Alterations in cholinergic and non-cholinergic neurotransmitter receptor densities in transgenic Tg2576 mouse brain with beta-amyloid plaque pathology.

Cholinergic deficits in Alzheimer's disease are accompanied by a number of alterations in other transmitter systems including glutamate, noradrenaline and serotonin, suggesting the involvement also of other neurotransmitter systems in the pathogenesis of the disease. To address the question whether beta-amyloid may contribute to these deficits, brain tissue from transgenic Tg2576 mice with Alzheimer plaque pathology at ages of 5 (still no significant plaque load) and 17 months (moderate to high cortical beta-amyloid plaque load) were examined for a number of cholinergic and non-cholinergic markers. Transgenic mice with no significant plaque load demonstrated reduced hemicholinium-3 (HCh-3) binding to choline uptake sites in anterior brain regions as compared to non-transgenic littermates, while in aged transgenic mice with high number of plaque deposits decreased HCh-3 binding levels were accompanied by increased vesicular acetylcholine transporter binding in selected cortical brain regions. In aged transgenic mice GABA(A), NMDA, AMPA, kainate, and beta-adrenergic as well 5-HT(1A)- and 5-HT(2A)-receptor binding levels were hardly affected, whereas alpha(1)- and alpha(2)-adrenoceptor binding was increased in selected cerebral cortical regions as compared to non-transgenic littermates. The development of changes in both cholinergic and non-cholinergic markers in transgenic Tg2576 mouse brain already before the onset of progressive plaque deposition provides in vivo evidence of a modulatory role of soluble beta-amyloid on cortical neurotransmission and may be referred to the deficits in learning and memory observed in these mice also before significant plaque load.     

Glial cell expression of hepatocyte growth factor in vitreoretinal proliferative disease

The hepatocyte growth factor (HGF) has been crucially implicated in the development of proliferative retinal diseases; however, it is unclear whether retinal glial cells express or respond to HGF. Therefore, we examined the expression of HGF and of the receptor for HGF, c-Met, by immunohistochemical costaining with glial fibrillary acidic protein (GFAP) in epiretinal membranes of patients with proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR), respectively. Furthermore, it was determined whether cells of the human retinal glial cell line, MIO-M1, secrete HGF protein, and whether HGF stimulates proliferation and chemotaxis, and secretion of the vascular endothelial growth factor (VEGF). Neuroretinas of patients with PVR express elevated mRNA level for HGF in comparison to control retinas. In epiretinal membranes of patients with PVR or PDR, immunoreactivity for HGF and for c-Met, respectively, partially colocalized with immunoreactivity for GFAP. Fetal bovine serum and basic fibroblast growth factor, but not heparin-binding epidermal or platelet-derived growth factors, evoked HGF secretion by cultured retinal glial cells. HGF displayed only a marginal effect on cell proliferation while it stimulated chemotaxis. HGF promoted the secretion of VEGF, via activation of the phosphatidylinositol-3 kinase. It is concluded that glial cells in epiretinal membranes express both HGF protein and c-Met receptors. The results suggest an autocrine/paracrine role of HGF in glial cell responses during proliferative vitreoretinal disorders as well as in retinal neovascularization, by stimulating of VEGF release.     

Decreased differentiation of erythroid cells exacerbates ineffective erythropoiesis in beta-thalassemia

In β-thalassemia, the mechanism driving ineffective erythropoiesis (IE) is insufficiently understood. We analyzed mice affected by β-thalassemia and observed, unexpectedly, a relatively small increase in apoptosis of their erythroid cells compared with healthy mice. Therefore, we sought to determine whether IE could also be characterized by limited erythroid cell differentiation. In thalassemic mice, we observed that a greater than normal percentage of erythroid cells was in S-phase, exhibiting an erythroblast-like morphology. Thalassemic cells were associated with expression of cell cycle–promoting genes such as EpoR, Jak2, Cyclin-A, Cdk2, and Ki-67 and the antiapoptotic protein Bcl-X_L. The cells also differentiated less than normal erythroid ones in vitro. To investigate whether Jak2 could be responsible for the limited cell differentiation, we administered a Jak2 inhibitor, TG101209, to healthy and thalassemic mice. Exposure to TG101209 dramatically decreased the spleen size but also affected anemia. Although our data do not exclude a role for apoptosis in IE, we propose that expansion of the erythroid pool followed by limited cell differentiation exacerbates IE in thalassemia. In addition, these results suggest that use of Jak2 inhibitors has the potential to profoundly change the management of this disorder.     

Cyp7b, a novel brain cytochrome P450, catalyzes the synthesis of neurosteroids 7α-hydroxy dehydroepiandrosterone and 7α-hydroxy pregnenolone

Steroids produced locally in brain (neurosteroids), including dehydroepiandrosterone (DHEA), influence cognition and behavior. We previously described a novel cytochrome P450, Cyp7b, strongly expressed in rat and mouse brain, particularly in hippocampus. Cyp7b is most similar to steroidogenic P450s and potentially could play a role in neurosteroid metabolism. To examine the catalytic activity of the enzyme mouse Cyp7b cDNA was introduced into a vaccinia virus vector. Extracts from cells infected with the recombinant showed NADPH-dependent conversion of DHEA (K_m, 13.6 μM) and pregnenolone (K_m, 4.0 μM) to slower migrating forms on thin layer chromatography. The expressed enzyme was less active against 25-hydroxycholesterol, 17β-estradiol and 5α-androstane-3β,17β-diol, with low to undetectable activity against progesterone, corticosterone, and testosterone. On gas chromatography and mass spectrometry of the Cyp7b metabolite of DHEA the retention time and fragmentation patterns were identical to those obtained with authentic 7α-hydroxy DHEA. The reaction product also comigrated on thin layer chromatography with 7α-hydroxy DHEA but not with 7β-hydroxy DHEA; when [7α-³H]pregnenolone was incubated with Cyp7b extracts the extent of release of radioactivity into the medium suggested that hydroxylation was preferentially at the 7α position. Brain extracts also efficiently liberated tritium from [7α-³H]pregnenolone and converted DHEA to a product with a chromatographic mobility indistinguishable from 7α-hydroxy DHEA. We conclude that Cyp7b is a 7α-hydroxylase participating in the synthesis, in brain, of neurosteroids 7α-hydroxy DHEA, and 7α-hydroxy pregnenolone.     

The impact of video-assisted thoracic surgery (VATS) in patients with suspected advanced ovarian malignancies and pleural effusions

OBJECTIVES: We previously reported our initial experience of patients with suspected advanced ovarian cancer and moderate to large pleural effusions who underwent video-assessed thoracic surgery (VATS) before planned abdominal exploration. The objective of this study was to report the surgical findings and management of patients who underwent VATS in an update of our experience. METHODS: We performed a retrospective review of all patients with suspected advanced ovarian cancer and moderate to large pleural effusions who underwent VATS for assessment of extent of intrathoracic disease at our institution between 6/01 and 8/05. RESULTS: Twenty-three patients with a median age of 61 years (range, 36-79) were identified. VATS was performed for right-sided effusions in 17 patients (74%), and a median of 1350 ml (400-3700 ml) of pleural fluid was drained. VATS demonstrated macroscopic disease in 15 (65%) patients, with nodules >1 cm in 11/15 (73%), and nodules <1 cm in 4/15 (27%). Macroscopic intrathoracic disease was found in 4/10 (40%) patients with negative cytology. Intrathoracic cytoreduction was performed in 3/11 patients (27%) with intrathoracic disease >1 cm. After VATS, 12/23 patients (52%) underwent primary surgical management, with cytoreduction to < or =1 cm achieved in 11/12 patients (92%). The other eleven patients received primary chemotherapy after undergoing diagnostic laparoscopy alone (4/11) or no further abdominal exploration (7/11). Nine of these patients proceeded to interval cytoreduction, while 2 had pathology demonstrating upper gastrointestinal and lymphoma primaries at the time of VATS. Final diagnosis of primary site of disease included: ovary, 14 (61%); endometrial, 2 (9%); dual ovarian/endometrial primaries, 1 (4%); fallopian tube, 1 (4%); primary peritoneal, 1 (4%); other, 4 (17%). Overall, findings at VATS altered primary surgical management in 11/23 (48%) patients. CONCLUSIONS: Sixty-five percent of patients with suspected advanced ovarian cancer and moderate to large pleural effusions had gross intrathoracic disease identified at VATS, with the majority (11/15, 73%) having disease >1 cm in diameter. Use of VATS allows for assessment of intrathoracic disease and may help identify candidates for primary cytoreductive surgery and possible intrathoracic cytoreduction versus neoadjuvant chemotherapy.     

Production of agglutinators and rheumatoid factors in plasma cells of rheumatoid and nonrheumatoid synovial tissues

Immunohistochemical studies were performed in synovial tissues from 40 patients with rheumatoid arthritis (RA), 9 with juvenile rheumatoid arthritis (JRA), 7 with psoriatic arthritis, and 4 with various rheumatic diseases. Overall synthesis of IgG– and/or IgM–rheumatoid factor (RF) was found in all patients with seropositive RA and JRA, in 75% of patients with seronegative RA, and in 1 patient with psoriatic arthritis. Agglutinator producing cells were found in 77% of the samples from seropositive RA and in 44% and 56% from seronegative RA and JRA patients, respectively. The percentage of IgG plasma cells synthesizing one or more of the 5 types of agglutinators studied was approximately 10% of plasma cells synthesizing IgG–RF. Intercellular and intracellular immune complex deposits were also found in patients with seropositive and seronegative RA and JRA. These findings suggest that synthesis of agglutinators by synovial tissue plasma cells of RA and JRA patients is a distinct—but definitely less prominent—function than that of RF synthesis.     

Influence of interleukin 10 and transforming growth factor-beta on T cell stimulation through allogeneic retinal pigment epithelium cells in vitro

The effect of interleukin 10 (IL-10) and transforming growth factor-beta (TGF-beta) on the immunological capability of human retinal pigment epithelium (RPE) cells was tested by measuring the proliferation of allogeneic T cells after stimulation through the RPE cells in vitro. For this, human RPE cells were activated with different concentrations of interferon-gamma (0, 50, 100 and 250 U/ml) used as stimulator cells. The following T cell proliferation was measured with a tetrazolium-based colorimetric assay over a period of up to 4 days. Our results show that RPE cells are able to stimulate allogeneic T cells in the presence or absence of professional antigen-presenting cells (macrophages). Additionally, the proliferation could be significantly decreased by IL-10 or TGF-beta. The maximum of suppression, especially by TGF-beta, was found after 48 and 72 h of incubation. This shows that the antigen-presenting properties of human RPE cells can be altered in culture.