Tumour-associated macrophages correlate with poor prognosis in myxoid liposarcoma and promote cell motility and invasion via the HB-EGF-EGFR-PI3K/Akt pathways.

BACKGROUND: Myxoid liposarcoma (MLS) is the second most common subtype of liposarcoma, and metastasis occurs in up to one-third of cases. However, the mechanisms of invasion and metastasis remain unclear. Tumour-associated macrophages (TAMs) have important roles in tumour invasion, metastasis, and/or poor prognosis. The aim of this study was to investigate the relationship between TAMs and MLS. METHODS: Using 78 primary MLS samples, the association between clinical prognosis and macrophage infiltration was evaluated by immunochemistry. The effects of macrophages on cell growth, cell motility, and invasion of MLS cell lines were investigated in vitro. In addition, clinicopathological factors were analysed to assess their prognostic implications in MLS. RESULTS: Higher levels of CD68-positive macrophages were associated with poorer overall survival in MLS samples. Macrophage-conditioned medium enhanced MLS cell motility and invasion by activating epidermal growth factor receptor (EGFR), with the key ligand suggested to be heparin-binding EGF-like growth factor (HB-EGF). The phosphoinositide 3-kinase/Akt pathway was mostly involved in HB-EGF-induced cell motility and invasion of MLS. The expression of phosphorylated EGFR in MLS clinical samples was associated with macrophage infiltration. In addition, more significant macrophage infiltration was associated with poor prognosis even in multivariate analysis. CONCLUSIONS: Macrophage infiltration in MLS predicts poor prognosis, and the relationship between TAMs and MLS may be a new candidate for therapeutic targets of MLS.

Recurrent peritoneal inclusion cysts successfully treated with oral contraceptives: a report of two cases.

OBJECTIVE: To examine whether conservative treatment with oral contraceptives is effective in the shrinkage of a peritoneal inclusion cyst (PIC). This is a case report of two patients with a PIC that developed after gynecological surgery. CASES: Both cases were suspected of a PIC based on the medical history, laboratory data, and image findings. It was difficult in differentiate a PIC from an ovarian tumor. Surgery was chosen at first. However, PICs in both cases recurred after surgery and were treated with oral contraceptives as a conservative treatment. PICs shrank after the treatment of oral contraceptives in both cases. CONCLUSION: Due to the high rate of recurrence following surgery, conservative treatment is recommended to treat PICs. Hormone therapy using oral contraceptives seems to have some therapeutic benefit for the PICs.

Inhibitory effects of (-)-epigallocatechin-3-gallate from green tea on the growth of Babesia parasites.

(-)-Epigallocatechin-3-gallate (EGCG) is the major tea catechin and accounts for 50-80% of the total catechin in green tea. (-)-Epigallocatechin-3-gallate has antioxidant, anti-inflammatory, anti-microbial, anti-cancer, and anti-trypanocidal activities. This report describes the inhibitory effect of (-)-Epigallocatechin-3-gallate on the in vitro growth of bovine Babesia parasites and the in vivo growth of the mouse-adapted rodent babesia B. microti. The in vitro growth of the Babesia species was significantly (P<0.05) inhibited in the presence of micromolar concentrations of EGCG (IC50 values=18 and 25 microM for B. bovis, and B. bigemina, respectively). The parasites showed no re-growth at 25 microM for B. bovis and B. bigemina in the subsequent viability test. The drug significantly (P<0.05) inhibited the growth of B. microti at doses of 5 and 10 mg/kg body weight, and the parasites completely cleared on day 14 and 16 post-inoculation in the 5 and 10 mg/kg treated groups, respectively. These findings highlight the potentiality of (-)-Epigallocatechin-3-gallate as a chemotherapeutic drug for the treatment of babesiosis.

GTP hydrolysis by Ran occurs at the nuclear pore complex in an early step of protein import.

Mediated import of proteins into the nucleus involves multiple cytosolic factors, including the small GTPase Ran. Whether Ran functions by interacting with other cytosolic proteins or components of the nuclear pore complex has been unclear. Furthermore, the precise transport step where Ran acts has not been determined. To address these questions, we have analyzed the binding interactions of Ran using permeabilized cells and isolated nuclear envelopes. By light and electron microscope immunolocalization, we have found that Ran accumulates specifically at the cytoplasmic surface of the nuclear pore complex when nuclear import in permeabilized cells is inhibited by nonhydrolyzable analogs of GTP. Ran associates with a peripheral pore complex region that is similar to the area where transport ligands accumulate by depletion of ATP, which arrests an early step of transport. Binding studies with isolated nuclear envelopes in the absence of added cytosol indicate that Ran-GTP directly interacts with a pore complex protein. Using blot overlay techniques, we detected a single prominent polypeptide of isolated nuclear envelopes that binds Ran-GTP. This corresponds to the 358-kD protein RanBP2, a Ran binding pore complex protein recently identified by two-hybrid screening. Thus, RanBP2 is likely to constitute the Ran-GTP-binding site detected at the cytoplasmic periphery of the pore complex. These data support a model in which initial ligand binding to the nuclear pore complex occurs at or near RanBP2, and that hydrolysis of GTP by Ran at this site serves to define commitment to the nuclear import pathway.

When overexpressed, a novel centrosomal protein, RanBPM, causes ectopic microtubule nucleation similar to gamma-tubulin.

A novel human protein with a molecular mass of 55 kD, designated RanBPM, was isolated with the two-hybrid method using Ran as a bait. Mouse and hamster RanBPM possessed a polypeptide identical to the human one. Furthermore, Saccharomyces cerevisiae was found to have a gene, YGL227w, the COOH-terminal half of which is 30% identical to RanBPM. Anti-RanBPM antibodies revealed that RanBPM was localized within the centrosome throughout the cell cycle. Overexpression of RanBPM produced multiple spots which were colocalized with gamma-tubulin and acted as ectopic microtubule nucleation sites, resulting in a reorganization of microtubule network. RanBPM cosedimented with the centrosomal fractions by sucrose- density gradient centrifugation. The formation of microtubule asters was inhibited not only by anti- RanBPM antibodies, but also by nonhydrolyzable GTP-Ran. Indeed, RanBPM specifically interacted with GTP-Ran in two-hybrid assay. The central part of asters stained by anti-RanBPM antibodies or by the mAb to gamma-tubulin was faded by the addition of GTPgammaS-Ran, but not by the addition of anti-RanBPM anti- bodies. These results provide evidence that the Ran-binding protein, RanBPM, is involved in microtubule nucleation, thereby suggesting that Ran regulates the centrosome through RanBPM.

Altered growth of human colon cancer cell lines disrupted at activated Ki-ras.

Point mutations that activate the Ki-ras proto-oncogene are presented in about 50 percent of human colorectal tumors. To study the functional significance of these mutations, the activated Ki-ras genes in two human colon carcinoma cell lines, DLD-1 and HCT 116, were disrupted by homologous recombination. Compared with parental cells, cells disrupted at the activated Ki-ras gene were morphologically altered, lost the capacity for anchorage-independent growth, grew more slowly both in vitro and in nude mice, and showed reduced expression of c-myc. Thus, the activated Ki-ras gene plays a key role in colorectal tumorigenesis through altered cell differentiation and cell growth.

Molecular and immunological characterization of Babesia gibsoni and Babesia microti heat shock protein-70.

Serological immunoscreening was used to identify a gene encoding heat shock protein-70 from Babesia gibsoni (BgHSP-70) that showed high homology with HSP-70s from other apicomplexan parasites. This gene corresponded to a full-length cDNA containing an open reading frame of 1968 bp predicted to result in a 70-kDa mature protein consisting of 656 amino acids. Analysis of the expression levels of BgHSP-70 indicated elevated transcription from cultured parasites incubated at 40 degrees C for 1 h, but not at 30 degrees C. Interestingly, antiserum raised against recombinant BgHSP-70 protein reacted specifically not only with a 70-kDa protein of B. gibsoni but also with a corresponding native protein of B. microti (BmHSP-70), indicating the high degree of conservation of this protein. The BmHSP-70 gene was then isolated and characterized and the immunoprotective properties of recombinant BgHSP-70 (rBgHSP-70) and rBmHSP-70 were compared in vitro and in vivo. Both proteins had potent mitogenic effects on murine and canine mononuclear cells as evidenced by high proliferative responses and IFN-gamma production after stimulation. Immunization regimes in BALB/c and C57BL/6 mice using rBgHSP-70 and rBmHSP-70 elicited high antibody levels, with concurrent significant reductions in peripheral parasitaemias. Taken together, these results emphasize the potential of HSP-70s as a molecular adjuvant vaccine.

Renal Infiltration of Macrophages in Canine and Feline Chronic Kidney Disease.

During the progression of chronic kidney disease (CKD), macrophage infiltration is a crucial event leading to tubulointerstitial fibrosis. In the present study, macrophages infiltrating renal tissue in dogs and cats with CKD were analysed immunohistochemically. Iba-1 was used as a pan-macrophage marker, CD204 was used as a marker of M2 macrophages and tumour necrosis factor (TNF)-α was used as a marker of M1 macrophages. Signals for Iba1 and CD204 were observed in the interstitium of all tested kidney samples. In dogs, the signals were diffusely scattered. In cats, both diffuse and focal signals were observed. Cells that were positive for Iba1 and CD204 were also observed in the tubular lumina in cats. Co-expression of Iba1 and CD204 was also observed in the infiltrating cells by immunofluorescence labelling, and these cells were negative for TNF-α. By quantitative analysis, the indices for Iba1- and CD204-positive cells were significantly correlated with the concentrations of plasma creatinine and/or urea and the extent of interstitial fibrosis in both dogs and cats. These results demonstrated that renal infiltration of M2 macrophages plays an important role in the progression of CKD in dogs and cats. The distribution pattern of the kidney-infiltrating macrophages was unique in cats and may be associated with a cat-specific renal fibrotic process.

Duodenal expression of antimicrobial peptides in dogs with idiopathic inflammatory bowel disease and intestinal lymphoma.

Although antimicrobial peptides (AMPs) play an integral role in the regulation of intestinal microbiota and homeostasis, their expression in canine gastrointestinal diseases, including idiopathic inflammatory bowel disease (IBD) and intestinal lymphoma, remains unknown. The objective of this study was to investigate the intestinal expression of AMPs in dogs with IBD or intestinal lymphoma. IBD was diagnosed in 44 dogs, small cell intestinal lymphoma in 25 dogs, and large cell intestinal lymphoma in 19 dogs. Twenty healthy beagles were used as normal controls. Duodenal mRNA expression of six representative AMPs - lactoferrin, lysozyme, cathelicidin, secretory leukocyte peptidase inhibitor (SLPI), bactericidal/permeability increasing protein (BPI), and canine beta defensin (CBD103) - was quantified by real-time reverse transcription polymerase chain reaction. The relative expression of BPI, lactoferrin, and SLPI was significantly higher in dogs with IBD and intestinal lymphomas than in healthy controls. Interestingly, the expression patterns of AMPs differed between dogs with IBD and those with intestinal lymphomas, especially small cell lymphoma. Increased expression of BPI differentiated IBD from dogs with small cell intestinal lymphoma, with a sensitivity of 93.2%, a specificity of 100%, and an area under the curve of 0.955. These results suggest that the expression patterns of AMP aid in the diagnosis of canine IBD and intestinal lymphoma, although it remains uncertain whether the altered AMP expression is the cause or effect of mucosal inflammation.

EphB2 guides axons at the midline and is necessary for normal vestibular function.

Mice lacking the EphB2 receptor tyrosine kinase display a cell-autonomous, strain-specific circling behavior that is associated with vestibular phenotypes. In mutant embryos, the contralateral inner ear efferent growth cones exhibit inappropriate pathway selection at the midline, while in mutant adults, the endolymph-filled lumen of the semicircular canals is severely reduced. EphB2 is expressed in the endolymph-producing dark cells in the inner ear epithelium, and these cells show ultrastructural defects in the mutants. A molecular link to fluid regulation is provided by demonstrating that PDZ domain-containing proteins that bind the C termini of EphB2 and B-ephrins can also recognize the cytoplasmic tails of anion exchangers and aquaporins. This suggests EphB2 may regulate ionic homeostasis and endolymph fluid production through macromolecular associations with membrane channels that transport chloride, bicarbonate, and water.

Forward signaling mediated by ephrin-B3 prevents contralateral corticospinal axons from recrossing the spinal cord midline.

To investigate Eph-ephrin bidirectional signaling, a series of mutations were generated in the ephrin-B3 locus. The absence of both forward and reverse signaling resulted in mice with mirror movements as typified by a hopping locomotion. The corticospinal tract was defective as axons failed to respect the midline boundary of the spinal cord and bilaterally innervated both contralateral and ipsilateral motor neuron populations. A second mutation that expresses a truncated ephrin-B3 protein lacking its cytoplasmic domain did not lead to hopping, indicating that reverse signaling is not required for corticospinal innervation. Ephrin-B3 is concentrated at the spinal cord midline, while one of its receptors, EphA4, is expressed in postnatal corticospinal neurons as their fibers pathfind down the contralateral spinal cord. Our data indicate ephrin-B3 functions as a midline-anchored repellent to stimulate forward signaling in EphA4-expressing axons.

C3 contributes to the cross-protective immunity induced by Babesia gibsoni phosphoriboprotein P0 against a lethal B. rodhaini infection.

We have studied the impact of complement component 3 (C3) deficiency on the progression of lethal Babesia rodhaini infection in immune mice. A B. gibsoni ribosomal phosphoprotein P0 (BgP0) previously reported to be a cross-protective antigen against Babesia infection was used to immunize C57BL/6 wild-type (WT) and C3-deficient (C3-/-) mice. Test mice were immunized intraperitoneally (i.p.) with recombinant BgP0 (rBgP0), while controls either were immunized with PBS or did not receive any immunization. Following the immunization regime, test WT mice induced a specifically strong humoral response consisting of mixed immunoglobulins IgG1 and IgG2 associated with high production of IFN-gamma in the supernatant of splenocytes. While test C3-/- mice had significantly decreased total IgG, IgG1 and IgG2b responses, the secretions of IL-12 and IFN-gamma tended to be lower than those in WT mice. Furthermore, partial protection was only observed in rBgP0-immunized WT mice but not in C3-/- mice or controls. Indeed, rBgP0-immunized WT mice showed significant reductions in the initiation of parasitaemia correlated with delayed mortalities and considerable survival rates. Taken together, our results indicate that cross-protection was impaired in C3-/- mice in view of the decrease in the antibody responses and cytokine production and the high susceptibility to infection.

Changes in Renal Peritubular Capillaries in Canine and Feline Chronic Kidney Disease.

Renal capillary rarefaction is a crucial event that leads to tubulointerstitial damage during the progression of chronic kidney disease (CKD). In the present study, changes in CD34-positive renal capillaries were investigated in dogs and cats with CKD. A significant decrease in CD34-positive capillaries was observed in canine diseased kidneys, even at the early stage of disease. In cats, CD34-positive capillaries were well preserved in the diseased kidneys, with no link to the severity of CKD. Renal capillary rarefaction might be a trigger event that leads to the progression of CKD in dogs, rather than in cats.

Association of the PCK2 Gene Polymorphism With New-onset Glucose Intolerance in Japanese Kidney Transplant Recipients.

BACKGROUND: New-onset diabetes mellitus after transplantation (NODAT) is a risk factor for both cardiovascular disease and poor graft survival after kidney transplantation (KTx). In this study, we identified single-nucleotide polymorphisms (SNPs) in genes involved in glucose metabolism and examined the correlation between these SNPs and glucose intolerance after KTx. METHODS: Thirty-eight patients with normal glucose tolerance before KTx were included in this study. Patients with plasma glucose levels of >140 mg/dL at 120 minutes on the 75-g oral glucose tolerance test at 1 year after KTx were classified as having new-onset impaired glucose tolerance (NIGT). We identified 8 SNPs in 7 genes that are involved in glucose metabolism among the patients included in this study, and compared the prevalence rate of NIGT among SNPs in each gene. RESULTS: Of the 38 patients, 11 (28.9%) were diagnosed with NIGT. For rs4982856 in the PCK2 gene, the distribution of genotypes among the total patient population was as follows: T/T, 12 (31.6%); T/C, 22 (57.9%); and C/C, 4 (10.5%). Seven of 11 patients with NIGT had the T/T genotype of rs4982856, whereas only 5 of 27 patients with normal glucose tolerance had this genotype. The T allele frequency of the rs4982856 was significantly higher in the NIGT group than in the normal group (81.8 vs 52.8%, respectively; P = .015). CONCLUSION: Our study indicates that the T allele of the rs4982856 SNP in the PCK2 gene may be a risk factor for glucose intolerance after KTx.

Transcranial Doppler Ultrasound Examination in Dogs with Suspected Intracranial Hypertension Caused by Neurologic Diseases.

BACKGROUND: Transcranial Doppler ultrasound examination (TCD) is a rapid, noninvasive technique used to evaluate cerebral blood flow and is useful for the detection of intracranial hypertension in humans. However, the clinical usefulness of TCD in diagnosing intracranial hypertension has not been demonstrated for intracranial diseases in dogs. OBJECTIVES: To determine the association between the TCD variables and intracranial hypertension in dogs with intracranial diseases. ANIMALS: Fifty client-owned dogs with neurologic signs. METHODS: Cross-sectional study. All dogs underwent TCD of the basilar artery under isoflurane anesthesia after magnetic resonance imaging (MRI). Dogs were classified into 3 groups based on MRI findings: no structural diseases (group I), structural disease without MRI evidence of intracranial hypertension (group II), and structural disease with MRI evidence of intracranial hypertension (group III). The TCD vascular resistance variables (resistive index [RI], pulsatility index [PI], and the ratio of systolic to diastolic mean velocity [Sm/Dm]) were measured. RESULTS: Fifteen, 22, and 13 dogs were classified into groups I, II, and III, respectively. Dogs in group III had significantly higher Sm/Dm (median, 1.78; range, 1.44-2.58) than those in group I (median, 1.63; range, 1.43-1.75) and group II (median, 1.62; range, 1.27-2.10). No significant differences in RI and PI were identified among groups. CONCLUSIONS AND CLINICAL IMPORTANCE: Our findings suggest that increased Sm/Dm is associated with MRI findings of suspected intracranial hypertension in dogs with intracranial diseases and that TCD could be a useful tool to help to diagnose intracranial hypertension.

Diversity of LEF/TCF action in development and disease.

Lymphoid enhancer factor/T cell factor proteins (LEF/TCFs) mediate Wnt signals in the nucleus by recruiting beta-catenin and its co-activators to Wnt response elements (WREs) of target genes. This activity is important during development but its misregulation plays a role in disease such as cancer, where overactive Wnt signaling drives LEF/TCFs to transform cells. The size of the LEF/TCF family is small: approximately four members in vertebrates and one orthologous form in flies, worms and hydra. However, size belies complexity. The LEF/TCF family exhibits extensive patterns of alternative splicing, alternative promoter usage and activities of repression, as well as activation. Recent work from numerous laboratories has highlighted how this complexity has important biological consequences in development and disease.

Increased frequency of antigen-specific CD8(+) cytotoxic T lymphocytes infiltrating an Epstein-Barr virus-associated gastric carcinoma.

Gastric adenocarcinomas carrying Epstein-Barr virus (EBV) are known to be accompanied by massive lymphocyte infiltration. To characterize the tumor-infiltrating lymphocytes (TILs), we isolated and cultured such cells from a surgically resected EBV-associated gastric carcinoma. They were found to be positive for CD3, CD8, T-cell receptor beta chain, and cytotoxic molecules. The isolated TILs consisted of human leukocyte antigen (HLA) class I-restricted CD8(+) cytotoxic T lymphocytes (CTLs), which killed autologous EBV-transformed cells (but not phytohemagglutinin blast cells) and recognized HLA-A24 as restriction molecules. However, the TILs did not recognize known EBV antigenic peptides presented by HLA-A24 molecules, nor HLA-A24(+) fibroblasts infected with vaccinia recombinant virus expressing each of the EBV latent proteins. EBV(+) gastric carcinomas do not express conventional target proteins of EBV-specific CTLs, and the data suggest that some cellular proteins may be involved in the strong T-cell response to EBV-associated gastric carcinoma. In addition, our data suggest that class I-restricted, antigen-specific CD8(+) CTLs are specifically expanded within EBV(+) gastric carcinoma tissue.