[German Translation and Validation of the Patient-Oriented Questionnaire "Thumb Disability Examination" (TDX) for Specific Diagnosis and Follow-Up of Thumb Carpometacarpal Joint Osteoarthritis]. / Deutsche Übersetzung und Validierung des patienten-orientierten Fragebogens "Thumb Disability Examination" (TDX) zur spezifischen Diagnostik und Verlaufskontrolle bei Rhizarthrose.

BACKGROUND: Patient-reported outcome measures (PROMs) are becoming increasingly important in today's patient care. Unfortunately, the most common PROMs in hand surgery are very time-consuming and usually do not cover the actual, diagnosis-specific complaints of the patients. For diagnosis and follow-up of thumb carpometacarpal joint osteoarthritis, Noback et al. developed and validated the Thumb Disability Examination (TDX) questionnaire. This 20-item questionnaire specifically assesses daily living limitations and pain as well as patient-reported satisfaction of thumb function. The aim of this study is to validate the German translation of the TDX, the Thumb Disability Examination - German (TDX-G), and to compare it with the German-language versions of the MHQ and qDASH, which are currently used as gold standard. MATERIAL UND METHODS: Translation and back-translation were performed in accordance with accepted guidelines. For statistical validation of the TDX-G, 30 consecutive patients with thumb carpometacarpal joint osteoarthritis were interviewed under standardised conditions. Internal consistency was calculated using Cronbach's alpha. Test-retest reliability was determined in 10 patients who completed the TDX-G twice at an interval of 2 weeks. The validity of the TDX-G was determined by calculating the correlation coefficients of the TDX-G with the MHQ and qDASH, subjective pain sensation (NRS), and hand strength levels (coarse and pinch strength). In addition, the time to collect each questionnaire was compared. RESULTS: The TDX-G has high internal consistency (Cronbach's alpha 0.932) and test-retest reliability (intraclass correlation coefficient 0.963 [0.850-0.991]). There is a significant correlation between TDX-G and MHQ (- 0.782; p<0.001) and qDASH (0.833; p<0.001). All questionnaires correlate significantly with pain on exertion and pinch force, with the TDX-G having the highest correlation in each case. Significantly less time is needed to record the TDX-G (110±28 s) than to record the MHQ (413±98 s). CONCLUSION: The TDX-G is a reliable tool for diagnosing and monitoring the progression of thumb carpometacarpal joint osteoarthritis. It can be used in both patient care and clinical research and accurately mirrors the symptoms.

Cross-sectional field study comparing hippocampal subfields in patients with post-traumatic stress disorder, major depressive disorder, post-traumatic stress disorder with comorbid major depressive disorder, and adjustment disorder using routine clinical data.

Background: The hippocampus is a central brain structure involved in stress processing. Previous studies have linked stress-related mental disorders, such as post-traumatic stress disorder (PTSD) and major depressive disorder (MDD), with changes in hippocampus volume. As PTSD and MDD have similar symptoms, clinical diagnosis relies solely on patients reporting their cognitive and emotional experiences, leading to an interest in utilizing imaging-based data to improve accuracy. Our field study aimed to determine whether there are hippocampal subfield volume differences between stress-related mental disorders (PTSD, MDD, adjustment disorders, and AdjD) using routine clinical data from a military hospital. Methods: Participants comprised soldiers (N = 185) with PTSD (n = 50), MDD (n = 70), PTSD with comorbid MDD (n = 38), and AdjD (n = 27). The hippocampus was segmented and volumetrized into subfields automatically using FreeSurfer. We used ANCOVA models with estimated total intracranial volume as a covariate to determine whether there were volume differences in the hippocampal subfields cornu ammonis 1 (CA1), cornu ammonis 2/3 (CA2/3), and dentate gyrus (DG) among patients with PTSD, MDD, PTSD with comorbid MDD, and AdjD. Furthermore, we added self-reported symptom duration and previous psychopharmacological and psychotherapy treatment as further covariates to examine whether there were associations with CA1, CA2/3, and DG. Results: No significant volume differences in hippocampal subfields between stress-related mental disorders were found. No significant associations were detected between symptom duration, psychopharmacological treatment, psychotherapy, and the hippocampal subfields. Conclusion: Hippocampal subfields may distinguish stress-related mental disorders; however, we did not observe any subfield differences. We provide several explanations for the non-results and thereby inform future field studies.

Anti-AGO1 Antibodies Identify a Subset of Autoimmune Sensory Neuronopathy.

BACKGROUND AND OBJECTIVES: Autoantibodies (Abs) improve diagnosis and treatment decisions of idiopathic neurologic disorders. Recently, we identified Abs against Argonaute (AGO) proteins as potential autoimmunity biomarkers in neurologic disorders. In this study, we aim to reveal (1) the frequency of AGO1 Abs in sensory neuronopathy (SNN), (2) titers and IgG subclasses, and (3) their clinical pattern including response to treatment. METHODS: This retrospective multicentric case/control study screened 132 patients with SNN, 301 with non-SNN neuropathies, 274 with autoimmune diseases (AIDs), and 116 healthy controls (HCs) for AGO1 Abs through ELISA. Seropositive cases were also tested for IgG subclasses, titers, and conformation specificity. RESULTS: AGO1 Abs occurred in 44 patients, comprising significantly more of those with SNN (17/132 [12.9%]) than those with non-SNN neuropathies (11/301 [3.7%]; p = 0.001), those with AIDs (16/274 [5.8%]; p = 0.02), or HCs (0/116; p < 0.0001). Ab titers ranged from 1:100 to 1:100,000. IgG subclass was mainly IgG1, and 11/17 AGO1 Ab-positive SNN (65%) had a conformational epitope. AGO1 Ab-positive SNN was more severe than AGO1 Ab-negative SNN (e.g., SNN score: 12.2 vs 11.0, p = 0.004), and they more frequently and more efficiently responded to immunomodulatory treatments than AGO1 Ab-negative SNN (7/13 [54%] vs 6/37 [16%], p = 0.02). Regarding the type of treatments more precisely, this significant difference was confirmed for the use of IV immunoglobulins (IVIg) but not for steroids or second-line treatments. Multivariate logistic regression adjusted for potential confounders showed that AGO1 Ab positivity was the only predictor of response to treatment (OR 4.93, 1.10-22.24 95% CI, p = 0.03). DISCUSSION: Although AGO Abs are not specific for SNN, based on our retrospective data, they may identify a subset of cases with SNN with more severe features and a possibly better response to IVIg. The significance of AGO1 Abs in clinical practice needs to be explored on a larger series.

Conformation-stabilizing ELISA and cell-based assays reveal patient subgroups targeting three different epitopes of AGO1 antibodies.

Autoantibodies (Abs) are biomarkers for many disease conditions and are increasingly used to facilitate diagnosis and treatment decisions. To guarantee high sensitivity and specificity, the choice of their detection method is crucial. Via cell-based assays, we recently found 21 patients with neurological diseases positive for antibodies against argonaute (AGO), 10 of which having a neuropathy (NP). Here, we established a simple and conformation-sensitive ELISA with the aim to distinguish between AGO1 Abs against conformational epitopes and non-conformational epitopes and to reveal further characteristics of AGO1 antibodies in NP and autoimmune disease (AID). In a retrospective multicenter case/control and observational study, we tested 434 patients with NP, 274 disease controls with AID, and 116 healthy controls (HC) for AGO1 Abs via conformation-stabilizing ELISA. Seropositive patients were also tested for conformation-specificity via comparative denaturing/stabilizing ELISA (CODES-ELISA), CBA positivity, AGO1 titers and IgG subclasses, and AGO2 reactivity. These parameters were statistically compared among different epitope-specific patient groups. We found Abs in 44 patients, including 28/434 (6.5%) NP, 16/274 (5.8%) AID, and 0/116 (0%) HC. Serum reactivity was consistently higher for AGO1 than AGO2. Globally among the 44 AGO1 Abs-positive patients, 42 were also tested in CBA for AGO1 Abs positivity and 15 (35.7%) were positive. Furthermore, 43 were tested for conformation-specificity and 32 (74.4%) bound a conformational epitope. Among the subgroups of highly positive patients (ELISA z-score >14) with sera binding conformational epitopes (n=23), 14 patient sera were also CBA positive and 9 bound a second conformational but CBA-inaccessible epitope. A third, non-conformational epitope was bound by 11/43 (15.6%). Among the epitope-specific patient subgroups, we found significant differences regarding the Abs titers, IgG subclass, and AGO2 reactivity. When comparing AGO1 Abs-positive NP versus AID patients, we found the conformation-specific and CBA inaccessible epitope significantly more frequently in AID patients. We conclude that 1) conformational ELISA was more sensitive than CBA in detecting AGO1 Abs, 2) serum reactivity is higher for AGO1 than for AGO2 at least for NP patients, 3) AGO1 Abs might be a marker-of-interest in 6.5% of NP patients, 4) distinguishing epitopes might help finding different patient subgroups.

The prognostic role of early tumor shrinkage in patients with hepatocellular carcinoma undergoing immunotherapy.

BACKGROUND: Early tumor shrinkage (ETS) has been identified as a promising imaging biomarker for patients undergoing immunotherapy for several cancer entities. This study aimed to validate the potential of ETS as an imaging biomarker for patients undergoing immunotherapy for hepatocellular carcinoma (HCC). METHODS: We screened all patients with HCC that received immunotherapy as the first or subsequent line of treatment at our tertiary care center between 2016 and 2021. ETS was defined as the reduction in the sum of the sizes of target lesions, between the initial imaging and the first follow-up. The ETS was compared to the radiologic response, according to the modified response evaluation criteria in solid tumors (mRECIST). Furthermore, we evaluated the influence of ETS on overall survival (OS), progression-free survival (PFS), and the alpha-fetoprotein (AFP) response. RESULTS: The final analysis included 39 patients with available cross-sectional imaging acquired at the initiation of immunotherapy (baseline) and after 8-14 weeks. The median ETS was 5.4%. ETS was significantly correlated with the response according to mRECIST and with the AFP response. Patients with an ETS ≥10% had significantly longer survival times after the first follow-up, compared to patients with < 10% ETS (15.1 months vs. 4.0 months, p = 0.008). Additionally, patients with both an ETS ≥10% and disease control, according to mRECIST, also had significantly prolonged PFS times after the initial follow-up (23.6 months vs. 2.4 months, p < 0.001). CONCLUSION: ETS was strongly associated with survival outcomes in patients with HCC undergoing immunotherapy. Thus, ETS is a readily assessable imaging biomarker that showed potential for facilitating a timely identification of patients with HCC that might benefit from immunotherapy.

Anti-FGFR3 antibody epitopes are functional sites and correlate with the neuropathy pattern.

Antibodies against FGFR3 define a subgroup of sensory neuropathy (SN). The aim of this study was to identify the epitope(s) of anti-FGFR3 autoantibodies and potential epitope-dependent clinical subtypes. Using SPOT methodology, five specific candidate epitopes, three in the juxtamembrane domain (JMD) and two in the tyrosine kinase domain (TKD), were screened with 68 anti-FGFR3-positive patients and 35 healthy controls. The identified epitopes cover 6/15 functionally relevant sites of the protein. Four patients reacted with the JMD and 11 with the TKD, partly even in a phosphorylation-state dependent manner. The epitope could not be identified in the others. Patients with antibodies recognizing TKD exhibited a more severe clinical and electrophysiological impairment than others.

[A mathematical model to estimate the number of unreported SARS-CoV-2 infections in the early phase of the pandemic using Germany and Italy as examples]. / Ein mathematisches Modell zur Schätzung der Dunkelziffer von SARS-CoV-2-Infektionen in der Frühphase der Pandemie am Beispiel Deutschland und Italien.

BACKGROUND: Especially in the early phase, it is difficult to obtain reliable figures on the spread of a pandemic. The effects of the COVID-19 pandemic and the associated comprehensive but incomplete data monitoring provide a strong reason to estimate the number of unreported cases. AIM: The aim of this paper is to present a simple mathematical model that allows early estimation of the number of unregistered cases (underreporting). MATERIAL AND METHODS: Prevalences of reported infections in different age groups are combined with additional assumptions on relative contact rates. From this, a corrected prevalence is derived for each age group, which can then be used to estimate the number of unreported cases. RESULTS: Our model derives for Germany in mid-April 2020 about 2.8 times more total infections than registered cases. For Italy, the model results in a factor of 8.3. The case mortalities derived from this are 0.98% for Germany and 1.51% for Italy, which are much closer together than the case mortalities of 2.7% and 12.6% derived purely from the number of reports available at that time. CONCLUSION: The number of unreported SARS-CoV-2-infected cases derived from the model can largely explain the difference in observations in case mortalities and of conditions in the early phase of the COVID-19 pandemic in Germany and Italy. The model is simple, fast, and robust to implement, and can respond well when the reporting numbers are not representative of the population in terms of age structure. We suggest considering this model for efficient and early estimations of unreported case numbers in future epidemics and pandemics.

Argonaute Autoantibodies as Biomarkers in Autoimmune Neurologic Diseases.

OBJECTIVE: To identify and characterize autoantibodies (Abs) as novel biomarkers for an autoimmune context in patients with central and peripheral neurologic diseases. METHODS: Two distinct approaches (immunoprecipitation/mass spectrometry-based proteomics and protein microarrays) and patients' sera and CSF were used. The specificity of the identified target was confirmed by cell-based assay (CBA) in 856 control samples. RESULTS: Using the 2 methods as well as sera and CSF of patients with central and peripheral neurologic involvement, we identified Abs against the family of Argonaute proteins (mainly AGO1 and AGO2), which were already reported in systemic autoimmunity. AGO-Abs were mostly of immunoglobulin G 1 subclass and conformation dependent. Using CBA, AGO-Abs were detected in 21 patients with a high suspicion of autoimmune neurologic diseases (71.4% were women; median age 57 years) and only in 4/856 (0.5%) controls analyzed by CBA (1 diagnosed with small-cell lung cancer and the other 3 with Sjögren syndrome). Among the 21 neurologic patients identified, the main clinical presentations were sensory neuronopathy (8/21, 38.1%) and limbic encephalitis (6/21, 28.6%). Fourteen patients (66.7%) had autoimmune comorbidities and/or co-occurring Abs, whereas AGO-Abs were the only autoimmune biomarker for the remaining 7/21 (33.3%). Thirteen (61.9%) patients were treated with immunotherapy; 8/13 (61.5%) improved, and 3/13 (23.1%) remained stable, suggesting an efficacy of these treatments. CONCLUSIONS: AGO-Abs might be potential biomarkers of autoimmunity in patients with central and peripheral nonparaneoplastic neurologic diseases. In 7 patients, AGO-Abs were the only biomarkers; thus, their identification may be useful to suspect the autoimmune character of the neurologic disorder. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that AGO-Abs are more frequent in patients with autoimmune neurologic diseases than controls.

H3F3A-mutated giant cell tumour of bone without giant cells-clinical presentation, radiology and histology of three cases.

AIMS: Giant cell tumour of bone (GCTB) is histologically defined as a lesion containing reactive giant cells and a neoplastic mononuclear cell population; in up to 92% of cases, GCTB is characterised by a specific mutation of the histone gene H3F3A. The cellular composition ranges from giant-cell-rich to giant-cell-poor. The diagnosis of GCTB can be challenging, and several other lesions need to be excluded, e.g. aneurysmal bone cysts, non-ossifying fibromas, chondroblastomas, brown tumours, and giant-cell-rich osteosarcomas. Our aim was to analyse the clinical history, imaging, molecular pathology and histology of three H3F3A-mutated bone tumours without detectable giant cells. None of the patients received denosumab therapy. METHODS AND RESULTS: Diagnostic material was obtained by curettage or resection and/or biopsy. Common histomorphological features of all three reported lesions were fibrocytic, oval cells in a background of osteoid and an absence of multinuclear giant cells as confirmed with CD68 immunohistochemistry. We used immunohistochemistry and Sanger sequencing to demonstrate positivity for the H3.3 p.G34W mutation. Differential diagnoses were systematically excluded on the basis of histomorphology, immunohistochemistry, and fluorescence in-situ hybridisation. The imaging (radiography, computed tomography, and magnetic resonance imaging) for all three cases is presented and discussed. CONCLUSIONS: We believe that these GCTBs without giant cells expand one end of the heterogeneous range of GCTB. Because of the lack of giant cells, correct diagnosis of GCTB is challenging or even impossible on histological grounds alone. In these cases, detection of the characteristic H3F3A mutation (G34W-specific antibody RM263 or sequencing) is extremely helpful for diagnosing those lesions without giant cells as giant cell tumours of bone.

CIDP Antibodies Target Junction Proteins and Identify Patient Subgroups: An Autoantigenomic Approach.

OBJECTIVE: To discover systemic characteristics in the repertoires of targeted autoantigens in chronic inflammatory demyelinating polyneuropathy (CIDP), we detected the entire autoantigen repertoire of patients and controls and analyzed them systematically. METHODS: We screened 43 human serum samples, of which 22 were from patients with CIDP, 12 from patients with other neuropathies, and 9 from healthy controls via HuProt Human Proteome microarrays testing about 16,000 distinct human bait proteins. Autoantigen repertoires were analyzed via bioinformatical autoantigenomic approaches: principal component analysis, analysis of the repertoire sizes in disease groups and clinical subgroups, and overrepresentation analyses using Gene Ontology and PantherDB. RESULTS: The autoantigen repertoires enabled the identification of a subgroup of 10/22 patients with CIDP with a younger age at onset and a higher frequency of mixed motor and sensory CIDP. IV immunoglobulin therapy responders targeted 3 times more autoantigens than nonresponders. No CIDP-specific autoantibody is present in all patients; however, anchoring junction components were significantly targeted by 86.4% of patients with CIDP. There are potential novel CIDP-specific autoantigens such as the myelination- or axo-glial structure-related proteins actin-related protein 2/3 complex subunit 1B, band 4.1-like protein 2, cadherin-15, cytohesin-1, epidermal growth factor receptor, ezrin, and radixin. CONCLUSIONS: The repertoire of targeted autoantigens of patients with CIDP differs in a systematic degree from those of controls. Systematic autoantigenomic approaches can help to understand the disease and to discover novel bioinformatical tools and novel autoantigen panels to improve diagnosis, treatment, prognosis, or patient stratification.

Proper definition of the set of autoantibody-targeted antigens relies on appropriate reference group selection.

Autoimmune diseases are frequently associated with autoantibodies. Recently, large sets of autoantibody-targeted antigens ("autoantigen-omes") of patient and control sera have been revealed, enabling autoantigen-omic approaches. However, statistical standards for defining such autoantigen-omes are lacking. The z-score indicates how many standard deviations an antigen reactivity of a given sample is from the mean reactivity of the corresponding antigen in a reference group. Hence, it is a common measure to define significantly positive reactivity in autoantigen profiling approaches. Here, we address the risk of biased analyses resulting from unbalanced selection of the reference group. Three study groups were selected. Patients-of-interest were chronic inflammatory demyelinating polyneuropathy (CIDP); controls were other neuropathies (ONP); and healthy controls (HC). Each serum was screened for significant autoantigen reactivity using HuProt™ protein arrays. We compared three possible selections of reference groups for statistical z-score calculations: method#1, the control groups (ONP + HC); method #2, all groups together; method #3, the respective other groups (e.g., CIDP + HC for the ONP autoantigen-ome). The method selection seriously affected the size of the autoantigen-omes. Method #1 introduced a bias favoring significantly more antigens per patient in the CIDP group (for z >4: 19 ±â€¯3 antigens) than in the control groups (ONP: 2 ±â€¯1; HC: 0 ±â€¯0). The more balanced methods #2 and #3 did not result in significant differences. This contribution may help to avoid interpretation biases and to develop guidelines for population studies revealing autoantigen-omes via high throughput studies such as protein microarrays, immunoprecipitation with mass spectrometry, or phage display assays.

Clinical characterisation of sensory neuropathy with anti-FGFR3 autoantibodies.

OBJECTIVE: Sensory neuropathies (SNs) are often classified as idiopathic even if immunological mechanisms can be suspected. Antibodies against the intracellular domain of the fibroblast growth factor receptor 3 (FGFR3) possibly identify a subgroup of SN affecting mostly the dorsal root ganglion (DRG). The aim of this study was to identify the frequency of anti-FGFR3 antibodies and the associated clinical pattern in a large cohort of patients with SN. METHODS: A prospective, multicentric, European and Brazilian study included adults with pure SN. Serum anti-FGRF3 antibodies were analysed by ELISA. Detailed clinical and paraclinical data were collected for each anti-FGFR3-positive patient and as control for anti-FGFR3-negative patients from the same centres ('center-matched'). RESULTS: Sixty-five patients out of 426 (15%) had anti-FGFR3 antibodies, which were the only identified autoimmune markers in 43 patients (66%). The neuropathy was non-length dependent in 89% and classified as sensory neuronopathy in 64%, non-length-dependent small fibre neuropathy in 17% and other neuropathy in 19%. Specific clinical features occurred after 5-6 years of evolution including frequent paresthesia, predominant clinical and electrophysiological involvement of the lower limbs, and a less frequent mixed large and small fibre involvement. Brazilians had a higher frequency of anti-FGFR3 antibodies than Europeans (36% vs 13%, p<0.001), and a more frequent asymmetrical distribution of symptoms (OR 169, 95% CI 3.4 to 8424). CONCLUSIONS: Anti-FGFR3 antibodies occur in a subgroup of SN probably predominantly affecting the DRG. Differences between Europeans and Brazilians could suggest involvement of genetic or environmental factors.

40 years Western blotting: A scientific birthday toast.

The Western blot technique was first published 40 years ago. Since then, it has been mentioned in the titles, abstracts, and keywords of more than 400,000 PubMed-listed publications. This anniversary represents an opportunity to look back to its origins, to address the question as to whether the technique is outdated and heading toward being replaced, and to point out current improvements. Publications within the last ten years indicate Western blotting may be the most used protein-analytical technique. Although no decline in its usage is visible, the method has been moving out of the spotlight.

Autoantigenomics: Holistic characterization of autoantigen repertoires for a better understanding of autoimmune diseases.

Autoimmune diseases are mostly characterized by autoantibodies in the patients' serum or cerebrospinal fluid, representing diagnostic or prognostic biomarkers. For decades, research has focused on single autoantigens or panels of single autoantigens. In this article, we advocate to broaden the focus by addressing the entire autoantigen repertoire in a systemic "omics-like" way. This approach aims to capture the enormous biodiversity in the sets of targeted antigens and pave the way toward a more holistic understanding of the concerted character of antibody-related humoral immune responses. Ongoing technological progress permits high-throughput screenings of thousands of autoantigens in parallel, e.g., via protein microarrays, phage display, or immunoprecipitation with mass spectrometry. We argue that the time is right for combining omics and autoantibody screening approaches into "autoantigenomics" as a novel omics subcategory. In this article, we introduce the concept of autoantigenomics, describe its roots and application options, and demarcate the method from related holistic approaches such as systems serology or immune-related transcriptomics and proteomics. We suggest the following extendable method set to be applied to autoantigen repertoires: (1) principal component analysis, (2) hierarchical cluster analysis, (3) partial least-square discriminant analysis or orthogonal projections to latent structures discriminant analysis, (4) analysis of the repertoire sizes in disease groups and clinical subgroups, (5) overrepresentation analyses using databases like those of Gene Ontology, Reactome Pathway, or DisGeNET, (6) analysis of pathways that are significantly targeted by specific repertoires, and (7) machine learning approaches. In an unsupervised way, these methods can identify clusters of autoantigens sharing certain functional or spatial properties, or clusters of patients comprising clinical subgroups potentially useful for patient stratification. In a supervised way, these methods can lead to prediction models that may eventually assist diagnosis and prognosis. The untargeted autoantigenomics approach allows for the systematic survey of antibody-related humoral immune responses. This may enhance our understanding of autoimmune diseases in a more comprehensive way compared to current single or panel autoantibodies approaches.

Targeting cyclin-dependent kinases for the treatment of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a devastating disease with poor prognosis and limited therapeutic options. We screened for pathways that may be responsible for the abnormal phenotype of pulmonary arterial smooth muscle cells (PASMCs), a major contributor of PAH pathobiology, and identified cyclin-dependent kinases (CDKs) as overactivated kinases in specimens derived from patients with idiopathic PAH. This increased CDK activity is confirmed at the level of mRNA and protein expression in human and experimental PAH, respectively. Specific CDK inhibition by dinaciclib and palbociclib decreases PASMC proliferation via cell cycle arrest and interference with the downstream CDK-Rb (retinoblastoma protein)-E2F signaling pathway. In two experimental models of PAH (i.e., monocrotaline and Su5416/hypoxia treated rats) palbociclib reverses the elevated right ventricular systolic pressure, reduces right heart hypertrophy, restores the cardiac index, and reduces pulmonary vascular remodeling. These results demonstrate that inhibition of CDKs by palbociclib may be a therapeutic strategy in PAH.

Rapid clearance of cellular debris by microglia limits secondary neuronal cell death after brain injury in vivo.

Moderate or severe traumatic brain injury (TBI) causes widespread neuronal cell death. Microglia, the resident macrophages of the brain, react to injury by migrating to the lesion site, where they phagocytose cellular debris. Microglial phagocytosis can have both beneficial (e.g. debris clearance) and detrimental (e.g. respiratory burst, phagoptosis) consequences. Hence, whether the overall effect of microglial phagocytosis after brain injury in vivo is neuroprotective or neurotoxic is not known. Here, we establish a system with which to carry out dynamic real-time analyses of the mechanisms regulating cell death after brain injury in vivo We show that mechanical injury to the larval zebrafish brain induces distinct phases of primary and secondary cell death. Excitotoxicity contributes to secondary cell death in zebrafish, reflecting findings from mammals. Microglia arrive at the lesion site within minutes of injury, where they rapidly engulf dead cells. Importantly, the rate of secondary cell death is increased when the rapid removal of cellular debris by microglia is reduced pharmacologically or genetically. In summary, our results provide evidence that microglial debris clearance is neuroprotective after brain injury in vivo.

Clearance by Microglia Depends on Packaging of Phagosomes into a Unique Cellular Compartment.

Phagocytic immune cells such as microglia can engulf and process pathogens and dying cells with high efficiency while still maintaining their dynamic behavior and morphology. Effective intracellular processing of ingested cells is likely to be crucial for microglial function, but the underlying cellular mechanisms are poorly understood. Using both living fish embryos and mammalian macrophages, we show that processing depends on the shrinkage and packaging of phagosomes into a unique cellular compartment, the gastrosome, with distinct molecular and ultra-structural characteristics. Loss of the transporter Slc37a2 blocks phagosomal shrinkage, resulting in the expansion of the gastrosome and the dramatic bloating of the cell. This, in turn, affects the ability of microglia to phagocytose and migrate toward brain injuries. Thus, this work identifies a conserved crucial step in the phagocytic pathway of immune cells and provides a potential entry point for manipulating their behavior in development and disease.

Completing the Immunological Fingerprint by Refractory Proteins: Autoantibody Screening via an Improved Immunoblotting Technique.

PURPOSE: Identifying autoantigens of serological autoantibodies requires expensive methods, such as protein microarrays or IP+MS. Thus, sera are commonly pre-screened for interesting immunopatterns via immunocytochemistry/immunohistochemistry. However, distinguishing immunopatterns can be difficult and intracellular antigens are less accessible. Therefore, a simple and cheap immunoblot screening able to distinguish immunopatterns and to detect refractory proteins is presented. EXPERIMENTAL DESIGN: Five steps of immunoblotting-based autoantigen screening are revised: (1) choice of protein source, (2) protein extraction, (3) protein separation, (4) protein transfer, (5) antigen detection. Thereafter, 52 patients' sera with chronic inflammatory demyelinating polyneuropathy (CIDP) and 45 controls were screened. RESULTS: The protein source impacts the detected antigen set. Steps 2-4 can be adapted for refractory proteins. Furthermore, longitudinal cutting of protein lanes saves ≥75% of time and material and allows for exact comparison of band patterns. As the latter are individually specific and temporarily constant, we call them "immunological fingerprints". In a proof-of-principle, a 155 kDa immunoband was detected with two anti-neurofascin-155-positive CIDP sera and two further immunobands (120/220 kDa) specific to a subgroup of 3-6 of 52 CIDP patients. CONCLUSIONS AND CLINICAL RELEVANCE: Adapted immunoblotting is a cheap and simple method for accurate serum screening including refractory and intracellular antigens.

Poor transcript-protein correlation in the brain: negatively correlating gene products reveal neuronal polarity as a potential cause.

Transcription, translation, and turnover of transcripts and proteins are essential for cellular function. The contribution of those factors to protein levels is under debate, as transcript levels and cognate protein levels do not necessarily correlate due to regulation of translation and protein turnover. Here we propose neuronal polarity as a third factor that is particularly evident in the CNS, leading to considerable distances between somata and axon terminals. Consequently, transcript levels may negatively correlate with cognate protein levels in CNS regions, i.e., transcript and protein levels behave reciprocally. To test this hypothesis, we performed an integrative inter-omics study and analyzed three interconnected rat auditory brainstem regions (cochlear nuclear complex, CN; superior olivary complex, SOC; inferior colliculus, IC) and the rest of the brain as a reference. We obtained transcript and protein sets in these regions of interest (ROIs) by DNA microarrays and label-free mass spectrometry, and performed principal component and correlation analyses. We found 508 transcript|protein pairs and detected poor to moderate transcript|protein correlation in all ROIs, as evidenced by coefficients of determination from 0.34 to 0.54. We identified 57-80 negatively correlating gene products in the ROIs and intensively analyzed four of them for which the correlation was poorest. Three cognate proteins (Slc6a11, Syngr1, Tppp) were synaptic and hence candidates for a negative correlation because of protein transport into axon terminals. Thus, we systematically analyzed the negatively correlating gene products. Gene ontology analyses revealed overrepresented transport/synapse-related proteins, supporting our hypothesis. We present 30 synapse/transport-related proteins with poor transcript|protein correlation. In conclusion, our analyses support that protein transport in polar cells is a third factor that influences the protein level and, thereby, the transcript|protein correlation. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* and *Open Data* because it provided all relevant information to reproduce the study in the manuscript and because it made the data publicly available. The data can be accessed at https://osf.io/ha28n/. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.