Philip Hartjen

Expansion of HIV-specific T follicular helper cells in chronic HIV infection

HIV targets CD4 T cells, which are required for the induction of high-affinity antibody responses and the formation of long-lived B cell memory. The depletion of antigen-specific CD4 T cells during HIV infection is therefore believed to impede the development of protective B cell immunity. Although several different HIV-related B cell dysfunctions have been described, the role of CD4 T follicular helper (TFH) cells in HIV infection remains unknown. Here, we assessed HIV-specific TFH responses in the lymph nodes of treatment-naive and antiretroviral-treated HIV-infected individuals. Strikingly, both the bulk TFH and HIV-specific TFH cell populations were significantly expanded in chronic HIV infection and were highly associated with viremia. In particular, GAG-specific TFH cells were detected at significantly higher levels in the lymph nodes compared with those of GP120-specific TFH cells and showed preferential secretion of the helper cytokine IL-21. In addition, TFH cell expansion was associated with an increase of germinal center B cells and plasma cells as well as IgG1 hypersecretion. Thus, our study suggests that high levels of HIV viremia drive the expansion of TFH cells, which in turn leads to perturbations of B cell differentiation, resulting in dysregulated antibody production.

Comprehensive Analysis of Frequency and Phenotype of T Regulatory Cells in HIV Infection: CD39 expression of FoxP3+ T regulatory cells correlates with progressive disease.

ABSTRACT There are conflicting data about the frequency and role of regulatory T cells (Tregs) during the course of HIV infection. Peripheral blood of a large cohort of HIV-infected patients (n = 131) at different stages of disease, including 15 long-term nonprogressors and 21 elite controllers, was analyzed to determine the frequency and phenotype of Tregs, defined as CD4+, CD25high, CD127low, FoxP3high cells. A significantly increased relative frequency of Tregs within the CD4+ compartment of HIV+ patients compared to that of healthy controls (P < 0.0001) was observed. Additionally, the relative frequency of Tregs directly correlated with HIV viral load and inversely with CD4+ counts. However, the absolute Treg number was reduced in HIV-infected patients versus healthy controls (P < 0.0001), with the exception of elite controllers (P > 0.05). The loss of absolute Treg numbers coincided with rising markers of immune activation (P < 0.0006). The initiation of antiviral therapy significantly increased absolute Treg numbers (P < 0.0031). We find that the expression of CD39, a newly defined ectonucleotidase with immunomodulatory functions on Tregs, correlated with progressive HIV disease, HIV viral load, and immune activation. Of note, when tested in peripheral blood mononuclear cells of healthy volunteers, the in vitro capacity to suppress T-cell proliferation was limited to CD4+, CD25high, CD39+ T cells. Interestingly, Tregs of elite controllers exhibited not only the highest expression of CCR5, CTLA-4, and ICOS but also the lowest level of CD39. The data presented here reconcile the seemingly contradictory results of previous studies looking at Tregs in HIV and highlight the complexity of Treg-mediated immunoregulation during human viral infections.

Highly Significant Antiviral Activity of HIV-1 LTR-Specific Tre-Recombinase in Humanized Mice

Stable integration of HIV proviral DNA into host cell chromosomes, a hallmark and essential feature of the retroviral life cycle, establishes the infection permanently. Current antiretroviral combination drug therapy cannot cure HIV infection. However, expressing an engineered HIV-1 long terminal repeat (LTR) site-specific recombinase (Tre), shown to excise integrated proviral DNA in vitro, may provide a novel and highly promising antiviral strategy. We report here the conditional expression of Tre-recombinase from an advanced lentiviral self-inactivation (SIN) vector in HIV-infected cells. We demonstrate faithful transgene expression, resulting in accurate provirus excision in the absence of cytopathic effects. Moreover, pronounced Tre-mediated antiviral effects are demonstrated in vivo, particularly in humanized Rag2−/−γc−/− mice engrafted with either Tre-transduced primary CD4+ T cells, or Tre-transduced CD34+ hematopoietic stem and progenitor cells (HSC). Taken together, our data support the use of Tre-recombinase in novel therapy strategies aiming to provide a cure for HIV.

CD161+ MAIT cells are severely reduced in peripheral blood and lymph nodes of HIV-infected individuals independently of disease progression

Mucosal-associated invariant T (MAIT) cells are characterized by the combined expression of the semi-invariant T cell receptor (TCR) Vα7.2, the lectin receptor CD161, as well as IL-18R, and play an important role in antibacterial host defense of the gut. The current study characterized CD161+ MAIT and CD161–TCRVα7.2+ T cell subsets within a large cohort of HIV patients with emphasis on patients with slow disease progression and elite controllers. Mononuclear cells from blood and lymph node samples as well as plasma from 63 patients and 26 healthy donors were analyzed by multicolor flow cytometry and ELISA for IL-18, sCD14 and sCD163. Additionally, MAIT cells were analyzed after in vitro stimulation with different cytokines and/or fixed E.coli. Reduced numbers of CD161+ MAIT cells during HIV infection were detectable in the blood and lymph nodes of all patient groups, including elite controllers. CD161+ MAIT cell numbers did not recover even after successful antiretroviral treatment. The loss of CD161+ MAIT cells was correlated with higher levels of MAIT cell activation; an increased frequency of the CD161–TCRVα7.2+T cell subset in HIV infection was observed. In vitro stimulation of MAIT cells with IL-18 and IL-12, IL-7 and fixed E.coli also resulted in a rapid and additive reduction of the MAIT cell frequency defined by CD161, IL-18R and CCR6. In summary, the irreversible reduction of the CD161+ MAIT cell subset seems to be an early event in HIV infection that is independent of later stages of the disease. This loss appears to be at least partially due to the distinctive vulnerability of MAIT cells to the pronounced stimulation by microbial products and cytokines during HIV-infection.

Assessment of the range of the HIV-1 infectivity enhancing effect of individual human semen specimen and the range of inhibition by EGCG

Recently, it has been shown that human ejaculate enhances human immunodeficiency virus 1 (HIV-1) infectivity. Enhancement of infectivity is conceived to be mediated by amyloid filaments from peptides that are proteolytically released from prostatic acid phosphatase (PAP), termed Semen-derived Enhancer of Virus Infection (SEVI). The aim of this study was to test the range of HIV-1 infectivity enhancing properties of a large number of individual semen samples (n = 47) in a TZM-bl reporter cell HIV infection system. We find that semen overall increased infectivity to 156% of the control experiment without semen, albeit with great inter- and intraindividual variability (range -53%-363%). Using transmission electron microscopy, we provide evidence for SEVI fibrils in fresh human semen for the first time. Moreover, we confirm that the infectivity enhancing property can be inhibited by the major green tea ingredient epigallocatechin-3-gallate (EGCG) at non-toxic concentrations. The median inhibition of infection by treatment with 0.4 mM EGCG was 70.6% (p < 0.0001) in our cohort. Yet, there were substantial variations of inhibition and in a minority of samples, infectivity enhancement was not inhibited by EGCG treatment at all. Thus, topical application of EGCG may be a feasible additional measure to prevent the sexual transmission of HIV. However, the reasons for the variability in the efficacy of the abrogation of semen-mediated enhancement of HIV-1 infectivity and EGCG efficacy have to be elucidated before therapeutic trials can be conducted.

Optimized in vitro procedure for assessing the cytocompatibility of magnesium-based biomaterials.

Magnesium (Mg) is a promising biomaterial for degradable implant applications that has been extensively studied in vitro and in vivo in recent years. In this study, we developed a procedure that allows an optimized and uniform in vitro assessment of the cytocompatibility of Mg-based materials while respecting the standard protocol DIN EN ISO 10993-5:2009. The mouse fibroblast line L-929 was chosen as the preferred assay cell line and MEM supplemented with 10% FCS, penicillin/streptomycin and 4mM l-glutamine as the favored assay medium. The procedure consists of (1) an indirect assessment of effects of soluble Mg corrosion products in material extracts and (2) a direct assessment of the surface compatibility in terms of cell attachment and cytotoxicity originating from active corrosion processes. The indirect assessment allows the quantification of cell-proliferation (BrdU-assay), viability (XTT-assay) as well as cytotoxicity (LDH-assay) of the mouse fibroblasts incubated with material extracts. Direct assessment visualizes cells attached to the test materials by means of live-dead staining. The colorimetric assays and the visual evaluation complement each other and the combination of both provides an optimized and simple procedure for assessing the cytocompatibility of Mg-based biomaterials in vitro.

Reduced CD161+ MAIT cell frequencies in HCV and HIV/HCV co-infection: Is the liver the heart of the matter?

To the Editor: Previously, it has been hypothesized that the ubiquitous loss of mucosal-associated invariant T (MAIT) cells in HIV-1 monoinfection leads to a leakage of the intestinal barrier and increased microbial translocation [1]. Furthermore, increased susceptibility of HIV patients to tuberculosis (TB) co-infection is possibly also a result of the decrease of MAIT cell frequencies in the lung [2]. In analogy, Tang et al. characterized the relative frequency and number of different lymphocyte populations in the liver. Indeed, up to 15% of intrahepatic lymphocytes were MAIT cells [3]. These high intrahepatic MAIT cell frequencies were also confirmed in two subsequent studies [4,5]. Barathan et al. recently comprehensively studied the phenotypic characteristics of peripheral cluster of differentiation (CD)161 TCR-Va7.2 MAIT cells in a cross-sectional cohort of chronic hepatitis C virus (HCV) infected patients and healthy controls, altogether demonstrating that the frequency of CD161 MAIT cells was significantly decreased and the remaining MAIT cells showed increased signs of immune exhaustion and chronic immune activation [6]. The results of the recent studies indicate that loss and functional impairment of MAIT cells play a role in the pathogenesis of HCV and lead at the same time to a new set of questions. First of all, it is not clear whether peripheral MAIT cell frequencies recover after successful HCV therapy. Another unsolved question is whether MAIT cells simply migrate from the periphery to the liver and are detectable at increased frequencies at this inflammatory site in HCV infection. Furthermore, increased microbial translocation has been discussed as one of the reasons for a faster disease progression in HIV/HCV infection in comparison to HCV mono-infection [7,8] and it is interesting to speculate whether an additional loss of MAIT cells due to the concomitant HIV infection could be at least partially causative for the unfavorable course of liver disease in HIV/HCV co-infected patients.

From bench to application: Current practices in tissue engineering and its realisation at maxillofacial units in Germany, Austria and Switzerland

Over the last 20 years, the highly interdisciplinary field of tissue engineering (TE) has become an established subspecialty in research facilities all over the world. Numerous methods and protocols are available for various research intentions and aims, but there are no data indicating which of these methods and resources are generally used. This study is an overview of the resources and methods that are commonly applied in TE research in general, and in the field of oral and maxillofacial surgery (OMFS) in Germany, Austria and Switzerland. The DÖSAK collaborative group for TE developed a detailed questionnaire and collected information from participating university hospitals in these three countries. We evaluated the availability of research facilities, in vitro realisation and in vivo designs for animal studies in these departments. 11 units who replied, out of 35 we contacted, conducted research on bone regeneration in interdisciplinary research facilities. 10 departments used xenogeneic and alloplastic scaffolds for in vitro and in vivo applications. In this case, the most commonly utilised trademarks were Bio-Oss(®) and CERASORB(®). 9 units used osteoblasts (73%) and 10 proliferation assays in vitro, whereas rats served as the standard animal model for histology/immunohistochemistry in 6. All research units were interested in establishing a platform for research exchange and communication. This study shows that tissue engineering is well established and highly accepted in most participating university hospitals and research facilities. The presented data, together with data published in a foregoing paper will help arrange more readily available standardised procedures for further investigations.

THE NTPASE/HELICASE DOMAIN OF HEPATITIS C VIRUS NONSTRUCTURAL PROTEIN 3 INHIBITS PROTEIN KINASE C INDEPENDENTLY OF ITS NTPASE ACTIVITY

Helicase motif VI is a short arginine-rich motif within the NTPase/helicase domain of the non-structural protein 3 (NS3) of the hepatitis C virus (HCV). We previously demonstrated that it reduces the catalytic activity and intracellular shuttling of protein kinase C (PKC). Thus, NS3-mediated PKC inhibition may be involved in HCV-associated hepatocellular carcinoma (HCC). In this study, we expand on our earlier results, which were obtained in experiments with short fragments of NS3, to show for the first time that the catalytically active, longer C-terminal NTPase/helicase of NS3 acts as a potent PKC inhibitor in vitro. PKC inhibition assays with the NTPase-inactive mutant NS3h-D1316A revealed a mixed type kinetic inhibition pattern. A broad range of 11 PKC isotypes was tested and all of the PKC isotypes were inhibited with IC50-values in the low micromolar range. These findings were confirmed for the wild-type NTPase/helicase domain in a non-radiometric PKC inhibition assay with ATP regeneration to rule out any effect of ATP hydrolysis caused by its NTPase activity. PKCα was inhibited with a micromolar IC50 in this assay, which compares well with our result for NS3h-D1316A (IC50 = 0.7 μM). In summary, these results confirm that catalytically active NS3 NTPase/helicase can act in an analogous manner to shorter NS3 fragments as a pseudosubstrate inhibitor of PKC.

Biocompatibility and Osseointegration of Titanium Implants with a Silver-Doped Polysiloxane Coating: An In Vivo Pig Model

PURPOSE To test the antimicrobial properties, surface topography, reaction of surrounding tissue (biocompatibility), and osseointegration of ultrathin implant surfaces containing polysiloxane and nanoscaled silver particles. MATERIALS AND METHODS Implants with polysiloxane coating and nanoscaled silver particles (Ag/SiOxCy; HyProtect, Bio-Gate) were compared with implants with polysiloxane coating alone and with noncoated (grit-blasted and acid-etched) implants. A total of 72 implants were inserted into the calvaria of eight domestic pigs (nine implants each, three of each type). After 3 months, histologic sections were evaluated for inflammatory cell infiltration and bone implant contact. RESULTS Roughness parameters did not differ between all three implant types. The Ag/SiOxCy coating exhibited a good antimicrobial effect in vitro and no sign of inflammatory cell infiltration in vivo. The noncoated implants demonstrated 10.85% and 14.48% more bone contact than the polysiloxane-coated implants (P = .003) and the Ag/SiOxCy‑coated implants (P ≤ .001), respectively. Osseointegration was not significantly different between the Ag/SiOxCy‑coated and polysiloxane-coated implants (P = .72). CONCLUSION The osseointegration capability of the Ag/SiOxCy-coated implants was equal to that of the polysiloxane-coated implants but less than that of the grit-blasted and acid-etched implants. Because of the biocompatibility of the polysiloxane coating, further studies should be conducted in load-bearing models and in the oral cavity to investigate the antimicrobial effect of the embedded silver clusters.