Herpes simplex virus vector-mediated expression of Bcl-2 protects spinal motor neurons from degeneration following root avulsion

Proximal axotomy in adult animals results in delayed death of motor neurons. Features characteristic of both necrosis and apoptosis have been described in motor neurons of the spinal cord following proximal avulsion of the ventral roots. We have previously demonstrated that a genomic herpes simplex virus (HSV)-based vector expressing the anti-apoptotic peptide Bcl-2 protects dopaminergic neurons of the substantia nigra from neurotoxin-induced apoptotic cell death and preserves the neurotransmitter phenotype of those cells. In this study we examined whether the same vector could protect adult rat lumbar motor neurons from cell death following proximal ventral root avulsion. Injection of the Bcl-2-expressing vector 1 week prior to root avulsion increased the survival of lesioned motor neurons, determined by retrograde Fluorogold labeling, by 50%. The Bcl-2-expressing vector did not preserve choline acetyltransferase neurotransmitter phenotype of the lesioned cells. These results shed light on the mechanism of cell death following axonal injury, and have implications for developing an effective treatment for the clinical problem of proximal root avulsion.     

Herpes simplex virus mediated nerve growth factor expression in bladder and afferent neurons: potential treatment for diabetic bladder dysfunction

PURPOSE: Diabetic cystopathy resulting from sensory neuropathy may potentially be treated by direct gene therapy. It has been suggested that nerve growth factor (NGF) has an ameliorative effect in preventing the death in diabetes of afferent dorsal root ganglion neurons, which control bladder function. We investigated NGF gene transfer to the bladder and bladder afferent pathways for treating diabetic cystopathy. We used replication competent and replication defective herpes simplex virus type 1 (HSV-1) vectors that express a functionally active form of the beta-subunit of mouse NGF (beta-NGF) to examine the level and duration of therapeutic gene expression after administration of the vectors. MATERIALS AND METHODS: NGF expression during acute (3 days) and latent (21 days) infections was assessed by enzyme-linked immunosorbent assay (ELISA) and immunohistochemical testing after the injection of 1 x 106 to 1 x 108 pfu HSV-NGF expression vectors into the bladder wall of adult rats. RESULTS: HSV vectors with the strong human cytomegalovirus immediate early promoter used to drive beta-NGF gene expression exhibited increased NGF 3 days after infection in the bladder and L6 to S1 dorsal root ganglia, where bladder afferent neurons are located. ELISA analysis revealed that NGF in the bladder tissue and dorsal root ganglia was increased 7 to 9 and 2 to 4-fold, respectively, over the control vector. Increased NGF expression in L6 to S1 dorsal root ganglia neurons was also detected by immunohistochemical staining with antiNGF antibodies. Extended NGF expression was detected by ELISA 21 days after injection. Replication defective vectors containing HSV-1 latency promoter (LAP-2) driving NGF expressed NGF in the bladder and dorsal root ganglia 21 days after bladder injection. ELISA analysis confirmed an approximate 2 to 3-fold increase of NGF expression in the bladder and L6 to S1 dorsal root ganglia. CONCLUSIONS: The NGF gene may be transferred and expressed in the bladder and bladder afferent pathways using HSV vectors. To our knowledge our study represents the first demonstration of the effectiveness of gene therapy for altering neurotrophic expression in visceral sensory neurons. This technique of gene transfer may be useful for treating certain types of neurogenic bladder dysfunction, such as diabetic cystopathy, in which decreased NGF transport may be a causative factor.     

Cytogenetic and molecular genetic analysis of tumorigenic human bronchial epithelial cells induced by radon alpha particles

To establish a cell culture model for lung carcinogenesis, independent populations of the human papillomavirus 18-immortalized human bronchial epithelial cell line BEP2D were treated with high linear energy transfer radon-simulated alpha-particles, expanded and xenotransplanted into Nu/Nu mice. Six independent cell lines were established from tumors that developed from three separate radiation treatments as follows: treatment (Tx) 1 (30 cGy--two doses), H2BT, Tx 2 (30 cGy-- single dose), R30T1L, R30T2 and R30T3L, Tx 3 (30 cGy--single dose), H1ATN and H1ATBA1. Cytogenetic analysis revealed common changes in all tumor lines: loss of the Y chromosome (ch), one of three copies of ch8, one of three copies of ch14, and one of two copies of ch4p16-pter and ch11p15-pter. Analysis of polymerase chain reaction-amplified short tandem repeats of informative loci confirmed the loss of chY in all lines and loss of heterozygosity (LOH) at eight loci spanning the length of ch8 in all lines from Tx's 1 and 2. Our data support previous studies indicating the presence of tumor suppressor genes on ch8. LOH also was confirmed on ch14 at locus D14S306 in all cell lines from Tx 2 and in one of two lines from Tx 3. This region, 14q12-q13, may contain changes in one of the five known somatostatin receptor genes (SSTR1). No LOH was detected at any of the informative loci tested for on ch4 or ch11.      

Mycobacterial infection in an inner city children's hospital

Childhood tuberculosis is perceived by many as a disease of the past. Experience in a children's hospital serving a deprived population suggested that tuberculosis and other mycobacterial infections were not declining in clinical practice. Fifty three tuberculous and 11 atypical mycobacterial infections were identified between 1978 and 1992. There was no decline in tuberculosis and nine of the 11 atypical infections occurred in the last five years. Altogether 40% of cases of tuberculosis were in non-Asian children; 32% had arrived in the UK or visited family overseas in the previous year; and 38% had a history of tuberculosis contact, usually a close adult relative. Nationally, the previous decline in tuberculosis in all ages has reversed. In the local health districts in London's east end, childhood tuberculosis has also stopped declining and seems to be increasing. It is regrettable that BCG vaccination has been abolished by some districts in the UK, against current recommendations. Childhood tuberculosis is still common in the practice described here, including among children who do not fall into conventionally recognised high risk groups. Inner city dwellers and junior doctors are both highly mobile populations, adding to the risk that paediatricians, particularly those in training, may encounter tuberculosis with little or no previous experience of the condition.     

MR imaging of blood vessels using three-dimensional reconstruction: methodology

Multisection, dual-echo magnetic resonance (MR) transaxial images of blood vessels contain both anatomic and qualitative information about flow. Even so, the images are produced as a series of two-dimensional tomographic sections from which full visualization of connected structures is difficult. A computer algorithm was developed that automatically detects flowing blood based on pixel intensity and calculated T2 and provides reconstructed views of vessels while analyzing and displaying flow characteristics. Images of abdominal vessels, aortic aneurysms, and the heart were encoded by flow and color to demonstrate depth. In addition, these data were reconstructed to derive a more accurate assessment of patency. With this technique, transaxial images can be used to analyze flow patterns, determine patent areas, and visualize all levels of vessels in a single image.     

Role of HSD11B2 polymorphisms in essential hypertension and the diuretic response to thiazides

BACKGROUND: The renal 11beta-hydroxysteroid dehydrogenase type 2 (11beta HSD2) enzyme inactivates 11-hydroxy steroids in the kidney, thereby protecting the nonselective mineralocorticoid (MR) receptor from occupation by glucocorticoids. Loss-of-function mutations in the gene encoding 11beta HSD2 (HSD11B2) result in overstimulation of the MR and cause salt-sensitive hypertension. METHODS: We have investigated the role of HSD11B2 in hypertension in 377 genetically homogeneous essential hypertensives from North Sardinia. RESULTS: Thirty of these patients displayed increased urinary cortisol metabolite ratios (greater than or equal to 2) (tetrahydrocortisol [THF]+allotetrahydrocortisol [aTHF]/tetrahydrocortisone [THE]) reflecting a mild reduction in 11beta HSD2 activity. No mutations in HSD11B2 were detected in these patients. All 377 patients were genotyped for a CA repeat microsatellite in intron 1 of HSD11B2 and a G534A polymorphism in exon 3 of HSD11B2. CA repeat length was associated with the (THF+aTHF)/THE ratio, which in turn was significantly related to PRA levels. No associations were found between the G354A polymorphism and the other parameters. There were no differences in blood pressure (BP) levels between HSD11B2 genotypes, but in a subgroup of 91 patients that underwent diuretic therapy, CA repeat length was strongly associated with the BP response to hydrochlorothiazide. CONCLUSION: This study highlights the role of this HSD11B2 polymorphism in sodium handling and is consistent with a role in the BP response to thiazide diuretics.     

In vitro thymidine kinase/ganciclovir-based suicide gene therapy using replication defective herpes simplex virus-1 against leukemic B-cell malignancies (MCL,HCL, B-CLL)

A replication defective herpes simplex virus-1 was evaluated as a therapeutic vector. Mantle cell lymphoma (MCL), hairy cell leukemia (HCL), and B-cell chronic lymphocytic leukemia (B-CLL) were chosen because leukemic cells were collectable from peripheral bloods in these diseases. Cells from six MCL, one HCL, and nine B-CLL were infected in vitro with T0Z.1 at 3 multiplicity of infection (MOI). Herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV)-mediated suicide gene therapy showed 14.7% of mean tumor killing against leukemic B-cell malignancies. The mean tumor-killing effects were 8.7 and 17.1% in MCL and B-CLL, respectively. The effect against HCL was 29%. The study indicates that herpes simplex virus (HSV)-based gene therapy might be an effective strategy.     

Protective effect of HSV-mediated gene transfer of nerve growth factor in pyridoxine neuropathy demonstrates functional activity of trkA receptors in large sensory neurons of adult animals

The distinct distribution of trkA receptors on small neurons and trkC receptors on large neurons in the dorsal root ganglion correlates with the dependence of these two classes of neurons on nerve growth factor and neurotrophin-3, respectively, for survival during development. In adult animals, the distribution of high affinity neurotrophin (trk) is complex and overlapping; neurotrophins are not required for cell survival, but may influence cell phenotype and the response to injury. In order to test the functional activity of trkA receptors in the sensory ganglia of adult animals in vivo, we examined the ability of a nerve growth factor-expressing recombinant replication-defective herpes simplex virus-based vector to prevent the selective degeneration of large sensory fibres caused by intoxication with pyridoxine. Transduction of dorsal root ganglion neurons in vivo by subcutaneous inoculation of the nerve growth factor-expressing vector prevented the development of pyridoxine-induced neuropathy measured by electrophysiological, morphological and behavioural measures. These results demonstrate a functional activity of trkA receptors expressed on large neurons in the dorsal root ganglion in mature animals; this observation has important implications for the choice of neurotrophic factors for treatment of peripheral nerve disease.     

Eating in larger groups increases food consumption

OBJECTIVE: To determine whether children's food consumption is increased by the size of the group of children in which they are eating. DESIGN: Crossover study. SETTING: University based preschool. PARTICIPANTS: 54 children, aged 2.5-6.5 years. INTERVENTIONS: Each child ate a standardised snack in a group of three children, and in a group of nine children. MAIN OUTCOME MEASURES: Amount each individual child consumed, in grams. RESULTS: Amount eaten and snack duration were correlated (r = 0.71). The association between group size and amount eaten differed in the short (<11.4 min) versus the long (> or =11.4 min) snacks (p = 0.02 for the interaction between group size and snack duration). During short snacks, there was no effect of group size on amount eaten (16.7 (SD 11) g eaten in small groups vs 15.1 (6.6) g eaten in large groups, p = 0.42). During long snacks, large group size increased the amount eaten (34.5 (16) vs 26.5 (13.8), p = 0.02). The group size effect was partially explained by a shorter latency to begin eating, a faster eating rate and reduced social interaction in larger groups. CONCLUSIONS: Children consumed 30% more food when eating in a group of nine children than when eating in a group of three children during longer snacks. Social facilitation of food consumption operates in preschool-aged children. The group size effect merits consideration in creating eating behaviour interventions.