Systemic sclerosis sera affect fibrillin-1 deposition by dermal blood microvascular endothelial cells: therapeutic implications of cyclophosphamide

Introduction

Systemic sclerosis (SSc) is a connective tissue disorder characterized by endothelial cell injury, autoimmunity and fibrosis. The following three fibrillin-1 alterations have been reported in SSc. (1) Fibrillin-1 microfibrils are disorganized in SSc dermis. (2) Fibrillin-1 microfibrils produced by SSc fibroblasts are unstable. (3) Mutations in the FBN1 gene and anti-fibrillin-1 autoantibodies have been reported in SSc. Fibrillin-1 microfibrils, which are abundantly produced by blood and lymphatic microvascular endothelial cells (B-MVECs and Ly-MVECs, respectively), sequester in the extracellular matrix the latent form of the potent profibrotic cytokine transforming growth factor β (TGF-β). In the present study, we evaluated the effects of SSc sera on the deposition of fibrillin-1 and microfibril-associated glycoprotein 1 (MAGP-1) and the expression of focal adhesion molecules by dermal B-MVECs and Ly-MVECs.

Methods

Dermal B-MVECs and Ly-MVECs were challenged with sera from SSc patients who were treatment-naïve or under cyclophosphamide (CYC) treatment and with sera from healthy controls. Fibrillin-1/MAGP-1 synthesis and deposition and the expression of α_vβ₃ integrin/phosphorylated focal adhesion kinase and vinculin/actin were evaluated by immunofluorescence and quantified by morphometric analysis.

Results

Fibrillin-1 and MAGP-1 colocalized in all experimental conditions, forming a honeycomb pattern in B-MVECs and a dense mesh of short segments in Ly-MVECs. In B-MVECs, fibrillin-1/MAGP-1 production and α_vβ₃ integrin expression significantly decreased upon challenge with sera from naïve SSc patients compared with healthy controls. Upon challenge of B-MVECs with sera from CYC-treated SSc patients, fibrillin-1/MAGP-1 and α_vβ₃ integrin levels were comparable to those of cells treated with healthy sera. Ly-MVECs challenged with SSc sera did not differ from those treated with healthy control sera in the expression of any of the molecules assayed.

Conclusions

Because of the critical role of fibrillin-1 in sequestering the latent form of TGF-β in the extracellular matrix, its decreased deposition by B-MVECs challenged with SSc sera might contribute to dermal fibrosis. In SSc, CYC treatment might limit fibrosis through the maintenance of physiologic fibrillin-1 synthesis and deposition by B-MVECs.     

Biodegradability of Food-Associated Extracellular Polysaccharides

Exopolysaccharides (EPSs) produced by lactic acid bacteria, which are common in fermented foods, are claimed to have various beneficial physiological effects on humans. Although the biodegradability of EPSs is important in relation to the bioactive properties, knowledge on this topic is limited. Therefore, the biodegradability of eight EPSs, six of which were produced by lactic acid bacteria, was compared with microorganisms from human feces or soil. EPS-degradation was determined from the decrease in polysaccharide-sugar concentration and by high-performance size exclusion chromatography (HPSEC). Xanthan, clavan, and the EPSs produced by Streptococcus thermophilus SFi 39 and SFi 12 were readily degraded, in contrast to the EPSs produced by Lactococcus lactis ssp. cremoris B40, Lactobacillus sakei 0-1, S. thermophilus SFi20, and Lactobacillus helveticus Lh59. Clearly, the susceptibility of exopolysaccharides to biological breakdown can differ greatly, implying that the physiological effects of these compounds may also vary a lot. Received: 23 August 1999 / Accepted: 5 October 1999     

Genomic analysis reveals the biotechnological ability of Enterococcus italicus to produce glutathione

Through the analysis of the recently available genome shotgun sequence of Enterococcus italicus DSM 15952^T type strain (Accession PRJNA61487, ID 61487), we found the presence of a gene encoding a bifunctional enzyme, termed γ-GCS-GS or GshF, involved in glutathione production and not influenced by feedback inhibition. The gshF gene exhibited high nucleotide and amino acid sequence similarity to other reported sequences from the Enterococcus genus and was constitutively expressed both in osmotic shock or in common cultural conditions. Several experimental studies concerning the culture medium, physiological stress, cell extract obtainment, and scaling-up showed that in selected conditions E. italicus was able to accumulate up to 250 μM of intracellular glutathione, which represented the main thiol group present into the cells. This is the first report regarding the production of glutathione by E. italicus, a species that could be used as a safe adjunct culture for glutathione-enriched dairy foods.     

PYK2 is overexpressed in chronic lymphocytic leukaemia: A potential new therapeutic target

Chronic Lymphocytic Leukaemia (CLL) is the most common adult B-cell leukaemia and despite improvement in patients' outcome, following the use of targeted therapies, it remains incurable. CLL supportive microenvironment plays a key role in both CLL progression and drug resistance through signals that can be sensed by the main components of the focal adhesion complex, such as FAK and PYK2 kinases. Dysregulations of both kinases have been observed in several metastatic cancers, but their role in haematological malignancies is still poorly defined. We characterized FAK and PYK2 expression and observed that PYK2 expression is higher in leukaemic B cells and its overexpression significantly correlates with their malignant transformation. When targeting both FAK and PYK2 with the specific inhibitor defactinib, we observed a dose–response effect on CLL cells viability and survival. In vivo treatment of a CLL mouse model showed a decrease of the leukaemic clone in all the lymphoid organs along with a significant reduction of macrophages and of the spleen weight and size. Our results first define a possible prognostic value for PYK2 in CLL, and show that both FAK and PYK2 might become putative targets for both CLL and its microenvironment (e.g. macrophages), thus paving the way to an innovative therapeutic strategy.     

Modeling human osteosarcoma in mice through 3AB-OS cancer stem cell xenografts

Osteosarcoma is the second leading cause of cancer‐related death for children and young adults. In this study, we have subcutaneously injected—with and without matrigel—athymic mice (Fox1nu/nu) with human osteosarcoma 3AB‐OS pluripotent cancer stem cells (CSCs), which we previously isolated from human osteosarcoma MG63 cells. Engrafted 3AB‐OS cells were highly tumorigenic and matrigel greatly accelerated both tumor engraftment and growth rate. 3AB‐OS CSC xenografts lacked crucial regulators of beta‐catenin levels (E‐cadherin, APC, and GSK‐3beta), and crucial factors to restrain proliferation, resulting therefore in a strong proliferation potential. During the first weeks of engraftment 3AB‐OS‐derived tumors expressed high levels of pAKT, beta1‐integrin and pFAK, nuclear beta‐catenin, c‐Myc, cyclin D2, along with high levels of hyperphosphorylated‐inactive pRb and anti‐apoptotic proteins such as Bcl‐2 and XIAP, and matrigel increased the expression of proliferative markers. Thereafter 3AB‐OS tumor xenografts obtained with matrigel co‐injection showed decreased proliferative potential and AKT levels, and undetectable hyperphosphorylated pRb, whereas beta1‐integrin and pFAK levels still increased. Engrafted tumor cells also showed multilineage commitment with matrigel particularly favoring the mesenchymal lineage. Concomitantly, many blood vessels and muscle fibers appeared in the tumor mass. Our findings suggest that matrigel might regulate 3AB‐OS cell behavior providing adequate cues for transducing proliferation and differentiation signals triggered by pAKT, beta1‐integrin, and pFAK and addressed by pRb protein. Our results provide for the first time a mouse model that recapitulates in vivo crucial features of human osteosarcoma CSCs that could be used to test and predict the efficacy in vivo of novel therapeutic treatments. J. Cell. Biochem. 113: 3380–3392, 2012. © 2012 Wiley Periodicals, Inc.     

Biomarkers of Good EULAR Response to the B Cell Depletion Therapy in All Seropositive Rheumatoid Arthritis Patients: Clues for the Pathogenesis

Objective

To find out whether a high number of auto-antibodies can increase the probability of a “good-EULAR response” and to identify the possible biomarkers of response in seropositive rheumatoid arthritis (RA) patients undergoing the B cell depletion therapy (BCDT).

Patients and Methods

One hundred and thirty-eight patients with long standing RA (LSRA), 75% non or poorly responsive to one or more TNFα blockers, all seropositive for at least one autoantibody (AAB) (RF-IgM, RF-IgA, RF-IgG, anti-MCV, ACPA-IgG, ACPA-IgA, ACPA-IgM) received one full course of BCDT. The major outcomes (moderate or good-EULAR response) were assessed after 6 months of therapy. The IL6 and BAFF levels were also determined.

Results

At a 6-month follow-up, 33 (23.9%) of the RA patients achieved a good EULAR response. Having up to 5-AABs positivity increased the chances for treatment response. After a logistic regression analysis, however, only 4 baseline factors arose as associated with a good-EULAR response: no steroid therapy (OR = 6.25), a lymphocyte count <1875/uL (OR = 10.74), a RF-IgG level >52.1 IU/ml (OR = 8.37) and BAFF levels <1011 pg/ml (OR = 7.38). When all the AABs, except for RF-IgM and ACPA-IgG, were left in the analysis, the two final predictors were no-steroid therapy and low lymphocyte count.

Discussion

The number of AABs increased the chances of being a “good-EULAR” responder. The only predictors, however, at the baseline of a good response in this seropositive cohort of RA patients were 2 simple variables – no steroids and lymphocyte count – and two laboratory assays – IgG-RF and BAFF.     

Combination treatment using thymosin α1 and interferon after cyclophosphamide is able to cure Lewis lung carcinoma in mice

Summary A combination treatment with thymosin 1 (200 µg/kg) for 4 days, followed by a single injection of murine interferon / (3 × 10⁴ international units/mouse), starting 2 days after cyclophosphamide treatment (200 mg/kg, single injection) demonstrated a dramatic and rapid disappearance of tumor burden in mice bearing Lewis lung carcinoma (3LL) tumor. The effectiveness of this new chemoimmunotherapy protocol was evident even on the long-term survival in a high percentage of animals, and was statistically significant when compared to treatment with the single agents in conjunction with chemotherapy or to chemotherapy itself. The same combination immunotherapy treatment strongly stimulated natural killer activity and cytotoxicity against autologus 3LL tumor cells in 3LL-tumor-bearing mice treated with cyclophosphamide, whereas treatments with each agent singly did not alter or only slightly modified the cytotoxic activity towards Yac-1 or 3LL target cells. Selective depletion with antibodies showed that killer cells stimulated by combination chemoimmunotherapy treatment bear phenotypic characteristics of asialo-GM1-positive cells. A histological study has shown a high number of infiltrating lymphoid cells in the tumors obtained from mice treated with combination chemoimmunotherapy.     

Organization and nucleotide sequence of the genes for ribosomal protein S2 and elongation factor Ts in Spirulina Platensis

A 6.5 kb region from the genome of the cyanobacterium Spirulina platensis was cloned using as a probe the Escherichia coli gene for ribosomal protein S2. Sequence analysis revealed, in this region, the presence of the gene for ribosomal protein S2 and part of the gene for the elongation factor Ts (EF-Ts). The arrangement rpsB-spacer-tsf resembles that reported for E. coli. The deduced amino acid sequences of the platensis S2 and EF-Ts show significant homology with the E. coli counterparts.     

Comparative analysis of inner cavities and ligand migration in non-symbiotic AHb1 and AHb2

This study reports a comparative analysis of the topological properties of inner cavities and the intrinsic dynamics of non-symbiotic hemoglobins AHb1 and AHb2 from Arabidopsis thaliana. The two proteins belong to the 3/3 globin fold and have a sequence identity of about 60%. However, it is widely assumed that they have distinct physiological roles. In order to investigate the structure–function relationships in these proteins, we have examined the bis-histidyl and ligand-bound hexacoordinated states by atomistic simulations using in silico structural models. The results allow us to identify two main pathways to the distal cavity in the bis-histidyl hexacoordinated proteins. Nevertheless, a larger accessibility to small gaseous molecules is found in AHb2. This effect can be attributed to three factors: the mutation Leu35(AHb1) → Phe32(AHb2), the enhanced flexibility of helix B, and the more favorable energetic profile for ligand migration to the distal cavity. The net effect of these factors would be to facilitate the access of ligands, thus compensating the preference for the fully hexacoordination of AHb2, in contrast to the equilibrium between hexa- and pentacoordinated species in AHb1. On the other hand, binding of the exogenous ligand introduces distinct structural changes in the two proteins. A well-defined tunnel is formed in AHb1, which might be relevant to accomplish the proposed NO detoxification reaction. In contrast, no similar tunnel is found in AHb2, which can be ascribed to the reduced flexibility of helix E imposed by the larger number of salt bridges compared to AHb1. This feature would thus support the storage and transport functions proposed for AHb2. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.