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Biomedical subjects

L S Rodkey

Publications and source records attributed to L S Rodkey.

At least 19 recordsLinked to original sources
PubMed1998

An animal model for hemolytic disease of the fetus or newborn in New Zealand white and New Zealand red rabbits: newborn effects.

OBJECTIVE: Our purpose was to study the neonatal effects of red blood cell alloimmunization in a rabbit model. STUDY DESIGN: Eighteen does were alloimmunized to incompatible red blood cells. Does were bred twice, once with a homozygous buck of incompatible blood type and once with a homozygous buck of compatible blood type. Fetal blood sampling was undertaken on day 27 of gestation (term 28 to 31 days). Does were delivered on day 30 and the neonatal pups were anesthetized. Direct cardiac samplings were performed for hemoglobin, reticulocyte count, and direct Coombs' test. Hepatic, splenic, and renal wet weights were measured. RESULTS: Twenty-two pregnancies (12 compatible and 10 incompatible) were studied. Neonatal hemoglobin was higher in the compatible litters (11.1 gm/dL [7.7 to 12.6 gm/dL] vs 4.9 gm/dL [2.1 to 9.1 gm/dL], P <.001), whereas no difference could be detected between the respective reticulocyte counts (34.0/100 red blood cells [27.3 to 36.1/100 red blood cells] vs 32.6/100 red blood cells [26.8 to 43.5/100 red blood cells], P =.55). The direct Coombs' assay was negative in 23 pups from 8 compatible litters and false positive (weakly positive result) in 2 pups of a ninth compatible litter. The Coombs' assay was positive in all 22 incompatible pups tested. Hepatosplenomegaly was noted in affected pups but not in controls. CONCLUSIONS: A disease analogous to human hemolytic disease of the newborn can be induced in the rabbit neonate.

Animals↗
PubMed1997

DNA hydrolysis by monoclonal anti-ssDNA autoantibody BV 04-01: origins of catalytic activity.

Monoclonal anti-DNA autoantibody BV 04-01 catalyzed hydrolysis of DNA in the presence of Mg2+ ions. DNA hydrolyzing activity was associated with BV 04-01 IgG, Fab, and SCA 04-01 proteins. Pronounced cleavage specificity for both ss and dsDNA was observed with efficient hydrolysis of the C-rich region of the oligonucleotide A7C7ATATAGCGCGT7 as well as preference for cleavage within CG-rich regions of double-stranded DNA. Data on specificity of ssDNA hydrolysis and kinetic data obtained from wild-type SCA 04-01 and two SCA 04-01 mutants (L32Phe and L27dHis) were used to model the catalytically active antibody site utilizing the previously resolved X-ray structure of (dT)3 liganded Fab 04-01. The resulting model suggested that BV 04-01 activates the target phosphodiester bond by induction of conformational strain. In addition, the antibody-DNA complex contained a potential Mg2+ ion coordination site composed of the L32Tyr and L27dHis amino acid side chains and a DNA 3'-phosphodiester group. Induction of strain and metal coordination could be constituents of a mechanism by which this antibody catalyzed DNA hydrolysis. Sequence data for BV 04-01 VH and VL genes suggested that the proposed catalytic antibody active site was germ-line encoded. This observation suggests the hypothesis that catalytic activity might represent an important but unspecified function of some antibody molecules.

Animals↗
PubMed1997

Stable solid-phase Rh antigen.

Numerous investigators have attempted to isolate the Rh antigens in a stable, immunologically reactive form since the discovery of the Rh system over 56 years ago. We report here a successful and reproducible approach to solubilizing and adsorbing the human Rh antigen(s) to a solid-phase matrix in an antigenically active form. Similar results were obtained with rabbit A/D/F red blood cell antigens. The antigen preparation was made by dissolution of the red blood cell membrane lipid followed by fragmentation of the residual cytoskeleton in an EDTA solution at low ionic strength. The antigenic activity of the soluble preparations was labile in standard buffers but was stable in zwitterionic buffers for extended periods of time. Further studies showed that the antigenic activity of these preparations was enhanced, as was their affinity for plastic surfaces, in the presence of acidic zwitterionic buffers. Adherence to plastic surfaces at low pH maintained antigenic reactivity and specificity for antibody was retained. The data show that this approach yields a stable form of antigenically active human Rh D antigen that could be used in a red blood cell-free assay for quantitative analysis of Rh D antibody and for Rh D antibody immunoadsorption and purification.

Animals↗
PubMed1995

An animal model for hemolytic disease of the fetus and newborn. I. Alloimmunization techniques.

OBJECTIVE: Our purpose was to establish an animal model for hemolytic disease of the fetus and newborn by developing red blood cell alloimmunization techniques in the rabbit. STUDY DESIGN: Twenty-six nonpregnant New Zealand White or Red does underwent blood typing to identify them as homozygous at the HgA or HgF red blood cell antigen locus. Alloimmunization to incompatible red blood cells was attempted through a series of subcutaneous injections using complete then incomplete Freund's adjuvant. RESULTS: Successful induction of an antibody response occurred in 96% of cases. The median response in FF rabbits was 2560 (range 40 to 10,240), whereas the response in AA does was 2560 (range 320 to 20,480). These responses were not statistically different (p = 0.77). Responses were categorized as poor, moderate, or good. No difference was noted between FF and AA does in distribution of the categories of response (p = 0.53). CONCLUSION: Red blood cell alloantibodies of high titer can be induced successfully in the rabbit.

Animals↗
PubMed1995

An animal model for hemolytic disease of the fetus and newborn. II. Fetal effects in New Zealand rabbits.

OBJECTIVE: The addition of ultrasonography and ultrasonographically directed fetal blood sampling was attempted in an effort to study the fetal effects of red blood cell alloimmunization in a rabbit model. STUDY DESIGN: Nineteen New Zealand does were alloimmunized to incompatible red blood cells. Sensitized does were bred twice, once with a homozygous buck of incompatible blood type and once with a homozygous buck of compatible blood type. Ultrasonographic examinations were performed on days 20 and 27 of gestation (term 28 to 31 days). Fetal blood sampling was undertaken on day 27 of gestation, and hematologic data were compared between compatible and incompatible litters. RESULTS: A total of 41 pregnancies occurred in 19 does. Fetal hemoglobin was higher in the compatible litters (9.7 gm/dl vs 5.8 gm/dl, p < 0.001), whereas no difference could be detected between the respective reticulocyte counts (31.9 vs 36.0/100 red blood cells, p = 0.2). Hydrops fetalis was noted in none of 18 compatible litters versus 12 of 19 incompatible litters (p < 0.01). CONCLUSION: A disease analogous to human hemolytic disease of the newborn can be induced in the rabbit fetus.

Animals↗
PubMed1995

Molecular mimicry between Fc receptor and S peplomer protein of mouse hepatitis virus, bovine corona virus, and transmissible gastroenteritis virus.

We have previously demonstrated molecular mimicry between the S peplomer protein of mouse hepatitis virus (MHV) and Fc gamma R (Fc gamma R). A monoclonal antibody (MAb) to mouse Fc gamma R (2.4G2 anti-Fc gamma R MAb), purified rabbit immunoglobulin, but not their F(ab')2 fragments, as well as mouse and rat IgG, immunoprecipitated (1) recombinant S peplomer protein expressed by a vaccinia virus recombinant in human, rabbit, and mouse cells, and (2) natural S peplomer protein from cells infected with several strains of MHV and MHV escaped mutants. We report here results of studies documenting molecular mimicry between Fc gamma R and S peplomer protein of viruses representing three distinct antigenic subgroups of the Coronaviridae. We have shown a molecular mimicry between the S peplomer protein of bovine corona virus (BCV) and Fc gamma R. The 2.4G2 anti-Fc gamma R MAb, rabbit IgG, but not its F(ab')2 fragments, as well as homologous bovine serum, free of anti-BCV antibodies, immunoprecipitated S peplomer protein of BCV (Mebus strain). In contrast, we did not find molecular mimicry between S peplomer protein of human corona virus (HCV-OC43) and Fc gamma R. Although the OC43 virus belongs to the same antigenic group as MHV and BCV, MAb specific for human Fc gamma RI or Fc gamma RII and purified human IgG1, IgG2, and IgG3 myeloma proteins did not immunoprecipitate the S peplomer protein from HCV-OC43-infected RD cells. In addition, we did demonstrate molecular mimicry between the S peplomer protein of porcine transmissible gastroenteritis virus (TGEV) and Fc gamma R. TGEV belongs to the second antigenic subgroup of coronaviridae.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Development of crosslinked polyamines suitable for synthesizing complex ampholytes for isoelectric focusing.

Commercially available ampholytes used in isoelectric focusing applications vary widely from source to source in their resolving power. This study was initiated to develop alternative ampholyte formulations for high resolution preparative and analytical isoelectric focusing. Initial IR spectroscopy studies showed that divalent acid esters would efficiently crosslink available polyamines with complete consumption of ester. Fast atom bombardment mass spectroscopic analysis of resulting crosslinked polyamines showed extensive structural heterogeneity of the resulting polyamine mixture. Conversion of the polyamine mixture to functional zwitterions using alpha,beta-unsaturated carboxylic acids yielded mixtures giving smooth pH gradients in acrylamide gel isoelectric focusing. Further analysis of these mixtures in immobilized pH gradients showed increases in heterogeneity of available carrier species over similar zwitterion mixtures made using only commercially available polyamine monomers. The mixtures were also more heterogeneous than commercially available ampholytes when analyzed by picric acid precipitation in immobilized pH gradients.

Buffers↗
PubMed1992

Direct detection of carrier ampholytes in immobilized pH gradients using picric acid precipitation.

A protocol is described for monitoring the heterogeneity of end products of organic syntheses yielding amphoteric molecules containing two or more amino groups. This protocol was found to be a valuable aid in synthesis of carrier ampholytes for specific isoelectric focusing applications. This method does not depend on the ampholytes themselves to dictate the conditions under which they are analyzed. Carrier ampholytes have been found previously to be insoluble in picric acid and the insolubility property was not dependent upon the pI of individual ampholyte species. This insolubility property was exploited in the protocol. Immobilized pH gradients were used to focus the carrier ampholytes. Ampholytes were then visualized in situ by picric acid precipitation. The data shows that the protocol is useful for analyzing the results of chemical manipulations for enhancing the resolution of carrier ampholytes. A direct relationship was shown between carrier ampholyte heterogeneity as demonstrated by this protocol and the resolution of complex protein mixtures in isoelectric focusing gels. Picric acid formed visible precipitates with a variety of organic compounds which contained more than one amino group.

Acrylamides↗
PubMed1992

Microheterogeneity of S-glycoprotein of mouse hepatitis virus temperature-sensitive mutants.

Mouse hepatitis virus (MHV) strain JHM (MHV-JHM) is a neurotropic coronavirus that causes acute fatal encephalomyelitis in 75-99% of infected mice. The surviving animals may subsequently develop demyelinating disease. We compared the S peplomer protein of the wild type (wt) and five temperature-sensitive (ts) mutants of MHV-JHM. In contrast with the wt, none of these five cause fatal disease (mortality less than 10%). Three of these ts mutants did not induce any demyelinating disease, a fourth caused demyelinating disease in 5% of the animals and a fifth, designated ts8, exhibited strong demyelinating properties and caused demyelination in 99% of the animals. SDS-PAGE analysis revealed no differences in the molecular weight of S peplomer protein of wt or ts MHV-JHM mutants. However, isoelectric focusing of the S protein of these five ts mutants and the wt MHV-JHM, followed by transfer to nitrocellulose sheets and immunoblotting with anti-S specific antibody revealed significant differences in the microheterogeneity of the S protein.

Animals↗
PubMed1992

Ultrasound-guided blood sampling of rabbit fetuses.

A rabbit animal model for hemolytic disease of the newborn has been previously described. However, evaluating the effects of this disease was limited to histologic and hematologic examinations of liveborn kitlings. To assess the feasibility of in utero blood sampling, we performed ultrasound-guided cardiac sampling of 50 fetuses in 16 New Zealand White does on days 26 and 27 of gestation. The overall rate of successful sampling was 80%. The procedure-related mortality declined to 35% by the third phase of the study. The mean (+/- SD) hematocrit (%) and reticulocyte values (#/100 RBCs) on day 26 were 26.3 +/- 3.3 and 35.6 +/- 5.1, respectively; values on day 27 were 31.3 +/- 4.9 and 27.5 +/- 7.6. The results of this study suggest that hematologic data can be obtained from rabbit fetuses in the majority of cases with only moderate fetal loss.

Animals↗
PubMed1990

Comparative studies of recycling isoelectric focusing and continuous flow electrophoresis: separation of proteins with minor charge differences.

Continuous flow zone electrophoresis (CFE) and recycling isoelectric focusing (RIEF) are two of the alternative formats for fluid phase preparative isolation of biological products in liquid separation media. The McDonnell Douglas CFE system has been used for both ground-based and microgravity separations. The ground-based McDonnell Douglas CFE and RIEF were compared for the ability to resolve mixtures of proteins with known charge differences. Mixtures of 1) cytochrome c, myoglobin, and ovalbumin or 2) beta-lactoglobulin and ovalbumin were used to evaluate the resolving capabilities of CFE and RIEF. Following separation, fractions were analyzed by determining absorbance at 280 nm and by analytical isoelectric focusing (IEF) using Coomassie Brilliant Blue or silver staining to detect focused proteins. Both CFE and RIEF apparently separated the components of both mixtures into individual peaks, separated by fractions which contained little or no detectable protein. Coomassie-stained analytical IEF gels supported this finding. However, when separated proteins were analyzed by silver staining of the analytical gels, the separation of ovalbumin from beta-lactoglobulin by CFE was not complete. Ovalbumin was free of beta-lactoglobulin but beta-lactoglobulin was contaminated by trace amounts of ovalbumin. RIEF clearly separated each protein with no detectable contamination. These data demonstrate the superiority of RIEF over CFE for resolution of protein mixtures having only minor charge differences. RIEF may be more efficient due to the documented electrodissociation of noncovalent protein:protein complexes which occurs during RIEF separations.

Cytochrome c Group↗
PubMed1990

Induction and immunochemical properties of a novel anti-antibody.

This paper describes results which characterize an induced antibody in normal outbred rabbits which we have, for convenience, called parareactant (PR). PR resulting from autoimmunization of rabbits with either keyhole limpet hemocyanin-anti-tetanus toxoid F(ab')2 or with tetanus toxoid-anti-tetanus toxoid F(ab')2 complexes was studied. PR activity was directed solely to autologous, homologous or heterologous F(ab')2 fragments regardless of their specificity. PR failed to react with intact antibodies or with antigen-antibody complexes consisting of homologous antibody bound to specific antigen. Radioimmunoassay and ELISA inhibition assays showed that reactivity between PR and autologous anti-tetanus toxoid F(ab')2 or homologous anti-bovine serum albumin F(ab')2 fragments was specifically inhibited with antigen. Anti-allotypic antibodies specific for a2 and b6 markers strongly inhibited binding of 125I-anti-micrococcal carbohydrate F(ab')2 (a2, b6) with PR (a3, b4, b5). PR specificity thus appears to be directed against non-idiotypic determinants present in Fv regions. Affinity immunoblotting was used to analyze clonality of PR in the sera collected from individual rabbits during the course of an active immune response. PR-positive sera displayed clonally restricted spectrotype patterns. PR molecules were predominantly IgG with isoelectric points of 5.9-6.8. These results strongly suggest that these PR molecules are coded by a small number of V region genes.

Animals↗
PubMed1990

Free flow cell electrophoresis using zwitterionic buffer.

Studies of a zwitterionic buffer formulated for cell electrophoresis were done using the McDonnell-Douglas Continuous Flow Electrophoresis System. Standard buffers were analyzed for their stability in the electrical field and the results showed that both buffers tested were inherently unstable. Further, titration studies showed that the standards buffers buffered poorly at the pH employed for electrophoresis. The zwitterionic buffer buffered well at its nominal pH and was shown to be stable in the electrical field. Comparative studies of the buffer with standard cell separation buffers using formalin fixed rabbit and goose red blood cells showed that the zwitterionic buffer gave better resolution of the fixed cells. Studies with viable hybridoma cells showed that buffer Q supported cell viability equal to Hank's Balanced Salt Solution and that hybridoma cells in different stages of the growth cycle demonstrated reproducible differences in electrophoretic mobility.

Animals↗
PubMed1989

Adoptive immunity transferred by naive donor cells immunized in vitro.

Speedy restoration of immune responsiveness in bone marrow recipients has been the objective of studies in which the donor was immunized so that specific immunologic memory could be transferred adoptively and selectively. Using unrelated rabbits, matched for major histocompatibility antigens but mismatched for their immunoglobulin allotypes, it could be shown that recipients of lymphoid cells from naive donors became B cell chimeras but did not use donor-derived B cells for their antibody responses to test antigens. In contrast, cells from donors primed for such antigens dominated antibody production in recipients in response to specific challenge. Clonal restriction in such adoptive responses was demonstrated. We now show that the induction of effective memory in cells from naive donors can be achieved in vitro during the preparation of donor cells for transfer to the recipient. Early challenge of the recipient enhances expression of the transferred immune response quantitatively and also results in the establishment or preservation of a larger diversity of clones from the donor.

Animals↗