A comparison of the pathogenicity of avian and swine H5N1 influenza viruses in Indonesia

Highly pathogenic avian influenza H5N1 viruses are circulating in many countries. We recently discovered that these viruses have been transmitted to pigs on multiple occasions in Indonesia. To investigate whether avian H5N1 influenza viruses adapted to mammals through their introduction into pigs, we examined the growth of avian and swine isolates in cell culture and compared their pathogenicity in mice. We found that swine isolates were less virulent to mice than avian isolates, suggesting that the viruses became attenuated during their replication in pigs. Continuous surveillance of H5N1 viruses among pigs is clearly warranted.     

Inhibition of Prostaglandin D Synthase Suppresses Muscular Necrosis

Duchenne muscular dystrophy is a fatal muscle wasting disease that is characterized by a deficiency in the protein dystrophin. Previously, we reported that the expression of hematopoietic prostaglandin D synthase (HPGDS) appeared in necrotic muscle fibers from patients with either Duchenne muscular dystrophy or polymyositis. HPGDS is responsible for the production of the inflammatory mediator, prostaglandin D₂. In this paper, we validated the hypothesis that HPGDS has a role in the etiology of muscular necrosis. We investigated the expression of HPGDS/ prostaglandin D₂ signaling using two different mouse models of muscle necrosis, that is, bupivacaine-induced muscle necrosis and the mdx mouse, which has a genetic muscular dystrophy. We treated each mouse model with the HPGDS-specific inhibitor, HQL-79, and measured both necrotic muscle volume and selected cytokine mRNA levels. We confirmed that HPGDS expression was induced in necrotic muscle fibers in both bupivacaine-injected muscle and mdx mice. After administration of HQL-79, necrotic muscle volume was significantly decreased in both mouse models. Additionally, mRNA levels of both CD11b and transforming growth factor β1 were significantly lower in HQL-79-treated mdx mice than in vehicle-treated animals. We also demonstrated that HQL-79 suppressed prostaglandin D₂ production and improved muscle strength in the mdx mouse. Our results show that HPGDS augments inflammation, which is followed by muscle injury. Furthermore, the inhibition of HPGDS ameliorates muscle necrosis even in cases of genetic muscular dystrophy.Duchenne muscular dystrophy (DMD) is one of the most common types of muscular dystrophy, affecting approximately 1 out of 3500 boys.¹ Progressive muscular dystrophy in DMD is caused by membrane vulnerability,² which results from a defect in the muscle protein dystrophin,^3,4 but the precise pathophysiology of the disease progression is not known. There is still no complete cure for this disastrous disease, albeit gene transfer has been extensively tried in mammalian models. Glucocorticoids^5,6 and their analogs⁷ are effective in suppressing the disease only to some degree. In DMD, these steroids reduce the infiltration of inflammatory cells into the muscle⁸ and down-regulate the expression of genes involved in the immune response.⁹ These data suggest inflammation may play a role in the progression of the disease.Earlier we reported the expression of hematopoietic prostaglandin (PG) D synthase (HPGDS), the enzyme responsible for the production of PGD₂,¹⁰ in necrotic muscle fibers, mainly in the focus of grouped necrosis, in patients with DMD or polymyositis.¹¹ We recently reported that overproduction of PGD₂ produced by HPGDS aggravates inflammation and causes profound tissue damage in twitcher, a genetic demyelinating mouse model.¹² The biosynthesis of PGs was also suppressed by glucocorticoids, via suppression of enzymes in the overall synthesis of PGs including phospholipase A₂ and cyclooxygenase. PGD₂ mediates inflammatory responses through two specific receptors, DP1¹³ and DP2,¹⁴ causing peripheral vasodilatation, augmentation of vascular permeability, and chemotaxis.¹⁵ Based on these findings, we hypothesized that HPGDS augments the inflammation that is followed by the muscle injury, especially in the foci of grouped necrosis. Here, using bupivacaine hydrochloride (BPVC)-induced muscle necrosis, where sequences of muscle necrosis are similar to that of progressive muscular dystrophy,¹⁶ and the mdx mouse as a DMD model, we clarified the role of PGD₂ in the pathogenesis and investigated the therapeutic potentials of blockade of HPGDS/PGD₂/DP signaling on the muscular necrosis.     

Akt1 in murine chondrocytes controls cartilage calcification during endochondral ossification under physiologic and pathologic conditions

Objective

To examine the role of the phosphoinositide‐dependent serine/threonine protein kinase Akt1 in chondrocytes during endochondral ossification.

Methods

Skeletal phenotypes of homozygous Akt1‐deficient (Akt1^−/−) mice and their wild‐type littermates were compared in radiologic and histologic analyses. An experimental osteoarthritis (OA) model was created by surgically inducing instability in the knee joints of mice. For functional analyses, we used primary costal and articular chondrocytes from neonatal mice and mouse chondrogenic ATDC5 cells with retroviral overexpression of constitutively active Akt1 or small interfering RNA (siRNA) for Akt1.

Results

Among the Akt isoforms (Akt1, Akt2, and Akt3), Akt1 was the most highly expressed in chondrocytes, and the total level of Akt protein was decreased in Akt1^−/− chondrocytes, indicating a dominant role of Akt1. Akt1^−/− mice exhibited dwarfism with normal proliferative and hypertrophic zones but suppressed cartilage calcification in the growth plate compared with their wild‐type littermates. In mice with surgically induced OA, calcified osteophyte formation, but not cartilage degradation, was prevented in the Akt1^−/− joints. Calcification was significantly suppressed in cultures of Akt1^−/− chondrocytes or ATDC5 cells overexpressing siRNA for Akt1 and was enhanced in ATDC5 cells overexpressing constitutively active Akt1. Neither proliferation nor hypertrophic differentiation was affected by the gain or loss of function of Akt1. The expression of ANK and nucleotide pyrophosphatase/phosphodiesterase 1, which accumulate pyrophosphate, a crucial calcification inhibitor, was enhanced by Akt1 deficiency or siRNA for Akt1 and was suppressed by constitutively active Akt1.

Conclusion

Our findings indicate that Akt1 in chondrocytes controls cartilage calcification by inhibiting pyrophosphate during endochondral ossification in skeletal growth and during osteophyte formation in OA.

     

Association of the FAM167A–BLK region with systemic sclerosis

Objective

An association of single‐nucleotide polymorphisms (SNPs) in the FAM167A (previously referred to as C8orf13)–BLK region with systemic lupus erythematosus (SLE) has been demonstrated in Caucasians and in Asians. Recent studies have shown that many genes, including IRF5, STAT4, and PTPN22, are shared susceptibility genes in multiple autoimmune diseases. We undertook the current study to examine whether the FAM167A–BLK region is also associated with susceptibility to systemic sclerosis (SSc).

Methods

Japanese patients with SSc (n = 309) and healthy controls (n = 769) were enrolled in a 2‐tiered case–control association study. In tier 1, 124 patients and 412 controls were tested to determine association of 16 tag SNPs encompassing the FAM167A–BLK region with SSc. In tier 2, an additional 185 patients and 357 controls were analyzed for SNP rs13277113.

Results

Two haplotype blocks that correspond approximately to FAM167A and BLK were observed. In tier 1 of the study, the rs13277113A allele in the BLK block exhibited the most significant association with SSc after correction for multiple testing (permutated P = 0.024). Two SNP haplotypes formed by rs13277113 and the most significant SNP in the FAM167A block did not exhibit stronger association. When samples from tier 1 and tier 2 were combined, the rs13277113A allele was significantly associated with SSc (odds ratio 1.45 [95% confidence interval 1.17–1.79], P = 6.1 × 10⁻⁴). Association or a tendency toward association of rs13277113A with SSc was observed regardless of a patient's autoantibody profile or whether a patient had diffuse cutaneous or limited cutaneous SSc.

Conclusion

Our findings indicate that the rs13277113A allele is associated not only with SLE but also with SSc and that the FAM167A–BLK region is a common genetic risk factor for both SLE and SSc.

     

A case of local-recurring breast cancer under long term-treatment by capecitabine

A 57-year-old woman had undergone a modified radical mastectomy at another medical institute. Six years after the resection, a tumor was confirmed and diagnosed as a recurrent metastasis in the right margin of the breastbone. The patient underwent chemoradiation therapy at the other medical institute and was then transferred to our institute, where treatment was continued. Four years after the recurrence, chemotherapy with capecitabine was started. Over the past 6 years, the capecitabine dose has not been changed and no signs of local recurrence have been seen. The values of biomarkers are normal. No adverse events, such as hand-foot syndrome, have occurred. Early during the capecitabine treatment, grade 2 elevations in the patient's GOT and GPT values were observed. However, these values recovered immediately without supportive therapy. At present, this patient is continuing to receive capecitabine treatment without experiencing any serious adverse events.     

Clusterin is a potential molecular predictor for ovarian cancer patient's survival: targeting Clusterin improves response to paclitaxel

Background

Clusterin is a cytoprotective chaperone protein involved in numerous physiological processes, carcinogenesis, tumor growth and tissue remodelling. The purpose of this study was to investigate whether clusterin (CLU), an antiapoptotic molecule, could be a potential predictor molecule for ovarian cancer and whether or not targeting this molecule can improve survival of ovarian cancer patients.

Methods

Clusterin expression was compared between ten primary and their recurrent tumors from same patients immunohistochemically. We analyzed prognostic significance of CLU expression in another 47 ovarian cancer tissue samples by immunohistochemistry. We used small interference RNA to knock down CLU in the chemo-resistant ovarian cancer cell lines. KF-TX and SKOV-3-TX, paclitaxel-resistant ovarian cancer cells, were established from parental KF and SKOV-3 chemo-sensitive cell lines, respectively. Either siRNA or second generation antisense oligodeoxynucleotide against CLU (OGX-011), which is currently evaluated in clinical phase II trials in other cancer s, was used to modulate sensitivity to paclitaxel (TX) in ovarian cancer cells in vitro. Cellular viability assay, FACS analysis and annexin V staining were used to evaluate the comparative effect of CLU knocking down in ovarian cancer cells.

Results

Immunohistochemical analysis of CLU expression in primary ovarian cancer tissue specimens and their recurrent counterparts from same patients demonstrated higher expression of CLU in the recurrent resistant tumors compared with their primary tumors. High expression of CLU by immunohistochemistry among 47 surgical tissue specimens of early-stage (stage I/II) ovarian cancer, who underwent complete cytoreduction as a primary surgery, significantly related to poor survival, while none of other clinicopathological factors analyzed were related to survival in this patient cohort. Secretory CLU (s-CLU; 60 KDa) expression was upregulated in TX-resistant ovarian cancer cells compared to parental cells. Transfection of siRNA or OGX-011 clearly reduced CLU expression. Cell viability assay, FACS analysis and annexin V staining demonstrated that targeting CLU expression by siRNA or OGX-011 sensitized ovarian cancer cells to TX.

Conclusion

We conclude that CLU could be a potential molecular target to predict survival while targeting this s-CLU may improve survival of patients with ovarian cancer.     

Interventional endoscopic ultrasonography for pancreatic cancer

Endoscopic ultrasonography (EUS) represents the combination of endoscopy and intraluminal ultrasonography. This allows use of a high-frequency transducer (5-20 MHz) that, due to the short distance to the target lesion, provides ultrasonographic images of higher resolution than those obtained from other imaging modalities, including multiple-detector-row-computed tomography, magnetic resonance imaging, and positron emission tomography. EUS is now a widely accepted modality for diagnosing pancreatic diseases. However, the most important limitation of EUS has been the lack of specificity in differentiating between benign and malignant changes. In 1992, EUS-guided fine needle aspiration (FNA) of lesions in the pancreas head was introduced into clinical practice, using a curved linear-array echoendoscope. Since then, EUS has evolved from EUS imaging to EUS-FNA and wider applications. Interventional EUS for pancreatic cancer includes EUS-FNA, EUS-guided fine needle injection, EUS-guided biliary drainage and anastomosis, EUS-guided celiac neurolysis, radiofrequency ablation, brachytherapy, and delivery of a growing number of anti-tumor agents. This review focuses on interventional EUS, including EUS-FNA and therapeutic EUS for pancreatic cancer.