Towards a role of interleukin-32 in atherosclerosis.

BACKGROUND: IL-32 has been previously shown to promote inflammation in rheumatoid arthritis patients and to contribute to IL-1ß-induced ICAM-1 as well as other proinflammatory cytokines synthesis in human umbilical endothelial cells (HUVECs). Given the high rate of atherosclerosis in RA, these observations suggest that IL-32 may be involved in the inflammatory pathways of atherosclerosis. METHODS: mRNA and protein levels of IL-32 were determined in human atherosclerotic arterial vessel wall tissue by quantitative real-time PCR and immunohistochemistry. HUVEC and M1/M2 macrophages were stimulated with proinflammatory cytokines and TLR ligands to assess IL-32 mRNA induction. Human THP1 macrophages were transduced with AdIL-32γ, to investigate induction of several proatherosclerotic mediators. Finally, aortas from IL-32γ transgenic mice were studied and compared with aortas from age-matched wild-type mice. RESULTS: IL-32 expression was detectable in human atherosclerotic arterial vessel wall, with the expression of IL-32ß and IL-32γ mRNA significantly enhanced. TLR3-ligand Poly I:C in combination with IFNγ were the most potent inducers of IL-32 mRNA expression in both HUVEC and M1/M2 macrophages. Adenoviral overexpression of IL-32γ in human THP1 macrophages resulted in increased production of CCL2, sVCAM-1, MMP1, MMP9, and MMP13. The IL-32γ transgenic mice chow a normal fat diet exhibited vascular abnormalities resembling atherosclerosis. CONCLUSIONS: IL-32 acts as a proinflammatory factor and may be implicated in the inflammatory cascade contributing to atherosclerosis. By promoting the synthesis of matrix metalloproteinases, it may further contribute to plaque instability. Further studies are warranted to investigate whether IL-32 may serve as a potential therapeutic target in fighting atherosclerosis.

In utero cigarette smoke exposure impairs somatic and lung growth in BALB/c mice.

The aim of this study was to assess whether in utero tobacco smoke exposure alone affects early-life lung growth and development. Pregnant BALB/c mice were exposed to cigarette smoke from six cigarettes per day, or air, from day 8 to 20 of gestation. At 2 weeks of age, pups were weighed and had their lung volumes and lung mechanics measured. Pups born from mothers exposed to cigarette smoke (CS pups; n=17) were significantly lighter (6.76 ± 0.76 versus 7.72 ± 0.68 g) and had lower lung volumes (0.123 ± 0.02 versus 0.149 ± 0.02 mL) than control pups (n=20). Respiratory mechanics were adversely impacted by cigarette smoke exposure. CS pups had higher baseline airway resistance, tissue damping and tissue elastance. These differences were largely due to lower lung volumes. Both tissue damping and elastance were increased excessively in CS pups at high transrespiratory pressures, while other parameters were not affected. There were no histological differences between groups. In utero tobacco smoke exposure significantly affects growth and development in BALB/c mice. These impacts may partially explain the susceptibility of infants born to smoking mothers to early respiratory disease and chronic respiratory disease as adults.

Cyanide in bronchoalveolar lavage is not diagnostic for Pseudomonas aeruginosa in children with cystic fibrosis.

Early detection of the cyanobacterium Pseudomonas aeruginosa in the lungs of young children with cystic fibrosis (CF) is considered the key to delaying chronic pulmonary disease. We investigated whether cyanide in bronchoalveolar lavage (BAL) fluid could be used as an early diagnostic biomarker of infection. Cyanide was measured in 226 BAL samples (36 P. aeruginosa infected) obtained from 96 infants and young children with CF participating in an early surveillance programme involving annual BAL. Cyanide was detected in 97.2% of P. aeruginosa infected and 60.5% of uninfected samples. Cyanide concentrations were significantly higher in BALs infected with P. aeruginosa (median (25th-75th percentile) 27.3 (22.1-33.3) µM) than those which were not (17.2 (7.85-23.0) µM, p<0.001). The best sensitivity, specificity, positive and negative predictive values were obtained with a cut-off concentration of 20.6 µM, and were 83%, 66%, 32% and 96%, respectively. Neutrophil number in BAL was a significant predictor of cyanide concentration (p<0.001). Cyanide concentration can distinguish between P. aeruginosa infected and uninfected BALs as a group, but not individually; therefore, cyanide is a poor diagnostic biomarker of P. aeruginosa infection. Cyanide levels in BAL are related to the level of neutrophilic inflammation.

Adoptive immunotherapy for cancer: the next generation of gene-engineered immune cells.

Adoptive cellular immunotherapy involving transfer of tumor-reactive T cells has shown some notable antitumor responses in a minority of cancer patients. In particular, transfer of tumor-infiltrating lymphocytes has resulted in long-term objective responses in patients with advanced melanoma. However, the inability to isolate sufficient numbers of tumor-specific T cells from most malignancies has restricted the broad utility of this approach. An emerging approach to circumvent this limitation involves the genetic modification of effector cells with T cell receptor (TCR) transgenes or chimeric single-chain variable fragment (scFv) receptors that can specifically redirect T cells to tumor. There has been much progress in the design of TCR and scFv receptors to enhance the antigen-specific activation of effector cells and their trafficking and persistence in vivo. Considerable effort has been directed toward improving the safety of this approach and reducing the immunogenicity of the receptor. This review discusses the latest developments in the field of adoptive immunotherapy using genetically modified immune cells that have been transduced with either TCR or scFv receptor transgenes and used in preclinical and clinical settings as anticancer agents.

The effect of the initiation of renal replacement therapy on lipid profile and oxidative stress during the first 6 months of treatment.

BACKGROUND: Disturbed lipoproteins and increased oxidative stress are two of the "non-traditional" cardiovascular risk factors in chronic renal failure. There are very few prospective data of the influence of dialysis on these two factors. In the present study we investigated the effects of the initiation of both hemo- and peritoneal dialysis therapy on lipoproteins and parameters of LDL oxidation. METHODS: In this prospective cohort study, we assessed lipoproteins, plasma lipid peroxides and in vitro copper-induced LDL oxidation in 46 patients with end-stage renal disease prior to the start of dialysis and after 6 months of treatment with either hemodialysis (n=33) or peritoneal dialysis (n=13). RESULTS: After 6 months of treatment with hemodialysis there was an increase in total cholesterol (4.6+/-1.1 vs. 5.0+/-1.3 mmol/l; p<0.05) and triglycerides (2.0+/-0.9 vs. 2.8+/-1.6 mmol/l; p<0.03). In the peritoneal dialysis group the lipoproteins did not change. Regarding lipid peroxides and in vitro copper-induced LDL oxidation, also no changes were observed after 6 months of treatment in both groups. CONCLUSION: Dyslipidemia aggravates after 6 months of hemodialysis but not after 6 months of peritoneal dialysis. During this period, no net effects on oxidative stress were demonstrated.

Results of an international round robin for the quantification of serum non-transferrin-bound iron: Need for defining standardization and a clinically relevant isoform.

Non-transferrin-bound iron (NTBI) appears in the circulation of patients with iron overload. Various methods to measure NTBI were comparatively assessed as part of an international interlaboratory study. Six laboratories participated in the study, using methods based on iron mobilization and detection with iron chelators or on reactivity with bleomycin. Serum samples of 12 patients with hereditary (n=11) and secondary (n=1) hemochromatosis were measured during a 3-day analysis using 4 determinations per sample per day, making a total of 144 measurements per laboratory. Bland-Altman plots for repeated measurements are presented. The methods differed widely in mean serum NTBI level (range 0.12-4.32mumol/L), between-sample variation (SD range 0.20-2.13mumol/L and CV range 49.3-391.3%), and within-sample variation (SD range 0.02-0.45mumol/L and CV range 4.4-193.2%). The results obtained with methods based on chelators correlated significantly (R(2) range 0.86-0.99). On the other hand, NTBI values obtained by the various methods related differently from those of serum transferrin saturation (TS) when expressed in terms of both regression coefficients and NTBI levels at TS of 50%. Recent studies underscore the clinical relevance of NTBI in the management of iron-overloaded patients. However, before measurement of NTBI can be introduced into clinical practice, there is a need for more reproducible protocols as well as information on which method best represents the pathophysiological phenomenon and is most pertinent for diagnostic and therapeutic purposes.

Characteristics of an avirulent Campylobacter jejuni strain and its virulence-enhanced variants.

The virulence of Campylobacter jejuni for 11-day-old chick embryos is associated with the ability to invade the chorio-allantoic membrane, to resist phagocytosis and to survive and proliferate in vivo. The pathogenicity of a well characterised avirulent C. jejuni strain was enhanced by passaging it intravenously and chorio-allantoically through chick embryos. The resulting isogenic variants had greatly increased ability to survive in vivo. In this study, the morphological and cell-surface characteristics of the avirulent parental strain were compared with those of the more virulent variants to determine whether pathogenicity was associated with one or more cell-surface constituents. Changes associated with the increased virulence of the two variants included alterations in cultural and cellular morphology, loss of flagella, expression of a new outer-membrane protein, alterations in cell-surface carbohydrates and decreases in cell-surface hydrophobicity.

Virulence of Campylobacter jejuni for chicken embryos is associated with decreased bloodstream clearance and resistance to phagocytosis.

The 11-day-old chicken embryo has been shown to be a useful animal model for comparing the virulence of human isolates of Campylobacter jejuni. Virulence in this system is associated with the ability to invade the chorioallantoic membrane and to survive and proliferate in vivo. In this study, the survival and multiplication of C. jejuni in the embryonic host was investigated. It was possible to enhance the virulence of a relatively avirulent C. jejuni strain by passaging it intravenously through the embryos. The resulting isogenic variants demonstrated enhanced abilities to survive in vivo but were still unable to invade when inoculated onto the chorioallantoic membrane. The bloodstream clearance of C. jejuni was studied, and virulent, but not avirulent, strains persisted and multiplied both in the bloodstream and in embryonic liver. Virulent strains also were cleared significantly more slowly from the bloodstream of adult BALB/c mice after intravenous challenge than were avirulent strains. C. jejuni strains which were cleared slowly in vivo were also ingested slowly in vitro by mouse peritoneal macrophages. Clearance studies in mice pretreated with cobra venom factor demonstrated that opsonization by serum complement was not a prerequisite for clearance of campylobacters from the murine bloodstream.

Influence of iron on growth, morphology, outer membrane protein composition, and synthesis of siderophores in Campylobacter jejuni.

Three human isolates of Campylobacter jejuni were grown in a biphasic culture medium with and without the addition of a synthetic chelator to induce iron limitation. Cells grown in low-iron medium exhibited slower growth rates and altered cellular morphology. Increased numbers of longer, more filamentous forms were seen in Gram-stained smears. Three proteins, with apparent Mrs of 82,000, 76,000, and 74,000, were consistently present in the outer membrane of cells grown in low-iron medium. At least one of these proteins (76,000 to 74,000) was exposed on the cell surface. A bioassay was used to look for the production of siderophores by these and other strains of C. jejuni. Seven of 26 strains tested produced detectable amounts of siderophores. Growing strains at 42 degrees C failed to suppress siderophore synthesis or to alter the outer membrane protein profiles of iron-starved cells. The ability of three strains to utilize exogenously supplied siderophores for growth in low-iron medium was also examined. All three strains were able to utilize enterochelin and ferrichrome, but none utilized aerobactin, rhodotorulic acid, or desferrioxamine B. The effect of iron on the virulence of C. jejuni for 11-day-old chicken embryos inoculated via the chorioallantoic membrane was also determined.

The chicken embryo as a model for campylobacter invasion: comparative virulence of human isolates of Campylobacter jejuni and Campylobacter coli.

Eleven-day-old chicken embryos were used to compare the relative virulence of minimally passaged human isolates of Campylobacter jejuni and Campylobacter coli. Graded doses of bacteria were inoculated onto the chorioallantoic membrane, and 50% lethal doses were calculated at 72 h postinfection. Strains varied markedly in their ability to invade the chorioallantoic membrane and kill the embryos. The 50% lethal doses varied by about 6 logs for 25 strains of C. jejuni, and by 2 logs for 5 strains of C. coli. Although both outbred and inbred embryos were employed in the study, the latter were found to be more susceptible to infection with most strains. All isolates were screened for plasmid DNA, but there was no apparent relationship between plasmid content and virulence of strains for the embryos. Neither could virulence be associated with the production of siderophores by the strains. The ability of selected strains of C. jejuni to invade the liver of embryos was also studied. The number of campylobacters culturable from the liver was found to be inversely related to the 50% lethal dose of the strain. By inoculating 11-day-old embryos intravenously, it was possible to demonstrate that a strain of C. jejuni which was poorly virulent after chorioallantoic inoculation was relatively noninvasive. Invasiveness alone, however, could not fully account for the lethality of two highly virulent strains of C. jejuni administered by the intravenous route. Finally, there was no correlation between motility and virulence in this model system.

Endotoxin treatment inhibits glucocorticoid induction of hepatic enzymes at a late induction step.

Bacterial endotoxin inhibits the glucocorticoid induction of several hepatic enzymes including phosphoenolpyruvate carboxykinase (PEPCK). Experiments were performed to elucidate the mechanism of this inhibition by examination of the early events in the glucocorticoid induction process. At a dose of endotoxin 2-to 10-fold greater than that required to inhibit the induction of PEPCK activity, no effect on the entry of glucocorticoids into hepatocytes or their ability to form complexes with glucocorticoid receptors could be measured. Binding data showed no effect of endotoxin treatment on the association or dissociation kinetics of the steroid-receptor binding reaction. Scatchard analysis revealed no effect on the affinity and number of hepatic glucocorticoid receptor binding sites, indicating that down-regulation of receptors is not responsible for inhibition of induction. Finally, activation of receptor complexes was unaffected as well by endotoxin treatment. We conclude from these data that endotoxin does not act at the early events in the glucocorticoid induction process and must therefore intervene at a subsequent step.

Inhibition of hepatic phosphoenolpyruvate carboxykinase by avian reticuloendotheliosis viruses.

Severe weight loss is associated with many malignant diseases of humans and animals. Avian reticuloendotheliosis viruses (RE viruses) induce runting in experimentally infected chickens. Chickens infected with a replication-competent RE virus, reticuloendotheliosis-associated virus, weighed 30-50% less than control birds at the time of death. Chickens infected with reticuloendotheliosis virus, a replication-defective acute leukemia virus, weighed 30% less than the controls. The runting induced by RE viruses does not occur because of reduced food intake. Activities of phosphoenolpyruvate carboxykinase, a key gluconeogenic enzyme in the liver, were reduced approximately 40 and 50%, respectively, by infection with reticuloendotheliosis-associated virus and reticuloendotheliosis virus. RE virus infection, however, did not affect the hepatic pyruvate carboxylase activity, indicating that inhibition of phosphoenolpyruvate carboxykinase is not due to a general inhibition of all liver enzymes. Birds given injections of UV-inactivated RE viruses or reticuloendotheliosis virus-transformed, non-virus-producing tumor cells also exhibited a reduction in phosphoenolpyruvate carboxykinase activity.

Viral persistence and abnormalities of the central nervous system after congenital infection of sheep with border disease virus.

Lambs congenitally infected with border disease (BD) virus and sheep exposed to BD virus as adults were studied for one year to determine the pathogenesis of congenital exposure compared with adult exposure to the virus. Persistent BD virus was isolated in tissue culture and detected by immunofluorescence of the peripheral white blood cells, urine, and cerebrospinal fluid of lambs with congenital BD up to one year of age. These animals had no detectable serum neutralizing antibody response to the virus for the same interval. BD virus antigen was also detected by immunofluorescence in many central nervous system tissues of these lambs with congenital BD. Dysmyelination and glial proliferation in the central nervous system and microencephaly were noted in the lambs with congenital BD, and these lesions appeared to remain the same over a 12-month period.

The role of gut flora and animal passage in the colonisation of adult mice with Campylobacter jejuni.

Adult female mice were given drinking water containing tobramycin 0.05 mg/ml for a week. After a further day without antibiotic they were inoculated intragastrically with one of three strains of Campylobacter jejuni. Colonisation of the gastrointestinal tract was judged by culturing faecal pellets. Tobramycin-treated mice differed from untreated animals in that many more of them discharged infected pellets, and their pellets contained 5- greater than 300 times more campylobacters. Colonisation could be prevented by inoculating the tobramycin-treated animals intragastrically, 24 h before the administration of C. jejuni, with a bacterial suspension prepared from normal faecal pellets. Coliforms, lactobacilli, the two in combination, and anaerobes grown from faecal pellets were not effective in preventing colonisation. Most of the C. jejuni were found in the large intestine of the tobramycin-fed mice. The persistence of colonisation of six dams nursing C. jejuni-infected offspring ranged from 10 to at least 29 weeks.

Pathogenesis of atoxinogenic strains of gram--negative bacteria--a hypothesis.

Textbooks of medical microbiology attribute to endotoxin a primary pathogenic role in diseases caused by nontoxinogenic gram negative bacteria. There is little experimental data to support this claim while some observations indicate the contrary. The possibility cannot be ignored that our understanding of the biological effects of endotoxin is based on the administration of endotoxin as a bolus rather than as a continuous release in small amounts over a period of hours or days as probably occurs during an infection. Results are described that clearly show how different the response can be under the two conditions. It is suggested, moreover, that mediators formed primarily by macrophages stimulated by endotoxin may contribute significantly to the pathogenesis of these diseases.

Inactivated bluetongue virus vaccine in lambs: differential serological responses related to breed.

A mixed breed flock of lambs, consisting of Suffolks, Hampshires, Columbias and Finnish breeds, were vaccinated with binary ethylenimine inactivated blue-tongue virus (BTV) serotypes 11, 17 and a mixture of 11 and 17 in aluminum hydroxide. Agar gel precipitin antibodies were used as an indicator of immunity. Sero-conversion of Hampshires and Suffolk lambs was poor at 43% as compared to 84% in the Columbia and Finn lambs. These results indicate a breed difference in immunological response to inactivated BTV vaccine.

Intestinal colonization of neonatal animals by Campylobacter fetus subsp. jejuni.

Neonatal mice (2.3 to 2.8 g) were inoculated intragastrically with different human isolates of Campylobacter fetus subsp. jejuni. At weekly intervals thereafter, mice were sacrificed and dilution plate counts were performed on segments of the gastrointestinal tract. Mice were uniformly colonized by some strains for 2 weeks, whereas other strains were being cleared at that time. One strain (BO216) persisted in some mice for 3 weeks. The greatest number of organisms (10(7)) was recovered from the cecum and large intestine. The small intestine had from 10(2) to 10(5) colony-forming units. Colonization of the stomach was not found consistently. One strain killed 13% of the infected mice. Deaths occurred between 1 and 5 days postinfection. Two other strains killed a smaller percentage of challenged animals, and two additional strains killed none. Retarded weight gain was noticed in some, but not all, of the infected mice. The intestines of neonatal rats and rabbits were colonized much the same as those of mice, whereas hamsters were resistant to colonization. Preweanling mice, up to about 6.5 to 7.0 g, could be colonized with C. fetus subsp. jejuni after intragastric challenge, but weanling mice of larger weight (9.8 g) and young adult mice (18.3 g) could not. Scanning electron photomicrographs of the lower ileum showed campylobacters in and below the dried mucous gel that lines the intestines. The use of this model for additional studies is discussed.

Persistence and spread of Candida albicans after intragastric inoculation of infant mice.

Infant mice have been shown previously to be a useful model for the study of gastrointestinal (GI) and systemic candidosis. In this study, the virulence of four strains of Candida albicans was compared in intragastrically inoculated infants and in adult mice inoculated intravenously. The four strains differed in their ability to kill both infant and adult mice. A smaller inoculum was required to kill adult mice inoculated intravenously. Neonates could not be inoculated intravenously. The ability of the strains to spread systemically from and to persist for long periods of time in the digestive tract was also examined in intragastrically inoculated infants. The yeast cells spread to liver, lungs, kidneys, and spleen within 30 min postinoculation. Yeast were not detectable in the lungs or in blood from the pleural cavity up to 15 min post-inoculation, thus making it unlikely that systemic spread resulted from faulty inoculation or from aspiration. The region where C. albicans crossed the GI tract of infant mice was visualized histologically in the upper third of the small intestine. The four strains varied in their ability to persist for long periods in the GI tract, in the rate at which they appeared systemically, and in ability to kill infant mice. Three of the four strains colonized the gut for up to 10 weeks postinoculation without use of any compromising agents.