Ansgar W. Lohse

Induction of cytokine production in naive CD4+ T cells by antigen-presenting murine liver sinusoidal endothelial cells but failure to induce differentiation toward Th1 cells

BACKGROUND & AIMS Murine liver sinusoidal endothelial cells (LSECs) constitutively express accessory molecules and can present antigen to memory Th1 CD4(+) T cells. Using a T-cell receptor transgenic mouse line, we addressed the question whether LSECs can prime naive CD4(+) T cells. METHODS Purified LSECs were investigated for their ability to induce activation and differentiation of naive CD4(+) T cells in comparison with bone marrow-derived antigen-presenting cells and macrovascular endothelial cells. Activation of T cells was determined by cytokine production. LSECs were further studied for expression of interleukin (IL)-12 by reverse-transcription polymerase chain reaction, and the unique phenotype of LSECs was determined by flow cytometry. RESULTS We provide evidence that antigen-presenting LSECs can activate naive CD62Lhigh CD4(+) T cells. Activation of naive CD4(+) T cells by LSECs occurred in the absence of IL-12. In contrast, macrovascular endothelial cells from aorta could not activate naive CD4(+) T cells. The unique functional characteristics of microvascular LSECs together with a unique phenotype (CD4(+), CD11b+, CD11c+, CD80(+), CD86(+)) make these cells different from macrovascular endothelial cells. Furthermore, LSECs did not require in vitro maturation to activate naive CD4(+) T cells. Most importantly, LSECs failed to induce differentiation toward Th1 cells, whereas conventional antigen-presenting cell populations induced a Th1 phenotype in activated CD4(+) T cells. Upon restimulation, CD4(+) T cells, which were primed by antigen-presenting LSECs, expressed interferon gamma, IL-4, and IL-10, which is consistent with a Th0 phenotype. Exogenous cytokines (IL-1beta, IL-12, or IL-18) present during T-cell priming by antigen-presenting LSECs could not induce a Th1 phenotype, but neutralization of endogenously produced IL-4 during T-cell priming led to a reduced expression of IL-4 and IL-10 by CD4(+) T cells upon restimulation. The addition of spleen cells to cocultures of LSECs and naive CD4(+) T cells during T-cell priming led to differentiation of T cells toward a Th1 phenotype. CONCLUSIONS The ability of antigen-presenting LSECs to induce cytokine expression in naive CD4(+) T cells and their failure to induce differentiation toward a Th1 phenotype may contribute to the unique hepatic microenvironment that is known to promote tolerance.

A Case of Severe Ebola Virus Infection Complicated by Gram-Negative Septicemia

Ebola virus disease (EVD) developed in a patient who contracted the disease in Sierra Leone and was airlifted to an isolation facility in Hamburg, Germany, for treatment. During the course of the illness, he had numerous complications, including septicemia, respiratory failure, and encephalopathy. Intensive supportive treatment consisting of high-volume fluid resuscitation (approximately 10 liters per day in the first 72 hours), broad-spectrum antibiotic therapy, and ventilatory support resulted in full recovery without the use of experimental therapies. Discharge was delayed owing to the detection of viral RNA in urine (day 30) and sweat (at the last assessment on day 40) by means of polymerase-chain-reaction (PCR) assay, but the last positive culture was identified in plasma on day 14 and in urine on day 26. This case shows the challenges in the management of EVD and suggests that even severe EVD can be treated effectively with routine intensive care.

Faldaprevir and Deleobuvir for HCV Genotype 1 Infection

BACKGROUND Interferon-free regimens would be a major advance in the treatment of patients with chronic hepatitis C virus (HCV) infection. METHODS In this phase 2b, randomized, open-label trial of faldaprevir (a protease inhibitor) and deleobuvir (a nonnucleoside polymerase inhibitor), we randomly assigned 362 previously untreated patients with HCV genotype 1 infection to one of five groups: faldaprevir at a dose of 120 mg once daily and deleobuvir at a dose of 600 mg three times daily, plus ribavirin, for 16, 28, or 40 weeks (TID16W, TID28W, or TID40W, respectively); faldaprevir at a dose of 120 mg once daily and deleobuvir at a dose of 600 mg twice daily, plus ribavirin, for 28 weeks (BID28W); or faldaprevir at a dose of 120 mg once daily and deleobuvir at a dose of 600 mg three times daily, without ribavirin, for 28 weeks (TID28W-NR). The primary end point was a sustained virologic response 12 weeks after the completion of therapy. RESULTS The primary end point was met in 59% of patients in the TID16W group, 59% of patients in the TID28W group, 52% of patients in the TID40W group, 69% of patients in the BID28W group, and 39% of patients in the TID28W-NR group. The sustained virologic response 12 weeks after the completion of therapy did not differ significantly according to treatment duration or dosage among ribavirin-containing regimens. This response was significantly higher with TID28W than with TID28W-NR (P=0.03). Rates of a sustained virologic response 12 weeks after the completion of therapy were 56 to 85% among patients with genotype 1b infection versus 11 to 47% among patients with genotype 1a infection and 58 to 84% among patients with IL28B CC versus 33 to 64% with non-CC genotypes. Rash, photosensitivity, nausea, vomiting, and diarrhea were the most common adverse events. CONCLUSIONS The rate of a sustained virologic response 12 weeks after the completion of therapy was 52 to 69% among patients who received interferon-free treatment with faldaprevir in combination with deleobuvir plus ribavirin. (Funded by Boehringer Ingelheim; SOUND-C2 ClinicalTrials.gov number, NCT01132313.).

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.

Ectopic expression of neural autoantigen in mouse liver suppresses experimental autoimmune neuroinflammation by inducing antigen-specific Tregs

Tregs are important mediators of immune tolerance to self antigens, and it has been suggested that Treg inactivation may cause autoimmune disease. Therefore, immunotherapy approaches that aim to restore or expand autoantigen-specific Treg activity might be beneficial for the treatment of autoimmune disease. Here we report that Treg-mediated suppression of autoimmune disease can be achieved in vivo by taking advantage of the ability of the liver to promote immune tolerance. Expression of the neural autoantigen myelin basic protein (MBP) in the liver was accomplished stably in liver-specific MBP transgenic mice and transiently using gene transfer to liver cells in vivo. Such ectopic MBP expression induced protection from autoimmune neuroinflammation in a mouse model of multiple sclerosis. Protection from autoimmunity was mediated by MBP-specific CD4+CD25+Foxp3+ Tregs, as demonstrated by the ability of these cells to prevent disease when adoptively transferred into nontransgenic mice and to suppress conventional CD4+CD25- T cell proliferation after antigen-specific stimulation with MBP in vitro. The generation of MBP-specific CD4+CD25+Foxp3+ Tregs in vivo depended on expression of MBP in the liver, but not in skin, and occurred by TGF-beta-dependent peripheral conversion from conventional non-Tregs. Our findings indicate that autoantigen expression in the liver may generate autoantigen-specific Tregs. Thus, targeting of autoantigens to hepatocytes may be a novel approach to prevention or treatment of autoimmune diseases.

Mycophenolate mofetil as second line therapy in autoimmune hepatitis

INTRODUCTION:In patients with autoimmune hepatitis, efficient immunosuppressive therapy is essential to avoid progression to cirrhosis. There is no established second line therapy for patients failing standard therapy with steroids and azathioprine. The aim of this study was to examine the possible role of mycophenolate mofetil (MMF) as second line treatment of autoimmune hepatitis (AIH).PATIENTS AND ETHODS:We were able to identify 37 patients (29 women, 8 men) with AIH proven according to International AIH Group criteria who failed standard therapy. One patient on MMF was excluded due to non-compliance. A total of 28 of 36 patients had experienced side effects necessitating stop of treatment. One patient stopped azathioprine due to pregnancy. A total of nine patients did not respond sufficiently to azathioprine. A total of four patients with a treatment duration of 3 months or less because of severe side effects were considered as intolerant to MMF. Remission was defined as aspartate transaminase (ASP) < twice upper normal limit (UNL).RESULTS:Of 36 patients on MMF included in the analysis, 14 patients (39%) experienced remission. A total of 22 patients (61%) did not respond sufficiently to MMF. The response rate to MMF was dependent on the cause of treatment cessation of azathioprine. Of eight patients with prior nonresponse to azathioprine, six (75%) did not respond to MMF and only two (25%) reached biochemical remission. Of 28 patients with azathioprine intolerance in 16 (57%) patients, the response to MMF was insufficient and in 12 patients (43%) remission was reached. The difference did not reach statistical significance due to the relatively small numbers included.CONCLUSION:In the light of its good tolerability, MMF seems to be an alternative for patients who could not tolerate azathioprine previously. However, our data suggest that a majority of patients fail MMF particularly if they are switched because of an insufficient response to azathioprine.

Humanized chimeric uPA mouse model for the study of hepatitis B and D virus interactions and preclinical drug evaluation

No specific drugs are currently available against hepatitis delta virus (HDV), a defective virus leading to the most severe form of chronic viral hepatitis in man. The lack of convenient HDV infection models has hampered the development of effective therapeutics. In this study, naïve and hepatitis B virus (HBV) chronically infected humanized uPA/SCID mice were employed to establish a small animal model of HBV/HDV coinfection and superinfection. For preclinical antiviral drug evaluation, the GMP version of the myristoylated preS‐peptide (Myrcludex‐B), a lipopeptide derived from the pre‐S1 domain of the HBV envelope, was applied to prevent de novo HBV/HDV coinfection in vivo. Virological parameters were determined at serological and intrahepatic level both by real‐time polymerase chain reaction (PCR) and by immunohistochemistry. Establishment of HDV infection was highly efficient in both HBV‐infected and naïve chimeric mice with HDV titers rising up to 1 × 10E9 copies/mL. Notably, HDV superinfection led to a median 0.6log reduction of HBV viremia, which although not statistically significant suggests that HDV may hinder HBV replication. In the setting of HBV/HDV simultaneous infection, a majority of human hepatocytes stained HDAg‐positive long before HBV spreading was completed, confirming that HDV can replicate intrahepatically also in the absence of HBV infection. Furthermore, the increase of HBV viremia and intrahepatic cccDNA loads was significantly slower than in HBV mono‐infected mice. Treatment with the HBV entry inhibitor Myrcludex‐B, efficiently hindered the establishment of HDV infection in vivo. Conclusion: We established an efficient model of HBV/HDV infection to exploit mechanisms of viral interference in human hepatocytes and to test the efficacy of an HDV‐entry inhibitor in vivo. (HEPATOLOGY 2011)

Broadly directed virus-specific CD4+ T cell responses are primed during acute hepatitis C infection, but rapidly disappear from human blood with viral persistence

Early after symptom onset, HCV-specific CD4+ T cell responses are primed and detectable in patients regardless of clinical outcome, but without early antiviral therapy these T cells become exhausted or deleted in chronically infected patients.

Efficacy of the Protease Inhibitor BI 201335, Polymerase Inhibitor BI 207127, and Ribavirin in Patients With Chronic HCV Infection

BACKGROUND & AIMS Therapeutic regimens are being developed for patients with hepatitis C virus (HCV) infection that do not include the combination of peginterferon alfa and ribavirin. We investigated the antiviral effect and safety of BI 201335 (an inhibitor of the NS3/4A protease) and BI 207127 (an inhibitor of the NS5B non-nucleoside polymerase) with ribavirin. METHODS Thirty-two treatment-naïve patients with chronic HCV genotype 1 infection were randomly assigned to groups that were given 400 mg or 600 mg BI 207127 3 times daily plus 120 mg BI 201335 once daily and 1000 to 1200 mg/day ribavirin for 4 weeks. The primary efficacy end point was virologic response (HCV RNA level <25 IU/mL at week 4). Thirty-two patients received treatment; 31 completed all 4 weeks of assigned combination therapy. RESULTS In the group given BI 207127 400 mg 3 times daily, the rates of virologic response were 47%, 67%, and 73% at days 15, 22, and 29; a higher rate of response was observed in patients with genotype-1b compared with genotype-1a infections. In the group given BI 207127 600 mg 3 times daily, the rates of virologic response were 82%, 100%, and 100%, respectively, and did not differ among genotypes. One patient in the group given 400 mg 3 times daily had virologic breakthrough (≥1 log(10) rebound in HCV RNA) at day 22. The most frequent adverse events were mild gastrointestinal disorders, rash, and photosensitivity. There were no severe or serious adverse events; no patients discontinued therapy prematurely. CONCLUSIONS The combination of the protease inhibitor BI 201335, the polymerase inhibitor BI 207127, and ribavirin has rapid and strong activity against HCV genotype-1 and did not cause serious or severe adverse events.

Hepatitis B Virus Limits Response of Human Hepatocytes to Interferon-α in Chimeric Mice

BACKGROUND & AIMS Interferon (IFN)-α therapy is not effective for most patients with chronic hepatitis B virus (HBV) infection for reasons that are not clear. We investigated whether HBV infection reduced IFN-α-mediated induction of antiviral defense mechanisms in human hepatocytes. METHODS Human hepatocytes were injected into severe combined immune-deficient mice (SCID/beige) that expressed transgenic urokinase plasminogen activator under control of the albumin promoter. Some mice were infected with HBV; infected and uninfected mice were given injections of human IFN-α. Changes in viral DNA and expression of human interferon-stimulated genes (ISGs) were measured by real-time polymerase chain reaction, using human-specific primers, and by immunohistochemistry. RESULTS Median HBV viremia (0.8log) and intrahepatic loads of HBV RNA decreased 3-fold by 8 or 12 hours after each injection of IFN-α, but increased within 24 hours. IFN-α activated expression of human ISGs and nuclear translocation of signal transducers and activators of transcription-1 (STAT1) in human hepatocytes that repopulated the livers of uninfected mice. Although baseline levels of human ISGs were slightly increased in HBV-infected mice, compared with uninfected mice, IFN-α failed to increase expression of the ISGs OAS-1, MxA, MyD88, and TAP-1 (which regulates antigen presentation) in HBV-infected mice. IFN-α did not induce nuclear translocation of STAT1 in HBV-infected human hepatocytes. Administration of the nucleoside analogue entecavir (for 20 days) suppressed HBV replication but did not restore responsiveness to IFN-α. CONCLUSIONS HBV prevents induction of IFN-α signaling by inhibiting nuclear translocation of STAT1; this can interfere with transcription of ISGs in human hepatocytes. These effects of HBV might contribute to the limited effectiveness of endogenous and therapeutic IFN-α in patients and promote viral persistence.