Pneumocystis carinii organisms obtained from rats, ferrets, and mice are antigenically different.

Pneumocystis carinii infections were developed in animals immunosuppressed by dexamethasone treatment either from activation of latent infection (ferret) or by transtracheal inoculation with P. carinii-infected lung tissue from the homologous species (rat or mouse). Convalescent-phase antisera were obtained by stopping dexamethasone treatment after 2 to 4 weeks and allowing animals 5 to 8 weeks for recovery. P. carinii harvests from infected lungs were purified by differential filtration, solubilized in buffer containing urea, sodium dodecyl sulfate (SDS), and 2-mercaptoethanol, subjected to SDS-polyacrylamide gel electrophoresis, and blotted to polyvinylidene difluoride sheets for Western immunoblot analysis. These lung preparations are hereafter referred to as P. carinii antigens. Convalescent-phase antisera from each animal species were reacted on Western blots of P. carinii antigens prepared from organisms isolated from rats, ferrets, or mice. Each combination of P. carinii antigens and antisera from the same species of animal reacted with three or more P. carinii antigen proteins. Convalescent-phase mouse antisera reacted with P. carinii antigens from mice but not rats or ferrets. Convalescent-phase rat antisera reacted with P. carinii antigens from rats and mice but not ferrets, and convalescent-phase ferret antisera showed reactions with ferret and mouse P. carinii antigens but not rat antigens. These findings indicate antigenic differences among P. carinii strains infecting these animals.

Immunologic comparisons of Pneumocystis carinii strains obtained from rats, ferrets, and mice using convalescent sera from the same sources.

Pneumocystis carinii (Pc) infections were developed in animals immunosuppressed by dexamethasone treatment either from activation of latent infection (ferret) or trans-tracheal inoculation of Pc obtained from infected lungs of the homologous species (rat, mouse). Convalescent antisera were obtained by stopping dexamethasone treatment after 2-4 wk and allowing 5-8 wk for recovery. Parasites from infected lungs were purified by differential filtration, solubilized in loading buffer, subjected to sodium dodecyl sulfate- polyacrylamide gel electrophoresis, and blotted to polyvinylidene fluoride sheets for Western analysis. Antisera from each animal species were reacted on Western blots of antigens from rat, ferret, and mouse. Each combination of antigen and antibody from the same species of animal showed reaction with 5 or more bands of Pc antigen. Convalescent mouse antibody did not react with rat or ferret antigens. Convalescent rat antibody reacted with a mouse antigen at about 66 kDa but not with ferret antigen, and convalescent ferret antibody showed minimal, probably non-specific reactions with both rat and mouse antigens. Variations in reactions indicate antigenic differences in Pc strains infecting these animals.

Cloning and DNA sequence of the omc gene encoding the outer membrane protein-macromolecular complex from Neisseria gonorrhoeae.

The omc gene, encoding the outer membrane protein-macromolecular complex (OMP-MC), was cloned in two pieces from Neisseria gonorrhoeae 2686. The 5' fragment of the omc gene included a promoter sequence, as indicated by its unregulated expression in Escherichia coli. Attempts to reconstruct an intact omc gene were unsuccessful, suggesting that expression of the complete OMP-MC protein was toxic to E. coli. Complete sequence determination revealed a coding sequence of 2,133 nucleotides; the deduced amino acid sequence indicated a mature protein of 687 amino acids with an NH2-terminal signal peptide of 24 amino acids. Analysis of the deduced amino acid sequence revealed that the NH2-terminal half of OMP-MC is generally hydrophilic, while the COOH-terminal portion contains alternating hydrophobic and hydrophilic regions. Serological analyses demonstrated that the NH2-terminal portion of OMP-MC is exposed on the gonococcal surface and the COOH-terminal portion is membrane associated.

Accumulation of chlamydial lipopolysaccharide antigen in the plasma membranes of infected cells.

The presence of a chlamydia-specified antigen associated with the plasma membrane of infected cell lines was demonstrated by indirect immunofluorescence staining with a monoclonal antibody, designated 47A2, specific for the chlamydial genus-specific lipopolysaccharide (LPS) antigen. Staining of HeLa, L-929, and McCoy cells infected with the L2 or F serovar of Chlamydia trachomatis was observed either without fixation or following aldehyde fixation and brief drying. The 47A2-reactive antigen appeared to be present on the plasma membrane, on bleb-like structures on the host cell surface, and on proximal processes of neighboring uninfected cells. Antibodies to chlamydial protein antigens such as the major outer membrane protein produced no surface staining under similar conditions. Membrane vesicles elaborated from infected cells were enriched for the 47A2-reactive antigen. Superinfection of chlamydia-infected cells with vesicular stomatitis virus, an enveloped virus which buds from the plasma membrane, allowed purification of progeny virions that were enriched with chlamydial LPS. These results are consistent with the presence of chlamydial LPS in the plasma membranes of infected host cells.

Relative importance of estrogen and progesterone receptor assays as prognostic indicators in primary breast cancer: a short-term study.

This short-term study of the relative importance of estrogen and progesterone receptors shows that progesterone receptor correlates better than estrogen receptor with tumor recurrence regardless of lymph-node status. Life-table analysis has effectively identified only two groups of patients that may be classified by progesterone receptor status alone. Progesterone-receptor negativity correlated well with tumors of histological Grade III; estrogen-receptor positivity correlated with Grade I and II tumors. The earlier recurrence of Grade III breast tumors may explain why progesterone receptor is a better prognostic indicator than estrogen receptor in short-term studies.

Antigenic analysis of the major outer membrane protein of Chlamydia trachomatis with murine monoclonal antibodies.

We prepared monoclonal antibodies against prototype strains of the 15 serovars of Chlamydia trachomatis and identified a subset of reagents that reacted with the major outer membrane protein(s) (MOMPs) of one or more serovars. We then determined the specificities of these anti-MOMP monoclonal antibodies by radioimmunoassay and immunoblot assays against the 15 serovars of C. trachomatis and a C. psittaci strain. We identified 14 different anti-MOMP antibody specificities, including serovar-, several orders of subspecies-, and species-specific determinants. In addition, one antibody reacted with all C. trachomatis serovars and a C. psittaci strain, indicating the presence of a genus-specific epitope on MOMP. Many of the cross-reactions of the subspecies-specific antibodies were similar to those previously reported by use of the microimmunofluorescence technique. We also observed a number of cross-reactions that were unexpected but consistent with data derived by the microimmunofluorescence test. All antibodies, except the genus-specific antibodies, reacted with whole elementary bodies in a radioimmunoassay, suggesting surface exposure of the epitopes. These data confirm and extend previous observations that MOMPs among C. trachomatis serovars are antigenically complex and diverse. In addition, these data indicate that the cross-reaction patterns of some monoclonal antibodies directed against MOMP are similar to those detected by the microimmunofluorescence test and are consistent with the hypothesis that such determinants are contained within MOMPs.

Production of monoclonal antibody against Pneumocystis carinii by using a hybrid of rat spleen and mouse myeloma cells.

It has been difficult to obtain pure Pneumocystis carinii antigen either from cultures or from infected lungs for use in producing a specific antibody against P. carinii. This report describes an approach toward producing a monoclonal antibody that bypasses the antigen purification steps. P. carinii infection was developed in Sprague-Dawley rats by the method of immunosuppression with cortisone. The infected lungs were homogenized, and the homogenate was used to immunize Sprague-Dawley rats. Rat spleen cells were then fused with SP2/0 mouse myeloma cells. Hybridoma clones were screened for antibody production against P. carinii by immunoperoxidase staining techniques and by enzyme-linked immunosorbent assay, using as antigens homogenates of normal rat lung, homogenates of P. carinii-infected rat lung, and harvests of P. carinii grown with WI-38 cells. Out of six hybridoma clones obtained that produced antibodies against P. carinii, one was able to produce ascitic fluid. This monoclonal antibody reacted with two P. carinii antigens with masses of about 35,000 and 65,000 daltons in P. carinii-infected lungs and three proteins with masses of about 35,000, 65,000, and 110,000 daltons in P. carinii that was harvested from a WI-38 cell culture.

Serovar determination of Chlamydia trachomatis isolates by using type-specific monoclonal antibodies.

A panel of 15 monoclonal antibodies was prepared that could distinguish among the 15 serovars of Chlamydia trachomatis. Twelve of these antibodies were specific for a single serovar (A, B, C, D, E, F, G, H, I, K, L1, and L2) and three were specific for two serovars (B/Ba, C/J, and C/L3). Ten of the serovar-specific and two of the bispecific antibodies were shown by immunoblotting to recognize epitopes on the major outer membrane protein. These data provide evidence that such epitopes are closely correlated with and may be partly responsible for the antigenic variations detected by microimmunofluorescence that distinguish the currently recognized serovars. When used in a radioimmunoassay, these antibodies correctly identified the serovar of 17 strains that had been serotyped by the microimmunofluorescence test. In addition, we found that the chlamydial antigen derived from 1.0 cm2 of an infected HeLa cell monolayer was sufficient to allow serotyping with these antibodies. Thus, these monoclonal antibodies may provide a rapid and reliable alternative to mouse immunization and microimmunofluorescence for serotyping of clinical isolates.

Purification and antigenic relatedness of proteins II of Neisseria gonorrhoeae.

Gonococcal proteins II from three strains were purified by chromatofocusing, and antisera was raised against them. These antisera were examined by immunoblotting to explore the antigenic relatedness of proteins II of seven different strains. The strongest reactions of the antisera were with the homologous proteins II. The antiserum against the proteins II of one strain also reacted with the proteins II present in all of the heterologous strains, whereas the antisera against the proteins II of two other strains showed little cross-reactivity with heterologous proteins II. Monoclonal antibodies produced against the three proteins II of strain F62 were specific for homologous proteins II and recognized epitopes unique to each individual protein II. These studies confirm the extensive intra- and interstrain variability in the antigenic structure of these proteins.

External quality assessment of urinary pregnancy oestrogen assay: further experience in the United Kingdom.

Four years' experience with external quality assessment of urinary pregnancy oestrogen and creatinine assays using a revised scheme design, incorporating frequent distributions, individualised reports and running scores based on variance index scores, is described. During this period there was a rapid and substantial improvement in interlaboratory agreement, which appears attributable in part to these improvements in scheme design. The derivation and use of a new index, the SDBIS (standard deviation of the bias index score), which assesses variability of bias, are described. Investigations showed that urine-based calibration materials could improve between-laboratory agreement; there were only minor differences in performance between method groups.

The distribution among ophidian venoms of a toxin isolated from the venom of the Mojave rattlesnake (Crotalus scutulatus scutulatus).

A toxin analogous to Mojave toxin or protein K' was isolated from venom of the Mojave rattlesnake (Crotalus s. scutulatus) by anion exchange and gel permeation chromatography. This toxin has an apparent native molecular weight of 20,000-22,000, a subunit molecular weight of 14,000 and a pI of 4.9-5.0. The i.p. LD50 is 0.094 mg/kg for mice. A wide variety of ophidian venoms (crotaline, viperine, elapid, hydrophid and colubrid) were examined for the presence of this toxin using Ouchterlony, immunoelectrophoresis, ELISA and Western transfer. High concentrations were found in 4 of 6 C. scutulatus venom samples, 2 of 3 C. durissus samples and samples from C. viridis concolor and C. tigris. A moderate concentration was found in 1 of 3 C. durissus samples and low to trace concentrations in 1 C. durissus sample, 1 C. scutulatus sample, 2 of 12 C. atrox samples and a Trimeresurus flavoviridis sample, the latter being the only instance of detection of the toxin in a snake other than a rattlesnake. The toxin appears in at least two phylogenetic lines of rattlesnakes, and its geographic distribution in North American rattlesnake species resembles a mosaic.

Conservation of peptide structure of outer membrane protein-macromolecular complex from Neisseria gonorrhoeae.

The structural conservation of an outer membrane protein of Neisseria gonorrhoeae called OMP-MC (outer membrane protein-macromolecular complex) was investigated by determining the isoelectric point and amino-terminal amino acid sequence of the protein and by using high-performance liquid chromatography for comparative tryptic peptide mapping. The 76,000-dalton subunits generated by reduction and alkylation of the native 800,000-dalton complex from six test strains focused in ultrathin gels as bands of restricted heterogeneity at an approximate pI of 7.6. Dansyl chloride labeling indicated that all strains shared glycine as the amino-terminal amino acid. Sequence analysis of OMP-MC from two strains revealed no amino acid differences within the first 11 residues. Dual-label peptide maps revealed an extremely high degree of conservation of peptide structure. The results indicate that (i) OMP-MCs isolated from various strains of N. gonorrhoeae share structural homology and (ii) the 800,000-dalton complex is a homopolymer composed of 10 to 12 apparently identical 76,000-dalton subunits.

Evidence for the hormonal regulation of the multimolecular forms of serum alkaline phosphatase.

PIP: The relationship between serum alkaline phosphatase (AP) activity and hormonal status was investigated in normally menstruating women. oral contraceptive (OC) users, and in women attending an infertility clinic and in early and late pregnancy. Reliable measurement of small differences in serum AP is now possible as a result of improvements in faractionation, sample handling techniques, and precision in AP assays. The centrifugal analyzer method used in these studies gives a within-batch precision of 0.4% compared to the 4-5% precision obtained with eaarlier methods. The maximun divergence in levels of circulating AP was noted in the 30-35year age group, which corresponds to the peak 24 hour urinary excretion of estrogen between days 10-20 of the menstrual cycle. Only limited changes of liver-bone AP were demonstrated in the luteal and menstrual phases of the normal cycle, although a marked increase in intestinal AP was noted during menses. Serum AP increased during menses in OC users. The removal of suppression on AP produces an increase during days 21-27. Among women attending an infertility clinic, those with lower hormone levels had a wider range of serum AP activity and a greater incidence of intestinal AP. The finding that ptients with progesterone levels under 7.5 nmol/1 with normal levels of estradiol has raised serum AP levels suggests that progesterone may modulate the estrogenic effect on serum AP levels. Finally, an inverse relationship was found between intestinal AP and levels of estradiol and progesterone in different phases of the menstrual cycle and in early and late pregnancy. These results suggest that small or large increases in estrogen increase pinocytosis, which reduces the level of the circulating glycoprotein AP by cell binding.^ieng

An enzyme-linked immunoassay for detection of North American pit viper venoms.

We describe a method for immunodetection of North American pit viper venoms in clinical materials. Antibody-enzyme conjugates prepared against venoms of the western diamondback rattlesnake (Crotalus atrox), Mojave rattlesnake (Crotalus scutulatus), and copperhead (Agkistrodon contortrix) detect homologous venoms in concentrations of 0.1-.01 mcg/ml using a double antibody sandwich technique. Venoms of 10 additional species of U.S. pit vipers were detected in concentrations of 10 mcg/ml or less. Venoms of 4 species could not be detected at levels likely to be encountered in clinical situations. There are extensive cross-reactions between venoms of certain species, hence specific identification of a given venom cannot always be made. Venom usually can be detected at injection sites of experimental animals receiving intramuscular doses of 0.5-1.5 mg of venom but can rarely be detected in urine or plasma specimens. Venom was readily detected in specimens from experimental animals bitten by pit vipers of 6 species. The method is relatively rapid, simple, and inexpensive.

Strain distribution in extents of lysozyme resistance and O-acetylation of gonococcal peptidoglycan determined by high-performance liquid chromatography.

The extent of lysozyme resistance and O-acetylation of purified peptidoglycan (PG) from 20 strains of Neisseria gonorrhoeae was examined to determine how widespread these properties are among various subsets of gonococcal isolates. To determine digestibility by lysozyme, we treated [3H]- or [14C]glucosamine-labeled PG with hen egg white lysozyme (HEW-LZ) and determined the size distribution of HEW-LZ soluble PG at the completion of the reaction by molecular-sieve high-performance liquid chromatography, using a Varian TSK SW2000 column, a method that proved considerably more efficient than traditional chromatography for fractionating low-molecular-weight PG fragments solely on the basis of size. The extent of HEW-LZ resistance was expressed as the percentage of PG that was larger in size than disaccharide peptide tetramers (including insoluble PG removed by centrifugation). The percent O-acetylation was determined by converting insoluble PG totally to uncross-linked monomers by the combined action of Chalaropsis B muramidase followed by Escherichia coli endopeptidase and then quantitating radioactivity in O-acetylated and non-O-acetylated monomers after paper chromatography. The PG of the vast majority (19 of 20) of gonococcal strains examined was extensively HEW-LZ resistant (range, 40 to 60% larger than tetramers) and extensively O-acetylated (range, 34 to 52%). Only the PG of strain RD5 (highest rate of PG turnover among gonococci so far examined and the prototype of gonococci having O-acetyl-deficient PG) had greatly reduced O-acetylation (15%) and exhibited virtually no HEW-LZ resistance (2% larger than tetramers). Extensive HEW-LZ resistance and O-acetylation were apparently not associated specifically with (i) a given type of colonial variant (piliated versus nonpiliated or opaque versus transparent), (ii) a given type of clinical isolate (local versus disseminated), (iii) the extent of laboratory passage, or (iv) (with the possible exception of penicillin-resistant strain FA102) the presence of one or more genetic loci governing antibiotic resistance among members of an isogenic set of gonococci. From this survey, we conclude that lysozyme resistance and extensive O-acetylation of PG are widespread among gonococci and, thus, that most strains are potential sources of hydrolase-resistant PG that conceivably could persist as macromolecular fragments in vivo.

Amniotic fluid alkaline phosphatase, gamma-glutamyltransferase, and 5'-nucleotidase activity from 13 to 40 weeks' gestation, and alkaline phosphatase as an index of fetal lung maturity.

Reference ranges for amniotic fluid alkaline phosphatase, gamma-glutamyltransferase, and 5-nucleotidase are described from 13 to 40 weeks' gestation. Gamma-glutamyltransferase and 5-nucleotidase activities peak early in the second trimester and then decrease to low values. Alkaline phosphatase shows a similar pattern of activity from 13 to 29 weeks' gestation, but thereafter activity increases to term; this late increase is mainly related to the heat-labile particulate form of alkaline phosphatase. Total and heat-labile alkaline phosphatase alone or expressed as a ratio with gamma-glutamyltransferase can be used with or as an alternative to lecithin/sphingomyelin ratios in the investigation of fetal lung maturity. A total alkaline phosphatase activity of 0.36 mukat/L and an alkaline phosphatase/gamma-glutamyltransferase ratio greater than 2 indicate pulmonary maturity.