Strongyloides ratti infection induces transient nematode-specific Th2 response and reciprocal suppression of IFN-gamma production in mice.

Over one-third of the world population is infected with parasitic helminths, Strongyloides ssp. accounting for approximately 30-100 million infected people. In this study, we employ the experimental system of murine Strongyloides ratti infection to investigate the interaction of this pathogenic nematode with its mammalian host. We provide a comprehensive kinetic description of the immune response to S. ratti infection that was reflected by induction of antigen-specific IgM and IgG1, mast cell activation and a Th2-like cytokine response. T cells derived from infected mice displayed an increased IL-3, IL-4, IL-5, IL-13 and IL-10 response to CD3-engagement in comparison with T cells derived from naïve mice. The IFN-gamma response to CD3-engagement that was well detectable in T cells derived from naïve mice, however, was suppressed in T cells derived from infected mice. Both, the induction of the S. ratti-specific Th2 response and the suppression of pro-inflammatory cytokines were transient and observed in strict correlation to the course of infection and the number of infective larvae used. Finally, comparing artificial infections induced by subcutaneous injection of larvae to natural infections, we observed similar antigen-specific T cell responses although the natural infection led to a significantly lower worm burden.

Rat sperm acrosin: cDNA sequence, derived primary structure and phylogenetic origin.

Rat preproacrosin primary structure as predicted from a 1431 nucleotide (nt) cDNA indicates that the molecule is synthesized as a preproenzym consisting of a putative 19 amino acid signal sequence, a 23 amino acid light chain and finally a 395 amino acid heavy chain. Functional domains like the catalytic triad (His-70, Asp-124, Ser-222) are highly conserved not only between the available acrosin primary structures of different mammals but also in comparison with other serine proteinases. Number of amino acid exchanges and the degree in amino acid identity between the different serine proteinases and rat acrosin leads to the assumption that acrosin is one of the early descendants within the phylogenetic tree of the serine proteinase superfamily.

Genetic diversity and recombination of murine noroviruses in immunocompromised mice.

Murine noroviruses (MNV) are newly identified pathogens which infect laboratory mice. In this study, we found a high prevalence (64.3%) of MNV in various breeding colonies of immunocompromised, transgenic and wild-type mouse lines. All mice survived infection with no signs of clinical disease. Faeces samples were collected from animals housed in two separate laboratory mouse colonies in Berlin, Germany, and screened using quantitative reverse transcription (RT)-PCR. We have determined the complete nucleotide sequences of 3 novel MNV strains. Furthermore, we sequenced two subgenomic regions within open reading frames (ORFs) 1 and 2 that are suitable for genotyping. Sequence analysis of the full-length and partial genomes obtained from naturally infected mice yielded valuable data on genetic diversity of murine noroviruses. The discordance of genotype affiliation of some MNVs shown in ORF1 and ORF2 suggests intertypic recombination events in vivo.

Endothelial cells are involved in the vasodilatory response to hypoxia.

The role of endothelial cells in the dilatory response of arteries to hypoxia was studied in vitro using perfused arterial segments of rat and dog. The pO2 of the intra- and extraluminal perfusate could be varied separately. Intraluminal hypoxia (pO2 of 40 mmHg) induced a dilation irrespective of extraluminal pO2 level. On the contrary extraluminal hypoxia could not elicit a dilation during intraluminal normoxic perfusion. Dilation during extraluminal hypoxia could only be induced if the segment was not intraluminally perfused. The dilatory response to intraluminal hypoxia was abolished after enzymatical or mechanical removal of the endothelium. While theophylline and lipoxygenase inhibitors did not influence this endothelium-induced dilation, a significant reduction of the response could be observed after incubation with indomethacin. These results support the concept that prostacyclin (PGI2) might be involved in the hypoxic endothelium-induced dilation.

Acrosin, the peculiar sperm-specific serine protease.

The sperm enzyme acrosin has long been known as one of the key enzymes in the mammalian fertilization process. Elucidation of primary structures of preproacrosin from various species have allowed a deeper insight into the structural organization and the complex evolution of the sperm proteinase acrosin. In addition to the typical elements of serine proteases, the acrosin molecule possesses one novel domain that might convey DNA-binding properties.

Mouse preproacrosin: cDNA sequence, primary structure and postmeiotic expression in spermatogenesis.

The primary structure of mouse preproacrosin was deduced by nucleotide sequencing of cDNA clones isolated from a mouse testis cDNA library. The largest cDNA, with 1373 bp, consists of a 11-bp 5'untranslated sequence, a 1254-bp open reading frame terminated by a TGA triplet and a 105-bp 3' untranslated end, including one potential polyadenylation signal. The NH2-terminus of the polypeptide contains a hydrophobic 15-amino acid signal peptide. This cleavable signal sequence is followed by 403 amino acids, representing the acrosin light and the heavy chain of 23 and 380 amino acid residues, respectively. The proteolytic active site segments His, Asp and Ser are part of the heavy chain, as well as a proline-rich COOH-terminus, which is not present in any other serine proteinase studied so far. Furthermore the postmeiotic expression of the preproacrosin gene during mouse spermatogenesis was studied.

Genes from two intermoult puffs in Drosophila virilis polytene chromosomes are differentially transcribed during larval development.

Genes from two Drosophila virilis intermoult puffs were isolated by microcloning. From puff 16A on the X-chromosome a 2.9 kb DNA fragment was obtained, which hybridizes with three transcripts. Two of them represent the mRNAs for larval glue proteins. They are found in different abundancies in third larval instar salivary glands, but also in minor amounts in midgut and in fat body. In puff 55E on chromosome III two genes were identified. They are transcribed exclusively in salivary glands during all three larval instars. Therefore, their products must be related to another gland-specific function, which is sustained throughout larval life.

Molecular cloning of human preproacrosin cDNA.

Complementary DNA-clones for human preproacrosin have been isolated from a human testis cDNA library in lambda gt11. The nucleotide sequence of the 1402 bp cDNA insert includes a 20 bp 5' noncoding region, an open reading frame of 1263 bp corresponding to 421 amino acids (45.9 kdalton), and a 105 bp 3' untranslated region. The deduced amino acid sequence is compared with that recently evaluated from a cDNA clone for boar preproacrosin. The sequence identity is 70%; the leader sequence, the catalytic triad (His, Asp, Ser; which is characteristic for serine proteinases) and the positions of the cysteine residues crosslinking the light and the heavy chain of the active enzyme, acrosin, are conserved in both species. At the C-terminal end, a proline-rich sequence is present in both species; this may represent the species-specificity of acrosin.

Nucleotide sequences and expression of cDNA clones for boar and bull transition protein 1 and its evolutionary conservation in mammals.

During spermatogenesis, the nucleoproteins undergo several dramatic changes as the germinal cells differentiate to produce the mature sperm. With nuclear elongation and condensation, the histones are replaced by basic spermatidal transition proteins, which are themselves subsequently replaced by protamines. We have isolated cDNA clones for one of the transition proteins, namely for TP1, of bull and boar. It turned out that TP1 is a small, but very basic protein with 54 amino acids (21% arginine, 19% lysine) and is highly conserved during mammalian evolution at the nucleotide as well as at the amino-acid level. Gene expression is restricted to the mammalian testis, and the message first appears in round spermatids. Thus production of TP1 is an example of haploid gene expression in mammals. The size of the mRNA for TP1 was found to be identical in 11 different mammalian species at around 600 bp. Hybridization experiments were done with cDNAs from boar and bull, respectively. The positive results in all mammalian species give further evidence for the conservation of the TP1 gene during mammalian evolution and its functional importance in spermatid differentiation.

Nucleotide sequence of a cDNA encoding rat protamine and the haploid expression of the gene during rat spermatogenesis.

The nucleotide sequence of a 342-base cDNA encoding the rat protamine has been determined. This insert, isolated from a rat testis cDNA library, encodes a polypeptide of 50 amino acids of which 29 are arginine 9 are cysteine and 2 are tyrosine. The insert contains the complete 3'-noncoding region of 170 bases and 18 bases of the 5'-noncoding region. Hybridization of the protamine cDNA with the RNA prepared from testes of prepubertal and sexually mature rats revealed that protamine mRNA is first detectable as a 600 nucleotide long molecule in the 35-day old testis containing around 15% of round spermatids but not in testis of younger animals. The RNA of 50-day old and sexually mature rats was found to contain a second protamine mRNA which is around 500 nucleotides in length. Hybridization of the protamine cDNA with the RNA of isolated spermatids of the mature testis resulted in 2 prominent hybridization signals (600 and 500 bp) while the faint signal obtained with the RNA of pachytene spermatocytes (600 bp) was found to be due to contamination of the cell preparation by spermatids. After digestion of the mRNAs with ribonuclease H a single hybridization band even smaller than 500 nucleotides was obtained. As demonstrated on testis sections the transcripts are confined to the central layers of the tubuli seminiferi corresponding to the spatial arrangement of corresponding to the spatial arrangement of postmeiotic cells. The results indicate that the protamine gene in the rat is postmeiotically expressed and that the mRNA undergoes post-transcriptional processing that includes a reduction in molecular size with respect to the poly-(A)+ tail.

The lack of protamine 2 (P2) in boar and bull spermatozoa is due to mutations within the P2 gene.

The nuclei of spermatozoa in all mammals examined so far contain P1 protamine. A second protamine variant, protamine P2, has to date been isolated only from human and murine spermatozoa where it represents the major fraction of basic nuclear protein. In order to elucidate the reason for this unusual distribution of the protamine variants among mammals we have investigated the expression of protamine P2 in boar and bull. It can be shown that also in these species protamine 2 is transcribed and translated on low levels. Various mutational events though have altered the primary structure of the protein: In boar, a deletion of 8 aminoacids has removed a sequence motif from the amino-terminus of the molecule, which highly probable is of functional relevance. The bovine sequence, as a consequence of numerous point mutations has accumulated neutral and hydrophobic aminoacids which reduce the affinity of the protamine 2 to DNA.

Altered intestinal immune system but normal antibacterial resistance in the absence of P-selectin and ICAM-1.

ICAM-1 and P-selectin are adhesion molecules that regulate leukocyte migration, extravasation to inflammatory sites, and other immune cell interactions. T cell-mediated resistance against acute infection with Listeria monocytogenes and chronic infection with Mycobacterium bovis Calmette-Guérin bacillus was investigated in mutant mice lacking P-selectin and/or ICAM-1. Mice deficient in P-selectin (Psel-/-), ICAM-1 (ICAM-/-), or the combination of both (Psel-/- x ICAM-/-) showed normal bacterial clearance, comparable delayed-type hypersensitivity reactions, and equivalent memory T cell responses. Additionally, the distribution of alpahbeta vs gammadelta T lymphocyte populations was examined. Normal lymphocyte distributions were noted in thymus, spleen, and blood, whereas mutant mice showed marked alterations in the intestinal intraepithelial (i-IEL) and lamina propria lymphocytes. Differences in i-IEL populations were reflected functionally by differential lytic activities and cytokine productions of i-IEL populations from mutant mice. Despite these changes within the mucosal immune system of mutant mice, their resistance against oral infection with L. monocytogenes was apparently unimpaired. These findings demonstrate that P-selectin and ICAM-1 are critically involved in the shaping of lymphocyte populations of the gut but have only a minor influence on systemic and regional host defense against intracellular bacteria.

Requirement of NF-kappaB/Rel for the development of hair follicles and other epidermal appendices.

NF-kappaB/Rel transcription factors and IkappaB kinases (IKK) are essential for inflammation and immune responses, but also for bone-morphogenesis, skin proliferation and differentiation. Determining their other functions has previously been impossible, owing to embryonic lethality of NF-kappaB/Rel or IKK-deficient animals. Using a gene targeting approach we have ubiquitously expressed an NF-kappaB super-repressor to investigate NF-kappaB functions in the adult. Mice with suppressed NF-kappaB revealed defective early morphogenesis of hair follicles, exocrine glands and teeth, identical to Eda (tabby) and Edar (downless) mutant mice. These affected epithelial appendices normally display high NF-kappaB activity, suppression of which resulted in increased apoptosis, indicating that NF-kappaB acts as a survival factor downstream of the tumor necrosis factor receptor family member EDAR. Furthermore, NF-kappaB is required for peripheral lymph node formation and macrophage function.

Abdominal segmentation of the Drosophila embryo requires a hormone receptor-like protein encoded by the gap gene knirps.

The body pattern along the anterior-posterior axis of the insect embryo is thought to be established by two organizing centres localized at the ends of the egg. Genetic analysis of the polarity-organizing centres in Drosophila has identified three distinct classes of maternal effect genes that organize the anterior, posterior and terminal pattern elements of the embryo. The factors provided by these gene classes specify the patterns of expression of the segmentation genes at defined positions along the longitudinal axis of the embryo. The system responsible for organizing the posterior segment pattern is a group of at least seven maternal genes and the zygotic gap gene knirps (kni). Their mutant phenotype has adjacent segments in the abdominal region of the embryo deleted. Genetic analysis and cytoplasmic transplantation experiments suggested that these maternal genes are required to generate a 'posterior activity' that is thought to activate the expression of kni (reviewed in ref. 2). The molecular nature of the members of the posterior group is still unknown. Here we report the molecular characterization of the kni gene that codes for a member of the steroid/thyroid receptor superfamily of proteins which in vertebrates act as ligand-dependent DNA-binding transcription regulators.

Molecular cloning of preproacrosin and analysis of its expression pattern in spermatogenesis.

Complementary DNA clones for the boar preproacrosin have been isolated from a randomly primed testis cDNA library in lambda gt10 and from an oligo(dT)-primed testis cDNA in lambda gt11. The nucleotide sequence of the 1418-bp cDNA insert includes a 46-bp 5'-untranslated region, an open reading frame of 1248 bp corresponding to 416 amino acids (45.59 kDa) and a 121-bp 3'-untranslated region. The deduced amino acid sequence includes the active-site residues histidine, asparagine and serine of the catalytic triad of the serine proteinase super-family and is colinear with that determined by amino acid sequencing of the boar acrosin light chain and of a small region of the NH2-terminal sequence of the heavy chain. The preproacrosin cDNA contains at the 3' end a 381-bp sequence which codes for an amino acid sequence not yet found in any other serine proteinase. This amino acid sequence is rich in proline (42 out of 127 amino acids) and is suggested to be involved in the recognition and binding of the spermatozoa to the zona pellucida of the ovum. The mRNA for preproacrosin is synthesized as an approximately 1.6-kb-long molecule only in the postmeiotic stages of boar and bull spermatogenesis.

Autoimmune intestinal pathology induced by hsp60-specific CD8 T cells.

Due to their ubiquitous distribution and high degree of structural similarity, heat shock proteins (hsp) are potential target antigens in autoimmune diseases. Here, we describe induction of intestinal inflammation following transfer of hsp60-reactive CD8 T cells into mice. Inflammatory reactions were MHC class I dependent and developed primarily in the small intestine. IFN gamma and TNF alpha, as well as gut-derived hsp60, were elevated at sites of T cell infiltration. Intestinal lesions were drastically reduced in mice lacking receptors for TNF alpha. Pathology also developed in germ-free mice, indicating recognition of host-derived hsp60 by CD8 T cells. This report demonstrates that CD8 T cells with defined antigen specificity cause intestinal inflammation, emphasizing a link between infection and autoimmune disease.