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.

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.

Antiarthritic gold compounds effectively quench electronically excited singlet oxygen.

Although certain gold [Au(I)] compounds have been used effectively in the treatment of rheumatoid arthritis for some years, the molecular basis for such therapeutic action has been unclear. One possible mechanism of the action of Au(I) compounds is that they protect unsaturated membrane lipids and proteins against oxidative degradation caused by activated phagocytes that are not properly regulated. In this study it has been shown that superoxide ion (O-2.), a product of activated phagocytes, can be oxidized to electronically excited singlet oxygen (O1(2)delta g), an agent that is capable of peroxidation of unsaturated fatty acid derivatives. It has also been shown that antiarthritic Au(I) compounds are effective deactivators of O1(2)delta g with quenching constants on the order of 10(7) M-1 sec-1.

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.

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.

Mechanism of the negative inotropic action of leukotrienes C4 and D4 on isolated rat heart.

Leukotrienes C4 and D4 (LTC4 and LTD4), possible mediators of cardiac dysfunction during inflammatory injury, may depress cardiac function by reducing coronary flow or by exerting a negative effect directly on the myocardium. We used an isovolumic rat heart preparation perfused at constant pressure and measured left ventricular developed pressure (mmHg), coronary flow (ml.min-1), oxygen extraction, and myocardial oxygen consumption and delivery (mumol O2.[gramme dry weight]-1.min-1) during infusion of five doses of angiotensin II, LTC4, LTD4 (approximately 10 to approximately 300 pmol.min-1), and noradrenaline (400 to 2000 pmol.min-1), or perfusion with medium which contained calcium at half-concentration. LTC4 and LTD4 were equipotent with angiotensin. At low effective doses, increased oxygen extraction offset the decrease in oxygen delivery, maintaining a stable level of oxygen consumption and left ventricular developed pressure. At the highest doses, angiotensin, LTC4 and LTD4 reduced coronary flow from 21 to 15, 21 to 13, and 21 to 13 ml.min-1, respectively. And, despite greater oxygen extraction of 59%, 58% and 65% for angiotensin, LTC4 and LTD4, left ventricular developed pressure fell from a baseline of 120 mmHg to 113, 106 and 92, respectively. In contrast, low calcium perfusion reduced left ventricular developed pressure (126 to 92) and oxygen extraction (46 to 30%) without changing coronary flow or oxygen delivery. These results suggest that LTC4 and LTD4 are potent coronary vasoconstricting agents which depress cardiac function by limiting oxygen delivery.

Substrate specificity in short-chain phospholipid analogs at the active site of human synovial phospholipase A2.

The substrate specificity at the active site of recombinant human synovial fluid phospholipase A2 (hs-PLA2) was investigated by the preparation of a series of short-chain phospholipid analogs and measurement of their enzymatic hydrolysis at concentrations well below the critical micelle concentration. Substrates used in the study included 1,2-dihexanoylglycerophospholipids, 1,2-bis(alkanoylthio)glycerophospholipids, and 1-O-alkyl-2-(alkanoylthio)phospholipids. Turnover was observed for only a few of the 1,2-dihexanoylglycerophospholipids, and the rate of hydrolysis was very low, near the limit of detection of the assay. In contrast, selected 2-(alkanoylthio)-glycerophospholipids were hydrolyzed by hs-PLA2 at much higher rates at concentrations well below their critical micelle concentration (cmc). Thus, the 1,2-bis(hexanoylthio)glycerophosphatidylmethanol exhibits a k(cat)/K(M) = 1800 L mol-1 s-1. Over the calculated log P (cLogP) range of 3-9, cLogP and log(k(cat)/K(M) were linearly related for compounds with straight-chain sn-1 and sn-2 substituents. At comparable cLogP's, the sn-1 ethers and thioesters were hydrolyzed at comparable rates. A negative charge in the phosphate head group was required for enzyme activity. Unsaturation, aromaticity, and branching in the sn-2 substituent reduce turnover dramatically. The same structural modifications in the sn-1 substituent have less effect on turnover. Certain of these substrates, e.g., 1,2-bis(hexanoylthio)glycerophosphatidylmethanol, may be useful in assaying for active site inhibitors of PLA2. The structure--activity relationships established here for substrates should serve as a reference for the structure--activity relationships of substrate-based inhibitors.

Effect of giardia infection on nutritional status of preschool children.

Nutritional status of 34 children aged 24-72 months and infected with giardia was compared with that of 92 children matched for age, sex and income and 34 children matched for age, sex, income as well as birth order. Children without giardiasis were found to have better nutritional status in both comparisons. The difference was more pronounced in the second instance. Of the seven anthropometric parameters used for comparison, four were significantly lower (weight, weight for age, midarm circumference and midarm circumference for age), while two (height for age and weight for height) showed nonsignificant childhood malnutrition and that matching for birth order enhances the utility of case-control studies in children.

Fertility status of men following vaso-vasostomy.

PIP: In 1977, 114 vas reanastomoses were performed at the S.V.B.P. Hospital in Meerut, India. Follow-up of 74 of the cases indicated that 81.08% of the procedures were technically successful in that an adequate sperm count was reestablished; however, clinical success, as measured by the wife's subsequent pregnancy, was achieved in only 20.27% of the cases. Wives who did not conceive were gynecologically examined and no abnormalities were observed. In order to identify factors which contributed toward fertility restoration failure the investigators assessed the relationship between clinical and technical success and 1) the age of the patient; 2) the length of the interval between the vasectomy and the reversal procedure; and 3) the presence of sperm agglutinating and sperm immobilizing antibodies in the blood serum of the patients. The age of the patient was not related to either clinical or technical success. The length of the interval between vasectomy and reversal did not affect technical success; however, as the interval increased, clinical success declined. Serum samples were obtained from 86 of the 114 patients and from a control group of 24 fertile men and examined for the presence of sperm agglutinating and sperm immobilizing antibodies. The antibodies were absent in the sera of all but 1 of the controls; however, both types of antibodies were present in the sera of 32 of the patients and sperm agglutinating antibodies only were present in the sera of another 26 patients. The presence of the antibodies was not related to technical success but the presence of the antibodies did appear to have an adverse effect on clinical success. This relationship also appeared to be influenced by the length of the interval between vasectomy and vas reanastomosis. Major findings were presented in tabular form.^ieng

Compatibility of oral contraceptive with anti-tubercular chemotherapy in female pulmonary tuberculosis patients.

PIP: The compatibility of oral contraceptives with antitubercular chemotherapy and the effect of the contraceptives on regression of the tubercular lesions were studied in 100 16-40 year old female patients with bacteriologically confirmed pulmonary tuberculosis. Each patient was living with her husband and had 1 or more living children. Antitubercular treatment was given similarly to all. Half of the patients were also given Ovulen therapy. Of the 100 who started, 41 of those using Ovulen and 42 of the others completed the investigation. A patient in each group died of tuberculosis during the study. The overall antitubercular efficacy was similar in the 2 groups at the end of 1 year. Side effects from the oral contraceptive were minimal and caused no withdrawals. Menstrual abnormalities were improved in those taking the Ovulen. Weight gain was about twice as much and hemoglobin increased 3 times as much in the oral contraceptive users. A pregnancy occurred in the group taking the contraceptive; there were 4 pregnancies in the other group. Some early increases in blood pressures were found in the contraceptive users. It is concluded that oral contraceptives in female tuberculosis patients are compatible with antituberculosis drugs. Also, overall progress was improved by the therapy and a greater sense of well-being expressed.^ieng

Complete heart block complicating acute myocardial infarction.

Complete heart block (CHB) developed in 10.3% of patients with acute myocardial infarction (MI). It was more frequent among patients with inferior myocardial infarction compared to anterior myocardial infarction, but the mortality was significantly high among patients with anterior MI who developed CHB. A new classification into primary (P) and secondary (S) CHB is suggested by the sequence of events. The incidence and mortality of SCHB was significantly high when compared to the incidence and mortality of PCHB. Wide QRS complexes, a heart rate of less than 60/min, and syncopal attacks were the grave prognostic signs. Progression from a lesser degree of AV block and regression of CHB to a lesser degree of AV Block were both observed in 25.8% cases. Circulatory failure in the form of shock, hypotension, congestive heart failure, and left ventricular failure, alone or in combination was a factor in 17 of the 22 patients who died. Four of 5 patients who underwent transvenous pacing also died.

A study of disseminated intravascular coagulopathy in hepatic coma complicating acute viral hepatitis.

Thirty cases of viral hepatitis with and without coma and 15 normal healthy age and sex matched controls were studied for disseminated intravascular coagulopathy (DIC). Cases of viral hepatitis with coma showed strong evidence of DIC in the form of increased plasma FDP (240 +/- 190.64 dilution), diminished fibrinogen half life (48.1 +/- 15.3 hours) and increased fractional catabolic rate (46.13 +/- 14.7% per day) with normal Euglobulin clot lysis time and reduced fibrinogen (147.33 +/- 43.5%) and platelet count (1.27 +/- 0.324 lakhs/cm.). The possible role of heparin in the reversal of coagulation abnormalities have been discussed.

Strategies to optimize the impact of needle exchange programs.

In the United States today, half of all new HIV infections are injection drug use-associated, many of which are a result of the reuse and sharing of contaminated syringes. Thus, providing access to sterile syringes for injection drug users is an important part of preventing HIV transmission. Needle exchange programs (NEPs) have been established as one successful approach to providing sterile injection equipment. The medical literature shows that these programs are effective in decreasing both syringe sharing and HIV incidence in injection drug users. In addition, many NEPs are also beneficial because they provide other injection drug use-relevant services. There are several strategies that can be adopted in order to optimize the impact of needle exchange programs, at both the community and national levels. These include establishing NEPs in communities that need them, expanding and improving those that already exist, and implementing such programs on a larger national scale with the provision of federal funds.

Detection of bovine herpesvirus 1 (BHV-1) genomic sequences in bovine semen inoculated with BHV-1 by polymerase chain reaction.

Polymerase chain reaction (PCR) technique was applied to detect BHV-1 in bovine semen inoculated with BHV-1. The technique was found to be 10(6) times more sensitive than a non-isotopic dot-blot hybridization method in detecting viral genomic DNA. Of the three primer pairs used, the one chosen from glycoprotein gC appeared to be most sensitive as it could detect up to 0.01 TCID50 of BHV-1 in the semen. The technique could be useful in screening breeding bulls or samples of frozen semen prior to use in artificial insemination.

Mast cell reactivity in lymphoma: a preliminary communication.

Fifty nodes of Hodgkin's disease, 50 of non-Hodgkin's lymphoma and 20 of reactive hyperplasia revealed varying degree of mast cell reactivity. It was maximum in reactive lymph nodes, sinuses and interfollicular areas being infiltrated more than zones. The number of mast cells was more in nodular sclerosis variety of Hodgkin's disease than other Rye-subtypes, and more in Hodgkin's group than non-Hodgkin's, out of the latter, the diffuse histiocytic lymphoma revealed higher number. Analysis of a larger series and follow-up of these patients are indicated to establish the possible reactionary nature of mast cell reactivity in lymphomas, and the prognostic bearing, if any.

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.

A study of aluminum phosphide (AlP) poisoning with special reference to electrocardiographic changes.

Ninety patients with aluminum phosphide poisoning have been studied over a period of 3 years. Epigastric pain and vomiting were the common initial clinical features, followed 12 to 24 hours later by cardiogenic shock, oliguria, altered mental state and respiratory distress. Death occurred within 24 to 72 hours presumably due to poison-induced toxic chemical myocarditis as reflected by electrocardiographic changes. The overall mortality was 63.3%. Intravenous magnesium sulphate, probably due to its membrane stabilizing action, appears to be related to the reduction in mortality from 90% to 52% in the latter 62 cases.