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.

Predicting difficulty in extending the ileal pouch to the anus in restorative proctocolectomy: investigation of a simple predictive method using computed tomography.

AIM: This study aimed to assess the ability of preoperative axial computed tomography (CT) to predict surgical difficulty in bringing the ileal pouch to the level of the anus during restorative proctocolectomy (RPC). METHOD: Patients who underwent RPC with an ileal pouch-anal anastomosis (or ileal pouch-anal canal anastomosis) in our institution between January 2008 and April 2014 were enrolled. The patients were divided into two groups, including those in whom CT indicated potential difficulty in extending the pouch downwards (extension difficult (ED) group) and patients with no CT evidence of potential difficulty (normal group). The groups were compared for clinical factors and the thickness of the slices of CT showing the root of the superior mesenteric artery, the point of communication of the ileocaecal artery with the marginal artery (tICA) and the anal verge (AV). Receiver-operating characteristic analysis was performed, and a cut-off value was calculated for predicting the degree of difficulty in bringing the ileal pouch down to the anal canal. RESULTS: Thirty-four patients were entered in the study. The ED group included significantly taller patients and more with familial adenomatous polyposis than the normal group. The distance between tICA and AV was significantly longer in the ED group, with a cut-off of 21 cm giving a sensitivity of 100% and a specificity of 83.3%. CONCLUSION: The distance between tICA and AV measured by axial CT can be a useful predictor for the difficulty in bringing the ileal pouch down to the anus during RPC.

An internally and externally validated nomogram for predicting the risk of irinotecan-induced severe neutropenia in advanced colorectal cancer patients.

BACKGROUND: In Asians, the risk of irinotecan-induced severe toxicities is related in part to UGT1A1*6 (UGT, UDP glucuronosyltransferase) and UGT1A1*28, variant alleles that reduce the elimination of SN-38, the active metabolite of irinotecan. We prospectively studied the relation between the UGT1A1 genotype and the safety of irinotecan-based regimens in Japanese patients with advanced colorectal cancer, and then constructed a nomogram for predicting the risk of severe neutropenia in the first treatment cycle. METHODS: Safety data were obtained from 1312 patients monitored during the first 3 cycles of irinotecan-based regimen in a prospective observational study. In development of the nomogram, multivariable logistic regression analysis was used to test the associations of candidate factors to severe neutropenia in the first cycle. The final nomogram based on the results of multivariable analysis was constructed and validated internally using a bootstrapping technique and externally in an independent data set (n=350). RESULTS: The UGT1A1 genotype was confirmed to be associated with increased risks of irinotecan-induced grade 3 or 4 neutropenia and diarrhoea. The final nomogram included type of regimen, administered dose of irinotecan, gender, age, UGT1A1 genotype, Eastern Cooperative Oncology Group performance status, pre-treatment absolute neutrophil count, and total bilirubin level. The model was validated both internally (bootstrap-adjusted concordance index, 0.69) and externally (concordance index, 0.70). CONCLUSIONS: Our nomogram can be used before treatment to accurately predict the probability of irinotecan-induced severe neutropenia in the first cycle of therapy. Additional studies should evaluate the effect of nomogram-guided dosing on efficacy in patients receiving irinotecan.

The HLA-A*0201-restricted minor histocompatibility antigen HA-1H peptide can also be presented by another HLA-A2 subtype, A*0206.

HA-1(H) is one of the most attractive minor histocompatibility antigens (mHA) as a target for immunotherapy of hematopoietic malignancies, but HLA-A*0201 and HLA-B60 molecules capable of presenting HA-1(H)-derived peptides are less common in eastern Asian populations when compared with Caucasian populations. Therefore, an attempt was made to search for novel epitopes presented by HLA alleles other than those previously reported by generating CTL lines from patients undergoing HLA-identical, HA-1 disparate hematopoietic stem cell transplantation (hematopoietic SCT) by stimulation with a 29-mer HA-1(H) peptide spanning a central polymorphic histidine (His). Two CTL clones established were found to be restricted by HLA-A*0206, which is the second or third most common HLA-A2 subtype worldwide. Epitope mapping revealed that the clones recognized the same nonameric peptide as A*0201-restricted HA-1(H), VLHDDLLEA. This epitope was unexpected, since it does not contain any preferred anchor motifs for HLA-A*0206. However, an HLA peptide binding assay revealed stronger binding of this peptide to A*0206 than to A*0201. Interestingly, HLA-A*0206-restricted CTL clones could lyse both HLA-A*0206(+) and HLA-A*0201(+) targets (including leukemic blasts) that express HA-1(H) peptide endogenously, whereas an HLA-A*0201-restricted, HA-1(H)-specific CTL clone failed to lyse HLA-A*0206(+) targets. This finding will expand the patient population who can benefit from HA-1(H)-based immunotherapy.

Enhanced desensitization efficacy by liposomal conjugation of a specific antigen.

Since liposomes are known as strong adjuvants, we attempted to use liposomes in immunotherapy as adjuvants, and to achieve desensitization in pre-sensitized mice. At first, we sensitized mice with intraperitoneal injection of model antigen, 100 microg ovalbumin (OVA), with Alum and treated them with liposome composed of distearoylphosphatidylcholine (DSPC) and cholesterol (2:1 as a molar ratio), which was coupled with a small amount of OVA (10 microg OVA in 400 nmol DSPC and 200 nmol cholesterol-liposome was injected into 20 g mouse). It is well known that antigen-specific immunotherapy increases IgG blocking antibodies and decreases in IgE antibodies. The treatment with i.v. injection of OVA-liposome at days 8, 10, and 12 after sensitization strongly suppressed OVA-specific IgE production without affecting IgG level after the boost (100 microg OVA with Alum). Moreover, the treatment with high-density OVA-liposome (10 microg OVA in 80 nmol DSPC and 40 nmol cholesterol-liposome/20 g mouse) not only strongly suppressed IgE levels but also reduced IgG production after the boost of OVA-sensitized mice suggesting the importance of liposomal characteristic in desensitization immunotherapy. Next we reduced the dose of OVA-liposome and the desensitization effect was also observed at the dose of as low as 1 microg OVA on OVA-liposome/mouse. On the contrary, free OVA did not affect the production of both IgG and IgE levels. Biodistribution study indicated that OVA-liposome was highly accumulated in spleen of OVA-sensitized mice compared to control liposome at 3 h after i.v. injection. These results suggest that the liposomal OVA effectively interacts with and desensitizes immune cells, therefore, liposomes coupling with a certain antigen may be effective in allergy immunotherapy.

Hypertrophy of the extra-articular tendon of the long head of biceps correlates with the location and size of a rotator cuff tear.

AIMS: The aim of this study was to assess hypertrophy of the extra-articular tendon of the long head of biceps (LHB) in patients with a rotator cuff tear. PATIENTS AND METHODS: The study involved 638 shoulders in 334 patients (175 men, 159 women, mean age 62.6 years; 25 to 81) with unilateral symptomatic rotator cuff tears. The cross-sectional area (CSA) of the LHB tendon in the bicipital groove was measured pre-operatively in both shoulders using ultrasound. There were 154 asymptomatic rotator cuff tears in the contralateral shoulder. Comparisons were made between those with a symptomatic tear, an asymptomatic tear and those with no rotator cuff tear. In the affected shoulders, the CSAs were compared in relation to the location and size of the rotator cuff tear. RESULTS: The mean CSA was 21.0 mm2 (4 to 71) in those with a symptomatic rotator cuff tear, 19.9 mm2 (4 to 75) in those with an asymptomatic rotator cuff tear and 14.1 mm2 (5 to 43) in those with no rotator cuff tear. The mean CSA in patients with both symptomatic and asymptomatic rotator cuff tears was significantly larger than in those with no rotator cuff tear (p < 0.001). In the affected shoulders, there were significant differences between patients with more than a medium sized posterosuperior cuff tear and those with an antero-superior cuff tear. CONCLUSION: Regardless of the symptoms, there was significant hypertrophy of the extra-articular LHB tendon in patients with a rotator cuff tear. The values were significantly related to the size of the tear. Cite this article: Bone Joint J 2017;99-B:806-11.

Note: On the wavelength dependence of the intensity calibration factor of extreme ultraviolet spectrometer determined with profile measurement of bremsstrahlung continuum.

The absolute calibration factor of extreme ultraviolet spectroscopic instrument which has recently been determined from absolute radiation profile measurement of bremsstrahlung continuum has been investigated by comparing the calculated diffraction efficiency of grating. An overall tendency of the wavelength dependence of the calibration factor from 40 Å to 500 Å can be reproduced by that of the grating efficiency, especially the agreement between the measured calibration factor and the calculated grating efficiency has been found to be fairly good for the wavelength range 200 Å-500 Å.

Ultraviolet B-induced local immunosuppression of contact hypersensitivity is modulated by ultraviolet irradiation and hapten application.

The induction of contact hypersensitivity is suppressed when hapten is applied topically to an area irradiated by ultraviolet B (UVB). There is no standardized procedure to induce this local immunosuppression by UVB. We investigated the effects of the following factors on induction of dinitrofluorobenzene contact hypersensitivity in mice. UVB dose, divided UVB exposure, timing of sensitization after irradiation, hapten concentration, hapten volume (application area), sex, age, and simultaneous sensitization on UV-exposed and nonexposed skin. The suppression was enhanced by increasing the UVB dose. When 100 mJ/cm2 of UVB was irradiated, divided daily exposure (25 mJ x 4 d) was more suppressive than single exposure (100 mJ x 1 d). Sensitization 2 d after irradiation (100 mJ/cm2) induced suppression most effectively. When 25 microliters of dinitrofluorobenzene solution was applied to exposed skin, higher concentrations induced lower suppression. When the total dose of hapten was kept constant (92 micrograms), the application of lower concentrations to large areas (0.25%, 25 microliters) caused stronger suppression than higher concentrations (1%, 6.25 microliters) to small areas. Simultaneous sensitization on UV-exposed and nonexposed skin revealed less suppression than sensitization only on exposed skin. The suppression of contact hypersensitivity was significantly greater in young than in old mice. These results provide details that may be useful in designing studies involving immunosuppression by UVB radiation.

Phototoxicity and photoallergenicity of quinolones in guinea pigs.

Clinical reports indicate that the fluoroquinolone group of antibiotics can induce cutaneous photosensitivity reactions. In the present study, phototoxicity and photoallergenicity of quinolones including nalidixic acid (NA) norfloxacin (NFLX), ofloxacin (OFLX), enoxacin (ENX), ciprofloxacin (CPFX), lomefloxacin (LFLX), and tosufloxacin (TFLX) were experimentally examined in an in vivo system using the guinea pig. Phototoxicity of all quinolones tested was demonstrated after a single, oral administration of the drugs and subsequent exposure to long-wave ultraviolet (UVA) at a dose of 30 J/cm2. The phototoxic potencies were: ENX, LFLX > OFLX > NA, TFLX > NFLX, CPFX. Photoallergic reaction was also induced to LFLX and NA by pretreatment with cyclophosphamide, an immunoadjuvant. No cross-reactions in photoallergy were observed among quinolones. The photo-ingestion test was positive in photoallergically sensitized animals, while the photopatch test was negative. This is the first report which demonstrated experimentally the photoallergenicity of quinolones. Clinical features of the photosensitivity due to quinolones can be explained by the results of the present experiments.

The morphologic characteristics of dry skin in atopic dermatitis.

Uninvolved skin sites in 436 consecutive patients, 6 to 25 years old, with atopic dermatitis were observed during the winter months (from November to February). Ichthyosis vulgaris occurred in 133 patients. Of the 303 remaining patients, only 11 (4%) had generalized dry skin; 191 (63%) exhibited focal areas of dry skin; and 95 (33%) showed only normal-appearing skin. Microscopically, in 41 patients, dry skin associated with atopic dermatitis showed mild eczematous changes. Dry skin coexistent with ichthyosis in patients with atopic dermatitis revealed ichthyotic changes frequently superimposed on eczematous changes. We suggest that in patients with atopic dermatitis the presence of dry skin may reflect mild eczematous changes, a manifestation of concomitant ichthyosis, or a complex of both of these changes.

Diminished contact sensitivity response in vitiliginous skin.

Thirty patients with vitiligo (ten of the segmental type and 20 of the generalized type) were sensitized with dinitrochlorobenzene (DNCB) in a normal skin site on the upper medial aspect of the arm. Challenge tests with dinitrochlorobenzene were performed in vitiliginous patches and in normal skin sites. In vitiliginous patches diminished contact sensitivity reactions to dinitrochlorobenzene were noted in both patient groups, while in normal skin sites a normal delayed hypersensitivity response to the same antigen developed in the same patients. Tuberculin reactivity was not suppressed in vitiliginous lesions. We suggest that diminished contact reactivity in vitiliginous skin might be due to functional changes in Langerhans' cells, or to an alteration of carrier (skin) proteins in the lesions.

Bullous pemphigoid and antinuclear antibodies. Unmasking of basement membrane fluorescence in sodium chloride-treated substrates.

Immunoflourescent (IF) microscopic studies were done on a patient with bullous pemphigoid who had a high titer of antinuclear antibodies (ANAs). Direct IF microscopy showed deposition of lgG and C3 at the basement membrane zone (BMZ), while, by indirect IF microscopy, ANAs (but not circulating pemphigoid antibodies) were detected. The 2M sodium chloride (NaCl) solution-treated substrates, in which nuclear antigens were eluted, uncovered pemphigoid antibodies. Treatment of the substrates with NaCl solution unmasked BMZ fluorescence in another set of experiments in which a large amount of serum samples containing ANAs was added to serum samples containing only pemphigoid antibodies. These results suggest that pemphigoid antibodies are able to react to the BMZ in the presence of concomitant ANAs unless bind to the nuclear antigens.

T-cell subsets in drug-induced toxic epidermal necrolysis. Possible pathogenic mechanism induced by CD8-positive T cells.

A patient with bromisovalum-induced toxic epidermal necrolysis showed pronounced delayed hypersensitivity to bromisovalum by patch testing. Biopsy specimens from the cutaneous lesion and the site of the positive patch test reaction were analyzed and compared immunohistologically. The findings were similar: most of the mononuclear cells disposed along the dermoepidermal junction and migrating into the epidermis were CD8-positive lymphocytes, whereas the dermal inflammatory infiltrates were composed predominantly of CD4-positive lymphocytes. This case showed the potential usefulness of patch testing in evaluating cases of toxic epidermal necrolysis. We believe that delayed hypersensitivity plays a crucial role in the development of drug-induced toxic epidermal necrolysis. Furthermore, potential effector cells with phenotypic characteristics of CD8-positive lymphocytes (suppressor/cytotoxic T cells) seem to represent important mediators of the epidermal damage of the cutaneous lesion in our case.

Internal carotid artery aneurysm visualized during successful endovascular treatment of carotid embolism.

We herein present a case of an internal carotid artery embolism associated with a hidden internal carotid artery aneurysm. The aneurysm was visualized during successful endovascular treatment of the carotid embolism. In retrospect, the aneurysm was at risk of rupture during the procedure. In the endovascular treatment of cerebral embolism, the possibility that aneurysms are hidden by emboli must be borne in mind. Care should be taken not to injure unidentified arterial walls while advancing a catheter blindly.

Application of high energy shock waves to cancer treatment in combination with cisplatin and ATX-70.

BACKGROUND: The anti-tumor effects of high-energy shock waves (HESW) in combination with cisplatin (CDDP) and ATX-70, as a photosensitizer, were investigated in vitro and in vivo. METHODS: Active oxygen generation by HESW was studied by means of electron spin resonance (ESR) system. The anti-tumor effects of HESW in combination with cisplatin (CDDP) and ATX-70 was investigated in vitro and in vivo. RESULTS: HESW exposure induced singlet oxygen or hydroxyl radical. Adding ATX-70 enhanced the cell damage induced by HESW dose-dependently. Ascorbic acid inhibited the combined effect of HESW and ATX-70. Adding CDDP did not enhanced cell damage by HESW. The combined therapy of HESW, CDDP and ATX-70 showed further inhibition of tumor growth in vivo. CONCLUSIONS: These results indicate that HESW activated ATX-70, and that the anti-tumor effect of HESW and ATX-70 was caused by active oxygen generation. HESW is able to be utilized with any other cancer treatment.

Evolutionary changes of immunohistological characteristics of secondary lesions in pityriasis rosea.

In 15 patients with pityriasis rosea, we studied the evolutionary changes of the immunohistological characteristics of the secondary lesions. Many CD1a+ cells were seen in the epidermis and dermis of early lesions. In the well-developed lesions, the number of CD1a+ cells greatly increased in the dermis. In the late lesions, CD1a+ cells in the dermis significantly decreased as compared with the well-developed lesions. Early lesions showed a moderate T-cell infiltrate. In the well-developed lesions, the dermal T-cell infiltrate was dense, and the CD4 CD8 ratio was 2.9. The late lesions had a moderate T-cell infiltrate, in which the CD4 CD8 ratio significantly decreased as compared with the well-developed lesions. Thus, the relative decrease in CD4+ helper inducer cells during lesion regression, concomitant with a decrease in number of CD1a+ Langerhans cells, is in accordance with a broader concept of increased suppressor mechanisms during healing.

The distribution of IgG subclass autoantibodies in bullous pemphigoid analysed by immunofluorescence and immunoblotting.

The distribution of IgG subclasses in bullous pemphigoid (BP) autoantibodies in 14 BP sera and four biopsies was analysed by immunofluorescence (IF) and immunoblotting (IB). Three clones of monoclonal antibodies (MoAbs) to each IgG subclass were used. All 14 sera showed linear fluorescence in the basement membrane zone with IF, and 240 kDa and/or 180 kDa protein bands in human epidermal extract were detected by IB using a polyclonal antibody to total IgG. BP antibody in IgG4 subclass was found to be predominant, as it was detected most frequently and intensively in all positive sera and lesions studied by both techniques. In the IgG1 to IgG3 subclasses, a range of proportions of positive sera was obtained among MoAbs to the same IgG subclass in both techniques. However, one MoAb could detect IgG1 subclass BP antibody with a high frequency in both techniques. No difference in IgG subclass distribution of BP antibodies was observed during the course of the disease. In each serum, any IgG subclass of BP antibody recognized the identical BP antigen(s). These results suggest the predominance of IgG4 subclass and the possible presence of IgG1 subclass in BP antibodies.

Evidence on N-acetyltransferase allele-associated metabolism of hydrazine in Japanese workers.

Hydrazine (N2H4), which has been categorized as a weak carcinogen, is a chemical with the one of the largest production rates in Japan. We have investigated the effects of acetylation phenotypes on the metabolism of hydrazine. Genotypes of N-acetyl transferases, NAT2*, were determined using polymerase chain reaction for 297 male workers. Biological and exposure monitoring were also conducted. The rapid and intermediate acetylators accounted for 45% each, and the slow acetylators accounted for 10%. Biological half-lives were significantly different among the three acetylation phenotypes (analysis of variance, P < 0.05): 3.94+/-1.70 hours for slow acetylators, 2.25+/-0.37 hours for intermediate acetylators, and 1.86+/-0.67 hours for rapid acetylators. Among Japanese, rapid and intermediate acetylators are the major phenotypes, which is in sharp contrast with those among Caucasians. We conclude that biological monitoring should take genetic factors, which may vary dramatically among different populations, into account.

Noonan phenotype associated with intracerebral hemorrhage and cerebral vascular anomalies: case report.

A case of a 19-year-old man with multiple characteristics of the Noonan phenotype and a massive intracerebral hemorrhage in the left putaminal region is presented. The hemorrhage was removed surgically, and the patient made a good recovery and was finally able to walk unassisted. Postoperative cerebral angiograms revealed various vascular anomalies, including a small aneurysm. To the best of our knowledge, this is the second detailed case report of an association of the Noonan phenotype with cerebral vascular anomalies presenting intracerebral hemorrhage. The etiology of this syndrome remains unknown but the possible causes of this rare association are discussed.

Simultaneous determination of bromvalerylurea, bromodiethylacetylurea, and allylisopropylacetylurea in serum and urine by high-performance liquid chromatography with a multiwavelength UV detector and thin-layer chromatography.

A method for rapid detection and identification of bromvalerylurea (BVU), bromodiethylacetylurea (BDU), and allylisopropylacetylurea (AIU) in serum and urine by high-performance liquid chromatography (HPLC) with a multiwavelength UV detector after Sep-Pak C18 cartridge extraction is reported. A Jasco Finepak C18 reversed-phase column was used for the separation. Acetonitrile-distilled water (1:1, v/v) was used as a mobile phase. There was no significant absorption of the three hypnotics in the UV spectra (210-350 nm). However, the absorption of each was higher at the shorter wavelengths. The quantifications for the three hypnotics detected at 210 nm by the chromatogram were linear over the range 0.2-4 micrograms/mL and the detection limits of BVU, BDU, and AIU were 5, 10, and 10 ng as absolute amounts, respectively. The mean recovery yields of BVU, BDU, and AIU by Sep-Pak C18 cartridge extraction were 85.7 +/- 4.1, 98.6 +/- 2.2, and 95.1 +/- 3.5% (n = 5) in serum and 79.5 +/- 3.8, 95.7 +/- 1.8, and 93.0 +/- 4.2% (n = 5) in urine, respectively. An optimal system of thin-layer chromatography for the identification of the hypnotics is also discussed.