Amplification and overexpression of HER-2/neu in parotid gland salivary duct carcinoma. Immunohistochemical study and fluorescence in situ hybridization

Salivary duct carcinoma (SDC) is highly malignant salivary gland tumour with aggressive clinical behaviour, characterised by its histological resemblance to invasive ductal carcinoma of the breast. Amplification of gene HER-2/neu and overexpression of its gene product have been shown to have both prognostic and treatment implications in breast cancer. The reports concerning the expression of c-erbB2/HER-2/neu in salivary gland tumours are few and controversial. Thus, eleven cases of SDC were evaluated for HER-2/neu status using immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH). To the best of our knowledge, this is the first molecular genetic analysis of SDCs using FISH. HER-2/neu overexpression, identified as strong membrane staining, was observed in all but one case of SDC in majority of neoplastic cells while only four tumours, of nine cases analysed, revealed HER-2/neu gene amplification by means of FISH analysis. SDCs were associated with poor clinical outcome, 6 patients (55%) died of disseminated carcinoma within 4 to 44 months after therapy. There was no difference in outcome of patients with IHC positive-nonamplified and IHC positive-amplified tumours.     

Immortalized bovine pancreatic duct cells become tumorigenic after transfection with mutant k-ras

Mutation of the K-ras gene is thought to be an early and important event in pancreatic carcinogenesis. In order to study the role of this molecular alteration in the transition from the normal to the neoplastic pancreatic cell, bovine pancreatic duct cells were first immortalized by SV40 large T antigen (Ag) complementary (c)DNA transfection and then transfected with a mutated K-ras gene. As did primary duct cells, the immortalized duct cells (more than 100 passages) expressed cytokeratins, carbonic anhydrase type-II, cystic fibrosis transmembrane conductance regulator (CFTR), and multidrug resistance (mdr). They grew as a single layer after transplantation under plastic domes and formed three-dimensional structures resembling ducts when grown on Matrigel. Cell growth was stimulated by insulin, epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, but cells did not respond to gastrin and CCK-8. They did not form colonies in soft agar nor did they form tumors in nude mice. Immortalized cells transfected with mutated K-ras acquired the ability to form tumors after orthotopic injection into the nude mouse pancreas. It is concluded that SV 40 immortalized bovine pancreatic     

Influence of D-glucose on lipid solid support membrane system as attempt for biosensing of medically relevant molecules

The influence of D-glucose on a lipid solid support system with the aid of impedance spectrocopy as a preliminary attempt for the biosensing of medical relevant molecules was studied. In spite of some shortcomings, s-BLM's proved to be an appropriate model for the study of lipid membrane-D-glucose interactions. The shortcomings were the roughness of the metal support, and the lack of homogeneity in the monolayer or multilayer lipid structures.     

Cardiac troponins following repeated administration of an iron chelator--salicylaldehyde isonicotinoyl hydrazone (SIH)--in rabbits

Both cardiac troponin T (cTnT) and cardiac troponin I (cTnI) are considered to be reliable biomarkers with sufficient sensitivity and specificity for cardiac injury in the majority of laboratory animals. The aim of our study was to compare the diagnostic performance of cTnT and cTnI in three groups of rabbits: 1) control (saline 1 ml/kg i.v.); 2) Salicylaldehyde Isonicotinoyl Hydrazone--SIH (50 mg/kg, once weekly, i.p.; partially dissolved in 10% Cremophor solution); 3) 10% Cremophor solution in water (2 ml/kg i.v.). The drugs were given once a week, 10 administrations. The concentration of cTnT was measured using Elecsys Troponin T STAT Immunoassay (Roche). The concentration of cTnI was measured using AxSYM Troponin I (Abbott). The linear regression model was applied to see if there is a dependence between cTnT and cTnI. The coefficient of determination was not acceptable in all groups. The highest value of R2 was found in the control group (R2 = 0.424). We may conclude that in rabbits meaningful dependence between cTnT and cTnI was not found. According to our long-term experiences cTnT seems to be more suitable cardiomarker in rabbits in comparison with cTnI where the data are characterized by the large scatter.     

Separation and mapping of multiple genes that control IgE level in Leishmania major infected mice

The strain BALB/cHeA (BALB/c) is a high producer, and STS/A (STS) a low producer of IgE after Leishmania major infection. We analyzed this strain difference using 20 recombinant congenic (RC) BALB/c-c-STS/Dem (CcS/Dem) strains that carry different random subsets of 12.5% of genes of the strain STS on the BALB/c background. Strains CcS-16 and -20 exhibit a high and a low IgE level, respectively. In their F(2) hybrids with BALB/c we mapped nine Leishmania major response (Lmr) loci. Two of them we previously found to influence IgE level in CcS-5. IgE production in CcS-16 is controlled by loci on chromosomes 2, 10, 16 and 18 and in CcS-20 by loci on chromosomes 1, 3, 4, 5 and 8. The STS alleles of loci on chromosomes 1, 4, 5, 8 and 10 were associated with a low, whereas the STS alleles on chromosomes 16 and 18 with a high IgE production. The loci on chromosomes 2 and 3 have no apparent individual effect, but interact with the loci on chromosomes 10 and 1, respectively. The loci on chromosomes 10 and 18 were mapped in the regions homologous with the human regions containing genes that control total serum IgE and intensity of infection by Schistosoma mansoni, suggesting that some Lmr loci may participate in the pathways influencing atopic reactions and responses to several parasites. The definition of genes controlling anti-parasite responses will permit a better understanding of pathways and genetic diversity underlying the disease phenotypes.     

HLA-DPB1 typing by polymerase chain reaction amplification with sequence-specific primers

DPB1 is the second most polymorphic class II locus with currently 84 recognized alleles, i.e. DPB1*0101 to DPB1*8101. Most of the alleles have been described during the last few years using oligonucleotide and sequencing techniques and relatively little is known about the role and importance of the polymorphic residues as regards to the function of DP molecules. In the present study, polymerase chain reaction (PCR) primers were designed for identification of all the phenotypically different DPB1 alleles by PCR amplification with sequence-specific primers. Forty-eight standard genomic PCR reactions per sample were performed in order to achieve this resolution. Unique amplification patterns were obtained in 2983 of 3160 (94.4%) possible genotypes. The primers were combined so that only very rare genotypes gave rise to ambiguous patterns. Sixty-four Histocompatibility Workshop cell lines and 150 DNAs provided by the UCLA DNA exchange were investigated by the DPB1 primer set. All typing results were conclusive. Analysis of the distribution of DPB1 alleles was performed in 200 Caucasian samples, 100 African samples and 40 Oriental samples. The population study by the DPB1 PCR-SSP method showed a characteristic distribution of HLA-DPB1 alleles. Each ethnic group had one, or two, frequent DPB1 allele(s) and the frequency of homozygotes was high, suggesting that balancing selection does not appear to be affecting the evolution of the DPB1 locus.     

Local environmental factors determine hematopoietic differentiation of neural stem cells

Stem cells exhibit unique properties and hold high therapeutic promise, but factors influencing their differentiation after transplantation need to be recognized and defined for this promise to be fully met. Here, we demonstrate that endogenous colony-forming unit spleen (CFU-S) colonies are not generated in lethally irradiated mice transplanted with neural stem cells obtained from brain tissue of syngeneic donors. We investigated the proportion of transplanted neural stem cells that contributed to hematopoietic reconstitution and compared the distribution of transplanted cells in nonsplenectomized to that of splenectomized mice following sublethal whole-body irradiation. We also used clonogenic assays, colony assays, and histochemical analyses to explore conditions under which transplanted, beta-galactosidase-tagged neural stem cells underwent hematopoietic differentiation. Our results suggest that neural stem cells do undergo extramedullary hematopoiesis, even while no endogenous hematopoietic colonies develop in the spleen. Furthermore, we found that neural stem cells effectively colonized the bone marrow of splectomized recipients. We conclude that the hematopoietic differentiation of neural stem cells is highly dependent on the extramedullary environment. We also conclude that the bone marrow does not provide an environment supportive of hematopoietic differentiation by neural stem cells.     

Structure of synovial lymphatic capillaries in rheumatoid arthritis and juvenile idiopathic arthritis

The structure of lymphatic capillaries (LC) of the synovial membrane (SM) from patients with rheumatoid arthritis and juvenile idiopathic arthritis obtained by synovectomy was investigated by transmission electron microscopy. This method allows comparison of the structure of the same vessel under light and electron microscope and clear differentiation between lymphatic and blood capillaries and venules. Synovial LC were localized in the subintimal connective tissue of the SM in the vicinity of venules. The shape of some LC was irregular, suggesting edema of the interstitium. Lymphatic endothelium has extremely attenuated cytoplasm with the exception of the perinuclear region. Many nuclei of endothelial cells had distinct nucleoli. The basal lamina was discontinuous. The walls of LC showed close connection with the interstitium represented by anchoring filaments that were attached to the endothelial cells and to the surrounding connective tissue. In some LC connective tissue appeared to be disconnected from endothelium and gaps between their walls and the interstitium were seen. Mononuclear cells were accumulated adjacent to some LC. Specialized interendothelial junctions (endothelial microvalves) were observed in the LC walls. Their structure and function in the migration of cells and debris from synovial interstitium into LC lumina in rheumatoid arthritic synovium deserves further investigation. In the lumina of some of the LC lymphocytes, monocytes, macrophages, cell debris and enlarged endothelium were observed. Accumulation of such material may cause obstruction of tiny LC. We suggest that reported alterations of the fine synovial lymphatic vessels can contribute to the progression of the inflammatory process to chronicity.     

The epidemic wave of meningococcal disease in Spain in 1996-1997: probably a consequence of strain displacement

During 1996 and 1997 an epidemic wave of meningococcal disease took place in Spain. Initial studies described the antigenic expression of the epidemic strain as C:2b:P1.2,5 and proposed that it was a variant of the previously identified Spanish C:2b:non-subtypable epidemic strain. To clarify this hypothesis, 1036 C:2b:P1.2(5) and 76 C:2b:NST isolates obtained during 1992-1999 were analysed by pulsed-field gel electrophoresis. The majority of the C:2b:P1.2,5 and C:2b:P1.2 isolates showed one of two very closely related profiles. During the epidemic period, 80% of the C:2b:NST strains showed these two pulsotypes. However, before the epidemic wave, most of these C:2b:NST strains (60%) showed a profile that was found infrequently among C:2b:P1.2,5 and C:2b:P1.2 isolates. A similar evolution was observed in C:2b:P1.5 isolates. Thirty-four C:2b:P1.2(5) and 10 C:2b:NST isolates, exhibiting representative pulsotypes, were subjected to multi-locus sequence typing. Isolates belonging to both A4 and ET-37 lineages were identified. These data point to the possibility that the A4 cluster has displaced the ET-37 complex among serogroup C meningococci in Spain.     

Cell-cell and cell-matrix interactions during initial enamel organ histomorphogenesis in the mouse

Relationships between cell-cell/cell-matrix interactions and enamel organ histomorphogenesis were examined by immunostaining and electron microscopy. During the cap-bell transition in the mouse molar, laminin-5 (LN5) disappeared from the basement membrane (BM) associated with the inner dental epithelium (IDE), and nondividing IDE cells from the enamel knot (EK) underwent a tooth-specific segregation in as many subpopulations as cusps develop. In the incisor, the basement membrane (BM) in contact with EK cells showed strong staining for LN5 and integrin alpha 6 beta 4. LN5 seems to provide stable adhesion, while its proteolytic processing might facilitate cell segregation. In both teeth, immunostaining for antigens associated with desmosomes or adherens junctions was similar for EK cells and neighboring IDE cells. Outside the EK, IDE cell-BM interactions changed locally during the initial molar cusp delimitation and on the labial part of the incisor cervical loop. Conversely, cell-cell junctions stabilized the anterior part of the incisor during completion of morphogenesis. Time and space regulation of cell-matrix and cell-cell interactions might thus play complementary roles in allowing plasticity during tooth morphogenesis and stabilization at later stages of epithelial histogenesis.