Our multi-omic approach

Deutsches Ärzteblatt

The understanding and classification of inflammatory skin diseases is shifting from a historical-descriptive perspective to a molecular-pathophysiological one based on immune response patterns. These are derived from a few key immunological mediators, each of which induces its own characteristic clinical, histopathological, and molecular patterns in the skin. Methods: This discussion of the definition of the immune response patterns of inflammatory skin diseases is based on information from pertinent publications retrieved by a selective literature search. A systematic literature search was also conducted on the response of inflammatory skin diseases to treatment with specific biologic agents. Results: The described immune response patterns are: autoinflammation; type 1, cytotoxic; type 2a, eczematous; type 2b, blistering; type 3, psoriasiform; type 4a, fibrosing; and type 4b, granulomatous. Each signature can usually be treated in a targeted manner. In general, each therapeutic target structure is associated with an adequate treatment response if and only if the skin disease under treatment has the relevant signature type. Hardly any biomarkers are currently available for the determination of immune response patterns in routine clinical practice. Conclusion: The classification of inflammatory skin diseases by their immune response patterns opens up the prospect of specifically targeted immunotherapy for each immune response pattern regardless of the historical-descriptive disease entity. Targeting is intended to improve response rates. Initial findings suggest that this strategy is likely to succeed.

Nature Communications

Previous advances have identified immune pathways associated with inflammatory skin diseases, leading to the development of targeted therapies. However, there is a lack of molecular approaches that delineate these pathways at the individual patient level for personalized diagnostic and therapeutic guidance. Here, we conduct a cross-comparison of expression profiles from multiple inflammatory skin diseases to identify gene modules defining relevant immune pathways. Seven modules are identified, representing key immune pathways: Th17, Th2, Th1, Type I IFNs, neutrophilic, macrophagic, and eosinophilic. These modules allow the development of a molecular map with high diagnostic efficacy for inflammatory skin diseases and clinico-pathologically undetermined cases. Aligning dominant modules with treatment targets offers a rational framework for treatment selection, improving response rates in both treatment-naïve patients and non-responders to targeted therapies. Overall, our approach offers precision medicine for inflammatory skin diseases, utilizing transcriptional modules to support diagnosis and guide personalized treatment selection.

Journal of Investigative Dermatology

Highly effective targeted therapies are available to treat noncommunicable chronic inflammatory skin diseases. In contrast, the exact diagnosis of noncommunicable chronic inflammatory skin diseases is complicated by its complex pathogenesis and clinical and histological overlap. Particularly, the differential diagnosis of psoriasis and eczema can be challenging in some cases, and molecular diagnostic tools need to be developed to support a gold standard diagnosis. The aim of this work was to develop a real-time PCR-based molecular classifier to distinguish psoriasis from eczema in formalin-fixed and paraffin-embedded-fixed skin samples and to evaluate the use of minimally invasive microbiopsies and tape strips for molecular diagnosis. In this study, we present a formalin-fixed and paraffin-embedded–based molecular classifier that determines the probability for psoriasis with a sensitivity/specificity of 92%/100%, respectively, and an area under the curve of 0.97, delivering comparable results to our previous published RNAprotect-based molecular classifier. The psoriasis probability, as well as levels of NOS2 expression, positively correlated with the disease hallmarks of psoriasis and negatively with eczema hallmarks. Furthermore, minimally invasive tape strips and microbiopsies were effectively used to differentiate psoriasis from eczema. In summary, the molecular classifier offers broad usage in pathology laboratories as well as outpatient settings and can support the differential diagnosis of noncommunicable chronic inflammatory skin diseases on a molecular level using formalin-fixed and paraffin-embedded tissue, microbiopsies, and tape strips.

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Journal of Allergy and Clinical Immunology

Nummular eczema (NE) is a common chronic inflammatory skin disease characterized by multiple, pruritic, discoid-shaped lesions. Since the underlying immune mechanisms are not fully understood, it is unclear whether NE should be regarded as variant of atopic dermatitis (AD) or a distinct disease.

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Abstract 49th Annual Meeting of the Arbeitsgemeinschaft Dermatologische Forschung (ADF)

One year after inclusion in the cohort, the patient’s dermatologists increasingly tend to support the MCs assessment. At baseline, the proportion of congruent clinical diagnoses was 58.8% for eczema and increased up to 76.5% after 12 months. For psoriasis, dermatologists agreed on the MC at baseline in only 23% of the cases. This strongly increased up to 61.5% in the first year. The current status shows clear benefits of the MC in the diagnostic of psoriasis and eczema. Clinically and/or pathologically unclear cases can receive a decisive impulse for the correct diagnosis through this method, even in long persisting disease courses. This poses a serious advance in the available dermatological diagnostics.

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medRxiv

Highly specific and efficient drugs have been developed during the last two decades to treat non-communicable chronic inflammatory skin diseases (ncISD). Due to their specificity, these drugs are asking for precise diagnostic measures to attribute the most efficient treatment to each patient. Diagnosis, however, is complicated by the complex pathogenesis of ncISD and their clinical and histological overlap. Especially, precise diagnosis of psoriasis and eczema is difficult in special cases and molecular diagnostic tools need to be developed to support gold standard diagnosis of patients. In this line, we have developed a real-time based molecular classifier to distinguish psoriasis from eczema in RNA-later fixed skin samples. However, this type of skin sample is not regularly used in routine diagnostics. Therefore, we evaluated if the combination of NOS2 and CCL27 expression in lesional skin can be transferred to formalin-fixed paraffin embedded (FFPE) tissue. We present a FFPE-based molecular classifier (MC) that determines the probability for psoriasis with a specificity and sensitivity of 100% and 92%, respectively, and an area under the curve (AUC) of 0.97 delivering comparable results to the RNA-later based MC. The probability for psoriasis as well as the PCR result of NOS2 expression correlated positive with disease hallmarks of psoriasis and negative with eczema hallmarks. This tool now offers broad usage in pathology laboratories and can support diagnostic decision making on a molecular level.

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Journal of the German Society of Dermatology, 20 (5)

Contact dermatitis is an inflammation of the epidermis and dermis at the site of exposure triggered by external agents. The two main forms are irritant and allergic contact dermatitis, which cause significant health and socioeconomic costs in addition to a marked reduction in quality of life. The anamnesis and the clinical picture are decisive for the necessary diagnostic measures. The most accurate possible diagnostic classification of contact dermatitis by means of allergological testing is important for disease management, since not only classical eczema therapy but also avoidance of the exogenous triggering factors are of great importance here. The choice of therapy should be based on the acuity, clinical severity, morphology of the lesions and localization of the contact dermatitis. A combination of basic therapy, topical, physical, and systemic therapy adapted to the patient’s needs is required, whereby not all forms of therapy must be carried out simultaneously but can be used in a varying manner. Primary, secondary, and tertiary prevention strategies aim at the recognition of the triggering noxae or allergens with subsequent contact avoidance or minimization. The present S1-guideline on contact dermatitis is primarily intended to provide dermatologists, allergologists and physicians working in allergology and occupational dermatology with a decision-making aid for the selection and implementation of suitable and sufficient diagnostics, therapy, and prevention.

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Journal of the German Society of Dermatology, 20 (9)

Currently,  it  can  be  assumed  that  psoriasis  of  the  hands  often  cannot  be  reliably  identified  as  such  by  the  treating  dermatologist,  significantly  influencing  the  success  of  therapy.  Preliminary  results  indicate  that  the  Molecular  Classifier  contributes  to  optimized  diagnostics  (psoriasis  vs.  eczema).  Further  follow-up  studies  will  show  whether  this  also applies to other aspects such as healing and occupational status.

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Dermatologie in Beruf und Umwelt, 66(3)

In daily dermatological practice as in occupational dermatology, patients with psoriasis vulgaris and atopic eczema are seen frequently. These diseases may appear very similar in terms of morphology. Thus, the differentiation between nummular eczema and psoriatic plaque may form a challenge, equally between hand eczema and psoriasis manuum. So far, diagnostic classification was based on patient and family medical history, comorbidities, onset and course of disease, morphology and localization, and diagnostic tests such as allergy tests and dermatopathology. Especially the latter was not yet able to allow helpful classification. A new molecular disease classifier based on the expression of the genes NOS2 and CCL27 in lesional skin has been developed that improves the diagnostic classification of diseases such as eczema and psoriasis. This test is reliable in classical variations as well as in subtypes of the diseases. Our paper presents first experiences with this molecular classifier in a group of patients attending a clinic for occupational skin diseases. We hope that this classifier may in future be applied as a routine test in diagnostics and may possibly even allow prognostics about clinical course, comorbidities and optimal therapy. Being now a first simple diagnostic test for the differentiation between psoriasis and eczema, this classifier is most likely to achieve an important prospective role in times of personalized medicine in occupational dermatology.

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Experimental Dermatology, 25(10)

Novel specific therapies for psoriasis and eczema have been developed, and they mark a new era in the treatment of these complex inflammatory skin diseases. However, within their broad clinical spectrum, psoriasis and eczema phenotypes overlap making an accurate diagnosis impossible in special cases, not to speak about predicting the clinical outcome of an individual patient. Here, we present a novel robust molecular classifier (MC) consisting of NOS2 and CCL27 gene that diagnosed psoriasis and eczema with a sensitivity and specificity of >95% in a cohort of 129 patients suffering from (i) classical forms; (ii) subtypes; and (iii) clinically and histologically indistinct variants of psoriasis and eczema. NOS2 and CCL27 correlated with clinical and histological hallmarks of psoriasis and eczema in a mutually antagonistic way, thus highlighting their biological relevance. In line with this, the MC could be transferred to the level of immunofluorescence stainings for iNOS and CCL27 protein on paraffin‐embedded sections, where patients were diagnosed with sensitivity and specificity >88%. Our MC proved superiority over current gold standard methods to distinguish psoriasis and eczema and may therefore build the basis for molecular diagnosis of chronic inflammatory skin diseases required to establish personalized medicine in the field.

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Science Translational Medicine, 6(244)

Previous attempts to gain insight into the pathogenesis of psoriasis and eczema by comparing their molecular signatures were hampered by the high interindividual variability of those complex diseases. In patients affected by both psoriasis and nonatopic or atopic eczema simultaneously (n = 24), an intraindividual comparison of the molecular signatures of psoriasis and eczema identified genes and signaling pathways regulated in common and exclusive for each disease across all patients. Psoriasis-specific genes were important regulators of glucose and lipid metabolism, epidermal differentiation, as well as immune mediators of T helper 17 (TH17) responses, interleukin-10 (IL-10) family cytokines, and IL-36. Genes in eczema related to epidermal barrier, reduced innate immunity, increased IL-6, and a TH2 signature. Within eczema subtypes, a mutually exclusive regulation of epidermal differentiation genes was observed. Furthermore, only contact eczema was driven by inflammasome activation, apoptosis, and cellular adhesion. On the basis of this comprehensive picture of the pathogenesis of psoriasis and eczema, a disease classifier consisting of NOS2 and CCL27 was created. In an independent cohort of eczema (n = 28) and psoriasis patients (n = 25), respectively, this classifier diagnosed all patients correctly and also identified initially misdiagnosed or clinically undifferentiated patients.

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The New England Journal of Medicine

The simultaneous occurrence of psoriasis driven by type 1 helper T (Th1) cells and type 17 helper T (Th17) cells and atopic eczema dominated by type 2 helper T (Th2) cells is rare. Here, we describe three patients with co-occurring psoriasis and atopic eczema with an antagonistic course and distinct T-cell infiltrates in lesions from psoriasis and those from atopic eczema. Sensitized patients with psoriasis had a reaction to epicutaneous allergen challenge, with clinically and histologically verified eczema lesions containing a large number of allergen-reactive T cells. These findings support a causative role for T cells triggered by specific antigens in both psoriasis and atopic eczema. (Supported by the German Research Foundation and others.)

Lab Chip

Centrifugal microfluidics allows for miniaturization, automation and parallelization of laboratory workflows. The fact that centrifugal forces are always directed radially outwards has been considered a main drawback for the implementation of complex workflows leading to the requirement of additional actuation forces for pumping, valving and switching. In this work, we review and discuss the combination of centrifugal with pneumatic forces which enables transport of even complex liquids in any direction on centrifugal systems, provides actuation for valving and switching, offers alternatives for mixing and enables accurate and precise metering and aliquoting. In addition, pneumatics can be employed for timing to carry out any of the above listed unit operations in a sequential and cascaded manner. Firstly, different methods to generate pneumatic pressures are discussed. Then, unit operations and applications that employ pneumatics are reviewed. Finally, a tutorial section discusses two examples to provide insight into the design process. The first tutorial explains a comparatively simple implementation of a pneumatic siphon valve and provides a workflow to derive optimum design parameters. The second tutorial discusses cascaded pneumatic operations consisting of temperature change rate actuated valving and subsequent pneumatic pumping. In conclusion, combining pneumatic actuation with centrifugal microfluidics allows for the design of robust fluidic networks with simple fluidic structures that are implemented in a monolithic fashion. No coatings are required and the overall demands on manufacturing are comparatively low. We see the combination of centrifugal forces with pneumatic actuation as a key enabling technology to facilitate compact and robust automation of biochemical analysis.

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Lab Chip

In centrifugal microfluidics, dead volumes in valves downstream of mixing chambers can hardly be avoided. These dead volumes are excluded from mixing processes and hence cause a concentration gradient. Here we present a new bubble mixing concept which avoids such dead volumes. The mixing concept employs heating to create a temperature change rate (TCR) induced overpressure in the air volume downstream of mixing chambers. The main feature is an air vent with a high fluidic resistance, representing a low pass filter with respect to pressure changes. Fast temperature increase causes rapid pressure increase in downstream structures pushing the liquid from downstream channels into the mixing chamber. As air further penetrates into the mixing chamber, bubbles form, ascend due to buoyancy and mix the liquid. Slow temperature/pressure changes equilibrate through the high fluidic resistance air vent enabling sequential heating/cooling cycles to repeat the mixing process. After mixing, a complete transfer of the reaction volume into the downstream fluidic structure is possible by a rapid cooling step triggering TCR actuated valving. The new mixing concept is applied to rehydrate reagents for loop-mediated isothermal amplification (LAMP). After mixing, the reaction mix is aliquoted into several reaction chambers for geometric multiplexing. As a measure for mixing efficiency, the mean coefficient of variation ([C with combining macron][V with combining macron], n = 4 LabDisks) of the time to positivity (tp) of the LAMP reactions (n = 11 replicates per LabDisk) is taken. The [C with combining macron][V with combining macron] of the tp is reduced from 18.5% (when using standard shake mode mixing) to 3.3% (when applying TCR actuated bubble mixing). The bubble mixer has been implemented in a monolithic fashion without the need for any additional actuation besides rotation and temperature control, which are needed anyhow for the assay workflow.

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Chemical Society Reviews, 44(17)

Centrifugal microfluidics has evolved into a mature technology. Several major diagnostic companies either have products on the market or are currently evaluating centrifugal microfluidics for product development. The fields of application are widespread and include clinical chemistry, immunodiagnostics and protein analysis, cell handling, molecular diagnostics, as well as food, water, and soil analysis. Nevertheless, new fluidic functions and applications that expand the possibilities of centrifugal microfluidics are being introduced at a high pace. In this review, we first present an up-to-date comprehensive overview of centrifugal microfluidic unit operations. Then, we introduce the term “process chain” to review how these unit operations can be combined for the automation of laboratory workflows. Such aggregation of basic functionalities enables efficient fluidic design at a higher level of integration. Furthermore, we analyze how novel, ground-breaking unit operations may foster the integration of more complex applications. Among these are the storage of pneumatic energy to realize complex switching sequences or to pump liquids radially inward, as well as the complete pre-storage and release of reagents. In this context, centrifugal microfluidics provides major advantages over other microfluidic actuation principles: the pulse-free inertial liquid propulsion provided by centrifugal microfluidics allows for closed fluidic systems that are free of any interfaces to external pumps. Processed volumes are easily scalable from nanoliters to milliliters. Volume forces can be adjusted by rotation and thus, even for very small volumes, surface forces may easily be overcome in the centrifugal gravity field which enables the efficient separation of nanoliter volumes from channels, chambers or sensor matrixes as well as the removal of any disturbing bubbles. In summary, centrifugal microfluidics takes advantage of a comprehensive set of fluidic unit operations such as liquid transport, metering, mixing and valving. The available unit operations cover the entire range of automated liquid handling requirements and enable efficient miniaturization, parallelization, and integration of assays.

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Lab Chip

Stick-packaging of goods in tubular-shaped composite-foil pouches has become a popular technology for food and drug packaging. We miniaturized stick-packaging for use in lab-on-a-chip (LOAC) systems to pre-store and on-demand release the liquid and dry reagents in a volume range of 80–500 μl. An integrated frangible seal enables the pressure-controlled release of reagents and simplifies the layout of LOAC systems, thereby making the package a functional microfluidic release unit. The frangible seal is adjusted to defined burst pressures ranging from 20 to 140 kPa. The applied ultrasonic welding process allows the packaging of temperature sensitive reagents. Stick-packs have been successfully tested applying recovery tests (where 99% (STDV = 1%) of 250 μl pre-stored liquid is released), long-term storage tests (where there is loss of only <0.5% for simulated 2 years) and air transport simulation tests. The developed technology enables the storage of a combination of liquid and dry reagents. It is a scalable technology suitable for rapid prototyping and low-cost mass production.

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Lab Chip

We present a novel process flow enabling prototyping of microfluidic cartridges made out of polymer films. Its high performance is proven by implementation of a microfluidic genotyping assay testing 22 DNA samples including clinical isolates from patients infected by methicilin-resistant Staphylococcus aureus (MRSA). The microfluidic cartridges (disks) are fabricated by a novel process called microthermoforming by soft lithography (µTSL). Positive moulds are applied allowing for higher moulding precision and very easy demoulding when compared to conventional microthermoforming. High replication accuracies with geometric disk-to-disk variations of less than 1% are typical. We describe and characterise fabrication and application of microfluidic cartridges with wall thicknesses <188 µm thus enabling efficient thermocycling during real-time polymerase chain reaction (PCR). The microfluidic cartridges are designed for operation in a slightly modified commercial thermocycling instrument. This approach demonstrates new opportunities for both microfluidic developments and well-established laboratory instruments. The microfluidic protocol is controlled by centrifugal forces and divides the liquid sample parallely into independent aliquots of 9.8 µl (CV 3.4%, N = 32 wells). The genotyping assays are performed with pre-stored primers and probes for real-time PCR showing a limit of detection well below 10 copies of DNA per reaction well (N = 24 wells in 3 independent disks). The system was evaluated by 44 genotyping assays comprising 22 DNA samples plus duplicates in a total of 11 disks. The samples contained clinical samples of seven different genotypes of MRSA as well as positive and negative controls. The results are in excellent agreement with the reference in microtubes.

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