Expression of a secreted fibroblast growth factor binding protein-1 (FGFBP1) in angioproliferative Kaposi sarcoma

Objective: Kaposi’s sarcoma (KS) is an angioproliferative disease frequently seen in patients with the acquired immunodeficiency syndrome (AIDS). Previous studies suggest that the HIV-1 protein Tat and Fibroblast Growth Factor 2 (FGF-2) have synergistic angiogenic effects in AIDS-KS tumors. However, the mechanisms by which FGF-2 is released and activated in KS tumors are not clearly defined. We carried out this study to determine whether an FGFbinding protein (FGFBP1 or BP1) that enhances the angiogenic activity of FGF-2 is expressed in AIDS-KS tumors, and to define whether BP1, FGF-2, and HIV-Tat protein-protein interactions could play a potential clinically role in the pathogenesis of AIDS-KS. Methods: BP1 was localized in AIDS-KS lesions by immunohistochemistry and in situ hybridization studies. The binding of radiolabeled FGF-2 to His-tagged BP1 or the FGF-receptor 1 was assessed in the presence and absence of HIV-Tat and other viral proteins. Mice carrying tetracycline-regulated BP1 transgene mice were used to determine whether activation of BP1 during wound healing induces KS-like lesions. Results: BP1 expression was detected in AIDS-KS tumor keratinocytes, spindle cells, and infiltrating mononuclear cells. In addition, HIV-Tat competed for the binding of FGF-2 to immobilized BP1, but does not affect the interactions of FGF-2 with its high affinity receptor (FGFR-1). In contrast, two other HIV-proteins, Nef and gp120, did not affect the binding of FGF-2 to BP1 or to FGFR-1. Finally, up-regulation of BP1 expression in tetracycline-regulated –conditional BP1 transgenic mice subjected to skin wounds, induced KS-like skin lesions. Conclusion: Taking into consideration the results of previous studies showing that both HIV-Tat and BP1 enhance the mitogenic and angiogenic activity of locally-stored FGF-2, both in vitro and in vivo, our findings suggest a novel mechanism by which the release and activity of FGFs can be modulated in AIDS-KS tumors by HIV-Tat as well as BP1

Sequence-dependent Administration of Raloxifene and 5-Fluorouracil/Pemetrexed Protects against Pemetrexed Cytotoxicity in Human Bone Marrow

BACKGROUND: Pemetrexed (Alimta) is a new-generation multitargeted antifolate that inhibits several key enzymes in the de novo pathways of pyrimidine and purine biosynthesis, including thymidylate synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). Alimta has demonstrated antitumor activity in a broad array of human malignancies, e.g. breast, non-small cell lung cancer, malignant pleural mesothelioma and pancreatic, colorectal, gastric, bladder, head and neck cancer, and is currently in phase III clinical trials. It has been reported that a dose of 600 mg/m2 of pemetrexed showed toxicity to bone marrow and the gastrointestinal system. The aim of this investigation was to evaluate raloxifene (RAL) in combination with 5-fluorouracil (5-FU)/pemetrered multitargeted antifolate (MTA) to determine the most effective regimens and cellular mechanism of action to mitigate pemetrexed cytotoxicity in human bone marrow cells. MATERIALS AND METHODS: In order to determine the sequence-dependent interaction between MTA, 5-FU and RAL on proliferation, cell viability was carried out using the Quick Cell Proliferation Assay by exposing the HS-5 and MCF-7 cells to (i) MTA, 5-FU and RAL alone, or (ii) RAL 24 h prior to 5-FU followed 2 h later by MTA, or (iii) 5-FU 2 h prior to MTA followed 24 h later by RAL. RESULTS: The growth rate in MCF-7 in early RAL was 69 +/- 8.65% and late RAL was 36 +/- 4.6% of the control whereas in bone marrow early RAL was 78 +/- 8.65% and late RAL was 52 +/- 5.49% of the control. The late RAL exhibits significant protection against MTA cytotoxicity in bone marrow. The findings were further supported by cell flow cytometry, apoptosis and Western blot analysis data. CONCLUSION: This study suggests that sequence-dependent administration of RAL (5FU/MTA/RAL), in combination with 5-FU/MTA, protects against MTA toxicity in human bone marrow while maintaining the maximum inhibitory effect of pemetrexed in breast cancer

Raloxifene and Selective Cell Cycle Specific Agents: A Case for the Inclusion of Raloxifene in Current Breast Cancer Treatment Therapies

BACKGROUND: Breast cancer patients are at increased risk of osteoporosis. Contributing factors include age and/or chemotherapy. The selective estrogen modulator, raloxifene (RAL), effective in the prevention of breast cancer and approved for the treatment and prevention of osteoporosis, may prove beneficial in current breast cancer treatment modules. The purpose of this study was to evaluate RAL in combination with 5-fluorouracil (5-FU) and trimetrexate (TMX) to determine the most effective sequence in which to administer these cell cycle specific agents while taking into consideration the cellular mechanism of action. The goal was to maintain cytotoxicity to breast cancer cells and capitalize on the selective estrogen receptor modulatory effects of RAL. MATERIALS AND METHODS: MCF-7 cells were exposed to (i) TMX, 5-FU or RAL alone, or (ii) RAL 24 h prior to 5-FU followed 2 h later by TMX, or (iii) 5-FU 2 h prior to TMX followed 24 h later by RAL. The cell viability was determined using the Quick Cell Proliferation Assay. RESULTS: The growth rate of MCF- 7 cells exposed to early RAL was 68.25 +/- 4.11% that of the control, however, late RAL exposure produced a growth of 34.75 +/- 4.79% that of the control. Late RAL maintained the cytotoxicity of the regimen. The findings were further supported by cell flow cytometry and Western blot analysis data. CONCLUSION: RAL given prior to 5-FU/TMX significantly compromised cytotoxicity to breast cancer cells

Anaphylaxis caused by the ingestion of cultivated mushroom (Agaricus bisporus): Identification of allergen as mannitol

ABSTRACTBackgroundThe role of mushroom spores as inhalants in causing respiratory allergy has been well established. Although mushrooms are commonly used as food throughout the world, food allergy to mushrooms is not very common. A severe case of anaphylaxis in a 32-year-old woman who experienced facial edema and generalized urticaria minutes after eating mushroom curry is presented herein. The purpose of the present study was to identify the putative allergen in the cultivated mushroom Agaricus bisporus.MethodsA combination of biochemical fractionation/ analytical techniques (gel filtration, ultrafiltration, ion-moderated cation-exchange chromatography, high-pressure liquid chromatography and gas chromatography–mass spectrometry (GC-MS)) and allergy diagnostic tests (skin prick test (SPT), allergen-specific IgE) were used.ResultsThe SPT with mushroom extract was strongly positive; however, allergen-specific IgE could not be detected by enzyme-linked immunosorbent assay. The SPT was also positive with cooked, steamed or dried mushroom extracts, suggesting the presence of a heat-stable allergen. Gel filtration of mushroom extract on Sephadex G-25, as analyzed by SPT, indicated the presence of a low molecular weight (<1 kDa) allergen. Using ion-moderated cation-exchange chromatography, the allergen was isolated and identified as mannitol based on skin reactivity. Mannitol was confirmed by GC-MS analysis.ConclusionsThis is the first report of food allergy to cultivated mushroom A. bisporus and also the first report describing a low molecular weight allergen (mannitol) in mushroom

Childhood HIV-associated nephropathy: 36 years later.

HIV-associated nephropathy (HIVAN) predominantly affects people of African ancestry living with HIV who do not receive appropriate antiretroviral therapy (ART). Childhood HIVAN is characterized by heavy proteinuria and decreased kidney function. Kidney histology shows mesangial expansion, classic or collapsing glomerulosclerosis, and microcystic renal tubular dilatation leading to kidney enlargement. The pathogenesis of HIVAN involves the kidney recruitment of inflammatory cells and the infection of kidney epithelial cells. In addition, both viral and genetic factors play key roles in this disease. Modern ART has improved the outcome and decreased the prevalence of childhood HIVAN. However, physicians have had modest success providing chronic ART to children and adolescents, and we continue to see children with HIVAN all over the world. This article discusses the progress made during the last decade in our understanding of the pathogenesis and treatment of childhood HIVAN, placing particular emphasis on the mechanisms that mediate the infection of kidney epithelial cells, and the roles of cytokines, the HIV-Tat gene, and the Apolipoprotein-1 (APOL1) gene risk variants in this disease. In view of the large number of children living with HIV at risk of developing HIVAN, better prevention and treatment programs are needed to eradicate this disease

Angiopoietin-1 Prevents Severe Bleeding Complications Induced by Heparin-like drugs and Fibroblast Growth Factor-2 in Mice.

Critically ill children can develop bleeding complications when treated with heparin-like drugs. These events are usually attributed to the anticoagulant activity of these drugs. However, previous studies showed that fibroblast growth factor-2 (FGF-2), a heparin-binding growth factor released in the circulation of these patients, could precipitate intestinal hemorrhages in mice treated with the heparin-like drug pentosan polysulfate (PPS). Yet very little is known about how FGF-2 induces bleeding complications in combination with heparin-like drugs. Here, we examined the mechanisms by which circulating FGF-2 induces intestinal hemorrhages in mice treated with PPS. We used a well-characterized mouse model of intestinal hemorrhages induced by FGF-2 plus PPS. Adult FVB/N mice were infected with adenovirus carrying Lac-Z or a secreted form of recombinant human FGF-2, and injected with PPS, at doses that do not induce bleeding complications per se. Mice treated with FGF-2 in combination with PPS developed an intestinal inflammatory reaction that increased the permeability and disrupted the integrity of submucosal intestinal vessels. These changes, together with the anticoagulant activity of PPS, induced lethal hemorrhages. Moreover, a genetically modified form of the endothelial ligand angiopoietin-1 (Ang-1*), which has powerful antipermeability and anti-inflammatory activity, prevented the lethal bleeding complications without correcting the anticoagulant status of these mice. These findings define new mechanisms through which FGF-2 and Ang-1* modulate the outcome of intestinal bleeding complications induced by PPS in mice and may have wider clinical implications for critically ill children treated with heparin-like drugs

Urine samples harvested from HIV-infected children with renal diseases increase the permeability of cultured HGEc-1 through RhoA and Src mediated mechanisms.

<p><b>(A)</b> Urine samples harvested from HIV infected children with and without renal diseases (RD) were used (1:10 dilution) to stimulate monolayers of 5 hours-starved HGEc-1. The RhoA inhibitor: C3 transferase (20 ng/ml), was added 4 hrs before stimulation. The inhibitor of Src family kinase SU6656 (1 μM) and the ROCK inhibitor Y-27632 (10 μM) were added 1hr before stimulation. The data represent FITC-dextran permeability expresses as fold increase. <b>(B)</b> Overnight-starved HGEc-1 monolayer were treated with urine (1:20 dilution) for 5 min as described above and then harvested to assess the phosphorylation of RhoA, Rac1, MLC, and Src, as described in Methods. <b>(C)</b> The graphs show mean ± SEM values corresponding to three different Western blots that assessed the phosphorylation of Rho-A, Rac1, MLC, and Src, in cultured HGEc-1. Results were expressed in optical density units expressed as a ratio of the total activity. Values significantly different from the control group (HIV-N; n = 5) were marked with <i>asterisks</i> **p<0.01, and those significantly different from the HIV-RD group (n = 5) were marked with stars ★ p<0.05 and ★★ p<0.01 (for permeability), or crosses, + p <0.05 and ++ p<0.01 (for signaling).</p

Urine samples harvested from HIV-infected children with renal diseases increase the permeability of cultured podocytes cell line (P-2) through Rho-A and Src mediated mechanisms.

<p><b>(A)</b> Urine samples harvested from HIV infected children with and without renal diseases (RD) were used (1:10 dilution) to stimulate monolayers of 5 hours-starved P-2 cells. The data represent FITC-dextran permeability changes expressed as fold increase. <b>(B)</b> Overnight-starved P-2 monolayers were treated with urine (1:20 dilution) for 5 min as described above and then harvested to assess the phosphorylation of Rho-A, MLC, and Src, as described in Methods. <b>(C)</b> The graphs show mean ± SEM values corresponding to three different Western blots that assessed the phosphorylation of Rho-A, MLC, and Src in cultured podocytes. Results were expressed in optical density units expressed as a ratio of the total activity. Values significantly different from the serum free treated control cells (Control) were marked with <i>asterisk</i>, *p<0.05 and **p<0.01, and differences between HIV-RD (n = 5) and HIV-N (n = 5) urine samples, were marked with stars ★ p< 0.05 (for permeability) or crosses + p<0.05 (for signaling).</p

Rho-A activation increases the permeability of HGEc-1.

<p><b>(A)</b> Cultured HGEc-1 cells were transfected with different plasmids, pCEFL-mock, constitutively active RhoAQL (pCEFL-AU5-RhoAQL), or dominant negative mutant RhoAN19 (pCEFL-AU5-RhoAN19). Twenty-four hours later, the cells were treated with VEGF-A (50 ng/ml) + Tat (100 ng/ml) + Heparin (50 units/ml), all together, and exposed to FITC-dextran as described in methods. <b>(B)</b> In other experiments HGEc-1 cells were treated for 5 min as described above and then harvested to assess the phosphorylation of Rho-A, MLC, Src as described in Methods. Panel B shows representative western blots corresponding to the phosphorylation changes. <b>(C)</b> The graphs show mean ± SEM values corresponding to three different Western blots that assessed the phosphorylation of Rho-A, MLC and Src in cultured HGEc-1. Results were expressed in arbitrary optical density units as a ratio of the total activity. In each group, mock, RhoAQL and RhoAN19 transfected cells were treated with either serum free media (Controls) (-), or VEGF-A + Tat + Heparin (+). Groups that were significantly different from controls (-) were labeled with <i>asterisk</i>, *p<0.05 and **p<0.01. Cells transfected with constitutively active RhoAQL that were significantly different from mock or RhoAN19 cells, were labeled with crosses: + p <0.05 and ++ p<0.01.</p