Usefulness of specific anti-desmoglein 1 and 3 enzyme-linked immunoassay and indirect immunofluorescence in the evaluation of pemphigus activity.

OBJECTIVE: The objective was to assess the relationship between enzyme-linked immunoassay (ELISA) values of desmoglein (Dsg) 1 and Dsg3 antibodies and indirect immunofluorescence (IIF) values of anti-epithelial antibodies with disease activity in patients with pemphigus. PATIENTS AND METHODS: In a retrospective study, we analyzed 353 serum samples taken from 35 patients with pemphigus vulgaris (PV) and nine with pemphigus foliaceus (PF) during the course of the disease. In each sample, we measured anti-Dsg1 and anti-Dsg3 antibodies by ELISA. A receiver operating characteristics (ROC) curve was calculated to determine a cutoff value for anti-Dsg1 and anti-Dsg3 antibodies with optimal sensitivity and specificity. In 263 samples, we compared the ROC curves of anti-Dsg1 and anti-Dsg3 antibodies with the ROC curves of the IIF results. RESULTS: Activity of pemphigus was associated with a wide range of anti-Dsg1 and anti-Dsg3 antibody values. Levels of anti-Dsg1 antibodies showed a better relationship with cutaneous activity of pemphigus than levels of IIF anti-epithelial antibodies. The levels of IIF anti-epithelial antibodies showed a relationship with activity of mucosas similar to the levels of anti-Dsg3 antibodies. DISCUSSION: Abnormal values of anti-Dsg antibodies are not always associated with disease activity. ELISA detects both pathogenic and nonpathogenic anti-Dsg antibodies. CONCLUSIONS: Therapeutic strategies should not be based exclusively on anti-Dsg antibody values. Anti-Dsg1 antibodies showed a closer relationship with skin activity than IIF, while anti-Dsg3 antibodies showed a relationship with mucosal activity similar to the IIF test.

CD4+CD45RO+CD25-/lowCD127+: CD4+CD45RO+CD25hiCD127-/low Ratio in Peripheral Blood: A Useful Biomarker to Detect Cardiac Allograft Vasculopathy in Heart Transplanted Patients.

BACKGROUND: Cardiac allograft vasculopathy (CAV) is a major limitation in long-term graft survival after heart transplantation (HTx). Its prediction and detection at an early stage is a challenge because an accurate, minimally invasive blood test is lacking. The aim of this study was to analyze the relationship of Tact (CD4CD45ROCD25-CD127) cells, Th1 cells, and thymus-derived regulatory (Treg) (CD4CD45ROCD25CD127) cells in peripheral blood with the development of CAV in HTx patients. METHODS: First, we performed a cross-sectional study in 29 patients at least 2 years after HTx, 17 with CAV and 12 without CAV. We then prospectively followed a group of 38 patients for 2 years immediately after HTx surgery. In both groups, we analyzed the relationship between CAV and the effector-to-regulatory T cell ratio. RESULTS: In the cross-sectional study, patients with CAV showed statistically significant higher values of Th1-to-FoxP3Treg and Tact-to-CD127Treg ratios than non-CAV patients, with P less than 0.01 and P less than 0.001, respectively. Receiver operating characteristic curve analysis showed that the Tact:CD127Treg ratio was a potential biomarker of CAV, clearly discriminating CAV and non-CAV patients (area under curve [AUC] = 0.955; P = 0.001). In the prospective part of the study, we monitored the Th1:FoxP3Treg and Tact:CD127Treg ratios using the best tradeoff between anterior receiver operating characteristic sensitivity and specificity as a cutoff. Changes in the Tact:CD127Treg ratio were detected earlier than changes in the Th1:FoxP3Treg ratio. Both ratios were higher in HTx patients with CAV. CONCLUSION: The Tact-to-Treg ratio is a valuable follow-up marker to detect HTx patients at risk of developing CAV.

Efficiency of a solid-phase chemiluminescence immunoassay for detection of antinuclear and cytoplasmic autoantibodies compared with gold standard immunoprecipitation.

PURPOSE: The aim of this study was to compare the degree of agreement of a novel Zenit RA chemiluminescent immunoassay (CLIA) from A. Menarini Diagnostics (Florence, Italy) and the gold standard immunoprecipitation assay to screen for the presence of specific anti-U1snRNP, anti-Sm, anti-Ro/SS-A, anti-La/SS-B, anti-Jo-1((his)tRNA-Synthetase) and anti-Scl-70(Topo I) antibodies. MATERIALS AND METHODS: We studied 114 sera, 98 from patients with well-defined autoimmune connective tissue diseases and 16 from blood donor volunteers. All samples were fully characterized using the new chemiluminescent immunoassay and immunoprecipitation. In addition, all the samples were analyzed by indirect immunofluorescence (IIF) and anti-Scl-70(Topo I) antibodies were analyzed by immunoblot (IB) assay. Discrepant samples were analyzed using a commercial dot blot technique (Recomline from Mikrogen). The simple Kappa coefficient was used to measure the level of agreement between the results of Zenit RA CLIA and the gold standard. RESULTS: The Kappa agreement between Zenit RA CLIA and gold standard immunoprecipitation, as well as IB and IIFassays for the presence of anti-Scl-70(Topo I)(0.948) was excellent. The concordance between Zenit RA CLIA and gold standard immunoprecipitation for the presence of anti-U1snRNP (0.883), anti-Ro/SS-A (0.878), anti-Jo-1((his)tRNA-Synthetase) (0.791) and anti-Sm (0.786) was good, and excellent when the cut-off was raised to 14 U/ml (arbitrary units/ml). Between Zenit RA CLIA and gold standard immunoprecipitation for the presence of anti-La/SS-B, the Kappa agreement had a value of 0.689, but this improved to 0.775 when the cut-off was raised to14 U/ml. Precision was good based on the evaluation of replicate samples. Inter-assay coefficient variation was lower than 3.4 % (CV in %) in all the kits and <1.2 % (CV in  %) for intra-assay measurements. CONCLUSION: Our findings show that Zenit RA CLIA was specific and sensitive to detect anti-U1snRNP, anti-Sm, anti-Ro/SS-A, anti-La/SS-B, anti-Jo-1((his)tRNA-Synthetase) and anti-Scl70(Topo I) autoantibodies. This simple, fast and precise method can be a suitable option to analyze these autoantibody specificities.

Acquired bullous dermatosis associated with IgA multiple myeloma: a case report.

We present the case of a patient with IgA paraprotein who developed hemorrhagic subepidermal vesicles and bullae with numerous neutrophils. Direct immunofluorescence test (DIF) showed weak deposits of IgA lambda paraprotein at the dermal-epidermal junction and at the intercellular level in the basal layer of the epidermis, and stronger deposits in a perivascular and diffuse pattern in the dermis. Indirect immunofluorescence (IIF) test revealed the presence of circulating IgA lambda antibodies reacting with the intercellular space of monkey and guinea pig esophagus and human skin. A blood test revealed an IgA lambda paraprotein and multiple myeloma stage I(0) was diagnosed in a later hematological study. Dapsone was prescribed and cutaneous lesions improved. This is the second report of subepidermal vesicles and bullae with dermal deposits of IgA paraprotein appearing prior to diagnosis of an IgA multiple myeloma, and it is a unique case with circulating IgA lambda antibodies reacting with the intercellular space of epithelia.

Paraneoplastic pemphigus with negative direct immunofluorescence in epidermis or mucosa but positive findings in adnexal structures.

INTRODUCTION: Paraneoplastic pemphigus (PNP) is considered an autoimmune, multiorgan disease caused by antiplakin antibodies. We present three PNP patients who had negative epithelial direct immunofluorescence (DIF) findings in one or more biopsies. PATIENTS: An early lip biopsy of uninvolved oral epithelia in patient 1 was negative. A later biopsy from foreskin showed intense intercellular immunoglobulin G (IgG) deposits in the epithelia. In the early phase of the disease in patient 2, the intercellular fluorescence was negative in the epidermis, while intercellular IgG and C3 were observed in the sweat ducts. A later biopsy showed weak intercellular epidermal IgG and C3 fluorescence. Patient 3 showed intercellular IgG and/or C3 in follicular, sebaceous and sweat duct structures in several biopsies. No intercellular IgG or C3 was observed in the epithelia. DISCUSSION: The presence of immunoreactants in adnexal structures suggests that desmoplakins can be more strongly expressed in adnexa than in the epidermis, facilitating visualization of antibody deposits. CONCLUSIONS: Negative DIF findings in epithelia do not rule out the diagnosis of PNP, and the presence of IgG and/or C3 at the intercellular level of adnexal structures can help establish this diagnosis.

Enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence testing in a bullous pemphigoid and pemphigoid gestationis.

Enzyme-linked immunosorbent assay (ELISA) is an excellent tool for detection of circulating antibodies against the NC16A portion of BP180 antigen. We compared the sensitivity and specificity of a commercially available BP180-NC16a domain ELISA with that of an indirect immunofluorescence (IIF) testing in the evaluation of bullous pemphigoid (BP) and pemphigoid gestationis (PG), and analyzed the relationship between ELISA results and the presence of IgG deposition, in an epidermal or combined pattern, on direct immunofluorescence (DIF) testing of salt-split skin. ELISA was performed on serum from 28 patients (24 BP, 4 PG) and 50 controls. IIF testing was performed on serum from 27 patients and 98 controls. For the group of 28 patients with BP or PG, ELISA had a sensitivity of 93% and specificity of 96% (P < 0.001), while sensitivity was 74% and specificity 96% (P < 0.001) for IIF testing. In these patients, ELISA has a higher sensitivity than IIF testing, but similar specificity. Evaluation of controls who had IgG deposition on the dermal side of salt-split skin on DIF testing showed specificity for the ELISA of 100% (all four cases negative) and 80% for IIF testing (one of five positive). Positive ELISA correlated with a diagnosis of BP or PG only in patients who had IgG at the basement membrane zone (BMZ) by DIF testing. Overall, ELISA appears to have greater sensitivity and specificity for BP or PG than does IIF testing.

Bullous pemphigoid in a patient with psoriasis during the course of PUVA therapy: study by ELISA test.

A 65-year-old woman had a history of deep vein thrombosis and depression. Psoriasis was diagnosed in 1986 and various topical and systemic therapies, singly or in combination, were prescribed: tar, topical corticosteroids, cyclosporine, etretinate, and methotrexate. Two courses of oral and one course of bath psoralen plus UVA (PUVA) therapy (cumulative dose, 467 J/cm(2)) and UVB (2.96 J/cm(2)) had been given. In January 1999, she developed a flare of generalized psoriasis. In May 1999, therapy with PUVA (8-methoxypsoralen) plus topical acetonide triamcinolone 0.1% was initiated. At the time, she was taking acenocoumarol, lorazepam, and hydroxyzine chlorhydrate. In August 1999, at session 30, when the dose of UVA was 9 J/cm(2), and the total dose was 205 J/cm(2), a bulla appeared on the dorsum of the toe and was controlled with topical antibiotics. Five further sessions of PUVA were given and a generalized itching bullous eruption appeared all over the body. PUVA was stopped and the patient was hospitalized. On physical examination, extensive psoriatic plaques plus vesicles and bullae on the normal skin and on psoriatic lesions were observed all over the body (Fig. 1). Histopathologic study of a lesion showed a subepidermal vesicle containing fibrin, neutrophils, and a few eosinophils. No sunburn cells were observed (Fig. 2). The direct immunofluorescence (DIF) test of perilesional uninvolved skin revealed immunoglobulin G (IgG) (Fig. 3) and C3 at the dermal-epidermal junction. The DIF study using the patient's skin, previously treated with 1 m NaCl, localized the IgG at both the epidermal and dermal sides of the basement membrane zone (Fig. 4). Bullous pemphigoid (BP) was diagnosed and therapy with prednisone (60 mg/day) was started. The disease was well controlled in 3 weeks. The dose of prednisone was tapered and stopped 20 months later, without any recurrence. Study of the antibodies by the indirect immunofluorescence (IIF) test, using monkey esophagus and guinea pig as substrate, was positive at a titer of 1/160 in September 1999. The titer decreased to 1/10 in January 2000, and was negative in July 2000. An enzyme-linked immunosorbent assay (ELISA) test, performed using the commercial kit MBL, which identifies antibodies directed against epitopes of the extracellular fragment NC16 of antigen 2 of BP, was positive at 15 U/mL (normal value, < 9 U/mL) in September 1999, and negative in July 2000 (Table 1).

Anti-tRNP(ser)sec/SLA/LP autoantibodies. Comparative study using in-house ELISA with a recombinant 48.8 kDa protein, immunoblot, and analysis of immunoprecipitated RNAs.

BACKGROUND: Antibodies against tRNP((ser)sec) (ribonucleoproteins, RNP) have been described in our laboratory as markers of poor outcome in type 1 autoimmune hepatitis (AIH). The antigenic protein has been sequenced and cloned as a 48.8 kDa protein and identified with soluble liver antigen (SLA) and liver-pancreas (LP) antigen. The aim of this paper was to determine the best assay by which to detect these antibodies in type 1 AIH. METHODS: A simple and reliable enzyme linked immunoassay based on prokaryotically expressed protein was compared with an immunoblot assay using prokaryotically- and eukaryotically-expressed proteins and an assay based on immunoprecipitated RNAs from HeLa cell extracts. Eighty-one sera from 58 patients with type 1 AIH, 168 sera from patients with autoimmune diseases or chronic hepatitis C, and 60 sera from healthy subjects were similarly tested. RESULTS: The specificity of the assays was 100%, but the frequency of seropositivity was higher in the assay based on immunoprecipitated RNAs (44.4%) than in the enzyme-linked immunosorbent assay (ELISA) (16%) and the immunoblot assay with prokaryotically (12.34%) and eukaryotically (14.8%)-expressed protein. There were no clinical differences between the patients positive by ELISA, immunoblot assay, or immunoprecipitated RNAs. CONCLUSIONS: These results suggest that the analysis of the immunoprecipitated RNAs is the most useful, sensitive and specific method to detect anti-tRNP((ser)sec)/SLA/LP autoantibodies.

The IL-12 role in donor cell engraftment in a murine model of semiallogenic GVH disease with signs of autoimmune disease.

We analyzed the IL-12 effect in an autoimmune disease induced in a semiallogenic murine model of graft-vs-host disease (GVHD) Balb/c semiallogenic lymphoid cells i.v. infected in hybrid mice (Balb/c x A/J) F1 (CAF). IL-12 was administered 1 h before cell transplantation following two different protocols: (a) injecting 2 microg of mrIL-12 (murine recombinant IL-12) per mouse before the first semiallogenic cell injection; or (b) injecting the 2 microg of mrIL-12 fractionated in 5 days. ATh1 response was produced but an acute GVHD did not appear although differences in class I and II major histocompatibility complex (MHC) antigens were present. Four days after the semiallogenic cell transfer, IL-12 treated mice showed a marked reduction in the percent of spleen B cells compared with CAF1 control and CAF1 + Balb/c GVHD mice. After 5-6 months of follow-up, the donor cell chimerism increased significantly in spleen (70 +/- 31 vs. 43 +/- 31%) and in thymus. Flow cytometry of spleen lymphocytes demonstrated that donor chimerism was made up of TCD4, TCD8 and B lymphocytes and was higher in animals injected with IL-12. Moreover, CD8 T lymphocytes were 100% donor origin in the IL-12-injected group of GVHD animals and 50% origin in the IL-12-non-injected CAF1 + Balb/c group of animals. This paper shows that: (1) IL- 12 may play a role in the mechanisms of donor cell engraftment, probably produced by a CTL donor anti-host mechanism; (2) no acute GVHD was induced in spite of class I and II MHC differences; (3) IL-12 did not show any effect on the AR-like clinical signs of disease developed in this model of GVHD although histological subclinical signs were less frequent, and no glomerulonephritis was detected in the IL-12-treated GVHD mice.

Higher processing rates of Alu-containing sequences in kidney tumors and cell lines with overexpressed Alu-mRNAs.

Tumor cell growth and differentiation involve several molecular mechanisms that control gene expression and define specific genomic molecular profiles in cancer cells. Among these mechanisms, it has been shown that Alu-repetitive sequences are capable of regulating gene expression at transcriptional and posttranscriptional levels, and also of modulating cellular growth, differentiation and tumor suppression. Furthermore, repetitive sequences have also been implicated in alternative RNA splicing, although the specific mechanisms involved remain unknown. Nonetheless, exactly what the involvement of Alu-containing sequences in tumor cell growth and differentiation is or to what extent they might be related to tumorigenesis or to alternative splicing is not yet clear. In order to address some of these issues, we analyzed the level of expression of Alu-containing sequences in renal tumors and cell lines and their association with immunoprecipitated ribonucleoprotein splicing complexes in nuclear RNA fractions. Over-expression of Alu-containing sequences was detected in the poly(A)-RNA fractions of all analyzed tumors and cell lines. Furthermore, Alu-sequences were associated with tumor cell growth and differentiation and found overexpressed in purified small nuclear ribonucleoprotein fractions. Overall, our results suggest the involvement of Alu-sequences in the overexpression of Alu-containing-mRNAs in human tumors, and also higher processing rates of Alu-containing sequences at the spliceosome associated with tumor cell growth and differentiation.

Pénfigo paraneoplásico. Descripción de un caso y estudio de las subpoblaciones linfocitarias en el infiltrado inflamatorio / Paraneoplastic pemphigus. Description of a case and a study of lymphocyte subpopulations in the inflammatory tissue

Se presenta el caso de un paciente afecto de un linfoma folicular de bajo grado que en el curso de su enfermedad desarrolló úlceras en mucosa oral, en glande y prepucio. Las lesiones orales mostraron un patrón histológico de dermatitis de interfase y de acantólisis suprabasal. Solamente se detectaron depósitos de IgG en la sustancia intercelular del epitelio afecto de prepucio, pero no en la mucosa perilesional de labio inferior. Se detectaron anticuerpos circulantes dirigidos contra la sustancia intercelular que también reaccionaron con el epitelio de vejiga de rata. Por medio de la técnica de inmunoprecipitación fue posible comprobar que dichos anticuerpos reaccionaban frente a antígenos de 190 y 210 kD, que se han descrito forman parte del complejo del antígeno del pénfigo paraneoplásico. El estudio de las subpoblaciones linfocitarias del infiltrado inflamatorio demostró un predominio de linfocitos CD8, sin que se observara clonalidad en dicho infiltrado inflamatorio (AU)