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The placenta acts as physical and immunological barrier against the transmission of viruses and bacteria from mother to fetus. However, the specific mechanisms by which the placenta protects the developing fetus from viral and bacterial pathogens are poorly understood.
To identify placental peptides and small proteins protecting from viral and bacterial infections, we generated a peptide library from 10 kg placenta by chromatographic means. Screening the resulting fractions against Herpes-Simplex-Virus 2 HSV-2which is rarely transmitted through the placenta, in a cell-based system identified two adjacent fractions with ificant antiviral activity. Further rounds of chromatographic purification and anti-HSV-2 testing allowed to purify the bioactive peptide.
Full-length hemoglobin tetramer had no antiviral activity. HBB — did not impair infectivity by direct targeting of the virions but prevented HSV-2 infection at the cell entry level. Notably, HBB — has ly been identified as broad-spectrum antibacterial agent. We here additionally show, that HBB — also acts potently against Pseudomonas aeruginosa strains including a multi-drug resistant strain in a dose dependent manner, while full-length hemoglobin is inactive.
Interestingly, the antibacterial activity of HBB — was increased under acidic conditions, a hallmark of infection and inflammatory conditions. Indeed, we found that HBB — is released from the hemoglobin precursor by Cathepsin D and Napsin A, acidic proteases highly expressed in placental and other tissues.
We propose that upon viral or bacterial infection, the abundant hemoglobin precursor is proteolytically processed to release HBB —a broadly active antimicrobial innate immune defense peptide. The placenta is a fetal organ tightly interacting with maternal blood vessels to nourish and protect the fetus Robbins and Bakardjiev, Placental infections by pathogens are major causes of disease and represent a substantial source of human morbidity and mortality. The placenta is composed of villi that float in maternal blood and organize the exchange of substances between the mother and fetus.
At this interface bacteria and viruses may enter the placenta and be transmitted to the fetus. Thus, the placenta is an important transmission site for viral and bacterial infections, and therefore it is conceivable that this organ evolved antiviral and antibacterial innate immune defense mechanisms that prevent or restrict diaplacental pathogen transmission.
Indeed, only relatively few pathogens are capable of placental and fetal infections in humans Robbins and Bakardjiev, For example, most viral dia-placental transmissions are caused by Cytomegalovirus, Lymphocytic choriomeningitis virus, Parvovirus B19, Rubella Virus and Varicella zoster virus Robbins and Bakardjiev,but generally the frequencies are low.
More recently, it has also been shown that Zika virus ZIKV may cross the placenta and In search of Roan Mountain and relationship hsv2 the fetus which may result in severe developmental defects of the fetus Zanluca et al. However, for the majority of viral pathogens the placenta represents an impenetrable barrier Robbins and Bakardjiev, For example, Herpes simplex viruses HSV -1 and -2 have infected billions of people. In the US, neonatal infection with HSV occurs in 1 out of every —10, live births and is associated with serious morbidity and mortality, and leaves many survivors with permanent sequelae Brown et al.
Interestingly, most congenital HSV infections are transmitted during delivery whereas reports on transplacental transmission and fetal infection are extremely rare Koi et al. Likewise, as part of the innate immune system, the placenta plays an important role in prevention of bacterial infection during pregnancy Mor and Kwon, Trophoblast cells carry toll-like receptors and thus are able to recognize bacteria via pattern recognition receptors to elicit an immune response Abrahams et al.
Failure to prevent bacterial infection during pregnancy may result in the termination of pregnancy and preterm delivery. While bacterial infections have clearly been implicated In search of Roan Mountain and relationship hsv2 one of the most important reason for preterm deliveries, the specific bacterial species that are responsible remain elusive. Nevertheless, this situation underlines the importance of an intact placental barrier. The potent barrier function that prevents dia-placental transmission of pathogens is likely due to a lack of intercellular junctions of the syncytiotrophoblast comprising most of the maternofetal interface and an environment rich in innate immune defense molecules Robbins and Bakardjiev, One part of the innate immune response consists of antimicrobial peptides AMPs that are constantly expressed or released upon TLR stimulation Hertz et al.
Such peptide libraries are generated from pooled body fluids by chromatographic means and typically consist of — peptide fractions that contain all peptides and small proteins of the respective source material in a lyophilized and concentrated form.
Screening these libraries for antibacterial fractions and subsequent purification of the bioactive peptides allowed to identify, e. Thus, the concept of isolating novel AMPs from body fluids resulted in the identification of peptides that inhibited bacterial and viral infections by novel and unexpected mechanisms. Here, we sought to adapt this technology and generated a peptide library from human placenta to identify endogenous inhibitors that may restrict HSV-2 infection and bacterial pathogens.
Ten kilograms of human placenta were obtained from healthy individuals in a maternity ward of a local hospital and processed immediately after delivery. All subjects gave written informed consent in accordance with the Declaration of Helsinki. First, the organs were homogenized and peptides and small proteins extracted by an ice-cold acetic acid extraction procedure. Thereafter, the obtained peptides and proteins were separated by an ultrafiltration step cut-off: 50 kDa. The resulting peptides were then separated in a first dimension by means of their isoelectric point using cation exchange chromatography resulting in pH pool fractions 1—6.
Each pH pool was then further separated in a second dimension by peptide hydrophobicity using reversed phase chromatography resulting in a total of fractions as described in detail before Liepke et al. All values represent reporter gene activities derived from triplicates minus background activities derived from uninfected cells. The instrument is equipped In search of Roan Mountain and relationship hsv2 a 1. Positive ions are accelerated at 30 kV and laser shots are automatically accumulated per sample. Accuracy of mass measurement was 0. Native HBB — was isolated in large amounts from human placenta.
Infection rates were determined 1 or 2 day s later as described above. Figure 1. Identification of a C-terminal fragment of hemoglobin as HSV-2 inhibitor in a placenta-derived peptide library. The peaks correspond to HBB — Peptides were added to ELVIS cells at indicated concentrations and cells were infected after 30 min incubation. Figure 2. The hemoglobin fragment blocks an early step in the HSV-2 life cycle. A Indicated concentrations of HBB — were added to cells and incubated for 30 min.
Thereafter, cells were either infected directly with HSV-2 cell treatment or washed, supplemented with fresh medium and then infected wash. Additionally, virus was first exposed to HBB — for 3 h at indicated concentrations, and then these mixtures were used to infect cells resulting in a fold dilution of the inoculum.
After 3—5 days the virus was collected by centrifuging the cell supernatant for 3 min at g to remove cell debris. Two days later infection rates were determined with a cell-based ZIKV immunodetection assay. After an incubation of 5 min at room temperature, reaction was stopped with 0. Mixtures were used to inoculate Vero cells. Following a 2. Absorption was measured at nm and baseline was corrected at nm using a Vmax kinetic microplate reader. Digestion experiments were carried out with purified human hemoglobin Sigma H and recombinant or purified proteases.
Staining was then performed with GelCode Blue coll oidal coomassie overnight.
Destaining was done with ultrapure water until the background appeared clear, the gel then imaged in a LiCor Odyssey system. The CD al was recorded from to nm with a bandwidth of 1 nm. The data pitch was set to 0. Each sample was measured in three individual scans and the data were accumulated. Spectra are baseline corrected against MilliQ water, smoothened Means Movement, convolution width 17 and normalized. For visualization, spectral data was exported from the datafile using XCalibur Qual Browser 2.
Carbamidomethylated cysteine was considered as a fixed modification along with oxidation M as a variable modification. Following resuspension in 10 mM sodium phosphate buffer optical density was determined at nm OD nm. For LPS inhibition experiments, The samples were treated with HBB — of different concentrations or were mock treated with ddH 2 O. The fluorescent intensity of the samples was measured in a Tecan infinite M plate reader with an excitation wavelength of nm and an emission wavelength of nm.
The relative fluorescence of the samples was calculated by normalizing the peptide treated samples to the mock treated samples grown in the corresponding assay medium. The pelleted cells were fixed with 2. Ultra-thin sections 80 nm were collected on copper grids, contrasted with 0. Protein sequences were aligned using Clustal W 3 and residue conservation was determined using the Scorecons Server 4.
This convention follows that of Mirny and Shakhnovich In an attempt to identify endogenous antiviral and antibacterial peptides in placenta, we generated a peptide library from 10 kg of pooled homogenized placental tissue. Human tissue was obtained from healthy individuals in a maternity ward of a local hospital and was processed immediately after delivery.
Peptides were extracted by an ice-cold acetic acid extraction procedure and the obtained peptide and protein fractions In search of Roan Mountain and relationship hsv2 separated by ultrafiltration cut-off: 50 kDa. The peptide fraction was then chromatographically separated and resulted in approx.In search of Roan Mountain and relationship hsv2
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Localized and Systemic Immune Response in Human Reproductive Tract