Defective secretion of an immunoglobulin caused by mutations in the heavy chain complementarity determining region 2.

We have investigated four secretion-deficient antibodies (Abs) derived from a panel of 46 mutant T15 anti-phosphocholine Abs, all of which have point mutations in the heavy chain (H) complementarity determining region 2 (CDR2). The level of secretion for these four Abs was < 10% of wild type when expressed together with the T15 light chain (L) in either SP2/0 or P3X63Ag8.653 myeloma cells although normal levels of H and L chain mRNA were produced. Moreover, abundant intracellular H and L chain proteins were detected. Three of the four mutants had little or no assembled H and L complexes intracellularly whereas one had a significant amount of intracellular immunoglobulin (Ig) which was shown to be capable of binding Ag. Thus, we demonstrate for the first time that point mutations confined to CDR2 of the H chain variable (V) region can impede Ab assembly and secretion. We then introduced the same CDR2 mutations into a related H chain which is encoded by the same T15 VH gene but different diversity (D) and joining (J) genes. When these H chains were expressed with a non-T15 L chain, the resulting Abs were secreted normally. The results thus suggest that the effects of the CDR2 mutations on Ab secretion are dependent on their interactions with L and/or H chain D-J sequences. These results also reveal a novel mechanism that could contribute to B cell wastage.

Predictive models for carcinogenicity and mutagenicity: frameworks, state-of-the-art, and perspectives.

Mutagenicity and carcinogenicity are endpoints of major environmental and regulatory concern. These endpoints are also important targets for development of alternative methods for screening and prediction due to the large number of chemicals of potential concern and the tremendous cost (in time, money, animals) of rodent carcinogenicity bioassays. Both mutagenicity and carcinogenicity involve complex, cellular processes that are only partially understood. Advances in technologies and generation of new data will permit a much deeper understanding. In silico methods for predicting mutagenicity and rodent carcinogenicity based on chemical structural features, along with current mutagenicity and carcinogenicity data sets, have performed well for local prediction (i.e., within specific chemical classes), but are less successful for global prediction (i.e., for a broad range of chemicals). The predictivity of in silico methods can be improved by improving the quality of the data base and endpoints used for modelling. In particular, in vitro assays for clastogenicity need to be improved to reduce false positives (relative to rodent carcinogenicity) and to detect compounds that do not interact directly with DNA or have epigenetic activities. New assays emerging to complement or replace some of the standard assays include Vitotox, GreenScreenGC, and RadarScreen. The needs of industry and regulators to assess thousands of compounds necessitate the development of high-throughput assays combined with innovative data-mining and in silico methods. Various initiatives in this regard have begun, including CAESAR, OSIRIS, CHEMOMENTUM, CHEMPREDICT, OpenTox, EPAA, and ToxCast. In silico methods can be used for priority setting, mechanistic studies, and to estimate potency. Ultimately, such efforts should lead to improvements in application of in silico methods for predicting carcinogenicity to assist industry and regulators and to enhance protection of public health.

Activation of NOD2 in vivo induces IL-1beta production in the eye via caspase-1 but results in ocular inflammation independently of IL-1 signaling.

Nucleotide-binding and oligomerization domain 2 (NOD2) belongs to the emerging Nod-like receptor (NLR) family considered important in innate immunity. Mutations in NOD2 cause Blau syndrome, an inherited inflammation of eye, joints, and skin. Mutations in a homologous region of another NLR member, NALP3, cause autoinflammation, wherein IL-1beta plays a critical role. Here, we tested the hypothesis that IL-1beta is a downstream mediator of NOD2-dependent ocular inflammation. We used a mouse model of NOD2-dependent ocular inflammation induced by muramyl dipeptide (MDP), the minimal bacterial motif sensed by NOD2. We report that MDP-induced ocular inflammation generates IL-1beta and IL-18 within the eye in a NOD2- and caspase-1-dependent manner. Surprisingly, two critical measures of ocular inflammation, leukocyte rolling and leukocyte intravascular adherence, appear to be completely independent of IL-1 signaling effects, as caspase-1 and IL-1R1-deficient mice still developed ocular inflammation in response to MDP. In contrast to the eye, a diminished neutrophil response was observed in an in vivo model of MDP-induced peritonitis in caspase-1-deficient mice, suggesting that IL-1beta is not essential in NOD2-dependent ocular inflammation, but it is involved, in part, in systemic inflammation triggered by NOD2 activation. This disparity may be influenced by IL-1R antagonist (IL-1Ra), as we observed differential IL-1Ra levels in the eye versus plasma at baseline levels and in response to MDP treatment. This report reveals a new in vivo function of NOD2 within the eye yet importantly, distinguishes NOD2-dependent from NALP3-dependent inflammation, as ocular inflammation in mice occurred independently of IL-1beta.

Anterior uveitis accompanies joint disease in a murine model resembling ankylosing spondylitis.

BACKGROUND: Uveitis is often associated with a systemic inflammatory disease such as ankylosing spondylitis. Our understanding of the eye's susceptibility to immune-mediated uveitis as in the apparent absence of infection has been limited by a relative lack of experimental models. Here we sought to assess whether ocular inflammation occurs in a previously described murine model of proteoglycan-induced spondylitis, wherein mice develop progressive spondylitis, sacroiliitis and peripheral arthritis--features common to the clinical presentations of ankylosing spondylitis. METHODS: Using intravital microscopy we examined the ocular inflammatory response after the onset of arthritis in mice that overexpressed the T cell receptor (TCR) specific for a dominant arthritogenic epitope of cartilage proteoglycan [TCR-Tg (transgenic) mice] or BALB/c controls. RESULTS: Immunized TCR-Tg mice showed a significant increase in the number of rolling and adhering cells within the iris vasculature compared to adjuvant control mice. Cellular infiltration within the iris tissue, as assessed by intravital microscopy and histology, was also increased. Our initial temporal analysis has revealed that immunized TCR-Tg mice show a significant increase in intravascular inflammation by 2 weeks after immunization, but it diminishes at 4 weeks after immunization. CONCLUSIONS: Although these data are preliminary, this model has the potential to clarify the mechanisms accounting for the coexistence of eye and sacroiliac inflammation as occurs in patients with ankylosing spondylitis.

Prediction of pesticide acute toxicity using two-dimensional chemical descriptors and target species classification.

Previous modelling of the median lethal dose (oral rat LD50) has indicated that local class-based models yield better correlations than global models. We evaluated the hypothesis that dividing the dataset by pesticidal mechanisms would improve prediction accuracy. A linear discriminant analysis (LDA) based-approach was utilized to assign indicators such as the pesticide target species, mode of action, or target species - mode of action combination. LDA models were able to predict these indicators with about 87% accuracy. Toxicity is predicted utilizing the QSAR model fit to chemicals with that indicator. Toxicity was also predicted using a global hierarchical clustering (HC) approach which divides data set into clusters based on molecular similarity. At a comparable prediction coverage (~94%), the global HC method yielded slightly higher prediction accuracy (r2 = 0.50) than the LDA method (r2 ~ 0.47). A single model fit to the entire training set yielded the poorest results (r2 = 0.38), indicating that there is an advantage to clustering the dataset to predict acute toxicity. Finally, this study shows that whilst dividing the training set into subsets (i.e. clusters) improves prediction accuracy, it may not matter which method (expert based or purely machine learning) is used to divide the dataset into subsets.

Prediction of in vitro and in vivo oestrogen receptor activity using hierarchical clustering.

In this study, hierarchical clustering classification models were developed to predict in vitro and in vivo oestrogen receptor (ER) activity. Classification models were developed for binding, agonist, and antagonist in vitro ER activity and for mouse in vivo uterotrophic ER binding. In vitro classification models yielded balanced accuracies ranging from 0.65 to 0.85 for the external prediction set. In vivo ER classification models yielded balanced accuracies ranging from 0.72 to 0.83. If used as additional biological descriptors for in vivo models, in vitro scores were found to increase the prediction accuracy of in vivo ER models. If in vitro activity was used directly as a surrogate for in vivo activity, the results were poor (balanced accuracy ranged from 0.49 to 0.72). Under-sampling negative compounds in the training set was found to increase the coverage (fraction of chemicals which can be predicted) and increase prediction sensitivity.

MOAtox: A comprehensive mode of action and acute aquatic toxicity database for predictive model development.

The mode of toxic action (MOA) has been recognized as a key determinant of chemical toxicity and as an alternative to chemical class-based predictive toxicity modeling. However, the development of quantitative structure activity relationship (QSAR) and other models has been limited by the availability of comprehensive high quality MOA and toxicity databases. The current study developed a dataset of MOA assignments for 1213 chemicals that included a diversity of metals, pesticides, and other organic compounds that encompassed six broad and 31 specific MOAs. MOA assignments were made using a combination of high confidence approaches that included international consensus classifications, QSAR predictions, and weight of evidence professional judgment based on an assessment of structure and literature information. A toxicity database of 674 acute values linked to chemical MOA was developed for fish and invertebrates. Additionally, species-specific measured or high confidence estimated acute values were developed for the four aquatic species with the most reported toxicity values: rainbow trout (Oncorhynchus mykiss), fathead minnow (Pimephales promelas), bluegill (Lepomis macrochirus), and the cladoceran (Daphnia magna). Measured acute toxicity values met strict standardization and quality assurance requirements. Toxicity values for chemicals with missing species-specific data were estimated using established interspecies correlation models and procedures (Web-ICE; http://epa.gov/ceampubl/fchain/webice/), with the highest confidence values selected. The resulting dataset of MOA assignments and paired toxicity values are provided in spreadsheet format as a comprehensive standardized dataset available for predictive aquatic toxicology model development.

Comparison of global and mode of action-based models for aquatic toxicity.

The ability to estimate aquatic toxicity is a critical need for ecological risk assessment and chemical regulation. The consensus in the literature is that mode of action (MOA) based toxicity models yield the most toxicologically meaningful and, theoretically, the most accurate results. In this study, a two-step prediction methodology was developed to estimate acute aquatic toxicity from molecular structure. In the first step, one-against-the-rest linear discriminant analysis (LDA) models were used to predict the MOA. The LDA models were able to predict the MOA with 85.8-88.8% accuracy for broad and specific MOAs, respectively. In the second step, a multiple linear regression (MLR) model corresponding to the predicted MOA was used to predict the acute aquatic toxicity value. The MOA-based approach was found to yield similar external prediction accuracy (r(2) = 0.529-0.632) to a single global MLR model (r(2) = 0.551-0.562) fit to the entire training set. Overall, the global hierarchical clustering approach yielded a higher combination of accuracy and prediction coverage (r(2) = 0.572, coverage = 99.3%) than the other approaches. Utilizing multiple two-dimensional chemical descriptors in MLR models yielded comparable results to using only the octanol-water partition coefficient (log K(ow)).

HLA-DRB1*0102 is associated with TINU syndrome and bilateral, sudden-onset anterior uveitis but not with interstitial nephritis alone.

BACKGROUND: Tubulointerstitial nephritis and uveitis (TINU) syndrome is a rare form of uveitis. Previously, the authors had demonstrated a strong association of human leukocyte antigen (HLA) DRB1*0102 with TINU. Here, the authors performed HLA analysis on subjects with isolated bilateral sudden-onset uveitis (as in the TINU subtype) or with isolated tubulointerstitial nephritis (TIN). METHODS: Patients with sudden onset, anterior, bilateral uveitis not fulfilling a diagnosis of TINU were identified. Pathology reports were reviewed to identify subjects with biopsy-proven TIN. Molecular typing of the HLA-DRB1 gene was performed by the Luminex technology-based sequence-specific oligonucleotide (SSO) hybridisation method (One Lambda, Canoga Park, California). HLA-DRB1 allele frequencies were compared with normal published controls (http://www.ncbi.nlm.nih.gov/projects/gv/mhc/ihwg.cgi dbMHC Europe cohort) and the published TINU cohort (n=18). RESULTS: The authors included 28 subjects with uveitis and 14 with TIN. There was a significantly higher frequency of DRB1*0102 in the isolated uveitis cohort versus in normal controls (10.7% vs 0.6%, respectively, p<0.0001; RR 14.3 (6.9-29.8)). None of the nephritis patients showed this HLA subtype. Another association with HLA-DRB1*08 was seen in the isolated uveitis cohort with an allele frequency of 10.7% versus 2.7% in normal controls (p=0.0019; RR 4.0 (1.8-9.0)). In contrast, the HLA-DRB1*08 was not different from controls in the TINU cohort (allele frequency 2.8%, p=not significant). CONCLUSION: The incidence of HLA-DRB1*0102 is increased in sudden-onset bilateral anterior uveitis, as seen in patients with TINU. The same allele does not appear to occur in increased frequency in patients with isolated TIN. HLA DRB1*0102 might predispose to this subset of uveitis.

Gender and laterality affect recurrences of acute anterior uveitis.

AIM: Acute anterior uveitis (AAU) associated with HLA-B27 or axial spondyloarthritis (axial SpA) is primarily unilateral and recurrent. We tested the hypotheses that disease laterality and gender affected recurrences of AAU. METHODS: We studied 207 AAU subjects who were either HLA-B27 positive or had a verified history of axial SpA with documentation of the first uveitis episode. We recorded gender, laterality, duration, and time between episodes. RESULTS: Of 207 subjects, 126 (60.9%) had axial spondyloarthritis. Of the 179 with known HLA-B27 status, 174 (97.2%) were HLA-B27 positive. The initial episode of AAU occurred slightly more often in the right eye, 109 (52.6%), than in the left, 91 (44.0%) or bilaterally, 7 (3.4%), but the difference between right and left was not significant (p=0.23). Interestingly, 69.4% of subsequent episodes occurred in the same eye affected previously (95% CI 59.3%, 78.3%, p=0.0001). In subjects with recurrent AAU, the probability of being disease-free for one year was 38.9% (95% CI 29.1%, 52.0%) using Kaplan-Meier estimates. Univariate analyses showed that male gender (p=0.03) and AAU which recurred in the same eye (p=0.04) was associated with a shorter time interval between episodes. Multivariate analysis by the Cox proportional hazards model showed similar results. CONCLUSIONS: The initial episode of unilateral AAU associated with HLA-B27 or axial SpA randomly affects either eye. Subsequent episodes occur more often in the same eye previously affected. Male gender and history of unilateral AAU in the same eye are associated with a shortened time interval between relapses.

Activation of nucleotide oligomerization domain 2 exacerbates a murine model of proteoglycan-induced arthritis.

In addition to its role in innate immunity, nucleotide oligomerization domain 2 (NOD2) has been shown to play a suppressive role in models of colitis. Notably, mutations in NOD2 cause the inherited granulomatous disease of the joints called Blau syndrome, thereby linking NOD2 with joint disease as well. However, the role of NOD2 in joint inflammation has not been clarified. We demonstrate here that NOD2 is functional within the mouse joint and promotes inflammation, as locally or systemically administered muramyl dipeptide (MDP; the NOD2 agonist) resulted in significant joint inflammation that was abolished in NOD2-deficient mice. We then sought to investigate the role of NOD2 in a mouse model of inflammatory arthritis dependent on adaptive immunity using TCR-transgenic mice whose T cells recognized the dominant epitope of proteoglycan (PG). Mice immunized with PG in the presence of MDP developed a more severe inflammatory arthritis and histopathology within the joints. Antigen-specific activation of splenocytes was enhanced by MDP with respect to IFN-gamma production, which would be consistent with the Th1-mediated disease in vivo. Intriguingly, NOD2 deficiency did not alter the PG-induced arthritis, indicating that NOD2 does not play an essential role in this model of joint disease when it is not activated by MDP. In conclusion, we demonstrate that in a model of inflammatory arthritis dependent on T and B cell priming, NOD2 activation potentiates disease. However, the absence of NOD2 does not alter the course of inflammatory arthritis, in contrast to models of intestinal inflammation.

Posttonsillectomy vomiting. Ondansetron or metoclopramide during paediatric tonsillectomy: are two doses better than one?

This randomized, double blinded, placebo controlled, prospective study compared the anti-emetic efficacy of one preoperative dose of metoclopramide 0.25 mg.kg-1 intravenously or ondansetron 0.15 mg.kg-1 intravenously with two doses of the same drugs (second dose administered one h postoperatively) in 200 preadolescent children undergoing tonsillectomy with either isoflurane or propofol anaesthesia. The incidence of posttonsillectomy vomiting was significantly reduced (P < 0.005) by two doses of either metoclopramide or ondansetron (18% and 8%, respectively) compared with placebo (50%). No difference in posttonsillectomy vomiting exists between the children who received isoflurane and those who received a propofol infusion. Our results suggest that two doses of metoclopramide 0.25 mg.kg-1 intravenously, like two doses of ondansetron 0.15 mg.kg-1, are effective in reducing vomiting after tonsillectomy in children who have received either isoflurane or propofol anaesthesia.

Prediction of the acute toxicity (96-h LC50) of organic compounds to the fathead minnow (Pimephales promelas) using a group contribution method.

A group contribution method has been developed to correlate the acute toxicity (96-h LC50) to the fathead minnow (Pimephales promelas) for 397 organic chemicals. Multilinear regression and computational neural networks (CNNs) were used for model building. The models were able to achieve a fairly good correlation of the data (r2 > 0.9). The linear model, which included four specific interaction terms, provided a rapid means of predicting the toxicity of a compound. The CNN model was able to yield virtually the same predictions with or without the four interaction terms that were included in the multilinear model.

Propofol anesthesia reduces emesis and airway obstruction in pediatric outpatients.

This study was an authors comparison of the effects of and recovery from anesthesia in healthy, premedicated pediatric outpatients who received either inhaled anesthetics (group 1) or propofol (group 2). Group 1 (n = 68) averaged 3.8 +/- 0.2 yr and weighed 17.7 +/- 0.8 kg, whereas group 2 (n = 75) averaged 3.3 +/- 0.2 yr and weighed 16.3 +/- 0.6 kg. The incidence of vomiting in the Postanesthetic Care Unit (PACU) and from discharge to the first postoperative morning was lower in the group receiving propofol (0% and 18%) than in the group receiving volatile agents (7% and 34%, P < 0.05). The incidence of airway obstruction during induction of anesthesia was higher (34% vs 10%, P < 0.01) in children receiving inhaled agent. Withdrawal of the extremity with propofol injection occurred in 14 (19%) patients. Arterial blood pressure was higher at loss of consciousness, laryngoscopy, and tracheal intubation in group 2 (P < 0.01). The length of time from the end of surgery to extubation of the trachea, recovery scores, and length of time spent in the PACU and the Day Surgery Unit were the same in the two groups. Pain scores obtained in the PACU were not different. The data indicate that propofol can be used safely to induce and maintain anesthesia in healthy pediatric outpatients. This coupled with the low incidence of vomiting and airway obstruction in the propofol group suggests distinct and compelling reasons to consider using the drug in this patient population.

Inefficient assembly and intracellular accumulation of antibodies with mutations in V(H) CDR2.

We previously described secretion defects in four mutants of the murine anti-phosphocholine Ab, T15. The mutant heavy (H) chains had amino acid replacements in the V(H) complementarity-determining region 2 (HCDR2) and were expressed at normal intracellular levels. Here, the intracellular fate of the secretion-defective mutant heavy chains was investigated. Metabolic labeling demonstrated that the T15 wild-type Ab was secreted within a 4-h chase. In contrast, the mutant H chains accumulated with intracellular t(1/2) values ranging from 10 to 24 h. The mutant H chains were associated with increased levels of the molecular chaperones BiP and GRP94, and remained endoglycosidase H sensitive, suggesting retention in the endoplasmic reticulum. Assembly of the mutant H chains with T15 light (L) chain was arrested at the H2 and H2L intermediate stages of the T15 wild-type pathway (H2 --> H2L --> H2L2). Even though some assembly with L chain occurred, it was not as a secretion-competent H2L2 Ig moiety. The T15 L chains coexpressed with mutant H chains were degraded efficiently except for a minor L chain population with a long t(1/2) that was apparently protected at the H2L stage. To our knowledge, this is the first study demonstrating that intracellular half-lives of Ig H and L chains can be influenced by somatic mutations in HCDR2.

Ondansetron reduces the incidence and severity of poststrabismus repair vomiting in children.

This prospective, randomized, placebo-controlled, double-blinded study evaluated the antiemetic efficacy of ondansetron and metoclopramide in 90 ASA physical status I or II children, 2-17 yr of age, undergoing strabismus repair. After anesthetic induction and prior to eye muscle manipulation, subjects received normal saline 0.3 mL/kg (Group 1), metoclopramide 0.25 mg/kg (Group 2), or ondansetron 0.15 mg/kg (Group 3), intravenously. There were no differences between groups with respect to age, weight, gender, fluids received, number of eye muscles repaired, anesthetic technique, or time in the operating room. The incidence of vomiting in Groups 1, 2, and 3 was 50%, 27%, and 10% prior to discharge, and 67%, 53%, and 30% during the 24 h after surgery, respectively. The number of children vomiting prior to discharge and within 24 h of surgery was significantly reduced in Group 3 compared with Group 1 (P < 0.003 and P < 0.015, respectively). The number of vomiting episodes per patient in Groups 1, 2, and 3 was 1.1, 0.5, and 0.1 prior to discharge, and 4.5, 2.6, and 1.2 during the 24 h after surgery (P < 0.0005 and P < 0.004, respectively). Ondansetron 0.15 mg/kg intravenously after the induction of anesthesia reduces the incidence and severity of vomiting after strabismus repair both prior to discharge from the hospital and during the 24 h after surgery.

PKA-dependent regulation of mKv1.1, a mouse Shaker-like potassium channel gene, when stably expressed in CHO cells.

Potassium (K) channels are important regulators of cellular physiology and can themselves be modulated by phosphorylation. We have investigated the potential protein kinase A (PKA) regulation of mKv1.1, a mouse Shaker-like K channel gene, when it is expressed in stably transfected Chinese hamster ovary (CHO) cell lines. Whole-cell patch-clamp records show that expression of mKv1.1 gives rise to a rapidly activating, sustained K+ current, referred to classically as a delayed rectifier-type current. In order to study the effects of PKA, we compared cell lines transfected with mKv1.1 alone with lines cotransfected with both mKv1.1 and a plasmid encoding a dominant negative mutation in the regulatory subunit of PKA. These mutant regulatory subunits bind to endogenous catalytic subunits of PKA but do not respond to cAMP, thereby causing a chronic reduction in the basal PKA activity in these cells. We found that mKv1.1 current kinetics are unaltered but current density is 3.4-fold higher in the cell lines expressing mutant regulatory subunit than in lines expressing only mKv1.1. RNase protection assays indicate that levels of the specific RNA for mKv1.1 are increased almost twofold in the lines expressing mutant regulatory subunit over the lines expressing mKv1.1 only. Further, the levels of mKv1.1 protein, assayed using an mKv1.1 channel-specific antibody, are increased by almost a factor of 3 between the two types of cell lines. These results suggest that PKA can regulate mKv1.1 channel expression by changing steady-state levels of RNA and by other posttranscriptional mechanisms.

Heteromultimeric K+ channels in terminal and juxtaparanodal regions of neurons.

Voltage-gated potassium (K+) channels display a wide variety of conductances and gating properties in vivo. This diversity can be attributed not only to the presence of many K(+)-channel gene products, but also to the possibility that different K(+)-channel subunits co-assemble to form heteromultimeric channels in vivo. When expressed in Xenopus oocytes or transfected cells, K(+)-channel polypeptides assemble to form tetramers. Certain combinations of Shaker-like subunits have been shown to co-assemble, forming heteromultimeric channels with distinct properties. It is not known, however, whether K(+)-channel polypeptides form heteromultimeric channels in vivo. Here we describe the co-localization of two Shaker-like voltage-gated K(+)-channel proteins, mKv1.1 and mKv1.2, in the juxtaparanodal regions of nodes of Ranvier in myelinated axons, and in terminal fields of basket cells in mouse cerebellum. We also show that mKv1.1 and mKv1.2 can be coimmunoprecipitated with specific antibodies that recognize only one of them. These data indicate that the two polypeptides occur in subcellular regions where rapid membrane repolarization may be important and that they form heteromultimeric channels in vivo.