Circulating CD56dim natural killer cells and CD56+ T cells that produce interferon-γ or interleukin-10 are expanded in asymptomatic, E antigen-negative patients with persistent hepatitis B virus infection.

Infection with hepatitis B virus (HBV) can result in spontaneous resolution or chronic infection, which can remain asymptomatic or can progress to cirrhosis and/or hepatocellular carcinoma. The host immune response is thought to be a major determinant of the outcome of HBV infection and virus-specific cytotoxic T lymphocytes (CTL) can mediate immunity against the virus and cause liver pathology. Antigen-nonspecific innate lymphocytes may also contribute to HBV infection and liver disease, therefore, we examined the frequencies, phenotypes, cytolytic activities and cytokine profiles of circulating natural killer (NK) cells, CD1d-restricted invariant natural killer T (iNKT) cells and CD56(+) T cells in 102 asymptomatic HBV-infected patients and compared them with those in 66 uninfected control subjects. NK cells expressing low levels of CD56 (CD56(dim)) and CD56(+) T cells were significantly expanded in the circulation of HBV-infected patients compared with control subjects. CD1d expression and iNKT cell frequencies were similar in both groups. Despite these expansions, we did not detect augmented natural or cytokine-induced cytotoxicity in the HBV-infected subjects. All lymphocyte populations studied produced interferon-γ (IFN-γ) significantly more frequently when taken from HBV-infected patients compared with when taken from healthy controls. Additionally, NK cells from the patients more frequently produced interleukin-10. As our HBV-infected cohort consisted of asymptomatic patients with low viral loads, we propose that CD56(dim) NK cells and CD56(+) T cells control HBV infection by noncytolytic mechanisms.

PDBe: Protein Data Bank in Europe.

The Protein Data Bank in Europe (PDBe; pdbe.org) is a partner in the Worldwide PDB organization (wwPDB; wwpdb.org) and as such actively involved in managing the single global archive of biomacromolecular structure data, the PDB. In addition, PDBe develops tools, services and resources to make structure-related data more accessible to the biomedical community. Here we describe recently developed, extended or improved services, including an animated structure-presentation widget (PDBportfolio), a widget to graphically display the coverage of any UniProt sequence in the PDB (UniPDB), chemistry- and taxonomy-based PDB-archive browsers (PDBeXplore), and a tool for interactive visualization of NMR structures, corresponding experimental data as well as validation and analysis results (Vivaldi).

Altered natural killer cell subset distributions in resolved and persistent hepatitis C virus infection following single source exposure.

BACKGROUND: Natural killer (NK) cells may be impaired in patients with persistent hepatitis C virus (HCV) infection, but studies to date have yielded inconsistent findings due to patient and virus heterogeneity and difficulties obtaining appropriate controls. AIMS: To overcome these variables, we have examined numbers, phenotypes, cytotoxic activities and cytokine profiles of circulating NK cells from Irish women who acquired infection through administration of HCV genotype 1b-contaminated anti-D immunoglobulin from a single source and matched controls. RESULTS: Comparing 29 women who developed persistent infection with 21 who spontaneously resolved infection and 26 controls, we found that NK cell numbers were consistently lower in the persistently infected group (p = 0.02 and 0.002). This decrease was due to depletions of NK cells expressing low levels of CD56 (CD56(dim) NK cells; p = 0.004 and 0.0001), whilst CD56(bright) NK cells were expanded (p = 0.004 and 0.0001). Compared to HCV resolvers, CD56(dim) NK cells from persistently infected patients less frequently expressed CD16 and more frequently expressed NKG2A/C/E. These phenotypic changes did not significantly affect natural or interleukin-2-induced cytotoxicity by peripheral blood mononuclear cells against K562 and Daudi targets. Greater frequencies of CD56(bright) NK cells from chronic HCV patients produced interferon-gamma compared with HCV responders (p = 0.05) and controls (p = 0.0001) after phorbol ester stimulation in vitro. CONCLUSIONS: Alterations in NK subset distributions in chronic HCV infection may explain why previous reports of impaired NK cell functions were difficult to confirm. Altered NK cell functions may contribute to impaired cellular immune responses and chronicity of disease following HCV infection.

The Escherichia coli AmtB protein as a model system for understanding ammonium transport by Amt and Rh proteins.

The Escherichia coli ammonium transport protein (AmtB) has become the model system of choice for analysis of the process of ammonium uptake by the ubiquitous Amt family of inner membrane proteins. Over the past 6 years we have developed a range of genetic and biochemical tools in this system. These have allowed structure/function analysis to develop rapidly, offering insight initially into the membrane topology of the protein and most recently leading to the solution of high-resolution 3D structures. Genetic analysis has revealed a novel regulatory mechanism that is apparently conserved in prokaryotic Amt proteins and genetic approaches are also now being used to dissect structure/function relationships in Amt proteins. The now well-recognised homology between the Amt proteins, found in archaea, eubacteria, fungi and plants, and the Rhesus proteins, found characteristically in animals, also means that studies on E. coli AmtB can potentially shed light on structure/function relationships in the clinically important Rh proteins.

Three-dimensional domain swapping in the folded and molten-globule states of cystatins, an amyloid-forming structural superfamily.

Cystatins, an amyloid-forming structural superfamily, form highly stable, domain-swapped dimers at physiological protein concentrations. In chicken cystatin, the active monomer is a kinetic trap en route to dimerization, and any changes in solution conditions or mutations that destabilize the folded state shorten the lifetime of the monomeric form. In such circumstances, amyloidogenesis will start from conditions where a domain-swapped dimer is the most prevalent species. Domain swapping occurs by a rearrangement of loop I, generating the new intermonomer interface between strands 2 and 3. The transition state for dimerization has a high level of hydrophobic group exposure, indicating that gross conformational perturbation is required for domain swapping to occur. Dimerization also occurs when chicken cystatin is in its reduced, molten-globule state, implying that the organization of secondary structure in this state mirrors that in the folded state and that domain swapping is not limited to the folded states of proteins. Although the interface between cystatin-fold units is poorly defined for cystatin A, the dimers are the appropriate size to account for the electron-dense regions in amyloid protofilaments.

Near-infrared and nuclear magnetic resonance spectroscopic assessment of tissue energetics in an isolated, perfused canine hind limb model of dysoxia.

This controlled laboratory study examined the efficacy of near-infrared spectroscopy (NIRS) and 31P-nuclear magnetic resonance (NMR) spectroscopy in measuring regional tissue oxygenation in a isolated, perfused hind limb model of tissue dysoxia. Isolated hind limb perfusion was carried out in 20 mongrel dogs and oxygen delivery was varied by manipulating either hemoglobin concentration, oxygen saturation, or flow. Hind limbs from anesthetized mongrel dogs (n = 20) were separated and isolated perfusion performed. NIRS probes for recording relative O2 saturation of tissue hemoglobin (HbO2) and cytochrome a,a3 and NMR probes for measuring 31P-high energy phosphates were placed over the limb. Measurements of physiologic parameters, blood gases, lactate, NIRS values for HbO2 and cytochrome a,a3 redox state, and 31P-phosphate levels were recorded at set intervals throughout the experiment. Measures of tissue oxygen consumption (VO2) correlated with tissue oxygenation as measured by HbO2 and cytochrome a,a3 redox state (NIRS), as well as by 31P-high energy phosphate levels (NMR) throughout the experiment. Delivery-dependent tissue oxygenation was detected at a higher DO2 by NIRS than by VO2 or NMR. Tissue oxygenation as measured by NIRS and NMR shows excellent correlation with oxygen delivery in an isolated, perfused model of shock. NIRS may allow early detection of tissue dysoxia using rapid non-invasive techniques.

The solution structure of Rhodobacter sphaeroides LH1beta reveals two helical domains separated by a more flexible region: structural consequences for the LH1 complex.

Here, the solution structure of the Rhodobacter sphaeroides core light-harvesting complex beta polypeptide solubilised in chloroform:methanol is presented. The structure, determined by homonuclear NMR spectroscopy and distance geometry, comprises two alpha helical regions (residue -34 to -15 and -11 to +6, using the numbering system in which the conserved histidine residue is numbered zero) joined by a more flexible four amino acid residue linker. The C-terminal helix forms the membrane spanning region in the intact LH1 complex, whilst the N-terminal helix must lie in the lipid head groups or in the cytoplasm, and form the basis of interaction with the alpha polypeptide. The structure of a mutant beta polypeptide W(+9)F was also determined. This mutant, which is deficient in a hydrogen bond donor to the bacteriochlorophyll, showed an identical structure to the wild-type, implying that observed differences in interaction with other LH1 polypeptides must arise from cofactor binding. Using these structures we propose a modification to existing models of the intact LH1 complex by replacing the continuous helix of the beta polypeptide with two helices, one of which lies at an acute angle to the membrane plane. We suggest that a key difference between LH1 and LH2 is that the beta subunit is more bent in LH1. This modification puts the N terminus of LH1beta close to the reaction centre H subunit, and provides a rationale for the different ring sizes of LH1 and LH2 complexes.

Effect of harvesting protocol on performance of a hollow fiber bioreactor.

In this study, a bioreactor subject to Starling flow in closed shell batch harvest mode was compared to two forms of additional forced extracapillary (EC) space convection including EC circulation and EC cycling. Despite the presence of Starling flow as the dominant EC convection mechanism in the batch harvest system, the bioreactor start up was fairly good. However, the antibody productivity of the batch harvest system fell off rapidly after day 20 resulting in only 4.5 g of antibody produced. EC circulation with flow parallel to the fibers had a slightly better start up than the batch harvest. However, the antibody productivity also dropped after day 20 with EC circulation, resulting in only 7.5 g of antibody produced. EC cycling with flow both parallel and perpendicular to the fibers resulted in a start up similar to that of EC circulation. However, in contrast to the other two systems, antibody productivity in the EC cycling system was stable over the 60-day experiment resulting in the production of 23 g of antibody. These results demonstrate the importance of inducing the proper flow distribution in the EC space to allow consistent and stable production in hollow fiber bioreactors.

A light-harvesting antenna protein retains its folded conformation in the absence of protein-lipid and protein-pigment interactions.

The first study by nmr of the integral membrane protein, the bacterial light-harvesting (LH) antenna protein LH1 beta, is reported. The photosynthetic apparatus of purple bacteria contains two different kinds of antenna complexes (LH1 and LH2), which consist of two small integral membrane proteins alpha and beta, each of approximately 6 kDa, and bacteriochlorophyll and carotenoid pigments. We have purified the antenna polypeptide LH1 beta from Rhodobacter sphaeroides, and have recorded CD spectra and a series of two-dimensional nmr spectra. A comparison of CD spectra of LH1 beta observed in organic solvents and detergent micelles shows that the helical character of the peptide does not change appreciably between the two milieus. A significantly high-field shifted methyl signal was observed both in organic solvents and in detergent micelles, implying that a similar three-dimensional structure is present in each case. However, the 1H-nmr signals observed in organic solvents had a narrower line width and better resolution, and it is shown that in this case organic solvents provide a better medium for nmr studies than detergent micelles. A sequential assignment has been carried out on the C-terminal transmembrane region, which is the region in which the pigment is bound. The region is shown to have a helical structure by the chemical shift values of the alpha-CH protons and the presence of nuclear Overhauser effects characteristic of helices. An analysis of the amide proton chemical shifts of the residues surrounding the histidine chlorophyll ligand suggests that the local structure is well ordered even in the absence of protein-lipid and protein-pigment interactions. Its structure was determined from 348 nmr-derived constraints by using distance geometry calculations. The polypeptide contains an alpha-helix extending from Leu19 (position of cytoplasmic surface) to Trp44 (position of periplasmic surface). The helix is bent, as expected from the amide proton chemical shifts, and it is similar to the polypeptide fold of the previously determined crystal structure of Rhodopseudomonas acidophila Ac10050 LH2 beta (S. M. Prince et al., Journal of Molecular Biology, 1997, Vol. 268, pp. 412-423). It is concluded that the polypeptide conformation of this region may facilitate assembly of the LH complex.

Exposure of wild waterfowl to Mycoplasma anatis.

We developed an ELISA procedure to assess the presence of M. anatis-specific serum antibody in ducks. Sera from exposed and unexposed Pekin ducks (Anas platyrhynchos) were used to standardize the ELISA and to establish reference ranges to classify ELISA results as exposed or not exposed. We conducted serological surveys of female waterfowl in the central and eastern United States between 1988 and 1992 to assess the frequency of exposure in wild waterfowl. Adult breeding mallards (Anas platyrhynchos), wintering mallards, and black ducks (Anas rubripes) had high prevalences of exposure to M. anatis (25% to > 80%). In comparison, none of the breeding adult canvasbacks (Aythya valisineria) had serum antibody levels indicating exposure. Approximately 50% of the juvenile mallards and black ducks were exposed to M. anatis by 8 months of age, indicating high transmission rates among wild birds.

The occurrence of mycoplasmas in selected wild North American waterfowl.

We determined the prevalence of mycoplasma infection in breeding mallard (Anas platyrhynchos) and canvasback (Aythya valisineria) hens and their broods from the central United States (1988 to 1990); and wintering American black duck (Anas rubripes) and mallard hens from the eastern United States (1990 to 1993). Mycoplasmas were isolated by culturing tracheal swabs from 656 live birds and tissue samples from 112 dead waterfowl. Nine (18%) of 51 mycoplasma isolates were identified as Mycoplasma anatis; M. anatis was recovered from four mallards, a black duck, and a gadwall (Anas strepera) duckling. Nineteen (37%) of 51 mycoplasma isolates were identified as Mycoplasma cloacale; these isolates were obtained from mallard, canvasback, and black duck adults, and from a mallard duckling. Additional unspeciated mycoplasmas were isolated from mallards, black ducks, and one canvasback.

Diseases, parasites and survival of coyotes in south-central Georgia.

Serologic testing, radio-telemetry and post-mortem diagnostic evaluations were used to investigate survival and causes of mortality among 17 coyotes (Canis latrans) in south-central Georgia (USA). Prevalence of canine heartworm (Dirofilaria immitis) microfilariae was lower (P = 0.057) among fall-captured (22%) than among winter-captured (75%) coyotes. Prevalence of heartworm was higher among adults than juveniles in the fall, but no significant difference was detected between animals captured in winter. Antibodies were found against canine parvovirus (65%), canine parainfluenza virus (59%), infectious canine hepatitis virus (41%), and Toxoplasma gondii (18%). Antibodies were not found to Brucella canis, canine coronavirus, five serovars of Leptospira interrogans, or canine distemper virus. Seroprevalence of canine parvovirus was lower (P = 0.009) among fall-captured animals (33%) than winter-captured animals (100%). The Kaplan-Meier estimate of annual survival was 0.500 for all animals. Juvenile survival did not differ (P = 0.79) from adult survival, but male survival (S = 0.217) was lower (P = 0.11) than female survival (S = 0.804). Two of nine (22%) mortalities were human-caused, one was due to concurrent canine parvovirus and canine distemper virus infections, one animal died of trauma, two were considered natural mortalities of unknown cause, and no cause of death could be determined for the remaining three animals. Natural mortality may be significant for coyotes in south-central Georgia, although there was no apparent link between exposure to pathogens and the animals' subsequent fate in our small sample.