Robust neurite extension following exogenous electrical stimulation within single walled carbon nanotube-composite hydrogels.

UNLABELLED: The use of exogenous electrical stimulation to promote nerve regeneration has achieved only limited success. Conditions impeding optimized outgrowth may arise from inadequate stimulus presentation due to differences in injury geometry or signal attenuation. Implantation of an electrically-conductive biomaterial may mitigate this attenuation and provide a more reproducible signal. In this study, a conductive nanofiller (single-walled carbon nanotubes [SWCNT]) was selected as one possible material to manipulate the bulk electrical properties of a collagen type I-10% Matrigel™ composite hydrogel. Neurite outgrowth within hydrogels (SWCNT or nanofiller-free controls) was characterized to determine if: (1) nanofillers influence neurite extension and (2) electrical stimulation of the nanofiller composite hydrogel enhances neurite outgrowth. Increased SWCNT loading (10-100-µg/mL) resulted in greater bulk conductivity (up to 1.7-fold) with no significant changes to elastic modulus. Neurite outgrowth increased 3.3-fold in 20-µg/mL SWCNT loaded biomaterials relative to the nanofiller-free control. Electrical stimulation promoted greater outgrowth (2.9-fold) within SWCNT-free control. The concurrent presentation of electrical stimulation and SWCNT-loaded biomaterials resulted in a 7.0-fold increase in outgrowth relative to the unstimulated, nanofiller-free controls. Local glia residing within the DRG likely contribute, in part, to the observed increases in outgrowth; but it is unknown which specific nanofiller properties influence neurite extension. Characterization of neuronal behavior in model systems, such as those described here, will aid the rational development of biomaterials as well as the appropriate delivery of electrical stimuli to support nerve repair. STATEMENT OF SIGNIFICANCE: Novel biomedical devices delivering electrical stimulation are being developed to mitigate symptoms of Parkinson's, treat drug-resistant depression, control movement or enhance verve regeneration. Carbon nanotubes and other novel materials are being explored for novel nano-neuro devices based on their unique properties. Neuronal growth on carbon nanotubes has been studied in 2D since the early 2000s demonstrating increased outgrowth, synapse formation and network activity. In this work, single-walled carbon nanotubes were selected as one possible electrically-conductive material, dispersed within a 3D hydrogel containing primary neurons; extending previous 2D work to 3D to evaluate outgrowth within nanomaterial composites with electrical stimulation. This is the first study to our knowledge that stimulates neurons in 3D composite nanomaterial-laden hydrogels. Examination of electrically conductive biomaterials may serve to promote regrowth following injury or in long term stimulation.

[Hepatic portal venous gas: surgery or not surgery?]. / L'aéroportie: bloc ou pas bloc?

Finding hepatic portal venous gas with pneumatosis intestinalis on computed tomography (CT) represents diagnostic and therapeutic challenge. The intestinal necrosis, particularly associated with acute mesenteric ischemia, is the very first hypothesis to assess, with the underlying question of an urgent surgery. However, knowing the non-surgical causes that have been identified in the last decade seems necessary to better assess the risk-benefit ratio of emergency surgery. Among these causes, we report the case of the acute colonic pseudo-obstruction, also known as Ogilvie's syndrome, whose first line treatment is medical.

Algal extracellular products suppress Anabaena flos-aquae heterocyst spacing.

Intra- and interspecific chemical signals allow bacteria to respond to environmental conditions by regulating gene transcription. In cyanobacteria, gene products and the presence of fixed nitrogen regulate heterocyst frequency. In this paper, we describe a chemical made by a green alga, Chlamydomonas reinhardtii, that suppresses heterocyst formation in the co-occurring cyanobacterium, Anabaena flos-aquae. Cyanobacterial heterocyst frequencies were reduced in the presence of water-soluble, proteinase- and heat-resistant molecules greater than 15 kDa in molecular size. Green algal cells in all phases of growth made the suppressor. Ammonium and nitrate concentrations in the medium did not correlate with this change in phenotype. In addition, growth rate was not enhanced by the extracellular products. Therefore, C. reinhardtii extracellular products acted as a heterocyst inhibitor, not as a fixed nitrogen source. Chemical interactions between green algae and cyanobacteria influence heterocyst formation, an important consideration in understanding the outcome of competition between these organisms and the dynamics of phytoplankton communities.

Toxin-Producing Anabaena flos-aquae Induces Settling of Chlamydomonas reinhardtii, a Competing Motile Alga.

Toxin production is an adaptation that allows cyanobacteria in resource-limiting environments to ameliorate the effects of herbivory and competition with other phototrophs. We demonstrate that the cyanobacterial toxins anatoxin-a and microcystin-LR paralyze the motile green alga Chlamydomonas reinhardtii. In addition, both purified toxins and cyanobacterial extracellular products containing these toxins cause the alga to settle faster than in nontoxic media. In microcosm experiments, the presence of either the cyanobacterium or its extracellular products induce settling in the alga, similar to the response observed with the addition of both anatoxin-a and microcystin-LR. The cyanobacterial production of paralyzing toxins represents a novel mechanism for phytoplankton settling. This prokaryotic/eukaryotic chemical interaction may create a competitor-free zone for cyanobacteria in lake environments, predicating optimal conditions for a toxic cyanobacterial bloom.

Oral induction of anesthesia with droperidol and transmucosal carfentanil citrate in chimpanzees (Pan troglodytes).

Five chimpanzees (Pan troglodytes) initially received oral droperidol sedation (1.25 mg for a juvenile chimpanzee, body wt = 18.5 kg, and 2.5 mg for adults, body wt >20 kg, range: 18.5-71 kg) followed by transmucosal carfentanil administration at 2.0 microg/kg. This preinduction regimen was developed to produce heavy sedation or even light anesthesia in order to eliminate the need for or at least minimize the stress of darting with tiletamine/zolazepam at 3 mg/kg i.m. This study was designed to assess the safety and efficacy of transmucosal carfentanil. Once each animal was unresponsive to external stimuli, or at approximately 25 min (range 24-34 min) after carfentanil administration, naltrexone and tiletamine/zolazepam (N/T/Z) were combined into one intramuscular injection for anesthetic induction. Naltrexone was administered at 100 times the carfentanil dose in milligrams. For comparison, two chimpanzees received only droperidol, 2.5 mg p.o., followed by tiletamine/zolazepam, 3 mg/kg i.m. The preinduction period for all animals receiving carfentanil was characterized as smooth, with chimpanzees becoming gradually less active and less responsive to external stimuli. Two animals became very heavily sedated at 24 and 35 min, respectively, and were hand injected with N/T/Z. The other three chimpanzees became sternally recumbent but retained some response to stimuli, and N/T/Z was administered by remote injection with minimal response. Rectal body temperatures, pulse and respiratory rates, arterial oxygen hemoglobin saturation, and arterial blood gases were measured at initial contact (t = 0 min) and at 10-min intervals thereafter. Respiratory depression was present in all chimpanzees, regardless of protocol. Mean hemoglobin saturation was 91% for both groups. Mean partial pressure of oxygen, arterial values for carfentanil-treated and control animals were 64.4 +/- 7.6 and 63.5 +/- 6.0 at t = 0, respectively. Only the partial pressure of carbon dioxide, arterial (Paco2) and pH showed significant differences between treated and control animals. Mean Paco2 was greater and mean pH lower for the carfentanil-treated group compared with the controls at t = 0 (58.9 +/- 3.7 and 50.3 +/- 3.1 for Paco2 and 7.33 +/- 0.02 and 7.40 +/- 0.30 for pH, respectively). The results of this study suggest that oral droperidol followed by transmucosal carfentanil can be used effectively as a premedication regimen to produce profound sedation, which limits the stress of darting during parenteral anesthetic induction with tiletamine/zolazepam in chimpanzees. The main side effect of respiratory depression appears to be adequately managed by reversing the carfentanil at the time of induction.

Zinc-responsive dermatosis in a red wolf (canis rufus).

An 18-mo-old male red wolf (Canis rufus) presented with footpad hyperkeratosis, suppurative paronychia, distal limb pyoderma, and peripheral lymphadenopathy. Diet for the previous 11 mo consisted of a mixture of two commercially prepared dog foods with a mineral supplement containing primarily calcium. Culture of the draining tracts on the distal limbs yielded a mixed population of opportunistic bacteria. Histopathologic findings were consistent with a diagnosis of zinc deficiency. Medical therapy consisted of 15 mg/kg amoxicillin p.o. b.i.d. and 10 mg/kg zinc sulfate p.o. s.i.d. Calcium supplementation was discontinued. Clinical signs resolved by 10 wk after the initiation of treatment.

Emergi-paths and stroke teams: an emergency department approach to acute ischemic stroke.

In patients with early acute ischemic stroke (AIS), studies have shown improved recovery rates when thrombolytic therapy is appropriately initiated. However, in clinical practice, there are several barriers to rapid patient evaluation and drug administration. To facilitate the management of this population, an AIS clinical pathway, Emergi-path, was developed. Initiated at the time of the patients' arrival to the emergency department, Emergi-path provides a step-by-step guide for early care of AIS patients. A citywide stroke team plays an integral role in this process by responding to stroke codes. Implementation of an AIS pathway and activation of an organized team of stroke specialists can facilitate rapid evaluation and treatment of this high-risk population.

Green algal extracellular products regulate antialgal toxin production in a cyanobacterium.

Many bacterial genes and virulence factors are regulated by interbacterial and/or host-parasite chemical signals. We demonstrate that toxin production by a free-living freshwater cyanobacterium is regulated in part by the presence of extracellular products of a eukaryotic green alga. In growth experiments, extracellular products made by the cyanobacterium Anabaena flos-aquae contained both anatoxin and microcystin, and significantly reduced the yield of Chlamydomonas reinhardtii, a green alga. Based on experiments in which we added purified toxins to C. reinhardtii cultures, we believe that microcystin was responsible for the growth reduction. A. flos-aquae produced anatoxin constitutively when grown alone, but anatoxin concentration increased in the presence of C. reinhardtii elicitors. Microcystin accumulation depended on the growth phase; however, high concentrations of C. reinhardtii extracellular products completely inhibited microcystin accumulation. Our results demonstrate that cyanobacterial toxin production may be regulated by complex growth phase-dependent and environmental chemical cues, and suggest that secreted chemicals can mediate the outcome of competition between the cyanobacterium A. flos-aquae and the green alga C. reinhardtii.

Hiatal hernia and diaphragmatic eventration in a leopard (Panthera pardus).

A 1-yr-old male leopard (Panthera pardus) presented for intermittent anorexia, emaciation, and generalized muscle wasting. Plain radiographs, ultrasonography, and esophageal endoscopy led to a diagnosis of diaphragmatic eventration with probable concurrent hiatal hernia. An exploratory laparotomy confirmed both diagnoses, and surgical repair and stabilization were performed. After surgery, the leopard was maintained on small liquid meals for 4 days, with a gradual return to normal diet over 2 wk. By 4 wk after surgery, the leopard was eating well and gaining weight, and it showed no recurrence of clinical signs for 2 yr subsequently, becoming mildly obese.

A clinical trial of retroviral-mediated transfer of a rev-responsive element decoy gene into CD34(+) cells from the bone marrow of human immunodeficiency virus-1-infected children.

Genetic modification of hematopoietic stem cells with genes that inhibit replication of human immunodeficiency virus-1 (HIV-1) could lead to development of T lymphocytes and monocytic cells resistant to HIV-1 infection after transplantation. We performed a clinical trial to evaluate the safety and feasibility of this procedure, using bone marrow from four HIV-1-infected pediatric subjects (ages 8 to 17 years). We obtained bone marrow, isolated CD34(+) cells, performed in vitro transduction with a retroviral vector carrying a rev-responsive element (RRE) decoy gene, and reinfused the cells into these subjects with no evidence of adverse effects. The levels of gene-containing leukocytes in peripheral blood samples in the 1 year after gene transfer/cell infusion have been extremely low. These observations support the potential of performing gene therapy for HIV-1 using hematopoietic cells, but emphasize the need for improved gene transfer techniques.

Dermatophytosis in red pandas (Ailurus fulgens fulgens): a review of 14 cases.

Fourteen cases of dermatophytosis were identified from medical records of red pandas (Ailurus fulgens fulgens) housed at the Knoxville Zoo between 1980 and 1996. The median age of affected animals on initial presentation was 8.5 wk (3 wk-11 mo). Clinical signs included crusting, purulent exudate, alopecia, thickening of affected skin, ulceration, and necrosis. Seven animals had mild lesions with signs restricted to crusting and/or alopecia, and six animals had more severe infections, with ulceration, skin necrosis, and purulent exudate. Five of the severely affected pandas had tail involvement. The severity of disease affecting one individual was not recorded. Dermatophytosis was confirmed by culture, cytology, histopathology, or culture followed by histopathology. Microsporum gypseum was the only fungal organism cultured. Six animals were treated for mild disease, and all clinical signs resolved. Partial tail amputation was required as part of the treatment regimen for two of the six severely affected animals, and two others had ulcerated tail lesions that left circumferential scarring after resolution of infection. Itraconazole (5 mg/kg p.o. q 12-24 hr) was the most frequently used systemic antifungal agent in animals with severe lesions. All fungal infections resolved, although one panda died from unrelated causes early in the treatment period.

Open label tissue plasminogen activator for stroke: the Oregon experience.

BACKGROUND: Tissue plasminogen activator (t-PA) is the first effective treatment for stroke. This study sought to explore the outcome of patients treated with t-PA in the community after approval of its use in the treatment of stroke in June, 1996. METHODS: All patients with acute stroke within the 6-hospital Oregon Stroke Center network were screened for potential t-PA treatment. Baseline and 24-hour outcome assessments were performed with the use of the National Institutes of Health Stroke Scale (NIHSS) and computed tomography (CT); 3-month outcome was evaluated by using the Modified Rankin scale. RESULTS: Thirty-three patients who met the criteria for t-PA therapy were treated within 3 hours of symptom onset. All but 2 strokes were in the anterior circulation; 48.5% were cardioembolic. The NIHSS scores at 24 hours after administration of t-PA (mean, 14.7) showed modest gains from baseline NIHSS scores (mean, 16.6). Twelve patients (36%) had minimal or no deficits at 3 months. Three patients (9%), all of whom had baseline NIHSS scores of 20 or more, had symptomatic intracranial hemorrhages, 2 of which were fatal (6%). CONCLUSION: This study shows the feasibility of treating acute stroke with t-PA in the community. The percentage of fully recovered patients at 3 months mirrored those in the National Institute of Neurologic Disorders and Stroke (NINDS) trial.

Infection of human marrow stroma by human immunodeficiency virus-1 (HIV-1) is both required and sufficient for HIV-1-induced hematopoietic suppression in vitro: demonstration by gene modification of primary human stroma.

Patients with human immunodeficiency virus-1 (HIV-1) infection often present with bone marrow (BM) failure that may affect all hematopoietic lineages. It is presently unclear whether this failure reflects a direct viral impairment of the CD34+ hematopoietic progenitor cells or whether the virus affects the BM microenvironment. To study the effects of HIV-1 on the BM microenvironment, we examined the stromal cell monolayers in long-term BM culture (LTBMC), which are the in vitro equivalent of the hematopoietic microenvironment. We assessed the hematopoietic support function (HSF) of human stromal layers by determining the cellular proliferation and colony-forming ability of hematopoietic progenitors from BM cells grown on the stromal layers. We show that the HSF is reduced by in vitro infection of the human stromal cell layer by a monocytotropic isolate of HIV-1 (JR-FL). There is no loss of HSF when the stromal cell layer is resistant to HIV-1 replication, either using murine stromal cell layers that are innately resistant to HIV-1 infection or using human stromal cells genetically modified to express a gene that inhibits HIV-1 replication (an RRE decoy). Decreased HSF was seen using either human or murine hematopoietic cells, if the stromal cells were human cells that were susceptible to HIV-1 infection. These in vitro studies implicate HIV-1 replication in the stroma as the essential component causing decreased hematopoietic cell production in HIV-1 infection.

Inhibition of human immunodeficiency virus-1 (HIV-1) replication after transduction of granulocyte colony-stimulating factor-mobilized CD34+ cells from HIV-1-infected donors using retroviral vectors containing anti-HIV-1 genes.

Transfer of "anti-HIV-1 genes" into hematopoietic stem cells of human immunodeficiency virus-1 (HIV-1)-infected individuals may be a potent therapeutic approach to render mature cells arising from transduced stem cells resistant to the destructive events associated with HIV-1 infection. To determine the feasibility of gene therapy for acquired immunodeficiency syndrome in individuals already infected with HIV-1, granulocyte colony-stimulating factor mobilized peripheral blood CD34+ cells were isolated from HIV-1-infected individuals and transduced with retroviral vectors containing three different anti-HIV-1-genes: the Rev binding domain of the Rev Responsive Element (RRE decoy) (L-RRE-neo), a double hammerhead ribozyme vector targeted to cleave the tat and rev transcripts (L-TR/TAT-neo), and the trans-dominant mutant of rev (M10) (L-M10-SN). As a control, a vector mediating only neomycin resistance (LN) was used. After 3 days of transduction on allogeneic stroma in the presence of stem cell factor, interleukin-6 (IL-6), and IL-3, the cultures were G418-selected, and then challenged with HIV-1(JR-FL) and a primary HIV-1 isolate. Compared with the control cultures, the L-RRE-neo-, L-TR/TAT-neo-, and L-M10-SN-transduced cultures displayed up to 1,000-fold inhibition of HIV-1 replication after challenge with HIV-1(JR-FL) and the primary HIV-1 isolate. Growth of the hematopoietic cells in long-term bone marrow culture was not perturbed by the presence of any of the anti-HIV-1 genes. This study shows that anti-HIV-1 genes can be introduced into CD34+ cells from individuals already infected with HIV-1, and strongly inhibit HIV-1 replication in primary monocytes derived from the CD34+ progenitors.

Suitability of bone marrow from HIV-1-infected donors for retrovirus-mediated gene transfer.

Bone marrow samples from 21 human immunodeficiency virus type 1 (HIV-1)-infected subjects were evaluated for their suitability for retrovirus-mediated gene transduction with anti-HIV-1 genes. The percentages of CD34+ cells that could be isolated from the mononuclear fraction of bone marrow samples were determined. Fifteen of the 21 marrow samples had normal percentages of CD34+ cells isolated by immunomagnetic methods. All seven donors with CD4 counts > 100/mm3 had normal percentages of CD34+ cells; of 14 patients with low CD4 cell counts (< 100/mm3), 5 had reduced and 9 had normal percentages of CD34+ cells. Samples of the marrow were plated in a methylcellulose colony-forming unit (CFU) assay to determine the clonogenic capacity of the progenitor cells. Overall, the marrow samples from HIV-infected donors showed a 44% reduction in CFU derived from the mononuclear cell fraction and a 75% reduction in CFU derived from the isolated CD34+ cell fraction, when compared to marrow samples from uninfected donors. Isolated CD3+ cells were transduced with retroviral vectors containing various anti-HIV-1 genes to determine their susceptibility to gene transfer. Transduction of the clonogenic CD34+ cells by retroviral vectors did not differ among marrow samples from 13 HIV-1+ donors and 9 uninfected donors. Long-term bone marrow cultures established from the transduced CD34+ cells demonstrated equivalent survival of clonogenic progenitor cells from both HIV-1-infected and uninfected marrows. Toxicity from expression of the anti-HIV-1 genes was not observed; the percentages of clonogenic progenitor cells that survived in cultures transduced by vectors carrying anti-HIV-1 genes were similar to those transduced by the control LN vectors. Stromal cells cultured from marrow samples from HIV-1-infected donors showed similar growth kinetics, hematopoietic support function, and enhancement of retrovirus-mediated transduction of CD34+ cells as seen with stromal cells cultured from uninfected marrow donors. Semi-quantitative polymerase chain reaction (PCR) was performed before and after ex vivo transduction to determine the frequency of HIV-1-containing cells in the CD34+ cell preparations. Although HIV-1+ cells were present at low levels in the mononuclear cell fractions of some of the marrow samples, the CD34+ cell preparation from only one marrow sample contained detectable HIV-1 positive cells (< 1 positive cell/100,000 by PCR) prior to transduction. None of the CD34+ cell preparations contained detectable HIV-1 after transduction. These studies demonstrate that HIV-1-infected patients are candidates for retrovirus-mediated transduction of anti-HIV-1 genes in bone marrow gene therapy clinical trials.

Transduction of human CD34+ hematopoietic progenitor cells by a retroviral vector expressing an RRE decoy inhibits human immunodeficiency virus type 1 replication in myelomonocytic cells produced in long-term culture.

Genetic modification of hematopoietic stem cells with a synthetic "anti-human immunodeficiency virus type 1 (HIV-1) gene" which inhibits replication of HIV-1 may allow production of mature lymphoid and monocytic cells resistant to HIV-1 growth after autologous transplantation. Because productive HIV-1 replication requires binding of the Rev protein to the Rev-responsive element (RRE) within the viral transcripts for the HIV-1 structural proteins, anti-HIV-1 gene products which interfere with Rev-RRE interactions may inhibit HIV-1 replication. One such strategy involves overexpression of the RRE sequences in transcripts derived from retroviral vectors to act as decoys to sequester Rev protein and prevent its binding to the RRE element in HIV-1 transcripts. We developed an in vitro model to test the efficacy of this gene therapy approach in primary human hematopoietic cells. Human CD34+ hematopoietic progenitor cells from normal bone marrow or umbilical cord blood were transduced with retroviral vectors carrying RRE decoy sequences as part of a long terminal repeat-directed transcript expressing the neo gene (L-RRE-neo) or with a control vector expressing only the neo gene (LN). The transduced progenitors were allowed to differentiate into mature myelomonocytic cells which were able to support vigorous growth of the monocytotropic isolate of HIV-1, JR-FL. HIV-1 replication was measured in unselected cell populations and following G418 selection to obtain uniformly transduced cell populations. Inhibition of HIV-1 replication in the unselected cell cultures was between 50.2 and 76.7% and was highly effective (99.4 to 99.9%) in the G418-selected cultures. Progenitors transduced by either the L-RRE-neo vector or the control LN vector were identical with respect to hematopoietic growth and differentiation. These findings demonstrate the ability of an RRE decoy strategy to inhibit HIV-1 replication in primary human myelomonocytic cells after transduction of CD34+ progenitor cells, without adverse effects on hematopoietic cell function.

Effects of NO synthase inhibition on the muscular blood flow response to treadmill exercise in rats.

The functional role of nitric oxide (NO) release in regulating blood flow (BF) to exercising skeletal muscle was studied in conscious male Sprague-Dawley rats (603 +/- 28 g; n = 6). In this study, BF was measured using radiolabeled microspheres during treadmill exercise (10% grade, 20 m/min) before and after NO synthase (NOS) inhibition with NG-nitro-L-arginine methyl ester (30 mg/kg ia). After NOS inhibition, mean arterial blood pressure increased from resting baseline values and the duration of vasodilator responses to acetylcholine (ACh) injections (3.0 and 10.0 micrograms/kg ia) was diminished (P < 0.05), demonstrating reduced NOS function. During exercise, BF to the kidneys and organs of the gut was reduced after NOS inhibition. In addition, BF was reduced in 16 of the 28 individual hindquarter muscles or muscle parts. Moreover these reductions in BF were linearly correlated with the estimated sum of the percentage of fast-twitch oxidative glycolytic (FOG) and slow-twitch oxidative (SO) types of fibers found in each muscle [delta BF = -1.1 (%SO + %FOG) + 16.4; r = 0.88, P < 0.001]. These results suggest that NO-mediated vasodilation contributes to the BF responses within and among the muscles of the rat's hindquarters during exercise.

Fine mapping of the locus for nevoid basal cell carcinoma syndrome on chromosome 9q.

The nevoid basal cell carcinoma syndrome is an autosomal dominant disorder characterized primarily by multiple basal cell carcinomas, odontogenic keratocysts, and pits of the palms and soles. Tumor deletion studies and linkage analysis in Caucasians have revealed that the gene is on chromosome 9q. To further refine the location of the nevoid basal cell carcinoma syndrome locus, we tested linkage to this region in three families. Evaluation of recombinants suggested that the nevoid basal cell carcinoma syndrome locus lies in the interval defined distally by D9S127. Our data, together with existing published data defining D9S12 as a proximal flanking marker, refine the location of nevoid basal cell carcinoma syndrome to an 8.3-cM interval. Two of the families studied were African-American and show a notable variation in phenotypic expression in which affected individuals developed few skin cancers. However, despite clinical heterogeneity, our data are consistent with the hypothesis that the same locus is involved in these African-American families.