Gregory B. Wilson

Immunofixation after electrofocusing: Improved method for specific detection of serum proteins with determination of isoelectric points I. Immunofixation print technique for detection of alpha-1-protease inhibitor

An improved method is described for direct localization of human serum proteins in polyacrylamide gel with simultaneous determination of their isoelectric points (pI). The technique employs isoelectric focusing in thin-layer polyacrylamide gels to separate the serum proteins and the pH gradient is read at 4 degrees C with a dual-membrane surface microelectrode. Subsequently, the desired proteins are localized by immunofixation in the gel or by immunofixation-printing onto cellulose acetate strips soaked in specific antiserum. No sectioning of the electrofocused gel is necessary, and the entire technique can be completed in less than 14 h. When this method is applied to the detection of the genetic variants of alpha-1-antitrypsin (alpha-1-protease inhibitor) (A1Pi system), the results indicate that it can be used to specifically localize serum proteins whose pIs differ by as little as 0.01 pH units. The resolution afforded is especially evident in the analysis of A1Pi M variants.

Studies on cystic fibrosis using isoelectric focusing. I. An assay for detection of cystic fibrosis homozygotes and heterozygote carriers from serum.

Extract: We have developed a standardized biophysical assay for the rapid detection of individuals homozygous or heterozygous for cystic fibrosis (C/F). The assay employs isoelectric focusing in thin layer polyacrylamide gels to analyze microliter quantities of whole serum for the presence of a C/F factor protein and for deletions in a group of proteins called proteins B, C, and D (Fig. 1). A pH 5–10 gradient is used (Fig. 2) and each sample is screened using a serum volume which contains 300 μ g immunoglobulin G (IgG). Individuals homozygous or heterozygous for C/F are distinguished from normal unaffected individuals on the basis of the presence of a C/F factor protein band (Table 1). Heterozygous carriers for C/F are distinguished from C/F homozygotes 75% of the time, on the basis of a deletion in either band B, C, or D (Table 2).On the basis of screening 65 patients with cystic fibrosis. 61 heterozygous carriers for C/F, and 105 normal control subjects, it was concluded that no obvious correlation existed between either sex, age, or severity of the disease in the individual C/F patient, and the absolute presence or absence of the C/F factor. In addition, no correlation existed between sex or age and the presence of the C/F factor or deletions in proteins B, C, and D in the individual heterozygous carrier for C/F or normal control subjects. Analysis of serum samples from 68 patients with a variety of other diseases, many with clinical symptoms resembling those seen in the patient with cystic fibrosis (Table 3), indicated that the C/F factor protein described in this study appears to be diagnostic for C/F genotypes, with the possible exception of patients with certain types of leukemia.Speculation: The biophysical assay described in this report or a modification of it may prove to be a useful method for the routine detection of carriers of cystic fibrosis in the general population.

Fatal infantile cardiac glycogenosis with phosphorylase kinase deficiency and a mutation in the γ2-subunit of AMP-activated protein kinase

A 10-wk-old infant girl with severe hypertrophy of the septal and atrial walls by cardiac ultrasound, developed progressive ventricular wall thickening and died of aspiration pneumonia at 5 mo of age. Postmortem examination revealed ventricular hypertrophy and massive atrial wall thickening due to glycogen accumulation. A skeletal muscle biopsy showed increased free glycogen and decreased activity of phosphorylase b kinase (PHK). The report of a pathogenic mutation (R531Q) in the gene (PRKAG2) encoding the γ2 subunit of AMP-activated protein kinase (AMPK) in three infants with congenital hypertrophic cardiomyopathy, glycogen storage, and “pseudo PHK deficiency” prompted us to screen this gene in our patient. We found a novel (R384T) heterozygous mutation in PRKAG2, affecting an arginine residue in the N-terminal AMP-binding domain. Like R531Q, this mutation reduces the binding of AMP and ATP to the isolated nucleotide-binding domains, and prevents activation of the heterotrimer by metabolic stress in intact cells. The mutation was not found in DNA from the patients father, the only available parent, and is likely to have arisen de novo. Our studies confirm that mutations in PRKAG2 can cause fatal infantile cardiomyopathy, often associated with apparent PHK deficiency.

Studies on cystic fibrosis using isoelectric focusing. II. Demonstration of deficient proteolytic cleavage of alpha2-macroglobulin in cystic fibrosis plasma.

Extract: A protein with an isoelectric point (pI) of 5.48 was found to be deficient in plasma from most cystic fibrosis (CF) homozygotes and obligate heterozygote carriers for CF as compared with normal control plasma. Purification of the protein with a pI of 5.48 from normal plasma was performed using ammonium sulfate precipitation, DEAE-cellulose and CM-cellulose chromatography, Sephadex G-200 gel filtration, starch block clectrophoresis, and Sepharose 4B gel filtration. The purified protein migrated as a single band on polyacrylamide gel electrophoresis, and displayed a single arc on immunoelectrophoresis against polyvalent antiserum to whole human serum. Results from various techniques used in its characterization indicate that this protein is a fragment of α2-macroglobulin (α2M) which is derived from α2M by proteolytic cleavage of intact α2M subunits. Quantitation of α2M levels in plasma indicated no significant differences between levels of α2M in CF homozygote, obligate heterozygote carrier, or normal control plasma samples. Quantitation of arginine esterase activity in plasma treated with chloroform and ellagic acid indicated that both the total arginine esterase activity and that fraction of arginine esterase activity inhibited by soybean trypsin inhibitor (SBTI) were decreased in most CF homozygote and obligate heterozygote plasma samples relative to normal control values. The results of this study indicate that plasma samples from CF homozygotes and obligate heterozygote carriers for CF show deficient proteolytic cleavage of α2M as compared with normal control plasma, and suggest that a structural abnormality in α2M or a deficiency in plasma proteolytic activity may be responsible for this deficiency in proteolysis.Speculation: An abnormality in the binding affinity of α2M for plasma proteases may account for the presence of “factors” in CF homozygotes and obligate heterozygote carriers.

alpha-1-antitrypsin (Pi) phenotypes in a Finnish population.

alpha-1-antitrypsin (Pi) phenotypes of 548 normal Finnish blood donors were determined by isoelectric focusing in polyacrylamide gel. The frequencies obtained (M, 93.3%; FM, 0.4%, GM, 0.2%; MS, 3.5%; MZ, 2.7%) differed from those reported earlier for a more restricted Finnish population but agreed better with the frequencies reported for other Scandinavian populations. The disagreement with the earlier report was attributed to differences in sampling and laboratory techniques. The present study confirms an earlier finding of a sarcity of fast Pi variants in the Finnish population. One M variant was found. The isoelectric focusing technique used in this study has several advantages over the methods previously described for Pi typing.

Mycobacterium fortuitum pulmonary infection associated with an antigen-selective defect in cellular immunity.

In this study we describe the first example of a well documented case of pulmonary infection caused by Mycobacterium fortuitum shown to be associated with an antigen-selective defect in cell-mediated immunity to this organism. Immunologic parameters were evaluated before, during and after antibiotic treatment with amikacin. A defect in cellular immunity to purified protein derivative from Myco. fortuitum, shown to be antigen-selective as indicated by normal responsiveness to purified protein derivative from Mycobacterium tuberculosis and several other common recall antigens, accompanied the prolonged infection by this organism. During the first three months of treatment with amikacin, the patients clinical status improved coincident with the eradication of the organism from the sputum. During the next three months of therapy with amikacin, however, a generalized defect in cellular immunity developed, and the lung disease again progressed. The deteriorating clinical condition was presumably related to a generalized cellular immune anergy or hyporesponsiveness induced by the amikacin therapy. After three more months of treatment, the organism became resistant to the drug and reappeared in sputum cultures. Since amikacin therapy was discontinued, the patients general immune responsiveness returned to normal. He did, however, remain unresponsive to purified protein derivative from Mycobacterium fortuitum.

Treatment of Mycobacterium fortuitum pulmonary infection with “transfer factor” (TF): New methodology for evaluating TF potency and predicting clinical response☆

The successful clinical use of dialyzable leukocyte extracts (DLE) containing transfer factor (TF) has been hampered in the past by the lack of an in vitro assay for TF activity. We have shown previously that the leukocyte migration inhibition (LMI) assay can be used to detect and quantitate TF in DLE in vitro. The development of an in vitro assay for TF has in turn permitted the development of a new method for determining the TF potency in different preparations of DLE. We report herein the first application of the LMI assay to the clinical use of DLE. As an example we present a case study in which DLE was successfully employed to treat a patient with pulmonary disease associated with an “antigen-selective” defect in cell-mediated immunity to Mycobacterium fortuitum. The clinical applications of the LMI assay illustrated in this report include: (1) documentation of the antigen-selective defect in cell-mediated immunity; (2) selection of donors for the preparation of DLE for immunotherapy; (3) determination of the TF potency of different DLE preparations; (4) prediction of the patients in vivo response to DLE therapy; (5) determining dosages of DLE for immunotherapy and immunoprophylaxis; and (6) monitoring the patients response to treatment with DLE.

Letter to the Editor: Cystic Fibrosis Protein, a Confirmed Diagnostic Marker for Detecting Heterozygote Carriers: Significance in Relation to Future Screening and to a Proposed Prijnary Defect in Alpha 2 -Macroglobulin

Letter to the Editor: Cystic Fibrosis Protein, a Confirmed Diagnostic Marker for Detecting Heterozygote Carriers: Significance in Relation to Future Screening and to a Proposed Prijnary Defect in Alpha 2 -Macroglobulin

Ciliary dyskinesia factors in cystic fibrosis and asthma

CYSTIC fibrosis (CF) homozygotes and heterozygotes carriers for CF harbour a factor in their serum (termed CDF) which causes ciliary dyskinesia when applied to ciliated epithelial tissue derived from rabbit trachea1–4. Spock et al.1 reported that serum ciliary dyskinesia activity (CDA) was a specific marker for the CF gene. Conover et al.5 have reported that a CDA is also found in sera from individuals with various respiratory and autoimmune disorders; they have since suggested that the CF-ciliary dyskinesia factor was C3a (anaphylatoxin), and proposed that a defect or deficiency in the anaphylatoxin inactivator was the primary gene defect in cystic fibrosis6,7. Other laboratories have been unable to detect CF-CDF using a rabbit tracheal bioassay for the detection of the CF-CDF (refs 8–10). Using a modified rabbit tracheal bioassay, we have confirmed that serum from individuals with bronchial asthma and serum from homozygotes and heterozygotes for CF cause ciliary dyskinesia, whereas normal healthy control sera do not. Most individuals with asthma, however, were also found to have serum which causes a subsequent ciliostasis. All sera tested by bioassay for CFP by electrofocusing indicated that all CDA-positive sera except sera from seven of eight individuals with asthma were also positive for cystic fibrosis protein (CFP). The CFP is a small (molecular weight 5,000–9,000), positively charged compound and is found in sera from most homozygotes and heterozygotes for CF (refs 11–16). These two findings suggested that the CDAs in CF sera and in asthmatic sera could be due to two different substances. We have developed two simple chromatographic techniques which can separate CDAs in CF and asthmatic sera. Our results indicate that they may be useful in purifying a CF-specific CDA. Purification of this CF-CDA is a prerequisite for its biochemical characterisation and for elucidating its role in the pathophysiology of CF (ref. 17).

Improved method for detection of cystic fibrosis protein in serum using the LKB multiphor electrofocusing apparatus.

Summary: We have developed an improved method for the detection of cystic fibrosis protein (CFP). The method employs the LKB Multiphor to electrofocus whole serum, instead of the apparatus used in previous studies. Two basic modifications were necessary: (1) a pH 2.5–10.0 gradient instead of a pH 5–10 gradient, and (2) constant power for focusing the serum proteins instead of constant voltage. The first modification ensured adequate dissolution of the CFP-IgG complexes (or other precursor complexes which may liberate CFP). The second modification ensured a linear gradient (between pH 3.8 and pH 9.2), excellent resolution in the pH 8–9 region, and the separation of CFP within 2 hr without overheating of the gel.Electrofocusing with the LKB Multiphor permits the detection of CFP in as many as 24 serum samples per gel. Results obtained from the analysis of 31 cystic fibrosis, 28 obligate heterozygote carrier, and 28 normal control sera indicate that CFP can be reproducibly and accurately detected in sera using the LKB Multiphor.Speculation: Strict adherence to our methods of serum sample collection, processing, storage, and the methods described for the detection of CFP using the LKB Multiphor electrofocusing apparatus may ensure the successful use of our technique to detect CFP and to determine individuals with the CF gene.