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.

[Intravenous 3-day Helicobacter pylori eradication therapy is highly effective in patients with bleeding peptic ulcer]. / Alta eficacia erradicadora de la infección por Helicobacter pylori de una pauta terapéutica intravenosa de 3 días de duración en pacientes con úlcera péptica sangrante.

AIMS: To evaluate the efficacy of 3-day intravenous Helicobacter pylori eradication therapy in patients with bleeding peptic ulcer associated with H. pylori infection. MATERIAL AND METHOD: We studied 53 patients admitted to hospital with bleeding of the upper gastrointestinal tract due to peptic ulcer and positive urease test over a 12-month period. After endoscopic diagnosis, intravenous pantoprazole (40 mg/12 hours), metronidazole (500 mg/8 hours) and amoxicillin-clavulanic acid (1,000 mg/200 mg/8 hours) was administered for 72 hours. The efficacy of eradication therapy was evaluated by 13C-urea breath test at least 2 months after the end of treatment. RESULTS: Fifty-one patients were included. Of these, 40 had duodenal ulcer and 11 had gastric ulcer. H. pylori eradication was achieved in 87.5% of those with duodenal ulcers and in 63.6% of those with gastric ulcers (p = 0.066). No adverse reactions or episodes of bleeding recurrence were found and none of the patients withdrew from treatment. CONCLUSIONS: The ultra-short course eradication therapy used in this study is highly effective. Its efficacy is similar to that of oral treatment and it avoids certain problems such as adverse effects and adherence to treatment.

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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Regression of primary gastric B-cell mucosa-associated lymphoid tissue (MALT) lymphoma after eradication of Helicobacter pylori.

OBJECTIVE: primary gastric B-cell lymphoma of the mucosa-associated lymphoid tissue type has been linked to infection of the gastric mucosa with Helicobacter pylori. The eradication of this pathogen with antibiotics can lead to regression of this type of lymphoma. The objective of this study was to describe the clinical, endoscopic and histologic evolution in 6 patients with primary gastric B-cell lymphoma of the mucosa-associated lymphoid tissue type treated with eradication of H. pylori. METHOD: descriptive study of a retrospective case series. Patients with low-grade gastric mucosa-associated lymphoid tissue type lymphoma were initially treated with eradication therapy for H. pylori. We evaluated their clinical, endoscopic and histologic course in sequential follow-up visits after initial therapy. RESULTS: six patients who satisfied all selection criteria were studied. In five of six patients H. pylori was eradicated and the lymphoma showed regression within 6 to 24 months. In one patient transition of low-grade to high-grade gastric lymphoma occurred. CONCLUSIONS: these results, along with similar findings from other studies, suggest eradication of H. pylori should be the initial treatment of choice for low-grade B-cell gastric mucosa-associated lymphoid tissue type lymphoma in the early stages. The most appropriate duration of follow-up is unknown, but prolonged follow-up is indicated to detect recurrences.

Inhibition of human sperm-zona-free hamster oocyte binding and penetration by protein C inhibitor.

Protein C inhibitor is a heparin-dependent serine protease inhibitor present in plasma at about 0.08 mumol l-1. Protein C inhibitor inhibits activated protein C and other coagulation factors. Previously, we described the presence of high protein C inhibitor levels in human semen (3.1 mumol l-1) and showed potential roles of the inhibitor in human reproduction. Here, we show that protein C inhibitor is present in an active form in follicular fluid at about 0.1 mumol l-1 and that purified, functionally active human plasma-derived and inactive, semen-derived protein C inhibitor and a synthetic peptide derived from its sequence inhibited both binding and penetration of zona-free hamster oocytes by human sperm. The binding inhibition by protein C inhibitor was dose dependent, with 50% inhibition at 0.037 mumol l-1 inhibitor (45 +/- 17 sperm per egg versus 90 +/- 23 in control experiments). The inhibitor also blocked in a dose-dependent manner the penetration of zona-free hamster eggs by human sperm (20 +/- 7% fertilized eggs at 0.1 mumol l-1 protein C inhibitor versus 55 +/- 10% in control experiments). Polyclonal antiprotein C inhibitor or antipeptide antibodies partially abolished the effect of protein C inhibitor and peptide on the inhibition of the binding and penetration of zona-free hamster oocytes by human sperm. The effect of the protein C inhibitor was not dependent on its antiprotease activity since purified semen-derived protein C inhibitor which did not have antiprotease activity gave comparable results. We conclude that protein C inhibitor may be involved in human reproduction at several steps, including the fertilization process.

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.

Quantitative immunoassay for complexes of prostate-specific antigen with alpha2-macroglobulin.

We have developed two ELISAs for quantifying complexes of prostate-specific antigen (PSA) with alpha2-macroglobulin (alpha2M), using partially purified PSA:alpha2M complex as the calibrator. One ELISA was designed to evaluate )SA:alpha2M complex in fluids containing a huge excess of PSA over the amount of complex (semen-derived fluids), the other for use in fluids containing an excess of alpha2M over PSA (blood plasma). The range of the assays was 2-1000 micrograms/L for PSA complexed to alpha2M; the detection limit was 3 micrograms/: Intra- and interassay CVs were 7-13% and 11-17%, respectively, at complexed PSA concentrations of 6-500 micrograms/L. Seminal fluid from healthy men (n = 60) contained 5.2 +/- 2.6 micrograms/L PSA complexed with alpha2M. Prostatic and seminal vesicle fluids contained 6.5 +/- 2.9 ad 0.3 +/- 0.2 mg/L PSA complexed to alpha2M, respectively. When purified PSA was incubated with citrated plasma, between 45% and 65% of the added PSA was recovered as free PSA, whereas approximately 25% formed complexes with alpha2M, 10% complexed with alpha1-antichymotrypsin, and only 0.1-6% was complexed with protein C inhibitor. Of 30 patients with prostate disease, 20 showed detectable plasma PSA:alpha2M complexes; however, the potential diagnostic significance of this complex requires further investigation.

[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.

Prostate-specific antigen and its complexes with alpha 1-antichymotrypsin in the plasma of patients with prostatic disease.

OBJECTIVE: To improve the specificity and sensitivity of the prostate-specific-antigen (PSA) assay for the distinction between prostate cancer and benign prostate hyperplasia (BPH). METHODS: Two sensitive immunoassays, one that measures free PSA and PSA complexed to alpha 1-antichymotrypsin (alpha 1-ACT) with the same efficiency (PSAag assay) and another that specifically measures the complex between PSA and alpha 1-ACT, have been designed to measure the PSA forms in the plasma of 84 patients with prostate disease and in the seminal plasma from 60 healthy individuals. RESULTS: The proportion of plasma PSA in complex with alpha 1-ACT was significantly higher in the 34 patients with prostate cancer (89 +/- 12%, mean +/- SD; median, 91%) than in the 50 patients with BPH (71 +/- 12%; 73%) and did not correlate with the total amount of PSA. Normal seminal plasma (n = 60) had 2.1 +/- 0.6 mg/ml PSA, 175 +/- 62 microns/ml alpha 1-ACT and 9.6 +/- 3.4 micrograms/ml PSA: alpha 1-ACT complex. CONCLUSION: These results confirm that PSA: alpha 1-ACT may be a good marker for a differential diagnosis of carcinoma of the prostate and BPH.

Complexes of tissue kallikrein with protein C inhibitor in human semen and urine.

An ELISA was developed for quantifying the complex between tissue kallikrein (tKK) and protein C inhibitor (PCI) (tKK:PCI) in seminal plasma and urine. The ELISA used purified tKK:PCI complex as a standard and was specific for this complex with a detection limit of about 1.1 pM. Purified tKK:PCI complex was obtained from human urine and was 95% homogeneous as judged by SDS/PAGE. The 90-kDa band corresponding to the purified tKK:PCI complex reacted with anti-tKK and anti-PCI antibodies as judged by immunoblotting. Seminal plasma collected in the absence of extrinsic inhibitors contained 1.8 +/- 0.6 nM tKK:PCI complex and 4.7 +/- 2.8 nM immunoreactive tKK (mean +/- SD, n = 10), which indicates that about 28% of the total tKK immunoreactivity is forming complexes with PCI. When semen was collected in the presence of tKK inhibitors it had only about 6% of the tKK complexed to PCI. In vitro studies showed that the tKK:PCI complex formation in semen was accomplished in about 1 h and that heparin stimulated both the rate and the extent of complexation of tKK with PCI. Native urine showed low levels of tKK:PCI complex, but after dialysis urine had 0.17 +/- 0.05 nM complex. Formation of tKK:PCI complex in urine and semen was also demonstrated by immunoblotting. These results suggest that PCI is a physiological inhibitor of tKK and provide additional evidence of the involvement of PCI in human reproduction.

[A new method of studying gastric emptying in gastrectomized patients]. / Nuevo método de estudio del vaciado gástrico en pacientes gastrectomizados.

OBJECTIVE: We present a new method of assessment of gastric emptying in gastrectomized patients. PATIENTS AND METHODS: The gastric emptying of non digestible solids were studied in two groups of gastrectomized patients, the Billroth II (n = 25) and the Roux-en-Y (n = 36) to relate the gastric emptying with the postgastrectomy symptoms. RESULTS: The food retained in the gastric stump is related with significance with the presence (p = 0.01) and the absence (p = 0.02) of markers. The patients operated on with the Billroth II technique present a higher frequency of markers in the gastric stump (40% over 19.4% with the Roux-en-Y). The markers in the gastric stump are more common with the symptoms of pyrosis, nausea, bilious vomit, diarrhea and the postoperative gastritis of alkaline reflux. CONCLUSIONS: The method of gastric emptying of non digestible solids is a valid and reliable study of the gastric emptying and should be carried out in all candidates for endoscopy because it is harmless and easy to perform and evaluate.

[Value of endoscopy in the diagnosis of postoperative gastritis caused by alkaline reflux]. / Valor de la endoscopia en el diagnóstico de la gastritis postoperatoria por reflujo alcalino.

OBJECTIVE: A long term retrospective study comparing endoscopic lesions and their relationship with symptoms and histological lesions of postoperative Alkaline Reflux Gastritis (ARG) between partial gastrectomy Billroth II (n = 25) and the Roux-en-Y (n = 36) is presented. PATIENTS AND METHODS: In patients operated on with both techniques the clinical, endoscopic and histological aspects are studied. RESULTS: The patients operated on with the Billroth II technique present a higher frequency of bile in the gastric remnant and mucosal lesions in the endoscopic study (p < 0.001) compared with the Roux-en-Y group. Bile and mucosal lesions on the gastric stump are more common with symptoms and histological lesions of postoperative ARG. CONCLUSIONS: The endoscopic evaluation is a valid and reliable examination in the diagnosis and follow up of postoperative ARG.

Activation of the protein C pathway in acute sepsis.

To obtain quantitative information on the in vivo activation of the protein C system during the acute phase of sepsis, several components of the protein C pathway were studied in 18 patients. Blood samples were obtained one day after diagnosis (day 1) and, in 11 patients, also on the fourth and tenth days after diagnosis (days 4 and 10). On day 1, patients showed laboratory signs of haemostatic alterations such as positive fibrinogen/fibrin degradation products, and increased thrombin:antithrombin-III (TAT) complex levels. Compared with the control group, patients on day 1 had significantly decreased (p < 0.001) antigenic protein C (69 +/- 28%) and protein C inhibitor (PCI) (33 +/- 22%) whereas a significant increase in the levels of activated protein C (APC) complexed with alpha 1-antitrypsin (alpha 1AT) (APC:alpha 1AT, 26 +/- 15 ng/mL) and APC:PCI complex (3.0 +/- 2.0 ng/mL), and in the level of plasma kallikrein (KK) complexes with PCI (KK:PCI) (31 +/- 22 ng/mL) was observed. There was a positive correlation between APC:alpha 1AT and TAT complex levels (r = 0.597, p = 0.009). In the follow-up a trend toward normal values in antigenic protein C and PCI, and in APC:PCI and KK:PCI complex levels was found. However, PCI remained significantly decreased compared to normal values. C4b-binding protein, alpha 1AT, and TAT and APC:alpha 1AT complexes did not show any significant variations during the course of the disease, suggesting the contribution of the inflammatory and haemostatic responses, in spite of the good recovery of the patients. This study shows that in the course of sepsis, patients experience a generalized activation of the protein C pathway which was more prominent on day 1, resulting in the consumption of protein C and PCI and in the increase of APC:inhibitor complexes. Moreover, these data provide further evidence that KK:PCI circulating complexes occur in vivo.