A role for TNF-alpha and mucosal T helper-1 cytokines in the pathogenesis of Crohn's disease

Recent clinical studies of Crohn's disease patients demonstrated dramatic clinical responses following one i.v. infusion of a chimeric mAb to TNF-alpha (cA2). To assess the role of TNF-alpha in mucosal cytokine regulation, the effects of TNF-alpha on lamina propria mononuclear cell (LPMC) Th1 production were determined. Increased IFN-gamma production was demonstrated in anti-CD2-stimulated LPMC cultured in TNF-alpha. To determine the effects of cA2 on cytokine production, TNF-alpha- and IFN-gamma-producing cells were quantitated in LPMC from five Crohn's disease patients treated with cA2. In all four patients who demonstrated clinical and endoscopic improvement, decreased numbers of LPMC producing IFN-gamma and TNF-alpha following CD2/CD28 activation paralleled improvement in disease activity over 8 wk. In one patient who did not improve, increased numbers of TNF-alpha- and IFN-gamma-secreting LPMC were observed. In three of four responding patients, CD2/CD28-activated PBMC demonstrated increased IFN-gamma production over 8 wk. These observations suggest that TNF-alpha may be a cofactor for mucosal Th1 responses, and improvement in clinical parameters and intestinal inflammation induced by cA2 in Crohn's disease may be mediated by down-regulation of mucosal Th1 cytokines.     

Influence of iron-removal procedures on sequential electron transfer in photosynthetic bacterial reaction centers studied by transient EPR spectroscopy

Electron spin polarized electron paramagentic resonance (ESP EPR) spectra were obtained with deuterated iron-removed photosynthetic bacterial reaction centers (RCs) to specifically investigate the effect of the rate of primary charge separation, metal-site occupancy, and H-subunit content on the observed P865+QA- charge-separated state. Fe-removed and Zn-substituted RCs from Rb. sphaeroides R-26 were prepared by refined procedures, and specific electron transfer rates (kQ) from the intermediate acceptor H- to the primary acceptor QA of (200 ps)-1 vs (3-6 ns)-1 were observed. Correlation of the transient EPR and optical results shows that the observed slow kQ rate in Fe-removed RCs is H-subunit-independent, and, in some cases, independent of Fe-site occupancy as Zn2+ substitution does not ensure retention of the native kQ. In addition, shifts in the optical spectrum of P865 and differences in the high-field region of the Q-band ESP spectrum for Fe-removed RCs with slow kQ indicate possible structural changes near P865. The experimental X-band and Q-band spin-polarized EPR spectra for deuterated Fe-removed RCs where kQ is at least 15-fold slower at room temperature than the (200 ps)-1 rate observed for native Fe-containing RCs have different relative amplitudes and small g-value shifts compared to the spectra of Zn-RCs which have a kQ unchanged from native RCs. These differences reflect the trends in polarization predicted from the sequential electron transfer polarization (SETP) model [Morris et al. (1995) J. Phys. Chem. 99, 3854-3866; Tang et al. (1996) Chem. Phys. Lett. 253, 293-298]. Thus, SETP modeling of these highly resolved ESP spectra obtained with well-characterized proteins will provide definitive information about any light-induced structural changes of P865, H, and QA that occur upon formation of the P865+QA- charge-separated state.     

The practice of obstetrics in Mongolia

Sponsored by the US Department of State and the US Army Pacific as part of a Medical Readiness Training Exercise, 17 physicians from Tripler Army Medical Center traveled to Outer Mongolia during September 1995 to examine the practice of medicine in the country. The obstetrical care observed was delivered at Third Women's Hospital in Ulanbataar. Prenatal care in Mongolia is provided mainly by family physicians, with the family doctor visiting each pregnant patient every month for the first 5 months, increasing to weekly visits at 7 months. The patient is taken to an obstetrical or women's and children's hospital when she reaches term or goes into labor. The staff at Third Women's Hospital deliver approximately 2000 babies annually and perform approximately 1000 pregnancy terminations through sharp curettage. The cesarean section rate is reported to be 10%. The hospital has no laboratory or X-ray capability, but can send out for such tests on rare occasions. During labor, patients are fully ambulatory on an as-needed basis. Fetal monitoring is not routinely available, except for occasional auscultation, patients in labor were not given IV fluid, delivery rooms were bleak and with only antiquated equipment, lighting was poor, and gloves were reused until they were too tattered for reuse. The authors discuss their experience with 3 cesarean sections and 1 ectopic pregnancy performed while in the country.     

Immunologic events during the incubation period of hepatitis C virus infection: the role of antibodies to E2 glycoprotein. Multicentre Hemodialysis Cohort Study on Viral Hepatitis

BACKGROUND: The study of the sensitivity of screening assays is greatly facilitated by testing the sequential changes in seroconverting individuals. The aim of this study was to investigate the early immunologic response after hepatitis C virus (HCV) infection and to evaluate whether HCV envelope (E2) recombinant antigen would provide a significant increase in sensitivity for detection of anti-HCV. STUDY DESIGN AND METHODS: Twenty hemodialysis patients who were seroconverting to anti-HCV were included in this study. They were followed up for a mean period (+/- SD) of 10.5 +/- 3.3 months, in which 13 to 46 serum samples per case were collected. Each sample was tested for anti-HCV by second- and third-generation enzyme immunoassay (EIA-2 and EIA-3) and recombinant immunoblot assay (RIBA-3). E2 antibodies were tested by a prototype EIA in which E2 was expressed as a recombinant antigen in Chinese hamster ovary cells. RESULTS: Alanine aminotransferase elevation was observed in 18 of 20 cases. Reactivity against c100, c33c, c22, NS5, and E2 was detected in 15 (75%), 19 (95%), 15 (75%), 2 (10%), and 17 (85%) patients, respectively; c33c was the most immunogenic antigen, followed in descending order by E2, c22, c100, and NS5. E2 antibody reactivity resolved the two RIBA-3-indeterminate cases. However, there was no case in which E2 reactivity preceded all other HCV antigens. Anti-E2 was found to react in all patients of genotypes 1a, 1b, and 3a but in only 2 of 4 patients of genotype 4a. CONCLUSION: In this group of seroconverting individuals, E2 antigen was shown to be highly immunoreactive and did resolve some RIBA-3-indeterminate samples as being positive, on the basis of reactivity to multiple antigens, but it did not improve early detection of seroconversion.     

Modulation of translation initiation in rat skeletal muscle and liver in response to food intake

Protein synthesis is altered in both skeletal muscle and liver in response to nutritional status with food deprivation being associated with an inhibition of mRNA translation. In the present study, the effect of food-intake on the initiation of mRNA translation was examined in rats fasted for 18-h and then refed a complete diet. Fasting and refeeding caused alterations in translation initiation in both skeletal muscle and liver that were not associated with any detectable changes in the activity of eIF2B or in the phosphorylation state of eIF2 alpha. Instead, alterations in initiation were associated with changes in the phosphorylation state of eIF4E and/or the association of eIF4E with eIF4G as well as the eIF4E binding protein, 4E-BP1. In muscle from fasted rats, the amount of eIF4E present in an inactive complex with 4E-BP1 was increased 5-fold compared to freely fed control animals. One hour after refeeding a complete diet, the amount of 4E-BP1 bound to eIF4E was reduced to freely fed control values. Reduced association of the two proteins was the result of increased phosphorylation of 4E-BP1. Refeeding a complete diet also stimulated the binding of eIF4E to eIF4G to form the active eIF4F complex. In liver, the amount of eIF4E associated with eIF4G, but not the amount of eIF4E associated with 4E-BP1, was altered by fasting and refeeding. Furthermore, in liver, but not in skeletal muscle, fasting and refeeding resulted in modulation of the phosphorylation state of eIF4E. Overall, the results suggest that protein synthesis may be differentially regulated in muscle and liver in response to fasting and refeeding. In muscle, protein synthesis is regulated through modulation of the binding of eIF4E to eIF4G and in liver through modulation of both phosphorylation of eIF4E as well as binding of eIF4E to eIF4G.     

Human herpesvirus 8 variants in sarcoid tissues

BACKGROUND: The cause of sarcoidosis is unknown, although mycobacteria have been implicated. We examined sarcoid tissues for human herpesvirus 8 (HHV-8) in addition to mycobacterial genomic sequences. METHODS: Biopsy samples from 17 patients with sarcoidosis were studied (eight transbronchial, 27 lymph node, two skin, and two oral mucosa). We used tissues (n = 137) from 96 patients without sarcoidosis as negative controls. A nested PCR was applied to amplify a segment of open reading frame (ORF) 26 of the HHV-8 genome, and a heminested PCR was to amplify a segment of ORF 25 of HHV-8 and of the 16 S rRNA gene of mycobacteria. Differences in base sequences of the amplified fragments were resolved with single-strand conformation polymorphism and dideoxy sequencing. FINDINGS: HHV-8 ORF 26 DNA was detected in significantly higher proportions of sarcoid than of non-sarcoid tissue samples from lung (8/8 vs 0/54; p < 0.0001), lymph nodes (26/27 vs 6/29; p < 0.0001), skin (2/2 vs 0/17; p = 0.006), and oral tissues (2/2 vs 1/13; p = 0.029). 31 (82%) of the 38 ORF 26 DNA-positive sarcoid specimens were also positive for ORF 25 DNA. For mycobacteria-like 16 S rRNA DNA, the proportion positive was significantly higher in sarcoid than non-sarcoid tissues for lymph node samples (11/27 vs 2/29; p = 0.003) but not for other tissues (lung 3/8 vs 22/54; skin 2/2 vs 15/17; and oral tissues 1/2 vs 0/13). Overall, the prevalence of HHV-8 ORF 26 sequences was higher in sarcoid tissues than in non-sarcoid tissues (p < 0.0001). When patients whose tissues were included in a masked phase of the study were treated as units of analysis, eight of eight sarcoidosis patients were positive for HHV-8 ORF 26 DNA, compared with three of 56 control patients (p < 0.0001); for mycobacteria-like sequences, three of eight sarcoidosis patients were positive, compared with four of 56 controls (p = 0.0464). The HHV-8 ORF 26 sequences, ten of which were unique, could be segregated into four groups according to peptide motifs. In seven of nine patients from whom biopsy samples were taken from various sites, different sequences were recovered. The mycobacterial sequences amplified from sarcoid tissues were also varied, but none was homologous to those of known species. INTERPRETATION: Variant HHV-8 DNA sequences are found in a wide range of sarcoid but not non-sarcoid tissues. Mycobacteria-like 16 S rRNA sequences are more frequently present in sarcoid lymph nodes and not in other tissue types, but do not indicate infection by a particular mycobacterial species.     

Streptokinase entrapment in interdigitation-fusion liposomes improves thrombolysis in an experimental rabbit model

1. The 5-HT receptor involved in the effect of mucosal application of 5-HT to facilitate peristalsis was investigated in the isolated guinea pig ileum. 2. An application of 5-HT (3-100 microM) to the mucosal surface (by inclusion of 5-HT in the Krebs-Henseleit solution passing through the lumen of the ileum) caused a concentration related facilitation of peristalsis characterized by a reduction in the peristaltic threshold. 3. Peristalsis was not modified by methiothepine (0.1 microM), ritanserin (0.1 microM), ondansetron (5 microM), granisetron (1 microM) or SB 204070 (0.1 microM) administered alone to the mucosal surface. 4. The concentration-response curve to mucosally applied 5-HT was not altered by the mucosally applied 5-HT1/2 receptor antagonist methiothepine (0.1 microM), the 5-HT2 receptor antagonist ritanserin (0.1 microM) or the 5-HT4 receptor antagonist SB 204070 (0.1 microM). However, the mucosally applied 5-HT3 receptor antagonists ondansetron (5 microM) and granisetron (1 microM) shifted the response curves to mucosally applied 5-HT to the right in a parallel and surmountable manner. The pD2 values in the absence and presence of ondansetron were 5.42 +/- 0.07 and 4.12 +/- 0.10, respectively, (n = 6) and that of granisetron were 5.45 +/- 0.12 and 4.50 +/- 0.10 respectively, (n = 5). 5. Serosally applied ondansetron (5 microM) or granisetron (1 microM) had no effect on the concentration-response curve to mucosally applied 5-HT. However, the serosally applied ondansetron and granisetron antagonised the facilitatory effect of serosally applied 5-HT (10 microM) when administered in the presence of serosally applied SB 204070 (0.1 microM). 6. It is concluded that the facilitatory effect of mucosally applied 5-HT to reduce the peristaltic threshold in the guinea pig ileum is mediated via a 5-HT3 receptor located on the mucosal and not the serosal side of the ileum.     

Alteration in cell kinetics in control B6C3F1 mice infected with Helicobacter hepaticus

The discovery of Helicobacter hepaticus infection, H. hepaticus hepatitis, and increased incidence of liver tumors in control males from several recent National Toxicology Program B6C3F1 mouse carcinogenicity bioassays raised questions regarding the suitability of these bioassays for hazard identification. The purpose of this study was to determine if changes in cell proliferation and death at terminal sacrifice might be linked to the increased liver tumor incidences among control males. In control males, enhanced rates of hepatocyte proliferation, as assessed by immunostaining for proliferating cell nuclear antigen (PCNA), and apoptosis, as assessed from hematoxylin and eosin- and TUNEL-stained preparations, were seen in 3 bioassays with H. hepaticus hepatitis. One bioassay with H. hepaticus infection without attendant hepatitis and one bioassay without H. hepaticus or hepatitis did not have elevated rates of hepatocyte proliferation or apoptosis. There was no significant effect on PCNA cell proliferation indices or apoptosis in females. The present findings are indicative of a clear association between the presence of H. hepaticus infection with attendant hepatitis, increased cell proliferation and apoptosis, and increased incidences of hepatocellular neoplasia in males but not in females. Thus, the interpretation of liver tumor responses in H. hepaticus-infected studies is considered to be confounded in male mice. The lack of enhanced cell proliferation or hepatocellular neoplasia in control females suggests that bioassay results from females are valid for hazard identification. Furthermore, the absence of enhanced cell proliferation in lungs and kidneys of male and females suggests that neoplastic effects at these sites are not exacerbated by H. hepaticus infection.