Biomedical subjects
Publications and source records attributed to S Watkins.
In due course, magnetic resonance myocardial perfusion imaging will probably replace SPECT as the most widely used non-invasive method for detecting reversible myocardial ischaemia.
When a single brief visual flash is accompanied by two auditory bleeps, it is frequently perceived incorrectly as two flashes. Here, we used high field functional MRI in humans to examine the neural basis of this multisensory perceptual illusion. We show that activity in retinotopic visual cortex is increased by the presence of concurrent auditory stimulation, irrespective of any illusory perception. However, when concurrent auditory stimulation gave rise to illusory visual perception, activity in V1 was enhanced, despite auditory and visual stimulation being unchanged. These findings confirm that responses in human V1 can be altered by sound and show that they reflect subjective perception rather than the physically present visual stimulus. Moreover, as the right superior temporal sulcus and superior colliculus were also activated by illusory visual perception, together with V1, they provide a potential neural substrate for the generation of this multisensory illusion.
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The development of a strategy to deliver a gene to pulmonary endothelium will be useful for gene function study and for pulmonary gene therapy. Cationic lipidic vectors are efficient in gene transfer to pulmonary endothelium via the vascular route; however, gene expression is transient and lasts for only a few days. In this study, we show that pulmonary gene transfer via cationic lipidic vectors can be significantly improved using an Epstein-Barr virus (EBV)-based expression plasmid. Systemic administration of cationic liposomes followed by the EBV-based plasmid led to gene expression in the lung that lasted for more than 3 weeks. Prolonged and high levels of gene expression can also be obtained in primary mouse lung endothelial cells (MLEC) following lipofection with an EBV-based plasmid. These results suggest the utility of this gene transfer protocol in studying the expression of cloned genes in lung endothelial cells and in pulmonary gene therapy.
In the urinary bladder, the capsaicin-gated ion channel TRPV1 is expressed both within afferent nerve terminals and within the epithelial cells that line the bladder lumen. To determine the significance of this expression pattern, we analyzed bladder function in mice lacking TRPV1. Compared with wild-type littermates, trpv1(-/-) mice had a higher frequency of low-amplitude, non-voiding bladder contractions. This alteration was accompanied by reductions in both spinal cord signaling and reflex voiding during bladder filling (under anesthesia). In vitro, stretch-evoked ATP release and membrane capacitance changes were diminished in bladders excised from trpv1(-/-) mice, as was hypoosmolality-evoked ATP release from cultured trpv1(-/-) urothelial cells. These findings indicate that TRPV1 participates in normal bladder function and is essential for normal mechanically evoked purinergic signaling by the urothelium.
Somatic gene transfer to the pulmonary endothelium may be a useful strategy for modifying the phenotype of endothelium and/or vascular smooth muscle in disorders such as primary pulmonary hypertension, ARDS or pulmonary metastatic disease. Adenoviral (Ad) vectors, although highly efficient in liver gene transfer, have proven to be limited for pulmonary gene transfer with respect to efficiency, in part because of difficulty in assuring significant residence time in the lung and/or paucity of receptors for adenovirus on the endothelium. A recent study has shown that the use of a bispecific antibody to endothelial cells and Ad vectors efficiently redirects Ad vectors to pulmonary endothelium and improves gene expression in the lung. In this study, we report that pulmonary gene transfer by Ad vectors can also be improved significantly via the use of cationic liposomes. Preinjection of cationic liposomes followed by adenovirus led to a significant increase in the level of gene expression in the lung. The improvement in pulmonary gene transfer was associated with a decrease in the level of gene expression in the liver. Gene expression in the lung lasted for up to 2 weeks. This protocol, together with genetic modification of adenovirus, may prove to be useful for pulmonary gene transfer for the treatment of pulmonary diseases. This method may also be extended to pulmonary gene transfer using other types of viral vectors via vascular route.
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This study tested the hypothesis that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) antagonizes estrogen-induced hepatic lipid synthesis and metabolism in birds. Twenty immature male chickens (Gallus domesticus) were divided evenly into four groups: (1) vehicle control; (2) estrogen alone (1.0 mg/kg estradiol cypionate injected on three consecutive days); (3) TCDD alone (50 microg/kg injected on the fourth day); and (4) a combination of the estrogen and TCDD treatments. On day 14, liver samples were collected for quantitative fatty acid analysis by capillary gas chromatography. Birds treated with estrogen alone had increased total triacylglyceride concentrations with specific increases in the Delta9 desaturase products 16:1n7, 18:1n7, 18:1n9, and 20:1n9. In addition, estrogen treatment specifically increased 22:6n3 concentrations in both triacylglycerides and phospholipids. However, these increases in Delta9 desaturase products or 22:6n3 did not occur for birds treated with estrogen in combination with TCDD. TCDD and estrogen plus TCDD treatments increased phospholipid concentrations of the diet-derived polyunsaturated fatty acids 18:2n6, 18:3n6, 20:3n6, 18:3n3, and 20:5n3, although only the estrogen plus TCDD group had significantly increased total phospholipids. In cholesterol esters, all three treatments decreased concentrations of total fatty acids, saturated fatty acids, and Delta9 desaturase products compared to the control group.
Interleukin-12 (IL-12) mediates significant antitumor effects in animal models but associated with dose-dependent toxicity in human. To achieve local expression of IL-12 at the tumor site without systemic toxicity, we performed intra-arterial administration of fibroblasts genetically engineered to produce IL-12 protein with or without retrovirus (CRIP- IL-12 or 3T3-IL-12) in liver metastasis model. Rat breast cancer cells ( MADB - 106) were injected into the portal vein of syngeneic Fisher rats on day 0, and fibroblasts were injected into the hepatic artery on day 7. On day 21, liver weight and number of liver tumors were examined. As controls, CRIP cells expressing retrovirus carrying lacZ marker gene (CRIP-lacZ) or saline (Hanks balanced salt solution, HBSS) were injected. Administration of CRIP-IL-12 significantly reduced tumor metastasis in liver measured by number of foci (CRIP- IL-12: 45.2 +/- 36.7, CRIP-lacZ: >250, HBSS: >250, P<.05) and by liver weight (CRIP-IL-12: 13.0+/-2.5 g, CRIP-lacZ: 30.4+/-8.5 g, HBSS: 26.0+/-7.6 g, P<.05). 3T3-IL-12, which produced only IL-12 protein but not IL-12 retrovirus, also had significant antitumor effects equivalent to CRIP-IL-12. Intra-arterial injection of IL-12--producing fibroblasts into the liver may be an effective therapy for liver tumors reducing systemic toxicity, and could be developed for clinical application.
The authors report the case of a 29-week-gestation twin with very severe vascular compromise of both lower limbs secondary to premature twin-twin transfusion. A tissue plasminogen activator (tPA) infusion was used locally for 13 days with complete recovery of perfusion to both legs, demonstrated by serial angiograms, thereby avoiding bilateral amputation. There were no side effects as a result of the continuous administration of tPA. The authors therefore suggest that the benefits of thrombolysis from a local infusion of tPA in neonates may outweigh the potential risks. This case report thus supports the view that under certain circumstances infusion of tPA in neonates may offer significant benefits. J Pediatr Surg 36:654-656.
BACKGROUND/PURPOSE: Interleukin 12 (IL-12) is a potent proinflammatory cytokine that enhances cytotoxic T lymphocytes (CTL) and natural killer (NK) cell activity. The goal of these experiments was to assess whether adenoviral-mediated IL-12 expression by dendritic cells (DC) could induce an antitumor immune response in a murine model of neuroblastoma. METHODS: Syngeneic A/J mice were inoculated subcutaneously with 1 x 10(6) cells from a murine neuroblastoma-derived cell line (TBJ). Murine DC were transduced in vitro with adCMV-mIL-12, and 1 x 10(6) cells were injected intratumorally. Tumor growth in these mice was compared with control animals injected with enhanced green-filled protein (EGFP) transduced DC or saline. The role of CTL was evaluated through cytotoxicity assays. Immunohistochemical analysis of tumor, spleen, and lymph node was performed to characterize the behavior and fate of various populations of immune effector cells in these tissues. RESULTS: The tumors of mice injected with adIL-12 transduced DC all underwent complete regression over a 3-week period. Splenocytes isolated from mice 7 days after intratumoral injection of adIL-12 DC showed increased cytolytic activity relative to control animals in vitro. Immunohistochemistry of tumor and lymph tissue showed increased amounts of DC and T lymphocyte infiltration and a slight decrease in apoptosis relative to the control groups. CONCLUSIONS: Increased IL-12 production by DC induced a significant antitumor response in a poorly immunogenic murine neuroblastoma model. These results show the vital role of DC in the immunobiology of the disease, and that protection of these cells from tumor induced apoptosis is a critical aspect for immunotherapies treating this aggressive tumor.
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FSH binding to Sertoli cells is required for optimal production of sperm in mammals. The cAMP response element-binding protein (CREB) is a major mediator of FSH-induced changes in gene expression. To determine whether CREB is required for spermatogenesis, an adenovirus encoding a phosphorylation-defective CREB mutant (AdCREBm1) was used to inhibit CREB activity in Sertoli cells. Addition of AdCREBm1 to primary rat Sertoli cell cultures completely abolished induction of the CREB-regulated c-fos gene. Injection of an adenovirus encoding ss-galactosidase into the rat testis seminiferous tubules in vivo demonstrated that predominately Sertoli cells were infected by adenovirus. AdCREBm1-directed expression of CREBm1 in seminiferous tubules did not affect Sertoli cell viability, but resulted in the apoptosis of meiotic spermatocyte germ cells within 4 days of adenovirus injection into seminiferous tubules. Disrupted spermatogenesis, defined by at least a 75% reduction of round spermatids, was observed in 42 +/- 5.8% of seminiferous tubules 14 days after AdCREBm1 infection, whereas using this criteria, testes injected with a control adenovirus did not display disrupted spermatogenesis. These data demonstrate that AdCREBm1 causes apoptosis and elimination of germ cells and suggest that CREB is required to produce a Sertoli cell-derived factor that is critical for germ cell survival.
We examined the hypothesis that an excess accumulation of intramuscular lipid (IMCL) is associated with insulin resistance and that this may be mediated by the oxidative capacity of muscle. Nine sedentary lean (L) and 11 obese (O) subjects, 8 obese subjects with type 2 diabetes mellitus (D), and 9 lean, exercise-trained (T) subjects volunteered for this study. Insulin sensitivity (M) determined during a hyperinsulinemic (40 mU x m(-2)min(-1)) euglycemic clamp was greater (P < 0.01) in L and T, compared with O and D (9.45 +/- 0.59 and 10.26 +/- 0.78 vs. 5.51 +/- 0.61 and 1.15 +/- 0.83 mg x min(-1)kg fat free mass(-1), respectively). IMCL in percutaneous vastus lateralis biopsy specimens by quantitative image analysis of Oil Red O staining was approximately 2-fold higher in D than in L (3.04 +/- 0.39 vs. 1.40 +/- 0.28% area as lipid; P < 0.01). IMCL was also higher in T (2.36 +/- 0.37), compared with L (P < 0.01). The oxidative capacity of muscle determined with succinate dehydrogenase staining of muscle fibers was higher in T, compared with L, O, and D (50.0 +/- 4.4, 36.1 +/- 4.4, 29.7 +/- 3.8, and 33.4 +/- 4.7 optical density units, respectively; P < 0.01). IMCL was negatively associated with M (r = -0.57, P < 0.05) when endurance-trained subjects were excluded from the analysis, and this association was independent of body mass index. However, the relationship between IMCL and M was not significant when trained individuals were included. There was a positive association between the oxidative capacity and M among nondiabetics (r = 0.37, P < 0.05). In summary, skeletal muscle of trained endurance athletes is markedly insulin sensitive and has a high oxidative capacity, despite having an elevated lipid content. In conclusion, the capacity for lipid oxidation may be an important mediator of the association between excess muscle lipid accumulation and insulin resistance.
In obesity and type 2 diabetes, skeletal muscle has been observed to have a reduced oxidative enzyme activity, increased glycolytic activity, and increased lipid content. These metabolic characteristics are related to insulin resistance of skeletal muscle and are factors potentially related to muscle fiber type. The current study was undertaken to examine the interactions of muscle fiber type in relation to oxidative enzyme activity, glycolytic enzyme activity, and muscle lipid content in obese and type 2 diabetic subjects compared with lean healthy volunteers. The method of single-fiber analysis was used on vastus lateralis muscle obtained by percutaneous biopsy from 22 lean, 20 obese, and 20 type 2 diabetic subjects (ages 35+/-1, 42+/-2, and 52+/-2 years, respectively), with values for BMI that were similar in obese and diabetic subjects (23.7+/-0.7, 33.2+/-0.8, and 31.8+/-0.8 kg/m2, respectively). Oxidative enzyme activity followed the order of type I > type IIa > type IIb, but within each fiber type, skeletal muscle from obese and type 2 diabetic subjects had lower oxidative enzyme activity than muscle from lean subjects (P < 0.01). Muscle lipid content followed a similar pattern in relation to fiber type, and within each fiber type, muscle from obese and type 2 diabetic subjects had greater lipid content (P < 0.01). In summary, based on single-fiber analysis, skeletal muscle in obese and type 2 diabetic subjects mani-fests disturbances of oxidative enzyme activity and increased lipid content that are independent of the effect of fiber type.
The anti-inflammatory cytokine interleukin (IL)-10 suppresses inducible nitric oxide synthase (iNOS); therefore, NO production should increase in the absence of IL-10. Production of NO (as nitrite) by bronchoalveolar lavage cells of IL-10 knockout ((-/-)) mice was assessed after ovalbumin sensitization and airway challenge (S/C) and was compared with the IL-10-sufficient, wild-type (WT) C57Bl6. Eosinophil recruitment occurred in S/C WT and IL-10(-/-) mice, suggesting allergic airway inflammation. Alveolar macrophages (per g mouse) were unchanged (approximately 3x10(4) cells) with the exception of a doubling in the S/C IL-10(-/-) mice (approximately 6x10(4) cells, P<0.05). NO production (per million cells) was doubled in cells from S/C IL-10(-/-) (15.3 microM) mice compared with WT (7.6 microM, P<0.05). Inhibition of iNOS by L-N(5)-(1-iminoethyl)-ornithine reduced NO production in all S/C mice, confirming that the increase was a result of up-regulation of iNOS. We conclude that IL-10 is a critical cytokine regulating iNOS in murine airway cells and that its absence can lead to up-regulation of iNOS and development of allergic airway inflammation.
Oxidative stress can trigger neuronal cell death and has been implicated in several chronic neurological diseases and in acute neurological injury. Oxidative toxicity can be induced by glutamate treatment in cells that lack ionotrophic glutamate receptors, such as the immortalized HT22 hippocampal cell line and immature primary cortical neurons. Previously, we found that neuroprotective effects of geldanamycin, a benzoquinone ansamycin, in HT22 cells were associated with a down-regulation of c-Raf-1, an upstream activator of the extracellular signal-regulated protein kinases (ERKs). ERK activation, although often attributed strictly to neuronal cell survival and proliferation, can also be associated with neuronal cell death that occurs in response to specific insults. In this report we show that delayed and persistent activation of ERKs is associated with glutamate-induced oxidative toxicity in HT22 cells and immature primary cortical neuron cultures. Furthermore, we find that U0126, a specific inhibitor of the ERK-activating kinase, MEK-1/2, protects both HT22 cells and immature primary cortical neuron cultures from glutamate toxicity. Glutamate-induced ERK activation requires the production of specific arachidonic acid metabolites and appears to be downstream of a burst of reactive oxygen species (ROS) accumulation characteristic of oxidative stress in HT22 cells. However, inhibition of ERK activation reduces glutamate-induced intracellular Ca(2+) accumulation. We hypothesize that the precise kinetics and duration of ERK activation may determine whether downstream targets are mobilized to enhance neuronal cell survival or ensure cellular demise.