DC maturation and function are not altered by melanoma-derived immunosuppressive soluble factors.

BACKGROUND: Although melanoma can elicit robust tumor antigen-specific immune responses, advanced melanoma is associated with immune tolerance. We have previously described several mechanisms of melanoma-induced immunosuppression, including the skewing of the immune response towards a Th2 cytokine profile and the induction of regulatory T cells. Since dendritic cells (DCs) are potentially important players that can direct other cells of the immune system towards a cytotoxic, humoral, or regulatory phenotype, we hypothesized that melanoma-produced factors directly affect the maturation and function of DCs, influencing the nature and magnitude of the resulting immune response. MATERIALS AND METHODS: To test this hypothesis, immature myeloid-derived DCs (mdDCs) were derived with cytokines from CD14+ peripheral blood mononuclear cells (PBMCs) and exposed to 20% melanoma-conditioned media (MCM). After 2 d, the expression of maturation markers and the function of these mdDCs, measured by cytokine production, the amount of endocytosis, expression of the inhibitory molecule indoleamine 2,3-dioxygenase (IDO), and the ability to stimulate T cells were determined. RESULTS: We found that incubation with MCM did not inhibit the expression of maturation markers or IDO, the production of cytokines, the amount of antigen uptake, or the ability to induce T cell proliferation in mixed-lymphocyte reactions by mdDC. CONCLUSIONS: These results suggest that the immunosuppressive effects of melanoma-produced factors are independent of directly measurable changes in mdDC function or maturation in vitro.

Interaction of hepatocyte nuclear factors in transcriptional regulation of tissue specific hormonal expression of human multidrug resistance-associated protein 2 (abcc2).

Multidrug resistance-associated protein 2 (MRP2) (ABCC2) is an ATP-binding cassette membrane protein located primarily on apical surface of hepatocytes that mediates transport of conjugated xenobiotics and endogenous compounds into bile. MRP2 is highly expressed in hepatocytes, and at lower levels in small intestines, stomach and kidney. Previous reports have characterized mammalian MRP2 promoters, but none have established the molecular mechanism(s) involved in liver enriched expression. This study aims to investigate the mechanism of hepatic MRP2 regulation. A 2130 bp of MRP2 promoter was cloned from PAC-1 clone P108G1-7, to identify putative liver specific/hormone responsive functional DNA binding sites. Using deletion analysis, site specific mutagenesis and co-transfection studies, liver specific expression was determined. MRP2 promoter-LUC constructs were highly expressed in liver cell lines compared to non-liver cells. The region extending from -3 to+458 bp of MRP2 promoter starting from AUG contained the potential binding sites for CAATT box enhancer binding protein (C/EBP), hepatocytes nuclear factor 1, 3 and 4 (HNF1, HNF3, and HNF4. Only HNF1 and HNF4 co-transfection with MRP2 luciferase increased expression. Site specific mutational analysis of HNF1 binding site indicated an important role for HNF1alpha. HNF4alpha induction of MRP2 was independent of HNF1 binding site. C/EBP, HNF3, and HNF6 inhibited HNF1alpha while HNF4alpha induced MRP2 luciferase expression and glucocorticoids stimulated MRP2 expression. This study emphasizes the complex regulation of MRP2 with HNF1alpha and HNF4alpha playing a central role. The coordinated regulation of xenobiotic transporters and oxidative conjugation may determine the adaptive responses to cellular detoxification processes.

A new mechanism for generating TCR diversity: A TCR Jalpha-like gene that inserts partial nucleotide sequences in a D-gene manner.

delta-TCR genes of two gammadelta-T cell hybridomas were found to contain an identical 19-nt sequence in their non-germline N-regions. To determine if this sequence represented a third murine TCR Ddelta gene, genomic PCR was performed by using it as a primer together with primers for interspersed repetitive elements (IRE). Sequencing revealed that the 19-nt segment is part of a 61-nt gene with flanking 5' and 3' recombination signaling sequences (RSS). Southern blot analysis confirmed the presence of this 61-nt gene in the genome of several mouse strains. The gene is unusual in that the distal 24 nucleotides of its 3' RSS region are contributed by the 5' portion of a B2 IRE sequence that includes an apparently non-functional RNA splice site within the 3' nonamer sequence. It has sequence similarities with the Ddelta1 gene (81%) at its 5' end and with Jalpha genes (73%) overall. Tyramide-FISH analysis identified the gene to exist within or adjacent to the TCR alpha/delta locus on chromosome 14. Surveys of available TCR sequences reveal possible partial insertions of the 61-nt gene in other delta-TCR and in alpha-TCR gene sequences. Thus, the unique 61-nt gene is Jalpha gene-like in structure but D gene-like in function.

Hormonal regulation of hepatic multidrug resistance-associated protein 2 (Abcc2) primarily involves the pattern of growth hormone secretion.

Biliary excretion is the rate-limiting step in transfer of bilirubin, other organic anions, and xenobiotics across the liver. Multidrug resistance-associated protein 2 (Mrp2, Abcc2) is the major transporter for conjugated endo- and xenobiotic-conjugated compounds into bile. Hormones regulate bilirubin and xenobiotic secretion into bile, which have dimorphic differences. Therefore, we examined the possible role of sex steroids and growth hormone in the regulation of Mrp2. In approximately 8-wk-old rats, mRNA, transcriptional activity, and hepatic content of Mrp2 were selectively increased fourfold (P < 0.001) in females compared with males. In males, estrogens increased and testosterone decreased Mrp2 mRNA and protein, whereas no significant effect was measured in females, suggesting either a direct effect on the liver or an alteration in growth hormone secretory pattern. After hypophysectomy, Mrp2 mRNA was markedly reduced and the effects of estrogens and testosterone on Mrp2 were prevented, supporting the role of pituitary hormones in controlling Mrp2 expression. Mrp2 increased following growth hormone infusion in males. Mrp2 mRNA was decreased in growth hormone-deficient "Little" mice. Growth hormone infusions in hypophysectomized rats partially restored Mrp2 levels, whereas thyroxine addition returned Mrp2 mRNA and protein to basal levels. Morphology as well as biochemical measurements demonstrated that Mrp2 was localized to the bile canaliculus in equal density in both genders, whereas hormone replacements increased Mrp2 in hypophysectomized animals. In cultured hepatocytes, thyroxine did not have an effect, but growth hormone alone and combined with thyroxine increased Mrp2 mRNA levels. In conclusion, Mrp2 levels are regulated by the combination of thyroxine and different growth hormone secretory patterns.

Spectrum of genotype and clinical manifestations in cerebral cavernous malformations.

OBJECTIVE: Cerebral cavernous malformations (CCMs) are focal dysmorphic blood vessel anomalies predisposing individuals to hemorrhagic stroke and epilepsy. CCMs are sporadic or inherited as autosomal dominant disease with three known genes. The hypothesis that genetic heterogeneity would account for the remarkable variability in CCM manifestations was tested. METHODS: CCM cases were prospectively enrolled. Germline CCM1 gene mutations were sought in 89 CCM samples. Associations with clinical manifestations and lesion characteristics were made among 41 symptomatic familial cases, including one cohort of 26 cases with CCM1 mutations and a second cohort of 15 cases without identifiable CCM1 mutations. The 15 cases were screened for CCM2 and CCM3 mutations. RESULTS: CCM1 mutations were found in 34 out of 50 subjects with familial disease and in none of 39 sporadic CCM cases. CCM2 and CCM3 mutations were found in three out of 10 families screened without CCM1 mutations. Clinical manifestations in 22 Hispanic-American cases with identical CCM1 mutations were highly variable. Fewer CCM1 patients experienced hemorrhage than others with familial disease (P = 0.0139 for all cases and P = 0.0442 for symptomatic cases). Adjusting for sex and age improved the logistic regression model, suggesting decreased numbers of patients with hemorrhage in CCM1 familial disease (P = 0.003 for all cases and P = 0.014 for symptomatic cases). Hemorrhage differences were not related to size or number of lesions. CONCLUSION: Factors in addition to CCM1 germline mutation contribute to CCM clinical manifestations. However, this evidence suggests that familial cases with CCM1 mutations may have less severe clinical manifestations than other familial cases.

Differential gene expression in human cerebrovascular malformations.

OBJECTIVE: We sought to identify genes with differential expression in cerebral cavernous malformations (CCMs), arteriovenous malformations (AVMs), and control superficial temporal arteries (STAs) and to confirm differential expression of genes previously implicated in the pathobiology of these lesions. METHODS: Total ribonucleic acid was isolated from four CCM, four AVM, and three STA surgical specimens and used to quantify lesion-specific messenger ribonucleic acid expression levels on human gene arrays. Data were analyzed with the use of two separate methodologies: gene discovery and confirmation analysis. RESULTS: The gene discovery method identified 42 genes that were significantly up-regulated and 36 genes that were significantly down-regulated in CCMs as compared with AVMs and STAs (P = 0.006). Similarly, 48 genes were significantly up-regulated and 59 genes were significantly down-regulated in AVMs as compared with CCMs and STAs (P = 0.006). The confirmation analysis showed significant differential expression (P < 0.05) in 11 of 15 genes (angiogenesis factors, receptors, and structural proteins) that previously had been reported to be expressed differentially in CCMs and AVMs in immunohistochemical analysis. CONCLUSION: We identify numerous genes that are differentially expressed in CCMs and AVMs and correlate expression with the immunohistochemistry of genes implicated in cerebrovascular malformations. In future efforts, we will aim to confirm candidate genes specifically related to the pathobiology of cerebrovascular malformations and determine their biological systems and mechanistic relevance.