Diane M Spencer

The Effects of Heparins on the Liver: Application of Mechanistic Serum Biomarkers in a Randomized Study in Healthy Volunteers

Heparins have been reported to cause elevations in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) but have not been associated with clinically significant liver injury. The mechanisms underlying these benign laboratory abnormalities are unknown. Forty‐eight healthy men were randomized to receive subcutaneous injections of unfractionated heparin (UFH; 150 U/kg), enoxaparin sodium (1 mg/kg), dalteparin sodium (120 IU/kg), or adomiparin sodium (125 IU/kg; a novel heparin) every 12 h for 4.5 days. Asymptomatic elevations in serum ALT or AST were observed in >90% of the subjects. Elevations were also observed in the levels of serum sorbitol dehydrogenase (SDH), glutamate dehydrogenase (GLDH), miR‐122, high‐mobility group box‐1 protein (including the acetylated form), full‐length keratin 18, and DNA. Keratin 18 fragments, which are apoptosis biomarkers, were not detected. Biomarker profiles did not differ significantly across heparin treatments. We conclude that heparins as a class cause self‐limited and mild hepatocyte necrosis with secondary activation of an innate immune response.

The Expression of HMGB1 on Microparticles from Jurkat and HL-60 Cells Undergoing Apoptosis in vitro

HMGB1 is a highly conserved nuclear protein that displays important biological activities inside as well as outside the cell and serves as a prototypic alarmin to activate innate immunity. The translocation of HMGB1 from inside to outside the cell occurs with cell activation as well as cell death, including apoptosis. Apoptosis is also a setting for the release of cellular microparticles (MPs), which are small membrane‐bound vesicles that represent an important source of extracellular nuclear molecules. To investigate whether HMGB1 released from cells during apoptosis is also present on MPs, we determined the presence of HMGB1 on particles released from Jurkat and HL‐60 cells induced to undergo apoptosis in vitro by treatment with either etoposide or staurosporine; MPs released from cells undergoing necrosis by freeze–thaw were also characterized. As shown by both Western blot analysis and flow cytometry, MPs from apoptotic cells contain HMGB1, with binding by antibodies indicating an accessible location in the particle structure. These results indicate that HMGB1, like other nuclear molecules, can translocate into MPs during apoptosis and demonstrate another biochemical form of this molecule that may be immunologically active.

Assay variation in the detection of antinuclear antibodies in the sera of patients with established SLE

Objective The expression of antinuclear antibodies (ANA) is considered almost constant in systemic lupus erythematosus (SLE), although recent experience has suggested that many subjects with SLE considered for clinical trials are ANA negative at screening. The objective of this study is to determine whether assay variation can influence ANA detection in patients with established SLE. Methods Sera from 103 patients with established SLE were tested using three different immunofluorescence assays (IFA) for ANA determination. ANA determinations were also performed by an ELISA and bead-based multiplex assay. Results With IFA kits, the frequency of ANA negativity varied from 5 to 23 of 103 samples (4.9%–22.3%). The ELISA and multiplex assays showed that 12 (11.7%) and 14 (13.6%) samples were negative, respectively. Samples positive in all assays differed from those with discordant assay results in the frequency of historical anti–double-stranded DNA positivity and low complement levels at the time of blood sampling. Discussion These findings indicate that ANA negativity occurs in patients with established SLE although the frequency varies depending on the assay kit. Given the range of negativity with well-validated assays, these findings raise questions about whether ANA positivity should be employed to determine eligibility for clinical trials.

Effects of Progesterone and Estradiol Sex Hormones on the Release of Microparticles by RAW 264.7 Macrophages Stimulated by Poly(I:C)

ABSTRACT Microparticles (MPs) are small membrane-bound vesicles that display proinflammatory and prothrombotic properties. These particles can be released by macrophages stimulated by ligands of the Toll-like receptors (TLRs) in a process that depends on nitric oxide (NO) production. Since sex hormones can modulate macrophage responses, we investigated the effects of progesterone and estradiol on macrophage particle release in vitro, comparing the responses with those induced by the glucocorticoid dexamethasone. As a model system for particle release, RAW 264.7 cells were stimulated in vitro with poly(I:C), a ligand of TLR3. Microparticles were measured by flow cytometry, while NO was measured by the Griess reaction. As the results of these studies showed, progesterone but not estradiol can block particle release by RAW264.7 cells treated with poly(I:C); dexamethasone was also active. Furthermore, while progesterone and dexamethasone inhibited NO production under the same culture conditions, neither agent blocked the production of particles stimulated by the NO donors dipropylenetriamine NONOate {(z)-1-[N-(3-aminopropyl)-N-(3-ammoniopropyl)amino] diazen-1-ium-1,2-diolate} and (z)-1-[(2-aminoethyl)-N-(2-ammonioethyl)amino] diazen-1-ium-1,2-diolate. Studies using RU486 to assess the role of hormone receptors indicated that while this agent blocked the inhibition of particle and NO production by dexamethasone, it did not affect the inhibition by progesterone. Together, these results indicate that progesterone but not estradiol can inhibit particle release by stimulated macrophages and suggest a mechanism that may contribute to the immunomodulatory effects of this sex hormone.

The properties of microparticles from RAW 264.7 macrophage cells undergoing in vitro activation or apoptosis.

Microparticles (MPs) are small, membrane-bound vesicles that arise from dead and dying cells, and display pro-inflammatory and pro-thrombotic activity. As shown previously, the RAW 264.7 murine macrophage cell line can release MPs following stimulation with LPS or polyinosinic:polycytidylic acid [poly (I:C)], ligands of TLR4 and TLR3 respectively. To determine the relationship of these MPs to those released during apoptosis, the nucleic acid content of MPs from cultures stimulated with LPS or poly (I:C) was compared with the nucleic acid content of MPs from untreated cells or cells induced to undergo apoptosis by treatment with etoposide or staurosporine (STS). As results of these studies showed, MPs from activated, apoptotic and untreated cells had features in common, as demonstrated by binding of the nucleic acid dyes SYTO 13 and propidium iodide; molecular mass of DNA; and binding of monoclonal anti-DNA and anti-nucleosome Abs. While MPs from the different culture conditions all contained ribosomal RNA, ribosomal RNA from MPs from STS-treated cells showed cleavage and degradation. Taken together, these studies indicate that the nucleic acid content of MPs from activated and apoptotic cells have important similarities, suggesting that events during TLR activation may lead to apoptosis and subsequent MP release.

Response to: ‘Lack of standardization of ANA and implications for drug development and precision medicine’ by Mahler

We appreciate Dr Mahler’s1 comments on our paper, ‘Assay variation in the detection of antinuclear antibodies in the sera of patients with established SLE’,2 by Pisetsky and colleagues, and the thoughtful discussion on the technical issues that affect the testing for antinuclear antibodies (ANA) by immunofluorescence assays with HEp-2 cells (IFA). We agree that our sample size was relatively small and that we did not present confidence limits on the frequency of positive responses. Rather than attempting to revalidate the assays, we …

The release of microparticles and mitochondria from RAW 264.7 murine macrophage cells undergoing necroptotic cell death in vitro

Abstract Microparticles (MPs) are small membrane‐bound vesicles released from activated or dying cells. As shown previously, LPS stimulation of the RAW 264.7 macrophage cell line can induce MP release, with the caspase inhibitor Z‐VAD increasing the extent of this process. Since combined treatment of cells with LPS and Z‐VAD can induce necroptosis, we explored particle release during this form of cell death using flow cytometry to assess particle size, binding of annexin V and staining for DNA with propidium iodide (PI) and SYTO 13. The role of necroptosis was assessed by determining the effects of necrostatin, an inhibitor of RIP1, a kinase regulating this form of cell death. These studies demonstrated that, during necroptosis, RAW 264.7 cells release MPs that resemble those released from cells treated with staurosporine to induce apoptosis. The particles contained DNA as determined by binding of PI and SYTO 13, with PCR analysis demonstrating both chromosomal and mitochondrial DNA. The presence of mitochondria in the MP preparations was demonstrated by staining with MitoTracker Green. Flow cytometry indicated that purified mitochondria have properties of MPs. Together, these studies indicate that cells undergoing necroptosis can release MPs and that mitochondria can be components of MP preparations. HighlightsMacrophages undergoing necroptotic cell death release microparticles.Microparticles from necroptotic and apoptotic macrophages have similar properties.Microparticle preparations contain both nuclear and mitochondrial DNA.Microparticle preparations contain mitochondria staining with MitoTracker Green.Purified mitochondria behave as microparticles by flow cytometry. Abbreviations: MP: microparticle; LPS: lipopolysaccharide; NO: nitric oxide; HMGB1: high mobility group box 1; RIP1: receptor interacting serine/threonine kinase 1; LDH: lactate dehydrogenase; MTG: MitoTracker green; Z‐VAD: Z‐VAD‐FMK; TFAM: mitochondrial transcription factor A; TNF‐&agr;: tumor necrosis factor &agr;.

Response to: ‘Variation in antinuclear antibody detection by automated indirect immunofluorescence analysis’ by van Hoovels et al

We thank van Hoovels et al 1 for their comments on our article2 on the variability of antinuclear antibody (ANA) determinations; other letters also discussed ANA testing issues.3 4 Meroni et al noted the value of distinguishing staining patterns by immunofluorescence assays (IFAs).3 …