LPS-nonresponsive variants of mouse B cell lymphoma, 70Z/3: Isolation and characterization

We have used a genetic approach to study the differentiation of B lymphocytes. The cultured murine cell line 70Z/3 resembles pre-B cells in containing the heavy chain of the immunoglobulin IgM, , as an internal protein in the absence of light chain, L. However, overnight incubation with the B cell mitogen lipopolysaccharide (LPS) induces the cells to mature to a B lymphocyte-like state by the induction of L chain synthesis and the appearance of IgM on the cell surface. We have used immunoselection against surface-bound IgM to isolate LPS uninducible variants of 70Z/3. These fall into two complementation groups, LPS A and LPS B. LPS A variants predominated and were found at a frequency of 1/1200. These cells were completely unresponsive to LPS. LPS B was represented by a single variant in which a subset of cells was induced to display wild-type levels of membranebound IgM, and the proportion of induced cells increased with prolonged incubation with LPS. We detected no structural defects in either variant group, but LPS B may represent a defect in the decision to differentiate in response to LPS.     

Evaluation of Streptococcus pneumoniae Type XIV Opsonins by Phagocytosis-Associated Chemiluminescence and a Bactericidal Assay

The relative roles of serum factors required for opsonization of type XIV Streptococcus pneumoniae were investigated by means of luminol-enhanced chemiluminescence (CL), bactericidal, and immunofluorescence assays employing adult sera containing high (>1,000 ng of antibody nitrogen per ml) or low (<200 ng of antibody nitrogen per ml) antibody concentrations as determined by radioimmunoassay. Specific antibody concentration correlated directly with both total and heat-labile CL activity (P < 0.005) and with the bactericidal index (P < 0.05) at a serum concentration of 10%. The importance of specific antibody as an opsonin was confirmed by the abolition of CL activity and immunoglobulin immunofluorescence observed after absorption of heated sera with type XIV pneumococcal cells and by the dose response in CL and bactericidal activity observed with the addition of immunoglobulin G to hypogammaglobulinemic serum. A role for the classical complement pathway in opsonization was indicated by significantly greater CL integrals for high-antibody sera than for low-antibody sera depleted of factor D and by the bactericidal activity noted for untreated, but not magnesium ethylene glycol-bis(β-aminoethyl ether)-N,N-tetraacetic acid-chelated low-antibody sera. The alternative pathway contributed more than half of the CL activity of both high- and low-antibody sera. However, after magnesium ethylene glycol-bis(β-aminoethyl ether)-N,N-tetraacetic acid chelation, only sera with high antibody concentrations or agammaglobulinemic serum reconstituted with immunoglobulin G with high specific antibody levels supported significant bactericidal activity. Therefore, type-specific antibody and complement promote opsonization of type XIV S. pneumoniae, and this may occur via either complement pathway. These results suggest that CL is a suitable tool to delineate serum factors and their contribution to opsonization, but results must be related to other functional assays.     

A Nice development: The first joint meeting of the British and French Societies for Developmental Biology, 13-16th September, 2003, Nice, France.

Held this autumn on the beautiful Cote d'Azur, the first joint meeting of the BSDB and SFBD provided delegates with the perfect informal setting for discussion spanning a broad cross-section of Developmental Biology. Participants' interests were diverse, ranging from the implementation of genome-wide approaches aimed at identifying all the molecular components of cell proliferation, signalling, patterning, and morphogenesis, to those engaged in capturing mesmerising glimpses of the minute and intricate workings of the cell. The meeting considered a wide spectrum of model organisms, including the simple plant Arabidopsis, the invertebrates Dictyostelium, Caenorhabditis elegans, and Drosophila melanogaster, the ascidian Ciona intestinalis, and the vertebrates Xenopus, zebrafish, chick, and mouse. Such a diverse approach served to highlight both similarities and differences in the molecular mechanisms that govern embryonic development among different species. Here, we highlight a few aspects of the meeting that illustrate this point.     

Role of Adherence in the Pathogenesis of Neonatal Group B Streptococcal Infection

The ability of group B streptococci to attach to buccal epithelial cells from adult volunteers, healthy neonates, and infants with invasive group B streptococcal infection was assessed by using (3)H-labeled bacteria incubated at a bacteria-to-cells ratio of 1,000:1. Type III group B streptococcal clinical isolates adhered significantly better to the epithelial cells of healthy neonates than to those of adults (mean bacteria per cell of 31 versus 7, respectively; P < 0.005). In contrast, no statistically significant differences in adherence of type Ia or type II strains to cells of neonates and adults were noted. The adherence of strains isolated from 15 infants with invasive group B streptococcal infection was significantly greater to the cells of infected infants than to those of age-matched controls (mean bacteria per cell of 39 versus 18, respectively; P < 0.005). In contrast, no significant difference was noted in the adherence of a usually adherent type Ia strain and a nonadherent type III strain to the cells of infected infants compared with control infants. These results indicate that the serotype of group B streptococci with the greatest virulence for neonates (type III) adheres better to neonatal than to adult epithelial cells. Infants who develop invasive infection may have an increased number of epithelial cell surface receptor sites for attachment of group B streptococci, the bacteria may elaborate products which unmask receptor sites, or both.     

Molecular genetics of pseudoxanthoma elasticum: type and frequency of mutations in ABCC6

Pseudoxanthoma elasticum (PXE) is a systemic heritable disorder that affects the elastic tissue in the skin, eye, and cardiovascular system. Mutations in the ABCC6 gene cause PXE. We performed a mutation screen in ABCC6 using haplotype analysis in conjunction with direct sequencing to achieve a mutation detection rate of 97%. This screen consisted of 170 PXE chromosomes in 81 families, and detected 59 distinct mutations (32 missense, eight nonsense, and six likely splice-site point mutations; one small insertion; and seven small and five large deletions). Forty-three of these mutations are novel variants, which increases the total number of PXE mutations to 121. While most mutations are rare, three nonsense mutations, a splice donor site mutation, and the large deletion comprising exons 23-29 (c.2996_4208del) were identified as relatively frequent PXE mutations at 26%, 5%, 3.5%, 3%, and 11%, respectively. Chromosomal haplotyping with two proximal and two distal polymorphic markers flanking ABCC6 demonstrated that most chromosomes that carry these relatively frequent PXE mutations have related haplotypes specific for these mutations, which suggests that these chromosomes originate from single founder mutations. The types of mutations found support loss-of-function as the molecular mechanism for the PXE phenotype. In 76 of the 81 families, the affected individuals were either homozygous for the same mutation or compound heterozygous for two mutations. In the remaining five families with one uncovered mutation, affected showed allelic compound heterozygosity for the cosegregating PXE haplotype. This demonstrates pseudo-dominance as the relevant inheritance mechanism, since disease transmission to the next generation always requires one mutant allelic variant from each parent. In contrast to other previous clinical and molecular claims, our results show evidence only for recessive PXE. This has profound consequences for the genetic counseling of families with PXE.     

Dental abnormalities in individuals with pathogenic germline variation in DICER1

Pathogenic germline variation in the microRNA processing gene DICER1 gives rise to an autosomal dominant, tumor‐predisposition disorder. Conditional deletion of Dicer1 in murine dental epithelium shows that it controls tooth patterning, size, number, and shape. The human dental phenotype of people with germline pathogenic variation in DICER1 is unknown. DICER1‐carriers (n = 57) and family controls (n = 55) were evaluated at the NIH Clinical Center dental clinic as part of a comprehensive medical evaluation. Digital panoramic radiographs, bite‐wing radiographs, and oral photographs were collected. A single observer, blind to DICER1 status, reviewed the dental records and determined the presence or absence of 11 dental characteristics as described in the clinic notes, radiographs, or oral photographs. Subjective phenotypes were reviewed on radiographs by two examiners (blind to DICER1 status) for the presence or absence of the dental characteristics to reduce inconsistencies. By simple association, bulbous crown, periodontitis, and taurodontism were all significant (p < .05). Logistic regression with chi‐square maximum likelihood estimates showed that bulbous crown and periodontitis remained significant. Recognition of these phenotypes may aid identification of individuals and families at risk for DICER1‐associated neoplasms. These findings may also guide dental care for individuals with germline DICER1 pathogenic variation.     

Student's perceptions of a virtual PBL experience.

OBJECTIVE: Our purpose is to present findings regarding student attitudes towards a virtual PBL program used to standardize their pediatric clinical experience. DESCRIPTION: With funding provided by the Fund for the Improvement of Post-Secondary Education, we developed Project LIVE (Learning through Interactive Video Education), a CD-ROM/Web hybrid program that uses digital video cases to conduct "virtual" problem-based learning groups with students doing a clinical rotation in a remote setting. Cases were progressively disclosed by videos of patient/physician encounters on a CD-ROM. Groups of five students and a faculty facilitator collaborated, teaching each other within the discussion section of the program. We conducted a multifaceted evaluation of Project LIVE to study the impact of case modality or distance on student learning and attitudes. We placed students in one of three groups (1) a face-to-face group with a paper case (FFT), (2) a face-to-face group with a video case (FFV), and (3) a virtual group (VG) with the digital video case. We then studied student attitudes about the three teaching formats. Over a six-month period three education specialists, who were not a part of the development team, conducted eight focus groups lasting one hour to assess student attitudes about Project LIVE. No one from the project team was present during these groups, and an independent evaluator analyzed the notes taken by each focus group leader. DISCUSSION: Trends across the groups included the following: (1) Authenticity (video)-Students reported that the authenticity of the case was a critical feature and that, "seeing (videos) made learning more memorable." Virtual and FFV groups reported more confidence in their ability to recognize abnormal findings in their patients. "You can't expect to teach clinical exam skills with a piece of paper." (2) Use of time-Students from all groups believed the cases were a good use of their time and improved their ability to solve clinical problems. They said it gave them an opportunity to "get away from just doing and focus on learning." However, the virtual groups complained of the lack of "a barometer for how much is too much" time. Some students reported spending an average of eight to ten hours per case over the period of a week. (In contrast, face-to-face groups met for three hours.) (3) Modeling clinical reasoning-Students believed the cases were valuable in structuring their knowledge, conceptualizing how to handle difficult situations, distinguishing abnormal from normal physical examination findings, and collaborating with their peers and their mentor to develop critical thinking. "It forced us to be logical" and ". how to think through the process-it mimics the real setting." (4) Technical support-The responsiveness of the Project LIVE staff was essential in assisting students in troubleshooting problems. (5) Distance component-Students preferred to work through the cases in face-to-face groups but agreed that the virtual experience is "good if you are in the middle of nowhere." This program was enjoyed by students and gave us an approach to standardizing experiences across multiple clinical sites.     

Measuring contraceptive values: An alternative approach

Previous assessments of individuals' values for various contraceptive consequences have employed one of four methodologies: free elicitation, direct ratings, multiple regression, or factor analysis. All four methodologies are flawed because they produce group rather than individual values, rely on rating scales, and fail to incorporate information regarding consequence trade-offs. Axiomatic conjoint measurement is proposed as an alternative methodology and used to determine individuals' values for a selected set of contraceptive consequences at two stages of the family-planning career.Preparation of this paper was supported in part by Grants HD-10802 and HD-14403 from the National Institute of Child Health and Human Development. Appreciation is due the Statistical Computing Facility of the University of California at Berkeley. Requests for reprints should be sent to the Publications Librarian, Center for Research on Judgment and Policy, Muenzinger Psychology Building, University of Colorado, Boulder, Colorado 80309-0344.