Paradoxical sex-specific patterns of autoantibody response to SARS-CoV-2 infection.

BACKGROUND: Pronounced sex differences in the susceptibility and response to SARS-CoV-2 infection remain poorly understood. Emerging evidence has highlighted the potential importance of autoimmune activation in modulating the acute response and recovery trajectories following SARS-CoV-2 exposure. Given that immune-inflammatory activity can be sex-biased in the setting of severe COVID-19 illness, the aim of the study was to examine sex-specific autoimmune reactivity to SARS-CoV-2 in the absence of extreme clinical disease. METHODS: In this study, we assessed autoantibody (AAB) reactivity to 91 autoantigens previously linked to a range of classic autoimmune diseases in a cohort of 177 participants (65% women, 35% men, mean age of 35) with confirmed evidence of prior SARS-CoV-2 infection based on presence of antibody to the nucleocapsid protein of SARS-CoV-2. Data were compared to 53 pre-pandemic healthy controls (49% women, 51% men). For each participant, socio-demographic data, serological analyses, SARS-CoV-2 infection status and COVID-19 related symptoms were collected by  an electronic survey of questions. The symptoms burden score was constructed based on the total number of reported symptoms (N = 21) experienced within 6 months prior to the blood draw, wherein a greater number of symptoms corresponded to a higher score and assigned as more severe burden. RESULTS: In multivariable analyses, we observed sex-specific patterns of autoreactivity associated with the presence or absence (as well as timing and clustering of symptoms) associated with prior COVID-19 illness. Whereas the overall AAB response was more prominent in women following asymptomatic infection, the breadth and extent of AAB reactivity was more prominent in men following at least mildly symptomatic infection. Notably, the observed reactivity included distinct antigens with molecular homology with SARS-CoV-2. CONCLUSION: Our results reveal that prior SARS-CoV-2 infection, even in the absence of severe clinical disease, can lead to a broad AAB response that exhibits sex-specific patterns of prevalence and antigen selectivity. Further understanding of the nature of triggered AAB activation among men and women exposed to SARS-CoV-2 will be essential for developing effective interventions against immune-mediated sequelae of COVID-19.

Proteomic analysis to define predictors of treatment response to adalimumab or methotrexate in rheumatoid arthritis patients.

Seropositivity for anti-citrullinated peptide antibodies (ACPA) in patients with rheumatoid arthritis (RA), a chronic autoimmune arthritis, is associated with worse long-term disease outcomes. ACPA is ubiquitously tested in RA patients, but other autoantibodies exist (in both citrullinated and non-citrullinated form) which may provide additional information on RA subtypes and/or treatment response. We used a multiplex bead-based assay of 376 autoantibodies to test associations between these autoantibodies and treatment response in RA patients. Clusters of patients with similar autoantibody expression were defined and cluster membership was associated with treatment response. Thirty-four autoantibodies were differentially expressed in RA patients compared with healthy controls; citrullinated vimentin was associated with treatment response. A selection of citrullinated autoantibodies was found to be associated with treatment response in a subanalysis of ACPA-negative RA patients. Finer ACPA specificities in ACPA-negative RA patients may be predictive of treatment response and could represent a rich vein of future study.

High diagnostic accuracy of histone H4-IgG autoantibodies in systemic lupus erythematosus.

Objective: Diagnosis of SLE relies on the detection of autoantibodies. We aimed to assess the diagnostic potential of histone H4 and H2A variant antibodies in SLE. Methods: IgG-autoantibodies to histones H4 (HIST1H4A), H2A type 2-A (HIST2H2AA3) and H2A type 2-C (HIST2H2AC) were measured along with a standard antibody (SA) set including SSA, SSB, Sm, U1-RNP and RPLP2 in a multiplex magnetic microsphere-based assay in 153 SLE patients [85% female, 41 (13.5) years] and 81 healthy controls [77% female, 43.3 (12.4) years]. Receiver operating characteristic analysis was performed to assess diagnostic performance of individual markers. Logistic regression analysis was performed on a random split of samples to determine the additional value of histone antibodies in comparison with SA by likelihood ratio test and determination of diagnostic accuracy in the remaining validation samples. Results: Microsphere-based assay showed good interclass correlation (mean 0.85, range 0.73-0.99) and diagnostic performance in receiver operating characteristic analysis (area under the curve (AUC) range 84.8-93.2) compared with routine assay for SA parameters. HIST1H4A-IgG was the marker with the best individual diagnostic performance for SLE vs healthy (AUC 0.97, sensitivity 95% at 90% specificity). HIST1H4A-IgG was an independent significant predictor for the diagnosis of SLE in multivariate modelling (P < 0.0001), and significantly improved prediction of SLE over SA parameters alone (residual deviance 45.9 vs 97.1, P = 4.3 × 10-11). Diagnostic accuracy in the training and validation samples was 89 and 86% for SA, and 95 and 89% with the addition of HIST1H4A-IgG. Conclusion: HIST1H4A-IgG antibodies improve diagnostic accuracy for SLE vs healthy.

Comprehensive multiplexed autoantibody profiling of patients with advanced urothelial cancer.

BACKGROUND: Comprehensive profiling of autoantibodies (AAbs) in metastatic urothelial cancer (mUC) has not been performed to date. This may aid in diagnosis of UC, uncover novel therapeutic targets in this disease as well as identify associations between AAbs and response and toxicity to systemic therapies. METHODS: We used serum from patients with mUC collected prior to and after systemic therapy (immune checkpoint inhibitor (ICI) or platinum-based chemotherapy (PBC)) at Dana-Farber Cancer Institute. 38 age-matched and sex-matched healthy controls (HCs) from healthy blood donors were also evaluated. The SeroTag immuno-oncology discovery array (Oncimmune) was used, with quantification of the AAb reactivity toward 1132 antigens. Bound AAbs were detected using an anti-immunoglobulin G-specific detection antibody conjugated to the fluorescent reporter dye phycoerythrin. The AAb reactivity was reported as the median fluorescence intensity for each color and sample using a Luminex FlexMAP3D analyzer. Clinical outcomes of interest included radiographic response and development of immune-related adverse events (irAEs). Significance analysis of microarray was used to compare mUC versus HC and radiographic response. Associations with irAE were evaluated using a logistic regression model. P<0.05 was considered statistically significant. RESULTS: 66 patients were included with a median age of 68 years; 54 patients (82%) received ICI and 12 patients (18%) received PBC. Compared with HCs, AAbs against the cancer/testis antigens (CTAG1B, CTAG2, MAGEB18), HSPA1A, TP53, KRAS, and FGFR3 were significantly elevated in patients with mUC. AAbs against BRCA2, TP53, and CTNBB1 were associated with response, and those against BICD2 and UACA were associated with resistance to ICI therapy. AAbs against MITF, CDH3, and KDM4A were associated with development of irAEs in patient who received an ICI. A higher variance in pre-to-post treatment fold change in AAb levels was seen in patients treated with ICI versus PBC and was associated with response to ICI. CONCLUSIONS: This is the first report of comprehensive AAb profiling of patients with mUC and identified key AAbs that were elevated in patients with mUC versus HCs as well as AAbs associated with therapeutic response to ICI. These findings are hypothesis generating and further mechanistic studies evaluating humoral immunity in UC are required.

Comprehensive exploratory autoantibody profiling in patients with early rheumatoid arthritis treated with methotrexate or tocilizumab.

BACKGROUND: We sought to identify immunoglobin G autoantibodies predictive of early treatment response to methotrexate, the recommended first-line therapy for patients with newly diagnosed rheumatoid arthritis, and to the interleukin-6 receptor inhibitor biologic tocilizumab, initiated as the first disease-modifying anti-rheumatic drug. MATERIALS AND METHODS: In baseline sera of a subset of patients with newly diagnosed rheumatoid arthritis in the U-Act-Early study, selected based on specific responder/non-responder criteria using the Disease Activity Score assessing 28 joints (DAS28) within the first 20 weeks, we measured immunoglobin G antibody reactivity against 463 protein antigens and performed supervised cluster analysis to identify predictive autoantibodies for treatment response. The analysis subset comprised 56 patients in the methotrexate arm (22 responders, 34 non-responders) and 50 patients in the tocilizumab arm (34 responders, 16 non-responders). For comparison, these analyses were also performed in 50 age- and gender-matched healthy controls. RESULTS: Increased reactivity in responders versus non-responders was found in the methotrexate arm against two antigens-DOT1-like histone lysine methyltransferase (p = 0.009) and tropomyosin (p = 0.003)-and in the tocilizumab arm against one antigen-neuro-oncological ventral antigen 2 (p = 0.039). Decreased reactivity was detected against two antigens in the methotrexate arm-G1 to S phase transition 2 (p = 0.023) and the zinc finger protein ZPR1 (p = 0.021). Reactivity against the identified antigens was not statistically significant in either treatment arm for patients with rheumatoid factor-positive versus-negative or anti-cyclic citrullinated test-positive versus test-negative rheumatoid arthritis (p ≥ 0.06). CONCLUSIONS: Comprehensive profiling of baseline sera revealed several novel immunoglobin G autoantibodies associated with early treatment response to methotrexate and to tocilizumab in disease-modifying anti-rheumatic drug-naive patients with rheumatoid arthritis. These findings could eventually yield clinically relevant predictive markers, if corroborated in different patient cohorts, and may facilitate future benefit in personalised healthcare.

Profiling of IgG antibodies targeting unmodified and corresponding citrullinated autoantigens in a multicenter national cohort of early arthritis in Germany.

OBJECTIVE: To assess the diagnostic potential of IgG antibodies to citrullinated and corresponding native autoantigens in early arthritis. METHODS: IgG autoantibodies to 390 distinct unmodified and corresponding in vitro citrullinated recombinant proteins were measured by a multiplex assay in baseline blood samples from a German multicenter national cohort of 411 early arthritis patients (56.5 ± 14.6 years, 62.8% female). The cohort was randomly split into a training cohort (n = 329, 28.6% ACPA positive) and a validation cohort (n = 82, 32.9% ACPA pos.). The diagnostic properties of candidate antibodies to predict a subsequent diagnosis of rheumatoid arthritis (RA) as opposed to a non-RA diagnosis were assessed by receiver operating characteristics analysis and generalized linear modeling (GLM) with Bonferroni correction in comparison to clinically determined IgM rheumatoid factor (RF) and citrullinated peptide antibody (ACPA) status. RESULTS: Of 411 patients, 309 (75.2%) were classified as RA. Detection rates of antibody responses to citrullinated and uncitrullinated forms of the proteins were weakly correlated (Spearman's r = 0.13 (95% CI 0.029-0.22), p = 0.01). The concentration of 34 autoantibodies (32 to citrullinated and 2 to uncitrullinated antigens) was increased at least 2-fold in RA patients and further assessed. In the training cohort, a significant association of citrullinated "transformer 2 beta homolog" (cTRA2B)-IgG with RA was observed (OR 5.3 × 103, 95% CI 0.8 × 103-3.0 × 106, p = 0.047). Sensitivity and specificity of cTRA2B-IgG (51.0%/82.9%) were comparable to RF (30.8%/91.6%) or ACPA (32.1%/94.7%). Similar results were obtained in the validation cohort. The addition of cTRA2B-IgG to ACPA improved the diagnostic performance over ACPA alone (p = 0.026 by likelihood ratio test). CONCLUSIONS: cTRA2B-IgG has the potential to improve RA diagnosis in conjunction with RF and ACPA in early arthritis.

Comprehensive Longitudinal Surveillance of the IgG Autoantibody Repertoire in Established Systemic Lupus Erythematosus.

OBJECTIVE: To investigate the role of epitope spreading in established systemic lupus erythematosus (SLE). METHODS: IgG autoantibody reactivity with 398 distinct recombinant proteins was measured over a period of 6 years in 69 SLE patients and compared to that in 45 controls. Changes in mean fluorescence intensity (MFI), number of autoantibodies to distinct antigens, and reactivity with distinct clones of established antigenic targets (e.g., U1 RNP, Sm, and ribosomal P) representing epitope fine mapping were assessed. Linear mixed modeling, adjusted with Bonferroni correction for age and sex, was applied. RESULTS: The total number of autoantibodies, mean MFI, and number of autoantibodies in epitope fine mapping were higher in SLE patients compared to controls (P < 0.0001). The total number of antibodies to distinct autoantigens remained stable over time, while the mean MFI decreased over time in SLE (P < 0.021). SLE patients showed variable recognition of epitopes in fine mapping over time. In particular, in SLE patients, more clones of the U1 RNP complex were recognized at the time of new organ involvement (+0.65) (P = 0.007). Mean MFI was higher in patients with lupus nephritis (P = 0.047). The time-averaged MFIs of 22 individual autoantibodies (including double-stranded DNA [dsDNA]) were higher, after Bonferroni correction, in SLE (P < 0.0001). The MFIs of dsDNA and histone cluster 2 H3c were associated with scores on the Systemic Lupus Activity Measure (P < 0.0001). CONCLUSION: Longitudinal surveillance of the IgG autoantibody repertoire in established SLE reveals evidence of sustained breadth of autoantibody repertoire without significant expansion. Associations of disease activity with dsDNA and with histone H3 autoantibodies were confirmed.

Oncopeptidomics--a commentary on opportunities and limitations.

Cancer cells exhibit specific changes in protein expression and alterations in proteolytic activities. Peptides are capable of reflecting these pathological changes and are educible by dedicated analytical technologies. Oncopeptidomics can be defined as the comprehensive multiplexed analysis of endogenous peptides from a biological sample, under defined conditions, to discover probable valid peptide tumor biomarker. Here, mass spectrometry has shown its potential as a comprehensive peptide profiling tool. The efforts to arrive at diagnostically relevant biomarkers may have been underestimated. The establishment of novel cancer biomarkers will necessitate a multidisciplinary effort and presumably require a duration comparable to the drug development process. This review will address current concepts, new perspectives and the developmental process leading to clinically useful peptide tumor markers.

Sequential high-content profiling of the IgG-autoantibody repertoire reveals novel antigens in rheumatoid arthritis.

BACKGROUND: The aim was to identify novel diagnostic autoantibody candidates for rheumatoid arthritis (RA) by comprehensive screening for autoreactivity. METHOD: We incubated 5892 recombinant proteins coupled to fluorescent beads, with patients' sera for the detection of IgG-autoantibodies in three independent patient cohorts: A (n = 72 patients with established RA); B/B- (n = 116 patients with early RA (B) and n = 51 CCP-negative patients with early RA from B (B-)); and C (n = 184 patients with early seronegative RA), in comparison to matched healthy controls. Intersects of significantly increased autoantibodies as determined by the Mann-Whitney test were sought. RESULT: Screening of 5892 antigens in RA cohorts A and B, or the seronegative cohorts B- and C revealed intersects of 23 and 13 significantly increased autoantibodies, respectively. Reactivity to three antigens was increased in all cohorts tested: N-acetylglucosamine-1-phosphate transferase, gamma subunit (GNPTG), heterogeneous nuclear ribonucleoprotein A1-like 2 (HNRNPA1), and insulin-like growth factor binding protein 2 (IGFBP2). CONCLUSIONS: Comprehensive sequential screening for autoantibodies reveals novel candidates for diagnostic markers in both seropositive and seronegative RA and suggests new fields of research into the pathogenesis of RA.

The concept of functional peptidomics for the discovery of bioactive peptides in cell culture models.

Detection and purification of novel bioactive peptides from biological sources is a scientific task that led to a substantial number of important discoveries. One major laborious approach used is the repetitive stepwise separation of the test sample into several fractions followed by the determination of their bioactivity, until purity allows for sequence identification. We tested whether functional peptidomics, a combination of biological read-outs with differential peptide display (DPD) is a suitable strategy to isolate bioactive peptides at lower workload and with improved success. Additionally, we evaluated the use of DPD to monitor the processing status of proinsulin by inhibition of the insulin processing pathway. The rat insulinoma cell line INS-1 stimulated either with 2 mmol/l or 10 mmol/l glucose was used as model to generate differential peptide displays. In parallel, the bioactivity of the supernatants from the INS-1 cells was measured by glucose uptake and lipolysis assays using the adipocyte cell line 3T3-L1. We were able to quickly and elegantly trace the known activity of insulin to increase glucose uptake and inhibit lipolysis. Following re-chromatography of selected fractions, relevant peptides were identified by DPD and bioassays: the rat insulin-1 precursor and two different insulin peptides. We demonstrated in a semi-quantitative fashion that inhibition of proinsulin processing leads to accumulation of the insulin precursor, and reduced secretion of insulin-1. Thus, we conclude that DPD is an attractive support technology in peptide purification strategies aiming to identify bioactive compounds, and is superior to ELISA in discriminating between the processing status of insulin and its precursor.

Peptidomics biomarker discovery in mouse models of obesity and type 2 diabetes.

Type 2 diabetes mellitus (T2DM) is caused by the failure of the pancreatic beta-cell to secrete sufficient insulin to compensate a decreased response of peripheral tissues to insulin action. The pathological events causing beta-cell dysfunctions are only poorly understood and early markers that would predict islet function are missing. In contrast to immunoassays, unbiased proteomic technologies provide the opportunity to screen for novel marker protein and peptides of T2DM. An important subset of the proteome, peptides and peptide hormones secreted by the pancreas are deregulated in T2DM. The mass range of peptides and small proteins (1-20 kDa) is only sufficiently targeted by peptidomics, a combination of liquid chromatographic and mass spectrometric (MS) peptide analysis. Here, we describe the application of isotope label-free quantitative peptidomics to display and quantify relevant changes in the level of pancreatic peptides and peptide hormones in a preclinical model of T2DM, the Lep(ob)/Lep(ob) mouse. The amino acid sequence of statistical relevant top candidates was determined by MS/MS fragmentation or Edman degradation. The comparison of lean versus obese mice revealed increased levels of islet-specific peptides that can be divided into the following categories 1) the major islet peptide hormones insulin, amylin and glucagon; 2) proinsulin and C-peptide and 3) novel processing products of secretogranin, glucagon and amylin. Furthermore, we found increased levels of proteins and peptides implicated in zymogen granule maturation (syncollin) and nutritional digestion. In summary, our findings demonstrate that peptidomics is a valid approach to screen for novel peptide biomarkers.

Prerequisites for peptidomic analysis of blood samples: II. Analysis of human plasma after oral glucose challenge -- a proof of concept.

Mass spectrometric plasma analysis for biomarker discovery has become an exploratory focus in proteomic research: the challenges of analyzing plasma samples by mass spectrometry have become apparent not only since the human proteome organization (HUPO) has put much emphasis on the human plasma proteome. This work demonstrates fundamental proteomic research to reveal sensitivity and quantification capabilities of our Peptidomics technologies by detecting distinct changes in plasma peptide composition in samples after challenging healthy volunteers with orally administered glucose. Differential Peptide Display (DPD) is a technique for peptidomics studies to compare peptides from distinct biological samples. Mass spectrometry (MS) is used as a qualitative and quantitative analysis tool without previous trypsin digestion or labeling of the samples. Circulating peptides (< 15 kDa) were extracted from 1.3 mL plasma samples and the extracts separated by liquid chromatography into 96 fractions. Each fraction was subjected to MALDI MS, and mass spectra of all fractions were combined resulting in a 2D-display of > 2,000 peptides from each sample. Endogenous peptides that responded to oral glucose challenge were detected by DPD of pre-and post-challenge plasma samples from 16 healthy volunteers and subsequently identified by nESI-qTOF MS. Two of the 15 MS peaks that were significantly modulated by glucose challenge were subsequently identified as insulin and C-peptide. These results were validated by using immunoassays for insulin and C-peptide. This paper serves as a proof of principle for proteomic biomarker discovery down to the pM concentration range by using small amounts of human plasma.

Rolling Medical Practice: Ambulant Mobile Medical Care for Rural Areas.

We designed, constructed, and evaluated a mobile medical care vehicle called "Rollende Arztpraxis" (rolling medical practice, RMP) that delivers the full medical care of a general practitioner to increase medical care supply in rural areas. Six communities have been identified, where the RMP has been visited 501 times in 14 months. Two different schedules of stops and treatment times have been tested. We show that the RMP treated mainly elderly and multimorbid patients. An accompanying study showed high acceptance and satisfaction of treated patients and treating doctors. An economic evaluation of three different utilization models with three treatment modes each showed no financial sustainability. We show that ambulatory care in rural areas can be complemented by a mobile care unit, if legal and financial barriers can be overcome.

Structural domains required for channel function of the mouse transient receptor potential protein homologue TRP1beta.

Transient receptor potential proteins (TRP) are supposed to participate in the formation of store-operated Ca(2+) influx channels by co-assembly. However, little is known which domains facilitate the interaction of subunits. Contribution of the N-terminal coiled-coil domain and ankyrin-like repeats and the putative pore region of the mouse TRP1beta (mTRP1beta) variant to the formation of functional cation channels were analyzed following overexpression in HEK293 (human embryonic kidney) cells. MTRP1beta expressing cells exhibited enhanced Ca(2+) influx and enhanced whole-cell membrane currents compared to mTRP1beta deletion mutants. Using a yeast two-hybrid assay only the coiled-coil domain facilitated homodimerization of the N-terminus. These results suggest that the N-terminus of mTRP1beta is required for structural organization thus forming functional channels.

Rollende arztpraxis - first results of an ambulant mobile care model for rural areas.

German medical care is going to suffer from a decreasing number of general practitioners due to demographic change. We study if ambulatory care in rural areas can be complementary ensured by a mobile care unit. A medical care van - the "rolling medical practice" (RMP) - has been constructed based on care scenarios created for rural communities in northern Germany. Performance and acceptance of the RMP is evaluated by constant monitoring of anonymized medical documentation and questionnaires. The RMP is visiting six villages on two days a week in a three-week interval. It is constructed from a standard van with a custom box body fully equipped for general care needs. Actually treated care cases meet expectations and are acute as well as chronicle symptoms. Case numbers range from 6 to 50 visits in 5 month. We showed that almost full ranged mobile medical care, as supplement to general medical supply is possible.

In vivo profiling of DPP4 inhibitors reveals alterations in collagen metabolism and accumulation of an amyloid peptide in rat plasma.

Dipeptidyl peptidase 4 (DPP4) inhibitors represent a novel class of oral anti-hyperglycemic agents. The complete pharmacological profile of these protease inhibitors remains unclear. In order to gain deeper insight into the in vivo effects caused by DPP4 inhibition, two different DPP4 inhibitors (vildagliptin and AB192) were analyzed using differential peptide display. Wistar rats were treated with the DPP4 inhibitors (0.3mgkg(-1); 1mgkg(-1) or 3mgkg(-1) body weight) and DPP4 activity was measured before and at the end of the experiment. One hour after compound administration, blood plasma samples were collected to generate peptide displays and to subsequently identify differentially regulated peptides. A dose-dependent decrease in blood plasma DPP4 activity was measured for both inhibitors. DPP4 inhibition influenced collagen metabolism leading to depletion of collagen derived peptides (e.g. collagen alpha 1 (III) 521-554) and accumulation of related N-terminally extended collagen derived peptides (e.g. collagen alpha 1 (III) 519-554). Furthermore, the intact amyloid rat BRI (1-23) peptide was detected in plasma following in vivo DPP4 inhibition. DPP4 catalyzed cleavage kinetics of the BRI peptide were determined in vitro. The k(cat) and K(m) for cleavage by DPP4 were 5.2s(-1) and 14microM, respectively, resulting in a specificity constant k(cat)/K(m) of 0.36 x 10(6)s(-1)M(-1). Our results demonstrate that differential peptide analysis can be applied to monitor action of DPP4 inhibition in blood plasma. For the first time effects on basal collagen metabolism following DPP4 inhibition in vivo were demonstrated and the BRI amyloid peptide was identified as a novel DPP4 substrate.

Peptidomics analysis of human blood specimens for biomarker discovery.

This review addresses the concepts, limitations and perspectives for the application of peptidomics science and technologies to discover putative biomarkers in blood specimens. Peptidomics can be defined as the comprehensive multiplex analysis of endogenous peptides contained within a biological sample under defined conditions to describe the multitude of native peptides in a biological compartment. In addition to the discovery of disease associated biomarkers, an emerging field in peptidomics is the analysis of peptides to describe in vivo effects of protease inhibitors. The development and application of peptidomics technologies represent an arena of biomarker research that has the potential for adding significant clinical value.

Peptidomic analysis of breast cancer reveals a putative surrogate marker for estrogen receptor-negative carcinomas.

Estrogen-receptor status provides a major biomarker in breast cancer classification and has an important impact on prognosis and treatment options. The aim of this study was to investigate peptide profiles of invasive breast cancer with positive (n=39) and negative receptor status (n=41). Peptide profiles were generated by 'Differential Peptide Display', which is an offline-coupled combination of reversed-phase-HPLC and MALDI mass spectrometry. Mass spectrometric data were correlated with the immunohistochemically determined receptor state. Identification of peptides of interest was carried out by additional mass spectrometric methods (eg MALDI-TOF-TOF-MS-MS). Approximately 3000-7000 signals were detected per sample and thymosin alpha-1, an asparaginyl endopeptidase generated cleavage product of the ubiquitous acidic protein prothymosin-alpha, was found to differentiate the tumor samples according to their receptor status with the highest specificity. The concentration of Thymosin alpha-1 was found to be upregulated (n=37) in estrogen-negative cancer samples and downregulated (n=32) in estrogen-positive breast cancer samples. The expression of the precursor protein (Prothymosin-alpha) has been discussed previously as a prognostic factor in breast cancer. It is involved in the ER signal transduction pathway as an anti-coactivator-inhibitor. From our findings we conclude that Thymosin alpha-1 could serve as a surrogate marker in breast cancers and may indicate ER functionality.

B-cell chronic lymphocytic leukemia cells express a surface membrane phenotype of activated, antigen-experienced B lymphocytes.

B-cell chronic lymphocytic leukemia (B-CLL) is considered an accumulative disease of antigen-naive CD5(+) B lymphocytes that circulate in the resting state. However, to evaluate the possibility that B-CLL cells resemble antigen-experienced and activated B cells, we analyzed the expression of markers of cellular activation and differentiation on CD5(+)CD19(+) cells from B-CLL patients and from age-matched healthy donors. The leukemic cells from all B-CLL patients, including those that lack significant numbers of V gene mutations, bear the phenotype of activated B cells based on the overexpression of the activation markers CD23, CD25, CD69, and CD71 and the underexpression of CD22, Fcgamma receptor IIb, CD79b, and immunoglobulin D that are down-regulated by cell triggering and activation. Furthermore, these leukemic cells resemble antigen-experienced lymphocytes in the underexpression of molecules that are down-regulated by cell triggering and in the uniform expression of CD27, an identifier of memory B cells. A comparison of the phenotypes of B-CLL patients with and without immunoglobulin V gene mutations suggests that the 2 subgroups differ both in specific marker expression (CD69, CD71, CD62 L, CD40, CD39, and HLA-DR) and in the time since antigenic stimulation, based on the reciprocal relationship of CD69 and CD71 expression. These findings imply that the leukemic cells from all B-CLL cases (irrespective of V gene mutations) exhibit features of activated and of antigen-experienced B lymphocytes and that the B-CLL cells that differ in immunoglobulin V genotype may have different antigen-encounter histories.