Multispecificity of a recombinant anti-ras monoclonal antibody.

Recombinant monoclonal antibodies (Ab's) have widespread application as research tools, diagnostic reagents and as biotherapeutics. Whilst studying the cellular molecular switch protein m-ras, a recombinant monoclonal antibody to m-ras was generated for use as a research tool. Antibody genes from a single rabbit B cell secreting IgG to an m-ras specific peptide sequence were expressed in mammalian cells, and monoclonal rabbit IgG binding was characterized by ELISA and peptide array blotting. Although the monoclonal Ab was selected for specificity to m-ras peptide, it also bound to both recombinant full-length m-ras and h-ras proteins. The cross-reactive binding of the monoclonal Ab to h-ras was defined by peptide array blot revealing that the Ab showed preference for peptide sequences containing multiple positively charged amino acid residues. These data reinforce the concept of antibody multispecificity through multiple interactions of the Ab paratope with diverse polypeptides. They also emphasize the importance of immunogen and Ab selection processes when generating recombinant monoclonal Ab's.

Structures of Preferred Human IgV Genes-Based Protective Antibodies Identify How Conserved Residues Contact Diverse Antigens and Assign Source of Specificity to CDR3 Loop Variation.

The human Ab response to certain pathogens is oligoclonal, with preferred IgV genes being used more frequently than others. A pair of such preferred genes, IGVK3-11 and IGVH3-30, contributes to the generation of protective Abs directed against the 23F serotype of the pneumonococcal capsular polysaccharide of Streptococcus pneumoniae and against the AD-2S1 peptide of the gB membrane protein of human CMV. Structural analyses of Fab fragments of mAbs 023.102 and pn132p2C05 in complex with portions of the 23F polysaccharide revealed five germline-encoded residues in contact with the key component, l-rhamnose. In the case of the AD-2S1 peptide, the KE5 Fab fragment complex identified nine germline-encoded contact residues. Two of these germline-encoded residues, Arg91L and Trp94L, contact both the l-rhamnose and the AD-2S1 peptide. Comparison of the respective paratopes that bind to carbohydrate and protein reveals that stochastic diversity in both CDR3 loops alone almost exclusively accounts for their divergent specificity. Combined evolutionary pressure by human CMV and the 23F serotype of S. pneumoniae acted on the IGVK3-11 and IGVH3-30 genes as demonstrated by the multiple germline-encoded amino acids that contact both l-rhamnose and AD-2S1 peptide.

Characterization of pathogenic human monoclonal autoantibodies against GM-CSF.

The origin of pathogenic autoantibodies remains unknown. Idiopathic pulmonary alveolar proteinosis is caused by autoantibodies against granulocyte-macrophage colony-stimulating factor (GM-CSF). We generated 19 monoclonal autoantibodies against GM-CSF from six patients with idiopathic pulmonary alveolar proteinosis. The autoantibodies used multiple V genes, excluding preferred V-gene use as an etiology, and targeted at least four nonoverlapping epitopes on GM-CSF, suggesting that GM-CSF is driving the autoantibodies and not a B-cell epitope on a pathogen cross-reacting with GM-CSF. The number of somatic mutations in the autoantibodies suggests that the memory B cells have been helped by T cells and re-entered germinal centers. All autoantibodies neutralized GM-CSF bioactivity, with general correlations to affinity and off-rate. The binding of certain autoantibodies was changed by point mutations in GM-CSF that reduced binding to the GM-CSF receptor. Those monoclonal autoantibodies that potently neutralize GM-CSF may be useful in treating inflammatory disease, such as rheumatoid arthritis and multiple sclerosis, cancer, and pain.

Absence of M-Ras modulates social behavior in mice.

BACKGROUND: The molecular mechanisms that determine social behavior are poorly understood. Pheromones play a critical role in social recognition in most animals, including mice, but how these are converted into behavioral responses is largely unknown. Here, we report that the absence of the small GTPase M-Ras affects social behavior in mice. RESULTS: In their interactions with other males, Mras(-/-) males exhibited high levels of territorial aggression and social investigations, and increased fear-related behavior. They also showed increased mating behavior with females. Curiously, increased aggression and mating behaviors were only observed when Mras(-/-) males were paired with Mras(-/-) partners, but were significantly reduced when paired with wild-type (WT) mice. Since mice use pheromonal cues to identify other individuals, we explored the possibility that pheromone detection may be altered in Mras(-/-) mice. Unlike WT mice, Mras(-/-) did not show a preference for exploring unfamiliar urinary pheromones or unfamiliar isogenic mice. Although this could indicate that vomeronasal function and/or olfactory learning may be compromised in Mras(-/-) mice, these observations were not fully consistent with the differential behavioral responses to WT and Mras(-/-) interaction partners by Mras(-/-) males. In addition, induction of c-fos upon pheromone exposure or in response to mating was similar in WT and Mras (-/-) mice, as was the ex vivo expansion of neural progenitors with EGF. This indicated that acute pheromone detection and processing was likely intact. However, urinary metabolite profiles differed between Mras(-/-) and WT males. CONCLUSIONS: The changes in behaviors displayed by Mras(-/-) mice are likely due to a complex combination of factors that may include an inherent predisposition to increased aggression and sexual behavior, and the production of distinct pheromones that could override the preference for unfamiliar social odors. Olfactory and/or social learning processes may thus be compromised in Mras(-/-) mice.

Somatic diversity in CDR3 loops allows single V-genes to encode innate immunological memories for multiple pathogens.

The human Ab response to many common pathogens is oligoclonal, with restricted usage of Ig V-genes. Intriguingly, the IGVK3-11 and IGVH3-30 V-genes are repeatedly paired in protective Abs against the 23F polysaccharide of Streptococcus pneumoniae, as well as against the gB envelope protein of human CMV, where germline-encoded amino acids make key contacts with the gB protein. We constructed IgGs encoded by the germline IGVK3-11 and IGVH3-30 V-genes together with DNA encoding the respective CDR3 regions of the L chain and H chain found in a hypermutated anti-23F Ab. These IgGs encoded by germline V-genes bound specifically to 23F pneumococcal capsular polysaccharides with no reactivity to other serotypes of pneumococcal capsular polysaccharides or arrayed glycans and recognized L-rhamnose, a component of the 23F repeating subunit. IgGs encoded by this pair of germline V-genes mediated complement-dependent phagocytosis of encapsulated 23F S. pneumoniae by human neutrophils. Mutations in CDRL3 and CDRH3 had significant effects on binding. Thus, IGKV3-11 and IGHV3-30, depending on with which distinct DNA sequences encoding CDR3 they are recombined, can encode binding sites for protective Abs against chemically distinct Ags and thus, may encode innate immunological memory against human CMV and S. pneumoniae.

Germline V-genes sculpt the binding site of a family of antibodies neutralizing human cytomegalovirus.

Immunoglobulin genes are generated somatically through specialized mechanisms resulting in a vast repertoire of antigen-binding sites. Despite the stochastic nature of these processes, the V-genes that encode most of the antigen-combining site are under positive evolutionary selection, raising the possibility that V-genes have been selected to encode key structural features of binding sites of protective antibodies against certain pathogens. Human, neutralizing antibodies to human cytomegalovirus that bind the AD-2S1 epitope on its gB envelope protein repeatedly use a pair of well-conserved, germline V-genes IGHV3-30 and IGKV3-11. Here, we present crystallographic, kinetic and thermodynamic analyses of the binding site of such an antibody and that of its primary immunoglobulin ancestor. These show that these germline V-genes encode key side chain contacts with the viral antigen and thereby dictate key structural features of the hypermutated, high-affinity neutralizing antibody. V-genes may thus encode an innate, protective immunological memory that targets vulnerable, invariant sites on multiple pathogens.

Distinct structural features of caprin-1 mediate its interaction with G3BP-1 and its induction of phosphorylation of eukaryotic translation initiation factor 2alpha, entry to cytoplasmic stress granules, and selective interaction with a subset of mRNAs.

Caprin-1 is a ubiquitously expressed, well-conserved cytoplasmic phosphoprotein that is needed for normal progression through the G(1)-S phase of the cell cycle and occurs in postsynaptic granules in dendrites of neurons. We demonstrate that Caprin-1 colocalizes with RasGAP SH3 domain binding protein-1 (G3BP-1) in cytoplasmic RNA granules associated with microtubules and concentrated in the leading and trailing edge of migrating cells. Caprin-1 exhibits a highly conserved motif, F(M/I/L)Q(D/E)Sx(I/L)D that binds to the NTF-2-like domain of G3BP-1. The carboxy-terminal region of Caprin-1 selectively bound mRNA for c-Myc or cyclin D2, this binding being diminished by mutation of the three RGG motifs and abolished by deletion of the RGG-rich region. Overexpression of Caprin-1 induced phosphorylation of eukaryotic translation initiation factor 2alpha (eIF-2alpha) through a mechanism that depended on its ability to bind mRNA, resulting in global inhibition of protein synthesis. However, cells lacking Caprin-1 exhibited no changes in global rates of protein synthesis, suggesting that physiologically, the effects of Caprin-1 on translation were limited to restricted subsets of mRNAs. Overexpression of Caprin-1 induced the formation of cytoplasmic stress granules (SG). Its ability to bind RNA was required to induce SG formation but not necessarily its ability to enter SG. The ability of Caprin-1 or G3BP-1 to induce SG formation or enter them did not depend on their association with each other. The Caprin-1/G3BP-1 complex is likely to regulate the transport and translation of mRNAs of proteins involved with synaptic plasticity in neurons and cellular proliferation and migration in multiple cell types.

Microtubule-dependent association of AKAP350A and CCAR1 with RNA stress granules.

Recent investigations have highlighted the importance of subcellular localization of mRNAs to cell function. While AKAP350A, a multifunctional scaffolding protein, localizes to the Golgi apparatus and centrosomes, we have now identified a cytosolic pool of AKAP350A. Analysis of AKAP350A scaffolded complexes revealed two novel interacting proteins, CCAR1 and caprin-1. CCAR1, caprin-1 and AKAP350A along with G3BP, a stress granule marker, relocate to RNA stress granules after arsenite treatment. Stress also caused loss of AKAP350 from the Golgi and fragmentation of the Golgi apparatus. Disruption of microtubules with nocodazole altered stress granule formation and changed their morphology by preventing fusion of stress granules. In the presence of nocodazole, arsenite induced smaller granules with the vast majority of AKAP350A and CCAR1 separated from G3BP-containing granules. Similar to nocodazole treatment, reduction of AKAP350A or CCAR1 expression also altered the size and number of G3BP-containing stress granules induced by arsenite treatment. A limited set of 69 mRNA transcripts was immunoisolated with AKAP350A even in the absence of stress, suggesting the association of AKAP350A with mRNA transcripts. These results provide the first evidence for the microtubule dependent association of AKAP350A and CCAR1 with RNA stress granules.

FGF signals induce Caprin2 expression in the vertebrate lens.

The lens of the eye is derived from the non-neural ectoderm situated next to the optic vesicle. Fibroblast growth factor (FGF) signals play a major role at various stages of vertebrate lens development ranging from induction and proliferation to differentiation. Less is however known about the identity of genes that are induced by FGF activity within the lens. We have isolated and characterized mouse cytoplasmic activation/proliferation-associated protein-2 (Caprin2), with domains belonging to both the Caprin family and the C1q and tumour necrosis factor (TNF) super-family. Here we show that Caprin2 is expressed in the developing vertebrate lens in mouse and chick, and that Caprin2 expression is up-regulated in primary lens fiber cells, after the induction of crystallins the earliest known markers for differentiated lens fiber cells. Caprin2 is subsequently down-regulated in the centre of the lens at the time and at the position of the first fiber cell denucleation and terminal differentiation. In vitro analyses of lens fiber cell differentiation provide evidence that FGF activity emanating from neighboring prospective retinal cells is required and that FGF8 activity is sufficient to induce Caprin2 in lens fiber cells. These results not only provide evidence that FGF signals induce the newly characterized protein Caprin2 in the lens, but also support the general idea that FGF signals are required for lens fiber cell differentiation.

Analysis of clinical motor testing for adult patients with diagnosed ulnar neuropathy at the elbow.

OBJECTIVE: To compare the dichotomous results for 7 ulnar nerve clinical motor tests (Froment's sign, Wartenberg's sign, finger flexion sign, Jeanne's sign, crossed finger test, Egawa's sign, presence of clinical fasciculations) with motor nerve conduction velocity findings. DESIGN: A static group comparison design assessed for differences among dichotomous test outcomes with respect to motor nerve conduction velocity. SETTING: Five medical facilities throughout the United States provided data for this study. PARTICIPANTS: Records from participants (N=26) with diagnosed ulnar neuropathy at the elbow were included for data analysis. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Demographic data included age, sex, handedness, duration of symptoms, and the number of days between the clinical and electrodiagnostic exam. Other dependent variables included motor conduction velocity of the ulnar nerve, compound muscle action potential amplitude, and the dichotomous clinical motor test outcomes. RESULTS: Two motor signs, the presence of clinical fasciculations and a positive finger flexion sign, were identified more frequently (each present in 11 patients) than the other motor signs. An analysis of covariance revealed significant differences in motor nerve conduction velocity between positive and negative results for all the clinical motor tests except for the finger flexion sign. Significant chi-square analyses were found for the following comparisons: the presence of clinical fasciculations and Froment's sign, the finger flexion sign and the crossed finger test, Egawa's sign and Froment's sign, Warteberg's sign and Froment's sign, the crossed finger test and Froment's sign, and Egawa's sign and Wartenberg's sign. CONCLUSIONS: Some clinical motor tests are better than others at identifying early motor involvement, providing the rehabilitation professional some insight regarding the relative decrement of motor nerve conduction velocity when a selected test is positive.

The ability of 4 single-limb hopping tests to detect functional performance deficits in individuals with functional ankle instability.

STUDY DESIGN: Experimental laboratory testing using a cross-sectional design. OBJECTIVES: To determine if functional performance deficits are present in individuals with functional ankle instability (FAI) in 4 single-limb hopping tests, including figure-of-8 hop, side hop, 6-meter crossover hop, and square hop. BACKGROUND: Conflicting results exist regarding the presence of functional deficits in individuals with FAI. It is important to evaluate whether functional performance deficits are present in this population, as well as if subjective feelings of giving way can assist in identifying these deficits. METHODS: Sixty college students volunteered for this study. Thirty participants with unilateral ankle instability were placed in the FAI group and 30 participants with no history of ankle injuries were placed in the control group. The FAI group was subsequently further divided to indicate those that reported giving way during the functional test (FAI-GW) and those that did not (FAI-NGW). Time to complete each test was recorded and the mean of 3 trials for each test were used for statistical analysis. To identify performance differences, we used 4 mixed-design 2-way (side-by-group) ANOVAs, 1 for each hop test. A Tukey post hoc test was completed on all significant findings. RESULTS: We identified a significant side-by-group interaction for all 4 functional performance tests (P<.05). Specifically, for each functional performance test, the FAI limb performed significantly worse than the contralateral uninjured limb in the FAI-GW group. Additionally, the FAI limb in the FAI-GW group performed worse than the FAI limb in the FAI-NGW group, and the matched limb in the control group in 3 of the 4 functional performance tests. CONCLUSION: We found that functional performance deficits were present in participants with FAI who also experienced instability during the test. This difference was identified when comparing the FAI limb to the contralateral uninjured limb as well as control participants. However, the performance deficits identified in this study were relatively small. Future research in this area is needed to further evaluate the clinical meaningfulness of these findings. Finally, we found that limb dominance did not affect performance.

Severity of functional and mechanical ankle instability in an active population.

BACKGROUND: The purpose of this investigation was to evaluate the relationship between the severity of functional and mechanical ankle instability in physically active individuals. MATERIALS AND METHODS: Eighty college aged physically active individuals from a large university were recruited for this study. All subjects had unilateral functional ankle instability (FAI). FAI was defined as a history of at least two ankle sprains and a score less than or equal to 27 on the Cumberland Ankle Instability Tool (CAIT). The contralateral limb had no history of ankle injury or instability. Anterior displacement (mm) and talar tilt (degrees) were measured using the LigMaster joint arthrometer to identify mechanical ankle instability(MAI). Individuals were tested bilaterally and the maximum value attained during talar tilt and anterior displacement was used for statistical analysis. RESULTS: First we evaluated side-to-side differences in MAI in all subjects. We found no significant difference between the FAI and the non-FAI ankle for anterior displacement (t(1.79)=1.66, p = 0.10) or talar tilt (t(1.79)=-0.07, p=0.95). Secondly, we evaluated the relationship between the FAI and MAI measures and found no significant correlations between the severity of FAI and magnitude of anterior displacement(r=0.18, p=0.12) or talar tilt (r=0.09, p=0.42). CONCLUSION: This study demonstrated there was no side-to-side difference in MAI in individuals with unilateral functional ankle instability. Similarly, we also conclude there was no significant relationship between the severity of FAI and MAI. We feel that these findings suggest the symptoms of FAI may not be related to ankle joint laxity, but instead due to other factors associated with FAI.

Ground Reaction Forces in Ballet Differences Resulting from Footwear and Jump Conditions.

In the research devoted to ballet, ground reaction force (GRF) and shoe condition have been identified as possible risk factors for injury. Shoe conditions vary immensely between dancers and could indeed have significant impact on biomechanics and injury rates. Therefore, the objectives of this study were: 1. to investigate the maximal ground reaction force (GRFmax) when ballet dancers land from two jump conditions in pointe shoes, in flat technique shoes, and barefoot; and 2. to explore the effects that specific pointe shoe characteristics (shoe age, shank style) have on GRFmax. Twenty-one healthy female ballet majors in an elite college program volunteered for the study. All participants had similar years of classical ballet training (12.85 ± 2.37). For the study, they performed two ballet jumps, assemblé and grand jeté. Each jump was performed in the three shoe conditions mentioned previously. A total of 18 trials per subject were completed, with the order of jump type and shoe condition randomized. Each jump was landed on a force plate, and maximal GRFs were recorded. A repeated measures analysis of variance was calculated with two within subject factors, shoe type at three levels and jump type at two levels. Tukey's post hoc test was applied to significant findings. Alpha level was set a priori at p = 0.05. Results demonstrated no significant differences in GRFmax between the three shoe conditions; however, significant differences in GRFmax between the jump types were identified. Post-hoc testing revealed that when dancers performed the grand jeté jump, higher GRFmax was obtained compared to the assemblé jump. In conclusion, results of this study indicate that GRFmax varies between ballet jumps; however, it does not appear that shoe condition significantly affects GRFmax.

The mechanism of GM-CSF inhibition by human GM-CSF auto-antibodies suggests novel therapeutic opportunities.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that can stimulate a variety of cells, but its overexpression leads to excessive production and activation of granulocytes and macrophages with many pathogenic effects. This cytokine is a therapeutic target in inflammatory diseases, and several anti-GM-CSF antibodies have advanced to Phase 2 clinical trials in patients with such diseases, e.g., rheumatoid arthritis. GM-CSF is also an essential factor in preventing pulmonary alveolar proteinosis (PAP), a disease associated with GM-CSF malfunction arising most typically through the presence of GM-CSF neutralizing auto-antibodies. Understanding the mechanism of action for neutralizing antibodies that target GM-CSF is important for improving their specificity and affinity as therapeutics and, conversely, in devising strategies to reduce the effects of GM-CSF auto-antibodies in PAP. We have solved the crystal structures of human GM-CSF bound to antigen-binding fragments of two neutralizing antibodies, the human auto-antibody F1 and the mouse monoclonal antibody 4D4. Coordinates and structure factors of the crystal structures of the GM-CSF:F1 Fab and the GM-CSF:4D4 Fab complexes have been deposited in the RCSB Protein Data Bank under the accession numbers 6BFQ and 6BFS, respectively. The structures show that these antibodies bind to mutually exclusive epitopes on GM-CSF; however, both prevent the cytokine from interacting with its alpha receptor subunit and hence prevent receptor activation. Importantly, identification of the F1 epitope together with functional analyses highlighted modifications to GM-CSF that would abolish auto-antibody recognition whilst retaining GM-CSF function. These results provide a framework for developing novel GM-CSF molecules for PAP treatment and for optimizing current anti-GM-CSF antibodies for use in treating inflammatory disorders.

Location, location, timing: analysis of cytomegalovirus epitopes for neutralizing antibodies.

Attempts to produce a vaccine to human cytomegalovirus (HCMV) have failed. The principal target of the humoral immune response to HCMV is the viral envelope glycoprotein gB, which contains several immunodominant epitopes. Here we discuss human antibodies reacting with gB and offer an explanation as to why most humans make antibodies to an epitope that does not always elicit neutralizing antibodies. We suggest modifications to gB for an improved HCMV vaccine design.

Distinct mechanisms determine the patterns of differential activation of H-Ras, N-Ras, K-Ras 4B, and M-Ras by receptors for growth factors or antigen.

Although GTPases of the Ras family have been implicated in many aspects of the regulation of cells, little is known about the roles of individual family members. Here, we analyzed the mechanisms of activation of H-Ras, N-Ras, K-Ras 4B, and M-Ras by two types of external stimuli, growth factors and ligation of the antigen receptors of B or T lymphocytes (BCRs and TCRs). The growth factors interleukin-3, colony-stimulating factor 1, and epidermal growth factor all preferentially activated M-Ras and K-Ras 4B over H-Ras or N-Ras. Preferential activation of M-Ras and K-Ras 4B depended on the presence of their polybasic carboxy termini, which directed them into high-buoyant-density membrane domains where the activated receptors, adapters, and mSos were also present. In contrast, ligation of the BCR or TCR resulted in activation of H-Ras, N-Ras, and K-Ras 4B, but not M-Ras. This pattern of activation was not influenced by localization of the Ras proteins to membrane domains. Activation of H-Ras, N-Ras, and K-Ras 4B instead depended on the presence of phospholipase C-gamma and RasGRP. Thus, the molecular mechanisms leading to activation of Ras proteins vary with the stimulus and can be influenced by either colocalization with activated receptors or differential sensitivity to the exchange factors activated by a stimulus.

Autoreactivity of primary human immunoglobulins ancestral to hypermutated human antibodies that neutralize HCMV.

The human antibody response to the AD-2S1 epitope of glycoprotein B (gB) of human cytomegalovirus (HCMV) is dominated by a family of closely related somatically mutated antibodies. These antibodies neutralize viral infectivity and the genes encoding them are derived from two commonly used germ-line variable (V) region genes, IGHV3-30 and IGKV3-11. Recombination of these V genes with the appropriate junctional diversity generates genes that encode primary immunoglobulins that bind to AD-2S1. To further understand the initial primary immunoglobulin response to AD-2S1 we synthesized the germ-line-based ancestor of one such family of antibodies and showed that it bound gB at the AD-2S1 epitope. Here we show that the germ-line ancestor of a second family of antibodies likewise binds to gB. We further show that one of the ancestral primary immunoglobulins, but not the other, also recognized autoantigens. In contrast, the hypermutated derivatives did not demonstrate autoreactivity and minor structural changes in the primary immunoglobulin were sufficient to generate or abolish autoreactivity or to change specificity. Thus, our demonstration that the ancestor of a highly mutated, non-autoreactive antiviral IgG antibody binds nuclear and cell-surface autoantigens indicates for the first time that self-reactivity is not necessarily a barrier to development into a follicular B lymphocyte that undergoes antigen-initiated affinity maturation.

Expression of a constitutively active mutant of M-Ras in normal bone marrow is sufficient for induction of a malignant mastocytosis/mast cell leukemia, distinct from the histiocytosis/monocytic leukemia induced by expression of activated H-Ras.

Expression of constitutively activated M-Ras in normal murine bone-marrow cells was sufficient to induce the factor-independent, in vitro growth and differentiation of colonies of macrophages and neutrophils, and the generation of immortal lines of factor-independent mast cells, and, upon in vivo injection of the transduced cells, a fatal mastocytosis/mast-cell leukemia. In contrast, expression of constitutively activated H-Ras in bone-marrow cells resulted in the in vitro growth, in the absence of exogenous factors, of colonies that contained only macrophages and of lines of cells resembling dendritic cells, and, upon in vivo injection of the transduced cells, a fatal histiocytosis/monocytic leukemia. Macrophages generated by bone-marrow cells expressing activated M-Ras or activated H-Ras differed morphologically, the latter appearing more activated, a difference abrogated by an inhibitor of Erk activation. Inhibition of either Erk or PI3 kinase blocked the capacity of both activated M-Ras and activated H-Ras to support proliferation and viability. However, inhibition of p38 MAPK activity suppressed proliferation of bone-marrow cells expressing activated H-Ras, but enhanced that of bone-marrow cells expressing activated M-Ras. Thus, expression of either activated M-Ras or H-Ras in normal hematopoietic cells was sufficient for transformation but each resulted in the generation of distinct lineages of cells.

RIVETS: the recombinant immunoglobulin and viral epitope tag system.

The human monoclonal antibody 8F9 binds to a linear 10 amino acid epitope that is present within the N-terminal region of the gB envelope glycoprotein of HCMV. Here we show that this short sequence (ETIYNTTLKY) can function as a tag for the detection of recombinant proteins using antibody 8F9. The AD-2S1 tag was recognized by 8F9 whether present at the N- or C-terminus of recombinant proteins and tagged recombinant proteins could be quantified with multiple analytical techniques such as ELISA, western blotting, immunofluorescence and flow cytometry. Production of 8F9 using different constant regions or constant regions from different species enhances the convenience and range of use of this system which we term the Recombinant Immunoglobulin and Viral Epitope Tag System or RIVETS.

Prophylactic ankle braces and knee varus-valgus and internal-external rotation torque.

CONTEXT: Although prophylactic ankle bracing has been shown to be effective in reducing the incidence of ankle sprains,how these ankle braces might affect the other joints of the lower extremity is not clearly understood. OBJECTIVE: To determine the effects of a prophylactic ankle brace on knee joint varus-valgus and internal-external rotation torque during a drop landing onto a slanted surface. DESIGN: A repeated-measures design. SETTING: Biomechanics research laboratory. PATIENTS OR OTHER PARTICIPANTS: Twenty-four physically active college students. INTERVENTION(S): Participants were tested in a brace and no brace condition. MAIN OUTCOME MEASURE(S): We measured 3 dependent variables:(1) peak ankle inversion-eversion torque, (2) peak knee varus-valgus torque, and (3) peak knee internal-external rotation torque. A force plate was used to collect ground reaction force data, and 6 motion analysis cameras collected kinematic data during the unilateral drop landing. An adjustable bar was hung from the ceiling, and a slant board was positioned over the center of the force plate, so that the ankle of the participant's dominant leg would invert upon landing. Peak torque was measure din both the brace and no-brace conditions. The average of the peak values in 3 trials for both conditions was used for the statistical analysis. RESULTS: Ankle eversion torque was significantly greater in the brace condition (F1,23 19.75, P < .01). Knee external rotation torque was significantly greater in the brace condition(F1,23 4.33, P <.05). Valgus knee torque was smaller in the brace condition, but the difference was not statistically significant(F1,23 3.45, P .08). CONCLUSIONS: This study provides an important first step in understanding the effects of prophylactic ankle bracing on other joints of the lower extremity. We found that prophylactic ankle bracing did have an effect on knee torque when the subject was landing on a slanted surface. Specifically, knee external rotation torque increased when the ankle was braced.