Human serum promotes the proliferation but not the stemness genes expression of human adipose-derived stem cells

Recently human adipose-derived stem cells (ASCs) have shown much therapeutic potential in regenerative medicine. However, fetal bovine serum (FBS) used in culturing human cells may give risk to viral and prion transmission as well as immune rejection. Human serum (HS) is a safer growth supplement in human cell culture but its effects have not been well established. Therefore the objectives of this study were to compare the effects of HS versus FBS on the proliferation and stemness gene expression of ASCs. ASCs were cultured for 5 passages in medium supplemented with either 10% HS or 10% FBS. ASCs proliferation rate and viability were determined at every passage. Total RNA was extracted at passage 5 (P5) and quantitative PCR was carried out to determine the stemness gene expression level of SOX-2, Nanog3, BST-1, REX-1, ABCG2 and FGF-4. The results showed ASC cultured in 10% HS scored greater proliferation rates and viability compared to 10% FBS. ASCs proliferated significantly faster in 10% HS compared to 10% FBS at P2, P3, and P4 (p < 0.05). In quantitative gene expression analysis, ASCs cultured in 10% FBS showed a significant increase of BST-1, REX-1 and ABCG2 expression compared to 10% HS. In conclusion, HS promotes ASCs proliferation and viability but its ability to support the stemness property of ASCs was inferior to FBS.     

Optimization of embryo culture conditions in the production of cloned goat embryos, following somatic cell nuclear transfer

This study was initiated to investigate the effect of culture conditions on the in vitro development of cloned goat embryos, as well as the pregnancy and delivery success after embryo transfer. The fused embryos were randomly distributed to four culture groups: (i) 72 h in G1 medium then 96 h in G2 medium (group G1/G2); (ii) 168 h in mSOFBSA (group mSOFBSA); (iii) 72 h in mSOFBSA then 96 h in mSOFBSA supplemented with 10% FBS (group mSOFBSA-FBS); (iv) 168 h in mSOFBSA supplemented with 10% FBS (group mSOFFBS). A higher proportion of reconstructed embryos developed to the hatching blastocyst stage in the mSOFBSA group, compared to the G1/G2 group (30.8% vs. 11.4%; P < 0.05). Furthermore, supplementation of 10% FBS to mSOFBSA at the beginning of culture, or 72 h later both significantly improved the hatching rates, compared to the control group (48.9%, 51.2% vs. 28.2%, respectively; P < 0.05). The pregnancy rate following the transfer of one-cell stage embryos was higher than that of in vitro cultured blastocysts (32.0% vs. 6.9%; P < 0.05). However, no significant differences were observed regarding NT efficiency, based on the number of embryos transferred between the two groups (2.0% and 1.3%, respectively). Results suggest that cloned goat embryos cultured to the blastocyst stage in vitro in mSOFFBS may develop to term.     

MicroRNA-146a modulates human bronchial epithelial cell survival in response to the cytokine-induced apoptosis

MicroRNA plays an important role in cell differentiation, proliferation and cell death. The current study found that miRNA-146a was up-regulated in human bronchial epithelial cells (HBECs) in response to stimulation by TGF-ß1 plus cytomix (a mixture of IL-1ß, IFN-γ and TNF-α). TGF-ß1 plus cytomix (TCM) induced apoptosis in HBECs (3.4 ± 0.6% of control vs 83.1 ± 4.0% of TCM treated cells, p < 0.01), and this was significantly blocked by the miRNA-146a mimic (8.8 ± 1.5%, p < 0.01). In contrast, a miRNA-146a inhibitor had only a modest effect on cell survival but appeared to augment the induction of epithelial-mesenchymal transition (EMT) in response to the cytokines. The MicroRNA-146a mimic appears to modulate HBEC survival through a mechanism of up-regulating Bcl-XL and STAT3 phosphorylation, and by this mechanism it could contribute to tissue repair and remodeling.     

Growth and development of rabbit oocytes in vitro: Effect of fetal bovine serum concentration on culture medium

The objective was to develop a culture system that produced blastocyst stage embryos from rabbit oocytes grown in vitro. Two experiments were performed. First, various concentrations of fetal bovine serum (FBS, 0, 0.05, 0.5 and 5%) were used in the culture medium for in vitro growth (IVG) of oocytes recovered from follicles 200 to 299 μm in diameter. Intracytoplasmic sperm injection (ICSI) was performed on mature oocytes obtained after IVG for 8 days and in vitro maturation for 14 to 16 h. Rates of survival and pronuclear formation after ICSI were higher for oocytes grown in a medium with 0.05% FBS compared to oocytes grown in a medium lacking FBS (97.6 vs. 76.9%, 97.5 vs. 70%, P < 0.1). The rate of development to the blastocyst stage was also higher in the medium containing 0.05% FBS than in the medium lacking FBS (9.5 vs. 17.9%, P < 0.05). Next, using oocytes recovered from follicles 200 to 399 μm in diameter which were cultured in 0.05% FBS, oxygen consumption and the number of cells were analyzed. Blastocysts from oocytes grown in vitro with 0.05% FBS had reduced oxygen consumption and number of cells compared with those from ovulated oocytes (21.66 ± 4.54 × 10¹⁴ vs. 50.19 ± 4.61 × 10¹⁴ mol/sec, 244 ± 25 vs. 398 ± 24, P < 0.05). Rabbit oocytes grown in vitro with 0.05% FBS achieved pregnancy, but pregnancies were not maintained to term. In conclusion, the addition of 0.05% FBS to the culture medium for IVG improved developmental competence of rabbit oocytes grown in vitro.     

Sequential use of human-derived medium supplements favours cardiovascular tissue engineering

For clinical application of tissue engineering strategies, the use of animal-derived serum in culture medium is not recommended, because it can evoke immune responses in patients. We previously observed that human platelet-lysate (PL) is favourable for cell expansion, but generates weaker tissue as compared to culture in foetal bovine serum (FBS). We investigated if human serum (HS) is a better human supplement to increase tissue strength. Cells were isolated from venous grafts of 10 patients and expanded in media supplemented with PL or HS, to determine proliferation rates and expression of genes related to collagen production and maturation. Zymography was used to assess protease expression. Collagen contraction assays were used as a two-dimensional (2D) model for matrix contraction. As a prove of principle, 3D tissue culture and tensile testing was performed for two patients, to determine tissue strength. Cell proliferation was lower in HS-supplemented medium than in PL medium. The HS cells produced less active matrix metallo-proteinase 2 (MMP2) and showed increased matrix contraction as indicated by gel contraction assays and 3D-tissue culture. Tensile testing showed increased strength for tissues cultured in HS when compared to PL. This effect was more pronounced if cells were sequentially cultured in PL, followed by tissue culture in HS. These data suggest that sequential use of PL and HS as substitutes for FBS in culture medium for cardiovascular tissue engineering results in improved cell proliferation and tissue mechanical properties, as compared to use of PL or HS apart.     

Choosing the right type of serum for different applications of human adipose tissue–derived stem cells: influence on proliferation and differentiation abilities

Background aimsAdipose tissue–derived stem cells (ADSCs) are thought to have great potential in regenerative medicine. A xenoprotein-free culture and handling system is desirable. To date, there is only little and contradictory information about the influence of the different types of human serum on ADSC proliferation and differentiation.MethodsFirst, ADSCs were cultured in media containing regular human serum (HS plus) or fetal calf serum (FCS plus) with supplementation of growth factors for three passages. During passage 4, ADSC proliferative activity and adipogenic, osteogenic and chondrogenic differentiation ability was quantified. Second, ADSCs were cultured with three different human sera (regular human serum [HS], human serum from platelet-poor plasma [SPPP] or human serum from platelet-rich plasma [SPRP]) without supplementation of platelet-derived growth factor and assessed accordingly. The growth factor content of the different types of human sera was determined by means of multiplex protein assay and enzyme-linked immunosorbent assay.ResultsThe different sera did not affect ADSC doubling time significantly (P < 0.05). Specific glycerol-3-phosphat-dehydrogenase activity was significantly lower in cultures with SPRP (P < 0.01) compared with the other media compositions. Extracellular calcium deposition was significantly higher in cells differentiated in cultures with HS or SPPP compared with those with SPRP, HS plus or FCS (P < 0.01). Glycosaminoglycan content and collagen 2 were highest in cells cultured with SPRP (P < 0.001).ConclusionsCulturing ADSCs in human serum appears to be a reasonable and efficient alternative compared with FCS. With respect to the outcome of a sighted clinical application, it appears to be feasible to handle the cells in a serum suitable for the intended later use.     

Manufacturing of human Wharton's jelly stem cells for clinical use: selection of serum is important

BackgroundHuman Wharton's jelly–derived mesenchymal stromal cells (hWJSCs) have gained considerable attention for their use in cell therapy. Many of these applications would require manufacturing of millions of hWJSCs. It is, therefore, necessary to develop a Good Manufacturing Practice (GMP)-compliant hWJSC expansion protocol, allowing the generation of a large quantity of cells to meet both clinical and regulatory requirements. Here, we compared human platelet lysate (HPL) and human serum (HS) in supporting clinical-grade hWJSC expansion.MethodshWJSCs were successfully isolated from six different umbilical cords using GMP-compliant dissociation enzymes. Freshly isolated hWJSCs were cultured in media supplemented with 10% of one of the following sera: fetal bovine serum (FBS), HPL and HS. Properties of the expanded hWJSCs were analyzed.ResultsWe showed that GMP-compliant dissociation enzymes were as efficient as research-grade dissociation enzymes in isolating hWJSCs. hWJSC fresh cell yield and cell viability using HPL and HS supplementations were at greater advantages than FBS. Moreover, hWJSCs expanded in HPL and HS supplementations not only preserved classical MSCs phenotypes and differentiation potential to adipocytes, osteocytes and chondrocytes, they also enhanced the migration of skin fibroblasts. However, HS, unlike HPL, did not alter immunogenicity properties of hWJSCs. hWJSCs expanded in HS supplementation also exerted greater immunosuppressive action in inhibiting T-cell proliferation and increased extracellular matrix (ECM) gene expression, making them useful in tissue repair clinical application.ConclusionOur findings indicate that HS can be considered as a promising and safer alternative to FBS, and should be recommended for clinical-grade expansion of hWJSCs.     

Allergen-specific B cell subset responses in cow’s milk allergy of late eczematous reactions in atopic dermatitis

B cells have regulatory functions in immune responses. Antigen-specific responses of B cell subsets by allergen stimulation ex vivo were examined in milk allergy of late eczematous reactions. Eight milk allergy subjects and 13 milk tolerant subjects were selected by DBPCFC. PBMCs were stimulated by casein ex vivo and stained for B cell subsets using monoclonal antibodies. CD19+ B cells unchanged from 8.7 ± 3.8% to 8.0 ± 5.1% (p = 0.504, n = 8) in the milk allergy group and decreased in the milk tolerant group from 8.5 ± 3.2% to 5.0 ± 1.6% (p = 0.001, n = 13). The fraction of apoptotic B cells in B cells significantly decreased 4.4 ± 3.1% to 1.3 ± 0.4% (p = 0.027, n = 4) in the allergy group and insignificantly increased from 2.8 ± 0.6% to 5.4 ± 2.6% (p = 0.059, n = 6) in the milk tolerant group. CD5+ regulatory B1 cell% in B cells decreased in milk allergy subjects from 36.2 ± 5.0% to 31.0 ± 5.7% (p = 0.010) and unchanged in milk tolerant subjects from 41.6 ± 10.2% to 43.8 ± 10.0% (p = 0.413). IL-10 producing CD19+CD5+ regulatory B cell% in CD19+CD5+ regulatory B cells significantly decreased from 24.9 ± 6.5% to 13.8 ± 5.6% (p = 0.002, n = 5) by casein stimulation in milk allergy group and unchanged from 44.8 ± 11.3% to 43.9 ± 10.0% (p = 0.297, n = 5) in the milk tolerant group. B cell subset responses to IL-4 and IL-5 were also similar in both groups. B cell subset changes seemed to have diagnostic value. Exact immunologic roles of regulatory CD5+ B1 cells need further investigation.     

Optimization of the processing of porcine platelet-rich plasma and its application on human mesenchymal stem cell cultivation

Because of ethical and scientific controversy, the utilization of fetal bovine serum (FBS) for cell culture medium must be minimized. This study develops porcine platelet-rich plasma (P-PRP) as a FBS substitute for human mesenchymal stem cell (hMSC) cultivation. Concentrating porcine blood by serial centrifugation to obtain P-PRP leads to activation by different agonist combinations to stimulate the secretion of growth factors. The concentration of growth factor in P-PRP is significantly increased by activation (p < 0.05). The concentration of PDGF, KGF and TGF-β in activated P-PRP is significantly higher than that in FBS. Design-expert was used to decide whether Co−T+Ca−, Co+T−Ca−, and Co+T+Ca− are optimal agonist formulations. MSC cultivation shows that the attachment rate, proliferation rate and viability of P-PRP supplemented media are significantly higher than those for FBS-supplemented and commercial media (p < 0.05). The results demonstrate that P-PRP is an optimal FBS substitute that supports in vitro h-MSCs expansion for subsequent biomedical applications.     

Scd1 mammary-specific vector constructed and overexpressed in goat fibroblast cells resulting in an increase of palmitoleic acid and oleic acid

Stearoyl-CoA desaturase-1 (Scd1) is a rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids. Overexpression of Scd1 in transgenic animals would modify the nutritional value of ruminant-derived foods by increasing the monounsaturated fatty acid (MUFA) and decreasing the saturated fatty acid (SFA) content. The aim of this study was to develop an effective Scd1 vector that is specifically expressed in dairy goat mammary glands. We successfully amplified the goat full length Scd1 cDNA and evaluated its activity in goat ear skin-derived fibroblast cells (GEFCs) by lipid analysis. In addition, we constructed a mammary gland-specific expression vector and confirmed efficient expression of Scd1 in goat mammary epithelial cells (GMECs) by qRT-PCR and Western blot analysis. Fatty acid analysis showed that Scd1-overexpression resulted in an increase in levels of palmitoleic acid (16:1n-7) and oleic acid (18:1n-9), from 1.73 ± 0.02% to 2.54 ± 0.02% and from 27.25 ± 0.13% to 30.37 ± 0.04%, respectively (both p < 0.01) and the ratio of MUFA to SFA was increased. This work lays a foundation for the generation of Scd1 transgenic goats.     

Amphipathic polymer-mediated uptake of trehalose for dimethyl sulfoxide-free human cell cryopreservation

For stem cell therapy to become a routine reality, one of the major challenges to overcome is their storage and transportation. Currently this is achieved by cryopreserving cells utilising the cryoprotectant dimethyl sulfoxide (Me₂SO). Me₂SO is toxic to cells, leads to loss of cell functionality, and can produce severe side effects in patients. Potentially, cells could be frozen using the cryoprotectant trehalose if it could be delivered into the cells at a sufficient concentration. The novel amphipathic membrane permeabilising agent PP-50 has previously been shown to enhance trehalose uptake by erythrocytes, resulting in increased cryosurvival. Here, this work was extended to the nucleated human cell line SAOS-2. Using the optimum PP-50 concentration and media osmolarity, cell viability post-thaw was 60 ± 2%. In addition, the number of metabolically active cells 24 h post-thaw, normalised to that before freezing, was found to be between 103 ± 4% and 91 ± 5%. This was found to be comparable to cells frozen using Me₂SO. Although reduced (by 22 ± 2%, p = 0.09), the doubling time was found not to be statistically different to the non-frozen control. This was in contrast to cells frozen using Me₂SO, where the doubling time was significantly reduced (by 41 ± 4%, p = 0.004). PP-50 mediated trehalose delivery into cells could represent an alternative cryopreservation protocol, suitable for research and therapeutic applications.     

The effects of bovine serum albumin and fetal bovine serum on the development of pre- and postcleavage-stage bovine embryos cultured in modified CR2 and M199 media

The effects of protein supplementation on bovine embryo development in vitro was evaluated using a 4 × 2 factorial arrangement with ten replications. A total of 6438 oocytes collected from abattoir ovaries were used. Bovine serum albumin (BSA) and fetal bovine serum (FBS) were added in various combinations to simple (modified CR2) and complex (M199) media during culture of precleavage-stage IVM/IVF-derived ova from 18 h after insemination to 72 h and postcleavage-stage embryos after 72 h of culture. Cleavage rates did not differ (p > 0.05) between media supplemented with FBS or with BSA. However, the postcleavage development to the blastocyst stage of in vitro-derived bovine embryos is better in media supplemented with FBS than BSA. A greater (p < 0.05) proportion of cleaved occytes developed to blastocysts and hatched blastocysts in media supplemented with FBS during postcleavage culture. The percentage of embryos that stopped development at the morula stage was significantly (p < 0.05) greater in media supplemented with BSA during postcleavage culture. Viability of blastocysts produced in CR2 and M199 supplemented with FBS were further assessed by transfer to recipients. In CR2, 25 transferred blastocysts resulted in seven pregnancies and the birth of three normal calves. In M199, 24 transferred blastocysts resulted in five pregnancies and the birth of two normal calves. There was no difference (p > 0.05) in rate of embryo development between CR2 and M199.     

Characterisation of allergen-specific responses of IL-10-producing regulatory B cells (Br1) in Cow Milk Allergy

CD19+CD5+ regulatory B cells regulate immune responses by producing IL-10. IL-10-producing regulatory B cell (Br1) responses by allergen stimulation were investigated in human food allergy. Six milk allergy patients and eight milk-tolerant subjects were selected according to DBPCFC. PBMCs were stimulated by casein in vitro and stained for intracellular IL-10 and apoptosis. In response to allergen stimulation, Br1 decreased from 26.2 ± 18.3 to 15.5 ± 8.9% (p = 0.031, n = 6) in the milk allergy group and increased from 15.4 ± 9.0 to 23.7 ± 11.2% (p = 0.023, n = 8) in the milk-tolerant group. Apoptotic non-IL-10-producing regulatory B cells increased from 21.8 ± 9.3 to 38.0 ± 16.1% (p = 0.031, n = 6) in the milk allergy group and unchanged from 28.8 ± 13.8 to 28.0 ± 15.0% (p = 0.844, n = 8) in the milk-tolerant group. Br1 may be involved in the immune tolerance of food allergies by producing IL-10 and simultaneously undergoing apoptosis in humans. The exact roles for Br1 in immune tolerance needs to be further investigated.     

Effects of growth factors and kinase inhibitors on the properties of human adipose-stromal cells in different culture conditions

The best-fit environments for the proliferation and differentiation of human adipose tissue-derived stromal cells (hADSCs) may require specific media and stimuli. The characteristics of hADSCs cultured in different media might be different. We evaluated the effects of growth factors on the proliferation and differentiation of hADSCs and compared the effects of these growth factors on hADSCs cultured in different serum-contained media. The effects of kinase inhibitors on the proliferation of hADSCs were also examined. The combination of insulin-transferrin-selenium (ITS) and fibroblast growth factor (FGF) induced the greatest increase in proliferation, while the combination of ITS and epidermal growth factor (EGF) showed the second-greatest effect in the human serum (HS)-contained media. By contrast, in the fetal bovine serum (FBS)-contained media, ITS had little effect while FGF had better results than the combination of ITS and EGF. The inhibitory potency of the kinase inhibitors was different in the two different media. These results suggest that hADSCs may have different characteristics according to the culture conditions, and that extrapolations of results from animal studies, using FBS media, may not correlate to findings in human studies using HS.     

Application of F magnetic resonance to study the efficacy of fluorine labeled drugs in the three-dimensional cultured breast cancer cells

The cellular monitoring of tumor response to treatments is important for drug discovery and drug development in cancer therapy. We studied efficacy of Herceptin, a common breast cancer drug conjugated with a fluorine organic compound, perfluoro-15-crown-5-ether (PFCE) which easily forms biocompatible emulsions. Three new pharmaceutical forms of Herceptin, Herceptin/PFCE, Herceptin/PFCE/Lipoplex and Herceptin/PFCE/HydraLink were synthesized for the ex vivo study of their efficacy in breast cancer treatment. The emulsions were administered to 10⁹ cells mL⁻¹ of HER-2 positive human adenocarcinoma (MCF-7) cells and the same amount of human mammary epithelial cells (HMEC) cultured in three-dimensional (3D) geometry using hollow fiber bioreactor (HFB) device. Following drugs administration ex vivo, fluorine-19 magnetic resonance imaging (¹⁹F MRI) was applied for cells imaging to measure their viability and to study drug efficacy over 72 h. To ensure optimum drug tracking, HydraLink was used to provide stable binding affinity of emulsified Herceptin to receptor while cationic lipid (Lipofectamine) was used to enhance lipophilicity of the emulsions.After 72 h of treatment with Herceptin, Herceptin/PFCE, Herceptin/PFCE/Lipoplex and Herceptin/PFCE/HydraLink the viability of cells was 54 ± 2%, 49 ± 3%, 43 ± 5% and 42 ± 1%, respectively, as compared with control 93 ± 2%. The efficacy (EC₅₀) of Herceptin conjugated with emulsions was found to be 970 ± 13 μg mL⁻¹ for Herceptin/PFCE, 645 ± 11 μg mL⁻¹ for Herceptin/PFCE/Lipoplex, 678 ± 7 μg mL⁻¹ for Herceptin/PFCE/HydraLink and 1000 ± 3 μg mL⁻¹ for Herceptin. The results show that fluorine emulsions improved the efficacy of Herceptin and ¹⁹F signal intensity changes validated drug efficiency. The significant correlations between duration of treatments and MCF-7 cells viability were observed. While we studied breast cancer cells, the fluorine emulsions could be applied for treatment of other cancer cells overexpressing HER-2.     

Harvesting and cryopreservation of lymphatic endothelial cells for lymphatic tissue engineering

In order to provide a suitable source of cells for lymphatic tissue engineering, the present study was designed to investigate techniques for harvesting and cryopreservation of human dermal lymphatic endothelial cells (LECs) in vitro. The LECs were isolated from children’s foreskins and then cultured in endothelial growth medium-2 MV (EGM-2-MV) with 5% FBS. The second passage LECs were suspended in cryopreservation solution containing 40% FBS and 10% Me₂SO in EGM-2-MV, cooled to −80 °C at about 1 °C/min and stored in liquid nitrogen. Samples were thawed quickly in a 37 °C water bath, and the cryoprotectant was removed by serial elution. The membrane integrity of thawed LECs was determined by trypan blue staining exclusion, and their proliferation was evaluated using the MTT method. The expanded cells of two groups were identified using immunofluorescence staining and RT-PCR with lymphatic-specific markers such as Podoplanin and VEGFR-3. Uptake of fluorescent DiI-Ac-LDL and microtubular formation in three-dimensional cultures were used to detect the function of LECs. Flow cytometry was applied to identify cells and to measure the apoptosis rate as well. Cryopreservation resulted in a retrieval of 67 ± 4% and an intact cell rate of 80 ± 3%. The early apoptosis rate of thawed LECs (9.15 ± 0.34%) was higher than that of fresh control LECs (5.31 ± 0.23%). The growth curves of thawed LECs were similar to those of fresh LECs. The thawed LECs were propagated for at least 6-7 passages without alterations in phenotype and function. Highly purified LECs can be isolated by immunomagnetic beads from human dermis. The cryopreserved/thawed and recultivated LECs are proven to have high vitality and growth potential in vitro and may be considered suitable seed cells for lymphatic tissue engineering.     

Characterization of a new insect cell line (NTU-YB) derived from the common grass yellow butterfly, Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera) and its susceptibility to microsporidia

A new lepidopteran cell line, NTU-YB, was derived from pupal tissue of Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera). The doubling time of YB cells in TNM-FH medium supplemented with 8% FBS at 28 °C was 26.87 h. The chromosome numbers of YB cells varied widely from 21 to 196 with a mean of 86. Compared to other insect cell lines, the YB cells produced distinct esterase, malate dehydrogenase, and lactate dehydrogenase isozyme patterns. Identity of the internal transcribed spacer region-I (ITS-I) of YB cells to E. hecabe larvae was 96% and to Eurema blanda larvae (tissue isolated from head) was 81%. The YB cells were permissive to Nosema sp. isolated from E. blanda and the infected YB cells showed obvious cytopathic effects after 3 weeks post inoculation. The highest level of spore production was at 4 weeks post inoculation when cells were infected with the Nosema isolate, and spore production was 1.34 ± 0.9 × 10⁶ spore/ml. Ultrastructrual studies showed that YB cells can host in vitro propagation of the E. blanda Nosema isolate, and developing stages were observed in the host cell nuclei as observed in the natural host, E. blanda. The NTU-YB cell line is also susceptible to Nosema bombycis.     

The functional VNTR of IGH enhancer HS1.2 associates with human longevity and interacts with TNFA promoter diplotype in a population of Central Italy

The dysregulation of both immune and inflammatory responses occurring with aging is believed to substantially contribute to morbidity and mortality in humans. We have already reported the association of the functional Variable Number of Tandem Repeat (VNTR) at the Immunoglobulin heavy chain (IGH) enhancer HS1.2 with Immunoglobulin levels and with several autoimmune diseases. Herein we tested the association of the VNTR at the HS1.2 enhancer with human longevity, also evaluating the possible modulatory effect of TNFA promoter diplotype (rs361525/rs1800629). HS1.2 enhancer genotypes have been determined for 193 unrelated healthy individuals from Central Italy divided into two groups: Group 1 (18–84 yrs, mean age 56.8 ± 19.4) and Group 2 (85–100 yrs, mean age 93.0 ± 3.5). Homozygous subjects for *2 allele were significantly disadvantaged in reaching higher life-expectancy (OR = 0.457, p = 0.021). A significant interaction between TNFA promoter diplotype status, HS1.2 2/2 genotype and the two Groups was found (p = 0.014). Of note, TNFA − 308A allele seems to exert a protective effect in HS1.2 2/2 carriers. These results support the hypothesis of an important role of HS1.2 VNTR in the puzzle of the immune-system regulation, evidenced also by the potential interaction with TNFA. Moreover, the previous results showing the association of HS1.2 *2 allele with inflammatory phenomena are consistent with the hypothesis that this allele is a detrimental factor in reaching advanced age.     

An ouabain-like factor is secreted from immortalized hypothalamic cells in an aldosterone-dependent manner

Ouabain-like factor (OLF) modulates blood pressure via sodium pump inhibition in the central nervous system and in the peripheral circulation. Ouabain-like factor (OLF) is thought to be produced in the adrenal gland and hypothalamus, and it may relate locally to the renin-angiotensin-aldosterone system. However, the evidence for the latter was obtained from in vivo experiments using animals. In the present study, we investigated ouabain production in the immortalized hypothalamic cell line N1. First, cell culture supernatant was collected from the immortalized hypothalamic cell line N1 at 0.5, 4, 8, and 24 h. A newly developed enzyme-linked immunosorbent assay (ELISA) that used anti-ouabain antibody showed that immunoreactivity in the supernatant was increased significantly at 24 vs. 0.5 h (0.01 ± 0.004 vs. 0.16 ± 0.033 pmol/mg protein, p < 0.01). A combination of HPLC and ELISA was used to characterize N1 cell-derived OLI, showing that the highest peak of OLI had the same retention time as authentic ouabain. Thereafter, N1 cells were cultured with (1-10 μM) aldosterone, and supernatant was collected after 24 h of culture. In addition, N1 cells were cultured with 5 μM eplerenone, a mineralocorticoid receptor blocker, plus aldosterone. OLI was significantly increased in the supernatant of the cells cultured with 10 μM aldosterone (0.40 ± 0.078 pmol/mg protein), and this increase was abolished by the addition of the aldosterone antagonist eplerenone (0.12 ± 0.030 pmol/mg protein). These data suggest that the immortalized hypothalamic N1 cells secrete OLF and that aldosterone stimulates its secretion via mineralocorticoid receptors.