Flexible and sensitive method to functionally validate tumor-specific receptors via activation of NFAT

Tumor-specific receptors may provide effective tools for anti-tumor immunogene therapy. However, the functional analysis of primary human T cells engrafted with tumor-specific receptors is laborious and emphasizes the need for a fast and sensitive method to validate such receptors. To this end, we have set up a Jurkat T cell-based reporter gene assay, and tested receptors with various formats, i.e., receptors based on either a monoclonal antibody (mAb), a full-length T cell receptor (fl-TCR)β or a chimeric (ch-)TCRβ, and various antigen specificities for their ability to mediate tumor-specific activation of nuclear factor of activated T cells (NFAT). The mAb-based receptor specifically mediates NFAT activation after stimulation with tumor antigen-positive target cells. The observed receptor-mediated NFAT responses were validated by the use of ligand- and receptor-specific mAbs, as well as cyclosporin A (CsA) and a dominant negative mutant of NFAT. Furthermore, anti-TCR mAbs, peptide-loaded tumor cells and antigen-positive tumor cells all resulted in specific NFAT activation in TCR/CD8 co-transduced Jurkat T cells, irrespective of the TCR format used. Importantly, receptor-mediated NFAT responses parallel tumor-specific cytolysis and TNF production of receptor-transduced primary human T lymphocytes. In fact, inhibition of NFAT activation compromises the immune responses of primary human T lymphocytes, pointing to a central involvement of NFAT in anti-tumor T cell responses. Taken together, receptor-mediated activation of NFAT constitutes a representative measure of anti-tumor T cell responses, and the genetically modified Jurkat T cells provide a flexible and sensitive tool with which to select rapidly tumor-specific (chimeric) receptors for immunogene therapy.     

Cytogenetic map of common bean (Phaseolus vulgaris L.)

A cytogenetic map of common bean was built by in situ hybridization of 35 bacterial artificial chromosomes (BACs) selected with markers mapping to eight linkage groups, plus two plasmids for 5S and 45S ribosomal DNA and one bacteriophage. Together with three previously mapped chromosomes (chromosomes 3, 4, and 7), 43 anchoring points between the genetic map and the cytogenetic map of the species are now available. Furthermore, a subset of four BAC clones was proposed to identify the 11 chromosome pairs of the standard cultivar BAT93. Three of these BACs labelled more than a single chromosome pair, indicating the presence of repetitive DNA in their inserts. A repetitive distribution pattern was observed for most of the BACs; for 38% of them, highly repetitive pericentromeric or subtelomeric signals were observed. These distribution patterns corresponded to pericentromeric and subtelomeric heterochromatin blocks observed with other staining methods. Altogether, the results indicate that around half of the common bean genome is heterochromatic and that genes and repetitive sequences are intermingled in the euchromatin and heterochromatin of the species.     

Mitochondrial recombination increases with age in Podospora anserina

With uniparental inheritance of mitochondria, there seems little reason for homologous recombination in mitochondria, but the machinery for mitochondrial recombination is quite well-conserved in many eukaryote species. In fungi and yeasts heteroplasmons may be formed when strains fuse and transfer of organelles takes place, making it possible to study mitochondrial recombination when introduced mitochondria contain different markers. A survey of wild-type isolates from a local population of the filamentous fungus Podospora anserina for the presence of seven optional mitochondrial introns indicated that mitochondrial recombination does take place in nature. Moreover the recombination frequency appeared to be correlated with age: the more rapidly ageing fraction of the population had a significantly lower linkage disequilibrium indicating more recombination. Direct confrontation experiments with heterokaryon incompatible strains with different mitochondrial markers at different (relative) age confirmed that mitochondrial recombination increases with age. We propose that with increasing mitochondrial damage over time, mitochondrial recombination - even within a homoplasmic population of mitochondria - is a mechanism that may restore mitochondrial function.     

The effects on offspring of premature parturition

The time of parturition defines the length of the intrauterine period of fetal life, a requisite to achieve adequate adaptation to the external environment. Immaturity, a condition whose severity is inversely related to the length of pregnancy, is the main determinant of the increased morbidity and mortality associated with preterm birth. Despite great advances in medical technology and expertise, mainly after the introduction of the neonatal intensive care units, only one- to two-thirds of infants from the subsets with lower birthweight/gestational age reach survival at discharge. Distinct major neurological and sensorial sequelae, including cerebral palsy, retinopathy of prematurity, and hearing loss, as well as reduced neuropsychological abilities, leading to deficient academic achievement and deterioration of several aspects of health status, are still highly prevalent among the most immature children. Interestingly, decreasing mortality rates, which are not followed by detectable increases of disability, are being observed in recent years. Future advances may be expected from clinical and basic research on uterine contractility and cervical softening. Also, changes in reproductive technology procedures, a main factor in the incidence of multiple pregnancies, and a more refined approach to obstetric care, compose some of the clinical interventions which may reduce the problem.     

MMTV-cre-mediated fur inactivation concomitant with PLAG1 proto-oncogene activation delays salivary gland tumorigenesis in mice

Proprotein convertases are serine endoproteases implicated in the proteolytic processing of a large variety of regulatory proteins. An important role of proprotein convertases in tumorigenic processes has been suggested by various studies. In this study, the role of the proprotein convertase furin in PLAG1 proto-oncogene-induced salivary gland tumorigenesis was investigated. PLAG1 overexpression in salivary glands has previously been shown to result in salivary gland tumors in 100% of mice within 5 weeks after birth. MMTV-cre-mediated inactivation of fur without over-expression of PLAG1 caused smaller but histologically normal salivary glands. Moreover, the lymph nodes close to the salivary glands were enlarged, and histology showed that they had activated follicles. When genetic ablation of 1 or 2 alleles of fur and overexpression of the PLAG1 transgene were simultaneously achieved, a significant delay in tumorigenesis was observed. Collectively, these results suggest an important role for furin in PLAG1-induced salivary gland tumorigenesis in mice.     

BRCA1 mutation analysis of 41 human breast cancer cell lines reveals three new deleterious mutants

Germ line mutations of the BRCA1 gene confer a high risk of breast cancer and ovarian cancer to female mutation carriers. The BRCA1 protein is involved in the regulation of DNA repair. How specific tumor-associated mutations affect the molecular function of BRCA1, however, awaits further elucidation. Cell lines that harbor BRCA1 gene mutations are invaluable tools for such functional studies. Up to now, the HCC1937 cell line was the only human breast cancer cell line with an identified BRCA1 mutation. In this study, we identified three other BRCA1 mutants from among 41 human breast cancer cell lines by sequencing of the complete coding sequence of BRCA1. Cell line MDA-MB-436 had the 5396 + 1G>A mutation in the splice donor site of exon 20. Cell line SUM149PT carried the 2288delT mutation and SUM1315MO2 carried the 185delAG mutation. All three mutations were accompanied by loss of the other BRCA1 allele. The 185delAG and 5396 + 1G>A mutations are both classified as pathogenic mutations. In contrast with wild-type cell lines, none of the BRCA1 mutants expressed nuclear BRCA1 proteins as detected with Ab-1 and Ab-2 anti-BRCA1 monoclonal antibodies. These three new human BRCA1 mutant cell lines thus seem to be representative breast cancer models that could aid in further unraveling of the function of BRCA1.     

Physical activity and risk of endometrial cancer: the European prospective investigation into cancer and nutrition

The etiologic role of physical activity in endometrial cancer risk remains unclear given the few epidemiologic studies that have been conducted. To investigate this relation more fully, an analysis was undertaken in the European prospective investigation into cancer and nutrition (EPIC). During an average 6.6 years of follow-up, 689 incident endometrial cancer cases were identified from an analytic cohort within EPIC of 253,023 women. Cox proportional hazards models were used to estimate the associations between type of activity (total, occupational, household, recreational) and endometrial cancer risk. For total activity, women in the highest compared with the lowest quartile of activity had a risk of 0.88 (95% confidence interval (95% CI=0.61-1.27). No clear associations between each type of activity and endometrial cancer risk were found for the total study population combined. Associations were more evident in the stratified results, with premenopausal women who were active versus inactive experiencing a risk of 0.66 (95% CI=0.38-1.14) overall. Among premenopausal women, for household and recreational activities the risk estimates in the highest as compared with the lowest quartiles were, respectively, 0.48 (95% CI=0.23-0.99) and 0.78 (95% CI=0.44-1.39). No effect modification by body mass index, hormone replacement therapy, oral contraceptive use or energy intake was found. This study provides no evidence of a protective effect of increased physical activity in endometrial cancer risk in all women but some support for a benefit among premenopausal women. The relative risk reductions are most apparent for household activities.     

Long-term structural and functional consequences of cardiac ischaemia-reperfusion injury in vivo in mice

The short-term (<24 h) consequences of oxidative stress induced by ischaemia-reperfusion (IR) have been studied extensively in the mouse heart. However, much less is known about the long-term effects inflicted by a brief ischaemic period on the murine heart. We therefore examined the structural and functional consequences of a 30 min ischaemic period after 2 and 8 weeks of reperfusion and compared these to the effects induced by permanent occlusion of the left anterior descending coronary artery (LAD). The latter procedure resulted in transmural myocardial infarcts of about 52% of the left ventricle. In contrast, the single 30 min ischaemic period led to infarct sizes of about 13% of the left ventricle (range, 4-23%) at 2 and 8 weeks after reperfusion. Maximal cardiac contractility responses (+dP/dt) to dobutamine infusion and volume loading were depressed at 2, but not at 8 weeks after IR. The restoration of cardiac contractility at 8 weeks after IR was associated with a significant 20% enlargement of the end-diastolic volume and 16% increase of the left ventricular wall thickness. These changes in cardiac geometry were less pronounced at 2 weeks after IR. Histological examination revealed that the IR injury was associated with prominent calcification. At 2 and at 8 weeks after IR, 25 +/- 5 and 38 +/- 5% of the injured area was calcified as observed in 69 and 73% of the animals, respectively. After permanent occlusion of the LAD, calcification was not observed and healing of the affected area was characterized by thinning and dilatation of the infarcted myocardium. These data indicate that, in mice, a single 30 min period of ischaemia reduced ventricular contractility up to at least 2 weeks after reperfusion. However, 8 weeks after IR, cardiac function was restored by eccentric hypertrophy associated with calcification of the injured ventricular wall.     

Medicinal mushrooms in supportive cancer therapies: an approach to anti-cancer effects and putative mechanisms of action

Medicinal mushrooms have been valued as natural sources of bioactive compounds since times immemorial and have been recognized as potential immunomodulating and anti-cancer agents. Their consumption has consistently been shown to have beneficial effects on human health. Cancer is a generic term for several types of diseases that can be chronic and are responsible for a large number of deaths worldwide. Although there has been considerable progress in modern cancer therapy research, difficulties in understanding the molecular behavior of various types of cancers and the numerous side effects experienced by patients from treatments means that this whole subject area is still problematic. Thus, biological immunotherapy using natural bioactive compounds as supportive treatments in conventional cancer therapies has become in vogue. Bioactive metabolites isolated from medicinal mushrooms have shown potential successes in cancer treatment as biological immunotherapeutic agents that stimulate the immune system against cancer cells. They also act as an effective source of anti-cancer agents, capable of interfering with cellular signal transduction pathways linked to cancer development and progression. In this review we compile available data on the characteristics of medicinal mushrooms that appear to be particularly effective as biological immunotherapeutic agents. Major consideration is given to biological constituents and the putative mechanisms of action by which bioactive compounds act on the human body. Consideration is also given to the benefits that have been claimed for the use of mushrooms in treating cancer and the future prospects of using medicinal mushrooms as potent supportive candidate bioagents for treatment of cancers is discussed.     

Specific immunization strategies against oxidized low-density lipoprotein: A novel way to reduce nonalcoholic steatohepatitis in mice

Nonalcoholic steatohepatitis (NASH) is characterized by hepatic lipid accumulation combined with inflammation, which can ultimately progress into cirrhosis. Recently, we demonstrated that deletion of scavenger receptors (SRs) CD36 and SR-A in hematopoietic cells reduced hepatic inflammation. In addition to uptake of modified lipoproteins, CD36 and SR-A are also involved in other functions that can activate the inflammatory response. Therefore, the actual trigger for SR activation during NASH is unclear. Here, we hypothesized that hepatic inflammation is triggered by recognition of oxidized LDL (oxLDL) by Kupffer cells (KCs). To inhibit recognition of oxLDL by KCs, low-density lipoprotein receptor (Ldlr(-/-) ) mice were immunized with heat-inactivated pneumococci, which were shown to induce the production of anti-oxLDL immunoglobulin M (IgM) antibodies, due to molecular mimicry with oxLDL. The mice received a high-fat, high-cholesterol diet during the last 3 weeks to induce NASH. Immunization with pneumococci increased anti-oxLDL IgM levels and led to a reduction in hepatic inflammation, as shown by reduced macrophage, neutrophil, and T cell infiltration, and reduced gene expression of tumor necrosis factor (Tnf), interleukin-6 (Il-6), interleukin-1β (Il-1b), monocyte chemoattractant protein 1 (Mcp1), and fibrosis-related genes. In immunized mice, KCs were smaller and showed fewer cholesterol crystals compared with nonimmunized mice. Conclusion: Antibodies to oxLDL play an important role in the pathogenesis of NASH. Therefore, the potential of phosphorylcholine-based vaccination strategies as a novel tool for the prevention and therapy of NASH should be tested in the future. (HEPATOLOGY 2012;56:894-903).