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An immunoblot assay was tested to evaluate its ability to diagnose human hydatidosis and to analyse the reactivity of hydatid patients' sera with the subunits of the 2 major Echinococcus granulosus antigens (5 and B). In all, 308 sera were examined: 166 sera from patients with clinically diagnosed hydatidosis, 100 sera from healthy control subjects, and 42 sera from patients with diseases other than hydatidosis. The sensitivity of the method was 90%, as compared to 78% with the immunoelectrophoresis/ double diffusion test for antigen 5. No reactivity was found with 15 sera from patients with schistosomiasis, 7 sera from patients with trichinellosis, or 20 sera from patients with non-parasitic diseases. Analysis of serum reactivities showed the presence in all positive sera of antibodies directed against the 39 kDa molecule of the antigen 5 complex. A lower reactivity (55% of all hydatid sera) was observed with the subunits of the antigen B complex.

This study describes a 42-year-old male born in Tunisia, who presented with vertebral hydatidosis which was initially misdiagnosed as Pott's disease. The correct diagnosis was finally made utilizing CT and MRI imaging and confirmation of the diagnosis was made possible by molecular and immunochemical characterization of the content of vertebral cyst fluid.

Given the lack of a serological test specific for Behcet's disease, its diagnosis rests upon clinical criteria. The clinical diagnosis is nevertheless difficult because the disease manifestations vary widely, especially at the onset of disease. The aim of this study was to identify molecules specifically recognized by serum autoantibodies in patients with Behcet's disease and to evaluate their diagnostic value. We screened a cDNA library from human microvascular endothelial cells with serum IgG from two patients with Behcet's disease and isolated a reactive clone specific to the carboxy-terminal subunit of Sip1 (Sip1 C-ter). Using ELISA, we measured IgG, IgM and IgA specific to Sip1 C-ter in patients with various autoimmune diseases characterized by the presence of serum anti-endothelial cell antibodies, such as Behcet's disease, systemic lupus erythematosus, systemic sclerosis and various forms of primary vasculitis, as well as in patients with diseases that share clinical features with Behcet's disease, such as inflammatory bowel disease and uveitis. IgM immunoreactivity to Sip1 C-ter was significantly higher in patients with Behcet's disease and in patients with primary vasculitis than in the other groups of patients and healthy subjects tested (P < 10-4 by Mann-Whitney test). ELISA detected IgG specific to Sip1 C-ter in sera from 11/56 (20%) patients with Behcet's disease, IgM in 23/56 (41%) and IgA in 9/54 (17%). No sera from patients with systemic lupus erythematosus, systemic sclerosis, inflammatory bowel disease, uveitis or healthy subjects but 45% of sera from patients with primary vasculitis contained IgM specific to Sip1 C-ter. Serum levels of soluble E-selectin, a marker of endothelial activation and inflammation, correlated with levels of serum IgM anti Sip-1 C-ter in patients with Behcet's disease (r = 0.36, P = 0.023). In conclusion, Sip1 C-ter is a novel autoantigen in Behcet's disease. IgM specific to Sip1 C-ter might be useful in clinical practice as an immunological marker of endothelial dysfunction in vasculitis.

Anti-endothelial-cell antibodies are associated with psychiatric manifestations in systemic lupus erythematosus (SLE). Our primary aim in this study was to seek and characterize molecules that behave as endothelial autoantigens in SLE patients with psychiatric manifestations. By screening a cDNA library from human umbilical artery endothelial cells with serum from an SLE patient with psychosis, we identified one positive strongly reactive clone encoding the C-terminal region (C-ter) of Nedd5, an intracytoplasmatic protein of the septin family. To evaluate anti-Nedd5 serum immunoreactivity, we analyzed by ELISA specific IgG responses in 17 patients with SLE and psychiatric manifestations (group A), 34 patients with SLE without psychiatric manifestations (group B), 20 patients with systemic sclerosis, 20 patients with infectious mononucleosis, and 35 healthy subjects. IgG specific to Nedd5 C-ter was present in 14 (27%) of the 51 SLE patients. The mean optical density value for IgG immunoreactivity to Nedd5 C-ter was significantly higher in patients of group A than in those of group B, those with infectious mononucleosis, or healthy subjects (0.17 ± 0.14 vs, respectively, 0.11 ± 0.07, P = 0.04; 0.11 ± 0.06, P = 0.034; and 0.09 ± 0.045, P = 0.003, on Student's t-test). Moreover, IgG immunoreactivity to Nedd5 C-ter was significantly higher in patients with systemic sclerosis than in patients of group B or healthy subjects (0.18 ± 0.18 vs, respectively, 0.11 ± 0.07, P = 0.046; and 0.09 ± 0.045, P = 0.003). The percentage of patients with anti-Nedd5 C-ter serum IgG was higher in group A than in group B (8 (47%) of 17, vs 6 (17%) of 34, P = 0.045, on Fisher's exact test). In order to clarify a possible mechanism by which Nedd5 might be autoantigenic, we observed that Nedd5 relocated from cytoplasm to the plasma membrane of EAhy926 endothelial cells after apoptotic stimuli. In conclusion, Nedd5 is a novel autoantigen of potential clinical importance that could be successfully used for a more thorough investigation of the pathogenesis of psychiatric manifestations in SLE. Although anti-Nedd5 autoantibodies are not specific to SLE, they are significantly associated with neuropsychiatric SLE and may represent immunological markers of psychiatric manifestations in this pathology.

Oropharyngeal washings (Ophs) from 27 HIV infected patients (18 with P. carinii pneumonia, PCP, and 9 without PCP) were examined for P. carinii using morphological staining and DNA amplification with PCR-SHELA and nested PCR methods. The comparison of these techniques shows that 1. the amplification of P. carinii DNA is more sensitive than (and as specific as) morphological staining; 2. PCR-SHELA is less sensitive than (and as specific as) nested PCR.

To understand the way of reinfection of Pneumocystis carinii we have analyzed the genetic variation at the internal transcribed spacer (ITS) in DNA samples from bronchoalveolar lavage fluid of Italian HIV patients who had multiple episodes of P.carinii pneumonia. The presence of the same and/or a new type in both episodes suggest the possible occurrence of both reactivation of a previously acquired infection and reinfection from an exogenous source. Furthermore the occurrence of two different types in the same episode indicate that a mixed infection is common.

Antibodies to the three major components of the complex called soluble extractable nuclear antigen (ENA) were detected by passive hemagglutination (HA), counterimmunoelectrophoresis (CIE) and double immunodiffusion (DI) in 256 patients with connective tissue diseases. Anti-ENA antibodies were demonstrated by all the three employed methods in only 44.9% of the cases. These methods were not able to detect all antibodies to these antigens or any single specificity; CIE was however the most sensitive method for anti-RNP and HA for anti-Sm antibodies, while DI was the most suitable technique for serum samples with multiple anti-ENA specificities. Only in less than 50% of the cases the specificity detected by HA was comparable with that given by CIE or DI. Hence, for detecting anti-ENA antibodies a combination of these methods should be maintained, at least until more precise and reliable methods will become available.

SUMMARY The relation of interferon-gamma (IFN-γ), IL-4, IL-10 production and specific IgE, total IgG, IgG subclass expression to the effectiveness of pharmacological treatment in human hydatid disease (Echinococcus granulosus infection) was evaluated in 27 hydatid patients divided into four clinical groups according to their response to albendazole/mebendazole therapy (full, partial, low and non-responders). After parasite antigen stimulation, peripheral blood mononuclear cells (PBMC) from full responders produced significantly more IFN-γ (P= 0·038), significantly less IL-4 (P= 0·001) and less IL-10 than PBMC from non-responders. PBMC from partial and low responders produced intermediate cytokine concentrations. ELISA determining immunoglobulin production showed that sera from all non-responders had IgE and IgG4 antibodies, both regulated by IL-4. In contrast to IgG4, IgE decreased rapidly in full responders. Full responders also showed the highest percentage of IgG3 reactions. Qualitative analysis of total IgG responses in hydatid patients’ sera determined by immunoblotting showed that binding profiles to hydatid cyst fluid antigen differed in the four groups of treated patients. Non-responders had the highest percentage of reactions to all subunits of antigens 5 and B, and full responders had the highest percentage of reactions to antigen 5 alone. The high IFN-γ production associated with a lack of IL-4 and low IL-10 production in the full responders, and vice versa the high IL-4 and IL-10 production associated with lack of or low IFN-γ production in the non-responders implies Th1 cell activation in protective immunity and Th2 cell activation in susceptibility to hydatid disease. IgE may be a useful marker of therapeutic success in hydatid patients with pretreatment specific IgE antibodies. IgG subclass responses and differential immunoglobulin subclass binding pattern to hydatid antigens may also be useful in the immunosurveillance of hydatid disease.

Summary. DNA amplification by the polymerase chain reaction (PCR) is a promising method for the detection of Pneumocystis carinii in immunosuppressed patients. The sensitivity and specificity of the PCR technique has been assessed in comparison with the immunofluoresence method (IF) on bronchoalveolar lavage fluid (BALF). Results correlated in 43 (78·8%) of 52 cases studied. P. carinii PCR gave positive results with BALF from all 32 patients found to have P. carinii pneumonia (PCP); IF gave positive results with 26 of them. PCR was more sensitive and as specific as IF. However, at the present time, we do not believe that it is clinically useful for detection of P. carinii in BALF samples. P. carinii DNA amplification by PCR should be reserved for testing IF-negative BALF samples from patients judged clinically to have PCP.

Data reported by Dr. Sing et al. ([3][1]) about Pneumocystis carinii carriage in immunocompetent adults with primary pulmonary disorders differ partially from our results. We have analyzed 81 bronchoalveolar lavage fluid samples (BALs) from 78 patients (37 male and 41 female; mean age, 57.8 years

A nested PCR which amplified a portion of the mitochondrial large-subunit rRNA gene of Pneumocystis carinii was used to detect P. carinii DNA in blood from patients with P. carinii pneumonia. P. carinii DNA was not detected in serum and was detected at low levels of blood cells.

Two amplification steps were made to detect Pneumocystis carinii DNA by polymerase chain reaction (PCR). pAZ102-E and pAZ102-H (standard PCR), pAZ102-L2 (sense), and pAZ102-E (antisense) (two-step PCR) were used as primers. The amplification products were analyzed by ethidium bromide. After the two-step PCR, ethidium bromide detected all samples positive by oligohybridization after one amplification step. Our two-step PCR is a rapid, cost-effective, and clinically suitable method for the detection of P. carinii infection.