[Rose-scented geranium essential oil from Algeria (Pelargonium graveolens L'Hérit.): Assessment of antioxidant, anti-inflammatory and anticancer properties against different metastatic cancer cell lines]. / Essence aromatique du Géranium Odorant (Pelargonium graveolens L'Hérit.) d'Algérie : exploration des propriétés antioxydante, anti-inflammatoire et anticancéreuse (anti-angiogénique et cytotoxique), in vitro et in ovo, vis-à-vis de différentes lignées cellulaires cancéreuses métastasiques.

INTRODUCTION: The study of natural products is one of the strategies implemented for the discovery of new compounds that can be used in cancer therapy. Aromatic herbs and medicinal plants found in Algeria and their anti-angiogenesis and cytotoxic potentials against cancer have not been much explored. OBJECTIVES: Our work aimed to evaluate the antioxidant, anti-inflammatory and anticancer properties of the essential oil (EO) extracted from rose-scented geranium (Pelargonium graveolens) and its major (citronellol) and characteristic (linalool) constituents. RESULTS: The chemical composition of EO was determined with chromatographic analysis and revealed the presence of citronellol as the major compound (25.84%). A strong chelating power of terpene alcohols (IC50=1.58±0.23mg/mL for citronellol) was found, with a significant difference (P<0.05) compared with the standard antioxidants used (L-ascorbic acid and butylated hydroxyanisole). The EO is distinguished by an interesting anti-inflammatory effect with the lowest IC50 (4.63±0.3mg/mL), and it constitutes a good stabilizer of the erythrocyte membrane. Citronellol also exhibited the best anti-inflammatory effect (IC50=0.74±0.09mg/mL). We also assessed the anticancer effect of EO on two main pathways involved in cancer development, angiogenesis and cell proliferation, using in ovo bioassays with a chorio-allantoic membrane (CAM) of chicken eggs and in vitro assays of its cytotoxicity on different metastatic breast cancer (MDA-MB-231), gastric (AGS) and melanoma (MV3) cell lines. In the CAM model, the density of micro-vessels is 75±10 in the group supplemented with EO compared to 140±9 for the control group (b-FGF). In addition, the EO significantly reduced the number of newly formed vessels. The cytotoxicity was evaluated using the cell proliferation inhibition method and cell viability was measured using the MTT test. Results revealed that the treatment of cancer lines with different concentrations of EO reduces the rate of cell viability in a dose-dependent manner. EO showed the greatest cytotoxicity on the AGS line with an inhibition rate of 92.87±0.13% at the highest dose (4µL/mL), followed by the MV3 line (88.76±0.96%). CONCLUSION AND PROSPECTS: Data demonstrated that rose-scented geranium EO has an antitumor potential on metastatic cancer cell lines. It is distinguished by its antiproliferative, anti-angiogenic, and anti-inflammatory activities. Medicinal plants might contain new molecules, with new structures, which could become lead candidate among future anticancer drugs.

Lycopene nanoparticles promotes osteoblastogenesis and inhibits adipogenesis of rat bone marrow mesenchymal stem cells.

OBJECTIVE: Lycopene is a carotenoid and antioxidant with potent singlet oxygen quenching ability that reduces oxidative stress and promotes bone health. However, the cellular mechanisms by which lycopene influences bone metabolism are not known. MATERIALS AND METHODS: The present study investigated the effects of lycopene nanoparticles on the differentiation of rat bone marrow-derived mesenchymal stem cells into osteoblasts or adipocytes. RESULTS: In osteogenic medium, lycopene supplementation dose-dependently enhanced osteoblast differentiation, as evidenced by the transcription of Alpl, Runx2, Col1a1, Sp7, and Bglap, higher alkaline phosphatase activity, osteocalcin secretion and extracellular matrix mineralisation seen with Alizarin red S staining, and increased haem oxygenase levels. By contrast, lycopene in adipogenic medium inhibited adipocyte differentiation evidenced by decreases in the transcription of Tnfsf11, Tnfrsf11b, Pparg, Lpl, and Fabp4 and reduced fat accumulation observed by Oil Red O staining. CONCLUSIONS: Lycopene nanoparticles may promote bone health and are considered as a potential candidate for the prevention and/or treatment of bone loss conditions.

Avoiding COVID-19 complications with diabetic patients could be achieved by multi-dose Bacillus Calmette-Guérin vaccine: a case study of beta cells regeneration.

Diabetes mellitus (DM) is one of the major risk factors for COVID-19 complications as it is one of the chronic immune-compromising conditions especially if patients have uncontrolled diabetes, poor HbA1c and/or irregular blood glucose levels. Diabetic patients' mortality rates with COVID-19 are higher than those of cardiovascular or cancer patients. Recently, Bacillus Calmette-Guérin (BCG) vaccine has shown successful results in reversing diabetes in both rats and clinical trials based on different mechanisms from aerobic glycolysis to beta cells regeneration. BCG is a multi-face vaccine that has been used extensively in protection from tuberculosis (TB) and leprosy and has been repositioned for treatment of bladder cancer, diabetes and multiple sclerosis. Recently, COVID-19 epidemiological studies confirmed that universal BCG vaccination reduced morbidity and mortality in certain geographical areas. Countries without universal policies of BCG vaccination (Italy, Nederland, USA) have been more severely affected compared to countries with universal and long-standing BCG policies that have shown low numbers of reported COVID-19 cases. Some countries have started clinical trials that included a single dose BCG vaccine as prophylaxis from COVID-19 or an attempt to minimize its side effects. This proposed research aims to use BCG vaccine as a double-edged weapon countering both COVID-19 and diabetes, not only as protection but also as therapeutic vaccination. The work includes a case study of regenerated pancreatic beta cells based on improved C-peptide and PCPRI laboratory findings after BCG vaccination for a 9 year old patient. The patient was re-vaccinated based on a negative tuberculin test and no scar at the site of injection of the 1st BCG vaccination at birth. The authors suggest and invite the scientific community to take into consideration the concept of direct BCG re-vaccination (after 4 weeks) because of the reported gene expressions and exaggerated innate immunity consequently. As the diabetic MODY-5 patient (mutation of HNF1B, Val2Leu) was on low dose Riomet® while eliminating insulin gradually, a simple analytical method for metformin assay was recommended to ensure its concentration before use as it is not approved yet by the Egyptian QC labs.

Emerging therapies for Parkinson's disease: from bench to bedside.

The prevalence of Parkinson's disease (PD) increases with age and is projected to increase in parallel to the rising average age of the population. The disease can have significant health-related, social, and financial implications not only for the patient and the caregiver, but for the health care system as well. While the neuropathology of this neurodegenerative disorder is fairly well understood, its etiology remains a mystery, making it difficult to target therapy. The currently available drugs for treatment provide only symptomatic relief and do not control or prevent disease progression, and as a result patient compliance and satisfaction are low. Several emerging pharmacotherapies for PD are in different stages of clinical development. These therapies include adenosine A2A receptor antagonists, glutamate receptor antagonists, monoamine oxidase inhibitors, anti-apoptotic agents, and antioxidants such as coenzyme Q10, N-acetyl cysteine, and edaravone. Other emerging non-pharmacotherapies include viral vector gene therapy, microRNAs, transglutaminases, RTP801, stem cells and glial derived neurotrophic factor (GDNF). In addition, surgical procedures including deep brain stimulation, pallidotomy, thalamotomy and gamma knife surgery have emerged as alternative interventions for advanced PD patients who have completely utilized standard treatments and still suffer from persistent motor fluctuations. While several of these therapies hold much promise in delaying the onset of the disease and slowing its progression, more pharmacotherapies and surgical interventions need to be investigated in different stages of PD. It is hoped that these emerging therapies and surgical procedures will strengthen our clinical armamentarium for improved treatment of PD.

International clinical practice guidelines for the treatment and prophylaxis of venous thromboembolism in patients with cancer.

BACKGROUND: Guidelines addressing the management of venous thromboembolism (VTE) in cancer patients are heterogeneous and their implementation has been suboptimal worldwide. OBJECTIVES: To establish a common international consensus addressing practical, clinically relevant questions in this setting. METHODS: An international consensus working group of experts was set up to develop guidelines according to an evidence-based medicine approach, using the GRADE system. RESULTS: For the initial treatment of established VTE: low-molecular-weight heparin (LMWH) is recommended [1B]; fondaparinux and unfractionated heparin (UFH) can be also used [2D]; thrombolysis may only be considered on a case-by-case basis [Best clinical practice (Guidance)]; vena cava filters (VCF) may be considered if contraindication to anticoagulation or pulmonary embolism recurrence under optimal anticoagulation; periodic reassessment of contraindications to anticoagulation is recommended and anticoagulation should be resumed when safe; VCF are not recommended for primary VTE prophylaxis in cancer patients [Guidance]. For the early maintenance (10 days to 3 months) and long-term (beyond 3 months) treatment of established VTE, LMWH for a minimum of 3 months is preferred over vitamin K antagonists (VKA) [1A]; idraparinux is not recommended [2C]; after 3-6 months, LMWH or VKA continuation should be based on individual evaluation of the benefit-risk ratio, tolerability, patient preference and cancer activity [Guidance]. For the treatment of VTE recurrence in cancer patients under anticoagulation, three options can be considered: (i) switch from VKA to LMWH when treated with VKA; (ii) increase in LMWH dose when treated with LMWH, and (iii) VCF insertion [Guidance]. For the prophylaxis of postoperative VTE in surgical cancer patients, use of LMWH o.d. or low dose of UFH t.i.d. is recommended; pharmacological prophylaxis should be started 12-2 h preoperatively and continued for at least 7-10 days; there are no data allowing conclusion that one type of LMWH is superior to another [1A]; there is no evidence to support fondaparinux as an alternative to LMWH [2C]; use of the highest prophylactic dose of LMWH is recommended [1A]; extended prophylaxis (4 weeks) after major laparotomy may be indicated in cancer patients with a high risk of VTE and low risk of bleeding [2B]; the use of LMWH for VTE prevention in cancer patients undergoing laparoscopic surgery may be recommended as for laparotomy [Guidance]; mechanical methods are not recommended as monotherapy except when pharmacological methods are contraindicated [2C]. For the prophylaxis of VTE in hospitalized medical patients with cancer and reduced mobility, we recommend prophylaxis with LMWH, UFH or fondaparinux [1B]; for children and adults with acute lymphocytic leukemia treated with l-asparaginase, depending on local policy and patient characteristics, prophylaxis may be considered in some patients [Guidance]; in patients receiving chemotherapy, prophylaxis is not recommended routinely [1B]; primary pharmacological prophylaxis of VTE may be indicated in patients with locally advanced or metastatic pancreatic [1B] or lung [2B] cancer treated with chemotherapy and having a low risk of bleeding; in patients treated with thalidomide or lenalidomide combined with steroids and/or chemotherapy, VTE prophylaxis is recommended; in this setting, VKA at low or therapeutic doses, LMWH at prophylactic doses and low-dose aspirin have shown similar effects; however, the efficacy of these regimens remains unclear [2C]. Special situations include brain tumors, severe renal failure (CrCl<30 mL min(-1) ), thrombocytopenia and pregnancy. Guidances are provided in these contexts. CONCLUSIONS: Dissemination and implementation of good clinical practice for the management of VTE, the second cause of death in cancer patients, is a major public health priority.

International clinical practice guidelines for the treatment and prophylaxis of thrombosis associated with central venous catheters in patients with cancer.

BACKGROUND: Although long-term indwelling central venous catheters (CVCs) may lead to pulmonary embolism (PE) and loss of the CVC, there is lack of consensus on management of CVC-related thrombosis (CRT) in cancer patients and heterogeneity in clinical practices worldwide. OBJECTIVES: To establish common international Good Clinical Practices Guidelines (GCPG) for the management of CRT in cancer patients. METHODS: An international working group of experts was set up to develop GCPG according to an evidence-based medicine approach, using the GRADE system. RESULTS: For the treatment of established CRT in cancer patients, we found no prospective randomized studies, two non-randomized prospective studies and one retrospective study examining the efficacy and safety of low-molecular-weight heparin (LMWH) plus vitamin K antagonists (VKAs). One retrospective study evaluated the benefit of CVC removal and two small retrospective studies were on thrombolytic drugs. For the treatment of symptomatic CRT, anticoagulant treatment (AC) is recommended for a minimum of 3 months; in this setting, LMWHs are suggested. VKAs can also be used, in the absence of direct comparisons of these two types of anticoagulants in this setting [Guidance]. The CVC can be kept in place if it is functional, well-positioned and non-infected and there is good resolution under close surveillance; whether the CVC is kept or removed, no standard approach in terms of AC duration has been established [Guidance]. For the prophylaxis of CRT in cancer patients, we found six randomized studies investigating the efficacy and safety of VKA vs. placebo or no treatment, one on the efficacy and safety of unfractionnated heparin, six on the value of LMWH, one double-blind randomized and one non randomized study on thrombolytic drugs and six meta-analyses of AC and CVC thromboprophylaxis. Type of catheter (open-ended like the Hickman(®) catheter vs. closed-ended catheter with a valve like the Groshong(®) catheter), its position (above, below or at the junction of the superior vena cava and the right atrium) and method of placement may influence the onset of CRT on the basis of six retrospective trials, four prospective non-randomized trials, three randomized trials and one meta-analysis. In light of these data: use of AC for routine prophylaxis of CRT is not recommended [1A]; a CVC should be inserted on the right side, in the jugular vein, and distal extremity of the CVC should be located at the junction of the superior vena cava and the right atrium [1A]. CONCLUSION: Dissemination and implementation of these international GCPG for the prevention and treatment of CRT in cancer patients at each national level is a major public health priority, needing worldwide collaboration.

Pro- and anti-angiogenic agents.

The vascular endothelium has been characterized in every organ system, and is described as a selective permeable barrier and as a dynamic and disseminated organ with endocrine function. These activities have been shown to result from the interactions of ligands with membrane-bound receptors as well as through specific junctional proteins and receptors that govern cell-cell interactions. The endothelial cells' movement (e.g., angiogenesis) has been hypothesized to occur following the release of stimuli that could promote the formation of new blood vessels. Angiogenesis has also been reported to be the continued expansion of the vascular tree in avascular regions, as a result of the sprouting of endothelial cells from existing vessels. Most commonly, angiogenesis has been characterized during wound healing and tumour growth. Herein we summarize and discuss the latest results from fundamental laboratory research aimed at proving a link between the proliferation of cancer and angiogenesis, as well as the new rationale around novel pro- and anti-angiogenic molecules.

Biomarkers of inflammation, growth factor, and coagulation activation in patients with sickle cell disease.

Acute painful crisis is a common sequela that can cause significant morbidity and negatively impact the quality of life of patients with sickle cell disease (SCD). Plasma levels of several chemokines and cytokines including tumor necrosis factor-α (TNF-α), interleukin 1ß (IL-1ß), IL-6, IL-8, monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1α (MIP-1α), and interferon γ (IFN-γ) in patients with SCD showed a distinct and statistically significant rise either during painful crisis or at steady state. Plasma levels of various growth factors, including human vascular endothelial growth factor (VEGF), human basic fibroblast growth factor (FGF), and human hepatocyte growth factor (HGF), showed a sustained 2- to 3-fold increase either during painful crisis or at steady state in patients with SCD. Furthermore, plasma levels of the biomarker d-Dimer, a marker of hypercoagulation, showed a 2- to 3-fold increase either during painful crisis or at steady state in patients with SCD as compared to that in healthy participants, suggesting an increased risk of thrombosis.

Greasing the wheels of managing overweight and obesity with omega-3 fatty acids.

The epidemic of overweight and obesity around the world and in the US is a major public health challenge, with 1.5 billion overweight and obese adults worldwide, and 68% of US adults and 31% of US children and adolescents overweight or obese. Obesity leads to serious health consequences, including an increased risk of type 2 diabetes mellitus and heart disease. Current preventive and medical treatments include lifestyle modification, medication, and bariatric surgery in extreme cases; however, they are either not very efficacious or are very expensive. Obesity is a complex condition involving the dysregulation of several organ systems and molecular pathways, including adipose tissue, the pancreas, the gastrointestinal tract, and the CNS. The role of the CNS in obesity is receiving more attention as obesity rates rise and treatments continue to fail. While the role of the hypothalamus in regulation of appetite and food intake has long been recognized, the roles of the CNS reward systems are beginning to be examined as the role of environmental influences on energy balance are explored. Omega-3 polyunsaturated fatty acids are essential nutrients that play a beneficial role in several disease processes due to their anti-inflammatory effects, modulation of lipids, and effects on the CNS. Omega-3 fatty acids, specifically EPA and DHA, have shown promising preliminary results in animal and human studies in the prevention and treatment of obesity. Given their effects on many of the pathways involved in obesity, and specifically in the endocannabinoid and mesocorticolimbic pathways, we hypothesize that EPA and DHA supplementation in populations can reduce the reward associated with food, thereby reduce appetite and food intake, and ultimately contribute to the prevention or reduction of obesity. If these fatty acids do harbor such potential, their supplementation in many parts of the world may hold great promise in reducing the global burden of obesity.

Tetraiodothyroacetic acid (tetrac) and nanoparticulate tetrac arrest growth of medullary carcinoma of the thyroid.

CONTEXT: Tetraiodothyroacetic acid (tetrac) blocks angiogenic and tumor cell proliferation actions of thyroid hormone initiated at the cell surface hormone receptor on integrin alphavbeta3. Tetrac also inhibits angiogenesis initiated by vascular endothelial growth factor and basic fibroblast growth factor. OBJECTIVE: We tested antiangiogenic and antiproliferative efficacy of tetrac and tetrac nanoparticles (tetrac NP) against human medullary thyroid carcinoma (h-MTC) implants in the chick chorioallantoic membrane (CAM) and h-MTC xenografts in the nude mouse. DESIGN: h-MTC cells were implanted in the CAM model (n = 8 per group); effects of tetrac and tetrac NP at 1 microg/CAM were determined on tumor angiogenesis and tumor growth after 8 d. h-MTC cells were also implanted sc in nude mice (n = 6 animals per group), and actions on established tumor growth of unmodified tetrac and tetrac NP ip were determined. RESULTS: In the CAM, tetrac and tetrac NP inhibited tumor growth and tumor-associated angiogenesis. In the nude mouse xenograft model, established 450-500 mm(3) h-MTC tumors were reduced in size over 21 d by both tetrac formulations to less than the initial cell mass (100 mm(3)). Tumor tissue hemoglobin content of xenografts decreased by 66% over the course of administration of each drug. RNA microarray and quantitative real-time PCR of tumor cell mRNAs revealed that both tetrac formulations significantly induced antiangiogenic thrombospondin 1 and apoptosis activator gene expression. CONCLUSIONS: Acting via a cell surface receptor, tetrac and tetrac NP inhibit growth of h-MTC cells and associated angiogenesis in CAM and mouse xenograft models.

Tetraidothyroacetic acid (tetrac) and tetrac nanoparticles inhibit growth of human renal cell carcinoma xenografts.

Renal cell carcinoma is the most lethal of the common urologic malignancies, with no available effective therapeutics. Tetrac (tetraiodothyroacetic acid) is a deaminated analogue of L-thyroxine (T(4)) that blocks the pro-angiogenesis actions of T(4) and 3, 5, 3'-triiodo-L-thyronine as well as other growth factors at the cell surface receptor for thyroid hormone on integrin alphavbeta3. Since this integrin is expressed on cancer cells and also on endothelial and vascular smooth cells, the possibility exists that Tetrac may act on both cell types to block the proliferative effects of thyroid hormone on tumor growth and tumor-related angiogenesis. To test this hypothesis, we determined the effect of Tetrac on tumor cell proliferation and on related angiogenesis of human renal cell carcinoma (RCC). We used two models: tumor cell implants in the chick chorioallantoic membrane (CAM) system and xenografts in nude mice. To determine the relative contribution of the nuclear versus the plasma membrane action of Tetrac, we compared the effects of unmodified Tetrac to Tetrac covalently linked to poly (lactide-co-glycolide) as a nanoparticle (Tetrac NP) that acts exclusively at the cell surface through the integrin receptor. In the CAM model, Tetrac and Tetrac NP (both at 1 microg/CAM) arrested tumor-related angiogenesis and tumor growth. In the mouse xenograft model, Tetrac and Tetrac NP promptly reduced tumor volume (p<0.01) when administered daily for up to 20 days. Animal weight gain was comparable in the control and treatment groups. Overall, the findings presented here provide evidence for the anti-angiogenic, and anti-tumor actions of Tetrac and Tetrac NP and suggest their potential utility in the treatment of renal cell carcinoma.

AlphaIIbbeta3 priming and clustering by orally active and intravenous integrin antagonists.

BACKGROUND: Drugs that block platelet-platelet and platelet-fibrin interactions via the alpha(IIb)beta(3) (glycoprotein IIb/IIIa) receptor are used daily in patients undergoing percutaneous coronary interventions. Along with expected increases in spontaneous bleeding, clinical trials have revealed a surprising increase in thrombosis when these drugs are used without other anticoagulants. A better understanding of their mechanisms can minimize these risks. OBJECTIVES: This study tested the hypothesis that interventions designed to block fibrinogen binding inevitably leave the alpha(IIb)beta(3) receptor in an activated state. It compared the effects on platelet function and alpha(IIb)beta(3) conformation of the orally active compounds orbofiban and roxifiban, the i.v. agents eptifibatide and tirofiban, and echistatin, an arginine-glycine-aspartate (RGD) disintegrin. METHODS: The integrin antagonist concentrations required to saturate platelets and to block platelet-platelet and platelet-fibrin interactions were determined by flow cytometry, aggregometry, and clot-based adhesion assays, respectively. Analytical ultracentrifugation measured each antagonist's effects on the solution structure of alpha(IIb)beta(3). Fluorescence anisotropy provided equilibrium and kinetic data for integrin:antagonist interactions. RESULTS: Both orally active drugs bound more tightly and inhibited platelet aggregation and adhesion to fibrin more effectively than echistatin. Analytical ultracentrifugation yielded this order for perturbing alpha(IIb)beta(3) conformation (priming) and promoting oligomerization (clustering): echistatin > eptifibatide > orbofiban > tirofiban > roxifiban. Roxifiban was also most effective at disrupting the rapidly forming/slowly dissociating alpha(IIb)beta(3):echistatin complex. CONCLUSIONS: Our results suggest that the same molecular mechanisms that enable glycoprotein IIb/IIIa inhibitors to bind tightly to the alpha(IIb)beta(3) receptor and block fibrinogen binding contribute to their ability to perturb the resting integrin's conformation, thus limiting the safety and efficacy of both oral and i.v. integrin antagonists.

Synthetic oligosaccharide stimulates and stabilizes angiogenesis: structure-function relationships and potential mechanisms.

To determine the proangiogenesis effect of series of saccharides and a synthetic oligosaccharide and potential mechanisms, an in vitro 3-dimensional endothelial cell sprouting (3D-ECS) assay and the chick chorioallantoic membrane (CAM) model were used. We demonstrated that a sulfated oligosaccharide significantly promotes the endothelial capillary network initiated by vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF). Furthermore, although the capillary network initiated by VEGF and b-FGF lasts no more than 7 days, addition of a sulfated oligosaccharide significantly amplifies angiogenesis and stabilizes the capillary network of new blood vessels. In the CAM model, sulfated oligosaccharide also stimulated angiogenesis. In both the CAM and the 3D-ECS assay, structure-function studies reveal that increased saccharide chain length up to the hexa- to decasaccharide show optimal proangiogenesis efficacy. In addition, the sulfation and molecular shape (branched vs linear) of oligosaccharide are important for sustained proangiogenesis efficacy. Data indicate that chemically defined synthetic oligosaccharides can play an important role in regulation of capillary structure and stability, which may contribute to future advances in therapeutic angiogenesis. The proangiogenesis efficacy of an oligosaccharide is mediated via integrin alphavbeta3 and involves mitogen-activated protein kinase signaling mechanisms.

Elevation of plasma von Willebrand factor and tumor necrosis factor-a in obese subjects and their reduction by the low molecular weight heparin tinzaparin.

AIM: Increased plasma-soluble von Willebrand factor (vWF) level, a marker of vascular endothelial cell dysfunction, is a predictor of atherosclerotic cardiovascular disease. We compared associations between vWF level and markers of inflammation as well as the effects of LMWH in obese as compared to healthy human subjects. METHODS: Plasma samples were obtained from healthy volunteers (n=32) and obese subjects (n=12) before and after administration of a single subcutaneous dose of tinzaparin, given at 75 IU/kg once a day, a deep vein thrombosis prophylaxis dose. Plasma samples were analyzed for vWF and tumor necrosis factor-alfa (TNF-alfa) using specific and sensitive ELISA. RESULTS: Obese subjects showed relatively higher plasma levels of TNF-alfa compared with normal-weight subjects. Regression analysis showed that plasma vWF levels to be directly associated with the presence of higher plasma levels of TNF-alfa in these obese subjects. Tinzaparin significantly reduced elevated plasma levels of both vWF and TNF-a levels (P<0.01). CONCLUSIONS: Plasma values of vWF and TNF-alfa are higher in obese than in normal-weight individuals. Treatment with tinzaparin lowers plasma levels of TNF-alfa in both obese and normal-weight subjects. The levels of vWF were higher in obese subjects than in normal-weight ones, which might be due to the higher levels of circulating TNF-alfa. Tinzaparin reduced vWF levels in these obese subjects.

Antithrombotics in thrombosis and cancer.

Many cancer patients have a hypercoagulable state, with recurrent thrombosis due to the impact of cancer cells and chemotherapy or radiotherapy on the coagulation cascade. Studies have demonstrated that unfractionated heparin (UFH) or its low molecular weight fractions interfere with various processes involved in tumour growth and metastasis. These include fibrin formation; binding of heparin to angiogenic growth factors, such as basic fibroblast growth factor (FGF2) and vascular endothelial growth factor (VEGF); modulation of tissue factor; and perhaps other more important modulatory mechanisms, such as enhanced tissue factor pathway inhibitor (TFPI) release and inhibition of various matrix-degrading enzymes. Clinical trials have suggested a clinically relevant effect of low molecular weight heparin (LMWH), as compared to UFH, on the survival of cancer patients with deep vein thrombosis. Similarly, the impact of warfarin on the survival of cancer patients with thromboembolic disorders was demonstrated. Studies from our laboratory demonstrated a significant role for LMWH, warfarin, anti-VIIa, and LMWH-releasable TFPI on the regulation of angiogenesis, tumour growth, and tumour metastasis. Thus, modulation of tissue factor/VIIa non-coagulant activities by LMWH, warfarin, anti-VIIa, or TFPI might be a useful therapeutic method for the inhibition of angiogenesis associated with human tumour growth and metastasis. Additionally, antiplatelet drugs could have an impact on tumour metastasis, and the combination of antiplatelets and anticoagulants at adjusted doses might provide greater benefits to cancer patients.

Increased numbers of opioid expressing inflammatory cells do not affect intra-articular morphine analgesia.

BACKGROUND: Both locally expressed beta-endorphin (END) and low doses of morphine relieve pain within inflamed knee joints. Here we examined whether enhanced inflammation and END expression within the synovial tissue of patients undergoing arthroscopic knee surgery might shift the analgesic dose-response curve of intra-articular (i.a.) morphine. METHODS: Following IRB approval and informed consent, patients were randomly assigned to the following i.a. treatments at the end of surgery: group I (n=39), isotonic saline; group II (n=40), 1 mg morphine hydrochloride; group III (n=48), 2 mg morphine hydrochloride; group IV (n=39), 4 mg morphine hydrochloride. Postoperative pain intensity was assessed by the visual analogue scale (VAS), by the time to first analgesic request and by the supplemental piritramide consumption. Synovial specimens from each patient were stained for the presence of inflammatory cells and END and were discriminated into groups with low versus high numbers of these cells. Differences between groups were statistically analyzed by chi(2), anova and mancova where appropiate. RESULTS: Patient characteristics and VAS scores did not differ between groups. Total postoperative piritramide consumption decreased and the time to first analgesic request increased significantly with increasing doses of i.a. morphine (P<0.05, anova and linear regression). These dose-response relationships were not different between patients with low versus high numbers of inflammatory and END-containing synovial cells (P>0.05, mancova). CONCLUSIONS: The dose-response relationship of i.a. morphine analgesia is not shifted by enhanced inflammation and END expression within synovial tissue. Thus, the presence of END within inflamed synovial tissue does not seem to interfere with i.a. morphine analgesia.

Antimetastatic effect of tinzaparin, a low-molecular-weight heparin.

The importance of coagulation activation in cancer patients is suggested by the clinical finding of hypercoagulability, experimental enhancement of metastasis and angiogenesis by coagulation factors such as tissue factor (TF) and thrombin and the possible antitumor effects of anticoagulant agents. Tinzaparin is a low-molecular-weight heparin (LMWH) with a relatively high molecular weight distribution and high sulfate to carboxylate ratio. In addition to its ability to inhibit thrombin and factor Xa, tinzaparin is particularly effective at releasing endothelial tissue factor pathway inhibitor (TFPI), the natural inhibitor of both procoagulant and non-coagulant effects of TF. The present study was undertaken to investigate the effect of tinzaparin on lung metastasis using a B16 melanoma model in experimental mice. Tinzaparin's anticoagulant effect in mice and its ability to release TFPI from human endothelial cells at various time points were demonstrated. Subcutaneous (s.c.) injection of tinzaparin (10 mg kg-1) 4 h before intravenous administration of melanoma cells (2.0 x 105) markedly (89%) reduced lung tumor formation (3 +/- 2) compared with controls (31 +/- 23; P < 0.001). In a second group of animals, tinzaparin (10 mg kg-1, s.c.) administered daily for 14 days following the initial (pretumor cell) dose, before assessment of lung seeding, reduced tumor formation by 96% (P < 0.001). No bleeding problems were observed in any of the tinzaparin-treated animals, despite a 4-fold prolongation of the whole blood clotting time after a single s.c. dose of tinzaparin (10 mg kg-1). Administration of tumor cells (2 x 106) caused a rapid and significant fall in platelet count 15 min after injection (a sensitive marker of intravascular coagulation) in controls (939 +/- 37 vs. 498 +/- 94 x 106 mL-1, P < 0.01), but this was prevented by tinzaparin treatment (921 +/- 104 x 106 mL-1). These data provide further experimental evidence to support the potential for LMWH as antimetastatic agents.

Inhibition of angiogenesis by antibody blocking the action of proangiogenic high-molecular-weight kininogen.

Previously we demonstrated that domain 5 (D5) of high-molecular-weight kininogen (HK) inhibits neovascularization in the chicken chorioallantoic membrane (CAM) assay and further found that kallikrein cleaved HK (HKa) inhibited FGF2-and VEGF-induced neovascularization, and thus was antiangiogenic. In this study, we sought to demonstrate whether uncleaved HK stimulates neovascularization and thus is proangiogenic. The chick chorioallantoic membrane was used as an in ovo assay of angiogenesis. Low-molecular-weight kininogen stimulates angiogenesis, indicating that D5 is not involved. Bradykinin stimulates neovascularization equally to HK and LK and is likely to be responsible for the effect of HK. A murine monoclonal antibody to HK (C11C1) also recognizes a similar component in chicken plasma as detected by surface plasmon resonance. Angiogenesis induced by FGF2 and VEGF is inhibited by this monoclonal antibody and is a more potent inhibitor of neovascularization induced by VEGF than an integrin alphavbeta3 antibody (LM 609). Our postulate that C11C1 inhibits the stimulation of angiogenesis by HK was confirmed when either C11C1 or D5 completely inhibited angiogenesis in the CAM induced by HK. Growth of human fibrosarcoma (HT-1080) on the CAM was inhibited by GST-D5 and C11C1. These results indicate HK is proangiogenic probably by releasing bradykinin and that a monoclonal antibody directed to HK could serve as an antiangiogenic agent with a potential for inhibiting tumor angiogenesis and other angiogenesis-mediated disorders.

Opioid peptide-expressing leukocytes: identification, recruitment, and simultaneously increasing inhibition of inflammatory pain.

BACKGROUND: Inflammatory pain can be effectively controlled by an interaction of opioid receptors on peripheral sensory nerve terminals with opioid peptides released from immune cells upon stressful stimulation. To define the source of opioid peptide production, we sought to identify and quantify populations of opioid-containing cells during the course of Freund's complete adjuvant-induced hind paw inflammation in the rat. In parallel, we examined the development of stress-induced local analgesia in the paw. METHODS: At 2, 6, and 96 h after Freund's complete adjuvant inoculation, cells were characterized by flow cytometry using a monoclonal pan-opioid antibody (3E7) and antibodies against cell surface antigens and by immunohistochemistry using a polyclonal antibody to beta-endorphin. After magnetic cell sorting, the beta-endorphin content was quantified by radioimmunoassay. Pain responses before and after cold water swim stress were evaluated by paw pressure thresholds. RESULTS: In early inflammation, 66% of opioid peptide-producing (3E7+) leukocytes were HIS48+ granulocytes. In contrast, at later stages (96 h), the majority of 3E7+ immune cells were ED1+ monocytes or macrophages (73%). During the 4 days after Freund's complete adjuvant inoculation, the number of 3E7+ cells increased 5.6-fold (P < 0.001, Kruskal-Wallis test) and the beta-endorphin content in the paw multiplied 3.9-fold (P < 0.05, Kruskal-Wallis test). In parallel, cold water swim stress-induced analgesia increased by 160% (P < 0.01, analysis of variance). CONCLUSIONS: The degree of endogenous pain inhibition is proportional to the number of opioid peptide-producing cells, and distinct leukocyte lineages contribute to this function at different stages of inflammation. These mechanisms may be important for understanding pain in immunosuppressed states such as cancer, diabetes, or AIDS and for the design of novel therapeutic strategies in inflammatory diseases.