Amniotic Membrane Cultures Research Articles

Time to reconsider antibiotic prophylaxis in women with prolonged rupture of membranes: The trend of Enterobacteriaceae in peripartum infections.

The proportion of neonatal early-onset sepsis (EOS) by gram-negative bacteria has increased. The authors examined bacterial distribution in the amniotic membrane cultures of women with peripartum fever (PPF) and related perinatal outcomes. This retrospective study covered the period 2011 to 2019. The primary outcomes were Enterobacteriaceae-positive birth culture rates in women with PPF and the trend of ampicillin resistance. Maternal and neonatal outcomes were compared between women with group B Streptococcus (GBS) and Enterobacteriaceae-positive isolates. Bacterial distribution was also compared according to rupture of membrane (ROM) duration. Among 621 women with PPF, the positive birth culture rate was 52%. Increasing prevalences of ampicillin-resistant Enterobacteriaceae (81%) were noted. Positive birth cultures were associated with maternal bacteremia (P = 0.017) and neonatal EOS (P = 0.003). Prolonged ROM ≥18 h was associated with increased risk for Enterobacteriaceae-positive cultures, while intrapartum ampicillin and gentamicin were associated with lower risk. Enterobacteriaceae-positive compared with GBS-positive birth cultures were associated with adverse maternal and neonatal outcomes. Positive birth cultures were related to maternal bacteremia and neonatal sepsis. Adverse outcomes were more prevalent among women with Enterobacteriaceae-positive versus GBS-positive birth cultures. Prolonged ROM is a risk factor for Enterobacteriaceae-positive birth cultures among women with PPF. Antibiotic prophylaxis treatment for prolonged ROM should be reconsidered.

Reconsidering the Current Preterm Premature Rupture of Membranes Antibiotic Prophylactic Protocol.

The purpose of our study was to determine whether the current antibiotic regimen for preterm premature rupture of membranes (PPROM) is adequate for covering the current causative agents and sensitivities of chorioamnionitis and early-onset neonatal sepsis. During a 3-year period, we retrieved the results from placental and amniotic membrane cultures obtained at delivery in cases of maternal fever, chorioamnionitis, and PPROM, and from blood cultures obtained from neonates with early-onset sepsis (EOS) in three participating hospitals. Sensitivity of pathogens to antimicrobial agents was performed using routine microbiologic techniques. There were 1,133 positive placental or amniotic cultures, 740 (65.3%) were from gram-negative Enterobacteriaceae. There were 27 neonates diagnosed with EOS with positive blood cultures. Aerobic Enterobacteriaceae accounted for 14 cases (52%) and group B streptococcus for 7 cases (26%). Of the Escherichia coli and Klebsiella sp., only 38% were sensitive to ampicillin. Local pathogens and their antibiotic sensitivity profiles should be explored every few years and an effective antibiotic protocol chosen to cover the main pathogens causing chorioamnionitis and EOS. Consideration should be made for changing ampicillin in women with PPROM to a regimen with better coverage of gram-negative Enterobacteriaceae.

Keratocan Expression of Murine Keratocytes Is Maintained on Amniotic Membrane by Down-regulating Transforming Growth Factor-β Signaling

Keratocytes in the corneal stroma express keratan sulfate-containing proteoglycans including cornea-specific keratocan. On plastic dishes, human, bovine, and rabbit keratocytes lose their characteristic dendritic morphology and keratocan expression when cultured in serum-containing media. Herein, we demonstrated that murine keratocytes also acquired a fibroblastic shape and lost keratocan expression after first passage when cultured on plastic in the presence of serum. In contrast, cells expanded on human amniotic membrane (AM) stromal matrix maintained a three-dimensional dendritic morphology and expressed keratocan mRNA and protein for at least 8 passages before senescence. When keratocytes were cultured on AM, the promoter activity of transforming growth factor (TGF)-beta2 and TGF-beta receptor II was down-regulated as compared with that on plastic. Furthermore, cells on AM continuously retained Smad 2 and Smad 4 in the cytoplasm and did not express alpha-smooth muscle actin, even when 10 ng/ml TGF-beta1 was added in a serum-free medium for up to 5 days. In parallel to such down-regulation of TGF-beta signaling, keratocan promoter-driven ECFP expression was observed in cells cultured either on AM in the presence of serum or on plastic containing serum treated with a neutralizing antibody to TGF-beta. Collectively, these results indicate that down-regulation of Smad-mediated TGF-beta signaling is an important mechanism for cultured keratocytes to maintain a normal phenotype while continuously expanded in a serum-containing medium. This strategy of suppressing TGF-beta signaling, achieved by AM stromal matrix in part via suppression of TGF-beta gene transcription, can be used to expand keratocytes in culture without the use of AM in the future.

Expansion of Conjunctival Epithelial Progenitor Cells on Amniotic Membrane

Amniotic membrane (AM) reconstructed human conjunctival surfaces recover a goblet cell density higher than normal. Cultured rabbit conjunctival epithelial cells (RCE) on AM preferentially exhibit non-goblet epithelial differentiation. It was thus wondered if conjunctival progenitor cells that might have been preserved during ex vivo expansion on AM can still differentiate into conjunctival non-goblet epithelial and goblet cells under the influence of mesenchymal cells.Fourteen day old AM cultures of RCE were subcutaneously implanted in Balb/c athymic mice for 11 days and processed for PAS staining and immunostaining with monoclonal antibodies to conjunctival goblet cell mucin (MUC5AC, AM3), glycocalyx (AMEM2), cornea specific cytokeratins K3 (AE5) and K12 (AK2) and basal cell specific cytokeratin K14. Cell cycle kinetics were measured by BrdU labelling for 1 or 7 days. The 7 day labelled RCE were chased for 14 days in the same primary culture.After subcutaneous implantation, conjunctival non-goblet epithelial cells increased stratification and formed occasional cysts. The resultant epithelial phenotype was conjunctival with many PAS-positive, MUC5AC-positive, and AM3-positive goblet cells, AMEM2-positive suprabasal and superficial cells, and K14-positive basal cells, but was not corneal (negative to AE5 and AK2 staining). Twenty four hr BrdU labelling showed a labelling index of 42.5%. A higher labelling index or 69% was noted after continuous BrdU labelling for 7 days. A large number of label retaining basal cells with a labelling index of 84% were noted following 14 days of chase.Conjunctival epithelial progenitor cells for goblet and non-goblet cell differentiation are preserved by AM in vitro as evidenced by being able to differentiate into goblet cells in a permissive stromal environment, and being slow-cycling, and label retaining. This information is useful for future ex vivo expansion of conjunctival epithelial stem cells for conjunctival surface reconstruction.

Toxic shock syndrome after laminaria insertion

BACKGROUND: Laminaria tents used to facilitate surgical abortion are rarely associated with significant infectious morbidity. CASE: A parous woman in midpregnancy had laminaria placed in her cervix followed by a second set after 24 hours. Eight hours later, she presented with dyspnea, hives, fever, tachycardia, and hypotension. Antibiotic treatment was initiated and a dilation and evacuation procedure was performed. Amniotic membrane cultures showed a heavy growth of Staphylococcus aureus with staphylococcal enterotoxin C expression, compatible with toxic shock syndrome. CONCLUSION: Laminaria cervical dilation might be associated with toxic shock syndrome.

Toxic Shock Syndrome After Laminaria Insertion

In Brief BACKGROUND Laminaria tents used to facilitate surgical abortion are rarely associated with significant infectious morbidity. CASE A parous woman in midpregnancy had laminaria placed in her cervix followed by a second set after 24 hours. Eight hours later, she presented with dyspnea, hives, fever, tachycardia, and hypotension. Antibiotic treatment was initiated and a dilation and evacuation procedure was performed. Amniotic membrane cultures showed a heavy growth of Staphylococcus aureus with staphylococcal enterotoxin C expression, compatible with toxic shock syndrome. CONCLUSION Laminaria cervical dilation might be associated with toxic shock syndrome. Laminaria used for cervical dilation might be associated with toxic shock syndrome.

Study on Risk Factors for Transplacental Viral Infections; Effect of Bacterial Factors and Double Viral Infections on Virus Replication in Placenta and Amniotic Membranes

Among risk factors for vertical transmission of HIV there are listed concomitant viral and bacterial infections. Therefore the influence on the viruses replication in human placenta and amniotic membrane cultures of double viral infection with two unrelated viruses—encephalomyocarditis (EMCV) and vesicular stomatitis virus (VSV)—was studied and compared with the replication of the viruses in single virus infection (EMCV or VSV) in the same organ cultures. Additionally effect of bacterial factors—lipopolysaccharide (LPS) Escherichia coli and sonicated Treponema pallidum antigens (Tpa)—on VSV replication in the same culture system was studied and compared with VSV replication in untreated explants. Two effects were observed in double-virus infected cultures and also in bacterial factors treated cultures: inhibition and stimulation of virus replication. The kind of effect in the both cases was dependent on the presence or absence of innate antiviral immunity. In virus-sensitive organs double infected or treated with LPS or Tpa, inhibition of virus titer (2–5 log TCID50/ml) was observed. In the organs expressing the innate immunity, stimulation (1–4 log TCID50/ml) of virus replication was noticed. Contribution of endogenous TNFα in both reactions (stimulation and inhibition) was confirmed using antibodies against the TNF.

Suppression of transforming growth factor-beta isoforms, TGF-beta receptor type II, and myofibroblast differentiation in cultured human corneal and limbal fibroblasts by amniotic membrane matrix.

Down-regulation of the transforming growth factor-beta (TGF-beta) signaling system is a strategy for preventing scarring during wound healing. Human corneal and limbal fibroblasts were cultured on the stromal matrix side of preserved human amniotic membrane. The levels of TGF-beta1, beta2, and beta3 and TGF-beta type II receptor transcripts and TGF-beta1 and beta2 proteins were suppressed as early as 8 hr and more dramatically at 24 hr after contact with an amniotic membrane. This suppressive effect was accompanied by down-regulation of alpha-smooth muscle actin, EDA spliced form of fibronectin, and integrin alpha5. It persisted even when challenged by 10 ng/ml TGF-beta1. In contrast with their counterparts grown on plastic or in collagen gel, such suppression in amniotic membrane cultures remained complete after 1 week of culturing. Cells cultured on amniotic membrane showed significantly reduced [3H]-thymidine incorporation compared to cells cultured on plastic and displayed no DNA fragmentation. These results reveal a novel mechanism by which the TGF-beta signaling system, DNA synthesis, and subsequent myofibroblast differentiation can be suppressed by an amnionic membrane matrix. This action explains in part the antiscarring results of amniotic membrane transplantation used for ocular surface reconstruction, a surgical technique applicable to other subspecialties. It may also explain in part why fetal wound healing is scarless.

Mill Hill Strain of Parainfluenza 1 Virus Is a Potent Human Interferon Inducer

The interferon (IFN)-inducing capacities of the widely used Cantell strain of Sendai and the Mill Hill strain of parainfluenza 1 were compared. The Mill Hill strain proved to be a more potent inducer of IFN than the Cantell strain when tested in human peripheral blood leukocytes and amniotic membrane cultures. The Mill Hill strain did not produce defective particles readily, nor did these particles appear necessary for IFN induction. The Mill Hill strain of parainfluenza 1 virus appears superior to the Cantell strain of Sendai virus for the production of natural human IFN.

A microbiologic and clinical study of placental inflammation at term

This study describes the relationships between histologically evident inflammation of the placenta, membranes, and umbilical cords, and correlates these lesions with clinical outcome and with amniotic fluid and amniotic membrane cultures in pregnancies at risk for clinical infection. The overall frequency of inflammation in 123 placentas was 85.45%. There were good interrelationships between inflammatory lesions at various sites. Membrane infiltrates of 3+ were seen in 90.9% of intra-amniotic infection cases, but in only 18.4% of asymptomatic patients (P less than .001). Conversely, 76.9% of patients with 3+ inflammation had intra-amniotic infection, whereas only 7% with lesser degrees of inflammation had intra-amniotic infection. Inflammation of any degree (1-3+) had a low specificity (28%) for febrile maternal outcome. Bacteria were recovered in 117 (95.1%) of the amniotic fluids. The grade of histologic lesions was associated with total colony count of bacteria in amniotic fluid (P less than .05) and with high-virulence bacteria in amniotic fluid (P less than .05), and Mycoplasma hominis in amniotic fluid (P less than .05). Bacteria were found in 67.6% and mycoplasmas in 18.6% of amniotic membrane cultures. Chlamydia trachomatis was not recovered in any of 35 amniotic membrane cultures. No significant correlation was seen between organisms in the amniotic membrane and histologic inflammation. Thus, positive amniotic fluid culture results are associated with histologic inflammation, and may cause the inflammatory response. A logistic regression model revealed that predictors of histologic inflammation include maternal diagnosis, amniotic fluid colony count, and M hominis in amniotic fluid.

Inclusion body formation with Newcastle disease and mumps viruses in cultures of chick embryo cells

Twelve strains of Newcastle disease virus (NDV) were grown in roller cultures of chick embryo chorioallantoic membrane (CAM). In each case cytopathogenic effects including eosinophilic cytoplasmic inclusion body formation accompanied viral multiplication. On repeated subculture the virus continued to multiply and to produce the same cytopathogenic changes. Inclusion body formation also was demonstrated in CAM cultures for the third, thirty-ninth, and eighty-seventh egg passages of one strain of mumps virus. This virus continued to multiply and to produce inclusions on subculture. An early egg passage of one other mumps strain failed to produce inclusions. Changes demonstrated for NDV and mumps strains in chick embryo amniotic membrane cultures were similar to those in CAM. The effects of both Newcastle and mumps viruses were prevented by specific antiserum. Other representative myxoviruses failed to produce inclusions in these tissue culture systems.