High frequency of nonclassical steroid 21-hydroxylase deficiency

Nonclassical steroid 21-hydroxylase deficiency is an autosomal recessive disorder that is defined by clinical and hormonal criteria that distinguishes it from the classical 21-hydroxylase deficiency. No estimates of the gene frequency of nonclassical 21-hydroxylase deficiency, also called attenuated, late-onset, acquired, and cryptic adrenal hyperplasia, have been published thus far. Here, we have used HLA-B genotype data in families containing multiple members affected with nonclassical 21-hydroxylase deficiency together with the results of quantitative hormonal tests to arrive at estimates of gene and disease frequencies for this disorder. We found nonclassical 21-hydroxylase deficiency to be a far more common disorder than classical 21-hydroxylase deficiency, which occurs in 1/8,000 births. The prevalence of the disease in Ashkenazi Jews was 3.7%; in Hispanics, 1.9%; in Yugoslavs, 1.6%; in Italians, 0.3%; and in the diverse Caucasian population, 0.1%. The gene for nonclassical 21-hydroxylase deficiency is in genetic linkage disequilibrium with HLA-B14 in Ashkenazi Jews, Hispanics, and Italians, but not in Yugoslavs or in a diverse, non-Jewish, Caucasian group. The penetrance of nonclassical 21-hydroxylase deficiency gene in the HLA-B14 containing haplotypes was incomplete. Thus, nonclassical 21-hydroxylase deficiency is probably the most frequent autosomal recessive genetic disorder in man and is especially frequent in Ashkenazi Jews, Hispanics, Italians, and Yugoslavs.     

Identification and characterization of genus-specific epitopes of Serpulina species using monoclonal antibodies

Four murine monoclonal antibodies (mAbs) designated as C9E8, A10, G12, and G8 which recognized both Serpulina hyodysenteriae and S. innocens were produced and characterized. The mAbs reacted with whole cell antigens in ELISA, indirect immunofluorescence and immunoblot assays. The mAbs did not show any cross reactivity in rapid dot ELISA or immunoblot assay with Leptospira icterohemorrhagiae, Campylobacter jejuni and Escherichia coli. Treatment of whole cell suspension with proteinase K and sodium periodate indicated that the reacting epitopes of the mAbs were protein in nature. The genus-specific antigens were identified as heat-stable proteins with molecular weight in the range of 26 to 45 kDa. Immunofluorescence and immunogold labelling studies showed that the antibody-binding epitopes were exposed on the outer-surface of the spirochaetal cell wall. The mAbs inhibited growth of reference strains of both S. hyodysenteriae and S. innocens in vitro but failed to cause agglutination. The detection of spirochaetal forms directly in fecal smears or paraffin-embeded tissue sections from experimentally infected pigs indicated that such mAbs were potentially useful for the diagnosis of swine spirochaetosis. This is the first report of mAbs identifying and characterizing common antigens of porcine Serpulina.     

Characterisation of epitopes of type 1 fimbriae of Salmonella using monoclonal antibodies specific for SEF21 fimbriae of Salmonella enteritidis

Monoclonal antibodies (mAbs) were used to identify and characterise epitopes of type 1 (SEF21) fimbriae of Salmonella enteritidis. The distribution of the epitopes among salmonellas and other enterobacteria was investigated, as well as the influence of growth media and temperatures on their expression. At least four different epitope clusters were identified on SEF21 fimbriae of S. enteritidis. Two of these clusters were associated with fimbrial haemagglutinins that were either common to all salmonellae tested, or restricted only to S. enteritidis and S. dublin. The four epitope clusters were identified on type 1 fimbriae of most Salmonella serotypes, as well as non-haemagglutinating type 2 fimbriae of S. pullorum and S. gallinarum, and on many other enterobacterial species. The expression of the epitopes was affected by growth conditions.     

Conventional molecular diagnosis of steroid 21-hydroxylase deficiency using mismatched primers and polymerase chain reaction

We tested a conventional method based on polymerase chain reaction (PCR) and specific primers with one mismatched base at the 3' end to introduce restriction enzyme sites in order to detect mutations of the CYP21 gene without radioisotope. Using this method, the intron 2 mutation causing aberrant splicing of mRNA (In2G) and the exon 4 mutation (Ile->Asn, Ex4) in the CYP21 gene were analyzed. The nonsense mutation in exon 8 (Ex8NON) of the CYP21 gene was also investigated by PCR and subsequent restriction enzyme digestion. The mismatched primers successfully amplified the CYP21 gene containing the In2G and the Ex4 mutation sites, and the presence of these two mutations could be determined by restriction enzyme digestion after PCR. We used this new method to study 33 patients. Twenty-five of these patients were found to have at least one mutation (In2G and/or Ex4 mutation). By enzyme digestion after PCR, the Ex8NON mutation was also identified (7 out of 33 patients). In conclusion, we have developed a new method to detect point mutations in the CYP21 gene. This method was proved to be sensitive and rapid for the detection of the mutations studied. Therefore, this method is suitable for clinical genetic diagnosis.     

Numerous PCR-RFLPs within the porcine CYP21 (steroid 21-hydroxylase) gene

Mesenteric cysts are rare intra-abdominal tumors with an incidence around one case per 100,000 hospital admissions. The clinical presentation is variable; patients may be asymptomatic or present with either acute or chronic abdominal pain. Physical examination commonly demonstrates a smooth, round and mobile abdominal mass. Differential diagnosis includes any abdominal cyst or tumor. Laboratory tests are usually helpless. Ultrasonography and CT scans are the best diagnostic tools. The treatment of choice is the total resection of the cyst, which is regularly performed by open surgery. This paper reports a case of a mesenteric cyst successfully resected by laparoscopy, and addresses the possible uses of this approach.     

Specificities of anti-sialyl-Tn and anti-Tn monoclonal antibodies generated using novel clustered synthetic glycopeptide epitopes

The fine specificities of MAbs generated using novel synthetic clustered STn and Tn glycopeptides as immunogens were compared with the anti-TAG-72 antibodies B72.3 and CC49. Hapten inhibition experiments demonstrated the specificity of several of the MAbs for STn and Tn expressed on ovine submaxillary mucin and tumor derived MUC-1 mucin. Amongst the STn specific MAbs only the B195.3 MAb shows absolute dependence on the presence of sialic acid and specificity to the simple disaccharide NANAA alpha2-6-GalNAc. Identification of tumor associated carbohydrate epitopes in cluster and monomer configurations are possible using MAbs detecting the defined structure specificities described herein.     

False negative results observed in anti-thyroid peroxidase autoantibody determination by competitive radioimmunoassays using monoclonal antibodies

OBJECTIVE: Anti-thyroid peroxidase autoantibody (anti-TPO) and anti-thyroid microsomal antibody (anti-M) are strictly related, but discrepancies are sometimes observed. The aim of this study was to assess the incidence and to identify the causes of these discrepancies. DESIGN AND ANTIBODY MEASUREMENTS: Anti-M by passive hemagglutination and anti-TPO by two competitive monoclonal antibody-assisted radioimmunoassays (RIA-1 and RIA-2) were measured in 10,103 sera from 4232 subjects (663 male, 3569 female) screened for thyroid disease. RESULTS: Anti-TPO and anti-M correlated quite well (r = 0.7 and p < 0.0001 by RIA-1: r = 0.74 and p < 0.0001 by RIA-2), with discrepancies mostly limited to sera with low antibody titers. After exclusion of the latter samples, anti-TPO were detected in only 79 (1.4%) out of 5317 anti-M negative sera, but were undetectable in a more consistent proportion (130/2880 = 4.5%) of sera from patients with autoimmune thyroid disease and positive anti-M. In 61 sera of the latter group, anti-TPO was measured by a non-competitive RIA (RIA-3). Forty-one (67.7%) were positive by RIA-3, suggesting the presence of anti-TPO not competing with the monoclonal antibodies of RIA-1 and RIA-2. The remaining 20 sera had undetectable anti-TPO also by RIA-3. Nineteen (95%) of these sera had positive anti-thyroglobulin (anti-Tg) autoantibody and preincubation with thyroglobulin inhibited the agglutination reaction of anti-M tests. CONCLUSION: Anti-TPO by competitive monoclonal antibody-assisted RIA is negative in a minority of sera of patients with autoimmune thyroid disease and positive anti-M. This could be accounted for by anti-Tg producing false positives in the anti-M assay and by a subset of anti-TPO not competing with the monoclonal antibodies in the RIA. When autoimmune thyroid disease is suspected on clinical grounds, a negative anti-TPO test with a competitive RIA should be confirmed always by a non-competitive assay.     

Identification of novel drug resistance-associated proteins by a panel of rat monoclonal antibodies

It is well established that the rabbit corpus luteum (CL) function depends upon endogenous oestradiol, the major source of which in the rabbit ovary is considered to be the ovarian follicles. The absence of oestradiol formation by the rabbit CL has been previously reported. In a hyperstimulated pseudopregnant rabbit model used in our laboratory which developed a large number of corpora lutea in response to chorionic gonadotrophin (eCG)/hCG, we observed the survival of corpora lutea in vivo, and normal levels of plasma progesterone throughout pseudopregnancy (PP), despite the scarcity or the absence of follicles as a source of the luteotrophic hormone. Measurement of oestradiol in the plasma indicated that it was at high levels and correlated with the number of corpora lutea. This led us to investigate the luteal origin of oestradiol in this model. PP was induced in rabbits by i.m. injection of 200 IU eCG daily for 2 days followed on day 4 by i.m. injection of 200 IU hCG (day 0 of PP). Luteal tissue obtained at days 5, 9 and 12 of PP and cultured for 24 h synthesized oestradiol and testosterone in addition to progesterone. However, under the same conditions, follicles had limited capacity to secrete oestradiol. The presence of an aromatase activity in luteal tissue was confirmed when exogenous testosterone was added to the culture medium. P450aromatase (P450arom) mRNA was found in luteal tissue at days 5, 9 and 12 of PP. Small or large luteal cells, obtained by enzymatic digestion of the tissue followed by centrifugation in a Percoll density gradient, were cultured during several days with or without gonadotrophin or dibutyryl cAMP (dbcAMP). Both types of cells secreted oestradiol. In small cells and luteal tissue, aromatase activity was stimulated (1.5-2-fold) by hCG and dbcAMP. Large cells exhibited a greater capacity to aromatize testosterone than small cells, but aromatase activity was not modified by hCG or by dbcAMP. FSH had no effect on aromatase activity of either luteal cell type. This intrinsic luteal tissue aromatase capacity and the absence of premature regression of corpora lutea despite the limited support of follicular oestrogen, suggest an autocrine and luteotrophic role for this luteal oestrogen.     

Identification of a domain containing B-cell epitopes in hepatitis C virus E2 glycoprotein by using mouse monoclonal antibodies

BACKGROUND: Bacterial resistance to aminoglycoside antibiotics occurs primarily through the expression of modifying enzymes that covalently alter the drugs by O-phosphorylation, O-adenylation or N-acetylation. Aminoglycoside phosphotransferases (APHs) catalyze the ATP-dependent phosphorylation of these antibiotics. Two particular enzymes in this class, APH(3')-IIIa and AAC(6')-APH(2"), are produced in gram-positive cocci and have been shown to phosphorylate aminoglycosides on their 3' and 2" hydroxyl groups, respectively. The three-dimensional structure of APH (3')-IIIa is strikingly similar to those of eukaryotic protein kinases (EPKs), and the observation, reported previously, that APH(3')-IIIa and AAC(6')-APH(2") are effectively inhibited by EPK inhibitors suggested the possibility that these aminoglycoside kinases might phosphorylate EPK substrates. RESULTS: Our data demonstrate unequivocally that APHs can phosphorylate several EPK substrates and that this phosphorylation occurs exclusively on serine residues. Phosphorylation of Ser/Thr protein kinase substrates by APHs was considerably slower than phosphorylation of aminoglycosides under identical assay conditions, which is consistent with the primary biological roles of the enzymes. CONCLUSIONS: These results demonstrate a functional relationship between aminoglycoside and protein kinases, expanding on our previous observations of similarities in protein structure, enzyme mechanism and sensitivity to inhibitors, and suggest an evolutionary link between APHs and EPKs.     

Analysis of thermal stability of soya globulins using monoclonal antibodies

Previous research on spatial memory indicated that memories of small layouts were orientation dependent (orientation specific) but that memories of large layouts were orientation independent (orientation free). Two experiments investigated the relation between layout size and orientation dependency. Participants learned a small or a large 4-point path (Experiment 1) or a large display of objects (Experiment 2) and then made judgments of relative direction from imagined headings that were either the same as or different from the single studied orientation. Judgments were faster and more accurate when the imagined heading was the same as the studied orientation (i.e., aligned) than when the imagined heading differed from the studied orientation (i.e., misaligned). This alignment effect was present for both small and large layouts. These results indicate that location is encoded in an orientation-dependent manner regardless of layout size.     

Mapping immunoreactive epitopes in the human peripheral nervous system using human monoclonal anti-GM1 ganglioside antibodies

A series of monoclonal IgM anti-GM1 ganglioside antibodies has been cloned from peripheral blood lymphocytes of patients with multifocal motor neuropathy and Guillain-Barré syndrome. In solid-phase immunoassay, the antibodies react with GMI, and also in differing degrees to the structurally related glycolipids asialo-GM1 (GA1) and GD1b. Here we describe the binding patterns of six human anti-GM I antibodies to epitopes within the human nervous system. Antibodies were observed to bind to motor neurons and spinal grey matter, dorsal and ventral spinal roots, dorsal root ganglion neurons, nodes of Ranvier, neuromuscular junctions and skeletal muscle. The distribution of immunoreactive epitopes, which included sensory structures, extended beyond those sites conventionally regarded as pathologically affected in anti-GM1 antibody-associated motor nerve syndromes. This undermines a model of disease pathogenesis based solely on antigen distribution. Factors other than the presence or absence of antigen, such as the local ganglioside topography, antibody penetration into, and pathophysiological vulnerability of a particular site may also influence the clinicopathological outcome of anti-GM1 antibody-mediated autoimmune attack.     

Characterization of steroid 21-hydroxylase gene mutations in a oligosymptomatic form of congenital adrenal hyperplasia: family study

BACKGROUND: 21-hydroxilase deficiency accounts for over 90% of all cases of congenital adrenal hyperplasia (CAH). There is a non-negligible incidence of both severe and nonclassical forms of this genetic disorder. Enzyme deficiency is due to mutations in the gene encoding adrenal 21-hydroxylase (named CYP 21B) and is inherited in an autosomical recesive way. Complete or partial impairment of enzyme activity has been correlated with the different clinical forms of the disease. PATIENTS AND METHODS: In the present paper CYP 21B gene analysis results obtained in a family with two kindred affected by a nonclassical form of the disease are shown. Clinical assessment of these two kindred showed a very mild form of the disease, whereas biochemical results suggested a late-onset partial 21-hydroxylase deficiency. Genotyping for deletions and 10 point mutations in the CYP 21B gene was performed by Southern blot analysis and polymerase chain reaction (PCR) allele-specific oligonucleotide (ASO) hybridation technique. RESULTS: Molecular genetic analysis performed in the two affected patients and two further relatives allowed us to detect the presence of different mutations in the two alleles of the CYP 21B gene. One of these mutations was severe (655G) and came from maternal line, whereas the other was mild (Val281Leu) and originated in paternal line. CONCLUSION: Molecular genetic analysis allows the possibility of finding severe (and non-expected) mutations in patients with clinically mild and late-onset forms of the 21-hydroxylase deficiency.     

Two monoclonal antibodies from the platelet panel recognize sheep plasma fibrinogen

PCR techniques were applied for the detection of mycoplasma DNA and pestivirus RNA to 43 lots of live viral vaccines (measles, mumps, rubella, and oral poliomyelitis) produced by six manufacturers in Japan. Although mycoplasma DNA was not detected in any of the vaccines tested, pestivirus RNA was detected in 12 lots (28%). The incidence of contamination among the four viral vaccines was in the range of 20 to 37%, and the incidence among the six manufacturers varied from 0 to 56%.     

Passive protection of gnotobiotic calves using monoclonal antibodies directed at different epitopes on the fusion protein of bovine respiratory syncytial virus

Two neutralizing, fusion-inhibiting bovine monoclonal antibodies (MAbs; B4 and B13) directed at different epitopes on the fusion protein of respiratory syncytial virus (RSV) protected the lungs of gnotobiotic calves from RSV infection. The MAbs were administered intratracheally 24 h before the calves were challenged with bovine RSV. A third, nonneutralizing, non-fusion-inhibiting but complement-fixing MAb, B1, provided no significant protection against infection, and the disease was not exacerbated. Pneumonic consolidation and mean virus titer in lung 7 days after challenge were significantly lower in calves given the fusion-inhibiting MAbs than in either control calves or those given MAb B1. The proliferative bronchiolitis with syncytial formation and widespread distribution of RSV antigen in the lower respiratory tract of the B1-treated and control calves were indistinguishable and typical of experimental bovine RSV infection. Syncytia were markedly absent, and little or no viral antigen was detected in either the B4- or B13-treated calves.     

Production of monoclonal antibodies using a secretion capture report web

We describe a novel method for the production of monoclonal antibodies using a secretion capture report web (SCRW). Following HAT selection in bulk culture, individual hybridomas are encapsulated in biotinylated agarose drops. Antibody secreted by the hybridoma is captured within the agarose drop using an avidin bridge and biotinylated anti-mouse immunoglobulin. The secreted antibody is detected by a fluorescent reporter which can be either a second anti-mouse antibody or an antigen. The binding of the reporter can be quantitated and the desired hybridoma directly cloned by flow cytometry. Multiparameter (i.e., two-color) reporter analysis can also be used to selectively enrich and clone rare hybridomas secreting antibodies directed to unique epitopes. The method allows the characterization of thousands of clones per second and the isolation of hundreds of clones per day.     

Prediction of epitopes and production of monoclonal antibodies against gastric H,K-ATPase

Monoclonal antibodies (mAbs) were produced against gastric H,K-ATPase using a theoretical and experimental strategy based on prediction of linear epitopes by molecular modelling followed by production of anti-peptide antibodies. By analysing the alpha subunit sequence, we predicted several epitopes corresponding to amino acids K519-L533, E543-Y553 and S786-L798 and produced monoclonal antibodies HK519, HK543 and HK786. All three react against gastric H,K-ATPase in RaLISA, immunohistochemistry and Western blots demonstrating that they recognize the native and the SDS-denatured ionic pump and that the epitopes are located at the surface of the native ATPase. Antibody Kd are in the range 6-10x10(-8) M. Monoclonal antibody HK519 is a competitive inhibitor of ATP, in agreement with ATP binding to K519. Neither mAb 543, nor mAb 786 inhibit the ATPase activity. Monoclonal antibody 95111, whose epitope is mapped between residues C529 and E561, competes with mAb HK543 but not with the other two. We suggest that the 95111 epitope is overlapping or very close to the HK543-553 sequence. Induction of E1 conformer by binding FITC to K519 increases the number of mAb 95111 and mAb HK543 epitopes but not that of mAb 786, supporting the fact that the fragment E543-Y553 changes accessibility, maybe during the E1-E2 transconformation.     

Neutralizing monoclonal antibodies against listeriolysin: mapping of epitopes involved in pore formation

Six different mouse monoclonal antibodies (MAbs) and a specific rabbit polygonal antibody were raised against listeriolysin. Four of the MAbs also recognized seeligeriolysin, and five cross-reacted with ivanolysin. The hemolytic activity could be neutralized by the polygonal antibody as well as by five of the MAbs. None of the neutralizing antibodies interfered with the binding of listeriolysin to the cellular membrane. The epitopes recognized by the MAbs were localized by using overlapping synthetic peptides between positions 59 and 279, a region hitherto not implicated in mediating hemolytic activity.     

Production of mouse monoclonal anti-idiotypic antibodies specific to epitopes of tumor-associated mucin TAG-12

The tumor-associated mucin-glycoprotein TAG-12 is strongly expressed in approximately 96% of all breast cancer patients and nearly 68% of all ovarian cancers. The experimental results of this work indicated that humoral immune response against TAG-12 is possible. Immunization with anti-idiotypic monoclonal antibodies produces this response. In this experiment, anti-idiotypic monoclonal antibodies represent the internal image of a specific epitope on TAG-12. Monoclonal antibody (MAb) 12H12 was selected to produce anti-idiotypic antibodies (anti-Ids) because of its high reactivity with TAG-12. Syngeneic murine anti-Ids were developed by immunization of BALB/c mice with the 12H12-Fab-KLH conjugate. A competitive assay with purified TAG-12 was utilized to identify anti-Ids with mirror image function. Two MAbs with "internal image" specificity were selected, 5H8 and 5H2. Two New Zealand White rabbits were immunized with 5H8. Serum samples tested 6 weeks after the initial immunization showed comparable titers against TAG-12. The binding capacities of the rabbit sera to different human breast as well as nonbreast cancer cell lines demonstrated strong binding with TAG-12-positive breast cancer cell lines. Competitive inhibition assays demonstrate that Ab3 and purified TAG-12 totally inhibit the binding of 12H12 antibody to TAG-12-positive cells. No inhibition was detectable with unrelated MAbs or normal mouse immunoglobulin. Binding assays with polyclonal Ab3 serum and several human cancer cell lines showed reactivity to nearly every tested cell line. Soluble TAG-12 showed no inhibition, indicating that this binding is due to a different set of idiotypes. Anti-Id 5H8 elicited an immune response to TAG-12. Utilization of anti-Id as a vaccine against the breast cancer-associated tumor antigen TAG-12 was successfully demonstrated in a xenogeneic animal model.     

Naturally occurring mutants of human steroid 21-hydroxylase (P450c21) pinpoint residues important for enzyme activity and stability

Three mutants (deletion of E196, G291S, and R483P) of steroid 21-hydroxylase (P450c21) from patients with inherited congenital adrenal hyperplasia had reduced activity toward progesterone and 17-hydroxyprogesterone after transient expression in cultured mammalian cells. In addition, both the E196 deletion and the R483P mutant had shorter half-lives than the wild-type enzyme, whereas the half-life of the G291S mutant was comparable with that of the normal protein. These results directly link the clinical situation with the three mutations and suggest that G291 is important for the catalytic activity of P450c21.