HLA antigens and anti-neutrophil cytoplasmic antibodies (ANCA) in inflammatory bowel disease.

HYPOTHESIS AND OBJECTIVES: the hypothesis of this study is that genes involved in the regulation of the immune system, expressed by HLA antigens and anti-neutrophil cytoplasmic antibodies (ANCA), could be determinants of disease susceptibility and behavior in inflammatory bowel disease (IBD). MATERIAL AND METHOD: seventy patients with a diagnosis of inflammatory bowel disease, 46 with ulcerative colitis and 24 with Crohn"s disease were included. HLA class I (A and B) and II (DR) antigens were studied by serological techniques. Detection of ANCA was carried out in all patients by an indirect immunofluorescence method. The relative frequencies of HLA antigens were compared with a control group made up of 156 blood donors. The control group for the ANCA study was made up of 100 individuals. RESULTS: we found a significant increased frequency of HLA-DR2 in patients with ulcerative colitis. No significant differences were found between patients with Crohn"s disease and controls regarding HLA typing. We detected a significant increase of HLA-DR3 in extensive forms of ulcerative colitis. Detection of ANCA was positive in 46% of the patients with ulcerative colitis and in 12% of the patients with Crohn"s disease (p <0.05). We observed an increased frequency of ANCA in patients with UC and HLA-DR2 (p = 0.15). CONCLUSIONS: the association found between HLA-DR3 and extensive forms of ulcerative colitis provides evidence of genetic heterogeneity. The relationship between ANCA and HLA phenotype (although not significant) supports this concept.

Influence of age on clinical and immunological characteristics of newly diagnosed type 1 diabetic patients.

The aim of this study was to analyse the immunological and clinical characteristics of a group of patients at the onset of type 1 diabetes and to determine if these findings are age related. For this purpose, 68 newly diagnosed type 1 diabetes mellitus patients referred to our hospital between 1997 and 1999 were studied; 42 were adults (mean age 24+/-3.5 years) and 26 children (mean age 6.1+/-4 years). Autoantibody markers islet cell antibodies, glutamic acid decarboxylase antibodies (GADA) and tyrosine phosphatase antibodies (IA-2A), pancreatic reserve (glucagon test) and HbA1c were determined. Some clinical characteristics, such as mode of presentation and insulin requirements, were also analysed. Type 1 diabetes mellitus was found to be autoimmune in 83.8% of the patients and idiopathic in 16.2%, without significant differences between adults and children. In the whole autoimmune group, GADA was more prevalent in adults and IA-2A more frequent in children. On the other hand, adults showing autoimmune markers developed ketosis more frequently and needed higher insulin doses at diagnosis, while children did not exhibit clinically significant differences associated with the presence or absence of antibodies. In conclusion, in children the presence of autoimmune markers is not related to the mode of presentation or characteristics of type 1 diabetes. In adults, however, the autoimmune group presents with more-severe clinical disease than antibody negative patients. Age at onset seems to be an important parameter in the natural history of type 1 diabetes and must be taken into account in epidemiological or intervention studies.

Utilidad clínica de los anticuerpos anticitoplasma de neutrófilo con especial referencia a la fibrosis quística / Clinical usefulness of antineutrophil cytoplasmic antibodies with special reference to cystic fibrosis

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Response to treatment with interferon-alfa in patients with chronic hepatitis C and high titers of -M2, -M4 and -M8 antimitochondrial antibodies.

UNLABELLED: The prevalence of antimitochondrial antibodies (AMA) in chronic hepatitis C is 2%; titers of AMA are usually low (< 1:40). The prevalence decreases to 0.5% when the results are verified by determination of the M2 subtype (anti-M2, ELISA). In patients in whom both hepatitis C virus (HCV) and AMA are present, the therapeutic decision to give interferon-alfa is complicated, because AMA may be 'real', and if it reflects primary biliary cirrhosis, cholestasis can be triggered or exacerbated. This does not occur when AMA positivity results from induction by hepatotropic C virus; however, this is rarely the case when AMA titers are high (> 1:160). OBJECTIVE: to undertake a preliminary analysis of the submitochondrial profile of AMA in three patients with chronic hepatitis C and positive AMA titers (> 1:160). METHODS: we determined antibodies to submitochondrial particles (subtypes) -M2, -M4 and -M8 by ELISA, complement binding (CB) and western immunoblotting with Immunoblot-M2 or WIB-M2 (immunoreactive bands). RESULTS: two patients were positive for mitochondrial subtypes by ELISA (IgG/IgM subclass) and CB (ELISA M2 470/365 in patient 1 and 600/1370 in patient 2; M4 490/1200 in patient 2. CB M2 1:128, M4 1:64, M8 1:64 in patient 1, M2 1:128 in patient 2). Immunoreactive epitopes (bands) were detected with WIB-M2 for 70, 56, 51, 45 and 36-kDa molecules. Interferon-alfa treatment was unsuccessful, with biochemical exacerbation of cholestasis. In contrast, the patient with no submitochondrial particles according to ELISA, CB and WIB-M2 results responded favorable to this drug. CONCLUSION: these preliminary results suggest that analyses to detect antibodies to submitochondrial particles (-M2, -M4 and -M8 subtypes) and -M2-immunoreactive epitopes in patients with chronic hepatitis C and AMA titers > 1:160 facilitates the diagnosis of primary biliary cirrhosis, and establishes a contraindication for treatment with interferon-alfa despite the presence of HCV infection.

[Significance of the determination of antineutrophil cytoplasmic antibodies (ANCA) in ulcerative colitis and Crohn's disease]. / Significado de la determinación de anticuerpos frente al citoplasma de los neutrófilos (ANCA) en la colitis ulcerosa y en la enfermedad de Crohn.

We have studied prospectively 126 consecutive patients recruited with a known diagnosis of ulcerative colitis (UC; n = 78) and Crohn's disease (CD; n = 48) for anti-neutrophil cytoplasmatic antibodies (ANCA) by indirect immunofluorescence (IFI). Forty-six percent of UC and 18% of CD patients were found positive. The sensitivity and specificity for UC diagnosis were 0.46 and 0.81, respectively. We evaluated the pattern of IFI exhibited (perinuclear: pANCA and cytoplasmatic: cANCA). cANCA was found in 77% of CD and in only 30% of UC patients (p = 0.01). Sera from all CD patients were positive at a 1:20 dilution (and not at higher dilution) and it occurred in only in 14 UC patients (30%). Positive sera were also tested to characterize the antigen specificity by enzyme-linked immunosorbent assay (ELISA) but the antigenic nature of ANCA could not be identified in most cases. No differences were found between ANCA positive and ANCA negative patients regarding colonic extension (UC) or colonic involvement (CD), activity and colectomy. We conclude that ANCA may be a helpful diagnostic test in UC patients but it not seems to be important as a marker of activity. ANCA positivity can reflect disease heterogeneity in UC patients, perhaps discriminating those with immunologic disturbances.

[Chronic autoimmune hepatitis following cholestatic hepatitis caused by droxicam]. / Hepatitis crónica autoinmune después de una hepatitis colestásica inducida por droxicam.

Droxicam in a nonsteroid antiinflammatory from the oxicam family which acts as a pro-drug, being transformed into pyroxicam after being hydrolized in the stomach and has induced several cases of cholestatic or mixed hepatitis. A clinical observation in which droxicam provoked initial cholestatic hepatitis which later developed into chronic autoimmune hepatitis is presented. It has been postulated that, after causing cholestatic hepatitis by hypersensitivity and within the context of a previous autoimmune entity such as vitiligo, this drug triggered a silent autoimmune liver disease which was demonstrated clinical, analytical and histopathological manifestations 18 months later and required permanent immunosuppressive treatment.

Circulating auto-antibodies in Ménière's disease.

In the last years, it has been claimed that patients suffering from Menière's disease exhibit Circulating Immune Complexes, as well as free antibodies specifically directed against type II collagen, a component of the mammal inner ear. Furthermore, by means of repeated injections of type II collagen to the guinea-pig. Menière's disease has been reproduced. Taken together, all these features strongly suggest that Menière's disease fulfills most of the postulates that Witebsky established for the classification of auto-immune diseases. However, in spite of being a very well known marker of auto-immune conditions, non organ nor species specific Circulating Auto-Antibodies (CAA) have not been reported, to our knowledge, in Meniere's disease. Based upon this fact, Anti-Nuclear, Anti-Mitochondrial, Anti-Smooth Muscle, Anti-LKM and Anti-Parietal Gastric Cells Auto-Antibodies have been investigated in 49 patients diagnosed as having Meniere's disease according to the criteria of the AAOO. All of them were asymptomatic at the time of the study. Seventy eight serum samples from Normal Healthy Subjects (NHS) constituted the control group. CAA were tested by indirect immunofluorescence on rat organs according to the method described by Coons. Additionally, the seric levels of C3, and C4 as well as the haemolytic capacity (CH-50) of the serum samples were tested. Furthermore, Circulating Immune Complexes (CIC) as well as the Complement-Mediated Mechanisms involved in the clearance of CIC (Complement-Mediated Solubilization and Inhibition of the Immune Precipitation) were investigated in parallel.(ABSTRACT TRUNCATED AT 250 WORDS)

Complement mediated solubilization: role of the complement in the clearance of circulating immune-complexes.

In 1975, Miller and Nussenzweig described a new function of the Complement (C'): the Capacity of this substance to solubilize "in vitro" antigen/antibody complexes previously formed. Subsequent studies of these authors and other researches (particularly Takahashi and his group) demonstrated that this process is mediated by activation of the alternative way of C', although it is faster when the classic way is involved. Basically, thanks to the Takahashi and group studies, it has been possible to know in detail the intimate mechanism of this phenomenon, usually known demonstrated that the key of all this process lies in the generation, through the C' alternative way, of numerous molecules of C3b that deposit over the antigen/antibody complexes (Ag/Ac), causing the breakage of the necessary mesh for such complexes to precipitate, which leads to formation of small immunocomplexes (IC) very rich in C3b which turn soluble again. ICs solubilized in this way suffer important physicochemical and biological changes, and transform into substances without inflammatory power that are not able to activate C' nor to interact with C3b receptors present in a great number of blood cells. However, they are captured by Kupffer cells in the hepatic sinusoids and quickly catabolized. CMS is closely related with another similar phenomenon, the Immuno-Precipitation inhibition (IPI), described by Schiferli and his group in the early 80's.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of two different anti-factor VIII/von Willebrand factor antibodies of the IgA class in a hemophilic patient with a polyclonal factor VIII inhibitor of the IgG class.

A hemophilic patient treated with Factor VIII (F.VIII) concentrates developed a F.VIII inhibitory activity. The patient's plasma showed 2 anti-F.VIII/von Willebrand Factor (vWF) antibodies (Abs) of the IgG and IgA class respectively. The specific anti-F.VIII/vWF Abs were isolated and resulted to be IgG (20 micrograms/ml) and IgA (35 micrograms/ml). The plasma was subsequently fractionated by Protein A-Sepharose and the IgG and IgA containing fractions were separately analyzed for anti-F.VIII activity. Both fractions exhibited F.VIII inhibitory activity, but that corresponding to the IgA was lower than expected. Regarding the possible existence of a non-inhibitor Ab in the IgA containing sample, plasma was processed through an immunoadsorbent to which a F.VIII devoid vWF preparation had been previously coupled and further fractionated by Protein A-Sepharose, obtaining 2 IgA fractions. One of them exhibited F.VIII inhibitory activity while the other, which reacted with the F.VIII devoid vWF preparation, did not. Therefore, this latter one was considered as a true anti-vWF Ab. The IgG and IgA inhibitors were polyclonal and the IgG one was composed of the 4 IgG subclasses.

Immunological characterization of factor VIII inhibitors by a sensitive micro-ELISA method.

A micro-ELISA method for the immunological characterization of factor VIII (F. VIII) inhibitors is described. Microplates sensitized with purified monospecific anti-von Willebrand factor (vWF) IgG were firstly incubated with a commercial F. VIII concentrate and then with the samples containing the F. VIII inhibitors to be characterized. Peroxidase conjugated monospecific antisera were used to determine the Ig class and the light chain type. Alkaline phosphatase conjugated monoclonal antibodies were employed to investigate the IgG subclasses. The F. VIII inhibitors from 8 hemophiliacs and 1 patient with systemic lupus erythematosus (SLE) were characterized. All of them belonged to the IgG class but one case simultaneously exhibited another inhibitor of the IgA class. Eight inhibitors analyzed for light chains resulted to be polyclonal. In 7 cases the inhibitors belonged solely to the IgG4 subclass while in the other 2 cases contained all the 4 IgG subclasses. The method appears to be simple, accurate and highly sensitive (detecting as low as 0.156 Bethesda Units/ml) for the immunological characterization of F. VIII inhibitors.

[A fast and simple method for obtaining specific anti-von Willebrand factor IgG]. / Un método rápido y sencillo para la obtención de IgG específica anti factor von Willebrand.

Date expired commercial F.VIII concentrates (1.000 I.U. in 15 ml) were chromatographed through a Sepharose 4B in Tris-HCI (0.15 M, pH = 7.4) column. The void volume of this column was found to constitute an enriched vWF preparation. Half a milliliter of this preparation (protein content: 200 micrograms/ml) was mixed (1: 3) with Freund's complete adyuvant and administered to two rabbits by intramuscular route. A total of 3 doses were administered at days 1, 15 and 21 and the 2 animals were bled at day 30. The serum samples so obtained were pooled and analysed (DGD and IEP) for anti-vWF and anti-NHP activity. Strong anti-vWF specificity could be demosntrated. However, since a weak anti-fibrinogen and anti-IgG contaminant activity was also detected, the pool was filtered through a column containing normal human plasma coupled to a periodate activated Sephacryl S-1.000 immunoadsorbent. The filtrate, which was found to contain only a strong anti-vWF activity, was again processed by affinity chromatography through another Sephacryl immunoadsorbent containing a commercial F.VIII concentrate of the same lot employed for purifying the vWF. The eluate so obtained demonstrated to be constituted only by a pure rabbit IgG with strong anti-vWF specificity and was completely devoid of activity against any other human plasmastic protein. The method was found to be rapid, simple and economic, yielding as much as 5 mg of specific IgG for every 10 ml of antiserum so processed.

A study of lymphocyte populations and subpopulations in hemophiliacs and their relationships with age and consumption of lyophilized factor VIII concentrates.

Peripheral blood lymphocyte populations and subpopulations of 154 hemophiliacs treated with factor VIII concentrates were studied. The patients had significantly increased percentages of leukocytes and lymphocytes and also absolute numbers of the lymphocyte populations in cells/mm3. The increases in cell counts in the two major T cell subpopulations (T4 and T8) were unbalanced, leading to a significant decrease in the T4/T8 ratio. There was no correlation between the T4/T8 ratio and the consumption of factor VIII during the year prior to the study. The values of these T cell subpopulations and of the T4/T8 ratio significantly correlated with the patient's age. There were no analytical or clinical data for any of the patients indicating AIDS.

A standardized method for measuring anti-F VIII: C inhibitors in haemophilia A by coagulation inhibition in agarose gel.

Antibodies against factor-VIII coagulant activity can appear in haemophilic patients and, although infrequently, can affect individuals not suffering from Haemophilia A. The Oxford and Bethesda methods are presently the most commonly used techniques for measuring these antibodies. Both methods are time-consuming and not suitable for the screening of large risk groups. An appropriate method for screening these coagulation inhibitors is that described by P. Bird in 1975. It is based on inhibition of the coagulation produced when plasma samples containing inhibitors diffuse in agarose gels mixed with normal platelet rich plasma (PRP). However, this technique is highly dependent on the variability derived from the use of PRP (amount of coagulant factor-VIII, number of platelets, etc.). In an attempt to avoid these disadvantages, Bird's method has been modified by using standardized commercial reagents (lyophilized plasma with 100% factor-VIII coagulant activity, purified fibrinogen, and platelet Factor 3) instead of PRP. The sensitivity reaches 0.8 Bethesda units and the correlation with the Bethesda method is r = 0.964, p less than 0.001. This newly developed method is as simple as Bird's, and appears to be at least, as accurate and reproducible as the Bethesda method.