Ectopic expression of FSH receptor isoforms in neoplastic but not in endothelial cells from pancreatic neuroendocrine tumors.

FSH receptor (FSHR) expression is restricted to gonads, where it drives FSH-dependent cell differentiation; in addition, FSHR plays an important role in the regulation of ovarian angiogenesis. Recently, FHSR expression has been shown in blood vessels of various tumors. However, pancreatic neuroendocrine tumors (p-NET), which have high-degree blood supply, were not included in that study. The aim of this study was to evaluate FSHR expression in p-NET. FSHR expression was evaluated in tumor samples from 30 patients with p-NET by immunohistochemistry and Western blot; fluorescence microscopy was used to localize FSHR in specific cells from tissue samples. von Willebrand factor (vWF) and chromograninA (chrA) was used as blood vessel and NET cells marker, respectively, to co-localize FSHR. FSHR expression was detected in all p-NET by immunohistochemistry. Western blot confirmed FSHR expression on p- NET although different FSHR isoforms, ranging from 240 kD to 55 kD were found in the samples studied. Surprisingly, FSHR co-localized with chrA but not with vWF, suggesting that neoplastic cells of neuroendocrine origin rather than blood vessels expressed FSHR. No relationship was found between degree of FSHR expression and histology of p-NET. FSHR may be aberrantly expressed in neoplastic cells from p-NET and not in tumor blood vessels; however, its biological significance as well as its clinical relevance remains to be elucidated.

Vaginal lactoferrin in asymptomatic patients at low risk for pre-term labour for shortened cervix: cervical length and interleukin-6 changes.

A total of 3,324 singleton pregnant women were screened for pre-term delivery and 128 women were finally randomised and analysed for outcome showing borderline cervical length (25-29 mm) and elevated cervico-vaginal interleukin 6 levels. To verify if vaginal administration of lactoferrin might have an influence on these variables, two groups of 64 patients were formed. Study cases were submitted to lactoferrin for 21 days; controls received no treatment. An inverse relation was found between interleukin 6 levels and cervical length. On day 30 from the beginning of the treatment, study cases showed a decrease in interleukin 6 levels and an increase in cervical length. A greater number of women with regular uterine contractions and reduced cervical consistency before the 37th week of gestation were found in the controls. Our data show that lactoferrin could play a role in reducing the number of women at risk for pre-term birth for shortened cervical length and elevated interleukin 6 levels.

Dendritic cell functional improvement in a preclinical model of lentiviral-mediated gene therapy for Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency caused by the defective expression of the WAS protein (WASP) in hematopoietic cells. It has been shown that dendritic cells (DCs) are functionally impaired in WAS patients and was(-/-) mice. We have previously demonstrated the efficacy and safety of a murine model of WAS gene therapy (GT), using stem cells transduced with a lentiviral vector (LV). The aim of this study was to investigate whether GT can correct DC defects in was(-/-) mice. As DCs expressing WASP were detected in the secondary lymphoid organs of the treated mice, we tested the in vitro and in vivo function of bone marrow-derived DCs (BMDCs). The BMDCs showed efficient in vitro uptake of latex beads and Salmonella typhimurium. When BMDCs from the treated mice (GT BMDCs) and the was(-/-) mice were injected into wild-type hosts, we found a higher number of cells that had migrated to the draining lymph nodes compared with mice injected with was(-/-) BMDCs. Finally, we found that ovalbumin (OVA)-pulsed GT BMDCs or vaccination of GT mice with anti-DEC205 OVA fusion protein can efficiently induce antigen-specific T-cell activation in vivo. These findings show that WAS GT significantly improves DC function, thus adding new evidence of the preclinical efficacy of LV-mediated WAS GT.

T-DM1 versus pertuzumab, trastuzumab and a taxane as first-line therapy of early-relapsed HER2-positive metastatic breast cancer: an Italian multicenter observational study.

BACKGROUND: The current standard first-line treatment of human epidermal growth factor receptor 2 (HER2)-positive (+) metastatic breast cancer is the combination of pertuzumab, trastuzumab and a taxane (P + T + taxane), while standard second-line is ado-trastuzumab-emtansine (T-DM1). The registration trial of pertuzumab, however, did not include early-relapsing patients, defined as patients experiencing tumor relapse ≤12 months from the end of (neo)adjuvant anti-HER2 therapy. Conversely, the pivotal trial of T-DM1 included some patients relapsing ≤6 months after the end of (neo)adjuvant trastuzumab. Thus, a proportion of early-relapsing patients are currently eligible to receive T-DM1 as first-line treatment. Nevertheless, no direct comparison exists between the two regimens in this clinical setting. PATIENTS AND METHODS: We retrospectively compared T-DM1 versus P + T + taxane as first-line treatment in two cohorts of early-relapsing patients in an Italian 'real-world' setting, involving 14 public health care institutions. The primary endpoint was progression-free survival. Secondary endpoints included patients' characterization, overall survival and post-progression survival. Univariate and multivariate analyses were carried out. All tests were two-sided and a P ≤ 0.05 was considered statistically significant. RESULTS: Among 1252 screened patients, 75 met the inclusion criteria. Forty-four (58.7%) received P + T + taxane and 31 (41.3%) received T-DM1. The two cohorts showed similar characteristics of aggressiveness and no significant differences in treatment history. T-DM1, compared with P + T + taxane was associated with worse progression-free survival (adjusted hazard ratio: 2.26, 95% confidence interval: 1.13-4.52, P = 0.021) and overall survival (adjusted hazard ratio: 3.95, 95% confidence interval: 1.38-11.32, P = 0.010), irrespective of previous (neo)adjuvant treatment, age, hormone receptors status, time-to-relapse (≤6 months or within 6-12 months) and presence of visceral/brain metastases. No differences were observed in post-progression survival (P = 0.095). CONCLUSIONS: Our study suggests superiority for P + T + taxane over T-DM1 as up-front treatment of early-relapsing HER2+ metastatic breast cancer, which merits further assessment in larger and prospective trials.

Persistence of reactivity against the 45 k Da glycoprotein in late trichinellosis patients.

Over the years, the opinions of clinicians on the existence of the so-called chronic trichinellosis or late sequelae of infection have differed. However, the persistence of a humoral immune response against Trichinella in these late-stage patients has been confirmed using specific tests such as the competitive inhibition assay (CIA). We evaluated sera from late-stage trichinellosis patients (2--8 years from acute infection), for their reactivity against Trichinella spiralis antigens. The following tests were carried out: (i) indirect immunofluorescence assay (IFA), performed on muscle sections from mice, 30 days following synchronous infection by intramuscular injection with T. spiralis newborn larvae (NBL); (ii) enzyme immunoassay, employing a synthetic beta-tyvelose antigen conjugated to bovine serum albumin (BSA-Ag); and (iii) western blot (WB) with both an "in house" kit and a commercial kit. The results of IFA obtained by confocal laser microscopy showed that sera reacted against both surface and internal structures of L(1) larvae but at varying levels. Employing the synthetic antigen, EIA showed that 50% of sera tested were positive for the presence of specific antibodies against beta-tyvelose. By WB, all sera were reactive with the 45 k Da glycoprotein (45 gp). These data suggest that reactivity against the beta-tyvelosylated 45 gp persists even in very late stages of human trichinellosis.

Yolk utilization in stick insects entails the release of vitellin polypeptides into the perivitelline fluid.

This study investigates the developmental fate of vitellin (Vt) polypeptides generated by limited proteolysis in an insect embryo. To this end, a number of polyclonal (pAb) and monoclonal antibodies (mAb) were raised against the yolk sac and the perivitelline fluid of late embryos of the stick insect Carausius morosus. Two dimensional immuno gel electrophoresis and Western blotting demonstrate that polypeptides resulting from Vt processing are present both in the yolk sac and the perivitelline fluid. At the confocal microscope, different labelling patterns were detected in the ooplasm depending on the stage of development attained by the embryo. At early developmental stages, label is associated with large unsegmented portions of the fluid ooplasm. During embryonic development, the fluid ooplasm is gradually transformed into yolk granules by intervention of vitellophages. Prior to dorsal closure, the yolk sac is separated from the perivitelline fluid by interposition of serosa cells (the so called serosa membrane). Several mAbs raised against the perivitelline fluid react specifically with this membrane suggesting that the release of Vt polypeptides from the yolk sac occurs by intracellular transit through the serosa cells. By immunocytochemistry, gold label appears associated with the cell surface and a number of vacuoles of the serosa membrane. These data are interpreted as suggesting that Vt polypeptides resulting from limited proteolysis in stick insect embryos are not exhaustively degraded within the yolk sac, but are instead transferred transcytotically to the perivitelline fluid through the serosa membrane.

Uterine fibroids: protocols of integrated medical/surgical treatment.

Uterine fibroids are the most common solid pelvic tumor in women and in these last years their management has been deeply reviewed. An optimal integration of GnRH analogue treatment with different surgical techniques require an adequate work-up for the specific problems of each single patient. For a correct management of these patients, we divided them into 4 categories on the basis of symptoms and specific objectives to persuasive: a) perimenopausal patients; b) young patients, symptomatic or with large myomas, with no wish for more children; c) young patients, symptomatic or with large myomas, wishing to preserve fertility; d) infertile patients or patients with history of repeated miscarriages. Our suggested work-up is the following: a careful USGraphic evaluation of fibroid size and localization, a transvaginal doppler examination of the uterine blood supply, a complete haematochemical analysis, a hysteroscopy guided biopsy and a complete bone evaluation. On the basis of the above mentioned evaluations for each group we will use a different therapeutic approach in regard of either length of medical treatment (for tree to six months, for one or more cycles) or necessity of surgical treatment (with the possibility to get a natural menopausal), or different surgical techniques (operative hysteroscopy and/or laparoscopy, laparotomic myomectomy, vaginal or abdominal hysterectomy.

Vaginal administration of gemeprost for preoperative cervical dilatation in non pregnant patients.

The use of Gemeprost vaginal suppositories has been evaluated in a trial for induction of the cervical dilatation in non pregnant women. 30 voluntary patients, 22 nulliparous and 8 pluriparous, had to be subjected to biopsy of the endometrium; 24 were treated for the control of sterility and 6 for menstrual perimenopausal disorders. The biopsies of the sterility control were effected in the second half of the cycle, generally without having recourse to narcosis. A single Gemeprost pessary containing 1 mg of 16, 16-dimethyl-trans-delta 2 PGE1 methyl ester was intravaginal administered, deeply into the posterior fornix, 3 hours before the biopsy. The success rate was 86.66 (26 pts.) with an average dilatation of 5.38 H (Hegar) +/- 0.75 SD. For 4 patients (13.33%) having a dilatation less than 4 H, it was necessary to complete the dilatation mechanically. All the observed side effects presented a modest intensity: cephalalgia 6.6% (no. 2), gastralgia 3.3% (no. 1), vaginal burning 6.6% (no. 2). No significant variation of vital function parameters was recorded. In conclusion this type of preparation of the cervix has permitted us to achieve a more gradual dilatation and to prevent the traumata of the cervico-isthmic system due to forced mechanical dilatations by the exclusive use of Hegar's dilators.

[Dacron-EXS prosthesis in axillo-femoral bypass]. / La protesi di Dacron-EXS nel distretto axillo-femorale.

Twenty-eight axillary by-pass were performed in 27 patients using a dacron externally supported prosthesis. This group of patients has been compared with two other groups in which a dacron knitted (20 p.) or a PTFE (55 p.) prosthesis were implanted. A three years patency has been respectively: 66.6% (EXS), 57.7 (PTFE), 45.5 (Knitted Velour).

Atosiban vs ritodrine used prophylactically with cerclage in ICSI pregnancies to prevent pre-term birth in women identified as being at high risk on the basis of transvaginal ultrasound scan.

Our objective was to compare the effectiveness and safety of atosiban and ritodrine, in pregnancies obtained by intracytoplasmic sperm injection (ICSI) undergoing cervical cerclage. Data from a prospective study were compared with those from a retrospective study. Sixteen ICSI pregnant women, 20-24 weeks' gestation and maternal age >18 years, received atosiban (bolus dose 6.75 mg i.v., followed by 300 microg/min i.v. for 3 h and 100 microg/min i.v. for 45 h). Cervical cerclage was performed 3 h after starting atosiban. The control group (group B) of 16 ICSI pregnant women were matched and received ritodrine hydrochloride (100-350 microg/min) for 48 h. Cervical cerclage was performed after 24 h. Pre-term rupture of membranes occurred within 48 h of cervical cerclage in one woman receiving atosiban and in four women receiving ritodrine. There was no significant difference in terms of pregnancies not delivered at 48 h (short-term tocolysis) and at 7 days (long-term tocolysis). However, there was a significantly higher incidence of maternal tachycardia with ritodrine compared with atosiban (p < 0.001). The mean gestational age at delivery was significantly higher for atosiban compared with ritodrine (36 vs 33 weeks; p < 0.001). The neonatal outcome was poorer for ritodrine than atosiban, as there were very low birth weight infants (p = 0.008), resulting in lower Apgar scores (p = 0.005) and there were more neonates requiring a long stay in the neonatal intensive care unit (p = 0.005). We conclude that atosiban is associated with a significantly lower incidence of maternal tachycardia and improved neonatal outcome compared with ritodrine.

Peroxisome proliferator-activated receptor (PPAR)gamma is highly expressed in normal human pituitary gland.

OBJECTIVE: Expression of peroxisome proliferator-activated receptor (PPAR)gamma in normal pituitary seems to be restricted to ACTH-secreting cells. The aim of the study was to evaluate the expression of PPARgamma in normal human pituitary tissue and to study its localization in the pituitary secreting cells. MATERIALS AND METHODS: Normal pituitary tissue samples were obtained form 11 patients with non-secreting adenoma who underwent surgical excision of the tumor. Expression of PPARgamma was evaluated by immunostaining and western blotting; localization of PPARgamma in each pituitary secreting cell lineage was evaluated by double immunofluorescence using confocal microscopy. Pituitary non-functioning adenomas served as Controls. RESULTS: PPARgamma was highly expressed in all pituitary samples with a (mean +/- SD) 81 +/- 6.5% of stained cells; expression of PPARgamma was confirmed by western blotting. Non-functioning pituitary adenomas had 74 +/- 11% PPARgamma positive cells. Expression of PPARy was either in cytoplasm or nuclei. In addition, treatment of GH3 cells, with a PPARgamma ligand was associated with traslocation of the receptor from cytoplasm into the nucleus. Double immunostaining revealed that every pituitary secreting cell (GH, TSH, LH, FSH, PRL and ACTH) had PPARgamma expressed. DISCUSSION: The present study demonstrated that PPARgamma is highly expressed in every normal pituitary secreting cell lineage. It can translocate into the nucleus by ligand binding; however, its role in pituitary hormone regulation remains to be elucidated.

Fetal cerebral haemodynamic adaptation: a progressive mechanism? Pulsed and color Doppler evaluation.

The importance of studying, by Doppler ultrasound, the cerebral haemodynamics to monitor the fetal response to the hypoxia is well known, but there is not a general agreement about the anatomical landmarks for the middle cerebral artery. Seventy-one normal fetuses and fifteen IUGR fetuses were studied. The umbilical artery and the middle cerebral artery (MCA) were evaluated by color Doppler ultrasound. The well-known decrease of the pulsatility index from the umbilical artery was observed throughout pregnancy. This velocimetric pattern did not occur in the IUGR fetuses. Four IUGR fetuses showed the ARED (absent or reversed and diastolic flow) pattern. The MCA was evaluated at the origin (M1) and at the distal tract (M2). PI values from M1 and M2 decreased during the pregnancy. A significant difference was detected between M1 and M2 PI values from the 26th to the 37th week of gestation. The M1 brain sparing effect was detected in the IUGR fetuses. Two ARED fetuses, observed during labor, showed the M2 sparing effect. The different Doppler patterns found in M1 and M2 could be due to the functional differences existing between these tracts. As a matter of fact, M1 and M2 supply different parts of the fetal brain, which develop in different periods of fetal life. These findings, if ulteriorly confirmed, could offer new perspectives for the monitoring of high risk fetuses.

A fat body-derived protein is selectively sulfated during transit to ovarian follicles in the stick insect Carausius morosus.

A monoclonal antibody raised against ovarian follicles of the stick insect Carausius morosus reacted with two related polypeptides of 157 and 85 kDa in both the ovary and the hemolymph. In vitro cultured fat body proved capable of releasing the 157-kDa polypeptide into the culture medium and processing it to the lower-molecular-weight polypeptide of 85 kDa. This was further demonstrated by in vitro exposure to [35S]methionine. Under the same culturing conditions, ovarian follicles proved incapable of synthesizing and/or secreting the 85-kDa polypeptide. However, in vivo [35S]methionine-labeled ovarian follicles released both polypeptides when cultured in vitro for up to 24 hr. Vitellogenin polypeptides were labeled in vivo following exposure to [3H]glucosamine, while 157- and 85-kDa polypeptides were labeled only in ovarian follicles exposed in vivo to sodium [35S]sulfate. Under in vitro conditions, the 157-kDa polypeptide could be labeled with sodium [35S]sulfate only if ovarian follicles were cocultured with fat body. No sulfation occurred in fat body or ovarian follicles cultured separately. These experiments suggest that the 157-kDa polypeptide is a fat body-derived polypeptide that is sulfated upon transfer to the ovarian follicle.

Angiogenesis: a new diagnostic aspect of obstetric and gynecologic echography.

An ultrasounds triplex system (pulsed Doppler, Color mode, B mode) has been employed to study ovarian and uterine circulatory changes in fertile, infertile and postmenopausal patients. PI and mean percentage variation of PI mean values have been used. No difference has been detected in uterine haemodynamics between fertile women and those infertile patients getting pregnant by IVF-ET procedure. Patients who did not obtain the pregnancy showed different uterine and endometrial Color Doppler patterns. Furthermore, a typical endometrial blood flow has been detected both in fertile patients and infertile patients who get pregnant. Color Doppler seems to be an useful tool in the monitoring of fibroids blood supply and the uterine blood supply, especially for the clinical monitoring of GnRH analogues therapies. The technique has been also employed for the study of ectopic pregnancy. The evaluation of both uterine and throphoblast haemodynamics has resulted in a good indicator of the pregnancy progression. Concerning the ovarian pathology the color Doppler evaluation has been useful to differentiate ovarian malignant cysts from inflammatory or disfunctional masses. Nevertheless, the most interesting results have been obtained from the study of endometrial and ovarian tumors. Indeed, in the most cases, a mass supply blood flow has been detected, although in some cases no angiogenetic color flow has been found. No difference has been found in uterine artery velocimetry between malign or benign pathologies. Furthermore, histological data supporting our findings are presented.

Vitellin polypeptide pathways in late insect yolk sacs.

A panel of monoclonal antibodies was raised against late yolk sacs of the stick insect Carausius morosus and tested by immunoblotting to establish the extent vitellin polypeptides are processed proteolytically during embryonic development. Cryosections of late yolk sacs were also examined by confocal laser microscopy to determine how vitellin cleavage products become spatially distributed amongst yolk granules during the same developmental period. Distinct labelling patterns were obtained on yolk granules depending on: (1) the nature of the proteolytic processing; (2) the origin of vitellin cleavage products; and ultimately (3) their molecular sizes. Monoclonal antibodies raised against vitellin cleavage products resulting from proteolytic processing appeared to label: (1) the entire volume of many yolk granules; (2) their limiting membrane; or (3) a number of small vesicles interposed between larger yolk granules. On the other hand, monoclonal antibodies against vitellin cleavage products that remain invariant throughout development appeared to label either the serosa membrane or the cytosolic space comprised between adjacent yolk granules. Data are interpreted as indicating that vitellin cleavage products may leak out from the yolk granules, gain access to the cytosolic space of the vitellophages and eventually percolate through the serosa membrane enclosing the yolk sac.

Native vitellins are modified during ovarian development in the stick insect Carausius morosus (Br.).

Vitellins from ovarian follicles and newly laid eggs of the stick insect Carausius morosus were examined by ion exchange chromatography on a HPLC Mono Q column. Under these conditions, vitellins from newly laid eggs resolved as two distinct peaks, referred to as VtA and VtB, that eluted at 8.5 and 12.0 min, respectively. On native gels, both VtA and VtB separated into two different variant forms (VtA' and VtA", VtB' and VtB"). By two-dimensional gel electrophoresis, VtA' and VtA" were shown to contain polypeptides A1, A2 and A3. On the other hand, VtB' and VtB" appeared to comprise polypeptides B1 and B2 and B1, A1, A2, B2 and A3*, respectively. A similar Vt polypeptide composition was also observed by size-exclusion chromatography of vitellins from newly laid eggs. Vitellins from early vitellogenic ovarian follicles resolved into a single chromatographic peak at 7.5 min that coeluted with a major peak from the hemolymph of egg-laying females. Ovarian follicles progressively more advanced in development exhibited a more complex chromatographic profile, consisting of three separate peaks. By two-dimensional gel immunoelectrophoresis, vitellins from ovarian follicles appeared to consist of two closely related, immunologically cross-reacting antigens that gradually shifted apart as ovarian development proceeded to completion. By size-exclusion chromatography, each Vt from ovarian follicles was shown to consist of a unique set of polypeptides different from those listed above. Single ovarian follicles were fractionated into yolk granules and yolk fluid ooplasm and tested by immunoblotting against Mab 12. Under these conditions, VtA variant forms in yolk granules and yolk fluid ooplasm reacted differently. Sections from ovarian follicles in different developmental stages were exposed to Mab 12 and stained with a peroxidase-conjugated, goat anti-mouse antibody. Regardless of the developmental stage attained, staining for peroxidase was restricted to free yolk granules, suggesting that native vitellins in stick insects are structurally modified upon fusion into the yolk fluid ooplasm.

Correlation of Doppler and placental immunohistochemical features in normal and intrauterine growth-retarded fetuses.

The aim of this study was to correlate and compare Doppler and anatomical placental findings obtained from 48 normal and 35 intrauterine growth-retarded (IUGR) fetuses. The IUGR group consisted of 19 fetuses from pregnancies complicated by pre-eclampsia and 16 from healthy mothers. Color Doppler evaluation of umbilical, spiral and uterine arteries was performed. Placental specimens from both normal and growth-retarded fetuses were obtained at the time of delivery. Placental specimens were evaluated using histochemical and immunohistochemical techniques. A progressive decrease in the pulsatility index was observed in umbilical, spiral and uterine arteries throughout pregnancy in the normal-growth fetuses. High umbilical artery pulsatility index values were obtained in 29 out of the 35 growth-retarded fetuses, six of them showing absent or reversed end-diastolic umbilical artery flow pattern. A total of 13 IUGR fetuses showed high resistance uterine artery flow velocity waveforms. Increased pulsatility index values were obtained from the spiral arteries of 16 growth-retarded fetuses. Abnormal histological and histochemical placental patterns were observed in all the growth-retarded fetuses with umbilical artery Doppler abnormalities. The presence of a peculiar dendritic cell subpopulation, strongly resembling the Langerhans cells, expressing the HLA-DR+/CD1+ phenotype, was detected in all growth-retarded fetuses, whether there was maternal pathology or not. Our data show uterine and spiral artery data as being ineffective in the monitoring of IUGR fetuses. The placental extracellular matrix seems to play an important role in the regulation of the umbilical circulation. The presence of CD1+ cells as a sign of a possible immunological mechanism in the pathogenesis of the intrauterine growth retardation is discussed.

Serosa membrane plays a key role in transferring vitellin polypeptides to the perivitelline fluid in insect embryos.

In mid-embryogenesis, the stick insect Carausius morosus comes to be comprised of three distinct districts: the embryo proper, the yolk sac and the perivitelline fluid. A monolayered epithelium, the so-called serosa membrane, encloses the yolk sac and its content of vitellophages and large yolk granules. During embryonic development, the yolk sac declines gradually in protein concentration due to Vt polypeptides undergoing limited proteolysis to yield a number of Vt cleavage products of lower molecular weights. mAbs 1D1 and 5H11 are monoclonal antibodies raised against some of the Vt cleavage products generated by this process in the yolk sac. At the confocal microscope, antibody fluorescence is initially associated with a few yolk granules, while it is gradually displaced in the cytosolic spaces of the vitellophages. With the proceeding of embryonic development, label appears also in the serosa membrane in the form of clustered dots. At the ultrastructural level, gold particles are initially associated with the vitellophages that are labeled on a few yolk granules and in the cytosolic space flanking the yolk granules. Subsequently, the serosa cells become labeled on vesicles close to the yolk granules or just underneath the plasma membrane. Inside the serosa cells, label is also associated with granules budding from the Golgi apparatus, but never with the intercellular channels percolating the serosa membrane. These observations are interpreted as indicating that Vt cleavage products leak out from the yolk granules into the cytosolic spaces of the vitellophages and are eventually transferred to the perivitelline fluid via transcytosis through the serosa cells.

Different growth conditions for peripheral blood endothelial progenitors.

PURPOSE: To compare different growth conditions for endothelial progenitor cells (EPCs) from peripheral blood mononuclear cells (PBMNCs). METHODS AND MATERIALS: PBMNCs of healthy volunteers were cultured on fibronectin as follows: M199 with VEGF, bFGF, IGF-I; the same medium with bovine retina-derived extract (RDE); freshly isolated or depleted of adherent cells PBMNCs in HUVEC conditioned medium; DiI-stained PBMNCs with HUVECs (1:4 ratio) in Ml99 with RDE. PBMNCs were analysed by FACS using mAbs for endothelial markers. EPCs migration was determined using a modified Boyden chamber assay and VEGF as chemoattractant. EPCs were seeded alone or with HUVECs on Matrigel to assess in vitro angiogenesis. RESULTS: With growth factors, numerous cell clusters appeared within 1 week. Spindle-shaped and attached cells sprouted, differentiating in endothelial cell (EC)-like cells within 2 weeks and forming cobblestone-like monolayers within 3 weeks. With RDE, numerous large cell clusters appeared within 1 week, but the number of cells with an EC morphology decreased during culture. FACS confirmed the endothelial phenotype and attached cells were able to migrate in response to VEGF. When nonadherent cells were cultured in HUVEC conditioned medium, they proliferated readily and EPCs were induced while freshly isolated cells neither proliferated nor induced EPCs. FACS analysis of the cocultures showed the presence of double-labeled PBMNCs expressing endothelial antigens. Capillary-like structures were observed on Matrigel only from cocultures and PBMNCs were able to incorporate in these networks. CONCLUSIONS: PBMNCs are able to differentiate in EPCs when stimulated with appropriate culture conditions (growth factors, HUVEC conditioned medium, HUVECs).