Developmental changes in concentrations and distributions of neurotrophins in the monkey cerebellar cortex

Neurotrophins are involved in the survival, differentiation, migration and neurite outgrowth of various neuronal populations. Neurotrophins and their receptors are widely expressed in the developing cerebellum of various experimental animals. To gain some insight into the possible roles played by these molecules in monkey cerebellum, we examined the protein levels of BDNF, NT-4/5 and NT-3 and distributions of those neurotrophins and TrkC, a high affinity receptor for NT-3, in the cerebellum of developing macaque monkeys using ELISAs and immunohistochemical methods. We found that the level of BDNF increased during development, while the level of NT-3 was higher during embryonic stages and decreased toward adulthood. The level of NT-4/5 increased from embryonic stages to infant stages and gradually declined with age. Among the three neurotrophins, BDNF immunoreactivity was found in all kinds of cerebellar neurons, including all inhibitory interneurons, throughout the postnatal periods examined, indicating that BDNF may be an essential factor for the maintenance of cerebellar neural functions. The Bergmann glial fibers and the internal part of the external granule cell layer were strongly NT-3 immunopositive at the early postnatal stages, and more weakly immunoreactive toward adulthood. In addition, we found that the premigratory precursors of the granule cells were TrkC immunopositive at early postnatal stages. These findings suggest that NT-3 in Bergmann glial fibers may be involved in the migration of the premigratory granule cells.     

Inhibition of endoplasmic reticulum-associated degradation in CHO cells resistant to cholera toxin,Pseudomonas aeruginosa exotoxin A,and ricin

Many plant and bacterial toxins act upon cytosolic targets and must therefore penetrate a membrane barrier to function. One such class of toxins enters the cytosol after delivery to the endoplasmic reticulum (ER). These proteins, which include cholera toxin (CT), Pseudomonas aeruginosa exotoxin A (ETA), and ricin, move from the plasma membrane to the endosomes, pass through the Golgi apparatus, and travel to the ER. Translocation from the ER to the cytosol is hypothesized to involve the ER-associated degradation (ERAD) pathway. We developed a genetic strategy to assess the role of mammalian ERAD in toxin translocation. Populations of CHO cells were mutagenized and grown in the presence of two lethal toxins, ETA and ricin. Since these toxins bind to different surface receptors and attack distinct cytoplasmic targets, simultaneous acquisition of resistance to both would likely result from the disruption of a shared trafficking or translocation mechanism. Ten ETA- and ricin-resistant cell lines that displayed unselected resistance to CT and continued sensitivity to diphtheria toxin, which enters the cytosol directly from acidified endosomes, were screened for abnormalities in the processing of a known ERAD substrate, the Z form of alpha1-antitrypsin (alpha1AT-Z). Compared to the parental CHO cells, the rate of alpha1AT-Z degradation was decreased in two independent mutant cell lines. Both of these cell lines also exhibited, in comparison to the parental cells, decreased translocation and degradation of a recombinant CTA1 polypeptide. These findings demonstrated that decreased ERAD function was associated with increased cellular resistance to ER-translocating protein toxins in two independently derived mutant CHO cell lines.     

Regulation of the TSC pathway by LKB1: evidence of a molecular link between tuberous sclerosis complex and Peutz-Jeghers syndrome

Tuberous sclerosis complex (TSC) and Peutz-Jeghers syndrome (PJS) are dominantly inherited benign tumor syndromes that share striking histopathological similarities. Here we show that LKB1, the gene mutated in PJS, acts as a tumor suppressor by activating TSC2, the gene mutated in TSC. Like TSC2, LKB1 inhibits the phosphorylation of the key translational regulators S6K and 4EBP1. Furthermore, we show that LKB1 activates TSC2 through the AMP-dependent protein kinase (AMPK), indicating that LKB1 plays a role in cell growth regulation in response to cellular energy levels. Our results suggest that PJS and other benign tumor syndromes could be caused by dysregulation of the TSC2/mTOR pathway.     

Role of oxidative stress,angiogenesis and chemo-attractant cytokines in the pathogenesis of ischaemic protection induced by remote ischaemic conditioning: Study of a human model of ischaemia-reperfusion induced vascular injury

Aims

We explored the effect of remote ischaemic conditioning (RIC) on endothelial function and on circulating mediators.

Intermittent preventive sulfadoxine-pyrimethamine treatment of primigravidae reduces levels of plasma immunoglobulin G, which protects against pregnancy-associated Plasmodium falciparum malaria

Pregnancy-associated malaria (PAM) is an important cause of maternal and neonatal suffering. It is caused by Plasmodium falciparum capable of inhabiting the placenta through expression of particular variant surface antigens (VSA) with affinity for proteoglycans such as chondroitin sulfate A. Protective immunity to PAM develops following exposure to parasites inhabiting the placenta, and primigravidae are therefore particularly susceptible to PAM. The adverse consequences of PAM in primigravidae are preventable by intermittent preventive treatment (IPTp), where women are given antimalarials at specified intervals during pregnancy, but this may interfere with acquisition of protective PAM immunity. We found that Kenyan primigravidae receiving sulfadoxine-pyrimethamine IPTp had significantly lower levels of immunoglobulin G (IgG) with specificity for the type of parasite-encoded VSA-called VSA(PAM)-that specifically mediate protection against PAM than did women receiving a placebo. VSA(PAM)-specific IgG levels depended on the number of IPTp doses received and were sufficiently low to be of clinical concern among multidose recipients. Our data suggest that IPTp should be extended to women of all parities, in line with current World Health Organization recommendations.     

Expression of vascular endothelial growth factor in human gastric carcinomas

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a secreted protein which may play a pivotal role in tumor-associated microvascular angiogenesis and hyperpermeability. The expression of mRNA for VEGF was examined in eight gastric carcinoma cell lines and 30 gastric carcinoma tissues as well as corresponding normal mucosa. All the cell lines expressed VEGF mRNA at various levels that correlated well with the amounts of VEGF secreted into the condition medium. The expression of VEGF mRNA by TMK-1 cells was increased by the treatment of epidermal growth factor (EGF) or interleukin-1α (IL-1α), whereas it was decreased by the treatment of interferon-β (IFN-β). In gastric carcinoma tissues, the level of VEGF mRNA in primary tumors was higher than that in the corresponding normal mucosas in six (46%) of 13 well-differentiated adenocarcinomas and in two (12%) of 17 poorly differentiated adenocarcinomas, respectively. Vessel counts in well-differentiated adenocarcinomas had a tendency to be higher than those in poorly differentiated adenocarcinomas. In well-differentiated adenocarcinomas, the levels of VEGF mRNA expression tended to be higher in carcinomas of advanced stage than in early stage carcinomas. Both in situ mRNA hybridization and immunohistochemistry demonstrated the presence of VEGF expression within the tumor cells. These results suggest that VEGF may confer angiogenesis and progression of human gastric carcinomas, especially of the well-differentiated type.     

Identification of an HLA-DQ6-derived peptide recognized by mouse MHC class I H-2Db-restricted CD8+ T cells in HLA-DQ6 transgenic mice

Summary CD8⁺ T cells from C57BL/6(B6) mice show cytotoxicity to B cell blasts prepared from syngeneic transgenic mice expressing HLA-DQ6 molecules in a mouse MHC class I H-2D^b restricted manner. Although these results suggest that CD8⁺ T cells recognize peptides derived from DQ6 molecule bound to H-2D^b on target cells, no direct evidence so far has been obtained. To clarify this, we synthesized 23 peptides corresponding to DQ6α orβ chain and carrying the motifs of D^b-binding peptides, and examined their capacity to induce cytotoxicity in the CD8⁺ T cell line. We show here that DQA1-2, one of these peptides, induced cytotoxicity of the CD8⁺ T cells when this peptide was pulsed to H-2D^b expressing target cells, as efficiently as HLA-DQ6 expressing target cells did. Thus, our results suggest that DQA1-2 can be naturally processed from DQ6 molecules and recognized by the CD8⁺ T cells in the context of H-2D^b molecules. These results suggest that allogeneic HLA class II molecules are involved in the rejection not only as the ligand for T cell receptor of alloreactive CD4⁺ T cells but also as self-peptides bound to HLA class I molecules recognized by CD8⁺ T cells.     

Structural and replicative forms of mitochondrial DNA in tissues from adult and senescent BALB/c mice and Fischer 344 rats

Age-related changes in the structure and replication of mitochondrial DNA (mtDNA) were investigated in different organs from young adult (9–10 months' old) and senescent (28–29 months' old) BALB/c mice and Fischer 344 rats. Total mtDNA from brain, heart, kidney and liver was isolated by centrifugation in ethidium bromide—CsCl gradient and examined for the occurrence of complex forms and replicative intermediates by electron microscopy. The frequency of catenated mtDNA (interlinked molecules containing two or more circular units) varied from about 2.5% to 5% in adult tissues and showed a small increase in the majority of senescent organs. The frequency of double-sized circular molecules, or circular dimers, was very low in adult tissues, with an average of about 0.04% in mice and 0.1% in rats. The frequency of circular dimers increased with aging to 1.9% in mouse brain and 1.5% in rat kidney, with smaller increases (0.4% and 0.7%) in heart mtDNA from both species; there was no significant increase in the other organs. It is suggested that the increase in the frequency of circular dimer mtDNA reflects an overall deterioration of tissue physiology rather than intrinsic senescent changes in the mitochondria. The frequencies and types of the various replicative forms of mtDNA varied significantly according to tissue but not according to species or donor age. The only exception was a significant increase in the frequency of larger replicative forms in senescent mouse liver, to about 20% compared with 12% in adult liver, suggesting an age-related change in the rate of mtDNA replication and/or turnover in this organ.     

IFN-alpha enhances CD40 ligand-mediated activation of immature monocyte-derived dendritic cells

Type I IFN are immune modulatory cytokines that are secreted during early stages of infection. Type I IFN bridge the innate and the adaptive immune system in humans and mice. We compared the capacity of type I and II IFN to induce the functional maturation of monocyte-derived dendritic cells (MoDC). Extending our earlier observation that type I IFN promote DC maturation, we report that these cytokines also enhance DC differentiation by augmenting CD40 ligand (CD40L)-induced cytokine secretion by MoDC. Type I IFN alone were poor inducers of MoDC maturation as compared with other stimuli. They up-regulated the expression of HLA-DR, CD80, CD86, partially CCR7 but not CD83, partially reduced antigen-uptake function, increased the levels of IL-12p35 mRNA, and prolonged surface expression of peptide-MHC class I complexes for presentation to cytotoxic T lymphocytes, but did not induce migration towards CCL21 chemokine. However, type I IFN were potent co-factors for CD40L-mediated function. Here, they enhanced CD40L-mediated IL-6, IL-10 and IL-12p70 secretion. Furthermore, when combined with IL-1beta and/or IL-4, IFN-alpha2a type I IFN increased CD40L-mediated IL-12p70 production by 2- to 3-fold, and biased the IL-12 p40/p70 ratio towards the IFN-gamma inducing p70 heterodimer, this correlating with higher levels of IFN-gamma secretion by allogeneic T cell subsets and NK cells. Our results suggest that the rapid expression of CD40L, IFN and IL-1beta at sites of infection and inflammation can act in concert on immature DC, thereby linking innate and adaptive immune responses. In this way, type I IFN play a dual role as DC maturation factors and enhancers of CD40L-mediated DC activation.