Heart rate variability: comparison among devices with different temporal resolutions.

Several devices based on ECG can acquire beat-to-beat intervals, and some of these devices may be used for heart rate variability (HRV) analysis. Most of these devices and their methods to acquire the beat-to-beat intervals need to be validated for HRV analysis by comparing them against traditional methods. Some of these methods have low temporal resolution, which may be sufficient for certain studies. HRV analysis parameters obtained from two methods differing in temporal resolution were compared to the HRV analysis parameters obtained from a referent method (ECG). One of the methods had a high resolution (<1 ms) and the other method had a low resolution (10 ms). Seven healthy male volunteers participated in the study. The beat-to-beat intervals were collected simultaneously from the three methods and the HRV parameters derived from them were used for our comparisons. The Pearson product-moment correlations were used, which demonstrated an excellent correlation (r > 0.99) in time domain, frequency domain and some non-linear HRV measures. The HRV measures were further analyzed using the one-way repeated-measures analysis of variance (ANOVA) and statistically significant differences were observed in some of the HRV parameters obtained from the low-resolution method, especially the mean RR (p < 0.001) and the mean heart rate (p < 0.001). Since the same software was used for HRV analysis of the three methods, any differences were due to the temporal resolution of the RR intervals measured by the methods. Overall, both the methods correlated very well with the referent method among healthy volunteers in resting conditions and may be used by researchers for HRV studies.

Marked elevation of serum interleukin-6 in patients with cholangiocarcinoma: validation of utility as a clinical marker.

OBJECTIVE: To determine if the serum level of interleukin-6 (IL-6) was elevated in patients with hepatic malignancies or correlated with radiologic tumor burden. SUMMARY BACKGROUND DATA: High serum levels of IL-6 signify an adverse prognosis in many patients with cancer. IL-6 is a growth factor for bile duct epithelium. METHODS: Using bioactive and enzyme-linked immunosorbent assays, serum level of IL-6 was measured in 35 healthy adults and in 60 patients presenting for definitive management of cholangiocarcinoma (CC) (15 patients), hepatocellular carcinoma (HCC) (14), metastatic colorectal cancer (MCRC) (26), and benign biliary disease (BBD) (5). Patients with clinical conditions known to raise the serum level of IL-6 were excluded. Tumor burden was calculated from concurrent computed tomography scans. IL-6 levels were measured 2 weeks after resection in 3 CC patients. Secretion of IL-6 was examined in 3 human CC cell lines. RESULTS: An elevated level of bioactive IL-6 was detected in every patient with CC and in 13 of 14 patients with HCC, 14 of 26 patients with MCRC, 2 of 5 patients with BBD, and 3 of 35 healthy adults. Median and mean levels of bioactive IL-6 were higher in CC than in other neoplasms (p < 0.026) and for all tumor groups differed from healthy adults (p < or = 0.026). IL-6 level was elevated more often in primary than in secondary liver neoplasms (p = 0.02), distinguished patients with CC or MCRC from BBD (p = 0.014 and 0.031, respectively), correlated with tumor burden in CC (p < 0.001), and dropped sharply after CC resection. CC line SG231 secreted bioactive IL-6. CONCLUSIONS: In selected patients, a high serum level of IL-6 marks patients with CC and correlates with tumor burden both before and after resection. IL-6 levels are elevated in patients with other liver neoplasms and may distinguish patients with hepatic malignancies from those with benign disease.

FLT3 ligand induces the generation of functionally active dendritic cells in mice.

FLT3 ligand (FL) is a recently described hematopoietic growth factor that stimulates the proliferation and differentiation of hematopoietic progenitors. We have investigated the effect of FL on murine hematopoiesis and dendritic cell (DC) generation and accumulation in lymphoid tissues and liver in vivo and in vitro evaluating the morphologic, phenotypic, and functional characteristics of these DC. We have observed extramedullary hematopoiesis in the mouse spleen with all lineages of hematopoietic cells represented after the administration of FL. Injection of FL results in a time-dependent and reversible accumulation of DC in the spleen, bone marrow, lymph nodes, and liver. Both flow cytometry and immunohistochemistry revealed a significant accumulation of DC in these tissues. Results of mixed leukocyte reaction suggested that these cells, isolated from murine bone marrow or spleen, were active as antigen presenting cells. Furthermore, cultivation of splenic and marrow cells with GM-CSF and IL-4 gave rise to large numbers of functionally active mature DC. Thus, the results of this study suggest that FL is a promising growth factor that stimulates the generation of large number of DC and may be a useful cytokine for the immunotherapy of cancer.

A comparison of locoregional depot and systemic preparations of 9-aminocamptothecin for treatment of liver metastases in a rat tumor model: superior antitumor activity of sustained-release preparation.

BACKGROUND: Locoregional therapy of hepatic-metastatic disease may overcome the limitations of systemic therapy by allowing tumor drug dose intensification without increasing systemic toxicity. This may result in improved efficacy. We have demonstrated that the novel topoisomerase I inhibitor 9-Aminocamptothecin (9-AC) when dissolved in Ethiodol acts as a sustained release preparation, with good antitumor activity. Its benefit as a depot hepatic intraarterial (i.a.) therapy for hepatic metastases was compared to systemic therapy with an aqueous colloidal dispersion (CD) preparation of 9-AC in a rat model, since a lack of demonstrable benefit of the locoregional therapy, would argue against further clinical evaluation. METHODS: Fisher rats underwent direct intraportal injection of 2 x 10(5) MADB106 adenocarcinoma cells and were treated 6 to 7 days later with: (a) bolus i.a. 9-AC (60 micrograms) in 60 microliters of Ethiodol; (b) bolus i.a. CD/9-AC (60 micrograms) in 200 microliters water; (c) bolus i.a. Ethiodol only (60 microliters), or (d) CD/9-AC (60 micrograms) in 200 microliters water, via a mini-osmotic pump pumping at 1 microliter/hr for 7 days intraperitoneally (i.p.). Livers were harvested 10 to 12 days later, and the number of metastases on the surface were counted blindly. RESULTS: Bolus hepatic i.a. 9-AC/Ethiodol was found to be significantly superior in reducing the number of hepatic metastases, when compared to the aqueous CD preparation administered in the same manner, or by continuous infusion via mini-osmotic pump i.p. (p < 0.01). Systemic therapy was also associated with substantial toxicity. CONCLUSIONS: These results suggest that locoregional therapy of hepatic neoplasms with 9-AC/Ethiodol would be associated with clinical efficacy far exceeding that associated with its systemic administration.

Treatment of hepatic-metastatic colorectal cancer with a chemotherapeutic emulsion: interim results of a phase I trial.

BACKGROUND: Hepatic arterial infusion of 5-fluoro-2-deoxyuridine (FUdR) is associated with a 60% response rate among previously untreated patients who have hepatic-metastatic colorectal cancer. One obstacle to further dose escalation has been concomitant hepatic toxicity. We are evaluating a FUdR-containing chemotherapeutic emulsion to further dose intensify therapy without associated toxicity. METHODS: The in vitro pharmacokinetics of the emulsion were determined using high-pressure liquid chromatography (HPLC). The rate at which FUdR is released from emulsion into an overlying aqueous phase was determined in static and dynamic assays. Fifteen patients with hepatic-metastatic colorectal cancer were treated with intrahepatic arterial infusions of emulsion on a phase I dose-escalating clinical protocol. Serum collection determined systemic drug levels using HPLC. RESULTS: In vitro studies demonstrate that FUdR is slowly released from emulsion into overlying aqueous medium. The emulsion serves as a depot for FUdR. Therapy was well tolerated. Emulsion was sequestered in the liver after infusion in all treated patients. CONCLUSIONS: This Ethiodol-based, oil-in-water emulsion serves as a sustained-release preparation of FUdR. An Ethiodol-based oil-in-water emulsion is a clinically effective vehicle for delivering FUdR to hepatic-metastatic colorectal tumors.

Surface aminopeptidase activity of rat natural killer cells. I. Biochemical and biological properties.

Aminopeptidase (AP) activity on rat natural killer (NK) cells was found to have the following characteristics: (1) the activity was surface associated and not secreted, as determined by extracellular location of product and by the cessation of hydrolysis of substrate upon removal of the cells from the medium. (2) The activity was linear with respect to time and cell number. (3) The enzymatic activity on splenocytes and on the NK leukemia cell line CRNK-16, but not on IL-2 activated NK (A-NK) cells, was sensitive to trypsin treatment. (4) The AP activity on intact cells had a broad pH dependency with optimal activity at slightly alkaline pH but lower activity at acidic pH. (5) There was a preference for neutral substrates and essentially no activity towards acidic substrates. (6) Enzymatic activity was inhibited in the presence of the AP inhibitors bestatin and amastatin, and in the presence of the chelator, 1,10 phenanthroline, indicating the involvement of a metalloprotease. (7) Culture of A-NK cells with bestatin resulted in a decrease in cytotoxicity against YAC-1 and P815 targets. Amastatin treatment caused only a slight decrease in cytotoxicity against YAC-1 targets, but a significant decrease in cytotoxicity against P815 targets. (8) Treatment of A-NK cultures with specific inhibitors of APases caused an increase in expression of CD2 (an increase from 20-80% with bestatin and an increase from 25-35% in the presence of amastatin). These results provide the first evidence for the existence of APases on the surface of NK cells and suggest a role for these enzymes in the regulation of cytotoxic activity and of CD2 surface expression.

Functional heterogeneity between NKR-P1bright/Lycopersicon esculentum lectin (L.E.)bright and NKR-P1bright/L.E.dim subpopulations of rat natural killer cells.

In this report, we present data on heterogeneity of rat NK cells utilizing a combination of antibody and lectin-binding characteristics. Among NKR-P1bright NK cells, two discrete populations characterized as Lycopersicon esculentum lectin (L.E.)bright (60 to 80%) and L.E.dim (20 to 40%) were identified by flow cytometry. Comparison of the morphology of sorted NKR-P1bright/L.E.bright and NKR-P1bright/L.E.dim cells indicated that both were greater than 90% LGL. An analysis of the functional capabilities of the sub-populations indicated that NKR-P1bright/L.E.bright NK cells were more efficient in lysis of YAC-1 target cells (1743 LU20/10(7) cells) than were NKR-P1bright/L.E.dim cells (504 LU20/10(7) cells). Conversely, NKR-P1bright/L.E.dim NK cells were much more efficient at lysis of antibody-sensitized erythrocytes (antibody-dependent cellular cytotoxicity (ADCC)) (1412 LU20/10(7) cells) than were NKR-P1bright/L.E.bright cells (165 LU20/10(7) cells). Lysis of antibody sensitized P815 target cells yielded similar results as NKR-P1bright/L.E.dim cells and NKR-P1bright/L.E.bright cells had 905 LU20/10(7) and 189 LU20/10(7), respectively. Additional experiments indicated that NKR-P1bright/L.E.bright NK cells had the capacity to trigger lytic activity via NKR-P1 whereas NKR-P1bright/L.E.dim NK cells did not. NKR-P1bright/L.E.bright sorted cells had a greater capacity to form conjugates with YAC-1 target cells than did NKR-P1bright/L.E.dim sorted cells. Conversely, NKR-P1bright/L.E.dim NK cells were demonstrated to form E-A rosettes whereas the NKR-P1bright/L.E.bright NK cells were not. Additional experiments indicated that tomato lectin itself was not responsible for the differences in reverse ADCC activity or ADCC activity among the subsets. However, lysis of YAC-1 target cells was modulated to some degree by the lectin. These data indicate that NKR-P1bright/L.E.bright and NKR-P1bright/L.E.dim subpopulations of rat NK cells have different capacities for: 1) triggering through NKR-P1; and 2) E-A rosette formation and lysis of antibody-sensitized target cells by ADCC.

Natural killer cell cytolytic granule-associated enzymes. I. Purification, characterization, and analysis of function of an enzyme with sulfatase activity.

An enzyme with sulfatase activity has been isolated from the granules of a rat NK leukemia cell line, CRNK-16. The enzyme has been purified from crude preparation, with a specific activity of 52 nmol/min/mg of protein, by DEAE ion exchange and Con A-Sepharose affinity chromatography, resulting in a specific activity of 230 nmol/min/mg of protein. The molecular mass of the purified enzyme was estimated to be 40 kDa by gel filtration chromatography at pH 7.4, but the enzyme had the ability to complex to molecular masses of greater than 300 kDa at low pH when crude granule extract was used as the starting sample, suggesting that it associates with other granule components. The enzyme was determined to be an arylsulfatase by its ability to (a) hydrolyze p-nitrophenyl sulfate (Km = 26.0 mM) and p-nitrocatechol sulfate (pNC sulfate) (Km = 1.1 mM) and (b) be inhibited by sulfite (Ki = 6.0 x 10(-7) M), sulfate (Ki = 1 x 10(-3) M), and phosphate (Ki = 4 x 10(-5) M) in a competitive manner. The pH optimum for enzymatic activity was determined to be 5.6. The role of this enzyme in cytolytic function was investigated by examining the effect of its substrates and inhibitors on granule- and cell-mediated lysis. pNC sulfate was shown to cause a dose-dependent inhibition of target cell lysis by isolated cytolytic granules (complete inhibition at 12.5 mM). Sulfite induced an incomplete inhibition (50% at 1 mM), whereas phosphate was essentially without inhibitory effect. Sulfate, on the other hand, altered lytic activity in a biphasic manner, inasmuch as it induced an inhibition of lysis at high concentrations and an increase of lysis at low concentrations. Cell-mediated lysis was inhibited by pNC sulfate in a dose-dependent fashion at concentrations greater than 2.5 mM, with nearly complete inhibition at 50 mM. Sulfate also altered the lytic activity by intact cells in a biphasic manner, although the effect was much less pronounced. Sulfite and phosphate caused only a 30% inhibition of lytic activity. These results suggest that the sulfatase enzyme is involved in NK cytolytic function, presumably at the lethal hit stage.

Alterations in cell-surface carbohydrates of rat large granular lymphocytes associated with interleukin-2 activation.

The activation of large granular lymphocytes (LGLs)/natural killer (NK) cells with interleukin-2 (IL-2) has been shown to increase the ability of these cells to lyse NK-resistant tumor target cells. Activated LGLs, termed LAK (lymphokine-activated killer) cells, have been demonstrated to be of therapeutic value in vivo against metastatic tumors. The mechanism by which IL-2 induces broadened cytolytic capability, as well as the molecular basis of target recognition and killing by the activated cells has not yet been elucidated. Since carbohydrate moieties have been demonstrated to be of possible significance in the cytolytic cascade of a variety of effector cells, the current study was undertaken to determine if the activation of LGLs with IL-2 is accompanied by an alteration of cell-surface carbohydrates. Two-color flow cytometry was performed to identify LGL/NK cells in populations of nylon wool-nonadherent splenic mononuclear cells and to assess the binding of various lectins to activated as well as nonactivated LGLs. Increases were observed in the binding of four lectins to LGLs after IL-2 activation; Triticum vulgaris (wheat germ agglutinin), Phytolacca americana (pokeweed mitogen), Lycopersicon esculentum (tomato lectin), and Griffonia simplicifolia I-B4 (GSI-B4). The wheat germ, pokeweed, and tomato lectins recognize complex carbohydrates structure consisting of GlcNAc(Bl,4GlcNAc)n while GSI-B4 recognizes alpha-D-galactose terminal end groups. Lectin binding to the activated LGLs was homogenous (i.e., flow cytometry revealed only a single population of fluorescent cells). Lectin binding to LGLs prior to activation was more heterogeneous, however, the tomato lectin uniquely revealed a bimodal distribution of receptors. These data indicate that LGL/NK cells from the rat are heterogeneous in their ability to bind specific lectins, and that IL-2 activation of these cells results in altered expression of specific cell-surface carbohydrates.

Monoclonal antibody probes for the niaD specified subunit in the NADPH-nitrate reductase from Aspergillus nidulans.

In Aspergillus nidulans, the nitrate assimilatory pathway is regulated by a variety of agents, one being the autogenous enzyme nitrate reductase. A major subunit of the enzyme which is specified by the niaD structural gene and is implicated in autogenous control exhibits both nitrate inducible diaphorase activity and ammonium repression. The former was used to test the extent to which alterations in the niaD specified protomer might affect its formation in selected niaD point and deletion mutants. Enzyme preparations from the wild type and mutant strains were compared on the basis of nitrate inducible co-activities and their reaction to specific monoclonal antibodies (MABS). Proteins in partially purified mycelial extracts were subjected to Western blot analyses with three MABs to functional native enzyme. Extracts of niaD point mutants exhibited nitrate induced co-activities which matched those of the wild type while those from deletion mutants were diminished or totally inactive. Nitrate reductase, from the wild type and specific cofactor mutants, shares an epitope common to both the monomeric and dimeric form in the case of one MAB, and exhibits epitopes unique to one or the other form in the case of the other two forms. Enzyme-antibody interaction occurs with or without inhibition of catalytic activity depending on the MAB involved.