Igor Y. Pavlov

High Soluble CD30, CD25, and IL-6 May Identify Patients with Worse Survival in CD30+ Cutaneous Lymphomas and Early Mycosis Fungoides

Histopathology alone cannot predict outcome of patients with CD30+ primary cutaneous lymphoproliferative disorders (CD30CLPD) and early mycosis fungoides (MF). To test the hypothesis that serum cytokines/cytokine receptors provide prognostic information in these disorders, we measured soluble CD30 (sCD30), sCD25, and selected cytokines in cell cultures and sera of 116 patients with CD30CLPD and 96 patients with early MF followed up to 20 years. Significant positive correlation was found between sCD30 levels and sCD25, CD40L, IL-6, and IL-8, suggesting CD30+ neoplastic cells secrete these cytokines, but not Th2 cytokines. In vitro studies confirmed sCD30, sCD25, IL-6 and IL-8 are secreted by CD30CLPD-derived cell lines. CD30CLPD patients with above normal sCD30 and sCD25 had worse overall and disease-related survivals, but only sCD30 retained significance in Cox models that included advanced age. High sCD30 also identified patients with worse survival in early MF. Increased IL-6 and IL-8 correlated with poor disease-related survival in CD30CLPD patients, We conclude that: (1) neoplastic cells of some CD30CLPD patients do not resemble Th2 cells, (2) high serum sCD30, sCD25, IL-6, and perhaps IL-8 levels may provide prognostic information useful for patient management.

Identification and validation of shrimp-tropomyosin specific CD4 T cell epitopes.

BACKGROUND Shellfish allergy is an immune-mediated adverse reaction to allergenic shellfish and is responsible for significant morbidity and mortality. CD4 T cell responses play an important role in the pathophysiological mechanisms of sensitization and in production of IgE. OBJECTIVE We sought to identify and validate CD4 T cell shrimp tropomyosin-derived epitopes and characterize CD4 T cell responses in subjects with a clinical history of shellfish allergy. METHOD Using an in vitro MHC-peptide binding assay, we screened 91 overlapping peptides and identified 28 epitopes with moderate and strong binding capacities; 3 additional peptides were included based on MHC binding prediction score. These peptides were then examined in proliferation and cytokine release assays with T cells from allergic subjects. RESULT 17 epitopes restricted to DRB(∗)01:01, DRB1(∗)03:01, DRB1(∗)04:01, DRB1(∗)09:01, DQB1(∗)02:01, DQB1(∗)03:02 and DQB1(∗)05:01 alleles were identified and validated by both the MHC binding and the functional assays. Two peptides showed specificities to more than one MHC class II allele. We demonstrated that these peptides exert functional responses in an epitope specific manner, eliciting predominantly IL-6 and IL-13. CONCLUSION The identified epitopes are specific to common MHC class II alleles in the general population. Our study provides important data for the design of peptide-based immunotherapy of shrimp-allergic patients.

Post-transplant increased levels of serum sCD30 is a marker for prediction of kidney allograft loss in a 5-year prospective study.

BACKGROUND Levels of sCD30 represent a biomarker for early outcome in kidney transplantation. The purpose of this study was to determine the role of sCD30 levels for prediction of graft loss in the late post-transplant period. METHODS Sera were collected immediately pre-transplant and yearly thereafter for up to 5-year post-transplant in 37 primary renal transplant recipients. Levels of serum sCD30 were tested using a fluorescent microsphere assay. RESULTS Levels of sCD30 significantly decreased after transplantation and remained normal in 34 patients without graft loss up to 5-year post-transplant. Elevated levels of serum sCD30 preceded the increase of serum creatinine in patients with subsequent graft loss. CONCLUSIONS Elevated levels of serum sCD30 post-transplant might be a marker for predicting subsequent graft loss in the post-transplant period.

Penaeus monodon tropomyosin induces CD4 T-cell proliferation in shrimp-allergic patients.

Shellfish allergy affects approximately 2% of the population and can cause immediate hypersensitivity reactions such as urticaria, swelling, difficulty breathing, and, in some cases, anaphylaxis. Tropomyosin is the major shrimp allergen and binds IgE in two-thirds of patients. A total of 38 shrimp-allergic patients and 20 negative control subjects were recruited and evaluated on the basis of history, skin prick testing, specific immunoglobulin E (IgE) levels, and peripheral blood mononuclear cell proliferation in response to shrimp tropomyosin or shrimp tropomyosin-derived peptides. Of the classically allergic patients by history, 59% tested positive for serum shrimp IgE antibodies. Of patients with shrimp-specific IgE in sera, 70% also had significant IgE levels specific for shrimp tropomyosin. Peripheral blood mononuclear cells from classically shrimp-allergic patients proliferated in a dose-dependent manner in response to to tropomyosin. In addition, a T-cell line derived from a shrimp-allergic patient proliferated specifically in response to tropomyosin-derived peptides. These studies suggest a strategy for immunotherapy using a tropomyosin-derived T-cell epitope vaccination.

Patient result median monitoring for clinical laboratory quality control

BACKGROUND Statistical process control is foundational in laboratory medicine. It typically uses artificial control specimens and can detect some, but not all, analytical defects. A practical, robust method to more directly detect trends in patient results, such as monitoring mean or median patient results, is desirable. METHODS We generated a simulated set of laboratory results from a normal distribution, and also downloaded sequential patient results for serum sodium and CA 19-9. For each of the three data sets we calculated the standard error of the mean and estimated the standard error of the median by bootstrapping. RESULTS The standard error of the mean is a practical, easily calculated summary statistic that can be used to construct control charts. The standard error of the median, cannot be reliably estimated without using bootstrap methods, but is more resistant to outliers. Our study confirms a simple relationship between the variance of the median and the variance of the mean, i.e., for Gaussian distributions, Var[Median]/Var[Mean]=π/2. We also confirm that for skewed distributions, the median is more stable than the mean, implying Var[Median]/Var[Mean]<1. Finally, we establish a sample size of 200 individual patient results as sufficient for monitoring medians for data from approximately Gaussian distributions. CONCLUSIONS Monitoring patient result medians represents a practical, statistically self-consistent approach to laboratory quality control.

Binding of anti-HLA class I antibody to endothelial cells produce an inflammatory cytokine secretory pattern.

Current methods are inadequate for the diagnosis of early chronic allograft rejection. The goal of this study was to determine whether ligation of anti‐HLA antibodies to endothelial cells is associated with a distinctive cytokine secretory pattern. Human iliac artery endothelial cells (HIAEC) cultured in vitro were incubated with w6/32, an anti‐HLA class I mAb. Culture supernatants collected daily for up to 4 days were tested for secretion of 13 cytokines using a multiplexed fluorescent microsphere immunoassay. Culture of HIAEC with medium containing mAb w6/32 supported the growth of HIAEC during the 4‐day study period. Levels of the pro‐inflammatory cytokines IL‐1β, IL‐6, IL‐8, and TNF‐α became significantly increased in supernatants of HIAEC incubated with the mAb w6/32. We conclude that ligation of anti‐HLA class I antibodies to HLA class I antigens in endothelial cells initiates an acute inflammatory process and detecting an inflammatory cytokine secretory pattern might be useful to diagnose sub‐clinical chronic allograft rejection. J. Clin. Lab. Anal. 23:157–160, 2009.

Prognostic factors and risk stratification in early mycosis fungoides

Abstract Available demographic, clinical, histologic, immunohistochemical and laboratory findings, including serum cytokine/cytokine receptor levels, obtained at initial evaluation in a cohort of 33 patients with mycosis fungoides (MF) at stages I–IIA who had subsequent progression of disease were compared against 70 stage-matched cases of MF without observed progression. Significant factors that correlated with both disease progression and overall survival were: (1) presence of large Pautrier microabscesses (10 or more atypical lymphocytes), (2) presence of atypical lymphocytes with hyperchromatic or vesicular nuclei in the dermal infiltrate, (3) less than 20% CD8 + cells in the dermal infiltrate and (4) above normal (> 122 U/mL) serum immunoglobulin E (IgE) level. Combination of these factors was used to construct prognostic groupings which, if validated, might be useful to identify patients with clinically early MF at highest risk for disease progression and poor outcome.

Development and Validation of a Fluorescent Microsphere Immunoassay for Soluble CD30 Testing

ABSTRACT Testing for soluble CD30 (sCD30), an indicator of Th2 immune response, is a useful prognostic marker in solid organ transplantation, lymphoproliferative disorders, autoimmunity, and various parasitic diseases. In this study we report the development and validation of a fluorescent microsphere immunoassay for the detection of sCD30 in serum, plasma, and culture supernatants. The dynamic range of this assay is 1 to 400 ng/ml, and the rate of recovery of various concentrations of recombinant sCD30 ranges from 97 to 116% (average recovery, 105%). The test showed a high degree of precision in both intra-assay and interassay studies (coefficients of variation, as high as 7% and 8%, respectively), with a sensitivity of 1 ng/ml. The normal reference range calculated for a cohort of 151 healthy individuals was 1 to 29 ng/ml. The clinical usefulness of the sCD30 fluorescent microsphere immunoassay was demonstrated by showing that levels of sCD30 have a positive correlation with specimens containing high titers of anti-double-stranded DNA antibodies and high titers of immunoglobulin G against Leishmania species. Given the multiplexing potential of the sCD30 fluorescent microsphere immunoassay reported in this study, it is expected that testing of sCD30 concentrations along with those of other cytokines will become an important diagnostic tool for selected immunological and inflammatory diseases where Th2-type cytokine responses have been reported.

Clinical laboratory application of a reporter-gene assay for measurement of functional activity and neutralizing antibody response to infliximab

BACKGROUND TNF-α antagonists such as infliximab are effective for the treatment of inflammatory bowel disease and other inflammatory and autoimmune diseases. Development of an immune response and subsequent neutralizing antibodies against these protein-based drugs is a major impediment that contributes to therapeutic failure, or adverse effects such as hypersensitivity reactions. As opposed to empirical dose-escalation strategies, rational and cost-effective evaluation of clinical non-responsiveness includes measurement of serum drug levels, and detection of drug-specific antibodies. We present the validation and 2-y experience using a functional, cell-based reporter gene assay (RGA) developed for measuring the biological activity and antibody response to serum infliximab. METHODS The RGA was used to test 4699 clinical specimens from patients suspected of therapeutic failure. In contrast to binding assays, which detect an overall antibody response, the RGA specifically detects those antibodies that have drug-neutralizing function, and thus, poses higher risk for therapeutic failure. RESULTS The RGA presented here is currently the only functional clinical test available to measure serum infliximab activity and neutralizing antibodies. CONCLUSIONS Due to its accuracy and precision, and suitability for high-throughput testing, this robust platform can be applied to any TNF-α antagonist, providing an invaluable tool for the clinical management of patients with treatment failure.

Validation of Cytomegalovirus Immune Competence Assays for the Characterization of CD8 + T Cell Responses Posttransplant

Cytomegalovirus (CMV) infection is one of the most important infectious complications of transplantation. Monitoring CMV-specific CD8 T cell immunity is useful for predicting active CMV infection and for directing targeted antiviral therapy. In this study, we examined four basic parameters for validation of CMV-specific tetramer staining and peptide stimulation assays that cover five most frequent HLA class I alleles. We also examined the potential use of CMV-specific CD8+ T cell numbers and functional and cytolytic responses in two autologous HSCT recipients treated for multiple myeloma. The coefficient of variation (CV %) of the precision within assays was 3.1−24% for HLA-tetramer staining, 2.5−47% for IFN-γ, and 3.4−59.7% for CD107a/b production upon peptide stimulation. The precision between assays was 5−26% for tetramer staining, 4−24% for IFN-γ, and 5−48% for CD107a/b. The limit of detection was 0.1−0.23 cells/μL of blood for tetramer staining, 0−0.23 cell/μL for IFN-γ, and 0.11−0.98 cells/μL for CD107a/b. The assays were linear and specific. The reference interval with 95% confidence level was 0−18 cells/μL for tetramer staining, 0−2 cells/μL for IFN-γ, and 0–3 cells/μL for CD107a/b. Our results provide acceptable measures of test performance for CMV immune competence assays for the characterization of CD8+ T cell responses posttransplant measured in the absolute cell count per μL of blood.