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Aims: Tissue transglutaminase (tTG) was recently identified as the major autoantigen in coeliac disease. The aim of this multicentre study was to evaluate the impact of a new immunoenzymatic assay for the detection of IgA anti-tGT antibodies. Methods: Seventy four Italian and French clinical laboratories participated in this study; anti-tTG IgA with an enzyme linked immunosorbent assay (ELISA) method using guinea pig liver extract as the coating antigen, anti-endomysium IgA autoantibodies (EMA), and total serum IgA were determined in 7948 patients, 1162 of whom had coeliac disease (737 untreated cases and 425 on a gluten free diet). A proportion of the sera were then sent to a reference laboratory for anti-tTG retesting with an ELISA method using recombinant human tTG antigen. Results: Seven thousand four hundred and fifty eight (93.8%) sera were EMA/antiguinea pig tTG concordant (positive or negative); 490 (6.2%) were non-concordant. The sensitivity of EMA and antiguinea pig tTG in the 737 untreated patients with coeliac disease was 92.1% and 94.8%, respectively, and the specificity was 99.8% and 99.2%, respectively. Retesting of the discordant sera showed that of the 162 sera classified as EMA negative/antiguinea pig tTG positive, only 49 were positive for human recombinant anti-tTG, and that 39 of these were also EMA positive. Furthermore, of the 36 sera classified as EMA positive/antiguinea pig tTG negative, only two were confirmed as EMA positive. Conclusions: The antiguinea pig tTG assay is more sensitive but less specific than EMA, whereas the antihuman recombinant tTG assay is far more specific and just as sensitive as antiguinea pig tTG. Testing for EMA presents considerable interpretative problems and is difficult to standardise.

Allergy to crustacean shellfish is one of the most common IgE-mediated food allergies, and tropomyosin has been identified as the major allergen. However, not all subjects affected by this allergy are IgE-positive to tropomyosin.To evaluate whether sera of patients with shrimp allergy but negative for tropomyosin react to other allergen(s); and to evaluate the role such allergen(s) may play in cross-reactivity between crustaceans and house dust mites (HDMs).Three different pools of sera-one from subjects with shellfish allergy and HDMs positivity, but negative for recombinant and native tropomyosin (rPen a 1 and nPen m 1) (Pool 2); a second from subjects with tropomyosin and HDMs positivity (Pool 1); and the last from subjects allergic only to HDMs (Pool 3) were submitted to immunoblotting. Subsequently, a 20 kDa protein- enriched fraction of shrimp extract was used at two different concentrations (10 and 100 microg/mL) to pre-absorb the Pool 2 serum and to evaluate, by ELISA assay, the level of inhibition on shrimp and HDMs-coated wells, respectively.The Pool 2 serum showed IgE reactivity against a 20 kDa component. Its pre-absorption with an enriched fraction of 20 kDa protein caused an inhibition of 56% in IgE binding to shrimp extract at a concentration of 100 microg/mL, and of 14% and 35% to HDMs extract at concentrations of 10 and 100 microg/mL, respectively, as measured by ELISA assay.The 20 kDa component seems to be a new crustacean allergen and it could play a role in cross-reactivity with HDMs.

The transglutaminase enzyme (tTG) is the target antigen of autoantibodies found in the serum of patients suffering from coeliac disease (CD). The most physiologically important function of tTG is to deamidate the glutamine residues of the gliadin peptides contained in corn gluten, and convert them into glutamic acid; this modification makes the gliadin negatively charged, allowing it to bind with HLA-DQ2/DQ8 antigens, with consequent exposure of the neopeptides to recognition by the T cells. In persons genetically predisposed to CD, the development of a T and B immune response leads to synthesis of anti-tTG IgA antibodies and of proinflammatory cytokines, resulting in chronic inflammation and progressive destruction of the intestinal mucosa. The anti-tTG antibodies of class IgA are a very sensitive and specific marker for CD, while the anti-tTG antibodies of class IgG can be used to diagnose CD in subjects with an IgA deficiency. Anti-tTG antibodies recognize the same antigen as anti-endomysial antibodies (EMA), from which they only differ in terms of detection method. EMA IgA antibodies are tested by the indirect immunofluorescence method on cryostatic sections of monkey oesophagus. The EMA IgA test is highly specific, but less sensitive than IgA anti-tTG, and should therefore preferably be used as a confirmation test. Anti-tTG testing can also be used to monitor CD patients following a diet in which the autoantibodies gradually decline, until they disappear.

Castleman's is an uncommon lymphoproliferative disorder secondary to lymphoid follicle hyperplasia and marked capillary proliferation with endothelial hyperplasia. This illness can be associated with glomerulonephritis (GN). Here, we report a case with steroid-dependent nephrotic syndrome secondary to proliferative mesangial glomerulonephritis in a patient with Castleman's disease, that was diagnosed several years before. Considering the involvement of IL-6 in Castleman's disease we treated the patient with thalidomide obtaining the remission of the nephrotic syndrome. Our experience suggests a possible role of thalidomide in the treatment of glomerular pathology when a role of IL-6 is hypothesized.

We evaluated the diagnostic and analytical performance of the Coupled Particle Light Scattering technology applied to the detection of anti-topoisomerase I (anti-Scl70) and anti-CENP-B autoantibodies.The Scl70 antigen was obtained by recombinant DNA procedures using a prokaryotic expression system; CENP-B was a Baculovirus-expressed recombinant protein. Anti-centromere and anti-Scl70 antibodies were assayed in serum samples from 288 patients, of whom 123 had systemic sclerosis/scleroderma and 165 constituted the control groups (including patients with other connective tissue diseases, viral infections, Lyme disease, and healthy subjects).The sensitivity was 98.8% (confidence interval, 96-101%) for anti-Scl70 and 100% (99.6-100%) for anti-CENP-B; the specificity was 99.0% (98-100%) and 100% (99.9-100%) for anti-Scl70 and anti-CENP-B, respectively. The intra-assay coefficient variations (CV) ranged from 3.8 to 9.2% for anti-Scl70, and from 2.8 to 8.0% for anti-CENP-B. Inter-assay CVs were 8.1 to 12.0% for anti-Scl70, and 4.7 to 10.5% for anti-CENP-B. In 3 patients, coexpression of both antibodies was observed.The results of this study demonstrate that the light scattering technology is also applicable to the detection of autoantibodies to intracellular antigens for the diagnosis of autoimmune rheumatic diseases.

Assay of the anti-thyroperoxidase (TPO), anti-thyroglobulin (Tg), and anti-TSH receptor antibodies constitutes the basis of the laboratory diagnostic work-up for autoimmune thyroid diseases. However, although these antibodies have been routinely assayed in serum for more than 40 years with increasingly reliable methods, it still exists a wide analytical variability that influences their correct diagnostic use. We analysed the biochemical, physiopathological and clinical aspects of the thyroid antigen-anti body systems, and propose the following guidelines for using autoantibody tests and analytical assay methods: a) assay of anti-TPO with third generation ultrasensitive method should constitute the main test for the diagnosis of autoimmune thyroid diseases. Assay of anti-M antibodies should be considered obsolete because even purified microsome preparations contain traces of contaminants (Tg and other antigens) that make the test aspecific; b) assay of anti-Tg antibodies may be limited to patients with suspect AITD and negative for anti-TPO antibodies, and patients undergoing thyroglobulin assay, because anti-Tg autoantibodies may interfere in the assay of the molecule in the immunometric test; c) assay of the anti-TSH receptor antibodies is used to diagnose Basedow's disease, and atrophic chronic thyroiditis; d) it is appropriate to use normal reference values adjusted for age and gender.

The ability of immunometric methods to identify anti-topoisomerase I (Scl70) antibodies is controversial. We wished to quantify the performance of the currently available commercial systems for the assay of anti-topoisomerase I antibodies in a large multicenter study.Fifty Italian clinical laboratories analyzed 36 serum samples: 27 from individuals with scleroderma/systemic sclerosis, and 9 from a control group. The scleroderma/systemic sclerosis samples were positive in our laboratories by both ELISA and immunoblot (IB), and the control samples were negative. The laboratories used 42 immunoenzymatic (ELISA), 21 IB, 3 counterimmunoelectrophoresis, and 2 dot-blot methods, produced by 23 different manufacturers.We obtained 2389 results. The ELISA methods showed 99.2% specificity and 97.2% sensitivity for detection of anti-Scl70 antibodies. For IB methods, specificity was 97.6% and sensitivity was 96.1%. The Western-blot method had poor analytical specificity (27% false positives for anti-extractable nuclear antigen antibodies other than anti-Scl70).Excluding Western blots, commercial ELISA and IB reagents as used in clinical laboratories have a sensitivity and a specificity95% for determination of anti-Scl70 antibodies.

This study was designed to assess the analytical sensitivity and rate of agreement between commercial methods and reagents, among the most used in Italy for the detection of autoantibodies to extractable nuclear antigens (ENA). Sixty-eight serum samples from patients with clinically diagnosed systemic rheumatic diseases were aliquoted and distributed to 4 hospital laboratories; three ELISA (Elias, Shield, Inova) and 1 immunoblot method (Euroimmun) were used. Overall agreement between the test reagents, for each anti-ENA specificity, was 69.1% for Ro/SSA, 83.3% for La/SSB, 70.6% for RNP, 73.5% for Sm, 91.1% for Jo1, and 82.3% for Scl70. Lack of specificity (i.e., false positive reactions) was the most important cause of low concordance. When the data were analysed according to the clinical diagnosis, total agreement and specificity improved. However, a significant difference in terms of sensitivity was observed in the SLE group (30 sera) for RNP (positivity ranged from 20% to 43%) and for Sm (from 7% to 37%), and in the Sjögren's syndrome group (13 sera) for anti-La/SSB (from 8% to 38%). Comparable data were obtained for anti-Ro/SSA (from 70% to 77%) both in the SLE and the Sjögren's syndrome group. Sensitivity of all 4 reagents was good in detecting anti-Scl70 autoantibodies in the 8 patients with diffuse systemic sclerosis, as well as anti-Jo1 autoantibody in the 5 polymyositis patients, with a 100% and a 95% agreement, respectively. These data suggest the need of a better standardization of commercial reagents and analytical procedures, and the opportunity that every laboratory should perform anti-ENA determination by at least two different methods, since none of the methods tested was completely reliable in detecting all anti-ENA autoantibody specificities.

To define whether Sjögren's syndrome (SS) patients have lymphocytopenia compared to healthy controls, and to assess which lymphocyte subset might be involved. The presence of any concurrent infection was recorded.A cohort of ten consecutive patients with SS was studied, and the results were compared with ten sex- and age-matched controls (C).In SS, a significant cytopenia of CD4+ (679 +/- 339 vs 1110 +/- 222 cells/mm3, p0.005) and an even more impressive decrease in the CD4+CD45 RA+ (242 +/- 154 vs 491 +/- 190 cells/mm3, p0.005) subset was observed. An absolute CD4 lymphocytopenia (CD4+300 cells/mm3) was seen in two patients. In one patient an unusual finding was the expansion of a double positive population of CD4+CD8+ lymphocytes. No striking relationship with any particular infection was shown. A retrospective review of the absolute CD4+ cytopenia in 54 consecutive SS cases revealed a prevalence of 5.5%.Some SS patients have T lymphocytopenia which mainly affects the CD4+CD45 RA+ subset. Occasional cases with absolute CD4 lymphocytopenia may also be observed. These patients show some evidence of mild recurrent or chronic, but never severe, opportunistic infections.

The definitive demonstration of the clonal origin of an expanded B or T cell population is achieved by the molecular analysis of B or T cell receptor gene rearrangement. However, in recent years techniques that are both sensitive and relatively easy to carry out have been introduced as the first step to the diagnosis of clonality. These include immunochemical analysis of serum and urine to characterize their monoclonal immunoglobulin components, and immunophenotypic studies of peripheral blood or tissue cells. The most widely used techniques are reviewed here, with particular emphasis on the clinical applications of immunofixation electrophoresis and multiparametric cytofluorimetric procedures.

Background: Between November 2000 and February 2001 a study has been performed by eight Italian hospital centers with the aim of verifying the usefulness of Calprest® (detection of fecal calprotectin) as a marker of bowel inflammation. Preliminary data are reported. Objective: To define the use of fecal calprotectin as a marker of intestinal inflammation, to assess whether it might help in differentiating between IBS and IBD, and its role in IBD. Subjects: Healthy population (51), active Crohn (16), non active or under remission Crohn (11), active ulcerative Colitis (8), non active or under remission ulcerative Colitis (13), pathological Controls of various nature (79). Results: The calculated cut-of value was 65 μgr/gr. 42/51 healthy subjects were negative. 16/16 patients with active CD were all positive. 8/8 patients with RCU were also positive. Conclusion: Calprest® might become an important tool in the in-vitro diagnosis of patients referred to the gastroenterology departments. The results achieved in this study demonstrate that a negative test result is an index of absence of inflammatory bowel disease. High Calprest® values (fecal calprotectin) are present, as highlighted by this study, in patients with inflammatory bowel disease.

An assessment was made on emergency laboratory test ordering at Udine General Hospital (Italy) to investigate the reasons for the excessive number of requested tests. All the orders for emergency laboratory tests during one week in June 1990 were studied. For each test the time of the order was recorded for every day of the week. The most important aspect of our investigation is the distribution of the orders during the day: in fact, test orders reached two peaks, the upper between 7 and 11 a.m., and the lower between 3 and 6 p.m. The analysis of the record cards showed that 42% of the orders were inappropriate. These preliminary findings were very useful in making the consensus on a Quality Assurance program easier, to improve the use of the Emergency Laboratory by doctors and nurses. Some preliminary results in the Emergency Medicine Department confirmed the validity of this program.

The authors investigated the effects of radiation therapy on the immune system by studying lymphocyte subsets and other parameters in 32 patients undergoing radiation therapy for solid cancer. With monoclonal antibody techniques, we studied both T- and B-lymphocytes; cell suspensions were analyzed by means of a Facs Spectrum III Ortho (Ortho-Diagnostic) unit. The first control was performed right after the beginning of radiotherapy, when the dose to the patients was 50 Gy or higher. The second control was performed at 40 Gy because all patients received this dose. 30% of the patients exhibited lymphopenia from the beginning of the study; at 40 Gy the number of T-lymphocytes was low and helper/suppressor ratio was altered. A variable response of B-cells was observed, although all patients exhibited restoration of normal values at 6 months. Four patients only suffered from side-effects: a patient with tongue cancer presented oral mycosis, and a woman--treated for breast cancer--presented vaginal mycosis. Two cases of cystitis were also observed, after 18 Gy, in patients with uterine carcinoma undergoing pelvic irradiation. Disease progression was observed in 2 patients with head and neck cancer, while 3 patients died from lung cancer progression. Another one, with head and neck cancer, died because of heart failure.

Bei einem Knaben mit einer Pseudopubertas praecox wurde ein androgenproduzierender Hodentumor entfernt. Praoperativ war die Harnausscheidung der gesamten 17-Ketosteroide, von Androsteron und Atiocholanolon, der 11-Oxy-17-Ketosteroide, insbesondere von 11β-Hydroxy-androsteron, von Pregnandiol, Pregnantriol sowie der Oestrogene abnorm erhoht und fiel nach der Operation auf Normwerte ab. Nach Inkubation von Glucose allein mit Tumorschnitten wurden 11β-Hydroxy-Δ4-androstendion undΔ4-Androstendion gefunden, nach Inkubation vonΔ4-Androstendion: 11β-Hydroxy-Δ4-androstendion, Testoseron und Adrenosteron, jedoch keine Oestrogene, nach Inkubation von Desoxycorticosteron: Corticosteron und 17α-Hydroxy-desoxy-corticosteron. Im V. spermatica-Blut wurden grose MengenΔ4-Androstendion, ferner 17α-Hydroxy-progesteron, 11β-Hydroxy-Δ4-androstendion und Testosteron nachgewiesen.

Bei Meerschweinchen mit Zerstorungsherden im Bereich des mittleren Hypothalamus wurde im Testversuch mit Diphtherietoxin die Reaktion des Nebennierenrindengewebes, das Verhalten des Zellkernvolumens der Zona fasciculata und die Ausscheidung der 17-Hydroxycorticosteroide im Harn gepruft und mit dem Verhalten von Normaltieren nach Diphtherietoxin verglichen.

1. Periphere Penicillingaben bewirken beim Meerschweinchen, jedoch nicht bei der Ratte nach einer charakteristischen Latenzzeit eine todliche Enteritis. Diese ist durch schwere Durchblutungsstorungen der Darmwand, massive Exsudation und nachfolgende Zottennekrose gekennzeichnet. Die Enteritisentstehung kann durch die Kontaktnahme Penicillin—Darmwand nicht erklart werden. 2. Nach intracerebraler Injektion kleinster Penicillinmengen tritt beim Meerschweinchen, wiederum nach einer Latenzzeit, eine schwere Enteritis, bei der Ratte dagegen eine vorubergehende Durchblutungsstorung der Darmwand auf. Bei beiden Tierarten bewirkt der Kontakt Penicillin—Zentralnervensystem ein schweres Krampf-syndrom. 3. Auf Grund verschiedenartiger Versuchsanordnungen kann die Entstehung der fur die Enteritisgenese masgeblichen Durchblutungsstorung der Darmwand auf eine zentral-nervale Erregungswirkung des Penicillins zuruckgefuhrt werden. 4. Nach den Versuchen vonKoch und Mitarbeiter reichert sich intramuskular gegebenes Penicillin im Gehirn des Meerschweinchens an, nicht aber in dem der Ratte. Der Unterschied ist durch zwei Faktoren bedingt: Verschiedene Durchlassigkeit der Bluthirnschranke und differentes Penicillinabbauvermogen der Hirnsubstanz bei beiden Tierarten. 5. Die Enteritis des Meerschweinchens ist daher ein Modell einer zentral ausgelosten Erkrankung der Darmschleimhaut. Der Entstehungsmechanismus last sich in die TheorieRickers einordnen. 6. Die Nebennierenrinde des Meerschweinchens entwickelt nach Penicillinverabreichung die typischen Zeichen der progressiven Transformation Dies ist erklarbar durch die beim Meerschweinchen nach Penicillin entstehende Stress-Situation.

Sera from 55 patients with positivity for anti-HTLV III antibodies, including 24 "healthy" subjects, 29 LAS and 2 AIDS, were examined by means of high resolution agarose gel electrophoresis and immunofixation, in order to evaluate the occurrence of monoclonal-oligoclonal immunoglobulin patterns. Monoclonal-oligoclonal immunoglobulins were found in 20.8% of "healthy" subjects and in 72.4% of LAS patients. Furthermore, a lower but significant prevalence of monoclonal-oligoclonal immunoglobulins was found in a control group including patients with high-titre positivity for anti-CMV and anti-EBV antibodies. Considering the consistent association between EBV-CMV and HTLV III infection, it is concluded that at least a part of monoclonal-oligoclonal immunoglobulins found in LAS-AIDS patients are likely to be induced by these associated viral infections. On the other hand, the finding of monoclonal-oligoclonal immunoglobulins in "healthy" HTLV III-positive subjects points out a possible direct stimulatory effect of HTLV III on B-cell lineage.

The following conclusions which show the importance of time of medical treatment resulted from the examination of 288 testicular biopsies (including 46 controls) by statistical analysis in correlation to age. 1. There is no difference in the number of spermatogonia per tubule cross section of descended and undescended testes in children from birth to 2 years of age. 2. The number of spermtogonia in descended testes is not different in children from birth to 2 years in comparison to children aged 2-6 years. 3. A statistically significant decrease of spermatogonia takes place in the undescended testes of children from 2-6 years in comparison to children from birth to 2 years. 4. The difference in the number of spermatogonia of descended and undescended testes increases in children above the age of 2. It must be concluded from this study that the pathological alterations of the germinative epithelium increase in children beyond the age of 2. Assuming that pathological alterations of the germinative epithelium are caused by the dystopy of the testes, medical treatment should be carried out before the child is more than 2 years old.

The study of T lymphocyte subpopulations defined by monoclonal antibodies in a group of patients with rheumatoid arthritis and a control group indicated no significant differences in the percentages of total T lymphocytes (OKT3+) or helper-inducer lymphocytes (OKT4+). However there was a significant diminution in suppressor-cytoxic lymphocytes (OKT8+) in patients with active forms of the disease. This diminution led to an increase in the proportion of OKT4+/OKT8+ in patients with active R.A. forms compared to both those in remission and the control group.

In an attempt to investigate an aspect of the immunoregulatory mechanisms operating in alcoholic hepatic cirrhosis with ascites we have enumerated T-cell subsets in the peripheral blood and ascitic fluid of 16 patients with this disease. The peripheral blood analysis revealed a reduction of OKT3+, OKT4+ and OKT8+ cells compared to those of the control group (p less than 0.001) and an increase of OKT4+/OKT8+ ratio. In the ascitic fluid the OKT4+/OKT8+ ratio, compared to that of the peripheral blood, was decreased (p less than 0.001) because of increase of OKT8+ cell proportion. We suggest that the sequestrated splanchnic OKT8+ subset might be represented by suppressor lymphocytes transuding from the liver.

Using monoclonal antibodies of the OKT series we studied the blood T lymphocyte subsets of 12 patients with Primary Sjögren's Syndrome, 15 with Rheumatoid Arthritis associated Sjögren's Syndrome and 10 normal controls. We found a marked reduction of T lymphocytes both in Primary and Secondary Sjögren's Syndrome compared with normal controls (p less than 0.001). While no difference was noted in the number of helper-inducer T cells between Primary and Secondary Sjögren's Syndrome, a more important decrease of suppressor-cytotoxic cells was demonstrated in Secondary Sjögren's Syndrome (p less than 0.01). These data may contribute to a better understanding of the disease.

During a five years survey of unselected serum protein electrophoreses in a general hospital, 2347 sera (0.95%) were found to be positive for one or more monoclonal immunoglobulins. Class and type distribution of monoclonal immunoglobulins (characterized by means of immunofixation electrophoresis) mostly corresponded with the literature data. Nevertheless, higher incidence and lower mean age at detection were observed for IgG monoclonal components. This finding is probably related to the discovery of a large number of micro-monoclonal immunoglobulins with no association with B-lymphoproliferative disorders, mostly belonging to the IgG class and affecting younger people. The need is stressed for a widespread standardization of the techniques employed for detection and characterization of monoclonal immunoglobulins, in order to obtain more comparable results.