Endometrial immunocompetent cells in proliferative and secretory phase of normal menstrual cycle.

BACKGROUND: Menstruation was presented as a result of inflammatory process. The total and relative numbers of the endometrial immunocompetitive cells vary during the different phases of the menstrual cycle. The aim of this morphological study is to make a contribution to understanding different distribution of leukocyte types during proliferative and secretory phase of normal menstrual cycle. MATERIALS AND METHODS: The study included 40 women (20 in proliferative and 20 in secretory phase of the menstrual cycle). Exploratory curettage performed as preoperative preparation due to uterine myomas. Immunophenotyping was performed by immunoalkaline phosphatase (APAAP) using monoclonal antibodies: CD15, CD20, CD30, CD45RO, CD56, CD57 and CD68. The results were statistically analysed using SPSS 20.0 software. RESULTS: Natural killer (NK) cells are dominant during secretory, and CD45RO T lymphocytes are dominant during proliferative phase of the menstrual cycle. During the secretory phase of menstrual cycle, leukocytes make 30% of total endometrial cells. NK cells (CD56+ bright subpopulation), activated T lymphocytes, macrophages and B lymphocytes significantly increase in their number during the secretory phase of menstrual cycle. CONCLUSIONS: Significant changes in endometrial leukocyte populations during proliferative and secretory phase of the menstrual cycle are emphasized. Changes in dominance of different leukocyte subpopulations are determined by hormonal and microenvironmental changes in modulatory factors that have not yet been fully explained.

Ascites in Ovarian Carcinoma - Reliability and Limitations of Cytological Analysis.

OBJECTIVE: The objective of this study was to examine the validity of ascitic fluid cytology in the detection of pathological findings, to examine the percentage of false positive and false negative results in the cytology of ascitic fluid and to determine the validity of peritoneal cytology in relation to the histopathological type of the ovarian tumour. METHODS: This retrospective study included 170 peritoneal cytology findings. The study was conducted from January 2010 to December 2012. The experimental group included 76 cytology findings obtained from patients diagnosed with ovarian carcinoma, whereas the control group was composed of 94 cytology findings of benign ovarian tumours and liver cirrhosis ascites. The patients with ovarian carcinoma had grades III, as well as grades I and IIc but only in cases where operative and pathological finding indicated a ruptured or perforated tumour capsule. RESULTS: The sensitivity of peritoneal cytology is 68.92%, specificity is 93.61%, positive predictive value is 89.65% and negative predictive value is 78.57%. In 30.02% of patients, the peritoneal cytology showed false negative results, while in 6.38%, the results were false positive. The highest percentage of false negative findings was 77%, found in endometrioid carcinoma. CONCLUSION: Peritoneal cytology of ascitic fluid is highly specific but has relatively low sensitivity, particularly in the case of endometrioid ovarian carcinoma. In order to increase sensitivity, peritoneal cytology should be combined with monoclonal antibodies and other biochemical and immunohistochemical markers.

Differences between molecular mechanisms involved in the regulation of haptoglobin gene expression during the acute phase response and dietary restriction.

Haptoglobin is a glycoprotein involved in the acute phase response. Previously we reported that haptoglobin gene expression was up-regulated during dietary restriction in young female rats. The present study aimed at determining whether chronic dietary restriction affects haptoglobin blood levels through changing levels and/or activities of IL-6-related transcription factors STAT and C/EBP in the liver as is the case during the acute phase response. To this end, we compared a female Wistar rat model of 50% 6-week-long dietary restriction with the standard laboratory model for the acute phase response induced by turpentine administration. During the turpentine-induced acute phase response, the transitory 5.4-fold increase of rat haptoglobin expression was accompanied by a prominent rise of serum IL-6 concentration and the increased binding of STAT3 and 35kD C/EBPbeta/LAP transcription factors to the haptoglobin gene hormone-responsive element. Results obtained after immunoblotting and DNA affinity chromatography (using hormone-responsive element) suggest that the stable 1.7-fold increase of serum haptoglobin level during dietary restriction was the result of increased amounts and activities of constitutive transcription factors C/EBPalpha and STAT5b, and to a smaller extent of STAT3. When dietary restriction rats were administered turpentine, a 8.7-fold increase in haptoglobin expression was followed by a considerable increase in the amount and hormone-responsive element binding activity of STAT3 but not 35kD C/EBPbeta/LAP. We concluded that haptoglobin gene up-regulation during chronic dietary restriction was regulated by different mechanisms than during the acute phase response, and that it depended on the amount(s) and activit(ies) of transcription factor(s) that characterize low-grade inflammatory conditions.

A West Nile virus (WNV) recombinant canarypox virus vaccine elicits WNV-specific neutralizing antibodies and cell-mediated immune responses in the horse.

Successful vaccination against West Nile virus (WNV) requires induction of both neutralizing antibodies and cell-mediated immune responses. In this study, we have assessed the ability of a recombinant ALVAC-WNV vaccine (RECOMBITEK WNV) to elicit neutralizing antibodies and virus-specific cell-mediated immune responses in horses. In addition, we examined whether prior exposure to ALVAC-WNV vaccine would inhibit B and cell-mediated immune responses against the transgene product upon subsequent booster immunizations with the same vaccine. The results demonstrated that the recombinant ALVAC-WNV vaccine induced neutralizing antibodies and prM/E insert-specific IFN-gamma(+) producing cells against WNV in vaccinated horses. Prior exposure to ALVAC-WNV vaccine did not impair the ability of horses to respond to two subsequent booster injections with the same vaccine, although anti-vector-specific antibody and cell-mediated immune responses were induced in vaccinated horses. This report describes, for the first time, the induction of antigen-specific cell-mediated responses following vaccination with an ALVAC virus recombinant vaccine encoding WNV antigens. Moreover, we showed that both WNV-specific IFN-gamma producing cells and anti-WNV neutralizing antibody responses, are not inhibited by subsequent vaccinations with the same vector vaccine.

Effective priming of foals born to immune dams against influenza by a canarypox-vectored recombinant influenza H3N8 vaccine.

A classical limitation of early life immunization is the interference by maternally derived antibodies, which are known to inhibit the immune response to modified-live and killed vaccines. Several studies have convincingly shown that even minute amounts of maternally derived antibodies against equine influenza can strongly interfere with successful vaccination of foals born to immune mares. In this study we evaluated the response of foals born to vaccinated mares to immunization with a canarypox-vectored recombinant vaccine against equine influenza virus H3N8. The recombinant vaccine was able to efficiently prime foals in the presence of maternally derived immunity against influenza as was evidenced by a clear anamnestic antibody response when a secondary vaccination with the same vaccine was performed. The canarypox-vectored recombinant influenza vaccine therefore offers a unique opportunity to overcome the limitations of early life vaccination in the face of maternally derived immunity in foals.

Acute-phase related binding ability of p53 for the hormone response element of the haptoglobin gene in adult rats.

Interaction between transcription factor p53 and the hormone response element (HRE) of the haptoglobin (Hp) gene in adult rat liver was studied. We detected a sequence homologous to the p53 consensus DNA-binding site in the regulatory promoter element of the Hp gene. DNA-affinity chromatography, followed by Western immunoblot analysis with an antibody to p53 indicated that components of the nuclear extract possessed the same antigen determinants as p53. While p53 was identified in both control and acute-phase (AP) samples, DNA-binding affinity for the Hp gene HRE was detected only in the nuclear extract prepared from rats undergoing the AP response. Whether either as an inducible or as a constitutive transcription factor, p53 could be involved in the transcriptional regulation of the Hp gene in adult rat liver.

Expression of C/EBP delta in rat liver during development and the acute-phase response.

Using Western analysis, C/EBP delta was established in the nuclear extract and nuclear matrix throughout rat liver development and in the adult. During the acute-phase response (APR), C/EBP delta increased in the nuclear extract but remained unchanged in the nuclear matrix of fetal and postnatal rats, whereas it increased in both the nuclear extract and nuclear matrix of the adult. The solubility partitioning of gene regulatory proteins in the nucleus is important for their functioning (Uskokovic et al. 2002). The obtained different solubility partitioning profiles of C/EBP delta suggest that its activity is regulated by different mechanisms during development and in the adult.

Acute-phase-dependent binding affinity of C/EBPbeta from the nuclear extract and nuclear matrix towards the hormone response element of the alpha2-macroglobulin gene in rat hepatocytes.

Interactions of nuclear extract and nuclear matrix proteins from rat hepatocytes with the hormone response element of the alpha2-macroglobulin gene were studied. By Western and South-Western blot analysis we have shown the presence of C/EBPbeta in the examined nuclear fractions as well as its increased binding affinity to the examined gene sequence during the acute-phase response. The results suggest that both nuclear protein fractions could participate in the transcriptional regulation of the alpha2-macroglobulin gene in the rat hepatocytes.

The protein composition of the hepatocyte nuclear matrix is differentiation-stage specific.

The protein composition of hepatocyte nuclear matrices was examined in rats from the 16th day of gestation to 75 days after birth (adult). An overall increase in size of the nuclear matrix was accompanied by quantitative and qualitative changes in its protein content. Quantitative changes of the major proteins of the peripheral lamina surrounding the isolated nuclear matrix were detected. By Western analysis we established that in pre- and postnatal nuclear matrices the relative concentrations of lamin C were greater than lamin A. After birth, the relative concentrations of both lamins progressively increased. In the adult nuclear matrix, the concentration of lamin A was greater than lamin C. In contrast, the relative concentrations of lamin B remained unchanged throughout development and growth. The relative concentrations of two nuclear matrix-associated regulatory proteins studied changed with development and growth: transcription factor C/EBPalpha isoforms, which were detected during the gestation period, increased notably after the first postnatal day, attaining a maximum at the adult stage; the high concentrations of the proliferating cell nuclear antigen (PCNA) perceptibly decreased after the 21st prenatal day. Changes in the composition of the nuclear matrix protein suggest that this structure coordinates nuclear functioning during cell differentiation.

C/EBPalpha and C/EBPbeta are persistently associated with the rat liver nuclear matrix throughout development and the acute phase response.

The partitioning of C/EBPalpha and C/EBPbeta on the nuclear matrix structure was examined during the different transcriptional activities accompanying liver development and the acute phase (AP) response. The presence of C/EBPalpha and C/EBPbeta was established on the nuclear matrix. Their relative concentrations on the matrix always reflected the developmental stage- and AP-related fluctuations observed in the nuclear extract. Thus, they progressively increased as development proceeded, whereas during the AP response, C/EBPalpha decreased and C/EBPbeta increased. In addition, the levels of both transcription factors were always notably higher in the nuclear matrix than in the extracts. We conclude that the observed changes and overall enrichment of the nuclear matrix with regulatory proteins is a reflection of the importance of such interactions for the in vivo functioning of C/EBP proteins.