A gp41 MPER-specific llama VHH requires a hydrophobic CDR3 for neutralization but not for antigen recognition

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10

Potent and Broad Neutralization of HIV-1 by a Llama Antibody Elicited by Immunization

Llamas (Lama glama) naturally produce heavy chain–only antibodies (Abs) in addition to conventional Abs. The variable regions (VHH) in these heavy chain–only Abs demonstrate comparable affinity and specificity for antigens to conventional immunoglobulins despite their much smaller size. To date, immunizations in humans and animal models have yielded only Abs with limited ability to neutralize HIV-1. In this study, a VHH phagemid library generated from a llama that was multiply immunized with recombinant trimeric HIV-1 envelope proteins (Envs) was screened directly for HIV-1 neutralization. One VHH, L8CJ3 (J3), neutralized 96 of 100 tested HIV-1 strains, encompassing subtypes A, B, C, D, BC, AE, AG, AC, ACD, CD, and G. J3 also potently neutralized chimeric simian-HIV strains with HIV subtypes B and C Env. The sequence of J3 is highly divergent from previous anti–HIV-1 VHH and its own germline sequence. J3 achieves broad and potent neutralization of HIV-1 via interaction with the CD4-binding site of HIV-1 Env. This study may represent a new benchmark for immunogens to be included in B cell–based vaccines and supports the development of VHH as anti–HIV-1 microbicides

From DNA sequence to application: possibilities and complications

The development of sophisticated genetic tools during the past 15 years have facilitated a tremendous increase of fundamental and application-oriented knowledge of lactic acid bacteria (LAB) and their bacteriophages. This knowledge relates both to the assignments of open reading frames (ORF’s) and the function of non-coding DNA sequences. Comparison of the complete nucleotide sequences of several LAB bacteriophages has revealed that their chromosomes have a fixed, modular structure, each module having a set of genes involved in a specific phase of the bacteriophage life cycle. LAB bacteriophage genes and DNA sequences have been used for the construction of temperature-inducible gene expression systems, gene-integration systems, and bacteriophage defence systems. The function of several LAB open reading frames and transcriptional units have been identified and characterized in detail. Many of these could find practical applications, such as induced lysis of LAB to enhance cheese ripening and re-routing of carbon fluxes for the production of a specific amino acid enantiomer. More knowledge has also become available concerning the function and structure of non-coding DNA positioned at or in the vicinity of promoters. In several cases the mRNA produced from this DNA contains a transcriptional terminator-antiterminator pair, in which the antiterminator can be stabilized either by uncharged tRNA or by interaction with a regulatory protein, thus preventing formation of the terminator so that mRNA elongation can proceed. Evidence has accumulated showing that also in LAB carbon catabolite repression in LAB is mediated by specific DNA elements in the vicinity of promoters governing the transcription of catabolic operons. Although some biological barriers have yet to be solved, the vast body of scientific information presently available allows the construction of tailor-made genetically modified LAB. Today, it appears that societal constraints rather than biological hurdles impede the use of genetically modified LAB.

Using intervention mapping to develop a home-based parental-supervised toothbrushing intervention for young children

BACKGROUND: Dental caries in young children is a major public health problem impacting on the child and their family in terms of pain, infection and substantial financial burden on healthcare funders. In the UK, national guidance on the prevention of dental caries advises parents to supervise their child's brushing with fluoride toothpaste until age 7. However, there is a dearth of evidence-based interventions to encourage this practice in parents. The current study used intervention mapping (IM) to develop a home-based parental-supervised toothbrushing intervention to reduce dental caries in young children. METHODS: The intervention was developed using the six key stages of the IM protocol: (1) needs assessment, including a systematic review, qualitative interviews, and meetings with a multi-disciplinary intervention development group; (2) identification of outcomes and change objectives following identification of the barriers to parental-supervised toothbrushing (PSB), mapped alongside psychological determinants outlined in the Theoretical Domains Framework (TDF); (3) selection of methods and practical strategies; (4) production of a programme plan; (5) adoption and implementation and (6) Evaluation. RESULTS: The comprehensive needs assessment highlighted key barriers to PSB, such as knowledge, skills, self-efficacy, routine setting and behaviour regulation and underlined the importance of individual, social and structural influences. Parenting skills (routine setting and the ability to manage the behaviour of a reluctant child) were emphasised as critical to the success of PSB. The multi-disciplinary intervention development group highlighted the need for both universal and targeted programmes, which could be implemented within current provision. Two intervention pathways were developed: a lower cost universal pathway utilising an existing national programme and an intensive targeted programme delivered via existing parenting programmes. A training manual was created to accompany each intervention to ensure knowledge and standardise implementation procedures. CONCLUSIONS: PSB is a complex behaviour and requires intervention across individual, social and structural levels. IM, although a time-consuming process, allowed us to capture this complexity and allowed us to develop two community-based intervention pathways covering both universal and targeted approaches, which can be integrated into current provision. Further research is needed to evaluate the acceptability and sustainability of these interventions

Enhancement of Polymeric Immunoglobulin Receptor Transcytosis by Biparatopic VHH

The polymeric immunoglobulin receptor (pIgR) ensures the transport of dimeric immunoglobulin A (dIgA) and pentameric immunoglobulin M (pIgM) across epithelia to the mucosal layer of for example the intestines and the lungs via transcytosis. Per day the human pIgR mediates the excretion of 2 to 5 grams of dIgA into the mucosa of luminal organs. This system could prove useful for therapies aiming at excretion of compounds into the mucosa. Here we investigated the use of the variable domain of camelid derived heavy chain only antibodies, also known as VHHs or Nanobodies®, targeting the human pIgR, as a transport system across epithelial cells. We show that VHHs directed against the human pIgR are able to bind the receptor with high affinity (∼1 nM) and that they compete with the natural ligand, dIgA. In a transcytosis assay both native and phage-bound VHH were only able to get across polarized MDCK cells that express the human pIgR gene in a basolateral to apical fashion. Indicating that the VHHs are able to translocate across epithelia and to take along large particles of cargo. Furthermore, by making multivalent VHHs we were able to enhance the transport of the compounds both in a MDCK-hpIgR and Caco-2 cell system, probably by inducing receptor clustering. These results show that VHHs can be used as a carrier system to exploit the human pIgR transcytotic system and that multivalent compounds are able to significantly enhance the transport across epithelial monolayers

A novel seven-octapeptide repeat insertion in the prion protein gene (PRNP) in a Dutch pedigree with Gerstmann–Sträussler–Scheinker disease phenotype: comparison with similar cases from the literature

Human prion diseases can be sporadic, inherited or acquired by infection and show considerable phenotypic heterogeneity. We describe the clinical, histopathological and pathological prion protein (PrPSc) characteristics of a Dutch family with a novel 7-octapeptide repeat insertion (7-OPRI) in PRNP, the gene encoding the prion protein (PrP). Clinical features were available in four, neuropathological features in three and biochemical characteristics in two members of this family. The clinical phenotype was characterized by slowly progressive cognitive decline, personality change, lethargy, depression with anxiety and panic attacks, apraxia and a hypokinetic-rigid syndrome. Neuropathological findings consisted of numerous multi- and unicentric amyloid plaques throughout the cerebrum and cerebellum with varying degrees of spongiform degeneration. Genetic and molecular studies were performed in two male family members. One of them was homozygous for valine and the other heterozygous for methionine and valine at codon 129 of PRNP. Sequence analysis identified a novel 168 bp insertion [R2–R2–R2–R2–R3g–R2–R2] in the octapeptide repeat region of PRNP. Both patients carried the mutation on the allele with valine at codon 129. Western blot analysis showed type 1 PrPSc in both patients and detected a smaller ~8 kDa PrPSc fragment in the cerebellum in one patient. The features of this Dutch kindred define an unusual neuropathological phenotype and a novel PRNP haplotype among the previously documented 7-OPRI mutations, further expanding the spectrum of genotype–phenotype correlations in inherited prion diseases

Health-related quality of life of Canadian children and youth prenatally exposed to alcohol

BACKGROUND: In Canada, the incidence of Fetal Alcohol Spectrum Disorder (FASD) has been estimated to be 1 in 100 live births. Caused by prenatal exposure to alcohol, FASD is the leading cause of neuro-developmental disabilities among Canadian children, and youth. Objective: To measure the health-related quality of life (HRQL) of Canadian children and youth diagnosed with FASD. METHODS: A prospective cross-sectional study design was used. One-hundred and twenty-six (126) children and youth diagnosed with FASD, aged 8 to 21 years, living in urban and rural communities throughout Canada participated in the study. Participants completed the Health Utilities Index Mark 3 (HUI3). HUI3 measures eight health attributes: vision, hearing, speech, ambulation, dexterity, emotion, cognition, and pain. Utilities were used to measure a single cardinal value between 0 and 1.0 (0 = all-worst health state; 1 = perfect health) to reflect the global HRQL for that child. Mean HRQL scores and range of scores of children and youth with FASD were calculated. A one-sample t-test was used to compare mean HRQL scores of children and youth with FASD to those from the Canadian population. RESULTS: Mean HRQL score of children and youth with FASD was 0.47 (95% CI: 0.42 to 0.52) as compared to a mean score of 0.93 (95% CI: 0.92 to 0.94) in those from the general Canadian population (p < 0.001). Children demonstrated moderate to severe dysfunction on the single-attributes of cognition and emotion. CONCLUSION: Children and youth with FASD have significantly lower HRQL than children and youth from the general Canadian population. This finding has significant implications for practice, policy development, and research

Health-related quality-of-life measures for long-term follow-up in children after major trauma

Objective: Our objective was to review measures of health-related quality of life (HRQL) for long-term follow up in children after major trauma and to determine the measures that are suitable for a large age range, reliable and valid, and cover a substantial amount of the domains of functioning using the International Classification of Functioning, Disability, and Health (ICF) of the World Health Organization (WHO). Methods: The Medline and EMBASE databases were searched in all years up to October 2007 for generic HRQL measures suitable for children aged 5-18 years old and validated in English or Dutch. Measures were reviewed with respect to the age range for which the measure was suitable and reliability, validity, and content related to the ICF. Results: The search resulted in 1,235 hits and 21 related articles. Seventy-nine papers met the inclusion criteria, describing in total 14 measures: Child Health and Illness Profile Adolescent and Child Edition (CHIP-AE/CE), Child Health Questionnaire Child and Parent Forms (CHQCF87/PF50/PF28), DISABKIDS, Functional Status II (FS II)(R), Health Utilities Index Mark 2 (HUI 2), KIDSCREEN 52/27, KINDL, Pediatric Quality of Life Inventory (PedsQL), TNO Institute of Prevention and Health and the Leiden University Hospital (TNO-AZL), TNO-AZL Children’s Quality Of Life (TACQOL), and Youth Quality of Life Instrument-Research Version (YQOL-R). Measures that were suitable for a large age range were CHQ-PF50/PF28, DISABKIDS, FS II(R), HUI 2, KIDSCREEN, PedsQL, and TACQOL. All measures had moderate to good psychometric properties, except for CHQ-PF50/PF28, KINDL, and TACQOL, which had either low internal consistency or bad test-retest reliability. The measures that covered more than six chapters of the ICF domains were CHIP-AE/CE, CHQ-CF87/PF50, DISABKIDS, KIDSCREEN-52, PedsQL, and TACQOL. Conclusions: DISABKIDS, KIDSCREEN 52, and Peds-QL are suitable for long-term follow-up measurement of HRQL in children after major trauma. They cover a large age range, have good psychometric properties, and cover the ICF substantially

Development, Problem Behavior, and Quality of Life in a Population Based Sample of Eight-Year-Old Children with Down Syndrome

OBJECTIVE: Children with Down syndrome (DS) have delayed psychomotor development. We investigated levels of development, problem behavior, and Health-Related Quality of Life (HRQoL) in a population sample of Dutch eight-year-old children with DS. Developmental outcomes were compared with normative data of eight-year-old children from the general population. METHOD: Over a three-year-period all parents with an eight-year-old child with DS were approached by the national parent organization. Developmental skills were assessed by means of the McCarthy Scales of Children's Ability. To measure emotional and behavioral problems we used the Child Behavior Checklist. HRQoL was assessed with the TNO-AZL Children's Quality of Life questionnaire. Analyses of variance were applied to compare groups. RESULTS: A total of 337 children participated. Mean developmental age was substantially lower than mean calendar age (3.9 years, SD 0.87 and 8.1 years, SD 0.15 respectively). Mean developmental age was significantly lower among boys than girls (3.6 (SD 0.85) and 4.2 years (SD 0.82) respectively; p<0.001). Compared with the general population, children with DS had more emotional and behavioral problems (p<0.001). However on the anxious/depressed scale, they scored significantly more favorably (p<0.001). Significantly lower HRQoL scores for the scales gross motor skills, autonomy, social functioning and cognitive functioning were found (p-values<0.001). Hardly any differences were observed for the scales physical complaints, positive and negative emotions. CONCLUSION: Eight-year-old children with DS have an average developmental delay of four years, more often have emotional and behavioral problems, and have a less favorable HRQoL compared with children from the general population