Computerized image analysis as a tool to quantify infiltrating leukocytes: a comparison between high- and low-magnification images.

The purpose of the present study was to establish a rapid and reproducible method for quantification of tissue-infiltrating leukocytes using computerized image analysis. To achieve this, the staining procedure, the image acquisition, and the image analysis method were optimized. Because of the adaptive features of the human eye, computerized image analysis is more sensitive to variations in staining compared with manual image analysis. To minimize variations in staining, an automated immunostainer was used. With a digital scanner camera, low-magnification images could be sampled at high resolution, thus making it possible to analyze larger tissue sections. Image analysis was performed by color thresholding of the digital images based on values of hue, saturation, and intensity color mode, which we consider superior to the red, green, and blue color mode for analysis of most histological stains. To evaluate the method, we compared computerized analysis of images with a x100 or a x12.5 magnification to assess leukocytes infiltrating rat brain tumors after peripheral immunizations with tumor cells genetically modified to express rat interferon-gamma (IFN-gamma) or medium controls. The results generated by both methods correlated well and did not show any significant differences. The method allows efficient and reproducible processing of large tissue sections that is less time-consuming than conventional methods and can be performed with standard equipment and software.(J Histochem Cytochem 49:1073-1079, 2001)     

Nitric oxide synthase inhibitor and IL-18 enhance the anti-tumor immune response of rats carrying an intrahepatic colon carcinoma

The role of nitric oxide (NO) produced by adherent spleen cells in the systemic immunosuppression developing in tumor-bearing hosts was investigated. After therapeutic immunization of rats carrying an intrahepatic colon carcinoma, H1D2, the spleen cell antitumor immune responsiveness was analyzed. Compared to parallel immunized tumor-free rats, tumor-bearing rats (TB rats) had a greatly reduced proliferative T-cell response to wild-type tumor stimulator cells. The TB rats had a depressed proliferative response to anti-CD3 and to the superantigen SEA. TB rats with small tumors had a stronger response to IL-18-producing H1D2 stimulator cells than to wild type H1D2 cells. This was not the case with TB rats carrying larger tumors. Also the IFN-gamma production and cytotoxicity against the wild-type tumor cells and the NK sensitive YAC cells were depressed in spleen cells of TB rats after 5-day restimulation with wild-type tumor cells. A part of this immunosuppression was mediated by adherent spleen cells, mostly consisting of macrophages. An important mode of action appears to involve their production of an enhanced level of nitric oxide, since the competitive nitric oxide synthase (NOS) inhibitor L-NAME could partially counteract the suppression in vitro. We conclude that NOS inhibitors in combination with immunostimulatory cytokines, such as IL-18, could be useful tools to enhance anti-tumor immune responses in TB rats and therefore to increase the efficiency of immunotherapies.     

Glial progenitor-like phenotype in low-grade glioma and enhanced CD133-expression and neuronal lineage differentiation potential in high-grade glioma

Background

While neurosphere- as well as xenograft tumor-initiating cells have been identified in gliomas, the resemblance between glioma cells and neural stem/progenitor cells as well as the prognostic value of stem/progenitor cell marker expression in glioma are poorly clarified.

Methodology/Principal Findings

Viable glioma cells were characterized for surface marker expression along the glial genesis hierarchy. Six low-grade and 17 high-grade glioma specimens were flow-cytometrically analyzed for markers characteristics of stem cells (CD133); glial progenitors (PDGFRα, A2B5, O4, and CD44); and late oligodendrocyte progenitors (O1). In parallel, the expression of glial fibrillary acidic protein (GFAP), synaptophysin and neuron-specific enolase (NSE) was immunohistochemically analyzed in fixed tissue specimens. Irrespective of the grade and morphological diagnosis of gliomas, glioma cells concomitantly expressed PDGFRα, A2B5, O4, CD44 and GFAP. In contrast, O1 was weakly expressed in all low-grade and the majority of high-grade glioma specimens analyzed. Co-expression of neuronal markers was observed in all high-grade, but not low-grade, glioma specimens analyzed. The rare CD133 expressing cells in low-grade glioma specimens typically co-expressed vessel endothelial marker CD31. In contrast, distinct CD133 expression profiles in up to 90% of CD45-negative glioma cells were observed in 12 of the 17 high-grade glioma specimens and the majority of these CD133 expressing cells were CD31 negative. The CD133 expression correlates inversely with length of patient survival. Surprisingly, cytogenetic analysis showed that gliomas contained normal and abnormal cell karyotypes with hitherto indistinguishable phenotype.

Conclusions/Significance

This study constitutes an important step towards clarification of lineage commitment and differentiation blockage of glioma cells. Our data suggest that glioma cells may resemble expansion of glial lineage progenitor cells with compromised differentiation capacity downstream of A2B5 and O4 expression. The concurrent expression of neuronal markers demonstrates that high-grade glioma cells are endowed with multi-lineage differentiation potential in vivo. Importantly, enhanced CD133 expression marks a poor prognosis in gliomas.     

Conservation of highly repetitive DNA in cetaceans

It is controversial whether odontocetes (toothed whales) and mysticetes (whalebone whales) have a common ancestry. Cetacean karyological uniformity, which is unique among mammalian orders, suggests a monophyletic origin; however, several anatomical authorities have maintained that odontocetes and mysticetes are diphyletic. We investigated the issue using Southern blot hybridization. Two labelled restriction fragment probes from the DNA of the sei whale (a mysticete) were hybridized to restricted DNA of cetacean species representing all extant families except the Eschrichtiidae, the gray whales. The probes hybridized to specific restriction fragments in all odontocete and mysticete materials. Hybridizations showed preservation of hybridization homologies and a striking conservation of the length of highly repeated DNA sequences. The results are compatible with a common ancestry of odontocetes and mysticetes.     

Effects of transforming growth factor β1 expression in a rat colon carcinoma: growth inhibition, leukocyte infiltration and production of interleukin-10 and tumor necrosis factor α

The cytokine transforming growth factor β-1 (TGFβ1), was transfected into a TGFβ1-negative rat colon carcinoma. The growth of isografts of TGFβ1-expressing tumors was compared to that of vector control transfectants. The TGFβ1 transfectant grew significantly more slowly after intrahepatic isografting than did vector control and wild-type tumors. The TGFβ1-transfected tumor tissue had significantly greater infiltration of both CD4⁺ and CD8⁺ T lymphocytes than did the vector control tumor. The tumor-infiltrating leukocytes (TIL) from TGFβ1-transfected tumor secreted significantly more of the cytokines interleukin-10 (IL-10) and tumor necrosis factor α (TNFα) than did TIL from the vector control tumor. The TGFβ1 transfectant also demonstrated a significantly slower outgrowth in immunodeficient SCID mice, supporting a non-T-lymphocyte-dependent mechanism for the tumor retardation. In SCID mice, the TGFβ1-transfected tumor demonstrated significantly greater infiltration of both granulocytes and macrophages than did the vector control transfectant. We also demonstrated a direct inhibitory effect of rat TNFα on tumor proliferation in vitro. These results suggest that TGFβ1 induces a local secretion of immunomodulating cytokines and that this may influence monocytes, lymphocytes and granulocytes to retard tumor outgrowth. Received: 7 July 1999 / Accepted: 12 August 1999     

Influence of chronic and acute spinal cord injury on skeletal muscle Na+-K+-ATPase and phospholemman expression in humans

Na(+)-K(+)-ATPase is an integral membrane protein crucial for the maintenance of ion homeostasis and skeletal muscle contractibility. Skeletal muscle Na(+)-K(+)-ATPase content displays remarkable plasticity in response to long-term increase in physiological demand, such as exercise training. However, the adaptations in Na(+)-K(+)-ATPase function in response to a suddenly decreased and/or habitually low level of physical activity, especially after a spinal cord injury (SCI), are incompletely known. We tested the hypothesis that skeletal muscle content of Na(+)-K(+)-ATPase and the associated regulatory proteins from the FXYD family is altered in SCI patients in a manner dependent on the severity of the spinal cord lesion and postinjury level of physical activity. Three different groups were studied: 1) six subjects with chronic complete cervical SCI, 2) seven subjects with acute, complete cervical SCI, and 3) six subjects with acute, incomplete cervical SCI. The individuals in groups 2 and 3 were studied at months 1, 3, and 12 postinjury, whereas individuals with chronic SCI were compared with an able-bodied control group. Chronic complete SCI was associated with a marked decrease in [(3)H]ouabain binding site concentration in skeletal muscle as well as reduced protein content of the α(1)-, α(2)-, and β(1)-subunit of the Na(+)-K(+)-ATPase. In line with this finding, expression of the Na(+)-K(+)-ATPase α(1)- and α(2)-subunits progressively decreased during the first year after complete but not after incomplete SCI. The expression of the regulatory protein phospholemman (PLM or FXYD1) was attenuated after complete, but not incomplete, cervical SCI. In contrast, FXYD5 was substantially upregulated in patients with complete SCI. In conclusion, the severity of the spinal cord lesion and the level of postinjury physical activity in patients with SCI are important factors controlling the expression of Na(+)-K(+)-ATPase and its regulatory proteins PLM and FXYD5.     

Effects of exercise on mitogen- and stress-activated kinase signal transduction in human skeletal muscle

Exercise/contraction is a powerful stimulator of mitogen-activated protein (MAP) kinase cascades in skeletal muscle. Little is known regarding the physiological activation of enzymes downstream of MAP kinase. We investigated whether acute exercise results in activation of mitogen- and stress-activated kinases (MSK) 1 and 2, p90 ribosomal S6 kinase (p90rsk), and MAP kinase-activated protein kinase 2 (MAPKAPK2). Muscle biopsies were obtained from healthy volunteers before, during, and after 60 min one-leg cycle ergometry, from exercising and resting legs. MSK1 and MSK2 activities were increased 400-500% and 200-300%, respectively, in exercised muscle (P < 0.05 vs. rest). A dramatic increase in activity of p90rsk (MAPKAPK1) (>2,500%), and to a lesser extent MAPKAP2 (300%), was noted with exercise (P < 0.05 vs. rest). MSK1, MSK2, p90rsk, and MAPKAP2 activities were sustained throughout exercise. Exercise-induced activation of these enzymes was limited to working muscle, indicating that local rather than systemic factors activate these signaling cascades. Thus physical exercise leads to activation of multiple enzymes downstream of MAP kinase.     

Mysticete (baleen whale) relationships based upon the sequence of the common cetacean DNA satellite.

The genomes of all extant cetaceans are characterized by the presence of the so-called common cetacean DNA satellite. In the mysticetes (whalebone whales) the repeat length of the satellite is 1,760 bp. In the odontocetes (toothed whales), other than the family Delphinidae, the repeat length is usually approximately 1,740 bp. The Delphinidae are characterized by a repeat length of approximately 1,580 bp. It has been shown in odontocetes that the satellite evolves in concert and that differences between species, with respect to the sequence of the satellite, correspond reasonably well to their evolutionary distances. In the present study the sequence of the satellite was determined in three repeats in each of seven mysticete species, and a consensus for each species established. Parsimony and neighbor-joining analyses based upon sequences of all repeats showed that the primary evolutionary distinction among the mysticetes is between the Balaenidae sensu stricto (i.e., the bowhead whale and the right whale) and all remaining species, including the pygmy right whale, a species that usually has been included in the Balaenidae. The comparisons also showed that the humpback whale and the gray whale were approximately equidistant from the blue whale and the fin whale (genus Balaenoptera). Concerted evolution of the satellite was also demonstrated among the mysticetes, but it appeared to evolve more slowly in the mysticetes than in the odontocetes.     

Multivariate intralocus sexual conflict in seed beetles

Intralocus sexual conflict (IaSC) is pervasive because males and females experience differences in selection but share much of the same genome. Traits with integrated genetic architecture should be reservoirs of sexually antagonistic genetic variation for fitness, but explorations of multivariate IaSC are scarce. Previously, we showed that upward artificial selection on male life span decreased male fitness but increased female fitness compared with downward selection in the seed beetle Callosobruchus maculatus. Here, we use these selection lines to investigate sex‐specific evolution of four functionally integrated traits (metabolic rate, locomotor activity, body mass, and life span) that collectively define a sexually dimorphic life‐history syndrome in many species. Male‐limited selection for short life span led to correlated evolution in females toward a more male‐like multivariate phenotype. Conversely, males selected for long life span became more female‐like, implying that IaSC results from genetic integration of this suite of traits. However, while life span, metabolism, and body mass showed correlated evolution in the sexes, activity did not evolve in males but, surprisingly, did so in females. This led to sexual monomorphism in locomotor activity in short‐life lines associated with detrimental effects in females. Our results thus support the general tenet that widespread pleiotropy generates IaSC despite sex‐specific genetic architecture.     

DNA cloning and hybridization in deer species supporting the chromosome field theory

The Cervidae show the largest variation in chromosome number found within any mammalian family. The eight species of deer which are the subject of this study vary in chromosome number from 2n = 70 to 2n = 6. Three species of Bovidae are also included since they belong to a closely related family. Digestion of nuclear DNAs with the restriction endonucleases Hae III, Hpa II, Msp I, Eco RI, Xba I, Pst I and Bam HI reveals that there is a series of highly repetitive sequences forming similar band patterns in the different species. There are two bands (1100 and 550 base pairs) which are common to all species although the two families separated more than 40 million years ago. To obtain information on the degree of homology among these conserved sequences we isolated a Bam HI restriction fragment of approximately 770 base pairs from red deer DNA. This sequence was 32P labeled and hybridized by the Southern blot technique with DNAs cleaved with Bam HI, Eco RI, Hpa II and Msp I. Moreover, the same sequence was cloned in the plasmid vector pBR322 nick translated with 32P and hybridized with the DNAs of 8 species of Cervidae and 3 of Bovidae. The same cloned probe was labeled with 3H and hybridized in situ with the metaphase chromosomes of red deer (2n = 68) and Muntiacus muntjak (2n = 7 male). Homologies are still present between the highly repetitive sequences of the 8 species of Cervidae despite the drastic reorganization that led to extreme chromosome numbers. Moreover, the cloned DNA sequence was found to occupy the same position, in the proximal regions of the arms, in both red deer (2n = 68) and M. muntjak (2n = 7 male) chromosomes. The ribosomal RNA genes and the centromeres in these species have also maintained their main territory despite the drastic chromosome reorganization. These results are experimental confirmation of the chromosome field theory which predicted that each DNA sequence has an optimal territory within the centromere-telomere field and tends to occupy this same territory following chromosome reorganization.