TLR4 mutations are associated with endotoxin hyporesponsiveness in humans.

There is much variability between individuals in the response to inhaled toxins, but it is not known why certain people develop disease when challenged with environmental agents and others remain healthy. To address this, we investigated whether TLR4 (encoding the toll-like receptor-4), which has been shown to affect lipopolysaccharide (LPS) responsiveness in mice, underlies the variability in airway responsiveness to inhaled LPS in humans. Here we show that common, co-segregating missense mutations (Asp299Gly and Thr399Ile) affecting the extracellular domain of the TLR4 receptor are associated with a blunted response to inhaled LPS in humans. Transfection of THP-1 cells demonstrates that the Asp299Gly mutation (but not the Thr399Ile mutation) interrupts TLR4-mediated LPS signalling. Moreover, the wild-type allele of TLR4 rescues the LPS hyporesponsive phenotype in either primary airway epithelial cells or alveolar macrophages obtained from individuals with the TLR4 mutations. Our findings provide the first genetic evidence that common mutations in TLR4 are associated with differences in LPS responsiveness in humans, and demonstrate that gene-sequence changes can alter the ability of the host to respond to environmental stress.

Is an abbreviated bronchial challenge with histamine valid?

Investigators have validated an abbreviated protocol for testing nonspecific bronchial reactivity with methacholine. We performed a similar validation study with histamine, another bronchoprovocative agent known to induce airflow obstruction. Histamine is pharmacologically distinct from methacholine and, under some circumstances, may provide specific clinical and investigative advantages to methacholine. Twenty-four patients with a clinical history of asthma underwent bronchoprovocative testing using the standard histamine airway protocol recommended by the American Academy of Allergy, Committee on Standardization of Bronchoprovocation. In addition, two abbreviated histamine challenge protocols were tested using the same administration and testing equipment. The abbreviated protocols involved fewer dilutions and dosages of histamine than the standard histamine protocol but covered the same range of cumulative doses. The two abbreviated protocols differed only in the intervals for determination of FEV1 between doses of histamine (30 s vs 3 min). The sequence of these three protocols was randomized for each study subject and each airway challenge was separated by one week. The two abbreviated protocols took significantly less time to administer than the standard protocol--18 min vs 30 min vs 44 min. Both the provocative dose to cause a 20 percent decline in the FEV1 (PD20 FEV1) and the slope of the dose-response curve were not significantly different between the standard protocol and either of the two abbreviated protocols. Moreover, a high degree of agreement was observed between the two abbreviated protocols and the standard histamine protocol for both the PD20 FEV1 and the slope of the dose-response curve. These findings indicate that similar estimates of bronchial reactivity are obtained from either of the abbreviated protocols when compared with the standard histamine protocol.

Pentoxifylline does not alter the response to inhaled grain dust.

Pentoxifylline (PTX) has been shown to reduce sepsis-induced neutrophil sequestration in the lung and inhibit endotoxin-mediated release of tumor necrosis factor-alpha (TNF-alpha). Previously, we have shown that endotoxin appears to be the principal agent in grain dust causing airway inflammation and airflow obstruction following grain dust inhalation. To determine whether PTX affects the physiologic and inflammatory events following acute grain dust inhalation, 10 healthy, nonsmoking subjects with normal airway reactivity were treated with PTX or placebo (PL) followed by corn dust extract (CDE) inhalation (0.08 mL/kg), using a single-blinded, crossover design. Subjects received PTX (1,200 mg/d) or PL for 4 days prior to CDE inhalation and 400 mg PTX or PL on the exposure day. Both respiratory symptoms and declines in FEV1 and FVC occurred following CDE exposure in both groups, but there were no significant differences in the frequency of symptoms or percent declines from baseline in the FEV1 and FVC at any of the time points measured in the study. Elevations in peripheral blood leukocyte and neutrophil concentrations and BAL total cell, neutrophil, TNF-alpha, and interleukin-8 concentrations were measured 4 h following exposure to CDE in both the PTX- and PL-treated subjects, but no significant differences were found between treatment groups. These results suggest that pretreatment with PTX prior to inhalation of CDE, in the doses used in this study, does not alter the acute physiologic or inflammatory events following exposure to inhaled CDE.

Nasal lavage cellularity, grain dust, and airflow obstruction.

To evaluate the clinical utility of nasal lavage (NL), we performed post-work shift NL on 172 grain workers and 78 postal worker control subjects. The grain worker group included a higher percentage of current smokers (25.7% vs 16.7%) and a lower percentage of former smokers (21.15% vs 35.9%) compared with the postal workers. The control subjects included more female workers and were slightly older than the grain workers. Compared with the postal workers, the grain workers were exposed to significantly greater concentrations of total dust (0.1 +/- 0.0 vs 6.8 +/- 1.4 mg/m3; mean +/- SEM) and total endotoxin (4.3 +/- 0.8 vs 2,372.4 +/- 653.8 endotoxin units/m3). NL from gain workers showed a higher concentration of total cells (55,000 +/- 14,000 vs 25,000 +/- 5,000 cells per milliliter; p=0.03), a higher concentration of squamous epithelial cells (17,029.0 +/- 4,177 .0 vs 7,103.7 +/- 1,479.8 cells per milliliter; p=0.03), and a higher concentration of neutrophils (40,058.0 +/- 12,803.2 vs 17,891.0 +/- 3,822.3 cells per milliliter; p=0.10) compared with postal workers. Importantly, these differences in NL cellularity between grain workers and postal workers were observed within the three strata of smokers. To further assess the importance of total cells, squamous epithelial cells, and neutrophils in the NL fluid of grain workers, we investigated the relationship between these cell concentrations and (1) measures of dust and endotoxin exposure during the work shift. (2) spirometric measures of airflow obtained immediately before the NL, and (3) work-related respiratory symptoms. The concentration of total cells, the concentration of squamous epithelial cells, or the concentration of neutrophils in the NL was not associated with ambient levels of dust or endotoxin, with baseline or cross-shift changes in lung function, or with work-related respiratory symptoms. These findings suggest that increased NL cellularity may be seen in workers exposed to high dust levels. However, the NL cellularity does not appear to be associated with ambient concentrations of dusts or endotoxins, with signs of airflow obstruction, or with work-related respiratory symptoms.

Asbestos-induced pleural fibrosis and impaired exercise physiology.

To further assess the clinical significance of asbestos-induced pleural fibrosis, we performed cardiopulmonary exercise testing in 90 subjects who were exposed to asbestos. Of the 82 subjects without an abnormal resperate exercise, 35 had normal pleura, 33 had circumscribed pleural plaques, and 14 had diffuse pleural thickening. Interstitial fibrosis (International Labor Organization [ILO]. > or = 1/10) was present in 14 of 35 subjects with normal pleura, 13 of 33 subjects with circumscribed pleural plaques, and 2 of 14 subjects with diffuse pleural thickening. Although pleural fibrosis did not appear to be related to impaired respiratory function with exercise in our entire cohort, this finding was confounded by a higher proportion of interstitial fibrosis in subjects with normal pleura. In fact, among study subjects without asbestosis, significant decreases in gas exchange (higher VD/VT and increased alveolar-arterial oxygen pressure difference) were observed at maximal exercise among subjects with pleural fibrosis. Interestingly, neither a higher respiratory rate nor a lower VT/FVC ratio was observed among those with pleural fibrosis, suggesting that the mechanical effects of pleural fibrosis on the chest wall do not explain the increased VD/VT. Using multivariate analyses to control for potential confounders, regression models showed that pleural plaques (p = 0.04) and diffuse pleural thickening (p = 0.03) were independently associated with significant increases in dead space ventilation (VD/VT) with maximal exercise. These findings indicate that asbestos-induced pleural fibrosis is independently associated with decrements in gas exchange with maximal exercise and suggest that interstitial lung disease, not detected on the routine chest x-ray film, may be responsible for this abnormal response to exercise.

Grain dust and endotoxin inhalation challenges produce similar inflammatory responses in normal subjects.

STUDY OBJECTIVE: To compare the physiologic and inflammatory response following inhalation of corn dust extract (CDE) and lipopolysaccharide (LPS) solutions in normal subjects. DESIGN: Randomized, double-blind crossover design. PARTICIPANTS: Fourteen healthy, nonatopic, nonasthmatic, never-smoking volunteers. INTERVENTIONS: On separate visits, subjects underwent a series of four inhalation challenges to LPS or CDE, each containing either a high (6 micrograms/mL) or low (0.9 microgram/mL) endotoxin concentration, and administered at equal Xolumes. RESULTS: Chest tightness, cough, dyspnea, and sputum production were experienced following both LPS and CDE exposures and with similar frequency at both high and low endotoxin concentrations. LPS and CDE inhalations caused acute declines in FEV1, and the changes in FEV1 from baseline following exposure to both inhalants were not significantly different at both high and low endotoxin concentrations. Following exposure to the high-endotoxin LPS and CDE, no consistent differences in total cell and cytokine (tumor necrosis factor-alpha [TNF-alpha], interleukin-1 beta [IL-1 beta], IL-6, IL-8) concentrations were seen between exposures, although the neutrophil concentration was greater following the LPS exposure (p = 0.01). BAL cellularity and cytokine concentrations following the low-endotoxin LPS and CDE exposure revealed no differences, except for IL-1 beta, which was greater following LPS exposure (p = 0.05). The high-endotoxin LPS and CDE exposures resulted in greater increases in BAL neutrophils and cytokines in comparison to its respective low-endotoxin exposure. CONCLUSIONS: At exposure levels of endotoxin, LPS and CDE result in similar symptoms, changes in airflow, and increases in BAL inflammatory cells and mediators. Moreover, the physiologic and inflammatory response to LPS and CDE appears to be related to the exposure level of endotoxin.

The effect of of glucocorticoids on grain dust-induced airway disease.

STUDY OBJECTIVES: To determine the effect of glucocorticoids on grain dust-induced airflow obstruction and airway inflammation. DESIGN: Randomized controlled trial. SETTING: University hospital. PARTICIPANTS: Health volunteers. INTERVENTIONS: Two randomized, placebo-controlled trials, each studying 10 healthy volunteers who were pretreated with either triamcinolone acetonide (Azmacort) oral inhaler 4 puffs twice daily (800 microg daily) for 7 consecutive days or IV hydrocortisone (3 microg/kg/min) as a 14-h continuous infusion, then subjected to a controlled inhalation exposure to corn dust extract (CDE) (endotoxin exposure dose of 3 microg/kg). A single-blind, crossover study design was performed for each trial enrolling 10 healthy, lifetime nonsmokers, with no history of lung disease or environmental exposure to grain dust. MEASUREMENTS AND RESULTS: Following each inhalation exposure to CDE, spirometry was performed at regular intervals and BAL was performed at 4 h. Both treatment and placebo groups demonstrated significant decrements in spirometry and increments in BAL cellularity following CDE inhalation compared with placebo. Inhaled steroid treatment resulted in a significantly higher FEV1 only at the 2-h time point following CDE inhalation with no significant differences observed in the BAL total cell concentration or cellular differential compared with placebo. IV hydrocortisone treatment resulted in a significantly higher FEV1 and FVC between 2 and 4 h after CDE inhalation, as well as significant reductions in the BAL total cell, macrophage, and eosinophil concentrations. Interestingly, the concentration of tumor necrosis factor-alpha and interleukin-8 in the BAL fluid was also decreased following treatment with IV glucocorticoids. CONCLUSIONS: These results demonstrate that glucocorticoids, administered IV and perhaps by inhalation, have a mildly protective effect on airflow obstruction and airway inflammation induced by inhalation of grain dust.

Bronchial hyperreactivity is associated with enhanced grain dust-induced airflow obstruction.

Bronchial hyperreactivity (BHR) is associated with the presence of airway inflammation in asthma and is seen in individuals occupationally exposed to grain dust. To better understand the relationship between BHR and pulmonary inflammation after grain dust exposure, we carried out an inhalation challenge to corn dust extract (CDE) on seven subjects with BHR [a 20% or greater decrease in forced expiratory volume in 1 s (FEV(1)) compared with diluent FEV(1) with a cumulative dose of histamine

Chromosome damage and sister chromatid exchanges in lymphocyte cultures from patients with two primary cancers.

Sister chromatid exchanges (SCEs) and chromosome damage were scored in lymphocyte cultures from 11 patients with two or more primary cancers and were compared with normal controls. None of the patients had a constitutional chromosome anomaly, but six showed evidence of chromosome instability, which could not be accounted for by treatment, expressed either as elevated SCE frequency or increased nonspecific chromosome damage and chromosome loss. Chromosome damage included major rearrangements as well as deletions and gaps. The possibility of common mechanisms in chromosome instability leading to susceptibility to a heterogeneous group of primary cancers is discussed.

Variation of SCE frequency in lymphocyte cultures from patients with Hodgkin's disease before, during, and after treatment.

Seven patients with newly-diagnosed Hodgkin's disease, having standard mustine-vinblastine-prednisolone-procarbazine therapy, had a large and regular pattern of rise in SCE frequency in their peripheral lymphocytes, which apparently started to fall before the end of the course (32 wk). In contrast, SCE frequency fell below normal in the lymphocytes of nine patients having mantle radiotherapy. Twenty-eight patients studied 2-13 yr after initial chemotherapy or radiotherapy for Hodgkin's disease had normal or near normal SCE frequencies.

The heterogeneity of the 5q--chromosome marker in refractory anemia.

A patient presenting with an unusual case of refactory anemia is described in whom acquired markers originating from chromosomes No. 5 and 8 are identified by G- and sequential C-banding. Comparison with various published illustrations of chromosome No. 5 deletions in this general broad category of disease raises the possibility that karyotypic misclassification in cases of absent or substandard banding (typical of bone marrow) may help to explain the considerable variation in size and morphology of the 5q--marker.

An unusual karyotype in preleukemia.

A case of a myeloproliferative disorder classified as preleukemia is described in which the patient developed a single, complicated, abnormal karyotype in 100% of the bone marrow cells (45, XY, -2, -5, -7, -8, -11, -12, -13, -14, + t(2;5), +t(11;12), +t(16;17), +17, plus three or four dicentric markers). The possible significance of these unusual, if not unique, chromosome changes is discussed in relation to hematologic and clinical findings and with particular reference to the existing nonrandom patterns of chromosome changes in myeloproliferative disorders and acute nonlymphoblastic leukemias.

Reciprocal translocation and the Philadelphia chromosome.

We examined metaphases from three patients with chronic myeloid leukaemia and a typical Philadelphia chromosome with one chromosome 9 as the recipient to determine whether the 9q+22q- translocation is reciprocal. Good quality G-banded photographs of the chromosomes concerned were subjected to light absorption density analysis. This provided enlarged tracings corresponding to the relevant chromosome regions and so facilitated accurate measurement. This technique has unambiguously shown that the typical Philadelphia chromosome results from a reciprocal translocation and that probably no material is gained or lost in the exchange. Furthermorein a total of six patients for whom sequential G and C banding was performed, the chromosome 9 with the largest block of centromeric heterochromatin received the translocated material. We offer tentative explanations for this curious observation.

De novo translocation heterozygote with three reciprocal translocations.

An extremely rare case of a child with three balanced reciprocal translocations involving six different autosomes is described. These abnormalities have apparently arisen de novo and seem to have only relatively minor phenotypic effects. The meiotic possibilities are discussed and cytogenetic markers suggest that the damage may have occurred in a paternal gamete.

The prenatal detection of a familial pericentric inversion of chromosome 19.

A familial pericentric inversion of chromosome 19 was detected prenatally in a young primagravida. As far as the authors are aware there are no previous published reports of this rare abnormality being ascertained in this manner. The problems of counselling such a family are considered.

Chorion in culture.

This study is based on the processing of 115 samples of pure chorion. The different types of colony morphology, together with cytogenetic analysis, indicate the predisposition of chorion cells to artefacts of culture. Chorion survives longer than other foetal tissues and can be cultured when those are dead or contaminated (e.g. in macerated foetuses and missed abortions) making it a suitable and popular tissue for cytogenetic purposes. However, our results indicate that it is slow in culture and prone to chromosome variation so that pseudomosaicism frequently arises and may lead to misdiagnosis. We therefore suggest that cytogenetic analysis of spontaneous abortion material is interpreted with caution in cases where chorion is the only tissue cultured, especially when mosaicism is found.

Cytogenetic analysis in 100 spontaneous abortions in North-East Scotland.

The results of an extremely detailed cytogenetic analysis on 100 spontaneous abortions occurring in the Aberdeen area over a 3 year period is presented. A comparison with other published series reveals that there is a higher culture success rate (93%) accompanied by a lower incidence of trisomy and a higher incidence of triploidy, although the percentage of chromosomally abnormal foetuses in first trimester abortions is comparable with other studies. However, these apparent discrepancies can largely be explained on the basis of gestational age and sampling variation.

Structural rearrangements in the parents of children with primary trisomy 21.

A retrospective cytogenetic study was carried out on the parents of children with regular trisomy 21 Down's syndrome. In a total of 128 parents referred routinely to our laboratory after the birth of their affected child, three structural abnormalities, a reciprocal translocation and two pericentric inversions not involving chromosome 21, were detected. This is about 10 times the frequency expected based on current figures from consecutive newborn studies. In addition, the brother of one of nine older people with trisomy 21 referred for cytogenetic analysis for the first time was found to have a reciprocal translocation. This supports the contention made by others that an interchromosomal effect does exist in man. It is suggested that centres who routinely analyse the parents of their trisomy 21 referrals in an unbiased fashion should review their records. They will almost certainly contain useful information regarding the possible existence of this phenomenon and may even contain clues as to its nature. In addition to its undoubted scientific value, such data should prove useful in the genetic counselling of carriers of structural rearrangements.

Cytogenetic study of 10 cases of infectious mononucleosis.

Cytogenetic analysis of 10 cases of infectious mononucleosis has revealed increased damage in the form of chromosome breakage and aberrations typical of viral infection, but in addition a few cells were consistently found to harbour a deleted number 22, similar tsponse to the mitogen phytohaemagglutinin is suggested. This inhibitor appears to have a reversible action on the patient's own lymphocytes but no inhibitory effect on control lymphocytes.

Variation in the Philadelphia chromosome.

Cytogenetic study of 17 cases of chronic myeloid leukaemia has shown that the Philadelphia chromosome is a variable entity, differing in size and banding pattern between individuals.