Cryptosporidium parvum is a protozoan parasite that infects the gastrointestinal epithelium and causes diarrheal disease worldwide. Innate epithelial immune responses are key mediators of the host's defense to C. parvum. MicroRNAs (miRNAs) regulate gene expression at the posttranscriptional level and are involved in regulation of both innate and adaptive immune responses. Using an in vitro model of human cryptosporidiosis, we analyzed C. parvum-induced miRNA expression in biliary epithelial cells (i.e., cholangiocytes). Our results demonstrated differential alterations in the mature miRNA expression profile in cholangiocytes following C. parvum infection or lipopolysaccharide stimulation. Database analysis of C. parvum-upregulated miRNAs revealed potential NF-κB binding sites in the promoter elements of a subset of miRNA genes. We demonstrated that mir-125b-1, mir-21, mir-30b, and mir-23b-27b-24-1 cluster genes were transactivated through promoter binding of the NF-κB p65 subunit following C. parvum infection. In contrast, C. parvum transactivated mir-30c and mir-16 genes in cholangiocytes in a p65-independent manner. Importantly, functional inhibition of selected p65-dependent miRNAs in cholangiocytes increased C. parvum burden. Thus, we have identified a panel of miRNAs regulated through promoter binding of the NF-κB p65 subunit in human cholangiocytes in response to C. parvum infection, a process that may be relevant to the regulation of epithelial anti-microbial defense in general.

MicroRNAs (miRNAs), small non-coding regulatory RNAs that regulate gene expression at the post-transcriptional level, are master regulators of a wide array of cellular processes. Altered miRNA expression could be a determinant of disease development and/or progression and manipulation of miRNA expression represents a potential avenue of therapy. Exosomes are cell-derived extracellular vesicles that promote cell–cell communication and immunoregulatory functions. These “bioactive vesicles” shuttle various molecules, including miRNAs, to recipient cells. Inappropriate release of miRNAs from exosomes may cause significant alterations in biological pathways that affect disease development, supporting the concept that miRNA-containing exosomes could serve as targeted therapies for particular diseases. This review briefly summarizes recent advances in the biology, function, and therapeutic potential of exosomal miRNAs.

LincRNAs are long non-coding transcripts (>200 nt) from the intergenic regions of annotated protein-coding genes. One of the most highly induced lincRNAs in macrophages upon TLR ligation is lincRNA-Cox2, which has recently been shown to mediate both the activation and repression of distinct classes of immune genes in innate immune cells. We report here that lincRNA-Cox2 located at chromosome 1 proximal to the prostaglandin-endoperoxide synthase 2 (Ptgs2/Cox2) gene is an early-primary inflammatory gene controlled by NF-κB signaling in murine macrophages. Functionally, lincRNA-Cox2 is required for the transcription of NF-κB-regulated late-primary inflammatory response genes stimulated by bacterial lipopolysaccharide. Specifically, lincRNA-Cox2 is assembled into the SWI/SNF (SWItch/Sucrose NonFermentable) complex in cells after lipopolysaccharide stimulation. This resulting lincRNA-Cox2/SWI/SNF complex can modulate the assembly of NF-κB subunits to the SWI/SNF complex, and ultimately, SWI/SNF-associated chromatin remodeling and transactivation of the late-primary inflammatory response genes in macrophages in response to microbial challenge. Therefore, our data indicate a new regulatory role of NF-κB-induced lincRNA-Cox2 to act as a co-activator of NF-κB for the transcription of late-primary response genes in innate immune cells through modulation of epigenetic chromatin remodeling.

Insulin-dependent (type 1) diabetes mellitus (T1D) onset is mediated by individual human genetics as well as undefined environmental influences such as viral infections. The group B coxsackieviruses (CVB) are commonly named as putative T1D-inducing agents. We studied CVB replication in nonobese diabetic (NOD) mice to assess how infection by diverse CVB strains affected T1D incidence in a model of human T1D. Inoculation of 4-or 8-week-old NOD mice with any of nine different CVB strains significantly reduced the incidence of T1D by 2-to 10-fold over a 10-month period relative to T1D incidences in mock-infected control mice. Greater protection was conferred by more-pathogenic CVB strains relative to less-virulent or avirulent strains. Two CVB3 strains were employed to further explore the relationship of CVB virulence phenotypes to T1D onset and incidence: a pathogenic strain (CVB3/M) and a nonvirulent strain (CVB3/GA). CVB3/M replicated to four-to fivefold-higher titers than CVB3/GA in the pancreas and induced widespread pancreatitis, whereas CVB3/GA induced no pancreatitis. Apoptotic nuclei were detected by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay in CVB3/M-infected pancreata but not in CVB3/GA-infected pancreata. In situ hybridization detected CVB3 RNA in acinar tissue but not in pancreatic islets. Although islets demonstrated inflammatory infiltrates in CVB3-protected mice, insulin remained detectable by immunohistochemistry in these islets but not in those from diabetic mice. Enzyme-linked immunosorbent assay-based examination of murine sera for immunoglobulin G1 (IgG1) and IgG2a immunoreactivity against diabetic autoantigens insulin and HSP60 revealed no statistically significant relationship between CVB3-protected mice or diabetic mice and specific autoimmunity. However, when pooled sera from CVB3/M-protected mice were used to probe a Western blot of pancreatic proteins, numerous proteins were detected, whereas only one band was detected by sera from CVB3/GA-protected mice. No proteins were detected by sera from diabetic or normal mice. Cumulatively, these data do not support the hypothesis that CVB are causative agents of T1D. To the contrary, CVB infections provide significant protection from T1D onset in NOD mice. Possible mechanisms by which this virus-induced protection may occur are discussed.The group B coxsackieviruses (CVB; family Picornaviridae, genus Enterovirus, species group B coxsackievirus; six serotypes, CVB1 to -6) are among the best studied of human enteroviruses (102). The CVB genome is a single strand of positive sense RNA 7,400 nucleotides in length that encodes 11 proteins in a single open reading frame (89). The CVB have been associated with diverse human diseases, among the more serious of which are myocarditis, pancreatitis, and aseptic meningitis. The CVB have been soundly implicated as causes of human myocarditis (1, 26, 42, 60-62, 73, 74, 108, 109) and pancreatitis (2,41,54,58,66,107) and, furthermore, cause these diseases readily i...

Group B coxsackieviruses (CVB) are believed to trigger some cases of human type 1 diabetes (T1D), although the mechanism by which this may occur has not been shown. We demonstrated previously that inoculation of young nonobese diabetic (NOD) mice with any of several different CVB strains reduced T1D incidence. We also observed no evidence of CVB replication within islets of young NOD mice, suggesting no role for CVB in T1D induction in the NOD mouse model. The failure to observe CVB replication within islets of young NOD mice has been proposed to be due to interferon expression by insulin-producing beta cells or lack of expression of the CVB receptor CAR. We found that CAR protein is detectable within islets of young and older NOD mice and that a CVB3 strain, which expresses murine IL-4, can replicate in islets. Mice inoculated with the IL-4 expressing CVB3 chimeric strain were better protected from T1D onset than were mock-infected control mice despite intraislet viral replication. Having demonstrated that CVB can replicate in healthy islets of young NOD mice when the intraislet environment is suitably altered, we asked whether islets in old prediabetic mice were resistant to CVB infection. Unlike young mice in which insulitis is not yet apparent, older NOD mice demonstrate severe insulitis in all islets. Inoculating older prediabetic mice with different pathogenic CVB strains caused accelerated T1D onset relative to control mice, a phenomenon that was preceded by detection of virus within islets. Together, the results suggest a model for resolving conflicting data regarding the role of CVB in human T1D etiology.

Enteroviruses can induce human myocarditis, which can be modeled in mice inoculated with group B coxsackieviruses (CVB) and in which CVB evolve to produce defective, terminally deleted genomes. The 5' non-translated region (NTR) was enzymatically amplified from heart tissue of a fatal case of enterovirus-associated myocarditis in Japan in 2002. While no intact 5' viral genomic termini were detected, 5' terminal deletions ranged in size from 22 to 36 nucleotides. Sequence of the 5' third of this viral genome is of a modern strain, closely related to CVB2 strains isolated in Japan in 2002. A CVB3 chimera containing the 5' NTR with a 22 nt deletion produced progeny virus upon transfection of HeLa cells. When the 5' 22 nucleotide deletion was repaired, the virus induced myocarditis in mice and replicated like wild type virus in murine heart cells. This is the first report of these naturally-occurring defective enteroviral genomes in human myocarditis.

Type 1 diabetes (T1D) is an autoimmune disease in which the immune system mounts an attack on the host's insulin-producing beta cells. Because most cases of T1D cannot be attributed only to individual genetics, it is strongly inferred that there is a significant environmental contribution, such as infection, impacting disease development. The human enteroviruses (HEV) are common picornaviruses often implicated as triggers of human T1D, although precisely which of the numerous HEV may be involved in human T1D development is unknown. Experiments using non-obese diabetic (NOD) mice, commonly used to model T1D, show that induction of T1D by HEV infection in NOD mice is a multifactorial process involving both the virus and the host. Interestingly, results demonstrate that HEV infection of NOD mice can also induce long-term protection from T1D under certain conditions, suggesting that a similar mechanism may occur in humans. Based upon both experimental animal and observational human studies, we postulate that HEV have a dual role in T1D development and can either cause or prevent autoimmune disease. Whichever outcome occurs depends upon multiple variables in the host-virus equation, many of which can be deduced from results obtained from NOD mouse studies. We propose that the background to the sharply rising T1D incidences observed in the 20th century correlates with increased levels of hygiene in human societies. Viewing T1D in this perspective suggests that potential preventative options could be developed.

The precise factors involved in the development of a progressive motor dysfunction, a hallmark of immune-mediated demyelinating diseases such as multiple sclerosis, are not well defined. The ability to identify neurologic deficits that result in impaired motor performance early in disease may allow for the identification of therapeutic interventions that slow or eliminate the progression toward a permanent dysfunction. Here we describe the use of three objective, quantitative functional assays (spontaneous activity box, rotarod, and footprint analysis) to detect early neurologic deficits following the initiation of a demyelinating disease with Theiler's murine encephalomyelitis virus (TMEV). The results show that the assays are capable of revealing neurologic deficits at the early stages of the demyelinating disease process. These findings are the first to objectively characterize neurologic function in an animal model of progressive CNS demyelination.