The use of Tween 20 in immunoblotting assays for the detection of autoantibodies in connective tissue diseases.

Autoantibodies directed against intracellular antigens can be detected by immunoblotting (IB). Due to its high sensitivity this technique has many advantages, but it can give misleading results when the specific bands are weak or blurred against the background staining. To decrease background staining, non-ionic detergents (Tween 20, Triton X-100, Nonidet P-40) are generally used as blocking agents. Moreover, these agents appear to have a renaturating action towards proteins and antigens. Tween 20 has a more pronounced renaturating effect on proteins than other detergents and thereby improves antigen-antibody binding. To evaluate the effect of Tween 20 on specific autoantibody detection by IB, we tested the sera of 162 patients with connective tissue diseases (CTDs) by adding this detergent at certain steps of the IB assay. We found that the use of Tween 20 in the IB procedure significantly improved the binding of autoantibodies to Jo-1, Scl70, (U1)RNP 68 kDa and C, Sm B/B' and D. Moreover, it increased the sensitivity for the detection of anti-Sm D peptide in systemic lupus erythematosus (SLE) sera with no decrease in specificity. In contrast, the addition of Tween 20 significantly decreased the binding of autoantibodies specific for ribosomal P proteins, La/SSB, Ro/SSA, but not the overall sensitivity and specificity of the method. We conclude that the addition of Tween 20 to standard IB is advantageous for anti-nuclear antigen antibody detection and improves the sensitivity of the method in revealing anti-Sm-positive sera in SLE. However, Tween 20 is not recommended for the detection of anti-cytoplasmic antibodies.

Serum IgA and gold toxicity in rheumatoid arthritis: lack of predicting value.

The previously reported predictive value of serum IgA for gold toxicity was investigated by measuring such immunoglobulins in 114 patients affected with rheumatoid arthritis and treated with gold salts over a period of 36 months. Side effects were observed in 41 cases (35.9%) (toxic group), mostly within the first year of treatment. Basal levels of IgA were normal in all but 2 patients who maintained low levels throughout the follow-up but did not show any toxic effects. Before therapy and during gold salt administration no difference in serum IgA was noted between the toxic and the non-toxic group. After 6 months of therapy a significant decrease (p less than 0.05) in serum IgA (although never below normal limits) was detected in the toxic group as compared to both the basal values of the same group and the values of the non-toxic group at the same control. Moreover, we did not find any difference in serum IgA between toxic patients with and without mucocutaneous reactions. In our experience the monitoring of serum IgA is not useful in predicting gold toxicity.

The influence of intra-articular hyaluronic acid on PGE2 and cAMP of synovial fluid.

Prostaglandin (PG)E2, cyclic adenosine monophosphate (cAMP), white blood cells (WBC), total protein (TP), total complement activity (CH50) and beta-2-microglobulin (beta-2-m) were measured at baseline and after eight days in the synovial fluid (SF) of 16 patients affected with knee-joint effusion due to various arthropathies. The volume of SF was also calculated. Eight patients--4 with rheumatoid arthritis (RA), 2 with recurrent monoarthritis (RM) and 2 with osteoarthritis (OA) were randomly allocated to the treatment with intra-articular injection of Hyalgan (HA, Na-hyaluronate, 20 mg/2 ml), while eight patients having similar arthropathies--4 RA, 2 RM and 2 OA--were not treated (control group). In the patients treated with HA a significant reduction of SF volume (from 28.5 +/- 5.1 ml to 20.5 +/- 4.0 ml; p less than 0.02) and PGE2 (from 96.1 +/- 22.7 pg/ml to 66.2 +/- 14.5 pg/ml; p less than 0.05) was found, whereas cAMP concentration was significantly increased (from 4.5 +/- 0.7 pmol/ml to 7.2 +/- 1.2 pmol/ml; p less than 0.05). No significant variations were observed in the control group. Moreover, no differences in WBC count, TP and beta-2-m and CH50 were found in either group. These data could suggest an anti-inflammatory effect of HA that appears to be mediated by PG-inhibition as well as cAMP stimulation.

Mineral metabolism and bone mineral content in rheumatoid arthritis. Effect of corticosteroids.

An evaluation of mineral metabolism was performed in 41 patients with RA and the pertinent data were compared to bone mineral content in patients either untreated or treated with different doses of corticosteroids. Our study confirms that osteoporosis is a common finding even in rheumatoid patients never treated with corticosteroids. Moreover, in patients treated with such drug the loss of bone mineral content was related to the dosage rather than to the length of treatment. In all cases no overt biochemical derangement was observed. According to our study, parathyroid hormone does not seem to influence the development of osteoporosis in rheumatoid arthritis, while a relative deficiency of calcitonin along with an inadequate vitamin D metabolism could play some role.

Juvenile onset of primary Sjögren's syndrome: report of 10 cases.

OBJECTIVE: Ten new cases with primary Sjögren's syndrome (pSS) whose disease began before age 16 are described. Special attention is paid both to the follow-up and treatment of this condition. METHODS: Cases with juvenile pSS were retrospectively identified from our series of 180 pSS patients. Ocular, salivary, and extraglandular manifestations as well as a full laboratory evaluation including HLA-DR typing were retrieved. RESULTS: A disease prevalence of 5.5% (10 cases, 8 female and 2 male) was found in our series. The mean age at onset was 11.0 years, but the disease started at the age of 4 in 2 patients. At onset, parotid swelling was found in 6 cases and extraglandular manifestations in 3. Throughout the follow-up period (mean 48.6 months from the time of diagnosis), the clinical picture was similar to that of pSS in adults, but oral involvement was generally milder. Extraglandular manifestations were always present but never severe. Pertinent laboratory abnormalities (e.g. rheumatoid factor, polyclonal hypergammaglobulinemia, leukopenia, increased ESR, ANA and anti-SSA/SSB antibodies) were found in all patients. Specifically, ANA and anti-SSA were always positive. Moreover, in our cases histocompatibility antigens HLA-DR3 and DR 52 were closely associated with the disease. Clinical outcome was difficult to predict; however, no serious complications have been observed so far. We obtained good results with low-dose steroids and/or hydroxychloroquine, especially with regard to the extraglandular manifestations and laboratory abnormalities. CONCLUSION: We confirm that juvenile pSS is not a rare condition. It closely resembles pSS in adults except for the extremely high prevalence of recurrent parotitis and immunological findings.

Fructose-induced hyperuricemia in psoriasis and psoriatic arthritis.

Acute intravenous infusion of fructose was given to 30 normal subjects and 27 normo-uricemic patients affected by psoriasis, 12 with cutaneous involvement only and 15 with psoriatic arthritis. Serum uric acid was measured before and after infusion. A significantly lower increase in serum uric acid levels was found in psoriatic patients in comparison to controls, and the increase rate appeared to be significant only in controls. Moreover, the overall fructose-induced hyperuricemia was significantly lower in the group of patients with psoriatic arthritis than in normal subjects. Since the fructose-induced increase of serum uric acid is most probably achieved by an augmented turnover of preformed purine nucleotides, it is suggested that in normouricemic patients with psoriasis the "pool" of purine nucleotides is lower than normal. Such a condition seems to be more evident in psoriatic arthritis.

Outcome of fifty-five newborns of antiphospholipid antibody-positive mothers treated with calcium heparin during pregnancy.

OBJECTIVE: The outcome of 55 infants born to 53 antiphospholipid antibody (aPL)-positive mothers treated during pregnancy with calcium heparin is described. METHODS: The clinical state of the children was evaluated immediately after delivery by a clinical examination, and a neonatological check-up was performed no later than 24 hours after birth. Neonates with problems were transferred to the neonatal intensive care unit. After their discharge from hospital the clinical state of the babies was followed by means of interviews with the pediatricians and mothers for a period varying between 1.33 and 5.66 years (mean 2.51 +/- 0.92 SD). RESULTS: The newborns comprised 30 females and 25 males, including 2 sets of twins, delivered between the 25th and 40th weeks of gestation (mean 36.69 +/- 2.91 SD). They had a mean birth weight of 2.828 g +/- 706.50 SD (range 800-4.000) and a mean Apgar score at 5 minutes of 9.60 +/- 0.68 SD (range 7-10). Soon after delivery, 12 children (21.81%) were admitted to the neonatal intensive care unit for periods varying between 2 and 120 days (mean 30.33 +/- 33.40 SD), after which the clinical course was normal. All of these neonates suffered from complications exclusively due to prematurity. Malformations and signs of thrombosis or other aPL-related disorders were not observed in any of the newborns. During the follow-up, none of the diseases suffered by the 55 children differed from those of the normal pediatric population; in particular, aPL-related manifestations were never observed. CONCLUSION: These data indicate the absence of aPL-related problems in the offspring of aPL-positive mothers treated during pregnancy with calcium heparin.

Gastric involvement in primary Sjögren's syndrome.

Gastric involvement was investigated in twenty Italian patients with primary Sjögren's syndrome (pSS). Gastric complaints were present in 11 cases (55%) and endoscopic abnormalities in 10 (50%) including 2 cases with active duodenal ulcer. Only two patients (10%) showed moderate chronic atrophic gastritis (AG), while most (85%) had superficial gastritis (SG). No correlations were found among endoscopy, histology and gastric symptoms. Mean serum group I pepsinogen (PG I) levels were significantly higher (p < 0.01) and PG I concentrations in the fundus of the stomach were significantly lower (p < 0.05) in pSS patients than in a matched control group of dyspeptic subjects. Serum and antral gastrin levels were elevated in 3 cases with pSS (15%) including the two with AG, although the mean levels were not different from the controls. Antibodies to gastric parietal cells (PCA) were detected in two cases (10%) including 1 with AG. The present study contradicts previous reports claiming that AG with hypopepsinogenemia is a prominent feature in Sjögren's syndrome. We suggest that, at least in Italian patients, pSS is often associated with SG and high PG I levels.

Hyper-IGD syndrome: a new case treated with colchicine.

We report a new case of hyper-IgD syndrome, a recently described disease characterized by recurrent episodes of fever with headache, bilateral cervical lymphadenopathy and, more rarely, abdominal pain and diarrhoea. Polyclonal increase of serum IgD is the most important laboratory finding. Etiopathogenesis and differences with familial Mediterranean fever are discussed. Moreover, good results obtained with colchicine treatment are also reported.

Psoriatic arthritis exacerbated by Salmonella infection.

Psoriatic arthritis (PsA) is an inflammatory joint disease in which environmental factors, particularly trauma and infections, are thought to play an important role. The authors describe the case of a patient with a mild and long-untreated form of PsA which was severely exacerbated by Salmonella typhimurium infection. This case confirms the importance of infectious agents in the occurrence and course of PsA.

Gynaecological aspects of primary Sjogren's syndrome.

UNLABELLED: Female patients affected with Sjogren's Syndrome (SS) frequently describe symptoms such as vaginal dryness and dyspareunia; however, only a few controlled studies have regarded clinical involvement of the female external genitalia. OBJECTIVE: The present study was undertaken in order to: (1) Evaluate the involvement of external genitalia in a large number of female patients affected with primary SS (pSS) by semi-quantitative methods covering subjective symptoms and clinical evaluation. (2) Compare pSS patients with a matched healthy control group (pre- and post-menopausal women were separately studied). (3) Correlate the gynaecological involvement with salivary and lacrimal abnormalities in pSS patients. METHODS: We evaluated 36 patients with primary SS (18 pre- and 18 post-menopausal women) and 43 healthy controls using a questionnaire regarding vulvar and vaginal dryness and a complete gynaecological examination. Subsequently, three scores related to vulvar and cervical status plus a global score were obtained. In primary SS patients, salivary and lacrimal involvement was also evaluated. RESULTS: Dyspareunia was present in 61% and vaginal dryness in 55% of SS patients versus 39% and 33% of healthy controls. No significant differences regarding gynaecological scores were found between SS patients and controls, in both pre- and post-menopausal women, nor correlation was observed between gynaecological and lacrimal or salivary involvement. CONCLUSIONS: Our data suggest that although SS patients frequently complain of dyspareunia and vaginal dryness they do not greatly differ from healthy subjects in regard to some major gynaecological aspects.

[Detection of serum anti-B/B' UsnRNP antibodies in patients with connective tissue diseases by immunoblotting]. / Determinazione all'immunoblotting degli anticorpi sierici anti-B/B' UsnRNP nei pazienti con connettiviti.

OBJECTIVE: To investigate the reliability of the immunoblot method in the detection of serum immunoreactivity towards the B/B' polypeptides of U small nuclear ribonucleoproteins (UsnRNP) and to assess the significance of these antibodies in connective tissue disease (CTD) patients. METHODS: We tested the sera of 348 patients with CTD (101 SLE, 51 systemic sclerosis, 53 primary Sjögren's syndrome, 27 poly/dermatomyositis, 15 rheumatoid arthritis and 101 overlap CTD), of 31 matched healthy subjects and 13 patients with primary Epstein-Barr virus (EBV) infection with high titre of IgG anti-EBV antibodies. IgG anti-UsnRNP antibodies were determined by immunoblotting on nuclear extract from Raji cells (an EBV-immortalised human B lymphoid cell line) and Jurkat cells (a human T lymphoid cell line). Anti-dsDNA antibodies were detected by indirect immunofluorescence on Crithidia luciliae and anti-ENA by counterimmunoelectrophoresis. Anti-dsDNA activity and avidity were measured in SLE sera by ELISA with Scatchard analysis. Results were statistically analysed by chi-square and Mann-Whitney tests. RESULTS: A high frequency of anti-B/B' antibodies was found in the sera of CTD patients, confined to SLE (54.4%) and overlap CTD with SLE features (55,2%). Anti-B/B' immune reactivity was closely associated with other anti-UsnRNP specificities, gel precipitating anti-nRNP and anti-P antibodies. Nine out of 15 (60%) anti-B/B' positive/anti-ENA negative lupus sera on Raji blots were confirmed to be positive also on Jurkat blots. The sera from patients with EBV infection provided, on Raji blots, completely different band patterns from those obtained with auto-immune sera. CONCLUSIONS. The Sm B/B' proteins are the predominant or, at least, the most frequently targeted antigens of the UsnRNP auto-immune response in SLE and "lupus-like" overlap CTD. Moreover, anti-B/B' is diagnostically specific for CTD with SLE features. Immunoblotting on human B lymphoid cells is a reliable method, in terms of sensitivity and specificity, for the detection of anti-Sm B/B' antibodies.

[Quality of life in systemic lupus erythematosus] / La qualità della vita nel lupus eritematoso sistemico.

The SF-20 and the SF-36 are the most frequently used questionnaires for assessing the quality of life in SLE patients. The SF-36 is actually considered the most suitable for this disease, due to the inclusion of fatigue, a manifestation frequently observed in SLE patients. Using these instruments, it has been clearly demonstrated that patients with SLE have a worse quality of life than healthy people of the same age. Some aspects of daily life, like physical activity, job, social relationship and vitality, are particularly affected. In the majority of studies, an inverse relation between quality of life and disease activity has been observed. The influence the damage has on the quality of life is more complex, since a greater number of variables are involved. In fact, the amount of damage largely depends on the organ involved and on functional impairment resulting from it. To explain the variability in the quality of life among different patients, it is important to consider, besides the clinical complaints, the psycho-social dimension of each person. In fact, some SLE patients, unlike others, cope well with the disease. People behave differently when faced with critical situations, i.e. after being diagnosed with a chronic disease; their reaction depends on the degree of support they receive from family, friends and colleagues, and from the different strategies of coping, that they use.

[Imaging in major salivary gland diseases] / Diagnostica per immagini nelle malattie delle ghiandole salivari maggiori.

Most of the salivary glands diseases are characterized only by a few distinct clinical patterns. Medical history and clinical examination are still considered of great relevance. However, in order to obtain a definite diagnosis, imaging techniques are required in most of the cases. Salivary glands ultrasonography (US) is the technique to be used as the first because US can easily differentiate calculosis, inflammatory diseases and tumors. Sonography is also frequently needed to perform needle aspiration or biopsy (FNAC). Sialography should be used essentially for assessing chronic sialoadenitis as well as Sjögren's syndrome. At present, Magnetic Resonance sialography should be preferred because of the greater sensibility in diagnosing inflammatory diseases of the salivary glands. It allows to evaluate both intraglandular oedema and nodules, so that incannulation of the salivary duct is not required. Computer Tomography (CT) and Magnetic Resonance imaging (MR) are useful when neoplasm are suspected, particularly if deep areas of the gland, which cannot be visualized by US, are involved. Sequential scintigraphy is currently employed for assessing the functional status of all the 4 major salivary glands and evaluating the chronic evolution of glandular damage.