Evaluation of the microbial wet-vacuum system (M-Vac®) for DNA sampling from rough, porous substrates, and its compatibility with fully automated platforms.

DNA retrieval methods traditionally used during forensic evidence recovery including swabbing and tape lifting, can have limited effectiveness when used on porous, rough substrates such as bricks and carpet. This is possibly due to the DNA material being dispersed and unreachable for surface sampling techniques. In this evaluation we investigated the effectiveness of the Microbial Wet-Vacuum System (M-Vac®; M-Vac® Systems, Inc., Sandy, UT), as it has been reported to retrieve greater amounts of DNA material from challenging exhibits. A four-stage evaluation was conducted, starting with seeding carpet and brick substrates with a known donor's saliva in two dilutions and comparing the DNA recovery of tape lifting, swabbing, and the M-Vac®. A victim struggle scenario on carpet was then mimicked to compare trace DNA recovery by each method. Two mock scenarios were also conducted; a shirt was submerged in a creek bed for a period of five days to sample for the wearer's DNA, and a car boot was sampled to assess the possibility of recovering a victim's DNA amongst background DNA from the usual car occupants. Finally, the compatibility of the M-Vac® sampling process was optimised for the fully automated DNA lysis and extraction platforms used in the NSW (Australia) jurisdiction by comparing filter subsampling methods. The results from the study were mixed. For bricks, none of the collection methods were effective in retrieving DNA. On carpet, the M-Vac® retrieved the greatest quantities of DNA from the saliva-seeded samples, however, tape lifts outperformed all methods for 'touch' DNA recovery. The M-Vac® retrieved the greatest amount of DNA from the t-shirt recovered from a creek bed as it was able to retrieve the embedded DNA. The final mock case car boot scenario resulted in greater victim DNA recovery from tape lifts, with the M-Vac® more likely to recover mixtures too weak and/or complex to be interpreted. Finally, operational considerations regarding the compatibility of the M-Vac® system with fully automated DNA lysis and extraction are discussed. Considering the substantial time and cost to deploy the M-Vac®, it is recommended to be utilised in casework only after swabbing and tape lifting methods have failed to yield sufficient DNA material, where the substrate properties would likely benefit from the M-Vac's® niche capabilities for retrieving embedded DNA, and low levels of background DNA may be anticipated.

ADAM17-Mediated Reduction in CD14++CD16+ Monocytes ex vivo and Reduction in Intermediate Monocytes With Immune Paresis in Acute Pancreatitis and Acute Alcoholic Hepatitis.

Impaired immune responses and increased susceptibility to infection characterize acute inflammatory conditions such as pancreatitis and alcoholic hepatitis and are major causes of morbidity and mortality. However, the mechanisms that drive this apparent immune paresis remain poorly understood. Monocytes mediate host responses to damage and pathogens in health and disease, and three subsets of monocytes have been defined based on CD14 and CD16 expression. We sought to determine the changes in monocyte subsets in acute pancreatitis (AP) and acute alcoholic hepatitis (AAH), together with functional consequences and mechanisms that underlie this change. Peripheral blood mononuclear cells (PBMCs) from patients with AP or AAH were compared with healthy controls. Monocyte subsets were defined by HLA-DR, CD14, and CD16 expression. Changes in surface and intracellular protein expression and phosphorylation were determined by flow cytometry. Phenotype and function were assessed following stimulation with lipopolysaccharide (LPS) or other agonists in the presence of specific inhibitors of TNFα and a disintegrin and metalloproteinase 17 (ADAM17). Patients with AP and AAH had reduced CD14++CD16+ intermediate monocytes compared to controls. Reduction of intermediate monocytes was recapitulated ex vivo by stimulating healthy control PBMCs with Toll-like receptor (TLR) agonists LPS, flagellin or polyinosilic:polycytidylic acid (poly I:C). Stimulation caused shedding of CD14 and CD16, which could be reversed using the ADAM17 inhibitor, TMI005 but not direct inhibitors of TNFα, a known ADAM17-target. Culturing PBMCs from healthy controls resulted in expansion of intermediate monocytes, which did not occur when LPS was in the culture medium. Cultured intermediate monocytes showed reduced expression of CX3CR1, CCR2, TLR4, and TLR5. We found reduced migratory responses, intracellular signaling and pro-inflammatory cytokine production, and increased expression of IL-10. Stimulation with TLR agonists results in ADAM17-mediated shedding of phenotypic markers from CD16+ monocytes, leading to apparent "loss" of intermediate monocytes. Reduction in CD14++CD16- monocytes and increased CD14++CD16+ is associated with altered responses in functional assays ex vivo. Patients with AP and AAH had reduced proportions of CD14++CD16+ monocytes and reduced phosphorylation of NFκB and IL-6 production in response to bacterial LPS. Together, these processes may contribute to the susceptibility to infection observed in AP and AAH.

Successful expansion of functional and stable regulatory T cells for immunotherapy in liver transplantation.

Strategies to prevent organ transplant rejection whilst minimizing long-term immunosuppression are currently under intense investigation with regulatory T cells (Tregs) nearing clinical application. The clinical trial, ThRIL, recently commenced at King's College London, proposes to use Treg cell therapy to induce tolerance in liver transplant recipients, the success of which has the potential to revolutionize the management of these patients and enable a future of drug-free transplants. This is the first report of the manufacture of clinical grade Tregs from prospective liver transplant recipients via a CliniMACS-based GMP isolation technique and expanded using anti-CD3/CD28 beads, IL-2 and rapamycin. We report the enrichment of a pure, stable population of Tregs (>95% CD4(+)CD25(+)FOXP3(+)), reaching adequate numbers for their clinical application. Our protocol proved successful in, influencing the expansion of superior functional Tregs, as compared to freshly isolated cells, whilst also preventing their conversion to Th17 cells under pro-inflammatory conditions. We conclude with the manufacture of the final Treg product in the clinical research facility (CRF), a prerequisite for the clinical application of these cells. The data presented in this manuscript together with the much-anticipated clinical results from ThRIL, will undoubtedly inform the improved management of the liver transplant recipient.

Acute liver failure.

Acute liver failure (ALF) is a rare critical illness with high mortality whose successful management requires early recognition and effective initial management. Though it may result from a wide variety of causes, in the UK and much of the developed world most cases result from paracetamol-induced hepatotoxicity, and administration of antidotal N-acetyl cysteine at first recognition is key. Involvement of local critical care services should occur at an early stage for stabilisation, monitoring and supportive care with parallel discussion with specialist liver centres to identify those patients who may benefit from transfer. Prognostic criteria are applied to identify patients for emergency liver transplantation, and candidates for surgery are prioritised on waitlisting schemes. Outcomes now approach that of elective surgery. However, the majority of cases, and particularly those with paracetamol-induced disease, recover with supportive medical care alone. Overall outcomes for patients with ALF have improved dramatically over the last three decades, but mortality remains unacceptable and further advances in care are required.

Acute alcoholic hepatitis and cellular Th1 immune responses to alcohol dehydrogenase.

BACKGROUND: Alcoholic hepatitis is characterised by florid hepatic inflammation, liver failure, and death within 28 days in 35% of patients. We recently showed proliferative peripheral blood mononuclear cell (PBMC) responses to alcohol dehydrogenase (ADH) in patients with alcohol-related cirrhosis, associated with T-helper-type 1 (Th1) immunity and disease severity. We aimed to define whether ADH-specific cellular immunity is present in alcoholic hepatitis. METHODS: PBMCs were collected from 15 patients with alcoholic hepatitis (modified Maddrey's discriminant function >32), nine with alcohol-related cirrhosis (long-term alcohol abstinence), and three healthy controls. 25 overlapping peptides, spanning the human ADH ß1 subunit, were constructed. Proliferation to ADH peptides (1 × 10(5) cells per well, cultured with 10 mM peptides for 7 days) was assessed by (3)H-thymidine incorporation. A stimulation index (SI) of 2·5 or more was regarded as positive. ELISA measured concentrations of interferon γ (IFNγ), interleukin (IL) 17, and IL4 from supernatant. FINDINGS: PBMCs from seven of 15 patients with alcoholic hepatitis recognised one to three ADH peptides (SI ≤5·7). IFNγ (mean 390·9 pg/mL [SE 31·4]) was detected in 48% of wells, IL17 (20·1 [3 ·4]) in 15%, and IL4 (90·5 [9·3]) in 14%. PBMCs from six of the nine patients with alcohol-related cirrhosis recognised one to five peptides (SI ≤5·2). IFNγ (360·7 [58·9], p>0·05) was detected in 31% of wells, IL17 (57·7 [10·9], p=0·0006) in 19%, and IL4 (219·7 [11·2], p=0·0012) in 28%. PBMCs from two healthy controls recognised one to two peptides (SI ≤3·1); all cytokine levels were below baseline. INTERPRETATION: Proliferative anti-ADH immune responses in alcoholic hepatitis focused on individual epitopic regions. Predominance of proinflammatory Th1 responses was more pronounced in alcoholic hepatitis than in alcoholic-related cirrhosis. This finding requires investigation of targeted therapies to inhibit Th1 immunity in alcoholic hepatitis. FUNDING: Wellcome Trust.

Autoantibody and human leukocyte antigen profiles in children with autoimmune liver disease and their first-degree relatives.

OBJECTIVE: Familial clustering of juvenile autoimmune liver disease (AILD), including autoimmune hepatitis and autoimmune sclerosing cholangitis (ASC), is rare, despite a high prevalence of autoimmune disorders in AILD families. METHODS: To investigate this discrepancy, we measured autoantibodies diagnostic for AILD, anti-nuclear, anti-smooth muscle, anti-liver kidney microsomal type 1, anti-liver cytosol type 1, and anti-soluble liver antigen antibodies, and human leukocyte antigen profiles in 31 patients and 65 of their first-degree relatives (FDR). The autoantibody profile was compared with that of 42 healthy subjects (HS). RESULTS: Autoantibodies were detected in 71% (22/31) patients. Anti-nuclear antibody or anti-smooth muscle antibody were present in 4/65 FDR (6.2%). HS were negative for all autoantibodies. The frequencies of homozygous HLA DRB1*0301 (DR3) genes and haplotype A1-B8-DR3 were higher in the patients (25% and 43%) than in FDR (9% and 27%) and HS (0% and 16%). The frequencies of disease-protective genes DR4 and/or DR15 were lower in the patients (25%) than in FDR (42%) and HS (42%). Only 1 family contained 2 patients with AILD, 1 with ASC and 1 with primary sclerosing cholangitis. Both patients possessed A1-B8-DR3 genes, the ASC being homozygous and the primary sclerosing cholangitis heterozygous. Six FDR had nonhepatic autoimmune disorders, none being autoantibody positive. CONCLUSIONS: Homozygosity for DR3 plays a major role in the predisposition to juvenile AILD. Diagnostic autoantibodies for AILD are rare among patients' FDR and not linked to clinical manifestation of AILD.

Alcohol dehydrogenase-specific T-cell responses are associated with alcohol consumption in patients with alcohol-related cirrhosis.

UNLABELLED: Patients with alcohol-related liver disease (ALD) have antibodies directed to alcohol dehydrogenase (ADH), anti-ADH titers being associated with disease severity and active alcohol consumption. ADH-specific T-cell responses have not been characterized. We aimed to define anti-ADH cellular immune responses and their association with active alcohol consumption and disease severity. Using cultures of peripheral blood mononuclear cells (PBMCs) from 25 patients with alcohol-related cirrhosis (ARC; 12 were actively drinking or abstinent for <6 months, and 13 were abstinent for >6 months) and hepatic mononuclear cells (HMCs) from 14 patients with ARC who were undergoing transplantation, we investigated T-cell reactivity to 25 overlapping peptides representing the full human ADH protein (beta 1 subunit). ADH-specific peripheral T-cell responses were assessed by the quantification of T-cell proliferation and cytokine production and were correlated with the clinical course. In active alcohol consumers, proliferative T-cell responses targeted ADH31-95 and other discontinuous sequences in the ADH peptide, whereas only one sequence was targeted in abstinents. ADH peptides induced the production of interferon-γ (IFN-γ), interleukin-4 (IL-4), and IL-17. IL-4 production was lower in active drinkers versus abstinents, and IL-17 production was higher. Peptides inducing IFN-γ production outnumbered those inducing T-cell proliferation. The intensity of the predominantly T helper 1 (Th 1) responses directly correlated with disease severity. Similar to PBMCs in abstinents, ADH peptides induced weak T-cell proliferation and a similar level of IL-4 production in HMCs but less vigorous Th 1 and T helper 17 responses. CONCLUSION: This suggests that Th 1 responses to ADH in ARC are induced by alcohol consumption. A Th 1/T helper 2 imbalance characterizes T-cell responses in active drinkers with ARC, whereas IL-4 production prevails in abstinents. This identifies new targets for immunoregulatory therapies in ALD patients for halting detrimental effector T-cell responses, which may encourage liver fibrogenesis and progression to end-stage liver disease.

Polymorphisms in ABCB11 and ATP8B1 Associated with Development of Severe Intrahepatic Cholestasis in Hodgkin's Lymphoma.

We report a young man presenting with jaundice and severe debilitating intrahepatic cholestasis 7 months before the diagnosis of Hodgkin's lymphoma. Serum gamma-glutamyl transferase (GGT) activity was not raised. Liver biopsy demonstrated deficiency of canalicular GGT and bile salt export pump expression, which suggested "benign" recurrent intrahepatic cholestasis. Direct sequencing of genomic DNA was therefore undertaken to look for mutations in ATP8B1 and ABCB11. Cholestasis and pruritus are well recognized presenting features of Hodgkin's lymphoma. However, striking in this case is that the intrahepatic cholestasis presented and resolved 7 months before the diagnosis. Furthermore, 4 polymorphisms were identified in ATP8B1 in this patient-c.696T > C (rs319438), c.811A > C (rs319438), c.2855G > A (rs1296811) and c.3454G > A (rs222581)-and two polymorphisms in ABCB11-c.1331T > C (rs2287622) and c.3084A > G (rs497692); 2 of which have been associated with intrahepatic cholestasis of pregnancy. We therefore postulate that these polymorphisms predisposed this patient to the development of intrahepatic cholestasis within the abnormal pro-inflammatory cytokine milieu typical for Hodgkin's lymphoma. This case shows for the first time that some polymorphisms in ABCB11 and ATP8B1 may predispose to the development of intrahepatic cholestasis in Hodgkin's lymphoma. It also demonstrates the importance of close clinical surveillance for the development of Hodgkin's lymphoma in patients presenting with unexplained intrahepatic cholestasis.

Parenteral methotrexate for patients with Crohn's disease: how to develop a high-quality and safe self-administration service.

Parenteral methotrexate is recommended for patients with Crohn's disease who have failed treatment with thiopurines. There is no good evidence for the use of oral methotrexate, yet patients frequently receive this due to the difficulties associated with prescribing and administering an unlicensed, cytotoxic drug. We present our experience of developing a local service to provide our patients with the option to self-administer parenteral methotrexate in a safe and structured manner at home.