Effects of chemotherapy treatment with doxorubicin on right ventricular function in dogs.

Left ventricular dysfunction in dogs after the administration of doxorubicin (DOX) has been extensively examined. However, the effects of DOX on right ventricular (RV) function remain unknown. Therefore, the present study investigated whether the chemotherapy treatment with DOX decreases RV function. Twelve dogs (five with multicentric lymphoma, four with hemangiosarcoma, two with thyroid cancer, and one with lung adenocarcinoma) that received at least two doses of DOX were prospectively enrolled. Echocardiography and the measurement of troponin I were performed prior to each administration of DOX and approximately one month after the last administration. Right ventricular function was assessed by the RV fractional area change and RV Tei index. Two (n=4), three (n=3), four (n=3), and five (n=2) doses of DOX were administered. While no significant differences were observed in the RV fractional area change, the RV Tei index was significantly impaired after two doses of DOX. Troponin I level significantly increased after four doses. Cumulative doses of DOX correlated with the RV Tei index (r=0.77, P<0.001). The present results demonstrated that the chemotherapy treatment with DOX decreased RV function in a dose-dependent manner in dogs.

Single-cell and spatial transcriptomics analysis of human adrenal aging.

OBJECTIVE: The human adrenal cortex comprises three functionally and structurally distinct layers that produce layer-specific steroid hormones. With aging, the human adrenal cortex undergoes functional and structural alteration or "adrenal aging", leading to the unbalanced production of steroid hormones. Given the marked species differences in adrenal biology, the underlying mechanisms of human adrenal aging have not been sufficiently studied. This study was designed to elucidate the mechanisms linking the functional and structural alterations of the human adrenal cortex. METHODS: We conducted single-cell RNA sequencing and spatial transcriptomics analysis of the aged human adrenal cortex. RESULTS: The data of this study suggest that the layer-specific alterations of multiple signaling pathways underlie the abnormal layered structure and layer-specific changes in steroidogenic cells. We also highlighted that macrophages mediate age-related adrenocortical cell inflammation and senescence. CONCLUSIONS: This study is the first detailed analysis of the aged human adrenal cortex at single-cell resolution and helps to elucidate the mechanism of human adrenal aging, thereby leading to a better understanding of the pathophysiology of age-related disorders associated with adrenal aging.

Steroids-producing nodules: a two-layered adrenocortical nodular structure as a precursor lesion of cortisol-producing adenoma.

BACKGROUND: The human adrenal cortex consists of three functionally and structurally distinct layers; zona glomerulosa, zona fasciculata (zF), and zona reticularis (zR), and produces adrenal steroid hormones in a layer-specific manner; aldosterone, cortisol, and adrenal androgens, respectively. Cortisol-producing adenomas (CPAs) occur mostly as a result of somatic mutations associated with the protein kinase A pathway. However, how CPAs develop after adrenocortical cells acquire genetic mutations, remains poorly understood. METHODS: We conducted integrated approaches combining the detailed histopathologic studies with genetic, RNA-sequencing, and spatially resolved transcriptome (SRT) analyses for the adrenal cortices adjacent to human adrenocortical tumours. FINDINGS: Histopathological analysis revealed an adrenocortical nodular structure that exhibits the two-layered zF- and zR-like structure. The nodular structures harbour GNAS somatic mutations, known as a driver mutation of CPAs, and confer cell proliferative and autonomous steroidogenic capacities, which we termed steroids-producing nodules (SPNs). RNA-sequencing coupled with SRT analysis suggests that the expansion of the zF-like structure contributes to the formation of CPAs, whereas the zR-like structure is characterised by a macrophage-mediated immune response. INTERPRETATION: We postulate that CPAs arise from a precursor lesion, SPNs, where two distinct cell populations might contribute differently to adrenocortical tumorigenesis. Our data also provide clues to the molecular mechanisms underlying the layered structures of human adrenocortical tissues. FUNDING: KAKENHI, The Uehara Memorial Foundation, Daiwa Securities Health Foundation, Kaibara Morikazu Medical Science Promotion Foundation, Secom Science and Technology Foundation, ONO Medical Research Foundation, and Japan Foundation for Applied Enzymology.

Dectin-2 Deficiency Promotes Proinflammatory Cytokine Release From Macrophages and Impairs Insulin Secretion.

Pancreatic islet inflammation plays a crucial role in the etiology of type 2 diabetes (T2D). Macrophages residing in pancreatic islets have emerged as key players in islet inflammation. Macrophages express a plethora of innate immune receptors that bind to environmental and metabolic cues and integrate these signals to trigger an inflammatory response that contributes to the development of islet inflammation. One such receptor, Dectin-2, has been identified within pancreatic islets; however, its role in glucose metabolism remains largely unknown. Here we have demonstrated that mice lacking Dectin-2 exhibit local inflammation within islets, along with impaired insulin secretion and ß-cell dysfunction. Our findings indicate that these effects are mediated by proinflammatory cytokines, such as interleukin (IL)-1α and IL-6, which are secreted by macrophages that have acquired an inflammatory phenotype because of the loss of Dectin-2. This study provides novel insights into the mechanisms underlying the role of Dectin-2 in the development of islet inflammation.

The role of heat shock protein 90 in the proliferation of Babesia gibsoni in vitro.

The present study investigated the role of heat shock protein 90 (HSP90) in the proliferation and survival of Babesia gibsoni in vitro. To detect the effect on the entry of B. gibsoni into host erythrocytes, the parasite was incubated with an antibody against B. gibsoni HSP90 (BgHSP90) for 24 h. The results of this experiment demonstrated that both the incorporation of [3H]hypoxanthine into the nucleic acids of B. gibsoni and the number of parasites were not altered, indicating that an anti-BgHSP90 antibody did not directly inhibit the entry of the parasite into erythrocytes. Moreover, two HSP90 inhibitors, geldanamycin (GA) and tanespimycin (17-AAG), were used to evaluate the function of BgHSP90. GA and 17-AAG decreased both the incorporation of [3H]hypoxanthine and the number of infected erythrocytes, suggesting that BgHSP90 plays important roles in DNA synthesis and the proliferation of B. gibsoni. The effect of 17-AAG on the parasites was weaker than that of GA. Additionally, the effect of GA on the survival and superoxide generation of canine neutrophils was assessed. The survival of canine neutrophils was not affected. The superoxide generation was strongly suppressed by GA. This result indicated that GA inhibited the function of canine neutrophils. Additional studies are necessary to elucidate the role of BgHSP90 in the proliferation of the parasite.

Vitamin C transporter SVCT1 serves a physiological role as a urate importer: functional analyses and in vivo investigations.

Uric acid, the end product of purine metabolism in humans, is crucial because of its anti-oxidant activity and a causal relationship with hyperuricemia and gout. Several physiologically important urate transporters regulate this water-soluble metabolite in the human body; however, the existence of latent transporters has been suggested in the literature. We focused on the Escherichia coli urate transporter YgfU, a nucleobase-ascorbate transporter (NAT) family member, to address this issue. Only SLC23A proteins are members of the NAT family in humans. Based on the amino acid sequence similarity to YgfU, we hypothesized that SLC23A1, also known as sodium-dependent vitamin C transporter 1 (SVCT1), might be a urate transporter. First, we identified human SVCT1 and mouse Svct1 as sodium-dependent low-affinity/high-capacity urate transporters using mammalian cell-based transport assays. Next, using the CRISPR-Cas9 system followed by the crossing of mice, we generated Svct1 knockout mice lacking both urate transporter 1 and uricase. In the hyperuricemic mice model, serum urate levels were lower than controls, suggesting that Svct1 disruption could reduce serum urate. Given that Svct1 physiologically functions as a renal vitamin C re-absorber, it could also be involved in urate re-uptake from urine, though additional studies are required to obtain deeper insights into the underlying mechanisms. Our findings regarding the dual-substrate specificity of SVCT1 expand the understanding of urate handling systems and functional evolutionary changes in NAT family proteins.

The effect of atovaquone on the mitochondrial membrane potential of Babesiagibsoni.

Atovaquone (ATV) has a growth inhibitory effect against Babesia gibsoni. The target site is considered mitochondria, as in the case of Plasmodium spp.; ATV would collapse the mitochondrial membrane potential. B. gibsoni has also reported that single nucleotide polymorphisms in cytochrome b of mitochondria are involved in ATV susceptibility. However, the details are still unknown. The study aim was to measure the mitochondrial membrane potential of B. gibsoni and evaluate the effect of ATV alone and combined with proguanil (PG) on the mitochondrial membrane potential. As a result of exposure of wild-type B. gibsoni to ATV alone, the number of cells with decreased mitochondrial membrane potential increased. When wild-type B. gibsoni was exposed to the ATV + PG combination, the peak value of mitochondrial membrane potential was larger than that when exposed to ATV alone. It was suggested that ATV alone affects the mitochondrial membrane potential of B. gibsoni, and the effect is enhanced by the combination of ATV and PG. The effect of ATV was weakened for B. gibsoni having reduced sensitivity to ATV (B. gibsoni with M121I), and the effect was not enhanced by the combination of ATV and PG. Although we still need to elucidate the mechanism of ATV and PG for B. gibsoni, these results strongly suggests that the target of ATV for B. gibsoni is also cytochrome b of mitochondria.

Whole Transcriptome Profiling of Adrenocortical Tumors Using Formalin-Fixed Paraffin-Embedded Samples.

Whole transcriptome profiling is a promising technique in adrenal studies; however, whole transcriptome profiling of adrenal disease using formalin-fixed paraffin-embedded (FFPE) samples has to be further explored. The aim of this study was to evaluate the utility of transcriptome data from FFPE samples of adrenocortical tumors. We performed whole transcriptome profiling of FFPE and fresh frozen samples of adrenocortical carcinoma (ACC, n = 3), aldosterone-producing adenoma (APA, n = 3), and cortisol-producing adenoma (CPA, n = 3), and examined the similarity between the transcriptome data. We further examined whether the transcriptome data of FFPE samples could be used to distinguish tumor types and detect marker genes. The number of read counts was smaller in FFPE samples than in fresh frozen samples (P < 0.01), while the number of genes detected was similar (P = 0.39). The gene expression profiles of FFPE and fresh frozen samples were highly correlated (r = 0.93, P < 0.01). Tumor types could be distinguished by consensus clustering and principal component analysis using transcriptome data from FFPE samples. In the differential expression analysis between ACC and APA-CPA, known marker genes of ACC (e.g., CCNB2, TOP2A, and MAD2L1) were detected in FFPE samples of ACC. In the differential expression analysis between APA and CPA, known marker genes of APA (e.g., CYP11B2, VSNL1, and KCNJ5) were detected in the APA of FFPE samples. The results suggest that FFPE samples may be a reliable alternative to fresh frozen samples for whole transcriptome profiling of adrenocortical tumors.

Comparison of vascular endothelial growth factor/vascular endothelial growth factor receptor 2 expression and its relationship to tumor cell proliferation in canine epithelial and mesenchymal tumors.

The vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) signaling pathway plays an important role in tumor angiogenesis. VEGFR2 is expressed not only in vascular endothelial cells but also in tumor cells; however, the relationship of VEGF/VEGFR2 expression and tumor proliferation has yet to be elucidated. In addition, since several studies have reported that VEGFR2 inhibitors are more effective against epithelial tumors than mesenchymal tumors, there may be a difference in VEGF/VEGFR2 expression between epithelial and mesenchymal tumors. The purpose of this study was to elucidate differences in VEGF/VEGFR2 expression between epithelial and mesenchymal tumors and the relationship of VEGF/VEGFR2 expression and proliferation in canine tumor cells. We assessed 29 epithelial and 21 mesenchymal canine tumors for microvessel density (MVD), mRNA transcription levels of von Willebrand Factor (vWF) and endoglin, expression of VEGF, VEGFR2, and phosphorylated VEGFR2 (pVEGFR2), and proliferation index (PI) using real-time reverse transcription polymerase chain reaction and immunohistochemistry. VEGFR2 expression on vascular endothelial cells, MVD, and mRNA transcription levels of vWF and endoglin were not significantly different between the two groups. However, expression of VEGF, VEGFR2, and pVEGFR2 was higher in epithelial tumors (P<0.01). Moreover, PI correlated with pVEGFR2 expression in only epithelial tumors (P<0.01, Rs=0.543). These results suggest that the activity of VEGF/VEGFR2 signaling in tumor cells is raised in epithelial tumors, and that this signaling pathway may be related to tumor cell proliferation in epithelial tumors.

Three cases of canine babesiosis caused by Babesia odocoilei-like parasites in Japan.

Babesia odocoilei-like parasites were first reported in 2003, and their virulence and hosts remain unknown. We report three cases of dogs with canine babesiosis in Iwate Prefecture. Since Iwate Prefecture area is an area of Japan where canine babesiosis is not endemic, we suspected that these cases of canine babesiosis were caused by B. odocoilei-like parasites. In the present study, we tried to identify the Babesia species that caused these cases of canine babesiosis. To classify Babesia parasites, the heat shock protein 70 (HSP70) gene was examined. Accordingly, we cloned and analyzed the HSP70 gene sequences of B. odocoilei-like parasites from three Ixodes ovatus ticks. It was determined that the nucleotide sequence of the HSP70 gene of the B. odocoilei-like parasites was not consistent with that of B. odocoilei, which suggests that these parasites were from a different species than B. odocoilei. Second, we identified the Babesia species that infected the three dogs by using the HSP70 gene and 18S rRNA. A partial HSP70 gene of B. odocoilei-like parasites was detected in the three dogs, but that of B. gibsoni was not detected. Additionally, a partial sequence of 18S rRNA of B. odocoilei-like parasites was detected in two dogs. These results demonstrated that two dogs were certainly infected with B. odocoilei-like parasites and that one dog was probably infected with B. odocoilei-like parasites. Therefore, these dogs were diagnosed with canine babesiosis due to the presence of B. odocoilei-like parasites. As there were only three cases, additional cases are needed to confirm our findings.

Substantial anti-gout effect conferred by common and rare dysfunctional variants of URAT1/SLC22A12.

OBJECTIVES: Gout, caused by chronic elevation of serum uric acid levels, is the commonest form of inflammatory arthritis. The causative effect of common and rare variants of ATP-binding cassette transporter G2 (ABCG2/BCRP) on gout risk has been studied, but little attention has been paid to the effect of common (rs121907892, p.W258X) and rare variants of urate transporter 1 (URAT1/SLC22A12) on gout, despite dysfunctional variants of URAT1 having been identified as pathophysiological causes of renal hypouricaemia. METHODS: To address this important but overlooked issue, we investigated the effects of these URAT1 variants on gout susceptibility, using targeted exon sequencing on 480 clinically defined gout cases and 480 controls of Japanese males in combination with a series of functional analyses of newly identified URAT1 variants. RESULTS: Our results show that both common and rare dysfunctional variants of URAT1 markedly decrease the risk of gout (OR 0.0338, reciprocal OR 29.6, P = 7.66 × 10-8). Interestingly, we also found that the URAT1-related protective effect on gout eclipsed the ABCG2-related causative effect (OR 2.30-3.32). Our findings reveal only one dysfunctional variant of URAT1 to have a substantial anti-gout effect, even in the presence of causative variants of ABCG2, a 'gout gene'. CONCLUSION: Our findings provide a better understanding of gout/hyperuricaemia and its aetiology that is highly relevant to personalized health care. The substantial anti-gout effect of common and rare variants of URAT1 identified in the present study support the genetic concept of a 'Common Disease, Multiple Common and Rare Variant' model.

Repeatable and objective method for evaluating angiogenesis using real-time RT-PCR of endoglin expression in canine tumours.

Anti-angiogenic therapy is a cancer treatment strategy targeting new blood vessel formation. Microvessel density (MVD) is a histopathological method for evaluating angiogenesis and endoglin is used as an activated endothelial marker in human medicine. The assessment of the treatment effect using MVD is difficult because it is a non-repeatable method. To develop a repeatable method for evaluating angiogenesis, we investigated correlations among MVD, mRNA transcription levels of endothelial markers and angiogenesis factors, and confirmed the agreement of mRNA transcription levels between tissue samples and small samples obtained by fine needle aspiration (FNA). The various types of spontaneous tumours were collected from 51 dogs. MVD was assessed by immunostaining for von Willebrand factor (vWF). mRNA transcription levels of vWF, endoglin, vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR2) were analysed using real-time reverse transcription polymerase chain reaction (real-time RT-PCR). There were significant correlations between MVD and mRNA transcription levels of vWF, endoglin and VEGFR2. VEGFR2 was more strongly correlated with endoglin (P <.01, Rs = 0.649) than vWF (P <.01, Rs = 0.512), indicating that angiogenesis can be evaluated more accurately by the measurement of mRNA transcription levels of endoglin. The mRNA transcription levels in tissue and FNA samples were strongly correlated, suggesting that evaluating angiogenesis using FNA samples is possible. In conclusion, we developed a repeatable and objective method for angiogenesis evaluation using mRNA transcription levels of endothelial markers by FNA sampling.

Successful Outcome in an Adult Patient with Influenza-associated Hemorrhagic Shock and Encephalopathy Syndrome.

A 50-year-old woman presented with coma and hemorrhagic shock. A rapid influenza antigen test revealed influenza A infection; other laboratory examinations ruled out any other suspected infections. She was diagnosed with hemorrhagic shock and encephalopathy syndrome (HSES) induced by influenza A. She was administered methylprednisolone pulse therapy and peramivir. Subsequently, she was discharged without any sequelae. Only a few cases of influenza-induced HSES have been reported, and the clinical outcomes were very poor. We herein report a successfully treated adult case of influenza-induced HSES and review this rare syndrome.

Case Report: Disseminated Mycobacterium kansasii Disease in a Patient with Anti-Interferon-Gamma Antibody.

Disseminated nontuberculous mycobacterial (NTM) infections usually occur in severely immunosuppressed patients. These infections may also occur in previously immunocompetent patients with acquired anti-interferon-gamma antibodies (anti-IFN-γ Abs). A previously healthy 33-year-old man presented with a 3-week history of cough and fever. Chest computed tomography showed air-space consolidation in the middle lobe of the right lung and enlargement of the supraclavicular, mediastinal, and hilar lymph nodes. Tissue samples obtained via mediastinoscopy showed granuloma formation with acid-fast bacteria; cultures from the tissue revealed Mycobacterium kansasii. Accordingly, a diagnosis of disseminated M. kansasii disease was made. The positive control tested negative in the QuantiFERON-TB Gold In-tube test, suggesting the presence of anti-IFN-γ Abs. The ELISA test for anti-IFN-γ Abs demonstrated an increased titer. Antimycobacterial drug treatments were initiated after diagnosis. His symptoms improved over 2 months, and he remains well on outpatient management. Disseminated M. kansasii disease is a very rare condition suggestive of immunosuppression. Testing for anti-IFN-γ antibodies might be important in all cases of disseminated M. kansasii disease.

Retrospective study on intercurrent pancreatitis with Babesia gibsoni infection in dogs.

Babesia rossi infection has been reported to be associated with the high prevalence of pancreatitis in dogs. In this study, we retrospectively investigated whether pancreatitis occurs in B. gibsoni-infected dogs. The clinical manifestations, and hematological and serum biochemical examination results, including canine pancreatic-specific lipase (cPL), in 20 B. gibsoni-infected dogs were analyzed. The cPL concentration exceeded 400 µg/l in only 2 dogs, and they were suspected of having pancreatitis. Although the cPL concentration did not correlate with the degree of anemia or the level of parasitemia, it correlated with the band neutrophil count, platelet count, and blood urea nitrogen (BUN) level. Our study suggested that the prevalence of pancreatitis is lower among B. gibsoni-infected dogs than B. rossi-infected dogs.

Retreatment With Anti-PD-L1 Antibody in Advanced Non-small Cell Lung Cancer Previously Treated With Anti-PD-1 Antibodies.

AIM: To evaluate the efficacy and safety of re-treatment with anti-programmed death (PD)-L1 antibody (atezolizumab) after anti-PD-1 antibody (nivolumab/pembrolizumab) treatment in advanced non-small cell lung cancer (NSCLC) patients. PATIENTS AND METHODS: We retrospectively reviewed 18 NSCLC patients who received atezolizumab after anti-PD-1 antibody treatment. Data on patient characteristics, number of cycles of anti-PD-1 antibody and atezolizumab, regimens between anti-PD-1 antibody and atezolizumab, best response, and immune-related adverse events (irAEs) were collected and analyzed. RESULTS: Nine patients a had high (≥50%) PD-L1 expression. The median number of cycles of atezolizumab was 3 (range=2-7). The median progression-free survival was 2.9±1.8 months. Seven (38.9%) and 11 (61.1%) patients had stable and progressive disease, respectively. No patient achieved partial or complete response. There were no significant differences in the occurrence of irAEs between anti-PD-1 antibodies and atezolizumab. CONCLUSION: Preliminary results showed that patients previously treated with anti PD-1 antibodies received only limited benefit from subsequent atezolizumab.

Epidemiological evaluation of cats associated with feline polycystic kidney disease caused by the feline PKD1 genetic mutation in Japan.

Feline polycystic kidney disease (PKD), an inherited autosomal dominant disease, has been reported to occur mostly in Persian or Persian related cats, and to be associated with a mutation from C to A at position 10063 in exon 29 of the feline PKD1 gene (PKD1 mutation). Many clinical cases have been recognized in Japan, but the mutation rate in cats has not been reported. The objective of this study was to determine epidemiological characteristics and clinical features in cats with the PKD1 mutation. Referring veterinarians sent blood samples of 377 cats for the PKD1 gene evaluation. The blood samples were from 159 cats with renal cysts confirmed by ultrasonography, 60 cats without renal cysts, and 158 cats that did not undergo ultrasonography. In total, 150 cats carried the PKD1 mutation and the signalment, site and number of renal cysts, and results of blood test were evaluated in cats with the PKD1 mutation. The breeds with the highest rate of the PKD1 mutation were Persian (46%), Scottish Fold (54%) and American Shorthair cats (47%). However, mixed breed cats also showed high rates of the PKD1 mutation. Of cats with the mutation, the incidence of high plasma creatinine (≥1.6 mg/dl) was greater in cats ≥3 years old, although a few cats ≥9 years of age had low plasma creatinine (<1.6 mg/dl). The coincidence of renal and hepatic cysts was 12.6%, with the high prevalence in Persian cats (31%).

Suspected eccrine adenocarcinoma on footpad of the right hindlimb in a dog.

A 12-year-old, male miniature dachshund has an ulcer on the footpad of the right hind limb. Despite treatment for longer than 6 months, the ulcer did not heal. Biopsy of the lesion was done to make a definitive diagnosis. Histologically, there were lumens containing weakly eosinophilic fluid surrounded by tumor cells with a similar circular pale nucleus and distinct nucleoli that showed some variation in size. Immunohistochemically, the tumor cells were positive for cytokeratin (AE1/AE3) and vimentin, were negative for S100 and p63. A poorly differentiated eccrine adenocarcinoma was diagnosed. Treatment was started with toceranib, an anti-angiogenic agent, and enlargement of the lesion was not observed during the administration period.

The clinical benefits of immune checkpoint inhibitor for thymic carcinomas ∼experience of single public hospital in Japan∼.

Thymic carcinomas is rare and highly aggressive carcinoma. Most patients with them are diagnosed as being at surgically unresectable stages due to it. There are several reports which showed the effect of chemotherapy, however, it is controversial. Recently, immune checkpoint inhibitors have changed conventional chemotherapy due to their effect against various types of cancers. We administered nivolumab, anti-Programmed Cell Death (PD)-1 antibody, to four patients with unresectable thymic carcinomas who had previously undergone conventional chemotherapy. A histopathology on tumors from these patients revealed the presence of squamous cell carcinoma and PD-L1 high expression. After treatment with nivolumab, it seemed to be beneficial to all patients; The best clinical responses of 3 patients were partial response and that of the other one was stable disease. None of them experienced severe immune-related adverse events. Our results suggest the potential benefits of using these inhibitors to treat thymic carcinomas in real world clinical setting as is the cases in recent clinical trials for the evaluation of immune checkpoint inhibitors for the treatment of thymic carcinoma.

Nivolumab-induced severe acute kidney injury with a long latent phase in a patient with non-small-cell lung cancer: A case report.

In the treatment of immunotherapy with immune checkpoint inhibitors, we often experience immune-related adverse event which manifest most frequently as a skin disorder, and very rarely as a renal disorder. In our manuscript, we report the case of a 71-year-old man with nivolumab-induced severe acute kidney injury (AKI) in which the time from treatment initiation to the onset of AKI was the longest among the previously reported cases (377 days). Prolonged follow-up is therefore warranted to detect late-onset AKI.