Novel paramyxovirus associated with severe acute febrile disease, South Sudan and Uganda, 2012.

In 2012, a female wildlife biologist experienced fever, malaise, headache, generalized myalgia and arthralgia, neck stiffness, and a sore throat shortly after returning to the United States from a 6-week field expedition to South Sudan and Uganda. She was hospitalized, after which a maculopapular rash developed and became confluent. When the patient was discharged from the hospital on day 14, arthralgia and myalgia had improved, oropharynx ulcerations had healed, the rash had resolved without desquamation, and blood counts and hepatic enzyme levels were returning to reference levels. After several known suspect pathogens were ruled out as the cause of her illness, deep sequencing and metagenomics analysis revealed a novel paramyxovirus related to rubula-like viruses isolated from fruit bats.

Advance Care Planning: A Story of Trust Within the Family.

As the family usually plays a central role at the end of life, the quality of family relationships may influence how individuals approach advance care planning (ACP). Our study investigates the associations of trust in relatives with regard to end-of-life (EOL) issues-used as a proxy measure of family relationship quality-with individuals' engagement in EOL discussions, advance directive (AD) awareness, approval and completion, and designation of a healthcare proxy. Using nationally representative data of adults aged 55 years and over from wave 6 (2015) of the Survey of Health, Ageing, and Retirement in Europe (SHARE) in Switzerland (n = 1911), we show that complete trust in relatives is related to higher engagement in ACP. Subject to patient consent, the family should, therefore, be included in the ACP process, as such practice could enhance patient-centered EOL care and quality of life at the end of life.

Toll-like receptor 4 differentially regulates epidermal growth factor-related growth factors in response to intestinal mucosal injury.

Epiregulin (EPI) and amphiregulin (AR) are epidermal growth factor receptor (EGFR) ligands implicated in mucosal repair and tumorigenesis. We have shown that Toll-like receptor 4 (TLR4) induces intestinal epithelial cell (IEC) proliferation by activating EGFR through AR expression. We examined whether TLR4 differentially regulates expression of EGFR ligands in response to mucosal injury. The human IEC line SW480 was examined expression of EGFR ligands, EGFR phosphorylation, and proliferation in response to lipopolysaccharide (LPS). Small-interfering RNA (siRNA) was used to block TLR4. Neutralizing antibodies to EGFR ligands were used to examine inhibition of LPS-dependent EGFR activation. Acute colitis and recovery were examined in the mice given 2.5% dextran sodium sulfate (DSS). Colonic secretion of EPI and AR was analyzed by enzyme-linked immunosorbent assay. LPS selectively induces EPI and AR but not other EGFR ligands. LPS induced early EPI mRNA expression between 30 min and 24 h. The neutralizing antibodies to EPI and AR prevented activation of EGFR by LPS. LPS induces IEC proliferation (200%, P=0.01) in 24 h but blocking EPI and AR significantly decreased proliferation. In vivo, mucosal EPI and AR expression are significantly decreased in TLR4(-/-) mice (P=0.02) compared to wild-type mice during acute colitis. EPI and AR exhibit different kinetics in response to mucosal damage: EPI expression is upregulated acutely at day 7 of DSS, but falls during recovery at day 14. By contrast, a sustained upregulation of AR expression is seen during mucosal injury and repair. We show that TLR4 regulates EPI and AR expression and that both these EGFR ligands are necessary for optimal proliferation of IEC. The diverse kinetics of EPI and AR expression suggest that they function in distinct roles with respect to acute injury vs repair. Our results highlight the role of bacterial sensing for IEC homeostasis and may lead to targeted therapy for mucosal healing and prevention of tumorigenesis.

Molecular characteristics of antibodies bearing an anti-DNA-associated idiotype.

Anti-double-stranded DNA antibodies are the hallmark of the disease systemic lupus erythematosus and are believed to contribute to pathogenesis. While a large number of anti-DNA antibodies from mice with lupus-like syndromes have been characterized and their variable region genes sequenced, few human anti-DNA antibodies have been reported. We describe here the variable region gene sequences of eight antibodies produced by Epstein-Barr virus (EBV)-transformed B cells that bear the 3I idiotype, an idiotype expressed on anti-DNA antibodies and present in high titer in patients with systemic lupus. The comparison of these antibodies to the light chains of 3I+ myeloma proteins and serum antibodies reveals that EBV transformation yields B cells producing antibodies representative of the expressed antibody repertoire. The analysis of nucleotide and amino acid sequences of these antibodies suggests the first complementarity determining region of the light chain may be important in DNA binding and that paradigms previously generated to account for DNA binding require modification. The understanding of the molecular genetics of the anti-DNA response requires a more complete description of the immunoglobulin germ line repertoire, but data reported here suggest that somatic diversification is a characteristic of the anti-DNA response.

The role of prostaglandin E2 (PGE 2) in toll-like receptor 4 (TLR4)-mediated colitis-associated neoplasia.

BACKGROUND: We have previously found that TLR4-deficient (TLR4-/-) mice demonstrate decreased expression of mucosal PGE 2 and are protected against colitis-associated neoplasia. However, it is still unclear whether PGE 2 is the central factor downstream of TLR4 signaling that promotes intestinal tumorigenesis. To further elucidate critical downstream pathways involving TLR4-mediated intestinal tumorigenesis, we examined the effects of exogenously administered PGE 2 in TLR4-/- mice to see if PGE 2 bypasses the protection from colitis-associated tumorigenesis. METHOD: Mouse colitis-associated neoplasia was induced by azoxymethane (AOM) injection followed by two cycles of dextran sodium sulfate (DSS) treatment. Two different doses of PGE 2 (high dose group, 200 microg, n = 8; and low dose group, 100 microg, n = 6) were administered daily during recovery period of colitis by gavage feeding. Another group was given PGE 2 during DSS treatment (200 microg, n = 5). Inflammation and dysplasia were assessed histologically. Mucosal Cox-2 and amphiregulin (AR) expression, prostanoid synthesis, and EGFR activation were analyzed. RESULTS: In control mice treated with PBS, the average number of tumors was greater in WT mice (n = 13) than in TLR4-/- mice (n = 7). High dose but not low dose PGE 2 treatment caused an increase in epithelial proliferation. 28.6% of PBS-treated TLR4-/- mice developed dysplasia (tumors/animal: 0.4 +/- 0.2). By contrast, 75.0% (tumors/animal: 1.5 +/- 1.2, P < 0.05) of the high dose group and 33.3% (tumors/animal: 0.3 +/- 0.5) of the low dose group developed dysplasia in TLR4-/- mice. Tumor size was also increased by high dose PGE 2 treatment. Endogenous prostanoid synthesis was differentially affected by PGE 2 treatment during acute and recovery phases of colitis. Exogenous administration of PGE 2 increased colitis-associated tumorigenesis but this only occurred during the recovery phase. Lastly, PGE 2 treatment increased mucosal expression of AR and Cox-2, thus inducing EGFR activation and forming a positive feedback mechanism to amplify mucosal Cox-2. CONCLUSIONS: These results highlight the importance of PGE 2 as a central downstream molecule involving TLR4-mediated intestinal tumorigenesis.

Molecular analysis of the human immunoglobulin V lambda II gene family.

The 8.12 idiotype characterizes a subpopulation of anti-DNA antibodies in patients with systemic lupus erythematosus (SLE). The idiotype is present on lambda light chains and has previously been shown to be exclusively encoded by V lambda II light chains. RFLP analysis of the V lambda II gene family has shown the family to consist of 10 to 15 members. Thus far, the sequences of seven V lambda II germline genes are reported in the literature with one of these a pseudogene. To identify the V lambda II genes that encode 8.12 positive antibodies and to further characterize the V lambda II family, germline V lambda II clones were derived from a patient with SLE. Two libraries were constructed: a genomic DNA library and a library of PCR-derived V lambda II gene products obtained using a conserved V lambda II leader region primer and a primer for the nonamer region 3' of the coding sequence. We now describe seven new germline genes, two of which are pseudogenes. Comparison of V lambda II germline genes to sequences of 8.12 positive light chains produced by EBV-transformed B cell lines show that all 8.12 positive light chains are encoded by a limited number of highly homologous members of the V lambda II family. 8.12 negative V lambda II encoded light chains also derive from a limited number of V lambda II genes, suggesting that only a subset of the apparently available V lambda II genes are commonly expressed.

Lupus-specific antibodies reveal an altered pattern of somatic mutation.

The F4 idiotype is a heavy chain determinant expressed almost exclusively on IgG immunoglobulins and is highly associated with specificity for double-stranded DNA. Since high-titered F4 expression is present predominantly in sera of patients with systemic lupus erythematosus (SLE), we thought F4+ IgG antibodies might constitute a useful subset of immunoglobulins in which to investigate lupus-specific alterations in variable (V) region gene expression or in the process of somatic mutation. This molecular analysis of F4+ B cell lines generated from lupus patients demonstrates that despite the strong association of F4 reactivity with specificity for native DNA, there is no apparent VH gene restriction. Furthermore, VH gene segments encoding these antibodies are also used in protective immune responses. An examination of the process of somatic mutation in F4+ antibodies showed no abnormality in frequency of somatic mutation nor in the distribution of mutations in complementarity-determining regions or framework regions. However, there was a decrease in targeting of mutations to putative mutational hot spots. This subtle difference in mutations present in these antibodies may reflect an intrinsic defect in mutational machinery or, more likely, altered state of B cell activation that affects the mutational process and perhaps also negative selection.

Molecular characterization of a somatically mutated anti-DNA antibody bearing two systemic lupus erythematosus-related idiotypes.

We report the molecular characterization of 2A4, an IgG, DNA-binding antibody bearing the 3I and F4 idiotypes which are associated with anti-DNA antibodies in serum of patients with systemic lupus erythematosus (SLE). The antibody is produced by an EBV-transformed B cell line derived from a patient with multiple myeloma whose myeloma protein is also an IgG, 3I-reactive, F4-reactive, DNA-binding immunoglobulin, although the 2A4 antibody does not itself represent the myeloma protein. The 2A4 heavy chain is encoded by a VH4 gene, a D-D gene fusion and the JH6 gene; the light chain is derived from a Vk1 gene and the Jk2 gene. This is the first human antibody shown to have a CDR3 encoded by a D-D fusion. DNA sequence analysis of the 2A4 VH gene together with a Southern blot of genomic DNA probed with a 2A4 VH-specific oligonucleotide strongly suggest it to be somatically mutated. The data provide evidence that human autoantibodies can be products of somatically mutated genes and suggest that the 2A4 antibody may reflect the selective pressure of antigen.

High frequency of autoantibodies bearing cross-reactive idiotopes among hybridomas using VH7183 genes prepared from normal and autoimmune murine strains.

Hybridomas obtained by in vitro stimulation with lipopolysaccharides (LPS) of BALB/c, MRL/lpr, and NZB splenocytes were selected for expression of VH7183 by hybridization using slot blotting. Northern blot analysis showed that the majority of hybrids produce a full length message complementary to the VH7183 probe. The frequency of VH7183 hybridomas was significantly higher in NZB mice as compared with BALB/c mice. Using multiple binding assays, 60% of the total antibodies encoded by VH7183 were specific for self-epitopes. Finally, the vast majority express cross-reactive idiotypes borne by autoantibodies of various specificities.

Generation and analysis of clonal IgM- and IgG-producing human B cell lines expressing an anti-DNA-associated idiotype.

This study describes a methodology for generating stable, cloned, EBV-transformed IgG- and IgM-producing human B cell lines. Using these lines we have characterized immunoglobulin V gene utilization in an anti-DNA-associated idiotypic system. The 31 anti-DNA-associated idiotype is encoded preferentially by the VK1 gene family, and, in all probability, reflects a germ line gene-encoded framework determinant. Analysis of these lines indicates that the DNA-binding antibodies produced by B cell lines from SLE patients may differ from DNA binding myeloma proteins and from natural autoantibodies.

Shared idiotypes and restricted immunoglobulin variable region heavy chain genes characterize murine autoantibodies of various specificities.

The study of the Ig variable region heavy chain (VH) genes used to encode antibodies specific for self-epitopes from murine hybridomas showed that three VH families are primarily utilized: VH J558, the largest family, and VH QPC52 and VH 7183, the families most proximal to the Ig joining region heavy chain genes. These monoclonal autoantibodies express cross-reactive idiotopes shared by rheumatoid factors and antibodies specific for Sm. The expression of these idiotypes is independent of major histocompatibility complex and Ig constant region heavy chain haplotypes, self-antigen specificity, and even the VH gene family utilized. Though the experiments described here are limited to murine autoantibodies, similarities exist between murine and human autoimmune diseases. Studies that aim to investigate the relationship between VH gene expression and the presence of cross-reactive idiotypes among human autoantibodies should enable us to better understand the mechanisms of autoimmunity and self-tolerance.

Assessing Student Satisfaction with Face-to-Face Synchronous Distance Education in a Dental Hygiene Program.

As universities and colleges seek to reach more students in efficient ways, the use of synchronous distance education (SDE) can be an alternative to traditional classrooms. This study focused on face-to-face SDE, in which classrooms equipped with interactive synchronous technologies allow students in both classrooms and the professor to synchronously see and hear one another. The aims of the study were to aid educators in understanding student concerns, determine whether face-to face SDE was sacrificing overall student satisfaction, and investigate whether satisfaction improved as the program matured. This mixed-methods study utilized a convenience sample of two cohorts of dental hygiene students (n=122) in one program: Cohort 1, which graduated in 2014 as the first class to experience face-to-face SDE; and Cohort 2, which graduated in 2015. The response rate for the two cohorts was 95%. Perceptions of face-to-face SDE versus traditional classroom experiences and characteristics of face-to-face SDE were measured using pre- and post-program surveys. The results showed no difference in student perceptions and expectations pre-course vs. post-course, although Cohort 2 had a more positive perception of SDE than did Cohort 1 (p<0.001). Perceptions of characteristics related to the classroom setting and instructor satisfaction were overall positive (p<0.001). The qualitative data suggested that technological support and faculty familiarity with SDE were substantial influences on students' satisfaction. Overall, there was no significant difference in satisfaction with face-to-face SDE when students compared it to their previous classroom experiences.

[French recommendations on control measures to reduce the infectious risk in immunocompromised patients]. / Quelles mesures pour maîtriser le risque infectieux chez les patients immunodéprimés ? Recommandations formalisées d'experts.

The increase use of immunosuppressive treatments in patients with solid cancer and/or inflammatory diseases requires revisiting our practices for the prevention of infectious risk in the care setting. A review of the literature by a multidisciplinary working group at the beginning of 2014 wished to answer the following 4 questions to improve healthcare immunocompromised patients: (I) How can we define immunocompromised patients with high, intermediate and low infectious risk, (II) which air treatment should be recommended for this specific population? (III) What additional precautions should be recommended for immunocompromised patients at risk for infection? (IV) Which global environmental control should be recommended? Based on data from the literature and using the GRADE method, we propose 15 recommendations that could help to reduce the risk of infection in these exposed populations.

Pathogenic anti-DNA antibodies in SLE: idiotypic families and genetic origins.

We have adopted an idiotypic approach to study the double stranded DNA (dsDNA) binding antibodies of systemic lupus erythematosus (SLE). Three anti-idiotypic reagents, 8.12, 3I, and F4, identify cross reactive idiotypes that are each expressed on anti-dsDNA antibodies in the sera of many patients with SLE. These idiotypic antibodies are implicated in the pathogenesis of SLE as they are present in immune complex deposits in the kidneys of patients with SLE glomerulonephritis. The autoantibody associated idiotypes are also expressed on antibodies that do not bind DNA. We are investigating the origin of the pathogenic anti-dsDNA antibodies of SLE by comparing the autoantibodies, the antibodies to foreign antigens, and the myeloma proteins that express each SLE associated idiotype. In conjunction with serological analysis of these idiotypic systems, molecular genetic studies indicate that both the 8.12 and the 3I autoantibody associated idiotypes may be germline encoded, while the F4 idiotype is generated by somatic mutation. The data further suggest that the antigenic specificity of the pathogenic anti-DNA antibodies of SLE is acquired through somatic mutation of germline immunoglobulin genes. By studying the regulation of genes capable of encoding pathogenic autoantibodies, in both SLE patients and non-autoimmune individuals, we may be able to elucidate the pathogenesis of autoimmune disease and begin to design more effective therapeutic interventions.

Analysis of the V kappa I family: germline genes from an SLE patient and expressed autoantibodies.

Our studies of anti-DNA antibodies in systemic lupus erythematosus have demonstrated a preferential use of the V kappa I family to encode light chains of antibodies that express the anti-DNA associated 3I idiotype. This idiotype is present on a high percentage of anti-DNA antibodies in approximately 80% of SLE patients1,2. In this study, we employed PCR to obtain V kappa I germline genes from a lupus patient in order to address the following questions: Do the V kappa I germline genes of an individual with autoimmune disease differ from those of healthy individuals? What V kappa I genes are used to encode autoantibodies and are they used to encode protective antibodies also? Does the V kappa I gene family display peculiarities in V gene segment rearrangement or somatic mutation? Our analysis shows that the coding region sequences of germline genes of an autoimmune individual are highly homologous to those of non-autoimmune individuals. In addition, the same germline genes can be utilized to encode antibodies to both exogenous and self antigens. While rearranged V kappa genes are ordinarily derived from the J kappa proximal region of the V kappa locus, V kappa I genes encoding autoantibodies derive primarily from the J kappa distal region. It is not yet clear if this applies equally to V kappa I encoded antibodies directed to foreign antigen.

Analysis of V kappa I and V lambda II light chain genes in the expressed B-cell repertoire.

Our previous studies of anti-DNA antibodies in SLE have demonstrated a preferential use of V kappa I and V lambda II gene families to encode light chains of antibodies that express the anti-DNA-associated 3I and 8.12 idiotypes, respectively. In this study, we employed PCR to obtain V kappa I and V lambda II germline genes from lupus patients in order to compare the germline genes to genes encoding expressed V kappa I and V lambda II light chains and to analyze the extent of somatic mutation among autoantibodies that derive from these light chain families. Our analysis shows that the germline repertoire among all persons (autoimmune and healthy) is comparable and that somatic mutation is used to diversify autoantibodies as well as anti-microbial antibodies. We have observed that autoantibodies encoded by V kappa I and V lambda II genes have a higher number of amino acid replacements in CDRs than autoantibodies encoded by other VL gene families. In addition, there may be subtle differences in V gene usage that distinguish the V kappa I-encoded light chains from other expressed V kappa light chains.

Possible mechanisms of autoantibody production.

Recent advances in the understanding of the ontogeny of the normal B cell response and of the molecular mechanisms that are used to generate a diverse B cell repertoire have resulted in new approaches to the study of autoimmune diseases. B cell lines with autoantibody specificity can easily be generated from normal individuals. These low affinity and generally polyspecific "natural autoantibodies" have features of a B cell response prior to antigenic stimulation and are encoded by germline or relatively unmutated genes. Pathogenic autoantibodies from autoimmune individuals on the other hand, appear to be higher affinity antibodies that have features of an antigen selected response. The relationship between these two different classes of autoantibodies remains to be determined. Our studies of anti-DNA antibodies in human SLE have revealed that anti-DNA antibodies from unrelated patients share dominant cross-reactive idiotypes. Analysis of monoclonal anti-DNA antibodies bearing two SLE related idiotypes, 3I and F4, have indicated to us that DNA binding activity is acquired by somatic mutation, suggesting that these autoantibodies are not germline encoded but require antigenic stimulation and T cell help. Molecular analysis of genes encoding 3I reactive light chains from a panel of EBV transformed B cell lines have revealed that 3I reactive light chains are nearly all encoded by a member of the VK 1 gene family. Thus for this idiotypic system, there is restricted gene usage to encode anti-DNA antibodies. Further molecular analysis may reveal the structural features that determine idiotype reactivity and autoreactivity and may help determine what features of these genes could account for their preferential expression in SLE patients and their family members.(ABSTRACT TRUNCATED AT 250 WORDS)