Severe Neuro-COVID is associated with peripheral immune signatures, autoimmunity and neurodegeneration: a prospective cross-sectional study

Growing evidence links COVID-19 with acute and long-term neurological dysfunction. However, the pathophysiological mechanisms resulting in central nervous system involvement remain unclear, posing both diagnostic and therapeutic challenges. Here we show outcomes of a cross-sectional clinical study (NCT04472013) including clinical and imaging data and corresponding multidimensional characterization of immune mediators in the cerebrospinal fluid (CSF) and plasma of patients belonging to different Neuro-COVID severity classes. The most prominent signs of severe Neuro-COVID are blood-brain barrier (BBB) impairment, elevated microglia activation markers and a polyclonal B cell response targeting self-antigens and non-self-antigens. COVID-19 patients show decreased regional brain volumes associating with specific CSF parameters, however, COVID-19 patients characterized by plasma cytokine storm are presenting with a non-inflammatory CSF profile. Post-acute COVID-19 syndrome strongly associates with a distinctive set of CSF and plasma mediators. Collectively, we identify several potentially actionable targets to prevent or intervene with the neurological consequences of SARS-CoV-2 infection

β-CATENIN N-TERMİNAL BÖLGE DEĞİŞİKLİKLERİNİN NEOPLASTİK SÜREÇLERDEKİ ROLÜNÜN ARAŞTIRILMASI

Uzun, S, Investigation of The Role of β-catenin N-terminal Domain Alterations in Neoplastic Processes, Hacettepe University Graduate School of Health Sciences Tumor Biology and Immunology Program Doctor of Philosophy Thesis, Ankara, 2021. β-catenin is a multifunctional protein and located at the center of Wnt pathway. Intracellular localization and level of β-catenin are strictly controlled by special amino acid residues located at its N-terminal domain. These amino acid residues are encoded by the third exon (exon 3) of CTNNB1 (β-catenin encoding gene) and the mutations altering this region lead to neoplastic transformation by changing the amino acid sequence of the N-terminal domain. Our research group has recently defined an immunohistochemistry-based approach that can detect CTNNB1 exon 3 mutations. The primary purpose of this study is to evaluate the CTNNB1 exon 3 alterations in neoplastic diseases of unknown pathogenesis by using this immunohistochemistry-based approach. For this purpose, β-catenin N-terminal domain alterations were evaluated with this method in the cases of sclerosing angiomatoid nodular transformation (SANT) of the spleen, a rare vascular lesion of the spleen. As a result of immunohistochemical staining, findings suggesting the presence of exon 3 mutations were obtained. These findings were confirmed with polymerase chain reaction and Sanger sequencing. The secondary purpose of this study is to evaluate the post-translational modifications of the β-catenin N-terminal domain by using the immunohistochemical staining method that we have described. For this purpose, the intracellular localization of β-catenin phosphorylated at the amino acids S33, S37 and T41 (phospho-S33/S37/T41-β-catenin) was investigated using immunohistochemical and immunofluorescent staining methods, and it was shown that this protein could accumulate in the nucleus of the colon cancer cells. By evaluating protein lysates of colorectal cancer patients with Western Blot method, it was also suggested that low molecular weight β-catenin forms might be formed after proteolytic cleavage of N-terminal and C-terminal regions of β-catenin.Uzun, S, β-catenin N-Terminal Bölge Değişikliklerinin Neoplastik Süreçlerdeki Rolünün Araştırılması, Hacettepe Üniversitesi Sağlık Bilimleri Enstitüsü Tümör Biyolojisi ve İmmünolojisi Programı Doktora Tezi, Ankara, 2021. β-catenin, Wnt sinyal yolağının merkezinde yer alan çok fonksiyonlu bir proteindir. β-catenin’in hücre içi yerleşimi ve seviyesi, N-terminal bölgesinde yer alan özel bir amino asit dizisi sayesinde sıkı bir şekilde kontrol edilmektedir. Bu amino asit dizisi CTNNB1’in (β- catenin’i kodlayan gen) üçüncü ekzonu (ekzon 3) tarafından kodlanmaktadır ve bu ekzonda görülen mutasyonlar N-terminal bölgede yer alan amino asit dizisini değiştirerek neoplastik transformasyona neden olmaktadır. Araştırma grubumuz tarafından CTNNB1 ekzon 3 mutasyonlarının tespit edilmesini sağlayan immünohistokimyasal boyama temelli bir yöntem tanımlanmıştır. Bu tez çalışmasının birincil amacı, tanımladığımız yöntemi kullanarak patogenezi bilinmeyen neoplastik hastalıklarda CTNNB1 ekzon 3 mutasyonlarının araştırılmasıdır. Bu kapsamda, dalağın nadir görülen vasküler lezyonlarından biri olan sklerozan anjiomatoid nodüler transformasyon (SANT) olgularında tanımladığımız yöntemle N-terminal bölge değişiklikleri incelenmektedir. İmmünohistokimyasal boyama sonucunda ekzon 3 mutasyonu varlığını düşündüren bulgular elde edilmektedir. Bu bulgular polimeraz zincir reaksiyonu ve Sanger sekanslama ile doğrulanmaktadır. Tez çalışmasının ikincil amacı ise tanımladığımız immünohistokimyasal boyama yöntemini kullanarak β- catenin’in N-terminal bölgesini etkileyebilecek post-translasyonel modifikasyonların araştırılmasıdır. Bu amaç doğrultusunda immünohistokimyasal ve immünofloresan boyama yöntemleri kullanılarak S33, S37 ve T41 amino asitlerinden fosforile olan β- catenin’in (fosfo-S33/S37/T41-β-catenin) hücre içi yerleşimi incelenmektedir ve bu proteinin kolon kanseri hücrelerinin çekirdeğinde birikebileceği gösterilmektedir. Ek olarak kolorektal kanser protein lizatlarında Western Blot yöntemi ile N-terminal ve C-terminal bölgesinden proteolitik olarak kesilmiş, düşük moleküler ağırlığı sahip β- catenin formlarının bulunabileceği ileri sürülmektedir

A Case Of Disseminated Hypopigmented Keratoses Improved With Oral Acitretin

Disseminated hypopigmented keratosis is a distinct clinical entity and only few cases have been reported so far. Here, we present a 21-year-old man with almost 10-year history of hypopigmented, nonfollicular, keratotic lichenoid papules occurring on the extensor surfaces of the extremities, back and lumber region. Histopathological examination showed orthohyperkeratosis, irregular acanthosis, and sporadic papillomatosis with a normal amount of melanin and number of melanocytes. In addition, no marked inflammation or melanophages were seen. In order to exclude other possible causes, we performed laboratory tests and radiological examination which were all found to be normal. As the clinical and histopathological features of our patient were taken into account, it was considered to be compatible with the diagnosis of disseminated hypopigmented keratoses. So far, only topical therapies have been used with failure in the previously reported cases except one patient. Considering the extensive lesions, we treated the present patient with 5% salicylic acid in addition to oral acitretin and significant regression in all lesions was achieved, particularly on the keratosis.PubMe

Loss of CTNNB1 exon 3 in sclerosing angiomatoid nodular transformation of the spleen

Sclerosing angiomatoid nodular transformation (SANT) is a rare vascular lesion of the spleen. Although several hypotheses have been suggested, the etiopathogenesis of SANT remains unknown. It is also unclear whether SANT is a reactive or a neoplastic lesion. Since CTNNB1 (beta-catenin gene) exon 3 mutations were frequently detected in some rare fibrovascular lesions, we aimed to investigate the presence of oncogenic CTNNB1 mutations in SANT cases. For this purpose, 7 cases of SANT with typical histopathological features were retrieved. First, the presence of CTNNB1 exon 3 alterations was examined with a recently described immunohistochemistry-based method. Then, the findings were confirmed with polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), and Sanger sequencing. In all cases, immunochemistry of beta-catenin gave a staining pattern that was suggestive of exon 3 alteration; however, no missense mutations were found in any case at the CTNNB1 exon 3 hotspot region. Subsequently, we screened for large interstitial deletions of CTNNB1 exon 3 which revealed short PCR products in three cases. Sequencing confirmed that these cases had large interstitial deletions, resulting in loss of the entire exon 3 of CTNNB1. In the remaining four cases, loss of exon 3 was documented at the cDNA level, although genomic deletion was not identified. These results demonstrate that loss of CTNNB1 exon 3 and stabilization of beta-catenin with activation of Wnt signaling pathway might have a significant role in the pathogenesis of SANT. Through this study, we provided important evidence for the neoplastic nature and pathogenesis of this disorder

An immunohistochemical approach to detect oncogenic CTNNB1 mutations in primary neoplastic tissues.

The Wnt/β-catenin signaling pathway is dysregulated in different types of neoplasms including colorectal cancer (CRC). Aberrant activation of this signaling pathway is a key early event in the development of colorectal neoplasms, and is mainly caused by loss of function mutations in Adenomatous Polyposis Coli (APC), and less frequently by β-catenin stabilization mutations via missense or interstitial genomic deletions in CTNNB1. In this study, we have defined an immunohistochemical algorithm to dissect Wnt pathway alterations in formalin-fixed and paraffin-embedded neoplastic tissues. Basically, consecutive sections of tumor specimens were stained by immunohistochemistry with two different monoclonal antibodies against β-catenin: one (anti-active β-catenin antibody) recognizes hypo-phosphorylated β-catenin and the other recognizes the total pool of β-catenin. We validated the strategy in the HCT116 CRC cell line which has an in-frame deletion of β-catenin serine 45, and then studied human tumor microarrays containing colon adenomas, CRCs, solid pseudopapillary neoplasms of the pancreas as well as the whole tissue sections of CRCs, desmoid fibromatosis, and pilomatrixoma of the skin. In some tumors, we found strong β-catenin cytoplasmic and/or nuclear staining with the total β-catenin antibody but no staining with the anti-active β-catenin antibody. This was inferred to be an altered/mutant β-catenin staining pattern. All six colon adenomas of the 126 total adenomas studied for the altered/mutant β-catenin staining pattern had presumptively pathogenic point mutations or deletions in CTNNB1. Four of 10 CRCs with the alterated/mutant β-catenin staining pattern studied in depth, from 181 total CRCs from tissue microarray, had pathogenic CTNNB1 mutations. The frequencies of CTNNB1 alterations in non-colonic tumors with altered/mutant β-catenin staining ranged between 46 and 100%. Our results demonstrate that the immunohistochemical approach described here can detect oncogenic forms of β-catenin in primary tissue samples and can also highlight other tumors with presumptive novel defects activating the Wnt/β-catenin pathway

Genetic and epigenetic analysis of hepatocellular adenomas with atypical morphological features.

BACKGROUND Hepatocellular adenoma (HCA) is a rare liver tumor, which can have atypical morphological features such as cytological atypia, pseudo-glandular architecture, and altered reticulin framework. Little is known about the genetic and epigenetic alterations of such HCAs and whether they show the alterations classically found in HCC or in HCA without atypical morphology. METHODS We analyzed five HCAs with atypical morphological features and one HCA with transition to HCC. Every tumor was subtyped by immunohistochemistry, sequenced by a targeted NGS panel and analyzed on a DNA methylation microarray. RESULTS Subtyping of the five HCAs with atypical features revealed 2 β-catenin mutated HCA (b-HCA), 2 β-catenin mutated inflammatory HCA (b-IHCA), and 1 sonic hedgehog activated HCA (shHCA). None of them showed mutations typically found in HCC, such as e.g. TERT or TP53 mutations. The epigenomic pattern of HCAs with atypical morphological features clustered with reference data for HCAs without atypical morphological features but not with HCC. Similarly, phylo-epigenetic trees using the DNA methylation data reproducibly showed, that HCAs with morphological atypia are much more similar to non-malignant samples than to malignant samples. Finally, atypical HCAs showed no relevant copy number variations (CNV). CONCLUSIONS In our series, mutational, DNA methylation, as well as CNV analyses supported a relationship of atypical HCAs with non-atypical HCAs rather than with HCC. Therefore, in cases with difficult differential diagnosis between HCC and HCA, it might be advisable to perform targeted sequencing and/or combined methylation/copy number profiling

Genetic and epigenetic analysis of hepatocellular adenomas with atypical morphological features

BACKGROUND Hepatocellular adenoma (HCA) is a rare liver tumor, which can have atypical morphological features such as cytological atypia, pseudo-glandular architecture, and altered reticulin framework. Little is known about the genetic and epigenetic alterations of such HCAs and whether they show the alterations classically found in HCC or in HCA without atypical morphology. METHODS We analyzed five HCAs with atypical morphological features and one HCA with transition to HCC. Every tumor was subtyped by immunohistochemistry, sequenced by a targeted NGS panel and analyzed on a DNA methylation microarray. RESULTS Subtyping of the five HCAs with atypical features revealed 2 β-catenin mutated HCA (b-HCA), 2 β-catenin mutated inflammatory HCA (b-IHCA), and 1 sonic hedgehog activated HCA (shHCA). None of them showed mutations typically found in HCC, such as e.g. TERT or TP53 mutations. The epigenomic pattern of HCAs with atypical morphological features clustered with reference data for HCAs without atypical morphological features but not with HCC. Similarly, phylo-epigenetic trees using the DNA methylation data reproducibly showed, that HCAs with morphological atypia are much more similar to non-malignant samples than to malignant samples. Finally, atypical HCAs showed no relevant copy number variations (CNV). CONCLUSIONS In our series, mutational, DNA methylation, as well as CNV analyses supported a relationship of atypical HCAs with non-atypical HCAs rather than with HCC. Therefore, in cases with difficult differential diagnosis between HCC and HCA, it might be advisable to perform targeted sequencing and/or combined methylation/copy number profiling

miR-579-3p Controls Hepatocellular Carcinoma Formation by Regulating the Phosphoinositide 3-Kinase-Protein Kinase B Pathway in Chronically Inflamed Liver.

Chronic liver inflammation causes continuous liver damage with progressive liver fibrosis and cirrhosis, which may eventually lead to hepatocellular carcinoma (HCC). Whereas the 10-year incidence for HCC in patients with cirrhosis is approximately 20%, many of these patients remain tumor free for their entire lives. Clarifying the mechanisms that define the various outcomes of chronic liver inflammation is a key aspect in HCC research. In addition to a wide variety of contributing factors, microRNAs (miRNAs) have also been shown to be engaged in promoting liver cancer. Therefore, we wanted to characterize miRNAs that are involved in the development of HCC, and we designed a longitudinal study with formalin-fixed and paraffin-embedded liver biopsy samples from several pathology institutes from Switzerland. We examined the miRNA expression by nCounterNanostring technology in matched nontumoral liver tissue from patients developing HCC (n = 23) before and after HCC formation in the same patient. Patients with cirrhosis (n = 26) remaining tumor free within a similar time frame served as a control cohort. Comparison of the two cohorts revealed that liver tissue from patients developing HCC displayed a down-regulation of miR-579-3p as an early step in HCC development, which was further confirmed in a validation cohort. Correlation with messenger RNA expression profiles further revealed that miR-579-3p directly attenuated phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) expression and consequently protein kinase B (AKT) and phosphorylated AKT. In vitro experiments and the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology confirmed that miR-579-3p controlled cell proliferation and cell migration of liver cancer cell lines. Conclusion: Liver tissues from patients developing HCC revealed changes in miRNA expression. miR-579-3p was identified as a novel tumor suppressor regulating phosphoinositide 3-kinase-AKT signaling at the early stages of HCC development