Histologische, immunhistochemische und molekularbiologische Charakterisierung retrovirusinduzierter, muriner hämatopoietischer Neoplasien

Histological, immunhistochemical and molecular biological characterisation of retrovirus-induced murine haematopoietic neoplasms The murine leukaemia virus Akv has lymphomagenic properties in newborn inbred NMRI mice. The enhancer in the U3 long terminal repeat sequence of the Akv genome is a major determinant of retroviral lymphomagenesis. In this study, the pathogenicity of wild-type Akv, Akv1-99, an Akv lacking one 99 bp repeat in the U3 sequence, and a panel of ten Akv1-99 mutants containing mutations in the different transcription factor binding sites, was analysed. The retrovirus-induced haematopoietic tumours were classified according to the Bethesda Proposals (94, 138), based on histological and immunohistochemical analyses, and IgH gene rearrangements. All viruses were pathogenic. Tumour incidence ranged from 90 % to 100 %. The mutant viruses, however, differed in their latency periods and tissue specificity. Akv induced diffuse large B-cell lymphomas (32.5 %), follicular B-cell lymphomas (22.5 %), splenic marginal zone lymphomas (5 %), small B-cell lymphomas (5 %), and plasmacytomas (35 %). The mutant viruses induced almost exclusively plasmacytomas (up to 100 %). The latency periods of three mutant viruses were comparable to that of Akv; six mutant viruses showed a shorter latency (five latency periods were significantly shorter), and two mutant viruses with an additional mutation in the EGRE binding site induced tumours after significantly prolonged latency. These results suggest that mutations of transcription factor binding sites in the Akv enhancer maintained the pathogenicity of the viruses but resulted in a smaller spectrum of haematopoietic tumours with different impact on the latency. Comprehensive histological, immunohistochemical, and molecular analyses of 323 retrovirus-induced haematopoietic tumours (83.8 % plasmacytomas, 6.9 % diffuse large B-cell lymphomas, 4.5 % follicular B-cell lymphomas, 4.2 % splenic marginal zone lymphomas, 1.2 % small B-cell lymphomas, 1.2 % acute myeloid leukaemias, and 0.9 % precursor-T-cell lymphoblastic lymphomas) illustrated the importance of transcription factor binding sites of Akv and its impact on viral virulence and disease specificity. This model facilitates the dissection of disease-determining sequences in the retroviral genome and discloses differences and similarities between human and murine haematopoietic neoplasms.Das murine Leukämievirus Akv erzeugt in neugeborenen NMRI-Mäusen Lymphome. Der innerhalb des Long Termial Repeats in der U3-Sequenz lokalisierte Enhancer von Akv, wird als wesentlicher Regulator der Tumorinduktion angesehen. In dieser Studie wurde die Pathogenität des Wildtyp Akv, des Akv1-99 (einem Akv mit nur einer 99 bp langen Sequenz in der U3-Region) sowie von zehn Akv1-99 Mutanten, die zusätzlich eine Reihe von Basensubstituierungen in verschiedenen Transkriptionsfaktor-Bindungsstellen aufwiesen, analysiert. Nach der Etablierung eines immunhistochemischen Antikörperpanels zur Diagnostik muriner hämatopoietischer Neoplasien und der Etablierung der auf einer PCR basierenden IgH-Fragmentanalyse, wurden die retrovirusinduzierten Tumore entsprechend den "Bethesda Proposals" (94, 138) klassifiziert. Alle untersuchten Viren waren pathogen, jedoch zeigten die Mutanten veränderte Latenzzeiten und Gewebsspezifität. Akv induzierte diffus großzellige B-Zell-Lymphome (32,5 %), follikuläre B-Zell-Lymphome (22,5 %), splenische Marginalzonen-Lymphome (5 %), kleinzellige B-Zell-Lymphome und Plasmozytome (35 %). Virusmutanten induzierten fast ausschließlich Plasmozytome (bis 100 %). Drei Mutanten hatten eine dem Wildtypvirus vergleichbare Latenzzeit. Sechs Mutanten zeigten eine kürzere Latenzzeit als das Wildtypvirus (davon waren bei fünf Mutanten die Latenzzeiten signifikant kürzer). Beide Doppelmutanten, die den Bereich EGRE mit einschlossen, zeigten, im Vergleich zum Wildtypvirus, eine signifikant verlängerte Latenzzeit. Die histologische, immunhistochemische und molekularbiologische Charakterisierung der 323 analysierten hämatopoietischen Neoplasien erbrachte 83,8 % Plasmozytome, 6,9 % diffus großzellige B-Zell-Lymphome, 4,5 % follikuläre B-Zell-Lymphome, 4,2 % splenische Marginalzonen-Lymphome, jeweils 1,2 % kleinzellige B-Zell-Lymphome und akute myeloische Leukämien und 0,9 % Vorläufer-T-Zell lymphoblastische Lymphome. Diese Ergebnisse weisen auf die Bedeutung der Transkriptionsfaktor-Bindungsstellen im Enhancer für die Pathogenität und Gewebsspezifität von Akv hin. Dieses Modell ist geeignet, Gemeinsamkeiten und Unterschiede zwischen murinen und humanen hämatopoietischen Neoplasien und deren Klassifikation detailliert herauszuarbeiten

Impairment of alternative splice sites defining a novel gammaretroviral exon within gag modifies the oncogenic properties of Akv murine leukemia virus

<p>Abstract</p> <p>Background</p> <p>Mutations of an alternative splice donor site located within the <it>gag </it>region has previously been shown to broaden the pathogenic potential of the T-lymphomagenic gammaretrovirus Moloney murine leukemia virus, while the equivalent mutations in the erythroleukemia inducing Friend murine leukemia virus seem to have no influence on the disease-inducing potential of this virus. In the present study we investigate the splice pattern as well as the possible effects of mutating the alternative splice sites on the oncogenic properties of the B-lymphomagenic Akv murine leukemia virus.</p> <p>Results</p> <p>By exon-trapping procedures we have identified a novel gammaretroviral exon, resulting from usage of alternative splice acceptor (SA') and splice donor (SD') sites located in the capsid region of <it>gag </it>of the B-cell lymphomagenic Akv murine leukemia virus. To analyze possible effects <it>in vivo </it>of this novel exon, three different alternative splice site mutant viruses, mutated in either the SA', in the SD', or in both sites, respectively, were constructed and injected into newborn inbred NMRI mice. Most of the infected mice (about 90%) developed hematopoietic neoplasms within 250 days, and histological examination of the tumors showed that the introduced synonymous <it>gag </it>mutations have a significant influence on the phenotype of the induced tumors, changing the distribution of the different types as well as generating tumors of additional specificities such as <it>de novo </it>diffuse large B cell lymphoma (DLBCL) and histiocytic sarcoma. Interestingly, a broader spectrum of diagnoses was made from the two single splice-site mutants than from as well the wild-type as the double splice-site mutant. Both single- and double-spliced transcripts are produced <it>in vivo </it>using the SA' and/or the SD' sites, but the mechanisms underlying the observed effects on oncogenesis remain to be clarified. Likewise, analyses of provirus integration sites in tumor tissues, which identified 111 novel RISs (retroviral integration sites) and 35 novel CISs (common integration sites), did not clearly point to specific target genes or pathways to be associated with specific tumor diagnoses or individual viral mutants.</p> <p>Conclusion</p> <p>We present here the first example of a doubly spliced transcript within the group of gammaretroviruses, and we show that mutation of the alternative splice sites that define this novel RNA product change the oncogenic potential of Akv murine leukemia virus.</p

The Icsbp locus is a common proviral insertion site in mature B-cell lymphomas/plasmacytomas induced by exogenous murine leukemia virus

AbstractICSBP (interferon consensus sequence binding protein)/IRF8 (interferon regulatory factor 8) is an interferon gamma-inducible transcription factor expressed predominantly in hematopoietic cells, and down-regulation of this factor has been observed in chronic myelogenous leukemia and acute myeloid leukemia in man. By screening about 1200 murine leukemia virus (MLV)-induced lymphomas, we found proviral insertions at the Icsbp locus in 14 tumors, 13 of which were mature B-cell lymphomas or plasmacytomas. Only one was a T-cell lymphoma, although such tumors constituted about half of the samples screened. This indicates that the Icsbp locus can play a specific role in the development of mature B-lineage malignancies. Two proviral insertions in the last Icsbp exon were found to act by a poly(A)-insertion mechanism. The remaining insertions were found within or outside Icsbp. Since our results showed expression of Icsbp RNA and protein in all end-stage tumor samples, a simple tumor suppressor function of ICSBP is not likely. Interestingly, proviral insertions at Icsbp have not been reported from previous extensive screenings of mature B-cell lymphomas induced by endogenous MLVs. We propose that ICSBP might be involved in an early modulation of an immune response to exogenous MLVs that might also play a role in proliferation of the mature B-cell lymphomas

An intranasal live-attenuated SARS-CoV-2 vaccine limits virus transmission

The development of effective SARS-CoV-2 vaccines has been essential to control COVID-19, but significant challenges remain. One problem is intramuscular administration, which does not induce robust mucosal immune responses in the upper airways—the primary site of infection and virus shedding. Here we compare the efficacy of a mucosal, replication-competent yet fully attenuated virus vaccine, sCPD9-ΔFCS, and the monovalent mRNA vaccine BNT162b2 in preventing transmission of SARS-CoV-2 variants B.1 and Omicron BA.5 in two scenarios. Firstly, we assessed the protective efficacy of the vaccines by exposing vaccinated male Syrian hamsters to infected counterparts. Secondly, we evaluated transmission of the challenge virus from vaccinated and subsequently challenged male hamsters to naïve contacts. Our findings demonstrate that the live-attenuated vaccine (LAV) sCPD9-ΔFCS significantly outperformed the mRNA vaccine in preventing virus transmission in both scenarios. Our results provide evidence for the advantages of locally administered LAVs over intramuscularly administered mRNA vaccines in preventing infection and reducing virus transmission

A non-transmissible live attenuated SARS-CoV-2 vaccine

Live attenuated vaccines (LAVs) administered via the mucosal route may offer better control of the COVID-19 pandemic than non-replicating vaccines injected intramuscularly. Conceptionally, LAVs have several advantages, including presentation of the entire antigenic repertoire of the virus, and the induction of strong mucosal immunity. Thus, immunity induced by LAV could offer superior protection against future surges of COVID-19 cases caused by emerging SARS-CoV-2 variants. However, LAVs carry the risk of unintentional transmission. To address this issue, we investigated whether transmission of a SARS-CoV-2 LAV candidate can be blocked by removing the furin cleavage site (FCS) from the spike protein. The level of protection and immunity induced by the attenuated virus with the intact FCS was virtually identical to the one induced by the attenuated virus lacking the FCS. Most importantly, removal of the FCS completely abolished horizontal transmission of vaccine virus between cohoused hamsters. Furthermore, the vaccine was safe in immunosuppressed animals and showed no tendency to recombine in vitro or in vivo with a SARS-CoV-2 field strain. These results indicate that removal of the FCS from SARS-CoV-2 LAV is a promising strategy to increase vaccine safety and prevent vaccine transmission without compromising vaccine efficacy

Diverse Hematological Malignancies Including Hodgkin-Like Lymphomas Develop in Chimeric MHC Class II Transgenic Mice

A chimeric HLA-DR4-H2-E (DR4) homozygous transgenic mouse line spontaneously develops diverse hematological malignancies with high frequency (70%). The majority of malignancies were distributed equally between T and B cell neoplasms and included lymphoblastic T cell lymphoma (LTCL), lymphoblastic B cell lymphoma (LBCL), diffuse large B cell lymphoma (DLBCL), the histiocyte/T cell rich variant of DLBCL (DLBCL-HA/T cell rich DLBCL), splenic marginal zone lymphoma (SMZL), follicular B cell lymphoma (FBL) and plasmacytoma (PCT). Most of these neoplasms were highly similar to human diseases. Also, some non-lymphoid malignancies such as acute myeloid leukemia (AML) and histiocytic sarcoma were found. Interestingly, composite lymphomas, including Hodgkin-like lymphomas, were also detected that had CD30+ Hodgkin/Reed-Sternberg (H/RS)-like cells, representing a tumor type not previously described in mice. Analysis of microdissected H/RS-like cells revealed their origin as germinal center B cells bearing somatic hypermutations and, in some instances, crippled mutations, as described for human Hodgkin lymphoma (HL). Transgene integration in an oncogene was excluded as an exclusive driving force of tumorigenesis and age-related lymphoma development suggests a multi-step process. Thus, this DR4 line is a useful model to investigate common molecular mechanisms that may contribute to important neoplastic diseases in man

Single-cell-resolved interspecies comparison shows a shared inflammatory axis and a dominant neutrophil-endothelial program in severe COVID-19

A key issue for research on COVID-19 pathogenesis is the lack of biopsies from patients and of samples at the onset of infection. To overcome these hurdles, hamsters were shown to be useful models for studying this disease. Here, we further leverage the model to molecularly survey the disease progression from time-resolved single-cell RNA sequencing data collected from healthy and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected Syrian and Roborovski hamster lungs. We compare our data to human COVID-19 studies, including bronchoalveolar lavage, nasal swab, and postmortem lung tissue, and identify a shared axis of inflammation dominated by macrophages, neutrophils, and endothelial cells, which we show to be transient in Syrian and terminal in Roborovski hamsters. Our data suggest that, following SARS-CoV-2 infection, commitment to a type 1- or type 3-biased immunity determines moderate versus severe COVID-19 outcomes, respectively

Mutation of All Runx (AML1/Core) Sites in the Enhancer of T-Lymphomagenic SL3-3 Murine Leukemia Virus Unmasks a Significant Potential for Myeloid Leukemia Induction and Favors Enhancer Evolution toward Induction of Other Disease Patterns

SL3-3 murine leukemia virus is a potent inducer of T-lymphomas in mice. Using inbred NMRI mice, it was previously reported that a mutant of SL3-3 with all enhancer Runx (AML1/core) sites disrupted by 3-bp mutations (SL3-3dm) induces predominantly non-T-cell tumors with severely extended latency (S. Ethelberg, J. Lovmand, J. Schmidt, A. Luz, and F. S. Pedersen, J. Virol. 71:7273-7280, 1997). By use of three-color flow cytometry and molecular and histopathological analyses, we have now performed a detailed phenotypic characterization of SL3-3- and SL3-3dm-induced tumors in this mouse strain. All wild-type induced tumors had clonal T-cell receptor β rearrangements, and the vast majority were CD3(+) CD4(+) CD8(−) T-lymphomas. Such a consistent phenotypic pattern is unusual for murine leukemia virus-induced T-lymphomas. The mutant virus induced malignancies of four distinct hematopoietic lineages: myeloid, T lymphoid, B lymphoid, and erythroid. The most common disease was myeloid leukemia with maturation. Thus, mutation of all Runx motifs in the enhancer of SL3-3 severely impedes viral T-lymphomagenicity and thereby discloses a considerable and formerly unappreciated potential of this virus for myeloid leukemia induction. Proviral enhancers with complex structural alterations (deletions, insertions, and/or duplications) were found in most SL3-3dm-induced T-lymphoid tumors and immature myeloid leukemias but not in any cases of myeloid leukemia with maturation, mature B-lymphoma, or erythroleukemia. Altogether, our results indicate that the SL3-3dm enhancer in itself promotes induction of myeloid leukemia with maturation but that structural changes may arise in vivo and redirect viral disease specificity to induction of T-lymphoid or immature myeloid leukemias, which typically develop with moderately shorter latencies

Northern blot hybridizations with an ecotropic specific probe and a probe of RNA isolated from NIH 3T3 cells chronically infected with the viruses listed above each lane

<p><b>Copyright information:</b></p><p>Taken from "Impairment of alternative splice sites defining a novel gammaretroviral exon within modifies the oncogenic properties of Akv murine leukemia virus"</p><p>http://www.retrovirology.com/content/4/1/46</p><p>Retrovirology 2007;4():46-46.</p><p>Published online 6 Jul 2007</p><p>PMCID:PMC1936429.</p><p></p> The sizes of the full-length transcript (unspliced) and the single-spliced transcript are indicated at the left. The arrow indicates splice product C. For verification of integrity and concentration of the loaded RNA, the original ethidium bromide stained agarose gel exposing 18S and 28S rRNAs is shown below

Pathogenicity of Akv and derived splice site mutants in inbred NMRI mice

<p><b>Copyright information:</b></p><p>Taken from "Impairment of alternative splice sites defining a novel gammaretroviral exon within modifies the oncogenic properties of Akv murine leukemia virus"</p><p>http://www.retrovirology.com/content/4/1/46</p><p>Retrovirology 2007;4():46-46.</p><p>Published online 6 Jul 2007</p><p>PMCID:PMC1936429.</p><p></p> Shown are the cumulative incidences of tumor development related to age of injected mice (in days)