Circulating tumour cells as a biomarker for diagnosis and staging in pancreatic cancer.

BACKGROUND: Current diagnosis and staging of pancreatic ductal adenocarcinoma (PDAC) has important limitations and better biomarkers are needed to guide initial therapy. We investigated the performance of circulating tumour cells (CTCs) as an adjunctive biomarker at the time of disease presentation. METHODS: Venous blood (VB) was collected prospectively from 100 consecutive, pre-treatment patients with PDAC. Utilising the microfluidic NanoVelcro CTC chip, samples were evaluated for the presence and number of CTCs. KRAS mutation analysis was used to compare the CTCs with primary tumour tissue. CTC enumeration data was then evaluated as a diagnostic and staging biomarker in the setting of PDAC. RESULTS: We found 100% concordance for KRAS mutation subtype between primary tumour and CTCs in all five patients tested. Evaluation of CTCs as a diagnostic revealed the presence of CTCs in 54/72 patients with confirmed PDAC (sensitivity=75.0%, specificity=96.4%, area under the curve (AUROC)=0.867, 95% CI=0.798-0.935, and P<0.001). Furthermore, a cut-off of ⩾3 CTCs in 4 ml VB was able to discriminate between local/regional and metastatic disease (AUROC=0.885; 95% CI=0.800-0.969; and P<0.001). CONCLUSION: CTCs appear to function well as a biomarker for diagnosis and staging in PDAC.

NOTES spin-off for the therapeutic gastroenterologist: natural orifice surgery.

Natural orifice transluminal endoscopic surgery (NOTES) has ushered in a new era in flexible endoscopy. Over the past decade, modest advances have been made in addressing the fundamental challenges of NOTES surgery including transluminal access and closure techniques, and advancement of NOTES-specific technology. Despite these encouraging initial efforts significant obstacles to widespread acceptance of NOTES as a surgical option persist. Moreover, due to the well-documented safety and efficacy of laparoscopic techniques, the question remains as to the best candidate NOTES procedure. Presently, interest has shifted from true NOTES to hybrid procedures and single incision laparoscopic surgery, due to the lure of more immediate success. Additionally, there is also a growing awareness of the potential applications of natural orifice surgery techniques to the present field of therapeutic endoscopy. Research into transluminal access and closure has born several techniques and devices that are now being explored in endoscopic procedures such as full-thickness resection, endoscopic myotomy, direct endoscopic pancreatic necrosectomy and bariatric endoscopy. Such NOTES "spin-off" procedures are expanding the armamentarium of today's therapeutic endoscopists, and a growing body of literature suggests that they will play a significant role in the evolution of therapeutic endoscopy in the future.

Differences in the control of breathing between Himalayan and sea-level residents.

We compared the control of breathing of 12 male Himalayan highlanders with that of 21 male sea-level Caucasian lowlanders using isoxic hyperoxic ( = 150 mmHg) and hypoxic ( = 50 mmHg) Duffin's rebreathing tests. Highlanders had lower mean +/- s.e.m. ventilatory sensitivities to CO(2) than lowlanders at both isoxic tensions (hyperoxic: 2.3 +/- 0.3 vs. 4.2 +/- 0.3 l min(1) mmHg(1), P = 0.021; hypoxic: 2.8 +/- 0.3 vs. 7.1 +/- 0.6 l min(1) mmHg(1), P < 0.001), and the usual increase in ventilatory sensitivity to CO(2) induced by hypoxia in lowlanders was absent in highlanders (P = 0.361). Furthermore, the ventilatory recruitment threshold (VRT) CO(2) tensions in highlanders were lower than in lowlanders (hyperoxic: 33.8 +/- 0.9 vs. 48.9 +/- 0.7 mmHg, P < 0.001; hypoxic: 31.2 +/- 1.1 vs. 44.7 +/- 0.7 mmHg, P < 0.001). Both groups had reduced ventilatory recruitment thresholds with hypoxia (P < 0.001) and there were no differences in the sub-threshold ventilations (non-chemoreflex drives to breathe) between lowlanders and highlanders at both isoxic tensions (P = 0.982), with a trend for higher basal ventilation during hypoxia (P = 0.052). We conclude that control of breathing in Himalayan highlanders is distinctly different from that of sea-level lowlanders. Specifically, Himalayan highlanders have decreased central and absent peripheral sensitivities to CO(2). Their response to hypoxia was heterogeneous, with the majority decreasing their VRT indicating either a CO(2)-independent increase in activity of peripheral chemoreceptor or hypoxia-induced increase in [H(+)] at the central chemoreceptor. In some highlanders, the decrease in VRT was accompanied by an increase in sensitivity to CO(2), while in others VRT remained unchanged and their sub-threshold ventilations increased, although these were not statistically significant.

Upper extremity arteriovenous dialysis fistula resulting in cavernous sinus arterialized blood flow.

Arterialized blood flow in the cavernous sinus may result from carotid-cavernous fistula or dural venous fistula. We encountered an unusual case of arterialized blood flow in the cavernous sinus on MR angiography resulting from arterialized retrograde venous flow in the internal jugular vein. This abnormal flow originated from an upper extremity dialysis arteriovenous fistula in the presence of central venous occlusion. The patient's symptoms of visual disturbance resolved after the central venous occlusion was treated with stent placement.

Resistance to L1210 mouse leukemia cells in moderately protein-malnourished BALB/c mice treated in vivo with thymosin fraction V.

Moderate protein malnutrition retarded the i.p. proliferation of L1210 mouse leukemia cells in BALB/c mice. The increased resistance against leukemia cell growth in protein-malnourished mice was correlated with increased in vitro mitogenic responsiveness of spleen lymphocytes to phytohemagglutinin and increased levels of serum corticosterone but could not be correlated with altered development of splenic lymphocyte-mediated cytotoxicity. The increased resistance against leukemia cells in well-fed mice treated with thymosin alone could not be correlated with an increase in any of these parameters. Treatment with Thymosin Fraction V further increased the resistance of protein-malnourished mice to i.p. leukemia cell growth. The increased resistance of these mice to tumor cell growth was correlated with increased splenic lymphocyte mitogenic responsiveness to phytohemagglutinin, elevated serum corticosterone levels, and a slight increase in lymphocyte-mediated cytotoxicity 14 days after tumor challenge. For 7 days after the last treatment, protein-malnourished mice had reduced serum corticosterone levels. Nevertheless, the serum corticosterone levels were still higher than normal in these mice.

Effects of dietary retinyl palmitate or 13-cis-retinoic acid on the promotion of tumors in mouse skin.

The present study was designed to determine the effects of dietary 13-cis-retinoic acid and retinyl palmitate on mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). Female CD-1 mice were initiated with 150 nmol of 7,12-dimethylbenz(a)anthracene and promoted twice weekly with 8 nmol of TPA. Diets supplemented with retinyl palmitate to yield 60,000 or 200,000 IU or 700,000 for 5 wk followed by 350,000 IU per kg of diet (700,000/350,000) fed to mice during tumor promotion resulted in 9%, 37%, and 65% inhibition of the papilloma yield, respectively, at 21 wk of promotion. Although topical applications of 13-cis-retinoic acid have been almost as effective as retinoic acid in preventing the appearance of mouse skin tumors, dietary 13-cis-retinoic acid at 200,000 or 700,000 IU per kg of diet resulted in no reduction in papilloma yield but did result in a dose-dependent decrease in the tumor burden (weight of tumors per mouse). Therefore, dietary retinyl palmitate yielded a dose-dependent inhibition of the number and weight of tumors promoted by TPA, whereas dietary 13-cis-retinoic acid resulted in a decrease in weight but not in number of tumors promoted by TPA.

Immunomodulation by immunopeptides and autoantibodies in aging, autoimmunity, and infection.

The operation of the immune system is a complex orchestration of specific self and non-self-recognition capacities mediated by cells of the innate system acting in coordination with T and B lymphocytes in a series of processes modulated by cytokines. We provide evidence for a natural immunomodulatory system involving autoantibodies directed against a controlling segment of T cell receptor Vbeta chains that downregulate production of stimulatory cytokines balanced by the peptides which in turn upregulate inflammatory activities mediated by TH1-type helper cells. TCR Vbeta-derived peptides effective in retrovirally induced immunosupression could also reverse the effects of immunosenescence in aged mice by restoring the balance of TH1- and TH2-type immunity and the resistance of the animals to cardiac pathology caused by infection with coxsackievirus. An unexpected finding was an adaptive role of the T cells from peptide-treated mice in remodeling damaged hearts by increasing net collagen synthesis by cardiac fibroblasts.

T-cell receptor-derived peptides in immunoregulation and therapy of retrovirally induced immunosuppression.

Retrovirally infected humans and mice showed progressive acquired immunodeficiency accompanied by the production of elevated levels of autoantibodies directed against T-cell receptor variable-domain epitopes. Epitope mapping analyses indicated that a major determinant recognized was defined by a 16-mer peptide containing the entire CDR1 segment and part of the FR2 region of human Vbeta8, and that both species showed reactivity to the same sequence. Either prophylactic or therapeutic administration of this peptide to retrovirus-infected C57/BL/6 mice normalized the balance of T(H)1- and T(H)2-type helper activity and restored the resistance to infection by the opportunistic parasite Cryptosporidium. Administration of the peptide did not generate significantly increased levels of autoantibody, but had a profound effect on T-cell activity as well as other aspects of inflammation, including NK-cell activity. A 16-mer derived from the Jbeta sequence showed similar functional effects on T cells from retrovirus-infected mice. Direct binding of the VbetaCDR1 peptide to recombinant TCR Valpha/Vbeta constructs, as well as to IgM natural autoantibodies, suggests that the cell surface receptor for the peptide is the alpha/beta TCR on T cells and surface IgM in B cells. The Vbeta CDR1 peptide stimulated division of murine splenocytes in vitro, stimulated the production of the T(H)1 cytokine IL-2, and synergized with the T-cell mitogen concanavalin A in proliferation and IL-2 production. These studies indicate that administration of peptides derived from T-cell receptor variable domains to animals immunosuppressed as a result of retroviral infection has a profound immunomodulatory effect enhancing overall T-cell functional capacity, particularly with respect to the cytokine production characteristic of T(H)1-type cells. Our studies are interpreted in the context of other recent investigations of immunomodulatory peptides.

Modulation of tumor necrosis factor and gamma interferon production by cocaine and morphine in aging mice infected with LP-BM5, a murine retrovirus.

The actions of retroviral infections, aging, and cocaine and morphine injection on cytokine production were investigated in C57BL/6 female mice. Retroviral infection with LP-BM5 murine leukemia virus was further developed as a model of murine acquired immunodeficiency syndrome (AIDS). The effects of cocaine and morphine on gamma-interferon (IFN) and tumor necrosis factor (TNF) production in vivo and with isolated spleen cells were measured by a sandwich enzyme-linked immunosorbent assay (ELISA) method. Serum IFN was generally not detected in any group except mice injected with saline and young mice infected with LP-BM5 virus. Splenocytes from mice with murine AIDS produced less IFN when stimulated in vitro by ConA. In aged mice, IFN production by spleen cells was severely suppressed by retroviral infection. Cocaine had a tendency to suppress IFN production by stimulated cells in vitro. Morphine tended to reduce IFN production by spleen cells from retrovirally infected animals. The serum TNF level in mice with murine AIDS was elevated creating higher levels in morphine and morphine plus cocaine treated uninfected mice while cocaine injection eliminated serum TNF. When stimulated in vitro by lipopolysaccharides (LPS), splenocytes from mice with murine AIDS also produced more TNF than uninfected controls. TNF production in vitro and in vivo was significantly increased by retroviral infection. Therefore, results indicate that cocaine and retroviral infection modulate TNF and IFN production.

Vitamin E supplementation with interferon-gamma administration retards immune dysfunction during murine retrovirus infection.

Murine retrovirus infection induces loss of vitamin E and immune dysfunction with loss of cytokine production by T-helper cells. Therefore interferon-gamma (IFN-gamma) was given during dietary vitamin E supplementation to effectively prevent murine retrovirus-induced immunosuppression, cytokine dysregulation, and development of murine AIDS. Administration of IFN-gamma during vitamin E supplementation significantly prevented development of retrovirus-induced suppression of splenic natural killer cell activity and T cell proliferation. It also significantly slowed retrovirus-induced elevation of T helper (Th) 2 cytokine [interleukin (IL)-4, IL-5, and IL-10] production and monokine (IL-6 and tumor necrosis factor-alpha) secretion by splenocytes. The treatment also prevented loss of Th1 cytokine (IL-2 and IFN-gamma) secretion by splenocytes from retrovirus-infected mice alleviating splenomegaly and hypergammaglobulinemia. The combined therapy had an additive therapeutic impact. It was more effective than IFN-gamma treatment or vitamin E supplementation alone in delaying the development of retrovirus-induced immunosuppression with its cytokine dysregulation.

Enhancement of the expression of activation markers on human peripheral blood mononuclear cells by in vitro culture with retinoids and carotenoids.

Retinoids (retinol, retinal, retinoic acid, retinyl beta-glucuronide, and 13-cis retinoic acid) and carotenoids (beta-carotene and canthaxanthin) were evaluated for their immunomodulatory effects on human peripheral blood T-lymphocyte subpopulations and natural killer (NK) cells. Peripheral blood mononuclear cells (PBMC) from healthy young volunteers were isolated and incubated for 72 hours at various levels of retinoids and carotenoids including a physiological concentration (10(-8) M). Expression of surface antigens for total T cells, T-helper and T-suppressor cells, and activation markers (transferrin receptor, HLA-Dr antigen, and interleukin 2 receptor) were analyzed with an EPICS V flow cytometer. Retinoic acid and 13-cis retinoic acid (13-cRA) produced significant increases in the percentage of cells with markers for total T-helper cells, with a minimal effect on percentage of lymphocytes with markers for NK cells. However, beta-carotene (BC), canthaxanthin (CTX), and retinyl beta-glucuronide (RBG) dramatically increased the percentage of PBMC with markers for NK cells and produced a smaller increase in lymphocytes with surface antigens identifying them as T-helper cells. Furthermore, retinol and retinal did not show significant change either in the percentage of lymphocytes with markers for T-helper cells or in the helper/suppressor ratio. An increase in the expression of HLA-Dr antigen and transferrin receptors was greater when cells were incubated with 13-cRA than with either BC, CTX, or RBG, while carotenoids produced a greater increase in the expression of IL-2 receptors than 13-cRA. Our study indicates that both retinoids and carotenoids might be activating different subpopulations of immune cells.

Beta-carotene stimulates human leukocytes to secrete a novel cytokine.

The effect of beta-carotene on cytokine production by human peripheral blood leukocytes was tested. Beta-carotene stimulated the secretion of a novel cytotoxic cytokine when peripheral blood cells were exposed to carotenoid concentrations between 10(-6) and 10(-10) M. Beta-carotene-treated supernatants caused the cytolysis of four out of the six human tumor cell lines tested. Low level toxicity was also observed when normal diploid fibroblast lines were exposed to beta-carotene-treated leukocyte supernatants. The cytotoxic activity elicited by beta-carotene was found to be distinct from characterized cytokines based on both antisera neutralization and target cell specificity studies. This study demonstrates that beta-carotene can induce human leukocytes to secrete one or more cytokines that can manifest cytotoxic activity against human tumor cells in vitro.

Cellular immune functions of adults treated with a daily, long-term, low dose of 13-cis retinoic acid.

The effects of long-term consumption of 13-cis retinoic acid (13-cRA) on cellular immune functions were measured in young, adult volunteers. The retinoid was administered for 9 months at about 0.13 mg/kg/day. The mean 8AM concentrations of 13-cRA ranged between 30 and 60 ng/ml of serum throughout the study. Corticosteroid levels in plasma decreased significantly throughout treatment, declining from 15.2 ug/dL to 9.1 mg/dL (p less than 0.05). T-cell mitogenesis stimulated by PHA or A Con A was not significantly affected, although this parameter was slightly depressed during the first 2 months of treatment. The percentage of B-lymphocytes tended to decrease during treatment and returned to normal after cessation of 13-cRA (p less than 0.05), while the percentage of T-cells as measured by E-rosette and by fluorescent antibody tagging of surface antigens did not change. The percentage of non T-cells tended to increase slightly during treatment.

Production of tumor necrosis factor alpha by resident and activated murine macrophages.

Alveolar macrophages (Am phi s), resident peritoneal macrophages (RPm phi s), and thioglycolate-elicited peritoneal macrophages (TGPm phi s) were isolated from C57BL/6 mice and incubated with lipopolysaccharide (LPS), stimulated cell supernatant, or recombinant interferon gamma (IFN-gamma) for 24 h. Tumor necrosis factor (TNF) in cell-free supernatants was measured by enzyme-linked immunosorbent assay. Amo phi s incubated with 10(3) ng/ml LPS produced 50 times more TNF than RPm phi s and 5 times more than TGPm phi s, and LPS alone induced maximum TNF production by Am phi s. Stimulated cell supernatant or recombinant IFN-gamma alone did not induce TNF production. A combination of LPS with stimulated cell supernatant or IFN-gamma had only a limited synergistic effect on TNF production by Am phi s. However, both LPS and stimulated cell supernatant or recombinant IFN-gamma induced maximum TNF production by RPm phi s and TGPm phi s. TGPm phi s showed greater sensitivity to LPS and stimulated cell supernatant or IFN-gamma with regard to TNF production than the other macrophage populations investigated.

Effects of selenium in vitro on human T-lymphocyte functions and K-562 tumor cell growth.

In vitro E-rosette formation, lymphocyte mitogenesis, and natural killer (NK) cell activity of human blood lymphocytes were strongly inhibited by high concentrations (10(-4) M) of sodium selenite, sodium selenate, and selenium dioxide. Lower concentrations (10(-5) or 10(-7) M) also inhibited E-rosette formation and natural killer cell activity against K-562 tumor cells. Lymphocyte transformation induced by concanavalin A (con A) or pokeweed mitogen (PWM) was also inhibited by all selenium compounds tested, but only at the highest concentrations (10(-5) and 10(-4) M). There was depression of the total number of viable lymphocytes following incubation with selenium dioxide only at a high concentration (10(-4) M). Interferon production was enhanced at lower levels (10(-9) to 10(-6)M) of selenium dioxide while a higher concentration (10(-5) and 10(-4)M) appeared to inhibit its production. The mechanism of inhibition by selenium compounds (10(-4) M) is due, in part, to the decrease of viable lymphocytes. It is unclear how other and lower concentrations (10(-7) or 10(-9) M) of selenium compounds inhibit E-rosette formation, NK activity, or K-562 tumor cell growth.

Nutraceutical and antioxidant effects of a delphinidin-rich maqui berry extract Delphinol®: a review.

Anthocyanins represent water-soluble flavonoid species, commonly found in higher plants, the richest plant source representing berries. While all anthocyanins present with antioxidant activity, the delphinidins represent the most potent antioxidant anthocyanin species owed to largest number of hydroxyl groups in the B-ring. The richest known natural source of delphinidins is the maqui berry (Aristotelia chilensis) from which an extract Delphinol®, standardized to 25% delphinidin, is commercially available. Delphinol® significantly reduces oxidative stress (oxidized LDL and F2-isoprostane) and blood glucose in controlled clinical trials. In human umbilical vein endothelium delphinidins concentration-dependently decrease intracellular oxygen radicals. Furthermore, delphinidins increase endothelial nitric oxide synthase expression and decreases expression of vaso-constrictory endothelin-1. Delphinidins inhibit the expression of cell adhesion molecules ICAM and VCAM, thus counteracting vascular inflammatory situations. Furthermore, delphinidins decrease platelet activity and may contribute to thrombosis prevention. Research on delphinidins showed improved endothelial function with elevated endothelial NO generation, lowered platelet aggregability and anti-inflammatory vascular effects. Delphinidins dose-dependently inhibit NF-κB-, activator protein-1- as well as COX-2 expression in UV-exposed epidermis. Delphinidins are found to be internalized into keratinocytes and pre-clinical investigations show significant UV-photo-protective 1effects with topical application of 40 nM delphinidin, both when applied prior to UV exposure as well as after exposure. Delphinidins may counteract skin-aging due to inhibition of UV-induced expression of matrix metalloproteinase in fibroblasts. In a rodent osteoporosis model delphinidin was found to inhibit differentiation of osteoclasts, resulting in an inhibited bone demineralization, while other anthocyanins were ineffective. Future research on Delphinol® and delphinidins may be expected to identify further health benefits.

Alcohol, cancer, and immunomodulation.

There is a great deal of epidemiological evidence indicating that chronic, excessive alcohol consumption is a major risk factor for cancers in humans. However, the experimental basis for the increased cancer risk associated with alcohol intake is not clear. Since it appears that ethanol alone is not carcinogenic, ethanol effects must be explained in terms of its modifying the actions of other causal agents. Current studies indicate that ethanol and its congeners may act as tumor promoters, thereby enhancing the effect of initiating carcinogens from the environment. Available evidence also shows that ethanol is immunosuppressive. Clearly, cirrhosis due to high, prolonged alcohol intake is an indicator of the immunosuppressive effects of ethanol. It is less clear that more moderate intakes of alcohol could have as profound an effect on immune systems. However, changes do occur yielding alterations in lymphocyte sensitivity to alcohol in vitro and in cell development, as shown by increased NK cell function at low concentrations. Since other conditions, such as cytotoxic drugs which suppress cellular immune functions, are clearly associated with increased cancer risk. It is intriguing to think that prolonged exposure to ethanol-induced immunosuppression may be a cofactor in the promotion of cancer. The tumor promotion may take place via a variety of mechanisms as discussed in this paper, including reduced host defenses by direct effects of ethanol, its metabolites, and/or malnutrition. It may be beneficial to test methods for immunostimulation in prolonged alcohol abusers, where cessation of use is unsuccessful or residual immunosuppression remains, to reduce the risk of development or growth of initiated tumors.

Solar ultraviolet-induced erythema in human skin and nuclear factor-kappa-B-dependent gene expression in keratinocytes are modulated by a French maritime pine bark extract.

The procyanidin-rich French maritime pine bark extract Pycnogenol (PBE) has been investigated for its effect in protecting human skin against solar UV-simulated light-induced erythema. Twenty-one volunteers were given an oral supplementation of Pycnogenol: 1.10 mg/kg body weight (b. wt.)/d for the first 4 weeks and 1.66 mg/kg b. wt./d for the next 4 weeks. The minimal erythema dose (MED) was measured twice before supplementation (baseline MED), once after the first 4 weeks of supplementation, and a last time at the end of the study. The UVR dose necessary to achieve 1 MED was significantly increased during PBE supplementation. Since the activation of the pro-inflammatory and redox-regulated transcription factor NF-kappaB is thought to play a major role in UVR-induced erythema, the effect of PBE was also investigated in the human keratinocyte cell line HaCaT. PBE, added to the cell culture medium, inhibited UVR-induced NF-kappaB-dependent gene expression in a concentration-dependent manner. However, NF-kappaB-DNA-binding activity was not prevented, suggesting that PBE affects the transactivation capacity of NF-kappaB. These data indicate that oral supplementation of PBE reduces erythema in the skin. Inhibition of NF-kappaB-dependent gene expression by PBE possibly contributes to the observed increase in MED.

Effect of renutrition on humoral and cell-mediated immunity in severely malnourished children.

Forty-three Colombian children suffering from either kwashiorkor (21), combined protein-calorie malnutrition (11), or maramus (11) were hospitalized and provided a high protein, high calorie diet for 4 to 5 wk. Improvement in clinical and nutritional status was accompanied by significant increases in levels of serum immunoglobulins G and M and C3 complement and by significant decreases in serum immunoglobulin A concentrations, especially in infants with kwashiorkor. Skin test reactions to purified protein derivative and candidin improved during renutrition. Lymphocyte blastogenesis after stimulation in vitro with phytohemagglutinin and pokeweed mitogen increased rapidly during hospitalization. After 1 yr posttreatment, cell-mediated immune responses, both in vivo and in vitro, had diminished. These results indicate that some aspects of the immune response are affected to a different degree in kwashiorkor, maramus, and combined malnutrition. Short-term nutritional rehabilitation has a differential effect on the long-term restoration of various aspects of immunity.