Evaluation of Bacillus anthracis and Yersinia pestis sample collection from nonporous surfaces by quantitative real-time PCR.

AIM: We will validate sample collection methods for recovery of microbial evidence in the event of accidental or intentional release of biological agents into the environment. METHODS AND RESULTS: We evaluated the sample recovery efficiencies of two collection methods - swabs and wipes - for both nonvirulent and virulent strains of Bacillus anthracis and Yersinia pestis from four types of nonporous surfaces: two hydrophilic surfaces, stainless steel and glass, and two hydrophobic surfaces, vinyl and plastic. Sample recovery was quantified using real-time qPCR to assay for intact DNA signatures. We found no consistent difference in collection efficiency between swabs or wipes. Furthermore, collection efficiency was more surface-dependent for virulent strains than nonvirulent strains. For the two nonvirulent strains, collection efficiency was similar between all four surfaces, albeit B. anthracis Sterne exhibited higher levels of recovery compared to Y. pestis A1122. In contrast, recovery of B. anthracis Ames spores and Y. pestis CO92 from the hydrophilic glass or stainless steel surfaces was generally more efficient compared to collection from the hydrophobic vinyl and plastic surfaces. CONCLUSIONS: Our results suggest that surface hydrophobicity may play a role in the strength of pathogen adhesion. The surface-dependent collection efficiencies observed with the virulent strains may arise from strain-specific expression of capsular material or other cell surface receptors that alter cell adhesion to specific surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings contribute to the validation of standard bioforensics procedures and emphasize the importance of specific strain and surface interactions in pathogen detection.

Respuesta inmune sistémica y mucosal contra Neisseria meningitidis B inducida por estrategia de vacunación simultànea mucosal y parental

Immunization is one of the most successful and cost-effective health interventions ever. Immunization have been helping to reduce child mortality, improving maternal health and combating infectious diseases. In spite of its, undisputed past success and promising future, however, immunization remains an unfinished agenda because of them inadequate coverage. Several factors have been largely responsible of a difficulty to attain immunization coverage and have been recognized as a problems of current vaccines, such as: the number of dose, excessive use of parenteral route, a small number of adjuvants approve for use in human, higher reactogenicity and unavailability against intracellular pathogens, infected or altered cells and scanty feasibility to combined more than one antigen in the same formulation. For bacterial meningitis WHO estimates that 1,2 million cases occur annually and Neisseria meningitidis is the etiological agent in more than 40 percent of these cases although some meningococcal vaccines are available. To bear in mind these principals problems, a novel protocol for vaccination against N meningitidis called Single Time Vaccination Strategy (SinTimVaS) is proposed. Using female BALB/c mice, we induce systemic and mucosal immune responses against N meningitidis with only one parenteral and one mucosal dose at the same time, employing the Finlay Adjuvants derivate from N meningitidis, AFPL1 and AFCo1, respectively. In conclusion, SinTimVaS could increase the vaccination coverage and reduce the time-cost of vaccine campaigns, adding the possibility to increase the herd immunity by mucosal specific response induction(AU)

Differences in genotypes of Helicobacter pylori from different human populations.

DNA motifs at several informative loci in more than 500 strains of Helicobacter pylori from five continents were studied by PCR and sequencing to gain insights into the evolution of this gastric pathogen. Five types of deletion, insertion, and substitution motifs were found at the right end of the H. pylori cag pathogenicity island. Of the three most common motifs, type I predominated in Spaniards, native Peruvians, and Guatemalan Ladinos (mixed Amerindian-European ancestry) and also in native Africans and U.S. residents; type II predominated among Japanese and Chinese; and type III predominated in Indians from Calcutta. Sequences in the cagA gene and in vacAm1 type alleles of the vacuolating cytotoxin gene (vacA) of strains from native Peruvians were also more like those from Spaniards than those from Asians. These indications of relatedness of Latin American and Spanish strains, despite the closer genetic relatedness of Amerindian and Asian people themselves, lead us to suggest that H. pylori may have been brought to the New World by European conquerors and colonists about 500 years ago. This thinking, in turn, suggests that H. pylori infection might have become widespread in people quite recently in human evolution.

Flow cytometric, phase-resolved fluorescence measurement of propidium iodide uptake in macrophages containing phagocytized fluorescent microspheres.

BACKGROUND: Spectral interference (overlap) from phagocytosed green-yellow (GY) microspheres in the flow cytometric, red fluorescence emission measurement channel causes errors in quantifying damaged/dead alveolar macrophages by uptake of propidium iodide. METHODS: Particle burdens of uniform GY fluorescent microspheres phagocytosed by rat alveolar macrophages and the discrimination of damaged/dead cells as indexed by propidium iodide uptake were assessed with conventional and phase-sensitive flow cytometry. RESULTS: The fluorescence spectral emission from phagocytosed microspheres partly overlapped the propidium iodide red fluorescence emission and interfered with the measurement of damaged/dead cells when using conventional flow cytometry without subtractive compensation. This caused errors when estimating the percentage of nonviable, propidium iodide-positive, phagocytic macrophages. The interference was eliminated by employing phase-sensitive detection in the red fluorescence measurement channel based on differences in fluorescence lifetimes between the fluorescent microspheres and propidium iodide. Intrinsic cellular autofluorescence, whose fluorescence lifetime is approximately the same as that of the phagocytosed microspheres, also was eliminated in the phase-sensitive detection process. Because there was no detectable spectral interference of propidium iodide in the green fluorescence (phagocytosis) measurement channel, conventional fluorescence detection was employed. CONCLUSIONS: Phase-resolved, red fluorescence emission measurement eliminates spectral overlap errors caused by autofluorescent phagocytes that contain fluorescent microspheres in the analyses of propidium iodide uptake. Cytometry 39:45-55, 2000. Published 2000 Wiley-Liss, Inc.

Amiloidosis secundaria: características clínicas / Clinical characteristic: secundary amyloidosis

Objetivo: determinar las características clínicas de la amiloidosis secundaria. Material y métodos: Se realizó un estudio retrospectivo de 115 pacientes con diagnóstico histopatológico de amiloidosis secundaria entre 1964 y 1997, determinados por biopsia renal (97.5 por ciento) o de glándula salival menor (2.5 por ciento). Resultados: como enfermedades determinantes se encontraron TBC pulmonar (90.43 por ciento), bronquiectasias (6.08 por ciento), osteomielitis (1.74 por ciento). Las manifestaciones clínicas fueron: edema en 98.2 por ciento, proteinuria 100 por ciento, e hipotensión en 30.43 por ciento. La proteinuria en 24 horas fluctuó entre 1.26 y 3.23 gr. El rango en que fluctuó la albúmina sérica fue de 0.6 a 3 gr/dl. Se encontró función renal normal en 5 por ciento e insuficiencia renal en 95 por ciento de los pacientes

Antimicrobial susceptibility of Helicobacter pylori determined by the E test using tetrazolium egg yolk agar.

Metronidazole and tetracycline E tests were compared to an agar dilution method for the antimicrobial susceptibility testing of Helicobacter pylori. Sixteen strains were tested by using tetrazolium egg yolk (TEY) agar. The characteristic E test inhibition ellipse was clearer on TEY agar than on standard blood agar and gave results comparable to those of the agar dilution test. The use of TEY medium is preferable to that of blood agar medium in E test MIC determinations for H. pylori.

Helicobacter pylori populations in Peruvian patients.

Helicobacter pylori is an extremely diverse species. The characterization of strains isolated from individual patients should give insights into colonization and disease mechanisms and bacterial evolution. We studied H. pylori isolates from patients in the Japanese-Peruvian Polyclinic in Lima, Peru, by determining metronidazole susceptibility or resistance and by random amplified polymorphic DNA (RAPD) fingerprinting (a measure of overall genotype). Strains isolated from several biopsy specimens from each of 24 patients were studied. Both metronidazole-susceptible and -resistant strains were isolated from 13 patients, whereas strains of more than one RAPD type were isolated from only seven patients. We propose that the homogeneity in RAPD fingerprints for strains isolated from most persons reflects selection for particular H. pylori genotypes during chronic infection in individual hosts and the human diversity in traits that are important to this pathogen. Carriage of related metronidazole-resistant and -susceptible strains could reflect frequent metronidazole use in Peru and alternating selection for resistant and susceptible phenotypes during and after metronidazole therapy.

Rapid recurrence of Helicobacter pylori infection in Peruvian patients after successful eradication. Gastrointestinal Physiology Working Group of the Universidad Peruana Cayetano Heredia and The Johns Hopkins University.

Helicobacter pylori is associated with gastritis, peptic ulcer disease, and gastric cancer. Since gastric cancer is common in Peru, eradication of H. pylori may help to reduce the occurrence of gastric cancer. This study involved three randomized trials to determine the efficacy of four different triple-drug therapy regimens. The most successful regimen was furazolidone combined with bismuth subsalicylate and amoxicillin, which eradicated infection in 82% of patients. Patients successfully treated were followed every 2-3 months to determine the recurrence rate of H. pylori infection. Of 105 patients with H. pylori eradication documented by pathology and culture, 52% (55) returned for follow-up endoscopy, and in 73% (40) of these 55 the infection recurred during the 8-month follow-up period. Thirty-five patients from whom H. pylori was eradicated and who were tested for antibodies to H. pylori remained consistently seropositive. Rapid recurrence of H. pylori infection after successful eradication suggests that measures other than antimicrobial therapy are needed to fight H. pylori in developing countries.

Metronidazole and clarithromycin resistance in Helicobacter pylori determined by measuring MICs of antimicrobial agents in color indicator egg yolk agar in a miniwell format. The Gastrointestinal Physiology Working Group of Universidad Peruana Cayetano Heredia and the Johns Hopkins University.

Resistance of Helicobacter pylori to metronidazole often causes failure of commonly used combination drug treatment regimens. We determined the MICs of metronidazole and clarithromycin against 18 H. pylori strains from Peru using tetrazolium egg yolk (TEY) agar. The MIC results obtained by agar dilution with petri dishes were compared with the results found through a miniwell format. The results of the two protocols for measuring drug susceptibility differed by no more than 1 dilution in all cases. On TEY agar, bright-red H. pylori colonies were easy to identify against a yellow background. Sixty-one percent (11 of 18) of the strains were resistant to metronidazole (MIC, > or = 4 micrograms/ml) and 50% (9 of 18) were resistant to clarithromycin (MIC, > or = 0.125 micrograms/ml), whereas none (0 of 5) of the strains tested were resistant to tetracycline (MIC, > or = 1 micrograms/ml). Thus, the prevalence of metronidazole and clarithromycin resistance in Peru is higher than that in developed regions of the world. The miniwell plate with TEY agar allows easy H. pylori colony identification, requires about one-third less of the costly medium necessary for petri dish assaying, conserves space, and yields MICs equivalent to those with agar dilution in petri dishes.

Stimulation of rat and murine alveolar macrophage proliferation by lung fibroblasts.

Increases in alveolar macrophage (AM) number occur during chronic inflammation and pulmonary fibrosis. Although the underlying mechanism(s) for such increases remain poorly understood, the overall process is known to involve the local proliferation of the AM. In the present study, we report that AM lavaged from the lungs of rats and mice proliferate in vitro when grown atop lung fibroblasts (LF) or when they are cultured in the presence of LF-conditioned media. Using murine AM and LF, we additionally show that the LF-derived mitogenic cytokines for the AM are macrophage colony-stimulating factor (M-CSF) and granulocyte/macrophage colony-stimulating factor (GM-CSF). Our findings suggest that LF, via the production of M-CSF and GM-CSF, may play an important role in regulating the size of the AM population during chronic inflammatory/fibrogenic lung disorders, and that the complex cytokine network that results in pulmonary fibrogenesis may involve a "coupled reciprocity" between the lung's AM and LF.

Alveolar and interstitial macrophage populations in the murine lung.

Pulmonary macrophages (PM) exist in two general anatomical compartments in the lower respiratory tract: the alveolar space (alveolar macrophages, AM) and the interstitium (interstitial macrophages, IM). We determined the relative contribution that macrophages in each of these compartments make to the size of the total PM population found in the lungs of C3H/OUJ mice, while also evaluating how efficiently bronchoalveolar lavage (BAL) removes AM from the murine lung. These objectives were accomplished by combining extensive BAL with subsequent mechanical and enzymatic dissociation of the lungs in conjunction with in situ and in vitro phagocytic assays involving opsonized erythrocytes (EA) to identify mononuclear phagocytes. On average, 2.5 x 10(6) cells were recovered by extensive BAL, and approximately 78% of these cells ingested EA in vitro. To determine the efficiency of BAL in removing PM from the alveolar space, EA were instilled intratracheally into intact lungs, which had been removed from the chest cavity, and allowed to incubate for 60 min; this was followed by exhaustive BAL and subsequent lung digestion. After these procedures, approximately 4% of the dissociated lung cells contained EA, indicating that these cells were alveolar in origin but had not been removed despite extensive BAL. Subtraction of these AM from the total EA+ cells in lung cell suspensions following a second in vitro incubation with EA indicated that approximately 37% of all PM were within the interstitium. These results suggest that, while AM comprise the majority of lung macrophages, IM constitute a larger component of the total PM population in murine lungs than previously reported. In addition, this study, like several previous investigations using other species, indicates that a significant proportion of AM remain in the lung despite attempts to remove them with BAL. Accordingly, residual AM significantly contaminate the IM population present in murine lung cell suspensions even after extensive lavage.

Airway intra-luminal macrophages: evidence of origin and comparisons to alveolar macrophages.

Airway intra-luminal macrophages (AI-LM) are a little-studied subpopulation of pulmonary macrophages that are located on the surfaces of the conducting airways of the lower respiratory tract. In this study, we: (1) developed a flow cytometric approach by which AI-LM can be viably isolated in high purity from cell suspensions obtained by airway washings; (2) comparatively examined various functional, biochemical, biophysical, and morphologic features of the rat's AI-LM and alveolar macrophage (AM) phenotypes, and (3) investigated the origin of the AI-LM in the rat. Airway cells were harvested from the tracheas of adult Fischer-344 rats, and AM were obtained from the lungs by conventional bronchopulmonary lavage or via prosthetic airway circuits that supplanted the removed tracheas. Flow cytometric analyses of lavaged airway cells revealed that the AI-LM fell within the range of the electro-optical phenotype of AM, and subsequent cell-sorting experiments demonstrated that virtually all viable AI-LM could be sorted from contaminating airway epithelial cells in greater than 95% purity based on their electro-optical characteristics, e.g., electronic volume, axial light loss, 90 degrees light scatter, and blue and green autofluorescence signals. In Fc gamma receptor-mediated phagocytic studies, approximately 90% of AM engulfed opsonized erythrocytes (EIgG) whereas only 60% of the AI-LM were able to do so. Comparisons of the numbers of EIgG in phagocytic AM and in phagocytic AI-LM indicated the AI-LM were less phagocytic. Densitometric analyses of sorted AI-LM and of sorted AM stained for acid phosphatase, nonspecific esterase, and beta-glucuronidase indicated that the activities of these enzymes were generally less in the AI-LM than in the AM. Morphometric comparisons of sorted AM and of sorted AI-LM showed that the AI-LM were generally larger than the AM and that the surfaces and nuclei of the AI-LM were more regular than those of the AM. The AI-LM were found to strongly label with the monoclonal antibody ED1, which recognizes an antigen on the surfaces of rat AM, but the AI-LM did not label with the monoclonal antibody ED2, which recognizes an antigen on the surfaces of rat peribronchial and pulmonary perivascular macrophages. Over the course of alveolar phase clearance of a lung burden of polystyrene microspheres, the frequency distributions of the particles in AI-LM and in AM were found to be virtually identical.(ABSTRACT TRUNCATED AT 400 WORDS)

Migratory behaviors of alveolar macrophages during the alveolar clearance of light to heavy burdens of particles.

We investigated the unstimulated and stimulated migratory activities of lavaged alveolar macrophages (AMs) in vitro over the course of alveolar clearance of three different mass lung burdens of microspheres. Our intent was to uncover potentially important relationships between the migratory behaviors of the AM and the retention kinetics of particles. Groups of adult, male Fischer-344 rats were intratracheally instilled with approximately 86 micrograms (low burden, LB), approximately 1 mg (medium burden, MB), or approximately 3.7 mg (high burden, HB) of polystyrene microspheres (2.13 microns diameter), or with carrier vehicle (phosphate buffered saline, PBS) alone. The lung retention kinetics of the particles were determined over an approximately 170 day period. On days 14, approximately 57, and approximately 85, lavaged AMs were assessed for their abilities to migrate through 5-microns pore membranes in response to inactivated rat serum (unstimulated condition) and yeast-activated rat serum (stimulated condition). The retention characteristics of the three burdens could be satisfactorily described by two-component, negative exponential equations. The kinetics of retention of the LB and MB were similar, although some evidence indicated the MB slightly retarded the lung clearance process. Deposition of the HB resulted in more marked prolongations of both the early, more rapid, and the slower, longer term components of alveolar clearance. The unstimulated migration indices of AMs from the particle-instilled lungs were generally not significantly different from those of AMs from PBS-instilled lungs except for a significant increase in the migration indices of LB AMs at the last assay time. The stimulated migration indices of MB and HB AMs were significantly decreased on assay days 14 and approximately 57. On day approximately 85, however, the migration indices of LB, MB, and HB AMs were all increased above the PBS AMs. Comparisons of the frequency distributions of particles in the unstimulated and stimulated AM that migrated to those in corresponding parent AM populations consistently indicated a preferential migration of particle-free AMs and of AMs with lesser loads of microspheres. The overall results of this study suggest that the unstimulated and stimulated migratory activities of particle-laden AMs are depressed in vitro. Our results also suggest that the migratory activities of generally particle-free AMs may be enhanced over a prolonged period of time following the deposition of particles in the lung.

Proliferation of rat and human lung fibroblasts following exposure to prostaglandin E2.

Numerous investigators have reported that exogenous prostaglandin E2 (PGE2) can inhibit human lung fibroblast proliferation in vitro. Yet various lines of evidence derived from clinical and experimental studies suggest that PGE2 may not be of major importance in inhibiting fibroblast proliferation in vivo. We examined the effects of exogenously-supplied PGE2 on the in vitro proliferation of HFL-1 human lung fibroblasts and rat lung fibroblasts derived from Fischer 344 rats using a multisample assay system that provided a detailed kinetic picture of PGE2 effects on fibroblast proliferation. Exogenously supplied PGE2 (5-5000 ng/ml) had no effect on the proliferation of actively cycling or initially quiescent subconfluent populations of rat lung fibroblasts. In contrast, initially quiescent subconfluent or confluent cultures of HFL-1 cells that were treated with 50-5000 ng/ml PGE2 exhibited a dose-dependent, transitory inhibition of division when stimulated to return to a state of active proliferation. Once division resumed, the cells divided at the rate of the PGE2-free control condition, even in the continued presence of the prostaglandin. This initial postinhibitory resumption of division was not attributable to the emergence of a PGE2-resistant subpopulation. Thus, although exogenously supplied PGE2 indeed inhibits proliferation of human pulmonary fibroblasts in vitro, the duration of the inhibition appears to be much shorter than previously suspected.

Alveolar macrophage-particle relationships during lung clearance.

Retention kinetics for insoluble particles that deposit in the lung oftentimes resemble a multicomponent process during alveolar clearance, with each component appearing to follow simple first-order kinetics. Inasmuch as alveolar macrophages (AM) are thought to play an important role in particle removal from the lung, a study was undertaken to examine particle-AM relationships during the clearance of particles to obtain information on potential AM mechanisms that could provide the underlying bases for the lung retention kinetics of the particles. Adult, Fischer 344 rats were intratracheally instilled with 1.6 x 10(7) (approximately 86 micrograms) polystyrene microspheres (approximately 2 microns diam). On Days 7, 14, 57, 85, and 176 thereafter, subgroups were killed, their lungs were lavaged, recovered cells (greater than 95% AM) were counted, the frequency distribution of the particles among the AM was determined (e.g., zero, 1 to 2, 3 to 4 particles/AM), and the total numbers of particles lavaged were estimated. The lavaged lungs were solubilized, and unlavaged particles were also counted. The sums of the lavaged and unlavaged particles were used to estimate retained lung burdens at each postinstillation time. The lung retention data followed a pattern consistent with the sum of two negative exponential components, i.e., an earlier, more rapid component and a slower, longer term component. The rates at which the AM disappeared from a given particle category also were biphasic for AM that contained up to 14 microspheres. The rates of both the earlier and longer term components of such disappearance were found to increase with increasing AM burdens. Over an AM burden range of 1 to 10 microspheres, the proportion of AM that disappeared via rapid components also increased as the particle burden defining an AM category increased. At higher particle burdens, the proportion of AM that disappeared by an early component appeared to markedly diminish; an early component for AM disappearance was no longer resolvable for AM that contained greater than 15 microspheres. The net effect of these phenomena was that retained lung burdens over time became progressively contained in AM with lesser burdens of particles. The results from this study suggest that the rate(s) of translocation of particle-containing AM from the lung during lung clearance may be related to their individual particulate burdens. These findings, however, are also consistent with a gradual redistribution of particles among the lung's AM population over time concurrent with AM removal from the lung. Regardless, the biphasic nature of the lung retention data qualitatively was generally evident for particle-containing AM as well.

Leukocytic responses to the intrapleural deposition of particles, particle-cell associations, and the clearance of particles from the pleural space compartment.

Polystyrene microspheres (2.02 micron diameter) were instilled in the pleural space compartment (PSC) of the Fischer 344 rat. Rats that were administered carrier vehicle only, and untreated rats served as controls. On d 1, 6, 14, and 28 following the instillations, the pleural free cells were harvested by pleural lavage and the major cell phenotypes retrieved were quantitated to determine how the pleural free cell population was altered by the particles. Concurrent with these studies, quantitative analyses were performed to determine the numbers of particles lavaged from the pleural space at the above sacrifice times, and these particle retention data were used to estimate particle translocation rates from the PSC. Deposition of the particles in the PSC brought about an early recruitment of polymorphonuclear leukocytes (PMN) and an enlargement in the size of the pleural mononuclear phagocyte (PMP) population. PMN numbers substantially decreased by d 6, but continued to remain elevated over the course of the study. The early increase in PMP appeared to subside by d 6, but again increased thereafter. Other notable changes were decreases in the size of the pleural eosinophil population at later times after particle deposition, and an approximate 200-fold increase in lymphocytes by d 28. The particles were cleared from the PSC with a biphasic pattern. The most rapid phase, which accounted for the clearance of greater than or equal to 80% of the particles, had a t1/2 = 0.3 d and the slower component had a t1/2 = 6 d. Most of the particles were translocated from the PSC to the retrosternal, caudal mediastinal tissue. The slower phase of particle clearance appeared to be macrophage-mediated, suggesting the t1/2 for macrophages in the PSC is also approximately 6 d.

Flow cytometric characterization of alveolar macrophages.

Phenotypes of lung free cells (FC) harvested from Fischer-344 rats by episodic lavage were characterized by flow cytometry. Parameters evaluated included electronic volume (EV), axial light loss (ALL), 90 degrees light scatter (LS), blue autofluorescence (BA), and green-yellow autofluorescence (G-YA). Three phenotypic populations, FC-A, FC-B, and FC-C were identified by their differing LS characteristics. FC-C represented 90% of the cells and were exclusively alveolar macrophages. Two subpopulations in FC-C, FC-CI and FC-CII, were further distinguished by their unique ALL features. The morphologic appearances of these subpopulations by light microscopy clearly differed in sorted preparations. Based on their patterns of autofluorescence, these FC-CI and FC-CII phenotypes were found to be composed of eight subpopulations. In FC populations harvested during further lavage episodes of the same lungs, the relative contributions of FC-CI to the FC-C subpopulation decreased as FC-CII correspondingly increased. This study demonstrates 1) that subpopulations of lavaged AM can be categorized according to their optical phenotypes by flow cytometry and 2) that the relative frequency of retrieval of some phenotypes depends on how exhaustively the lungs are lavaged. With regard to the latter, bronchoalveolar lavage does not randomly sample the underlying AM population in the alveolar compartment.

Translocation of particles to the tracheobronchial lymph nodes after lung deposition: kinetics and particle-cell relationships.

One of the pathways for particle clearance from the lung involves the translocation of particles from the alveoli to the tracheobronchial lymph nodes. Details of the mechanisms involved in this translocation process have not been well delineated. In the present study, we: assessed the kinetics of particle transfer to the tracheobronchial lymph nodes in the rat over a 30-day period following the intrapulmonary deposition of 4 X 10(8), 1.9 micron, fluorescent polystyrene microspheres; utilized multiparameter flow cytometric technology to quantitatively differentiate between "free" and cell-associated particles that accumulated in the lymph nodes; and assessed the role of alveolar macrophages (AM) in carrying particles to the lymph nodes by comparing the distributions of particles in lavaged AM with distributions of particles found in nodal mononuclear phagocytes (NMP). The accumulation of particles, most of which were extracellular, in the nodes was biphasic with the most rapid phase occurring within the first 24 hr. Over the Day 1-30 period, the numbers of particles in the lymph nodes increased linearly to approximately equal to 1.2 X 10(6) microspheres, or approximately equal to 0.3% of the originally instilled lung burden. The percentages of the nodal particles that were associated with NMP over the course of the study were inversely proportional to nodal particulate burdens, even though the percentage of cells with engulfed particles increased; the percentage of NMP asymptotically approached a maximum value over the range of nodal burdens of 6-12 X 10(5) particles. The distributions of the microspheres in the NMP were essentially identical on Days 1, 14, and 30. Major differences in the distributions of particles in lavaged AM and NMP were not consistent with the notion that the latter represented translocated AM.

Pulmonary macrophages: alveolar and interstitial populations.

The sizes of the alveolar macrophage (AM) and interstitial macrophage (IM) populations in the lungs of adult Fischer-344 rats were determined during steady state. AM labeled with opsonized erythrocytes during an in situ phagocytic assay were lavaged from excised lungs. The lungs were then dispersed into single-cell suspensions with collagenase and mechanical agitation, and the remaining mononuclear phagocytes were identified following a second labeling step. The size of the AM population was 1.3 X 10(7) cells, or approximately equal to 3% of the total lung cell population. The AM were negative for cytoplasmic myeloperoxidase granules. The size of the IM population was 8 X 10(6) cells, or approximately equal to 2% of the total lung cell population. IM were also negative for myeloperoxidase and, like AM, demonstrated marked Fc gamma R-mediated phagocytic activity. The high cell yields (approximately equal to 4 X 10(8) cells/lung; viability, greater than 85%) and the percentages of type II cells (11%) and ciliated epithelial cells (less than 0.5%) indicated the enzymatic dispersion method resulted in a highly efficient and representative sampling of the lung parenchyma. The collagenase method used in this study to disperse the lung cells into single-cell suspensions, in conjunction with additional cell separation techniques, may be of potential use for isolating enriched populations of IM, as well as other lung cell types, for in vitro study.