Evaluation and optimization of a latex agglutination assay for detection of cholera toxin and Escherichia coli heat-labile toxin.

The effectiveness of a latex agglutination assay kit for the detection of Escherichia coli heat-labile toxin and cholera toxin was determined for the identification of natural isolates of the corresponding enteric pathogens in Southeast Asia. By selection of the appropriate culture media, the sensitivity of the assay was improved from 90.6% (for the detection of heat-labile toxin) and 75% (for the detection of cholera toxin) to 100%, and the results were confirmed with bioassays and DNA hybridization assays for both clinical and environmental isolates.     

Effect of biphenyl dimethyl dicarboxylate on the humoral immunosuppression by ethanol

The present study was undertaken to investigate the effect of biphenyl dimethyl dicarboxylate (PMC) on the humoral immunosuppression by ethanol (EtOH) in ICR mice. PMC at a dose of 6 mg/kg was orally administered to mice daily for 28 consecutive days, and the control mice were given vehicle. Mice treated with EtOH were given freely with 20% EtOH instead of water. The results of this study are summarized as follows; a gain of body weight and the relative weights of spleen and liver were significantly increased by combination of PMC and EtOH, as compared with those in mice treated with EtOH alone. Splenic plaque forming cells (PFC) and hemagglutination (HA) titers to sheep red blood cells (SRBC), and the secondary IgG antibody response to bovine serum albumin (BSA) were decreased by the treatment of EtOH alone, then restored to normal level by PMC treatment. The elevations of serum glutamic-pyruvic transaminase (S-GPT) and total protein levels caused by EtOH were reduced to normal level by the combination of PMC and EtOH. In addition, lower serum albumin and A/G ratio were also increased to normal level. These findings indicate that PMC has a protective effect against EtOH-induced humoral immunosuppression.     

Mesh-independent prediction of skin burns injury

This paper presents a robust finite element model (FEM) with multiple-layers of varying properties for investigation of burn effects on human skin during a burning process resulting from exposure of skin surface to a contact heat source and a hot moving fluid. Henriques' theory of skin burns is used in conjunction with two-dimensional Pennes bioheat transfer equation for determining the spatial and temporal extent of burn injury. The model developed is a two-dimensional axisymmetric model in cylindrical coordinates. The various tissue layers account for changing thermal properties with respect to skin anatomy. A finite element scheme that uses the backward Euler method is used to solve the problem. The injury processes of skin subsequent to the removal of the heat source (post-burn) will also be inspected. The mesh employed in this model consists of a high density of nodes and elements in which a thorough mesh convergence study was done. A comparison of the transient temperature field computed by this model against Diller's results using the FE technique with a comparatively coarse mesh of 125 elements and experimental data by Orgill et al. has been done in the present study. It concluded that improved accurate solutions have been performed using the robust model developed due to the achievement of a mesh-independent solution.     

Quality control of storage phosphor digital radiography systems

Quality control (QC) of storage phosphor devices is important in assuring that the image information entered into an Image management and communication (IMAC) system is sufficient for diagnosis. QC of storage phosphor digital radiography systems is complex because of the self-corrective nature of the image-processing software used in these machines. Currently, one must produce hard copy to perform adequate QC. Inspection of images with reject analysis and inspection of cassettes and imaging plates has helped us in our QC program. For those QC tests using control limits, the appropriate settings for these limits are unknown. Starting approximations are given. Recommended tests are described.     

Cytokine production in lethal and non-lethal murine malaria

Levels of IFN-gamma, IL-2 and IL-4 were measured in vitro during the course of non-lethal Plasmodium chabaudi adami and lethal P. chabaudi strain 1309 infections in BALB/cByJ mice. Spleen cells from mice infected with the non-lethal Plasmodium had a higher initial response to P. chabaudi antigens than mice infected with P. chabaudi strain 1309, as determined by measuring all three lymphokines. We conclude that both Th1 and Th2 subsets of T helper lymphocytes are activated during P. chabaudi adami infection but that these responses are suppressed in mice infected with the more virulent P. chabaudi strain 1309.     

Prospective study of community-acquired rotavirus infection.

We determined titers of group A rotavirus common antibodies and neutralizing antibodies against serotypes 1 to 4 of prototype human rotavirus (HRV) in cord blood and serum specimens obtained from 38 infants at 4-month intervals from birth until 2 years of age. Nineteen of the infants developed one episode of HRV diarrhea each, and they were matched by age and birth weight with the other 19 infants, who did not develop HRV diarrhea during the follow-up period. We estimated the incidence rate of HRV infection for the two groups of infants combined to be a minimum of 1.34 episodes per infant per year, which is 22 times more common than the occurrence of overt disease caused by the virus in this community. The infection occurred constantly throughout the first 2 years of infancy, whereas all but one of the 19 episodes of overt disease occurred before 12 months of age. Seven of these overt episodes were preceded by at least one episode of subclinical infection earlier, and the other seven were probably due to primary HRV infection. The remaining five episodes occurred before 4 months of age, so that we could not ascertain whether they were due to primary infections because of the presence of maternal antibodies. We showed that levels of HRV antibodies in serum specimens obtained before clinical onset of diarrhea varied widely, and, for most infants in the diarrheal group, levels of these antibodies were similar to those in the serum specimens obtained at the same times from the corresponding age- and birth weight-matched control infants. Nevertheless, the age at which overt disease caused by HRV was most prevalent coincided with the time when the maternal antibodies had declined to low levels but the infants had not yet acquired high titers of these antibodies in their sera.     

Antigens of virulent and attenuated Rickettsia tsutsugamushi.

We studied the antigens present in L929 mouse fibroblast or rabbit testicular cells, which had been infected or not with a prototype strain of Rickettsia tsutsugamushi, the causal agent of scrub typhus, and its attenuated variant. Immunoblotting revealed four antigens, designated 1, 1a, 2 and 3, which appeared to be specifically associated with infection with this organism. Antigens 1 and 1a had similar mol. wt of about 60 kD and antigen 2 and 3 had mol. wts of 45 kD and 28 kD respectively. Whereas antigen 1a, 2 and 3 were common to infection with either the virulent or the attenuated strains of the organism, antigen 1 was only detected in cells infected with the virulent strain and was reactive only with the antiserum raised against cells infected with that strain. In addition, two antigens were also detected by crossed immunoelectrophoresis, one of which was similarly associated with infection with the virulent strain as antigen 1, while the other was common to infection with either of the strains. It seems that the antigenic cross reaction between the two strains may account, in part at least, for the protection of mice against infection with the virulent strain afforded by the attenuated strain, while the loss or modification of antigen 1 might be associated with attenuation of the organism with respect to its virulence to mice.     

Effect of cyclic adenosine 3'',5''-monophosphate antagonists on endotoxin-induced inhibition of human neutrophil chemotaxis.

We reported previously that Escherichia coli endotoxin inhibited human neutrophil chemotaxis toward C5a. This effect of endotoxin was antagonized by anti-inflammatory steroids. We now report that dibutyryl cyclic adenosine 3',5'-monophosphate, prostaglandin E1, isoproterenol, and cholera toxin also antagonize the suppression of chemotaxis by endotoxin. Each compound inhibited the effect of endotoxin in a dose-dependent fashion. To be effective, each compound except cholera toxin had to be present at the time of endotoxin challenge. Furthermore, propranolol blocked the protective effect of isoproterenol against endotoxin but not the protective effect of dibutyrl cyclic adenosine 3',5'-monophosphate or prostaglandin E1. Dibutyryl cyclic guanosine 3',5'-monophosphate, adenosine 5'-monophosphate, phenylephrine, prostaglandin F2 alpha, and carbachol did not modify the suppression of chemotaxis by endotoxin. Anti-inflammatory steroids and dibutyryl cyclic adenosine 3',5'-monophosphate are thought to stabilize phospholipids in certain cell membranes. This phospholipid-stabilizing action may contribute, at least in part, to the protective effect against endotoxin-mediated suppression of neutrophil chemotaxis.     

Optimization of 3-D hepatocyte culture by controlling the physical and chemical properties of the extra-cellular matrices

Hepatocytes are anchorage-dependent cells sensitive to microenvironment; the control of the physicochemical properties of the extra-cellular matrices may be useful to the maintenance of hepatocyte functions in vitro for various applications. In a microcapsule-based 3-D hepatocyte culture microenvironment, we could control the physical properties of the collagen nano-fibres by fine-tuning the complex-coacervation reaction between methylated collagen and terpolymer of hydroxylethyl methacrylate-methyl methacrylate-methylacrylic acid. The physical properties of the nano-fibres were quantitatively characterized using back-scattering confocal microscopy to help optimize the physical support for hepatocyte functions. We further enhanced the chemical properties of the collagen nano-fibres by incorporating galactose onto collagen, which can specifically interact with the asialoglycoprotein receptor on hepatocytes. By correlating a range of collagen nano-fibres of different physicochemical properties with hepatocyte functions, we have identified a specific combination of methylated and galactosylated collagen nano-fibres optimal for maintaining hepatocyte functions in vitro. A model of how the physical and chemical supports interplay to maintain hepatocyte functions is discussed.     

Toxicity and adaptive immune response to intracellular transgenes delivered by helper-dependent vs. first generation adenoviral vectors

The host immune response to intracellular transgenes delivered by helper-dependent (HDV) vs. first generation (FGV) adenoviral vectors has been relatively unstudied. Previous studies showed short-term correction of bovine and murine argininosuccinate synthetase (ASS) deficiency after first generation adenoviral-mediated liver gene therapy. To determine whether the host adaptive immune response against the intracellular transgene human ASS (hASS) contributed to loss of gene expression in this setting, the same vector (FGV-CAG-hASS) was injected into Rag-/- (immunodeficient) mice. As in wild-type C57BL/6 (B6) mice, Rag-/- mice also showed significant loss of hASS expression and vector by week 4 post-injection, with concomitant elevation of liver enzymes and disruption of liver architecture. Therefore, direct toxicity due to vector rather than adaptive immune response against hASS primarily accounted for loss of expression with FGVs. In contrast to hASS, beta-galactosidase is strongly immunogenic and activates the host adaptive immune response. Loss of transgene expression was observed in B6 mice with either a FGV or a HDV expressing beta-galactosidase. However, the drop in gene expression observed with the HDV was primarily due to the adaptive immune response, since both beta-galactosidase expression and vector genome were sustained in immunodeficient mice treated with HDV. As expected, with weakly immunogenic hASS, vector genome and hASS expression were sustained with a HDV in spite of ubiquitous expression of the transgene. Therefore, viral gene expression is a primary determinant of intermediate and chronic toxicities at day 3 and week 4 post-injection. However, even in the absence of viral gene expression, strongly immunogenic intracellular transgenes can stimulate clearance of transduced hepatocytes.