Diagnostic evaluation of a new combined HIV p24 antigen and anti-HIV1/2/O screening assay.

To evaluate a new fourth generation assay for simultaneous detection of antibodies to the human immunodeficiency virus (HIV) 1 and 2 and HIV p24 antigen in daily routine we tested 675 sera obtained from 673 patients and compared the results to conventional antibody tests. In 546 uninfected patients the rate of unspecific reactivities was slightly higher in the new screening assay as compared to conventional antibody assays (1.1% vs. 0.4%). All 121 sera derived from patients with known HIV infection were detected correctly. In six patients from whom sera were obtained during early seroconversion the fourth generation ELISA was positive in three cases, while conventional third generation tests still were negative. In patients negative for HIV antibodies and low amounts of p24 antigen less than 100 pg/ml also the fourth generation ELISA remained negative. Thus, this new assay permits earlier detection of HIV infection and reduces the diagnostic window. It is a reliable tool for routine diagnosis of HIV, especially in blood donors and patients with high risk behavior.

Shortening of the diagnostic window with a new combined HIV p24 antigen and anti-HIV-1/2/O screening test.

Because antibodies to the human immunodeficiency virus (HIV) are absent in the very early phase of HIV infection, there remains a slight residual risk for HIV transmission by blood donations by viremic but antibody negative donations. To shorten the diagnostic window between infection and the detection of antibodies, Enzygnost HIV Integral (Dade Behring, Germany) was developed. With this new test, HIV p24 antigen and HIV antibodies can be detected simultaneously in a single test. In a multicenter study the new screening assay has been compared with various tests that detect only HIV antibodies or HIV p24 antigen and with assays which permit a simultaneous detection of HIV antigen and HIV antibodies. The new assay showed 100% sensitivity for the detection of antibodies to HIV-1, groups M (n=1102) and O (n=55), and HIV-2 (n=289). In 23 out of 52 seroconversion panels, seroconversion was detected 2-18 days earlier with the new combined antigen/antibody test compared to single antibody tests. All samples from a viral load panel (n=451), all samples containing p24 antigen (n=302), and all but one of the cell culture supernatants (n=38) infected with various HIV-1 subtypes or HIV-2 were identified reliably by the new test. The specificity of the assay for 4002 unselected blood donors was 99.78% initially and 99.80% after retesting. Potentially interfering factors had no systematic influence on specificity. By testing for p24 antigen, which is present prior to the onset of antibody production in some cases of recent HIV infection, the new assay reduces the diagnostic window as compared to third generation screening assays, thus permitting an earlier diagnosis of HIV infection.

Blood-borne cancer cells--quo vadis?

The detection of blood-borne cancer cells may help in clinical staging and further understanding of cancer metastasis. We developed a cytokeratin-based immunomagnetic method to isolate epithelium-derived cells from the circulating blood of patients. The number of cell clusters positive for cytokeratin/prostate-specific antigen (PSA) from the peripheral blood of prostate cancer patients and cytokeratin/p185c-erbB-2 from the peripheral blood of breast cancer patients has been related to stage of the disease. Breast cancer patients who presented cytokeratin/p185c-erbB-2-positive cell clusters showed a decrease in such cells under adriamycin adjuvant therapy with Further molecular characterization by a highly sensitive microsatellite multiplex-PCR enabled reproducible detection of microsatellite alterations. The impact of these individually targeted results may contribute to an individual diagnostic and therapeutic strategy.

In vitro torque-deformation characteristics of orthodontic polycarbonate brackets.

The purpose of this study was to investigate the torque-deformation characteristics of the following four types of polycarbonate brackets: (1) pure polycarbonate, PPC (anterior Miura, RMO, Denver, Colo.), (2) ceramic reinforced polycarbonate, CRPC (Silkon bracket, American, Sheboygan, Wis.), (3) metal slot reinforced polycarbonate, MRPC (Plastic bracket, Tella Tech, Miami, Fla.), and (4) metal slot and ceramic reinforced polycarbonate, MCRPC (Spirit, Ormco, Glendora, Calif.). A stainless steel bracket, (Mini Diamond, Ormco, Glendora, Calif.), was used as a control. Ten brackets of each type were tested. Each bracket was bonded to a porcelain tooth and engaged in a torquemeter. The tooth-bracket assembly was made stationary by embedding it in die stone. Torsion was applied to the bracket at 4 degrees per minute and the resultant torque (grams.centimeters) and deformation (degree) were measured. For optimum labiolingual tooth movement for a maxillary incisor at 175 grams . centimeters, the amount of angular deflection necessary for the different polycarbonate brackets was the following: (a) 15 degrees for MRPC, (b) 17 degrees for MRPC, (c) 24 degrees for CRPC, and (d) > 30 degrees for PPC. The amount of deformation at this deflection was the least for MRCP followed by MCRPC, CRCP, and PPC. When compared with the stainless steel bracket, all polycarbonate brackets showed significantly (p < 0.0001) higher deformation and lower torque. Within the polycarbonate group, there was a significant difference (p < 0.0001) between each bracket for both measurements. The MRPC produced the highest torque and lowest deformation values followed by the MCRPC, CRCP, and PPC. It appears that only the metal slot reinforced brackets are clinically capable of torquing teeth sufficiently.

Cytogenetic studies on human breast carcinomas.

Cytogenetic studies were performed on cell material obtained from surgical specimens of 50 human breast carcinomas and from 61 cancerous effusions of 46 patients. Classical cytogenetic analyses of numerical chromosome changes and marker chromosomes revealed the non-random involvement of chromosomes #X and #22 as monosomics, of chromosomes #3, #7, and #19 as trisomics, and chromosome #1 (particularly p 13 to q 12) in marker formation. Karyotypic evolution was followed in vitro and in vivo and showed a highly individualistic pattern of stability and variability. In addition, a systematic screening for the presence of cytogenetic equivalents of gene amplification (double minutes 'DM', homogeneously staining regions 'HSR') was carried out. A high incidence of DM-positive cases was detected in primary tumors (48%) as well as in metastatic cells from effusions (40%), with the frequency of DM-containing metaphases ranging from 1 to 100% in the positive cases. This finding supports the assumption of the fundamental biological importance of gene amplification in human solid tumors. Furthermore, chromosome breakage and micronuclei were observed in breast carcinoma cells as an apparent consequence of therapy-independent mutability.

Effect of monoclonal antibodies to the attachment-tip on experimental Mycoplasma pneumoniae infection of hamsters. A preliminary report.

Mycoplasma pneumoniae, an important human pathogen, is an extra-cellular parasite, colonizing mucosal surfaces. Attachment to epithelial cells of the host is therefore an important mechanism of pathogenicity, and inhibition of adhesion might protect the host. M. pneumoniae predominantly adheres with a special organelle, the attachment-tip, to host cells. In vitro studies confirmed the observation that monoclonal antibodies (MOAB) to the tip inhibited adherence to erythrocytes. In animal experiments, high numbers of virulent M. pneumoniae exposed for 4 h at 37 C to MOAB and kept in suspension with MOAB 1:100 were inoculated intranasally into hamsters. A significant reduction in the lung lesion score, but not in the numbers of organisms in lung tissue or wash fluid of the upper respiratory tract, was seen in hamsters 14 days after inoculation of MOAB-treated organisms, as compared with controls. These observations, although preliminary, may have implications for the understanding of pathogenesis and for vaccine development.

Mycoplasma attachment to solid surfaces: a review.

Mycoplasma attachment to glass in a protein-containing environment requires energization of the cells, probably to provide more accessibility of binding sites. The substance mediating attachment is of protein nature. Studies with monoclonal antibodies on M. pneumoniae suggest a concentration of the binding sites at the tip structure.

Analysis of polypeptides of mutants of Mycoplasma pneumoniae that lack the ability to haemadsorb.

Haemadsorption-negative mutants of Mycoplasma pneumoniae were isolated which varied in their capacity to adsorb erythrocytes of various animal species suggesting adherence to erythrocytes is mediated by different binding mechanisms. Trypsin treatment of the wild-type strain resulted in loss of haemadsorbing activity; several polypeptides, some of which regenerated with haemadsorbing activity following further incubation, were also trypsin sensitive. The haemadsorption-negative mutants could be divided into two groups according to their polypeptide pattern. In the first group (11 mutants) the PAGE pattern was identical to that of the wild-type strain. The second group comprised 7 mutants which differed from the wild-type by lack of one or more polypeptides with molecular weights of 190 000, 90 000 or 40 000. During growth attachment to glass was weak or absent in the mutants. Surface hydrophobicity as measured by hydrophobic-interaction chromatography was nearly comparable in mutants and parent strain.

Influence of cell shape and surface charge on attachment of Mycoplasma pneumoniae to glass surfaces.

Attachment of Mycoplasma pneumoniae to glass is reduced in the presence of protein, and fatty acid-free bovine serum albumin is more effective than Cohn fraction V. Cultures in the early log phase (pH 7.45 to 7.25) and cultures in the stationary or decline phase (pH 6.9 to 6.4) were more sensitive to this inhibiting effect of protein-containing buffer. Treatment of the glass surface with bovine serum albumin, concanavalin A, or polylysine reduced attachment of the mycoplasma cells. The inhibiting effects of both proteins in buffer or on the glass surface could be overcome by the addition of glucose. Modification of the mycoplasma surface charge by blocking of carboxyl groups or neutralization of ionic lipids by tetracaine altered the attachment level, whereas fibronectin and its corresponding antiserum were without effect. The results suggest that the mycoplasma interaction with glass is a complex multifactorial process. In protein-free buffer both hydrophobic and electrostatic forces are involved; in protein-containing fluid, other factors seem to be involved. The energy required for this type of attachment could be necessary for maintenance of cell shape or synthesis of polypeptides.

Attachment of mycoplasmas to host cell membranes.

Pathogenic mycoplasmas rarely invade the tissues or bloodstream. Their adherence to epithelial cell surface, the first stage in disease, involves protein binding sites on the mycoplasmal cell membrane and receptors on the host cell membrane. Strong evidence indicates that Mycoplasma gallisepticum and Mycoplasma pneumoniae adhere with the aid of sialic acid residues on host cells, but the data do not preclude participation by other host-cell membrane components. Several studies indicate that these mycoplasmas adhere by blebs or terminal structures; others suggest that binding occurs via other cell areas. Scanning electron microscopy suggests tight interaction between these mycoplasmas and red blood cell membranes, causing imprints resembling those from interaction of viruses with red blood cells. Because sialoglycoproteins are major sites for attachment of M. pneumoniae to respiratory epithelium and red blood cells, glycophorin--the major sialoglycoprotein of human red blood cells--was the ligand used in affinity chromatography for isolation of the binding sites specific for sialic acid receptors from M. pneumoniae membranes solubilized by detergents. The fraction eluted with 0.2% sodium dodecylsulfate from the glycophorin-Sepharose column, highly enriched with two proteins, exhibited high binding capacity to glycophorin-Sepharose beads and lower binding capacity to human red blood cells. The latter capacity was nearly abolished by glycophorin, but not by its hydrophobic moiety.

Adherence of mycoplasmas: phenomena and possible role in the pathogenesis of disease.

Mycoplasma pneumoniae attaches to a variety of surfaces. Adherence to inert surfaces such as glass requires an intact energy metabolism. Interaction with sheep erythrocytes occurs via a binding protein on the mycoplasma surface. The protein reacts with a receptor containing sialic acid. Adherence to other erythrocytes may involve different mechanisms. Different results have been reported on interaction with tissue cells. The various mechanisms probably cooperate and thereby facilitate the colonization of the human respiratory tract.

Attachment of killed Mycoplasma gallisepticum cells and membranes to erythrocytes.

To correlate viability with attachment capacity, Mycoplasma gallisepticum cell harvested at different growth phases and treated by various agents were tested for their capacity to attach to human erythrocytes. The results show that viability per se is not essential for M. gallisepticum attachment to erythrocytes, as cells killed by ultraviolet irradiation anmd membranes isolated by lysing M. gallisepticum cells by various means retained attachment capacity. However, treatment of the mycoplasmas by protein-denaturing agents, such as heart, glutaraldehyde, or prolonged exposure to low pH, drastically affected or even abolished attachment, supporting the protein nature of the mycoplasma membrane components responsible for specific binding to the sialoglycoprotein receptors on the erythrocytes.

Adherence of mycoplasmas to cells and inert surfaces: phenomena, experimental models and possible mechanisms.

Mycoplasmas are typical surface parasites colonizing the mucous membranes of animals and man. Efficient adherence mechanisms are therefore a prerequisite for survival and, in some species, for pathogenicity. The lack of a rigid cell wall enables the mycoplasmas, in contrast to bacterial mechanisms, to actively rearrange their surface structure in a vertical or horizontal direction. The energy requirement for attachment of M. pneumoniae in inert surfaces strongly suggests such a mechanism, although no supporting morphological data are yet available. There seem to exist different kinds of adherence mechanisms depending on the species of mycoplasma and the host involved. The receptors of sheep erythrocytes for M. pneumoniae, M. gallisepticum and M. dispar are neuraminidase-sensitive, whereas those for M. hominis and M. salivarium are not, but are protease-sensitive. On the other hand the receptors of rabbit red blood cells for M. pneumoniae and M. dispar are neuraminidase-resistant. The binding sites on the mycoplasma surface too differ in some properties. Data on M. pneumoniae suggest a protein as major constituent of the binding mechanism. The results of all studies are to some extent also dependent on the method used to examine adherence. Most work was done with either hemagglutination and hemadsorption or with attachment to cells and organ cultures. A special experimental system is provided by the adherence of some species to glass or plastic surfaces. On this model the role of energy metabolism could be studied in more detail. Further strategy of research must include biochemical methods as well as morphological and immunological approaches.

Role of energy metabolism in Mycoplasma pneumoniae attachment to glass surfaces.

Attachment values of Mycoplasma pneumoniae to glass are normally very low when tested in buffer containing bovine serum albumin (10 mg/ml). However, the addition of one of the metabolizable sugars glucose, fructose, or mannose increased attachment more than 10-fold. The effect was dose dependent with a distinct optimum at about 0.25 mg/ml. Higher concentrations reduced this effect. Not only the sugars themselves but also the products of their catabolism, pyruvate and phosphoenolpyruvate, enhanced attachment. Pyruvate was effective in the same range of concentrations as the sugars, whereas phosphoenolpyruvate enhanced attachment at a significantly lower concentration (0.001 mg/ml). Higher levels of these substances also resulted in a decrease of attachment. The glucose-induced increase could be partially inhibited by glucose analogs, especially by 3-O-methyl-glucopyranoside, and by various inhibitors or glycolysis. Furthermore, attachment was strongly reduced by the uncoupling agents carbonylcyanide m-chlorophenylhydrazone and 2,4-dinitrophenol, as well as by dicyclohexylcarbodiimide, an inhibitor of the membrane-bound Mg2+-adenosine triphosphatase, whereas the ionophore valinomycin increased attachment by about 30%. These findings provide strong evidence for coupling between the attachment process of M. pneumoniae to glass and the utilization of metabolic energy.

Adherence of Mycoplasma pneumoniae to glass surfaces.

Attachment of M. pneumoniae to glass was quantitated in an experimental system enabling the settling down of [3H]palmitic acid-labeled cells onto glass cover slips. Attachment of mycoplasmas suspended in buffer increased with temperature, decreased with higher ionic strength, and showed a maximum at about pH 5.5. The findings suggest a participation of ionic bonds in the attachment process. Trypsin did not detach glass-bound mycoplasmas, and treatment of the cells with glutaraldehyde did not reduce their attachment to glass, suggesting that membrane components other than proteins may be involved in the attachment. Low concentrations (up to 20 mg/ml) of bovine serum albumin buffer. However, during the next few hours, attachment increased far above the bovine serum albumin control. This marked increase was reduced by more than half in the presence of chloramphenicol. Increased attachment was also observed when glucose (0.1 to 2 mg/ml) was added to the bovine serum albumin-containing buffer. The findings suggest different mechanisms for the attachment in protein-free buffer and in growth medium or glucose-containing bovine serum albumin buffer, respectively. The latter apparently requires metabolic activity of the mycoplasmas.

Adherence of erythrocytes to Mycoplasma pneumoniae.

The human pathogen Mycoplasma pneumoniae adheres to a variety of cells, including erythrocytes. A hemadsorption technique was developed to quantitate adherence by photometric measurement of lysates of erythrocytes that attached to sheets of M. pneumoniae grown in cups of Linbro plates. Attachment of sheep erythrocytes (SE) increased with higher ionic strength, was unaffected by minor pH variations (6 to 9), and was blocked by anti-M. pneumoniae antiserum, but was not inhibited by a variety of sugars, amino acids, and bovine serum albumin. The reaction was time and temperature dependent. The temperature curve showed peaks at 14 and 28 degrees C with untreated SE but only one peak at about 38 degrees C with glutaraldehyde-treated SE. The temperature dependence indicated involvement of either metabolic or membrane activities in the binding process. Trypsin treatment of the M. pneumoniae sheet abolished adherence of SE but was only partially effective with human erythrocytes and noneffective with rabbit erythrocytes. The binding capacity of the mycoplasma cells for SE was restored by incubation in growth medium for 3 to 4 h; this restoration was inhibited by 10 mug of chloramphenicol per ml. Neuraminidase treatment of SE removed their attachment capacity but had no effect on attachment of rabbit erythrocytes and only a slight effect on attachment of human erythrocytes. Pretreatment of M. pneumoniae with neuraminic acid partially blocked the adherence of SE, whereas rabbit erythrocyte attachment was not affected. Attached SE could be detached by trypsin, but not by neuraminidase. For human and rabbit erythrocytes, the results suggest binding mechanisms other than the interaction between neuraminidase-sensitive receptors and protein-containing binding sites shown for SE.

Threonine deaminase from a nonsense mutant of Escherichia coli requiring isoleucine or pyridoxine: evidence for half-of-the-sites reactivity.

The mutant IP7 of Escherichia coli B requires isoleucine or pyridoxine for growth as a consequence of a mutation in the gene coding for biosynthetic threonine deaminase. The mutation of IP7 was shown to be of the nonsense type by the following data: (1) reversion to isoleucine prototrophy involves the formation of external suppression at a high frequency, as shown by transduction experiments; and (ii) the isoleucine requirement is suppressed by lysogenization with a phage carrying the amber suppressor su-3. Cell extracts of the mutant strain contain a low activity of threonine deaminase. The possibility that this activity is biodegradative was ruled out by kinetic experiments. The mutant threonine deaminase was purified to homogeneity by conventional procedures. The enzyme is a dimer of identical subunits of an approximate molecular weight of 43,000 (Grimminger and Feldner, 1974), whereas the wild-type enzyme is a tetramer of 50,000-dalton subunits (Calhoun et al., 1973; Grimminger et al., 1973). The mutant enzyme is not inhibited by isoleucine and does not bind isoleucine, as shown by equilibrium dialysis experiments. Pyridoxal phosphate enhances the maximum catalytic activity of the mutant enzyme by a factor of five, whereas the wild-type enzyme is not affected. In wild-type and mutant threonine deaminase the ratio of protein subunits and bound pyridoxal phosphate is 2:1. The activation of threonine deaminase from strain IP7 is due to a second coenzyme binding site, as shown by (i) spectrophotometric titration of the enzyme with pyridoxal phosphate and by (ii) measurement the pyridoxal phosphate content of the enzyme after sodium borohydride reduction of the protein. The observation of one pyridoxal phosphate binding site per peptide dimer in the wild-type enzyme and of two binding sites per dimer in the mutant strongly suggests that one of the potential sites in the wild-type enzyme is masked by allosteric effects. The factors responsible for the half-of-the-sites reactivity of the coenzyme sites appear to be nonoperative in the mutant protein.

Threonine deaminase from a mutant requiring isoleucine or pyridoxine: inability to form a tetrameric state.

Threonine deaminase from a mutant of Escherichia coli growing alternatively with isoleucine or pyridoxine is a dimer, whereas the wild-type enzyme is a tetramer.