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1.
Immunotherapy ; 14(2): 115-133, 2022 02.
Article in English | MEDLINE | ID: mdl-34783257

ABSTRACT

Aim: The aim of this work is to utilize a gene expression procedure to safely express systemic IL-12 and evaluate its effects in mouse tumor models. Materials & methods: Secondary lymphoid organs and tumors from EL4 and B16 tumor-bearing mice were analyzed by supervised and unsupervised methods. Results: IL-12 cDNA induced systemic IL-12 protein levels lower than the tolerated dose in patients. Control of tumor growth was observed in subcutaneous B16 and EL4 tumors. Systemic IL-12 expression induced a higher frequency of both total tumor-infiltrated CD45+ cells and proliferative IFN-γ+CD8+ T cells along with a lower frequency of CD4+FOXP3+ and CD11b+Gr-1+ cells. Conclusion: This approach characterizes the systemic effects of IL-12, helping to improve treatment of metastases or solid tumors.


Lay abstract IL-12 has emerged as a potent cytokine in mediating antitumor activity in preclinical models of cancer. However, this antitumor response has not yet been translated into the clinic because of toxic side effects. The aim of our work is to analyze the effects of IL-12 in mouse tumor models. We demonstrate that one injection of IL-12 cDNA can induce systemic IL-12 levels in serum even lower than the tolerated dose in patients. At this dose, an efficient control of tumor growth can be observed. We found a higher frequency of both total tumor-infiltrated leukocytes and IFN-γ-producing CD8+ T cells along with a lower frequency of regulatory CD4+FOXP3+ and CD11b+Gr1+ cells. Our work demonstrates that IL-12 cDNA can safely be used to treat cancer.


Subject(s)
Adjuvants, Immunologic/therapeutic use , DNA, Complementary/blood , Interleukin-12/therapeutic use , Lymphoma/drug therapy , Melanoma, Experimental/drug therapy , Animals , Disease Models, Animal , Gene Expression , Interleukin-12/blood , Lymphoma/blood , Lymphoma/immunology , Melanoma, Experimental/blood , Melanoma, Experimental/immunology , Mice , Mice, Inbred C57BL , Signal Transduction
2.
Br Poult Sci ; 60(6): 649-658, 2019 Dec.
Article in English | MEDLINE | ID: mdl-31469320

ABSTRACT

1. A previous whole-genome association analysis has identified the motilin receptor gene (MLNR), which regulates gastrointestinal motility and gastric emptying, as a candidate gene related to chicken growth.2. MLNR mRNA was expressed in all tissues tested, and the expression level in digestive tissues was greater than in other tissues. Expression levels in the pancreas, duodenum and glandular stomach at day old and one, two and three weeks of age indicated a possible correlation with the digestive system. This suggested that the MLNR gene plays a central role in gastrointestinal tract function and affects the growth and development of chickens. Moreover, there was a significant difference in expression in the glandular stomach tissue between Ross 308 and Gushi chickens at six weeks of age.3. Re-sequencing revealed an 86-bp insertion/deletion polymorphism in the downstream region of the MLNR gene. The mutation locus was genotyped in 2,261 individuals from nine different chicken breeds. MLNR expression levels in the glandular stomach of chickens with DD genotypes were greater than those in chickens with the ID and II genotypes. The DD genotype was the most dominant genotype in commercial broiler's (Ross 308 and Arbor Acres broilers), and the D allele frequency in these breeds exceeded 91%. The deletion mutation tended towards fixation in commercial broilers.4. Association with growth and carcass traits analysed in a Gushi-Anka F2 intercrossed population, showed that the DD genotype was significantly associated with the greatest growth and carcass trait values, whereas values associated with the II genotype were the lowest in the F2 reciprocal cross chickens.5. The results suggest that the mutation is strongly associated with growth related traits and it is likely to be useful for marker-assisted selection of chickens.


Subject(s)
Chickens/genetics , INDEL Mutation , Receptors, Gastrointestinal Hormone/genetics , Receptors, Neuropeptide/genetics , Animals , Chickens/anatomy & histology , Chickens/growth & development , Crosses, Genetic , DNA, Complementary/blood , DNA, Complementary/isolation & purification , Duodenum/metabolism , Female , Gastric Emptying/genetics , Gastric Mucosa/metabolism , Gastrointestinal Motility/genetics , INDEL Mutation/genetics , Male , Pancreas/metabolism , RNA, Messenger/analysis , RNA, Messenger/biosynthesis
3.
Carcinogenesis ; 37(1): 10-17, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26498247

ABSTRACT

Lynch syndrome is an autosomal dominant disorder that predisposes carriers of DNA mismatch repair (MMR) gene mutations to early-onset cancer. Germline testing screens exons and splice sites for mutations, but does not examine introns or RNA transcripts for alterations. Pathogenic mutations have not been detected in ~30% of suspected Lynch syndrome cases with standard screening practices. We present a 38-year-old male with a clinicopathological and family history consistent with Lynch syndrome, including loss of MSH2 expression in his tumor. Germline testing revealed normal MSH2 coding sequence, splice sites and exon copy number, however, cDNA sequencing identified an aberrant MSH2 transcript lacking exons 2-6. An inversion PCR on germline DNA identified an ~18kb unbalanced, paracentric inversion within MSH2, with breakpoints in a long terminal repeat in intron 1 and an Alu repeat in intron 6. The 3' end of the inversion had a 1.2 kb deletion and an 8 bp insertion at the junction with intron 6. Screening of 55 additional Australian patients presenting with MSH2-deficient tumors who were negative in germline genetic tests for MSH2 mutations identified another inversion-positive patient. We propose an Alu-mediated recombination model to explain the origin of the inversion. Our study illustrates the potential value of cDNA screening to identify patients with cryptic MMR gene rearrangements, clarifies why standard testing may not detect some pathogenic alterations, and provides a genetic test for screening individuals with suspected Lynch syndrome that present with unexplained MSH2-deficient tumors.


Subject(s)
Colorectal Neoplasms, Hereditary Nonpolyposis/genetics , Exons , MutS Homolog 2 Protein/genetics , Sequence Inversion , Adult , Amino Acid Sequence , Base Sequence , Colorectal Neoplasms, Hereditary Nonpolyposis/blood , DNA Mutational Analysis/methods , DNA, Complementary/blood , DNA, Complementary/genetics , DNA, Neoplasm/blood , DNA, Neoplasm/genetics , Gene Rearrangement , Germ-Line Mutation , Humans , Male , Molecular Sequence Data , Pedigree
4.
PLoS One ; 10(3): e0116373, 2015.
Article in English | MEDLINE | ID: mdl-25768837

ABSTRACT

Manipulation of gene expression to invoke loss of function (LoF) or gain of function (GoF) phenotypes is important for interrogating complex biological questions both in vitro and in vivo. Doxycycline (Dox)-inducible gene expression systems are commonly used although success is often limited by high background and insufficient sensitivity to Dox. Here we develop broadly applicable platforms for reliable, tightly controlled and reversible Dox-inducible systems for lentiviral mediated generation of cell lines or FLP Recombination-Mediated Cassette Exchange (RMCE) into the Collagen 1a1 (Col1a1) locus (FLP-In Col1a1) in mouse embryonic stem cells. We significantly improve the flexibility, usefulness and robustness of the Dox-inducible system by using Tetracycline (Tet) activator (Tet-On) variants which are more sensitive to Dox, have no background activity and are expressed from single Gateway-compatible constructs. We demonstrate the usefulness of these platforms in ectopic gene expression or gene knockdown in multiple cell lines, primary neurons and in FLP-In Col1a1 mouse embryonic stem cells. We also improve the flexibility of RMCE Dox-inducible systems by generating constructs that allow for tissue or cell type-specific Dox-inducible expression and generate a shRNA selection algorithm that can effectively predict potent shRNA sequences able to knockdown gene expression from single integrant constructs. These platforms provide flexible, reliable and broadly applicable inducible expression systems for studying gene function.


Subject(s)
Doxycycline/pharmacology , Lentivirus/genetics , Recombination, Genetic , Animals , Cell Line , Cell Line, Tumor , Collagen Type I/genetics , Collagen Type I, alpha 1 Chain , DNA, Complementary/blood , DNA, Complementary/genetics , Gene Expression Regulation/drug effects , Gene Knockdown Techniques , Gene Transfer Techniques , HEK293 Cells , Humans , Mice , Mutagenesis, Insertional/methods , RNA, Small Interfering/biosynthesis , RNA, Small Interfering/genetics
5.
Ann Hematol ; 94(6): 1025-32, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25623380

ABSTRACT

Acute graft-versus-host disease (aGVHD) has become the important complication post-allogeneic hematopoietic stem cell transplantation. Abnormally activated T cells might play an important role in the pathogenesis of aGVHD. But its exact mechanism remains poorly understood. T cell immune response cDNA 7 (TIRC7) has been identified to be essential in T cell activation; however, the role of TIRC7 in aGVHD remains unclear. The purpose of this study was to measure the expression of TIRC7 and T helper (Th) cells in patients with aGVHD before and after treatment. We showed that TIRC7 levels in aGVHD patients were higher than those of healthy controls and markedly declined after treatment. The levels of IFN-γ (Th1), IL-17 (Th17), and IL-22 (Th22) were in accordance with the grade of aGVHD. In addition, TIRC7 levels were also associated with the severity of aGVHD. In conclusion, TIRC7 might be involved in the pathogenesis of aGVHD and TIRC7 level might be an indicator to evaluate the response of patients with aGVHD to treatment.


Subject(s)
DNA, Complementary/blood , Graft vs Host Disease/blood , Graft vs Host Disease/diagnosis , Vacuolar Proton-Translocating ATPases/blood , Acute Disease , Adolescent , Adult , Biomarkers/blood , DNA, Complementary/immunology , Female , Gene Expression Regulation , Graft vs Host Disease/immunology , Humans , Immunity, Cellular/physiology , Male , Middle Aged , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Vacuolar Proton-Translocating ATPases/biosynthesis , Young Adult
6.
Int Urol Nephrol ; 45(4): 1023-8, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23779229

ABSTRACT

OBJECTIVES: To investigate whether plasma cell-free DNA (cfDNA) or its integrity could differentiate prostate cancer from benign prostate hyperplasia (BPH) in patients with serum prostate-specific antigen (PSA) ≥ 4 ng/ml. METHODS: Ninety-six patients with prostate cancer and 112 patients with BPH were enrolled. cfDNA levels in plasma before prostate biopsy were quantified by real-time PCR amplification of ALU gene (product size of 115 bp), and quantitative ratio of ALU (247 bp) to ALU (115 bp) reflected the integrity of cfDNA. RESULTS: In patients with serum PSA ≥ 4 ng/ml, there were significant differences in plasma cfDNA or its integrity between the patients with prostate cancer (19.74 ± 4.43, 0.34 ± 0.05) and patients with BPH (7.36 ± 1.58, 0.19 ± 0.03; P < 0.001, P < 0.001). Prostate cancer could be differentiated with a sensitivity of 73.2 % and a specificity of 72.7 % by cfDNA (AUC = 0.864). The integrity of cfDNA had a sensitivity of 81.7 % and a specificity of 78.8 % for the distinguishing prostate cancer from BPH (AUC = 0.910). CONCLUSIONS: cfDNA and its integrity could be applied to differentiate prostate cancer from BPH in patients with serum PSA ≥ 4 ng/ml.


Subject(s)
DNA, Complementary/blood , Prostate-Specific Antigen/blood , Prostatic Hyperplasia/blood , Prostatic Neoplasms/blood , Aged , Area Under Curve , Biomarkers/blood , Biopsy, Needle , Cohort Studies , Diagnosis, Differential , Humans , Immunohistochemistry , Male , Middle Aged , Oligonucleotide Array Sequence Analysis , Plasma Cells , Prostatic Hyperplasia/diagnosis , Prostatic Hyperplasia/pathology , Prostatic Neoplasms/diagnosis , Prostatic Neoplasms/pathology , ROC Curve , Real-Time Polymerase Chain Reaction , Retrospective Studies , Sensitivity and Specificity
7.
Exp Parasitol ; 131(1): 40-4, 2012 May.
Article in English | MEDLINE | ID: mdl-22417971

ABSTRACT

Loop-mediated isothermal amplification (LAMP) has been increasingly used for diagnosis and quantification of pathogens. Since the Bst DNA polymerase used in this assay is highly resistant to PCR inhibitors present in blood, direct analysis of blood samples without DNA or RNA extraction is possible. Indeed, the presence of Plasmodium chabaudi specific nucleic acids was easily detectable using primer sets for P. chabaudi 18S rRNA and the cir 1 mRNA. Despite the fact that primers for cir 1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and actin II mRNAs were used that spanned an intron, selective amplification of mRNA in the presence of contaminating genomic DNA was not possible. Optimization of the reaction temperature could only improve discrimination when low complexity templates (target sequences cloned in a plasmid vector) were used. Placing different LAMP primers across intron exon boundaries did not prevent amplification in the absence of reverse transcriptase. Probably due to the high A+T content and low number of introns only a very limited number of possible primer sets spanning introns could be identified in the target genes and no reaction conditions could be established that would allow quantification of RNA levels in the presence of DNA directly from blood samples.


Subject(s)
DNA, Complementary/blood , DNA, Protozoan/blood , Malaria/diagnosis , Nucleic Acid Amplification Techniques/methods , Plasmodium chabaudi/genetics , Animals , DNA Primers/chemistry , DNA, Complementary/isolation & purification , DNA, Protozoan/isolation & purification , DNA, Ribosomal/blood , DNA, Ribosomal/isolation & purification , Malaria/blood , Malaria/parasitology , Male , Mice , Nucleic Acid Amplification Techniques/standards , Plasmodium chabaudi/isolation & purification , RNA, Ribosomal, 18S/genetics , Restriction Mapping , Temperature
8.
Curr HIV Res ; 9(5): 276-88, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21861823

ABSTRACT

Mucosal transmission of HIV predominately occurs during sexual intercourse or breast-feeding and generally results in a successful infection from just one or few founder virions. Here we assessed the impact of viral inoculum size on both viral and immune events within two groups of Rhesus macaques that were non-traumatically, orally inoculated with either multiple low (1000 to 4000 TCID(50)) or high (100,000 TCID(50)) doses of SIV. In agreement with previous studies, more diverse SIV variants were observed in macaques following infection with high dose oral SIV compared to a low dose challenge. In peripheral blood cells, the immune gene transcript levels of CXCL9, IFNγ, TNFα and IL10 remained similar to uninfected macaques. In contrast, OAS and CXCL10 were upregulated following SIV infection in both the high and low dosed macaques, with a more rapid kinetics (detectable by 7 days) following the high SIV dose challenge. In peripheral lymph nodes, an increase in CXCL10 was observed irrespective of viral dose while CXCL9 and OAS were differentially regulated in the two SIV dosed groups. Magnetic bead sorting of CD3+, CD14+ and CD3- /CD14- cells from peripheral blood identified the increase in OAS expression primarily within CD14+ monocytes, whereas the CXCL10 expression was primarily in CD3+ T cells. These findings provide insights into the impact of SIV challenge dose on viral and innate immune factors, which has the potential to inform future SIV/HIV vaccine efficacy trials in which vaccinated hosts have the potential to be infected with a range of viral challenge doses.


Subject(s)
Cytokines/blood , Immunity, Innate , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/immunology , Administration, Oral , Animals , Antigens, CD/analysis , Blood Cells/immunology , DNA, Complementary/blood , Disease Models, Animal , Genotype , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/virology , Lymph Nodes/immunology , Macaca mulatta , RNA, Messenger/metabolism , RNA, Viral/blood , Real-Time Polymerase Chain Reaction , Simian Immunodeficiency Virus/genetics , T-Lymphocytes/immunology , Up-Regulation , Viral Load
9.
J Immunol ; 186(4): 2454-61, 2011 Feb 15.
Article in English | MEDLINE | ID: mdl-21242514

ABSTRACT

Our previous study showed that a combination of a plasmid-expressing Flt3 ligand (pFL) and CpG oligodeoxynucleotides (CpG ODN) as a combined nasal adjuvant elicited mucosal immune responses in aged (2-y-old) mice. In this study, we investigated whether a combination of pFL and CpG ODN as a nasal adjuvant for a pneumococcal surface protein A (PspA) would enhance PspA-specific secretory-IgA Ab responses, which could provide protective mucosal immunity against Streptococcus pneumoniae infection in aged mice. Nasal immunization with PspA plus a combination of pFL and CpG ODN elicited elevated levels of PspA-specific secretory-IgA Ab responses in external secretions and plasma in both young adult and aged mice. Significant levels of PspA-specific CD4(+) T cell proliferative and PspA-induced Th1- and Th2- type cytokine responses were noted in nasopharyngeal-associated lymphoreticular tissue, cervical lymph nodes, and spleen of aged mice, which were equivalent to those in young adult mice. Additionally, increased numbers of mature-type CD8, CD11b-expressing dendritic cells were detected in mucosal inductive and effector lymphoid tissues of aged mice. Importantly, aged mice given PspA plus a combination of pFL and CpG ODN showed protective immunity against nasal S. pneumoniae colonization. These results demonstrate that nasal delivery of a combined DNA adjuvant offers an attractive possibility for protection against S. pneumoniae in the elderly.


Subject(s)
Adjuvants, Immunologic/administration & dosage , Aging/immunology , DNA, Complementary/administration & dosage , Immunoglobulin A, Secretory/biosynthesis , Membrane Proteins/genetics , Nasal Mucosa/immunology , Oligodeoxyribonucleotides/administration & dosage , Pneumococcal Infections/immunology , Adjuvants, Immunologic/blood , Animals , Antibodies, Bacterial/biosynthesis , Bacterial Proteins/administration & dosage , Bacterial Proteins/immunology , Cells, Cultured , CpG Islands/immunology , DNA, Complementary/blood , DNA, Complementary/immunology , Drug Combinations , Humans , Immunoglobulin A, Secretory/physiology , Membrane Proteins/administration & dosage , Membrane Proteins/blood , Mice , Nasal Mucosa/metabolism , Nasal Mucosa/microbiology , Oligodeoxyribonucleotides/blood , Oligodeoxyribonucleotides/immunology , Pneumococcal Infections/microbiology , Pneumococcal Infections/prevention & control , Streptococcus pneumoniae/immunology
10.
Prion ; 4(2): 87-93, 2010.
Article in English | MEDLINE | ID: mdl-20424511

ABSTRACT

Alu DNA elements were long considered to be of no biological significance and thus have been only poorly defined. However, in the past Alu DNA elements with well-defined nucleotide sequences have been suspected to contribute to disease, but the role of Alu DNA element transcripts has rarely been investigated. For the first time, we determined in a real-time approach Alu DNA element transcription in buffy coat cells isolated from the blood of humans suffering from sporadic Creutzfeldt-Jakob disease (sCJD) and other neurodegenerative disorders. The reverse transcribed Alu transcripts were amplified and their cDNA sequences were aligned to genomic regions best fitted to database genomic Alu DNA element sequences deposited in the UCSC and NCBI data bases. Our cloned Alu RNA/cDNA sequences were widely distributed in the human genome and preferably belonged to the "young" Alu Y family. We also observed that some RNA/cDNA clones could be aligned to several chromosomes because of the same degree of identity and score to resident genomic Alu DNA elements. These elements, called paralogues, have purportedly been recently generated by retrotransposition. Along with cases of sCJD we also included cases of dementia and Alzheimer disease (AD). Each group revealed a divergent pattern of transcribed Alu elements. Chromosome 2 was the most preferred site in sCJD cases, besides chromosome 17; in AD cases chromosome 11 was overrepresented whereas chromosomes 2, 3 and 17 were preferred active Alu loci in controls. Chromosomes 2, 12 and 17 gave rise to Alu transcripts in dementia cases. The detection of putative Alu paralogues widely differed depending on the disease. A detailed data search revealed that some cloned Alu transcripts originated from RNA polymerase III transcription since the genomic sites of their Alu elements were found between genes. Other Alu DNA elements could be located close to or within coding regions of genes. In general, our observations suggest that identification and genomic localization of active Alu DNA elements could be further developed as a surrogate marker for differential gene expression in disease. A sufficient number of cases are necessary for statistical significance before Alu DNA elements can be considered useful to differentiate neurodegenerative diseases from controls.


Subject(s)
Alu Elements , Creutzfeldt-Jakob Syndrome/blood , Creutzfeldt-Jakob Syndrome/genetics , Base Sequence , Blood Buffy Coat/pathology , Blood Buffy Coat/physiology , Case-Control Studies , Chromosome Mapping , DNA, Complementary/blood , DNA, Complementary/genetics , Gene Expression Regulation , Gene Frequency , Humans , Molecular Sequence Data , RNA/blood , RNA/genetics , Reverse Transcription , Sequence Alignment
11.
Transplantation ; 88(7): 942-51, 2009 Oct 15.
Article in English | MEDLINE | ID: mdl-19935467

ABSTRACT

BACKGROUND: Acute graft rejection is an important clinical problem in renal transplantation and an adverse predictor for long-term graft survival. Peripheral blood biomarkers that provide evidence of early graft rejection may offer an important option for posttransplant monitoring, optimize the utility of graft biopsy, and permit timely and effective therapeutic intervention to minimize the graft damage. METHODS: In this feasibility study (n=58), we have used gene expression profiling in a case-control design to compare whole blood samples between normal subjects (n=20) and patients with (n=11) or without (n=22) biopsy-confirmed acute rejection (BCAR) or borderline changes (n=5). RESULTS: A total of 183 probe sets representing 160 genes were differentially expressed (false discovery rate [FDR] <0.01) between subjects with or without BCAR, from which linear discriminant analysis and cross-validation identified an initial gene signature of 24 probe sets, and a more refined set of 11 probe sets found to classify subject samples correctly. Cross-validation suggested an out-of-sample sensitivity of 73% and specificity of 91% for identification of samples with or without BCAR. An increase in classifier gene expression correlated closely with acute rejection during the first 3 months posttransplant. Biological evaluation indicated that the differentially expressed genes encompassed processes related to immune response, signal transduction, and cytoskeletal reorganization. CONCLUSION: Preliminary evidence indicates that gene expression in the peripheral blood may yield a relevant measure for the occurrence of BCAR and offer a potential tool for immunologic monitoring. These results now require confirmation in a larger cohort.


Subject(s)
Gene Expression Profiling , Genomics , Graft Rejection/genetics , Kidney Transplantation/pathology , Acute Disease , Antibodies, Monoclonal/therapeutic use , Basiliximab , Biopsy , Case-Control Studies , DNA, Complementary/blood , DNA, Complementary/genetics , Discriminant Analysis , Follow-Up Studies , Humans , Immunosuppressive Agents/therapeutic use , Kidney Transplantation/immunology , Phenotype , Prospective Studies , RNA/blood , RNA/genetics , Recombinant Fusion Proteins/therapeutic use , Reproducibility of Results , Time Factors
12.
Arthritis Rheum ; 60(7): 2193-200, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19565506

ABSTRACT

OBJECTIVE: To identify the autoantigen recognized by the autoantibody that is associated with clinically amyopathic dermatomyositis (C-ADM) and rapidly progressive interstitial lung disease (ILD). METHODS: An anti-CADM-140 antibody-positive prototype serum sample was used to screen a HeLa cell-derived complementary DNA (cDNA) library. Selected cDNA clones were further evaluated by immunoprecipitation of their in vitro-transcribed and in vitro-translated products using anti-CADM-140 antibody-positive and anti-CADM-140 antibody-negative sera. The lysates of COS-7 cells transfected with the putative antigen were similarly tested. An enzyme-linked immunosorbent assay (ELISA) to detect the anti-CADM-140 antibody was established using a recombinant CADM-140 antigen, and its specificity and sensitivity for C-ADM and rapidly progressive ILD were assessed in 294 patients with various connective tissue diseases. RESULTS: By cDNA library screening and immunoprecipitation of in vitro-transcribed and in vitro-translated products, we obtained a cDNA clone encoding melanoma differentiation-associated gene 5 (MDA-5). The anti-CADM-140 antibodies in patients' sera specifically reacted with MDA-5 protein expressed in cells transfected with full-length MDA-5 cDNA, confirming the identity of MDA-5 as the CADM-140 autoantigen. The ELISA, using recombinant MDA-5 protein as the antigen, showed an analytical sensitivity of 85% and analytical specificity of 100%, in comparison with the "gold standard" immunoprecipitation assay, and was useful for identifying patients with C-ADM and/or rapidly progressive ILD. CONCLUSION: Given that RNA helicase encoded by MDA-5 is a critical molecule involved in the innate immune defense against viruses, viral infection may play an important role in the pathogenesis of C-ADM and rapidly progressive ILD. Moreover, our ELISA using recombinant MDA-5 protein makes detection of the anti-CADM-140 antibody routinely available.


Subject(s)
Autoantigens/immunology , DEAD-box RNA Helicases/immunology , Dermatomyositis/immunology , Lung Diseases, Interstitial/immunology , RNA Helicases/immunology , Animals , Antibodies, Anti-Idiotypic/blood , Antibodies, Anti-Idiotypic/immunology , Autoantigens/blood , COS Cells , Case-Control Studies , Chlorocebus aethiops , DEAD-box RNA Helicases/blood , DEAD-box RNA Helicases/genetics , DNA, Complementary/blood , DNA, Complementary/genetics , Dermatomyositis/blood , Disease Progression , Humans , Interferon-Induced Helicase, IFIH1 , Lung Diseases, Interstitial/blood , RNA Helicases/blood , RNA Helicases/genetics , Sensitivity and Specificity , Transfection
13.
J Vet Med Sci ; 71(2): 189-94, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19262030

ABSTRACT

Adiponectin is an adipokine that is specifically expressed in adipose tissues, directly sensitizes the body to insulin via specific receptors and its decreased plasma concentration is responsible for insulin resistance in obese humans. Diabetes is an important problem also in veterinary medicine, and feline diabetes is very similar to human type 2 diabetes, in which obesity is an important risk factor. In the present study, We obtained cDNA clones corresponding to feline adiponectin and adiponectin receptor 1 (AD-R1), whose nucleotide and deduced amino acid sequences were highly identical to those of other species, especially, the extra-cellular domain of feline AD-R1 was almost identical to that of human AD-R1. Adiponectin mRNA was exclusively detected in the adipose tissue, but AD-R1 was in all tissues tested in this study. Next, plasma samples were collected from 22 cats visiting veterinary practices. They were divided to 2 groups based on a five-point scale body condition score (BCS), such as normal group (BCS ranged from 2.5 through 3.5) and obese group (BCS ranged from 4.0 through 5.0). Plasma adiponectin in obese cats (7.2 +/- 1.5 microg/ml) was significantly lower than that of normal cats (18.0 +/- 3.2 microg/ml). These results suggest that adiponectin may be responsible for insulin function also in the cat, and it can be a target molecule for treatment of obesity and diabetes in cats.


Subject(s)
Adiponectin/blood , Adiponectin/genetics , Diabetes Mellitus, Type 2/veterinary , Insulin/blood , Obesity/veterinary , Adipose Tissue/metabolism , Amino Acid Sequence , Animals , Cats , DNA, Complementary/blood , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/physiopathology , Enzyme-Linked Immunosorbent Assay , Insulin/genetics , Molecular Sequence Data , Molecular Structure , Obesity/blood , Obesity/physiopathology , Polymerase Chain Reaction , RNA, Messenger/blood , RNA, Messenger/genetics , Receptors, Adiponectin/analysis , Risk Factors
14.
Trans R Soc Trop Med Hyg ; 102(3): 284-7, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18243259

ABSTRACT

In developing countries as many as 50% of patients for whom a transfusion is indicated are at risk of dying immediately if transfusion is withheld. It is therefore important that blood transfusion is made as safe as possible. This study was designed to assess the safety of blood transfusion in two large blood banks in Ibadan, Nigeria. Aliquots of 250 samples already screened and passed as negative for HIV-1 and -2 were collected from each of the blood banks. Samples were tested for the presence of HIV-1 antigen (ELAVIA Ag I) and the antigen-positive samples tested for the presence of specific HIV-1 antibodies by Western blot (BioRad, France). All antigen-positive samples were also subjected to PCR. HIV-1 antigen was detected in 6 (1.2%) of the 500 samples, of which 4 (0.8%) and 3 (0.6%) were Western blot-indeterminate and PCR-positive, respectively. Transfusion of HIV-contaminated blood may be contributing significantly to the spread of the virus in Nigeria. There is therefore an urgent need for an organized blood-banking system with facilities for more sensitive assays for the detection of HIV in blood to prevent transmission through transfusion.


Subject(s)
Blood Transfusion , HIV Antigens/blood , HIV Infections/prevention & control , HIV Seronegativity/immunology , Blood Banks/standards , Blood Donors , Blood Transfusion/standards , Blotting, Western/methods , DNA, Complementary/blood , HIV Antibodies/blood , HIV Infections/transmission , HIV-1/immunology , Humans , Nigeria , Polymerase Chain Reaction/methods
15.
Hum Mutat ; 29(1): 106-12, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17722006

ABSTRACT

Autosomal dominant optic atrophy (adOA) is most commonly caused by mutations in the OPA1 gene. There is a considerable allelic heterogeneity among adOA-associated OPA1 mutations, however these mutations have mostly been identified and studied only at the genomic DNA level. Here we report the identification of 22 novel OPA1 mutations and their analysis at the cDNA level along with 15 already known OPA1 mutations. We found that 18 of these mutations cause splice defects that involve either skipping of the adjacent exon or the activation of a cryptic splice site. We also observed a reduced level of the mutant transcript in several adOA subjects. Allele-specific quantification of the transcript steady-state level was performed for 13 different OPA1 mutations applying pyrosequencing to a RT-PCR amplified cSNP (c.2109C>T) in OPA1. Using this new assay we could demonstrate that the majority of OPA1 mutations that lead to a premature termination codon (PTC) undergo nonsense-mediated mRNA decay (NMD). Mutant transcript levels were reduced between 1.25- and 2.5-fold and varied between PTC containing mutations, and between subjects. Our results emphasize the value of cDNA analysis in the characterization of OPA1 mutations and further strengthen the model of haploinsufficiency as a major pathomechanism in OPA1-associated adOA.


Subject(s)
Codon, Nonsense/genetics , DNA, Complementary/metabolism , GTP Phosphohydrolases/genetics , Mutation , Optic Atrophy, Autosomal Dominant/genetics , Alleles , DNA Mutational Analysis/methods , DNA, Complementary/blood , DNA, Complementary/genetics , Genetic Variation , Humans , Optic Atrophy, Autosomal Dominant/blood
16.
Anticancer Res ; 27(5A): 3227-33, 2007.
Article in English | MEDLINE | ID: mdl-17970065

ABSTRACT

We previously performed SEREX (serological identification of antigens by recombinant expression cloning) using the sera of patients with esophageal squamous cell carcinoma (SCC), and isolated a variant clone (AK093616) of ubiquitin-conjugating enzyme E21 (UBE2I). This clone was tentatively designated as UBE2I-v5 and analyzed for biological function by transient transfection of the cDNA into activated Ha-ras-transformed NIH3T3 (ras-NIH) mouse fibroblasts. Chemosensitivity to 92 cytotoxic drugs was compared between UBE2I-v5-transfected cells and the parental ras-NIH cells. The UBE2I-v5-transfected cells were more sensitive than the parental cells to anticancer drugs such as vincristine (VCR), mitoxantrone (MIT) and etoposide (VP16). The regression analysis of the total chemosensitivity pattern of UBE2I-vS-transfected cells revealed that the function of UBE2I-v5 was positively related to RPA2 (replication protein A2), Rho-GDI (Rho guanine nucleotide dissociation inhibitor a), FUS (putative tumor suppressor) and TKT (transketolase) but negatively related to Per-1 (period-I), Ran (nuclear Ras-related protein), PTEN (phosphatase and tensin homolog), C/EBPalpha (CCAAT/enhancer binding protein a) and the tumor suppressor p53. Thus, it is possible that UBE21-v5 plays a role in carcinogenesis by suppressing the function of CIEBPa and/or p53 via RPA2-like activity.


Subject(s)
Antineoplastic Agents/pharmacology , Fibroblasts/drug effects , Fibroblasts/enzymology , Ubiquitin-Conjugating Enzymes/physiology , Animals , Carcinoma, Squamous Cell/blood , Carcinoma, Squamous Cell/enzymology , Carcinoma, Squamous Cell/genetics , DNA, Complementary/blood , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Esophageal Neoplasms/blood , Esophageal Neoplasms/enzymology , Esophageal Neoplasms/genetics , Etoposide/pharmacology , Fibroblasts/physiology , Genes, ras , Mice , Mitoxantrone/pharmacology , NIH 3T3 Cells , Transfection , Ubiquitin-Conjugating Enzymes/biosynthesis , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/metabolism , Vincristine/pharmacology
17.
J Rheumatol ; 30(12): 2524-8, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14719188

ABSTRACT

OBJECTIVE: To examine whether the expression of intact CD1d, a critical molecule for the presentation of glycolipid antigens to natural killer T (NKT) cells, and its variants differs between patients with autoimmune diseases including rheumatoid arthritis (RA) and healthy subjects. Recently, we identified 8 different CD1d variants, generated by alternative splicing. V1 lacking exon 4 (CD1d without beta2 microglobulin, beta2m) and V2 lacking exons 4 and 5 (soluble CD1d) may be functional molecules, because the antigen binding sites (exons 2 and 3) are intact. METHODS: Peripheral blood mononuclear cells (PBMC) from 44 patients with autoimmune disease (RA 19, systemic lupus erythematosus, SLE 10, Sjögren's syndrome, SS 15) and 15 healthy controls were separated and complementary (c)DNA was prepared. The expression of intact CD1d on PBMC was detected by flow cytometry. Alternatively spliced CD1d variants were quantified by TaqMan PCR using polymerase chain reaction with confronting 2-pair primers (PCR-CTPP) based amplification. RESULTS: The mean (+/- SEM) transmembrane and beta2m binding site deleted CD1d mRNA level in 19 patients with RA (2.0 +/- 0.33) was significantly lower than in 15 controls (6.9 +/- 2.08; p < 0.05), whereas there were no differences in beta2m deleted variants and intact CD1d mRNA. CONCLUSION: Our findings suggest that low expression of soluble CD1d variants might play a role in the formation of symptoms or pathogenesis of RA.


Subject(s)
Antigens, CD1/genetics , Arthritis, Rheumatoid/genetics , Autoimmune Diseases/blood , Alternative Splicing , Antigens, CD1/metabolism , Antigens, CD1d , Arthritis, Rheumatoid/metabolism , DNA, Complementary/analysis , DNA, Complementary/blood , Flow Cytometry , Humans , Killer Cells, Natural/metabolism , Polymerase Chain Reaction , Protein Isoforms/genetics , Taq Polymerase/metabolism
19.
Blood ; 101(7): 2514-20, 2003 Apr 01.
Article in English | MEDLINE | ID: mdl-12468426

ABSTRACT

The ligation of programmed death-ligand 1 (B7-H1) to T cells results in the preferential production of interleukin 10 (IL-10). We investigated if B7-H1 would be up-regulated in HIV infection, a disease characterized by increased IL-10 production, by measuring B7-H1, B7-1 (CD80), and B7-2 (CD86) expression and mRNA in 36 HIV-infected patients and in 22 healthy controls (HCs). Results showed that (1) B7-H1 expression and mRNA are augmented in cells of HIV patients; (2) increased IL-10 production in these patients is largely induced by B7-H1-expressing CD14(+) cells; (3) an inverse correlation is detected between B7-H1 expression and CD4 counts, whereas the up-regulation of B7-H1 is directly associated with HIV plasma viremia; (4) antiviral therapy results in the parallel down modulation of IL-10 production and B7-H1 expression/synthesis; and (5) B7-H1/CD80 and B7-H1/CD86 mRNA ratios are increased in peripheral blood mononuclear cells (PBMCs) of HIV patients compared with HCs. B7-H1 synthesis and expression are up-regulated in HIV infection, and the degree of dysregulation correlates with the severity of disease. Aberrant antigen presentation by antigen-presenting cells (APCs) that exhibit increased B7-H1 expression and IL-10 production in HIV infection could be responsible for T-lymphocyte unresponsiveness and loss of protective immunity. B7-H1 is a surrogate marker potentially involved in AIDS disease progression.


Subject(s)
B7-1 Antigen/biosynthesis , Blood Proteins , HIV Infections/etiology , HIV Infections/metabolism , Peptides , Antigens, CD , Antiretroviral Therapy, Highly Active , Antiviral Agents/pharmacology , B7-1 Antigen/genetics , B7-H1 Antigen , Biomarkers/blood , Case-Control Studies , DNA, Complementary/blood , Disease Progression , HIV Infections/pathology , Humans , Interleukin-10/biosynthesis , Interleukin-10/genetics , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/pathology , Membrane Glycoproteins , RNA, Messenger/blood , Up-Regulation/drug effects
20.
Oncogene ; 21(51): 7817-23, 2002 Nov 07.
Article in English | MEDLINE | ID: mdl-12420218

ABSTRACT

Quantification of circulating cancer cells in whole blood samples by real time quantitative RT-PCR might be of clinical value for monitoring therapeutic effectiveness. In colon cancer patients, carcinoembrynic antigen (CEA) and cytokeratin 20 (CK20) have been frequently used for RT-PCR based tumor cell detection, but the specificity in particular for CEA has been questioned. In this study, we compared real-time RT-PCR for CEA and CK20 and analysed patients with metastatic disease (n=32) and healthy volunteers (n=17). CK20 mean values were elevated in cancer patients (P<0.001) and defined a subgroup (38%) who showed CK20 levels at least 100-fold above the highest value of the healthy control group. In contrast, only two cancer patients (6%) showed elevated CEA levels. Samples of the healthy control group showed exclusively a CEA-PCR product of 79 degrees C melting temperature. Thirty per cent of the colon cancer patients showed an additional product of 82 degrees C melting temperature. The 82 degrees C product was identical with the amplification product of CEA-cDNA and cDNA from different colon cancer cell lines. Colon cancer cells were spiked into normal blood in 10-fold dilutions that resulted in a dose dependent shift of the melt curve from 79 degrees C to the 82 degrees C. Sequencing of the PCR products showed that white blood cells express a splice variant of CEA, which hinders detection of tumor cell cDNA in whole blood samples. Our findings have implications for the use of CEA as a diagnostic molecule (e.g. by RT-PCR). The discovery of a physiologically expressed CEA splice variant might lead to a better understanding of the biological function of CEA and its family members.


Subject(s)
Adenocarcinoma/blood , Biomarkers, Tumor/blood , Carcinoembryonic Antigen/analysis , Colonic Neoplasms/blood , Gene Expression Regulation, Neoplastic , Neoplasm Proteins/blood , Neoplastic Cells, Circulating , Protein Isoforms/blood , RNA Splicing , Reverse Transcriptase Polymerase Chain Reaction , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Base Sequence , Binding, Competitive , Biomarkers, Tumor/biosynthesis , Biomarkers, Tumor/genetics , Carcinoembryonic Antigen/biosynthesis , Carcinoembryonic Antigen/genetics , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Computer Systems , DNA, Complementary/blood , False Positive Reactions , Hot Temperature , Humans , Intermediate Filament Proteins/blood , Keratin-20 , Leukocytes/metabolism , Molecular Sequence Data , Neoplasm Metastasis , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Neoplasm, Residual , Protein Denaturation , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Reagent Kits, Diagnostic , Sensitivity and Specificity , Sequence Alignment , Sequence Homology, Nucleic Acid , Tumor Cells, Cultured
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