Conference Scene: Personalized medicine: changing the healthcare paradigm.

The US Air Force Patient-Centered Precision Care Genomic Medicine Research Program hosted a symposium entitled 'Changing the Health Care Paradigm' on 27 September 2011 in Laurel, MD, USA. The program provided a unique opportunity for subject matter experts to participate in critical discussions on the current and potential outlook for the implementation of genome-informed healthcare. The implications for patient privacy, data management and security revealed the ethical, legal and social issues faced by clinicians, patients and policy-makers. Highlights from these discussions are summarized in this article.

Immunodiagnostics: evaluation of functional T-cell immunocompetence in whole blood independent of circulating cell numbers.

The need for a systematic approach for immune function monitoring has becoming increasingly apparent in the past decade due to the rapid expansion of the development and use of immunomodulatory drug therapies and vaccines. While there has been a great deal of progress in the development of methodologies for evaluating and enumerating T-lymphocyte responses to infection and cancer, the translation of these assays into the clinical setting has remained seemingly elusive. This is likely due to inherent difficulties in the standardization and validation of cell-based assays. Here, we describe a novel assay that measures ATP production in CD4(+) T-lymphocytes in response to stimulation. Results from the test, unlike absolute cell counts, assess the functional response of lymphocytes. Clinical utility of the assay has been demonstrated in managing immunosuppression in solid organ transplant recipients such that adverse events such as infection and rejection can be avoided. The need for a global immune response test in the clinical setting of transplantation and the value of such a test in post-transplant management is discussed. Furthermore, additional applications of this assay for monitoring diseases that impact immune function including autoimmunity and infection are considered.

Longitudinal analysis of simian immunodeficiency virus (SIV) replication in the lungs: compartmentalized regulation of SIV.

BACKGROUND: Before the onset of AIDS, replication of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) in the lungs is considered to be latent. When and how virus replication is controlled in the lungs is unclear. In the present study, we examine virus replication in the lungs and in cells recovered from bronchoalveolar lavage (BAL) samples in a comprehensive, longitudinal analysis of an SIV/macaque model. METHODS: Gene-specific RNA and DNA were quantitated by polymerase chain reaction (PCR) and by real-time reverse-transcription PCR (RT-PCR). Alveolar macrophages were isolated using Dynabeads CD14 (Invitrogen). Expression of CCAAT/enhancer-binding protein beta (C/EBP beta ) isoforms was examined by Western blot analysis. RESULTS: SIV replication occurred in the lungs during acute infection and correlated with plasma viral load. Innate immune responses involving interferon- beta and the dominant-negative isoform of C/EBP beta were induced at this time. SIV RNA expression was suppressed in the lungs during asymptomatic infection, when no correlation existed with plasma viral load until SIV RNA levels rebounded again during late-stage disease. Modulation of viral RNA levels in BAL cells reflected RNA levels in lung tissue throughout each phase of infection. CONCLUSION: Quantitation of SIV RNA in BAL cells provides a consistent surrogate assessment of virus replication in lung tissue. Innate immune responses contribute to compartmentalized suppression of acute SIV replication in the lungs.

Morphine withdrawal dramatically reduces lymphocytes in morphine-dependent macaques.

The immune effects of chronic opiate exposure and/or opiate withdrawal are not well understood. The results of human studies with opiate abusers are variable and may not be able to control for important factors such as subjects' drug histories, health and nutritional status. Nonhuman primate models are necessary to control these important factors. A model of opiate dependence in macaques was developed to study the effects of opiate dependence and withdrawal on measures of immune function. Four pigtailed macaques drank a mixture of morphine (20 mg/kg/session) and orange-flavored drink every 6 h for several months. During stable morphine dependence, absolute numbers of neutrophils, monocytes and lymphocytes did not change relative to pre-morphine levels. However, there was a significant decrease in the absolute number and percentage of natural killer (NK) cells in morphine dependence. Either precipitated withdrawal or abstinence for 24 h resulted in behavioral withdrawal signs in all animals. Absolute lymphocyte counts decreased and absolute netrophil counts increased significantly in withdrawal, relative to levels during morphine dependence. Lymphocyte subset (CD4+, CD8+, CD20+) cells were also decreased in absolute numbers with little change in their percentage distributions. There was, however, a significant increase in the percentage of NK cells in withdrawal relative to levels during morphine dependence. This study demonstrates the usefulness of voluntary oral self-dosing procedures for maintaining morphine dependence in nonhuman primates and demonstrates that the morphine withdrawal syndrome includes large alterations in blood parameters of immune system function, including nearly 50% reduction in numbers of CD4+, CD8+ and CD20+ cells.

The central nervous system is a viral reservoir in simian immunodeficiency virus--infected macaques on combined antiretroviral therapy: a model for human immunodeficiency virus patients on highly active antiretroviral therapy.

This study used a simian immunodeficiency virus (SIV)-macaque model to determine whether virus persists in the central nervous system (CNS) of human immunodeficiency virus (HIV)-infected individuals in which plasma viral load has been suppressed by highly active antiretroviral therapy. SIV-infected macaques were treated with two reverse transcriptase inhibitors: PMPA (q- R-(2-phosphonomethoxypropyl)adenine)which does not cross the blood-brain barrier, and FTC (beta-2('),3(')-dideoxy-3 thia-5-fluorocytidine), which does. Viral DNA and RNA were quantitated in the brain after 6 months of suppression of virus replication in blood and cerebrospinal fluid (CSF). Viral DNA was detected in brain from all macaques, including those in which peripheral viral replication had been suppressed either by antiretroviral therapy or host immune responses. Significant neurological lesions were observed only in one untreated macaque that had active virus replication in the CNS. Expression of the inflammatory markers, major histocmopatibility complex (MHC) II and CD68 was significantly lower in macaques treated with PMPA/FTC. Thus, although antiretroviral treatment may suppress virus replication in the periphery and the brain and reduce CNS inflammation, viral DNA persists in the brain despite treatment. This suggests that the brain may serve as a long-term viral reservoir in HIV-infected individuals treated with antiretroviral drugs that suppress virus replication.

SIV-specific T lymphocyte responses in PBMC and lymphoid tissues of SIV-infected pigtailed macaques during suppressive combination antiretroviral therapy.

There is currently no SIV macaque model in which the effects of combination antiretroviral therapy on tissue immune responses and latent reservoirs have been measured. This study was performed to define the impact of combination therapy on the specificity and distribution of the T lymphocyte response in multiple tissue compartments. Pigtailed macaques (Macaca nemestrina) were infected with SIV/17E-Fr and treated with combination antiretroviral therapy consisting of 9-R-(2-phosphonomethoxypropyl)adenine (PMPA) and beta-2',3'-dideoxy-3'-thia-5-fluorocytidine (FTC). The SIV-specific T lymphocyte response was measured in peripheral blood, spleen and several lymph nodes at necropsy by IFN-gamma Elispot analysis. Two animals (one treated, one untreated) had high acute peak viremia, which was associated with lower SIV-specific T lymphocyte responses in the peripheral blood and lymphoid tissues. In the treated animal, viremia was controlled to low or undetectable for the study duration, and virus-specific responses remained low. The untreated animal remained viremic throughout the study and developed clinical symptoms of AIDS. In contrast, the two animals that had lower acute peak viremia (one treated, one untreated) had more robust T lymphocyte responses, and controlled viral replication. Virus-specific responses were detected in the treated animal despite 6 months of suppressive therapy. These data suggest that in this model, in the context of acute peak viremia and weak T cell responses, combination therapy may be essential to control virus replication and disease progression. Conversely, in the setting of low initial viremia and robust T lymphocyte responses, treatment does not have a detrimental effect on the immune response.

Resting CD4+ T lymphocytes but not thymocytes provide a latent viral reservoir in a simian immunodeficiency virus-Macaca nemestrina model of human immunodeficiency virus type 1-infected patients on highly active antiretroviral therapy.

Despite suppression of viremia in patients on highly active antiretroviral therapy (HAART), human immunodeficiency virus type 1 persists in a latent reservoir in the resting memory CD4(+) T lymphocytes and possibly in other reservoirs. To better understand the mechanisms of viral persistence, we established a simian immunodeficiency virus (SIV)-macaque model to mimic the clinical situation of patients on suppressive HAART and developed assays to detect latently infected cells in the SIV-macaque system. In this model, treatment of SIV-infected pig-tailed macaques (Macaca nemestrina) with the combination of 9-R-(2-phosphonomethoxypropyl)adenine (PMPA; tenofovir) and beta-2',3'-dideoxy-3'-thia-5-fluorocytidine (FTC) suppressed the levels of plasma virus to below the limit of detection (100 copies of viral RNA per ml). In treated animals, levels of viremia remained close to or below the limit of detection for up to 6 months except for an isolated "blip" of detectable viremia in each animal. Latent virus was measured in blood, spleen, lymph nodes, and thymus by several different methods. Replication-competent virus was recovered after activation of a 99.5% pure population of resting CD4(+) T lymphocytes from a lymph node of a treated animal. Integrated SIV DNA was detected in resting CD4(+) T cells from spleen, peripheral blood, and various lymph nodes including those draining the gut, the head, and the limbs. In contrast to the wide distribution of latently infected cells in peripheral lymphoid tissues, neither replication-competent virus nor integrated SIV DNA was detected in thymocytes, suggesting that thymocytes are not a major reservoir for virus in pig-tailed macaques. The results provide the first evidence for a latent viral reservoir for SIV in macaques and the most extensive survey of the distribution of latently infected cells in the host.

Cross-clade T lymphocyte-mediated immunity to HIV type 1: implications for vaccine design and immunodetection assays.

Identifying immunodominant regions of HIV-1 that are recognized by CD8(+) T lymphocytes in infected individuals may be important for the design and evaluation of candidate HIV-1 vaccines, particularly for developing countries. In this study, cryopreserved peripheral blood mononuclear cells (PBMCs) from 15 chronically HIV-1-infected U.S. volunteers were screened for HIV-1 Gag-specific T lymphocyte interferon gamma production in an enzyme-linked immunospot (ELISpot) assay matrix format, using overlapping HIV-1 subtype A, B, and C Gag peptide pools. In the initial matrix screen, responses to HIV-1 subtype B Gag were detected in 11 of 15 (73%) of seropositive individuals and in none of 4 HIV-1-seronegative controls. There were differences in both the breadth and magnitudes of the responses observed in the matrix assay. Responses varied in breadth, ranging from broad responses (more than four peptides) of moderate magnitude (<100 spot-forming cells [SFCs]/10(5) PBMCs) to narrowly focused (two or fewer peptides), more potent responses (>150 SFC/10(5) PBMCs). Responses to A, B, and C clade peptides of HIV-1 Gag revealed that all responders to subtype B peptides were also found to recognize corresponding peptides from at least one of the other clades. The ability to recognize cross-clade peptides with one or two amino acid substitutions relative to the B clade peptide was both peptide and patient dependent. Overall, our results show that the ELISpot matrix algorithm described here may be an efficient approach for characterizing cross-clade CD8(+) T cell responses in either seropositive individuals or in seronegative HIV-1 vaccine recipients.

Association of Gag-specific T lymphocyte responses during the early phase of human immunodeficiency virus type 1 infection and lower virus load set point.

T lymphocyte responses to human immunodeficiency virus (HIV) type 1 Gag were measured in 9 patients by interferon-gamma enzyme-linked immunospot assay at 3 time points within 12 months of infection. Patients with early recognition of HIV-1 Gag had lower subsequent HIV-1 load set points, as measured during the first 2 years of infection, compared with those of patients with undetectable Gag-specific responses (median, 4.27 vs. 5.05 log(10) HIV-1 RNA copies/mL, respectively; P=.028). An inverse correlation existed between the magnitude of the Gag-specific responses and the HIV-1 load set point (r=-0.733; P=.025). Early sustained T lymphocyte responses to HIV-1 Gag may be important for the establishment of virus load set point.