Mapping adolescent brain change reveals dynamic wave of accelerated gray matter loss in very early-onset schizophrenia

Neurodevelopmental models for the pathology of schizophrenia propose both polygenetic and environmental risks, as well as early (pre/perinatal) and late (usually adolescent) developmental brain abnormalities. With the use of brain mapping algorithms, we detected striking anatomical profiles of accelerated gray matter loss in very early-onset schizophrenia; surprisingly, deficits moved in a dynamic pattern, enveloping increasing amounts of cortex throughout adolescence. Early-onset patients were rescanned prospectively with MRI, at 2-year intervals at three time points, to uncover the dynamics and timing of disease progression during adolescence. The earliest deficits were found in parietal brain regions, supporting visuospatial and associative thinking, where adult deficits are known to be mediated by environmental (nongenetic) factors. Over 5 years, these deficits progressed anteriorly into temporal lobes, engulfing sensorimotor and dorsolateral prefrontal cortices, and frontal eye fields. These emerging patterns correlated with psychotic symptom severity and mirrored the neuromotor, auditory, visual search, and frontal executive impairments in the disease. In temporal regions, gray matter loss was completely absent early in the disease but became pervasive later. Only the latest changes included dorsolateral prefrontal cortex and superior temporal gyri, deficit regions found consistently in adult studies. These emerging dynamic patterns were (i) controlled for medication and IQ effects, (ii) replicated in independent groups of males and females, and (iii) charted in individuals and groups. The resulting mapping strategy reveals a shifting pattern of tissue loss in schizophrenia. Aspects of the anatomy and dynamics of disease are uncovered, in a changing profile that implicates genetic and nongenetic patterns of deficits.     

The U3 portion of feline leukemia virus DNA identifies horizontally acquired proviruses in leukemic cats.

The presence and location of DNA sequences related to the U3 and U5 portions of the infectious exogenous feline leukemia virus (FeLV) long terminal repeat (LTR) in various cat DNAs have been determined by hybridization experiments. In uninfected cat DNAs, the U5 LTR segment from the Gardner-Arnstein strain B virus is present at approximately 150 copies per cell. This level is approximately 10-fold greater than that of endogenous internal FeLV sequences. The U5 sequences differ in copy number and, to some extent, in location from one animal to another. For any one animal, the sequence organization of the U5 segments is the same among different tissues, showing that the pattern is inherited through the germ line. Most importantly, the viral U3 LTR probe hybridizes only very weakly with uninfected cat DNAs. Both the U3 and the U5 regions of the LTR from the Gardner-Arnstein strain of virus cross-hybridize with DNA derived from four other infectious FeLVs representing A, B, and C subtypes. Thus, the C3 region may be used as a probe for studying the number and location of exogenously acquired FeLV proviruses in infected cat tissues. In some cases exogenously acquired proviruses are present in unique sites in the genome of virus-positive cat lymphosarcomas, indicating a monoclonal origin for the tumor. In other tumors, the proviral sequences are randomly distributed over many sites. Lymphosarcomas of virus-negative cats have no exogenous U3 sequences despite epidemiological evidence of an association of virus-negative leukemia with exposure to FeLV.     

Heterogeneous expression of cytokeratins in metastatic mammary adenocarcinoma cells in vitro and in vivo

The expression of cytokeratins was investigated in rat 13762NF adenocarcinoma cell lines and clones growing in vitro and in vivo. The anti-cytokeratin monoclonal antibody PKK1 used in this study recognized one cytokeratin with a molecular weight of 54,000 in these cells. Immunofluorescence staining of cultured tumor cells with PKK1 detected cytokeratins in less than 1% of the tumor cells isolated from the locally growing parental tumors, but intense cytokeratin staining in the form of fibrils and networks was found in the cell cultures derived from spontaneous metastases. There were variable degrees of staining with the anti-cytokeratin antibody in cultured cells from metastases, indicating phenotypic diversity in the expression of cytokeratins. Staining with PKK1 of frozen sections from locally growing tumors obtained after injecting the cells into the mammary fat pads of syngeneic rats also demonstrated considerable heterogeneity in cytokeratin expression. The anti-cytokeratin reagent stained only a few, isolated cells in frozen sections of tumors established from parental tumor cells, while this reagent stained intensely cells in local tumors and their metastases established from tumor cells cultured from spontaneous lymph node or lung metastases.     

Coexpression of cytokeratins characteristic for myoepithelial and luminal cell lineages in rat 13762NF mammary adenocarcinoma tumors and their spontaneous metastases.

We used immunohistochemical procedures to study the cellular expression and distribution of cytokeratins (CKs) in rat 13762NF mammary adenocarcinoma cells growing at mammary fat pad sites and at spontaneous lymph node and lung sites. In order to establish CK distribution in normal rat mammary epithelia, immature, resting, and lactating rat mammary glands were probed with a panel of monospecific antibodies that recognize individual CKs. Basal/myepithelial cells were distinguished by expression of CKs 5 and 14 and coexpression of vimentin from luminal cells, which expressed CKs 8, 18, and 19. Antibody to CK 7 recognized luminal epithelium of immature and resting, but not lactating, mammary glands. Myoepithelial cells of lactating mammary gland were weakly recognized by antibodies to CKs 7 and 19. Tumors formed by cell lines and clones derived from parental 13762NF tumor (MTPa, MTC, MTA, and MTF7) were not recognized by any of the anti-CK antibodies. Only vimentin was expressed in these tumors and their metastases. In tumors and metastases generated from cell lines and clones derived from lymph node (MTLY) and lung metastases (MTLn2 and MTLn3) of the 13762NF tumor we observed heterogeneous CK phenotypes. Expression of CKs 5 and 18 was greatly reduced or lacking, while CK 14 was coexpressed with CKs 7, 8, and 19 with or without vimentin. Tumors from the highly metastatic clone MTLn3 had a dominant cellular phenotype, expressing CKs 7, 8, 14, and 19 and vimentin, a pattern that did not match normal mammary epithelia, whether luminal, basal/myoepithelial, or the dual-phenotype stem cell, in which CKs 5, 8, 14, and 18 were coexpressed. MTLn3 lymph node and lung metastases expressed the same cellular phenotype as the s.c. growing MTLn3 tumor. The results appear to contradict the belief that malignant mammary tumors may be distinguished from benign tumors or hyperplastic growths by the lack of basal/myoepithelial markers.     

Gene expression profiling in non-small cell lung cancer: from molecular mechanisms to clinical application.

Non-small cell lung cancer (NSCLC) is the most common cause of premature death from malignant disease in western countries. A better understanding of the molecular mechanisms underlying NSCLC etiology, pathogenesis, and therapeutics will lead to improved clinical outcomes. Recent technological advances in gene expression profiling (in particular, with cDNA and oligonucleotide microarrays) allow the simultaneous analysis of the expression of thousands of genes. In this review, the technology of global gene expression profiling is discussed, and the progress made thus far with it in NSCLC is reviewed. A new molecular classification of NSCLC has been developed, which has provided important insights into etiology and pathogenesis. Other studies have found potential biomarkers for NSCLC that may be of use in diagnosis, screening, and assessing the effectiveness of therapy. Finally, advances have been made in the understanding of the molecular mechanisms of NSCLC progression and the molecular mechanisms of action of currently used cytotoxic drugs. This may facilitate the improvement of current therapeutics and the identification of novel targets. Taken together, these advances hold the promise of an improved understanding of the molecular biology of NSCLC and its treatment, which in turn will lead to improved outcomes for this deadly disease.     

Mechanism of cell entry and toxicity of an affinity- purified lectin from Ricinus communis and its differential effects on normal and virus-transformed fibroblasts.

An affinity-purified plant lectin from Ricinus communis (RCAII) was shown to exhibit differential toxicity toward SV40-transformed 3T3 fibroblasts grown in vitro. When macromolecular synthesis was examined in SV3T3 and 3T3 cells, RCAII suppressed cell protein synthesis in the transformed line at lower concentrations (1/50 to 1/100) compared to the 3T3 line, and these effects were blocked by the RCAII inhibitors D-galactose or lactose. RNA and DNA synthesis and L-leucine transport were relatively unaffected by RCAII concentrations (greater than 1 mug/ml) that completely suppressed protein synthesis in both cell lines. The RCAII-mediated inhibition of cell protein synthesis required incubation times longer than 60 min, but quantitative cell binding studies with 125-I-RCAII indicated that the lectin binds to maximal levels in approximately 5 to 10 min, even at 4 degrees. During 10-min labeling experiments with 125-I-RCAII (1 mug/ml), it was demonstrated that the cell-bound lectin could be almost quantitatively removed from cells up to an additional 15 min after labeling without subsequent inhibition of protein synthesis. However, longer incubation times (greater than 30 min) after RCAII cell labeling and washing resulted in incomplete removal of cell-bound lectin (less than 20 to 30% of cell-bound lectin could be removed after a 60-min incubation). The longer incubation times (greater than 60 min) also resulted in almost complete inhibition of protein synthesis. Ferritin-conjugated RCAII (ferritin-RCAII) was used to follow the fate of the cell-bound lectin. Ferritin-RCAII bound rapidly (less than 10 min) to SV3T3 cell surfaces and could be blocked from labeling with lactose. After a 10-min incubation at 4 degrees in ferritin-RCAII solutions, the ferritin label was exclusively located at the extracellular surface in a random distribution. After washing and incubation at 37 degrees, the ferritin-RCAII induced clustering of its receptors (15 to 30 min) and eventually induced endocytosis (30 to 60 min). Further incubation (greater than 60 min) resulted in a predominantly intracellular localization of ferritin-RCAII inside endocytotic vesicles and free in the cell cytoplasm. That RCAII acts directly on protein synthesis after cell entry was confirmed with rabbit reticulocyte and mouse Krebs II ascites S30 cell-free protein synthesis system in diameter wit     

Adhesive, invasive, and growth properties of selected metastatic variants of a murine large-cell lymphoma

The adhesive, invasive, and growth properties of parental murine large-cell lymphoma cells of low metastatic potential (RAW117-P) were compared to in-vivo-selected sublines of high metastatic potential to liver (RAW117-H10) or lung (RAW117-L17). Using small (approximately 0.5 mm3) pieces of syngeneic organ tissue (lung, liver, kidney) we found that RAW117-L17 cells selectively attached to and invaded lung tissue, whereas RAW117-H10 cells preferentially attached to and invaded liver tissue. We measured adhesion to microvessel endothelial cells established from syngeneic lung and liver and found that the RAW117-L17 cells bound to lung microvessel endothelial cells at significantly higher rates than the other lines, and RAW117-H10 and -L17 cells attached to hepatic sinusoidal endothelial cells at significantly faster rates than RAW117-P cells. Such organ specificity of adhesion was not found at the level of the subendothelial matrix, and the rates of adhesion of RAW117 cells to subendothelial matrix were lower than to endothelial cells. RAW117 cells of low or high metastatic potential bound to immobilized extracellular matrix components, such as fibronectin, at high rats, but adhesion to laminin or collagen IV was minimal. Previous studies indicated that RAW117 lines could proliferate in vitro in certain organ-conditioned media under limiting serum conditions. We therefore examined the ability of a purified paracrine lung growth factor (LDGF-1) to stimulate growth of RAW117 cells in limiting serum-containing medium. The high lung-colonizing L17 line was stimulated to proliferate by LDGF-1 at faster rates than the other lines. The data support Paget's hypothesis that the organ specificity of tumor metastasis is determined by specific tumor cell and host properties.     

Effects of chemotherapeutic drugs on platelet and metastatic tumor cell-endothelial cell interactions as a model for assessing vascular endothelial integrity

An in vitro assay for examining the sublethal effects of chemotherapeutic agents on vascular endothelial integrity is described. Using vascular endothelial cell monolayers, the kinetics of binding of radiolabeled platelets or metastatic tumor cells were altered when endothelial cells were pretreated for 2 hr with low, clinically relevant concentrations of certain drugs. Electron microscopic examination by scanning electron microscopy revealed that these same drugs caused endothelial cell retraction and exposure of subendothelial matrix. Platelets and tumor cells were found bound only to the exposed areas of subendothelial matrix. Some drugs (bleomycin, 1,3-bis(2-chloroethyl)-1-nitrosourea, vincristine) induced rapid endothelial cell retraction and increased platelet and tumor cell binding to exposed subendothelial matrix, while one of the drugs tested (Adriamycin) caused delayed (1 to 3 days after a 2-hr drug treatment) endothelial cell retraction and increased cell binding. Of the drugs tested, only 5'-fluoro-2'-deoxyuridine which interferes with DNA replication failed to induce endothelial cell retraction and increased tumor cell and platelet binding. The results suggest that certain drug effects on the vascular endothelium can be assessed using the vascular endothelial cell monolayer model.