Ignacio Sanz

Emory University, Atlanta, Georgia, United States

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Publications (24)121.84 Total impact

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    ABSTRACT: Flow cytometry datasets from clinical trials generate very large datasets and are usually highly standardized, focusing on endpoints that are well defined apriori. Staining variability of individual makers is not uncommon and complicates manual gating, requiring the analyst to adapt gates for each sample, which is unwieldy for large datasets. It can lead to unreliable measurements, especially if a template-gating approach is used without further correction to the gates. In this article, a computational framework is presented for normalizing the fluorescence intensity of multiple markers in specific cell populations across samples that is suitable for high-throughput processing of large clinical trial datasets. Previous approaches to normalization have been global and applied to all cells or data with debris removed. They provided no mechanism to handle specific cell subsets. This approach integrates tightly with the gating process so that normalization is performed during gating and is local to the specific cell subsets exhibiting variability. This improves peak alignment and the performance of the algorithm. The performance of this algorithm is demonstrated on two clinical trial datasets from the HIV Vaccine Trials Network (HVTN) and the Immune Tolerance Network (ITN). In the ITN data set we show that local normalization combined with template gating can account for sample-to-sample variability as effectively as manual gating. In the HVTN dataset, it is shown that local normalization mitigates false-positive vaccine response calls in an intracellular cytokine staining assay. In both datasets, local normalization performs better than global normalization. The normalization framework allows the use of template gates even in the presence of sample-to-sample staining variability, mitigates the subjectivity and bias of manual gating, and decreases the time necessary to analyze large datasets. © 2013 International Society for Advancement of Cytometry.
    Cytometry Part A 12/2013; · 3.71 Impact Factor
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    ABSTRACT: 9G4(+) IgG Abs expand in systemic lupus erythematosus (SLE) in a disease-specific fashion and react with different lupus Ags including B cell Ags and apoptotic cells. Their shared use of VH4-34 represents a unique system to understand the molecular basis of lupus autoreactivity. In this study, a large panel of recombinant 9G4(+) mAbs from single naive and memory cells was generated and tested against B cells, apoptotic cells, and other Ags. Mutagenesis eliminated the framework-1 hydrophobic patch (HP) responsible for the 9G4 idiotype. The expression of the HP in unselected VH4-34 cells was assessed by deep sequencing. We found that 9G4 Abs recognize several Ags following two distinct structural patterns. B cell binding is dependent on the HP, whereas anti-nuclear Abs, apoptotic cells, and dsDNA binding are HP independent and correlate with positively charged H chain third CDR. The majority of mutated VH4-34 memory cells retain the HP, thereby suggesting selection by Ags that require this germline structure. Our findings show that the germline-encoded HP is compulsory for the anti-B cell reactivity largely associated with 9G4 Abs in SLE but is not required for reactivity against apoptotic cells, dsDNA, chromatin, anti-nuclear Abs, or cardiolipin. Given that the lupus memory compartment contains a majority of HP(+) VH4-34 cells but decreased B cell reactivity, additional HP-dependent Ags must participate in the selection of this compartment. This study represents the first analysis, to our knowledge, of VH-restricted autoreactive B cells specifically expanded in SLE and provides the foundation to understand the antigenic forces at play in this disease.
    The Journal of Immunology 10/2013; · 5.52 Impact Factor
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    ABSTRACT: Objective: To determine the prevalence anti-apoptotic cell antibodies with the 9G4+ idiotype (9G4+) and the relationship between this reactivity and other known 9G4+ specificities and disease activity. Methods: Sera from 60 SLE patients and 40 healthy control donors were incubated with apoptotic Jurkat cells and assayed for binding of 9G4+ antibodies by flow cytometry. These samples were also tested for 9G4+ reactivity against naïve B cells and total IgG and IgM anti-apoptotic cell antibody (AACA) reactivity. Results: 9G4+ antibodies bound late apoptotic cells in a majority of SLE patients (60%) but only 2 healthy control sera. Among samples with global IgM or IgG AACA, samples with 9G4+ AACA predominated. Patients with high levels of 9G4+ AACA were more likely to have active disease and this remained true even in patients with IgG AACA, or anti-dsDNA. Patients with lupus nephritis were also more likely to have 9G4+ AACA. While 9G4+ reactivity to apoptotic cells often coincided with anti-B cell reactivity, some samples had distinct anti-apoptotic cell or anti-B cell reactivity. Conclusion: 9G4+ antibodies represent a major species of anti-apoptotic cell antibodies in SLE serum and this autoreactivity is associated with disease activity. The anti-apoptotic cell reactivity of 9G4+ antibodies can be separated from the germline VH4-34 encoded anti-B cell autoreactivity. Our results indicate that apoptotic cells are an important antigenic source in SLE that positively select B cells with intrinsic autoreactivity against other self-antigens. This selection of 9G4+ B cells by apoptotic cells may represent an important step in disease progression. © 2013 American College of Rheumatology.
    Arthritis & Rheumatology 08/2013; · 7.48 Impact Factor
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    ABSTRACT: Abstract Systemic lupus erythematosus is a chronic autoimmune disease of complex clinical presentation and etiology and is likely influenced by numerous genetic and environmental factors. While a large number of susceptibility genes have been identified, the production of antibodies against a distinct subset of nuclear proteins remains a primary distinguishing characteristic in disease diagnosis. However, the utility of autoantibody biomarkers for disease sub-classification and grouping remains elusive, in part, because of the difficulty in large scale profiling using a uniform, quantitative platform. In the present study serological profiles of several known SLE antigens, including Sm-D3, RNP-A, RNP-70k, Ro52, Ro60, and La, as well as other cytokine and neuronal antigens were obtained using the luciferase immunoprecipitation systems (LIPS) approach. The resulting autoantibody profiles revealed that 88% of a pilot cohort and 98% of a second independent cohort segregated into one of two distinct clusters defined by autoantibodies against Sm/anti-RNP or Ro/La autoantigens, proteins often involved in RNA binding activities. The Sm/RNP cluster was associated with a higher prevalence of serositis in comparison to the Ro/La cluster (P = 0.0022). However, from the available clinical information, no other clinical characteristics were associated with either cluster. In contrast, evaluation of autoantibodies on an individual basis revealed an association between anti-Sm (P = 0.006), RNP-A (P = 0.018) and RNP-70k (P = 0.010) autoantibodies and mucocutaneous symptoms and between anti-RNP-70k and musculoskeletal manifestations (P = 0.059). Serologically active, but clinically quiescent disease also had a higher prevalence of anti-IFN-α autoantibodies. Based on our findings that most SLE patients belong to either a Sm/RNP or Ro/La autoantigen cluster, these results suggest the possibility that alterations in RNA-RNA-binding protein interactions may play a critical role in triggering and/or the pathogenesis of SLE.
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    ABSTRACT: Mimetics of conformational protein epitopes have broad applications, but have been difficult to identify using conventional peptide phage display. The tenth type III domain of human fibronectin (FNfn10) has two extended, randomizable surface-exposed loops and might be more amenable to the identification of such mimetics. We therefore selected a library of FNfn10 clones, randomized in both loops (15 residues in all), for binding to monoclonal antibodies (MAb) that recognize the HIV-1 envelope glycoprotein. Anti-idiotypic monobodies (αIMs) mimicking both "linear" epitopes (2F5 and 4E10 MAbs) and conformational epitopes (b12 and VRC01 MAbs) were generated. αIMs selected against 2F5 and 4E10 frequently displayed sequence homology to the corresponding linear native epitopes. In the case of b12 and VRC01, we expected that the two constrained loop domains of FNfn10 would both contribute to complex conformational interactions with target antibodies. However, mutagenesis studies revealed differences from this simple model An αIM selected against b12 was found to bind its cognate antibody via only a few residues within the BC loop of FNfn10, with minimal contribution from the FG loop. Unexpectedly, this was sufficient to generate a protein that engaged its cognate antibody in a manner very similar to HIV-1 Env, and with a strong K(D) (43 nM). In contrast, an αIM selected against VRC01 engaged its cognate antibody in a manner that was dependent on both BC and FG loop sequences. Overall, these data suggest that the FNfn10 scaffold can be used to identify complex structures that mimic conformational protein epitopes. ααα
    Biochemistry 02/2013; · 3.38 Impact Factor
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    ABSTRACT: Potent HIV-1 specific broadly neutralizing antibodies (BNA) are uncommon in HIV infected individuals, and have proven hard to elicit by vaccination. Several, isolated monoclonal BNA are polyreactive and also recognize self-antigens, suggesting a breach of immune tolerance in persons living with HIV (PLWH). Persons with systemic lupus erythematosus (SLE) often have elevated levels of autoreactive antibodies encoded by the VH4-34 heavy chain immunoglobulin gene whose protein product can be detected by the 9G4 rat monoclonal antibody. We have recently found that levels of these "9G4+" antibodies are also elevated in PLWH. However, the putative autoreactive nature of these antibodies and the relationship of such reactivities with HIV neutralization have not been investigated. We therefore examined the autoreactivity and HIV neutralization potential of 9G4+ antibodies from PLWH. Results show that 9G4+ antibodies from PLWH bound to recombinant HIV-1 envelope (Env) and neutralized viral infectivity in vitro, whereas 9G4+ antibodies from persons with SLE did not bind to Env and failed to neutralize viral infectivity. In addition, while 9G4+ antibodies from PLWH retained the canonical anti-i reactivity that mediates B cell binding, they did not display other autoreactivities common to SLE 9G4+ antibodies, such as binding to cardiolipin and DNA and had much lower reactivity with apoptotic cells. Taken together, these data indicate that the autoreactivity of 9G4+ antibodies from PLWH is distinct from that of SLE patients, and therefore, their expansion is not due to a general breakdown of B cell tolerance but is instead determined in a more disease-specific manner by self-antigens that become immunogenic in the context of, and possibly due to HIV infection. Further studies of 9G4+ B cells may shed light on the regulation of B cell tolerance and interface between the generation of specific autoreactivities and the induction of antiviral immunity in persons living with HIV.
    PLoS ONE 01/2013; 8(12):e85098. · 3.73 Impact Factor
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    ABSTRACT: Objective. To determine the requirement for protein kinase C-β (PKC?β) in lupus development in Sle mice and to explore the potential of targeting PKCβ as a therapeutic strategy for lupus. Method. Congenic mice bearing the disease loci Sle1 or Sle1 and Sle3, which develop lupus with different severities, were crossed with PKCβ-deficient mice. The effect of PKCβ deficiency on lupus development was analyzed. In addition, the effects of a PKCβ-specific inhibitor, Enzastaurin, on the survival of B cells from lupus mice and human 9G4+ B cells, as well as in vivo treatment of the inhibitor in Sle mice on lupus development were investigated.Results. PKCβ deficiency abrogated clinical lupus phenotypes including autoantibodies, proteinuria, and histological features of lupus nephritis. Immunologically, there were remarkable decreases in spleen size and in peritoneal B1 cells, reduced activated CD4 T cells, and normalized CD4/CD8 ratios. Functionally, PKCβ deficiency induced an anergic B cell phenotype. Strikingly, PKCβ deficiency preferentially inhibited autoreactive plasma cells and autoantibodies in lupus mice, and PKCβ inhibition enhanced apoptosis of both lupus B cells from Sle mice and human autoreactive B cells (9G4+). Importantly, treatment of the Sle lupus mice with the PKCβ?specific inhibitor Enzastaurin prevented the disease development. Conclusion. Our study identifies PKCβ as a central mediator of SLE pathogenesis, suggesting that PKCβ? represents a promising therapeutic target for the treatment of SLE. Moreover, our results indicate the feasibility of using PKCβ inhibitor for lupus treatment. © 2012 American College of Rheumatology.
    Arthritis & Rheumatology 12/2012; · 7.48 Impact Factor
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    ABSTRACT: The diagnosis of primary Sjögren's syndrome (pSS) is difficult due to the lack of specific laboratory and clinical tests. As an initial step for the global discovery of changes in the abundance of parotid salivary proteins in pSS, a pooled sample was compared to that from healthy control subjects by multidimensional protein identification technology (MudPIT). A total of 1246 proteins were identified by MudPIT. The abundance of 477 of these proteins did not change, 529 were only detected in either the pSS or HC sample, while 206 of these proteins were significantly upregulated ≥ twofold and 34 were downregulated ≤ 0.5. Ingenuity Pathway Analyses of differentially expressed proteins identified by MudPIT resulted in the identification of 100 significant pathways. The same samples were quantified in parallel using RP MS. Fifty-eight of 71 proteins identified by RP overlapped with MudPIT results. Five proteins were further analyzed by targeted label-free quantification to confirm the similar relative differential expression observed by RP and MudPIT approaches. The present study supports the use of MS for global discovery and validation of marker proteins for improved and early diagnosis of pSS.
    Proteomics 08/2012; 12(19-20):3113-20. · 4.43 Impact Factor
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    ABSTRACT: A major goal in the study of autoimmune disease is the identification of biomarkers of disease to allow early diagnosis and initiation of treatment. The production of autoantibodies is the key feature of most autoimmune disease, so much effort has focused on characterizing the antigens reactive with these antibodies. However, even for the most well understood autoimmune diseases like rheumatoid arthritis and systemic lupus erythematosus, identification of antigens that detect autoantibodies in all patients have yet to be discovered. We describe a novel strategy for deriving mimotopes to disease-specific serum antibodies by selecting anti-idiotypic monobodies from a large molecular diversity library. Monobodies are derived by partial randomization of two surface exposed loops of a fibronectin domain scaffold in a phage display vector. The phage library is selected for binding to serum antibodies using a subtractive panning strategy. We evaluated this strategy by selecting the monobody library on a pool of serum immunoglobulin derived from a group of rheumatoid arthritis patients and evaluated selected clones for multi-patient reactivity and specificity for rheumatoid arthritis. The use of the fibronectin scaffold to derive stable, easy to produce molecular probes for diagnosis of autoimmune disease could be of significant value in improving diagnostic assays for virtually any disease that exhibits a characteristic immune response.
    Methods 07/2012; · 3.64 Impact Factor
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    ABSTRACT: Diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin lymphoma, remains a partially curable disease. Genetic alterations affecting components of NF-κB signaling pathways occur frequently in DLBCL. Almost all activated B cell-like (ABC) DLBCL, which is the least curable group among the 3 major subtypes of this malignancy, and a substantial fraction of germinal center B cell-like (GCB) DLBCL exhibit constitutive NF-κB pathway activity. It has been demonstrated that ABC-DLBCL cells require such activity for proliferation and survival. Therefore, inhibition of NF-κB activation in DLBCL may provide an efficient and targeted therapy. In screening for small-molecule compounds that may inhibit NF-κB activation in DLBCL cells, we identified a compound, NSC697923, which inhibits the activity of the ubiquitin-conjugating (E2) enzyme Ubc13-Uev1A. NSC697923 impedes the formation of the Ubc13 and ubiquitin thioester conjugate and suppresses constitutive NF-κB activity in ABC-DLBCL cells. Importantly, NSC697923 inhibits the proliferation and survival of ABC-DLBCL cells and GCB-DLBCL cells, suggesting the Ubc13-Uev1A may be crucial for DLBCL growth. Consistently, knockdown of Ubc13 expression also inhibited DLBCL cell survival. The results of the present study indicate that Ubc13-Uev1A may represent a potential therapeutic target in DLBCL. In addition, compound NSC697923 may be exploited for the development of DLBCL therapeutic agents.
    Blood 07/2012; 120(8):1668-77. · 9.06 Impact Factor
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    ABSTRACT: Systemic lupus erythematosus is a chronic autoimmune disease of complex clinical presentation and etiology and is likely influenced by numerous genetic and environmental factors. While a large number of susceptibility genes have been identified, the production of antibodies against a distinct subset of nuclear proteins remains a primary distinguishing characteristic in disease diagnosis. However, the utility of autoantibody biomarkers for disease sub-classification and grouping remains elusive, in part, because of the difficulty in large scale profiling using a uniform, quantitative platform. In the present study serological profiles of several known SLE antigens, including Sm-D3, RNP-A, RNP-70k, Ro52, Ro60, and La, as well as other cytokine and neuronal antigens were obtained using the luciferase immunoprecipitation systems (LIPS) approach. The resulting autoantibody profiles revealed that 88% of a pilot cohort and 98% of a second independent cohort segregated into one of two distinct clusters defined by autoantibodies against Sm/anti-RNP or Ro/La autoantigens, proteins often involved in RNA binding activities. The Sm/RNP cluster was associated with a higher prevalence of serositis in comparison to the Ro/La cluster (P = 0.0022). However, from the available clinical information, no other clinical characteristics were associated with either cluster. In contrast, evaluation of autoantibodies on an individual basis revealed an association between anti-Sm (P = 0.006), RNP-A (P = 0.018) and RNP-70k (P = 0.010) autoantibodies and mucocutaneous symptoms and between anti-RNP-70k and musculoskeletal manifestations (P = 0.059). Serologically active, but clinically quiescent disease also had a higher prevalence of anti-IFN-α autoantibodies. Based on our findings that most SLE patients belong to either a Sm/RNP or Ro/La autoantigen cluster, these results suggest the possibility that alterations in RNA-RNA-binding protein interactions may play a critical role in triggering and/or the pathogenesis of SLE.
    PLoS ONE 01/2012; 7(2):e32001. · 3.73 Impact Factor
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    ABSTRACT: The induction of a broadly neutralizing antibody (BNAb) response against HIV-1 would be a desirable feature of a protective vaccine. Vaccine strategies thus far have failed to elicit broadly neutralizing antibody responses; however a minority of HIV-infected patients do develop circulating BNAbs, from which several potent broadly neutralizing monoclonal antibodies (mAbs) have been isolated. The findings that several BNmAbs exhibit autoreactivity and that autoreactive serum antibodies are observed in some HIV patients have advanced the possibility that enforcement of self-tolerance may contribute to the rarity of BNAbs. To examine the possible breakdown of tolerance in HIV patients, we utilized the 9G4 anti-idiotype antibody system, enabling resolution of both autoreactive VH4-34 gene-expressing B cells and serum antibodies. Compared with healthy controls, HIV patients had significantly elevated 9G4+ serum IgG antibody concentrations and frequencies of 9G4+ B cells, a finding characteristic of systemic lupus erythematosus (SLE) patients, both of which positively correlated with HIV viral load. Compared to the global 9G4-IgD--memory B cell population, the 9G4+IgD--memory fraction in HIV patients was dominated by isotype switched IgG+ B cells, but had a more prominent bias toward "IgM only" memory. HIV envelope reactivity was observed both in the 9G4+ serum antibody and 9G4+ B cell population. 9G4+ IgG serum antibody levels positively correlated (r = 0.403, p = 0.0019) with the serum HIV BNAbs. Interestingly, other serum autoantibodies commonly found in SLE (anti-dsDNA, ANA, anti-CL) did not correlate with serum HIV BNAbs. 9G4-associated autoreactivity is preferentially expanded in chronic HIV infection as compared to other SLE autoreactivities. Therefore, the 9G4 system provides an effective tool to examine autoreactivity in HIV patients. Our results suggest that the development of HIV BNAbs is not merely a consequence of a general breakdown in tolerance, but rather a more intricate expansion of selective autoreactive B cells and antibodies.
    PLoS ONE 01/2012; 7(4):e35356. · 3.73 Impact Factor
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    ABSTRACT: To advance our understanding and treatment of disease, research immunologists have been called-upon to place more centralized emphasis on impactful human studies. Such endeavors will inevitably require large-scale study execution and data management regulation ("Big Biology"), necessitating standardized and reliable metrics of immune status and function. A well-known example setting this large-scale effort in-motion is identifying correlations between eventual disease outcome and T lymphocyte phenotype in large HIV-patient cohorts using multiparameter flow cytometry. However, infection, immunodeficiency, and autoimmunity are also characterized by correlative and functional contributions of B lymphocytes, which to-date have received much less attention in the human Big Biology enterprise. Here, we review progress in human B cell phenotyping, analysis, and bioinformatics tools that constitute valuable resources for the B cell research community to effectively join in this effort.
    Frontiers in Immunology 01/2012; 3:302.
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    ABSTRACT: B lymphocyte involvement in systemic lupus erythematosus has been recognized for several decades, mainly in the context of autoantibody production. Both mouse and human studies reveal that different types of antibody responses, as well as antibody-independent effector functions can be ascribed to distinct subpopulations (subsets) of circulating B cells. Characterizing human B cell subsets can advance the field of autoimmunity even further by establishing B cell signatures associated with disease severity, progression, and response-to-treatment. For this purpose, we have developed specialized B cell reagent panels for multiparameter flow cytometry, and combine their use with advanced bioinformatics strategies that together will likely be advantageous for improving the characterization, prognosis, and for possibly improving treatment regimens of chronic inflammatory diseases such as lupus.
    Methods in molecular biology (Clifton, N.J.) 01/2012; 900:109-34. · 1.29 Impact Factor
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    ABSTRACT: As a group, rheumatoid arthritis (RA) patients exhibit increased risk of infection, and those treated with anti-tumor necrosis factor (TNF) therapy are at further risk. This increased susceptibility may result from a compromised humoral immune response. Therefore, we asked if short-term effector (d5-d10) and memory (1 month or later) B cell responses to antigen were compromised in RA patients treated with anti-TNF therapy. Peripheral blood samples were obtained from RA patients, including a subset treated with anti-TNF, and from healthy controls to examine influenza-specific responses following seasonal influenza vaccination. Serum antibody was measured by hemagglutination inhibition assay. The frequency of influenza vaccine-specific antibody secreting cells and memory B cells was measured by EliSpot. Plasmablast (CD19+IgD-CD27hiCD38hi) induction was measured by flow cytometry. Compared with healthy controls, RA patients treated with anti-TNF exhibited significantly decreased influenza-specific serum antibody and memory B cell responses throughout multiple years of the study. The short-term influenza-specific effector B cell response was also significantly decreased in RA patients treated with anti-TNF as compared with healthy controls, and correlated with decreased influenza-specific memory B cells and serum antibody present at one month following vaccination. RA patients treated with anti-TNF exhibit a compromised immune response to influenza vaccine, consisting of impaired effector and consequently memory B cell and antibody responses. The results suggest that the increased incidence and severity of infection observed in this patient population could be a consequence of diminished antigen-responsiveness. Therefore, this patient population would likely benefit from repeat vaccination and from vaccines with enhanced immunogenicity.
    Arthritis research & therapy 12/2011; 13(6):R209. · 4.27 Impact Factor
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    ABSTRACT: Simple and non-invasive saliva-based diagnostics may be useful for the identification, understanding, and monitoring of autoimmune and infectious diseases. Previously, Luciferase Immunoprecipitation Systems (LIPS) were used for sensitive detection of patient serum autoantibodies in Sjögren's Syndrome (SjS), a chronic autoimmune disease affecting the salivary and lacrimal glands. Here we explored the ability of LIPS to diagnose SjS based on IgG autoantibodies in patient saliva. From LIPS testing, anti-Ro60 autoantibodies were detected in the saliva of 70% (19/27) of SjS patients with 96% specificity. Positive anti-Ro60 autoantibodies were also found in 70% of the matched serum samples (96% specificity). LIPS detected Ro52 autoantibodies in the saliva and serum of 67% of SjS patients with 100% specificity. Overall, the autoantibody titers in saliva were approximately 4000-fold lower by volume than serum, but still distinguished seropositive patients from controls. These results suggest that LIPS salivary-based testing for SjS autoantibodies is a practical alternative to serum and compatible with point-of-care testing.
    Journal of dental research 01/2011; 90(4):445-9. · 3.46 Impact Factor
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    ABSTRACT: Establishing long-term allograft acceptance without the requirement for continuous immunosuppression, a condition known as allograft tolerance, is a highly desirable therapeutic goal in solid organ transplantation. Determining which recipients would benefit from withdrawal or minimization of immunosuppression would be greatly facilitated by biomarkers predictive of tolerance. In this study, we identified the largest reported cohort to our knowledge of tolerant renal transplant recipients, as defined by stable graft function and receiving no immunosuppression for more than 1 year, and compared their gene expression profiles and peripheral blood lymphocyte subsets with those of subjects with stable graft function who are receiving immunosuppressive drugs as well as healthy controls. In addition to being associated with clinical and phenotypic parameters, renal allograft tolerance was strongly associated with a B cell signature using several assays. Tolerant subjects showed increased expression of multiple B cell differentiation genes, and a set of just 3 of these genes distinguished tolerant from nontolerant recipients in a unique test set of samples. This B cell signature was associated with upregulation of CD20 mRNA in urine sediment cells and elevated numbers of peripheral blood naive and transitional B cells in tolerant participants compared with those receiving immunosuppression. These results point to a critical role for B cells in regulating alloimmunity and provide a candidate set of genes for wider-scale screening of renal transplant recipients.
    The Journal of clinical investigation 06/2010; 120(6):1836-47. · 15.39 Impact Factor
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    ABSTRACT: Upon vaccination, B cells differentiate into antibody secreting cells (ASCs) that migrate via the circulation to tissues. The kinetics of this response and the relationship of circulating ASCs to protective antibody titers have not been completely explored. Influenza-specific and total-IgG ASCs were enumerated by Elispot and flow cytometry daily in the blood in 6 healthy adults after trivalent influenza vaccination (TIV). Peak H1-specific IgG ASC frequencies occurred variably from day 5 to 8 and correlated with the fold-rise rise in hemagglutination inhibition (HAI titers); r=0.91, p=0.006. H3-specific IgG ASC frequencies correlated less well, perhaps due to a mismatch of the H3 protein in the vaccine and that used in the Elispot assay. Peak frequencies of vaccine-specific and total-IgG ASCs were 0.3% and 0.8%, respectively, of peripheral blood mononuclear cells (PBMC). Peak TIV-, H1-, H3-, and total-IgG ASC frequencies were 1736+/-1133, 626+/-520, 592+/-463, and 4091+/-2019 spots/10(6) PBMC, respectively. Peak TIV-, H1-, and H3-specific IgG ASC of total-IgG ASC frequencies constituted 63%+/-21, 26%+/-10, 22%+/-17, respectively. After immunization with inactivated influenza vaccine the peak in influenza-specific ASC frequencies is variable but correlates well with the magnitude of protective HAI responses.
    Vaccine 03/2010; 28(20):3582-7. · 3.77 Impact Factor
  • Andreea Coca, Ignacio Sanz
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    ABSTRACT: The critical role of B cells in the pathogenesis of systemic lupus erythematosus and Sjogren's syndrome has provided a strong rationale to specifically target B cells. This review summarizes recent advances in the field of B cell depletion in systemic lupus erythematosus and Sjögren's syndrome. Reports of successful B cell depletion therapy in refractory SLE have continued to surface over the last year. The accumulation of positive results therefore stands in stark contrast to the recent reports that two phase III randomized placebo controlled trials employing B cell depletion with rituximab in nonlupus and lupus nephritis (Explorer and Lunar, respectively) did not achieve. Multiple reasons, including trial design, limitations of outcome instruments and sort follow-up have been invoked to explain these disconcerting results. In the representative studies addressing B cell depletion in lupus in the last year, complete and partial remission in lupus nephritis has been achieved in 60-89% of cases. Improvements in the British Isles Lupus Assessment Group (BILAG) and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) scores were associated with decrease in anti-dsDNA and increase in complement levels. B cell depletion seemed quite efficacious also in pediatric SLE. While more definitive studies are still lacking for primary Sjogren's syndrome, incidental reports indicating potential efficacy have also been recently published. Despite the disappointing results of Explorer and Lunar trials, other evidence continues to be published in support of the notion that B cell depletion could be useful for patients with refractory disease, including lupus nephritis, and antibody-mediated cytopenias, possibly in combination with other immunosuppressant medication.
    Current opinion in rheumatology 08/2009; 21(5):483-8. · 4.60 Impact Factor
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    ABSTRACT: The rise in influenza-specific neutralizing antibody levels is proceeded by a burst of antigen-specific antibody secreting cells (ASC) or plasmablasts identified in peripheral blood approximately 5-10 days post immunization. Blood antigen-specific ASC may function as an immune marker of vaccine responses in comparison to the pre- and post-neutralizing titers; however, some have questioned whether there is adequate survival of ASC isolated from peripheral blood after freezing, making multi-center vaccine trials difficult. Here, we demonstrate similar frequencies of influenza-specific ASC from fresh and frozen peripheral blood mononuclear cells (PBMC). Influenza Hemagglutinin (HA) H1, H3, and H7-specific ASC IgG ELISpots frequencies were compared from the same fresh and frozen PBMC 7 days after 2006 Trivalent Influenza Vaccine (TIV) in 10 young healthy subjects. H1-, H3-, and H7-specific IgG ASC spots/10(6) from fresh PBMC on day 7 were 229+/-341, 98+/-90, and 6+/-11 respectively. Total IgG ASC spots/million PBMC pre- and 7-day post-vaccination were 290+/-188 (0.029% PBMC) and 1691+/-836 (0.17% PBMC) respectively. There was no difference in the H1 -H3-, and total specific ASC IgG ELISpot frequencies from the fresh versus frozen PBMC on day 7 (p=0.43, 0.28, 0.28 respectively). These results demonstrate feasibility of testing whether antigen-specific ASC from frozen PBMC are an early biomarker of long-term antibody responses in multi-center vaccine trials.
    Journal of Immunological Methods 12/2008; 340(1):42-7. · 2.23 Impact Factor

Publication Stats

473 Citations
121.84 Total Impact Points

Institutions

  • 2013
    • Emory University
      • Division of Rheumatology
      Atlanta, Georgia, United States
  • 2009–2013
    • University of Rochester
      • Division of Allergy, Immunology and Rheumatology
      Rochester, New York, United States
  • 2011–2012
    • University Center Rochester
      Rochester, Minnesota, United States