Citrullinated inter-alpha-trypsin inhibitor heavy chain 4 in arthritic joints and its potential effect in the neutrophil migration.

The citrullinated inter-alpha-trypsin inhibitor heavy chain 4 (cit-ITIH4) was identified as its blood level was associated with the arthritis score in peptide glucose-6-phosphate-isomerase-induced arthritis (pGIA) mice and the disease activity in patients with rheumatoid arthritis (RA). This study aimed to clarify its citrullination pathway and function as related to neutrophils. In pGIA-afflicted joints, ITIH4 and cit-ITIH4 levels were examined by immunohistochemistry (IHC), immunoprecipitation (IP) and Western blotting (WB), while peptidylarginine deiminase (PAD) expression was measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), IHC and immunofluorescent methods. The pGIA mice received anti-lymphocyte antigen 6 complex locus G6D (Ly6G) antibodies to deplete neutrophils and the expression of cit-ITIH4 was investigated by WB. The amounts of ITIH4 and cit-ITIH4 in synovial fluid (SF) from RA and osteoarthritis (OA) patients were examined by I.P. and W.B. Recombinant ITIH4 and cit-ITIH4 were incubated with sera from healthy volunteers before its chemotactic ability and C5a level were evaluated using Boyden's chamber assay and enzyme-linked immunosorbent assay (ELISA). During peak arthritic phase, ITIH4 and cit-ITIH4 were increased in joints while PAD4 was over-expressed, especially in the infiltrating neutrophils of pGIA mice. Levels of cit-ITIH4 in plasma and joints significantly decreased upon neutrophil depletion. ITIH4 was specifically citrullinated in SF from RA patients compared with OA patients. Native ITIH4 inhibited neutrophilic migration and decreased C5a levels, while cit-ITIH4 increased its migration and C5a levels significantly. Cit-ITIH4 is generated mainly in inflamed joints by neutrophils via PAD4. Citrullination of ITIH4 may change its function to up-regulate neutrophilic migration by activating the complement cascade, exacerbating arthritis.

Brillouin Light Scattering by Magnetic Quasivortices in Cavity Optomagnonics.

A ferromagnetic sphere can support optical vortices in the form of whispering gallery modes and magnetic quasivortices in the form of magnetostatic modes with nontrivial spin textures. These vortices can be characterized by their orbital angular momenta. We experimentally investigate Brillouin scattering of photons in the whispering gallery modes by magnons in the magnetostatic modes, zeroing in on the exchange of the orbital angular momenta between the optical vortices and magnetic quasivortices. We find that the conservation of the orbital angular momentum results in different nonreciprocal behavior in the Brillouin light scattering. New avenues for chiral optics and optospintronics can be opened up by taking the orbital angular momenta as a new degree of freedom for cavity optomagnonics.

Cavity Optomagnonics with Spin-Orbit Coupled Photons.

We experimentally implement a system of cavity optomagnonics, where a sphere of ferromagnetic material supports whispering gallery modes (WGMs) for photons and the magnetostatic mode for magnons. We observe pronounced nonreciprocity and asymmetry in the sideband signals generated by the magnon-induced Brillouin scattering of light. The spin-orbit coupled nature of the WGM photons, their geometrical birefringence, and the time-reversal symmetry breaking in the magnon dynamics impose the angular-momentum selection rules in the scattering process and account for the observed phenomena. The unique features of the system may find interesting applications at the crossroad between quantum optics and spintronics.

Foam sclerotherapy for spouting haemorrhage in patients with varicose veins.

The bleeding from various veins can be intense and may be mistaken for arterial haemorrhage. Several fatal cases are reported due to delay of treatment and inappropriate first aid. We describe five cases of haemorrhage from varicose veins that were treated with foam sclerotherapy. Polidocanol foam was injected in the various veins using ultrasound guidance. There was no recurrence of haemorrhage in any patient during the 17.4 months follow-up period. Foam sclerotherapy can be performed easily in an out-patient clinic setting. This method is an ideal therapy for haemorrhage from varicose veins because it mitigates problematic varicose veins.

Spraguea (Microsporida: Spraguidae) infections in the nervous system of the Japanese anglerfish, Lophius litulon (Jordan), with comments on transmission routes and host pathology.

Anglerfish from the genus Lophius are a globally important commercial fishery. The microsporidian Spraguea infects the nervous system of these fish resulting in the formation of large, visible parasitic xenomas. Lophius litulon from Japan were investigated to evaluate the intensity and distribution of Spraguea xenomas throughout the nervous system and to assess pathogenicity to the host and possible transmission routes of the parasite. Spraguea infections in L. litulon had a high prevalence; all fish over 403 mm in standard length being infected, with larger fish usually more heavily infected than smaller fish. Seventy percent of all fish examined had some gross visible sign of infection. The initial site of development is the supramedullary cells on the dorsal surface of the medulla oblongata, where all infected fish have parasitic xenomas. As the disease progresses, a number of secondary sites typically become infected such as the spinal, trigeminal and vagus nerves. Fish with infection in the vagus nerve bundles often have simultaneous sites of infection, in particular the spinal nerves and along the ventral nerve towards the urinary bladder. Advanced vagus nerve infections sometimes form xenomas adjacent to kidney tissue. Spraguea DNA was amplified from the contents of the urinary bladders of two fish, suggesting that microsporidian spores may be excreted in the urine. We conclude that supramedullary cells on the hindbrain are the primary site of infection, which is probably initiated at the cutaneous mucous glands where supramedullary cells are known to extend their peripheral axons. The prevalence of Spraguea infections in L. litulon was very high, and infections often extremely heavy; however, no associated pathogenicity was observed, and heavily infected fish were otherwise normal.

Harvesting epithelial keratinocyte sheets from temperature-responsive dishes preserves basement membrane proteins and improves cell survival in a skin defect model.

Cultured epithelial autograft (CEA) therapy has been used in clinical applications since the 1980s. However, there are some issues related to this treatment that still remain unsolved. Enzymatic treatment is typically used in the collection of epithelial keratinocyte sheets, but it tends to break the adhesion and basement membrane proteins. It is thought that the loss of proteins after enzymatic treatment is responsible for the poor survival of transplanted cell sheets. Our laboratory has developed a temperature-responsive culture dish that does not require enzymatic treatment to harvest the cells. In this study, we compare morphological and survival results from rat epithelial keratinocyte cell sheets harvested by temperature-reducing treatment (TT sheets) against cell sheets harvested by enzymatic (dispase) treatment (DT sheets). TT sheets preserve keratin structure in better conditions and express higher levels of collagen IV and laminin 5 than DT sheets. In order to evaluate cell sheet survival after transplantation, we created an in vivo transplant model. Keratinocyte sheets obtained from GFP-positive animals were transplanted into athymic rats. The survival rate 7 days after transplantation of TT sheet was higher than that of DT sheets. Collagen IV and Laminin 5 expression was observed in the TT sheet transplantation group. These results indicate that the remaining basement membrane proteins are important for initial attachment and cell survival. We believe that the cell sheet harvesting method using temperature-responsive culture dishes provides superior cell survival and can solve one of the roadblocks in CEA therapy. Copyright © 2016 John Wiley & Sons, Ltd.

Up-regulation of CD44 expression by tumor necrosis factor-alpha is neutralized by interleukin-10 in Langerhans cells.

CD44 is a principal cell-surface receptor for hyaluronate and is found on a wide variety of cells. CD44 plays an important role in lymphocyte homing, lymphohemopoiesis, and T-cell activation as well as in cell motility and migration. CD44 is expressed on the cell surface of epidermal Langerhans cells (LC), and is one of the candidates for molecules that are involved in the migratory capability of LC, but little is known about its regulatory properties. We examined the modulatory effects of tumor necrosis factor (TNF)-alpha and interleukin (IL)-10 on the CD44 expression in LC. We found 1) that TNF-alpha significantly up-regulated the expression of CD44 in a concentration-dependent manner, 2) that IL-10 down-regulated the expression of CD44 in a concentration-dependent manner, 3) that the effect of TNF-alpha or IL-10 was readily detectable as early as 24 h after the initiation of culture, and 4) that the simultaneous addition of TNF-alpha and IL-10 mutually neutralized the effect of each other. These data suggest that in the epidermal microenvironment the expression of CD44 in LC may be reciprocally regulated by TNF-alpha and IL-10, both of which are known to be produced by surrounding keratinocytes.

Immunosuppressive effects of azelastine hydrochloride on contact hypersensitivity and T-cell proliferative response: a comparative study with FK-506.

Azelastine hydrochloride (AZE) is an anti-allergic drug that inhibits the release of various chemical mediators from mast cells. We compared the immunosuppressive effects of AZE and FK-506 in vivo and in vitro. Topical application of AZE strongly inhibited the efferent phase of contact hypersensitivity, as did application of FK-506. In in vitro experiments, we found that 1) the suppression by AZE on interleukin (IL)-2 production from splenic T cells was partial and considerably large amounts of IL-2 were still produced, even in the presence of 10(-5) M of AZE, which was in sharp contrast to the observed marked inhibition of [3H]-TdR incorporation; 2) AZE significantly inhibited the phorbol myristate acetate-induced IL-2 responsiveness; 3) AZE did not inhibit the IL-2 receptor alpha expression of activated T cells; and 4) the significant inhibitory action was still observed even when AZE was added at 48 h after the initiation of culture. In regard to FK-506, we found that 1) FK-506 completely blocked the production of IL-2; 2) exogeneous IL-2 consistently restored the FK-506-induced inhibition; 3) FK-506 affected the phorbol myristate acetate-induced IL-2 responsiveness very little, if any; and 4) the significant suppression was observed only when FK-506 was added within 24 h after the initiation of culture. Thus, AZE exerts its in vitro immunosuppressive activity preferentially by interfering with the IL-2 responsiveness, with partial inhibition of IL-2 production. Conversely, FK-506 acts as a strong inhibitor of IL-2 production without a prominent effect on IL-2 responsiveness. The immunosuppressive activity of AZE shown in vitro may also be operative in vivo and may be applicable for topical use.

gp100 mRNA is more sensitive than tyrosinase mRNA for RT-PCR amplification to detect circulating melanoma cells in peripheral blood of melanoma patients.

Two different melanocyte-specific mRNAs are studied as markers for circulating melanoma cells in vitro using the human melanoma cell line G361 and in vivo using blood samples from Japanese melanoma patients at different clinical stages. These mRNAs encode tyrosinase, the most essential enzyme for melanin synthesis, and gp100, a melanosomal matrix glycoprotein recognized by monoclonal antibody HMB-45. We used reverse-transcription polymerase chain reaction (RT-PCR) to detect tyrosinase mRNA and gp100 mRNA in peripheral blood. Since melanocytes would not normally be present in peripheral blood, the detection of those transcripts should indicate the presence of circulating melanoma cells. RT-PCR detection of these two mRNAs was highly sensitive and specific. Our in vitro study showed that as few as 10 melanoma cells in 0.125 ml normal blood could be detected. In in vivo study, 130 blood samples from 55 melanoma patients gave positive and variably sensitive results, whereas no samples from healthy controls or patients with other cancers gave positive results. Tyrosinase mRNA was not detected in any of the melanoma patients. gp100 mRNA was detected in 12 of 55 melanoma patients, in none of five stage I patients (0%), in four of 26 stage II patients (15.4%), in one of six stage III patients (16. 7%) and in seven of 18 stage IV patients (38.9%). Thus gp100 mRNA is a more sensitive marker for detecting circulating melanoma cells compared with tyrosinase mRNA.

Migration of Langerhans cells in an in vitro organ culture system: IL-6 and TNF-alpha are partially responsible for migration into the epidermis.

Although it is well established that epidermal Langerhans cells (LC) originate from bone marrow, little is known about the mechanism of this migration into the epidermis from bone marrow. In order to clarify the mechanism of this migration, we constructed an in vitro model. LC were depleted by daily topical application of clobetazole propionate (CP) solution onto the ear of Balb/c mice. Seven days later, ear skin was cut off, separated and co-cultured dermal-side-up with syngeneic (Balb/c), semisyngeneic ((C3H x Balb/c)F1), or allogeneic (C3H) epidermal cells (EC) for 3 days. We found (1) that a marked migration of donor LC into the recipient epidermis was observed in the LC-depleted skin, (2) that only syngeneic LC actively migrated into the recipient epidermis; however, the migration of semisyngeneic and allogeneic LC was detected at very low levels, (3) that the migratory capacity of donor LC was directly proved by a biolabeling technique using donor EC labeled with PKH-26, and (4) that anti-IL-6 and anti-TNF-alpha antibodies inhibited the migration of donor LC into the recipient epidermis. These data demonstrate that the resident LC have the potential to traffic through the dermis into the epidermis in a highly syngeneic-specific fashion, and that IL-6 and TNF-alpha are partially responsible for promoting this migration.

A comparative study on Langerhans cells in lymph nodes with dermatopathic lymphadenopathy and histiocytosis X cells.

It was elucidated that the majority of DC were LC which were positive for CD 1a, but negative for PCNA, and possessed BG in the lymph nodes with DPL. On the other hand, HCX cells were almost always positive for PCNA. From this point of view, it can be speculated that LC in the lymph nodes of the DPL are non dividing mature cells and migrate from the skin lesion. HCX cells which were positive for CD 4 may be more immature cells than LC in DPL, and may be pathological cells which can divide in the foci.

Differentiation of Langerhans cells from interdigitating cells using CD1a and S-100 protein antibodies.

The present study shows that Langerhans cells can be differentiated from interdigitating cells at the light microscopic level. Superficial lymph nodes and skin taken from necropsies and the lymph nodes of dermatopathic lymphadenopathy (DPL) were used for this experiment. Sections of lymph node and skin were embedded using the acetone, methyl benzoate and xylene (AMeX) method and dendritic cells were immunostained with anti S-100 protein antibody (S-100, and OKT-6 (CD1a) using the restaining method. Langerhans cells in the skin were positive for both CD1a and S-100. Dendritic cells positive for both CD1a and S-100, and dendritic cells positive for S-100, but not for CD1a were observed in superficial lymph nodes. In normal superficial lymph nodes, there were more interdigitating cells than Langerhans cells. The majority of the dendritic cells in the DPL were Langerhans cells. We conclude that the S-100 and CD1a positive cells are Langerhans cells, and the S-100 positive-CD1a negative cells are interdigitating cells.

Interferon-gamma-induced HLA-DR, but not ICAM-1, expression of human keratinocytes is down-regulated by calmodulin antagonist.

Interferon-gamma (IFN-gamma) has been shown to induce or enhance the expression of MHC class II and intercellular adhesion molecule-1 (ICAM-1) in a variety of human and murine cell types, including epidermal keratinocytes (KC). However, the expression of MHC class II and ICAM-1 molecules induced by IFN-gamma is not necessarily coordinated. We investigated the inhibitory effects of the calmodulin antagonist, W-7, and its chlorine deficient inactive analogue, W-5, on the expression of MHC class II (HLA-DR) and ICAM-1 by human KC incubated with IFN-gamma. We found that the IFN-gamma-induced expression of HLA-DR was reproducibly and dose-dependently inhibited by W-7. However, the expression of ICAM-1 was highly resistant to the inhibitory effects of W-7. Neither HLA-DR nor ICAM-1 expression was affected by W-5. These data suggest that the IFN-gamma-induced HLA-DR, but not ICAM-1, expression is mediated, if not exclusively, by calmodulin in human KC.

Studies on intranuclear inclusions and nuclear grooves in papillary thyroid cancer by light, scanning electron and transmission electron microscopy.

OBJECTIVE: To successively examine intranuclear inclusions and nuclear grooves in the same papillary thyroid cancer specimens using a light microscope (LM), scanning electron microscope (SEM) and transmission electron microscope (TEM). STUDY DESIGN: We stained cells by the Papanicolaou method after fixation in 1.25% glutaraldehyde for LM and then attempted to observe them successively by SEM-TEM after fixation in 2% paraformaldehyde and 2% osmium tetroxide. RESULTS: On SEM, intranuclear inclusions were observed as elevated parts, like hills, and nuclear grooves were observed as deep fissures or shallow cracks, sometimes with a few in one cell. On TEM, both intranuclear inclusions and nuclear grooves seemed formed by the nuclear membranes. Intranuclear inclusions also possessed cytoplasm and/or cytoplasmic organelles within some expanded areas in the nuclear grooves. CONCLUSION: It was evident from our three-step technique that intranuclear inclusions and nuclear grooves were essentially the same structures.

Influence of dietary fiber on the bioavailability of zinc in rats.

Young male albino rats were fed ad libitum semipurified diet supplemented with or without 5% of purified dietary fiber (cellulose, agar-agar, pectin, chitin or chitosan) for 31 days. Each test diet was carefully prepared in order to contain zinc at the level of 8 ppm from zinc acetate. In rats fed diets containing 5% of dietary fiber except chitosan, food consumption was higher than the control. The body weight gain of rats fed diets containing cellulose, pectin or chitin was higher than the control. However, food intake and body weight gain of rats fed the diet containing 5% of chitosan were definitely lower than not only the other fiber including diet groups but also the control group. When dietary fiber was added at 5% level to diet, zinc absorption was not changed to a considerable degree. But, the apparent zinc absorption of rats fed the agar-agar diet was 70%. On the other hand, zinc absorption in the pectin diet group was about 10% higher than the control. The total amount of zinc in tibia or femur of rats fed non-fiber diets was a little higher than that of rats fed fiber diets.

The Clinical Significance of TachoComb, a Fibrin Adhesive in Sheet Form.

Recent improvements in surgical techniques and procedures have been accompanied by attempts to develop adhesives to seal wounds. In the 1940s, Young et al., in animal studies, attempted to connect nervous tissue with a fibrin adhesive, however, as separation and purification of blood constituents were still in a primitive state, their efforts did not progress to clinical usage.

[Preclinical and clinical studies on the efficacy of bifonazole in patients with tinea pedis at 10 years after approval. Part 1. Susceptibility to bifonazole of clinical isolates of dermatophytes].

An investigation was carried out to determine whether or not here had been any changes in the susceptibility of clinically isolated strains of Trichophyton metagrophytes and Trichophyton rubrum (both leading causes of tinea) to bifonazole, an imidazole derivative and antifungal for topical use. Susceptibility was measured in 107 strains of these fungi isolated from clinical samples during a study on the treatment of tinea pedis with Mycospor cream in 1995, 42 strains isolated and stored in 1990, and 39 strains isolated and stored prior to development of the drug. The results are as follows: (1) There was no distinct difference in the susceptibility to bifonazole of T. mentagrophytes strains isolated before 1986 and those isolated in 1990 or 1995. (2) T. rubrum strains isolated before 1986 were slightly more susceptible to bifonazole than those isolated in 1995, while the 1990 strains were slightly less susceptible than the 1995 strains, but the difference was not significant. (3) The highest MICs of bifonazole for all the T. mentagrophytes and T. rubrum strains isolated from before 1986 and those in 1995 were relatively low, being 2.5 micrograms/ml and 1.25 micrograms/ml, respectively. These results suggest that no resistance or reduced susceptibility to bifonazole has emerged among clinical isolates of dermatophytes since the development of the drug.

[Fundamental and clinical studies on the efficacy of bifonazole in patients with tinea pedis at 10 years after approval. Part 2. Clinical evaluation].

The usefulness of bifonazole (Mycospor), a topical imidazole antifungal agent approved 10 years ago, was evaluated for the treatment of tinea pedis. Mycospor cream was applied by 141 patients with tinea pedis once daily for 4 233ks, and the clinical efficacy and adverse reactions (as well as any correlations with susceptibility of isolates and the mycological activity of the agent against these isolates) were studied. The results were then compared to those of a previous study. The following results were obtained. 1. Mycological activity Mycological examination results became negative in 63.2% (36/57) of the patients with plantar tinea pedis, in 94.1% (32/34) of those with interdigital tinea pedis, and in 74.7% (68/91) of all tinea pedis patients. 2. Mycological activity and MIC No correlation was found between the MICs of bifonazole against the pathogenic fungi and the rate of eradication on mycological examination. 3. Improvement of symptoms The improvement rates for local symptoms were 82.5% for plantar tinea pedis, 85.7% for interdigital tinea pedis, and 83.7% for all tinea pedis. 4. Clinical efficacy Good clinical efficacies were found in 61.4% of the patients with plantar tinea pedis, in 88.6% of those with interdigital tinea pedis, and in 71.7% of all patients. 5. Safety Regarding adverse reactions, what seemed to be contact dermatitis was reported in 5 out of 127 cases (3.9%). The reaction decreased or disappeared in all cases. 6. Usefulness Mycospor was found to be useful in 64.9% of patients with plantar tinea pedis, in 88.6% of those with interdigital tinea pedis, and in 73.9% of all tinea pedis patients. 7. Comparison with former results The results obtained in the present clinical study were comparable to those obtained in patients with tinea pedis treated in a double-blind comparative study conducted during the development of as a new topical antifungal agent. From the above results, Mycospor cream was confirmed to be still useful, although it has been used widely for the topical treatment of cutaneous mycoses in the past 10 years since its approval.

[Can ulinastatin be an effective inhibitor of human polymorphonuclear granulocyte elastase under severe stressed state?].

The inhibitory effect of ulinastatin (UST), an intrinsic human trypsin inhibitor was investigated on the activity of polymorphonuclear granulocyte elastase (PMNE) with or without alpha 1-protease inhibitor (alpha 1-PI) using the in vitro models. The results of the dodecyl-sulfate-electrophoresis (SDS-PAGE), indicated that splitting-action of crude granulocyte enzyme solution on the plasma fibronectin was inhibited by concentration-dependent UST of an equivalent value for treatment of stressed state. The PMNE-UST complex was competitively replaced by PMNE-alpha 1-PI complex in vitro models of inflammatory focus and circulation, hence UST was weaker than alpha 1-PI in its binding-affinity for PMNE. The PMNE activity was directly inhibited by UST in the inflammatory focus and circulation with or without alpha 1-PI.