Platelet Inhibition Prevents NLRP3 Inflammasome Activation and Sepsis-Induced Kidney Injury.

Platelets, cellular mediators of thrombosis, are activated during sepsis and are increasingly recognized as mediators of the immune response. Platelet activation is significantly increased in sepsis patients compared to ICU control patients. Despite this correlation, the role of activated platelets in contributing to sepsis pathophysiology remains unclear. We previously demonstrated NOD-like receptor protein 3 inflammasome (NLRP3) inflammasome activation in sepsis-induced platelets from cecal-ligation puncture (CLP) rats. Activated platelets were associated with increased pulmonary edema and glomerular injury in CLP vs. SHAM controls. In this study, we investigated whether inhibition of platelet activation would attenuate NLRP3 activation and renal and pulmonary injury in response to CLP. CLP was performed in male and female Sprague Dawley (SD) rats (n = 10/group) to induce abdominal sepsis and SHAM rats served as controls. A subset of CLP animals was treated with Clopidogrel (10 mg/kg/day, CLP + CLOP) to inhibit platelet activation. At 72 h post-CLP, platelet activation and NLRP3 inflammasome assembly were evaluated, IL-1ß and IL-18 were measured in plasma, and tissues, renal and pulmonary pathology, and renal function were assessed. Activated platelets were 7.8 ± 3.6% in Sham, 22 ± 6% in CLP and significantly decreased to 14.5 ± 0.6% in CLP + CLOP (n = 8-10/group, p < 0.05). NLRP3 inflammasome assembly was inhibited in platelets of CLP + CLOP animals vs. CLP. Significant increases in plasma and kidney IL-1ß and IL-18 in response to CLP were decreased with Clopidogrel treatment. Renal injury, but not lung histology or renal function was improved in CLP + CLOP vs. CLP. These data provide evidence that activated platelets may contribute to sepsis-induced renal injury, possibly via NLRP3 activation in platelets. Platelets may be a therapeutic target to decrease renal injury in septic patients.

Developmental changes in methylation of spermatogenesis-specific genes include reprogramming in the epididymis.

We have determined the status of DNA methylation at specific sites in three spermatogenesis-specific genes, Pgk-2, ApoA1 and Oct-3/4, throughout the development and differentiation of male germ cells in the mouse. We observed a specific demethylation event in the Pgk-2 gene in prospermatogonia at about the time of birth, about 10 days before the onset of transcription which first occurs in primary spermatocytes. All three genes were unmethylated in adult spermatogenic cells in the testis, but were remethylated in mature spermatozoa in the vas deferens. Surprisingly, we found that this remethylation is part of the process of sperm maturation which occurs in the epididymis.

Gamete-specific methylation correlates with imprinting of the murine Xist gene.

We have investigated the potential role of DNA methylation as a regulator of imprinted Xist expression in mouse preimplantation embryos. The active paternal allele was found to be unmodified in sperm at CpG loci near the 5' end of the gene transcription unit. In contrast, on the inactive maternal allele, these same sites are initially methylated in the oocyte and then remain modified in the early embryo. In the male germ line, these methyl moieties are removed during spermatogenesis, and this occurs before the programmed reactivation of Xist in the testis. This represents a clear-cut example of a potential methylation imprinting signal that is reprogrammable and gamete derived.

A role for nuclear NF-kappaB in B-cell-specific demethylation of the Igkappa locus.

The immunoglobulin kappa gene is specifically demethylated during B-cell maturation in a process which utilizes discrete cis-acting modules such as the intronic kappa enhancer element and the matrix attachment region (MAR). While any MAR sequence is sufficient for this reaction, mutation analysis indicates that tissue specificity is mediated by kappaB binding sequences within the kappa intronic enhancer. The plasmacytoma cell line S107 lacks kappaB binding activity and fails to demethylate the kappa locus. However, B-cell specific demethylation is restored by the introduction of an active kappaB binding protein gene relB. This represents the first demonstration of a trans-acting factor involved in cell-type-specific demethylation, and suggests that the same protein-DNA recognition system used for transcription may also contribute to the earlier developmental events that bring about activation of the kappa locus.

DNA methylation represses transcription in vivo.

DNA in somatic tissue is characterized by a bimodal pattern of methylation, which is established in the animal through a series of developmental events. In the mouse blastula, most DNA is unmethylated, but after implantation a wave of de novo methylation modifies most of the genome, excluding the majority of CpG islands, which are mainly associated with housekeeping genes. This genomic methylation pattern is broadly maintained during the life of the organism by maintenance methylation, and generally correlates with gene expression. Experiments both in vitro and in vivo indicate that methylation inhibits transcription. It has not yet been possible, however, to determine the role of DNA methylation on specific sequences during normal development. Cis-acting regulatory elements and trans-acting factors appear to be involved in both stage- and tissue-specific demethylation processes. Sp1-like elements have a key role in protecting the CpG island of Aprt (encoding adenine phosphoribosyltransferase) from de novo methylation, and when these elements are specifically mutated, the Aprt CpG island becomes methylated in transgenic mice. We have now characterized an embryo-specific element from the CpG island sequence upstream of Aprt that can protect itself from de novo methylation in transgenic mice as well as reduce methylation of flanking sequences. We placed this element on a removable cassette adjacent to a human HBB (encoding beta-globin) reporter and generated a transgene whose methylation pattern can be switched in vivo. Analysis of globin transcription in this system showed that methylation in cis inhibits gene expression in a variety of tissues, indicating that DNA modification may serve as a global genomic repressor.

The imprinting box of the Prader-Willi/Angelman syndrome domain.

A subset of mammalian genes is monoallelically expressed in a parent-of-origin manner. These genes are subject to an imprinting process that epigenetically marks alleles according to their parental origin during gametogenesis. Imprinted genes can be organized in clusters as exemplified by the 2-Mb domain on human chromosome 15q11-q13 and its mouse orthologue on chromosome 7c (ref. 1). Loss of this 2-Mb domain on the paternal or maternal allele results in two neurogenetic disorders, Prader-Willi syndrome (PWS) or Angelman syndrome (AS), respectively. Microdeletions on the paternal allele share a 4.3-kb short region of overlap (SRO), which includes the SNRPN promoter/exon1, cause PWS and silence paternally expressed genes. Microdeletions on the maternal allele share a 0.88-kb SRO located 35 kb upstream to the SNRPN promoter, cause AS and alleviate repression of genes on the maternal allele. Individuals carrying both AS and PWS deletions on the paternal allele show a PWS phenotype and genotype. These observations suggest that cis elements within the AS-SRO and PWS-SRO constitute an imprinting box that regulates the entire domain on both chromosomes. Here we show that a minitransgene composed of a 200-bp Snrpn promoter/exon1 and a 1-kb sequence located approximately 35 kb upstream to the SNRPN promoter confer imprinting as judged by differential methylation, parent-of-origin-specific transcription and asynchronous replication.

Perioral volumization using a temperature controlled fractionated radiofrequency device.

BACKGROUND AND AIMS: Cosmetic rejuvenation of the perioral area can be challenging due to a mix of skin laxity and volumetric loss. Current techniques including fillers, neurotoxins, and non-ablative and ablative resurfacing have several drawbacks and can create a stiff, box-shaped, unnatural appearance. Aside from filler, these techniques do not address deeper volume deficiency. Temperature controlled fractionated radiofrequency (FRF) provides consistent formation of new collagen, elastin, and hyaluronic acid. This has been reported to provide long-lasting results for the treatment of skin laxity and volume loss of the face and neck with a single treatment, but it has not been previously reported for perioral rejuvenation. PATIENTS/METHODS: We present a series of seven patients who were treated with perioral FRF, and we assessed their results objectively using a published, validated scale for lower face laxity. RESULTS: All of the patients in this case series showed improvement on the Facial Laxity Rating (FLR) scale after FRF treatment. CONCLUSIONS: This case series represents the first reported use of temperature controlled FRF for perioral rejuvenation. FRF is a promising option for low-risk, natural perioral rejuvenation with long-lasting results and few risks.

Generalized essential telangiectasia treated with PDL.

Generalized essential telangiectasia (GET) is a rare, clinically benign condition but a source of cosmetic concern for affected patients. There is a dearth of publications and known treatment options for GET. This case report reviews the clinical course of a 54-year-old woman who presented with a long-standing history of telangiectatic patches on her dorsal feet and ankles with progressive spread to the lower extremities consistent with GET. The patient proceeded with two pulsed dye laser (PDL) treatments and had complete resolution of her skin findings maintained at her 1.5-year follow-up appointment.

A Pilot Study Evaluation of 3-Dimensional Imaging in Cosmetic Breast Augmentation: Results of a Single Surgeon 3.5-Year Retrospective Study Using the BREAST-Q Questionnaire.

BACKGROUND: Traditional methods of breast implant size selection provide limited ability to demonstrate postoperative outcomes. Three-dimensional (3D) imaging provides an opportunity for improved patient evaluation, surgical planning, and evaluation of postoperative breast appearance. OBJECTIVES: The authors hypothesized that preoperative 3D imaging for patients undergoing breast augmentation would improve patient satisfaction and understanding of expected surgical outcomes. METHODS: A retrospective review of patients undergoing breast augmentation by a single surgeon over a 3.5-year period was performed. Patients presenting after the VECTRA was purchased had preoperative 3D imaging, while patients presenting before this did not. Eligible patients received a BREAST-Q questionnaire designed for postoperative evaluation of breast augmentation. They also received a second survey that evaluated expected vs actual breast outcomes. RESULTS: In total, 120 surveys were mailed and 61 patients (50.8%) returned the survey. The 3D imaged group had improved BREAST-Q scores regarding satisfaction with outcome, surgeon, and physical well-being compared with the group that did not. The imaged group also had higher size, shape, and overall breast correlation scores, confidence in implant size selection scores, and communication with surgeon scores. The differences between the 2 groups were not statistically significant. CONCLUSIONS: Three-dimensional imaging is a valuable tool in breast surgery. Although this study showed improvement in patient satisfaction and predicted outcome scores in the 3D imaged group, the results were not statistically significant. With the majority of patients reporting that they would choose 3D imaging, it appears to instill confidence in patients regarding both surgeon and implant selection.

NLRP3 inflammasome inhibition attenuates sepsis-induced platelet activation and prevents multi-organ injury in cecal-ligation puncture.

Sepsis is characterized by organ dysfunction due to a dysregulated immune response to infection. Currently, no effective treatment for sepsis exists. Platelets are recognized as mediators of the immune response and may be a potential therapeutic target for the treatment of sepsis. We previously demonstrated that NLRP3 inflammasome activation in sepsis-induced activated platelets was associated with multi-organ injury in the cecal-ligation puncture (CLP) rat model of sepsis. In this study, we tested the hypothesis that inhibition of NLRP3 would inhibit platelet activation and attenuate multi-organ injury in the CLP rat. CLP (n = 10) or Sham (n = 10) surgery were performed in male and female Sprague-Dawley rats. A subset of CLP rats were treated with MCC950 (50mg/kg/d), a specific NLRP3 inhibitor (CLP+MCC950, n = 10). At 72 hrs. post-CLP, blood and organs were harvested for analysis of platelet activation, NLRP3 activation, inflammation and end organ damage. Platelet activation increased from 8±0.8% in Sham to 16±1% in CLP, and was reduced to 9±1% in CLP+M rats (p<0.05). NLRP3 activation was also increased in platelets of CLP vs Sham. NLRP3 expression was unchanged in kidney and lung after CLP, but Caspase 1 expression and IL-1ß were increased. MCC950 treatment attenuated NLRP3 activation in platelets. Plasma, kidney, and lung levels of NLRP3 inflammasome associated cytokines, IL-1ß and IL-18, were significantly increased in CLP compared to Sham rats. Inhibition of NLRP3 normalized cytokine levels. Glomerular injury, pulmonary edema, and endothelial dysfunction markers were increased in CLP rats vs Sham. MCC950 treatment significantly decreased renal and pulmonary injury and endothelial dysfunction in CLP+M. Our results demonstrate a role for NLRP3 in contributing to platelet activation and multi-organ injury in sepsis.

Active genes are sensitive to deoxyribonuclease I during metaphase.

The active exogenous murine leukemia virus sequences of mouse cells growing in culture are preferentially digested by deoxyribonuclease I in metaphase chromosomes. As determined by nuclear nick translation, all of the gene sequences of these cells active during interphase are in a deoxyribonuclease I-sensitive conformation during metaphase. This method of nick translation can therefore be used to label chromosomes in situ in order to visualize the active regions of the genome.

A Plate-based Cytotoxicity Assay for the Assessment of Rat Placental Natural Killer Cell Cytolytic Function.

It is well known that decidual natural killer (NK) cells play a critical role in establishment and maintenance of normal pregnancy. Recent studies have demonstrated an altered population of circulating and decidual NK cells in women who suffer from adverse pregnancy complications such as recurrent miscarriage and preeclampsia. Studies from our group have shown that hypertension in pregnancy is associated with an increased population of activated NK cells in the placenta based on the expression of surface activation markers. This manuscript provides a detailed protocol to assess the cytotoxic function of NK cells isolated from placentas in a preeclampsia-like animal model of surgically induced placental ischemia. The following steps are described in detail: generation of single cell suspension, NK cell isolation, ex vivo stimulation, effector:target cell co-culture, and the cytotoxicity assay.

NLRP3 inflammasome activation in platelets in response to sepsis.

Sepsis is a complex syndrome characterized by organ dysfunction and a dysregulated immune host response to infection. There is currently no effective treatment for sepsis, but platelets have been proposed as a potential therapeutic target for the treatment of sepsis. We hypothesized that the NLRP3 inflammasome is activated in platelets during sepsis and may be associated with multiorgan injury in response to polymicrobial sepsis. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in 12- to 13-week-old male Sprague-Dawley rats. The necrotic cecum was removed at 24 h post-CLP. At 72 h post-CLP, activated platelets were significantly increased in CLP versus Sham rats. Colocalization of NLRP3 inflammasome components was observed in platelets from CLP rats at 72 h post-CLP. Plasma, pulmonary, and renal levels of IL-1ß and IL-18 were significantly higher in CLP rats compared to Sham controls. Soluble markers of endothelial permeability were increased in CLP versus Sham. Renal and pulmonary histopathology were markedly elevated in CLP rats compared to Sham controls. NLRP3 is activated in platelets in response to CLP and is associated with inflammation, endothelial permeability and multiorgan injury. Our results indicate that activated platelets may play a role to cause multiorgan injury in sepsis and may have therapeutic potential for the treatment of sepsis multiorgan injury.

Role of DNA methylation in the regulation of transcription.

DNA methylation plays an important role in the regulation of gene expression during development. Methyl moieties at CpG residues suppress transcription by affecting DNA-protein interactions, thus altering the accessibility of genes to trans-acting factors in the cell. Because it works in cis, this mechanism is important in the control of X inactivation and genomic imprinting.

Imprinting: focusing on the center.

Recent studies have focused on the identification of imprinting centers and on the elucidation of the mechanisms by which they control imprinting. These studies begin to shed light on the means by which imprinting marks are established in the gametes and on the various molecular strategies utilized to execute differential expression of the two parental alleles.

Chromosome structure and eukaryotic gene organization.

The DNA in the eukaryotic nucleus is highly compacted but well organized into distinct regional units. Chromosomal bands are characterized by their structure and distinctive replication time. They are subdivided into chromatin loops which serve as functional domains that have discrete boundary elements and can be regulated during development.

DNA methylation: a molecular lock.

In mammals, the promoters of expressed genes are generally unmethylated, whereas those of genes that are not expressed are methylated. Two recent papers help to explain the mechanism by which methylation modulates gene expression.

Allele-specific expression patterns of interleukin-2 and Pax-5 revealed by a sensitive single-cell RT-PCR analysis.

Autosomal genes that are subject to random allelic inactivation (RAI), like imprinted genes [1] and genes subject to X-inactivation [2], require mechanisms that dictate the differential transcriptional regulation of two sequence-identical alleles. RAI genes include olfactory receptor genes [3], and the various genes encoding antigen receptors on lymphocytes (immunoglobulin genes, T cell receptor genes and NK receptor genes [4] [5] [6] [7]). These observations raise the possibility that other genes might be similarly regulated. Moreover, an interesting possibility is that certain genes might be monoallelically expressed in some cells and biallelically expressed in others. Recently, reports of monoallelic expression of interleukin-2 (IL-2) [8] [9] and IL-4 [10] [11] have raised the possibility that the cytokine gene family may be subject to monoallelic expression. Another report suggests that the gene encoding the transcription factor Pax-5, which is involved in B-cell (and cerebellar) development [12] [13], is also subject to monoallelic expression [14]. Using a novel single-cell reverse transcription-polymerase chain reaction (RT-PCR) approach, we have analyzed the IL-2 and Pax-5 genes in mice. We found that IL-2 is monoallelically transcribed in some T cells and biallelically transcribed in others, raising interesting questions regarding cytokine gene regulation. Additionally, our analyses suggest that Pax-5 is consistently biallelically transcribed. Thus, the IL-2 gene and other cytokine genes may be regulated in a stochastic manner that results in 0, 1 or 2 alleles of a given cytokine gene expressed in each T cell. This type of regulation could account for the wide variety of different combinations of cytokine genes expressed in individual T cells and therefore plays a role in the generation of T cells with a range of different effector functions.

Developmental regulation of immune system gene rearrangement.

Rearrangement of antigen receptors in the immune system is mediated through the action of complex enhancers which function in both a developmentally stage-specific and a cell-type-specific manner to demethylate DNA, open chromatin structure and tether the recombination machinery to one preferred allele at each locus.