Sequential Vacc-4x and romidepsin during combination antiretroviral therapy (cART): Immune responses to Vacc-4x regions on p24 and changes in HIV reservoirs.

OBJECTIVES: The REDUC clinical study Part B investigated Vacc-4x/rhuGM-CSF therapeutic vaccination prior to HIV latency reversal using romidepsin. The main finding was a statistically significant reduction from baseline in viral reservoir measurements. Here we evaluated HIV-specific functional T-cell responses following Vacc-4x/rhuGM-CSF immunotherapy in relation to virological outcomes on the HIV reservoir. METHODS: This study, conducted in Aarhus, Denmark, enrolled participants (n = 20) with CD4>500 cells/mm3 on cART. Six Vacc-4x (1.2 mg) intradermal immunizations using rhuGM-CSF (60 µg) as adjuvant were followed by 3 weekly intravenous infusions of romidepsin (5 mg/m2). Immune responses were determined by IFN-γ ELISpot, T-cell proliferation to p24 15-mer peptides covering the Vacc-4x region, intracellular cytokine staining (ICS) to the entire HIVGag and viral inhibition. RESULTS: The frequency of participants with CD8+ T-cell proliferation assay positivity was 8/16 (50%) at baseline, 11/15 (73%) post-vaccination, 6/14 (43%) during romidepsin, and 9/15 (60%)post-romidepsin. Participants with CD8+ T-cell proliferation assay positivity post-vaccination showed reductions in total HIV DNA post-vaccination (p = 0.006; q = 0.183), post-latency reversal (p = 0.005; q = 0.183), and CA-RNA reductions post-vaccination (p = 0.015; q = 0.254). Participants (40%) were defined as proliferation 'Responders' having ≥2-fold increase in assay positivity post-baseline. Robust ELISpot baseline responses were found in 87.5% participants. No significant changes were observed in the proportion of polyfunctional CD8+ T-cells to HIVGag by ICS. There was a trend towards increased viral inhibition from baseline to post-vaccination (p = 0.08). CONCLUSIONS: In this 'shock and kill' approach supported by therapeutic vaccination, CD8+ T-cell proliferation represents a valuable means to monitor functional immune responses as part of the path towards functional HIV cure.

Fitness to drive in cognitive impairment--a quantitative study of GPs' experience.

Assessing fitness to drive is part of the role of general practitioners. Cognitive impairment may affect an individual's ability to drive safely. The aims of our study were to question GPs about their experience of assessing patients with cognitive impairment for driving fitness and to explore their attitudes to this role. We carried out a quantitative cross-sectional anonymous postal survey of 200 GPs in counties Galway, Mayo and Roscommon. Ethical approval was obtained from the Irish College of General Practitioners. Data was analysed using Epi Info. The response rate was 62.5% (n=125). 86 (68.8%) GPs used guidelines when assessing fitness to drive in cognitive impairment. 83 (66.4%) respondents formally assess cognitive function. 52 (41.6%) GPs would certify someone as fit to drive with verbal restrictions. 102 (81.6%) respondents feel confident in assessing fitness to drive. 98 (78.4%) GPs have referred patients for further assessment.

Stem cell collection in patients with multiple myeloma: impact of induction therapy and mobilization regimen.

Lenalidomide is an active treatment for multiple myeloma (MM) and is increasingly used as part of the initial treatment of this disease. Recent reports have suggested decreases in the number of CD34+ cells collected and increases in the failure rate among patients whose initial therapy contained lenalidomide when mobilized with G-CSF alone. A retrospective data analysis of 364 patients with MM who underwent stem cell mobilization and attempted harvest at the Hospital of the University of Pennsylvania between January 2002 and December 2007 was performed. Forty-three of the patients received lenalidomide in their induction regimen, and were mobilized with either CY and G-CSF or G-CSF alone. The number of apheresis cycles and the failure rate were lower, whereas the mean number of collected stem cells was higher in patients who were mobilized with CY and G-CSF in comparison with G-CSF alone. This suggests that lenalidomide does not prevent the harvest of adequate numbers of CD34 cells for autologous stem cell transplant, but mobilization with G-CSF and CY may be required to obtain adequate numbers of stem cells. Finally, in our study, the number of lenalidomide cycles did not correlate with stem cell yield.

cis Expression of DC-SIGN allows for more efficient entry of human and simian immunodeficiency viruses via CD4 and a coreceptor.

DC-SIGN is a C-type lectin expressed on dendritic cells and restricted macrophage populations in vivo that binds gp120 and acts in trans to enable efficient infection of T cells by human immunodeficiency virus type 1 (HIV-1). We report here that DC-SIGN, when expressed in cis with CD4 and coreceptors, allowed more efficient infection by both HIV and simian immunodeficiency virus (SIV) strains, although the extent varied from 2- to 40-fold, depending on the virus strain. Expression of DC-SIGN on target cells did not alleviate the requirement for CD4 or coreceptor for viral entry. Stable expression of DC-SIGN on multiple lymphoid lines enabled more efficient entry and replication of R5X4 and X4 viruses. Thus, 10- and 100-fold less 89.6 (R5/X4) and NL4-3 (X4), respectively, were required to achieve productive replication in DC-SIGN-transduced Jurkat cells when compared to the parental cell line. In addition, DC-SIGN expression on T-cell lines that express very low levels of CCR5 enabled entry and replication of R5 viruses in a CCR5-dependent manner, a property not exhibited by the parental cell lines. Therefore, DC-SIGN expression can boost virus infection in cis and can expand viral tropism without affecting coreceptor preference. In addition, coexpression of DC-SIGN enabled some viruses to use alternate coreceptors like STRL33 to infect cells, whereas in its absence, infection was not observed. Immunohistochemical and confocal microscopy data indicated that DC-SIGN was coexpressed and colocalized with CD4 and CCR5 on alveolar macrophages, underscoring the physiological significance of these cis enhancement effects.

Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding.

The study of early events in the human immunodeficiency virus type 1 (HIV-1) life cycle can be limited by the relatively low numbers of cells that can be infected synchronously in vitro. Although the efficiency of HIV-1 infection can be substantially improved by centrifugal inoculation (spinoculation or shell vial methods), the underlying mechanism of enhancement has not been defined. To understand spinoculation in greater detail, we have used real-time PCR to quantitate viral particles in suspension, virions that associate with cells, and the ability of those virions to give rise to reverse transcripts. We report that centrifugation of HIV-1(IIIB) virions at 1,200 x g for 2 h at 25 degrees C increases the number of particles that bind to CEM-SS T-cell targets by approximately 40-fold relative to inoculation by simple virus-cell mixing. Following subsequent incubation at 37 degrees C for 5 h to allow membrane fusion and uncoating to occur, the number of reverse transcripts per target cell was similarly enhanced. Indeed, by culturing spinoculated samples for 24 h, approximately 100% of the target cells were reproducibly shown to be productively infected, as judged by the expression of p24(gag). Because the modest g forces employed in this procedure were found to be capable of sedimenting viral particles and because CD4-specific antibodies were effective at blocking virus binding, we propose that spinoculation works by depositing virions on the surfaces of target cells and that diffusion is the major rate-limiting step for viral adsorption under routine in vitro pulsing conditions. Thus, techniques that accelerate the binding of viruses to target cells not only promise to facilitate the experimental investigation of postentry steps of HIV-1 infection but should also help to enhance the efficacy of virus-based genetic therapies.

The dendritic cell-T cell milieu of the lymphoid tissue of the tonsil provides a locale in which SIV can reside and propagate at chronic stages of infection.

Previous studies described the presence of numerous human immunodeficiency virus (HIV)-positive cells within and just beneath the mucosal surfaces of the tonsillar tissue of HIV-1-infected individuals. The virus-positive cells were most abundant in the dendritic cell (DC)-T cell rich areas of the lymphoepithelia lining the crypts, and consisted of multinucleated syncytia that contained DCs. This suggested that such cells within the tonsillar tissue might represent a site for chronic virus replication in infected individuals. Using the simian immunodeficiency virus (SIV)-macaque system, we chose to study further the viral distribution within the tonsillar tissue of animals infected via the vaginal route 8-10 months earlier. Our initial studies demonstrated that in situ hybridization (ISH)-positive DCs and T cells could be identified within the genital mucosa and draining lymph nodes of these infected animals even at this chronic stage of infection. Here we specifically examined the distal mucosa-associated lymphoid tissues of the tonsil. ISH-positive cells were mostly restricted to the DC-rich T cell areas of the underlying lymphoid tissue. However, T cells were the most commonly infected cell type and virus-positive cells were rarely found within the epithelia. In isolated cell suspensions, ISH-positive lymphocytes were often tightly associated with ISH-negative DCs, although few ISH-positive lymphocytes were often tightly associated with ISH-negative DCs, although few ISH-positive DCs could be identified within these clusters. Therefore, the naturally occurring DC-T cell milieu of the lymphoid tissue of the tonsil provides a locale in which SIV can reside and propagate on a chronic basis, even many months after the animals were infected by virus crossing the genital mucosa.

The orodental status of a group of elderly in-patients: a preliminary assessment.

OBJECTIVE: To provide a preliminary assessment of the orodental status and dental treatment requirements of a group of elderly in-patients. DESIGN: Cross-sectional. SETTING: Acute Care of the Elderly and Stroke Rehabilitation units at teaching hospitals in Merseyside. SUBJECT: 150 patients aged 58 to 94 years, in which a history could be validated at interview. INTERVENTION: Questionnaire administered by dentist and clinical examination. MAIN OUTCOME MEASURES: Registration with a dentist, prosthetic status and difficulties with dentures, denture hygiene and identification marking, dental treatment needs and evidence of mucosal pathology. RESULTS: Only 27% of patients claimed registration with a dentist. Three quarters of the patients were edentulous and 66 patients wore full dentures; 18 had no prostheses. Difficulties were experienced by one quarter of patients with upper dentures, compared with a half of lower denture wearers. Of the dentures available for inspection, 61% had removable soft debris, 66% were left out at night and 75% were cleaned by the patient, whilst on the ward. No dentures had evidence of identification marking. Of the 39 partially dentate patients, 75% required interventive dental treatment. Denture stomatitis was diagnosed in 29% of patients and 19 had evidence of benign mucosal pathology. CONCLUSIONS: The orodental status of this group of elderly in-patients was poor, with a high proportion being edentulous. Few were registered with a dentist and denture hygiene was inadequate. Lack of identification marking is a matter of concern. Closer liaison between hospital staff responsible for elderly in-patients is required, to improve the orodental health and quality of life of this medically compromised group of patients.

Dendritic cells from skin and blood of macaques both promote SIV replication with T cells from different anatomical sites.

The SIV-macaque system offers the opportunity to study the pathogenesis and immune aspects of a primate retroviral infection in which immunodeficiency also develops, much like HIV infection in humans. Since it is known that human dendritic cells (DCs) are involved in HIV replication, mature cytokine-generated DCs obtained from precursors in the blood and skin-derived DCs were isolated from healthy rhesus macaques and compared with respect to their ability to support SIV infection. Here, it is shown for both skin- and blood-derived DCs that i) virus production depends on both DCs and T cells, ii) this occurs similarly with T cells from blood, skin, spleen, or lymph nodes, and iii) DCs can transmit virus equally to syngeneic and allogeneic T cells. No differences between DCs from skin or blood were observed. Therefore, the easily accessible blood-derived DCs of macaques provide an appropriate population to study the role of DCs in immunodeficiency virus infection.

Immunophenotypic characterization of simian immunodeficiency virus-infected dendritic cells in cervix, vagina, and draining lymph nodes of rhesus monkeys.

Significant progress has been made in understanding the biology of heterosexual transmission of HIV by utilizing the simian immunodeficiency virus (SIV)/rhesus monkey animal model. Our previous studies have shown that SIV-infected cells within the stratified squamous epithelium of the vagina have a dendritic morphology. However, the type of cell infected was not conclusively determined. The purpose of the present study was to immunophenotype the SIV-infected cells in the lower reproductive tract and genital lymph nodes of the female rhesus monkey. Vagina, cervix, and iliac lymph node from eight chronically SIV-infected adult female monkeys were examined for this study. None of the animals had histologic evidence of opportunistic infections or genital tract pathogens other than SIV. Combined in situ hybridization and immunohistochemistry were used to detect SIV RNA and to determine the immunophenotype of SIV-infected cells in tissue sections and cytospin preparations of cells from the tissues. We now show that SIV-infected cells were most common in iliac lymph node and that the majority of infected cells in the lymph node were T lymphocytes. SIV-infected macrophages, Langerhans' cells, and dendritic cells were also found in the lymph node. SIV-infected cells were found within the epithelium and lamina propria of the vagina. Although most of the infected cells were T cells, a significant proportion (approximately 40%) of the SIV-infected cells in cytospin preparations from explant cultures of vagina and cervix were Langerhans' cells. SIV-infected T cells in the lower genital tract were commonly associated with focal mononuclear cell infiltrates. SIV-infected macrophages were rarely found in the genital tract. The present study provides the first direct demonstration that Langerhans' cells and dendritic cells in the genital tract and lymph nodes are infected with SIV in vivo. Thus, dendritic cells, in general, and Langerhans' cells, in particular, are important reservoirs for HIV/SIV replication in vivo.

Generation of monocyte-derived dendritic cells from precursors in rhesus macaque blood.

While the dendritic cells (DCs) of mouse and man have been extensively studied, until recently those of non-human primates remained poorly characterized. We present a method for generating large numbers of DCs from precursors in rhesus macaque blood, based on techniques developed for human blood. For 7 days, a T cell-depleted population of mononuclear cells was cultured in 1% human plasma with GM-CSF and IL-4, both to initiate DC differentiation and to inhibit macrophage development. On day 7, 50% of the culture medium was replaced with a monocyte-conditioned medium (MCM), which is required for the final maturation of the DCs into potent stimulators of the allogeneic MLR. Between 0.5 and 1.0 x 10(6) DCs can be generated from 20 ml of rhesus macaque blood. We compared these cytokine-generated DCs to the adherent macrophages present in the same cultures. Cytokine-generated DCs were considerably more potent at stimulating allogeneic T cells than adherent macrophages. Furthermore, the DCs had a distinct morphology and phenotype, with long processes, high levels of p55, and a characteristic perinuclear collection of intracellular CD68. In contrast, adherent macrophages expressed very low levels of p55, and high diffuse levels of CD68. Macaque DCs generated by this method may be useful in vaccine development and for studies of SIV pathogenesis.

Efficient interaction of HIV-1 with purified dendritic cells via multiple chemokine coreceptors.

HIV-1 actively replicates in dendritic cell (DC)-T cell cocultures, but it has been difficult to demonstrate substantial infection of purified mature DCs. We now find that HIV-1 begins reverse transcription much more efficiently in DCs than T cells, even though T cells have higher levels of CD4 and gp120 binding. DCs isolated from skin or from blood precursors behave similarly. Several M-tropic strains and the T-tropic strain IIIB enter DCs efficiently, as assessed by the progressive formation of the early products of reverse transcription after a 90-min virus pulse at 37 degrees C. However, few late gag-containing sequences are detected, so that active viral replication does not occur. The formation of these early transcripts seems to follow entry of HIV-1, rather than binding of virions that contain viral DNA. Early transcripts are scarce if DCs are exposed to virus on ice for 4 h, or for 90 min at 37 degrees C, conditions which allow virus binding. Also the early transcripts once formed are insensitive to trypsin. The entry of a M-tropic isolates is blocked by the chemokine RANTES, and the entry of IIIB by SDF-1. RANTES interacts with CCR5 and SDF-1 with CXCR4 receptors. Entry of M-tropic but not T-tropic virus is ablated in DCs from individuals who lack a functional CCR5 receptor. DCs express more CCR5 and CXCR4 mRNA than T cells. Therefore, while HIV-1 does not replicate efficiently in mature DCs, viral entry can be active and can be blocked by chemokines that act on known receptors for M- and T-tropic virus.

Difficulties in obtaining monoclonal antibodies to subsets of human leukocytes, using neonatal tolerance induction in mice.

Many antigens are shared between different types of human leukocytes. In an effort to obtain new lineage-specific monoclonal antibodies, particularly antibodies to dendritic cells, we attempted to tolerize newborn mice to one type of leukocyte and then immunize the adults with another. We found that T cells, either unstimulated T cells or T blasts, were more effective at inducing neonatal tolerance than non-T cells or B cell lines. However, the tolerance that was achieved was not restricted to T cells, since we could not elicit from the tolerized mice a specific antibody response to a B cell line or to blood dendritic cells. Here we describe several efforts, all unsuccessful, to achieve cell specific immune responses in tolerant mice. The parameters we considered included the type of cell used to tolerize neonatal mice, the regimen of injections for inducing tolerance or eliciting immunity, and the use of several different adjuvants.

Susceptibility of dendritic cells to HIV-1 infection in vitro.

We review recent work on the extent of HIV-1 infection of dendritic cells (DCs) and the consequences of exposure to virus. The reported levels of infection of DCs from blood have varied from "explosive" to "undetectable." The only study that used sorted DCs demonstrated little if any infectability, which may not be surprising given the very low levels of CD4 on the populations that were studied. HIV-1-pulsed, highly purified DCs function as potent antigen-presenting cells during the mixed leukocyte reaction and responses to superantigens. At the same time that the HIV-1-pulsed DCs stimulate CD4+ T cells in DC-T clusters, the virus is transferred to the responding lymphocytes and a vigorous productive infection of the T cells takes place. This pool of transferable HIV-1 is short lived in cultured human blood DCs and likely reflects the capacity of these cells to internalize and recycle vesicles in the endocytic pathway, as revealed with experiments using 0.1-micron fluorescent latex beads. Current efforts are directed to analyzing the interaction of HIV-1 with several populations of DCs that express higher levels of CD4. These include DCs studied in fresh, uncultured blood, as well as skin, thymus, and tonsil DCs. In each case, entry and reverse transcription of HIV-1 are seen, but again, coculture with T cells is required for a productive infection to take place. We conclude that DCs could play a critical role in the pathogenesis of HIV-1 infection, but that the interaction with CD4+ T cells is a critical variable in analyzing the extent of productive infection and its consequences.

Human blood contains two subsets of dendritic cells, one immunologically mature and the other immature.

Two subsets of dendritic cells, differing in T-cell stimulatory function, have been purified directly from human blood. Both subsets are positive for major histocompatibility complex (MHC) class II expression and negative for lineage-specific antigens (e.g. CD3, CD14, CD16, CD19 negative), but are separated by exploiting differences in expression of the beta 2-integrin, CD11c. The CD11c-negative subset is functionally immature, requiring monocyte-derived cytokines to develop into typical dendritic cells. The CD11c-positive subset has potent T-cell stimulating activity and expresses the activation antigen CD45RO, unlike its immature counterpart. However, these mature cells only develop typical dendritic morphology and high levels of MHC proteins and adhesins after a period of culture independent of exogenous cytokines. Although the freshly isolated mature dendritic cells resemble monocytes in cytospin preparations, the former lack CD14 and have a much stronger primary T-cell stimulatory capacity. We hypothesize that the CD11c-negative immature cells are marrow-derived precursors to tissue dendritic cells, such as epidermal Langerhans' cells, while the CD11c-positive cells are derived from tissues where they have been activated by antigen, and are en route to the spleen or lymph nodes to stimulate T-cell responses there.

Dendritic cells freshly isolated from human blood express CD4 and mature into typical immunostimulatory dendritic cells after culture in monocyte-conditioned medium.

A procedure has been developed to isolate dendritic cells to a high degree of purity from fresh blood. Prior enrichment methods have relied upon an initial 1-2-d culture period. Purified fresh isolates lack the characteristic morphology, phenotype, and immunostimulatory function of dendritic cells. The purified cells have the appearance of medium sized lymphocytes and express substantial levels of CD4, but lack the T cell molecules CD3, CD8, and T cell receptor. When placed in culture, the cells mature in a manner resembling the previously described, cytokine-dependent maturation of epidermal dendritic cells (Langerhans cells). The cells enlarge and exhibit many cell processes, express much higher levels of major histocompatibility complex class II and a panel of accessory molecules for T cell activation, and become potent stimulators of the mixed leukocyte reaction. Among the many changes during this maturation process are a fall in CD4 and the appearance of high levels of B7/BB1, the costimulator for enhanced interleukin 2 production in T cells. These changes are not associated with cell proliferation, but are dependent upon the addition of monocyte-conditioned medium. We suggest that the freshly isolated CD4-positive blood dendritic cells are recent migrants from the bone marrow, and that subsequent maturation of the lineage occurs in tissues in situ upon appropriate exposure to cytokines.

Tolerizing mice to human leukocytes: a step toward the production of monoclonal antibodies specific for human dendritic cells.

Despite several attempts to isolate a mAb specific for human dendritic cells, none currently exists. Recent attempts have utilized an improved dendritic cell purification method to prepare immunogens and a rapid two-color flow cytometric screening procedure that allows large numbers of hybridoma supernatants to be examined in each fusion. Yet these improvements have also failed, yielding only hybridomas that bind "shared" antigens expressed by both dendritic cells and other leukocytes. Dendritic cells express many shared antigens, including CD45 [leukocyte common antigen], CD40, leukocyte [beta 2] integrins CD11a and CD11c, CD54 [ICAM-1], CD44 [Pgp-1], CD58 [LFA-3], and the B7/BB1 antigen. Therefore, we are attempting to bias the immune response toward rarer, dendritic cell-specific clones by tolerizing or immunosuppressing our animals to shared antigens. In one approach, adult mice held in barrier cages are injected with "nondendritic" cells and cyclophosphamide [CP], in order to ablate responding "nonspecific" B cell clones. Fifteen days after the last dose of CP, they are challenged with nondendritic cells. A week later they are bled, and serum antibody titers against nondendritic cells are determined by FACS, in order to demonstrate tolerance compared to controls injected with CP alone. In the second approach, neonatal mice are injected with human T lymphoblasts at birth, followed by boosting at 1 week. In adulthood, they are challenged sequentially with sheep erythrocytes [sRBC], then with T blasts, to demonstrate that they can respond to unrelated cells but not to tolerogenic cells. One week after each kind of challenge, mice are bled and serum antibody levels are determined for treated and sham-injected mice. When these two approaches were compared, CP led only to nonspecific immunosuppression, while neonatal injections produced selective, antigen-specific nonresponsiveness to the tolerizing T blasts.