Troy D. Jaskowski

Analysis of Proinflammatory and Anti-Inflammatory Cytokine Serum Concentrations in Patients With Multiple Sclerosis by Using a Multiplexed Immunoassay

We examined cytokines and other inflammatory markers in serum samples from 833 patients with multiple sclerosis and 117 healthy control subjects. A multiplexed immunoassay was used to assess the concentrations of 13 cytokines/inflammatory markers: interferon (IFN)-γ; interleukins (ILs)-1β, 2, 4, 5, 6, 8, 10, 12, and 13; tumor necrosis factor (TNF)-α; IL-2 receptor; and soluble CD40 ligand. Significant increases between patients and control subjects were found for IFN-γ (mean, 7.5 vs 0.4 pg/mL; P = .0002), IL-2 (mean 5.7 vs 1.0 pg/mL; P =.0002), IL-1β (mean, 23.0 vs 11.3 pg/mL; P ≤ .0001), TNF-α (mean, 4.1 vs 1.2 pg/mL; P = .01), IL-4 (mean, 1.4 vs 0.1 pg/mL; P ≤ .0001), IL-10 (mean, 16.8 vs 7.5 pg/mL; P = .03), and IL-13 (mean, 4.5 vs 0.8 pg/mL; P ≤ .0001). Profiling cytokines in multiple sclerosis may help to identify mechanisms involved in the pathogenesis of the disease, aid in monitoring the disease course and in evaluating responses to specific therapies, and, potentially, lead to new therapies directed at cytokines or their receptors.

Determination of Cytokine Responses Using a Multiplexed Fluorescent Microsphere Immunoassay

We used a multiplexedfluorescent microsphere immunoassay to develop a sandwich capture assay to assess simultaneously the production of thymus helper (TH) 1- and TH2-type cytokines in tissue culture supernatant obtained from stimulated peripheral blood mononuclear cells. The assay then was used to assess the cytokine production of patients with hyperimmunoglobulinemia E syndrome and in cord blood from neonates. The multiplexed assay has a reportable range of less than 10 to 50,000 pg/mL. For linearity and recovery studies, R2 values for the 6 cytokines ranged from 0.988 to 0.999 for samples spiked with known concentrations of recombinant cytokine standards and for patient samples. The assay showed good specificity, with little cross-reactivity between cytokines. Results from supernatants of Staphylococcus aureus-stimulated peripheral blood mononuclear cells obtainedfrom 6 patients with hyperimmunoglobulinemia E syndrome showed significantly less interferon (IFN)-gamma production than cells from healthy control subjects. Cord blood cells from neonates produced significantly less interleukin 12 and IFN-gamma than cells from adults in group B streptococci-stimulated mononuclear cells. The fluorescent multiplexed microsphere immunoassay can be used to quantitate multiple cytokines from 1 sample and should be useful for further understanding of the cytokine role in disease.

Evaluation of Multiplexed Fluorescent Microsphere Immunoassay for Detection of Autoantibodies to Nuclear Antigens

ABSTRACT Antibodies to extractable nuclear antigens (ENA) are found in a variety of collagen vascular diseases. Determining the individual specificities of these antibodies is extremely useful in establishing the disease diagnosis and in some cases the prognosis. With a multiplexed fluorescent microsphere immunoassay, reactivity to five of the most diagnostically useful ENA was measured in 249 serum samples, including samples from 56 patients previously documented to have systemic lupus erythematosus (SLE). Results of the multiplexed assay were compared to results from established ENA enzyme-linked immunosorbent assays (ELISAs), and the agreement, sensitivity, and specificity, respectively, for the five ENA evaluated were as follows: SSA, 99.1, 100.0, and 98.8%; SSB, 98.6, 88.9, and 99.5%; Sm, 97.6, 95.8, and 97.9%; RNP, 97.2, 92.7, and 98.8%; Scl-70, 93.6, 50.0, and 99.0%. In the 56 confirmed SLE patients, the frequency of significant concentrations of autoantibodies with the multiplexed assay was 21.4% for SSA, 7.1% for SSB, 10.7% for Sm, 32.1% for RNP, and 0% for Scl-70. The new flow cytometric bead-based multiplexed assay showed excellent correlation with the well-established single-analyte ELISA methods for four of five the ENA markers investigated in this study. The most notable discrepancies between the two assays were for the Scl-70 antigen, which was most often resolved in favor of the multiplexed assay. Our studies show that the multiplexed microsphere-based immunoassay is a sensitive and specific method for the detection and semiquantitation of ENA antibodies in human sera.

Elevation of IgA anti-epidermal transglutaminase antibodies in dermatitis herpetiformis

Background  Dermatitis herpetiformis (DH) is a papulovesicular eruption caused by ingestion of gluten. It is characterized by the deposition of IgA in the dermal papillae. IgA antibodies directed at tissue transglutaminase (TG2) are elevated in gluten‐sensitive diseases including DH and coeliac disease (CD). More recently, antibodies directed at epidermal transglutaminase (TG3) were identified in patients with DH, and this may be the dominant autoantigen in this disease.

IgA Anti-Epidermal Transglutaminase Antibodies in Dermatitis Herpetiformis and Pediatric Celiac Disease

leprosy through their functional roles in antigen presentation and inhibition of T-cell responses. Although predisposing major histocompatibility complex alleles may exhibit inefficient antigen presentation, the LYP-Trp620 allele may have a pathogenic role in the hyporesponsiveness of T cells owing to anomalies in early T-cell signaling, resulting in clinical manifestations of leprosy. Contrary to our expectations, a significantly higher number of tuberculoid patients had PTPN22 1858CT, suggesting that there may be early T-cell defects in these patients. This results in a compromised immune response to the infectious agent, which manifests in the milder form of the disease. Most healthy people exposed to M. leprae are resistant to the infection, and with an effective immune response they do not develop the disease (Bongiorno et al., 2008). Because the CT genotype accounts for only 15–16% of lepromatous and tuberculoid patients, other genes involved in the downregulation of T-cell responses, such as CTLA-4 and Foxp3, should be investigated for additional host factors that may be detrimental in conferring anergy to M. leprae antigens in lepromatous leprosy patients.

Multiplex analysis of heterophil antibodies in patients with indeterminate HIV immunoassay results

We hypothesized that heterophil antibodies reactive with animal proteins used in blot preparation caused nonspecific staining (NSS) on HIV Western blot (WB) studies, causing indeterminate results. We analyzed samples showing NSS on HIV WB using a multiplexed immunoassay to simultaneously measure IgG antibodies to animal IgG (bovine, goat, sheep, mouse) and bovine serum albumin. Heterophil antibodies reactive with IgG from several animal species were detected in 23 (49%) of 47 samples showing NSS on HIV WB; 15 positive samples demonstrated antibodies to all 5 antigens. Similar IgG heterophil antibodies were detected in only 2 (8%) of 24 control samples. Of the HIV WB samples with a positive HIV-1 enzyme-linked immunosorbent assay (ELISA) result at the time of WB testing (11/47), heterophil antibodies were found in 8 (73%) of 11. Preabsorption with bovine, goat, and sheep IgG removed heterophil antibodies detected by the multiplexed assay and, in some cases, eliminated reactivity in ELISA and WB testing. Heterophil antibodies are associated with indeterminate HIV immunoassay results and are an important cause of false-positive HIV ELISA results. Multiplexed immunoassays provide a powerful tool for screening patients for heterophil antibodies and resolving possible false-positive results.

Comparison of Four Enzyme Immunoassays With a Western Blot Assay for the Determination of Type-Specific Antibodies to Herpes Simplex Virus

Most current enzyme immunoassays (EIAs) differentiate inadequately between types 1 and 2 herpes simplex virus (HSV) antibodies since significant cross-reactivity exists. We compared 4 IgG type-specific EIAs using a Western blot assay for resolution of discrepant results. The Diamedix had sensitivities of 100% for types 1 and 2 but specificities of only 71% and 61%, respectively. The cross-reactivity rate was 82% in positive samples tested. For HSV types 1 and 2, the Zeus sensitivities were 92% and 98%, respectively; specificities were 72% and 79%, respectively; the cross-reactivity rate was 54%. For HSV types 1 and 2, the Wampole sensitivities were 98% and 95%, respectively; specificities were 68% and 85%, respectively; the cross-reactivity rate was 47%. For HSV types 1 and 2, the Meridian sensitivities were 98% and 90%, respectively; specificities were 96% and 100%, respectively; no cross-reactivity was found between positive samples tested. While the Diamedix, Zeus, and Wampole assays showed good sensitivity, they lacked type specificity. The Meridian EIA offers the highest specificity along with no observed cross-reactivity. This EIA may be an easier, reliable alternative to Western blot for the determination of HSV type-specific antibodies.

Dermatitis Herpetiformis Sera or Goat Anti–Transglutaminase-3 Transferred to Human Skin-Grafted Mice Mimics Dermatitis Herpetiformis Immunopathology

Dermatitis herpetiformis (DH) is characterized by deposition of IgA in the papillary dermis. However, indirect immunofluorescence is routinely negative, raising the question of the mechanism of formation of these immune deposits. Sárdy et al. (2002. J. Exp. Med. 195: 747–757) reported that transglutaminase-3 (TG3) colocalizes with the IgA. We sought to create such deposits using passive transfer of Ab to SCID mice bearing human skin grafts. IgG fraction of goat anti-TG3 or control IgG were administered i.p. to 20 mice. Separately, sera from seven DH patients and seven controls were injected intradermally. Biopsies were removed and processed for routine histology as well as direct immunofluorescence. All mice that received goat anti-TG3 produced papillary dermal immune deposits, and these deposits reacted with both rabbit anti-TG3 and DH patient sera. Three DH sera high in IgA anti-TG3 also produced deposits of granular IgA and TG3. We hypothesize that the IgA class anti-TG3 Abs are directly responsible for the immune deposits and that the TG3 is from human epidermis, as this is its only source in our model. These deposits seem to form over weeks in a process similar to an Ouchterlony immunodiffusion precipitate. This process of deposition explains the negative indirect immunofluorescence results with DH serum.

Screening for IgG Antinuclear Autoantibodies by HEp-2 Indirect Fluorescent Antibody Assays and the Need for Standardization

We evaluated 5 commercially available HEp-2 antinuclear antibody (ANA) indirect fluorescent antibody (IFA) assays using patient serum samples from 45 patients with rheumatoid arthritis, 50 with systemic lupus erythematosus (SLE), 35 with scleroderma, 20 with Sjögren syndrome, 10 with polymyositis, and 100 healthy control subjects. In addition, 12 defined serum samples from the Centers for Disease Control and Prevention and 100 patient serum samples sent to ARUP Laboratories (Salt Lake City, UT) for ANA IFA testing were also examined (n = 372). Standardization among the HEp-2 IFA assays occurred when they exhibited the same titer ± 1 doubling dilution. Agreement of the 5 assays was 78%. Within the specific groups of serum samples, agreement ranged from 44% in scleroderma serum samples to 93% in healthy control subjects, with 72% agreement in the SLE group. Variations in slide and substrate quality were also noted (ie, clarity, consistency of fluorescence, cell size, number and quality of mitotic cells). Along with subjectivity of interpretation, HEp-2 IFA assays are also vulnerable to standardization issues similar to other methods for ANA screening.

Autoantibodies against phosphatidylserine, prothrombin and phosphatidylserine–prothrombin complex: Identical or distinct diagnostic tools for antiphospholipid syndrome?

BACKGROUND To clarify the association and diagnostic utility of measuring antiphosphatidyl serine (aPS), antiprothrombin (aPT) and the antiphosphatidyl serine/prothrombin complex (aPS/PT) antibodies in patients with antiphospholipid syndrome (APS) and recurrent pregnancy loss (RPL). METHOD We measured IgG and IgM anti-aPS/PT (2 different kits) as well as the aPS and aPT autoantibodies by ELISA. All subjects were also tested for the presence of Lupus anticoagulant (LA), IgG and IgM anti-cardiolipin (aCL) and anti-beta-2 glycoprotein I (ass(2)GPI), and these served as the gold standard unless otherwise indicated. Two groups of patients were studied: (i) APS with RPL and/or thrombosis (n=62); (ii) RPL only (n=66) and a group of healthy women with successful pregnancies (WSP; n=30). RESULTS The area under curve (AUC) analyses demonstrated significant differences between the aPS/PT (0.980) and aPT (0.850) assays (p=0.002) with no significant differences between the 2 aPS/PT kits or the aPS/PT and aPS assays. The overall agreement between the tested aPL antibodies and lupus anticoagulant (LA) for the APS cohort was very poor but associations between aPL assays were substantial (kappa>0.5) as determined by Cohen kappa analysis. CONCLUSIONS Our data show a lack of association between aPS/PT antibodies and LA in APS patients with recurrent pregnancy loss. In the evaluation of these patients, there may be redundancy in testing all three markers as the aPT and aPS assays formed part of the aPS/PT antibody repertoire.