The monoclonal antibody Alz-50, used to reveal cytoskeletal changes in Alzheimer's disease, also reacts with a large subpopulation of somatostatin neurons in the normal human hypothalamus and adjoining areas

The monoclonal antibody Alz-50 is directed against Alzheimer's disease-related modified tau proteins and reveals cytoskeletal changes, i.e. neurofibrillary tangles and dystrophic neurites. The present study shows that, in the hypothalamus of non-demented control subjects, this same antibody gives a distinctive staining pattern of a subpopulation of somatostatin neurons and beaded fibers. Furthermore, Alz-50 occasionally recognizes somatostatin-containing cell bodies and dystrophic neurite-like fibers in the (neuritic) senile plaques of AD patients. These observations have direct consequences for the interpretation of Alz-50 staining in diagnostic usage and for the assessment of Alzheimer's disease-like changes induced by β-amyloid in experimental animal brains. On dot spotting, Alz-50 was found to bind to a number of fragments from the somatostatin precursor, of which somatostatin_15–28 stained best. Preadsorption of Alz-50 by somatostatin_15–28, as well as other specificity tests, failed, however, to provide any clue to the nature of the unknown compound(s) stained in the control hypothalamus.     

emm Gene distribution among erythromycin-resistant and -susceptible Italian isolates of Streptococcus pyogenes

The phenotypes and genetic determinants for macrolide resistance were determined for 167 erythromycin-resistant Streptococcus pyogenes strains. A cMLS phenotype was shown in 18% of the erythromycin-resistant strains, while inducible resistance was apparent in 31% and the M phenotype was apparent in 50%. The emm gene type of this set of resistant isolates and that of 48 erythromycin-sensitive isolates were determined. emm2 and emm48 were recorded only in the resistant strains of the M phenotype, while approximately all of the strains harboring the emm22 gene had the cMLS phenotype. More than 80% of the emm89-positive strains had the iMLS phenotype, and the same portion of emm4 strains presented the M phenotype. emm3 is recorded only among sensitive strains. The distribution of frequencies of the genetic determinant for the virulence factor M protein was significantly different both among organisms of different types of resistance and between resistant and sensitive populations of S. pyogenes under study.     

Biopathological profile of multiple synchronous homolateral and bilateral breast cancers

It still needs to be verified whether multiple syncronous homolateral and bilateral breast cancers represent intramammary spread of a single tumor or two or more separate neoplastic events. To clarify this problem, we studied the biopathological profile of 46 homolateral and 20 bilateral cases. The cancers were always surgically removed and processed at the same time. The expression of estrogen receptors (ER), progesterone receptors (PR), MIB 1, p53, and c-erbB-2 was determined. Computer-assisted image analysis (CAS 200) was used to evaluate ER, PR, MIB 1, and p53. The histological concordance was 95.6% in homolateral and 50% in bilateral cases. The immunophenotype profile of multiple homolateral neoplasms showed a concordance between 93.47% for ER and 78.26% for p53. The results were statistically significant for all parameters except for p53. In bilateral cancers, there was a significant statistical concordance for ER. These data strongly suggest that both mechanisms may exert an influence and, in particular, that in the majority of homolateral carcinomas, there may be intramammary spread of tumor cells. In multiple bilateral tumors, however, the great diversity of the histological aspects and the differences in the immunophenotype pattern suggest that the vast majority of these may constitute independent multiple events.     

Erythromycin resistance in italian isolates of Streptococcus pyogenes and correlations with pulsed-field gel electrophoresis analysis

Erythromycin resistance among Streptococcus pyogenes strains has been reported in Italy at high rates during the last few years. A total of 152 erythromycin-resistant isolates of this species from southern Italian regions were characterized for the macrolide-resistance phenotype and screened by PCR for the corresponding genetic determinant. A close correlation was found between these phenotypic/genotypic data concerning macrolide resistance and results of Sma I macrorestriction fragment patterns (PFGE) analysis. In fact, the vast majority of the isolates assigned to individual PFGE classes mostly belonged to a single phenotype of macrolide resistance. All untypeable isolates belonged to the M phenotype. Twenty-two distinct PFGE types were recognized, of which 11 were recorded in only one isolate (one-strain type); about 50% of typeable isolates fell into five type clusters and 70% in seven. The increased erythromycin resistance among Italian isolates of S. pyogenes does not appear to be due to the spread of a single clone, but results indicate that the majority of group A streptococci examined are probably spread from a limited number of clones.     

PCR m typing: a new method for rapid typing of group a streptococci

A new approach for the M-typing of Streptococcus pyogenes is reported. Oligonucleotide primers were used in a PCR to amplify the N-terminal region of the emm gene. The presence of the PCR amplification product is associated with the corresponding M serotype. This technique offers potential advantages over other molecular typing methods.     

Electrochemical functionalization of gold and silicon surfaces by a maleimide group as a biosensor for immunological application

In the present study we investigated the preparation of biofunctionalized surfaces using the direct electrochemical grafting of maleimidophenyl molecules with subsequent covalent immobilization of specific peptide to detect target antibody, thereby extending the application of the biosensing systems towards immunodiagnostics. Para-maleimidophenyl (p-MP) functional groups were electrochemically grafted on gold and silicon surfaces from solutions of the corresponding diazonium salt. A specially synthesized peptide modified with cysteine (Cys-peptide) was then immobilized on the p-MP grafted substrates by cross-linking between the maleimide groups and the sulfhydryl group of the cysteine residues. Accordingly, the Cys-peptide worked as an antigen that was able to bind specifically the target antibody (anti-GST antibody), while it was non-sensitive to a negative contrast antibody (i.e. anti-Flag β). The immobilization of both specific and non-specific antibodies on the Cys-peptide-modified surfaces was monitored by infrared spectroscopic ellipsometry, a quartz crystal microbalance integrated in flow injection analysis system and potentiometric response. The results obtained clearly demonstrated that the direct modification of a surface with maleimidophenyl provides a very simple and reliable way of preparing biofunctionalized surfaces suitable for the construction of immunological biosensors.     

In vitro study of cell survival following dynamic MLC intensity-modulated radiation therapy dose delivery

The possibility of reduced cell kill following intensity-modulated radiation therapy (IMRT) compared to conventional radiation therapy has been debated in the literature. This potential reduction in cell kill relates to prolonged treatment times typical of IMRT dose delivery and consequently increased repair of sublethal lesions. While there is some theoretical support to this reduction in cell kill published in the literature, direct experimental evidence specific to IMRT dose delivery patterns is lacking. In this study we present cell survival data for three cell lines: Chinese hamster V79 fibroblasts, human cervical carcinoma, SiHa and colon adenocarcinoma, WiDr. Cell survival was obtained for 2.1 Gy delivered as acute dose with parallel-opposed pair (POP), irradiation time 75 s, which served as a reference; regular seven-field IMRT, irradiation time 5 min; and IMRT with a break for multiple leaf collimator (MLC) re-initialization after three fields were delivered, irradiation time 10 min. An actual seven-field dynamic MLC IMRT plan for a head and neck patient was used. The IMRT plan was generated for a Varian EX or iX linear accelerator with 120 leaf Millenium MLC. Survival data were also collected for doses 1X, 2X, 3X, 4X, and 5x 2.1 Gy to establish parameters of the linear-quadratic equation describing survival following acute dose delivery. Cells were irradiated inside an acrylic cylindrical phantom specifically designed for this study. Doses from both IMRT and POP were validated using ion chamber measurements. A reproducible increase in cell survival was observed following IMRT dose delivery. This increase varied from small for V79, with a surviving fraction of 0.8326 following POP vs 0.8420 following uninterrupted IMRT, to very pronounced for SiHa, with a surviving fraction of 0.3903 following POP vs 0.5330 for uninterrupted IMRT. When compared to IMRT or IMRT with a break for MLC initialization, cell survival following acute dose delivery was significantly different, p < 0.05, in three out of six cases. In contrast, when cell survival following IMRT was compared to that following IMRT with a break for MLC initialization the difference was always statistically insignificant. When projected to a 30 fraction treatment, dose deficit to bring cell survival to the same value as in POP was calculated as 4.1, 24.9, and 31.1 Gy for V79, WiDr, and SiHa cell lines, respectively. The dose deficit did not relate to the alpha/beta ratio obtained in this study for the three cell lines. Clinical data do not show reduction in local control following IMRT. Possible reasons for this are discussed. The obtained data set can serve as a test data set for models designed to explore the effect of dose delivery prolongation/fractionation in IMRT on radiation therapy outcome.     

Alanine Mutagenesis of the Primary Antigenic Escape Residue Cluster,C1, of Apical Membrane Antigen 1

Antibodies against apical membrane antigen 1 (AMA1) inhibit invasion of Plasmodium merozoites into red cells, and a large number of single nucleotide polymorphisms on AMA1 allow the parasite to escape inhibitory antibodies. The availability of a crystal structure makes it possible to test protein engineering strategies to develop a monovalent broadly reactive vaccine. Previously, we showed that a linear stretch of polymorphic residues (amino acids 187 to 207), localized within the C1 cluster on domain 1, conferred the highest level of escape from inhibitory antibodies, and these were termed antigenic escape residues (AER). Here we test the hypothesis that immunodampening the C1 AER will divert the immune system toward more conserved regions. We substituted seven C1 AER of the FVO strain Plasmodium falciparum AMA1 with alanine residues (ALA). The resulting ALA protein was less immunogenic than the native protein in rabbits. Anti-ALA antibodies contained a higher proportion of cross-reactive domain 2 and domain 3 antibodies and had higher avidity than anti-FVO. No overall enhancement of cross-reactive inhibitory activity was observed when anti-FVO and anti-ALA sera were compared for their ability to inhibit invasion. Alanine mutations at the C1 AER had shifted the immune response toward cross-strain-reactive epitopes that were noninhibitory, refuting the hypothesis but confirming the importance of the C1 cluster as an inhibitory epitope. We further demonstrate that naturally occurring polymorphisms that fall within the C1 cluster can predict escape from cross-strain invasion inhibition, reinforcing the importance of the C1 cluster genotype for antigenic categorization and allelic shift analyses in future phase 2b trials.The merozoite stage of Plasmodium falciparum is a highly specialized form of the parasite that selectively invades human red blood cells. Although the exact mechanism of invasion is still under investigation, the expression of apical membrane antigen 1 (AMA1) appears to be an absolute requirement for successful invasion (31). Monoclonal and polyclonal antibodies against AMA1 inhibit invasion in vitro, and immunization with recombinant AMA1 protects against live parasite challenge in animal models of malaria (10a, 18, 28).P. falciparum AMA1 vaccines based on two laboratory strains, 3D7 and FVO, are being studied for efficacy in human trials (23, 27, 30). One of the major concerns in further development of the AMA1 vaccine is that ∼10% of its 622 amino acids are polymorphic (2). Strain specificity of vaccine-induced AMA1 antibodies has been observed by enzyme-linked immunosorbent assay (ELISA) and in a functional assay of parasite growth and invasion inhibition (growth inhibition assay [GIA]) (15, 24). Allelic replacement experiments show that sequence polymorphism within AMA1 causes antigenic escape (14), and the extent of escape correlates with sequence distance between the vaccine and target strain (18).The crystal structure of AMA1 shows that it contains two PAN domains, with loops extending outwards from its central core (1, 22). The loops contain the majority of the polymorphic residues of AMA1 and surround a highly conserved hydrophobic trough. Residues within the trough have been implicated in the binding of AMA1 to AMA1-associated proteins on the merozoite (5). The location of the polymorphic loops surrounding the trough is highly suggestive of its role of providing diversity to a functional region of AMA1, preventing the binding of inhibitory antibodies. Using chimeric proteins to specifically deplete strain-specific antibodies against 3D7 strain AMA1, in a growth inhibition assay (GIA), we showed that certain polymorphic sites conferred escape upon the FVO strain parasite from invasion inhibitory anti-3D7 AMA1 antibodies. These polymorphic residues were termed “antigenic escape residues” (AER). The majority of AER in the 3D7-FVO model mapped to domain 1 (10), and within domain 1, the highest escape per residue was conferred by 7 polymorphic sites located on a linear stretch of sequence between residues 187 and 207. This polymorphic cluster was termed the C1′ cluster (10). It contains the three most polymorphic residues of AMA1, as follows: residue 187 is located on loop Ic, and residues 197 and 200 are located on the adjacent C1-L loop (also termed loop Id).Given that the polymorphic residues within the C1 cluster are the primary determinants of strain specificity of AMA1, it is likely that protein engineering strategies targeted to C1 AER could be used to modulate the cross-reactivity of AMA1 antibodies. We hypothesized that if the immunogenicity of the C1 AER could be reduced, this might result in an increased antibody response to non-strain-specific cross-reactive epitopes. In this study, seven 3D7-FVO polymorphic differences within the C1 cluster were replaced on the FVO AMA1 protein with alanine residues. The 3D7-FVO escape model was then used to study the effect of alanine mutagenesis on the quantity and quality of the induced antibodies. This strategy resulted in a measurable shift in the immune response away from the C1 region but did not enhance the generation of cross-strain-reactive antibodies that were inhibitory to parasite invasion.     

Bordetella pertussis Clones Identified by Multilocus Variable-Number Tandem-Repeat Analysis

Multilocus variable-number tandem-repeat analysis (MLVA) of 316 Bordetella pertussis isolates collected over 40 years from Australia and 3 other continents identified 66 MLVA types (MTs), including 6 predominant MTs. Typing of genes encoding acellular vaccine antigens showed changes that may be vaccine driven in 2 MTs prevalent in Australia.