Immunology in the Clinic Review Series; focus on host responses: T cell responses to herpes simplex viruses.

Herpes virus infections are chronic and co-exist with acquired immune responses that generally prevent severe damage to the host, while allowing periodic shedding of virus and maintenance of its transmission in the community. Herpes simplex viruses type 1 and 2 (HSV-1, HSV-2) are typical in this regard and are representative of the viral subfamily Alphaherpesvirinae, which has a tropism for neuronal and epithelial cells. This review will emphasize recent progress in decoding the physiologically important CD8(+) and CD4(+) T cell responses to HSV in humans. The expanding data set is discussed in the context of the search for an effective HSV vaccine as therapy for existing infections and to prevent new infections.

Inhibition of cytokine-stimulated thymic lymphocyte proliferation by fatty acids: the role of eicosanoids.

The effect of individual fatty acids on the proliferation of thymic lymphocytes in response to interleukin-1 (IL-1) was investigated. Proliferation was estimated by measuring [3H]thymidine incorporation into the acid insoluble fraction of the thymocytes. A concentration-dependent inhibition (in the range 1-100 microM) in the IL-1-stimulated proliferation was observed with the C20 fatty acids dihomo-gamma-linolenic acid (DGLA), arachidonic acid and eicosapentaenoic acid (EPA). A less pronounced concentration-dependent inhibitory response was observed with the C18 fatty acids linoleic acid, alpha-linolenic acid and gamma-linolenic acid. Palmitic acid and oleic did not have any effect on either basal or IL-1-stimulated proliferation at concentrations up to 100 microM. The potencies of each fatty acid for this effect at a concentration of 100 microM were: arachidonic acid > EPA > or = DGLA > linoleic acid. DGLA, arachidonic acid and EPA also attenuated IL-2-stimulated proliferation. The inhibitory action of these fatty acids was not mediated by conversion to prostaglandins or other eicosanoids as the cyclooxygenase inhibitor, ketoprofen and NDGA did not alter their action. Incubation of thymocytes with radiolabelled DGLA and EPA followed by reverse-phase HPLC analysis revealed that DGLA is predominantly converted to a more polar metabolite which is not PGE1 whereas EPA does not appear to be converted to any other detectable metabolite. The data indicate that the inhibitory actions of fatty acids on cell proliferation do not occur as a result of conversion to other metabolites but may be direct effects. The inhibition of cytokine-stimulated lymphocyte proliferation by unsaturated fatty acids would imply that they may attenuate cell-mediated immune reactions.

Expression of an inducible nitric oxide synthase gene in rainbow trout Oncorhynchus mykiss.

Using oligonucleotide primers based on mammalian nitric oxide synthases (NOS), expression of an inducible NOS (iNOS) gene was detected in head kidney and gill tissue of bacterially-challenged rainbow trout. Three overlapping fragments were amplified by RT-PCR and used to construct a contiguous sequence of 1410bp, with high nucleotide homology to iNOS in birds (61%) and mammals (57-59%). The nucleotide sequence translated in one reading frame to produce a partial peptide containing 470 amino acids, with 69-71% amino acid homology with mammalian iNOS, 81% homology with chicken iNOS and 85% homology with a partial (492bp) goldfish iNOS sequence. In vitro stimulation of head kidney macrophages with LPS also induced expression of the trout iNOS RNA, with optimal expression seen using 20-50 microg/ml LPS at 2h to 6h post-stimulation. The evolutionary and functional significance of the trout iNOS sequence are discussed.

Cloning and sequencing of caspase 6 in rainbow trout, Oncorhynchus mykiss, and analysis of its expression under conditions known to induce apoptosis.

The rainbow trout caspase 6 gene has been cloned and sequenced. The open reading frame consisted of 906bp, which translated into a protein of 302 amino acids, containing the caspase active site pentapeptide (QACRG) and the caspase family signature (HADADCFVCVFLSHG). Amino acids involved in catalysis and those known to form the P1 carbohydrate binding pocket were conserved. Phylogenetic tree analysis showed a tight grouping with other known caspase 6 genes. Conserved aspartic acid residues at positions 33, 191 and 202 suggested that this molecule is produced as a proenzyme that is subsequently cleaved to release active subunits, with the region between Asp-191 and Ala-203 acting as a linker that is cleaved out. RT-PCR analysis revealed that the trout caspase 6 gene was expressed in brain, blood, gill, liver, head kidney and spleen. Addition of LPS or cortisol to head kidney leucocyte cultures had no effect upon caspase 6 expression. However, addition of LPS after preincubation with cortisol increased expression relative to control cultures. Incubation with RU486 abrogated this effect, confirming it was mediated via glucocorticoid receptors. Lastly, a confinement stress in vivo increased caspase 6 expression. The data are discussed with respect to the immunoregulatory role of apoptosis in fish immune responses.

TGF-beta3 exists in bony fish.

A partial nucleotide sequence of transforming growth factor-beta3 (TGF-beta3) has been isolated from the Siberian sturgeon (Acipenser baeri), rainbow trout (Oncorhynchus mykiss) and European eel (Anguilla anguilla), confirming a ubiquitous presence in the ray-finned (Actinopterygian) bony fish. The bony fish TGF-beta3 is highly conserved, with some 83-84% nucleotide identity (coding region) and 90-95% predicted amino acid identity to known homeotherm TGF-beta3's. Far lower homologies are apparent with other known TGF-beta isoforms in fish (e.g. 64-66% and 81-82% amino acid identity to trout TGF-beta 1/5 and carp TGF-beta2 respectively). Phylogenetic tree analysis showed that the fish TGF-beta3's clustered with the known homeotherm TGF-beta3's. The relatively tight clustering of TGF-beta1, TGF-beta2 and TGF-beta3 was in contrast to the TGF-beta5's, which are clearly a more heterogenous group.

Genes for three different isoforms of transforming growth factor-beta are present in plaice (Pleuronectes platessa) DNA.

Although transforming growth factor-beta (TGF-beta) genes have been described in several species of fish, whether an individual fish possesses more than one member of this multigene family has yet to be established. During this study, three DNA fragments were isolated from the plaice (Pleuronectes platessa) by homology cloning. Sequence analysis revealed that each fragment closely resembled a distinct member of the TGF-beta family. Each putative plaice TGF-beta clustered individually with a different TGF-beta subgroup during phylogenetic analysis suggesting that these may be the plaice homologues of vertebrate TGF-beta 1/4/5, -beta 2 or -beta 3. The first direct evidence for the presence of multiple TGF-beta genes in a single fish species is presented.

A partial sequence for nitric oxide synthase from a goldfish (Carassius auratus) macrophage cell line.

Expression of inducible nitric oxide synthase (iNOS) mRNA was detected in a recently developed goldfish macrophage cell line by RT-PCR, using degenerate primers designed against conserved nucleotide motifs within the different mammalian isoforms of NOS. Increased expression of iNOS poststimulation with LPS was found, and suggests that it is a functional enzyme in goldfish macrophages, supporting the view that iNOS regulation is pretranslational. The nucleotide sequence translated in one reading frame with no stop codons to produce a partial peptide containing 164 amino acids, with highest homology (85%) to a recently identified rainbow trout iNOS sequence. The peptide translation also gave an insight into the conservation of binding motifs, since two cofactor binding sites were present in the amplified PCR product (FMN and calmodulin). In addition, a 42 aa motif present in the region just upstream of the FMN binding motif of mammalian endothelial and neuronal NOS isoforms was absent in the translation, in agreement with every published sequence for iNOS. Finally, the translation was used to construct an unrooted phylogenetic tree.

Isolation of the first piscine transforming growth factor beta gene: analysis reveals tissue specific expression and a potential regulatory sequence in rainbow trout (Oncorhynchus mykiss).

The nucleotide sequence of a rainbow trout transforming growth factor beta (TGF-beta) peptide is presented, which translates into a 382 amino acid precursor molecule containing a 20 amino acid leader and a mature peptide of 112 amino acids. The mature peptide has nine conserved cysteines and a conserved proline (position 36) and glycine (position 46), all characteristics of TGF-beta superfamily molecules. Within the precursor region are three glycosylation sites, two in common with known TGF-beta s, an integrin binding site (RGD) and the tetrabasic peptide cleavage site (RKKR). The full 3' untranslated region (UTR) consists of 542 nucleotides with a polyadenylation signal 16 nucleotides upstream of the poly(A) tail. The 5' UTR contains an open reading frame with the potential to encode an eleven amino acid peptide, which may have significance for regulation of TGF-beta translation. A wide tissue distribution of TGF-beta message was detected by RT-PCR; in blood leukocytes, kidney macrophages, brain, gill, and spleen tissue but not liver. A phylogenetic tree reveals the trout TGF-beta sequence is most related to xenopus TGF-beta 5, with these sequences and that of chicken TGF-beta 4 grouping with mammalian TGF-beta 1 s. The impact of the trout sequence on current theories of TGF-beta isotype evolution is discussed.

Cloning and expression analysis of rainbow trout Oncorhynchus mykiss tumour necrosis factor-alpha.

A rainbow trout (Oncorhynchus mykiss) gene for tumor necrosis factor (TNF) has been cloned and sequenced. The cDNA contains an open reading frame of 738 nucleotides that translate into a 246 amino-acid putative peptide, with a 5' untranslated region (UTR) of 140 bp and a 3' UTR of 506 bp. Two potential N-linked glycosylation sites exist in the translation. The genomic sequence measures 2007 bp and contains three introns that intercept four coding exons. Expression studies using RT-PCR have shown that the trout TNF gene is constitutively expressed in the gill and kidney of unstimulated fish. Trout TNF expression could be up-regulated by stimulation of isolated head kidney leucocytes with lipopolysaccharide (LPS). Similarly, stimulation of a trout macrophage cell line (RTS11) with LPS resulted in an increased transcript level, as did incubation with recombinant trout interleukin (IL)-1 beta. The optimal timing for induction of TNF expression in trout macrophages was determined using recombinant trout IL-1 beta, where a clear induction was apparent by 2 h and peaked at 4 h. Evidence that this TNF gene is equivalent to mammalian TNF-alpha is discussed.