HIV-1 Infection of Long-Lived Hematopoietic Precursors In Vitro and In Vivo.

Latent reservoirs in human-immunodeficiency-virus-1 (HIV-1)-infected individuals represent a major obstacle in finding a cure for HIV-1. Hematopoietic stem and progenitor cells (HSPCs) have been described as potential HIV-1 targets, but their roles as HIV-1 reservoirs remain controversial. Here we provide additional evidence for the susceptibility of several distinct HSPC subpopulations to HIV-1 infection in vitro and in vivo. In vitro infection experiments of HSPCs were performed with different HIV-1 Env-pseudotyped lentiviral particles and with replication-competent HIV-1. Low-level infection/transduction of HSPCs, including hematopoietic stem cells (HSCs) and multipotent progenitors (MPP), was observed, preferentially via CXCR4, but also via CCR5-mediated entry. Multi-lineage colony formation in methylcellulose assays and repetitive replating of transduced cells provided functional proof of susceptibility of primitive HSPCs to HIV-1 infection. Further, the access to bone marrow samples from HIV-positive individuals facilitated the detection of HIV-1 gag cDNA copies in CD34+ cells from eight (out of eleven) individuals, with at least six of them infected with CCR5-tropic HIV-1 strains. In summary, our data confirm that primitive HSPC subpopulations are susceptible to CXCR4- and CCR5-mediated HIV-1 infection in vitro and in vivo, which qualifies these cells to contribute to the HIV-1 reservoir in patients.

Tumours involving the retrobulbar space in cats: 37 cases.

OBJECTIVES: The aim of this multicentre retrospective study was to describe the clinical presentation, imaging findings, diagnosis and outcomes of cats with retrobulbar neoplasia. METHODS: A total of 37 cats that were diagnosed with retrobulbar neoplasia and underwent advanced imaging were recruited from searches of the clinical records of two referral hospitals. All cats had neoplasia confirmed via cytology or histopathology. Data relating to the signalment, presentation, results of investigations, treatment and outcome were recorded. A review of imaging studies was performed where possible. RESULTS: In total, 23 cases (62%) were presented with respiratory signs. Exophthalmos was the most common ophthalmological examination finding, present in 18 cases (49%). Thirty-two cases (86%) had secondary extension of neoplasia to the retrobulbar space (most commonly from the nasal cavities), present in 20 cases (54%), of which 12 were lymphoma. In cases where contrast was administered, 28/35 (80%) had contrast-enhancing masses. Orbital extension was detected in 21 cases (57%), exophthalmos in 22 (59%), globe deformation in 12 (32%) and local lymphadenomegaly in 22 (61%). In total, 36 (97%) retrobulbar tumours were malignant. Thoracic imaging, where it was performed, was concerning for metastasis in 8/25 cases (31%), with abdominal imaging suggestive of metastasis in 5/12 (42%). The most common diagnosis was lymphoma with 19 cases (51%), with nasal lymphoma representing 12 of these, followed by carcinoma in 10 (27%). The median survival time, for cases where death was recorded, was 85 days (range 1-263 days). CONCLUSIONS AND RELEVANCE: To the authors' knowledge, this is the largest study of neoplasia affecting the feline retrobulbar space. Retrobulbar tumours in cats are overwhelmingly malignant, and commonly due to secondary extension of tumours originating elsewhere. Lymphoma, particularly arising from the nasal cavities, was the most common cause. Cats presenting with signs suggestive of retrobulbar disease should be assessed for disease affecting any of the structures of the head.

Changes in retinal vascular diameters in senior and geriatric cats in association with variation in systemic blood pressure.

OBJECTIVES: Early diagnosis of arterial hypertension is essential to prevent target organ damage. In humans, retinal arteriolar narrowing predicts hypertension. This blinded prospective observational study investigated the retinal vessel diameters in senior and geriatric cats of varying systolic blood pressure (SBP) status and evaluated retinal vascular changes in hypertensive cats after treatment. METHODS: Cats with a median age of 14 years (range 9.1-22 years) were categorised into five groups: group 1, healthy normotensive (SBP <140 mmHg; n = 40) cats; group 2, pre-hypertensive (SBP 140-160 mmHg; n = 14) cats; group 3, cats with chronic kidney disease (CKD) and normotensive (n = 26); group 4, cats with CKD and pre-hypertensive (n = 13); and group 5, hypertensive cats (SBP >160 mmHg, n = 15). Colour fundus images (Optibrand ClearView) were assessed for hypertensive lesions. Retinal vascular diameters and bifurcation angles were annotated and calculated using the Vascular Assessment and Measurement Platform for Images of the Retina annotation tool (VAMPIRE-AT). When available, measurements were obtained at 3 and 6 months after amlodipine besylate treatment. RESULTS: Ten hypertensive cats had retinal lesions, most commonly intraretinal haemorrhages and retinal exudates. Arteriole and venule diameters decreased significantly with increasing age (-0.17 ± 0.05 pixels/year [P = 0.0004]; -0.19 ± 0.05 pixels/year). Adjusted means ± SEM for arteriole and venule diameter (pixels) were 6.3 ± 0.2 and 8.9 ± 0.2 (group 1); 7.6 ± 0.3 and 10.1 ± 0.4 (group 2); 6.9 ± 0.2 and 9.5 ± 0.3 (group 3); 7.4 ± 0.3 and 10.0 ± 0.4 (group 4); and 7.0 ± 0.3 and 9.8 ± 0.4 (group 5). Group 1 arteriole and venule diameters were significantly lower than those of groups 2 and 4. Group 2 arteriole bifurcation angle was significantly narrower than those of groups 1 and 3. Post-treatment, vessel diameters decreased significantly at 3 and 6 months in seven hypertensive cats. CONCLUSIONS AND RELEVANCE: Increased age was associated with reduced vascular diameters. Longitudinal studies are required to assess if vessel diameters are a risk indicator for hypertension in cats.

The Hard Way towards an Antibody-Based HIV-1 Env Vaccine: Lessons from Other Viruses.

Although effective antibody-based vaccines have been developed against multiple viruses, such approaches have so far failed for the human immunodeficiency virus type 1 (HIV-1). Despite the success of anti-retroviral therapy (ART) that has turned HIV-1 infection into a chronic disease and has reduced the number of new infections worldwide, a vaccine against HIV-1 is still urgently needed. We discuss here the major reasons for the failure of "classical" vaccine approaches, which are mostly due to the biological properties of the virus itself. HIV-1 has developed multiple mechanisms of immune escape, which also account for vaccine failure. So far, no vaccine candidate has been able to induce broadly neutralizing antibodies (bnAbs) against primary patient viruses from different clades. However, such antibodies were identified in a subset of patients during chronic infection and were shown to protect from infection in animal models and to reduce viremia in first clinical trials. Their detailed characterization has guided structure-based reverse vaccinology approaches to design better HIV-1 envelope (Env) immunogens. Furthermore, conserved Env epitopes have been identified, which are promising candidates in view of clinical applications. Together with new vector-based technologies, considerable progress has been achieved in recent years towards the development of an effective antibody-based HIV-1 vaccine.

Clinical outcome of concomitant vs interrupted BRAF inhibitor therapy during radiotherapy in melanoma patients.

This corrects the article DOI: 10.1038/bjc.2017.85.

Optimization of the EC26-2A4 Epitope in the gp41 Membrane Proximal External Region Targeted by Neutralizing Antibodies from an Elite Controller.

The analysis of patient derived HIV neutralizing antibodies (nAbs) and their target epitopes in the viral envelope (Env) protein provides important basic information for vaccine design. In this study we optimized an epitope, EC26-2A4, that is targeted by neutralizing antibodies from an elite controller (EC26) and localizes in the membrane-proximal external region from the gp41 transmembrane protein. Due to its overlap with the epitope of the first generation broadly neutralizing monoclonal Ab (mAb) 2F5 associated with autoreactivity, we first defined the minimal core epitope reacting with antibodies from EC26 plasma, but not with mAb 2F5. The optimized minimal epitope, EC26-2A4ΔM, was able to induce neutralizing antibodies in vaccinated mice. We further analyzed the frequency of antibodies against the EC26-2A4ΔM peptide in HIV-positive patient sera from a treated cohort and an untreated long-term nonprogressor (LTNP) cohort. Interestingly, 27% of the LTNP sera reacted with the peptide, whereas only 9% showed reactivity in the treated cohort. Although there was no association between the presence of antibodies against the EC26-2A4ΔM epitope and viral load or CD4 count in these patients, the CD4 nadir in the treated cohort was higher in patients positive for EC26-2A4ΔM antibodies, in particular in patients having such antibodies at an early and a late timepoint after infection.

Cutaneous, gastrointestinal, hepatic, endocrine, and renal side-effects of anti-PD-1 therapy.

BACKGROUND: Anti-programmed cell death receptor-1 (PD-1) antibodies represent an effective treatment option for metastatic melanoma as well as for other cancer entities. They act via blockade of the PD-1 receptor, an inhibitor of the T-cell effector mechanisms that limit immune responses against tumours. As reported for ipilimumab, the anti-PD-1 antibodies pembrolizumab and nivolumab can induce immune-related adverse events (irAEs). These side-effects affect skin, gastrointestinal tract, liver, endocrine system and other organ systems. Since life-threatening and fatal irAEs have been reported, adequate diagnosis and management are essential. METHODS AND FINDINGS: In total, 496 patients with metastatic melanoma from 15 skin cancer centers were treated with pembrolizumab or nivolumab; 242 side-effects were described in 138 patients. In 116 of the 138 patients, side-effects affected the skin, gastrointestinal tract, liver, endocrine, and renal system. Rare side-effects included diabetes mellitus, lichen planus, and pancreas insufficiency due to pancreatitis. CONCLUSION: Anti-PD1 antibodies can induce a plethora of irAEs. The knowledge of them will allow prompt diagnosis and improve the management resulting in decreased morbidity.

Neurological, respiratory, musculoskeletal, cardiac and ocular side-effects of anti-PD-1 therapy.

BACKGROUND: Anti-programmed cell death 1 (PD-1) antibodies represent an effective treatment option for metastatic melanoma and other cancer entities. They act via blockade of the PD-1 receptor, an inhibitor of the T-cell effector mechanisms that limit immune responses against tumours. As reported for ipilimumab, the anti-PD-1 antibodies pembrolizumab and nivolumab can induce immune-related adverse events (irAEs). These side-effects can involve skin, gastrointestinal tract, liver, the endocrine system and other organ systems. Since life-threatening and fatal irAEs have been reported, adequate diagnosis and management are essential. METHODS AND FINDINGS: In total, 496 patients with metastatic melanoma from 15 skin cancer centres were treated with pembrolizumab or nivolumab. Two hundred forty two side-effects in 138 patients have been analysed. In 77 of the 138 patients side-effects affected the nervous system, respiratory tract, musculoskeletal system, heart, blood and eyes. Not yet reported side-effects such as meningo-(radiculitis), polyradiculitis, cardiac arrhythmia, asystolia, and paresis have been observed. Rare and difficult to manage side-effects such as myasthenia gravis are described in detail. CONCLUSION: Anti-PD-1 antibodies can induce a plethora of irAEs. The knowledge of them will allow prompt diagnosis and improve the management resulting in decreased morbidity.

Systemic therapy of metastatic melanoma.

For patients with metastatic melanoma, there are currently several effective therapeutic options. The BRAF inhibitors vemurafenib and dabrafenib are characterized by rapid tumor control and high response rates. In combination with one of the two MEK inhibitors trametinib and cobimetinib, they achieve response rates (CR + PR, complete plus partial remissions) of 70%, while delaying the development of treatment resistance, as well as a median overall survival of > 2 years with tolerable side effects. Showing long-term survival rates of approximately 20%, the anti-CTLA-4 antibody ipilimumab is the first substance that has led to a significant prolongation of overall survival in patients with metastatic melanoma. However, delayed treatment response and severe immune-mediated side effects may pose limitations to its therapeutic benefit. Usually well tolerated, anti-PD-1 antibody monotherapy using nivolumab and pembrolizumab has yielded response rates (CR + PR) of up to 45% and one-year survival rates of > 70%. The combination of ipilimumab and nivolumab has shown response rates of up to 58% and a median progression-free survival of > 11 months. While this combination is expected to result in a rapid and long-lasting response, this potential benefit comes at the expense of a high level of toxicity. Strategies for treatment sequencing and treatment combinations are currently being investigated in clinical studies. Overall, the prognosis for patients with metastatic melanoma has significantly improved. With long-term survival a possibility, not only acute but also long-term therapeutic side effects must be taken into account.

In Silico Prediction and Experimental Confirmation of HA Residues Conferring Enhanced Human Receptor Specificity of H5N1 Influenza A Viruses.

Newly emerging influenza A viruses (IAV) pose a major threat to human health by causing seasonal epidemics and/or pandemics, the latter often facilitated by the lack of pre-existing immunity in the general population. Early recognition of candidate pandemic influenza viruses (CPIV) is of crucial importance for restricting virus transmission and developing appropriate therapeutic and prophylactic strategies including effective vaccines. Often, the pandemic potential of newly emerging IAV is only fully recognized once the virus starts to spread efficiently causing serious disease in humans. Here, we used a novel phylogenetic algorithm based on the informational spectrum method (ISM) to identify potential CPIV by predicting mutations in the viral hemagglutinin (HA) gene that are likely to (differentially) affect critical interactions between the HA protein and target cells from bird and human origin, respectively. Predictions were subsequently validated by generating pseudotyped retrovirus particles and genetically engineered IAV containing these mutations and characterizing potential effects on virus entry and replication in cells expressing human and avian IAV receptors, respectively. Our data suggest that the ISM-based algorithm is suitable to identify CPIV among IAV strains that are circulating in animal hosts and thus may be a new tool for assessing pandemic risks associated with specific strains.

Re-engineering vesicular stomatitis virus to abrogate neurotoxicity, circumvent humoral immunity, and enhance oncolytic potency.

As cancer treatment tools, oncolytic viruses (OV) have yet to realize what some see as their ultimate clinical potential. In this study, we have engineered a chimeric vesicular stomatitis virus (VSV) that is devoid of its natural neurotoxicity while retaining potent oncolytic activity. The envelope glycoprotein (G) of VSV was replaced with a variant glycoprotein of the lymphocytic choriomeningitis virus (LCMV-GP), creating a replicating therapeutic, rVSV(GP), that is benign in normal brain but can effectively eliminate brain cancer in multiple preclinical tumor models in vivo. Furthermore, it can be safely administered systemically to mice and displays greater potency against a spectrum of human cancer cell lines than current OV candidates. Remarkably, rVSV(GP) escapes humoral immunity, thus, for the first time, allowing repeated systemic OV application without loss of therapeutic efficacy. Taken together, rVSV(GP) offers a considerably improved OV platform that lacks several of the major drawbacks that have limited the clinical potential of this technology to date.

Peptides as drugs: from screening to application.

Peptides are ideally suited to mimic natural ligands and thereby function in an antagonistic or agonistic way. Furthermore they are able to physiologically disrupt functional complexes due to their small size and specific binding properties. Proteins form homo- or heteromeric (macro)molecular complexes and intricate networks by interacting with small molecules, peptides, nucleic acids or other proteins. On average, five interaction partners have been estimated for any given cellular protein, illustrating the complexity of the formed 'interactomes' and the impact of their investigation. Many protein-protein interactions are mediated by hot-spots, which comprise only a small part of the large binding interface but account for 80% of the binding energy. Thus, these hot spots provide an 'Achilles heel' for pharmaceutical interventions aiming at the disruption of functional protein-protein complexes. Methods to select peptides for defined target structures include display technologies on phages, ribosomes or yeast, and the yeast-two-hybrid system. Once selected, these peptides can be optimized for their binding affinity using peptide arrays. Stabilization of biologically unstable peptides is achieved by the introduction of non-natural amino acids to form so-called peptidomimetics that are resistant to cellular proteases. Moreover, lipocalins and peptide aptamers represent scaffolded binding structures with unique binding characteristics and enhanced stability. In case of extracellular targets, like cell surface receptors or pathogens in patients` plasma, peptide inhibitors have direct access. Addressing intracellular targets with peptides is more difficult since short hydrophilic peptides generally do not cross plasma membranes on their own. However, intracellular uptake of peptides can be achieved by coupling to carrier systems like liposomes or nanoparticles or upon fusion to a protein transduction domain. Alternatively, peptides may be expressed within cells after transduction with viral vectors in a gene therapy setting. This review will summarize the broad potential of peptides as drugs, with special emphasis on peptides which inhibit protein-protein interactions.

Comparison of three humanized mouse models for adoptive T cell transfer.

BACKGROUND: Humanized mouse models for adoptive T cell transfer are important for preclinical efficacy and toxicity studies. However, common xenograft models using immunodeficient mice have so far failed to efficiently support the homing of human T cells to secondary lymphoid tissues. METHODS: We established a new mouse model for the adoptive transfer of genetically-modified (gm) T cells using conditioned BALB/c mice. Conditioning involved cyclophosphamide injections, lethal irradiation and radioprotection with bone marrow from immunodeficient mice. We compared repopulation kinetics and the quality of grafts in these modified Trimera (mT3) mice with immunodeficient BALB/c Rag2(-/-) interleukin (IL)2 receptor gamma (rg) knockout (DKO) and NOD/LtSz-scid IL2rg(-/-) (NSG) recipient mouse strains. RESULTS: DKO mice showed only marginal engraftment until onset of graft-versus-host disease, whereas mT3 and NSG were repopulated with comparable kinetics. However, T cell repertoire and human cytokine profiles suggest a xenoreactivity-driven gm T cell expansion in mT3 mice, whereas NSG mice were characterized by an initial homeostatic proliferation. Morphological analysis revealed high levels of human gm T cell infiltration in the spleen and liver of all three mouse strains. However, mT3 mice provided the strongest homing of human gm T cells to mucosal sites. Additionally, mT3 mice were the only model with macroscopically visible superficial inguinal lymph nodes. These lymph nodes strongly supported the homing of gm T cells. CONCLUSIONS: In the present study, we give proof-of-concept that wild-type mice can accept gm T cell grafts while providing secondary lymphoid structures. Despite limitations, mT3 mice are a valid alternative for applications that specifically rely on improved secondary lymphoid structures.

Semireplication-competent vesicular stomatitis virus as a novel platform for oncolytic virotherapy.

Among oncolytic viruses, the vesicular stomatitis virus (VSV) is especially potent and a highly promising agent for the treatment of cancer. But, even though effective against multiple tumor entities in preclinical animal models, replication-competent VSV exhibits inherent neurovirulence, which has so far hindered clinical development. To overcome this limitation, replication-defective VSV vectors for cancer gene therapy have been tested and proven to be safe. However, gene delivery was inefficient and only minor antitumor efficacy was observed. Here, we present semireplication-competent vector systems for VSV (srVSV), composed of two trans-complementing, propagation-deficient VSV vectors. The de novo generated deletion mutants of the two VSV polymerase proteins P (phosphoprotein) and L (large catalytic subunit), VSVΔP and VSVΔL respectively, were used mutually or in combination with VSVΔG vectors. These srVSV systems copropagated in vitro and in vivo without recombinatory reversion to replication-competent virus. The srVSV systems were highly lytic for human glioblastoma cell lines, spheroids, and subcutaneous xenografts. Especially the combination of VSVΔG/VSVΔL vectors was as potent as wild-type VSV (VSV-WT) in vitro and induced long-term tumor regression in vivo without any associated adverse effects. In contrast, 90% of VSV-WT-treated animals succumbed to neurological disease shortly after tumor clearance. Most importantly, even when injected into the brain, VSVΔG/VSVΔL did not show any neurotoxicity. In conclusion, srVSV is a promising platform for virotherapeutic approaches and also for VSV-based vector vaccines, combining improved safety with an increased coding capacity for therapeutic transgenes, potentially allowing for multipronged approaches.

A motif unique to the human DEAD-box protein DDX3 is important for nucleic acid binding, ATP hydrolysis, RNA/DNA unwinding and HIV-1 replication.

DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication.

Pseudotyping vesicular stomatitis virus with lymphocytic choriomeningitis virus glycoproteins enhances infectivity for glioma cells and minimizes neurotropism.

Vesicular stomatitis virus (VSV)-based oncolytic virotherapy has the potential to significantly improve the prognosis of aggressive malignancies such as brain cancer. However, VSV's inherent neurotoxicity has hindered clinical development so far. Given that this neurotropism is attributed to the glycoprotein VSV-G, VSV was pseudotyped with the nonneurotropic envelope glycoprotein of the lymphocytic choriomeningitis virus (LCMV-GP→VSV-GP). Compared to VSV, VSV-GP showed enhanced infectivity for brain cancer cells in vitro while sparing primary human and rat neurons in vitro and in vivo, respectively. In conclusion, VSV-GP has a much wider therapeutic window than VSV and is thus more suitable for clinical applications, especially in the brain.

Peptide ligands selected with CD4-induced epitopes on native dualtropic HIV-1 envelope proteins mimic extracellular coreceptor domains and bind to HIV-1 gp120 independently of coreceptor usage.

During HIV-1 entry, binding of the viral envelope glycoprotein gp120 to the cellular CD4 receptor triggers conformational changes resulting in exposure of new epitopes, the highly conserved CD4-induced (CD4i) epitopes that are essential for subsequent binding to chemokine receptor CCR5 or CXCR4. Due to their functional conservation, CD4i epitopes represent attractive viral targets for HIV-1 entry inhibition. The aim of the present study was to select peptide ligands for CD4i epitopes on native dualtropic (R5X4) HIV-1 envelope (Env) glycoproteins by phage display. Using CD4-activated retroviral particles carrying Env from the R5X4 HIV-1 89.6 strain as the target, we performed screenings of random peptide phage libraries under stringent selection conditions. Selected peptides showed partial identity with amino acids in the extracellular domains of CCR5/CXCR4, including motifs rich in tyrosines and aspartates at the N terminus known to be important for gp120 binding. A synthetic peptide derivative (XD3) corresponding to the most frequently selected phages was optimized for Env binding on peptide arrays. Interestingly, the optimized peptide could bind specifically to gp120 derived from HIV-1 strains with different coreceptor usage, competed with binding of CD4i-specific monoclonal antibody (MAb) 17b, and interfered with entry of both a CCR5 (R5)-tropic and a CXCR4 (X4)-tropic Env pseudotyped virus. This peptide ligand therefore points at unique properties of CD4i epitopes shared by gp120 with different coreceptor usage and could thus serve to provide new insight into the conserved structural details essential for coreceptor binding for further drug development.

Regulation of HIV-1 infection in cells derived from purified CD34+ cells through manipulation of APOBEC3G expression.

The cytidine deaminase APOBEC3G has been identified as an antiviral host factor that combats HIV-1. The protein was found to be present in HIV-1 target cells such as macrophages and dendritic cells. The antiviral state of these cells has been partially attributed to a G→A hypermutation of the HIV genome caused by APOBEC3G during reverse transcription. However, the viral infectivity factor (Vif) counteracts this antiviral mechanism by inducing the inactivation of APOBEC3G. In this study, we tested the effect of APOBEC3G expression on the HIV-1 infection of cells derived from purified CD34+ cells that have been transduced with lentiviral vectors containing APOBEC3G and/or shRNAs directed against APOBEC3G and then have been differentiated before infection with HIV-1. In cell lines, the infection was strongly inhibited after upregulation of APOBEC3G. The infection could then be rescued after transducing these cells with shRNAs targeting APOBEC3G. In cells derived from purified CD34+ cells a strong inhibition of the HIV-1 infection was observed in both a Vif defective HIV-1 virus and the corresponding wild-type HIV-1 virus with Vif. Our data implies that when APOBEC3G is expressed high enough, it can escape the inhibition from Vif, thereby exerting its antiviral activity.

Inhibition of HIV-1 replication by small interfering RNAs directed against glioma pathogenesis related protein (GliPR) expression.

BACKGROUND: Previously, we showed that glioma pathogenesis related protein (GliPR) is induced in CEM T cells upon HIV-1 infection in vitro. To examine whether GliPR plays a role as HIV dependency factor (HDF), we tested the effect of GliPR suppression by siRNA on HIV-1 replication. RESULTS: Induction of GliPR expression by HIV-1 was confirmed in P4-CCR5 cells. When GliPR was suppressed by siRNA, HIV-1 replication was significantly reduced as measured by HIV-1 transcript levels, HIV-1 p24 protein levels, and HIV-1 LTR-driven reporter gene expression, suggesting that GliPR is a cellular co-factor of HIV-1. Microarray analysis of uninfected HeLa cells following knockdown of GliPR revealed, among a multitude of gene expression alterations, a down-regulation of syndecan-1, syndecan-2, protein kinase C alpha (PRKCA), the catalytic subunit beta of cAMP-dependent protein kinase (PRKACB), nuclear receptor co-activator 3 (NCOA3), and cell surface protein CD59 (protectin), all genes having relevance for HIV-1 pathology. CONCLUSIONS: The up-regulation of GliPR by HIV-1 and the early significant inhibition of HIV-1 replication mediated by knockdown of GliPR reveal GliPR as an important HIV-1 dependency factor (HDF), which may be exploited for HIV-1 inhibition.

[Memorandum III "methods for health services research", Part 1 [authors: H. Pfaff, G. Glaeske, E. A. M. Neugebauer, M. Schrappe] and Part 2 [authors: E. A. M. Neugebauer, A. Icks, M. Schrappe] of the Deutsches Netzwerk Versorgungsforschung e.V.: A summary]. / Memorandum III "Methoden für die Versorgungsforschung", Teil 1 [Verfasser: H. Pfaff, G. Glaeske, E. A. Neugebauer, M. Schrappe] und Teil 2 [Verfasser: E. A. Neugebauer, A. Icks, M. Schrappe] des Deutschen Netzwerks Versorgungsforschung e.V. : Eine zusammenfassende Darstellung.

This article presents an overview of the recently published first two parts of the Memorandum III "Methods for Health Services Research" discussed and approved by the member societies of the German Network Health Services Research [Deutsches Netzwerk Versorgungsforschung e.V.]. Part one of this memorandum covers methodical principles and minimum standards for the subject areas of health services research "Epidemiological Methods for Health Services Research", "Methods for Organisational Health Services Research" and "Methods for Quality of Life Research", the second part the topics "Methods of Health Economic Evaluation" and "Registries for the Health Services Research". The Memorandum is addressed to health services researchers and to reviewers who are planning, conducting, publishing studies as well as evaluating research proposals and publications. Assurance of quality and increase of the health services research are the aims of the Memorandum III. According to the advanced knowledge in health services research the Memorandum needs regular updates. Therefore the Memorandum has to be understood as "work in progress".