The yield of procalcitonin and Interleukin-6 in predicting intraamniotic infection in the presence of intrapartum fever: A pilot study.

Intrapartum fever (IF) accompanied by either maternal or foetal tachycardia, elevated WBC, or purulent discharge is classified as "suspected triple 1", the hallmark of intraamniotic infection (IAI). Poor specificity of the clinical diagnosis of IAI results, in retrospect, in the unnecessary treatment of most parturients and neonates. We studied the yield of specific acute phase reactants (APRs): procalcitonin, CRP, IL-6, in detecting bacterial IAI among parturients classified as "suspected triple 1" (cases) compared to afebrile parturients (controls). Procalcitonin, CRP, and IL-6 were all significantly elevated in the cases compared to the controls, yet this by itself was not sufficient for an additive effect in detecting a bacterial infection among parturients clinically diagnosed with "suspected triple 1", as demonstrated by the poor area under the receiver operating characteristic curve of all three APRs.

The Role of Innate Immune System in the Human Amniotic Membrane and Human Amniotic Fluid in Protection Against Intra-Amniotic Infections and Inflammation.

Intra-amniotic infection and inflammation (IAI) affect fetal development and are highly associated with preterm labor and premature rupture of membranes, which often lead to adverse neonatal outcomes. Human amniotic membrane (hAM), the inner part of the amnio-chorionic membrane, protects the embryo/fetus from environmental dangers, including microbial infection. However, weakened amnio-chorionic membrane may be breached or pathogens may enter through a different route, leading to IAI. The hAM and human amniotic fluid (hAF) respond by activation of all components of the innate immune system. This includes changes in 1) hAM structure, 2) presence of immune cells, 3) pattern recognition receptors, 4) cytokines, 5) antimicrobial peptides, 6) lipid derivatives, and 7) complement system. Herein we provide a comprehensive and integrative review of the current understanding of the innate immune response in the hAM and hAF, which will aid in design of novel studies that may lead to breakthroughs in how we perceive the IAI.

Necrotizing funisitis is an indicator that intra-amniotic inflammatory response is more severe and amnionitis is more frequent in the context of the extension of inflammation into Wharton's jelly.

OBJECTIVE: Necrotizing funisitis (NF) is defined as the presence of an arc (i.e., crescent/band/ring/halos) of infiltrated neutrophils and/or associated debris in Wharton's jelly (WJ) of umbilical-cord (UC). However, no information exists about the comparison in intra-amniotic inflammatory-response (IAIR) and inflammation in extra-placental membranes between the presence and absence of NF in the context of inflammation in WJ among spontaneous preterm births (PTBs). The objective of current study is to examine this issue. MATERIALS AND METHODS: We examined IAIR and the frequency of amnionitis according to the progression of inflammation in UC (i.e. stage-1, umbilical phlebitis [inflammation in umbilical-vein(UV)] only; stage-2, involvement of at least one umbilical-artery[UA] and either the other UA or UV without extension into WJ; stage-3, the extension of inflammation into WJ without NF; stage-4, the extension of inflammation into WJ with NF) in 120singleton spontaneous PTBs (<37weeks). IAIR was gauged by AF MMP-8 (ng/ml) within 3days before birth. RESULTS: 1) Stage-1, stage-2, stage-3, and stage-4 were present in 20%(24/120), 6%(7/120), 61%(73/120), and 13%(16/120) of cases respectively; 2) AF MMP-8 continuously increased (stage-1 vs. stage-2 vs. stage-3 vs. stage-4; median[ng/ml], range[ng/ml]; 207.2[16.8-1196.5] vs. 444.1[8.5-2608.0] vs. 458.8[0.4-3116.7] vs. 1859.7[912.3-5304.8]; Spearman's rank correlation-test, α = 0.454, P = 0.006), and the frequency of increased AF MMP-8 (≥854.1 ng/ml) elevated (stage-1 vs. stage-2 vs. stage-3 vs. stage-4; 13%[1/8] vs. 33%[1/3] vs. 32%[6/19] vs. 100%[5/5]; Linear-by-linear-association, P = 0.012) with the progression of inflammation in UC; 3) Moreover, there was a stepwise increase in the frequency of amnionitis according to the progression of inflammation in UC (stage-1, 33%[8/24]; stage-2, 43%[3/7]; stage-3, 62%[45/73]; stage-4, 81%[13/16]; Linear-by-linear-association, P = 0.001). CONCLUSION: NF is an indicator that IAIR is more severe and amnionitis is more frequent in the context of the extension of inflammation into WJ. Therefore, current study confirms that NF is the most advanced stage in the progression of inflammation within UC.

Assessment of Antimicrobial Activity of Dehydrated Amniotic Membrane in Infectious Keratitis: A Small Retrospective Case Series and In Vitro Study.

PURPOSE: To describe a small case series of infectious keratitis with poor visual outcomes after amniotic membrane (AM) placement and to prospectively evaluate whether AM demonstrates antibacterial activity in vitro against pathogens commonly isolated from infectious corneal ulcers. METHODS: A retrospective case series and in vitro study of antibacterial activity of dehydrated AM using disk diffusion and measurement of inhibitory zones for bacterial assessment and inverted microscopy analysis for Acanthamoeba sp. growth. RESULTS: Three cases of known etiology infectious keratitis are described where the clinical presentation worsened after treatment with AM. In vitro analysis of dehydrated AM, with and without a soft contact lens, demonstrated no inhibition of growth against Pseudomonas aeruginosa or Streptococcus pneumoniae. There was minimal growth inhibition of Staphylococcus aureus, although these zones of inhibition were much smaller than that surrounding the positive control. For Acanthamoeba sp., solubilized, dehydrated AM did not alter cyst density. CONCLUSIONS: In an in vitro analysis, dehydrated AM did not provide evidence for a potentially clinically meaningful antibacterial effect against organisms commonly isolated from corneal ulcers.

High Mobility Group Box 1 in Pig Amniotic Membrane Experimentally Infected with E. coli O55.

Intra-amniotic infections (IAI) are one of the reasons for preterm birth. High mobility group box 1 (HMGB1) is a nuclear protein with various physiological functions, including tissue healing. Its excessive extracellular release potentiates inflammatory reaction and can revert its action from beneficial to detrimental. We infected the amniotic fluid of a pig on the 80th day of gestation with 1 × 104 colony forming units (CFUs) of E. coli O55 for 10 h, and evaluated the appearance of HMGB1, receptor for glycation endproducts (RAGE), and Toll-like receptor (TLR) 4 in the amniotic membrane and fluid. Sham-infected amniotic fluid served as a control. The expression and release of HMGB1 were evaluated by Real-Time PCR, immunofluorescence, immunohistochemistry, and ELISA. The infection downregulated HMGB1 mRNA expression in the amniotic membrane, changed the distribution of HMGB1 protein in the amniotic membrane, and increased its level in amniotic fluid. All RAGE mRNA, protein expression in the amniotic membrane, and soluble RAGE level in the amniotic fluid were downregulated. TLR4 mRNA and protein expression and soluble TLR4 were all upregulated. HMGB1 is a potential target for therapy to suppress the exaggerated inflammatory response. This controlled expression and release can, in some cases, prevent the preterm birth of vulnerable infants. Studies on suitable animal models can contribute to the development of appropriate therapy.

Human Amnion Epithelial Cells (AECs) Respond to the FSL-1 Lipopeptide by Engaging the NLRP7 Inflammasome.

Context and Objectives: Inflammation is the leading mechanism involved in both physiological and pathological rupture of fetal membranes. Our aim was to obtain a better characterization of the inflammasome-dependent inflammation processes in these tissues, with a particular focus on the nucleotide-binding oligomerization domain (NOD)-like receptor, pyrin domain containing protein 7 (NLRP7) inflammasome. Methods: The presence of NLRP7 inflammasome actors [NLRP7, apoptosis-associated speck-like protein containing a CARD domain (ASC), and caspase-1] was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) in human amnion and choriodecidua at the three trimesters and at term. The protein concentrations were then determined by enzyme-linked immunosorbent assay in term tissues, with or without labor. The presence of Mycoplasma salivarium and Mycoplasma fermentans in human fetal membranes was investigated using a PCR approach. Human amnion epithelial cells (AECs) were treated for 4 or 20 h with fibroblast-stimulating lipopeptide-1 (FSL-1), a M. salivarium-derived ligand. Transcripts and proteins quantity was then measured by RT-quantitative PCR and Western blotting, respectively. NLRP7 and ASC colocalization was confirmed by immunofluorescence. Western blots allowed analysis of pro-caspase-1 and gasdermin D cleavage. Results: NLRP7, ASC, and caspase-1 transcripts were expressed in both sheets of human fetal membranes during all pregnancy stages, but only ASC protein expression was increased with labor. In addition, M. salivarium and M. fermentans were detected for the first time in human fetal membranes. NLRP7 and caspase-1 transcripts, as well as NLRP7, ASC, and pro-caspase-1 protein levels, were increased in FSL-1-treated AECs. The NLRP7 inflammasome assembled around the nucleus, and pro-caspase-1 and gasdermin D were cleaved into their mature forms after FSL-1 stimulation. Conclusion: Two new mycoplasmas, M. salivarium and M. fermentans, were identified in human fetal membranes, and a lipopeptide derived from M. salivarium was found to induce NLRP7 inflammasome formation in AECs.

Comparação dos meios de preparação e preservação de membrana amniótica humana para uso no tratamento de doenças da superfície ocular / Comparison of the preparation and preservation techniques of amniotic membrane used in the treatment of ocular surface diseases

Resumo Atualmente a membra amniótica (MA) tem obtido importância devido à comprovada capacidade de reduzir inflamação, auxiliar a cicatrização e epitelização, possuindo propriedades antimicrobianas e antivirais, além de baixa imunogenicidade. As indicações de seu uso na oftalmologia têm aumentado muito nas duas últimas décadas. Objetivo: Descrever a estrutura básica e as propriedades biológicas da MA em relação aos componentes da sua matriz extracelular e fatores de crescimento, as consequências de diferentes técnicas empregadas na sua preservação e esterilização, métodos para remoção do epitélio e a comparação dos custos dos diferentes meios de conservação atualmente empregados. Métodos: Pesquisa nas bases de dados do Portal da Biblioteca Virtual em Saúde (BVS), Pubmed, Cochrane, Scielo e Lilacs com as palavras-chave: membrana amniótica, transplante, reconstrução da córnea, doenças da conjuntiva. Resultados: A literatura é vasta na descrição dos efeitos de diversos agentes e técnicas na preparação da MA, dentre elas sua preservação, esterilização e desepitelização. A membrana desnuda tem sido a escolha para a reconstrução da superfície ocular, pois facilita a cicatrização. Em relação aos agentes conservantes, o glicerol é o meio mais utilizado mundialmente pelo baixo custo e facilidade de manuseio. Conclusão: A comparação das diversas técnicas nos guia na elaboração de protocolos de preparo da MA para uso oftalmológico. A membrana desnuda facilita a cicatrização em relação a com células epiteliais. O glicerol é o meio de conservação mais utilizado pelo baixo custo e facilidade de manuseio.

Abstract Currently, the amniotic membrane (AM) has obtained importance due to its ability to reduce inflammation, helping in the healing and epithelialization processes, having antimicrobial and antiviral properties and low immunogenicity. Its indications in ophthalmology have increased considerably in the past two decades. Objective: To describe the basic structure and biological properties of the AM, the components of the extracellular matrix and growth factors, the consequences of different techniques used in its preservation, and sterilization methods for the epithelium removal. To compare the costs of the different preservation solutions currently employed. Study design: literature review. Methods: Research in BVS databases, PubMed, Cochrane, Scielo and Lilacs with keywords: amniotic membrane transplantation, corneal reconstruction, conjunctival diseases. Results: The literature is vast in describing the effects of different agents and techniques used in the preparation of MA, including its preservation, sterilization and desepithelization. The naked membrane is the choice to reconstruct the ocular surface, as it facilitates the healing course. Regarding the preservatives, glycerol is the most used worldwide due its low cost and easy handling. Conclusion: Comparing different techniques guides us in developing a MA preparation protocol for ophthalmic use. The naked membrane facilitates the healing process compared with the presence of epithelial cells. The glycerol is the most used preservation method because of its low cost and easy handling.

Validation and assessment of an antibiotic-based, aseptic decontamination manufacturing protocol for therapeutic, vacuum-dried human amniotic membrane.

Amniotic membrane (AM) is used to treat a range of ophthalmic indications but must be presented in a non-contaminated state. AM from elective caesarean sections contains natural microbial contamination, requiring removal during processing protocols. The aim of this study was to assess the ability of antibiotic decontamination of AM, during processing by innovative low-temperature vacuum-drying. Bioburden of caesarean section AM was assessed, and found to be present in low levels. Subsequently, the process for producing vacuum-dried AM (VDAM) was assessed for decontamination ability, by artificially loading with Staphylococcus epidermidis at different stages of processing. The protocol was highly efficient at removing bioburden introduced at any stage of processing, with antibiotic treatment and drying the most efficacious steps. The antibacterial activity of non-antibiotic treated AM compared to VDAM was evaluated using minimum inhibitory/biocidal concentrations (MIC/MBC), and disc diffusion assays against Meticillin-resistant Staphylococcus aureus, Meticillin-resistant S. epidermidis, Escherichia coli, Pseudomonas aeruginosa and Enterococcus faecalis. Antibacterial activity without antibiotic was low, confirmed by high MIC/MBC, and a no inhibition on agar lawns. However, VDAM with antibiotic demonstrated effective antibacterial capacity against all bacteria. Therefore, antibiotic decontamination is a reliable method for sterilisation of AM and the resultant antibiotic reservoir is effective against gram-positive and -negative bacteria.

Effects of 2 preparation methods and long-term storage on structural integrity and bacterial loads of equine amnion.

OBJECTIVE: To determine the influence of tissue preparation and long-term storage methods on structural integrity and risk of bacterial contamination of equine amnion. STUDY DESIGN: Prospective experimental investigation SAMPLE POPULATION: Amniotic membranes from 8 healthy mares (n = 440 tested samples). METHODS: Samples for baseline bacteriology and histology were taken after removal of debris. The remaining tissue was divided and processed with 0.05% chlorhexidine or 2% iodine/0.25% acetic acid. Processed amnion samples were assigned to 1 of 9 combinations of storage media (saline, chlorhexidine, acetic acid) and temperature (4 °C, -20 °C, -80 °C). Samples were submitted for quantitative bacteriology and histopathology at 1 week, 4 weeks, and 3, 6, 9, and 12 months. RESULTS: Baseline bacterial levels ranged from <200 to > 150 000 colony-forming units (cfu)/mL. None of the potentially pathogenic bacteria in baseline samples were subsequently cultured throughout the study. Nonpathogenic bacteria (median 20 cfu/mL), most commonly Bacillus sp, were cultured sporadically across storage conditions. Tissue architecture was minimally affected histologically by processing protocol, storage temperature, or storage duration. CONCLUSION: The 2 processing protocols tested here resulted in minimal bacterial contamination or loss of structural integrity of equine amnion stored for up to 12 months at 4 °C, -20 °C, or -80 °C. CLINICAL SIGNIFICANCE: Amnion collected during the foaling season may be stored for up to 12 months without significant bacterial contamination or structural alterations.

Human ß-defensin-1: A natural antimicrobial peptide present in amniotic fluid that is increased in spontaneous preterm labor with intra-amniotic infection.

PROBLEM: Human ß-defensins (HBDs) are antimicrobial peptides that participate in the soluble innate immune mechanisms against infection. Herein, we determined whether HBD-1 was present in amniotic fluid during normal pregnancy and whether its concentrations change with intra-amniotic inflammation and/or infection. METHOD OF STUDY: Amniotic fluid was collected from 219 women in the following groups: (a) midtrimester who delivered at term (n = 35); (b) term with (n = 33) or without (n = 17) labor; (c) preterm labor with intact membranes who delivered at term (n = 29) or who delivered preterm with (n = 19) and without (n = 29) intra-amniotic inflammation and infection or with intra-amniotic inflammation but without infection (n = 21); and (d) preterm prelabor rupture of membranes (pPROM) with (n = 19) and without (n = 17) intra-amniotic inflammation/infection. Amniotic fluid HBD-1 concentrations were determined using a sensitive and specific ELISA kit. RESULTS: (a) HBD-1 was detectable in all amniotic fluid samples; (b) amniotic fluid concentrations of HBD-1 were changed with gestational age (midtrimester vs term no labor), being higher in midtrimester; (c) amniotic fluid concentrations of HBD-1 were similar between women with and without spontaneous labor at term; (d) among patients with spontaneous preterm labor, amniotic fluid concentrations of HBD-1 in women with intra-amniotic inflammation/infection and in those with intra-amniotic inflammation without infection were greater than in women without intra-amniotic inflammation or infection who delivered preterm or at term; and (e) the presence of intra-amniotic inflammation and infection in patients with pPROM did not change amniotic fluid concentrations of HBD-1. CONCLUSION: HBD-1 is a physiological constituent of amniotic fluid that is increased in midtrimester during normal pregnancy and in the presence of culturable microorganisms in the amniotic cavity. These findings provide insight into the soluble host defense mechanisms against intra-amniotic infection.

Cervical fluid interleukin 6 and intra-amniotic complications of preterm prelabor rupture of membranes.

OBJECTIVE: To determine if cervical fluid interleukin (IL)-6 concentrations in women with preterm prelabor rupture of membranes (PPROM) allows identification of microbial invasion of the amniotic cavity (MIAC) and/or intra-amniotic inflammation (IAI). METHODS: One hundred forty-four women with singleton pregnancies complicated by PPROM were included in this prospective cohort study. Cervical and amniotic fluids were collected at the time of admission and concentrations of IL-6 were measured using an ELISA and point-of-care test, respectively. Cervical fluid was obtained using a Dacron polyester swab and amniotic fluid was obtained by transabdominal amniocentesis. MIAC was diagnosed based on a positive PCR result for Ureaplasma species, M. hominis, and/or C. trachomatis and/or by positivity for the 16 S rRNA gene. IAI was defined as amniotic fluid point-of-care IL-6 concentrations ≥745 pg/mL. The women were assigned to four subgroups based on the presence of MIAC and/or IAI: microbial-associated IAI (both MIAC and IAI), sterile IAI (IAI alone), MIAC alone, and without either MIAC or IAI. RESULTS: (1) Women with microbial-associated IAI had higher cervical fluid IL-6 concentrations (median 560 pg/mL) than did women with sterile IAI (median 303 pg/mL; p = .001), women with MIAC alone (median 135 pg/mL; p = .0004), and women without MIAC and IAI (median 180 pg/mL; p = .0001). (2) No differences were found in cervical fluid IL-6 concentrations among women with sterile IAI, with MIAC alone, and without MIAC and IAI. (3) A positive correlation was observed between cervical fluid IL-6 concentrations and the amount of Ureaplasma species in amniotic fluid (copies DNA/mL; rho = 0.57, p < .0001). (4) A weak positive correlation was detected between cervical and amniotic fluid IL-6 concentrations (rho = 0.33, p < .0001). CONCLUSIONS: The presence of microbial-associated IAI is associated with the highest cervical fluid IL-6 concentrations. Cervical IL-6 can be helpful in the identification of microbial-associated IAI.

A self-assembled clavanin A-coated amniotic membrane scaffold for the prevention of biofilm formation by ocular surface fungal pathogens.

Amniotic membrane (AM) is frequently used in ophthalmologic surgery for rapid ocular surface reconstruction. Sometimes it may create a major problem with associated infections after biofilm formation over the membrane. To overcome this problem, AM was coated with the antimicrobial peptide clavanin A. The antifungal activity of clavanin A in the native and self-assembled form was determined against the common ocular surface pathogens Candida albicans, Aspergillus fumigatus, Alternaria sp. and Fusarium sp. Biofilm formation over the coated surface was significantly reduced in comparison with the uncoated membrane. The coated membrane revealed effectiveness in terms of biocompatibility, cell attachment colonization when tested in non-cancerous 3T3 and human embryonic kidney (HEK)-293 cell lines. Clavanin A-coated AM also exhibited excellent physical, morphological and antifungal characteristics, indicating potential applicability for ocular surface infection control.

Chlamydia trachomatis Is Responsible for Lipid Vacuolation in the Amniotic Epithelium of Fetal Gastroschisis.

BACKGROUND: Vacuolated amniotic epithelium with lipid droplets in gastroschisis placentas is an unusual finding. Mass spectrometry of lipid droplets identified triglycerides, ester-linked to an unusual pattern of fatty acids. We hypothesize that these findings result from a Chlamydia trachomatis infection during the periconceptional period. The rising incidence of chlamydia infections has paralleled the increasing prevalence of gastroschisis among women less than 25 years of age. Histologically, young women are at greatest risk for a chlamydia infection due to their immature columnar epithelium, the preferential site for attachment of Chlamydia trachomatis infectious particle (elementary body). METHODS: Chlamydia trachomatis survive in an inclusion, relying on its host to acquire essential nutrients, amino acids, and nucleotides for survival and replication. If essential nutrients are not available, the bacteria cannot replicate and may be trafficked to the lysosome for degradation or remain quiescent, within the inclusion, subverting innate immunologic clearance. RESULTS: Chlamydiae synthesize several lipids (phosphatidylethanolamine, phosphatidylserine, and phosphoatidylglycerol); however, their lipid content reveal eukaryotic lipids (sphingomyelin, cholesterol, phosphatidylcholine, and phosphatidylinositol), evidence that chlamydiae "hijack" host lipids for expansion and replication. CONCLUSION: The abnormal amniotic epithelial findings are supported by experimental evidence of the trafficking of host lipids into the chlamydiae inclusion. If not lethal, what harm will elementary bodies inflict to the developing embryo? Do these women have a greater pro-inflammatory response to an environmental exposure, whether cigarette smoking, change in partner, or a pathogen? Testing the hypothesis that Chlamydia trachomatis is responsible for amniotic epithelium vacuoles will be a critical first step. Birth Defects Research 109:1003-1010, 2017. © 2017 Wiley Periodicals, Inc.

Twenty-four percent of patients with clinical chorioamnionitis in preterm gestations have no evidence of either culture-proven intraamniotic infection or intraamniotic inflammation.

BACKGROUND: Recent studies on clinical chorioamnionitis at term suggest that some patients with this diagnosis have neither intraamniotic infection nor intraamniotic inflammation. A false-positive diagnosis of clinical chorioamnionitis in preterm gestation may lead to unwarranted preterm delivery. OBJECTIVE: We sought to determine the frequency of intraamniotic inflammation and microbiologically proven amniotic fluid infection in patients with preterm clinical chorioamnionitis. STUDY DESIGN: Amniocentesis was performed in singleton pregnant women with preterm clinical chorioamnionitis (<36 weeks of gestation). Amniotic fluid was cultured for aerobic and anaerobic bacteria and genital mycoplasmas and assayed for matrix metalloproteinase-8 concentration. Microbial invasion of the amniotic cavity was defined as a positive amniotic fluid culture; intraamniotic inflammation was defined as an elevated amniotic fluid matrix metalloproteinase-8 concentration of >23 ng/mL. Nonparametric and survival techniques were used for analysis. RESULTS: Among patients with preterm clinical chorioamnionitis, 24% (12/50) had neither microbiologic evidence of intraamniotic infection nor intraamniotic inflammation. Microbial invasion of the amniotic cavity was present in 34% (18/53) and intraamniotic inflammation in 76% (38/50) of patients. The most common microorganisms isolated from the amniotic cavity were the Ureaplasma species. Finally, patients without microbial invasion of the amniotic cavity or intraamniotic inflammation had significantly lower rates of adverse outcomes (including lower gestational age at delivery, a shorter amniocentesis-to-delivery interval, acute histologic chorioamnionitis, acute funisitis, and significant neonatal morbidity) than those with microbial invasion of the amniotic cavity and/or intraamniotic inflammation. CONCLUSION: Among patients with preterm clinical chorioamnionitis, 24% had no evidence of either intraamniotic infection or intraamniotic inflammation, and 66% had negative amniotic fluid cultures, using standard microbiologic techniques. These observations call for a reexamination of the criteria used to diagnose preterm clinical chorioamnionitis.

Amniotic membrane in ophthalmology: properties, preparation, storage and indications for grafting-a review.

The use of amniotic membrane in ophthalmic surgery and other surgical procedures in the fields of dermatology, plastic surgery, genitourinary medicine and otolaryngology is on the increase. Furthermore, amniotic membrane and its epithelial and mesenchymal cells have broad use in regenerative medicine and hold great promise in anticancer treatment. Amniotic membrane is a rich source of biologically active factors and as such, promotes healing and acts as an effective material for wound dressing. Amniotic membrane supports epithelialization and exhibits anti-fibrotic, anti-inflammatory, anti-angiogenic and anti-microbial features. Placentas utilised in the preparation of amniotic membrane are retrieved from donors undergoing elective caesarean section. Maternal blood must undergo serological screening at the time of donation and, in the absence of advanced diagnostic testing techniques, 6 months postpartum in order to cover the time window for the potential transmission of communicable diseases. Amniotic membrane is prepared by blunt dissection under strict aseptic conditions, then is typically transferred onto a nitrocellulose paper carrier, usually with the epithelial side up, and cut into multiple pieces of different dimensions. Amniotic membrane can be stored under various conditions, most often cryopreserved in glycerol or dimethyl sulfoxide or their mixture with culture medium or buffers. Other preservation methods include lyophilisation and air-drying. In ophthalmology, amniotic membrane is increasingly used for ocular surface reconstruction, including the treatment of persistent epithelial defects and non-healing corneal ulcers, corneal perforations and descemetoceles, bullous keratopathy, as well as corneal disorders with associated limbal stem cell deficiency, pterygium, conjunctival reconstruction, corneoscleral melts and perforations, and glaucoma surgeries.

Neutrophil Extracellular Traps in the Amniotic Cavity of Women with Intra-Amniotic Infection: A New Mechanism of Host Defense.

OBJECTIVE: Neutrophil extracellular traps (NETs) control microbial infections through their antimicrobial activities attributed to DNA, histones, granules, and cytoplasmic proteins (eg, elastase). Intra-amniotic infection is characterized by the influx of neutrophils into the amniotic cavity; therefore, the aim of this study was to determine whether amniotic fluid neutrophils form NETs in this inflammatory process. METHODS: Amniotic fluid samples from women with intra-amniotic infection (n = 15) were stained for bacteria detection using fluorescent dyes. Amniotic fluid neutrophils were purified by filtration. As controls, neutrophils from maternal blood samples (n = 3) were isolated by density gradients. Isolated neutrophils were plated onto glass cover slips for culture with and without 100 nM of phorbol-12-myristate-13-acetate (PMA). NET formation was assessed by 4',6-diamidino-2-phenylindole (DAPI) staining and scanning electron microscopy. Different stages of NET formation were visualized using antibodies against elastase and histone H3, in combination with DAPI staining, by confocal microscopy. Finally, maternal or neonatal neutrophils were added to amniotic fluid samples from women without intra-amniotic infection (n = 4), and NET formation was evaluated by DAPI staining. RESULTS: (1) NETs were present in the amniotic fluid of women with intra-amniotic infection; (2) all of the amniotic fluid samples had detectable live and dead bacteria associated with the presence of NETs; (3) in contrast to neutrophils from the maternal circulation, amniotic fluid neutrophils did not require PMA stimulation to form NETs; (4) different stages of NET formation were observed by co-localizing elastase, histone H3, and DNA in amniotic fluid neutrophils; and (5) neither maternal nor neonatal neutrophils form NETs in the amniotic fluid of women without intra-amniotic infection. CONCLUSION: NETs are detectable in the amniotic fluid of women with intra-amniotic infection.

Infections in the tissue material and their impact on the loss of transplants in the Laboratory of in vitro Cell and Tissue Culture with Tissue Bank in the years 2011-2015.

Radiation sterilization eliminates microbiological infections but causes the degradation of the cell factor. The negative result of microbiological examination for tissue transplants is one of the conditions for approval for distribution in patients. The study attempts to verify impact of the presence of microbes onto material for transplant loss. In the 2011-2015 period, we analyzed 293 donors of skin and amnion. Microbiological sampling was performed. The total of 21 strains of bacteria, molds and fungi was identified in collected tissue. The widest spectrum of strains was found in skin (17), followed by amnia (8). The total number of positive findings was 147 and was again highest in skin (129), while the number of positive findings in amnia was 18 only. The general percentage of fungal infections was very low. The presence of fungal strains was only observed in allogeneic skin (2%). Large number of microorganisms isolated from the skin before sterilization was observed, so it seems impossible to use allogeneic intravital skin. However, the intravital application of allogeneic amnion obtained from cesarean section remains to be considered.

Membrane Vesicles of Group B Streptococcus Disrupt Feto-Maternal Barrier Leading to Preterm Birth.

Infection of the genitourinary tract with Group B Streptococcus (GBS), an opportunistic gram positive pathogen, is associated with premature rupture of amniotic membrane and preterm birth. In this work, we demonstrate that GBS produces membrane vesicles (MVs) in a serotype independent manner. These MVs are loaded with virulence factors including extracellular matrix degrading proteases and pore forming toxins. Mice chorio-decidual membranes challenged with MVs ex vivo resulted in extensive collagen degradation leading to loss of stiffness and mechanical weakening. MVs when instilled vaginally are capable of anterograde transport in mouse reproductive tract. Intra-amniotic injections of GBS MVs in mice led to upregulation of pro-inflammatory cytokines and inflammation mimicking features of chorio-amnionitis; it also led to apoptosis in the chorio-decidual tissue. Instillation of MVs in the amniotic sac also resulted in intrauterine fetal death and preterm delivery. Our findings suggest that GBS MVs can independently orchestrate events at the feto-maternal interface causing chorio-amnionitis and membrane damage leading to preterm birth or fetal death.

Regulatory T cell frequencies are increased in preterm infants with clinical early-onset sepsis.

The predisposition of preterm neonates to invasive infection is, as yet, incompletely understood. Regulatory T cells (Tregs ) are potential candidates for the ontogenetic control of immune activation and tissue damage in preterm infants. It was the aim of our study to characterize lymphocyte subsets and in particular CD4(+) CD25(+) forkhead box protein 3 (FoxP3)(+) Tregs in peripheral blood of well-phenotyped preterm infants (n = 117; 23 + 0 - 36 + 6 weeks of gestational age) in the first 3 days of life in comparison to term infants and adults. We demonstrated a negative correlation of Treg frequencies and gestational age. Tregs were increased in blood samples of preterm infants compared to term infants and adults. Notably, we found an increased Treg frequency in preterm infants with clinical early-onset sepsis while cause of preterm delivery, e.g. chorioamnionitis, did not affect Treg frequencies. Our data suggest that Tregs apparently play an important role in maintaining maternal-fetal tolerance, which turns into an increased sepsis risk after preterm delivery. Functional analyses are needed in order to elucidate whether Tregs have potential as future target for diagnostics and therapeutics.

Amniotic fluid calreticulin in pregnancies complicated by the preterm prelabor rupture of membranes.

OBJECTIVE: This study aimed to determine the amniotic fluid calreticulin concentrations in women with the preterm prelabor rupture of membranes (PPROM) based on the microbial invasion of the amniotic cavity (MIAC), intraamniotic inflammation (IAI) and microbial-associated IAI. METHODS: One hundred sixty-eight women with singleton pregnancies were included in this study. Amniotic fluid samples were obtained by transabdominal amniocentesis and were assayed for calreticulin concentrations by ELISA. IAI was defined as an amniotic fluid interleukin-6 concentration > 745 pg/ml. Microbial-associated IAI was defined as the presence of both MIAC and IAI. RESULT: Women with MIAC (with MIAC: median 54.4 ng/ml, versus without MIAC: median 32.6 ng/ml; p < 0.0001), IAI (with IAI: median 66.8 ng/ml, versus without IAI: median 33.0 ng/ml; p < 0.0001) and microbial-associated IAI (with microbial-associated IAI: median 82.5 ng/ml, versus without microbial-associated IAI: median 33.7 ng/ml; p < 0.0001) had higher concentrations of calreticulin than women without these complications. An amniotic fluid calreticulin concentration of 81.4 ng/ml was found to be the best cutoff point for identifying women with microbial-associated IAI. CONCLUSIONS: The presence of microbial-associated IAI is associated with increased amniotic fluid calreticulin concentrations. Calreticulin seems to be a promising marker for the early identification of PPROM complicated by microbial-associated IAI.