Delivery of the Brainshuttle™ amyloid-beta antibody fusion trontinemab to non-human primate brain and projected efficacious dose regimens in humans.

There are few treatments that slow neurodegeneration in Alzheimer's disease (AD), and while therapeutic antibodies are being investigated in clinical trials for AD treatment, their access to the central nervous system is restricted by the blood-brain barrier. This study investigates a bispecific modular fusion protein composed of gantenerumab, a fully human monoclonal anti- amyloid-beta (Aß) antibody under investigation for AD treatment, with a human transferrin receptor 1-directed Brainshuttle™ module (trontinemab; RG6102, INN trontinemab). In vitro, trontinemab showed a similar binding affinity to fibrillar Aß40 and Aß plaques in human AD brain sections to gantenerumab. A single intravenous administration of trontinemab (10 mg/kg) or gantenerumab (20 mg/kg) to non-human primates (NHPs, Macaca fascicularis), was well tolerated in both groups. Immunohistochemistry indicated increased trontinemab uptake into the brain endothelial cell layer and parenchyma, and more homogeneous distribution, compared with gantenerumab. Brain and plasma pharmacokinetic (PK) parameters for trontinemab were estimated by nonlinear mixed-effects modeling with correction for tissue residual blood, indicating a 4-18-fold increase in brain exposure. A previously developed clinical PK/pharmacodynamic model of gantenerumab was adapted to include a brain compartment as a driver of plaque removal and linked to the allometrically scaled above model from NHP. The new brain exposure-based model was used to predict trontinemab dosing regimens for effective amyloid reduction. Simulations from these models were used to inform dosing of trontinemab in the first-in-human clinical trial.

Vaccine-induced CD8 T cells are redirected with peptide-MHC class I-IgG antibody fusion proteins to eliminate tumor cells in vivo.

Simulating a viral infection in tumor cells is an attractive concept to eliminate tumor cells. We previously reported the molecular design and the in vitro potency of recombinant monoclonal antibodies fused to a virus-derived peptide MHC class I complex that bypass the peptide processing and MHC loading pathway and directly displays a viral peptide in an MHC class I complex on the tumor cell surface. Here, we show that a vaccination-induced single peptide-specific CD8 T cell response was sufficient to eliminate B16 melanoma tumor cells in vivo in a fully immunocompetent, syngeneic mouse tumor model when mice were treated with mouse pMHCI-IgGs fusion proteins targeting the mouse fibroblast activation protein. Tumor growth of small, established B16 lung metastases could be controlled. The pMHCI-IgG had similar potency as an analogous pan-CD3 T-cell bispecific antibody. In contrast to growth control of small tumors, none of the compounds controlled larger solid tumors of MC38 cancer cells, despite penetration of pMHCI-IgGs into the tumor tissue and clear attraction and activation of antigen-specific CD8 T cells inside the tumor. pMHCI-IgG can have a similar potency as classical pan-T-cell recruiting molecules. The results also highlight the need to better understand immune suppression in advanced solid tumors.

The Contorsbody, an antibody format for agonism: Design, structure, and function.

The careful design of the antibody architecture is becoming more and more important, especially when the purpose is agonism. We present the design of a novel antibody format that is able to promote receptor dimerization and induce signal transduction resulting in cell proliferation. Mono-specific bivalent Y-shape IgGs made of two light chains and two heavy chains are engineered into single chain dimers of two modified heavy chains, resulting in the fixation of the two Fab fragments along the Fc dimerizing moiety. By this, an antagonist of the Her-receptor family, Trastuzumab, is re-formatted into an agonist by simply incorporating the original binding motif into a different geometrically and sterically constrained conformation. This novel format, named Contorsbody, retains antigen binding properties of the parental IgGs and can be produced by standard technologies established for recombinant IgGs. Structural analyses using molecular dynamics and electron microscopy are described to guide the initial design and to confirm the Contorsbody as a very compact molecule, respectively. Contorsbodies show increased rigidity compared to IgGs and their Fab moieties are positioned parallel and adjacent to each other. This geometry has an increased potential to trigger cell surface antigen or receptor 'cis'-dimerization without 'trans'-bridging of cells or mere receptor blockade.

Variable heavy-variable light domain and Fab-arm CrossMabs with charged residue exchanges to enforce correct light chain assembly.

Technologies for the production of bispecific antibodies need to overcome two major challenges. The first one is correct heavy chain assembly, which was solved by knobs-into-holes technology or charge interactions in the CH3 domains. The second challenge is correct light chain assembly. This can be solved by engineering the Fab-arm interfaces or applying the immunoglobulin domain crossover approach. There are three different crossovers possible, namely Fab-arm, constant domain and variable domain crossovers. The CrossMabCH1-CL exchange does not lead to the formation of unexpected side products, whereas the CrossMabFab and the CrossMabVH-VL formats result in the formation of typical side products. Thus, CrossMabCH1-CL was initially favored for therapeutic antibody development. Here, we report a novel improved CrossMab design principle making use of site-specific positional exchanges of charged amino acid pairs in the constant domain of these CrossMabs to enable the correct light chain assembly in the CrossMabVH-VL and improvements for the CrossMabFab design.

Magnetic resonance tissue phase mapping demonstrates altered left ventricular diastolic function in children with chronic kidney disease.

BACKGROUND: Echocardiographic examinations have revealed functional cardiac abnormalities in children with chronic kidney disease. OBJECTIVE: To assess the feasibility of MRI tissue phase mapping in children and to assess regional left ventricular wall movements in children with chronic kidney disease. MATERIALS AND METHODS: Twenty pediatric patients with chronic kidney disease (before or after renal transplantation) and 12 healthy controls underwent tissue phase mapping (TPM) to quantify regional left ventricular function through myocardial long (Vz) and short-axis (Vr) velocities at all 3 levels of the left ventricle. RESULTS: Patients and controls (age: 8 years-20 years) were matched for age, height, weight, gender and heart rate. Patients had higher systolic blood pressure. No patient had left ventricular hypertrophy on MRI or diastolic dysfunction on echocardiography. Fifteen patients underwent tissue Doppler echocardiography, with normal z-scores for mitral early diastolic (VE), late diastolic (VA) and peak systolic (VS) velocities. Throughout all left ventricular levels, peak diastolic Vz and Vr (cm/s) were reduced in patients: Vzbase -10.6 ± 1.9 vs. -13.4 ± 2.0 (P < 0.0003), Vzmid -7.8 ± 1.6 vs. -11 ± 1.5 (P < 0.0001), Vzapex -3.8 ± 1.6 vs. -5.3 ± 1.6 (P = 0.01), Vrbase -4.2 ± 0.8 vs. -4.9 ± 0.7 (P = 0.01), Vrmid -4.7 ± 0.7 vs. -5.4 ± 0.7 (P = 0.01), Vrapex -4.7 ± 1.4 vs. -5.6 ± 1.1 (P = 0.05). CONCLUSION: Tissue phase mapping is feasible in children and adolescents. Children with chronic kidney disease show significantly reduced peak diastolic long- and short-axis left ventricular wall velocities, reflecting impaired early diastolic filling. Thus, tissue phase mapping detects chronic kidney disease-related functional myocardial changes before overt left ventricular hypertrophy or echocardiographic diastolic dysfunction occurs.

Characterization of a re-engineered, mesothelin-targeted Pseudomonas exotoxin fusion protein for lung cancer therapy.

Mesothelin overexpression in lung adenocarcinomas correlates with the presence of activating KRAS mutations and poor prognosis. Hence SS1P, a mesothelin-targeted immunotoxin, could offer valuable treatment options for these patients, but its use in solid tumor therapy is hampered by high immunogenicity and non-specific toxicity. To overcome both obstacles we developed RG7787, a de-immunized cytotoxic fusion protein comprising a humanized SS1 Fab fragment and a truncated, B-cell epitope silenced, 24 kD fragment of Pseudomonas exotoxin A (PE24). Reactivity of RG7787 with sera from immunotoxin-treated patients was >1000 fold reduced. In vitro RG7787 inhibited cell viability of lung cancer cell lines with picomolar potency. The pharmacokinetic properties of RG7787 in rodents were comparable to SS1P, yet it was tolerated up to 10 fold better without causing severe vascular leak syndrome or hepatotoxicity. A pharmacokinetic/pharmacodynamic model developed based on NCI-H596 xenograft studies showed that for RG7787 and SS1P, their in vitro and in vivo potencies closely correlate. At optimal doses of 2-3 mg/kg RG7787 is more efficacious than SS1P. Even large, well established tumors (600 mm(3)) underwent remission during three treatment cycles with RG7787. Also in two patient-derived lung cancer xenograft models, Lu7336 and Lu7187, RG7787 showed anti-tumor efficacy. In monotherapy two treatment cycles were moderately efficacious in the Lu7336 model but showed good anti-tumor activity in the KRAS mutant Lu7187 model (26% and 80% tumor growth inhibition, respectively). Combination of RG7787 with standard chemotherapies further enhanced efficacy in both models achieving near complete eradication of Lu7187 tumors.

A New Class of Bifunctional Major Histocompatibility Class I Antibody Fusion Molecules to Redirect CD8 T Cells.

Bifunctional antibody fusion proteins engaging effector T cells for targeted elimination of tumor cells via CD3 binding have shown efficacy in both preclinical and clinical studies. Different from such a polyclonal T-cell recruitment, an alternative concept is to engage only antigen-specific T-cell subsets. Recruitment of specific subsets of T cells may be as potent but potentially lead to fewer side effects. Tumor-targeted peptide-MHC class I complexes (pMHCI-IgGs) bearing known antigenic peptides complexed with MHC class I molecules mark tumor cells as antigenic and utilize the physiologic way to interact with and activate T-cell receptors. If, for example, virus-specific CD8(+) T cells are addressed, the associated strong antigenicity and tight immune surveillance of the effector cells could lead to efficacious antitumor treatment in various tissues. However, peptide-MHC class I fusions are difficult to express recombinantly, especially when fused to entire antibody molecules. Consequently, current formats are largely limited to small antibody fragment fusions expressed in bacteria followed by refolding or chemical conjugation. Here, we describe a new molecular format bearing a single pMHCI complex per IgG fusion molecule characterized by enhanced stability and expression yields. This molecular format can be expressed in a full immunoglobulin format and can be designed as mono- or bivalent antibody binders. Mol Cancer Ther; 15(9); 2130-42. ©2016 AACR.

Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry.

The quadroma antibody represents the first attempt to produce a bispecific heterodimeric IgG antibody by somatic fusion of 2 hybridoma cells each expressing monoclonal antibodies with distinctive specificities. However, because of random heavy and light chain pairing, the desired functional bispecific antibody represents only a small fraction of the protein produced. Subsequently, the knobs-into-holes (KiH) approach was developed to enforce correct heavy chain heterodimerization. Assuming equimolar expression of 4 unmodified chains comprising 2 heavy and 2 light chains, the statistical distribution of all paired combinations can be calculated. With equimolar expression as the goal, we transfected HEK cells with 1:1:1:1 plasmid ratios and analyzed the protein A affinity-purified antibodies from the quadroma and KiH approaches qualitatively and quantitatively with regard to the estimated relative amounts of the products using electrospray quadrupole time-of-flight mass spectrometry. Our results show that all expected species are formed, and that, within the methodological limits, the species distribution in the mixtures corresponds approximately to the statistical distribution.

Left ventricular mass and systolic function in children with chronic kidney disease-comparing echocardiography with cardiac magnetic resonance imaging.

BACKGROUND: Increased left ventricular mass (LVM) is an important risk marker of uremic cardiovascular disease. Calculation of LVM by echocardiography (Echo) relies on geometric assumptions and in adults on hemodialysis overestimates LVM compared to cardiac magnetic resonance (CMR). We compare both techniques in children with chronic kidney disease (CKD). METHODS: Concurrent Echo and CMR was performed in 25 children with CKD (14 after kidney transplantation) aged 8-17 years. RESULTS: Compared to normal children, CMR-LVM was increased (standard deviation score (SDS) 0.39 ± 0.8 (p = 0.03)), stroke volume and cardiac output decreased (SDS -1.76 ± 1.1, p = 0.002 and -1.11 ± 2.0, p = 0.001). CMR-LVM index but not Echo-LVMI correlated to future glomerular filtration rate (GFR) decline (r = -0.52, p = 0.01). Mean Echo-LVM was higher than CMR-LVM (117 ± 40 vs. 89 ± 29 g, p < 0.0001), with wide limits of agreement (-6.2 to 62.8 g). The Echo-CMR LVM difference increased with higher Echo-LVMI (r = 0.77, p < 0.0001). Agreement of classifying left ventricular hypertrophy was poor with Cohen's kappa of 0.08. Mean Echo and CMR-ejection fraction differed by 1.42% with wide limits of agreement (-12.6 to 15.4%). CONCLUSIONS: Echo overestimates LVM compared to CMR, especially at higher LVM. Despite this, CMR confirms increased LVM in children with CKD. Only CMR-LVMI but not Echo-LVMI correlated to future GFR decline.

In vitro and in vivo activity of the low-immunogenic antimesothelin immunotoxin RG7787 in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, and new therapies are needed. RG7787 is a novel low-immunogenic antimesothelin recombinant immunotoxin (RIT), engineered to overcome the limitations of SS1P, a RIT now in clinical trials. In vitro activity was evaluated on five established PDAC cell lines (KLM-1, AsPC-1, BxPC-3, Panc 3.014, and PK-1) and on PDAC cells directly established from a patient tumor (GUMC108). RG7787 had subnanomolar IC50s in most cell lines, and was significantly more active than SS1P in GUMC108, KLM-1, and Panc 3.014 cells. GUMC108 was most sensitive, with RG7787 killing >99% of the cells. In a subcutaneous KLM-1 xenograft mouse model, two cycles of 3 × 2.5 mg/kg RG7787 QOD combined with two cycles of 1 × 50 mg/kg paclitaxel induced near-complete responses, with all tumors regressing below 5 mm(3) within 30 days after therapy was initiated (>95% decrease) and no significant growth increase for at least another 3 weeks. RG7787 alone gave limited but significant regressions and paclitaxel by itself arrested tumor growth. Quantifying the uptake of Alexa Fluor 647-labeled RG7787 in tumors showed that the RIT reached only 45% of KLM-1 cells, accounting in part for the limited responses. Paclitaxel did not improve RG7787 uptake, which thus cannot explain the beneficial effect of the combination therapy. In conclusion, RG7787 has high cytotoxic activity on PDAC cell lines as well as on primary patient cells. In vivo, this novel RIT gives durable near-complete tumor responses when combined with paclitaxel. RG7787 merits further evaluation for the treatment of PDAC.

Deciphering the hormonal signalling network behind the systemic resistance induced by Trichoderma harzianum in tomato.

Root colonization by selected Trichoderma isolates can activate in the plant a systemic defense response that is effective against a broad-spectrum of plant pathogens. Diverse plant hormones play pivotal roles in the regulation of the defense signaling network that leads to the induction of systemic resistance triggered by beneficial organisms [induced systemic resistance (ISR)]. Among them, jasmonic acid (JA) and ethylene (ET) signaling pathways are generally essential for ISR. However, Trichoderma ISR (TISR) is believed to involve a wider variety of signaling routes, interconnected in a complex network of cross-communicating hormone pathways. Using tomato as a model, an integrative analysis of the main mechanisms involved in the systemic resistance induced by Trichoderma harzianum against the necrotrophic leaf pathogen Botrytis cinerea was performed. Root colonization by T. harzianum rendered the leaves more resistant to B. cinerea independently of major effects on plant nutrition. The analysis of disease development in shoots of tomato mutant lines impaired in the synthesis of the key defense-related hormones JA, ET, salicylic acid (SA), and abscisic acid (ABA), and the peptide prosystemin (PS) evidenced the requirement of intact JA, SA, and ABA signaling pathways for a functional TISR. Expression analysis of several hormone-related marker genes point to the role of priming for enhanced JA-dependent defense responses upon pathogen infection. Together, our results indicate that although TISR induced in tomato against necrotrophs is mainly based on boosted JA-dependent responses, the pathways regulated by the plant hormones SA- and ABA are also required for successful TISR development.

Crystal structure of an anti-Ang2 CrossFab demonstrates complete structural and functional integrity of the variable domain.

Bispecific antibodies are considered as a promising class of future biotherapeutic molecules. They comprise binding specificities for two different antigens, which may provide additive or synergistic modes of action. There is a wide variety of design alternatives for such bispecific antibodies, including the "CrossMab" format. CrossMabs contain a domain crossover in one of the antigen-binding (Fab) parts, together with the "knobs-and-holes" approach, to enforce the correct assembly of four different polypeptide chains into an IgG-like bispecific antibody. We determined the crystal structure of a hAng-2-binding Fab in its crossed and uncrossed form and show that CH1-CL-domain crossover does not induce significant perturbations of the structure and has no detectable influence on target binding.

Mycorrhiza-induced resistance and priming of plant defenses.

Symbioses between plants and beneficial soil microorganisms like arbuscular-mycorrhizal fungi (AMF) are known to promote plant growth and help plants to cope with biotic and abiotic stresses. Profound physiological changes take place in the host plant upon root colonization by AMF affecting the interactions with a wide range of organisms below- and above-ground. Protective effects of the symbiosis against pathogens, pests, and parasitic plants have been described for many plant species, including agriculturally important crop varieties. Besides mechanisms such as improved plant nutrition and competition, experimental evidence supports a major role of plant defenses in the observed protection. During mycorrhiza establishment, modulation of plant defense responses occurs thus achieving a functional symbiosis. As a consequence of this modulation, a mild, but effective activation of the plant immune responses seems to occur, not only locally but also systemically. This activation leads to a primed state of the plant that allows a more efficient activation of defense mechanisms in response to attack by potential enemies. Here, we give an overview of the impact on interactions between mycorrhizal plants and pathogens, herbivores, and parasitic plants, and we summarize the current knowledge of the underlying mechanisms. We focus on the priming of jasmonate-regulated plant defense mechanisms that play a central role in the induction of resistance by arbuscular mycorrhizas.

Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies.

We describe a generic approach to assemble correctly two heavy and two light chains, derived from two existing antibodies, to form human bivalent bispecific IgG antibodies without use of artificial linkers. Based on the knobs-into-holes technology that enables heterodimerization of the heavy chains, correct association of the light chains and their cognate heavy chains is achieved by exchange of heavy-chain and light-chain domains within the antigen binding fragment (Fab) of one half of the bispecific antibody. This "crossover" retains the antigen-binding affinity but makes the two arms so different that light-chain mispairing can no longer occur. Applying the three possible "CrossMab" formats, we generated bispecific antibodies against angiopoietin-2 (Ang-2) and vascular endothelial growth factor A (VEGF-A) and show that they can be produced by standard techniques, exhibit stabilities comparable to natural antibodies, and bind both targets simultaneously with unaltered affinity. Because of its superior side-product profile, the CrossMab(CH1-CL) was selected for in vivo profiling and showed potent antiangiogenic and antitumoral activity.

Prognostic and predictive relevance of CA-125 at primary surgery of ovarian cancer.

INTRODUCTION: Despite radical surgery and chemotherapy, most patients with ovarian cancer develop recurrence and die due to progressive disease. To stratify patients for optimal therapy, prognostic and predictive factors are needed. We examined the role of pre- and postoperative CA-125 in this context. METHODS: A total of 231 patients with primary ovarian cancer who presented for surgery at our institution between 1996 and 2004 were included in this study (25% FIGO stage I/II and 75% FIGO stage III/IV). The prognostic and predictive values of CA-125 serum concentrations before and after surgery as well as their correlation with clinicopathological variables were analyzed. RESULTS: Median preoperative CA-125 was 61.6 kU/l (9-1,867 kU/l) in stage I/II patients and 533.15 kU/l (10-22,617 kU/l) in stage III/IV patients. Before surgery, 67% of stage I/II patients and 96% of stage III/IV patients had elevated CA-125 (>35 kU/l). There was a significant decrease in CA-125 after surgery in both patient cohorts (61.6-43.4 kU/l, P = 0.001 and 533.15-92.3 kU/l, P < 0.001, respectively). Furthermore, in stage III/IV patients with complete or so-called optimal (<1 cm residual disease) debulking, preoperative CA-125 levels were significantly lower than in patients with residual disease >1 cm (P = 0.01, P = 0.009, respectively). Neither CA-125 concentration before surgery nor its decrease was prognostically relevant for recurrence and survival at any stage. However, in stage III/IV patients, a high postoperative CA-125 was associated with shorter progression-free survival (P = 0.024). CONCLUSIONS: Although CA-125 serum levels differ significantly before and after surgery in early and advanced-stage ovarian cancer and preoperative CA-125 values correlate with surgical outcome in advanced-stage disease, we could not determine a preoperative cutoff value for prediction of the surgical result. A prognostic relevance was only observed for postoperative CA-125 in stage III/IV patients.

Tumor volume of growth hormone-secreting pituitary adenomas during treatment with pegvisomant: a prospective multicenter study.

CONTEXT: Clinical and biochemical remission in acromegaly can frequently be achieved with the recombinant GH receptor antagonist pegvisomant, even when other treatments fail. However, increases in tumor volume have been reported. OBJECTIVE: Because previous studies suffer from inhomogenous magnetic resonance imaging (MRI) protocols, this prospective study examined the long-term course of adenoma volume during pegvisomant therapy by standardized MRI. DESIGN: Five centers in Germany participated. High-resolution MRI was performed at baseline and 6, 12, and 24 months after enrollment. SETTING/PATIENTS: Patients were outpatients, and pegvisomant is third-line therapy in most of the cases. MAIN OUTCOME MEASURES: The primary end point was tumor volume at 24 month follow-up, measured by a single, double-blinded rater. RESULTS: Forty-five of 61 patients completed 24 months' follow-up (73.8%). Tumor volume increase greater than 25% during the study was observed in three of 61 patients (4.9%), all during the first year of enrollment. All three patients had had octreotide treatment before initiation of pegvisomant; none of them had had radiotherapy. All volumetric findings were comparable with clinical radiological interpretations. ANOVA revealed no significant change in tumor volume after 24 months (n = 45). CONCLUSIONS: This study shows that pegvisomant therapy infrequently coincides with tumor growth during long-term treatment of acromegaly. Because all significant tumor volume increases occurred during the first year, these changes might correlate to the change of medication and thus be the result of a rebound from somatostatin-induced shrinkage.

Prognostic significance of CA-125 in the management of patients with recurrent epithelial ovarian carcinoma selected for secondary cytoreduction.

BACKGROUND: Despite radical surgical and chemotherapeutic treatment of ovarian cancer, the majority of patients develop recurrence and die due to progressive disease. Routine measurement of the tumor marker CA-125 is often used in the follow-up management. However, the role of preoperative CA-125 as a prognostic factor before secondary cytoreduction of relapsed ovarian cancer has not been determined. PATIENTS AND METHODS: CA-125 serum concentration and relevant clinico-pathological variables were analyzed regarding their potential prognostic impact in patients selected for secondary cytoreduction of recurrent epithelial ovarian cancer. RESULTS: In total, 48 patients underwent secondary cytoreduction at the University Medical Center Hamburg-Eppendorf between 1996 and 2004 and 36 patients were evaluable for serum CA-125 concentration. Median age was 60 years (range 30-78 years) and median relapse-free survival before secondary cytoreduction was 18 months. The median time to progression after secondary surgery was 22 months (range 1-100 months), and median overall survival was 26 months (range 1-100 months). Serum CA-125 at the time of secondary cytoreduction was elevated (>35 kU/L) in 30 of 36 patients (81%) with a median of 212 kU/L (range 6-3866 kU/L). Multivariate analysis did not reveal a prognostic significance for preoperative CA-125. The only independent prognostic factors of improved survival were progression-free interval before secondary cytoreduction (p=0.047) and minimal residual disease after secondary cytoreduction (p=0.024). CONCLUSION: Although most patients had elevated serum CA-125 at the time of secondary cytoreductive surgery, CA-125 had no prognostic relevance.

Education and a standardized management protocol improve the assessment and management of asthma in the emergency department.

STUDY OBJECTIVE: To evaluate the effect of a standardized management protocol on acute asthma care in the emergency department (ED). METHOD: We conducted a before-after study regarding acute asthma management. Deficiencies in acute asthma care over a time period of 19 month (January 1997- October 1998) were identified. Subsequently a management protocol consisting of an assessment sheet and written guidelines for the initial management of acute asthma in the emergency department, was developed. In addition, physicians and nurses of the emergency department were informed about the recommendations given in the guidelines, and instructed in peak-flow meter use. The assessment sheet was introduced in January 2002 and posted at several locations in the emergency department. Between February 2002 and August 2003 the acute asthma consultations in the emergency department were consecutively registered. Data on medical history, physical examination and objective measurements of airflow obstruction, as well as data on treatment and assessment of the response to therapy were collected. In addition, medication and instructions at discharge were reviewed and compared with the results before the introduction of the assessment sheet. RESULTS: The first group consisted of patients seen between January 1997 and October 1998; the second group consisted of all patients seen between February 2002 and August 2003 (104 vs 273 patients respectively). Both groups had a similar gender distribution (56% females in the first group vs 53% females in the second group) and the mean age of both groups was also alike (median 33 vs 36 years). Most patients had a known history of asthma (76% in the first group vs 70% in the second group). The self-referral rate was high in both groups (86% vs 96% respectively). Blood pressure and pulse rate were reported in the majority of patients (95% vs 98% respectively), whereas the respiratory rates were reported in 14% of patients in the first group vs 65% of patients in the second group. The introduction of the assessment sheet led to an increased measurement of initial airflow obstruction (53% of patients in the first group vs 96% of patients in the second group) as well as repeated measures under treatment (36% of patients in the first group vs 85% of patients in the second group). Repeated inhalations with short-acting inhaled beta-agonists, and use of systemic corticosteroid therapy at admission and at discharge increased significantly (from 31% to 84%, 43% to 68% and 37% to 70% respectively). CONCLUSION: The assessment and management of patients presenting to the emergency department with acute asthma can be improved with a guideline based management protocol, and by educating physicians and nurses in the management of acute asthma.