Standard Washing Procedure Research Articles

Measurement of Trogocytosis: Quantitative Analyses Validated with Rigorous Controls.

Trogocytosis is a process in which receptors on acceptor cells remove and internalize cognate ligands from donor cells. Trogocytosis has a profound and negative impact on mAb-based cancer immunotherapy, as seen in the treatment of chronic lymphocytic leukemia (CLL) with CD20 mAbs, such as rituximab (RTX) and ofatumumab (OFA). Our clinical observations of RTX/OFA-mediated loss of the CD20 target from circulating CLL cells have been replicated in our in vitro studies. Here we describe flow cytometry and fluorescence microscopy experiments, which demonstrate that acceptor cells, such as monocytes/macrophages that express FcγR, remove and internalize both antigen and donor cell-bound cognate IgG mAbs for several different mAb-donor cell pairs. Fluorescent mAbs and portions of the plasma cell membrane are transferred from donor cells to acceptor cells, which include the THP-1 monocytic cell line as well as freshly isolated monocytes. We describe rigorous controls to validate the reactions and eliminate dissociation or internalization as alternative mechanisms. Trogocytosis is likely to contribute to neutropenia, thrombocytopenia, and liver damage associated with use of antibody-drug conjugates. The methods we have described should allow for examination of strategies focused on blocking trogocytosis and its adverse effects. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Trogocytosis of mAb-opsonized donor cells mediated by adherent THP-1 cells Alternate Protocol: Application of fluorescence microscopy to examine THP-1 cell-mediated trogocytosis Support Protocol 1: Alexa labeling of mAbs and determination of F/P ratios Support Protocol 2: Standard washing procedure Support Protocol 3: Labeling and opsonization of cells Basic Protocol 2: Trogocytosis mediated by human monocytes as acceptor cells Support Protocol 4: Isolation of human monocytes Basic Protocol 3: Trogocytosis mediated by THP-1 cells in solution Support Protocol 5: Retinoic acid treatment of THP-1 cells Support Protocol 6: Culturing of SCC-25, BT-474, MOLT-4 and THP-1 cell lines.

Multifunctional finishing of cotton fabric by electron beam radiation synthesized silver nanoparticles

A radiation induced in-situ synthesis of silver particles in cotton fabrics, padded with different initial concentration of an aqueous alcoholic silver nitrate solution, has been accomplished by exposing them to different doses of electron beam (EB) radiation in an inert environment. The amount of synthesized silver particles has been studied as the function of initial solution concentration and EB radiation doses. The treated fabrics were characterized for the surface morphology with the aid of Scanning Electron Microscopy (SEM). The homogeneous and uniform coating of silver particles having an average size of 81 nm was observed on the fibre surface. The quantitative deposition of silver nanoparticles was estimated by inductively coupled plasma‒optical emission (ICP‒OES) spectroscopy technique. Their quantity was found to vary with concentration of the solution used for padding and EB doses, specifically more on the former. In this connection, deposited nanoparticles produced visual colour change in cotton fabrics with light to deeper shade which could be controlled primarily by concentration of solution used for padding. A reflectance and transmittance spectrum in the UV‒Vis region confirmed UV radiation blocking property of the fabric that also dependents on the solution concentration used for padding. Fabric samples exhibited antibacterial behaviour against two different microorganisms, S. Aureus and K. Pneuomonia. Ultraviolet protection (UPF) and antibacterial properties remained similar even after ten washing cycles, carried out as per standard washing procedure. This functional finishing did not adversely affect the air permeability and bending length properties of the fabric to a significant level but introduced a positive change of increased total crease recovery angle.

French firefighters exposure to Benzo[a]pyrene after simulated structure fires.

Firefighters have occupational exposure to toxic compounds during firefighting, but not only. Surface contamination of equipment has never been studied in French firefighters. This study measured the surface load in benzo [a]pyrene (BaP), a carcinogenic polycyclic aromatic hydrocarbon, on the outer surface of fire jackets, personal protective equipment and tools used by firefighters after a live fire training in a closed environment. The effectiveness of a standard jacket washing procedure on BaP contamination was assessed. A single training session was responsible for a BaP deposit of 113.75 ± 45.03μg/m2 on exposed fire jacket material. After a single session, the deposit of BaP found on PPE and tools was measured on different surfaces ranged from 12 to 157 μg/m2. After multiple training sessions, a cumulative effect was suspected. The current PPE cleaning and maintenance procedures does not appear to effectively reduce contamination. French firefighters' exposure during in a live-fire training session in fire simulator is responsible for exposure to BaP. The estimated load of BaP on the outer surface of fire jackets could potentially have acute and chronic effects if absorbed in one's body. Further studies are needed to better understand individual French firefighters' exposure and determine appropriate measures to prevent contamination. It will be also be necessary find maintenance procedures that significantly reduce the BaP load.

Wash resistance and bio-efficacy of Olyset® Plus, a long-lasting insecticide-treated mosquito net with synergist against malaria vector, Anopheles stephensi.

To determine the wash resistance of Olyset® Plus using World Health Organization Pesticide Evaluation Scheme standard washing procedure and to assess the value of knock down and mortality rates of Anopheles stephensiat different regimens of long lasting insecticide treated nets washings. The study was conducted at the Bioassay Laboratory of Culicidae Insectary, School of Public Health, Tehran University of Medical Sciences, Iran. The net was made of polyester impregnated with permethrin and piperonyl butoxide at a ratio of 2:1. The washing resistance was assessed using Le Chat® soap and a shaker incubator set at a speed of 155 r/min, 30°C for 10min. The cone bioassay test was carried out according to World Health Organization recommended guideline with tolerant field strain of female Anopheles stephensi to pyrethroids. The knockdown and mortality rates of female mosquitoes exposed to Olyset® Plus from un-washed nets to 2 washings were 79.7% and 88.8% respectively. Mortality was dropped to zero while active ingredient estimated 0.532μg/100cm2 to 0.481μg/100cm2 after 15 washings. A positive correlation was seen between residues of permethrin on nets, knockdown rate and mortality rate of female Anopheles stephensi exposed to different regimes of washed Olyset® Plus (r=0.954, P=0.001). It is recommended that a preliminary survey conducted on resistance level of Anopheles vectors before the distribution of Olyset® Plus in malaria endemic communities.

Detection of Zinc in Nail Samples of Iron Welders

The current study was conducted to quantify zinc in nail samples of iron welders from different areas of Lahore, Pakistan. This study intended to assess nutritional deficiencies of zinc in welders. As nails serve a beneficial biomarker of concentrations of trace elements, hence in the present study the nails of welders were used for monitoring. The total number of nail samples collected from workers of iron welding shops, were 40. In a standardized washing procedure the nail samples were scrapped and cleaned of dust particles with nonionic detergent (Triton X-100) and then nail samples were digested afterwards in acid mixture. The concentration of zinc was evaluated by atomic absorption spectrophotometer. The results revealed that concentration of zinc in nail samples ranged 0.297 – 1.718 mg/kg and averaged at 0.88±0.39 mg/kg which is below the ideal zinc levels in nail samples. Correlation of zinc (mg/kg) was significant with age (0.214< 0.5), weight (0.320< 0.5) and Body Mass Index (0.268< 0.5) of the welders, while a weak correlation of zinc (mg/kg) was found with height (0.042< 0.5) of the welders.

Contamination of commonly consumed raw vegetables with soil transmitted helminth eggs in Mazandaran province, northern Iran

Soil-transmitted helminth (STH) infections are responsible for significant burden of morbidity and mortality worldwide. Consumption of raw vegetables without proper washing is one of the major routes of such infections. We evaluate the prevalence of STH contamination in commonly used vegetables in Mazandaran province, northern Iran. A total of 772 fresh raw vegetables were obtained from retail markets. Each sample was divided into two groups. One group was used as the unwashed sample and the second group was washed with standard washing procedures. Then, samples were examined for helminth eggs by using standard methods. Data analysis was performed using SPSS20. The overall prevalence of STHs was 14.89% (115/772). The rate of STH contamination was significantly higher in warm seasons (20.5%, 79/386) than in cold seasons (9.32%, 36/386) among the unwashed vegetables (OR=2.50; CI 95%=1.64–3.8; P<0.001). No parasites were observed in standard washed samples (OR=271.40; CI 95%=16.84–4373.64; P<0.001). Prevalence of STH contamination was significantly higher in leafy vegetables than root vegetables (OR=1.67; CI 95%=1.09–2.55; P<0.05). The prevalence of STHs species in all the vegetables were as follows: Ascaris lumbricoides (3.36%), Trichuris trichiura (2.2%), hookworms (2.9%), Toxocara spp. (1.68%), Trichostrongylus spp. (1.55), Taenia sp. (0.9%) and Hymenolepis nana (2.2%). The results of the present study emphasized that vegetables are potential risk factor for transmission of helminth infection to human in northern Iran. It is necessary that health authorities trained the consumers to proper and standard washing of vegetables before consumption.

Efectos del lavado del arroz enriquecido en su contenido de selenio

&lt;p&gt;El arroz es un alimento básico en la dieta de los costarricenses. Alrededor del 45% del arroz que se consume se produce localmente y el resto se importa de Estados Unidos y Argentina. En Costa Rica, el cáncer de próstata es el tumor que con mayor frecuencia se diagnostica en hombres. Las investigaciones apoyan el efecto del selenio como antioxidante en la prevención de ese tipo de cáncer. Se estima que cada persona consume 130 gramos de arroz por tiempo de alimentación. El Ministerio de Salud, ente rector en materia de salud pública, considerando que el arroz se consume en los tres tiempos de alimentación, decretó en 2006 el enriquecimiento del grano nacional e importado con 107 μg/ kg de selenio. Es costumbre lavar el arroz antes de cocinarlo, sin seguir las recomendaciones que se incluyen en el empaque del producto. En esta investigación se estudió el efecto del lavado sobre el contenido de selenio y se determinó que disminuye entre 51% y 79% para diferentes tipos de arroz, con un procedimiento estándar de lavado. La técnica utilizada para la medición de selenio fue la espectroscopía de absorción atómica, en la modalidad de generación de hidruros, y la digestión de las muestras se realizó con ácido nítrico en horno de microondas.&lt;/p&gt;

Isolated human and animal stratum corneum as a partial model for the 15 steps of percutaneous absorption: emphasizing decontamination, part II

Cutaneously directed chemical warfare agents can elicit significant morbidity and mortality. The optimization of prophylactic and therapeutic interventions counteracting these agents is crucial, and the development of decontamination protocols and methodology of post dermal exposure risk assessments would be additionally applicable to common industrial and consumer dermatotoxicants. Percutaneous (PC) penetration is often considered a simple one-step diffusion process but presently consists of at least 15 steps. The systemic exposure to an agent depends on multiple factors and the second part of this review covers absorption and excretion kinetics, wash and rub effects, skin substantivity and transfer, among others. Importantly, the partitioning behavior and diffusion through the stratum corneum (SC) of a wide physicochemical array of compounds shows that many compounds have approximately the same diffusion coefficient which determines their percutaneous absorption in vivo. After accounting for anatomical variation of the SC, the penetration flux value of a substance depends mainly on its SC/vehicle partition coefficient. Additionally, the SC acts as a 'reservoir' for topically applied molecules, and tape stripping methodology can quantify the remaining chemical in the SC which can predict the total molecular penetration in vivo. The determination of ideal decontamination protocols is of utmost importance to reduce morbidity and mortality. However, even expeditious standard washing procedures post dermal chemical exposure often fails to remove chemicals. The second part of this overview continues to review percutaneous penetration extending insights into the complexities of penetration, decontamination and potential newer assays that may be of practical importance.

Isolated human/animal stratum corneum as a partial model for 15 steps in percutaneous absorption: emphasizing decontamination, Part I

Since the advent of World War II, governments and laboratories have made a concerted effort to improve prophylactic and therapeutic interventions counteracting cutaneously directed chemical warfare agents (CWA), and by inference, common industrial and consumer dermatotoxicants. In vitro percutaneous penetration assays, first utilized by Tregear in the 1940s and presently in various modifications, have been fundamental to this effort. Percutaneous penetration, often considered a simple one-step diffusion process, consists of at least 15 steps. The first part of this review covers the initial steps related to absorption and excretion kinetics, vehicle characteristics, and tissue disposition. Importantly, the partitioning behavior and stratum corneum (SC) diffusion by a wide physicochemical array of compounds shows that many compounds have similar diffusion coefficients determining their percutaneous absorption in vivo. After accounting for anatomical SC variation, the penetration flux value of a substance depends mainly on its SC/vehicle partition coefficient. Additionally, the SC acts as a 'reservoir' for topically applied molecules and application of tape stripping has been found to quantify the chemical remaining in the SC which can predict total molecular penetration in vivo. Decontamination is of particular concern and even expediting standard washing procedures after dermal chemical exposure often fails to remove chemicals. This overview summarizes knowledge of percutaneous penetration extending insights into the complexities of penetration, decontamination and potential newer assays that may be of practical importance.

Prevalence of parasitic contamination in vegetables used for raw consumption in Shahrekord, Iran: Influence of season and washing procedure

This study aimed to determine the prevalence of intestinal parasites in raw vegetables consumed in Shahrekord, Iran; and efficacy of season and washing procedures to remove parasites from the vegetables. A total of 304 field vegetable samples composed of parsley, spearmint, scallion, basil, coriander, dill, cress, leek, tarragon, radish and purslane were purchased from the vegetable markets. Each sample was divided into three groups. One group was used as the unwashed sample and the second and third groups were washed with traditional and standard washing procedures, respectively. Intestinal parasites were detected in 32.6% of unwashed, 1.3% of traditionally washed and not in any standard washed samples (P < 0.001). The parasites were detected in unwashed vegetable samples included Ascaris lumbricoides eggs (14.1%), Taeniid spp. eggs (9.2%), Toxocara spp. eggs (3.3%), Trichostrongylus spp. eggs (4.3%), Giardia spp. cysts (8.2%) and non-pathogenic protozoan Entamoeba coli cysts (9.2%). The rate of parasitic contamination in different seasons was found to be 55% in spring, 45.5% in summer, 14.9% in autumn and 12.3% in winter (P < 0.005). The results highlight the potential of unwashed raw vegetables in transmission of intestinal parasites to human, and also emphasize the use of standard washing procedure (proper washing and disinfecting) before consumption of vegetables instead of traditional procedure (quick washing without disinfecting).

Efficacy of the International Embryo Transfer Society (IETS) washing procedure for rendering oocytes matured in vitro free of bovine viral diarrhea virus (BVDV)

To ensure the freedom of embryos from pathogenic agents prior to embryo transfer (ET), a specific sanitary washing procedure has been recommended by the International Embryo Transfer Society (IETS). In the present study, the efficacy of removing the bovine viral diarrhea virus (BVDV) from cumulus-free matured oocytes at the stage of extruded first polar body (N = 240) was evaluated, using the IETS-recommended 10 sequential wash procedure, after exposure in vitro to BVDV type 2 (strain PA-131, 1 × 105.2 TCID50/mL for 1 h). In general, the percentage of contaminated oocytes was reduced (P < 0.03) after the first two washes. Nevertheless, after 10 washes, approximately 20% of oocytes still remained infectious or contaminated with virus, as detected by the virus isolation test (VI) and quantitative reverse transcription PCR (qRT-PCR) of viral RNA (on average 13 copies/oocyte). Similarly, a higher percentage of positive washing fluid samples were detected in the first three washes (50–100%). The six subsequent washes had lower but variable proportions of fluid samples contaminated with infectious virus. We concluded that the standard washing procedure may not render all oocytes free from the infectious virus adhered to the zona pellucida (ZP), and application of an additional method of oocyte disinfection was warranted to ensure nontransmission of BVDV to recipients by embryos derived from infected oocytes.

An investigation into the possibility of bluetongue virus transmission by transfer of infected ovine embryos

Bluetongue (BT), a disease that affects mainly sheep, causes economic losses owing to not only its deleterious effects on animals but also its associated impact on the restriction of movement of livestock and livestock germplasm. The causative agent, bluetongue virus (BTV), can occur in the semen of rams and bulls at the time of peak viraemia and be transferred to a developing foetus. The risk of the transmission of BTV by bovine embryos is negligible if the embryos are washed according to the International Embryo Transfer Society (IETS) protocol. Two experiments were undertaken to determine whether this holds for ovine embryos that had been exposed to BTV. Firstly, the oestrus cycles of 12 ewes were synchronised and the 59 embryos that were obtained were exposed in vitro to BTV-2 and BTV-4 at a dilution of 1 x 102.88 and 1 x 10 3.5 respectively. In the second experiment, embryos were recovered from sheep at the peak of viraemia. A total of 96 embryos were collected from BTV-infected sheep 21 days after infection. In both experiments half the embryos were washed and treated with trypsin according to the IETS protocol while the remaining embryos were neither washed nor treated. All were tested for the presence of BTV using cell culture techniques. The virus was detected after three passages in BHK-21 cells only in one wash bath in the first experiment and two unwashed embryos exposed to BTV-4 at a titre of 1 x 10 3.5 . No embryos or uterine flush fluids obtained from viraemic donors used in the second experiment were positive for BTV after the standard washing procedure had been followed. The washing procedure of the IETS protocol can thus clear sheep embryos infected with BTV either in vitro or in vivo .

An investigation into the possibility of bluetongue virus transmission by transfer of infected ovine embryos

Bluetongue (BT), a disease that affects mainly sheep, causes economic losses owing to not only its deleterious effects on animals but also its associated impact on the restriction of movement of livestock and livestock germplasm. The causative agent, bluetongue virus (BTV), can occur in the semen of rams and bulls at the time of peak viraemia and be transferred to a developing foetus. The risk of the transmission of BTV by bovine embryos is negligible if the embryos are washed according to the International Embryo Transfer Society (IETS) protocol. Two experiments were undertaken to determine whether this holds for ovine embryos that had been exposed to BTV. Firstly, the oestrus cycles of 12 ewes were synchronised and the 59 embryos that were obtained were exposed in vitro to BTV-2 and BTV-4 at a dilution of 1 x 102.88 and 1 x 10(3.5) respectively. In the second experiment, embryos were recovered from sheep at the peak of viraemia. A total of 96 embryos were collected from BTV-infected sheep 21 days after infection. In both experiments half the embryos were washed and treated with trypsin according to the IETS protocol while the remaining embryos were neither washed nor treated. All were tested for the presence of BTV using cell culture techniques. The virus was detected after three passages in BHK-21 cells only in one wash bath in the first experiment and two unwashed embryos exposed to BTV-4 at a titre of 1 x 103.5. No embryos or uterine flush fluids obtained from viraemic donors used in the second experiment were positive for BTV after the standard washing procedure had been followed. The washing procedure of the IETS protocol can thus clear sheep embryos infected with BTV either in vitro or in vivo.

Mixing history affects gluten protein recovery, purity, and glutenin re-assembly capacity from optimally developed flour–water batters

Batters, from three wheat cultivars, were mixed up to their maximal consistency ( t peak ) at different mixing speeds ( N) and flour/water ratios [Auger, F., Morel, M.H., Lefebvre, J., Dewilde, M., Redl, A., 2008. A parametric and microstructural study of the formation of gluten network in mixed flour–water batter. Journal of Cereal Science 48, 349–358]. Gluten and starch were extracted from those batters using a process which included two successive steps: dilution and sieving. In order to reveal the specific influence of the mixing step, a standardized gentle washing and sieving procedure was selected. Mixing the batters at t peak guaranteed a high and stable gluten protein recovery (ca. 82%) irrespective of mixing conditions. SE-HPLC analysis of protein, from flours and batters sampled at t peak , demonstrated that mixing led to the almost total breakdown of the unextractable glutenin polymers (ca. 80%), whereas their re-assembly occurred during gluten extraction. The extent of glutenin re-assembly in gluten was influenced by the batter mixing history and was mainly related to the number of mixing rotations ( N.t peak ). Gluten protein contents were also found related to N.t peak . We proposed that the leaching of starch from the batter during gluten extraction was controlled by the elasticity of the protein network, i.e. the gluten content in unextractable glutenin. An innovating scheme relating the glutenin re-assembly capacity to the irreversible thiol protein oxidation is proposed.

Production of Virus-Free Seronegative Pups from Murine Embryos Arising from In Vitro Fertilization with Mouse Minute Virus-Exposed Spermatozoa

In the present study, the risk of transmission of mouse minute virus (MMV) to recipients of murine embryos arising from in vitro fertilization (IVF) of oocytes with MMV-exposed spermatozoa and to resulting pups was evaluated. Also, the time of seroconversion of recipients and pups was investigated. To achieve this goal, IVF of oocytes with cryopreserved spermatozoa from the inbred C3HeB/FeJ mouse strain was performed, and the resulting embryos were transferred to suitable Swiss recipients. Three groups were investigated: 1) oocytes or the developing embryos were continuously exposed to 10(4) TCID(50) MMVp per milliliter in the fertilization (human tubal fluid [HTF]), culture (KSOM), and embryo transfer (M2) media (positive control); 2) oocytes and spermatozoa were exposed to MMVp in the HTF medium only and transferred after a standard washing procedure with 10 washing steps in virus-free KSOM and M2; and 3) oocytes and spermatozoa were exposed to virus-free HTF, KSOM, and M2 (negative control). To detect antibodies to MMV in recipients and progeny, serological analyses were performed by ELISA on Days 14, 21, 28, and 42, and on Days 42 and 63, respectively, after embryo transfer. The presence of MMV in the washing drops was analyzed by PCR and an in vitro infectivity assay, while organs of some recipients and pups were analyzed by PCR. Using 10(4) of the tissue culture infective dose of MMVp per millilitre in the fertilization medium only, the present results demonstrate that 10 washing steps in the IVF-ET procedure are sufficient to remove the virus to a noninfectious dose, producing MMV-free seronegative recipients and pups. As such, there is minimal risk of transmission of MMV to recipients and pups if spermatozoa become contaminated with such viral loads.

Risk Assessment of Mouse Hepatitis Virus Infection via In Vitro Fertilization and Embryo Transfer by the Use of Zona-Intact and Laser-Microdissected Oocytes1

The aim of this study was to estimate the risk of mouse hepatitis virus (MHV) transmission by the in vitro fertilization and embryo transfer (IVF-ET) procedure. In addition, resistance to infection of zona-intact and laser-microdissected oocytes was compared. For this purpose, infectious mouse hepatitis virus, a common viral pathogen in mouse facilities, was used. Oocytes having an intact or laser-microdissected zona pellucida were incubated for fertilization in media containing MHV-A59 and resulting embryos were transferred to the oviduct of specific pathogen-free (SPF) Swiss recipients. The oocytes were divided into three experimental groups: 1) zona-intact oocytes continuously exposed to MHV in fertilization (HTF), culture (KSOM), and embryo transfer (M2) media; 2) zona-intact oocytes exposed to MHV in HTF medium and transferred after a standard washing procedure with virus-free KSOM and M2; and 3) laser-microdissected oocytes exposed to MHV in HTF medium and transferred after a standard washing procedure with virus-free KSOM and M2. Respective serum samples of embryo recipients and their offspring were tested for MHV antibodies using ELISA. In experiment 1, 10 out of 14 embryo recipients seroconverted to MHV and only their offspring (8 of 19) received maternal antibodies. In experiments 2 and 3, MHV antibodies were detected neither in the recipients nor in the offspring. These results indicate, for the first time, that even if the zona pellucida is partially disrupted by laser microdissection, the transmission of MHV-A59 can be avoided by correctly performed washing steps in the IVF-ET procedure.

Gender Difference of in vivo Skin Surface pH in the Axilla and the Effect of a Standardized Washing Procedure with Tap Water

The purpose of our study was to evaluate the axillary skin surface pH and explore potential gender-related differences together with the influence of a washing procedure in healthy subjects. After a run-in period, the skin surface pH was measured in vivo in 10 men and 10 women under standardized conditions in three distinct locations of each axilla (at baseline and up to 6 h after washing). Potential interfering influences were thoroughly excluded. Our study revealed a statistically significant difference in axillary skin surface pH between men and women with more acidic values in females (p ≤ 0.001). The baseline axillary pH before washing was 6.58 ± 0.63 (right) and 6.67 ± 0.65 (left) in men compared to 5.8 ± 0.53 (right) and 5.94 ± 0.62 (left) in women. The difference between the right and left armpit was not statistically significant (p > 0.05) in any group. After standardized washing procedures with pure tap water, the mean axillary pH decreased significantly in women at all measurement times [lowest value 60 min after washing: 5.51 ± 0.7 (right) and 5.64 ± 0.7 (left)], while it slightly increased in men [highest value 240 min after washing: 6.7 ± 0.59 (right) and 6.78 ± 0.69 (left)]. In summary, there is a gender difference in axillary skin surface pH. Washing with water further increased the difference between male and female pH values.

Ureaplasma parvum and Ureaplasma urealyticum are detected in semen after washing before assisted reproductive technology procedures

Objective: To investigate the prevalence of ureaplasmas in semen and washed semen and to explore their effect on semen andrology variables. Design: Prospective study. Setting: In vitro fertilization (IVF) unit of a private hospital. Patient(s): Three hundred forty-three men participating in an assisted reproductive technology (ART) treatment cycle. Main outcome measure(s) The prevalence of ureaplasmas in semen and washed semen tested by culture, polymerase chain reaction assays, and indirect immunofluorescent antibody assays. Result(s): Ureaplasmas were detected in 73 of 343 (22%) semen samples and 29 of 343 (8.5%) washed semen samples. Ureaplasmas adherent to the surface of spermatozoa were demonstrated by indirect immunofluorescent antibody testing. Ureplasma parvum serovar 6 (36.6%) and U. urealyticum (30%) were the most prevalent isolates in washed semen. A comparison of the semen andrology variables of washed semen ureaplasma positive and negative groups demonstrated a lower proportion of nonmotile sperm in men ureaplasma positive for washed semen. Conclusion(s): Ureaplasmas are not always removed from semen by a standard ART washing procedure and can remain adherent to the surface of spermatozoa.

Selection of scFv phages on intact cells under low pH conditions leads to a significant loss of insert-free phages.

Display of functional antibody fragments on the surface of filamentous bacteriophages allows fast selection of specific phage antibodies against a variety of target antigens. However, enrichment of single chain variable fragment (scFv)-displaying phages is often hampered by the abundance of bacteriophages lacking antibody fragments. Moderate adhesive binding activities and production advantages of these "empty" phages results in their subsequent enrichment during selection on target cells. To date, very limited effort has been made to develop strategies removing nonspecific binding phages during the selection processes. To efficiently reduce insert-free phages when panning on intact cells, we increased the washing stringency by lowering the pH of the buffer with citric acid. Under standard washing procedures (pH 7.4), only approximately 73% of recovered phages were insert-free after three rounds of selection. Using stringent washing procedures (pH 5.0), approximately 12% of recovered phages contained no scFv. Using this protocol, we have cloned an antibody fragment from a mouse/human hybridoma cell line directed against the disialoganglioside GD2. This study confirms that selection of phage antibodies on cells is efficiently enhanced by assays augmenting the stringency to remove nonspecific binding phages.

Washing or washing and trypsin treatment is ineffective for removal of noncytopathic bovine viral diarrhea virus from bovine oocytes or embryos after experimental viral contamination of an in vitro fertilization system

The objectives of this study were to investigate the association of oocytes and embryos with bovine viral diarrhea virus (BVDV) in an IVF system and the possibility of rendering them free of infectious virus by sequential washing (10 washes) or by washing and trypsin (0.25%, 90 sec) treatment. In a series of experiments, a noncytopathic strain of BVDV (New York-1; 10 6 TCID 50/ml) was introduced into the maturation, fertilization or embryo culture medium during the IVF procedure. The virus was isolated 1) from all 40 samples containing unfertilized washed or unwashed oocytes (n = 360); 2) from 46% of 52 samples of Day 7 embryos (n = 307) produced by IVF, which were washed 10 times; 3) from 66% of the 44 samples of unwashed embryos (n = 325); and 4) from 50% of the 20 samples of embryos (n = 111) exposed to washing and trypsin treatment. In addition, 5 heifers inoculated intravenously with single unwashed Day 7 embryos developed antibodies to BVDV. Three of 5 other heifers seroconverted following inoculation with single embryos that had been washed 10 times. Two of 4 heifers inoculated intravenously with 2 trypsin-treated embryos seroconverted, while the remaining 2 heifers which received a single embryo did not develop antibodies. In summary, our present study shows that under in vitro conditions, BVDV may be associated with groups of unwashed and washed oocytes and embryos, and may persist through the IVF process. The standard washing procedure with or without trypsin treatment is not fully effective in rendering IVF embryos free of BVDV.