Absence of microsatellite instability in extramammary Paget's disease.

Background: Deficiency of DNA mismatch repair (MMR) induces microsatellite instability (MSI). Pembrolizumab, an antibody targeting PD-1 (an immune checkpoint inhibitor), is more effective against MMR-deficient tumours than against MMR-proficient tumours. The status of MMR is a useful biomarker for predicting the effectiveness of pembrolizumab administration. Although the status of MMR has attracted attention in skin tumours, there are few reports on MSI in extramammary Paget's disease (EMPD). Objectives: To evaluate the status of MMR in patients with EMPD. Materials & Methods: One hundred one patients with EMPD were included. MMR status of the genomic DNA of each subject was analysed using Promega panel (approved as a companion diagnostic agent for the administration of pembrolizumab). Results: MSI testing showed the occurrence rates of MSI-high (more than two markers are unstable), MSI-low (one marker is unstable) and MSS (all markers are stable) tumour tissues were 0% (0/101), 1.0% (1/101) and 99.0% (100/101), respectively. Conclusion: The status of MMR may not be useful for the potential therapeutic application of pembrolizumab.

The Warburg effect and tumour immune microenvironment in extramammary Paget's disease: overexpression of lactate dehydrogenase A correlates with immune resistance.

BACKGROUND: Extramammary Paget's disease (EMPD) is a rare malignant skin cancer. One of the hallmarks of cancers, including EMPD, is an enhancement of aerobic glycolysis, which is also known as the Warburg effect. In the last step of glycolysis, the enzyme lactate dehydrogenase A (LDHA) catalyzes the conversion of pyruvate to lactic acid, the accumulation of which contributes to the creation of an acidic tumour microenvironment. This in turn results in immunosuppression in various types of cancers. However, the contribution of these pathways has not been well-studied in EMPD. OBJECTIVE: To investigate the significance of the Warburg effect and its contribution to the tumour immune microenvironment in EMPD. METHODS: The mRNA expression levels of molecules involved in glycolysis and immune-related cytokines were examined by ddPCR. The number of immune cells was assessed by immunohistochemistry (IHC). RESULTS: The levels of two glycolytic enzymes, HK2 and LDHA, in tumour tissues were significantly increased compared to those in paired-normal tissues. IHC analyses revealed increased numbers of PD-L1+ , PD-1+ , CD163+ M2 macrophages, Iba1+ macrophages and Foxp3+ Tregs that were associated with high LDHA levels in EMPD. ddPCR demonstrated that multiple cytokines including IL-4, IL-6, IL-10, TGF-ß and CCL-2 were upregulated and associated with high LDHA levels in EMPD. Statistical analyses showed that IL-6 mRNA expression correlated with the number of CD163+ , Iba-1+ and Foxp3+ cells. CONCLUSION: The Warburg effect contributes to immunomodulation in the tumour microenvironment and further elucidation may lead to better understanding of the pathogenesis of EMPD.

Efferent connections of the anterior hypothalamic nucleus: a biocytin study in the cat.

The efferent connections of the anterior hypothalamic nucleus (AH) were examined using biocytin as anterograde tracer in the cat. The results provide several new findings in addition to confirming earlier observations. In the hypothalamus, the AH projections terminated mainly in the medial regions which are related to the defensive, reproductive and feeding behaviors, and autonomic functions. Moreover, we found dense patches of the AH terminals in the medial preoptic area and ventromedial hypothalamic nucleus, which suggests the existence of modular connections between sub-regions of each nucleus. In addition, the AH projected to regions which may be related to the emotional and autonomic responses, i.e., such regions in the amygdala, midline thalamus, septum, subthalamus, and midbrain. The data suggest that the AH may play an important role in the autonomic functions and behaviors between animals, and thus may play a key role in the defensive behavior elicited in the medial preoptic area and ventromedial hypothalamic nucleus.

Distribution and co-localization of nitric oxide synthase and argininosuccinate synthetase in the cat hypothalamus.

Argininosuccinate synthetase (ASS) and nitric oxide synthase (NOS) comprise part of the cyclic metabolic pathway to produce nitric oxide (NO). ASS is one of the arginine synthesis enzymes which synthesizes argininosuccinate from aspartate and citrulline, and NOS forms NO and citrulline from arginine. This study examines the localization of ASS and NOS in the cat hypothalamus using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and immunohistochemistry against ASS and NOS. NADPH-d positive and/or ASS-immunoreactive neurons were localized in the following areas: the anterior hypothalamic area, the anterior hypothalamic nucleus, the supraoptic nucleus, the suprachiasmatic nucleus, the periventricular complex, the paraventricular nucleus, the parvocellular nucleus, the lateral hypothalamic area, the dorsomedial hypothalamic nucleus, the dorsal hypothalamic area, the posterior hypothalamic area, and the supramammillary nucleus. NOS and ASS double-labeled neurons were found in the anterior hypothalamic area, the supraoptic nucleus, the central part of the paraventricular nucleus of the hypothalamus, the lateral hypothalamic area, ventral part of the parvocellular hypothalamic nucleus, the posterior hypothalamic area, and the supramammillary nucleus. Double-labeled neurons in the hypothalamus comprised 20.7-32.0% of ASS-immunoreactive neurons and 10.2-26.3% of NOS-immunoreactive neurons. The results suggest the existence of the 'NO cycle' in situ and the physiological importance of NO and argininosuccinate in several regions of the cat hypothalamus.

Analysis of properties of monoclonal antibodies to transmissible gastroenteritis virus using TO-163 strain.

Nineteen monoclonal antibodies (MAbs), reactive in enzyme-linked immunosorbent assay (ELISA), with porcine transmissible gastroenteritis (TGE) virus TO-163 were obtained. Of these MAbs, 5 showed neutralizing (NT) activity (x 3,200 to 25,600) against TO-163. One of the MAbs which had NT activity showed hemagglutination inhibition activity (x 5,120) too. 14 hybridomas of polypeptide specificity against TO-163 strain were developed from which 11, 2, and 1 were specific for protein E2, N, and E1, respectively. Immunofluorescence staining patterns in TGE virus-infected cells reacted with MAbs were divided into three groups (types I, II and III). The fluorescence staining of E2 specific MAbs having NT activity were limited to the perinuclear area. All MAbs having NT activity showed the same fluorescence staining pattern.