Macrophages and Natural Killer Cells Characteristics in Variously Colored Endometriotic Lesions: A Cross-Sectional Analytic Study.

Background: Dysregulation of the immune response contribute to a significant role in endometriosis. This research examined macrophages and natural killer (NK) cells numbers in endometriotic lesions and their association with the different lesion colors: red, black, and white. To investigate the amount of the CD68 and CD56 in eutopic endometrium and different type of the endometriotic lesions. Materials and Methods: A cross-sectional analytic study was conducted. Women suspected endometriosis requiring laparoscopic surgery between July 2016 and January 2017 were recruited. Their lesions were classified as red, black, or white and these lesions were excised by standard laparoscopic surgery. Twenty-four endometriotic lesions from each color group were obtained from 45 women who met the inclusion criteria. One type of lesion was collected from 25 women. Two different lesion types and three-color lesion types were collected from the same women in 13 and 7 subjects, respectively. Immunohistochemistry staining with anti-human mouse cluster of differentiation (CD) 68 monoclonal antibody for macrophages and mouse anti-human CD56 monoclonal antibody for NK cells were performed. Results: The number of CD68 macrophages in red lesions was higher than in black and white lesions [median (25th-75th percentile); 10 (5-19.4), 0 (0-6.9), 0 (0-2.5) cells per mm2, respectively, adjusted P=0.001 for red vs. black lesions and red vs. white lesions, and adjusted P=1.000 for black and white lesions]. The number of CD56 NK cells was not significantly different among red, black, and white lesions [median (25th-75th percentile; 5 (2-16.5), 3.8 (0-14.4), 1.3 (0-6.9) respectively, adjusted P=1.000 for red vs. black lesions and black vs. white lesions, and adjusted P=0.617 for red vs. white lesions]. Conclusion: The dynamic changes in the immune cells in ectopic endometrium were specific to the macrophages but not to the NK cells, as demonstrated by the highest number of CD68 macrophages in the red lesion, the earliest established ectopic endometrium. NK cells in endometriosis may have a role in the uterus.

Identification and localization of growth factor genes in the sea cucumber, Holothuria scabra.

The sea cucumber Holothuria scabra is both an economically important species in Asian countries and an emerging experimental model for research studies in regeneration and medicinal bioactives. Growth factors and their receptors are known to be key components that guide tissue repair and renewal, yet validation of their presence in H. scabra has not been established. We performed a targeted in silico search of H. scabra transcriptome data to elucidate conserved growth factor family and receptor genes. In total, 42 transcripts were identified, of which 9 were validated by gene cloning and sequencing. The H. scabra growth factor genes, such as bone morphogenetic protein 2A (BMP 2A), bone morphogenetic protein 5-like (BMP5-like), neurotrophin (NT) and fibroblast growth factor 18 (FGF18), were selected for further analyses, including phylogenetic comparison and spatial gene expression using RT-PCR and in situ hybridization. Expression of all genes investigated were widespread in multiple tissues. However, BMP 2A, BMP5-like and NT were found extensively in the radial nerve cord cells, while FGF18 was highly expressed in connective tissue layer of the body wall. Our identification and expression analysis of the H. scabra growth factor genes provided the molecular information of growth factors in this species which may ultimately complement the research in regenerative medicine.

Molecular characterization of a vitellogenesis-inhibiting hormone (VIH) in the mud crab (Scylla olivacea) and temporal changes in abundances of VIH mRNA transcripts during ovarian maturation and following neurotransmitter administration.

The vitellogenesis-inhibiting hormone (VIH), also known as gonad-inhibiting hormone, is a neuropeptide hormone in crustaceans that belongs to the crustacean hyperglycemic hormone (CHH)-family peptide. There is regulation vitellogenesis by VIH during gonad maturation in crustaceans. A full-length Scylla olivacea VIH (Scyol-VIH) was identified through reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. The open reading frame consists of 378 nucleotides, which encodes a 126-amino acid precursor protein, including a 22-residue signal peptide and a 103-amino acid mature peptide in which 6 highly conserved cysteine residues are present. There was expression of the Scyol-VIH gene in immature female Scylla olivacea in the eyestalk, brain and ventral nerve cord. The Scyol-VIH gene expression was localized to the eyestalk X-organ, brain neuronal clusters 6 and 11, and in multiple neuronal clusters of the ventral nerve cord. The relative abundance of Scyol-VIH mRNA transcript in the eyestalk was relatively greater in immature stage females, then decreased as ovarian maturation progressed. Furthermore, eyestalk Scyol-VIH increased after dopamine (5 µg/g BW) injection. The present research provides fundamental information about Scyol-VIH and its potential effect in controlling reproduction.

Differential expressions of estrogen and progesterone receptors in endometria and cyst walls of ovarian endometrioma from women with endometriosis and their responses to depo-medroxyprogesterone acetate treatment.

BACKGROUND: Depo-medroxyprogesterone acetate (DMPA) is an injectable progestin contraceptive that provides a highly effective reduction of pelvic pain in women with endometriosis. Despite its wide use to treat pain associated with endometriosis, its precise mechanisms of action remain unclear. The aims of this study were to investigate the differential expressions of estrogen receptors (ERs), and progesterone receptors (PRs) in endometria and ovarian endometrioma cyst walls of women with endometriosis with and without DMPA treatment. METHODS: Endometria and cyst walls of endometrioma were obtained from 25 to 45 year-old women who suffered from endometriosis and had ovarian endometrioma with the size ≥3 cm. The expression levels of ERs and PRs and the numbers of ER- and PR-positive cells before and after treatment with DMPA were evaluated by Western blot, real-time PCR, and immunohistochemistry. RESULTS: The levels of ERα and ERß expression, their corresponding mRNAs, and numbers of ERα- and ERß-immunoreactive cells in stroma and glands of endometria of the DMPA group were significantly decreased when compared with those of the untreated groups (p < 0.05). In contrast, the levels of PRA/B expression and numbers of PRA/B positive cells in stroma and number of PRB positive cells in stroma and endometrial glands were significantly increased in endometria of the DMPA group when compared with those of the untreated groups. However, in cyst wall the expression levels of these proteins, their corresponding mRNAs, and immonoractive cells were low compared to those in endometria, and DMPA-treatment did not cause any significant changes in these parameters. CONCLUSION: These data indicated that DMPA could upregulate the expressions of PRA/B and down-regulate ERα and ERß in endometria but not in cyst walls from women with endometriosis.

DMPA Suppresses Cell Proliferation and Enhances Cell Apoptosis of Eutopic Endometrium in Women with Endometriosis: A Randomized Controlled Study.

Background: Although Depo-medroxyprogesterone acetate (DMPA), an injectable contraceptive progestin, is very effective for pain relief and prevention of recurrence in women with endometriosis, there is no report on the mechanism of this medication about cell proliferation and apoptosis. Objective: To investigate the effects of DMPA on cell proliferation and apoptosis in the eutopic endometrium of women with endometriosis. Material and Method: A randomized controlled study was conducted in 28 women with endometriosis. The DMPA-treated group included 14 women who were scheduled to undergo laparoscopic surgery after 150 mg of DMPA injections. The control group included 14 women who were scheduled to undergo the surgery without DMPA injection. The endometrial tissue was obtained from each woman by endometrial aspiration before surgery. The ELISA formats of PCNA and the quantitative colorimetric analysis of TUNEL were used for estimating cell proliferation and apoptosis of the eutopic endometrium. Results: There were no differences in the women characteristics between the two groups. The relative level of cell proliferation was significantly less in the DMPA than the control groups (1.08±0.57 vs. 1.73±0.50, p = 0.014). Whereas the relative level of cell apoptosis was greater in the DMPA group than that in the control group (1.12±0.36 vs. 0.82±0.39, p = 0.034). Conclusion: Three months of 150 mg DMPA treatment could suppress cell proliferation and enhance cell apoptosis of the eutopic endometrium of women with endometriosis.

Monoclonal antibody against recombinant Fasciola gigantica cathepsin L1H could detect juvenile and adult cathepsin Ls of Fasciola gigantica.

Cathepsin Ls (CatLs), the major cysteine protease secreted by Fasciola spp., are important for parasite digestion and tissue invasion. Fasciola gigantica cathepsin L1H (FgCatL1H) is the isotype expressed in the early stages for migration and invasion. In the present study, a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1H (rFgCatL1H) was produced by hybridoma technique using spleen cells from BALB/c mice immunized with recombinant proFgCatL1H (rproFgCatL1H). This MoAb is an immunoglobulin (Ig)G1 with κ light chain isotype. The MoAb reacted specifically with rproFgCatL1H, the native FgCatL1H at a molecular weight (MW) 38 to 48 kDa in the extract of whole body (WB) of metacercariae and newly excysted juvenile (NEJ) and cross-reacted with rFgCatL1 and native FgCatLs at MW 25 to 28 kDa in WB of 2- and 4-week-old juveniles, adult, and adult excretory-secretory (ES) fractions by immunoblotting and indirect ELISA. It did not cross-react with antigens in WB fractions from other parasites, including Gigantocotyle explanatum, Paramphistomum cervi, Gastrothylax crumenifer, Eurytrema pancreaticum, Setaria labiato-papillosa, and Fischoederius cobboldi. By immunolocalization, MoAb against rFgCatL1H reacted with the native protein in the gut of metacercariae and NEJ and also cross-reacted with CatL1 in 2- and 4-week-old juveniles and adult F. gigantica. Therefore, FgCatL1H and its MoAb may be used for immunodiagnosis of both early and late fasciolosis in ruminants and humans.

The in vitro anthelmintic effects of plumbagin on newly excysted and 4-weeks-old juvenile parasites of Fasciola gigantica.

The effect of plumbagin (PB, 5-hydroxy-2-methyl-1,4-naphthoquinone) against newly excysted juveniles (NEJs) and 4-weeks-old immature parasites of Fasciola gigantica were compared with triclabendazole (TCZ). The anthelmintic efficacy of 1, 10 and 100µg/ml of PB or TCZ following incubation in vitro for 1-24h was compared using a combination of relative motility (RM), survival index (SI) and larval migration inhibition (LMI) assays for parasite viability. The RM and SI values of the PB-treated group decreased at a more rapid rate than the TCZ-treated group. For NEJs, the decreased RM values were first observed at 1h incubation with 1µg/ml PB, and 90% of flukes were killed at 24h. In contrast, in TCZ-treated groups a 10-fold higher concentration of TCZ (10µg/ml) resulted in only 9% dead parasites after 24h incubation. In 4-weeks-old juvenile parasites, PB reduced the RM value at 10µg/ml with 100% of flukes dead after 3h, while TCZ decreased RM values at the concentration of 100µg/ml but with only 5% of flukes killed at 24h. NEJs treated with PB exhibited 88%, 99% and 100% of LMIs at the concentrations of 1, 10 and 100µg/ml, respectively. NEJs incubated with TCZ have an LMI of only 32% at the highest concentration of 100µg/ml. Similarly PB had a significantly greater killing of immature 4weeks juvenile stages than TCZ at all concentrations; however, 4-weeks-old juvenile parasites were more resistant to killing by PB or TCZ at all concentrations when compared to NEJs. Further studies were carried out to investigate the alterations of the parasite tegument by scanning electron microscope (SEM). PB caused similar tegumental alterations in 4-weeks-old juveniles as those observed in TCZ treatment but with greater damage at comparative time points, comprising of swelling, blebbing and rupture of the tegument, loss of spines, and eventual erosion, lesion and desquamation of the total tegument. These data indicate that PB had a greater fasciolicidal effect against immature stages of F. gigantica parasites than TCZ and warrant further studies for use as a potential new anthelmintic against Fasciola infections.

Vaccine potential of recombinant cathepsin B against Fasciola gigantica.

In Fasciola gigantica, cathepsin Bs, especially cathepsin B2 and B3 are expressed in early juvenile stages, and are proposed to mediate the invasion of host tissues. Thus they are thought to be the target vaccine candidates that can block the invasion and migration of the juvenile parasite. To evaluate their vaccine potential, the recombinant cathepsin B2 (rFgCatB2) and cathepsin B3 (rFgCatB3) were expressed in yeast, Pichia pastoris, and used to immunize mice in combination with Freund's adjuvant to evaluate the protection against the infection by F. gigantica metacercariae, and the induction of immune responses. Mice immunized with both recombinant proteins exhibited high percent of parasite reduction at 60% for rFgCatB2 and 66% for rFgCatB3. Immunization by both antigens induced continuously increasing levels of IgG1 and IgG2a with a higher level of IgG1 isotype, indicating the mixed Th1/Th2 responses with Th2 predominating. When examined individually, the higher levels of IgG1 and IgG2a were correlated with the lower numbers of worm recoveries. Thus, both cathepsin B2 and cathepsin B3 are plausible vaccine candidates whose potential should be further tested in large economic animals.

Production and characterization of a monoclonal antibody against recombinant saposin-like protein 2 of Fasciola gigantica.

A monoclonal antibody (MoAb) against recombinant Fasciola gigantica saposin-like protein 2 (rFgSAP-2) was produced by hybridoma technique using spleen cells from BALB/c mice immunized with rFgSAP-2. This MoAb is an IgG1, κ light chain isotype. By immunoblotting and indirect ELISA, the MoAb reacted specifically with rFgSAP-2, the natural FgSAP-2 at 10kDa in whole body (WB) and excretory-secretory (ES) fractions of F. gigantica. It did not cross react with antigens in WB fractions from other parasites, including Opisthorchis viverrini, Schistosoma mansoni which are human parasites, Haemonchus placei, Setaria labiato-papillosa, Eurytrema pancreaticum, Cotylophoron cotylophorum, Fischoederius cobboldi, Gigantocotyle explanatum, Gastrothylax crumenifer, and Paramphistomum cervi which are ruminant parasites. By immunohistochemistry, the FgSAP-2 protein was localized only in the cytoplasm of caecal epithelial cells of 4-week-old juvenile and adult stages, but not in metacercariae, newly excysted juvenile (NEJ), 2- and 3-week-old juveniles. This finding indicated that FgSAP-2 is an abundantly expressed parasite protein that is released into the ES, hence SAP-2 and its MoAb may be used for immunodiagnosis of ruminant and human fasciolosis.

Fasciola gigantica: production and characterization of a monoclonal antibody against recombinant cathepsin B3.

A number of monoclonal antibodies (MoAbs) against a recombinant cathepsin B3 (rCatB3) of Fasciola gigantica were produced in BALB/c mice. Reactivity and specificity of these MoAbs were assessed by indirect ELISA and immunoblotting techniques. Six stable clones, namely 1C4, 1E9, 2E5, 2F9, 5B4, 5D7 were obtained. All MoAbs reacted with rCatB3 at molecular weight (MW) 37 kDa as well as the glycosylated peptide at 55-75 kDa and with the native CatB3 at MW 37 kDa in WB extracts of metacercariae (Met) and newly excysted juveniles (NEJ). It was found to be IgG(1) and λ light chain isotypes. Immunolocalization of CatB3 in metacercariae, NEJ, 4-week-old juvenile and adult F. gigantica performed by immunoperoxidase technique by using these MoAbs as probes indicated that CatB3 was present in high concentration in the caecal epithelium and caecal lumen of the Met and NEJ, but not in the 4-week-old juvenile and adult fluke. The MoAbs show no cross-reactions with antigens of other parasites including Gigantocotyl explanatum, Eurytrema pancreaticum, Paramphistomum cervi, Schistosoma spindale, S. mansoni, Haemonchus placei and Setaria labiato-papillosa. Thus, it is possible that these MoAbs could be a good candidate for immunodiagnosis of fasciolosis.