Analysis of host responses to Mycobacterium tuberculosis antigens in a multi-site study of subjects with different TB and HIV infection states in sub-Saharan Africa.

BACKGROUND: Tuberculosis (TB) remains a global health threat with 9 million new cases and 1.4 million deaths per year. In order to develop a protective vaccine, we need to define the antigens expressed by Mycobacterium tuberculosis (Mtb), which are relevant to protective immunity in high-endemic areas. METHODS: We analysed responses to 23 Mtb antigens in a total of 1247 subjects with different HIV and TB status across 5 geographically diverse sites in Africa (South Africa, The Gambia, Ethiopia, Malawi and Uganda). We used a 7-day whole blood assay followed by IFN-γ ELISA on the supernatants. Antigens included PPD, ESAT-6 and Ag85B (dominant antigens) together with novel resuscitation-promoting factors (rpf), reactivation proteins, latency (Mtb DosR regulon-encoded) antigens, starvation-induced antigens and secreted antigens. RESULTS: There was variation between sites in responses to the antigens, presumably due to underlying genetic and environmental differences. When results from all sites were combined, HIV- subjects with active TB showed significantly lower responses compared to both TST(-) and TST(+) contacts to latency antigens (Rv0569, Rv1733, Rv1735, Rv1737) and the rpf Rv0867; whilst responses to ESAT-6/CFP-10 fusion protein (EC), PPD, Rv2029, TB10.3, and TB10.4 were significantly higher in TST(+) contacts (LTBI) compared to TB and TST(-) contacts fewer differences were seen in subjects with HIV co-infection, with responses to the mitogen PHA significantly lower in subjects with active TB compared to those with LTBI and no difference with any antigen. CONCLUSIONS: Our multi-site study design for testing novel Mtb antigens revealed promising antigens for future vaccine development. The IFN-γ ELISA is a cheap and useful tool for screening potential antigenicity in subjects with different ethnic backgrounds and across a spectrum of TB and HIV infection states. Analysis of cytokines other than IFN-γ is currently on-going to determine correlates of protection, which may be useful for vaccine efficacy trials

Clinical implications of thymidylate synthetase, dihydropyrimidine dehydrogenase and orotate phosphoribosyl transferase activity levels in colorectal carcinoma following radical resection and administration of adjuvant 5-FU chemotherapy

<p>Abstract</p> <p>Bckground</p> <p>A number of studies have investigated whether the activity levels of enzymes involved in 5-fluorouracil (5-FU) metabolism are prognostic factors for survival in patients with colorectal carcinoma. Most reports have examined thymidylate synthetase (TS) and dihydropyrimidine dehydrogenase (DPD) in unresectable or metastatic cases, therefore it is unclear whether the activity of these enzymes is of prognostic value in colorectal cancer patients treated with radical resection and adjuvant chemotherapy with 5-FU.</p> <p>Methods</p> <p>This study examined fresh frozen specimens of colorectal carcinoma from 40 patients who had undergone curative operation and were orally administered adjuvant tegafur/uracil (UFT) chemotherapy. TS, DPD and orotate phosphoribosyl transferase (OPRT) activities were assayed in cancer tissue and adjacent normal tissue and their association with clinicopathological variables was investigated. In addition, the relationships between TS, DPD and OPRT activities and patient survival were examined to determine whether any of these enzymes could be useful prognostic factors.</p> <p>Results</p> <p>While there was no clear relationship between pathological findings and TS or DPD activity, OPRT activity was significantly lower in tumors with lymph node metastasis than in tumors lacking lymph node metastasis. Postoperative survival was significantly better in the groups with low TS activity and/or high OPRT activity.</p> <p>Conclusion</p> <p>TS and OPRT activity levels in tumor tissue may be important prognostic factors for survival in Dukes' B and C colorectal carcinoma with radical resection and adjuvant chemotherapy with UFT.</p

Homeostatic and pathogenic roles of GM3 ganglioside molecular species in TLR4 signaling in obesity

Innate immune signaling via TLR4 plays critical roles in pathogenesis of metabolic disorders, but the contribution of different lipid species to metabolic disorders and inflammatory diseases is less clear. GM3 ganglioside in human serum is composed of a variety of fatty acids, including long-chain (LCFA) and very-long-chain (VLCFA). Analysis of&nbsp;circulating levels of human serum GM3 species from patients at different stages of insulin resistance and chronic inflammation&nbsp;reveals that levels of VLCFA-GM3 increase significantly in metabolic disorders, while LCFA-GM3 serum levels decrease. Specific GM3 species also correlates with disease symptoms. VLCFA-GM3 levels increase in the adipose tissue of obese mice, and this is blocked in TLR4-mutant mice. In cultured monocytes, GM3 by itself has no effect on TLR4 activation; however, VLCFA-GM3 synergistically and selectively enhances TLR4 activation by LPS/HMGB1, while LCFA-GM3 and unsaturated VLCFA-GM3 suppresses TLR4 activation. GM3 interacts with the extracellular region of TLR4/MD2 complex to modulate dimerization/oligomerization. Ligand-molecular docking analysis supports that VLCFA-GM3 and LCFA-GM3 act as agonist and antagonist of TLR4 activity, respectively, by differentially binding to the hydrophobic pocket of MD2. Our findings suggest that VLCFA-GM3 is a risk factor for TLR4-mediated disease progression

Diversity of sympathetic vasoconstrictor pathways and their plasticity after spinal cord injury

Sympathetic vasoconstrictor pathways pass through paravertebral ganglia carrying ongoing and reflex activity arising within the central nervous system to their vascular targets. The pattern of reflex activity is selective for particular vascular beds and appropriate for the physiological outcome (vasoconstriction or vasodilation). The preganglionic signals are distributed to most postganglionic neurones in ganglia via synapses that are always suprathreshold for action potential initiation (like skeletal neuromuscular junctions). Most postganglionic neurones receive only one of these “strong” inputs, other preganglionic connections being ineffective. Pre- and postganglionic neurones discharge normally at frequencies of 0.5–1 Hz and maximally in short bursts at <10 Hz. Animal experiments have revealed unexpected changes in these pathways following spinal cord injury. (1) After destruction of preganglionic neurones or axons, surviving terminals in ganglia sprout and rapidly re-establish strong connections, probably even to inappropriate postganglionic neurones. This could explain aberrant reflexes after spinal cord injury. (2) Cutaneous (tail) and splanchnic (mesenteric) arteries taken from below a spinal transection show dramatically enhanced responses in vitro to norepinephrine released from perivascular nerves. However the mechanisms that are modified differ between the two vessels, being mostly postjunctional in the tail artery and mostly prejunctional in the mesenteric artery. The changes are mimicked when postganglionic neurones are silenced by removal of their preganglionic input. Whether or not other arteries are also hyperresponsive to reflex activation, these observations suggest that the greatest contribution to raised peripheral resistance in autonomic dysreflexia follows the modifications of neurovascular transmission

Phase I/II study of S-1 combined with irinotecan for metastatic advanced gastric cancer

A dose-escalation study of irinotecan (CPT-11) combined with S-1, an oral dihydropyrimidine dehydrogenase inhibitory fluoropyrimidine, was performed to determine the maximum-tolerated dose (MTD), recommended dose (RD), dose-limiting toxicities (DLTs), and objective response rate (RR) in advanced gastric cancer (AGC). S-1 was administered orally at 80 mg m−2 day−1 from day 1 to 14 of a 28-day cycle and CPT-11 was given intravenously on day 1 and 8 at an initial dose of 70 mg m−2 day−1, stepping up to 100 mg m−2. The treatment was repeated every 4 weeks, unless disease progression was observed. In the phase I portion, the MTD of CPT-11 was presumed to be 100 mg m−2, because 66.6% of patients (two of three) developed DLTs. All three patients at the initial RD of CPT-11 (90 mg m−2) experienced grade 4 haematological or grade 3 nonhaematological toxicities at second course, followed by the dose reduction of CPT-11 from the third course. Considering safety and the ability to continue treatment, the final RD was determined to be 80 mg m−2. In the phase II portion, 42 patients including seven patients in the final RD phase I portion were evaluated. The median treatment course was five (range: 1–13). The incidences of severe (grade 3–4) haematological and nonhaematological toxicities were 19 and 10%, respectively, but all were manageable. The RR was 62% (26 of 42, 95% confidence interval: 47.2–76.6%), and the median survival time was 444 days. Our phase I/II trial showed S-1 combined with CPT-11 is effective for AGC and is well tolerated, with acceptable toxicity

Multimarker RT–PCR assay for the detection of minimal residual disease in sentinel lymph nodes of breast cancer patients

The presence of metastases in lymph nodes is the most powerful prognostic factor in breast cancer patients. Routine histological examination of lymph nodes has limited sensitivity for the detection of breast cancer metastases. The aim of the present study was to develop a multimarker reverse transcriptase–polymerase chain reaction (RT—PCR) assay for the detection of minimal residual disease in sentinel nodes of breast cancer patients. RNA was extracted from 30 sentinel lymph nodes (SLN) obtained from 28 patients, three primary breast cancers (positive controls), three lymph nodes from patients with benign diseases, and peripheral blood lymphocytes of 10 healthy volunteers (negative controls). RT–PCR was performed using the following markers; cytokeratin (CK)-19, NY-BR-1 and mammaglobin B. RT–PCR results were compared to enhanced histopathologic examination and immunohistochemistry (IHC). All three positive controls showed strong PCR amplification for all three markers. None of the 13 negative controls was amplified by any of the three markers. Among the 30 SLN analysed, breast cancer metastases were detected in six SLNs by routine histology, in eight by IHC and in 15 by RT–PCR. We conclude that a multimarker RT–PCR assay probing for NY-BR-1, mammaglobin-B, and CK-19 is more sensitive compared to enhanced pathologic examination. This method may prove to be of value in breast cancer staging and prognosis evaluation

Linking Changes in Epithelial Morphogenesis to Cancer Mutations Using Computational Modeling

Most tumors arise from epithelial tissues, such as mammary glands and lobules, and their initiation is associated with the disruption of a finely defined epithelial architecture. Progression from intraductal to invasive tumors is related to genetic mutations that occur at a subcellular level but manifest themselves as functional and morphological changes at the cellular and tissue scales, respectively. Elevated proliferation and loss of epithelial polarization are the two most noticeable changes in cell phenotypes during this process. As a result, many three-dimensional cultures of tumorigenic clones show highly aberrant morphologies when compared to regular epithelial monolayers enclosing the hollow lumen (acini). In order to shed light on phenotypic changes associated with tumor cells, we applied the bio-mechanical IBCell model of normal epithelial morphogenesis quantitatively matched to data acquired from the non-tumorigenic human mammary cell line, MCF10A. We then used a high-throughput simulation study to reveal how modifications in model parameters influence changes in the simulated architecture. Three parameters have been considered in our study, which define cell sensitivity to proliferative, apoptotic and cell-ECM adhesive cues. By mapping experimental morphologies of four MCF10A-derived cell lines carrying different oncogenic mutations onto the model parameter space, we identified changes in cellular processes potentially underlying structural modifications of these mutants. As a case study, we focused on MCF10A cells expressing an oncogenic mutant HER2-YVMA to quantitatively assess changes in cell doubling time, cell apoptotic rate, and cell sensitivity to ECM accumulation when compared to the parental non-tumorigenic cell line. By mapping in vitro mutant morphologies onto in silico ones we have generated a means of linking the morphological and molecular scales via computational modeling. Thus, IBCell in combination with 3D acini cultures can form a computational/experimental platform for suggesting the relationship between the histopathology of neoplastic lesions and their underlying molecular defects

Long-term retinal PEDF overexpression prevents neovascularization in a murine adult model of retinopathy

Neovascularization associated with diabetic retinopathy (DR) and other ocular disorders is a leading cause of visual impairment and adult-onset blindness. Currently available treatments are merely palliative and offer temporary solutions. Here, we tested the efficacy of antiangiogenic gene transfer in an animal model that mimics the chronic progression of human DR. Adeno-associated viral (AAV) vectors of serotype 2 coding for antiangiogenic Pigment Epithelium Derived Factor (PEDF) were injected in the vitreous of a 1.5 month-old transgenic model of retinopathy that develops progressive neovascularization. A single intravitreal injection led to long-term production of PEDF and to a striking inhibition of intravitreal neovascularization, normalization of retinal capillary density, and prevention of retinal detachment. This was parallel to a reduction in the intraocular levels of Vascular Endothelial Growth Factor (VEGF). Normalization of VEGF was consistent with a downregulation of downstream effectors of angiogenesis, such as the activity of Matrix Metalloproteinases (MMP) 2 and 9 and the content of Connective Tissue Growth Factor (CTGF). These results demonstrate long-term efficacy of AAV-mediated PEDF overexpression in counteracting retinal neovascularization in a relevant animal model, and provides evidence towards the use of this strategy to treat angiogenesis in DR and other chronic proliferative retinal disorders