Thomas M Kündig's research while affiliated with University of Zurich and other places

Vaccines generally require T lymphocytes for B-cell activation and immunoglobulin class switching in response to peptide or protein antigens. In the absence of T cells, limited IgG class switch takes place, germinal centers are short-lived, and the B cells lack memory. Here, immunization of mice with liposomes containing 15mer peptides and monophosphoryl lipid A (MPLA) as adjuvant, induced T-cell independent (TI) IgG class switch within three days, as well as germinal center formation. The antibody responses were long-lived, strictly dependent on Toll-like receptor 4 (TLR4) signaling, partly dependent on Bruton’s tyrosine kinase (BTK) signal transmission, and independent of signaling through T-cell receptors, MHC class II and inflammasome. The antibody response showed characteristics of both TI type 1 and TI type 2. All IgG subclasses could be boosted months after primary immunization, and the biological function of the secreted antibodies was demonstrated in murine models of allergic anaphylaxis and of bacterial infection. Moreover, antibody responses after immunization with peptide- and MPLA-loaded liposomes could be triggered in neonatal mice and in mice receiving immune-suppressants. This study demonstrates T-cell independent endogenous B-cell memory and recall responses in vivo using a peptide antigen. The stimulation of these antibody responses required a correct and dense assembly and administration of peptide and adjuvant on the surface of liposomes. In the future, TI vaccines may prove beneficial in pathological conditions in which T-cell immunity is compromised through disease or medicines or when rapid, antibody-mediated immune protection is needed.

Inflammasomes comprise a group of protein complexes with fundamental roles in the induction of inflammation. Upon sensing stress factors, their assembly induces the activation and release of the pro-inflammatory cytokines interleukin (IL)-1β and -18 and a lytic type of cell death, termed pyroptosis. Recently, CARD8 has joined the group of inflammasome sensors. The carboxy-terminal part of CARD8, consisting of a function-to-find-domain (FIIND) and a caspase activation and recruitment domain (CARD), resembles that of NLR family pyrin domain containing 1 (NLRP1), which is recognized as the main inflammasome sensor in human keratinocytes. The interaction with dipeptidyl peptidases 8 and 9 (DPP8/9) represents an activation checkpoint for both sensors. CARD8 and NLRP1 are activated by viral protease activity targeting their amino-terminal region. However, CARD8 also has some unique features compared to the established inflammasome sensors. Activation of CARD8 occurs independently of the inflammasome adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), leading mainly to pyroptosis rather than the activation and secretion of pro-inflammatory cytokines. CARD8 was also shown to have anti-inflammatory and anti-apoptotic activity. It interacts with, and inhibits, several proteins involved in inflammation and cell death, such as the inflammasome sensor NLRP3, CARD-containing proteins caspase-1 and -9, nucleotide-binding oligomerization domain containing 2 (NOD2), or nuclear factor kappa B (NF-κB). Single nucleotide polymorphisms (SNPs) of CARD8, some of them occurring at high frequencies, are associated with various inflammatory diseases. The molecular mechanisms underlying the different pro- and anti-inflammatory activities of CARD8 are incompletely understood. Alternative splicing leads to the generation of multiple CARD8 protein isoforms. Although the functional properties of these isoforms are poorly characterized, there is evidence that suggests isoform-specific roles. The characterization of the functions of these isoforms, together with their cell- and disease-specific expression, might be the key to a better understanding of CARD8’s different roles in inflammation and inflammatory diseases.

During recent years, RNA therapeutics have begun to make a substantial impact in the clinic, with the approval of the siRNA-based therapeutic Patisiran in 2018, and of the two mRNA SARS-CoV-2 vaccines, BNT162b2 and mRNA-1273 in 2021. A key to the success of these therapeutics lies in the lipid-based delivery system. The therapeutic RNAs are encapsulated in lipid nanoparticles (LNPs), which protect against enzymatic degradation and efficiently deliver the RNA across the cell membrane into the cytosol. Thereby, the method used for LNP synthesis and its lipid composition are crucial aspects that decide the efficacy of the LNP-RNA hetero system. Here we provide a detailed guide for the simple preparation of LNP-encapsulated mRNA vaccines.

The recent success of the synthetic mRNA-based anti-COVID-19 vaccines has demonstrated the broad potential of the mRNA platform for applications in medicine, thanks to the combined efforts of a small community that has vastly improved key determinants such as design and formulation of synthetic mRNA during the past three decades. However, the cost of production and sensitivity to enzymatic degradation are still limiting the broader application of synthetic mRNA for therapeutic applications. The increased interest in mRNA-based technologies has spurred a renaissance for circular RNA (circRNA), as the lack of free 5′ and 3′ ends substantially increases resistance against enzymatic degradation in biological systems and does not require expensive cap analogs, as translation is controlled by an Internal Ribosome Entry Site (IRES) sequence. Thus, it can be expected that circRNA will play an important role for future mRNA therapeutics. Here we provide a detailed guide to the production of synthetic circRNA.

Background Intralymphatic immunotherapy (ILIT) represents a promising novel approach treating allergic diseases. However, no standardized procedures or recommendations have been established or reported, despite the recognized fact that treatment efficacy relies on the ability to inject the allergen intranodally. Objective We aim to provide a critical appraisal of ILIT as a method of allergen immunotherapy and to deliver practical recommendations for accurate ILIT. Methods One hundred and seventy‐three ILIT injections were performed in 28 (47%) women and 32 (53%) men with median age of 29 years (21–59). The injections were ultrasound‐guided and recorded for retrospective analysis with respect to injection location, needle visibility, medication release, and patient characteristics. Results The results show that the correct positioning of the needle within the lymph node (LN) was most critical. If the whole length of the needle bevel was not inserted into the LN, substance backflush into the interstitium was observed. Selecting a more superficial LN and inserting the needle at a smaller angle towards the LN significantly improved needle visibility in the ultrasound. Longitudinal results showed that continuous practice significantly correlated with improved needle visibility and more accurate ILIT injections. Conclusion Based on our results and practical experience, we propose several recommendations for LN selection and the correct handling of ultrasound probe and needle. We are confident that ILIT standardization and training will be important as to meet the goals of good safety and efficacy of ILIT.

Objectives Peanut allergy is an IgE-mediated food allergy that is associated with asthma in certain patients. With increasing prevalence, its great impact on the quality of life, and a lack of treatment options, the need for new therapy options is a given. Hence, models for research and development are required. This study aimed to establish a murine model of allergic airway inflammation induced by peanut allergens. Methods C3H mice were sensitised by intraperitoneal injections of peanut allergen extract and challenged by an intranasal application of the same extract. The assessment of airway inflammation involved the analysis of immune cells in the bronchoalveolar lavage fluid as measured by flow cytometry. Inflammatory reactions in the lung tissue were also studied by histology and quantitative PCR. Moreover, peanut-specific immune responses were studied after re-stimulation of spleen cells in vitro . Results Sensitisation led to allergen-specific IgE, IgA, and IgG1 seroconversion. Subsequent nasal exposure led to allergic airway inflammation as manifested by structural changes such as bronchial smooth muscle hypertrophy, mucus cell hyperplasia, infiltration of eosinophil cells and T cells, as well as an upregulation of genes expressing IL-4, IL-5, IL-13, and IFN-γ. Upon re-stimulation of splenocytes with peanut allergen, increased secretion of both T-helper type 2 (Th2) and Th1 cytokines was observed. Conclusion We successfully established a peanut-associated asthma model that exhibited many features characteristic of airway inflammation in human patients with allergic asthma. The model holds potential as a tool for investigating novel therapeutic approaches aimed at preventing the development of allergic asthma.

Due to their full differentiation capacity in vitro, the culture of human primary keratinocytes (HPKs) represents a physiological model for answering basic biological and dermatological research questions, including those related to skin diseases and the investigation of treatment options. When modified with the CRISPR/Cas9 gene editing approach and cultivated in organotypic 3D epidermal equivalents (EEs), these human cells have the potential to replace established mouse models. However, even when cultivated on feeder cells, HPKs have only a low proliferation capacity in 2D culture, limiting their application potential. This is particularly true for CRISPR/Cas9-modified HPKs, whose generation commonly requires selection of targeted cells, negatively affecting their lifespan. Here, we describe a robust protocol for the rapid, simple, and efficient generation of single- and multi-gene CRISPR/Cas9 knockout HPKs by electroporation of ribonucleoprotein (RNP) complexes, which comprise one or multiple guide RNAs (gRNAs) and Cas9 protein. Unlike DNA transfection or virus-based targeting strategies, electroporation of RNPs represents a targeting approach that minimizes immunological and toxic side effects. Using efficient gRNAs results in the generation of HPKs with a high yield of knockout cells, allowing for their immediate use in experiments without requiring the laborious process of selecting targeted cells or maintaining a feeder cell culture. Furthermore, the use of RNPs and their delivery via electroporation minimizes off-target and other unspecific effects, preventing unintended genomic alterations. Most importantly, CRISPR/Cas9 knockout HPKs generated with this protocol have the ability to form a fully differentiated epidermis in 3D, thus facilitating the understanding of specific protein functions in a highly physiological human skin model. Alternatively, this approach proves valuable for generating models of mono- or polygenic skin diseases via knockouts, providing insights into the underlying molecular mechanisms and facilitating the development of novel therapeutic approaches.

Introduction: Erythema nodosum (EN) is the most common form of panniculitis that predominantly affects the shins. While EN in atypical sites has been described by many authors, there are currently only case studies published on this topic. This study aimed to evaluate clinical differences between patients suffering from EN on the shins, compared to patients with EN in atypical locations. Methods: We analyzed 105 patients in a retrospective, single-center study at a university hospital in Switzerland. Typical EN was defined as lesions, found only on the lower legs, while atypical EN as lesions on the upper legs, trunk, arms, or face, only or in addition to lesions on the lower legs. The patients were assessed for age, gender, dermatologic history, time until first medical consultation, time to diagnosis, and time until remission. Further, etiology, symptoms, and applied therapies were investigated. Findings were then compared between the typical and atypical EN cohorts. Results: Overall, we included 70 patients (37.99 ± 15.67 [3-81] years) with EN solely on the shins and 35 patients (41.27 ± 16.85 [9-76] years) with EN on other locations. Interestingly, time until diagnosis was significantly shorter in atypical EN (p = 0.034, 1.14 ± 4.68 vs. 0.46 ± 1.14 months). Time to remission was similar in both groups (3.61 ± 2.73 vs. 3.05 ± 2.86 months, respectively). Sarcoidosis was the only etiologic factor significantly more frequent in atypical EN compared to typical EN (23% vs. 9%, p = 0.042). Besides that, solely subtle differences were seen regarding etiology, gender, age at onset, course of the disease, and symptoms. Conclusions: Our study suggests that only minor alterations between both study populations exist. Significant differences were found in time to diagnosis (shorter for atypical EN), as well as in sarcoidosis as an etiologic factor (more frequent in atypical EN). While adalimumab was only prescribed in atypical EN cases, prognosis seems to be similar for typical and atypical EN (similar time to remission, similar amount of reoccurring cases). Due to the limited sample size, however, our study population may have been too small to detect the relevant differences, and bigger studies may be needed.

Background Psoriasis is a disease that often requires prolonged systemic treatment. It is important to determine the safety of available therapies. There is currently little insight into sex‐specific differences in the safety of systemic psoriasis therapies. Objectives To examine the real‐world, long‐term safety of systemic psoriasis therapies with sex stratification in drug‐related adverse events (ADRs). Methods Ten‐year data from adults with moderate‐to‐severe psoriasis requiring systemic treatment (conventional systemic therapies [CST], biologics) were obtained from the Swiss psoriasis registry (SDNTT). ADRs were categorized according to the international terminology Medical Dictionary for Regulatory Activities (MedDRA). Safety was assessed by calculating event rates per 100 patient‐years (PY). We used descriptive statistics for patient and disease characteristics, and binomial and t ‐tests to compare treatment groups and sex. Results In total, 791 patients (290 females) were included with a mean age of 46 years. 358 (45%) received CSTs and 433 (55%) biologics; both groups had similar baseline characteristics except for more joint involvement in patients using biologics (26.86% vs. 14.8%, p < 0.0001). CSTs were associated with a 2.2‐fold higher ADR rate (40.43/100 PY vs. 18.22/100 PY, p < 0.0001) and an 8.0‐fold higher drug‐related discontinuation rate than biologics (0.16/PY vs. 0.02/PY, p < 0.0001). Trends showed non‐significant higher serious adverse event rates per 100 PY for biologics (8.19, CI 6.87–9.68) compared to CSTs (7.08, CI 5.39–9.13) ( p = 0.3922). Sex stratification revealed a significantly higher overall ADR rate for all treatments in females (1.8‐fold for CSTs [57.30/100 PY vs. 31.69/100 PY] and 2.0‐fold for biologics [27.36/100 PY vs. 13.9/100 PY], p < 0.0001), and drug‐related discontinuation rates for most CSTs in females. Conclusion Females were associated with a significantly higher rate of ADRs and drug‐related discontinuation rates. Sex stratification should be taken into consideration when designing studies in the patient‐tailored management of psoriasis.

Background Psoriatic arthritis (PsA) is a prevalent comorbidity among patients with psoriasis, heavily contributing to their burden of disease, usually diagnosed several years after the diagnosis of psoriasis. Objectives To investigate the predictability of psoriatic arthritis in patients with psoriasis and to identify important predictors. Methods Data from the Swiss Dermatology Network on Targeted Therapies (SDNTT) involving patients treated for psoriasis were utilized. A combination of gradient-boosted decision trees and mixed models was used to classify patients based on their diagnosis of PsA or its absence. The variables with the highest predictive power were identified. Time to PsA diagnosis was visualized with the Kaplan-Meier method and the relationship between severity of psoriasis and PsA was explored through quantile regression. Results A diagnosis of psoriatic arthritis was registered at baseline of 407 (29.5%) treatment series. 516 patients had no registration of PsA, 257 patients had PsA at inclusion, and 91 patients were diagnosed with PsA after inclusion. The model’s AUROCs was up to 73.7%, and variables with the highest discriminatory power were age, PASI, physical well-being, and severity of nail psoriasis. Among patients who developed PsA after inclusion, significantly more first treatment series were classified in the PsA-group, compared to those with no PsA registration. PASI was significantly correlated with the median burden/severity of PsA ( P = .01). Conclusions Distinguishing between patients with and without PsA based on clinical characteristics is feasible and even predicting future diagnoses of PsA is possible. Patients at higher risk can be identified using important predictors of PsA.