High glucose exposure promotes proliferation and in vivo network formation of adipose-tissue-derived microvascular fragments.

High glucose concentrations have been shown to activate endothelial cells and promote angiogenesis. In the present study, it was investigated whether high glucose concentrations could improve the vascularisation capacity of adipose-tissue-derived microvascular fragments (ad-MVF). Ad-MVF were isolated from the epididymal fat pads of donor mice and cultivated for 24 h in University of Wisconsin (UW) solution supplemented with vehicle or 30 mM glucose. Protein expression, morphology, viability and proliferation of the cultivated ad-MVF were analysed by means of proteome profiler mouse angiogenesis array, scanning electron microscopy and immunohistochemistry. Additional cultivated ad-MVF were seeded on to collagen-glycosaminoglycan scaffolds to study their in vivo vascularisation capacity in the dorsal skinfold chamber model by intravital fluorescence microscopy, histology and immunohistochemistry. In vitro, high glucose exposure changed the protein expression pattern of ad-MVF with endoglin, interleukin (IL)-1ß and monocyte chemoattractant protein (MCP)-1 as the most up-regulated pro-angiogenic factors. Moreover, high glucose exposure induced the formation of nanopores in the ad-MVF wall. In addition, ad-MVF contained significantly larger numbers of proliferating endothelial and perivascular cells while exhibiting a comparable number of apoptotic cells when compared to vehicle-treated controls. In vivo, scaffolds seeded with high-glucose-exposed ad-MVF exhibited an improved vascularisation and tissue incorporation. These findings demonstrated that the exposure of cultivated ad-MVF to high glucose concentrations is a promising approach to improve their in vivo performance as vascularisation units for tissue engineering and regenerative medicine.

Experimental infections in humans-historical and ethical reflections.

Vaccine efficacy and prophylactic treatment of infections are tested best when the vaccinated or treated individual is challenged through deliberate infection with the respective pathogen. However, this trial design calls for particular ethical caution. Awareness of the history of challenge trials is indispensable, including trials that were problematic or even connected to abuse. We briefly introduce historical aspects of experimental infections in humans and the ethical debate around them and give estimates of the numbers of volunteers participating in human experimental infection models. Challenge models can offer a great chance and benefit for the development of medical interventions to fight infectious diseases, but only when they are appropriately controlled and regulated.

L'efficacité des vaccins et le traitement prophylactique des infections sont mieux testés lorsque l'individu vacciné ou traité est exposé par le biais d'une infection délibérée par l'agent pathogène concerné. Cependant, cette conception d'essai appelle à une prudence éthique particulière. Il est indispensable de connaître l'histoire des essais cliniques, y compris des essais qui se sont avérés problématiques ou même liés à des abus. Nous présentons brièvement les aspects historiques des infections expérimentales chez l'homme et le débat éthique autour d'eux et donnons des estimations du nombre de volontaires participant à des modèles d'infection expérimentale humaine. Les modèles d'exposition peuvent offrir une grande chance et un avantage pour le développement d'interventions médicales pour lutter contre les maladies infectieuses, mais uniquement lorsqu'elles sont contrôlées et réglementées de manière appropriée.

Generation of co-culture spheroids as vascularisation units for bone tissue engineering.

Cell spheroids represent attractive building units for bone tissue engineering, because they provide a three-dimensional environment with intensive direct cell-cell contacts. Moreover, they allow for co-culture of both osteoblasts and vessel-forming cells, which may markedly increase their survival and vascularisation after transplantation. To test this hypothesis, we generated co-culture spheroids by aggregating different combinations of primary human osteoblasts (HOB), human dermal microvascular endothelial cells (HDMEC) and normal human dermal fibroblasts (NHDF) using the liquid overlay technique. Mono-culture spheroids consisting either of HOB or HDMEC served as controls. After in vitro characterisation, the different spheroids were transplanted into dorsal skinfold chambers of CD1 nu/nu mice to study in vivo their viability and vascularisation over a 2-week observation period by means of repetitive intravital fluorescence microscopy and immunohistochemistry. In vitro, co-culture spheroids containing HDMEC rapidly formed dense tubular vessel-like networks within 72 h and exhibited a significantly decreased rate of apoptotic cell death when compared to mono-culture HDMEC spheroids. After transplantation, these networks interconnected to the host microvasculature by external inosculation. Of interest, this process was most pronounced in HOB-HDMEC spheroids and could not further be improved by the addition of NHDF. Accordingly, HOB-HDMEC spheroids were larger when compared to the other spheroid types. These findings indicate that HOB-HDMEC spheroids exhibit excellent properties to preserve viability and to promote proliferation and vascularisation. Therefore, they may be used as functional vascularisation units in bone tissue engineering for the seeding of scaffolds or for the vitalisation of non-healing large bone defects.

Pantoprazole, a proton pump inhibitor, delays fracture healing in mice.

Proton pump inhibitors (PPIs), which are widely used in the treatment of dyspeptic problems, have been shown to reduce osteoclast activity. There is no information, however, on whether PPIs affect fracture healing. We therefore studied the effect of the PPI pantoprazole on callus formation and biomechanics during fracture repair. Bone healing was analyzed in a murine fracture model using radiological, biomechanical, histomorphometric, and protein biochemical analyses at 2 and 5 weeks after fracture. Twenty-one mice received 100 mg/kg body weight pantoprazole i.p. daily. Controls (n = 21) received equivalent amounts of vehicle. In pantoprazole-treated animals biomechanical analysis revealed a significantly reduced bending stiffness at 5 weeks after fracture compared to controls. This was associated with a significantly lower amount of bony tissue within the callus and higher amounts of cartilaginous and fibrous tissue. Western blot analysis showed reduced expression of the bone formation markers bone morphogenetic protein (BMP)-2, BMP-4, and cysteine-rich protein (CYR61). In addition, significantly lower expression of proliferating cell nuclear antigen indicated reduced cell proliferation after pantoprazole treatment. Of interest, the reduced expression of bone formation markers was associated with a significantly diminished expression of RANKL, indicating osteoclast inhibition. Pantoprazole delays fracture healing by affecting both bone formation and bone remodeling.

Correlation of T cell response and bacterial clearance in human volunteers challenged with Helicobacter pylori revealed by randomised controlled vaccination with Ty21a-based Salmonella vaccines.

BACKGROUND: Helicobacter pylori remains a global health hazard, and vaccination would be ideal for its control. Natural infection appears not to induce protective immunity. Thus, the feasibility of a vaccine for humans is doubtful. METHODS: In two prospective, randomised, double-blind, controlled studies (Paul Ehrlich Institute application nos 0802/02 and 1097/01), live vaccines against H pylori were tested in human volunteers seronegative for, and without evidence of, active H pylori infection. Volunteers (n = 58) were immunised orally with Salmonella enterica serovar Typhi Ty21a expressing H pylori urease or HP0231, or solely with Ty21a, and then challenged with 2x10(5) cagPAI(-) H pylori. Adverse events, infection, humoral, cellular and mucosal immune response were monitored. Gastric biopsies were taken before and after vaccination, and postchallenge. Infection was terminated with antibiotics. RESULTS: Vaccines were well tolerated. Challenge infection induced transient, mild to moderate dyspeptic symptoms, and histological and transcriptional changes in the mucosa known from chronic infection. Vaccines did not show satisfactory protection. However, 13 of 58 volunteers, 8 vaccinees and 5 controls, became breath test negative and either cleared H pylori (5/13) completely or reduced the H pylori burden (8/13). H pylori-specific T helper cells were detected in 9 of these 13 (69%), but only in 6 of 45 (13%) breath test-positive volunteers (p = 0.0002; Fisher exact test). T cells were either vaccine induced or pre-existing, depending on the volunteer. CONCLUSION: Challenge infection offers a controlled model for vaccine testing. Importantly, it revealed evidence for T cell-mediated immunity against H pylori infection in humans.

Anaemia and malaria in Yanomami communities with differing access to healthcare.

Inequitable access to healthcare has a profound impact on the health of marginalised groups that typically suffer an excess burden of infectious disease morbidity and mortality. The Yanomami are traditionally semi-nomadic people living in widely dispersed communities in Amazonian Venezuela and Brazil. Only communities living in the vicinity of a health post have relatively constant access to healthcare. To monitor the improvement in the development of Yanomami healthcare a cross-sectional survey of 183 individuals was conducted to investigate malaria and anaemia prevalence in communities with constant and intermittent access to healthcare. Demographic and clinical data were collected. Malaria was diagnosed by microscopy and haemoglobin concentration by HemoCue. Prevalence of malaria, anaemia, splenomegaly, fever and diarrhoea were all significantly higher in communities with intermittent access to healthcare (anaemia 80.8% vs. 53.6%, P<0.001; malaria 18.2% vs. 6.0%, P=0.013; splenomegaly 85.4% vs.12.5%, P<0.001; fever 50.5% vs. 28.6%, P=0.003; diarrhoea 30.3% vs.10.7% P=0.001). Haemoglobin level (10.0 g/dl vs. 11.5 g/dl) was significantly associated with access to healthcare when controlling for age, sex, malaria and splenomegaly (P=0.01). These findings indicate a heavy burden of anaemia in both areas and the need for interventions against anaemia and malaria, along with more frequent medical visits to remote areas.

Malaria diagnosis under field conditions in the Venezuelan Amazon.

To improve practical, accurate diagnosis of malaria in the Amazon rainforest of Venezuela, two rapid diagnostic tests (RDT) (OptiMAL-IT) and FalciVax) and a laboratory light microscope, used in the field with a battery-operated head lamp as an external light source, were evaluated against the standard laboratory microscope procedure for malaria detection. One hundred and thirty-six Yanomami patients were studied for the presence of malaria parasites. Thirty-three patients (24%) were positive for malaria (Plasmodium falciparum, P. vivax, P. malariae). Twenty-one (64%) of the positive patients had <100 parasites/microl. Both RDTs showed poor sensitivity (24.2% for OptiMAL-IT) and 36.4% for FalciVax) but good specificity (99% both for OptiMAL-IT) and FalciVax). Field and laboratory microscopy showed sensitivities of 94% and 91%, respectively. The kappa coefficient was 0.90, indicating a high agreement between field and laboratory microscopy. We conclude that (i) adequate slide reading cannot be substituted by either of the two RDTs in the Venezuelan Amazon and (ii) the use of a light source such as that described above makes slide reading more feasible than hitherto in remote areas without electricity.

Elevated bronchoalveolar lavage fluid histamine levels in allergic asthmatics are associated with methacholine bronchial hyperresponsiveness.

Using a sensitive single isotope enzymatic assay we measured bronchoalveolar lavage (BAL) fluid histamine in asymptomatic normal (nonallergic), allergic rhinitic, and allergic asthmatic subjects. Normal subjects were found to have little or no detectable amounts of histamine in BAL fluid (11 +/- 11 pg/ml), and few BAL fluid mast cells. In comparison, the allergic rhinitics and allergic asthmatics had much higher amounts of BAL fluid histamine (113 +/- 53 and 188 +/- 42 pg/ml, respectively), and a significantly greater number of BAL fluid mast cells. Furthermore, despite having equivalent baseline pulmonary function values, allergic asthmatics with BAL fluid histamine levels greater than 100 pg/ml required only 7 +/- 2 breath units of methacholine to induce a 20% drop in forced expiratory volume in 1 s (FEV1) (PD20FEV1) while asthmatics with BAL fluid histamine levels less than 100 pg/ml required 49 +/- 19 breath units (P less than 0.05). These data suggest that allergic asthmatics have ongoing lung mast cell degranulation that might contribute to the etiology of airway hyperresponsiveness.

Vaccines for preventing smallpox.

BACKGROUND: Smallpox was eradicated by 1980, but its possible use as a bioweapon has rekindled interest in the development of protective vaccines. Therefore, stockpiled calf lymph-derived vaccines and recently developed cell-cultured vaccines have been investigated to contribute information to smallpox emergency response plans, while newer (non-replication competent) vaccines are developed. OBJECTIVES: To assess the effects of smallpox vaccines in preventing the disease, in inducing immunity, and in regard to adverse events. SEARCH STRATEGY: In December 2006, we searched the Cochrane Infectious Diseases Group Specialized Register, CENTRAL (The Cochrane Library 2006, Issue 4), MEDLINE, EMBASE, LILACS, and Current Controlled Trials, and handsearched Index Medicus. We also searched three databases of vaccine safety in December 2005. SELECTION CRITERIA: Randomized controlled trials of smallpox vaccines versus placebo, other smallpox or non-smallpox vaccine, no intervention, or different dose of the same vaccine in people receiving smallpox vaccination irrespective of age. DATA COLLECTION AND ANALYSIS: Both authors independently assessed trial quality and extracted data. We combined dichotomous data using relative risk with a random-effects model. MAIN RESULTS: Ten trials involving 2412 participants were included. The vaccines investigated were calf-lymph derived first-generation vaccines (Dryvax, APVS, Lancy-vaxina, Lister), and cell-cultured second-generation vaccines (ACAM, CCSV). Vaccines were investigated in different dilutions. All undiluted vaccines induced a reaction in 95% of people vaccinated in terms of pustule and immunogenicity. Also 1:10 dilutions were fully efficient when the starting concentration was defined. Serious adverse events were reported in 1% to 2% of the volunteers. Fever was observed in 11% to 22% of participants, and headache in roughly half of the participants. Fever was less frequent when new vaccines were administered, but rates of headache were similar in new and old vaccines. AUTHORS' CONCLUSIONS: The evidence shows that stockpiled vaccines have maintained their immunogenicity and new cell-cultured vaccines are similar to stockpiled vaccines in terms of vaccination success rate and immunogenicity. First- and second-generation vaccines diluted to at least 1:10 are as effective as undiluted vaccine in terms of clinical success rate and immunogenicity. Dilution did not reduce the frequency of adverse events. Success rate and immunogenicity were similar in naive and previously vaccinated persons, but there were fewer adverse events in previously vaccinated persons. The rate of adverse events found in this review reveals the need for further development and improvement of smallpox vaccines.

Osteogenic differentiation of immature osteoblasts: Interplay of cell culture media and supplements.

Differentiation of immature osteoblasts to mature osteoblasts in vitro initially was induced by supplementing the medium with ß-gylcerophosphate and dexamethasone. Later, ascorbic acid, vitamin D3, vitamin K3 and TGFß1 were used in varying concentrations as supplements to generate a mature osteoblast phenotype. We tested the effects of several combinations of cell culture media, seeding protocols and osteogenic supplements on osteogenic differentiation of human primary osteoblasts. Osteogenic differentiation was analyzed by staining alkaline phosphatase (ALP) with 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) and by von Kossa staining of deposited calcium phosphate. The combinations of culture media and supplements significantly influenced osteogenic differentiation, but the seeding protocol did not. Staining of ALP and calcium phosphate could be achieved only if our own mix of osteogenic supplements was used in combination with Dulbecco's modified Eagle medium or if a commercial mix of osteogenic supplements was used in combination with osteoblast growth medium. Especially for von Kossa, we observed great variations in the staining intensity. Because osteogenic differentiation is a complex process, the origin of the osteoblasts, cell culture media and osteogenic supplements should be established by preliminary experiments to achieve optimal differentiation. Staining of ALP or deposited calcium phosphate should be supplemented with qRT-PCR studies to learn more about the influence of specific supplements on osteogenic markers.

Large-scale preparation of a DNA fragment containing the strong promoter A1 of the phage T7.

A procedure has been developed to isolate DNA fragments on a large scale. A DNA fragment of 130 base-pairs containing the strong promoter A1 of the phage T7 was purified to homogeneity in amounts of 10 mg. The procedure includes the rapid purification of gram amounts of plasmid DNA, a new, simple method to separate small DNA fragments from the vector by a phenol/water partitioning system, and a liquid-liquid PEG-dextran partition chromatography for the final purification of the fragment. The fragment was cloned in two vector systems: The vector pDS1, to1+ (1), containing an efficient terminator downstream from the promoter integration site, gives high yields, 3-4 mg plasmid DNA per liter medium. In the plasmid pWH802 (2), which is not specially designed for the amplification of a strong promoter, the integration of the promoter was possible but the yield decreased by a factor of about 50. The stability of the inserts was tested in both systems. Monomeric inserts were stable in both plasmids, multimeric inserts up to a tetramer were only stable in pWH802. Only one orientation of the fragment was found.

Nucleation of RNA chain formation by Escherichia coli DNA-dependent RNA polymerase.

We have studied the early steps in RNA synthesis. The kinetic behaviour of the nascent RNA, having chain lengths between 3 and 11 bases, and the transcription fidelity were analysed using the bacteriophage T7 A1 promoter. By moving the stop-inducing base at position +12 in the wild-type template in single base steps upstream, a set of closely related templates was constructed which allowed stalling of the complexes in the registers 11, 10, 9 and 8. Using this set of templates sigma-factor release was determined. It occurs when RNA synthesis has proceeded to base position +9. Analysis of the RNA synthesis both with and without heparin yielded the following results: there are three kinds of complexes, (a) the well-known abortively transcribing complex, which is present until the RNA has reached a length of 5 bases, (b) an intermediate complex having RNA chain lengths between 6 and 8 bases, which is stably bound but has high forward as well as back reaction rates, (c) complexes with RNA chains consisting of more than 8 bases, which are stably bound and do not contain the sigma-factor. In general, the likelihood of chain elongation and the stability of the complexes increases with increasing RNA chain length in the early stages of RNA synthesis. Also the transcription fidelity increases correspondingly. Lack of fidelity leads to additional RNA products during the abortive state of transcription. "Read through" of RNA polymerase at stall positions of +8 to +11 also result from misincorporation.

Thyrotrophin-releasing hormone test: an adverse reaction.

The side effects of intravenous (IV) administration of protirelin (thyrotrophin-releasing hormone [TRH] are usually mild and transient. The loss of consciousness and apical heart tones occurred in two young patients after protirelin injection. The mechanism for the reactions in these patients is not known, but the absence of heart sounds in the presence of no palpable pulse and no obtainable blood pressure should now be included among the adverse effects that may occur after administration of protirelin IV.

Bacteremia in diabetic patients: comparison of incidence and mortality with nondiabetic patients.

We determined the incidence of bacteremia and associated mortality in diabetic and nondiabetic patients in the four major hospitals of one metropolitan area over the 5-yr period 1977-1981. Mortality rates, based on episodes of bacteremia, were similar in diabetic and nondiabetic patients in most instances. Diabetic patients experienced lower mortality rates from Enterobacteriaceae bacteremia compared with nondiabetic patients; this finding was explained by a greater tendency for diabetic patients to have Escherichia coli bacteremia due to community-acquired urinary tract infection. However, the incidence of bacteremia due to all microorganisms was increased twofold in diabetic patients and the incidence of Enterobacteriaceae bacteremia was increased threefold. Because of their increased incidence of bacteremia, diabetic patients in this population were nearly twice as likely to die as a result of bacteremia compared with nondiabetic patients. Thus, the frequent occurrence of bacteremia among patients with diabetes mellitus represents a significant problem.

Intravenous or intramuscular anti-HBs immunoglobulin for the prevention of hepatitis B reinfection after orthotopic liver transplantation.

To prevent reinfection with hepatitis B virus after orthotopic liver transplantation, patients receive long-term intravenous anti-HBs immunoprophylaxis. We compared the pharmacokinetics of intravenously and intramuscularly administered commercially available hepatitis B virus immunoglobulins. The study group consisted of 12 patients on immunoprophylaxis after orthotopic liver transplantation, who were Hbs antigen negative; 11 were anti-HBe positive and one was HBe positive. The patients first received intravenous immunoglobulin, and six of them were then transferred to intramuscular immunoglobulin. Our findings show that with fortnightly intramuscular application of 1000 IU of anti-HBs, reproducible and stable antibody titers above 100 IU of anti-HBs can be achieved. Side effects of intramuscular immunoprophylaxis are minimal and the method is safe. The switch from intravenous (1500 IU of anti-HBs) to intramuscular (1000 IU of anti-HBs) reduced the cost of immunoprophylaxis by more than 50%.

Pneumococcal bacteremia in infants and children: a ten-year experience at the Cook County Hospital with special reference to the pneumococcal serotypes isolated.

In a ten-year period we identified 305 hospitalized children with a pneumococcal bacteremia. From these children 293 pneumococcal isolates were serotyped, and 90% belonged to a group of 11 "prevalent serotypes." These 11 serotypes were the prevalent serotypes isolated from children in all disease categories, as well as from children with sickle-cell disease. No more than 1% of the isolates belonged to any one of the other serotypes. A pneumococcal vaccine effective against these 11 prevalent serotypes should be optimal for use in children. Our highest case fatality rates were noted in children with meningitis (13%) and children with sickle-cell disease (20%). A polysaccharide pneumococcal vaccine might not have prevented most of our pneumococcal meningitis, as 80% of these children were less than 1 year old, an age when polysaccharide vaccines are poor antigens. On the other hand, many of our children with sickle-cell disease acquired their pneumococcal bacteremia at an older age and should have benefitted from such a vaccine.

Site of airflow obstruction during early and late phase asthmatic responses to allergen bronchoprovocation.

Chronic asthma and late asthmatic responses (LAR) are associated with local inflammation which might be expected to produce airflow obstruction in small airways and to increase nonspecific airway reactivity. In contrast, early asthmatic responses (EAR) are primarily bronchospastic and probably involve more central airways. We challenged 17 nonsmoking, mildly asthmatic atopic subjects with allergen bronchoprovocation and measured changes in spirometry (FEV1) over the next 24 hours. Each subject also performed a helium-oxygen (He-O2) flow-volume loop before challenge (baseline), during the EAR, and six hours and 24 hours after challenge to measure the effect of gas density on flow rates at midvital capacity. Twelve subjects had both an EAR and a LAR; five subjects had only a LAR. Of these 17 subjects, 15 were initially density dependent, while only two were density independent. During the EAR, 13 percent of the density dependent population had significant decreases in delta Vmax 50 percent; 47 percent had significant decreases during LAR. The He-O2 flow data analyzed at specified time points after challenge revealed significant decreases in the mean delta Vmax 50 percent at six hours in those who had only a LAR (p less than 0.01). In those who had a dual airway response, density dependence increased during the EAR, but decreased at six and significantly at 24 hours (p less than 0.05) postchallenge. There was a strong trend for the severity of the LAR (measured by changes in FEV1) to be directly related to the total decrease in delta Vmax 50 percent during the LAR. We conclude that late asthmatic responses occur frequently after a single antigenic bronchial challenge and can be associated with persistent symptoms. The LAR were often associated with a decrease in density dependence of maximal expiratory airflow, and therefore, may involve small airways.

Upper airway obstruction presenting as exercise-induced asthma.

A patient presented with a ten-year history of exercise-induced wheezing. After trials of metaproterenol and cromolyn failed to improve her symptoms, she was observed during exercise. She proved to have inspiratory stridor caused by collapse of the posterior aryepiglottic folds over the vocal cords during inspiration only following exercise. Symptoms primarily improved with physical conditioning. Further improvement came after treatment of her chronic rhinitis and post-nasal drip with a steroid nasal spray.

Prevalence of bronchial hyperresponsiveness in highly trained athletes.

Previous studies indicated that the prevalence of symptomatic asthma is about 4 to 7 percent. No similar studies exist to suggest the prevalence of asthma in highly trained competitive athletes, since asthma is thought to be an uncommon disease in this population. We became concerned, therefore, when a large number of football players developed symptoms consistent with asthma during preparation in California for the Rose Bowl in December 1981. We studied the team and found 12 percent of the football players admitted to a history of asthma, whereas none of the members of the university basketball team and 7 percent of a group of sophomore medical students and physician assistant students gave a history of asthma. Furthermore, 19 percent of the football players indicated that at some time they had chest tightness, cough, wheezing, or prolonged shortness of breath after exercise; 12 percent of the basketball players and 37 percent of the students indicated such a history. We examined each of these three groups for non-specific bronchial hyperresponsiveness to inhaled methacholine using a modified methacholine bronchoprovocation (MBP) challenge and found that 76 of 151 (50 percent) football players tested had positive tests; 76 percent of those with symptoms had positive results of inhalation tests and 47 percent of those with minimal or no symptoms had positive test results. In addition, four of 16 (25 percent) basketball players and 69 of 167 (41 percent) students had positive MBP tests. These studies indicate that bronchial hyperresponsiveness to inhaled methacholine is much more common in these young adults than has previously been suspected.

Bronchoalveolar lavage of allergic asthmatic patients following allergen bronchoprovocation.

In these studies, we describe the use of bronchoalveolar lavage (BAL) to study local changes following aerosol bronchoprovocation (BPC) and environmental exposure to antigen in mildly symptomatic asthmatic patients. The BAL was performed in 12 atopic subjects "out of season," and in five normal subjects at baseline, less than or equal to 4, or 24 hours following BPC. Five asthmatic patients were also lavaged during seasonal exposure to allergen. The BAL cells were examined with light and transmission electron microscopy. Bronchoprovocation, by itself, resulted in an average maximal decrease in FEV1 of 13 percent just prior to BAL. There was no significant decrease in FEV1 as a result of BAL. Within four hours after BPC, the number of neutrophils was significantly greater in BAL compared to baseline (1.5 +/- 0.6 X 10(5) vs 3.4 +/- 1.7 X 10(5) cells; p less than 0.01), and the number of eosinophils was significantly greater within four hours and at 24 hours when compared to baseline values (0.4 +/- 0.3 X 10(5) vs 1.9 +/- 0.7 X 10(5) vs 1.2 +/- 0.4 X 10(5) cells; p less than 0.02). Transmission electron micrographs of BAL from lungs of asthmatic patients revealed degranulation of mast cells and loss of core material from eosinophil granules following challenge with aerosolized allergen or with spontaneous environmental exposure. These studies show that in carefully selected, mildly symptomatic asthmatic subjects, BPC and BAL may be useful to evaluate pathogenetic mechanisms in allergic bronchial asthma.