Immunological and Antiviral Responses After Therapeutic DNA Immunization in Chronic Hepatitis B Patients Efficiently Treated by Analogues

A substudy of a phase I/II, prospective, multicenter clinical trial was carried out to investigate the potential benefit of therapeutic vaccination on hepatitis B e antigen-negative patients with chronic hepatitis B (CHB), treated efficiently with analogues. Patients were randomized in 2 arms, one receiving a hepatitis B virus (HBV) envelope DNA vaccine, and one without vaccination. At baseline, HBV-specific interferon (IFN)-γ–producing T cells were detected in both groups after in vitro expansion of peripheral blood mononuclear cells. Vaccine-specific responses remained stable in the vaccine group, whereas in the control group the percentage of patients with HBV-specific IFN-γ–producing T cells decreased over time. The vaccine-specific cytokine-producing T cells were mostly polyfunctional CD4⁺ T cells, and the proportion of triple cytokine-producer T cells was boosted after DNA injections. However, these T-cell responses did not impact on HBV reactivation after stopping analogue treatment. Importantly, before cessation of treatment serum hepatitis B surface antigen (HBsAg) titers were significantly associated with DNA or HBsAg clearance. Therapeutic vaccination in CHB patients with persistent suppression of HBV replication led to the persistence of T-cell responses, but further improvements should be searched for to control infection after treatment discontinuation.     

The HIV-1 reservoir in eight patients on long-term suppressive antiretroviral therapy is stable with few genetic changes over time

The source and dynamics of persistent HIV-1 during long-term combinational antiretroviral therapy (cART) are critical to understanding the barriers to curing HIV-1 infection. To address this issue, we isolated and genetically characterized HIV-1 DNA from naïve and memory T cells from peripheral blood and gut-associated lymphoid tissue (GALT) from eight patients after 4–12 y of suppressive cART. Our detailed analysis of these eight patients indicates that persistent HIV-1 in peripheral blood and GALT is found primarily in memory CD4⁺ T cells [CD45RO⁺/CD27(^+/−)]. The HIV-1 infection frequency of CD4⁺ T cells from peripheral blood and GALT was higher in patients who initiated treatment during chronic compared with acute/early infection, indicating that early initiation of therapy results in lower HIV-1 reservoir size in blood and gut. Phylogenetic analysis revealed an HIV-1 genetic change between RNA sequences isolated before initiation of cART and intracellular HIV-1 sequences from the T-cell subsets after 4–12 y of suppressive cART in four of the eight patients. However, evolutionary rate analyses estimated no greater than three nucleotide substitutions per gene region analyzed during all of the 4–12 y of suppressive therapy. We also identified a clearly replication-incompetent viral sequence in multiple memory T cells in one patient, strongly supporting asynchronous cell replication of a cell containing integrated HIV-1 DNA as the source. This study indicates that persistence of a remarkably stable population of infected memory cells will be the primary barrier to a cure, and, with little evidence of viral replication, this population could be maintained by homeostatic cell proliferation or other processes.Combinational, antiretroviral therapy (cART) effectively suppresses but does not eradicate HIV-1 infection (1). Persistent low-level HIV-1 can still be detected in plasma (2–7) and cellular reservoirs (8–10) even after several years of suppressive cART, and cessation of current treatments invariably results in resumption of viral replication. Resting-memory CD4⁺ T cells are a well-defined reservoir of HIV-1, and the reservoir is established when an activated CD4⁺ T cell becomes infected by HIV-1 but transitions to a resting state (9) or perhaps when resting cells are infected directly (11–13). Central and transitional memory T cells have recently been identified as major contributors to the HIV-1 reservoir in the memory T-cell population (14). Naïve T cells have also been demonstrated to contain HIV-1 DNA in patients on suppressive therapy, although at a lower infection frequency than the memory T-cell population (15). In addition, many other cell types, including monocyte/macrophages, have been proposed to play a role in HIV-1 persistence (reviewed in ref. 16). These long-lived HIV-1–infected cells have been detected in peripheral blood. Several studies, however, suggest that the reservoir is largely established and maintained in lymphoid tissues, and that the infected cells circulating in blood may not be representative of the population of infected cells in tissue. For example, the majority of lymphocytes are sequestered in the gastrointestinal tract, and gut-associated lymphoid tissue (GALT) has been shown to be a major viral reservoir in patients on suppressive antiretroviral therapy (17–22).In addition to the persistence of long-lived, latently infected cells, low-level viral replication has been proposed as a mechanism that maintains HIV-1 during cART. If complete viral replication cycles persist, despite suppressive antiretroviral therapy, this would lead to de novo cellular infection and a constant replenishment of the viral reservoir. Investigations into whether HIV-1 replication continues during suppressive therapy have been carried out with peripheral blood and GALT samples but have led to potentially contradictory results. Some studies have found an absence of genetic evolution in viral reservoirs (23–29) and no reduction of plasma RNA during intensification of cART (30, 31), suggesting that cART is effective in preventing viral replication in these anatomical sites. In contrast, increased numbers of 2-long terminal repeat circles in peripheral blood mononuclear cells and decreased amounts of unspliced HIV-1 RNA in CD4⁺ T cells isolated from the terminal ileum have been reported during raltegravir intensification, supporting the concept that some viral replication can occur despite suppressive cART (32, 33). Thus, the role of on-going replenishment via cycles of replication as a cause of persistence is not fully understood.To investigate the source and dynamics of HIV-1 reservoirs in peripheral blood and GALT, we sorted and genetically characterized intracellular HIV-1 from subsets of memory T cells, naïve T cells, and myeloid cells from these two compartments from eight patients who had been on suppressive therapy with undetectable viral loads (<40–75 copies/mL) for 4–12 y: five who initiated therapy during acute/early infection and three who initiated therapy during chronic infection. Our aim was to investigate the nature of the infected cell population during cART and explore the role of HIV-1 replication, as reflected by nucleotide sequence substitutions in maintaining this reservoir. Our study revealed that both memory T cells and naïve T cells harbor HIV-1 DNA after long-term suppressive therapy, and the infection frequency of these T cells was higher in patients treated during chronic infection compared with patients treated during early infection. In-depth phylogenetic analysis revealed little or no change in viral structure or divergence over time within the viral sequences isolated from the different T-cell populations compared with sequences isolated from plasma collected just before initiation of cART, indicating lack of on-going replication during long-term suppressive therapy.     

The usefulness of F-18 deoxyglucose whole-body positron emission tomography (PET) for re-staging of renal cell cancer

PURPOSE: The use of whole-body PET for re-staging of renal cell carcinoma has not been investigated. The aim of the current study was to examine the diagnostic accuracy and clinical usefulness of whole-body PET imaging for re-staging of renal cell cancer. PATIENTS AND METHODS: Clinical PET was performed for re-staging in 36 patients with advanced renal cell cancer. Written reports of imaging studies (including CT, MRI, US, plain film and bone scan), patient history, and extensive chart notes were used to define the clinical stage before PET (pre-PET stage). The written PET report was used to define the clinical stage after PET (PET stage). Reports were used to determine the accuracy of PET for re-staging renal cell cancer and for defining biopsy proven lesions. Clinical parameters and biopsy proven lesions served as reference for the accuracy of PET for re-staging renal cell cancer. RESULTS: PET classified the clinical stage correctly in 32/36 patients (89%) and was incorrect in 4/36 (11%) (sensitivity and specificity: 87% and 100%). In 20 patients, 25 suspicious lesions were biopsied within 3.2 +/- 6.7 months of the PET study. Of these, 17 were malignant and 8 were benign. PET correctly classified 21/25 (84%) of the biopsied lesions (sensitivity and specificity: 88% and 75%). CONCLUSION: PET re-stages renal cell cancer with a diagnostic accuracy of 89%. Its diagnostic accuracy for classifying biopsy proven anatomic lesions as malignant or benign was 84%. These findings suggest that PET is useful in characterizing anatomic lesions of unknown significance in patients with renal cell cancer.     

间硝苯地平对血管紧张素Ⅱ促进血管平滑肌细胞增殖和蛋白质合成的影响

以培养血管平滑肌细胞（ｖａｓｃｕｌａｒｓｍｃｏｔｈｍｕｓｃｌｅｃｅｌｌ，ＶＳＭＣ）为模型，观察了间硝苯地平（ｍ－ｎｉｆｅｄｉｐｉｎｅ，ｍ－Ｎｉｆ）对血管紧张素Ⅱ（ａｎｇｉｏｔｅｎｓｉｎⅡ，ＡＮＧⅡ）促进ＶＳＭＣ增殖和蛋白质合成的影响。结果表明，ｍ－Ｎｉｆ抑制ＡＮＧⅡ（１００ｎｍｏｌ·Ｌ￣（－１））引起ＶＳＭＣ［￣３Ｈ］ｔｈｙｍｉｄｉｎｅ和［￣３Ｈ］ｌｅｕｃｉｎｅ参入，并呈剂量依赖性。ｍ－Ｎｉｆ（２×１０￣（－６）ｍｏｌ·Ｌ￣（－１））可抑制ＡＮＧⅡ对ＶＳＭＣ的刺激、ＤＮＡ及蛋白质合成速率，分别降低了４６％，５８％，５３％。提示ｍ－Ｎｉｆ可抑制ＡＮＧⅡ对ＶＳＭＣ增殖和蛋白合成的促进作用     

Involvement of viral procapsid in the RNA synthesis and maturation of poliovirus

The procapsids of poliovirus were found to associate with the viral RNA replicative complex in the smooth membrane fraction. Solubilization of the membrane in the presence of high concentrations of salt dissociates the procapsids from the replicative intermediate; solubilization and RNase treatment result in the same dissociation. Nascent viral RNA synthesized in vitro in cell-free extracts was found to sediment as a provirion-like particle. In guanidine-treated cells in which viral RNA synthesis was inhibited, procapsids were preferentially lost from the smooth membrane. It is suggested that the procapsid is associated with the nascent RNA of the replicative complex in the smooth membrane and that this association is necessary for the continual synthesis of viral RNA.