Preferential rearrangement of V kappa 4 gene segments in pre-B cell lines.

Examination of the in vitro V kappa gene rearrangements of murine adult bone marrow-derived pre-B cell lines reveals that 21 of 25 (84%) cell lines have rearranged a member of the V kappa 4 family. In contrast, analysis of two V kappa cDNA libraries prepared from LPS-stimulated adult spleen cells indicates that only 17% of the Ig kappa cDNAs contain sequences belonging to the V kappa 4 gene family. Half of the pre-B cell lines examined also share an 8-kbp BamHI reciprocal product (rp). However, these rp do not involve the same V kappa gene, indicating that conserved BamHI sites exist 3' of some V kappa genes. This rp is also readily detected in DNA from normal adult spleen cells, suggesting that the in vitro rearrangements examined in this study are representative of kappa rearrangements that occur in vivo. We suggest that, unlike the diverse V kappa repertoire expressed by mature B cells, the germline V kappa segments involved in initial rearrangements of the Ig kappa locus are highly restricted, and that an initial V kappa 4 rearrangement is probably followed by other, more random recombination events.

Strain variation in the frequency of Abelson murine leukemia virus-transformed fetal liver pre-B cells bearing complete immunoglobulin heavy chain rearrangements.

Fetal liver Abelson pre-B cell lines obtained from CBA/Tufts.xid and (CBA/Tufts.xid x CBA/Tufts)F1 mice have complete VDJH rearrangements on at least one allele. Such high frequencies of VDJH rearrangements have previously been observed in adult derived but not fetal liver derived Abelson pre-B cell lines. Genetic analyses suggest that CBA/Tufts.xid carries an autosomal dominant gene(s) that determines the predominance of VDJH rearrangements among transformants. This autosomal gene(s) might affect the intrinsic development of the early B cell lineage in the fetus or the fetal microenvironment, expanding pre-B cells of the "more mature" VDJH phenotype.

The organization of the mouse Igh-V locus. Dispersion, interspersion, and the evolution of VH gene family clusters.

We have constructed a panel of Abelson murine leukemia virus-transformed pre-B cells to study the organization of the mouse VH gene families. Based on the analyses of VH gene deletions on 51 chromosomes with VH gene rearrangements, we have inferred a map order of the Igh locus that holds for both the Igha and Ighb haplotypes. We show that members of each VH gene family are generally clustered, although three family clusters (VHS107, VH36-60, VGAM3.8) are dispersed in two or three subregions of the locus. Members of two VH gene families, VHQ52 and VH7183, are extensively interspersed and map within the same subregion. An examination of the distribution of VH group members (VH II, I, and III) within the locus suggests that two major duplications may, in part, explain the dispersed pattern of VH family clusters. The relationship of VH organization and functional expression is discussed in terms of position-dependent and complexity-driven models.

Molecular characterization of antibodies bearing Id-460. II. Molecular basis for Id-460 expression.

Id-460+ immunoglobulins can be induced in vivo by immunization with dinitrophenyl (DNP) or P. pneumotropica and form two nonoverlapping groups of antibodies with respect to antigen binding specificity. In this study, using Id-460+ antibodies of differing antigen binding specificities, we compared on the molecular genetic level the five gene segment combinations (VH, DH, JH, VL, and JL) that encode the variable regions of these idiotype-positive immunoglobulins. The Id-460 determinant appears to be a conformational or combinatorial determinant encoded by VH460 and VK1 crosshybridizing genes. DH, JH, and JK gene segments appear to have no measurable effect upon expression of Id-460. Finally, antigen binding specificity does not appear to simply localize to any particular gene segment but may in part be the result of somatic mutation and/or VDJH junctional sequences, whose length correlates roughly with antigen binding specificity.

Biological markers to establish a relationship between the health status of the St. Lawrence River yellow perch (Perca flavescens) with a gradient of anthropogenic disturbances.

Since the mid-1990s, the decline of the yellow perch population of Lake Saint-Pierre (hereinafter LSP) in Quebec, Canada has been the subject of several research programs. The combined effect of habitat deterioration, the presence of invasive species, and poor water quality negatively affected the yellow perch population in this lake. In 2013, we sampled yellow perch (larvae, juveniles and adults) at six sites along the St. Lawrence River representing a gradient of increasing human influences from upstream to downstream and measured several biomarkers including retinoid compounds (vitamin A). In the most contaminated sites (LSP, north and south shores), we found that retinoid stores were decreased in all three stages of development. To corroborate these results and to test other biomarkers, we once again sampled yellow perch (adults only) from the same sites. Results from our 2014 and 2015 samplings confirmed that LSP yellow perch appeared to be at a disadvantage compared to fish from upstream populations. Individuals from LSP have lower acetylcholinesterase (AChE) activity as well as lower retinoid levels in liver and plasma. These fish were also marked by lower levels of antioxidants such as lycopene and vitamin E. A discriminant analysis of this set of results confirmed that the yellow perch of the LSP could be easily discriminated from those of the other sites (2014 and 2015) on the basis of liver retinoid and, to a lesser extent, of the liver tocopherol and protein concentration of the muscle, as well as AChE activity and DROH (all-trans-3,4-dehydroretinol) measured in plasma.

Junctional diversification in the generation of the precursor of a discrete immune response.

Phosphocholine (PC)-specific antibodies that arise in the mouse in response to Proteus morganii (PM) and use V1-DFL16.1-JH1 are characterized by a number of recurring mutations. Most striking is an invariant A for G substitution in codon 95 of VH which results in an asparagine instead of aspartate at that position. Because of the apparent importance of this substitution in an anti-PC(PM) response, we wanted to determine the molecular basis for this base change. A cDNA library derived from pre-immune splenic B cells was examined for the frequency of VDJ containing the A substitution at 95 and the presence of additional point mutations in these sequences. Six different cDNA were isolated which contained an A substitution at the VD junction (frequency 0.00009); a seventh positive cDNA could not be examined. The V segments of four of these cDNA matched known germline genes and were, therefore, unmutated. Two others closely matched V in families whose members have not all been characterized, hence, it is not known whether the mutations observed are somatic or germline in origin. Sequences of 35 cDNA clones, containing the same V segment but differing in D, J and junctional nucleotides, revealed no mutations. These results indicate that the A substitution generated at codon 95 is most likely a product of V-DJ joining.

Germline diversity of the expressed BALB/c VhJ558 gene family.

Although the mouse immunoglobulin heavy chain (Igh) locus contains 15 heavy chain V (Vh) gene families, at least half of the Vh gene segments are members of the VhJ558 family. This large Vh gene family represents the least characterized germline coding regions of any of the mouse antigen receptor loci and the contribution of individual VhJ558 genes to the preimmune repertoire is poorly understood. In fact, relatively few germline VhJ558 sequences have been reported for BALB/c, the foundation strain for mouse immunoglobulin genetics and the prototypic strain of the Igh(a) haplotype. Here we present a database consisting of 66 sequences estimated to represent one-half of the total number of functional BALB/c VhJ558 genes. Our results indicate that a subset of the VhJ558 genes is highly expressed in the preimmune repertoire, with just nine Vh sequences accounting for nearly 50% of the VhJ558 heavy chains expressed by splenic B cells. We show that this disparity in the expressed Vh gene repertoire is not due to the position of the Vh genes relative to the Dh cluster or to multiple germline copies of the highly expressed VhJ558 genes. Together, these data constitute the first detailed analysis of functional BALB/c VhJ558 genes, demonstrate a striking bias in the use of particular VhJ558 genes in the preimmune repertoire, and provide sufficient information to study the regulation of the Dh-distal region of the Igh-V locus at the level of individual genes.

Progesterone receptors and ventilatory stimulation by progestin.

Progestin is thought to be a ventilatory stimulant but its effectiveness in raising ventilation is variable in humans and other species. We hypothesized that the level of progesterone receptors was an important determinant of the ventilatory response to progestin. Since estradiol induces progesterone receptor formation, we compared the ventilatory effect of the synthetic progestin medroxyprogesterone acetate (MPA) given in combination with estradiol with the effects of estradiol alone, MPA alone, or vehicle (saline) in ovariectomized rats. Animals receiving MPA alone had low numbers of progesterone receptors (2.43 pmol/g uterine wt) and had no change in ventilation, arterial Pco2, or Po2. MPA administration raised ventilation 23 +/- 5%, lowered arterial Pco2 3.2 +/- 0.9 Torr (both P less than 0.01) and tended to raise arterial Po2 when given in combination with estradiol to animals with increased numbers of progesterone receptors (4.85 pmol/g uterine wt). Estradiol alone produced the highest number of progesterone receptors (12.3 pmol/g uterine wt) but had no effect on ventilation or arterial Pco2 and decreased arterial Po2. Combined estradiol plus MPA treatment produced a greater fall in arterial Pco2 than did treatment with MPA alone, estradiol, or saline (all P less than 0.05). These results suggest that both an elevation in progestin levels and progesterone receptor numbers are required to stimulate ventilation.

Maternal hypoxic ventilatory response, ventilation, and infant birth weight at 4,300 m.

To test the hypothesis that increased hypoxic ventilatory responsiveness (HVR) raised maternal ventilation and arterial oxygenation during high-altitude pregnancy and related to the birth weight of the offspring, we studied 21 residents of Cerro de Pasco, Peru (4,300 m), while eight of them were 36 +/- 0 wk pregnant and 15 of them 13 +/- 0 wk postpartum. HVR was low in the nonpregnant women (mean +/- SE shape parameter A = 23 +/- 8) but increased nearly fourfold with pregnancy (A = 87 +/- 17). The increase in HVR appeared to account for the 25% rise in resting ventilation with pregnancy (delta VE observed = 2.4 +/- 0.7 l/min BTPS vs. delta VE predicted from delta HVR = 2.6 +/- 1.7 l/min BTPS, P = NS). Hyperoxia decreased ventilation in the pregnant women (P less than 0.01) to levels similar to those measured when nonpregnant. The increased ventilation of pregnancy raised arterial O2 saturation (SaO2) from 83 +/- 1 to 87 +/- 0%, and SaO2 was correlated positively with HVR in the pregnant women. The rise in SaO2 compensated for a 0.9 g/100 ml decrease in hemoglobin concentration to preserve arterial O2 content at levels present when nonpregnant. Cardiac output in the 36th wk of pregnancy did not differ significantly from values measured postpartum. The increase in HVR correlated positively with infant birth weight. An increase in HVR may be an important contributor to increased maternal ventilation with pregnancy and infant birth weight at high altitude.

Computer-assisted pedicle screw fixation. A feasibility study.

STUDY DESIGN: We evaluated a computer-assisted surgical tool for inserting pedicle screws. OBJECTIVES: This study reviewed the feasibility, usefulness, and accuracy of the proposed tool. SUMMARY OF BACKGROUND DATA: Reviews documented neurovascular damage caused by screw misplacement. Currently, screw hole position is assessed by radiologic means and curette palpation. METHODS: Three sheep vertebrae and one artificial object were reconstructed three-dimensionally from computed tomography scan slices. At surgery, the surgeon's movements were displayed relative to the three-dimensional vertebrae on a computer screen. The tool was used to detect pedicles and to verify the position of drilled holes. In our laboratory, we calculated the system's accuracy by taking measurements on the artificial object. RESULTS: All pedicles were identified with the computer. Five of the six drilled hole positions were correctly represented. An accuracy of 4.5 mm +/- 1.1 mm RMS (root of the mean squared) and 1.6 degrees +/- 1.2 degrees were calculated. CONCLUSIONS: Results suggested the proposed system could be useful for pedicle detection and assessing the intravertebral location of a drilled hole. The proposed system could be used for many different orthopedic procedures where structures are hidden from the surgeon's view.

Registration and geometric modelling of the spine during scoliosis surgery: a comparison study of different pre-operative reconstruction techniques and intra-operative tracking systems.

During scoliosis instrumentation surgery, it is difficult for surgeons fully to track vertebral motion in 3D, because only the posterior elements of the spine are exposed. Different intra-operative modelling approaches are evaluated using a registration technique that matches intra-operative measurements with a 3D pre-operative model of the spine. Two tracking systems (magnetic digitiser and mechanical arm) and two pre-operative reconstruction techniques (multiplanar radiography and CT scan) are sequentially combined to build four intra-operative models. Their accuracy is assessed by comparison with the pre-operative geometry. The most minimally invasive approach (multiplanar radiographic reconstruction and magnetic digitiser) has an accuracy of 5.9 mm in translation, and errors on vertebral rotations are 4.4 degrees, 6.7 degrees and 5.0 degrees in the frontal, sagittal and transverse planes, respectively. With CT scan reconstruction, the accuracy is significantly increased by about 2 mm in translation and as much as 4.5 degrees for vertebral rotations in the sagittal plane. For the mechanical arm, the accuracy is increased by less than 1 mm in translation and 1 degree for vertebral rotations. CT scan is the most accurate reconstruction technique, but its use for long spinal segments is generally not allowed because of the high radiation exposure. Multiplanar radiographic reconstruction may be an alternative solution for long spinal segments when great accuracy is not necessary. Considering the small increase in accuracy and its awkwardness, the use of the mechanical arm may not be appropriate during surgical manoeuvres.

Fiber characterization in a stationary ultrasonic field.

An instrument has been developed to characterize the mean dimensions of softwood fiber samples. It is based on the phenomenon that particles of cylindrical shape diluted in water and shorter than one fourth of the acoustic wavelength migrate to nodal planes of acoustic radiation pressure and reorient parallel to these planes when subjected to a stationary ultrasonic field. As the resonator operating frequency is 72 kHz, fibers up to 5 mm in length can be measured. The time evolution of the fiber suspension during ultrasonic excitation is monitored with a collimated beam of light. Scattered light signals collected off-axis in the plane perpendicular to the acoustic nodal planes are shown to be a function of the weighted average fiber length. Results are presented for pulp samples in the average fiber length range of 0.2 to 3 mm. It was found that there is a region where the scattered light is linearly related to concentration. Acoustooptical measurements obtained at initial concentration in this linear region, for all fractions, have shown that the longer the average length from screen classifier is, the faster the layer formation is. Since the fiber length the radius are proportional for a wood species, this observation is in agreement with the theoretical prediction.

Community values in Vermont health planning.

KIE: This is one of a set of six short articles, grouped under the umbrella title "Grassroots bioethics revisited: health care priorities and community values," with a very brief introduction by Bruce Jennings. The articles focus on the involvement of community health decisions projects with public policy issues of access to health care, allocation of resources, setting health care priorities, cost containment, and rationing.^ieng

[Evaluation of peroperative measurement system in the follow-up of vertebral displacements]. / Evaluation d'un système de mesures per-opératoires pour le suivi de déplacements vertébraux.

A computer assisted surgery system has been developed to quantify the vertebral displacements induced by the Cotrel-Dubousset instrumentation and to visualize in 3-D the spine at different per-operative steps. This spinal surgery is realized to correct idiopathic scoliosis. Per-operative measurements are obtained by using a magnetic digitizer. Five points per thoracic vertebra and six points per lumbar vertebra are digitized. The method proposed estimates the location of the digitized points on a computer graphics 3-D model of vertebrae by a point-to-surface matching algorithm. The 3-D models are constructed before the surgery using multiplanar radiographic reconstruction technique (2 or 3 views) and geometric modeling methods. Computer tools permit the 3-D visualization of the spine peroperatively and the evaluation of clinical indices such as Cobb angles and vertebral rotations. The system includes 3 principal error sources: 2) reconstruction error; 2) digitizer error (digitizer precision) and 3) point-to-surface matching error. In order to validate the overall system, measurements have been simulated for 2 vertebrae and 2 digitizers of different precision, taking onto account the reconstruction error. For 5 and 6 digitized points per vertebra, the matching algorithm gives a RMS error of respectively 3.0 and 2.0 mm.

Regulation of thymus-independent responses: unresponsiveness to a second challenge of TNP-Ficoll is mediated by hapten-specific antibodies.

CBA mice immunized with the TI-2 antigen TNP-Ficoll are unresponsive to a second challenge of the same antigen. In addition, spleen cells from unresponsive mice fail to respond to any dose of TNP-Ficoll in vitro. This hapten-specific unresponsive state is T cell independent and is due to hapten-specific IgM-mediated inhibition of B cell triggering. The mechanism of inhibition, although unknown, is unlikely to be antigen masking. In vitro responses to the TI-1 antigen TNP-LPS are not inhibited by concentrations of anti-TNP antibody, which completely suppress responses to TNP-Ficoll.

Molecular cloning of the primary IgH repertoire: a quantitative analysis of VH gene usage in adult mice.

The generation of the primary antibody repertoire requires the somatic rearrangement of germline gene segments. It is not known, however, whether all functional V and J gene segments have an equal probability of contributing to this initial set of antibody specificities. To address this issue, we have examined the relative utilization of VH and JH gene segments of the mouse. We have constructed VH cDNA phage libraries from C mu transcripts obtained from polyclonally activated spleen cells of the BALB/c and C57BL/6 strains. We show that probes specific for either one, two or three functional VH gene segments hybridize to cDNAs at frequencies directly proportional to the number of functional germline VH genes detected by each probe. In contrast, the representation of 10 VH gene families within each library indicates that certain families are under-represented relative to their estimated germline gene number. These families must either have extraordinary proportions of nonfunctional genes or are influenced by as yet unidentified regulatory mechanisms or constraints on rearrangement.