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.

Serum cell-free DNA levels are a useful marker for extramammary Paget disease.

BACKGROUND: Although carcinoembryonic antigen (CEA) and cytokeratin 19 fragment (CYFRA) are useful markers for extramammary Paget disease (EMPD), serum CEA and CYFRA levels are not elevated in most patients with EMPD without metastasis. Cell-free (cf)DNA has attracted attention as an indicator of clinical conditions in several cancers. OBJECTIVES: To identify further useful biomarkers for the detection of EMPD, including early lesions, and to study the clinical implications of cfDNA in EMPD. METHODS: cfDNA were isolated from serum of patients with EMPD with and without metastasis, and from healthy volunteers. Serum extracts were amplified using polymerase chain reaction. RESULTS: Serum cfDNA levels were significantly elevated in patients with EMPD with or without metastasis compared with those in healthy controls. Serum cfDNA was a better diagnostic marker for the presence of EMPD than serum CYFRA. Moreover, the postoperative serum cfDNA levels were significantly lower than those from the preoperative samples, and the change in serum cfDNA levels reflected the clinical courses of patients with EMPD treated with chemotherapy. CONCLUSIONS: Taking the evidence together, serum cfDNA levels may be a useful marker for diagnosis and disease progression in EMPD. What's already known about this topic? Serum levels of carcinoembryonic antigen (CEA) and cytokeratin 19 fragment (CYFRA) are not elevated in most patients with extramammary Paget disease (EMPD) without metastasis. Cell-free (cf)DNA has attracted attention as an indicator of clinical conditions in several cancers. There are few reports of the clinical implications of cfDNA in dermatology. What does this study add? Serum cfDNA levels were significantly elevated in patients with EMPD with or without metastasis compared with those in healthy controls. Postoperative serum cfDNA levels were significantly lower than those from the preoperative samples. Changes in serum cfDNA levels reflected the clinical courses of patients with EMPD treated with chemotherapy. What is the translational message? Serum cfDNA levels in patients with EMPD are a useful marker for the detection of EMPD, including localized EMPD. Changes in serum cfDNA levels in an individual patient may reflect the clinical course of EMPD.

Energy expenditure during walking in amputees after disarticulation of the hip. A microprocessor-controlled swing-phase control knee versus a mechanical-controlled stance-phase control knee.

We have compared the energy expenditure during walking in three patients, aged between 51 and 55 years, with unilateral disarticulation of the hip when using the mechanical-controlled stance-phase control knee (Otto Bock 3R15) and the microprocessor-controlled pneumatic swing-phase control knee (Intelligent Prosthesis, IP). All had an endoskeletal hip disarticulation prosthesis with an Otto Bock 7E7 hip and a single-axis foot. The energy expenditure was measured when walking at speeds of 30, 50, and 70 m/min. Two patients showed a decreased uptake of oxygen (energy expenditure per unit time, ml/kg/min) of between 10.3% and 39.6% when using the IP compared with the Otto Bock 3R15 at the same speeds. One did not show any significant difference in the uptake of oxygen at 30 m/min, but at 50 and 70 m/min, a decrease in uptake of between 10.5% and 11.6% was found when using the IP. The use of the IP decreased the energy expenditure of walking in these patients.

Fluence to effective dose conversion coefficients calculated for monoenergetic electrons up to 200 MeV in partial exposure geometries.

Organ doses and effective doses were calculated for monoenergetic electrons from 0.1 to 200 MeV using the EGS4 Monte Carlo simulation code and the MIRD-5 human phantom in various non-uniform exposure geometries: anterior-posterior (AP) and posterior-anterior (PA). Below 1 MeV, the skin is the main contributor to the effective dose conversion coefficients for each exposure geometry; however, above 1 MeV the calculations showed that the effective doses of partial exposures depended on the incident electron energy, the place and the size of the exposure on the body.

Treatment of chronic lymphocytic leukaemia in three dogs with melphalan and prednisolone.

Three adult dogs with chronic lymphocytic leukaemia (CLL) were successfully treated with melphalan and prednisolone. Based on the immunophenotypic analysis of leukaemic cells, two dogs were diagnosed with B cell CLL and one dog was tentatively diagnosed as having T cell CLL. One dog with B cell CLL had IgM monoclonal gammopathy. The clinical signs and haematological abnormalities associated with CLL in the three dogs improved with the administration of cytoreductive melphalan (3 to 5 mg/m2/day) and prednisolone (4.3 to 30 mg/m2/day) for eight to 210 days. There were no severe adverse effects except a mild increase in plasma alkaline phosphatase activity. Melphalan and prednisolone therapy may achieve remission with few side effects in dogs with CLL.

%VO2max as an indicator of prosthetic rehabilitation outcome after dysvascular amputation.

In this study, independent ambulation of at least 100 metres with/without a cane was regarded as successful prosthetic rehabilitation. The subjects were classified into two groups according to this criterion at the time of discharge. The successful group attained this performance, the other group failed to reach this level. The successful group included 8 unilateral trans-femoral amputees aged 72.2 +/- 2.1 years who underwent amputation at more than 70 years, and succeeded in walking with a prosthesis. The group which failed included 9 unilateral trans-femoral amputees aged 63.2 +/- 2.1 years who underwent amputation between the ages of 60-65 years, and had great difficulty in walking with a prosthesis. The purpose of this research was to investigate whether or not %VO2max as an indicator of physical fitness is useful in predicting prosthetic rehabilitation outcome after dysvascular amputation by comparing these two groups. Evaluation of physical fitness was conducted before the subjects began prosthetic rehabilitation. Information about each subject before fitting with a prosthesis was collected retrospectively from clinical charts made during admission. The successful group were capable of strenuous exercise, reaching the intensity of 50% VO2max or more. In the group which failed only one reached the intensity of 50% VO2max. The working capacity of 50% VO2max or greater would appear to be a valid initial guideline level of physical fitness at which an amputee can expect to succeed in walking with a prosthesis. Apart from physical fitness, a lesser number of comorbidity, good ability to stand on the remaining leg, and a strong will to walk were found to be important factors contributing to successful prosthetic rehabilitation. This study also showed that age alone was not an important factor.

The importance of tail temperature monitoring during tail-flick test in evaluating the antinociceptive action of volatile anesthetics.

BACKGROUND: Tail-flick (TF) latency can be influenced by tail-skin temperature (TT), and treatments that raise TT can mimic hyperalgesia on a TF test. As volatile anesthetics can raise TT via heat redistribution, their antinociceptive action can be hidden or obscured in a TF test. We tested the hypothesis that TT monitoring improves the efficiency of TF tests in evaluating the antinociceptive action of volatile anesthetics. METHODS: The relationship between TT and TF latency was first explored under varied TTs in 12 rats. Then, TT and TF latency were measured before and during isoflurane exposure (1.2%). In the low temperature group (n=6), rats were prewarmed mildly to increase TT during isoflurane exposure. In the high temperature group (n=6), rats were prewarmed enough to prevent a TT increase during isoflurane exposure. RESULTS: There was a highly significant correlation between TT and TF latency, that is, TF latency decreased as TT increased. In the low temperature group, there was a significant increase in TT during isoflurane exposure, while an increase in TF latency did not reach statistical significance. Tail-flick latency corrected by a change in TT showed a significant increase. In the high temperature group, TF latency increased significantly during isoflurane exposure without an increase in TT. CONCLUSIONS: Isoflurane inhalation can induce an increase in TT, which can obscure its antinociceptive action as evaluated by a TF test. Monitoring TT during a TF test is important to efficiently evaluate the antinociceptive action of volatile anesthetics.

Nitric oxide targets bronchiolar epithelial cells in murine cytomegalovirus-associated disease in lungs that are free of the virus.

At 4 weeks after intraperitoneal (i.p.) injection of 0.2LD50 (50% lethal dose) of murine cytomegalovirus (MCMV) in adult BALB/c mice, productive virus and the viral DNA were detected only in the salivary glands, but not in the lungs. A single i.p. injection of anti-CD3 mAb to these mice provoked pulmonary lesions, which included a thickening of the interstitium and peribronchiolar areas, accompanied with a cellular infiltration in those areas. As a result, half of the mice died. In a histochemical analysis with anti-nitrotyrosine polyclonal Ab, bronchiolar epithelial cells were stained with this Ab, thus demonstrating that peroxynitrite, which was biochemically derived from nitric oxide (NO), injured those cells. Similarly, when T cells of iNOS+/+ mice, which had been infected with MCMV 4 weeks before, were activated by a single injection of anti-CD3 mAb, 37.5% of the mice died. Nitrotyrosine was also detected in the bronchiolar epithelial cells in these mice. In contrast, none of MCMV-infected iNOS-/- mice died after the anti-CD3 injection. No pathological changes were noted in the histological findings of the lungs of those mice. An intranasal injection of bacterial superantigen, staphylococcal enterotoxin B (SEB), demonstrated the same histopathological changes in the lungs and mortality in BALB/c mice as those in mice i.p. injected with anti-CD3. Therefore, T-cell responsiveness to stimulation with anti-CD3 or a superantigen was presumably modified by MCMV infection. MCMV-associated pneumonitis in the present study was thus mediated not by a direct viral attack but by iNOS-derived NO, which can be induced by the cytokines from the T cells. In addition, it was demonstrated that the NO produced by the cytokine-mediated pathway targeted bronchiolar epithelial cells.

Effective doses and organ doses in the MIRD-5 phantom exposed to monoenergetic 0.1 MeV to 200 MeV electrons in the LAT direction.

Organ doses and effective doses were calculated using the EGS-4 Monte Carlo simulation code and a MIRD-5 mathematical human phantom placed in a vacuum. For broad right and left lateral beams of monoenergetic (0.1-200 MeV) electrons, conversion coefficients from the incident fluence to organ dose, to effective dose, and to effective dose equivalent were obtained. There were no clear differences between the conversion coefficients in the case of left-lateral (LLAT) and right-lateral (RLAT) irradiation. Therefore, when investigating lateral geometries for electron exposure, it is not necessary to evaluate both directions independently. In general, conversion coefficients for lateral irradiation (LAT) were smaller than those for AP and PA. The difference between the AP and PA conversion coefficients and LAT became smaller with increasing incident energy; at 200 MeV the conversion coefficients were almost independent of the irradiation geometry. The agreement between the results of the present study and those of other studies was acceptable within the statistical uncertainties.

Enkephalin release and opioid receptor activation does not mediate the antinociceptive or sedative/hypnotic effects of nitrous oxide.

In previous studies using Fos expression as a marker of neuronal activation, we showed that nitrous oxide (N(2)O) activates bulbospinal noradrenergic neurons in rats and that destruction of these neuronal pathways leads to loss of N(2)O antinociceptive action. Based on previous rat studies it has been proposed that these noradrenergic neurons are activated through opioid receptors through the release of endogenous opioid ligands in the periaqueductal gray. Using mice with a disrupted preproenkephalin gene (Penk2 -/-) and the opioid receptor antagonist naltrexone, we investigated the role of enkephalinergic mechanisms and opioid receptor activation in the behavioral and bulbospinal neuron responses to N(2)O in mice. The antinociceptive response to N(2)O was investigated using the tail-flick, hot-plate, and von Frey assays, the sedative/hypnotic response was measured using rotarod and loss of righting reflex, and bulbospinal neuronal activation was assessed with pontine Fos immunostaining. No differences were observed between wild-type and Penk2 -/- mice for the antinociceptive, sedative/hypnotic, and pontine neuronal activation effects of N(2)O. Similarly, naltrexone did not block N(2)O-induced antinociception, sedation, or hypnosis. We conclude that neither enkephalin nor opioid receptors participate in either the antinociceptive or the sedative/hypnotic actions of N(2)O in mice.

Black tea extract, thearubigin fraction, counteracts the effect of tetanus toxin in mice.

The aim of this study was to find an inactivating substance for tetanus toxin in natural foodstuff. Tetanus toxin (4 micrograms/ml) abolished indirect twitches in In vitro mouse phrenic nerve-diaphragm preparations within 2.5 hr. Hot water infusion of black tea mixed with tetanus toxin blocked the inhibitory effect of the toxin. Mixing the toxin with thearubigin fraction extracted from black tea infusion produced an identical result. Furthermore, thearubigin fraction mixed with the toxin protected against the in vivo paralytic effect of the toxin. Thearubigin fraction had no protective effect on other toxins, such as tetrodotoxin and saxitoxin. The specific binding of [125I]tetanus toxin to rat cerebrocortical synaptosomes was inhibited by mixing iodinated toxin with thearubigin fraction. These results imply that thearubigin fraction counteracts the effect of tetanus toxin by binding with toxin, and also suggest that this fraction may be able to apply for prophylaxis of tetanus.

Effect of endurance training program based on anaerobic threshold (AT) for lower limb amputees.

We have already reported that the one-leg cycling test driven by the subject's sound leg as the exercise load test is effective in measuring the anaerobic threshold (AT) of unilateral lower limb amputees. The aim of this research is to investigate whether or not endurance training based on each subject's AT gained from the one-leg cycling test is useful in improving the physical fitness of lower limb amputees. The test subjects were all unilateral transfemoral amputees comprising a group of 14 undertaking endurance training and a control group of 10. The form of endurance training is driving an ergometer with the sound limb only in the same way as the load test. The training program was designed so that the subjects would exercise at a target heart rate corresponding to AT point for 30 minutes per day, 3-5 days each week for 6 weeks. After the training periods, in the training subjects the AT and maximum oxygen uptake (__O2max) increased significantly. The rate of increase averaged 36.5%, 26.0%, respectively, compared to their levels before the training. On the contrary, no changes occurred in the control subjects. These results suggest that our chosen training program based on each subject's AT is effective in improving the physical fitness of lower limb amputees.

[Anesthetic management of a patient with gunshot injury in the neck and the chest].

We present a case of a 27-year-old man with gunshot injury in the neck and the chest. On admission, he had an entry wound in the neck and his chest radiograph showed left hemopneumothorax. Nasal endotracheal intubation and chest drainage were immediately performed. Angiography revealed pseudoaneurysm of the left carotid artery and fistula between the artery and the innominate vein. The patient showed progressive severe facial edema due to the fistula. Anesthesia was induced and maintained with fentanyl and sevoflurane. The carotid artery was repaired with an autologous saphenous vein graft. Although one lung ventilation (OLV) was requested for partial resection of the left lung, replacing the endotracheal tube was impossible due to severe facial edema. OLV was successfully performed by blocking the left main trunchus with a 7 Fr Fogarty catheter placed under fiberscopic monitoring. The patient recovered without any serious complications. Prompt and proper airway management is required in gun shot injury of the neck and chest.