Publisher Summary Early studies pointed to the directions for subsequent investigations and there has been continuing identification of other circulating autoantibodies, characterization of their respective cellular antigens, and demonstration of the relationship between autoantibodies and clinical syndromes. The antibodies identified in these studies have been used extensively by investigators in molecular and cell biology as powerful probes for understanding the precursor messenger RNA (mRNA) splicing. Autoantibodies designated SS-A or Ro and SS-B or La were identified in patients with Sjogren's syndrome (SS) and SLE, and have been shown to target intracellular proteins that may be involved with regulation of RNA polymerase III function. Investigators in the field had long perceived that these spontaneously occurring auto-antibodies in human disease would turn out to be useful reagents in cell biology. It was also appreciated that it was necessary to characterize the molecular nature of the auto-antigens so that in addition to the immunochemical properties, their structure and function might be determined. One of the purposes of this chapter is to show that the new molecular biology of cellular antigens and auto-antibodies could now be providing insights into comprehending some features of autoimmunity. In addition, there appears to be a need for the synthesis of the wealth of information that has accumulated and an evaluation of what it might signify.

Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology

Proliferating cell nuclear antigen (PCNA) is a 36 kD nuclear protein associated with the cell cycle A monoclonal antibody, PC10, that recognizes a fixation and processing resistant epitope has been used to investigate its tissue distribution Nuclear PCNA immunoreactivity is found in the proliferative compartment of normal tissues PCNA immunoreactivity is induced in lectin stimulated peripheral blood mononuclear cells in parallel with bromodeoxyuridine incorporation and the number of cells with PCNA immunoreactivity is reduced by induction of differentiation in HL60 cells In non-Hodgkin's lymphomas a linear relation between Ki67 and PCNA staining was demonstrated These data suggest that in normal tissues and lymphoid neoplasms, PCNA immunolocalization can be used as an index of cell proliferation However, in some forms of neoplasia, including breast and gastric cancer and in vitro cell lines, the simple relation between PCNA expression and cell proliferation is lost In some breast and pancreatic tumours there is apparent deregulation of PCNA with increased expression in tissues adjacent to the tumours The over-expression in some tumours and in adjacent morphologically normal tissue may represent autocrine or paracrine growth factor influence on PCNA gene expression

Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: An index of cell proliferation with evidence of deregulated expression in some, neoplasms

The proliferative compartment of stratified squamous epithelia consists of stem and transient amplifying (TA) keratinocytes. Some polypeptides are more abundant in putative epidermal stem cells than in TA cells, but no polypeptide confined to the stem cells has yet been identified. Here we show that the p63 transcription factor, a p53 homologue essential for regenerative proliferation in epithelial development, distinguishes human keratinocyte stem cells from their TA progeny. Within the cornea, nuclear p63 is expressed by the basal cells of the limbal epithelium, but not by TA cells covering the corneal surface. Human keratinocyte stem and TA cells when isolated in culture give rise to holoclones and paraclones, respectively. We show by clonal analysis that p63 is abundantly expressed by epidermal and limbal holoclones, but is undetectable in paraclones. TA keratinocytes, immediately after their withdrawal from the stem cell compartment (meroclones), have greatly reduced p63, even though they possess very appreciable proliferative capacity. Clonal evolution (i.e., generation of TA cells from precursor stem cells) is promoted by the sigma isoform of the 14-3-3 family of proteins. Keratinocytes whose 14-3-3σ has been down-regulated remain in the stem cell compartment and maintain p63 during serial cultivation. The identification of p63 as a keratinocyte stem cell marker will be of practical importance for the clinical application of epithelial cultures in cell therapy as well as for studies on epithelial tumorigenesis.

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p63 identifies keratinocyte stem cells

https://www.cell.com/cell/pdf/0092-8674(92)90518-H.pdf

D Type Cyclins Associate with Multiple Protein Kinases and the DNA Replication and Repair Factor PCNA

Pulse-chase experiments have revealed that cyclin, the auxiliary protein of DNA polymerase-delta, is stable during the transition from growth to quiescence in 3T3 cells. Immunoblotting together with immunofluorescence analysis has shown that the amount of cyclin after 24 h of quiescence is 30-40% of that of growing cells and that it presents a nucleoplasmic staining. Immunofluorescence studies show the existence of two populations of cyclin during the S phase, one that is nucleoplasmic as in quiescent cells and is easily extracted by detergent, and another that is associated to specific nuclear structures. By using antibromodeoxyuridine immunofluorescence to detect the sites of DNA synthesis, it was shown that the staining patterns of the replicon clusters and their order of appearance throughout the S phase are identical to those observed for cyclin. Two-dimensional gel analysis of Triton-extracted cells show that 20-30% of cyclin remains associated with the replicon clusters. This population of cyclin could not be released from the nucleus using high-salt extractions. This demonstrates that cyclin is tightly associated to the sites of DNA replication and that it must have a fundamental role in DNA synthesis in eukaryotic cells.

/pdf/existence-of-two-populations-of-cyclin-proliferating-cell-3cz7n97r9g.pdf

Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites.

Identification of the cellular proteins whose expression is regulated during the cell cycle in normal cells is essential for understanding the mechanisms involved in the control of cell proliferation. A nuclear protein called cyclin of relative molecular mass 36,000 (Mr 36K), whose synthesis correlates with the proliferative state of the cell, has been identified in several cell types of human, mouse, hamster and avian origin. The rate of cyclin synthesis is very low in quiescent cells and increases several fold after serum stimulation shortly before DNA synthesis. Immunofluorescence and autoradiography studies have shown that the nuclear staining patterns of cyclin during S phase have a sequential order of appearance and a clear correlation can be found between DNA synthesis and cyclin positive nuclei. The proliferating cell nuclear antigen (PCNA) and cyclin have many common properties and it has been shown that these two are identical. Recently a protein which is required by DNA polymerase-delta for its catalytic activity with templates having low primer/template ratios has been isolated from calf thymus. We report here that cyclin and the auxiliary protein of DNA polymerase-delta are identical.

Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta.

Snakebite is a neglected tropical disease that causes high rates of global mortality and morbidity. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Despite polyclonal antibody-based antivenoms being the mainstay life-saving therapy for snakebite, they are associated with limited cross-snake species efficacy, as there is often extensive toxin variation between snake venoms, including those used as immunogens for antivenom production. This restricts the therapeutic utility of any antivenom to certain geographical regions. In this study, we explored the feasibility of using recombinantly expressed toxins as immunogens to stimulate focused, pathology-specific, antibodies in order to broadly counteract specific toxins associated with snakebite envenoming. Three snake venom serine proteases (SVSP) toxins, sourced from geographically diverse and medically important viper snake venoms, were successfully expressed in HEK293F mammalian cells and used for murine immunisation. Analyses of the resulting antibody responses revealed that ancrod and RVV-V stimulated the strongest immune responses, and that experimental antivenoms directed against these recombinant SVSP toxins, and a mixture of the three different immunogens, extensively recognised and exhibited immunological binding towards a variety of native snake venoms. While the experimental antivenoms showed some reduction in abnormal clotting parameters stimulated by the toxin immunogens and crude venom, specifically reducing the depletion of fibrinogen levels and prolongation of prothrombin times, fibrinogen degradation experiments revealed that they broadly protected against venom- and toxin-induced fibrinogenolytic functional activities. Overall, our findings further strengthen the case for the use of recombinant venom toxins as supplemental immunogens to stimulate focused and desirable antibody responses capable of neutralising venom-induced pathological effects, and therefore potentially circumventing some of the limitations associated with current snakebite therapies.

/pdf/exploring-the-utility-of-recombinant-snake-venom-serine-1eivg9xt.pdf

Exploring the Utility of Recombinant Snake Venom Serine Protease Toxins as Immunogens for Generating Experimental Snakebite Antivenoms

Snakebite is a neglected tropical disease that causes high rates of global mortality and morbidity. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Despite polyclonal antibody-based antivenoms being the mainstay life-saving therapy for snakebite, they are associated with limited cross-snake species efficacy, as there is often extensive toxin variation between snake venoms, including those used as immunogens for antivenom production. This restricts the therapeutic utility of any antivenom to certain geographical regions. In this study, we explored the feasibility of using recombinantly expressed toxins as immunogens to stimulate focused, pathology-specific, antibodies to broadly counteract specific toxins associated with snakebite envenoming. Three snake venom serine proteases (SVSP) toxins, sourced from geographically diverse and medically important viper snake venoms were successfully expressed in HEK293F mammalian cells and used for murine immunisation. Analyses of the resulting antibody responses revealed that ancrod and RVV-V stimulated the strongest immune responses, and that experimental antivenoms directed against these recombinant SVSP toxins, and a mixture of the three different immunogens, extensively recognised and exhibited immunological binding towards a variety of native snake venoms. While the experimental antivenoms showed some reduction in abnormal clotting parameters stimulated by the toxin immunogens and crude venom, specifically reducing the depletion of fibrinogen levels and prolongation of prothrombin times, fibrinogen degradation experiments revealed they broadly protected against venom- and toxin-induced fibrinogenolytic functional activities. Overall, our findings further strengthen the case for the use of recombinant venom toxins as supplemental immunogens to stimulate focused and desirable antibody responses capable of neutralising venom-induced pathological effects, and therefore potentially circumventing some of the limitations associated with current snakebite therapies.

Exploring the utility of recombinantly expressed snake venom serine protease toxins as immunogens for generating experimental snakebite antivenoms

Many clinically important viruses, including influenza A, SARS-CoV-1, adenoviruses, and DNA tumour viruses such as Kaposi’s sarcoma herpesvirus use multivalent binding to sialic acid (SA) to infect cells, or to modulate immune responses through interactions with sialylated attachment factors that facilitate virus infectivity and/or host survival. Molecular scaffolds rich in SA that bind virions with high avidity may therefore be useful as anti-infective medicines. We generated a panel of 12 of these molecules using fragment-crystallisable scaffolds in CHO-S cells that are rich in SA. The viral surface protein of influenza A virus (IAV), haemagglutinin, binds SA for cell entry, and so we tested the activity of these compounds against this virus. Two of the sialylated Fc-molecules reduced IAV haemagglutination activity by up to 64-fold. However, the same molecules enhanced virus infectivity of A549 cultured cells. To explain the increased viral titres, we postulated that sialylated Fcs may be anti-inflammatory. However, sialylated Fc multimers were instead pro-inflammatory; they induced chemokine/cytokine responses from differentiated human THP-1 derived macrophages, including raised IL-8 and MIP-1α/β, that mimicked responses driven by universal type I interferon. Steric targeting of SA to block virus entry may therefore have unexpected effects in target cells that currently preclude their use for medical intervention.

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Synthetically engineered IgG1 antibody Fc fragments presenting influenza A virus receptor sialic acid inhibit viral haemagglutination activity, but enhance virus replication in cultured A549 cells

Patricia A. Blundell

Papers

Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta.

Exploring the Utility of Recombinant Snake Venom Serine Protease Toxins as Immunogens for Generating Experimental Snakebite Antivenoms

Exploring the utility of recombinantly expressed snake venom serine protease toxins as immunogens for generating experimental snakebite antivenoms

Synthetically engineered IgG1 antibody Fc fragments presenting influenza A virus receptor sialic acid inhibit viral haemagglutination activity, but enhance virus replication in cultured A549 cells