Development of a monoclonal antibody specific for beta/A4 amyloid in Alzheimer's disease brain for application to in vivo imaging of amyloid angiopathy.

We evaluated the efficacy of murine monoclonal antibodies (Mabs) targeted to beta/A4 amyloid for development of procedures for the in vivo identification of amyloid angiopathy (AA) in Alzheimer's disease (AD). Mabs to beta/A4 amyloid were prepared and screened for effectiveness in visualizing AA and senile plaques in postmortem AD brain sections. They were assessed again after enzymatic cleavage to produce Fab fragments and after labeling with 99mTc using a diamide dimercaptide ligand system. Modified and radiolabeled Fab fragments retained activity and specificity towards amyloid-laden blood vessels and senile plaques. A highly specific murine Mab, 10H3, was identified and characterized that fulfills criteria necessary for the development of a diagnostic imaging agent. Expansion and adaptation of these strategies may provide the methods and materials for the noninvasive analysis of AA in living patients, and permit assessment of the contribution of AA to the clinical and pathological features of AD.     

Multiple genetic alterations in malignant metastatic insulinomas

Proto-oncogenes, growth factors/receptors, and tumour suppressor genes were analysed in malignant metastatic insulinomas. Normal pancreas showed only a moderate immunoreaction for c-myc proto-oncogene and a strong reaction for insulin. Benign insulinomas were slightly or moderately positive for transforming growth factor a (TGFα), weakly positive for epidermal growth factor receptor (EGF-R), and strongly positive for c-myc and insulin. In malignant insulinomas, besides a strong immunoreaction for c-myc and TGFα, activation of c-K-ras and overexpression of p53 protein were found. Insulin reaction was moderate or strong. Three out of six malignant insulinomas displayed a c-K-ras point mutation at codon 12. All mutations were guanine to cytosine transversion, resulting in amino acid substitution, glycine to arginine. Mutations were present in metastatic insulinomas only. Patients with mutated c-K-ras oncogene had overexpression of p53 protein as well as c-myc and TGFα overexpression. Our results support the view that malignant progression is a consequence of more than one genetic lesion and suggest that activation of myc, TGFα, and ras genesα plays a role in a multistep process of tumour progression, perhaps serving as an initiating event.     

Use of a group-reactive and other monoclonal antibodies in an enzyme immunodot assay for identification and presumptive serotyping of aquatic birnaviruses.

A panel of monoclonal antibodies (MAbs) was prepared and used to develop an enzyme immunodot assay for the rapid identification and presumptive serotyping of aquatic birnaviruses. Comparison of the reaction patterns of these MAbs with representative virus isolates indicated that one MAb recognizes a serogroup-reactive epitope and can therefore be used for identification of all serogroup A aquatic birnaviruses, the predominant serotype worldwide. Other MAbs exhibited more restrictive specificities, permitting the presumptive serotyping of viruses of the three recognized serotypes and the identification of some individual strains. This assay, in which MAbs are used, is more efficient in terms of time, cost, and case of performance and provides significant advantages in specificity and standardization compared with currently used tests.     

Cure of human carcinoma xenografts by a single dose of pretargeted yttrium-90 with negligible toxicity

A covalent conjugate (NR-LU-10/SA) was prepared between streptavidin (SA) and NR-LU-10, a mAb that binds an antigen expressed on the surface of most human carcinomas. NR-LU-10/SA was injected into nude mice bearing human tumor xenografts. Injection of biotinylated galactosyl-human serum albumin reduced the circulating levels of conjugate by 95%. Subsequent administration of (90)Y-1,4,7, 10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-biotin achieved peak uptake at the tumor within 2 hr while >80% of the radioactivity was eliminated in the urine. A single dose of 600-800 microCi of (90)Y-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-biotin produced cures in 10/10 mice with established (>200 mm(3)) s.c. human small cell lung or colon cancer xenografts and 8/10 cures in mice with human breast cancer xenografts without significant toxicity.     

Invasive cribriform breast carcinomas in patients with grade 1 and stage IIA (T2 N0 M0) breast cancer strongly express the v3 and v6, but not the v4 isoforms of the metastatic marker CD44

Patients with breast carcinomas of the invasive cribriform (IC) histological type often have excellent prognosis. Nevertheless, prognostic markers such as CD44v3, v4, and v6 isoforms have never been evaluated in this histological type. Cases seen between 1996 and 2006 at two major public hospitals in Kuwait were reviewed. We selected the cases which still had enough tissue in the paraffin blocks, had pure rather than mixed typical histological type, did not receive hormonal or any other type of therapy prior to or at the time the tumor was excised, and which were grade 1, and stage IIA (T2, N0, M0). This is to control for confounding factors that could affect the degree of tumor expression of the above isoforms. Sections were immunostained using a highly sensitive roxidase-anti-peroxidase kit, and scoring of immunostaining was performed in a semi-quantitative manner as established in the literature. An extensive expression of the CD44v3 and v6 isoforms was seen in 83.3% of the IC tumours, while 83.3% lacked the expression of the v4 isoform. A significant association between the histological type and degree of expression of CD44 isoforms was found only with the v3 isoform. The degree of expression of the v3 isoform was significantly different in the IC tumors as compared to the papillary, invasive lobular, and invasive ductal (NOS) ones. There was a significant negative correlation (rs= - 0.201) between the expression of the v4 and v6 isoforms. IC tumors seem to have a strong expression of the prognostic markers v3 and v6 isoforms of the transmembrane molecule CD44, and to lack the expression of the v4 isoform.     

Release of [(3)H]-L-glutamate by stimulation of nicotinic acetylcholine receptors in rat cerebellar slices

This is a neurochemical study which shows that nicotine acting through alpha7-containing nicotinic acetylcholine receptors promotes the release of [(3)H]-glutamate from rat cerebellar slices. Release evoked by half maximal concentration of nicotine (100 microM) was blocked by alpha-bungarotoxin and in a calcium-free medium, suggesting an effect mediated by an alpha7 receptor. Dihydro-beta-erythroidine and mecamylamine were effective only at very high concentrations, excluding the participation of heteromeric receptors. The effect of nicotine was partially blocked by inhibitors of glutamatergic receptors DL-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione, indicating a glutamate-induced glutamate release. Nicotine-evoked response was dependent on activation of tetrodotoxin sensitive sodium channels. Therefore, here we show that glutamate released by stimulation of alpha7-containing nicotinic receptors, located preterminal and/or postsynaptically, evokes a further glutamate release in adult rat cerebellar slices.     

Frequency of loss of heterozygosity of the NF2 gene in schwannomas from Croatian patients

Aim. To identify gross deletions in the NF2 gene in a panel of schwannomas from Croatian patients in order to establish their frequencies in Croatian population. Methods. Changes of the NF2 gene were tested by polymerase chain reaction/loss of heterozygosity (LOH) using two microsatellite markers, D22S444 and D22S929. Results. The analysis with both markers demonstrated that 43.75% of schwannomas exhibited LOH of the NF2 gene. The D22S444 region exhibited 45.5% of LOHs and the D22S929 region exhibited 14.3% of LOHs. Four LOHs were found in Antoni B, 2 in Antoni A, and 1 in Antoni A and B type tumors. Conclusion. The frequency of changes observed in Croatian patients is broadly similar to that reported in other populations and thus confirms the existing hypothesis regarding the tumorigenesis of schwannomas and contributes to schwannoma genetic profile helping us to better understand its etiology and treatment.     

Analysis of protein kinase C delta (PKC delta) expression in endometrial tumors

Endometrial cancer is the most common gynecologic malignancy in the United States. However, its underlying molecular mechanisms are poorly understood; and few prognostic indicators have been identified. The protein kinase C (PKC) family has been shown to regulate pathways critical to malignant transformation; and in endometrial tumors, changes in PKC expression and activity have been linked to a more aggressive phenotype and poor prognosis. We have recently shown that PKC delta is a critical regulator of apoptosis and cell survival in endometrial cancer cells; however, PKC delta levels in endometrial tumors had not been determined. We used immunohistochemistry to examine PKC delta protein levels in normal endometrium and endometrioid carcinomas of increasing grade. Normal endometrium exhibited abundant nuclear and cytoplasmic staining of PKC delta confined to glandular epithelium. In endometrial tumors, decreased PKC delta expression, both in intensity and fraction of epithelial cells stained, was observed with increasing tumor grade, with PKC delta being preferentially lost from the nucleus. Consistent with these observations, endometrial cancer cell lines derived from poorly differentiated tumors exhibited reduced PKC delta levels relative to well-differentiated lines. Treatment of endometrial cancer cells with etoposide resulted in a translocation of PKC delta from cytoplasm to nucleus concomitant with induction of apoptosis. Decreased PKC delta expression, particularly in the nucleus, may compromise the ability of cells to undergo apoptosis, perhaps conferring resistance to chemotherapy. Our results indicate that loss of PKC delta is an indicator of endometrial malignancy and increasing grade of cancer. Thus, PKC delta may function as a tumor suppressor in endometrial cancer.     

Diet-induced obesity leads to pro-inflammatory alterations to the vitreous humour of the eye in a rat model

Objective and design

The purpose of this study was to investigate if diet-induced obesity (DIO) and subsequent low-level systemic inflammation would result in local increases in pro-inflammatory mediators in the vitreous humour (VH) of the eyes of rats.

Methods

Sixteen male Sprague–Dawley rats were fed a high-fat/high-sucrose (n?=?9) or chow control-diet (n?=?7) for 12-weeks. RT-qPCR was conducted on RNA from VH cells and a 27-plex Luminex^® Assay was conducted on VH fluid and serum.

Results

Increased protein levels for IL-1β, IL-6, and IL-18 in both serum and VH fluid were observed. VH protein levels for IL-13 and IL-17 were also increased. All mediators significantly increased in VH fluid were also positively correlated with percent body fat. Increased mRNA levels in VH cells for an oxidative stress molecule were accompanied by decreased mRNA levels for an antioxidant scavenger, suggesting an antioxidant/oxidant imbalance in the VH with DIO. In addition, decreased mRNA levels for TRAIL, FAS-L and TGF-β, molecules associated with immune privilege, were also significantly depressed.

Conclusions

DIO-related metabolic disturbances disrupt VH homeostasis in a manner that reflects development of a pro-inflammatory environment. Prolonged exposure to such an environment may lead to overt pathologies with compromised eye function.