Microsatellite analysis of early stage (Ia-IIb) uterine cervical squamous carcinoma

Cervical cancer is the most common gynecologic malignancy of the developing world. The oncogenic role of human papilloma virus (HPV) is well known. Attention is now focusing on the complicit genetic changes, which allow progression of these tumors. Regarding these changes, deletion of tumor suppressor genes (loss of heterozygosity [LOH]) is the preferred pathway of progression with only a subset manifesting microsatellite instability (MSI). Implicated loci include 3p14.1-22. Several studies suggest that the mutator phenotype in cervical cancer may correlate with higher grade tumors, more advanced disease stage, and poor outcome. Unlike colorectal cancer, in which an inverse relationship has been demonstrated between microsatellite instability and loss of heterozygosity, cervical cancers expressing MSI have been found to coexpress LOH at other loci. In this study we analyzed 8-microsatellite loci including p53, DCC, APC, the MMR gene hMLH1 and 2 regions of interest on chromosome 3 in a high-risk population group in which HPV infection is endemic.     

Desensitization of macrophages to stimuli which induce secretion of superoxide anion. Down-regulation of receptors for phorbol myristate acetate

The ability of cultivated mouse peritoneal macrophages (M phi) to release superoxide anion (O-2) after repeated stimulation by phorbol myristate acetate (PMA) or serum-treated zymosan (STZ) has been studied. After a maximal first stimulus bacillus Calmette-Guérin (BCG)-activated M phi released high levels of O-2, 2-fold more than thioglycollate-elicited M phi and the response ceased within 4 h. Both populations either responded again to a second challenge or displayed a refractory state which varied in duration and selectivity. Desensitization by STZ pretreatment was transient and selective whereas PMA could render M phi refractory for 3 days to PMA alone or to both agents, depending on the amount of PMA used and the conditions of stimulation. PMA induced a selective loss of specific saturable receptors for [3H]phorbol dibutyrate, a closely related agent, and receptor activity recovered with the ability to release O-2. Loss of receptors did not account for concomitant loss of the response to STZ after nonselective deactivation. Such M phi were fully viable and able to endocytose various soluble and particulate ligands vigorously, but without stimulation of the hexose monophosphate shunt or release of O-2. Our studies indicate that M phi activities can be profoundly altered by prior stimulation, that specific receptors play a role in ligand-induced desensitization and that agents such as PMA can selectively eliminate the cells' ability to generate a second respiratory burst.     

Use of a T cell-specific monoclonal antibody, T10B9, in a novel allogeneic stem cell transplantation protocol for hematologic malignancy high-risk patients.

To reduce the toxicity of traditional conditioning regimens for allogeneic stem cell transplantation (allo-SCT), we used single-agent chemotherapy conditioning with either busulfan (total cumulative dose, 16 mg/kg) or melphalan (200 to 240 mg/m 2 ), followed by the anti-T cell-specific monoclonal antibody T10B9 (MEDI-500) daily for 3 days. T cell-replete SCT was performed from HLA-identical sibling donors. Acute graft-versus-host disease (aGVHD) prophylaxis consisted of 7 additional days of T10B9 and delayed onset of cyclosporine (ie, on day +4 or +5). Twenty-six high-risk hematologic malignancy patients were entered onto this study. All 24 patients who survived longer than 8 days engrafted, although 1 patient experienced late graft failure. Deaths occurred in 21 of 26 patients because of infection (n = 7), progression/recurrence of primary disease (n = 6), aGVHD (n = 4), regimen-related toxicity (n = 1), and other causes (n = 3). Five of these patients are enjoying disease-free survival with a median survival of 1193 days after allo-SCT. The conditioning regimen induced modulation of surface expression of CD3 (but not CD4 or CD8) and was associated with decreasing tumor necrosis factor-alpha (but not interleukin-6) serum levels. In conclusion, single-agent chemotherapy conditioning with T10B9 produced durable engraftment and long-term survival in some patients who would not have qualified for a traditional allo-SCT.     

Committing embryonic stem cells to early endocrine pancreas in vitro

A panel of genetic markers was used to assess the in vitro commitment of murine embryonic stem (ES) cells toward the endoderm-derived pancreas and to distinguish insulin-expressing cells of this lineage from other lineages such as neuron, liver, and yolk sac. There are two nonallelic insulin genes in mice. Neuronal cells express only insulin II, whereas the pancreas expresses both insulin I and II. Yolk sac and fetal liver express predominately insulin II, small amounts of insulin I, and no glucagon. We found that ES-derived embryoid bodies cultured in the presence of stage-specific concentrations of monothio-glycerol and 15% fetal calf serum, followed by serum-free conditions, give rise to a population that expresses insulin I, insulin II, pdx-1 (a pancreas marker), and Sox17 (an endoderm marker). Immunohistochemical staining shows intracellular insulin particles, and its de novo production was confirmed by staining for C-peptide. Most, but not all, of the insulin+ or C-peptide+ cells coexpress glucagon, demonstrating a differentiation pathway to pancreas rather than yolk sac or fetal liver. Addition of beta-cell specification and differentiation factors activin beta B, nicotinamide, and exendin-4 to later-stage culture increased insulin-positive cells to 2.73% of the total population, compared with the control culture, which gave rise to less than 1% insulin-staining cells. These findings suggest that stepwise culture manipulations can direct ES cells to become early endocrine pancreas.     

Norepinephrine triggers release of glial ATP to increase postsynaptic efficacy

Glial cells actively participate in synaptic transmission. They clear molecules from the synaptic cleft, receive signals from neurons and, in turn, release molecules that can modulate signaling between neuronal elements. Whether glial-derived transmitters can contribute to enduring changes in postsynaptic efficacy, however, remains to be established. In rat hypothalamic paraventricular nucleus, we demonstrate an increase in the amplitude of miniature excitatory postsynaptic currents in response to norepinephrine that requires the release of ATP from glial cells. The increase in quantal efficacy, which likely results from an insertion of AMPA receptors, is secondary to the activation of P2X(7) receptors, an increase in postsynaptic calcium and the activation of phosphatidylinositol 3-kinase. The gliotransmitter ATP, therefore, contributes directly to the regulation of postsynaptic efficacy at glutamatergic synapses in the CNS.     

Rat sponge implant model: a new system for evaluating angiogenic gene transfer

Therapeutic angiogenesis, either by protein injection or gene therapy, holds considerable promise for the treatment of coronary and peripheral artery diseases. Given the large number of angiogenic genes available, a simple, well defined, standard system to compare the relative angiogenic efficacy of such genes would be valuable. We have employed a replication-deficient adenovirus vector (complete E1a-, partial E1b- and partial E3-) to deliver the beta-galactosidase (beta-gal, AdLacZ) reporter gene or the human VEGF121 gene (AdGV VEGF121.10) to a rat sponge implant model of angiogenesis. beta-gal staining results reveal a transfection efficiency as high as 60% 24 h after 2x1010 particle units AdLacZ injection. Our results also indicate that a single injection of 2x1010 particle units of AdGVVEGF121.10 in the sponge results in >10, 000 pg VEGF protein expression per milligram of sponge tissue 24 h later. VEGF121 protein concentrations decreased 10-fold within 3 days and 100-fold within 7 days after injection. Significant VEGF121 protein levels were still detectable 14 days after initial virus injection. The high level of gene transfection efficiency was accompanied by enhanced angiogenesis in the sponge, a tissue devoid of any vessels before implantation. Compared to control (AdNull: adenovirus vector without the VEGF gene), AdGVVEGF121.10 induced a 2- to 3-fold up-regulation of angiogenesis at 7 and 14 days post vector injection as determined by both increased capillary number and increased tissue ingrowth. The angiogenic effects of AdGVVEGF121. 10 were dose-related in this model system. These findings demonstrate a dose-related angiogenic response to adenovirus-mediated gene therapy in this model.