CLS advanced degrees and career enhancement. Part 1--Comparison of career data.

OBJECTIVE: Determine whether recipients of clinical laboratory science (CLS) advanced degrees (MS) experience greater career achievements than their baccalaureate level (BS) colleagues. DESIGN: Two similar questionnaires were used-one for certified or licensed CLS professionals who had earned advanced CLS degrees (MS); the other for matched BS CLS colleagues. SETTING: Five academic programs that conduct both National Accrediting Agency for Clinical Laboratory Sciences accredited CLS education and CLS MS degree programs participated. PARTICIPANTS: The number of survey respondents was 220, 117 with advanced CLS degrees and 103 BS level controls. There were 99 matched pairs, i.e., 198 individuals were matched for gender, residence region, and years of experience. MAIN OUTCOME MEASURES: Careers of BS vs. MS respondents were statistically compared, e.g., fractions with managerial level jobs, relative earnings increases per year, numbers of publications and reports, and other professional contributions. RESULTS: Compared to their BS degree controls, MS degree respondents had more managerial level jobs (62% MS; 36% BS), a higher frequency of job change (once per 4.3 years MS; once per 5.9 years BS), and a higher increase per year of earnings (9.1% MS; 8.1% BS). A greater percentage of the MS degree graduates (77%) than the BS level controls (33%) had authored external publications; the responses related to authorship of institutional reports and procedures were less different-84% MS and 64% BS. Professional contributions to their institutions or profession were cited slightly more frequently by the MS graduates (65%) than by the BS level controls (55%). CONCLUSION: Compared to their matched BS level CLS colleagues, CLS MS degree recipients had greater job mobility, greater management authority, higher salary, and more numerous professional contributions.

CLS advanced degrees and career enhancement. Part 2--A comparison of perceptions.

OBJECTIVE: To determine whether recipients of clinical laboratory science (CLS) advanced degrees (MS) perceive greater career enhancement value related to earning an advanced degree than is perceived by their baccalaureate level (BS) colleagues. DESIGN: Two questionnaires were used-one for certified or licensed CLS professionals who had earned MS CLS degrees; the other for matched BS CLS colleagues. SETTING: Five academic programs that conduct both National Accrediting Agency for Clinical Laboratory Sciences accredited CLS education and CLS MS degree programs participated. PARTICIPANTS: The number of survey respondents was 220 (117-MS; 103-BS level controls). The groups were matched for gender, residence region, and years of experience. MAIN OUTCOME MEASURES: The primary outcome measurements were the perceived benefits of having a CLS MS degree, the reasons for and against obtaining a CLS MS degree, and the overall evaluation of CLS degree programs at both levels. RESULTS: The highest perceived benefit of having a CLS MS degree was the same in both groups, "enhanced self esteem and confidence". The highest priority motivation of MS degree recipients for obtaining a CLS advanced degree was "personal satisfaction". The highest priority reason of the BS group for not obtaining a CLS advanced degree was "family obligation". In both levels of degree programs the subject most commonly cited as needing modification was laboratory management. CONCLUSION: The results indicate that CLS professionals who have CLS MS degrees perceive a greater career enhancement value of advanced CLS degrees than their BS level colleagues.

Neutrophil function screening in patients with chronic granulomatous disease by a flow cytometric method.

Neutrophils from patients with chronic granulomatous disease (CGD) fail to produce a significant oxidative burst following stimulation. We have evaluated the use of flow cytometry and the dye 2',7'-dichlorofluorescein diacetate (DCF) for routine screening for deficiencies of neutrophil oxidative burst. A range for DCF fluorescence for phorbol myristate acetate stimulated and non-stimulated neutrophils was established based on data from 52 healthy adults. Samples from three patients with suspected neutrophil dysfunction, three patients with X-linked CGD, and one patient with autosomal recessive (AR) CGD were evaluated with both the DCF assay and the quantitative nitroblue tetrazolium dye reduction (NBT) test. For the DCF test, the ratio of mean fluorescence intensity of stimulated to non-stimulated neutrophils was less than 5 for CGD patients and from 16 to greater than 50 for healthy individuals. With the DCF test, two populations of neutrophils could be identified in samples from four carriers of X-linked CGD, although two carriers of AR CGD had NBT and DCF results in the normal range. Our data suggest the DCF test is a sensitive and convenient method for detecting CGD.

Immunization procedures for E. coli proteins.

Three immunization procedures were compared for the production of antibodies to the minor components of a complex E. coli protein (ECP) mixture: a conventional protocol and two methods that allow for the selective in vitro (cascade) or in vivo (passive) depletion of highly immunogenic proteins. An indirect ELISA showed that a maximum ELISA antibody titer was obtained with all the procedures 60 d after immunization. Analysis of these antisera by two-dimensional SDS-PAGE immunoblots, however, demonstrated that antibody reactivity to minor components in the mixture was not achieved until 112 d. This analysis also showed that a marked improvement in antibody response to minor components was obtained with the cascade immunization procedure. The mean titer and spectrum of antibody reactivity was similar for each group, and suggested that, although some individual variation was noted, the improvements observed were the result of the protocol used. Thus, for these ECPs, and multiple antigen mixtures in general, the preferred immunization protocol should employ at least three hosts and utilize the cascade immunization of Thalhamer and Freund. Characterization of the resulting antisera is best performed by use of silver stained two-dimensional SDS-PAGE and immunoblotting.

Development of a microsphere-based fluorescent immunoassay and its comparison to an enzyme immunoassay for the detection of antibodies to three antigen preparations from Candida albicans.

A sensitive assay for the simultaneous detection of multiple serum antibodies by flow cytometry was developed. Polystyrene microspheres of 5, 7 and 9.3 micron in diameter were used as solid supports for the attachment of three different antigen preparations from Candida albicans. These antigens were a whole cell extract; a cytoplasmic protein extract and a cell wall polysaccharide. Microsphere-associated fluorescence was quantitated by flow cytometry, with the different sized microspheres analyzed separately using electronic volume gating. This procedure allowed for different antigen-coated microspheres with discrete sizes to be analyzed independently for immunofluorescence. The assay detected antibody levels in human serum at dilutions up to 10(-6) and provided complete discrimination, using all three antigen preparations, between antibody levels seen in healthy subjects and those seen in patients suspected of having a systemic Candida infection. A standard enzyme immunoassay (EIA) failed to provide complete discrimination between healthy subjects and patient samples: at least 17% of patient values fell within the healthy subject range using all three antigen preparations. The microsphere assay which allowed for the simultaneous detection of multiple antibodies, has increased dynamic range over EIA and provides for better discrimination of patients from healthy subjects in comparison to EIA. Precise quantitation of antibodies is possible and the rapid analysis of thousands of microspheres markedly enhances the statistical accuracy of the assay. We suggest this assay is likely to have many other important applications in immunologic testing.

Suppression of the delayed-type hypersensitivity and cell-mediated immune responses to Listeria monocytogenes induced by Pseudomonas aeruginosa.

Pseudomonas aeruginosa-mediated suppression of the immune response to Listeria monocytogenes was investigated in mice. Because delayed-type hypersensitivity (DTH) footpad swelling to L. monocytogenes was suppressed equally in lipopolysaccharide-responsive and -hyporesponsive mouse strains, the lipopolysaccharide component of P. aeruginosa could not have been the suppressive agent. Mucoid P. aeruginosa cells were no more suppressive than their nonmucoid revertants; therefore, mucoid coating was not an additional immunosuppressive element. Interleukin-1 and macrophage inhibitory factor production to L. monocytogenes and clearance of L. monocytogenes from mouse spleens were all decreased by prior Pseudomonas infection, indicating that cell-mediated immunity, as well as DTH, was decreased to a sublethal Listeria dose. The timing of Pseudomonas exposure relative to Listeria sensitization was varied. P. aeruginosa injected 24 or 6 h before or at the same time as L. monocytogenes depressed DTH to Listeria challenge 7 days later. Animals treated in this way could not respond to reinfection with L. monocytogenes at 13 days. P. aeruginosa administered to L. monocytogenes-sensitized mice at the time of footpad challenge was suppressive, but these mice responded normally upon reinfection. It appears that P. aeruginosa induced two types of suppression to L. monocytogenes: a transient suppression, affecting DTH challenge but not resensitization, and a longer lasting suppression that did not permit mice exposed to P. aeruginosa at the time of Listeria sensitization to respond to subsequent Listeria exposure.

Decreased delayed-type hypersensitivity and increased protection to Listeria monocytogenes seen in mice infected with mucoid and nonmucoid Pseudomonas aeruginosa.

Infection of mice with Pseudomonas aeruginosa, washed and unwashed, mucoid and nonmucoid, altered subsequent immunity to Listeria monocytogenes. Mice were protected against lethal doses of L. monocytogenes yet exhibited decreased delayed-type hypersensitivity footpad swelling to sublethal doses. The mucoid coating of mucoid P. aeruginosa, an important pathogen in chronic bronchopulmonary disorders, imparted no additional immunomodulating capabilities to P. aeruginosa.

Evaluation of Pseudomonas aeruginosa exotoxin A and elastase as virulence factors in acute lung infection.

Acute Pseudomonas aeruginosa pneumonia was established in guinea pigs by intratracheal instillation of bacteria. Challenge strains included PAO-1, a strain known to produce exotoxin A, alkaline protease, and elastase, and several PAO-1 mutants deficient in either biologically active exotoxin A or elastase production. Survival, intrapulmonary killing of bacteria, and blood cultures were compared among the groups. Strains of P. aeruginosa deficient in active elastase production appeared to be less virulent than the parent strain and were more easily cleared from the lung. Opposite results were obtained for the exotoxin A-deficient mutants. These data suggest that elastase, but not exotoxin A, was an important virulence factor during acute pneumonia due to P. aeruginosa.

Dose-dependent effect of glucocorticosteroids on pulmonary defenses in a steroid-resistant host.

An experimental model of Listeria monocytogenes pneumonia was employed in order to study the pathogenesis of lung infection with a facultative intracellular pathogen in normal and steroid-treated hosts. Guinea pigs, which resemble humans as a "steroid-resistant" species, were treated with week-long regimens of cortisone acetate or saline. Cortisone regimens were 100 mg/kg/day (low-dose) or 200 mg/kg/day (high-dose). Lungs were then infected with Listeria monocytogenes, and groups were compared for survival as well as intrapulmonary killing of Listeria. A dose-dependent defect in pulmonary resistance to Listeria was observed among the steroid-treated animals, with survivals of 67% for the low-dose group and 0% for the high-dose group. Similarly, acquired in vivo pulmonary resistance to Listeria was diminished in steroid-treated animals, as reflected by reduced intrapulmonary killing and a tendency for systemic dissemination of Listeria. Numbers of T-lymphocytes in blood (p less than 0.001) and lungs (p less than 0.001) were significantly reduced in cortisone-treated animals. In addition, alveolar macrophages obtained from high-dose-treated animals displayed a 47% reduction in listericidal activity. It is concluded that glucocorticosteroid administration causes a dose-dependent reduction in pulmonary defenses to intracellular pathogens in the steroid-resistant host, and that suppression of both acquired local immunity as well as nonimmune defense mechanisms occurs.

Potentiation of MIF production by lymphocytes of Trichinella spiralis-infected mice.

In order to further investigate the mechanism involved in the immunopotentiation following Trichinella spiralis infection, mice were injected intraperitoneally with either heat-killed or viable bacillus Calmette-Guérin (BCG) 28 days after oral infection with 200 nematode larvae. Delayed-type hypersensitivity (DTH) was assessed 28 days later by measuring levels of migration inhibitor factor (MIF) elicited by Old Tuberculin challenge. Sensitization with killed BCG produced only negligible amounts of MIF; however, prior T. spiralis infection resulted in significantly increased titers of MIF, reaching levels induced by viable BCG. The data indicated that a lymphokine component may be involved in addition to non-specific mechanisms previously proposed.

Active immunization with lipopolysaccharide Pseudomonas antigen for chronic Pseudomonas bronchopneumonia in guinea pigs.

Chronic respiratory infection with Pseudomonas aeruginosa is a leading clinical problem among patients with cystic fibrosis. Because antimicrobial agents are usually ineffective in eradicating these infections, additional therapeutic or prophylactic measures should be considered. In this study, an experimental guinea pig model of chronic Pseudomonas aeruginosa bronchopneumonia was utilized to determine whether active immunization with lipopolysaccharide (LPS) P. aeruginosa antigen may favorably influence the course of this infection. Experimental pneumonia was established by tracheobronchial instillation of suspensions of microscopic agar beads, which were impregnated with viable P. aeruginosa. After 4 wk of infection, the geometric mean (reciprocal) passive hemagglutinating Pseudomonas antibody titer was 185+/-1.3, and lungs contained 16.8+/-4 x 10(3) colony-forming units Pseudomonas/ml of lung homogenate. Pseudomonas immunization, given prior to a 4-wk infection, resulted in significantly higher passive hemagglutinating titers (474+/-1.4; P < 0.05), lower numbers of viable Pseudomonas in lung tissues (2.4+/-0.6 x 10(3); P < 0.01), and reduced histopathology in lungs. In contrast, providing Pseudomonas immunization to animals 2 wk after pulmonary infection was established, offered no apparent benefit. Likewise, no protection was afforded by prophylactic immunization with a non-Pseudomonas LPS antigen (Escherichia coli J5 vaccine). Using a Raji cell assay, modified to detect circulating immune complexes in vaccinated and infected guinea pig sera, there was no evidence that active immunization increased the frequency of circulating immune complexes in infected guinea pigs. It is concluded that prophylactic immunization with Pseudomonas LPS antigen may confer protection from subsequent Pseudomonas bronchopneumonia, but that immunization during established infection is not beneficial.

Influence of mucoid coating on clearance of Pseudomonas aeruginosa from lungs.

Pulmonary infection with mucoid strains of Pseudomonas aeruginosa in present in the majority of cystic fibrosis patients with chronic lung disease. It has been postulated that this mucoid coating may act to decrease lung clearance of Pseudomonas by limiting access of phagocytes, antibodies, and antibiotics to the bacteria. To determine whether mucoid coating of Pseudomonas might decrease intrapulmonary killing, groups of guinea pigs were infected with intrabronchial instillations of equivalent numbers of mucoid and nonmucoid Pseudomonas. For this study, mucoid strains of Pseudomonas were obtained from cystic fibrosis sputa and passaged on blood agar plates to obtain their nonmucoid revertants. Animals were then sacrificed at timed intervals after infection, and quantitative cultures were performed on lung homogenates. In all cases, mucoid challenge strains retained their mucoid morphology after passage in guinea pig lungs. No difference in killing of mucoid and nonmucoid Pseudomonas could be detected at 6, 24, or 48 h after lung infection. Further challenge studies used guinea pigs that were either prevaccinated with lipopolysaccharide P. aeruginosa vaccine or else treated with tobramycin sulfate after infection. Nonvaccinated or untreated controls had reduced intrapulmonary killing of Pseudomonas compared with vaccinees or treated groups (P < 0.02 and P < 0.01, respectively). However, there were no differences in pulmonary killing of mucoid and nonmucoid Pseudomonas in the presence of either specific antibodies or antibiotic. We conclude from these studies that mucoid coating of Pseudomonas does not selectively impede mechanisms of intrapulmonary killing in guinea pig lungs.

Dose dependence of Trichinella spiralis-induced immunopotentiation.

Previous reports studying the immunological and histopathological sequelae following infection with Trichinella spiralis indicated that this nematode can affect the host's immune capability to heterologous antigens. The present investigation was aimed at determining the dose of T. spiralis larvae required for development of maximum delayed-type hypersensitivity (DTH) against heat-killed bacillus Calmette-Guérin (BCG). Mice were injected intraperitoneally with 4 x 10(6) bacteria 28 days after oral intubation with 0, 50, 100, 200, 300 or 400 viable T. spiralis larvae. Groups were tested in vivo for DTH 28 days after administration of nonviable BCG by the footpad swelling assay. Prior infection with 200 nematode larvae resulted in 34 of 42 animals (81%) developing positive 24-hour DTH reactions. When these data were compared to those seen in the other experimental and control groups, the 200 larval dose appeared to effect maximal immunopotentiation in this system.

Effect of Trichinella spiralis infection on delayed hypersensitivity to heterologous antigens.

Studies were initiated to determine the effects of the different phases of the nematode life cycle on the delayed-type hypersensitivity (DTH) capabilities of the host. Mice were injected intraperitoneally with 4 X 10(6) viable or heat-killed BCG at 0, 3, 7, 14 and 28 days after oral infection with 200 Trichinella spiralis larvae. Groups were tested for DTH 28 days after BCG inoculation by the footpad swelling assay. Infection with T. spiralis was found to suppress the response in mice administered viable BCG 0 or 3 days later. Mice injected with viable bacteria 7 days after T. spiralis regained the ability to develop in vivo DTH reactions against old tuberculin. Groups administered viable BCG 14 and 28 days after nematode infection yielded potentiated DTH responses, when compared with control mice. Animals infected with T. spiralis and later injected with heat-killed BCG required a longer interval to develop specific immunopotentiation. Maximum immunopotentiation seemed to be related to the presence of larvae in muscle tissue (28 days). These findings suggest that the effect of T. spiralis on the immune capabilities of the host is dependent on the different anatomical locations of the parasite.