NK-active cytokines IL-2, IL-12, and IL-15 selectively modulate specific protein kinase C (PKC) isoforms in primary human NK cells.

Natural killer (NK) cell function is largely modulated by growth factors and cytokines. In particular, interleukin (IL)-2, IL-12, and IL-15 have major effects on the proliferative and cytotoxic activities of NK cells against tumor and virus-infected cells. It is thought that the members of the protein kinase C (PKC) family of serine/threonine kinases play an important role in mediating the pleiotropic effects of cytokines on their target cells. We have investigated the downstream effects generated in purified human NK cells by IL-2, IL-12, and IL-15 on PKCalpha and PKCepsilon--a canonical and a novel isoform of PKC, respectively. By means of Western blotting, PKC activity assays, and immunofluorescence performed on highly purified preparations of primary human NK cells, we demonstrate that: 1) the three cytokines have similar effects on PKCalpha and PKCepsilon activities; 2) whereas PKCepsilon activity is induced by cytokine stimulation, PKCalpha activity is inhibited; and 3) both the induction of PKCepsilon and the inhibition of PKCalpha functional activity are relatively early events in NK cells, while longer cytokine stimulations do not generate significant variations in enzyme activity, suggesting that the activation of both the canonical and novel isoforms of PKC are events required in the early phases of cytokine-induced NK cell stimulation.     

Allergic challenge-elicited lipid bodies compartmentalize in vivo leukotriene C4 synthesis within eosinophils

Eosinophils are an important source of leukotriene (LT)C(4), which can be synthesized within lipid bodies-cytoplasmic organelles where eicosanoid formation may take place. Allergy-driven lipid body formation and function have never been investigated. Here, we studied the in vivo induction and role of lipid bodies within eosinophils recruited to sites of allergic inflammation. Using two murine models of allergic inflammation (asthma and pleurisy), we verified that parallel to the eosinophil influx, allergic challenge also induced lipid body formation within recruited eosinophils. Neutralizing antibodies to eotaxin/CCL11, RANTES/CCL5, or CCR3 partially inhibited lipid body formation within recruited eosinophils in the allergic pleurisy model. Likewise, intrapleural administration of RANTES or eotaxin also induced significant influx of eosinophils loaded with lipid bodies. By immunolabeling, we detected the presence of a key enzyme involved in the leukotriene metabolism-5-lipoxygenase-within eosinophil lipid bodies formed in vivo after allergen challenge. Furthermore, specific immunolocalization of newly formed LTC(4) demonstrated that lipid bodies were the sites of formation of this eicosanoid within infiltrating eosinophils. Therefore, allergic inflammation triggers in vivo formation of new lipid bodies within infiltrating eosinophils, a phenomenon largely mediated by eotaxin/RANTES acting via CCR3 receptors. Such in vivo allergen-driven lipid bodies function as intracellular compartments of LTC(4) synthesis.     

Four-color flow cytometric analysis of peripheral blood donor cell chimerism

Passenger leukocytes have been demonstrated to play significant roles in initiating and also regulating immune reactions after organ transplantation. Reliable techniques to detect donor leukocytes in recipients after organ transplantation are essential to analyze the role, function, and behavior of these leukocytes. In this report we describe a simple, reliable method to detect donor cells with low frequencies using peripheral blood samples. Detection of small numbers of major histocompatibility complex (MHC) mismatched cells was first studied using four-color flow cytometry in artificially created cell mixtures. By selecting the CD45(+) population and simultaneous staining with several leukocyte lineage markers (CD3, CD4, CD8, CD56, and CD19), MHC-mismatched leukocytes were consistently detected in cell suspensions prepared from directly stained whole blood samples with a threshold sensitivity as low as 0.1%-0.2%. When the fresh peripheral blood mononuclear cells were separated by conventional Ficoll gradient purification, similar, but slightly lower levels of donor cells were detected. Blood samples obtained 1-5 months after liver, kidney, and intestine transplants revealed that the kind of organ allograft influenced levels and lineage pattern of the circulating donor cells. This procedure provided a simple and reliable method in determining early chimerism in transplant recipients. However, the detection of MHC-mismatched leukocytes of all lineages was much lower when frozen peripheral blood mononuclear cells were used.     

Nkx2.5‐negative myocardium of the posterior heart field and its correlation with podoplanin expression in cells from the developing cardiac pacemaking and conduction system

Recent advances in the study of cardiac development have shown the relevance of addition of myocardium to the primary myocardial heart tube. In wild‐type mouse embryos (E9.5–15.5), we have studied the myocardium at the venous pole of the heart using immunohistochemistry and 3D reconstructions of expression patterns of MLC‐2a, Nkx2.5, and podoplanin, a novel coelomic and myocardial marker. Podoplanin‐positive coelomic epithelium was continuous with adjacent podoplanin‐ and MLC‐2a‐positive myocardium that formed a conspicuous band along the left cardinal vein extending through the base of the atrial septum to the posterior myocardium of the atrioventricular canal, the atrioventricular nodal region, and the His‐Purkinje system. Later on, podoplanin expression was also found in the myocardium surrounding the pulmonary vein. On the right side, podoplanin‐positive cells were seen along the right cardinal vein, which during development persisted in the sinoatrial node and part of the venous valves. In the MLC‐2a‐ and podoplanin‐positive myocardium, Nkx2.5 expression was absent in the sinoatrial node and the wall of the cardinal veins. There was a mosaic positivity in the wall of the common pulmonary vein and the atrioventricular conduction system as opposed to the overall Nkx2.5 expression seen in the chamber myocardium. We conclude that we have found podoplanin as a marker that links a novel Nkx2.5‐negative sinus venosus myocardial area, which we refer to as the posterior heart field, with the cardiac conduction system. Anat Rec, 290:115–122, 2007. © 2006 Wiley‐Liss, Inc.     

The pulmonary biology of isoprostanes

Isoprostanes were first recognized as convenient markers of oxidative stress, but their powerful effects on a variety of cell functions are now also being increasingly appreciated. This is particularly true of the lung, which is comprised of a wide variety of different cell types (smooth muscle, innervation, epithelium, lymphatics, etc.), all of which have been shown to respond to exogenously applied isoprostanes. In this review, we summarize these biological responses in the lung, and also consider the roles that isoprostanes might play in a range of pulmonary clinical disorders.     

Trisomy 8 in myelodysplasia and acute leukemia is constitutional in 15-20% of cases.

The trisomy 8 found in malignancies may derive from a constitutional trisomy 8 mosaicism (CT8M), and in these cases the trisomy itself may be regarded as the first mutation in a multistep carcinogenetic process. To assess the frequency of CT8M in hematological dysplastic and neoplastic disorders with trisomy 8, an informative sample of 14 patients was collected. The data ascertained included chromosome analyses of fibroblast cultures and of PHA-stimulated blood cultures in patients with normal blood differential count, as well as possible CT8M clinical signs. One patient showed trisomy 8 in all cell types analyzed and undoubtedly has a CT8M; a second patient consistently showed trisomy 8 in PHA-stimulated blood cultures when no immature myeloid cells were present in blood and should be considered as having CT8M; a third patient, with Philadelphia-positive chronic myelocytic leukemia, was more difficult to interpret, but the possibility that she had CT8M is likely. A few clinical signs of CT8M were also present in these three patients. Our data indicate that the frequency of CT8M in hematological dysplastic and neoplastic disorders with trisomy 8 is approximately 15-20%.     

Oxidative stress response involving induction of protective enzymes in Candida dubliniensis.

Candida dubliniensis is a yeast species closely related to Candida albicans, but in contrast to C. albicans, limited information is available on the virulence factors of this important fungal pathogen. The objective of the present study was to determine if this species was able to evoke an adaptive response to oxidants. C. dubliniensis, treated with a low concentration of either H(2)O(2) or methyl viologen (a superoxide generating agent), mounts an adaptive response that results in increased survival against lethal doses of both oxidants. This response was characterized by the induction of enzymes with known antioxidant function. C. dubliniensis strains were less resistant to oxidants than C. albicans, displaying higher susceptibility to their toxic effects. The adaptive response described here might be responsible, among other factors, for the ability of this pathogen to cause infections in individuals with impaired immunity.     

<Emphasis Type="Italic">Trypanosoma evansi</Emphasis> in inbred and Swiss-Webster mice: distinct aspects of pathogenesis

Trypanosoma evansi (Trypanosomatidae, Kinetoplastida) is a salivarian trypanosomatid that infects eight mammal orders spread over America, Europe and Asia. In Brazil, T. evansi is the etiological agent of Mal de Cadeiras, a horse disease very often described in the region known as Pantanal do Mato Grosso. Few data concerning the genetic diversity and biology of subpopulations of T. evansi that circulate in Brazil are available. The factors that modulate the interaction of this parasite with its hosts also remain to be elucidated. Here we evaluated the course of experimental infection of six T. evansi isolates derived from domestic and wild animals in Swiss-Webster mice and three Mus musculus lineages. The follow-up included biological, immunological as well as biochemical and hematological parameters. The same isolates as well as three others were characterized by pulsed-field electrophoresis. Our results showed that T. evansi isolates displayed significant differences regarding behavior and morbidity patterns in the distinct mouse lineages. Nevertheless, these differences could not be correlated with pulsed-field electrophoresis profiles. Indeed, concerning this molecular marker, only microheterogeneity was observed. Moreover, we observed that the outcome of the infection is defined by both host genetic background and peculiarities (virulence factors) of the distinct T. evansi isolates. Anemia and hypoglycemia were the only features that could be observed in all mouse lineages, independently of the inoculated T. evansi subpopulation. In addition, our data also show that Mus musculus is a suitable model host for the study of the different pathogenetic features of T. evansi infection.     

Effects of selective NMDA and non-NMDA blockade in the nucleus accumbens on the plus-maze test

Effect of blocking N-methyl-D-aspartic acid (NMDA) and non-NMDA-glutamatergic receptors on performance in the plus-maze was studied in male rats bilaterally cannulated into the nucleus accumbens (Acc). Rats were divided into seven groups that received either 1 microl injections of saline, (+/-)2-amino-7-phosphonoheptanoic acid (AP-7, 0.2, 0.5, or 1 microg) or 2,3 dioxo-6-nitro-1,2,3,4,tetrahydrobenzo-(f)quinoxaline-7-sulphonamide disodium (NBQX, 0.2, 0.5, or 1 microg) 15 min before testing. Time spent in open arm, time per entry, end arrivals, open, closed, and total arm entries, relationship between open-, closed-, and total arm entries, rearing, face-, head-, and body grooming, and number of fecal boli were recorded. Time spent in the open arm increased under AP-7 (0.5 and 1 microg; P<.01) and NBQX (1 microg; P<.05) treatment, whereas time per entry was increased only with AP-7 (1 microg; P<.05). Open arm entries were increased by the intermediate doses of AP-7 (0.5 microg; P<.01) and NBQX (0.5 microg; P<.05); end arrivals were increased by the intermediate dose of AP-7 (0.5 microg/1 microl, P<.05). The frequency of rearing, grooming, and closed arm entries was not affected by the treatment. We conclude that NMDA and non-NMDA-glutamatergic blockade in the Acc lead to a behavioral disinhibition of cortical influences with the median doses, but that at higher doses the blockers have an anxiolytic-like effect.