Polymorphisms of the TLR4 gene: Risk factor for chronicity and severity in oral vectorial Chagas disease.

Chagas disease is one of the parasitic infections with the greatest socio-economic impact in Latin America. In Venezuela, epidemiological data has shown different sources of infection, such as the vectorial route by oral transmission. Given the importance of the TLR4 gene in the innate immune response triggered by infection with Trypanosoma cruzi, this work analyses the role of TLR4 polymorphisms and its possible effect on cytokine expression. Genomic DNA was extracted from the peripheral blood of patients from the main outbreak of oral Chagas disease in Venezuela (n = 90), as well as from a group of healthy individuals (n = 183). Subsequently, peripheral blood was also extracted from individuals with different TLR4 haplotypes and then stimulated with LPS to determine the cytokine concentration by ELISA. The internalization of TLR4 was evaluated by flow cytometry. In comparison to healthy individuals, the analysis showed a significantly increased frequency of the Asp/Gly genotype in symptomatic patients. Also, observed a correlation of the 299/399 haplotype with a significant decrease in cytokine concentration and disease severity. Finally, the parasites' trypomastigotes cause the internalization or negative regulation of TLR4. The variants of TLR4 associated with low production of cytokines may be a risk factor for chronicity and severity (cardiac involvement) in oral vectorial Chagas disease.

Detection of a new reaction by-product in BDDE cross-linked autoclaved hyaluronic acid hydrogels by LC-MS analysis.

BACKGROUND: Hyaluronic acid (HA), a naturally occurring polysaccharide, is used in the production of dermal fillers for esthetic purposes. As it has a few days of half-life in human tissues, HA-based dermal filler is chemically modified to increase its lifetime in the body. The most common modification used in commercial HA-based filler is the cross-linking of HA chains using 1,4-butanediol diglycidyl ether (BDDE) as cross-linking agent. Residual, or unreacted, BDDE is considered nontoxic when it is <2 parts per million (ppm); therefore, the quantification of residual BDDE in the final dermal filler is mandatory to ensure the safety of the patients. MATERIALS AND METHODS: The present study describes the detection and characterization of one by-product of the cross-linking reaction between BDDE and HA in alkaline conditions by combining both liquid chromatography and mass spectroscopy (LC-MS). RESULTS: After different analyses, it was found that the alkaline conditions and the high temperatures employed to sterilize the HA-BDDE hydrogel promote the formation of this new by-product, a "propene glicol-like" compound. LC-MS analysis confirmed that this by-product have the same monoisotopic mass as that of BDDE, a different retention time (tR), and also a different UV absorbance (λ=200 nm) pattern. Unlike BDDE, it was observed in the LC-MS analysis that this by-product had a higher detection at 200 nm in the same assay conditions. CONCLUSION: These results suggest that this new compound does not have an epoxide on its structure. The discussion is open to assess the risk of this new by-product found in the production of HA-BDDE hydrogels (HA dermal fillers) for commercial purposes.

In vitro study of RRS® Silisorg CE Class III medical device composed of silanol: effect on human skin fibroblasts and its clinical use.

INTRODUCTION: Silanol (organic silicon) has been used for decades in the treatment of skin photoaging as it stabilizes and maintains skin structures through hydrogen bonding electrostatic interaction with extracellular matrix (ECM) proteins or glycosaminoglycans. Organic silicon-based products are often presented as silanol derivatives which are currently associated to other structural molecules such as orthohydroxybenzoate, carboxymethyl theophylline alginate, ascorbate, acetyltyrosine, sodium lactate or mannuronate. Consequently, organic silicon formulations may differ substantially between the medical devices available on the market, which may result in additional effect on the skin. Therefore, there is a real need for a better characterization of the products in terms of their action on human skin and in vitro skin model. MATERIALS AND METHODS: In this in vitro study, the effect of RRS® Silisorg was analyzed. RRS® Silisorg is a dermal implant (CE Class III medical device) containing monomethylsilanol mannuronate associated to an antioxidant resveratrol. Skin fibroblast viability and capacity to induce the production of key ECM genes were evaluated in the presence of different concentrations of RRS® Silisorg. The key ECM genes selected were collagen type I, elastin and hyaluronan synthase type 2 (HAS2), which is the cellular enzyme responsible for high-molecular weight hyaluronic acid (HA) production. Viability was evaluated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and expression was quantified by quantitative polymerase chain reaction. RESULTS: RRS® Silisorg increased fibroblast gene expression of HAS2 in the first 24 hours, 25 times in the presence of 1 mg/mL of solution, followed by a collagen type I gene expression (4.7 times) and elastin expression (2.5 times) increase after 48 hours. CONCLUSION: These results demonstrate that the silanol-based medical device RRS® Silisorg sustains HA, collagen and elastin production in human skin fibroblasts in vitro.

In vitro study of RRS HA injectable mesotherapy/biorevitalization product on human skin fibroblasts and its clinical utilization.

Mesotherapy/biorevitalization with hyaluronic acid (HA) is a treatment approach currently used for skin rejuvenation. Various products with a wide range of polycomponent formulations are available on the market. Most of these formulations contain noncross-linked HA in combination with a biorevitalization cocktail, formed by various amounts of vitamins, minerals, amino acids, nucleotides, coenzymes, and antioxidants. Although ingredients are very similar among the different products, in vitro and clinical effects may vary substantially. There is a real need for better characterization of these products in terms of their action on human skin or in vitro skin models. In this study, we analyzed the effect of the RRS(®) (Repairs, Refills, Stimulates) HA injectable medical device on human skin fibroblasts in vitro. Skin fibroblast viability and its capacity to induce the production of key extracellular matrix were evaluated in the presence of different concentrations of RRS HA injectable. Viability was evaluated through colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay, and key extracellular matrix genes, type I collagen and elastin, were quantified by quantitative polymerase chain reaction. Results demonstrated that RRS HA injectable could promote human skin fibroblast viability (+15%) and increase fibroblast gene expression of type I collagen and elastin by 9.7-fold and 14-fold in vitro, respectively. These results demonstrate that mesotherapy/biorevitalization products can, at least in vitro, effectively modulate human skin fibroblasts.

Thrombin selectively induces transcription of genes in human monocytes involved in inflammation and wound healing.

Thrombin is essential for blood coagulation but functions also as a mediator of cellular signalling. Gene expression microarray experiments in human monocytes revealed thrombin-induced upregulation of a limited subset of genes, which are almost exclusively involved in inflammation and wound healing. Among these, the expression of F3 gene encoding for tissue factor (TF) was enhanced indicating that this physiological initiator of coagulation cascade may create a feed-forward loop to enhance blood coagulation. Activation of protease-activated receptor type 1 (PAR1) was shown to play a main role in promoting TF expression. Moreover, thrombin induced phosphorylation of ERK1/2, an event that is required for expression of thrombin-regulated genes. Thrombin also increased the expression of TF at the protein level in monocytes as evidenced by Western blot and immunostaining. Furthermore, FXa generation induced by thrombin-stimulated monocytes was abolished by a TF blocking antibody and therefore it is entirely attributable to the expression of tissue factor. This cellular activity of thrombin provides a new molecular link between coagulation, inflammation and wound healing.

The degree of BCR and NFAT activation predicts clinical outcomes in chronic lymphocytic leukemia.

B-cell antigen receptor (BCR)-mediated signaling plays a critical role in chronic lymphocytic leukemia (CLL) pathogenesis and gives an in vitro survival advantage to B cells isolated from patients with unfavorable prognostic factors. In this study, we undertook to elucidate the signaling intermediates responsible for this biologic alteration. In responding cells only, in vitro BCR engagement triggers global phosphorylation of Syk, activation of phospholipase Cγ2, and intracellular calcium mobilization, reflecting competency of BCR signaling. The calcium-calcineurin-dependent transcription factor NFAT2 is up-regulated and to some extent constitutively activated in all CLL B cells. In contrast, its DNA-binding capacity is enhanced on IgM stimulation in responding cells only. NFAT inhibition using the VIVIT peptide prevents induction of CD23 target gene and IgM-induced survival, converting responding cells to unresponsive status. At the opposite, ionomycin-induced NFAT activity allows survival of nonresponding cells. These results demonstrate that the functional heterogeneity relies on variability of protein levels establishing BCR-dependent thresholds and NFAT-dependent activation. Finally, status of the BCR-NFAT pathway for each patient reveals its relevance for CLL clinical outcome and points out to BCR-NFAT intermediates as promising functional therapeutic targets.

Unexpectedly high frequency of European parentage in Venezuelan patients with chronic lymphocytic leukemia.

There is insufficient information on the characteristics of chronic lymphocytic leukemia (CLL) in Latin American patients. Immunoglobulin variable-region heavy-chain (IGVH) gene usage and mutation status and prognostic factors were investigated in patients resident in Venezuela. The most frequently used IGVH family genes were: VH3 > VH1 > VH4 > VH5, with a high incidence of IGVH1.69 and IGVH3.21 genes, and 55.2% of IGVH genes were mutated. Analysis of HCDR3 (third complementarity-determining region of the heavy chain) revealed that 24% of Venezuelan HCDR3s belonged to a CLL stereotyped HCDR3. Results for prognostic factors were similar to those reported previously for Caucasian populations. Interestingly, we found an over-representation of people of European extraction among Venezuelan patients with CLL, suggesting the possibility of a higher frequency of susceptibility genes for CLL in Europeans in comparison with Latin American mestizos.

CD16 cross-linking induces increased expression of CD56 and production of IL-12 in peripheral NK cells.

Human NK cells are classified into two populations according to the intensity of CD56 surface expression, as well as possession of CD16, FcRIII. CD56(dim)CD16(bright) make up 90% circulating NK cells, whereas CD56(bright)CD16(-/dim) comprises the remaining 10%. Here we report that peripheral NK cells upon CD16 cross-linking up-regulates the expression of activating markers and receptors such as CD25, CD69, NKp44, NKp30, CD40L and the intensity of CD56 expression. Additionally, co-culturing immature DCs with CD16 activated NK cells was found to significantly increase the expression of maturation markers on DCs. These results suggest that CD16 cross-linking on resting peripheral blood NK cells triggers the activation of these cells and induces the appearance of CD56(bright) NK cells. The latter were found capable of producing pro-inflammatory cytokines, IFN-gamma and TNF-alpha and notably IL-12.

STAT1-dependent IgG cell-surface expression in a human B cell line derived from a STAT1-deficient patient.

STAT1 is a key effector of cytokines involved in the resistance to pathogens; its identified transcriptional targets mediate the innate immune response involved in the defense against viruses and bacteria. Little is known about the role of STAT1 in adaptive immunity, including its impact on BCR or surface Ig expression. Analysis of this point is difficult in humans, as STAT1 deficiency is extremely rare. SD patients die early in childhood from a severe immunodeficiency. Herein, a SD B cell line obtained from a SD patient was compared with a B cell line from a STAT1-proficient subject in search of differences in surface Ig expression. In this SD B cell line, a complete absence of surface IgG was noted. The mRNA encoding the surface form of IgG was detected only in STAT1-proficient B cells; the mRNAs encoding the secreted and the surface forms were detected in SD and STAT1-proficient B cells. Re-expression of STAT1 in SD B cells restored surface IgG expression and a functional BCR. Conversely, shRNA silencing of STAT1 in B cells reduced considerably the expression of the surface IgG. Although limited to one B cell line, these results suggest that STAT1 may play an essential role in surface IgG expression in human B cells. Possible mechanisms involve regulation of mRNA splicing, transcription, or both. These observations extend the role of STAT1 further in adaptive immunity, including the regulation of BCR expression.

Down-regulation of CXCR4 and CD62L in chronic lymphocytic leukemia cells is triggered by B-cell receptor ligation and associated with progressive disease.

Progressive cases of B-cell chronic lymphocytic leukemia (CLL) are frequently associated with lymphadenopathy, highlighting a critical role for signals emanating from the tumor environment in the accumulation of malignant B cells. We investigated on CLL cells from 30 untreated patients the consequence of B-cell receptor (BCR) triggering on the membrane expression of CXCR4 and CD62L, two surface molecules involved in trafficking and exit of B-lymphocytes from lymph nodes. BCR stimulation promoted a strictly simultaneous down-regulation of CXCR4 and CD62L membrane expression to a variable extent. The variable BCR-dependent decrease of the two proteins was strikingly representative of the heterogeneous capacity of the CLL cells to respond to BCR engagement in a given patient. Functionally, cells down-regulating CXCR4 and CD62L in response to BCR engagement displayed a reduction in both migration toward CXCL12 and adhesion to lymphatic endothelial cells. Remarkably, the ability of CLL cells to respond to BCR ligation was correlated with unfavorable prognostic markers and short progression-free survival. In conclusion, BCR signaling promotes decrease of CXCR4 and CD62L membrane expression in progressive cases only. These results are consistent with the hypothesis that BCR-mediated signaling pathways favor accumulation of a proliferative pool within the lymph nodes of progressive CLL cases.

Novel function of STAT1beta in B cells: induction of cell death by a mechanism different from that of STAT1alpha.

Alternate splicing of STAT1 produces two isoforms: alpha, known as the active form, and beta, previously shown to act as a dominant-negative factor. Most studies have dealt with STAT1alpha, showing its involvement in cell growth control and cell death. To examine the specific function of either isoform in cell death, a naturally STAT1-deficient human B cell line was transfected to express STAT1alpha or STAT1beta. STAT1alpha, expressed alone, enhanced cell death, potentiated the fludarabine-induced apoptosis, and enhanced the nuclear location, the phosphorylation, and the transcriptional activity of p53. Unexpectedly, STAT1beta, expressed alone, induced cell death through a mechanism that was independent of the nuclear function of p53. Indeed, in STAT1beta-expressing B cells, p53 was strictly cytoplasmic where it formed clusters, and there was no induction of the transcriptional activity of p53. These data reveal a novel role of STAT1beta in programmed cell death, which is independent of p53.

Survival response to B-cell receptor ligation is restricted to progressive chronic lymphocytic leukemia cells irrespective of Zap70 expression.

Despite very similar gene expression profiles, the clinical course of B-cell chronic lymphocytic leukemia (B-CLL) is heterogeneous. Immunoglobulin VH (IgVH) mutational status and expression of B-cell receptor (BCR) signaling mediators have been associated with disease progression. However, the consequences of BCR engagement on cell survival and evolution of the disease remain unclear. We show here that B-CLL cell survival is dependent on the threshold of BCR stimulation induced by immobilized antibody, in contrast to soluble anti-mu F(ab)'2 antibody, which leads to apoptosis. Measurement of metabolic activity and apoptotic response discriminated two subgroups. "Nonresponders" showed low metabolic activity and unmodified apoptotic response upon BCR stimulation. In contrast, "responders" exhibited increased metabolic activity and inhibition of spontaneous apoptosis. This survival advantage was associated to a BCR-dependent activation profile leading to induction of cyclin D2/cyclin-dependent kinase 4 (cdk4) expression and G1 cell cycle progression. The ability to respond to BCR ligation correlated with an unfavorable clinical course and allowed to define an additional group of patients among IgVH-mutated cases exhibiting a risk of progression. Remarkably, we show that Zap70 expression was neither mandatory nor sufficient to generate downstream survival signals and cyclin D2/cdk4 up-regulation. In conclusion, BCR engagement has a significant effect on B-CLL cell survival, activation, and G1 progression. Furthermore, our results provide new insights in the physiopathology of progressive IgVH-mutated cases.

AC133+ umbilical cord blood progenitors demonstrate rapid self-renewal and low apoptosis.

Umbilical cord blood (UCB) provides immediate access to haemopoietic stem/progenitor cells (HSPC) but low cell number restricts use in full adult bone marrow reconstitution. This study investigated early ex vivo expansion kinetics of UCB AC133+ cells (2-4 x 10(4)/ml), mononuclear cells (MNC, 1-2 x 10(6)/ml) and AC133negative cells (AC133(neg), 2-4 x 10(4)/ml) in stroma-free 8 d liquid culture (fetal bovine serum-supplemented Iscove's-modified Dulbecco's medium (IMDM) with either 'K36EG'[c-Kit ligand, interleukin 3 (IL-3), IL-6, erythropoietin, granulocyte colony-stimulating factor] or 'TPOFL' (thrombopoietin, Flt-3 ligand). Cell enumeration, apoptosis assay and AC133/CD34/CD38 antigen immunophenotyping were performed at d 0, 3, 5 and 8. All three cell populations went through a proliferation lag phase between d 3 and d 5. AC133+ cells recovered better from lag phase with significantly higher fold increase (FI) when compared with MNC and AC133(neg) populations (K36EG FI: 15.04 +/- 5.46; TPOFL FI: 8.59 +/- 2.92, P < 0.05). After 8 d, populations lacking AC133+ cells were significantly more inclined to undergo apoptosis under proliferative conditions (P < 0.01). Also, when compared with K36EG, 8 d TPOFL-expanded AC133+ cells encompassed a significantly higher percentage of AC133+ and CD34+ early HSPC (K36EG: 20.50 +/- 2.36; TPOFL: 47.00 +/- 7.69; P < 0.05). In conclusion, TPOFL synergism demonstrated the potential for AC133+ HSPC ex vivo expansion inducing self-renewal, early HSPC maintenance and promoting cell survival status.