A cutoff thyroglobulin value suggestive of distant metastases in differentiated thyroid cancer patients.

Serum thyroglobulin is used as part of the early postoperative assessment of differentiated thyroid cancer (DTC) since there is a clear relationship between an increased risk of recurrence and persistent disease after initial treatment and high postoperative stimulated thyroglobulin (ps-Tg) values. Thus, although ps-Tg above 10-30 ng/mL is considered an independent predictor of worse prognosis, the value that is associated with distant metastases is not defined. Thus, this was our objective. We selected 655 DTC patients from a nuclear medicine department database (Irmandade Santa Casa de Misericórdia de São Paulo, Brazil). All patients had received total thyroidectomy and radioactive iodine (RAI) therapy and had ps-Tg values higher than 10 ng/mL with negative anti-thyroglobulin antibodies. Then, we selected patients who presented post-therapy whole-body scan with pulmonary and/or bone uptake but with no mediastinum or cervical uptake. Patients with negative findings on functional imaging or any doubt on lung/bone uptake were submitted to additional exams to exclude another non-thyroid tumor. Of the 655 patients, 14.3% had pulmonary and 4.4% bone metastases. There was a significant difference in ps-Tg levels between patients with and without metastases (P<0.001). The cutoff value of ps-Tg was 117.5 ng/mL (sensitivity: 70.2%; specificity: 71.7%) for those with lung metastasis, and 150.5 ng/mL (sensitivity: 79.3%; specificity: 85%) for those with bone metastasis. The cutoff value for patients with eitherpulmonary or bone metastasis was 117.5 ng/mL (sensitivity: 70.2%; specificity: 83.7%). Our findings demonstrated that ps-Tg could predict distant metastasis in DTC patients. We identified a cutoff of 117.5 ng/mL with a high negative predictive value of 93.7%.

A cutoff thyroglobulin value suggestive of distant metastases in differentiated thyroid cancer patients

Serum thyroglobulin is used as part of the early postoperative assessment of differentiated thyroid cancer (DTC) since there is a clear relationship between an increased risk of recurrence and persistent disease after initial treatment and high postoperative stimulated thyroglobulin (ps-Tg) values. Thus, although ps-Tg above 10-30 ng/mL is considered an independent predictor of worse prognosis, the value that is associated with distant metastases is not defined. Thus, this was our objective. We selected 655 DTC patients from a nuclear medicine department database (Irmandade Santa Casa de Misericórdia de São Paulo, Brazil). All patients had received total thyroidectomy and radioactive iodine (RAI) therapy and had ps-Tg values higher than 10 ng/mL with negative anti-thyroglobulin antibodies. Then, we selected patients who presented post-therapy whole-body scan with pulmonary and/or bone uptake but with no mediastinum or cervical uptake. Patients with negative findings on functional imaging or any doubt on lung/bone uptake were submitted to additional exams to exclude another non-thyroid tumor. Of the 655 patients, 14.3% had pulmonary and 4.4% bone metastases. There was a significant difference in ps-Tg levels between patients with and without metastases (P<0.001). The cutoff value of ps-Tg was 117.5 ng/mL (sensitivity: 70.2%; specificity: 71.7%) for those with lung metastasis, and 150.5 ng/mL (sensitivity: 79.3%; specificity: 85%) for those with bone metastasis. The cutoff value for patients with eitherpulmonary or bone metastasis was 117.5 ng/mL (sensitivity: 70.2%; specificity: 83.7%). Our findings demonstrated that ps-Tg could predict distant metastasis in DTC patients. We identified a cutoff of 117.5 ng/mL with a high negative predictive value of 93.7%.

Changes in the aromatic profile of espresso coffee as a function of the grinding grade and extraction time: a study by the electronic nose system.

The changes in chemical attributes and aromatic profile of espresso coffee (EC) were studied taking into account the extraction time and grinding level as independent variables. Particularly, using an electronic nose system, the changes of the global aromatic profile of EC were highlighted. The results shown as the major amounts of organic acids, solids, and caffeine were extracted in the first 8 s of percolation. The grinding grade significantly affected the quality of EC probably as an effect of the particle size distribution and the percolation pathways of water through the coffee cake. The use of an electronic nose system allowed us to discriminate the fractions of the brew as a function of the percolation time and also the regular coffee obtained from different grinding grades. Particularly, the aromatic profile of a regular coffee (25 mL) was significantly affected by the grinding level of the coffee grounds and percolation time, which are two variables under the control of the bar operator.

[Distortion product otoacoustic emissions in newborns treated by ototoxic drugs]. / Etude des produits de distorsions acoustiques chez les nouveau-nés traités par ototoxiques.

OBJECTIVE: The aim of this prospective longitudinal study is to research the amplitude of distortion product otoacoustic emissions caused by the ototoxic drugs used, between the end of the administration and from 15 to 40 days after its use. METHODS: It was a prospective longitudinal study composed by term and preterm newborns from the Santo André city hospital, in the period from July 2003 to September 2004. The first evaluation occurred on the hospital discharge day. Three groups were evaluated: control group with 33 term and healthy newborns; term study group with 19 term newborns with more than 37 weeks exposed to amikacin and/or vancomycin; and preterm study group with 15 preterm newborns from 32 to 37 weeks exposed to the same ototoxic. The newborns did not present risk factors for hearing loss according to the JCIH, 2000 concomitant to the neonatal infection. All newborns were evaluated at a corrected gestational age greater than 37 weeks. The otoacoustic emissions amplitudes obtained at the hospital discharge were compared to the ones obtained from 15 to 40 days after the discharge. RESULTS: The otoacoustic emissions amplitudes of the preterm study group were smaller than the amplitudes of the control group and the term study group in both moments of the test. The amplitude of the newborns' otoacoustic emissions increased in the second moment of the test. The otoacoustic emissions amplitudes of the control group in the second moment of the test were similar to the term study group in the first moment of the research. CONCLUSION: There are the increase of the distortion product otoacoustic emissions amplitude from the discharge moment until 15 to 40 days after in the post-natal period. The exposure to amikacin and vancomycin on the recommended dose by Neofax, 2003/2004 did not alter the amplitude of the emissions in the newborns without risk indicators concomitant with neonatal infection.

Transforming growth factor-beta1 transcriptionally activates CD34 and prevents induced differentiation of TF-1 cells in the absence of any cell-cycle effects.

A number of cytokines modulate self-renewal and differentiation of hematopoietic elements. Among these is transforming growth factor beta1 (TGF-beta1), which regulates cell cycle and differentiation of hematopoietic cells, but has pleiotropic activities depending on the state of responsiveness of the target cells. It has been previously shown by us and other authors that TGF-beta1 maintains human CD34(+) hematopoietic progenitors in an undifferentiated state, independently of any cell cycle effects, and that depletion of TGF-beta1 triggers differentiation accompanied by a decrease in CD34 antigen expression. In the present work, we show that exogenous TGF-beta1 upregulates the human CD34 antigen in the CD34(+) cell lines TF-1 and KG-1a, but not in the more differentiated CD34(-) cell lines HL-60 and K-562. We further studied this effect in the pluripotent erythroleukemia cell line TF-1. Here, TGF-beta1 did not effect cell growth, but induced transcriptional activation of full-length CD34 and prevented differentiation induced by differentiating agents. This effect was associated with nuclear translocation of Smad-2, activation of TAK-1, and with a dramatic decrease in p38 phosphorylation. In other systems TGF-beta1 has been shown to activate a TGF-beta-activated kinase 1 (TAK1), which in turn, activates p38. The specific inhibitor of p38 phosphorylation, SB202190, also increased CD34 RNA expression, indicating the existence of a link between p-38 inhibition by TGF-beta1 and CD34 overexpression. Our data demonstrate that TGF-beta1 transcriptionally activates CD34 and prevents differentiation of TF-1 cells by acting independently through the Smad, TAK1 and p38 pathways, and thus provide important clues for the understanding of hematopoietic development and a potential tool to modify response of hematopoietic cells to mitogens or differentiating agents.

The use of 99mTc-phytate for sentinel node mapping in melanoma, breast cancer and vulvar cancer: a study of 100 cases.

Sentinel node mapping reduces surgical morbidity and allows the use of more accurate tumour staging techniques. Radionuclide studies are preferentially performed using small colloids, which have limited availability in our country. The possibility of using phytate for sentinel node mapping was raised because of the similarity between its biodistribution and that of nanocolloids in the reticulo-endothelial system. In this paper we evaluated the use of 99mTc-phytate for sentinel node mapping, correlating the histopathological results with the status of the rest of the lymph node chain in different malignant tumours. A total of 100 patients were studied. group 1 consisted of 62 patients with breast cancer, group 2 of 20 patients with melanoma and group 3 of 18 patients with vulvar carcinoma. Lymph node scintigraphy was carried out after injecting 99mTc-phytate subdermally, and the sentinel node projection was marked on the skin. After 18-24 h, intraoperative sentinel node localisation was performed using a gamma probe (combined with visual localisation using patent blue dye) in 75 patients, and lymph node dissection was then carried out. Radionuclide scintigraphy identified the sentinel node in 98% of all studies. Intraoperative detection using the gamma probe was equally efficient: group 1=93% (38/41), group 2=95% (18/19) and group 3=100% (15/15). The sentinel node was involved in 41%, 31% and 20% of cases in groups 1, 2 and 3, respectively. Among the patients with positive nodes, the sentinel node was the only one affected in 53% of group 1, 50% of group 2 and 67% of group 3 cases. The method's negative predictive value was 91% in group 1 and 100% in the other groups. One false-negative study occurred in a patient who had a multifocal tumour and an intraparenchymatous lymph node; another occurred in a patient with a macroscopically affected node found during surgery. There were no side-effects related to the 99mTc-phytate. It is concluded that scintigraphic and intraoperative sentinel node identification was satisfactorily performed using 99mTc-phytate. The results were comparable to those previously described in the literature using other radiopharmaceuticals. Easy availability and low cost justify the use of phytate in our practice.

Quercetin abrogates taxol-mediated signaling by inhibiting multiple kinases.

Cell cycle block in G(2)/M initiates apoptosis, but the mechanism of this signaling cascade are largely unknown. The microtubule-perturbing agent Taxol has multiple effects on this signaling pathway and is a potent inducer of apoptosis. The specific pathways activated by low, clinically relevant concentrations of the drug are still largely unknown and are dependent on cell type and drug concentration. In this work, we have investigated why HeLa cells respond to Taxol by undergoing complete apoptosis, whereas MCF-7 cells remain in an intermediate phase with reduced death. Three phases were distinguished in these apoptotic pathways. The initial phase characterized by cellular detachment is followed by a second phase which includes the onset of apoptotic morphology, and p38 and Bcl-2 phosphorylation. These two phases are common to both cell lines. HeLa cells then proceed to the third and final execution phase, which culminates in death, whereas MCF-7 cells do not progress. Interestingly, the isoflavonoid Quercetin, a known general kinase inhibitor and an antioxidant, was able to prevent the onset of Taxol-induced cellular detachment and to protect from cell death. Moreover, it blocked Taxol-induced phosphorylation of p38 and Bcl-2, and prevented a Taxol-induced change in relative mobility of the apoptosis signal-regulating kinase 1 (Ask1). Our data elucidate the signaling pathways activated by Taxol at low clinically relevant concentrations.

T-cell apoptosis induced by granulocyte colony-stimulating factor is associated with retinoblastoma protein phosphorylation and reduced expression of cyclin-dependent kinase inhibitors.

Peripheral blood progenitor cells (PBPC) mobilized by granulocyte colony-stimulating factor (G-CSF) promptly engraft allogeneic recipients after myeloablative chemotherapy for hematologic malignancies. Surprisingly, no exacerbation of acute graft-vs-host disease has been observed despite a 10-fold higher T-cell content in PBPC compared with bone marrow allografts. Because G-CSF can suppress T-cell proliferation in response to mitogens and enhance their activation-induced apoptosis, we examined the molecular mechanisms underlying G-CSF-induced immune dysfunction. Normal allogeneic lymphocytes were challenged with phytohemagglutinin in the presence of serum collected after G-CSF administration (postG) to healthy PBPC donors, and the expression of key components of the cell cycle and apoptotic machineries was investigated by flow cytometry and Western blotting. Lymphocyte stimulation was associated with collapse of mitochondrial transmembrane potential, hypergeneration of reactive oxygen intermediates, and activation of caspase-3 and DNA fragmentation. Lymphocytes were arrested in a G(1)-like phase of the cell cycle, as measured by G(1)-phase cyclin expression and bromodeoxyuridine (BrdUrd) incorporation. Cell tracking experiments confirmed the occurrence of a lower number of population doublings in postG compared with preG cultures. Unexpectedly, the phosphorylation state of the protein encoded by the retinoblastoma susceptibility gene (pRB) was unaltered in postG cultures, and the inhibition of cell cycle progression occurred without the recruitment of the cyclin-dependent kinase inhibitors p15(INK4B), p16(INK4A), and p27(Kip1). We eventually evaluated the ability of antioxidant/cytoprotectant agents to prevent the G-CSF-induced mitochondrial dysfunction and inhibition of cell cycle progression. Of interest, both N-acetylcysteine and amifostine reduced apoptotic cell death by 45% on average, inhibited the activation/processing of caspase-3, and increased BrdUrd incorporation in postG cultures. Based on these experimental findings, a model is proposed in which T-cell activation in the presence of serum immunoregulatory factor(s) induced by G-CSF is associated with a molecular phenotype mimicking the G(1)-S transition and consisting of pRB phosphorylation, lack of CDKI recruitment, and reduced cyclin-E expression. The putative relationship between lymphocyte mitogenic unresponsiveness and apoptosis induction would occur at the level of key molecules shared by the cell cycle and apoptotic machineries. Whether the G-CSF-mediated modulation of lymphocyte functions in vitro is beneficial in transplantation medicine remains to be determined.

CD105 (endoglin) expression on hematopoietic stem/progenitor cells.

Endoglin (CD105) is a component of the transforming growth factor-beta (TGF-beta) receptor (TGF-betaR) complex. Together with betaglycan, CD105 is considered as a TGF-betaR accessory molecule (also called TGF-betaRIII), but its functions in the receptor-ligand interactions are still poorly understood. A small subset of human CD34+ hematopoietic stem/progenitor cells that has phenotypic and functional features suggestive of very primitive hematopoietic cells expresses the CD105 antigen. CD34+/CD105+ cells recirculate in the peripheral blood of mobilized subjects and can be purified by immunomagnetic isolation strategies. The hematopoietic potential of these CD34+/CD105+ cells appears to be sustained by a combination of hematopoietic and non-hematopoietic cytokines, which comprises Flt3 ligand, erythropoietin, interleukin-15 and vascular endothelial growth factor. Endogenous TGF-beta1 is a crucial factor for the maintenance of CD34+/CD105+ immaturity acting through positive modulation of both CD105 and CD34 molecules in the absence of relevant effects on the cell cycle profile. CD105 is absent on very primitive CD34-/lineage-/CD45+ (CD34-Lin-) human hematopoietic cells isolated from cord blood. However, in vitro exposure of CD34-Lin- cells to exogenous TGF-beta1 causes the appearance of a discrete population of CD34+/CD105+ cells. Collectively, available data on CD105 expression and function in primitive hematopoiesis indicate that this molecule could cooperate with the dissociation of TGF-beta1 cell cycle effects from its other effects on cell survival and differentiation.

nm23 Protein Expression by Western Blotting in Patients with Epithelial Ovarian Carcinoma.

Western blotting is utilized to detect and quantify the amount of a specific protein in a complex mixture of proteins and at the same time to determine its molecular weight. In particular, by Western blotting it is also possible to detect and discriminate the two isoforms of nm23, H1, and H2 (1, 2). nm23-H1 is about 18.5 kD (3) and nm23-H2 is 17 kD (4), hence, they migrate as two distinct bands on a standard SDS polyacrylamide gel. Because the two isoforms are very highly conserved (4), they retain most of the same epitopes; thus any polyclonal antibody raised against one of the two isoforms or a monoclonal antibody against a conserved epitope will recognize both nm23-H1 and -H2 at the same time. Analysis by immunohistochemistry, on the other hand would require two distinct reactions with two specific antibodies for H1 and H2 raised against the least conserved epitopes to avoid crossreaction.

The use of (99m)Tc-phytate for sentinel node mapping in melanoma, breast cancer and vulvar cancer: a study of 100 cases.

Sentinel node mapping reduces surgical morbidity and allows the use of more accurate tumour staging techniques. Radionuclide studies are preferentially performed using small colloids, which have limited availability in our country. The possibility of using phytate for sentinel node mapping was raised because of the similarity between its biodistribution and that of nanocolloids in the reticulo-endothelial system. In this paper we evaluated the use of (99m)Tc-phytate for sentinel node mapping, correlating the histopathological results with the status of the rest of the lymph node chain in different malignant tumours. A total of 100 patients were studied. group 1 consisted of 62 patients with breast cancer, group 2 of 20 patients with melanoma and group 3 of 18 patients with vulvar carcinoma. Lymph node scintigraphy was carried out after injecting (99m)Tc-phytate subdermally, and the sentinel node projection was marked on the skin. After 18-24 h, intraoperative sentinel node localisation was performed using a gamma probe (combined with visual localisation using patent blue dye) in 75 patients, and lymph node dissection was then carried out. Radionuclide scintigraphy identified the sentinel node in 98% of all studies. Intraoperative detection using the gamma probe was equally efficient: group 1=93% (38/41), group 2=95% (18/19) and group 3=100% (15/15). The sentinel node was involved in 41%, 31% and 20% of cases in groups 1, 2 and 3, respectively. Among the patients with positive nodes, the sentinel node was the only one affected in 53% of group 1, 50% of group 2 and 67% of group 3 cases. The method's negative predictive value was 91% in group 1 and 100% in the other groups. One false-negative study occurred in a patient who had a multifocal tumour and an intraparenchymatous lymph node; another occurred in a patient with a macroscopically affected node found during surgery. There were no side-effects related to the (99m)Tc-phytate. It is concluded that scintigraphic and intraoperative sentinel node identification was satisfactorily performed using (99m)Tc-phytate. The results were comparable to those previously described in the literature using other radiopharmaceuticals. Easy availability and low cost justify the use of phytate in our practice.

High cyclin-dependent kinase inhibitors in Bcl-2 and Bcl-xL-expressing CD34+-proliferating haematopoietic progenitors.

We have previously described the isolation of primitive, slow-proliferating progenitors from normal, circulating CD34+ cells by using the fluorescent dye 5-6-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE). CFDA-SE(bright) (primitive) and CFDA-SE(dim) (differentiating) cells were isolated following cytokine stimulation on the basis of their different proliferation rates. In the present work we analysed the expression levels of a number of proteins involved with differentiation, proliferation and survival/apoptosis in CFDA-SE(bright)/CD34+/slow-proliferating cells that were previously defined as progenitors capable of differentiating into different lineages. The aim of this work was to gain a better understanding of our model system in order to define some of the important parameters that regulate differentiation in haematopoietic progenitors. GATA-1 and PU.1 RNA levels were similar in freshly isolated (d 0) CD34+ and in CFDA-SE(bright) (bright) cells, whereas they increased in CFDA-SE(dim) (dim) cells. Accordingly, Nm23 was expressed at higher levels in bright cells. Moreover, bright cells had higher p21WAF1/CIP1, p27KIP1 and p16Ink4 protein levels than dim cells. Consistently, Cdc2 and Cdk2 kinase activity was much higher in the dim than in the slower proliferating bright cells. C-myc and p53 levels were higher in bright cells than in d 0 CD34+ and dim cells, and so was Bcl-xL, which followed the trend we have previously described for Bcl-2. Thus, bright cells, despite having a higher proliferation rate than the starting d 0 CD34+ population, have strikingly elevated levels of cyclin-dependent kinase inhibitors, which are likely to also act as inhibitors of differentiation.

Modulation of bcl-2 and p27 in human primitive proliferating hematopoietic progenitors by autocrine TGF-beta1 is a cell cycle-independent effect and influences their hematopoietic potential.

Primitive, proliferating hematopoietic progenitors (defined as cytokine low-responding primitive progenitors; CLRPP), isolated from human CD34+ cells, expressed endoglin (CD105) and produced transforming growth factor-beta1 (TGF-beta1). Culture of CLRPP in serum-free conditions with anti-TGF-beta1 monoclonal antibody produced a substantial decrease in bcl-2 protein/RNA levels and a significant reduction of cloning and long-term culture-initiating cell (LTC-IC) activities. GATA-1 and PU.1 RNA levels were significantly up-regulated in anti-TGF-beta1-treated CLRPP, which generated an increased number of cells expressing CD15/CD11b/glycophorin-A. The described effects of TGF-beta1 neutralization were observed in the absence of any relevant effect on cell cycle; number of cell divisions; p53, c-myc, and p21 RNA levels; bcl-xL and bax protein levels; and c-myc/p16/p21/p107/Rb cell cycle-related protein levels. A relevant increase in p27 protein levels was observed in anti-TGF-beta1-treated CLRPP, suggesting a role for p27 in the regulation of the hematopoietic potential. The present study on human progenitors and previously reported data on TGF-beta1 knockout mice suggest that, at the autocrine level, the cell cycle inhibitor TGF-beta1 plays an important role in regulating the survival and differentiation of primitive proliferating hematopoietic progenitors by cell cycle-independent mechanisms.

The fusion protein MEN 11303 (granulocyte-macrophage colony stimulating factor/erythropoietin) acts as a potent inducer of erythropoiesis.

OBJECTIVE: A fusion protein made of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO), referred to as MEN 11303, has been tested for biologic activity using mobilized CD34(+) cells. METHODS AND RESULTS: MEN 11303 and a combination of GM-CSF/EPO produced the same amount of colony-forming unit granulocyte-macrophage (CFU-GM), of burst-forming unit erythroid (BFU-E), and of multipotent CFU-mixed. After 15 days, liquid cultures of CD34(+) cells exposed to MEN 11303 yielded a total cell number larger than that obtained with an equimolar mixture of GM-CSF and EPO, with a clear prevalence of cells exhibiting an erythroid phenotype. A colony-forming cell assay established from CD34(+) cells precultured with MEN 11303 for 7 days yielded a greater amount of BFU-E than GM-CSF/EPO combination. Exposing CD34(+) cells to MEN 11303 for 7 days in liquid culture resulted in higher recoveries of cells expressing a comparatively less differentiated hematopoietic phenotype and of long-term culture initiating cells. A cell-based binding-competition assay using the human EPO-receptor (EPO-R) transfected murine Ba/F3EPOR cell line showed that MEN 11303 bound to EPO-R with a sixfold lower affinity but induced a more sustained receptor phosphorylation. MEN 11303 supported the growth of Ba/F3EPOR cells more efficiently than EPO and remained detectable in the spent culture medium for a longer time. CONCLUSIONS: MEN 11303 and the combination of GM-CSF/EPO are equally potent in recruiting hematopoietic progenitors into cycle, but the fusion protein is superior in promoting the expansion of committed erythroid percursors. Primitive hematopoiesis is less affected by MEN110303 than GM-CSF/EPO combination. Part of these effects may reflect the peculiar interaction of the EPO moiety of MEN 11303 with the EPO-R.

Bcl-2, bax, bcl-x(L) and bcl-x(S) expression in neoplastic and normal cervical tissue.

We simultaneously assessed bcl-2, bax, bcl-x(L) and bcl-x(S) expression levels by Western blotting on 53 primary untreated cervical cancers and 15 normal samples. Bcl-2 showed a trend to be lower in neoplastic than in normal samples (P<0.01), while no significant difference was observed for bax and bcl-x(L). Bcl-x(S) was barely detectable in only a few samples. Interestingly, in cervical cancer, bcl-2 and bcl-x(L) were directly correlated (P<0. 01). A significant association of bcl-2 levels with age (P<0.021) and menopausal status (P<0.041) in cervical cancer patients as well as in control patients was observed. Bcl-2, bax and bcl-x(L) levels in responding and non-responding patients were not differently distributed. Bcl-2, bax and bcl-x(L) are likely to play a role in the natural history of cervical tumors, but their clinical significance in predicting response to treatment and clinical outcome needs long-term follow-up studies.

CD34+/CD105+ cells are enriched in primitive circulating progenitors residing in the G0 phase of the cell cycle and contain all bone marrow and cord blood CD34+/CD38low/- precursors.

A subset of circulating CD34+ cells was found to express CD105 antigen. Sorting experiments showed that most granulocyte-macrophage colony-forming units (GM-CFU) and burst-forming units - erythroid (BFU-E) were retained in the CD34+/CD105- fraction, whereas rare GM-CFU/BFU-E were generated from CD34+/CD105+ cells. Megakaryocytic aggregates were entirely retained in the CD34+/CD105+ fraction. Neutralizing doses of an anti-TGF-beta1 antibody demonstrated CD34+/CD105+ cells capable of colony-forming activity without any significant effect on CD34+/CD105- cells. Cloning of secondary colonies revealed that CD34+/CD105+ cells had a significantly higher secondary cloning efficiency than CD34+/CD105- cells. CD34+/CD105+ cells had a significantly higher long-term culture-initiating cell (LTC-IC) frequency than CD34+/CD105- cells. Kinetic analysis showed that 75% of CD34+/CD105+ cells consisted of DNA 2n G0Ki-67- cells whereas 82% of CD34+/CD105- were DNA 2n G1Ki-67+ cells, and this latter subset showed a RNA content consistently higher than CD34+/CD105+ cells. CD34+/CD105+ progenitors were CD25+, whereas CD34+/CD105- contained a small CD25+ subset. Three-colour analysis of bone marrow and cord blood CD34+ cells demonstrated that all the CD34+/CD38low/- primitive precursors were contained in CD34+/CD105+ cells. Extensive characterization of these CD105+ precursors indicated that they have biological properties associated with primitive haematopoietic precursors.

Bcl-2, Bax, Bcl-x(L) and Bcl-x(S) expression in neoplastic and normal endometrium.

The two anti-apoptotic proteins Bcl-2 and Bcl-x(L), and the two pro-apoptotic proteins Bax and Bcl-x(S) were measured by Western blotting in 51 neoplastic and 8 normal endometrial samples. The corresponding mRNA levels were analyzed by semiquantitative reverse transcriptase-polymerase chain reaction in a subgroup of 19 endometrial carcinomas. Neoplastic tissues had higher amounts of Bcl-2 protein than normal tissues (p < 0.051). Bcl-x(L) followed the same trend since its levels were higher in tumor than in normal samples (p < 0.048). Interestingly, Bcl-2 and Bcl-x(L) protein content showed a trend towards an inverse correlation (r = 0.27, p < 0.052). mRNA and protein levels directly correlated only with Bcl-2 (r = 0.63, p < 0.0032). Despite the fact that the amounts of Bcl-2, Bax and Bcl-x(L) proteins in the neoplastic population were not significantly differently distributed according to the clinicopathological features of the patients, the differences observed between normal and neoplastic samples suggest that these proteins may play a role in endometrial carcinoma: long-term follow-up studies will be required to confirm this hypothesis.

Expression of bcl-2, bax-xL, and bcl-xS in endometrial and cervical tissues.

We assessed the protein expression levels of bcl-2, bax, bcl-xL, and bcl-xS in a group of 51 endometrial cancers and 8 normal samples as well as in 59 cervical neoplasms and in 15 normal cervical tissues. Neoplastic endometria (median, 1.30 absorbance units [AU]; range, 0.13-7.26 AU) had slightly higher bcl-2 levels than did normal tissue (median, 0.83 AU; range, 0.29-1.90 AU; P < .068), whereas bcl-2 was lower in neoplastic (median, 3.59 AU; range, 0.13-19.86 AU) than in normal cervical samples (median, 8.45 AU; range, 2.09-15.04 AU; P < .010). Bcl-xL levels were higher in endometrial carcinoma (median, 1.23 AU; range, 0.03(4.29 AU) than in normal tissues (median, 0.56 AU; range, 0.46-1.48 AU; P < .048), whereas no significant difference was observed in cervical tissues. Bax levels did not show any variation in either system. The protein bcl-xS was marginally detectable in only a few samples. In endometrial carcinoma, bcl-2 and bcl-xL levels were correlated inversely (r = -0.27; P < .054), whereas in cervical cancer, they were correlated directly (r = +0.40; P < .002). The different expression patterns of bcl-2 family members in endometrial and cervical tissues confirm the hypothesis of a strictly tissue-specific regulation of these proteins.

Morphometric dual-energy X-ray absorptiometry of the spine: report of a large series and correlation with axial bone mineral density.

We studied vertebral morphometry and its relation to bone mineral density (BMD) in normal Brazilian women (n = 605). All women (age 22-97 years) were ambulatory and healthy. A lateral spine scan was done for morphometric X-ray absorptiometry using an imaging densitometer. In 429 of these women, BMD of the spine and proximal femur also were measured using dual-energy X-ray absorptiometry. All women were white with mean (+/- 1 SD) age of 53.7 (+/- 9.5) years. About 21% of the women over 50 years had a T score for spine BMD lower than -2.5 SD, and 7% had a femoral neck BMD below this osteoporosis threshold. Vertebral heights (anterior, HA; middle, HM; and posterior, HP) and ratios (HA/HP and HM/HP) were assessed. There was no systematic difference between younger (20-49 years) and older (50+ years) women in heights or ratios. The vertebral heights were normalized for those observed in each individual case for the L2-L4 sequence. This normalization was adequate for all vertebral heights; the Z score averaged about +0.1. The average Z score for HA/HP was +0.01, but that for the HM/HP was -0.72, indicating that the latter ratio might differ from the reference population used (white American and European women). We observed a small positive correlation between vertebral heights and spine or femur BMD, but this was due entirely to the influence of body size on BMD. On a group basis, the HM/HP showed a significant association with axial BMD; the 1 SD difference between the lowest and highest quartile was associated with a difference of 8-15% (0.5-1.0 SD) in axial BMD.