Genetically fused charged peptides induce rapid crystallization of proteins.

We utilized electrostatic interaction to induce rapid crystallization of streptavidin. Simply mixing streptavidins possessing either a positively or negatively charged peptide at their C-terminus generated diffraction-quality crystals in a few hours. We modified the streptavidin crystals with fluorescent molecules using biotin, demonstrating the concept of protein crystals as functional biomaterials.

Evaluation of the efficacy and safety of the combination of gemcitabine and nedaplatin for elderly patients with advanced non-small-cell lung cancer.

OBJECTIVE: The aim of the present study was to retrospectively assess the safety and efficacy of the combination of gemcitabine and nedaplatin in elderly patients with advanced non-small-cell lung cancer (NSCLC). METHODS: Patients ≥75 years with previously untreated NSCLC who underwent chemotherapy consisting of gemcitabine (800 mg/m(2) on days 1 and 8) and nedaplatin (80 mg/m(2) on day 1) every 3 weeks were retrospectively analyzed. RESULTS: Of the 35 patients, 28 were men and 7 were women, with a mean age of 78 years (range 75-87); 10 patients had stage IIIB disease and 25 patients had stage IV disease. The overall response rate was 45.7% (95% confidence interval 28.8-63.4). The median survival time was 14 months (range 3-44). Grade 3-4 toxicities included neutropenia in 74.3%, thrombocytopenia in 48.6%, anemia in 34.3%, hepatic dysfunction in 11.4%, and infection in 2.9%. There were no treatment-related deaths. There were no differences in response rate and survival between patients aged 75-79 years and patients ≥80 years, although grade 3-4 thrombocytopenia and anemia were significantly more frequent in patients ≥80 years. CONCLUSION: Our results suggest that the combination of gemcitabine and nedaplatin is effective and well tolerated for selected elderly patients with advanced NSCLC.

Asthma programmes in diverse regions of the world: challenges, successes and lessons learnt.

International surveys have demonstrated that asthma is still underdiagnosed and undertreated in many parts of the world. Despite improvements in the standard of asthma care delivered in many areas, as evidenced by improved global asthma mortality data, much information on projects and programmes undertaken in resource-limited regions of the world is not in the public domain. The aim of this report is to review projects and programmes in diverse regions around the world so that health care providers, planners and consumers may draw on the successes, failures and lessons learnt. Such real world experiences may contribute to achieving Global Initiative for Asthma goals of asthma control. Asthma projects and programmes in Argentina, Australia, Brazil, China, Japan, Mexico, Philippines, Russia, South Africa and Turkey were discussed by a group of experts in asthma care, the Advancing Asthma Care Network, from their respective countries, over a course of three satellite meetings in 2010. Collective analyses consistently identified low rates of dissemination and implementation of national and international treatment guidelines, low levels of continuing medical education and training of primary health care professionals and access and distribution of inhaled corticosteroids to be major barriers that are critical to the overall success of a national asthma management programme. In the less developed asthma programmes, under-recognition and undertreatment further limited the success of the programmes. Evidence from well-established national asthma management programmes suggests that establishment of a successful programme entails a logical progression through specific developmental stages, starting with political/stakeholder endorsement and commitment, followed by epidemiological evaluation, evaluation of disease burden, evaluation of access to care and best therapy, and finally optimisation and maintenance therapy for individual patients.

Induction of insulin-like growth factor-I by interleukin-17F in bronchial epithelial cells.

BACKGROUND: Increased expression of IL-17F has been noted in the airway of asthmatic patients, but its role in asthma has not been fully elucidated. Insulin-like growth FACTOR-I (IGF-I) is known to be involved in airway remodelling and inflammation, while its regulatory mechanisms remain to be defined. OBJECTIVE: To further clarify the biological function of IL-17F, we investigated whether IL-17F is able to regulate the expression of IGF-I in bronchial epithelial cells. METHODS: Bronchial epithelial cells were stimulated with IL-17F in the presence or absence of T-helper type 2 cytokines. Various kinase inhibitors were added to the culture to identify the key signalling events leading to the expression of IGF-I, in conjunction with the use of short interfering RNAs (siRNAs) targeting mitogen- and stress-activated protein kinase (MSK) 1, p90 ribosomal S6 kinase (p90RSK), and cyclic AMP response element-binding protein (CREB). RESULTS: IL-17F significantly induced IGF-I gene and protein expression, and co-stimulation with IL-4 and IL-13 augmented its production. MAP kinase kinase (MEK) inhibitors and the Raf1 kinase inhibitor significantly inhibited IGF-I production, and the combination of PD98059 and Raf1 kinase inhibitor showed further inhibition. Overexpression of Raf1 and Ras dominant-negative mutants inhibited its expression. MSK1 inhibitors significantly blocked IL17F-induced IGF-I expression. Moreover, transfection of the siRNAs targeting MSK1, p90RSK, and CREB blocked its expression. CONCLUSIONS: In bronchial epithelial cells, IL-17F is able to induce the expression of IGF-I via the Raf1-MEK1/2-ERK1/2-MSK1/p90RSK-CREB pathway in vitro.

Effect of tiotropium bromide on airway inflammation and remodelling in a mouse model of asthma.

BACKGROUND: Tiotropium bromide, a long acting muscarinic receptor inhibitor, is a potent agent for patients with bronchial asthma as well as chronic obstructive pulmonary disease. OBJECTIVE: The aim of this study was to evaluate whether tiotropium bromide can inhibit allergen-induced acute and chronic airway inflammation, T helper (Th)2 cytokine production, and airway remodelling in a murine model of asthma. METHODS: Balb/c mice were sensitized and challenged acutely or chronically to ovalbumin (OVA). The impact of tiotropium bromide was assessed using these mice models by histologic, morphometric, and molecular techniques. Moreover, the effect of tiotropium bromide on Th2 cytokine production from purified human peripheral blood mononuclear cells (PBMCs) was assessed. RESULTS: Treatment with tiotropium bromide significantly reduced airway inflammation and the Th2 cytokine production in bronchoalveolar lavage fluid (BALF) in both acute and chronic models of asthma. The levels of TGF-beta1 were also reduced by tiotropium bromide in BALF in a chronic model. The goblet cell metaplasia, thickness of airway smooth muscle, and airway fibrosis were all significantly decreased in tiotropium bromide-treated mice. Moreover, airway hyperresponsiveness (AHR) to serotonin was significantly abrogated by tiotropium bromide in a chronic model. Th2 cytokine production from spleen cells isolated from OVA-sensitized mice was also significantly inhibited by tiotropium bromide and 4-diphenylacetoxy-N-methylpiperidine methiodide, which is a selective antagonist to the M3 receptor. Finally, treatment with tiotropium bromide inhibited the Th2 cytokine production from PBMCs. CONCLUSION: These results indicate that tiotropium bromide can inhibit Th2 cytokine production and airway inflammation, and thus may reduce airway remodelling and AHR in a murine model of asthma.

Necrotizing fasciitis caused by Cryptococcus neoformans in a patient with pemphigus vegetans.

Cryptococcosis occurs most often in immunocompromised people. The cutaneous features of cryptococcosis include papules, pustules, nodules, subcutaneous swelling, abscesses, molluscum contagiosum-like or tumour-like lesions, cellulitis, blisters, ulcers and very rarely, necrotizing fasciitis (NF). NF is a destructive soft-tissue infection that is most typically caused by group A streptococci or by a combination of facultative and anaerobic bacteria. We present the case of a 55-year-old woman with pemphigus vegetans, who developed cryptococcal NF in the legs. She had been treated with immunosuppressants including plasmapheresis and pulse therapy with steroid and cyclophosphamide. Cryptococcal NF localized to the legs is very rare. Because diagnosis and treatment of cryptococcal infection is often delayed, clinicians should be aware of the possibility of cryptococcal infection when antibacterial therapy is not effective in an immunocompromised patient.

Clinical features of microdeletion 9q22.3 (pat).

Congenital anomaly syndromes manifesting overgrowth are rare, and only a small number of recognized or defined conditions are known to be associated with overgrowth. Some of them are related to genomic imprinting as a genetic cause. We report a girl who showed pre- and postnatal overgrowth who was found to have a 2.3-Mb deletion of 9q22.32 involving PTCH1, the gene responsible for Gorlin syndrome (nevoid basal cell carcinoma syndrome), by array-comparative genomic hybridization analysis. Clinical re-evaluation according to the diagnostic criteria was performed after identification of the PTCH1 deletion, and the patient was then diagnosed as having Gorlin syndrome. Further delineation involved unusual features including cerebellar dysplasia, an ectopic meninx on her shoulder, and an intraorbital hemangioma. Overgrowth is not a common finding in Gorlin syndrome. We reviewed 23 patients reported to have a 9q22 deletion. Five patients, including our patient, had overgrowth and loss of the paternal allele.

Preventing oxidative stress: a new role for XBP1.

Antioxidant molecules reduce oxidative stress and protect cells from reactive oxygen species (ROS)-mediated cellular damage and probably the development of cancer. We have investigated the contribution of X-box-binding protein (XBP1), a major endoplasmic reticulum stress-linked transcriptional factor, to cellular resistance to oxidative stress. After exposure to hydrogen peroxide (H(2)O(2)) or a strong ROS inducer parthenolide, loss of mitochondrial membrane potential (MMP) and subsequent cell death occurred more extensively in XBP1-deficient cells than wild-type mouse embryonic fibroblast cells, whereas two other anticancer agents induced death similarly in both cells. In XBP1-deficient cells, H(2)O(2) exposure induced more extensive ROS generation and prolonged p38 phosphorylation, and expression of several antioxidant molecules including catalase was lower. Knockdown of XBP1 decreased catalase expression, enhanced ROS generation and MMP loss after H(2)O(2) exposure, but extrinsic catalase supply rescued them. Overexpression of XBP1 recovered catalase expression in XBP1-deficient cells and diminished ROS generation after H(2)O(2) exposure. Mutation analysis of the catalase promoter region suggests a pivotal role of CCAAT boxes, NF-Y-binding sites, for the XBP1-mediated enhancing effect. Taken together, these results indicate a protective role of XBP1 against oxidative stress, and its positive regulation of catalase expression may at least in part account for this function.

Alterações do plano oclusal decorrentes do tratamento ortopédico com o Bionatorde Balters em pacientes com má-oclusão classe II, divisão 1a / Occlusal plane changes in patients with Class II, division 1a treated with Balters’ Bionator

Objetivo: Esse estudo avaliou alterações no plano oclusal decorrentes do tratamento de pacientes com má oclusão de Classe II e retrognatismo mandribular por meio do bionator de Balters. Material e Método: a amostra utilizada neste estudo retrospectivo foi coletada no acervo de documentação ortodôntica do curso de especialização da Universidade Paulista – UNIP, e consta de 60 teleradiografias (sendo 30 tomadas realizadas antes do tratamento e 30 após) em norma lateral de 30 indivíduos portadores de má-oclusão Classe II, divisão 1a, brasileiros, de ambos os sexos, com idade média de 9 anos e 2 meses ao iniciarem o tratamento. As avaliações cefalométricas foram realizadas em dois tempos, T1 (antes do tratamento) e T2 (após tratamento ortopédico). Foram avaliados os ângulos Ba-Na/plano palatino, Se-Na/plano palatino e plano palatino/plano oclusal funcional. Resultado: Apenas a variável plano palatino/plano oclusal apresentou diferença estatiscamente siginificante quando comparadas antes e após o tratamento (p<0,05). Conclusão: Os resultados obtidos demonstraram uma estabilidade significativa do plano palatino em relação aos planos Ba-Na e Se-Na e uma rotação no sentido anti-horário do plano oclusal funcional, estatisticamente significante em relação ao plano palatino.

A case of Varicella zoster virus polyneuropathy: involvement of the glossopharyngeal and vagus nerves mimicking a tumor.

A 36-year-old woman presented with glossopharyngeal and vagus nerve palsy, which proved to be herpes zoster based on the high titers of Varicella zoster virus antibody in her serum. Thin-section T1-weighted images with contrast media demonstrated swelling and distinct contrast enhancement of the glossopharyngeal and vagus complex, mimicking a tumor. Following MR imaging, the size of the nerve complex returned to normal; however, the contrast enhancement remained longer than the symptoms.

Inhibitory effects of suplatast tosilate on the differentiation and function of monocyte-derived dendritic cells from patients with asthma.

BACKGROUND: Dendritic cells (DCs) are antigen-presenting cells that efficiently activate T cells. OBJECTIVE: We examined the effects of suplatast tosilate, which prevents T-helper type 2 responses, on the differentiation and function of monocyte-derived DCs (moDCs). METHODS: DCs were differentiated in vitro from peripheral monocytes from patients with asthma by the addition of granulocyte macrophage colony-stimulating factor and IL-4 in the presence or absence of suplatast tosilate. Cell surface molecules (CD1a, CD14, CD80, CD83, CD86, HLA-DR) on immature and mature DCs were analysed with flow cytometry, and the secretion of CC chemokine ligand (CCL)17 (thymus and activation-regulated chemokine), IL-12p70, IL-12p40, and IL-10 was measured with an ELISA. We also studied the proliferative responses of allogeneic CD4(+) T cells from healthy subjects to DCs differentiated in the presence of suplatast tosilate. In addition, the production of IFN-gamma and IL-5 by CD4(+) T cells after coculture with untreated DCs or suplatast tosilate-treated DCs was measured with ELISA. RESULTS: Suplatast tosilate significantly inhibited the expression of CD1a, CD80, and CD86 on immature DCs and of CD1a, CD80, CD83, and CD86 on mature DCs. Suplatast tosilate also significantly inhibited the secretion of CCL17, IL-12p70, and IL-12p40; however, the secretion of IL-10 was not affected. The proliferative responses of allogeneic CD4(+) T cells to suplatast tosilate-treated DCs were suppressed. Moreover, suplatast tosilate-treated DCs had an impaired capacity to stimulate CD4(+) T cells to produce IFN-gamma and IL-5. CONCLUSION: Suplatast tosilate inhibits the differentiation, maturation, and function of moDCs.

Tonsillectomy in the treatment of pediatric Henoch-Schönlein nephritis.

The exact pathophysiology of HSN remains to be elucidated. Hence, a therapeutic strategy that enables curative treatments for all the various grades of HSN patients has yet to be established. We report our experience performing tonsillectomy combined with steroid therapy for 16 pediatric proteinuric Henoch-Schönlein nephritis (HSN) patients. All patients exhibited hematuria and proteinuria in their first HSN attack with the mean age of onset 7.7 years (range 4.75 - 13.9 years). Nine patients were diagnosed with clinically severe HSN presenting with massive proteinuria (> 1 g/m(2)/day). Renal biopsy findings performed in 6 patients were Grade II (3), Grade III (2) and Grade IV (1) according to the International Study of Kidney Diseases in childhood classification. Tonsillectomy was performed after 1-4 cycles of methylprednisolone pulses during oral prednisolone (0.5 - 1.5 mg/kg/day) therapy. In 2 patients, oral cyclophosphamide therapy was added before the tonsillectomy. The interval between the onset of HSN and tonsillectomy was 97.4 +/- 24.5 days (range 27 424 days). In all patients, proteinuria had disappeared by 6 months after the tonsillectomy and the urine findings had normalized. The interval between therapy initiation and complete remission was 9.6 +/- 2.0 months (range 2 - 26 months). Over follow-up periods of 4.9 +/- 0.6 years (range 2.2 - 9.3 years), no recurrences of Henoch-Schonlein purpura or HSN were observed. There was a significant correlation between early tonsillectomy performance and decreased time until normalization of the urine findings, indicating that the tonsils may have pivotal roles in the initiation and progression of HSN. Their elimination might promote the reversal of nephritis. Although this study is retrospective, we suggested that tonsillectomy at an early stage of HSN may be beneficial by shortening the period of illness and contributing to clinical recovery. Randomized controlled trials will be needed to confirm this supposition.

A polymorphism of the aldehyde dehydrogenase 2 gene is a risk factor for multiple lacunar infarcts in Japanese men: the Takahata Study.

The objective of the present study was to examine the association between a polymorphism of the aldehyde dehydrogenase 2 (ALDH2) gene and lacunar infarcts of the brain. We conducted a population-based, cross-sectional study on residents from two age groups (61- and 72-year olds). A total of 376 subjects participated in the study, which included brain magnetic resonance image and genetic analysis of the ALDH2 gene. Of the 61- and 72-year-old subjects, 46.4% and 64.3%, respectively, had one or more lacunar infarcts. The average number of infarcts also increased from 2.0 to 2.8 in men and from 2.3 to 3.5 in women. No significant association between the ALDH2 genotype and the presence of lacunar infarction (> or =1) was found. However, in subjects with lacunar infarction, the genotype of ALDH2 *1/*1 was associated with a larger number of the lesion ['single' versus 'multiple' odds ratio (OR) 3.73, 95%CI: 1.43-9.74] in men. The OR was comparable even after adjusting for alcohol consumption, tobacco habits, age, hypertension, hypercholesterolemia, and diabetes mellitus (DM) (OR 3.88; 95% CI: 1.10-13.66). In women, there was no significant association between the ALDH2 genotypes and lacunar infarcts. The present study revealed that the ALDH2 *1/*1 genotype was significantly associated with the prevalence of multiple lacunar infarcts in Japanese men.

Nonsteroidal anti-inflammatory drugs and oxidative stress in cancer cells.

Nonsteroidal antiinflammatory drugs (NSAIDs) induce apoptosis in a variety of cancer cells, including those of colon, prostate, breast and leukemia. In addition, the classical NSAIDs sulindac and aspirin are promising chemopreventive agents against colon cancer. NSAIDs inhibit cyclooxygenases (COX) preventing the formation of prostaglandins, prostacyclin and thromboxane. NSAIDs also exert other biological effects, including generation of reactive oxygen species (ROS) and inhibition of NF-kappaB-mediated signals. Despite many suggested mechanisms for their anticancer effects, it remains uncertain how they induce cell cycle arrest and apoptosis in cancer cells. Furthermore, there is little information on the selectivity of NSAIDs-mediated anticancer effects, although this is one of the most important issues in cancer therapy. Increased understanding of the biological basis for the anticancer activity of NSAIDs and their selectivity is essential for future therapeutic advances. In this paper, we propose that increased ROS generation is one of the key mechanisms for NSAIDs-mediated anticancer effects on various cancer cells.

Induction of apoptosis and cellular senescence in mice lacking transcription elongation factor, Elongin A.

Elongin A is a transcription elongation factor that increases the overall rate of mRNA chain elongation by RNA polymerase II. To gain more insight into the physiological functions of Elongin A, we generated Elongin A-deficient mice. Elongin A homozygous mutant (Elongin A(-/-)) embryos demonstrated a severely retarded development and died at between days 10.5 and 12.5 of gestation, most likely due to extensive apoptosis. Moreover, mouse embryonic fibroblasts (MEFs) derived from Elongin A(-/-) embryos exhibited not only increased apoptosis but also senescence-like growth defects accompanied by the activation of p38 MAPK and p53. Knockdown of Elongin A in MEFs by RNA interference also dramatically induced the senescent phenotype. A study using inhibitors of p38 MAPK and p53 and the generation of Elongin A-deficient mice with p53-null background suggests that both the p38 MAPK and p53 pathways are responsible for the induction of senescence-like phenotypes, whereas additional signaling pathways appear to be involved in the mediation of apoptosis in Elongin A(-/-) cells. Taken together, our results suggest that Elongin A is required for the transcription of genes essential for early embryonic development and downregulation of its activity is tightly associated with cellular senescence.

Synthetic double-stranded RNA induces multiple genes related to inflammation through Toll-like receptor 3 depending on NF-kappaB and/or IRF-3 in airway epithelial cells.

BACKGROUND: We hypothesized that synthetic double-stranded (ds)RNA may mimic viral infection and induce expression of genes related to inflammation in airway epithelial cells. OBJECTIVE: We analysed what gene was up-regulated by synthetic dsRNA poly I : C and then focused this study on the role of Toll-like receptor 3 (TLR3), a receptor of dsRNA and its transcriptional pathway. METHODS: Airway epithelial cell BEAS-2B and normal human bronchial epithelial cells were cultured in vitro. Expression of targets RNA and protein were analysed by PCR and ELISA. Localization of TLR3 expression in the cells was analysed with flow cytometry. To analyse the role of TLR3 and transcription factors, knockdown of these genes was performed with short interfering RNA (siRNA). RESULTS: Real-time PCR revealed that poly I : C significantly increased the expression of mRNAs for chemokines IP-10, RANTES, LARC, MIP-1alpha, IL-8, GRO-alpha and ENA-78 and cytokines IL-1beta, GM-CSF, IL-6 and the cell adhesion molecule ICAM-1 in both cell types. Increases in protein levels were also observed. Expression of these genes was significantly inhibited in BEAS-2B cells in which TLR3 expression was knocked down. However, pre-treatment with anti-TLR3 mAb, which interferes with the function of TLR3 expressed on the cell surface, did not inhibit the genes expression and these data were concordant with the results that TLR3 was expressed inside airway epithelial cells. The study of siRNA for NF-kappaB and IRF3 showed that they transduce the signal of poly I : C, but their roles were different in each target gene. CONCLUSION: TLR3 is expressed inside airway epithelial cells and transduces synthetic dsRNA signals. These signals may increase expression of inflammatory cytokines, chemokines and ICAM-1 through activation of transcription factors NF-kappaB and/or IRF3 in airway epithelial cells.

Peroxisome proliferator activated receptor gamma in colonic epithelial cells protects against experimental inflammatory bowel disease.

INTRODUCTION: Peroxisome proliferator activated receptor gamma (PPARgamma) is expressed in epithelial cells, macrophage, and T and B lymphocytes. Ligand induced activation of PPARgamma was reported to attenuate colitis activity but it is not clear whether this protection is mediated by epithelial or leucocyte PPARgamma. METHODS: Mice with targeted disruption of the PPARgamma gene in intestinal epithelial cells, generated using a villin-Cre transgene and floxed PPARgamma allele and designated PPARgamma(DeltaIEpC), were compared with littermate mice having only the PPARgamma floxed allele with no Cre transgene that expressed PPARgamma in the gut, designated PPARgamma(F/F). Colitis was induced by administering dextran sodium sulphate (DSS) and the two mouse lines compared for typical symptoms of disease and expression of inflammatory cytokines. RESULTS: PPARgamma(DeltaIEpC) mice displayed reduced expression of the PPARgamma target genes ADRP and FABP in the gut but were otherwise normal. Increased susceptibility to DSS induced colitis, as defined by body weight loss, colon length, diarrhoea, bleeding score, and altered histology, was found in PPARgamma(DeltaIEpC) mice in comparison with PPARgamma(F/F) mice. Interleukin (IL)-6, IL-1beta, and tumour necrosis factor alpha mRNA levels in colons of PPARgamma(DeltaIEpC) mice treated with DSS were higher than in similarly treated PPARgamma(F/F) mice. The PPARgamma ligand rosiglitazone decreased the severity of DSS induced colitis and suppressed cytokine production in both PPARgamma(F/F) and PPARgamma(DeltaIEpC) mice. CONCLUSIONS: These studies reveal that PPARgamma expressed in the colonic epithelium has an endogenous role in protection against DSS induced colitis and that rosiglitazone may act through a PPARgamma independent pathway to suppress inflammation.