Preclinical evaluation of radiation and systemic, RGD-targeted, adeno-associated virus phage-TNF gene therapy in a mouse model of spontaneously metastatic melanoma.

The incidence of melanoma in the United States continues to rise, with metastatic lesions notoriously recalcitrant to therapy. There are limited effective treatment options available and a great need for more effective therapies that can be rapidly integrated in the clinic. In this study, we demonstrate that the combination of RGD-targeted adeno-associated virus phage (RGD-AAVP-TNF) with hypofractionated radiation therapy results in synergistic inhibition of primary syngeneic B16 melanoma in a C57 mouse model. Furthermore, this combination appeared to modify the tumor microenvironment, resulting in decreased Tregs in the draining LN and increased tumor-associated macrophages within the primary tumor. Finally, there appeared to be a reduction in metastatic potential and a prolongation of overall survival in the combined treatment group. These results indicate the use of targeted TNF gene therapy vector with radiation treatment could be a valuable treatment option for patients with metastatic melanoma.

Mitigated Tregs and augmented Th17 cells and cytokines are associated with severity of experimental autoimmune neuritis.

Experimental autoimmune neuritis (EAN), an animal model of human Guillain-Barré syndrome, has long been considered as a T helper (Th) 1 cell-mediated autoimmune disorder. However, deficiency of IFN-γ, a signature Th1 cytokine, aggravated EAN, with features of elevated production of IL-17A, despite an alleviated systemic Th1 immune response. We hypothesized that Th17 cells and their cytokines might play a pathogenic role in EAN. To further clarify the roles of these Th and regulatory T cell (Treg) cytokines in the pathogenesis of EAN and their interrelationship, we investigated the expression of Th1/Th2/Th17/Treg cytokines in EAN in this study. We found that the levels of Th17 cells and IL-17A in cauda equina (CE)-infiltrating cells and splenic mononuclear cells (MNCs) as well as in serum paralleled the disease evolution, which increased progressively during the initiation stage and reached higher value at the peak of EAN. The same pattern was also noticed for the expression of IL-22. The diverse expression profiles of FoxP3, IL-17 receptors A and C were seen in CE-infiltrating cells and splenic MNCs in EAN. These findings indicate a major pro-inflammatory role of Th17 cells and IL-17A in the pathogenesis of EAN. Therapeutic interventions may be focused upon inhibiting Th17 cells and their cytokines in the early phase of EAN, so as to delay and suppress clinical signs of the disease, which has relevance for future studies on pathogenesis and treatment of GBS in humans.

Frondoside A enhances the antiproliferative effects of gemcitabine in pancreatic cancer.

Pancreatic cancer has a very poor prognosis. While gemcitabine is the mainstay of therapy and improves quality of life, it has little impact on survival. More effective treatments are desperately needed for this disease. Frondoside A is a triterpenoid glycoside isolated from the Atlantic sea cucumber, Cucumaria frondosa. Frondoside A potently inhibits pancreatic cancer cell growth and induces apoptosis in vitro and in vivo. The aim of the present study was to investigate whether frondoside A could enhance the anti-cancer effects of gemcitabine. Effects of frondoside A and gemcitabine alone and in combination on proliferation were investigated in two human pancreatic cancer cell lines, AsPC-1 and S2013. To investigate possible synergistic effects, combinations of low concentrations of the two drugs were used for a 72 h treatment period in vitro. Growth inhibition was significantly greater with the drug combinations than their additive effects. Combinations of frondoside A and gemcitabine were tested in vivo using the athymic mouse model. Xenografts of AsPC-1 and S2013 cells were allowed to form tumours prior to treatment with the drugs alone or in combination for 30 days. Tumours grew rapidly in placebo-treated animals. Tumour growth was significantly reduced in all treatment groups. At the lowest dose tested, gemcitabine (4 mg/kg/dose), combined with frondoside A (100 µg/kg/day) was significantly more effective than with either drug alone. To conclude: The present data suggest that combinations of frondoside A and gemcitabine may provide clinical benefit for patients with pancreatic cancer.

Blockade of inhibitors of apoptosis (IAPs) in combination with tumor-targeted delivery of tumor necrosis factor-α leads to synergistic antitumor activity.

In the current study, we examined whether the combination of tumor vasculature-targeted gene therapy with adeno-associated virus bacteriophage-tumor necrosis factor-α (AAVP-TNF-α) and/or the orally administered LCL161, an antagonist of inhibitors of apoptosis proteins (IAPs), enhanced antitumor efficacy without systemic toxicity. M21 human melanoma xenografts were grown subcutaneously in nude mice. Mice were treated according to one of four treatment regimens: AAVP-TNF-α alone (AAVP-TNF-α plus sodium acetate-acetic acid (NaAc) buffer) via tail vein injection; LCL161 alone (phosphate-buffered saline (PBS) plus LCL161) via oral gavage; AAVP-TNF-α plus LCL161; and PBS plus NaAc Buffer as a control group. Tumor volume, survival and toxicity were analyzed. AAVP trafficking and TNF-α production in vivo were detected on days 7 and 21 by real-time PCR, enzyme-linked immunosorbent assay and immunofluorescence. The levels of apoptosis and activation of caspases were assessed on days 7 and 21 by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling) and immunofluorescence assays. Our results showed that the combination of AAVP-TNF-α and LCL161 significantly inhibited tumor growth and prolonged survival in mice with melanoma xenografts. The combination of AAVP-TNF-α and LCL161 was also significantly more effective than either agent alone, showing a synergistic effect without systemic toxicity.

High dose epoetin beta in the first weeks following renal transplantation and delayed graft function: Results of the Neo-PDGF Study.

Erythropoietin promotes nephroprotection in animal models of ischemia-reperfusion injury. Neorecormon and Prevention of Delayed Graft Function (Neo-PDGF) is a French open-label multicenter randomized study to evaluate the effect of high doses of epoetin beta (EPO-beta) during the first 2 weeks of renal transplantation on renal function in patients at risk for delayed graft function (DGF). One hundred and four patients were included in the study. Patients randomized in treatment group (A) received four injections of EPO-beta (30.000 UI each), given before surgery and at 12 h, 7 days and 14 days posttransplantation. Patients randomized in control group (B) did not receive EPO-beta. Immunosuppression included induction with basiliximab and maintenance therapy with steroids, mycophenolate mofetil and tacrolimus. At 1 month posttransplant, the estimated glomerular filtration rate (MDRD formula) was 42.5 +/- 19.0 mL/min in the EPO-beta group and 44.0 +/- 16.3 mL/min in the control group (p = ns). The frequency of DGF was similar in both groups (32% vs. 38.8%; p = ns). No difference in the incidence of serious adverse events was observed. (ClinicalTrials.gov number, NCT00815867.).

Chromatographic separation of antiviral/anticancer nucleoside reverse transcriptase inhibitor drugs.

This paper discusses the current methods used for quantitative determination of analogues of nucleotide reverse transcriptase inhibitors (NtRTIs) in body fluids, cells, and tissues. Nucleoside reverse transcriptase inhibitors (NRTIs) prodrugs given to AIDS/herpes/cancer patients conjugate with phosphates at the site of their action. Separation of phosphorylated NRTIs is generally performed by reversed-phase chromatography. After separation, plasma NRTIs can be detected using a variety of methods, including immunoassay through monitoring of UV absorbance, fluorescence, and mass spectrometry. The most recent development in the field of detection of plasma NtRTIs shows a tendency toward the use double- or triple-focusing mass spectrometry, the most specific and sensitive monitoring technique.

Medicinal chemistry of antiviral/anticancer prodrugs subjected to phosphate conjugation.

Certain xenobiotics are given in the "prodrug" form. Either the human body, or one compartment of the body, or the targeted virus itself metabolizes the prodrug into its active form. The bioprecursor form of drugs is used for a wide variety of reasons, namely: to make drug penetration into the target organ (mainly to the brain through the blood-brain-barrier) possible, eliminate unpleasant taste, alter (either increasing or decreasing) the half life of the active component or supply more than one active components to the body.

Chronic lymphocytic leukemia: a hazardous condition before kidney transplantation.

Long-term survival of patients with chronic lymphocytic leukemia (CLL) is over 10 years, and such patients are thus potential kidney recipients in the case of superimposed end-stage renal disease. However, the renal and patient outcome in this condition is unknown. We report the charts of four patients with CLL who were engrafted in France with a deceased-donor kidney and underwent routine triple immunosuppressive therapy. The results show that these patients developed severe infectious episodes (fatal in one case) and tumoral complications including rapid progression of CLL in two cases. Moreover, the graft may be infiltrated and damaged by monoclonal B cells: one patient lost his graft 14 months after transplantation. Various therapeutic options (modifications of the immunosuppressive regimen, anti-CD20 antibodies, irradiation of the graft) showed little (if any) efficacy. Therefore, we believe that CLL is a too hazardous condition to envisage solid organ transplantation with a routine immunosuppressive regimen, and we propose a more appropriate approach.

The role of IL-12 and TNF-alpha in AIDP and AMAN.

BACKGROUND: Acute inflammatory demyelinating polyradiculoneuropathy (AIDP) and acute motor axonal neuropathy (AMAN) have been described as two major subtypes of Guillain-Barré syndrome (GBS); however, the possible difference of their immune-inflammatory pathogenesis remains unclear. METHODS: In this study, by using FACS and enzyme-linked immunosorbent assays analyses, the role of Th1 cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-12 (IL-12) and their receptors on peripheral blood mononuclear cells (PBMCs) and in serum concentrations were investigated in AIDP and AMAN. RESULTS: The results showed enhanced IL-12, IL-12R1 in AIDP and TNF-alpha in AMAN during the acute phase, as well as increased TNF-alpha and TNFR1 during the plateau phase of AIDP. Intravenous high dose immunoglobulin decreased IL-12R1 expression on cells in AIDP, but increased TNF-alpha and TNFR2 in AMAN. DISCUSSION: Our data suggest that IL-12 promotes disease development in AIDP and in contrast to previously inflammatory assumptions, TNF-alpha may play double roles in GBS. The anti-inflammatory role of TNF-alpha realized through TNFR2 in AMAN is possibly a therapeutic mechanism in the IVIg treatment of AMAN.

Distribution of neuroendocrine cells in the small and large intestines of the one-humped camel (Camelus dromedarius).

The distribution and relative frequency of neuroendocrine cells in the small and large intestines of one-humped camel were studied using antisera against 5-hydroxytryptamine (5-HT), cholecystokinin (CCK-8), somatostatin (SOM), peptide tyrosine tyrosine (PYY), gastric inhibitory polypeptide (GIP), neuronal nitric oxide synthase (nNOS), gastrin releasing peptide (GRP), substance P (SP), and neurokinin A (NKA). Among these cell types, CCK-8 immunoreactive (IR) cells were uniformly distributed in the mucosa, while others showed varied distribution in the villi or crypts of the small intestine. Immunoreactive cells like 5HT, CCK-8, and SOM showed peak density in the villi and crypts of the small intestine and in the colonic glands of the large intestine, while cells containing SP were discerned predominately in the crypts. 5-HT, CCK-8 and SOM cells were mainly flask-shaped and of the open-variety, while PYY and SP immunoreactive cells were mainly rounded or basket-shaped and of the closed variety. Basically the distribution pattern of the endocrine cells in the duodenum, jejunum and colon of the one-humped camel is similar to that of other mammals. Finally, the distribution of these bioactive agents may give clues as to how these agents aid in the function of the intestinal tract of this desert animal.

Streptozotocin-induced diabetic nephropathy in rats: the role of inflammatory cytokines.

The role of inflammatory cytokines in the pathogenesis of diabetic nephropathy has been studied in streptozotocin-induced diabetic rats. Rat kidneys were examined by light and electron microscopy and kidney homogenates were also analyzed by Western blot and flow cytometry for the expression of markers of inflammation namely, CD4+ and CD8+ T cells, macrophages, MHC classes I and II, the proinflammatory cytokines tumor necrosis factor-alpha, interferon-gamma and nitric oxide (NO). Light and electron microscope examination revealed infiltration of mononuclear cells throughout the renal parenchyma, with the glomeruli being more severely affected especially at 8 months after disease induction. Western blot and flow cytometric analyses revealed the infiltrating cells to be CD4+ T cells, CD8+ T cells and macrophages. Western blot analyses also revealed increased expression of the proinflammatory and Th1 cytokines tumor necrosis factor-alpha, interferon-gamma as well as nitric oxide. Using flow cytometry, we have shown that the difference in expression of CD4+ T cells in control and diabetic kidneys is more significant at 1 month than at 8 months, while expression of CD8+ T cells is more significant at 8 months. We speculate therefore that diabetic nephropathy is probably initiated and driven by a Th1 process. CD8+ T cells, however, become more significant at later stages of the disease when tissue loss is evident. Since NO induction also occurs only after 8 months, we hypothesize that NO might be significant for the later stages of the disease. Our data implicate inflammation in the pathogenesis of diabetic nephropathy in view of the overexpression of the proinflammatory cytokines TNF-alpha and IFN-gamma and the cells that secrete them in the early and late phases of the disease.

Distribution of insulin like growth factor-1 (IGF-1) and its receptor in the intestines of the one-humped camel (Camelus dromedarius).

The distribution of insulin-like growth factor-1 (IGF-1) and its receptor in the gut of the one-humped camel (Camelus dromedarius) were studied by immunohistochemistry and quantitative receptor autoradiography. IGF-1-IR cells occurred mainly in the lamina propria and epithelium of the small intestine, while in the large intestine positive cells were seen in the columnar cells of the epithelial layer of colonic glands. IGF-I was also discernible in the muscularis externa of the intestines. Autoradiography revealed a higher concentration of receptors in the mucosa compared to the muscular layer. With regard to the mucosa, the highest density of receptors was discernible in the duodenum. Immunohistochemistry revealed the main sites of the receptors to be the lamina propria, epithelia of the crypts and the villi of intestines. Double immunofluorescence studies with combined antisera to IGF-I and its receptor showed that the ligand and its receptor usually occurred within the same cell in the mucosa. A few cells with varied profiles immunoreacted to either the ligand or the receptor but not to both. Cells with varied profiles immunoreacted to antiserum of the receptors but not to the ligand in the muscle layer. Thus IGF-1 might be acting on its receptor via both an autocrine and paracrine modes in the camel mucosa. In the muscularis layer, IGF-1 may be acting by different mechanisms. Our data demonstrate that unlike all other mammals studied, the camel contains a high concentration of IGF-1 receptors in the duodenal mucosa compared to other parts of the camel gut. It also possesses a higher concentration of the receptor in its mucosa compared to the muscle layer. We speculate that this might be a significant feature necessary for the regenerative ability of the duodenal mucosa in the one-humped camel.

Estrogen and progesterone treatment: effects on muscarinic M(4) receptor subtype in the rat brain.

We investigated the effect of ovariectomy (OVX) and hormonal treatment for 10 weeks by estradiol and progesterone on muscarinic M(4) receptor subtype in different brain areas of female rats. Moreover, motor activity of OVX and hormone-treated rats was measured by automated open field exploration boxes. Receptor quantification in the hippocampus, frontal cortex, parietal cortex, amygdala and hypothalamus was done by receptor autoradiography using a selective ligand for muscarinic M(4) receptors. Ovariectomy up-regulated M(4) receptors in the dentate gyrus, CA1, CA3, frontal cortex and hypothalamus whereas the estrogen treatment restored M(4) binding to that of the sham group. Progesterone treatment had no effect on the ovariectomy-induced up-regulation of M(4) receptors. Ovariectomy significantly decreased the exploratory activity of the rats compared to the sham group. Estrogen treatment restored the exploratory behavior of the ovariectomized rats to that of the sham group whereas the progesterone-treated rats were less alert to the surrounding when compared to the sham and estrogen supplemented rats. The effect of estrogen on the hippocampal muscarinic M(4) receptor subtype is a novel finding and may have functional significance for cholinergic receptors especially in relation to postmenopausal memory problems and neurodegenerative disease like Alzheimer's disease.

Increased levels of somatostatin in rat ankles with adjuvant arthritis.

Levels of somatostatin were investigated in the ankles and spinal cords of rats suffering from acute and chronic adjuvant arthritis. As measured by radioimmunoassay, somatostatin showed significantly higher concentrations only in chronic arthritic ankles. No significant difference was observed in somatostatin levels between the spinal cords of normal and arthritic groups. Using immunohistochemical labeling and electron microscopy, we observed increased somatostatin labeling in the mature bone matrix, monocytes, and polymorphonuclear cells of bone marrow and macrophage-like synovial cells of chronically arthritic rats. Understanding the mechanism(s) which lead to increased somatostatin in chronic arthritic joints may result in more effective treatment methods.

The effect of ovariectomy and ovarian steroid treatment on growth hormone and insulin-like growth factor-I levels in the rat femur.

Growth hormone (GH) and insulin-like growth factor-I (IGF-I) are known to play an important role in bone metabolism. The regulation of plasma levels of GH and IGF-I by ovarian steroids is well known, however, their effect on local GH and IGF-I is still unclear. In this study, we investigated the effect of ovariectomy and ovarian steroid treatment on the femur GH and IGF-I levels as well as on bone density in the rat. Nine month-old rats were ovariectomized (OVX) or sham-operated (SHAM) and 9 weeks after the surgery they were treated with daily s.c. injections of either 17beta-estradiol (OVX + E), progesterone (OVX + P), or vehicle (OVX + V) for another 10 weeks. GH and IGF-I levels in the femur extracts were measured by specific radioimmunoassay (RIA). Ovariectomy decreased GH and had no effect on IGF-I levels. Estradiol treatment increased femur GH and IGF-I levels compared to SHAM rats. Progesterone restored GH and increased IGF-I levels. Ovariectomy decreased, estrogen restored and progesterone partially restored femur bone density. Our results demonstrate that ovariectomy and ovarian steroids modulate the levels of GH and IGF-I in the bone of aged OVX rats. However, these effects appear to be limited to supraphysiological concentrations of estradiol and progesterone.

Inhibition of acetylcholine muscarinic M(1) receptor function by the M(1)-selective ligand muscarinic toxin 7 (MT-7).

MT-7 (1 - 30 nM), a peptide toxin isolated from the venom of the green mamba Dendroaspis angusticeps and previously found to bind selectively to the muscarinic M(1) receptor, inhibited the acetylcholine (ACh)-stimulated [(35)S]-guanosine-5'-O-(3-thio)triphosphate ([(35)S]-GTPgammaS) binding to membranes of Chinese hamster ovary (CHO) cells stably expressing the cloned human muscarinic M(1) receptor subtype. MT-7 failed to affect the ACh-stimulated [(35)S]-GTPgammaS binding in membranes of CHO cells expressing either the M(2), M(3) or M(4) receptor subtype. In N1E-115 neuroblastoma cells endogenously expressing the M(1) and M(4) receptor subtypes, MT-7 (0.3 - 3.0 nM) inhibited the carbachol (CCh)-stimulated inositol phosphates accumulation, but failed to affect the CCh-induced inhibition of pituitary adenylate cyclase activating polypeptide (PACAP) 38-stimulated cyclic AMP accumulation. In both CHO/M(1) and N1E-115 cells the MT-7 inhibition consisted in a decrease of the maximal agonist effect with minimal changes in the agonist EC(50) value. In CHO/M(1) cell membranes, MT-7 (0.05 - 25 nM) reduced the specific binding of 0.05, 1.0 and 15 nM [(3)H]-N-methylscopolamine ([(3)H]-NMS) in a concentration-dependent manner, but failed to cause a complete displacement of the radioligand. Moreover, MT-7 (3 nM) decreased the dissociation rate of [(3)H]-NMS by about 5 fold. CHO/M(1) cell membranes preincubated with MT-7 (10 nM) and washed by centrifugation and resuspension did not recover control [(3)H]-NMS binding for at least 8 h at 30 degrees C. It is concluded that MT-7 acts as a selective noncompetitive antagonist of the muscarinic M(1) receptors by binding stably to an allosteric site.

Intracerebroventricular met-enkephalin administration modulates adjuvant arthritis.

The purpose of this study was to investigate the anti-inflammatory properties of intracerebroventricular met-enkephalin (met-enk) administration in an animal model of arthritis. Adjuvant arthritis was induced in rats by intradermal inoculation of mycobacterium butyricum and the effects of intraventricular met-enk+thiorphan (enkephalinase inhibitor) were studied. Treatment was initiated either simultaneously with the bacterial inoculation (preventive group) or on post-inoculation day 17 after the appearance of inflammation (treatment group). The degree of inflammation was evaluated by measuring the diameter and the circumference of the ankle joint immediately before the sacrifice (day 31) and by histologic examination of ankle joint sections. The results of this study revealed that combined intraventricular injections of met-enk+thiorphan reduced the arthritic-like inflammation in the preventive group as well as in the treatment group. These findings suggest that centrally applied met-enk+thiorphan may suppress the development adjuvant arthritis as well as the symptoms of manifest arthritis. Thus central met-enk may be involved in both hypothalamic pituitary adrenal axis and immune forms of stress-induced modulation.

Neuromuscular effects of two types of fracture treatment.

Immobilisation causes denervation-like changes in the motor endplates, decreases the content of IGF-I, and increases the number of IGF-I receptors in the spinal cord. In the rat we investigated whether similar changes occur after a fracture of the midshaft of the femur which had been treated by intramedullary fixation with adequate or undersized pins. A more pronounced reduction in muscle wet weight was seen after fixation by undersized pins as well as decreased ash density of the ipsilateral tibia which did not completely return to normal within the 12-week experimental period. The nicotinic cholinergic receptors in the motor endplates of tibialis anterior were increased (p < 0.01) and there was a significant increase (p < 0.02) in IGF-I receptors in the lumbar spinal cord ipsilateral to the fracture after treatment by undersized nails. These changes may be associated with the impaired proprioception, co-ordination and motor activity which are sometimes seen after fractures.

Hypophysectomy enhances interleukin-1beta, tumor necrosis factor-alpha, and interleukin-10 mRNA expression in the rat brain.

Although the effects of various cytokines as regulators of hormone synthesis and production are well documented, the role for pituitary hormones as modulators of cytokine synthesis is not fully understood. In this study, we investigated the effect of pituitary hormones' depletion on cytokine synthesis after short- (21 days) and long- (35 days) term hypophysectomy (ST-HX and LT-HX, respectively). The expresssion of the proinflammatory cytokine interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) and the anti-inflammatory cytokines IL-10 and transforming growth factor-beta (TGF-beta) in the rat brain was studied using in situ hybridization. Our results indicate that IL-1beta mRNA-expressing cells were significantly upregulated at day 21 in hypophysectomized rats compared to sham-operated controls. This enhanced expression was also detected later at day 35 post hypophysectomy. However, TNF-alpha mRNA expression was significantly increased only at the later sampling interval. IL-10 mRNA-expressing cells were increased after long-term hypophysectomy compared to controls. TGF-beta mRNA-expressing cells were not increased after hypophysectomy. In conclusion, these results suggest a role for pituitary hormones in IL-1beta, TNF-alpha, and IL-10 synthesis.

Selectivity profile of muscarinic toxin 3 in functional assays of cloned and native receptors.

By using acetylcholine-induced stimulation of [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding to membrane G proteins as a functional assay of the cloned human m1-m4 muscarinic receptor subtypes stably expressed in Chinese hamster ovary cells, muscarinic toxin 3 (MT3) was found to block the m4 receptor with a potency (pA2 = 8.33) much higher than those displayed at the m1 (pA2 = 6.78), m3 (pA2 = 6.3), and m2 (pA2 < 6.3) subtypes. In N1E-115 cells, which have been reported to express m4 receptors coupled to inhibition of cAMP, MT3 potently antagonized the carbachol-induced inhibition of adenylyl cyclase with a pA2 of 8. 81 and displayed monophasic inhibitory curves. Unexpectedly, in NG108-15 cells, known to express only m4 receptors, MT3 counteracted the carbachol inhibition of adenylyl cyclase with a lower potency (pA2 = 7.60) and showed a biphasic inhibitory curve, suggesting the participation of both m4 and m2 receptors. This possibility was supported by radioligand binding data showing that MT3 failed to completely displace the binding of [3H]N-methylscopolamine to NG108-15 cell membranes and by reverse transcription-polymerase chain reaction analysis, revealing the presence of mRNAs for both m4 and m2 receptor subtypes. These data demonstrate that MT3 possesses a high functional receptor selectivity for both the cloned and native m4 receptors and that in cell systems containing m4 and m2 receptors coupled to a common response, the toxin constitutes a powerful tool to resolve the relative contribution by each receptor subtype.