Pharmacokinetics and clinical efficacy of cyclosporine treatment of dogs with steroid-refractory inflammatory bowel disease

The usual treatment of dogs with inflammatory bowel disease (IBD) consists of administration of immunosuppressive doses of steroids. However, some dogs are refractory to steroid treatment and pose a significant challenge to the veterinarian. Because cyclosporine A (cyA) has been shown to be effective in steroid-resistant IBD in humans, the purpose of this study was to investigate the pharmacokinetics and clinical efficacy of PO cyA treatment in dogs with steroid-refractory IBD (n = 14). All dogs were treated with cyA 5 mg/kg PO q24h for a period of 10 weeks. A clinical activity score was assigned to assess severity of clinical signs before and after treatment. The total number of infiltrating lymphocytes and T cells in duodenal biopsies were assessed before and after treatment in 9 dogs. In addition, serum concentration of cyA was measured in 8 dogs over a 24-hour period. Pharmacokinetic profiles in dogs with IBD were similar to those of healthy dogs. Improvement of clinical signs was observed in 12 of 14 dogs with IBD. Median clinical activity score after treatment with cyA was significantly reduced from a median score of 9 to a median score of 5 (P = 0.001). T cell numbers in duodenal biopsies were significantly decreased after treatment from a median +/- 95% range in the villous region of 28 (19-30) cells/10,000 microm2 before versus 7 (0-10)/10,000 microm2 after treatment, P = 0.01; and from a median +/- 95% range number in the crypt region of 15 (6-23) cells/10,000 microm2 before versus 4 (0-9)/10,000 microm2 after treatment, P = 0.02, implying T cell lysis as a possible mechanism of action. In conclusion, based on this small study, cyA appears to be an effective alternative drug in dogs with IBD that are refractory to immunosuppressive doses of steroids.

Prospective study of fetal DNA in serum and disease activity during pregnancy in women with inflammatory arthritis

OBJECTIVE: Rheumatoid arthritis (RA) usually improves during pregnancy and recurs postpartum. Fetal cells and cell-free DNA reach the maternal circulation during normal pregnancy. The present study investigated dynamic changes in levels of fetal DNA in serum from women with RA and inflammatory arthritis during and after pregnancy to test the hypothesis that the levels of circulating fetal DNA correlate with arthritis improvement. METHODS: Twenty-five pregnant patients were prospectively studied. A real-time quantitative polymerase chain reaction panel targeting unshared, paternally transmitted HLA sequences, a Y chromosome-specific sequence, or an insertion sequence within the glutathione S-transferase M1 gene was used to measure cell-free fetal DNA. Results were expressed as fetal genomic equivalents per milliliter (gE/ml) of maternal serum. Physical examinations were conducted during and after pregnancy. RESULTS: Levels of fetal DNA in women with improvement in or remission of arthritis were higher than those in women with active disease, especially in the third trimester. Overall, an inverse relationship between serum fetal DNA levels and disease activity was observed (P < 0.001). Serum fetal DNA increased with advancing gestation, reaching median levels of 24 gE/ml (range 0-334), 61 gE/ml (range 0-689), and 199 gE/ml (range 0-2,576) in the first, second, and third trimesters, respectively, with fetal DNA clearance observed postpartum. Arthritis improvement was initially noted in the first trimester for most patients, increased further or was sustained with advancing gestation, and was active postpartum. CONCLUSION: Changes in serum fetal DNA levels correlated with arthritis improvement during pregnancy and recurrence postpartum. Immunologic mechanisms by which pregnancy might modulate RA activity are described.

Serological and DNA-based evaluation of Chlamydia pneumoniae infection in inflammatory bowel disease

OBJECTIVES: Chlamydia has been associated with autoimmune diseases, but a link between chlamydial infection and the aetiopathogenesis of inflammatory bowel disease (IBD) remains controversial. In this study we assessed the relationship between chlamydial infection and IBD, as evidenced by serological measurement and DNA analysis of mucosal biopsy specimens. PATIENTS AND METHODS: The sera of 78 patients with Crohn's disease (CD), 24 patients with ulcerative colitis (UC), 73 healthy family members, and 20 healthy controls were tested for anti-C. pneumoniae IgG titres. A subgroup consisting of 13 UC and 39 CD patients was screened for the presence of chlamydial DNA on 42 inflamed versus 30 non-inflamed biopsy specimens and for mutations of their NOD2/CARD15 gene. RESULTS: Anti-C. pneumoniae IgG antibodies were found in the sera of 32 (41%) patients with CD, 11 (46%) patients with UC, 35 (48%) of unaffected family members, and nine (45%) unrelated healthy controls. Thirty-five percent of the control, 18% CD and 24% UC biopsy specimens contained C. pneumoniae DNA. In CD, however, C. pneumoniae DNA was significantly more frequently found in inflamed (27%) versus non-inflamed (8%) biopsy specimens (P < 0.05, Fisher's exact test). The frequencies of NOD2/CARD15 mutations were 33% for CD patients with C. pneumoniae DNA compared to 47% for CD patients without C. pneumoniae DNA. CONCLUSION: We found no marked differences in respect to anti-C. pneumoniae serum IgG or C. pneumoniae DNA between healthy controls and patients with IBD. However, in CD patients, inflamed tissue specimens contained significantly more likely C. pneumoniae DNA compared with biopsies from unaffected areas. Thus C. pneumoniae is unlikely to be of pathogenic importance in IBD while it may still influence local clinical manifestations.

LRH-1-mediated glucocorticoid synthesis in enterocytes protects against inflammatory bowel disease

Liver receptor homolog-1 (LRH-1) is a nuclear receptor involved in intestinal lipid homeostasis and cell proliferation. Here we show that haploinsufficiency of LRH-1 predisposes mice to the development of intestinal inflammation. Besides the increased inflammatory response, LRH-1 heterozygous mice exposed to 2,4,6-trinitrobenzene sulfonic acid show lower local corticosterone production as a result of an impaired intestinal expression of the enzymes CYP11A1 and CYP11B1, which control the local synthesis of corticosterone in the intestine. Local glucocorticoid production is strictly enterocyte-dependent because it is robustly reduced in epithelium-specific LRH-1-deficient mice. Consistent with these findings, colon biopsies of patients with Crohn's disease and ulcerative colitis show reduced expression of LRH-1 and genes involved in the production of glucocorticoids. Hence, LRH-1 regulates intestinal immunity in response to immunological stress by triggering local glucocorticoid production. These findings underscore the importance of LRH-1 in the control of intestinal inflammation and the pathogenesis of inflammatory bowel disease.

Surfactant proteins SP-B and SP-C and their precursors in bronchoalveolar lavages from children with acute and chronic inflammatory airway disease

BACKGROUND: The surfactant proteins B (SP-B) and C (SP-C) are important for the stability and function of the alveolar surfactant film. Their involvement and down-regulation in inflammatory processes has recently been proposed, but their level during neutrophilic human airway diseases are not yet known. METHODS: We used 1D-electrophoresis and Western blotting to determine the concentrations and molecular forms of SP-B and SP-C in bronchoalveolar lavage (BAL) fluid of children with different inflammatory airway diseases. 21 children with cystic fibrosis, 15 with chronic bronchitis and 14 with pneumonia were included and compared to 14 healthy control children. RESULTS: SP-B was detected in BAL of all 64 patients, whereas SP-C was found in BAL of all but 3 children; those three BAL fluids had more than 80% neutrophils, and in two patients, who were re-lavaged later, SP-C was then present and the neutrophil count was lower. SP-B was mainly present as a dimer, SP-C as a monomer. For both qualitative and quantitative measures of SP-C and SP-B, no significant differences were observed between the four evaluated patient groups. CONCLUSION: Concentration or molecular form of SP-B and SP-C is not altered in BAL of children with different acute and chronic inflammatory lung diseases. We conclude that there is no down-regulation of SP-B and SP-C at the protein level in inflammatory processes of neutrophilic airway disease.

Comparison of interferon-gamma release assay versus tuberculin skin test for tuberculosis screening in inflammatory bowel disease

OBJECTIVES: Reactivation of latent tuberculosis (TB) in inflammatory bowel disease (IBD) patients treated with antitumor necrosis factor-alpha medication is a serious problem. Currently, TB screening includes chest x-rays and a tuberculin skin test (TST). The interferon-gamma release assay (IGRA) QuantiFERON-TB Gold In-Tube (QFT-G-IT) shows better specificity for diagnosing TB than the skin test. This study evaluates the two test methods among IBD patients. METHODS: Both TST and IGRA were performed on 212 subjects (114 Crohn's disease, 44 ulcerative colitis, 10 indeterminate colitis, 44 controls). RESULTS: Eighty-one percent of IBD patients were under immunosuppressive therapy; 71% of all subjects were vaccinated with Bacille Calmette Guérin; 18% of IBD patients and 43% of controls tested positive with the skin test (P < 0.0001). Vaccinated controls tested positive more often with the skin test (52%) than did vaccinated IBD patients (23%) (P = 0.011). Significantly fewer immunosuppressed patients tested positive with the skin test than did patients not receiving therapy (P = 0.007); 8% of patients tested positive with the QFT-G-IT test (14/168) compared to 9% (4/44) of controls. Test agreement was significantly higher in the controls (P = 0.044) compared to the IBD group. CONCLUSIONS: Agreement between the two test methods is poor in IBD patients. In contrast to the QFT-G-IT test, the TST is negatively influenced by immunosuppressive medication and vaccination status, and should thus be replaced by the IGRA for TB screening in immunosuppressed patients having IBD.

Combined alpha-methylacyl coenzyme A racemase/p53 analysis to identify dysplasia in inflammatory bowel disease

Identification of dysplasia in inflammatory bowel disease represents a major challenge for both clinicians and pathologists. Clear diagnosis of dysplasia in inflammatory bowel disease is sometimes not possible with biopsies remaining "indefinite for dysplasia." Recent studies have identified molecular alterations in colitis-associated cancers, including increased protein levels of alpha-methylacyl coenzyme A racemase, p53, p16 and bcl-2. In order to analyze the potential diagnostic use of these parameters in biopsies from inflammatory bowel disease, a tissue microarray was manufactured from colons of 54 patients with inflammatory bowel disease composed of 622 samples with normal mucosa, 78 samples with inflammatory activity, 6 samples with low-grade dysplasia, 12 samples with high-grade dysplasia, and 66 samples with carcinoma. In addition, 69 colonoscopic biopsies from 36 patients with inflammatory bowel disease (28 low-grade dysplasia, 8 high-grade dysplasia, and 33 indefinite for dysplasia) were included in this study. Immunohistochemistry for alpha-methylacyl coenzyme A racemase, p53, p16 and bcl-2 was performed on both tissue microarray and biopsies. p53 and alpha-methylacyl coenzyme A racemase showed the most discriminating results, being positive in most cancers (77.3% and 80.3%) and dysplasias (94.4% and 94.4%) but only rarely in nonneoplastic epithelium (1.6% and 9.4%; P < .001). Through combining the best discriminators, p53 and alpha-methylacyl coenzyme A racemase, a stronger distinction between neoplastic tissues was possible. Of all neoplastic lesions, 75.8% showed a coexpression of alpha-methylacyl coenzyme A racemase and p53, whereas this was found in only 4 of 700 nonneoplastic samples (0.6%). alpha-methylacyl coenzyme A racemase/p53 coexpression was also found in 10 of 33 indefinite for dysplasia biopsies (30.3 %), suggesting a possible neoplastic transformation in these cases. Progression to dysplasia or carcinoma was observed in 3 of 10 p53/alpha-methylacyl coenzyme A racemase-positive, indefinite-for-dysplasia cases, including 1 of 7 cases without and 2 of 3 cases with p53 mutation. It is concluded that combined alpha-methylacyl coenzyme A racemase/p53 analysis may represent a helpful tool to confirm dysplasia in inflammatory bowel disease.