Subtype analysis of Blastocystis isolates in Swedish patients.

Blastocystis is a genetically diverse and widespread intestinal parasite of animals and humans with controversial pathogenic potential. At least nine subtypes of Blastocystis have been found in humans. The genetic diversity of Blastocystis was examined in stool samples from 68 patients from the Stockholm area, Sweden. Blastocystis was identified by light microscopy, and subtyped by sequencing the 5'-end of the small subunit ribosomal RNA gene. Five Blastocystis subtypes were identified in the 63 patients whose samples were successfully subtyped: ST1 (15.9%), ST2 (14.3%), ST3 (47.6%), ST4 (20.6%), and ST7 (1.6%). ST3 was more common in males compared to females (P=0.049). Comparative molecular analysis of Blastocystis sequences revealed intra-subtype variations within the identified subtypes with the exception of ST4. Among ST4 sequences in this study, as well as in the majority of human GenBank sequences, a limited genetic diversity was found compared to what was found among the other common subtypes (ST1, ST2 and ST3). The relative prevalence of ST4 in this study was comparable to the overall distribution of ST4 in European cohorts (16.5%). This contrasts with the sparse reports of ST4 in studies from other continents, which may indicate that the distribution of this subtype is geographically heterogeneous.

A rotatable electron spectrometer for multicoincidence experiments.

We have developed a rotatable hemispherical spectrometer with good energy and angular resolution, which can be positioned with the lens axis arbitrarily within a solid angle of 1 pi. The collection angle of the emitted electrons with respect to the polarization axis of the light is set by means of a three-axes goniometer, operating under vacuum. An important requirement for this setup was the possibility to perform coincidences between the electron analyzed by the spectrometer and one or several other particles, such as ions, electrons, or photons. The lens system and the hemispheres have been designed to accommodate such experimental demands, regarding parameters such as the resolving power, the acceptance angle, or the width of the kinetic energy window which can be recorded for a given pass energy. We have chosen to detect the impact position of the electron at the focal plane of the hemispherical analyzer with a delay line detector and a time-to-digital converter as acquisition card rather than using a conventional charge-coupled device camera.

The early ontogeny of neuronal nitric oxide synthase systems in the zebrafish.

To examine a putative role for neuronal nitric oxide synthase (nNOS) in early vertebrate development we investigated nNOS mRNA expression and cGMP production during development of the zebrafish Danio rerio. The nNOS mRNA expression in the central nervous system (CNS) and periphery showed a distinct spatio-temporal pattern in developing zebrafish embryo and young larvae. nNOS mRNA expression was first detected at 19 h postfertilisation (h.p.f.), in a bilateral subpopulation of the embryonic ventrorostral cell cluster in the forebrain. The number of nNOS mRNA-expressing cells in the brain slowly increased, also appearing in the ventrocaudal cell cluster from about 26 h.p.f., and in the dorsorostral and hindbrain cell cluster and in the medulla at 30 h.p.f. A major increase in nNOS mRNA expression started at about 40 h.p.f., and by 55 h.p.f. the expression constituted cell populations in differentiated central nuclei and in association with the proliferation zones of the brain, and in the medulla and retina. In parts of the skin, nNOS mRNA expression started at 20 h.p.f. and ended at 55 h.p.f. Between 40 and 55 h.p.f., nNOS mRNA expression started in peripheral organs, forming distinct populations after hatching within or in the vicinity of the presumptive swim bladder, enteric ganglia, and along the alimentary tract and nephritic ducts. Expression of nNOS mRNA correlated with the neuronal differentiation pattern and with the timing and degree of cGMP production. These studies indicate spatio-temporal actions by NO during embryogenesis in the formation of the central and peripheral nervous system, with possible involvement in processes such as neurogenesis, organogenesis and early physiology.

Parallel DNA template preparation using a vacuum filtration sample transfer device.

Solid-phase techniques have facilitated the handling of biochemical analytes. This has stimulated the development of systems by which large sample panels can be analyzed with high levels of security and quality. We describe a sample transfer device based on the principle of vacuum filtration, which enables parallel handling of 96 samples of analytes bound to Sepharose beads. The tool was employed for strand separation of DNA samples, by attracting the beads to filter probes while passing them between the reagent solutions. The samples were analyzed using Pyrosequencing technology and proved to yield genotyping results of high quality. The presented sample preparation procedure provides an important link in the development of integrated systems for rapid genetic analysis at a low cost. In addition, the same filter could be reused extensively with very low risk for detectable cross-contamination between assays and without any reduction in processing capacity, thus further reducing the cost per analyzed sample.

Mouse retina explants after long-term culture in serum free medium.

The neonatal mouse retina remains viable as an explant in serum-supplemented growth media for more than 4 weeks. Interpretation of drug effects on this tissue is compromised by the enigmatic composition of the serum. We sought to remove this ambiguity by culturing neonatal as well as late postnatal mouse retina in serum-free nutrient medium. In this study three important observations were made, (1) there is histotypic development of neonatal as well as preservation of late postnatal mouse retinal structure during long-term culture in serum-free medium, although the late postnatal tissue tends to show some loss of cells in the outer nuclear layer. (2) Protein expression in explant photoreceptor cells was similar to that in the litter-matched ones, except for green cone opsin and interphotoreceptor retinoid-binding protein, although mRNA of the latter is present at similar amounts as in age-matched in vivo controls. (3) Cells of the inner retina stained by antibodies to calcium-binding proteins display some novel sprouting of processes. The results show that the mouse retina can be cultured as an explant for more than 4 weeks in a serum-free medium. This represents an important step forward because, (1) the possibility of interference of drug effects by unknown serum factors has been eliminated; and (2) the spent culture medium can be analyzed to investigate biomolecules released by the retina in vitro.

Expression of pineal ultraviolet- and green-like opsins in the pineal organ and retina of teleosts.

In teleostean bony fishes, studies on the adults of various species have shown that pineal photoreceptors are maximally sensitive to short- and middle-wavelength light, possibly utilising both rod-like and pineal-specific opsins. Until recently, however, very little was known about the pineal opsins present in embryonic and larval teleosts and their relationships to opsins expressed by retinal photoreceptors. Our immunocytochemical studies have revealed that, in Atlantic halibut, herring and cod, pineal photoreceptors express principal phototransduction molecules during embryonic life before they appear in retinal photoreceptors. In cDNA from embryonic and adult halibut, we identified two partial opsin gene sequences, HPO1 and HPO4, with highest homology to teleost green and ultraviolet cone opsins (72-83% and 71-83% amino acid identity, respectively). In halibut, these opsins are expressed in the pineal organ of embryos and appear in the retina of larvae. Our recent in situ hybridisation studies with RNA probes for HPO1 and HPO4 demonstrate the presence of green-like opsin mRNAs in the pineal organ and the retina of herring, cod, turbot, haddock, Atlantic salmon, zebrafish and three species of cichlid, and of ultraviolet opsins in the retinas of zebrafish, Atlantic salmon, turbot and the three cichlid species. We conclude that the halibut pineal organ appears to have the potential for both ultraviolet and green photosensitivity from the embryonic stage and that the retina may acquire the same potential during the larval stages. In the other teleosts studied, although both pineal and retinal photoreceptors seem to utilise a green-like opsin from the larval stage, ultraviolet photoreception appears to be restricted to the retina.

Identification and distribution of nitric oxide synthase in the brain of adult zebrafish.

Nitric oxide (NO) is proposed to be involved in developmental and plastic processes. We investigated the presence and distribution of nitric oxide synthase (NOS) in the zebrafish (Danio rerio) using molecular and histochemical techniques. A partial gene sequence corresponding to the neuronal NOS isoform (nNOS) was identified, and in situ hybridization revealed cellular nNOS mRNA expression throughout the brain of adult zebrafish, distributed in distinct central nuclei and in proliferation zones. NOS immunoreactivity and nicotinamide adenine dinucleotide phosphate diaphorase activity partly coincided with the nNOS mRNA expression, however was present also in additional neuronal and non-neuronal cell types. The results indicate the occurrence of different NOS isoforms in the adult brain, of which nNOS may participate in neurotransmission, and in mechanisms related to the continuous growth and neuronal plasticity of the teleost brain.

Analysis of potential causes of positive microbiological cultures in tissue donors.

The purpose of this statistical analysis is to determine what factors are the major contributors to bacterial contamination of recovered human cadaveric tissue. In this study we analyzed factors that could contribute to an increased bacterial bioburden from recovered tissues using the following independent variables: (1) the physical recovery environment; (2) recovery before or after an autopsy; (3) the length of time from death to recovery; (4) the cause of death; (5) the length of time to complete recovery; (6) the number of staff involved with the tissue recovery; and (7) the impact of organ and skin recovery on musculoskeletal contamination rates.In these analyses we used analysis of variance of main effects on data from seven tissue banks. The scale of the analysis included 1036 donors each having multiple cultures to better control for the inherent large variation in this type of data. We looked at several dependent variables. The dependent variable that was most useful was 'percent positive cultures'.The results of the combined data differed from analyzing the tissue banks individually. The differences in each tissue bank's procedures and techniques were responsible for most of the variability. Depending on how the data was organized, statistically significant increases in bioburden were seen with: (1) recoveries after autopsy; (2) location of the recovery; (3) length of time taken for a recovery; (4) size of the recovery team; and (5) the impact of organ and skin recovery on musculoskeletal contamination rates.In conclusion, statistical analysis of recovery cultures can be a powerful tool that may be used to indicate problems within any bank's recovery procedures or techniques.

Human cadaver skin viability for in vitro percutaneous absorption: storage and detrimental effects of heat-separation and freezing.

PURPOSE: For decades, human cadaver skin has been banked and utilized by hospitals for burn wounds and to study percutaneous absorption and transdermal delivery. Skin storage maintenance and confirmation of skin viability is important for both uses, especially for the absorption process where the in vivo situation is simulated. METHODS: Our system uses dermatomed human cadaver skin immediately placed in Eagles MEM-BSS, and refrigerated after donor death, then transferred to the laboratory and placed in Eagles MEM-BSS with 50 micrograms/ml gentamicin at 4 degrees C for storage. RESULTS: Skin viability, determined by anaerobic metabolism where glucose is converted to lactose, was highest (p < 0.000) during the 18 hours of the first day after donor death, decreased some 3-fold by day 2 (p < 0.000), but then maintained steady-state viability through day 8. Viability then decreased by approximately one-half by day 13. Thus, using the above criteria, human skin will sustain viability for 8 days following donor death in this system. Heat-treated (60 degrees C water for one minute) and heat-separated epidermis and dermis lose viability. CONCLUSIONS: Human skin viability can be maintained for absorption studies. It is recommended that this system be used, and that heat-separation and skin freezing not be used, in absorption studies where skin viability and metabolism might be contributing factors to the study.

Role of the pineal organ in the photoregulated hatching of the Atlantic halibut.

The timing of hatching in the Atlantic halibut (Hippoglossus hippoglossus) has been suggested to be regulated by environmental light conditions. However, the photosensory organ that perceives the triggering light has not been identified. In the present study, we investigated the morphogenesis of the pineal organ and the neurochemical differentiation of photoreceptors in the pineal organ and the retina of the Atlantic halibut during embryonic development. Immunocytochemical techniques were used for detection of integral protein components of the phototransduction process: opsins, arrestin (S-antigen) and alpha-transducin. We also studied the expression of serotonin (5-HT), a precursor of the neurohormone melatonin known to be synthesized by pineal photoreceptors. In the pineal anlage, opsin immunoreactive (ir) cells appear at 11 days post-fertilization (pf), arrestin, alpha-transducin and serotonin ir cells appear at 14 days pf; hatching took place 15 days pf. The retina contained no immunoreactive cells in embryos or in newly hatched larva. During this period, the pineal anlage is morphologically discernible only as a wedge-shaped region in the diencephalic roof, where elongated cells are aligned with their long axes converging toward a centrally located presumptive pineal lumen. The results show that the pineal photoreceptors contain serotonin and molecules involved in the phototransduction cascade before hatching. We suggest that the pineal organ has the capacity to perceive and mediate photic information before hatching in halibut embryos, and may thereby influence the timing of hatching.

Impaired B cell maturation in mice lacking Bruton's tyrosine kinase (Btk) and CD40.

Mutations in Bruton's tyrosine kinase (Btk) gene, in mice, result in reduced numbers and responses of peripheral B cells. Surface Ig-mediated signaling is defective in Btk mutant B cells as they do not proliferate upon slg cross-linking and lack thymus-independent (TI) type II responses. Signals through sIg and CD40 play a critical role in B cell maturation. To investigate the consequences of the lack of both Btk and CD40 on B cell development and function, mice were generated that were homozygous for targeted mutations in the Btk and the CD40 genes (BtkMCD40M). The CD40 mutation (CD40M) had a synergistic effect on the BtkM defects. In BtkMCD40M mice the number of B cells was reduced 3- to 4-fold compared to BtkM mice and mature B cells (IgMlow/IgDhigh) were virtually absent; serum levels of all Ig isotypes were diminished; and antibody responses to TI-I TI-II and thymus-dependent antigens were impaired. Furthermore, although wild-type BtkM and CD40M mice produced germinal centers in response to TI-I antigen, the BtkMCD40M mice did not. Maturational and functional B cell defects in BtkMCD40M mice may result from a combination of intrinsic B cell defects, lack of CD40L-dependent T cell help and microenvironmental defects. These data suggest that signals through Btk and CD40 are necessary for the production and maintenance of the mature B cell.

Inhibition of human lymphocyte transformation by the macrocyclic trichothecenes roridin A and verrucarin A.

The effect of in vitro exposure to the macrocyclic trichothecenes roridin A and verrucarin A on human lymphocyte transformation was evaluated in the mitogen-induced blastogenesis assay. Both compounds were capable of inhibiting stimulation of B- and T-cell subsets by a mitogen panel that included leukoagglutinin, concanavalin A, and pokeweed mitogen. Doses of roridin A and verrucarin A which inhibited [3H]thymidine uptake by 50%, as averaged from this mitogen panel, were 20 and 9 pg/ml, respectively. Verrucarol, a compound which results from base hydrolysis of macrocyclic trichothecenes, had no effect on blastogenesis at levels up to 5 X 10(5) pg/ml, indicating that an intact macrocyclic ring was essential for the potent inhibitory activity of roridin A and verrucarin A. The toxicity of these two compounds was extraordinary relative to that reported for non-macrocyclic trichothecenes and could not be predicted quantitatively from previous structure-activity studies on the toxic and biochemical properties of the trichothecenes.

Suppression of immune response in the B6C3F1 mouse after dietary exposure to the Fusarium mycotoxins deoxynivalenol (vomitoxin) and zearalenone.

The effect that dietary exposure to the naturally-occurring Fusarium graminearum toxins deoxynivalenol (DON) and zearalenone (ZEA) may have on immune function was assessed in the B6C3F1 mouse. Dietary DON depressed the plaque-forming response to sheep red blood cells, the delayed hypersensitivity response to keyhole limpet haemocyanin and the ability to resist Listeria monocytogenes. Listerial resistance was similarly decreased in control mice fed restricted diets comparable to the dietary restriction caused by DON-induced feed refusal, whereas equivalent food restriction did not decrease the plaque or delayed hypersensitivity responses. ZEA ingestion decreased resistance to L. monocytogenes but did not affect splenic plaque-forming or delayed hypersensitivity responses. Resistance to Listeria was reduced to a greater extent by co-administration of DON and ZEA than by DON alone, whereas the ability of DON to inhibit the delayed hypersensitivity response was significantly lessened in the presence of ZEA. While effects on resistance to Listeria and delayed hypersensitivity were detectable in mice ingesting the mycotoxins for 2-3 wk, these effects disappeared upon extension of the feeding period to 8 wk. In contrast, some effect on the plaque-forming response was detectable with both the 2- and the 8-wk period of mycotoxin ingestion. Immunosuppression can thus result from ingestion of F. graminearum-infected agricultural staples, the suppression being attributable to interactions between direct immunotoxic effects of DON and ZEA and nutritional effects associated with DON-induced food refusal.

Comparison of acute toxicities of deoxynivalenol (vomitoxin) and 15-acetyldeoxynivalenol in the B6C3F1 mouse.

The acute toxic effects of deoxynivalenol (DON) and 15-acetyldeoxynivalenol (15-ADON) were compared in the B6C3F1 female mouse after oral and intraperitoneal exposure. Using the abbreviated procedure of Lorke (Archs Toxicol. 1983, 54, 275), LD50 values for DON were estimated to be 78 mg/kg (oral) and 49 mg/kg (ip) whereas the LD50 values for 15-ADON were 34 mg/kg (oral) and 113 mg/kg (ip). Acute doses of these toxins resulted in extensive necrosis of the gastro-intestinal tract, bone marrow and lymphoid tissues, and focal lesions in kidney and cardiac tissue. The minimum doses required for these histopathological effects were consistent with LD50 estimations. The results indicate that 15-ADON was more or less toxic than DON depending on the route of administration. Risk assessments for DON should therefore consider the potential for 15-ADON occurrence and toxicity in food and feed.

Decreased feed consumption and body-weight gain in the B6C3F1 mouse after dietary exposure to 15-acetyldeoxynivalenol.

15-Acetyldeoxynivalenol (15-ADON), a biosynthetic precursor of deoxynivalenol (DON), was extracted from rice cultures of Fusarium graminearum R6576 and purified. Growing female B6C3F1 mice were fed semi-purified diets containing 0, 0.5, 2.0 and 5.0 ppm 15-ADON over 56 days and assessed for effects on feed intake, body-weight gain, terminal organ weights and blood clotting function. A significant reduction in feed intake was observed at the 5.0-ppm level after 44 days, whereas reduced rates of weight gain were found to occur at the 5.0-ppm level after only 16 days. Terminal liver, kidney and spleen weights were significantly lower in mice consuming the 5.0-ppm diet when compared with controls. Dietary 15-ADON at the 0.5- and 2.0-ppm levels did not show significant effects on weight gain, feed intake or organ weights. Although mice treated with 15-ADON had significantly decreased bleeding times, other measurements of clotting function indicated no differences between the control and treated groups. Results indicated that 15-ADON was only slightly less toxic than DON and that chronic manifestations of dietary 15-ADON were similar to those found previously for DON. Future risk assessments for DON should therefore include consideration of 15-ADON occurrence and toxicity.

Effects of 8-week exposure of the B6C3F1 mouse to dietary deoxynivalenol (vomitoxin) and zearalenone.

Weanling female B6C3F1 mice were fed semi-purified diets containing 0, 0.5, 2.0, 5.0, 10.0 or 25.0 ppm (mg/kg) deoxynivalenol (DON) over 8 wk and were assessed for effects on feed intake, body-weight gain, terminal organ weights, histopathology, haematology and serum immunoglobulin levels. To determine whether DON effects were potentiated by the oestrogen zearalenone (ZEA), a mycotoxin frequently found to occur with DON in cereals, two additional groups of mice were fed diets containing either 10 ppm ZEA or 10 ppm ZEA plus 5 ppm DON. The rate of body-weight gain was significantly reduced (P less than 0.01) for all mice consuming feed containing 2.0 ppm or more of DON, whereas only the mice ingesting the diet containing 25 ppm DON showed a significantly decreased (P less than 0.01) rate of feed consumption. Gross and histopathological evaluation of thymus, spleen, liver, kidney, uterus, small intestine, colon, heart, brain, lungs and bone marrow from control and all mycotoxin-exposed mice revealed that these tissues were normal in appearance and in histological architecture. DON-amended diets did however, cause dose-dependent decreases in the terminal organ weights recorded (thymus, spleen, liver, kidney and brain). In the DON-treated groups, statistically significant dose-dependent decreases in the counts of total circulating white blood cells were associated with an increase in polymorphonuclear neutrophils and a decrease in lymphocytes and monocytes. Dietary DON caused a dose-dependent decrease in serum IgM but, in contrast, a dose-dependent increase in serum IgA. In none of the above instances was 10 ppm ZEA shown to act synergistically or antagonistically with 5 ppm DON. Since dietary DON at levels as low as 2.0 ppm exerted significant effects on the growing B6C3F1 female mouse, future approaches should include studies of the mechanisms by which this mycotoxin affects nutrient utilization and modifies the normal immune response.

Assessment of pentachlorophenol toxicity in newborn calves: clinicopathology and tissue residues.

Newborn Holstein bull calves were fed either analytical pentachlorophenol (aPCP) or technical pentachlorophenol (tPCP) for 6 wk to establish and compare the clinical and pathologic manifestations of toxicity. Four groups of three calves/group were each fed either 1 or 10 mg X (kg body weight)-1 X d-1 of either aPCP or tPCP. A fifth group served as control. Dosages of both PCP preparations were normalized to contain equal concentrations of PCP. Toxic effects were observed only at the 10 mg/kg dose in the tPCP-treated calves. These effects included decreased body weight gain, anorexia, decreased serum protein concentration, elevated serum gamma glutamyl transferase, and decreased triiodothyronine (T3) and thyroxine (T4) concentrations. Histologic lesions included cortical atrophy in the thymus and squamous metaplasia and hyperkeratous changes in the Meibomian gland of the eyelid. Thyroid function, which was assessed in vivo by measuring the rate of T3 and T4 production over 4 h after thyrotropin-releasing hormone (TRH)-challenge, was not impaired suggesting an extrathyroidal site of toxic action. Although serum chemistry indicators were suggestive of hepatic injury there were no discernable lesions. Organ weight analyses were inconclusive but there was a tendency toward enlargement of liver, kidneys and thyroid and decreased weight of lungs, spleen and thymus. A toxic effect clearly related to PCP and not its contaminants was depressed active transport of p-aminohippurate measured in kidney slices in vitro. Steady state concentrations of PCP in serum were about 40 and 90 ppm for the 1 and 10 mg/kg groups, respectively. Concentrations of PCP among the major organs were comparable.

Relation of 8-ketotrichothecene and zearalenone analog structure to inhibition of mitogen-induced human lymphocyte blastogenesis.

The 50% effective doses of fusarenon X, nivalenol, deoxynivalenol, and 15-acetyldeoxynivalenol required to reduce [3H]thymidine uptake in mitogen-stimulated human lymphocytes by 50% were 18, 72, 140, and 240 ng/ml, respectively. These results indicated that lymphotoxicity of 8-ketotrichothecenes decreased according to the C-4 substituent order acetyl greater than hydroxyl greater than hydrogen, whereas acetylation of position C-15 of deoxynivalenol caused a slight decrease in in vitro toxicity. The 50% effective doses for zearalenone, alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol were 3,500, 6,300, 36,000, 3,750, and 33,000 ng/ml, respectively, suggesting that a keto group or alpha-hydroxyl at the position C-6' contributed to the lymphotoxicity of the parent molecule. The inhibitory effects of zearalenone analogs observed in the blastogenesis assay did not correlate with the estrogenic potencies of these compounds. All 8-ketotrichothecenes and zearalenone analogs tested were capable of inhibiting B- and T-cell subsets stimulated by a mitogen panel of leukoagglutinin, concanavalin A, and pokeweed mitogen.

Bovine neutrophils treated with chemotactic agents: morphologic changes.

Neutrophils were isolated from the peripheral blood of cattle. After the neutrophils were incubated with zymosan-activated serum, the neutrophils changed from spherical to a bipolar shape. Ninety percent of the neutrophils became bipolar in 5 to 10 minutes. Bacterial cell filtrate and casein also induced bipolar shape changes in neutrophils, but N-formyl-L-methionyl-L-leucinyl-L-phenylalanine did not. The neutrophil-shape-change response was a rapid in vitro assay to evaluate early chemotactic events.