Pim-1 protein levels are strongly up-regulated by Th1-specific cytokines in all of these cell types. Taken together, our results suggest that pim genes and their protein products are involved in the early differentiation process of T helper cells.     

Distribution of dengue-2 antigens by electron immunocytochemistry.

The distribution of dengue-2 antigens was studied in infected monkey kidney cells (LLC MK2) using an indirect, horseradish peroxidase-conjugated immunoglobulin technique. This procedure allowed both light and electron microscopic examination of serial-step sections of individual cells cut in a plane perpendicular to the monolayer. Both virion and nonvirion antigens were identified on the plasma membrane with this technique. A diffuse cytoplasmic reaction product was also present. The intensity and distribution of the cytoplasmic reaction product was related to disruption of the plasma membrane.     

Attempts to isolate virus from human trigeminal and facial ganglions

Herpes virus hominis type 1 was isolated from the trigeminal ganglion (ganglion semilunare, gasservian) in three out of 20 randomly selected autopsies. Two of the three patients had been treated with immunosuppressive or cytostatic agents. Clinical signs of herpes infection were not observed during the previous 6 months. No virus was isolated from the facial ganglion (geniculate ganglion) in the same 20 cases. The findings are discussed in relation to the viral etiology of acute peripheral facial palsy.     

Further studies on the aberrant crossed visual corticotectal pathway in rats

Summary An aberrant crossed corticotectal pathway can be generated by removal of one visual cortex and the contralateral superior colliculus from newborn rats. This aberrant crossed corticotectal projection arises from the pyramidal neurons located in layer V of the visual cortex and terminates in a spatially orderly manner in the appropriate laminae of the cortically deafferented contralateral colliculus. Comparable results cannot be reproduced by unilateral collicular lesions alone. The significance of these findings and the possible mechanisms involved in the formation of the aberrant pathway are discussed and compared with the retinotectal system.The research was supported by USPHS Grant EY-00596 from the National Institutes of Health     

Placentally derived prostaglandin E2 acts via the EP4 receptor to inhibit IL-2-dependent proliferation of CTLL-2 T cells

A number of immunomodulatory molecules are present in the placenta, including cytokines, prostaglandins, progesterone and indoleamine 2,3-dioxygenase. An undefined factor capable of down-regulating T-cell activity has recently been reported [1] as being produced by short-term cultures of placental fragments. By careful repetition of these studies we have confirmed that chorionic villi isolated from term placenta produce a low molecular weight, heat stable factor capable of inhibiting the IL-2-dependent proliferation of mouse CTLL-2 cells. This activity was not due, however, to a previously unknown immunosuppressive molecule, but rather to prostaglandin E2 (PGE2). Expression of cyclooxygenase (COX)-2 was detected in the syncytiotrophoblast of chorionic villi explants using immunohistochemistry. Culture of the explants in the presence of the COX-1/COX--2 inhibitors indomethacin and diclofenac, or with the COX-2-selective inhibitor DFP, blocked the production of the immunosuppressive factor. The immunosuppressive activity was restored by adding PGE2 to the supernatants obtained from diclofenac-inhibited explants. A number of different receptors are involved in mediating the biological effects of prostaglandins. By utilizing selective antagonists of individual receptors, we have established that the immunosuppressive effect of PGE2 on CTLL-2 cells is exerted via the EP4 receptor. Thus, addition of an EP4-selective antagonist, but not of EP1 or EP3 antagonists, abolished the immunosuppressive effect of PGE2 on CTLL-2 cells. This may have implications for attempts to selectively manipulate T-cell responses.     

Comparison of the activation of the Ca2+ release-activated Ca2+ current I CRAC to InsP3 in Jurkat T-lymphocytes, pulmonary artery endothelia and RBL-1 cells

In many electrically non-excitable cells, Ca2+ entry is mediated predominantly by the store-operated Ca2+ influx pathway. The best-characterised store-operated Ca2+ current is the Ca2+ release-activated Ca2+ current (ICRAC). It is generally believed that high concentrations of intracellular Ca2+ buffer are required to measure ICRAC, due to Ca2+-dependent inactivation of the channels. Recently, we have recorded robust ICRAC in rat basophilic leukaemia (RBL-1) cells at physiological levels of Ca2+ buffering when stores were depleted by inhibition of the sarcoplasmic/ endoplasmic reticulum Ca2+-activated adenosine triphosphatase (SERCA) pumps. However, the second messenger inositol 1,4,5-trisphosphate (InsP3) was not able to evoke the current under such conditions, despite inducing substantial Ca2+ release. We have therefore suggested that a threshold exists within the Ca2+ stores which has to be overcome for macroscopic ICRAC to activate. To establish whether this is a specific feature of ICRAC in RBL-1 cells or whether it is a more general phenomenon, we investigated whether a threshold is also seen in other cell-types used to study store-operated Ca2+ entry. In Jurkat-T lymphocytes, ICRAC is activated weakly by InsP3 in the presence of low concentrations of Ca2+ buffer, whereas the current is large when SERCA pumps are blocked simultaneously, as in RBL-1 cells. Although the electrophysiological properties of ICRAC in the Jurkat cell are very similar to those of RBL-1 cells, the Na+ conductance in the absence of external divalent cations is quite different. Unexpectedly, we failed consistently to record any store-operated Ca2+ current in macrovascular pulmonary artery endothelia whereas robust ICRAC was seen under the same conditions in RBL-1 cells. Our results show that ICRAC has a similar profile of activation in the presence of physiological levels of Ca2+ buffering for Jurkat T-lymphocytes and RBL-1 cells, indicating that the threshold mechanism may be a general feature of ICRAC activation. Because ICRAC in pulmonary artery endothelia is, at best, very small, additional Ca2+ influx pathways may also contribute to agonist-induced Ca2+ entry.     

Rapid isolation of K88+ Escherichia coli by using immunomagnetic particles.

Superparamagnetic polymer particles precoated with sheep anti-mouse immunoglobulin G were coated with immunoglobulin G2 monoclonal antibodies to the K88 antigen of Escherichia coli (MAb-K88). These immunomagnetic particles (IMP) were used for isolation and identification of K88 antigen-positive (K88+) E. coli. The bacteria presenting the K88 antigen were easily isolated in almost pure culture from a mixed culture of five different O serogroups of E. coli. Nonspecific binding of K88 antigen-negative (K88-) E. coli to the IMP was not observed. The sensitivity of the test to detect K88+ E. coli was found to be 4,000 CFU/ml with fluorescence microscopy. When bacteria attached to the MAb-K88 IMP were grown on blood agar, about 20% of the initial number of CFU was recovered. The test is promising as a rapid method for isolation and identification of K88+ E. coli from a mixed culture.