Sensitization of enteric neurons to morphine by HIV-1 Tat protein.

BACKGROUND: Gastrointestinal (GI) dysfunction is a major cause of morbidity in acquired immunodeficiency syndrome (AIDS). HIV-1-induced neuropathogenesis is significantly enhanced by opiate abuse, which increases proinflammatory chemokine/cytokine release, the production of reactive species, glial reactivity, and neuronal injury in the central nervous system. Despite marked interactions in the gut, little is known about the effects of HIV-1 in combination with opiate use on the enteric nervous system. METHODS: To explore HIV-opiate interactions in myenteric neurons, the effects of Tat ± morphine (0.03, 0.3, and 3 µM) were examined in isolated neurons from doxycycline- (DOX-) inducible HIV-1 Tat(1-86) transgenic mice or following in vitro Tat 100 nM exposure (>6 h). KEY RESULTS: Current clamp recordings demonstrated increased neuronal excitability in neurons of inducible Tat(+) mice (Tat+/DOX) compared to control Tat-/DOX mice. In neurons from Tat+/DOX, but not from Tat-/DOX mice, 0.03 µM morphine significantly reduced neuronal excitability, fast transient and late long-lasting sodium currents. There was a significant leftward shift in V(0.5) of inactivation following exposure to 0.03 µM morphine, with a 50% decrease in availability of sodium channels at -100 mV. Similar effects were noted with in vitro Tat exposure in the presence of 0.3 µM morphine. Additionally, GI motility was significantly more sensitive to morphine in Tat(+) mice than Tat(-) mice. CONCLUSIONS & INFERENCES: Overall, these data suggest that the sensitivity of enteric neurons to morphine is enhanced in the presence of Tat. Opiates and HIV-1 may uniquely interact to exacerbate the deleterious effects of HIV-1-infection and opiate exposure on GI function.

Chromosome aberration induction is dependent on the spatial distribution of energy deposition through a cell nucleus.

The importance of the spatial distribution of energy deposition through the nucleus in determining the resultant chromosome rearrangements was investigated using fluorescent in situ hybridisation technique following either uniform or partial irradiation of HF19 human fibroblast cells with low-LET 1.5 keV ultrasoft X-rays. Irradiations were performed with and without a copper irradiation mask with a Poisson distribution of micron-sized holes immediately below the irradiation dish and the results are compared with previous results obtained following exposure to a Poisson distribution of alpha particles. For the same radiation quality, the spatial distribution of energy deposition within the nucleus was found to be important in determining the ultimate biological response, with an increased ratio of complex-to-simple aberrations observed for partial compared to uniform irradiation. Comparisons between low-LET ultrasoft X-rays and high-LET alpha particles indicate that the sub-micron clustering of damage along the alpha particle track is more important than just the total number of double-strand breaks produced.

The first case of Pasteurella canis bacteremia: a cirrhotic patient with an open leg wound.

INTRODUCTION: Severe human infections caused by the Pasteurella species are typically seen following animal bites. P. canis is a species that rarely affects humans and has never been found in systemic infections. Here, we report the first documented case of P. canis bacteremia in an infected human, thought to be caused by a dog lick to an open leg wound.

Innate immune recognition of, and response to, Clostridium sordellii.

Clostridium sordellii, an anaerobic pathogen, has recently been associated with rapidly fatal infections following medically induced abortions and injecting drug use. Patients with C. sordellii infection display few signs of inflammation such as fever, or redness and pain at the site of infection. We hypothesized that this could be due to reduced recognition of the organism by Toll-like receptors (TLRs) of the innate immune system. An ELAM-NF-kappaB luciferase reporter system in TLR-transfected HEK cells was used to measure TLR-dependent recognition of washed, heat-killed C. sordellii and other pathogenic clostridial species. Results demonstrated that all clostridia were well recognized by TLR2 alone and that responses were greatest when TLR2 was co-expressed with TLR6. Further, isolated human monocytes produced the pro-inflammatory cytokine TNFalpha and the immunoregulator IL-10 in response to C. sordellii. In addition, C. sordellii-stimulated monocytes produced 30% less TNFalpha following treatment with an anti-TLR2 blocking antibody. These data demonstrate that innate immune recognition of, and response to, cell-associated components of C. sordellii and other clostridial pathogens are mediated by TLR2 in combination with TLR6. We conclude that the characteristic absence of inflammatory signs and symptoms in C. sordellii infection is not related to inadequate immune detection of the organism, but rather is attributable to a species-specific immune system dysfunction that remains to be elucidated.

Hereditary multi-infarct dementia of the Swedish type is a novel disorder different from NOTCH3 causing CADASIL.

Several hereditary small vessel diseases (SVDs) of the brain have been reported in recent years. In 1977, Sourander and Wålinder described hereditary multi-infarct dementia (MID) in a Swedish family. In the same year, Stevens and colleagues reported chronic familial vascular encephalopathy in an English family bearing a similar phenotype. These disorders have invariably been suggested to be cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) but their genetic identities remain unknown. We used molecular, radiological and neuropathological methods to characterize these disorders. Direct DNA sequencing unexpectedly confirmed that affected members of the English family carried the R141C mutation in the NOTCH3 gene diagnostic of CADASIL. However, we did not detect any pathogenic mutations in the entire 8091 bp reading frame of NOTCH3 or find clear evidence for NOTCH3 gene linkage in the Swedish DNA. This was consistent with the lack of hyperintense signals in the anterior temporal pole and external capsule in Swedish subjects upon magnetic resonance imaging. We further found no evidence for granular osmiophilic material in skin biopsy or post-mortem brain samples of affected members in the Swedish family. In addition, there was distinct lack of NOTCH3 N-terminal fragments in the cerebral microvasculature of the Swedish hereditary MID subjects compared to the intense accumulation in the English family afflicted with CADASIL. Several differences in arteriosclerotic changes in both the grey and white matter were also noted between the disorders. The sclerotic index values, density of collagen IV immunoreactivity in the microvasculature and number of perivascular macrophages were greater in the English CADASIL samples compared to those from the Swedish brains. Multiple approaches suggest that the Swedish family with hereditary MID suspected to be CADASIL has a different novel disorder with dissimilar pathological features and belongs to the growing number of genetically uncharacterized familial SVDs.

Experimental techniques for studying bystander effects in vitro by high and low-LET ionising radiation.

Ionising radiation can induce responses within non-exposed neighbouring (bystander) cells which potentially have important implications on the estimates of risk from low dose or low dose rate exposures of ionising radiations. A range of strategies have been developed for investigating bystander effects in vitro for both high-LET alpha particles or low-LET ultrasoft X rays using either partial shielding (grids, half-shields and slits) or by using a co-culture system where two physically separated populations of cells can be cultured together, allowing one population of cells to be irradiated while the second population remains unirradiated. The techniques described provide a useful tool to study bystander effects and complement microbeam studies. Studies using these systems show significant increases in the unirradiated bystander cells for various end points including the induction of chromosomal instability in haemopoetic stem cells and transformation in CGL1 cells.

Keeping up with the neighbours--measuring the bystander response.

Ionising radiation can induce responses within non-exposed neighbouring (bystander) cells, which potentially have important implications on the estimates of risk at environmentally relevant doses. Using human skin fibroblasts (AG1522), a range of methods were used to investigate the nature of the signal(s) arising from the exposed cells. The signal(s) can be transmitted by direct cell-cell communication (investigated by using partial dish irradiations) or by medium-borne factors (a co-culture system where two monolayers share the same medium but only one monolayer is exposed to ionising radiation). CDKN1A was found to be up-regulated in both directly exposed and non-exposed cells. The data suggest that direct cell-cell communication dominates for these confluent cells, with medium-borne factors also contributing.

Clostridium sordellii infection: epidemiology, clinical findings, and current perspectives on diagnosis and treatment.

Clostridium sordellii infections pose difficult clinical challenges and are usually fatal. Most commonly, these infections occur after trauma, childbirth, and routine gynecological procedures, but they have recently been associated with medically induced abortions and injection drug use. We report 2 fatal cases, one of which was associated with minor trauma, and the other of which was associated with normal childbirth, and we summarize the clinical features of 43 additional cases of reported C. sordellii infection. Of these 45 cases, 8 (18%) were associated with normal childbirth, 5 (11%) were associated with medically induced abortion, and 2 (0.4%) were associated with spontaneous abortion. The case-fatality rate was 100% in these groups. Ten (22%) of the C. sordellii infections occurred in injection drug users, and 50% of these patients died. Other cases of C. sordellii infection (in 19 patients [43%]) occurred after trauma or surgery, mostly in healthy persons, and 53% these patients died. Overall, the mortality rate was 69% (31 of 45 patients). Eighty-five percent of all patients with fatal cases died within 2-6 days of initial infection, and nearly 80% of fatal cases developed leukemoid reactions. Rapid diagnostic tests and improved treatments are needed to reduced the morbidity and mortality associated with this devastating infection.

Clostridium perfringens phospholipase C-induced platelet/leukocyte interactions impede neutrophil diapedesis.

Clostridium perfringens gas gangrene is a fulminant necrotizing infection in which inflammatory cells are notably absent from infected tissues but are often massed within adjacent vessels. It has been shown that C. perfringens phospholipase C (PLC) stimulates formation of large intravascular platelet/leukocyte complexes and that PLC-induced activation of platelet gpIIbIIIa plays a major role. In vivo, such aggregates contribute to microvascular thrombosis and ischaemic necrosis of tissue. However, the effects of adherent platelets on neutrophil diapedesis have not been established. The present work investigated (1) the contribution of platelet P-selectin (CD62P) to PLC-induced cellular complex formation and (2) the effects of platelet adhesion on neutrophil diapedesis. The effects of anti-gpIIbIIIa and anti-CD62P strategies on PLC-induced complex formation were measured by flow cytometry and followed by light microscopy. Both platelet gpIIbIIIa and CD62P contributed to the formation of platelet/leukocyte complexes. Specifically, gpIIbIIIa mediated the formation of large platelet/platelet aggregates that were tethered to the leukocyte principally via CD62P. Neutrophil diapedesis, quantified by a transendothelial cell migration assay and visualized by electron microscopy, was significantly reduced (>60%) by the adherence of large platelet aggregates. It was concluded that the absence of a tissue inflammatory response in C. perfringens gas gangrene is due, in part, to impaired neutrophil mobility caused by large aggregates of adherent platelets induced by PLC. Further, an adjunctive immunotherapeutic strategy targeting both gpIIbIIIa and CD62P may improve the tissue inflammatory response, prevent vascular occlusion, maintain tissue viability, and reduce the need for radical amputation in patients with clostridial gas gangrene.

Increased complexity of radiation-induced chromosome aberrations consistent with a mechanism of sequential formation.

Complex chromosome aberrations (any exchange involving three or more breaks in two or more chromosomes) are effectively induced in peripheral blood lymphocytes (PBL) after exposure to low doses (mostly single particles) of densely ionising high-linear energy transfer (LET) alpha-particle radiation. The complexity, when observed by multiplex fluorescence in situ hybridisation (m-FISH), shows that commonly four but up to eight different chromosomes can be involved in each rearrangement. Given the territorial organisation of chromosomes in interphase and that only a very small fraction of the nucleus is irradiated by each alpha-particle traversal, the aim of this study is to address how aberrations of such complexity can be formed. To do this, we applied theoretical "cycle" analyses using m-FISH paint detail of PBL in their first cell division after exposure to high-LET alpha-particles. In brief, "cycle" analysis deconstructs the aberration "observed" by m-FISH to make predictions as to how it could have been formed in interphase. We propose from this that individual high-LET alpha-particle-induced complex aberrations may be formed by the misrepair of damaged chromatin in single physical "sites" within the nucleus, where each "site" is consistent with an "area" corresponding to the interface of two to three different chromosome territories. Limited migration of damaged chromatin is "allowed" within this "area". Complex aberrations of increased size, reflecting the path of alpha-particle nuclear intersection, are formed through the sequential linking of these individual sites by the involvement of common chromosomes.

Diabetic ketoacidosis associated with aripiprazole.

BACKGROUND: Atypical antipsychotics have become the mainstay of management of schizophrenia and other psychotic disorders due to low risk of extrapyramidal symptoms. However, postmarketing data has reported atypical antipsychotic agents being associated with hyperglycaemia and diabetic ketoacidosis (DKA). We believe this to be the first published report of hyperglycaemia and DKA with the newest atypical antipsychotic agent, aripiprazole. CASE REPORT: A 34-year-old African-American female with schizophrenia presented to the emergency department with nausea, vomiting, and malaise for 3-4 days shortly after initiation of aripiprazole therapy. Initial laboratory results revealed significant hyperglycaemia with metabolic acidosis. The patient received treatment for DKA with an intravenous insulin infusion and fluid replacement. Isophane insulin suspension (NPH insulin) was begun immediately following the insulin drip and continued upon discharge from the hospital. Outpatient follow-up information was not available. CONCLUSION: To our knowledge, this is the first case report of aripiprazole associated with hyperglycaemia and DKA. This case is striking in that DKA occurred 4 days following initiation of aripiprazole and the patient had rapid resolution of symptoms and normalization of laboratory values upon discontinuation of aripiprazole. It is important that health-care providers monitor for hyperglycaemia when prescribing atypical antipsychotics including aripiprazole.

Bound PCNA in nuclei of primary rat tracheal epithelial cells after exposure to very low doses of plutonium-238 alpha particles.

Bystander effects from ionizing radiation have been detailed for a number of cell systems and a number of end points. We wished to use a cell culture/ex vivo rat model of respiratory tissue to determine whether a bystander effect detected in culture could also be shown in a tissue. Examination by immunofluorescence techniques of tracheal cell cultures after exposure to very low doses of alpha particles revealed a large proportion of cells with proliferating cell nuclear antigen (PCNA) bound in their nuclei. PCNA was selected as an end point because it is involved in both DNA repair and the changes in cell cycle that are typical of many reported bystander effects. Maximum response can be detected in up to 28% of the cells in sub-confluent cultures with a dose of only 2 mGy. At this dose less than 2% of the cell nuclei have experienced a particle traversal and less than 6% of the cells have experienced an alpha-particle traversal through either their nucleus or some part of their cytoplasm. The hypothesis that this bystander response in nontargeted cells is mediated through secreted factor(s) is presented, and supporting evidence was found using partial irradiation and co-culture experiments. Examination of the effect with excised pieces of trachea demonstrated a response similar to that seen in culture.

M type 1 and 3 group A streptococci stimulate tissue factor-mediated procoagulant activity in human monocytes and endothelial cells.

Streptococcal toxic shock syndrome (StrepTSS) is an invasive infection characterized by marked coagulopathy, multiple organ failure, and rapid tissue destruction and is strongly associated with M type 1 and 3 group A streptococci (GAS). Initiation of the coagulation cascade with formation of microvascular thrombi contributes to multiple organ failure in human cases of gram-negative bacteremia; however, little is known regarding the mechanism of coagulopathy in StrepTSS. Thus, we investigated the abilities of several strains of M type 1 and 3 GAS isolated from human cases of StrepTSS to stimulate production of tissue factor (TF), the principal initiator of coagulation in vivo. Washed, killed M type 1 and 3 GAS, but not M type 6 GAS, elicited high-level TF-mediated procoagulant activity from both isolated human monocytes and cultured human umbilical vein endothelial cells. M type 1 GAS consistently elicited higher levels of TF from monocytes than did M type 3 GAS. GAS-induced TF synthesis in monocytes did not correlate with production of tumor necrosis factor alpha or interleukin-8. Conversely, M type 3 GAS were consistently more potent than M type 1 GAS in stimulating endothelial cell TF synthesis. These results demonstrate that (i) M type 1 and 3 strains of GAS are potent inducers of TF synthesis, (ii) GAS-induced TF synthesis is not simply an epiphenomenon of cytokine generation, and (iii) induction of TF in endothelial cells and monocytes may be M type specific. In total, these findings suggest that a novel interaction between GAS and host cells contributes to the observed coagulopathy in StrepTSS.

Is the increased relative biological effectiveness of high LET particles due to spatial or temporal effects? Characterization and OER in V79-4 cells.

The efficiency of producing biological damage varies with radiation quality. Conventional explanations rely on spatial differences in the radiation track structure; generally however there are also very large temporal differences in delivery of the radiation at the cellular level. High-LET radiation normally deposits substantial amounts of energy by individual heavily ionizing tracks on a timescale of the order of picoseconds. By contrast each low-LET radiation track deposits a small amount of energy. Many of these tracks, distributed over the whole cell, are required to deliver an equivalent dose to a high-LET track and they are usually delivered over much longer timescales (typically seconds) during which chemical, biochemical and biological processes are occurring. In this paper the design, characterization and initial application of a high-brightness, laser-plasma ultrasoft x-ray source is described. This has been used to investigate the importance of the temporal differences by irradiating mammalian cells with an energy deposition with spatial properties of low-LET radiation and temporal properties similar to high-LET radiation. The present system delivers a typical dose, to the incident surface of the cells, of 0.12 Gy per pulse delivered in <10 ps. The capabilities of the x-ray source were tested by determining the survival of V79-4 hamster cells irradiated with picosecond pulses of ultrasoft x-rays under aerobic and anaerobic conditions, which were found to be consistent with previously published non pulsed data with x-rays of similar energy. These results support the expectation that the disappearance of an oxygen effect for high-LET radiation particles is due to their spatial properties rather than the very short timescale of each particle traversal. For other effects, particularly non-targeted phenomena such as induced genomic instability, expectations may be less clear cut.

Biological effectiveness of isolated short electron tracks: V79-4 cell inactivation following low dose-rate irradiation with Al(K) ultrasoft X-rays.

PURPOSE: To investigate the biological effect of single, isolated, short electron tracks (<70 nm) relevant to practical human exposures to low-linear energy transfer radiation. MATERIALS AND METHODS: An irradiation rig was constructed that allowed environmentally controlled, protracted irradiations with an individually prescribed dose to up to 20 samples over a period of days. Inactivation of V79-4 mammalian cells by Al(K) ultrasoft X-rays was studied at high and low dose-rates with a maximum exposure time of 42 h. RESULTS: A significant increase in clonogenic survival was observed at the higher doses when the exposure time was increased from <6 min to 21 h, with no further increase observed for 42-h exposures. Despite the short range of the low-energy electrons produced (<70 nm), significant cell inactivation was observed for these low dose-rate exposures. CONCLUSIONS: The results are consistent with the hypothesis that even individual tracks can be biologically effective.

Streptococcal toxic shock syndrome.

Perhaps more noteworthy than the emergence of Streptococcal toxic shock syndrome (StrepTSS) is its persistence for a period of more than 15 years in most geographical areas and an actual increase in incidence in some regions. Early diagnosis remains a problem, and aggressive surgery often cannot be avoided. The continuing rates of mortality and morbidity indicate the need for novel approaches to diagnosis and treatment.

Cell inactivation and double-strand breaks: the role of core ionizations, as probed by ultrasoft X rays.

The large RBE (approximately 7) measured for the killing of Chinese hamster V79 cells by 340 eV ultrasoft X rays, which preferentially ionize the K shell of carbon atoms (Hervé du Penhoat et al., Radiat. Res. 151, 649-658, 1999), was used to investigate the location of sensitive sites for cell inactivation and the physical modes of action of radiation. The enhancement of the RBE above the carbon K-shell edge either may indicate a high intrinsic efficiency of carbon K-shell ionizations (due, for example, to a specific physical or chemical effect) or may be related to the preferential localization of these ionizations on the DNA. The second interpretation would indicate a strong local (within 3 nm) action of K-shell ionizations and consequently the importance of a direct mechanism for radiation lethality (without excluding an action in conjunction with an indirect component). To distinguish between these two hypotheses, the efficiencies of core ionizations in DNA atoms (phosphorus L-shell, carbon K-shell, and oxygen K-shell ionizations) to induce damages were investigated by measuring their capacities to produce DNA double-strand breaks (DSBs). The effect of photoionizations in isolated DNA was studied using pBS plasmids in a partially hydrated state. No enhancement of the efficiency of DSB induction by carbon K-shell ionizations compared to oxygen K-shell ionizations was found, supporting the hypothesis that it is the localization of these carbon K-shell events on DNA which gives to the 340 eV photons their high killing efficiency. In agreement with this interpretation, cell inactivation and DSB induction, which do not appear to be correlated when expressed in terms of yields per unit dose in the sample, exhibit a rather good correlation when expressed in terms of efficiencies per core event in the DNA. These results suggest that core ionizations in DNA, through core-hole relaxation in conjunction with localized effects of spatially correlated secondary and Auger electrons, may be the major critical events for cell inactivation, and that the resulting DSBs (or a constant fraction of these DSBs) may be a major class of unrepairable lesions.