Bone Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Ameliorated Lipopolysaccharide-Induced Lung Injury Via the miR-21-5p/PCSK6 Pathway.

Acute lung injury (ALI) is a life-threatening disease that currently lacks a cure. Although stem cell-derived small extracellular vesicles (sEVs) have shown promising effects in the treatment of ALI, their underlying mechanisms and responsible components have yet to be identified. Proprotein convertase subtilisin/kexin type 6 (PCSK6) is a gene involved in inflammation and a potential target of miR-21-5p, a microRNA enriched in stem cell-derived sEVs. The current study investigated the role of PCSK6 in lipopolysaccharide (LPS)-induced ALI and its interaction with miR-21-5p. Notably, our results showed that PCSK6 expression was positively correlated with LPS stimulation. Knockdown of PCSK6 ameliorated LPS-induced inhibition of proliferation and upregulation of permeability in human BEAS-2B cells, whereas PCSK6 overexpression displayed the opposite effects. BEAS-2B cells were able to actively internalize the cocultured bone mesenchymal stem cell (MSC)-derived sEVs (BMSC-sEVs), which alleviated the cell damage caused by LPS. Overexpressing PCSK6, however, eliminated the therapeutic effects of BMSC-sEV coculture. Mechanistically, BMSC-sEVs inhibited PCSK6 expression via the delivery of miR-21-5p, which is directly bound to the PCSK6 gene. Our work provides evidence for the role of PCSK6 in LPS-induced ALI and identified miR-21-5p as a component of BMSC-derived sEVs that suppressed PCSK6 expression and ameliorated LPS-induced cell damage. These results reveal a novel molecular mechanism for ALI pathogenesis and highlight the therapeutic potential of using sEVs released by stem cells to deliver miR-21-5p for ALI treatment.

Club Cell TRPV4 Serves as a Damage Sensor Driving Lung Allergic Inflammation.

Airway epithelium is the first body surface to contact inhaled irritants and report danger. Here, we report how epithelial cells recognize and respond to aeroallergen alkaline protease 1 (Alp1) of Aspergillus sp., because proteases are critical components of many allergens that provoke asthma. In a murine model, Alp1 elicits helper T (Th) cell-dependent lung eosinophilia that is initiated by the rapid response of bronchiolar club cells to Alp1. Alp1 damages bronchiolar cell junctions, which triggers a calcium flux signaled through calcineurin within club cells of the bronchioles, inciting inflammation. In two human cohorts, we link fungal sensitization and/or asthma with SNP/protein expression of the mechanosensitive calcium channel, TRPV4. TRPV4 is also necessary and sufficient for club cells to sensitize mice to Alp1. Thus, club cells detect junction damage as mechanical stress, which signals danger via TRPV4, calcium, and calcineurin to initiate allergic sensitization.

Phase I study on safety and pharmacokinetics of a novel influenza endonuclease inhibitor, AL-794 (JNJ-64155806), following single- and multiple-ascending doses in healthy adults.

BACKGROUND: This double-blind, first-in-human Phase I study evaluated pharmacokinetics, safety and tolerability of AL-794 (prodrug of ALS-033719), a potent endonuclease inhibitor of influenza A and B in healthy volunteers. METHODS: Healthy adult volunteers were randomized to AL-794 (50-2,000 mg single ascending doses, fasting) or placebo (5 cohorts, n=6:2 AL-794: placebo/cohort) in part 1, and AL-794 (50-600 mg multiple ascending doses, twice-daily, fed or fasted) or placebo (3 cohorts, n=8:2 AL-794: placebo/cohort) for 7 days in part 2. In part 3, 8 healthy volunteers from part 1 received 450 mg AL-794 (n=6) or placebo (n=2) following a high-fat meal. All dosing was done with an oral suspension. Blood and urine samples for pharmacokinetics were collected at scheduled times and analysed for ALS-033719 and ALS-033927 (inactive glucuronide) plasma concentrations using LC-MS/MS. RESULTS: ALS-033719 plasma concentrations increased dose proportionately up to 150 mg but less than proportionately above 150 mg. Steady-state was generally achieved by the third dose. ALS-033719 exposure increased following administration with a standard meal (19%-33%) or high-fat meal (3-3.6-fold). ALS-033927 was the major metabolite observed. Renal elimination was negligible (0.2%). Seventeen AL-794-treated healthy volunteers reported ≥1 treatment-emergent adverse event (TEAE; part 1: n=6, 24%; part 2: n=11, 69%). The most common TEAEs were headache (part 1: n=3; part 2: n=5) and dizziness (part 1: n=2; part 2: n=6). CONCLUSIONS: AL-794 up to 200 mg twice daily achieved ALS-033719 exposures which are expected to be efficacious and were generally tolerated. Further studies are planned to characterize safety and antiviral activity.

The Allergen Der p3 from House Dust Mite Stimulates Store-Operated Ca2+ Channels and Mast Cell Migration through PAR4 Receptors.

The house dust mite is the principal source of perennial aeroallergens in man. How these allergens activate innate and adaptive immunity is unclear, and therefore, there are no therapies targeting mite allergens. Here, we show that house dust mite extract activates store-operated Ca2+ channels, a common signaling module in numerous cell types in the lung. Activation of channel pore-forming Orai1 subunits by mite extract requires gating by STIM1 proteins. Although mite extract stimulates both protease-activated receptor type 2 (PAR2) and PAR4 receptors, Ca2+ influx is more tightly coupled to the PAR4 pathway. We identify a major role for the serine protease allergen Der p3 in stimulating Orai1 channels and show that a therapy involving sub-maximal inhibition of both Der p3 and Orai1 channels suppresses mast cell activation to house dust mite. Our results reveal Der p3 as an important aeroallergen that activates Ca2+ channels and suggest a therapeutic strategy for treating mite-induced asthma.

Sensitization to 10 mites in a tropic area. Der p and Der f are important risk factor for sensitization to other mites from Pyroglyphidae, Acaridae, Chortoglyphidae, and Glyciphagidae families.

BACKGROUND: Much is known about the frequency of sensitization to Blomia tropicalis, Dermatophagoides pteronyssinus and Dermatophagoides farinae, although less is known about sensitization to other species and their possible interactions. OBJECTIVE: In patients with allergic manifestations, to evaluate the frequency of sensitization to 10 species of mites in a tropical area and their possible interactions. METHODS: Cross-sectional study. Sensitization was evaluated by skin tests. A generalized linear Poisson regression model with robust variance was used. Based on the sensitization probability reasons and social networking analysis, explorations of relationship for 10 mites were performed. RESULTS: 147 patients were included. The highest sensitization was found to mites' family Pyroglyphidae (> 70 %) and less frequently was the Glycyphagidae family (< 50 %). Sensitization to any mites significantly increased the likelihood of sensitization to others. Sensitization to Der f or Der p increased, more than 20 times the likelihood of sensitization to other mites of the Pyroglyphidae family and more than 10 times to mites from other families. Sensitization to mites from Glycyphagidae, Chortoglyphidae or Acaridae family also increased the risk of sensitization to other mites but less than 5 times. CONCLUSION: Sensitization to mites is frequent in tropical area. Pyroglyphidae sensitization is the main risk factor for polysensitization with other mites from Glycyphagidae, Chortoglyphidae or Acaridae. These results must be considered at diagnosis and treatment of allergy diseases.

Association of Prostate Cancer Risk Variants with TMPRSS2:ERG Status: Evidence for Distinct Molecular Subtypes.

BACKGROUND: Numerous genetic variants have been confirmed as prostate cancer risk factors. These variants may confer susceptibility to the development of specific molecular alterations during tumor initiation and progression. The TMPRSS2:ERG gene fusion occurs in roughly 50% of prostate cancers. Genetic risk variants may influence the development of this fusion. We sought to determine whether prostate cancer risk variants are differentially associated with TMPRSS2:ERG fusion-positive and negative cancer. METHODS: In the Health Professionals Follow-up Study and Physicians' Health Study Tumor Cohort, we evaluated the associations of 39 prostate cancer risk SNPs with TMPRSS2:ERG fusion status, measured by ERG protein expression. Logistic regression was performed to generate OR and 95% confidence intervals. The primary outcome was ERG(+) (n = 227) versus ERG(-) (n = 260) prostate cancer. A secondary outcome was ERG(+) or ERG(-) cancer versus controls without cancer. RESULTS: Six of 39 SNPs were significantly associated (P < 0.05) with ERG(+) versus ERG(-) disease. Three SNPs were exclusively associated with the risk of ERG(+), one with risk of ERG(-), and two with associations trending in opposite directions for ERG(+) and ERG(-) Only two significant SNPs would be expected by chance. CONCLUSIONS: Prostate cancer genetic risk variants are differentially associated with the development of ERG(+) and ERG(-) prostate cancer. IMPACT: Our findings suggest the molecular process of prostate carcinogenesis may be distinct for men with different underlying genetic predisposition. When examining risk factors for prostate cancer, the integration of molecular subtypes may enhance understanding of the etiology of this disease. Cancer Epidemiol Biomarkers Prev; 25(5); 745-9. ©2016 AACR.

Intravitreal inhibition of complement C5a reduces choroidal neovascularization in mice.

PURPOSE: To investigate the influence of complement component C5a inhibition on laser-induced choroidal neovascularization (CNV) in mice using a C5a specific L-aptamer. METHODS: In C57BL/6 J mice CNV was induced by argon-laser, C5a-inhibitor (NOX-D20) was intravitreally injected in three concentrations: 0.3, 3.0, and 30 mg/ml. The unPEGylated derivate (NOX-D20001) was applied at 3.0 mg/ml; the vehicle (5 % glucose) was injected in controls. Vascular leakage was evaluated using fluorescence angiography, CNV area was examined immunohistochemically. Activated immune cells surrounding the CNV lesion and potential cytotoxicity were analyzed. RESULTS: Compared to controls, CNV areas were significantly reduced after NOX-D20 injection at a concentration of 0.3 and 3.0 mg/ml (p = 0.042; p = 0.016). NOX-D20001 significantly decreased CNV leakage but not the area (p = 0.007; p = 0.276). At a concentration of 30 mg/ml, NOX-D20 did not reveal significant effects on vascular leakage or CNV area (p = 0.624; p = 0.121). The amount of CD11b positive cells was significantly reduced after treatment with 0.3 and 3.0 mg/ml NOX-D20 (p = 0.027; p = 0.002). No adverse glial cell proliferation or increased apoptosis were observed at effective dosages. CONCLUSIONS: Our findings demonstrate that the targeted inhibition of complement component C5a reduces vascular leakage and neovascular area in laser-induced CNV in mice. NOX-D20 was proven to be an effective and safe agent that might be considered as a therapeutic candidate for CNV treatment. The deficiency of activated immune cells highlights promising new aspects in the pathology of choroidal neovascularization, and warrants further investigations.

Pharmacokinetics and toxicology of a fibroblast activation protein (FAP)-activated prodrug in murine xenograft models of human cancer.

BACKGROUND: As carcinoma progresses, the stroma undergoes a variety of phenotypic changes, including the presence of carcinoma-associated fibroblasts (CAFs) that express fibroblast activation protein (FAP). FAP is a post-prolyl endopeptidase whose expression in a healthy adult is largely restricted to the cancer-associated stroma. FAP-targeted prodrugs with a 100-fold greater therapeutic window over the parent compound were previously generated. METHODS: Prodrugs and non-cleavable controls were incubated in the presence of FAP. Plasma and tumor half-lives (t1/2) of the full-length and active forms of the prodrugs were determined using LCMS. Biodistribution studies of prodrug activation were performed. Histopathological analysis of tissues from treated animals were compared to vehicle-treated controls. Toxicity and efficacy studies were performed in human breast (MDA-MB-231 and MCF-7) and prostate (LNCaP) cancer xenografts models. RESULTS: These FAP-activated prodrugs have a significantly slower clearance from tumor tissue than the circulation (∼12 vs. ∼4.5 hr). Micromolar concentrations of active drug persist in the tumor. Active drug is detected in non-target tissues; however, histopathologic evaluation reveals no evidence of drug-induced toxicity. A FAP-activated prodrug (ERGETGP-S12ADT) inhibits tumor growth in multiple human breast and prostate cancer xenograft models. The anti-tumor effect is comparable to that observed with docetaxel, but results in significantly less toxicity. CONCLUSION: FAP-activated prodrugs are a viable strategy for the management of prostate and other cancers. These prodrugs exhibit less toxicity than a commonly used chemotherapeutic agent. Further refinement of the FAP cleavage site for greater specificity may reduce prodrug activation in non-target tissues and enhance clinical benefit.

Consumption of gluten with gluten-degrading enzyme by celiac patients: a pilot-study.

AIM: To assesses the safety and efficacy of Aspergillus niger prolyl endoprotease (AN-PEP) to mitigate the immunogenic effects of gluten in celiac patients. METHODS: Patients with initial diagnosis of celiac disease as confirmed by positive serology with subtotal or total villous atrophy on duodenal biopsies who adhere to a strict gluten-free diet (GFD) resulting in normalised antibodies and mucosal healing classified as Marsh 0 or I were included. In a randomised double-blind placebo-controlled pilot study, patients consumed toast (approximately 7 g/d gluten) with AN-PEP for 2 wk (safety phase). After a 2-wk washout period with adherence of the usual GFD, 14 patients were randomised to gluten intake with either AN-PEP or placebo for 2 wk (efficacy phase). Measurements at baseline included complaints, quality-of-life, serum antibodies, immunophenotyping of T-cells and duodenal mucosa immunohistology. Furthermore, serum and quality of life questionnaires were collected during and after the safety, washout and efficacy phase. Duodenal biopsies were collected after the safety phase and after the efficacy phase. A change in histological evaluation according to the modified Marsh classification was the primary endpoint. RESULTS: In total, 16 adults were enrolled in the study. No serious adverse events occurred during the trial and no patients withdrew during the trial. The mean score for the gastrointestinal subcategory of the celiac disease quality (CDQ) was relatively high throughout the study, indicating that AN-PEP was well tolerated. In the efficacy phase, the CDQ scores of patients consuming gluten with placebo or gluten with AN-PEP did not significantly deteriorate and moreover no differences between the groups were observed. During the efficacy phase, neither the placebo nor the AN-PEP group developed significant antibody titers. The IgA-EM concentrations remained negative in both groups. Two patients were excluded from entering the efficacy phase as their mucosa showed an increase of two Marsh steps after the safety phase, yet with undetectable serum antibodies, while 14 patients were considered histologically stable on gluten with AN-PEP. Also after the efficacy phase, no significant deterioration was observed regarding immunohistological and flow cytometric evaluation in the group consuming placebo compared to the group receiving AN-PEP. Furthermore, IgA-tTG deposit staining increased after 2 wk of gluten compared to baseline in four out of seven patients on placebo. In the seven patients receiving AN-PEP, one patient showed increased and one showed decreased IgA-tTG deposits. CONCLUSION: AN-PEP appears to be well tolerated. However, the primary endpoint was not met due to lack of clinical deterioration upon placebo, impeding an effect of AN-PEP.

Occupational asthma in female factory worker resulting from exposure to savinase in dishwashing tablets-a case study.

OBJECTIVES: Savinase is one of the endopeptidases widely used in washing detergents. Its ability to cause respiratory allergy has been known. Up to now, most cases of occupational asthma (OA) to savinase have been described among workers involved in the manufacture of laundry detergents. We present a case study of 51-year-old female worker of a dishwashing tablets factory, who had been packaging ready-made tablets into foil wrappers for 4 years and developed respiratory symptoms, such as cough, dyspnoea and wheezing. METHODS: A number of clinical procedures were performed, including the clinical examination, routine laboratory tests, evaluation of total and allergen-specific serum IgE (asIgE) to enzymes, skin prick tests for common allergens, rest spirometry, inhalation methacholine challenge test and a single-blind, placebo-controlled specific inhalation challenge test (SICT) with dishwashing tablets. RESULTS: Clinical findings and results of routine laboratory tests were within normal limits. Baseline nonspecific bronchial hyperreactivity was revealed. In patient's serum blood we found significantly elevated asIgE to savinase. Decline of FEV1 and PEF in late phase of asthmatic reaction was observed during the specific challenge test. The patient reported chest tightness between 5-12 hours after exposure to dishwashing tablet ingredients. Cytological assessment of an induced sputum revealed increase in the percentage of eosinophils 24 hours after specific challenge in comparison to values noted before the SICT. CONCLUSIONS: Positive clinical response to the challenge confirmed in objective method tests validated the diagnosis of OA.

Acute kidney injury during therapy with an antisense oligonucleotide directed against PCSK9.

Antisense oligonucleotides have been explored widely in clinical trials and generally are considered to be nontoxic for the kidney, even at high concentrations. We report a case of toxic acute tubular injury in a healthy 56-year-old female volunteer after a pharmacologically active dose of a locked nucleic acid antisense oligonucleotide was administered. The patient received 3 weekly subcutaneous doses of experimental drug SPC5001, an antisense oligonucleotide directed against PCSK9 (proprotein convertase subtilisin/kexin type 9) that is under investigation as an agent to reduce low-density lipoprotein cholesterol levels. Five days after the last dose, the patient's serum creatinine level increased from 0.81 mg/dL at baseline (corresponding to an estimated glomerular filtration rate [eGFR] of 78 mL/min/1.73 m(2)) to 2.67 mg/dL (eGFR, 20 mL/min/1.73 m(2)), and this increase coincided with the presence of white blood cells, granular casts, and minimal hematuria on urine microscopy. The patient's serum creatinine level peaked at 3.81 mg/dL (eGFR, 13 mL/min/1.73 m(2)) 1 week after the last oligonucleotide dose. Kidney biopsy showed multifocal tubular necrosis and signs of oligonucleotide accumulation. Upon conservative treatment, the patient's serum creatinine level gradually decreased and reached her baseline level 44 days after the last oligonucleotide was administered. The patient recovered fully and kidney function was normal at every follow-up visit.

Allergic asthma caused by exposure to bacterial alpha-amylase Termamyl®.

BACKGROUND: Airborne enzymes behave as potent respiratory allergens. Till date, allergic disorders caused by genetically engineered enzymes widely used in the industry, have not been reported. RESULTS AND CONCLUSIONS: We describe a worker employed in the detergent industry who developed asthma and rhinitis from IgE-mediated sensitization to the thermostable endo-alpha-amylase Termamyl® and to the protease Savinase®. This is the first report showing that Termamyl® elicits allergic respiratory disorders in humans.

[Biological significance of IgA1 proteases].

The IgA1 proteases are a group of proteolytic enzymes, which are produced by pathogenic bacteria that infect and colonize mucosal surfaces. This group of proteolytic enzymes was found to cleave specific peptide bonds within the sequence TPPTPSPSTPPTPSPS (T, P and S are threonine, proline and serine residues, respectively) found in the hinge region of human IgA1. Several findings support the role of IgA1 protease, for example, its ability to cleave human LAMP1 (hLAMP1), TNF-RII, the CD8 molecule of T lymphocytes and granulocyte-macrophage colony-stimulating factor (GM-CSF), synaptobrevin II, hormone human chorionic gonadotropin, and its ability to exhibit important immunomodulatory properties, etc. , in particular the induction of proinflammatory cytokines. The IgA1 proteases have been found to instigate part of the T cell inflammatory response, especially to stimulate the release of cytokines such as tumour necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and interleukin-8 (IL-8). All these suggest that this enzyme plays a significant role in pathogenesis. There are many other researches to explore new biological treatments of diseases using the biological characteristics of IgA1 protease.

Brown recluse spider (Loxosceles reclusa) envenomation leading to acute hemolytic anemia in six adolescents.

Loxosceles reclusa (brown recluse spider) bites often cause local envenomation reactions; however, acute hemolysis from systemic loxoscelism is rare. To highlight this important diagnostic consideration for unexplained hemolysis in areas endemic for brown recluse spiders, we report on 6 adolescents with acute hemolytic anemia from presumed L reclusa bites.

Characterization of water-in-oil microemulsion for oral delivery of earthworm fibrinolytic enzyme.

Earthworm fibrinolytic enzyme (EFE-d, Mr 24177), a water-soluble protein, is clinically used for the management of cardiovascular diseases. However, this protein drug has a very low oral bioavailability because of its low oil/water partitioning, low membrane permeability and unstable nature in harsh gastric juice. This study explored the possibility of absorption and efficacy enhancement for EFE-d through the delivery of the water-in-oil (w/o) microemulsions. The w/o microemulsion consisting of Labrafac CC, Labrasol, Plurol Oleique CC 497 and saline (54/18/18/10, % w/w) was developed and characterized, including conductivity, viscosity, particle size and in vitro membrane permeability. The w/o microemulsion and the control solution of EFE-d were administered intraduodenally (or orally) to rats. The w/o microemulsion possessed a higher intestinal membrane permeability in vitro as well as a higher absorption and efficacy in vivo, when compared to control solution. The intraduodenal bioavailability of EFE-d for microemulsions was 208-fold higher than that of control solution and the absolute bioavailability was 17.55%. Meanwhile, there was no tissue damage of the intestinal mucosa found after oral multiple-dose administration of the EFE-d microemulsion to rats. These findings indicated that the w/o microemulsion may represent a safe and effective oral delivery system for hydrophilic bioactivity macromolecules.

[Regenerative medicine of skeletal muscle].

In the dystrophin-deficient mdx mice, an animal model of Duchenne muscular dystrophy (DMD), damaged skeletal muscles are efficiently regenerated and thus the animals thrive. The phenotypic differences between DMD patients and mdx mice suggest the existence of factors that modulate the muscle wasting in the mdx mice. To identify these factors, we searched for mRNAs affected by the mdx mutation using cDNA microarrays with newly established skeletal muscle cell lines derived from mdx and normal mice. We found that genes encoding thymosin beta4, frizzled related protein 2 (FRP2), and regeneration-associated muscle protease (RAMP) are up-regulated in skeletal muscle of mdx mice. Thymosin beta4 was induced in both regenerating muscle fibers and inflammatory cells after muscle injury. It stimulated migration and chemotaxis of myoblasts. FRP2 was dramatically induced upon muscle injury. RNA interference-mediated knockdown of FRP2 mRNA in myoblasts resulted in a massive cell death. Thus FRP2 may enhance the survival rate of myoblasts in the regenerative regions. RAMP mRNA was specifically induced in the regenerating areas of injured skeletal muscle. Expression of RAMP and FRP2 was much lower in individual muscle cell lines derived from biopsy specimens from several DMD patients compared to in a normal muscle cell line. Above results suggest that thymosin beta4, FRP2, and RAMP may play roles in the regeneration of skeletal muscle and that down-regulation of these molecules could be involved in the progression of DMD in humans.

Study on OELs for enzyme-containing detergent in China.

This study is aimed at setting occupational exposure levels for total detergent dust and enzymes in detergent industries. The study population consisted of 795 workers from four enzyme-containing detergent manufacturing plants (A1, A2, B1 and B2), and 156 control workers from an electronic assembly factory. Work environment monitoring was conducted using high volume of air sampler fro measuring the concentration of total dust (mg/m3), and analyzing the level of enzyme (ng/m3) by ELISA method. A standard questionnaires, pulmonary function test, and skin prick test are used to assess health effects. The levels of detergent total dust varied from 0.2 mg/m3 to 12.54 mg/m3. For enzyme levels, in A1, B1 and B2, the concentration ranged from non-detectable to 9.92 ng/m3 and in A2, the concentration was analyzed by enzyme activity methods and was expressed as Gu/m3 (1 Gu/m3 = 16 ng/m3). The concentration is between 0.16-31.36 ng/m3. Non-specific irritation rates in exposed workers were significantly higher than that in controls. Based on the data collected from A1, B1 and control plants, 95% benchmark dose lower bound were calculated as 1.17 mg/m3. The difference of pulmonary function between exposed workers and controls is not significant. The results of SPT showed that neither Savinase- nor Alcalase-induced sensitization was found in controls. The prevalence rates of sensitization for Savinase and Alcalase were ranged between 3.2% and 31% in all enzyme-containing detergent manufacturers investigated. No case of occupational asthma was observed. For total dust, 1 mg/m3 is suggested as permissible concentration-time weighted average (PC-TWA), and 2 mg/m3 as permissible concentration-short term exposure limit (PC-STEL). For the enzyme Subtilisins, 15 ng/m3 is suggested as PC-TWA, and 30 ng/m3 as PC-STEL.

Exposure levels of asthmatic children to allergens, endotoxins, and serine proteases in a tropical environment.

A cross-sectional study was conducted in Bayamón, Puerto Rico, to identify and quantify indoor allergens, serine proteases, and bacterial endotoxin present in homes of asthmatic children. A total of 126 dust samples from houses were obtained from the entire mattress and bedside floor. Most of the patients had detectable levels of mite, cockroach, cat, and dog allergens. Mold allergens were found only in bedside floor dust samples. Mouse allergens were not detected. Forty-two percent, 36.5%, and 1.8% of the patients demonstrated exposures to sensitizing levels of mite, Bla g 1 and cat allergens, respectively. The percentage of patients exposed to high levels of allergens capable of triggering asthma symptoms was 33.3% and 26.4% for mite and Bla g 1 allergens. Only dog allergen, bacterial endotoxin, elastase, and trypsin were associated with asthma symptoms. Eighty-nine percent of the asthmatic children were exposed to endotoxin concentrations greater than 100 EU/mg dust, and more than half of the patients were exposed to high levels of serine proteases. Our study indicates that indoor concentrations of allergens traditionally associated with asthma symptoms and severity may not be applicable in tropical environments and highly ventilated households. In fact, in the study population, endotoxins, dog allergen, and serine proteases may play a dominant role in the induction of asthma symptoms.