The Local Complement Activation on Vascular Bed of Patients with Systemic Sclerosis: A Hypothesis-Generating Study

ObjectiveThe role of complement system in the pathogenesis of systemic sclerosis (SSc) has been debated during the last decade but an evident implication in this disease has never been found. We carried out an explorative study on SSc patients to evaluate the expression of soluble and local C5b-9 complement complex and its relation with a complement regulator, the Membrane Cofactor Protein (MCP, CD46) on skin vascular bed as target distinctive of SSc disease. We also analyzed two polymorphic variants in the complement activation gene cluster involving the MCP region.MethodsC5b-9 plasma levels of SSc patients and healthy subjects were analyzed by ELISA assay. Archival skin biopsies of SSc patients and controls were subjected to immunofluorescence analysis to detect C5b-9 and MCP on vascular endothelial cells. The expression of MCP was validated by immunoblot analysis with specific antibody. Polymorphic variants in the MCP gene promoter were tested by a quantitative PCR technique-based allelic discrimination method.ResultsEven though circulating levels of C5b-9 did not differ between SSc and controls, C5b-9 deposition was detected in skin biopsies of SSc patients but not in healthy subjects. MCP was significantly lower in skin vessels of SSc patients than in healthy controls and was associated with the over-expression of two polymorphic variants in the MCP gene promoter, which has been related to more aggressive phenotypes in other immune-mediated diseases.ConclusionsOur results firsty document the local complement activation with an abnormal expression of MCP in skin vessels of SSc patients, suggesting that a subset of SSc patients might be exposed to more severe organ complications and clinical evolution due to abnormal local complement activation.     

Reliable and Accurate CD4+ T Cell Count and Percent by the Portable Flow Cytometer CyFlow MiniPOC and “CD4 Easy Count Kit-Dry”, as Revealed by the Comparison with the Gold Standard Dual Platform Technology

Background

An accurate and affordable CD4+ T cells count is an essential tool in the fight against HIV/AIDS. Flow cytometry (FCM) is the “gold standard” for counting such cells, but this technique is expensive and requires sophisticated equipment, temperature-sensitive monoclonal antibodies (mAbs) and trained personnel. The lack of access to technical support and quality assurance programs thus limits the use of FCM in resource-constrained countries. We have tested the accuracy, the precision and the carry-over contamination of Partec CyFlow MiniPOC, a portable and economically affordable flow cytometer designed for CD4+ count and percentage, used along with the “CD4% Count Kit-Dry”.

Materials and Methods

Venous blood from 59 adult HIV+ patients (age: 25–58 years; 43 males and 16 females) was collected and stained with the “MiniPOC CD4% Count Kit-Dry”. CD4+ count and percentage were then determined in triplicate by the CyFlow MiniPOC. In parallel, CD4 count was performed using mAbs and a CyFlow Counter, or by a dual platform system (from Beckman Coulter) based upon Cytomic FC500 (“Cytostat tetrachrome kit” for mAbs) and Coulter HmX Hematology Analyzer (for absolute cell count).

Results

The accuracy of CyFlow MiniPOC against Cytomic FC500 showed a correlation coefficient (CC) of 0.98 and 0.97 for CD4+ count and percentage, respectively. The accuracy of CyFlow MiniPOC against CyFlow Counter showed a CC of 0.99 and 0.99 for CD4 T cell count and percentage, respectively. CyFlow MiniPOC showed an excellent repeatability: CD4+ cell count and percentage were analyzed on two instruments, with an intra-assay precision below ±5% deviation. Finally, there was no carry-over contamination for samples at all CD4 values, regardless of their position in the sequence of analysis.

Conclusion

The cost-effective CyFlow MiniPOC produces rapid, reliable and accurate results that are fully comparable with those from highly expensive dual platform systems.     

Inaccuracy of Venous Point-of-Care Glucose Measurements in Critically Ill Patients: A Cross-Sectional Study

Introduction

Current guidelines and consensus recommend arterial and venous samples as equally acceptable for blood glucose assessment in point-of-care devices, but there is limited evidence to support this recommendation. We evaluated the accuracy of two devices for bedside point-of-care blood glucose measurements using arterial, fingerstick and catheter venous blood samples in ICU patients, and assessed which factors could impair their accuracy.

Methods

145 patients from a 41-bed adult mixed-ICU, in a tertiary care hospital were prospectively enrolled. Fingerstick, central venous (catheter) and arterial blood (indwelling catheter) samples were simultaneously collected, once per patient. Arterial measurements obtained with Precision PCx, and arterial, fingerstick and venous measurements obtained with Accu-chek Advantage II were compared to arterial central lab measurements. Agreement between point-of-care and laboratory measurements were evaluated with Bland-Altman, and multiple linear regression models were used to investigate interference of associated factors.

Results

Mean difference between Accu-chek arterial samples versus central lab was 10.7 mg/dL (95% LA -21.3 to 42.7 mg/dL), and between Precision PCx versus central lab was 18.6 mg/dL (95% LA -12.6 to 49.5 mg/dL). Accu-chek fingerstick versus central lab arterial samples presented a similar bias (10.0 mg/dL) but a wider 95% LA (-31.8 to 51.8 mg/dL). Agreement between venous samples with arterial central lab was the poorest (mean bias 15.1 mg/dL; 95% LA -51.7 to 81.9). Hyperglycemia, low hematocrit, and acidosis were associated with larger differences between arterial and venous blood measurements with the two glucometers and central lab. Vasopressor administration was associated with increased error for fingerstick measurements.

Conclusions

Sampling from central venous catheters should not be used for glycemic control in ICU patients. In addition, reliability of the two evaluated glucometers was insufficient. Error with Accu-chek Advantage II increases mostly with central venous samples. Hyperglycemia, lower hematocrit, acidosis, and vasopressor administration increase measurement error.     

An efficient protocol for micropropagation of <Emphasis Type="Italic">Melaleuca alternifolia</Emphasis> Cheel

Melaleuca alternifolia is cultivated for the production of an essential oil useful in the cosmetic and pharmaceutical industries. Despite the economic importance of this species, there is little knowledge about its in vitro propagation. The aim of this study was to establish an efficient protocol for micropropagation of M. alternifolia. With the goal of in vitro multiplication by axillary shoot proliferation, both solid and liquid MS and WPM media were tested with supplementation with BA at 0, 0.55, 1.11, 2.22, 3.33, and 4.44 μM. The best result for shoot multiplication was obtained when either 0.55 μM BA was added into solid MS medium or 1.11 μM BA was added into liquid MS medium, with 5.6 and 11.8 shoots per explant generated, respectively. On solid or liquid WPM medium supplemented with 0.55 μM BA, the proliferation rates were 5.5 and 4.7, respectively. Three auxins (NAA, IAA, and IBA) were tested at 0.53 and 2.64 μM during the rooting stage. Several sucrose concentrations (15, 30, and 45 g L⁻¹) were compared to a sucrose-free medium. Rooting performances on four culture media were then compared: MS, half-strength MS (MS/2), MS + activated charcoal (AC), and MS/2 + AC. The results showed that auxin addition to culture medium is not necessary for in vitro rooting. Rooted microcuttings from different culture media were acclimatized in a greenhouse, and the survival percentage was evaluated. All shoots cultured in an auxin-free MS medium supplemented with sucrose (30 g L⁻¹) produced roots, and all plants survived during acclimatization. Activated charcoal added in rooting medium reduced rooting rates.     

Jejunal Creatine Absorption: What is the Role of the Basolateral Membrane?

The mechanism of the intestinal creatine absorption is not well understood. Previous studies have established the involvement of a CT1 carrier system in jejunal apical membrane. The current research was aimed at completing the picture of creatine absorption. To investigate the process supporting creatine exit from enterocyte, basolateral membrane vesicles isolated from rat jejunum were used. The presence of various symport and antiport mechanisms was searched and a NaCl-dependent electrogenic transport system for creatine was evidenced, which shares some functional and kinetic features with the apical CT1. However, Western blot and immunohistochemical experiments ruled out the presence of a CT1 transporter in the basolateral membrane. Further studies are required to identify the basolateral transport mechanism. However, in the in vivo conditions, the NaCl gradient is inwardly directed, therefore such a mechanism cannot energetically mediate the exit of creatine from the cell into the blood during the absorptive process, but rather it may drive creatine into the enterocyte. To shed more light on the creatine absorption process, a possible creatine movement through the paracellular pathway has been examined using the jejunal tract everted and incubated in vitro. A linear relationship between creatine transport and concentration was apparent both in the mucosa-to-serosa and serosa-to-mucosa directions and the difference between the two slopes suggests that paracellular creatine movement by solvent drag may account for transintestinal creatine absorption. As a matter of fact, when transepithelial water flux is reduced by means of a mucosal hypertonic solution, the opposite creatine fluxes tend to overlap. The findings of the present study suggest that paracellular creatine movement by solvent drag may account for transintestinal creatine absorption.     

4-hydroxynonenal and TGF-beta1 concur in inducing antiproliferative effects on the CaCo-2 human colon adenocarcinoma cell line

4-Hydroxynonenal (HNE) has been demonstrated to exert its antiproliferative effect by up-regulating the c-Jun-N-terminal kinase (JNK), a member of the mitogen-activated protein kinase family (MAPKs). Transforming growth factor-beta1 (TGF-beta1) is the major negative regulatory factor in controlling cell proliferation, and Smads are its intracellular transducers. Recent data on human colon adenocarcinoma has shown a low HNE content paralleled by a marked alteration of TGF-beta1 levels within the tumor mass. The two events appear related because of the demonstrated marked ability of HNE to up-regulate expression and synthesis of TGF-beta1; the combined decreases of HNE and TGF-beta1 found in cancer cells provide a favorable condition for neoplastic progression. Furthermore, HNE is likely able to interact with the cytokine to enhance apoptosis and increase intracellular reactive oxygen species (ROS) formation in the CaCo-2 colon carcinoma cell line. The probable mechanism whereby HNE and TGF-beta1 interact to induce apoptosis is through cross-talk between the main signaling pathways of the two molecules (JNK and Smads), and the observed ROS production might only contribute to amplifying the apoptotic pathways. The network between the two signaling pathways here involved is now under investigation.     

Oxysterol-induced up-regulation of MCP-1 expression and synthesis in macrophage cells

To investigate the proinflammatory potential of cholesterol and cholesterol oxidation products (oxysterols), which are present in oxidized low-density lipoproteins, foam cells, and fibrotic plaque, we used an in vitro model mimicking the challenge of macrophage cells by the cholesterol accumulating within the central core of atheroma. A biologically representative oxysterol mixture was shown to be potentially able to sustain a chronic inflammatory process within the vascular wall by up-regulating the expression of defined proinflammatory genes. In particular, expression and synthesis of the major chemokine for monocytes/macrophages, namely monocyte chemotactic protein-1 (MCP-1), were consistently increased when cells of the macrophage lineage (U937 cell line) were incubated with this mixture. On the contrary, an identical concentration of unoxidized cholesterol in no case modified expression or synthesis of the chemokine. Up-regulated expression and synthesis of MCP-1 by the oxysterol mixture was clearly dependent on a net increment of phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor kappaB (NF-kappaB) nuclear binding. The results indicate that cholesterol may contribute to the progression of atherosclerotic lesions by strongly up-regulating crucial proinflammatory factors like MCP-1, but only after having been oxidized to oxysterols.     

4-Hydroxynonenal signalling to apoptosis in isolated rat hepatocytes: the role of PKC-delta

4-Hydroxynonenal, a significant aldehyde end product of membrane lipid peroxidation with numerous biochemical activities, has consistently been detected in various human diseases. Concentrations actually detectable in vivo (0.1-5 microM) have been shown to up-regulate different genes and modulate various enzyme activities. In connection with the latter aspect, we show here that, in isolated rat hepatocytes, 1 microM 4-hydroxynonenal selectively activates protein kinase C-delta, involved in apoptosis of many cell types; it also induces very early activation of Jun N-terminal kinase, in parallel increasing activator protein-1 DNA-binding activity in a time-dependent manner and triggering apoptosis after only 120 min treatment. These phenomena are likely protein kinase C-delta-dependent, being significantly reduced or annulled by cell co-treatment with rottlerin, a selective inhibitor of protein kinase C-delta. We suggest that 4-hydroxynonenal may induce apoptosis through activation of protein kinase C-delta and of Jun N-terminal kinase, and consequent up-regulation of activator protein-1 DNA binding.     

c-Jun N-terminal kinase upregulation as a key event in the proapoptotic interaction between transforming growth factor-beta1 and 4-hydroxynonenal in colon mucosa

Cells of colonic mucosa are sensitive to the Smad-mediated growth-inhibitory effect of transforming growth factor-beta1 (TGF-beta1). Another important cell growth inhibitor is the polyunsaturated lipid peroxidation end product, 4-hydroxynonenal (HNE), which triggers apoptosis through c-Jun N-terminal kinase (JNK) activation. Interestingly, a close association between TGF-beta1 and HNE was found in the progression of human colon cancer, with concentration of both molecules inversely related to the malignancy. We investigated the cross talk between Smads and JNK signal transduction pathways in inducing apoptosis. To this purpose TGF-beta1 and HNE were added singly or in combination to CaCo-2 human colon adenocarcinoma cells. The cotreatment induced a marked enhancement of apoptosis and of JNK and Smad4 activities much more than either individual molecule. Cell preincubation with the JNK inhibitor SP600125 significantly prevented JNK and Smad4 enhancement and, subsequently, the cooperative proapoptotic effect was abolished. The primary role of JNK activity in TGF-beta1/HNE cooperative signaling was fully confirmed in a second set of experiments by using JNKi I, a more selective kinase inhibitor. Hence, in tumor cells becoming resistant to TGF-beta1-mediated growth inhibition, increased induction of the remaining TGF-beta1 pathways by interaction with other antiproliferative molecules, such as HNE, could help in inhibiting tumor growth.