Presence of cyclophilin A in synovial fluids of patients with rheumatoid arthritis.

Cyclophilins have been suggested to act as leukocyte chemotactic factors produced in the course of inflammation. Therefore we looked for the presence of cyclophilins in the synovial fluids (SF) from patients with rheumatoid arthritis (RA). Peptidyl prolyl cis-trans isomerase activity (PPIase) was measured in SF from knee punctures of 26 patients with RA and five patients with knee osteoarthritis (OA). PPIase was detected in SF from RA patients, but not in samples from OA patients. Enzyme activity was sensitive to inhibition by cyclosporin A (IC50 = 28-50 nM). Estimated concentrations of the SF-derived cyclophilin based on the enzyme activity were in the range of 11 to 705 nM. The presence of cyclophilin in the SF showed disease correlation; its concentration correlated with the number of cells in the SF (r = 0.91, P < 0.0001) and with the percentage of neutrophils in the cellular infiltrate and was higher in more acute cases of joint swelling. In immunoblots of partially purified preparations of SF from RA patients, an approximately 18-kD protein band reacted with polyclonal antibodies that recognize cyclophilin A and B, but not with antibodies specific for cyclophilin B. Sequencing of this protein revealed identity of the NH2-terminal amino acids with those of human cyclophilin A. The finding is unexpected since cyclophilin B rather than A is generally regarded as the secreted isoform, the presence of cyclophilin A being confined to the cytoplasm. Our data support the hypothesis that cyclophilins may contribute to the pathogenesis of inflammatory diseases, possibly by acting as cytokines. This may offer a possible explanation of the effectiveness of cyclosporin A in RA, in addition to the known immunosuppressive effects of the drug.

New insights into mechanisms of atrial fibrillation.

Although atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice, precise mechanisms that lead to the onset and persistence of AF have not completely been elucidated. Over the last decade, outstanding progress has been made in understanding the complex pathophysiology of AF. The key role of ectopic foci in pulmonary veins as a trigger of AF has been recognized. Furthermore, structural remodeling was identified as the main mechanism for AF persistence, confirming predominant role of atrial fibrosis. Systemic inflammatory state, oxidative stress injury, autonomic balance and neurohormonal activation were discerned as important modifiers that affect AF susceptibility. This new understanding of AF pathophysiology has led to the emergence of novel therapies. Ablative interventions, renin-angiotensin system blockade, modulation of oxidative stress and targeting tissue fibrosis represent new approaches in tackling AF. This review aims to provide a brief summary of novel insights into AF mechanisms and consequent therapeutic strategies.

Neutrophil attractant protein-1-immunoglobulin G immune complexes and free anti-NAP-1 antibody in normal human serum.

After obtaining data indicating the presence of a neutrophil attractant protein-1 (NAP-1)-IgG complex in normal human serum, we developed sandwich ELISAs that could quantify NAP-1 and NAP-1-IgG in mixtures of the two moieties. The ELISA for free NAP-1 used a monoclonal capture antibody that did not bind NAP-1-IgG. The ELISA for NAP-1-IgG was based on omission of the anti-NAP-1 detection antibody (required for the free NAP-1 ELISA) and on interaction of phosphatase-conjugated anti-human IgG with the human NAP-1-IgG complex. Gel filtration of immunoaffinity-purified NAP-1-IgG showed that the bulk of the complex comprised a single IgG. Binding between NAP-1 and antibody is strong, since 8 M urea at neutral or alkaline pH did not release NAP-1. However, at pH 2.0 in 9 M urea approximately 15% of the total NAP-1 could be dissociated from the complex. NAP-1-IgG was detected in 18 of 26 sera from normal humans. The mean serum concentration was 58 ng of IgG-bound NAP-1/ml, with an SEM of 16 and a range from undetectable to 247 ng/ml. NAP-1-IgG concentrations in paired sera drawn at a 1-mo interval were remarkably constant. Using an ELISA for free NAP-1 with a detection limit of 200 pg/ml, we found no free NAP-1 in the 26 sera. Free anti-NAP-1-IgG autoantibody was found in 9 of 26 sera by direct ELISA. IgG anti-NAP-1 of all nine sera was polyclonal, comprising both kappa and lambda isotypes; predominant subclasses were IgG2 and IgG3. NAP-1-IgG did not compete with 125I-NAP-1 for binding to neutrophils, which suggests that IgG anti-NAP-1 is a molecular trap that prevents binding of NAP-1 to neutrophils after it diffuses from production sites into the circulation.

Intracardiac echocardiography in electrophysiology.

Intracardiac echocardiography (ICE) broadens the spectrum of echocardiographic techniques. Modern 10F sector echocardiographic catheters introduced into the right atrium allow high quality imaging of all cardiac structures, including pulse and continuous wave Doppler and/or color Doppler. The main indication for ICE appears to be monitoring of catheter ablation of complex arrhythmic substrates such atrial fibrillation, postincisional tachycardias and ventricular tachycardias. The other important role of ICE is the early diagnosis and prevention of complications during ablation procedures. These include those occurring during transseptal catheterization, damage to cardiac structures, left atrial thrombus formation, pulmonary venous stenosis, esophageal injury and pericardial effusion.

In vitro activity of inhibitors of late stages of the replication of HIV in chronically infected macrophages.

Because of the importance of macrophages in the pathogenesis of the disease caused by HIV, we investigated the efficacy of various anti-HIV drugs in human primary macrophages acutely or chronically infected by this virus. The results obtained for acutely infected macrophages show that dideoxynucleosides (AZT, ddI, and ddC), interferon-alpha and -gamma, mismatched double-stranded RNA, Tat inhibitor, phosphorothioate antisense, and inhibitors of HIV protease, all significantly inhibit virus replication at concentrations far below those toxic for the cells. However, in macrophages in which proviral DNA is already integrated (chronically infected macrophages), only the three inhibitors of HIV protease induced significant virus inhibition at concentrations 100 or more times higher than those effective in acutely infected macrophages. Treatment of macrophages with macrophage colony-stimulating factor does not affect the anti-HIV efficacy of protease inhibitors. These results suggest that therapeutic strategies with activity for macrophages, including inhibitors of HIV protease, are worth pursuing in patients with HIV infection.

HIV proteinase inhibitors containing 2-aminobenzylstatine as a novel scissile bond replacement: biochemical and pharmacological characterization.

Derivation of the 2-aminobenzylstatine containing HIV-1 proteinase (PR) inhibitor I led to a series of compounds with considerably improved antiviral activity, the most potent derivatives inhibiting HIV-1 with IC50 values below 25 nM. This was achieved by the combination of several structural modifications, most prominently by introduction of a benzimidazole heterocycle into the inhibitor. The mode of action of the 2-aminobenzylstatine PR inhibitors was demonstrated to be inhibition of gag precursor processing. The antiviral efficacy of the PR inhibitors was demonstrated in various cell lines, in primary T4 lymphocytes and in monocytes. The most potent compound (XI) inhibited replication of several HIV-1 clinical isolates in primary cells with IC50 values of 8 to 23 nM. The analysis of the pharmacokinetic behaviour of compounds I and VII revealed blood half-lives in rodents in the range of about 1.5 h. Compound I also showed appreciable oral uptake in mice (18%), but yielded no detectable blood levels in rats after oral administration. Benzimidazole containing compounds like VII were not orally bioavailable to a significant extent, neither in mice nor in rats. Thus, while introduction of a benzimidazole group into the PR inhibitors was a successful structural modification with regard to antiviral activity in cell culture, it completely abolished oral bioavailability.

CagA/cytotoxic strains of Helicobacter pylori and interleukin-8 in gastric epithelial cell lines.

AIMS: To investigate: (1) whether Helicobacter pylori directly induces interleukin-8 (IL-8) message expression and protein secretion in established gastric epithelial cell lines; and (2) if CagA/cytotoxin positive and negative strains of H pylori differ in their ability to induce epithelial IL-8. METHODS: Gastric epithelial cell lines were co-cultured with H pylori NCTC 11637 and 10 clinical isolates (four cytotoxic, six non-cytotoxic) and secreted IL-8 was measured by enzyme linked immunosorbent assay (ELISA). Specific induction of gastric epithelial IL-8 mRNA was examined by reverse transcription and polymerase chain reaction (RT-PCR) amplification. RESULTS: H pylori (NCTC 11637) induced IL-8 secretion from three gastric epithelial cell lines (KATO-3, ST42, AGS) but not from MKN 45 (gastric) or intestinal (SW480, HT29) cell lines. H mustelae did not stimulate IL-8 secretion from KATO-3, ST42, and AGS cells. H pylori induced IL-8 secretion was reduced by heat killing, sonication, freeze thawing or formalin fixation of the bacteria. CagA/cytotoxin positive strains of H pylori induced significantly higher IL-8 secretion than CagA/cytotoxin negative strains in the three positive gastric epithelial cell lines (KATO-3, ST42: p < 0.01; AGS: p < 0.02). A significant increase (p < 0.01) in the expression of IL-8 mRNA relative to G3PDH mRNA was observed in KATO-3 cells after three hours of co-culture with CagA/cytotoxin positive strains. CONCLUSIONS: H pylori directly increases gastric epithelial IL-8 mRNA expression and IL-8 protein secretion in a strain specific manner. Induction of epithelial IL-8 by CagA/cytotoxin positive strains is likely to result in neutrophil chemotaxis and activation and thus mucosal damage. These observations on epithelial IL-8 may explain the association between CagA/cytotoxin positive strains and gastroduodenal disease.

Interleukin-8 expression in Helicobacter pylori infected, normal, and neoplastic gastroduodenal mucosa.

AIMS: To investigate the expression of interleukin-8 (IL-8) in Helicobacter pylori infected normal and neoplastic gastroduodenal mucosa, and in established gastric cancer cell lines. METHODS: Immunofluorescence techniques were used to localise IL-8 in cryosections of gastric (n = 25) and duodenal (n = 17) endoscopic biopsy specimens an in resected gastric tumour tissue samples from 16 patients. Two gastric cancer cell lines (Kato 3 and MKN 45) were examined for IL-8 protein expression by immunofluorescence and for the presence of IL-8 mRNA by reverse transcription followed by the polymerase chain reaction (RT-PCR). RESULTS: IL-8 was localised to the epithelium in histologically normal gastric mucosa, with particularly strong expression in the surface cells. IL-8 expression was also a feature of surface epithelium in the duodenal bulb, but was much reduced in the second part of the duodenum. In chronic H pylori-associated gastritis gastritis gastric epithelial IL-8 expression was increased and expression of IL-8 within the lamina propria was evident. By contrast, large areas of IL-8 negative epithelium were observed in the body mucosa of a subject with Ménétrier's disease. In gastric carcinoma the tumour cells were positive for IL-8. IL-8 was also detected by immunofluorescence in unstimulated Kato 3 and MKN 45 cells, and constitutive IL-8 gene expression in these cell lines was confirmed by detection of IL-8 mRNA by RT-PCR. CONCLUSIONS: Immunoreactive IL-8, a potent neutrophil chemotactic and activating factor, is present in the epithelium of both normal and inflamed gastric mucosa with increased expression in the latter. There is site dependent variation in epithelial IL-8 expression within the gastroduodenal mucosa. The expression of the pro-inflammatory cytokine IL-8 in gastric carcinoma cells may influence peritumoural cellular infiltrates.

Serum crosslaps in comparison to serum osteocalcin and urinary bone resorption markers.

In this study we evaluated the routine practice and clinical application of serum crosslaps to urinary-crosslaps, -N-telopeptide-related fraction of type 1 collagen, -deoxpyridinoline, -totalpyridinoline and serum osteocalcin. The utility of both the serum and urine immunoassays for bone formation and resorption marker were tested in a cohort of 593 female and male patients from our outpatient clinic for osteology and rheumatology and compared to important osteoporosis risk factors like age, gender, E2 deficiency, bone density and chronic renal failure. The biochemical maker of bone formation, serum osteocalcin exhibit significant correlations to all five tested serum and urinary markers of bone resorption (p < 0.0001) crosswise to all different groups of patients. The group of chronic renal failure patients showed no significant correlation between the tested bone turnover parameters and the serum creatinine level except a significant increase and correlation for serum crosslaps and for the ratio of serum and urinary crosslaps. Associations between the age of the patients and the markers of bone turnover were rather poor. We found a significant, negative association between serum and urinary bone turnover markers and bone density and were interested, whether in patients with bone density < 2.5 SD an enhanced bone turnover could be detected in the same way as for E2 deficiency. Applying a discriminant analysis it was possible to discriminate between the patient with BD < 2.5 SD and those with BD > 1.0 SD with a sensitivity of 70% and a specificity of 65% using serum crosslaps. In case of urinary crosslaps the discriminatory power was slightly lower (sensitivity: 65.6%, specificity: 67.5%) and for serum osteocalcin the discriminatory power was negligible higher (sensitivity: 79%, specificity: 56%). The highly significant correlation between the urinary and serum crosslinked peptides by ELISA and serum osteocalcin supports the concept that these respective indices of bone formation and resorption both in urine and serum reflect a coupled process in vivo with sensitivity and specificity to pathological bone density, estrogen deficiency and chronic renal failure.

[Hypersensitivity vasculitis causing an acute abdomen]. / Hypersensitivitätsvaskulitis als Ursache eines akuten Abdomens.

The members of a family of four persons suffered acute gastroenteritis after eating a meal consisting of chicken. While three of them recovered rapidly, the 18-year old son developed an acute abdomen which had to be treated surgically and led to a complicated stay at the intensive care unit. Intraoperatively, a mild insignificantly inflamed appendix and an obscure segmental inflammatory process of the small bowel with local peritonitis were seen; this required an appendectomy and a peritoneal lavage. The development of bacterial peritonitis with multiple organ dysfunction required several surgical revisions with an open abdominal toilet treatment. Histological examination of the resected appendix specimen showed a severe primary fibrinoid necrotizing vasculitis with epitheloid-granulomatous reaction. Diseases such as Panenteritis nodosa, Wegener's disease and Churg-Strauss's syndrome were excluded by negative serology. By a process of exclusion, a hypersensitivity vasculitis was diagnosed and treated successfully with a high-dose cortisone regime.

Biochemical markers in menopausal women.

Markers of bone formation (osteocalcin and C-Terminal Propeptide of Type I Collagen [CICP]) and of resorption (Crosslaps, total pyridinoline [Pyd] and deoxypyridinoline [Dpd]) as well as female sex hormones (estradiol [E2], follicle stimulating hormone [FSH] and luteinizing hormone [LH]) were measured in 237 women aged 44-66 years coming for the first medical examination to the outpatient clinic of menopause at the Kaiser-Franz-Josef-Hospital, Vienna. All women (0.5-5.0 years since cessation of menses) selected were not taking medications other than hormone replacement therapy in 52 cases (21.9%) and did not have diseases known to affect bone and mineral metabolism. The best correlation was found between Dpd and Pyd (r = 0.63, p = 0.0001), followed by Crosslaps and Dpd (r = 0.47, p = 0.0001). Only weak but significant correlations between E2 and Crosslaps (r = 0.21, p < 0.0001) as well as E2 and osteocalcin (r = 0.16, p = 0.0007) were observed, 53% of the 237 women suffered from a severe E2 deficiency (E2 < 10.0 ng/L). In these patients Crosslaps (approx. +48%) and osteocalcin (+22%) were significantly higher (p < 0.0001) compared to those with E2 concentrations > 10 ng/L. Women with E2 concentrations > 10 ng/L were further subdivided into women with and without sex hormone replacement therapy, whereby no statistical differences in any of the biochemical markers could be observed between these both groups. In conclusion, we could clearly demonstrate that in postmenopausal women suffering from severe E2 deficiency (E2 < 10 ng/L) Crosslaps and osteocalcin are significantly increased, indicating in principle a clear correlation between E2 deficiency and these markers of bone turnover.

Cranioventral Subluxation of the Odontoid Process With Accompanying Neo(pseudo)arthrosis in Rheumatoid Arthritis.

Cervical spine involvement is a common feature in the course of long existing rheumatoid arthritis (RA).We describe a rare type of vertical subluxation with pronounced cranioventral disposition of the odontoid process and neo(pseudo)arthrosis between the skull and the tip of the odontoid processus. This 74-year-old women with RA for 40 years had progressive neck symptoms over the previous 5 years. She no longer had signs of active synovitis. Plain roentgenographs performed in a neutral position and full flexion and extension gave information about neither the osseous integrity nor the subluxation of the dens axis. Computed tomography and MR imaging techniques established the nature of the atlantoaxial and atlantooccipital joint involvement. During 2 years, this rare cranio-ventral subluxation with neo(pseudo)arthrosis seems to be rather solid, and follow up CT-images demonstrate no signs of progressive migration or suggestion of immediate fracture risk. Pronounced reduction of cervical spine mobility by long standing destructive cases of RA should always raise suspicions for cervical subluxation at the atlantoaxial level, irrespective of serological and clinical signs of rheumatoid arthritis. Vertical subluxation as seen in this patient may be missed on routine x-rays.

Successful ablation of ventricular tachycardia after correction of tetralogy of Fallot.

We present a case of a patient who, after correction of tetralogy of Fallot (TOF), experienced runs of ventricular tachycardia (VT). Mapping of the aortic root showed that the critical component of the reentry was located within the noncoronary cusp. The potential explanations of such an unusual isthmus location may be the presence of myocardial extensions in the aortic root or the close vicinity of the right ventricle (RV) to the noncoronary cusp, since in TOF the aorta overrides the RV. Our case highlights the advantage of using electroanatomic mapping systems together with intracardiac echocardiography in such complex cases.

Clinical outcome of salmon calcitonin nasal spray treatment in postmenopausal women after total hip arthroplasty.

OBJECTIVE: The increasing rate of hip fractures is giving rise to a number of socio-economic problems for the aging community. In addition to being unable to resume their previous living habits, many patients fail to achieve full functional recovery after the fractures. Total hip arthroplasty (THA) is a successful operation for the majority of patients with all forms of hip fractures, being performed increasingly often throughout the world. Revision rates for THA range up to 20% per year. Aseptic loosening is the reason for 75% of the revisions. An additional problem post-THA is the rate of heterotopic soft tissue calcification after THA, resulting in severely impaired function, pain, and a reduced range of hip movement. SUBJECTS: In an open study, 37 women who had undergone cementless THA after accidental hip fractures were treated twice daily with 200 IU of salmon calcitonin nasal spray for 12 months. Simultaneously the patients received one bag of 1,000 mg calcium plus 880 IU vitamin D daily throughout the treatment period of 1 year. A parallel group of 38 women with a similar clinical status in terms of hip fractures and cementless THA were treated with only one bag of 1,000 mg calcium plus 880 IU vitamin D daily through the treatment period. RESULTS: The results of this 12-month clinical trial show that 200 IU of salmon calcitonin nasal spray per day significantly improves the clinical outcome of postmenopausal elderly women following THA. Treatment with a salmon calcitonin nasal spray significantly reduces bone turnover, loss of bone density, and pain. The functional status of the patients was improved and the risk of falling reduced by rehabilitation during the observation period of 12 months. Additionally, calcitonin promoted the repair of hip fractures and was associated with a significantly lesser rate of refractures as well as periprosthetic ossifications. CONCLUSION: The increasing revision rate for THA during the first year and the patient's problem of resuming their previous living habits are the main foci of our study. Calcitonin nasal spray seems to cause few side effects. The additive treatment appears to improve the clinical outcome of THA in elderly postmenopausal women.

[Epidemiology, incidence and sex specific differences in primary gout]. / Epidemiologie, Häufigkeit und geschlechts-spezifische Unterschiede in der primären Gicht.

Gout is common disease with a worldwide distribution. The major risk factor for the development of gout is sustained asymptomatic hyperuricemia. Primary prevention of gout can be achieved through lifestyle changes including weight loss, restricting protein and calorie intake, limiting alcohol consumption and optimal treatment of hypertension.

[Uric acid crystals and chemotactic cytokines--pathogenesis of an acute gout attack]. / Harnsäurekristalle und chemotaktische Zytokine--Die Pathogenese des akuten Gichtanfalls.

The identification of monosodium urate crystals in synovial fluid is essential for the definite diagnosis of gout. Neutrophils interact with urate crystals to produce inflammatory reactions associated with acute arthritis. Crystals activate monocyte-macrophages and synoviocytes to produce high levels of proinflammatory cytokines, like TNF alpha und interleukin-1 and different chemokines. High levels of interleukin-8, a powerful neutrophil attractant with activating properties, were demonstrated in the synovial fluid of patients with acute gout arthritis.

Health, safety and environmental protection in a biological research laboratory.

This manuscript summarizes the mandatory regulations for Health, safety and environmental protection (HSE) at the Novartis Forschungsinstitut-Vienna to ensure the safe and contained biological laboratory work especially with class II agents in the specialized biosafety level 2 (BL2) facilities available at this institute. These regulations apply to work practices conducted within these facilities; to special safe-containment features of these BL2 facilities; to containment and decontamination of biohazardous or potentially biohazardous materials of risk class II; and to the procedures in place to guarantee that these regulations are strictly carried out, and that only individuals with the appropriate training and approval have access to these facilities. The regulations governing BL2 facilities summarized here have been taken directly from CDC and NIH public documents; any special adaptations or additions to these regulations have been stated as such, in order to make these guidelines as transparent and nonredundant as possible.

Autoantibodies associated with rheumatic diseases.

The identification of circulating autoantibodies contributes to the correct diagnosis as well as to the follow-up of rheumatic diseases. Some autoantibodies are even included in diagnostic and classification criteria for these types of autoimmune diseases. There are several relatively specific screening and identification methods for the measurement of autoantibodies available. The type of assay crucially influences the diagnostic value of the parameters. In general, routine laboratories should prefer enzyme immunoassays (ELISA) using well characterized antigens, although ELISA tests tend to produce more false-positive and true weakly positive results, which reduce their positive predictive value. Therefore one should be aware that laboratory results can only be properly interpreted when there is a correlation with the clinical situation and when the limitations of the technologies used for autoantibody identification have been taken into consideration. A diagnostic algorithm consisting of screening and identification steps should be established by each laboratory in order to create a rational, evidence-based and cost-effective basis for the diagnosis of rheumatic diseases.