IL-2, IL-5, TNF-α and IFN-γ mRNA expression in epidermal keratinocytes of systemic lupus erythematosus skin lesions.

OBJECTIVE: To analyze cytokine gene expression in keratinocytes from patients with systemic lupus erythematosus (SLE). INTRODUCTION: Keratinocytes represent 95% of epidermal cells and can secrete several cytokines. METHODS: Keratinocytes were obtained by laser microdissection from 21 patients with SLE (10 discoid and 11 acute lesions) at involved and uninvolved sites. All patients were receiving a low/moderate prednisone dose and 18 were receiving chloroquine diphosphate. IL-2, IL-5, TNF-α and IFN-γ gene expression was evaluated by real-time PCR and expressed as the ratio (R) to a pool of skin samples from 12 healthy volunteers. RESULTS: Heterogeneity in cytokine gene expression was found among patients with SLE. Eighteen of 38 valid SLE samples (47%) presented overexpression (R>1) of at least one cytokine. Lesional skin samples tended to show higher cytokine expression than samples from uninvolved skin (p = 0.06). IL-5 and IFN-γ were the most commonly overexpressed cytokines. Samples with cytokine overexpression corresponded to more extensive and severe lesions. Prednisone dose did not differ between samples without cytokine overexpression (15.71±3.45 mg/day) and those with overexpressed cytokines (12.68±5.41 mg/day) (p = 0.216). Samples from all patients not receiving diphosphate chloroquine had at least one overexpressed cytokine. CONCLUSIONS: The heterogeneous keratinocyte cytokine gene expression reflects the complex immunological and inflammatory background in SLE. Patients with severe/extensive skin lesions showed a higher frequency of cytokine gene overexpression. Increased IFN-γ and IL-5 expression suggests that Th1 and Th2 cells are involved in SLE skin inflammation. The possibility that prednisone and antimalarial drugs may have contributed to low cytokine gene expression in some samples cannot be ruled out.

IL-2, IL-5, TNF-α and IFN-γ mRNA expression in epidermal keratinocytes of systemic lupus erythematosus skin lesions

OBJECTIVE: To analyze cytokine gene expression in keratinocytes from patients with systemic lupus erythematosus (SLE). INTRODUCTION: Keratinocytes represent 95 percent of epidermal cells and can secrete several cytokines. METHODS: Keratinocytes were obtained by laser microdissection from 21 patients with SLE (10 discoid and 11 acute lesions) at involved and uninvolved sites. All patients were receiving a low/moderate prednisone dose and 18 were receiving chloroquine diphosphate. IL-2, IL-5, TNF-α and IFN-γ gene expression was evaluated by real-time PCR and expressed as the ratio (R) to a pool of skin samples from 12 healthy volunteers. RESULTS: Heterogeneity in cytokine gene expression was found among patients with SLE. Eighteen of 38 valid SLE samples (47 percent) presented overexpression (R>1) of at least one cytokine. Lesional skin samples tended to show higher cytokine expression than samples from uninvolved skin (p = 0.06). IL-5 and IFN-γ were the most commonly overexpressed cytokines. Samples with cytokine overexpression corresponded to more extensive and severe lesions. Prednisone dose did not differ between samples without cytokine overexpression (15.71±3.45 mg/day) and those with overexpressed cytokines (12.68±5.41 mg/day) (p = 0.216). Samples from all patients not receiving diphosphate chloroquine had at least one overexpressed cytokine. CONCLUSIONS: The heterogeneous keratinocyte cytokine gene expression reflects the complex immunological and inflammatory background in SLE. Patients with severe/extensive skin lesions showed a higher frequency of cytokine gene overexpression. Increased IFN-γ and IL-5 expression suggests that Th1 and Th2 cells are involved in SLE skin inflammation. The possibility that prednisone and antimalarial drugs may have contributed to low cytokine gene expression in some samples cannot be ruled out.

Factors associated with the intensity of liver fibrosis in renal transplant patients with hepatitis B virus infection.

BACKGROUND: Hepatitis B may show a more aggressive course after kidney transplantation, but the factors associated with the progression of fibrosis in this group have not been identified. OBJECTIVES: To determine the influence of hepatitis B virus (HBV) viral load and host-related factors on the progression of hepatic fibrosis in hepatitis B virus-infected renal transplant recipients. PATIENTS AND METHODS: Renal transplant patients positive for HBV surface antigen (HBsAg) and submitted to a liver biopsy because of evidence of viral replication were included. Patients with advanced fibrosis (METAVIR F3-F4) were compared with patients with mild fibrosis (F0-F2) regarding sex, age, estimated time since infection, post-transplant time, donor type, history of renal transplantation, alanine aminotransferase, anti-hepatitis C virus, HBeAg and quantitative hepatitis B virus-DNA. Logistic regression analysis was applied to identify variables independently associated with more advanced fibrosis. RESULTS: Fifty-five patients (75% men, 41+/-11 years) with a mean post-transplant time of 5+/-4 years were included. HBeAg was detected in 67% of the patients and anti-hepatitis C virus in 35%. The median hepatitis B virus-DNA level was 2.8 x 10(8) copies/ml. Seventeen (31%) patients had advanced fibrosis. Using logistic regression analysis, the only variable that showed an independent association with more advanced stages of fibrosis was post-transplant time (P=0.03, odds ratio: 1.2, 95% confidence interval: 1.02-1.45). CONCLUSION: Hepatitis B virus viral load, although very high, and hepatitis B virus/hepatitis C virus coinfection are not related to the intensity of liver fibrosis in renal transplant patients infected with hepatitis B virus. Post-transplant time was the only factor independently associated with more advanced liver fibrosis, suggesting the influence of immunosuppression on the progression of liver disease in these patients.

Large-scale transcriptome analyses reveal new genetic marker candidates of head, neck, and thyroid cancer.

A detailed genome mapping analysis of 213,636 expressed sequence tags (EST) derived from nontumor and tumor tissues of the oral cavity, larynx, pharynx, and thyroid was done. Transcripts matching known human genes were identified; potential new splice variants were flagged and subjected to manual curation, pointing to 788 putatively new alternative splicing isoforms, the majority (75%) being insertion events. A subset of 34 new splicing isoforms (5% of 788 events) was selected and 23 (68%) were confirmed by reverse transcription-PCR and DNA sequencing. Putative new genes were revealed, including six transcripts mapped to well-studied chromosomes such as 22, as well as transcripts that mapped to 253 intergenic regions. In addition, 2,251 noncoding intronic RNAs, eventually involved in transcriptional regulation, were found. A set of 250 candidate markers for loss of heterozygosis or gene amplification was selected by identifying transcripts that mapped to genomic regions previously known to be frequently amplified or deleted in head, neck, and thyroid tumors. Three of these markers were evaluated by quantitative reverse transcription-PCR in an independent set of individual samples. Along with detailed clinical data about tumor origin, the information reported here is now publicly available on a dedicated Web site as a resource for further biological investigation. This first in silico reconstruction of the head, neck, and thyroid transcriptomes points to a wealth of new candidate markers that can be used for future studies on the molecular basis of these tumors. Similar analysis is warranted for a number of other tumors for which large EST data sets are available.

DNA-based typing of blood groups for the management of multiply-transfused sickle cell disease patients.

BACKGROUND: The usefulness of DNA genotyping for RBC antigens as a tool for the management of multiply-transfused patients with sickle cell disease (SCD) to overcome the limitations of hemagglutination assays was evaluated. STUDY DESIGN AND METHODS: Blood samples from 40 multiply-transfused SCD patients were studied by hemagglutination and by PCR-RFLP for antigens or genes in the Rh (D, C/c, E/e), Kell, Kidd, and Duffy systems. RESULTS: Discrepancies were found between hemaglutination and DNA typing test results in six patients: two were discrepant in Rh typing (one was D- by hemagglutination and RhD by DNA, and one was E+e- and RhEe by DNA), two were discrepant in Duffy typing [both were Fy(a+b-) and Fy(b)/Fy(b) by DNA], and four were discrepant in Kidd typing [Jk(a+b+) and Jk(b)/Jk(b) by DNA; two of these samples were also discrepant in Duffy]. Stored segments from blood units that had been recently transfused to these six recipients were phenotyped, confirming that the transfused RBCs were the source of the discrepancy between genotype and phenotype. CONCLUSION: DNA typing of blood groups by PCR-RFLP in peripheral blood WBCs contributes to the management of transfusions in SCD patients by allowing a more accurate selection of donor units.

Genotyping of Kell, Duffy, kidd and RHD in patients with B Thalassemia

A determinaçäo dos fenótipos Rh, Kell, Duffy e Kidd, associada ao ABO é utilizada para prevenir a aloimunizaçäo a antígenos eritrocitários e participam também no processo de identificaçäo de anticorpos nos pacientes com B talassemia. Todavia, a fenotipagem desses pacientes é trabalhosa e de difícil interpretaçäo. Nesta situaçäo, deve ser avaliada uma alternativa ao teste de hemaglutinaçäo para determinar o padräo antigênico dos pacientes. Utilizamos para tal fim o método PCR-RFLP. Foram preparados DNAs de 50 pacientes com Btalassemia que haviam sido anteriormente fenotipados pela hamglutinaçäo e testados para Kell, Kidd, Duffy/GATA mutaçäo por PCR-RFLP. RHD/näo-D foi analisado pelo tamanho do produto, do PCR associado à seqüência do gene RHD no intron 4 e exon 10/3' UTR. Os testes de genotipagem foram realizados sem o conhecimento dos resultados dos fenótipos. Para os RHD/näo-D, 47 foram RHD+ e RHD+/RHCE+, e 3 foram RHD- e RHD-/RHCE+. Para o Kell, 48 kk foram K2K2 e 2 Kk foram K1K2. Para o Duffy, das 44 amostras que haviam sido normais, GATA box, 8 Fy(a+b-)foram FYA/FYA, 15Fy(a+b-) foram FYB/FYB; e 19 Fy(a+b+) foram FYA/FYB; das outras 4 amostras, 3 foram FYA/FYB; homozigoto GATA mutaçäo. Duas amostras fenotipadas como Fy(a+b-), que eram normais GATA, apresentavam as mutaçöes 265T/298a e 2 amostras fenotipadas como Fy(a+b+) haviam sido genotipadas como FYA/FYB. Para o Kidd, 15Jk(a+b+) foram JKA/JKA, 12jk(aa-b+) foram JKB/JKB, e 20 jk(a+b+) haviam sido genotipadas como JKB/JKB. A genotipagem é mais acurada que a fenotipagem para determinaçäo de grupos sangüíneos em pacientes portadores de B talassemia poli transfundidos. A genotipagem nesses pacientes pode ser importante para selecionar hemácias antigenicamente negativas para transfusäo de glóbulos vermelhos.