Different populations of A(H1N1)pdm09 viruses in a patient with hemolytic-uremic syndrome.

Respiratory viral infections may have different impacts ranging from infection without symptoms to severe disease or even death though the reasons are not well characterized. A patient (age group 5-15 years) displaying symptoms of hemolytic uremic syndrome died one day after hospitalization. qPCR, next generation sequencing, virus isolation, antigenic characterization, resistance analysis was performed and virus replication kinetics in well-differentiated airway cells were determined. Autopsy revealed hemorrhagic pneumonia as major pathological manifestation. Lung samples harbored a large population of A(H1N1)pdm09 viruses with the polymorphism H456H/Y in PB1 polymerase. The H456H/Y viruses replicated much faster to high viral titers than upper respiratory tract viruses in vitro. H456H/Y-infected air-liquid interface cultures of differentiated airway epithelial cells did reflect a more pronounced loss of ciliated cells. A different pattern of virus quasispecies was found in the upper airway samples where substitution S263S/F (HA1) was observed. The data support the notion that viral quasispecies had evolved locally in the lung to support high replicative fitness. This change may have initiated further pathogenic processes leading to rapid dissemination of inflammatory mediators followed by development of hemorrhagic lung lesions and fatal outcome.

B-cell receptors of EBV-negative Burkitt lymphoma bind modified isoforms of autoantigens.

Burkitt lymphoma (BL) represents the most aggressive B-cell-lymphoma. Beside the hallmark of IG-MYC-translocation, surface B-cell receptor (BCR) is expressed, and mutations in the BCR pathway are frequent. Coincidental infections in endemic BL, and specific extra-nodal sites suggest antigenic triggers. To explore this hypothesis, BCRs of BL cell lines and cases were screened for reactivities against a panel of bacterial lysates, lysates of Plasmodium falciparum, a custom-made virome array and against self-antigens, including post-translationally modified antigens. An atypically modified, SUMOylated isoform of Bystin, that is, SUMO1-BYSL was identified as the antigen of the BCR of cell line CA46. SUMO1-BYSL was exclusively expressed in CA46 cells with K139 as site of the SUMOylation. Secondly, an atypically acetylated isoform of HSP40 was identified as the antigen of the BCR of cell line BL41. K104 and K179 were the sites of immunogenic acetylation, and the acetylated HSP40 isoform was solely present in BL41 cells. Functionally, addition of SUMO1-BYSL and acetylated HSP40 induced BCR pathway activation in CA46 and BL41 cells, respectively. Accordingly, SUMO1-BYSL-ETA' immunotoxin, produced by a two-step intein-based conjugation, led to the specific killing of CA46 cells. Autoantibodies directed against SUMO1-BYSL were found in 3 of 14 (21.4%), and autoantibodies against acetylated HSP40 in 1/14(7.1%) patients with sporadic Burkitt-lymphoma. No reactivities against antigens of the infectious agent spectrum could be observed. These results indicate a pathogenic role of autoreactivity evoked by immunogenic post-translational modifications in a subgroup of sporadic BL including two EBV-negative BL cell lines.

Impact of COVID-19 Pandemic on Emergency Inpatient Volume at a Tertiary Eye Care Center in Germany with Corneal Main Specialization.

BACKGROUND: To estimate the impact of the COVID-19 pandemic on emergency inpatient volume in a tertiary eye care center in Germany with corneal main subspecialization. MATERIAL AND METHODS: A retrospective review of ocular emergency patients who attended the inpatient unit of the Department of Ophthalmology of Saarland University, Homburg/Saar, Germany during the COVID-19 pandemic, between 1 March and 30 April 2020, in comparison to the same time period in 2019. For each subject, clinical history and surgical reports were reviewed. After 24 March 2020, PCR examinations for SARS-CoV-2 were performed from throat swab specimens in all patients using real-time RT-PCR. RESULTS: Totally, 135 patients were admitted in 2019 and 115 patients in 2020 as emergency cases. The patient age at the time of admission did not differ significantly between the two time periods (63.6 ± 17.9 years vs. 62.5 ± 19.6 years) (p = 0.792), but the average length of hospital stays increased significantly for 2020 (4.0 ± 3.6 vs. 4.4 ± 2.7 days, p = 0.043). The percentage of admissions due to acute corneal hydrops (0% vs. 3.5%) increased significantly from 2019 to 2020 (χ2 = 4.772, p = 0.028), however, there was not a significant difference between the two years for any other diagnosis (χ2 ≤ 3.564, p ≥ 0.059). From 2019 to 2020, the percentage of acute intravitreal anti-VEGF injections decreased significantly (7.9% vs. 1.3%, χ2 = 3.985, p = 0.045), but the proportion of other emergency surgeries did not differ between the two years (χ2 ≤ 3.617, p ≥ 0.057). COVID-19 PCR examination was performed in 66 (57.4%) cases in 2020 and all samples (100%) were negative. CONCLUSIONS: The COVID pandemic did not change emergency inpatient volume in our department, but duration of hospital stay was extended on average by 8 hours, mainly due to additional COVID-19-PCR examinations. The proportion of the most frequently performed surgeries did not change remarkably between 2019 and 2020, but with the introduction of Muraine's sutures in 2019, the percentage of admissions with acute corneal hydrops (with or without subsequent surgery) increased for 2020. No urgent surgery had to be postponed due to the COVID-19 pandemic at our department; all operations were performed successfully.

Protection strategy against outbreak of COVID-19 at a tertiary hematology-oncology: strengths and pitfalls.

Due to the worldwide COVID-19 outbreak it is mandatory for health care workers to develop containment strategies. Recently published data showed, that cancer patients might have a higher risk for severe course of the disease. We therefore developed a strategy of screening and containment for SARS-CoV-2 for hospitalized cancer patients. Our approach includes a temporary isolation in a so-called floating zone and testing strategy for screening of asymptomatic individuals by pooling of samples before RT-PCR amplification. Patients as far as health care professionals got tested twice a week. Nurses and physicians entered the floating zone with full body protection. Within 8 weeks we tested 418 individuals (professionals and patients) in total. Only 2 patients had COVID-19 without documented further transmission of SARS-CoV-2. We therefore think that our strategy might be a useful approach to protect inpatients with cancer at high risk for SARS-CoV-2 infection during this ongoing pandemic.

Challenging the prognostic impact of the new WHO and TNM classifications with special emphasis on HPV status in penile carcinoma.

The evidence concerning prognostic parameters for clinical decision-making in penile cancer is either weak or missing. We therefore analysed the prognostic value of the revised TNM and WHO classification systems on relapse and survival with special emphasis on HPV status. We collected clinical data and tissue samples of 121 patients from centres in Germany and Russia. HPV genotyping and p16INK4a immunostaining were performed. The histological subtype and TNM were reclassified by two experienced uropathologists. Survival analyses were performed by Kaplan-Meier estimator and log-rank test. Uni- and multivariable analyses were performed by Cox proportional hazard model and Fisher's exact test for contingency analysis. HPV status was not found to be an independent prognostic factor. Histological subtypes differ in prognosis with the best outcome found in warty and the worst in basaloid carcinomas. Patients with pT1b defined by poor differentiation or lymphovascular invasion (LVI) had the shortest metastasis-free survival compared with pT1a (log-rank, p = 0.02). Lymph node metastasis and LVI were significantly associated with poor metastasis-free, cancer-specific and overall survival and could be identified as the only independent prognostic parameters. Prognostic value of TNM could not be improved using the 8th versus the 7th edition. In contrast to HPV status, histological subtypes are of prognostic value and should be an essential part of pathologic reports. The impact of the HPV status needs to be analysed in a subtype-specific manner. Parameters describing lymphatic dissemination have the highest impact on prognosis. Inclusion of tumour grade and LVI into a single T-category (pT1b) seems questionable.

Expression of 3q oncogene SEC62 in atypical fibroxanthoma-immunohistochemical analysis of 41 cases and correlation with clinical, viral and histopathologic features.

Atypical fibroxanthoma (AFX) is a rare mesenchymal tumor with predominance in older male patients located mainly in chronically UV-exposed skin. Differentiation from clinically more aggressive pleomorphic dermal sarcoma (PDS) is still under debate and immunohistochemical markers are not available yet. An immunohistochemical study, including 41 cases of AFX was conducted to investigate the expression of 3q encoded oncogene SEC62 in AFX and determine the associations with histomorphologic, clinical and viral parameters. Our cohort displayed a mean of 79.9 years at the onset of the disease. In total, 90.2% (37/41) AFXs were located in the head and neck area, whereas, four were located at the extremities (9.7%). Tumor diameter ranged between 0.06 and 40 cm2 with a mean of 5.7 cm2. SEC62 expression was markedly increased in lesional tissue compared with the adjacent healthy squamous epithelium. We found significantly higher expression of SEC62 in cases of AFX with tumor necrosis. Tendency of higher Sec62-IRS-scores were found for tumors with higher Clark levels and a tumor size >5 cm2. Sec62 is involved in endoplasmic reticulum stress tolerance and cell migration, and has been identified as a novel prognostic marker for non-small cell lung cancer as well as head and neck squamous cell carcinoma. For the first time, to the best of our knowledge, we suggest a role of 3q oncogene SEC62 in AFX and discuss a potential prognostic relevance in cases of disputable AFX with unfavorable histomorphologic features and may initiate a discussion on Sec62 serving as discriminating marker between AFX and PDS.

Effect of 3q oncogenes SEC62 and SOX2 on lymphatic metastasis and clinical outcome of head and neck squamous cell carcinomas.

Chromosome 3q26 amplification represents a frequent alteration in head and neck squamous cell carcinomas (HNSCCs). Overexpression of 3q26 encoded genes SEC62 and SOX2 was detected in various cancers, including HNSCCs, indicating their potential function as oncogenes. In our study, we elucidated the function of SEC62 and SOX2 in HNSCC patients, with a main focus on their effect on lymphatic metastasis and patient survival. We analyzed SEC62 and SOX2 expression in tissue specimens from 65 HNSCC patients and 29 patients with cervical cancer of unknown primary (CUP); a higher SEC62 and lower SOX2 expression was observed in the lymph node metastases from HNSCC patients compared with the respective primary tumor. Lymph node metastases from CUP patients showed higher SEC62 and lower SOX2 expression compared with lymph node metastases from HNSCC patients. When proceeding from the N1 to the N3 stage, SEC62 expression in the lymph node metastases showed an increase and SOX2 expression showed a decrease. Moreover, both genes showed a highly significant relevance as prognostic biomarkers, with the worst prognosis for patients with high SEC62 and low SOX2 expression levels. In functional analyses, knockdown of SEC62 resulted in an inhibition of HNSCC cell migration while, conversely, SEC62 and SOX2 overexpression stimulated cell migration. Taken together, our study showed that the expression of the 3q oncogenes SEC62 and SOX2 affects lymphatic metastasis and cell migration in HNSCC and CUP patients and has a high prognostic relevance in these diseases.

Fatal multiorgan failure associated with disseminated herpes simplex virus-1 infection: a case report.

Herpes simplex virus type 1 (HSV-1) infections cause typical dermal and mucosal lesions in children and adults. Also complications to the peripheral and central nervous system, pneumonia or hepatitis are well known. However, dissemination to viscera in adults is rare and predominantly observed in immunocompromised patients. Here we describe the case of a 70-year-old male admitted with macrohematuria and signs of acute infection and finally deceasing in a septic shock with multi organ failure 17 days after admission to intensive care unit. No bacterial or fungal infection could be detected during his stay, but only two days before death the patient showed signs of rectal, orolabial and genital herpes infection. The presence of HSV-1 was detected in swabs taken from the lesions, oropharyngeal fluid as well as in plasma. Post-mortem polymerase chain reaction analyses confirmed a disseminated infection with HSV-1 involving various organs and tissues but excluding the central nervous system. Autopsy revealed a predominantly retroperitoneal diffuse large B-cell lymphoma as the suspected origin of immunosuppression underlying herpes simplex dissemination.

The NP9 protein encoded by the human endogenous retrovirus HERV-K(HML-2) negatively regulates gene activation of the Epstein-Barr virus nuclear antigen 2 (EBNA2).

Epstein-Barr virus (EBV) is a human tumour virus that efficiently growth-transforms primary human B-lymphocytes in vitro. The viral nuclear antigen 2 (EBNA2) is essential for immortalisation of B-cells and stimulates viral and cellular gene expression through interaction with DNA-bound transcription factors. Like its cellular homologue Notch, it associates with the DNA-bound repressor RBPJκ (CSL/CBF1) thereby converting RBPJκ into the active state. For instance, both EBNA2 and Notch activate the cellular HES1 promoter. In EBV-transformed lymphocytes, the RNA of the NP9 protein encoded by human endogenous retrovirus HERV-K(HML-2) Type 1 is strongly up-regulated. The NP9 protein is detectable both in EBV-positive Raji cells, a Burkitt's lymphoma cell line, and in IB4, an EBV-transformed human lymphoblastoid cell line. NP9 binds to LNX that forms a complex with the Notch regulator Numb. Therefore, the function of NP9 vis-à-vis Notch and EBNA2 was analysed. Here, we show that NP9 binds to EBNA2 and negatively affects the EBNA2-mediated activation of the viral C- and LMP2A promoters. In contrast, NP9 did neither interfere in the activation of the HES1 promoter by Notch nor the induction of the viral LMP1 promoter by EBNA2. In an electrophoretic mobility shift analysis, NP9 reduced the binding of EBNA2 to DNA-bound RBPJκ by about 50%. The down-regulation of EBNA2-activity by NP9 might represent a cellular defence mechanism against viral infection or could, alternatively, represent an adaptation of the virus to prevent excessive viral protein production that might otherwise be harmful for the infected cell.

Identification of novel Epstein-Barr virus microRNA genes from nasopharyngeal carcinomas.

MicroRNAs (miRNAs) represent a conserved class of small noncoding RNAs that are found in all higher eukaryotes as well as some DNA viruses. miRNAs are 20 to 25 nucleotides in length and have important regulatory functions in biological processes such as embryonic development, cell differentiation, hormone secretion, and metabolism. Furthermore, miRNAs have been implicated in the pathology of various diseases, including cancer. miRNA expression profiles not only classify different types of cancer but also may even help to characterize distinct tumor stages, therefore constituting a valuable tool for prognosis. Here we report the miRNA profile of Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) tissue samples characterized by cloning and sequencing. We found that all EBV miRNAs from the BART region are expressed in NPC tissues, whereas EBV miRNAs from the BHRF1 region are not found. Moreover, we identified two novel EBV miRNA genes originating from the BART region that have not been found in other tissues or cell lines before. We also identified three new human miRNAs which might be specific for nasopharyngeal tissues. We further show that a number of different cellular miRNAs, including miR-15a and miR-16, are up- or downregulated in NPC tissues compared to control tissues. We found that the tumor suppressor BRCA-1 is a target of miR-15a as well as miR-16, suggesting a miRNA role in NPC pathogenesis.

Epstein-Barr virus-encoded microRNA miR-BART2 down-regulates the viral DNA polymerase BALF5.

MicroRNAs (miRNAs) have been implicated in sequence-specific cleavage, translational repression or deadenylation of specific target mRNAs resulting in post-transcriptional gene silencing. Epstein-Barr virus (EBV) encodes 23 miRNAs of unknown function. Here we show that the EBV-encoded miRNA miR-BART2 down-regulates the viral DNA polymerase BALF5. MiR-BART2 guides cleavage within the 3'-untranslated region (3'UTR) of BALF5 by virtue of its complete complementarity to its target. Induction of the lytic viral replication cycle results in a reduction of the level of miR-BART2 with a strong concomitant decrease of cleavage of the BALF5 3'UTR. Expression of miR-BART2 down-regulates the activity of a luciferase reporter gene containing the BALF5 3'UTR. Forced expression of miR-BART2 during lytic replication resulted in a 40-50% reduction of the level of BALF5 protein and a 20% reduction of the amount of virus released from EBV-infected cells. Our results are compatible with the notion that EBV-miR-BART2 inhibits transition from latent to lytic viral replication.

Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) induces the expression of the cellular microRNA miR-146a.

MicroRNAs (miRNAs) are involved in sequence-specific cleavage, translational repression or deadenylation of specific target mRNAs resulting in post-transcriptional gene silencing. Epstein-Barr Virus (EBV) infection induces cellular non-coding (nc)RNAs e.g., the "vault" RNAs or miRNAs such as miR-21, miR-155 or miR-146a. MiR-146a is upregulated in various tumours and plays a role in innate immunity. We show that the EBV-encoded latent membrane protein 1 (LMP1) induces the expression of miR-146a via NFkappaB. LMP1 activates the miR-146a promoter but not a promoter with a mutation of the NFkappaB-response elements. Conversely, a LMP1-mutant deficient in NFkappaB-activation failed to activate the promoter. The "CAO"-LMP1 variant which has an increased potential to induce NFkappaB also showed a higher ability to activate the miR-146a promoter as compared to standard B95.8-LMP1. Northern blotting revealed high levels of miR-146a and miR-155 in the Burkitt's lymphoma cell line Jijoye which expresses LMP1 while the LMP1-deficient P3HR1 mutant derived from Jijoye expresses less miR-146a or miR-155. Likewise, EBV-latency type I Burkitt's lymphoma cells with low LMP1 levels also contain low levels of either miR-146a or miR-155 while their levels are increased in LMP1-expressing EBV-latency type III BL cells. Expression of LMP1 in P3HR1 cells upregulates miR-146a levels. Neither miR-146a nor miR-155 are detectable in BCBL-1 cells transformed by the Kaposi-Sarcoma Herpes virus (KSHV/HHV8). It is possible that the induction of miR-146a plays a role in the induction or maintenance of EBV latency by modulating innate immune responses to the virus infected host cell.

Biochemical characterisation of the proteins encoded by the DiGeorge critical region 6 (DGCR6) genes.

The DiGeorge critical region 6 (DGCR6) gene exists in two highly homologous copies (DGCR6 and DGCR6L) on chromosome 22q11 and is deleted in patients with velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS). The DGCR6 mRNA levels are increased in metastatic mammary tumour cells and regulate the expression of neighbouring genes at the 22q11 region. Newly developed monoclonal antibodies detected predominantly nuclear phosphoproteins of approximately 25 kDa, with low expression levels in the cytoplasm. Both proteins have half-lives of about 2.5 h. Exogenously expressed DGCR6 and DGCR6L migrated with slightly different mobility in SDS-gels in accordance with two immunoreactive bands observed for the endogenous proteins. DGCR6 is found at low levels in primary human fibroblasts or peripheral blood mononuclear cells, while tumour cells, B-cells transformed by EBV as well as activated normal human T cells, contain elevated levels of the proteins. The proteins are differentially expressed in mammalian tissues, with high protein levels in heart, liver and skeletal muscle. These observations are important as some patients with DGCR6 syndrome exhibit a T-cell deficiency and/or cardiac malformations. As the DGCR6 protein(s) influence gene expression in trans, we analysed the influence of DGCR6/DGCR6L on the Epstein-Barr virus-encoded oncoproteins EBNA2 and EBNA3c in the activation of the viral LMP1 promoter, as well as LMP1-mediated activation of NFkB, but found no effect in either setting.