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1.
J Natl Med Assoc ; 92(9): 430-5, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11052456

ABSTRACT

Oxyhemoglobin dissociation curves measure the most important function of red blood cells - the affinity for oxygen and its delivery to the tissues. This function may be deranged in sickle cell anemia and some other hemoglobinopathies. An automated oxyhemoglobin dissociation curve analyzer constructed dissociation curves in 55 patients with hemoglobinopathies and in 24 control subjects while maintaining constant temperature and pH. Sigmoid curves were converted to rectilinear ones using the Hill equation. Oxygen affinity of red cells was assessed by calculation of P50 (the PO2 at which hemoglobin is half saturated). Results revealed separation of oxyhemoglobin dissociation Hill plots according to phenotype but with wide variability. Mean oxygen affinity of fetal hemoglobin was greatest, whereas that of sickle hemoglobin was least. Other hemoglobins were intermediate. A positive correlation between decreased oxygen affinity and carboxyhemoglobin confirmed the decreased oxygen affinity of sickle hemoglobin and decreased oxygen affinity and increased diphosphoglycerate in red cells. Hill plots are less sensitive discriminators of oxygen affinity than traditional sigmoid dissociation curves and offer no particular advantage. Serial studies in a subset of three sickle cell anemia patients treated conservatively suggest automated oxyhemoglobin dissociation curves may be useful in assessment of effectiveness of newer therapies of sickle cell anemia after refinement of the method and studies of larger populations.


Subject(s)
Anemia, Sickle Cell/blood , Anemia, Sickle Cell/therapy , Oxyhemoglobins/analysis , Adult , Case-Control Studies , Female , Hemoglobinometry , Humans , Male
2.
Vaccine ; 18(13): 1273-81, 2000 Jan 18.
Article in English | MEDLINE | ID: mdl-10649629

ABSTRACT

Covalently linking protein to polysaccharides converts the anti-polysaccharide immune response from a T-cell independent response to one which is T-cell dependent. The organic cyanylating reagent 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) (Vaccine 14:190, 1996) has been used to activate polysaccharides, which can then be reacted with spacer reagents or directly with protein. We wished to explore ways in which proteins could be linked to CDAP-activated polysaccharides to conjugate in a more controlled and selective fashion. To this end, we examined the reaction of nucleophilic amino acids with CDAP-activated polysaccharides under basic and acidic conditions. We found that lysine, cysteine and histidine but not methionine, serine or tyrosine conjugated to CDAP-activated dextran. We also examined the reaction of various spacer reagents with CDAP-activated dextran as a function of pH. The addition of hexanediamine was highly pH dependent and maximal at pH 9.3. In contrast, the addition of adipic dihydrazide, which has a pKa of ca 2.5 was essentially independent of pH. By performing the conjugation reaction at pH 5, we were able to selectively couple hydrazides even in the presence of high concentrations of amines. Proteins derivatized with limited numbers of hydrazides could be conjugated to CDAP-activated polysaccharides at pH5, where the native protein was not reactive. Proteins could be derivatized with hydrazides on carboxyls using adipic dihydrazide and a water soluble carbodiimide or on amines using a mild two-step reaction. Tetanus toxoid-pneumococcal type 14 conjugates produced by coupling hydrazide-derivatized tetanus toxoid under acidic conditions induced anti-polysaccharide antibodies at titers comparable to that stimulated by conjugates produced using a basic coupling pH. Our data suggest that crosslinking was occurring only with the limited number of hydrazides on the protein and that we achieved limited and selective crosslinking between the protein and CDAP-activated polysaccharide. This work also demonstrates that CDAP-mediated conjugation to polysaccharides can be applied even to very pH sensitive proteins and polysaccharides.


Subject(s)
Cross-Linking Reagents/chemistry , Nitriles/immunology , Polysaccharides, Bacterial/chemistry , Proteins/immunology , Pyridinium Compounds/immunology , Vaccines, Conjugate/chemistry , Amines/chemistry , Animals , Haptens/chemistry , Haptens/immunology , Hydrazones/chemistry , Hydrogen-Ion Concentration , Mice , Mice, Inbred BALB C , Nitriles/chemistry , Polysaccharides, Bacterial/immunology , Proteins/chemistry , Pyridinium Compounds/chemistry , Solubility , Structure-Activity Relationship , Vaccines, Conjugate/immunology
3.
Vaccine ; 14(3): 190-8, 1996 Feb.
Article in English | MEDLINE | ID: mdl-8920699

ABSTRACT

Neonates have poor immune responses to type 2 T-cell independent antigens (TI-2), such as polysaccharides and immunization of human infants with these antigens does not induce protective levels of serum antibodies. Conjugating proteins to TI-2 antigens converts the immune response to one which is T-cell dependent. We used an organic cyanylating reagent, 1-cyano-4-dimethylaminopyridinium tetrafluroborate (CDAP), to activate polysaccharides, in water, and subsequently react them with hexanediamine, in preparation for coupling proteins to the polysaccharide. CDAP activation of polysaccharide is rapid (< 2 min) and efficient. CDAP can be used to activate polysaccharides of diverse chemical natures, including dextrans and pneumococcal types 6, 14, 19 and 23. The critical parameters in CDAP activation of polysaccharides were the reagent concentrations and the pH. Activation can be performed over a broad alkaline pH range, with an optimum of pH 9-10. Furthermore, proteins can be coupled to CDAP-activated polysaccharides without the use of a spacer. Direct conjugation of protein to CDAP-activated polysaccharides can be performed under mildly alkaline conditions (pH 7-9). These conditions allow CDAP to be used with alkaline-sensitive polysaccharides and proteins. Mice immunized with BSA-pneumococcal type 14 polysaccharides (Pn14) conjugates, prepared either by direct conjugation or via a spacer, had high anti-Pn14 and anti-BSA serum antibody IgG1 titers, whereas no IgG1 antibody was induced to the unconjugated components. The ease of use and mild activating conditions should prove of value in using CDAP to prepare conjugate vaccines, as well as other immunologically useful reagents.


Subject(s)
Bacterial Vaccines/chemistry , Nitriles/chemistry , Polysaccharides, Bacterial/chemistry , Pyridinium Compounds/chemistry , Streptococcus pneumoniae , Animals , Bacterial Vaccines/immunology , Dextrans/chemistry , Hydrogen-Ion Concentration , Mice , Mice, Inbred DBA , Nitriles/immunology , Polysaccharides, Bacterial/immunology , Pyridinium Compounds/immunology , Serum Albumin, Bovine/chemistry , Serum Albumin, Bovine/immunology , Vaccines, Conjugate/chemistry
4.
Biochem Biophys Res Commun ; 202(1): 271-7, 1994 Jul 15.
Article in English | MEDLINE | ID: mdl-7518676

ABSTRACT

To determine selenium's effects on glutathione S-transferase gene expression and enzyme activity, weanling rats were fed a selenium-deficient diet, or the same diet supplemented with 0.1 (control) or 2.0 mg selenium/kg diet as sodium selenite, for 91 days. Consumption of either the selenium-deficient or high selenium diet increased activity of glutathione S-transferase, measured with 1-chloro-2,4-dinitrobenzene as substrate, compared to the control diet. Transcription of genes for glutathione S-transferase subunits was unaffected by selenium intake. Steady state levels of mRNA for glutathione S-transferase subunits were affected variably by changes in selenium intake, depending upon the tissue and subunit examined. These results suggest that the biological effects of selenium may be due in part to its regulation of gene expression for glutathione S-transferase family enzymes.


Subject(s)
Diet , Gene Expression Regulation, Enzymologic/drug effects , Glutathione Transferase/biosynthesis , Sodium Selenite/pharmacology , Animals , Blotting, Northern , Glutathione Transferase/metabolism , Kidney/enzymology , Liver/enzymology , Macromolecular Substances , Male , RNA/analysis , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Selenium/deficiency , Sodium Selenite/administration & dosage , Transcription, Genetic/drug effects
5.
J Allergy Clin Immunol ; 78(4 Pt 1): 590-600, 1986 Oct.
Article in English | MEDLINE | ID: mdl-3771951

ABSTRACT

Inland areas of northern California have an intense grass pollination in the spring of each year. This is accompanied by a stirking rise in the incidence of asthma. We documented this relationship and designed a trial to test the efficacy of immunotherapy for grass-pollen asthma. Aeroallergen counts were performed on the roof of the allergy clinic of David Grant Medical Center from January 1981 to December 1984 by a gravity collector. These counts were compared to counts done on a Rotorod at a nearby hospital from July 1982 to September 1984. Climatologic factors were also tabulated. Visits for asthma and rhinitis to our emergency room and asthma admissions to our hospital were counted for the 4-year period. A randomized, double-blinded, placebo-controlled trial of immunotherapy with grass-pollen extract was performed from November 1984 to June 1985. Two groups of clinically and immunologically well-matched subjects were started on an accelerated preseasonal trial of immunotherapy. One group received a standardized grass extract, and the other group did not. Both groups received other extracts of aeroallergens to which they were skin test positive that occur locally in the spring and summer. This was done because of our dissatisfaction with a histamine placebo used in a previous pilot study. Symptom medication scores (SMS) and immunologic parameters were followed. For the 4-year period, grass-pollen count (GPC) correlated strongly with asthma emergency room visits (r = 0.90; p less than 0.001) and for rhinitis (r = 0.92; p less than 0.001). Asthma admissions also correlated strongly with GPC (r = 0.72; p less than 0.001). Other aeroallergens either did not correlate significantly or occurred in such small numbers that they could not be seriously considered. Rotorod counts supported these conclusions with the exceptions of some Basidiomycetes. Climatologic factors demonstrated no relationship to the incidence of asthma. Asthma SMS were lower in the grass-treated group, p less than 0.05. Rhinitis SMS were also lower but did not reach significance, p = 0.11. RGGI sIgE did not rise significantly in the grass-treated group but did in the placebo-treated group. RGGI sIgE rose in both groups, although to significantly higher levels in the grass-treated group, p less than 0.001. The asthma SMS were inversely related to increasing RGGI cumulative dose, p less than 0.10. Linear regression analysis of the dose-response scattergram suggests that a cumulative dose of approximately 90 micrograms of RGGI may be desirable.(ABSTRACT TRUNCATED AT 400 WORDS)


Subject(s)
Asthma/etiology , Desensitization, Immunologic , Poaceae/immunology , Seasons , Adult , Asthma/immunology , Asthma/therapy , California , Dose-Response Relationship, Immunologic , Female , Humans , Immunoglobulin E/analysis , Immunoglobulin G/analysis , Male , Rhinitis/epidemiology
6.
Ann Allergy ; 56(4): 331-4, 1986 Apr.
Article in English | MEDLINE | ID: mdl-3963525

ABSTRACT

To assess the relative risk of local and systemic reactions to injections of pollen extracts and to examine the predictive value of these local reactions for systemic reactions, a prospective survey of local and systemic reactions was performed from October 1981 to June 1983 at a large military allergy clinic. Four hundred and sixteen patients received 26,645 injections of pollen extracts. Of the total, 25,508 injections (95.7%) were not associated with reactions, 1,055 injections (4.0%) were associated with local reactions (669, less than 2 cm in diameter; 279, 2 to 5 cm; and 107 greater than 5 cm), and 82 injections (0.3%) were associated with systemic reactions. Seventy patients experienced 82 systemic reactions. Of these, 25 (36%) had no prior local reactions. Eighteen males and 52 females had systemic reactions. The sensitivity of local reactions as a predictor of systemic reactions was 64%, the specificity was 1.2%, the positive predictive value was 12%, and the negative predictive value was 14%. Proportionally, more women than men experienced systemic reactions, P less than 0.05. We made the following conclusions. Most injections of pollen extracts were not associated with local or systemic reactions. The majority of reactions were local. The presence of local reactions was not helpful in predicting which patients would develop systemic reactions. Women appeared to be at greater risk for systemic reactions than men.


Subject(s)
Immunotherapy/adverse effects , Plant Extracts/administration & dosage , Pollen , Erythema/etiology , Female , Humans , Male , Risk , Sex Ratio
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