Assessing neuropsychiatric symptoms in nursing home patients with dementia: reliability and Reliable Change Index of the Neuropsychiatric Inventory and the Cohen-Mansfield Agitation Inventory.

OBJECTIVE: The aim of the study was to estimate inter-observer and test-retest reliability of the Neuropsychiatric Inventory Nursing Home version (NPI-NH) and the Cohen-Mansfield Agitation Inventory (CMAI), and to establish their Reliable Change Index (RCI). Reliable Change methodology is a practical method for estimating the least change acquired in outcome measures. METHODS: Nursing home physicians and certified nurses assessed 105 patients with dementia (in five nursing homes) at baseline and after 2 weeks. Spearman rank correlations were calculated and Reliable Change Difference Scores (S(diff) (80)). RESULTS: NPI-NH inter-observer correlations ranged 0.14-0.70. NPI-NH test-retest correlations ranged 0.23-0.80. CMAI inter-observer correlations ranged -0.10 to 0.72. CMAI test-retest correlations ranged 0.32-1.00 (CMAI total score, ρ=0.89). S(diff) (80) for NPI-NH items ranged 1.7-5.0. A change of 11 points on the NPI-NH total score can be considered a true behavioral change. S(diff) (80) for CMAI total score was 8 and factor analysis based sub-scale scores physically aggressive behavior, physically non-aggressive behavior, and verbally agitated behavior were 3, 6, and 4, respectively. CONCLUSION: Reliability estimates and RCI for the NPI-NH were modest, seriously challenging its reliability and sensitivity to change over time. NPI-NH may only be useful for monitoring behavioral changes in individual patients with dementia, when symptoms are moderate to severe, or when effect sizes are large. Reliability of the CMAI was good, supporting its usefulness in clinical practice. Poor inter-observer agreement on behavioral observations poses a real challenge in nursing homes. Reliable scales are needed that include unambiguously formulated items.

Massive, sustained gammadelta T cell migration from the bovine skin in vivo.

In all species studied so far, gammadelta T cells are abundantly present in epithelia. The functions of these cells are largely unknown. Using a lymph duct cannulation method, which is only possible in large animals such as cattle, we show that large numbers of gammadelta T cells, but not alphabeta T cells, are constitutively present in pseudoafferent lymph draining bovine skin. The gammadelta T cells, which are present in pseudoafferent lymph, use Vgamma segments that are characteristic for bovine dermal gammadelta T cells, suggesting that these cells migrated from the skin. Further supporting the origin of these cells is the fact that fluorescent latex beads injected in the skin could be recovered in cells in the pseudoafferent lymph. The cannulation method is minimally invasive, and the lymph flow, which was sustained and remained essentially unaltered during 14 days, closely represents the in vivo situation. The gammadelta T cells could not be induced to produce IFN-gamma, TNF-alpha, and IL-10, and they did not express costimulatory molecules, IL-2 receptor, and MHC Class II molecules. The level of gammadelta T cell egress was 6.7x10(3) gammadelta T cells per cm2 skin per hour, which is enough to deplete all gammadelta T cells from the skin within 46 h. As this massive gammadelta T cell migration was observed during 14 days, constant replenishment of these cells must take place. Our data suggest that gammadelta T cells in tissues fulfill more than exclusively local functions.

Highly diverse TCR delta chain repertoire in bovine tissues due to the use of up to four D segments per delta chain.

Tissue-specific distribution of gammadelta TCRs with limited TCR diversity is a common phenomenon in species with a low percentage of gammadelta T cells like humans and mice. We set out to investigate whether this is also the case in cattle (Bos taurus), a species with high percentages of gammadelta T cells. Using a method that was independent of variable (V) segment-specific primers, we generated 65 unique TCR delta chain sequences. We found no evidence for preferential use of certain Vdelta segments in lymph node, skin, spleen, small intestine, large intestine, and blood. The delta chain CDR3 length distribution was very wide in each tissue, which was confirmed by spectratyping. The highly variable CDR3 length was due to the use of up to four diversity (D) segments by one bovine delta chain. Human and murine delta chains contain only one or two D segments. The five functional Ddelta segments that we describe here were identified at cDNA and genomic level, and are the first ruminant D segments described. Fourteen TCR delta chain sequences used novel Vdelta1 segments, and one expressed a novel member of the Vdelta3 family. The number of known functional Vdelta segments in cattle including these new ones is 42 now, but the total number may be much higher. A high number of Vdelta segments in combination with the use of up to four out of five D segments, and the possibility of using non-template encoded (N) nucleotides on either side of these, makes the potential bovine delta chain repertoire much bigger than any known TCR chain. This situation is quite different from the situation in humans and mice, and suggests that the differences between gammadelta high and gammadelta low species in distribution, diversity, and function of gammadelta T cells may be substantial.

Release of Major Peanut Allergens from Their Matrix under Various pH and Simulated Saliva Conditions-Ara h2 and Ara h6 Are Readily Bio-Accessible.

The oral mucosa is the first immune tissue that encounters allergens upon ingestion of food. We hypothesized that the bio-accessibility of allergens at this stage may be a key determinant for sensitization. Light roasted peanut flour was suspended at various pH in buffers mimicking saliva. Protein concentrations and allergens profiles were determined in the supernatants. Peanut protein solubility was poor in the pH range between 3 and 6, while at a low pH (1.5) and at moderately high pHs (>8), it increased. In the pH range of saliva, between 6.5 and 8.5, the allergens Ara h2 and Ara h6 were readily released, whereas Ara h1 and Ara h3 were poorly released. Increasing the pH from 6.5 to 8.5 slightly increased the release of Ara h1 and Ara h3, but the recovery remained low (approximately 20%) compared to that of Ara h2 and Ara h6 (approximately 100% and 65%, respectively). This remarkable difference in the extraction kinetics suggests that Ara h2 and Ara h6 are the first allergens an individual is exposed to upon ingestion of peanut-containing food. We conclude that the peanut allergens Ara h2 and Ara h6 are quickly bio-accessible in the mouth, potentially explaining their extraordinary allergenicity.

Intra- and inter-laboratory validation of an innovative huFcεRIα-RBL-2H3 degranulation assay for in vitro allergenicity assessment of whey hydrolysates.

Cow's milk-derived whey hydrolysates are milk substitutes for cow's milk allergic infants. Safety assessment of these hydrolysates is crucial. Currently, huFcεRIα-RBL-2H3 cells, sensitized with serum IgE from cow's milk allergic patients, are used to assess in vitro residual allergenicity. However, limited availability and high inter-lot variation of sera impede the standardization of safety testing. Recently, we generated an oligoclonal pool of chimeric human (chu)IgE antibodies against bovine ß-lactoglobulin (BLG) as an alternative for human serum. These antibodies demonstrated increased sensitivity, specificity and reproducibility. An inter-laboratory ring trial using our new degranulation assay with different whey-based hydrolysates was performed at four independent laboratories to investigate the robustness and reproducibility. RBL-2H3 cells expressing huFcεRIα were sensitized with our oligoclonal pool of anti-BLG chuIgE antibodies. The cells were subsequently incubated with an amino-acid based formula (AAF), two extensively hydrolyzed formulas (eHF) and three partially hydrolyzed formulas (pHF) to assess the degranulation upon challenge. Results demonstrated a very strong inter-laboratory correlation and the intra- and inter-laboratory variations were acceptable. The AAF and both eHFs showed no degranulation, whereas all pHFs demonstrated degranulation. The study showed that this degranulation assay is robust and reproducible within and between laboratories. This new in vitro degranulation assay seems predictive for allergenicity outcome and might therefore be considered as a relevant substitute for animal models.

Comparison of six commercial ELISA kits for their specificity and sensitivity in detecting different major peanut allergens.

Six commercial peanut enzyme-linked immunosorbent assay kits were assessed for their ability to recover peanut from the standard reference material 2387 peanut butter and also for their specificity in detecting four major peanut allergens, Ara h 1, Ara h 2, Ara h 3, and Ara h 6. The percentage recovery of peanut from peanut butter differed across different kits as well as at different sample concentrations. The highest recovery was observed with the Romer and R-Biopharm kits, while four other kits were found to underestimate the protein content of the reference peanut butter samples. Five of the kits were most sensitive in detecting Ara h 3 followed by Ara h 1, while hardly recognizing Ara h 2 and Ara h 6. The other kit showed the highest sensitivity to Ara h 2 and Ara h 6, while Ara h 1 and Ara h 3 were poorly recognized. Although Ara h 2 and Ara h 6 are known to be heat stable and more potent allergens, antisera specific to any of these four peanut proteins/allergens may serve as good markers for the detection of peanut residues.

International ring trial of the epidermal equivalent sensitizer potency assay: reproducibility and predictive-capacity.

This study describes the international ring trial of the epidermal-equivalent (EE) sensitizer potency assay. This assay does not distinguish a sensitizer from a non-sensitizer, but may classify known skin sensitizers according to their potency. It assesses the chemical concentration resulting in 50% cytotoxicity (EE-EC50) or the 2-fold increase in IL-1α (IL-1α2x). Four laboratories received 13 coded sensitizers. Reproducible results were obtained in each laboratory. A binary prediction model, EC50≥7 mg/ml=weak to moderate sensitizer and EC50<7 mg/ml=strong to extreme sensitizer had an accuracy of 77%. A superior EE (EC50 and IL-1α2x) correlation was observed with human in vivo DSA05 data compared to LLNA-EC3 data. Human in vivo NOEL and LLNA-EC3 data correlated to a similar extent to in vitro EE data. Our results indicate that this easily transferable EE potency assay is suitable for testing chemical allergens of unknown potencies and may now be ready for further validation, providing complementary potency information to other assays already undergoing validation for assessing skin sensitization potential.

Transfer of a two-tiered keratinocyte assay: IL-18 production by NCTC2544 to determine the skin sensitizing capacity and epidermal equivalent assay to determine sensitizer potency.

At present, the identification of potentially sensitizing chemicals is carried out using animal models. However, it is very important from ethical, safety and economic point of view to have biological markers to discriminate allergy and irritation events, and to be able to classify sensitizers according to their potency, without the use of animals. Within the Sens-it-iv EU Frame Programme 6 funded Integrated Project (LSHB-CT-2005-018681), a number of in vitro, human cell based assays were developed which, when optimized and used in an integrated testing strategy, may be able to distinguish sensitizers from non-sensitizers. This study describes two of these assays, which when used in a tiered strategy, may be able to identify contact sensitizers and also to quantify sensitizer potency. Tier 1 is the human keratinocyte NCTC2544 IL-18 assay and tier 2 is the Epidermal Equivalent potency assay. The aim of this study is to show the transferability of the two-tiered approach with training chemicals: 3 sensitizers (DNCB, resorcinol, pPD) and 1 non sensitizer (lactic acid) in tier 1 and 2 sensitizers with different potency in tier 2 (DNCB; extreme and resorcinol; moderate). The chemicals were tested in a non-coded fashion. Here we describe the transferability to naïve laboratories, the establishment of the standard operating procedure, critical points, acceptance criteria and project management. Both assays were successfully transferred to laboratories that had not performed the assays previously. The two tiered approach may offer an unique opportunity to provide an alternative method to the Local Lymph Node Assay (LLNA). These assays are both based on the use of human keratinocytes, which have been shown over the last two decades, to play a key role in all phases of skin sensitization.

Fast, antigen-saving multiplex immunoassay to determine levels and avidity of mouse serum antibodies to pertussis, diphtheria, and tetanus antigens.

To enhance preclinical evaluation of serological immune responses to the individual diphtheria, tetanus, and pertussis (DTP) components of DTP combination vaccines, a fast hexavalent bead-based method was developed. This multiplex immunoassay (MIA) can simultaneously determine levels of specific mouse serum IgG antibodies to P antigens P.69 pertactin (P.69 Prn), filamentous hemagglutinin (FHA), pertussis toxin (Ptx), and combined fimbria type 2 and 3 antigens (Fim2/3) and to diphtheria toxin (Dtx) and tetanus toxin (TT) in a single well. The mouse DTP MIA was shown to be specific and sensitive and to correlate with the six single in-house enzyme-linked immunosorbent assays (ELISAs) for all antigens. Moreover, the MIA was expanded to include avidity measurements of DTP antigens in a multivalent manner. The sensitivities of the mouse DTP avidity MIA per antigen were comparable to those of the six individual in-house avidity ELISAs, and good correlations between IgG concentrations obtained by both methods for all antigens tested were shown. The regular and avidity mouse DTP MIAs were reproducible, with good intra- and interassay coefficients of variability (CV) for all antigens. Finally, the usefulness of the assay was demonstrated in a longitudinal study of the development and avidity maturation of specific IgG antibodies in mice having received different DTP vaccines. We conclude that the hexaplex mouse DTP MIA is a specific, sensitive, and high-throughput alternative for ELISA to investigate the quantity and quality of serological responses to DTP antigens in preclinical vaccine studies.

Impaired long-term maintenance and function of Bordetella pertussis specific B cell memory.

Frequent occurrence of whooping cough in vaccinated populations suggests limited duration of vaccine-induced immunological memory. To investigate peculiarities in B cell memory specific for pertussis antigens P.69 pertactin (P.69 Prn), pertussis toxin (Ptx) and filamentous hemagglutinin (FHA), we monitored the induction and maintenance of specific serum IgG, long-lived bone marrow (BM)-derived plasma cell (PC) and splenic memory B cell (B(mem)) populations in a long-term preclinical vaccination model. Groups of BALB/c mice were primed and boosted (day 28) with a combined diphtheria (D), tetanus (T), acellular pertussis (aP) vaccine (DTaP) or whole cell pertussis (P) vaccine (DTP) and the immune status was followed over time. Levels of pertussis specific IgG, induced after primary and booster immunization, peaked at day 98 to decline thereafter. This was not paralleled by a decay, but rather an increase in BM resident specific PC, over time (>1 year). In contrast, splenic B(mem) peaked after booster immunization to decline till background levels. Late recall of immunological memory more than 1 year after primary and booster vaccination, however, did reveal a rapid proliferative response of pre-existing B(mem) but failed to evoke an anamnestic IgG response. A combination of waning P-antigen specific IgG production by PC and poor functions of the B(mem) compartment such as self-maintenance and anamnestic IgG responses could be a hallmark of waning pertussis immunity. A better understanding of the mechanisms of limited immunological memory to pertussis may help to improve current vaccines.

Massive, sustained γδ T cell migration from the bovine skin in vivo.

In all species studied so far, γδ T cells are abundantly present in epithelia. The functions of these cells are largely unknown. Using a lymph duct cannulation method, which is only possible in large animals such as cattle, we show that large numbers of γδ T cells, but not αß T cells, are constitutively present in pseudoafferent lymph draining bovine skin. The γδ T cells, which are present in pseudoafferent lymph, use Vγ segments that are characteristic for bovine dermal γδ T cells, suggesting that these cells migrated from the skin. Further supporting the origin of these cells is the fact that fluorescent latex beads injected in the skin could be recovered in cells in the pseudoafferent lymph. The cannulation method is minimally invasive, and the lymph flow, which was sustained and remained essentially unaltered during 14 days, closely represents the in vivo situation. The γδ T cells could not be induced to produce IFN-γ, TNF-α, and IL-10, and they did not express costimulatory molecules, IL-2 receptor, and MHC Class II molecules. The level of γδ T cell egress was 6.7 × 103 γδ T cells per cm2 skin per hour, which is enough to deplete all γδ T cells from the skin within 46 h. As this massive γδ T cell migration was observed during 14 days, constant replenishment of these cells must take place. Our data suggest that γδ T cells in tissues fulfill more than exclusively local functions.