Lymphangiogenesis and Lymphatic Zippering in Skin Associated with the Progression of Lymphedema.

Secondary lymphedema often develops after lymph node dissection or radiation therapy for cancer treatment, resulting in marked skin fibrosis and increased stiffness owing to insufficiency of the lymphatic system caused by abnormal structure and compromised function. However, little is known about the associated changes of the dermal lymphatic vessels. In this study, using the lower limb skin samples of patients with secondary lymphedema, classified as types 1-4 by lymphoscintigraphy, we first confirmed the presence of epidermal thickening and collagen accumulation in the dermis, closely associated with the progression of lymphedema. Three-dimensional characterization of lymphatic capillaries in skin revealed prominent lymphangiogenesis in types 1 and 2 lymphedema. In contrast, increased recruitment of smooth muscle cells accompanied by development of the basement membrane in lymphatic capillaries was observed in types 3 and 4 lymphedema. Remarkably, the junctions of dermal lymphatic capillaries were dramatically remodeled from a discontinuous button-like structure to a continuous zipper-like structure. This finding is consistent with previous findings in an infection-induced mouse model. Such junction tightening (zippering) could reduce fluid transport and cutaneous viral sequestration during the progression of lymphedema and might explain the aggravation of secondary lymphedema. These findings may be helpful in developing stage-dependent treatment of patients with lymphedema.

Pattern of Peri-Operative Antibiotic Use among Surgical Patients in a Regional Referral and Teaching Hospital in Uganda.

Background: Prolonged surgical antimicrobial prophylaxis (SAP) to prevent surgical site infection (SSI) is generally discouraged after completion of surgery. However, little is known about the pattern of peri-operative antibiotic use in resource-limited settings. We aimed to describe its use at a typical government hospital in Uganda. Methods: A study was originally conducted in a rural Ugandan regional referral and teaching hospital in 2014 and 2015 to improve hand hygiene practice and measure its impact on health-care-associated infections including SSI (WardGel study). This is a secondary analysis of the data from the WardGel study to assess the frequency of peri-operative antibiotic use among surgical patients. Results: Of 3,627 patients enrolled into the original study, 960 (26.5%) underwent surgery at the hospital and 907 patients (94.5%) received antibiotic agents during hospitalization. Of these, 880 patients (97.0%, of 907 patients) received antibiotic agents on the day of surgery. A combination of ceftriaxone and metronidazole was the most common regimen (609/907 patients, 67.1%). Thirty-six of 907 patients (4.0%) started and completed their antibiotic agents on the day of surgery. The mean length of antibiotic use during hospitalization was 3.5 days (standard deviation, 3.3). After adjusting for covariates, linear regression analysis showed an extra 1.9 days of antibiotic use post-operatively (95% confidence interval = 1.7-2.3). During the total 4,960 inpatient-days for those having surgery, there were 6,503 days of therapy (DOTs) of antibiotic agents and 1,649 antibiotic-free days (AFDs). Conclusions: Most patients received prolonged antibiotic therapy after surgery. Antimicrobial stewardship for SAP can play a major role in combating antimicrobial resistance in resource-limited settings.

Alcohol-based hand rub and incidence of healthcare associated infections in a rural regional referral and teaching hospital in Uganda ('WardGel' study).

Background: Good hand hygiene (HH) practice is crucial to reducing healthcare associated infections (HAIs). Use of alcohol-based hand rub (ABHR) at health facilities is strongly recommended but it is limited in Uganda. Data on the practice of HH and the incidence of HAIs is sparse in resource-limited settings. We conducted a quasi-experimental study to evaluate HH practices of health care providers (HCPs) utilizing locally made ABHR and the incidence of HAIs. Methods: HH compliance among HCPs and the incidence of HAIs were assessed at Mbale Regional Referral Hospital, a teaching hospital in rural Uganda. Inpatients from the obstetrics/gynecology (OBGYN), pediatric and surgical departments were enrolled on their day of admission and followed up during their hospital stay. The baseline (pre-intervention) phase of 12-weeks was followed by a 12-week intervention phase where training for HH practice was provided to all HCPs present on the target wards and ABHR was supplied on the wards. Incidence of HAIs and or Systemic Inflammatory Response Syndrome (SIRS) was measured and compared between the baseline and intervention phases. Multivariate survival analysis was performed to identify associated variables with HAIs/SIRS. Results: A total of 3335 patients (26.3%) were enrolled into the study from a total of 12,665 admissions on the study wards over a 24-week period. HH compliance rate significantly improved from 9.2% at baseline to 56.4% during the intervention phase (p < 0.001). The incidence of HAIs/SIRS was not significantly changed between the baseline and intervention phases (incidence rate ratio (IRR) 1.07, 95% CI: 0.79 - 1.44). However, subgroup analyses showed significant reduction in HAIs/SIRS on the pediatric and surgical departments (IRR 0.21 (95% CI: 0.10 - 0.47) and IRR 0.39 (95% CI: 0.16 - 0.92), respectively) while a significant increase in HAIs/SIRS was found on the OBGYN department (IRR 2.99 (95% CI: 1.92 - 4.66)). Multivariate survival analysis showed a significant reduction in HAIs/SIRS with ABHR use on pediatric and surgical departments (adjusted hazard ratio 0.26 (95% CI: 0.15 - 0.45)). Conclusions: To our knowledge, this study is one of the largest studies that address HAIs in Africa. During the 24-week study period, significant improvement in HH compliance was observed by providing training and ABHR. The intervention was associated with a significant reduction in HAIs/SIRS on the pediatric and surgical departments. Further research is warranted to integrate HAIs surveillance into routine practice and to identify measures to further prevent HAIs in resource limited settings. Trial registration: ClinicalTrials.gov NCT02435719, registered on 20 April, 2015 (retrospectively registered).

Development of novel in vitro photosafety assays focused on the Keap1-Nrf2-ARE pathway.

Although photoallergens require UV energy for antigen formation, the subsequent immune response is considered to be the same as in ordinary skin sensitization. Therefore, in vitro tests for skin sensitization should also be applicable for photoallergy testing. In this study, we examined whether activation of the Keap1 (Kelch-like ECH-associated protein 1)-Nrf2 (nuclear factor-erythroid 2-related factor 2)-ARE (antioxidant response element) pathway could be used to assess the photoallergenic potential of chemicals, using the reporter cell line AREc32 or KeratinoSens(TM) . First, we identified an appropriate UVA irradiation dose [5 J cm(-2) irradiation in phosphate-buffered saline (PBS)] by investigating the effect of UV irradiation on ARE-dependent gene induction using untreated or 6-methylcoumarin (6-MC)-treated cells. Irradiation of well-known photoallergens under this condition increased ARE-dependent gene expression by more than 50% compared with both vehicle and non-irradiated controls. When the cut-off value for detecting photoallergens was set at 50% induction, the accuracy of predicting photoallergenic/phototoxic chemicals was 70% in AREc32 cells and 67% in KeratinoSens(TM) cells, and the specificity was 100% in each case. We designate these assays as a photo-ARE assay and photo-KeratinoSens(TM) , respectively. Our results suggest that activation of the Keap1-Nrf2-ARE pathway is an effective biomarker for evaluating both photoallergenic and phototoxic potentials. Either of the above tests might be a useful component of a battery of in vitro tests/in silico methods for predicting the photoallergenicity and phototoxicity of chemicals. Copyright © 2015 John Wiley & Sons, Ltd.

Oncocytic Carcinoma of the Submandibular Gland: A Case Report.

Oncocytic carcinoma (OC) of the submandibular gland is extremely rare. A 76-year-old man complained of a painless tumor of the right neck. Ultrasonography demonstrated swelling in the lymph nodes of the neck, and fine-needle aspiration cytology of a node showed metastatic carcinoma with oncocytic features. Radical surgery revealed infiltrating carcinoma of the right submandibular gland with lymph node metastases (19/23). Tumor cells showed less atypia and had abundant eosinophilic cytoplasm, which stained deep blue with phosphotungstic acid-hematoxylin stain. The cells were immunohistochemically positive for cytokeratin 7 but negative for p63 and SOX10. We diagnosed the tumor as OC. Chemoradiotherapy was performed after surgery. The patient showed no sign of recurrence until 42 months after the operation, when lymph node swelling was detected in the mediastinum by computed tomography scanning. With no further treatment, the patient is alive with lymph node swellings in the mediastinum and pulmonary hilum 80 months after surgery.

In silico risk assessment for skin sensitization using artificial neural network analysis.

The sensitizing potential of chemicals is usually identified and characterized using in vivo methods such as the murine local lymph node assay (LLNA). Due to regulatory constraints and ethical concerns, alternatives to animal testing are needed to predict the skin sensitization potential of chemicals. For this purpose, an integrated evaluation system employing multiple in vitro and in silico parameters that reflect different aspects of the sensitization process seems promising. We previously reported that LLNA thresholds could be well predicted by using an artificial neural network (ANN) model, designated iSENS ver. 2 (integrating in vitro sensitization tests version 2), to analyze data obtained from in vitro tests focused on different aspects of skin sensitization. Here, we examined whether LLNA thresholds could be predicted by ANN using in silico-calculated descriptors of the three-dimensional structures of chemicals. We obtained a good correlation between predicted LLNA thresholds and reported values. Furthermore, combining the results of the in vitro (iSENS ver. 2) and in silico models reduced the number of chemicals for which the potency category was under-estimated. In conclusion, the ANN model using in silico parameters was shown to be have useful predictive performance. Further, our results indicate that the combination of this model with a predictive model using in vitro data represents a promising approach for integrated risk assessment of skin sensitization potential of chemicals.

Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays.

The sensitizing potential of chemicals is usually identified and characterized using in vivo methods such as the murine local lymph node assay (LLNA). Due to regulatory constraints and ethical concerns, alternatives to animal testing are needed to predict skin sensitization potential of chemicals. For this purpose, combined evaluation using multiple in vitro and in silico parameters that reflect different aspects of the sensitization process seems promising. We previously reported that LLNA thresholds could be well predicted by using an artificial neural network (ANN) model, designated iSENS ver.1 (integrating in vitro sensitization tests version 1), to analyze data obtained from two in vitro tests: the human Cell Line Activation Test (h-CLAT) and the SH test. Here, we present a more advanced ANN model, iSENS ver.2, which additionally utilizes the results of antioxidant response element (ARE) assay and the octanol-water partition coefficient (LogP, reflecting lipid solubility and skin absorption). We found a good correlation between predicted LLNA thresholds calculated by iSENS ver.2 and reported values. The predictive performance of iSENS ver.2 was superior to that of iSENS ver.1. We conclude that ANN analysis of data from multiple in vitro assays is a useful approach for risk assessment of chemicals for skin sensitization.

A novel technique for observing the internal ultrastructure of human chromosomes with known karyotype.

Observation of the internal ultrastructure of human chromosomes by transmission electron microscopy (TEM) has frequently been attempted in spite of the difficulties in detaching metaphase chromosome spreads from the glass slide for further processing. In this study we have used a method in which metaphase chromosome spreads were prepared on a flexible thermoplastic membrane (ACLAR) film. To assess chromosome identity, a diamidino-phenylindole staining and karyotying was first done using a conventional cytogenetic system. The chromosome spreads were then fixed with 1% osmium tetroxide, stained with freshly prepared 2% tannic acid, dehydrated, and flat-embedded in epoxy resin. The resin sheet was easily detachable and carried whole chromosome spreads. By this method, TEM observation of chromosomes from normal human lymphocytes allowed a thorough examination of the ultrastructure of centromeres, telomeres, fragile sites, and other chromosomal regions. Various ultrastructural patterns including thick electron dense boundaries, less dense internal regions, and extended chromatin loops at the periphery of the chromosomes were discernible. Application of the present method to chromosome research is expected to provide comprehensive information on the internal ultrastructure of different chromosomal regions in relation to function.

Regioselectivity in benzotropolone formation between catechins and proanthocyanidins.

A theaflavin-related benzotropolone pigment formed between procyanidin B-1 and (-)-epigallocatechin was synthesized enzymatically for the first time. The condensation occurred regioselectively at the extension (upper) unit of the procyanidin, probably due to a steric effect.

Occurrence of hyaline droplets in renal biopsy specimens: an ultrastructural study.

Hyaline droplets are apical cytoplasmic vesicles containing an accumulation of electron-dense amorphous materials surrounded by a unit membrane. Hyaline droplets may originate from apical vesicles after conversion to osmotic vesicles and loss of internally lined glycocalyx. They are found in the proximal tubular epithelium in biopsies from patients with renal diseases; however, their biological importance is not well understood. We reviewed ultrastructural pathology records of 140 renal biopsy patients to determine the occurrence and relevance of hyaline droplets. Of the cases, 14 (10%) showed the presence of hyaline droplets in proximal tubular epithelium. The distribution of cases were 8 of the 19 (42%) with minimal change nephritic syndrome, 2 of the 37 (5%) with IgA nephropathy, 2 of the 4 (50%) with membranous glomerulonephropathy, 1 of the 4 (25%) with tubulointerstitial nephritis, and 1 of the 1 (100%) with acute renal failure. The droplets were frequently found in male patients (86%), never in children, and were mostly associated with tubular necrosis (8 of 14 cases; 56%). Many hyaline droplets were observed in the cytoplasm of necrotic proximal tubular epithelial cells, and even when tubular necrosis was not evident, the proximal tubular epithelial cells containing hyaline droplets showed degenerated microvilli and decreased basal interdigitations. These results suggest that hyaline droplets could be one marker of renal tubular necrosis and a sign of functional disorder of protein reabsorption by degenerating proximal tubular epithelium.

NMR characterization of three-disulfide variants of lysozyme, C64A/C80A, C76A/C94A, and C30A/C115A--a marginally stable state in folded proteins.

Our earlier NMR study showed that a two-disulfide variant of hen lysozyme containing intra-alpha-domain disulfide bridges, C6-C127 and C30-C115, is partially folded, with the alpha domain tightly folded to the nativelike conformation and the beta domain flexible or unfolded. With a view that the formation of a third disulfide bridge is a key for the accomplishment of the overall chain fold, three-dimensional structures of three-disulfide variants of hen lysozyme lacking one disulfide bridge (C64A/C80A, C76A/C94A, and C30A/C115A) were studied in detail using NMR spectroscopy. Amide hydrogen exchange rates were measured to estimate the degree of conformational fluctuation in a residue-specific manner. The structure of C76A/C94A was found to be quite similar to that of the wild type, except for the peptide segment of residues 74-78. The structure of C64A/C80A was considerably disordered in the entire region of the loop (residues 62-79). Further, it was found that a network of hydrogen bonds within the beta sheet and the 3(10) helix in the beta domain were disrupted and fluctuating. In C30A/C115A, the D helix was unstructured and the interface of the B helix with the D helix was significantly perturbed. However, the structural disorder generated in the hydrophobic core of the alpha domain was prevented by the C helix from propagating toward the beta domain. A marginally stable state in folded proteins is discussed based on the structures remaining in each three-disulfide variant.

Expression of steroidogenic acute regulatory protein (StAR) and LH receptor in MA-10 cells.

LH stimulation is mediated by its own receptor at the first step of the cascade, after which intracellular cAMP increases to stimulate the transcription of steroidogenic acute regulatory protein (StAR) in mouse MA-10 Leydig tumor cells. StAR mediates the rate-limiting step of steroidogenesis, which is the transfer of cholesterol to the inner mitochondrial membrane. Northern blot analysis consistently revealed two major transcripts, of about 3.6 kb and 1.6 kb, that hybridized with rat StAR mRNA. In this culture, treatment with hCG led StAR mRNA levels to rapidly and strongly increase by 3 h. Parallel increases were observed in transcripts of both sizes. Compared to StAR mRNA expression, LH receptor mRNA levels gradually decreased and declined to 50% of control values between 6 and 12 h incubation. Compared to the control, StAR mRNA levels increased and LH receptor mRNA levels decreased in a dose-dependent manner in the presence of increasing concentrations of hCG (3-100 ng/ml) and of increasing concentrations of 8-Br-cAMP (0.2-2 mM) after 4 h incubation. Since the over production of steroid hormones might be toxic to the own cells, the LH signal transduction that stimulates steroidogenesis might concomitantly decrease the responsiveness of steroidogenesis to LH stimulation at the receptor level. This result should be further investigated to clarify the mechanism of LH receptor regulation and steroidogenesis.

Mechanisms of action of transforming growth factor beta on the expression of follicle-stimulating hormone receptor messenger ribonucleic acid levels in rat granulosa cells.

The present study was undertaken to identify the mechanisms underlying the effect of transforming growth factor (TGF) beta on FSH receptor (FSH-R) in rat granulosa cells. Compared to the control, the treatment of granulosa cells with TGFbeta (10 ng/ml) increased FSH-R mRNA transcripts (5.5 and 2.4 kilobases) in a time-dependent manner, with a maximum increase of approximately 2-fold at 48 h. We then investigated whether the effect of TGFbeta on FSH-R mRNA levels was the result of increased transcription and/or altered mRNA stability. To determine whether the FSH-R 5'-flanking region plays a role in directing FSH-R mRNA expression, the proximal area of the FSH-R 5'-flanking regions were inserted into an expression vector, pGL-Basic, which contains luciferase as the receptor gene, and the resulting plasmids were transiently transfected into rat granulosa cells. The FSH (30 ng/ml) significantly enhanced the activity of 1862 base pairs of the FSH-R 5'-flanking region, but treatment with TGFbeta did not significantly influence the activity induced by FSH. On the other hand, the decay curves for FSH-R mRNA transcript in primary granulosa cells showed a significant increase in half-life after the addition of TGFbeta. Transforming growth factor beta stimulates the expression of follistatin mRNA accumulation in a dose- and time-dependent manner. Treatment with activin produced a substantial increase in FSH-R mRNA level. Concurrent treatment with follistatin neutralized this activin effect on FSH-R mRNA, as reported, although concurrent treatment with follistatin did not affect TGFbeta-induced FSH-R mRNA. Therefore, the profile of the TGFbeta effect on FSH-R mRNA granulosa cells may be caused by the increased stability of FSH-R mRNA and insensitivity to the follistatin.

Effect of transforming growth factor beta on the expression of luteinizing hormone receptor in cultured rat granulosa cells.

The present study was undertaken in order to identify the mechanism underlying the effect of transforming growth factor-beta (TGFbeta) on LH receptor (LH-R) expression in rat granulosa cells. Treatment with FSH produced a substantial increase in LH-R mRNA level, and concurrent treatment with increasing concentrations of TGFbeta brought about dose-dependent increases in FSH-induced LH receptor mRNA. TGFbeta, either alone or in combination with FSH, did not affect intracellular cAMP levels. We then investigated whether the effect of TGFbeta and FSH on LH-R mRNA levels results in increased transcription and/or altered mRNA stability. To determine whether the LH receptor 5'-flanking region plays a role in directing LH receptor mRNA expression, the proximal area of the LH receptor 5'-flanking regions were inserted into an expression vector, pGL-Basic, which contains luciferase as the receptor gene, and the resulting plasmids were transiently transfected into rat granulosa cells. FSH (30 ng/ml) significantly enhanced the activity of 1389 base pairs of the LH receptor 5'-flanking region, but treatment with TGFbeta did not significantly influence the activity induced by FSH. On the other hand, the decay curves for LH-R mRNA transcript in primary granulosa cells showed a significant increase in half-life after the addition of TGFbeta.

Synthesis of theaflavin from epicatechin and epigallocatechin by plant homogenates and role of epicatechin quinone in the synthesis and degradation of theaflavin.

Oxidation products of (-)-epicatechin and (-)-epigallocatechin by treatment with homogenates of 62 plants belonging to 49 families were compared. Forty-six plants were capable of synthesizing theaflavin, a black tea pigment, regardless of whether they contained catechins. Loquat, Japanese pear, and blueberry had activities higher than that of fresh tea leaves after 5 h of treatment; furthermore, these plants oxidized theaflavin to theanaphthoquinone. An additional new metabolite, dehydrotheasinensin, was generated on treatment with fresh tea leaves, eggplant, and unripened Japanese orange. Evidence for the oxidation of epigallocatechin and theaflavin by electron transfer to epicatechin quinone was demonstrated in a time course study using bananas and trapping the quinone intermediates as glutathione conjugates.