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Biomedical subjects

J E Robinson

Publications and source records attributed to J E Robinson.

At least 73 records · Page 4Linked to original sources
PubMed1991

Biotransformation of cyclophosphamide in post-implantation rat embryo culture using maternal hepatocytes in co-culture.

The post-implantation rat embryo culture technique is employed to study embryotoxic effects of xenobiotic compounds in the absence of the maternal compartment. For compounds biotransformed in vivo the embryo culture technique must be adapted in order to mimick the in vivo effects. In the present study the possibility of co-culturing metabolically active maternal hepatocytes suspended in the standard culture system with rat serum as a medium was investigated. Cyclophosphamide (CP) was used as a model compound as it needs bioactivation to display embryotoxicity. Morphologic and histologic effects were studied. Neither hepatocytes nor CP alone affected embryo development, whereas in the presence of hepatocytes embryotoxicity was observed at 30 micrograms/ml CP. Embryotoxicity was decreased in the additional presence of metyrapone, a monoxygenase inhibitor. Hepatocyte suspensions prepared via slicing or perfusion of livers were equally effective. In conclusion, co-culture of embryos and suspended hepatocytes can be performed under optimal conditions for embryo development and in the presence of biotransforming activity.

Animals↗
PubMed1991

Conformational epitope on gp120 important in CD4 binding and human immunodeficiency virus type 1 neutralization identified by a human monoclonal antibody.

A human monoclonal antibody designated 15e is reactive with the envelope glycoprotein (gp120) of multiple isolates of human immunodeficiency virus type 1 (HIV-1). Antibody 15e also neutralizes HIV-1 with broad specificity and blocks gp120 binding to CD4. Characterization of the 15e epitope shows that it is conformation dependent and is distinct from previously recognized functional domains of gp120, suggesting that this epitope represents a novel site important for HIV-1 neutralization and CD4 binding. These findings have implications for the development of a vaccine for AIDS.

Antibodies, Monoclonal↗
PubMed1990

A novel enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to HIV-1 envelope glycoproteins based on immobilization of viral glycoproteins in microtiter wells coated with concanavalin A.

We have developed a novel method that greatly simplifies the preparation of solid-phase HIV-1 envelope glycoproteins for use in an ELISA that detects serum antibodies to HIV envelope antigens. This method utilizes concanavalin A absorbed to wells of microtiter plates to affinity immobilize detergent-solubilized viral glycoproteins released in culture fluids of HIV-1 infected cell lines grown in serum free medium. Antibodies binding to ConA-immobilized viral antigens are detected by peroxidase-conjugated antibodies and appropriate enzyme substrates. Unlike most commercial HIV ELISAs, which utilize gp120 depleted-purified virus as the source of antigens and thus favor detection of antibodies to core antigens, the ConA envELISA is highly sensitive for detecting antibodies to native gp120, as evidenced by the strong reactivity of gp120-specific human monoclonal antibodies. Our results also suggest that representation of gp41 in the assay varies and depends on which virus infected cell lines are used for antigen production. Since this assay accurately identified 14 HIV-1 antibody positive patient sera and no false positives were detected among 16 HIV-1 negative sera, the ConA envELISA shows promise as an inexpensive assay for the serologic diagnosis of HIV infections.

Animals↗
PubMed1990

Human IgG and IgM monoclonal antibodies against autologous melanoma produced by Epstein-Barr-virus-transformed B lymphocytes.

The serum antibody response to human melanoma has prognostic and potential physiological consequences. The specificity of the host B cell antibody response may be an important determinant of disease outcome. We have utilized Epstein-Barr virus (EBV) transformation to analyze the repertory of the host B cell response to melanoma. Production of antibody that binds selectively to autologous (eight cases) or allogeneic (four cases) short-term-cultured melanoma cells was assessed from EBV-transformed B lymphoblastoid cells. Forty-two cultures of EBV-transformed B cells that secreted IgM and 23 that secreted IgG antibodies gave patterns of differential reactivity with autologous or allogeneic melanoma. Antibody-forming B cells persisted in producing melanoma-reactive IgG and IgM for 8-21 weeks. Preselection of B cells by adsorption to tumor cell antigens before transformation enhanced the frequency of antibody secretion. The specificity of the antibody produced by the longest-producing culture appears to be restricted to a subset of melanomas. The patient from whom this tumor-restricted IgG-producing B cell was retrieved was unusual, having had a transient serum IgG of similar specificity, and having manifest a syndrome of vitiligo at the time of her development of serum antimelanoma antibody, followed by disease-free survival of resected recurrent metastatic melanoma to the present (more than 6 years). This study has given support to findings of conventional serology, revealing the production of melanoma-reactive antibody from B cells of patients who have demonstrable serological response to tumor.

Antibodies, Monoclonal↗
PubMed1990

Human monoclonal antibody that recognizes the V3 region of human immunodeficiency virus gp120 and neutralizes the human T-lymphotropic virus type IIIMN strain.

We describe a human IgG1 monoclonal antibody (N701.9b) derived by Epstein-Barr virus transformation of B cells from a human immunodeficiency virus-seropositive asymptomatic donor. This antibody was shown to recognize the principal neutralizing domain contained within the V3 region of gp120 of the MN strain of human immunodeficiency virus and MN-like strains, as determined by binding to the PB-1 fragment of MN gp120 and to synthetic peptides corresponding to the V3 region of MN and related virus strains. The epitope identified by monoclonal antibody N701.9b was mapped to a segment of V3 containing at least 7 amino acids (amino acids 316-322), which is located in the "tip" and "right" side of the V3 loop of the MN strain. Furthermore, this antibody manifested potent type-specific fusion-inhibitory activity against the MN strain but not against the IIIB or RF virus strains. This antibody also neutralized four virus isolates that had MN-like V3 region sequences and failed to neutralize three other strains containing unrelated V3 region sequences. Our findings confirm that the V3 region stimulates type-specific neutralizing antibody during natural human immunodeficiency virus infection in humans. The potential clinical use of this antibody is discussed.

Amino Acid Sequence↗
PubMed1990

Identification of conserved and variant epitopes of human immunodeficiency virus type 1 (HIV-1) gp120 by human monoclonal antibodies produced by EBV-transformed cell lines.

Using Epstein-Barr virus (EBV) transformation of B cells isolated from peripheral blood of two asymptomatic human immunodeficiency virus type 1-(HIV-1) infected subjects, we have produced four IgG1 human monoclonal antibodies (HMAbs) that bind to HIV-1 gp120, as determined by Western blot analysis. Two of these HMAbs, designated N70-1.5e and N70-2.3a, react with epitopes of gp120 expressed by all strains tested thus far, and therefore, appear to identify conserved epitopes. The other two HMAbs, K24-3b and N70-1.9b, identify variant epitopes; K24-3b binds to an epitope which is absent from two strains but heterogeneously expressed in eight other strains; N70-1.9b binds to an epitope that is found in relatively few strains. We also describe a novel immunoassay in which viral glycoproteins, produced by HIV-1-infected cells grown in serum-free medium, are affinity immobilized in Concanavalin A-coated wells of enzyme-linked immunosorbent assay (ELISA) plates. This method greatly facilitates the preparation of solid-phase HIV envelope glycoproteins from multiple virus strains and screening immunoassays based on this method are highly sensitive and effective in detecting antibodies to gp120.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Use of external shock-wave lithotripsy and adjuvant ursodiol for treatment of radiolucent gallstones. A national multicenter study.

A prospective multicenter trial was performed to evaluate the use of external shockwave lithotripsy (ESL) and adjuvant medical therapy for the treatment of gallstones. A Medstone STS lithotripter was used together with ursodiol. Two hundred twenty-three patients were treated under general anesthesia (75%) or with intravenous analgesia (25%). Initial treatments were on an inpatient basis, but as centers gained experience, outpatient treatments became more common. Stone fragmentation and clearance were greatest in patients with solitary gallstones less than 2 cm in diameter. In this group of patients, stone fragmentation occurred in 97% of patients, and the cumulative stone-free rates at three and six months were 54% and 90%, respectively. These results indicate that fragmentation of gallstones can be achieved by a dry shock-wave lithotripter and that stone clearance is induced more rapidly by external shock-wave lithotripsy and adjuvant ursodiol therapy than by ursodiol therapy alone.

Adult↗
PubMed1989

Circannual cycles of luteinizing hormone and prolactin secretion in ewes during prolonged exposure to a fixed photoperiod: evidence for an endogenous reproductive rhythm.

Circulating patterns of luteinizing hormone (LH) and prolactin (PRL) were monitored for 5 yr in ewes maintained either outdoors in natural conditions or indoors in a fixed, short photoperiod (8L:16D). The ewes were ovariectomized and each was treated with a Silastic implant containing estradiol to provide a fixed negative feedback signal to the reproductive neuroendocrine axis. Serum concentrations of LH and PRL were subjected to a statistical algorithm developed for the purpose of detecting hormone cycles. In ewes maintained outdoors, serum concentrations of both hormones underwent high amplitude cycles with a period no different from 365 days. Among ewes maintained in the fixed photoperiod, unambiguous cycles of LH and PRL persisted through the 5 yr of exposure to short days. Period of these cycles differed from 365 days. Further, the LH cycles became desynchronized among ewes housed together and desynchronized with respect to the LH cycles in ewes kept outdoors. These findings document the existence of an endogenous circannual rhythm of reproductive neuroendocrine function in ewes.

Animals↗
PubMed1989

Regulation of the onset of the breeding season of the ewe: importance of long days and of an endogenous reproductive rhythm.

Three experiments were conducted to evaluate the role that the increasing day lengths of late winter and spring play in timing the breeding season of the Suffolk ewe. In the first experiment, ewes were denied their normal complement of increasing day length by maintaining them on the photoperiod experienced at the winter solstice. This prevented the breeding season from occurring the subsequent autumn. In the second experiment, ewes were exposed to increases in day length at different time-intervals after the winter solstice: the normal time, later than normal or earlier than normal. Once the summer solstice photoperiod was reached, it was maintained until the end of the study. When increasing photoperiod was provided early, the breeding season was advanced; when it was provided late, reproduction was delayed. In the third experiment, ewes were exposed to a continuously increasing photoperiod matching the maximal rate of rise in natural conditions; this treatment was begun on the spring equinox and continued until mid-autumn. The steadily increasing photoperiod did not alter the time of reproductive onset in the autumn. These findings support the following conclusions for timing of the breeding season of the Suffolk ewe. (1) The lengthening photoperiod between the winter and summer solstices is required for the occurrence of the breeding season in the autumn. (2) The time of initial exposure to this lengthening photoperiod provides an important cue for determining when the reproductive period occurs. (3) The time of onset of the breeding season does not depend upon the decreasing photoperiod after the summer solstice, nor does it require the photoperiod to stop increasing as the summer solstice approaches. These findings have been incorporated into a conceptual model for temporal regulation of the annual reproductive cycle of the ewe. An important component of this model is a critical role for increasing photoperiod to initiate a process in the late winter-spring which ultimately leads to an obligatory reproductive onset in the autumn.

Animals↗
PubMed1988

Ultrastructure and synaptic organization of luteinizing hormone-releasing hormone (LHRH) neurons in the anestrous ewe.

Electron microscopic immunocytochemistry was employed to examine the ultrastructure of luteinizing hormone-releasing hormone (LHRH) neurons and their projections to the median eminence in the sheep brain. LHRH perikarya in the preoptic area of anestrous ewes are less innervated than nonimmunoreactive cells in the same sections, but still receive numerous synaptic inputs, primarily onto distal dendrites and small somatic protuberances. Axon terminals synapsing upon LHRH cells contain a combination of clear spherical vesicles and larger dense-core vesicles. Interestingly, LHRH cell bodies and dendrites are almost entirely surrounded by glial processes. These processes intervene between immunoreactive elements that at a light microscopic level appear to be in contact with each other. Thus no evidence was obtained at the ultrastructural level for contacts among adjacent LHRH cells or dendrites in the preoptic area. Synaptic inputs onto LHRH cell bodies and dendrites appear to penetrate this glial sheath. In contrast to the absence of contacts among LHRH cells in the preoptic area, individual LHRH terminals in the median eminence are often clustered in direct plasma membrane contact. Comparisons between animals of differing reproductive status are needed to determine whether alterations in synaptic inputs, glial ensheathment, or LHRH-LHRH appositions, may underlie seasonal changes in the activity of LHRH neurons.

Anestrus↗
PubMed1988

Timing the breeding season of the ewe: what is the role of daylength?

Photoperiod is the proximate cue which times the reproductive period of the sheep and the role of daylength as a "zeitgeber" is evaluated in this report. Although sheep are regarded as a short-day breeding species, the reproductive period of the Suffolk ewe begins on daylengths which are on average 2.5 hours longer than those on which it ends. The reasons for this apparent anomaly are considered. Experimental evidence is presented which demonstrates that the transitions between the breeding season and anoestrus are not actively generated by changing ambient photoperiod. Rather the ewe expresses an endogenous circannual rhythm of reproduction and the role of daylength is to entrain this reproductive rhythm to a period of 365 days. It appears that only portions of the annual cycle of photoperiod are necessary to synchronize the breeding season, however, additional photic cues may be required to produce a reproductive period of normal duration.

Animals↗
PubMed1988

Characteristics of the melatonin signal that provide the photoperiodic code for timing seasonal reproduction in the ewe.

The following is a progress report of our studies to identify important features of the circadian pattern of melatonin secretion which provide the photoperiodic code for daylength in regulating seasonal breeding in the Suffolk ewe. The first series of experiments evaluated two conceptual models of how melatonin codes for daylength: the circadian timing of the melatonin elevation as opposed to the length of the time melatonin is elevated during each 24-hr period (phase vs duration). Strong support has been gathered for the duration hypothesis. No evidence was obtained to support a role for phase; nevertheless, this hypothesis could not be discounted definitively. A second series of studies evaluated the importance of the previous melatonin pattern to the interpretation of a given melatonin signal. Evidence is presented that a fixed melatonin pattern can maintain a given reproductive response only for a limited length of time and that this response can be prolonged by appropriate changes in the melatonin pattern. Thus, change is an important feature of the melatonin signal. Further, the nature of the melatonin change appears to be crucial, specifically whether the nocturnal elevation increases or decreases in duration. Thus, transfer to a common photoperiod can promote either reproductive induction or arrest, depending upon whether the transfer leads to a decrease or increase in daylength. This has important ramifications to the photoperiodic timekeeping process in those species of mammals which utilize daylength to time their seasonal reproductive cycle.

Animals↗
PubMed1988

Importance of pituitary and neural actions of estradiol in induction of the luteinizing hormone surge in the ewe.

Two experiments were performed to test the importance of both pituitary and neural sites of action of estradiol in inducing the surge of luteinizing hormone (LH) in the ewe. Both experiments were conducted using an animal model in which pulsatile secretion of gonadotropin-releasing hormone (GnRH) and endogenous secretion of ovarian steroids were eliminated by ovariectomy during seasonal anestrus and treatment with Silastic implants which maintained a luteal-phase level of serum progesterone. The hormonal requirements for the surge were then evaluated by systematic application of GnRH and estradiol signals using pulsatile infusion pumps (for GnRH) and Silastic implants (for estradiol). In experiment 1, the circulating level of estradiol and frequency of GnRH pulses were increased either abruptly or progressively (i.e. mimicking the changes in the estrous cycle between luteolysis and just before the LH surge). Abrupt increments led to an LH surge in all ewes; progressive rises to the same absolute levels did not. However, sudden application of a further large increase in GnRH upon the progressive rise elicited an LH surge in every instance. In experiment 2, a GnRH pulse pattern known to be effective in inducing the LH surge was applied under conditions of differing estradiol concentration: no estradiol, basal estradiol, basal rising to peak estradiol. The GnRH signal elicited high-amplitude surges of LH only in the presence of a peak estradiol concentration. Our findings are consistent with the conclusion that two actions are required for a rise in estradiol to elicit a full-amplitude surge of LH in the ewe: an action on the brain to evoke a sudden increase in GnRH release and an action on the pituitary to maximize its response to GnRH.

Animals↗
PubMed1988

Endocrine responses to acute stress in castrated rams: no increase in oxytocin but evidence for an inverse relationship between cortisol and vasopressin.

Plasma concentrations of cortisol, oxytocin, vasopressin and prolactin were measured by radioimmunoassay in eight castrated rams subjected to handling (control), restraint or isolation. Determinations were also made of hematocrit and plasma osmolality. Blood was collected by jugular venipuncture 60 and less than 1 min before, and 5, 15, 30, 60 and 120 min after the start of treatment. Plasma cortisol was raised above pre-treatment levels during isolation and restraint but only transiently after handling. In the first 30 min of treatment, cortisol concentrations were higher following isolation than after restraint or handling and, in the final 60 min, levels were higher during both isolation and restraint than after handling. Oxytocin concentrations showed little change but vasopressin levels were reduced 15, 30 and 60 min after the start of isolation and were negatively correlated with those of cortisol. Plasma prolactin concentrations decreased during handling and restraint but remained unaltered after isolation. Osmolality and hematocrit decreased in all three treatment conditions. These findings indicate that stress does not alter oxytocin levels in sheep, but changes in vasopressin secretion and water balance may occur if cortisol release is greatly enhanced.

Animals↗