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At least 55 records · Page 3Linked to original sources
PubMed2006

Anti-HIV-1 immunotoxin 3B3(Fv)-PE38: enhanced potency against clinical isolates in human PBMCs and macrophages, and negligible hepatotoxicity in macaques.

Highly active antiretroviral therapy (HAART) against human immunodeficiency virus type 1 (HIV-1) infection dramatically suppresses viral load, leading to marked reductions in HIV-1 associated morbidity and mortality. However, infected cell reservoirs and low-level replication persist in the face of suppressive HAART, leading invariably to viral rebound upon cessation of treatment. Toxins engineered to target the Env glycoprotein on the surface of productively infected cells represent a complementary strategy to deplete these reservoirs. We described previously highly selective killing of Env-expressing cell lines by CD4(178)-PE40 and 3B3(Fv)-PE38, recombinant derivatives of Pseudomonas aeruginosa exotoxin A containing distinct targeting moieties against gp120. In the present report, we compare the in vitro potency and breadth of these chimeric toxins against multiple clinical HIV-1 isolates, replicating in biologically relevant primary human target cell types. In PBMCs, 3B3(Fv)-PE38 blocked spreading infection by all isolates examined, with greater potency than CD4(178)-PE40. 3B3(Fv)-PE38 also potently inhibited spreading HIV-1 infection in primary macrophages. Control experiments demonstrated that in both target cell types, most of the 3B3(Fv)-PE38 activity was due to selective killing of infected cells, and not merely to neutralization by the antibody moiety of the chimeric toxin. High-dose treatment of rhesus macaques with 3B3(Fv)-PE38 did not induce liver toxicity, whereas equivalent dosage of CD4(178)-PE40 induced mild hepatotoxicity. These findings highlight the potential use of 3B3(Fv)-PE38 for depleting HIV-infected cell reservoirs persisting in the face of HAART.

Animals↗
PubMed1991

Different types of allospecific CTL clones identified by their ability to recognize peptide loading-defective target cells.

Allospecific immune responses against the MHC of another individual are remarkably strong, due t a high number of responding T cell clones. Although it has been demonstrated that some allospecific cytotoxic T lymphocytes (CTL) recognize peptides presented by allogeneic MHC class I molecules, it has remained unclear whether MHC molecules can be recognized directly. We used the H-2b-derived murine lymphoma mutant RMA-S, which has a defect affecting peptide loading of class I molecules, to test whether recognition by allospecific CTL always requires the presence of peptides. Three types of anti-H-2Kb CTL clones can be distinguished by their ability to lyse RMA-S target cells. Type A CTL clones efficiently lyse these target cells, the lysis by type B CTL clones is inefficient, and type C clones fail to lyse RMA-S. Up-regulation of the levels of H-2Kb density improved lysis by type B clones, but did not lead to lysis by type C clones. Some type A and B CTL clones apparently can recognize class I molecules devoid of peptides, while others are likely to recognize peptides which are not affected by the presentation defect of RMA-S. We suggest that type C clones are specific for peptides which are not presented by the mutant cells. The results show that the majority of alloreactive CTL recognize peptide/MHC complexes, while some CTL behave as if they can recognize class I molecules in the absence of MHC-bound peptides.

Animals↗
PubMed1985

The human lymphokine-activated killer cell system. V. Purified recombinant interleukin 2 activates cytotoxic lymphocytes which lyse both natural killer-resistant autologous and allogeneic tumors and trinitrophenyl-modified autologous peripheral blood lymphocytes.

Culture of human peripheral blood lymphocytes (PBL) in purified natural or recombinant interleukin 2 in the absence of exogenous antigen or mitogen causes the differentiation of nonlytic precursor cells into lymphokine-activated killers (LAK). A titration of purified Jurkat IL-2 (BRMP, FCRC, NIH) IL-2 showed that the relatively low concentration of 5 U/ml was optimal for LAK activation. When the responding PBL were pretreated with either mitomycin C or gamma irradiation, LAK activation did not occur, indicating that proliferation, in addition to differentiation, is required. The spectrum of target cells susceptible to LAK lysis in a 4-hr chromium-51-release assay includes fresh NK-resistant tumor cells and trinitrophenyl (TNP)-modified autologous PBL. Unmodified PBL are not lysed. Cold target inhibition studies indicated that LAK lysis of autologous TNP-PBL is totally inhibited by fresh tumors cells, and that tumor lysis is inhibited by TNP-PBL. Additionally, allogeneic tumors totally inhibit lysis of autologous tumor cells in other cold target studies. These results demonstrate that the lytic activity expressed by LAK is not HLA restricted, is not limited to tumor cells, and is "polyspecific" as indicated by the cross-reactive recognition of multiple target cell types in these cold target inhibition studies.

Humans↗
PubMed1997

Rapid purification of glial cells using immunomagnetic separation.

By purifying glial cells from brain tissue containing a heterogeneous cell population, a number of interactions that define glial cell diversification and function within the central nervous system have been determined. The current methods for purifying glial cells, however, can be time consuming and costly. In the following study we have adapted the technique of immunomagnetic separation to separately enrich 0-2A progenitor cells and astrocytes from the rat central nervous system (CNS). In this procedure, tissue from the CNS was enzymatically dissociated and incubated in a primary antibody specific to a surface antigen found on the target cell type (e.g. A2B5 or RAN-2). The target cells were then immunologically coupled to magnetic beads, which were precoated with a secondary antibody specific to the primary, and then separated out from the heterogeneous cell population using a magnetic field. We found that the immunomagnetic separation procedure, which was completed within 2 h, produced a near pure population of glial cells (> 99%). This was confirmed by the absence of unbound cells in the bead-bound fraction. The identification and viability of bead-bound cells were established by culturing these cells and subsequently examining their morphology and antigenic expression. This study shows that glial cell types can be separated out of brain tissue to near purity using immunomagnetic separation. This simple procedure is reliable, inexpensive, and achieves levels of purity and viability comparable with currently available techniques of immunopanning and fluorescence-activated cell sorting, within a fraction of the time.

Animals↗
PubMed2004

Alveolar epithelial cells type II are major target cells for C. pneumoniae in chronic but not in acute respiratory infection.

Pulmonary presence of Chlamydia pneumoniae is associated with acute and chronic infections. We show that unapparent chlamydial infection in four out of 31 chronic obstructive pulmonary disease (COPD) patients (12.9%) is characterized by a significant increase in infected alveolar epithelial cells type II (18.2 +/- 3.5% vs. 2.3 +/- 0.9; IHC/ISH) compared to a newly established model of acute chlamydial infection (ACIM) in vital lung specimens from pulmonary lobectomy. Expression of cHSP60 demonstrated pathogen viability and virulence in the ACIM. We conclude that target cells differ in acute and chronic chlamydial infection and suggest the ACIM as a novel tool to analyze the host-pathogen-interactions in acute respiratory infections.

Acute Disease↗
PubMed2000

Aqueous humor stimulates the migration of human trabecular meshwork cells in vitro.

PURPOSE: Depletion of trabecular meshwork cell numbers is a feature of the outflow system in aging and in primary open-angle glaucoma. It is possible that migration stimulated by factors present in aqueous humor may contribute to the cell loss. This investigation assessed the chemoattractant potential of glaucomatous and nonglaucomatous human aqueous humor and fibronectin, one of its constituents, on a range of cultured trabecular meshwork cell lines. METHODS: Migration was assessed in 48-well modified Boyden chambers. The potential migratory stimulants were soluble fibronectin and glaucomatous and nonglaucomatous aqueous humor. The glaucomatous aqueous samples were collected from patients undergoing trabeculotomy for primary open-angle glaucoma and the normal aqueous from normal bovine eyes and patients undergoing cataract surgery. The target cell types were normal human and bovine meshwork cells grown from explants and two human transformed meshwork cell lines from a normal (HTM-5) and a glaucomatous (HTM-3) source. RESULTS: Soluble fibronectin stimulated all the target cells to migrate with an optimal concentration ranging from 1 to 30 microg/ml, and Zigmond Hirsch checkerboard analysis indicated that both chemotaxis and chemokinesis took place. All the aqueous humor samples stimulated migration of the meshwork cell lines at an optimal concentration of 200 microl/ml. Glaucomatous aqueous humor stimulated a greater migratory response than nonglaucomatous aqueous for two of the four target cell types (P < or = 0.03). Neutralization of the fibronectin content of nonglaucomatous and glaucomatous aqueous by addition of excess anti-fibronectin antibody indicated that fibronectin could account for 35% to 80% of the migratory activity of the aqueous. CONCLUSIONS: Aqueous humor contains potentially powerful chemoattractants for trabecular meshwork cells. The activity of one of these constituents, fibronectin, has been accounted for by this study. Glaucomatous aqueous appears to be as good and in some cases a better migratory stimulant than nonglaucomatous aqueous in vitro. The migratory evidence points to a trend that may help to explain cell loss in the aging meshwork and possibly some of the extra loss in primary open-angle glaucoma.

Adolescent↗
PubMed2004

Use of dual recombinant vaccinia virus vectors to assay viral glycoprotein-mediated fusion with transfection-resistant primary cell targets.

Fusion mediated by the human immunodeficiency virus type-1 (HIV-1) envelope (Env) glycoprotein and the cellular CD4/chemokine receptor complex is the first step in entry and is often analyzed in cell-cell fusion assays that require Env expression by recombinant vaccinia viruses and/or target cell transfection. Primary lymphocytes and macrophages are the principal targets for HIV-1 in vivo, but are poor substrates for transfection, and constructing recombinant vaccinia viruses expressing every novel or mutant env gene is laborious. This chapter describes a fusion assay using two recombinant vaccinia viruses that express distinct RNA polymerases suitable for transfection-resistant targets, such as primary human lymphocytes and macrophages. It also uses env genes contained in plasmid vectors, eliminating the need to construct recombinant vaccinia viruses to analyze each construct. Effector 293T cells are cotransfected with SP6-driven reporter gene and T7-driven env plasmids, then infected with recombinant vaccinia virus expressing T7 polymerase. Primary lymphocyte or macrophage targets are infected with recombinant vaccinia virus expressing SP6 polymerase. Fusion mediated by effector cell Env and endogenous CD4/coreceptors in target lymphocytes or macrophages enables SP6 polymerase-mediated reporter gene transactivation. This approach provides an efficient tool to study fusion mediated by multiple-cloned primary isolate or mutant HIV-1 env genes with the primary target cell types relevant to infection in vivo.

Cell Fusion↗
PubMed1986

Cytochemical evidence for different routes of gonadotropin-releasing hormone processing by large gonadotropes and granulosa cells.

The route and rate of internalization of GnRH were compared in studies of dispersed ovarian granulosa cells and large pituitary gonadotropes from fractions enriched by centrifugal elutriation. GnRH receptors were localized with the use of a biotinylated [D-Lys6]GnRH analog, followed by avidin gold or avidin-biotin-peroxidase complex stains. Both target cell types bound the [biotinyl-D-Lys6]GnRH (Bio-GnRH) in 1 min, and there were multiple patches of label on microvilli and coated or uncoated pits by 3 min. Quantification of the avidin-gold stains showed a significant increase in the number of labeled sites per cell profile at 3 min, followed by a decrease 15 min after exposure. No staining was seen in cells treated with medium only or with Bio-GnRH competing with a 100-fold excess of unlabeled [D-Lys6]GnRH. Internalization of the Bio-GnRH occurred during the first 3 min in both target cell types. However, the initial sites of processing appeared to be different. In granulosa cells, label was in vesicles and receptosomes (endosomes) and a few small multivesicular bodies. No stain was seen in the Golgi region for at least 15 min, at which time the stain was of low intensity. At later times (15-30 min), most of the label appeared in large multivesicular bodies. In contrast, gonadotropes exhibited labeling in Golgi complex cisternae, condensing vesicles, and immature granules as early as 3 min after exposure. Label was also seen on a subpopulation of granules in the cytoplasm and in a few multivesicular bodies. These comparative studies of two different target cells suggest that whereas the rates of internalization of GnRH are similar, the initial sites of processing may be different. Granulosa cells may degrade or separate the ligand from its receptor in multivesicular bodies. Large pituitary gonadotropes appear to use the Golgi complex route, and the processing may be associated with the formation of granules. The staining pattern correlates with early immunocytochemical studies that showed staining for GnRH on gonadotrope granules. We hypothesize that the granules may be sites for degradation of the ligand, separation of the ligand from its receptor, recycling of the receptor to the plasma membrane, or all three.

Animals↗
PubMed2004

Human and avian influenza viruses target different cell types in cultures of human airway epithelium.

The recent human infections caused by H5N1, H9N2, and H7N7 avian influenza viruses highlighted the continuous threat of new pathogenic influenza viruses emerging from a natural reservoir in birds. It is generally believed that replication of avian influenza viruses in humans is restricted by a poor fit of these viruses to cellular receptors and extracellular inhibitors in the human respiratory tract. However, detailed mechanisms of this restriction remain obscure. Here, using cultures of differentiated human airway epithelial cells, we demonstrated that influenza viruses enter the airway epithelium through specific target cells and that there were striking differences in this respect between human and avian viruses. During the course of a single-cycle infection, human viruses preferentially infected nonciliated cells, whereas avian viruses as well as the egg-adapted human virus variant with an avian virus-like receptor specificity mainly infected ciliated cells. This pattern correlated with the predominant localization of receptors for human viruses (2-6-linked sialic acids) on nonciliated cells and of receptors for avian viruses (2-3-linked sialic acids) on ciliated cells. These findings suggest that although avian influenza viruses can infect human airway epithelium, their replication may be limited by a nonoptimal cellular tropism. Our data throw light on the mechanisms of generation of pandemic viruses from their avian progenitors and open avenues for cell level-oriented studies on the replication and pathogenicity of influenza virus in humans.

Animals↗
PubMed1977

Testosterone and cellular response.

With the possible exception of male germ cells, the androgen responsiveness of all the target cell types in the mammalian body appears to be mediated by a single species of nuclear-cytosol androgen-receptor protein which is apparently specified by the X-linked Tfm locus. Although this receptor has the highest binding affinity to DHT, it can in the absence of DHT function with TES, albeit less efficiently. For the testosterone-dependent differentiation of embryonic organs, the androgen responsiveness of underlying mesenchymal cells appears to be more important than that of epithelial elements. Some of the androgen effects on apparent target cell types are through the indirect route involving the hypothalamopituitary axis.

Androgen-Insensitivity Syndrome↗
PubMed2003

Expression of cytokine signal transduction components in the postnatal mouse retina.

PURPOSE: Members of the ciliary neurotrophic factor (CNTF) family of cytokines have been shown to influence neuronal differentiation during retinal development and enhance cell survival in various retinal degeneration models. However, the cellular mechanism of CNTF signaling and the target cell types for CNTF in the developing retina remain unidentified. The purpose of this study is to characterize expression patterns of proteins involved in cytokine signal transduction in the mouse retina, thus to assess the potential responsiveness of different retinal cell types to CNTF-like cytokine signals. METHODS: The expression profiles of various cytokine signal transduction components, including receptor subunits CNTF receptor alpha (CNTFRa) and gp130, intracellular protein kinases, Jak2 and Tyk2, as well as latent transcription factors, STAT1 and STAT3, were determined by immunohistochemical staining of mouse retinal sections derived from different postnatal stages. In addition, the distribution of ERK was studied by immunofluorescent staining. RESULTS: In the neonatal retina, intense staining signals for gp130, CNTFRalpha, Jak2, Tyk2, STAT1, and STAT3 were present in the differentiated ganglion cell layer and the developing inner plexiform layer of the mouse retina. Detectable staining signals were also observed in the ventricular zone of the early postnatal mouse retina. From P5 to P10, cytokine signaling molecules also accumulated in the developing outer plexiform layer. In the adult retina, cytokine signaling components examined were localized to the ganglion cell layer, the inner nuclear layer, and the two plexiform layers. In addition, regions corresponding to the inner and/or outer segments of the photoreceptor cells showed positive staining for cytokine signaling components. In contrast, the ERK2 protein kinase was found throughout the neonatal retina. In the mature retina, ERK2 was concentrated in the ganglion cells and the inner plexiform layer, while a lesser expression of ERK2 was detected in the inner nuclear layer, the outer plexiform layers, and the outer nuclear layer. CONCLUSIONS: In the neonatal mouse retina, signaling components of the Jak-STAT pathway and ERK2 are differentially expressed. All cytokine signaling components included in this study are expressed in the differentiated inner retina as well as in cells occupying the ventricular zone, suggesting that both postmitotic neurons and proliferative progenitors may directly respond to CNTF-like cytokines during postnatal development. The distribution of cytokine signaling pathway components in the adult mouse retina is consistent with previous findings that ganglion cells and Müller glia are the primary target cell types for CNTF.

Animals↗
PubMed2005

Quantitative changes in metallothionein expression in target cell-types in the gills of turbot (Scophthalmus maximus) exposed to Cd, Cu, Zn and after a depuration treatment.

Turbot (Scophthalmus maximus) were exposed to two sublethal concentrations (1 and 10 mg metal/l) of cadmium (8.9 and 89 microM Cd), copper (15.26 and 152.6 microM Cu) and zinc (15.3 and 153 microM Zn) for 7 days, and afterwards were maintained depurating for 14 days. Immunoreactive metallothioneins (irMTs) and metal ions were localized in the branchial epithelium by immunohistochemistry (using an anti-Cod MT antibody) and autometallography (AMG), respectively. Metal ions were demonstrated by AMG as black silver deposits (BSD), mainly in mucocytes (MC) and to a lesser extent in the other branchial cell-types (respiratory cells (RC), chloride cells (CC) and basal layer cells (BLC)). Irrespective of the metal supplied, BSD were rapidly visualized in MC after 1 h of exposure. This accumulation did not increase with increasing exposure time and concentration. Metallothionein expression was mainly observed in mature CC in the interlamellar space for all exposure conditions and it was shown that all mature cells express the same amount of irMT. The number of CC exhibiting irMT in metal-exposed turbots increased following short exposure times (1 h-1 day) in the filament epithelium and following longer exposure times (1-7 days) in the secondary lamellae. Total levels of irMT in the gills (quantified by image analysis and densitometry) increased significantly in metal-exposed turbot and were related to increased exposure times. It can be concluded that the total content of irMT in the gills of metal-exposed turbot is governed by changes in the number of mature CC expressing the protein. The quantification of total irMT in branchial CC can be considered as a reliable biomarker of metal exposure since reflects changes in metal bioavailability. This approach based on cell-selective immunohistochemistry can be simplified by only quantifying the number of mature CC. In addition, the dramatic increase of CC in the gills that produces epithelial thickening of the FE enhances migration of CC up to the edge of the SL and provokes the hypertrophy and fusion of secondary lamellae can be considered as unspecific biomarkers of effect indicating disturbed health in turbot.

Analysis of Variance↗
PubMed2003

Change of porcine circovirus 2 target cells in pigs during development from fetal to early postnatal life.

Change of porcine circovirus 2 (PCV2) target cells during development from fetal to postnatal life in pigs was examined. PCV2 inoculation was performed in fetuses in utero at either 57, 75 or 92 gestational days and in piglets at 1 day of age. Twenty-one days after virus inoculation, PCV2-infected cells in the heart, lungs, liver, spleen and inguinal lymph nodes were localized and immuno-phenotyped by double-immunofluorescence labeling using different cell markers and PCV2-antibodies. During fetal life, viral antigens were detected in cardiomyocytes, hepatocytes and macrophages and infected cell numbers decreased with increasing fetal age at inoculation. The heart contained the highest number of infected cells and cardiomyocytes were the main target cell. Postnatally, macrophages were the only target cell type in different organs and infected cell numbers were similar to those of fetuses inoculated at 92 days of gestation. One piglet showed exceptionally high number of infected cells in different organs with values 13-513-fold higher compared to littermates. In this piglet, the majority of infected cells in lymphoid tissues could not be typed. This study reveals that PCV2 target cells change from cardiomyocytes, hepatocytes and macrophages during fetal life to only macrophages postnatally.

Animals↗
PubMed2001

Targeting of high-capacity adenoviral vectors.

High-capacity adenoviral (HC-Ad) vectors contain only the noncoding termini of the viral genome, can deliver large DNA fragments of up to 36 kb into target cells, and feature reduced toxicity and prolonged transgene expression in vivo. To enhance the potential of HC-Ad vectors to transduce specific cell types, we constructed a versatile infectious new helper virus plasmid that can be used readily to introduce peptide ligands into the HI loop of the fiber knob domain of Ad5-based HC-Ad vectors. Helper viruses with a 6x-His epitope or Arg-Gly-Asp (RGD) peptide insertion retained the full infectivity of the wild-type helper virus. The RGD-modified helper virus was used for production of a capsid-modified HC-Ad vector expressing beta-galactosidase. The RGD HC-Ad vector transduced the ovarian carcinoma cell lines SK-OV-3 and OVCAR-3 with 4- to 20-fold higher efficiency, compared to unmodified vectors. Transduction of both primary vascular smooth muscle cells as well as primary human endothelial cells was increased up to 15-fold with the RGD-modified vector. Competition experiments with recombinant knob protein and different RGD peptides indicated that the RGD-mediated transduction was Coxsackie and Adenovirus receptor (CAR)-independent and involved integrin alpha(v)beta(5). The use of fiber-modified helper viruses in the last amplification step of HC-Ad vector production allows for convenient and efficient targeting of these vectors towards different cell types. Targeting strategies will increase the spectrum of applications for HC-Ad vectors and will further add to their safety.

Adenoviridae↗
PubMed2025

Transcript-guided targeted cell enrichment for scalable single-nucleus RNA sequencing.

Large-scale single-cell atlases have revealed many aging- and disease-associated cell types, yet these populations are often underrepresented in heterogeneous tissues, limiting detailed molecular analyses. To address this, we developed EnrichSci-a scalable, microfluidics-free platform that combines hybridization chain reaction RNA fluorescence in situ hybridization (FISH) with combinatorial indexing to profile single-nucleus transcriptomes of target cell types with full gene-body coverage. Applied to oligodendrocytes in the aging mouse brain, EnrichSci uncovered aging-associated molecular dynamics across distinct oligodendrocyte subtypes, revealing both shared and subtype-specific gene expression changes. Additionally, we identified aging-associated exon-level signatures missed by conventional gene-level analyses, highlighting post-transcriptional regulation as a critical dimension of cell-state dynamics in aging. By coupling transcript-guided enrichment with a scalable sequencing workflow, EnrichSci provides a versatile approach to decode dynamic regulatory landscapes in diverse cell types from complex tissues.

Animals↗
PubMed1978

Antibody-dependent cell-mediated cytotoxicity to herpes simplex virus type 2 infected target cells in the course of cervical carcinoma.

The antibody-dependent cell-mediated cytotoxicity (ADCC) assay was used as a sensitive instrument to study if specific antibodies acting synergistically with lymphocytes to induce target cell destruction, could be detected in sera from patients with cervical carcinoma. Significant cytotoxicity to herpes simplex type 2 (HSV-2) infected target cells was observed in sera of 17 out of 23 (74%) surviving patients with cervical cancer who were followed with at least two serum samples. A rise in cytolytic activity was also observed in 11 out of 23 cases. Seven out of 14 tumor-bearing cervical cancer patients with progressing cervical lesions showed significant cytotoxicity, but four of the seven had lost activity in later sera. When 43 patients surviving the observation period of 24--60 months with no signs of residual tumors were compared with 23 severely ill cervical cancer patients, patients with less advanced cancer had significantly higher cytolytic activity.

Animals↗