Weight distribution in the sitting position in patients with paralytic scoliosis: pre- and postoperative evaluation.

Patients with paralytic scoliosis spend most of their time in the sitting position. The spinal deformity, pelvic obliquity and uneven weight distribution on the seating surface necessitates frequent seating adaptations in the wheelchair. In this prospective study, 45 wheelchair-bound patients were evaluated preoperatively and 43 postoperatively. The pre- and postoperative evaluation was done by an independent observer. Surgical correction was performed between 1993 and 1996. Assessments included sitting balance on a box; number of seating supports in the wheelchair; weight distribution on the seating surface, measured with a computerized EMED system; Cobb angle; hip dislocation; mediolateral translation of T1 and of the apex vertebra with reference to a perpendicular line drawn upwards from the spinal process of S1; and pelvic obliquity from a line drawn between the most proximal points in the iliac crests. X-rays for the measurement of Cobb angle and pelvic obliquity were performed in sitting position. Reference values for normal weight distribution on the seating surface were obtained for 27 normal subjects and revealed a mean value of 59% of weight supported on one side. A stepwise regression analysis on the preoperative results showed that pelvic obliquity and thoracolumbar/lumbar spinal imbalance explained weight distribution on the seating surface (R(2)=0.45). There were significant improvements in all variables except in sitting balance and imbalance of T1, 1 year postoperatively. When dividing the material into two subgroups, the results showed no significant difference in any of the assessed parameters of the scoliosis, pelvic obliquity, or sitting position between individuals with even (50-59% on one side) and those with uneven (60-100% on one side) weight distribution postoperatively. The results of the assessment showed a significant improvement after surgical correction, but the majority still had pelvic obliquity and uneven weight distribution in a sitting position. The weight distribution on the seating surface preoperatively was explained by thoracolumbar/lumbar spinal imbalance and pelvic obliquity, with R(2)= 0.45. There were no significant differences in any variables in comparisons between individuals with even weight distribution and those with uneven weight distribution. For the group with even weight distribution, however, the mean pelvic obliquity was 6 degrees and in the group with uneven weight distribution the mean pelvic obliquity was 12 degrees. Attention to seating surface and adjustment of seating position is needed for patients with paralytic scoliosis.

Separation of mistletoe lectins based on the degree of glycosylation using boronate affinity chromatography.

A mixture of two mistletoe lectins (MLs) has been separated according to the degree of glycosylation using boronate affinity chromatography. The mistletoe lectins, mistletoe lectin I (MLI) and mistletoe lectin III (MLIII) with degrees of glycosylation of 6.1 and 3.8%, respectively, were used in the investigation. MLI exhibited a higher retention time than MLIII due to its higher degree of glycosylation. Separation was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The developed method may lead to new applications for the boronate affinity technique, as well as provide an alternative separation method for MLs.

Affinity chromatography of neoglycoproteins.

Glycoproteins, as a class of biomolecules, exhibit much more heterogeneous structures than non-glycosylated proteins. They present a challenging area of research. Model glycoproteins with well-defined protein and carbohydrate structures are helpful in the search for high-resolution methods for the separation of glycoproteins. Neoglycoproteins, maltose-modified chymotrypsin and lactose-modified chymotrypsin, were synthesised by modifying chymotrypsin with maltose and lactose, respectively, using the reductive amination method. Boronate chromatography was applied to isolate the neoglycoproteins from non-glycosylated substances. The use of Tris-HCl as a shielding reagent during the boronate chromatography proved to be efficient in eliminating unwanted interactions between the boronate ligand and the peptide backbone of chymotrypsin. The retention time of neoglycoproteins on the boronate column was increased with increasing the degree of modification.

Activities and functional assessment 1 year after spinal fusion for paralytic scoliosis.

The results of spinal fusion in patients with paralytic deformities are usually presented as the correction of the deformity. When evaluating the surgical results in such patients, it is advantageous to classify the patients into subgroups because of the varying dysfunction and disabilities. The aim of this study was to evaluate the effect of spinal fusion in patients with paralytic scoliosis in relation to function in terms of Impairments, activities in terms of Disabilities, and dependence in terms of Handicaps 1 year postoperatively, with emphasis on subgroups. A total of 94 patients with paralytic scoliosis and 18 different diagnoses were evaluated 1 year after surgery. The patients were classified according to whether or not they could understand verbal instructions. The patients were also grouped according to the Scoliosis Research Society classification of diagnoses. A set of instruments was used with the variables classified according to WHO's International Classification of Impairments, Disabilities, and Handicaps (ICIDH). This consists of the patient's/relative's motives for surgery, persistent skin discoloration, ambulating or use of wheelchair, use of a brace, sitting balance, weight distribution on a sitting surface, angle of scoliosis, reaching, pain estimation, activities of daily living (ADL) Klein and Bell, care given, time spent resting, and seating supports. The set of instrument also included a follow-up questionnaire comprising 12 different areas, where the patients/relatives assessed the results of surgery. The study showed that spinal fusion in paralytic scoliosis led to showed improvements in the whole group of patients at the Impairment level in the angle of scoliosis, sitting balance, weight distribution, and reduced number of patients with persistent skin discoloration, and at the Handicap level in reduced time for resting during the day, reduced number of seating supports in the wheelchair, and in the use of a brace. Most of the parameters were unchanged. The results in the subgroups were almost the same as in the whole group, although pain and reaching at the Impairment level, and ADL at the Disability level, could not be measured in the patients who were unable to understand verbal instructions. The subjectively assessed results showed that seating posture was ranked positively irrespective of the motive for surgery. The study showed that the patients with paralytic scoliosis maintained or improved their function and level of independence in terms of Impairment and Handicap 1 year postoperatively. The subjective results assessed by the patients/relatives also showed a positive outcome of surgery. Weight distribution on a seating surface was improved, but still uneven, and with respect to better sitting balance and increasing time sitting in a wheelchair, this can involve a risk for pressure sores and needs further investigation. When introducing outcomes including the Disability level, one must take the importance of homogeneity in the groups into consideration.

Preoperative evaluation of activity and function in patients with paralytic scoliosis.

Preoperative evaluation of patients with paralytic scoliosis should take into account the consequences of surgery on the every day life of the patient. However, the parameters that are customarily used in these operations relate only to very narrow measures such as the angle of scoliosis or kyphosis. The aim of this study was to introduce a set of instruments appropriate for measuring both function and activities in paralytic scoliosis patients. The study took as its starting point the WHO International Classification of Impairments, Disabilities and Handicaps (ICIDH), in which an activity is described at the level of the individual and function at the level of the organ. A consecutive series of 100 paralytic scoliosis patients with 18 different diagnoses were evaluated preoperatively with a set of instruments that had been specially developed at Linköping hospital, in which the variables are classified according to the system used in the ICIDH. The set of instruments included general information and evaluation of activities and function--sitting, balance, weight distribution to sitting surface, angle of scoliosis, reaching, pain estimation, activities of daily living (ADL) Barthel and ADL Klein and Bell, care given, time spent resting, and seating supports). The results showed that patients with paralytic scoliosis constitute a heterogeneous group in activities and function. Even when the patients were grouped into four subgroups according to the Scoliosis Research Society Classification, they remained very heterogeneous. However, reaching, Klein and Bell Activities of Daily Living and pain could only evaluate patients who could understand verbal instructions. In those who could not, assessment relied more heavily on measures of function and level of dependence. It was concluded that the choice of assessment must be guided by the patient's ability to understand verbal instructions irrespective of his/her disorder. It is important to use the three levels--impairments, disabilities and handicaps-- in order to focus on the different outcomes in the two groups with respect to the patient's total situation.

Affinity precipitation of concanavalin A--studies of some underlying mechanisms using dynamic laser light scattering.

The final outcome of an affinity precipitation process will depend upon the efficiency of each individual stage involved: the formation of initial affinity complexes, the build-up of a precipitate and the elution of the target protein. Investigations on the first stage were done in this study utilizing a model system. The target protein was the lectin concanavalin A (Con A). Eudragit S-100, a reversibly soluble/insoluble polymer consisting of methyl methacrylate and methacrylic acid, to which the affinity ligand p-aminophenyl-alpha-D-glucopyranoside was coupled, served as the bifunctional ligand (ligand-Eudragit). Owing to the tetrameric structure of Con A, where each subunit has the ability to bind one sugar moiety, and to the multivalency of ligand-Eudragit, a network was formed between the Con A and ligand-Eudragit. It was possible to detect the initial soluble complexes formed by dynamic laser light scattering (DLLS) long before any precipitate could be analysed by transmittance measurements. The rate of complex formation was highly dependent on the ratio between lectin and ligand-Eudragit. It was further shown that the system did not reach equilibrium within the 110 min studied. When the complex formation was studied in the presence of glucose, the build-up rate was decreased to different degrees depending on the sugar concentration used. At high glucose concentrations the complex formation was completely inhibited.

Development of an instrument for clinical evaluation after surgery for neuromuscular scoliosis.

Surgical treatment for neuromuscular scoliosis is effective for most patients. Although those afflicted constitute a heterogeneous group, the aim of surgical treatment is approximately the same for all patients: a spine balanced in the coronal and sagittal planes over a level pelvis. Surgery results in a more stable and straighter spine, which should in turn improve performance in different activities. Previous evaluations of surgery for neuromuscular scoliosis reported in the literature have focused primarily on Cobb angles; there are very few studies dealing with the ability to perform various activities. A new tool for evaluation was developed in several steps, starting with a telephone interview with patients who had undergone surgery and a literature search. The evaluation instrument was then developed, followed by a pilot study and validation of new parts of the instrument. The instrument focuses on performance components and on activity performance. Eight items are evaluated before and after surgery. These data are complemented by a questionnaire administered to the patient or relatives at follow-up. The new parts of the instrument were developed specifically for patients with neuromuscular scoliosis, and the data obtained have been shown to have a high correlation with established measures of activities of daily living of daily living). They should therefore provide us with useful information concerning functional gains as a result of surgery as well as the effect of surgery on activity performance.

T cell dependence of the "natural" autoreactive B cell activation in the spleen of normal mice.

A high frequency of clonal precursor B cells producing lytic antibodies to syngeneic erythrocytes treated with bromelain (BMRC) is revealed in normal mouse spleen cells by lipopolysaccharide-driven limiting dilution analysis. All such specificities are recovered as activated blasts after density gradient fractionation, the "small lymphocyte" pool being depleted of anti-BMRC reactivities. In contrast, the spleens of athymic (nude) mice contain undetectably low frequencies of these specificities in either lymphocyte compartment. Transfer of relatively low numbers of normal syngeneic splenic T lymphocytes to adult nude mice restores the high frequency of anti-BMRC clonal precursor B cells, again in the activated, but not in the resting spleen cell fractions. Large total T cells are more than tenfold better than resting T cells in reconstitution potential, as are enriched CD4+ as compared to CD8+ cells which are practically devoid of activity in this respect. These results apply exclusively to B cells at a differentiative stage that allows for extensive clonal expansion, since there is a marked difference between the frequency of clonal precursors determined by limiting dilution analysis and the frequency of Ig-secreting plaque-forming cells of the same specificity, and induced by the same mitogen in short-term cultures. The implications of these findings for the physiology of autoreactivity and repertoire selection in the compartment of perinatal B cells are discussed.

"In vivo" activated splenic T cells are refractory to interleukin 2 growth "in vitro".

Naturally activated T lymphocytes present in normal mouse spleen were studied for direct reactivity to interleukin 2 (IL 2) and for binding of anti-IL 2 receptor (IL 2R) antibodies or radiolabeled IL 2. The majority of large-sized splenic T lymphocytes are IL 2R-; thus, at the most one third of large L3T4+ T cells and of large Lyt-2+ T lymphocytes bind (weakly) anti-IL 2R antibodies; furthermore, most IL 2R+ cells in the normal spleen are actually Lyt2-, L3T4-. Total large splenic lymphocytes do not express more than an average of 150 high-affinity IL 2R/cell. Such cell populations, as well as large T cells enriched from them, failed to proliferate exponentially in the presence of recombinant IL 2, but did not suppress the IL 2-dependent proliferative responses of small T cells activated in the same cultures. In vivo activation of small T cells, in concanavalin A-treated irradiated syngeneic hosts, also results in refractoriness to growth in recombinant IL 2. In addition, these cells remain insensitive to ligands such as concanavalin A, phorbol myristate acetate and ionomycin, in the presence or absence of IL 2. These results question the role of IL 2 as a growth factor of in vivo activated splenic T lymphocytes, or else indicate that most of these are terminally differentiated cells.

Mechanism of action of the new immunomodulator LS2616 on T cell responses.

Spleen cells from mice treated with LS2616 display a highly increased response to the polyclonal T cell lectin ConA. The total number of splenic T cells, and the relative ratios between L3T4+ and Lyt-2+ T cells were not altered by LS2616 treatment. By dissecting the overall ConA response it was found that the number of ConA-inducible, IL-2 reactive T cells was unaffected, while ConA-induced IL-2 production was enhanced after LS2616 treatment. Spleen cells from LS2616 treated mice, depleted of G10 adherent macrophages (M phi) and reconstituted with M phi from untreated mice displayed normal levels of ConA responses. M phi depleted spleen cells from untreated animals, cocultured with M phi enriched populations from LS2616 treated animals resulted in an increased ConA response. Furthermore, spleen cells from treated mice were found to be excellent stimulators for alloantigen-induced T cell responses; when used as responders in MLC, however, these cells were comparable to responders from non-treated animals. Taken together the results demonstrate that LS2616 exerts an immunostimulatory effect on M phi, which indirectly facilitates polyclonal and antigen-specific T cell responses. The possible implications of this observation on various immunoregulatory events are discussed.

The cytotoxic T-cell response to Ia antigens: lack of correlation between the level of specific cytotoxicity obtained in primary mixed lymphocyte reaction and the frequency of specific cytotoxic T lymphocyte.

We have investigated the cytotoxic response to allogeneic I-E and I-A antigens in bulk culture and limiting dilution experiments. A high degree of specific killing could be generated from unprimed T cells of I-E-negative strains upon stimulation with cells expressing I-E. In such conditions, cytotoxic T lymphocytes (CTL) were generated in the absence of a specific proliferative response. The frequency determinations by means of limiting dilution experiments showed that I-E-specific CTL precursors were much less frequent than the K and D specific precursors. The results suggest the existence of a population of I-E-specific CTL that failed to grow under conditions that allowed the growth of class I-specific CTL.

Development of a model system for analysing graft-versus-host-mediated immune suppression.

F1 hybrid mice injected with parental T lymphocytes undergo a graft-versus-host (GvH) reaction, one of the consequences being a highly depressed cytotoxic T-lymphocyte (CTL) potential against alloantigens and modified self antigens. In the present study we demonstrate that concanavalin A (Con A) can be used for analysing such GvH-mediated immune suppression. Thus, both alloantigen- and Con A-induced responses were reduced by approximately 80% in F1 suppressed (F1s) animals as compared to F1 control (F1c) mice. Although interleukin 2 (IL-2) production was found to be reduced by approximately 50% it did not account for the reduced CTL response in F1s mice. The addition of IL-2 to Con A-stimulated F1s spleen cell cultures did not reconstitute the response. The results suggest that the suppressive mechanism operates by preventing a large fraction of Lyt-2+ CTL precursors from acquiring IL-2 reactivity. However, a small fraction of CTL precursors, escaped the suppression and differentiated into effector CTL.

Autonomous activation of B and T cells in antigen-free mice.

The spleen of adult antigen-free mice contains a sizable proportion (5-15%) of activated cells in all lymphocyte sets, as marked by the membrane expression of immunoglobulins, L3T4 and Lyt-2 antigens. The frequency of activated cells is very high in early post-natal life, and reaches adult levels by 6 weeks of age when it is comparable to that observed in healthy unmanipulated mice raised in conventional conditions. The effector B cell compartment is quantitatively similar in antigen-free mice and specific pathogen-free mice, but the former is deficient in isotype diversification, since IgG- and IgA-secreting cells are drastically reduced. The effector T cell compartment is slightly reduced in number, but is equally competent in providing help or suppression of syngeneic B cells. The results indicate the existence of a compartment of the immune system displaying autonomous self-determined activity which is predominant early in life. This compartment, physically localized to the spleen, appears to be distinct from an antigen-dependent compartment which is essential for the development of peripheral lymphoid organs draining sites of "natural" environmental immunization.

Degeneracy of H-2 recognition by cytotoxic T lymphocytes: 10% of the total repertoire is "specific" for a given haplotype and up to 1% is self-H-2 reactive.

The frequency of specific cytotoxic T lymphocytes (CTL) precursors in the total CTL pool was analyzed in a lectin-driven limiting dilution system. We found that up to 10% of the expressed CTL repertoire in a normal mouse is "specific" for a given allogeneic H-2 haplotype. Split-well analysis under clonal conditions demonstrates that the antigens recognized by the effector CTL are H-2 encoded. A high frequency of CTL "specific" for self-H-2 antigens was revealed in all the experiments, accounting for about 1% of the total inducible pool of CTL. These results suggest a high degree of degeneracy of H-2 recognition by CTL and the immunocompetence of self-H-2-reactive CTL precursors in normal individuals.

Interleukin-2 (IL-2) and its receptor.

The interleukin-2 (IL-2) receptor system which is responsible for T-cell cycle progression is a unique hormone-receptor system in that both the ligand and the receptor need to be induced. In the present article I will review the biological and molecular characteristics of IL-2 and its receptor, as well as the differential triggering and growth requirements that L3T4+ (T4+) helper and Lyt-2+ (T8+) cytotoxic/suppressor T-cells display to respond. The various mechanisms of regulation operating to restrain clonal IL-2-dependent expansion of normal T-lymphocytes will be discussed.

Natural effector T lymphocytes in normal mice.

The "natural" T-cell activity in normal unimmunized mice was studied. By double-parameter fluorescence-activated cell sorter analysis, it was found that 5-10% of all splenic Lyt-2+ and L3T4+ lymphocytes are large, of which more than half are in mitotic cycle. In contrast with small resting cells of the same phenotype, activated (large) T cells isolated from normal mice are functional effector cells: L3T4+ large cells induce normal B lymphocytes into proliferation and antibody secretion, while large Lyt-2+ cells efficiently suppress B-lymphocyte responses. No effector cell cytolytic activity could be detected among naturally activated T cells. The significance of these findings for the internal activity in the normal immune system is discussed.

Detection of cell-mediated lympholysis determinants encoded by the I-A and I-E genes on ultraviolet-irradiated spleen cells.

We have analysed the effects of ultraviolet (UV) irradiation on the expression, by normal splenic cell populations, of cell-mediated lympholysis determinants encoded by the I-A and I-E genes of the H-2 complex. Cytotoxic T lymphocytes specific for class II antigens could be generated from unprimed responder mice when stimulated with UV-treated cells from I-A-and/or I-E-congenic mice in the presence of interleukin 2-containing medium. A cytotoxic T-cell line specific for I-Ek antigens was obtained and maintained upon stimulation with UV-irradiated spleen cells. Secondary Ia-specific responses were obtained with UV-irradiated stimulators in the absence of added soluble factors. The results show that the failure of UV-irradiated cells to stimulate primary cytotoxic T-cell responses is not due to a selective destruction of class II antigens but rather to the inactivation of a still undefined function of Ia-positive cells, other than interleukin-1 production, involved in the establishment of high-affinity interactions between the responding T helper cells and the stimulator cells.

Clonal specificity of concanavalin A-induced cytotoxic T lymphocytes.

The specificity of polyclonally induced cytotoxic T lymphocyte (CTL) precursors has been analyzed under clonal conditions. Primary clones of concanavalin A-inducible CTL, if tested on different targets, revealed that they could distinguish between (a) two different allogeneic tumor targets; (b) allogeneic and syngeneic tumor targets, and (c) syngeneic B blasts and tumor targets. No "nonspecific" CTL clones were generated under these culture conditions, and all clones were found to display classical immunological specificity.

Activation of Lyt-2+ T cells by antibodies towards brain-associated antigens. I. Accessory cell requirement and role of Fc receptors in the induction of reactivity to interleukin 2.

The present report describes a system where essentially all Lyt-2+ T cells are selectively activated by rabbit anti-mouse brain antibodies (RaMB) to interleukin 2 (IL 2) reactivity. High efficiency of RaMB-mediated induction was obtained by a 5 h incubation with antibodies at high cell density of Sephadex G-10-nonadherent spleen cells. No in situ production of IL 2 by RaMB-treated cells was detected, and proliferative responses were entirely dependent on exogeneous IL 2. RaMB-induced IL 2 reactivity was found to require accessory cells which are Fc receptor positive, and clearly distinct from those required to induce T cell proliferation in mixed lymphocyte cultures. We conclude that Lyt-2+ T cells are triggered to IL 2 reactivity by Fc receptor-mediated presentation of RaMB antibodies. The mechanism of induction by RaMB antibodies is discussed.