Spontaneous calcification in F344/Slc and F344/JCL rats

F344/Slc and F344/JCL rats 2 years of age were histologically examined for the incidence and distribution of calcification. In the male rats of both strains, calcification was observed in the testis, lung, brain, kidney, heart, aorta, cornea, prostate and seminal vesicle respectively. In the female F344/JCL rats, calcification appeared in the kidney, lung, cornea, brain, stomach, ovary and heart. Among these of both sexes, the lung was one of the most affected organs for calcification. On the other hand, calcification in the kidney was more severe and frequent in the females than in the males, suggesting that the sex may be one of enhancing factors for calcification.     

Dose-dependent recruitment of CD25+ and CD26+ T cells in a novel F344 rat model of asthma

The ovalbumin (OVA)-induced airway inflammation in rats is a commonly used model to explore the pathobiology of asthma. However, its susceptibility varies greatly between rat strains, and presently Brown Norway (BN) rats are preferentially used. Since recruitment of T cells to the lungs depends on the CD26 (dipeptidyl peptidase IV, DPPIV) expression, Fischer 344 strain (F344) rats are a highly relevant rat strain, in particular because CD26-deficient substrains are available. To establish a F344 rat model of asthma, we challenged F344 rats using different doses of aerosolized antigen (0%, 1%, 2.5%, 5%, and 7.5% OVA) and compared these effects with intratracheal instillation of OVA (1.5 mg/0.3 ml). Asthmoid responsiveness was determined by analysis of early airway responsiveness (EAR), antigen-specific IgE levels, as well as airway inflammation including the composition of T cell subpopulations in the bronchoalveolar lavage (BAL) and lung tissue with special respect to the T cell activation markers CD25 and CD26. Even low allergen doses caused allergen-specific EAR and increases of antigen-specific IgE levels. However, EAR and IgE levels did not increase dose dependently. Higher concentrations of OVA led to a dose-dependent increase of several immunological markers of allergic asthma including an influx of eosinophils, T cells, and dendritic cells. Interestingly, a dose-dependent increase of CD4(+)/CD25(+)/CD26(+) T cells was found in the lungs. Summarizing, we established a novel F344 rat model of aerosolized OVA-induced asthma. Thereby, we found a dose-dependent recruitment of cellular markers of allergic asthma including the activated CD4(+)/CD25(+)/CD26(+) T cell subpopulation, which has not been described in asthma yet.     

CD4 expression on activated NK cells: ligation of CD4 induces cytokine expression and cell migration

NK cells play an important role in the innate immune response. We have isolated NK cells from human lymphoid tissues and found that these cells express the CD4 molecule on their surface at levels higher than those found on peripheral blood NK cells. To study the functional role of the CD4 molecule on NK cells, we developed an in vitro system by which we are able to obtain robust CD4 expression on NK cells derived from blood. CD4+ NK cells efficiently mediate NK cell cytotoxicity, and CD4 expression does not appear to alter lytic function. CD4+ NK cells are more likely to produce the cytokines gamma-IFN and TNF-alpha than are CD4- NK cells. Ligation of CD4 further increases the number of NK cells producing these cytokines. NK cells expressing CD4 are also capable of migrating toward the CD4-specific chemotactic factor IL-16, providing another function for the CD4 molecule on NK cells. Thus, the CD4 molecule is present and functional on NK cells and plays a role in innate immune responses as a chemotactic receptor and by increasing cytokine production, in addition to its well-described function on T cells as a coreceptor for Ag responsive cell activation.     

Melanoma cell expression of CD200 inhibits tumor formation and lung metastasis via inhibition of myeloid cell functions

CD200 is a cell surface glycoprotein that functions through engaging CD200 receptor on cells of the myeloid lineage and inhibits their functions. Expression of CD200 has been implicated in a variety of human cancer cells including melanoma cells and has been thought to play a protumor role. To investigate the role of cancer cell expression of CD200 in tumor formation and metastasis, we generated CD200-positive and CD200-negative B16 melanoma cells. Subcutaneous injection of CD200-positive B16 melanoma cells inhibited tumor formation and growth in C57BL/6 mice but not in Rag1^−/−C57BL/6 mice. However, i.v. injection of CD200-positive B16 melanoma cells dramatically inhibited tumor foci formation in the lungs of both C57BL/6 and Rag1^−/−C57BL6 mice. Flow cytometry analysis revealed higher expression of CD200R in Gr1⁺ myeloid cells in the lung than in peripheral myeloid cells. Depletion of Gr1⁺ cells or stimulation of CD200R with an agonistic antibody in vivo dramatically inhibited tumor foci formation in the lungs. In addition, treatment with tumor antigen specific CD4 or CD8 T cells or their combination yielded a survival advantage for CD200 positive tumor bearing mice over mice bearing CD200-negative tumors. Taken together, we have revealed a novel role for CD200-CD200R interaction in inhibiting tumor formation and metastasis. Targeting CD200R may represent a novel approach for cancer immunotherapy.     

Mutation and expression of the p53 gene during chemical hepatocarcinogenesis in F344 rats

Inactivation of the p53 gene is one of the most frequent genetic alterations in carcinogenesis. We studied gene mutations, the mRNA expression of p53, and the accumulation of p53 protein in chemical hepatocarcinogenesis in rats. Samples consisting of 44 precancerous foci and 18 cancerous foci were collected by laser capture microdissection (LCM), and analyzed for mutations in rat p53 gene exons 5-8 by PCR-single-strand conformational polymorphism (PCR-SSCP). We found that 25 PCR-SSCP bands of exons 6/7 and 8 were altered in 22/62 (35.4%) LCM samples. Direct p53 gene sequencing showed that 20/62 (9 precancer, 11 cancer) (32.3%) LCM samples exhibited 34 point mutations. Ten LCM samples exhibited double or triple mutations in exons 6/7 and 8 simultaneously. A quantitative analysis of p53 mRNA showed that p53 mRNA peaked at an early stage (week 6) in the precancerous lesion, 20 times that of adjacent normal tissue, and returned to normal by week 23. Similar to precancer, p53 mRNA in cancer was five times as high as that of adjacent normal tissue at week 12, and was closer to normal at week 23. When p53 mRNA declined from a high to low, positive immunostaining for the p53 protein began to be seen in precancerous and cancerous foci, suggesting that the p53 protein had accumulated in these foci. Results show that p53 gene mutation is present in initial chemical hepatocarcinogenesis and p53 mRNA concentration is clearly elevated before gene mutation. Once the p53 gene has mutated, mRNA concentration progressively declines, suggesting that mutation leads to inactivation of the p53 gene.     

Sex differences in kidney gene expression during the life cycle of F344 rats

Background

The kidney functions in key physiological processes to filter blood and regulate blood pressure via key molecular transporters and ion channels. Sex-specific differences have been observed in renal disease incidence and progression, as well as acute kidney injury in response to certain drugs. Although advances have been made in characterizing the molecular components involved in various kidney functions, the molecular mechanisms responsible for sex differences are not well understood. We hypothesized that the basal expression levels of genes involved in various kidney functions throughout the life cycle will influence sex-specific susceptibilities to adverse renal events.

Methods

Whole genome microarray gene expression analysis was performed on kidney samples collected from untreated male and female Fischer 344 (F344) rats at eight age groups between 2 and 104 weeks of age.

Results

A combined filtering approach using statistical (ANOVA or pairwise t test, FDR 0.05) and fold-change criteria (>1.5 relative fold change) was used to identify 7,447 unique differentially expressed genes (DEGs). Principal component analysis (PCA) of the 7,447 DEGs revealed sex-related differences in mRNA expression at early (2 weeks), middle (8, 15, and 21 weeks), and late (104 weeks) ages in the rat life cycle. Functional analysis (Ingenuity Pathway Analysis) of these sex-different genes indicated over-representation of specific pathways and networks including renal tubule injury, drug metabolism, and immune cell and inflammatory responses. The mRNAs that code for the qualified urinary protein kidney biomarkers KIM-1, Clu, Tff3, and Lcn2 were also observed to show sex differences.

Conclusions

These data represent one of the most comprehensive in-life time course studies to be published, assessing sex differences in global gene expression in the F344 rat kidney. PCA and Venn analyses reveal specific periods of sexually dimorphic gene expression which are associated with functional categories (xenobiotic metabolism and immune cell and inflammatory responses) of key relevance to acute kidney injury and chronic kidney disease, which may underlie sex-specific susceptibility. Analysis of the basal gene expression patterns of renal genes throughout the life cycle of the rat will improve the use of current and future renal biomarkers and inform our assessments of kidney injury and disease.

     

NK cell lymphoma, nasal type, with massive lung involvement: a case report

Extranodal NK/T cell lymphoma, nasal type, is an Epstein–Barr virus-associated lymphoma that most commonly involves the nasal cavity and upper respiratory tract. Lung involvement by NK/T cell lymphoma is rare and seldom reported in the literature. We describe the unusual case of a 41-year-old male with NK cell lymphoma, nasal type, who presented with massive secondary lung involvement 2.5 years after the detection of a retroperitoneal mass. The diagnosis was made by open lung biopsy. Despite aggressive treatment, the patient died shortly after the initiation of therapy. Lung involvement by NK/T cell lymphoma occurs most commonly as part of widely disseminated disease and carries a poor prognosis for the patient. Novel agents and innovative therapies need to be developed for this aggressive lymphoma.     

Age and sex differences in kidney microRNA expression during the life span of F344 rats

Background

Growing evidence suggests that epigenetic mechanisms of gene regulation may play a role in susceptibilities to specific toxicities and adverse drug reactions. MiRNAs in particular have been shown to be important regulators in cancer and other diseases and show promise as predictive biomarkers for diagnosis and prognosis. In this study, we characterized the global kidney miRNA expression profile in untreated male and female F344 rats throughout the life span. These findings were correlated with sex-specific susceptibilities to adverse renal events, such as male-biased renal fibrosis and inflammation in old age.

Methods

Kidney miRNA expression was examined in F344 rats at 2, 5, 6, 8, 15, 21, 78, and 104 weeks of age in both sexes using Agilent miRNA microarrays. Differential expression was determined using filtering criteria of ≥1.5 fold change and ANOVA or pairwise t-test (FDR <5%) to determine significant age and sex effects, respectively. Pathway analysis software was used to investigate the possible roles of these target genes in age- and sex-specific differences.

Results

Three hundred eleven miRNAs were found to be expressed in at least one age and sex. Filtering criteria revealed 174 differentially expressed miRNAs in the kidney; 173 and 34 miRNAs exhibiting age and sex effects, respectively. Principal component analysis revealed age effects predominated over sex effects, with 2-week miRNA expression being much different from other ages. No significant sexually dimorphic miRNA expression was observed from 5 to 8 weeks, while the most differential expression (13 miRNAs) was observed at 21 weeks. Potential target genes of these differentially expressed miRNAs were identified.

Conclusions

The expression of 56% of detected renal miRNAs was found to vary significantly with age and/or sex during the life span of F344 rats. Pathway analysis suggested that 2-week-expressed miRNAs may be related to organ and cellular development and proliferation pathways. Male-biased miRNA expression at older ages correlated with male-biased renal fibrosis and mononuclear cell infiltration. These miRNAs showed high representation in renal inflammation and nephritis pathways, and included miR-214, miR-130b, miR-150, miR-223, miR-142-5p, miR-185, and miR-296*. Analysis of kidney miRNA expression throughout the rat life span will improve the use of current and future renal biomarkers and inform our assessments of kidney injury and disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s13293-014-0019-1) contains supplementary material, which is available to authorized users.     

MTA1-TJP1 interaction and its involvement in non-small cell lung cancer metastasis

Distant metastasis is the main cause of death in non-small cell lung cancer (NSCLC) patients. The mechanism of metastasis-associated protein 1(MTA1) in NSCLC has not been fully elucidated. This study aimed to reveal the mechanism of MTA1 in the invasion and metastasis of NSCLC.Bioinformatics analysis and our previous results showed that MTA1 was highly expressed in NSCLC tissues and correlated with tumor progression. Knockout of MTA1 by CRISPR/Cas9 significantly inhibited the migration and invasion of H1299 cells, but enhanced cell adhesion. Stable overexpression of MTA1 by lentivirus transfection had opposite effects on migration, invasion and adhesion of A549 cells. The results of in vivo experiments in nude mouse lung metastases model confirmed the promotion of MTA1 on invasion and migration. Tight junction protein 1 (TJP1) was identified by immunoprecipitation and mass spectrometry as an interacting protein of MTA1 involved in cell adhesion. MTA1 inhibited the expression level of TJP1 protein and weakened the tight junctions between cells. More importantly, the rescue assays confirmed that the regulation of MTA1 on cell adhesion, migration and invasion was partially attenuated by TJP1.In Conclusion, MTA1 inhibits the expression level of TJP1 protein co-localized in the cytoplasm and membrane of NSCLC cells, weakens the tight junctions between cells, and changes the adhesion, migration and invasion capabilities of cells, which may be the mechanism of MTA1 promoting the invasion and metastasis of NSCLC. Thus, targeting the MTA1-TJP1 axis may be a promising strategy for inhibiting NSCLC metastasis.     

Comparison of characteristics between F344 and Slc:Wistar rats--Slc:Wistar rats cannot be distinguished from the F344 strain

With respect to F344/DuCrj and Slc: Wistar rats, both widely used in Japan, it was found that there is a close similarity in the changes of body weights and survival rates, and in the organ distribution and incidence of spontaneous tumors. To examine the degree of homozygosity between F344 and Slc: Wistar strains, tumor transplantation and skin grafting were performed. The bladder carcinomas that originated from F344/DuCrj rats grew subcutaneously in the other F344 strains and Slc: Wistar rats, but did not grow in the other Wistar-derived strains. The skin grafts between F344/DuCrj or F344/NSlc and Slc: Wistar rats were accepted, but those between F344/DuCrj or Slc: Wistar and the other Wistar-derived strains were rejected. These results suggest that Slc: Wistar rats cannot be distinguished genetically from the F344 strain of rats.     

CD10 expression in non-small cell lung cancer.

CD10 is a cell surface endopeptidase that inactivates various potentially growth stimulatory peptides. In lung cancer cell lines this downregulation has been associated with increased proliferation. Downregulation of CD10 in lung cancer tissue is described, suggesting a potential role in carcinogenesis and a possible use of CD10 as a prognostic marker. We aimed to determine the rate of CD10 expression in our non-small cell lung cancer (NSCLC) collection and to clarify its correlation with clinicopathological parameters and patient survival. 114 NSCLC were analysed immunohistochemically using a monoclonal CD10 antibody (clone NCL-CD10-270) on an NSCLC tissue micro array. The staining was semiquantitatively scored. CD10 expression was observed in 19% of cases, without any significant association with tumour type, -size, -grading, nodal status, clinical stage, and patient survival time. We conclude that a diagnostic use of CD10 immunostaining in NSCLC is unlikely.     

人可溶性CD14基因克隆表达及功能研究

利用反转录PCR技术,从U937细胞总RNA中,扩增编码人可溶性CD14的基因序列,构建了重组表达质粒pEF1/HisC/sCD14^348aa;用脂质体转染法,实现了在真核细胞中的高效表达;用免疫亲和层析纯化表达产物,纯度达90％以上;PLS刺激U937细胞产生CD14的变化,证明了表达产物具有结合LPS的功能。     

Molecular cloning of rat NK target structure--the possibility of CD44 involvement in NK cell-mediated lysis

The nature of target molecules of natural killer (NK) cell-mediated lysis remains to be elucidated. As we previously reported, mAb 109 recognizes one of the tumor-associated antigens, designated as 109 antigen (Ag), expressed on the cell surface of rat fibrosarcomas W31 and W14, which are transformants of WFB (rat fetal fibroblast cell line) with H-ras oncogene. 109Ag was thought to be a target structure of NK cells since mAb 109 inhibited NK cell-mediated lysis against W31 and W14. Here, we demonstrate by molecular cloning that 109Ag is identical to rat CD44. Immunoprecipitation and immunoblotting studies also showed that mAb 109 and anti-rat CD44 mAb OX-50 recognize the same protein of W31 cell lysates with an 86 kDa molecular size. CD44 was suggested to be a target structure of NK cell-mediated lysis; however, rat CD44 cDNA transfection alone into CD44 null cell lines did not result in up-regulation of target cell susceptibility to NK cell-mediated lysis. Our results therefore indicated that CD44 may play a crucial role as one of the target structures in our rat fibrosarcoma system though the cell surface expression of CD44 alone does not affect NK susceptibility of the target cells.     

B cell induction of IL-13 expression in NK cells: role of CD244 and SLAM-associated protein

NK cells are an important component of the innate immune system that can also interact with B cells in a mutually productive manner. We have previously shown that activated B cells can induce NK cells to up-regulate their secretion of IFN-gamma. In this study, we show that B cells, and, particularly, marginal zone B cells, can, in addition, induce NK cells via direct cell-cell interactions to express mRNA encoding the Th2 cytokine IL-13. The induction of NK cell IL-13 mRNA expression requires the ligation of the CD244 receptor by the CD48 ligand on B cells via signaling pathways that depend upon expression of the X-linked lymphoproliferative disease gene product, SH2D1A/DSHP/SAP (SLAM-associated protein, or SAP) in NK cells. Thus, the positive signals attributed to the B cell activation of CD244 on murine NK cells appears to be more similar to the activity of CD244 on human cells. The induction of IL-13 mRNA by B cells may account for the effect of NK cells on the generation of Th2-type responses in the presence of some adjuvants.     

Deficiencies in gut NK cell number and function precede diabetes onset in BB rats

Defects in the intestinal immune system may contribute to the pathogenesis of autoimmune diseases. Intraepithelial lymphocytes represent a substantial fraction of gut-associated lymphocytes, but their function in mucosal immunity is unclear. A newly described population of NK cells that spontaneously secrete IL-4 and IFN-gamma is present in the intraepithelial lymphocyte compartment of the rat. We hypothesized that defects in the number or function of these cells would be present in rats susceptible to autoimmunity. We report that the number of NKR-P1A(+)CD3(-) intraepithelial NK (IENK) cells is deficient before onset of spontaneous autoimmune diabetes in diabetes-prone BB (BBDP) rats. The absolute number of recoverable IENK cells was only approximately 8% of that observed in WF rats. Bone marrow transplantation from histocompatible WF donors reversed the IENK cell deficiency (and prevented diabetes) in these animals, suggesting a hemopoietic origin for their IENK cell defect. Analysis of diabetes-resistant BB rats, which develop autoimmune diabetes only after perturbation of the immune system, revealed IENK cell numbers intermediate between that of BBDP and WF rats. IENK cells were selectively depleted during treatment to induce diabetes. Prediabetic BBDP and diabetes-resistant BB animals also exhibited defective IENK cell function, including decreased NK cell cytotoxicity and reduced secretion of IL-4 and IFN-gamma. IENK functional defects were also observed in LEW and BN rats, which are susceptible to induced autoimmunity, but not in WF, DA, or F344 rats, which are resistant. Defects in IENK cell number and function may contribute to the pathogenesis of autoimmune diseases including type 1 diabetes.     

Early high expression of IP-10 in F344 rats resistant to Sendai virus-induced airway injury

Weanling F344 and BN rats differ markedly in their susceptibility to Sendai virus-induced airway injury. Early gene expression that controls their differences in susceptibility remains poorly understood. In this study we combined suppressive subtractive hybridization and cDNA library array hybridization to identify genes differentially expressed in virus-susceptible BN and virus-resistant F344 rats during the first 3 days after inoculation. Differential expression of selected clones was further verified by quantitative RT-PCR. Seven virus-induced gene segments were identified. Of them, interferon-gamma-inducible protein 10 (IP-10), Mx1, and guanylate-binding protein-2 mRNA abundance in infected F344 rats was 201.5, 188.2, and 281.7% higher, respectively, than that of infected BN rats at 2 days after inoculation. In situ hybridization indicated that virus-induced IP-10 was expressed mainly in airway epithelial cells of F344 rats. Sendai virus infection can directly induce IP-10 expression in rat tracheal epithelial cells in vitro. IP-10 early high expression might contribute to the resistance to virus-induced airway disease in F344 rats by promoting Th1 responses and increasing antiviral activity.     

Differences of mu-opioid receptors between Lewis and F344 rats

F344 and Lewis rats show different responses to opioids in several experimental paradigms. In this study we have used the specific mu-opioid agonist DAMGO to find out if these differences could be attributed to heterogeneity of mu-opioid receptors. The density of [H3]DAMGO binding sites was similar in the brain cortex and spinal cord of both strains, but DAMGO affinity for mu-opioid receptors was higher in F344 tissues. Moreover, a parallel study of the effects of DAMGO on electrically-evoked twitches of isolated vasa deferentia revealed that this drug was also more effective in F344 preparations. These results suggest that mu-opioid receptors of F344 rats are more sensitive to pharmacological stimulation in vitro, which could be related to a higher drug affinity.     

Cloning and functional expression of the T cell activation antigen CD26.

A cDNA encoding the T cell activation Ag CD26 was isolated from human PHA-activated T cells by using an expression cloning method. The nucleotide sequence obtained predicts a protein of 766 amino acids of type II membrane topology, with six amino acids in the cytoplasmic region. The predicted amino acid sequence of the Ag was 85% homologous to that of the dipeptidyl peptidase IV enzyme isolated from rat liver. Derivatives of the human leukemic T cell line Jurkat transfected with a CD26 expression plasmid were established. Characterization of the CD26 Ag expressed by the transfected Jurkat cells revealed that the Ag could be immunoprecipitated as a 110-kDa molecule similar to that found on peripheral blood T cells and that the Ag had dipeptidyl peptidase IV activity. Functional analysis of these Jurkat transfectants showed that cross-linking of the CD26 and CD3 Ag with their respective antibodies resulted in enhanced intracellular calcium mobilization and IL-2 production. These results provide direct evidence that the CD26 Ag plays a role in T cell activation.     

Age-related changes in the haematology of female F344 rats

As little comprehensive baseline data are available on age-related haematological changes in genetically-defined rat strains, the haematology of female F344 rats is described in animals sampled at 2, 4, 8, 20, 66 and 121 weeks of age. Values for Hb, RBC and PCV increased from 2 weeks of age to reach adult levels at 8 weeks, whereas MCV, MCH and reticulocyte counts were high initially but decreased to reach the adult range at 8 weeks. Between 66 and 121 weeks, reticulocyte counts were significantly increased and values for MCHC significantly decreased. Lymphocytes were the predominant white cell type in each age group. The absolute numbers of neutrophils and lymphocytes showed slight variations between 2 and 66 weeks and both cell types increased significantly between 66 and 121 weeks. Platelet counts showed no overall age-related trends. Fibrinogen values increased from 2 weeks of age to reach the adult level at 8 weeks. One animal of the 14 sampled at 121 weeks showed changes in the blood, liver and spleen consistent with a diagnosis of lymphoid leukaemia.