IgA anti-actin antibodies ELISA in coeliac disease: a multicentre study.

BACKGROUND: Previous studies have demonstrated that serum anti-actin antibodies are a reliable marker of intestinal damage severity in coeliac disease. AIMS: To validate in a multicentre study the clinical usefulness of serum IgA anti-actin antibody ELISA and its possible use in monitoring intestinal mucosa lesions during gluten-free diet. PATIENTS AND METHODS: Four centres recruited 205 newly diagnosed coeliac disease patients with villous atrophy, 80 healthy controls and 81 "disease" controls. Twelve coeliac disease patients on gluten-free diet but with persistent symptoms underwent serum IgA anti-actin antibody assay and intestinal histology evaluation. IgA anti-actin antibody ELISA was performed with a commercial kit. All coeliac disease patients underwent intestinal histology study. RESULTS: IgA anti-actin antibodies showed a sensitivity of 80% and a specificity of 85% in the diagnosis of coeliac disease patients with villous atrophy. The area under the receiving operator curve for anti-actin antibodies was 0.873 [95% C.I. 0.805-0.899]. Serum anti-actin antibodies values were significantly higher in coeliac disease patients than in healthy or "disease" controls (P<0.0001). Serum anti-actin antibodies were positive in 41 of the 60 coeliac disease patients with mild intestinal histology lesions (69%) and in 123 of the 145 with severe lesions (85.3%) (P<0.05). There was a significant inverse correlation between anti-actin antibody values and the villi/crypts ratio (r=-0.423; P<0.0001). In the 12 coeliac disease patients on gluten-free diet who underwent re-evaluation as they were persistently symptomatic, intestinal histology showed three cases with persistent villous atrophy: all of these were positive for serum anti-actin antibodies ELISA, whereas both serum anti-tTG and EmAs were negative. The other nine patients showed normal intestinal villi and were negative for serum anti-actin antibodies. CONCLUSIONS: Anti-actin antibodies are a reliable marker of severe intestinal mucosa damage in coeliac disease patients and a simple ELISA technique offers an accurate method for their determination. These antibodies seem to be a very reliable marker of persistent intestinal damage in coeliac disease patients.

Gastrointestinal symptoms in infancy: a population-based prospective study.

BACKGROUND: During the first months of life, infants can suffer from many 'minor' gastroenterological disturbances. However, little is known about the frequency of these problems and the factors which predispose or facilitate their onset. AIMS: (a) To ascertain the frequency of the most common gastrointestinal symptoms in infants during the first 6 months after birth; (b) to evaluate the influence of some variables on the onset of the symptoms. STUDY DESIGN AND PATIENTS: Each of the 150 paediatricians distributed throughout Italy followed 20 consecutive infants from birth to 6 months. 2879 infants (1422 f, 1457 m) concluded the study. The presence of the following symptoms was evaluated: constipation, diarrhoea, vomiting, regurgitation, failure to thrive and prolonged crying fits (colic). Symptoms were recorded whenever the parents requested a clinical check-up or during a set monthly examination. RESULTS: 1582/2879 (54.9%) infants suffered from one of the gastrointestinal symptoms. Regurgitation was the most common disturbance (present in 23.1% of infants), followed by colic (20.5%), constipation (17.6%), failure to thrive (15.2%), vomiting (6%) and diarrhoea (4.1%). Low birth weight was the factor most frequently associated with the onset of gastrointestinal symptoms, followed by low gestational age. Feeding habits did not influence the onset of symptoms, with the exception of constipation, which was linked to a low frequency of breast-feeding. Ninety-three infants (3.2%) were hospitalised for one or more of the gastrointestinal symptoms which were considered. During the whole study period the type of formula-milk was changed in 60% of the infants with one or more gastrointestinal symptoms, and in 15.5% of the infants who did not suffer from any gastrointestinal troubles. CONCLUSIONS: Gastrointestinal symptoms are very common in infants during the first 6 months after birth. These symptoms required hospitalisation only in a small percentage of cases, but led to the prescription of a 'dietary' milk formula in approximately 60% of the cases. Low birth weight and low gestational age were the main factors influencing the onset of the symptoms.

Scrapie strain infection in vitro induces changes in neuronal cells.

PC12 cells, in the presence of nerve growth factor (NGF), support replication of the mouse-derived scrapie strains 139A and ME7, with the former yielding 100-1000-fold higher levels of infectivity. Infectivity remained cell-associated and cells did not show any gross morphological alterations, although changes were observed by electron microscopy in the form of an increased number of lipid droplets in 139A-infected cultures. Analysis of phospholipid metabolism in 139A infected cells indicated that scrapie replication did not change the inositol phosphate levels, but did stimulate phosphoinositide synthesis. Replication was not detected in PC12 cells infected with either the hamster-derived 263K or rat-derived 139R scrapie strains. Since scrapie-infected cultures did not exhibit cell death or any gross changes, any scrapie-induced effects would probably be manifested in nonvital cellular functions. When compared to controls, infection with the 139A scrapie strain resulted in decreased activity of the cholinergic pathway-related enzymes, as well as the GABA synthetic pathway; however, the adrenergic pathway was unaffected by scrapie infection. The effects of the 139A scrapie strain on the cholinergic system appeared to be dose-dependent and were first detected prior to the detection of scrapie agent replication in these cells. No neurotransmitter-related enzymatic changes were detected in 263K- or 139R-infected PC12 cells. The enzymatic changes observed in ME7-infected PC12 cells and in Chandler agent-infected mouse neuroblastoma cells suggest that the significant changes in neurotransmitter levels in cultures exhibiting low infectivity titers must involve factors other than, but not excluding, replication of the agent. The role of additional factors is also suggested in studies of protein kinase C activity in 139A- and 139R-infected PC12 cells. These studies emphasize the value of the PC12 cell model system in examining the scrapie strain-host cell interaction and, in addition, support the concept of variation among scrapie strains.

Iatrogenic Creutzfeldt-Jakob disease: an example of the interplay between ancient genes and modern medicine.

We tested DNA from 15 centrally infected cases of iatrogenic Creutzfeldt-Jakob disease (CJD) (dura mater or corneal homografts and stereotactic EEG electrodes), 11 peripherally infected cases (native human growth hormone or gonadotrophin), and 110 control individuals for the presence of mutations in the chromosome 20 amyloid gene. No patient or control had any of the known pathogenic point or insert mutations found in familial disease, but allelic homozygosity at polymorphic codon 129 was present in all but two (92%) of the 26 patients, compared with 54 (50%) of the 110 controls (p < 0.001). Pooled data from all identified and tested cases of iatrogenic disease yielded a worldwide total of 56 patients, of whom all but four were homozygous at codon 129 (p < 0.001). These findings support the thesis that homozygosity at codon 129 enhances susceptibility to iatrogenic infections of both central and peripheral origin, with evident implications for the population of dura mater homograft and pituitary hormone recipients whose lives have been complicated by the possibility of exposure to the infectious agent of CJD.

The natural history of adrenal function in autoimmune patients with adrenal autoantibodies.

Adrenal autoantibodies (AA) were found in 23 of 2571 (0.9%) patients with organ-specific autoimmune diseases, in one of 632 first-degree relatives of insulin-dependent diabetic patients, and in none of 375 normal controls. In AA-positive subjects the prevalence of human leucocyte antigens (HLA)-A1, -B8 and -DR3 was significantly higher with respect to the general population. Two groups were followed (15 subjects persistently positive for AA and 51 negative subjects) for a mean period of 3.2 years. Yearly tests were made for AA and adrenal function. Of the 15 subjects persistently positive for AA, six developed Addison's disease after a period varying from 6 months to 10 years. Of the 51 subjects initially negative, two became positive during follow-up, and one of these developed Addison's disease 15 months later. In contrast, all the remaining 49 persistently negative subjects maintained normal adrenal function tests. Overall, of the 17 positive subjects, seven (41%) developed Addison's disease, three (18%) showed various degrees of subclinical adrenocortical failure and the remaining seven maintained normal glandular function. In the positive patients the yearly incidence of detriment in adrenal function was 19%. Patients who developed Addison's disease showed significant association with HLA-B8 phenotype. The development from normal adrenocortical function to overt Addison's disease seemed to progress through four distinct stages of functional impairment: increased plasma renin activity with normal/low aldosterone (stage 1), low cortisol response after i.v. administration of ACTH (stage 2), increased ACTH (stage 3), and low basal cortisol (stage 4).(ABSTRACT TRUNCATED AT 250 WORDS)

[Addison's disease: principal clinical associations and description of natural history of the disease]. / Morbo di Addison: principali associazioni cliniche e descrizione della storia naturale della malattia.

From 1967 to 1988 we studied 75 cases of Addison's disease (AD). An autoimmune etiology was found in 68%, while previous tubercular infection was demonstrated in 21% of the cases; minor causes were involved in 3%, and in 8% of the cases the disease remained of unknown origin. Autoimmune adrenalitis has become the most frequent cause of AD as a consequence of decreased tubercular infection. Autoimmune AD prevailed in children or in young people, and tubercular AD in adults. The finding of calcifications in adrenals and/or other organs was the specific diagnostic sign for the identification of tubercular forms. On the other hand, the finding of circulating adrenal autoantibodies (AA) and/or other organ specific autoantibodies was the fundamental diagnostic marker of autoimmune forms. In 72% of the cases autoimmune AD is associated with other organ-specific autoimmune diseases. Subjects with AA but without clinical signs of hypoadrenalism were considered to have "potential AD", because they showed a high risk of developing clinical hypoadrenalism. This condition develops over a long period characterized by different stages of subclinical adrenal hypofunction. Steroid cell autoantibodies (StCA) are frequently detectable in patients with autoimmune AD, in whom they are markers of autoimmune premature ovarian failure. The study of StCA-positive cases without hypogonadism will be important to clarify whether or not these autoantibodies could be markers of potential autoimmune hypogonadism.

PCR detection of Salmonella typhimurium in pharmaceutical raw materials and products contaminated with a mixed bacterial culture using the BAX system.

The BAX system, a PCR-based assay, was evaluated for detecting Salmonella typhimurium in pharmaceutical raw materials and products contaminated with mixed bacterial cultures of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella typhimurium. Artificially contaminated samples were preenriched in lactose broth with and without Tween 20. After preenrichment, samples were analyzed by PCR and standard methods. Ten of 25 samples did not show presence of the specific Salmonella spp. 740-base pair DNA fragment. However, S. typhimurium was isolated and identified by standard methods from all 25 samples. To optimize S. typhimurium detection in PCR negative samples, lactose broth was replaced by buffered peptone water (BPW) as the preenrichment broth. When BPW was used, all 10 samples were PCR positive. BPW enrichments increased S. typhimurium growth resulting in rapid PCR detection. The presence of non-Salmonella bacteria influenced the performance of the PCR-based assay. Optimization of S. typhimurium PCR detection in mixed culture required the use of different preenrichment broths. However, the BAX system detected S. typhimurium within 27 hours while standard methods required 5-7 days.

Oral pathology in untreated coeliac [corrected] disease.

BACKGROUND: Many coeliac disease patients with atypical symptoms remain undiagnosed. AIM: To examine the frequency of oral lesions in coeliac disease patients and to assess their usefulness in making coeliac disease diagnosis. PATIENTS AND METHODS: One hundred and ninety-seven coeliac disease patients and 413 controls were recruited and the oral examination was performed. RESULTS: Forty-six out of 197 coeliac disease patients (23%) were found to have enamel defects vs. 9% in controls (P < 0.0001). Clinical delayed eruption was observed in 26% of the pediatric coeliac disease patients vs. 7% of the controls (P < 0.0001). The prevalence of oral soft tissues lesions was 42% in the coeliac disease patients and 2% in controls (P < 0.0001). Recurrent aphthous stomatitis disappeared in 89% of the patients after 1 year of gluten-free diet. Multi-logistic analysis selected the following variables as the most meaningful in coeliac disease patients: dental enamel defects (OR = 2.652 CI = 1.427-4.926) and soft tissue lesions (OR = 41.667, CI = 18.868-90.909). Artificial Neural Networks methodology showed that oral soft tissue lesions have sensitivity = 42%, specificity = 98% and test accuracy = 83% in coeliac disease diagnosis. CONCLUSIONS: The overall prevalence of oral soft tissue lesions was higher in coeliac disease patients (42%) than in controls. However, the positive-predictive value of these lesions for coeliac disease diagnosis was low.

Alterations in neurotransmitter-related enzyme activity in scrapie-infected PC12 cells.

Enzyme activities associated with the neurotransmitter pathways in nerve growth factor-treated, 139A scrapie strain-infected PC12 cells were examined. Since these cells show no morphological alterations during the time of agent replication, any scrapie-induced effects would have to be associated with non-vital cellular functions. When compared to controls, infection with the 139A scrapie strain resulted in decreased activity of the cholinergic pathway-related enzymes, choline acetyltransferase and acetylcholinesterase. However, the adrenergic pathway was unaffected by scrapie infection as evidenced by unaltered tyrosine hydroxylase activity, the putative rate-limiting enzyme in the synthesis of catecholamines. The effects of the 139A scrapie strain on the cholinergic system appeared to be dose-dependent and were first detected prior to the detection of scrapie agent replication in these cells. Furthermore, the altered enzymic activities observed were not the result of contaminating material in the scrapie brain homogenate because similar results were obtained when partially purified scrapie preparations were used as the inoculum. These scrapie agent-induced alterations in specific neuronal properties suggest a mechanism for the clinical manifestations observed in scrapie and perhaps other related central nervous system disorders.

Further characterization of scrapie replication in PC12 cells.

The rat pheochromocytoma cell line, PC12, undergoes neuron-like morphological, biochemical and electrophysiological differentiation, in the presence of low concentrations of nerve growth factor (NGF). NGF-treated PC12 cells have been shown previously to support 139A scrapie agent replication. In the present report we extended these findings and analysed the cellular conditions necessary for agent replication. Following the infection of differentiated PC12 cells, scrapie replicated to relatively high titres as determined by an incubation period assay. The removal of NGF, which causes the gradual dedifferentiation of PC12 cells, resulted in the inability of scrapie to replicate. The scrapie infectivity detected in PC12 cultures is cell-associated and not released into the medium. Cells in infected cultures did not show any change in morphology when compared to cells in mock-infected cultures. Titration studies of scrapie infectivity in PC12 cells have indicated that up to 4 LD50 units per cell can be obtained although a yield of 1 LD50 per cell was more common. Using an approximate m.o.i. of 1, only differentiated PC12 cells supported 139A scrapie agent replication when compared to two other differentiated, neuronal cell types, indicating that PC12 cells are more susceptible to agent replication. These studies support further the suitability of using differentiated PC12 cells as an in vitro model to study scrapie agent replication.

Concentration and distribution of infectivity and PrPSc following partial denaturation of a mouse-adapted and a hamster-adapted scrapie strain.

PrPSc is a specific protein marker for slow infectious diseases known as the transmissible subacute spongiform encephalopathies. Although PrPSc is closely associated with infectivity, it is not known if it is the infectious agent itself, a component of the agent or merely adventitiously associated with infectivity. In the present study we demonstrate that the resistance of PrPSc to partial denaturation and of infectivity to inactivation differs markedly for two scrapie strains. Proteinase K treatment or electrophoretic analysis of partially denatured PrPSc preparations reveal a dissociation between infectivity and demonstrable PrPSc. Our findings support other evidence that not all PrPSc is required for infectivity. Our studies combined with previous biological analyses suggest that PrPSc cannot be the sole component associated with the infectious agent.

Scrapie-infected spleens: analysis of infectivity, scrapie-associated fibrils, and protease-resistant proteins.

Scrapie-associated fibrils (SAF) and protease-resistant proteins (PrP) were isolated from spleens and brains of clinical animals (mice and hamsters) from three scrapie agent-host strain combinations, and their concentrations were compared with infectivity levels. The spleens of infected animals contained lower levels of infectivity, PrP, and SAF than did brains. Regardless of the route of infection, both SAF and infectivity were detected in spleen before brain. Infectivity increased in brains and spleens of 139A-infected mice before the detection and increase in SAF, suggesting that the synthesis of SAF and PrP may not be the limiting factor in agent replication. In contrast to those in ME7- and 263K-infected animals, the Western blot profiles for PrP from brain and spleen of 139A-infected mice exhibited distinct differences. Results indicate that SAF and PrP found in the spleens are both organ- and scrapie strain-specific.

Demonstration of scrapie strain diversity in infected PC12 cells.

Scrapie strain replication in the nerve growth factor-induced, differentiated PC12 cell culture system was examined. Differences in replication between mouse-derived agents were demonstrated, with the 139A scrapie strain yielding 100- to 1000-fold higher levels of infectivity than the ME7 scrapie strain. Replication was not detected in PC12 cells infected with either the hamster-derived 263K or rat-derived 139R scrapie strains. Studies on the neurotransmitters in infected PC12 cells demonstrated that the adrenergic pathway was unchanged but the cholinergic pathway was altered. Furthermore, the degree of alteration correlated with the level of scrapie strain replication. Comparison of infectivity titres and enzymatic changes in ME7-infected PC12 cells with those in Chandler agent-infected mouse neuroblastoma cells suggests that the significant changes in neurotransmitter levels in cultures exhibiting low titres of infectivity involve factors in addition to strain replication. The variation in the range of scrapie strain replication in PC12 cells is discussed in relationship to species barrier, cell targeting, genetic susceptibility and species strain specificity. These studies further emphasize the value of the PC12 cell model system in examining the scrapie strain-host cell interaction and in addition support the concept of variation among scrapie strains.

Detection of scrapie-associated fibrils (SAF) and SAF proteins from scrapie-affected sheep.

Scrapie-associated fibrils (SAF) were detected by negative-stain electron microscopy in the brains (by two different isolation procedures) and spleens of sheep naturally and experimentally infected with scrapie. Although the numbers of SAF varied from case to case, the yield of SAF from brains of naturally affected sheep was lower than that from experimentally affected sheep. SAF-specific, protease-resistant proteins (PrPs) were detected by silver staining and western blot analysis in most samples of brain from experimentally affected sheep. PrPs, however, could be detected in only a limited number of natural cases of sheep scrapie because of the lower yields of SAF. PrPs from sheep SAF appear biochemically and antigenically similar to PrPs from other species infected with unconventional agents. This study further establishes the unique association of SAF and PrPs with natural or experimentally induced scrapie in its natural host.