Review paper: Gene expression profiling in veterinary and human medicine: overview of applications and proposed quality control practices.

High throughput molecular analysis of veterinary tissue samples is being applied to a wide range of research questions aimed at improving survival, development of diagnostic assays, and improving the economics of commercial production of animal products. Many of these efforts also, implicitly or explicitly, have ramifications for the clinical care of humans and, potentially, animals. Here we provide an overview of applications of gene expression profiling in veterinary research and practice. We then focus on the current state of quality control and quality assurance efforts in gene expression profiling studies, underscoring lessons learned from such analysis of human samples. Finally, we propose practices aimed at ensuring the reliability and reproducibility of such assays. The implementation of quality assurance practices by a trained pathologist is an essential link in the chain of events leading ultimately to reliable and reproducible research findings and appropriate clinical care.

Neutralizing monoclonal antibodies to hepatocyte growth factor/scatter factor (HGF/SF) display antitumor activity in animal models.

The hepatocyte growth factor (HGF/SF) receptor, Met, regulates mitogenesis, motility, and morphogenesis in a cell type-dependent fashion. Activation of Met via autocrine, paracrine, or mutational mechanisms can lead to tumorigenesis and metastasis and numerous studies have linked inappropriate expression of this ligand-receptor pair to most types of human solid tumors. To prepare mAbs to human HGF/SF, mice were immunized with native and denatured preparations of the ligand. Recloned mAbs were tested in vitro for blocking activity against scattering and branching morphogenesis. Our results show that no single mAb was capable of neutralizing the in vitro activity of HGF/SF, and that the ligand possesses a minimum of three epitopes that must be blocked to prevent Met tyrosine kinase activation. In vivo, the neutralizing mAb combination inhibited s.c. growth in athymic nu/nu mice of tumors dependent on an autocrine Met-HGF/SF loop. Importantly, growth of human glioblastoma multiforme xenografts expressing Met and HGF/SF were markedly reduced in the presence of HGF/SF-neutralizing mAbs. These results suggest interrupting autocrine and/or paracrine Met-HGF/SF signaling in tumors dependent on this pathway is a possible intervention strategy.

Predictors of vaccination rates during a mass meningococcal vaccination program on a college campus.

Factors contributing to students' compliance with mass vaccination programs during meningococcal outbreaks have not been well described. A 1997 mass vaccination campaign at Michigan State University provided an opportunity to study such factors. Of 34,024 students in the target population, 17,538 (51.5%) were vaccinated in 5 days. Vaccination rates were higher for women (47.9%) than for men (43.1%) and higher for on-campus residents (65.3%) than for off-campus residents (35.6%). For each year of students' age beyond 19, the adjusted odds of vaccination were reduced by 0.82. Adjusted odds ratios for vaccination, with White students as the reference group at 1.0, were 1.33 for Asian American students, 0.97 (not significant) for Hispanic students, 0.82 for African American students, and 0.80 for Native American students. Students from the Colleges of Business, Engineering, Communication, and Natural Science had the highest vaccination rates; those from the College of Arts and Letters had the lowest rates.

A quantitative analysis of the development of the pyramidal tract in the cervical spinal cord in the rat.

A quantitative electron microscopic analysis was undertaken of the development of the pyramidal tract, at the level of the third cervical spinal segment, in rats ranging in age from the day of birth to three months old. The axon number was calculated as the product of axon density, determined in a systematic random sample of electron micrographs, and tract area. During the first postnatal week the tract contains thin unmyelinated axons and growth cones. Growth cones are abundant in neonatal rats, but can still be observed occasionally at the end of the first postnatal week, indicating a continuous addition of pyramidal tract axons during the first postnatal week. Myelination starts around P10. By the end of the first postnatal month approximately 50% of the axons have already been myelinated. Myelination proceeds during further maturation, but in the three month old rat 28% of the axons are still unmyelinated. The total number of axons increases rapidly after birth up to 153,000 at the fourth postnatal day. Subsequently, the number of axons is reduced by nearly 50% to 79,000 in the adult rat. The axon loss is most prominent during the second postnatal week, when 32,000 axons are eliminated, but continues for several weeks at a slower rate.

Immunocytochemical distribution of the protein kinase C substrate B-50 (GAP43) in developing rat pyramidal tract.

The neuron-specific phosphoprotein B-50 is a major substrate of kinase C in fetal nerve growth cones, neonatal neural and synaptosomal plasma membranes. B-50 is identical to a growth-associated protein GAP43. Similarly, increases in B-50 occur during rat brain development, neuronal differentiation and axon regeneration. To document the relation between the expression of B-50 and the outgrowth of central axons, we studied B-50 in the developing pyramidal tract in rats at postnatal days 2, 7 and 90 (P2, P7 and P90), at the third cervical spinal segment C3, using affinity-purified antibodies to B-50. At P2 and P7, when outgrowth of pyramidal tract fibers is occurring, B-50 immunoreactivity (BIR) is intense in these fibers. BIR is reduced from P2 to P7 in the ascending fiber tracts of the cuneatus and the gracilis, which develop earlier. At P90 when most of the dorsal funiculus fibers have reached their targets and many are myelinated, BIR is dramatically reduced. In agreement, a 10-fold decrease in B-50 content was measured at P90, as compared to P7. Therefore, our results indicate that B-50 is only expressed relatively abundant in axons of the funiculus posterior during outgrowth. By inference, B-50 may be a differentiating marker to detect elongating fibers.

On the development of the pyramidal tract in the rat. II. An anterograde tracer study of the outgrowth of the corticospinal fibers.

An anterograde tracer study has been made of the developing corticospinal tract (CST) in the rat using wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). Analysis of normal Rager stained material revealed that corticospinal axons reach upper cervical spinal cord levels at the day of birth (PO). Postnatal rats ranging in age from one (P1) to fourteen (P14) days received multiple WGA-HRP injections into the cortex of their left hemisphere and were allowed to survive for 24 h. The first labeled CST fibers caudally extend into the third thoracic spinal cord segment at P1; into the eighth thoracic segment at P3; into the first or second lumbar segment at P7 and into the second to third sacral segment at Pg. Thus the outgrowth of the leading 'pioneer' fibers of the CST is completed at P9 but later developing axons are continuously added even beyond P9. Quantitative analysis of the amount of label along the length of the outgrowing CST revealed a characteristic pattern of labeling varying with age. The most striking features of that pattern are: the formation of two standing peaks at the level of the cervical and lumbar enlargements respectively and the transient presence of a smaller running peak which moves caudally with the front of the outgrowing bundle. The standing peaks are ascribed to the branching of the axon terminals at both intumescences, whereas the running peak probably arises by the accumulation of tracer within the growth cones at the tips of the outgrowing CST axons. Factors such as the number of axons, the varying axon diameters, the branching collaterals, the presence of varicosities, the transport rate of the tracer, the uptake of the tracer at the injection site, which possibly may affect the amount of label present in both the entire bundle and in the individual axons are discussed. Current research is focused upon an analysis of the relation between the site of injection within the cortex and the pattern of labeling of the CST. A delay of two days was found between the arrival of the CST axons at a particular spinal cord level and their outgrowth into the adjacent spinal gray. However, combined HRP and electronmicroscopic experiments are necessary to determine the factors behind the maturation of the CST as well as the maturation of the spinal gray.

On the development of the pyramidal tract in the rat. I. The morphology of the growth zone.

An electron microscopic study has been made of the tip of the growing pyramidal tract in the rat. This part of the developing bundle, designated as the growth-zone, has been examined at the levels of the medulla oblongata and the third spinal segment at embryonic day 20 and on the day of birth, respectively. The tip of the pyramidal tract contains, apart from axons, numerous larger profiles. An analysis of serial sections revealed that these represent either growth cones or preterminal periodic varicosities. In the growth cones of the corticospinal axons three zones can be distinguished: a proximal "tubular", an intermediate "vesicular-reticular" and a distal "fine-granular" zone. As distinct from the classical descriptions the corticospinal growth cones end in a single or, less frequently, in two more or less parallel filopodia. None of the growth cones analyzed in this study showed multiple filopodia radiating from the terminal expansion as observed at the end of growing axons in tissue cultures and in developing spinal fibre tracts of nonmammalian vertebrates. As regards the varicosities, most of these structures are characterized by a light cytoplasmic density. Others, however, contain a denser cytoplasm, closely resembling that of the vesiculo-reticular part of growth cones.

On the development of the spinal cord of the clawed frog, Xenopus laevis. I. Morphogenesis and histogenesis.

The morphogenesis and histogenesis of the spinal cord of Xenopus were examined. The study encompasses the developmental period between stage 41 and stage 66 (stages according to Nieuwkoop and Faber 1967). This period can roughly be divided into three phases. From stage 50 up to stage 53 strong proliferation and rapid growth are the most striking features. This developmental phase is preceded and followed by less dynamic periods. From stage 41 up to stage 50 the rate of proliferation is relatively low. The numbers of cells in the matrix and in the mantle layer are very small. In the mantle layer two classes of early differentiated transient neurons can be distinguished: primitive giant sensory or Rohon-Beard cells and primitive motor neurons. From stage 46 onward the originally tube-shaped spinal cord swells at the thoracic level into a thoracic enlargement. After stage 50 the proliferation strongly increases until a maximum at stage 53. Concomitantly a considerable acceleration of growth takes place. The major part of the mitoses are always concentrated in the dorsal part of the matrix. From stage 51 onward the cervical and lumbar regions show much more mitoses than the thoracic part. Distinct cervical and lumbar enlargements develop and are going to mask the thoracic swelling of the cord. From stage 54 on proliferation continues on an increasingly low level. The period between stage 54 and stage 66 is characterized by differentiation of the spinal neuronal elements.

On the development of the spinal cord of the clawed frog, Xenopus laevis. II. Experimental analysis of differentiation and migration.

In order to determine the time and site of origin and the final location of various cell groups in the spinal cord, tadpoles of Xenopus laevis, ranging from stage 48 to stage 56 were treated with tritiated thymidine and sacrificed at various stages from 49 to 66 (stages according to Nieuwkoop and Faber (1967). From the poorly developed matrix at stage 48-49 not only ventral horn cells, but also neuroblasts of the intermediate zone and the dorsal horn arise. Both the matrix and the ventricle expand in a dorsal direction. From the well-developed matrix at stage 54, in which the mitotic activity is almost exclusively confined to its dorsal part, mainly cells of the dorsal horn develop. However, this later-stage matrix also gives rise to a considerable number of neuroblasts, which become located in the central parts of the intermediate zone and the ventral horn. Generally the later-born cells come to lie dorsomedially to the older ones. The neuroblasts of the lateral motor column, however, migrate through and settle ventrolaterally to their predecessors. Our observations do not support the basal plate-alar plate concept of His (1893).