Transplantation of gene-modified haematopoietic stem cells: Application and clinical considerations.

Autologous and allogeneic haematopoietic stem cell (HSC) transplantation has been performed in patients with various malignant and non-malignant haematological disorders for more than 50 years. Ex vivo gene modification of HSCs for autologous transplantation opens up new therapeutic avenues for genetic and infectious diseases. Major advances have been made over the last three decades with respect to gene modification of HSCs and transplantation strategies, ultimately culminating in the approval of two such therapies in Europe (Strimvelis for a rare primary immune deficiency, and LentiGlobin for beta-thalassaemia). Newer gene-modifying technologies and treatment regimens have also recently come to the fore, which hold great promise for the development of safer and more effective treatments. We provide an overview of the current state of gene-modified HSC therapies, highlighting success stories, limitations and important considerations for achieving successful translation of these therapies to the clinic.

Transplantation of gene-modified haematopoietic stem cells: Application and clinical considerations

Autologous and allogeneic haematopoietic stem cell (HSC) transplantation has been performed in patients with various malignant and non-malignant haematological disorders for more than 50 years. Ex vivo gene modification of HSCs for autologous transplantation opens up new therapeutic avenues for genetic and infectious diseases. Major advances have been made over the last three decades with respect to gene modification of HSCs and transplantation strategies, ultimately culminating in the approval of two such therapies in Europe (Strimvelis for a rare primary immune deficiency, and LentiGlobin for beta-thalassaemia). Newer gene-modifying technologies and treatment regimens have also recently come to the fore, which hold great promise for the development of safer and more effective treatments. We provide an overview of the current state of gene-modified HSC therapies, highlighting success stories, limitations and important considerations for achieving successful translation of these therapies to the clinic

Promising Role of Toll-Like Receptor 8 Agonist in Concert with Prostratin for Activation of Silent HIV.

The persistence of latently HIV-infected cells in patients under combined antiretroviral treatment (cART) remains the major hurdle for HIV eradication. Thus far, individual compounds have not been sufficiently potent to reactivate latent virus and guarantee its elimination in vivo. Thus, we hypothesized that transcriptional enhancers, in concert with compounds triggering the innate immune system, are more efficient in reversing latency by creating a Th1 supportive milieu that acts against latently HIV-infected cells at various levels. To test our hypothesis, we screened six compounds on a coculture of latently infected cells (J-lat) and monocyte-derived dendritic cells (MDDCs). The protein kinase C (PKC) agonist prostratin, with a Toll-like receptor 8 (TLR8) agonist, resulted in greater reversion of HIV latency than any single compound. This combinatorial approach led to a drastic phenotypic and functional maturation of the MDDCs. Tumor necrosis factor (TNF) and cell-cell interactions were crucial for the greater reversion observed. Similarly, we found a greater potency of the combination of prostratin and TLR8 agonist in reversing HIV latency when applying it to primary cells of HIV-infected patients. Thus, we demonstrate here the synergistic interplay between TLR8-matured MDDCs and compounds acting directly on latently HIV-infected cells, targeting different mechanisms of latency, by triggering various signaling pathways. Moreover, TLR8 triggering may reverse exhaustion of HIV-specific cytotoxic T lymphocytes that might be essential for killing or constraining the latently infected cells. IMPORTANCE: Curing HIV is the Holy Grail. The so-called "shock and kill" strategy relies on drug-mediated reversion of HIV latency and the subsequent death of those cells under combined antiretroviral treatment. So far, no compound achieves efficient reversal of latency or eliminates this latent reservoir. The compounds may not target all of the latency mechanisms in all latently infected cells. Moreover, HIV-associated exhaustion of the immune system hinders the efficient elimination of the reactivated cells. In this study, we demonstrated synergistic latency reversion by combining agonists for protein kinase C and Toll-like receptor 8 in a coculture of latently infected cells with myeloid dendritic cells. The drug prostratin stimulates directly the transcriptional machinery of latently infected cells, and the TLR8 agonist acts indirectly by maturing dendritic cells. These findings highlight the importance of the immune system and its activation, in combination with direct-acting compounds, to reverse latency.

TLR9 triggering in Burkitt's lymphoma cell lines suppresses the EBV BZLF1 transcription via histone modification.

Endemic Burkitt's lymphoma (BL) is considered to preferentially develop in equatorial Africa because of chronic co-infection with Epstein-Barr virus (EBV) and the malaria pathogen Plasmodium falciparum. The interaction and contribution of both pathogens in the oncogenic process are poorly understood. Earlier, we showed that immune activation with a synthetic Toll-like receptor 9 (TLR9) ligand suppresses the initiation of EBV lytic replication in primary human B cells. In this study we investigate the mechanism involved in the suppression of EBV lytic gene expression in BL cell lines. We show that this suppression is dependent on functional TLR9 and MyD88 signaling but independent of downstream signaling elements, including phosphatidylinositol-3 kinase, mitogen-activated protein kinases and nuclear factor-kappaB. We identified TLR9 triggering resulting in histone modifications to negatively affect the activation of the promoter of EBV's master regulatory lytic gene BZLF1. Finally, we show that P. falciparum hemozoin, a natural TLR9 ligand, suppresses induction of EBV lytic gene expression in a dose-dependent manner. Thus, we provide evidence for a possible interaction between P. falciparum and EBV at the B-cell level and the mechanism involved in suppressing lytic and thereby reinforcing latent EBV that has unique oncogenic potential.

Human T cell development and HIV infection in human hemato-lymphoid system mice.

Advances in generation of mice that on human hematopoietic stem and progenitor cell transplantation develop and maintain human hemato-lymphoid cells have fueled an already thriving field of research. We focus here on human T cell development and HIV infection in Rag2 -/- gamma(c) -/- mice transplanted as newborns with human CD34+ cord blood hematopoietic stem and progenitor cells.

[Risk of infection during treatment with tumor necrosis factor-alpha inhibitors]. / Risiko von Infektionen unter einer Behandlung mit Tumornekrosefaktor-alpha-lnhibitoren.

Tumor necrosis factor-alpha (TNF) is essential for immune defense. TNF plays a major role in the recruitment of inflammatory cells to the site of infection and in the formation and maintenance of granulomas. In addition, it plays a primary and detrimental role in chronic autoimmune diseases. Drugs that inhibit TNF are effective in the treatment of inflammatory rheumatic and autoimmune diseases. However, the three currently available TNF antagonists (etanercept, infliximab and adalimumab) decrease host resistance to granulomatous diseases such as tuberculosis. The incidence of tuberculosis in patients treated with TNF antagonists is higher than in the general population. There are a number of case reports describing the association of TNF-antagonists and the presentation of other infectious diseases such as histoplasmosis, listeriosis, coccidioidomycosis, candidiasis and aspergillosis. These case reports, however, are anecdotal. Nonetheless, patients treated with TNF antagonists are immunocompromised and infectious diseases are most likely more frequent and may present differently than expected. In this review, we describe the role of TNF in constraining infectious diseases, the difference between the three available TNF antagonists, and we discuss the relevant clinical data published in the literature as related to the risk of anti-TNF therapy for infectious diseases.

[HIV-1 vaccination--is there hope?]. / HIV-1 Impfung--gibt es Hoffnung?

The HIV pandemic continues. Today the number of HIV-infected people is estimated to be 39.4 million. Despite huge efforts it is unlikely that there will be an efficient HIV vaccine available in the near future. Until now, only two phase III HIV vaccination trials have been performed in man. However, transmission could not be prevented. The hurdle for a rational approach to generate a vaccine is the still incomplete knowledge about HIV pathogenesis and the high rate of mutations of HIV. In this review we discuss the distinct aspects of the immune response, which could be exploited for HIV vaccine strategies and describe current vaccine approaches.

Rapidly destructive Staphylococcus epidermidis endocarditis.

A 29-year-old man with rapidly destructive Staphylococcus epidermidis endocarditis after mitral valve reconstruction is presented. Resistance to rifampin and teicoplanin occurred during antibiotic treatment resulting in clinical failure and valve destruction. Subsequently, the patient was successfully treated, by combining valve replacement with antibiotic therapy including quinupristin/dalfopristin, levofloxacin, and vancomycin. In conclusion, S. epidermidis can cause rapid valve destruction with large vegetations, and combination of surgery and antibiotic therapy may be necessary.

Retrospective analysis of varicella zoster virus (VZV) copy DNA numbers in plasma of immunocompetent patients with herpes zoster, of immunocompromised patients with disseminated VZV disease, and of asymptomatic solid organ transplant recipients.

BACKGROUND: Varicella zoster virus (VZV) causes significant morbidity and mortality in immunocompromised patients. Subclinical reactivation has been described in solid organ recipients and has been associated with graft versus host disease in bone marrow transplantation. Newer studies assessing the prevalence and impact of subclinical VZV reactivation in solid organ transplant (SOT) recipients are lacking. METHODS AND RESULTS: In a first step we developed a highly sensitive quantitative polymerase chain reaction (qPCR) assay for VZV DNA with a detection limit of < or = 20 copies/mL. Using this assay, we retrospectively analyzed plasma samples of different patient groups for VZV DNA. VZV DNA was found in 10/10 plasma samples of immunocompetent patients with herpes zoster (VZV copy numbers/mL: mean+/-SEM 1710+/-1018), in 1/1 sample of a human immunodeficiency virus-infected patient with primary VZV disease (15,192 copies/mL) and in 4/4 plasma samples of immunocompromised patients with visceral VZV disease (mean of first value 214,214+/-178,572). All 108 plasma samples of asymptomatic SOT recipients off any antiviral therapy, randomly sampled over 1 year, were negative for VZV DNA. CONCLUSION: Our qPCR assay proved to be highly sensitive (100%) in symptomatic VZV disease. We did not detect subclinical reactivation in asymptomatic SOT recipients during the first post-transplant year. Thus, subclinical VZV reactivation is either a rare event or does not exist. These data need to be confirmed in larger prospective trials.

Impaired CD8+ T-cell reactivity against viral antigens in cancer patients with solid tumors.

BACKGROUND: Patients with hematological malignancies are at increased risk for various infections. In patients with solid cancer, a variety of immunosuppressive mechanisms affecting T-cell response are described. We hypothesized that patients with advanced solid tumors may exhibit an impaired recognition of viral antigens. To test this, the capability of CD8+ T cells to recognize recall antigens from influenza and vaccinia virus was compared in patients and healthy individuals. Since all patients and most of the healthy individuals had been vaccinated against vaccinia years ago, comparison of the two groups was expected to be especially informative with respect to distinct effector T-cell reactivity. MATERIALS AND METHODS: Our test population included 16 healthy individuals and 12 patients with advanced solid cancers who were currently not receiving chemotherapy. We stimulated peripheral blood mononuclear cells (PBMC) ex vivo with the well-characterized influenza A matrix 58-66 peptide and the immunogenic and HLA-A*0201 restricted peptide epitope SLSAYIIRV derived from the modified vaccinia virus Ankara (MVA). A specific CD8+ T-cell reactivity was determined by quantitative real-time polymerase chain reaction (qRT-PCR) measuring changes in interferon gamma (IFN-gamma) mRNA expression levels. RESULTS: We found that significantly fewer cancer patients than healthy individuals exhibited specific T-cell recognition of the vaccinia epitope (25% and 69%, respectively). In addition, strength of the T-cell responses against both viral peptides was significantly reduced in cancer patients. CONCLUSION: Patients with advanced tumors are Less likely to mount a T-cell response against viral epitopes. These findings may have implications for the design of immunotherapeutic interventions against virus-induced diseases, including tumors.

Alveolar echinococcosis of the liver in an adult with human immunodeficiency virus type-1 infection.

We describe a patient with human immunodeficiency virus type-1 (HIV) infection and alveolar echinococcosis (AE) with a focus on two messages. Despite being severely immunocompromised over years the patient exhibited a long-term asymptomatic course of AE. This is in clear contrast to reports describing accelerated courses of AE in immunocompromised patients. The patient had therapeutic mebendazole drug levels with only 1/10 of the normal drug dose. He was co-treated with protease inhibitors for his HIV infection. These drugs are known as strong inhibitors of cytochrome P450 3A4 (CYP3A4)-dependent metabolism. We speculate that benzimidazoles and protease inhibitors interfere at the CYP3A4-level. The first report of co-infection of HIV and accelerated AE was in a young girl with an extremely low CD4 cell count and an abrogated lymphoproliferative responsiveness to parasite antigen stimulation. Since the CD4 cell count in our patient remained in the range of 27-150 cells/microl, we speculate that there was a critical threshold of immunosupression for constraining AE. Initial treatment with albendazole for AE added to the current highly active antiretroviral treatment (HAART), and suppressive toxoplasmosis therapy became complicated by pancytopenia. After full recovery of the bone marrow, mebendazole was introduced with a new HAART and the previously prescribed toxoplasmosis maintenance therapy. Surprisingly, efficient mebendazole levels were achieved with an uncommonly low dose. These observations suggest that the benzimidazoles, albendazole and mebendazole, may interact with protease inhibitors, which are known for their strong inhibition of the CYP3A4.

CCTTT-repeat polymorphism of the inducible nitric oxide synthase is not associated with HIV pathogenesis.

Nitric oxide (NO) produced by the inducible form of nitric oxide synthase (iNOS) has bactericidal and virocidal effects. Although NO synthesis and iNOS expression in macrophages affect several aspects of human immunodeficiency virus (HIV) type-1 pathogenesis, their role in HIV disease remains largely unknown. In humans, the expression of iNOS is influenced by a functional CCTTT-repeat polymorphism in the promoter region of the gene. We investigated the association of this polymorphism with HIV pathogenesis in naive HIV-infected patients before the initiation of antiretroviral therapy. The allele frequencies of the iNOS CCTTT-repeat polymorphism were assessed by PCR in 857 patients from the Swiss HIV Cohort Study, including rapid progressors and long-term nonprogressors, and in 240 healthy volunteers. In HIV-infected patients, the initial viral load and the decline in total CD4 cells was calculated to estimate disease progression. Allele frequencies of the iNOS CCTTT-repeat polymorphism were similar between the HIV-infected and noninfected blood donors. In treatment-naive HIV-positive patients, there was no association of the iNOS polymorphism with viral load or with the course of CD4 cells. Regulation of iNOS expression by the functional CCTTT-polymorphism does not modify HIV pathogenesis.

Differential effects of p-glycoprotein and multidrug resistance protein-1 on productive human immunodeficiency virus infection.

P-glycoprotein (P-gp) and multidrug-resistance protein-1 (MRP-1) are adenosine triphosphate-binding cassette proteins that may decrease intracellular concentrations of anti-human immunodeficiency virus (HIV) drugs. After HIV-1(IIIB) infection, HIV-1 protein and infectious virus production were decreased by at least 70-fold in CEM cells overexpressing P-gp but were increased by at least 50-fold in CEM cells overexpressing MRP-1, compared with control CEM cells. After transfection with the HIV-1(IIIB) genome, cells overexpressing P-gp and MRP-1 expressed similar amounts of HIV protein. Selective inhibitors of MRP-1 and P-gp partially reversed the effect of these transporters in a concentration-dependent manner. P-gp preferentially associated with glycolipid-enriched membrane (GEM) domains, which may be an important site for cellular binding and egress of HIV. In contrast, MRP-1 was not preferentially found in GEM domains. These results suggest that the inhibition of HIV productive infection by P-gp and augmentation by MRP-1 occur predominantly at a preintegration step but act through different mechanisms.

Susceptibility of rat-derived cells to replication by human immunodeficiency virus type 1.

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.

Folate receptor-alpha is a cofactor for cellular entry by Marburg and Ebola viruses.

Human infections by Marburg (MBG) and Ebola (EBO) viruses result in lethal hemorrhagic fever. To identify cellular entry factors employed by MBG virus, noninfectible cells transduced with an expression library were challenged with a selectable pseudotype virus packaged by MBG glycoproteins (GP). A cDNA encoding the folate receptor-alpha (FR-alpha) was recovered from cells exhibiting reconstitution of viral entry. A FR-alpha cDNA was recovered in a similar strategy employing EBO pseudotypes. FR-alpha expression in Jurkat cells facilitated MBG or EBO entry, and FR-blocking reagents inhibited infection by MBG or EBO. Finally, FR-alpha bound cells expressing MBG or EBO GP and mediated syncytia formation triggered by MBG GP. Thus, FR-alpha is a significant cofactor for cellular entry for MBG and EBO viruses.

Impact of TNFalpha, LTalpha, Fc gammaRII and complement receptor on HIV-1 trapping in lymphoid tissue from HIV-infected patients.

OBJECTIVES: To investigate HIV trapping mechanisms in patients with acute infection and in asymptomatic individuals prior to and during antiretroviral therapy. To determine the role of complement receptor (CR), Fc gamma receptor II (Fc gammaRII), tumour necrosis factor alpha (TNFalpha), and lymphotoxin alpha (LTalpha) expression in HIV trapping efficiency. METHODS: Lymphoid tissues from three acutely HIV-infected patients and six asymptomatic, chronically HIV-infected patients collected prior to and during antiretroviral therapy were compared with lymphoid tissues from six HIV-seronegative subjects. HIV, TNFalpha and LTalpha RNA expression was detected and quantified by fluorescence in situ hybridization. CR, Fc gammaRII and HIV p24 antigen were detected and quantified by fluorescence immunohistochemistry. RESULTS: The amount of trapped HIV did not differ significantly between patients with acute HIV infection and asymptomatic individuals, and was independent of the presence of CR or Fc gammaRII expression. However, in patients with acute infection, the amount of trapped virus was correlated inversely with the number of HIV-infected cells (P = 0.0092) and with the size of the light zone (P = 0.037). In these patients, the number of TNFalpha-expressing cells was correlated inversely with the amount of trapped virus (P = 0.014) and positively correlated with the size of the light zone in germinal centers (P = 0.041). No correlations were observed between TNFalpha or LTalpha expression and Fc gammaRII or CR expression. CONCLUSION: This report provides the first evidence that in humans TNFalpha is involved in the development of lymphoid follicles, HIV trapping, and, consequently, in early host immune responses. A model is proposed for early events in patients during acute HIV infection.

Molecular function of the CD4 D1 domain in coreceptor-mediated entry by HIV type 1.

The surface molecule CD4 plays a key role in initiating cellular entry by the human immunodeficiency virus type 1 (HIV-1), and it is now recognized as acting synergistically with select chemokine receptors (coreceptors) in the infection process. The present study was undertaken to determine whether the extracellular region of CD4 is sufficient to induce fusion of HIV-1 virions with target cells in the absence of its anchoring function. Using pseudotype reporter viruses to quantitate infection, soluble CD4 (sCD4) was tested for its ability to induce fusion by viruses utilizing CCR5 as their coreceptor. We found that sCD4 was competent to replace membrane-bound CD4 to trigger infection mediated by several HIV-1 envelopes. Furthermore, in a comparison of the envelopes of HIV-1 NL4-3 and a chimera containing the gp120 V3 loop of Ba-L, the V3 region was found to be one factor affecting susceptibility to induction by sCD4. In addition, using truncated and mutant derivatives of sCD4, the amino-terminal D1 domain of CD4 was found to be necessary and sufficient for induction of fusion and to require an intact gp120-binding site for this activity. These results delineate determinants on CD4 and gp120 required for fusion induction in collaboration with a coreceptor, and suggest a mechanism whereby CD4 may contribute to viral infection in trans.