Diverse Target Cells Research Articles

Research progress on astrocyte-derived extracellular vesicles in the pathogenesis and treatment of neurodegenerative diseases.

Neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD), pose significant global health risks and represent a substantial public health concern in the contemporary era. A primary factor in the pathophysiology of these disorders is aberrant accumulation and aggregation of pathogenic proteins within the brain and spinal cord. Recent investigations have identified extracellular vesicles (EVs) in the central nervous system (CNS) as potential carriers for intercellular transport of misfolded proteins associated with neurodegenerative diseases. EVs are involved in pathological processes that contribute to various brain disorders including neurodegenerative disorders. Proteins linked to neurodegenerative disorders are secreted and distributed from cell to cell via EVs, serving as a mechanism for direct intercellular communication through the transfer of biomolecules. Astrocytes, as active participants in CNS intercellular communication, release astrocyte-derived extracellular vesicles (ADEVs) that are capable of interacting with diverse target cells. This review primarily focuses on the involvement of ADEVs in the development of neurological disorders and explores their potential dual roles- both advantageous and disadvantageousin the context of neurological disorders. Furthermore, this review examines the current studies investigating ADEVs as potential biomarkers for the diagnosis and treatment of neurodegenerative diseases. The prospects and challenges associated with the application of ADEVs in clinical settings were also comprehensively reviewed.

Mesenchymal Stem Cell-Derived Extracellular Vesicles and Their Therapeutic Potential.

Extracellular vesicles (EVs) are cell-derived membrane-bound nanoparticles, which act as shuttles, delivering a range of biomolecules to diverse target cells. They play an important role in maintenance of biophysiological homeostasis and cellular, physiological, and pathological processes. EVs have significant diagnostic and therapeutic potentials and have been studied both in vitro and in vivo in many fields. Mesenchymal stem cells (MSCs) are multipotent cells with many therapeutic applications and have also gained much attention as prolific producers of EVs. MSC-derived EVs are being explored as a therapeutic alternative to MSCs since they may have similar therapeutic effects but are cell-free. They have applications in regenerative medicine and tissue engineering and, most importantly, confer several advantages over cells such as lower immunogenicity, capacity to cross biological barriers, and less safety concerns. In this review, we introduce the biogenesis of EVs, including exosomes and microvesicles. We then turn more specifically to investigations of MSC-derived EVs. We highlight the great therapeutic potential of MSC-derived EVs and applications in regenerative medicine and tissue engineering.

Spatiotemporal Protein Atlas of Cell Death-Related Molecules in the Rat MCAO Stroke Model.

Ischemic stroke and cerebral infarction triggered by the blockage of blood supply can cause damage to the brain via a complex series of pathological changes. Recently, diverse therapies have emerged as promising candidates for the treatment of stroke. These treatments exert therapeutic effects by acting on diverse target molecules and cells in different time windows from the acute to chronic phases. Here, using immunohistochemistry, we show pathophysiological changes in the brain microenvironment at the hyperacute (within 6 h), acute (1~3 days), subacute (7 days), and chronic (1 month) phases following ischemic injury. Ischemic injury in rats was induced by occluding the middle cerebral artery and was validated by magnetic resonance imaging. The progression of damage to the brain was evaluated by immunohistochemistry for NeuN+ neurons, GFAP+ astrocytes, and Iba1+ microglia, and by the emergence of the cell death-related molecules such as AIF, FAF1, and activated caspase-3. Our data regarding the spatial and temporal information on pathophysiological changes may warrant the investigation of the timing of administration of therapeutic treatments in preclinical studies with an animal model of stroke.

Single-cell gene expression reveals a landscape of regulatory T cell phenotypes shaped by the TCR.

CD4+ T regulatory (Treg) cells are central to immune homeostasis, their phenotypic heterogeneity reflecting the diverse environments and target cells they regulate. To understand this heterogeneity, we combined single-cell RNAseq, activation reporter and TCR analysis to profile thousands of Tregs or Tconvs from mouse lymphoid organs or human blood. Treg and Tconv pools showed areas of overlap, as resting “furtive” Tregs with overall similarity to Tconv, or as a convergence of activated states. All Tregs express a small core of FoxP3-dependent transcripts, onto which additional programs are added less uniformly. Among suppressive functions, Il2ra and Ctla4 were quasi-constant, inhibitory cytokines being more sparsely distributed. TCR signal intensity didn’t affect resting/activated Treg proportions, but molded activated Treg programs. The main lines of Treg heterogeneity in mice were strikingly conserved in human blood. These results reveal unexpected TCR-shaped states of activation, providing a framework to synthesize previous observations about Treg heterogeneity.

Evolutionary radiation of cytotoxic effects in bivalve and gastropod haemolymph

A comparative immunological survey of cytotoxic properties of whole and cell-free haemolymph (plasma) was performed on four species of marine molluscs. Diverse target cells were used: auto- and heterologous mollusc haemocytes, starfish Asterias rubens coelomocytes, trematode Himasthla elongata rediae, and human erythrocytes. While all the four mollusc species were found to contain cytotoxic factors in their plasma, functional features of these molecules in bivalve and gastropod species are essentially different. Cell-free haemolymph of two clams, Mya arenaria and Mytilus edulis, contains pre-synthetized cytolytic complexes capable of immediate and generalized activation, leading to destruction of a broad range of target cells. On the contrary, two gastropod species, Buccinum undatum and Littorina littorea, possess haemolytic factors which display high specificity of recognition of receptor molecules on the target cells and slow cytolytic reaction. Overall, molecular strategies of cytolytic cascade activation in Bivalvia and Gastropoda can be described as low- and highly selective, respectively.

A Polymorphism within the Internal Fusion Loop of the Ebola Virus Glycoprotein Modulates Host Cell Entry.

The large scale of the Ebola virus disease (EVD) outbreak in West Africa in 2013-2016 raised the question whether the host cell interactions of the responsible Ebola virus (EBOV) strain differed from those of other ebolaviruses. We previously reported that the glycoprotein (GP) of the virus circulating in West Africa in 2014 (EBOV2014) exhibited reduced ability to mediate entry into two nonhuman primate (NHP)-derived cell lines relative to the GP of EBOV1976. Here, we investigated the molecular determinants underlying the differential entry efficiency. We found that EBOV2014-GP-driven entry into diverse NHP-derived cell lines, as well as human monocyte-derived macrophages and dendritic cells, was reduced compared to EBOV1976-GP, although entry into most human- and all bat-derived cell lines tested was comparable. Moreover, EBOV2014 replication in NHP but not human cells was diminished relative to EBOV1976, suggesting that reduced cell entry translated into reduced viral spread. Mutagenic analysis of EBOV2014-GP and EBOV1976-GP revealed that an amino acid polymorphism in the receptor-binding domain, A82V, modulated entry efficiency in a cell line-independent manner and did not account for the reduced EBOV2014-GP-driven entry into NHP cells. In contrast, polymorphism T544I, located in the internal fusion loop in the GP2 subunit, was found to be responsible for the entry phenotype. These results suggest that position 544 is an important determinant of EBOV infectivity for both NHP and certain human target cells.IMPORTANCE The Ebola virus disease outbreak in West Africa in 2013 entailed more than 10,000 deaths. The scale of the outbreak and its dramatic impact on human health raised the question whether the responsible virus was particularly adept at infecting human cells. Our study shows that an amino acid exchange, A82V, that was acquired during the epidemic and that was not observed in previously circulating viruses, increases viral entry into diverse target cells. In contrast, the epidemic virus showed a reduced ability to enter cells of nonhuman primates compared to the virus circulating in 1976, and a single amino acid exchange in the internal fusion loop of the viral glycoprotein was found to account for this phenotype.

Revisiting the identification and cDNA cloning of T cell-replacing factor/interleukin-5.

This is a perspective based on the paper “Cloning of complementary DNA encoding T cell-replacing factor and identity with B cell growth factor II,” by Kinashi et al. (1). We have been interested in understanding the molecular basis of T–B cell cooperation for antibody formation. Although many investigators had described a number of different soluble factors that appeared to have biological relevance to T–B cell interactions, molecular basis of such active substances remained unknown for a long period of time. In this perspective, I will briefly summarize the history of the initial discovery of T cell-replacing factor/B cell growth factor II that appeared to be involved in B cell growth and differentiation, and outline the discovery and characterization of interleukin-5. Studies of interleukin-5 have provided strong evidence that a single cytokine exerts a variety of activities on diverse target cells.

Abstract P1-07-11: ERa regulates epithelial-mesenchymal transition in breast cancers through suppressing Bmi1 expression

Abstract Background: Bmi1, a polycomb-group protein, maintains stem cell self-renewal and is frequently overexpressed in cancers. Upregulation of Bmi1 by Twist1 can promote tumor-initiating capability and Epithelial-mesenchymal transition (EMT) by repressing E-cadherin expression. It has been demonstrated that Post-EMT breast cancer cells express cancer stem cell (CSC) markers including Bmi1, but display decreased ERα expression. ERα is a ligand-activated nuclear hormone receptor that regulates the transcription of E2-responsive genes in diverse target cells. It's reported that ligand-activated ERα could repress slug transcription by binding to the half-site ERE element in its promoter, and then regulate E-cadherin and EMT. Herein, we hypothesized that ERα signaling might regulate E-cadherin and EMT through Bmi1. Methods: First, we determined Bmi1 and ERα expression at both mRNA and protein levels by quantitative RT-PCR and Western blot in various breast cancer cell lines. Further, we quantitatively analyzed mRNA and protein levels of Bmi1 as well as its down-stream genes including E-cadherin after silencing ERα in T47D cells with siRNA, or after overexpressing ERα in BT549 cells. The chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) were also performed to investigate whether ERα binds directly to Bmi1 promoter region. Luciferase assay with Bmi1 reporter was performed to ERα mediated regulation. To identify whether ERα regulates EMT, transwell and matrigel invasion, flow cytometry, mammosphere assay and wound healing assay were performed to invasion, metastases and characterize stem-cell properties of EMT. Finally, we analyzed the expression of ERα, Bmi1 and E-cadherin in human breast cancer specimens with immunohistochemistry. Results: The study showed that Bmi1 inversely correlated with ERα expression in different subtypes of breast cancer cells. We further confirmed that Bmi1 was upregulated at both mRNA and protein levels and E-cadherin was downregulated with siRNA against ERα in T47D cells, and Bmi1 was downregulated and E-cadherin was upregulated when overexpressing ERα in BT549 cells with qRT-PCR and western blot analyses. The present study also demonstrated that ERα could directly binds to the half-ERE region of the Bmi1 gene and repressing Bmi1 expression transcriptionally with CHIP/EMSA and reporter analyses. Overexpression of ERα in BT549 cell significantly decreased levels of migration of transwell and wounding healing, invasiveness, CD44high/CD24low population, and the capabilities of mammosphere formation. Conclusions: Our results demonstrated that ERα can suppress EMT through transcriptionally down-regulating Bmi-1 and its down-stream genes in human breast cancer cells. The inverse relationship between ERα and Bmi1 expression further supports the epithelial phenotype of ERα positive tumors or mesenchymal phenotype of ERα negative tumors. Our findings provide a novel mechanistic insight into how ERα regulates EMT, and is of valuable for developing biomarkers to predict prognosis and targeted therapies in breast cancers in the future. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-07-11.

UMG-lenti: novel dual-promoter lentiviral vectors for efficient transgene- and reporter protein expression in primitive haematopoietic progenitors

Lentiviral vectors are widely used to investigate the biological effects of regulatory proteins and candidate oncogenes by stably enforcing their expression in diverse target cells. In these studies it is essential to be able to monitor and/or isolate the cell subpopulation(s) infected by the virus, and hence expressing the relevant transgene. This can be achieved through the insertion in the vectors of sequences encoding selectable markers or reporter proteins whose expression can be revealed by flow cytometry. To ensure simultaneous expression of transgene and selectable/ reporter gene, intra-ribosomal entry site (IRES) sequences are often inserted between the two cDNAs to generate bi-cistronic mRNAs whose transcription is directed by a single promoter. However, depending on the different transgenes and on the target cell types, the expression of the cDNA located downstream of the IRES sequences (typically encoding the reporter protein) is frequently inconsistent and this makes the recognition of the infected, transgene-expressing cells, problematic. To circumvent this problem, we have designed novel lentiviral vectors (named UMG-LV5 and -LV6) where the expression of the transgene is driven by the UbiC promoter and that of the reporter protein, EGFP, by the 170-bp-long minimal promoter of the WASP gene. These vectors, harbouring two distinct transgenes (ZNF521 or MSI2), were tested in a variety of human haematopoietic cell lines as well as in primary human CFD34+ cells in comparison with the FUIGW vector that contains the expression cassette UbiC-transgene-IRES-EGFP. In all cells studied, infection with UMG-LV5 and -LV6 resulted in a transgene expression slightly lower than that of FUIGW, but a considerably stronger expression of EGFP, that allowed to easily distinguish the transduced cells from the non-transduced counterpart. These vectors are therefore valuable new tools for gene transfer-based studies in haematopoietic stem and progenitor cells. Supported by: AIRC, PhD Programme in Molecular Oncology and POR Calabria FSE 2007/2013 (HEMMAS project). Lentiviral vectors are widely used to investigate the biological effects of regulatory proteins and candidate oncogenes by stably enforcing their expression in diverse target cells. In these studies it is essential to be able to monitor and/or isolate the cell subpopulation(s) infected by the virus, and hence expressing the relevant transgene. This can be achieved through the insertion in the vectors of sequences encoding selectable markers or reporter proteins whose expression can be revealed by flow cytometry. To ensure simultaneous expression of transgene and selectable/ reporter gene, intra-ribosomal entry site (IRES) sequences are often inserted between the two cDNAs to generate bi-cistronic mRNAs whose transcription is directed by a single promoter. However, depending on the different transgenes and on the target cell types, the expression of the cDNA located downstream of the IRES sequences (typically encoding the reporter protein) is frequently inconsistent and this makes the recognition of the infected, transgene-expressing cells, problematic. To circumvent this problem, we have designed novel lentiviral vectors (named UMG-LV5 and -LV6) where the expression of the transgene is driven by the UbiC promoter and that of the reporter protein, EGFP, by the 170-bp-long minimal promoter of the WASP gene. These vectors, harbouring two distinct transgenes (ZNF521 or MSI2), were tested in a variety of human haematopoietic cell lines as well as in primary human CFD34+ cells in comparison with the FUIGW vector that contains the expression cassette UbiC-transgene-IRES-EGFP. In all cells studied, infection with UMG-LV5 and -LV6 resulted in a transgene expression slightly lower than that of FUIGW, but a considerably stronger expression of EGFP, that allowed to easily distinguish the transduced cells from the non-transduced counterpart. These vectors are therefore valuable new tools for gene transfer-based studies in haematopoietic stem and progenitor cells. Supported by: AIRC, PhD Programme in Molecular Oncology and POR Calabria FSE 2007/2013 (HEMMAS project).

Construction and Testing of orfA +/- FIV Reporter Viruses

Single cycle reporter viruses that preserve the majority of the HIV-1 genome, long terminal repeat-promoted transcription and Rev-dependent structural protein expression are useful for investigating the viral life cycle. Reporter viruses that encode the viral proteins in cis in this way have been lacking for feline immunodeficiency virus (FIV), where the field has used genetically minimized transfer vectors with viral proteins supplied in trans. Here we report construction and use of a panel of single cycle FIV reporter viruses that express fluorescent protein markers. The viruses can be produced to high titer using human cell transfection and can transduce diverse target cells. To illustrate utility, we tested versions that are (+) and (-) for OrfA, an FIV accessory protein required for replication in primary lymphocytes and previously implicated in down-regulation of the primary FIV entry receptor CD134. We observed CD134 down-regulation after infection with or without OrfA, and equivalent virion production as well. These results suggest a role for FIV proteins besides Env or OrfA in CD134 down-regulation.

Construction and characterization of efficient, stable and safe replication-deficient foamy virus vectors

As serious side effects affected recent virus-mediated gene transfer studies, novel vectors with improved safety profiles are urgently needed. In the present study, replication-deficient retroviral vectors based on feline foamy virus (FFV) were constructed and analyzed. The novel FFV vectors are devoid of almost the complete env gene plus the internal promoter - accessory bel gene cassette including the gene for the viral transcriptional transactivator Bel1/Tas. In these Bel1/Tas-independent vectors, expression of the lacZ (beta-galactosidase) marker gene is directed by the heterologous, constitutively active human ubiquitin C promoter (ubi). Env-transcomplemented vectors have un-concentrated titers of more than 10(5) transducing units/ml. The vectors allow efficient transduction of a broad array of diverse target cells, which can be increased by repeated vector exposure. However, the number of lacZ marker gene expressing cells decreased slightly upon serial passages of the transduced cells. Vectors carrying a self-inactivating (SIN) deletion of the TATA box and most parts of the viral promoter were not rescued by wt FFV whereas those with the intact or a partially deleted promoter were readily reactivated. This finding indicates that the viral promoters are in fact non-functional, pointing to a highly advantageous safety profile of these new FFV-ubi-lacZ-SIN vectors.

Structural separation of different extracellular activities in aminoacyl-tRNA synthetase-interacting multi-functional protein, p43/AIMP1

AIMP1 (previously known as p43) is first found as a factor associated with a macromolecular tRNA synthetase complex. However, it is also secreted and acts on diverse target cells such as endothelial cells, macrophages, and fibroblasts to control angiogenesis, inflammation, and dermal regeneration, respectively. We previously showed that AIMP1 induces the death of endothelial cell but proliferation of fibroblasts and activates macrophages. In this work, we found that elastase 2-cleaved AIMP1 retained its pro-apoptotic activity to endothelial cells but lost the growth-stimulatory activity to fibroblasts. To determine the functional domains responsible for each activity, we generated several deletion fragments of AIMP1 and compared the activities to the target cells. AIMP1 promoted endothelial cell death and caspase-3 activation through its 101–114 amino acid region, fibroblast proliferation through its 6–46 amino acid region, and endothelial migration through its 114–192 amino acid region as revealed by deletion mapping. Thus, this work revealed that AIMP1 uses different regions for its diverse extracellular activities.

BIOGENESIS, ARCHITECTURE, AND FUNCTION OF BACTERIAL TYPE IV SECRETION SYSTEMS

Type IV secretion (T4S) systems are ancestrally related to bacterial conjugation machines. These systems assemble as a translocation channel, and often also as a surface filament or protein adhesin, at the envelopes of Gram-negative and Gram-positive bacteria. These organelles mediate the transfer of DNA and protein substrates to phylogenetically diverse prokaryotic and eukaryotic target cells. Many basic features of T4S are known, including structures of machine subunits, steps of machine assembly, substrates and substrate recognition mechanisms, and cellular consequences of substrate translocation. A recent advancement also has enabled definition of the translocation route for a DNA substrate through a T4S system of a Gram-negative bacterium. This review emphasizes the dynamics of assembly and function of model conjugation systems and the Agrobacterium tumefaciens VirB/D4 T4S system. We also summarize salient features of the increasingly studied effector translocator systems of mammalian pathogens.

Mechanisms that explain the contraceptive action of progestin implants for women

Four different contraceptive implants for women, in the form of capsules or covered rods, that release either one of the synthetic progestins levonorgestrel, etonogestrel, Nestorone, or Elcometrine and nomegestrol acetate were considered. These progestins act by binding to their receptors located in diverse target cells, which are distributed along the hypothalamic-pituitary-gonadal-genital tract axis. These implants differ in the extent to which each one interferes with various steps of the reproductive process and in the intensity with which each one affects the same process along its effective life, but they have in common the ability to interfere with several key processes required for gamete encounter and fertilization. The steps they interfere with most are the ovulatory process, through partial or complete inhibition of the gonadotropin surge, and by changing the quality of cervical mucus; they restrict or suppress the access of fertile spermatozoa to the site of fertilization. Changes in endometrial development also occur, but this contribution to the contraceptive action is difficult to determine at the present time.

Molecular mechanisms of estrogen action: selective ligands and receptor pharmacology

Estrogens exert profound effects on the physiology of diverse target cells and these effects appear to be mediated by two estrogen receptor (ER) subtypes, ERα and ERβ. We have investigated how ER ligands, ranging from pure agonists to antagonists, interact with ERα and ERβ, and regulate their transcriptional activity on different genes. Mutational mapping-structure activity studies indicate that different residues of the ER ligand binding domain are involved in the recognition of structurally distinct estrogens and antiestrogens. We have identified from ligands of diverse structure, several particularly interesting ones that are high potency selective agonists via ERα and others that are full agonists through ERα while being full antagonists through ERβ. Antiestrogens such as hydroxytamoxifen, which are mixed agonist/antagonists through ERα, are pure antagonists through ERβ at estrogen response element-containing gene sites. Studies with ERα/β chimeric proteins reveal that tamoxifen agonism requires the activation function 1 region of ERα. Through two-hybrid assays, we have isolated an ER-specific coregulator that potentiates antiestrogen antagonist effectiveness and represses ER transcriptional activity. We have also focused on understanding the distinct pharmacologies of antiestrogen- and estrogen-regulated genes. Although antiestrogens are thought to largely act by antagonizing the actions of estrogens, we have found among several new ER-regulated genes, quinone reductase (QR), a detoxifying phase II antioxidant enzyme, that has its activity up-regulated by antiestrogens in an ER-dependent manner in breast cancer cells. This response is antagonized by estrogens, thus showing ‘reversed pharmacology’. Increased QR activity by antiestrogens requires a functional ER (ERα or ERβ) and is, interestingly, mediated via the electrophile response element in the QR gene 5′ regulatory region. The up-regulation of QR may contribute to the beneficial effects of tamoxifen, raloxifene, and other antiestrogens in breast cancer prevention and treatment. Estrogens rapidly up-regulate expression of several genes associated with cell cytoarchitectural changes including NHE-RF, the sodium hydrogen exchanger regulatory factor, also known as EBP50. NHE-RF/EBP50 is enriched in microvilli, and may serve as a scaffold adaptor protein in regulating early changes in cell architecture and signal transduction events induced by estrogen. Analyses of the regulatory regions of these primary response genes, and the antioxidant and other signaling pathways involved, are providing considerable insight into the mechanisms by which ligands, that function as selective estrogen receptor modulators or SERMs, exert their marked effects on the activities and properties of target cells. The intriguing biology of estrogens in its diverse target cells is thus determined by the structure of the ligand, the ER subtype involved, the nature of the hormone-responsive gene promoter, and the character and balance of coactivators and corepressors that modulate the cellular response to the ER–ligand complex. The continuing development of ligands that function as selective estrogens or antiestrogens for ERα or ERβ should allow optimized tissue selectivity of these agents for menopausal hormone replacement therapy and the treatment and prevention of breast cancer.

Mechanisms of action and cross-talk between estrogen receptor and progesterone receptor pathways.

The intriguing biology of estrogens and progestins in their diverse target cells is determined by the structure of the hormonal ligand, the receptor subtype or isoform involved, the nature of the hormone-responsive gene promoter, and the character and balance of coactivators and corepressors that modulate the cellular response to the receptor-ligand complex. Estrogens regulate the growth, differentiation, and functioning of diverse target tissues, both within and outside of the reproductive system. Most of the actions of estrogens appear to be exerted through the estrogen receptor (ER) of target cells, an intracellular receptor that is a member of a large superfamily of proteins, which function as ligand-activated transcription factors, regulating the synthesis of specific RNAs and proteins. To understand how the ER discriminates between estrogen ligands, which activate the ER, and antiestrogen ligands, which fail to effectively activate the ER, we have generated and analyzed human ERs with mutations or other alterations in portions of the receptor. These studies provide evidence for the promoter-specific and cell-specific actions of the estrogen-occupied and antiestrogen-occupied ER, highlight a regional dissociation of the hormone-binding and transcription activation functions in domain E of the receptor, and indicate that some of the contact sites of estrogens and antiestrogens in the ER are likely different. In addition, multiple interactions among different cellular signaling pathways are involved in the regulation of gene expression and cell proliferation by the ER. In several cell types, protein kinase activators and some growth factors enhance the transcriptional activity of the ER. Cyclic AMP also alters the agonist/antagonist balance of some antiestrogens. Estrogens, and antiestrogens to a lesser extent, as well as protein kinase activators and growth factors, increase phosphorylation of the ER and possibly other proteins involved in the ER-specific response pathway, suggesting that changes in cellular phosphorylation state will be important in determining the biologic activity of the ER and the effectiveness of antiestrogens as estrogen antagonists. The ER also has important interrelationships with the progesterone receptor (PR) system in modulation of biologic responses. Liganded PR-A and PR-B can each suppress estradiol-stimulated ER activity, with the magnitude of repression dependent on the PR isoform, progestin ligand, promoter, and cell type. These findings underscore the mounting evidence for the importance of interactions between members of the steroid hormone receptor family.

Factors affecting the measurement of cytokines in biological fluids: implications for their clinical measurement.

Cytokines, a term first used by Cohen, I are usually defined as soluble proteins, released by living cells of the host, which act to regulate host cell function. The first cytokines to be described had immunoregulatory effects, although it is now clear that this definition encompasses a wide variety of functions. A more precise definition is difficult because of the increasing number of such regulatory peptides which have been discovered. In general, cytokines are multifunctional molecules with diverse biological actions and target cells. Their main functions are usually local, modulating the behaviour of adjacent cells (paracrine) or the cell that secretes them (autocrine), but in some cases they have significant effects at distant sites. In normal conditions, cytokines are released in picomolar amounts, and only in certain pathological situations do they become detectable in significant amounts in peripheral blood. The term cytokines includes: the interleukins (interleukin-I to interleukin-14); tumour necrosis factor (TNF) a and (3; interferon (lFN) a, (3and -y; transforming growth factor (TGF) a and (3; chemokines (including monocyte chemoattractant protein and IL-8); haematopoietic growth factors; and others (see reviews/v' for discussion of individual cytokines). The most frequently measured cytokines are TNFa, interleukin-I (lL-I), interleukin-6 (I L-6)and more recently interleukin-8 (lL-8), all in the context of acute inflammation and infection. Measurements of IL-2 and soluble lL-2 receptor are proving of interest in the investigation of patients with immune-mediated disease and IL-5 in allergic disorders.

A common 43 K protein induced by glucocorticoids in a variety of cells and tissues

We have investigated the domain of adrenal steriod action in a variety of mammalian cells and tissue by high resolution two-dimensional gel electrophoresis and autoradiography. Following in vivo or in vitro treatment with steroids, minced tissue or cells were pulsed with [ 35S]methionine, and they newly synthesized polypeptides compared with controls by visual inspection of partial protein maps. We have found a similar protein ( M r ∼ 41-44K, pI ∼ 6.3–6.5) to be consistently increased by dexamethasone in 3 rat tissues and 9 human, bovine and rat cell lines. The protein was constitutively synthesized in all targets; quantitative measurements on the magnitude of response show a 2-fold induction by dexamethasone in all systems, in vivo and in vitro. Glucocorticoid-specific hormones but not sex steroids increase the rate of synthesis; deoxycorticosterone has agonist or antagonist effects on 43K synthesis in different systems studied. Co-electrophoresis experiments indicated that 43K proteins co-migrate in at least 3 rat cell lines from tissues with diverse functions. The functional significance of this common glucocorticoid-induced protein is unknown; its ubiquity and electrophoretic properties suggest a highly conserved, common indicator of glucocorticoid action in diverse target cells and tissues, rather than a tissue-specific response.

Some aspects of the modern concept of neurosecretion.

Certain glandular nerve cells communicate with effector cells by means of hormonal mediators (neurohormones). The production of neurohormones is restricted to this specialized group of glandular neurones, which possess glandular attributes as well as attributes of conventional neurons. These glandular neurons, because of their dual nature, are called ‘neurosecretory cells’ and their secretion is termed ‘neurosecretory material’. The identification of neurosecretion based upon the occurrence of apparent secretory inclusions in the neuron is not very reliable. The classical staining techniques of Gomori as well as newer Alcian blue and pseudocyanin methods are only suggestive rather than conclusive. Ultra structurally, the ‘classical’ neurosecretory neurons may be distinguished as having neurosecretory materials as prominent content of membrane-bound granules of varying size ranges and of varying, usually high, electron density. Intermingled with typical proteinaceous granules are small electron-lucent vesicles in dense aggregate. These are considered as packets of acetylcholine, helping in the release of neurohormones. The secretory material contains active principles regulating multiple and diverse target cells via a circular channel exemplified by polypeptides, e.g., vasopressin and oxytocin, bound to carrier proteins (neurophysins). Various physiologic traits, e.g. colour change in crustaceans, tanning of the integument of insects, water balance in vertebrates, growth and regeneration in annelids, liberation of the ‘tropic hormones’ from the adenohypophysis by the so-called ‘releasing factors’ are regulated by the neurohormones from the central nervous system.

Macrophage-mediated natrual cytotoxicity against various target cells in vitro. I. Macrophages from diverse anatomical sites and different strains of rats and mice.

Adherent, predominantly phagocytic, mononuclear cells expressing spontaneous cytotoxic activity against diverse target cells in vitro were present in various tissues of different strains of rats and mice. Cells with such natural killer capacity were thus everywhere readily available for mobilization and activation. The inherent spontaneous killer capacity of adherent mononuclear phagocytes can be abrogated by silica particles in vitro, and can be considerably enhanced by appropriate stimuli in vivo. Spontaneous cytolysis mediated by unstimulated mononuclear phagocytes was consistently manifested only after a lag phase of 12-20 h and was quite nonspecific; there was no cogent correlation between susceptibility to lysis and transformation.