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1.  Nodular regenerative hyperplasia of the liver associated with didanosine persists for years even after its interruption 
BMJ Case Reports  2011;2011:bcr0320113928.
The authors describe an HIV-positive patient with nodular regenerative hyperplasia of the liver with non-cirrhotic portal hypertension. Despite stopping the culprit drug, didanosine, the radiologic changes persisted for years. When evaluating liver pathologies, antiretroviral drugs must be included in the differential diagnosis, even when they have been stopped years ago.
doi:10.1136/bcr.03.2011.3928
PMCID: PMC3089926  PMID: 22696691
2.  RAG2−/−γc−/− Mice Transplanted with CD34+ Cells from Human Cord Blood Show Low Levels of Intestinal Engraftment and Are Resistant to Rectal Transmission of Human Immunodeficiency Virus▿  
Journal of Virology  2008;82(24):12145-12153.
Rectal transmission is one of the main routes of infection by human immunodeficiency virus type 1 (HIV-1). To efficiently study transmission mechanisms and prevention strategies, a small animal model permissive for rectal transmission of HIV is mandatory. We tested the susceptibility of RAG2−/−γc−/− mice transplanted with human cord blood hematopoietic stem cells to rectal infection with HIV. We rectally exposed these humanized mice to cell-free and cell-associated HIV. All mice remained HIV negative as assessed by plasma viral load. The same mice infected intraperitoneally showed high levels of HIV replication. In the gut-associated lymphatic tissue, we found disproportionately smaller numbers of human cells than in other lymphoid organs. This finding may explain the observed resistance to rectal transmission of HIV. To increase the numbers of local HIV target cells and the likelihood of HIV transmission, we treated mice with different proinflammatory stimuli: local application of interleukin-1β, addition of seminal plasma to the inoculum, or induction of colitis with dextran sodium sulfate. These procedures attracted some human leukocytes, but the transmission rate was still very low. The humanized mice showed low levels of human engraftment in the intestinal tract and seem to be resistant to rectal transmission of HIV, and thus they are an unsuitable model for this application.
doi:10.1128/JVI.01105-08
PMCID: PMC2593344  PMID: 18842716
3.  Humanized Mice Recapitulate Key Features of HIV-1 Infection: A Novel Concept Using Long-Acting Anti-Retroviral Drugs for Treating HIV-1 
PLoS ONE  2012;7(6):e38853.
Background
Humanized mice generate a lymphoid system of human origin subsequent to transplantation of human CD34+ cells and thus are highly susceptible to HIV infection. Here we examined the efficacy of antiretroviral treatment (ART) when added to food pellets, and of long-acting (LA) antiretroviral compounds, either as monotherapy or in combination. These studies shall be inspiring for establishing a gold standard of ART, which is easy to administer and well supported by the mice, and for subsequent studies such as latency. Furthermore, they should disclose whether viral breakthrough and emergence of resistance occurs similar as in HIV-infected patients when ART is insufficient.
Methods/Principal Findings
NOD/shi-scid/γcnull (NOG) mice were used in all experimentations. We first performed pharmacokinetic studies of the drugs used, either added to food pellets (AZT, TDF, 3TC, RTV) or in a LA formulation that permitted once weekly subcutaneous administration (TMC278: non-nucleoside reverse transcriptase inhibitor, TMC181: protease inhibitor). A combination of 3TC, TDF and TMC278-LA or 3TC, TDF, TMC278-LA and TMC181-LA suppressed the viral load to undetectable levels in 15/19 (79%) and 14/14 (100%) mice, respectively. In successfully treated mice, subsequent monotherapy with TMC278-LA resulted in viral breakthrough; in contrast, the two LA compounds together prevented viral breakthrough. Resistance mutations matched the mutations most commonly observed in HIV patients failing therapy. Importantly, viral rebound after interruption of ART, presence of HIV DNA in successfully treated mice and in vitro reactivation of early HIV transcripts point to an existing latent HIV reservoir.
Conclusions/Significance
This report is a unique description of multiple aspects of HIV infection in humanized mice that comprised efficacy testing of various treatment regimens, including LA compounds, resistance mutation analysis as well as viral rebound after treatment interruption. Humanized mice will be highly valuable for exploring the antiviral potency of new compounds or compounds targeting the latent HIV reservoir.
doi:10.1371/journal.pone.0038853
PMCID: PMC3374767  PMID: 22719966
4.  Recognition of Potentially Novel Human Disease-Associated Pathogens by Implementation of Systematic 16S rRNA Gene Sequencing in the Diagnostic Laboratory▿ †  
Journal of Clinical Microbiology  2010;48(9):3397-3402.
Clinical isolates that are difficult to identify by conventional means form a valuable source of novel human pathogens. We report on a 5-year study based on systematic 16S rRNA gene sequence analysis. We found 60 previously unknown 16S rRNA sequences corresponding to potentially novel bacterial taxa. For 30 of 60 isolates, clinical relevance was evaluated; 18 of the 30 isolates analyzed were considered to be associated with human disease.
doi:10.1128/JCM.01098-10
PMCID: PMC2937732  PMID: 20631113
5.  β1 Integrin Expression Increases Susceptibility of Memory B Cells to Epstein-Barr Virus Infection▿  
Journal of Virology  2010;84(13):6667-6677.
Epstein-Barr virus (EBV) uses nasal mucosa-associated lymphoid tissue (NALT) as a portal of entry to establish life-long persistence in memory B cells. We previously showed that naïve and memory B cells from NALT are equally susceptible to EBV infection. Here we show that memory B cells from NALT are significantly more susceptible to EBV infection than those from remote lymphatic organs. We identify β1 integrin, which is expressed the most by naïve B cells of distinct lymphoid origin and by memory B cells from NALT, as a mediator of increased susceptibility to infection by EBV. Furthermore, we show that BMRF-2-β1 integrin interaction and the downstream signal transduction pathway are critical for postbinding events. An increase of β1 integrin expression in peripheral blood memory B cells provoked by CD40 stimulation plus B-cell receptor cross-linking increased the susceptibility of non-NALT memory B cells to EBV infection. Thus, EBV seems to utilize the increased activation status of memory B cells residing in the NALT to establish and ensure persistence.
doi:10.1128/JVI.02675-09
PMCID: PMC2903285  PMID: 20427540
6.  Inadequate Clearance of Translocated Bacterial Products in HIV-Infected Humanized Mice 
PLoS Pathogens  2010;6(4):e1000867.
Bacterial translocation from the gut and subsequent immune activation are hallmarks of HIV infection and are thought to determine disease progression. Intestinal barrier integrity is impaired early in acute retroviral infection, but levels of plasma lipopolysaccharide (LPS), a marker of bacterial translocation, increase only later. We examined humanized mice infected with HIV to determine if disruption of the intestinal barrier alone is responsible for elevated levels of LPS and if bacterial translocation increases immune activation. Treating uninfected mice with dextran sodium sulfate (DSS) induced bacterial translocation, but did not result in elevated plasma LPS levels. DSS-induced translocation provoked LPS elevation only when phagocytic cells were depleted with clodronate liposomes (clodrolip). Macrophages of DSS-treated, HIV-negative mice phagocytosed more LPS ex vivo than those of control mice. In HIV-infected mice, however, LPS phagocytosis was insufficient to clear the translocated LPS. These conditions allowed higher levels of plasma LPS and CD8+ cell activation, which were associated with lower CD4+/CD8+ cell ratios and higher viral loads. LPS levels reflect both intestinal barrier and LPS clearance. Macrophages are essential in controlling systemic bacterial translocation, and this function might be hindered in chronic HIV infection.
Author Summary
HIV infection leads to continuous destruction of the body's immune defenses. Furthermore, disease progression is linked to heightened levels of immune activation. However, the underlying activating factors and their relationships to HIV pathogenesis are controversial. In patients with chronic HIV infection, bacteria and their products, such as lipopolysaccharide (LPS), translocate from the intestinal lumen into the systemic circulation. In the current study, we investigated the pathogenic potential of bacterial translocation in HIV-infected humanized mice. By modulating the amount of bacterial translocation in the mice, we determined that LPS elevation depends on intestinal barrier dysfunction and defective LPS clearance by macrophages. HIV-infected mice showed inadequate LPS clearance, leading to a cascade of uncontrolled bacterial translocation, T-cell activation, HIV replication, and T-cell loss. Our study highlights how important the interplay between different immune cells is for maintaining a healthy balance between immune activation with the goal to defend the body against microbes and detrimental activation that fuels HIV replication.
doi:10.1371/journal.ppat.1000867
PMCID: PMC2861703  PMID: 20442871
7.  Distinct Ex Vivo Susceptibility of B-Cell Subsets to Epstein-Barr Virus Infection According to Differentiation Status and Tissue Origin▿  
Journal of Virology  2008;82(9):4400-4412.
Epstein-Barr virus (EBV) uses tonsils as the portal of entry to establish persistent infection. EBV is found in various B-cell subsets in tonsils but exclusively in memory B cells in peripheral blood. The in vitro susceptibilities of B-cell subsets to EBV infection have been studied solely qualitatively. In this work, we examined quantitatively the in vitro susceptibilities of various B-cell subsets from different tissue origins to EBV infection. First, we established a centrifugation-based inoculation protocol (spinoculation) that resulted in a significantly increased proportion of infected cells compared to that obtained by conventional inoculation, enabling a detailed susceptibility analysis. Importantly, B-cell infection occurred via the known EBV receptors and infected cells showed EBV mRNA expression patterns similar to those observed after conventional inoculation, validating our approach. Tonsillar naïve and memory B cells were infected ex vivo at similar frequencies. In contrast, memory B cells from blood, which represent B cells from various lymphoid tissues, were infected at lower frequencies than their naïve counterparts. Immunoglobulin A (IgA)-positive or IgG-positive tonsillar memory B cells were significantly more susceptible to EBV infection than IgM-positive counterparts. Memory B cells were transformed with lower efficiency than naïve B cells. This result was paralleled by lower proliferation rates. In summary, these data suggest that EBV exploits the B-cell differentiation status and tissue origin to establish persistent infection.
doi:10.1128/JVI.02630-07
PMCID: PMC2293034  PMID: 18321980
8.  Anti-HIV Activity Mediated by Natural Killer and CD8+ Cells after Toll-Like Receptor 7/8 Triggering 
PLoS ONE  2008;3(4):e1999.
We previously found that triggering TLR7/8 either by single stranded HIV RNA or synthetic compounds induced changes in the lymphoid microenvironment unfavorable to HIV. In this study, we used selective TLR7 and 8 agonists to dissect their contribution to the anti-HIV effects. While triggering TLR7 inhibited efficiently HIV replication in lymphoid suspension cells from tonsillar origin, its effect was inconsistent in peripheral blood mononuclear cells (PBMC). In contrast, triggering TLR8 showed a very prominent and overall very consistent effect in PBMC and tonsillar lymphoid suspension cells. Depletion of dendritic cells (DC), Natural killer cells (NK) and CD8+ T-cells from PBMC resulted in the reversal of TLR8 induced anti-HIV effects. Especially noteworthy, depletion of either NK or CD8+ T-cells alone was only partially effective. We interpret these findings that DC are the initiator of complex changes in the microenvironment that culminates in the anti-HIV active NK and CD8+ effector cells. The near lack of NK and the low number of CD8+ T-cells in tonsillar lymphoid suspension cells may explain the lower TLR8 agonist's anti-HIV effects in that tissue. However, additional cell-type specific differences must exist since the TLR7 agonists had a very strong inhibitory effect in tonsillar lymphoid suspension cells. Separation of effector from the CD4+ target cells did not abolish the anti-HIV effects pointing to the critical role of soluble factors. Triggering TLR7 or 8 were accompanied by major changes in the cytokine milieu; however, it appeared that not a single soluble factor could be assigned for the potent effects.
These results delineate the complex effects of triggering TLR7/8 for an efficient antiviral defense. While the ultimate mechanism(s) remains unknown, the potent effects described may have therapeutic value for treating chronic viral diseases. Notably, HIV replication is blocked by TLR triggering before HIV integrates into the host chromosome which would prevent the establishment or maintenance of the latent reservoir.
doi:10.1371/journal.pone.0001999
PMCID: PMC2292240  PMID: 18431484
10.  CpG Oligodeoxynucleotides Block Human Immunodeficiency Virus Type 1 Replication in Human Lymphoid Tissue Infected Ex Vivo 
Journal of Virology  2004;78(22):12344-12354.
Oligodeoxynucleotides (ODNs) with immunomodulatory motifs control a number of microbial infections in animal models, presumably by acting through toll-like receptor 9 (TLR9) to induce a number of cytokines (e.g., alpha interferon and tumor necrosis factor alpha). The immunomodulatory motif consists of unmethylated sequences of cytosine and guanosine (CpG motif). ODNs without CpG motifs do not trigger TLR9. We hypothesized that triggering of TLR9 generates a cellular environment unfavorable for human immunodeficiency virus (HIV) replication. We tested this hypothesis in human lymphocyte cultures and found that phosphorothioate-modified ODN CpG2006 (type B ODNs) inhibited HIV replication nearly completely and prevented the loss of CD4+ T cells. ODNs CpG2216 and CpG10 (type A ODNs) were less effective. CpG2006 blocked HIV replication in purified CD4+ T cells and T-cell lines; CpG10 was ineffective in this setting, indicating that type A ODNs may inhibit HIV replication in CD4+ T-cell lines indirectly through a separate cell subset. However, control ODNs without CpG motifs also showed anti-HIV effects, indicating that these effects are nonspecific and not due to TLR9 triggering. The mechanism of action is not clear. CpG2006 and its control ODN blocked syncytium formation in a cell fusion-based assay, but CpG10, CpG2216, and their control ODNs did not. The latter types interfered with the HIV replication cycle during disassembly or reverse transcription. In contrast, CpG2006 and CpG2216 specifically induced cytokines critical to initiation of the innate immune response. In summary, the nonspecific anti-HIV activity of CpG ODNs, their ability to stimulate HIV replication in latently infected cells, potentially resulting in their elimination, and their documented ability to link the innate and adaptive immune responses make them attractive candidates for further study as anti-HIV drugs.
doi:10.1128/JVI.78.22.12344-12354.2004
PMCID: PMC525063  PMID: 15507621
11.  Progress Toward a Human CD4/CCR5 Transgenic Rat Model for De Novo Infection by Human Immunodeficiency Virus Type 1 
The development of a permissive small animal model for the study of human immunodeficiency virus type (HIV)-1 pathogenesis and the testing of antiviral strategies has been hampered by the inability of HIV-1 to infect primary rodent cells productively. In this study, we explored transgenic rats expressing the HIV-1 receptor complex as a susceptible host. Rats transgenic for human CD4 (hCD4) and the human chemokine receptor CCR5 (hCCR5) were generated that express the transgenes in CD4+ T lymphocytes, macrophages, and microglia. In ex vivo cultures, CD4+ T lymphocytes, macrophages, and microglia from hCD4/hCCR5 transgenic rats were highly susceptible to infection by HIV-1 R5 viruses leading to expression of abundant levels of early HIV-1 gene products comparable to those found in human reference cultures. Primary rat macrophages and microglia, but not lymphocytes, from double-transgenic rats could be productively infected by various recombinant and primary R5 strains of HIV-1. Moreover, after systemic challenge with HIV-1, lymphatic organs from hCD4/hCCR5 transgenic rats contained episomal 2–long terminal repeat (LTR) circles, integrated provirus, and early viral gene products, demonstrating susceptibility to HIV-1 in vivo. Transgenic rats also displayed a low-level plasma viremia early in infection. Thus, transgenic rats expressing the appropriate human receptor complex are promising candidates for a small animal model of HIV-1 infection.
doi:10.1084/jem.20011549
PMCID: PMC2193739  PMID: 11901198
HIV-1; transgenic rats; CD4; CCR5; macrophages
12.  Susceptibility of Rat-Derived Cells to Replication by Human Immunodeficiency Virus Type 1 
Journal of Virology  2001;75(17):8063-8073.
Progress in developing a small animal model of human immunodeficiency virus type 1 (HIV-1) disease would greatly facilitate studies of transmission, pathogenesis, host immune responses, and antiviral strategies. In this study, we have explored the potential of rats as a susceptible host. In a single replication cycle, rat cell lines Rat2 and Nb2 produced infectious virus at levels 10- to 60-fold lower than those produced by human cells. Rat-derived cells supported substantial levels of early HIV-1 gene expression, which was further enhanced by overexpression of human cyclin T1. Rat cells displayed quantitative, qualitative, and cell-type-specific limitations in the late phase of the HIV-1 replication cycle including relative expression levels of HIV-1 Gag proteins, intracellular Gag processing, and viral egress. Nb2 cells were rendered permissive to HIV-1 R5 viruses by coexpression of human CD4 and CCR5, indicating that the major restriction on HIV-1 replication was at the level of cellular entry. We also found that primary rat lymphocytes, macrophages, and microglia expressed considerable levels of early HIV-1 gene products following infection with pseudotyped HIV-1. Importantly, primary rat macrophages and microglia, but not lymphocytes, also expressed substantial levels of HIV-1 p24 CA and produced infectious virions. Collectively, these results identify the rat as a promising candidate for a transgenic small animal model of HIV-1 infection and highlight pertinent cell-type-specific restrictions that are features of this species.
doi:10.1128/JVI.75.17.8063-8073.2001
PMCID: PMC115050  PMID: 11483751
13.  Human Immunodeficiency Virus Type 1 Coreceptor Preferences Determine Target T-Cell Depletion and Cellular Tropism in Human Lymphoid Tissue 
Journal of Virology  2000;74(11):5347-5351.
The present study sought to determine how usage of coreceptors by human immunodeficiency virus type 1 dictates cell tropism and depletion of CD4+ T cells in human lymphoid tissues cultured ex vivo. We found that coreceptor preferences control the marked, preferential depletion of coreceptor-expressing CD4+ lymphocytes. In addition, there was a strong, but not absolute, preference shown by CXCR4-using strains for lymphocytes and by CCR5-using strains for macrophages.
PMCID: PMC110890  PMID: 10799612
14.  Distinct Mechanisms of Entry by Envelope Glycoproteins of Marburg and Ebola (Zaire) Viruses 
Journal of Virology  2000;74(10):4933-4937.
Since the Marburg (MBG) and Ebola (EBO) viruses have sequence homology and cause similar diseases, we hypothesized that they associate with target cells by similar mechanisms. Pseudotype viruses prepared with a luciferase-containing human immunodeficiency virus type 1 backbone and packaged by the MBG virus or the Zaire subtype EBO virus glycoproteins (GP) mediated infection of a comparable wide range of mammalian cell types, and both were inhibited by ammonium chloride. In contrast, they exhibited differential sensitivities to treatment of target cells with tunicamycin, endoglycosidase H, or protease (pronase). Therefore, while they exhibit certain functional similarities, the MBG and EBO virus GP interact with target cells by distinct processes.
PMCID: PMC112022  PMID: 10775638
15.  Viral Entry through CXCR4 Is a Pathogenic Factor and Therapeutic Target in Human Immunodeficiency Virus Type 1 Disease 
Journal of Virology  2000;74(1):184-192.
The chemokine receptors CCR5 and CXCR4 function as the principal coreceptors for human immunodeficiency virus type 1 (HIV-1). Coreceptor function has also been demonstrated for a variety of related receptors in vitro. The relative contributions of CCR5, CXCR4, and other putative coreceptors to HIV-1 disease in vivo have yet to be defined. In this study, we used sequential primary isolates and recombinant strains of HIV-1 to demonstrate that CXCR4-using (X4) viruses emerging in association with disease progression are highly pathogenic in ex vivo lymphoid tissues compared to CXCR4-independent viruses. Furthermore, synthetic receptor antagonists that specifically block CXCR4-mediated entry dramatically suppressed the depletion of CD4+ T cells by recombinant and clinically derived X4 HIV-1 isolates. Moreover, in vitro specificity for the additional coreceptors CCR3, CCR8, BOB, and Bonzo did not augment cytopathicity or diminish sensitivity toward CXCR4 antagonists in lymphoid tissues. These data provide strong evidence to support the concept that adaptation to CXCR4 specificity in vivo accelerates HIV-1 disease progression. Thus, therapeutic intervention targeting the interaction of HIV-1 gp120 with CXCR4 may be highly valuable for suppressing the pathogenic effects of late-stage viruses.
PMCID: PMC111527  PMID: 10590105
16.  V3 Recombinants Indicate a Central Role for CCR5 as a Coreceptor in Tissue Infection by Human Immunodeficiency Virus Type 1 
Journal of Virology  1999;73(3):2350-2358.
Binding of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 to both CD4 and one of several chemokine receptors (coreceptors) permits entry of virus into target cells. Infection of tissues may establish latent viral reservoirs as well as cause direct pathologic effects that manifest as clinical disease such as HIV-associated dementia. We sought to identify the critical coreceptors recognized by HIV-1 tissue-derived strains as well as to correlate these coreceptor preferences with site of infection and dementia diagnosis. To reconstitute coreceptor use, we cloned HIV-1 envelope V3 sequences encoding the primary determinants of coreceptor specificity from 13 brain-derived and 6 colon-derived viruses into an isogenic (NL4-3) viral background. All V3 recombinants utilized the chemokine receptor CCR5 uniformly and efficiently as a coreceptor but not CXCR4, BOB/GPR15, or Bonzo/STRL33. Other receptors such as CCR3, CCR8, and US28 were inefficiently and variably used as coreceptors by various envelopes. CCR5 without CD4 present did not allow for detectable infection by any of the tested recombinants. In contrast to the pathogenic switch in coreceptor specificity frequently observed in comparisons of blood-derived viruses early after HIV-1 seroconversion and after onset of AIDS, the characteristics of these V3 recombinants suggest that CCR5 is a primary coreceptor for brain- and colon-derived viruses regardless of tissue source or diagnosis of dementia. Therefore, tissue infection may not depend significantly on viral envelope quasispeciation to broaden coreceptor range but rather selects for CCR5 use throughout disease progression.
PMCID: PMC104480  PMID: 9971818
17.  Rabbit Cells Expressing Human CD4 and Human CCR5 Are Highly Permissive for Human Immunodeficiency Virus Type 1 Infection 
Journal of Virology  1998;72(7):5728-5734.
To evaluate the feasibility of using transgenic rabbits expressing CCR5 and CD4 as a small-animal model of human immunodeficiency virus type 1 (HIV) disease, we examined whether the expression of the human chemokine receptor (CCR5) and human CD4 would render a rabbit cell line (SIRC) permissive to HIV replication. Histologically, SIRC cells expressing CD4 and CCR5 formed multinucleated cells (syncytia) upon exposure to BaL, a macrophagetropic strain of HIV that uses CCR5 for cell entry. Intracellular viral capsid p24 staining showed abundant viral gene expression in BaL-infected SIRC cells expressing CD4 and CCR5. In contrast, neither SIRC cells expressing CD4 alone nor murine 3T3 cells expressing CCR5 and CD4 exhibited significant expression of p24. These stably transfected rabbit cells were also highly permissive for the production of virions upon infection by two other CCR5-dependent strains (JR-CSF and YU-2) but not by a CXCR4-dependent strain (NL4-3). The functional integrity of these virions was demonstrated by the successful infection of human peripheral blood mononuclear cells (PBMC) with viral stocks prepared from these transfected rabbit cells. Furthermore, primary rabbit PBMC were found to be permissive for production of infectious virions after circumventing the cellular entry step. These results suggest that a transgenic rabbit model for the study of HIV disease may be feasible.
PMCID: PMC110246  PMID: 9621031

Results 1-17 (17)