Localization of cathepsins D and B at the maternal-fetal interface and the invasiveness of the trophoblast during the postimplantation period in the mouse.

A survey of existing data suggests that trophoblast cells produce factors involved in extracellular matrix degradation. In this study, we correlated the expression of cathepsins D and B in the murine ectoplacental cone with the ultrastructural progress of decidual invasion by trophoblast cells. Both proteases were immunolocalized at implantation sites in lysosome-endosome-like compartments of trophoblast giant cells. Cathepsin D, but not cathepsin B, was also detected ultrastructurally in extracellular compartments surrounded by processes of the invading trophoblast containing extracellular matrix components and endometrial cell debris. The expression of cathepsins D and B by trophoblast cells was confirmed by RT-PCR in ectoplacental cones isolated from implantation chambers at gestation day 7.5. Our data addressed a positive relationship between the expression and presence of cathepsin D at the extracellular compartment of the maternal-fetal interface and the invasiveness of the trophoblast during the postimplantation period, suggesting a participation of invading trophoblast cells in the cathepsin D release. Such findings indicate that mouse trophoblast cells might exhibit a proteolytic ability to partake in the decidual invasion process at the maternal-fetal interface.

Fetal-placental hypoxia does not result from failure of spiral arterial modification in mice.

OBJECTIVES: To determine if fetal-placental hypoxia is a primary outcome of defective spiral artery remodeling. STUDY DESIGN: Pregnancies in Rag2(-/-)Il2rg(-/-) double knock-out mice, which fail to undergo normal physiological spiral arterial remodeling, were compared to syngeneic BALB/c control pregnancies. Mice at gestation day (gd)6, 8, 10, 12 and 18 were infused with Hypoxyprobe-1 before euthanasia to enable detection of cellular hypoxia by immunohistochemistry. RESULTS: In implantation sites of both phenotypes, trophoblast cells were reactive to Hypoxyprobe-1. No major differences were observed between the phenotypes in decidua or placenta at any gd or in gd18 fetal brain, lung, heart, liver or intestine or in maternal heart, brain, liver or spleen. Maternal kidneys from BALB/c were significantly hypoxic to Rag2(-/-)Il2rg(-/-) kidneys. CONCLUSIONS: In mice, lack of pregnancy-associated spiral artery remodeling does not impair oxygen delivery to the conceptus, challenging the concept that deficient spiral arterial remodeling leads to fetal hypoxia in human gestational complications such as preeclampsia and fetal growth restriction. The isolated hypoxic response of normal kidney has revealed that renal lymphocytes may have unique, tissue-specific regulatory actions on vasoconstriction that are pregnancy independent.

DBA-lectin reactivity defines natural killer cells that have homed to mouse decidua.

During normal mouse pregnancy, abundant numbers of uterine natural killer (uNK) cells differentiate at implantation sites and contribute to early, post-implantation endometrial angiogenesis and to spiral arterial modification. Mouse uNK cells are confidently recognized by light microscopy in tissue sections stained with special protocols. Classically, mouse uNK cells were identified as lymphocytes containing Periodic Acid Schiff's (PAS) reactive cytoplasmic granules. More recently, Dolichos biflorus lectin (DBA) reactions which stain not only the cytoplasmic granules but also the uNK cell membranes have been widely adopted. No lymphocytes in any tissues of virgin mice or external to the uterus of pregnant mice have DBA lectin reactivity equivalent to that of uNK cells; however, some uNK cells are now recognized as DBA-. Here, we describe a PAS/DBA lectin double staining protocol and assess the coincident staining of C57Bl/6J uNK cells from gestation day (gd)6, the first day of uNK cell abundance, to gd12, the day when Tunel+ nuclear senecence appears widely in uNK cells before their numerical decline. For these gd, PAS+ DBA- and PAS+DBA+ cells but not PAS-DBA+ cells were identified. Dual positive cells increased from 47% at gd6 to 85% at gd12. Transplantation of normal bone marrow into alymphoid mice who were subsequently mated revealed the uterus repopulated by doubly reactive PAS+DBA+ uNK cells (>95%). Thus, in normal pregnancies, most uNK cells appear to arise from progenitor cells that have homed to the uterus.

Time-course analyses addressing the acquisition of DBA lectin reactivity in mouse lymphoid organs and uterus during the first week of pregnancy.

Terminal differentiation of uterine natural killer (uNK) cells is induced in humans and mice at the time of endometrial decidualization. In mice, commitment to this lineage is first recognized at gestation day (gd)5.5 when small, non-granulated lymphocytes in the mesometrial decidua basalis become histochemically reactive with the lectin Dolichlos biflorus agglutinin (DBA). Transplantation experiments in mice have shown that the self-renewing progenitors of uNK cells are present in peripheral rather than uterine tissues. While lymphoid tissues of virgin mice lack DBA lectin reactive putative uNK cell precursors or progenitors, peripheral organs have not been systematically examined in female mice during early pregnancy while the uNK cell population is becoming established. Here we report such a study in gd0.5-7.5 random bred mice. Only mesenteric lymph nodes showed a transient gain in DBA lectin reactive lymphocytes between gd0.5 and 4.5. These cells had cytoplasmic but almost no surface DBA lectin reactivity. This study indicates that the decidual environment is unique and locally promotes DBA lectin surface expression in terminally differentiating uNK cells.

Subset classification of mouse uterine natural killer cells by DBA lectin reactivity.

Uterine Natural Killer (uNK) cells are a transient lymphocyte population found in the pregnant uteri of human and rodents. The pregnant uterine environment appears to influence migration, differentiation and suppression of the cytolytic activation of uNK cells but the mechanisms involved in these processes are not well understood. Similarities to circulating NK (cNK) cells are limited. The present study sought to discrimate uNK cells from cNK cells in mice by identification of a unique uNK cell marker. Dolichos biflorus (DBA) lectin, which has high selectivity for glycoconjugates containing N-acetyl D-galactosomine in the terminal position, reacted with the plasma membranes of mouse uNK cells. DBA lectin did not react with other uterine lymphocytes or with cNK cell surfaces in Swiss, CBA-J, C57BL/6, SJL, BALB/c, DBA-2 mice strains. DBA lectin staining was useful for both light and electron microscopy and distinguished 4 uNK cell subtypes that appear to be stages of differentiation. Quantitative evaluation of these 4 uNK cell subtypes over early to late gestational times showed dynamic changes between immature and mature forms in different compartments of the implantation sites and indicated the occurrence of microdomains in the uterus capable of controlling uNK cell proliferation and differentiation. This is the first report showing mouse uNK cells expressing specific molecules not found in other NK cells. Use of this reagent should enhance studies of earlier, non-granulated forms of uNK cells and provide new strategies for purification of mouse uNK cells for functional and molecular studies.

Functional analysis of murine uterine natural killer cells genetically devoid of oestrogen receptors.

Uterine Natural Killer (uNK) cell differentiation in vivo requires oestrogen (E) priming prior to progesterone (P). Hybridomas between uNK precursor and SP2/0 cells express message for E receptor (ER)alpha but nor PR. However, mature, rodent and human uNK cells lack these receptors. To functionally assess requirements for uNK cell expression of ERalpha or ERbeta during precursor differentiation, marrow was transplanted from either ERalpha(o/o) (alphaERKO) or ERbeta(o/o) (betaERKO) mice into alymphoid RAG-2(o/o)/gammac(o/o) females. Recipients were mated and their implantation sites were examined by light microscopy, morphometry and ultrastructure. High numbers of uNK cells were established from each donor strain. Graft-derived uNK cells were similar in number and morphology to uNK cells of normal mice, suggesting that neither alpha- nor beta-ER is required for uNK precursor cell differentiation. Induction of spiral artery modification in the transplant recipients indicated that graft-derived uNK cells had functional properties. A novel technique for rapid isolation of highly purified uNK cells from normal mice using Dolichos biflorus agglutinin (DBA) lectin-conjugated magnetic beads was employed to obtain RNA. Expression of alpha- and beta-ER was absent by RT-PCR from NK cells isolated from the uterus, supporting the conclusions from the in vivo study.

Role of Tamm-Horsfall protein in the binding and in vivo phagocytosis of type 1 fimbriated Escherichia coli by mouse peritoneal macrophages.

Tamm-Horsfall glycoprotein (THP) contains manno-oligosaccharides that are recognized by type 1 fimbriae (F1) of Escherichia coli. In the present study, we examined the in vivo phagocytic activity of mouse peritoneal macrophages after treatment of bacteria with THP. At low THP concentrations (12.5 microg/ml and 50 microg/ml) no significant difference was observed in the phagocytosis of E. coli F1+. However, at high THP concentrations (500 microg/ml and 1250 microg/ml) we obtained a reduction of bacterial phagocytosis by mouse peritoneal macrophages.

Role of Tamm-Horsfall protein in the binding and in vivo phagocytosis of type 1 fimbriated Escherichia coli by mouse peritoneal macrophages

Tamm-Horsfall glycoprotein (THP) contains manno-oligosaccharides that are recognized by type 1 fimbriae (F1) of Escherichia coli. In the present study, we examined the in vivo phagocytic activity of mouse peritoneal macrophages after treatment of bacteria with THP. At low THP concentrations (12.5 æg/ml and 50 æg/ml) no significant difference was observed in the phagocytosis of E. coli F1+. However, at high THP concentrations (500 æg/ml and 1250 æg/ml) we obtained a reduction of bacterial phagocytosis by mouse peritoneal macrophages

Role of peroxynitrite in macrophage microbicidal mechanisms in vivo revealed by protein nitration and hydroxylation.

The cytotoxins produced by phagocytic cells lacking peroxidases such as macrophages remain elusive. To elucidate macrophage microbicidal mechanisms in vivo, we compared the lesion tissue responses of resistant (C57Bl/6) and susceptible (BALB/c) mice to Leishmania amazonensis infection. This comparison demonstrated that parasite control relied on lesion macrophage activation with inducible nitric oxide synthase expression (iNOS), nitric oxide synthesis, and extensive nitration of parasites inside macrophage phagolysosomes at an early infection stage. Nitration and iNOS expression were monitored by confocal microscopy; nitric oxide synthesis was monitored by EPR. The main macrophage nitrating agent was shown to be peroxynitrite derived because parasite nitration occurred in the virtual absence of polymorphonuclear cells (monitored as peroxidase activity) and was accompanied by protein hydroxylation (monitored as 3-hydroxytyrosine levels). In vitro studies confirmed that peroxynitrite is cytotoxic to parasites whereas nitric oxide is cytostatic. The results indicate that peroxynitrite is likely to be produced close to the parasites and most of it reacts with carbon dioxide to produce carbonate radical anion and nitrogen dioxide whose concerted action leads to parasite nitration. In parallel, some peroxynitrite decomposition to the hydroxyl radical should occur due to the detection of hydroxylated proteins in the healing tissues. Consequently, peroxynitrite and derived radicals are likely to be important macrophage-derived cytotoxins.

Morphological and intracellular alterations induced by cytotoxin VT2y produced by Escherichia coli isolated from chickens with swollen head syndrome.

Recently, a novel verocytotoxin named VT2y was described which belongs to the STx family and is produced by Escherichia coli isolated from domestic poultry with swollen head syndrome (SHS). The VT2y toxin induced apoptosis in Vero, HeLa, CHO, CEF (primary chicken embryo fibroblast) and PCK (primary chicken kidney) cell lines. Morphological evidence (nuclear shrinkage, chromatin condensation and blebbing of the plasma membrane) of apoptosis could be distinguished in 15 min and was maximal at 1 h after treatment with VT2y. This was confirmed by the terminal dUTP nick-end-labeling (TUNEL) method.

Phospholipid containing choline histochemistry of mouse uterine epithelia during preimplantation stage.

The physiological role of high lipid content in endometrial cells during pregnancy has not been well established. In the present work we used histochemical techniques to analyze the total lipids and phospholipid containing choline (PCC) in the mouse uterine glandular and luminal epithelia during preimplantation stage. Sudan black histochemistry showed the highest intensity during the second day of pregnancy in both the basal and apical portions of luminal epithelium. Peaks of PCC staining were seen both in the luminal and glandular epithelia at the second and fifth days of pregnancy. Changes in localization and in the amount of lipid in the uterine epithelia suggest high mobility and metabolic rates of this substance, which may be related to morphological and/or functional changes occurring at the same time in the pregnant uterus. The increase and depletion timing of PCC content in the uterine epithelia during preimplantation stage, when uterine prostaglandin is also oscillating, suggest a possible involvement of PCC in prostaglandin biosynthesis. Therefore, the fate of lipid droplets found in the uterine epithelia may be related to critical changes of the pregnant endometrium, rather than the nourishment of developing embryos alone.

Adhesion of bovine enterotoxigenic Escherichia coli (ETEC) by type 1-like fimbriae.

Enterotoxigenic Escherichia coli (STa+) strains were isolated from adult bovine with diarrhea. These strains did not express any known ETEC-specific adhesins. Although hemagglutination with rat and sheep erythrocytes was observed in the presence of D-mannose (MRHA), these strains also showed mannose-sensitive hemagglutination (MSHA) with guinea-pig erythrocytes. Electron microscopic studies revealed the presence of fimbria-like structures (provisionally called "F43ms") on bacterial cells grown at 37 degrees C but not on cells grown at 18 degrees C. However, it was observed by SDS-PAGE that the J-1 strain (F43ms+) produces a protein similar to F1 fimbriae, and this strain hybridized with a DNA probe for F1 fimbriae. Immunogold-labelling techniques indicated that a rabbit anti-serum is specific for F43ms fimbrial structures, but not for Type 1 fimbriae. The immunofluorescence test carried out with semipurified F43ms on bovine brush borders suggests that the fimbria-like structures are responsible for the adhesion to bovine epithelial cells.

Light and electron microscopic radioautographic studies on the RNA synthesis of peri-implanting pregnant mouse uterus during activation of receptivity for blastocyst implantation.

The uterine receptivity for blastocyst implantation in the rodents is activated by nidatory estradiol and is maintained for a restricted period. To establish the variation of the RNA synthesis related to the activation of the uterine receptivity for blastocyst implantation, 1 hr. pulse 3H-uridine incorporation in the mouse uterus at time 0, 3, 6, 12 and 18 hrs. after nidatory estradiol treatment was analyzed by light (LM-RAG) and electron microscopic (EM-RAG) radioautography. The qualitative and quantitative differences of 3H-uridine incorporation in the luminal epithelium and endometrial stroma of three regions of the uterus were evaluated. In the LM-RAM, both the luminal epithelium and stromal cells around the conceptus showed a high incorporation 6 hrs. after estrogen treatment. After the peak of incorporation, the RNA synthesis both of the epithelial and stromal cells of antimesometrial side of the implantation site (AI) decreased abruptly and the grain count values become lower than seen in the other two sites. Both the epithelial and stromal cells of the IN sites showed a gradual but low increase of 3H-uridine incorporation through the time analyzed. The cells of the MI sites also showed a peak of incorporation 6 hrs. after estradiol treatment, but less intense than the AI sites. The EM-RAM showed the silver grains distributed over the cell structures related to the mRNA and rRNA metabolism both on the epithelial and stromal cells. The labeling were also seen on the trophoblastic cells, endothelial cells, smooth muscle cells of the myometrium and mesothelial cells. These results proved the strong influences of the estrogen on the pregnant uterus and showed the topological differences on the responses of the endometrium according to their relation to the implanting blastocyst, and suggest a time restricted responses of the endometrium for activation of uterine receptivity for blastocyst implantation.

Timely and topologically defined protein synthesis in the peri-implanting mouse endometrium revealed by light and electron microscopic radioautography.

Many efforts have been made to correlate the morphological and biochemical evidences with changes on physiological state of the uterus during activation of the implantation window. However, the exact mechanism involved in such a phenomenon remains to be determined. The present experiment used the radioautographic approach to determine whether, chronological and/or topological variations of proteins synthesis occur in the peri-implanting endometrium. Pregnant mice submitted to ovariectomy, received exogenous supply of nidatory estradiol. After 0 to 18 hrs. of time-lapsed estradiol effects, each animal received intraperitoneally, 1 hr. pulse 3H-leucine. The uterine fragments embedded in epoxy resin, were processed for light and electron microscope radioautography. The pattern of 3H-leucine incorporation in the luminal epithelium varied according to their relation with the blastocyst present in the uterine lumen. The highest incorporation ratio was seen in the epithelium just around the conceptus 6 hrs. after estradiol treatment, while in the cells localized at interimplantation site no peak of incorporation was seen. At ultrastructural level, cell organelles involved in protein synthesis were found to be labelled. Accumulation of silver grains occurred at apical portion of the epithelial cells showed accumulation of silver grains after 6 hrs. of estradiol treatment, but not on cell surface. The endometrial stromal cells localized around the blastocyst also showed a peak of 3H-leucine incorporation 6 hrs. after estradiol, but not in the cells localized at interimplantation sites. No increased labeling was seen on the components of extra-cellular matrix at ultrastructural level. The present radioautographic experiment showed that epithelial and stromal cells localized in the endometrium of implantation chamber, changed their pattern of protein synthesis under nidatory estradiol effects. This evidence suggests that the phenomenon of implantation window induced by estradiol, is not a systemic response of the whole pregnant endometrium. The activation involves only a specific population of the endometrial cells localized just around the conceptus.

Light and electron microscopic radioautography of DNA synthesis in the endometria of pregnant-ovariectomized mice during activation of implantation window.

Pregnant mice were ovariectomized at pre-implantation stage and exogenous nidatory estradiol was administered to evaluate the DNA synthesis of the endometrial cells during activation of uterine receptivity for blastocyst implantation. After 0, 3, 6, 12 and 18 hrs. of estradiol treatment, the animals received 3H-thymidine injection, sacrificed 1 hr. later, and the uteri were prepared for light and electron microscopic radioautography. At time 0, no labelled stromal or epithelial cells was found in the endometrium. According to the time-lapse after estradiol induction, a gradual increase of labelled stromal and endothelial cells was seen in the endometrium. The highest labeling index was observed at the antimesometrial side of the implantation sites and the lowest value was found at the interimplantation site. The cells found at mesometrial side of the implantation site showed an intermediate labeling index. Eighteen hrs. after estradiol treatment, the labelled stromal cells found near the implantation chamber resembled the morphology of decidual cells while those labelled cells localized at the interimplantation sites were similar to the fibroblast. The uterine luminal epithelial cells showed low DNA synthesis after estradiol treatment resulting in only a few labelled cells at the interimplantation sites and no labelled cells at the implantation sites. A similar labeling pattern was seen in the glandular epithelium. The distribution of labelled cells seen among the regions of pregnant endometrium under estradiol effect suggest that DNA synthesis related to uterine activation for blastocyst implantation is a focal reaction, where the luminal epithelium does nt proliferate while the stromal and endothelial cells around the conceptus increase the DNA synthesis to prepare the endometrial decidualization.

[Ambulatory cardiologic care: comparison between patients from a referral hospital and from community health centers]. / Atendimento cardiológico ambulatorial: comparação entre pacientes atendidos em hospital de referência e em centro de saúde comunitário.

PURPOSE--Comparison between patients from a cardiology referral center and those from community health facility. PATIENTS AND METHODS--564 (5.3%) of 10667 patients from the referral center--Instituto do Coração (InCor) and 105 (58.6%) of 169 from community health facility--Centro de de Santo Amaro (CSSA), São Paulo. RESULTS--217 (35.8%) patients in InCor and 27 (25.5%) in CSSA were younger than 40 years of age. Female patients were more frequent: 316 (56%) in InCor and 70 (66.7%) in CSSA. In InCor, 317 (56.2%) patients lived in São Paulo City and in CSSA all the patients live in the surroundings. Forty-three percent of the patients sought for medical attention in InCor by themselves, without medical referral. The diagnosis of heart disease was established in 81% of the patients in InCor and in 92.5% of the patients in CSSA. Other tests (besides electrocardiogram and chest roentgenogram) were considered to be indicated in 35% of the patients from InCor. The diagnoses were: a) coronary heart disease in 92 (20.1%) cases (InCor) and in 8 (8.2%) cases (CSSA); b) valvular heart disease in 46 (10.1%) cases (InCor) and in 9 (9.2%) cases (CSSA); c) mitral valve prolapse in 31 (6.8%) cases (InCor) and in 7 (7.1%) cases (CSSA); d) congenital heart disease in 10 (2.2%) cases (InCor) and in 1 (1%) case (CSSA); e) systemic arterial hypertension in 161 (35.2%) cases (InCor) and in 55 (56.1%) cases (CSSA); f) Chagas' infection or Chagas' heart disease in 44 (9.6%) cases (InCor) and in 8 (8.2%) cases (CSSA); g) cardiac rhythm disorders in 38 (8.3%) cases (InCor) and in 8 (8.2% cases (CSSA); h) other diseases in 33 (7.2%) cases (InCor) and in 1 (1%) (CSSA); i) diseases of the aorta in 2 (0.4%) cases (InCor) and in 1 (1%) (CSSA). Medical treatment was recommended to 71.5% of the patients from the InCor and to 92.5% of the patients from CSSA. CONCLUSION--Our data suggest a degree of similarity between the groups of patients cared for in a cardiology referral center in our city in relation to a community health facility. These data may be useful in planning optimal use of referral hospitals facilities in our city.