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PubMed1995

Autogenous intrasynovial and extrasynovial tendon grafts: an experimental study of pro alpha 1(I) collagen mRNA expression in dogs.

On the basis of recent evidence that the healing processes of tendon grafts are donor-tissue specific, in situ hybridization, using a 372 bp cDNA fragment complementary to a portion of pro alpha 1(I) collagen mRNA, was utilized to compare the cellular responses to transplantation exhibited by autogenous intrasynovial and extrasynovial flexor tendon grafts. Intrasynovial and extrasynovial tendons from the hindpaw were transferred to synovial sheaths in the forepaw of 12 mongrel dogs (24 tendons) and treated with immediate controlled passive motion. The tendon grafts were harvested at 2, 4, and 6 weeks, and each was divided into a proximal, central (8 mm), and distal portion. Sections from the central portion were embedded in paraffin and subjected to in situ hybridization, autoradiography, and staining; levels of procollagen mRNA then were assessed by microscopic examination. The two types of tendon grafts exhibited different levels of pro alpha 1(I) collagen mRNA expression at all three time points. Intrasynovial tendon grafts displayed no areas of increased type-I procollagen mRNA at 2, 4, and 6 weeks. The extrasynovial tendon grafts displayed increased surface levels of type-I procollagen mRNA at 2 and 4 weeks; the levels decreased to background levels by 6 weeks. The high levels of procollagen mRNA exhibited by the extrasynovial grafts suggest increased collagen synthetic activity, indicative of a cellular response to injury, whereas the preservation of low levels of expression in the intrasynovial grafts may signify a less inflammatory cellular response.

Animals↗

PubMed2007

Systemic LPS causes chronic neuroinflammation and progressive neurodegeneration.

Inflammation is implicated in the progressive nature of neurodegenerative diseases, such as Parkinson's disease, but the mechanisms are poorly understood. A single systemic lipopolysaccharide (LPS, 5 mg/kg, i.p.) or tumor necrosis factor alpha (TNFalpha, 0.25 mg/kg, i.p.) injection was administered in adult wild-type mice and in mice lacking TNFalpha receptors (TNF R1/R2(-/-)) to discern the mechanisms of inflammation transfer from the periphery to the brain and the neurodegenerative consequences. Systemic LPS administration resulted in rapid brain TNFalpha increase that remained elevated for 10 months, while peripheral TNFalpha (serum and liver) had subsided by 9 h (serum) and 1 week (liver). Systemic TNFalpha and LPS administration activated microglia and increased expression of brain pro-inflammatory factors (i.e., TNFalpha, MCP-1, IL-1beta, and NF-kappaB p65) in wild-type mice, but not in TNF R1/R2(-/-) mice. Further, LPS reduced the number of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra (SN) by 23% at 7-months post-treatment, which progressed to 47% at 10 months. Together, these data demonstrate that through TNFalpha, peripheral inflammation in adult animals can: (1) activate brain microglia to produce chronically elevated pro-inflammatory factors; (2) induce delayed and progressive loss of DA neurons in the SN. These findings provide valuable insight into the potential pathogenesis and self-propelling nature of Parkinson's disease.

Animals↗

PubMed1998

The expression of cytokine mRNA in the cauda equina of Lewis rats with experimental allergic neuritis.

Autoreactive CD4+ T cells can transfer experimental allergic neuritis (EAN) to naive recipients. In order to further analyze the role of these T cells and their corresponding cytokines in EAN, we studied the expression of mRNA for IFN-gamma, IL-4, and IL-10 in the cauda equina of rats with EAN using a quantitative competitive reverse transcriptase PCR method. Nerves were studied on days 0 (pre-immunization), 10 (disease onset), 13 (clinical progression), 16 (disease peak), as well as 20, 24, and 34 post immunization (recovery). IFN-gamma messages increased at disease onset and peaked at day 13 p.i. IL-10 message remained at a very low level at disease onset and surged at day 16. Both messages were low in recovery stage. IL-4 message was undetectable at any time point. These data suggest a pro-inflammatory role of IFN-gamma and anti-inflammatory role of IL-10 in EAN lesions. It is also possible that a clonal switch from Th1 to Th2 occurs in EAN lesions during the disease course.

Actins↗

PubMed1997

Studies on prolysostaphin processing and characterization of the lysostaphin immunity factor (Lif) of Staphylococcus simulans biovar staphylolyticus.

Lysostaphin is an extracellular glycylglycine endopeptidase produced by Staphylococcus simulans biovar staphylolyticus ATCC1362 that lyses staphylococcal cells by hydrolysing the polyglycine interpeptide bridges of the peptidoglycan. Renewed analysis of the sequence of the lysostaphin gene (lss), and the sequencing of the amino-terminus of purified prolysostaphin and of mature lysostaphin revealed that lysostaphin is organized as a preproprotein of 493 amino acids (aa), with a signal peptide consisting of 36 aa, a propeptide of 211 aa from which 195 aa are organized in 15 tandem repeats of 13 aa length, and a mature protein of 246 aa. Prolysostaphin is processed in the culture supernatant of S. simulans biovar staphylolyticus by an extracellular cysteine protease. Although prolysostaphin was staphylolytically active, the mature lysostaphin was about 4.5-fold more active. The controlled expression in Staphylococcus carnosus of lss and lss with deletions in the prepropeptide region indicated that the tandem repeats of the propeptide are not necessary for protein export or activation of Lss, but keep Lss in a less active state. Intracellularly expressed pro- and mature lysostaphin exert staphylolytic activity in cell-free extracts, but do not affect growth of the corresponding clones. We characterized a lysostaphin immunity factor gene (lif) which is located in the opposite direction to lss. The expression of lif in S. carnosus led to an increase in the serine/glycine ratio of the interpeptide bridges of peptidoglycan from 2 to 35%, suggesting that lysostaphin immunity depends on serine incorporation into the interpeptide bridge. If, in addition to lif, lss is co-expressed the serine/glycine ratio is further increased to 58%, suggesting that Lss selects for optimal serine incorporation. Lif shows similarity to FemA and FemB proteins, which are involved in the biosynthesis of the glycine interpeptide bridge of staphylococcal peptidoglycan. In contrast to that of Lif, the production of FemA and FemB in S. carnosus does not cause lysostaphin immunity. The putative tRNASer gene located downstream of lss had no recognizable influence on lysostaphin immunity. lss and lif are flanked by insertion sequences, suggesting that S. simulans biovar staphylolyticus received lif and lss by horizontal gene transfer.

Amino Acid Sequence↗

PubMed1991

Mitochondrial mutations restricting spontaneous translational frameshift suppression in the yeast Saccharomyces cerevisiae.

The +1 frameshift mutation, M5631, which is located in the gene (oxi1) for cytochrome c oxidase II (COXII) of the yeast mitochondrial genome, is suppressed spontaneously to a remarkably high extent (20%-30%). The full-length wild-type COXII produced as a result of suppression allows the mutant strain to grow with a "leaky" phenotype on non-fermentable medium. In order to elucidate the factors and interactions involved in this translational suppression, the strain with the frameshift mutation was mutated by MnCl2 treatment and a large number of mutants showing restriction of the suppression were isolated. Of 20 mutants exhibiting a strong, restricted, respiration-deficient (RD) phenotype, 6 were identified as having mutations in the mitochondrial genome. Furthermore, genetic analyses mapped one mutation to the vicinity of the gene for tRNA(Pro) and two others to a region of the tRNA cluster where two-thirds of all mitochondrial tRNA genes are encoded. The degree of restriction of the spontaneous frameshift suppression was characterized at the translational level by in vivo 35S-labeling of the mitochondrial translational products and immunoblotting. These results showed that in some of these mutant strains the frameshift suppression product is synthesized to the same extent as in the leaky parent strain. It is suggested that more than one +1 frame-shifted product is made as a result of suppression in these strains: one is as functional as the wild-type COXII, the other(s) is (are) nonfunctional and prevent leaky growth on non-fermentable medium. A possible mechanism for this heterogenous frameshift suppression is discussed.

Amino Acid Sequence↗

PubMed2004

SsrA tagging of Escherichia coli SecM at its translation arrest sequence.

SecM is expressed from the secM-secA operon and activates the expression of secA in response to secretion defects. The 3'-end of secM encodes an "arrest sequence," which can interact with the ribosomal exit tunnel, preventing complete secM translation under secretion-defective conditions. In a cis-acting manner, ribosome stalling enhances secA translation. Pro(166) is the last residue incorporated when SecM elongation is arrested. We report that secretion deficiencies lead to SsrA tagging of SecM after Pro(166), Gly(165), and likely Arg(163). Northern blot analysis revealed the presence of a truncated secM transcript, likely issued from a secM-secA cleavage. The level of secM transcripts was decreased either when secM translation was totally prevented or when Pro(166) was mutated. However, the accumulation of a truncated secM transcript required secM translation and was prevented when the SecM arrest sequence was inactivated by a point mutation changing Pro(166) to Ala. We suggest that ribosome pausing at the site encoding the arrest sequence is required for formation of the truncated secM mRNA. SsrA tagging affected neither the presence of the secM mRNA nor secA expression, even under translocation-defective conditions. It is therefore likely that SsrA tagging of SecM occurs only after cleavage of secM-secA mRNA within the secM open reading frame encoding the SecM arrest sequence. Accumulation of transcripts expressing arrested SecM generated growth inhibition that was alleviated by the SsrA tagging system. Therefore, SsrA tagging of SecM would rescue ribosomes to avoid excessive jamming of the translation apparatus on stop-less secM mRNA.

Adenosine Triphosphatases↗

PubMed2002

Electroporation-mediated pain-killer gene therapy for mononeuropathic rats.

The relatively low expression levels achieved from transferred genes have limited the application of nonviral vectors for gene transfer into the spinal cord in vivo. Thus, the aim of this study was to evaluate the efficacy of electroporation-mediated pro-opiomelanocortin (POMC) gene therapy for neuropathic pain using an animal model of chronic constrictive injury (CCI). Firstly, the optimal pulse characteristics (voltage, pulse duration, number of shocks) were investigated for in vivo electroporation-mediated gene transfer into the spinal cord. The electroporation process makes use of plasmid DNA, which expresses the POMC gene. Expression levels were evaluated in this study by Western blot. We conclude that the optimal conditions for electroporation are a pulse voltage of 200 V, 75-ms duration, 925-ms interval, for five iterations. Secondly, electroporation treatment for neuropathic pain was attempted on CCI rats using plasmid DNA that expresses the POMC gene. Intrathecal administrations of the POMC vector elevated spinal beta-endorphin levels, as manifested in a significantly elevated pain threshold for the CCI limbs. This result suggests that gene therapy for neuropathic pain using this novel technique is very efficacious, and thus shows promise for further clinical trials.

Analysis of Variance↗

PubMed2000

The trans-spliceosomal U4 RNA from the monogenetic trypanosomatid Leptomonas collosoma. Cloning and identification of a transcribed trna-like element that controls its expression.

U4 small nuclear RNA is essential for trans-splicing. Here we report the cloning of U4 snRNA gene from Leptomonas collosoma and analysis of elements controlling its expression. The trypanosome U4 RNA is the smallest known, it carries an Sm-like site, and has the potential for extensive intermolecular base pairing with the U6 RNA. Sequence analysis of the U4 locus indicates the presence of a tRNA-like element 86 base pairs upstream of the gene that is divergently transcribed to yield a stable small tRNA-like RNA. Two additional tRNA genes, tRNA(Pro) and tRNA(Gly), were found upstream of this element. By stable expression of a tagged U4 RNA, we demonstrate that the tRNA-like gene, but not the upstream tRNA genes, is essential for U4 expression and that the B box but not the A Box of the tRNA-like gene is crucial for expression in vivo. Mapping the 2'-O-methyl groups on U4 and U6 small nuclear RNAs suggests the presence of modifications in canonical positions. However, the number of modified nucleotides is fewer than in mammalian homologues. The U4 genomic organization including both tRNA-like and tRNA genes may represent a relic whereby trypanosomatids "hired" tRNA genes to provide extragenic promoter elements. The close proximity of tRNA genes to the tRNA-like molecule in the U4 locus further suggests that the tRNA-like gene may have evolved from a tRNA member of this cluster.

Animals↗

PubMed2000

The effects of acute and chronic stresses on vasotocin gene transcripts in the brain of the rainbow trout (Oncorhynchus mykiss).

Secretion of adrenocorticotropic hormone (ACTH) from the fish pituitary, which occurs in times of stress, is stimulated by several hypothalamic neuropeptides, one of which is arginine vasotocin (AVT). This study investigates whether gene expression for AVT is up-regulated during acute or chronic stress. Rainbow trout (Oncorhynchus mykiss) were subjected to one of two forms of acute stress-either 2 h confinement followed by 2 h recovery, or capture and transfer to low water for 2 min followed by 4 h recovery in their home tank before autopsy. In other experiments, these stresses were repeated daily for 5 or 6 days (chronic stress). Quantification of AVT transcript prevalence in the parvocellular and magnocellular neurones of the preoptic nucleus after in situ hybridization was used as a monitor of the AVT gene response to stress. The results showed that acute confinement, but apparently not brief low-water stress, significantly increased AVT transcript prevalence in a group of parvocellular perikarya. When applied repeatedly, both forms of stress caused habituation, such that the AVT hybridization signal remained at control or even lower levels despite elevated pro-opiomelanocortin transcripts in the corticotropes and raised plasma cortisol concentrations. The AVT hybridization signal in the magnocellular perikarya showed no significant response to either acute or chronic stress. The results support the idea that these parvocellular AVT neurones are involved in ACTH stimulation during acute stress, and that the system habituates to chronic stresses.

Animals↗

PubMed1995

Functional expression of the human insulin gene in a human hepatoma cell line (HEP G2).

To develop a model somatic gene therapy system for diabetes, a human hepatoma cell line (HEP G2) was transfected with a mammalian expression vector carrying the full-length human insulin cDNA. More proinsulin than insulin was released daily by the stably transformed cell line (HEP G2ins). However, on acute stimulation with 5mM 8-Br-cAMP and 10mM theophylline the HEP G2ins cells released predominantly insulin into the medium. The cells did not secrete insulin in response to glucose. Examination of acid-ethanol extracts confirmed insulin was preferentially being stored. Immunohistochemical analysis of the cells also showed (pro)insulin was being stored. Electron microscopy revealed large membrane-bound vacuoles, containing electron-dense material, which were not seen in control cells. Glucokinase activity and albumin secretion of the transfectants were unaltered from the controls. Five-minute pulse-chase labelling of the HEP G2ins cells with 3H-leucine confirmed insulin synthesis in the presence of 20mM glucose and 5mM 8-Br-cAMP. A dose-response curve for insulin synthesis was also generated to increasing concentrations of glucose with a half Vmax of 4.9mM. Our results show that the introduction of insulin cDNA into a human hepatoma cell line results in synthesis, storage and acute regulated insulin release and lend credence to the possibility of engineering a liver cell to secrete insulin acutely in response to physiological stimuli.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2004

UV-induced expression of key component of the tanning process, the POMC and MC1R genes, is dependent on the p-38-activated upstream stimulating factor-1 (USF-1).

Protection against UV-mediated DNA damage and the onset of oncogenesis is afforded by the tanning response in which UV irradiation triggers melanocytes to increase production of melanin that is then transferred to keratinocytes. A key component of the tanning process is the UV-mediated induction of the pro-opiomelanocortin (POMC) and MC1R genes encoding the alpha-melanocyte-stimulating hormone and its receptor, respectively, which play a crucial role in pigmentation by regulating the intracellular levels of cAMP. How these genes are regulated in response to UV irradiation is not known. Here we have shown that UV-induced activation of the POMC and MC1R promoters is mediated by p38 stress-activated kinase signaling to the transcription factor, upstream stimulating factor-1 (USF-1). Importantly, melanocytes derived from USF-1 -/- mice exhibit a defective UV response and fail to activate POMC and MC1R expression in response to UV irradiation. The results define USF-1 as a critical UV-responsive activator of genes implicated in protection from solar radiation.

Amino Acid Motifs↗

PubMed1989

Evolution of a tRNA operon in gamma purple bacteria.

Genomic DNA from eubacteria belonging to the gamma-3 subdivision of purple bacteria, as classified by Woese (C.R. Woese, Microbiol. Rev. 51:221-271, 1987), were probed with the argT operon of Escherichia coli encoding 5'-tRNA(Arg)-tRNA(His)-tRNA(Leu)-tRNA(Pro)-3'. The homologous operon from Vibrio harveyi was isolated and sequenced. Comparison of the five available sequences of this tRNA cluster from members of the families Enterobacteriaceae, Aeromonadaceae, and Vibrionaceae led to the conclusion that variations in different versions of this operon arose not only by point mutations but also by duplication and addition-deletion of entire tRNA genes. This data base permitted the formulation of a proposal dealing with the evolutionary history of this operon and suggested that DNA regions containing tRNA genes are active centers (hot spots) of recombination. Finally, since the operon from V. harveyi was not highly repetitive and did not contain tRNA pseudogenes, as in the Photobacterium phosphoreum operon, hybridization of genomic DNAs from different photobacterial strains with probes specific for the repeated pseudogene element was performed. We conclude that the phylogenetic distribution of the repetitive DNA is restricted to strains of P. phosphoreum.

Base Sequence↗

PubMed1983

Crosslinking of Escherichia coli 50S ribosomal subunits with chlorambucilyl oligoprolyl phenylalanyl-tRNA molecular rulers.

A series of P-site probes, chlorambucilyl-(Pro)n-Phe-tRNAPhe, were prepared and reacted with poly(U)-directed Escherichia coli MRE 600 ribosomes. Upon binding of the probes to ribosomes, 90% of the cpm bound were not released following subsequent interaction with puromycin. In the absence of poly(U) or in the presence of poly(C), binding was limited to the amount of cpm bound if ribosomes were incubated in the presence of puromycin before adding modified tRNA and poly(U). AcPhe-tRNAPhe was a competitive inhibitor of chlorambucilyl Phe-tRNAPhe. Binding to 50S subunits was strongly stimulated by poly(U), while binding to 30S subunits was not. Crosslinked 50S proteins were analyzed by two-dimensional gel electrophoresis. Crosslinking with molecular rulers containing zero prolines led to poly(U)-dependent labeling of L1 and L27. With rulers containing five prolines, L6, L25, L28, and the group L18,23,24 were labeled. Analysis of crosslinked ribosomal RNA on sucrose density gradients revealed almost no cpm in the 16S or 23S peaks, but only in the 5S peaks. This was observed with molecular rulers containing either zero or five proline residues.

Binding, Competitive↗

PubMed2003

Analysis of the candidate 8p21 tumour suppressor, BNIP3L, in breast and ovarian cancer.

Loss of heterozygosity (LOH) on the short arm of chromosome 8, at 8p12-p23, is one of the most frequent genetic events in both breast and ovarian cancer, suggesting the location of a shared tumour suppressor gene. Microcell-mediated chromosome transfer of chromosome 8 suppresses tumorigenicity and growth of colorectal and prostate cancer cell lines, further supporting the presence of a tumour suppressor gene on 8p. We have taken a candidate gene approach to try to identify this tumour suppressor gene at 8p12-p23. BNIP3L, which has sequence homology to pro-apoptotic proteins and the ability to suppress colony formation in soft agar, is located at 8p21, within a region of ovarian cancer LOH, breast cancer LOH and prostate cancer metastasis suppression. BNIP3L expression was assessed by both RT-PCR and Northern blot analysis in breast and ovarian cancer cell lines and found to be expressed at similar levels relative to expression in their respective normal epithelial cell lines. Genetic analysis of BNIP3L in 40 primary ovarian and 25 primary breast tumours identified one somatic, intronic mutation in one ovarian tumour, as well as several polymorphisms, including one resulting in an amino-acid substitution. These data suggest that BNIP3L is unlikely to be the target of 8p LOH in ovarian or breast cancer.

Apoptosis↗

PubMed2001

Methanococcus jannaschii prolyl-cysteinyl-tRNA synthetase possesses overlapping amino acid binding sites.

The protein translation apparatus of Methanococcus jannaschii possesses the unusual enzyme prolyl-cysteinyl-tRNA synthetase (ProCysRS), a single enzyme that attaches two different amino acids, proline and cysteine, to their cognate tRNA species. Measurement of the ATP-PP(i) exchange reaction revealed that amino acid activation, the first reaction step, differs for the two amino acids. While Pro-AMP can be formed in the absence of tRNA, Cys-AMP synthesis is tRNA-dependent. Studies with purified tRNAs indicate that tRNA(Cys) promotes cysteine activation. The k(cat) values of wild-type ProCysRS for tRNA prolylation (0.09 s(-1)) and cysteinylation (0.02 s(-1)) demonstrate that both aminoacyl-tRNAs are synthesized with comparable rates, the cysteinyl-tRNA synthetase activity being only 4.5-fold lower than prolyl-tRNA synthetase activity. Kinetic analysis of ProCysRS mutant enzymes, generated by site-directed mutagenesis, shows glutamate at position 103 to be critical for proline binding, and proline at position 100 to be involved in cysteine binding. The proximity in ProCysRS of amino acid residues affecting binding of either cysteine or proline strongly suggests that structural elements of the two amino acid binding sites overlap.

Acylation↗

PubMed2006

Pro-apoptotic effect of endogenous H2S on human aorta smooth muscle cells.

Cystathionine gamma-lyase (CSE) is a key enzyme in the trans-sulfuration pathway, which uses L-cysteine to produce hydrogen sulfide (H2S). The CSE/H2S system has been shown to play an important role in regulating cellular functions in different systems. In the present study, we overexpressed CSE in human aorta smooth muscle cells (HASMCs) using a recombinant defective adenovirus containing CSE gene (Ad-CSE). Infection of HASMCs with Ad-CSE resulted in a significant increase in the expression of CSE protein and H2S production. Ad-CSE transfection inhibited cell growth and stimulated apoptosis, as evidenced by cell viability assay, Hoechst 33258 staining, TUNEL, and caspase 3 activation. CSE-mediated apoptosis was associated with an increased ERK and p38 MAPK activation, up-regulation of p21(Cip/WAK-1), and down-regulation of cyclin D1 expression. After inhibiting endogenous background CSE gene expression, direct administration of H2S at 100 microM induced apoptosis of HASMCs. The other two endproducts of CSE-catalyzed enzymatic reaction, ammonium and pyruvate, failed to do so. These results demonstrate that overexpression of CSE stimulates SMC apoptosis due to an increased endogenous production of H2S. Adenovirus-mediated transfer of CSE gene may provide a novel therapeutic approach in treating vascular diseases linked to abnormal cellular proliferation and vascular remodeling.

Aorta↗

PubMed2026

The Tor pathway, ribosome concentration, and wobble decoding mediate inhibitory effects of the Leu-Pro CUC-CCG codon pair in Saccharomyces cerevisiae.

Translation elongation and efficiency are modulated by the genetic code, with reduced translation efficiency and slow translation caused by 17 inhibitory codon pairs in the yeast Saccharomyces cerevisiae Nine of these inhibitory pairs are functionally important as they are disproportionately strongly conserved within orthologous genes in Saccharomyces sensu stricto For three pairs, including CGA-CGA, inhibition is triggered by ribosome collisions followed by known quality control responses, but the mechanisms by which nine other pairs cause inhibition are unknown. Here, our examination of the molecular basis of inhibition by one such pair, the highly conserved Leu-Pro CUC-CCG codon pair, yielded four findings. First, inhibition is mediated by tRNALeu(UAG), which decodes CUC by a U•C wobble interaction and effectively competes with the nonessential Watson-Crick base-pairing tRNALeu(GAG) Second, despite nearly universal conservation of U33 in tRNAs, the C33 alteration in tRNALeu(GAG) does not significantly impair its function. Third, inhibition likely is mediated by ribosome collisions, as many suppressors bear mutations known or predicted to reduce ribosome concentration, and as local reduction in ribosome concentration suppresses inhibition. Thus, differences between CUC-CCG and CGA-CGA inhibition likely occur downstream from ribosome collisions. Fourth, we find a link between the metabolic state and CUC-CCG inhibition, as we find six suppressor mutations in SCH9, a downstream effector of TORC1 that mediates ribosome production. As Sch9 is inactive during starvation, causing reduced ribosome concentration, one biological function of inhibitory pairs may be to mediate a change in relative expression during starvation conditions.

Ribosomes↗

PubMed1997

Prevention of adenocarcinoma colon 26-induced cachexia by interleukin 10 gene transfer.

A s.c. injection of a mouse colon adenocarcinoma cell line, colon 26 clone 20, induced cachexia, as evidenced by progressive weight loss and severe hypoglycemia. Several lines of evidence indicate that a pro-inflammatory cytokine, interleukin 6 (IL-6), plays a major role, albeit partially, in the establishment of cachexia in this model. Because IL-10 can potentially inhibit the production of pro-inflammatory cytokines including IL-6, we evaluated the effects of IL-10 gene transfer on the establishment of cachexia. IL-6 transcript was detected at tumor sites of mice inoculated with parental or control vector transfectant cells, and serum IL-6 levels were markedly increased in these mice. The injection of parental cells into IL-6-deficient mice induced cachexia with elevated serum IL-6 levels comparable to wild-type mice, indicating that tumor cells are a major source of IL-6. The inoculation of IL-10-transfectant cells kept IL-10 mRNA expression at tumor sites and induced the elevation in serum IL-10 levels without affecting the growth rates of colon 26 cells both in vitro and in vivo. However, the implantation with IL-10-transfectant cells reduced the expression of IL-6 mRNA at the tumor sites and the elevation in serum IL-6 levels. Concomitantly, mice inoculated with IL-10-transfectant cells did not exhibit progressive weight loss, a reduction in food intake, or severe hypoglycemia, which was observed in mice inoculated with parental or control vector-transfectant cells. Collectively, these results suggest that IL-10 gene transfer prevented the occurrence of cachexia with a concomitant inhibition of IL-6 production at the tumor sites.

Animals↗