Carlo Pafumi

Nerve growth factor–endothelial cell interaction leads to angiogenesis in vitro and in vivo

Nerve growth factor (NGF) has important functions during embryonic development and on various tissues and organs under normal and pathological conditions during the extrauterine life. RT‐PCR analysis and immunological methods demonstrate that human umbilical vein endothelial cells (HUVECs) express the NGF receptors trkANGFR and p75NTR. NGF treatment caused a rapid phosphorylation of trkANGFR in HUVECs, determining a parallel increase of phosphorylated ERK1/2. Accordingly, NGF induced a significant increase in HUVEC proliferation that was abolished by the trkANGFR inhibitor K252a. Also, HUVECs express significant levels of NGF under standard culture conditions that were up‐regulated during serum starvation. Endogenous NGF was responsible for the basal levels of trkANGFR and ERK1/2 phosphorylation observed in untreated HUVEC cultures. Finally, NGF exerted a potent, direct, angiogenic activity in vivo when delivered onto the chorioallantoic membrane of the chicken embryo. The data indicate that NGF may play an important role in blood vessel formation in the nervous system and in several pathological processes, including tumors and inflammatory diseases. Unraveling mechanisms of NGF‐dependent angiogenesis could provide valuable tools for novel therapeutic approaches in antiangiogenic therapy.

Genetics of polycystic ovarian syndrome

Polycystic ovarian syndrome (PCOS) is a reproductive system disorder characterized by irregular menses, anovulation, clinical and/or biochemical signs of hyperandrogenism (hirsutism and/or acne), ovarian micropolycystic appearance and metabolic abnormalities, such as hyperinsulinaemia and obesity. The aetiopathogenesis of this syndrome is not well known. Several pathogenetic hypotheses have been proposed to explain the full array of symptoms and signs, but with elusive results. A genetic abnormality causing PCOS is supported by the observation that different members of the same family are often affected, and about half of the sisters of PCOS women have elevated serum testosterone concentrations. Therefore, the presence of gene abnormalities in women with PCOS has been widely explored in the attempt to establish whether their mutations or polymorphisms may cause PCOS. The main genes evaluated are those involved in steroidogenesis, steroid hormone effects, gonadotrophin release regulation and action, insulin secretion and action, and adipose tissue metabolism. Despite the vast body of literature produced, none of the genes evaluated seems to play a key role in PCOS pathogenesis. It is likely that PCOS may represent the final outcome of different, deeply inter-related genetic abnormalities that influence each other and perpetuate the syndrome.

Haematopoietic progenitors from umbilical cord blood.

Background/Aim: The aim of this study is to improve the obstetrician-based cord blood collection system and an efficient recovery of CD34+ haematopoietic progenitor stem cells. Methods: CD34+ cells were purified from total blood using a positive selection enrichment method, called Mini-Macs. Results: The final yield of CD34+ cells we obtained was 104 cells/ml, with a CD34+ purity of 99%. Conclusion: Our results confirm that, by using this method, it is possible to get a significant stem cell number, thus improving transplanting both peripheral stem cells and umbilical cord ones.

Divergent effects of corticotropin releasing hormone on endothelial cell nitric oxide synthase are associated with different expression of CRH type 1 and 2 receptors

Endothelium is a target for an array of factors involved in inflammation. Endothelial cells express receptors for CRH, a neuropeptide produced during inflammation. We report both the concentration‐dependent inhibitory effect of CRH upon cytokine‐stimulated nitrite release by H5V murine endothelioma cells, and its stimulatory one in HUVEC cells. Western blot analysis showed that CRH inhibits cytokine‐stimulated iNOS protein in H5V cells, and, instead, potentiated it in HUVEC cells. H5V cells expressed both CRH receptors (CRH‐R1 and R2) mRNAs, whereas HUVEC cells expressed the CRH‐R2 mRNA solely. CRH increased medium nitrites and iNOS protein expression in H5V cells pretreated with the selective CRH‐R1 antagonist CP 154,526. However, the selective CRH‐R2 antagonist anti‐Svg‐30 failed to produce similar effects. In fact, anti‐Svg‐30 inhibited CRH‐induced increase of nitrite release and iNOS expression in HUVEC cells. Our results confirm the activating role of CRH on endothelial cells, although it suggests its possible inhibitory role in the late phase of the inflammatory response. NO‐mediated effects of CRH on endothelial cells could be exploited in therapeutic strategies related to inflammatory and/or degenerative diseases.

Chromosome analysis of epididymal and testicular spermatozoa in patients with azoospermia.

Azoospermic patients can now father children once spermatozoa have been retrieved from the epididymis or the testis. However, there are concerns about the risk of chromosomal abnormalities since an increase in sperm aneuploidy rate has been reported in samples from patients with abnormal sperm parameters. The purpose of this study was therefore to evaluate the sperm aneuploidy and diploidy rates for chromosomes 8, 12, 18, X and Y in spermatozoa extracted from the epididymes (n=10) or the testes (n=6) of patients with azoospermia. Ejaculated spermatozoa of healthy men (n=14) served as control. Epididymal and testicular spermatozoa had an aneuploidy rate significantly higher than that found in ejaculated spermatozoa. The aneuploidy and diploidy rates of testicular spermatozoa were higher, but not significantly different, than those found in epididymal spermatozoa. This study has shown that azoospermic patients have an increased sperm aneuploidy rate. They should therefore be given appropriate genetic counselling before entering in-vitro fertilisation programs.

Umbilical cord blood collection in Cesarean section: a comparison before and after placental delivery.

Abstract Fetal stem cells transplants depend on nuclea-ted cells from fetal blood. This study was a prospective randomized trials to compare the collection of fetal blood by gravity into a bag containing anticoagulant, before and after delivery of the placenta. The obstetric and the newborn characteristics in the two group were not significantly different. The mean volume of fetal blood collected while the placental was still in utero was 74.93±7.1 ml as against 35.78±3.6 ml for collection of fetal blood after delivery of the placenta.

Uterine Myxoid Leiomyosarcoma Associated with Multiple Myomas in a Fertile Woman: A Case Report

A 37 year old woman was admitted to our university hospital for menometrorrhagia. The case history showed that the patient had menometrorrhagia for six months; moreover, during the abdominal examination we found a mass occupying the hipogastric and mesogastric area. The tumefaction was hard and it reached the level of the umbilicus. On combined vaginal-abdominal examination a mass on the anterior wall and multiple myomata were felt; the uterus was found to have been enlarged to the size equivalent to 18 weeks pregnancy; adnexa regular were felt. During the surgery multiple myomas were found. The largest, 10 cm diameter, was soft in consistence with a gelatinous structure. Total abdominal hysterectomy with preservation of adnexa was performed (Figure 1). Histopathological result gave evidence of myxoid leiomyosarcoma in the largest myoma, whereas the others fibroid nodes were without atypia.

Delivery Mode and Pelvic Floor Disfunction

Introduction: Pelvic floor disorders compromise quality of life for a lot of women of all ages. The prevalence of urinary incontinence (UI) is thought to range from 17 to 45% among adult women. The etiology is thought to be multifactorial. Traumatic damage to fascial and muscular support structures during childbirth may be, an important contributor to the development of UI and prolapse of pelvic organ (POP). The aim of this study is to consider the association between delivery mode and pelvic floor disorders (POP and UI) Materials and methods: A review of the literature was undertaken using the Medline and Popline CD Rom considering articles published from 1996 to 2011; additional sources were identified from references cited in relevant research articles. We studied articles concerning stress urinary incontinence, pregnancy, childbirth, pelvic prolapse were considered. Conclusion: Literature research confirms that anatomic and functional damages are linked with obstetric factors. Pregnancy may cause urinary incontinence and prolapse. However vaginal delivery is associated with a significant higher risk of urinary incontinence and pelvic defects. Caesarean section may protect from perineal risk of delivery but not from the damage due to the pregnancy itself. During pregnancy UI ranges from 31 to 39%, in post-partum ranges from 24,5 to 29% and from 5 to 8% after vaginal and caesarean respectively. Pelvic floor disorders ranges from 21 to 36% after instrumental operative delivery and from 9 to 21% in vaginal spontaneous delivery. Forceps is found out the most dangerous instrument for pelvic floor, followed by vacuum and vaginal delivery with tears. The consequences of a traumatic delivery affect quality of life and increasing late damages have to be considered.

VLA-2 and VLA-5 Cell adhesion molecules expression in CD34+ cells from Umbilical cord blood and from bone marrow

Background/Aims: Umbilical cord blood contains a large number of early hematopoietic cells with high proliferating capacity, that has been used as an alternative to bone marrow transplantation. The aim of this study is to investigate the number of two cell adhesion molecules in cord blood and in bone marrow. Methods: We investigated two integrins, named VLA-2 and VLA-5 (Very Late Appearing Antigen), expressed in the surface of CD34+ cells. The CD34+ cells, isolated with MACS CD34+ isolation kit, were labelled with the appropriate monoclonal antibodies. Results: Cell adhesion molecules showed highly expressed in both cord blood and bone marrow CD34+ cells. Conclusion: There are no significant differences between the two sources of CD34+ populations.

CD34 enrichment of umbilical cord blood.

OBJECTIVE We describe the relation between the clamping time and blood volume collected, plus two enrichment systems for CD34+ stem cells from umbilical cord blood, to achieve an excellent recovery of cells with a high proliferative ability and bone marrow reconstitution. METHOD After cord blood collection by an obstetrician, stem cell purification has been performed either with a combination of monoclonal antibodies, using the negative selection Stem Sep method, or with a positive cell selection based on their surface CD34 antigens, using the Mini Macs system. RESULTS An excellent recovery of hematopoietic progenitors: Burst Forming Unit Erythroid; Colony Forming Unit Granulocyte and Macrophage; and Colony Forming Unit Granulocyte, Erythroid, Monocyte and Macrophage, inversely related to the rising of clamping time, has been achieved with the Mini Macs system (54 % of colonies, with 90 % purity). With the Stem Sep method, recovery of hematopoietic progenitors was 35 % (with 80 % purity). CONCLUSIONS By an early clamping of umbilical cord blood, we obtained a higher number of CD34+ cells whose clonogenic activity increased with enrichment. This is particularly useful considering that the number of CD34+ stem cells contained in a unit of placental blood is enough for transplantation to a child, but not for an adult engraftment. Thus, using these methods, we can obtain a larger number of CD34+ stem cells, which increases the possibility of reducing graft-versus-host disease in adult patients, producing survival rates similar to those of transplantation of bone marrow from unrelated donors (Ref. 5).