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Biomedical subjects

D G Marsh

Publications and source records attributed to D G Marsh.

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

Simplified method for isolation of white cells from whole blood suitable for direct polymerase chain reaction.

A simple method for isolating mononuclear cells from whole blood is described. The procedure utilizes phytohemagglutinin to agglutinate the erythrocytes, separating white cells from whole blood in a very brief handling time. The isolated cells are readily subjected to DNA isolation simply by boiling, and the released DNA can be directly employed for the polymerase chain reaction analysis. The efficiency of this method is similar to other conventional methods, but less costly and less time-consuming. This method is particularly useful in analyzing DNA samples from the peripheral blood cells when the simplicity and low cost of the assay are preferable.

Base Sequence↗
PubMed1990

Aortic valve repair for aortic stenosis in adults.

The stenotic aortic valve was surgically repaired in 48 adults, 21 women and 27 men, aged 38 to 83 years. Five had congenital aortic stenosis (AS), with a mean aortic valve gradient and area of 58 +/- 23 mm Hg (standard deviation) and 0.54 +/- 0.13 cm2, respectively; 32 had senile AS with a mean aortic valve gradient and area of 43 +/- 20 mm Hg and 0.98 +/- 0.41 cm2; and 11 had rheumatic AS with a mean aortic valve gradient and area of 59 +/- 24 mm Hg and 0.47 +/- 0.15 cm2. Only 6 patients underwent isolated aortic valvoplasty, 11 underwent concomitant mitral valve procedure, and 34 underwent concomitant coronary revascularization. Repair consisted of decalcification in 33 patients and decalcification as well as commissurotomy in 15 patients. There were three hospital deaths, none related to the aortic valve. Only 2 patients (both rheumatic) did not improve clinically. During follow-up (mean, 64 +/- 41 months) aortic valve restenosis developed in 24% (10 patients, 3/5 congenital, 4/11 rheumatic, and 3/32 senile) at a mean of 64 +/- 28 months. Postoperative Doppler echocardiographic assessment of 21 patients with senile AS at 1.1 +/- 2.7 and 18.1 +/- 1.4 months showed significantly lower aortic valve gradient and improved area in comparison with preoperative values. At 36 +/- 2.7 months, aortic valve gradient and area were not significantly different than preoperative values, and at 58.5 +/- 2.6 months aortic valve gradient was 1.41 (p = 0.07) times the preoperative value. At 7 years, actuarial freedom from aortic valve-related symptoms of the patients with senile AS was 87%. We conclude that in select patients aortic valve repair results in excellent relief of AS. Late restenosis is expected and more likely to occur in the valves with congenital and rheumatic disease than in those with senile disease.

Adult↗
PubMed1990

Frequency of booster injections of allergoids.

In 1982, 43 ragweed-sensitive patients receiving maintenance injections of full doses of ragweed allergoid were selected for a study of the immunologic and clinical efficacy of booster injections only four times a year. These patients had participated for 2 to 7 years as part of a trial of mixes of up to four allergoids to common pollens in the mid-Atlantic area tailored to each patient's skin test sensitivity. They were divided into a group (21 patients) to receive injections every 3 months and a group (22 patients) to receive injections about every 6 weeks (eight injections per year). Patients were rerandomized after 1 year so that half of each original group switched to the alternate treatment, and this program was continued until after the ragweed season of 1985. Thirty-four patients were still under study the last year. Doses, per injection, were 100 allergoid units (1 allergoid unit equals 100 PNU) of each allergen in the mixture. Symptom scores during the 8 weeks of each of the four ragweed seasons were not significantly higher in the 3-month treated group. IgG antibody levels to Amb a I (antigen E) were followed until early 1984 and were not significantly different in the two groups, even though the 6-week treated patients received a two times higher cumulative dose per year. Rates of local and systemic reactions (percent of injections eliciting reactions) were not different in the groups, which means that the 3-month treated group had about half as many reactions by virtue of taking half as many injections.(ABSTRACT TRUNCATED AT 250 WORDS)

Allergens↗
PubMed1989

Complete primary structure of a Lolium perenne (perennial rye grass) pollen allergen, Lol p III: comparison with known Lol p I and II sequences.

The complete amino acid sequence of a Lolium perenne (rye grass) pollen allergen, Lol p III, determined by the automated Edman degradation of the protein and its selected fragments, is reported in this paper. Cleavage by enzymatic and chemical techniques established unambiguously the sequence for this 97-residue protein (Mr = 10,909), which lacks cysteine and shows no evidence of glycosylation. The sequence of Lol p III is very similar to that of another L. perenne allergen, Lol p II, which was sequenced recently; of the 97 positions in the two proteins, 57 are occupied by identical amino acids (59% identity). In addition, both allergens share a similar structure with an antibody-binding fragment of a third L. perenne allergen, Lol p I. Since human antibody responsiveness to all these three allergens is associated with HLA-DR3, and since the structure common to the three molecules shows high degrees of amphipathicity in Lol p II and III, we speculate that this common segment in the three molecules might contain or contribute to the respectively Ia/T-cell sites.

Allergens↗
PubMed1989

Epidemiology of insect venom sensitivity.

The prevalence of insect sting reaction and of venom sensitization in adults is unknown. We report the results of intake evaluation of a stratified random sample of a large adult population previously studied for the determinants of atopic disease. In 269 subjects, the prevalence of systemic allergic sting reactions was 3.3% and 26.5% had IgE antibodies to venom demonstrated by skin test or radioallergosorbent test. Asymptomatic sensitization (positive venom skin test) was observed in 15% of subjects with no history of an allergic sting reaction. Positive venom skin tests were more frequent in men, in those with positive skin tests to inhalant allergens, and in subjects aged 20 through 29 years. A positive venom skin test or radioallergosorbent test was more frequent in subjects who had been stung within the previous 3 years (35%) than in those stung more than 3 years before (20%). We conclude that both systemic allergic reactions to insect stings and asymptomatic sensitivity to venom are common and that most affected persons never seek medical advice. The significance of asymptomatic venom sensitization is unknown.

Adult↗
PubMed1989

Complete amino acid sequence of a Lolium perenne (perennial rye grass) pollen allergen, Lol p II.

The complete amino acid sequence of a Lolium perenne (rye grass) pollen allergen, Lol p II was determined by automated Edman degradation of the protein and selected fragments. Cleavage of the protein by enzymatic and chemical techniques established an unambiguous sequence for the protein. Lol p II contains 97 amino acid residues, with a calculated molecular weight of 10,882. The protein lacks cysteine and glutamine and shows no evidence of glycosylation. Theoretical predictions by Fraga's (Fraga, S. (1982) Can. J. Chem. 60, 2606-2610) and Hopp and Woods' (Hopp, T. P., and Woods, K. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 3824-3828) methods indicate the presence of four hydrophilic regions, which may contribute to sequential or parts of conformational B-cell epitopes. Analysis of amphipathic regions by Berzofsky's method indicates the presence of a highly amphipathic region, which may contain, or contribute to, an Ia/T-cell epitope. This latter segment of Lol p II was found to be highly homologous with an antibody-binding segment of the major rye allergen Lol p I and may explain why immune responsiveness to both the allergens is associated with HLA-DR3.

Amino Acid Sequence↗
PubMed1989

Molecular genetics of human immune responsiveness to allergens.

Specific immune responsiveness to certain highly purified allergens is significantly associated with particular HLA-D types. In one striking example, IgE and IgG responsiveness to the ragweed Amb V (Ra5) allergens is strongly associated with HLA-DR2/Dw2. We studied the HLA-D gene sequences of 67 Caucasoid subjects. All Dw2+ (Dw2 +/- in one case) high responders to Amb a V, before or after high-dose ragweed immunotherapy, possessed typical DRB1*2.2 and DRB3*2.2 (Dw2-associated DR beta) gene sequences. Their DQB1 genes all had typical Dw2-associated DQB1*1.2 sequences, with two exceptions (DQB1*1.21 and DQB1*1.18, that are usually associated with Dw21 and Dw18, respectively). The finding of these unusual sequence combinations in these Amb a V responders implicates a DR alpha beta I2.2 or a DR alpha beta III2.2 molecule as the major Amb a V Ir gene product. This conclusion is supported by our finding of one non-responder (after prolonged immunotherapy) who possessed the DQ1.2, but not the DR2.2, sequences. In ragweed-allergic subjects further data suggest that a DR2.2 molecule is generally a necessary and almost always a sufficient requirement for high Amb a V responsiveness. An Amb a V-specific T cell clone obtained from a Dw2+ subject was DR-restricted according to inhibition studies using anti-HLA-D monoclonal antibodies. Also, antigen-presenting cells that were DR2.2+ or DR2.12+, but not DR2.21+ or DR2.22+, were able to present antigen to the cloned T cells, implicating certain DR2 molecules as restriction elements. In similar studies using the rye grass allergen Lol p III we obtained preliminary data implicating the DR beta I sequence E9 YSTS13 (found in DR3, DR5 and DRw6 genotypes) in the recognition of a major Lol p III agretope. Since Lol p I and II are homologous to Lol p III in one region of their amino acid sequences the EYSTS sequence may also be involved in the recognition of similar agretopes of these molecules. These and other studies demonstrate the power of the allergy model for genetic and molecular studies of the human immune response.

HLA Antigens↗
PubMed1989

Immunotherapy decreases skin sensitivity to cat extract.

In 22 patients with cat asthma who were highly sensitive to cat, we compared, double-blind, the effects of immunotherapy with cat-hair and dander extract (11 patients) with effects of placebo (11 patients). Patients matched by intradermal skin test titration, leukocyte histamine release, and the doses of both cat extract and methacholine required for 20% fall in FEV1 were randomly assigned to one of the two treatment groups. Immunotherapy doses were increased to a maintenance dose of 4.56 Food and Drug Administration (FDA) units of Fel d I or if maintenance dose was less, to the highest tolerated dose. Before and during immunotherapy, we measured by intradermal titration the dose of cat extract, in FDA units of Fel d I equivalents, required for (1) 2+ end point (wheal diameter, greater than or equal to 10 mm; erythema diameter, 20 to 30 mm) and (2) 20 mm end point (erythema diameter, 20 mm). By prick skin test, we measured (1) the dose of cat extract that produced a wheal equal in area to that produced by histamine, 1 mg/ml, (2) the doses of cat extract that produced a wheal 22 mm2 in area, and (3) the sum of the wheal areas produced by five prick tests to 23.8, 7.1, 2.4, 0.7, and 0.2 FDA units per milliliter of Fel d I, respectively. After 1 year of treatment, in comparison to control patients, treated patients had significant increases in the ratio 1 year value/pretreatment value for the doses of cat extract required for intradermal test end points 1 and 2 (p less than 0.01), for prick test end point 1 (p less than 0.01), for end point 2 (p = 0.015), and significant decreases in this ratio for prick test end point 3 (p = 0.015). Treated patients also had significant increases in cat extract required for a 20% fall in FEV1 (p less than 0.01), in IgG antibodies toward whole cat extract, Fel d I and cat albumin (p less than 0.001), and in IgE antibodies toward whole cat extract, (p less than 0.01). Thus, a decrease in skin sensitivity to cat extract demonstrated by both intradermal and prick methods occurred in patients after 1 year of immunotherapy with cat extract at a time when bronchial sensitivity to cat extract was decreased and IgG and IgE antibodies toward cat extract were increased.

Allergens↗
PubMed1989

Responses to ragweed-pollen nasal challenge before and after immunotherapy.

To evaluate whether immunotherapy reduces mediator release after nasal challenge, we followed previously untreated patients with ragweed hay fever through 2 years (three seasons) of treatment. Eleven adult patients started immunotherapy after a season of symptom diaries and graded pretreatment challenges with 0.03, 0.3, 3.3, and 16.5 mg of ragweed pollen. Repeat challenges were performed when the treatment dosage reached 0.6, 12.4, and 24.8 micrograms of Amb a I (antigen E) equivalents per injection. After the 0.6 micrograms dose, there was little change, but after the larger doses, there was a significant reduction in histamine and tosylarginine methyl ester-esterase (TAME-esterase) release in two respects. More pollen was required before any mediator appeared, and the amount of mediator released at each stage of the challenge was reduced. There was no significant difference between the responses at the 12.4 and 24.8 micrograms treatment doses. Sneezing after challenge was not apparently changed after immunotherapy; however, patients' seasonal symptom-medication scores were reduced after treatment. These data set the optimal individual treatment dose of ragweed extract for immunotherapy at greater than 0.6 micrograms, but probably not more than 12.4 micrograms in terms of Amb a I equivalents.

Adolescent↗
PubMed1989

Task-related variation in airborne concentrations of laboratory animal allergens: studies with Rat n I.

To define airborne allergen exposure during various tasks with rats in a laboratory, concentrations of allergen Rat n I were measured by radioimmunoassay in extracts from filters in personal air sampling devices that were worn by laboratory workers while they were performing these tasks. The tasks included feeding, cage cleaning, handling, injection, surgery, and sacrifice. Median concentrations encountered during feeding or cleaning (21 ng/m3) and injection or handling (13 ng/m3) were higher than those associated with surgery or sacrifice (3.1 ng/m3; p less than 0.01). Area samples in animal-holding rooms contained 3.4 ng/m3 during animal handling and 2.3 ng/m3 at other times. Very low concentrations were found in air outside the handling room, in unused laboratories, or outside air. We concluded that certain tasks incur a higher risk of allergen exposure but that exposure may occur anywhere within an animal laboratory environment.

Air Pollutants, Occupational↗
PubMed1989

Association of class II DNA restriction fragments with responsiveness to Ambrosia artemisiifolia (short ragweed)-pollen allergen Amb a V in ragweed-allergic patients.

Human IgE and IgG antibody responsiveness to the short ragweed-pollen allergen Amb a V (formerly known as Ra5) has been found to be strongly associated with HLA-D specificities Dw2 and DR2 in ragweed-allergic white individuals. To study the molecular basis of these associations, restriction fragment length polymorphism (RFLP) mapping was performed on a group of 45 white ragweed-allergic patients with full-length HLA-DR beta, DQ beta, and DQ alpha cDNA probes. The data on 41 of these subjects were used for the purposes of statistical analysis. With the DR beta probe, we found that the presence of three polymorphic restriction fragments correlated with responsiveness to Amb a V and with the DR2 specificity, namely, a 6.5 kb Eco RI fragment, a 9.4 kb Hind III fragment, and a 2.2 kb Hind III fragment. The presence of four fragments detected with the DQ beta probe correlated with responsiveness to Amb a V and with Dw2 specificity: a 2.3 kb Eco RI fragment, a 13.0 kb Pst I fragment, a 2.9 kb Taq I fragment, and a 5.2 kb Eco RV fragment. The DR beta Eco RI 6.5 kb and the DQ beta Eco RI 2.3 kb fragments were studied in detail; the concordant presence of these fragments was even more strongly associated with responsiveness to Amb a V. Fifteen of 17 responders had both fragments, whereas only one of 24 nonresponders had both fragments (p = 5 x 10(-7). This is the first time that such an association has been found between a person's immune response to a well-defined antigen and a set of HLA class II DNA restriction fragments.

Adult↗
PubMed1989

Human immune responsiveness to Lolium perenne pollen allergen Lol p III (rye III) is associated with HLA-DR3 and DR5.

A well-characterized allergen of Lolium perenne (perennial rye grass) pollen, Lol p III, has been used as a model antigen to study the genetic control of the human immune response. Associations between HLA type and IgE or IgG antibody (Ab) responsiveness to Lol p III were studied in two groups of skin-test-positive Caucasoid adults (N = 135 and 67). We found by nonparametric and parametric analyses that immune responsiveness to Lol p III was significantly associated with HLA-DR3 and DR5. No association was found between any DQ type and immune responsiveness to Lol p III. Geometric mean IgE or IgG Ab levels to Lol p III were not different between B8+, DR3+ subjects and B8-, DR3+ subjects, showing that HLA-B8 had no influence on the association. Lol p III IgG Ab data obtained on subjects after grass antigen immunotherapy showed that 100% of DR3 subjects and 100% of DR5 subjects were Ab+. A comparison of all the available protein sequences of DRB gene products showed that the first hypervariable region of DR3 and DR5 (and DRw6), and no other region, contains the sequence Glu9-Tyr-Ser-Thr-Ser13. Our observations are consistent with the possibility that immune responsiveness to the allergen Lol p III is associated with this amino acid sequence in the first hypervariable region of the DR beta 1 polypeptide chain.

Adult↗
PubMed1989

Molecular genetics of human immune responsiveness to Lolium perenne (rye) allergen, Lol p III.

Lol p II and III are each about 11-kD protein allergens from the pollen of Lolium perenne (rye grass). We have found that human immune responses (IgE and IgG antibodies) to both proteins are significantly associated with HLA-DR3. In addition, the two proteins are cross-reactive with the antibodies in many human sera (about 84% human sera showed the cross-reactivity). We have determined greater than 90% of the amino acid sequences of the two proteins and found that they are at least 54% homologous. Berzofsky found that 75% of the 23 known T cell sites in various proteins had an amphipathic structure. Our analysis by the same method showed that both Lol p II and III have a major region of amphipathicity (at residues 61-67, Lol p III numbering) which might contain sites for binding to an Ia molecule and a T cell receptor. This region is identical between Lol p II and III, except for an Arg-Lys substitution, and could account, in part, for the DR3 association with responsiveness to both molecules. An interesting difference between the two proteins is that immune response to Lol p III is associated with DR5 (in addition to DR3), whereas no DR5 association is found in the case of Lol p II. One possibility is that Lol p III has an additional site which binds to the DR5 Ia molecule. Lol p III indeed has a second highly amphiphathic peptide, 24-30 (Lol p III 24 R P G D T L A 30), which is different and not amphipathic in Lol p II.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed1988

Immunotherapy for cat asthma.

In 22 patients with cat asthma who were highly sensitive to cat, we compared, double-blind, the effects of immunotherapy with cat-hair and dander extract (11 patients) with effects of placebo (11 patients). Patients were matched by the dose of the cat extract expressed in Food and Drug Administration (FDA) units of Fel d I (previously called cat allergen 1) required for end point reaction in intradermal skin test end point titration (STEPT), for in vitro leukocyte histamine release (LHR), and for the dose of cat extract producing a 20% fall in FEV1 (cat-extract PD20) in bronchoprovocation test. Patients were matched also for bronchoprovocation dose of methacholine producing a 20% fall in FEV1 (methacholine PD20). Patients were randomly assigned to one of two treatment groups. During immunotherapy, doses were increased to maintenance dose of 4.56 FDA units of Fel d I, or, if this were less, to the highest tolerated dose. Systemic reactions to cat-extract immunotherapy were mild and infrequent. Before and during immunotherapy, we measured (in FDA units of Fel d I) cat-extract PD20, cat-extract intradermal STEPT, cat-extract in vitro LHR, serum levels of cat IgG and cat IgE, and methacholine PD20. After they had received 1 year of immunotherapy, patients receiving cat extract, in comparison to patients receiving placebo, had decreased cat-extract PD20 (p less than 0.01), diminished responses to cat-extract intradermal STEPT (p less than 0.025), increased IgE antibodies toward cat extract (p less than 0.01), increased IgG antibodies toward cat extract, Fel d I, and cat albumin (p less than 0.001), but no significant change in cat-extract in vitro LHR or in methacholine PD20. We conclude that cat-extract immunotherapy was well tolerated, significantly decreased skin and bronchial responses to cat extract, and significantly increased IgE antibodies to cat extract and IgG antibodies to cat extract, Fel d I, and cat albumin.

Adult↗