[Show abstract][Hide abstract] ABSTRACT: A 33-year-old male, first time blood donor with a past history of HBV vaccination and no declared risk factors for HBV infection, was found with an absence of reactivity to HBV markers, HBV DNA fluctuation, and a strong and unremitting host cellular immune response to HBV detected over time. HBV genome sequencing identified a genotype F strain, subgenotype F2. This is the first case report that associated subgenotype F2 with occult HBV infection in Brazilian blood donors.
[Show abstract][Hide abstract] ABSTRACT: Providing blood units for patients with an antibody to a high-prevalence antigen or with multiple common antibodies is a constant challenge to the blood banks. Finding a compatible donor requires extensive screening, with incurs a large amount of investment. In this article, we share our experience of organizing a rare donor inventory with limited resources, we include the strategy used for finding rare donors, and we share the difficulties found during the implementation of the approach and the results obtained.
No preview · Article · Jan 2016 · Immunohematology / American Red Cross
[Show abstract][Hide abstract] ABSTRACT: Background:
Wr(a) is a low-incidence antigen, which is antithetical to the high prevalence red blood cell antigen, Wr(b). Anti-Wr(a) is a naturally occurring antibody that is found in approximately 1-2% of blood donors. The aim of this study was to determine the frequency of Wr(a) and anti-Wr(a) in Brazilian blood donors.
A total of 1662 Brazilian blood donors were molecularly analyzed using the SNaPshot methodology to determine the WR*A/B alleles and to predict the frequency of the Wr(a) antigen. To detect the anti-Wr(a), samples from 1049 blood donors were analyzed using a gel test with Wr(a+) red blood cells. The serum was treated with dithiothreitol (DTT) to determine the immunoglobulin classes. Immunoglobulin (Ig)-G isotype classification was performed in a gel test using the IgG1/IgG3 card. A monocyte monolayer assay was employed to predict the clinical significance of IgG anti-Wr(a).
Of the 1662 donors, only one sample had the DI*02.03 allele in heterozygous predicting the Wr(a+b+) phenotype. Anti-Wr(a) was detected in 34 (3.24%) samples, 64.7% in females and 35.3% in males. Regarding the immunoglobulin class, eight (23.5%) cases of anti-Wr(a) were classified as IgG and 26 (76.5%) as IgM. Of the eight cases of IgG anti-Wr(a), four were IgG1, two were IgG3 and three anti-Wr(a) were not IgG3 or IgG1, and thus probably IgG2 or IgG4. The results of the monocyte monolayer assay showed that IgG anti-Wr(a) might be of clinical significance.
This study shows a very low frequency (0.06%) of the Wr(a) antigen in Brazilian blood donors. Additionally, it shows that the frequency of anti-Wr(a) in this population is higher than previously reported.
Preview · Article · Sep 2015 · Revista Brasileira de Hematologia e Hemoterapia
[Show abstract][Hide abstract] ABSTRACT: Cellular and Molecular Immunology aims to report the dynamic progress being made in China and abroad in immunological research, and welcomes high-quality Research Articles, Reviews and Brief Reports across a broad range of topics including, but not limited to, clinical immunology, comparative immunology, immunobiology, immunogenetics, immunological techniques, immunopathology, immunopharmacology, infection immunology, neuroimmunology, transplantation immunology, tumor immunology, and veterinary immunology.
No preview · Article · May 2014 · Cellular & molecular immunology
[Show abstract][Hide abstract] ABSTRACT: Serologic resolution of Rh discrepancies due to partial D or weak D phenotypes is a frequent problem encountered during routine typing that can be solved by RHD genotyping because it provides better characterization of these variants. The objective of the current study was to develop algorithms for identification of D variants in multiethnic populations based on a logic sequence of molecular tests using a large number of atypical RhD specimens. Thus, a total of 360 blood samples with atypical D antigen expression were analyzed. A previously published multiplex polymerase chain reaction (PCR) procedure was performed and depending on multiplex PCR analysis, the associated RHCE allele, and D variant frequency in our population, an algorithm was developed composed of six flow charts using specific PCR-restriction fragment length polymorphism and/or specific exon sequencing. This strategy allowed the identification of 22 different variants with few assays and a much reduced cost. This study describes a simple and practical algorithm that we use to determine RHD genotypes in samples with unknown RHD. This strategy is relatively easy to implement and the algorithm can be adapted to populations with various ethnic backgrounds after an initial assessment of the type and frequency of D variants. Essentially, we demonstrate that sequencing of all RHD exons is not necessary for the identification of the majority of known D variants.
[Show abstract][Hide abstract] ABSTRACT: Background:
As an alternative to phenotyping, large-scale DNA-based assays, which are feasible for high-throughput donor red blood cell typing, were developed for determination of blood group polymorphisms. However, high-throughput genotyping platforms based on these technologies are still expensive and the inclusion of single nucleotide polymorphisms and analysis of the alleles depend on the manufacturer's determination. To overcome this limitation and in order to develop an assay to enable the screening of rare donors, we developed a SNaPshot assay for analysis of nine single nucleotide polymorphisms related to antigens that are difficult to assess using conventional serology.
Materials and methods:
The single polymerase chain reaction multiplex SNaPshot reaction was optimized to identify nine single nucleotide polymorphisms determining 16 alleles: KEL*3/KEL*4, KEL*6/KEL*7, DI*1/DI*2, DI*3/DI*4, YT*1/YT*2, CO*1/CO*2, DO*1/DO*2, DO*4, DO*5. We designed a single multiplex PCR with primers encompassing the blood group single nucleotide polymorphisms and performed an internal reaction with probe primers able to discriminate the alleles after fragment analysis. The SNaPshot assay was validated with 140 known alleles previously determined by PCR restriction fragment length polymorphism.
We were able to simultaneous detect nine single nucleotide polymorphisms defining 16 blood group alleles on an assay based on a multiplex PCR combined with a single base extension using genomic DNA.
This study demonstrates a robust genotyping strategy for conducting rare donor screening which can be applied in blood centers and could be an important tool for identifying antigen-negative donors and, therefore, for providing rare blood.
No preview · Article · Apr 2013 · Blood transfusion = Trasfusione del sangue
[Show abstract][Hide abstract] ABSTRACT: The Kell blood group system expresses high and low frequency antigens with the most important in relation to transfusion including the antithetic KEL1 and KEL2; KEL3 and KEL4; KEL6 and KEL7 antigens. Kell is a clinically relevant system, as it is highly immunogenic and anti-KEL antibodies are associated with hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. Although required in some situations, Kell antigen phenotyping is restricted due to technical limitations. In these cases, molecular approaches maybe a solution. This study proposes three polymerase chain reaction genotyping protocols to analyze the single nucleotide polymorphisms responsible for six Kell antithetic antigens expressed in a Brazilian population.
DNA was extracted from 800 blood donor samples and three polymerase chain reaction-restriction fragment length polymorphism protocols were used to genotype the KEL*1/KEL*2, KEL*3/KEL*4 and KEL*6/KEL*7 alleles. KEL*3/KEL*4 and KEL*6/KEL*7 genotyping was standardized using the NlaIII and MnlI restriction enzymes and validated using sequencing. KEL*1/KEL*2 genotyping was performed using a previously reported assay.
KEL genotyping was successfully implemented in the service; the following distribution of KEL alleles was obtained for a population from southeastern Brazil: KEL*1 (2.2%), KEL*2 (97.8%), KEL*3 (0.69%), KEL*4 (99.31%), KEL*6 (2.69%) and KEL*7 (97.31%). Additionally, two individuals with rare genotypes, KEL*1/KEL*1 and KEL*3/KEL*3, were identified.
KEL allele genotyping using these methods proved to be reliable and applicable to predict Kell antigen expressions in a Brazilian cohort. This easy and efficient strategy can be employed to provide safer transfusions and to help in rare donor screening.
Full-text · Article · Mar 2013 · Revista Brasileira de Hematologia e Hemoterapia
[Show abstract][Hide abstract] ABSTRACT: Background
Chronic hepatitis C (CHC) has emerged as a leading cause of cirrhosis in the U.S. and across the world. To understand the role of apoptotic pathways in hepatitis C virus (HCV) infection, we studied the mRNA and protein expression patterns of apoptosis-related genes in peripheral blood mononuclear cells (PBMC) obtained from patients with HCV infection.
The present study included 50 subjects which plasma samples were positive for HCV, but negative for human immunodeficiency virus (HIV) or hepatitis B virus (HBV). These cases were divided into four groups according to METAVIR, a score-based analysis which helps to interpret a liver biopsy according to the degree of inflammation and fibrosis. mRNA expression of the studied genes were analyzed by reverse transcription of quantitative polymerase chain reaction (RT-qPCR) and protein levels, analyzed by ELISA, was also conducted. HCV genotyping was also determined.
HCV infection increased mRNA expression and protein synthesis of caspase 8 in group 1 by 3 fold and 4 fold, respectively (p < 0.05). In group 4 HCV infection increased mRNA expression and protein synthesis of caspase 9 by 2 fold and 1,5 fold, respectively (p < 0.05). Also, caspase 3 mRNA expression and protein synthesis had level augumented by HCV infection in group 1 by 4 fold and 5 fold, respectively, and in group 4 by 6 fold and 7 fold, respectively (p < 0.05).
HCV induces alteration at both genomic and protein levels of apoptosis markers involved with extrinsic and intrinsic pathways.
[Show abstract][Hide abstract] ABSTRACT: Duffy or DARC (Duffy Antigen Receptor for Chemokines) is a glycosylated membrane protein that selectively binds angiogenic chemokines. Previous in vivo and in vitro studies of DARC function in cancer have associated DARC over expression with better prognosis, decreased metastatic potential, and inhibition of tumor-associated neovascularization. Another carcinogenesis-associated antigen is Lutheran or BCAM (basal cell adhesion molecule), a surface glycoprotein that acts as a receptor for the extracellular matrix protein, laminin. BCAM is a protein related to tumor progression; and, its over expression is associated with skin, ovarian and pancreatic cancers. We explored DARC and BCAM functions and investigated whether or not their expressions were altered in thyroid cancer. The expression of DARC and BCAM were evaluated by quantitative real-time PCR (qPCR) in a set of 18 normal thyroid tissues (NT), 15 follicular adenomas (FTA), 17 follicular carcinomas (FTC), and 122 papillary thyroid carcinomas (PTC), including 78 classical (CVPTC) and 44 follicular variant (FVPTC). RNA was isolated, reverse transcribed to cDNA, and used in qPCR reactions containing SYBR Green. The relative expression value was calculated using ribosomal protein S8 as an internal control. When we compared benign (NT and FTA) versus malignant samples (FTC, CVPTC and FVPTC) we observed a significant decrease of DARC and BCAM relative expression in malignant cases. Additionally, we correlated clinic-pathological features (tumor size, presence of metastasis, presence of lymphocyte infiltrate) with DARC and BCAM expression. We found a diminished expression of DARC in PTC samples, which was correlated with tumor size and presence of a lymphocyte infiltrate. We, also, found a correlation between decreased BCAM expression and tumor size or presence of metastasis. DARC and BCAM expression was associated with pathogenesis of thyroid carcinoma and correlated with clinical-pathological features.
No preview · Article · Nov 2012 · Blood Cells Molecules and Diseases
[Show abstract][Hide abstract] ABSTRACT: We have previously described a p.G533C substitution in the rearranged during transfection (RET) oncogene in a large family with medullary thyroid carcinoma. Here, we explore the functional transforming potential of RET p.G533C mutation.
Plasmids expressing RET mutants (p.G533C and p.C634Y) and RET wild type were stable transfected into a rat thyroid cell line (PCCL3). Biological and biochemical effects of RET p.G533C were investigated both in vitro and in vivo. Moreover, we report the first case of pheochromocytoma among the RET p.G533C-carriers in this Brazilian family and explore the RET mutational status in DNA isolated from pheochromocytoma.
Ectopic expression of RET p.G533C and p.C634Y activates RET/MAPK/ERK pathway at similar levels and significantly increased cell proliferation, compared with RET wild type. We additionally show that p.G533C increased cell viability, anchorage-independent growth, and micronuclei formation while reducing apoptosis, hallmarks of the malignant phenotype. RET p.G533C down-regulates the expression of thyroid specific genes in PCCL3. Moreover, RET p.G533C-expressing cells were able to induce liver metastasis in nude mice. Finally, we described two novel RET variants (G548V and S556T) in the DNA isolated from pheochromocytoma while they were absent in the DNA isolated from blood.
Our in vitro and in vivo analysis indicates that this mutation confers a malignant phenotype to PCCL3 cells. These findings, in association with the report of first case of pheochromocytoma in the Brazilian kindred, suggest that this noncysteine mutation may be more aggressive than was initially considered.
No preview · Article · Aug 2011 · Thyroid: official journal of the American Thyroid Association
[Show abstract][Hide abstract] ABSTRACT: Mounting evidence has indicated that ABI3 (ABI family member 3) function as a tumor suppressor gene, although the molecular mechanism by which ABI3 acts remains largely unknown.
The present study investigated ABI3 expression in a large panel of benign and malignant thyroid tumors and explored a correlation between the expression of ABI3 and its potential partner ABI3-binding protein (ABI3BP). We next explored the biological effects of ABI3 ectopic expression in thyroid and colon carcinoma cell lines, in which its expression was reduced or absent.
We not only observed that ABI3 expression is reduced or lost in most carcinomas but also that there is a positive correlation between ABI3 and ABI3BP expression. Ectopic expression of ABI3 was sufficient to lead to a lower transforming activity, reduced tumor in vitro growth properties, suppressed in vitro anchorage-independent growth and in vivo tumor formation while, cellular senescence increased. These responses were accompanied by the up-regulation of the cell cycle inhibitor p21 WAF1 and reduced ERK phosphorylation and E2F1 expression.
Our result links ABI3 to the pathogenesis and progression of some cancers and suggests that ABI3 or its pathway might have interest as therapeutic target. These results also suggest that the pathways through which ABI3 works should be further characterized.
[Show abstract][Hide abstract] ABSTRACT: We have previously shown that ARG2 expression was increased in most malignant thyroid tumors, but absent in benign lesions and normal tissues. Small interfering RNA knockdown was used to investigate the role of ARG2 in a thyroid carcinoma cell line. ARG2 knockdown decreased eNOS expression as well as the expression of eNOS-related genes (p21, Akt1, HIF-1, VEGF, and CAV1). ARG2 silencing changed tumor properties of thyroid cancer cells promoting apoptosis and reduced expression of cell proliferation markers. These results, coupled with enhanced nitric oxide production and elevated reactive oxygen species (ROS) levels, account for the altered intracellular redox environment. Genes related to either production (DUOX1 and NOX4) or catabolism (SODs) of ROS and reactive nitrogen species were negatively modulated by ARG2 knockdown. Additionally, a positive correlation of ARG2 with eNOS and related genes was investigated in thyroid tumors, further substantiating our in vitro findings. Our results suggest that ARG2 and eNOS may work in a coordinated manner and the underlying mechanism might be of major significance for thyroid tumorigenesis and/or tumor progression pathways. Fine modulation of ARG2, eNOS, and related genes may represent a potential source for targeted therapy of several cancer types.
Full-text · Article · Sep 2010 · Free Radical Biology and Medicine
[Show abstract][Hide abstract] ABSTRACT: The identification of follicular thyroid adenoma-associated transcripts will lead to a better understanding of the events involved in pathogenesis and progression of follicular tumours. Using Serial Analysis of Gene Expression, we identified five genes that are absent in a malignant follicular thyroid carcinoma (FTC) library, but expressed in follicular adenoma (FTA) and normal thyroid libraries.
NR4A1, one of the five genes, was validated in a set of 27 normal thyroid tissues, 10 FTAs and 14 FTCs and three thyroid carcinoma cell lines by real time PCR. NR4A1 can be transiently increased by a variety of stimuli, including lithium, which is used as adjuvant therapy of thyroid carcinoma with (131)I. We tested if lithium could restore NR4A1 expression. The expression of other genes potentially involved in the same signalling pathway was tested. To this end, lithium was used at different concentration (10 mm or 20 mm) and time (2 h and 24 h) and the level of expression was tested by quantitative PCR. We next tested if Lithium could affect cell growth and apoptosis.
We observed that NR4A1 expression was under-expressed in most of the FTCs investigated, compared with expression in normal thyroid tissues and FTAs. We also found a positive correlation between NR4A1 and FOSB gene expression. Lithium induced NR4A1 and FOSB expression, reduced CCDN1 expression, inhibited cell growth and triggered apoptosis in a FTC cell line.
NR4A1 is under-expressed in most of FTCs. The loss of expression of both NR4A1 and the Wnt pathway gene FOSB was correlated with malignancy. This is consistent with the hypothesis that its loss of expression is part of the transformation process of FTCs, either as a direct or indirect consequence of Wnt pathway alterations. Lithium restores NR4A1 expression, induces apoptosis and reduces cell growth. These findings may explain a possible molecular mechanism of lithium's therapeutic action.
[Show abstract][Hide abstract] ABSTRACT: Loss of ABI gene family member 3-binding protein (ABI3BP) expression may be functionally involved in the pathogenesis of cancer. Previous reports have indicated a loss of expression in lung cancer and a presumed role in inducing cellular senescence. We show here that ABI3BP expression is significantly decreased in most malignant thyroid tumors of all types. To better understand ABI3BP's role, we created a model by re-expressing ABI3BP in two thyroid cancer cell lines. Re-expression of ABI3BP in thyroid cells resulted in a decrease in transforming activity, cell growth, cell viability, migration, invasion, and tumor growth in nude mice. ABI3BP re-expression appears to trigger cellular senescence through the p21 pathway. Additionally, ABI3BP induced formation of heterochromatin 1-binding protein gamma-positive senescence-associated (SA) heterochromatin foci and accumulation of SA beta-galactosidase. The combination of a decrease in cell growth, invasion, and other effects upon ABI3BP re-expression in vitro helps to explain the large reduction in tumor growth that we observed in nude mice. Together, our data provide evidence that the loss of ABI3BP expression could play a functional role in thyroid tumorigenesis. Activation of ABI3BP or its pathway may represent a possible basis for targeted therapy of certain cancers.
Full-text · Article · Jul 2008 · Endocrine Related Cancer
[Show abstract][Hide abstract] ABSTRACT: Fine-needle aspiration (FNA) cytology, a standard method for thyroid nodule diagnosis, cannot distinguish between benign follicular thyroid adenoma (FTA) and malignant follicular thyroid carcinoma (FTC). Previously, using expression profiling, we found that a combination of transcript expression levels from DDIT3, ARG2, C1orf24, and ITM1 distinguished between FTA and FTC. The goal of this study was to determine if antibody markers used alone or in combination could accurately distinguish between a wider variety of benign and malignant thyroid lesions in fixed sections and FNA samples.
Immunohistochemistry was done on 27 FTA, 25 FTC, and 75 other benign and malignant thyroid tissue sections using custom antibodies for chromosome 1 open reading frame 24 (C1orf24) and integral membrane protein 1 (ITM1) and commercial antibodies for DNA damage-inducible transcript 3 (DDIT3) and arginase II (ARG2). FNA samples were also tested using the same antibodies. RNA expression was measured by quantitative PCR in 33 thyroid lesions.
C1orf24 and ITM1 antibodies had an estimated sensitivity of 1.00 for distinguishing FTA from FTC. For the expanded analysis of all lesions studied, ITM1 had an estimated sensitivity of 1.00 for detecting malignancy. Because all four cancer biomarkers did well, producing overlapping confidence intervals, not one best marker was distinguished. Transcript levels also reliably predicted malignancy, but immunohistochemistry had a higher sensitivity. Malignant cells were easily detected in FNA samples using these markers.
We improved this diagnostic test by adding C1orf24 and ITM1 custom antibodies and showing use on a wider variety of thyroid pathology. We recommend that testing of all four cancer biomarkers now be advanced to larger trials. Use of one or more of these antibodies should improve diagnostic accuracy of suspicious thyroid nodules from both tissue sections and FNA samples.
Preview · Article · Jul 2006 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: Alternative splicing is the differential processing of exon junctions to produce a new transcript variant from one gene. Some aberrant splicing, however, has been shown to be cancer specific. Identification of these specific splice variations will provide important insight into the molecular mechanism of normal cellular physiology as well as the disease processes. To gain knowledge about whether alternative splicing is linked to thyroid tumorigenesis, we used our prediction database to select targets for analysis. Fifteen putatively new alternative splicing isoforms were selected on the basis of their expression in thyroid libraries and/or their origin in genes previously associated with carcinogenesis. Using a set of 66 normal, benign, and malignant thyroid tissue samples, new splicing events were confirmed by RT-PCR for 13 of 15 genes (a validation rate of 87%). In addition, new alternative splicing isoforms not predicted by the system and not previously described in public databases were identified. Five genes (PTPN18, ABI3BP, PFDN5, SULF2, and ST5) presented new and/or additional unpredicted isoforms differentially expressed between malignant and benign or normal thyroid tissues, confirmed by sequencing. PTPN18, ABI3BP, and PFDN5 revealed a statistically significant differential splicing profile. In addition, real-time PCR analysis revealed that expression of an alternative PFDN5 variant was higher in malignant lesions than in benign lesions or normal tissues.
No preview · Article · Jun 2006 · Genes Chromosomes and Cancer