Mammalian transforming growth factor beta1 activated after ingestion by Anopheles stephensi modulates mosquito immunity

During the process of bloodfeeding by Anopheles stephensi, mammalian latent transforming growth factor beta1 (TGF-beta1) is ingested and activated rapidly in the mosquito midgut. Activation may involve heme and nitric oxide (NO), agents released in the midgut during blood digestion and catalysis of L-arginine oxidation by A. stephensi NO synthase (AsNOS). Active TGF-beta1 persists in the mosquito midgut to extended times postingestion and is recognized by mosquito cells as a cytokine. In a manner analogous to the regulation of vertebrate inducible NO synthase and malaria parasite (Plasmodium) infection in mammals by TGF-beta1, TGF-beta1 regulates AsNOS expression and Plasmodium development in A. stephensi. Together, these observations indicate that, through conserved immunological cross talk, mammalian and mosquito immune systems interface with each other to influence the cycle of Plasmodium development.     

The mutational spectrum of brachydactyly type C

Growth/differentiation factor-5 (GDF5), also known as cartilage-derived morphogenetic protein-1 (CDMP-1), is a secreted signaling molecule that participates in skeletal morphogenesis. Heterozygous mutations in GDF5, which maps to human chromosome 20, occur in individuals with autosomal dominant brachydactyly type C (BDC). Here we show that BDC is locus homogeneous by reporting a GDF5 frameshift mutation segregating with the phenotype in a family whose trait was initially thought to map to human chromosome 12. We also describe heterozygous mutations in nine additional probands/families with BDC and show nonpenetrance in a mutation carrier. Finally, we show that mutant GDF5 polypeptides containing missense mutations in their active domains do not efficiently form disulfide-linked dimers when expressed in vitro. These data support the hypothesis that BDC results from functional haploinsufficiency for GDF5.     

Preferential presentation of herpes simplex virus T-cell antigen by HLA DQA1*0501/DQB1*0201 in comparison to HLA DQA1*0201/DQB1*0201

The HLA DQA1 locus is polymorphic. Haplotypes containing HLA DQA1*0501, but not HLA DQA1*0201, together with HLA DQB1*0201 are associated with Grave's disease and celiac sprue. In this report, we demonstrate a functional correlate of DQA1 polymorphism. T cells infiltrating a herpes simplex virus (HSV) lesion from a HLA DQ 2,7 individual yielded a virusspecific CD4+ clone restricted by DQ2. Presentation of viral peptide and protein segregated with DQA1 allele, because cell lines bearing DQA1*0501/DQB1*0201 heterodimers presented antigen in proliferation and cytotoxicity assays much more efficiently than cell lines bearing DQA1*0201/DQB1*0201. Binding of viral peptide to cell lines bearing DQA1*0201, in comparison to DQA1*0501, was only moderately reduced and may not explain this effect. Truncation and substitution analyses of peptide binding and T-cell activation were performed to determine which viral peptide residues contacting TCR might therefore be presented in an altered conformation by DQA1*0201/DQB1*0201. Residues 432, 435, 437, 438, and 440 (position P1, P4, P6, P7, and P9) contributed to DQ2 binding, whereas residues 431, 433, 434, and 436 (positions P-1, P2, P3, and P5) contributed to TCR contact. Differential presentation of peptide by HLA DQ2 heterodimers varying at the DQA1 locus may have relevance to host defense and the pathogenesis of HLA DQ2-associated autoimmune diseases. Human Immunology 53, 195-205 (1997).     

High level expression of apoptosis inhibitor in hepatoma cell line expressing Hepatitis B virus

The serious result of hepatitis B (HBV) virus infection is development of hepatocellular carcinoma (HCC). However, the reason of development of HCC in HBV infected patients is still unclear. Recently, the suppression of cell apoptosis is found to relate with the development of cell carcinogenesis, therefore, the expression of apoptosis inhibitor in the virus related cancer line such as hepatoma cell line HepG2.215 was investigated. There are at least six Human apoptosis inhibitors (IAP) have been identified now. They are cIAP1, cIAP2, XIAP, NAPI, survivin and pIAP. Using gene-assay technology, we have recently compared the expression of IAPs in the HepG2.215 cells that persistently expresses Hepatitis B virus by integrated HBV genome with its parent cell line HepG2. The results suggest that there was obviously increase of cIAP2 and cIAP1 in the HepG2.215 cells versus HepG2 cells. Those observations imply a possibility of long time HBV infection could induce the over-expressing apoptosis inhibitors, furthermore, causing the liver cancer. The high expression of cIAP1 and cIAP2 in HBV expressing cells was confirmed by RT-PCR and Northern blot analysis. However, we did not find the change of NIAP and suvivin in HepG2.215 cells. In contrast, the expression of XIAP was down in the HepG2.215 cells comparing with HepG2 cells. How HBV triggers the over-expression of apoptosis inhibitor is unclear. Transient transfection of HepG2 cells with the plasmids expressing different HBV proteins such as S, M, L, X and core proteins did not give a decisive conclusion. Further study is going on now.     

Fenfluramine challenge, self-injurious behavior, and aggression in rhesus monkeys

Self-injurious behavior (SIB) and aggression have been linked to reduced serotonergic (5-HT) functioning in both humans and nonhuman primates. The present study examined serum prolactin and cortisol responses to the 5-HT releasing agent D,L-fenfluramine (FEN) in 24 individually housed rhesus monkeys (Macaca mulatta), 15 of which carried a veterinary record of self-wounding (SW). Subjects received two doses of FEN, 4 and 2 mg/kg, separated by an interval of at least 2 months. For control purposes, monkeys were given an intramuscular saline injection 1 week prior to each FEN challenge. The relationship between the hormonal responses to FEN, wounding history, the rates of self-directed biting and aggression were determined for each animal based on 100 five-minute observations conducted over a period of 12 months surrounding the challenge procedures. Prolactin and cortisol responses to FEN were unrelated either to wounding history or to rates of self-directed biting. However, there were significant inverse correlations between levels of aggression and the prolactin response to both doses of FEN. The present findings provide no evidence for reduced 5-HT system function in rhesus monkeys with SIB under the present challenge conditions. However, the results are consistent with a previously reported inverse relationship between serotonergic activity and aggression. Moreover, a dose-dependent response to FEN was observed only for prolactin, suggesting that this variable is more appropriate than cortisol as an endpoint for FEN challenge in monkeys.     

Role of lymphocyte activation products (LAP) in cell-mediated immunity. I. Preparation and partial purification of guinea-pig LAP

Partial purification of soluble products of guinea-pig lymphocyte activation (LAP) was undertaken by fractional precipitation with ammonium sulphate, ion-exchange and Sephadex chromatography, and by immune precipitation of inducing antigen and of contaminating serum protein. During these purification steps the activity of macrophage migration-inhibition factor (MIF) was concentrated up to 1300-fold and separated from inducing antigen and serum protein. An endpoint assay was devised for expressing antigen-induced MIF activity of LAP fractions as weights of material giving 30% inhibition of migration (MI₃₀ doses).

MIF activity precipitated between 50% and 80% saturated ammonium sulphate and eluted from DEAE-cellulose at pH 7·9 at intermediate salt concentrations (0·03–0·2 M phosphate). On Sephadex gel filtration MIF activity was concentrated in fractions of molecular weight range 56,000–82,000 with a smaller amount of activity eluting from 20,000–56,000. After immune precipitation of extraneous protein and elution from DEAE-cellulose, LAP material was found to have an MI₃₀ dose of 0·4 μg.

Materials representative of antigen and serum protein-depleted MIF were selected for intralymphatic injection in order to determine whether MIF-rich LAP fractions were able to induce paracortical distension in guinea-pig lymph nodes (see following paper).

     

Enhancement of gap junctional intercellular communication of normal human dermal fibroblasts cultured on polystyrene dishes grafted with poly-N-isopropylacrylamide

Technology developed to allow recovery of cells without enzyme treatment, involving a dish grafted with a thermoreactive polymer gel of poly-N-isopropylacrylamide (PIPAAm), was found to significantly enhance gap junctional intercellular communication (GJIC) in normal human dermal fibroblasts (NHDF cells). NHDF cells were cultured for 4 days on PIPAAm-grafted dishes irradiated with various doses of electron beams, and GJIC was assayed by the scrape-loading dye transfer method. The area of dye transfer was greater in the PIPAAm-grafted dishes than in the control culture dishes, indicating that the PIPAAm-grafted dishes enhanced the GJIC of NHDF cells. Connexin-43 (Cx43) expression was analyzed because Cx43 is considered to be a main component of the gap junctional channel. PIPAAm-grafted dishes irradiated with 100, 250, or 500 kGy of electron beams showed significantly enhanced expression of Cx43-NP, Cx43-P1, and especially Cx43-P2. Enhanced expression of Cx43-P2, a functional transmembrane protein, may be related to the promotion of GJIC. These results suggest that the PIPAAm-grafted dish not only enables the enzyme-free recovery of a cell monolayer for use in the construction of a three-dimensional artificial tissue, but also significantly contributes to the enhancement of GJIC, which may partly promote tissue strength on the surface of the PIPAAm-grafted dish.     

The architecture of variant surface glycoprotein gene expression sites in Trypanosoma brucei

Trypanosoma brucei evades the immune system by switching between Variant Surface Glycoprotein (VSG) genes. The active VSG gene is transcribed in one of approximately 20 telomeric expression sites (ESs). It has been postulated that ES polymorphism plays a role in host adaptation. To gain more insight into ES architecture, we have determined the complete sequence of Bacterial Artificial Chromosomes (BACs) containing DNA from three ESs and their flanking regions. There was variation in the order and number of ES-associated genes (ESAGs). ESAGs 6 and 7, encoding transferrin receptor subunits, are the only ESAGs with functional copies in every ES that has been sequenced until now. A BAC clone containing the VO2 ES sequences comprised approximately half of a 330 kb 'intermediate' chromosome. The extensive similarity between this intermediate chromosome and the left telomere of T. brucei 927 chromosome I, suggests that this previously uncharacterised intermediate size class of chromosomes could have arisen from breakage of megabase chromosomes. Unexpected conservation of sequences, including pseudogenes, indicates that the multiple ESs could have arisen through a relatively recent amplification of a single ES.