Neutron-mapping polymer flow: scattering, flow visualization, and molecular theory.

Flows of complex fluids need to be understood at both macroscopic and molecular scales, because it is the macroscopic response that controls the fluid behavior, but the molecular scale that ultimately gives rise to rheological and solid-state properties. Here the flow field of an entangled polymer melt through an extended contraction, typical of many polymer processes, is imaged optically and by small-angle neutron scattering. The dual-probe technique samples both the macroscopic stress field in the flow and the microscopic configuration of the polymer molecules at selected points. The results are compared with a recent "tube model" molecular theory of entangled melt flow that is able to calculate both the stress and the single-chain structure factor from first principles. The combined action of the three fundamental entangled processes of reptation, contour length fluctuation, and convective constraint release is essential to account quantitatively for the rich rheological behavior. The multiscale approach unearths a new feature: Orientation at the length scale of the entire chain decays considerably more slowly than at the smaller entanglement length.

Veterinary sources of nonrodent monoclonal antibodies: interspecific and intraspecific hybridomas.

The generation of monoclonal antibodies from species other than rats and mice has developed slowly over the last 20 years. The advent of antibody engineering and realization of the advantages of nonmurine antibodies, in terms of their superior affinities and specificities, and their potential as components of human and veterinary therapeutics has increased their relevance recently. There have been significant advances in the development of myeloma and heteromyeloma fusion partners. This is an opportune moment to consolidate experiences of MAb production across the range of species of veterinary interest and place it into context with other developments in the field of monoclonal antibodies. The background to the development of antibodies from species other than the mouse is discussed. The species and antigens used to date are reviewed, as are the methods and results reported. A suggested protocol is provided for first attempts to exploit the huge potential of this aspect of hybridoma technology and suggestions are made for its further expansion.

Accuracy of reporting of methicillin-resistant Staphylococcus aureus in a provincial quality control program: a 9-year study.

We report the results of a province-wide quality control program in which five methicillin-resistant Staphylococcus aureus strains were circulated to all Ontario laboratories (hospital, private, and public health laboratories) on nine occasions between 1980 and 1989. The level of expression of methicillin resistance in each of the isolates was determined by performing viable colony counts on serial dilutions of methicillin in agar, and each isolate was assigned to an expression class according to previous published criteria (A. Tomasz, S. Nachman, and H. Leaf, Antimicrob. Agents Chemother. 35:124-129, 1991). Over this time there was an improvement in the performance of laboratories in the recognition of three strains that were relatively easy to detect (strains B, C, and E). These strains were of expression class II, and 98% of laboratories reported correct identifications in 1986. Performance in identifying two strains (strains A and D) of expression class I remained poor. Strain A was circulated in two surveys in 1987 and 1989, and laboratories were sent a questionnaire requesting details of the methods used in those two surveys. The methods used by the laboratories were classified into three categories: disk diffusion, single-plate screening by agar incorporation, and automated methods, which included premanufactured MIC panels. Between the 1987 and 1989 surveys, there was no change in the performance of the disk diffusion test (60% correct on both occasions), but there was improvement in the sensitivity of the agar incorporation test (36% correct in 1987 and 84% correct in 1989) and in automated methods (43% correct in 1987 and 79% correct in 1989). Over a decade, there was overall improvement in the performance of laboratories in detecting easy-to-detect strains, but there were difficulties in detecting organisms of low expression class, and an organism of very low expression class should be designated as a control organism for routine testing of methicillin-resistant s. aureus isolates.

The preparation of an ovine monoclonal antibody to progesterone.

Peripheral blood mononuclear cells were collected from a sheep immunized against progesterone-11 alpha-hemisuccinate-ovalbumin. Following fusion with NS1 mouse myeloma or heteromyeloma cells, a large number of hybrid colonies was established. These were screened for the production of sheep antibodies to progesterone. Twenty-four cell lines were cloned and one was stabilized. This cell line, O/MP.1A9.D7B2, produced a high-affinity ovine immunoglobulin G1 (dissociation constant 4.8 pmol/l) with a high degree of specificity for progesterone. The antibody was substituted into a competitive enzyme-linked immunosorbent assay for the measurement of progesterone in bovine milk, originally established using an ovine polyclonal antibody, and the results were compared. The monoclonal antibody produced an assay with a lower limit of detection and a greater degree of discrimination than the polyclonal antiserum.

Pharmacokinetic studies of passively administered ovine anti-testosterone antibodies given to cattle by the subcutaneous and intravenous routes.

Passive immunization of cattle with ovine anti-testosterone antiserum can result in an increased ovulation rate, but the effect is variable and may be influenced by the route of administration. Investigations were made into the pharmacokinetics of these antibodies in cattle when given intravenously (i.v.), subcutaneously (s.c.) or via a combination of these two routes. Serum levels of free residual binding sites were measured by testosterone radioimmunoassay, whilst total circulating ovine IgG was determined using a specific enzyme-linked immunosorbent assay which shows no cross-reactivity with bovine IgG. The biological half-life of the administered antibodies was longer when it was calculated by measuring titre than when it was calculated by measuring IgG. Subcutaneous injection resulted in a significantly longer half-life of IgG than intravenous injection or the combined route, with a concomitant increase in the area under the curve. No significant differences between the half-lives as measured by titre were noted following the various routes of administration, but the mean value following s.c. injection was longest. The choice of route of administration of antiserum for passive immunization can be used to control the timing and duration of effective antibody levels. The results of the present study suggest that the s.c. or combined i.v. and s.c. routes are the preferred methods of passive immunization if an effect of long duration is required. It may be that it is the period over which the maximum level is maintained, rather than the absolute maximum level, which is important for successful immunomodulation of ovulation rate.

The production and application of non-rodent monoclonal antibodies in veterinary science.

The requirement for monoclonal antibodies derived from species other than rats and mice is becoming increasingly realised in veterinary, as well as human, medicine. This paper reviews current knowledge of the production of inter-species hybridomas (heterohybridomas) by the fusion of rodent myeloma cell lines with lymphocytes from species of veterinary importance. To date a number of monoclonal immunoglobulins derived from sheep, cattle, pig, rabbit, mink and primate species have been produced to a variety of different bacterial, viral and nematode pathogens as well as to blood group and MHC determinants and to hormones. The technique opens up a number of possibilities for the future; some of these applications are discussed in relation to the antibodies produced thus far.

Examination of the envelope glycoprotein of yellow fever vaccine viruses with monoclonal antibodies.

Two panels of envelope glycoprotein reactive monoclonal antibodies (mAbs) were prepared against yellow fever (YF) 17D vaccine viruses. Five mAbs were prepared against the World Health Organization 17D-204 avian leukosis virus-free secondary seed virus and eight mAbs against 17DD vaccine manufactured in Brazil. The majority of these mAbs were type-specific and displayed differing reactions in neutralization tests. One, B14, would only neutralize YF vaccine virus grown in invertebrate cells. Others would differentiate 17D-204 and 17DD vaccines, from different manufacturers, in neutralization tests when the viruses were grown in vertebrate cells. The data indicate that heterogeneity exists between the epitopes that elicit neutralizing antibody on YF vaccine from different manufacturers.

A bovine monoclonal antibody to oestrone/oestradiol prepared by a (murine x bovine) xbovine interspecies fusion.

A heterohybridoma was produced by the fusion of sensitized peripheral blood lymphocytes (PBLs) with a previously derived heteromyeloma, generated by the fusion of bovine PBLs with murine myeloma cells. The sensitized bovine PBLs were collected from a steer immunized with an oestradiol-ovalbumin conjugate. The cell lines resulting from the fusion were screened for the production of bovine antibodies to oestradiol. A stable heterohybridoma was isolated which secreted a bovine IgG1 to oestrone/oestradiol. The use of sensitized PBLs together with heteromyeloma fusion partners has proved to be a reliable and simple way of producing monoclonal antibodies against specific haptens.

Preparation of a bovine monoclonal antibody to testosterone by interspecies fusion.

This communication reports the first successful attempt to produce a hybridoma cell line secreting bovine immunoglobulin to a small hapten, starting with peripheral blood lymphocytes, rather than spleen or lymph node cells. A heteromyeloma line, sensitive to selective media, was made by fusing NS1/1-Ag4-1 mouse myeloma cells with bovine peripheral blood lymphocytes. This cell line was then fused with blood lymphocytes from a steer immunised with a testosterone immunogen. Cell cultures were screened using an ELISA specific for bovine antibodies to testosterone. Following repeated cloning, a cell line was established which secretes moderate levels of a specific, high affinity antibody to testosterone. This particular cell line has significant potential for veterinary application and the successful fusion demonstrates the possibilities of heteromyelomas for the development of non-murine monoclonal antibodies.

Preparation of ribonucleic acid probes specific for Bacteroides fragilis.

Nucleic acid probes promise to be very useful in the detection of infectious agents. Several RNA probes have been developed by random cloning of chromosomal DNA for the detection of Bacteroides fragilis. Four such probes prepared by transcription of inserts of 400 bp, 800 bp, 2.4 kbp, and 6 kbp all hybridized to denatured B. fragilis DNA on nitrocellulose, but did not bind to DNA from a variety of other Bacteroides spp. or from selected aerobic strains. Each probe readily detected five clinical isolates of B. fragilis and did not react with 11 clinical isolates representing four other species in the "B. fragilis group".

Production of an ovine monoclonal antibody to testosterone by an interspecies fusion.

Hybridomas were made between NS1/1-Ag4-1 mouse myeloma cells and peripheral blood lymphocytes from a sheep immunized with a testosterone-protein conjugate. ELISAs were developed to detect ovine IgG and specific ovine anti-testosterone and these were used to screen the colonies. Although the cells fused acceptably only 3% of colonies secreted detectable quantities of antibody. The antibody was secreted at low levels (2.5 ng/ml) but was of high affinity (KD = 7.63 x 10(-12) M/1). One line, O/M 4.22, continued to secrete in culture for four months until the termination of the experiment.

Steroidogenesis by gonads of a viviparous teleost, the sailfin molly (Poecilia latipinna), in vitro and in vivo.

Gonads of Poecilia latipinna transformed [3H]testosterone into a number of reduced and conjugated metabolites in high yield in vitro. In the male 5 beta-androstane-3 alpha, 17 beta-diol, 5 beta-androstane-3 beta, 17 beta-diol, and their "sulphates" were identified. The only 11-oxygenated androgen detected was a compound tentatively identified as 5 beta-androstane-3 beta, 11 beta, 17 beta-triol. In ovarian incubates androstenedione, 5 beta-androstane-3 alpha, 17 beta-diol and its glucuronide, testosterone glucuronide, and 5 beta-androstane-3 alpha, 17 beta-diol glucuronide were identified. Highest yields of the ovarian glucuronides coincided with the termination of vitellogenesis which may indicate a possible pheromonal role of these conjugates. In vivo plasma levels of estradiol in the female were correlated with vitellogenesis and fell markedly after castration or hypophysectomy. In males the plasma concentrations of testosterone, 11-ketotestosterone and 11 beta-hydroxytestosterone and their conjugates were variable and not apparently correlated with testicular weight, but they were reduced to undetectable levels by castration and hypophysectomy. The results suggest that 5 alpha- and 5 beta-reduced steroids may play a role in the reproductive endocrinology of P. latipinna and that measurements of only the "classical" steroid hormones in this and possibly other species may give only a partial and misleading picture of endocrine changes.

Direct control of the gonadotroph in a teleost, Poecilia latipinna. II. Neurohormones and neurotransmitters.

Pituitaries from male and female mollies were incubated with varying amounts of mammalian LH-RH, arginine vasotocin, dopamine, or serotonin for 18 hr. Ultrastructural differences between control and experimentally treated glands were used to define the direct effects of these neurohormones and neurotransmitters on the gonadotrophic cells of the adenohypophysis. The effects varied in intensity according to the sex and reproductive state of the donor animal. LH-RH stimulated gonadotrophin secretion by the gonadotrophs, as did vasotocin, although to a much lesser extent and with noticeable differences between the sexes. Dopamine inhibited secretion by basally active gonadotrophs and probably from active cells also, although to a lesser extent. Serotonin mildly stimulated secretion at all stages in both sexes. The results of this study indicate the possible involvement of neurohypophysial octapeptides and of monoamines in the direct control of the gonadotroph of Poecilia latipinna.

Direct control of the gonadotroph in a teleost, Poecilia latipinna: gonadal steroids.

Pituitaries from male and female fish were incubated with 1, 5, and 10 micrograms/ml of oestradiol-17 beta, testosterone, 17 alpha OH-progesterone, and 17 alpha,20 beta-dihydroxy-4-pregnan-3-one for 18 hr. Ultrastructural differences between control and experimentally treated glands were used to define the direct effects of these steroids on the gonadotropic cells of the adenohypophysis. The effects of the steroids differed according to sex and reproductive state of the donor animal. Oestradiol and testosterone stimulated gonadotropin secretion by active gonadotrophs, but inhibited it in inactive cells. Both the progesterones generally inhibited gonadotropin secretion although 17 alpha,20 beta-dihydroxy-4-pregnan-3-one had no effect on active gonadotrophs. The four steroids investigated all show potential for direct control of gonadotropin secretion in Poecilia latipinna although factors affecting the balance of these actions, and their relative importance in vivo, remain to be elucidated.

Compound heterozygosity for two genotypes of alpha-thalassemia-2: hematological, biosynthetic and DNA studies.

Hemoglobin and DNA gene analyses were carried out in two Black Canadian families. In Family Q, both the parents and the brother were found to be heterozygotes for alpha-thalassemia-2 with the following alpha-genotypes: -alpha 3.7/alpha alpha, -alpha 4.2/alpha alpha and -alpha 4.2/alpha alpha, respectively. In Family C, the mother was found to be a homozygote for alpha-thalassemia-2 with the alpha-genotype of -alpha 3.7/-alpha 3.7. In both families, the propositi were compound heterozygotes for alpha-thalassemia-2 with the alpha-genotype of -alpha 3.7/-alpha 4.2. The propositus in Family C was also a sickle cell trait carrier. The usefulness of DNA gene analyses in family studies of hemoglobinopathy was discussed.

Development and evaluation of a coculture technique for identification of anaerobic organisms.

We investigated a method to reduce oxygen tension and produce anaerobic conditions conveniently by coculture with a nonfermentative organism. The biochemical reactions of 94 strains representing 41 different species were determined using prereduced media and gas chromatography according to standard methods. The standard reactions were compared with those obtained by preparing similar media, modified by containing no cysteine or resazurin, under aerobic conditions. Preincubation of the modified biochemical media with a standard suspension of a culture of Acinetobacter lwoffi provided reducing conditions. In 1,449 individual tests using 22 different biochemicals, 1,349 (93%) of the reactions agreed. Only two misidentifications resulted when using the modified media, one each at the species and genus levels. Two strains of Actinomyces odontolyticus failed to grow in coculture.

Hemophilus vaginalis bacteremia.

During 1975--77 Hemophilus vaginalis bacteremia occurred post partum in eight previously healthy women. Seven had been admitted for delivery at term and one because of threatened abortion. Six underwent cesarean section. Post-partum pyrexia and neutrophilia were the main features. All the patients recovered uneventfully while receiving antibiotics. H. vaginalis is an infrequent agent of bacteremia; it affects predominantly women after obstetric trauma.

Interspecific relationships of antibiotic resistance in Staphylococcus sp.: isolation and comparison of plasmids determining tetracycline resistance in S. aureus and S. epidermidis.

Tetracycline resistance in Staphylococcus aureus and S. epidermidis was confirmed to be determined by plasmids of the same size. Digestion of plasmids from each strain with restriction endonucleases EcoR1, HindIII, and AluI showed a high degree of similarity in their DNA sequences. At least 10 cleavage sites which appear to be common to both plasmids were detected. An additional three cleavage sites appear to be unique to the S. epidermidis plasmid. Further, a survey of recent clinical isolates of tetracycline-resistant staphylococci detected 7 or 10 S. aureus strains and 8 of 9 S. epidermidis strains with plasmids which were of similar size to the purified reference plasmids and which, by hybridization, showed extensive DNA homology to the S. aureus reference plasmid DNA.