A pharmacological approach to thromboxane receptor antagonism

Thromboxane A2 (TxA2) appears to be an important mediator of ischemia and hypoxia. Despite its short half-life and the fact that it may not circulate in the blood until its values become quite high, TxA2 contributes to the pathogenesis of cardiopulmonary diseases (e.g., sudden death, myocardial ischemia, circulatory shock). It does so because it propagates its own formation by activating platelets and constricting blood vessels, thus activating more TxA2 and trapping it locally within an ischemic or hypoxic region. TxA2 concentrations in the extracellular fluid of lymph of ischemic regions may be much higher than that occurring in nonischemic, normally perfused regions. Specific and potent Tx receptor antagonists (TxRA) have recently become available for study. The TxRA are useful tools in the study of the pathophysiology of Tx-dependent disease processes and have been found to be effective in a variety of ischemic disorders including circulatory shock, myocardial ischemia, and sudden cardiopulmonary death. Moreover, inasmuch as early work indicates that these agents are both safe and effective in humans, Tx receptor antagonists may be employed as therapeutic agents in several cardiovascular disease states. Further investigation is necessary to clarify the role of TxRA as therapeutic agents.     

Quantitation of the effect of L-carnitine on the levels of acid-soluble short-chain acyl-CoA and CoASH in rat heart and liver mitochondria

The steady state levels of mitochondrial acyl-CoAs produced during the oxidation of pyruvate, alpha-ketoisovalerate, alpha-ketoisocaproate, and octanoate during state 3 and state 4 respiration by rat heart and liver mitochondria were determined. Addition of carnitine lowered the amounts of individual short-chain acyl-CoAs and increased CoASH in a manner that was both tissue- and substrate-dependent. The largest effects were on acetyl-CoA derived from pyruvate in heart mitochondria using either state 3 or state 4 oxidative conditions. Carnitine greatly reduced the amounts of propionyl-CoA derived from alpha-ketoisovalerate, while smaller effects were obtained on the branched-chain acyl-CoA levels, consistent with the latter acyl moieties being poorer substrates for carnitine acetyltransferase and also poorer substrates for the carnitine/acylcarnitine translocase. The levels of acetyl-CoA in heart and liver mitochondria oxidizing octanoate during state 3 respiration were lower than those obtained with pyruvate. The rate of acetylcarnitine efflux from heart mitochondria during state 3 (with pyruvate or octanoate as substrate, in the presence or absence of malate with 0.2 mM carnitine) shows a linear response to the acetyl-CoA/CoASH ratio generated in the absence of carnitine. This relationship is different for liver mitochondria. These data demonstrate that carnitine can modulate the aliphatic short-chain acyl-CoA/CoA ratio in heart and liver mitochondria and indicate that the degree of modulation varies with the aliphatic acyl moiety.     

Localization of transforming growth factor-beta at the human fetal-maternal interface: role in trophoblast growth and differentiation.

We examined the localization of transforming growth factor (TGF)-beta in first-trimester and term human decidua and chorionic villi and explored the role of this factor on the proliferation and differentiation of cultured trophoblast cells. Two antibodies, 1D11.16.8, a mouse monoclonal neutralizing antibody capable of recognizing both TGF-beta 1 and TGF-beta 2 and CL-B1/29, a rabbit polyclonal antibody capable of recognizing TGF-beta 2, were used to immunolocalize TGF-beta in fixed, paraffin-embedded, or fixed, frozen sections of placenta and decidua, providing similar results. Intense labeling was observed in the extracellular matrix (ECM) of the first-trimester decidua and cytoplasm of term decidual cells. Syncytiotrophoblast cell cytoplasm as well as the ECM in the core of the chorionic villi of both first-trimester and term placentas exhibited a moderate degree of labeling. Strong cytoplasmic labeling was observed in the cytotrophoblastic shell of the term placenta. To examine the role of TGF-beta on trophoblast proliferation and differentiation, early passage cultures of first-trimester and primary cultures of term trophoblast cells were established and characterized on the basis of numerous immunocytochemical and functional markers. These cells expressed cytokeratin, placental alkaline phosphatase, urokinase-type plasminogen activator, and pregnancy-specific beta glycoprotein, but not factor VIII or 63D3; they also produced hCG and collagenase type IV. Exposure of first-trimester trophoblast cultures to TGF-beta 1 significantly inhibited proliferation in a dose-dependent manner. An antiproliferative effect was also noted in the presence of TGF-beta 2. These effects were abrogated in the presence of the neutralizing anti-TGF-beta antibody (1D11.16.8) in a concentration-dependent manner. In a 3-day culture, exogenous TGF-beta 1 stimulated formation of multinucleated cells by the first trimester as well as term trophoblast cells. Addition of neutralizing anti-TGF-beta antibody to first-trimester trophoblast cells stimulated proliferation beyond control levels in a 24-h culture and reduced formation of multinucleated cells in a 3-day culture, indicating the presence of endogenous TGF-beta activity. These results indicate that TGF-beta produced at the human fetal-maternal interface plays a major regulatory role in the proliferation and differentiation of the trophoblast.     

Allocation of scarce resources in Africa during COVID‐19: Utility and justice for the bottom of the pyramid?

The COVID‐19 pandemic has raised important universal public health challenges. Conceiving ethical responses to these challenges is a public health imperative but must take context into account. This is particularly important in sub‐Saharan Africa (SSA). In this paper, we examine how some of the ethical recommendations offered so far in high‐income countries might appear from a SSA perspective. We also reflect on some of the key ethical challenges raised by the COVID‐19 pandemic in low‐income countries suffering from chronic shortages in health care resources, and chronic high morbidity and mortality from non‐COVID‐19 causes. A parallel is drawn between the distribution of severity of COVID‐19 disease and the classic “Fortune at the bottom of the pyramid” model that is relevant in SSA. Focusing allocation of resources during COVID‐19 on the ‘thick’ part of the pyramid in Low‐to‐Middle Income Countries (LMICs) could be ethically justified on utilitarian and social justice grounds, since it prioritizes a large number of persons who have been economically and socially marginalized. During the pandemic, importing allocation frameworks focused on the apex of the pyramid from the global north may therefore not always be appropriate. In a post‐COVID‐19 world, we need to think strategically about how health care systems can be financed and structured to ensure broad access to adequate health care for all who need it. The root problems underlying health inequity, exposed by COVID‐19, must be addressed, not just to prepare for the next pandemic, but to care for people in resource poor settings in non‐pandemic times.     

Human Spermatogenic Failure Purges Deleterious Mutation Load from the Autosomes and Both Sex Chromosomes,including the Gene DMRT1

Gonadal failure, along with early pregnancy loss and perinatal death, may be an important filter that limits the propagation of harmful mutations in the human population. We hypothesized that men with spermatogenic impairment, a disease with unknown genetic architecture and a common cause of male infertility, are enriched for rare deleterious mutations compared to men with normal spermatogenesis. After assaying genomewide SNPs and CNVs in 323 Caucasian men with idiopathic spermatogenic impairment and more than 1,100 controls, we estimate that each rare autosomal deletion detected in our study multiplicatively changes a man''s risk of disease by 10% (OR 1.10 [1.04–1.16], p<2×10⁻³), rare X-linked CNVs by 29%, (OR 1.29 [1.11–1.50], p<1×10⁻³), and rare Y-linked duplications by 88% (OR 1.88 [1.13–3.13], p<0.03). By contrasting the properties of our case-specific CNVs with those of CNV callsets from cases of autism, schizophrenia, bipolar disorder, and intellectual disability, we propose that the CNV burden in spermatogenic impairment is distinct from the burden of large, dominant mutations described for neurodevelopmental disorders. We identified two patients with deletions of DMRT1, a gene on chromosome 9p24.3 orthologous to the putative sex determination locus of the avian ZW chromosome system. In an independent sample of Han Chinese men, we identified 3 more DMRT1 deletions in 979 cases of idiopathic azoospermia and none in 1,734 controls, and found none in an additional 4,519 controls from public databases. The combined results indicate that DMRT1 loss-of-function mutations are a risk factor and potential genetic cause of human spermatogenic failure (frequency of 0.38% in 1306 cases and 0% in 7,754 controls, p = 6.2×10⁻⁵). Our study identifies other recurrent CNVs as potential causes of idiopathic azoospermia and generates hypotheses for directing future studies on the genetic basis of male infertility and IVF outcomes.     

Passive Immunization with Hypochlorite-oxLDL Specific Antibodies Reduces Plaque Volume in LDL Receptor-Deficient Mice

Aims

New strategies to overcome complications of cardiovascular diseases are needed. Since it has been demonstrated that atherosclerosis is an inflammatory disease, modulation of the immune system may be a promising approach. Previously, it was suggested that antibodies may confer protective effects on the development of atherosclerosis. In this study, we hypothesised that passive immunization with anti-oxLDL IgM antibodies specific for hypochlorite (HOCl) may be athero-protective in mice.

Methods and Results

Monoclonal mouse IgM antibodies were produced and the antibody with specificity for hypochlorite-oxLDL (HOCl-oxLDL) (Moab A7S8) was selected. VH sequence determination revealed that Moab A7S8 is a natural IgM antibody. Atherosclerosis in LDLr^−/− mice was induced by a perivascular collar placement around the right carotid artery in combination with feeding a high-fat diet. Subsequently, the mice were treated every six days with 500 µg Moab A7S8, non-relevant IgM or with PBS and the carotid arteries and aortic roots were studied for atherosclerosis. Passive immunization with this Moab A7S8 resulted in a significant reduced plaque volume formation in LDLr^−/− mice when compared with PBS treatment (P = 0.002 and P = 0.035). Cholesterol levels decreased by 20% when mice were treated with Moab A7S8 compared to PBS. Furthermore, anti-oxLDL specific IgM and IgG antibody production increased significantly in the Moab A7S8 treated mice in comparison with PBS treated mice.

Conclusion

Our data show that passive immunization with a natural IgM antibody, directed to HOCl-oxLDL, can reduce atherosclerotic plaque development. We postulate that specific antibody therapy may be developed for use in human cardiovascular diseases.     

Phenotypic Switching Induced by Damaged Matrix Is Associated with DNA Methyltransferase 3A (DNMT3A) Activity and Nuclear Localization in Smooth Muscle Cells (SMC)

Extracellular matrix changes are often crucial inciting events for fibroproliferative disease. Epigenetic changes, specifically DNA methylation, are critical factors underlying differentiated phenotypes. We examined the dependency of matrix-induced fibroproliferation and SMC phenotype on DNA methyltransferases. The cooperativity of matrix with growth factors, cell density and hypoxia was also examined. Primary rat visceral SMC of early passage (0–2) were plated on native collagen or damaged/heat-denatured collagen. Hypoxia was induced with 3% O₂ (balanced 5% CO₂ and 95% N₂) over 48 hours. Inhibitors were applied 2–3 hours after cells were plated on matrix, or immediately before hypoxia. Cells were fixed and stained for DNMT3A and smooth muscle actin (SMA) or smooth muscle myosin heavy chain. Illumina 450 K array of CpG sites was performed on bisulfite-converted DNA from smooth muscle cells on damaged matrix vs native collagen. Matrix exquisitely regulates DNMT3A localization and expression, and influences differentiation in SMCs exposed to denatured matrix +/− hypoxia. Analysis of DNA methylation signatures showed that Matrix caused significant DNA methylation alterations in a discrete number of CpG sites proximal to genes related to SMC differentiation. Matrix has a profound effect on the regulation of SMC phenotype, which is associated with altered expression, localization of DNMTs and discrete changes DNA methylation.     

DNA marker based identification of spontaneous hybrids between Pinus mugo and P. sylvestris at the Lithuanian sea‐side

The objective was to identify spontaneous hybrids between P. mugo and P. sylvestris using organelle DNA markers in sympatric zones at the sea‐side spit of Kursiu Nerija in western Lithuania. A field inventory was carried out over the entire Lithuanian part of the spit and 203 individuals morphologically intermediate between P. sylvestris and P. mugo were tested for their male parent with chloroplast DNA PCR‐RFLP markers and for their female parent with mitochondrial DNA PCR markers. Unfortunately, the mitochondrial DNA Nad7 marker failed to identify the female parent species in our study. However, the chloroplast DNA PCR‐RFLP marker revealed that out of 203 tested individuals only 23 had a paternity different than that indicated by morphology. Of these, 13 individuals were morphologically identified as P. sylvestris but possessed cpDNA of P. mugo (putative hybrids with P. sylvestris (female) ×P. mugo (male parent), and 10 individuals morphologically identified as P. mugo possessed cpDNA of P. sylvestris and may be hybrids with P. mugo (female) ×P. sylvestris (male parent). The remainder of the 177 individuals identified in the field inventory were were considered as pure species. In conclusion, our study indicates ongoing spontaneous hybridization between P. mugo and P. syvestris in Kursiu Nerija. Human impact via seed transfer on altered hybridization rates as well as evolutionary consequences are discussed.     

Pomalidomide Shows Significant Therapeutic Activity against CNS Lymphoma with a Major Impact on the Tumor Microenvironment in Murine Models

Primary CNS lymphoma carries a poor prognosis. Novel therapeutic agents are urgently needed. Pomalidomide (POM) is a novel immunomodulatory drug with anti-lymphoma activity. CNS pharmacokinetic analysis was performed in rats to assess the CNS penetration of POM. Preclinical evaluation of POM was performed in two murine models to assess its therapeutic activity against CNS lymphoma. The impact of POM on the CNS lymphoma immune microenvironment was evaluated by immunohistochemistry and immunofluorescence. In vitro cell culture experiments were carried out to further investigate the impact of POM on the biology of macrophages. POM crosses the blood brain barrier with CNS penetration of ~ 39%. Preclinical evaluations showed that it had significant therapeutic activity against CNS lymphoma with significant reduction in tumor growth rate and prolongation of survival, that it had a major impact on the tumor microenvironment with an increase in macrophages and natural killer cells, and that it decreased M2-polarized tumor-associated macrophages and increased M1-polarized macrophages when macrophages were evaluated based on polarization status. In vitro studies using various macrophage models showed that POM converted the polarization status of IL4-stimulated macrophages from M2 to M1, that M2 to M1 conversion by POM in the polarization status of lymphoma-associated macrophages is dependent on the presence of NK cells, that POM induced M2 to M1 conversion in the polarization of macrophages by inactivating STAT6 signaling and activating STAT1 signaling, and that POM functionally increased the phagocytic activity of macrophages. Based on our findings, POM is a promising therapeutic agent for CNS lymphoma with excellent CNS penetration, significant preclinical therapeutic activity, and a major impact on the tumor microenvironment. It can induce significant biological changes in tumor-associated macrophages, which likely play a major role in its therapeutic activity against CNS lymphoma. POM should be further evaluated in clinical trials.