Debunking the Concept of Dentinal Tubule Penetration of Endodontic Sealers: Sealer Staining with Rhodamine B Fluorescent Dye Is an Inadequate Method

The aim of this study was to investigate the suitability of rhodamine B dye staining of an epoxy resin sealer (AH Plus) and calcium-silicate-based sealers (Total Fill BC Sealer, BioRoot RCS) to represent the penetration depth of the sealers into dentinal tubules after root canal obturation. In a three-step process, (1) leaching of rhodamine B from sealers into a buffer solution, (2) passive penetration of leached rhodamine B into dentinal tubules, and (3) conformity of rhodamine B penetration assessed by confocal laser scanning microscopy (CLSM), and sealer penetration assessed by scanning electron microscopy (SEM), in root-canal-filled teeth, were evaluated. Rhodamine B dye massively leached out of Total Fill BC Sealer and BioRoot RCS into the phosphate-buffered saline (PBS). A pinkish coloration of AH Plus was found after contact with PBS. Leached rhodamine B dye passively penetrated dentinal tubules from all three sealers when placed on root dentin. No correlation was observed between sealer penetration in SEM and rhodamine B penetration in CLSM. Staining of sealers using rhodamine B is an inadequate method with which to evaluate sealer penetration depth into dentinal tubules, as it overestimates the penetration of sealers into root dentin tubules.     

Follicular cholangitis mimicking cholangiocarcinoma treated with right hepatectomy: A case report and review of published works

Follicular cholangitis is a new, rare disease that causes severe biliary stricture. We herein describe the findings from a resected case of follicular cholangitis, suggesting a distinct disease entity that causes benign biliary stricture. A 60‐year‐old man who was referred to our hospital due to elevated γ‐glutamyl transpeptidase levels and dilatation of the B8 bile duct. Although bile juice cytology and bile duct brushing cytology showed no malignancy, the dilatation was progressive. Therefore, right hepatectomy combined with caudate lobectomy was carried out on suspicion of intrahepatic cholangiocarcinoma. The wall of the resected bile duct was markedly thickened due to severe fibrosis under the mucosal layer. Histology of the mucosal epithelium indicated no malignancy. Infiltration of plasma cells characterized by remarkable formation of lymphoid follicles with germinal centers was observed around the bile ducts. The patient was diagnosed with follicular cholangitis based on histological findings. We thus observed a rare case of follicular cholangitis. This case and review of published reports suggest that, despite its rarity, follicular cholangitis should be considered at the differential diagnosis of biliary stricture. This case report could contribute to a better understanding of how to address this disease.     

A topical parathyroid hormone/parathyroid hormone-related peptide receptor antagonist stimulates hair growth in mice

One of the biggest hurdles in developing peptides for the treatment of hair growth disorders is that there has been no effective method of delivering them topically. Murine hair growth can be stimulated with ip injections of the PTH/PTHrP receptor antagonist, PTH (7-34). We sought to determine whether we could deliver PTH (7-34) topically and achieve stimulation of hair growth. We prepared a topical cream by mixing PTH (7-34) into a liposome vehicle (Novasome A). We applied the cream daily to the backs of 5-wk-old female SKH-1 hairless mice for 1 wk. After the 1 wk of treatment, there was marked stimulation of hair growth in the SKH-1 hairless mice. Relative to controls, mice treated with PTH (7-34) had 216% longer hairs (P < 0.001), 40% more visible hairs (P < 0.001), and 43% more hair follicles stained with 5-bromo-2'-deoxyuridine (P < 0.01). A unique aspect of skin is the possibility to directly target it via topical treatment. Our study is the first to report the hair-stimulating effect of a PTH/PTHrP receptor antagonist topically applied to skin in vivo. Thus, we introduce a novel paradigm to develop topical PTH analogs for treating disorders of hair growth.     

Effect of occlusive dressings on the stratum corneum water holding capacity.

Occlusion of the skin is used in clinical dermatology to promote wound healing and to increase the transcutaneous penetration of topically applied drugs. These effects are related to the degree of occlusion exerted and depend on the physicochemical nature of the dressing. We have evaluated the effects of four different materials on the skin barrier and the stratum corneum water holding capacity (WHC) using the Plastic Occlusion Stress Test (POST). The following materials were compared: hydrocolloid dressing, polyurethane film, polyethylene film, and a plastic chamber. These devices were applied on the volar forearm for 24 hours in 10 healthy volunteers (mean age 32 +/- 4 years). Upon their removal, the stratum corneum WHC, measured as skin surface water loss (SSWL), was recorded continuously for 25 minutes using an Evaporimeter. SSWL decay curves showed significant differences between the occlusive materials (analysis of variance, p less than 0.01). Higher SSWL values were recorded in sites occluded with the plastic chamber, whereas the polyurethane film resulted in poor occlusive capacity. Hydrocolloid dressing and polyethylene gave similar responses with higher WHC values compared to polyurethane (p less than 0.05). The relevance of these findings to clinical dermatology in terms of wound healing and drug absorption is discussed.     

Determination of secretion rates of estradiol, progesterone, oxytocin, and angiotensin II from tertiary follicles and freshly formed corpora lutea in freely moving sows

Two days before ovulation ovarian follicles of sows were implanted with microdialysis systems (MDS) which function like artificial capillaries with exteriorized inlets and outlets. Steroid hormones and paracrine acting factors such as oxytocin (OXT) and angiotensin II (AII) diffuse from ovarian tissue into the fluid, which is pumped through the MDS and collected in fractions. This allows determination of dynamic changes of estradiol (E2), progesterone (P), OXT, and AII secretion during the pre-, peri-, and postovulatory periods in freely moving sows. More than 80% of such implanted follicles ovulate and form competent corpora lutea (CL) allowing continuation of experimentation during the early luteal phase. Follicular E2 release increases before ovulation and decreases with increasing blood LH concentrations. Twenty to 30 h after beginning of the preovulatory LH surge P secretion increases gradually. Both peptides OXT and AII are released episodically by the preovulatory follicle. During the time of decreased E2 and not yet increased P secretion, i.e. during the periovulatory period, mean AII secretion was highest in comparison to the late follicular and early luteal phase. E2 remains measurable during the early luteal phase. OXT and AII were also topically applied into the follicular wall and after ovulation into the CL. AII had no effect on steroidogenesis of both structures. Although OXT was ineffective in the follicle, in young CL it stimulated P secretion. These results indicate that the MDS can be used to study late follicular and early luteal steroid and peptide secretion. The function of OXT and AII in the follicle remains obscure, whereas OXT has a luteotropic effect in young porcine CL.     

Clinical and histopathological study of “follicular cholangitis”: Sclerosing cholangitis with prominent lymphocytic infiltration masquerading as hilar cholangiocarcinoma

Aim: Sclerosing cholangitis which mimic hilar biliary carcinoma have not been fully categorized. In this study, we investigated the clinicopathological features of sclerosing cholangitis of unknown origin, so‐called “follicular cholangitis”. Methods: Patients who had undergone surgery of the proximal bile duct from 1993–2008 on suspicion of proximal bile carcinoma were evaluated. Of these, we investigated the cases compatible with follicular cholangitis. Then, we reviewed the previous reports of follicular cholangitis showing similar clinicopathological findings. Results: Among 176 patients, two who were diagnosed with benign sclerosing cholangitis of unknown origin shared the common histopathological findings compatible with follicular cholangitis. Histopathological findings showed dense fibrosis with prominent lymph follicles and germinal centers, being appropriate for neither primary sclerosing cholangitis nor immunoglobulin G4‐related sclerosing cholangitis. Review of previous published work revealed two other cases diagnosed with follicular cholangitis and showing histopathological findings similar to ours. All these cases were middle‐aged patients with no history of autoimmune disease with focal strictures mainly involved in the hilar bile duct, mimicking hilar cholangiocarcinoma. Conclusion: Benign sclerosing cholangitis has not been fully categorized, yet. Follicular cholangitis shows unique histopathological features and can be categorized as an independent entity.     

Colloidal, in vitro and in vivo anti-leishmanial properties of transfersomes containing paromomycin sulfate in susceptible BALB/c mice

The aim of this study was to develop transfersomal formulation with respect to dermal delivery of paromomycin sulfate (PM) for possible topical therapy of cutaneous leishmaniasis (CL). PM transfersomal formulations (PMTFs) with different percent of soy phosphatidylcholine, sodium cholate (Na-Ch) and ethanol were prepared and characterized for the size, zeta potential and encapsulation efficiency. The results showed that the most stable formulations with suitable colloidal properties were obtained by 2% Na-Ch which had average size of around 200nm. The in vitro permeation study using Franz diffusion cells fitted with mouse skin at 37°C for 24h showed that almost 23% of the PMTFs applied penetrated the mouse skin, and the amount retained in the skin was about 67% for both formulations; however, the percent of penetration and retention for PM conventional cream was 49 and 13, respectively. The 50% effective doses of PMTFs against Leishmania major promastigotes and amastigotes in culture were significantly less than cream and/or solution of PM. Selected PMTFs and empty transfersomes showed no cytotoxicity in J774 A.1 mouse macrophage cell line. Selected PMTFs was used topically twice a day for 4 weeks to treat L. major lesions on BALB/c mice, and the results showed a significantly (P<0.05) smaller lesion size in the mice in the treated groups than in the mice in the control groups, which received either empty transfersomes or phosphate-buffered saline (PBS) and also PM cream. The spleen parasite burden was significantly (P<0.01) lower in mice treated with selected PMTFs than in mice treated with PBS or control transfersomes, and PM cream. The results of this study showed that PMTFs prepared with 2% of Na-Ch with and without 5% ethanol might be useful as a candidate for the topical treatment of CL.     

Loss of synaptonemal complex protein-1, a synaptonemal complex protein, contributes to the initiation of follicular assembly in the developing rat ovary

In the rat ovary, germ and somatic cells become organized into primordial follicles 48-72 h after birth. Although several genes have been implicated in the control of early follicular growth, less is known about the factors involved in the formation of primordial follicles. Using the method of differential display of mRNAs, we found several genes differentially expressed at the time of follicular assembly. One of them encodes synaptonemal complex protein-1 (SCP1), a core component of the protein complex that maintains recombining chromosomes together during prophase I of the first meiotic division in germ cells. This association, evident during the pachytene stage, ends when chromosomal desynapsis begins in the diplotene stage at the end of prophase I. Oocytes become arrested in the diplotene/dictate stage before becoming enclosed into primordial follicles, suggesting that oocytes must complete meiotic prophase I before becoming competent to direct follicle assembly. We now show that attainment of the diplotene stage results in follicular formation. In developing rat ovaries, SCP1 mRNA expression is confined to oocytes and decreases precipitously within 24 h after birth, preceding the organization of primordial follicles. The premature loss of SCP1, achieved via treatment with an antisense oligodeoxynucleotide targeting SCP1 mRNA, resulted in more oocytes reaching the diplotene stage, as evidenced by a decrease in the number of oocytes containing germ cell nuclear antigen-1 (a nuclear protein whose expression ceases in diplotene) and an increase in the number of oocytes expressing MSY2 (a cytoplasmic Y box protein expressed in oocytes that have become arrested in diplotene). SCP1-deficient ovaries exhibited an increased number of newly formed follicles, suggesting that completion of meiotic prophase I endows oocytes with the ability to orchestrate follicular assembly.     

Effects of maturation-inducing hormone on heterologous gap junctional coupling in ovarian follicles of Atlantic croaker.

A previous ultrastructural study of heterologous (granulosa cell-oocyte) gap junction (GJ) contacts in ovarian follicles of Atlantic croaker suggested that these contacts disappear late during the process of resumption of oocyte meiosis. This observation suggested that, unlike scenarios proposed for a number of other species, uncoupling of GJ is not necessary for the onset of meiotic resumption in croaker follicles. However, the functionality of heterologous GJ contacts and the temporal association between maturation-inducing hormone (MIH)-induced changes in heterologous coupling and resumption of oocyte meiosis have not been examined in Atlantic croaker. These questions were addressed with a cell-cell coupling assay that is based on the transfer of a GJ marker, Lucifer Yellow, from oocytes to granulosa cells. Follicle-enclosed oocytes injected with Lucifer Yellow allowed transfer of the dye into the follicle cell layer, thus confirming that there is functional heterologous coupling between the oocyte and the granulosa cells. Dye transfer was observed in vitellogenic, full-grown/maturation-incompetent, and full-grown/maturation-competent follicles. Treatment of maturation-competent follicles with MIH caused a time-dependent decline in the number of follicles transferring dye. However, although GJ uncoupling in some of the follicles was observed before germinal vesicle breakdown (GVBD, index of meiotic resumption), about 50% of the follicles maintained the ability to transfer dye even after GVBD had occurred. Further, a known GJ inhibitor (phorbol 12-myristate 13-acetate) blocked heterologous GJ within a time frame similar to that seen with MIH but without inducing any of the morphological changes (including GVBD) associated with follicular maturation. In conclusion, uncoupling of heterologous GJ seems insufficient and unnecessary for the onset of meiotic resumption in ovarian follicles of Atlantic croaker.     

Nascent blood vessels in the skin arise from nestin-expressing hair-follicle cells

Besides forming hair shafts, the highly organized, metabolically vigorous hair follicle plays several crucial roles in skin architecture. The follicle contains a distinct population of presumptive follicular stem cells that express nestin, also a marker for neural stem cells. These nestin-expressing follicle cells are located principally in the follicular bulge region. Nestin-driven GFP (ND-GFP), transfected into mice, principally labels cells in the bulge region, which is consistent with the cells' being the stem cells of the hair follicle. We report here that ND-GFP also labels developing skin blood vessels that appear to originate from hair follicles and form a follicle-linking network. This is seen most clearly by transplanting ND-GFP-labeled vibrissa (whisker) hair follicles to unlabeled nude mice. New vessels grow from the transplanted follicle, and these vessels increase when the local recipient skin is wounded. The ND-GFP-expressing structures are blood vessels, because they display the characteristic endothelial-cell-specific markers CD31 and von Willebrand factor. This model displays very early events in skin angiogenesis and can serve for rapid antiangiogenesis drug screening.     

CD5‐positive follicular lymphoma: prognostic significance of this aberrant marker?

Follicular lymphomas are the most common of the clinically indolent non‐Hodgkin lymphomas. The immunophenotypic pattern of follicular lymphoma classically demonstrates the B‐cell markers CD19, CD20, CD22, CD79a, surface immunoglobulin and CD10. The tumour cells are usually negative for CD5. We described two cases of CD5‐positive follicular lymphoma. This finding has rarely been described. Our aim was to discuss two cases of CD5‐positive follicular lymphoma and review the published literature on the significance of CD5 expression. Bone marrow biopsies showing involvement with follicular lymphoma at our institution over the past 34 months were evaluated for the presence of CD5 positivity by flow cytometry and immunohistochemistry. Two of eight cases (25%) of the follicular lymphomas with marrow involvement at our institution were found to be CD5‐positive. Only 36 cases of CD5‐positive follicular lymphoma have previously been described in the literature. The clinical and therapeutic significance of this remains uncertain. More research into such cases may establish whether the presence of this aberrant marker bears prognostic significance.     

Endocrine control of clutch size in reptiles. V. FSH-induced follicular formation and growth in immature ovaries of Anolis carolinensis

The immature ovary of Anolis carolinensis can be divided into germinal bed, stromal, and extrastromal regions. The germinal bed is covered by a surface epithelium and contains oogonia, “naked” oocytes, and several primordial follicles of similar size. One primordial follicle leaves the germinal bed at one time, enters the stroma, and is surrounded by a vascular theca. The granulosa then differentiates into four cell types. A follicular size hierarchy exists in the stromal and extrastromal regions within one ovary, and extrastromal and larger stromal follicles alternate in size between ovaries. The proposal is made that the alternation in follicular size between ovaries is a product of the follicular dominance hierarchy within each ovary. Treatment of immature females with mammalian FSH stimulates growth of all stromal and extrastromal follicles in each ovary. This gonadotropin also causes recruitment of follicles into the size hierarchy by initiating formation of new follicles in the germinal bed and movement of one follicle at a time into the stroma. Gonadotropin also changes the number of some cell types in the granulosa and stimulates yolk deposition in larger follicles.     

Changes in aromatase activity in small ovarian follicles of the domestic fowl (Gallus domesticus) during growth and atresia

The tritium release assay for measuring aromatase activity was adapted to measure oestrogen production in intact small ovarian follicles of the domestic fowl. The activity was measured in three types of follicle classified as normal, atretic or grossly atretic. Normal follicles were distinguished from atretic by the presence of small haemorrhages on the surface of the atretic follicles. Grossly atretic follicles were identified by their deformed shape. The aromatase activity in normal and atretic follicles was related to follicular weight, the activity in atretic follicles being less than that in normal follicles. The aromatase activity in grossly atretic follicles was independent of follicular weight. The enzyme activity in this group of follicles was significantly less than that in either normal or atretic follicles. The significance of these results is discussed in relation to the induction of atresia within the small ovarian follicles of the domestic fowl.     

Quantitative analysis of the ovarian cycle of the lizard Lepidodactylus lugubris

Patterns of ovarian follicular growth and atresia, as well as changes in number of oogonia, primary oocytes, and primordial follicles, were studied in the gecko Lepidodactylus lugubris. Sizes of larger, growing follicles increased at the stage in the ovarian cycle when yolking of the largest follicle occurred. Atretic follicles were replaced by new growing follicles recruited into the follicular size hierarchy. The single germinal bed in each ovary, containing oogonia, oocytes, and primordial follicles, was most active during the vitellogenic period. The two ovaries of this species each ovulate one egg simultaneously, and each contains only five growing follicles, about 17% of which are atretic. In contrast, each ovary of other lizard species that ovulate two eggs simultaneously contains two germinal beds and a relatively high number of growing follicles. Extensive follicular atresia (>50%) reduces the number of ovulations to one in these species.     

Exogenous melatonin positively influences follicular dynamics, oocyte developmental competence and blastocyst output in a goat model

Abstract:  The role of melatonin in modulating mammalian reproduction is of particular interest; however, its effects on ovarian follicles and their oocytes still remain to be characterized. This study determined the influence of melatonin treatment on follicular growth patterns and on in vitro oocyte developmental competence. In a first experiment, the effects of melatonin supplementation on follicular dynamics were evaluated using daily transrectal ultrasonographies for 21 days, in 7 multiparous Sarda goats receiving a subcutaneous implant of 18 mg of melatonin and in 5 control untreated does. Melatonin caused more follicular waves (5.2 ± 0.2 versus 4 ± 0.3; P  < 0.05) as the waves were shortened at around 2 days when compared with the non-melatonin treated control goats ( P  < 0.001). Oocyte developmental competence was evaluated in a second experiment by applying procedures for in vitro embryo production. There were no significant differences in the total number of oocytes obtained from 6 control (n = 192) and 7 melatonin-treated (n = 265) goats given follicle stimulating hormone to induce follicular development. Differences in oocyte developmental competence between the two groups became evident after in vitro fertilization and culture; melatonin increased the rate of cleaved oocytes in comparison with control animals (82.5 versus 63.4%; P  < 0.001), advanced timing of embryo development and enhanced blastocyst output (31.5 versus 16.3%; P  < 0.01). However, blastocyst quality, as evaluated by cryotolerance and gene expression analysis, was not found to be different between the groups. In conclusion, in vivo melatonin treatment is beneficial for increasing ovarian follicle turnover and improving oocyte developmental competence and kinetics of the blastocyst.     

Exploration of the microbial anatomy of normal human skin by using plasmid profiles of coagulase-negative staphylococci: search for the reservoir of resident skin flora

The aerobic bacterial flora of the top 25 layers of the stratum corneum of normal human skin was characterized by sampling glabrous skin with contact plates and analyzing plasmid patterns of coagulase-negative staphylococci (SCN) by agarose gel electrophoresis. The number of colonies of SCN on the skin surface at 12 sites varied from 14 to 838. Removal of five keratinized layers by sequential stripping with cellophane tape reduced the number of colonies by 80% (median; range, 42%-91%). Counts remained constant during removal of 20 additional layers. SCN with six different plasmid patterns were identified at a site on the skin surface. After removal of 25 layers, colonies with a single pattern were clustered in one quadrant of the site. The site was sterilized and covered with a sterile dressing for 18 h. Colonies reappeared in the same quadrant of the site; six of seven had the same pattern seen 18 h previously. Observations at three other sites were similar. Reappearance of the same strain(s) of SCN following sterilization of the site suggests that the reservoir for normal resident skin flora is located below the stratum corneum, perhaps in hair follicles and ducts of sebaceous glands.     

Ketoconazole in experimental candidosis

A relatively large number of animal models of candidosis exist in which the efficacy of antifungal substances can be evaluated. These include models of candidosis of the skin, gastrointestinal tract, genital system, and internal organs in a variety of animal species. The efficacy of ketoconazole administered orally and topically was evaluated; when given orally in relatively low doses, ketoconazole was found to be efficacious in all of the experimental models used. Scanning and transmission electron micrographs of infected tissue demonstrated the rapidity with which Candida albicans was eradicated from the host after administration of ketoconazole.     

A Case of Follicular Bronchiolitis Associated with Asthma,Eosinophilia, and Increased Immunoglobulin E

A 49-year-old woman, who had been diagnosed with asthma, showed a bilateral diffuse pattern of small centrilobular nodules on CT. Laboratory data revealed peripheral eosinophilia and a marked increase in total serum IgE levels. The nodules detected on CT were initially considered to be associated with bronchiolar infiltration of eosinophils. Pathological findings from thoracoscopy revealed infiltration of eosinophils into the airway lumen and walls, goblet cell hyperplasia, and thickening of the basement membrane in large bronchi, consistent with asthma. However, hyperplastic lymphoid follicles with reactive germinal centers were observed along the bronchioles. The follicles had no evidence of monoclonality suggested by immunohistological analysis, and no remarkable infiltrates of eosinophils, suggesting follicular bronchiolitis (FB). After treatment with prednisolone, the small diffuse nodules improved markedly, and peripheral eosinophilia and total serum IgE levels also decreased. To the best of our knowledge, this is the first documented case report of FB associated with asthma, eosinophilia, and elevated IgE with a definite pathophysiological diagnosis.     

Ovarian responses in macaques to pulsatile infusion of follicle-stimulating hormone (FSH) and luteinizing hormone: increased sensitivity of the maturing follicle to FSH

This study investigated the relationship between plasma gonadotropin concentrations and the initiation and maintenance of preovulatory follicular growth in macaques. Eight adult cynomolgus monkeys were treated with a GnRH antagonist [AcD2Nal1-4ClDPhe2, DTrp3, DArg6, DAla10]GnRH X HOAc to block endogenous gonadotropin secretion. In four animals, a pulsatile infusion of human FSH and human LH (hLH) (one 3-min pulse/h) was initiated, and the amount of hFSH delivered per pulse was increased every 3-4 days until serum estradiol concentrations rose. Thereafter, the amount of FSH delivered per pulse was reduced by 12.5%/day for 5 days, whereas the amount of LH delivered per pulse was not altered. Results indicated that plasma FSH concentrations in the range of 15-20 mIU/ml were associated with the initiation of estrogen production; in addition, a progressive reduction in plasma FSH concentration to 8-10 mI/ml over the subsequent 5 days was accompanied by continued rises in estradiol concentrations and preovulatory follicular growth. In contrast, in four control animals, maintenance of plasma FSH concentrations at 8-10 mIU/ml for 13 days did not result in elevation in serum estradiol concentrations or antral follicular growth. These observations demonstrate that after stimulation by elevated FSH concentration, follicles can continue to mature in the presence of FSH concentrations which are unable to support the growth of less mature follicles. Thus, this may account for the mechanisms by which the maturing follicle continues to develop during the mid-through late follicular phase of the menstrual cycle, whereas other less mature follicles undergo atresia.