Pregnancy and conception rate after two intravaginal inseminations with dog semen frozen either with 5% glycerol or 5% ethylene glycol.

The primary goal of this study was to compare the effects of 5% ethylene glycol (EG) and 5% glycerol (G) on fertility of frozen-thawed dog semen following intravaginal insemination. The sperm-rich fraction of the ejaculate of three male dogs was collected, pooled and divided into two aliquots, and then frozen with a Tris-glucose-egg yolk-citric acid extender containing either 5% G or 5% EG. A total of 10 bitches were inseminated twice, five with G-frozen-thawed semen and five with EG-frozen-thawed semen; intravaginal inseminations were performed the 4th and the 5th day after the estimated LH peak; four straws, thawed in a 37 degrees C water bath for 1 min and diluted in a Tris buffer, were used for insemination (200 x 10(6) spermatozoa); the insemination dose was introduced in the cranial vagina of the bitch using a sterile plastic catheter. Ovariohysterectomy was performed in all bitches between days 29 and 31 after the calculated LH surge, and pregnancy status, and the number of conceptuses and corpora lutea were recorded. All bitches were pregnant. Neither the number of conceptuses, nor the ratio of conceptuses to corpora lutea (conception rate) was significantly different between groups. In this first screening, with a limited number of bitches, EG-frozen semen did not show a higher fertility than G-frozen semen when used for two intravaginal inseminations. Irrespective of the semen used, conception rate was 0.50.

Gene expression profiling of acute promyelocytic leukaemia identifies two subtypes mainly associated with flt3 mutational status.

Acute promyelocytic leukaemia (APL) is a well-defined disease characterized by a typical morphology of leukaemic cells, the presence of t(15;17) translocation and the unique sensitivity to the differentiating effect of all-trans retinoic acid. Nevertheless, some aspects are variable among APL patients, with differences substantially related to morphological variants, peripheral leukocytes count, the presence of a disseminated intravascular coagulopathy, different PML/RARalpha isoforms (long, variable or short) and Fms-like tyrosine kinase 3 (Flt3) mutations. In order to better define this variability, we investigated the gene expression profiles of 18 APL cases revealing, besides a high uniformity in gene expression pattern, the presence of few robust differences among patients able to identify, by an unsupervised analysis, two major clusters of patients characterized by different phenotypes (hypogranular M3v vs classical M3) and by the presence or absence of Flt3 internal tandem duplications (ITDs). Further supervised analysis confirmed that Flt3 status was the APL parameter best associated with these two subgroups. We identified, between Flt3 wild-type and Flt3-ITDs subsets, 147 differentially expressed genes that were involved in the cytoskeleton organization, in the cell adhesion and migration, in the proliferation and the coagulation/inflammation pathways as well as in differentiation and myeloid granules constitution suggesting a role of Flt3 mutations in the pathogenesis and clinical manifestations of APL.

Immunoglobulin gene mutations and frequent use of VH1-69 and VH4-34 segments in hepatitis C virus-positive and hepatitis C virus-negative nodal marginal zone B-cell lymphoma.

Nodal marginal zone B-cell lymphoma (NMZL) is actually considered as a distinct entity that must be distinguished from extra-nodal and splenic marginal zone lymphomas. To define the cell origin and the role of antigen stimulation we determined the nucleotide sequence of the tumor-related immunoglobulin heavy chain variable genes in 10 cases of NMZL. The results were also evaluated on the basis of the presence of chronic hepatitis C virus (HCV) infection. All 10 cases harbored VH somatic mutations with a sequence homology compared to the closest germline gene, ranging from 83.33 to 98.28%. Interestingly, different VH segments were preferentially used in HCV-positive and HCV-negative patients: three of five HCV-negative NMZLs used a VH4-34 segment joined with different D and JH segments whereas three of five HCV-positive NMZLs used a VH1-69 gene joined with a D3-22 and a JH4 segment, with very strong similarities in the CDR3s among the three different cases. These data indicate: 1) NMZL is derived from B cells that have experienced the germinal center reaction; 2) the preferential usage of a VH1-69 segment in the majority of the HCV-positive NMZL cases with similar CDR3s suggests the presence of a common antigen, probably a HCV antigen epitope, involved in the B-cell selection; and 3) the use of a VH4-34 segment suggests a role of yet unknown B-cell superantigen(s) in the selection of tumor B-cell precursors in HCV-negative NMZL.

The protective efficacy of surgical latex gloves against the risk of skin contamination: how well are the operators protected?

Latex gloves are used by surgical staff to avoid exposure to patient body fluids, thus reducing the risk of contracting bloodborne viral diseases, such as hepatitis C and HIV. We studied the efficacy of the surgical barrier provided by latex gloves, before and after use in the operating theater. The electrical conductivity, insulation and mechanical resistance of glove latex were investigated, using routine supplies of surgical gloves. Latex structure was assessed by scanning electron microscopy and by mercury intrusion porosimetry. Latex is subject to hydration, a phenomenon associated in the laboratory with the loss of its electrical insulation properties. Such glove latex properties were found to be highly variable, with latex hydration times varying between 2 and more than 30 min. Rapidly hydrating gloves showed increased permeability to methylene blue, associated with higher levels of porosity. Thirty min of surgical use was associated with measurable hydration of glove latex and a statistically significant loss of electrical and mechanical resistance, with rupture load decreasing by 24%. Electronic control of the insulation properties of gloves during surgery permits early detection of hydration, and allows prompt correction by glove change, before the gloves lose their electrical and mechanical competence.

Effects of pulsed electromagnetic fields on human chondrocytes: an in vitro study.

(3)H-thymidine incorporation was studied in cultured human nasal and articular chondrocytes exposed to low-energy, low-frequency pulsed electromagnetic fields (PEMFs) (75 Hz, 2.3 mT). The reverse transcriptase polymerase chain reaction (RT-PCR) analysis shows that human secondary chondrocytes derived from both nasal and articular cartilage express collagen type II mRNA, which is a specific marker of the chondrocyte phenotype. In a preliminary series of experiments, cells were exposed to PEMF for different time periods ranging from 6 to 30 hours (time-course), in medium supplemented with 10% or 0.5% fetal calf serum (FCS) and in serum-free medium. The ratios between the (3)H-thymidine incorporation in PEMFs and control cultures show an increase of the cell proliferation in cultures exposed to PEMFs when serum is present in the culture medium, whereas no effect was observed in serum-free conditions. The increase in DNA synthesis, induced by PEMFs, was then evaluated only at the times of maximum induction and the results were analyzed by the three-factor analysis of variance (ANOVA). The data presented in this study show that even if (3)H-thymidine incorporation is higher in nasal than in articular chondrocytes, PEMF induce an increase in the proliferation of both cell types. Moreover, the concentration of FCS in the culture medium greatly influences the proliferative response of human chondrocytes to the PEMF exposure. Though normal human osteoblast cells increase their proliferation when exposed to PEMFs if only 10% FCS is present in the medium, human chondrocytes are able to increase their cell proliferation when exposed to PEMFs in the presence of both 0.5% and 10% of FCS in the medium. The results obtained may help to explain the basic mechanisms of PEMF stimulation of fracture healing.

Missense mutations in the PML/RARalpha ligand binding domain in ATRA-resistant As(2)O(3) sensitive relapsed acute promyelocytic leukemia.

BACKGROUND AND OBJECTIVE: Acute promyelocytic leukemia is characterized by the chromosomal translocation t(15;17) which yields the fusion product PML/RARa. All-trans retinoic acid probably induces differentiation of atypical promyelocytes and clinical remission in APL patients by binding to the ligand binding domain (LBD) of the RARa portion of the PML-RARa chimeric protein. Structural alterations of the LBD of the PML/RARa have been revealed in ATRA-resistant APL cell lines and in a few APL patients with acquired clinical resistance to ATRA therapy. Two APL relapsed patients with clinical resistance to ATRA therapy were evaluated for the presence of nucleotide mutations in the LBD of PML/RARa gene and then treated with arsenic trioxide (As2O3). DESIGN AND METHODS: DNA fragments from the LBD of the PML/RARa chimeric transcript were obtained by reverse-transcribed polymerase chain reaction. Direct sequencing was performed by an unambiguous bi-directional automatic analysis. Samples representative of APL onset and relapse were analyzed from both patients. RESULTS: In both patients, at the ATRA-resistant relapse, a missense point mutation in the LBD of the PML/RARa gene was found. The mutations, absent at APL onset, led to an Arg272Gln and to an Arg276Trp amino acid substitution, according to the sequence of the RARa protein. Both patients had complete clinical and hematologic remission after treatment with As2O3. INTERPRETATION AND CONCLUSIONS: LBD missense mutations appear to be a significant mechanism of acquired ATRA-resistance in vivo, closely related to clinical APL relapse. The two cases reported here provide the first in vivo evidence of APL relapsed patients, who have become ATRA-resistant for molecular reasons, being sensitive to arsenic trioxide.

Protective effect of low frequency low energy pulsing electromagnetic fields on acute experimental myocardial infarcts in rats.

This series of experiments assesses the effect of exposure to low-frequency pulsing electromagnetic fields (PEMFs) in 340 rats with acute experimental myocardial infarcts. The left anterior descending artery was ligated with suture thread, and the rats underwent total body exposure to PEMFs until they were killed. Twenty-four hours after surgery, the necrotic area was evaluated by staining with triphenyltetrazolium chloride. A significant reduction of the necrotic area was observed in the animals exposed to PEMFs compared with the nonexposed controls. Exposure for up to 6 days does not appear to affect the area of necrosis, although in exposed animals an increase of vascular invasion of the necrotic area is observed: 24.3 % as against 11.3 % in controls. No effect on the necrotic area size from exposure was found when the left anterior descending artery was occluded for 60 min, followed by reperfusion. The results reported show that exposure to PEMFs is able to limit the area of necrosis after an acute ischemic injury caused by permanent ligation of the left anterior descending artery. These data are in agreement with the protective effect of PEMFs observed on acute ischemia in skin free flaps in rats and in cerebral infarcts in rabbits.

Antisense inhibition of c-fes proto-oncogene blocks PMA-induced macrophage differentiation in HL60 and in FDC-P1/MAC-11 cells.

To gain some insight into the role of c-fes in macrophage differentiation, we have analyzed the ability of HL60 leukemic promyelocytic cells and FDC-P1/MAC-11 murine myeloid precursor cells to differentiate in response to phorbol esters after inhibition of c-fes function. Fes inactivation has been obtained by using oligodeoxynucleotides (ODN) complementary to the 5' region of c-fes mRNA and to 5' splice junctions of c-fes primary transcript. After 5 days (d) in culture, in several separate experiments performed with different ODN preparations, a complete inhibition of c-fes expression was observed in HL60 and in FDC-P1/MAC-11 cells. No perturbation of cell growth was evident in our experimental conditions in both cell lines after c-fes inhibition. Furthermore, in HL60 cells lacking c-fes product, an almost complete downregulation of the alpha 4 beta 1 fibronectin receptor occurred. However, in both cell lines, the induction of macrophage differentiation by phorbol esters resulted in an almost complete maturation arrest as evaluated by morphological, cytochemical, immunological criteria, and by the cytofluorimetric cell cycle analysis. A loss of the adhesion capacity of both myeloid cell lines, when compared to terminally differentiated macrophages, was also observed. These results suggest that HL60 and FDC-P1/MAC-11 cells, when treated with phorbol 12-myristate 13-acetate, require c-fes protein expression to activate the genetic program underlying macrophage differentiation.

Interleukin-11 (IL-11) acts as a synergistic factor for the proliferation of human myeloid leukaemic cells.

Interleukin-11 is a stromal cells derived cytokine which stimulates the proliferation of primitive haemopoietic progenitor cells. For this paper we have studied the constitutive expression of IL-11 mRNA in a panel of wellknown leukaemic cell lines and samples from AML patients at diagnosis. Moreover, the same cellular populations were evaluated for their proliferative response to recombinant-human-(r-hu). IL-11 alone and combined with r-hu-IL-3, granulocyte-macrophage colony stimulating factor (GM-CSF) and stem cell factor (SCF, c-kit ligand). The colony-forming ability of HL60, K562, KG1 cells and eight fresh AML cell populations was assessed by a clonogenic assay in methylcellulose. In eight additional AML cases the number of S-phase leukaemic cells induced by IL-11 was determined by the bromodeoxyuridine (BRDU) incorporation assay after 3d of liquid culture. IL-11, as single cytokine, did not stimulate the colony formation of the three myeloid cell lines under serum-containing and serum-free conditions. In contrast, the proliferation of the leukaemic cells in response to IL-3, GM-CSF and SCF was enhanced by co-incubation with IL-11, and this effect was reversed in blocking experiments by the anti-IL-11 Moab. When tested on primary AML samples, IL-11 alone showed little, if any, proliferative activity. However, it increased the IL-3-dependent blast colony formation in eight out of eight cases and GM-CSF in seven cases. IL-11 also augmented synergistically the number of CFU-L stimulated by SCF in seven cases. A combination of three factors (IL-11, SCF and IL-3) yielded optimal colony formation. The BRDU studies showed the significant increase of AML cells in S-phase when IL-11 was combined with SCF, whereas the two CSF had no activity on their own. Positive interaction was also observed when IL-11 was added to IL-3 supplemented cultures in five out of eight cases tested. Reverse transcriptase-polymerase chain reaction amplification (RT-PCR) demonstrated the constitutive expression of IL-11 mRNA in all the cell lines and 11/12 AML samples studied at diagnosis. These results indicate that IL-11 is expressed in leukaemic myeloid cells and that their proliferation is regulated by the cytokine which acts as a synergistic factor.

All-trans-retinoic acid induces simultaneously granulocytic differentiation and expression of inflammatory cytokines in HL-60 cells.

All trans-retinoic acid (ATRA) can induce granulocytic differentiation both in vitro and in vivo, and its activity is mediated by the retinoic acid receptor-alpha (RAR-alpha). In the present study, we evaluated the ability of this inducer in HL-60 cells, to stimulate simultaneously granulocytic differentiation and the expression of the cytokines interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-3, IL-6, tumor necrosis factor-alpha (TNF-alpha), and stem cell factor (SCF). The level of expression of these cytokines in ATRA-treated HL-60 cells was compared with that observed in normal and lipopolysaccharide (LPS)-treated peripheral granulocytes. The results indicate that the expression of these cytokines is enhanced during differentiation so that the pattern observed in ATRA-treated HL-60 cells is close to that of LPS-stimulated normal granulocytes. In addition, tetra phorbol acetate (TPA)-treated HL-60 cells express several of the above listed cytokines. It is concluded that ATRA not only induces granulocytic differentiation of HL-60 cells, but also activation of these terminally differentiated cells. The activating cytokine expression in these cells appears related to the progress of the differentiation program induced by ATRA since normal granulocytes do not respond to this inducer by activation of the expression of these genes. Furthermore, the cytokine activation is a specific effect of ATRA, since DMSO does not have any stimulatory effect.

Antiapoptotic effect of c-fes protooncogene during granulocytic differentiation.

The c-fes protooncogene is expressed at high levels in the terminal stages of granulocytic differentiation. Its product, p92c-fes, exhibits a tyrosine-kinase activity and is involved in the cellular response to GM-CSF, but its role is not yet clarified. To study this problem, the c-fes protooncogene expression has been inhibited in HL60 cells and in fresh leukemic blast cells of Acute Promyelocytic Leukemia (APL) induced to differentiate with All-Trans-Retinoic Acid (ATRA). Inhibition of c-fes function was obtained by treatment of the cells with a specific antisense oligomer complementary to the 5' region of the c-fes mRNA. It was observed that the cells, rather then differentiate to granulocytes, underwent premature cell death showing the morphological and molecular characteristics of apoptosis. Superimposable results are obtained on blast cells from APL. It is possible to conclude that the loss of cell viability that occurs during the in vitro differentiation of myeloid cells, after the complete inhibition of c-fes expression and treatment with ATRA, is due to activation of programmed cell death rather than an accelerated differentiation. Our data suggest that the c-fes product is essential for the survival of myeloid cells during differentiation.

Inhibition of c-fes expression by an antisense oligomer causes apoptosis of HL60 cells induced to granulocytic differentiation.

The c-fes protooncogene is expressed at high levels in the terminal stages of granulocytic differentiation, but so far no definite function has been attributed to the product of this oncogene. To tackle this problem, the c-fes protooncogene expression has been inhibited in HL60 cells, and fresh leukemic promyelocytes of acute promyelocytic leukemia have been induced to differentiate with retinoic acid (RA) and dimethylsulfoxide (DMSO). Inhibition was obtained by incubating the cells with a specific c-fes antisense oligodeoxynucleotide. It was observed that the cells, rather than differentiating, underwent premature cell death showing the morphological and molecular characteristics of apoptosis. This process was inhibited by granulocyte and granulocyte/macrophage colony-stimulating factor, but not by interleukin 3 (IL-3), IL-6, or stem cell factor. Our present results demonstrate that the loss of cell viability that occurs during the in vitro differentiation of myeloid cells, after the complete inhibition of the c-fes gene product and treatment with RA-DMSO, is due to activation of programmed cell death. It is concluded that a possible role of the c-fes gene product is to exert an antiapoptotic effect during granulocytic differentiation.

Overexpression of c-kit in a leukemic cell population carrying a trisomy 4 and its relationship with the proliferative capacity.

The expression of c-kit and its ligand, the stem cell factor (SCF), was studied in five cases of acute myeloid leukemia. One of these had a trisomy of chromosome 4, where the c-kit oncogene is located. In this case, the c-kit oncogene was overexpressed, but matched by a low expression of its ligand, SCF. The molecular evaluation of the growth rate by c-myc and the histone H3 expression indicated that the growth fraction of this cell population was very low. In one of the other leukemic cell populations studied, characterized by a low expression of c-kit and an elevated expression of the SCF, the growth fraction was also very low. Our results suggest that at least for some receptor oncogenes, the simple overexpression cannot be taken as an indication that the oncogene is involved in the deregulation of cell proliferation.

Expression of interleukins 1, 3, 6, stem cell factor and their receptors in acute leukemia blast cells and in normal peripheral lymphocytes and monocytes.

Reverse transcriptase-polymerase chain reaction amplification (RT-PCR) and Southern blot analysis were used to evaluate ligand and receptor expression of interleukin 1 alpha (IL-1 alpha), interleukin 3 (IL-3), interleukin 6 (IL-6) and stem cell factor (SCF) in peripheral blood lymphocytes and monocytes and in several acute leukemia blast cell populations. Resting peripheral lymphocytes and monocytes expressed both ligand and receptor of the four cytokines at considerable levels. The leukemic blast cells of the M1-M4 phenotypes are characterized by almost complete lack of expression of IL-1 alpha, IL-3 and IL-6 and the constant and usually high expression of SCF. On the other hand, these myeloid blast cells express generally high levels of the four cytokine receptors. The data suggest that the regulation of the expression of IL-1 alpha, IL-3 and IL-6, at least in our limited number of leukemic cell populations studied, is independent of that of SCF. The results indicate that, at least in most of the leukemic myeloid blasts cells, the expression of SCF and its receptor, the c-kit oncogene, may permit an autocrine regulation of cell cycling.

Lymphocytes and low-frequency electromagnetic fields.

Human lymphocytes have been used by several researchers to investigate the biological effect of electromagnetic fields (EMF). EMF modulate the response by lymphocytes to lectin stimulation. The size and direction of the effect depends both on the lymphocyte physiology and on the physical parameters characterizing the EMF. Lymphocytes have also been used to investigate the genotoxicity of EMF exposure.

Abundance of the primary transcript and its processed product of growth-related genes in normal and leukemic cells during proliferation and differentiation.

The relative abundance of primary transcript and mature mRNA of the c-myc, calcyclin, S14 ribosomal protein, and rRNA genes was determined densitometrically after reverse transcriptase-polymerase chain reaction and Northern blotting analysis in resting and mitogen-stimulated lymphocytes, proliferating and terminally differentiated HL-60 cells, and leukemic blast cells. Transcription and processing of c-myc and rRNA gene transcripts increased proportionally after mitogen stimulation, whereas these processes were independent of cell cycling status in the case of the S14 gene. Normal lymphocytes showed an unexpectedly large amount of primary transcript of the calcyclin gene, whereas the corresponding mRNA was undetectable. The abundance of c-myc, calcyclin, and S14 mRNA in terminally differentiated HL-60 cells decreased sharply, compared to their proliferating counterparts. This decrease reflected post-transcriptional modulation, since the abundance of precursor remained essentially unchanged. After HL-60 differentiation, the 32S rRNA levels remained relatively high, whereas the 45S primary transcript almost disappeared. Leukemic blast cells displayed highly variable precursor/mRNA ratios of c-myc, calcyclin, and S14 genes but consistently high ratios of 32S to 45S RNA, suggesting that the cleavage rate of the 32S rRNA was sharply reduced in these cells, resulting in an accumulation of this molecule. These results suggest the importance of efficient processing of primary transcript to generate adequate functional mRNA, thus regulating gene expression. Furthermore, in terminally differentiated cells and leukemic blast cells a stabilization of the primary transcript without efficient processing can be observed. The role of the stabilization of the primary transcript in terminal differentiation is further supported by the results of run-off transcription, indicating a sharp decrease of c-myc and calcyclin transcription rate in retinoic acid/dimethyl sulfoxide-treated HL-60 cells.