Selective deletion of E3 ubiquitin ligase FBW7 in VE-cadherin-positive cells instigates diffuse large B-cell lymphoma in mice in vivo.

During the maturation of hematopoietic stem/progenitor cells (HSPCs) to fully differentiated mature B lymphocytes, developing lymphocytes may undergo malignant transformation and produce B-cell lymphomas. Emerging evidence shows that through the endothelial-hematopoietic transition, specialized endothelial cells called the hemogenic endothelium can differentiate into HSPCs. However, the contribution of genetic defects in hemogenic endothelial cells to B-cell lymphomagenesis has not yet been investigated. Here, we report that mice with endothelial cell-specific deletion of Fbw7 spontaneously developed diffuse large B-cell lymphoma (DLBCL) following Bcl6 accumulation. Using lineage tracing, we showed that B-cell lymphomas in Fbw7 knockout mice were hemogenic endothelium-derived. Mechanistically, we found that FBW7 directly interacted with Bcl6 and promoted its proteasomal degradation. FBW7 expression levels are inversely correlated with BCL6 expression. Additionally, pharmacological disruption of Bcl6 abolished Fbw7 deletion-induced B-cell lymphomagenesis. We conclude that selective deletion of E3 ubiquitin ligase FBW7 in VE-cadherin positive endothelial cells instigates diffuse large B-cell lymphoma via upregulation of BCL6 stability. In addition, the mice with endothelial cell-specific deletion of Fbw7 provide a valuable preclinical platform for in vivo development and evaluation of novel therapeutic interventions for the treatment of DLBCL.

AMPKα2 regulates fasting-induced hyperketonemia by suppressing SCOT ubiquitination and degradation.

Ketone bodies serve as an energy source, especially in the absence of carbohydrates or in the extended exercise. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a crucial energy sensor that regulates lipid and glucose metabolism. However, whether AMPK regulates ketone metabolism in whole body is unclear even though AMPK regulates ketogenesis in liver. Prolonged resulted in a significant increase in blood and urine levels of ketone bodies in wild-type (WT) mice. Interestingly, fasting AMPKα2-/- and AMPKα1-/- mice exhibited significantly higher levels of ketone bodies in both blood and urine compared to fasting WT mice. BHB tolerance assays revealed that both AMPKα2-/- and AMPKα1-/- mice exhibited slower ketone consumption compared to WT mice, as indicated by higher blood BHB or urine BHB levels in the AMPKα2-/- and AMPKα1-/- mice even after the peak. Interestingly, fasting AMPKα2-/- and AMPKα1-/- mice exhibited significantly higher levels of ketone bodies in both blood and urine compared to fasting WT mice. . Specifically, AMPKα2ΔMusc mice showed approximately a twofold increase in blood BHB levels, and AMPKα2ΔMyo mice exhibited a 1.5-fold increase compared to their WT littermates after a 48-h fasting. However, blood BHB levels in AMPKα1ΔMusc and AMPKα1ΔMyo mice were as same as in WT mice. Notably, AMPKα2ΔMusc mice demonstrated a slower rate of BHB consumption in the BHB tolerance assay, whereas AMPKα1ΔMusc mice did not show such an effect. Declining rates of body weights and blood glucoses were similar among all the mice. Protein levels of SCOT, the rate-limiting enzyme of ketolysis, decreased in skeletal muscle of AMPKα2-/- mice. Moreover, SCOT protein ubiquitination increased in C2C12 cells either transfected with kinase-dead AMPKα2 or subjected to AMPKα2 inhibition. AMPKα2 physiologically binds and stabilizes SCOT, which is dependent on AMPKα2 activity.

Pycard deficiency inhibits microRNA maturation and prevents neointima formation by promoting chaperone-mediated autophagic degradation of AGO2/argonaute 2 in adipose tissue.

PYCARD (PYD and CARD domain containing), a pivotal adaptor protein in inflammasome assembly and activation, contributes to innate immunity, and plays an essential role in the pathogenesis of atherosclerosis and restenosis. However, its roles in microRNA biogenesis remain unknown. Therefore, this study aimed to investigate the roles of PYCARD in miRNA biogenesis and neointima formation using pycard knockout (pycard-/-) mice. Deficiency of Pycard reduced circulating miRNA profile and inhibited Mir17 seed family maturation. The systemic pycard knockout also selectively reduced the expression of AGO2 (argonaute RISC catalytic subunit 2), an important enzyme in regulating miRNA biogenesis, by promoting chaperone-mediated autophagy (CMA)-mediated degradation of AGO2, specifically in adipose tissue. Mechanistically, pycard knockout increased PRMT8 (protein arginine N-methyltransferase 8) expression in adipose tissue, which enhanced AGO2 methylation, and subsequently promoted its binding to HSPA8 (heat shock protein family A (Hsp70) member 8) that targeted AGO2 for lysosome degradation through chaperone-mediated autophagy. Finally, the reduction of AGO2 and Mir17 family expression prevented vascular injury-induced neointima formation in Pycard-deficient conditions. Overexpression of AGO2 or administration of mimic of Mir106b (a major member of the Mir17 family) prevented Pycard deficiency-mediated inhibition of neointima formation in response to vascular injury. These data demonstrate that PYCARD inhibits CMA-mediated degradation of AGO2, which promotes microRNA maturation, thereby playing a critical role in regulating neointima formation in response to vascular injury independently of inflammasome activity and suggest that modulating PYCARD expression and function may represent a powerful therapeutic strategy for neointima formation.Abbreviations: 6-AN: 6-aminonicotinamide; ACTB: actin, beta; aDMA: asymmetric dimethylarginine; AGO2: argonaute RISC catalytic subunit 2; CAL: carotid artery ligation; CALCOCO2: calcium binding and coiled-coil domain 2; CMA: chaperone-mediated autophagy; CTSB: cathepsin B; CTSD: cathepsin D; DGCR8: DGCR8 microprocessor complex subunit; DOCK2: dedicator of cyto-kinesis 2; EpiAdi: epididymal adipose tissue; HSPA8: heat shock protein family A (Hsp70) member 8; IHC: immunohistochemical; ISR: in-stent restenosis; KO: knockout; LAMP2: lysosomal-associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; miRNA: microRNA; NLRP3: NLR family pyrin domain containing 3; N/L: ammonium chloride combined with leupeptin; PRMT: protein arginine methyltransferase; PVAT: peri-vascular adipose tissues; PYCARD: PYD and CARD domain containing; sDMA: symmetric dimethylarginine; ULK1: unc-51 like kinase 1; VSMCs: vascular smooth muscle cells; WT: wild-type.

Nicotine exacerbates atherosclerosis and plaque instability via NLRP3 inflammasome activation in vascular smooth muscle cells.

Rationale: Nicotine has been reported to be a strong risk factor for atherosclerosis. However, the underlying mechanism by which nicotine controls atherosclerotic plaque stability remain largely unknown. Objective: The aim of this study was to evaluate the impact of lysosomal dysfunction mediated NLRP3 inflammasome activation in vascular smooth muscle cell (VSMC) on atherosclerotic plaque formation and stability in advanced atherosclerosis at the brachiocephalic arteries (BA). Methods and Results: Features of atherosclerotic plaque stability and the markers for NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome were monitored in the BA from nicotine or vehicle-treated apolipoprotein E deficient (Apoe-/-) mice fed with Western-type diet (WD). Nicotine treatment for 6 weeks accelerated atherosclerotic plaque formation and enhanced the hallmarks of plaque instability in BA of Apoe-/- mice. Moreover, nicotine elevated interleukin 1 beta (IL-1ß) in serum and aorta and was preferred to activate NLRP3 inflammasome in aortic vascular smooth muscle cells (VSMC). Importantly, pharmacological inhibition of Caspase1, a key downstream target of NLRP3 inflammasome complex, and genetic inactivation of NLRP3 significantly restrained nicotine-elevated IL-1ß in serum and aorta, as well as nicotine-stimulated atherosclerotic plaque formation and plaque destabilization in BA. We further confirmed the role of VSMC-derived NLRP3 inflammasome in nicotine-induced plaque instability by using VSMC specific TXNIP (upstream regulator of NLRP3 inflammasome) deletion mice. Mechanistic study further showed that nicotine induced lysosomal dysfunction resulted in cathepsin B cytoplasmic release. Inhibition or knockdown of cathepsin B blocked nicotine-dependent inflammasome activation. Conclusions: Nicotine promotes atherosclerotic plaque instability by lysosomal dysfunction-mediated NLRP3 inflammasome activation in vascular smooth muscle cells.

Metabolic labeling of cardiomyocyte-derived small extracellular-vesicle (sEV) miRNAs identifies miR-208a in cardiac regulation of lung gene expression.

Toxoplasma gondii uracil phosphoribosyltransferase (UPRT) converts 4-thiouracil (4TUc) into 4-thiouridine (4TUd), which is incorporated into nascent RNAs and can be biotinylated, then labelled with streptavidin conjugates or isolated via streptavidin-affinity methods. Here, we generated mice that expressed T. gondii UPRT only in cardiomyocytes (CM UPRT mice) and tested our hypothesis that CM-derived miRNAs (CM miRs) are transferred into remote organs after myocardial infarction (MI) by small extracellular vesicles (sEV) that are released from the heart into the peripheral blood (PB sEV). We found that 4TUd was incorporated with high specificity and sensitivity into RNAs isolated from the hearts and PB sEV of CM UPRT mice 6 h after 4TUc injection. In PB sEV, 4TUd was incorporated into CM-specific/enriched miRs including miR-208a, but not into miRs with other organ or tissue-type specificities. 4TUd-labelled miR208a was also present in lung tissues, especially lung endothelial cells (ECs), and CM-derived miR-208a (CM miR-208a) levels peaked 12 h after experimentally induced MI in PB sEV and 24 h after MI in the lung. Notably, miR-208a is expressed from intron 29 of α myosin heavy chain (αMHC), but αMHC transcripts were nearly undetectable in the lung. When PB sEV from mice that underwent MI (MI-PB sEV) or sham surgery (Sham-PB sEV) were injected into intact mice, the expression of Tmbim6 and NLK, which are suppressed by miR-208a and cooperatively regulate inflammation via the NF-κB pathway, was lower in the lungs of MI-PB sEV-treated animals than the lungs of animals treated with Sham-PB sEV or saline. In MI mice, Tmbim6 and NLK were downregulated, whereas endothelial adhesion molecules and pro-inflammatory cells were upregulated in the lung; these changes were significantly attenuated when the mice were treated with miR-208a antagomirs prior to MI surgery. Thus, CM UPRT mice enables us to track PB sEV-mediated transport of CM miRs and identify an miR-208a-mediated mechanism by which myocardial injury alters the expression of genes and inflammatory response in the lung.

AMP-activated protein kinase alpha1 promotes tumor development via FOXP3 elevation in tumor-infiltrating Treg cells.

Overwhelming evidence indicates that infiltration of tumors by Treg cells with elevated levels of FOXP3 suppresses the host antitumor immune response. However, the molecular mechanisms that maintain high expression of FOXP3 in tumor-infiltrating Treg cells remain elusive. Here, we report that AMP-activated protein kinase alpha1 (AMPKα1) enables high FOXP3 expression in tumor-infiltrating Treg cells. Mice with Treg-specific AMPKα1 deletion showed delayed tumor progression and enhanced antitumor T cell immunity. Further experiments showed that AMPKα1 maintains the functional integrity of Treg cells and prevents interferon-γ production in tumor-infiltrating Treg cells. Mechanistically, AMPKα1 maintains the protein stability of FOXP3 in Treg cells by downregulating the expression of E3 ligase CHIP (STUB1). Our results suggest that AMPKα1 activation promotes tumor growth by maintaining FOXP3 stability in tumor-infiltrating Treg cells and that selective inhibition of AMPK in Treg cells might be an effective anti-tumor therapy.

Activation of AMPKα1 is essential for regulatory T cell function and autoimmune liver disease prevention.

Regulatory T cells (Treg cells) are crucial for maintaining immune tolerance. Compromising the regulatory function of Treg cells can lead to autoimmune liver disease. However, how Treg cell function is regulated has not been fully clarified. Here, we report that mice with AMP-activated protein kinase alpha 1 (AMPKα1) globally knocked out spontaneously develop immune-mediated liver injury, with massive lymphocyte infiltration in the liver, elevated serum alanine aminotransferase levels, and greater production of autoantibodies. Both transplantation of wild-type bone marrow and adoptive transfer of wild-type Treg cells can prevent liver injury in AMPKα1-KO mice. In addition, Treg cell-specific AMPKα1-KO mice display histological features similar to those associated with autoimmune liver disease, greater production of autoantibodies, and hyperactivation of CD4+ T cells. AMPKα1 deficiency significantly impairs Treg cell suppressive function but does not affect Treg cell differentiation or proliferation. Furthermore, AMPK is activated upon T cell receptor (TCR) stimulation, which triggers Foxp3 phosphorylation, suppressing Foxp3 ubiquitination and proteasomal degradation. Importantly, the frequency of Treg cells and the phosphorylation levels of AMPK at T172 in circulating blood are significantly lower in patients with autoimmune liver diseases. Conclusion: Our data suggest that AMPK maintains the immunosuppressive function of Treg cells and confers protection against autoimmune liver disease.

Western-style diet impedes colonization and clearance of Citrobacter rodentium.

Western-style diet (WSD), which is high in fat and low in fiber, lacks nutrients to support gut microbiota. Consequently, WSD reduces microbiota density and promotes microbiota encroachment, potentially influencing colonization resistance, immune system readiness, and thus host defense against pathogenic bacteria. Here we examined the impact of WSD on infection and colitis in response to Citrobacter rodentium. We observed that, relative to mice consuming standard rodent grain-based chow (GBC), feeding WSD starkly altered the dynamics of Citrobacter infection, reducing initial colonization and inflammation but frequently resulting in persistent infection that associated with low-grade inflammation and insulin resistance. WSD's reduction in initial Citrobacter virulence appeared to reflect that colons of GBC-fed mice contain microbiota metabolites, including short-chain fatty acids, especially acetate, that drive Citrobacter growth and virulence. Citrobacter persistence in WSD-fed mice reflected inability of resident microbiota to out-compete it from the gut lumen, likely reflecting the profound impacts of WSD on microbiota composition. These studies demonstrate potential of altering microbiota and their metabolites by diet to impact the course and consequence of infection following exposure to a gut pathogen.

Immune Clearance of Senescent Cells to Combat Ageing and Chronic Diseases.

Senescent cells are generally characterized by permanent cell cycle arrest, metabolic alteration and activation, and apoptotic resistance in multiple organs due to various stressors. Excessive accumulation of senescent cells in numerous tissues leads to multiple chronic diseases, tissue dysfunction, age-related diseases and organ ageing. Immune cells can remove senescent cells. Immunaging or impaired innate and adaptive immune responses by senescent cells result in persistent accumulation of various senescent cells. Although senolytics-drugs that selectively remove senescent cells by inducing their apoptosis-are recent hot topics and are making significant research progress, senescence immunotherapies using immune cell-mediated clearance of senescent cells are emerging and promising strategies to fight ageing and multiple chronic diseases. This short review provides an overview of the research progress to date concerning senescent cell-caused chronic diseases and tissue ageing, as well as the regulation of senescence by small-molecule drugs in clinical trials and different roles and regulation of immune cells in the elimination of senescent cells. Mounting evidence indicates that immunotherapy targeting senescent cells combats ageing and chronic diseases and subsequently extends the healthy lifespan.

Peroxynitrite-Mediated SIRT (Sirtuin)-1 Inactivation Contributes to Nicotine-Induced Arterial Stiffness in Mice.

Objective- Inhibition of SIRT (sirtuin)-1, a nicotinamide adenine dinucleotide-dependent protein deacetylase, is linked to cigarette smoking-induced arterial stiffness, but the underlying mechanisms remain largely unknown. The aim of the present study was to determine the effects and mechanisms of nicotine, a major component of cigarette smoke, on SIRT1 activity and arterial stiffness. Approach and Results- Arterial stiffness, peroxynitrite (ONOO-) formation, SIRT1 expression and activity were monitored in mouse aortas of 8-week-old C57BL/6 mice (wild-type) or Sirt1-overexpressing ( Sirt1 Super) mice with or without nicotine for 4 weeks. In aortas of wild-type mice, nicotine reduced SIRT1 protein and activity by ≈50% without affecting its mRNA levels. In those from Sirt1 Super mice, nicotine also markedly reduced SIRT1 protein and activity to the levels that were comparable to those in wild-type mice. Nicotine infusion significantly induced collagen I, fibronectin, and arterial stiffness in wild-type but not Sirt1 Super mice. Nicotine increased the levels of iNOS (inducible nitric oxide synthase) and the co-staining of SIRT1 and 3-nitrotyrosine, a footprint of ONOO- in aortas. Tempol, which ablated ONOO- by scavenging superoxide anion, reduced the effects of nicotine on SIRT1 and collagen. Mutation of zinc-binding cysteine 395 or 398 in SIRT1 into serine (C395S) or (C398S) abolished SIRT1 activity. Furthermore, ONOO- dose-dependently inhibited the enzyme and increased zinc release in recombinant SIRT1. Finally, we found SIRT1 inactivation by ONOO- activated the YAP (Yes-associated protein) resulting in abnormal ECM (extracellular matrix) remodeling. Conclusions- Nicotine induces ONOO-, which selectively inhibits SIRT1 resulting in a YAP-mediated ECM remodeling. Visual Overview- An online visual overview is available for this article.

Brain Infection by Hepatitis E Virus Probably via Damage of the Blood-Brain Barrier Due to Alterations of Tight Junction Proteins.

Extrahepatic injury, particularly neurologic dysfunctions such as Guillain-Barré syndrome, neurologic amyotrophy, and encephalitis/meningoencephalitis/myositis were associated with HEV infection, which was supported by both clinical and laboratory studies. Thus, it is crucial to figure out how the virus invades into the central nervous system (CNS). In this study, CNS lesions were determined in rabbits and Mongolian gerbils inoculated with genotype 4 HEV. Junctional proteins were detected in HEV infected primary human brain microvascular cells (HBMVCs). Viral encephalitis associated perivascular cuffs of lymphocytes and microglial nodules were observed in HEV infected rabbits. Both positive- and negative-strand of HEV RNA was detected in brain and spinal cord in rabbits intraperitoneally infected with HEV at 28 dpi (days postinoculation), but not in rabbits gavaged with HEV. HEV ORF2 protein was further examined in both brain and spinal cord sections of infected rabbits, with positive signals located mainly in neural cells and perivascular areas. Ultrastructural study showed thickened and reduplicated basement membranes of capillary endothelium in HEV RNA positive brain tissues. In vitro study showed loss of tight junction proteins including Claudin5, Occludin, and ZO-1 (zonula occludens-1) in HBMVCs inoculated with HEV for 48 h. These findings indicated that disruption of the blood-brain barrier (BBB) might be potential mechanisms of HEV invasion into the CNS. It provides new insights to further study HEV associated neurologic disorders and will be helpful for seeking potential therapeutics for HEV infection in the future.

Hepatitis E Virus Induces Brain Injury Probably Associated With Mitochondrial Apoptosis.

Hepatitis E virus (HEV) infection has been associated with extrahepatic manifestations, particularly neurological disorders. Although it has been reported that HEV infection induced hepatocyte apoptosis associated with mitochondria injury, activation of mitochondrial apoptotic pathway in the central nervous system during HEV infection was not clearly understood. In this study, the induction of mitochondrial apoptosis-associated proteins and pro-inflammatory cytokines were detected in HEV infected Mongolian gerbil model and primary human brain microvascular endothelial cells (HBMVECs). Mitochondrial exhibited fragments with loss of cristae and matrix in HEV infected brain tissue by transmission electron microscope (TEM). In vitro studies showed that expression of NADPH oxidase 4 (NOX4) was significantly increased in HEV infected HBMVECs (p < 0.05), while ATP5A1 was significantly decreased (p < 0.01). Expressions of pro-apoptotic proteins were further evaluated. Bax was significantly increased in both HEV infected brain tissues and HBMVECs (p < 0.01). In vivo studies showed that caspase-9 and caspase-3 were activated after HEV inoculation (p < 0.01), associated with PCNA overexpression as response to apoptosis. Cytokines were measured to evaluate tissue inflammatory levels. Results showed that the release of TNFα and IL-1ß were significantly increased after HEV infection (p < 0.01), which might be attributed to microglia activation characterized by high level of IBA1 expression (p < 0.01). Taken together, these data support that HEV infection induces high levels of pro-inflammatory cytokines, associated with mitochondria-mediated apoptosis. The results provide new insight into mechanisms of extra-hepatic injury of HEV infection, especially in the central nervous system.

Replication of hepatitis E virus in the ovary and promotion of oocyte apoptosis in rabbits infected with HEV-4.

Hepatitis E virus (HEV) infection can induce infertility and miscarriage in pregnant women and infect neonates through vertical transmission. However, the mechanism of infertility and vertical transmission remains unclear. In the present study, we evaluated the replication of HEV in the ovary and structural and molecular changes induced by HEV after intraperitoneal injection of HEV in rabbits. Positive- and negative-strand HEV RNA was detected in the ovaries at 28 and 49 days post-infection. Positive HEV open reading frames 2 and 3 signals were observed in the ovaries by immunohistochemistry staining. Histopathological changes of ovarian tissues were observed, including scattered cell necrosis and lymphocyte infiltration. The ratio of normal follicles decreased, whereas the ratio of atresia follicles increased in the HEV RNA-positive ovaries compared to the control group by counting the number of follicles at all levels. In addition, TUNEL results showed that apoptosis in follicle cells and oocytes was promoted by HEV infection. These results suggest that the ovary is one of the replication sites of HEV and that the expression of HEV RNA and antigen in ovarian tissue caused structural and molecular changes that promoted germ cell apoptosis. HEV can infect and replicate in the ovum at different stages, which is a novel mechanism for HEV vertical transmission.

Detection of Genotype 4 Swine Hepatitis E Virus in Systemic Tissues in Cross-Species Infected Rabbits.

Increasing evidence demonstrates that hepatitis E virus (HEV) can be transmitted across species. According to previous reports, swine HEV has two genotypes, genotype 3 and 4, and both can infect humans by the fecal-oral route. Thus, it is crucial for the control of HEV zoonotic transmission to evaluate the dynamics of viral shedding and distribution in different tissues during cross-species infection by HEV. In this study, rabbits were infected with genotype 4 swine HEV by the intraperitoneal route. The results showed that HEV RNA not only shed in the feces but also in the saliva of some rabbits during infection with swine HEV. Viremia appeared late after infection, and anti-HEV IgG was not obvious until the appearance of high viremia levels. After the rabbits were euthanized, a histopathological examination showed that the livers developed overt hepatitis accompanied by an elevation of alanine aminotransferase (ALT) and aspartate transaminase (AST). Furthermore, HEV RNA was detected in various tissues, especially in the salivary glands and tonsils. Subsequently, negative-stranded HEV RNA was practiced in tissues with positive HEV RNA, which demonstrated that HEV replicated in the tissues. Next, we harvested additional tissues from the liver, salivary gland, tonsil, spleen, thymus gland, lymph node and intestine, which are known as replication sites of swine HEV. Additionally, we also observed the HEV antigen distributed in the organs above through immunohistochemical staining. These results demonstrate that rabbits could be used as an animal model for researching cross-species infection of genotype 4 HEV. It is also noteworthy that HEV can shed in the saliva and presents the risk of droplet transmission. These new data provide valuable information for understanding cross-species infection by HEV.

Development of a vivo rabbit ligated intestinal Loop Model for HCMV infection.

BACKGROUND: Human Cytomegalovirus (HCMV) infections can be found throughout the body, especially in epithelial tissue. Animal model was established by inoculation of HCMV (strain AD-169) or coinoculation with Hepatitis E virus (HEV) into the ligated sacculus rotundus and vermiform appendix in living rabbits. The specimens were collected from animals sacrificed 1 and a half hours after infection. RESULTS: The virus was found to be capable of reproducing in these specimens through RT-PCR and Western-blot. Severe inflammation damage was found in HCMV-infected tissue. The viral protein could be detected in high amounts in the mucosal epithelium and lamina propria by immunohistochemistry and immunofluorescense. Moreover, there are strong positive signals in lymphocytes, macrophages, and lymphoid follicles. Quantitative statistics indicate that lymphocytes among epithlium cells increased significantly in viral infection groups. CONCLUSIONS: The results showed that HCMV or HEV + HCMV can efficiently infect in rabbits by vivo ligated intestine loop inoculation. The present study successfully developed an infective model in vivo rabbit ligated intestinal Loop for HCMV pathogenesis study. This rabbit model can be helpful for understanding modulation of the gut immune system with HCMV infection.