Modest Attenuation of HIV-1 Vpu Alleles Derived from Elite Controller Plasma

In the absence of antiretroviral therapy, infection with human immunodeficiency virus type 1 (HIV-1) can typically not be controlled by the infected host and results in the development of acquired immunodeficiency. In rare cases, however, patients spontaneously control HIV-1 replication. Mechanisms by which such elite controllers (ECs) achieve control of HIV-1 replication include particularly efficient immune responses as well as reduced fitness of the specific virus strains. To address whether polymorphisms in the accessory HIV-1 protein Vpu are associated with EC status we functionally analyzed a panel of plasma-derived vpu alleles from 15 EC and 16 chronic progressor (CP) patients. Antagonism of the HIV particle release restriction by the intrinsic immunity factor CD317/tetherin was well conserved among EC and CP Vpu alleles, underscoring the selective advantage of this Vpu function in HIV-1 infected individuals. In contrast, interference with CD317/tetherin induced NF-κB activation was little conserved in both groups. EC Vpus more frequently displayed reduced ability to downregulate cell surface levels of CD4 and MHC class I (MHC-I) molecules as well as of the NK cell ligand NTB-A. Polymorphisms potentially associated with high affinity interactions of the inhibitory killer immunoglobulin-like receptor (KIR) KIR2DL2 were significantly enriched among EC Vpus but did not account for these functional differences. Together these results suggest that in a subgroup of EC patients, some Vpu functions are modestly reduced, possibly as a result of host selection

Autoantibodies to intracellular “rods and rings” structures in two patients with autoimmune hepatitis treated with azathioprine

In vitro, inhibition of the synthesis of guanosine monophosphate (GMP) by various drugs such as ribavirin, acyclovir, azathioprine, and mycophenolic acid leads to the formation of subcellular structures in cultured cells. Autoantibodies targeting these cellular structures can be detected as “rods and rings” (RR) patterns by immunofluorescence. In vivo, autoantibodies to RR have been almost exclusively associated with hepatitis C virus patients treated with pegylated interferon-α and ribavirin. However, longitudinal data for other patient groups are scarce. Here, we reviewed 276 sequential immunofluorescence results from 127 patients with autoimmune hepatitis for the presence of RR patterns. Of 102 patients exposed to drugs known to induce RR in vitro, two patients under long-term azathioprine therapy were positive for this pattern. This is the first report of anti-RR in patients with autoimmune hepatitis and in patients treated with azathioprine

Update of the simplified criteria for autoimmune hepatitis: Evaluation of the methodology for immunoserological testing

Background & aims: The simplified criteria for the diagnosis of autoimmune hepatitis (AIH) include immunofluorescence testing (IFT) of antinuclear and smooth muscle autoantibodies (ANA and SMA) on rodent tissue sections. We aimed to establish scoring criteria for the implementation of ANA IFT on human epithelioma-2 (HEp-2) cells and ELISA-based testing. Methods: ANA and SMA reactivity of 61 AIH sera and 72 non-alcoholic fatty liver disease controls were separately assessed on tissue sections and HEp-2 cells to compare the diagnostic value at increasing titers. A total of 113 patients with AIH at diagnosis and 202 controls from 3 European centers were assessed by IFT as well as 3 different commercially available ANA ELISA and 1 anti-F-actin ELISA. Results: ANA assessment by IFT on liver sections had 83.6% sensitivity and 69.4% specificity for AIH at a titer of 1:40. On HEp-2 cells, sensitivity and specificity were 75.4% and 73.6%, respectively, at an adjusted titer of 1:160. Area under the curve (AUC) values of ANA ELISA ranged from 0.70-0.87, with ELISA coated with HEp-2 extracts in addition to selected antigens performing significantly better. SMA assessment by IFT had the highest specificity for the SMA-VG/T pattern and anti-microfilament reactivity on HEp-2 cells. ELISA-based anti-F-actin evaluation was a strong predictor of AIH (AUC 0.88) and performed better than SMA assessment by IFT (AUC 0.77-0.87). Conclusion: At adjusted cut-offs, both ANA IFT using HEp-2 cells and ELISA-based autoantibody evaluation for ANA and SMA are potential alternatives to tissue-based IFT for the diagnosis of AIH

Hepatocellular carcinoma: Intratumoral EpCAM-positive cancer stem cell heterogeneity identifies high-risk tumor subtype

Background!#!The translational interest in the intratumoral heterogeneity of hepatocellular carcinoma (HCC) has been increasing. The dismal prognosis of this pathology is linked to the features of the HCC harbouring cancer stem cells (CSC), represented by EpCAM-expression. However, the extent of the impact of intratumoral distribution of CSC-features, both on the recurrence after curative resection and on clinical outcome, remains unknown. To address this, we investigated the spatial heterogeneity of CSC-features with the aim of identifying the unique HCC patient subgroups amenable to adjuvant treatment.!##!Methods!#!We designed a tissue microarray (TMA) from patients who had received liver resection between 2011 and 2017. Tumor specimens were sampled at multiple locations (n = 3-8). EpCAM-positivity was assessed for intensity and proportion by applying a score dividing three groups: (i) negative (E-/-); (ii) heterogeneous (E-/+); and (iii) homogeneous (E+/+). The groups were further analysed with regard to time-to-recurrence (TTR) and recurrence-free-survival (RFS).!##!Results!#!We included 314 tumor spots from 69 patients (76.8% male, median age 66, liver cirrhosis/fibrosis 75.8%). The risk factors were alcohol abuse (26.2%), NASH (13.1%), HBV (15.5%), HCV (17.9%) and others (27.4%), representative of a typical Western cohort. E+/+ patients experienced significantly shorter TTR and RFS compared to E+/- and E-/- patients (TTR 5 vs. 19 months, p = 0.022; RFS 5 vs. 14 vs. 21 months, p = 0.016). Only homogeneous EpCAM-positivity correlated with higher AFP levels (> 400 ng/ml, p = 0.031).!##!Conclusions!#!Spatial heterogeneity of EpCAM-expression was markedly present in the cohort. Of note, only homogeneous EpCAM-expression correlated significantly with early recurrence, whereas heterogeneous EpCAM-expression was associated with clinical endpoints comparable to EpCAM-negativity. We identified a unique HCC subtype associated with a high risk of tumor recurrence

Maximum-likelihood phylogenetic tree, sequences alignment, expression and detection of plasma HIV RNA-derived Vpu clonal alleles.

<p><b>A</b>) Maximum-likelihood phylogenetic tree of HIV-1 Vpu alleles. EC-derived Vpus in red, CP-derived Vpus in blue, and control strain NL4.3 in black. <b>B</b>) Amino acid sequence alignment of the Vpu proteins analyzed generated using Clustal Omega (EMBL-EBI). HIV-1 NL4.3 Vpu on the top serves as reference sequence. Boxes indicate the position of functionally relevant residues and motifs. Different colors present the properties of the amino acid: red for small and aromatic, blue for acidic, green for residues with hydroxyl or sulfhydryl groups and magenta for positively charged residues. <i>C</i>: Expression and detection of Vpu alleles. Lysates of A3.01 cells transfected with Vpu.GFP expression plasmids were analyzed by Western blotting using antibodies against Vpu, GFP, and transferrin receptor (Tfr).</p

MHC-I and NTB-A downregulation by patient-derived Vpus.

<p>Surface MHC-I and NTB-A levels were analyzed by flow cytometry on A3.01 cells 48 h post transfection with the indicated Vpu.GFP expression constructs. <b>A</b>) Representative flow cytometry dot plots of gated living cells for MHC-I-APC (y-axis) vs. GFP (x-axis). The MFI (Y Geo Mean) of untransfected (left gate) and medium to high GFP-expressing (right gate) cells is indicated. MHC-I cell surface levels of transfected cells were normalized to untransfected cells in the same sample by calculating the ratio of the MFIs of right and left gates and expressed relative to that of NL4.3 Vpu that was arbitrarily set to 100%. <b>B</b>) MHC-I cell surface levels of A3.01 cells expressing patient derived Vpu proteins relative to NL4.3 Vpu. Shown are mean values of triplicate transfections with the indicated standard deviation. The result is representative of three independent experiments. <b>C</b>) Comparison of MHC-I cell surface levels in A3.01 cells expressing EC or CP Vpu alleles. Statistical significance was assessed using two-tailed Mann-Whitney U-Test (p = 0.02), bars represent median and interquartile ranges. <b>D</b>) Representative flow cytometry dot plots of gated living cells for NTB-A-APC (y-axis) vs. GFP (x-axis). The MFI (Y Geo Mean) of untransfected (left gate) and medium to high GFP-expressing (right gate) cells is indicated. NTB-A cell surface levels of transfected cells were normalized to untransfected cells in the same sample by calculating the ratio of the MFIs of right and left gates and expressed relative to that of NL4.3 Vpu that was arbitrarily set to 100%. <b>E</b>) NTB-A cell surface levels of A3.01 cells expressing patient derived Vpu proteins relative to NL4.3 Vpu. Shown are mean values of triplicate transfections with the indicated standard deviation. The result is representative of three independent experiments. <b>F</b>) Comparison of NTB-A cell surface levels in A301 cells expressing EC or CP Vpu alleles. Statistical significance was assessed using two-tailed Mann-Whitney U-Test (p = 0.01), bars represent median and interquartile ranges.</p

CD4 and CD317/tetherin downregulation by patient-derived Vpus.

<p>Surface CD4 and CD317/tetherin levels were analyzed by flow cytometry on A3.01cells 48 h post transfection with the indicated Vpu.GFP expression constructs. <b>A</b>) Representative flow cytometry dot plots of gated living cells for CD4-APC (y-axis) vs. GFP (x-axis). The MFI (Y Geo Mean) of untransfected (left gate) and medium to high GFP-expressing (right gate) cells is indicated. CD4 cell surface levels of transfected cells were normalized to untransfected cells in the same sample by calculating the ratio of the MFIs of left and right gates and expressed relative to that of NL4.3 Vpu that was arbitrarily set to 100%. <b>B</b>) CD4 cell surface levels of A3.01cells expressing patient-derived Vpu proteins relative to NL4.3 Vpu. Shown are mean values of triplicate transfections with the indicated standard deviation. The result is representative of three independent experiments. <b>C</b>) Comparison of CD4 cell surface levels in A3.01 cells expressing EC or CP Vpu alleles. Statistical significance was assessed using two-tailed Mann-Whitney U-Test (p = 0.02), bars represent median and interquartile ranges. <b>D</b>) Representative flow cytometry dot plots of gated living cells for CD317/tetherin-APC (y-axis) vs. GFP (x-axis). The MFI (Y Geo Mean) of untransfected (left gate) and medium to high GFP-expressing (right gate) cells is indicated. CD317/tetherin cell surface levels of transfected cells were normalized to untransfected cells in the same sample by calculating the ratio of the MFIs of left and right gates and expressed relative to that of NL4.3 Vpu that was arbitrarily set to 100%. <b>E</b>) CD317/tetherin cell surface levels of TZM-bl cells expressing patient derived Vpu proteins relative to NL4.3 Vpu. Shown are mean values of triplicate transfections with the indicated standard deviation. The result is representative of three independent experiments. <b>F</b>) Comparison of CD317 cell surface levels in A3.01 cells expressing EC or CP Vpu alleles. Statistical significance was assessed using two-tailed Mann-Whitney U-Test (p = 0.64), bars represent median and interquartile ranges.</p

Recovery of HIV-1 particle release by patient-derived Vpus.

<p>TZM-bl cells were transiently transfected with HIV-1NL4.3 Δvpu provirus plasmids and the indicated Vpu allele or controls. 48 h post transfection, the resulting viral supernatants were assayed for infectivity on TZM-bl indicator cells to determine the amount of infectious virions produced. TZM-bl cell lysates from one replicate of the assay were subjected to Western blot detection, and TZM-bl cells from the same replicate were harvested and assayed for cell-surface level of CD317/tetherin. <b>A</b>) The yield of infectious HIV-1 in the supernatant and cell-associated levels of GFP, p24CA, and p55Gag were analyzed. HIV-1 particle release in the supernatant was assessed by measuring the induction of Luciferase units (top) in infected TZM-bl cells. Values (y axis) are normalized to that of control NL4.3Δ<i>vpu</i>, which was set to 1, error bars represent the standard error of the mean (SEM) for three independent experiments. Western blot results show the expression level of GFP, p24 and p55 in one representative experiment. <b>B</b>) Comparison of enhancement of virion release mediated by EC and CP Vpu alleles. Statistical significance was assessed using two-tailed Mann-Whitney U-Test (p = 0.07), bars represent median and interquartile ranges. <b>C</b>) CD317/tetherin cell surface downregulation by patient Vpu. TZM-bl cells transfected with HIV-1 NL4.3 Δvpu and the indicated VpuIRESGFP plasmids were harvested 48 h post transfection and CD317/tetherin cell surface levels quantified by flow cytometry. The y-axis represents the relative CD317/tetherin cell surface levels remaining normalized to control cells transfected with NL4.3Δ<i>vpu</i> and IRESGFP (set to 100%). <b>D</b>) Comparison of CD317/tetherin cell surface levels in TZM-bl cells producing viral particles and expressing EC or CP Vpu alleles. Statistical significance was assessed using two-tailed Mann-Whitney U-Test (p = 0.29), bars represent median and interquartile ranges.</p

Antagonism of CD317/tetherin-induced NF-kB signaling.

<p><b>A</b>) CD317/tetherin-induced NF-κB activation by Vpu alleles. Luciferase activity of the NF-κB reporter was determined 40 h post transfection of 293T cells with expression plasmids for CD317/tetherin or filler, the indicated VpuIRESGFP expression plasmids and luciferase reporter plasmids. HIV-1 NL4.3 Vpu and HIV-1 M WITO Vpu served as controls. Mean values of 6–9 transfections are shown with the indicated standard deviation. <b>B</b>) Effect of EC and CP Vpu alleles on CD317/tetherin-induced NF-κB activation. Statistical significance was assessed using two-tailed Mann-Whitney U-Test (p = 0.29), bars represent median and interquartile ranges. <b>C</b>) Correlation of Vpu-mediated inhibition of CD317/tetherin-induced NF-κB activity and release of infectious virions. Statistical analyses were done using Spearman’s correlation.</p