Airway reactivity following repeated milk aspiration in rabbits.

Asthma and gastroesophageal reflux (GER) are commonly associated disorders. Microaspiration is one possible link between these processes. The purpose of this study was to assess methacholine reactivity following repeated small-volume aspiration such as may occur with GER. This was also correlated with airway cytology. Five weekly intratracheal instillations of either milk (N = 8) or saline sham controls (N = 7) in volumes of 0.25 mL/kg were performed in anesthetized rabbits. Transpulmonary pressure, flow, tidal volume, central airways resistance, and dynamic lung compliance were measured in anesthetized and paralyzed animals at baseline, after 2 and 5 weeks of instillation, and 3 weeks after the last instillation. Doubling concentrations of methacholine were given until a 50% or greater increase in resistance occurred (PC50R). Bronchial washings for cytological evaluation were performed after the physiologic measurements and before each instillation. There were no significant differences in airway reactivity between baseline and all subsequent observation points within each of the two groups. However, methacholine responsiveness was significantly higher in the milk group before the final instillation (PC50R = 5.84 vs. 12.97 mg/mL, P = 0.03) and at recovery (PC50R = 6.40 vs. 10.56 mg/mL, P = 0.047) when compared to saline controls. This was associated with a higher neutrophil percentage (P = 0.01) at 5 weeks, and eosinophil percentage (P = 0.05) at recovery in the bronchial wash specimens from the milk group. These results show that repeated small-volume aspiration of milk in rabbits causes persistent inflammation and is associated with greater airway reactivity when compared to sham controls. This inflammation was accompanied by either increased neutrophils or eosinophils in bronchial lavage specimens. These findings lend support to a possible role of microaspiration in association with increased airway reactivity in patients with GER.

High consumption of fatty fish from the Baltic Sea is associated with changes in human lymphocyte subset levels.

Fatty fish from the Baltic Sea accumulate immunotoxic persistent organochlorine compounds. In a previous study we found inverse correlations between such fish consumption and natural killer (NK) cell levels in a Swedish population. The present study concerns 68 Latvian subjects with high, low or intermediate fish consumption. High fish consumption correlated positively with B cell levels and CD4+/CD8+ ratios, but negatively with levels of cytotoxic (CD8+) T cells. Furthermore, NK cell levels correlated inversely with plasma selenium, one of several strong correlates with fish intake. A high fish diet includes a set of possible immunomodulating agents. It is presently not possible to pinpoint the cause for the observed subset deviations or to establish their possible biological importance.

Parameters of immunological competence in subjects with high consumption of fish contaminated with persistent organochlorine compounds.

Consumption of fatty fish species, like salmon and herring, from the Baltic Sea is an important source of human exposure to persistent organochlorine compounds, e.g. polychlorinated dioxins (PCDDs), dibenzofurans (PCDFs) and biphenyls (PCBs). Many of these compounds show immunotoxic and hepatotoxic effects in animals. We have now studied immunological competence, including lymphocyte subsets, in 23 males with a high consumption of fish from the Baltic Sea and in a control group of 20 males with virtually no fish consumption. The high consumers had lower proportions and numbers of natural killer (NK) cells, identified by the CD 56 marker, in peripheral blood than the non-consumers. Weekly intake of fatty fish correlated negatively with proportions of NK cells (rs = -0.32, P = 0.04). There were also, in a subsample of 11 subjects, significant negative correlations between numbers of NK cells and blood levels of a toxic non-ortho-PCB congener (IUPAC 126; rs = -0.68, P = 0.02) and a mono-ortho congener (IUPAC 118; rs = -0.76, P = 0.01). A similar correlation, in 12 subjects, was seen for p,p'-DDT (rs = -0.76, P = 0.01). The corresponding negative correlation, in 13 subjects, with blood levels of PCDD/Fs was not significant (rs = -0.57, P = 0.07). No significant association was seen between organic mercury in erythrocytes and NK cells. Fish consumption was not associated with levels of any other lymphocyte subset. Neither were there any correlations with plasma immunoglobulins or liver enzyme activities. Our study indicates that accumulation of persistent organochlorine compounds in high consumers of fatty fish may adversely affect NK cell levels.

Time course of lipid-laden pulmonary macrophages with acute and recurrent milk aspiration in rabbits.

High levels of lipid-laden macrophages (LLM) in bronchial washings have been associated with food aspiration. We studied the time course of appearance and clearance of LLM in rabbits undergoing either a single milk instillation, five weekly milk instillations or saline (control) instillations into the airways. Cells were obtained by bronchoalveolar lavage of intubated rabbits at uniform time intervals following the single or the last of five milk/saline instillations. LLM semi-quantitative indexes were derived using oil-red-O staining. Significantly elevated indexes were found in both milk groups 6 hr after milk instillation. In the single saline and milk instillation groups the indexes were not different beginning on the 4th day, and indexes from 8 of 9 rabbits had returned to baseline by the 6th day. However, indexes remained significantly elevated up to 17 days in the group receiving weekly milk instillations. Indexes from all rabbits in the repeat milk instillation group remained elevated for 12 days or longer. This group also developed increased numbers of binucleated macrophages. Quantitation of LLM in this model appears to be a sensitive indicator of recurrent lipid aspiration, these cells remaining in the airways for several days after the last aspiration event.

Comparison of the limulus amebocyte lysate test and gas chromatography-mass spectrometry for measuring lipopolysaccharides (endotoxins) in airborne dust from poultry-processing industries.

The lipopolysaccharide (endotoxin) content in airborne dust samples from three different poultry slaughterhouses was determined with both the chromogenic Limulus amebocyte lysate assay and gas chromatography-mass spectrometry analysis of lipopolysaccharide-derived 3-hydroxy fatty acids. Gram-negative cell walls were also measured by using two-dimensional gas chromatography/electron-capture analysis of diaminopimelic acid originating from the peptidoglycan. The correlation between the results of the Limulus assay and those of gas chromatography-mass spectrometry for determination of the lipopolysaccharide content in the dust samples was poor, whereas a good correlation was obtained between lipopolysaccharide and diaminopimelic acid concentrations with the gas chromatographic methods. The results suggest that it is predominantly cell-wall-dissociated lipopolysaccharides that are measured with the Limulus assay, whereas the gas chromatographic methods allow determination of total concentrations of lipopolysaccharide, including Limulus-inactive lipopolysaccharide, gram-negative cells, and cellular debris.

Health effects of exposure to endotoxins and organic dust in poultry slaughter-house workers.

Twenty-three dust-exposed shacklers in the hanging departments of four poultry slaughter-house plants were examined immediately before work on a Monday morning with a standardized interview, pulmonary function tests (VC and FEV1), and blood sampling for analysis of complement factors. The examinations were repeated immediately after work the same day. Further, the individual breathing zone levels of both total dust and endotoxins were monitored during the whole work-shift. Moreover, spot samples of airborne bacteria and fungi were collected. The mean level of total dust was 6.3 mg/m3 (range 0.4-15.3 mg/m3) and of endotoxins 0.40 micrograms/m3 (range 0.02-1.50 micrograms/m3). Total levels of 4 x 10(5)-4 x 10(6) cfu/m3 of airborne bacteria, mainly coagulase-negative staphylococcal strains, but only 500-4000 cfu/m3 of fungi were found in the hanging departments. An over-shift increase in respiratory symptoms was found, but none of the workers had experienced any symptoms indicating extrinsic allergic alveolitis (EAA) or organic dust toxic syndrome (ODTS). Further, mean over-shift decreases of VC (3.1%) and FEV1 (4.1%) were found, indicating a harmful effect on the bronchi. There were, however, no associations between these over-shift decreases and the individual time-weighted average breathing zone levels of either total dust or of endotoxins. No over-shift change in serum complement factors was observed.

Mitogenic properties of two distinct forms of toxic shock syndrome toxin-1 separated on hydroxyapatite by high-performance liquid chromatography.

The homogeneity of a purified staphylococcal toxic shock syndrome toxin-1 (TSST-1) was tested by high-performance methods. This preparation was homogenous in ion-exchange chromatography and isoelectric focusing (pI = 7.4), but was resolved into two distinct peaks by high-performance hydroxyapatite chromatography. Both components, TSST-1hA and TSST-1hB had similar molecular weights (22 kD) and amino acid compositions. TSST-1 did not dimerize or polymerize upon heating at 60 degrees C for 30 min or in solutions with pH varying from 4.0 to 8.5. TSST-1hA and TSST-1hB showed similar immunological reactivity to native TSST-1 goat polyclonal antibodies. TSST-1hA and TSST-1hB as well as staphylococcal enterotoxin A and staphylococcal exfoliative toxin were potent mitogens in lymphocyte proliferation assays. The lymphocyte proliferative response to 10 pg of TSST-1hB was comparable to a response elicited by 10 ng of TSST-1hA, suggesting that the former component is a more potent mitogen. Rabbit or goat polyclonal antibodies to native TSST-1 efficiently neutralized both TSST-1 components. Heat treatment at 80 degrees C for 15 min had minimal or no effect on the mitogenic properties of TSST-1hA and TSST-1hB.

Determination of homozygosity or heterozygosity for the G2m(n) allotype by a monoclonal, precipitating antibody.

Monoclonal antibody SH 21 was found to be a good anti-IgG2m(n) reagent both for the conventional typing (HA inhibition) and for precipitation in polyethylene glycol containing gel. Antibody SH 21, together with monoclonal antibody HP 6014 (anti-IgG2), was used for the discrimination between G2m(+n) homozygotes and heterozygotes in a double diffusion assay, where the two antibodies and a serum sample were placed in corners of a triangle. In the case of a homozygote SH 21 was able to stop the diffusion of IgG2 to a precipitation line, but some IgG2 of a heterozygote was able to diffuse beyond the precipitate, forming a crossed precipitation pattern. The validity of the typing was checked with serum samples from 54 families. All the 33 'obligatory heterozygotes' (G2m(+n) parent(s) of a G2m(-n) child, or the other way round) exhibited the crossed precipitate. The frequency of the G2mn allele in a Finnish population of 297 unrelated adults was 0.409.

Inhibition of ConA-induced proliferative response in human lymphocytes by podophyllotoxin and a detergent, Tween80.

Podophyllotoxin (CPH86), at concentrations of 5 ng/ml or more, inhibited [3H]-thymidine uptake of ConA-induced proliferation of human peripheral mononuclear blood cells. Tween80, the detergent included in the anti-inflammatory drug Proresid, containing podophyllum lignan derivatives, also inhibited lymphocyte proliferation in a dose-dependent manner. Combination of the two drugs did not, or did only weakly, potentiate the inhibitory effect of each drug.

Biological effects in a chemical factory with mutagenic exposure. I. Cytogenetic and haematological parameters.

Analysis in two independent laboratories demonstrated no significant differences in chromosome aberrations or micronuclei in lymphocytes from peripheral blood between workers in a chemical factory (exposed to a mixture of chemicals, such as piperazine, low levels of ethylene oxide and formaldehyde, aromatic nitrogen compounds, and other aromatic compounds) compared to unexposed control subjects. The chemical workers had significantly higher counts of lymphocytes (3.57 vs. 2.82 X 10(9)/l; P less than 0.001), eosinophils (0.27 vs. 0.20 X 10(9)/l; P = 0.005), and basophils (0.06 vs. 0.05 X 10(9)/l; P = 0.01) than the controls. Twenty workers exposed to piperazine had higher total numbers of B-lymphocytes than control subjects.

Binding of histamine-albumin conjugates to human lymphocytes: evidence for labelling of histamine H-1 but not H-2 receptors.

Histamine conjugated to human serum albumin (HSA) bound to a major proportion of human peripheral blood lymphocytes in a rosetting assay. In contrast, red cells coated with the control conjugates pentylamine-HSA and ethanolamine-HSA were unreactive. Amine conjugation by the carbodiimide method imposed a positive charge on the albumin molecule. However, the percentage histamine-HSA-binding lymphocytes was related to the amount of histamine coupled to the albumin molecule and not to differences in isoelectric point (pI) of the modified protein carriers. Four different histamine-albumin conjugates and one pentylamine-HSA conjugate inhibited lymphocyte binding of histamine-HSA, whereas ethanolamine-albumin, carbodiimide-reacted HSA and native albumin were not inhibitory. The histamine H-2 receptor antagonists cimetidine, ranitidine, metiamide, and burimamide lacked inhibitory effects. The histamine H-1 receptor antagonists clemastine, mepyramine, and diphenhydramine efficiently inhibited lymphocyte binding of the histamine-albumin conjugate with IC50 values of 0.2, 1, and 40 mM, respectively, indicating interactions with low affinity H-1 receptors.

C2 deficiency, moderately low IgG2 concentrations and lack of the G2m(23) allotype marker in a child with repeated bacterial infections.

C2 deficiency was demonstrated in an 11-year-old boy with a past history of recurrent purulent otitis media, pneumonia, H. influenzae meningitis and S. pneumoniae septicaemia. The major histocompatibility complex haplotypes present, A10, B18, DR2, BF*S, C2*QO, C4*A4, C4*B2 and A28, B18, DR2, BF*S, C2*Q0, C4*A4, C4*B2, were in accord with previous observations in C2 deficiency. The concentrations of C1q, C5, factor B and factor D were in the low normal range and the hemolytic activity of the alternative pathway was slightly decreased. In addition, the patient showed moderately low IgG2 concentrations and lacked the IgG2 subclass marker G2m(23). The findings indicate that the patient's susceptibility to bacterial infections may be due to C2 deficiency in combination with the presence of an IgG allotype associated with impaired antibody responses to carbohydrate antigens.

Recurrent aspiration in children: lipid-laden alveolar macrophage quantitation.

We prospectively evaluated bronchial washings from 45 patients aged 1 month to 25 years (mean 3.3 years) for the presence of lipid-laden macrophages. By grading the amount of intracellular oil-red-O-positive material, we determined a semiquantitative lipid-laden macrophage index for each patient. All patients observed to be definite aspirators (n = 22) had indexes greater than or equal to 86 (mean +/- SD, 139 +/- 46). All patients with no clinical suspicion of aspiration (n = 23) had indexes less than or equal to 72 (mean +/- SD, 21 +/- 20). Our results suggest that simply sighting lipid-laden macrophages in a nonspecific finding, but quantitation of these cells may be a very good test for recurrent aspiration of food substances in children. This test appears to be more sensitive than radiographic studies.

Correlation between deficiency of immunoglobulin subclass G3 and Gm allotype.

Gm allotypes were investigated in 63 Swedes: 46 females and 17 males, in whom serum IgG3 was below 0.35 g/l. Both monoclonal antibodies and polyclonal antisera were used for the quantification. Concentrations of the other IgG subclasses were within the age-related normal ranges. The distribution of the IgG1 genetic markers G1m(a,x,f) differed markedly from that observed in normal Swedes (p less than 0.001). Thus G1m(a) was present in 60 subjects as compared to an expected 36, and phenotype G1m(-f) in 34 subjects as against an expected 8. The mean IgG3 concentration was numerically lower in the G1m(-f) group than in the G1m(+f) cohort, and individuals with IgG3 levels 0.10 g/l were more frequent in the G1m(-f) group. Among Caucasians, G3mg is in linkage disequilibrium with G1ma and our interpretations is that the haplotype G1ma;ax G2m-n G3mg is markedly increased in individuals with IgG3 deficiency.

Production and characterization of rabbit antibodies against histamine.

Antisera were raised in rabbits against histamine conjugated to human serum albumin (HSA) by the carbodiimide (ECDI) method. The specificity of the antisera was studied in a radioimmunoassay using 125I-protein A for detection of IgG binding. The HIS-HSA antisera reacted with histamine-HSA conjugates prepared by either the carbodiimide or diisocyanate coupling procedure, as well as with carbodiimide-prepared histamine-ferritin and histamine-ovalbumin conjugates. On the contrary, the antisera were unreactive with unconjugated HSA, ECDI-reacted HSA, or HSA conjugated to ethanolamine or pentylamine. Free unconjugated histamine significantly inhibited antibody binding to histamine-HSA and 50% inhibition of antibody binding (IC50) was recorded at 3 mM histamine concn. On a histamine molar concn basis a much lower inhibitory potency of free histamine was recorded, as compared to histamine-protein conjugates (IC50 = 3 X 10(-6) mM). This probably reflected amplification of antibody binding to the multivalent ligand, but possibly also that the protein carrier adds some common features to the antigenic determinant. Histidine, ornithine, glutamine, asparagine, sterylamine and several other amino acids lacked inhibitory effects. Histamine H1 and H2 receptor antagonists inhibited histamine binding to the histamine antibodies. The antagonists varied in their affinity for the histamine antibodies and 50% inhibition of antibody binding was recorded in the range of 1-50 mM concn of the antagonists. Comparing one H1 and one H2 antagonist (diphenhydramine and cimetidine, respectively) two of the sera were preferentially inhibited by cimetidine whereas the third serum seemed to be more prone to inhibition by diphenhydramine.

Histamine in endocrine cells in the stomach. A survey of several species using a panel of histamine antibodies.

Antibodies to histamine were used to examine the localization of the amine in cells of the stomach and upper small intestine of a great variety of species, including cartilaginous and bony fish, amphibia, reptiles (lizard), birds (chicken) and a large number of mammals. In all species gastric histamine was localized in endocrine cells (invariably found in the epithelium) and mast cells (usually with an extra-epithelial localization). The endocrine cells were identified as such by immunostaining with antibodies to chromogranin A and the mast cells were identified by toluidine blue staining. Histamine-immunoreactive endocrine cells were found almost exclusively in the acid-producing part of the stomach; only rarely were such cells observed in the pyloric gland area. They were fairly numerous in the gastric mucosa of the two subclasses of fish as well as in the amphibia and reptile species studied. Here, the majority of the histamine-immunoreactive endocrine cells seemed to have contact with the gastric lumen (open type cells) and were located in the surface epithelium (certain fish only) or together with mucous neck cells at the bottom of the pits. In the chicken, histamine-immunoreactive endocrine cells were numerous and located peripherally in the deep compound glands. They were without contact with the lumen (closed type) and had long basal extensions ("paracrine" appearance), running close to the base of the oxyntic-peptic cells. In mammals, the number of histamine-immunoreactive endocrine cells in the stomach varied greatly. They were particularly numerous in the rat and notably few in the dog, monkey and man. In all mammals, the histamine-immunoreactive endocrine cells were of the closed type and located basally in the oxyntic glands. They often had a "paracrine" appearance with long basal processes. Histamine-storing mast cells, finally, were few in both subclasses of fish as well as in the amphibian species and in the lizard. They were fairly numerous in chicken proventriculus (beneath the surface epithelium), few in the oxyntic mucosa of mouse, rat and hamster, moderate in number in hedgehog, guinea-pig, rabbit, pig and monkey, and numerous in cat, dog and man.(ABSTRACT TRUNCATED AT 400 WORDS)

Recurrent otitis media: genetic immunoglobulin markers in children and their parents.

The likelihood that hereditary factors play a significant role in the development of recurrent acute otitis media (rAOM) in children has been suggested. The genetically determined immunoglobulin variants, Gm and Km, are useful tools for mapping out the genetic loci involved in antibody responses. Certain Gm and Km types, G2m(23) and Km(1), appear to be linked to genes which regulate the concentrations of antibodies to pneumococcal polysaccharide antigens in adults. Our aim was to identify such immunoglobulin markers in rAOM children, since these children have extremely low concentrations of IgG antibodies against the pneumococcal types associated with this disease. The markers G1m(1), G1m(2), G1m(3), G2m(23) and Km(1) were identified in 20 families, each comprising 1 parent and 1 child with a history of rAOM and 1 parent free from rAOM. In addition, G2m(23) was identified in 47 children without AOM. The distribution of Gm and Km markers between rAOM and healthy subjects did not differ significantly. If anything, rAOM children exhibited a high rate of the G2m(23) marker, whereas earlier observations in adults have demonstrated low responders to polysaccharide antigens to be preferentially G2m(-23). Our findings indicate that mechanisms responsible for the low concentrations of antibodies to pneumococcal polysaccharides in rAOM children may differ from those causing certain adults to be low responders when exposed to pneumococcal polysaccharides.