Prime/boost immunization with DNA and adenoviral vectors protects from hepatitis D virus (HDV) infection after simultaneous infection with HDV and woodchuck hepatitis virus.

Hepatitis D virus (HDV) superinfection of hepatitis B virus (HBV) carriers causes severe liver disease and a high rate of chronicity. Therefore, a vaccine protecting HBV carriers from HDV superinfection is needed. To protect from HDV infection an induction of virus-specific T cells is required, as antibodies to the two proteins of HDV, p24 and p27, do not neutralize the HBV-derived envelope of HDV. In mice, HDV-specific CD8(+) and CD4(+) T cell responses were induced by a DNA vaccine expressing HDV p27. In subsequent experiments, seven naive woodchucks were immunized with a DNA prime and adenoviral boost regimen prior to simultaneous woodchuck hepatitis virus (WHV) and HDV infection. Five of seven HDV-immunized woodchucks were protected against HDV infection, while acute self-limiting WHV infection occurred as expected. The two animals with the breakthrough had a shorter HDV viremia than the unvaccinated controls. The DNA prime and adenoviral vector boost vaccination protected woodchucks against HDV infection in the setting of simultaneous infection with WHV and HDV. In future experiments, the efficacy of this protocol to protect from HDV infection in the setting of HDV superinfection will need to be proven.

Cellular immune response to hepatitis C virus (HCV) in nonviremic blood donors with indeterminate anti-HCV reactivity.

BACKGROUND: Blood donors with indeterminate hepatitis C virus antibody (anti-HCV) reactivity are rejected from blood donation. As they are mostly nonviremic, the source of these reactions remains unclear. Reasons for such findings can be resolved HCV infections as well as unspecific antibody reactions. The aim of this study was to investigate HCV-specific T-cell response in blood donors to determine the reason for the weak antibody detection. STUDY DESIGN AND METHODS: Anti-HCV reactivity was tested in 72 blood donors initially diagnosed with an indeterminate HCV result by enzyme-linked immunosorbent assay and immunoblot. Cellular immune response was measured by proliferation assay and enzyme-linked immunosorbent spot analysis after stimulation with viral proteins core, NS3, and NS4. RESULTS: In 56% of donors anti-HCV reactivity was detectable in the screening assay whereas 72% had a reaction in the confirmation immunoblot. Forty-six percent of donors had a cellular immune response against HCV proteins. The response was most frequent to NS3 protein. CONCLUSION: In almost half of donors the indeterminate result in serologic testings could be explained by a previous resolved HCV infection as the pattern of T-cell response was similar to these patients. These findings indicate that HCV-specific antibodies disappear more rapidly after resolved infection than HCV-specific T cells. These results are important for counseling blood donors and patients with indeterminate serologic results.

O6-methylguanine-DNA-methyltransferase (MGMT) gene therapy targeting haematopoietic stem cells: studies addressing safety issues.

As haematopoietic stem cell gene therapy utilizing O(6)-methylguanine-DNA-methyltransferase has reached the clinical stage, safety-related questions become increasingly important. These issues concern insertional mutagenesis of viral vectors, the acute toxicity of pre-transplant conditioning protocols and in vivo selection regimens as well as potential genotoxic side effects of the alkylating drugs administered in this context. To address these questions, we have investigated toxicity-reduced conditioning regimens combining low-dose alkylator application with sublethal irradiation and have analysed their influence on engraftment and subsequent selectability of transduced haematopoietic stem cells. In addition, a strategy to monitor the acute and long-term genotoxic effects of drugs with high guanine-O(6) alkylating potential, such as chloroethylnitrosoureas or temozolomide is introduced. For this purpose, assays were implemented which allow an assessment of the generation and fate of primary drug-induced adducts as well as their long-term effect on chromosomal integrity at the single cell level.

Neonatal antibiotic treatment alters gastrointestinal tract developmental gene expression and intestinal barrier transcriptome.

The postnatal maturation of the gut, partially modulated by bacterial colonization, ends up in the establishment of an efficient barrier to luminal antigens and bacteria. The use of broad-spectrum antibiotics in pediatric practices alters the gut bacterial colonization and, consequently, may impair the maturation of the gut barrier function. To test this hypothesis, suckling Sprague-Dawley rats received a daily intragastric gavage of antibiotic (Clamoxyl; an amoxicillin-based commercial preparation) or saline solution from postnatal day 7 (d7) until d17 or d21. Luminal microbiota composition and global gene expression profile were analyzed on samples from small intestine and colon of each group. The treatment with Clamoxyl resulted in the almost-complete eradication of Lactobacillus in the whole intestine and in a drastic reduction of colonic total aerobic and anaerobic bacteria, in particular Enterobacteriacae and Enterococcus. The global gene expression analysis revealed that Clamoxyl affects the maturation process of 249 and 149 Affymetrix probe sets in the proximal and distal small intestine, respectively, and 163 probe sets in the colon. The expression of genes coding for Paneth cell products (defensins, matrilysin, and phospholipase A2) was significantly downregulated by the Clamoxyl treatment. A significant downregulation of major histocompatibility complex (MHC) class Ib and II genes, involved in antigen presentation, was also observed. Conversely, mast cell proteases expression was upregulated. These results suggest that early treatment with a large-spectrum antibiotic deeply affects the gut barrier function at the suckling-weaning interface, a period during which the gut is challenged by an array of novel food-borne antigens.

Donor- and recipient-derived immunity in ABO incompatible living-related liver transplantation.

This report describes how donor- and recipient-derived immunity was influenced by immunosuppressive treatment of ABO incompatibility (rituximab and immunoadsorption/plasmaphereses) in the long-term. We present an 8-year course of Hepatitis B virus (HBV) immunity, isohemagglutinins and B cell numbers. Whereas cellular HBV immunity was transferred from the HBV vaccinated donor (blood group A1) to the HBV naïve recipient (blood group 0), humoral HBV specific immune transfer was lacking. Starting at month 17 after transplantation, the recipient was vaccinated six times against HBV. Anti-HBs did not appear until the sixth vaccination at month 44. Immunoadsorption prior to transplantation reduced anti-A1 IgG titers from 256 to 2. Titers after transplantation remained low (⩽64). B cell numbers were below standard values up to month 26, then normalized and exceeded normal values from year 7 to 8 post transplantation. In conclusion, donor-derived B cell immunity was lost but recipient-derived immunity persisted after ABO incompatible transplantation.

Magnetic dipole configurations on honeycomb lattices: effect of finite size and boundaries.

Artificial dipolar spin-ice patterns have attracted much attention recently because of their rich configurations and excitations in the form of Dirac strings connecting magnetic monopoles. We have analysed the distribution of excitations in the form of strings and vertices carrying magnetic charges Q=±3q in honeycomb artificial spin-ice patterns. Two types of patterns are compared, those that terminate with open hexagons and those with closed hexagons. The dipole configurations and the frequency of spin-ice rule-violating Q=±3q vertices depend slightly on the boundary conditions of the pattern. Upon rotation of the patterns by 2π in a coercive magnetic field of 500 Oe, complete reversibility of the charge and string configuration is observed.

Adoptive immune transfer of hepatitis B virus specific immunity from immunized living liver donors to liver recipients.

BACKGROUND: Liver transplantation is often the ultimate option of therapy for chronically hepatitis B virus (HBV) infected patients. Prevention of reinfection is therapy intensive and cost-effective. Adoptive transfer of HBV-specific immunity with the liver from an immune living liver donor (LLD) could be a new approach to prevent reinfection. METHODS: Forty-six potential LLDs were vaccinated against HBV. Humoral (antibodies to hepatitis B virus surface antigen [anti-HBs]-titer) and cellular (IFN-gamma-ELISpot and proliferation-assay) immune responses were examined in donors after immunization and in recipients before and after transplantation. RESULTS: Anti-HBs-titers of up to 50,000 IU/L were detected in LLDs. Fourteen recipients received livers from these donors. We detected humoral immunity in one HBV-naïve recipient and in one chronically HBV-infected recipient after transplantation. A transfer of cellular immunity (SI>3) was seen in three recipients. These patients received livers from donors with high anti-HBs-titers of more than 9000 IU/L. Cellular immunity was also detected in the corresponding donors (SI >3 and spots >22). CONCLUSIONS: Our study demonstrates that HBV-specific humoral and cellular immunity can be transferred by liver transplantation after vaccination of the donors. The transfer of B-cell and T-cell immunity correlates with the magnitude of immune responses in the donor.

Nutritional approach to restore impaired intestinal barrier function and growth after neonatal stress in rats.

OBJECTIVES: Psychological stress during the neonatal period results in intestinal barrier dysfunction and growth alterations later in life. We aimed to restore impaired barrier function and growth rate by a nutritional intervention. METHODS: Male rat pups (n = 84) were assigned to 1 of 2 rearing conditions from postnatal day (PND) 2 to PND14: S, separated 3 h/d from their mothers, or H, 15 min/d handled controls. From PND15 to PND35, rats received a control diet or a similar diet adapted to contain arachidonic and docosahexaenoic acids, galacto- and fructo-oligosaccharides and Lactobacillus paracasei NCC2461. RESULTS: Maternal separation had only a minor impact on the measured gut barrier parameters at PND15, whereas it severely affected them at PND35. At this age, intestinal permeability to macromolecules was higher, mucin content in small intestinal tissues was lower and microbiota composition was altered in S compared with H animals. Feeding the adapted diet normalized the intestinal permeability, although it did not restore intestinal mucin content or microbiota. In addition, the adapted diet improved the growth rate recovery of the S animals after weaning and resulted in increased villus length in small intestine. CONCLUSION: Our results suggest that an adapted diet containing specific long-chain polyunsaturated fatty acids, prebiotics and probiotics can revert the negative imprinting of neonatal stress on both intestinal barrier function and growth.