Relationship of faecal calprotectin and long-term outcomes in Finnish patients with Crohn's disease: retrospective multi-centre chart review study.

Background and Aims: A retrospective non-interventional, multi-centre patient chart review study was conducted to investigate the association of faecal calprotectin (FC) 1 year (±2 months) after biological therapy initiation with composite event-free survival (CEFS) consisting of surgical procedures, corticosteroid initiation, treatment failure or dose increase in patients with Crohn's disease (CD). In addition, the correlations of FC and other tests of disease activity were assessed.Materials and methods: Data on Finnish CD patients initiating a biological therapy between 2010 and 2016, were collected. The association of FC and CEFS was analysed with Kaplan-Meier and Cox proportional hazard modelling. The correlations were tested with Pearson's test.Results: Biological therapy was initiated in 186 patients, of which 87 (46.8%) had FC results available at 1 year and 80 had follow-up exceeding 14 months. The characteristics of patients with and without FC results were similar. Patients with elevated FC (>250 µg/g) had a significantly increased risk of experiencing composite event (HR 3.4, 95% CI: 1.3-8.9; p = .013) when compared to patients with normal FC (FC ≤ 100). No such risk was observed in patients with intermediately increased FC level (100 µg/g < FC ≤ 250 µg/g) (HR 2.2 (95% CI: 0.8-6.2; p = .120). FC value had significant positive correlation with CRP, HBI and leukocyte values when measured at similar timepoints.Conclusions: Elevated level of FC approximately 1 year after the initiation of biological therapy was associated with an increased risk of either surgical procedures, corticosteroid initiation, treatment failure or dose increase (i.e. composite outcome) in patients with CD.

Distinguishing bacterial pathogens of potato using a genome-wide microarray approach.

A set of 9676 probes was designed for the most harmful bacterial pathogens of potato and tested in a microarray format. Gene-specific probes could be designed for all genes of Pectobacterium atrosepticum, c. 50% of the genes of Streptomyces scabies and c. 30% of the genes of Clavibacter michiganensis ssp. sepedonicus utilizing the whole-genome sequence information available. For Streptomyces turgidiscabies, 226 probes were designed according to the sequences of a pathogenicity island containing important virulence genes. In addition, probes were designed for the virulence-associated nip (necrosis-inducing protein) genes of P. atrosepticum, P. carotovorum and Dickeya dadantii and for the intergenic spacer (IGS) sequences of the 16S-23S rRNA gene region. Ralstonia solanacearum was not included in the study, because it is a quarantine organism and is not presently found in Finland, but a few probes were also designed for this species. The probes contained on average 40 target-specific nucleotides and were synthesized on the array in situ, organized as eight sub-arrays with an identical set of probes which could be used for hybridization with different samples. All bacteria were readily distinguished using a single channel system for signal detection. Nearly all of the c. 1000 probes designed for C. michiganensis ssp. sepedonicus, c. 50% and 40% of the c. 4000 probes designed for the genes of S. scabies and P. atrosepticum, respectively, and over 100 probes for S. turgidiscabies showed significant signals only with the respective species. P. atrosepticum, P. carotovorum and Dickeya strains were all detected with 110 common probes. By contrast, the strains of these species were found to differ in their signal profiles. Probes targeting the IGS region and nip genes could be used to place strains of Dickeya to two groups, which correlated with differences in virulence. Taken together, the approach of using a custom-designed, genome-wide microarray provided a robust means for distinguishing the bacterial pathogens of potato.

Low circulating soluble interleukin 2 receptor level predicts rapid response in patients with refractory rheumatoid arthritis treated with infliximab.

BACKGROUND: Treatment with infliximab induces a rapid therapeutic response in most patients with active rheumatoid arthritis. Factors predicting good response are not well known. OBJECTIVE: To study the predictive value of baseline level of soluble interleukin 2 receptor (sIL2R), a marker of lymphocyte activation, on the treatment response. METHODS: 24 patients with active rheumatoid arthritis received intravenous infusions of infliximab at study entry, at two weeks, at six weeks, and at eight week intervals thereafter. Outcome was evaluated at six weeks and 22 weeks. Clinical assessment and standard laboratory tests were made and the DAS28 disease activity score was calculated. Serum sIL2R level at entry was measured by automated immunoassay analyser (Immulite). The mean change in DAS28 score from entry to six weeks and 22 weeks was calculated and related to sIL2R level using baseline adjusted robust regression analysis. RESULTS: Baseline level of serum sIL2R (mean (SD), 621 (325) U/ml) did not correlate with baseline DAS28 score (r = 0.24 (95% confidence interval, -0.18 to 0.58)). At six weeks DAS28 scores improved, with a mean change of -2.53 (-3.08 to -1.98) (p<0.001). This change was predicted by low baseline sIL2R level (regression coefficient per 100 U/ml: 0.205 (0.003 to 0.407) (p = 0.047)). At 22 weeks the DAS28 scores improved, with a mean change of -2.26 (-2.75 to -1.77) (p<0.001). The change was not predicted by baseline sIL2R level. CONCLUSIONS: Low baseline sIL2R level may predict a rapid clinical response in patients with refractory rheumatoid arthritis treated with infliximab.

Immune activation in the small intestine in patients with rheumatoid arthritis.

OBJECTIVES: To determine whether inflammation in the gut associated immune system is activated in rheumatoid arthritis (RA). The expression of chemokine receptor- (CCR4, CCR5) and cytokine- (interleukin (IL)2, IL10, interferon gamma (IFNgamma), tumour necrosis factor alpha (TNFalpha), and transforming growth factor beta (TGFbeta)) specific mRNA in intestinal biopsy samples from patients with RA was examined. METHODS: Duodenal biopsy samples from 13 patients with RA and 15 control subjects were studied. The mRNA expression of CCR4, CCR5, IL2, IL10, IFNgamma, TNFalpha, and TGFbeta in intestinal biopsy samples was demonstrated by real time quantitative reverse transcriptase-polymerase chain reaction. RESULTS: The mRNA expression of CCR4, CCR5, and IL10 in intestinal biopsy samples was increased in patients with RA in comparison with control subjects (p = 0.001, p = 0.046, p = 0.019). No difference in the expression levels of IL2, IFNgamma, TNFalpha, or TGFbeta was seen between patients with RA and controls. CONCLUSIONS: The increased intestinal mRNA expression of IL10, CCR5, and CCR4 suggests that gut associated immune cells are activated in patients with RA.

Cytokine and chemokine receptor profile of peripheral blood mononuclear cells during treatment with infliximab in patients with active rheumatoid arthritis.

OBJECTIVES: To analyse immunological changes during treatment with a monoclonal anti-tumour necrosis factor alpha (TNFalpha) antibody, infliximab, in patients with rheumatoid arthritis (RA). METHODS: 25 patients with RA and 5 patients with other arthritides were studied during the first 6 weeks of treatment with infliximab. At the start of treatment and after 2 and 6 weeks, spontaneous expression of CCR3 and CCR5 on peripheral blood T cells and monocytes was studied by flow cytometry. The secretion and mRNA expression of interferon gamma (IFNgamma), interleukin (IL)4, IL5, and TNFalpha from phytohaemagglutinin (PHA) stimulated peripheral blood mononuclear cells was measured with an ELISA and RT-PCR. Plasma levels of C reactive protein, serum amyloid protein A, rheumatoid factor, and antibodies to filaggrin and citrullinated cyclic peptide were measured with an ELISA. RESULTS: The number of CD4 T cells and CD14 monocytes expressing CCR3 (p = 0.013, p = 0.009, respectively) and CD8 T cells expressing CCR5 (p = 0.040) as well as PHA stimulated secretion of IL4 and IFNgamma (p<0.05) increased during treatment in patients with RA. 15 (60%) patients with RA achieved clinical response (at least ACR20) during the first 2 weeks. The number of T cells expressing CCR3 and CCR5 was higher before treatment in non-responders than in responders (p<0.05). The number of T cells increased in responders. CONCLUSION: Increase in secretion of Th1 and Th2 cytokines together with induced expression of chemokine receptors on T cells and monocytes suggest restoration of peripheral cell mediated immunity and blockade of the accumulation of inflammatory cells in joints as response to treatment.

Peptidylarginine deiminase, the arginine to citrulline converting enzyme, is frequently recognized by sera of patients with rheumatoid arthritis, systemic lupus erythematosus and primary Sjögren syndrome.

OBJECTIVE: Antibodies to citrulline-containing epitopes of filaggrin are highly specific for rheumatoid arthritis (RA). We studied whether the enzyme peptidylarginine deiminase (PAD), responsible for the post-translational modification of peptide-bound arginine residues to citrulline, constitutes an antigen for patients with RA. METHODS: IgG antibodies to PAD were measured by enzyme-linked immunosorbent assay (ELISA) in sera from patients with RA, systemic lupus erythematosus (SLE), primary Sjögren syndrome (pSS), multiple sclerosis (MS) and healthy controls. RESULTS: Compared to healthy controls, raised levels of IgG antibodies to PAD were found in 50 of 57 recent-onset RA patients (88%) and in 40 (70%) of the same 57 patients 3 years later (p<0.0001 for both comparisons). Eleven of 51 (22%) patients with RA of long duration, 19/43 (44%) patients with SLE and 16/19 (84%) patients with pSS, but none of 20 patients with MS, had elevated anti-PAD levels. CONCLUSION: The arginine-citrulline converting enzyme PAD was recognized as a new antigen against which patients with inflammatory rheumatic diseases frequently show IgG class antibodies.

Molecular heterogeneity of the Jk(null) phenotype: expression analysis of the Jk(S291P) mutation found in Finns.

Polymerase chain reaction genotyping of 32 unrelated Jk(null) individuals originating predominantly from Polynesia and Finland indicated that all were homozygous for the JK*B polymorphism and that 17 of 32, including the 14 Polynesians, carried a 3'-acceptor splice site mutation of intron 5 that resulted in the skipping of exon 6 (called mutation Jk delta 6). The remaining 15 Jk(null) donors from Finland were homozygous for a new T871C transition resulting in a S291P amino acid substitution at a consensus N-glycosylation site of the Jk polypeptide. Transcription-translation assays revealed that the Jk(S291P) mutant was translated into a glycosylated component as efficiently as the wild-type Jk polypeptide (wt Jk)] in the presence of microsomes, thus indicating that the S291P mutation has no effect on the N-glycosylation pattern of the Jk protein. Expression studies in Xenopus oocytes revealed that the Jk(S291P) polypeptide functions as a urea transporter, but the transport activity and the membrane expression level of the mutant protein was reduced to a similar extent. A substantial fraction of the mutant protein was retained intracellularly suggesting that the transit to the plasma membrane was reduced, presumably because of the S-->P mutation. After transfection in erythroleukemia K562 cells the wild-type, but not the mutant, protein was efficiently expressed at the cell surface. Because the Jk(S291P) mutant polypeptide was not present in human red cells from Jk(null) individuals, expression data in the erythroid context clearly indicates that the S-->P mutation is the molecular basis of the Finnish Jk(null) phenotype. (Blood. 2000;96:1566-1573)

Mechanisms of coxsackievirus-induced damage to human pancreatic beta-cells.

Enteroviruses may be involved in the pathogenesis of insulin-dependent diabetes mellitus, either through direct beta-cell infection or as triggers of autoimmunity. In the present study we investigated the patterns of infection in adult human islet cell preparations (consisting of 56+/-14% beta-cells) by several coxsackieviruses. The cells were infected with prototype strains of coxsackievirus B (CBV) 3, 4, and 5 as well as coxsackievirus A9 (CAV-9). The previously characterized diabetogenic strain of coxsackievirus B4 (CBV-4-E2) was used as a reference. All viruses replicated well in beta-cells, but only CBVs caused cell death. One week after infection, the insulin response of the beta-cells to glucose or glucose plus theophylline was most severely impaired by CBV-3 and CBV-5 infections. CBV-4 also caused significant functional impairment, whereas CAV-9-infected cells responded like uninfected controls. After 2 days of infection, about 40% of CBV-5-infected cells had undergone morphological changes characteristic of pyknosis, i.e. highly distorted nuclei with condensed but intact chromatin. Both mitochondria and plasma membrane were intact in these cells. DNA fragmentation was found in 5.9+/-1.1% of CBV-5-infected beta-cell nuclei (2.1+/-0.3% in controls; P<0.01). CAV-9 infection did not induce DNA fragmentation. One week after infection the majority of infected cells showed characteristics of secondary necrosis. Medium nitrite and inducible nitric oxide synthase messenger ribonucleic acid levels were not significantly up-regulated by CBV infection. These results suggest that several enteroviruses may infect human beta-cells. The infection may result in functional impairment or death of the beta-cell or may have no apparent immediate adverse effects, as shown here for CAV-9. Coxsackie B viruses cause functional impairment and beta-cell death characterized by nuclear pyknosis. Apoptosis appears to play a minor role during a productive CBV infection in beta-cells.

Clavibacter michiganensis subsp. Sepedonicus Elicits a Hypersensitive Response in Tobacco and Secretes Hypersensitive Response-Inducing Protein(s).

ABSTRACT Strains of Clavibacter michiganensis subsp. sepedonicus, causal agent of bacterial ring rot of potato, showed marked differences in virulence on host plants. When infiltrated into tobacco leaves, virulent strains caused a rapid localized necrotic response (within 24 to 48 h) characteristic of the hypersensitive response (HR), whereas nonpathogenic strains did not. Concentrated cell-free culture supernatants (CCS) from virulent strains caused a necrotic reaction on tobacco, whereas CCS from nonpathogenic strains did not. The necrosis-inducing activity was heat stable and protease sensitive. Inhibitors of eukaryotic metabolism suppressed the necrotic reaction of tobacco to CCS. No necrotic response was observed when host plants were infiltrated with either cells or CCS from virulent strains. HR-inducing protein(s) from a virulent strain separated from the majority of other proteins on DEAE cellulose at 250 to 300 mM NaCl. Ammonium sulfate-precipitated proteins from a virulent strain produced a necrotic reaction at a total protein concentration of 18 mug/ml, whereas those from a nonpathogenic strain did not, even at a concentration of 180 mug/ml. We conclude that virulent strains of C. michiganensis subsp. sepedonicus elicit a typical HR in tobacco and secrete proteinaceous elicitor(s) of the nonhost HR.

Identification of biotinylated molecules using a baculovirus-expressed luciferase-streptavidin fusion protein.

A genetic fusion between streptavidin of Streptomyces avidinii and luciferase of Pyrophorus plagiophthalamus was constructed. The fusion protein was produced in the Sf9 insect cell line using the baculovirus expression vector system (BEVS). Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the proteins from cells infected with the recombinant virus, VL1393-LucGR-StreptAv, revealed that the fusion protein migrated with an apparent molecular weight of 75 kDa. Light emission measurements showed that the infected cells produced about 255 mg of the chimeric protein per liter of cell culture (127.5 micrograms/1 x 10(6) cells). Precipitation of the LucGR-StreptAv fusion protein with biotinylated acrylic beads as well as immunoblot analyses using biotinylated immunoglobulins indicated that both fusion moieties of the chimeric protein product were functional with respect to their physical and enzymatic activities.

Inhibition of nicotine-induced relaxation of the bovine retractor penis muscle by beta-adrenoceptor antagonists.

The relative potency in inhibiting nicotine-induced relaxation of the bovine retractor penis muscle (BRP) was estimated for the racemates of seven beta-adrenoceptor antagonists, both of the optical isomers of propranolol, and lidocaine. The order of potency of the drugs studied was (+)-propranolol greater than (-)-propranolol greater than propranolol greater than alprenolol greater than metoprolol greater than lidocaine greater than acebutolol greater than pindolol greater than sotalol greater than atenolol. It is concluded that the inhibition of the relaxation was not due to blockade of beta-adrenoceptors but to the nonspecific effects of the beta-adrenoceptor antagonists. It is also concluded that the neurotransmitter(s) which was (were) released from the non-adrenergic non-cholinergic inhibitory nerves in the BRP did not relax the muscle by activating the beta-adrenoceptors. It is suggested that the beta-adrenoceptor antagonists inhibited the release of the inhibitory neurotransmitter(s) by a mechanism which is significantly correlated to their lipophilicity.