Effect of herpes simplex virus vector-mediated interleukin-4 gene therapy on bladder overactivity and nociception.

We investigated the effects of replication-defective herpes simplex virus (HSV) vector expression of interleukin-4 (IL-4) on bladder overactivity and nociception. HSV vector expressing murine interleukin-4 (S4IL4) or the control vector expressing ß-galactosidase (SHZ) were injected to the rat bladder wall. At 1 week after viral injection, in cystometry performed under urethane anesthesia, the S4IL4-treated group did not show the intercontraction intervals reduction during intravesical administration of 10 nM resiniferatoxin (RTx). At 2 weeks after viral injection, behavioral studies were performed on vector-injected animals in an awakened state. Freezing behavior induced by 3 µM RTx, administered for 1 min into the bladder, was significantly suppressed in the S4IL4 group compared with the SHZ group. Murine IL-4 levels examined by ELISA were significantly increased in bladder and bladder afferent dorsal root ganglia at 2 weeks after viral injection. The expression of IL-1ß and IL-2 and bladder inflammatory responses were significantly suppressed in the RTx-irritated bladder of S4IL4-injected rats. These results indicate that HSV vector-mediated interleukin-4 expression in the bladder and bladder afferent pathways reduces the inflammatory response, bladder overactivity and nociceptive behavior induced by bladder irritation in the rat model. Therefore, IL-4 gene therapy could be a new strategy for treating urinary frequency and/or bladder pain.

HSV vector-mediated modification of primary nociceptor afferents: an approach to inhibit chronic pain.

Chronic pain is a serious medical condition with millions of sufferers for whom long-term therapies are either lacking or inadequate. Here we review the use of herpes simplex virus vectors as therapeutic tools to treat chronic pain by gene therapy. We describe an approach to inhibit chronic pain signaling whereby vector-mediated genes transferred to sensory nerves will modify the primary afferent nociceptor to prevent pain signaling to second-order nerves in the spinal cord. This approach may be used to reverse the chronic pain state of the nociceptor and could affect downstream pain-related changes in the central nervous system.

Herpes simplex virus vector-mediated gene delivery for the treatment of lower urinary tract pain.

Interstitial cystitis (IC)/painful bladder syndrome (PBS) is a painful debilitating chronic visceral pain disorder of unknown etiology that affects an estimated 1 million people in the United States alone. It is characterized by inflammation of the bladder that results in chronic pelvic pain associated with bladder symptoms of urinary frequency and urgency. Regardless of the etiology, IC/PBS involves either increased and/or abnormal activity in afferent nociceptive sensory neurons. Pain-related symptoms in patients with IC/PBS are often very difficult to treat. Both medical and surgical therapies have had limited clinical utility in this debilitating disease and numerous drug treatments, such as heparin, dimethylsulfoxide and amitriptyline, have proven to be palliative at best, and in some IC/PBS patients provide no relief whatsoever. Although opiate narcotics have been employed to help alleviate IC/PBS pain, this strategy is fraught with problems as systemic narcotic administration causes multiple unwanted side effects including mental status change and constipation. Moreover, chronic systemic narcotic use leads to dependency and need for dose escalation due to tolerance; therefore, new therapies are desperately needed to treat refractory IC/PBS. This has led our group to develop a gene therapy strategy that could potentially alleviate chronic pelvic pain using the herpes simplex virus-directed delivery of analgesic proteins to the bladder.

Herpes simplex virus vector-mediated gene delivery of glutamic acid decarboxylase reduces detrusor overactivity in spinal cord-injured rats.

We examined whether replication-defective herpes simplex virus (HSV) vectors encoding the 67 kDa form of the glutamic acid decarboxylase (GAD(67)) gene product, the gamma-aminobutyric acid (GABA) synthesis enzyme, can suppress detrusor overactivity (DO) in rats with spinal cord injury (SCI). One week after spinalization, HSV vectors expressing GAD and green fluorescent protein (GFP) (HSV-GAD) were injected into the bladder wall. Rats with SCI without HSV injection (HSV-untreated) and those injected with lacZ-encoding reporter gene HSV vectors (HSV-LacZ) were used as controls. Three weeks after viral injection, continuous cystometry was performed under awake conditions in all three groups. In the HSV-GAD group, the number and amplitude of non-voiding contractions (NVCs) were significantly decreased (40-45% and 38-40%, respectively) along with an increase in voiding efficiency, compared with HSV-untreated and HSV-LacZ groups, but micturition pressure was not different among the three groups. Intrathecal application of bicuculline partly reversed the decreased number and amplitude of NVCs, and decreased voiding efficiency in the HSV-GAD group. In the HSV-GAD group, GAD(67) mRNA and protein levels were significantly increased in the L6-S1 dorsal root ganglia (DRG) compared with the HSV-LacZ group, while 57% of DRG cells were GFP-positive, and these neurons showed increased GAD(67)-like immunoreactivity compared with the HSV-LacZ group. These results indicate that GAD gene therapy effectively suppresses DO after SCI predominantly through the activation of spinal GABA(A) receptors. Thus, HSV-based GAD gene transfer to bladder afferent pathways may represent a novel approach for treatment of neurogenic DO.

Age-related changes in glial fibrillary acidic protein mRNA in the mouse brain.

Several RNA sequences were tested for age-related changes in prevalence levels in the mouse cerebral cortex, hippocampus, and cerebellum. In all three regions, there were increased levels of RNA for glial fibrillary acidic protein, an astrocyte-specific protein, by RNA gel-blot analysis and by a solution hybridization assay. There was no change in glutamine synthetase mRNA level, another glial protein. The only other mRNA sequence which changed was Thy-1 antigen, a neuronal protein, which decreased slightly in the hippocampus. We conclude that with age there is an age-related increase in glial fibrillary acidic protein RNA prevalence potentially reflecting an increase in the size, number, and/or fibrous character of astrocytes.

Increases of glial fibrillary acidic protein in the aging female mouse brain.

Age-related increases of the astrocyte marker, glial fibrillary acidic protein (GFAP), were further resolved by in situ hybridization and immunocytochemistry in female C57BL/6J mice. The age groups represented the major stages of reproductive aging: young (5 months), middle-age (18 months), and old (23 and 26 months). GFAP mRNA and protein showed generalized increases in old mice. Major white fiber tracts, such as the corpus callosum, fimbria, stria terminalis, and optic tract, showed increased GFAP immunostaining and mRNA. Gray matter showed robust > or = twofold increases in GFAP mRNA with age, especially in the thalamus and hypothalamus, areas that expressed little GFAP in the young. These generalized age-related increases of GFAP in many brain regions imply the existence of a widespread stimulus for increased activity of astrocytes during aging.

Genomic organization and precise physical location of protein phosphatase 2A regulatory subunit A beta isoform gene on chromosome band 11q23.

Protein phosphatase 2A (PP2A) holoenzyme plays a critical role in cell-cycle control and growth-factor signaling, and is implicated in tumorigenesis. Because the protein phosphatase 2 regulatory subunit A beta isoform gene (PPP2R1B) maps within the critical region of hereditary paraganglioma (PGL1) on chromosomal band 11q23, we characterized its genomic structure and evaluated it as a candidate gene for PGL1. PPP2R1B has 15 exons spanning approx. 27kb genomic distance. We placed the exons on genomic EcoRI fragments and identified their flanking intronic sequences. The gene was oriented from telomere to centromere. Splice acceptor and donor sites of all introns conformed to the GT/AG rule. Northern analysis with a cDNA probe identified 2.5kb and 5.0kb transcript sizes. We identified an ATG initiation codon in a favorable context and mapped two transcription start sites 15bp and 66bp upstream of it. We also mapped a 3'-polyadenylation site 504bp downstream of the TGA stop codon, consistent with the 2.5kb transcript size. We did not detect germ-line mutations by single-stranded conformational polymorphism (SSCP) analysis or major rearrangements by Southern analysis in a set of PGL1 patients. In conclusion, we precisely mapped and characterized the structure of PPP2R1B and evaluated it as a candidate gene for PGL1.

Mice transgenic for a human amyloid precursor protein promoter-lacZ reporter construct.

Transgenic mouse lines were generated that expressed a 2-kb amyloid precursor protein (APP) promoter/beta-galactosidase reporter gene construction. In brain, hippocampal pyramidal neurons, neurons in the deeper layers of cerebral cortex, and neurons in several thalamic nuclei were heavily labeled by beta-galactosidase histochemistry. In general, molecular layers and white matter regions did not express the reporter gene. When compared with in situ hybridization for endogenous murine APP RNA, the striatum and outer layers of cerebral cortex had little reporter expression. Thus, the match between reporter expression and endogenous APP expression in brain was not perfect. A similar mismatch between the relative expression of the reporter gene and endogenous APP RNA distribution was found in homogenates from several organs. Although prior work in transgenic mice found similar mismatches in reporter gene distribution, none had tested the APP promoter construct in response to neuronal injury. Kainic acid injections successfully increased murine APP expression in the transgenic mice, but had no effect on the reporter gene expression. Based on these data and those collected by others, we conclude that the 2-kb region upstream of the APP transcription initiation site contains some elements responsible for the tissue-specific expression of this gene, but does not contain all the cis-acting elements sufficient for either the differential tissue distribution of this gene or the regulation of this gene subsequent to neural damage.

Hypothermia attenuates the normal increase in interleukin 1 beta RNA and nerve growth factor following traumatic brain injury in the rat.

Significant morbidity and mortality associated with traumatic brain injury (TBI) are allied with secondary posttrauma inflammatory complications. Hypothermia has been suggested as a possible treatment to lessen or suppress these inflammatory reactions. We report here that interleukin 1 beta, a cytokine responsible for initiating inflammatory cascades, is elevated in rat cortex within 6 h of TBI in the rat. Nerve growth factor (NGF) RNA and protein also increased subsequently, and NGF protein remained elevated for up to 7 days. Four hours of whole body hypothermia (32 degrees C), applied immediately after the TBI, attenuated the posttrauma increase in IL-1 beta RNA and eliminated the increase in NGF RNA and protein observed in cerebral cortex following TBI. Thus, hypothermia may be an effective therapy to diminish the posttrauma inflammatory cascade in the brain (as suggested by the decrease in IL-1 beta). However, the same treatment may hinder the brain's intrinsic repair mechanisms. Optimal treatment may, therefore, require supplemental administration of neurotrophic factors or other agents along with hypothermia.

Washington State's model of physician leadership in cardiac outcomes reporting.

BACKGROUND: In 1993, the cardiac surgery community in Washington State opposed an effort by the state Health Care Authority (HCA) to identify "centers of excellence" for selective contracting of coronary artery bypass grafting (CABG) procedures, and proposed an alternate model that would create a statewide cardiac outcomes registry under physician governance to be used by all institutions for internal quality improvement activities. METHODS: A prospective pilot data collection effort, which examined preoperative and postoperative patient-reported health status, served as the basis for evaluating the capacity of a physician-led organization to develop a collaborative atmosphere and facilitate universal hospital participation. RESULTS: A surgical steering group met on a regular basis and reached consensus on governance issues, protocols for standardized data collection, and policies regarding data dissemination. All 14 centers that performed bypass surgery in the state participated. Patients who were surveyed reported statistically significant improvements in physical, emotional, and anginal-specific health status after bypass surgery. Baseline patient characteristics and longitudinal outcomes were compared across institutions. CONCLUSIONS: Based on the feasibility of this collaborative outcomes reporting program, the HCA revised its policy regarding selective contracting and has helped to support an ongoing physician-led and -governed cardiac outcomes reporting system that is particularly notable for the subsequent integration of both CABG surgery and catheterization-based procedures into one standardized registry.

DMSO effects on synaptic facilitation and calcium dependence in bullfrog sympathetic ganglion.

Synaptic transmission in the bullfrog sympathetic ganglion was studied by means of extra- and intracellular recordings. DMSO (3-10%) caused a single orthodromic stimulus to generate a brief burst of repetitive postganglionic discharges. DMSO also partially reversed a preexisting transmission failure in low Ca2+ medium. Ganglia were exposed to gradual reductions in extracellular Ca2+, in the absence and in the presence of DMSO. The recorded amplitude of the postganglionic compound action potential (CAP) was plotted as a function of Ca2+ concentration. In the absence of DMSO transmission failed progressively as Ca2+ was reduced from 1.8 to 0.47 mM but DMSO (3% and 10%) shifted the curve of transmission failure to the left (lower Ca2+ concentration). DMSO inhibits ganglion cholinesterase activity, but this is not the mechanism of its facilitatory effect on Ca2+ entry, since physostigmine did not shift the curve of transmission failure in low Ca2+ to the left. The data suggest that DMSO maintained transmitter release in low Ca2+ by a direct, nonspecific enhancement of Ca2+ influx into the presynaptic nerve terminal.

Glial fibrillary acidic protein mRNA increases at proestrus in the arcuate nucleus of mice.

Glial fibrillary acidic protein (GFAP) increases during proestrus in astrocytes of the hypothalamic arcuate nucleus (ARC). These changes are associated with altered astrocyte-neuron contacts and synaptic remodelling, during preparation for the preovulatory gonadotrophin surge. This study of young C57BL/6J mice showed transient elevations of GFAP mRNA on proestrus in the ARC by in situ hybridization. Basal GFAP mRNA was regained within 18 h. We hypothesize that changes in astrocytic GFAP on proestrus result from elevations of GFAP mRNA that are, in turn, driven by ovarian secretions of estradiol.

Teaching clinical reasoning to second-year medical students.

In most U.S. medical schools, students begin clinical clerkships during their third year. They are expected to arrive prepared with the skills necessary to participate in patient-care activities, including data gathering, care coordination, and diagnostic reasoning. However, most students' training may inadequately prepare them to be proficient in diagnostic reasoning, and little time is available in the third year to develop these skills. Because of this inadequate preparation, students may become frustrated with many aspects of their clerkships. This paper describes a series of exercises developed by the author and implemented in this three years as a clinical tutor for second-year medical students in an introduction to clinical medicine course. The exercises are designed to accelerate the student's understanding of diagnostic reasoning, thereby better preparing them for the requirements of the third-year clerkship. The author describes how he uses this teaching approach and discusses in detail the method's central components. He notes that the six students he has worked with from 1993 to the present have responded very positively, and that further research is needed to explore possible long-term benefits of this teaching method.

Haloperidol treatment increases D2 dopamine receptor protein independently of RNA levels in mice.

Haloperidol, administered to mice in their drinking water, produced a 21% increase in striatal D2 dopamine receptor density after seven days of continuous exposure. The steady-state D2 receptor RNA prevalence was unaffected by this treatment, yet the RNA coding for preproenkephalin was elevated, as expected. These data indicate that the homologous up-regulation of dopamine receptor density by antipsychotic drugs proceeds by mechanisms other than changes in RNA abundance.

The treatment of the painful hip in cerebral palsy by total hip replacement or hip arthrodesis.

The painful dislocated or subluxated hip in the patient with cerebral palsy presents a difficult problem in management. Twenty-three patients with cerebral palsy who had a painful subluxated or dislocated hip with degenerative changes were operated on at The Hospital for Special Surgery. Eight patients had a unilateral hip arthrodesis. Six had a successful arthrodesis initially, resulting in relief of pain and return to the preoperative functional level. A pseudarthrosis developed in two patients, but both had successful revision surgery, one by a second arthrodesis and the other by a total hip replacement. Thirteen of the fifteen patients with a total hip replacement were pain-free and functioning at a level consistent with their over-all involvement. One patient had migration of the greater trochanter and slight bending of the femoral component, with persistent pain. A second patient had progressive loosening of the femoral component over a period of seven years, but continued to walk with minimum pain. Two patients had a recurrent dislocation, one requiring revision of the femoral component and the other, of the acetabular component. We think that the presence of cerebral palsy, even in the patient with severe involvement, is not a contraindication for either a hip arthrodesis or a total hip replacement for a painful deformed or degenerated hip. These patients should be treated for the pain in the hip, and the presence of the underlying neuromuscular disorder should not deter the appropriate treatment. For unilateral hip disease in patients who are unable to walk and in young, active patients, we recommend hip fusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Illness and efficiency of health services delivery in a district hospital.

Demographic data, medical problems and diagnosis, and efficiency of laboratory investigations and drug administration were evaluated in all patients admitted to an adult medical ward over a one month period at a district hospital in Kenya. The results show that the medical ward serves a poor, cosmopolitan population in the economically productive age range. Mental disorders (16%), symptoms and ill-defined conditions (16%), and infective and parasitic disease (15%) were the most common diagnoses. Out of 999 orders and/or results, 357 were delayed, interrupted, never done, or never received. Factors that affect medical education and efficiency of health services delivery at a district hospital are identified and discussed.

Herpes simplex virus RNAi and neprilysin gene transfer vectors reduce accumulation of Alzheimer's disease-related amyloid-beta peptide in vivo.

Accumulation of insoluble aggregates of amyloid-beta peptide (Abeta), a cleavage product of amyloid precursor protein (APP), is thought to be central to the pathogenesis of Alzheimer's disease (AD). Consequently, downregulation of APP, or enhanced clearance of Abeta, represent possible therapeutic strategies for AD. We generated replication-defective herpes simplex virus (HSV) vectors that inhibit Abeta accumulation, both in vitro and in vivo. In cell culture, HSV vectors expressing either (i) short hairpin RNA directed to the APP transcript (HSV-APP/shRNA), or (ii) neprilysin, an endopeptidase that degrades Abeta (HSV-neprilysin), substantially inhibited accumulation of Abeta. To determine whether these vectors showed similar activity in vivo, we developed a novel mouse model, in which overexpression of a mutant form of APP in the hippocampus, using a lentiviral vector (LV-APP(Sw)), resulted in rapid Abeta accumulation. Co-inoculation of LV-APP(Sw) with each of the HSV vectors showed that either HSV-APP/shRNA or HSV-neprilysin inhibited Abeta accumulation in this model, whereas an HSV control vector did not. These studies demonstrate the utility of HSV vectors for reducing Abeta accumulation in the brain, thus providing useful tools to clarify the role of Abeta in AD that may facilitate the development of novel therapies for this important disease.

Enhanced glial fibrillary acidic protein RNA response to fornix transection in aged mice.

The effects of age on basal and lesion-induced changes in astrocyte RNA messages reported to respond to neurodegeneration were examined in the mouse brain. The first study found an age-related increase in glial fibrillary acidic protein RNA throughout the brain. Other astrocyte RNAs remained generally stable with age. We hypothesize this increase is due to astrocytes undergoing a mild reaction to the small amount of synaptic degeneration occurring with usual aging. To test this theory, we used an experimental model of modest synaptic loss in the hippocampus by transecting the fimbria/fornix bundle in mice and examined the same series of messages. In situ hybridization revealed the expected increase in glial fibrillary acidic protein RNA after the lesion; however, we unexpectedly found that aged mice showed a greater magnitude of this response, which appeared to develop more slowly. There was no significant change in the hippocampus for any of the other messages, although responses were observed at the site of transection. This study supports the idea that the age-related increase in glial fibrillary acidic protein may be secondary to modest synaptic degeneration. We also demonstrated an exaggerated reactive astrocytic response in aged mice, which may be associated with age-related deficits in reactive synaptogenesis and behavioral recovery in normal aging.

GFAP RNA increases during a wasting state in old mice.

A prolonged wasting condition is often associated with morbidity in older humans. The effects that such a state has on the quality/quantity of RNA are not known. In initial attempts to develop an animal model for premortem wasting, we examined whole brain RNA from mice slowly approaching death from natural causes. Glial fibrillary acidic protein (GFAP) RNA showed a three-fold increase as detected by RNA gel-blot hybridization analysis. Five other RNA sequences were stable under these circumstances. We conclude that brain RNA changes are selective during a degenerating premortem state. Moreover, RNA sequence changes in conditions such as Alzheimer's disease should be considered in the context of the wasting condition of the individual and may not be due to a direct effect of the disease process.