Synthetic glycolipid-like constructs as tools for glycobiology research, diagnostics, and as potential therapeutics.

Function-spacer-lipid (FSL) constructs are amphiphilic molecules that are able to disperse in water and then self-assemble into cell membranes or onto solid surfaces. Modification of a biological or non-biological surface is very easy and achieved by simple contact of the surface with an appropriately buffered solution containing one or more FSLs. When the functional head group of the FSL is a glycan, glycan modified surfaces can be rapidly formed. Once cells, viruses, or solid surfaces are FSL modified with either simple or complex glycans, they can be used in vitro and/or in vivo to measure interactions with cells, viruses, antibodies, and lectins. FSLs have already been used in a variety of techniques including antibody specificity mapping, antibody/toxin neutralization, diagnostic assays, immune system manipulation, and animal modeling of transfusion reactions. FSLs offer the easiest and fastest method available to achieve a glycan-modified surface.

Blood group A(1) and A(2) revisited: an immunochemical analysis.

BACKGROUND AND OBJECTIVE: The basis of blood group A(1) and A(2) phenotypes has been debated for many decades, and still the chemical basis is unresolved. The literature generally identifies the glycolipid chemical differences between blood group A(1) and A(2) phenotypes as being poor or no expression of A type 3 and A type 4 structures on A(2) red cells, although this assertion is not unanimous. MATERIALS AND METHODS: Using purified glycolipids and specific monoclonal antibodies, we revisited the glycolipid basis of the A(1) and A(2) phenotypes. Purified glycolipids were extracted from four individual A(1) and four individual A(2) blood units. One blood unit from an A weak subgroup was also included. Monoclonal anti-A reagents including those originally used to define the basis of A(1) and A(2) phenotypes were used in a thin layer chromatography - enzyme immunoassay to identify the presence of specific glycolipids. RESULTS: A type 3 glycolipid structures were found to be present in large amounts in all phenotypes. In contrast, the A type 4 glycolipid structure was virtually undetectable in the A(2) phenotype, but was present in the A(1) and A subgroup samples. CONCLUSION: The major glycolipid difference between the A(1) and A(2) phenotypes is the dominance of A type 4 glycolipids in the A(1) phenotype.

A novel study of association between Neisseria gonorrhoeae and the human carbohydrate blood groups.

Previous studies of association of ABO blood groups with gonorrhea have shown contradictory results. Despite the interdependencies of ABO, Lewis, and secretor systems, none of the previous studies examined the combined effect of these systems on their proposed association with gonorrhea. This study attempted to redress that and used genotyping in addition to RBC phenotyping to determine correct tissue phenotypes. Samples from 131 gonorrhea-positive individuals and from 175 gonorrhea-negative individuals were typed for ABO and Lewis using routine antisera. Secretor and Lewis genotyping was performed to ensure accurate determination of ABO and Lewis phenotypes. Chi-square and probability values were used to examine whether there is an association of ABO, Lewis, and secretor systems with gonorrhea infection. Neither single nor combined statistical analysis of data sets yielded a significant association of ABO, Lewis, and secretor phenotypes with Neisseria gonorrhoeae. Nevertheless, this study is an example of the approach that should be taken when examining microbial associations with ABO antigens, in turn influenced by coexpression and modification by the interdependent systems of Lewis and secretor, in mucosal tissues.

Novel glycolipid variations revealed by monoclonal antibody immunochemical analysis of weak ABO subgroups of A.

BACKGROUND AND OBJECTIVES: The chemical basis of the subgroups of A is largely unknown. We used thin-layer chromatography immunochemical staining techniques together with a range of characterized monoclonal reagents to analyse glycolipids isolated from a variety of weak subgroups. MATERIALS AND METHODS: Glycolipids isolated from red cells collected from nine genetically defined individuals of the rare subgroups of A, including a novel A(3) allele (A(2) 539G>A) not described previously, were subjected to a highly sensitive thin-layer chromatographic immunochemical analysis. RESULTS: Semicharacterized monoclonal antibodies revealed that, in addition to the expected quantitative differences between common phenotypes and the weak subgroups, qualitative glycolipid differences (or at least an apparent qualitative basis), caused by major changes in the ratios of different structures exist. Specifically it was found that the weakest A-expressing samples (A(el) phenotype) appeared to express an unusual A structure in the 8-12 sugar region. Variable expression of several structures in one of the A weak samples were suggestive of novel blood group A structures. CONCLUSIONS: Although no structural characterization could be undertaken, the results are clearly indicative that the variant glycosyltransferases of the rare ABO subgroups are not only inefficient, but they may potentially synthesize novel ABO structures.

The influence of fatigue on trunk muscle responses to sudden arm movements, a pilot study.

OBJECTIVE: To examine fatigue induced changes in trunk muscle latencies following trunk muscle fatigue. DESIGN: A repeated measures within subject design.Background. Trunk muscle responses to sudden movements is of interest in clinical biomechanics and motor control. METHODS: Electromyographic profiles were recorded from transversus abdominis (finewire), internal oblique, rectus abdominis and external oblique and longissimus at the level of the 3rd lumbar vertebrae bilaterally. Four asymptomatic subjects performed arm-raising task using a visual cue before and after an isometric fatiguing trunk extension task. RESULTS: Feed-forward responses were not detected in all muscles for every trial. In general, following fatigue trunk muscle onset latencies occur earlier (left, P=0.0016; right, P=0.0475). CONCLUSIONS: Trunk muscle fatigue alters anticipatory postural adjustments in normal subjects. It remains unclear if there is a pattern for specific muscles changes between individuals and if these are reflected in individuals with low back pain. RELEVANCE: Trunk muscle fatigue and altered trunk muscles latencies to movement perturbations have been associated with low back pain. These findings suggest that there may be a link between centrally mediated response to isometric muscle fatigue and anticipatory motor control strategies.

Trunk muscle fatigue during a back extension task in standing.

There is some evidence that the fatiguing characteristics during isometric back extension tasks may assist in identifying differences between individuals with and without low back pain (LBP). During these tasks, especially in standing, other abdominal trunk muscles are also active. The abdominal trunk muscles acting across multiple segments of the lumbar spine function in isolation or in synergy to create flexion torques. It is suggested that co-activation patterns of the trunk muscles are able to control the axis of rotation of the extension torque and also provide multi-segmental stability of the spine. The purpose of this study was to examine the fatigue responses in 4 asymptomatic individuals to a sustained isometric extension task of the trunk muscles evaluating the shifts in the median frequency of the electromyographic (EMG) signal. This study suggests that in asymptomatic subjects, the more superficial abdominal muscles (External Oblique and Rectus Abdominis) increased in activity as the test progressed. There was large inter-individual variation in both amplitude and median frequency changes. Rectus abdominis and the back extensors demonstrated characteristics of fatigue during the task. Studies to test for any characteristic trends in whether specific trunk muscles fatigue in standing is a feature in chronic LBP, invites a formal investigation.

Molecular diversity in the biosynthesis of GI tract glycoconjugates. A blood-group-related chart of microorganism receptors.

This paper examines the potential of carbohydrate blood-group antigens present on mucosal surfaces in acting as receptors for microorganisms. Mucosal surfaces express significant amounts of carbohydrate blood-group antigens under the control of the Secretor, Lewis and ABO systems. The exact glycoconjugate profile an individual presents to the lumen is complex, and can only be correctly determined by a combination of serology and genotyping. We have isolated and structurally resolved the glycolipids expressed in the small intestine of group O individuals having various common or rare phenotypes. Using this information, we have been able to construct a biosynthetic pathway and propose that the type, size and glycotopes expressed, are controlled to a major extent by blood-group-related glycosyltransferases. Many of these glycotopes are potential receptors for microorganisms; some resemble tumour antigens, while others resemble the lipopolysaccharides of some pathogens. Although the origins of the blood-group glycosyltransferases remain uncertain, it is evident that they significantly diversify the mucosal glycotopes exposed to microbes; and therein may be found a potential explanation for their existence.

Attachment of fibronectin to poly(vinyl alcohol) hydrogels promotes NIH3T3 cell adhesion, proliferation, and migration.

Hydrogels have been used in biology and medicine for many years, and they possess many properties that make them advantageous for tissue engineering applications. Their high water content and tissue-like elasticity are similar to the native extracellular matrix of many tissues. In this work, we investigated the potential of a modified poly(vinyl alcohol) (PVA) hydrogel as a biomaterial for tissue engineering applications. First, the ability of NIH3T3 fibroblast cells to attach to PVA hydrogels was evaluated. Because of PVA's extremely hydrophilic nature, important cell adhesion proteins do not adsorb to PVA hydrogels, and consequently, cells are unable to adhere to the hydrogel. By covalently attaching the important cell adhesion protein fibronectin onto the PVA hydrogel surface, the rate of fibroblast attachment and proliferation was dramatically improved, and promoted two-dimensional cell migration. These studies illustrate that a fibronectin-modified PVA hydrogel is a potential biomaterial for tissue engineering applications.

Effect of stance width on multidirectional postural responses.

The effect of stance width on postural responses to 12 different directions of surface translations was examined. Postural responses were characterized by recording 11 lower limb and trunk muscles, body kinematics, and forces exerted under each foot of 7 healthy subjects while they were subjected to horizontal surface translations in 12 different, randomly presented directions. A quasi-static approach of force analysis was done, examining force integrals in three different epochs (background, passive, and active periods). The latency and amplitude of muscle responses were quantified for each direction, and muscle tuning curves were used to determine the spatial activation patterns for each muscle. The results demonstrate that the horizontal force constraint exerted at the ground was lessened in the wide, compared with narrow, stance for humans, a similar finding to that reported by Macpherson for cats. Despite more trunk displacement in narrow stance, there were no significant changes in body center of mass (CoM) displacement due to large changes in center of pressure (CoP), especially in response to lateral translations. Electromyographic (EMG) magnitude decreased for all directions in wide stance, particularly for the more proximal muscles, whereas latencies remained the same from narrow to wide stance. Equilibrium control in narrow stance was more of an active postural strategy that included regulating the loading/unloading of the limbs and the direction of horizontal force vectors. In wide stance, equilibrium control relied more on an increase in passive stiffness resulting from changes in limb geometry. The selective latency modulation of the proximal muscles with translation direction suggests that the trunk was being actively controlled in all directions. The similar EMG latencies for both narrow and wide stance, with modulation of only the muscle activation magnitude as stance width changed, suggest that the same postural synergy was only slightly modified for a change in stance width. Nevertheless, the magnitude of the trunk displacement, as well as of CoP displacement, was modified based on the degree of passive stiffness in the musculoskeletal system, which increased with stance width. The change from a more passive to an active horizontal force constraint, to larger EMG magnitudes especially in the trunk muscles and larger trunk and CoP excursions in narrow stance are consistent with a more effortful response for equilibrium control in narrow stance to perturbations in all directions.

Secretor genotyping for A385T, G428A, C571T, C628T, 685delTGG, G849A, and other mutations from a single PCR.

BACKGROUND: The secretor status of an individual is important for disease relationship studies, because it determines the presence of ABH blood group antigens in the gastrointestinal tract and bodily secretions. Routine serologic methods for determining secretor status are unreliable. Current strategies based on PCR for genotyping require relatively large amounts of DNA and have to be done as several separate experiments. STUDY DESIGN AND METHODS: A single, multiplex PCR technique followed by RFLP digestion with four restriction enzymes produced unique genotype profiles for most known secretor (FUT2) mutations. RESULTS: Samples from a range of individuals with common and rare secretor genotypes were analyzed. Each gave unique patterns that allowed secretor genotypes to be determined. CONCLUSION: By using the method described here and genomic DNA, a secretor genotype based on the FUT2 mutations A385T, G428A, C571T, C628T, 685delTGG, and G849A could be accurately determined.

Decrease in trunk muscular response to perturbation with preactivation of lumbar spinal musculature.

STUDY DESIGN: An experimental study of healthy subjects' trunk muscle responses to force perturbations at differing angles and steady state efforts. OBJECTIVES: To determine whether increased preactivation of muscles was associated with decreased likelihood of muscular activation in response to a transient force perturbation. SUMMARY OF BACKGROUND DATA: Trunk stability (ability to return to equilibrium position after a perturbation) requires the stiffness of appropriately activated muscles to prevent buckling and consequent "self-injury." Therefore, greater trunk muscle preactivation might decrease the likelihood of reflex muscle responses to small perturbations. METHODS: Each of 13 subjects stood in an apparatus with the pelvis immobilized. A harness around the thorax provided a preload and a force perturbation by a horizontal cable and a movable pulley attached to one of five anchorage points on a wall track surrounding the subject at angles of 0 degrees, 45 degrees, 90 degrees, 135 degrees, and 180 degrees to the forward direction. Subjects first equilibrated with a preload effort of nominally 20% or 40% of their maximum extension effort. Then a single full sine-wave force perturbation pulse of nominal amplitude, 7.5% or 15% of maximum effort, duration 80 milliseconds or 300 milliseconds, was applied at a random time, with three repeated trials of each test condition. The applied force (via a load cell) and the electromyographic activity of six right and left pairs of trunk muscles were recorded. Muscle responses were detected by two methods. 1) Shewhart method: electromyographic signal greater than "baseline" values by more than three standard deviations, and 2) Mean Electromyographic Difference method: mean electromyographic signal in a time window 25 to 150 milliseconds after the force perturbation greater than that in a 25- to 150-millisecond window before the perturbation. RESULTS: Lower preload efforts were associated with more muscle responses (overall mean response detection rate = 33% at low preload and 25% at high preload). Using the Shewhart method, there were significant differences by effort (P<0.05) for all abdominal muscles and for all left dorsal muscles except multifidus. Using the Mean Electromyographic Difference method, there were significant differences by effort (P<0.05) for the same dorsal muscles, but only for one of the abdominal muscles. CONCLUSIONS: Findings are consistent with the hypothesis that the spine can be stabilized by the stiffness of activated muscles, obviating the need for active muscle responses to perturbations.

Genomic characterization of the kidd blood group gene:different molecular basis of the Jk(a-b-) phenotype in Polynesians and Finns.

BACKGROUND: The clinically important Kidd (JK) blood group antigens are carried by the urea transporter in red cells. The rare Jk(a-b-) phenotype can be caused by homozygosity at the JK locus for a silent allele, JK: This phenotype has been recorded in many ethnic groups, but it is most abundant among people originating from the Polynesian Islands and Finland. The molecular basis for Jk(a-b-) is unknown in these populations. STUDY DESIGN AND METHODS: Blood samples from individuals of Swedish, Polynesian, and Finnish origin were collected and characterized by routine JK blood group serology and JK genotyping. Genomic DNA covering the exons and intervening introns of the JK gene coding region was amplified by polymerase chain reaction, and fragments were directly sequenced. RESULTS: Exon and partial intron sequences in the coding region of the JK gene were determined. Finnish and Polynesian Jk alleles were analyzed; the only deviations from consensus were a splice-site mutation (G-->A) in Polynesians, causing skipping of exon 6, and a T871C substitution predicted to disrupt a potential N-glyco-sylation motif (NSS-->NSP) in Finns. Methods for rapid detection of silent Jk alleles were developed for clinical application. CONCLUSION: Polynesians and Finns have two different molecular alterations in their Jk alleles, both of which can now be determined by polymerase chain reaction.

Leb glycolipids present in the lumen of the gastrointestinal tract of rats do not enter the plasma compartment.

We orally administered to rats several times more Leb glycolipids than is proportionally found in the gastrointestinal tract of humans. This was done in an effort to study two potential phenomena: the possibility that glycolipids in plasma may originate from glycolipids derived from the lumen of the gastrointestinal tract, and to investigate the potential to secondarily modify in vivo the glycolipid profile of gastrointestinal tract epithelial cells, a phenomenon clearly established for human erythrocytes, leukocytes, and platelets. We were able to establish that some of the orally administered glycolipids can be detected at the surface of the upper region mucosa of the gastrointestinal tract for more than 24 hours and are essentially excreted intact in stools in less than 72 hours. Some fecal degradation of the Leb glycolipids into Lea and H type 1 did occur. Although we clearly established that the glycolipids were present in the mucus layer adherent to the cell surface, we could not conclusively establish if the glycolipids had inserted into the epithelial cell membrane. This, however, could not be excluded. The fact that the fed glycolipids remained in the mucus layer of the upper region of the gastrointestinal tract for at least 24 hours may have some pharmacological value. Using sensitive techniques, including red cell serology, immunohistology, and immunochemistry of glycolipids isolated from plasma and red cells, there was no evidence that the fed Leb glycolipids reached the plasma compartment, thus suggesting that glycolipids present in the lumen of the gastrointestinal tract cannot reach the circulation.

EMG responses to maintain stance during multidirectional surface translations.

To characterize muscle synergy organization underlying multidirectional control of stance posture, electromyographic activity was recorded from 11 lower limb and trunk muscles of 7 healthy subjects while they were subjected to horizontal surface translations in 12 different, randomly presented directions. The latency and amplitude of muscle responses were quantified for each perturbation direction. Tuning curves for each muscle were examined to relate the amplitude of the muscle response to the direction of surface translation. The latencies of responses for the shank and thigh muscles were constant, regardless of perturbation direction. In contrast, the latencies for another thigh [tensor fascia latae (TFL)] and two trunk muscles [rectus abdominis (RAB) and erector spinae (ESP)] were either early or late, depending on the perturbation direction. These three muscles with direction-specific latencies may play different roles in postural control as prime movers or as stabilizers for different translation directions, depending on the timing of recruitment. Most muscle tuning curves were within one quadrant, having one direction of maximal activity, generally in response to diagonal surface translations. Two trunk muscles (RAB and ESP) and two lower limb muscles (semimembranosus and peroneus longus) had bipolar tuning curves, with two different directions of maximal activity, suggesting that these muscle can play different roles as part of different synergies, depending on translation direction. Muscle tuning curves tended to group into one of three regions in response to 12 different directions of perturbations. Two muscles [rectus femoris (RFM) and TFL] were maximally active in response to lateral surface translations. The remaining muscles clustered into one of two diagonal regions. The diagonal regions corresponded to the two primary directions of active horizontal force vector responses. Two muscles (RFM and adductor longus) were maximally active orthogonal to their predicted direction of maximal activity based on anatomic orientation. Some of the muscles in each of the synergic regions were not anatomic synergists, suggesting a complex central organization for recruitment of muscles. The results suggest that neither a simple reflex mechanism nor a fixed muscle synergy organization is adequate to explain the muscle activation patterns observed in this postural control task. Our results are consistent with a centrally mediated pattern of muscle latencies combined with peripheral influence on muscle magnitude. We suggest that a flexible continuum of muscle synergies that are modifiable in a task-dependent manner be used for equilibrium control in stance.

Control of stance during lateral and anterior/posterior surface translations.

The purpose of this study was to compare and contrast postural responses to lateral and A/P surface translations by quantifying joint positions, bilateral three-dimensional (3-D) ground reaction forces, and lower limb and trunk muscle electromyographic (EMG) activity. Subjects stood on a movable platform which was randomly translated in four different directions. The kinematic patterns in response to lateral and anterior/posterior (A/P) surface translations were similar in that there was a sequential displacement and reversal of the shank/thigh and then trunk segments. While the body center of mass (CoM) was displaced equally in response to lateral and A/P translations, equilibrium was maintained by redistributing the vertical forces and changing the shear forces exerted against the support surface. These force responses were bilaterally symmetrical for A/P translations but not for lateral translations. With respect to EMG activity, the first muscle activated was the proximal tensor fascia latae for lateral translations whereas the distal muscles were recruited first for A/P translations. Results from this study suggest that control of postural equilibrium may be similar for A/P and lateral translations, although specific differences in patterns may reflect various biomechanical constraints of the trunk and the lower extremities associated with the two planes of movement.

Damage control for devastating pelvic and extremity injuries.

This article focuses on some general principles of care and then discusses devastating pelvic injury secondary to both blunt and penetrating trauma. The authors describe the current approach to the mangled extremity and discuss indications for primary amputation.

Postural perturbations: new insights for treatment of balance disorders.

This article reviews the neural control of posture as understood through studies of automatic responses to mechanical perturbations. Recent studies of responses to postural perturbations have provided a new view of how postural stability is controlled, and this view has profound implications for physical therapy practice. We discuss the implications for rehabilitation of balance disorders and demonstrate how an understanding of the specific systems underlying postural control can help to focus and enrich our therapeutic approaches. By understanding the basic systems underlying control of balance, such as strategy selection, rapid latencies, coordinated temporal spatial patterns, force control, and context-specific adaptations, therapists can focus their treatment on each patient's specific impairments. Research on postural responses to surface translations has shown that balance is not based on a fixed set of equilibrium reflexes but on a flexible, functional motor skill that can adapt with training and experience. More research is needed to determine the extent to which quantification of automatic postural responses has practical implications for predicting falls in patients with constraints in their postural control system.

Serum lactate is not predicted by anion gap or base excess after trauma resuscitation.

OBJECTIVE: The inability to normalize lactate predicts death after trauma, but lactate may not be immediately available in every center. We postulated that, in a normal acid-base environment, lactate would correlate with the anion gap and the base excess of an arterial blood gas. METHODS: We studied 52 consecutive, invasively monitored patients with trauma admitted directly to the intensive care unit (ICU) from the emergency department or operating room in our level I center to determine whether base excess and anion gap could predict lactate. Lactate, base excess, and anion gap were recorded upon admission to the ICU and 8, 16, 24, 36, and 48 hours after admission. Correlation coefficients (r2) were calculated for the total patients, the 43 survivors, and the nine non-survivors. RESULTS: Serum lactate was significantly higher in nonsurvivors at 16 hours after post ICU admission (4.0 +/- 1.69 vs. 2.84 +/- 1.49, p < 0.05), and this trend persisted; the greatest difference was seen at 48 hours after admission (2.92 +/- 1.47 vs. 1.76 +/- 0.57, p < 0.001). There were no differences in base excess or anion gap between survivors and nonsurvivors. We found no consistent correlation between lactate versus anion gap, lactate versus base excess, or anion gap versus base excess. CONCLUSIONS: There is no correlation between lactate, base excess, and anion gap after initial resuscitation. Neither anion gap nor base excess was capable of predicting lactate; therefore, lactate must be directly measured. The lack of correlation of anion gap with base excess or lactate suggests the presence of unmeasured anions, an impairment in acid-base regulation after injury and resuscitation, or both.