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Biomedical subjects

L M Snyder

Publications and source records attributed to L M Snyder.

At least 37 records · Page 2Linked to original sources
PubMed1991

Increased cisplatin tissue levels with prolonged arterial infusion in the rat.

Prolonged arterial infusions of cisplatin (DDP) have been effective in the treatment of regionally confined malignancies. It is unclear whether the route or schedule of DDP administration was responsible for the observed therapeutic benefit. To resolve this issue, tumor and normal tissue platinum (Pt) levels were determined in rats bearing hind-limb rat mammary tumors after intravenous (IV) and intra-arterial (IA) DDP infusions of constant dose and varying lengths. Infusions of DDP at 6 mg/kg were conducted IA over 30 minutes, and 3, 6, 24, and 48 hours and IV over 30 minutes and 48 hours. After infusion, Pt concentrations in solubilized tissue homogenates were measured by flameless atomic absorption spectroscopy. Maximum tumor Pt levels were seen after 48-hour IA infusion (29.3 micrograms Pt/mg tissue). IA infusions of 24 hours or less resulted in significantly lower Pt levels. Maximum tumor Pt concentration after IV administration was only 0.98 micrograms/mg tissue (48-hour infusion). Muscle Pt levels adjacent to the tumor were highest in the IA infused extremities, but at the 48-hour interval, were 53-fold less than tumor levels. Tumor and adjacent muscle Pt levels were not significantly different from each other after IV administration. This study provides pharmacologic evidence that lengthening the duration of IA DDP infusion increases tumor levels of Pt over that of IV or rapid IA administrations. The benefit of prolonged IA DDP infusions is dependent upon both route and schedule of drug administration.

Animals↗
PubMed1990

Decreased in vivo survival of hydrogen peroxide-damaged baboon red blood cells.

In this study we attempt to establish the consequence of in vitro hydrogen peroxide (H2O2)-induced membrane damage as manifested by spectrin-hemoglobin (Sp-Hb) complex formation and decreased red blood cell (RBC) deformability to in vivo RBC survival in baboons. After exposure to 135 to 581 mumols/L H2O2 and reduction with dithiothreitol (DTE), baboon RBCs were infused into the animal, and the fraction of cells remaining in circulation after 24 hours and the lifespan of surviving cells were quantitated. In a dose-dependent fashion, a positive correlation was observed between in vitro membrane alterations and the 24-hour in vivo survival. While 12% of the control cells were removed from circulation in 24 hours, 23% were removed after treatment with 339 mumols/L H2O2, and 36% following exposure to 581 mumols/L H2O2. Pretreatment with carbon monoxide before exposure with H2O2 increased the survival of oxidized RBCs. RBCs not removed from circulation in the first 24 hours had a normal lifespan. Moreover, by selectively isolating biotin-labeled, peroxide-treated cells that survived the first 24-hour posttransfusion period, a significant decrease in Sp-Hb crosslinking was observed in these cells. These results suggest that a subpopulation of cells sensitive to oxidation were removed during the first 24 hours. To identify this population, the survival of density-fractionated RBCs exposed to oxidant stress was quantitated. No differences in either the 24-hour survival or RBC life span were observed between untreated low-density (MCHC less than or equal to 32g/dL) and high-density cells (MCHC greater than or equal to 37g/dL). However, striking differences were noted after treatment with 339 mumols/L H2O2, with the 24-hour survival of high-density cells showing a marked decrease compared with low-density cells. These data support our hypothesis that during peroxidative membrane damage, Hb oxidation initiates a sequence of events resulting in skeletal changes that lead to membrane alterations and, eventually, in vivo destruction, and that the dense, dehydrated cells are more susceptible to oxidant damage.

Animals↗
PubMed1990

High-performance tryptic mapping of recombinant bovine somatotropin.

Experiments are described that have lead to the development of a highly reproducible tryptic map of recombinant DNA derived bovine somatotropin (rbSt). Tryptic digestion of rbSt at 37 degrees C results in the formation of a precipitate. Preliminary characterization of the precipitate suggests that its formation is due to the association of intermediate tryptic fragments. An examination of the temperature dependence of the digestion has revealed that precipitate formation is inhibited when digestion is performed at 10 degrees C or less. The combination of a 5-mg sample, the use of highly purified trypsin, and digestion at 5 degrees C generate a tryptic map that exhibits an average 1.3% RSD (0.5-3.6%) for all anticipated fragments. Validation studies demonstrate that while the peak response precision is rugged to daily variation of operators or chromatographic systems, the fragment retention is not. This dictates that peaks be assigned by qualitative pattern recognition. Assay ruggedness in the peak response domain allows for the implementation of quantitative methods for the comparison of rbSt reference standard and sample tryptic maps. The assay is linear for all anticipated fragments within 50-150% of the operating range. Specificity is established by assay of pituitary somatotropins from other species and rbSt analogs produced by site-specific mutagenesis. The data demonstrate that all single amino acid substitutions examined are identified by using the technique. Assay sensitivity is validated for selected tryptic fragments through analysis of reference standard digests spiked with known amounts of rbSt analog digests. The data indicate that potential impurities of 3.2, 2.0, and 4.5% can be quantitated with statistical confidence in the tryptic fragments T1, T10, and T23 + 25, respectively.

Amino Acid Sequence↗
PubMed1989

Cation transport in oxidant-stressed human erythrocytes: heightened N-ethylmaleimide activation of passive K+ influx after mild peroxidation.

Normal and chronically dehydrated (hereditary xerocytosis) human red cells were subjected to mild peroxidative treatment (315 microM hydrogen peroxide (H2O2), 15 min) in the presence of azide. The subsequent expression of passive (ouabain-resistant) K+ transport activities was analyzed by measurement of 86Rb+ influx. Peroxidation of normal red cells did not affect basal K+ transport activity, but the increment in K+ influx elicited by 0.5 mM N-ethylmaleimide (NEM) was increased 3-fold. The enhanced K+ influx was chloride-dependent, but only partially inhibited by 0.1 mM furosemide. Stimulated activity declined progressively after NEM activation, but could be restored by a second NEM treatment. Prior conversion of hemoglobin to the carbonmonoxy form abolished the response to peroxide, while 200 microM butylated hydroxytoluene (BHT) exerted only partial inhibition, suggesting that the effect of H2O2 requires interaction of activated, unstable hemoglobin species with the membrane, but that lipid peroxidation is not sufficient. Peroxidation following NEM treatment also enhanced NEM activation, indicating that enhancement does not require altered NEM reactions with stimulatory or inhibitory sites. Passive K+ transport in hereditary xerocytosis red cells was not activated by NEM, with or without H2O2 pretreatment. The results demonstrate that modest peroxidative damage to red cells can heighten the activation of a transport system that is thought to be capable of mediating net K+ efflux and volume reduction in cells that express it. Models are proposed in which the effects of NEM, H2O2, cell swelling and other factors are mediated by conformational changes in a postulated subpopulation of anion channel (Band 3) molecules that bind the K+ transporter.

Butylated Hydroxytoluene↗
PubMed1989

Decreased amount of the Rh antigen D in hereditary spherocytosis (HS).

This study was done to determine whether hereditary spherocytosis (HS) red blood cells (RBC) have decreased amounts of Rh antigens. Initially we studied the RBC of five members of one family, two of whom had HS. Using automated quantitative haemagglutination tests, we demonstrated that HS RBC agglutinated less with Rh antisera of four specificities than did normal RBC, indicating that Rh antigens are decreased on HS RBC. In this family, the strength of other blood group antigens on HS RBC was estimated by manual titres and agglutination scores. No appreciable differences in the agglutination of HS and normal RBC were observed with non-Rh antisera. To assess the strength of the D antigen more accurately, the number of D sites was quantitated on the RBC of 19 individuals with HS and 11 of their healthy relatives. HS RBC had 9209 +/- 4084 (mean +/- SD) D sites, whereas the normal RBC had 15 394 +/- 5763 D sites. These two means were significantly different (P less than 0.01). HS RBC were also compared to normal RBC of unrelated individuals who had the same Rh phenotype. These analyses showed that HS RBC had about half of the normal number of D sites. Our data indicate that HS red cells have decreased amount of the Rh antigen D and probably also of other Rh antigens.

Female↗
PubMed1989

Canid modification of human remains: implications for time-since-death estimations.

Time-since-death estimations are usually based on physical decomposition of the corpse, insect succession, and contextual associations. The rates of change and succession are based on decomposition studies, most of which control access of scavengers to the corpse; however, many naturally exposed corpses are subject to scavenger modification. These modifications change the rate of decomposition, the pattern of insect succession, and the context of associations, thus altering estimations of time since death. A controlled feeding study with captive wolves and road-killed deer is pertinent to understanding canid scavenging and how scavenging may alter postmortem changes. During feeding, the wolves commonly dismember and devour the deer in a predictable sequence. Although there are some variations in the usual sequence, the carcass is always moved, and skeletal elements are separated, diminished in size and scattered. Scavenging must, therefore, be considered in estimating time since death.

Animals↗
PubMed1988

The role of membrane protein sulfhydryl groups in hydrogen peroxide-mediated membrane damage in human erythrocytes.

The formation of spectrin-hemoglobin complex following treatment of red cells with hydrogen peroxide (H2O2) has previously been shown to be associated with alterations in cell shape, decreased membrane deformability and increased recognition of modified cells by anti-IgM immunoglobulin in a phagocytic assay by monocytes. Prior treatment with carbon monoxide completely inhibited the H2O2-associated membrane changes, indicating a role for oxidized hemoglobin in the complex formation. Also, in a cell-free system, blockage of sulfhydryl (SH) groups on purified spectrin by N-ethylmaleimide significantly reduced the complex formation, suggesting a role for SH groups of spectrin in crosslinking process. The present study was undertaken to examine the role of SH blockade by N-ethylmaleimide on intact red cells undergoing oxidative damage. Pretreatment of erythrocytes with N-ethylmaleimide at concentrations ranging from 0.1 to 0.2 mM resulted in decreased lipid peroxidation and spectrin hemoglobin crosslinking. Moreover, pretreatment with N-ethylmaleimide resulted in less marked alterations in cell shape and membrane deformability as well as reduced recognition of peroxidized cells by antiglobulin serum. N-Ethylmaleimide treatment had no effect on methemoglobin formation. Studies with 14C-labeled N-ethylmaleimide showed that over 50% of N-ethylmaleimide was incorporated into spectrin. Pretreatment of cells with higher concentrations of N-ethylmaleimide (over 0.2 mM) was associated with membrane dysfunction independent of H2O2. These results imply that blocking of reactive SH groups leads to reduced interaction of spectrin with oxidized globin. These data, along with our prior observations, indicate that SH groups on spectrin play an important role in hemoglobin oxidation-induced formation of spectrin-hemoglobin complex and the resultant deleterious effects on membrane properties.

Electrophoresis, Polyacrylamide Gel↗
PubMed1988

Plasma ultrafiltration as successful therapy of rabbit VX-2 carcinoma.

It has been demonstrated that tumor-bearing animals elaborate a low molecular weight (less than 10,000 daltons) factor capable of inhibiting in vitro lymphocyte function. It was postulated that removal of this factor would have a favorable effect on host immune response that would translate into improved tumor control. A study was conducted in rabbits bearing the VX-2 carcinoma. Ultrafiltration (UF) was performed 10 days following IV tumor inoculation. UF was achieved by passing blood through an Amicon Diafilter (molecular weight cutoff 10 kD) positioned between the arterial and venous cannulae after heparinization. Two plasma volumes of ultrafiltrate were removed with continuous saline replacement. Two groups of animals received the nonspecific immunoadjuvant, Detox, at time of therapy. Survival in the UF group (N = 9) was compared to untreated tumor-bearing animals (N = 10), sham-operated animals (N = 6), animals receiving Detox (N = 7), and animals receiving UF plus Detox (N = 6). UF imparted a survival advantage when compared to controls (mean 35 days vs. 25 days, P less than .01). The sham group had survival identical to controls. Detox alone conferred minimal survival advantage (mean 29 days, P greater than .05). However, UF + Detox demonstrated maximal survival benefit (mean 40 days, P less than .01). We conclude that UF is an effective anticancer modality in this preclinical model. This study suggests that efforts aimed at eliminating suppressor molecules in cancer patients may be of benefit, especially when combined with biological response modifiers such as Detox.

Adjuvants, Immunologic↗
PubMed1988

The relationship between in vivo generated hemoglobin skeletal protein complex and increased red cell membrane rigidity.

In vitro induced oxidative damage to normal human RBCs has previously been shown to result in increased membrane rigidity as a consequence of the generation of a protein complex between hemoglobin and spectrin. In order to determine if in vivo generated hemoglobin-spectrin complexes may play a role in increased membrane rigidity of certain pathologic red cells, we measured both these parameters in membranes prepared from hereditary xerocytosis (Hx), sickle cell disease (Sc), and red cells from thalassemia minor (beta thal). Membranes were prepared from density-fractionated red cells, and membrane deformability was measured using an ektacytometer. Hemoglobin-spectrin complex was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis, as well as by Western blot analysis using a monoclonal antibody against the beta-subunit of hemoglobin. For these three types of pathologic red cells, progressive cellular dehydration was associated with increased membrane rigidity and increased content of hemoglobin-spectrin complex. Moreover, the increase in membrane rigidity appeared to be directly related to the quantity of hemoglobin-spectrin complex associated with the membrane. Our findings imply that hemoglobin-spectrin complex is generated in vivo, and this in turn results in increased membrane rigidity of certain pathologic red cells. The data further suggest that oxidative crosslinking may play an important role in the pathophysiology of certain red cell disorders.

Anemia, Sickle Cell↗
PubMed1987

Antiglobulin serum mediated phagocytosis of normal senescent and oxidized RBC: role of anti-IgM immunoglobulins in phagocytic recognition.

Fresh human monocytes usually do not recognize normal RBCs; however, in our newly developed assay antiglobulin-opsonized normal RBCs were phagocytized. Both anti-IgG and anti-IgM fractions present in the antiglobulin serum were involved but the major opsonin was anti-IgM. The anti-IgM opsonized mainly senescent RBCs and therefore could be used to discriminate young from senescent RBCs. The antiglobulin serum and monospecific anti-IgM increased opsonization of in vitro oxidized and desialylated RBCs, whereas trypsin-treatment of RBCs decreased phagocytosis. The material removed by trypsin from the RBCs surface inhibited the antiglobulin and monospecific anti-IgM phagocytic assay supporting the view that membrane associated elements crossreacted with anti-IgM. These results suggest that both internal cellular events and external removal of sialic acid play a role in the emergence of non-IgG covered epitopes on the surface of senescent and oxidized erythrocytes.

Antibodies, Anti-Idiotypic↗
PubMed1985

Modulation of mononuclear phagocyte cytotoxicity by alpha-tocopherol (vitamin E).

The in vitro effect of colloidal suspensions of alpha-tocopherol (alpha-T) on phorbolmyristate-acetate (PMA)-induced monocyte cytotoxicity and on antibody-dependent monocyte cytotoxicity (ADCC) was studied. We observed that 1) in the presence of alpha-T, the inhibition was twice as high in the PMA-induced assay than in ADCC; 2) monocytes preincubated with alpha-T were inhibitory in both assays but much less in ADCC, and 3) target erythrocytes preincubated with alpha-T decreased the cytotoxicity in the PMA-induced assay only. Since alpha-T preincubated monocytes showed a decreased release of H2O2 but not of O2-, we concluded that one of the mechanisms by which alpha-T decreased cytotoxicity could be decreased release of H2O2. Whereas the role of H2O2 was documented in the PMA-induced cytotoxicity, in ADCC non-oxidative injury seems more important. This is supported by 1) lesser inhibition of the assay with alpha-T preincubated monocytes; 2) lack of protection with alpha-T preincubated erythrocytes, and 3) mild inhibition with protease inhibitor.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1985

Demonstration of haemoglobin associated with isolated, purified spectrin from senescent human red cells.

Employing a direct and sensitive radioimmunoassay (RIA) we have confirmed the presence of haemoglobin associated with isolated, purified spectrin from senescent red cells. Haemoglobin associated with spectrin occurs in the highest amount in cells with an MCHC greater than 36 g/dl and is approximately 3% of the total spectrin extract. Spectrin from the young cells had the least haemoglobin, while an intermediate amount was found in unfractionated, whole red cells. The RIA results were in close approximation with estimation of the haemoglobin-spectrin complex obtained by carefully integrating the Coomassie blue stain profiles from 4% SDS PAGE in densitometric scans from isolated spectrin.

Densitometry↗
PubMed1985

Properties and characterization of vesicles released by young and old human red cells.

We have presently demonstrated morphologic differences between young and senescent red cells following 18 h of metabolic depletion in vitro. Young and old red cells both form echinocytes, whereas only young cells demonstrated myelin forms or microspheres. Furthermore, vesicles were released in greater quantities into the cell-free supernatant from young cells. Isolated vesicles from both young and old red cells contained lipids, intrinsic membrane proteins (especially band 3), and haemoglobin, and they were also enriched in acetylcholinesterase (AChE). Young cells produced more vesicles than old cells but the composition of the low density vesicles was similar except that haemoglobin-spectrin complex was found exclusively in vesicles from young cells. Oxidation of young red cells prior to metabolic depletion prevented both myelin formation and vesicle release.

Acetylcholinesterase↗
PubMed1985

Effect of hydrogen peroxide exposure on normal human erythrocyte deformability, morphology, surface characteristics, and spectrin-hemoglobin cross-linking.

To further define the conditions for forming spectrin-hemoglobin cross-linking in human erythrocyte membranes and to examine its possible effects on membrane function, we incubated normal human erythrocytes for up to 3 h in concentrations of H2O2, varying from 45 to 180 microM, in an azide phosphate buffer, pH 7.4. The chemical changes observed indicated that methemoglobin formation occurred early and at a low concentration (45 microM). Morphologic changes characterized by increased echinocyte formation occurred in a dose-dependent fashion. In addition, decreased cell deformability commensurate with increased membrane rigidity was found. Finally, an increase in cell recognition as determined by monocyte phagocytosis and adherence in vitro, as well as decreased phosphatidylcholine accessibility to bee venom phospholipase A2, was found in H2O2-treated erythrocytes compared with controls. Both of these latter changes were closely correlated with the extent of spectrin-hemoglobin cross-linking. In addition to these protein-mediated interactions, lipid peroxidation also occurred after H2O2 exposure, as shown by generation of fluorescent amino propene derivatives. The addition of the antioxidant, butylated hydroxytoluene, decreased the fluorescent derivatives, but did not prevent the effects on membrane function. This suggests that lipid peroxidation, though present, was not necessary for the membrane changes found. In contrast, spectrin-hemoglobin aggregation and the alterations in membrane function were completely prevented by prior exposure of the erythrocytes to carbon monoxide.

Carbon Monoxide↗
PubMed1985

Plasmapheresis in paroxysmal nocturnal hemoglobinuria aplastic crisis: clinical improvement and in vitro demonstration of plasma inhibitory activity on erythroid stem cells.

Plasmapheresis was used in a 51 year old woman with known PNH and aplastic crisis unresponsive to conventional treatment. A clinical and laboratory improvement was noted and an in vitro inhibition of BFU-E's obtained from patient's peripheral blood mononuclear cells was observed when autologous plasma or serum were added to cultures. The beneficial effect of plasmapheresis is though to be to the elimination of a plasma inhibitory factor responsible for the aplastic crisis in this PNH case that exerts its action to the committed erythroid progenitors as well.

Anemia, Aplastic↗
PubMed1983

Evaluation of methods using adherence to substrate and density gradient for the isolation of human monocytes.

A new method for monocyte isolation based on cell adherence to gelatin-coated plastic and dislodgement of the adhering monocytes at low temperature was compared with 5 other methods based on cell adherence to substrate and density gradient separation. All methods produced yields of monocytes ranging approximately between 50-70% with about the same degree of purity (less than 90%) except for the method using Percoll density gradient centrifugation where the purity of monocytes was about 80%. When lidocaine at different concentrations was used for cell dislodgement or Percoll density gradient for separation, phagocytosis, Fc receptor function and cytotoxicity were adversely affected, unlike in methods using EDTA or low temperature for dislodgement of the adherent cells. In monocyte chemotaxis assays the rate of migration was affected but not the number of migrating cells for all the isolation procedures investigated. Cell spreading function was apparently well maintained only when gelatin coated plastic was used for adherence and low temperature for cell dislodgement. These data indicate that the newly described method, similar to methods using EDTA for cell dislodgement, yielded relatively intact monocytes but unlike the latter method with better preserved cell spreading. Thus, this method can be considered for standardization to obtain pure monocyte populations from peripheral blood which then can be submitted for comprehensive biochemical and physiologic studies.

Cell Adhesion↗