Connexins are expressed in primary brain tumors and enhance the bystander effect in gene therapy

OBJECTIVE: Experimental brain tumor gene therapy with the herpes simplex virus thymidine kinase (HSV-tk) gene has demonstrated that not only HSV-tk transduced but surrounding non-HSV-tk transduced cells are killed when given ganciclovir. This so-called bystander effect has recently been shown to be dependent on connexin-mediated intercellular communication. To assess potential susceptibility to the bystander effect, we examined levels of connexin-26 and connexin-43 expression in a series of primary brain tumors. Connexin-26 expression has not previously been studied in primary brain tumors and connexin-43 expression has not been studied in nonastrocytic primary brain tumors. We also attempted to enhance the bystander effect in vitro by overexpressing connexin in tumor cells with high basal levels of connexin expression. METHODS: Western blot analysis and immunohistochemistry were used to determine levels of connexin-26 and connexin-43 expression in a series of primary brain tumors. Wild-type 9L gliosarcoma cells were transfected in vitro with the connexin-43 gene and the HSV-tk gene or the HSV-tk gene alone. The bystander effect of each transfectant was then assessed and compared. RESULTS: Most of the primary brain tumors tested, including low-grade astrocytomas, anaplastic astrocytomas, glioblastomas, oligodendrogliomas, gangliogliomas, meningiomas, and medulloblastomas, showed connexin-26 and connexin-43 expression. Bystander experiments revealed a significant enhancement of the bystander effect in the gliosarcoma cells transfected with connexin-43 and HSV-tk, as compared with gliosarcoma cells transfected with HSV-tk alone. CONCLUSION: Most primary brain tumors express connexin-26 and connexin-43. This suggests that most primary brain tumors may be susceptible to the bystander effect of HSV-tk gene therapy. The bystander effect can be enhanced in vitro by overexpression of connexin-43 in a cell line with a high basal level of connexin-43 expression.     

Experimental hepatobiliary fascioliasis in rabbits: a radiology-pathology correlation

RATIONALE AND OBJECTIVES: The authors sought to correlate the radiologic findings of hepatobiliary fascioliasis with pathologic features. METHODS: Serial ultrasound, computed tomography (CT), and magnetic resonance findings in seven rabbits with experimentally induced fascioliasis were obtained every other week. Direct cholangiogram was also obtained after the rabbits were killed. Radiology-pathology correlation was done in specimens. RESULTS: In the parenchymal phase (an acute phase of parenchymal invasion of a larva), CT showed subcapsular clustered areas of low attenuation. Magnetic resonance appearance was similar in shape but better than CT in characterizing the hemorrhagic nature of the lesion. Ultrasound findings were nonspecific in this phase. In the ductal phase (a stationary phase after residing in the bile duct), CT showed dilatation of central ducts with symmetric periportal hypoattenuation (periportal tracking). Magnetic resonance could not depict mild ductal dilatation. Ultrasound was most valuable in demonstrating the moving worm within the dilated duct. Pathologically, the hepatic parenchymal lesions consisted of a cluster of eosinophilic granulomas with hemorrhagic change (migratory tract of the flukes). Ductal changes were observed predominantly in the central bile ducts. Periportal lymphangiectasia was also noted. CONCLUSIONS: Computed tomography or magnetic resonance can demonstrate the characteristic evolutionary pattern of fascioliasis that reflects the unique life cycle of Fasciola hepatica. The role of ultrasound, although limited in the parenchymal phase, was most useful in the ductal phase in that it demonstrated the moving worms themselves.     

An unconventional role for cytoplasmic disulfide bonds in vaccinia virus proteins

Previous data have shown that reducing agents disrupt the structure of vaccinia virus (vv). Here, we have analyzed the disulfide bonding of vv proteins in detail. In vv-infected cells cytoplasmically synthesized vv core proteins became disulfide bonded in the newly assembled intracellular mature viruses (IMVs). vv membrane proteins also assembled disulfide bonds, but independent of IMV formation and to a large extent on their cytoplasmic domains. If disulfide bonding was prevented, virus assembly was only partially impaired as shown by electron microscopy as well as a biochemical assay of IMV formation. Under these conditions, however, the membranes around the isolated particles appeared less stable and detached from the underlying core. During the viral infection process the membrane proteins remained disulfide bonded, whereas the core proteins were reduced, concomitant with delivery of the cores into the cytoplasm. Our data show that vv has evolved an unique system for the assembly of cytoplasmic disulfide bonds that are localized both on the exterior and interior parts of the IMV.     

APIC position paper: hepatitis C exposure in the health care setting. Association for Professionals in Infection Control and Epidemiology, Inc

The Association for Professionals in Infection Control and Epidemiology, Inc (APIC), is a multidisciplinary, voluntary, international organization of professionals who practice infection control and the application of epidemiology in all health settings. APIC is an international leader in prevention and control of infection transmission.     

Simultaneous monitoring of cell number and metabolic activity of specific bacterial populations with a dual gfp-luxAB marker system

A dual marker system was developed for simultaneous quantification of bacterial cell numbers and their activity with the luxAB and gfp genes, encoding bacterial luciferase and green fluorescent protein (GFP), respectively. The bioluminescence phenotype of the luxAB biomarker is dependent on cellular energy status. Since cellular metabolism requires energy, bioluminescence output is directly related to the metabolic activity of the cells. By contrast, GFP fluorescence has no energy requirement. Therefore, by combining these two biomarkers, total cell number and metabolic activity of a specific marked cell population could be monitored simultaneously. Two different bacterial strains, Escherichia coli DH5alpha and Pseudomonas fluorescens SBW25, were chromosomally tagged with the dual marker cassette, and the cells were monitored under different conditions by flow cytometry, plate counting, and luminometry. During log-phase growth, the luciferase activity was proportional to the number of GFP-fluorescent cells and culturable cells. Upon entrance into stationary phase or during starvation, luciferase activity decreased due to a decrease in cellular metabolic activity of the population, but the number of GFP-fluorescing cells and culturable cells remained relatively stable. In addition, we optimized a procedure for extraction of bacterial cells from soil, allowing GFP-tagged bacteria in soil samples to be quantitated by flow cytometry. After 30 days of incubation of P. fluorescens SBW25::gfp/lux in soil, the cells were still maintained at high population densities, as determined by GFP fluorescence, but there was a slow decline in luciferase activity, implicating nutrient limitation. In conclusion, the dual marker system allowed simultaneous monitoring of the metabolic activity and cell number of a specific bacterial population and is a promising tool for monitoring of specific bacteria in situ in environmental samples.     

Synthesis and biological properties of novel pyridinioalkanoyl thiolesters (PATE) as anti-HIV-1 agents that target the viral nucleocapsid protein zinc fingers

Nucleocapsid p7 protein (NCp7) zinc finger domains of the human immunodeficiency virus type 1 (HIV-1) are being developed as antiviral targets due to their key roles in viral replication and their mutationally nonpermissive nature. On the basis of our experience with symmetrical disulfide benzamides (DIBAs; Rice et al. Science 1995, 270, 1194-1197), we synthesized and evaluated variants of these dimers, including sets of 4,4'- and 3,3'-disubstituted diphenyl sulfones and their monomeric benzisothiazolone derivatives (BITA). BITAs generally exhibited diminished antiviral potency when compared to their disulfide precursors. Novel, monomeric structures were created by linking haloalkanoyl groups to the benzamide ring through -NH-C(=O)- (amide) or -S-C(=O)- (thiolester) bridges. Amide-linked compounds generally lacked antiviral activity, while haloalkanoyl thiolesters and non-halogen-bearing analogues frequently exhibited acceptable antiviral potency, thus establishing thiolester benzamides per se as a new anti-HIV chemotype. Pyridinioalkanoyl thiolesters (PATEs) exhibited superior anti-HIV-1 activity with minimal cellular toxicity and appreciable water solubility. PATEs were shown to preferentially target the NCp7 Zn finger when tested against other molecular targets, thus identifying thiolester benzamides, and PATEs in particular, as novel NCp7 Zn finger inhibitors for in vivo studies.     

Repeated neonatal deaths in families with special reference to causes of death

It is recognized that one infant death in a family indicates an increased risk of death of a subsequent sibling. This study examines which cause of death of a sibling is related to the mortality of the younger sibling and when. Longitudinal vital events data from the maternal and child health and family planning (MCH-FP) project and the comparison areas in Matlab, Bangladesh, were used. Primary causes of 868 neonatal deaths and 624 post-neonatal deaths resulting from 18,865 singleton live births in 1989-92 and those (967 as neonates and 708 as post-neonates) of their immediate elder siblings were categorised into infectious and non-infectious diseases. Multinomial logistic regression was used to estimate the risk of younger siblings dying in each age period from infectious and non-infectious diseases given the age and cause of deaths of older siblings and controlling for other biosocial correlates of infant mortality. A neonatal death of non-infectious causes in a family was twice as likely to be followed by another one occurring at the same age from similar causes compared with a surviving infant followed by a neonatal death from non-infectious causes. The MCH-FP project, though successful in reducing the risk of neonatal and post-neonatal mortality from infectious diseases, did not reduce the risk of dying from non-infectious diseases.