Meshes in a mess: Mesenchymal stem cell-based therapies for soft tissue reinforcement.

Surgical meshes are frequently used for the treatment of abdominal hernias, pelvic organ prolapse, and stress urinary incontinence. Though these meshes are designed for tissue reinforcement, many complications have been reported. Both differentiated cell- and mesenchymal stem cell-based therapies have become attractive tools to improve their biocompatibility and tissue integration, minimizing adverse inflammatory reactions. However, current studies are highly heterogeneous, making it difficult to establish comparisons between cell types or cell coating methodologies. Moreover, only a few studies have been performed in clinically relevant animal models, leading to contradictory results. Finally, a thorough understanding of the biological mechanisms of mesenchymal stem cells in the context of foreign body reaction is lacking. This review aims to summarize in vitro and in vivo studies involving the use of differentiated and mesenchymal stem cells in combination with surgical meshes. According to preclinical and clinical studies and considering the therapeutic potential of mesenchymal stem cells, it is expected that these cells will become valuable tools in the treatment of pathologies requiring tissue reinforcement. STATEMENT OF SIGNIFICANCE: The implantation of surgical meshes is the standard procedure to reinforce tissue defects such as hernias. However, an adverse inflammatory response secondary to this implantation is frequently observed, leading to a strong discomfort and chronic pain in the patients. In many cases, an additional surgical intervention is needed to remove the mesh. Both differentiated cell- and stem cell-based therapies have become attractive tools to improve biocompatibility and tissue integration, minimizing adverse inflammatory reactions. However, current studies are incredibly heterogeneous and it is difficult to establish a comparison between cell types or cell coating methodologies. This review aims to summarize in vitro and in vivo studies where differentiated and stem cells have been combined with surgical meshes.

NK and MSCs crosstalk: the sense of immunomodulation and their sensitivity.

Several clinical trials are currently evaluating the safety and efficacy of mesenchymal stem cells (MSCs) in different diseases. The MSCs are guided by inflammatory mediators and specific interactions with immune cells occur, especially between MSCs and the cells of the innate immune system. At the present many of these interactions remain to be determined. This review summarizes the current knowledge in relation to the susceptibility of MSCs to NK cell mediated lysis as well as the immunomodulatory activity of MSCs and its control over NK cell function. Here we hypothesize that the inflammatory context will regulate the outcome of NK-MSC interactions.

Comparative phenotypic and molecular characterization of porcine mesenchymal stem cells from different sources for translational studies in a large animal model.

Mesenchymal stem cells have demonstrated their potentiality for therapeutic use in treating diseases or repairing damaged tissues. However, in some cases, the results of clinical trials have been disappointing or have not worked out as well as hoped. These disappointing results can be attributed to an inadequate or insufficient preclinical study. For medical and surgical purposes, the similarities between the anatomy of pig and human make this animal an attractive preclinical model. In this sense, for mesenchymal stem cell-based therapy, it is strongly necessary to have well characterized animal-derived mesenchymal stem cell lines to validate preclinical effectiveness of these cells. In this work, porcine mesenchymal stem cells (pMSCs) were isolated from bone marrow, adipose tissue and peripheral blood and compared in terms of differentiation potential, cell surface markers and gene expression. Our results demonstrated that the isolation and in vitro expansion protocols were feasible and effective. The data presented in this work are relevant because they provide an extensive phenotypic characterization; genetic study and differentiation behavior of the most commonly used stem cell lines for clinical practices. These pMSCs are widely available to scientists and could be a valuable tool to evaluate the safety and efficacy of adoptively transferred cells.

Human prostasomes express CD48 and interfere with NK cell function.

Prostasomes are small vesicles secreted by the prostate gland to the seminal fluid. In male fertility, the fusion of prostasomes to sperm has been associated with an enhancement of sperm function. Prostasomes also contribute to sperm protection within the female reproductive tract and immunologically relevant proteins have been identified on the prostasomal membrane that may prevent immune-mediated destruction of the sperm.The female genital tract is a potentially hostile environment for spermatozoa and a large number of NK cells have been found in the female reproductive tract. Since it has been suggested that NK cells may represent an important component of innate immunity in the female reproductive tract, we have analyzed the role of prostasomes in the regulation of NK cell activity.Flow cytometric analysis revealed that prostasomes expressed high levels of CD48, the ligand for the activating receptor CD244. The interactions between NK cells and purified prostasomes resulted in a decrease of CD244 expression. Moreover, the decreased NK cell activity observed in NK cells cultured in the presence of prostasomes suggests that prostasomes may immunomodulate the local environment within the female reproductive tract preventing immune-mediated sperm destruction and prolonging their survival rate.

Fluoroquinolone-resistant Brucella melitensis mutants obtained in vitro.

Three fluoroquinolone-resistant Brucella melitensis mutants were obtained after successive passages on norfloxacin-containing agar plates. The original strain was B. melitensis biovar 2 ATCC 23457. Mutants emerged at frequencies ranging between 1 x 10(-6) and 3 x 10(-8). The three mutants showed a gyrA Ala71-->Ser mutation as well as insertion of Ala340. No mutations were observed in the parC and gyrB genes. Minimum inhibitory concentrations (MICs) increased by four- to eight-fold from the wild-type to the first-step mutant for norfloxacin, ciprofloxacin, levofloxacin and moxifloxacin. MICs of the second-step mutant increased significantly only for norfloxacin and those for the third-step mutant for norfloxacin and ciprofloxacin, suggesting the involvement of some efflux mechanism.

Preparation and characterization of carbon-enriched coal fly ash.

Carbon-enriched fractions have been obtained from two coal fly ash (FA) samples. The FA came from two pulverized-coal fired power stations (Lada and Escucha, Spain) and were collected from baghouse filters. Sieving was used to obtain carbon-enriched fractions, which were further subjected to two beneficiation processes: acid demineralization using HCl and HF, and oil agglomeration using soya oil-water. Yield in weight after sieving, unburned carbon content, and several physicochemical characteristics of the obtained fractions were used to compare the performance of the beneficiation methods. Low carbon concentration was obtained by sieving, particularly in the case of Escucha FA. However, after acid demineralization or oil agglomeration, fractions containing unburned carbon in a range of 63% to 68% were obtained. These fractions showed differences in mineral phase composition and distribution depending on the FA and on the beneficiation method used. The textural properties of the obtained fractions varied as a function of their carbon content and the beneficiation method used. However, no significant differences in morphology of the carbonaceous particles were found.

Role of the carboxy-termini of tubulin on its chaperone-like activity.

Mutational analysis and the enzymatic digestion of many chaperones indicate the importance of both hydrophobic and hydrophilic residues for their unique property. Thus, the chaperone activity of alpha-crystallin is lost due to the substitution of hydrophobic residues or upon enzymatic digestion of the negatively charged residues. Tubulin, an eukaryotic cytoskeletal protein, exhibits chaperone-like activity as demonstrated by prevention of DTT-induced aggregation of insulin, thermal aggregation of alcohol dehydrogenase, betagamma-crystallin, and other proteins. We have shown that the tubulin lost its chaperone-like activity upon digestion of its negatively charged C-termini. In this article, the role of the C-terminus of individual subunits has been investigated. We observe that the digestion of C-terminus of beta-subunit with subtilisin causes loss of chaperone-like activity of tubulin. The contribution of C-terminus of alpha-subunit is difficult to establish directly as subtilisin cleaves C-terminus of beta-subunit first. This has been ascertained indirectly using a 14-residue peptide P2 having the sequence corresponding to a conserved region of MHC class I molecules and that binds tightly to the C-terminus of alpha-subunit. We have shown that the binding of P2 peptide to alphabeta-tubulin causes complete loss of its chaperone-like activity. NMR and gel-electrophoresis studies indicate that the P2 peptide has a significant higher binding affinity for the C-terminus of alpha-subunit compared to that of beta-subunit. Thus, we conclude that both the C-termini are necessary for the chaperone-like activity of tubulin. Implications for the chaperone functions in vivo have been discussed.

HLA-B2702 (77-83/83-77) peptide binds to beta-tubulin on human NK cells and blocks their cytotoxic capacity.

It has been described that peptides derived from a highly conserved region of the alpha1 helix of the first domain of HLA class I Ags exhibit immunomodulatory capacity blocking both T and NK cell cytotoxicity. In vivo treatment with these peptides prolongs survival of MHC-mismatched allografts. However, the molecular bases of these effects are still unclear. In this study, we further analyze the mechanisms by which the dimeric peptide HLA-B2702 (77-83/83-77) induces suppression of NK cell cytotoxicity. This peptide inhibits natural and redirected lysis mediated by NK cells without significantly affecting effector-target cell binding. We have also isolated and sequenced a protein that binds this inhibitory peptide, which structurally corresponds to beta-tubulin. Tubulin is the major protein of microtubules and is involved in target cell killing. Furthermore, B2702 peptide promotes GTP-independent tubulin assembly, producing aggregates that cannot be depolymerized by cold. Treatment of NK cells with Taxol or demecolcine, which interfere with microtubule organization, also prevents NK cell cytotoxicity. Taken together, these results support the hypothesis that the peptide B2702 (77-83/83-77) exerts its inhibitory effect on NK cell cytotoxicity by inducing polymerization of microtubules and interfering with their normal assembly/disassembly dynamics.

Increased expression of NK cell markers on T lymphocytes in aging and chronic activation of the immune system reflects the accumulation of effector/senescent T cells.

In humans, CD56, CD161, CD16, CD94 or CD57 represent prototypic markers of NK cells, although they are also found on a subset of CD8+ T cells. We propose here that the expression of NK receptors on CD8+ T cells can be considered a marker of cytotoxic effector T cells that are expanded in vivo after antigenic activation leading to extensive proliferation. The persistence of antigen will lead to loss of co-stimulatory molecules, telomere shortening and defective IL-2 production, changes that define the state replicative senescence in T lymphocytes. The majority of these "effector/senescent" T lymphocytes are CD8+, CD45RA+, CD11a(bright), CD28-, CD27-, CD62L- and CCR7-. They are cytotoxic T cells with strong expression of intracytoplasmic perforin and granzyme, but with low proliferative capacity and defective IL-2 production. Many of these characteristics are shared by the recently defined "effector/memory" T cells, being mainly distinguished by the absence of CD45RA expression on the memory cells. The expression of NK receptor in these effector cells will probably contribute to the regulation of their cytotoxic function. Expansion of cells with these characteristics can be found not only in the elderly but also in other clinical conditions involving chronic activation of the immune system such as viral infections, rheumatic and autoimmune diseases or tumors. Another subset of T cells that expresses the NK receptors is the alpha-galactosyl-ceramide specific T cell subset defined by the expression of canonical Valpha24JalphaQ TCR, recognition of CD1d and secretion of high amounts of IL-4 and IFN-gamma. However, the changes observed in the expression of NK-R on T cells associated to immunosenescence can not be attributed to expansion of this particular T cell subset, although alterations in the number and function of these cells have been demonstrated in some autoimmune diseases.

Expression of killer inhibitory receptors on cytotoxic cells from HIV-1-infected individuals.

Dysfunction of cytotoxic activity of T and natural killer (NK) lymphocytes is a main immunological feature in patients with AIDS, but its basis are not well understood. It has been recently described that T and NK cell-mediated cytotoxicity can be regulated by HLA killer inhibitory receptors (KIR). In this work, we have determined on cytotoxic T cells and NK cells from HIV-1-infected individuals the expression of the following KIR molecules: p58, p70, and ILT2 (immunoglobulin-like family KIR) as well as CD94 and NKG2A (C-lectin-type family KIR). With some exceptions, no significant changes were found on the expression of immunoglobulin-like KIR in either CD8+ or CD56+ cells. Interestingly, the percentages of CD8+ and CD56+ cells expressing CD94 were significantly increased in these individuals. We also show that, in vitro, IL-10 up-regulates CD94 expression on CD8+ and CD56+ cells obtained from normal individuals, suggesting that the augmented expression observed in HIV-infected individuals could be related to the high levels of IL-10 previously described in HIV-1-infected individuals.

Cryptosporidium parvum infection in experimentally infected mice: infection dynamics and effect of immunosuppression.

The effect of mouse strain, age, sex, and the size of infective dose on the susceptibility to infection with the coccidium Cryptosporidium parvum Tyzzer, 1912 was determined using several murine models. Mice were infected with C. parvum oocysts originally of cervine origin, maintained by repeat passage in calves. All mice in the experimental groups proved susceptible to infection, though this resulted asymptomatic in all cases. C. parvum infection in BALB/c and Porton mice exhibited some variation. BALB/c mice demonstrated a longer prepatent period than Porton mice. They also produced a greater oocyst output over the patent period, though the differences were not statistically significant. Differences were observed between mice infected at either 3 or 4 weeks of age. Prepatent period was shorter in those mice infected at 3 weeks of age, reaching 100% infection rate by day 7 post-inoculation. The patent period was longer in younger mice showing that age at time of infection can modify the oocyst shedding profile. However, no sex related differences in the course of infection were observed. The effect of different infective doses of oocysts was analysed. The three doses used (10(4), 10(5), 10(6)) proved infective for all mice, there were no statistical differences in either prepatent or patent periods, or in the oocyst shedding profiles. Experimental cryptosporidiosis was also induced in cyclophosphamide-immunosuppressed mice. Cyclophosphamide was orally administered by stomach tube at a dose of 50 mg/kg/day starting 10 days before the intragastric inoculation of 10(6) oocysts of C. parvum per mouse and continuing until the end of the experiment. Immunosuppressed mice had a shorter prepatent period, remained infected longer and shed more oocysts than immunocompetent mice. Immunosuppression produced high mortality rates; during the course of the experiment 44% of immunosuppressed-infected and 30% of immunosuppressed-uninfected mice died. There were no deaths in the untreated groups. Differences in the clinical course of the infection were also observed between immunosuppressed and immunocompetent mice; however, some mice recovered without immunosuppression withdrawal.

Interactions with multiple peptide ligands determine the fate of developing thymocytes.

Thymocytes are positively or negatively selected depending on interactions between their T cell receptors (TCR) and peptides presented by major histocompatibility complex molecules. We have previously shown that apoptosis of thymocytes from an alpha beta TCR-transgenic mouse (F5), induced by antigenic peptide, can be inhibited specifically by an antagonist peptide variant in an in vitro culture model. We have now extended these experiments by demonstrating that the antagonist peptide can inhibit natural negative selection of maturing thymocytes, induced by endogenously expressed antigen, in fetal thymic organ cultures (FTOC). This inhibition resulted in the rescue and maturation of thymocytes that would otherwise have been deleted. Mature T cells generated in these cultures were able to respond to antigen by producing limited quantities of interferon-gamma, but unlike T cells from control FTOC, they required exogenous interleukin-2 to generate cytolytic effector cells. Interestingly, the antagonist peptide also accelerated the development of F5 thymocytes in the absence of the negatively selecting ligand. These data suggest that the developmental fate of a thymocyte may be determined by the recognition of multiple distinct peptide ligands during thymic selection. Alterations in the profiles of selecting peptides presented in the thymus would thus have profound effects on the size and autoreactive potential of the T cell repertoire generated.

Susceptibility and resistance to antigen-induced apoptosis in the thymus of transgenic mice.

Injection of TCR transgenic mice with antigenic peptide results in the deletion of immature thymocytes expressing the transgenic TCR. We have analyzed this process in mice transgenic for a TCR (F5) that recognizes a peptide from the influenza nucleoprotein (NP68). To determine whether deletion of immature thymocytes is the result of specific recognition of the antigenic peptide by the thymocytes or mature T cell activation, bone marrow chimeric mice were generated using a mixture of cells from F5 transgenic and nontransgenic mice. Injection of these mice with antigenic peptide leads to the preferential depletion of F5 transgenic thymocytes, whereas nontransgenic thymocytes remain largely unaffected. Furthermore, exposure of F5 fetal thymic lobes to peptide leads to thymocyte deletion even though no mature single positive T cells are present at this stage. These data suggest that Ag-induced death of immature thymocytes is due to peptide-specific recognition, although activated mature T cells appear to potentiate such deletion. Further administration of antigenic peptide to F5 mice results in the appearance of double-positive thymocytes that are resistant to Ag or anti-CD3-induced apoptosis. These data suggest a change in the ability of the cells to signal through the TCR-CD3 complex, resembling the state of anergy induced in peripheral T cells following chronic exposure to Ag.

Characterization of secretory IgA responses in mice infected with Cryptosporidium parvum.

Mice inoculated at 5, 21 and 28 days of age with 10(6) or 10(7) Cryptosporidium parvum oocysts became infected but did not exhibit any clinical signs of disease. Specific IgA antibodies were detected in faecal extracts from all infected mice by an indirect immunofluorescent assay. These antibodies first appeared between 11 and 37 days post-infection (dpi) and persisted until the end of the experiment at 55 dpl. They appeared earlier in older mice than in newborn mice. Reduction and resolution of oocyst shedding was not directly related, however, to IgA antibody levels in infected mice. Reactive C. parvum antigens were identified by immunoblotting techniques using faecal and serum samples from infected mice. IgA copro-antibodies reacted specifically with two antigens of 26 and 33 kDa, which were also identified by IgG antibodies in mouse serum. The role of these antibodies in the resolution of infections and the subsequent protection against challenge is unknown.

The agonist-antagonist balance in positive selection.

Peptide antigens expressed in the thymus, in combination with self major histocompatibility complex molecules play a crucial role in thymocyte selection and shaping of the mature T-cell repertoire. Here, it is proposed that a single thymocyte may be exposed to numerous different peptide ligands as it matures, such that its fate is determined by the sum of signals produced by these interactions.

In vitro positive selection of alpha beta TCR transgenic thymocytes by a conditionally immortalized cortical epithelial clone.

Development of mature CD4 and CD8 single-positive T cells requires a process known as positive selection, which depends on the specific recognition of self-peptide-MHC complexes on thymic stromal cells by immature CD4+CD8+ thymocytes. We have used an in vitro reaggregate system to study the positive selection of thymocytes by conditionally immortalized thymic epithelial clones. Thymocytes from mice transgenic for the F5 alpha beta TCR, specific for a peptide from the influenza nucleoprotein in the context of H-2Db, are positively selected in the H-2b MHC background, but fail to mature in mice expressing the H-2q haplotype. Development of embryonic day 15 F5 H-2q transgenic thymocytes was followed in reaggregate cultures supplemented with H-2b-expressing epithelial clones. A conditionally immortalized cortical epithelial clone, derived from H-2Kb-tsA58 transgenic mice, was found to be as efficient as freshly isolated thymic stromal cells in positively selecting CD8 transgenic thymocytes. In contrast, an H-2b-expressing kidney epithelial clone did not augment positive selection above background levels, implying that the effect of the thymic epithelial clone was not merely the presentation of selecting MHC molecules. Mature transgenic thymocytes generated in reaggregate cultures were able to differentiate into functionally competent cytotoxic T cells. This model provides an important in vitro system for the detailed study of the specific molecular interactions leading to positive selection of developing thymocytes.

Articulación temporomandibular: síndromes dolorosos miofaciales / Temporomadibular joint: myofacial painful syndromes

Los sindromes dolorosos de la articulación temporomandibular (ATM) se presentan muy frecuentemente en la práctica medica diaria y en muchas ocasiones pasa desapercibido este diagnostico debido a la gran variabilidad de su presentación clinica, ya que generalmente no causa sintomas articulares locales sino un gran espectro de síntomas en estructuras relaciónadas con esta articulación. En el presente articulo se presenta inicialmente una revision de la anatomia básica de la ATM, la clasificación de las disfunciones de la ATM y factores predisponentes para estos trastornos; posteriormente se hace enfasis en el diagnostico de estos sindromes, basándose en la historia clinica, examen fisico complementado con la imagenologia; finalmente se revisan las diversas modalidades terapeuticas, desde el manejo ultraconservador hasta las diversas opciones quirurgicas

Polyethylene glycol-modified IL-2 abrogates superantigen-induced anergy without affecting peripheral clonal deletion in vivo.

As compared with the native molecule, recombinant human interleukin-2 that is modified by covalently attached polyethylene glycol residues (IL-2-PEG) exhibits a markedly enhanced half-life in vivo, thus facilitating its biological evaluation. We have characterized the effect of IL-2-PEG on the Staphylococcus aureus enterotoxin B (SEB)-induced tolerance of peripheral SEB-reactive (V beta 8+) T cells. Treatment with sublethal doses of IL-2-PEG does not modulate (inhibit or enhance) the SEB-triggered apoptosis and deletion of V beta 8+ T cells. In contrast, in vivo treatment with IL-2-PEG partially abolishes the SEB-triggered anergy of V beta 8+ T cells, i.e., the failure to proliferate in response to SEB in vitro. To abolish SEB-triggered anergy, IL-2-PEG must act for an extended period in vivo; short term treatment in vivo (2 days) or exposure of anergic T cells to IL-2 in vitro fails to reconstitute proliferative responses. Moreover, the effect of in vivo treatment with IL-2-PEG on lymphokine production by anergic T cells is partial. IL-2-PEG restores IL-4-dependent autocrine proliferation in response to SEB but does not reestablish defective IL-2 production. These data are compatible with the notion that IL-2 is a regulator of postdeletional rather than deletional T cell tolerance.

Effects of different antigenic microenvironments on the course of CD8+ T cell responses in vivo.

The influence of microenvironment on the course of CD8 + T cell responses in vivo was investigated by injecting H-2Kb-specific T cells from donor TCR transgenic (TCR-Tg) mice into H-2kb-Tg mice. H-2Kb expression in recipients was either ubiquitous (CBK mice) or restricted to myeloid and erythroid cells (K beta mice). Donor T cells proliferated as extensively and acquired similar surface phenotypes in spleen of both recipient types. Thus, neither the restricted pattern of H-2Kb expression nor the significantly reduced level of H-2Kb expression by myeloid cells in Kbeta recipients affects the ability of the splenic microenvironment to prime T cell proliferation in vivo. However, an unsustained burst of cytolytic activity was generated rapidly in spleen of CBK recipients, whereas relatively little cytolytic activity was generated in K beta spleen. This indicates that effector T cells were not generated efficiently in spleen of Kbeta recipients even though extensive T cell proliferation was taking place in this microenvironment. Furthermore, activated donor T cells dispersed rapidly throughout primary and secondary lymphoid organs of Kbeta recipients, whereas few T cells migrated from spleen in CBK recipients. Consequently, the course of CD8+ T cell responses and the anatomical distribution of activated T cells are profoundly influenced by the nature of the antigenic microenvironment encountered in vivo. We conclude that T cells rapidly proliferate and acquire new tissue-homing characteristics but do not differentiate into cytolytic effector cells at the site of priming when they encounter myeloid cells expressing low levels of antigen in vivo.

Intestinal and extraintestinal cryptosporidiosis in AIDS patients.

In a prospective study in AIDS patients with chronic diarrhea, the overall prevalence of intestinal cryptosporidiosis was 15.6% (43/275). The prevalence was higher in homosexual patients (33.3%) than in intravenous drug abusers (10.6%) (p < 0.001). Extraintestinal infection was present in 30% (13/43) of the patients with known intestinal cryptosporidiosis. Eight of the 13 (61.5%) patients with extraintestinal cryptosporidiosis had Cryptosporidium in the bile and 7 of 13 (16.28%) had it in the sputum. Of the seven patients with Cryptosporidium in the sputum, four had respiratory symptoms and an abnormal chest radiograph, although another pulmonary pathogen was isolated simultaneously. Two other patients from whom Cryptosporidium was the sole respiratory pathogen isolated had no respiratory symptoms and normal chest radiographs. The seventh patient had pulmonary symptoms, interstitial infiltrate on chest radiograph and excessive activity on a pulmonary Gallium scan; Cryptosporidium was the only organism detected in induced sputum and bronchoalveolar lavage specimens. The mean CD4+ lymphocyte count in patients with extraintestinal cryptosporidiosis was 55 cells/mm3.