Carbon-infiltrated carbon nanotubes inhibit the development of Staphylococcus aureus biofilms.

Staphylococcus aureus forms biofilms that cause considerable morbidity and mortality in patients who receive implanted devices such as prosthetics or fixator pins. An ideal surface for such medical devices would inhibit biofilm growth. Recently, it was reported that surface modification of stainless steel materials with carbon-infiltrated carbon nanotubes (CICNT) inhibits the growth of S. aureus biofilms. The purpose of this study was to investigate this antimicrobial effect on titanium materials with CICNT coated surfaces in a variety of surface morphologies and across a broader spectrum of S. aureus isolates. Study samples of CICNT-coated titanium, and control samples of bare titanium, a common implant material, were exposed to S. aureus. Viable bacteria were removed from adhered biofilms and quantified as colony forming units. Scanning electron microscopy was used to qualitatively analyze biofilms both before and after removal of cells. The CICNT surface was found to have significantly fewer adherent bacteria than bare titanium control surfaces, both via colony forming unit and microscopic analyses. This effect was most pronounced on CICNT surfaces with an average nanotube diameter of 150 nm, showing a 2.5-fold reduction in adherent bacteria. Since S. aureus forms different biofilm structures by isolate and by growth conditions, we tested 7 total isolates and found a significant reduction in the biofilm load in six out of seven S. aureus isolates tested. To examine whether the anti-biofilm effect was due to the structure of the nanotubes, we generated an unstructured carbon surface. Significantly more bacteria adhered to a nonstructured carbon surface than to the 150 nm CICNT surface, suggesting that the topography of the nanotube structure itself has anti-biofilm properties. The CICNT surface possesses anti-biofilm properties that result in fewer adherent S. aureus bacteria. These anti-biofilm properties are consistent across multiple isolates of S. aureus and are affected by nanotube diameter. The experiments performed in this study suggest that this effect is due to the nanostructure of the CICNT surface.

Comparison of Intracellular Transcriptional Response of NHBE Cells to Infection with SARS-CoV-2 Washington and New York Strains.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in Wuhan, China in December 2019 and caused a global pandemic resulting in millions of deaths and tens of millions of patients positive tests. While studies have shown a D614G mutation in the viral spike protein are more transmissible, the effects of this and other mutations on the host response, especially at the cellular level, are yet to be fully elucidated. In this experiment we infected normal human bronchial epithelial (NHBE) cells with the Washington (D614) strain or the New York (G614) strains of SARS-CoV-2. We generated RNA sequencing data at 6, 12, and 24 hours post-infection (hpi) to improve our understanding of how the intracellular host response differs between infections with these two strains. We analyzed these data with a bioinformatics pipeline that identifies differentially expressed genes (DEGs), enriched Gene Ontology (GO) terms and dysregulated signaling pathways. We detected over 2,000 DEGs, over 600 GO terms, and 29 affected pathways between the two infections. Many of these entities play a role in immune signaling and response. A comparison between strains and time points showed a higher similarity between matched time points than across different time points with the same strain in DEGs and affected pathways, but found more similarity between strains across different time points when looking at GO terms. A comparison of the affected pathways showed that the 24hpi samples of the New York strain were more similar to the 12hpi samples of the Washington strain, with a large number of pathways related to translation being inhibited in both strains. These results suggest that the various mutations contained in the genome of these two viral isolates may cause distinct effects on the host transcriptional response in infected host cells, especially relating to how quickly translation is dysregulated after infection. This comparison of the intracellular host response to infection with these two SARS-CoV-2 isolates suggest that some of the mechanisms associated with more severe disease from these viruses could include virus replication, metal ion usage, host translation shutoff, host transcript stability, and immune inhibition.

Characterization of Staphylococcus aureus biofilms via crystal violet binding and biochemical composition assays of isolates from hospitals, raw meat, and biofilm-associated gene mutants.

Staphylococcus aureus (SA) is a gram-positive coccus and an opportunistic pathogen of humans. The ability of SA to form biofilms is an important virulence mechanism because biofilms are protected from host immune responses and antibiotic treatment. This study examines the relative biofilm strength of a variety of hospital and meat-associated strains of SA, using a crystal violet (CV) staining assay. Biofilms were treated with either DNase or proteinase K prior to CV staining, and compared to mock-treated results, to better understand the biochemical composition. Biofilm polysaccharide concentration was also measured using the phenol sulfuric-acid assay which was normalized to base biofilm strength. We found that hospital-associated isolates have biofilms that bind significantly more CV than for meat isolates and are significantly more protein and polysaccharide-based while meat isolates have significantly more DNA-based biofilms. This study also investigates the effects that biofilm-related genes have on biofilm formation and composition by analyzing specific transposon mutants of genes previously shown to play a role in biofilm development. agrA, atl, clfA, fnbA, purH, and sarA mutants produce significantly weaker biofilms (bind less CV) as compared to a wild-type control, whereas the acnA mutant produces a significantly stronger biofilm. Biofilms formed from these mutant strains were treated (or mock-treated) with DNase or proteinase K and tested with phenol and sulfuric acid to determine what role these genes play in biofilm composition. The acnA, clfA, fnbA, and purH mutants showed significant reduction in biofilm staining after either proteinase K or DNase treatment, agrA and sarA mutants showed significant biofilm reduction after only proteinase K treatment, and an atl mutant did not show significant biofilm reduction after either proteinase K or DNase treatment. These data suggest that biofilms that form without acnA, clfA, fnbA, and purH are DNA- and protein-based, that biofilms lacking agrA and sarA are mainly protein-based, and biofilms lacking atl are mainly polysaccharide-based. These results help to elucidate how these genes affect biofilm formation and demonstrate how mutating biofilm-related genes in SA can cause a change in biofilm composition.

Chikungunya virus time course infection of human macrophages reveals intracellular signaling pathways relevant to repurposed therapeutics.

Background: Chikungunya virus (CHIKV) is a mosquito-borne pathogen, within the Alphavirus genus of the Togaviridae family, that causes ~1.1 million human infections annually. CHIKV uses Aedes albopictus and Aedes aegypti mosquitoes as insect vectors. Human infections can develop arthralgia and myalgia, which results in debilitating pain for weeks, months, and even years after acute infection. No therapeutic treatments or vaccines currently exist for many alphaviruses, including CHIKV. Targeting the phagocytosis of CHIKV by macrophages after mosquito transmission plays an important role in early productive viral infection in humans, and could reduce viral replication and/or symptoms. Methods: To better characterize the transcriptional response of macrophages during early infection, we generated RNA-sequencing data from a CHIKV-infected human macrophage cell line at eight or 24 hours post-infection (hpi), together with mock-infected controls. We then calculated differential gene expression, enriched functional annotations, modulated intracellular signaling pathways, and predicted therapeutic drugs from these sequencing data. Results: We observed 234 pathways were significantly affected 24 hpi, resulting in six potential pharmaceutical treatments to modulate the affected pathways. A subset of significant pathways at 24 hpi includes AGE-RAGE, Fc epsilon RI, Chronic myeloid leukemia, Fc gamma R-mediated phagocytosis, and Ras signaling. We found that the MAPK1 and MAPK3 proteins are shared among this subset of pathways and that Telmisartan and Dasatinib are strong candidates for repurposed small molecule therapeutics that target human processes. The results of our analysis can be further characterized in the wet lab to contribute to the development of host-based prophylactics and therapeutics.

Alphaviruses: Host pathogenesis, immune response, and vaccine & treatment updates.

Human pathogens belonging to the Alphavirus genus, in the Togaviridae family, are transmitted primarily by mosquitoes. The signs and symptoms associated with these viruses include fever and polyarthralgia, defined as joint pain and inflammation, as well as encephalitis. In the last decade, our understanding of the interactions between members of the alphavirus genus and the human host has increased due to the re-appearance of the chikungunya virus (CHIKV) in Asia and Europe, as well as its emergence in the Americas. Alphaviruses affect host immunity through cytokines and the interferon response. Understanding alphavirus interactions with both the innate immune system as well as the various cells in the adaptive immune systems is critical to developing effective therapeutics. In this review, we summarize the latest research on alphavirus-host cell interactions, underlying infection mechanisms, and possible treatments.

Evaluation of a Surrogate Enzyme-Linked Immunosorbent Assay-Based Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) cPass Neutralization Antibody Detection Assay and Correlation With Immunoglobulin G Commercial Serology Assays.

CONTEXT.­: Emerging evidence shows correlation between the presence of neutralization antibodies (nAbs) and protective immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently available commercial serology assays lack the ability to specifically identify nAbs. An enzyme-linked immunosorbent assay-based nAb assay (GenScript cPass neutralization antibody assay) has recently received emergency use authorization from the Food and Drug Administration. OBJECTIVE.­: To evaluate the performance characteristics of this assay and compare and correlate it with the commercial assays that detect SARS-CoV-2-specific immunoglobulin G (IgG). DESIGN.­: Specimens from SARS-COV-2 infected patients (n = 124), healthy donors obtained prepandemic (n = 100), and patients with non-coronavirus disease 2019 (COVID-19) respiratory infections (n = 92) were analyzed using this assay. Samples with residual volume were also tested on 3 commercial serology platforms (Abbott, Euroimmun, Siemens). Twenty-eight randomly selected specimens from patients with COVID-19 and 10 healthy controls were subjected to a plaque reduction neutralization test. RESULTS.­: The cPass assay exhibited 96.1% (95% CI, 94.9%-97.3%) sensitivity (at >14 days post-positive PCR), 100% (95% CI, 98.0%-100.0%) specificity, and zero cross-reactivity for the presence of non-COVID-19 respiratory infections. When compared with the plaque reduction assay, 97.4% (95% CI, 96.2%-98.5%) qualitative agreement and a positive correlation (R2 = 0.76) was observed. Comparison of IgG signals from each of the commercial assays with the nAb results from plaque reduction neutralization test/cPass assays displayed greater than 94.7% qualitative agreement and correlations with R2 = 0.43/0.68 (Abbott), R2 = 0.57/0.85 (Euroimmun), and R2 = 0.39/0.63 (Siemens), respectively. CONCLUSIONS.­: The combined data support the use of cPass assay for accurate detection of the nAb response. Positive IgG results from commercial assays associated reasonably with nAbs presence and can serve as a substitute.

Artesunate Regulates Neurite Outgrowth Inhibitor Protein B Receptor to Overcome Resistance to Sorafenib in Hepatocellular Carcinoma Cells.

The multireceptor tyrosine kinase inhibitor sorafenib is a Food and Drug Administration-approved first-line drug for the treatment of advanced liver cancer that can reportedly extend overall survival in patients with advanced hepatocellular carcinoma (HCC). Primary and acquired resistance to sorafenib are gradually increasing however, leading to failure of HCC treatment with sorafenib. It is therefore crucial to study the potential mechanism of sorafenib resistance. The results of the current study indicate that neurite outgrowth inhibitor protein B receptor (NgBR) is overexpressed in cultured sorafenib-resistant cells, and that its expression is negatively correlated with the sensitivity of liver cancer cells to sorafenib. Artesunate can inhibit the expression of NgBR, and it may block sorafenib resistance. Herein we report that sorafenib treatment in combination with artesunate overcomes HCC resistance to sorafenib alone in a cell culture model.

Influences on Attitudes Regarding Potential COVID-19 Vaccination in the United States.

The COVID-19 pandemic continues to ravage the world, with the United States being highly affected. A vaccine provides the best hope for a permanent solution to controlling the pandemic. However, to be effective, a vaccine must be accepted and used by a large majority of the population. The aim of this study was to understand the attitudes towards and obstacles facing vaccination with a potential COVID-19 vaccine. To measure these attitudes a survey was administered to 316 respondents across the United States by a survey corporation. Structural equation modeling was used to analyze the relationships of several factors with attitudes toward potential COVID-19 vaccination. Prior vaccine usage and attitudes predicted attitudes towards COVID-19 vaccination. Assessment of the severity of COVID-19 for the United States was also predictive. Approximately 68% of all respondents were supportive of being vaccinated for COVID-19, but side effects, efficacy and length of testing remained concerns. Longer testing, increased efficacy and development in the United States were significantly associated with increased vaccine acceptance. Messages promoting COVID-19 vaccination should seek to alleviate the concerns of those who are already vaccine-hesitant. Messaging directed at the benefits of vaccination for the United States as a country would address the second predictive factor. Enough time should be taken to allay concerns about both short- and long-term side effects before a vaccine is released.

Novel monoclonal antibodies against thymidine kinase 1 and their potential use for the immunotargeting of lung, breast and colon cancer cells.

BACKGROUND: Thymidine kinase 1 (TK1) is a pyrimidine salvage pathway enzyme that is up-regulated in malignant tissues and elevated in the serum of cancer patients. While TK1 has been well established as a tumor biomarker, little has been done to explore its potential as a tumor target. Recently, we reported the membrane expression of TK1 on malignant cells, but not on normal cells. This study explores the possible use of monoclonal antibodies for the targeting of membrane associated TK1 in lung, breast, colon and prostate cancer cells. METHODS: We generated and evaluated a panel of monoclonal antibodies against six different epitopes exposed in the tetrameric form of TK1. Antibodies were developed with hybridoma technology and validated with Western blot, siRNA TK1 knockdown, enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The therapeutic potential of the antibodies was evaluated in vitro in antibody-dependent cell-mediated-cytotoxicity (ADCC) experiments. RESULTS: Binding of the antibodies to TK1 was confirmed by Western blot in purified recombinant protein, cancer serum, and cell lysate. After a TK1 knockdown was performed, a reduction of TK1 expression was observed with five antibodies. Using indirect ELISA, we identified 3B2E11, 9C10, 7H2, 3B4, 8G2 among the most sensitive antibodies (LOD = 10.73-66.9 pg/ml). Surface expression of TK1 on the membrane of various cancer cell lines was analyzed with flow cytometry. Antibodies 8G2, 3B4, 7HD and 5F7G11 detected TK1 on the membrane of various cancer cell lines, including lung, prostate, colon and breast. No significant binding was detected on normal lymphocytes. Increased cytolysis of lung (~ 70%. p = 0.0001), breast (~ 70%, p = 0.0461) and colon (~ 50% p = 0.0216) cancer cells by effector cells was observed when anti-TK1 antibodies were added during ADCC experiments. CONCLUSIONS: The antibodies developed showed potential to be used to detect and target TK1 on the membrane of various tumor cells. The targeting of TK1 in malignant cells using monoclonal antibodies may be a feasible approach for the elimination of high TK1 expressing tumor cells.

A comparison of Chikungunya virus infection, progression, and cytokine profiles in human PMA-differentiated U937 and murine RAW264.7 monocyte derived macrophages.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes rash, fever and severe polyarthritis that can last for years in humans. Murine models display inflammation and macrophage infiltration only in the adjacent tissues at the site of inoculation, showing no signs of systemic polyarthritis. Monocyte-derived macrophages are one cell type suspected to contribute to a systemic CHIKV infection. The purpose of this study was to analyze differences in CHIKV infection in two different cell lines, human U937 and murine RAW264.7 monocyte derived macrophages. PMA-differentiated U937 and RAW264.7 macrophages were infected with CHIKV, and infectious virus production was measured by plaque assay and by reverse transcriptase quantitative PCR at various time points. Secreted cytokines in the supernatants were measured using cytometric bead arrays. Cytokine mRNA levels were also measured to supplement expression data. Here we show that CHIKV replicates more efficiently in human macrophages compared to murine macrophages. In addition, infected human macrophages produced around 10-fold higher levels of infectious virus when compared to murine macrophages. Cytokine induction by CHIKV infection differed between human and murine macrophages; IL-1, IL-6, IFN-γ, and TNF were significantly upregulated in human macrophages. This evidence suggests that CHIKV replicates more efficiently and induces a much greater pro-inflammatory cytokine profile in human macrophages, when compared to murine macrophages. This may shed light on the critical role that macrophages play in the CHIKV inflammatory response.

Antibiotic resistance is lower in Staphylococcus aureus isolated from antibiotic-free raw meat as compared to conventional raw meat.

The frequent use of antibiotics contributes to antibiotic resistance in bacteria, resulting in an increase in infections that are difficult to treat. Livestock are commonly administered antibiotics in their feed, but there is current interest in raising animals that are only administered antibiotics during active infections. Staphylococcus aureus (SA) is a common pathogen of both humans and livestock raised for human consumption. SA has achieved high levels of antibiotic resistance, but the origins and locations of resistance selection are poorly understood. We determined the prevalence of SA and MRSA in conventional and antibiotic-free (AF) meat products, and also measured rates of antibiotic resistance in these isolates. We isolated SA from raw conventional turkey, chicken, beef, and pork samples and also from AF chicken and turkey samples. We found that SA contamination was common, with an overall prevalence of 22.6% (range of 2.8-30.8%) in conventional meats and 13.0% (range of 12.5-13.2%) in AF poultry meats. MRSA was isolated from 15.7% of conventional raw meats (range of 2.8-20.4%) but not from AF-free meats. The degree of antibiotic resistance in conventional poultry products was significantly higher vs AF poultry products for a number of different antibiotics, and while multi-drug resistant strains were relatively common in conventional meats none were detected in AF meats. The use of antibiotics in livestock contributes to high levels of antibiotic resistance in SA found in meat products. Our results support the use of AF conditions for livestock in order to prevent antibiotic resistance development in SA.

The Kaposi's sarcoma-associated herpesvirus viral interleukin 6 gene affects metastasis and expression of B cell markers in a murine xenograft model.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-causing virus in humans, primarily affecting AIDS patients. KSHV causes a range of cancers including Kaposi's sarcoma, pleural effusion lymphoma and multicentric Castleman's disease. Current methods available for treating these cancers are relatively ineffective, and new targets for therapy are needed. The KSHV viral homolog of interleukin-6 gene (vIL-6) may play a significant role in tumor development and may serve as a new anti-cancer target, but its role in tumor formation is only partially understood. Here, a novel animal model was used to study how vIL-6 affects tumor development. Highly immune-deficient Rag2-/-γc-/- mice were transplanted with an immortalized human B cell line (BJAB) harboring either wild-type (WT) KSHV or a mutant strain lacking vIL-6 ΔvIL-6). Solid tumors developed and total tumor mass and the number of tumors were characterized. The vIL-6 gene had no significant impact on tumor mass, but significantly more tumors were detected when vIL-6 was present. Significant differences in expression of B cell markers in cells from extracted tumors were detected based upon the presence of vIL-6. B cell markers in tumor cells were also compared to the same cell type in culture, prior to xenotransplantation; B cell markers were mostly downregulated during tumor formation and these changes did not differ based upon the presence of vIL-6. The only marker that significantly increased in expression during tumor development was CD30. Tumor blood vessels were quantified to determine if more angiogenesis occurred with vIL-6-expressing virus, but there was no significant difference. These data indicate that vIL-6 plays a role in KSHV tumor formation in B cells in vivo. Further investigation into how vIL-6 manipulates CD30 expression may shed insight into KSHV oncogenesis, and may identify how vIL-6 can be targeted.

TALEN gene editing takes aim on HIV.

Transcription activator-like effector nucleases (TALENs) are one of several types of programmable, engineered nucleases that bind and cleave specific DNA sequences. Cellular machinery repairs the cleaved DNA by introducing indels. In this review, we emphasize the potential, explore progress, and identify challenges in using TALENs as a therapeutic tool to treat HIV infection. TALENs have less off-target editing and can be more effective at tolerating HIV escape mutations than CRISPR/Cas-9. Scientists have explored TALEN-mediated editing of host genes such as viral entry receptors (CCR5 and CXCR4) and a protein involved in proviral integration (LEDGF/p75). Viral targets include the proviral DNA, particularly focused on the long terminal repeats. Major challenges with translating gene therapy from bench to bedside are improving cleavage efficiency and delivery, while minimizing off-target editing, cytotoxicity, and immunogenicity. However, rapid improvements in TALEN technology are enhancing cleavage efficiency and specificity. Therapeutic testing in animal models of HIV infection will help determine whether TALENs are a viable HIV treatment therapy. TALENs or other engineered nucleases could shift the therapeutic paradigm from life-long antiretroviral therapy toward eradication of HIV infection.

Piracy on the molecular level: human herpesviruses manipulate cellular chemotaxis.

Cellular chemotaxis is important to tissue homeostasis and proper development. Human herpesvirus species influence cellular chemotaxis by regulating cellular chemokines and chemokine receptors. Herpesviruses also express various viral chemokines and chemokine receptors during infection. These changes to chemokine concentrations and receptor availability assist in the pathogenesis of herpesviruses and contribute to a variety of diseases and malignancies. By interfering with the positioning of host cells during herpesvirus infection, viral spread is assisted, latency can be established and the immune system is prevented from eradicating viral infection.

Isolation and Host Range of Bacteriophage with Lytic Activity against Methicillin-Resistant Staphylococcus aureus and Potential Use as a Fomite Decontaminant.

Staphylococcus aureus (SA) is a commensal bacterium and opportunistic pathogen commonly associated with humans and is capable of causing serious disease and death including sepsis, pneumonia, and meningitis. Methicillin-resistant SA (MRSA) isolates are typically resistant to many available antibiotics with the common exception of vancomycin. The presence of vancomycin resistance in some SA isolates combined with the current heavy use of vancomycin to treat MRSA infections indicates that MRSA may achieve broad resistance to vancomycin in the near future. New MRSA treatments are clearly needed. Bacteriophages (phages) are viruses that infect bacteria, commonly resulting in death of the host bacterial cell. Phage therapy entails the use of phage to treat or prevent bacterial infections. In this study, 12 phages were isolated that can replicate in human SA and/or MRSA isolates as a potential way to control these infections. 5 phage were discovered through mitomycin C induction of prophage and 7 others as extracellular viruses. Primary SA strains were also isolated from environmental sources to be used as tools for phage discovery and isolation as well as to examine the target cell host range of the phage isolates by spot testing. Primary isolates were tested for susceptibility to oxacillin in order to determine which were MRSA. Experiments were performed to assess the host range and killing potential of newly discovered phage, and significant reductions in bacterial load were detected. We explored the utility of some phage to decontaminate fomites (glass and cloth) and found a significant reduction in colony forming units of MRSA following phage treatment, including tests of a phage cocktail against a cocktail of MRSA isolates. Our findings suggest that phage treatment can be used as an effective tool to decontaminate human MRSA from both hard surfaces and fabrics.

Development of human B cells and antibodies following human hematopoietic stem cell transplantation to Rag2(-/-)γc(-/-) mice.

Humanized mice represent a valuable model system to study the development and functionality of the human immune system. In the RAG-hu mouse model highly immunodeficient Rag2(-/-)γc(-/-) mice are transplanted with human CD34(+) hematopoietic stem cells, resulting in human hematopoiesis and a predominant production of B and T lymphocytes. Human adaptive immune responses have been detected towards a variety of antigens in humanized mice but both cellular and humoral immune responses tend to be weak and sporadically detected. The underlying mechanisms for inconsistent responses are poorly understood. Here, we analyzed the kinetics of human B cell development and antibody production in RAG-hu mice to better understand the lack of effective antibody responses. We found that T cell levels in blood did not significantly change from 8 to 28 weeks post-engraftment, while B cells reached a peak at 14 weeks. Concentrations of 3 antibody classes (IgM, IgG, IgA) were found to be at levels about 0.1% or less of normal human levels, but human antibodies were still detected up to 32 weeks after engraftment. Human IgM was detected in 92.5% of animals while IgG and IgA were detected in about half of animals. We performed flow cytometric analysis of human B cells in bone marrow, spleen, and blood to examine the presence of precursor B cells, immature B cells, naïve B cells, and plasma B cells. We detected high levels of surface IgM(+) B cells (immature and naïve B cells) and low levels of plasma B cells in these organs, suggesting that B cells do not mature properly in this model. Low levels of human T cells in the spleen were observed, and we suggest that the lack of T cell help may explain poor B cell development and antibody responses. We conclude that human B cells that develop in humanized mice do not receive the signals necessary to undergo class-switching or to secrete antibody effectively, and we discuss strategies to potentially overcome these barriers.

Recent developments in animal models for human herpesvirus 6A and 6B.

Progress in the identification of suitable animal models for human herpesvirus (HHV)-6A and HHV-6B infections has been slow. Recently, new models have been established, mainly for HHV-6A, which reproduce some pathological features seen in humans. Neuroinflammatory signs were observed in infected marmosets and CD46-transgenic mice; although viral replication was not prominent, persistence of viral DNA and specific immunologic responses were detected, suggesting an immune-mediated pathogenic mechanism. Pig-tailed macaques showed robust viral replication concomitant with acute-phase symptoms, and provided a model to study the effects of HHV-6A on AIDS progression. In humanized mice, viral replication was less evident, but infection led to T-cell alterations. Altogether, these recent developments have opened new perspectives for studying the pathogenic role of HHV-6A in humans.

Modelling of human herpesvirus infections in humanized mice.

The human herpesviruses (HHVs) are remarkably successful human pathogens, with some members of the family successfully establishing infection in the vast majority of humans worldwide. Although many HHV infections result in asymptomatic infection or mild disease, there are rare cases of severe disease and death found with nearly every HHV. Many of the pathogenic mechanisms of these viruses are poorly understood, and in many cases, effective antiviral drugs are lacking. Only a single vaccine exists for the HHVs and researchers have been unable to develop treatments to cure the persistent infections associated with HHVs. A major hindrance to HHV research has been the lack of suitable animal models, with the notable exception of the herpes simplex viruses. One promising area for HHV research is the use of humanized mouse models, in which human cells or tissues are transplanted into immunodeficient mice. Current humanized mouse models mostly transplant human haematopoietic stem cells (HSCs), resulting in the production of a variety of human immune cells. Although all HHVs are thought to infect human immune cells, the beta- and gammaherpesviruses extensively infect and establish latency in these cells. Thus, mice humanized with HSCs hold great promise to study these herpesviruses. In this review, we provide a historical perspective on the use of both older and newer humanized mouse models to study HHV infections. The focus is on current developments in using humanized mice to study mechanisms of HHV-induced pathogenesis, human immune responses to HHVs and effectiveness of antiviral drugs.

Humanized mice as a model to study human hematopoietic stem cell transplantation.

Hematopoietic stem cell (HSC) transplantation has the potential to treat a variety of human diseases, including genetic deficiencies, immune disorders, and to restore immunity following cancer treatment. However, there are several obstacles that prevent effective HSC transplantation in humans. These include finding a matched donor, having a sufficient number of cells for the transplant, and the potency of the cells in the transplant. Ethical issues prevent effective research in humans that could provide insight into ways to overcome these obstacles. Highly immunodeficient mice can be transplanted with human HSCs and this process is accompanied by HSC homing to the murine bone marrow. This is followed by stem cell expansion, multilineage hematopoiesis, long-term engraftment, and functional human antibody and cellular immune responses. As such, humanized mice serve as a model for human HSC transplantation. A variety of conditions have been analyzed for their impact on HSC transplantation to produce humanized mice, including the type and source of cells used in the transplant, the number of cells transplanted, the expansion of cells with various protocols, and the route of introduction of cells into the mouse. In this review, we summarize what has been learned about HSC transplantation using humanized mice as a recipient model and we comment on how these models may be useful to future preclinical research to determine more effective ways to expand HSCs and to determine their repopulating potential in vivo.

Human herpesvirus 6A infection and immunopathogenesis in humanized Rag2⁻/⁻ γc⁻/⁻ mice.

Although serious human diseases have been correlated with human herpesvirus 6A (HHV-6A) and HHV-6B, the lack of animal models has prevented studies which would more definitively link these viral infections to disease. HHV-6A and HHV-6B have recently been classified as two distinct viruses, and in this study we focused specifically on developing an in vivo model for HHV-6A. Here we show that Rag2⁻/⁻γc⁻/⁻ mice humanized with cord blood-derived human hematopoietic stem cells produce human T cells that express the major HHV-6A receptor, CD46. Both cell-associated and cell-free viral transmission of HHV-6A into the peritoneal cavity resulted in detectable viral DNA in at least one of the samples (blood, bone marrow, etc.) analyzed from nearly all engrafted mice. Organs and cells positive for HHV-6A DNA were the plasma and cellular blood fractions, bone marrow, lymph node, and thymic samples; control mice had undetectable viral DNA. We also noted viral pathogenic effects on certain T cell populations. Specific thymocyte populations, including CD3⁻ CD4⁺ CD8⁻ and CD3⁺ CD4⁻ cells, were significantly modified in humanized mice infected by cell-associated transmission. In addition, we detected significantly increased proportions of CD4⁺ CD8⁺ cells in the blood of animals infected by cell-free transmission. These findings provide additional evidence that HHV-6A may play a role in human immunodeficiencies. These results indicate that humanized mice can be used to study HHV-6A in vivo infection and replication as well as aspects of viral pathogenesis.