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.

Localization of Toll-like Receptor (TLR) 2 and TLR4 mRNA in the Colorectal Mucosa of Miniature Dachshunds with Inflammatory Colorectal Polyps.

Inflammatory colorectal polyps (ICRPs) are characterized by the formation of multiple or solitary polyps with marked neutrophil infiltration in the colorectal area, and are speculated to be a novel form of breed-specific canine idiopathic inflammatory bowel disease (IBD). In human IBD, toll-like receptor (TLR) 2 and TLR4 have been reported to be involved in the pathogenesis of the disease. The aim of this study was to evaluate the expression of TLR2 and TLR4 mRNA in the colorectal mucosa of dogs with ICRPs by in-situ hybridization using an RNAscope assay. Samples of inflamed colorectal mucosa (n = 5) and non-inflamed mucosa (n = 5) from miniature dachshunds (MDs) with ICRPs and colonic mucosa from healthy beagles (n = 5) were examined. TLR2 and TLR4 hybridization signals were localized to the colorectal epithelium, inflammatory cells and fibroblasts in the inflamed colorectal mucosa of affected dogs. The signals were significantly greater in inflamed colorectal epithelium compared with non-inflamed epithelium of MDs with ICRPs and healthy beagles (P <0.05). These results suggest that increased expression of TLR2 and TLR4 mRNA in the inflamed colorectal mucosa results from not only inflammatory cell infiltration, but also the upregulation of TLR2 and TLR4 mRNA in the colonic epithelium.

Hypoxia-Inducible Factor-1α Expression in Kidney Transplant Biopsy Specimens After Reperfusion Is Associated With Early Recovery of Graft Function After Cadaveric Kidney Transplantation.

BACKGROUND: Ischemia/reperfusion injury during kidney transplantation (KTx) delays allograft recovery. Hypoxia-inducible factor-1α (HIF-1α) is the key regulator of the protective response to ischemia/reperfusion injury. We evaluated the impact of the HIF-1α signaling pathway on allograft recovery during cadaveric KTx. METHODS: Between 1996 and 2015, 46 patients underwent cadaveric KTx. The expression levels of HIF-1α-related proteins, including phosphoinositide 3-kinase, phosphorylated (p)-Akt, p-mammalian target of rapamycin, p-Eukaryotic translation initiation factor 4E, p-S6 ribosomal protein, and HIF-1α, were immunohistochemically evaluated and semi-quantitatively scored in graft biopsy specimens after 1 hour of revascularization. Ten kidney biopsy specimens collected during donor nephrectomy for living KTx were used as controls. Delayed graft function (DGF) was defined as the need for dialysis within 1 week of KTx. We compared the staining scores of each protein and several clinical parameters between patients with and those without DGF. RESULTS: Expression levels of all six proteins in specimens after revasculization were elevated compared with those in controls. Thirty-five patients had DGF. Expression levels of PI3K, p-AKT, p-mTOR, p-eIF4E, and HIF-1α were significantly higher in patients without DGF than in those with DGF. Univariate analysis identified expression levels of p-Akt, p-S6, and HIF-1α, in addition to donor type (heart beating/non-heart beating), cold ischemic time, and donor age as significant predictors of DGF. Of these, only expression levels of HIF-1α and donor type were independently associated with DGF in multivariate analysis. CONCLUSIONS: Up-regulation of HIF-1α in allografts after reperfusion may be a predictor of early recovery after cadaveric KTx.

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.

Inhibitory effects of maternal smoking on the development of severe retinopathy of prematurity.

PURPOSE: In this study, the effects of maternal smoking along with other clinical risk factors in developing severe retinopathy of prematurity (ROP) were evaluated. DESIGN: A case-control study. METHODS: Records of newborn infants with an estimated postmenstrual age of 32 weeks or less (n=86) were reviewed. ROP grading was evaluated in accordance with the International Classification of Retinopathy of Prematurity. Severe ROP was diagnosed when it progressed to stage 3 with plus disease. The factors were first evaluated using a univariate logistic regression analysis between the groups of severe and non-severe ROP, followed by a multivariate logistic regression analysis using STATA version 10 and R version 2.71. RESULTS: A low birth weight, a long duration of artificial ventilation and oxygen supplementation, presence of chronic lung disease, and absence of maternal smoking were found to be significantly associated with severe ROP in the univariate logistic regression analysis. In the multivariate logistic regression analysis, maternal smoking was revealed as a significant factor independently associated with the incidence of severe ROP. CONCLUSIONS: An inhibitory effect of maternal smoking against developing severe ROP is suggested. The mechanism by which smoking may reduce the incidence of severe ROP needs to be further investigated.

Validation of the Japanese version of EORTC QLQ-CR38.

PURPOSE: The objective of this study was to test the reliability and validity of the Japanese version of the European Organization for Research and Treatment of Cancer (EORTC) Colorectal Cancer-Specific Quality of Life Questionnaire (QLQ-CR38). METHODS: The questionnaire was tested among 109 colorectal cancer patients on several occasions. The timing was prior to treatment with radiotherapy or chemotherapy, during treatment and 3 months following the second assessment. For purpose of test-retest reliability, a subgroup of patients completed the QLQ-CR38 1 week following the third assessment. RESULTS: Multitrait scaling analysis confirmed the hypothesised scale structure of both the function scales and the symptom scales except female sexual problem scale. Cronbach's alpha coefficients for seven of the nine scales exceeded the 0.7 criterion at one or both assessments. The test-retest reliability for all scales except three symptom scales was 0.67 or higher. On the basis of known-groups comparisons, four of 46 comparisons distinguished between patients differing in disease stage, initial and on-treatment performance status and presence or absence of a stoma. Additionally, these scales detected change over time as a function of change in performance status and treatment-induced change. CONCLUSION: These preliminary results suggest that the Japanese EORTC QLQ-CR38 may be a reliable and valid supplementary measure of quality of life in colorectal cancer patients.

Cyclin-dependent kinase inhibitors block erythrocyte invasion and intraerythrocytic development of Babesia bovis in vitro.

SUMMARYCyclin-dependent kinases (CDKs) are essential for the regulation of the eukaryotic cell cycle. A number of chemicals, which selectively inhibit the CDK activities, have been synthesized for the development of anti-cancer drugs. This report describes the inhibitory effect of purine derivatives known to be CDK inhibitors on the asexual growth of Babesia bovis. The 4 compounds, roscovitine, purvalanol A, CGP74514A, and CDK2 Inhibitor II, showed significantly suppressive effects on the in vitro growth of B. bovis. Three (roscovitine, purvalanol A, and CDK2 Inhibitor II) showed an inhibitory effect on the early stages of intraerythrocytic development of B. bovis. CGP74514A (CDK1-specific inhibitor) blocked the erythrocyte invasion by merozoites. Our data suggest the chemotherapeutic potential of the CDK inhibitors for babesiosis, and the target molecules of the compounds would participate in the process of successful erythrocyte invasion or intraerythrocytic development of B. bovis.

Number of positive lymph nodes independently affects long-term survival after resection in patients with ampullary carcinoma.

AIM: The nodal status is an established prognostic factor in ampullary carcinoma. The aim of this study was to compare the prognostic power of the anatomic location of positive nodes with that of the number of positive nodes. METHODS: Of 73 consecutive patients treated for ampullary carcinoma, 62 underwent pancreaticoduodenectomy with regional lymphadenectomy. A survival analysis of these 62 patients by nodal status was conducted retrospectively. A total of 1942 lymph nodes taken from the patients were examined histologically for metastasis. The location of positive regional nodes was classified into 4 categories, according to the Japanese staging system. The number of positive regional nodes was recorded for each patient. The median follow-up period was 124 months. RESULTS: Nodal disease was found in 31 patients, of whom 23 had 1-3 positive regional nodes and 8 had >or=4 positive regional nodes. Univariate analysis revealed that both the location (p<0.0001) and the number (p<0.0001) of positive nodes were significant prognostic factors. Multivariate analysis revealed that the number of positive nodes was an independent prognostic factor (p=0.007), while the location failed to remain as an independent variable. The median survival time was 59 months with a 5-year survival rate of 48% in patients with 1-3 positive nodes, whereas all patients with >or=4 positive nodes died of the disease within 29 months of resection (p=0.0001). CONCLUSION: The number, not the location, of positive regional lymph nodes independently affects long-term survival after resection in patients with ampullary carcinoma.

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.

A rare case of recurrent alpha-fetoprotein-producing gastric cancer without re-elevation of serum AFP.

We report an extremely rare case of recurrent alpha-fetoprotein (AFP)-producing gastric cancer without re-elevation of serum AFP. The patient was a 78-year-old woman with AFP-producing gastric cancer, a rare type of gastric adenocarcinoma. A Borrmann III gastric tumour was surgically resected and AFP-producing gastric cancer was diagnosed based on high levels of serum AFP (705.44 ng/ml) and immunohistochemical examination of the tumour. The serum AFP level decreased to the normal range after resection without any sign of recurrence by imaging, but the patient developed local recurrence of the cancer and died 13 months after surgery. No re-elevation of serum AFP levels was observed after recurrence. Although serum AFP levels are believed to be useful for follow-up in the post-operative period, the possibility that serum AFP levels do not always correlate with the extent of the cancer should be kept in mind.

Clinical significance of Ki-67 proliferation index in disease progression and prognosis of patients with resected colorectal carcinoma.

BACKGROUND: The prognostic role of proliferation markers in patients with colorectal neoplasia is inconclusive. The aim of this study was to evaluate proliferation markers in patients with colorectal carcinoma, to relate these to standard clinicopathological findings and to investigate their potential role in the prediction of long-term survival. METHODS: Archived formalin-fixed, paraffin-embedded tissue samples from a cohort of 106 patients who underwent curative resection for colorectal carcinoma were analysed to determine a Ki-67 (MIB-1)-derived proliferation index (PI) using a double immunostaining technique. The relationship between PI and standard clinicopathological variables was assessed and its association with long-term survival evaluated. RESULTS: A significant association was found between PI and tumour (T) status (P = 0.001), lymph node (N) involvement (P = 0.0098), the presence of distant metastases (P < 0.010) and advanced stage of disease (P < 0.001). On multivariate analysis, PI was shown to be an independent prognostic factor for long-term survival (hazard ratio 2.1 (95 per cent confidence interval 1.1 to 4.1); P = 0.032). CONCLUSION: Cell proliferation is significantly associated with tumour progression and may be used to identify patients with a predicted adverse outcome after resection of colorectal carcinoma.

High risk of gallbladder carcinoma in elderly patients with segmental adenomyomatosis of the gallbladder.

The clinical significance of adenomyomatosis of the gallbladder remains unclear. This study aimed to clarify the relationship between segmental adenomyomatosis and gallbladder carcinoma, and to elucidate the histogenesis of gallbladder carcinoma associated with segmental adenomyomatosis. A total of 4,560 consecutive patients underwent cholecystectomy. The specimens were examined grossly and histologically. Adenomyomatosis of the gallbladder was divided into segmental, fundal, and diffuse types. Sixty noncancerous gallbladders with segmental adenomyomatosis were examined for epithelial metaplasia. The incidence of gallbladder carcinoma was higher in patients with segmental adenomyomatosis (22/334, 6.6%) than in those without (181/4226, 4.3%; P=0.049). This difference was more marked among patients equal to or older than 60 years of age (15/96,15.6% versus 147/2407, 6.1%, respectively; P<0.001). The other types of adenomyomatosis did not show any significant increases in the incidence of gallbladder carcinoma. In all 22 patients with both segmental adenomyomatosis and carcinoma, the tumors developed only in the fundal mucosa. Epithelial metaplasia was more marked in the fundal mucosa of segmental adenomyomatosis than in the neck mucosa (P=0.003). Segmental adenomyomatosis is a high-risk condition for gallbladder carcinoma, especially in elderly patients. Epithelial metaplasia appears to be related to increased carcinogenesis in the fundal mucosa of segmental adenomyomatosis.

Hepatitis viral status affects the pattern of intrahepatic recurrence after resection for hepatocellular carcinoma.

AIM: To define whether the patterns of intrahepatic recurrence after resection for hepatocellular carcinoma differ according to hepatitis viral status. METHODS: One hundred and eleven patients undergoing a curative resection for hepatocellular carcinoma were divided into three groups: the C-viral group (n=55), which tested positive for hepatitis C antibody; the B-viral group (n=32), which tested positive for hepatitis B surface antigen; and the non-B non-C (NBNC) group (n=24), which tested negative for both hepatitis B surface antigen and hepatitis C antibody. The long-term outcomes were analyzed retrospectively. RESULTS: The pattern of development of intrahepatic recurrence differed between the NBNC group and the other groups: the cumulative probability of intrahepatic recurrence reached a plateau at 2.4 years after resection in the NBNC group, while it continued to increase steadily in the hepatitis viral groups. The C-viral group showed a higher incidence of intrahepatic recurrence than the other groups by univariate (P=0.0306) and multivariate (relative risk=1.69, P=0.0429) analyses. Multiple intrahepatic recurrent lesions were more common in the C-viral group (P=0.0457). CONCLUSIONS: Multicentric carcinogenesis in the remnant liver was less common in the NBNC group than in hepatitis viral groups. Hepatitis C virus infection is a significant risk factor for intrahepatic recurrence after resection and is also associated with multiple intrahepatic recurrent lesions.

Ultrastructural studies on the pheromone-producing cells in the silkmoth, Bombyx mori: formation of cytoplasmic lipid droplets before adult eclosion.

In Bombyx mori, pheromone-producing cells accumulate a number of lipid droplets in the cytoplasm preceding the production of the sex pheromone, bombykol. The process of lipid droplet formation in the pheromone-producing cells was investigated by using light and electron microscopy. Light microscopy revealed that the lipid droplets appeared from 2 days before adult eclosion and dramatic accumulation took place between 2 days and 1 day before eclosion. Electron microscopical studies revealed that smooth endoplasmic reticulum and numerous vesicles, their sizes being less than 1 microm, were detectable 2 days before eclosion, and some vesicles were fused with mitochondria at this stage. These characteristic changes in the pheromone-producing cells suggest that fatty acyl-CoA synthesis following de novo fatty acid synthesis takes place at this time. Involutions in the basal plasma membrane of the cells occurred throughout the observed period, which were extensive on the day before adult eclosion. Besides extensive basal involutions, immature lipid droplets appeared and then mature fully electron-dense lipid droplets were observed on the day of adult eclosion. These ultrastructural observations, combined with recent physiological studies suggest, that the basal involutions presumably reflect the uptake of lipidic components required for the construction of lipid droplets, the function of which is to store the bombykol precursor and to provide it for bombykol biosynthesis in response to pheromonotropic stimuli by pheromone biosynthesis activating neuropeptide (PBAN).

Characterisation of the gene encoding a protective antigen from Babesia microti identified it as eta subunit of chaperonin containing T-complex protein 1.

Passive immunisations with a monoclonal antibody termed 1-5H showed a partial but significant inhibition of parasitaemia against Babesia microti challenge infection. By immunoscreening with 1-5H, a clone (termed p58 gene) was obtained from a cDNA expression library of B. microti and the complete nucleotide sequence was determined. A protein homology search showed significant amino acid identities to the eta subunit of the chaperonin containing T-complex protein 1 (CCT) of human (59%), mouse (58%) and Plasmodium falciparum (62%). Genomic analyses indicated that the p58 gene is present as a single copy gene and contains a total of approximately 400-bp introns in the genome of B. microti. The mAb 1-5H recognised a 58-kDa protein of B. microti and was found to cross-react with a 60-kDa protein of Babesia rodhaini. These results suggest the possibility that the p58 protein is the CCT eta subunit of B. microti and functions as a chaperonin.