Limited Long-Term Impact of Insect Venom Immunotherapy on the Micro-RNA Landscape in Whole Blood.

OBJECTIVE: To perform a genome-wide characterization of changes in microRNA (miRNA) expression during the course of venom immunotherapy (VIT). METHODS: miRNA was isolated from the whole-blood of 13 allergic patients and 14 controls, who experienced no allergic reaction upon stings by honeybees and wasps. We analyzed 2549 miRNAs from the whole blood of these patients prior to VIT and 12 months after the start of VIT. The results for differential expression obtained on a microarray platform were confirmed by quantitative real-time PCR. Out of the 13 patients, 8 had a negative allergic reaction with VIT, thus indicating that this approach was successful. RESULTS: By comparing time points before and 12 months after ultrarush VIT, correlation analysis and principal component analysis both indicated a limited effect of VIT on the overall miRNA expression pattern. Volcano plot analysis based on raw P values revealed few deregulated miRNAs, most of which were increasingly expressed after VIT as compared with before VIT. Based on the 50 most altered miRNAs, no clear clustering was observed before or after VIT. CONCLUSIONS: Our results indicate an overall reduced effect of VIT on the miRNA expression pattern in whole blood.

Influence of hypoxia on the power-duration relationship during high-intensity exercise.

We investigated the influence of hypoxia on the asymptote (critical power, CP) and the curvature constant (W') of the hyperbolic power-duration relationship, as measured by both conventional and all-out testing procedures. 13 females completed 5 constant-power prediction trials and a 3-min all-out test to estimate CP and W', in both normoxia (N) and moderate hypoxia (H; FiO2=0.13). CP was significantly reduced in hypoxia compared to normoxia when estimated by conventional (H:132±17 vs. N:175±25 W; P<0.001) and all-out methods (H:134±23 vs. N:172±30 W; P<0.01). The W' was not significantly different in hypoxia compared to normoxia when established by conventional (H:12.3±2.7 vs. N:13.2±2.2 kJ) and all-out methods (H:12.0±2.6 vs. N:12.5±1.4 kJ). Estimates of CP and W' obtained with conventional and all-out methods were not significantly different either in normoxia or hypoxia. There was a significant relationship between the % change in CP relative to V̇(O2peak) and the % change in W' in normoxia compared to hypoxia (r=0.83, P<0.001; conventional test). Changes in the W' in hypoxia are related to changes in the CP relative to V̇(O2peak), suggesting that the W' may not be defined simply as an 'anaerobic' energy store.

[The case of severe craniocervical dislocation due to rheumatoid arthritis]. / Przypadek powaznej dyslokacji pogranicza czaszkowo-kregoslupowego na tle reumatoidalnego zapalenia stawów.

UNLABELLED: The authors describe a case of craniocervical dislocation secondary to rheumatoid arthritis producing important canal narrowing: ventrally by migrated odontoid and dorsally by posterior arch of C-1 with medullary compression. Symptoms of hyperreflexia, spasticity and left hemiparesis with Babinski sign were present. SURGICAL PROCEDURE: transoral odontoidectomy was performed followed by suboccipital approach, C-1 laminectomy and occipitocervical fixation (Olerud device and bone graft). Outcome with neurologic improvement. CONCLUSIONS: Transoral odontoidectomy combined with occipitocervical decompression and fixation is effective approach for treatment of severe craniocerebral dislocation. Its advantages: ventral and dorsal decompression combined with immediate stabilisation.

[Fixating techniques of craniocervical junction: selection criteria]. / Metody stabilizacji pogranicza czaszkowo-kregoslupowego--kryteria doboru.

Adequate choice of fixation technique at craniocervical junction depends on many factors: anatomical conditions at fusion site (e.g. anterior dislocations of the odontoid and rupture of the transverse ligament are contraindications for direct odontoid screw fixation. Sublaminar wiring and interlaminar clamps are useless in case of deficiency of posterior bony elements of C1 and C2 whether a result of laminectomy or destruction), bone quality (osteopenic bone is contraindication for screw techniques either transarticular or transpedicular). Enclosing of occipital bone into instrumentation may be difficult in wire and clamping techniques. In contrast screw techniques allow for easy grip the occipital bone. Screw techniques seem ideal in cases requiring enclosing of the occipital bone. The fusion rate at C1/C2 level seems independent of fixation techniques. When supplemented with external immobilization even biomechanically inferior wiring or interlaminar clamping provide nearly 100 rate of fusion. Screw techniques are technically demanding but they seem the method of choice when occipital bone is to be enclosed in instrumentation.

[Trans-sternal approach to the cervicothoracic junction]. / Dojscie przez mostek do pogranicza szyjno-piersiowego kregoslupa.

Cervicothoracic junction and upper thoracic spine down to T4 can be reached through anterior approach via sternotomy. Transsternal approach is the best route to gain access to lesions localized within vertebral bodies of the upper thoracic spine allowing for their resection, interbody fusion and replacement with bone cement. Consecutive modifications of transsternal approach evolved toward less extensive osteotomy from full median sternotomy, through manubriotomy with clavicle resection and partial lateral manubriotomy. Less extensive modifications provide limited lateral exposure of the spine and are more demanding technically. We present two cases of upper thoracic spine tumours operated on through full medial sternotomy. We believe that median sternotomy has several advantages over less extensive modifications: it is technically simple to perform for trained thoracic surgeon, safer as it provides better exposure of the mediastinum and thus sufficient control of great vessels including subclavian ones, gives better exposure of T3, T4 and even T5 vertebral bodies, allows perpendicular sight and attack to anterior surface of the upper thoracic spine and therefore good visualizing of the posterior longitudinal ligament and dura, do not destabilize shoulder girdle nor affect function of the upper limb. Additional caudal exposure of the thoracic spine as down as T5 can be obtained by dissecting a plane between the brachiocephalic vein, vena cava superior and ascending aorta.

Sphingomyelinase treatment induces ATP-independent endocytosis.

ATP hydrolysis has been regarded as a general requirement for internalization processes in mammalian cells. We found, however, that treatment of ATP-depleted macrophages and fibroblasts with exogenous sphingomyelinase (SMase) rapidly induces formation of numerous vesicles that pinch off from the plasma membrane; the process is complete within 10 min after adding SMase. By electron microscopy, the SMase-induced vesicles are approximately 400 nm in diameter and lack discernible coats. 15-30% of plasma membrane is internalized by SMase treatment, and there is no detectable enrichment of either clathrin or caveolin in these vesicles. When ATP is restored to the cells, the SMase-induced vesicles are able to deliver fluid-phase markers to late endosomes/lysosomes and return recycling receptors, such as transferrin receptors, back to the plasma membrane. We speculate that hydrolysis of sphingomyelin on the plasma membrane causes inward curvature and subsequent fusion to form sealed vesicles. Many cell types express a SMase that can be secreted or delivered to endosomes and lysosomes. The hydrolysis of sphingomyelin by these enzymes is activated by several signaling pathways, and this may lead to formation of vesicles by the process described here.

The distal pathway of lipoprotein-induced cholesterol esterification, but not sphingomyelinase-induced cholesterol esterification, is energy-dependent.

The stimulation of the intracellular cholesterol esterification pathway by atherogenic lipoproteins in macrophages is a key step in the development of atheroma foam cells. The esterification pathway can also be stimulated by hydrolysis of cell-surface sphingomyelin by the enzyme sphingomyelinase (SMase). In both cases, intracellular cholesterol transport to the cholesterol esterifying enzyme, acyl-CoA:cholesterol O-acyltransferase (ACAT), is thought to be critical, although the mechanism of cholesterol transport is not known. In this report, we explore two fundamental properties of the cholesterol esterification pathway, namely its dependence on energy and the effect of other treatments that block membrane vesicle trafficking. After the atherogenic lipoprotein, beta-very low density lipoprotein (beta-VLDL), was internalized by macrophages and hydrolyzed in lysosomes, the cells were depleted of energy by treatment with sodium azide and 2-deoxyglucose or by permeabilization. Under these conditions, which allowed equal beta-VLDL-cholesteryl ester hydrolysis, cholesterol esterification was markedly decreased in the energy-depleted cells. This effect was not due to blockage of lysosomal cholesterol export. In the permeabilized cell system, energy repletion restored beta-VLDL-induced cholesterol esterification. Remarkably, stimulation of cholesterol esterification by SMase was not inhibited by energy depletion. Energy depletion also inhibited beta-VLDL-induced, but not SMase-induced, cholesterol esterification in Chinese hamster ovary cells. Similar experiments were carried out using N-ethylmaleimide, low potassium medium, or inhibitors of phosphatidylinositol 3-kinase, each of which blocks intracellular membrane vesicle trafficking. These treatments also inhibited beta-VLDL-induced, but not SMase-induced, cholesterol esterification. Finally, we show here that SMase treatment of cells leads to an increase in plasma membrane vesiculation that is relatively resistant to energy depletion. In summary, the stimulation of cholesterol esterification by lipoproteins, but not by SMase, is energy-dependent, N-ethylmaleimide-sensitive, and blocked by both low potassium and phosphatidylinositol 3-kinase inhibitors. The affected step or steps are distal to cholesterol export from lysosomes and not due to direct inhibition of the ACAT enzyme. Thus, the mechanisms involved in lipoprotein-induced versus SMase-induced cholesterol esterification are different, perhaps due to the involvement of energy-dependent vesicular cholesterol transport in the lipoprotein pathway and a novel, energy-independent vesicular transport mechanism in the SMase pathway.

Fatty acid elongation in the biosynthesis of (Z)-10-heptadecen-2-one and 2-tridecanone in ejaculatory bulb microsomes of Drosophila buzzatii.

A fatty acid chain elongation process is involved in incorporation of saturated and unsaturated fatty acyl-CoA esters into 2-tridecanone and (Z)-10-heptadecen-2-one by Drosophila buzzatii. The microsomal fraction from mature male ejaculatory bulbs is chain-length specific and requires malonyl-CoA (or acetyl-CoA, if acetyl-CoA carboxylase were present) for the chain elongation step to 2-ketones. Decarboxylation of the proposed intermediate beta-ketoacid results in 2-ketone biosynthesis. Incubation of the microsomes with the acetyl-CoA carboxylase inhibitor avidin indicated that acetyl-CoA carboxylase was present in the microsomal preparations; however, washing of the microsomal preparation removed the acetyl-CoA carboxylase activity. Fatty acyl-CoA esters were also chain elongated to produce fatty acids two and four carbons longer, suggesting that the enzymes for normal fatty acid chain elongation are also present in the microsomal fraction from ejaculatory bulbs. How much, if any, of this fatty acid chain elongation system is used for 2-ketone biosynthesis is yet to be determined.

Interferon-gamma down-regulates the lipoprotein(a)/apoprotein(a) receptor activity on macrophage foam cells. Evidence for disruption of ligand-induced receptor recycling by interferon-gamma.

Cholesterol loading of macrophages, such as occurs in atheroma foam cells, has recently been shown to upregulate a novel receptor activity that mediates the internalization degradation of the atherogenic lipoprotein, lipoprotein(a) (Lp(a)), and its protein moiety, apoprotein(a), (apo(a)). Herein, the regulation of this receptor activity by macrophage activation and interferon-gamma (IFN-gamma) was investigated. Compared with control foam cells, 125I-recombinant-apo(a) (r-apo(a)) degradation assayed after 5 h of incubation was 3-6-fold less in foam cells derived from thioglycollate- or concanavalin A-elicited mouse peritoneal macrophages. In vitro treatment of foam cells derived from resident mouse peritoneal macrophages or from human monocyte-derived macrophages with IFN-gamma also led to a substantial decrease in the ability of these cells to degrade 125I-rapo(a); similar results were obtained with 125I-Lp(a). In contrast, IFN-gamma-treated foam cells that were incubated for 10 min with 125I-r-apo(a) and then chased for 2 h in the absence of ligand degraded similar amounts of 125I-r-apo(a) as untreated foam cells. To reconcile these data, we hypothesized that the apo(a) receptor activity undergoes ligand-induced recycling and that IFN-gamma disrupts this recycling. To test this idea, control and IFN-gamma-treated foam cells were incubated for 10 min with unlabeled r-apo(a), and then 125I-r-apo(a) receptor activity was assayed at various times thereafter. Untreated foam cells showed clear evidence of ligand-induced recycling of the apo(a) receptor activity, whereas recycling was markedly diminished in the IFN-gamma-treated foam cells. Thus, by disrupting ligand-induced receptor recycling, IFN-gamma leads to down-regulation of the foam cell Lp(a)/apo(a) receptor activity. Since T cells are known to be present in atherosclerotic lesions, these findings raise the possibility that the degradation by atheroma foam cells of Lp(a) and other possible ligands for the receptor may be reversibly regulated by IFN-gamma.

Macrophage foam cell lipoprotein(a)/apoprotein(a) receptor. Cell-surface localization, dependence of induction on new protein synthesis, and ligand specificity.

Understanding the interaction of the atherogenic lipoprotein, lipoprotein(a) [Lp(a)], with macrophages may provide important insight into the physiology and pathophysiology of this lipoprotein. We have recently shown that cholesterol loading of macrophages, such as occurs in atheroma foam cells, leads to marked upregulation of a novel receptor activity for native Lp(a) and its plasminogen-like protein component, apoprotein(a) [apo(a)]. We show here that the Lp(a)/apo(a) receptor activity on cholesterol-loaded macrophages is trypsin sensitive, indicating that a cell-surface protein is involved and that the upregulation by cholesterol loading requires new protein synthesis. Ligand studies revealed that the foam cell receptor activity recognizes Lp(a) containing both small and large isoforms of apo(a) as well as rhesus monkey Lp(a), which contains an inactive kringle-4(37) (K4(37) lysine-binding domain. Elastase degradation products of plasminogen did not compete for 125I-labeled recombinant apo(a) [125I-r-apo(a)] internalization and degradation by foam cells, indicating that the K4(37) sequence, as well as the K5 and "protease" domains of apo(a), are not sufficient for receptor interaction. Consistent with these data, the degradation of 125I-r-apo(a) was completely blocked by an anti-Lp(a) polyclonal antibody that does not cross-react with plasminogen. Furthermore, the multiple sialic residues of apo(a) are also not involved in receptor interaction, since desialylated r-apo(a) interacted with foam cells as well as native r-apo(a). In contrast, reduced and denatured r-apo(a) was degraded by foam cells only slightly better than by control cells [28% increased degradation by foam cells versus 450% for native r-apo(a)], suggesting that the upregulated receptor activity recognizes certain secondary and tertiary structural features of apo(a).(ABSTRACT TRUNCATED AT 250 WORDS)

46XY siblings with inadequate virilization and CNS deficiency.

Familial expression of inadequate virilization of 46XY siblings is often reported as an isolated anomaly. We recently evaluated two families with 2 siblings who had a 46XY karyotype, ambiguous genitalia or micropenis, facial anomalies and mental retardation. There is no evidence of gonadotropin deficiency, defects of steroidogenesis, or androgen insensitivity. While there was a testosterone response to human chorionic gonadotropin stimulation in all 3 tested, gonadotropin levels were elevated in 2 of the infants suggestive of faulty seminiferous tubules, 1 of whom later had elevated luteinizing hormone levels. These kindreds may represent a new syndrome with either an X-linked recessive or sex-limited autosomal dominant form of inheritance, with partial testicular failure, multiple congenital anomalies, and mental retardation.