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Functional T cell exhaustion occurs during chronic viral infection or in tumor settings. Beyer et al. report that chronic inflammation mediated by the cytokine TNF is responsible for this dysfunction and that blockade of this pathway restores immune system–mediated control of viral infection.

ABSTRACTPosaconazole is a commonly used antifungal for the prophylaxis and treatment of invasive fungal infections. We previously demonstrated that the intracellular concentration of posaconazole in peripheral blood mononuclear cells (PBMCs) and polymorphonuclear neutrophils (PMNs) was greatly increased compared to the plasma concentration. As these professional phagocytes are crucial to combat fungal infections, we set out to investigate if and how, beneficial or deleterious, this high loading of intracellular posaconazole impacts the functional capacities of these cells. Here, we show that high intracellular concentrations of posaconazole do not significantly impact PMN and monocyte-derived macrophage function in vitro. In particular, killing capacity and cytoskeletal features of PMN, such as migration, are not affected, indicating that these cells serve as vehicles for posaconazole to the site of infection. Moreover, since posaconazole as such slowed the germination of Aspergillus fumigatusconidia, infected neutrophils released less reactive oxygen species (ROS). Based on these findings, we propose that the delivery of posaconazole by neutrophils to the site of Aspergillusspecies infection warrants control of the pathogen and preservation of tissue integrity at the same time.

ABSTRACTTherapeutic drug monitoring (TDM) of antifungal plasma concentrations is increasingly recommended. However, data on antifungal concentrations in the other compartments of the peripheral blood are limited. Hence, we collected 23 blood samples from 14 patients receiving posaconazole for prophylaxis of fungal infections. These samples were separated by double-discontinuous Ficoll-Hypaque density gradient centrifugation. The intracellular posaconazole concentrations of the obtained cells, i.e., the peripheral blood mononuclear cells (PBMCs), polymorphonuclear leukocytes (PMNs), and red blood cells (RBCs), were determined by liquid chromatography-tandem mass spectrometry. The intracellular concentrations of the PBMCs and PMNs were significantly higher than those of surrounding media (P< 0.001). The ratios between the intracellular and extracellular concentrations (C/E) were 22.5 ± 21.2, 7.66 ± 6.50, and 0.09 ± 0.05 for the PBMCs, PMNs, and RBCs, respectively. Posaconazole reaches high concentrations within human PBMCs and PMNs and is, to a lesser extent, present in RBCs. The high intracellular concentrations might contribute to posaconazole efficacy and distribution.

ABSTRACTA rapid turnaround is a prerequisite of therapeutic drug monitoring (TDM). For antifungals, this need is still unmet, since hardly any method has been established to simultaneously quantitate concentrations of different antifungal classes. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed allowing quantitation of anidulafungin (ANF), caspofungin (CSF), isavuconazole (ISC), micafungin (MCF), posaconazole (PSC), and voriconazole (VRC). Quantitation was successful with diluted plasma samples, peripheral blood mononuclear cells (PBMC), polymorphonuclear leukocytes (PMN), and erythrocytes (RBC). A triple quadrupole mass spectrometer in selected reaction monitoring mode was used with positive electrospray ionization. Cells and calibration standards were extracted with acetonitrile containing internal standard. Internal standards were a CSF derivate for echinocandins and itraconazole for triazoles. Chromatographic separation of the supernatant was achieved by a gradient method facilitating a BetaBasic C4 column. Analytes were quantified in a single 8-min run. Calibration curves were linear and fitted using least squares with a weighting factor of the reciprocal concentration. Limits of detection (ng/ml) were ANF, 8.3; CSF, 31.5; ISC, 1.5; MCF, 97.7; PSC, 3.3; and VRC, 1.4. The lower limits of quantitation (ng/ml) were ANF, 64; CSF, 108; ISC, 4.5; MCF, 160; PSC, 10; and VRC, 4.2. Intraday precisions ranged from 6.3% to 8.8% for azoles and 8.8% to 15.4% for echinocandins. Intraday and interday accuracies (percent bias) of all analytes were within 13.8%. The method was established as standard practice for the quantitation of intracellular antifungal concentrations and optimizes TDM by applying a rapid single method for 6 antifungals.

Background: Despite the availability of 31 antiretroviral agents or fixed-dose combinations in the United States and European Union, there is a continuing need for antiretroviral agents with high genetic barriers to resistance, simple dosing schedules, and favorable tolerability and safety profiles. Vicriviroc is a small-molecule chemokine receptor antagonist that inhibits the binding of R5-tropic HIV-1 to host cells at the CCR5 co-receptor, thus preventing viral entry. Objective: To present an evidence-based assessment of the clinical efficacy, pharmacokinetics, and safety profile of vicriviroc. Method: We discuss available peer-reviewed publications as well as preliminary data presented at relevant scientific meetings. Results/conclusions: Vicriviroc has a favorable pharmacokinetic profile with a half-life that enables once-daily dosing. Minimal drug interactions have been demonstrated with other available antiretrovirals. Early clinical trials have established the safety of vicriviroc in both treatment-naive and treatment-experienced R5-tropic HIV-1 infected individuals. A Phase II study in treatment-experienced patients demonstrated early efficacy of 30 mg vicriviroc in a regimen containing a ritonavir-boosted protease inhibitor (PI/r). Phase III studies using the 30-mg PI/r dosing paradigm in R5-tropic treatment-experienced patients have completed 48 weeks, but data are not yet available. These results will further elucidate the role of vicriviroc in the treatment of HIV-1 infected individuals.

Antiretroviral therapy in human immunodeficiency virus infection is occasionally associated with poor immunologic responses despite full suppression of viral replication. As some combinations of nucleoside analogues (NA) have been associated with paradoxical depletion of CD4 T- cells, we postulated that depleting the antiretroviral regimen of NAs would improve quantitative immunological parameters. In a longitudinal prospective study we quantified CD4 Tcells after removing NAs from antiretroviral therapy. The NA for regimen consisted of atazanavir (300mg qd), saquinavir (1000mg bid), and ritonavir (100mg qd) in 14 patients with immunologic failure despite undetectable plasma HIV-RNA (CD4 T-cells < 250 cells/L (<17) HIV RNA, < 50 copies/mL). Additionally, we assessed the state of immunologic activation markers (CD38HLA-DR on CD4 and CD8 T-cells) by flow cytometry. The regimen was well tolerated. During the 48 week study CD4 T-cell counts improved significantly (mean and ± SEM [standard error of mean], baseline: 174/L (12.4) [15, 5.8], week 24: 232/L (14) [26, 5.3], week 48: 267/L (15.4) [34, 4.3]) with preservation of full viral suppression (p<0.05). Activation parameters of CD4 T-cells, but not of CD8 T-cells, decreased significantly. This treatment strategy may represent an option for patients with poor immunologic responses to antiretroviral therapy despite undetectable viremia.

Antiretroviral therapy in human immunodeficiency virus infection is occasionally associated with poor immunologic responses despite full suppression of viral replication. As some combinations of nucleoside analogues (NA) have been associated with paradoxical depletion of CD4+ T- cells, we postulated that depleting the antiretroviral regimen of NAs would improve quantitative immunological parameters. In a longitudinal prospective study we quantified CD4+ Tcells after removing NAs from antiretroviral therapy. The NA for regimen consisted of atazanavir (300mg qd), saquinavir (1000mg bid), and ritonavir (100mg qd) in 14 patients with immunologic failure despite undetectable plasma HIV-RNA (CD4+ T-cells < 250 cells/μL ( < 17%) HIV RNA, < = 50 copies/mL). Additionally, we assessed the state of immunologic activation markers (CD38+HLA-DR+ on CD4+ and CD8+ T-cells) by flow cytometry. The regimen was well tolerated. During the 48 week study CD4+ T-cell counts improved significantly (mean and ± SEM [standard error of mean], baseline: 174/μL (12.4%) [15, 5.8%], week 24: 232/μL (14%) [26, 5.3%], week 48: 267/μL (15.4%) [34, 4.3%]) with preservation of full viral suppression (p < 0.05). Activation parameters of CD4+ T-cells, but not of CD8+ T-cells, decreased significantly. This treatment strategy may represent an option for patients with poor immunologic responses to antiretroviral therapy despite undetectable viremia.

Carrier-mediated transport across cell membranes is an important determinant of activity, resistance, and toxicity of chemotherapeutic agents including antiretroviral (ARV) drugs (ARDs). The organic cation transporters (OCTs) 1 and 2 have been implicated in the translocation of different cationic drugs but so far were insufficiently tested for interactions with ARDs. Here, we assessed among cationic drugs commonly used in human immunodeficiency virus (HIV) therapy inhibitors and substrates of OCTs, and analyzed the tissue distribution of OCTs and their expression in lymph nodes (LNs), the primary intracellular target of HIV and ARDs. Inhibitors were identified by measuring the attenuated uptake of the radiolabeled model substrate 1-methyl-4-phenylpyridinium into OCT-transfected human embryonic kidney-293 cells in the presence of ARDs. Substrates were identified by measuring OCT-specific intracellular accumulation using liquid chromatography/tandem mass spectrometry. Inhibitory drugs were (in order of increasing potency): nelfinavir < ritonavir < saquinavir < indinavir < trimethoprim < pentamidine, with consistently lower IC50values determined for OCT1. Substrates with highest transport efficacy (Vmax/Km) were lamivudine (OCT1, 8 µl/mg protein/min; OCT2, 4.4 µl/mg protein/min) and zalcitabine (OCT1, 4.1 µl/mg protein/min; OCT2, 2.6 µl/mg protein/min). Using quantitative real-time polymerase chain reaction, a marked expression level of OCT1 was detected in human samples of liver, ovary, prostate, and testis, and of OCT2 in kidney, colon, heart, skeletal muscle, and testis. Expression of OCTs in LNs was low in HIV-negative control individuals but dramatically increased in HIV-infected persons. These data suggest that drug interactions about the OCTs may be relevant for the ARV therapy, in particular by influencing drug accession to infected tissues and hepatic or renal elimination.

Zusammenfassung: Anamnese und klinischer Befund:: Eine 63-jhrige Patientin stellte sich mit Verdacht auf Reaktivierung einer Lymphknotentuberkulose (LK-Tbc) in der Klinik der Autoren vor. Eine im Vorjahr diagnostizierte LK-Tbc war erfolgreich behandelt worden. Klinisch zeigten sich akut eine Verschlechterung des Allgemeinzustands, zervikale Lymphknoten-(LK-)Schwellungen, Nachtschwei, Dyspnoe und eine oberflchliche Inflammation der rechten Brust.Untersuchungen:: Computertomographie von Hals, Thorax und Abdomen mit Kontrastmittel: ausgedehnte LK-Vergrerung zervikal, axillr, mediastinal und retroperitoneal. Multiple intrapulmonale nodulre Lsionen mit einem Durchmesser bis zu 6 mm. Ausgeprgter Pleuraerguss rechts.Therapie und Verlauf:: Unter der Annahme einer Reaktivierung der vorbekannten LK-Tbc wurde die Patientin initial mit einer erweiterten antituberkulsen Therapie behandelt. Nach klinischer Progredienz erfolgte eine erneute LK-Biopsie aus der linken Axilla, welche nunmehr den Nachweis eines Morbus Hodgkin vom Mischtyp mit teils histiozytr nekrotisierender, teils tuberkuloider Begleitreaktion ohne Nachweis surefester Stbchen erbrachte. Die daraufhin eingeleitete Chemotherapie nach dem ABVD-Schema fhrte zu einer raschen Verbesserung der klinischen Beschwerdesymptomatik.Schlussfolgerung:: Sowohl bei dem Verdacht als auch bei gesicherter Diagnose einer LK-Tbc sollte differentialdiagnostisch ein Lymphom ausgeschlossen werden. Dieser Abwgung kommt insofern eine besondere Bedeutung zu, als der Morbus Hodgkin charakteristischerweise mit einem T-Zell-Defekt assoziiert ist, welcher zu einer Tbc prdestiniert.

Microsporidia are obligate intracellular protozoan parasites that emerged as major opportunistic pathogens in humans since the onset of the AIDS pandemic. In the present study, we investigated whether FCM is a useful method for the quantitation of intracellular microsporidian spores in cultured cells.Microsporidia (Encephalitozoon cuniculi) were grown in cell cultures and various cell‐lines were coincubated with microsporidian spores at different multiplicities of infection, as well as for different periods of time. After permeabilization of the cells, intracellular spores were stained with a polyclonal anti‐E. cuniculi serum and a FITC‐labeled secondary antibody. Stained cells were analyzed on a flow cytometer and results were compared with those of fluorescence microscopy.Noninfected cells showed a lower fluorescence, while the relative fluorescence observed for infected cells was significantly higher. The cell population with the more intense fluorescence, representing cells with internalized microsporidian spores, increased with the multiplicity of infection as well as over time. Results of FCM and fluorescence microscopy were in excellent agreement for all experiments.We have developed a flow cytometric assay to detect and quantify cells with intracellular microsporidian spores. This method is easy to use, highly reproducible, and should be useful for future research. © 2004 Wiley‐Liss, Inc.

Hodgkin’s lymphoma is the most common non-AIDS-defining tumor diagnosed in HIV-infected patients. Although the introduction of highly active antiretroviral therapy (HAART) led to a decreased incidence of several malignancies among HIV-infected patients, the incidence of HIV-associated Hodgkin’s lymphoma (HIV-HL) has been persistent in recent years. Its unusually aggressive tumor behavior includes a higher frequency of unfavorable histologic subtypes, high stage and extranodal involvement by the time of presentation and poor therapeutic outcome, in comparison with Hodgkin’s lymphoma outside the HIV setting. Treatment of HIV-HL is challenging considering the underlying immunodeficiency caused by HIV itself and may increase the risk of opportunistic infections by inducing further immunosuppression. To address this delicate vulnerability of the HIV-infected host, tailored regimens, which are less aggressive than standard regimens for HIV-negative hosts, have been applied to achieve tumor control. The introduction of HAART has opened a new perspective in the treatment of HIV-associated malignancies. The improved control of HIV infection and the subsequently improved survival rates of HIV-infected patients has changed the goal from tumor control to cure and new treatment approaches with more potent regimens need to be evaluated to improve survival and quality of life in HIV-HL.

Organisms belonging to the Mycobacterium aviumcomplex (MAC) cause life‐threatening bacteremia in immunocompromised patients. Monocytes and macrophages are thought to be responsible for ingestion and killing of MAC. However, it has been suggested that neutrophils may play a role in the early immune response to MAC infection. Here, neutrophils in autologous plasma were incubated (at 0 and 37°C) withM. aviumlabeled with Auramine O, a potent fluorochrome. Neutrophil phagocytosis was measured by flow cytometry. Neutrophils incubated at 37°C showed an increase in fluorescence over time with a maximum at 15 min, whereas neutrophils on ice showed no time‐dependent increase in FL1. At 15 min Fl 1 at 37°C was twice as high as FL1 at 0°C. Examination under the fluorescent microscope showed multiple intracellular fluorescent mycobacteria. Results in nine independent experiments showed time‐dependent decrease of colony‐forming units in neutrophil‐associated live M. avium. Significant killing was observed within 30 min and was complete by 120 min. Observation by electron microscopy clearly confirmed the presence of intraphagosomal MAC, both intact and with evidence of degradation. These data demonstrate that MAC is rapidly phagocytized and killed by human neutrophils. The newly established flow cytometry method should be useful in further studies of neutrophil function and of the role of G‐CSF and other cytokines in MAC disease.

ABSTRACTThe capacity of 20 Mycobacterium aviumcomplex isolates to multiply intracellularly in human monocyte-derived macrophages was assessed and correlated to the clinical relevance of each isolate and its reactivity with several candidate genetic virulence markers. The strongest correlation with a virulence phenotype was found for a conserved coding sequence of the macrophage-induced genemigidentified by a specific migrestriction fragment length polymorphism type.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Hallek: Mundipharma: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Celgene: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Roche: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Boehringher Ingelheim: Honoraria, Other: Speakers Bureau and/or Advisory Boards; Pharmacyclics: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; AbbVie: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Gilead: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Janssen: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding.

Accumulating evidence from animal models demonstrates that antigen presentation by B cells plays a crucial role during the natural immune response as well as in several important diseases. While resting B cells are poor antigen-presenting cells they can acquire strong immunostimulatory properties after activation via receptors such as CD40. Studies in animal models suggest that ex vivo generated CD40-actived B cells could serve a promising platform of cellular cancer immunotherapy. However, our understanding of the function of antigen presentation by B cells in human remains limited. This is partly due to the lack of well-defined cell surface markers that can be used to identify distinct immunostimulatory B cell subsets. We hypothesized that antigen-presenting B cells in humans would be characterized by a similar phenotype than in vitro generated CD40-activated B cells. Using a transcriptomic approach combined with flow cytometric phenotypic screening, we were able to show that CD40-activated human B cells can be distinguished from resting B cells by high expression of costimulatory receptors and low expression of BCR-associated coreceptors such as CD21 and FcγRIIB. Here, we report that CD21lowCD86posB cells represent a distinct B lymphocyte subpopulation with potent immunostimulatory capacity. This antigen-presenting B-cell subset possesses phenotypic and functional features of strong antigen-presenting cells, i.e. they express high levels of costimulatory molecules and inflammatory adhesion molecules and low levels of ITIM-bearing inhibitory receptors. In vitro, purified human CD21lowCD86posB cells induced stronger T cell proliferation than the other three subsets, which are defined by the markers CD21 and CD86. Since Epstein Barr Virus (EBV) transforms human B cells with the help of the viral protein LMP1, which mimics CD40-mediated activation of B cells, we first studied patients with replicative EBV infection. We found that CD21lowCD86posB cells were expanded in severely immunosuppressed patients with EBV-reactivation. Analysis of CD21lowCD86posB lymphocytes in blood samples of healthy volunteers and patients with rheumatoid arthritis revealed that this subpopulation was increased in inflammatory conditions. One week following vaccination there was an increase in the frequency of this B-cell subset. Furthermore, compared to healthy controls CD21lowCD86posB cells were significantly elevated in the blood of patients with rheumatoid arthritis (RA). Interestingly, the CD21lowCD86posB-cell subset made up the majority of the B lymphocytes in synovial fluid of RA patients with inflammatory knee effusion. In summary, we demonstrate that CD21lowCD86posB cells are a novel antigen-presenting B-cell subset, which is expanded in inflammatory conditions. Our findings suggest that this subset might represent a promising therapeutic target for the treatment of inflammatory diseases, such as rheumatoid arthritis, were antigen presentation by B cells plays a pathogenetic role.

Microsporidia are obligate intracellular protozoan parasites that infect a broad range of vertebrates and invertebrates. They are increasingly recognized as human pathogens, especially in patients infected with human immunodeficiency virus (HIV). Organisms of the genus Encephalitozoon have been implicated as a major cause of disseminated microsporidian infections in persons with AIDS. Until recently, E. hellem was the only Encephalitozoon species confirmed by antigenic or nucleic acid methods to have infected humans. We describe the clinical course and morphological features of a case of disseminated microsporidian infection with Encephalitozoon cuniculi in an HIV-infected patient with chronic sinusitis, rhinitis, and keratoconjunctivitis. Parasites were found in conjunctival swab, nasal discharge, sputum, urine, stool, and duodenal biopsy specimens, but no pulmonary, renal, or gastrointestinal symptoms were documented. The patient was treated with albendazole (400mg po b.i.d.), resulting in complete remission of his ocular and nasal symptoms, and microsporidian spores disappeared from all sites. To our knowledge, this case is only the second of E. cuniculi infection in humans that has been confirmed by either antibody- or nucleic acid-based methods, and it is the first in which an Encephalitozoon species has been found in the intestinal tract of a human. Microsporidiosis is an important emerging opportunistic infection in HIV-infected patients and, as documented in this report, has an expanding clinicopathologic spectrum.