Evidence That Intracellular Stages of Leishmania major Utilize Amino Sugars as a Major Carbon Source

Intracellular parasites, such as Leishmania spp, must acquire suitable carbon sources from the host cell in order to replicate. Here we present evidence that intracellular amastigote stages of Leishmania exploit amino sugars in the phagolysosome of mammalian macrophages as a source of carbon and energy. L. major parasites are capable of using N-acetylglucosamine and glucosamine as primarily carbon sources and contain key enzymes required for conversion of these sugars to fructose-6-phosphate. The last step in this pathway is catalyzed by glucosamine-6-phosphate deaminase (GND), which was targeted to glycosomes via a canonical C-terminal targeting signal when expressed as a GFP fusion protein. Mutant parasites lacking GND were unable to grow in medium containing amino sugars as sole carbohydrate source and rapidly lost viability, concomitant with the hyper-accumulation of hexosamine-phosphates. Expression of native GND, but not a cytosolic form of GND, in Δgnd parasites restored hexosamine-dependent growth, indicating that toxicity is due to depletion of glycosomal pools of ATP. Non-lethal increases in hexosamine phosphate levels in both Δgnd and wild type parasites was associated with a defect in promastigote metacyclogenesis, suggesting that hexosamine phosphate levels may influence parasite differentiation. Promastigote and amastigote stages of the Δgnd mutant were unable to replicate within macrophages and were either completely cleared or exhibited reduced lesion development in highly susceptible Balb/c mice. Our results suggest that hexosamines are a major class of sugars in the macrophage phagolysosome and that catabolism of scavenged amino sugars is required to sustain essential metabolic pathways and prevent hexosamine toxicity

Association of antigen processing machinery and HLA class I defects with clinicopathological outcome in cervical carcinoma

HLA class I loss is a significant mechanism of immune evasion by cervical carcinoma, interfering with the development of immunotherapies and cancer vaccines. We report the systematic investigation of HLA class I and antigen processing machinery component expression and association with clinical outcome. A tissue microarray containing carcinoma lesions from 109 cervical carcinoma patients was stained for HLA class I heavy chains, β2-microglobulin, LMP2, LMP7, LMP10, TAP1, TAP2, ERAP1, tapasin, calreticulin, calnexin and ERp57. A novel staining evaluation method was used to ensure optimal accuracy and reliability of expression data, which were correlated with known clinicopathological parameters. Partial HLA class I loss was significantly associated with decreased 5-years overall survival (61% vs. 83% for normal expression; P < 0.05) and was associated with decreased 5-years disease-free survival (DFS) (65% vs. 82% for normal expression; P = 0.05). All APM components except LMP10, calnexin and calreticulin were down-regulated in a substantial number of cases and, except ERAP1, correlated significantly with HLA class I down-regulation. LMP7, TAP1 and ERAP1 loss was significantly associated with decreased overall and (except LMP7) DFS (P < 0.05 and 0.005, respectively). ERAP1 down-regulation was an independent predictor for worse overall and DFS in multivariate analysis (HR 3.08; P < 0.05 and HR 2.84; P < 0.05, respectively). HLA class I and APM component down-regulation occur frequently in cervical carcinoma, while peptide repertoire alterations due to ERAP1 loss are a major contributing factor to tumour progression and mortality

Clinical impact of HLA class I expression in rectal cancer

Contains fulltext : 69499.pdf (publisher's version ) (Open Access)PURPOSE: To determine the clinical impact of human leukocyte antigen (HLA) class I expression in irradiated and non-irradiated rectal carcinomas. EXPERIMENTAL DESIGN: Tumor samples in tissue micro array format were collected from 1,135 patients. HLA class I expression was assessed after immunohistochemical staining with two antibodies (HCA2 and HC10). RESULTS: Tumors were split into two groups: (1) tumors with >50% of tumor cells expressing HLA class I (high) and (2) tumors with < or =50% of tumor cells expressing HLA class I (low). No difference in distribution or prognosis of HLA class I expression was found between irradiated and non-irradiated patients. Patients with low expression of HLA class I (15% of all patients) showed an independent significantly worse prognosis with regard to overall survival and disease-free survival. HLA class I expression had no effect on cancer-specific survival or recurrence-free survival. CONCLUSIONS: Down-regulation of HLA class I in rectal cancer is associated with poor prognosis. In contrast to our results, previous reports on HLA class I expression in colorectal cancer described a large population of patients with HLA class I negative tumors, having a good prognosis. This difference might be explained by the fact that a large proportion of HLA negative colon tumors are microsatellite instable (MSI). MSI tumors are associated with a better prognosis than microsatellite stable (MSS). As rectal tumors are mainly MSS, our results suggest that it is both, oncogenic pathway and HLA class I expression, that dictates patient's prognosis in colorectal cancer. Therefore, to prevent confounding in future prognostic analysis on the impact of HLA expression in colorectal tumors, separate analysis of MSI and MSS tumors should be performed

Construction and characterization of a fusion protein of single-chain anti-CD20 antibody and human beta-glucuronidase for antibody-directed enzyme prodrug therapy

The CD20 antigen is an attractive target for specific treatment of B-cell lymphoma. Antibody-directed enzyme prodrug therapy (ADEPT) aims at the specific activation of a nontoxic prodrug at the tumor site by an enzyme targeted by a tumor-specific antibody such as anti-CD20. We constructed a fusion protein of the single-chain Fv anti-CD20 mouse monoclonal antibody (MoAb) 1H4 and human beta-glucuronidase for the activation of the nontoxic prodrug N-[4-doxorubicin-N-carbonyl(-oxymethyl) phenyl] O-beta-glucuronyl carbamate to doxorubicin at the tumor site. The cDNAs encoding the light- and heavy-chain variable regions of 1H4 were cloned, joined by a synthetic sequence encoding a 18-amino acid linker and fused to human beta-glucuronidase by a synthetic sequence encoding a 6-amino acid linker. An antibody-enzyme fusion protein-producing cell line was established by transfection of the construct into human embryonic kidney 293/EBNA cells. The yield of active fusion protein was 100 ng/mL transfectoma supernatant. Antibody affinity, antibody specificity, and enzyme activity were fully retained by the fusion protein. Immunoprecipitation and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) showed that the fusion protein has a relative molecular weight (Mw) of 100 kD under denaturing conditions. Gel filtration analysis indicated that the enzymatically active form of the fusion protein is a tetramer with an Mw of approximately 400 kD. The nontoxic prodrug N-[4-doxorubicin-N-carbonyl(-oxymethyl) phenyl] O-beta-glucuronyl carbamate was hydrolyzed by the fusion protein at a hydrolysis rate similar to that of human beta-glucuronidase. When the fusion protein was specifically bound to Daudi lymphoma cells, the prodrug induced similar antiproliferative effects as doxorubicin. Thus, it is feasible to construct a eukaryotic fusion protein consisting of a single chain anti-CD20 antibody and human beta-glucuronidase for future use in the activation of anticancer prodrugs in B-cell lymphoma. (C) 1998 by The American Society of Hematology

PimE is a polyprenol-phosphate-mannose-dependent mannosyltransferase that transfers the fifth mannose of phosphatidylinositol mannoside in mycobacteria

C1 - Journal Articles RefereedPhosphatidylinositol mannosides (PIMs) are a major class of glycolipids in all mycobacteria. AcPIM2, a dimannosyl PIM, is both an end product and a precursor for polar PIMs, such as hexamannosyl PIM (AcPIM6) and the major cell wall lipoglycan, lipoarabinomannan (LAM). The mannosyltransferases that convert AcPIM2 to AcPIM6 or LAM are dependent on polyprenol-phosphate-mannose (PPM), but have not yet been characterized. Here, we identified a gene, termed pimE that is present in all mycobacteria, and is required for AcPIM6 biosynthesis. PimE was initially identified based on homology with eukaryotic PIG-M mannosyltransferases. PimE-deleted Mycobacterium smegmatis was defective in AcPIM6 synthesis, and accumulated the tetramannosyl PIM, AcPIM4. Loss of PimE had no affect on cell growth or viability, or the biosynthesis of other intracellular and cell wall glycans. However, changes in cell wall hydrophobicity and plasma membrane organization were detected, suggesting a role for AcPIM6 in the structural integrity of the cell wall and plasma membrane. These defects were corrected by ectopic expression of the pimE gene. Metabolic pulse-chase radiolabeling and cell-free PIM biosynthesis assays indicated that PimE catalyzes the alpha1,2-mannosyl transfer for the AcPIM5 synthesis. Mutation of an Asp residue in PimE that is conserved in and required for the activity of human PIG-M resulted in loss of PIM-biosynthetic activity, indicating that PimE is the catalytic component. Finally, PimE was localized to a distinct membrane fraction enriched in AcPIM4-6 biosynthesis. Taken together, PimE represents the first PPM-dependent mannosyl-transferase shown to be involved in PIM biosynthesis, where it mediates the fifth mannose transfer

Increased expression of tumor-associated antigens in pediatric and adult ependymomas: implication for vaccine therapy

Despite surgery and radiotherapy, as many as 50 % of children with ependymomas will suffer from tumor recurrences that will ultimately lead to death. Our group’s initial peptide-based glioma vaccine targeting EphA2, IL-13Rα2, and Survivin, which are overexpressed in pediatric gliomas, has shown promise in its initial phase of testing. We therefore investigated whether EphA2, IL-13Raα2, Survivin, and, additionally, Wilms’ Tumor 1 (WT1), are overexpressed in pediatric ependymomas to determine if a similar immunotherapy approach could be applicable. Immunohistochemistry was performed using antibodies specific for EphA2, IL-13Rα2, Survivin, and WT1 on paraffin-embedded specimens from 19 pediatric and 13 adult ependymomas. Normal brain and ependyma were used for background staining controls. Negative staining was defined as no staining or staining equaling the background intensity in normal brain tissues. In the 19 pediatric cases, 18 (95 %) demonstrated positive staining for EphA2, 16 (84 %) for IL-13Rα2, 18 (95 %) for Survivin, and only 7 (37 %) for WT1. Only 3 of 19 cases were positive for two or fewer tumor-associated antigens (TAAs); 16 of 19 cases were positive for three or more TAAs. In the 13 adult cases, all 13 demonstrated positive staining for EphA2, IL-13Rα2, and Survivin. Only 2 of 13 cases (15 %) demonstrated positive staining for WT1. All adult specimens were positive for three or more TAAs. Some ependymomas showed patchy variability in intensity. Pediatric and adult ependymomas frequently express EphA2, IL-13Rα2, and Survivin. This provides the basis for the utilization of an established multiple peptide vaccine for ependymoma in a clinical trial setting