Supervisors info:
Στυλιανοπούλου Φωτεινή, Καθηγήτρια, Τμήμα Νοσηλευτικής ΕΚΠΑ
Σταματάκης Αντώνιος, Αναπλ. Καθηγητής, Τμήμα Νοσηλευτικής ΕΚΠΑ
Γαϊτανάκη Αικατερίνη, Καθηγήτρια, Τμήμα Βιολογίας ΕΚΠΑ
Summary:
Perinatal transmission of hepatitis B virus has been a serious concern to the scientific community worldwide, due to the increased rates of vertical transmission from the mother to her child, while it predisposes the infant to develop chronicity and to have poor response to antiviral therapies. In this study, placental tissue samples from mothers HBV-carriers were examined, who nevertheless gave birth to healthy children. The conjecture which arose was that the placenta uses a virus-neutralizing mechanism, independent of the viral load which the mother carries.
Aim of thesis: The main hypothesis of this thesis is that T-cytotoxic lymphocytes play a dual role in prophylaxis of the fetus during a pregnancy, where the mother is a hepatitis B-carrier. Our goal was to demonstrate that CD8(+) cells preserve the fetus from both tranplacental HBV infection and their own cytotoxicity. For this purpose, the characteristics of the T-cell subpopulations were examined between two groups: pregnant women with low viral load (LVL) and pregnant women with high viral load (HVL).
Materials and Methods: Fifty-three placenta tissue samples and three methods were used to perform this study: Hematoxylin-Eosin histochemical staining, Immunohistochemistry (HBsAg HBcAg, anti-CD3, anti-CD4 and anti-CD8) and dual immunofluorescence (CD8 & CD28, CD8 & anti-Fas, CD8 & anti-Fas ligand), by using specific monoclonal and polyclonal antibodies in order to categorize the T-cell subpopulations located in the decidua and the chorionic villi, as well as to investigate the existence of an apoptotic process.
Results: Immunohistochemistry results revealed the presence of HBV in the trophoblast cells of the placenta. As far as for T-cell subpopulations, a statistical difference in the expression of CD4(+) cells was shown in the villi, while a statistical tendency was shown in the decidua. In both histological sites of the placenta, the LVL group had statistically more CD4(+) lymphocytes than the HVL group.
Regarding CD8(+) cells, the results revealed a statistically significant difference in the deciduas, where the LVL group had more CD8(+) lymphocytes than the HVL group.
The results of the double immunofluorescence, showed a difference between the percentage of CD8(+) cells which were CD8(+)Fas(+), as well as those which were CD8(+)Fas Ligand(+), compared to the percentage of CD8(+)CD28(+) cells. CD8(+) cells expressing Fas and Fas Ligand pro-apoptotic molecules were at least five times more than those expressing the CD28 survival molecule. Furthermore, the percentage of CD8(+)CD28(+) double positive cells was observed to be low in both experimental groups.
Regarding the comparison of the two groups, statistically significant differences were found in the decidua. Analytically, the LVL group exhibited a higher percentage of lymphocytes expressing CD8(+)CD28(+) and a higher percentage of lymphocytes expressing CD8(+)Fas Ligand(+) than the HVL group, whereas the HVL group exhibited a greater proportion of CD8(+)Fas(+) simultaneous expression in lymphocytes than the LVL group.
1st Conclusion: The tendency of CD8(+) lymphocytes in the decidua to be less in mothers with high viral load, combined with the predominance of CD8(+)Fas(+) in that group, could lead to the conclusion that apoptosis is amplified slightly more in the high viraemia group.
2nd Conclusion: CD8(+) cells exhibit greater Fas Ligand expression (pre-apoptotic molecule), than CD28 expression (survival molecule). Therefore, we assume that T-cytotoxic cells activate the apoptosis of both trophoblastic cells and amongst them, in order to protect the fetus from increased activation and possible cytotoxicity that could harm pregnancy.
To sum up, the apoptosis of the CD8(+) cells is significantly promoted in the placenta and T-cytotoxic lymphocytes induce apoptosis for the prophylaxis of the fetus from hepatitis B infection by vertical transmission from the mother, as well as from their own activation.
Keywords:
Hepatitis B, HBV Virus, placenta, lymphocytes, apoptosis, perinatal transmission, parafin samples, immunohistochemistry, immunofluorescence, CD8, CD4, CD28, FAS, FAS LIGAND, Aspioti
