Belgian recommendations on ANA, anti-dsDNA and anti-ENA antibody testing.

Autoantibodies to nuclear antigens, i.e. antinuclear antibodies (ANA), antibodies to double-stranded DNA (dsDNA) and extractable nuclear antigens (ENA), are useful as diagnostic markers for a variety of autoimmune diseases. In March 2010, the Belgian national External Quality Assessment Scheme sent a questionnaire on ANA, anti-dsDNA and anti-ENA antibody testing designed by the Dutch EASI (European Autoimmunity Standardization Initiative) team, to all clinical laboratories performing ANA testing. Virtually all laboratories completed the questionnaire (97·7%, 127/130). This paper discusses the results of this questionnaire and provides valuable information on the state-of-the-art of ANA, anti-dsDNA and anti-ENA antibody testing as practiced in the Belgian laboratories. In addition, this work presents practical recommendations developed by the members of the advisory board of the scheme as a result of the outcome of this study.

[Pathophysiology of cholesteatoma]. / La physiopathogénie des cholestéatomes.

OBJECTIVE: To describe the development of cholesteatoma using current knowledge. METHOD: Review of the literature. RESULTS: Cholesteatoma describes a mass of keratin (skin) in the middle ear which consists of a perimatrix and matrix. There are at least three kinds of cholesteatoma in the middle ear one resulting from invagination (retraction's pocket), another from migration and the last one from congenital inclusion. Cholesteatoma needs three successive inflammatory phases, the first leading to a retraction pocket, the second leading to pathology of the epidermis and of the floor of the external auditory canal and the third is the actual phase of cholesteatoma with invasion and middle ear auto-destruction with bone resorption. In this last phase, many factors play a role, collagenasis, osteoclats, cytokines, NO, bacteria and their biofilm and rupture of the retraction pocket. CONCLUSION: Cholesteatoma is an inflammatory disease of the ear caracterised by bone resorption. Current research is starting to appreciate the important role the immune system plays in the pathophysiology of cholesteatoma.

Prospective screening for biopsy proven coeliac disease, autoimmunity and malabsorption markers in Belgian subjects with Type 1 diabetes.

AIMS: To determine prospectively the prevalence of biopsy proven coeliac disease (CD) in an adult Type 1 diabetic population from Belgium with regards to associated auto-immunity and malabsorption. METHODS AND RESULTS: Determination in 400 Type 1 diabetic patients of serum anti-endomysial and/or anti-transglutaminase auto-antibodies. All subjects with abnormal serology underwent an intestinal biopsy. Ten patients (2.5%) had positive antibodies. Diagnosis of CD was confirmed by an intestinal biopsy. Eight patients were symptom-free, although laboratory findings suggesting malabsorption were prominent in the presence of CD [microcytic anaemia, iron and folate deficiencies, low levels of 25(OH)vitamin D3, calcium and cholesterol]. Other auto-immune conditions, especially vitiligo, were found in patients with CD. CONCLUSIONS: Asymptomatic coeliac disease occurs frequently in adult Type 1 diabetic patients, and is often associated with subclinical malabsorption. Screening should be part of routine evaluation, to implement life-long dietary gluten avoidance.

COCH5B2 is a target antigen of anti-inner ear antibodies in autoimmune inner ear diseases.

OBJECTIVE: This study was designed to identify the 58-kDa inner ear protein against which the sera of some patients with idiopathic, progressive sensorineural hearing loss or Ménière's disease strongly react. BACKGROUND: We and other groups have previously demonstrated that a 58-kDa protein extracted from guinea pig or bovine inner ear tissue is a target of antibodies in serum samples from some patients with autoimmune inner ear diseases. METHODS: After separation of inner ear proteins by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, the bands corresponding to 58 kDa were localized and excised from the gel. The concentrated protein was then digested with trypsin, and the peptide fragments were separated by high-pressure liquid chromatography. Three fractions were subjected to amino acid sequencing by the classic Edman degradation. RESULTS: The sequence of a stretch of 14 amino acids of the first fragment was identical to that of amino acids 526 to 539 of the COCH5B2 protein. The sequences of 11 and 10 amino acids of the second and third fragments, respectively, also were identical to residues 417 to 427 and 396 to 405 of the COCH5B2 protein. These data, together with two-dimensional gel electrophoresis followed by Western blot experiments, confirmed that the 58-kDa inner ear protein is the COCH5B2 protein. DISCUSSION: These findings indicate that the 58-kDa target protein of antibodies in serum samples of patients with autoimmune inner ear diseases is the COCH5B2 protein, a molecule that is highly and specifically expressed in the cochlea and vestibule.

Inner ear autoantibodies and their targets in patients with autoimmune inner ear diseases.

Immunological mechanisms are thought to play an important role in the pathogenesis of some cochleo-vestibular diseases. This study attempts to present further evidence of autoantibodies reactive against guinea pig inner ear proteins found in patients with autoimmune inner ear diseases (AIED) and specifically identifies the main target antigens of these antibodies. Sera from 110 patients with a clinical diagnosis of either rapidly progressive sensorineural hearing loss (n = 32). Ménière's disease (n = 41), sudden deafness (n = 6) or other aetiologies of hearing loss (n = 11) were screened by the Western blot technique. Forty-four percent of the patients' sera had antibodies to several inner ear proteins, of which the 30, 42 and 68 kDa proteins were found to be the most reactive. These highly reactive proteins were identified by gas-phase micro sequencing after digestion with trypsin and separation of peptide fragments by high-performance liquid chromatography. A partial sequence of each protein was determined. These data, together with those obtained from 2-dimensional gel electrophoresis followed by Western blotting, demonstrated that the 30 and 42 kDa inner ear proteins are the major peripheral myelin protein P0 and the beta-actin protein, respectively, while sequence analysis indicated that the 68 kDa protein is novel. These findings further support the hypothesis that several populations of antibodies may contribute to the enhanced immunological activity of AIED patients. They also add a new dimension to our knowledge of AIED and may open new avenues in the development of simple serological assays, which are easier to perform and more rapid than Western blotting.

No increased serum levels of antifood antibodies in patients with Ménière's disease.

The etiology of Ménière's disease (MD) is still unknown, but it is likely to be multifactorial, one of the factors being an immunological causation. Antifood allergens as well as anti-baker's yeast antibodies are humoral factors that may be linked with allergenic disorders and other autoimmune conditions. To determine their possible role in MD activity, we investigated 29 MD sera for the presence of antibodies against gliadin, beta-lactoglobulin, albumin, ovalbumin, soya, and Dermatophagoides pteronyssinus and Saccharomyces cerevisiae strains using an ELISA technique. The patients were compared with 29 healthy individuals matched for sex and age. A serum was regarded as positive if the absorbance was two standard deviations higher than values obtained with sera from healthy subjects. Historical data, including factors which the patients believed to provoke their Ménière's symptoms, were obtained from patients' questionnaires. MD patients showed no significant symptoms of allergenic disorders suggesting allergies when compared to controls (p > 0.05). IgG and IgA antibody levels were not significantly raised in MD patients as compared with healthy controls (p > 0.05) for gliadin, beta-lactoglobulin, soya, albumin, ovalbumin, and D. pteronyssinus and S. cerevisiae strains. These data do not convincingly support a hypothesis of increased serum levels of antifood antibodies in patients with MD, as very few patients were antibody positive.

No evidence of auditory dysfunction in guinea pigs immunized with myelin P0 protein.

Recent data have focused on the peripheral nerve myelin glycoprotein P0 as a putative autoantigen involved in the autoimmune etiology of some cases of Meniere's disease, idiopathic sensorineural hearing loss and sudden deafness. To determine whether antibodies to myelin P0 can alter cochlear function, 13 healthy guinea pigs were immunized with purified porcine myelin P0 while 10 controls were injected with saline water. The animals were then evaluated for evidence of evolving inner ear disease using immunological, electrophysiological and morphological methods. Twenty-six experimental ears were tested weekly with a brainstem auditory evoked potential technique for a period of 4 months and were compared to 20 control ears. Uniformly, all P0-sensitized guinea pigs showed antibodies to myelin protein P0 as evidenced by ELISA. Clinical signs of inflammatory demyelination were not discernible in P0-sensitized guinea pigs and all the animals were qualitatively normal. No significant increase of evoked potential thresholds was found in the P0-sensitized animals when compared to controls (P>0.05). Peak latencies of waves I, II, III, IV and V and inter-peak latencies in P0-sensitized guinea pigs did not significantly differ from those of controls (P>0.05). Histological sections of inner ear and peripheral nerves were free of disease in both groups. These findings indicate that the sole presence of antibodies to myelin P0 in the sera of guinea pigs or patients suspected of having autoimmune inner ear diseases is unlikely to elicit auditory abnormalities and that additional factors are necessary for the pathogenic development of these disorders.

Autoimmune sensorineural hearing loss in young patients: an exploratory study.

OBJECTIVES: P0 protein is expressed exclusively in myelinating Schwann cells of the peripheral nervous system. In a previous study from our laboratory, 27% of patients with sensorineural hearing loss (SNHL) had antibodies to P0 protein in their serum. The purpose of the present exploratory study was to examine the relationship between the clinical presentation of SNHL among children and young adults (age range, 5-30 y) and the presence of serum anti-P0 antibodies. STUDY DESIGN: The data were collected by retrospective questionnaires from Belgian otolaryngologists. METHODS: Patients were divided for comparison into two groups according to the presence or absence of anti-P0 antibodies. RESULTS: Analyses of clinical data and audiometric results indicated that a progressive hearing loss was more frequently recorded in the patients in the anti-P0 antibody-positive group (82% [14 of 17]) than in those in the anti-P0 antibody-negative group (35% [6 of 17]) (P <.005). CONCLUSIONS: Thus, in the age group in the present study, autoimmune SNHL (as measured in the present study by the presence of anti-P0 antibodies) is more frequently associated with progressive than with sudden hearing loss. The implications of this finding for preventive screening of hearing loss in children and young adults are discussed.

Identification of beta-actin as a candidate autoantigen in autoimmune inner ear disease.

It has been shown that sera from patients with autoimmune inner ear disease contain antibodies to several inner ear antigens. We report here the characterization of the 42-43 kDa protein against which a significant number of patients' sera react strongly. After separation of inner ear proteins from guinea-pig cochleas by SDS-PAGE, the band corresponding to the 42-43 kDa protein was digested with trypsin and the peptide fragments were separated by high-performance liquid chromatography. Two fractions were then subjected to amino acid sequencing by the classical automated Edman degradation. The sequence of a stretch of 15 amino acids of the first fragment was identical to that of amino acids 148-162 of beta-actin. The sequence of the 10 amino acids of the second fragment was also identical to beta-actin. On Western blots, monoclonal antibody directed against beta-actin reacted with the inner ear 42-43 kDa proteins. The serum samples from the patients and the monoclonal antibody reacted with the non-muscle actin used as antigen in Western blotting. Immunoblot analysis of inner ear proteins after two-dimensional gel electrophoresis showed a spot, corresponding to the region of the 43 kDa as compared to the protein standards. On the basis of these data it is concluded that the target 42-43 kDa protein for antibodies in sera of patients with autoimmune inner ear disease is beta-actin, a molecule, which has important and numerous functions inside cells. This is the first report to identify the cytoskeletal protein beta-actin as a candidate autoantigen in autoimmune inner ear disease.

Clinical significance of antiproteinase 3 antibody positivity in cANCA-positive patients.

We addressed the clinical significance of antiproteinase 3 (anti-PR3) antibody (Ab) positivity by reviewing the files of 79 patients whose serum contained antineutrophil cytoplasmic antibodies with a cytoplasmic staining pattern (cANCA) and had been tested for anti-PR3 reactivity. Vasculitis was present in most (22/35) cANCA+ PR3+ patients but in only a few (5/44) cANCA+ PR3- patients, thereby suggesting that anti-PR3 Ab positivity in cANCA+ patients is more indicative of vasculitis than cANCA positivity alone. Noteworthy, one-third of cANCA+ PR3+ patients -- those with anti-PR3 Ab titres lower than 100 U/ml -- did not suffer from vasculitis. Anti-PR3 reactivity in vasculitis patients was only weakly associated with Wegener's granulomatosis (WG), as nine out of 22 cANCA+ PR3+ vasculitis patients (41%) did not fulfil the ACR classification criteria for WG. There was no correlation between anti-PR3 Ab titres and disease activity at diagnosis. However, titres measured when patients were in remission were much lower than initial values. Taken together, our results indicate that anti-PR3 Ab positivity should be interpreted in its clinical context.

[Rectal leiomyosarcoma. 4 new cases]. / Léiomyosarcome du rectum. Quatre nouvelles observations.

We report four cases of leiomyosarcoma of the rectum suspected by endoscopic ultrasonography. Three patients were treated by local excision and one by abdominoperineal resection. An excision of the mass via a Kraske's approach was used. Leiomyosarcoma confined to the rectum wall can be treated by local excision. Endosonography can provide exact estimation of the lesion and is of great value in selecting the appropriate treatment. The treatment is surgical excision with wide margins. The histological stage and the presence or absence of metastases determine the therapeutic. Two patients in our series underwent radiation therapy. Chemotherapeutic agents including doxorubicin have had beneficial effect on recurrence or survival, only for higher grade sarcomas.

Right nonrecurrent inferior laryngeal nerve and arteria lusoria: the diagnostic and therapeutic implications of an anatomic anomaly. Review of 17 cases.

The authors report 17 cases of a right non-recurrent inferior laryngeal n. (NRILN) observed during 15 years of practice of thyroid and parathyroid surgery. In their last two cases, the existence of an aberrant right subclavian a., constantly associated with NRILN, was confirmed by MRI angiography. On the basis of the literature and their own experience, the authors review the incidence of this double anomaly, its embryologic explanation and its anatomic and surgical importance. They stress the diagnostic factors and the therapeutic implications, very different in children and adults, of a particular vascular anomaly whose outcome is little understood.

[Combination of colorectal tumor and Streptococcus bovis endocarditis. Apropos of 3 cases]. / Association d'une tumeur colo-rectale et d'une endocardite à Streptococcus bovis. A propos de trois observations.

The authors report about 3 cases of bovis endocarditis revealing a colic carcinoma. This morbidity confirms the need for routine digestive investigations in endocarditis due to group D streptococci and eventually a liver disease.

[Comments on emergency treatment of biliary lithiasis in patients over 75 years of age. Apropos of 157 cases]. / Réflexions sur le traitement des urgences de la maladie biliaire lithiasique chez les sujets de plus de 75 ans. A propos de 157 observations.

Surgery remains the ideal emergency treatment for biliary lithiasis in elderly subjects despite perioperative morbidity and mortality. Minimally invasive techniques appear promising but require assessment. The aim of this work was to determine the usefulness of these techniques and evaluate outcome in a series of 157 patients over 75 years of age who were hospitalized in an emergency setting of complicated biliary lithiasis from January 1990 to December 1996. There were 103 women and 54 men, mean age 82 years. The patients' general status was evaluated according to the ASA classification; 66% of the patients were ASA III, IV or V. Diagnoses at admission were acute cholecystitis (n = 71, 45%), angiocholitis (n = 50, 31%) subintrant hepatic colic (n = 17, 10.8%), pancreatitis (n = 10, 6%), isolated jaundice (n = 2), peritonitis (n = 2) and occlusion (n = 5). Within 24 hours of admission, 7 patients underwent emergency surgery, and the 150 others were given medical treatment. Among these 150 patients, cure was considered to have been achieved with medical treatment alone in 41 (subsequent surgery being required in only one 6 months later), semi-emergency was performed in 17, and a minimally invasive procedure was performed in the 92 others (echo-guided percutaneous cholecystostomy in 42, endoscopic sphincterotomy in 50) followed by a subsequent operation in 29. In the 103 patients (65.5%) in this series who did not undergo surgery, mortality was 3.8% and in the 54 patients (34.5%) who did, mortality was 15%, but this rate was only 6.9% when the open procedure followed a minimally invasive technique. Surgical treatment of complicated biliary disease remains the ideal therapy but indications should be carefully weighed in these elderly fragilized subjects. Under surgical observation, abstention from surgery or use of minimally invasive techniques can play an important role in the therapeutic strategy aimed at lowering perioperative mortality.

[Digestive cancers and choroid metastasis. Diagnostic circumstances and prognostic value apropos of 2 cases]. / Cancers digestifs et métastases choroïdiennes. Circonstances du diagnostic et intérêt pronostique à propos de 2 observations.

We report two observations of malignant gastric and pancreatic tumors with choroid metastasis. Clinical course was rapidly and spontaneously unfavorable.

Myelin protein Po as a potential autoantigen in autoimmune inner ear disease.

We have previously shown that a 30,000 Mr protein extracted from guinea pig inner ear tissue is recognized by autoantibodies present in the serum of patients suffering from autoimmune inner ear disease. This protein was localized in the modiolus and in the organ of Corti. We have now identified this protein by a combination of microsequencing and matrix-assisted laser desorption ionization time-of-flight mass spectrometry of its tryptic peptides. A partial sequence of the protein was thereby determined. These data and 2-dimensional gel electrophoresis followed by immunoblotting experiments showed that the 30,000 Mr inner ear antigen is the major peripheral myelin protein Po. This suggests that protein Po may be an important autoantigen in autoimmune inner ear disease.

HLA class II-associated genetic susceptibility in idiopathic progressive sensorineural hearing loss.

To investigate the association between genes in the major histocompatibility complex and inner ear disease susceptibility at the DNA level, high-resolution genotyping for HLA class II (HLA-DR, -DQ, -DP) was performed by polymerase chain reaction-sequence-specific oligonucleotide reverse dot blot and polymerase chain reaction-restriction fragment length polymorphism analysis in 34 patients with idiopathic progressive sensorineural hearing loss (PSHL) and in 214 controls. The frequencies of DRB1*0301, DRB3*0101, DQB1*0201, and DPB1*0401 were significantly increased in patients with idiopathic PSHL compared with controls. The DQB1*0301 allele was significantly decreased in the patients. A linkage disequilibrium was probably responsible for the concomitant increase of both DRB1*0301 and DRB3*0101 alleles in patients. The increase of DQB1*0201 in patients was associated with the DRB1*0301 allele. In addition, the telomeric DPB1*0401 allele may act as an independent risk factor. The DQB1*0301 allele may have a protective role in the pathogenesis of idiopathic PSHL. These results suggest that the specific HLA class II gene products may confer susceptibility or resistance to idiopathic PSHL.

Guinea pig inner ear antigens: extraction and application to the study of human autoimmune inner ear disease.

In this study, the authors attempted to develop a method of extracting guinea pig inner ear antigens for otoimmunological research, and to investigate the distribution of the antigens in the various structures of the inner ear. The antigens were extracted either from the entire or from various parts of the guinea pig inner ear. These antigens were separated on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Western blot techniques were used to test sera from patients with inner ear disease against guinea pig inner ear protein extracts. It found that the various molecular weight antigens in the inner ear were associated with the different structures of the inner ear. The sera of 37.5% (N = 80) of patients reacted with two bands (30 and 58 kd) of the guinea pig inner ear immunoblots. The 58 kd band was not specific to the inner ear, but instead was also found in the immunoblots of other guinea pig tissues (brain, lung, and liver). This study suggests that the various antigens of interest could be further extracted and purified from the corresponding locations of the inner ear.