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.     

Pregnancy following treatment of symptomatic myomas with laparoscopic bipolar coagulation of uterine vessels

BACKGROUND: Laparoscopic bipolar coagulation of uterine vessels (LBCUV) has been employed for women with symptomatic uterine myomas, but its effect on subsequent pregnancy has not been characterized. METHODS: Four-hundred and twenty-three women entered the study between March 1999 and December 2001. Of these, 142 women (33.6%) were under the age of 40 years at the time of LBCUV, 36 of whom (36/142, 25.3%) were sexually active without contraception. In a prospective study of 142 patients (<40 years old) undergoing LBCUV for symptomatic myomas, 15 women became pregnant (17 total pregnancies) and were evaluated by physical and ultrasound examinations. RESULTS: The volume of the dominant myoma was 117.4 +/- 118.4 and 36.8 +/- 56.8 cm(3) before and after LBCUV respectively. Volume of the dominant myoma after pregnancy was 46.2 +/- 76.7 cm(3) (mean +/- SD). There was a significant difference in myoma volume before and after LBCUV (P = 0.002), but no significant difference in myoma volume when comparing post-partum size with post-LBCUV size (P = 0.269). Pregnancy outcomes included seven miscarriages in the first trimester and one premature rupture of membrane (PPROM). Although the other pregnancies were regarded as uncomplicated, only two women were delivered of normal neonates as the other seven pregnancies were terminated secondary to patient request. CONCLUSIONS: The pregnancy and term pregnancy rates in sexually active women without contraception were 41.6% (15/36) and 5.6% (2/36) respectively. Because a relatively high rate (7/17, 41.2%) of early miscarriages was observed, we recommend that this procedure be employed only for women who do not desire additional children.     

Interaction of plasma fibronectin (pFN) with membranous constituents of peritoneal exudate cells and pulmonary macrophages

The prominent role of plasma fibronectin (pFN) in the host defense system as an opsonin for gelatin (collagen)-coated colloids has been established. In the present study we investigated the interaction of pFN and membrane isolates from cells devoid of collagen, as well as several tissues. In a liver slice assay system it was shown that subcellular membrane fractions from lung macrophages, peritoneal exudate cells, spleen, tests, and liver were able to competitively inhibit the pFN-mediated uptake of 125I-gelatin coated latex beads (gLtx) at low concentrations. Endocytosis of 125I-labeled membrane isolates by macrophage monolayers was also promoted by addition of pFN. In an attempt to characterize the membrane component(s) interacting with pFN, it was found that mild extraction procedure with 1 M KBr could release a significant amount of this inhibitory activity. Further studies demonstrated that the agent(s) responsible for inhibition of gLtx uptake was heat sensitive, not altered by trypsin treatment, and did not contain actin, a protein known to interact with pFN. This work indicates that pFN interacts specifically with an as yet unknown membrane component(s) and that such interaction will promote clearance of cellular debris by macrophages. This suggests that pFN may be an important opsonin for the reticuloendothelial system in clearance of collagenous and noncollagenous cellular debris once they are exposed to interact with it.     

Production of stable anti-digoxin Fv in Escherichia coli.

We have created a bacterial expression-export system and have used it to express (14 mg l-1) the variable region fragment (Fv) of an anti-digoxin antibody (26-10) in Escherichia coli. The expression-export plasmid contains a T7 promoter and the E. coli signal sequences ompA [Movva et al., J. biol. Chem. 255, 27-29 (1980)] and phoA [Inouye et al., J. Bacteriol. 149, 434-439 (1982)] fused to heavy chain (VH) and light chain (VL) variable region sequences to generate an artificial cistron. The 26-10 Fv protein made using this system was soluble, unlike many other expression systems which produce insoluble proteins in the form of inclusion bodies. The 26-10 VH and VL proteins were cleaved at their mature N-termini and exported into the bacterial periplasm where they could be easily extracted and affinity purified on ouabain-Sepharose. 26-10 Fv bound to digoxin with similar affinity and specificity as the whole 26-10 antibody (Ka for Fv, 1.3 x 10(9) M-1, Ka for IgG, 7 x 10(9) M-1). 26-10 Fv appears to be remarkably stable in comparison with other Fv fragments. The half-life for chain dissociation of 26-10 Fv was 48 hr compared to the reported 1.5 hr half-life of McPC603 Fv. We present the proton NMR spectra of the 26-10 Fv as preliminary evidence that this expression-export system can be used to facilitate the analysis of the solution structure of 26-10 Fv by NMR.     

Persistent infection of SARS coronavirus in colonic cells in vitro

Severe acute respiratory syndrome coronavirus (SARS-CoV) can produce gastrointestinal symptoms. The intestinal tract is the only extrapulmonary site where viable viruses have been detected. This study examined seven established human intestinal cell lines, DLD-1, HCT-116, HT-29, LoVo, LS-180, SW-480 and SW-620, for their permissiveness to SARS-CoV infection. The results showed that only LoVo cells were permissive to SARS-CoV infection as evident by positive findings from indirect immunofluorescence staining for intracellular viral antigens, in situ hybridization for intracellular viral RNA, and electron microscopy for intracellular viral particles. In contrast to Vero cells, SARS-CoV did not produce cytopathic effects on LoVo cells. However, LoVo cells were found to be highly permissive for productive infection with a high viral titre (>3 x 10(7) viral copies/ml) produced in culture supernatant following a few days of incubation. SARS-CoV established a stable persistent chronic infection that could be maintained after multiple passages. Being a cell line of human origin, LoVo cells could be a useful in vitro model for studying the biology and persistent infection of SARS-CoV. Our results on the expression of angiotensin-converting enzyme 2 (ACE2), a recently identified cellular receptor for SARS-CoV, in these cell lines indicated that it might not be the sole determinant for cells to be susceptible to SARS-CoV infection.     

A randomized, double blind, placebo-controlled study to investigate the use of conscious sedation in conjunction with paracervical block for reducing pain in termination of first trimester pregnancy by suction evacuation

BACKGROUND: This study evaluated the role of conscious sedation in pain relief during termination of first trimester pregnancy by suction evacuation (SE) under local anaesthesia. METHODS: A hundred women undergoing SE before 12 weeks gestation were randomized by computer using the sealed envelope method to receive placebo (saline) or conscious sedation (2 mg midazolam and 25 microg fentanyl) i.v. 5 min before cervical dilatation. Paracervical block was given to all patients, 2 min later. Pain scores during and after SE, post-operative side-effects and satisfaction level were compared. RESULTS: No statistically significant differences in pain scores were found between the two groups. Post-operative side-effects such as dizziness (P = 0.015) and drowsiness (P < 0.001) were significantly more severe in the conscious sedation group. However, patients in the conscious sedation group reported better satisfaction levels than the control group (P = 0.003). CONCLUSION: The use of conscious sedation significantly improved patient satisfaction during termination of first trimester pregnancy by SE under local anaesthesia, despite a lack of improvement in pain relief and the presence of increased severe dizziness/drowsiness in the post-operative period.     

Concentrations of endometrial protein PP 14 and CA-125 in uterine flushings performed in natural and stimulated cycles

BACKGROUND: Impaired implantation in assisted reproduction cycles with high serum estradiol (E(2)) concentrations may be attributed to abnormal endometrial development. This study compared concentrations of endometrial proteins in uterine flushings of infertile patients between natural and stimulated cycles. METHODS: Patients received a standard regimen of ovarian stimulation. Seven days after the LH surge in natural cycles or the hCG injection in stimulated cycles, uterine flushings were performed by slowly injecting and aspirating normal saline through a paediatric Foley catheter. Natural cycles were considered as group A whereas stimulated cycles with serum E(2) <20 000 pmol/l and serum E(2) >20 000 pmol/l were classified as groups B and C respectively. PP 14 and CA-125 in uterine flushings were measured and expressed per total protein content. RESULTS: Concentrations of the total protein, PP 14 and CA-125 in the uterine flushings were similar among the three groups. PP 14 per total protein in the uterine flushings was significantly correlated with serum E(2) on the day of hCG (r = 0.459; P = 0.009) in natural cycles only but not in stimulated cycles. CONCLUSION: There was no significant difference between natural and stimulated cycles in concentrations of PP 14 and CA-125 in uterine flushings performed in the mid-luteal phase.