Technetium-99m-labeled monoclonal antibodies for immunoscintigraphy. Simplified preparation and evaluation

Ascorbic acid incubated with monoclonal antibodies (22 degrees C, 60 min, pH 6.5) at a molar ratio of 3500:1, reduced 2.7 +/- 0.2% of the available disulfides to sulfhydryl groups that strongly bind 99mTc, and provided greater than 95% labeling efficiency for several IgM, IgG and F(ab')2 antibodies. The colloid formation was consistently less than 3% and the stability of the tracer when challenged with DTPA and cysteine was excellent. The immunospecificity of labeled antibodies as determined by immobilized specific antigen assay was 84 +/- 1% for IgM and 82.6 +/- 1.1% for IgG antibodies. For in vivo evaluation in mice bearing experimental abscesses and tumors, corresponding 125I-labeled antibodies served as controls. The liver uptake was similar (P = 0.76 and P = 0.12) for 99mTc or 125I labeled antinuclear antibody TNT-1 in mice bearing abscesses as well as for 99mTc-TNT-1-F(ab')2 and 125I-TNT-1-F(ab')2 in mice bearing tumors. Higher but statistically insignificant (P = 0.08, 0.18, and 0.73) urinary excretion was noted for 99mTc-antibodies. For corresponding 99mTc- and 125I-labeled antibodies, the abscess to muscle ratios (3.3 +/- 0.5 vs. 3.4 +/- 0.8) and tumor to muscle ratios (10.04 +/- 4.4 vs. 10.54 +/- 3.0) were similar. The high 99mTc-TNT-1-F(ab')2 uptake permitted excellent scintigraphic visualization of tumors whereas the nonspecific 99mTc-HSA did not (tumor/muscle ratio: 2.4 +/- 0.3). This method is simple, reliable, and adaptable to an instant labeling technique.     

Tumour necrosis factor production and cell-mediated immunity in anorexia nervosa.

Fourteen patients with anorexia nervosa (AN) were studied for the production of tumour necrosis factor (TNF), the activation of the interferon (IFN) system and cell-mediated cytotoxicity (CMC) and the results were compared with 16 age-matched healthy women. AN patients had significantly increased spontaneous TNF production by peripheral blood mononuclear cells (PBMC) in vitro (16 +/- 5 U/ml versus 4 +/- 3 U/ml in the control group; P less than 0.05), although no TNF was detectable in the plasma from either group. TNF production in vitro, following stimulation of PBMC by phytohaemagglutinin (PHA) or tumour cells, was similar in AN patients and controls; however, lipopolysaccharide (LPS) induced TNF production was found to be lower in AN (P less than 0.1). CMC was significantly lower in AN patients (4 +/- 2 versus 10 +/- 3 in controls, expressed as lytic units/10(6) cells; P less than 0.05), but no difference could be found between AN and controls in IFN activity as reflected by the level of the IFN-induced enzyme 2'-5' oligoadenylate synthetase (2-5A) in PBMC. Beta-endorphins in the plasma were higher in the AN group (P less than 0.05) but these levels could not be correlated to those of IFN, CMC or TNF. Defective CMC and increased TNF production by PBMC in patients with anorexia nervosa may possibly result from the nutritional deficiencies and neuroendocrine abnormalities associated with the disease, and may contribute to the pathophysiology of AN.     

Kinetics of serum soluble tumour necrosis factor receptor (TNF-R) type-I and type-II after a single interferon-alpha (IFN-alpha) injection in chronic hepatitis C.

Circulating soluble TNF receptors, which act as TNF inhibitors, increase following the administration of IFN-alpha. Whether this is due to a direct IFN action or to indirect mechanisms involving the release of other cytokines is unclear. The kinetics of serum IFN, TNF, IL-6, IL-10, soluble TNF receptor type-I (sTNF-RI) and sTNF-RII were evaluated by enzyme immunoassays in 11 patients with chronic hepatitis C, following the first dose of recombinant human IFN-alpha2b (3 MU given subcutaneously). sTNF-RI concentrations paralleled IFN concentrations, rising from a mean +/- s.e.m. value of 3.5 +/- 0.3 ng/ml at baseline to a peak value of 5.5 +/- 0.5 ng/ml after 9 h, followed by a return to 4.1 +/- 0.4 ng/ml after 24 h (P = 0.0001). sTNF-RII concentrations, which were 7.6 +/- 0.5 ng/ml at baseline, fell initially to 6.9 +/- 0.5 ng/ml, to reach a peak at 24 h of 9.0 +/- 0.7 ng/ml (P < 0.0001). In contrast, the concentrations of TNF, IL-6 and IL-10 fluctuated with no significant changes at any time point. The area under the curve (AUC) of incremental IFN values had a strong positive correlation with the AUC of incremental sTNF-RI values (r = 0.75, P < 0.01). In patients with hepatitis C, IFN concentrations reached after a single dose of IFN were paralleled by correlationally increased concentrations of sTNF-RI, which are a much better marker of administered IFN than sTNF-RII, IL-6 or IL-10.     

Increased uptake of 99mTc-HMPAO in necrotic brain tumors.

99mTc complex of hexamethylpropylene amine oxime (99mTc-HMPAO), which has been used as a tracer for regional cerebral blood flow (rCBF), has been shown to localize in primary brain tumors with wide spectrum of its uptake. The causes of the wide spectrum of tumor uptake, however, has not been understood in detail. We performed autoradiographic study with this agent to get further knowledge about HMPAO distribution in 10 cases of transplanted rat gliomas. Eight cases of rat gliomas without tumor necrosis, showed decreased uptake of 99mTc-HMPAO in the autoradiography (average tumor/normal (T/N) uptake ratio: 0.75, range: 0.40-0.90). On the other hand, two cases with tumor necrosis revealed increased uptakes of this agent in central necrotic area. T/N uptake ratios of these two cases were 1.23 and 1.42, respectively. In addition, three patients with histologically proven glioblastoma with tumor necrosis were studied after administration of 20mCi 99mTc-HMPAO. Two out of three patients showed higher uptake of 99mTc-HMPAO in tumor necrotic area than the contralateral area. Our findings suggest that the necrotic area of brain tumor may retain 99mTc-HMPAO and causes an increased uptake.     

Cytokine expression in labial salivary glands from patients with primary Sj?gren's syndrome.

The presence and distribution of 7 cytokines was examined immunohistologically in labial salivary gland (LSG) specimens from patients with primary Sj?gren's syndrome (SS) and control subjects. The cytokines interleukin (IL)-1 beta IL-6, tumor necrosis factor (TNF) alpha and interferon (IFN) gamma were identified in defined parts of LSG from patients but not in the corresponding parts of control glands: (a) LSG acinar epithelium expressed IL-1 beta, (b) blood vessels located in both normal LSG stroma and within lymphocytic infiltrates expressed IL-1 beta, IL-6 and IFN gamma, and (c) lymphocytic infiltrates expressed IL-1 beta, IL-6 and TNF alpha. All four cytokines were expressed by salivary ducts within both patient and control specimens, but with generally greater intensity in patients. IL-1 alpha, IL-4 and TNF beta (lymphotoxin) could not be detected in any of the specimens from patients or controls. The locations of cytokines in LSG suggests possible mechanisms of immunologically mediated parenchymal damage in primary SS.     

Augmentation of Antimetastatic Activity of Interferon and Tumor Necrosis Factor by Heparin

Interferon (IFN) and tumor necrosis factor (TNF) suppress the development of experimental metastasis and when used together, TNF and IFN show synergistic activity. However, the use of TNF is limited by its ability to initiate inappropriate hemostasis. Hemostatic effects are exaggerated by the procoagulant activity of certain tumor cell lines. Therapy with anticoagulants are indicated to block the effects of tumor cell products as well as chemotherapeutic side effects. Heparin is a glycosaminoglycan with diverse biological activity, including the ability to rapidly accelerate the inactivation of active clotting factors. the present studies have explored the therapeutic effects of combining heparin with TNF or interferon on experimental metastasis in mice using a melanoma cell line (B16BL6). Our data indicate that continued heparinization augments the antitumor activity of both interferon and TNF. Alterations of the hemostatic and immune systems play a role in the producing the observed effect.     

Rickettsia australis infection: a murine model of a highly invasive vasculopathic rickettsiosis.

A mouse model of spotted fever group rickettsiosis, in which disease results from disseminated rickettsial infection of endothelial cells and vascular damage, was developed by intravenous inoculation of 6- to 8-week-old, male, Balb/c mice with Rickettsia australis. Animals developed progressively severe vasculitis, interstitial pneumonia, and multifocal hepatic necrosis. These lesions correlated with early disseminated infection of endothelial cells followed by growth and invasion of rickettsiae into perivascular cells. The dose of 2 x 10(6) organisms was uniformly lethal. Serum interleukin- (IL) 1, IL-6, and interferon (IFN) increased by day 3 and tumor necrosis factor (TNF) on day 5. TNF, IL-6, and IFN declined on day 7. Spleen cells responded to Rickettsia australis antigen by producing IFN, TNF, IL-1, and IL-6 on day 5, followed by lower quantities of these cytokines on day 7. Despite the production of antibodies, IFN, TNF, IL-1, and IL-6, a lethal outcome occurred frequently. A decreased ability to secrete IL-2 suggests an element of infection-associated immunosuppression.     

In vivo analysis of intracellular thallium-201 accumulation in skeletal muscle of the rat.

The specific accumulation of the K(+)-analogue Tl+ (201Tl+) in muscle after intramuscular injection was analysed by gamma spectrometry in vivo of rat hamstring muscles. A mixture (0.1 ml) of 201Tl+ (thallous+ chloride-) and 99mTc-pertechnetate- (Na+ pertechnetate-) was given, by which 99mTc-pertechnetate- served as a reference substance with negligible intracellular accumulation. After 30 min 8.9 +/- 5.8% of injected 99mTc-pertechnetate- remained in the muscle and 49 +/- 10% of 201Tl+ (+/- SD, n = 18). The difference between 201Tl+ and 99mTc-pertechnetate- at 30 min was taken as a measure of the intracellular 201Tl+ accumulation, which was 40% of the initial amount of 201Tl+. The half-time of the calculated intracellular 201Tl+ accumulation was 4.9 +/- 1.9 min. In the presence of ouabain (1.0 mM in the injectate) the intracellular 201Tl+ accumulation was 25 +/- 10% (n = 7), that is ouabain decreased the intracellular 201Tl+ accumulation by 38% (P = 0.0035). Non-radioactive Tl+ (1.0 mM Tl-acetate in the injectate) inhibited the uptake by 35% (P = 0.0013). Ouabain did not significantly affect the half-time for the Tl+ uptake. An increase in [K+] of the injectate from 0 to 5 mM had no significant effect. Insulin (0.2 units in the injectate) had no effect. It is concluded that the specific Tl(+)-accumulating properties of muscle fibres can be studied with the present in vivo technique, which can provide information about the Na-K-ATPase activity and the membrane potential of muscle fibres.     

Augmentation of Antimetastatic Activity of Interferon and Tumor Necrosis Factor by Heparin

Abstract

Interferon (IFN) and tumor necrosis factor (TNF) suppress the development of experimental metastasis and when used together, TNF and IFN show synergistic activity. However, the use of TNF is limited by its ability to initiate inappropriate hemostasis. Hemostatic effects are exaggerated by the procoagulant activity of certain tumor cell lines. Therapy with anticoagulants are indicated to block the effects of tumor cell products as well as chemotherapeutic side effects. Heparin is a glycosaminoglycan with diverse biological activity, including the ability to rapidly accelerate the inactivation of active clotting factors. the present studies have explored the therapeutic effects of combining heparin with TNF or interferon on experimental metastasis in mice using a melanoma cell line (B16BL6). Our data indicate that continued heparinization augments the antitumor activity of both interferon and TNF. Alterations of the hemostatic and immune systems play a role in the producing the observed effect.     

Effect of tuftsin and its retro-inverso analogue on the release of interferon (IFN-gamma) and tumor necrosis factor (TNF-alpha) by human leucocytes.

The aim of this work was to demonstrate whether natural tuftsin or a retro-inverso (r.i.) analogue may induce interferon (IFN) and tumor necrosis factor (TNF) in peripheral-blood-mononuclear-cells (PBMC). For this purpose tuftsin or its analogue were added at different molar concentrations to PBMC and the supernatants were tested for IFN and TNF activity. Both cytokines were released after 12 hours incubation with r.i. tuftsin at an optimum concentration of 10(-10) M. Under the same conditions no activity was observed in the presence of natural tuftsin. In comparison to natural tuftsin the stimulatory activity of this tuftsin analogue is likely to be due to its high stability.     

Plasma cytokine concentration and the cytokine producing ability of whole blood cell cultures from healthy females with pharmacologically induced hyperprolactinemia

We investigated the in vitro effect of domperidone-induced hyperprolactinemia on plasma cytokine concentration and blood leukocyte cytokine production in healthy female volunteers. No changes were found in the plasma concentration of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-4, IL-10, IL-6 and IL-13 during hyperprolactinemia when compared with control values. Using unseparated blood leukocytes, we found that the spontaneous production of IL-6 (4-8 h) and transforming growth factor (TGF)-beta 1 (2-4 h) was significantly decreased and that the in vitro stimulated production of IFN-gamma (2-8 h) and TNF (4 h) was significantly increased compared with control. Our data concerning the increased IFN-gamma and TNF producing capacity of unseparated leukocytes during pharmacologically induced hyperprolactinemia strongly support the possibility that the lymphocyte production of these cytokines can be rapidly amplified by prolactin via a priming mechanism.     

Differential regulation of cytokine production in lipopolysaccharide tolerance in mice.

We investigated the pattern of down-regulation of cytokine production in endotoxin (lipopolysaccharide [LPS]) tolerance. A 4-day treatment with LPS (35 micrograms per mouse) was followed by a challenge on day 6 with one more injection of LPS. Circulating tumor necrosis factor (TNF) and interleukin-6 (IL-6) could not be induced (> 99% inhibition) by LPS in LPS-tolerant mice; colony-stimulating factor (CSF) was also down-regulated by more than 95%, whereas interferon (IFN) and IL-1 syntheses were only partially inhibited. To study the mechanism of cytokine down-regulation in tolerance, we attempted to reverse the tolerant state by pretreatment with phorbol 12-myristate 13-acetate (PMA) (4 micrograms per mouse) 10 min before the LPS challenge. PMA completely restored IL-6 production and partially that of CSF. PMA had no effect on IFN production and inhibited the induction of IL-1. TNF production was also not restored by PMA. To investigate the role of endogenously produced cytokines in the development of LPS tolerance, we administered IL-6, TNF, or IL-1 alpha, using the same treatment schedule as that for LPS. Whereas IL-6 had no effect, IL-1 alpha or TNF induced partial tolerance to LPS in terms of inhibition of LPS-stimulated TNF and IL-6 production. However, a full LPS-tolerant state could not be induced by administration of recombinant cytokines, suggesting the existence of additional mechanisms, such as a loss of LPS receptors or changes in release of soluble binding proteins.     

Kinetics of receptor-mediated uptake and processing of interferon-alpha 2a and tumor necrosis factor-alpha by human tumor cells

The kinetics of uptake and processing of recombinant human interferon-alpha 2a (IFN) and recombinant human tumor necrosis factor-alpha (TNF) were studied in human epithelial tumor cell lines differing in sensitivity to growth inhibition by IFN and TNF. Concentrations of [125I]IFN or [125I]TNF at the cell surface and internalized by confluent cell monolayers incubated at 37 degrees C were measured as a function of time. Cells incubated with [125I]IFN exhibited transient maxima of surface-associated and internalized [125I]IFN after 1-2 hr of incubation followed by a steady-state attained after approximately 6 hr of exposure to [125I]IFN. Concentration of [125I]TNF associated with the plasma membrane displayed a maximum within 20 min of incubation. Internalized radioactivity increased with time and did not achieve a steady state. We analyzed the time-dependent concentrations of membrane-associated and internalized cytokines by use of a compartmental model. This model describes changes in concentrations of free surface receptors, of ligand-receptor complex at the membrane and of internalized ligand and contains a term for receptor recycling. The rate constants for concentration changes were evaluated by fitting model functions to data. The best fits for IFN were obtained without receptor recycling. The best-fit values for the endocytotic rate constant (ke,IFN) varied among cell lines from 2.4 x 10(-4) to 7.8 x 10(-4) sec-1. To obtain fits to time-dependent concentrations of surface-associated and internalized [125I]TNF, introduction of a term for receptor recycling was required. Best-fit values for ke,TNF ranged among cell lines from 8.4 x 10(-4) to 2.5 x 10(-3) sec-1. For every cell line, the value of ke,TNF was larger than the value of ke,IFN. We tested the significance of these differences by substituting ke,IFN for ke,TNF as fixed parameters in fits to data for TNF and v.v., respectively. Under these conditions, fits were significantly worse. Our data indicate that recycling is insignificant in kinetics of Type-I IFN receptor; recycling is necessary to explain the kinetics of TNF receptor. Upon binding, TNF is taken up by cells faster than IFN and is eliminated from cells more slowly than IFN.     

Macrophage activating factors produced in the course of murine tularemia: effect on multiplication of microbes.

Primary F. tularensis infection in mice induces the production of macrophage activating factors (MAFs) by spleen cells. The stimulation of macrophage cytolytic activity (MAF-c) and hydrogen peroxide production (MAF-H2O2) dominates between days 7 and 10 in the course of tularemia. Three various pools of active fractions (10-11, 14-15, 25-28) were fractionated by two-step chromatography. Typical for 10-11 and 14-15 is MAF-c activity whereas in 25-28 prevails MAF-H2O2. Initial concentrated supernatant (day 7 of infection) and individual fractions have been used to raise antibodies KI (anti 10-11) and KII (anti 14-15). Neutralization reactions with specific antibodies indicate the presence of tumor necrosis factor alpha (TNF alpha) in 14-15 (44% inhibitable), interferon gamma (IFN gamma) and interleukin 2 (IL 2) in 25-28 (65% and 30% neutralization, respectively). Utilizing KI and KII, 99% and 90% inhibition of cytolytic activity is reached in 10-11 and 14-15, respectively, in spite of non-specific cross reaction. Western blot analysis of proteins in supernatant on day 7 detects, besides TNF alpha, further protein bands (13, 15.5, 52 and 72 kDa) that seem to be associated with macrophage activation. Significant protective effect against in vivo multiplication of tularemic microbes indicates a certain role of TNF alpha, however, cooperation of other molecules is worth to be taken into consideration.     

99m Tc 标记单克隆抗体Au14-1对荷宫颈癌小鼠的放射免疫显像研究

目的和方法：应用99mTc直接法标记抗小鼠宫颈癌（U14）单克隆抗体Au14-1,对荷瘤Km小鼠进行放射性分布和放射免疫显像的实验观察,探讨用99mTc－An14-1作宫颈癌导向诊断和导向治疗的可能性。结果：（1）99mTc－Au14-1注射后12h肿瘤灶已有放射性聚集,24h肿瘤组织的％ID／g值达8．79,呈明显特异性浓聚;（2）除肾脏外,各脏器的T／NT值在2．02~6．71之间,SPECT图像12h已见肿瘤显影,24h肿瘤影像更为清晰。结论：99TcTc－An14-1的体内分布与显像结果相一致,表明An14-1在体内具有良好的宫颈癌导向定位作用。     

Characterization of a tumor necrosis factor-alpha inhibitor activity in cancer patients.

To evaluate the feasibility of tumor necrosis factor-alpha (TNF-alpha) treatment of lung cancer patients, we chose the malignant cells contained in their pleural effusions as a first convenient target. We found, however, that a TNF-alpha inhibitor (TNF-alpha I) activity was present in both patient sera and pleural fluids. We therefore compared the TNF-alpha I activity present in patients with benign or malignant pleural effusions using a bioassay of TNF-alpha inhibition and partially characterized it. A high TNF-alpha I activity characterizes cancer patients with sera levels twice as high as the control level measured for blood bank donors (2.54 +/- 1.28 versus 1.19 +/- 0.38) and with even higher levels in pleural fluids (3.75 +/- 1.83). In contrast, patients with benign pleural effusions present similar levels of TNF-alpha I activity, at about the control level, in both their sera and pleural fluids (1.37 +/- 0.98 versus 1.16 +/- 0.85). A high TNF-alpha I activity is consistently found in cancer patients but is only released in vitro by leukocytes. It is most likely related to recently purified TNF-alpha inhibitors that, as soluble shed fragments of TNF receptors, may function as traps for TNF molecules. This study suggests that tumors may evade TNF cytotoxic action by modulating systemic levels of TNF and implies a reassessment of TNF therapy in cancer patients.     

Lack of protection against bacterial infections in patients with advanced cancer treated by biologic response modifiers.

A survey of patients with advanced cancer treated by biologic response modifiers (BRMs), including (i) recombinant interleukin-2 and lymphokine-activated killer cells, (ii) recombinant interleukin-2 and alpha interferon, and (iii) tumor necrosis factor, was done. A total of 52 patients were reviewed. A total of 73 courses of BRMs were administered. Prior to the initiation of therapy, all patients were infection free and not receiving antibiotics. Twelve patients developed bacteremia during treatment with these BRMs. Five of these 12 patients had catheter-related bacteremia. Six patients had bacteremic infections without an obvious source, and one patient had a urinary tract infection with bacteremia. Staphylococcus epidermidis accounted for six of the isolates. Other organisms were Staphylococcus aureus, group B streptococci, viridans group streptococci, and gram-negative bacilli. This was an unexpectedly high incidence of bacterial infections in patients treated with BRMs. These BRMs have been previously shown to be efficacious against infections (by bacteria and other intracellular organisms) in experimental animals. In this study BRMs did not influence host defense mechanisms or offer protection against bacterial infections.     

Protein A of Staphylococcus aureus evokes Th1 type response in mice.

Protein A (PA) of Staphylococcus aureus is known to elicit several cytokines such as IFN gamma, TNF alpha and IL1. However, it has not been delineated yet as to which differentiation pathway lymphocytes follow after stimulation by PA. In this report, we attempted to collect such evidences. Cytokines, such as IFN gamma, IL2, IL4, IL6, IL10, TNF alpha, IL1alpha and IL1beta were measured in serum by ELISA. Our results show that 1 microg dose of PA stimulates the production of IFN gamma (115 +/- 5 pg/ml), TNF alpha (250 +/- 8 pg/ml) and IL1alpha (100 +/- 5 pg/ml) as compared to control levels of, 22 +/- 2, 20 +/- 2 and 35 +/- 3 pg/ml respectively whereas IL2 and IL1beta secretion were less (beyond the lower detection limit of the kit and 25 +/- 1 pg/ml, respectively) as compared to control (28 +/- 2 and 52 +/- 4 pg/ml, respectively). Larger dose of PA (10 microg) increases the expression of IL2 (75 +/- 3 pg/ml), TNF alpha (1380 +/- 120 pg/ml), IL1alpha (495 +/- 10 pg/ml) and IL1beta (110 +/- 7 pg/ml) as compared to controls described above. We also observed that 1 microg dose of PA decreases IL4, IL6 and IL10 secretion to 9 +/- 1, 10 +/- 1 and 10 +/- 2 pg/ml, respectively, whereas 10 microg dose also decreased them to 11 +/- 1, 12 +/- 2 and 30 +/- 4 pg/ml, respectively as compared to the background controls, i.e. 50 +/- 5, 50 +/- 2 and 215 +/- 9 pg/ml respectively. The ratio of IFN gamma to IL4 increased and the peak value at 4 h, came to 13 +/- 1 and 9.6 +/- 0.5 with 1 microg and 10 microg PA, respectively, which is an established parameter indicating a Th1 type response. Flow cytometry analysis of CD4+/CD8+ cells, and c-myc protein expression by splenocytes indicate that 1 microg dose of PA causes 2-fold increase of CD4+ cells with no change in CD8+ cells, and 10-fold increase in c-myc protein, whereas 10 microg dose increases CD4+ cells 4-fold, CD8+ cells 3-fold and c-myc protein 100-fold. The cell cycle data shows an induction of apoptosis in thymocytes and splenocytes with the large dose (10 microg), whereas the 1 microg dose does not show any apoptosis. This report indicates that a Th1 response is induced in mice, after PA inoculation at a dose of 1 microg animal. Thus, cytokine mediated therapeutic strategies should consider the fact that an induction of large concentration of some cytokines might become detrimental to the host.     

Evolution of plasminogen activator inhibitor type 1 in patients with septic shock — correlation with cytokine concentrations

Serum concentrations of plasminogen activator inhibitor type 1 (PAI-1), of tumor necrosis factor alpha (TNFα), interferon alpha (IFNα), interferon gamma (IFNγ), interleukin 1 β (IL-β and interleukin 6 (IL-6) were measured in 51 patients with septic shock. PAI-1 concentrations at onset of shock correlated with those of TNFα (Spearman's rank correlation coefficient: r=0.383; p=0.007) and of IL-6 (r=0.529; p=0.0002), weakly with those of IFNγ (r=0.262; p=0.06), but not with ILB and IFNα (r=0.063 and -0.081, respectively). Within one day after admission PAI-1 concentrations decreased significantly (Wilcoxon signed rank test, p<0.0001) from 360ng/ml (median, 10–90% range: 54 6000ng/ml) to 144ng/ml (45–1200). IL-6 levels also decreased significantly (<0.0001) during the first day, whereas TNFot levels did not change. Extreme PAI-1 levels (>550ng/ml) at study entry were associated with a high mortality (12 out of 16 died within a week). PAI-1 concentrations decreased in all 11 patients (and normalised in 9) that survived the first 24 h. Despite normalisation of PAI-1, 7 of these patients died within a week, whereas of the 35 patients with initial PAI-1 levels below 550 ng/ml, only 3 died within a week.In conclusion, in patients and septic shock, PAI-1 levels at study entry correlate strongly with TNFα and IL-6. At study entry extreme PAI-1 levels are associated with a poor prognosis, but normalisation of PAI-1 within a day does not improve the prognosis.     

Elevation of creatine in red blood cells in vegetarians and nonvegetarians after creatine supplementation.

The purpose of this study was to examine the effect of a 5-day creatine (CR) supplementation period on red blood cell (RBC) CR uptake in vegetarian and nonvegetarian young women. Blood samples were collected from lacto-ovo vegetarians (VG, n = 6, age 21.8 +/- 1.9 yrs) and nonvegetarians (NV, n = 6, age 21.7 +/- 1.9 yrs) before and after a 5-day CR loading period (0. 3g CR/kg lean body mass/day), and from a control group of nonvegetarians (NV, n = 5, age 22.0 +/- 0.7 yrs) who did not supplement with creatine. RBC and plasma samples were analyzed for the presence of creatine. Significant increases (p < .05) in RBC and plasma CR levels were found for vegetarians and nonvegetarians following supplementation. The initial RBC CR content was significantly lower (p < .05) in the vegetarian group. There was no significant difference between vegetarians and nonvegetarians in final RBC CR content, suggesting that a ceiling had been reached. As the uptake into both muscle and RBC is moderated by creatine transporter proteins, analysis of the uptake of CR into RBC may reflect the uptake of CR into muscle, offering an alternative to biopsies.