Differentiation Profile Of Cells Research Articles

Autoantibodies-Abzymes with Phosphatase Activity in Experimental Autoimmune Encephalomyelitis Mice.

The exact mechanisms of MS (multiple sclerosis) evolution are still unknown. However, the development of EAE (experimental autoimmune encephalomyelitis simulating human MS) in C57BL/6 mice occurs due to the violation of bone marrow hematopoietic stem cell differentiation profiles, leading to the production of toxic for human autoantibody splitting MBP (myelin basic protein), MOG (mouse oligodendrocyte glycoprotein), five histones, DNA, and RNA. Here, we first analyzed the changes in the relative phosphatase activity of IgGs from C57BL/6 mice blood over time, corresponding to three stages of EAE: onset, acute, and remission. Antibodies have been shown to catalyze the hydrolysis of p-nitrophenyl phosphate at several optimal pH values, mainly in the range of 6.5-7.0 and 8.5-9.5. During the spontaneous development of EAE, the most optimal value is pH 6.5. At 50 days after the birth of mice, the phosphatase activity of IgGs at pH 8.8 is 1.6-fold higher than at pH 6.5. During spontaneous development of EAE from 50 to 100 days, an increase in phosphatase activity is observed at pH 6.5 but a decrease at pH 8.8. After mice were immunized with DNA-histone complex by 20 and 60 days, phosphatase activity increased respectively by 65.3 and 109.5 fold (pH 6.5) and 128.4 and 233.6 fold (pH 8.8). Treatment of mice with MOG at the acute phase of EAE development (20 days) leads to a maximal increase in the phosphatase activity of 117.6 fold (pH 6.5) and 494.7 fold (pH 8.8). The acceleration of EAE development after mice treatment with MOG and DNA-histone complex results in increased production of lymphocytes synthesizing antibodies with phosphatase activity. All data show that IgG phosphatase activity could be essential in EAE pathogenesis.

IL-7 producing immunotherapy improves ex vivo T cell functions of immunosenescent patients, especially post hip fracture

ABSTRACT Following acute stress such as trauma or sepsis, most of critically ill elderly patients become immunosuppressed and susceptible to secondary infections and enhanced mortality. We have developed a virus-based immunotherapy encoding human interleukin-7 (hIL-7) aiming at restoring both innate an adaptative immune homeostasis in these patients. We assessed the impact of this encoded hIL-7 on the ex vivo immune functions of T cells from PBMC of immunosenescent patients with or without hip fracture. T-cell ex vivo phenotyping was characterized in terms of senescence (CD57), IL-7 receptor (CD127) expression, and T cell differentiation profile. Then, post stimulation, activation status, and functionality (STAT5/STAT1 phosphorylation and T cell proliferation assays) were evaluated by flow cytometry. Our data show that T cells from both groups display immunosenescence features, express CD127 and are activated after stimulation by virotherapy-produced hIL-7-Fc. Interestingly, hip fracture patients exhibit a unique functional ability: An important T cell proliferation occurred compared to controls following stimulation with hIL-7-Fc. In addition, stimulation led to an increased naïve T cell as well as a decreased effector memory T cell proportions compared to controls. This preliminary study indicates that the produced hIL-7-Fc is well recognized by T cells and initiates IL-7 signaling through STAT5 and STAT1 phosphorylation. This signaling efficiently leads to T cell proliferation and activation and enables a T cell “rejuvenation.” These results are in favor of the clinical development of the hIL-7-Fc expressing virotherapy to restore or induce immune T cell responses in immunosenescent hip fracture patients.

Autoantibody-Abzymes with Catalase Activity in Experimental Autoimmune Encephalomyelitis Mice.

The exact mechanisms of the evolution of multiple sclerosis are still unknown. At the same time, the development in C57BL/6 mice of experimental autoimmune encephalomyelitis (EAE, simulating human multiple sclerosis) happens as a result of the violation of bone marrow hematopoietic stem cell differentiation profiles integrated with the production of toxic auto-antibodies splitting the basic myelin protein, myelin oligodendrocyte glycoprotein (MOG), histones, and DNA. It has been shown that IgGs from the plasma of healthy humans and autoimmune patients oxidize many different compounds due to their peroxidase (H2O2-dependent) and oxidoreductase (H2O2-independent) activities. Here, we first analyzed the changes in the relative catalase activity of IgGs from C57BL/6 mice blood plasma over time at different stages of the EAE development (onset, acute, and remission phases). It was shown that the catalase activity of IgGs of 3-month-old mice is, on average, relatively low (kcat = 40.7 min-1), but it increases during 60 days of spontaneous development of EAE 57.4-fold (kcat = 2.3 × 103 min-1). The catalase activity of antibodies increases by a factor of 57.4 by 20 days after the immunization of mice with MOG (kcat = 2.3 × 103 min-1), corresponding to the acute phase of EAE development, and 52.7-fold by 60 days after the treatment of mice with a DNA-histone complex (kcat = 2.1 × 103 min-1). It is the acceleration of the EAE development after the treatment of mice with MOG that leads to the increased production of lymphocytes synthesizing antibodies with catalase activity. All data show that the IgGs' catalase activity can play an essential role in reducing the H2O2 concentration and protecting mice from oxidative stress.

Retinal drusen in patients with chronic myeloproliferative blood cancers are associated with an increased proportion of senescent T cells and signs of an aging immune system.

The cause of age-related macular degeneration (AMD) is unknown, but evidence indicates that both innate and adaptive immunity play a role in the pathogenesis. Our recent work has investigated AMD in patients with myeloproliferative neoplasms (MPNs) since they have increased drusen and AMD prevalence. We have previously found increased levels of chronic low-grade inflammation (CLI) in MPN patients with drusen (MPNd) compared to MPN patients with normal retinas (MPNn). CLI and AMD are both associated with aging, and we, therefore, wanted to study immunosenescence markers in MPNd, MPNn, and AMD. The purpose was to identify differences between MPNd and MPNn, which might reveal novel information relevant to drusen pathophysiology and thereby the AMD pathogenesis. Our results suggest that MPNd have a T cell differentiation profile resembling AMD and more effector memory T cells than MPNn. The senescence-associated-secretory-phenotype (SASP) is associated with effector T cells. SASP is thought to play a role in driving CLI seen with advancing age. Senescent cells with SASP may damage healthy tissue, including the eye tissues affected in AMD. The finding of increased effector cells in MPNd could implicate a role for adaptive immunity and senescent T cells together with increased CLI in drusen pathophysiology.

IL-21 and IFN\u03b1 therapy rescues terminally\xa0differentiated NK cells and limits SIV reservoir in ART-treated macaques

Unlike HIV infection, which progresses to AIDS absent suppressive anti-retroviral therapy, nonpathogenic infections in natural hosts, such African green monkeys, are characterized by a lack of gut microbial translocation and robust secondary lymphoid natural killer cell responses resulting in an absence of chronic inflammation and limited SIV dissemination in lymph node B-cell follicles. Here we report, using the pathogenic model of antiretroviral therapy-treated, SIV-infected rhesus macaques that sequential interleukin-21 and interferon alpha therapy generate terminally differentiated blood natural killer cells (NKG2a/clowCD16+) with potent human leukocyte antigen-E-restricted activity in response to SIV envelope peptides. This is in contrast to control macaques, where less differentiated, interferon gamma-producing natural killer cells predominate. The frequency and activity of terminally differentiated NKG2a/clowCD16+ natural killer cells correlates with a reduction of replication-competent SIV in lymph node during antiretroviral therapy and time to viral rebound following analytical treatment interruption. These data demonstrate that African green monkey-like natural killer cell differentiation profiles can be rescued in rhesus macaques to promote viral clearance in tissues.

Bacteroides thetaiotaomicron relieves colon inflammation by activating aryl hydrocarbon receptor and modulating CD4+T cell homeostasis

Inflammatory bowel disease (IBD) is a form of nonspecific chronic intestinal inflammation associated with gut microbiome dysbiosis. Modulating the composition of the intestinal flora may be a viable means of alleviating such inflammatory pathology. Bacteroides thetaiotaomicron (B. thetaiotaomicron) is a symbiotic intestinal microbe that has been associated with IBD, although the mechanistic basis for this association remains to be clarified. In this present study, we determined that B. thetaiotaomicron can alleviate colonic inflammation through mechanisms associated with the modulation of tryptophan metabolism and T cell subsets within inflamed intestinal tissues. Specifically, we found that B. thetaiotaomicron promotes the preferential differentiation of anti-inflammatory Treg/Th2 cells while suppressing the relative differentiation of pro-inflammatory Th1/Th17 cells, thereby decreasing inflammation within the colon. At a molecular level, B. thetaiotaomicron treatment was linked to altered CpG methylation within the Foxp3 promoter that was associated with enhanced Treg cell functionality. In a murine dextran sulfate sodium (DSS) colitis model system, B. thetaiotaomicron increased the levels of the aryl hydrocarbon receptor (AHR) ligands indole metabolites-indole acetic acid (IAA) and indole propionic acid (IPA), thereby increasing AHR activation that is related to changes of transcription factor expression profiles within T cells. In summary, our data suggest that B. thetaiotaomicron can activate AHR and modulate CD4+ T cell differentiation profiles in a murine DSS colitis model system, suggesting that this bacterium may be of therapeutic relevance for the treatment of IBD.

Soluble silica stimulates osteogenic differentiation and gap junction communication in human dental follicle cells

Several studies have indicated that dietary silicon (Si) is beneficial for bone homeostasis and skeletal health. Furthermore, Si-containing bioactive glass biomaterials have positive effects on bone regeneration when used for repair of bone defects. Si has been demonstrated to stimulate osteoblast differentiation and bone mineralisation in vitro. However, the mechanisms underlying these effects of Si are not well understood. The aim of the present study was to investigate the effects of soluble Si on osteogenic differentiation and connexin 43 (CX43) gap junction communication in cultured pluripotent cells from human dental follicles (hDFC). Neutral Red uptake assay demonstrated that 25 μg/ml of Si significantly stimulated hDFC cell proliferation. Dosages of Si above 100 μg/ml decreased cell proliferation. Alizarin Red staining showed that osteogenic induction medium (OIM) by itself and in combination with Si (25 μg/ml) significantly increased mineralisation in hDFC cultures, although Si alone had no such effect. The expression of osteoblast-related markers in hDFC was analysed with RT-qPCR. OSX, RUNX2, BMP2, ALP, OCN, BSP and CX43 genes were expressed in hDFC cultured for 1, 7, 14 and 21 days. Expression levels of BMP-2 and BSP were significantly upregulated by OIM and Si (25 μg/ml) and were also induced by Si alone. Notably, the expression levels of OCN and CX43 on Day 21 were significantly increased only in the Si group. Flow cytometric measurements revealed that Si (50 μg/ml) significantly increased CX43 protein expression and gap junction communication in hDFC. Next-generation sequencing (NGS) and bioinformatics processing were used for the identification of differentially regulated genes and pathways. The influence of OIM over the cell differentiation profile was more prominent than the influence of Si alone. However, Si in combination with OIM increased the magnitude of expression (up or down) of the differentially regulated genes. The gene for cartilage oligomeric matrix protein (COMP) was the most significantly upregulated. Genes for the regulator of G protein signalling 4 (RGS4), regulator of G protein signalling 2 (RGS2), and matrix metalloproteinases (MMPs) 1, 8, and 10 were also strongly upregulated. Our findings reveal that soluble Si stimulates Cx43 gap junction communication in hDFC and induces gene expression patterns associated with osteogenic differentiation. Taken together, the results support the conclusion that Si is beneficial for bone health.

Polybenzimidazole nanofibers for neural stem cell culture

Neurodegenerative diseases compromise the quality of life of an increasing number of people in the world's aging population. While diagnosis is possible, no effective treatments are available. Strong efforts are needed to develop new therapeutic approaches, namely, in the areas of tissue engineering and deep brain stimulation. Conductive polymers are the ideal material for these applications owing to the positive effect of conducting electricity on the neural cell's differentiation profile. This novel study assessed the biocompatibility of polybenzimidazole (PBI) as electrospun fibers and after being doped with different acids. First, doped films of PBI were used to characterize the materials' contact angle and electroconductivity. After this, fibers were electrospun and characterized by scanning electron microscopy, Fourier-transform infrared spectroscopy, and thermogravimetric analysis. Neural stem cell (NSC) proliferation was assessed, and their growth rate and morphology on different samples were determined. Differentiation of NSCs on PBI-β-camphorsulfonic acid (CSA) fibers was also investigated, and gene expression (SOX2, NES, GFAP, and Tuj1) was assessed through immunochemistry and quantitative real-time polymerase chain reaction. All the samples tested were able to support NSC proliferation without significant changes on the cell's typical morphology. Successful differentiation of NSCs toward neural cells on PBI-CSA fibers was also achieved. This promising PBI fibrous scaffold material is envisioned to be used in neural cell engineering applications, including scaffolds and in vitro models for drug screening and electrodes.

Controlling fluid flow to improve cell seeding uniformity.

Standard methods for seeding monolayer cell cultures in a multiwell plate or dish do not uniformly distribute cells on the surface. With traditional methods, users find aggregation around the circumference, in the centre, or a combination of the two. This variation is introduced due to the macro scale flow of the cell seeding suspension, and movement of the dish before cells can settle and attach to the surface. Reproducibility between labs, users, and experiments is hampered by this variability in cell seeding. We present a simple method for uniform and user-independent the cell seeding using an easily produced uniform cell seeder (UCS) device. This allows precise control of cell density in a reproducible manner. By containing the cell seeding suspension in a defined volume above the culture surface with the UCS, fluctuations in cell density are minimised. Seeding accuracy, as defined by the actual cell density versus the target seeding density is improved dramatically across users with various levels of expertise. We go on to demonstrate the impact of local variation in cell density on the lineage commitment of human embryonic stem cells (hESCs) towards pancreatic endoderm (PE). Variations in the differentiation profile of cells across a culture well closely mirror variations in cell density introduced by seeding method–with the UCS correcting variations in differentiation efficiency. The UCS device provides a simple and reproducible method for uniform seeding across multiple culture systems.

Peripheral immune cell profile and activation status in chronic HCV mono- and HCV/HIV co-infected patients

Background: Due to similar routes of viral transmission, chronic HCV and HIV co-infection is frequent. Since it has been reported that both viruses perturbs immune homeostasis, our aim was to evaluate the peripheral immune cell moiety in HCV mono- and HCV/HIV co-infected patients to determine if co-infection alters HCV immune profile. Methods & Materials: Liver biopsies (47 = HCV treatment naïve, 13 = HCV/HIV only HAART) and concomitant blood samples were analysed. In peripheral blood: 1) frequency of total T cells(CD3 + ), T helper(Th, CD3 + CD4 + ), cytotoxic T lymphocytes(CTL, CD3 + CD8 + ) and Natural Killer cells(NK, CD3-CD56 + CD16 + ); 2) CTL and Th differentiation status [naïve (CD27 + CD45RA + ), central-memory (CD27 + CD45RA-), effector-memory(CD27-CD45RA-), effector(CD27-CD45RA + ) and activated (DR + )]; 3) frequencies of Th17(CD4 + IL17A + ), Th1(CD4 + IFNg + ) and Treg(CD4 + CD25hiFoxp3 + ) and 4) NK and CTL degranulation activity (CD107a) were evaluated by FACS. Non-infected donors (n = 33) were included. Inflammatory activity and fibrosis were assessed in liver biopsies using the modified Knodell scoring system and METAVIR. Results: While HCV patients did not display differences in lymphocyte frequencies compared to donors, co-infected patients showed the lowest Th(donor vs HCV/HIV p < 0.001, HCV vs HCV/HIV p < 0.001), the highest CTLs(donor vs HCV/HIV p < 0.001, HCV vs HCV/HIV p < 0.001) and the lowest NK(donor vs HCV/HIV p < 0.01, HCV vs HCV/HIV p < 0.05) frequencies. Absolute numbers confirmed these results. Moreover, only Th17 absolute numbers were diminished in co-infection (donor vs HCV/HIV p < 0.05). Regarding T cell differentiation status in HCV patients compared to donors: Th cells showed low naïve (p < 0.01) but high effector memory (p < 0.05) and activated cells (p < 0.01) frequencies; CTL showed low naïve (p < 0.01) but high activated cell (p < 0.01) frequencies. Although, HCV/HIV displayed a similar profile to HCV, Th and CTL activation was higher in HCV/HIV than HCV (p < 0.001). Degranulation activity of CTL and NK cells showed no differences between groups. Concerning to liver damage, any of the evaluated immune cell parameters displayed association with fibrosis or hepatitis severity. Conclusion: Although in chronic HCV infection the lymphocyte frequency was not modified, an altered T cell differentiation profile was evident. The co-infected condition distorted lymphocyte frequencies and enhanced T cell activation. This deteriorated immune profile may condition T cell response against antigens, including vaccines.

Bone marrow-derived inflammatory and steady state DCs are different in both functions and survival

Dendritic cells (DCs) contain heterogeneous populations, with classical DCs developed at steady state and monocyte-derived DCs mobilized under inflammatory conditions, although their total numbers in vivo are scares. To obtain enough quantity for immunological study or clinical application, we have previously established that bone marrow-derived DCs in the presence of Flt-3L (FL-DCs) or GM-CSF (GM-DCs) in vitro are equivalent to the steady state DCs and inflammatory DCs in vivo respectively. What difference, however, exists between these two most commonly used culture systems in DC functions and survival, and how does it correlate to the division of works by their corresponding counterparts in vivo remain ill-defined. In this study, we found that GM-DCs of inflammatory nature were more phagocytic, potent at inducing Th2 and Th17 differentiation, and had longer survival rate, whereas FL-DCs of steady state characters were stronger T cell activator and better at directing Th1 differentiation. Mechanistically, NO production induced by the LPS-activated GM-DCs could partly explain for their failure to improve T cell proliferation, and the distinct T cell differentiation profiles and viability demonstrated by the two types of DCs were underpinned by their preferential secretion of T cell polarizing cytokines and expression of anti-apoptotic genes. Such disparate functionalities and survival potentials of steady state and inflammatory DCs in vitro fit in well with their respective roles in vivo in particular immunological settings and have serious implications in translational applications.

Cyclosporin A enhances neurogenesis in the dentate gyrus of the hippocampus

Neural precursor cells (NPCs) in the adult mammalian brain demonstrate potential in applications of neural repair in the CNS. Recent work has shown that cyclosporin A (CsA), a commonly used immunosuppressive drug, expands the size of the NPC pool in the subventricular region by promoting cell survival. We asked if CsA had similar effects on NPCs in the dentate gyrus (DG) of the hippocampus, leading to increased neurogenesis. We used the in vitro and in vivo assays to examine CsA's effect on the size of the NPC pool and the proliferation and differentiation profile of cells within the DG. We found that CsA increases the numbers of NPCs and enriches for neurogenic NPCs in vitro. Similarly, in vivo systemic administration of CsA for 7days increases the size of the NPC pool in the DG observed through increases in numbers of proliferating cells and newborn neurons. Consistent with CsA's pro-survival effects, we have shown that CsA enhances the survival of newborn cells that normally undergo cell death over the time of infusion. Together these findings support the hypothesis that CsA administration promotes neurogenesis and may have implications for neural repair.

Microsphere-Based Scaffolds Carrying Opposing Gradients of Chondroitin Sulfate and Tricalcium Phosphate.

Extracellular matrix (ECM) components, such as chondroitin sulfate (CS) and tricalcium phosphate, serve as raw materials, and thus spatial patterning of these raw materials may be leveraged to mimic the smooth transition of physical, chemical, and mechanical properties at the bone-cartilage interface. We hypothesized that encapsulation of opposing gradients of these raw materials in high molecular weight poly(d,l-lactic-co-glycolic acid) (PLGA) microsphere-based scaffolds would enhance differentiation of rat bone marrow–derived stromal cells. The raw material encapsulation altered the microstructure of the microspheres and also influenced the cellular morphology that depended on the type of material encapsulated. Moreover, the mechanical properties of the raw material encapsulating microsphere-based scaffolds initially relied on the composition of the scaffolds and later on were primarily governed by the degradation of the polymer phase and newly synthesized ECM by the seeded cells. Furthermore, raw materials had a mitogenic effect on the seeded cells and led to increased glycosaminoglycan (GAG), collagen, and calcium content. Interestingly, the initial effects of raw material encapsulation on a per-cell basis might have been overshadowed by medium-regulated environment that appeared to favor osteogenesis. However, it is to be noted that in vivo, differentiation of the cells would be governed by the surrounding native environment. Thus, the results of this study demonstrated the potential of the raw materials in facilitating neo-tissue synthesis in microsphere-based scaffolds and perhaps in combination with bioactive signals, these raw materials may be able to achieve intricate cell differentiation profiles required for regenerating the osteochondral interface.

Immunohistochemistry in Dermatopathology: A Review of the Most Commonly Used Antibodies (Part I)

Dermatopathology includes a long list of disorders, some of which have very similar histopathology. Immunohistochemistry is an important auxiliary tool for diagnosis and differential diagnosis, and for predicting the outcome of many skin tumors. It is also the main technique for determining the origin of a tissue or the differentiation of neoplastic cells. In many cases, immunohistochemistry provides a more accurate diagnosis of the different processes that infiltrate the skin. This review examines the role of immunohistochemistry in studying the differentiation and biological behavior of the majority of tumors that can involve the skin. We review the immunoperoxidase techniques, discuss the utility of the most commonly used antibodies, and highlight a number of diagnostic problems in which immunohistochemistry may be very useful. In each case, the goal is to reach a specific and definitive diagnosis. In the first part of this review, we examine the antibodies that determine the different cell-differentiation profiles of skin tumors.

Inmunohistoquímica en dermatopatología: revisión de los anticuerpos utilizados con mayor frecuencia (parte i)

Dermatopathology includes a long list of disorders, some of which have very similar histopathology. Immunohistochemistry is an important auxiliary tool for diagnosis and differential diagnosis, and for predicting the outcome of many skin tumors. It is also the main technique for determining the origin of a tissue or the differentiation of neoplastic cells. In many cases, immunohistochemistry provides a more accurate diagnosis of the different processes that infiltrate the skin. This review examines the role of immunohistochemistry in studying the differentiation and biological behavior of the majority of tumors that can involve the skin. We review the immunoperoxidase techniques, discuss the utility of the most commonly used antibodies, and highlight a number of diagnostic problems in which immunohistochemistry may be very useful. In each case, the goal is to reach a specific and definitive diagnosis. In the first part of this review, we examine the antibodies that determine the different cell-differentiation profiles of skin tumors.

High CD8+ T Cell Activation Marks a Less Differentiated HIV-1 Specific CD8+ T Cell Response that Is Not Altered by Suppression of Viral Replication

BackgroundThe relationship of elevated T cell activation to altered T cell differentiation profiles, each defining features of HIV-1 infection, has not been extensively explored. We hypothesized that anti-retroviral suppression of T cell activation levels would lead to alterations in the T cell differentiation of total and HIV-1 specific CD8+ T cell responses among recently HIV-1 infected adults.Methodology/Principal FindingsWe performed a longitudinal study simultaneously measuring T cell activation and maturation markers on both total and antigen-specific T cells in recently infected adults: prior to treatment; after the initiation of HAART; and after treatment was halted. Prior to treatment, HIV-1 Gag–specific CD8+ T cells were predominantly of a highly activated, intermediate memory (CD27+CD28−) phenotype, while CMV pp65-specific CD8+ T cells showed a late memory (CD27−CD28−), low activation phenotype. Participants with the highest fraction of late memory (CD27−CD28−) HIV-1-specific CD8+ T cells had higher CD4+ T cell counts (rho = +0.74, p = 0.004). In turn, those with the highest fraction of intermediate memory (CD27+ CD28−) HIV-1 specific CD8+ T cells had high total CD8+ T cell activation (rho = +0.68, p = 0.01), indicating poorer long-term clinical outcomes. The HIV-1 specific T cell differentiation profile was not readily altered by suppression of T cell activation following HAART treatment.Conclusions/SignificanceA more differentiated, less activated HIV-1 specific CD8+ T cell response may be clinically protective. Anti-retroviral treatment initiated two to four months after infection lowered T cell activation but had no effect on the differentiation profile of the HIV-1-specific response. Intervention during the first month of acute infection may be required to shift the differentiation phenotype of HIV-1 specific responses to a more clinically favorable profile.

Proliferation and Differentiation of Human Stromal Cells Derived from Fat Tissue on Different Culture Substrates

The objective of this study is to investigate the proliferation and differentiation of stromal cells derived from human fat tissues cultured on substrates with different surface properties. In addition, the similar investigation was performed for the cells proliferated in different concentrations of basic fibroblast growth factor (bFGF). The culture substrates include several polymer films with different water wettabilities, glass or a cell culture plate, and that coated with collagen type I or IV, gelatin, and bFGF. The proliferation profiles of cells were influenced by the type of culture substrates and the growth factor concentration. A larger number of cells proliferated was observed for substrates with the water wettability around 80o, while the cell number was significantly larger for every protein-coated substrate. The rate of cell proliferation became maximum in a bFGF concentration of 1,000 ng/mL. The bFGF concentration used for cell proliferation affected the differentiation profile of cells proliferated. The stromal cells proliferated in 1 ng/mL bFGF were osteogenically differentiated to the strongest and fastest extent among those in other growth factor doses. The alkaline phosphatase (ALP) activity of cells increased with the increased cell number although the activity per cells was indentical, irrespective of the substrates type. The strongest adipogenic differentiation was observed for cells proliferated in 1,000 ng/mL bFGF and the differentiation induction was maintained for a long time period. No clear dependence of the cell number on adipogenesis was observed. For chondrogenic differentiation, the bFGF concentration had no influence on the glycosaminoglycans (GAG) amount. These findings indicate that the proliferation and differentiation of human fat tissue-derived stromal cells are influenced by the culture substrate and the concentration of bFGF used for proliferation.

Effect of culture substrate and fibroblast growth factor addition on the proliferation and differentiation of human adipo-stromal cells

The objective of this study is to investigate the proliferation and differentiation of stromal cells derived from human adipose tissues cultured on substrates with different surface properties. In addition, a similar investigation was performed on cells proliferated in different concentrations of basic fibroblast growth factor (FGF-2). The culture substrates include several polymer films with different water wettabilities, glass or a cell-culture plate, and that coated with collagen type I or IV, gelatin and FGF-2. The proliferation profiles of cells were influenced by the type of culture substrate and the growth factor concentration. A larger number of proliferated cells was observed for substrates with a water contact angle around 80°, while the cell number was significantly larger for every protein-coated substrate. The rate of cell proliferation became maximal at a FGF-2 concentration of 1000 ng/ml. The FGF-2 concentration used for cell proliferation affected the differentiation profile of cells proliferated. Stromal cells, proliferated in 1 ng/ml FGF-2, were osteogenically differentiated to the strongest and fastest extent among those in other growth factor doses. The alkaline phosphatase (ALP) activity of cells increased with the increased cell number, although the activity per cell was identical, irrespective of the substrate type. The strongest adipogenic differentiation was observed for cells proliferated in 1000 ng/ml FGF-2 and the differentiation induction was maintained for a long time period. No clear dependence of the cell number on adipogenesis was observed. These findings indicate that the proliferation and differentiation of human adipose tissue-derived stromal cells are influenced by the culture substrate and the concentration of FGF-2 used for proliferation.

PULMONARY EFFECTS OF ULTRAFINE AND FINE AMMONIUM SALTS AEROSOLS IN HEALTHY AND MONOCROTALINE-TREATED RATS FOLLOWING SHORT-TERM EXPOSURE

In the present study the effects of a 3-day inhalation exposure to model compounds for ambient particulate matter were investigated: ammonium bisulfate, ammonium ferrosulfate, and ammonium nitrate, all components of the secondary aerosol fraction of ambient particulate matter (PM), and carbon black (CB, model aerosol for primary PM). The objective of this study was to test the hypothesis that secondary model aerosols exert acute pulmonary adverse effects in rats, and that rats with pulmonary hypertension (PH), induced by monocrotaline (MCT), are more sensitive to these components than normal healthy animals. An additional aim was to test the hypothesis that fine particles exert more effects than ultrafines. Healthy and PH rats were exposed to ultrafine (mass median diameter [MMD] ≈ 0.07-0.10 µm; 4 × 105 particles/cm3) and fine (MMD ≈ 0.57-0.64 µm; 9 × 10 3 particles/cm 3) ammonium aerosols during 4 h/day for 3 consecutive days. The mean mass concentrations ranged from 70 to 420 µg/m 3, respectively, for ultrafine ammonium bisulfate, nitrate, and ferrosulfate and from 275 to 410 µg/m 3 for fine-mode aerosols. In an additional experiment, simultaneous exposure to a fine CB aerosol (0.6 µm; 2-9 mg/m 3) and ammonium nitrate (0.4-18 mg/m 3) was performed. Bronchoalveolar lavage fluid (BALF) analysis and histopathological examination were performed on animals sacrificed 1 day after the last exposure. Histopathology of the lungs did not reveal test atmosphere-related abnormalities in either healthy or PH rats exposed to the ammonium salts, or to a combination of CB + nitrate. Alveolar macrophages in rats exposed to CB only revealed the presence of black material in their cytoplasm. There were no signs of cytotoxicity due to the aerosol exposures (as measured with lactate dehydrogenase [LDH], protein, and albumin contents in BALF). Macrophages were not activated after MCT treatment or the test atmospheres, since no changes were observed in N -acetyl glucosaminidase (NAG). Cell differentiation profiles were inconsistent, partly caused by an already present infection with Haemophilus sp. However, we believe that the test atmospheres did not affect cell differentiation or total cell counts. The results show that at exposure levels of ammonium salts at least one order of magnitude higher than ambient levels, marked adverse health effects were absent in both healthy and PH rats.

Transforming growth factor beta1 involvement in the conversion of fibroblasts to smooth muscle cells in the rabbit bladder serosa.

In an attempt to identify the growth factors or cytokines involved in the serosal thickening that occurs in rabbit bladder subjected to partial outflow obstruction, the following growth factors--transforming growth factor beta1, platelet-derived growth factor, epidermal growth factor, granulocyte colony-stimulating factor and granulocyte-monocyte colony-stimulating factor--were delivered separately onto the serosal surface of the intact bladder via osmotic minipumps. The proliferative/differentiative cellular response of the rabbit bladder wall was evaluated by bromodeoxyuridine incorporation and immunofluorescence staining with a panel of monoclonal antibodies to cytoskeletal proteins (desmin, vimentin, keratins 8 and 18 and non-muscle myosin) and to smooth muscle (alpha-actin, myosin and SM22) proteins. Administration of the transforming growth factor, but not of the other growth factors/cytokines, was effective in inducing serosal thickening. Accumulating cells in this tissue were identified as myofibroblasts, i.e. cells showing a mixed fibroblast-smooth muscle cell differentiation profile. The phenotypic pattern of myofibroblasts changed in a time-dependent manner: 21 days after the growth factor delivery, small bundles of smooth muscle cells were found admixed with myofibroblasts, as occurs in the obstructed bladder. These 'ectopic' muscle structures displayed a variable proliferating activity and expressed an immature smooth muscle cell phenotype. The complete cellular conversion to smooth muscle cells was not achieved if transforming growth factor beta1 was delivered to fibroblasts of subcutaneous tissue. These findings suggest a tissue-specific role for this growth factor in the cellular conversion from myofibroblast to smooth muscle cells.