Actions of endothelin-1 on swine ovarian (granulosa) cells.

We have investigated the regulatory actions of endothelin-1 (ET-1) on inositol phosphate accumulation, cytosolic free Ca2+ ion concentrations ([Ca2+]i), and basal and FSH-stimulated progesterone and cAMP accumulation by swine granulosa cells in serum-free cultures. ET-1 induced a rapid stimulation of phosphoinositide hydrolysis in populations of granulosa cells, as inferred by the rapid appearance of soluble inositol polyphosphates in response to ET-1 exposure. At the single cell level, fura-2 videomicroscopy was used to measure [Ca2+]i in individual granulosa cells. We observed cell-cell variability in the threshold concentration of ET-1 required to induce a rise in [Ca2+]i. More than 75% of granulosa cells responded to maximal doses of ET-1. The following parameters of [Ca2+]i were influenced by ET-1 concentration: percentage of responding cells, lag time for the onset of response, amplitude, and kinetics of the response. Two types of ET-1-mediated [Ca2+]i rises were observed. One type exhibited rapid Ca2+ kinetics, reaching at least a 2-fold increase above basal (spike phase) within 1-10 sec and returning to a new steady state (plateau phase) 2 min after onset. The other mode of response had slower [Ca2+]i kinetics, in which 50 sec or more were required to double [Ca2+]i, which remained at this level throughout the observation period (2.5 min). These responses to ET-1 were specific and were not initiated by vasopressin or tumor necrosis factor-alpha. In cell population studies using monolayer cultures of swine granulosa cells, ET-1 inhibited FSH-stimulated accumulation of progesterone and cAMP. The ET-1-mediated inhibition of FSH-stimulated accumulation of progesterone required at least 4 h of ET-1 exposure. The ET-1-mediated inhibition of both the FSH-stimulated accumulation of progesterone and cAMP after 24-h incubation was mimicked by an activator of protein kinase-C, phorbol 12-myristate 13-acetate, but not by an inactive phorbol. These observations in either single cells or populations of swine ovarian (granulosa) cells are consistent with a possible regulatory role of an ET-1-activated intracellular signaling pathway involving inositol phosphates, [Ca2+]i, and protein kinase-C in the mammalian granulosa cell.     

Fc gamma receptor II (CD32) on malignant B cells influences modulation induced by anti-CD19 monoclonal antibody

Antigenic modulation is one of many factors determining the effectiveness of monoclonal antibody (MoAb)-mediated therapy. To select the isotype of a CD19 MoAb most suitable for radioimmunotherapy of patients with B-cell malignancies, we studied the influence of MoAb isotype on modulation, after binding of the MoAb to different cell-line cells. The CD19-IgG1 MoAb was found to induce modulation of CD19 antigens on Daudi cell line cells more rapidly than did its IgG2a switch variant. We provide evidence that this difference in modulation rate is caused by the expression of Fc gamma receptor II (Fc gamma RII) on these cells. Experiments aimed at elucidating the mechanism of Fc gamma RII involvement in modulation induction by CD19-IgG1 showed that Fc gamma RII did not comodulate with CD19 MoAbs. However, cocrosslinking of CD19 and Fc gamma RII with CD19-IgG1 MoAb resulted in enhanced calcium mobilization in Daudi cells. This increased signal induction accompanies the enhanced capping and subsequent modulation of CD19 antigens. Because Fc gamma RII is expressed in varying densities on malignant B cells in all differentiation stages, our results have implications for the MoAb isotype most suitable for use in MoAb-based therapy of patients with B-cell malignancies.     

Hodgkin and Reed-Sternberg cells harbor alterations in the major tumor suppressor pathways and cell-cycle checkpoints: analyses using tissue microarrays

Tumoral cells in Hodgkin lymphoma (HL) display an increased growth fraction and diminished apoptosis, implying a profound disturbance of the cell cycle and apoptosis regulation. However, limitations of molecular techniques have prevented the analysis of the tumor suppressor pathways and cell-cycle checkpoints. Tissue microarray (TMA) is a powerful tool for analyzing a large number of molecular variables in a large series of tumors, although the feasibility of this technique has not yet been demonstrated in heterogeneous tumors. The expression of 29 genes regulating the cell cycle and apoptosis were analyzed by immunohistochemistry and in situ hybridization in 288 HL biopsies using TMA. The sensitivity of the technique was validated by comparing the results with those obtained in standard tissue sections. The results revealed multiple alterations in different pathways and checkpoints, including G1/S and G2/M transition and apoptosis. Striking findings were the overexpression of cyclin E, CDK2, CDK6, STAT3, Hdm2, Bcl2, Bcl-X(L), survivin, and NF-kappaB proteins. A multiparametric analysis identified proteins associated with increased growth fraction (Hdm2, p53, p21, Rb, cyclins A, B1, D3, and E, CDK2, CDK6, SKP2, Bcl-X(L), survivin, STAT1, and STAT3), and proteins associated with apoptosis (NF-kappaB, STAT1, and RB). The analysis also demonstrated that Epstein-Barr virus (EBV)-positive cases displayed a characteristic profile, confirming the pathogenic role of EBV in HL. Survival probability depends on multiple biologic factors, including overexpression of Bcl2, p53, Bax, Bcl-X(L), MIB1, and apoptotic index. In conclusion, Hodgkin and Reed-Sternberg cells harbor concurrent and overlapping alterations in the major tumor suppressor pathways and cell-cycle checkpoints. This appears to determine the viability of the tumoral cells and the clinical outcome.     

Valvuloplasty with balloon catheter in biologic prosthesis. Reality or illusion

We performed catheter balloon valvuloplasty (CBV) on 8 stenotic operatively-excised bioprosthetic valves (2 Hancock and 6 Ionescu Shiley). Pathology of valves before CBV included degenerative changes: commissural fusion by mounds of calcific deposits (2 valves), fibrotic and focally calcified leaflets (7 valves) and stiff and thick valves (1 valve). Inflation of the balloon resulted in commissural splitting (2 valves), leaflet cracks and fractures (3 valves). Removal of the deflated balloon catheter was associated with debris dislodgement (3 valves). In one case the valve was unable to close with potential for acute regurgitation. Thus, CBV of bioprosthetic valves can split fused commissures by similar mechanisms as in native valves. CBV may fracture calcific deposits causing acute emboli. It can also disrupt the leaflets causing acute insufficiency. The findings suggest a limited role of CBV in the treatment of stenotic bioprosthetic valves in mitral and aortic position.     

Use of three‐dimensional power doppler sonography in the diagnosis of uterine arteriovenous malformation and follow‐up after uterine artery embolization: Case report and brief review of literature

Arteriovenous malformations (AVM) of the uterus can cause life‐threatening hemorrhage. Unexplained, heavy vaginal bleeding in a reproductive age woman should raise suspicion for an AVM. Here a 37‐year‐old woman had increasingly severe vaginal bleeding for 15 days. Serum β‐hCG was elevated. Two‐dimensional transvaginal ultrasound suggested retained products of conception. Before dilation and curettage (D&C), color Doppler and three‐dimensional (3D) power Doppler demonstrated findings indicative of uterine AVM. A bilateral uterine artery embolization was performed without complications. Three months after uterine artery embolization, 3D power Doppler ultrasonography found complete resolution of the AVM. This case illustrates the importance of assessing both gray‐scale and 3D power Doppler, and the ability of postprocedure Doppler to assess resolution. © 2014 Wiley Periodicals, Inc. J Clin Ultrasound 43:327–334, 2015     

Genetic diversity of 3′ region of glycoprotein D gene of bovine herpesvirus 1 and 5

Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) are closely related alphaherpesviruses of cattle. While BoHV-1 is mainly associated with respiratory/genital disease and rarely associated with neurological disease, BoHV-5 is the primary agent of meningoencephalitis in cattle. The envelope glycoprotein D of alphaherpesviruses (BoHV-1/gD1 and BoHV-5/gD5) is involved in the early steps of virus infection and may influence virus tropism and neuropathogenesis. This study performed a sequence analysis of the 3′ region of gD gene (gD3′) of BoHV-1 isolates recovered from respiratory/genital disease (n = 6 and reference strain Cooper) or from neurological disease (n = 7); and from seven typical neurological BoHV-5 isolates. After PCR amplification, nucleotide (nt) sequencing, and aminoacid (aa) sequence prediction; gD3′ sequences were compared, identity levels were calculated, and selective pressure was analyzed. The phylogenetic reconstruction based on nt and aa sequences allowed for a clear differentiation of BoHV-1 (n = 14) and BoHV-5 (n = 7) clusters. The seven BoHV-1 isolates from neurological disease are grouped within the BoHV-1 branch. A consistent alignment of 346 nt revealed a high similarity within each viral species (gD1 = 98.3 % nt and aa; gD5 = 97.8 % nt and 85.8 % aa) and an expected lower similarity between gD1 and gD5 (73.7 and 64.1 %, nt and aa, respectively). The analysis of molecular evolution revealed an average negative selection at gD3′. Thus, the phylogeny and similarity levels allowed for differentiation of BoHV-1 and BoHV-5 species, but not further division in subspecies. Sequence analysis did not allow for the identification of genetic differences in gD3′ potentially associated with the respective clinical/pathological phenotypes, yet revealed a lower level of gD3′ conservation than previously reported.