Use of multivariate statistical methods to identify immunochemical cross‐reactants

Quantitative competition immunoassays with appropriate combinations of antibodies give consistent dose‐response patterns which may be used to identify and estimate amounts of cross‐reacting compounds. Previously reported methods of analyzing cross‐reaction patterns include multiple regression, principal components analysis and minimum estimates of variance (MEV). Four other techniques which are preferable in theory have been surveyed: discriminant analysis (DA), maximum likelihood estimates (MLE), classification and regression trees (CART), and computational neural networks (NN). MLE and simple back‐propagation neural networks can estimate the concentration, as well as the identity, of individual compounds. These four methods worked well with unfitted, unscaled data from monoclonal assays of triazines, phenylureas and avermectins. Immunoassays must be properly designed to provide adequate data for pattern recognition. Cross‐reactivity pattern analysis will make multi‐analyte, multi‐antibody immunoassays feasible for many applications in toxicology and hazard assessment.     

Molecular characterization of severe α-Thalassemias causing hydrops fetalis in Taiwan

α-Thalassemia hydrops fetalis is a common disorder in Taiwan. The condition causes perinatal death and many maternal obstetrical complications. In order to determine the molecular defects of this condition in Chinese, 87 unrelated families with this disorder were collected in the past 4 years. The molecular defects were studied by Southern blotting and DNA hybridization with φζ1-globin gene and LO (a 0.4 kb BamHI/EcoRI fragment in the 5′ flanking region of the ζ2-globin gene) probes. Eighty-one (93.1%) fetuses had homozygous Southeast Asian deletion (– –^SEA/– –^SEA). Five (5.7%) fetuses were compound heterozygotes for the Southeast Asian deletion and Thailand deletion (– –^SEA/– –^THAI). The remaining fetus was a compound heterozygote for the Southeast Asian deletion and an uncharacterized nondeletional defect (– –^SEA/(αα)Th). The molecular defects of α-thalassemia hydrops fetalis in Chinese are heterogeneous. This fact has important implications for genetic counseling and prenatal diagnosis.     

Alterations in the genomes of avian sarcoma viruses

We have identified polypeptides specific to region Elb (map position [mp] 4.6–112) of adenovirus 2 (Ad2) that are synthesized in six lines of Ad-transformed rat or human cells (F17, F4, T2C4, 8617, 5RK clone I, 293), and in Ad2 early infected KB cells. [³⁵S]Methionine-labeled polypeptides were immunoprecipitated using antisera against F17 cells, an Ad2-transformed rat cell line that retains only El. To determine whether they are viral coded, these polypeptides were compared by tryptic peptide mapping with polypeptides translated in vitro from Ela-specific mRNA (mp 1.3–4.5) and Elb-specific mRNA. Polypeptides of 19,000 daltons early infected KB cells. The 19K, 20K, and 53K could be translated from Elb-specific mRNA and thus are coded by Elb. The 19K was precipitated from all transformed cell lines, the 20K was immunoprecipitated from F4, 8617, and T2C4 cells, and the 53K was immunoprecipitated from F4, 8617, T2C4, and 293 cells. These results suggest that the 19K, and perhaps the 20K and 53K, may be important in adenovirus-induced cell transformation. The 20K and 53K share methionine-containing tryptic peptides with each other, but not with the 19K. These results, together with the Ad2 Elb DNA sequence (T. Gingeras and R. Roberts, personal communication), suggest that 19K is translated in a different reading frame from 53K and 20K.     

C3d antiglobulin haemagglutination of human red blood cells. A demonstration of two types of cell-bound C3d by means of typsin digestion.

Washed human red blood cells from blood collected in EDTA were tested by Auto-Analyzer for a percentage of maximum antiglobulin haemagglutination (AH) using monospecific antisera to human C3d and C3c. The cells from normal persons were found to be agglutinated by anti-C3d but not by anti-C3c. To a fixed dilution of antiserum, the normal C3d AH values (X +/- 2 SD) were 34 +/- 19% for adult cells (n = 29) and 14 +/- 19% for cord cells (n = 19); the difference was significant (P less than 0.0001). By pretreatment of these cells with trypsin the C3d AH was either completely abolished or markedly reduced. Its difference between the adult and cord cells was eliminated as the observed values were 4 +/- 7% and 3 +/- 4% respectively (P = 0.15). The supernatant fluid of cell-trypsin mixture, treated with trypsin inhibitors, was found to be inhibitory to C3d AH but not to C3c AH. In contrast, the AH of C3d-coated red blood cells resulting from complement fixation in vivo (ie, cold agglutinin disease) or in vitro (eg, sucrose water reaction) was resistant to trypsin treatment. The difference between the trypsin-sensitive and trypsin-resistant cell-bound C3d is postulated to be at its attachment mechanism to the cell membranes. In addition, both the advantage and limitation of using trypsinised cells for C3d antiglobulin tests are demonstrated.     

Number of C-bands of human isochromosome Xqi and relation to 45,X mosaicism

With combination of C and G banding techniques, three morphologically different types of isochromosome for the long arm of X (Xqi) have been identified, i.e. those with one C-band and symmetrical banding patterns of both arms, those with two C-bands and symmetrical banding patterns of two arms, and those with two C-bands but with asymmetrical banding patterns of two arms. The last type is a heterogeneous group with various different asymmetrical patterns. We have studied 6 cases of Xqi with C, G, and Q banding: 3 showed one C-band and symmetrical arms and all these were without 45,X mosaicism; the other 3 cases showed two C-bands, 2 of the cases having symmetrical arms and being mosaic for 45X/46,XXqi/47,XXqiXqi (those 2 were a pair of identical twins). One other had asymmetrical arms and was mosaic for 45,X/46,XXqi. Including our 6 cases, there have been a total of 30 reported cases of Xqi with C and G banding studies. Two-thirds of Xqi's were found to have 2 C-bands and one-third to have 1 C-band. Mosaicism was found in 85% of Xqi's with 2 C-bands and in only 44% of Xqi's with 1 C-band. Apparently, Xqi's with 2 C-bands have a greater tendency for anaphase lag and mitotic nondisjunction. Several possible mechanisms for the formation of the different types of Xqi's are discussed.     

Plurihormonal pituitary adenomas: immunostaining of all pituitary hormones is mandatory for correct classification

AIMS: We studied the clinicopathological characteristics of plurihormonal pituitary adenomas. METHODS AND RESULTS: The study material included 167 plurihormonal adenomas, which consisted of 31% of the surgically removed pituitary adenomas that we collected during a 12-year period. The mean age of patients with plurihormonal adenoma was 45.7 years (range 13-75 years). There were 86 men and 81 women. All tumours were fully classified by immunohistochemical staining for seven pituitary hormones or subunits. Thirty immunohistochemical subtypes of plurihormonal adenomas were recognized. Hormonal symptoms were present in 70% of patients, while serum hormonal levels were increased in 89% of patients. Most patients had symptoms related to only one of the hormones and only 7% of patients had symptoms related to two hormones. The most common hormonal symptom was acromegaly (50%); symptoms related to hyperprolactinaemia ranked second (20%). Double immunostaining of all the possible combinations of the hormones was performed in 30 selected tumours, and they all showed mixtures of hormones in individual adenoma cells in any hormonal combinations studied. The latter finding supported the view that plurihormonal adenomas are monomorphous adenomas. CONCLUSIONS: Plurihormonal adenomas are common pituitary adenomas. Immunohistochemical staining of all pituitary hormones is mandatory for correct classification.     

Enzymatic characterization of Aeromonas hydrophila complex by the API ZYM system

Enzymatic characterization of 48 Aeromonas hydrophila complex isolates from various sources was determined with the API ZYM system (Analytab Products, Plainview, N.Y.). All isolates lacked valine and cystine aminopeptidases, chymotrypsin, alpha-mannosidase, alpha-fucosidase, alpha-galactosidase, and beta-glucuronidase but possessed caprylate esterase-lipase, leucine aminopeptidase, acid phosphatase, phosphoamidase, and N-acetyl-beta-glucosidase. Variability was found in the presence of alkaline phosphatase, butyrate esterase, myristate lipase, trypsin, beta-galactosidase, alpha-glucosidase, and beta-glucosidase. No significant differences were evident among the enzymatic profiles of isolates from various sources.     

Human leukaemic T cells with complement receptors

Peripheral blood lymphocytes from a patient (EP) with lymphosarcoma cell leukaemia reported previously, had been found capable of forming sheep erythrocyte rosettes, reacting with T cell-specific antiserum and carrying surface immunoglobulins (Ig), IgM and IgD. It was suggested that the surface Ig were generated by leukaemic T cells due to activation of genes controlling synthesis of surface Ig. We here present evidence that these lymphocytes also carry complement receptors of B cells as detected by bovine erythrocyte–anti-bovine serum–complement complexes and by complement-coated zymosan. This study firmly establishes the presence of dual surface markers for T and B cells on the same leukaemic lymphocytes.     

Filamentous tau in oligodendrocytes and astrocytes of transgenic mice expressing the human tau isoform with the P301L mutation

We recently reported a transgenic mouse line (JNPL3) that expresses mutant (P301L) tau and develops neurofibrillary tangles composed of filamentous tau aggregates. Here we show that these mice have abnormal tau filaments not only in neurons, but also in oligodendrocytes and astrocytes. Similar results were detected in another transgenic line (JNPL2+3+) that expresses the longest human tau isoform with the P301L mutation. The ultrastructure of the tau filaments and immunoreactivity with tau and ubiquitin antibodies were similar in glia and neurons. Given similarities of the lesions in the mice to human neuronal and glial inclusions, these transgenic mice appear to be a valuable model to study pathogenesis of the neurodegenerative tauopathies.     

Isolation and characterization of recombinant Escherichia coli clones secreting a 24-kilodalton antigen of Treponema pallidum.

Escherichia coli clones containing Treponema pallidum DNA in the pUC8 vector and secreting a 24-kilodalton antigen of T. pallidum have been isolated. Both syphilitic human and syphilis-immune rabbit sera reacted with the recombinant p24 antigen, indicating that an equivalent protein in T. pallidum is capable of eliciting antibody responses during natural infections. The p24 antigen of T. pallidum was identified by using two-dimensional gel electrophoresis and immunoblotting with monospecific anti-p24 serum. We tentatively concluded that this cloned antigen is a secreted protein or a labile or minor component of T. pallidum because (i) p24 was secreted by the recombinant E. coli cells; (ii) recombinant p24 in E. coli cells was processed into several smaller species with molecular masses ranging from 12 to 20 kilodaltons, which correlate well with the masses of secreted antigens described by others; and (iii) p24 protein appeared to be highly antigenic during natural infections, but only a very small amount of this antigen was associated with or retained by the purified organisms. The possible role of the p24 protein in determining the growth characteristics of T. pallidum is suggested by the ability of recombinant p24 to induce growth changes in E. coli cells. All E. coli colonies expressing the p24 polypeptide exhibited a flat and rough colony morphology and a filamentous growth pattern that were different from those of other E. coli cells. The DNA sequence coding for the p24 polypeptide is located on a 1.7-kilobase-pair BamHI fragment of the T. pallidum genomic DNA and is absent in the nonpathogenic Treponema phagedenis DNA. However, any possible relationship between the p24 antigen and the virulence of T. pallidum remains to be determined. In preliminary studies, rabbits immunized with the purified p24 were not protected from the infection with live T. pallidum organisms.