Lsc is required for marginal zone B cells, regulation of lymphocyte motility and immune responses.

Lsc (the murine homolog of human p115 Rho GEF) is a member of the Dbl-homology family of GTP exchange factors and is a specific activator of Rho. Lsc is activated by the G alpha(13) subunit of heterotrimeric G proteins and contains a regulator of G protein signaling domain that downmodulates G alpha(12) and G alpha(13). Lsc is expressed primarily in the hematopoietic system and links the activation of G alpha(12) and G alpha(13)-coupled receptors to actin polymerization in B and T cells. Lsc is essential for marginal zone B (MZB) cell homeostasis and for the generation of immune responses. Although Lsc-deficient lymphocytes show reduced basal motility, MZB cells show enhanced migration after serum activation. Thus, Lsc is a critical regulator of MZB cells and immune functions.

Compensation between Vav-1 and Vav-2 in B cell development and antigen receptor signaling.

Vav-1 and Vav-2 are closely related Dbl-homology GTP exchange factors (GEFs) for Rho GTPases. Mutation of Vav-1 disrupts T cell development and T cell antigen receptor-induced activation, but has comparatively little effect on B cells. We found that combined deletion of both Vav-1 and Vav-2 in mice resulted in a marked reduction in mature B lymphocyte numbers. Vav-1(-/-)Vav-2(-/-) B cells were unresponsive to B cell antigen receptor (BCR)-driven proliferation in vitro and to thymus-independent antigen in vivo. BCR-stimulated intracellular calcium mobilization was greatly impaired in Vav-1(-/-)Vav-2(-/-) B cells. These findings establish a role for Vav-2 in BCR calcium signaling and reveal that the Vav family of GEFs is critical to B cell development and function.

The oncogene product Vav is a crucial regulator of primary cytotoxic T cell responses but has no apparent role in CD28-mediated co-stimulation.

The guanine nucleotide-exchange factor Vav is a regulator of antigen-mediated cytoskeletal reorganization required for receptor clustering, proliferation and thymic selection. Moreover, Vav has been identified as a major substrate in the CD28 signal transduction pathway and overexpression of Vav enhances TCR-mediated IL-2 secretion in T cells. Here we show that CD3- plus CD28-mediated proliferation and IL-2 production were reduced in vav gene-deficient T cells. However, Vav had no apparent role in phorbol 12-myristate 13-acetate-plus CD28-mediated proliferation and IL-2 production, suggesting that Vav acts downstream of the TCR/CD3 complex. In vivo, Vav expression was crucial to generate primary vesicular stomatitis virus (VSV)-specific cytotoxic T cell responses. In contrast, vav-/- mice exhibited a reduced but significant footpad swelling after lymphocytic choriomeningitis virus (LCMV) infections and mounted a measurable primary cytotoxic T cell response to LCMV. Upon in vitro restimulation, cytotoxic T cell responses of both VSV- and LCMV-infected mice reached near normal levels. Our data provide the first genetic evidence that Vav is an important effector molecule that relays antigen receptor signaling to IL-2 production and activation of cytotoxic T cells.

The guanine-nucleotide exchange factor Vav is a crucial regulator of B cell receptor activation and B cell responses to nonrepetitive antigens.

The proto-oncogene product Vav is required for receptor clustering, proliferation, and differentiation of T cells, and Vav was identified as a substrate in the TCR and B cell receptor signaling pathway. The role of Vav in B cell responses to Ag challenge in vivo is not known. In this study, we show that Vav regulates B cell proliferation following in vitro activation of Ag receptors, but Vav has no apparent role in CD40-, IL-4-, or LPS-induced B cell activation. Increased degrees of Ag receptor cross-linking can partially reverse the proliferative defect in the anti-IgM response of vav-/- B cells. In vivo, vav-/- mice mounted protective antiviral IgM and IgG responses to infections with vesicular stomatitis virus and recombinant vaccinia virus expressing the vesicular stomatitis virus glycoprotein, which harbor repetitive surface epitopes that directly cross-link the Ag receptor and activate B cells in the absence of T cell help. vav-/- B cells also responded normally to the polyvalent, repetitive hapten Ag trinitrophenyl (TNP)-Ficoll that effectively cross-links B cell receptors. However, vav-/- mice failed to mount immune responses to the nonrepetitive, T cell-dependent hapten Ag (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP)-OVA. These results provide the first genetic evidence on the role of the guanine exchange factor Vav in immune responses to viral infections and antigenic challenge in vivo, and suggest that Vav adjusts the threshold for Ag receptor-mediated B cell activation depending on the nature of the Ag.

Vav links antigen-receptor signaling to the actin cytoskeleton.

The haematopoietic-specific Rho-family guanine-nucleotide exchange factor Vav is a regulator of lymphocyte antigen receptor signaling leading to proliferation of B and T cells, generation of the B1 cell lineage and IL-2 production and maturation in T cells. The specific role it plays in these events, however, has not yet been resolved. Recent findings suggest that Vav is recruited to activated antigen receptors and requires both tyrosine phosphorylation and the presence of activating phospholipids for catalytic activity towards Rho-family GTPases. Studies form vav-deficient mice show that in response to antigen receptor activation, Vav is not essential for activation of JNK kinase pathways, but is required for actin polymerisation and T cell capping. We discuss Vav function in the light of these new findings.

Vav is a regulator of cytoskeletal reorganization mediated by the T-cell receptor.

BACKGROUND: Vav is a guanine-nucleotide exchange factor for the Rho-like small GTPases RhoA, Rac1 and Cdc42, which regulate cytoskeletal reorganization and activation of stress-activated protein kinases (SAPK/JNKs). Vav is expressed in hematopoietic cells and is phosphorylated in T and B cells following activation of various growth factor or antigen receptors. Vav interacts with several signaling molecules in T cells, but the functional relevance of these interactions is established only for Slp76: they cooperate to induce activity of the transcription factor NF-AT and interleukin-2 expression. We have investigated the role of Vav in T cells by generating vav-/- mice. RESULTS: Mice deficient for vav were viable and healthy, but had impaired T-cell development. In vav-/- T cells, in response to activation of the T-cell receptor (TCR), cell cycle progression, induction of NF-ATc1 activity, downregulation of the cell-cycle inhibitor p27Kip1, interleukin-2 production, actin polymerization and the clustering of TCRs into patches and caps--a cytoskeletal reorganization process--were defective. TCR-mediated activation of mitogen-activated protein kinase and SAPK/JNK was unaffected. Ca2+ mobilization was impaired in vav-/- thymocytes and T cells. In wild-type cells, Vav constitutively associated with the cytoskeletal membrane anchors talin and vinculin. In the absence of Vav, phosphorylation of Slp76, Slp76-talin interactions, and recruitment of the actin cytoskeleton to the CD3 zeta chain of the TCR co-receptor were impaired. CONCLUSIONS: Vav is a crucial regulator of TCR-mediated Ca2+ flux, cytoskeletal reorganization and TCR clustering, and these are required for T-cell maturation, interleukin-2 production and cell cycle progression.

Regulation of the mating pheromone and invasive growth responses in yeast by two MAP kinase substrates.

BACKGROUND: In the budding yeast Saccharomyces cerevisiae, components of a single mitogen-activated protein (MAP) kinase pathway transduce two distinct signals, each of which activates an independent developmental programme: peptide mating pheromones initiate the mating response, whereas nutrient limitation initiates filamentous growth. One of the MAP kinases in this pathway, Fus3, triggers mating but antagonizes filamentous growth, while the other, Kss 1, preferentially triggers filamentous growth. Both kinases activate the same transcription factor, Ste 12, which can stimulate gene expression specific to each of the developmental programmes. The precise mechanism by which these MAP kinases activate Ste 12, however, is not clear. RESULTS: Two newly identified proteins, Rst 1 and Rst 2 (also known as Dig1 and Dig2), were found to associate physically with Fus3 and Ste12. Rst1 and Rst2 were prominent substrates in kinase reactions of Fus3 immune complexes from pheromone-treated cells. Association of Fus3 with Ste12 required Rst1 and Rst2, and activation of Fus3 by pheromone caused release of Ste12 from the Fus3 complex. Although rst1 and rst2 single mutants had no obvious phenotype, both filamentous growth and mating-specific gene expression were constitutive in rst1 rst2 double mutants. The phenotype of rst1 rst2 cells required Ste12 function, but did not require the function of upstream kinases. Consistent with Rst1 and Rst2 having a role in Ste12 regulation, both proteins were localized to the nucleus. CONCLUSIONS: Rst1 and Rst2 repress the mating and filamentous growth responses of S. cerevisiae by directly inhibiting Ste12. Activation of Fus3 or Kss1 may cause phosphorylation-dependent release of Ste12 from Rst1/Rst2 and thereby activate Ste12-dependent transcription.

Genetic instability of chromosome 3 in HPV-immortalized and tumorigenic human keratinocytes.

The HPV-1811 cell line is derived from primary human foreskin keratinocytes that have been transfected with human papilloma virus type 18. At late passage, these cells produce invasive squamous cell carcinomas when injected into nude mice. A striking, but unstable, aberration of chromosome 3 occurs very early after establishment of the culture; a consistent rearrangement is observed concomitant with tumorigenicity. Using molecular cytogenetic techniques, we characterized the complex development of this aberration. A whole chromosome probe to this chromosome was made by linker-adapter PCR amplification of a single flow-sorted chromosome. Hybridization of this probe to normal metaphase chromosomes revealed the der (3) to be composed of chromosome 3, distal 13q, and 21q22. Hybridization of a 3q subtelomeric probe and a glycoprotein V probe which maps to 3qter indicated that this locus is duplicated in the final form of the chromosome, but that much instability occurs prior to its establishment. The ETS2 oncogene, which maps to 21q22, is translocated to the der(3) when the cell line becomes tumorigenic, but not prior to this time. Early-passage cells which have been induced to become tumorigenic by exposure to the carcinogen nitrosomethylurea also have the localization of the ETS2 at 3qter.

A pilot study to test the influence of specific prosthetic features in preventing trans-tibial amputees from walking like able-bodied subjects.

The purpose of this pilot investigation was to develop a method to test the influence of specific prosthetic features in preventing trans-tibial amputees from walking like able-bodied subjects. An able-bodied subject was fitted with a patellar-tendon-bearing orthosis incorporating several features of an amputee's prosthesis. Kinetic, kinematic and metabolic data were collected as features were systematically removed from the orthosis. While wearing the orthosis the gait of the able-bodied subject closely simulated trans-tibial amputee gait kinematically, kinetically and metabolically. Although it was obvious that the various prosthetic features influenced the kinetics and kinematics of gait, they were difficult to quantify with only a single subject. However, the two features which appeared to have the largest influence in preventing trans-tibial amputees from walking like able-bodied subjects were patellar tendon loading and a solid ankle.

A comparison of oxygen consumption during walking between children with and without below-knee amputations.

BACKGROUND AND PURPOSE: Differences, if any, in energy costs during walking of children with below-knee amputations (BKAs) and those of children without amputations have not been quantified. The purpose of this investigation was to compare measures of heart rate and oxygen consumption during walking (1) between children with BKAs and long residual limbs and children with BKAs and short residual limbs and (2) between children with BKAs and children without amputations. SUBJECTS: Twenty-four children volunteered to participate in this investigation. Ten of the children, aged 6 to 18 years, had BKAs, and 14 children, aged 6 to 17 years, were without amputations. METHODS: The subjects walked for 2 minutes at each of the following four speeds: (1) chosen walking speed (CWS), (2) 20% below CWS, (3) 20% above CWS, and (4) fixed speed of 1.2 m/s. Heart rate and oxygen uptake were measured at each speed. RESULTS: The results indicated (1) that there were no significant differences between children with long residual limbs and those with short residual limbs; (2) that oxygen consumption was 15% greater for children with BKAs compared with children without amputations; (3) that there were no differences in heart rates between children without amputations and those with BKAs or within children with BKAs; and (4) that children with BKAs did not choose speeds different from their peers without amputations, regardless of stump length. CONCLUSION AND DISCUSSION: The results indicated that children with BKAs had higher energy needs for walking than children who had no amputation. Whether the increased energy needs prevent or inhibit children with BKAs from having a lifestyle comparable to that of children without amputations is currently unknown and warrants further research. [Herbert LM, Engsberg JR, Tedford KG, Grimston SK. A comparison of oxygen consumption during walking between children with and without below-knee amputations.

Normative ground reaction force data for able-bodied and trans-tibial amputee children during running.

The purpose of this investigation was to develop normative ground reaction force data for able-bodied (AB) and trans-tibial amputee (TTA) children during running. Two hundred AB (mean age 9.4 years, range 7-12) and 21 TTA (mean age 11.1 years, range 5-17) children ran (2.2 m/s +/- 10%) over a force platform. Ground reaction force data were normalized, averaged within groups and plotted to produce force-time curves characterizing the different leg types (i.e. able-bodied, non-prosthetic and prosthetic). In addition, discrete variables characterizing the leg type differences were determined. One way ANOVA determined significant differences between variables and a TukeyB Post Hoc analysis defined which variables were significantly different (p < 0.05). Results generally indicated differences between the three leg types with the non-prosthetic leg indicating greater forces than the prosthetic and AB legs. The results of this investigation provide normative ground reaction force data for both AB and TTA children during running and can be used for comparison with other groups of children.

Normative ground reaction force data for able-bodied and below-knee-amputee children during walking.

We determined normative ground reaction force data for able-bodied (AB) and below-knee-amputee (BKA) children during walking. Twenty-two BKA and 225 able-bodied children walked over two adjacent force platforms. Average normalized force-time curves were determined for legs of the children for subjective comparisons, and discrete variables were determined for statistical comparisons. No significant differences existed between (a) right and left legs, (b) gender, and (c) age for the AB children. BKA children had an asymmetrical gait pattern with a dominant role of the nonprosthetic limb. This dominant role was related to a greater rate of loading, magnitude of loading, impulse, and time of loading as compared with prosthetic limbs and with the limbs of AB children.

Weight distribution of below-knee amputee and able-bodied children during standing.

The purpose of this investigation was to compare weight distributions of a relatively large number of below-knee (BK) amputee and able-bodied children during two different standing positions. Twenty-one BK amputees and 200 able-bodied children volunteered as subjects for this investigation. Each child stood on a pressure plate and three sets of trial data were collected. One set of trial data was collected with both feet together on the pressure plate and two were collected with feet placed 20cm apart. The total force applied by each foot to the pressure plate was normalised by dividing by subject weight to yield foot force to body weight ratios. Data were separated into forefoot and rearfoot areas, force for the forefoot area was then calculated and normalised by dividing by total foot force to yield forefoot to whole-foot force ratios. Ratios for the two foot placement conditions and for non-prosthetic, prosthetic, dominant, and non-dominant feet were compared using paired t-tests (p < 0.05). Results indicated that: 1) BK amputee children placed more weight on their non-prosthetic limb than their prosthetic limb, yet this was not different from able-bodied children in respect of weight distribution between dominant and non-dominant limbs; 2) approximately 90% of the load on the prosthetic foot was placed on the forefoot; and 3) the load on the non-prosthetic foot was evenly distributed between the forefoot and rearfoot like that of able-bodied children. It was concluded that except for substantially more weight on the forefoot of the prosthetic leg BK amputee children stood in the same way as able-bodied children.

Center of mass location and segment angular orientation of below-knee-amputee and able-bodied children during walking.

This investigation compared the center of mass (COM) locations and segment angular orientations of the gait of below-knee-amputee (BKA) children to those of able-bodied (AB) children. Eleven AB children (mean age, 8.4 years) and three BKA children (mean age, 7.5 years) volunteered to participate as subjects. Film data (100 frames per second) of a typical walking stride, were collected for the children during four experimental sessions held at six-month intervals. Two 16mm cameras were used to obtain frontal and lateral views of the subjects. The same approximate rate of walking (1.2m/s +/- 10%) was enforced for all testing sessions and at least three trials of data were collected for each subject during each session. Segmental endpoints of each subject from the film of each camera were digitized and the direct linear transformation (DLT) method was used to obtain three-dimensional segmental location-time data for a complete stride. These data were then used to determine whole body COM locations and angular orientations for the segments. Discrete values describing normalized locations at touchdown, midsupport, and takeoff were determined for whole body COM and the angular orientation of the trunk, thighs, and legs. In the sagittal plane the COM was lower and more anterior for the BKA children when compared to that of the AB children. This was primarily due to the greater forward flexion of the trunk.(ABSTRACT TRUNCATED AT 250 WORDS)

Concentric and eccentric quadriceps torque in pre-adolescent males.

The purpose of this study was to evaluate the applicability of using the Kin Com (Chattecx Corp., Chattanooga, TN) isokinetic machine to measure concentric and eccentric quadriceps torque in a group of 12 healthy male volunteers aged 10-12 years. Each individual was tested by an experienced physiotherapist using a 60 degree per second velocity mode according to our standardized protocol. Average and peak torque values for concentric and eccentric contractions of the quadriceps were recorded; based upon the best of three maximum effort trials on each lower extremity. Retesting was performed on a randomly selected sub-group in an identical manner two weeks later. Our results showed no statistically significant difference between the original and retest values using the method error of repeated measurements and paired t-test analyses. Eccentric peak torque was greater on average than concentric. This was significant with p-values of 0.01 for the non-dominant quadriceps and 0.002 for the dominant side (paired t-test). There was no significant difference between the dominant and non-dominant sides. In conclusion, eccentric muscle testing has been reliably quantitated for the first time in children. This study has shown a practical and reproducible method of quantitative muscle strength assessment.