Validation of an Argentine version of Lupus Quality of Life questionnaire.

OBJECTIVE: To determine reproducibility and validity of an Argentine version of the Lupus Quality of Life questionnaire (LupusQoL) and to determine cut-off values in the questionnaire. MATERIALS AND METHODS: One hundred and forty-seven systemic lupus erythematosus patients (American College of Rheumatology 1982/1997) were assessed from April 2014 to July 2014. Demographic and socioeconomic variables were collected, as well as SELENA/SLEDAI, Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index Score, comorbidities and treatment data. Patients completed LupusQoL-Argentine version and European Quality of Life Questionnaire (EuroQoL-5D). Internal consistency and reliability were examined. Convergent validity with EuroQoL-5D was assessed through analysis of latent classes, which established homogeneous categories from the responses of each domain of LupusQoL and for the total. RESULTS: Out of 147 patients, 93.2% were female, mean age 36.4 ± 11.1 years, mean disease duration 2.7 ± 9 years, mean SELENA/SLEDAI 2.7 ± 3 points. The cut-off point that defined good or bad quality of life was 0.739 for EuroQoL 5D and 63 for LupusQoL. Cut-off values for each LupusQoL domain were also defined, creating two classes in each of them. There was moderate to high concordance to classify quality of life (Kappa = 0.74, 95% confidence interval = 0.54, 0.95). CONCLUSION: The Argentine version of LupusQoL is a valid, reliable and reproducible instrument to assess quality of life. In this study, cut-off points that allow the classification of patients regarding whether they have good or bad quality of life are established for the first time.

Chemotactic and anaphylatoxic fragment cleaved from the fifth component of guinea pig complement.

The fifth component of guinea pig complement, with a sedimentation coefficient 7.8S, is cleaved by sensitized sheep erythrocytes treated with the first four components of complement into two fragments with sedimentation coefficients of 7.4S and 1.5S. The smaller fragment, with a molecular weight of about 15,000, possesses chemotactic activity for rabbit polymorphonuclear leukocytes, as well as anaphylatoxic activity for guinea pig ileum.

Maintenance of spontaneous lumbar curve correction following thoracic fusion of main thoracic curves in adolescent idiopathic scoliosis.

AIMS: The aims of our study were to provide long-term information on the behaviour of the thoracolumbar/lumbar (TL/L) curve after thoracic anterior correction and fusion (ASF) and to determine the impact of ASF on pulmonary function. PATIENTS AND METHODS: A total of 41 patients (four males, 37 females) with main thoracic (MT) adolescent idiopathic scoliosis (AIS) treated with ASF were included. Mean age at surgery was 15.2 years (11 to 27). Mean follow-up period was 13.5 years (10 to 18). RESULTS: For the TL/L curve, the mean curve flexibility evaluated with supine pre-operative bending radiographs was 78.6% (standard deviation 16.5%), with no significant loss of correction observed. On comparing patients with an increase of the TL/L curve increase (> 4º, n = 9, 22%) to those without, significant differences were observed in the correction rate of the MT curve at the final follow-up (p = 0.011), correction loss of the MT curve (p = 0.003) and the proportion of patients who had semi-rigid instrumentation (p = 0.003). Pre-operative percentage predicted forced vital capacity (%FVC) was 80%, dropping to 72% at final follow-up (p < 0.001). The Scoliosis Research Society questionnaire score was not significantly different between patients with and without a TL/L curve increase (p = 0.606). Spontaneous lumbar curve correction (SLCC) was maintained up to 18 years following selective ASF in most patients and demonstrated significant correlation with maintenance of MT curve correction. CONCLUSION: Maintenance of MT curve correction using rigid instrumentation provided stable SLCC over time. An observed 8% decrease in %FVC indicates that ASF should be reserved for patients with no or only mild pulmonary impairment. Cite this article: Bone Joint J 2016;98-B:997-1002.

Membrane damage by a toxin from the sea anemone Stoichactis helianthus. II. Effect of membrane lipid composition in a liposome system.

In the first paper of this series, it was shown that a toxin from the sea anemone Stoichactis helianthus increased the permeability of black lipid membranes due to transmembrane channel formation. In the present study, we have used liposomes to examine the reactivity of the toxin with different phospholipids. Membrane damage was assessed by measuring the release of 86Rb+ and 14C-labeled membrane lipid. For the different lipids, the rank order of marker release was: sphingomyelin greater than C18 : 2 phosphatidylcholine greater than C18 : 1 phosphatidylcholine greater than C18 : 0 phosphatidylcholine greater than C16 : 0 phosphatidylcholine = C14 : 0 phosphatidylcholine. In C14 : 0 and C16 : 0 phosphatidylcholine liposomes there was no 14C-labeled lipid release and only 13 to 16% 86 Rb+ release which corresponds to the 86Rb+ content in the outermost aqueous shell of multilamellar liposomes. This indicates that membrane damage was limited to the outermost bilayer. In liposomes prepared with the other lipids, the extent of release of both markers increased proportionately with the length and the degree of unsaturation of the lipids' acyl side chains. Spingomyelin liposomes were the most susceptible with 47% of the 14C-labeled lipid marker and 90% of the 86Rb+ marker being released. The large extent of 14C-labeled lipid release is attributed to a detergent-like activity of the toxin which presumably is due to the amphipathic nature of the protein. Thus, the toxin can inflict membranrtance of one mechanism or the other apparently varies depending on membrane structure and lipid composition.

Size distribution and stability of the trans-membrane channels formed by complement complex C5b-9.

We have performed double marker sieving experiments with molecules ranging from ca. 0.5-3 nm dia. in order to evaluate the size distribution of the channels formed by complement in resealed sheep erythrocyte ghosts. Evidence is presented that marker release through the channels reached equilibrium between the ghosts and the extracellular fluid in a period of 3 hr and that the channels are stable at 37 degrees C for this period of time. Under these experimental conditions we have observed a differential in the endpoint release of inositol and sucrose, which indicates that some of the ghosts carried channels measuring between 0.7 and 0.9 nm dia. No differential was observed between release of sucrose and raffinose (0.9 and 1.1 nm mol. dia., respectively). Comparisons between sucrose and inulin (0.9 and 3 nm mol. dia, respectively) showed a difference in marker release. Also, there was substantial release of inulin, indicating the presence of channels above 3 nm in dia. Hence, the present data indicate formation of channels in three size ranges, namely, 0.7-0.9 nm, 0.9-3 nm and greater than 3 nm.

Complement lysis of nucleated cells: effect of temperature and puromycin on the number of channels required for cytolysis.

We have previously shown that lysis of a nucleated mammalian cell requires several complement channels unlike lysis of erythrocytes and that this difference is due primarily to rapid elimination of channels from the plasma membrane. We have now investigated this problem further by studying the rate of channel elimination at low temp, the osmotic fragility of the cells, and the effectiveness of the membrane-associated ion pumps. When complement channels were formed for 3 min at 37 degrees C, followed by prolonged incubation at 2 degrees C, the C6 lytic dose-response curves indicated that a single channel was required for lysis of a cell, whereas multiple channels were required when the entire process was carried out at 30 degrees C. The shift from multi- to one-hit lytic behavior can be explained by the drastic reduction in the rate of channel elimination at low temp. C6 lytic dose-response curves with puromycin-treated cells were also found to display one-hit behavior, but, in this case, the rate of channel elimination was reduced only about 35-40% (which would not suffice to explain the one-hit lytic characteristics). However, cell death was more extensive for puromycin-treated cells than normal cells after incubation in buffers of low ionic strength, suggesting that an increase in osmotic fragility may be a contributing factor in the shift from multi- to one-hit behavior. Blocking of the membrane-associated Na+/K+-ATPases with ouabain did not affect the multi-channel requirement. Presumably, this means that the ion pumping rate does not significantly influence the number of channels required for lysis.

Two mechanisms of cell-mediated cytotoxicity: Ca++ transport modulation by lymphotoxin and transmembrane channel formation by antibody and nonadherent spleen cells.

Lymphotoxin is a protein with a MW of 45,000 daltons derived from activated lymphocytes that kills target cells nonspecifically. Kinetic studies indicate that there is a lag period of about 4 hours before cytotoxicity becomes apparent, even at high concentrations of lymphotoxin. Therefore, the role of lymphotoxin in cell-mediated cytotoxicity would be restricted to situations in which more rapid mechanisms are not operative. It has found that lymphotoxin increases the rate of 45Ca++ uptake by the mouse L-cells used as targets. This effect and the cytotoxicity are abrogated by ouabain. A lymphotoxin-resistent L-cell mutant did not display the 45Ca++ uptake effect. It is not known whether the Ca++ effect is primary or secondary. Neutralization experiments with anti-lymphotoxin have indicated that there are at least two distinct pathways by which immune lymphocytes can destroy target cells in vitro--one that involves secretion of a nonspecific soluble factor, i.e., lymphotoxin, and another that probably requires intimate contact between the plasma membranes of the target and killer cells. This "membrane contact" mechanism may involve formation of channels in the target cell membranes. The transmembrane channel concept is a working hypothesis that is based on experiments by Henkart and Blumenthal in which it was found that antibody and lymphocytes jointly produce ion-conducting channels in planar bilayers of "oxidized cholesterol." In order to supplement and extend this approach we have made an exploratory study of 86Rb+ and 51Cr marker release from lecithin/cholesterol/dicetyl phosphate liposomes by antibody and nonadherent mouse spleen cells. Evidence is presented indicating that the antibody and cells cause direct synergistic marker release from liposomes into the fluid medium. This indicates that they have the capacity to damage phospholipid bilayers. Hence, it seems worthwhile to conduct further studies of the liposome model in order to uncover the mechanism of membrane damage and to assess its relevance to cell-mediated cytotoxicity.

Concordancia de los nuevos criterios ACR/EULAR y los ACR 1980 para la clasificación de la esclerosis sistémica / Concordance of the new ACR/EULAR criteria and the ACR 1980 for the classification of systemic sclerosis

Objetivo: Evaluar cumplimiento, y así mismo concordancia y discordancia de los criterios de clasificación de Esclerosis Sistémica (ES) ACR/EULAR 2013 y ACR 1980 en pacientes con diagnóstico clínico de la enfermedad. Método: Se incluyeron 169 pacientes con diagnóstico de Esclerosis Sistémica. Resultados: El 72,2 por ciento cumplía los criterios ACR 1980, y el 99,4 por ciento (168 pacientes) cumplía los criterios ACR/EULAR 2013. La concordancia absoluta de toda la muestra fue 72,7 por ciento, para el subtipo limitado 35,2 por ciento, y 100 por ciento el difuso. Se subanalizaron los pacientes con limitada que sólo cumplían criterios ACR/EULAR 2013, y se comparó con el resto de las limitadas. Los primeros presentaron en forma estadísticamente significativa menor esclerodactilia distal a MCF, menor presencia de úlceras digitales y pitting scars, menor afectación intersticial pulmonar, y mayor daño microvascular en la capilaroscopia. Conclusión: Los nuevos criterios de clasificación de Esclerosis Sistémica serían más adecuados para detectar esclerodermias limitadas, siendo dicho hallazgo estadísticamente significativo.

Objective: To evaluate the performance, and likewise concordance and discordance of the classification criteria of Systemic Sclerosis ACR/EULAR 2013 and ACR 1980 in a group of patients with clinical diagnosis of SSc. Methods: We enrolled 169 patients with diagnosis of Systemic Sclerosis. Results: 72.2 percent met the 1980 ACR criteria, and 99.4 percent met the ACR/EULAR 2013 criteria. The absolute agreement of the entire sample was 72.7 percent, 35.2 percent for the limited subtype, and 100 percent for the diffuse. Those patients with limited subtype who only met the ACR/EULAR 2013 criteria were compared with the rest of limited patients. The first group had statistically significantly lower sclerodactyly distal to MCF, lower presence of digital ulcers and pitting scars, less interstitial lung involvement, and greater abnormal nail fold capillaries. Conclusion: The new classification criteria for systemic sclerosis seem to be more suitable for detecting limited scleroderma. In the present study, statistically significant discrepancy was found in the limited subtype.

Mechanism of cytolysis by complement.

The attack of complement is directed against the lipid moiety of the cell membrane; a single lesion at the site of fixation of complement proteins C5-C9 is responsible for lysis of a cell. There are two hypothetical models for the generation of this membrane lesion. The first of these, designated the leaky-patch model, postulates either direct enzymatic attack or enzymatic generation of a lytic substance by C5-C9. As a result, the phospholipid bilayer of the membrane would be disrupted and a leaky patch permitting passage of water and salt would appear. However, this hole would persist only as long as enzymatic action continues. Thus, the leaky-patch model would not produce a stable hole, and for this reason it is considered an unlikely mechanism. The second hypothesis, termed the doughnut model, describes a structural concept for creating a hydrophilic passage through the hydrophobic phospholipid bilayer of the membrane. In essence, this would be a rigid and hollow structure, like a doughnut, with a hydrophobic exterior, which is inserted into the phospholipid bilayer of the cell membrane in such a way that its hollow hydrophilic core becomes a channel through which salt and water can exchange freely between the interior of the cell and the extracellular environment. The late-acting complement proteins C5-C9 are the most probable source of the structural components of the doughnut. A combination of the leaky-patch and doughnut models may represent the most likely mechanism.

Studies on the mechanism of action of guinea pig lymphotoxin. I. Membrane active substances prevent target cell lysis by lymphotoxin.

The effect of various pharamacologic agents on lysis of L cells by guinea pig lymphotoxin (LT) was studied. Among the many drugs tested, only those that affect plasma membrane functions were found to interfere with the cytolytic action of LT. Dimethyl sulfoxide or lidocaine potentiated L cell resistance against lysis. Stronger protection was provided by ouabain. Addition of ouabain to cells previously injured by LT was also effective in reducing cell death. Attempts to detect metabolic disturbances in cells before LT cytolysis were unsuccessful. The biosynthetic rate of DNA, RNA, or protein, and the total cellular content of ATP were not significantly changed in LT-treated cells. The results suggest that LT disturbs some plasma membrane functions of the target cell, perhaps ion transport systems, and consequently induces ionic imbalances between the intra-and extracellular milieu which eventually cause cell death.

Studies on the mechanism of action of guinea pig lymphotoxin. II. Increase of calcium uptake rate in LT-damaged target cells.

In the previous paper it was suggested that the primary action of guinea pig lymphotoxin (LT) involved creation of ionic imbalances within the target L cells. The nature of these ionic disturbances is explored in this paper. The exogenous addition of CaCl2, but not KCl or NaCl, inhibited the cytotoxic action of LT. Cellular uptake rates of 45Ca++, but not 86Rb+, increased in LT-damaged L cells. The factor responsible for increasing the 45Ca++ uptake rate cochromatographed on a hydroxyapatite column with the cytotoxic activity of LT. Ouabain prevented the LT-mediated lysis and, concomitantly, depressed the LT-induced increase of 45Ca++ uptake rate. The LT-damaged L cells excluded trypan blue to the same extent as the normal cells. The addition of LT to and LT-resistant L cell mutant affected neither the 45Ca++ uptake rate nor the viability. From these observations, damage to the calcium transport system in the L cell plasma membrane is proposed as a mechanism of LT action.

Life-span and size of the trans-membrane channel formed by large doses of complement.

To evaluate the life-span and size of trans-membrane channels in complement (C) treated membranes, resealed erythrocyte ghosts containing trapped native protein markers, as well as residual hemoglobin, were treated with anti-Forssman antibody and large doses of guinea pig C. Ovalbumin and hemoglobin were released slowly through the channels so produced, whereas human serum albumin was not. Release of hemoglobin was not blocked by extracellular bovine serum albumin. Release of hemoglobin continued for at least 72 hr at 4 degrees C. Semi-logarithmic plots of ovalbumin or hemoglobin release showed gradual diminution of the rate constant, which indicates slow loss of channels during the experimental period. These experiments demonstrate that the channels produced in erythrocyte ghost membranes by large C doses have a long, although finite, life-span. Their effective diameter is al least 55 A on the basis of ovalbumin and hemoglobin release, and not more than 150 A, since serum albumin was not released. However, an upper limit of 100 A would be more reasonable in light of electronmicroscopic observations by others. These results are compatible with the doughnut model.

Penetration of C8 and C9 in the C5b-9 complex across the erythrocyte membrane into the cytoplasmic space.

We have developed a technique in which transglutaminase is used to measure the penetration of terminal complement proteins across the erythrocyte membrane into the cytoplasmic space. Penetration of a given terminal complement protein into the cytoplasmic space was assessed by labeling the protein of interest with radioactive iodine, forming the complement channel using the labeled protein, adding transglutaminase to only one side of the membrane, and allowing the enzyme to cross-link the susceptible proteins on that side of the membrane. Cross-linking was assessed by measuring the increase in molecular weight of the appropriate molecule on sodium dodecyl sulfate gels under reducing conditions. The results of these experiments indicate that C8 and C9 are rapidly cross-linked to high molecular weight from either the interior or the exterior of the membrane. In order to determine whether the cross-linking mediated by enzyme on the interior was occurring from within the ghosts and not via enzyme that had leaked into the extracellular medium, experiments were performed with dimethylcasein in the extracellular medium. In the presence of this protein, cross-linking of C8 and C9 from outside was negligible. Hence, if cross-linking occurs when transglutaminase is trapped inside the ghosts, it cannot be due to leakage of enzyme, but must be attributable to cross-linking from the inside. The results show that C9 definitely penetrated across the membrane into the intracellular space. With respect to C8, statistical evaluation indicates that C8 probably penetrated into the intracellular space.

The relationship between channel size and the number of C9 molecules in the C5b-9 complex.

We have recently shown by dose-response analyses with resealed erythrocyte ghosts that the channel formed by complement is a monomer of C5b-9 of the composition C5b61C71C81C9n, in which n = 1 for channels permitting passage of sucrose (0.9 nm molecular diameter) and n = 2 for channels allowing transit of inulin (3 nm molecular diameter) (1). We have now continued these experiments and expanded them by including ribonuclease A (molecular diameter, 3.8 nm) as a marker to assess whether additional C9 molecules enlarge the functional C5b-9 channel. Our results show that formation of C5b-9 channels displays one-hit characteristics with respect to C5b6 when tested by transmembrane passage of inulin or ribonuclease A. By contrast, analysis of dose-response curves of C9 indicate that n = 2-3 for channels allowing transit of inulin and n = 4 for channels allowing transit of ribonuclease A. We have also performed sieving experiments with ghosts carrying C5b-7 and containing two small markers, inositol and sucrose. Dose-response curves for C8 were performed in the presence of excess C9 to ensure conversion of all C5b-8 to C5b-9 channels. The results indicate that small channels (approximately 0.8 nm effective diameter) are not formed at high C9 multiplicity, thus confirming the results obtained with the larger markers, i.e., increase of C9 input leads to formation of larger channels.

Effect of agents that produce membrane disorder on lysis of erythrocytes by complement.

To evaluate the effect of membrane lipid acyl-chain packing on the efficiency of cell lysis by complement, we have studied membrane modulation by 2-(2-methoxy)-ethoxyethyl-8-(cis-2-n-octylcyclopropyl)-octanoate (A2C) and by myristoleyl alcohol, the cis isomer of a C14:1 aliphatic alcohol. These substances are known to increase the membrane lipid disorder by virtue of the bend in their acyl chains, which is believed to loosen the phospholipid acyl-chain packing. We have found that both of these compounds markedly enhance the lysis of erythrocytes by the terminal complement proteins C5b-9. The enhancing effect by A2C is operative in the formation of erythrocytes carrying complement components C5b, C6, and C7, as well as in the subsequent reactions with complement components C8 and C9. We have also found that A2C-treated erythrocytes bind C5b6 to a measurable extent, whereas untreated erythrocytes do not. We attribute this to a shift in the partition equilibrium of C5b6 toward membrane association, which would improve lytic efficiency. The increase of membrane lipid disorder by these agents would also be expected to increase insertion of hydrophobic peptides from C7, C8, and C9, with consequent gain in lytic efficiency. Treatment of erythrocytes with sublytic doses of NaDodSO4, or Triton X-100 did not enhance lysis by C5b-9 appreciably, suggesting that enhancement of lysis by C5b-9 is not a general property of amphiphiles.

On the lysis of paroxysmal nocturnal hemoglobinuria erythrocytes by complement: dual role of C3b.

The efficiency of cytolysis by the terminal complement proteins C5b-9 can be markedly enhanced by C3b molecules bound on the target cell membrane (Hammer et al. 1976). This enhancement was shown to be proportional to the number of C3b molecules on the cell membrane. The present experiments have shown that the hemolytic efficiency of the complement membrane attack system is two to five times greater on paroxysmal nocturnal hemoglobulinuria erythrocytes (PNHE) than on normal human E. This difference is attribute to a derivative of C3, probably C3b, on PNHE since it was abolished by anti-C3 but not by anti-C2. The efficiency of C5b-9 to lyse PNHE was only partially decreased by C3b inactivator and beta 1 H, indicating that the C3b on PNHE is not readily inactivated by its regulatory proteins. Furthermore, cells from a single severely affected patient consumed 3-fold more C5b6 than normal human E yet concommitantly measured membrane fluidity was normal. From these observations we conclude that cell-bound C3b on PNHE serves two functions: (a) it increases the hemolytic efficiency of membrane attack components of the complement system; and (b) it provides sites for assembly of the alternative pathway convertases.