Ultrasound-guided transurethral urinary bladder biopsy using an endoscopic biopsy forceps in dogs: 27 cases (2016-2019).

OBJECTIVES: To describe an ultrasound-guided transurethral bladder biopsy technique using endoscopic forceps and its results in dogs of different sizes with different lesion locations. MATERIALS AND METHODS: Medical records of dogs that underwent ultrasound-guided transurethral bladder biopsy with endoscopic forceps were retrospectively reviewed. Patient signalment, lesion location, use of urinary catheter as a guide, outcome of the procedure and histopathology results were retrieved. RESULTS: Twenty-seven dogs underwent this procedure. Biopsy samples were successfully obtained in 23 dogs. Insertion of the endoscopic forceps without a urinary catheter allowed the procedure to be performed in patients with a small urethral diameter without complication. The procedure was unsuccessful in dogs with a urethral diameter smaller than the outer diameter of the biopsy forceps (i.e. 1.8 mm), either due to small patient size or obstructive urethral lesion. All biopsy samples allowed histopathological diagnosis. No complications were reported after the procedure. CLINICAL SIGNIFICANCE: This non-invasive biopsy technique should be considered in patients with bladder lesions in which histopathological diagnosis is needed, especially when endoscopic examination is not feasible. By use of the Doppler mode, biopsy retrieval was safe also when the lesion ​was highly vascularised.

Evaluation of urinary and serum level of chemokine (C-C motif) ligand 2 as a potential biomarker in canine urothelial tumours.

Chemokine (C-C motif) ligand 2 (CCL2) is a chemotactic cytokine recruiting monocytes, releasing growth factors and promoting adhesion in vascular endothelium. Elevated serum and urinary CCL2 levels and expression of its receptor (CCR2) have been associated with tumorigenesis in human urinary malignancies. CCL2 implication has not been investigated in canine urothelial carcinoma. The aim of this study was to evaluate CCL2 serum and urine levels (measured by ELISA) in dogs with urothelial carcinoma or non-neoplastic urinary tract disease. CCL2 serum and urine levels were significantly higher in diseased dogs compared with healthy dogs (P < 0.001). Dogs with carcinoma had significantly higher serum and urine CCL2 levels (P = 0.001) than healthy dogs. Dogs with metastases showed significantly lower serum and urine CCL2 levels compared with the non-metastasised tumour group (P = 0.007). CCL2 as a diagnostic marker for urothelial carcinoma held a sensitivity of 95.2% and a specificity of 38.2% in the urine. As a staging marker, sensitivity was 85.7% and specificity was 57.1% with a positive predictive value of 75.7% and a negative predictive value of 71.9%. Further investigation is needed to define the role of CCL2 as a prognostic marker in canine urothelial carcinoma.

RUNX3 is oncogenic in natural killer/T-cell lymphoma and is transcriptionally regulated by MYC.

RUNX3, runt-domain transcription factor, is a master regulator of gene expression in major developmental pathways. It acts as a tumor suppressor in many cancers but is oncogenic in certain tumors. We observed upregulation of RUNX3 mRNA and protein expression in nasal-type extranodal natural killer (NK)/T-cell lymphoma (NKTL) patient samples and NKTL cell lines compared to normal NK cells. RUNX3 silenced NKTL cells showed increased apoptosis and reduced cell proliferation. Potential binding sites for MYC were identified in the RUNX3 enhancer region. Chromatin immunoprecipitation-quantitative PCR revealed binding activity between MYC and RUNX3. Co-transfection of the MYC expression vector with RUNX3 enhancer reporter plasmid resulted in activation of RUNX3 enhancer indicating that MYC positively regulates RUNX3 transcription in NKTL cell lines. Treatment with a small-molecule MYC inhibitor (JQ1) caused significant downregulation of MYC and RUNX3, leading to apoptosis in NKTL cells. The growth inhibition resulting from depletion of MYC by JQ1 was rescued by ectopic MYC expression. In summary, our study identified RUNX3 overexpression in NKTL with functional oncogenic properties. We further delineate that MYC may be an important upstream driver of RUNX3 upregulation and since MYC is upregulated in NKTL, further study on the employment of MYC inhibition as a therapeutic strategy is warranted.

Enhancement of Satellite Cell Transplantation Efficiency by Leukemia Inhibitory Factor.

BACKGROUND AND OBJECTIVES: Cell transplantation is a promising therapy for several muscle diseases, including Duchenne muscular dystrophy. Satellite cells are stem cells in skeletal muscle that provide an important cell source for transplantation therapy. However, culture of satellite cells in vitro causes them to lose their undifferentiated state, associated with reduced transplantation efficiency. It is therefore necessary to develop optimal culture conditions for maintaining the undifferentiated state of satellite cells. METHODS: Primary satellite cells were cultured with or without leukemia inhibitory factor (LIF). The expression of undifferentiation and differentiation markers, and the transplantation efficiency were analyzed. RESULTS: LIF-treated satellite cells showed increased expression of Pax7, and enhanced transplantation efficiency in a mouse model of Duchenne muscular dystrophy. CONCLUSIONS: Our study showed that the treatment with LIF effectively maintained the undifferentiated state of satellite cells, and enhanced their transplantation efficiency. These results will contribute to the optimization of culture conditions for cell transplantation therapy.

Sequential Changes in Cortical Excitation during Orthodontic Treatment.

Cortical excitation responding to periodontal ligament (PDL) stimulation is observed in the rat primary somatosensory (S1), secondary somatosensory, and insular oral region of the cortex (S2/IOR), which are considered to process somatosensation, including nociception. Our previous studies have demonstrated that excitatory propagation induced by PDL stimulation is facilitated in S1 and S2/IOR 1 d after experimental tooth movement (ETM), and tetanic stimulation of IOR induces long-term potentiation of cortical excitatory propagation consistently. These findings raise the possibility that ETM induces neuroplastic changes, and as a result, facilitation of cortical excitation would be sustained for weeks. However, no information is available about the temporal profiles of the facilitated cortical responses. We estimated PDL stimulation-induced cortical excitatory propagation in S1 and S2/IOR of rats by optical imaging 1 to 7 d after ETM of the maxillary first molar. ETM models showed facilitated cortical excitatory propagation in comparison with controls and sham groups 1 d after ETM, but the facilitation gradually recovered to the control level 3 to 7 d after ETM. Sham groups that received wire fixation without orthodontic force tended to enhance cortical responses, although the differences between controls and sham groups were almost insignificant. We also examined the relationship between cortical responses and expression of inflammatory cytokines, interleukin (IL)-1ß and tumor necrosis factor (TNF)-α, in PDL of the first molar. The peak amplitude of optical signals responding to PDL stimulation tended to be increased in parallel to the number of IL-1ß and TNF-α immunopositive cells, suggesting that, at least in part, the enhancement of cortical responses is induced by PDL inflammation. These findings suggest that ETM-induced facilitation of cortical excitatory propagation responding to PDL stimulation 1 d after ETM recovers to the control level within a week. The time course of the facilitated cortical responses is comparable to that of pain and discomfort induced by clinical orthodontic treatments.

Successful treatment of cytomegalovirus enteritis after unrelated allogeneic stem cell transplantation by the infusion of ex vivo-expanded CD4+ lymphocytes derived from the recipient's peripheral blood donor cells.

Adoptive immunotherapies have been developed for antiviral agent-refractory cytomegalovirus (CMV) disease after stem cell transplantation (SCT). However, the application of such strategies is limited, particularly in terms of need for donor cooperation regarding blood sampling and inaccessibility in the setting of cord blood transplantation. Herein, we describe the first successful treatment of antiviral agent-refractory CMV enteritis after allogeneic SCT by the infusion of ex vivo-expanded donor-derived CD4(+) lymphocytes obtained from the recipient's peripheral blood.

Severe hyponatremia caused by syndrome of inappropriate secretion of antidiuretic hormone developed as initial manifestation of human herpesvirus-6-associated acute limbic encephalitis after unrelated bone marrow transplantation.

Severe hyponatremia is a critical electrolyte abnormality in allogeneic stem cell transplantation (allo-SCT) recipients and >50% of cases of severe hyponatremia are caused by the syndrome of inappropriate secretion of antidiuretic hormone (SIADH). Here, we present a patient with rapidly progressive severe hyponatremia as an initial sign and symptom of human herpesvirus-6-associated post-transplantation acute limbic encephalitis (HHV-6 PALE) after allo-SCT. A 45-year-old woman with acute lymphoblastic leukemia received unrelated bone marrow transplantation from a one locus-mismatched donor at the DR locus. On day 21, she developed a generalized seizure and loss of consciousness with severe hyponatremia, elevated serum antidiuretic hormone (ADH), and decreased serum osmolality. A high titer of HHV-6 DNA was detected in cerebrospinal fluid. Treatment with foscarnet sodium and hypertonic saline was started with improvement of neurological condition within several days. Although an elevated serum ADH, low serum osmolality, and high urinary osmolality persisted for 2 months, she had no other recurrent symptoms of encephalitis. Our experience suggests that hyponatremia accompanied by SIADH should be recognized as a prodromal or concomitant manifestation of HHV-6 PALE, and close monitoring of serum sodium levels in high-risk patients for HHV-6 PALE is necessary for immediate diagnosis and treatment initiation.

ERK is involved in tooth-pressure-induced Fos expression in Vc neurons.

Discomfort and pain encountered during orthodontic treatment are major problems for patients, but the details of the underlying neural processes and molecular mechanisms are not well-understood. Here we show that noxious tooth mechanical pressure induced by orthodontic elastics resulted in a rapid and transient activation of extracellular signal-regulated protein kinase (ERK) in the trigeminal spinal subnucleus interpolaris and caudalis transition zone (Vi/Vc), trigeminal spinal subnucleus caudalis (Vc), and upper cervical spinal cord (Vc/C2). The phosphorylated ERK (pERK) was observed in neurons but not in astroglia and microglia. Single-plane scanning analysis indicated that the pERK was localized to the nucleus of Vc neurons. In addition, the tooth mechanical pressure led to Fos expression in the pERK-positive Vc neurons that would be suppressed by intrathecal administration of an MEK1/2 inhibitor (PD98059). Taken together, these findings suggest that activation of the ERK signaling cascade following noxious mechanical pressure on the teeth regulates Fos expression in Vc neurons and may thereby contribute to pain associated with orthodontic treatment.

Safe placement techniques for self-drilling orthodontic mini-implants.

Self-drilling mini-implants are being used more frequently as an orthodontic anchorage, but the placement torque of self-drilling mini-implants can easily become excessive in the thick, mandibular cortical bone. The purpose of this study is to examine a safe self-drilling placement technique that provides adequate placement torque for orthodontic mini-implants. The mini-implants were placed using self-drilling and pre-drilling methods into the ribs of pigs. Specimens were classified into two groups, thin and thick, with cortical bone thicknesses of 1.2 ± 0.02 and 2.0 ± 0.03 mm, respectively, and used to model the human maxillary and the mandibular bones. The peak mini-implant placement torque value was measured and the surrounding cortical bone was observed histologically. In the mandible model, the torque in the self-drilling and pre-drilling groups exceeded 10 N cm, except in one case which had a 1.3 mm diameter pilot hole. Histology revealed cracks in the surrounding cortical bone in the groups whose torque value was 10 N cm or more. Therefore, when using the self-drilling technique to place a 1.6mm diameter mini-implant in the mandibular alveolar bone, it is preferable to drill a 1.3mm diameter pilot hole first.

Structures and immunolocalization of Na+, K+ -ATPase, Na+ /H+ exchanger 3 and vacuolar-type H+ -ATPase in the gills of blennies (Teleostei: Blenniidae) inhabiting rocky intertidal areas.

The structure and immunolocalization of the ion transporters Na(+) ,K(+) -ATPase (NKA), Na(+) /H(+) exchanger (NHE3) and vacuolar-type H(+) -ATPase (VHA) were examined in the gills of teleosts of the family Blenniidae, which inhabit rocky shores with vertical zonation in subtropical seas. These features were compared among the following species with different ecologies: the amphibious rockskipper blenny Andamia tetradactylus, the intertidal white-finned blenny Praealticus tanegasimae and the purely marine yaeyama blenny Ecsenius yaeyamaensis. Light and electron microscopic observations indicated that thick gill filaments were arranged close to each other and alternately on two hemibranches of a gill arch in the opercular space of A. tetradactylus. Many mucous cells (MC) and mitochondrion-rich cells (MRC) were present in the interlamellar regions of the gill filament. An immunohistochemical study demonstrated that numerous NKA, NHE3 and some VHA were located predominantly on presumed MRCs of gill filaments and at the base of the lamellae. Analyses using serial (mirror image) sections of the gills indicated that only a few NKA immunoreactive cells (IRC) were colocalized with VHA on some MRCs in the filaments. In the gills of P. tanegasimae, NKA- and NHE3-IRCs were observed in the interlamellar region of the filaments and at the base of the lamellae. VHA-IRCs were located sparsely on the lamellae and filaments. In the gills of E. yaeyamaensis, the lamellae and filaments were thin and straight, respectively. MCs were located at the tip as well as found scattered in the interlamellar region of gill filaments. NKA-, NHE3- and VHA-IRCs were moderately frequently observed in the filaments and rarely on the lamellae. This study shows that the structure and distribution of ion transporters in the gills differ among the three blennid species, presumably reflecting their different ecologies.

Development and external validation of a nomogram for predicting cancer probability at initial prostate biopsy using the life expectancy- and prostate volume-adjusted biopsy scheme.

BACKGROUND: We previously reported the diagnostic efficacy of the age- and prostate volume-adjusted prostate biopsy method (the adjusted biopsy method). Here, we developed a new nomogram for predicting cancer probability at initial biopsy using the adjusted method. METHODS: Between 2002 and 2010, 1059 Japanese men with PSA levels between 1.1 and 40 ng ml(-1) and biopsied for the first time using the adjusted method at Gunma University Hospital were enrolled. All subjects underwent digital rectal examination (DRE) and transrectal ultrasonography (TRUS). Data from the initial 849 subjects were used for development of the nomogram and those from the final 210 subjects were used for internal validation. External validation was conducted using data from two affiliated hospitals where the same adjusted biopsy method was used. The nomogram was developed through logistic regression analysis, and predictive accuracy and performance characteristics were assessed using the area under the curve (AUC) of the receiver operating characteristics and calibration plots. Furthermore, we compared the predictive accuracy of the newly developed nomogram with the 'Prostate Risk Indicator' using the development data set, as well as the two external data sets. RESULTS: The AUC of the logistic regression-based nomogram was significantly higher than those of any single clinical parameter. External validation showed significant correlations with the present model. The AUC-receiver operating characteristic of the 'Prostate Risk Indicator' was the second largest following the new nomogram using the development data set and one external data set and almost equal to the new nomogram using the other external data set. CONCLUSIONS: Nevertheless the present model does not include somewhat subjective findings on TRUS abnormality, which is necessary for the estimation by 'Prostate Risk Indicator', the predictive accuracy of the present simple nomogram could be excellent enough to contribute to accurate shared decision-making between doctors and men who are candidates for the adjusted biopsy scheme.

The role of microRNA-150 as a tumor suppressor in malignant lymphoma.

MicroRNA (miRNA; miR) is a class of small regulatory RNA molecules, the aberrant expression of which can lead to the development of cancer. We recently reported that overexpression of miR-21 and/or miR-155 leads to activation of the phosphoinositide 3-kinase (PI3K)-AKT pathway in malignant lymphomas expressing CD3(-)CD56(+) natural killer (NK) cell antigen. Through expression analysis, we show in this study that in both NK/T-cell lymphoma lines and samples of primary lymphoma, levels of miR-150 expression are significantly lower than in normal NK cells. To examine its role in lymphomagenesis, we transduced miR-150 into NK/T-cell lymphoma cells, which increased the incidence of apoptosis and reduced cell proliferation. Moreover, the miR-150 transductants appeared senescent and showed lower telomerase activity, resulting in shortened telomeric DNA. We also found that miR-150 directly downregulated expression of DKC1 and AKT2, reduced levels of phosphorylated AKT(ser473/4) and increased levels of tumor suppressors such as Bim and p53. Collectively, these results suggest that miR-150 functions as a tumor suppressor, and that its aberrant downregulation induces continuous activation of the PI3K-AKT pathway, leading to telomerase activation and immortalization of cancer cells. These findings provide new insight into the pathogenesis of malignant lymphoma.