Evaluating evidence for atrophic scarring treatment modalities.

INTRODUCTION: Atrophic scars cause significant patient morbidity. Whilst there is evidence to guide treatment, there does not appear to be a systematic review to analyse the efficacy of treatment options. OBJECTIVES: To retrieve all evidence relating to atrophic scar treatment and evaluate using the Clinical Evidence GRADE score in order to allow clinicians to make evidence-based treatment choices. METHOD: Searches were performed in Medline, EMBASE, CINHL and Cochrane to identify all English studies published evaluating treatment of atrophic scars on adults excluding journal letters. Each study was allocated a GRADE score based on type of study, quality, dose response, consistency of results and significance of results. The end score allowed categorisation of evidence into high, moderate, low or very low quality. RESULTS: A total of 41 studies were retrieved from searches including randomised controlled trials, observational studies, retrospective analyses and case reports of which 7% were allocated a high-quality score, 10% a moderate score, 7% a low score and 75% a very low score. Treatment modalities included ablative laser therapy, non-ablative laser therapy, autologous fat transfer, dermabrasion, chemical peels, injectables, subcision, tretinoin iontophoresis and combination therapy. CONCLUSION: There is a paucity of good-quality clinical evidence evaluating treatment modalities for atrophic scarring. Evidence supports efficacy of laser, surgery and peel therapy. Further biomolecular research is required to identify targeted treatment options and more randomised controlled trials would make the evidence base for atrophic scar treatment more robust.

A simple idea for approximating the volume of filler needed for chest-wall contour-defects.

Fillers can be used to correct contour-defects. Due to the irregularities of defects, estimating the volume of filler required can be difficult. This frequently results in surgeons taking a step-wise approach to filler-injection, usually occurring over different appointments. Using a patient with pectus excavatum as an example, we provide a simple tip as to estimating the volume of filler required. In this case, normal saline was poured into the chest-wall defect until the adequate level was reached (from the lateral aspect), while noting how much liquid was used. The patient then had a comparable volume of filler injected.

Quality-control analytical methods: endotoxins: essential testing for pyrogens in the compounding laboratory, part2.

Ensuring that the endotoxin burden in sterile preparations is within allowable limits is one of the greatest challenges faced by compounding pharmacists. Today, endotoxin analyses can be performed in the pharmacy with a test kit or accomplished by sending samples to a contract testing laboratory. Both types of screening are discussed in this article, and information that enables compounders to determine the preferred method of endotoxin testing is provided. A brief history of endotoxin testing is presented, and the advantages and disadvantages of pyrogen screening with either an in-house test kit or the services of a contract testing laboratory are explored. Accurate screening for pyrogens ensures that only sterile formulations containing a safe level of endotoxins are dispensed. An essential task in sterile compounding, endotoxin testing safeguards patients against the morbidity and mortality that can result from treatment with a contamination preparation.

Resistance of human alveolar macrophages to Bacillus anthracis lethal toxin.

The etiologic agent of inhalational anthrax, Bacillus anthracis, produces virulence toxins that are important in the disease pathogenesis. Current studies suggest that mouse and human macrophages are susceptible to immunosuppressive effects of one of the virulence toxins, lethal toxin (LT). Thus a paradigm has emerged that holds that the alveolar macrophage (AM) does not play a significant role in the innate immune response to B. anthracis or defend against the pathogen as it is disabled by LT. This is inconsistent with animal models and autopsy studies that show minimal disease at the alveolar surface. We examined whether AM are immunosuppressed by LT. We found that human AM were relatively resistant to LT-mediated innate immune cytokine suppression, MEK cleavage, and induction of apoptosis as compared with mouse RAW 264.7 macrophages. Mouse AM and murine bone marrow-derived macrophages were also relatively resistant to LT-mediated apoptosis despite intermediate sensitivity to MEK cleavage. The binding component of LT, protective Ag, does not attach to human AM, although it did bind to mouse AM, murine bone marrow-derived macrophages, and RAW 264.7 macrophages. Human AM do not produce significant amounts of the protective Ag receptor anthrax toxin receptor 1 (TEM8/ANTXR1) and anthrax toxin receptor 2 (CMG2/ANTXR2). Thus, mature and differentiated AM are relatively resistant to the effects of LT as compared with mouse RAW 264.7 macrophages. AM resistance to LT may enhance clearance of the pathogen from the alveolar surface and explain why this surface is relatively free of B. anthracis in animal models and autopsy studies.

Gene expression profiling of human alveolar macrophages infected by B. anthracis spores demonstrates TNF-alpha and NF-kappab are key components of the innate immune response to the pathogen.

BACKGROUND: Bacillus anthracis, the etiologic agent of anthrax, has recently been used as an agent of bioterrorism. The innate immune system initially appears to contain the pathogen at the site of entry. Because the human alveolar macrophage (HAM) plays a key role in lung innate immune responses, studying the HAM response to B. anthracis is important in understanding the pathogenesis of the pulmonary form of this disease. METHODS: In this paper, the transcriptional profile of B. anthracis spore-treated HAM was compared with that of mock-infected cells, and differentially expressed genes were identified by Affymetrix microarray analysis. A portion of the results were verified by Luminex protein analysis. RESULTS: The majority of genes modulated by spores were upregulated, and a lesser number were downregulated. The differentially expressed genes were subjected to Ingenuity Pathway analysis, the Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis, the Promoter Analysis and Interaction Network Toolset (PAINT) and Oncomine analysis. Among the upregulated genes, we identified a group of chemokine ligand, apoptosis, and, interestingly, keratin filament genes. Central hubs regulating the activated genes were TNF-alpha, NF-kappaB and their ligands/receptors. In addition to TNF-alpha, a broad range of cytokines was induced, and this was confirmed at the level of translation by Luminex multiplex protein analysis. PAINT analysis revealed that many of the genes affected by spores contain the binding site for c-Rel, a member of the NF-kappaB family of transcription factors. Other transcription regulatory elements contained in many of the upregulated genes were c-Myb, CP2, Barbie Box, E2F and CRE-BP1. However, many of the genes are poorly annotated, indicating that they represent novel functions. Four of the genes most highly regulated by spores have only previously been associated with head and neck and lung carcinomas. CONCLUSION: The results demonstrate not only that TNF-alpha and NF-kappab are key components of the innate immune response to the pathogen, but also that a large part of the mechanisms by which the alveolar macrophage responds to B. anthracis are still unknown as many of the genes involved are poorly annotated.

Bacillus anthracis peptidoglycan stimulates an inflammatory response in monocytes through the p38 mitogen-activated protein kinase pathway.

We hypothesized that the peptidoglycan component of B. anthracis may play a critical role in morbidity and mortality associated with inhalation anthrax. To explore this issue, we purified the peptidoglycan component of the bacterial cell wall and studied the response of human peripheral blood cells. The purified B. anthracis peptidoglycan was free of non-covalently bound protein but contained a complex set of amino acids probably arising from the stem peptide. The peptidoglycan contained a polysaccharide that was removed by mild acid treatment, and the biological activity remained with the peptidoglycan and not the polysaccharide. The biological activity of the peptidoglycan was sensitive to lysozyme but not other hydrolytic enzymes, showing that the activity resides in the peptidoglycan component and not bacterial DNA, RNA or protein. B. anthracis peptidoglycan stimulated monocytes to produce primarily TNFalpha; neutrophils and lymphocytes did not respond. Peptidoglycan stimulated monocyte p38 mitogen-activated protein kinase and p38 activity was required for TNFalpha production by the cells. We conclude that peptidoglycan in B. anthracis is biologically active, that it stimulates a proinflammatory response in monocytes, and uses the p38 kinase signal transduction pathway to do so. Given the high bacterial burden in pulmonary anthrax, these findings suggest that the inflammatory events associated with peptidoglycan may play an important role in anthrax pathogenesis.

Regulation of the prostaglandin enzymatic system by estradiol and progesterone in nonpregnant sheep cervix.

In the present study, we examined the in vivo effects of estradiol (E(2)) and progesterone on cyclooxygenase (COX) 2, prostaglandin F synthase (PTGFS, also known as PGFS), and membrane-associated prostaglandin E synthase 1 (mPTGES1) expression at both mRNA and protein levels using a nonpregnant ovariectomized (OVX) sheep model. Sixteen ewes were OVX shortly after ovulation. After 40 days, ewes were treated with saline (Cont, n=5), or E(2) infused intravenously for 2 days (50 microg/day, n=5) or intravaginal progesterone (P) sponges for 10 days (0.3 g P, n=6). Cervical COX2, PTGFS, and mPTGES1 mRNA and protein were quantified by northern and western blot analyses respectively. In situ hybridization and/or immunocytochemistry were used to localize the cellular distribution of COX2, PTGFS, and mPTGES1 mRNAs and proteins. COX2 mRNA abundance increased significantly in the cervix after E(2) treatment (P<0.05). However, progesterone was a more potent stimulator than E(2) of COX2 mRNA and protein abundance in the cervix (P<0.01). In contrast, PTGFS and mPTGES1 mRNA and protein concentrations did not change after E(2) or progesterone treatment (P>0.05). COX2, PTGFS, and mPTGES1 mRNA and protein were only localized in cervical glandular epithelial cells. This study shows that increased cervical COX2 mRNA and protein, but not PTGFS and mPTGES1 mRNA and protein, were associated with E(2) and progesterone treatment in nonpregnant sheep. More strikingly, progesterone was a more potent stimulator of cervical COX2 expression than E(2). The expression of COX2, PTGFS, and mPTGES1 mRNA and/or protein was confined in the cervical glandular epithelial cells of nonpregnant sheep.

Human lung innate immune response to Bacillus anthracis spore infection.

Bacillus anthracis, the causative agent of inhalational anthrax, enters a host through the pulmonary system before dissemination. We have previously shown that human alveolar macrophages participate in the initial innate immune response to B. anthracis spores through cell signal-mediated cytokine release. We proposed that the lung epithelia also participate in the innate immune response to this pathogen, and we have developed a human lung slice model to study this process. Exposure of our model to B. anthracis (Sterne) spores rapidly activated the mitogen-activated protein kinase signaling pathways ERK, p38, and JNK. In addition, an RNase protection assay showed induction of mRNA of several cytokines and chemokines. This finding was reflected at the translational level by protein peak increases of 3-, 25-, 9-, 34-, and 5-fold for interleukin-6 (IL-6), tumor necrosis factor alpha, IL-8, macrophage inflammatory protein 1alpha/beta, and monocyte chemoattractant protein 1, respectively, as determined by an enzyme-linked immunosorbent assay. Inhibition of individual pathways by UO126, SP600125, and SB0203580 decreased induction of chemokines and cytokines by spores, but this depended on the pathways inhibited and the cytokines and chemokines induced. Combining all three inhibitors reduced induction of all cytokines and chemokines tested to background levels. An immunohistochemistry analysis of IL-6 and IL-8 revealed that alveolar epithelial cells and macrophages and a few interstitial cells are the source of the cytokines and chemokines. Taken together, these data showed the activation of the pulmonary epithelium in response to B. anthracis spore exposure. Thus, the lung epithelia actively participate in the innate immune response to B. anthracis infection through cell signal-mediated elaboration of cytokines and chemokines.

Bacillus anthracis spores stimulate cytokine and chemokine innate immune responses in human alveolar macrophages through multiple mitogen-activated protein kinase pathways.

Contact with the human alveolar macrophage plays a key role in the innate immune response to Bacillus anthracis spores. Because there is a significant delay between the initial contact of the spore with the host and clinical evidence of disease, there appears to be temporary containment of the pathogen by the innate immune system. Therefore, the early macrophage response to Bacillus anthracis exposure is important in understanding the pathogenesis of this disease. In this paper, we studied the initial events after exposure to spores, beginning with the rapid internalization of spores by the macrophages. Spore exposure rapidly activated the mitogen-activated protein kinase signaling pathways extracellular signal-regulated kinase, c-Jun-NH2-terminal kinase, and p38. This was followed by the transcriptional activation of cytokine and primarily monocyte chemokine genes as determined by RNase protection assays. Transcriptional induction is reflected at the translational level, as interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, and tumor necrosis factor alpha (TNF-alpha) cytokine protein levels were markedly elevated as determined by enzyme-linked immunosorbent assay. Induction of IL-6 and TNF-alpha, and, to a lesser extent, IL-1alpha and IL-1beta, was partially inhibited by the blockade of individual mitogen-activated protein kinases, while the complete inhibition of cytokine induction was achieved when multiple signaling pathway inhibitors were used. Taken together, these data clearly show activation of the innate immune system in human alveolar macrophages by Bacillus anthracis spores. The data also show that multiple signaling pathways are involved in this cytokine response. This report is the first comprehensive examination of this process in primary human alveolar macrophages.

Regulation of membrane-associated prostaglandin E2 synthase 1 in pregnant sheep intrauterine tissues by glucocorticoid and estradiol.

This study was designed to determine the regulatory effect of glucocorticoid and estradiol on expression of ovine intrauterine membrane-associated prostaglandin E(2) synthase 1 (mPTGES1) in late gestation and at labor. For gestational and labor groups, 16 pregnant ewes from 95-147 d gestational age (dGA) and four pregnant ewes at spontaneous term labor were used. The fetal glucocorticoid group, 14 pregnant ewes at 123-125 dGA with fetuses, was divided into the following groups: after sham adrenalectomy (n = 5), adrenalectomy (n = 4), and adrenalectomy with fetal cortisol replacement to late gestation levels (n = 5). For the maternal glucocorticoid group, nine pregnant ewes were treated with saline (n = 4) and three courses of maternal dexamethasone (n = 5). For the estradiol group, 10 pregnant ewes at 119-121 dGA were treated with sesame oil (n = 5) or estradiol (n = 5) to produce labor levels of estradiol in maternal plasma. Endometrial, myometrial, and placental mRNA and proteins were analyzed by Northern and Western blot and immunocytochemistry for mPTGES1. Data were analyzed by Student's t test and ANOVA. There was a significant increase of placental mPTGES1 in late gestation. Glucocorticoids, given to the mother or fetus, significantly stimulated mPTGES1 in placenta. mPTGES1 was elevated only in the endometrium during spontaneous term labor and after estradiol treatment. The mPTGES1 was localized in the myometrial smooth muscle cells, endometrial stromal cells, and placental trophoblast cells. Our study suggested that increased expression of placental mPTGES1 throughout late gestation might result from the increased fetal and maternal circulating glucocorticoids, whereas elevated maternal plasma estradiol concentration might be responsible for the induced mPTGES1 expression in the endometrium during labor.

Iatrogenic fetal injury.

BACKGROUND: Iatrogenic fetal injury during cesarean delivery is a serious but underreported complication. CASE: A distal iatrogenic amputation of a digit occurred during a cesarean delivery. CONCLUSION: Obstetricians should be aware of this potential complication.

Glutamatergic dysfunction in OCD.

The role of glutamatergic dysfunction in the pathophysiology of OCD has hardly been explored despite recent reports implicating glutamatergic dysfunction in OCD. We decided to investigate CSF glutamate levels in adult OCD probands compared to psychiatrically normal controls. In total, 21 consenting psychotropic drug-naïve adult OCD patients, diagnosed using SCID-IV-CV, and 18 consenting psychiatrically normal controls with age within 10 years of age of the patients, who did not have any history of head injury or neurological illness, were included into the study. Aseptically collected and stored CSF samples obtained from the patients and control subjects were used for glutamate estimation, which was carried out by a modification of the procedure described by Lund (1986). CSF glutamate (micromol/l) level was found to be significantly higher [F(1,31)=6.846, p=0.014] in OCD patients (47.12+/-4.25) compared to control subjects (41.36+/-3.63) on analysis of covariance. There was no effect of gender, age, duration of illness, Y-BOCS score, or CGI-S score on CSF glutamate levels. Our study provides preliminary evidence implicating glutamatergic excess in the pathophysiology of OCD, which needs to be further explored by studies from other centers involving larger sample sets from different age groups.

Sufficient progesterone-priming prior to estradiol stimulation is required for optimal induction of the cervical prostaglandin system in pregnant sheep at 0.7 gestations.

The purposes of this study were to determine the separate and interactive functions of progesterone and estradiol in regulating the cervical prostaglandin (PG) system in pregnant sheep at 0.7 gestations. At 106-108 days of gestational age (dGA), ewes were treated with vehicle for 14 days (n = 5) or vehicle for 12 days followed by estradiol 5 mg twice a day, intramuscularly for 2 days (n = 5) or progesterone 100 mg, twice a day, intramuscularly for 14 days (n = 5) or progesterone 100 mg twice a day, intramuscularly for 10 days and then 2 days vehicle followed by estradiol 5 mg twice a day intramuscularly for 2 days (n = 5). At 121-123 dGA, cervical tissues were obtained under halothane anesthesia. Cervical RNA and protein were extracted and analyzed for prostaglandin-endoperoxide synthase 2 (COX2), two PGE(2) receptors, PTGER2 and PTGER4, and estrogen receptor alpha (ESR1) by Northern and Western blot analysis. Immunocytochemistry and in situ hybridization were applied to localize cellular distribution of COX2, PTGER2, and PTGER4 in the cervix. Data were analyzed by ANOVA. COX2 and PTGER4 mRNAs and proteins were increased (P < 0.05) in ewes treated with combined estradiol and progesterone but not in ewes treated with estradiol or progesterone alone compared with controls. ESR1 mRNA was increased in ewes treated with progesterone and estradiol plus progesterone. In contrast, PTGER2 mRNA and protein remained the same after all treatments. COX2 mRNA and protein were localized only in cervical glandular epithelial cells, whereas PTGER2 and PTGER4 were localized in both cervical glandular epithelial and smooth muscle cells. In conclusion, these data suggest that additional progesterone priming at 0.7 gestations synergizes with estradiol to induce cervical COX2, PTGER4, and ESR1 and support our hypothesis that stimulation of the cervical PG system by estradiol is optimized by sufficient progesterone priming in the pregnant sheep cervix.

Prostaglandin mediates premature delivery in pregnant sheep induced by estradiol at 121 days of gestational age.

The experiments reported here were designed for both in vivo and in vitro approaches in the same animals to obtain a better picture of the role of estrogen in the control of parturition. Chronically catheterized pregnant ewes were treated with vehicle (n = 5) or estradiol (n = 6), 5 mg twice a day, im for 2 d starting at d 119 of gestation. Maternal and fetal plasma estradiol, progesterone, and cortisol were measured by RIA and maternal plasma prostaglandin (PG) F2alpha was measured by enzyme immunoassay. Intrauterine PG H synthase 2 mRNA and protein and placental P450(c17)alpha hydroxylase mRNA were determined by Northern, in situ hybridization, Western blot analysis, and immunocytochemistry. Data were analyzed by ANOVA. Five of six estradiol-treated ewes delivered their fetuses within 48 h; however, the placenta was still retained 5-6 h after fetal delivery. Both maternal plasma estradiol and PGF2 alpha increased significantly in the estradiol-treated group. Maternal and fetal plasma progesterone and cortisol were not altered in either group. There were significant increases of PGH synthase 2 mRNA and protein in myometrium, endometrium, and maternal placenta but not in fetal placenta in estradiol-treated ewes. Placental P450(c17)alpha hydroxylase mRNA was not detectable in vehicle or estradiol-treated groups. Estradiol can, in the absence of increase in plasma cortisol, stimulate uterine PG production and induce labor, resulting in fetal delivery in the sheep. Failure of placental delivery after estradiol treatment suggests that estradiol alone is insufficient to stimulate some of the key changes required to complete delivery at the stage of gestation studied.

Fibroblasts play a regulatory role in the control of pigmentation in reconstructed human skin from skin types I and II.

Human melanocytes in monolayer culture are extremely dependent on a wide range of soluble signals for their proliferation and melanogenesis. The advent of three-dimensional models of reconstructed skin allows one to ask questions of how these cells are regulated within a setting which more closely approximates normal skin. The purpose of this study was to investigate to what extent melanocytes within a reconstructed skin model are sensitive to regulation by dermal fibroblasts, basement membrane (BM) proteins and the addition of alpha-melanocyte-stimulating hormone (alpha-MSH). Sterilized acellular de-epidermized dermis (prepared to retain BM proteins or deliberately denuded of BM by enzymatic treatment) from skin type I or II was reconstituted with fibroblasts, melanocytes and keratinocytes. In all but one case (9/10), cell donors were skin type I or II. The presence of BM antigens was found to be necessary for positional orientation of the melanocytes; in the absence of BM, melanocytes moved into the upper keratinocyte layer pigmenting spontaneously. Addition of fibroblasts suppressed the extent of spontaneous pigmentation of melanocytes within this model. Neither alpha-MSH nor cholera toxin induced pigmentation in this model despite the fact that melanocytes clearly had the ability to synthesize pigment.