Cytokine gene polymorphisms moderate illness severity in infants with respiratory syncytial virus infection

Illness severity and frequency of complications in infants with respiratory syncytial virus (RSV) infection may be influenced by the local elaboration of cytokines. Cytokine gene polymorphisms moderate severity of illness in various inflammatory and infectious diseases. We performed cytokine genotyping on 77 infants hospitalized with confirmed RSV infection to determine whether specific cytokine gene polymorphisms are associated with illness severity or complications. DNA was extracted from buccal brushings and assayed for tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-6 and IL-10, and transforming growth factor (TGF)-beta1 genotypes using polymerase chain reaction-sequence-specific primer technology. Clinical outcomes consisted of severity scores of lower respiratory illness, blood oxygen saturation, lengths of oxygen supplementation, and intensive care unit (ICU) and hospital stays, and the presence or absence of pneumonia and otitis media. IFN-gamma genotype was related to severity of lower respiratory illness, duration of ICU stay, and frequency of otitis media. Additionally, IL-6 genotype was related to the length of oxygen (O(2)) supplementation and hospital stay, IL-10 genotype to the frequency of pneumonia, and TGF-beta1 genotype to O(2) saturations at presentation. There were no associations between TNF-alpha genotype and any of the outcome parameters. These results demonstrate that certain cytokine gene polymorphisms contribute to illness severity and complications during RSV infection in infants. If future prospective studies confirm these observations, cytokine genotyping may be a useful tool for identifying "at risk" infants who may benefit from the selective use of preventive or early intervention treatments for RSV.     

Folic acid and homocysteine affect neural crest and neuroepithelial cell outgrowth and differentiation in vitro.

The beneficial effect of additional folic acid in the periconceptional period to prevent neural tube defects, orofacial clefts, and conotruncal heart defects in the offspring has been shown. Folate shortage results in homocysteine accumulation. Elevated levels of homocysteine have been related to neural tube defects. We studied the behavior of neuroepithelial cells and cranial and cardiac neural crest cells in vitro. Neural tube explants were cultured for 24 and 48 hr in medium after addition of folic acid and/or homocysteine. Folic acid addition increased neuroepithelial cell outgrowth and increased neural crest cell differentiation into nerve and smooth muscle cells. Addition of homocysteine increased neural crest cell outgrowth and migration from the neural tube and inhibited neural crest cell differentiation. Our findings suggest that neural tube defects caused by folate deficiency and hyperhomocysteinemia develop due to increased neuroepithelial to neural crest cell transformation. This increased transformation leads to a shortage of neuroepithelial cells in the neural tube. Defects in orofacial and conotruncal development are explained by abnormal differentiation of neural crest cells in the presence of high homocysteine concentrations. Our findings supports a critical role for folic acid and homocysteine in the development of neural tube defects and neural crest related heart malformations.     

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.     

Study of carrier frequency of Warsaw breakage syndrome in the Ashkenazi Jewish population and presentation of two cases

Warsaw breakage syndrome (WABS), caused by bi‐allelic variants in the DDX11 gene, is a rare cohesinopathy characterized by pre‐ and postnatal growth retardation, microcephaly, intellectual disability, facial dysmorphia, and sensorineural hearing loss due to cochlear hypoplasia. The DDX11 gene codes for an iron–sulfur DNA helicase in the Superfamily 2 helicases and plays an important role in genomic stability and maintenance. Fourteen individuals with WABS have been previously reported in the medical literature. Affected individuals have been of various ethnic backgrounds with different pathogenic variants. We report two unrelated individuals of Ashkenazi Jewish descent affected with WABS, who are homozygous for the c.1763‐1G>C variant in the DDX11 gene. Their phenotype is consistent with previously reported individuals. RNA studies showed that this variant causes an alternative splice acceptor site leading to a frameshift in the open reading frame. Carrier screening of the c.1763‐1G>C variant in the Jewish population revealed a high carrier frequency of 1 in 68 in the Ashkenazi Jewish population. Due to the high carrier frequency and the low number of affected individuals, we hypothesize a high rate of miscarriage of homozygous fetuses and/or subfertility for carrier couples. If the carrier frequency is reproducible in additional Ashkenazi Jewish populations, we suggest including DDX11 to Ashkenazi Jewish carrier screening panels.     

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.