Standard calibration curves are used to determine the concentrations of SLPI in infant saliva and breast milk. The effect of HIV exposure on the concentration of SLPI and E/tr-2 protein levels in breast milk.
INTRODUCTION
Exclusive breastfeeding (EBF) reduces the risk of postnatal HIV transmission compared to mixed feeding (Kuhn et al. 2007). Breastfed infants acquire HIV by ingesting the virus in breast milk (Coutsoudis et al. 1999), although this route of infection is inefficient.
HIV EPIDEMIC
HIV STRUCTURE AND THE MECHANISM OF ACTION
The virus uses coreceptors CXCR4 and CCR5 to gain access to CD4+ T cells (Bleul et al. 1997). The fusion causes the release of viral RNA into the host cell cytoplasm (Wilen et al. 2012) as depicted in figure 1.2.2.2.
HIV TRANSMISSION
- MOTHER TO CHILD HIV TRANSMISSION
The inhibitory effect exerted by human milk is due to various innate factors dispersed in mucosal secretions, which are discussed in detail in section 1.4 (Farquhar et al. 2002).
INFANT IMMUNE SYSTEM
Neonatal immunity shows a reduced ability to mount effective immune responses against invading pathogens due to insufficient production of essential immune factors such as pro-inflammatory cytokines TNF-. This results in an increased susceptibility to infection by invading bacterial and viral pathogens and is therefore highly dependent on breast milk immune protection. Breastfeeding also promotes the development of the neonatal immune system due to the bioactive components found in breast milk.
BREAST MILK
B REAST MILK BIOACTIVE COMPONENTS
Breast milk also contains antimicrobial peptides such as lysozyme, defensins and lactoferrin (Henrick et al. 2017). Lactoferrin has a high affinity for iron and therefore inhibits the proliferation of iron-dependent pathogenic bacteria (Spadaro et al. 2008).
THE ORAL MUCOSAE AND IMMUNOLOGICAL PROTECTION AGAINST PATHOGENS
PROTECTIVE IMMUNOLOGICAL PROPERTIES IN ORAL SECRETIONS
Also included in oral secretion are histatins, peptides that have antimicrobial activity against gram-negative bacteria. These peptides exert their antimicrobial activity by neutralizing the LPS of bacterial external membranes (Kavanagh & Dowd 2004).
HIV AND INNATE FACTORS IN SALIVA AND BREAST MILK
- WHEY ACIDIC PROTEINS (WAP)
- SECRETORY LEUKOCYTES PROTEASE INHIBITORS (SLPI)
- PROPERTIES OF SLPI AND ELAFIN/TRAPPIN-2
- ANTI-MICROBIAL AND ANTI-INFLAMMATORY ACTIVITY
- TISSUE REMODELLING AND WOUND HEALING
- ANTI-VIRAL ACTIVITY
- ANTI-HIV MECHANISMS OF SLPI AND E/TR-2
Similar to SLPI, the C-terminal domain of Elafin blocks tissue proteolysis by neutrophils (Moreau et al. 2008). SLPI and E/tr-2 play a significant role in tissue remodeling and wound healing (Doumas et al. 2005).
STUDY HYPOTHESIS
STUDY AIMS AND OBJECTIVES
MATERIALS & METHODS
- STUDY DESIGN
- STUDY SITE
- STUDY PARTICIPANTS
- CLINICAL ASSESSMENTS
- FEEDING PRACTICE
- ETHICAL CONSIDERATIONS
- SAMPLE COLLECTION AND PROCESSING
- INFANT SALIVA
- MATERNAL BREAST MILK
- SANDWICH ELISA USED TO MEASURE INNATE PROTEINS
- HUMAN SLPI QUANTIKINE ELISA
- E/TR-2- HUMAN TRAPPIN-2/ELAFIN DUO SET ELISA KIT
- STATISTICAL ANALYSIS
- OPTIMIZATION OF ELISA METHODOLOGY
- SPIKE AND RECOVERY
- LINEARITY TESTING
- LEVY-JENNINGS QUALITY CONTROL CHART
After the 60 seconds, the swab is removed from the child's mouth and placed back in the saliva bag. Maternal breast milk was collected in 50 ml sterile tubes (minimum 5 ml, maximum 15 ml per breast), transported on ice to the laboratory and processed within 4 hours of collection. The aqueous layer of human milk was used to measure in vitro innate immunological proteins with anti-HIV activity.
To measure SLPI in the study samples, 100 µl of Test Diluent RD1Q was added to each well. Substrate solution (200 µl) was added to each well and the plate was incubated for 20 minutes at RT in the dark. Stop solution (50 µl) was added to each well and the plate was incubated for 10 minutes at room temperature until a color change was observed, i.e.
After washing, 300 µl blocking buffer was added to each well and the plate was incubated at RT for 1 h. Standards were prepared according to the manufacturer's recommendations (Appendix 2) and added to their respective wells. 100 μl of working dilution of Streptavidin-HRP was added to each well, the plate was covered with new adhesive strips and incubated for 20 min at RT in the dark.
RESULTS
ASSAY OPTIMIZATION RESULTS
We measured the concentration of SLPI and E/tr-2 in infant saliva and breast milk using ELISA, as described in Section 2.6. The results show that the SLPI can be quantified in infant saliva and breast milk using 1:80 and 1:32 (Appendix 3) dilution factors, respectively. The results also show that E/tr-2 can be measured in infant saliva and breast milk at 1:128 and 1:64, respectively (Appendix 3).
A standard calibration curve used to determine concentrations of E/tr-2 in infant saliva and breast milk.
MUCOSAL FLUIDS CONTAIN MEASURABLE CONCENTRATIONS OF SLPI AND E/TR-2
The measured concentrations for SLPI and E/tr-2 of each of the control samples on all plates were all within two standard deviations (Upper Control Limit (UCL)) and -2 standard deviation (Lower Control Limit (LCL)) of the mean (Figure 3.3 (a) and 3.3(b)). Both ELISA kits were now used to measure concentrations of SLPI and E/tr-2 in infant breast milk and saliva samples.
STUDY PARTICIPANTS CHARACTERISTICS ACCORDING TO HIV STATUS
THE CONCENTRATION OF INNATE PROTEINS CHANGES OVERTIME IN BREAST MILK
- CONCENTRATIONS OF SLPI AND E/TR-2 IN MATERNAL BREAST MILK DECREASE
- SLPI AND E/TR-2 CONCENTRATIONS IN INFANT SALIVA DO NOT SHOW A
- MATERNAL HIV STATUS DOES NOT IMPACT THE CONCENTRATION OF SLPI AND
Next, SLPI and E/tr-2 concentrations were measured over 36 weeks in infant saliva from both HIV-exposed uninfected (HEU) and HIV-unexposed (HU) infants. SLPI and E/tr-2 concentrations were compared between HIV-infected (HIV+) and HIV-uninfected (HIV-) breast milk. EXPOSURE OF PETS TO MODERN HIV DIFFERENTIALLY AFFECTS CONCENTRATIONS OF SLPI AND E/TR-2 IN PETS SALIVA.
We next assessed whether changes in SLPI and E/tr-2 concentration in infant saliva were influenced by maternal HIV status or infant exposure to HIV. These results suggest that the concentration of SLPI in breast milk and infant saliva is influenced by the infant's HIV status/exposure to HIV. However, the concentration of E/tr-2 in infant saliva and breast milk does not appear to be affected by maternal HIV or infant exposure.
The effect of HIV exposure on the concentration of SLPI and E/tr-2 protein levels in infant saliva. Figures a and b represent SLPI and E/tr-2 in the saliva of HEU (red circles) and HU (green circles) infant saliva, respectively. INFLUENCE OF FEEDING PRACTICES ON THE CONCENTRATION OF SLPI AND E/TR-2 IN SALIVA.
THE IMPACT OF FEEDING PRACTICE ON THE CONCENTRATION OF SLPI AND E/TR-2
- THE CONCENTRATION OF SLPI IN EBF INFANT SALIVA INCREASES OVER TIME
- MATERNAL FEEDING MODE DOES NOT IMPACT SLPI AND E/TR-2
THE CONCENTRATION OF SLPI IN EBF-SALIVA INCREASED WITH TIME COMPARED TO EFF-BAABAS. We analyzed changes in SLPI and E/tr-2 concentrations in the saliva of EBF versus EFF infants'. SLPI and E/tr-2 are constitutively expressed in mucosal fluids, therefore changes in E/tr-2 concentrations over time in breast-fed and formula-fed infant saliva were also investigated.
These results suggest that, at least in HIV-exposed infants, exclusive breastfeeding significantly increases the concentration of SLPI over time. On the other hand, SLPI concentrations decreased over time in saliva of exclusively formula-fed infants, although this was not statistically significant. Changes in SLPI and E/tr-2 protein levels in HIV-exposed Exclusively Breastfed (EBF)/None Exclusively Breastfed and HIV-exposed Exclusively Formula Fed (EFF)/None Exclusively Formula Fed (NEFF) infant saliva.
After observing changes in analyte concentrations in breast-fed and formula-fed infants' saliva over time, it was subsequently assessed whether the concentration of SLPI and E/tr-2 was influenced by exclusive breastfeeding or exclusive formula feeding (Figure 3.6.2). The impact of nutrition on the concentration of SLPI and E/tr-2 in HIV-exposed uninfected infant saliva. The red circles represent Exclusively Breast Fed (EBF)/None Exclusively Breastfed (NEBF) and the orange circles represent Exclusively Formula Fed (EFF)/None Exclusively Formula Fed (NEFF) infant saliva.
ASSOCIATION OF SLPI AND E/TR-2 LEVELS IN MATERNAL BREAST MILK WITH
- MATERNAL BREAST MILK E/TR-2 CONCENTRATIONS DO NOT CORRELATE WITH
Spearman correlation plot of SLPI levels in breast milk versus baby saliva of all groups. Spearman correlation plot of SLPI levels comparing the two variables in HIV+ve and HEU at week 36, respectively. g). Therefore, we tried the concentration of SLPI and E/tr-2 in maternal breast milk (HIV-infected and non-infected), in saliva of breastfed and formula-fed infants (HIV-exposed and HIV-unexposed). comparable.
First, the concentrations of SLPI and E/tr-2 in the mother's breast milk and the infant's saliva were measured over time. To our knowledge, this study is the first to assess E/tr-2 concentrations in breast milk and infant saliva from birth to 9 months of age. THE INFLUENCE OF HIV EXPOSURE STATUS ON THE CONCENTRATION OF CONGENITAL PROTEINS IN INFANT'S MILK AND SALIVA.
In this study, the concentration of SLPI in maternal breast milk was not affected by. The data suggest that the concentration of E/tr-2 in breast milk and infant saliva is not affected by HIV infection or exposure to the virus. This suggests that the production of SLPI and E/tr-2 in the baby's saliva is independent of breast milk.
APPENDICES
90
From the stock 55.6 ul stock was added to 99445 ul PBS, to make a working solution of concentration 0.4 ug/ml. 100 µl of capture antibody per well was used to coat the plate in each. The plate was sealed using provided sealant and incubated at room temperature overnight.
2.5 ml of reagent diluent concentrate was added to 22.5 ml of dH2O to make 1% reagent diluent. To prepare 600 ml of wash buffer, 24 ml of the wash buffer concentrate was added to 576 ml of dH2O. A working solution of Streptavidin-HRP was prepared by making a 1:200 dilution of HRP in Reagent Diluent as directed on the vial.
After optimization, 128-fold dilution was selected as an appropriate dilution to measure SLPI analyte in infant saliva, Table 3.1.3. After optimization, 64-fold dilution was selected as an appropriate dilution to measure SLPI analyte in maternal breast milk. Table 3.1.4.
Secretory leukocyte protease inhibitor: a critical mediator of anti-inflammatory responses in acetaminophen-induced acute liver failure. Secreted leukocyte protease inhibitor in colostrum and breast milk is not a major determinant of protection from early postnatal transmission of HIV. Salivary secretion Leukocyte protease inhibitor is associated with reduced human immunodeficiency virus type 1 transmission through breast milk.
Secretory leukocyte protease inhibitor: A human salivary protein displaying anti-human immunodeficiency virus 1 Activity In vitro monocytes lymphocytes * SLPI * retrovirus * saliva. Secretory leukocyte protease inhibitor: a human salivary protein that exhibits anti-human immunodeficiency virus 1 activity in vitro. Multifaceted roles of human elafin and secretory leukocyte proteinase inhibitor (SLPI), two serine protease inhibitors of the chelonianin family.
Concentrations of secretory leukocyte protease inhibitor (SLPI) in cervical mucus of women with normal menstrual cycles. Phospholipid scrablases 1 and 4 are cellular receptors for leukocyte secretory protease inhibitor and interact with CD4 at the plasma membrane. Receptors for secretory leukocyte protease inhibitor and interact with CD4 on the plasma membrane L.
