Mycobacterium tuberculosis Is Able To Accumulate and Utilize Cholesterol

It is expected that the obligatory human pathogen Mycobacterium tuberculosis must adapt metabolically to the various nutrients available during its cycle of infection, persistence, and reactivation. Cholesterol, which is an important part of the mammalian cytoplasmic membrane, is a potential energy source. Here, we show that M. tuberculosis grown in medium containing a carbon source other than cholesterol is able to accumulate cholesterol in the free-lipid zone of its cell wall. This cholesterol accumulation decreases the permeability of the cell wall for the primary antituberculosis drug, rifampin, and partially masks the mycobacterial surface antigens. Furthermore, M. tuberculosis was able to grow on mineral medium supplemented with cholesterol as the sole carbon source. Targeted disruption of the Rv3537 (kstD) gene inhibited growth due to inactivation of the cholesterol degradation pathway, as evidenced by accumulation of the intermediate, 9-hydroxy-4-androstene-3,17-dione. Our findings that M. tuberculosis is able to accumulate cholesterol in the presence of alternative nutrients and use it when cholesterol is the sole carbon source in vitro may facilitate future studies into the pathophysiology of this important deadly pathogen.Mycobacterium tuberculosis, the causative agent of tuberculosis, is a very successful pathogen that infects one-third of the human population (21). Only 10% of primary infected individuals develop active disease during their lifetimes. Tubercle bacilli are able to persist in a dormant state, from which they may reactivate and induce the contagious disease state (13). In asymptomatic hosts, M. tuberculosis exists in reservoirs called granulomas, which are cellular aggregates that restrict bacterial spreading (40). Granulomas are organized collections of mature macrophages that exhibit a certain typical morphology and that arise in response to persistent intracellular pathogens (1, 4). Pathogenic mycobacteria can induce the formation of foamy macrophages filled with lipid-containing bodies; these have been postulated to act as a secure, nutrient-rich reservoir for tubercle bacilli (31). Moreover, M. tuberculosis DNA has been detected in fatty tissues surrounding the kidneys, as well as those of the stomach, lymph nodes, heart, and skin. Tubercle bacilli are able to enter adipocytes, where they accumulate within intracytoplasmic lipid inclusions and survive in a nonreplicating state (26). In vivo, it is expected that M. tuberculosis adapts metabolically to nutrient-poor conditions characterized by glucose deficiency and an abundance of fatty acids (25, 26). The presence of a complex repertoire of lipid metabolism genes in the genome of M. tuberculosis suggests that lipids, including steroids, are important alternative carbon and energy sources for this pathogen (7).One attractive potential alternative nutrient that is readily available in the mammalian host is cholesterol, a major sterol of the plasma membrane. The presence of cholesterol in lipid rafts is required in order for microorganisms to enter the intracellular compartment (14). Studies have shown that cholesterol is essential for the uptake of mycobacteria by macrophages, and it has been found to accumulate at the site of M. tuberculosis entry (2, 12, 30). Moreover, cholesterol depletion overcomes the phagosome maturation block experienced by Mycobacterium avium-infected macrophages (10).It is well known that cholesterol can be utilized by fast-growing, nonpathogenic mycobacteria (5, 20, 22), but it was previously thought that pathogenic mycobacteria might not be able to use cholesterol as a carbon and energy source (3). Recently, however, bioinformatic analysis identified a cassette of cholesterol catabolism genes in actinomycetes, including the M. tuberculosis complex (41). Microarray analysis of Rhodococcus sp. grown in the presence of cholesterol revealed the upregulation of 572 genes, most of which fell within six clearly discernible clusters (41). Most of the identified genes had significant homology to known steroid degradation genes from other organisms and were distributed within a single 51-gene cluster that appears to be very similar to a cluster present in the genome of M. tuberculosis (41). Many of the cholesterol-induced genes had been previously selected by transposon site hybridization analysis of genes that are essential for survival of tubercle bacilli (33) and/or are upregulated in gamma interferon-activated macrophages (37, 42). It was also demonstrated that the M. tuberculosis complex can grow on mineral medium with cholesterol as a primary source of carbon (27, 41). Moreover, the growth of tubercle bacilli on cholesterol was significantly affected by knockout of the mce4 gene, which encodes an ABC transporter responsible for cholesterol uptake (24, 27). Earlier studies had shown that disruption of mce4 attenuated bacterial growth in the spleens of infected animals that had developed adaptive immunity (17, 35).In the present study, we demonstrate for the first time that M. tuberculosis utilizes cholesterol via the 4-androstene-3,17-dione/1,4-androstadiene-3,17-dione pathway (AD/ADD) and that this process requires production of an intact KstD enzyme. We also show that tubercle bacilli growing in medium containing an alternative carbon source can accumulate cholesterol in the free-lipid zone of their cell walls, and this accumulation affects cell wall permeability.     

Mutations in CNNM4 Cause Recessive Cone-Rod Dystrophy with Amelogenesis Imperfecta

Cone-rod dystrophies are inherited dystrophies of the retina characterized by the accumulation of deposits mainly localized to the cone-rich macular region of the eye. Dystrophy can be limited to the retina or be part of a syndrome. Unlike nonsyndromic cone-rod dystrophies, syndromic cone-rod dystrophies are genetically heterogeneous with mutations in genes encoding structural, cell-adhesion, and transporter proteins. Using a genome-wide single-nucleotide polymorphism (SNP) haplotype analysis to fine map the locus and a gene-candidate approach, we identified homozygous mutations in the ancient conserved domain protein 4 gene (CNNM4) that either generate a truncated protein or occur in highly conserved regions of the protein. Given that CNNM4 is implicated in metal ion transport, cone-rod dystrophy and amelogenesis imperfecta may originate from abnormal ion homeostasis.     

The immunomodulatory sphingosine 1-phosphate analog FTY720 reduces lesion size and improves neurological outcome in a mouse model of cerebral ischemia

Cerebral ischemia is accompanied by fulminant cellular and humoral inflammatory changes in the brain which contribute to lesion development after stroke. A tight interplay between the brain and the peripheral immune system leads to a biphasic immune response to stroke consisting of an early activation of peripheral immune cells with massive production of proinflammatory cytokines followed by a systemic immunosuppression within days of cerebral ischemia that is characterized by massive immune cell loss in spleen and thymus. Recent work has documented the importance of T lymphocytes in the early exacerbation of ischemic injury. The lipid signaling mediator sphingosine 1-phosphate-derived stable analog FTY720 (fingolimod) acts as an immunosuppressant and induces lymphopenia by preventing the egress of lymphocytes, especially T cells, from lymph nodes. We found that treatment with FTY720 (1 mg/kg) reduced lesion size and improved neurological function after experimental stroke in mice, decreased the numbers of infiltrating neutrophils, activated microglia/macrophages in the ischemic lesion and reduced immunohistochemical features of apoptotic cell death in the lesion.     

Identification of conserved modes of expression profiles during hippocampal development and neuronal differentiation in vitro

Gene expression profiles can be regarded as sums of simpler modes, analogous to the modes of a vibrating violin string. Decomposition of temporal gene expression profiles into modes by singular value decomposition (SVD) was reported before, but the question as to what degree the SVD modes can be interpreted in terms of biology remains open. We report and compare the results of SVD of published datasets from hippocampal development, neuronal differentiation in vitro, and a control time-series hippocampal dataset. We demonstrate that the first SVD mode reflects the magnitude of expression, interpretable on the Affymetrix platform. In the datasets from gene profiling of hippocampal development and neuronal differentiation, the second mode reflects a monotonous change in expression, either up- or down-regulation, in the time course of experiment. We demonstrate that the top two SVD modes are conserved between datasets and therefore, likely reflect properties of the underlying system (gene expression in hippocampus) rather than of a particular experiment or dataset. Our results also indicate that the magnitude of expression, and the direction of change in expression during hippocampal development, are uncorrelated, suggesting that they are regulated by largely independent mechanisms.     

Induction of monocyte antitumor response by human cancer cells transduced with TNF-GFP fusion gene: possible implications for immunotherapy of cancer

This study was undertaken to determine how human pancreatic cancer (HPC-4) cells transduced with the TNF-GFP fusion gene (TLG) alter the antitumor response of human monocytes in vitro and whether they could act as an antitumor vaccine. In our model, HPC-4 cells were transduced with retroviral vector harboring TLG gene and designated as HPC-4(TLG). The TLG protein expression was confirmed by Western blot and flow cytometry analysis. Monocytes were co-cultured with transduced and control HPC-4 cells. The secretion of TNF, IL-10 and IL-12 was measured by ELISA. The cytotoxicity of monocytes against HPC-4 cells was determined by MTT test. The results show that the HPC-4(TLG) cells expressed membrane-bound, intracellular and secretory TLG protein. When cultured with HPC-4(TLG) cells, monocytes released a higher amount of TNF, but IL-10 and IL-12 secretion was inhibited. The pre-exposure of monocytes to HPC-4(TLG), but not to HPC-4, cells did not decrease TNF nor increase IL-10 production, thus not leading to monocyte deactivation. Also, the antitumor cytotoxicity of monocytes stimulated with HPC-4(TLG) was not downregulated, which occurred when non-transduced HPC-4 cells were used. In conclusion, compared to parental HPC-4 cells, TLG gene transduced HPC-4 cells induced stronger antitumor response of monocytes in vitro and prevented deactivation of monocytes.     

The prognostic significance of the immunohistochemical expression of P53 and BCL-2 in endometrial cancer

The objective of this study was to verify the frequency of P53 and BCL-2 immunohistochemical expression in 98 patients with endometrial carcinoma, and to correlate it with clinical stage and patient survival. A significant difference was found regarding the frequency of P53 expression when comparing type I and II tumors (23.7% and 54.5%, respectively; p = 0.006). A positive correlation was observed between P53 immunoexpression and patient survival in type I and II tumors (p = 0.009 and p = 0.036, respectively). BCL-2 expression was significantly more frequent in early clinical stages in both types of endometrial cancer (p 〈 0.001 and 0.002) and correlated with a decrease in overall survival in type I endometrial cancer (p = 0.014). Thus, the prognostic value of these biomarkers in endometrial cancer needs to be further investigated.     

Combined Use of Crystalline Sodium Salt and Polymeric Precipitation Inhibitors to Improve Pharmacokinetic Profile of Ibuprofen through Supersaturation

To maximize the pharmacological effect of a pain reliever such as ibuprofen, early onset of action is critical. Unfortunately, the acidic nature of ibuprofen minimizes the amount of drug that can be solubilized under gastric conditions and would be available for immediate absorption upon entry into the intestine. Although the sodium salt of ibuprofen has higher solubility, rapid conversion from the salt to the poorly soluble free acid phase occurs under gastric conditions. Therefore, the combination of the highly soluble sodium salt form of ibuprofen with polymers was evaluated as an approach to prolong supersaturation of ibuprofen during the disproportionation of the salt. Binary combinations of ibuprofen sodium with polymers resulted in the identification of several formulations that demonstrated high degrees and extended durations of supersaturation during in vitro dissolution experiments. These formulations included HPMC, polyvinyl pyrrolidone-vinyl acetate copolymer (PVP-VA64), methylcellulose (MC), and hydroxypropyl cellulose (HPC). The in vitro supersaturation observed with these ibuprofen-polymer formulations translated to an increase in C_max and an earlier T_max for the PVP-VA64, MC, and HPC formulations relative to ibuprofen only controls when administered orally to rats under fasted conditions. Based on these observations, combining ibuprofen sodium with polymers such as PVP-VA64, MC, or HPC is a viable formulation approach to prolong supersaturation in the stomach and enable an optimized pharmacokinetic profile in vivo where rapid onset of action is desired.