DGGE-based detection method for Quahog Parasite Unknown (QPX).

Quahog Parasite Unknown (QPX) is a significant cause of hard clam Mercenaria mercenaria mortality along the northeast coast of the United States. It infects both wild and cultured clams, often annually in plots that are heavily farmed. Subclinically infected clams can be identified by histological examination of the mantle tissue, but there is currently no method available to monitor the presence of QPX in the environment. Here, we report on a polymerase chain reaction (PCR)-based method that will facilitate the detection of QPX in natural samples and seed clams. With our method, between 10 and 100 QPX cells can be detected in 1 l of water, 1 g of sediment and 100 mg of clam tissue. Denaturing gradient gel electrophoresis (DGGE) is used to establish whether the PCR products are the same as those in the control QPX culture. We used the method to screen 100 seed clams of 15 mm, and found that 10 to 12% of the clams were positive for the presence of the QPX organism. This method represents a reliable and sensitive procedure for screening both environmental samples and potentially contaminated small clams.

Effects of streptovaricins and their degradation products on RNA-directed DNA polymerase of Rauscher leukemia virus.

The activities of streptovaricin complexes, streptovaricins, streptovals, and streptovarinic degradation products were elevated against RNA-directed DNA polymerases of Rauscher leukemia virus, DNA-dependent DNA polymerase of bacterial and mammalian cells, and DNA-dependent RNA polymerases of mammalian origin. The activities of streptovaricins were also listed for comparison purposes. The effects of streptovaricin complexes on viral DNA polymerases varied significantly from lot to lot, and streptovaricin complex lot 7 was the most active. All the streptovals and streptovaricin degradation products except varicinal A showed a marked improvement (twofold to tenfold) in activity against the viral enzyme over the parent streptovaricins. None of these compounds, however, displayed any significant effect on either the DNA polymerase of L1210 leukemia cells and Escherichia coli or the RNA polymerase of isolated nuclei of mouse liver. As a result of tests in these systems, some specific inhibitors of RNA-directed DNA polymerases of Rauscher leukemia virus were selected.

A micro immunoassay method for measuring IgG antibodies using staphylococcal protein-A.

A micro radioimmunoassay procedure has been developed for assaying IgG antibodies to ragweed and grass allergen components. The method offers a rapid and reproducible procedure for estimating antibodies in human sera and is well adapted for monitoring immunological responses following hyposensitisation therapy.

Assessment of anti-streptokinase antibody levels in human sera using a microradioimmunoassay procedure.

A high capacity, quantitative radioimmunoassay procedure has been developed to measure IgG antibodies to streptokinase (SK) in human serum. Results showed that the use of low concentrations of 125I-SK (100 ng/ml) and ambient temperature incubation conditions minimised degradation of the target labelled antigen and afforded antibody binding values which could be confidently related to the native intact SK protein. SK-complexed to plasminogen was also shown to retain the equivalent antigenic activity of native SK. Comparison of the absolute anti-SK levels with streptokinase resistance titres demonstrated that the two measurements of antibody correlated statistically but afforded quantitatively poor agreements. A survey of IgG levels to SK in sera from 93 normal healthy volunteers showed that all individuals displayed readily measurable anti-SK antibodies, with some 80% having IgG concentration capable of binding in excess of 1 microgram SK per ml serum.

Immunological properties of chemically produced fragments of rye grass pollen extract.

Two fragment pools, one of MW greater than 10,000 and the other of MW between 1,000 and 10,000, were prepared by the sequential treatment of rye grass pollen extract with cyanogen bromide (cleavage at Met-X) and 2-nitro-5-thiocyanobenzoic acid (cleavage at X-Cys). Electrophoretic analysis of the two pools showed that none of the major components of the whole extract remained intact. Characterisation of the two fragment pools by radioimmunoassay showed that whilst they both lost the ability to bind to human anti-rye IgE antibodies, they largely retained their reactivity towards both human and mouse anti-rye IgG antibodies. In addition, the higher molecular weight pool retained its ability to stimulate extract-specific T cells, after accessory cell processing. This separation of the immunological properties of rye grass pollen extract by chemical cleavage is seen as a basis for the development of novel immunotherapy agents.

Nutrient Acquisition and Population Growth of a Mixotrophic Alga in Axenic and Bacterized Cultures.

Axenic growth of a mixotrophic alga, Ochromonas sp., was compared in several inorganic and organic media, and in the presence of live bacteria under nutrient-replete and low-nutrient conditions. Axenic growth in the light was negligible in inorganic media with or without the addition of glucose. Addition of vitamins increased growth rate, but average cell size declined, resulting in no net increase in biomass. Supplementing axenic cultures with a more complex organic substrate resulted in moderate growth and higher maximal abundance (and biomass) than in the inorganic media with added vitamins. The absence of light did not greatly affect population growth rate in the presence of complex dissolved organic compounds, although cell size was significantly greater in the light than in the dark. The highest growth rates for the alga (up to 2.6 d-1) were measured in treatments containing live bacteria. Increases in cell number of Ochromonas sp. in the presence of bacterial prey were similar in the light and dark, although chloroplast and cell sizes differed. Bacterial abundance was reduced and dissolved phosphorus and ammonia were rapidly released in bacterized cultures in the light and dark, indicating high rates of bacterial ingestion and suggesting an inability of the alga to store or utilize N and P in excess of the quantities required for heterotrophic growth. Low-nutrient conditions in the presence of bacteria were promoted by adding glucose to stimulate bacterial growth and the uptake of N and P released by algal phagotrophy. Subsequent decreases in dissolved N and P following the addition of glucose corresponded to a second period of rapid growth of the alga in both light and dark. This result, combined with evidence for slow axenic growth of this strain, indicated that nutrient acquisition for this species in the presence of bacteria was accomplished primarily via ingestion of bacteria.

Poor response to a recombinant hepatitis B vaccine in dialysis patients.

Eighty-three dialysis patients were inoculated with 20 micrograms of the recombinant derived hepatitis B vaccine Engerix-B at o, I and 6 months. Twenty-seven (32.5%) became seropositive for anti-HBs antibody after the third inoculation. Of the 56 non-responders, 48 received a 40 micrograms booster dose of vaccine 6 weeks after completion of the initial course and a further eight seroconverted. Six months after the third inoculation only 18/71 patients retested (25.3%) had demonstrable antibodies. We were unable to identify clinical or laboratory parameters separating responders from non responders to the vaccine. We recommend regular checks of anti-HBs status of vaccinated patients as it cannot be assumed that even initial responders retain their immunity. Those infection control procedures known to have decreased the incidence of hepatitis B infection in dialysis units should not be relaxed.

Quantitative analysis of emetine and cephaeline by reversed-phase high performance liquid chromatography with fluorescence detection.

A versatile method for the quantitation of emetine and cephaeline in biological samples is described. Two milliliters of samples containing N-propylprocainamide as the internal standard are buffered to pH 9 and extracted with n-butyl chloride. After subsequent back extraction into 0.01 M hydrochloric acid, a portion of the acid layer is analyzed by reversed-phase high performance liquid chromatography with fluorescence detection. Routinely, the minimum level of detection for both drugs is 5 ng/mL and linearity is demonstrated from 5 to 2500 ng/mL.

Determination of temperature and cooling rate which induce cold shock in stallion spermatozoa.

Experiments were conducted to determine temperatures between 24 and 4 degrees C at which stallion spermatozoa are most susceptible to cold shock damage. Semen was diluted to 25x10(6) spermatozoa/ml in a milk-based extender. Aliquots of extended semen were then cooled in programmable semen coolers. Semen was evaluated by computerized semen analysis initially and after 6, 12, 24, 36 and 48 hours of cooling. In Experiment 1A, semen was cooled rapidly (-0.7 degrees C/minute) from 24 degrees C to either 22, 20, 18 or 16 degrees C; then it was cooled slowly (-0.05 degrees C/minute) to a storage temperature of 4 degrees C. In Experiment 1B, rapid cooling proceeded from 24 degrees C to either 22, 19, 16, or 13 degrees C, and then slow cooling occurred to 4 degrees C. Initiating slow cooling at 22 or 20 degrees C resulted in higher (P<0.05) total and progressive motility over the first 24 hours of cooling than initiating slow cooling at 16 degrees C. Initiation of slow cooling at 22 or 19 degrees C resulted in higher (P<0.05) total and progressive motility over 48 hours of cooled storage than initiation of slow cooling at 16 or 13 degrees C. In Experiment 2A, semen was cooled rapidly from 24 to 19 degrees C, and then cooled slowly to either 13, 10, 7 or 4 degrees C, at which point rapid cooling was resumed to 4 degrees C. Resuming the fast rate of cooling at 7 degrees C resulted in higher (P<0.05) total and progressive motility at 36 and 48 hours of cooled storage than resuming fast cooling at 10 or 13 degrees C. In Experiment 2B, slow cooling proceeded to either 10, 8, 6 or 4 degrees C before fast cooling resumed to 4 degrees C. There was no significant difference (P>0.05) at most storage times in total or progressive motility for spermatozoa when fast cooling was resumed at 8, 6 or 4 degrees C. In Experiment 3, cooling units were programmed to cool rapidly from 24 to 19 degrees C, then cool slowly from 19 to 8 degrees C, and then resume rapid cooling to storage temperatures of either 6, 4, 2 or 0 degrees C. Storage at 6 or 4 degrees C resulted in higher (P<0.05) total and progressive motility over 48 hours of storage than 0 or 2 degrees C.

Effect of dose of GnRH analog on ovulation in mares.

Proper timing of insemination for optimal conception is accomplished by frequent palpations per rectum, by ultrasonography of the preovulatory follicle and/or by treatment with hCG or GnRH. Sustained release of GnRH from implants has been shown to hasten ovulation. Therefore, 2 studies were conducted to evaluate the efficacy of a GnRH analog, deslorelin, for hastening ovulation in nonlactating cyclic mares. The GnRH implant was 2.3x3.7 mm and released deslorelin for 2 to 3 days. In Experiment 1, 60 nonlactating, cycling mares were assigned to 1 of 5 doses: 0, 1.2, 1.7, 2.2 and 2.7 mg per implant. Mares were assigned sequentially on the first day of estrus (Day 1). Ovaries were examined per rectum and with ultrasonography every 12 h until ovulation. Once the mares obtained a follicle>30 mm, they were injected subcutaneously with a GnRH implant. The mares were inseminated every other day during estrus with semen from 1 of 3 stallions. Pregnancy was determined with ultrasonography. Experiment 2, 40 nonlactating, cyclic mares were assigned to 1 of 5 treatments (same treatments as in Experiment 1). Data were obtained on interval to ovulation, duration of estrus and pregnancy rates at 12, 18 and 35 d after ovulation. Time to ovulation was shorter (P<0.05) in GnRH-treated mares than in control mares in the Experiment 1. Mean time to ovulation was 68, 49, 48, 47, 44 h in Experiment 1, and 91, 66, 58, 46, 58 h in Experiment 2 for mares given 0, 1.2, 1.7, 2.2 and 2.7 mg/mare in the 2 trials. Averaged for both experiments, the proportion of mares ovulating within 48 h of treatment was 40, 75, 85, 90 and 90% for 0, 1.2, 1.7, 2.2 and 2.7 mg/mare. For both experiments, there was no effect of GnRH on pregnancy rate. In summary, a subcutaneous implant containing GnRH analog induced ovulation in most mares by 48 h of injection, and there was no advantage of doses higher than 2.2 mg/mare.

Progesterone and estradiol in biodegradable microspheres for control of estrus and ovulation in mares.

Progesterone and estradiol 17-beta in poly (DL-lactide) microspheres were used to control estrus and ovulation in mares after luteolysis was induced by prostaglandin F(2)infinity. Mares were given a single intramuscular injection of biodegradable poly (DL-lactide) microspheres, 1 day following prostaglandin treatment, containing no hormones (control), 0.625 g progesterone and 50 mg estradiol (low dose), 1.25 g progesterone and 100 mg estradiol (medium dose), or 1.875 g progesterone and 150 mg estradiol (high dose; n=15 mares per group). Mares treated with the low dose had significantly longer intervals (P<0.05) to estrus and ovulation than the control mares; however, low dose mares had shorter intervals (P<0.05) to estrus than high dose mares and shorter intervals to ovulation than medium and high dose mares. Regression analysis indicated that the medium dose was sufficient for maximizing interval to ovulation while the high dose maximized interval to estrus. All groups of mares exhibited similar (P>0.05) post-treatment estrus lengths. A clinical response scoring system based on synchrony of both estrus and ovulation within a treatment group was also used to measure the effectiveness of treatments on control of estrus and ovulation. Clinical response scores did not differ (P>0.05) among treatment groups. Mares were randomly assigned for insemination at the beginning of the first post-treatment estrus. Rates for embryo recovery performed by uterine lavage 7 days post-ovulation did not differ (P>0.05) among groups. Concentrations of serum progesterone increased in mares receiving progesterone and estradiol microspheres. At 10 to 14 days post-injection of microspheres, progesterone concentrations were higher (P<0.05) and remained above 1 ng/ml in the mares receiving the high dose. Progesterone concentrations were also higher (P<0.05) on Days -3 to -1 (Day 0 = day of post-treatment ovulation) in mares receiving the high dose when compared to control mares. Gonadotropin concentrations were suppressed (P<0.05) in the medium and high dose groups.

Geldanamycin promotes premature mitotic entry and micronucleation in irradiated p53/p21 deficient colon carcinoma cells.

P53 wild-type and p53-null or mutant cells undergo a G(2)-phase cell-cycle arrest in response to ionizing radiation (IR). In this study we examined the effect of heat-shock protein 90 (HSP90) inhibitor, geldanamycin (GA), on IR-induced G(2) arrest in human colon adenocarcinoma cells with different p53 status. We show that GA treatment abrogates IR-induced G(2)-phase arrest in cells null or mutant for p53. Specifically, GA treatment pushed irradiated p53 signaling-defective cells into a premature mitosis characterized by aberrant mitotic figures, increased gammaH2AX expression and formation of micronucleated cells. Cells expressing wild-type p53 were resistant to GA-induced G(2) checkpoint abrogation. Notably, GA treatment decreased levels of G(2) regulatory proteins Wee1 and Chk1, and inhibitory phosphorylation of Cdc2, independent of p53 status. Further investigation identified p21 as the potential downstream effector of p53 that mediates resistance to G(2) checkpoint abrogation. Clonogenic survival studies demonstrated higher sensitivity to GA alone or combination IR plus GA treatment in p53 and p21-null cells. Collectively, these data demonstrate potential mechanisms through which HSP90 inhibition can enhance the effects of ionizing radiation in p53-compromised cancer cells. Combination IR plus HSP90 inhibitor therapies may be particularly useful in treating cancers that lack wild-type p53.

Proliferation of mouse lymphocytes to rye grass pollen extract: effect of inflammatory exudate cells.

The ability of inflammatory peritoneal exudate cells to modify a murine lymphoproliferative response to rye grass pollen extract was investigated. Popliteal and mesenteric lymph node cells from Balb/c mice immunised with either aqueous rye or rye grass emulsified in complete Freund's adjuvant were co-cultured with 2 X 10(3)-10(5) macrophage or polymorphonuclear neutrophil (PMN)-enriched exudate cells. There was a dose-dependent increase in the response to rye grass antigen with increasing numbers of PMN. A similar enhancement was seen with up to 10(4) macrophages per culture; higher numbers were inhibitory. Lymph node cells from rye grass or ovalbumin-immune mice that had been challenged with a combination of sensitising antigen plus 10(7) PMN gave an increased antigen-specific response in vitro. Neither antigen alone, PMN nor a mixture of PMN plus heterologous antigen was active. Adoptive transfer of a similar number of peritoneal macrophages plus antigen was also without effect. These results are in accord with the suggestion that cells obtained from acute inflammatory lesions can directly influence the activity of immunocompetent lymphocytes.

Antibody responses of mice to intragastric and parenterally administered aeroallergens.

Intragastric administration of aeroallergens (pollen extract)-primed mice to produce transient serum IgE antibody responses following subsequent parenteral stimulation while the same initial dose of extract, given parenterally, did not have this effect. In previously immunized animals, intragastric administration of pollen extract was found to enhance systemic antibody production. These observations indicate that exposure of gut-associated lymphoid tissue to aeroallergens can have a profound effect on subsequent reaginic antibody production. This procedure provides a useful model for studying IgE responses to allergens without the complication of an initial injection with adjuvant. A combination of parenteral immunization with oral administration may therefore offer a convenient immunotherapeutic manoeuvre for patients with seasonal rhinitis/asthma.

Accessory cell response to rye grass pollen extract in mice, guinea pigs and man.

The ability of murine, guinea-pig and human accessory cells to function in antigen presentation has been assayed by a T-cell proliferative response to adherent cells pulsed with rye grass pollen extract. A population of phagocytic, esterase-positive, plastic-adherent splenocytes from non-immune Balb/c mice briefly incubated with solubilized rye antigen were capable of stimulating syngeneic T lymphocytes from immune mice in an antigen-specific manner. Accessory cell function was H-2 restricted and required Ia-positive cells. Treatment with a monoclonal anti-Ia antibody impaired proliferation, whereas the presence of polyclonal rabbit anti-rye antibody increased activity. Guinea-pig peritoneal and alveolar adherent cells pulsed with up to 1 mg/ml antigen increased the response of rye-immune lymph node T cells in vitro. Proliferation was dose-dependent, despite less than 0.1% of the available antigen being cell-associated, and could be inhibited by treating the accessory cells with trypsin, sodium iodoacetate and chloroquine. Adherent human peripheral blood cells treated with rye antigen supported the proliferation of non-adherent and nylon wool-enriched mononuclear cells obtained from both grass pollen-sensitive donors and from individuals lacking either detectable serum IgE antibody or a positive skin test to rye antigen.

Chemical modification of crude timothy grass pollen extract. I. Antigenicity and immunogenicity changes following amino group modification.

Glutaraldehyde modification was found to reduce the allergenic potency of crude timothy pollen extracts yet even highly substituted materials retained the capability of inducing the formation of allergen-specific antibody in animals. Experiments showed that these antibodies were capable of blocking skin test reactions to native timothy allergens in pollen-sensitive human volunteers. The value of glutaraldehyde-modified allergen extracts for use in desensitization vaccine therapy is discussed.

Antibody response profiles to components of rye grass pollen extract in various strains of mice.

Using a combination of immunoprecipitation and polyacrylamide gel electrophoresis techniques, patterns of antibody responses to a number of components in rye pollen extract have been studied in hyperimmune mouse sera. Common laboratory strains of mice were investigated, including AKR, C3H/He, SwR, Ist, Asn, Balb/c, C57BL10, DBA2 and BD1:F1 hybrids. All strains responded vigorously to the immunisation protocol used (rye pollen extract/alum followed by a booster dose in aqueous media) when tested with non-discriminating assay procedures such as haemagglutination or passive cutaneous anaphylaxis directed against the whole rye pollen extract. However, markedly variable antibody responses were observed between strains and between murine and human sera when considered in relation to individual rye extract components. Particularly noteworthy was the relatively poor response of all mice to the low molecular weight rye antigen component (11,000 daltons). This material was readily detected by human sera derived from pollenosis patients. Notable also was the fact that no single rye component was detected by all mice sera. These results illustrate the complex serological response induced by heterogeneous antigen mixtures, such as grass pollen extracts.

Physicochemical and immunochemical characterization of allergenic proteins from rye-grass (Lolium perenne) pollen prepared by a rapid and efficient purification method.

Three fractions of rye-grass (Lolium perenne) pollen extract have been isolated by preparative isoelectric focusing (i.e.f.) and characterized in terms of physicochemical and immunochemical properties. The purified components were designated 'R7' and 'R14' on the basis of their positions in relation to other rye-grass pollen extract components on SDS/polyacrylamide-gel electrophoresis and their apparent molecular masses were assessed as 31 and 11 kDa respectively. On i.e.f., R14 split into two components, one acidic (pI 5.0) and one basic (pI 9.0), termed 'R14a' and 'R14b' respectively, and R7 focused at pI 5.8. R7 and R14a were shown to be allergenic by skin-prick test and all three components were recognized by rye-grass-pollen-specific human IgE. On SDS/polyacrylamide-gel electrophoresis and i.e.f., R7 behaved in a manner identical with that shown by an authentic sample of Rye I and gave an amino acid analysis similar to published data [Johnson & Marsh (1966) Immunochemistry 3, 91-100] for Rye group-I isoallergens; the amino acid sequence of the first 27 N-terminal amino acids was also determined. Physicochemical analysis revealed that R14a was equivalent to Rye II and 14b to Rye III. Preparative i.e.f. followed by gel-permeation chromatography proved to be a rapid and efficient method for purifying the allergenic components of Rye I (R7), Rye II (R14a) and Rye III (R14b) from rye-grass pollen extract.

Synergism between recombinant human interferon and nucleoside antiviral agents against herpes simplex virus: examination with an automated microtiter plate assay.

An automated, quantitative, cytopathic effect (CPE) inhibition assay with human fibroblasts in 96-well microtiter plates was used to examine the combination of recombinant human interferon-alpha (rIFN-alpha A) and acyclovir, vidarabine, or dihydroxypropoxymethyl guanine against herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) in vitro. Fifty percent CPE (CPE50) end points, calculated from optical density readings of crystal violet-stained monolayers in an automated spectrophotometer, represented 1.7 log reduction in viral yield (50-fold or 98% decrease). Using CPE50 end points of drugs alone and in combination, we defined synergism, additivism, or antagonism with an isobologram plot and a combination index equation. The combinations of rIFN-alpha A plus acyclovir and rIFN-alpha A plus dihydroxypropoxymethyl guanine were highly synergistic against both HSV-1 and HSV-2, whereas the combination of rIFN-alpha A plus vidarabine was additive to mildly synergistic. Combinations of antiviral agents synergistic in cell cultures should be pursued with further studies in animal models of human viral disease and potentially in clinical trials.

Suppression of murine IgE responses with amino acid polymer/allergen conjugates. V. By intranasal administration.

Serum IgE antibody responses were generated in mice by intranasal exposure to grass pollen extract. Primary IgE responses were suppressed by the concomitant intranasal administration of a conjugate of polysarcosine and pollen extract which has been shown to be a potent tolerogen when given parenterally. Partial suppression of boosted IgE responses was observed when the conjugate was applied intranasally with a secondary challenge of unmodified extract. The data suggest that clinical schedules of intranasal application of tolerogenic conjugates can be devised to bring about specific IgE suppression.