Dariusz Leszczynski

Impact of class II major histocompatibility complex antigen expression on the immunogenic potential of isolated rat vascular endothelial cells.

We have investigated the immunogenic potential of rat heart vascular endothelial cells by their ability to induce an accelerated rejection of a relevant heart allograft, and related the immunogenic potential to the expression of class II major histocompatibility complex (MHC) antigens on the endothelial cell surface. Only 12% of freshly isolated rat vascular endothelial cells express class II antigens in serum-free medium, and the level of expression is low as judged by immunoperoxidase staining and/or the ability of endothelial cells to bind staphylococci to the cell surface after treatment with monoclonal antibodies to the class II molecule. On the other hand, 99% of the endothelial cells under the same conditions express class I, and the level of expression is high. The class II antigen expression of vascular endothelial cells can be upregulated to more than 98% by recombinant gamma-interferon in vitro—and, con

Differences in the carcinogenic evaluation of glyphosate between the International Agency for Research on Cancer (IARC) and the European Food Safety Authority (EFSA)

The International Agency for Research on Cancer (IARC) Monographs Programme identifies chemicals, drugs, mixtures, occupational exposures, lifestyles and personal habits, and physical and biological agents that cause cancer in humans and has evaluated about 1000 agents since 1971. Monographs are written by ad hoc Working Groups (WGs) of international scientific experts over a period of about 12 months ending in an eight-day meeting. The WG evaluates all of the publicly available scientific information on each substance and, through a transparent and rigorous process,1 decides on the degree to which the scientific evidence supports that substances potential to cause or not cause cancer in humans. For Monograph 112,2 17 expert scientists evaluated the carcinogenic hazard for four insecticides and the herbicide glyphosate.3 The WG concluded that the data for glyphosate meet the criteria for classification as a probable human carcinogen . The European Food Safety Authority (EFSA) is the primary agency of the European Union for risk assessments regarding food safety. In October 2015, EFSA reported4 on their evaluation of the Renewal Assessment Report5 (RAR) for glyphosate that was prepared by the Rapporteur Member State, the German Federal Institute for Risk Assessment (BfR). EFSA concluded that ‘glyphosate is unlikely to pose a carcinogenic hazard to humans and the evidence does not support classification with regard to its carcinogenic potential’. Addendum 1 (the BfR Addendum) of the RAR5 discusses the scientific rationale for differing from the IARC WG conclusion. Serious flaws in the scientific evaluation in the RAR incorrectly characterise the potential for a carcinogenic hazard from exposure to glyphosate. Since the RAR is the basis for the European Food Safety Agency (EFSA) conclusion,4 it is critical that these shortcomings are corrected. EFSA concluded ‘that there is very limited evidence for an association between glyphosate-based formulations …

Protein kinase C-regulated production of prostacyclin by rat endothelium is increased in the presence of lipoxin A4

Prostacyclin is generated by cultured rat endothelial cells. Compound blocking activity of protein kinase C and cyclic nucleotide‐dependent protein kinases (H7) and compound blocking interaction between Ca2+ and calmodulin (W7) diminish generation of prostacyclin in rat endothelial cells. These compounds give a synergistic effect when they are introduced to the endothelial cell cultures simultaneously. Compound HA1004, an inhibitor ofcAMP‐ and cGMP‐dependent protein kinases has no effect on prostacyclin generation. Lipoxin A4, a potent direct stimulator of protein kinase C, rapidly induces prostacyclin generation in rat endothelium in a dose‐ and time‐dependent fashion. Lipoxin A4‐induced generation of prostacyclin can be inhibited by H7 and W7 but not by HA1004. Lipoxin B4 has no significant effect on prostacyclin generation in rat endothelium. In conclusion, our results demonstrate that generation of prostacyclin by rat endothelial cells is regulated via a pathway involving protein kinase C and Ca2+.

Radiation proteomics: A brief overview

Acute biological effects caused by the exposure to high doses of radiation, either ionizing or nonionizing, are relatively well‐known but the delayed effects, occurring decades after exposure, are difficult to predict. The knowledge of the acute and delayed effects of the low doses of ionizing radiation (e.g. bystander effect) or nonionizing radiation (e.g. radiation emitted by wireless communication devices) is not yet reliably established. Often the acute effects of low doses are small and difficult to discover and replicate in scientific studies. Chronic effects of prolonged exposures to low‐dose radiation for decades are virtually unknown and often not possible to predict on the basis of the knowledge gained from acute exposures to high doses of radiation. Physiological significance of the biological effects induced by low doses of radiation is not known. The same lack of predictability of outcomes applies to the delayed effects of high‐dose radiation exposures. Proteomics, supplemented with other “omics” techniques, might be the best way forward to find out the target molecules of radiation, the biomarkers of radiation exposure and the physiological and health significance of the acute and delayed biological effects caused by the exposures to high‐ and low‐dose radiation. However, the currently available database of radiation effects on proteomes is far too small to be useful in formulation of new hypotheses concerning health consequences of radiation exposures.

Leukotriene B4 increases the lymphocyte binding to endothelial cells.

Leukotrienes are potent mediators of local microvascular environment. Leukotriene B4 treatment of cultured endothelium increases the binding of lymphocytes to endothelial cell monolayers within minutes. This effect is dose‐dependent and reversible upon removal of the leukotriene. Pretreatment of lymphocytes slightly decreases the binding and pretreatment of both lymphocytes and endothelium with leukotriene B4 prior to the adherence assay did not alter the binding. These results suggest that leukotriene B4 regulates exclusively the vascular side, but not the white cell side of this interaction.

Localization and Turnover Rate of Rat Renal ‘Dendritic’ Cells

The turnover rate of rat renal dendritic cells was analysed by irradiation and bone marrow transplantation and by visualizing the dendritic cells in frozen sections of renal tissue via double indirect immunofluorescence and peroxidase assays. Interstitial dendritic cells disappear from rat renal tissue shortly after irradiation; after 7 days they can be seen again. However, since the renal proximal tubular cells also simultaneously lose and regain their class II contents, we assume that irradiation has resulted in the disappearance of the Ia antigen, not the dendritic cells proper. After allogeneic bone marrow transplantation, bone marrow‐derived dendritic cells appear in renal tissue between days 7 and 10, indicating a relatively fast turnover rate of these cells in vivo.

Effect of granulocyte-macrophage colony stimulating factor on endothelial antigenicity

We have investigated if recombinant granulocyte-macrophage colony stimulating factor (GM-CSF), alone or in concert with recombinant gamma interferon, affects the endothelial cell expression of class I major histocompatibility complex antigen. Results obtained show that the GM-CSF increases class I expression on the endothelial cell in a time- and dose-dependent manner. The effect of interferon gamma on class I expression is diminished in the presence of GM-CSF, cAMP, or prostaglandin E2, but is increased in the presence of cGMP. N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), an inhibitor of cAMP- and cGMP-dependent protein kinases, abolished GM-CSF-induced class I expression, while indomethacin increased it. When added to the endothelial cell cultures together with interferon gamma GM-CSF, HA 1004 as well as indomethacin abolished the inhibitory effect of GM-CSF on interferon gamma-induced class I expression. The results suggest that GM-CSF diminishes effect of interferon gamma on class I major histocompatibility complex expression on the endothelial cell by inducing production of rostacyclin. This, in turn, induces cAMP as a second messenger, which then leads to the events inhibiting expression of class I major histocompatibility complex antigen.

Eicosanoids are regulatory molecules in γ‐interferon‐induced endothelial antigenicity and adherence for leucocytes

When the endothelial cells (ECs) were stimulated with γ‐interferon (gIFN) in the presence of methylprednisolone (MP) or prostaglandin E2 (PGE2), MP enhanced gIFN‐induced Ia antigen expression, whereas PGE2 inhibited it. On the other hand, while PGE2 had no effect on leucocyte binding to ECs, MP entirely inhibited it. By using selective inhibitors of the cyclo‐oxygenase pathway (indomethacin, IM) and the 5‐lipoxygenase pathway (L651.392), we found that addition of IM to gIFN‐stimulated ECs enhanced Ia expression but had no effect on leucocyte adherence to ECs. Instead, addition of L651.392 to gIFN‐stimulated ECs partially reduced leucocyte adherence to ECs but had no effect on Ia expression. Pretreatment of the ECs or leucocytes or both with monoclonal anti‐class II antibody, had no effect on gIFN‐induced leucocyte binding to ECs. These findings suggest that gIFN‐induced endothelial cell antigenicity and leucocyte adherence are regulated independently of each other by different molecular pathways. Moreover, arachidonic acid metabolites appear to be the regulatory molecules in gIFN effects on the ECs.

Turnover of Dendritic Cells in Rat Heart

Taking advantage of the high class II (I‐region‐associaled) antigen content of the tissue dendritic cells and monoclonal antibodies directed in ihc backbone determinants of the bone marrow donor strain, we have investigated the location and analysed the turnover rate of tissue dendritic cells in rat heart. Low numbers of class II‐expressing, factor VII‐negative, nonphagocytic cells with dendritic appearance were observed between the heart muscle fibres. After irradiation with 960 rad, these cells were no longer visible but they reappeared (in lower numbers) on day 20. indicating that they are relatively radioresistant but the antigen expression is radiosensitive. Transplantation of allogeneic bone marrow demonstrated that similar cells appeared from the transplanted bone marrow on day 10, and that these cells populated the heart at a maximum of 20‐25 days after transplantation. This indicates a relatively rapid turnover rate, comparable with the turnover rate of dendritic cells in rat kidney and mouse lymphoid tissue.

Evidence that thymectomized, bone marrow-reconstituted rats do not reject their allografts.

We have investigated the reasons why thymectomized, bone marrow—reconstituted (B) rats do not reject their allografts, by comparing the structure of inflammation and functions of inflammatory cells in nonrejecting allografts to rejecting allografts in normal control recipients. The results demonstrate that B recipients mount a specific cellular response towards the graft. The response in B recipients differs from that in normal controls by a smaller intensity of inflammation, fewer blast cells, and activated mononuclear phagocytes in the inflammatory infiltrate, as well as a delay in the appearance of specific donor-directed lytic activity in the graft. B rats also have fewer blast cells and an inverted CD4/8 ratio in the spleen. There is no obvious absence of any given cell type or cellular function in the graft inflammatory infiltrate. In light of these results no cell type responsible for allograft nonrejection can be pinpointed.