Voltage-dependent sodium and calcium currents in acutely isolated adult rat trigeminal root ganglion neurons

Voltage-dependent sodium (INa) and calcium (ICa) currents in small (<30 microM) neurons from adult rat trigeminal root ganglia were characterized with a standard whole cell patch-clamp technique. Two types of INa showing different sensitivity to tetrodotoxin (TTX) were recorded, which showed marked differences in their activating and inactivating time courses. The activation and the steady-state inactivation kinetics of TTX-resistant INa were more depolarized by about +20 and +30 mV, respectively, than those of TTX-sensitive INa. Voltage-dependent ICa was recorded under the condition that suppressed sodium and potassium currents with 10 mM Ca2+ as a charge carrier. Depolarizing step pulses from a holding potential of -80 mV evoked two distinct inward ICa, low-voltage activated (LVA) and high-voltage activated (HVA) ICa. LVA ICa was first observed at -60 to -50 mV and reached a peak at about -30 mV. Amiloride (0.5 mM) suppressed approximately 60% of the LVA ICa, whereas approximately 10% of HVA ICa was inhibited by the same concentration of the amiloride. LVA ICa was far less affected by the presence of external Cd2+ or the replacement of Ca2+ by 10 Ba2+ than HVA ICa. The omega-conotoxin GVIA (omega-CgTx), an N-type ICa blocker, suppressed approximately 65% of the whole cell HVA ICa at the concentration of 1 microM. The omega-CgTx-resistant HVA ICa was sensitive to nifedipine (10 microM), a dihydropyridine (DHP) calcium channel antagonist, which produced an additional blockade by approximately 25% of the drug-free control ( approximately 70% of the omega-CgTx-resistant ICa). The combination of 10 microM nifedipine and 1 microM omega-CgTx left approximately 13% of the drug-free control ICa unblocked. The DHP agonist S(-)-BayK8644 (5 microM) shifted the activation of the HVA ICa to more negative potentials and increased its maximal amplitude. Additionally, S(-)-BayK8644 caused the appearance of a slowed component of the tail current. These results clearly demonstrate that the presence of two types of sodium channels, TTX sensitive and resistant, and three types of calcium channels, T, L, and N type, in the small-sized adult rat trigeminal ganglion neurons.     

Modulation of CD8 and CD3 by HIV or HIV antigens

To investigate whether human immunodeficiency virus (HIV)-1 and HIV-1 antigens modulate surface and cytoplasmic CD8 or CD3, as well as CD4, we used cell permeabilization reagents, surface/cytoplasmic fluorescent staining, multiparameter flow cytometric techniques and an in vitro culture system in which relatively few lymphocytes are actively infected with HIV. Human peripheral blood lymphocytes were: not stimulated, not stimulated but HIV-inoculated, phytohaemagglutinin (PHA)-stimulated, PHA/HIV-inoculated (PHA/HIV), or placed into media with soluble gp120, Rev or Nef. HIV inoculation and Nef had striking modulatory effects on CD8. The cytoplasmic CD8 median fluorescent intensity (MFI) of positive lymphocytes was lower for cells in unstimulated/HIV-infected cultures than unstimulated cultures (44 versus 62% of ex vivo value, P = 0.032) and lower for cells in PHA/HIV cultures than in PHA cultures (56 versus 100% of ex vivo, P = 0.041). The surface CD8 MFI values for Nef were significantly lower than the ex vivo value (75% of ex vivo, P = 0.006). At days 2-7 of culture, Rev was associated with slight reductions in surface CD4 MFI (58% of ex vivo versus 78% of ex vivo for unstimulated cultures, P = 0.047) and greater effects on cytoplasmic CD3 MFI (131 versus 179% of ex vivo for unstimulated cultures, P = 0.035), and surface CD8 MFI (70% of ex vivo, P = 0.006 versus ex vivo value). The globality of Rev's effects suggests these are related to a shared processing pathway, i.e. not due to direct interaction with CD3, CD4 and CD8; the effects of HIV inoculation and Nef on CD8 expression appear to be more CD8 specific. Because CD8 is essential for cytotoxic T-cell function, its down-modulation could inhibit this activity, including anti-HIV cytotoxicity. Given the critical roles of CD3 and CD8 in T-lymphocyte signal transduction and antigen responsiveness, the effects of HIV, Rev and Nef on these molecules have clinically significant implications concerning the pathogenesis and treatment of HIV.     

Carbon plasma immersion ion implantation of nickel-titanium shape memory alloys

Nickel-titanium (NiTi) shape memory alloys possess super-elasticity in addition to the well-known shape memory effect and are potentially suitable for orthopedic implants. However, a critical concern is the release of harmful Ni ions from the implants into the living tissues. We propose to enhance the corrosion resistance and other surface and biological properties of NiTi using carbon plasma immersion ion implantation and deposition (PIII&D). Our corrosion and simulated body fluid tests indicate that either an ion-mixed amorphous carbon coating fabricated by PIII&D or direct carbon PIII can drastically improve the corrosion resistance and block the out-diffusion of Ni from the materials. Our tribological tests show that the treated surfaces are mechanically more superior and cytotoxicity tests reveal that both sets of plasma-treated samples favor adhesion and proliferation of osteoblasts.     

Tissue engineering of arteries by directed remodeling of intact arterial segments

Traditional approaches to generating tissue-engineered arteries in vitro rely on expansion of cells in culture to seed appropriate scaffolds. In most envisioned applications, small autologous blood vessels would be harvested and used as a source for these cells. We propose that small autologous arteries, not the cells derived from them, may be an attractive starting point for engineered arteries. This approach capitalizes on the ability of intact arteries to grow and remodel in response to chronic changes in their mechanical environment. Carotid arteries from juvenile (approximately 30-kg) pigs were stretched longitudinally in an ex vivo perfusion system over 9 days. This resulted in a 40% increase in artery length at physiological longitudinal stress and a 20 +/- 3% increase when unstressed. Control arteries were perfused for 9 days ex vivo at their physiological loaded length. Control and elongated arteries displayed native appearance (macroscopic and histological), excellent viability (cellularity and mitochondrial activity), normal vasoactivity, and similar mechanical properties (ultimate stress and ultimate strain) as compared with freshly harvested arteries. Growth, as opposed to just redistribution of existing mass, contributed to elongation as evidenced by an increase in artery weight. Results on elongation of arteries from neonatal and adolescent pigs are also presented and discussed.     

Differences in extent of genetic introgression between sympatric Culex pipiens and Culex quinquefasciatus (Diptera: Culicidae) in California and South Africa

Comparisons of five morphological characters, 12 enzyme electrophoresis profiles, and Wolbachia pipientis infection rates were used to characterize populations of members of the Culex pipiens L. complex in California and South Africa. In South Africa, male phallosome DV/D ratio, male maxillary palp index, branching of siphonal seta 1a, the enzyme locus Mdhp-1, and W. pipientis infection rates proved highly diagnostic for separating Culex quinquefasciatus from Cx. pipiens phenotypes. In Johannesburg, where sympatric members of the Cx. pipiens complex were analyzed as one population, a significant Wahlund Effect was observed in the enzyme loci such as Ao, 6-Pgdh, Mdh-2, and Pgm. In California, all populations of the Cx. pipiens complex were in Hardy Weinberg equilibrium at all polymorphic enzyme loci examined. Additionally, in California, all populations had similar W. pipientis infection rates and appeared morphologically identical (except for DV/D ratio, in extreme north and south). These findings indicate that in South Africa, Cx. pipiens and Cx. quinquefasciatus remain as genetically distinct populations and behave as separate species. Conversely, in California, there is considerable genetic introgression between Cx. pipiens and Cx. quinquefasciatus, and they behave as a single species.     

Development and characterisation of neutralising monoclonal antibody to the SARS-coronavirus

There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103 mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.     

Changes of interleukin expression correlate with Helicobacter pylori infection and lymph node metastases in gastric carcinoma.

Helicobacter pylori (HP) infection induces expression of IL-8 and IL-10 in benign gastric epithelium. This study compared the expression of cytokines in CD4+ and CD8+ lymphocyte subsets of peripheral blood lymphocytes (PBL), benign mucosal lymphocytes (ML), and tumor infiltrative lymphocytes (TIL) as well as in the benign and malignant epithelial cells of the same patient, with respect to the presence of HP infection, lymph node metastases, and tumor histologic type. The mRNA of the cytokines was measured by a semiquantitative RT-PCR method. The levels were ranked and compared using the Wilcoxon sign-ranked test. Compared with CD8+ ML, the CD8+ TIL expresses higher levels of IL-6 and IL-8 but lower level of IL-4 in patients with lymph node metastases. In patients with HP infection, expression of IL-8 and IL-10 was higher in the gastric carcinoma cells than in the benign epithelial cells while expression of IL-6 and IL-8 were higher in CD8+ TIL than CD8+ ML. Overexpression of IL-8 in HP associated gastric carcinomas suggested that they might have arisen from HP-infected epithelial cells.     

Synergistic antimicrobial effect of cefotaxime and minocycline on proinflammatory cytokine levels in a murine model of Vibrio vulnificus infection.

BACKGROUND AND PURPOSE: Vibrio vulnificus causes primary bacteremia and necrotizing wound infection, leading to high morbidity and mortality in humans. This study aimed to evaluate the antimicrobial effect of cefotaxime and minocycline on proinflammatory cytokine levels in a murine model of V. vulnificus infection. METHODS: We investigated the dynamics of proinflammatory cytokines and their modulation by antimicrobial agents using a murine model of V. vulnificus infection. The change in cytokine levels was followed over a time course to identify the antimicrobial activity of the drugs against V. vulnificus. BALB/c female mice were challenged with an intraperitoneal infection using a clinical invasive isolate of Vv05191, and their cytokine levels were assayed over various time points. RESULTS: Serum levels of tumor necrosis factor-alpha, interleukin (IL)-1 beta, and IL-6 post-infection were found to be inoculum dose-dependent and positively correlated to the subsequent fatality rate in the infected mice. With an inoculum of 6.6 x 10(6) colony-forming units and intraperitoneal administration of cefotaxime, minocycline, or both, the serum and peritoneal fluid cytokine levels increased and then declined gradually. Comparison of the 3 antimicrobial regimens revealed that the magnitude of reduction in cytokine levels was greatest in mice treated with cefotaxime-minocycline combination. Moreover, the peritoneal fluid cytokine level in the combination group was significantly lower than that in the groups treated with minocycline or cefotaxime alone. CONCLUSIONS: The current results support the superiority of the combination therapy in treating invasive V. vulnificus infections.     

Comparison of Gadomer-17 and Gd-DTPA in image quality of contrast-enhanced MR angiographies using flow phantom model

The purpose of this study was to compare the image quality of 3D-TOF MR angiography (MRA) using Gadomer-17 with that using Gd-DTPA in a flow phantom model, and to present preliminary data about the proper dose concentration of Gadomer-17. In the visual analysis of vessel conspicuity, we compared the quality of pre- and post-contrast MIP images. For quantitative analysis, the signal intensities were measured in the axial base 3D-TOF images, and then the relative contrast enhancement was calculated. The results of our studies were that: 1. Maximal signal intensities were obtained at 1 mmol/L of Gadomer-17 and 4 mmol/L of Gd-DTPA. 2. Flow-related signal loss was decreased by Gd-DTPA proportional to the concentration, but Gadomer-17 did not show such a dose accumulative effect. In conclusion, after comparing the results of Gd-DTPA, it was clear that improved MRA images and higher signal intensities of vessels were obtained when lower concentrations of Gadomer-17 were used.