Response eQTLs, chromatin accessibility, and 3D chromatin structure in chondrocytes provide mechanistic insight into osteoarthritis risk.

Osteoarthritis (OA) poses a significant healthcare burden with limited treatment options. While genome-wide association studies (GWAS) have identified over 100 OA-associated loci, translating these findings into therapeutic targets remains challenging. Integrating expression quantitative trait loci (eQTL), 3D chromatin structure, and other genomic approaches with OA GWAS data offers a promising approach to elucidate disease mechanisms; however, comprehensive eQTL maps in OA-relevant tissues and conditions remain scarce. We mapped gene expression, chromatin accessibility, and 3D chromatin structure in primary human articular chondrocytes in both resting and OA-mimicking conditions. We identified thousands of differentially expressed genes, including those associated with differences in sex and age. RNA-seq in chondrocytes from 101 donors across two conditions uncovered 3782 unique eGenes, including 420 that exhibited strong and significant condition-specific effects. Colocalization with OA GWAS signals revealed 13 putative OA risk genes, 10 of which have not been previously identified. Chromatin accessibility and 3D chromatin structure provided insights into the mechanisms and conditional specificity of these variants. Our findings shed light on OA pathogenesis and highlight potential targets for therapeutic development. Highlights: ∘ Comprehensive analysis of sex- and age-related global gene expression in human chondrocytes revealed differences that correlate with osteoarthritis ∘ First response eQTLs in chondrocytes treated with an OA-related stimulus ∘ Deeply sequenced Hi-C in resting and activated chondrocytes helps connect OA risk variants to their putative causal genes ∘ Colocalization analysis reveals 13 (including 10 novel) putative OA risk genes.

Status and future developments for downstream processing of biological products: Perspectives from the Recovery XIX yield roundtable discussions.

Governments and biopharmaceutical organizations aggressively leveraged expeditious communication capabilities, decision models, and global strategies to make a COVID-19 vaccine happen within a period of 12 months. This was an unusual effort and cannot be transferred to normal times. However, this focus on a single vaccine has also led to other treatments and drug developments being sidelined. Society expects the pharmaceutical industry to provide an uninterrupted supply of medicines. However, it is often overlooked how complex the manufacture of these compounds is and what logistics are required, not to mention the time needed to develop new drugs. The overarching theme, therefore, is patient access and how we can help ensure access and extend it to low- and middle-income countries. Despite unceasing efforts to make medications available to all patient populations, this must never be done at the expense of patient safety. A major fraction of the costs in biopharmaceutical manufacturing are for drug discovery, process development, and clinical studies. Infrastructure costs are very difficult to quantify because they often depend on whether a greenfield facility or an existing, depreciated facility is used or adapted for a new product. To accelerate process development concepts of platform process and prior knowledge are increasingly leveraged. While more traditional protein therapeutics continue to dominate the field, we are also experiencing the exciting emergence and evolution of other therapeutic formats (bispecifics, tetravalent mAbs, antibody-drug conjugates, enzymes, peptides, etc.) that offer unique treatment options for patients. Protein modalities are still dominant, but new modalities are being developed that can be learned from including advanced therapeutics-like cell and gene therapies. The industry must develop a model-based strategy for process development and technologies such as continuous integrated biomanufacturing must be adopted. The overall conclusion is that the pandemic pace was unsustainable, focused on vaccine delivery at the expense of other modalities/disease targets, and had implications for professional and personal life (work-life balance). Routinely reducing development time from 10 years to 1 year is nearly impossible to achieve. Environmental aspects of sustainable downstream processing are also described.

A survey of awareness of diagnosis and treatment of rare diseases among healthcare professionals and researchers in India.

Rare diseases (RDs) are those that affect a small fraction of the total population. In India, where resources are scarce, the healthcare infrastructure and policy framework are focused on mitigating diseases that affect a large number of people. As a result,many cases ofRDs remain unreported, undiagnosed, and untreated. To understandthe currentlevel of RD awareness among healthcare professionals (HCPs) and researchers, as they are key stakeholders in diagnosis, treatment, policy making, and drug development, we conducted a survey based on identification of an RD, time for diagnosis, treatment options, and relationship with family history and geographic location. The survey was divided into two parts, one for researchers and the other for healthcare professionals, each consisting of 22 questions. We observed that among all our respondents, 31% of researchers and 29% of HCPs did not know the time required for diagnosis of a rare disease they mentioned in the survey. They identified the importance of family history but failed to realize its association with geographic location. The results from the exploratory study can provide information for enabling larger studies to develop recommendations and policies that can improve awareness about RDs in healthcare communities.

3D chromatin structure in chondrocytes identifies putative osteoarthritis risk genes.

Genome-wide association studies have identified over 100 loci associated with osteoarthritis risk, but the majority of osteoarthritis risk variants are noncoding, making it difficult to identify the impacted genes for further study and therapeutic development. To address this need, we used a multiomic approach and genome editing to identify and functionally characterize potential osteoarthritis risk genes. Computational analysis of genome-wide association studies and ChIP-seq data revealed that chondrocyte regulatory loci are enriched for osteoarthritis risk variants. We constructed a chondrocyte-specific regulatory network by mapping 3D chromatin structure and active enhancers in human chondrocytes. We then intersected these data with our previously collected RNA-seq dataset of chondrocytes responding to fibronectin fragment, a known osteoarthritis trigger. Integration of the 3 genomic datasets with recently reported osteoarthritis genome-wide association study variants revealed a refined set of putative causal osteoarthritis variants and their potential target genes. One of the putative target genes identified was SOCS2, which was connected to a putative causal variant by a 170-kb loop and is differentially regulated in response to fibronectin fragment. CRISPR-Cas9-mediated deletion of SOCS2 in primary human chondrocytes from 3 independent donors led to heightened expression of inflammatory markers after fibronectin fragment treatment. These data suggest that SOCS2 plays a role in resolving inflammation in response to cartilage matrix damage and provides a possible mechanistic explanation for its influence on osteoarthritis risk. In total, we identified 56 unique putative osteoarthritis risk genes for further research and potential therapeutic development.

Estrogen Stimulation of Pleiotrophin Enhances Osteoblast Differentiation and Maintains Bone Mass in IGFBP-2 Null Mice.

Insulin-like growth factor binding protein-2 (IGFBP-2) stimulates osteoblast differentiation but only male Igfbp2 null mice have a skeletal phenotype. The trophic actions of IGFBP-2 in bone are mediated through its binding to receptor tyrosine phosphatase beta (RPTPß). Another important ligand for RPTPß is pleiotrophin (PTN), which also stimulates osteoblast differentiation. We determined the change in PTN and RPTPß in Igfbp2-/- mice. Analysis of whole bone mRNA in wild-type and knockout mice revealed increased expression of Ptn. Rptpß increased in gene-deleted animals with females having greater expression than males. Knockdown of PTN expression in osteoblasts in vitro inhibited differentiation, and addition of PTN to the incubation medium rescued the response. Estradiol stimulated PTN secretion and PTN knockdown blocked estradiol-stimulated differentiation. PTN addition to IGFBP-2 silenced osteoblast stimulated differentiation, and an anti-fibronectin-3 antibody, which inhibits PTN binding to RPTPß, inhibited this response. Estrogen stimulated PTN secretion and downstream signaling in the IGFBP-2 silenced osteoblasts and these effects were inhibited with anti-fibronectin-3. Administration of estrogen to wild-type and Igfbp2-/- male mice stimulated an increase in both areal bone mineral density and trabecular bone volume fraction but the increase was significantly greater in the Igfbp2-/- animals. Estrogen also stimulated RPTPß expression in the null mice. We conclude that loss of IGFBP-2 expression is accompanied by upregulation of PTN and RPTPß expression in osteoblasts, that the degree of increase is greater in females due to estrogen secretion, and that this compensatory change may account for some component of the maintenance of normal bone mass in female mice.

Engineered Cartilage from Human Chondrocytes with Homozygous Knockout of Cell Cycle Inhibitor p21.

Osteoarthritis (OA) is a highly prevalent disease with limited treatment options. The search for disease-modifying OA therapies would benefit from a more comprehensive knowledge of the genetic variants that contribute to chondrocyte dysfunction and the barriers to cartilage regeneration. One goal of this study was to establish a system for producing engineered cartilage tissue from genetically defined primary human chondrocytes through genome editing and single-cell expansion. This process was utilized to investigate the functional effect of biallelic knockout of the cell cycle inhibitor p21. The use of ribonucleoprotein (RNP) CRISPR/Cas9 complexes targeting two sites in the coding region of p21 resulted in a high frequency (16%) of colonies with homozygous p21 knockout. Chondrogenic pellet cultures from expanded chondrocytes with complete loss of p21 produced more glycosaminoglycans (GAG) and maintained a higher cell number. Single-cell-derived colonies retained the potential for robust matrix production after expansion, allowing for analysis of colony variability from the same population of targeted cells. The effect of enhanced cartilage matrix production in p21 knockout chondrocytes persisted when matrix production from individual colonies was analyzed. Chondrocytes had lower levels of p21 protein with further expansion, and the difference in GAG production with p21 knockout was strongest at early passages. These results support previous findings that implicate p21 as a barrier to cartilage matrix production and regenerative capacity. Furthermore, this work establishes the use of genome-edited human chondrocytes as a promising approach for engineered tissue models containing user-defined gene knockouts and other genetic variants for investigation of OA pathogenesis. Impact Statement This work provides two important advances to the field of tissue engineering. One is the demonstration that engineered cartilage tissue can be produced from genetically defined populations of primary human chondrocytes. While CRISPR/Cas-9 genome editing has been extensively used in cell lines that divide indefinitely, this work extends the technique to an engineered tissue model system to support investigation of genetic changes that affect cartilage production. A second contribution is the finding that chondrocytes with p21 knockout synthesized more cartilage matrix tissue than unedited controls. This supports the continued investigation of p21 as a potential barrier to effective cartilage regeneration.

Controlled induction and targeted elimination of p16INK4a-expressing chondrocytes in cartilage explant culture.

Cellular senescence is a phenotypic state that contributes to age-related diseases through the secretion of matrix-degrading and inflammatory molecules. An emerging therapeutic strategy for osteoarthritis (OA) is to selectively eliminate senescent cells by initiating apoptosis. This study establishes a cartilage explant model of senescence induction and senolytic clearance using p16Ink4a expression as a biomarker of senescence. Growth-factor stimulation of explants increased the expression of p16Ink4a at both the mRNA and protein levels. Applying this culture system to cartilage from p16tdTom reporter mice (a knockin allele with tdTomato fluorescent protein regulated by the endogenous p16Ink4a promoter) demonstrated the emergence of a p16-high population that was quantified using flow cytometry for tdTomato. Cell sorting was used to separate chondrocytes based on tdTomato fluorescence and p16-high cells showed higher senescence-associated ß-galactosidase activity and increased gene expression of the senescence-associated secretory phenotype as compared with p16-low cells. The potential for effective senolysis within the cartilage extracellular matrix was assessed using navitoclax (ABT-263). Navitoclax treatment reduced the percentage of p16-high cells from 17.9 to 6.1% (mean of 13 matched pairs; P < 0.001) and increased cleaved caspase-3 confirmed apoptotic activity. Together, these findings establish a physiologically relevant cartilage explant model for testing the induction and elimination of senescent chondrocytes, which will support investigations of senolytic therapy for OA.-Sessions, G. A., Copp, M. E., Liu, J.-Y., Sinkler, M. A., D'Costa, S., Diekman, B. O. Controlled induction and targeted elimination of p16INK4a-expressing chondrocytes in cartilage explant culture.

IGFBP-2 stimulates calcium/calmodulin-dependent protein kinase kinase 2 activation leading to AMP-activated protein kinase induction which is required for osteoblast differentiation.

Insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins-2 (IGFBP-2) function coordinately to stimulate osteoblast differentiation. Induction of AMP-activated protein kinase (AMPK) is required for differentiation and is stimulated by these two factors. These studies were undertaken to determine how these two peptides lead to activation of AMPK. Enzymatic inhibitors and small interfering RNA were utilized to attenuate calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) activity in osteoblasts, and both manipulations resulted in failure to activate AMPK, thereby resulting in inhibition of osteoblast differentiation. IGFBP-2 and IGF-I stimulated an increase in CaMKK2, and inhibition of IGFBP-2 binding its receptor resulted in failure to induce CaMKK2 and AMPK activation. Injection of a peptide that contained the IGFBP-2 receptor-binding domain into IGFBP-2-/- mice activated CaMKK2 and injection of a CaMKK2 inhibitor into normal mice inhibited both CamKK2 and AMPK activation in osteoblasts. We conclude that induction of CaMKK2 by IGFBP-2 and IGF-I in osteoblasts is an important signaling event that occurs early in differentiation and is responsible for activation of AMPK, which is required for optimal osteoblast differentiation.

Stability of the adeno-associated virus 8 reference standard material.

Adeno-associated virus (AAV) vectors are extensively used for gene therapy clinical trials. Accurate and standardized titration methods are essential for characterizing and dosing AAV-based drugs and thus to assess their safety and efficacy. To this end, the Reference Standard Materials (RSM) working group generated standards for AAV serotype 2 and serotype 8. The AAV8RSM (ATCC® VR-1816™) was deposited to the American Type Culture Collection in 2014 and is available to the scientific community. Here, three independent laboratories of the RSM working group provide stability data of the AAV8RSM 2 years after the initial characterization and after container relabeling performed at the ATCC. The AAV8RSM showed constant titers across experimental conditions: 1.48 ± 0.62 × 1012 vector genome (vg)/ml, 9.38 ± 11.4 × 108 infectious units (IU)/ml and 5.76 ± 2.39 × 1011 total particles (p)/ml as determined by qPCR, TCID50 and ELISA, respectively. Additionally, the AAV8RSM capsid protein integrity assessed by SDS-PAGE was equivalent to the original analyses. In conclusion, the AAV8RSM titers remained stable for two years under appropriate storage conditions ( <-70° C). The use of RSM is strongly recommended and endorsed by regulatory agencies to normalize laboratory internal controls and to provide accurate titration of AAV vectors lots.

Mast cell corticotropin-releasing factor subtype 2 suppresses mast cell degranulation and limits the severity of anaphylaxis and stress-induced intestinal permeability.

BACKGROUND: Psychological stress and heightened mast cell (MC) activation are linked with important immunologic disorders, including allergy, anaphylaxis, asthma, and functional bowel diseases, but the mechanisms remain poorly defined. We have previously demonstrated that activation of the corticotropin-releasing factor (CRF) system potentiates MC degranulation responses during IgE-mediated anaphylaxis and psychological stress through corticotropin-releasing factor receptor subtype 1 (CRF1) expressed on MCs. OBJECTIVE: In this study we investigated the role of corticotropin-releasing factor receptor subtype 2 (CRF2) as a modulator of stress-induced MC degranulation and associated disease pathophysiology. METHODS: In vitro MC degranulation assays were performed with bone marrow-derived mast cells (BMMCs) derived from wild-type (WT) and CRF2-deficient (CRF2-/-) mice and RBL-2H3 MCs transfected with CRF2-overexpressing plasmid or CRF2 small interfering RNA. In vivo MC responses and associated pathophysiology in IgE-mediated passive systemic anaphylaxis and acute psychological restraint stress were measured in WT, CRF2-/-, and MC-deficient KitW-sh/W-sh knock-in mice. RESULTS: Compared with WT mice, CRF2-/- mice exhibited greater serum histamine levels and exacerbated IgE-mediated anaphylaxis and colonic permeability. In addition, CRF2-/- mice exhibited increased serum histamine levels and colonic permeability after acute restraint stress. Experiments with BMMCs and RBL-2H3 MCs demonstrated that CRF2 expressed on MCs suppresses store-operated Ca2+ entry signaling and MC degranulation induced by diverse MC stimuli. Experiments with MC-deficient KitW-sh/W-sh mice systemically engrafted with WT and CRF2-/- BMMCs demonstrated the functional importance of MC CRF2 in modulating stress-induced pathophysiology. CONCLUSIONS: MC CRF2 is a negative global modulator of stimuli-induced MC degranulation and limits the severity of IgE-mediated anaphylaxis and stress-related disease pathogenesis.

Frontline Science: Corticotropin-releasing factor receptor subtype 1 is a critical modulator of mast cell degranulation and stress-induced pathophysiology.

Life stress is a major risk factor in the onset and exacerbation of mast cell-associated diseases, including allergy/anaphylaxis, asthma, and irritable bowel syndrome. Although it is known that mast cells are highly activated upon stressful events, the mechanisms by which stress modulates mast cell function and disease pathophysiology remains poorly understood. Here, we investigated the role of corticotropin-releasing factor receptor subtype 1 (CRF1) in mast cell degranulation and associated disease pathophysiology. In a mast cell-dependent model of IgE-mediated passive systemic anaphylaxis (PSA), prophylactic administration of the CRF1-antagonist antalarmin attenuated mast cell degranulation and hypothermia. Mast cell-deficient KitW-sh/W-sh mice engrafted with CRF1-/- bone marrow-derived mast cells (BMMCs) exhibited attenuated PSA-induced serum histamine, hypothermia, and clinical scores compared with wild-type BMMC-engrafted KitW-sh/W-sh mice. KitW-sh/W-sh mice engrafted with CRF1-/- BMMCs also exhibited suppressed in vivo mast cell degranulation and intestinal permeability in response to acute restraint stress. Genetic and pharmacologic experiments with murine BMMCs, rat RBL-2H3, and human LAD2 mast cells demonstrated that although CRF1 activation did not directly induce MC degranulation, CRF1 signaling potentiated the degranulation responses triggered by diverse mast cell stimuli and was associated with enhanced release of Ca2+ from intracellular stores. Taken together, our results revealed a prominent role for CRF1 signaling in mast cells as a positive modulator of stimuli-induced degranulation and in vivo pathophysiologic responses to immunologic and psychologic stress.

Sexual dimorphism in the mast cell transcriptome and the pathophysiological responses to immunological and psychological stress.

BACKGROUND: Biological sex plays a prominent role in the prevalence and severity of a number of important stress-related gastrointestinal and immune-related diseases including IBS and allergy/anaphylaxis. Despite the establishment of sex differences in these diseases, the underlying mechanisms contributing to sex differences remain poorly understood. The objective of this study was to define the role of biological sex on mast cells (MCs), an innate immune cell central to the pathophysiology of many GI and allergic disorders. METHODS: Twelve-week-old C57BL/6 male and female mice were exposed to immunological stress (2 h of IgE-mediated passive systemic anaphylaxis (PSA)) or psychological stress (1 h of restraint stress (RS)) and temperature, clinical scores, serum histamine, and intestinal permeability (for RS) were measured. Primary bone marrow-derived MCs (BMMCs) were harvested from male and female mice and analyzed for MC degranulation, signaling pathways, mediator content, and RNA transcriptome analysis. RESULTS: Sexually dimorphic responses were observed in both models of PSA and RS and in primary MCs. Compared with male mice, female mice exhibited increased clinical scores, hypothermia, and serum histamine levels in response to PSA and had greater intestinal permeability and serum histamine responses to RS. Primary BMMCs from female mice exhibited increased release of ß-hexosaminidase, histamine, tryptase, and TNF-α upon stimulation with IgE/DNP and A23187. Increased mediator release in female BMMCs was not associated with increased upstream phospho-tyrosine signaling pathways or downstream Ca2+ mobilization. Instead, increased mediator release in female MCs was associated with markedly increased capacity for synthesis and storage of MC granule-associated immune mediators as determined by MC mediator content and RNA transcriptome analysis. CONCLUSIONS: These results provide a new understanding of sexual dimorphic responses in MCs and have direct implications for stress-related diseases associated with a female predominance and MC hyperactivity including irritable bowel syndrome, allergy, and anaphylaxis.

Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR.

Clinical trials using recombinant adeno-associated virus (rAAV) vectors have demonstrated efficacy and a good safety profile. Although the field is advancing quickly, vector analytics and harmonization of dosage units are still a limitation for commercialization. AAV reference standard materials (RSMs) can help ensure product safety by controlling the consistency of assays used to characterize rAAV stocks. The most widely utilized unit of vector dosing is based on the encapsidated vector genome. Quantitative polymerase chain reaction (qPCR) is now the most common method to titer vector genomes (vg); however, significant inter- and intralaboratory variations have been documented using this technique. Here, RSMs and rAAV stocks were titered on the basis of an inverted terminal repeats (ITRs) sequence-specific qPCR and we found an artificial increase in vg titers using a widely utilized approach. The PCR error was introduced by using single-cut linearized plasmid as the standard curve. This bias was eliminated using plasmid standards linearized just outside the ITR region on each end to facilitate the melting of the palindromic ITR sequences during PCR. This new "Free-ITR" qPCR delivers vg titers that are consistent with titers obtained with transgene-specific qPCR and could be used to normalize in-house product-specific AAV vector standards and controls to the rAAV RSMs. The free-ITR method, including well-characterized controls, will help to calibrate doses to compare preclinical and clinical data in the field.

Biochemical analysis of the multifunctional vaccinia mRNA capping enzyme encoded by a temperature sensitive virus mutant.

Prior biochemical analysis of the heterodimeric vaccinia virus mRNA capping enzyme suggests roles not only in mRNA capping but also in early viral gene transcription termination and intermediate viral gene transcription initiation. Prior phenotypic characterization of Dts36, a temperature sensitive virus mutant affecting the large subunit of the capping enzyme was consistent with the multifunctional roles of the capping enzyme in vivo. We report a biochemical analysis of the capping enzyme encoded by Dts36. Of the three enzymatic activities required for mRNA capping, the guanylyltransferase and methyltransferase activities are compromised while the triphosphatase activity and the D12 subunit interaction are unaffected. The mutant enzyme is also defective in stimulating early gene transcription termination and intermediate gene transcription initiation in vitro. These results confirm that the vaccinia virus mRNA capping enzyme functions not only in mRNA capping but also early gene transcription termination and intermediate gene transcription initiation in vivo.

Vaccinia virus protein A3 is required for the production of normal immature virions and for the encapsidation of the nucleocapsid protein L4.

Maturation of the vaccinia virion is an intricate process that results in the organization of the viroplasm contained in immature virions into the lateral bodies, core wall and nucleocapsid observed in the mature particles. It is unclear how this organization takes place and studies with mutants are indispensable in understanding this process. By characterizing an inducible mutant in the A3L gene, we revealed that A3, an inner core wall protein, is important for formation of normal immature viruses and also for the correct localization of L4, a nucleocapsid protein. L4 did not accumulate in the viral factories in the absence of A3 and was not encapsidated in the particles that do not contain A3. These data strengthen our previously suggested hypothesis that A3 and L4 interact and that this interaction is critical for proper formation of the core wall and nucleocapsid.

Evaluation of a side population of canine lymphoma cells using Hoechst 33342 dye.

Cancer stem cell (CSC) research has increased exponentially to gain further insight into the mechanisms underlying both carcinogenesis and chemotherapy resistance. The present study was performed to explore the potential value of a side population (SP) assay for identifying and characterizing putative CSCs among canine lymphoma cells. Canine lymphoma cells from cell lines and clinical samples were subjected to the SP assay consisting of Hoechst 33342 staining and subsequent flow cytometric analysis. The SP assay revealed various amounts of a SP fraction among the canine lymphoma cells. The percentages of SP were not affected by inhibitors of membrane transporters, verapamil hydrochloride, or fumitremorgin C. Most of the canine lymphoma cells expressed high levels of Bmi-1 and membrane transporter proteins such as ABCG2 and phosphorylated (p)-glycoprotein. This investigation lays the groundwork for further studies of the biological behaviors and molecular characteristics of CSCs in cases of canine lymphoma.

Biochemical and biophysical properties of a putative hub protein expressed by vaccinia virus.

H5 is a constitutively expressed, phosphorylated vaccinia virus protein that has been implicated in viral DNA replication, post-replicative gene expression, and virus assembly. For the purpose of understanding the role of H5 in vaccinia biology, we have characterized its biochemical and biophysical properties. Previously, we have demonstrated that H5 is associated with an endoribonucleolytic activity. In this study, we have shown that this cleavage results in a 3'-OH end suitable for polyadenylation of the nascent transcript, corroborating a role for H5 in vaccinia transcription termination. Furthermore, we have shown that H5 is intrinsically disordered, with an elongated rod-shaped structure that preferentially binds double-stranded nucleic acids in a sequence nonspecific manner. The dynamic phosphorylation status of H5 influences this structure and has implications for the role of H5 in multiple processes during virus replication.

Bone marrow-derived cells participate in the formation of normal and neoplastic lung stroma.

UNLABELLED: BACKGROUND/ AIM: The present study was performed in order to investigate the distribution of transplanted bone marrow-derived cells (BMDCs) in normal and neoplastic tissues. MATERIALS AND METHODS: A microchimeric mouse model harboring traceable BMDCs was created by injecting labeled BMDCs into adenovirus-expressing Cre (AdenoCre)-infected, irradiated, LSL-K-ras(G12D) p53(fl/fl) mice. RESULTS: In the normal mice, transplanted cells were detected in the femur, tibia, lung, heart, kidney and spleen of recipient mice. The transplanted BMDCs were detected predominantly in the connective tissue around the smaller bronchioles of normal lung parenchyma. In the lung tumor-bearing mice, the transplanted BMDCs were detected within the tumor tissue and more abundantly in the connective tissue adjacent to the tumor mass. CONCLUSION: This study provides morphological evidence of a microchimeric experimental system that may promote further research into the role of BMDCs in the carcinogenic process and for the development of a novel therapy aimed at targeting stromal cells and, eventually, tumor growth.

First complete and productive cell culture model for members of the genus Iridovirus.

Chilo iridescent virus (CIV; the type strain of the genus Iridovirus) replicates productively in larvae of the boll weevil, Anthonomus grandis. This study focuses on characterizing productive infections of a boll weevil cell line, BRL-AG-3A (AG3A), starting with CIV reared in the waxworm, Galleria mellonella. We show that CIV can be continually and productively passaged to high titer in AG3A cells. The replication of larval-derived CIV in AG3A was analyzed by observing viral DNA replication and restriction endonuclease digestion profiles, morphogenesis, and infectivity using TCID(50) assays with AG3A as an indicator cell line. The data showed that virus passaged in the AG3A host is stable. AG3A cells are more efficient than previously utilized CF-124T cells from Choristoneura fumiferana. This system constitutes a superior model for cellular and molecular studies on CIV; it represents the first complete, productive cell culture model for the replication of CIV or any member of the genus Iridovirus.

A virion-associated protein kinase induces apoptosis.

Apoptosis and inhibition of host gene expression are often associated with virus infections. Many viral polypeptides modulate apoptosis by direct interaction with highly conserved apoptotic pathways. Some viruses induce apoptosis during late stages of the infection cycle, while others inhibit apoptosis to facilitate replication or maintain persistent infection. In previous work, we showed that Chilo iridescent virus (CIV) or CIV virion protein extract induces apoptosis in spruce budworm and cotton boll weevil cell cultures. Here, we characterize the product of a CIV gene (iridovirus serine/threonine kinase; istk) with signature sequences for S/T kinase and ATP binding. ISTK appears to belong to the superfamily, vaccinia-related kinases (VRKs). The istk gene was expressed in Pichia pastoris vectors. Purified ISTK (48 kDa) exhibited S/T kinase activity. Treatment with ISTK induced apoptosis in budworm cells. A 35-kDa cleavage product of ISTK retaining key signature sequences was identified during purification. Pichia-expressed 35-kDa polypeptide, designated iridoptin, induced apoptosis and inhibition of host protein synthesis in budworm and boll weevil cells. A mutation in the ATP-binding site eliminated both kinase and apoptosis activity of iridoptin, suggesting that kinase activity is essential for induction of apoptosis. Analysis with custom antibody confirmed that ISTK is a structural component of CIV particles. This is the first demonstration of a viral kinase inducing apoptosis in any virus-host system and the first identification of a factor inducing apoptosis or host protein shutoff for the family Iridoviridae.