Different pathways of molecular pathophysiology underlie cognitive and motor tauopathy phenotypes in transgenic models for Alzheimer's disease and frontotemporal lobar degeneration.

A poorly understood feature of the tauopathies is their very different clinical presentations. The frontotemporal lobar degeneration (FTLD) spectrum is dominated by motor and emotional/psychiatric abnormalities, whereas cognitive and memory deficits are prominent in the early stages of Alzheimer's disease (AD). We report two novel mouse models overexpressing different human tau protein constructs. One is a full-length tau carrying a double mutation [P301S/G335D; line 66 (L66)] and the second is a truncated 3-repeat tau fragment which constitutes the bulk of the PHF core in AD corresponding to residues 296-390 fused with a signal sequence targeting it to the endoplasmic reticulum membrane (line 1; L1). L66 has abundant tau pathology widely distributed throughout the brain, with particularly high counts of affected neurons in hippocampus and entorhinal cortex. The pathology is neuroanatomically static and declines with age. Behaviourally, the model is devoid of a higher cognitive phenotype but presents with sensorimotor impairments and motor learning phenotypes. L1 displays a much weaker histopathological phenotype, but shows evidence of neuroanatomical spread and amplification with age that resembles the Braak staging of AD. Behaviourally, the model has minimal motor deficits but shows severe cognitive impairments affecting particularly the rodent equivalent of episodic memory which progresses with advancing age. In both models, tau aggregation can be dissociated from abnormal phosphorylation. The two models make possible the demonstration of two distinct but nevertheless convergent pathways of tau molecular pathogenesis. L1 appears to be useful for modelling the cognitive impairment of AD, whereas L66 appears to be more useful for modelling the motor features of the FTLD spectrum. Differences in clinical presentation of AD-like and FTLD syndromes are therefore likely to be inherent to the respective underlying tauopathy, and are not dependent on presence or absence of concomitant APP pathology.

Monocyte chemotactic protein-1 secreted by primary breast tumors stimulates migration of mesenchymal stem cells.

PURPOSE: Major barriers to effective adenovirus-based gene therapy include induction of an immune response and tumor-specific targeting of vectors. The use of mesenchymal stem cells (MSC) as systemic delivery vehicles for therapeutic genes has been proposed as a result of their combined ability to home in on the tumor site and evade the host immune response. This study is aimed at investigating factors mediating homing of human MSCs to breast cancer primary cultures and cell lines in vitro and in vivo. EXPERIMENTAL DESIGN: Fluorescently labeled MSCs were given to mice bearing breast cancer xenografts, and tumor tissue was harvested to detect MSC engraftment. MSC migration in response to primary breast tumors in vitro was quantified, and chemokines secreted by tumor cells were identified. The role of monocyte chemotactic protein-1 (MCP-1) in cell migration was investigated using antibodies and standards of the chemokine. Serum MCP-1 was measured in 125 breast cancer patients and 86 healthy controls. RESULTS: Engrafted MSCs were detected in metastatic breast tumors in mice after systemic administration. There was a significant increase in MSC migration in response to primary breast tumor cells in vitro (6-fold to 11-fold increase). Tumor explants secreted a variety of chemokines including GROalpha, MCP-1, and stromal cell-derived factor-1alpha. An MCP-1 antibody caused a significant decrease (37-42%) in MSC migration to tumors. Serum MCP-1 levels were significantly higher in postmenopausal breast cancer patients than age-matched controls (P < 0.05). CONCLUSIONS: These results highlight a role for tumor-secreted MCP-1 in stimulating MSC migration and support the potential of these cells as tumor-targeted delivery vehicles for therapeutic agents.

Mechanisms of disease and injury: utilization of mutants, monoclonals, and molecular methods.

Rapid advances in our ability to localize and quantify macromolecular changes in health and disease are being brought about by the availability of genetically altered animals (mutants), purified reagents such as monoclonal antibodies, and new molecular methods. Targeted gene deletion (knockouts) and gene insertions (transgenics) in animals can allow identification of the importance and function of macromolecules. Monoclonal antibodies and fluorescent labels coupled with advances in microscopy provide exacting and multi-dimensional information about localization and cellular changes in proteins, carbohydrates, and lipids using immunohistochemistry, fluorescent activated cell sorting, and immunoprecipitation. Similarly, new applications of molecular methods can be used to identify and localize nucleic acids in tissues via in situ hybridization, polymerase chain reaction (PCR), reverse transcription (RT) PCR, differential display RT-PCR, RNase protection assays, and microchip arrays. The ligand for CD40 (CD40L), an important immunoregulatory molecule, is an example of the successful application of mutants, monoclonal antibodies, and molecular methods to cloning and biological characterization of new molecules. CD40L knockout mice, monoclonal antibodies, and several molecular methods were used to identify mutations in CD40L as the genetic basis for hyper-IgM syndrome in humans, to provide new insights into the pathobiology of Pneumocystis carinii infection, and to evaluate CD40L for immunotherapy of tumors and opportunistic infections.

Dopamine transporter (Dat) and synaptic vesicle amine transporter (VMAT2) gene expression in the substantia nigra of control and Parkinson's disease.

The cellular expression of DAT mRNA and VMAT2 mRNA was investigated in sections of the human post-mortem substantia nigra in control and Parkinson's disease tissue using in situ hybridisation techniques. Short synthetic oligodeoxynucleotides were used to detect these gene transcripts at the cellular level. In the control human nigra, high levels of expression were seen in all sub-divisions of the substantia nigra, especially within medial regions. By contrast, the level of expression of both DAT mRNA and VMAT2 mRNA was markedly reduced in Parkinson's disease; these reductions in hybridisation signal were associated with (i) a marked loss of dopamine-containing cells in the substantia nigra, and (ii) a reduction in both DAT and VMAT2 signal per cell in the remaining pigmented neurones. These disease-related decreases in the cellular abundance of both DAT and VMAT2 gene transcripts in the surviving cells of the parkinsonian nigra may reflect compensatory changes in catecholamine signalling or may be a consequence of neuronal dysfunction.

Cellular localisation and co-expression of somatostatin receptor messenger RNAs in the human brain.

Genes for five high affinity somatostatin receptors, named sst1-5, have been cloned recently. In this study we describe the tissue distribution and cellular localisation of mRNA encoding sst1, sst3 and sst4 receptors in the human cerebellum, frontal cortex (Brodmann's area 11) and hippocampus. RT-PCR and in situ hybridisation studies indicated a distinct, but partially overlapping pattern of expression of the receptor mRNAs. In situ hybridisation studies using co-expression techniques with probes for sst1, sst3 and sst4 receptor mRNA on paraffin sections revealed the presence of neurones expressing more than one somatostatin receptor mRNA type in both the hippocampus and pyramidal cells of layer V of the frontal cortex.

Dopamine D1 receptor, D2 receptor, proenkephalin A and substance P gene expression in the caudate nucleus of control and schizophrenic tissue: a quantitative cellular in situ hybridisation study.

The cellular expression of the mRNAs encoding the dopamine D1 receptor, dopamine D2 receptor and the neuropeptides enkephalin and substance P was determined in fresh frozen sections of human post-mortem caudate nucleus from control and schizophrenic brains using the technique of radioactive in situ hybridisation coupled with computer-assisted image analysis. Measurements of silver grain densities and mean cross-sectional somatic areas revealed no significant differences in the expression of any of these four gene transcripts. Further, cell count estimates revealed that each of these four mRNAs was expressed by approximately 20% of caudate cells (neurones and glia) in both control and schizophrenic tissue. These data demonstrate that the cellular expression of the dopamine D1 and D2 receptors and the neuropeptides enkephalin and substance P mRNAs are stable post mortem and that the relative cellular abundance of these mRNAs is not altered in the caudate nucleus of schizophrenic brains when compared to controls. These findings draw into focus the possible sites of action of clinically prescribed neuroleptics and suggest that chronic neuroleptic treatment of patients displaying negative schizophrenic symptoms may 're-set' an underlying neurochemical imbalance within the caudate nucleus.

Expression of messenger RNA for somatostatin receptor subtype 4 in adult rat brain.

The distribution and cellular localisation of somatostatin receptor subtype 4 (SSTR4) was investigated in the adult rat brain using the technique of in situ hybridisation with subtype specific oligonucleotide probes. Somatostatin receptor subtype 4 was found mainly in the hippocampus CA1 > CA2 > CA3 pyramidal cells and in the pyramidal cells in layers (IV-VI) of the cerebral cortex. Reverse transcription-PCR with SSTR4 specific primers confirmed the tissue distribution revealed by in situ hybridisation.

The molecular cloning and expression of a human synaptic vesicle amine transporter that suppresses MPP+ toxicity.

A synaptic vesicle amine transporter cDNA, termed hSVAT, has been isolated by the reverse transcription and polymerase chain reaction (PCR) technique from human substantia nigra and subsequent screening of a human substantia nigra library. The hSVAT sequence obtained is highly homologous to the rat SVAT sequence (92% homology) and is essentially identical to the human sequence identified recently by Surratt and colleagues [33]. This labelled hSVAT cDNA detected a single band (approximately 5.0 kb) when used as a probe for Northern analysis of human nigral RNA extract. In situ hybridization studies using hSVAT specific antisense oligonucleotides showed a strong hybridization signal concentrated over the cells of the substantia nigra pars compacta. This cDNA sequence when expressed in chinese hamster ovary (CHO) cells conferred resistance to MPP+ the toxic metabolite of MPTP and cells containing it accumulated dopamine.

The initial suppression of bacterial growth in a salmonella infection is mediated by a localized rather than a systemic response.

Mice were infected intravenously with two antibiotic resistance tagged variants of the same S. typhimurium strain given in close succession, or simultaneously with strains of different virulence. The first manifestation of acquired resistance--suppression of exponential bacterial growth in liver and spleen--occurred independently for the different strains in the same individuals, implying that it is due to localized rather than systemic events. This early suppression of bacterial growth was ablated by whole body X-irradiation (800R), whereas the immediately preceding phase of exponential growth (Ity controlled innate resistance) was not affected. Transfer of spleen cell suspensions from infected mice into syngeneic recipients conferred protection by suppressing the growth of an intravenous challenge. Pre-treatment of the suspensions to deplete them of macrophages abolished their protective capacity, while depletion of T-cells did not. Mice deficient in T-cells by adult thymectomy and anti-T-cell monoclonal antibody treatment were able to suppress the growth of an intravenous challenge. Taken collectively, the present data show that the very early phase of acquired resistance to salmonellae, essential for survival, is not the result of systemically developing resistance but a localized event at the site of infection.

Expression of the innate resistance gene Ity in mouse Kupffer cells infected with Salmonella typhimurium in vitro.

Early innate resistance to salmonellae in mice is controlled by the chromosome 1 gene Ity which regulates the in vivo net growth rate of bacteria in the RES by an unknown mechanism. It similarly controls innate resistance to Leishmania donovani, BCG and Mycobacterium lepraemurium. Murine Kupffer cell cultures were infected with virulent Salmonella typhimurium and followed for 12 h. Multiresistant organisms were used so that the antibiotics in the medium could not interfere with the results; extracellular bacteria were removed by repeated washes. Monolayers from resistant Ityr (C3H/He, CBA, A/J) and Ityr/s (B10 x A/J)F1 mice resisted infection with S. typhimurium C5 better than those from susceptible Itys (DBA/1, BALB/c, B10.M) mice, which were progressively lost from the culture plates at a faster rate than resistant monolayers. Organisms were clearly visible inside large vacuoles in the macrophages. The results confirm and amplify results of others on salmonella-infected peritoneal and splenic macrophages, and support the view that the Ity gene is expressed as a function of macrophages.

Natural resistance to salmonellae in mice: control by genes within the major histocompatibility complex.

Determinations of 50% lethal dose (LD50) values in H-2 congenic B10 lines showed that late-emerging resistance (postimmune response phase) to salmonellae of intermediate virulence was less in H-2b and H-2d than in H-2a, H-2k, and H-2f mice. Association of resistance to H-2 was confirmed by backcross analysis, and LD50 determinations on H-2 recombinant haplotype strains showed that resistance maps to the I-E subregion. Bacterial growth curves in liver and spleen showed that susceptible mice carried bacteria for longer in the reticuloendothelial system than did resistant mice and that susceptible mice showed greater splenomegaly. Association of resistance and susceptibility to H-2 was not different when sister transductant salmonellae expressing somatic antigens O4 and O9 were used. Thus a gene(s) within the major histocompatibility complex controls natural resistance to salmonellae in mice by influencing the ability to clear bacteria from the reticuloendothelial system in the later phase of the infection, and the immunodominant O antigen cannot be solely involved.

Cardiac rehabilitation: evaluation and intervention less than 6 weeks after myocardial infarction.

Twenty-nine patients having myocardial infarction (MI) of recent origin (6 weeks or less) were evaluated and treated using a program in which assessment and progression were based not on generalized data but on the individual responses of each patient. Fourteen patients had complications including left ventricular impairment, continuing ischemia or rhythm disturbances, and 15 did not. Cardiac tolerance for the common self-care activities, walking and light exercise, was objectively defined by 3 levels of functional monitoring. The physician, occupational therapist and physical therapist evaluated the patient's responses to mild exertion, utilizing a 12-lead electrocardiogram (ecg), physical examination, 24-hour monitoring with a portable ecg, self-care evaluation and a modified treadmill screening test. Patient performance was assessed at each stage of testing and individualized activity levels were determined in accordance with cardiac responses. Thus patients progressed at their optimum rate with safety and without loss of time, ie, artificially induced invalidism. Following the 1st self-care evaluation, 3 patients were ordered to bedrest for further medical treatment while 26 were cleared for ward activity. When their conditions improved the 3 patients were reevaluated and began the program. The 15 patients without complications had appropriate responses to activity and proceeded to a mild exercise program with minimal observation. Of the 14 with complications, 5 experienced a temporary program interruption and 2 were dropped from the program secondary to severity of complications. The remaining 7 were progressed in the same manner as those without complication.