B‐cell capacity for differentiation changes with age

BackgroundAge‐related immune deficiencies are thought to be responsible for increased susceptibility to infection in older adults, with alterations in lymphocyte populations becoming more prevalent over time. The loss of humoral immunity in ageing was attributed to the diminished numbers of B cells and the reduced ability to generate immunoglobulin.AimsTo compare the intrinsic B‐cell capacity for differentiation into mature plasma cells (PCs), between young and old donors, using in vitro assays, providing either effective T‐cell help or activation via TLR engagement.MethodsB cells were isolated from healthy individuals, in younger (30–38 years) and older (60–64 years) donors. An in vitro model system of B‐cell differentiation was used, analysing 5 differentiation markers by flow cytometry, under T‐dependent (TD: CD40/BCR stimulation) or T‐independent (TI: TLR7/BCR activation) conditions. Antibody secretion was measured by ELISA and gene expression using qPCR.ResultsTI and TD differentiation resulted in effective proliferation of B cells followed by their differentiation into PC. B‐cell‐executed TI differentiation was faster, all differentiation marker and genes being expressed earlier than under TD differentiation (day 6), although generating less viable cells and lower antibody levels (day 13). Age‐related differences in B‐cell capacity for differentiation were minimal in TD differentiation. In contrast, in TI differentiation age significantly affected proliferation, viability, differentiation, antibody secretion and gene expression, older donors being more efficient.ConclusionAltogether, B‐cell differentiation into PC appeared similar between age groups when provided with T‐cell help, in contrast to TI differentiation, where multiple age‐related changes suggest better capacities in older donors. These new findings may help explain the emergence of autoantibodies in ageing.     

Phylogenetic distribution in the genus Mus of t-complex-specific DNA and protein markers: inferences on the origin of t-haplotypes

We have examined the phylogenetic distribution of two t-specific markers among representatives of various taxa belonging to the genus Mus. The centromeric TCP-1a marker (a testicular protein variant specific for all t-haplotypes so far studied) has also been apparently detected in several non-t representatives of the Mus IVA, Mus IVB, and probably M. cervicolor species. By contrast, a t-specific restriction- fragment-length polymorphism allele (RFLP) of the telomeric alpha- globin pseudogene DNA marker alpha-psi-4 was found only in animals belonging to the M. musculus-complex species either bearing genuine t- haplotypes or, like the M. m. bactrianus specimen studied here, likely to do so. This t-specific alpha-psi-4 RFLP allele was found to be as divergent from the RFLP alleles of the latter, non-t, taxonomical groups as it is from Mus 4A, Mus 4B, or M. spretus ones. These results suggest the presence of t-haplotypes and of t-specific markers in populations other than those belonging to the M. m. domesticus and M. m. musculus subspecies, implying a possible origin for t-haplotypes prior to the radiation of the most recent offshoot of the Mus genus (i.e., the spretus/domesticus divergence), some 1-3 Myr ago.      

Reconstitution and transphosphorylation of TGF-beta receptor complexes.

Transforming growth factor-beta (TGF-beta) signals by contacting two distantly related transmembrane serine/threonine kinases called receptors I (T beta R-I) and II (T beta R-II). TGF-beta binds to T beta R-II, which is a constitutively active kinase and this complex recruits T beta R-I, causing its phosphorylation and signal propagation to downstream substrates. The biochemical properties of this interaction were analyzed with reconstituted receptor systems. T beta R-I and T beta R-II baculovirally expressed at high levels in insect cells have the ligand binding properties of receptors expressed in mammalian cells, and form a complex in which T beta R-I phosphorylation is dependent on the kinase activity of T beta R-II. Furthermore, T beta R-I and T beta R-II can form a complex in vitro, and their cytoplasmic domains can specifically interact in a yeast two-hybrid system. In vitro complex formation with catalytically active T beta R-II is necessary and sufficient for T beta R-I phosphorylation, which within this complex does not require the catalytic activity of T beta R-I, thus mimicking T beta R-I phosphorylation in intact cells. In addition, T beta R-I phosphorylated in vitro remains associated with T beta R-II. These results suggest that T beta R-I and T beta R-II have affinity for each other, however, the ligand is required for stable complex formation under physiological conditions. Once formed, this complex is sufficient for T beta R-I phosphorylation by T beta R-II.     

Carryover effects from environmental change in early life: An overlooked driver of the amphibian extinction crisis?

Ecological carryover effects, or delayed effects of the environment on an organism's phenotype, are central predictors of individual fitness and a key issue in conservation biology. Climate change imposes increasingly variable environmental conditions that may be challenging to early life-history stages in animals with complex life histories, leading to detrimental physiological and fitness effects in later life. Yet, the latent nature of carryover effects, combined with the long temporal scales over which they can manifest, means that this phenomenon remains understudied and is often overlooked in short-term studies limited to single life-history stages. Herein, we review evidence for the physiological carryover effects induced by elevated ultraviolet radiation (UVR; 280–400 nm) as a potential contributor to recent amphibian population declines. UVR exposure causes a suite of molecular, cellular and physiological consequences known to underpin carryover effects in other taxa, but there is a lack of research linking embryonic and larval UVR exposures to fitness consequences post-metamorphosis in amphibians. We propose that the key impacts of UVR on disease-related amphibian declines are facilitated through carryover effects that bridge embryonic and larval UVR exposure with potential increased disease susceptibility post-metamorphosis. We conclude by identifying a practical direction for the study of ecological carryover effects in amphibians that could guide future ecological research in the broader field of conservation physiology. Only by addressing carryover effects can many of the mechanistic links between environmental change and population declines be elucidated.     

Regulation of expression of the 3β-hydroxysteroid dehydrogenases of human placenta and fetal adrenal

The appropriate expression of 3β-hydroxysteroid dehydrogenase/Δ^5→4-isomerase (3β-HSD) is vital for mammalian reproduction, fetal growth and life maintenance. Several isoforms of 3β-HSD, the products of separate genes, have been identified in various species including man. Current investigations are targeted toward defining the processes that regulate the levels of specific isoforms in various steroidogenic tissues of man. High levels of expression of 3β-HSD were observed in placental tissues. It has been generally considered that the multinucleated syncytiotrophoblastic cells are the principal sites of 3β-HSD expression and, moreover, that 3β-HSD expression is intimately associated with cyclic AMP-promoted formation of syncytia. Herein we report the presence of 3β-HSD immunoreactive and mRNA species in uninucleate cytotrophoblasts in the chorion laeve, similar to that in syncytia but not cytotrophoblast placenta. In vitro, 3β-HSD levels in chorion laeve cytotrophoblasts were not increased with time nor after treatment with adenylate cyclase activators, whereas villous cytotrophoblasts spontaneously demonstrated progressive, increased 3β-HSD expression. Moreover, 3β-HSD synthesis appeared to precede morphologic syncytial formation. Thus high steroidogenic enzyme expression in placenta is not necessarily closely linked to formation of syncytia. Both Western immunoblot and enzymic activity analyses also indicated that the 3β-HSD expressed in these cytotrophoblastic populations was the 3β-HSD type I gene product (M_r, 45K) and not 3β-HSD type II (M_r, 44K) expressed in fetal testis. In cultures of fetal zone and definitive zone cell of human fetal adrenal, 3β-HSD expression was not detected until ACTH was added. ACTH, likely acting in a cyclic AMP-dependent process, induced 3β-HSD type II activity and mRNA expression. The higher level of 3β-HSD mRNA in definitive zone compared with fetal zone cells was associated with parallel increases in cortisol secretion relative to dehydroepiandrosterone sulfate formation.     

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

Stimulation of folliculogenesis in domestic cats with human FSH and LH

Folliculogenesis in response to exogenous stimulation by human urinary follicle stimulating hormone (huFSH) and human menopausal gonadotropin (hMG) was evaluated in the domestic queen (Felis catus). The role of LH and/or FSH in folliculogenesis was examined by measuring concentrations of estradiol 17beta (E(2)) and progesterone (P) in the serum. Additionally, changes in the number and size of follicles from before the administration of exogenous hormones to surgical oocyte collection were monitored. Findings indicated that in queens receiving huFSH or hMG followed by human chorionic gonadotropin (hCG) to induce ovulation, the numbers of follicles from 1 to 3 mm increase with statistical significance (P<0.005) from before the initiation of treatment to surgical collection of oocytes. Although E(2) concentrations in cats receiving hMG increased above baseline by the third exogenous hormone injection, mean E(2) concentrations did not increase in the groups that received both huFSH and hCG, or hCG only, until after the administration of hCG. This suggests that the exogenous administration of LH contained in both hMG and hCG was necessary for E(2) to rise to levels associated with estrus.     

Roles of creatine in the regulation of cardiac protein synthesis

The observation that increased muscular activity leads to muscle hypertrophy is well known, but identification of the biochemical and physiological mechanisms by which this occurs remains an important problem. Experiments have been described (5, 6) which suggest that creatine, an end product of contraction, is involved in the control of contractile protein synthesis in differentiating skeletal muscle cells and may be the chemical signal coupling increased muscular activity and the increased muscular mass. During contraction, the creatine concentration in muscle transiently increases as creatine phosphate is hydrolyzed to regenerate ATP. In isometric contraction in skeletal muscle for example, Edwards and colleagues (3) have found that nearly all of the creatine phosphate is hydrolyzed. In this case, the creatine concentration is increased about twofold, and it is this transient change in creatine concentration which is postulated to lead to increased contractile protein synthesis. If creatine is found in several intracellular compartments, as suggested by Lee and Vissher (7), local changes in concentration may be greater then twofold. A specific effect on contractile protein synthesis seems reasonable in light of the work of Rabinowitz (13) and of Page et al. (11), among others, showing disproportionate accumulation of myofibrillar and mitochondrial proteins in response to work-induced hypertrophy and thyroxin-stimulated growth. Previous experiments (5, 6) have shown that skeletal muscles cells which have differentiated in vitro or in vivo synthesize myosin heavy-chain and actin, the major myofibrillar polypeptides, faster when supplied creatine in vitro. The stimulation is specific for contractile protein synthesis since neither the rate of myosin turnover nor the rates of synthesis of noncontractile protein and DNA are affected by creatine. The experiments reported in this communication were undertaken to test whether creatine selectively stimulates contractile protein synthesis in heart as it does in skeletal muscle.     

Microengineered systems with iPSC-derived cardiac and hepatic cells to evaluate drug adverse effects

Hepatic and cardiac drug adverse effects are among the leading causes of attrition in drug development programs, in part due to predictive failures of current animal or in vitro models. Hepatocytes and cardiomyocytes differentiated from human induced pluripotent stem cells (iPSCs) hold promise for predicting clinical drug effects, given their human-specific properties and their ability to harbor genetically determined characteristics that underlie inter-individual variations in drug response. Currently, the fetal-like properties and heterogeneity of hepatocytes and cardiomyocytes differentiated from iPSCs make them physiologically different from their counterparts isolated from primary tissues and limit their use for predicting clinical drug effects. To address this hurdle, there have been ongoing advances in differentiation and maturation protocols to improve the quality and use of iPSC-differentiated lineages. Among these are in vitro hepatic and cardiac cellular microsystems that can further enhance the physiology of cultured cells, can be used to better predict drug adverse effects, and investigate drug metabolism, pharmacokinetics, and pharmacodynamics to facilitate successful drug development. In this article, we discuss how cellular microsystems can establish microenvironments for these applications and propose how they could be used for potentially controlling the differentiation of hepatocytes or cardiomyocytes. The physiological relevance of cells is enhanced in cellular microsystems by simulating properties of tissue microenvironments, such as structural dimensionality, media flow, microfluidic control of media composition, and co-cultures with interacting cell types. Recent studies demonstrated that these properties also affect iPSC differentiations and we further elaborate on how they could control differentiation efficiency in microengineered devices. In summary, we describe recent advances in the field of cellular microsystems that can control the differentiation and maturation of hepatocytes and cardiomyocytes for drug evaluation. We also propose how future research with iPSCs within engineered microenvironments could enable their differentiation for scalable evaluations of drug effects.     

Magnetic core-shell nanoparticles for drug delivery by nebulization

Background

Aerosolized therapeutics hold great potential for effective treatment of various diseases including lung cancer. In this context, there is an urgent need to develop novel nanocarriers suitable for drug delivery by nebulization. To address this need, we synthesized and characterized a biocompatible drug delivery vehicle following surface coating of Fe₃O₄ magnetic nanoparticles (MNPs) with a polymer poly(lactic-co-glycolic acid) (PLGA). The polymeric shell of these engineered nanoparticles was loaded with a potential anti-cancer drug quercetin and their suitability for targeting lung cancer cells via nebulization was evaluated.

Results

Average particle size of the developed MNPs and PLGA-MNPs as measured by electron microscopy was 9.6 and 53.2 nm, whereas their hydrodynamic swelling as determined using dynamic light scattering was 54.3 nm and 293.4 nm respectively. Utilizing a series of standardized biological tests incorporating a cell-based automated image acquisition and analysis procedure in combination with real-time impedance sensing, we confirmed that the developed MNP-based nanocarrier system was biocompatible, as no cytotoxicity was observed when up to 100 μg/ml PLGA-MNP was applied to the cultured human lung epithelial cells. Moreover, the PLGA-MNP preparation was well-tolerated in vivo in mice when applied intranasally as measured by glutathione and IL-6 secretion assays after 1, 4, or 7 days post-treatment. To imitate aerosol formation for drug delivery to the lungs, we applied quercitin loaded PLGA-MNPs to the human lung carcinoma cell line A549 following a single round of nebulization. The drug-loaded PLGA-MNPs significantly reduced the number of viable A549 cells, which was comparable when applied either by nebulization or by direct pipetting.

Conclusion

We have developed a magnetic core-shell nanoparticle-based nanocarrier system and evaluated the feasibility of its drug delivery capability via aerosol administration. This study has implications for targeted delivery of therapeutics and poorly soluble medicinal compounds via inhalation route.