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Background/aims: Adefovir dipivoxil (adefovir) effectively inhibits both wild-type and lamivudine-resistant hepatitis B virus (HBV) replication. The development of adefovir resistance is both delayed and infrequent compared with lamivudine resistance. The aim of this study was to characterize the serologic, biochemical, and virologic response to adefovir, and to explore the factors affecting initial virologic response (IVR, defined as a decrease in serum HBV below 4 log10copies/mL after 6 month of treatment) and adefovir resistance in lamivudine resistant HBV-infected patients., Methods: This study population comprised 76 patients with lamivudine-resistance who had received adefovir for more than 12 months between March 2004 and December 2006. The adefovir-resistant mutant was assayed at 6 months and 12 months during adefovir administration. Restriction-fragment mass polymorphism analysis was used for detecting YMDD and adefovir mutants., Results: After adefovir administration, an IVR was observed in 31% of the patients with lamivudine resistance. Factors associated with an IVR were HBeAg negativity (P=0.04) and the presence of liver cirrhosis (P=0.04). Age, sex, pretreatment levels of alanine aminotransferase and aspartate aminotransferase, pretreatment HBV DNA levels, presence of precore mutation, and type of YMDD mutants were not related to an IVR during adefovir treatment. The prevalence of adefovir resistance was 5% and 13% at 6 months and 12 months after therapy, respectively. Mixed infection of the precore mutant was a risk factors for the emergence of adefovir resistance (P=0.01)., Conclusions: Lamivudine-resistant HBV patients exhibiting HBeAg negativity and liver cirrhosis were more likely to achieve an IVR after adefovir therapy. Adefovir resistance was associated with mixed infection of the precore mutant.

In a previous study, we generated a murine hepatitis B virus (HBV)-neutralizing monoclonal antibody (mAb), KR127, that binds to an epitope (amino acids 37-45, NSNNPDWDF) of the preS1 antigen. Furthermore, an epitope tag, S1 (NANNPDWDF), was developed for protein tagging. The aim of the present study was to develop a high-affinity antibody to the same preS1 epitope. Mice were immunized with the N-terminal domain of human thrombopoietin fused to the S1 tag (nTPO-S1), and a phage-displayed chimeric Fab library was constructed and screened by panning against nTPO-S1. A high-affinity antibody (3-34) was selected that binds to the preS1 antigen. The IgG molecules of 3-34 showed approximately nine-fold higher affinity (K(D) 1.2 nM) for preS1 compared with KR127 (K(D) 10.4 nM), competed with KR127 for binding to the epitope, and bound to HBV particles. This study provides a simple and efficient way to develop a high-affinity antibody to a defined epitope by phage display of an immune antibody library.

The present study sought to learn about risk perceptions held by parents of preschool fire-setters. A 41-item survey was distributed to 60 parents whose children, aged 6 years and younger, had previously set fires and who were involved in intervention programmes throughout the US. Most parents did not think their children would play with matches/lighters, or knew how to use these items, although some had witnessed their children playing with matches/lighters previously. Most parents reported having taken precautions to keep matches/lighters out of reach and also educating their children about fire. Regardless, children not only set fires, but in 40% of cases climbed to access the match/lighter. Parents' perceptions of their children's proclivity for fire play were not consistent with their actual fire-play behaviour. Parents underestimated the likelihood that their children would play with matches/lighters. Although most reportedly undertook preventative measures aimed at thwarting fire play, these strategies were ineffective. Traditionally relied upon precautionary techniques, such as storing lighters out of reach and discussing the dangers of fire, were not sufficient to stem interest and resultant fire play.

Thrombopoietin (TPO) is a megakaryocyte growth and differentiation factor that is currently being investigated as a therapeutic for cancer patients undergoing myelosuppressive chemotherapy. We generated monoclonal antibodies (MAbs) specific for human thrombopoietin (hTPO) by genetic immunization using an hTPO expression plasmid and an adjuvant plasmid that encodes mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). All genetically immunized mice exhibited a high humoral immune response. Splenocytes from these mice were used to generate hybridomas. Two MAbs, designated 2B9A10 and 4C16B15 (of IgG1 and IgG3 isotypes, respectively), were subsequently selected and produced. They specifically recognized and precipitated recombinant hTPO produced by mammalian cells and were effective in sandwich enzyme-linked immunosorbent assays (ELISAs) for hTPO quantitation. Our results demonstrate that these MAbs should be useful for purification and quantitation of hTPO in clinical and laboratory settings.

In bioequivalence trials, one often considers two or more generic products with the original one. The 3 x 3 crossover design can be adopted to evaluate the two generic candidates with a brand name drug, rather than conducting two separate 2 x 2 crossover trials. Dropouts, however, are more likely to occur due to various administrative reasons when we consider a higher order crossover design. A modified method, which was originally given by Chow and Shao (1997), is extended to compare two generic products with a reference in the incomplete 3 x 3 crossover design. A simulation study and discussion are also presented.

Lethal factor (LF) is a component of anthrax lethal toxin (LeTx). We generated anti-LF murine monoclonal antibodies (MAbs) that show LeTx-neutralizing activity in vitro and in vivo. Anti-LF MAbs were generated by immunization with recombinant LF, and the MAbs showing LeTx-neutralizing activity in vitro were selected. Two MAbs with the highest affinities, 5B13B1 (dissociation constant [K(d)], 2.62 nM) and 3C16C3 (K(d), 8.18 nM), were shown to recognize the same or closely overlapping epitopes on domain III of LF. The 50% inhibitory concentration of 5B13B1 (0.21 microg/ml) was approximately one-third that of 3C16C3 (0.63 microg/ml) in the in vitro LeTx-neutralization assay. The 5B13B1 antibody, which had the highest neutralizing activity, provided perfect protection against LeTx challenge in an in vivo LeTx neutralization assay using Fisher 344 rats. In addition, the antibody showed pre- and postexposure prophylactic effects in the animal experiments. This is the first report that an MAb binding to domain III of LF has neutralizing activity against LeTx. The 5B13B1 antibody may be useful in prophylaxis against anthrax poisoning.

Human cervical cancer oncogene (HCCR) was identified and appeared to function as a negative regulator of p53 gene. The objective of this study was to validate HCCR expression as a candidate marker for human hepatocellular carcinoma. HCCR epitope was identified as Y(355)LGTRR(360). According to immunofluorescence study, HCCR was predominantly localized in the plasma membrane and cytoplasm of hepatocellular carcinoma. HCCR proteins were overexpressed in the tumorous compared with the nontumorous cirrhosis tissues. However, HCCR was not detected in normal liver tissue. Concentration of HCCR protein in the serum was measured in a total of 570 subjects, and comparisons were made to alpha-fetoprotein. Serological studies revealed 78.2% sensitivity of HCCR (cutoff value, 15 microg/ml), which was significantly higher than 64.6% of alpha-fetoprotein (P = 0.0098) and 95.7% specificity for hepatocellular carcinoma. Forty of 52 (76.9%) patients with carcinoma negative for alpha-fetoprotein showed positive values for HCCR. A positive rate of 69.2% in carcinoma patients with tumor sizes <2 cm was found to be a higher rate than measurement of alpha-fetoprotein. Furthermore, HCCR expression was also detected in liver cirrhosis at an intermediate level between carcinoma and normal groups, which gave 88.1% sensitivity and 79.0% specificity using 8 microg/ml as a cutoff value. In summary, the HCCR assay may have an advantage over the alpha-fetoprotein assay in that it is elevated according to disease progression from liver cirrhosis to carcinoma, and it is more frequently positive in patients with early, small hepatocellular carcinoma.

Background: Human lung surfactant protein D (hSP-D) belongs to the collectin family of C-type lectins and participates in the innate immune surveillance against microorganisms in the lung through recognition of carbohydrate ligands present on the surface of pathogens. The involvement of this protein in innate immunity and the allergic response make it the subject of much interest., Results: We have determined the crystal structure of a trimeric fragment of hSP-D at 2.3 A resolution. The structure comprises an alpha-helical coiled-coil and three carbohydrate-recognition domains (CRDs). An interesting deviation from symmetry was found in the projection of a single tyrosine sidechain into the centre of the coiled-coil; the asymmetry of this residue influences the orientation of one of the adjacent CRDs. The cleft between the three CRDs presents a large positively charged surface., Conclusions: The fold of the CRD of hSP-D is similar to that of the mannan-binding protein (MBP), but its orientation relative to the alpha-helical coiled-coil region differs somewhat to that seen in the MBP structure. The novel central packing of the tyrosine sidechain within the coiled-coil and the resulting asymmetric orientation of the CRDs has unexpected functional implications. The positively charged surface might facilitate binding to negatively charged structures, such as lipopolysaccharides.

Porcine fetal pancreases (PFP) obtained from 4 pregnant sows were pooled, minced into 1 mm3 fragments and studied in organ culture for up to 30 days to determine tissue viability and insulin production in vitro. After 7-9 days in culture, some of these explants were transplanted into euglycemic, N:NIH-nu(s) nude recipient mice, and studied histologically over 69 days following grafting. Using RPMI 1640 supplemented with 5% fetal calf serum as the culture medium in 90% air/10% CO2, it was found that explants were viable with insulin production detected in vitro, which was maximal at day 7 (197 +/- 18.9 mU/L, n = 11), and gradually declined thereafter. By 22 days, insulin levels were less than 60.1 +/- 28.5 mU/L (n = 6). Histology of the explants showed viable tissue with evidence of mitoses present in insulin-positive cells at day 16 in vitro. Beyond this time, tissue viability diminished. Explants transplanted into euglycemic nude mice did not undergo rejection during the observation period of 69 days. Grafts remained viable with evidence of an increase in mitotic activity in the endocrine tissue on immunoperoxidase staining. These preliminary investigations confirm that pancreatic explants from porcine fetuses can be maintained in culture for up to 16 days. Such explants, when transplanted under the kidney capsule of euglycemic, nude mice, did not undergo necrosis, but remained viable, with evidence of mitoses in the islet tissue.

An isolated liver perfusion circuit was developed to study the xenogeneic reaction of human blood to porcine liver, and investigate the feasibility of using such a system for liver dialysis in fulminant hepatic failure. Three experimental groups were studied: a control group where pooled porcine blood was perfused through pig liver (n = 12), a xenogeneic group where banked human blood was perfused through porcine liver (n = 23), and a modified xenogeneic group where decomplemented human blood was perfused through porcine liver (n = 4). The following parameters of liver function were assessed: liver function tests, serum electrolytes, bile and ascites production. In addition, liver histology was assessed at the start and completion of each perfusion experiment. Control experiments established that the use of low perfusion pressures (mean inlet pressure of 29.5 +/- 7.4 mmHg), and low haematocrit of 20.5 +/- 8.9% (n = 14), enabled five hour perfusions to be consistently achieved with maintenance of normal acid base and electrolyte balance. Bile production over 5 hours was 18.8 +/- 8.0 ml in controls (n = 5) and 17.0 +/- 6.7 ml in the xenogeneic (human-pig) circuit (n = 11) (NS). Ascites production was 235.8 +/- 157.3 ml/hr in controls (n = 5) and 205 +/- 142.0 ml/hr in the xenogeneic circuit (n = 7) (NS).(ABSTRACT TRUNCATED AT 250 WORDS)

The human fetal pancreas is a potential source of islets for transplantation into insulin-dependent diabetic patients. In this study, 35 human fetal pancreas obtained from prostaglandin-induced abortions (12-26 weeks gestation), were placed in culture to determine their capacity to secrete insulin over 30 days. Culture media were sampled twice weekly for insulin and histology was performed serially. Of the 35 pancreases cultured, six were lost due to bacterial contamination, five discarded due to undetectable levels of insulin in culture, nine are still under study, whilst 15 pancreases have been cultured for one month, and insulin studies completed. Three patterns of insulin release were observed: (a) progressive decline (n = 6), indicating non-viable tissue at the onset; (b) delayed decline, indicating significant tissue damage before organ culture (n = 5); and (c) insulin production in vitro over 30 days (n = 4), with viable islets detected histologically. Factors such as gestational age and cold ischaemia time did not correlate with the pattern of insulin secretion observed. This was probably due to a more important variable, not easily assessed, of the period of intrauterine (warm) ischemia. These data suggest: (1) that a small number of fetal pancreases procured from prostaglandin-induced abortuses do yield islets which remain viable in culture over 30 days, and (2) the functional status of islets can be monitored in vivo by measuring insulin secretion, thereby providing a means of identifying tissue suitable for transplantation.