Erratum - Analysis of extracellular superoxide dismutase and Akt in ascending aortic aneurysm with tricuspid or bicuspid aortic valve.

This correct the article published on European Journal of Histochemistry 2014;58:200-206 doi: 10.4081/ejh.2014.2383.

Allopurinol adherence among patients with gout: an Italian general practice database study.

AIMS: Allopurinol is used as long-term therapy to reduce the occurrence of gout flares. This study estimated the impact of patient adherence to allopurinol on hyperuricaemia (serum uric acid levels, sUA > 6 mg/dl) and the identification of non-adherence predictors. METHODS: The Italian Health Search-CSD Longitudinal Patient Database was accessed to identify outpatients aged ≥ 18 years with gout and prescribed with allopurinol during the years 2002-2011. Patients with a proportion of days covered ≥ 80% were considered adherent to allopurinol. Data on sUA levels over the first year of therapy were categorised in three time-windows (30-89; 90-149; 150-365 days). Logistic regressions were used to estimate the association between adherence and hyperuricaemia, as well as non-adherence predictors. RESULTS: A total of 3727 patients were included. In the interval 0-29 days, the proportion of patients adherent to allopurinol was 45.9%, while up to 89, 149 and 365 days the percentages were 16.7%, 10.0% and 3.2%, respectively. The proportions of hyperuricaemic patients for each time-window were 43.1%, 42.4%, 32.6% and 59.0%, 64.0%, 66.4% among adherent and non-adherent patients, respectively. In the multivariable analysis, adherence was associated with a significant lower risk of hyperuricaemia. The adjusted ORs were 0.49 (95% CI: 0.33-0.73), 0.40 (95% CI: 0.24-0.67) and 0.23 (95% CI: 0.15-0.34) for the first, second and third time-window, respectively. Patients with hypertension (adjusted OR = 0.64, 95% CI: 0.42-0.99) and history of gout flares (adjusted OR = 0.55, 95% CI: 0.32-0.95) were significantly adherent to allopurinol. CONCLUSIONS: Adherence monitoring in patients with gout is pivotal to ensure the effectiveness of therapy. To gain a better patient adherence, the communication between physicians and patients should be improved.

Analysis of extracellular superoxide dismutase and Akt in ascending aortic aneurysm with tricuspid or bicuspid aortic valve.

Ascending aortic aneurysm (AsAA) is a consequence of medial degeneration (MD), deriving from apoptotic loss of smooth muscle cells (SMC) and fragmentation of elastin and collagen fibers. Alterations of extracellular matrix structure and protein composition, typical of medial degeneration, can modulate intracellular pathways. In this study we examined the relevance of superoxide dismutase (SOD3) and Akt in AsAA pathogenesis, evaluating their tissue distribution and protein levels in ascending aortic tissues from controls (n=6), patients affected by AsAA associated to tricuspid aortic valve (TAV, n=9) or bicuspid aortic valve (BAV, n=9). The results showed a significant reduction of SOD3, phospho-Akt and Akt protein levels in AsAA tissues from patients with BAV, compared to controls, whereas the differences observed between controls and patients with TAV  were not significant. The decreased levels of SOD3 and Akt in BAV aortic tissues are associated with decreased Erk1/Erk2 phosphorylation and MMP-9 levels increase. The authors suggest a role of decreased SOD3 protein levels in the progression of AsAA with BAV and a link between ECM modifications of aortic media layer and impaired Erk1/Erk2 and Akt signaling in the late stages of the aortopathy associated with BAV.

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.

Analysis of extracellular superoxide dismutase in fibroblasts from patients with systemic sclerosis.

Systemic sclerosis (SSc) is a chronic disease of connective tissue characterized by vascular damage, autoantibody production and extensive fibrosis of skin, skeletal muscles, vessels and visceral organs. Fibrosis is a biological process involving inflammatory response and reactive oxygen species (ROS) accumulation leading to fibroblast activation. Extracellular superoxide dismutase (SOD3), a copper and zinc superoxide dismutase, which is expressed in selected tissues, is secreted into the extracellular space and catalyzes the dismutation of superoxide radical to hydrogen peroxide and molecular oxygen. Moreover, SOD3 is associated to inflammatory responses in some experimental models. In this paper we analysed, by RT-PCR and immunofluorescence, SOD3 expression and intracellular localization in dermal fibroblasts from both healthy donors and patients affected by diffuse form of SSc. Moreover, we determined SOD3 enzymatic activity in fibroblast culture medium with the xanthine/xanthine oxidase method. Increased expression of SOD3 mRNA was detected in systemic sclerosis fibroblasts (SScF), as compared to control healthy fibroblasts (HF), and SOD3 immunofluorescence staining displayed a characteristic pattern of secretory proteins in both HF and SScF. Superoxide dismutase assay demonstrated that SOD3 enzymatic activity in SScF culture medium is four times more than in HF culture medium. These data suggest that an alteration in SOD3 expression and activity could be associated to SSc fibrosis.

Neuropeptide expression in the airways of COPD patients and smokers with normal lung function.

Neurogenic mechanisms seem to play a role in the pathogenesis of chronic obstructive pulmonary disease (COPD), as suggested by a number of in vitro data. However, few studies have investigated the presence of neuropeptides in the airways of patients with COPD, and they have yielded conflicting results. The aim of this study is to compare the expression of the neuropeptide substance P (SP), vasoactive intestinal peptide (VIP), and neuropeptide Y (NPY) in the airways of smokers with and without COPD. Surgical lung samples were obtained from 15 smokers with COPD and 16 smokers with normal lung function, who underwent lobectomy for a solitary lung carcinoma. Airway expression and distribution of SP, VIP, and NPY were identified by immunohistochemistry and analyzed by a computerized image analysis system. Compared to smokers with normal lung function, COPD patients exhibited an increased immunoreactivity for SP and VIP, paralleled by a decreased NPY expression in the epithelium and glands, and a decreased expression of all these three neuropeptides in the smooth muscle layer. Therefore, in the present study we have documented a different expression and distribution of the neuropeptides SP, VIP, and NPY in the airways of smokers with and without COPD. These findings suggest a possible involvement of such neuropeptides in the pathogenesis of some changes occurring in COPD.

In vitro cultured progenitors and precursors of cardiac cell lineages from human normal and post-ischemic hearts.

The demonstration of the presence of dividing primitive cells in damaged hearts has sparked increased interest about myocardium regenerative processes. We examined the rate and the differentiation of in vitro cultured resident cardiac primitive cells obtained from pathological and normal human hearts in order to evaluate the activation of progenitors and precursors of cardiac cell lineages in post-ischemic human hearts. The precursors and progenitors of cardiomyocyte, smooth muscle and endothelial lineage were identified by immunocytochemistry and the expression of characteristic markers was studied by western blot and RT-PCR. The amount of proteins characteristic for cardiac cells (alpha-SA and MHC, VEGFR-2 and FVIII, SMA for the precursors of cardiomyocytes, endothelial and smooth muscle cells, respectively) inclines toward an increase in both alpha-SA and MHC. The increased levels of FVIII and VEGFR2 are statistically significant, suggesting an important re-activation of neoangiogenesis. At the same time, the augmented expression of mRNA for Nkx 2.5, the trascriptional factor for cardiomyocyte differentiation, confirms the persistence of differentiative processes in terminally injured hearts. Our study would appear to confirm the activation of human heart regeneration potential in pathological conditions and the ability of its primitive cells to maintain their proliferative capability in vitro. The cardiac cell isolation method we used could be useful in the future for studying modifications to the microenvironment that positively influence cardiac primitive cell differentiation or inhibit, or retard, the pathological remodeling and functional degradation of the heart.

HLA-DRB1*0402 haplotypes without DQB1*0302 in Venezuelan patients with pemphigus vulgaris.

The two basic forms of autoimmune intraepidermal blistering diseases, pemphigus vulgaris (PV) and pemphigus foliaceus (PF), affect different layers of the skin, have different symptoms and target different antigens. We have defined human leukocyte antigen (HLA)-DRB1-DQB1 alleles and haplotypes in a case-control study of 66 non-Jewish patients attending a public reference Hospital over the past 10 years. The control group consisted of 101 matched individuals tested also by medium to high-resolution polymerase chain reaction-sequence-specific oligonucleotide with primers and probes from the 12th and 13th International Histocompatibility Workshop. Patients and controls were descendants of three-generation individuals born in the country. Among the patients, 49 had PV, 50% showed predominantly mucosal involvement, 50% showed predominantly the cutaneous clinical phenotype and 17 had PF. Statistically significant HLA-DR frequency differences between patients with PV and controls were found only for DRB1*0402 and DRB1*1401 [odds ratio (OR) = 27.22, confidence interval (CI) 94.7-7.82, P= 1.1 x 10(-14) and OR = 46.56, CI 801.4-2.70 P= 7.5 x 10(-6), respectively]. Both alleles were also increased in the patients with PF compared with the controls (OR = 7.0, P= 0.038 and OR = 21.64, P= 0.009, respectively), but the significance of the difference did not resist Bonferroni correction. Haplotype analysis showed that DRB1*0402 was always present with DQB1*0302 and DRB1*1401 with DQB1*0503, but no independent effect of the DQB1*0302 in the former haplotype was evident. Our results support the hypothesis that the DRB1*0402 without DQB1*0302 is the most relevant HLA-DRB1 allele responsible for the pathogenesis of pemphigus in Venezuelan patients with PV and discard the DQB1*0302 influence observed in other populations.

Human leukocyte antigen class I and MICA haplotypes in a multicase family with Cladophialophora carrionii chromoblastomycosis.

Previous studies carried out in an endemic semiarid region northwest of Venezuela at Falcon State have shown a prevalence of 15.4/1000 of chromoblastomycosis following traumatisms with xenophile vegetation infected with Cladophialophora carrionii. We performed high-resolution DNA typing of human leukocyte antigen (HLA)-A, -B and -C and major histocompatibility complex class I chain related gene A (MICA) alleles and segregation analysis in 49 members of one extended family with 12 affected individuals, who have lived for approximately 70 years in this endemic zone. None of the alleles, haplotypes or genotypes is shared by all the patients. No deviation from the expected HLA haplotype distribution or association of chromoblastomycosis with HLA-A, -B and -C haplotypes was observed. Further, a haplotype-sharing transmission/disequilibria testing of 11 nuclear families did not give enough evidence to claim linkage (P = 0.398), suggesting that genes located in the short arm of chromosome 6 may not be relevant in the immune response toward infection with C. carrionii in this Venezuelan endemic zone. Deleted MICA alleles on HLA-B*4802 haplotypes were present among several members of the extended family, but only two of them were affected.

Basal lamina structural alterations in human asymmetric aneurismatic aorta.

Basal lamina (BL) is a crucial mechanical and functional component of blood vessels, constituting a sensor of extracellular microenvironment for endothelial cells and pericytes. Recently, an abnormality in the process of matrix microfibrillar component remodeling has been advocated as a mechanism involved in the development of aortic dilation. We focused our attention on BL composition and organization and studied some of the main components of the Extracellular Matrix such as Tenascin, Laminins, Fibronectin, type I, III and IV Collagens. We used surgical fragments from 27 patients, submitted to operation because of aortic root aneurysm and 5 normal aortic wall specimens from heart donors without any evidence for aneurysmal or atherosclerotic diseases of the aorta. Two samples of aortic wall were harvested from each patient, proximal to the sinotubular junction at the aortic convexity and concavity. Each specimen was processed both for immunohistochemical examination and molecular biology study. We compared the convexity of each aortic sample with the concavity of the same vessel, and both of them with the control samples. The synthesis of mRNA and the levels of each protein were assessed, respectively, by RT-PCR and Western Blot analysis. Immunohistochemistry elucidated the organization of BL, whose composition was revealed by molecular biology. All pathological samples showed a wall thinner than normal ones. Basal lamina of the aortic wall evidentiated important changes in the tridimensional arrangement of its major components which lost their regular arrangement in pathological specimens. Collagen I, Laminin alpha2 chain and Fibronectin amounts decreased in pathological samples, while type IV Collagen and Tenascin synthesis increased. Consistently with the common macroscopic observation that ascending aorta dilations tend to expand asymmetrically, with prevalent involvement of the vessel convexity and relative sparing of the concavity, Collagen type IV is more evident in the concavity and Tenascin in the convexity.

Differential expression and cytoplasm/membrane distribution of endoglin (CD105) in human tumour cell lines: Implications in the modulation of cell proliferation.

Endoglin (CD105, an accessory component of the TGF-beta receptor complex) expression and distribution on different human tumour cells and its role in cellular proliferation were evaluated. We examined: 1) sixteen human carcinoma cell lines, 2) eight human sarcoma cell lines, 3) five miscellaneous tumour cell lines. HECV (endothelial cells) were employed as a positive control for endoglin expression. Normal Human Dermal Fibroblasts (NHDF) and 293 cells (epithelial kidney cells) were used as normal controls for connective and epithelial tissues, respectively. The results showed that CD105 was poorly expressed in the majority of human carcinoma cells (10/16), whereas it was highly expressed in most human sarcoma cells (7/8), and differently expressed by miscellaneous tumour cell lines. These data reflect endoglin expression by the normal counterparts of tumour cell lines, i.e. NHDF and 293 cells. However, CD105 levels in sarcoma cell lines, even though consistently lower than in NHDF, were significantly higher than those observed in carcinoma cells. Interestingly, CD105 presented a strong expression in the cytoplasm of MDA-MB-453 (breast carcinoma), NPA (papillary thyroid carcinoma), COLO-853 (melanoma) and SaOS-2 (osteosarcoma), but was weakly expressed on their cell membrane. This differential expression in the cytoplasm and on the membrane of some tumour cells, suggests a complex mechanism of translocation for this protein. The analysis of clonal growth in soft agar of some cell lines, characterized by high CD105 expression, showed an increased colony formation potential that was antagonized by the addition of anti-CD105 blocking mAb. The results indicated that endoglin is differentially expressed in human carcinoma and sarcoma cells and its overexpression modulates the proliferative rate of human solid tumour cells. Moreover, these data suggest that CD105 is involved in the regulation of TGF-beta effects in human solid malignancies, and therefore it could play an important role in tumour diagnosis and treatment.

Genetic variability of Apolipoprotein E in different populations from Venezuela.

The genetic variation at the Apolipoprotein E locus (APOE) is an important determinant of plasma lipids and has been implicated in various human pathological conditions. The objective of the present study was to estimate the distribution of APOE alleles in five Venezuelan communities: two Amerindian tribes (Bari and Yucpa), one Negroid population from Curiepe, one Caucasoid population from Colonia Tovar and the Mestizo urban population living in Caracas. The APOE*3 allele was the most common allele in all populations studied. However, a significant increase in the APOE*2 allele frequency in the Mestizo (18.96%) and Negroid (16.25%) populations was found. Similar to results reported in other Native American populations we have found that the APOE*2 allele is completely absent in the Bari and Yucpa Amerindians. Frequencies found in the Colonia Tovar population are in agreement with those reported in the population of Germany, indicating a high degree of relatedness. The results support the notion that the distribution of the APOE alleles shows ethnic variability.

Different titanium surfaces modulate the bone phenotype of SaOS-2 osteoblast-like cells.

Commercially pure titanium implants presenting a relatively smooth, machined surface or a roughened endosseous surface show a large percentage of clinical success. Surface properties of dental implants seem to affect bone cells response. Implant topography appears to modulate cell growth and differentiation of osteoblasts affecting the bone healing around the titanium implant. The aim of the present study was to examine the effects of 1cm diameter and 1mm thick titanium disks on cellular morphology, adhesion and bone phenotypic expression of human osteoblast-like cells, SaOS-2. SaOS-2 cells were cultured on commercially 1 cm pure titanium disks with three different surface roughness: smooth (S), sandblasted (SB) and titanium plasma sprayed (TPS). Differences in the cellular morphology were found when they were grown on the three different surfaces. An uniform monolayer of cells recovered the S surface, while clusters of multilayered irregularly shaped cells were distributed on the rough SB and TPS surfaces. The adhesion of SaOS-2 cells, as measured after 3h of culture, was not affected by surface roughness. ECM components such as Collagen I (CoI), Fibronectin (FN), Vitronectin (VN) and Tenascin (TN) were secreted and organized only on the SB and TPS surfaces while they remained into the cytoplasm on the S surfaces. Osteopontin and BSP-II were largely detected on the SB and TPS surfaces, while only minimal production was observed on the S ones. These data show that titanium surface roughness affects bone differentiation of osteoblast like-cells, SaOS-2, indicating that surface properties may be able to modulate the osteoblast phenotype. These observations also suggest that the bone healing response around dental implants can be affected by surface topography.

Is the CCR5-59029-G/G genotype a protective factor for cardiomyopathy in Chagas disease?

Investigated were two CCR5 gene polymorphisms, the CCR5 Delta 32 deletion and the pCCR5 59029 A-->G promoter point mutation, in 107 ethnically mixed Venezuelan patients serologically positive for Trypanosoma cruzi (34 asymptomatic, 38 arrhythmic, 35 cardiomyopathic). No difference in the distribution of CCR5 Delta 32 among asymptomatic and symptomatic patients was found. We have observed an increase of the 59029-G phenotype among asymptomatic compared with symptomatic chagasic patients (68% vs. 58%), in agreement with previously reported data (57% vs. 31%). This frequency difference, although not statistically significant, is more marked when the 59029-G allele is present in homozygous form. However, a similar distribution of the G/G genotype is present among asymptomatic patients and patients with heart failure. Because it has been reported that the 59029G/G genotype associates with lower CCR5 expression, 37% of our T. cruzi-infected patients with heart failure are genetically predisposed to express low levels of CCR5 on the surface of CD8(+) T cells, contrary to what would be expected if an inflammatory response is required for severe cardiac damage. If confirmed, the possible protection that might be conferred by the G/G genotype may be due to the existence of other genes in linkage disequilibria.

Colonia Tovar: the history of a semi-isolated Venezuelan population of German ancestry described by HLA class I genes.

The history of Colonia Tovar is very complex, being the home of descendants of only a small fraction of immigrants arriving to the South American continent from a specific region of Germany, with a restricted number of founders, small population size and consanguineous mating, experiencing isolation for 100 years, with later migrations, a low rate of population growth and a high mean number of children per couple. How complex is its genetic structure? Do the highly polymorphic HLA genes reflect its history and confirm the story of this population described by other genes? Several studies have been made in this population, but we describe for the first time the HLA Class I variability in the population of Colonia Tovar using PCR-SSOP. Random genetic drift, founder effect and gene flow could explain the HLA allele and haplotype frequencies observed in this population but alleles at the class I loci were insufficient to identify the German origin of the community established through history. This agrees with findings obtained testing other genetic systems (ACP, AK, ESD, G6PD, GLO, PGM, PGD, ALB, CP, HP, TF), but the HLA-typing results indicate that the original gene pool has been diluted due to gene flow from the surrounding Mestizo population.

Behavior of SaOS-2 cells cultured on different titanium surfaces.

Surface properties may affect the clinical outcome of titanium dental implants. The aim of the present study was to investigate the effects of 3 different titanium surfaces-smooth (S), sandblasted (SB), and titanium plasma-sprayed (TPS)-on proliferation, differentiation, and apoptosis of human osteoblast-like cells, SaOS-2. Cell proliferation was significantly (p < 0.05) higher on the S surface, and synthesis of extracellular matrix proteins was more abundant on TPS and SB than on S surfaces. Analysis of integrin receptors showed a higher expression of alpha2, alpha5, alphaVbeta3, and ss1 on TPS as compared with SB and S surfaces. An increase in alkaline phosphatase activity was detected only on SB and TPS surfaces. Analysis of cell apoptosis did not demonstrate any significant difference among the 3 different surfaces. The results indicate that titanium surface topography affects proliferation and differentiation of osteoblast-like SaOS-2 cells, suggesting that surface properties might be important for bone response around dental implants in vivo.

Altered expression of integrins in RSV-transformed chick epiphyseal chondrocytes.

Chondrocytes have been shown to express both in vivo and in vitro a number of integrins of the beta1-, beta3- and beta5-subfamilies (Biorheology 37 (2000) 109). Normal and v-Src-transformed chick epiphyseal chondrocytes (CEC) display different adhesion properties. While normal CEC with time in culture tends to increase their adhesion to the substrate by organizing focal adhesions and actin stress fibers, v-Src-transformed chondrocytes display a refractile morphology and disorganization of actin cytoskeleton. We wondered whether the reduced adhesion and spreading of v-Src-transformed chondrocytes could be ascribed to changes in integrin expression and/or function. Integrin expression by normal CEC is studied and compared to v-Src-transformed chick chondrocytes, using monoclonal and polyclonal antibodies to integrins alpha- and beta-chains. We show the presence of alpha1-, alpha3-, alphav-, alpha6-, beta1- and beta3-chains on CEC, with very low levels of alpha2- and alpha5-chains. Alphav chain associates with multiple beta subunits in normal and transformed chondrocytes. With the exception of alpha1- and alpha2-chains, the levels of the integrin chains analyzed are higher in transformed chondrocytes as compared with normal chondrocytes. In spite of the increased levels of integrin expression, transformed chondrocytes exhibit loss of focal adhesion and actin stress fibers and low adhesion activity on several extracellular matrix constituents. These observations raise the possibility that, in addition to its effects on global pattern of integrin expression, v-Src can influence integrin function in chondrocytes.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces the osteoblastic differentiation of the human osteosarcoma cell line SaOS-2.

The Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) is a hematopoietic growth factor that regulates the in vitro and in vivo proliferation and differentiation of hematopoietic cells through the interaction with a specific heterodimeric receptor complex (GM-CSFR), consisting of an alpha and a beta chain with molecular weights of 80 and 120 KDa, respectively. We have studied the expression of the GM-CSFR (alpha chain) on the surface of the human osteosarcoma cell line SaOS-2 and the in vitro effects of different concentrations (10, 100, and 200 ng/ml) of GM-CSF on GM-CSFR expression and the biological activity of SaOS-2 cells. Our data show that SaOS-2 cells express GM-CSFR and that GM-CSF can down-regulate the expression of its own receptor on these cells. Furthermore, to evaluate the biological effects of GM-CSF on SaOS-2 cells, we have investigated cell proliferation and differentiation of these cells treated with different doses of the growth factor through: (1) a morphological analysis of typical osteoblast differentiation markers such as osteopontin and BSP-II; (2) measurement of alkaline phosphatase (ALP) activity; (3) production of bone ECM components (collagen I, fibronectin, tenascin, and laminin); (4) production of interleukin-6 (IL-6) and osteocalcin in the culture medium. The results show that the in vitro treatment of SaOS-2 cells with recombinant human GM-CSF causes a decreased cell proliferation and an increased production of osteopontin, BSP-II, ALP, IL-6, and most but not all ECM components. These findings suggest that GM-CSF can regulate proliferation and differentiation of osteoblast-like SaOS-2 cells and could also play an unexpected role in the maturation of bone tissue.

Effect of human granulocyte macrophage-colony stimulating factor on differentiation and apoptosis of the human osteosarcoma cell line SaOS-2.

We investigated the effects of human granulocyte macrophage-colony stimulating factor (GM-CSF) on the relation between differentiation and apoptosis in SaOS-2 cells, an osteoblast-like cell line. To determine the relationship between these cellular processes, SaOS-2 cells were treated in vitro for 1, 7 and 14 days with 200 ng/mL GM-CSF and compared with untreated cells. Five nM insulin-like growth factor (IGF-I) and 30 nM okadaic acid were used as negative and positive controls of apoptosis, respectively. Effects on cell differentiation were determined by ECM (extracellular matrix) mineralization, morphology of some typical mature osteoblast differentiation markers, such as osteopontin and sialoprotein II (BSP-II), and production of bone ECM components such as collagen I. The results showed that treatment with GM-CSF caused cell differentiation accompanied by increased production of osteopontin and BSP-II, together with increased ECM deposition and mineralization. Flow cytometric analysis of annexin V and propidium iodide incorporation showed that GM-CSF up-regulated apoptotic cell death of SaOS-2 cells after 14 days of culture in contrast to okadaic acid, which stimulated SaOS-2 apoptosis only during the early period of culture. Endonucleolytic cleavage of genomic DNA, detected by "Aúladdering analysis"Aù, confirmed these data. The results suggest that GM-CSF induces osteoblastic differentiation and long-term apoptotic cell death of the SaOS-2 human osteosarcoma cell line, which in turn suggests a possible in vivo physiological role for GM-CSF on human osteoblast cells.

Preeclampsia: a multifactorial disease resulting from the interaction of the feto-maternal HLA genotype and HCMV infection.

PROBLEM: To clarify the possible influence of human leukocyte antigen (HLA) mother-child genotypes and human cytomegalo virus (HCMV) presence on the development of preeclampsia. METHODS OF STUDY: One hundred and four DNA samples from mothers with preeclampsia, mothers with a normal history of pregnancies and their neonates were tested by polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSOP) for HLA-A, -G, -DRB1, -DQA1, -DQB1 alleles. The HCMV sequences were analyzed using a PCR-SSOP method and the four primers described by Chou (Chou S: J Clin Microb 1992; 30:2307-2310). RESULTS: Compared with their respective controls, a significant increase of DRB1*07 among neonates (P(c) = 0.05) and of DRB1*07 and/or DRB1*06 among pre-eclamptic mothers (P(c) = 0.003, RR = 8,5) was found. When HCMV sequences were detected in pre-eclamptic mothers carrying those phenotypes the RR increased up to 40. Furthermore, the fetal inheritance of a maternal HLA-G*0104 increased the risk for the appearance of the disease (RR = 30; P = 0.025). CONCLUSION: The results suggest that the presence of alleles HLA-G*0104, DRB1*07/06, HCMV sequences and the fetal inheritance of maternal G*0104, should be considered as conditioning factors for the development of preeclampsia.