Water in cellulose: evidence and identification of immobile and mobile adsorbed phases by 2H MAS NMR.

The organization of water molecules adsorbed onto cellulose and the supramolecular hydrated structure of microfibril aggregates represents, still today, one of the open and complex questions in the physical chemistry of natural polymers. Here, we investigate by 2H MAS NMR the mobility of water molecules in carefully 2H-exchanged, and thereafter re-dried, microcrystalline cellulose. By subtracting the spectral contribution of deuteroxyls from the spectrum of hydrated cellulose, we demonstrate the existence of two distinct 2H2O spectral populations associated with mobile and immobile water environments, between which the water molecules do not exchange at the NMR observation time scale. We conclude that those two water phases are located at differently-accessible adsorption sites, here assigned to the cellulose surfaces between and within the microfibril aggregates, respectively. The superior performance of 2H MAS NMR encourages further applications of the same method to other complex systems that expose heterogeneous hygroscopic surfaces, like wood cell walls.

Validity of NMR pore-size analysis of cultural heritage ancient building materials containing magnetic impurities.

NMR relaxation time distributions, obtained with laboratory and portable devices, are utilized to characterize the pore-size distributions of building materials coming from the Roman remains of the Greek-Roman Theatre of Taormina. To validate the interpretation of relaxation data in terms of pore-size distribution, comparison of results from standard and in situ NMR experiments with results of the mercury intrusion porosimetry (MIP) has been made. Although the pore-size distributions can be obtained by NMR in terms of either longitudinal (T(1)) or transverse (T(2)) relaxation times distributions, the shorter duration of the T(2) measurement makes it, in principle, preferable, although the determination of T(2) distributions is not necessarily an easy alternative to finding T(1) distributions. Among other things, the T(1) distribution is almost independent of the inhomogeneity of the magnetic field, while the T(2) distribution is strongly influenced by it. This paper was aimed at answering two questions: what are the validity limits to interpret NMR data in terms of pore-size distributions and whether the portable device can successfully be applied as a non-destructive and non-invasive tool for in situ NMR analysis of building materials, particularly those of Cultural Heritage interest.

Water absorption kinetics in different wettability conditions studied at pore and sample scales in porous media by NMR with portable single-sided and laboratory imaging devices.

NMR relaxation time distributions of water (1)H obtained by a portable single-sided surface device have been compared with MRI internal images obtained with a laboratory imaging apparatus on the same biocalcarenite (Lecce Stone) samples during capillary water uptake. The aim of this work was to check the ability of NMR methods to quantitatively follow the absorption phenomenon under different wettability conditions of the internal pore surfaces. Stone wettability changes were obtained by capillary absorption of a chloroform solution of Paraloid PB72, a hydrophobic acrylic resin frequently used to protect monuments and buildings, through one face of each sample. Both relaxation and imaging data have been found in good quantitative agreement each other and with masses of water determined by weighing the samples. In particular the Washburn model of water capillary rise applied to the imaging data allowed us to quantify the sorptivity in both treated and untreated samples. Combining relaxation and imaging data, a synergetic improvement of our understanding of the water absorption kinetics at both pore and sample scales is obtained. Since relaxation data have been taken over the course of time without interrupting the absorption process, simply by keeping the portable device on the surface opposite to the absorption, the results show that the single-sided NMR technique is a powerful tool for in situ evaluation of water-repellent treatments frequently used for consolidation and/or protection of stone artifacts.

Mechanisms of immunological tolerance loss versus erythrocyte self-antigens and autoimmune hemolytic anemia.

Recent studies on animal and human autoimmune hemolytic anemia (AIHA) suggest that immunological tolerance loss toward red blood cells (RBC) self-antigens may be originate by different, non-mutually exclusive, mechanisms. According to now available data the identified mechanisms may be: ignorance against RBC self-antigens; molecular mimicry; polyclonal T and/or B cells activation; errors in central or peripheral tolerance; immunoregulatory disorders including cytokine network alteration. In some patients with AIHA, stimulation of PMBC by synthetic Rh peptides indicate that ignorant T and/or B cell clones may recognize cryptic RBC self-antigens. AIHA associated with bacterial or viral infections seems to be produced by polyclonal T and/or B cells activation against foreign antigens which mimic protein or carbohydrate epitopes on RBC. Polyclonal activation of host B cell clones by donor alloreactive T cells causes the AIHA in chronic GVHD. As the tolerance loss is concerned, experiments on mouse lines expressing a transgene with autoantibody activity against murine RBC have shown that non-deleted peripheral B cell clones may produce RBC autoantibodies. In humans a genetic defect of Fas/FasL autoreactive lymphocytes apoptosis may be associated to AIHA. Immunoregulatory disorders due to depletion of CD4+ CD25+ T cells or Th1/Th2 cytokines imbalance may induce autoimmune diseases. In mice AIHA may be induced or improved by cytokines or anticytokine antibodies administration. In NZB/W mice and human AIHA there is an increased production of Th2 cytokines as IL4 and IL10 but INF-gamma reduced production. In addition in human AIHA has been shown a downregulation of IL12 and therefore, an IL10/IL12 immunoregulatory circuit imbalance which might facilitate the RBC autoantibodies production.

Cytokine network in autoimmune haemolytic anaemia: new probable targets for therapy.

AIMS: Several immunological mechanisms seems to be similar in cancer and autoimmune disease. Studying interleukins production and proliferative response in autoimmune haemolytic anaemia (AIHA), it is possible to observe that manipulation of IL-10/IL-12 balance can have profound effect on the incidence of autoimmune diseases and this might be useful for the control of AIHA. METHODS: Respective role of IL-2, IL-4, IFN-gamma, IL-10 and IL-12 in non-cancer associated AIHA were investigated by examining the spontaneous and mitogen-induced (OKT3 or LPS) synthesis of these cytokines in PBMC cultures by ELISA methods. RESULTS: Our results affirmed that AIHA is a disease which exhibited an increased basal synthesis of IL-4 and decreased levels of IFN-gamma by AIHA PBMC compared with controls and then there is a basal increase of Th2 cytokines. Th1-type cytokine decrease in basal state occurred in parallel with an increase of constitutive IL-10 production and a IL-12 decrease. CONCLUSIONS: Decreased production of Th1-type cytokines and the production of autoantibodies in AIHA may be secondary to the imbalance between IL-10 and IL-12 and then the neutralisation of IL-10 may be efficacious in diminishing the clinical pathology associated with Th2 subset prevalence. In the same way, the treatment with IL-12 could offer a second and independent level of blockade against the consequences of the over B cell activation associated with AIHA and sometimes with cancer.

Th1 and Th2 cytokine modulation by IL-10/IL-12 imbalance in autoimmune haemolytic anaemia (AIHA).

Recent studies about autoimmune diseases in animal models and in humans focused their attention on lymphocyte activation and in vitro cytokine production. The respective contribution of the Th1 and Th2 cytokines to the pathogenesis of autoimmune diseases is still a matter of debate. In this study the role of IL-2, IL-4, IFN-gamma, IL-10 and IL-12 cytokines were investigated by examining their spontaneous and mitogen-induced (OKT3 and PHA or LPS) synthesis and T-cells proliferative response by peripheral blood mononuclear cells to determine their role in the pathogenesis of AIHA. Thirteen patients affected by AIHA, idiopathic or associated with other diseases, and 13 healthy subjects, randomly selected from a group of blood donors, were investigated. This study indicated that AIHA is characterised by increased basal synthesis of IL-4 and decreased levels of IFN-gamma compared with healthy controls (p < 0,01). These results suggest that there is a basal decrease of Th1 cytokine and an increase of the Th2 ones. Enhanced IL-2 levels in AIHA patients are likely due to the necessity of a T-cell proliferation stimulus rather than produced as Th1 prevalent stimulation. Furthermore, it has been observed a significant increase in IL-12 production in LPS stimulated cultures from healthy controls, but not in AIHA patients, that shows IL-10 increased levels, which could cause a secondary decrease in IFN-gamma production and a stimulation of Th2 differentiation. These observations indicate that decreased production of Th1-type cytokines and prevalent Th2 ones leading to autoantibodies production in AIHA may be secondary to the imbalance between IL-10 and IL-12. These results strongly suggest that manipulation of the cytokine network, i.e. IL-10/IL-12 balance, maintained by cells of the innate immune system, can have a strong effect on the incidence of AIHA and their modulation might be useful for a therapeutic control of the disorder.

Th2 cytokine role in autoimmune haemolytic anaemia (AIHA) pathogenesis.

BACKGROUND: AIHA is characterised by the destruction of antibody-coated red blood cells, but the mechanism that initiates the production of autoantibodies remains unclear. We have studied the proliferative response and the spontaneous and mitogen-induced (PHA and OKT3) synthesis of IFN-g, IL-2 and IL-4 by peripheral blood mononuclear cells from patients with AIHA before any treatment to investigate the activation of Th1 and Th2 subsets. METHODS: Thirteen AIHA patients, both idiopathic and associated with other diseases, were studied by ELISA methods and H3 thymidine incorporation to determine in vitro cytokine production and T cell proliferative response, respectively. RESULTS: Under basal conditions the proliferative response induced in peripheral blood mononuclear cells was enhanced in AIHA patients suggesting a basal state of hyperactivation. This increase in proliferative response can be related to the basal enhanced levels of IL-2 (p<0.04), a key cytokine, that regulates the growth, differentiation and function of lymphocytes. High IL-2 levels in AIHA patients supernatants, with or without OKT3 stimulation, justify the high basal activation of T lymphocytes. Under basal conditions levels of IFN-g are decreased and IL-4 levels are increased. CONCLUSIONS: AIHA is characterised by the destruction of antibody-coated red blood cells, but the mechanism that initiates the production of autoantibodies remains unclear. But looking to this data it is possible to suppose that there is a prevalent contribution of Th2 lymphocytes to the pathogenesis of AIHA.

Enhanced IL-10 production in vitro by monocytes in autoimmune haemolytic anaemia.

Our previous studies on autoimmune haemolytic anaemia (AIHA) have shown a hyperactivation concerning cytokine production in T and B lymphocytes obtained from AIHA patients. In this study the production of interleukin (IL)-10, basal and stimulated by lipopolysaccharide (LPS), was determined in cultured monocytes obtained from patients affected by AIHA, either idiopathic or associated with other autoimmune diseases, e.g. idiopathic thrombocytopenic purpura (ITP), systemic lupus erythematosus (SLE), myastenia gravis (MG). In cultured AIHA monocytes the IL-10 basal production (mean+/-SD) was increased in all but one patient, compared to the controls, 125.96+/-76.10 pg/mL and 19.18+/-15.80 pg/mL respectively. Specifically, LPS stimulation was able to induce a higher IL-10 production by monocytes in two AIHA cases, whereas in five patients there was no difference and in two cases the LPS stimulated IL-10 production was even decreased compared to the levels observed in the controls. Interestingly in the latter two cases AIHA was associated with other autoimmune diseases (ITP, SLE). These results indicate a constitutively higher basal IL-10 production in monocytes from AIHA patients. The increased level of IL-10 could play an important role in the modified pathways of the immune response in AIHA.