A phase I study of panobinostat and ruxolitinib in patients with primary myelofibrosis (PMF) and post--polycythemia vera/essential thrombocythemia myelofibrosis (post--PV/ET MF).

Ruxolitinib, a selective JAK1/JAK2 inhibitor, is the current first line therapy for myelofibrosis (MF), which reduces symptomatology and splenomegaly, but does not clearly modify disease course. Panobinostat, a histone deacetylase inhibitor, was shown to be safe and tolerable in phase I and II trials and demonstrated clinical activity in approximately a third of treated patients. Combination therapy of ruxolitinib and panobinostat showed synergistic activity in a preclinical MF model, which prompted clinical evaluation of this combination in both ruxolitinib naïve and treated MF patients. Herein, we report the results of an investigator-initiated, dose escalation, phase I trial of ruxolitinib and panobinostat in 15 patients with primary MF and post-polycythemia vera/essential thrombocythemia MF. This combination treatment proved to be safe and tolerable without dose limiting thrombocytopenia and a maximum tolerated dose of both agents in combination was not determined. The majority of patients maintained stable disease with this combination treatment and 40 % attained a clinical improvement (spleen n = 5, anemia n = 1) by modified IWG-MRT at the end of 6 cycles. This is one of the first attempts of rationally designed, JAK inhibitor-based, combination therapy studies and exemplifies the feasibility of such an approach in patients with advanced MF.

Liquid biopsy assay for lung carcinoma using centrifuged supernatants from fine-needle aspiration specimens.

INTRODUCTION: Tumor mutation profiling is standard-of-care in lung carcinoma patients. However, comprehensive molecular profiling of small specimens, including core needle biopsy (CNB) and fine-needle aspiration (FNA) specimens, may often be inadequate due to limited tissue. Centrifuged FNA supernatants, which are typically discarded, have emerged recently as a novel liquid-based biopsy for molecular testing. In this study, we evaluate the use of lung carcinoma FNA supernatants for detecting clinically relevant mutations. METHODS: Supernatants from lung carcinoma FNA samples (n = 150) were evaluated. Samples were further analyzed using next-generation sequencing (NGS) and ultrasensitive droplet digital PCR (ddPCR). Mutation profiles in a subset of samples were compared with results derived from paired tissue samples from the same patient (n = 67) and available plasma liquid biopsy assay (n = 45). RESULTS: All 150 samples yielded adequate DNA and NGS were carried out successfully on 104 (90%) of 116 selected samples. Somatic mutations were detected in 82% of the samples and in 50% of these patients a clinically relevant mutation was identified that would qualify them for targeted therapy or a clinical trial. There was high overall concordance between the mutation profiles of supernatants and the corresponding tissue samples, with 100% concordance with concurrent FNA and 96% with concurrent CNB samples. Comparison of actionable driver mutations detected in supernatant versus plasma samples showed 84% concordance. CONCLUSIONS: FNA supernatants can provide a valuable specimen source for genotyping lung carcinoma especially in patients with insufficient tumor tissue, thereby reducing multigene mutation profiling failure rates, improving turnaround times, and avoiding repeat biopsies.

Enhancing antimicrobial stewardship by strengthening the veterinary drug regulatory framework.

Antimicrobial resistance is a major and growing public health threat. Recently, Health Canada introduced multiple regulatory changes to strengthen the oversight of antimicrobial drugs for veterinary use. These changes aim specifically at increasing control over importation of veterinary drugs and active pharmaceutical ingredients, mandatory reporting of antimicrobial sales data from manufacturers, importers and compounders and facilitating access to low risk veterinary health products. Additional policy changes under existing authorities are also being made to enhance veterinary supervision of antimicrobial use and to remove production claims for food animals from labels of medically important antimicrobial drugs. These important interlinked initiatives are aimed towards enhancing antimicrobial stewardship in Canada to preserve the effectiveness of existing antimicrobials and to protect the health of Canadians.

Laparoscopic ventral/incisional hernia repair: a single centre experience of 1,242 patients over a period of 13 years.

BACKGROUND AND PURPOSE: Laparoscopic technique is now well established for ventral/incisional hernia repair. However several issues such as optimal fixation technique, occult hernias, management of inadvertent enterotomies, postoperative seromas and recurrence require appraisal. METHODS: A single centre retrospective review of 1,242 patients between January 1992 and June 2005 is described. All patients had laparoscopic ventral/incisional hernia repair (LVIHR) following a standardised protocol by five consultants and fellows in a dedicated minimal access surgery unit of a tertiary care hospital. RESULTS: LVIHR was completed in 1,223 patients (98.5%). The average BMI was 32, mean defect size was 26.2 cm(2), mean operating time was 81 min and mean hospital stay was 1.9 days. The mean mesh to hernia ratio was 37.5. Occult hernias were observed in 203 (16.3%) patients and inadvertent enterotomies occurred in 21 (1.7%) patients. Mortality occurred in two patients, pulmonary embolism and cardiac dysrhythmia being the respective reasons. The most common sequel was early seroma formation (25%). Chronic pain occurred in 14.7% patients. Recurrence rate was 4.4%, which was associated with a higher BMI, use of staplers as fixation device, multiple defects and recurrent hernias. The mean follow up was 5.4 years; (range 2.4-10 years). The follow up rate was 78%. CONCLUSION: LVIHR leads to low recurrence rates and low rates of wound and mesh infection. Occult hernias are diagnosed and optimally treated laparoscopically. However, chronic pain remains an unresolved issue.

Limited-conversion technique: a safe and viable alternative to conversion in laparoscopic ventral/incisional hernia repair.

BACKGROUND: During laparoscopic ventral/incisional hernia repair (LVIHR), conversion to conventional (open) technique is required when safe adhesiolysis is not possible, incarcerated bowel in hernial sac cannot be reduced or for repair of iatrogenic enterotomies. A formal laparotomy in these circumstances entails significant morbidity due to factors such as wound infection, prolonged immobility, and longer hospital stay. MATERIALS AND METHODS: During a period between 1994 and 2007, 1,503 LVIHRs were performed at our centre following a standardized protocol by five consultants and fellows. Out of these, 6 patients had a formal laparotomy in the initial part of our experience and 26 patients had a limited conversion to facilitate completion of LVIHR. We have devised the term "limited conversion" for the procedure wherein bowel reduction/adhesiolysis/enterotomy repair was performed through a small targeted skin incision. This was followed by laparoscopic placement of intraperitoneal mesh. RESULTS: Conversion to an open procedure was required in 32 (2.1%) out of 1,503 LVIHR procedures. Twenty-six patients underwent a limited conversion and completion of the repair by laparoscopy. All but one of these patients had intraperitoneal placement of mesh by laparoscopic route. The wound complication rate was 3.8% (one patient), the mean hospital stay was 2.1 days, and mean operative time was 124 min. CONCLUSION: Limited conversion offers a safe alternative to a formal laparotomy in patients with bowel incarcerated in hernial sacs or in patients requiring extensive bowel adhesiolysis. Patient morbidity is reduced due to the targeted skin incision whilst retaining several advantages of a minimal access approach viz. laparoscopic evaluation of the entire abdominal wall and placement of a large intraperitoneal prosthesis.

Effects of prostaglandin E2 and lipopolysaccharide on osteoclastogenesis in RAW 264.7 cells.

INTRODUCTION: Prostaglandins (PGs) can act on both hematopoietic and osteoblastic lineages to enhance osteoclast formation. METHODS: We examined PGE2 stimulated osteoclastogenesis in RAW 264.7 cells and the role of endogenous PGE2 in lipopolysaccharide (LPS) stimulated osteoclastogenesis. RESULTS: RANKL (1-100 ng/ml) increased formation of osteoclasts, defined as tartrate resistant acid phosphatase multinucleated cells, with peak effects at 30 ng/ml. Addition of PGE2 (0.01-1.0 microM) to RANKL (30 ng/ml) dose dependently increased osteoclast number 30-150%. Use of NS-398 (0.1 microM) or indomethacin (Indo, 1.0 micro M) to block endogenous PG synthesis had little effect on the response to RANKL alone but significantly decreased the response to PGE2. Addition of LPS (100 ng/ml) to RANKL increased osteoclast number 50%, and this response was significantly decreased by NS-398 and Indo. RANKL and PGE2 produced small, additive increases in COX-2 mRNA levels, while LPS produced a larger increase. PG release into the medium was not increased by RANKL and PGE2 but markedly increased by LPS. CONCLUSION: We conclude that RANKL stimulated osteoclastogenesis can be enhanced by PGE2 and LPS though direct effects on the hematopoietic cell lineage and that these effects may be mediated in part by induction of COX-2 and enhanced intracellular PG production.

A prospective comparison of four techniques for diagnosis of paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder with altered expression of glycosylphosphatidylinositol (GPI)-anchored proteins, resulting in the increased susceptibility of erythrocytes to complement-mediated lysis. This study compared the available laboratory methods for detection of PNH cells and evaluated their utility in routine clinical practice. Fifty patients were evaluated by flow cytometric immunophenotyping (FCMI) using CD55 and CD59 monoclonal antibodies, PNH gel card test (GCT), Ham test and sucrose lysis test (SLT). A PNH clone was detectable in erythrocytes in 14 (28%) patients by FCMI, 13 (26%) by GCT and 10 (20%) by Ham test and SLT. The GCT and lytic tests showed 100% specificity and sensitivity was 92.8% and 71.1%, respectively. The GCT results correlated with type III cells (positive for > or =3.21% type III cells) and lytic test results correlated with CD59(-) type III cells (positive for > or =5% CD59(-) type III cells). The GCT and lytic tests were comparable in their sensitivity to detect type II cells (positive for > or =18.5% type II cells). Among the available methods, FCMI is most sensitive, can quantify and delineate PNH cells with differential expression of GPI-anchored proteins. The GCT is a useful screening tool as it is fairly sensitive, easy to perform and interpret. Well-standardized lytic tests are fairly reliable as screening tests.

Day care laparoscopic cholecystectomy: a feasibility study in a public health service hospital in a developing country.

BACKGROUND: Day care laparoscopic cholecystectomy (DCLC) has been shown to be safe in centers with adequate infrastructure for day care surgery in economically advanced countries. However, the feasibility of applying this concept in public health service hospitals in less developed and developing nation needs to studied. Unique protocols need to be developed and tested, taking into account local conditions and infrastructural constraints. PATIENTS AND METHODS: Patients less than 60 years old, graded I and II on the American Society of Anesthesiologists (ASA) physical status score, living within one hour traveling time and willing to make their own arrangements for a return to hospital in case of problems, were selected for DCLC. RESULTS: 291 cases (78%) out of 373 laparoscopic cholecystectomies done in one calendar year were found suitable for DCLC. The most common cause for omitting from DCLC was that the patient lived out of the defined area (57%). Four of 291 (1.3%) cases were cancelled due to medical condition; 270/287 (96.1%) were discharged the same evening as surgery; 6 patients were converted to open surgery; and 11 did not meet the necessary discharge criteria. Eight of 270 (2.9%) required readmission out of which 3 (1.1%) required intervention. Overall, incidence of complication rate was 3.4%. Analysis of data showed that results were comparable to previously published studies, hence extrapolating that inclusion and discharge criteria used in the study are valid. However, there are certain social constraints which hinder truly universal application of DCLC. CONCLUSIONS: DCLC is a safe and technically feasible concept, even in public health service centers without dedicated ambulatory surgery units. It has potential for much economical and social benefit in these countries.

Immune response and protective efficacy in sheep immunised with hydroxylamine-inactivated bluetongue virus vaccine.

Vero cell adapted bluetongue virus (BTV), serotype 18 (BTV-18) was inactivated with hydroxylamine and adjuvanted with aluminium hydroxide (Al[OH](3)) gel, saponin, or Al(OH)(3) gel/saponin prior to immunisation of sheep. Group-specific non-neutralising antibodies to BTV were detected in all vaccinated sheep as soon as seven days after vaccination. However, before challenge, serotype-specific neutralising antibodies were not detected in vaccinated sheep. Upon challenge with homologous live BTV, there was a reduction in delayed type of hypersensitivity response in control sheep but not in the vaccinated animals. All vaccinated sheep had significant (P<0.01) reductions in their clinical reaction index (CRI) and duration of viraemia.

Effect of oophorectomy on expression of calcium sensing receptor mRNA in rat duodenal mucosa.

Calcium sensing receptor (CaR) in duodenal mucosa may be involved in active calcium absorption. Estrogen deficiency results in decreased intestinal calcium absorption. Effects of bilateral oophorectomy (OVX) have been studied on calcium homeostasis, bone mineral density (BMD) and CaR mRNA levels in duodenal mucosa at 4 weeks in adult female Sprague Dawley rats and compared with those in sham-operated and control group. There was no significant change in serum corrected calcium, inorganic phosphorous, calcidiol and intact parathyroid hormone in all the three groups. OVX rats had a significant decline in serum estrogen (E2) levels and alkaline phosphatase. They also had a significant decrease in BMD (DXA) at lumbar spine in vivo, and proximal and distal tibia in vitro while there was no significant change in serum E2 and BMD parameters in sham-operated and control rats. Northern blot analysis revealed no significant change in the CaR mRNA expression in duodenal mucosa in all three groups. The results suggests that CaR mRNA expression in duodenal mucosa is not affected by physiological circulating concentrations of estradiol in rats.

Short-term androgen deprivation does not alter CaR and VDR mRNA expression in duodenal mucosa in male rats.

Androgen deprivation is associated with decline in intestinal calcium absorption. The effect of androgen on CaR and VDR intestinal mucosa has not yet been studied. Calcium homeostasis, a real bone mineral density (aBMD, dual energy X-ray absorptiometry) and expression of CaR and VDR mRNA in duodenal mucosa of orchidectomized (ORX) and sham operated (Sham) adult Sprague Dawley rats at 4 week have been studied. There was no significant difference in serum calcium, alkaline phosphatase, calcidiol and calcitriol levels between both the groups. Serum testosterone (T) (ng/dl) and inorganic phosphorous (iP) (mg/dl) levels were significantly lower in ORX rats. As compared to sham rats, ORX rats had significant decline in in-vitro aBMD at proximal, middle and distal tibia, proximal, mid and distal femur and femoral neck (P < 0.05). Northern blot analysis revealed no significant alteration in the CaR and VDR mRNA expression in duodenal mucosa in ORX rats. CaR and VDR mRNA expression in duodenal mucosa is therefore, not affected by physiological concentrations of testosterone in rats.

Spirocyclic nonpeptide glycoprotein IIb-IIIa antagonists. Part 1: design of potent and specific 3,9-diazaspiro[5.5]undecanes.

The synthesis and biological activity of novel glycoprotein IIb-IIla antagonists containing the 3,9-diazaspiro[5.5]undecane nucleus are described. The potent activity of these compounds as platelet aggregation inhibitors demonstrates the utility of the spirocyclic structures as a central template for nonpeptide RGD mimics.

Spirocyclic nonpeptide glycoprotein IIb-IIIa antagonists. Part 2: design of potent antagonists containing the 3-azaspiro[5.5]undecanes.

The synthesis and biological activity of novel glycoprotein IIb-IlIa anatagonists containing 3-azaspiro[5.5]undec-9-yl nucleus are described. The potent activity of these compounds as platelet aggregation inhibitors demonstrates the utility of the monoazaspirocyclic structure as central template for nonpeptide RGD mimics.