The phosphatidylinositol 3-kinase/protein kinase B signaling pathway is activated by lipoteichoic acid and plays a role in Kupffer cell production of interleukin-6 (IL-6) and IL-10

Sepsis caused by gram-positive bacteria lacking lipopolysaccharide (LPS) has become a major and increasing cause of mortality in intensive-care units. We have recently demonstrated that the gram-positive-specific bacterial cell wall component lipoteichoic acid (LTA) stimulates the release of the proinflammatory cytokines in Kupffer cells in culture. In the present study, we have started to assess the signal transduction events by which LTA induces the production of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and the anti-inflammatory cytokine IL-10 in rat Kupffer cells. LTA was found to trigger phosphorylation of mitogen-activated protein kinases (MAPK) (p38 MAPK and ERK 1/2) and protein kinase B (PKB). Compared to LPS, LTA was more potent in inducing PKB phosphorylation after 40 min, although we found that the cytokine responses were similar. For both bacterial molecules, blocking phosphatidylinositol 3-kinase (PI3-K; Ly294002) or Janus kinase 2 (JAK-2; AG490) particularly affected the induction of IL-6 and IL-10 release, whereas TNF-alpha levels were strongly reduced by inhibition of Src family tyrosine kinases (PP2). All three cytokines were reduced by inhibition of p38 MAPK (SB202190) or the broad-range tyrosine kinase inhibitor genistein, whereas IL-6 release was particularly blocked by inhibition of ERK 1/2 (PD98059). Divergences in the regulatory pathways controlling TNF-alpha, IL-10, and IL-6 production in Kupffer cells following LPS or LTA stimulation may create a basis for understanding how the balance between pro- and anti-inflammatory cytokines is regulated in the liver following infections by gram-positive or gram-negative bacteria.     

Spontaneous reanastomosis between lymphatic vessels following syngeneic transplantation of the small intestine in the rat

Abstract Spontaneous lymphvascular reanastomosis (SLR) following small bowel transplantation in rats is of clinical relevance for the resorption of long chain fatty acids. Detailed morphological and molecular data concerning the process of lymphvascular reanastomosis are not available in the literature. In this study SLR was investigated using microradiology and scanning electron microscopy. Between the 8^th and 21^st postoperative days following transplantation SLR does not occur between the intestinal trunk of the transplant and the thoracic duct of the recipient. Instead, an indirect connection was observed between the inserted advential lymphatic vessels of the mesenteric artery and lymphatic vessels of the aorta or ductus deferens, which are connected with the thoracic duct.     

Optimisation of the CT h4S bioassay for detection of human interleukin-4 secreted by mononuclear cells stimulated by phytohaemaglutinin or by human leukocyte antigen mismatched mixed lymphocyte culture

Limiting dilution analysis has been used in the context of allogeneic bone marrow transplantation to determine anti-recipient interleukin-2 (IL-2) producing helper T lymphocyte precursor (HTLp) frequencies, which in several studies have been predictive of graft-versus-host disease (GVHD). Recently high anti-recipient IL-4 producing HTLp frequencies have been reported and associated with a decreased risk of GVHD. The aim of the present study was to define the optimal conditions for combined determination of IL-2 and IL-4 producing anti-recipient HTLp frequencies. We have optimised the CT.h4S bioassay with regards to specificity, sensitivity, detection limit, and reproducibility. We have found the optimal assay conditions to be 1 x 10 (4) CT.h4S cells/well deprived of IL-4 for 24 h and preincubated for 7 h followed by 18 h of incubation with tritiated methyl-thymidine. In this setting the CT.h4S bioassay detects 5 pg/ml of human recombinant IL-4 with no detection of IL-2 in concentrations below 500 pg/ml. We have found 72 h of culture optimal for detection of IL-2 and IL-4 produced by human mononuclear cells (MNC) in response to stimulation with phytohaemaglutinin and for detection of IL-2 in human leukocyte antigen (HLA)-mismatched mixed leukocyte culture (MLC). An interindividual variation in cytokine accumulation was demonstrated for IL-4 but not for IL-2. With the use of 5x10(4) responder cells/well no IL-4 could be detected in HLA-mismatched MLC between days 1 and 16. The lack of IL-4 detection was not due to high amounts of soluble IL-4 receptor. With the use of 1x10(6) responder cells/well in HLA-mismatched MLC, we found limited IL-4 accumulation still increasing at day 12. We conclude that the CT.h4S bioassay is a reliable and specific method for quantification of IL-4 accumulation in cultures of human MNC. The difference in optimal timing for IL-2 (day 3) and IL-4 (>/=day 12) detection and evidence of very low IL-4 producing HTLp frequencies makes the relevance of a combined IL-2/IL-4 HTLp assay questionable.     

Ultrastructural localization of gold in macrophages and mast cells exposed to aurothioglucose

The ultrastructural localization of gold in resting, stimulated, and activated mouse peritoneal cells exposed in vivo and in vitro to aurothioglucose was examined by a photochemical technique. Activated macrophages from herpes simplex virus type 2 infected mice showed the heaviest accumulation of gold, located in phagolysosomes. Gold was also visualized in granules of mast cells and in nuclei of disintegrated cells. No gold was found in polymorphonuclear leucoctyes and in lymphocytes. The mechanism by which gold is taken up into the cells is discussed.     

Autocrine motility factor (neuroleukin, phosphohexose isomerase) induces cell movement through 12-lipoxygenase-dependent tyrosine phosphorylation and serine dephosphorylation events

Autocrine motility factor (AMF) is one of the motility cytokines regulating tumor cell migration, therefore identification of the signaling pathway coupled with it has critical importance. Previous studies revealed several elements of this pathway predominated by lipoxygenase-PKC activations but the role for tyrosine kinases remained questionable. Motility cytokines frequently have mitogenic effect as well, producing activation of overlapping signaling pathways therefore we have used B16a melanoma cells as models where AMF has exclusive motility effect. Our studies revealed that in B16a cells AMF initiated rapid (1–5 min) activation of the protein tyrosine kinase (PTK) cascade inducing phosphorylation of 179, 125, 95 and 40/37 kD proteins which was mediated by upstream cyclo- and lipoxygenases. The phosphorylated proteins were localized to the cortical actin-stress fiber attachment zones in situ by confocal microscopy. On the other hand, AMF receptor activation induced significant decrease in overall serine-phosphorylation level of cellular proteins accompanied by serine phosphorylation of 200, 90, 78 and 65 kd proteins. The decrease in serine phosphorylation was independent of PTKs, PKC as well as cyclo- and lipoxygenases. However, AMF induced robust translocation of PKCα to the stress fibers and cortical actin suggesting a critical role for this kinase in the generation of the motility signal. Based on the significant decrease in serine phosphorylation after AMF stimulus in B16a cells we postulated the involvement of putative serine/threonine phosphatase(s) upstream lipoxygenase and activation of the protein tyrosine kinase cascade downstream cyclo- and lipoxygenase(s) in the previously identified autocrine motility signal. This revised version was published online in July 2006 with corrections to the Cover Date.     

Seroresponses to human papillomavirus types 16, 18, 31, 33, and 45 virus-like particles in South African women with cervical cancer and cervical intraepithelial neoplasia

The aim of the study was to determine the prevalence of antibodies to human papillomavirus (HPV) types 16, 18, 31, 33, and 45 in woman in Cape Town with cervical intraepithelial neoplasia (CIN) (n = 95), cervical cancer (n = 40), female blood donors (n = 95) and children (n = 110). The enzyme-linked immunosorbent assay (ELISA) made use of baculovirus synthesised HPV virus like particles (VLPs) as antigen. Antibodies to at least one HPV type were detected in sera from 75% of cancer patients, 71.6% of CIN patients, 44.2% of blood donors and 27.3% of children. Sera from 95 women with CIN were compared with age-matched female blood donors. There was a significant association of seropositivity to VLP-16 (P = 0.006) and VLP-45 (P = 0.008) with CIN compared with the blood donors. There was also a significant difference in the seropositivity of women with CIN to any of the five virus-like particle (VLP) types compared to the blood donors (P = 0.0002: OR = 3.2). Thirty-nine of sixty-nine (56.5%) women with CIN were found to be HPV-16 DNA positive. The average age of women in this group that were VLP-16 seropositive was 34 years and those found to be VLP-16 seronegative was 52 years of age. Antibodies to all five VLP types were detected in these populations, thus an ideal vaccine should induce protection from infection by a wide range of HPV types.     

Sensitivity of FL cells to polio- and adenoviruses

Summary Poliovirus plaque counts on the FL strain of human amnion cells were almost double the counts on rhesus monkey kidney for the same virus preparation, repeatedly assayed over more than six months. FL cells gave more consistent results in virus assay than monkey kidney cells. Plaque counts obtained with FL cells were 2 1/2 to 5 times higher than those obtained with HeLa, Chang's conjunctiva and Detroit-6 cells.Using the chick embryo-adapted MEF-1 strain of poliovirus, FL cells and primary human amnion cells reacted similarly in respect to plaque count and morphology, while no distinct plaques were seen on monkey kidney or HeLa monolayers under comparable conditions. For infectivity assays of adenovirus suspensions based on cytopathogenic effect, FL cells were found suitable, although no plaque formation was obtained.Aided by American Cancer Society Grant E-82 and a grant from the National Foundation.     

Difference in allelic expression of the CLCN1 gene and the possible influence on the myotonia congenita phenotype

Mutations in the CLCN1 gene, encoding a muscle-specific chloride channel, can cause either recessive or dominant myotonia congenita (MC). The recessive form, Becker's myotonia, is believed to be caused by two loss-of-function mutations, whereas the dominant form, Thomsen's myotonia, is assumed to be a consequence of a dominant-negative effect. However, a subset of CLCN1 mutations can cause both recessive and dominant MC. We have identified two recessive and two dominant MC families segregating the common R894X mutation. Real-time quantitative RT-PCR did not reveal any obvious association between the total CLCN1 mRNA level in muscle and the mode of inheritance, but the dominant family with the most severe phenotype expressed twice the expected amount of the R894X mRNA allele. Variation in allelic expression has not previously been described for CLCN1, and our finding suggests that allelic variation may be an important modifier of disease progression in myotonia congenita.