NLRC3, a member of the NLR family of proteins, is a negative regulator of innate immune signaling induced by the DNA sensor STING.

Stimulator of interferon genes (STING, also named MITA, MYPS, or ERIS) is an intracellular DNA sensor that induces type I interferon through its interaction with TANK-binding kinase 1 (TBK1). Here we found that the nucleotide-binding, leucine-rich-repeat-containing protein, NLRC3, reduced STING-dependent innate immune activation in response to cytosolic DNA, cyclic di-GMP (c-di-GMP), and DNA viruses. NLRC3 associated with both STING and TBK1 and impeded STING-TBK1 interaction and downstream type I interferon production. By using purified recombinant proteins, we found NLRC3 to interact directly with STING. Furthermore, NLRC3 prevented proper trafficking of STING to perinuclear and punctated region, known to be important for its activation. In animals, herpes simplex virus 1 (HSV-1)-infected Nlrc3(-/-) mice exhibited enhanced innate immunity and reduced morbidity and viral load. This demonstrates the intersection of two key pathways of innate immune regulation, NLR and STING, to fine tune host response to intracellular DNA, DNA virus, and c-di-GMP.

Contemporary issues in anaphylaxis and the evolution of epinephrine autoinjectors: What will the future bring?

BACKGROUND: Food allergy and anaphylaxis appear to be increasing in the United States, especially in young children, and preparedness is paramount to successful emergency management in the community. Although the treatment of choice for anaphylaxis is epinephrine delivered by autoinjection, some devices are challenged by less user-friendly designs or pose the risk of injury, especially in young patients. Human factors engineering has played a larger role in the development of more recent epinephrine autoinjector technologies and will continue to play a role in the evolution and future design of epinephrine autoinjectors. OBJECTIVE: To discuss contemporary issues related to the identification and management of anaphylaxis, current and future epinephrine autoinjector design, and unmet needs for the treatment of special populations, namely, young children weighing less than 15 kg. METHODS: The literature was reviewed and select articles retrieved to support expert clinical opinions on the need for improved recognition of anaphylaxis, epinephrine autoinjector design, and unmet needs in special populations. RESULTS: Anaphylaxis may be underrecognized and poorly defined in infant- and toddler-aged children, current devices may not be adequate to safely treat these patients (ie, inappropriate needle length), and health care professionals may not be aware of these issues. CONCLUSION: As epinephrine autoinjector technology continues to evolve, device characteristics that promote safe, user-friendly experiences and give clinicians and their patients confidence to successfully treat anaphylaxis during an emergency, without injury, will be favored.

An important role for mitochondrial antiviral signaling protein in the Kaposi's sarcoma-associated herpesvirus life cycle.

UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) has been shown to be recognized by two families of pattern recognition receptors (PRRs), Toll-like receptors (TLRs) and NOD-like receptors (NLRs). Here we show that MAVS and RIG-I (retinoic acid-inducible gene 1), an RLR family member, also have a role in suppressing KSHV replication and production. In the context of primary infection, we show that in cells with depleted levels of MAVS or RIG-I, KSHV transcription is increased, while beta interferon (IFN-ß) induction is attenuated. We also observed that MAVS and RIG-I are critical during the process of reactivation. Depletion of MAVS and RIG-I prior to reactivation led to increased viral load and production of infectious virus. Finally, MAVS depletion in latent KSHV-infected B cells leads to increased viral gene transcription. Overall, this study suggests a role for MAVS and RIG-I signaling during different stages of the KSHV life cycle. IMPORTANCE: We show that RIG-I and its adaptor protein, MAVS, can sense KSHV infection and that these proteins can suppress KSHV replication following primary infection and/or viral reactivation.

The viral interferon regulatory factors of kaposi's sarcoma-associated herpesvirus differ in their inhibition of interferon activation mediated by toll-like receptor 3.

Kaposi's sarcoma-associated herpesvirus (KSHV) infection is correlated with three human malignancies and can establish lifelong latent infection in multiple cell types within its human host. In order to establish and maintain infection, KSHV utilizes multiple mechanisms to evade the host immune response. One such mechanism is the expression of a family of genes with homology to cellular interferon (IFN) regulatory factors (IRFs), known as viral IRFs (vIRFs). We demonstrate here that KSHV vIRF1, -2, and -3 have a differential ability to block type I interferon signaling mediated by Toll-like receptor 3 (TLR3), a receptor we have previously shown to be activated upon KSHV infection. vIRF1, -2, and -3 inhibited TLR3-driven activation of IFN transcription reporters. However, only vIRF1 and vIRF2 inhibited increases in both IFN-ß message and protein levels following TLR3 activation. The expression of vIRF1 and vIRF2 also allowed for increased replication of a virus known to activate TLR3 signaling. Furthermore, vIRF1 and vIRF2 may block TLR3-mediated signaling via different mechanisms. Altogether, this report indicates that vIRFs are able to block IFN mediated by TLRs but that each vIRF has a unique function and mechanism for blocking antiviral IFN responses.

Latent Kaposi's sarcoma-associated herpesvirus infection of monocytes downregulates expression of adaptive immune response costimulatory receptors and proinflammatory cytokines.

Kaposi's sarcoma-associated herpesvirus (KSHV) infection is associated with the development of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. We report the establishment of a monocytic cell line latently infected with KSHV (KSHV-THP-1). We profiled viral and cytokine gene expression in the KSHV-THP-1 cells compared to that in uninfected THP-1 cells and found that several genes involved in the host immune response were downregulated during latent infection, including genes for CD80, CD86, and the cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß). Thus, KSHV minimizes its immunological signature by suppressing key immune response factors, enabling persistent infection and evasion from host detection.

Activation of plasmacytoid dendritic cells by Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with multiple human malignancies, including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Following primary infection, KSHV typically goes through a brief period of lytic replication prior to the establishment of latency. Plasmacytoid dendritic cells (pDCs) are the major producers of type 1 interferon (IFN), primarily in response to virus infection. Toll-like receptors (TLRs) are key components of the innate immune system, and they serve as pathogen recognition receptors that stimulate the host antiviral response. pDCs express exclusively TLR7 and TLR9, and it is through these TLRs that the type 1 interferon response is activated in pDCs. Currently, it is not known whether KSHV is recognized by pDCs and whether activation of pDCs occurs in response to KSHV infection. We now report evidence that KSHV can infect human pDCs and that pDCs are activated upon KSHV infection, as measured by upregulation of CD83 and CD86 and by IFN-α secretion. We further show that induction of IFN-α occurs through activation of TLR9 signaling and that a TLR9 inhibitor diminishes the production and secretion of IFN-α by KSHV-infected pDCs.

Toll-like receptor signaling controls reactivation of KSHV from latency.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. Like other herpesviruses, KSHV establishes life-long latency in the human host with intermittent periods of reactivation. Physiological triggers of herpesviral reactivation are poorly defined. Toll-like receptors (TLRs) recognize pathogens and are vital for the host innate immune response. We screened multiple TLR agonists for their ability to initiate KSHV replication in latently infected PEL. Agonists specific for TLR7/8 reactivated latent KSHV and induced viral lytic gene transcription and replication. Furthermore, vesicular stomatitis virus (VSV), a bonafide physiological activator of TLR7/8, also reactivated KSHV from latency. This demonstrates that secondary pathogen infection of latently infected cells can reactivate KSHV. Human herpesviruses establish life-long latency in the host, and it is plausible that a latently infected cell will encounter multiple pathogens during its lifetime and that these encounters lead to episodic reactivation. Our findings have broad implications for physiological triggers of latent viral infections, such as herpesviral reactivation and persistence in the host.

Evaluation of the Onclarity HPV assay on the high-throughput COR system.

Background: Here we compare the performance of the high-throughput BD COR System (COR) to the Viper LT System (Viper) using the BD Onclarity HPV assay.Research Design and Methods: Remnant clinical specimens, contrived specimens in SurePath (BD) and PreservCyt (Hologic) media, and prospective clinical specimens in BD Cervical Brush Diluent (CBD) were tested. Outcomes included intra-laboratory agreement of Onclarity results on COR and inter-system agreement between COR and Viper.Results: Onclarity reproducibility on COR resulted in standard deviation and correlation of variation of Ct values ranging from 0.14 to 1.98 and 0.49% to 2.15%, respectively, for contrived specimens, and 0.9-3.08 and 2.89-9.21%, respectively, for clinical specimens. In the COR and Viper clinical agreement study, OPA for Onclarity ranged from 97.1%-98.9%, depending on the collection media type. PPA values for pooled, HPV(+) specimens at low positive (C95), and moderate positive (3XC95) target concentrations were ≥95.0% and 100%, respectively; PPA values associated with HPV 16, 18, 31, 45, 33/58, 52, 35/39/68, 51, and 56/59/66, individually, ranged from 93.8%-100%.Conclusions: Onclarity performance on COR is equivalent to Viper, and is accurate and reproducible for detection of all high-risk HPV genotypes, with a throughput of 330 results from a single 8-hour shift.

Extended HPV Genotyping to Compare HPV Type Distribution in Self- and Provider-Collected Samples for Cervical Cancer Screening.

BACKGROUND: Primary high-risk human papillomavirus (hr-HPV) testing of self-collected cervico-vaginal swabs could increase cervical cancer screening coverage, although triage strategies are needed to reduce unnecessary colposcopies. We evaluated the use of extended hr-HPV genotyping of self-collected samples for cervical cancer screening. METHODS: We recruited women ages 25-65 years at two colposcopy clinics in North Carolina between November 2016 and January 2019, and obtained self-collected cervico-vaginal samples, provider-collected cervical samples, and cervical biopsies from all enrolled women. Self- and provider-collected samples were tested for 14 hr-HPV genotypes using the Onclarity Assay (Becton Dickinson). We calculated hr-HPV genotype-specific prevalence and assessed agreement between results in self- and provider-collected samples. We ranked the hr-HPV genotypes according to their positive predictive value (PPV) for the detection of cervical intraepithelial neoplasia (CIN) grade 2 or higher (CIN2+). RESULTS: A total of 314 women participated (median age, 36 years); 85 women (27%) had CIN2+. More women tested positive for any hr-HPV on self-collected (76%) than on provider-collected samples (70%; P = 0.009) with type-specific agreement ranging from substantial to almost perfect. HPV-16 was the most common genotype in self-collected (27%) and provider-collected samples (20%), and HPV-16 prevalence was higher in self- than provider-collected samples (P < 0.001). In self- and provider-collected samples, HPV-16 had the highest PPV for CIN2+ detection. CONCLUSIONS: Overall sensitivity for CIN2+ detection was similar for both sample types, but the higher HPV-16 prevalence in self-collected samples could result in increased colposcopy referral rates. IMPACT: Additional molecular markers might be helpful to improve the triage of women who are hr-HPV positive on self-collected samples.

Genome size, cell size, and the evolution of enucleated erythrocytes in attenuate salamanders.

Within the salamander family Plethodontidae, five different clades have evolved high levels of enucleated red blood cells, which are extremely unusual among non-mammalian vertebrates. In each of these five clades, the salamanders have large genomes and miniaturized or attenuated body forms. Such a correlation suggests that the loss of nuclei in red blood cells may be related, in part, to the interaction between large genome size and small body size, which has been shown to have profound morphological consequences for the nervous and visual systems in plethodontids. Previous work has demonstrated that variation in both the level of enucleated cells and the size of the nuclear genome exists among species of the monophyletic plethodontid genus Batrachoseps. Here, we report extensive intraspecific variation in levels of enucleated red blood cells in 15 species and provide measurements of red blood cell size, nucleus size, and genome size for 13 species of Batrachoseps. We present a new phylogenetic hypothesis for the genus based on 6150bp of mitochondrial DNA sequence data from nine exemplar taxa and use it to examine the relationship between genome size and enucleated red blood cell morphology in a phylogenetic framework. Our analyses demonstrate positive direct correlations between genome size, nucleus size, and both nucleated and enucleated cell sizes within Batrachoseps, although only the relationship between genome size and nucleus size is significant when phylogenetically independent contrasts are used. In light of our results and broader studies of comparative hematology, we propose that high levels of enucleated, variably sized red blood cells in Batrachoseps may have evolved in response to rheological problems associated with the circulation of large red blood cells containing large, bulky nuclei in an attenuate organism.

HIV integrase inhibitors block replication of alpha-, beta-, and gammaherpesviruses.

The catalytic site of the HIV integrase is contained within an RNase H-like fold, and numerous drugs have been developed that bind to this site and inhibit its activity. Herpes simplex virus (HSV) encodes two proteins with potential RNase H-like folds, the infected cell protein 8 (ICP8) DNA-binding protein, which is necessary for viral DNA replication and exhibits recombinase activity in vitro, and the viral terminase, which is essential for viral DNA cleavage and packaging. Therefore, we hypothesized that HIV integrase inhibitors might also inhibit HSV replication by targeting ICP8 and/or the terminase. To test this, we evaluated the effect of 118-D-24, a potent HIV integrase inhibitor, on HSV replication. We found that 118-D-24 inhibited HSV-1 replication in cell culture at submillimolar concentrations. To identify more potent inhibitors of HSV replication, we screened a panel of integrase inhibitors, and one compound with greater anti-HSV-1 activity, XZ45, was chosen for further analysis. XZ45 significantly inhibited HSV-1 and HSV-2 replication in different cell types, with 50% inhibitory concentrations that were approximately 1 µM, but exhibited low cytotoxicity, with a 50% cytotoxic concentration greater than 500 µM. XZ45 blocked HSV viral DNA replication and late gene expression. XZ45 also inhibited viral recombination in infected cells and ICP8 recombinase activity in vitro. Furthermore, XZ45 inhibited human cytomegalovirus replication and induction of Kaposi's sarcoma herpesvirus from latent infection. Our results argue that inhibitors of enzymes with RNase H-like folds may represent a general antiviral strategy, which is useful not only against HIV but also against herpesviruses. Importance: The herpesviruses cause considerable morbidity and mortality. Nucleoside analogs have served as effective antiviral agents against the herpesviruses, but resistance can arise through viral mutation. Second-line anti-herpes drugs have limitations in terms of pharmacokinetic properties and/or toxicity, so there is a great need for additional drugs for treatment of herpesviral infections. This study showed that the HIV integrase inhibitors also block herpesviral infection, raising the important potential of a new class of anti-herpes drugs and the prospect of drugs that combat both HIV and the herpesviruses.

Tousled-like kinases modulate reactivation of gammaherpesviruses from latency.

Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to human malignancies. The majority of tumor cells harbor latent virus, and a small percentage undergo spontaneous lytic replication. Both latency and lytic replication are important for viral pathogenesis and spread, but the cellular players involved in the switch between the two viral life-cycle phases are not clearly understood. We conducted a small interfering RNA (siRNA) screen targeting the cellular kinome and identified Tousled-like kinases (TLKs) as cellular kinases that control KSHV reactivation from latency. Upon treatment of latent KSHV-infected cells with siRNAs targeting TLKs, we saw robust viral reactivation. Knockdown of TLKs in latent KSHV-infected cells induced expression of viral lytic proteins and production of infectious virus. TLKs were also found to play a role in regulating reactivation from latency of another related oncogenic gammaherpesvirus, Epstein-Barr virus. Our results establish the TLKs as cellular repressors of gammaherpesvirus reactivation.

Toll-like receptor sensing of human herpesvirus infection.

Toll-like receptors (TLRs) are evolutionarily conserved pathogen sensors that constitute the first line of defense in the human immune system. Herpesviruses are prevalent throughout the world and cause significant disease in the human population. Sensing of herpesviruses via TLRs has only been documented in the last 10 years and our understanding of the relationship between these sentinels of the immune system and herpesvirus infection has already provided great insight into how the host cell responds to viral infection. This report will summarize the activation and modulation of TLR signaling in the context of human herpesvirus infections.

Inhibition of the inflammasome response by a viral protein that interacts with NLRs.

Pattern recognition receptors (PRRs) constitute the first line of host defense against bacterial, fungal and viral pathogens. Upon sensing microbial infection, PRRs initiate a cascade of signal transduction and transcriptional events to induce the production of inflammatory cytokines. As a result, many pathogens have evolved to evade PRR detection and activation in order to establish a successful infection. In a recent report, we described how a viral protein named Orf63 encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) inhibits activation of several members of a family of PRRs called NLRs (nucleotide-binding and oligomerization, leucine-rich repeat) by functionally inhibiting the NLR response. This resulted in reduced NLR-dependent pro-inflammatory cytokine secretion and cell death. Moreover, Orf63 was essential in the KSHV lifecycle. Thus, our work suggests KSHV has evolved to encode a functional homolog of NLR proteins in an effort to suppress the host inflammatory response.

Discovery of a viral NLR homolog that inhibits the inflammasome.

The NLR (nucleotide binding and oligomerization, leucine-rich repeat) family of proteins senses microbial infections and activates the inflammasome, a multiprotein complex that promotes microbial clearance. Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to several human malignancies. We found that KSHV Orf63 is a viral homolog of human NLRP1. Orf63 blocked NLRP1-dependent innate immune responses, including caspase-1 activation and processing of interleukins IL-1ß and IL-18. KSHV Orf63 interacted with NLRP1, NLRP3, and NOD2. Inhibition of Orf63 expression resulted in increased expression of IL-1ß during the KSHV life cycle. Furthermore, inhibition of NLRP1 was necessary for efficient reactivation and generation of progeny virus. The viral homolog subverts the function of cellular NLRs, which suggests that modulation of NLR-mediated innate immunity is important for the lifelong persistence of herpesviruses.

Acute silver toxicity in aquatic animals is a function of sodium uptake rate.

On the basis of these facts about freshwater fish and invertebrates: (i) the Na+ turnover is a physiological process associated with the gill membranes; (ii) the key mechanism of acute silver toxicity consists of reduction in Na+ uptake by blockade of gill Na+,K+-ATPase; (iii) the mass-specific surface area of the gills depends on animal body mass; and (iv) the gill surface is also the major site of Na+ loss by diffusion, we hypothesized that whole body Na+ uptake rate (i.e., turnover rate) and secondarily body mass would be good predictors of acute silver toxicity. Results obtained from toxicological (LC50 of AgNO3) and physiological (22Na uptake rate) tests performed on juvenile fish (rainbow trout, Oncorhynchus mykiss), early juvenile and adult crayfish (Cambarusdiogenes diogenes), and neonate and adult daphnids (Daphnia magna) in moderately hard water of constant quality support the above hypothesis. Therefore, sensitivity to AgNO3, in terms of either total measured silver or free Ag+, was reliably predicted from the whole body Na+ uptake rate in animals with body mass ranging over 6 orders of magnitude (from micrograms to grams). A positive log-log correlation between acute AgNO3 toxicity and body mass of the same species was also observed. Furthermore, the whole body Na+ uptake rate was inversely related to body mass in unexposed animals. The combination of these last two results explains why the small animals in this study were more sensitive to Ag+ than the larger ones. Taken together, these results clearly point out the possibility of incorporating the Na+ uptake rate into the current version of the Biotic Ligand Model to improve the predictive capacity of this model. In the absence of information on Na+ uptake rate, then body mass may serve as a surrogate.