Human lung extracellular matrix hydrogels resemble the stiffness and viscoelasticity of native lung tissue.

Chronic lung diseases such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) are associated with changes in extracellular matrix (ECM) composition and abundance affecting the mechanical properties of the lung. This study aimed to generate ECM hydrogels from control, severe COPD [Global Initiative for Chronic Obstructive Lung Disease (GOLD) IV], and fibrotic human lung tissue and evaluate whether their stiffness and viscoelastic properties were reflective of native tissue. For hydrogel generation, control, COPD GOLD IV, and fibrotic human lung tissues were decellularized, lyophilized, ground into powder, porcine pepsin solubilized, buffered with PBS, and gelled at 37°C. Rheological properties from tissues and hydrogels were assessed with a low-load compression tester measuring the stiffness and viscoelastic properties in terms of a generalized Maxwell model representing phases of viscoelastic relaxation. The ECM hydrogels had a greater stress relaxation than tissues. ECM hydrogels required three Maxwell elements with slightly faster relaxation times (τ) than that of native tissue, which required four elements. The relative importance (Ri) of the first Maxwell element contributed the most in ECM hydrogels, whereas for tissue the contribution was spread over all four elements. IPF tissue had a longer-lasting fourth element with a higher Ri than the other tissues, and IPF ECM hydrogels did require a fourth Maxwell element, in contrast to all other ECM hydrogels. This study shows that hydrogels composed of native human lung ECM can be generated. Stiffness of ECM hydrogels resembled that of whole tissue, while viscoelasticity differed.

Extracellular Vesicles from Adipose Tissue-Derived Stromal Cells Stimulate Angiogenesis in a Scaffold-Dependent Fashion.

BACKGROUND: The extracellular vesicles (EVs) secreted by adipose tissue-derived stromal cells (ASC) are microenvironment modulators in tissue regeneration by releasing their molecular cargo, including miRNAs. However, the influence of ASC-derived extracellular vesicles (ASC-EVs) on endothelial cells (ECs) and vascularisation is poorly understood. The present study aimed to determine the pro-angiogenic effects of ASC-EVs and explore their miRNA profile. METHODS: EVs were isolated from normoxic and hypoxic cultured ASC conditioned culture medium. The miRNA expression profile was determined by miRseq, and EV markers were determined by Western blot and immunofluorescence staining. The uptake dynamics of fluorescently labelled EVs were monitored for 24 h. ASC-EVs' pro-angiogenic effect was assessed by sprouting ex vivo rat aorta rings in left ventricular-decellularized extracellular matrix (LV dECM) hydrogel or basement membrane hydrogel (Geltrex®). RESULTS: ASC-EVs augmented vascular network formation by aorta rings. The vascular network topology and stability were influenced in a hydrogel scaffold-dependent fashion. The ASC-EVs were enriched for several miRNA families/clusters, including Let-7 and miR-23/27/24. The miRNA-1290 was the highest enriched non-clustered miRNA, accounting for almost 20% of all reads in hypoxia EVs. CONCLUSION: Our study revealed that ASC-EVs augment in vitro and ex vivo vascularisation, likely due to the enriched pro-angiogenic miRNAs in EVs, particularly miR-1290. Our results show promise for regenerative and revascularisation therapies based on ASC-EV-loaded ECM hydrogels.

Angiogenesis is promoted by hypoxic cervical carcinoma-derived extracellular vesicles depending on the endothelial cell environment.

INTRODUCTION: Cancer needs perfusion for its growth and metastasis. Cancer cell-derived extracellular vesicles (CA-EVs) alter the tumor microenvironment (TME), potentially promoting angiogenesis. We hypothesize that conditions in the tumor, e.g., hypoxia, and in the target cells of the TME, e.g., nutrient deprivation or extracellular matrix, can affect the angiogenic potential of CA-EVs, which would contribute to explaining the regulation of tumor vascularization and its influence on cancer growth and metastasis. METHODS: CA-EVs were isolated and characterized from cervical carcinoma cell lines HeLa and SiHa cultured under normoxia and hypoxia, and their angiogenic potential was evaluated in vitro in three endothelial cells (ECs) lines and aortic rings, cultured in basal (growth factor-reduced) or complete medium. RESULTS: Hypoxia increased EV production 10-100 times and protein content 2-4 times compared to normoxic CA-EVs. HeLa-EVs contained six times more RNA than SiHa-EVs, and this concentration was not affected by hypoxia. Treatment with CA-EVs increased tube formation and sprouting in ECs and aortic rings cultured in basal medium and long-term stabilized the stablished vascular networks formed by ECs cultured in complete medium. CONCLUSION: Hypoxia differentially affects CA-EVs in a cell line-dependent manner. The cellular environment (nutrient availability and extracellular matrix scaffold) influences the effect of CA-EV on the angiogenic potential of ECs.

Endothelial plasticity across PTEN and Hippo pathways: A complex hormetic rheostat modulated by extracellular vesicles.

Vascularization is a multifactorial and spatiotemporally regulated process, essential for cell and tissue survival. Vascular alterations have repercussions on the development and progression of diseases such as cancer, cardiovascular diseases, and diabetes, which are the leading causes of death worldwide. Additionally, vascularization continues to be a challenge for tissue engineering and regenerative medicine. Hence, vascularization is the center of interest for physiology, pathophysiology, and therapeutic processes. Within vascularization, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and Hippo signaling have pivotal roles in the development and homeostasis of the vascular system. Their suppression is related to several pathologies, including developmental defects and cancer. Non-coding RNAs (ncRNAs) are among the regulators of PTEN and/or Hippo pathways during development and disease. The purpose of this paper is to review and discuss the mechanisms by which exosome-derived ncRNAs modulate endothelial cell plasticity during physiological and pathological angiogenesis, through the regulation of PTEN and Hippo pathways, aiming to establish new perspectives on cellular communication during tumoral and regenerative vascularization.

Hyaluronic acid-recombinant gelatin gels as a scaffold for soft tissue regeneration.

An array of different types of hyaluronic acid (HA)- and collagen-based products is available for filling soft-tissue defects. A major drawback of the current soft-tissue fillers is their inability to induce cell infiltration and new tissue formation. Our aim is to develop novel biodegradable injectable gels which induce soft tissue regeneration, initially resulting in integration and finally replacement of the gel with new autologous tissue. Two reference gels of pure HA, monophasic HA-1 and micronised HA-2, were used. Furthermore, both gels were mixed with recombinant gelatin (RG) resulting in HA-1+RG and HA-2+RG. All gels were subcutaneously injected on the back of rats and explanted after 4 weeks. Addition of RG to HA-1 resulted in stroma formation (neovascularisation and ECM deposition) which was restricted to the outer rim of the HA-1+RG gel. In contrast, addition of RG to HA-2 induced stroma formation throughout the gel. The RG component of the gel was degraded by macrophages and giant cells and subsequently replaced by new vascularised tissue. Immunohistochemical staining showed that the extracellular matrix components collagen I and III were deposited throughout the gel. In conclusion, this study shows the proof of principle that addition of RG to HA-2 results in a novel injectable gel capable of inducing soft tissue regeneration. In this gel HA has a scaffold function whereas the RG component induces new tissue formation, resulting in proper vascularisation and integration of the HA-2+RG gel with the autologous tissue.

High stretch induces endothelial dysfunction accompanied by oxidative stress and actin remodeling in human saphenous vein endothelial cells.

The rate of the remodeling of the arterialized saphenous vein conduit limits the outcomes of coronary artery bypass graft surgery (CABG), which may be influenced by endothelial dysfunction. We tested the hypothesis that high stretch (HS) induces human saphenous vein endothelial cell (hSVEC) dysfunction and examined candidate underlying mechanisms. Our results showed that in vitro HS reduces NO bioavailability, increases inflammatory adhesion molecule expression (E-selectin and VCAM1) and THP-1 cell adhesion. HS decreases F-actin in hSVECs, but not in human arterial endothelial cells, and is accompanied by G-actin and cofilin's nuclear shuttling and increased reactive oxidative species (ROS). Pre-treatment with the broad-acting antioxidant N-acetylcysteine (NAC) supported this observation and diminished stretch-induced actin remodeling and inflammatory adhesive molecule expression. Altogether, we provide evidence that increased oxidative stress and actin cytoskeleton remodeling play a role in HS-induced saphenous vein endothelial cell dysfunction, which may contribute to predisposing saphenous vein graft to failure.

Endo180 and MT1-MMP are involved in the phagocytosis of collagen scaffolds by macrophages and is regulated by interferon-gamma.

Subcutaneously implanted disks of hexamethylenediisocyanate or glutaraldehyde cross-linked sheep collagen (referred to as HDSC and GDSC, respectively) in mice show large differences in degradation rate. Although comparable numbers of macrophages are seen in HDSC and GDSC, phagocytosis of collagen by macrophages occurred only in GDSC. The molecular mechanisms involved in the phagocytosis of collagen by macrophages are essentially unknown. Immunofluorescence and RT-PCR showed that Endo180 was expressed in GDSC only. TissueFaxs showed that Endo180 co-localized with MT1-MMP on F4÷80 positive cells, which is likely responsible for the phagocytosis in GDSC. RT-PCR further showed that Endo180 expression correlated with high levels of IFN-γ mRNA. In vitro, IFN-γ induced the expression Endo180 and MT1-MMP in murine macrophages cultured on collagen type I (although too high levels of IFN-γ dampened the expression of Endo180 and MT1-MMP). Moreover, the expression of Endo180 and MT1-MMP induced by IFN-γ can be inhibited through IL-10. The differences in microenvironment between GDSC and HDSC (high IFN-γ and low IL-10 levels in GDSC, low IFN-γ and high IL-10 levels in HDSC) provide an explanation why phagocytosis of collagen by macrophages is only seen in GDSC. In summary, we show for the first time that the IFN-γ dependent co-expression of Endo180 and MT1-MMP on macrophages coincides with collagen phagocytosis, thus providing evidence that the mechanism of collagen phagocytosis operating in the foreign body reaction by macrophages is comparable with the mechanism of intracellular collagen degradation by fibroblasts seen under physiological conditions.

CD34+ cells augment endothelial cell differentiation of CD14+ endothelial progenitor cells in vitro.

Neovascularization by endothelial progenitor cells (EPC) for the treatment of ischaemic diseases has been a topic of intense research. The CD34(+) cell is often designated as EPC, because it contributes to repair of ischaemic injuries through neovascularization. However, incorporation of CD34(+) cells into the neovasculature is limited, suggesting another role which could be paracrine. CD14(+) cells can also differentiate into endothelial cells and contribute to neovascularization. However, the low proliferative capacity of CD14(+) cell-derived endothelial cells hampers their use as therapeutic cells. We made the assumption that an interaction between CD34(+) and CD14(+) cells augments endothelial differentiation of the CD14(+) cells. In vitro, the influence of CD34(+) cells on the endothelial differentiation capacity of CD14(+) cells was investigated. Endothelial differentiation was analysed by expression of endothelial cell markers CD31, CD144, von Willebrand Factor and endothelial Nitric Oxide Synthase. Furthermore, we assessed proliferative capacity and endothelial cell function of the cells in culture. In monocultures, 63% of the CD14(+)-derived cells adopted an endothelial cell phenotype, whereas in CD34(+)/CD14(+) co-cultures 95% of the cells showed endothelial cell differentiation. Proliferation increased up to 12% in the CD34(+)/CD14(+) co-cultures compared to both monocultures. CD34-conditioned medium also increased endothelial differentiation of CD14(+) cells. This effect was abrogated by hepatocyte growth factor neutralizing antibodies, but not by interleukin-8 and monocyte chemoattractant protein-1 neutralizing antibodies. We show that co-culturing of CD34(+) and CD14(+) cells results in a proliferating population of functional endothelial cells, which may be suitable for treatment of ischaemic diseases such as myocardial infarction.

Expression of EpCAM is up-regulated during regeneration of renal epithelia.

The human epithelial cell adhesion molecule (hEpCAM) is involved in epithelial morphogenesis and repair of epithelial tissues. We hypothesized that changes in hEpCAM expression in vivo correlate with regeneration of renal epithelia after ischaemia/reperfusion injury (IRi). Unilateral IRi was performed on kidneys of hEpCAM transgenic mice. Changes in hEpCAM expression were investigated by quantitative RT-PCR in renal cortex and medulla dissected by laser dissection microscopy and expression patterns of hEpCAM in regenerating kidneys were assessed by immunohistochemistry. The mechanism of hEpCAM promoter activation was investigated in vitro, by real-time bioluminescent imaging in HK-2 cells and in primary tubular epithelial cells (PTECs) subjected to hypoxia and reoxygenation. In vivo, the transcription of the human epcam gene significantly increased in the renal cortex during tubular re-epithelialization (p < 0.01). Moreover, the number of tubuli that expressed hEpCAM protein more than doubled in the renal cortex during regeneration. De novo expression of hEpCAM was detected in the S1 segments of proximal tubuli. Under hypoxic conditions in vitro, activity of the hEpCAM promoter was up-regulated two-fold in the HK-2 proximal epithelial cell line. Moreover, both in primary proximal epithelial cells and in HK-2 cells, hEpCAM protein expression was increased after hypoxia and reoxygenation. The significant up-regulation of hEpCAM during post-ischaemic renal regeneration in vivo and during in vitro hypoxia indicates that hEpCAM expression is associated with renal regeneration.

Perivascular scaffolds loaded with adipose tissue-derived stromal cells attenuate development and progression of abdominal aortic aneurysm in rats.

Abdominal aortic aneurysm (AAA) is the pathological dilation and weakening of the abdominal aorta wall. Inflammation, degradation of the extracellular matrix (ECM) and loss of smooth muscle cells and skewing of their function are pivotal in AAA pathology. We developed a recombinant collagen-based patch (RCP) to provide structural integrity and deliver Adipose tissue-Derived Stromal Cells (ASC) for repair. Patches supported adhesion and function as well as proliferation of ASC. ASC-loaded RCPs or bare patches, applied around the aorta after AAA induction in rats, both maintained structural integrity of the aortic wall at time of explant (2w). However, wall thinning, accompanied by loss of elastin fibers and loss of medial SMC, was only attenuated in ASC-loaded RCP-treated AAA rats. Interestingly, this coincided with migration of ASC into the media and a reduced influx of macrophages. We hypothesize that the medially-migrated ASC dampened or skewed the adverse innate immunity and thus suppressed SMC apoptosis, phenotypic skewing and elastin degradation. We conclude that the periadventitial delivery of ASC with RCP suppresses development and progression of AAA, which is has an expected future clinical benefit in combination with an appropriate early screening program of patients at risk for aneurysms. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2494-2506, 2018.

Material dependent differences in inflammatory gene expression by giant cells during the foreign body reaction.

Multinucleated giant cells (GCs) are often observed in the foreign body reaction against implanted materials. The in vivo function of GCs in this inflammatory process remains to be elucidated. GCs degrade collagen implants in rats and may also orchestrate the inflammatory process via the expression and secretion of modulators, such as cytokines and chemokines. In this study, we show that the gene expression of PMN chemoattractants, CXCL1/KC and CXCL2/MIP-2, is high in GCs micro-dissected from explanted Dacron, cross-linked collagen (HDSC), and bioactive ureido-pyrimidinone functionalized oligocaprolactone (bioactive PCLdiUPy). Conversely, the gene expression levels of TGFbeta and pro-angiogenic mediators VEGF and FGF were found to be low in these GCs as compared with the expression levels in total explants. GCs in bioactive PCLdiUPy displayed high cytokine and angiogenic mediator expression compared with GCs isolated from the two other studied materials, whereas chemokine gene expression in GCs isolated form HDSC was low. Thus, GCs adopt their expression profile in response to the material that is encountered.

The carcinoma-specific epithelial glycoprotein-2 promoter controls efficient and selective gene expression in an adenoviral context.

Adenoviral vectors are widely used in cancer gene therapy. After systemic administration however, the majority of the virus homes to the liver and the expressed transgene may cause hepatotoxicity. To restrict transgene expression to tumor cells, tumor- or tissue-specific promoters are utilized. The tumor antigen epithelial glycoprotein-2 (EGP-2), also known as Ep-CAM, is expressed in many cancers from different epithelial origins. In this study, the EGP-2 promoter was shown to restrict the expression of luciferase and thymidine kinase in an adenoviral context in different cell lines. In vivo, the EGP-2 promoter mediated efficient expression of luciferase in tumors but showed a 3-log lower activity in liver tissue when compared with the cytomegalovirus (CMV) promoter. Similarly, the EGP-2 promoter mediated specific cell killing after ganciclovir treatment in EGP-2-positive cells. Moreover, in vivo, this treatment regiment did not cause any rise in the liver enzymes aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT), demonstrating absence of liver toxicity. In contrast, CMV-mediated expression of thymidine kinase in combination with ganciclovir treatment resulted in high ASAT and ALAT values. This study demonstrates the value of the EGP-2 promoter to restrict transgene expression to a broad range of tumor types, thereby preventing liver toxicity.

Chemokine scavenging by the human cytomegalovirus chemokine decoy receptor US28 does not inhibit monocyte adherence to activated endothelium.

The human cytomegalovirus has found smart ways to exploit the chemokine network in order to subvert immune attack. Chemokines trigger the arrest and firm adhesion of inflammatory cells to the vascular wall. Scavenging of chemokines by viral decoy receptors, such as US28, might prevent arrest of leukocytes to the vascular wall and impair an antiviral immune response. We determined the effect of chemokine scavenging by endothelium-expressed signaling mute US28 (US28R129A) on static monocyte adhesion. Despite the chemokine scavenging capacity of US28R129A, expression of this construct by endothelial cells was insufficient to disrupt leukocyte adhesion to cytokine-activated monolayers. Our results suggest that the concentrations of chemokines that trigger firm leukocyte adhesion are too high to be efficiently scavenged by viral chemokine decoy receptors like US28. From the results of this experimental model a role for US28 in viral immune evasion by chemokine scavenging would appear therefore unlikely.

The epithelial glycoprotein 2 (EGP-2) promoter-driven epithelial-specific expression of EGP-2 in transgenic mice: a new model to study carcinoma-directed immunotherapy.

The human pancarcinoma-associated epithelial glycoprotein-2 (EGP-2), a M(r) 38,000 transmembrane antigen also known as 17-1A or Ep-CAM, is commonly used for targeted immunotherapy of carcinomas because it is strongly expressed by most carcinomas. EGP-2 is, however, also expressed in most normal epithelia. To evaluate anti-EGP-2-directed treatment-associated effects on tumors and on EGP-2-positive normal tissue, we generated EGP-2-expressing transgenic mice. A 55-kb DNA fragment consisting of the 14-kb genomic coding sequence of the human EGP-2 gene with approximately 10-kb-upstream and approximately 31-kb-downstream sequences was isolated and used to direct EGP-2 expression in an epithelium-specific manner. In the EGP-2 transgenic mice, EGP-2 appeared to be specifically expressed in all of those epithelial tissues that also express EGP-2 in humans, whereas all of the other tissues were negative. The specific in vivo localization of the i.v. administered anti-EGP-2 monoclonal antibody MOC31 was studied in EGP-2-positive and -negative tumors induced s.c. in this EGP-2 transgenic mouse model. Immunohistochemical analysis showed specific localization of MOC31 in the EGP-2-positive tumors but not in the EGP-2-negative tumors. No anti-EGP-2 monoclonal antibody localization was observed in any of the EGP-2-positive normal mouse tissues, which indicated a limited in vivo accessibility. In conclusion, an EGP-2 transgenic mouse model has been generated that expresses the EGP-2 antigen as in humans and, therefore, can serve as a model to evaluate the efficacy and safety of a variety of anti-EGP-2-based immunotherapeutic modalities in both tumors and normal tissue.

Cancer immunotherapy: insights from transgenic animal models.

A wide range of strategies in cancer immunotherapy has been developed in the last decade, some of which are currently being used in clinical settings. The development of these immunotherapeutical strategies has been facilitated by the generation of relevant transgenic animal models. Since the different strategies in experimental immunotherapy of cancer each aim to activate different immune system components, a variety of transgenic animals have been generated either expressing tumor associated, HLA, oncogenic or immune effector cell molecule proteins. This review aims to discuss the existing transgenic mouse models generated to study and develop cancer immunotherapy strategies and the variable results obtained. The potential of the various transgenic animal models regarding the development of anti-cancer immunotherapeutical strategies is evaluated.

Cytomegalovirus infection increases the expression and activity of ecto-ATPase (CD39) and ecto-5'nucleotidase (CD73) on endothelial cells.

We describe enhanced expression and enzymatic activity of ecto-ATPase and ecto-5'nucleotidase on CMV infected endothelial cells as compared to uninfected cells. These ectoenzymes play a major role in modulation of platelet activation and aggregation. Furthermore, adenosine has a modulatory effect upon inflammation. Addition of ATP, ADP or AMP to cultures of CMV infected or uninfected endothelial cells revealed increased turnover of AMP in CMV infected endothelial cells. In addition, the superoxide production by stimulated polymorphonuclear cells was inhibited in the presence of CMV infected endothelial cells as compared to uninfected cells, probably due to the enhanced activity of ecto-5'nucleotidase and associated to production of adenosine.

Expression of recombinant proteinase 3, the autoantigen in Wegener's granulomatosis, in insect cells.

Proteinase 3 (PR3) is the major autoantigen for anti-neutrophil cytoplasmic antibodies (ANCA) in patients with Wegener's granulomatosis. Little is known about the major antigenic sites on PR3. To facilitate epitope mapping, PR3 was cloned in insect cells using a baculovirus expression system. Four different sequences of the PR3 cDNA were amplified by PCR: two clones containing the pro-peptide of PR3 with or without a His-tag (rproPR3-his and rproPR3, respectively) and two clones without the pro-peptide and with or without a His-tag (rPR3-his and rPR3, respectively). The PR3 sequences were cloned behind the polyhedrin promoter and the honeybee melittin signal peptide enabling secretion of rPR3. Plasmids were transposed into the genome of baculovirus, and wild types as well as PR3-containing virus genomes were transfected into Sf21 insect cells. All four rPR3 variants were secreted into the medium and were recognized by anti-neutrophil PR3 rabbit serum and by at least two anti-PR3 monoclonal antibodies. Mature forms of PR3 were recognized by almost all patient sera, whereas the pro-forms of PR3 were recognized by 14 of 18 PR3-ANCA sera tested. On SDS-PAGE, the four rPR3 forms migrated at approximately 32 kDa. RPR3-his and rproPR3-his could be purified by means of this His-tag. In conclusion, especially the mature rPR3s are well recognized by PR3-ANCA sera. The presence of a C-terminal His-tag facilitated purification of His-tagged rPR3. Thus, rPR3 expressed in insect cells can be used as a tool for diagnostic tests as well as for epitope mapping studies.

Plasma lactoferrin levels are decreased in end-stage AIDS patients.

The antimicrobial protein lactoferrin (Lf) is present in plasma and in mucosal secretions. Using ELISA we analysed plasma and saliva of HIV-infected patients, patients with AIDS, and healthy controls for the presence of secreted Lf. The plasma Lf levels of AIDS patients (classification C3) were significantly lower (p < 0.001) as compared to asymptomatic and symptomatic HIV infected patients, or controls. In addition, plasma Lf levels closely correlated with neutrophilic granulocyte counts in the HIV-infected patients. Thus, basal plasma Lf levels are likely the result of Lf release by neutrophilic granulocytes. The Candida titres present in the oral cavity were determined in a part of the HIV-infected patient group. As it appeared, the presence of this opportunistic pathogen always coincided with low levels of salivary Lf levels. We conclude that Lf, as part of the nonspecific immune system, might play an important role in the first line of defense against opportunistic microbial infections in AIDS patients.

Antiviral activities of lactoferrin.

Lactoferrin (LF) is an iron binding glycoprotein that is present in several mucosal secretions. Many biological functions have been ascribed to LF. One of the functions of LF is the transport of metals, but LF is also an important component of the non-specific immune system, since LF has antimicrobial properties against bacteria, fungi and several viruses. This review gives an overview of the present knowledge about the antiviral activities and, when possible, the antiviral modes of action of this protein. Lactoferrin displays antiviral activity against both DNA- and RNA-viruses, including rotavirus, respiratory syncytial virus, herpes viruses and HIV. The antiviral effect of LF lies in the early phase of infection. Lactoferrin prevents entry of virus in the host cell, either by blocking cellular receptors, or by direct binding to the virus particles.

Double-stranded RNA mycoviruses in mycelium of Pleurotus ostreatus.

Mycelium of Pleurotus ostreatus var. florida with a decreased growth rate contained seven double-stranded RNA segments and isometrical virus particles with diameters of 24 and 30 nm. Mycelium with a normal growth rate lacked dsRNA. Protoclones from virus-containing mycelium contained one to seven of these dsRNA segments in varying concentrations. The exact correlation between slow growth and the presence of dsRNA molecules could not be established. Infection of virus-free protoplasts with PEG-precipitated virus particles resulted in mycelium that stably maintained the 2.4 kbp dsRNA.