Insights into the molecular basis of gastric mucosa as a first step for using Raman microscopy in paediatrics.

Recent advances in endoscopic technology have allowed detailed observation of the gastric mucosa, including Raman microscopy and spectroscopy. To explore the possibilities for future diagnostic use, we discuss the measurements and molecular markers found in this tissue. The Raman spectra of 16 samples of antral mucosa and 16 samples of corpus gastric mucosa obtained from healthy donors were analysed. A stable protocol for measuring reproducible spectra was established. These data suggest that many biomarkers can be used for the rapid analysis of metabolic states and future investigations into the pathogenesis of gastrointestinal diseases.

Kono-S anastomosis in Crohn's disease: initial experience in pediatric patients.

PURPOSE: Children diagnosed with Crohn's disease (CD) often undergo ileocecal resection (ICR) during childhood. Anastomotic recurrence is a frequent finding following this procedure. Data addressing the effect of the anastomosis type on disease recurrence are scarce in the pediatric population. The Kono-S anastomosis has shown promise in reducing endoscopic, clinical, and surgical recurrence rates in adults. We aimed to report our experience with Kono-S anastomosis in children, focusing on its feasibility and postoperative complications. METHODS: We retrospectively analyzed pediatric CD patients who underwent ICR with Kono-S anastomosis between August 2022 and May 2023. Data on demographics, clinical characteristics, surgery, hospitalization, and follow-up including colonoscopy were collected. Complications were classified using the Clavien-Dindo classification. RESULTS: Twelve patients (7 females, 58.3%) were included. Six (50%) of the patients had the B3 luminal form of the disease (according to Paris classification). Median surgery duration was 174 (interquartile range [IQR] 161-216) minutes. Anastomosis creation took a median of 62 (IQR, 54.5-71) minutes. Median hospitalization length was 6 (IQR 4-7) days. No short- or mid-term complications were observed. Median follow-up duration was 9.5 (IQR 6.8-12) months. CONCLUSION: According to our results, Kono-S anastomosis is safe and feasible in pediatric CD patients, with no observed postoperative complications. These findings support the potential benefit of using Kono-S anastomosis as a treatment approach in children with CD.

Faecal Bacteriome and Metabolome Profiles Associated with Decreased Mucosal Inflammatory Activity Upon Anti-TNF Therapy in Paediatric Crohn's Disease.

BACKGROUND AND AIMS: Treatment with anti-tumour necrosis factor α antibodies [anti-TNF] changes the dysbiotic faecal bacteriome in Crohn's disease [CD]. However, it is not known whether these changes are due to decreasing mucosal inflammatory activity or whether similar bacteriome reactions might be observed in gut-healthy subjects. Therefore, we explored changes in the faecal bacteriome and metabolome upon anti-TNF administration [and therapeutic response] in children with CD and contrasted those to anti-TNF-treated children with juvenile idiopathic arthritis [JIA]. METHODS: Faecal samples collected longitudinally before and during anti-TNF therapy were analysed with regard to the bacteriome by massively parallel sequencing of the 16S rDNA [V4 region] and the faecal metabolome by 1H nuclear magnetic resonance imaging. The response to treatment by mucosal healing was assessed by the MINI index at 3 months after the treatment started. We also tested several representative gut bacterial strains for in vitro growth inhibition by infliximab. RESULTS: We analysed 530 stool samples from 121 children [CD 54, JIA 18, healthy 49]. Bacterial community composition changed on anti-TNF in CD: three members of the class Clostridia increased on anti-TNF, whereas the class Bacteroidia decreased. Among faecal metabolites, glucose and glycerol increased, whereas isoleucine and uracil decreased. Some of these changes differed by treatment response [mucosal healing] after anti-TNF. No significant changes in the bacteriome or metabolome were noted upon anti-TNF in JIA. Bacterial growth was not affected by infliximab in a disc diffusion test. CONCLUSIONS: Our findings suggest that gut mucosal healing is responsible for the bacteriome and metabolome changes observed in CD, rather than any general effect of anti-TNF.

A Narrative Review of Cytokine Networks: Pathophysiological and Therapeutic Implications for Inflammatory Bowel Disease Pathogenesis.

Inflammatory bowel disease (IBD) is a lifelong inflammatory immune mediated disorder, encompassing Crohn's disease (CD) and ulcerative colitis (UC); however, the cause and specific pathogenesis of IBD is yet incompletely understood. Multiple cytokines produced by different immune cell types results in complex functional networks that constitute a highly regulated messaging network of signaling pathways. Applying biological mechanisms underlying IBD at the single omic level, technologies and genetic engineering enable the quantification of the pattern of released cytokines and new insights into the cytokine landscape of IBD. We focus on the existing literature dealing with the biology of pro- or anti-inflammatory cytokines and interactions that facilitate cell-based modulation of the immune system for IBD inflammation. We summarize the main roles of substantial cytokines in IBD related to homeostatic tissue functions and the remodeling of cytokine networks in IBD, which may be specifically valuable for successful cytokine-targeted therapies via marketed products. Cytokines and their receptors are validated targets for multiple therapeutic areas, we review the current strategies for therapeutic intervention and developing cytokine-targeted therapies. New biologics have shown efficacy in the last few decades for the management of IBD; unfortunately, many patients are nonresponsive or develop therapy resistance over time, creating a need for novel therapeutics. Thus, the treatment options for IBD beyond the immune-modifying anti-TNF agents or combination therapies are expanding rapidly. Further studies are needed to fully understand the immune response, networks of cytokines, and the direct pathogenetic relevance regarding individually tailored, safe and efficient targeted-biotherapeutics.

Sustainability of biologic treatment in paediatric patients with Crohn's disease: population-based registry analysis.

BACKGROUND: We aimed to evaluate the predictors of sustainability of biologic drugs for paediatric patients with Crohn's disease (CD). METHODS: The Czech National Prospective Registry of Biologic and Targeted Therapy of Inflammatory Bowel Disease (CREdIT) was used to identify the biologic treatment courses in paediatric patients with CD. Mixed-effects Cox models and propensity score analyses were employed to evaluate predictors of treatment sustainability. RESULTS: Among the 558 observations of 473 patients, 264 were treated with adalimumab (47%), 240 with infliximab (43%), 41 with ustekinumab (7%), and 13 with vedolizumab (2%). Multivariable analysis revealed higher discontinuation risk with infliximab compared to adalimumab (HR = 0.600, 95%CI 0.389-0.926), both overall and in first-line treatment (HR = 0.302, 95%CI 0.103-0.890). Infliximab versus adalimumab was associated with shorter time to escalation (HR = 0.094, 95%CI 0.043-0.203). Propensity-score analysis demonstrated lower sustainability of infliximab (HR = 0.563, 95%CI 1.159-2.725). The time since diagnosis to treatment initiation (HR = 0.852, 95%CI 0.781-0.926) was the most important predictor. Baseline immunosuppressive therapy prolonged sustainability with infliximab (HR = 2.899, 95%CI 1.311-6.410). CONCLUSIONS: Given the results suggesting shorter sustainability, the need for earlier intensification and thus higher drug exposure, and the greater need for immunosuppression with infliximab than with adalimumab, the choice of these drugs cannot be considered completely equitable. IMPACT: Our study identified predictors of sustainability of biologic treatment in paediatric patients with Crohn's disease, including adalimumab (versus infliximab), early initiation of biologic treatment, and normalised baseline haemoglobin levels. Infliximab treatment was associated with earlier intensification, higher drug exposure, and a greater need for immunosuppression. Parents and patients should be fully informed of the disadvantages of intravenous infliximab versus adalimumab during the decision-making process. This study emphasises the importance of not delaying the initiation of biologic therapy in paediatric patients with Crohn's disease.

CCR7 acts as both a sensor and a sink for CCL19 to coordinate collective leukocyte migration.

Immune responses rely on the rapid and coordinated migration of leukocytes. Whereas it is well established that single-cell migration is often guided by gradients of chemokines and other chemoattractants, it remains poorly understood how these gradients are generated, maintained, and modulated. By combining experimental data with theory on leukocyte chemotaxis guided by the G protein-coupled receptor (GPCR) CCR7, we demonstrate that in addition to its role as the sensory receptor that steers migration, CCR7 also acts as a generator and a modulator of chemotactic gradients. Upon exposure to the CCR7 ligand CCL19, dendritic cells (DCs) effectively internalize the receptor and ligand as part of the canonical GPCR desensitization response. We show that CCR7 internalization also acts as an effective sink for the chemoattractant, dynamically shaping the spatiotemporal distribution of the chemokine. This mechanism drives complex collective migration patterns, enabling DCs to create or sharpen chemotactic gradients. We further show that these self-generated gradients can sustain the long-range guidance of DCs, adapt collective migration patterns to the size and geometry of the environment, and provide a guidance cue for other comigrating cells. Such a dual role of CCR7 as a GPCR that both senses and consumes its ligand can thus provide a novel mode of cellular self-organization.

Short- and Long-Interval Prime-Boost Vaccination with the Candidate Vaccines MVA-SARS-2-ST and MVA-SARS-2-S Induces Comparable Humoral and Cell-Mediated Immunity in Mice.

The COVID-19 pandemic caused significant human health and economic consequences. Due to the ability of SARS-CoV-2 to spread rapidly and to cause severe disease and mortality in certain population groups, vaccines are essential for controlling the pandemic in the future. Several licensed vaccines have shown improved protection against SARS-CoV-2 after extended-interval prime-boost immunizations in humans. Therefore, in this study, we aimed to compare the immunogenicity of our two Modified Vaccinia virus Ankara (MVA) based COVID-19 candidate vaccines MVA-SARS-2-S and MVA-SARS-2-ST after short- and long-interval prime-boost immunization schedules in mice. We immunized BALB/c mice using 21-day (short-interval) or 56-day (long-interval) prime-boost vaccination protocols and analyzed spike (S)-specific CD8 T cell immunity and humoral immunity. The two schedules induced robust CD8 T cell responses with no significant differences in their magnitude. Furthermore, both candidate vaccines induced comparable levels of total S, and S2-specific IgG binding antibodies. However, MVA-SARS-2-ST consistently elicited higher amounts of S1-, S receptor binding domain (RBD), and SARS-CoV-2 neutralizing antibodies in both vaccination protocols. Overall, we found very comparable immune responses following short- or long-interval immunization. Thus, our results suggest that the chosen time intervals may not be suitable to observe potential differences in antigen-specific immunity when testing different prime-boost intervals with our candidate vaccines in the mouse model. Despite this, our data clearly showed that MVA-SARS-2-ST induced superior humoral immune responses relative to MVA-SARS-2-S after both immunization schedules.

Pulmonary tularaemia in a female adolescent with inflammatory bowel disease receiving infliximab: Do not miss the diagnosis.

We report the first described case of pulmonary tularaemia in the pediatric patient receiving infliximab for ulcerative colitis. We highlight the importance of considering Francisella tularensis in diagnostically challenging cases of persistent respiratory symptoms to facilitate early diagnosis and adequate therapy. The TCR-γδ + DN T cells are gaining important role in clinical practice. Polymerase chain reaction assays and serology guarantee early recognition.

Prediction of thiopurine failure in pediatric Crohn's disease: pediatric IBD Porto group of ESPGHAN.

BACKGROUND: Maintaining of remission early in the disease course of Crohn's disease (CD) is essential and has major impact on the future prognosis. This study aimed to identify baseline predictors to develop model allowing stratification of patients who will not benefit from long-term azathioprine (AZA) treatment and will require more intensive therapy. METHODS: This study was designed to develop clinical prediction rule using retrospective data analysis of pediatric CD patients included in prospective inception cohort. Clinical relapse was defined as necessity of re-induction of remission. Sequence of Cox models was fitted to predict risk of relapse. RESULTS: Out of 1190 CD patients from 13 European centers, 441 were included, 50.3% patients did not experience clinical relapse within 2 years of AZA treatment initiation. Median time to relapse was 2.11 (CI 1.59-2.46) years. Of all the tested parameters available at diagnosis, six were significant in multivariate analyses: C-reactive protein (p = 0.038), body mass index Z-score >0.8 SD (p = 0.002), abnormal sigmoid imaging (p = 0.039), abnormal esophageal endoscopy (p = 0.005), ileocolonic localization (p = 0.023), AZA dose in specific age category (p = 0.031). CONCLUSIONS: Although the possibility of predicting relapse on AZA treatment appears limited, we developed predictive model based on six baseline parameters potentially helpful in clinical decision. IMPACT: The possibility of predicting relapse on AZA treatment appears to be possible but limited. We identified six independent predictors available at diagnosis of early AZA/6-MP treatment failure in pediatric CD patients. Using combination of these factors, a model applicable to clinical practice was created. A web-based tool, allowing estimation of individual relapse risk in pediatric CD patients on a particular therapeutic regimen, has been developed.

One fold, many functions-M23 family of peptidoglycan hydrolases.

Bacterial cell walls are the guards of cell integrity. They are composed of peptidoglycan that provides rigidity to sustain internal turgor and ensures isolation from the external environment. In addition, they harbor the enzymatic machinery to secure cell wall modulations needed throughout the bacterial lifespan. The main players in this process are peptidoglycan hydrolases, a large group of enzymes with diverse specificities and different mechanisms of action. They are commonly, but not exclusively, found in prokaryotes. Although in most cases, these enzymes share the same molecular function, namely peptidoglycan hydrolysis, they are leveraged to perform a variety of physiological roles. A well-investigated family of peptidoglycan hydrolases is M23 peptidases, which display a very conserved fold, but their spectrum of lytic action is broad and includes both Gram- positive and Gram- negative bacteria. In this review, we summarize the structural, biochemical, and functional studies concerning the M23 family of peptidases based on literature and complement this knowledge by performing large-scale analyses of available protein sequences. This review has led us to gain new insight into the role of surface charge in the activity of this group of enzymes. We present relevant conclusions drawn from the analysis of available structures and indicate the main structural features that play a crucial role in specificity determination and mechanisms of latency. Our work systematizes the knowledge of the M23 family enzymes in the context of their unique antimicrobial potential against drug-resistant pathogens and presents possibilities to modulate and engineer their features to develop perfect antibacterial weapons.

Bronchial Asthma as a Cardiovascular Risk Factor: A Prospective Observational Study.

Introduction: Asthma as a chronic inflammatory disorder has been suggested as a risk factor for endothelial dysfunction (ED), but studies on the association between asthma and cardiovascular disease (CVD) risk are limited. Background: We assessed associations of ED with the severity of asthma, eosinophilic inflammation, lung function, and asthma control. Methods: 52 young asthmatics (median age of 25.22 years) and 45 healthy individuals were included. Demographic, clinical, and laboratory findings were recorded. We evaluated microvascular responsiveness by recording the reactive hyperemia index (RHI) indicating post-occlusive peripheral endothelium-dependent changes in vascular tone using the Itamar Medical EndoPAT2000. VCAM-1, ADMA, high-sensitive CRP (hsCRP), and E-selectin were measured. Results: Asthmatics had considerably lower RHI values (p < 0.001) with a dynamic decreasing trend by asthma severity and higher hsCRP levels (p < 0.001). A substantial increase in hsCRP and E-selectin with asthma severity (p < 0.05) was also observed. We confirmed a higher body mass index (BMI) in asthmatics (p < 0.001), especially in women and in severe asthma. Conclusions: We demonstrated the progression of CVD in asthmatics and the association of the ongoing deterioration of ED with the inflammatory severity, suggesting that the increased risk of CVD in young asthmatics is dependent on disease severity. The underlying mechanisms of risk factors for CVD and disease control require further study.

Stabilized recombinant SARS-CoV-2 spike antigen enhances vaccine immunogenicity and protective capacity.

The SARS-CoV-2 spike (S) glycoprotein is synthesized as a large precursor protein and must be activated by proteolytic cleavage into S1 and S2. A recombinant modified vaccinia virus Ankara (MVA) expressing native, full-length S protein (MVA-SARS-2-S) is currently under investigation as a candidate vaccine in phase I clinical studies. Initial results from immunogenicity monitoring revealed induction of S-specific antibodies binding to S2, but low-level antibody responses to the S1 domain. Follow-up investigations of native S antigen synthesis in MVA-SARS-2-S-infected cells revealed limited levels of S1 protein on the cell surface. In contrast, we found superior S1 cell surface presentation upon infection with a recombinant MVA expressing a stabilized version of SARS-CoV-2 S protein with an inactivated S1/S2 cleavage site and K986P and V987P mutations (MVA-SARS-2-ST). When comparing immunogenicity of MVA vector vaccines, mice vaccinated with MVA-SARS-2-ST mounted substantial levels of broadly reactive anti-S antibodies that effectively neutralized different SARS-CoV-2 variants. Importantly, intramuscular MVA-SARS-2-ST immunization of hamsters and mice resulted in potent immune responses upon challenge infection and protected from disease and severe lung pathology. Our results suggest that MVA-SARS-2-ST represents an improved clinical candidate vaccine and that the presence of plasma membrane-bound S1 is highly beneficial to induce protective antibody levels.

Engineering and evaluation of thermostable IsPETase variants for PET degradation.

Polyethylene terephthalate (PET) is a mass-produced petroleum-based synthetic polymer. Enzymatic PET degradation using, for example, Ideonella sakaiensis PETase (IsPETase) can be a more environmentally friendly and energy-saving alternative to the chemical recycling of PET. However, IsPETase is a mesophilic enzyme with an optimal reaction temperature lower than the glass transition temperature (T g) of PET, where the amorphous polymers can be readily accessed for enzymatic breakdown. In this study, we used error-prone PCR to generate a mutant library based on a thermostable triple mutant (TM) of IsPETase. The library was screened against the commercially available polyester-polyurethane Impranil DLN W 50 for more thermostable IsPETase variants, yielding four variants with higher melting points. The most promising IsPETaseTMK95N/F201I variant had a 5.0°C higher melting point than IsPETaseTM. Although this variant showed a slightly lower activity on PET at lower incubation temperatures, its increased thermostability makes it a more active PET hydrolase at higher reaction temperatures up to 60°C. Several other variants were compared and combined with selected previously published IsPETase mutants in terms of thermostability and hydrolytic activity against PET nanoparticles and amorphous PET films. Our findings indicate that thermostability is one of the most important characteristics of an effective PET hydrolase.

Severe abdominal pain: an atypical initial and leading symptom preceding skin rash in Henoch-Schönlein purpura.

Henoch-Schönlein purpura (HSP) is the most common vasculitis in childhood and is clinically characterised by purpura, abdominal pain, arthritis and renal involvement. Scarcely, some patients with HSP may not always show visible rash and can present with insidious abdominal symptoms. We present two patients: an 8-year-old boy who was initially considered as having infectious diarrhoea and mesenteric lymphadenitis, then intussusception, appendicitis, appendicopathia oxyuriaca and post-operative ileus. However, he was finally diagnosed with HSP, as the typical rash appeared 10 days after onset of abdominal symptoms. The second patient was a 5-year-old boy with recurrent vomiting, abdominal pain and mild dehydration, where swollen joints and typical rash appeared on day 3. Both patients were successfully managed with orally administered corticosteroids. The patients did not have any further consequences of HSP.

Intranasal Delivery of MVA Vector Vaccine Induces Effective Pulmonary Immunity Against SARS-CoV-2 in Rodents.

Antigen-specific tissue-resident memory T cells (Trms) and neutralizing IgA antibodies provide the most effective protection of the lungs from viral infections. To induce those essential components of lung immunity against SARS-CoV-2, we tested various immunization protocols involving intranasal delivery of a novel Modified Vaccinia virus Ankara (MVA)-SARS-2-spike vaccine candidate. We show that a single intranasal MVA-SARS-CoV-2-S application in mice strongly induced pulmonary spike-specific CD8+ T cells, albeit restricted production of neutralizing antibodies. In prime-boost protocols, intranasal booster vaccine delivery proved to be crucial for a massive expansion of systemic and lung tissue-resident spike-specific CD8+ T cells and the development of Th1 - but not Th2 - CD4+ T cells. Likewise, very high titers of IgG and IgA anti-spike antibodies were present in serum and broncho-alveolar lavages that possessed high virus neutralization capacities to all current SARS-CoV-2 variants of concern. Importantly, the MVA-SARS-2-spike vaccine applied in intramuscular priming and intranasal boosting treatment regimen completely protected hamsters from developing SARS-CoV-2 lung infection and pathology. Together, these results identify intramuscular priming followed by respiratory tract boosting with MVA-SARS-2-S as a promising approach for the induction of local, respiratory as well as systemic immune responses suited to protect from SARS-CoV-2 infections.

Sequential and Switchable Patterning for Studying Cellular Processes under Spatiotemporal Control.

Attachment of adhesive molecules on cell culture surfaces to restrict cell adhesion to defined areas and shapes has been vital for the progress of in vitro research. In currently existing patterning methods, a combination of pattern properties such as stability, precision, specificity, high-throughput outcome, and spatiotemporal control is highly desirable but challenging to achieve. Here, we introduce a versatile and high-throughput covalent photoimmobilization technique, comprising a light-dose-dependent patterning step and a subsequent functionalization of the pattern via click chemistry. This two-step process is feasible on arbitrary surfaces and allows for generation of sustainable patterns and gradients. The method is validated in different biological systems by patterning adhesive ligands on cell-repellent surfaces, thereby constraining the growth and migration of cells to the designated areas. We then implement a sequential photopatterning approach by adding a second switchable patterning step, allowing for spatiotemporal control over two distinct surface patterns. As a proof of concept, we reconstruct the dynamics of the tip/stalk cell switch during angiogenesis. Our results show that the spatiotemporal control provided by our "sequential photopatterning" system is essential for mimicking dynamic biological processes and that our innovative approach has great potential for further applications in cell science.

Immunogenicity and efficacy of the COVID-19 candidate vector vaccine MVA-SARS-2-S in preclinical vaccination.

Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has emerged as the infectious agent causing the pandemic coronavirus disease 2019 (COVID-19) with dramatic consequences for global human health and economics. Previously, we reached clinical evaluation with our vector vaccine based on modified vaccinia virus Ankara (MVA) against the Middle East respiratory syndrome coronavirus (MERS-CoV), which causes an infection in humans similar to SARS and COVID-19. Here, we describe the construction and preclinical characterization of a recombinant MVA expressing full-length SARS-CoV-2 spike (S) protein (MVA-SARS-2-S). Genetic stability and growth characteristics of MVA-SARS-2-S, plus its robust expression of S protein as antigen, make it a suitable candidate vaccine for industrial-scale production. Vaccinated mice produced S-specific CD8+ T cells and serum antibodies binding to S protein that neutralized SARS-CoV-2. Prime-boost vaccination with MVA-SARS-2-S protected mice sensitized with a human ACE2-expressing adenovirus from SARS-CoV-2 infection. MVA-SARS-2-S is currently being investigated in a phase I clinical trial as aspirant for developing a safe and efficacious vaccine against COVID-19.

Obstetric Ultrasonography to Detect Fetal Abnormalities in a Mouse Model for Zika Virus Infection.

In 2015 Zika virus (ZIKV) emerged for the first time in South America. The following ZIKV epidemic resulted in the appearance of a clinical phenotype with microcephaly and other severe malformations in newborns. So far, mechanisms of ZIKV induced damage to the fetus are not completely understood. Previous data suggest that ZIKV may bypass the placenta to reach the fetus. Thus, animal models for ZIKV infection are important to facilitate studies about ZIKV infection during pregnancy. Here, we used ultrasound based imaging (USI) to characterize ZIKV induced pathogenesis in the pregnant Type I interferon receptor-deficient (IFNAR-/-) mouse model. Based on USI we suggest the placenta to be a primary target organ of ZIKV infection enabling ZIKV spreading to the fetus. Moreover, in addition to direct infection of the fetus, the placental ZIKV infection may cause an indirect damage to the fetus through reduced uteroplacental perfusion leading to intrauterine growth retardation (IUGR) and fetal complications as early as embryonic day (ED) 12.5. Our data confirmed the capability of USI to characterize ZIKV induced modifications in mouse fetuses. Data from further studies using USI to monitor ZIKV infections will contribute to a better understanding of ZIKV infection in pregnant IFNAR-/- mice.

Characterization of EGF-guided MDA-MB-231 cell chemotaxis in vitro using a physiological and highly sensitive assay system.

Chemotactic cell migration is a central mechanism during cancer cell invasion and hence metastasis. In order to mimic in vivo conditions, we used a three-dimensional hydrogel matrix made of collagen I and a stable gradient-generating chemotaxis assay system, which is commercially available (µ-Slide Chemotaxis) to characterize epidermal growth factor (EGF)-induced chemotaxis of the human breast cancer cell line MDA-MB-231. Surprisingly, chemotactic effects of EGF on MDA-MB-231 cells could neither be observed in the standard growth medium DMEM/F-12 supplemented with 10% serum nor in starvation medium. In contrast, after adapting the cells to the serum-free growth medium UltraCULTURETM, significant chemotactic effects could be measured with high sensitivity. The extremely time-stable linear gradients, generated in the chemotaxis chamber, led to consistent directional migration of MDA-MB-231 cells. Dose-response experiments showed increased directional and kinetic response of MDA-MB-231 cells towards stable gradients of EGF. While EGF-guided directional migration (chemotaxis) was highly concentration-dependent with the highest response at 1.5 nM/mm EGF, we found that the chemokinetic effect induced by EGF was concentration-independent. Both, blocking the ligand-binding domain of the EGF receptor by an antibody (monoclonal anti-EGFR antibody 225) and inhibition of its kinase domain by a small molecule inhibitor (AG1478) led to a reduction in EGF-induced directed migration. The high sensitivity of the assay even allowed us to observe synergistic effects in EGF-receptor inhibition using a combination of low doses of both inhibitor types. Those results validate the fact that EGF is a potent guidance cue for MDA-MB-231 cell migration and help to understand the mechanism behind chemotaxis-driven cancer metastasis.