Cell phenotypes as activity biomarkers in patients with Systemic Lupus Erythematosus

Abstract The pathogenesis of systemic lupus erythematosus (SLE) is complex. Few studies in Brazilian population have addressed cell phenotypes associated with immunological responses and their associations with SLE activity. The aim of this study is to investigate cell phenotypes associated to SLE diagnosis, treatment and activity. Twenty-eight SLE female patients (17 inactive, 11 active) and 10 healthy women were included in this study. Markers of natural killer (Nk), T and B cells in peripheral blood were evaluated by flow cytometry. Nkt cells were decreased only in SLE active patients. Activated CD4+, regulatory T FoxP3+ and B cells were decreased in both active and inactive SLE patients, compared to control group. The data corroborate the disruption of immune regulatory response in SLE patients and suggest phenotipic changes as possible biomarkers of SLE activity.

Correlation between active disease and hypercoagulability state in patients with systemic lupus erythematosus.

OBJECTIVE: This study has investigated whether high levels of Reticulocytes-C4d (R-C4d) and Platelets-C4d (P-C4d) reflecting recent activity in SLE patients are correlated with changes in natural anticoagulation components, coagulation activation and endothelial injury markers. METHODS: This study included three groups: 1) healthy women (control, n = 30); 2) women with low activity of the disease (SLEDAI 2 K ≤ 4, n = 30); 3) women with active disease (moderate or high activity) (SLEDAI 2 K > 4, n = 30). Median fluorescence intensity (MFI) of R-C4d and P-C4d were determined by flow cytometry using double labeling with specific monoclonal antibodies. Endothelial injury and hypercoagulability were evaluated by measuring Thrombomodulin and D-dimer levels. RESULTS: Higher MFI index of R-C4d were related to the recent activity of SLE, and higher expression of P-C4d indicated an elevated risk of thrombotic complications. Increased levels of soluble thrombomodulin and D-dimer were observed in patients with active SLE. CONCLUSION: R-C4d is helpful to monitor early disease activity and PC4-d may be an important tool to detect a prothrombotic phenotype in SLE. Elevated levels of D-dimer and thrombomodulin add value to P-C4d data and corroborate a hypercoagulable profile in women with SLE, contributing to an increased prothrombotic risk associated with inflammation.

Identification of potential biomarkers for systemic lupus erythematosus diagnosis using two-dimensional differential gel electrophoresis (2D-DIGE) and mass spectrometry.

Systemic lupus erythematosus (SLE) is an autoimmune disease of the connective tissue with a large spectrum of clinical manifestations. Immune deregulation leads to autoantibody and immune complexes overproduction, complement activation, and persistent tissue inflammation. Considering that the current diagnosis depends on the interpretation of the complex criteria established by the American College of Rheumatology and that the disease course is characterized by unpredictable activations and remissions, each patient develops different manifestations, and therefore, the discovery of specific biomarkers is urgently required. Therefore, this study aimed to identify putative biomarkers for active and inactive SLE potentially capable in distinguishing laboratorial SLE from other autoimmune diseases. The 2D-DIGE proteomics technique was used to evaluate the differential abundance of proteins between patients with active SLE, inactive SLE, patients with other autoimmune disease, and healthy individuals. Six proteins showed increased abundance in active SLE (A) and inactive SLE (I) compared to the C and O groups, but not between groups A and I. There were two transthyretin (TTR) fragments or proteins with a structure similar to TTR (accession numbers: PDB: 1GKO_A and 2PAB_A), retinol-binding protein 4 (RBP4) isoform X1 (no information in databases such as UNIPROT), and antibody fragments. Two proteins, APO-AIV and SP-40,40, were upregulated in group A than in O and C and in group I versus C, but not in group I versus O. Therefore, we suggest these proteins to be considered as candidates for the diagnosis of SLE.

Quality of bevacizumab (Avastin®) repacked in single-use glass vials for intravitreal administration.

PURPOSE:: Avastin® (bevacizumab) is an anti-vascular endothelial growth factor (VEGF) monoclonal antibody given as an off-label drug by intravitreal administration for treatment of ocular diseases. The drug's clinical application and its cost-benefit profile has generated demand for its division into single-use vials to meet the low volume and low-cost doses necessary for intraocular administration. However, the safety of compounding the drug in single-use vials is still under discussion. In this study, the stability and efficacy of Avastin® repacked in individual single-use glass vials and glass ampoules by external compounding pharmacies were evaluated. METHODS:: Polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography (SEC), dynamic light scattering (DLS), and turbidimetry were selected to detect the formation of aggregates of various sizes. Changes in bevacizumab biological efficacy were investigated by using an enzyme-linked immunosorbent assay (ELISA). RESULTS:: Repacked and reference bevacizumab showed similar results when analyzed by PAGE. By SEC, a slight increase in high molecular weight aggregates and a reduction in bevacizumab monomers were observed in the products of the three compounding pharmacies relative to those in the reference bevacizumab. A comparison of repacked and reference SEC chromatograms showed that the mean monomer loss was ≤1% for all compounding pharmacies. Protein aggregates in the nanometer- and micrometer-size ranges were not detected by DLS and turbidimetry. In the efficacy assay, the biological function of repacked bevacizumab was preserved, with <3% loss of VEGF binding capacity relative to that of the reference. CONCLUSION:: The results showed that bevacizumab remained stable after compounding in ampoules and single-use glass vials; no significant aggregation, fragmentation, or loss of biological activity was observed.

A Leishmania murine model to evaluate the immunomodulatory properties of Pythium insidiosum proteins.

Pythium insidiosum immunomodulatory vaccine (PiV) has been tested in clinical and experimental pythiosis. Previous data showed that P. insidiosum immunogens have the ability to switch the Th2 immune response, normally in place during pythiosis, to a curative Th1 response. Pythiosis cannot be reproduced in experimental rodents with the exception of rabbits, and thus thorough evaluation of PiV´s immunomodulatory properties has been limited by the lack of a compatible inbred mouse model. In this study, we took advantage of the murine BALB/c Leishmania infection model, where infected mice produce a Th2 response, to evaluate the PiV Th2 to Th1 immunomodulatory potential. Twenty-one days following challenge with L. major, large cutaneous granulomas developed in control mice, consistent with the expected Th2 response. In contrast, Leishmania-induced cutaneous lesions in PiV-immunized mice were minimal or absent. Flow cytometry analysis of spleen cells from mice immunized with PiV and subsequently challenged with L. major displayed more CD4+ and CD8+ cells than the control group. Moreover, spleen cells from mice that were immunized with PiV then challenged with L. major secreted high levels of IFN-γ, with a moderate IL-2, IL-4, and IL-10 mixed cytokine profile upon in vitro re-stimulation with PiV. Anti-P. insidiosum IgG1 in immunized animals was present at low titers suggesting a minor immunological role for this Ig isotype in this model. Our preliminary data showed that BALB/c mice challenged with L. major represent an attractive model in which to study PiV´s immunomodulatory properties.

Quality of bevacizumab (Avastin®) repacked in single-use glass vials for intravitreal administration / Avaliação da qualidade do bevacizumabe (Avastin®) fracionado em frascos de vidro de dose única para administração intravítrea

ABSTRACT Purpose: Avastin® (bevacizumab) is an anti-vascular endothelial growth factor (VEGF) monoclonal antibody given as an off-label drug by intravitreal administration for treatment of ocular diseases. The drug's clinical application and its cost-benefit profile has generated demand for its division into single-use vials to meet the low volume and low-cost doses necessary for intraocular administration. However, the safety of compounding the drug in single-use vials is still under discussion. In this study, the stability and efficacy of Avastin® repacked in individual single-use glass vials and glass ampoules by external compounding pharmacies were evaluated. Methods: Polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography (SEC), dynamic light scattering (DLS), and turbidimetry were selected to detect the formation of aggregates of various sizes. Changes in bevacizumab biological efficacy were investigated by using an enzyme-linked immunosorbent assay (ELISA). Results: Repacked and reference bevacizumab showed similar results when analyzed by PAGE. By SEC, a slight increase in high molecular weight aggregates and a reduction in bevacizumab monomers were observed in the products of the three compounding pharmacies relative to those in the reference bevacizumab. A comparison of repacked and reference SEC chromatograms showed that the mean monomer loss was ≤1% for all compounding pharmacies. Protein aggregates in the nanometer- and micrometer-size ranges were not detected by DLS and turbidimetry. In the efficacy assay, the biological function of repacked bevacizumab was preserved, with <3% loss of VEGF binding capacity relative to that of the reference. Conclusion: The results showed that bevacizumab remained stable after compounding in ampoules and single-use glass vials; no significant aggregation, fragmentation, or loss of biological activity was observed.

RESUMO Objetivos: Avastin® (bevacizumabe) é um anticorpo monoclonal inibidor do fator de crescimento endotelial de vasos (VEGF) utilizado "off-label" por meio de administração intravítrea para o tratamento de doenças oculares. A sua aplicação clínica associada ao custo-benefício do medicamento gerou uma demanda para seu fracionamento em frascos de dose única para utilização pela via intraocular. No entanto, a segurança do fracionamento do anticorpo em frascos de dose única ainda é alvo de discussão. Neste trabalho, a estabilidade e a eficácia do Avastin® fracionado em frascos ou ampolas de vidro de dose unitária por farmácias de manipulação do mercado foram avaliadas. Métodos: As técnicas de eletroforese em gel de poliacrilamida (PAGE), cromatografia por exclusão de tamanho (SEC), espalhamento dinâmico da luz (DLS) e turbidimetria foram empregadas para avaliar a formação de agregados de diferentes tamanhos. Alterações na atividade biológica do bevacizumabe foram estudadas utilizando ELISA. Resultados: Amostras referência e do bevacizumabe fracionado apresentaram resultados semelhantes quando analisado por gel de poliacrilamida. Por cromatografia por exclusão de tamanho, um pequeno aumento na quantidade de agregados de alta massa molar seguido de uma redução nos monômeros do bevacizumabe foram observados para as amostras das três farmácias de manipulação quando comparado ao referência. A comparação dos cromatogramas mostrou uma quantidade de redução do monômero inferior a 1% para todas as amostras fracionadas. Por espalhamento dinâmico da luz e turbidimetria, não foram detectados agregados de proteína na faixa de tamanho de micrômetro e nanômetro. No ensaio de eficácia, o bevacizumabe fracionado preservou sua função biológica pois apresentou menos de 3% de perda na capacidade de ligação ao VEGF quando comparado ao referência. Conclusão: Este estudo sugere que o bevacizumabe se mantem estável após fracionamento em ampolas e frascos de vidro de dose unitária pois não foram observadas agregação e/ou fragmentação de proteínas e perda de atividade biológica em quan tidades significativas.

Impaired fibrinolysis in angiographically documented coronary artery disease.

Impaired fibrinolysis may predispose to coronary artery disease (CAD). Hypofibrinolysis due to high levels of plasminogen activator inhibitor-1 (PAI-1) has been reported in CAD. A novel regulator of fibrinolytic activity, thrombin activatable fibrinolysis inhibitor (TAFI), has attracted attention in recent years. It acts by blocking the formation of a ternary complex of plasminogen, fibrin, and tissue plasminogen activator (t-PA). Previously ambiguous results regarding TAFI levels have been reported in CAD. We measured plasma levels of PAI-1 and TAFI antigen in 123 patients with age ranging from 40 to 65 years who had been submitted to coronary angiography and assessed the association of these markers with the extent of stenosis in three groups: angiographically normal artery (NAn), mild to moderate atheromatosis (MA), and severe atheromatosis (SA). Plasma levels of PAI-1 were increased in patients with severe atheromatosis compared to mild/moderate atheromatosis or to normal patients (66.60, 40.50, and 34.90 ng/mL, resp.; P < 0.001). For TAFI no difference was found between different groups. When patients were grouped in only two groups based on clinical cut-off point for intervention (stenosis less than or above 70%) we found increased plasma levels for PAI-1 (37.55 and 66.60 ng/mL, resp.; P < 0.001) and decreased plasma levels for TAFI (5.20 and 4.53 µg/mL, resp.; P = 0.04) in patients with stenosis above 70%. No difference was found in PAI-1 or TAFI levels comparing the number of affected vessels. Conclusion. As evidenced by a raised level of PAI-1 antigen, one can suggest an impaired fibrinolysis in stable CAD, although no correlation with the number of affected vessels was found. Curiously, a decreased plasma level of total TAFI levels was observed in patients with stenosis above 70%. Further studies measuring functional TAFI are required in order to elucidate its association with the extent of degree of atheromatosis.

Comparison of cytokine levels and metabolic parameters of stored platelet concentrates of the Fundação Hemominas, Belo Horizonte, Brazil.

BACKGROUND: Prolonged storage of platelets could improve availability and logistical management and decrease wastage. Immunobiochemical methods can be used to guarantee the quality of platelets after prolonged storage. OBJECTIVE: The aim of this study was to compare storage-related changes in buffy coat-derived platelet concentrations versus platelet-rich plasmal. METHODS: Units of whole blood were drawn using a quadruple-bag blood container system. Platelet-rich plasma and buffy coat prepared from whole blood following standard methods were stored for 9 days. During this period test samples were aseptically collected for analysis on Days 1, 2, 3, 5, 7 and 9. RESULTS: The highest CD42b expression was greater than 95%. The percentage of CD62p was significantly lower than the CD42b expression. The pH remained fairly stable during storage. Measurement of pO(2) and pCO(2) showed that oxygen levels were significantly higher than carbon dioxide levels. There were no significant differences in bicarbonate levels, glucose consumption and lactate production between the groups. The swirling effect with platelet-rich plasma samples decreased after 5 days of storage and after 7 days of storage for buffy coat samples. There was a significant twenty-fold increase in the mean IL-1ß after 5 days of storage for both groups. Slight increases in IL-6 and IL-8 levels were seen at 5 days. CONCLUSION: The quality of platelet concentrates remained acceptable during 7 days of storage in respect to the swirling effect, pH and platelet activation. There were no significant differences between buffy coat-derived platelets and platelet-rich plasma in this study.

Comparison of cytokine levels and metabolic parameters of stored platelet concentrates of the Fundação Hemominas, Belo Horizonte, Brazil

BACKGROUND: Prolonged storage of platelets could improve availability and logistical management and decrease wastage. Immunobiochemical methods can be used to guarantee the quality of platelets after prolonged storage. OBJECTIVE: The aim of this study was to compare storage-related changes in buffy coat-derived platelet concentrations versus platelet-rich plasma. METHODS: Units of whole blood were drawn using a quadruple-bag blood container system. Platelet-rich plasma and buffy coat prepared from whole blood following standard methods were stored for 9 days. During this period test samples were aseptically collected for analysis on Days 1, 2, 3, 5, 7 and 9. RESULTS: The highest CD42b expression was greater than 95%. The percentage of CD62p was significantly lower than the CD42b expression. The pH remained fairly stable during storage. Measurement of pO2 and pCO2 showed that oxygen levels were significantly higher than carbon dioxide levels. There were no significant differences in bicarbonate levels, glucose consumption and lactate production between the groups. The swirling effect with platelet-rich plasma samples decreased after 5 days of storage and after 7 days of storage for buffy coat samples. There was a significant twenty-fold increase in the mean IL-1β after 5 days of storage for both groups. Slight increases in IL-6 and IL-8 levels were seen at 5 days. CONCLUSION: The quality of platelet concentrates remained acceptable during 7 days of storage in respect to the swirling effect, pH and platelet activation. There were no significant differences between buffy coat-derived platelets and platelet-rich plasma in this study.

Comparative evaluation of phenol and thimerosal as preservatives for a candidate vaccine against American cutaneous leishmaniasis.

For decades thimerosal has been used as a preservative in the candidate vaccine for cutaneous leishmaniasis, which was developed by Mayrink et al. The use of thimerosal in humans has been banned due to its mercury content. This study addresses the standardization of phenol as a new candidate vaccine preservative. We have found that the proteolytic activity was abolished when the test was conducted using the candidate vaccine added to merthiolate (MtVac) as well as to phenol (PhVac). The Montenegro's skin test conversion rates induced by MtVac and by PhVac was 68.06% and 85.9%, respectively, and these values were statistically significant (p < 0.05). The proliferative response of peripheral mononuclear blood cells shows that the stimulation index of mice immunized with both candidate vaccines was higher than the one in control animals (p < 0.05). The ability of the candidate vaccines to induce protection in C57BL/10 mice against a challenge with infective Leishmania amazonensis promastigotes was tested and the mice immunized with PhVac developed smaller lesions than the mice immunized with MtVac. Electrophoresis of phenol-preserved antigen revealed a number of proteins, which were better preserved in PhVac. These results do in fact encourage the use of phenol for preserving the immunogenic and biochemical properties of the candidate vaccine for cutaneous leishmaniasis.

Comparative evaluation of phenol and thimerosal as preservatives for a candidate vaccine against American cutaneous leishmaniasis

For decades thimerosal has been used as a preservative in the candidate vaccine for cutaneous leishmaniasis, which was developed by Mayrink et al. The use of thimerosal in humans has been banned due to its mercury content. This study addresses the standardization of phenol as a new candidate vaccine preservative. We have found that the proteolytic activity was abolished when the test was conducted using the candidate vaccine added to merthiolate (MtVac) as well as to phenol (PhVac). The Montenegro's skin test conversion rates induced by MtVac and by PhVac was 68.06 percent and 85.9 percent, respectively, and these values were statistically significant (p < 0.05). The proliferative response of peripheral mononuclear blood cells shows that the stimulation index of mice immunized with both candidate vaccines was higher than the one in control animals (p < 0.05). The ability of the candidate vaccines to induce protection in C57BL/10 mice against a challenge with infective Leishmania amazonensis promastigotes was tested and the mice immunized with PhVac developed smaller lesions than the mice immunized with MtVac. Electrophoresis of phenol-preserved antigen revealed a number of proteins, which were better preserved in PhVac. These results do in fact encourage the use of phenol for preserving the immunogenic and biochemical properties of the candidate vaccine for cutaneous leishmaniasis.

Assessment of chemiluminescence and PCR effectiveness in relation to conventional serological tests for the diagnosis of Chagas' disease.

While testing 414 sera for the diagnosis of Chagas' disease, the conventional reactions of indirect hemagglutination, indirect immunofluorescence and the immunosorbent assay showed a sensitivity of 95.7%, 100% and 98.2% and a specificity of 98%, 98% and 96.4%, respectively, and an excellent association using Fisher's exact test. Chemiluminescence presented 100% sensitivity and 89.6% specificity, while PCR showed 100% specificity and 1.2% sensitivity. It is believed that the three conventional serological reactions are still adequate for diagnosing Chagas' disease.

Peripheral blood mononuclear cells immunophenotyping in pulmonary tuberculosis patients before and after treatment.

Tuberculosis (TB) is a lung disease caused by Mycobacterium tuberculosis. The interaction between the bacillus and the host may lead to a protective cellular immune response. In the present study, we propose the "in vitro" evaluation of this cellular immune response in patients with tuberculosis before and after chemotherapic treatment. Eleven patients with TB and 9 asymptomatic subjects with tuberculin skin test negative (TST-) (purified protein derivative (PPD)

Assessment of chemiluminescence and PCR effectiveness in relation to conventional serological tests for the diagnosis of Chagas' disease

While testing 414 sera for the diagnosis of Chagas' disease, the conventional reactions of indirect hemagglutination, indirect immunofluorescence and the immunosorbent assay showed a sensitivity of 95.7 percent, 100 percent and 98.2 percent and a specificity of 98 percent, 98 percent and 96.4 percent, respectively, and an excellent association using Fisher's exact test. Chemiluminescence presented 100 percent sensitivity and 89.6 percent specificity, while PCR showed 100 percent specificity and 1.2 percent sensitivity. It is believed that the three conventional serological reactions are still adequate for diagnosing Chagas' disease.

No exame de 414 soros, para o diagnóstico da doença de Chagas, as reações convencionais de hemaglutinação indireta, imunofluorescência indireta e o ensaio imunoenzimático mostraram, respectivamente, uma sensibilidade de 95,7 por cento, 100 por cento e 98,2 por cento e uma especificidade de 98 por cento, 98 por cento e 96,4 por cento e excelente associação usando teste exato de Fisher. A quimioluminescência apresentou 100 por cento de sensibilidade, 89,6 por cento de especificidade e a PCR 100 por cento de especificidade e 1,2 por cento de sensibilidade. Acredita-se que as três reações sorológicas convencionais ainda são suficientes para o diagnóstico da doença de Chagas.

Immuno-biochemical evaluations of phenol and thimerosal as antigen preservatives in Montenegro skin test.

Montenegro skin test (MST) represents the main complementary diagnostic test for tegumentary leishmaniases (TL) in endemic regions. Most antigen formulations used for the MST contain thimerosal as preservative. The Food and Drug Administration (FDA), however, recommended reducing or eliminating thimerosal from vaccines and other biological reagents and the Agência Nacional de Vigilância Sanitária (ANVISA) in Brazil, prohibited the use of mercurial compounds in immunobiologicals. In the search for an alternative stabilizer, phenol and thimerosal were tested as antigen preservatives in MST. Formulations were tested when fresh and after a 12-month storage at 4 degrees C in TL confirmed mice and human patients, and were evaluated for protein constitution by SDS-PAGE, Western blot and anti-gp63 ELISA. In mice, a decrease in the diagnostic effectiveness in merthiolate formulation was observed after a 12-month storage. SDS-PAGE, Western blot and anti-gp63 ELISA analyses showed a degradation of antigen proteins in both formulations after 12-month storage and that phenol-preserved antigen was quantitatively and qualitatively better than the merthiolate-preserved one. In patients, the average of induration diameter was larger in fresh antigens (p<0.05). However, storage time did not jeopardize their diagnostic capacity. No non-specific reactions produced by phenol or merthiolate were observed neither in humans nor in mice. Phenol could be a good alternative to replace the merthiolate in MST, and despite the proteolytic activity, antigens remain viable for at least 12 months.

Vaccination of C57BL/10 mice against cutaneous Leishmaniasis using killed promastigotes of different strains and species of Leishmania.

Antigenic extracts from five Leishmania stocks were used to vaccinate C57BL/10 mice. The Leishvacin(R) and PH8 monovalent vaccine yielded the highest IFN-gamma levels in the supernatants of spleen cell culture from vaccinated animals. Each single strain immunized group showed evidence of protective immunity six months after the challenge with promastigotes of Leishmania (Leishmania) amazonensis. No differences were detected between the vaccinated groups. It can be concluded that vaccines composed of single Leishmania stocks can provide protection to C57BL/10 mice against L. (L.) amazonensis infection.

Vaccination of C57BL/10 mice against cutaneous leishmaniasis using killed promastigotes of different strains and species of Leishmania

Antigenic extracts from five Leishmania stocks were used to vaccinate C57BL/10 mice. The Leishvacin® and PH8 monovalent vaccine yielded the highest IFN-gamma levels in the supernatants of spleen cell culture from vaccinated animals. Each single strain immunized group showed evidence of protective immunity six months after the challenge with promastigotes of Leishmania (Leishmania) amazonensis. No differences were detected between the vaccinated groups. It can be concluded that vaccines composed of single Leishmania stocks can provide protection to C57BL/10 mice against L. (L.) amazonensis infection

Assessment of immunity induced in mice by glycoproteins derived from different strains and species of Leishamnia

A comparative study was undertaken on the immunogenic properties of 63kDa glycoproteins obtained from five different strains/species of Leishmania and assessed in C57BL/10 mice. The humoral immune response was assessed by ELISA against the five different antigens of the immunized animals. The cellular immune response was derived from Leishmania. The response was found to be species-specific in all of determined by means of the cytokine profiles secreted by the spleen cells of immunized animals. The presence of y-IFN and IL-2, and the absence of IL-4 in the supernatants of cells stimulated by L. amazonensis antigen established that the cellular response is of Th1 type. The five glycoproteins tested were equally effective in protecting C57BL/10 mice against challenge by L. amazonensis. About 50 por cento of the immunized animals were protected for six months.