5'-Nor-3-Deaza-1',6'-Isoneplanocin, the Synthesis and Antiviral Study.

The arbocyclic nucleosides aristeromycin and neplanocin have been studied as a source for new antiviral agents. A convenient synthesis of C-5'-truncated 3-deaza-1',6'-isoneplanocin, which combines the features of antiviral candidates 5'-noraristeromycin and 3-deaza-1',6'-isoneplanocin is reported from (-)-cyclopentenone to give the two C-4' epimers of 5'-nor-3-deaza isoneplanocin. Antiviral assays showed activity against the JC virus (EC50 = 1.12 µM for (4'R)-8; EC50 = 59.14 µM for (4'S)-7) and inactivity of both compounds against several DNA and RNA viruses. Both compounds lacked cytotoxicity.

Towards a Multivariate Biomarker-Based Diagnosis of Autism Spectrum Disorder: Review and Discussion of Recent Advancements.

An ever-evolving understanding of autism spectrum disorder (ASD) pathophysiology necessitates that diagnostic standards also evolve from being observation-based to include quantifiable clinical measurements. The multisystem nature of ASD motivates the use of multivariate methods of statistical analysis over common univariate approaches for discovering clinical biomarkers relevant to this goal. In addition to characterization of important behavioral patterns for improving current diagnostic instruments, multivariate analyses to date have allowed for thorough investigation of neuroimaging-based, genetic, and metabolic abnormalities in individuals with ASD. This review highlights current research using multivariate statistical analyses to quantify the value of these behavioral and physiological markers for ASD diagnosis. A detailed discussion of a blood-based diagnostic test for ASD using specific metabolite concentrations is also provided. The advancement of ASD biomarker research promises to provide earlier and more accurate diagnoses of the disorder.

Representing Whom? U.K. Health Consumer and Patients' Organizations in the Policy Process.

This paper draws on nearly two decades of research on health consumer and patients' organizations (HCPOs) in the United Kingdom. In particular, it addresses questions of representation and legitimacy in the health policy process. HCPOs claim to represent the collective interests of patients and others such as relatives and carers. At times they also make claims to represent the wider public interest. Employing Pitkin's classic typology of formalistic, descriptive, symbolic, and substantive representation, the paper explores how and in what sense HCPOs represent their constituencies. We found that policymakers themselves are less concerned with formal mechanisms adopted by groups and are more concerned with credibility, in particular whether HCPOs carry the confidence of their constituents. While some concerns about legitimacy remain, particularly in relation to funding from commercial interests, we argue that HCPOs bring a unique perspective to the policy process and to focus purely on formalistic representation provides only a partial understanding of their representative role and a constrained view of their collective moral claims.

Prevention better than cure? Health consumer and patients' organisations and public health.

Previous studies of groups representing patients, users and carers in the UK indicate that their principal focus is on health care and treatment services. In recent years, UK government policy has emphasised prevention and health promotion as part of its wider public health agenda. This paper investigates how this might have affected health consumer and patients' organisations (HCPOs) by presenting findings from an online survey of 312 UK groups undertaken in the summer of 2010. The sample was identified using the publicly available membership lists of a number of large alliance organisations. The survey achieved a 39% response rate. The findings suggest that the main focus of lobbying and campaigning remains on health care rather than public health issues. However, a significant minority of groups stated they prioritised public health campaigning and lobbying. Possible explanations for engagement with the public health agenda include: the presence of public health on the government's agenda, the influence of other, more powerful interests in the health policy process, and growing awareness among groups of health promotion, prevention and environmental causes of illness. The paper argues that although HCPOs may be more active in the public health policy arena, more research is needed - notably with regard to their relationship with devolved governments, EU institutions and the current UK government's public health and voluntary sector agenda. The reasons why some groups engage more than others with the public health agenda also merit closer investigation. Moreover, not enough is currently known about the influence of HCPOs on the public health agenda and policies in this field.

Borrelia burgdorferi RST1 (OspC type A) genotype is associated with greater inflammation and more severe Lyme disease.

Evidence is emerging for differential pathogenicity among Borrelia burgdorferi genotypes in the United States. By using two linked genotyping systems, ribosomal RNA intergenic spacer type (RST) and outer surface protein C (OspC), we studied the inflammatory potential of B. burgdorferi genotypes in cells and patients with erythema migrans or Lyme arthritis. When macrophages were stimulated with 10 isolates of each RST1, RST2, or RST3 strain, RST1 (OspC type A)-stimulated cells expressed significantly higher levels of IL-6, IL-8, chemokine ligand (CCL) 3, CCL4, tumor necrosis factor, and IL-1ß, factors associated with innate immune responses. In peripheral blood mononuclear cells, RST1 strains again stimulated significantly higher levels of these mediators. Moreover, compared with RST2, RST1 isolates induced significantly more interferon (IFN)-α, IFN-γ, and CXCL10, which are needed for adaptive immune responses; however, OspC type I (RST3) approached RST1 (OspC type A) in stimulating these adaptive immune mediators. Similarly, serum samples from patients with erythema migrans who were infected with the RST1 genotype had significantly higher levels of almost all of these mediators, including exceptionally high levels of IFN-γ-inducible chemokines, CCL2, CXCL9, and CXCL10; and this pronounced inflammatory response was associated with more symptomatic infection. Differences among genotypes were not as great in patients with Lyme arthritis, but those infected with RST1 strains more often had antibiotic-refractory arthritis. Thus, the B. burgdorferi RST1 (OspC type A) genotype, followed by the RST3 (OspC type I) genotype, causes greater inflammation and more severe disease, establishing a link between spirochetal virulence and host inflammation.

Analysis of Borrelia burgdorferi genotypes in patients with Lyme arthritis: High frequency of ribosomal RNA intergenic spacer type 1 strains in antibiotic-refractory arthritis.

OBJECTIVE: Most of the Borrelia burgdorferi genotypes have been isolated from erythema migrans (EM) skin lesions in patients with Lyme disease. OspC type K strains, which are 16S-23S ribosomal RNA intergenic spacer type 2 (RST2) strains, are most commonly recovered, but a higher percentage of OspC type A strains (RST1), the next most commonly recovered type, is detectable in blood. The goal of this study was to determine the B burgdorferi genotypes in the joints of patients with Lyme arthritis. METHODS: Joint fluid samples from 124 patients seen over a 30-year period were analyzed for OspC types by semi-nested polymerase chain reaction (PCR) and sequencing, and for RSTs by nested PCR and restriction fragment length polymorphism analysis. These results were correlated with clinical outcome. RESULTS: OspC and RST genotypes were identified in 49 of the 124 joint fluid samples (40%). In these 49 samples, OspC type K strains (RST2) were identified in 21 samples (43%), OspC type A strains (RST1) were identified in 11 samples (22%), and 8 other OspC types and all 3 RSTs were identified among the remaining 17 samples (35%). However, among the 17 patients who had been treated with antibiotics according to current guidelines, all 7 patients who were infected with RST1 strains had antibiotic-refractory arthritis, compared with 4 of 6 patients infected with RST2 strains and only 1 of 4 infected with RST3 strains (P = 0.03). CONCLUSION: Most of the B burgdorferi genotypes, particularly OspC type K (RST2), were identified in the joint fluid of patients with Lyme arthritis, and the genotype frequencies found in joints reflected those in EM skin lesions. However, RST1 strains were most frequent in patients with antibiotic-refractory arthritis. Our results help to further the understanding of the differential pathogenicity of strains of B burgdorferi.

Strong IgG antibody responses to Borrelia burgdorferi glycolipids in patients with Lyme arthritis, a late manifestation of the infection.

In this study, the membrane lipids of B. burgdorferi were separated into 16 fractions; the components in each fraction were identified, and the immunogenicity of each fraction was determined by ELISA using sera from Lyme disease patients. Only the 2 glycolipids, acylated cholesteryl galactoside (ACG, BbGL-I) and monogalactosyl diacylglycerol (MgalD, BbGL-II), were immunogenic. Early in the infection, 24 of 84 patients (29%) who were convalescent from erythema migrans and 19 of the 35 patients (54%) with neuroborreliosis had weak IgG responses to purified MgalD, and a smaller percentage of patients had early responses to synthetic ACG. However, almost all of 75 patients with Lyme arthritis, a late disease manifestation, had strong IgG reactivity with both glycolipids. Thus, almost all patients with Lyme arthritis have strong IgG antibody responses to B. burgdorferi glycolipid antigens.

Arthritogenicity of Borrelia burgdorferi and Borrelia garinii: comparison of infection in mice.

Arthritogenicity, as determined by joint swelling and synovial histology, was compared between or within two Borrelia genospecies that cause Lyme arthritis in humans. The spirochete burden in bladder tissue (a site of spirochete persistence) was documented by quantitative polymerase chain reaction, and immune responses were analyzed. In C3H/HeJ mice, three B. burgdorferi isolates and two of the three B. garinii isolates induced severe arthritis and swelling. Previous designation as invasive or noninvasive B. garinii, or RNA spacer type of B. burgdorferi did not determine arthritis severity induced by isolates. Compared with the other five isolates, the B. garinii PBi isolate induced significantly less arthritis, a lower humoral immune response, and persisted at a much lower level in bladder tissue. However, B. garinii PBi isolates induced similar Borrelia antigen-specific inflammatory T cell responses from the local draining lymph node. Thus, diverse B. burgdorferi and B. garinii isolates were highly arthritogenic in immune competent mice.

Higher mRNA levels of chemokines and cytokines associated with macrophage activation in erythema migrans skin lesions in patients from the United States than in patients from Austria with Lyme borreliosis.

BACKGROUND: Erythema migrans (EM) is caused primarily by Borrelia afzelii in Europe and solely by Borrelia burgdorferi in the United States. B. burgdorferi infection in the United States has previously been associated with faster expansion of EM lesions and with more associated symptoms, compared with B. afzelii infection in Europe. However, reasons for these differences are not yet known. METHODS: We determined the Borrelia species infecting 67 US or Austrian patients with EM. The clinical pictures and chemokine and cytokine mRNA levels in lesional skin were then compared in the 19 B. burgdorferi-infected US patients and the 37 B. afzelii-infected Austrian patients, the 2 largest groups. RESULTS: The 19 B. burgdorferi-infected US patients had faster-expanding EM lesions and a median of 4 associated signs and symptoms, whereas the 37 B. afzelii-infected Austrian patients had slower-expanding lesions and usually did not experience associated symptoms. Compared with the EM lesions of B. afzelii-infected Austrian patients, those of B. burgdorferi-infected US patients had significantly higher mRNA levels of chemokines associated with activation of macrophages, including chemoattractants for neutrophils (CXCL1), macrophages (CCL3 and CCL4), and T helper 1 cells (CXCL9, CXCL10, and CXCL11). In addition, compared with the EM lesions of Austrian patients, the EM lesions of US patients tended to have higher mRNA levels of the macrophage-associated proinflammatory cytokines interleukin 1beta and tumor necrosis factor alpha, and they had significantly higher mRNA expression of the antiinflammatory cytokines interleukin 10 and transforming growth factor beta. CONCLUSIONS: The EM lesions of B. burgdorferi-infected US patients expanded faster, were associated with more symptoms, and had higher mRNA levels of macrophage-associated chemokines and cytokines than did the EM lesions of B. afzelii-infected Austrian patients.

Long-term storage of DNA-free RNA for use in vaccine studies.

RNA replicons represent potential vaccine delivery vehicles, but are considered too unstable for such use. This study examined the recovery, integrity and function of in vitro transcribed replicon RNA encoding hepatitis C virus (HCV) proteins. To remove residual template DNA, the RNA was digested with TURBO DNase followed by RNeasy DNase set and purified through an RNeasy column. The RNA was freeze-dried in distilled water or trehalose, stored under nitrogen gas for up to 10 months and analyzed at different time points. The recovery of RNA stored at < or = 4 degrees C that was freeze-dried in distilled water varied between 66% to zero of that recovered from RNA freeze-dried in 10% trehalose, a figure that depended on the duration of storage. In contrast, the recovery of the RNA stored in trehalose was consistently high for all time points. After recovery, both RNAs were translationally competent and expressed high levels of proteins after transfection, although the level of expression from the trehalose-stored RNA was consistently higher. Thus the addition of trehalose permitted stable storage of functional RNA at 4 degrees C for up to 10 months and this permits the development of RNA vaccines, even in developing countries where only minimum storage conditions (e.g., 4 degrees C) can be achieved.

Chemokine signatures in the skin disorders of Lyme borreliosis in Europe: predominance of CXCL9 and CXCL10 in erythema migrans and acrodermatitis and CXCL13 in lymphocytoma.

The three skin disorders of Lyme borreliosis in Europe include erythema migrans, an acute, self-limited lesion; borrelial lymphocytoma, a subacute lesion; and acrodermatitis chronica atrophicans, a chronic lesion. Using quantitative reverse transcription-PCR, we determined mRNA expression of selected chemokines, cytokines, and leukocyte markers in skin samples from 100 patients with erythema migrans, borrelial lymphocytoma, or acrodermatitis chronica atrophicans and from 25 control subjects. Chemokine patterns in lesional skin in each of the three skin disorders included low but significant mRNA levels of the neutrophil chemoattractant CXCL1 and the dendritic cell chemoattractant CCL20 and intermediate levels of the macrophage chemoattractant CCL2. Erythema migrans and particularly acrodermatitis lesions had high mRNA expression of the T-cell-active chemokines CXCL9 and CXCL10 and low levels of the B-cell-active chemokine CXCL13, whereas lymphocytoma lesions had high levels of CXCL13 and lower levels of CXCL9 and CXCL10. This pattern of chemokine expression was consistent with leukocyte marker mRNA in lesional skin. Moreover, using immunohistologic methods, CD3(+) T cells and CXCL9 were visualized in erythema migrans and acrodermatitis lesions, and CD20(+) B cells and CXCL13 were seen in lymphocytoma lesions. Thus, erythema migrans and acrodermatitis chronica atrophicans have high levels of the T-cell-active chemokines CXCL9 and CXCL10, whereas borrelial lymphocytoma has high levels of the B-cell-active chemokine CXCL13.

Defining target antigens for CD25+ FOXP3 + IFN-gamma- regulatory T cells in chronic hepatitis C virus infection.

The mechanism behind the apparent lack of effective antiviral immune responses in chronic hepatitis C virus (HCV) patients is poorly understood. It remains unclear if natural regulatory T cells (Treg) contribute to the induction and maintenance of HCV persistence. We herein report for the first time that CD25(high)IFN-gamma(-)FOXP3(high) Tregs can be rapidly induced by culturing peripheral blood mononuclear cells (PBMCs) of HCV-positive patients with HCV protein-derived peptides. The HCV-specific Tregs, generally CD4(+)CD45RO(+), did not proliferate in response to HCV peptide and failed to produce interferon (IFN)-gamma, in distinct contrast to antiviral effector cells. Stimulation of healthy donor PBMCs with HCV peptides did not result in CD25 and FOXP3 upregulation above non-antigen background. To further investigate the antigen specificity of these potentially disease-associated natural Tregs, CD25(+) cells were isolated from PBMCs, labeled with carboxyfluorescein diacetate succinimidylester and added back to CD25-depleted PBMCs, and the co-cultures were then stimulated with individual peptides derived from the HCV core protein. We found that the actual peptide that can stimulate Treg varied between patients, but within any given subject only a small number of the peptides were able to stimulate Treg, suggesting the existence of dominant Treg epitopes. Although functional experiments for these peptides are ongoing in our laboratory, data presented here suggests that HCV-specific natural Tregs are abundant in infected individuals, in contrast to the extremely low frequency of anti-HCV effector T cells, supporting the view that natural Treg may be implicated in host immune tolerance during HCV infection.

Borrelia burgdorferi genetic markers and disseminated disease in patients with early Lyme disease.

Three genetic markers of Borrelia burgdorferi have been associated with disseminated disease: the OspC type, the 16S-23S rRNA intergenic spacer type (RST), and vlsE. Here, we modified previous methods so as to identify the three markers by PCR and restriction fragment length polymorphism in parallel, analyzed B. burgdorferi isolates from erythema migrans (EM) skin lesions in 91 patients, and correlated the results with evidence of dissemination. OspC type A was found approximately twice as frequently in patients with disseminated disease, whereas type K was identified approximately twice as often in those without evidence of dissemination, but these trends were not statistically significant. The remaining seven types identified were found nearly equally in patients with or without evidence of dissemination. RST 1 strains were significantly associated with dissemination (P=0.03), whereas RST 2 and RST 3 strains tended to have an inverse association with this outcome. The vlsE gene was identified in all 91 cases, using primer sets specific for an N-terminal sequence of B. burgdorferi strain B31 (vlsEB31) or strain 297 (vlsE297), but neither marker was associated with dissemination. Specific combinations of the three genetic markers usually occurred together. OspC type A was always found with RST 1 and vlsEB31, type K was always identified with RST 2 and more often with vlsE297, and types E and I were almost always found with RST 3 and equally often with vlsEB31 and vlsE297. We conclude that B. burgdorferi strains vary in their capacity to disseminate, but almost all strains isolated from EM lesions sometimes caused disseminated disease.

RNA loading of leukemic antigens into cord blood-derived dendritic cells for immunotherapy.

The manipulation of dendritic cells (DCs) ex vivo to present tumor-associated antigens for the activation and expansion of tumor-specific cytotoxic T lymphocytes (CTLs) attempts to exploit these cells' pivotal role in immunity. However, significant improvements are needed if this approach is to have wider clinical application. We optimized a gene delivery protocol via electroporation for cord blood (CB) CD34(+) DCs using in vitro-transcribed (IVT) mRNA. We achieved > 90% transfection of DCs with IVT-enhanced green fluorescent protein mRNA with > 90% viability. Electroporation of IVT-mRNA up-regulated DC costimulatory molecules. DC processing and presentation of mRNA-encoded proteins, as major histocompatibility complex/peptide complexes, was established by CTL assays using transfected DCs as targets. Along with this, we also generated specific antileukemic CTLs using DCs electroporated with total RNA from the Nalm-6 leukemic cell line and an acute lymphocytic leukemia xenograft. This significant improvement in DC transfection represents an important step forward in the development of immunotherapy protocols for the treatment of malignancy.

Prospects for dendritic cell vaccination in persistent infection with hepatitis C virus.

Although hepatitis C virus (HCV) is classified in the Hepacivirus genus in the family Flaviviridae, it is unlike most of the other members of this family due to its propensity to cause persistent infections. This persistent infection eventually results in chronic liver disease, cirrhosis and hepatocellular carcinoma in a proportion of infected individuals. It has been difficult to examine correlates of clearance or persistence because most acute phase HCV infections are subclinical or result in symptoms which are non-specific; consequently, acute infections are not generally recognised and patients often present many years later with persistent infection and accompanying chronic liver disease. Nevertheless, seminal studies, performed during the acute phase, have identified a number of factors which are likely to influence the outcome of infection, although it is possible that the mechanism is multifactorial. One of these factors is impairment of dendritic cell function by a mechanism resulting from expression of an HCV protein(s) in these cells. This may be a major factor in the failure of the immune response to expand after HCV infection, leading to persistence. Nevertheless, it may be possible to overcome this defect by autologous transfusion of HCV antigen-loaded, mature dendritic cells and the purpose of this review is to highlight the need and general approaches for developing dendritic cell-based immunotherapy for HCV infection.

A ferredoxin from the thermohalophilic bacterium Rhodothermus marinus.

A [3Fe-4S](1+/0) ferredoxin was isolated from the thermohalophilic and strict aerobic bacterium Rhodothermus marinus. It is a small protein, with an apparent molecular mass of 9 kDa. Its N-terminal amino acid sequence reveals the capability of binding two tetranuclear clusters. However, upon purification, it contains a single [3Fe-4S](1+/0), with an unusually low reduction potential of -650 mV, determined by cyclic voltammetry at pH 7.6. [1H]NMR spectroscopy shows that the protein contains a single, homogeneous, trinuclear centre. When purified under anaerobic conditions, the EPR [3Fe-4S](1+/0) centre signal is also observed. However, it can now be reduced by dithionite and a new signal attributed to a [4Fe-4S](2+/1+) cluster develops. This can also be observed upon reconstitution of the prosthetic groups. The function of this ferredoxin in R. marinus is still unknown but it is very sensitive to oxygen, an unexpected characteristic for a protein from an aerobic organism. The thermodynamic stability of the R. marinus ferredoxin was also investigated and was shown to be high. Thermal and chemical unfolding reactions appear as single, cooperative transitions. The midpoint (T(m)) for thermally induced unfolding is 102+/-2 degrees C (pH 7). Unfolding induced by the chemical denaturant guanidine hydrochloride (GuHCl) shows a transition midpoint at 5.0 M GuHCl (pH 7.0, 20 degrees C). The iron-sulfur cluster degrades upon polypeptide unfolding, resulting in an irreversible denaturation process.