Organization, barrier function and antimicrobial lipids of the oral mucosa.

As one moves from the skin across the vermilion region of the lip and into the oral cavity, the oral mucosa is encountered. The oral mucosa consists of connective tissue known as the lamina propria covered by a stratified squamous epithelium. In the regions of the hard palate and gingiva, the epithelium is keratinized like the epidermis. In the buccal region, the floor of the mouth and the underside of the tongue, the epithelium is non-keratinized. The epithelium on the dorsum of the tongue is a specialized epithelium, but can be approximated as a mosaic of keratinized and non-keratinized epithelia. The non-keratinized epithelial regions do not produce a stratum corneum. Nuclei with intact DNA are retained in the superficial cells. In all regions, the outer portions of the epithelium provide a protective permeability barrier, which varies regionally. Antimicrobial lipids at the surfaces of the oral mucosa are an integral part of innate immunity.

Sphingoid bases are taken up by Escherichia coli and Staphylococcus aureus and induce ultrastructural damage.

Sphingoid bases found in the outer layers of the skin exhibit antimicrobial activity against gram-positive and gram-negative bacteria. We investigated the uptake of several sphingoid bases by Escherichia coli and Staphylococcus aureus, and assessed subsequent ultrastructural damage. E. coli and S. aureus were incubated with D-sphingosine, dihydrosphingosine, or phytosphingosine at ten times their MIC for 0.5 and 4 h, respectively, to kill 50% of viable bacteria. Treated bacterial cells were immediately prepared for SEM, TEM, and analyzed for lipid content by QTLC. E. coli and S. aureus treated with sphingoid bases were distorted and their surfaces were concave and rugate. Significant differences were observed in the visual surface area relative to controls for both E. coli and S. aureus when treated with dihydrosphingosine and sphingosine (p < 0.0001) but not phytosphingosine. While sphingoid base-treated S. aureus exhibited disruption and loss of cell wall and membrane, E. coli cytoplasmic membranes appeared intact and the outer envelope uncompromised. Both E. coli and S. aureus cells contained unique internal inclusion bodies, likely associated with cell death. QTLC demonstrated extensive uptake of sphingoid bases by the bacteria. Hence, sphingoid bases induce both extracellular and intracellular damage and cause intracellular inclusions that may reflect lipid uptake.

The emerging role of peptides and lipids as antimicrobial epidermal barriers and modulators of local inflammation.

Skin is complex and comprised of distinct layers, each layer with unique architecture and immunologic functions. Cells within these layers produce differing amounts of antimicrobial peptides and lipids (sphingoid bases and sebaceous fatty acids) that limit colonization of commensal and opportunistic microorganisms. Furthermore, antimicrobial peptides and lipids have distinct, concentration-dependent ancillary innate and adaptive immune functions. At 0.1-2.0 µM, antimicrobial peptides induce cell migration and adaptive immune responses to coadministered antigens. At 2.0-6.0 µM, they induce cell proliferation and enhance wound healing. At 6.0-12.0 µM, they can regulate chemokine and cytokine production and at their highest concentrations of 15.0-30.0 µM, antimicrobial peptides can be cytotoxic. At 1-100 nM, lipids enhance cell migration induced by chemokines, suppress apoptosis, and optimize T cell cytotoxicity, and at 0.3-1.0 µM they inhibit cell migration and attenuate chemokine and pro-inflammatory cytokine responses. Recently, many antimicrobial peptides and lipids at 0.1-2.0 µM have been found to attenuate the production of chemokines and pro-inflammatory cytokines to microbial antigens. Together, both the antimicrobial and the anti-inflammatory activities of these peptides and lipids may serve to create a strong, overlapping immunologic barrier that not only controls the concentrations of cutaneous commensal flora but also the extent to which they induce a localized inflammatory response.

Two antigenically distinct subtypes of human immunodeficiency virus type 1: viral genotype predicts neutralization serotype.

At least five distinct genetic subtypes (genotypes) of human immunodeficiency virus type 1 (HIV-1) have been identified by DNA sequencing. Current vaccine candidates are based on virus strains from North America and Europe that represent only one subtype. The extent to which distinct genotypes of HIV-1 correspond to antigenically distinguishable serotypes is largely unknown and may be critically important to vaccine design. Cross-neuralization studies were done with viruses and plasma from two different genotypes. Based on neutralization susceptibility, 10 primary HIV-1 isolates from Thailand and the United States were classified into one of two antigenic subtypes that correlated with viral genotype. The existence of serotypes of HIV-1 suggests that a broadly effective vaccine may have to include strains from multiple subtypes. Neutralization of these primary HIV-1 isolates differed substantially from results with laboratory strains. Future neutralization studies using primary isolates and multiple genotypes may be important for assessment of HIV-1 antigenic diversity.

Late manifestation of autosomal-recessive polycystic kidney disease in two sisters.

We report on 2 siblings with autosomal-recessive polycystic kidney disease, diagnosed at the ages of 14 and 18 years, respectively. Clinical findings and differential diagnosis, especially for autosomal-dominant polycystic kidney disease, are given. The consequences for genetic counselling are discussed.

An evaluation of a C-reactive protein assay using a rate immunonephelometric procedure.

By using 20 repetitive assays at three levels of C-reactive protein control serum, within-run and day-to-day precision of a rate immunonephelometric system was established. The precision was excellent in each case. Linearity of the system between 1.8 and 20 mg/dl was established at a coefficient of correlation of 0.999 by using serial dilutions of C-reactive protein control serum. When the immunonephelometric assay was compared with electroimmunodiffusion and radial immunodiffusion assays, coefficients of correlation of 0.986 and 0.993, respectively, were obtained. The lower limit of sensitivity of the immunonephelometric assay was 1.8 mg/dl. The relationship of clinical significance to the performance of the C-reactive protein test system using rate immunonephelometry is discussed.

Lymphocyte-mediated cytotoxicity microassay by cytofluorography.

A cytofluorometric method for measuring lymphocyte-mediated cytotoxicity is described which employs acridine orange and ethidium bromide stains. These stains permits the rapid (approximately 45 sec per sample) differential enumeration of viable cells and are therefore valuable in the preparation and performance of a cytotoxicity assay. Reproducibility of viable target cell counts is good and, statistically, the same mean result is obtained when the cytofluorometric and an accepted radioisotopic method are compared. It is found that the method employing radioisotopic label ([125I]iododeoxyuridine) non-specifically killed 80% of the target cells, whereas the cytofluorographic method does not result in non-specific kill. Therefore, the cytofluorometric method reflects in vivo conditions more accurately. The cytofluorometric method also provides a more rapid and less technically demanding assay than those previously described.

Automated differential cell counting of viable cells.

A rapid, automated method of differentially counting viable cells by ethidium bromide-acridine orange cytofluorography is described (Takasugi 1971). The method is shown to have comparability, equal precision, and sensitivity to the standard trypan blue, visual method. The method is applicable to microcytotoxicity testing.

Intracoronary myocardial perfusion imaging: patterns in patients with coronary artery disease.

Myocardial imaging following the intracoronary injection of radiolabeled particles is used to identify transmural scars in patients being evaluated for coronary atherosclerosis. Selective imaging of the microcirculation derived from each major coronary vessel is accomplished using a dual radionuclide technique. This report illustrates the various normal and abnormal imaging patterns encountered in patients with coronary artery disease. The regional myocardial nomenclature proposed by the American Heart Association Council on Cardiovascular Surgery is used. Correlation of the nuclear study with the contrast arteriogram and ventriculogram is essential for identifying both transmural scars and regions of collateral circulation. The procedure is safe and can be performed during routine coronary angiography.

Quantitation of "acute-phase proteins" postoperatively. Value in detection and monitoring of complications.

Serial determinations of serum C-reactive protein are helpful in the detection and monitoring of postoperative complications associated with inflammation and/or tissue necrosis. The serum C-reactive protein level begins to increase within sex hours after operation, peaks on the second day, and by the third postoperative day begins to decrease toward the preoperative level. In cases with surgical complications involving inflammation, serum C-reactive protein levels remain elevated and do not show a decline on the third postoperative day. Serum levels of other "acute-phase proteins," such as alpha-1 acid glycoprotein, ceruloplasmin, alpha-1 antitrypsin, and haptoglobin, were found to increase in response to surgical procedures, but subsequent to the increase in C-reactive protein. These other proteins offer no additional information in monitoring the postoperative acutephase response,

Lymphocyte blastoid transformation assay by cytofluorography.

This rapid, automated, micromethod quantitates blastoid transformation using a supra vital stain, acridine orange, in quantitating the ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) before and after cell stimulation. Only 0.2 ml of culture containing 1.0 X 10(5) to 1.5 X 10(5) lymphocytes is required to obtain results every 5 minutes. These results include a total cell count and cell counts of the populations with increased RNA (I-RNA), and increased DNA (I-DNA). The I-RNA and I-DNA lymphocyte populations, as determined by the cytofluorometric method, correlate well with the accepted H3 uridine and C14 thymidine uptake methodology. The phytohemagglutinin dose response measurements by the 2 methods also correlate well. Blast cell counts, by Wright-Giemsa staining, showed near identity to the I-RNA cell population.

Improved measurement of erythrocyte volume distribution by aperture-counter signal analysis.

In aperture counters, particles in fluid suspension flow through a small orifice or aperture, causing a change in the electrical resistance of the aperture. This change is sensed by an external electronic circuit and translated into a voltage pulse, the signal height of which is proportional to the volume of the particle in the aperture. These signal pulses are collated into a spectrum of pulse heights by a multichannel pulse-height analyzer. The channel number (voltage increment) spectrum is proportional to the volume distribution of the particles sensed. A problem is that pulse height not only depends on cell volume, but also on the orientation and shape of the particle sensed and the current density along the path taken by the particle through the aperture. Uneven current density exists, primarily at the aperture entrance and exit, close to the wall. Orientation and shape of particles are altered near the wall by the unbalanced shear forces there. Toward the center of the aperture, the shear forces act so as not to induce continuous change in the orientation of the particles sensed. Thus introduction into the pulse-height spectrum of pulses that do not show a good proportionality to volume is primarily caused by particles that are traveling near the aperture wall. Residence time in the aperture for a particle traveling near the wall will be longer than that for a particle traveling down the center of the aperture, because of the smaller fluid velocity near the wall. Duration of the signal pulse created by a particle traveling near the wall will be correspondingly greater. We discuss an electronic filter to remove from the pulse-height spectrum those pulses that appear to result from particles traveling near the wall and the effect of the filter on the measured signal height and hence the volume distribution of erythrocytes. Use of this technique to characterize erythrocytes by volume distribution is described.

Acomparative study of two methods of urine preservation.

In preparation for the conduct of biochemical experiments in the Skylab Orbital Workshop a study was performed on the stability of various chemical constituents in urine in 2 different techniques for preservation and storage. Urine samples were either vacuum dried or frozen and maintained in storage at minus 20 degrees for periods of up to 10 weeks. The urinary constituents studied included aldosterone, antidiuretic hormone, epinephrine, norepinephrine, urea, nitrogen, creatine, hydroxyproline, 17-hydroxycorticosteroids, calcium, sodium potassium, chloride, magnesium and phosphate. Some degradation of urinary compounds was observed after both treatments. The rate and variability of destruction following the vacuum drying treatment, however, was greater than for freezing. It was concluded that only the freezing treatment could be used to preserve with predictable loss the urinary samples which would be returned to earth following the conclusion of each Skylab flight.

Skylab experiment results: hematology studies.

These studies were designed and coordinated to evaluate specific aspects of man's immunologic and hematologic systems which might be altered by or respond to the space flight environment. The biochemical functions investigated included cytogenetic damage to blood cells, immune resistance to disease, regulation of plasma and red cell volumes, metabolic processes of the red blood cell, and physical chemical aspects of red blood cell functions. Only minor changes were observed in the functional capacity of erythrocytes as determined by measuring the concentrations of selected intracellular enzymes and metabolites. Tests of red cell osmotic regulation indicated some elevation in the activity of the metabolic dependent Na-K pump, with no significant alterations in the cellular Na and K concentrations or osmotic fragility. A transient shift in red cell specific-gravity profile was observed on recovery, possibly related to changes in cellular water content. Measurements of hemoconcentration (hematocrit, hemoglobin concentration, red cell count) indicated significant fluctuations postflight, reflecting observed changes in red cell mass and plasma volume. There was no apparent reticulocytosis during the 18 days following the first manned Skylab mission in spite of a significant loss in red cell mass. However, the reticulocyte count and index did increase significantly 5 to 7 days after completion of the second, longer duration, flight. There were no significant changes in either the while blood cell count or differential. However, the capacity of lymphocytes to respond to an in vitro mitogenic challenge was repressed postflight, and appeared to be related to mission duration. The cause of this repression is unknown at this time. Only minor differences were observed in plasma protein patterns. In the second mission there were changes in the proteins involved in the coagulation process which suggested a hypercoagulative condition.