Dissociative fugue in the elderly.

Dissociative fugue is a rarely reported diagnostic entity. It is one of the least understood and yet clinically one of the most fascinating disorders in Mental Health. Here, we describe a case of fugue in a 62-year-old housewife who was brought to our hospital with pockets of memory loss. This case illustrates the need for timely referrals, which could channelize valuable professional time and help avoid expensive and unnecessary investigations.

Enhanced protective response and immuno-adjuvant effects of porcine GM-CSF on DNA vaccination of pigs against Aujeszky's disease virus.

This study was conducted to investigate whether the co-delivery of DNA encoding porcine cytokines would enhance a protective immune response in pigs to a Pseudorabies virus (PRV; or Aujeszky's disease virus) DNA vaccine. Aujeszky's disease in pigs results in respiratory and nervous symptoms with important economic losses. To evaluate cytokine effects, eukaryotic expression vectors were constructed for porcine GM-CSF, IL-2 and IFN-gamma. cDNA for each of these cytokines was inserted under the control of a CMV promoter in the pcDNA3 plasmid and cytokine expression was confirmed after DNA transfection in various mammalian cell cultures by bioassays (GM-CSF and IL2) and ELISA (IFN-gamma). Pigs were vaccinated by single intramuscular injection with plasmid DNA encoding PRV gB and gD along with various combinations of cytokine plasmid constructs. Pig serum was tested for the production of antibody by isotype specific anti-PRV ELISA. Pigs were then challenged with the highly virulent PRV strain NIA3 on day 21 after vaccination. The survival and growth rate of pigs were monitored for seven days after the viral challenge. The co-administration of GM-CSF plasmid increased the immune response induced by gB and gD PRV DNA vaccine. This immune response was characterized by an earlier appearance of anti-PRV IgG2, a significantly enhanced anti-PRV IgG1 and IgG2 antibody response, a significantly decreased and shortened viral excretion in nasal swabs and an improved protection to the viral challenge. In contrast, the co-administration of porcine IL-2 or IFN-gamma had no adjuvant effects. Our results thus demonstrate for the first time that the application of porcine GM-CSF gene in a DNA vaccine formulation can exert immuno-adjuvant and protective effects with single vaccination in the natural host pig against Aujeszky's disease.

Development of a trispecific antibody conjugate that directs two distinct tumor-associated antigens to CD64 on myeloid effector cells.

A trispecific F(ab')3 antibody conjugate (TAC) with specificities for the Fc gamma receptor I (Fc gamma RI/CD64), the epidermal growth factor receptor (EGFR) and the HER2/neu antigen has been developed to redirect effector cell-mediated cytotoxicity against cancer cells expressing both or either of the tumor-associated antigens. The TAC was constructed in two steps using the sulfhydryl-specific cross-linker o-phenylenedimaleimide (o-PDM). In step one, a bispecific antibody was prepared by linking the Fab' fragments of mAb m22 (a murine IgG1 specific for Fc gamma RI) and mAb H425 (a humanized IgG1 antibody recognizing EGFR). The conjugation efficiency was about 60%. In the second step, the Fab' fragment of mAb 520C9, a murine IgG1 specific for HER2/neu, was coupled to the bispecific antibody made in step one. About 40% of the bispecific conjugate was derivatized to form the trispecific antibody. The purity of the TAC was more than 90% after gel filtration purification. The TAC was characterized in vitro for its ability to bind specifically to all the three antigens and to kill target cells expressing the tumor antigens. In contrast to bispecific conjugates that can only target cells expressing either of the tumor antigens, the TAC was able to bind both the antigens more efficiently in cell-binding assays and to kill tumor cells expressing EGFR and HER2/neu antigens.

Characterization of a novel bispecific antibody that mediates Fcgamma receptor type I-dependent killing of tumor-associated glycoprotein-72-expressing tumor cells.

A bispecific antibody was made by chemical conjugation of Fab' fragments from humanized antibodies specific for tumor-associated glycoprotein-72 (TAG-72) and high-affinity immunoglobulin receptor, FcgammaA receptor type I (FcgammaRI). The purified anti-TAG-72 x anti-FcgammaRI (HCC49xH22) bispecific antibody had an approximate Mr of 111,000, consistent with a F(ab')2, and bound specifically to KLEB and LS174T tumor cell lines, which express the TAG-72 tumor antigen. Furthermore, HCC49x H22 was shown to simultaneously bind to KLEB cells and a soluble FcgammaRI fusion protein, demonstrating the bifunctional nature of the molecule. Using IFN-gamma-treated monocytes as effector cells, concentrations of the bispecific antibody in the range of 1-10,000 ng/ml mediated specific lysis of TAG-72-positive tumor cells. In contrast, the bispecific antibody did not promote antibody-dependent cellular cytotoxicity of a cell line that was negative for TAG-72 antigen. Importantly, the antibody-dependent cellular cytotoxicity activity of the bispecific antibody was significantly greater than that of the monoclonal antibody HCC49. These in vitro data indicate that the humanized bispecific antibody HCC49xH22 has the appropriate specificity and functional activity for further evaluation as potential immunotherapy for TAG-72-positive malignancies.

Cytolytic and cytostatic properties of an anti-human Fc gammaRI (CD64) x epidermal growth factor bispecific fusion protein.

A bispecific fusion protein (H22-EGF) that binds simultaneously to the epidermal growth factor receptor (EGF-R) and to the high affinity receptor for the Fc portion of human IgG, Fc gammaRI (CD64), has been successfully constructed and expressed. For this construction, genomic DNA encoding the Fd fragment of humanized anti-Fc gammaRI mAb, H22, which binds Fc gammaRI at an epitope that is distinct from the Fc binding site, was fused to cDNA encoding human epidermal growth factor (EGF), a natural ligand for EGF-R. The resulting H22Fd-EGF-expressing vector was transfected into a myeloma cell line that was transfected previously with a vector containing DNA encoding the H22 kappa-light chain. SDS-PAGE analysis of purified H22-EGF demonstrated that the fusion protein was secreted predominantly as H22Fab'-EGF monomer (approximately 55 kDa), even though a free Cys residue exists in the hinge region of the H22 Fab' component. Using a novel bispecific flow cytometry-binding assay, we demonstrated that the purified bispecific fusion protein, H22-EGF, was able to bind simultaneously to soluble Fc gammaRI and EGF-R-expressing cells. H22-EGF inhibited the growth of EGF-R-overexpressing tumor cells and mediated dose-dependent cytotoxicity of these cells in the presence of Fc gammaRI-bearing cytotoxic effector cells. These results suggest that this fusion protein may have therapeutic utility for EGF-R-overexpressing malignancies.

Development of a bispecific F(ab')2 conjugate against the complement receptor CR3 of macrophages and a variant CD44 antigen of rat pancreatic adenocarcinoma for redirecting macrophage-mediated tumor cytotoxicity.

A bispecific F(ab')2 antibody conjugate (BAC) was constructed against the complement receptor CR3 of macrophages and variant CD44 (CD44v6) antigen of rat pancreatic adenocarcinoma cells to redirect macrophage-mediated tumor cytotoxicity. The Fab' fragments of monoclonal antibodies (mAb) 1.1ASML and OX42, recognizing the CD44v6 and the CR3 antigens respectively, were chemically coupled at the hinge region using 5,5'-dithiobis(2-nitrobenzoate). The BAC was characterized in vitro for its specific, dual binding capacity to CD44v6 and CR3 antigens. Although the monovalence of the BAC resulted in lower avidities to both the antigens as expected, it was still able to form stable cross-linkages between tumor cells and macrophages in culture leading to the formation of "clump-like" cell aggregates. The in vitro and in vivo tumor-targeting capacity of the BAC was compared with that of the parental antitumor mAb 1.1ASML, which mediates tumor killing by antibody-dependent cell cytotoxicity. These results showed that, even though the bivalent mAb 1.1ASML did not mediate stable cross-linking of target and effector cells, its Fc-receptor-mediated killing of tumor cells was more effective when compared to the BAC. Thus, this study strongly supports the hypothesis that firm persistent binding between effector and target cells per se is not as important as the choice of trigger molecule used for macrophage activation to redirect their tumor cytotoxic potential effectively.

A novel smooth muscle-specific enhancer regulates transcription of the smooth muscle myosin heavy chain gene in vascular smooth muscle cells.

Transient DNA transfection analysis of 5' end deletion mutants of the rabbit smooth muscle myosin heavy chain (SMHC) gene promoter was performed in primary cultures of rabbit vascular smooth muscle cells (VSMC). A positive element located at position -1,332 upstream of the transcription start site consistently gave the highest relative chloramphenicol acetyltransferase (CAT) activity (6.3 +/- 1.5-fold over the minimal SMHC promoter), suggesting that inclusion of the extra 107-base pair (bp) DNA fragment between -1,332 and -1,225 could significantly enhance CAT activity in VSMC. Transfection of mutants into several muscle and nonmuscle cell lines did not show any significant CAT activity above control, showing that factors unique to smooth muscle cells were required for SMHC expression. Gel shift analysis indicated that multiple factors interacted with the 107-bp element, two of which appeared to show smooth muscle specificity. Tests of enhancer function in transfected VSMC indicated that the 107-bp fragment behaved as a classical enhancer, i.e. independently of position and orientation. These results indicate that a novel DNA element may regulate the tissue-restricted expression of the SMHC gene and provides the first example of a role for a smooth muscle-specific enhancer in VSMC.

Development of a bispecific monoclonal antibody against a gallium-67 chelate and the human melanoma-associated antigen p97 for potential use in pretargeted immunoscintigraphy.

A bispecific monoclonal antibody (bsmAb) has been developed against the human melanoma-associated antigen p97 and an octahedral gallium chelate (Ga-HBED) using the hybrid hybridoma technology. As tetradomas were expected to produce a maximum of ten different molecular species of immunoglobulins, the bispecific antibody was purified from this mixture by consecutive protein A affinity and cation-exchange chromatographic techniques. Although it was established by sodium dodecyl sulphate/polyacrylamide gel electrophoresis that the heavy (H) and light (L) chains of the two parental immunoglobulins were mismatched in the bispecific antibody, results from cell enzyme-linked immunosorbent assay indicated significant dual specific binding to both the melanoma cells and 67Ga-HBED. Other in vitro techniques further confirmed that the bsmAb Bi 5-56-II-17 still retained about 30%-40% simultaneous binding capacity to both the antigens, as would have been expected in a bsmAb that has ideally matched H and L chains. Preliminary in vivo experiments using nude mice bearing the human melanoma xenografts showed that the bsmAb Bi 5-56-II-17 was able to target the radioactive gallium chelate to the tumours twice as efficiently compared to the monospecific, bivalent gallium chelate antibody.

Expression of CD44 isoforms carrying metastasis-associated sequences in newborn and adult rats.

Expression of a splice variant of CD44, recognised by the monoclonal antibody (Mab) 1.1ASML, confers metastatic potential to non-metastasising tumour cells (Cell 1991, 65, 13-24). To explore whether the metastasis-associated variant of CD44 (CD44v) is expressed under physiological conditions, tissues of newborn and adult rats were stained with the Mab 1.1ASML. The 1.1ASML epitope is, indeed, expressed on the basal layer of the epidermis and the hair follicles as well as on cryptic epithelia in the gut. In addition, ductal epithelia of the pancreatic gland of newborn rats express CD44v. This pattern of expression differs from that of standard lymphocyte CD44 (CD44s). The anti-CD44s mAB Ox50 predominantly stains connective tissue. Although different variants of CD44 may express the epitope recognised by 1.1ASML, cells expressing CD44v share properties with metastasising tumour cells: the stage of proliferation and a restricted degree of mobility. Thus, during metastatic progression tumour cells may reactivate the expression of gene segments which serve highly specialised functions in embryonic and adult tissues.

Increased efficiency of gene transfection in primary cultures of adult rat hepatocytes stimulated to proliferate: a comparative study using the lipofection and the calcium phosphate precipitate methods.

Transfection of the beta-galactosidase gene in quiescent cultures of adult rat hepatocytes with the calcium phosphate precipitate or the lipofection methods gave a higher level of beta-galactosidase gene expression with the lipofection than with the calcium phosphate precipitate method, but the transfection efficiency was weak in both cases. Transfection of hepatocytes stimulated to proliferate before transfection either in vivo by partial hepatectomy or in vitro by epidermal growth factor was more efficient than transfection of quiescent hepatocytes, and the lipofection method gave better results than the calcium phosphate precipitate method.