Implementation of various approaches to study the prevalence, incidence and progression of disseminated neoplasia in mussel stocks.

Marine mussel production is of substantial economic interest in numerous coastal areas worldwide, making crucial the study of pathologies that affect them. Disseminated neoplasia (DN) has recently been suggested to be linked to blue mussel, Mytilus edulis, mortality outbreaks observed in France since 2014, although the evidence remains indirect. In order to improve DN detection and monitoring, we compared the sensitivity of four diagnostic tools, namely haemocytology, histology, flow cytometry, and genetics. Haemocytological examination gave the best results in sensitivity and had the advantage of being non-invasive, allowing disease progression to be followed in affected mussels. Using this approach, we showed that DN progression is usually slow, and we provide evidence of remission events. We observed a high diversity of forms and mitotic features of neoplastic cells located in the vesicular connective tissue but rarely in the haemolymph. Circulating cells occur as four main types but are homogenous in morphology and DNA content within a single individual. Polyploidy proved very high, from 8 N to 18 N. Genetic analysis of haemolymph DNA showed that a Mytilus trossulus genetic signal was associated with almost all the DN cases here diagnosed by haemocytological examination, regardless of the DN type. This result corroborates DN is a transmissible cancer that first originated in a M. trossulus host and subsequently crossed into M. edulis. No pre-neoplastic conditions were detectable. The prevalence of the disease was quite low, which, together with the low morbidity observed in the lab, suggest DN is unlikely to be the direct cause of mortality outbreaks in France.

Comparison of two real-time PCR methods for detection of ostreid herpesvirus 1 in the Pacific oyster Crassostrea gigas.

The real-time polymerase chain reaction (PCR) is considered to be a suitable tool for nucleic acid quantitation because it is accurate, rapid and reliable. The reference protocol for quantitation of ostreid herpesvirus 1 in Pacific oysters Crassostrea gigas is based on a Sybr(®) Green real-time PCR developed by the IFREMER laboratory. The Frank Duncombe Departmental Laboratory has developed an alternative protocol based on TaqMan(®) chemistry (alternative technique). The quantitation limits were 1000 and 18UG/mg of tissues for the reference method and alternative protocols, respectively, and the latter protocol has a detection limit of 6UG/mg of tissues. The aim of this study was to compare the two protocols using DNA samples obtained from 210 spat. The kappa index (0.41) indicated a moderate concordance between the protocols, according to the measures of Landis and Koch. All samples that were positive by the reference protocol were also positive by the alternative protocol. Of the 76 samples that were negative by the reference protocol, 49 were positives by the alternative protocol. In conclusion, the alternative protocol is an improvement of the reference protocol in terms of sensitivity, specificity and rapidity (<3h).

Minimal antigenicity of intron A in human recipients demonstrated by three analytical methods.

Antibodies to recombinant human interferon alpha 2b (Intron A) were detected in only a small number of 101 Intron A recipients. This group of cancer patients received Intron A for a mean treatment time of 4.3 months and were selected from disease categories in which subjects were expected to be immunocompetent. Three methods for the detection of antibodies were employed: (a) a bioassay measuring the neutralizing activity of the sera for the antiviral action of interferon alpha 2b, (b) a radioimmunological assay measuring the ability of the sera to prevent the detection of interferon alpha 2b by an immunoradiometric assay (IRMA), and (c) an enzyme-linked immunosorbent assay (ELISA) that measures the binding of immunoglobulins to interferon alpha 2b attached to a solid support. Three of the 101 patients developed neutralizing activity during treatment. Two of these exhibited low neutralizing titers of 1:6-1:9 and were unreactive in the IRMA and ELISA, while only one was positive by bioassay, IRMA, and ELISA. An additional seven patients were positive only in the ELISA. Six of these were borderline positive, i.e., the posttreatment:pretreatment ratio was less than or equal to 5. The results of this study confirm that Intron A is minimally antigenic in human subjects.

Pharmacokinetics of interferon alpha-2b in healthy volunteers.

In a three-way crossover design, 12 healthy male volunteers received 5 X 10(6) IU/m2 body surface area interferon alpha-2b(IFN alpha-2b) by intravenous (IV) infusion over 30 minutes, intramuscular (IM) injections, and subcutaneous (SC) injections. Blood and urine samples were collected at specified times, and analysis of IFN alpha-2b concentrations was performed by immunoradiometric assay. "Flulike" symptoms were the most frequently reported adverse experiences and were independent of the route of administration. After a 30-minute IV infusion, IFN alpha-2b disappeared rapidly from serum, with distribution and elimination phase half-lives of 0.1 hour and 1.7 hours, respectively. Interferon alpha-2b was absorbed slowly after IM and SC administration, with similar absorption half-lives of 5.8 and 5.5 hours, respectively. The observed maximal concentrations after IM and SC administration were 42.1 IU/mL at six hours and 45.8 IU/mL at eight hours, respectively. Interferon alpha-2b was eliminated with half-lives of 2.2 hours after IM administration and 2.9 hours after SC administration. The areas under the serum concentration-time curves for the SC and IM doses were higher than those obtained for the IV infusion. Measurable amounts of IFN alpha-2b were not found in urine regardless of the route of administration.

Low incidence of serum neutralizing factors in patients receiving recombinant alfa-2b interferon (Intron A).

The development of serum neutralizing factors against recombinant alfa-2b interferon (Intron A) was reviewed in a large clinical experience. In 537 patients receiving systemic therapy, neutralizing factors developed in only 13 (2.4 percent). In 1,326 patients who received intranasal administration and 154 with intralesional administration, the incidence was less than 1 percent. Patients in whom antibody developed had no predisposing characteristics that could be identified, no particular types of patients with cancer had a high rate of neutralizing factors, and two of 10 patients with cancer in whom neutralizing factor developed were still able to show clinical responses. In patients in whom neutralizing factor was present, there was no discernible difference in the incidence or severity of interferon side effects as compared with patients who had no demonstrable neutralizing factor levels. This form of recombinant alpha-2 interferon appears to have a very low antigenic potential.

Immunoradiometric assay of a recombinant human alpha-2 interferon (SCH 30500).

A sensitive immunoradiometric assay for recombinant alpha-2 interferon (IFN-alpha 2; SCH 30500) was developed by using the monoclonal antibody NK2. A polystyrene bead with polyclonal sheep anti-human IFN-alpha (Hu IFN-alpha) antibody adsorbed to it formed the solid phase of the assay. The monoclonal antibody, labeled with 125I, reacted with the IFN-alpha 2 which bound to the polyclonal antibody on the bead. The assay was sensitive, capable of measuring less than 5 IU/ml, and it was precise. Coefficients of variation were less than 10%, often less than 5%. Interassay coefficients of variation for the same sample were also less than 10%. Specificity of the monoclonal antibody for IFN-alpha 2 was greater than for Hu IFN-alpha produced by leukocytes. In specificity studies it was necessary to add at least 200 IU of Hu IFN-alpha per ml to 25 IU of IFN-alpha 2 per ml before the Hu IFN-alpha affected the accuracy of the assay of IFN-alpha 2. Accuracy as determined by correlation studies with the antiviral cytopathic effect assay was very high. Results of the two assays usually agreed within 15%. This correlation was maintained in studies of IFN-alpha 2 after storage under accelerated degradation conditions, when the IFN was cleaved into smaller sequences of amino acids by cyanogen bromide and when the IFN was carboxymethylated.

Evaluation of the in-vivo efficacy of Sch 34343.

Sch 34343 showed a linear dose response (with respect to AUCs) in mice following both intravenous and subcutaneous administration. It was 100% bioavailable following subcutaneous administration. Peak serum levels, AUCs, beta-phase half-life and recovery of Sch 34343 from the urine of mice indicated that it was similar to cephalothin and cefamandole. In experimental mouse infections, against Gram-negative strains, Sch 34343 was more active than cephalothin, equal to or more active than cefamandole and cefoxitin, but less active than latamoxef (moxalactam) and cefotaxime following single or multiple dose therapy. It was the most active compound against Staphylococcus. Sch 34343 was equally active against strains sensitive to beta-lactams and strains producing beta-lactamases. In an anaerobic abscess model in mice, Sch 34343 was more active than cefoxitin and clindamycin against Bacteroides fragilis. In Escherichia coli meningitis in rabbits, it cured rabbits with a single intravenous dose of 50 mg/kg.

Pharmacokinetics of Sch 34343 in rats and dogs.

The pharmacokinetics of 14C-Sch 34343 were studied in rats and dogs following intravenous and intramuscular dosing. In both species, it was rapidly absorbed after intramuscular dosing. The serum AUC for total radioactivity and for intact drug after intramuscular dosing were similar to those obtained after intravenous dosing. Following both routes of drug administration, the elimination of half-life (T 1/2 beta) was 7 min in rats and 25-32 min in dogs. Following intravenous dosing of 14C-Sch 34343 to rats, radioactivity in tissues disappeared rapidly with time indicating no tissue accumulation. Highest concentrations of radioactivity were seen in the kidney. Liver, lung, skin and heart appeared to have concentrations of radioactivity similar to those of blood. Sch 34343 was excreted rapidly and primarily into the urine in both rats and dogs. After either route of dosing, urinary excretion of total radioactivity ranged from 84 to 93% and that of intact Sch 34343 from 41 to 51% of the dose, respectively. In addition, the effect of pretreatment with probenecid on the pharmacokinetics in rats and dogs and in anephric rats were also evaluated. Pretreatment with probenecid prolonged the elimination half-life in both rats and dogs. Anephric rats had a longer half-life than normal rats.

Interspecies pharmacokinetic scaling of Sch 34343.

Pharmacokinetic parameters of Sch 34343 have been determined for mice, rats, rabbits, monkeys, dogs and humans and correlated among species as an exponential function of body weight. The pertinent pharmacokinetic parameters tested are apparent and steady-state volumes of distribution, total body clearance, elimination phase half-life, and mean residence time. This study showed that the extrapolation of animal data to humans on a new investigational drug, Sch 34343, can be potentially useful.

A radioimmunologic technique to screen for antibodies to alpha-2 interferon.

A radioimmunologic technique has been developed to screen sera of persons receiving human alpha-2 interferon for the presence of specific antibodies to alpha-2 interferon. The method is sensitive and easy to perform. It tests the ability of the sera to neutralize alpha-2 interferon and prevent the interferon from being detected by an immunoradiometric assay. The results obtained using this technique are in good agreement with an anti-viral, cytopathic effect assay. Using the immunological technique, the sera from more than 1000 individuals who had received different doses of alpha-2 interferon by one or more of various routes of administration were tested. Twenty-five sera representing 14 individuals gave a positive or possibly positive reaction in the assay. Three of the 14 individuals were positive prior to receipt of alpha-2 interferon. Another 3 had reverted to negative when tested a few months later. Of the remaining 8, only 4 developed titers greater than 100 neutralizing units/ml. Hence approximately 1% of the alpha-2 interferon recipients may have produced neutralizing antibodies.

High-pressure liquid chromatographic method for determination of Sch 29482 in human serum.

A high-pressure liquid chromatographic method was developed for quantitative determination in human serum of a new penem antibiotic known as Sch 29482 (5R,6S,8R-2-ethylthio-6(1-hydroxyethyl)penem-3-carboxylic acid). The method involves serum extraction at an acid pH with ether, followed by separation on a reverse-phase column and UV light detection at 254 nm. This technique produced a good linear relationship between the peak height ratio and the Sch 29482 concentration, which ranged from 0.09 to 35.64 micrograms/ml. In addition, this procedure proved to be quite specific for Sch 29482, since all beta-lactam antibiotics tested did not interfere with the assay. For high concentrations (greater than 0.5 micrograms/ml), the mean values obtained from the high-pressure liquid chromatographic method correlated very well (r = 0.997) with those obtained from a microbiological assay. This method is accurate and reproducible, with a sensitivity of about 0.09 micrograms per ml of serum. It is useful for monitoring serum drug levels in animals and humans and can also be used for drug pharmacokinetic studies in humans.

Compatibility of netilmicin sulfate injection with commonly used intravenous injections and additives.

The compatibility and stability of netilmicin sulfate in a concentration of 3 mg/ml in 37 intravenous injections and in 5% dextrose and 0.9% sodium chloride injection with 26 commonly used additives were studied. Compatibility between an intravenous administration set and selected admixtures were also evaluated. The admixtures were stored at 4 and 25 degrees C for seven days. After mixing and on days 1, 3, and 7, all admixtures were evaluated for netilmicin potency, pH, color, osmolarity, and clarity. A microbiological assay was used to measure netilmicin potency. No changes in netilmicin potency, pH, osmolarity, color, or clarity were observed in any of the 37 netilmicin sulfate admixtures. Similarly, no changes in pH, osmolarity, or clarity were seen in the admixtures of netilmicin sulfate with a second additive. Netilmicin activity was retained for seven days in 22 of the 26 admixtures with a second additive. Netilmicin sulfate in admixtures with multivitamin injection or vitamin B complex (Upjohn) was stable for only one day; with diphenhydramine hydrochloride or neostigmine methylsulfate, for only three days. No incompatibilities between the intravenous infusion set and admixtures of netilmicin sulfate were apparent. Netilmicin sulfate injection is compatible and stable for at least seven days stored at 4 and 25 degrees C when mixed in 37 intravenous injections, and when mixed individually with 22 additives in 5% dextrose and 0.9% sodium chloride injection.

High-pressure liquid chromatographic method for determination of rosaramicin in humans.

A high-pressure liquid chromatographic method has been developed for the quantitative determination of rosaramicin in serum. This procedure involves addition of an internal standard, adjustment to alkaline pH, treatment with potassium carbonate, ether extraction, and a reverse-phase column separation with acetonitrile-acetate buffer mixture as the mobile phase. This technique produces a good linear relationship between the peak height ratio and the rosaramicin concentration. In addition, this method has proven to be quite specific for rosaramicin, since many of its derivatives tested do not interfere with the assay. The method is accurate and reproducible with a sensitivity of about 0.01 microgram of rosaramicin per ml of serum. It may be useful in monitoring drug levels in serum of patients and also for the pharmacokinetic studies of the drug in humans.

Method for qualitative determination of 2-deoxystreptamine.

We have developed a qualitative method to assay for the presence of 2-deoxystreptamine in hydrolysates of crude aminoglycoside preparations using a 2-deoxystreptamine-requiring idiotrophic mutant. The assay involves (i) incubation of a 2-deoxystreptamine-requiring mutant with 1 mg of a hydrolyzed preparation from a crude unknown antibiotic mixture per ml, and (ii) examination of the resultant incubation mixture for production of antibiotic(s) by disk assay.

New polyene antifungal antibiotic produced by a species of Actinoplanes.

A new species of Actinoplanes, which has been deposited with the designation NRRL 5325 at the Northern Utilization Research and Development Division of the U. S. Department of Agriculture, produces a polyene antifungal complex designated as Sch 16656. The complex, consisting of one major and three minor components, is isolated from the fermentation broth by a solvent extraction procedure and purified by precipitation methods. The major component is a heptaene and is highly active in vitro and in vivo against Candida albicans. It is active also against strains of Torulopsis and is significantly more potent orally than candicidin in mice against Candida infections.

Comparison of enzymatic and microbiological gentamicin assays.

The correlation coefficient between the rapid enzymatic and the overnight microbiological assays for 211 urine and serum specimens was 0.96. The 95% confidence limits yielded a correlation coefficient between 0.92 and 0.98. Both methods tended to underestimate the amount of a gentamicin added to urine. When only serum samples were considered, the predicted value obtained from the linear regression analysis of either method was within 0.57 mug/ml 99% of the time. This high degree of positive correlation will permit safe rapid adjustment of individualized patient gentamicin dosages.

Biological activity of Sch 14342, an aminoglycoside antibiotic coproduced in the gentamicin fermentation.

Sch 14342 is an aminoglycoside antibiotic coproduced as a minor component in the gentamicin fermentation. Sch 14342 was found to have the same antibacterial spectrum as gentamicin in vitro and in vivo, and was approximately one-third as active in mouse protection tests. Sch 14342 relative to gentamicin was one-third as toxic in acute tests in mice, one-eighth as toxic in renal toxicity tests in dogs, and an estimated one-tenth as toxic in cat ataxia tests. Sch 14342 possesses a significantly improved therapeutic index relative to gentamicin with reference to ataxia potential and renal toxicity.