Dysregulated circadian rhythm pathway in human osteoarthritis: NR1D1 and BMAL1 suppression alters TGF-ß signaling in chondrocytes.

OBJECTIVES: Circadian rhythm (CR) was identified by RNA sequencing as the most dysregulated pathway in human osteoarthritis (OA) in articular cartilage. This study examined circadian rhythmicity in cultured chondrocytes and the role of the CR genes NR1D1 and BMAL1 in regulating chondrocyte functions. METHODS: RNA was extracted from normal and OA-affected human knee cartilage (n = 14 each). Expression levels of NR1D1 and BMAL1 mRNA and protein were assessed by quantitative PCR and immunohistochemistry. Human chondrocytes were synchronized and harvested at regular intervals to examine circadian rhythmicity in RNA and protein expression. Chondrocytes were treated with small interfering RNA (siRNA) for NR1D1 or BMAL1, followed by RNA sequencing and analysis of the effects on the transforming growth factor beta (TGF-ß) pathway. RESULTS: NR1D1 and BMAL1 mRNA and protein levels were significantly reduced in OA compared to normal cartilage. In cultured human chondrocytes, a clear circadian rhythmicity was observed for NR1D1 and BMAL1. Increased BMAL1 expression was observed after knocking down NR1D1, and decreased NR1D1 levels were observed after knocking down BMAL1. Sequencing of RNA from chondrocytes treated with NR1D1 or BMAL1 siRNA identified 330 and 68 significantly different genes, respectively, and this predominantly affected the TGF-ß signaling pathway. CONCLUSIONS: The CR pathway is dysregulated in OA cartilage. Interference with circadian rhythmicity in cultured chondrocytes affects TGF-ß signaling, which is a central pathway in cartilage homeostasis.

Relationship between sun-protection factor and application thickness in high-performance sunscreen: double application of sunscreen is recommended.

BACKGROUND: High-performance sunscreen protects both healthy consumers and photosensitive patients from strong ultraviolet (UV) exposure. The sun-protection factor (SPF), which indicates the efficacy of UV protection, is determined using a prescribed sunscreen application thickness of 2.0 mg/cm(2). Therefore, users should apply at least 2.0 mg/cm(2) of sunscreen to obtain the level of UV protection expected from a product. In most cases, however, users apply insufficient amounts of sunscreen. AIM: To determine the amount of sunscreen applied under specific conditions, and the relationship between application thickness and SPF value in high-performance sunscreen. METHODS: The amount of applied sunscreen was calculated under practical conditions and conditions that directed a double application. The SPF values of high-performance sunscreen applied at three thicknesses (2.0, 1.0 and 0.5 mg/cm(2)) were determined according to the international SPF testing method. RESULTS. The relationship between SPF value and application thickness correlated in a logarithmic curve. The mean application thickness under practical conditions was approximately 1 mg/cm(2), and directing subjects to use a double application increased the application thickness to nearly 2 mg/cm(2). CONCLUSION: Encouraging a double application of sunscreen will help users apply products at a thickness sufficient to achieve expected SPF efficacy. We recommend that guidance on double application of sunscreen should be posted in public locations where sunscreen is likely to be in use.

Red ear syndrome presenting with vestibular migraine: case study and review of the literature.

BACKGROUND: Red ear syndrome is a rare disorder in which the colour of the ear suddenly becomes red, with discomfort, pain and a burning sensation. This paper reports a case of primary red ear syndrome presenting with vestibular migraine. CASE REPORT: A 39-year-old woman from Bangladesh reported dizziness and repeated headaches experienced since 18 years of age. She initially attended our hospital with dizziness aged 34 years. When dizzy, the colour of her right ear sometimes became red. Therefore, she was diagnosed with red ear syndrome with vestibular migraine. CONCLUSION: This patient experienced repeated episodes of a red ear with discomfort, leading to the diagnosis of red ear syndrome. In addition, she had repeated dizziness and headaches, and was also diagnosed with vestibular migraine. The diagnosis of red ear syndrome with vestibular migraine should be considered in cases of dizziness and headache with recurrent redness of the ear.

Induction of hypertrophic chondrocyte-like phenotypes by oxidized LDL in cultured bovine articular chondrocytes through increase in oxidative stress.

OBJECTIVE: It has been reported that the lectin-like oxidized low-density lipoprotein (Ox-LDL) receptor 1 (LOX-1) is expressed by chondrocytes in osteoarthritis (OA) cartilage and that Ox-LDL binding to LOX-1 increases intracellular oxidative stress in cultured bovine articular chondrocytes (BACs). It was recently demonstrated that reactive oxygen species (ROS) induce hypertrophic differentiation of chondrocytes in the growth plate. It has also been shown that activated chondrocytes in OA have hypertrophic chondrocyte-like phenotypes. The purpose of this study was to determine whether Ox-LDL induces hypertrophic chondrocyte-like phenotypes in BACs. DESIGN: Changes in type X collagen (COL10) and runt-related transcription factor 2 (Runx2) mRNA expression in BACs after Ox-LDL stimulation were investigated using real-time polymerase chain reaction (PCR). Western blotting and immunofluorescent cell staining were used to investigate changes in protein level. The antioxidant N-acetyl cysteine (NAC) was used to ascertain whether oxidative stress is involved in COL10 and Runx2 expression. We induced LOX-1 knockdown cells using small interfering RNA (siRNA) to examine the receptor specificity of Ox-LDL. RESULTS: COL10 expression was upregulated by Ox-LDL in a time- and dose-dependent manner. Immunofluorescent staining showed that Ox-LDL increased COL10 production in the extracellular matrix. Ox-LDL-induced upregulation of COL10 was suppressed by pretreatment with NAC and siRNA. Expression of Runx2 was upregulated by Ox-LDL and H(2)O(2), and these effects were suppressed by NAC pretreatment. CONCLUSION: Ox-LDL binding to LOX-1 induces a hypertrophic chondrocyte-like phenotype through oxidative stress, indicating that Ox-LDL plays a role in the degeneration of cartilage.

Oxidized LDL binding to LOX-1 enhances MCP-1 expression in cultured human articular chondrocytes.

OBJECTIVE: It has been suggested that oxidized low-density lipoprotein (ox-LDL) has some roles in progression of osteoarthritis. The purpose of this study is to investigate whether ox-LDL binding to lectin-like ox-LDL receptor 1 (LOX-1) enhances monocyte chemoattractant protein 1 (MCP-1) expression in cultured human articular chondrocytes (HACs). METHOD: The time course and dose response of MCP-1 mRNA expression and MCP-1 protein release into medium following ox-LDL stimulation were investigated using quantitative Real time PCR (delta-delta Ct method) and enzyme-linked immunosorbent assay (ELISA), respectively. To examine the receptor specificity of ox-LDL action, HACs were preincubated with anti-human LOX-1 monoclonal antibody (TS92). RESULTS: A time-course study revealed that MCP-1 mRNA expression increased 5.09+/-0.86 fold 12h after ox-LDL stimulation compared to time-0. ox-LDL stimulation increased MCP-1 protein level in conditioned medium in a time-dependent manner. Increased MCP-1 level was evident 6h after stimulation, reaching 830+/-91 pg/ml at 24h (33+/-8 pg/ml at time-0). Dose responses of MCP-1 expression were also evident in mRNA and protein levels. Pretreatment with TS92 markedly suppressed these stimulating effects of ox-LDL, although that with non-specific IgG did not. Native LDL did not affect MCP-1 expression. CONCLUSION: Our results suggest that ox-LDL enhances MCP-1 expression in HACs and supports the hypothesis that ox-LDL is involved in cartilage degeneration.

Requirement for etoposide in the treatment of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis.

PURPOSE: We sought to identify the clinical variables most critical to successful treatment of Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH). PATIENTS AND METHODS: Among the factors tested were age at diagnosis (< 2 years or > or = 2 years), time from diagnosis to initiation of treatment with or without etoposide-containing regimens, timing of cyclosporin A (CSA) administration during induction therapy, and the presence or absence of etoposide. RESULTS: By Kaplan-Meier analysis, the overall survival rate for the entire cohort of 47 patients, most of whom had moderately severe to severe disease, was 78.3% +/- 6.7% (SE) at 4 years. The probability of long-term survival was significantly higher when etoposide treatment was begun less than 4 weeks from diagnosis (90.2% +/- 6.9% v 56.5% +/- 12.6% for patients receiving this agent later or not at all; P <.01, log-rank test). Multivariate analysis with the Cox proportional hazards model demonstrated the independent prognostic significance of a short interval from EBV-HLH diagnosis to etoposide administration (relative risk of death for patients lacking this feature, 14.1; 95% confidence interval, 1.16 to 166.7; P =.04). None of the competing variables analyzed had significant predictive strength in the Cox model. However, concomitant use of CSA with etoposide in a subset of patients appears to have prevented serious complications from neutropenia during the first year of treatment. CONCLUSION: We conclude that early administration of etoposide, preferably with CSA, is the treatment of choice for patients with EBV-HLH.

Adenoviral infection in hematopoietic stem cell transplantation: early diagnosis with quantitative detection of the viral genome in serum and urine.

Early diagnosis and prompt introduction of effective therapy are imperative to manage systemic, often fatal adenoviral (AdV) disease following hematopoietic stem cell transplantation (SCT). We evaluated the usefulness of real-time polymerase chain reaction (PCR) in the diagnosis of AdV disease in SCT recipients. Seven SCT recipients, including three with AdV disease, were retrospectively evaluated for AdV genome detection. In serum specimens, the AdV genome was detected at >10(3) copies/ml in the pre-SCT period in two of the five recipients studied. These two patients subsequently developed AdV disease. The three patients with AdV disease had high levels of >10(5) copies/ml during the 4-6 weeks post-SCT period. In none of these patients was the AdV genome detected in urine specimens in pre-SCT period. However, three recipients with detectable urinary levels during the period 1-2 weeks post-SCT subsequently developed AdV disease. Regarding the outcome, two of the three patients with AdV disease died of progressive renal failure. Our results suggest that quantitative determination of the AdV genome in serum and urine is useful to identify patients at high risk of developing AdV disease. Prospectively applied, these measures are expected to improve the dismal outcome of AdV disease in SCT recipients.

Acute renal failure due to adenovirus-associated obstructive uropathy and necrotizing tubulointerstitial nephritis in a bone marrow transplant recipient.

Management of post-transplant complications caused by severe adenoviral infection remains a major therapeutic challenge. A 17-year-old male who had undergone bone marrow transplantation for the treatment of acute lymphoblastic leukemia developed complete anuria following hemorrhagic cystitis 34 days after the transplant procedure. The computed tomogram scan revealed bilateral hydronephrosis, indicating acute renal failure because of obstructive uropathy. The emergency procedure of percutaneous nephrostomy caused massive bleeding in the left kidney, which eventually required a nephrectomy. Adenovirus-positive severe necrotizing tubulointerstitial nephritis was the histopathological diagnosis. Post-transplant acute renal failure because of hydronephrosis, which could be complicated by adenovirus-induced renal parenchymal disease, is of great concern and may cause significant problems with interventional treatment.

Structures of oligomannoside chains of alpha-mannosidase from porcine kidney.

Lysosomal acid alpha-mannosidase from porcine kidney was found to contain mannose (4.8%), galactose (0.9%), fucose (0.5%), N-acetylglucosamine (3.1%), and mannose 6-phosphate (0.1%). Approximately 50% of the total hexose of the oligosaccharide chains could be released by endo-beta-N-acetylglucosaminidase-H (endo-H). They were predominantly neutral, oligomannoside-type oligosaccharides containing 5, 6, and 9 mannose residues, respectively, in the centesimal ratio of 36:25:34. 500-MHz 1H-NMR spectroscopy in conjunction with sequential exoglycosidase digestion of the reduced compounds revealed that each of the three fractions consisted of a single isomer only; the Man9 compound has the following structure: (Formula: see tex). The Man6-compound lacks Man residues D1, D2, and D3, while the Man5-compound lacks Man-C as well. In addition to the neutral ones, some (5%) phosphorylated oligomannoside-type oligosaccharides were obtained. The endo-H resistant glycopeptides were subjected to hydrazinolysis. Approximately 60% of the oligosaccharides released by hydrazine were found to be of rather small size; their composition can be represented asMan2-3GlcNAc[Fuc]0-1GlcNAcol. The remaining 40% consist of larger-size galactose-containing, N-acetyllactosamine-type oligosaccharides. Studies involving sequential exoglycosidase digestion and 500-MHz 1H-NMR spectroscopy performed on the highly purified small-sized compounds revealed the following four structures for the endo-H-resistant oligosaccharides: (Formula: see text).

Treatment strategies for Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH).

In Epstein-Barr virus (EBV) infection, the virus immortalizes B lymphocytes and cytotoxic T lymphocytes (CTLs) are directed toward both latent and lytic viral antigens expressed on EBV-infected B-cells. Various EBV-associated diseases occur as a result of this disruption of immune surveillance. In the majority of EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) cases, the major cell types containing EBV DNA are not B-cells, but clonally proliferating T-cells or NK-cells. Proliferation of these cells produces severe immune reactions in the host, and the clinical features related to massive cytokine production at the onset of disease are unique and distinct from other EBV-associated diseases. In the treatment of EBV-HLH, therapeutic infusion of EBV-specific CTLs appears to be ineffective, and eradication of EBV-containing cells is useful but not sufficient to save lives, because of high incidence of acute mortality due to cytokine-induced multiple organ failure and neutropenia-associated opportunistic infections. The optimal treatment strategy for this disease consists of three steps: (1) control of cytokine storm including coagulopathy and multiple organ failure, (2) control of opportunistic infections, and (3) eradication of clonally proliferating EBV-containing T- or NK- cells by immunochemotherapy and, if necessary, hemopoietic stem cell/bone marrow transplantation (SCT/BMT).

Molecular analysis of latent membrane protein 1 in patients with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in Japan.

Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is considered to be an oncoprotein because it is crucial for B-lymphocyte transformation. Since a 30 base pair (bp) deletion in the carboxy-terminal portion of the LMP1 gene was found in a CAO cell line derived from nasopharyngeal carcinoma containing EBV, an association between EB viral genetic alteration and tumorigenicity has been postulated. In this study we have analyzed LMP1 DNA isolated from 10 Japanese patients with EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH). In all HLH patients, we found the 30 bp deletion and 4-8-tandem repeats of the sequence DNGPQDPDNTD in the LMP1 gene. Furthermore, detailed amino acid (aa) sequence analysis revealed that 7 aa substitutions identical to those found in CAO-LMP1 but not in B95.8 cell line-LMP1 were found in all the HLH cases. NF-kappaB assay revealed that HLH-LMP1 activated NF-kappaB significantly more than that of B95.8-LMP1 (p=0.032). We conclude that EBV from all of our HLH cases shared common genetic characteristics with EBV obtained from the CAO cell line, which is distinct from the wild-type EBV isolated from the B95.8 cell line. These data suggest that the mutational changes of the LMP1 gene may play an important role in the pathogenesis of these fatal EBV-related disorders.

Recent developments in the management of haemophagocytic lymphohistiocytosis.

Over the past two decades, the underlying pathophysiology of haemophagocytic lymphohistiocytosis (HLH) (synonyms: haemophagocytic syndrome, macrophage activation syndrome) has been well recognised. Cytokine storm plays a major role, which derives from an inappropriate immune reaction caused by proliferating and activated T-cell or natural killer (NK) cells associated with macrophage activation and inadequate apoptosis of immunogenic cells. Many biological parameters reflecting activity of disease or response to treatment have been identified, in particular, serum ferritin has been confirmed to be one of the markers for HLH. The common types of HLH consist of non-hereditary (acquired) infection-associated disease such as Epstein-Barr virus (EBV)-haemophagocytic lymphohistiocytosis (HLH) and hereditary (familial) disease such as FHL, in which, at the molecular level, dysfunctional perforin was clarified. Regarding the therapeutic strategies, prompt differential diagnosis of underlying disease is essential and choice of treatment should be based on the risk (low or high) of prognosis, where either cyclosporin A, steroids or iv. immunoglobulin (IVIG) may be indicated as initial treatment for low-risk patients, with etoposide-containing regimens for high-risk patients. Significant improvement of prognosis has been obtained by incorporating intensive supportive care at the disease onset and prompt introduction of immunosuppressants to control cytokine storm. Subsequent immunochemotherapy and haemopoietic stem cell transplantation have contributed significantly to further improve survival of hereditary and refractory HLH patients.

Fulminant and relentless cutaneous necrosis with excruciating pain caused by calciphylaxis developing in a patient undergoing peritoneal dialysis.

A 50-year-old Japanese female with chronic renal failure who had been on continuous ambulatory peritoneal dialysis developed fulminant systemic cutaneous necrosis that began as painful livedo reticularis-like skin lesions on her thighs. Because of disseminated vascular calcification within the muscular layer of her lower limbs, we eventually diagnosed her with calciphylaxis. The skin necrosis progressed rapidly, and she died of sepsis and pneumonia on the 53rd hospital day. In addition to her long-lasting severe hyperparathyroidism and extremely elevated serum phosphorus and calcium levels, mechanical, frictional stimulation inflicted on the local skin and administration of corticosteroids were suspected to have precipitated the calciphylaxis. Our lack of awareness of this disease in its early stages resulted in our missing the chance to do a parathyroidectomy that might have changed the course. It is important to know the clinical features of this rare disease in order to make a diagnosis as early as possible.

[The shape of posterior staphyloma in high myopia].

In 105 eyes of 68 highly myopic patients with focal chorioretinal atrophy in posterior staphyloma, the shape of the staphyloma and the process of backward elongation were studied. Indirect binocular ophthalmoscopy (and in some cases slit-lamp biomicroscopy of the fundus.) was used to observe the staphyloma. The results were as follows: 1) In 72.4% of all the eyes, patchy atrophy (part or whole) protruded or became dented in the staphyloma. The protrusion and the dent did not correlate with age. The refraction of eyes which had protruded patchy atrophy was significantly stronger than those which had dented patchy atrophy. The axial length of eyes which had protruded patchy atrophy was also significantly longer. 2) The protrusion and the dent were localized in patchy atrophy with a diameter of over 1.5 PD. 3) In 80.3% of peripapillary chorioretinal atrophic cases, atrophy protruded or became dented in the staphyloma. 4) In 43.8% the limits of the location of retinal blood vessels formed part of the borderlines of the staphyloma. 5) In 28.6%, steps were created in the staphyloma by the limits of the location of retinal blood vessels. As the staphyloma progresses, patchy atrophy, annular-type myopic crescents, and the location of retinal blood vessels influence the shape of the staphyloma.

[Pregnancy obtained by in vitro fertilization (IVF) with epididymal spermatozoa in obstructive azoospermia: report of two cases].

Epididymal spermatozoa aspiration was performed in two cases of obstructive azoospermia. After the procedure, in both cases, the patient's wives obtained twin pregnancies by this method in conjunction with in vitro fertilization (IVF) and embryo transfer (ET). The patient's wife in one case had a normal delivery. The patient's wife in the other case, however, aborted artificially because she had cerebral infarction. Epididymal spermatozoa aspiration proved efficacious for male sterility in cases of obstructive azoospermia.

Marked species differences in the bioavailability of midazolam in cynomolgus monkeys and humans.

The bioavailability (F) of midazolam in cynomolgus monkeys (0.02) was markedly lower than that in humans (0.24-0.46) and the reason for this difference in F between the two species was investigated. Based on the area under the plasma concentration-time curve after intravenous and intraportal infusion to cynomolgus monkeys, the hepatic availability (F(h)) was estimated as 0.66. The fraction of dose absorbed (F(a)) estimated from the single-pass intestinal perfusion method was 1.0 in cynomolgus monkeys. The intestinal availability (F(g) = F/F(a)/F(h)) was calculated as 0.03 in cynomolgus monkeys. Since the F(a) of midazolam has been reported to be almost 1.0 in humans, F(h) and F(g) were calculated as 0.33-0.76 and 0.46-1.00 when the reference values for hepatic blood flow (1026-1530 ml h(-1) kg(-1)) were used. In conclusion, the main reason for low F in cynomolgus monkeys was the markedly higher first-pass intestinal metabolism seen in cynomolgus monkeys compared with humans.

Pharmacokinetics of barnidipine hydrochloride, a new dihydropyridine calcium channel blocker, in the rat, dog and human.

1. The pharmacokinetics of a new calcium antagonist barnidipine hydrochloride, a stereochemically pure enantiomer, was studied after intravenous and oral dosing to the rat and dog, and oral to man. 2. After intravenous dosing, plasma concentrations of barnidipine hydrochloride declined bi-exponentially with the terminal half-lives of 0.6 h in the rat and 4.1 h in the dog. The blood clearance was 5.2 l/h/kg in the rat and 3.3 l/h/kg in the dog, and was comparable with hepatic blood flow in both species. 3. After oral dosing, plasma concentrations of barnidipine hydrochloride peaked rapidly (0.3-0.4 h in the rat and dog, 1.0-1.6 h in man). Cmax and AUC rose non-linearly with increasing doses in all three species. 4. The absolute bioavailability was low (11-18% in the rat and 6-9% in the dog), suggesting a marked first-pass metabolism.

Metabolism and pharmacokinetics of barnidipine hydrochloride, a calcium channel blocker, in man following oral administration of its sustained release formulation.

1. The metabolism and pharmacokinetics of barnidipine hydrochloride, a 1, 4-dihydropyridine calcium antagonist were evaluated following single oral administration of a sustained release formulation (SR) capsule comprising of quick and slow release pellets to healthy male volunteers. 2. Various metabolites were identified and quantitated by newly established GC-MS analytical methods. Major metabolites were the hydrolyzed product of the benzyl-pyrrolidinyl ester (M-3) in plasma and its oxidized pyridine product (M-4) in plasma and urine. The pyridine form of unchanged barnidipine and the N-debenzylated product were observed as minor metabolites. Therefore, the primary metabolic pathways in man are (a) hydrolysis of the benzylpyrrolidine ester, (b) N-debenzylation, and (c) oxidation of the dihydropyridine ring. 3. When the SR and normal capsules were administered at a dose of 10 mg to six subjects in a crossover design, AUC 0-infinity of unchanged drug, M-3 and 4 in each subject receiving the SR were 97 +/- 15, 85 +/- 31 and 76 +/- 21% respectively of those subjects receiving the normal formulation. The sum of the excretion of urinary metabolites for the SR formulation was 65 +/- 6% of that for the normal formulation. These data suggest that the absorption of the SR formulation is slightly reduced but that its bioavailability is comparable to that of the normal formulation.