Antibody to a 145-kilodalton outer membrane protein has bactericidal activity and protective activity against experimental bacteremia caused by a Brazilian purpuric fever isolate of Haemophilus influenzae biogroup aegyptius. The Brazilian Purpuric Fever Study Group.

The immunologic basis for protection against Brazilian purpuric fever, a septicemic infection associated with Haemophilus influenzae biogroup aegyptius bacteremia, is unknown. Passive immunization of infant rats with antiserum to whole bacterial cells of the homologous strain protects them from experimental bacteremia following bacterial challenge. In immunoblotting, antibody to a 145-kDa protein (P145) was present in protective antisera but not in nonprotective antisera. As judged by analysis of the antibodies eluted from whole bacterial cells and the agglutination of bacteria by antisera to P145, this protein is surface exposed. We prepared monospecific rat antisera to this protein by three methods: (i) immunization with whole bacterial cells and absorption with a Brazilian purpuric fever strain not expressing P145, (ii) immunization with gel-purified P145, and (iii) immunization with a P145-expressing transformant of a laboratory H. influenzae strain expressing this protein and absorption of the antiserum with the laboratory H. influenzae strain. These antisera had low antilipooligosaccharide antibody titers, were reactive only with P145, and had bactericidal activity in vitro. Following passive immunization, these antisera partially protected infant rats from bacteremia resulting from intraperitoneal challenge with bacteria. As assessed by immunoblotting, pooled adult human sera contained antibodies reactive with P145. Antibody to P145 may contribute to protection against Brazilian purpuric fever.     

Monoclonal antibodies to fungi of significance to the quality of foods and feeds

Monoclonal antibodies (MAbs) were raised against Penicillium aurantiogriseum var. melanoconidium. Cross‐reactivity studies were carried out against 12 ‘field’ and 27 ‘storage’ fungi. Two MAbs (MAbs 32 and 37) reacted with all of the fungi tested and a third (MAb 38) with 38 out of the 39 fungi (although weakly with some). Monoclonal antibodies 32 and 37, but not MAb 38, were found to react to a degree with ‘clean’ barley. Enzyme‐linked immunosorbent assay (ELISA) using MAb 38 and extracts of barley inoculated with the homologous antigen showed a clear relationship between absorbance and amount of fungal growth. It is suggested that these MAbs could be used in a broad‐spectrum assay to detect fungi of significance to the quality of foods and feeds.     

Development of immunofiltration assay by light addressable potentiometric sensor with genetically biotinylated recombinant antibody for rapid identification of Venezuelan equine encephalitis virus

A genetically biotinylated single chain fragment variable antibody (scFv) against Venezuelan equine encephalitis virus (VEE) was applied in a system consisting of an immunofiltration enzyme assay (IFA) with a light addressable potentiometric sensor (LAPS) for the rapid identification of VEE. The IFA involved formation of an immunocomplex sandwich consisting of VEE, biotinylated antibody, fluoresceinated antibody and streptavidin, capture of the sandwich by filtration on biotinylated membrane, and labeling of the sandwich by anti-fluorescein urease conjugate. The concentration ratio of biotinylated to fluoresceinated antibodies was investigated and optimized. By the IFA/LAPS assay, the limit of detection (LOD) of VEE was approximately 30 ng/ml, similar to that achieved when chemically biotinylated monoclonal antibody (mAb) was applied. Total assay variance of the IFA/LAPS assay for both intra- and inter-assay precision was less than 20%. Assay accuracy was measured by comparing VEE concentrations estimated by IFA/LAPS standard curve to those obtained by conventional protein assay. VEE concentrations were found to differ by no more than 10%. The IFA/LAPS assay sensitivity was approximately equal to that of a conventional enzyme-linked immunosorbent assay (ELISA) utilizing polystyrene plates and a chromogenic substrate; however, less time and effort were required for performance of the IFA/LAPS assay. More importantly, use of genetically biotinylated scFv in the IFA/LAPS assay obviates the need for chemical biotinylation of antibody with resultant possible impairment of the antigen-binding site. Furthermore, the potential for batch-to-batch variability resulting from inequality in the number of biotin molecules labeled per antibody molecule is eliminated.     

Accumulation of filamentous tau in the cerebral cortex of human tau R406W transgenic mice

Missense mutations of the tau gene cause autosomal dominant frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), an illness characterized by progressive personality changes, dementia, and parkinsonism. There is prominent frontotemporal lobe atrophy of the brain accompanied by abundant tau accumulation with neurofibrillary tangles and neuronal cell loss. Using a hamster prion protein gene expression vector, we generated several independent lines of transgenic (Tg) mice expressing the longest form of the human four-repeat tau with the R406W mutation associated with FTDP-17. The TgTauR406W 21807 line showed tau accumulation beginning in the hippocampus and amygdala at 6 months of age, which subsequently spread to the cortices and subcortical areas. The accumulated tau was phosphorylated, ubiquitinated, conformationally changed, argyrophilic, and sarcosyl-insoluble. Activation of GSK-3beta and astrocytic induction of mouse tau were observed. Astrogliosis and microgliosis correlated with prominent tau accumulation. Electron microscopic examination revealed the presence of straight filaments. Behavioral tests showed motor disturbances and progressive acquired memory loss between 10 to 12 months of age. These findings suggested that TgTauR406W mice would be a useful model in the study of frontotemporal dementia and other tauopathies such as Alzheimer's disease (AD).     

Pre-clinical Study of <Superscript>213</Superscript>Bi Labeled PAI2 for the Control of Micrometastatic Pancreatic Cancer

Purpose: The urokinase plasminogen activator (uPA) and its receptor (uPAR) are expressed by pancreatic cancer cells and can be targeted by the plasminogen activator inhibitor type 2 (PAI2). We have labeled PAI2 with ²¹³Bi to form the alpha conjugate (AC), and have studied its in vitro cytotoxicity and in vivo efficacy. Methods and Materials: The expression of uPA/uPAR on pancreatic cell lines, human pancreatic cancer tissues, lymph node metastases, and mouse xenografts were detected by immunohistochemistry, confocal microscopy, and flow cytometry. Cytotoxicity was assessed by the MTS and TUNEL assay. At 2 days post-cancer cell subcutaneous inoculation, mice were injected with AC by local or systemic injection. Results: uPA/uPAR is strongly expressed on pancreatic cancer cell lines and cancer tissues. The AC can target and kill cancer cells in vitro in a concentration-dependent fashion. Some 90% of TUNEL positive cells were found after incubation with 1.2 MBq/ml of AC. A single local injection of ~222 MBq/kg 2 days post-cell inoculation can completely inhibit tumor growth over 12 weeks, and an intraperitoneal injection of 111 MBq/kg causes significant tumor growth delay. Conclusions: ²¹³Bi-PAI2 can specifically target pancreatic cancer cells in vitro and inhibit tumor growth in vivo. ²¹³Bi-PAI2 may be a useful agent for the treatment of post-surgical pancreatic cancer patients with minimum residual disease.     

HLA PROFILE OF ETHNIC PAKISTANI POPULATION GROUPS

Acute rejection is a major determinant of chronic allograft dysfunction and graft survival. This study evaluated the effect of basiliximab (Simulect^®), a 156-kDa chimeric monoclonal antibody (human and murine) directed against the alpha chain of the interleukin (IL)-2 receptor of human lymphocytes, on acute rejection in pediatric renal transplantation. Data were collected from two pediatric renal transplantation centers. Forty transplantations (22 males and 18 females; mean age 14.8±3.6 years) were performed between 1996 and 2001. Twelve of the grafts came from cadaveric donors and 28 from living-related donors. Twenty-four of the patients were on hemodialysis, 15 were on peritoneal dialysis, and one case was a pre-emptive transplantation. All patients were placed on triple-drug immunosuppression [prednisolone + (azathioprine or mycophenolate mofetil) +(cyclosporine or tacrolimus)]. Basiliximab was also administered in 17 cases. The respective rates of biopsy-proven acute rejection in the basiliximab group and the standard-regimen group were 0% vs. 17.4% ( P >0.05) at 1 month post-transplantation; 0% vs. 26.1% ( P <0.05) at 3 months; and 0% vs. 26.1% ( P <0.05) at 6 months. Thirty and 16 patients had completed 1- and 3-year follow ups, respectively, at the time of writing; the 1- and 3-year graft survival rates were 96% (29/30) and 81% (13/16), respectively.
Basiliximab significantly reduced the rates of acute rejection at 3- and 6 months post-pediatric renal transplantation. It was well tolerated by all patients, and caused no significant adverse effects. The effect of basiliximab on long-term graft survival and chronic allograft dysfunction deserves further investigation.