Poly I:C accelerates development of diabetes mellitus in diabetes-prone BB rat.

We developed a new experimental model of accelerated diabetes mellitus in the genetically susceptible diabetes-prone BB rat with the administration of the IFN-alpha inducer poly I:C. With this model, there was both an increased incidence and accelerated onset of insulin-dependent-diabetes in poly I:C-treated animals compared with saline-treated controls. All twelve rats administered poly I:C (5 micrograms/gm body weight 3 times/wk) developed diabetes by 57 days of age (100%) compared with 1 of 27 (3.7%) saline-treated controls. Furthermore, the development of diabetes was accelerated in the poly I:C-treated group (mean age +/- SE at onset 52.8 +/- 0.58 days) compared with saline-treated controls (89.3 +/- 2.4 days, P less than 0.01). Additionally, poly I:C-treated rats had higher mean serum IFN-alpha levels than saline-treated rats at weeks 2 and 3 of treatment (210 vs. 27 and 183 vs. 25 U/ml, respectively, P less than 0.001). Poly I:C treatment of 5 Wistar rats, the parental strain, which is not susceptible to diabetes, did not result in insulitis, diabetes, or hyperglycemia. The histopathologic findings of insulitis and decreased immunoreactive islet insulin in poly I:C-accelerated diabetic BB rats and in BB rats with spontaneous diabetes suggest a similar pathophysiology.

Gender-related variations and interaction of human neutrophil cyclooxygenase and oxidative burst metabolites.

Gender-related variations in human neutrophil membrane bound oxidative metabolism were evaluated employing the calcium ionophore A23187. These included the measurement of cyclooxygenase metabolites of arachidonate by specific RIA determination of thromboxane B2 (TxB2), prostaglandin E2 (PGE2), and 6-Keto PGF1 (6KPGF) as well as the initiation of the oxidative burst by the quantitative evaluation of superoxide (O-2) reduction of nitroblue tetrazolium (NBT). Neutrophils from women generated 30% less TxB2 and PGE2 than those obtained from men. In contrast, the neutrophils from women demonstrated relatively higher O-2 production with a cyclic pattern of both TxB2 and O-2 which correlated with their menstrual cycle. The elevated O-2 generation appeared to inversely correlate with TxB2 production. Further, introduction of an intracellular oxygen centered radical (OCR) scavenger, sodium benzoate, for the hydroxyl (.OH) radical was observed to affect cyclooxygenase metabolism in a dose-response manner. At higher concentrations of sodium benzoate, i.e., 10(-2) M, TxB2 production was inhibited; in contrast, 10(-3) M sodium benzoate enhanced neutrophil TxB2 generation which was particularly marked during times of increased oxidative burst activity, i.e. O-2 production. We conclude that the decreased production of cyclooxygenase metabolites observed in neutrophils from women in part derive from an increased oxidative burst activity. This suggests that a regulatory mechanism may exist between the neutrophil membrane bound oxidative system(s) involving oxygen centered radicals generated during both the oxidative burst and prostaglandin cyclic endoperoxide reduction. Further, these gender-related differences may be partially attributable to variations in circulating endogenous sex steroids.

Developmental aspects of pulmonary defenses in children.

It is now generally accepted that maturational immaturity of lung defense system(s) exists in the young infant and child which, together with other maturational deficiencies of immunologic systems, contribute to their undue susceptibility to infections, many of which are associated with pulmonary infections. The alveolar macrophage (AM), of central importance to lung defense, has been studied extensively in the neonatal rabbit by our group and in the human, by others. Collectively, the results indicate small numbers of morphologically and functionally immature AMs prior to birth followed by a dramatic increase within the first 24 hr. This increase coincides with the large release from type II cells of surfactant, which not only may be involved in the functional maturation of developing AMs but also, if present in large quantities, may lead to decreased activity of these cells. There is also an age-related increase in chemotactic and microbicidal activity of AMs during maturation. Deficiencies in bone marrow pools, complement and cytokines (IFN-gamma, TNF-alpha) as well as IgG isotype switching are known to occur in the neonate which may contribute to increased susceptibility to infection and which are amenable to immune interventions.

Current concepts of immune interventions in children with respiratory diseases.

In addition to their role in oxygen transport and ventilation, the lungs serve as an important defense function consisting of nonspecific and specific components. The nonspecific factors include aerodynamic filtration, mucociliary apparatus, bronchoalveolar fluid flow and lymphohematogenous drainage (anatomic systems) as well as phagocytosis and inflammation; the specific factors include B-cell immunity (IgG, A, M, D and E) and T-cell immunity (cell-mediated immunity). In the lungs, specialized lymphoid tissues in contact with epithelium (bronchus-associated lymphoid tissues; BALT) function in local antibody (secretory IgA) and cell-mediated immunity responses to foreign antigens. Based upon these considerations, a number of therapeutic interventions have been developed to enhance various components of lung defense. These include substances which enhance both nonspecific elements (leukocyte transfusion, plasma, nonspecific immunostimulants, e.g., immunoactive bacterial extracts) as well as specific elements (vaccines, intravenous gammaglobulin, plasma, interferons, cytokines). The need for further development and utilization of new immune interventions is underscored by the large number of infants and children who suffer from recurrent respiratory infections, who have either maturational immaturity (e.g. small for gestational age newborn), genetically determined (e.g. cystic fibrosis) or acquired defects (e.g. AIDS) of lung defense mechanisms. The emergence of antibiotic-resistant bacterial organisms, e.g. Streptococcus pneumoniae, poses an additional need for new immune interventions.

Functional and ultrastructural changes in alveolar macrophages from rabbits colonized with Bordetella bronchiseptica.

Alveolar macrophages from rabbits colonized with Bordetella bronchiseptica in their respiratory tract exhibited significant decreases in cell adherence, phagocytic uptake, and bactericidal activity compared with macrophages from uncolonized animals. These dysfunctions were accompanied by ultrastructural changes, including a decrease in overall cell density, a vacuolation of the rough endoplasmic reticulum, and an increase in organelle-poor cell surface projections.

Proliferation of alveolar macrophages in hyperoxia.

The effect of oxygen on the proliferative response of alveolar macrophages (AMs) was investigated. Alveolar macrophages cultured in hyperoxic atmosphere (95% O2 + 5% CO2) for 18 hours showed increased incorporation of [3H]-thymidine and proliferation in contrast to those cultured in a control atmosphere (95% air + 5% CO2). The proliferating cell was shown to be a macrophage by morphology, esterase staining, and phagocytic ability. The results suggest an oxygen-induced proliferation of AMs that may play a critical role in AM influx into the alveoli particularly at times of hyperoxia, eg, the neonatal period.

Chemotactic and candidacidal responses of rabbit alveolar macrophages during postnatal development and the modulating roles of surfactant in these responses.

Chemotactic responses of alveolar macrophages from 1-, 7-, and 28-day-old rabbits to various concentrations of endotoxin-activated serum and n-formyl-methionyl-phenylalanine were tested utilizing both blind well and agarose plate assay systems. A dramatic increase in both the chemotactic response and responsiveness to various concentrations of chemoattractant was observed during postnatal maturation. The pattern of result was similar with both methods of assay. An age-related increase was also found to occur in the candidacidal activity of alveolar macrophages in contrast to their phagocytic uptake, which showed no age-related increases. Furthermore, the decreased function of macrophages from newborn animals correlated with a morphologically and biochemically less mature cell population which contained large amounts of phagocytosed surfactant-related material. Moreover, pretreatment of macrophages from 7- and 28-day-old animals with vesicles of surfactant-related material resulted in decreases in both chemotactic and candidacidal activity, with a paradoxical increase in their phagocytic activity. The resulting activities were similar to those of macrophages from 1-day-old animals treated with buffer alone. These data suggest that there is an age-related increase in the chemotactic and candidacidal activity of alveolar macrophages during maturation and that the decreased activity of macrophages from newborn animals is related in part to the large amount of surfactant-related material present at that time.

Chemotactic responses of various differentiational stages of neutrophils from human cord and adult blood.

Chemotactic response and responsiveness, that is, the ability to respond to various concentrations of the chemoattractant zymosan-activated serum was measured for cord and adult blood neutrophils. Chemotactic response of neutrophils from adult blood was two to seven times greater than that of neutrophils from cord blood. In addition, neutrophils from cord blood showed poor response to concentrations of 25% chemoattractant or less, compared to those from adults, which responded to concentrations of 10%. Further, it was found that approximately 30% of neutrophils in cord blood were the band form compared to only 9% in adult. Based upon this, a simple method was devised to assay mean migrating distance of various differentiational stages of neutrophils incorporating both distance and magnitude of their responses. The results of these assays showed that all differentiational stages of cord blood neutrophils have a mean migrating distance less than those of adults. In addition, the band form from both cord and adult blood had a mean migrating distance less than the polymorphonuclear form. Adherence studies revealed that all differentiational stages of neutrophils adhered in a similar manner, and no difference was detected between cells from cord and adult blood. Assays to test the ability of cord blood to produce chemotactic activity when activated revealed 50% less activity than that obtained from adult serum which further decreased with dilution of attractant. These data suggest that both the cellular and humoral systems involved in chemotactic responses are less in cord blood compared to adult blood.

Maturation of the rabbit alveolar macrophage during animal development. III. Phagocytic and bactericidal functions.

Phagocytic and bactericidal function of rabbit alveolar macrophages (AMs) lavaged from animals during the course of postnatal maturation was studied. Staphylococcus aureus and a temperature-sensitive mutant of Escherichia coli, which could not replicate at 37 degrees during the functional assays, were employed as test bacteria. Assays of the phagocytic capacity of AMs from rabbits of various age groups revealed no significant differences either in the percentage of AMs which took up bacteria (79-90%) or in the number of bacteria taken up per AM (Table 1). In contrast, bactericidal activity of AMs was found to increase with increasing animal age. No bactericidal activity was detected in AMs from newborn animals (Figs. 1 and 2), whereas AMs from 7-day-old animals exhibited at least a bacteristatic activity against S. aureus (Fig. 1) and AMs from 28-day-old rabbits showed marked bactericidal activity, essentially the same as that of AMs from adult rabbits. Adult AMs killed 75% of the S. aureus and 60% of the E. coli within 120 min (Figs. 1 and 2).

Ideal target organism for quantitative bactericidal assays.

We have developed a target organism which permits quantitative bactericidal assays. The organism is an Escherichia coli mutant which cannot grow at the temperature of the assay (37 degrees C), but retains full colony-forming potential for subsequent quantitation at 25 degrees C. We show that quantitative data on the bactericidal capacity of polymorphonuclear leukocytes and alveolar macrophages can be obtained when this mutant is used as a target. The procedure used to generate the strain is described in detail and should be applicable to many bacterial species. Characterization of the properties of the mutant indicates that it has a strong potential for use in other in vivo and in vitro investigations of host responses to microbial invasion.

Maturation of the rabbit alveolar macrophage during animal development. I. Perinatal influx into alveoli and ultrastructural differentiation.

Rabbit alveolar macrophages (AM's) were collected by tracheobronchial lavege at sequential times during animal development. The total number of cells recovered by this technique was found to increase markedly shortly before birth (Fig. 4). This apparent influx of macrophages into the alveoli continued during the first postnatal week, and, at a reduced rate, throughout the first postnatal month of life (Fig. 3). Ultrastructurally, AM's of the prenatal period resembled their monocyte precursors, and contained modest numbers of lysosomes and mitochondria, scant lamellae of ribosome-studded endoplasmic reticulum (RER), and a small Golgi apparatus (Fig. 12). A considerable amount of phagocytosed material was observed in these AM's, and consisted largely of cellular debris and two forms of surfactant-related phospholipids, termed tubular and lamellar myelin (Figs. 12-15). The quantity of these ingested phospholipids increased dramatically shortly after birth, in correlation with the known release of similar material from type II pneumocytes at this time. (Figs. 16 and 19). During the first postnatal week AM's showed a considerable increase in number of mitochondria and in the development of the RER and Golgi apparatus (Fig. 22). Increased accumulations of lipid droplets were also noted during this period. Ingested material continued to consist largely of surfactant-related phospholipids, but was less abundant at this time. By 28 days after birth, AM's were nearly morphologically mature (Fig. 25). They showed large numbers of lysosomes and mitochondria, and well developed RER and Golgi apparatuses. Ingested phospholipid material was still visualized, and the incomplete degradation of this material appeared to give rise to the dense intraphagolysosomal whorls characteristic of the mature AM.

Age-dependent susceptibility of neonatal rats to group B streptococcal type III infection: correlation of severity of infection and response of myeloid pools.

A distinct age-dependent susceptibility to group B streptococcus type III (GBS) was demonstrated, utilizing a neonatal rat model. The most dramatic changes in susceptibility occurred within the first 7 days of postnatal life. To further investigate this susceptibility, experiments were performed utilizing two age groups of rats: (i) animals within the first 24 h of life (NB) and (ii) 7-day-old animals (7d). The infective dosage used was 10(4) GBS per g of body weight, a dose lethal to 100% of NB but only to 15% of 7d. The responses of the myeloid cells in the peripheral blood, spleen, and bone marrow were evaluated at intervals during the first 24 h post-GBS infection. The susceptibility of the NB to GBS appeared to be associated with a number of events, including smaller base-line levels of myeloid elements particularly in the bone marrow, a lag of at least 2 h in their initial response to infection, and an inability to maintain the myeloid pools. The band form of neutrophils appeared to be the predominant cell type in both total number and rapidity of response to infection. Moreover, an initial depletion of this band form was seen in both groups, which returned to base-line levels with recovery in 7d but persisted until death in NB animals. Similarly, shifts in numbers of peripheral nucleated erythrocytes appeared to reflect changes in the myeloid storage pools, with numbers of nucleated erythrocytes significantly decreasing in 7d animals with recovery in contrast to persistence in NB until death. Therefore, shifts in these cells in peripheral blood during infection appear to reflect the state of myeloid storage pools which parallel disease outcome.

Genetically stable temperature-sensitive mutants of Salmonella typhi induce protection in mice.

Temperature-sensitive (ts) mutants of Salmonella typhi were isolated following treatment with nitrosoguanidine, and characterized with respect to cut-off temperature, ts phenotype and reversion frequency. Linkage of the ts mutations to selectable chromosomal markers was established by generalized transduction with bacteriophage phage Vi I, and the appropriate antibiotic resistances were transduced into the ts mutants. Multiple mutant S. typhi were then constructed by combining three independent ts mutations in one strain, utilizing linkage of three of the mutations to erythromycin-, streptomycin- and methylglyoxal-resistance. Several recombinants are genetically stable, with calculated reversion rates of less than 10(-22), and induce both protection from intraperitoneal challenge with the virulent parental wild-type S. typhi in mice and the formation of antibodies to the somatic O-9 and O-12, the flagellar H and the capsular Vi antigens.

Study of the effects of vacuuming on the concentration of dust mite antigen and endotoxin.

BACKGROUND: Dust mite antigens are major sources of allergens in house dust and together with endotoxin, a proinflammatory component of gram negative bacteria also found in house dust, are important causes of tissue injury involved in the pathogenesis and severity of allergic diseases, eg, asthma. OBJECTIVE: To determine the effectiveness of vacuuming in reducing the quantity of dust mite antigens, Dermatophagoides pteronyssinus (Der p and Der p2) and endotoxin using a quantitative ELISA assay and to correlate results with those obtained using a qualitative rapid dipstick method for Der p2. METHODS: Four specimens of house dust were collected using a Kirby Model G5 vacuum cleaner with a Micron Magic Filtration system from an approximately 54" x 18" standardized area of rug from each of 20 homes at 4 time intervals over a 6-week period, ie, a baseline specimen #1 at 0 week; specimen #2 at 1 week; specimen #3 at 5 weeks (1 month after specimen #2); specimen #4 at 6 weeks (1 week after specimen #3). Three intervals were compared, ie, period 1-2 (1 week), period 2-3 (1 month), and period 3 to 4 (1 week). The concentrations of Der p1 and Der p2 were determined in dust samples using a standard ELISA assay and the concentration of endotoxin was detected using a limulus amebocyte lysate assay. Concentrations of Der p2, determined by the standard ELISA assay, were compared to those in the same samples determined by a rapid dipstick method. RESULTS: A wide range of values for total weight of unprocessed dust (0.3 to 59 g, X = 8.7) and finely sieved dust (0.1 to 19 g, X = 3) from all specimens were found. In finely sieved dust specimens the mean concentrations of Der p1, Der p2 and endotoxin were 775, 1310, and 3836 ng/g of dust, respectively. Following weekly vacuuming there was an increase in concentration of Der p1, Der p2, and endotoxin in 20%, 35%, and 63% of the houses, respectively, compared to in monthly vacuuming in which increases were seen in 65%, 50%, and 63% of the houses, respectively. In contrast, there was a decrease in concentration of Der p1, Der p2 and endotoxin with weekly vacuuming in 43%, 60%, and 37% of the houses respectively versus in monthly vacuuming in 15%, 35%, and 37% of the houses respectively. A correlation coefficient of 0.7 was found for the concentration of Der p2 in 37/40 samples tested detected using the ELISA method compared with rapid dipstick assay. CONCLUSION: The results of this study support the effectiveness of vacuuming on the reduction of dust mite antigens (Der p1 and Der p2) ie, Der p2 > Der p1. This reduction was more pronounced with weekly compared with monthly vacuuming. No reduction in the concentration of endotoxin was found. A good correlation was found between results obtained by ELISA and rapid dipstick assay for Der p2.

Alternative routes of immunization for prevention of infectious diseases: a new paradigm for the 21st century.

The prevention of infectious diseases by the use of vaccines represents one of medicine's greatest triumphs during the 20th century. This era has witnessed the global eradication of smallpox as a result of Jennerian cowpox vaccination, the elimination of paralytic poliomyelitis from the western hemisphere, and within 5-10 years the anticipated eradication of poliomyelitis worldwide as a result of the poliovirus vaccines. Next slated for worldwide eradication is measles, the great killer of infants and children, which each year extracts a global mortality of one million victims. Throughout the 20th century the percutaneous (i.e., subcutaneous or intramuscular) route has almost exclusively been the preferred way to administer vaccines. However, as a result of several important scientific discoveries made during the 20th century, including new tissue-culturing techniques, the development of recombinant DNA technology, and genetic sequencing, a whole new generation of tailor-made modern vaccines has become available, including DNA vaccines and transgenic plant vaccines. Moreover, it became apparent that alternative routes of administration of vaccines, such as by aerosol immunization and transcutaneous skin patches, might be more appropriate and more effective than immunization via the parenteral route. This paper describes some of the recent advances relating to alternative methods of immunization and will focus primarily on the development and use of respiratory aerosolized vaccines.

Inflammatory responses to Pseudomonas aeruginosa and Staphylococcus aureus in the murine lung.

The changes in pulmonary cell population in response to aerosols containing either Pseudomonas aeruginosa or Staphylococcus aureus were studied in a murine model. The lungs of inbred DBA/2J mice received an inoculum of 2 X 10(5) colony-forming units of the microorganism and lung lavages were performed at various time intervals thereafter. P. aeruginosa aerosols produced an immediate decrease in the number of resident alveolar macrophages (AM), followed by a two-waved recruitment of cells into the respiratory tract; the first wave was composed of polymorphonuclear leukocytes (PMN) and the second of monocyte-like peroxidase-positive AM. The change in cell populations was transient and returned to baseline values within a week after aerosolization. In contrast, aerosolized S. aureus initially induced a slight increase in mononuclear cells, and by 60 min after aerosol exposure, the cell population was not different from that of control animals.