Ingolfur Johannessen

Human cord blood-derived cells can differentiate into hepatocytes in the mouse liver with no evidence of cellular fusion

BACKGROUND & AIMS Studies have indicated that stem cells have unexpected plasticity and can differentiate down a multitude of nonhematopoietic cell lineages in rodents. Our aim was to identify whether human cord blood cells, which are a rich source of stem cells, would be able to differentiate into hepatocytes when infused into nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice. We also wanted to test whether such differentiated cells were the result of cellular fusion or true stem cell transdifferentiation. METHODS Unsorted mononuclear cell preparations of human cord blood were infused into sublethally irradiated NOD-SCID mice. After death, immunohistologic analysis of murine livers was performed using human specific hepatocyte, biliary, and endothelial markers. Fluorescent in situ hybridization (FISH) for mouse and human DNA was also performed. RESULTS We show that human cord blood cells have the ability to engraft into NOD-SCID liver and become mature hepatocytes. We were unable to identify any biliary or endothelial differentiation. Furthermore, we do not detect any evidence of cell fusion in any of the human cells found in the mouse liver, suggesting that human cord blood cells are capable of true transdifferentiation into hepatocytes in vivo. CONCLUSIONS We conclude that hepatocytes can derive from human cord blood cells when infused into NOD-SCID mice in the absence of fusion. The demonstration that human stem cell differentiation can occur in this murine model permits comprehensive study of human stem cell plasticity in vivo.

Enhanced Apoptotic Cell Clearance Capacity and B Cell Survival Factor Production by IL-10-Activated Macrophages: Implications for Burkitt’s Lymphoma

Burkitt’s lymphoma (BL) is typified by frequent tumor cell apoptosis and significant macrophage infiltration. Since BL cells have an inherent tendency to undergo apoptosis at a high rate, we reasoned that macrophages in BL are functionally enhanced in at least two activities that have implications for tumor pathogenesis: 1) engulfment of apoptotic cells, an anti-inflammatory process known to suppress immune responses, and 2) production of BL cell survival factors that limit the extent of tumor cell apoptosis. In this study, we show that the microenvironment of BL is rich in the pleiotropic cytokine IL-10, which can be produced by both tumor cells and macrophages, and that IL-10-activated human macrophages have enhanced capacity to engulf apoptotic cells in vitro. This was found to be dependent on the macrophage tethering receptor of apoptotic cells, CD14. Furthermore, IL-10-activated macrophages were found to produce markedly higher levels of the B cell survival factor, B cell-activating factor of the TNF family/B lymphocyte stimulator (BAFF/BLyS) than macrophages matured in the absence of IL-10. Coculture of macrophages with BL cells further enhanced BAFF secretion. Significantly, we show that enhancement of BL cell survival by IL-10-activated macrophages is mediated by a BAFF-dependent component and that BAFF is produced at high levels by tumor-associated macrophages in situ. These results indicate that macrophages, regulated by IL-10, have the potential to promote BL pathogenesis, first, through suppression of antitumor immunity following enhanced engulfment of apoptotic tumor cells and, second, through increased production of tumor cell growth/survival factors.

In vivo models for Epstein–Barr virus (EBV)-associated B cell lymphoproliferative disease (BLPD)

EBV infects B lymphocytes in vivo and establishes a life‐long persistent infection in the host. The latent infection is controlled by EBV‐specific MHC class 1‐restricted CTL. Immunosuppression reduces CTL activity, and this facilitates outgrowth of EBV+ve B cell lymphoproliferative disease (BLPD). BLPD are aggressive lesions with high mortality.

High risk of cytomegalovirus infection following solid organ transplantation despite prophylactic therapy.

Cytomegalovirus infection (CMV) in solid organ transplant recipients is a major clinical problem. The aim of this study was to evaluate the incidence of CMV infection and its association with mortality during the first year after transplantation in a large solid organ transplant cohort at the Royal Infirmary of Edinburgh between January 2006 and April 2009. Data including the use of CMV prophylaxis, nature of CMV disease, treatment and deceased date (when appropriate) was collected retrospectively using hospital databases and patient notes for all transplanted patients with detectable CMV viraemia. The outcomes between recipients of kidney and liver transplants in the four CMV donor/recipient serostatus categories (D+R+, D−R−, D+R−, D−R+) were compared. A total of 428 individuals were included. Despite the administration of valganciclovir prophylaxis, CMV disease (syndrome or end‐organ involvement) was diagnosed within the year of transplantation in the D+R−‐group in 31.3% of liver and 19.2% of kidney recipients. All D+R− transplant recipients that received CMV‐prophylaxis presented with late‐onset CMV disease. Furthermore, the rate of CMV disease in the D+R+‐group was markedly higher in renal graft recipients compared to liver recipients (22% vs. 5%). The highest mortality was observed among the D+R+ liver and kidney graft recipients with CMV infection. The high incidence of late‐onset CMV disease in D+R− transplant recipients receiving CMV prophylaxis demonstrates that CMV disease remains an important problem after organ transplantation. Furthermore, the surprisingly high mortality in the D+R+‐transplant patients with CMV viraemia highlights the need for proactive monitoring of this group. J. Med. Virol. 85:893–898, 2013.

Essential role for T cells in human B-cell lymphoproliferative disease development in severe combined immunodeficient mice

Epstein–Barr virus (EBV)‐positive B‐cell lymphoproliferative disease (BLPD)‐like lesions develop in severe combined immunodeficient (SCID) mice inoculated with peripheral blood mononuclear cells (PBMCs) from EBV‐seropositive donors. We used this model to investigate the pathogenesis of EBV‐associated BLPD. Tumour incidence fell from 81% to 11% when only B cells were inoculated, suggesting a key role for T cells in tumour formation. This was further underlined by the reduction in tumour incidence from 76% to 7% when PBMCs were depleted of CD4 positive (+ve) helper T cells. Tumour outgrowth was also reduced when PBMC were depleted of CD8 +ve, CD45RA +ve or CD45RO +ve T cells. The majority of PBMC‐derived tumours analysed by reverse transcriptase–polymerase chain reaction (RT–PCR) expressed mRNA for interleukin (IL) 2, 4, 6, 10 and interferon (IFN) γ. This is the cytokine pattern seen in activated T cells and includes B‐cell growth factors. In situ hybridization studies confirmed that the tumour cells themselves express the growth factors, which is consistent with autocrine‐stimulated tumour growth.

Evolution of the hepatitis E virus hypervariable region

The presence of a hypervariable (HVR) region within the genome of hepatitis E virus (HEV) remains unexplained. Previous studies have described the HVR as a proline-rich spacer between flanking functional domains of the ORF1 polyprotein. Others have proposed that the region has no function, that it reflects a hypermutable region of the virus genome, that it is derived from the insertion and evolution of host sequences or that it is subject to positive selection. This study attempts to differentiate between these explanations by documenting the evolutionary processes occurring within the HVR. We have measured the diversity of HVR sequences within acutely infected individuals or amongst sequences derived from epidemiologically linked samples and, surprisingly, find relative homogeneity amongst these datasets. We found no evidence of positive selection for amino acid substitution in the HVR. Through an analysis of published sequences, we conclude that the range of HVR diversity observed within virus genotypes can be explained by the accumulation of substitutions and, to a much lesser extent, through deletions or duplications of this region. All published HVR amino acid sequences display a relative overabundance of proline and serine residues that cannot be explained by a local bias towards cytosine in this part of the genome. Although all published HVRs contain one or more SH3-binding PxxP motifs, this motif does not occur more frequently than would be expected from the proportion of proline residues in these sequences. Taken together, these observations are consistent with the hypothesis that the HVR has a structural role that is dependent upon length and amino acid composition, rather than a specific sequence.

Rash and arthralgia caused by hepatitis E

A 52-year-old woman came to her general practitioner with pain, swelling, and stiff ness in her shoulders, elbows, hips, knees, and ankles. She also had bilateral retro-orbital pain, eye discharge, and headache for a week, and loss of appetite for 4 days. 2 days before her appointment, she developed a maculopapular, non-itchy rash all over her body. She was a non-smoker and consumed moderate amounts of alcohol. The general practitioner requested baseline blood tests including full blood count, liver and renal function tests, rheumatoid factor, auto-antibodies and complements, prescribed analgesia, and referred the patient to a rheumatologist. When reviewed by the rheumatologist 2 weeks later, the patient’s left second and third metacarpophalangeal joints were tender. There was no evidence of conjunctivitis, rash, or synovitis. Radiological analyses of her hands and feet were unremarkable. Investigations requested by the general practitioner were reviewed by the rheumatologist: liver function tests were abnormal (alanine amino transferase 1025 U/L [10–50 U/L]; alkaline phosphatase 177 U/L [40–125 U/L]; gamma glutamyl transferase 224 U/L [5–35 U/L]). Bilirubin was normal (11 μmol/L) (3–21 μmol/L). Ferritin (1593 μg/L) (15–200 μg/L), C-reactive protein (25 mg/L) (0–5 mg/L) and erythrocyte sedimentation rate (39 mm/h) (3–15 mm/h) were raised. Her full blood count, renal function, and coagulation profi le were within normal limits. Complement C3 was raised (1·7 g/L) (0·81–1·57 g/L) but C4 was normal (0·23 g/L) (0·13–0·39 g/L). Rheumatoid factor, anti-nuclear/anti-smooth muscle antibodies were not detected, whereas anti-dsDNA (17·5 IU/mL) (0–15 IU/mL), extractable nuclear antigen, antimitochondrial antibody (187·1 IU/mL), and anti-Ro (SS-A) antibody (68·7 U/mL) were detected. Hepatitis B surface antigen and hepatitis B core total antibody were not detected. HIV and hepatitis C virus screening tests were requested by the rheumatologist. In view of abnormal liver function tests, further tests were added to exclude other causes of viral hepatitis. Hepatitis A IgM was undetectable. There was serological evidence of past cytomegalovirus and Epstein-Barr virus infections. Hepatitis E virus (HEV) IgM and IgG were detectable; however, HEV RNA was not detected in the sample sent by the rheumatologist. The blood sample sent previously by the general practitioner was retrospectively investigated and acute hepatitis E was diagnosed on the basis of detectable HEV RNA and evolving seroconversion. HEV RNA was sequenced (ORF2 region as described previously) and identifi ed as genotype 3. During the ensuing 6 weeks, the patient’s liver function returned to normal, and clinical signs and symptoms resolved. On subsequent interview by the local Health Protection Team, the patient did not report any travel outside Scotland during the incubation period (previous 9 weeks). Furthermore, no specifi c association with food or other risk factors could be established. Most (98%) HEV infections are asymptomatic. Symptomatic individuals typically have non-specifi c symptoms such as myalgia, arthralgia, weakness, and vomiting. Some develop jaundice with transaminitis. Acute hepatitis E presenting as polyarthritis with longlasting viraemia (up to 7 months) has been reported in an individual who travelled to Egypt during the incubation period. By contrast, viraemia was short lived in our case. Until recently, HEV infection in developed countries was thought to be travel-associated. However, autochthonous HEV, a zoonotic infection caused predominantly by HEV genotype 3, is now recognised in many developed countries. We have previously reported autochthonous hepatitis E genotype 3 from Scotland, with an individual infected with multiple strains of HEV genotype 3. Recently, HEV genotype 3 has been detected in a substantial proportion of mussels harvested along the west and east coasts of Scotland, suggesting a possible transmission route, although a direct epidemiological link is yet to be established. Since HEV screening is not routine in most UK clinical laboratories, acute hepatitis E is probably underreported. Our case highlights the variable presentation of acute hepatitis E and the importance of hepatitis E screening in individuals with transaminitis, irrespective of travel history.

Non-correlation of in vivo and in vitro parameters of Epstein-Barr virus persistence suggests heterogeneity of B cell infection

Following primary infection Epstein-Barr virus (EBV) establishes a persistent infection which is maintained for the life-time of the host. EBV can be found in a small number of circulating B cells, but the nature of the virus-cell interaction has not been fully established. Several assay systems are used to quantify persistent EBV infection, including PCR amplification of EBV DNA and spontaneous outgrowth of lymphoblastoid cell lines in culture. More recently, outgrowth of EBV-positive B cell tumours in severe combined immunodeficient (SCID) mice inoculated with peripheral blood mononuclear cells (PBMC) from normal EBV-seropositive donors has also been used to study B cell infection in vivo. In the present study we have compared the results of these two biological assay systems with PCR detection of EBV DNA and a regression assay as a measure of host T cell immunity to EBV. PBMC from ten normal EBV-seropositive donors were studied and although each test gave consistent results on repeat assays, no correlation was found between any of the assays tested. This result suggests that each assay measures a separate aspect of EBV persistence in B cells, and indicates a previously unrecognized degree of heterogeneity in the B cell population in which EBV resides.

Acute viral hepatitis - Should the current screening strategy be modified?

BACKGROUND The epidemiology of viral hepatitis has changed. Since the introduction of safe and effective vaccines for hepatitis A and B in 1980s, the incidence of acute infections caused by these viruses has been declining in the UK. At the same time, hepatitis E virus (HEV) has been recognised as an increasingly important cause of acute hepatitis, but testing is not widely available. OBJECTIVES The aim of this study was to establish the viral causes of acute hepatitis, and use that data to modify the current diagnostic algorithm. STUDY DESIGN A Cognos search was performed to collate subjects tested for HAV, HBV, HCV, HEV, EBV and CMV between June 2010 and December 2012. Information included virological result and their ALT level if done within 5 days from virological testing. RESULTS From 3462 subjects with suspected acute viral hepatitis, only 25% had biochemical evidence of acute hepatitis (n=854; ALT>100IU/l). The frequency of detection of acute HEV infection (25/409) was over 31-times higher than that of HAV (6/3462), and 7-times higher than that of HBV (24/3462). Most cases of acute HAV, HEV, EBV and CMV infections presented with abnormal ALT levels. Most EBV infections were associated with lymphadenopathy (23/34); in comparison most of CMV infections were not associated with lymphadenopathy (18/22). CONCLUSIONS HEV screening should be included in the initial testing panel for acute hepatitis and screening at least for HAV and HEV might be limited to those with abnormal ALT levels.

Expansion in scid mice of Epstein-Barr virus-associated post- transplantation lymphoproliferative disease biopsy material

Post-transplant lymphoproliferative disease (PTLD) biopsy material is rarely available in adequate quantity for research. Therefore, the present study was designed to expand biopsy material in scid mice. Epstein-Barr virus (EBV)+ve PTLD samples from five transplant patients were established in scid mice. PCR analysis of immunoglobulin gene rearrangements demonstrated that four of the five biopsies (80%) gave rise to scid tumours which represented the original tumour cell clones. Immunophenotyping showed that these four biopsies (and all scid tumours) expressed all EBV latent genes and a B lymphoblast phenotype; <or=26% T cells were found in the biopsy material whereas scid tumours showed a paucity of T lymphocytes. RT-PCR analysis revealed expression of IL-2, -4, -6, -10 and IFN-gamma in all tumour material, suggesting key roles for these factors in tumour growth. The results show that EBV+ve PTLD material can be expanded in scid mice giving rise to quantities of homogeneous malignant tissue sufficient for research studies.