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.

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.

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.

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.

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.

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.

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.

Retained T-cell reactivity of rye grass pollen extract following cleavage with cyanogen bromide and nitrothiocyanobenzoic acid.

Rye grass pollen extract was fragmented by sequential treatments with cyanogen bromide and 2-nitro-5-thiocyanobenzoic acid, and a fraction containing fragments of molecular weight greater than 10,000 Mr was isolated. The in vitro reactivity of the extract with specific IgE was extensively reduced by fragmentation. Less reduction in activity was shown either by skin testing or by inhibition of an extract-specific IgG-binding assay. Reactivity with, and ability to induce, extract-specific mouse T cells were retained by the fragment preparation, and the ability to cause transformation of lymphocytes from atopic donors was unchanged. Fragments did not induce extract-specific IgG antibody in mice, were unable to stimulate the production of T-helper cells which could collaborate in an adoptive cell-transfer system, and did not induce delayed hypersensitivity reactions in guinea pigs. The possibility that such T-cell-reactive modified allergens (T'allergoids) might be used to stimulate selectively T-cell subsets and, therefore, could be used to advantage in immunotherapy is discussed.

Induction of allergen-specific T cells by conjugates of N-formyl-methionyl-leucyl-phenylalanine and rye grass pollen extract.

A conjugate of the biologically active peptide N-formyl-methionyl-leucyl-phenylalanine and rye grass pollen extract (F-MLP/rye), previously shown to react with rye grass pollen extract-specific T cells, induced the formation of allergen-specific T cells in mice. Lymph node cells prepared from mice immunized with either native extract or F-MLP/rye gave an enhanced response to unmodified rye grass pollen allergens in vitro. Syngeneic spleen macrophages were able to present the unmodified allergens to T cells obtained from both groups of mice causing their proliferation in vitro. Conjugation of the peptide into the extract brought about an extensive reduction in its reactivity with grass pollen-specific human IgE, and a loss of its ability to induce specific IgG antibody in guinea-pigs. A state of delayed hypersensitivity specific for rye grass pollen extract was produced in guinea-pigs by immunization with either the F-MLP/rye or unmodified extract. It is concluded that conjugates such as F-MLP/rye or other T' allergoids could be used as probes to investigate whether changes in T cell activity are important in immunotherapy.

Induction of T-helper cell activity by fragments of rye grass pollen extract produced by digestion with chymotrypsin.

Rye grass pollen extract was digested by chymotrypsin to produce fragments with a molecular weight below 10,000, as demonstrated by polyacrylamide gel electrophoresis. Chymotryptic fragments did not react with either human or mouse IgG antibodies specific for rye grass pollen, nor did they induce an antibody response in mice with specificity for the parent extract. However, macrophage-presented fragments retained the ability to react with rye-specific T cells in a lymphoproliferation assay. Furthermore, these fragments induced the development of splenocytes capable of supporting dinitrophenyl specific antibody production. This implies that the fragments still react with, and induce, rye grass pollen extract-specific T-helper cells. The possibility that such fragments might have potential for use in immunotherapy for the specific treatment of allergy is discussed.

Immunogenicity in guinea pigs and tolerance in grass pollen-sensitive volunteers of enteric-coated grass pollen allergens.

An acid-insoluble, methacrylic acid, methyl methacrylate copolymer (Eudragit L-100) was used to give an enteric coating to a grass pollen extract in order to protect it against gastric degradation. Substantial protection against the degradative effects of simulated gastric secretion was demonstrated using this preparation which was well tolerated by grass pollen-allergic volunteers. The enteric-coated allergen induced a greater secondary antibody response than did an aqueous presentation when administered orally to guinea pigs which had been primed previously by subcutaneous injection. This result indicates that an effective hyposensitisation regimen could consist of a short series of initial parenteral injections, followed by an oral course of the protected allergen.

T cell reactivity of conjugates of N-formyl-methionyl-leucyl-phenylalanine and rye-grass pollen allergens.

Conjugates of rye-grass pollen extract and N-formyl-methionyl-leucyl-phenylalanine were prepared using an activated form of the tripeptide. Introduction of the peptide into the extract brought about an extensive reduction of reactivity with grass-pollen-specific IgE, as measured by RAST inhibition. Despite this loss, guinea pig alveolar macrophages and murine splenic macrophages readily presented the conjugates to T lymphocytes specific for grass pollen allergens and caused their proliferation in vitro.

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.

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.

Suppression of murine IgE responses with amino acid polymer/allergen conjugates. IV. Suppressive activities in established IgE model systems.

Conjugates of poly-N-methylglycine (polysarcosine) and grass pollen extracts, previously found to be capable of suppressing immature IgE antibody responses in mice, were shown to be highly effective at inhibiting the capacity of immune splenocytes to produce a secondary response in sub-lethally irradiated recipient animals. Anamnestic IgE responses in mice primed without adjuvant were also suppressed, but the effects were modest and of short duration. The predictive value of murine models for selecting clinically appropriate specific IgE suppressive agents and treatment schedules that might be successfully employed for clinical use are discussed.

Suppression of murine IgE responses with amino acid polymer/allergen conjugates. III. Activity in vitro.

Conjugates of poly-N-methylglycine (polysarcosine) and grass pollen allergen extracts, which have been previously shown to suppress murine IgE responses, were examined for their ability to modify lymphocyte activity in vitro. Allergen-specific T lymphocytes obtained from Balb/c mice gave a reduced response to syngeneic accessory cells pulsed with conjugates of polysarcosine-allergen compared with the response found using equivalent concentrations of native extract. Pretreatment of accessory cells with either polysarcosine or polysarcosine-allergen conjugates did not impair their subsequent ability to present grass pollen extract to immune T cells. Incubation of allergen-specific spleen cells with polysarcosine-allergen conjugates, but not with polysarcosine or allergen alone, resulted in specific cell-mediated suppression which significantly reduced proliferation in vitro. This activity was sensitive to treatment of cells with anti-T-lymphocyte antisera plus complement. Spleen cells obtained from animals immunised with allergen and taken 21 days after intravenous treatment with polysarcosine-allergen conjugates, a regimen that suppressed IgE antibody production, did not proliferate in the presence of grass pollen extract and failed to suppress a secondary lymphoproliferative response in vitro. Spleen cells obtained from similarly treated animals 3 days after the final polysarcosine-allergen injection responded to pollen extract in culture and, additionally, impaired a secondary response. The results suggest that the reduced IgE response found in animals treated with polysarcosine-allergen conjugates may be due, in part, to the generation of a short-lived antigen-specific T cell suppression.

Human IgG antibody responses to grass pollen antigens: effects of immunotherapy and comparison of serum and secretory profiles.

IgG antibody responses, induced by individual temperate grass pollen allergens in atopic subjects, have been studied by an SDS-PAGE immunoprecipitation technique. These were shown to be of a complex nature and variable between individuals. Profiles obtained with serum and nasal secretions from the same individual were qualitatively similar. Immunotherapy with a glutaraldehyde-modified grass pollen extract, Pollinex, increased the level of specific IgG to three clinically significant grass pollen allergens but did not markedly change the overall pattern of response.

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.

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.