Enolase 1 of Candida albicans binds human CD4+ T cells and modulates naïve and memory responses

To obtain a better understanding of the biology behind life-threatening fungal infections caused by Candida albicans, we recently conducted an in silico screening for fungal and host protein interaction partners. We report here that the extracellular domain of human CD4 binds to the moonlighting protein enolase 1 (Eno1) of C. albicans as predicted bioinformatically. By using different anti-CD4 monoclonal antibodies, we determined that C. albicans Eno1 (CaEno1) primarily binds to the extracellular domain 3 of CD4. Functionally, we observed that CaEno1 binding to CD4 activated lymphocyte-specific protein tyrosine kinase (LCK), which was also the case for anti-CD4 monoclonal antibodies tested in parallel. CaEno1 binding to naïve human CD4⁺ T cells skewed cytokine secretion toward a Th2 profile indicative of poor fungal control. Moreover, CaEno1 inhibited human memory CD4⁺ T-cell recall responses. Therapeutically, CD4⁺ T cells transduced with a p41/Crf1-specific T-cell receptor developed for adoptive T-cell therapy were not inhibited by CaEno1 in vitro. Together, the interaction of human CD4⁺ T cells with CaEno1 modulated host CD4⁺ T-cell responses in favor of the fungus. Thus, CaEno1 mediates not only immune evasion through its interference with complement regulators but also through the direct modulation of CD4⁺ T-cell responses.     

The pharmacokinetics of high-dose epirubicin and of the cardioprotector ADR-529 given together with cyclophosphamide, 5-fluorouracil,and tamoxifen in metastatic breast-cancer patients

A high-pressure liquid chromatographic method for determination of the bisdioxopiperazine derivative ADR-529 (ICRF-187), a compound proven effective in protection against anthracycline-induced cardiotoxicity, has been developed. The limit of quantitation was 5 ng/ml using a narrow-bore 5-m silica column and UV detection. The method was used for determination of pharmacokinetic profiles of ADR-529 after a 3-weekly i.v. administration of different doses of ADR-529 (600–1000 mg/m²) together with different doses of epirubicin (E, 60–100 mg/m²), fixed-dose cyclophosphamide (C, 600 mg/m²), fixed-dose 5-fluorouracil (F, 600 mg/m²), and daily administration of tamoxifen (T, 30 mg; CEF-T) in the treatment of patients with metastatic breast cancer. Pharmacokinetic parameters for epirubicin were also determined. The aim of the study was to determine (1) whether the pharmacokinetics of ADR-529 as part of a combination with CEF-T changes with increasing doses of ADR-529 and increasing doses of epirubicin and (2) whether the pharmacokinetics of epirubicin in the same combinations is altered with the administration of increasing doses of ADR-529. A total of 82 patients were included. A crossover study including 16 of the patients showed no significant difference in epirubicin pharmacokinetic parameters when epirubicin was given with or without concomitant administration of ADR-529. Apart from minor changes in the distributional half-lives, the pharmacokinetic parameters of epirubicin were not altered with increasing doses of ADR-529, nor were the pharmacokinetic parameters of ADR-529 itself. Escalating doses of epirubicin did not significantly alter the pharmacokinetic parameters of ADR-529 with the exception of a 30% increase in the terminal half-life and a decrease in total body clearance when the epirubicin dose was raised from 60 to 100 mg/m². We conclude that concomitant administration of ADR-529 does not alter the distribution and elimination of epirubicin in doses suitable for preventing the anthracycline-induced cardiotoxicity.     

Response of multicellular tumor spheroids to liposomes containing TNF-α

Summary This publication describes a new model to investigate the influence of tumor necrosis factor- (TNF-) on a three-dimensional glial cell aggregate under defined, standardized, reproducible conditions using the glioma cell line A 172.The cells are initially grown as normal monolayer culture until they reach a cell density of up to 1×10⁶. Subsequently they are grown as spheroids by the liquid overlay technique. Spheroids grown in this way were divided into ten groups of more than 50 cell aggregates. Three groups were coincubated with free TNF- in increasing dosages (100 ng/ml, 200 ng/ml and 1000 ng/ml); three groups were incubated with empty liposomes (0.2 mg/ml, 0.4 mg/ml and 2 mg/ml); three groups received liposomes which had been loaded with TNF-, and one group, which received no treatment, served as control.The diameter of the spheroids ranged from 80 m to 350 m. There was no significant difference in growth between the 3 groups treated with free TNF-. Comparing spheroids treated with TNF- with those which had been coincubated with empty liposomes, there was a significant difference (p<0.001) in growth, which correlated with the amount of liposomes. Similarly, free TNF- had a significantly (P<0.001) stronger growth-inhibiting effect as compared to liposomes loaded with TNF-. Comparing the groups treated with liposomes only to those treated with liposomes loaded with TNF-, the latter exhibited a more marked (although not significantly) growth-inhibiting effect.The preliminary conclusion is that the major growth-inhibiting effect seems to be mediated by the liposomes. This phenomenon is in agreement with results obtained in monolayer cultures.     

Use of limited color-flow duplex for a carotid screening project.

BACKGROUND: Although the efficacy of carotid endarterectomy for asymptomatic carotid stenosis has been established, no cost-effective approach for identification of these patients has yet been devised. The purpose of this study was to develop a limited carotid duplex screening examination to be utilized for the detection of asymptomatic carotid stenoses. METHODS: Carotid screening examinations employed rapid identification of the carotid bifurcation using color-flow duplex imaging and an immediate Doppler-derived velocity of the segment of the internal carotid artery with the most turbulent flow. Complete examinations were then finished using well-established protocols in our accredited vascular laboratory. A total of 512 patients were referred for complete studies based upon standard indications. Criteria for at least a 50% internal carotid artery stenosis on the complete examination was defined as a peak systolic velocity (PSV) of at least 125 cm/sec. Receiver operator characteristic (ROC) curves were then constructed to identify the optimal screening velocity criteria as compared with the final results on the complete examination. RESULTS: Five screening examinations were technically limited yielding a total of 507 patients with 1,014 carotid arteries available for analysis. Comparison of screening examinations versus complete examinations for a PSV of 125 cm/sec yielded sensitivity 86%, specificity 98%, positive predictive value (PPV) 95%, and a negative predictive value (NPV) 93%. ROC analysis identified a "cut point" of 115 cm/sec on the screening examinations to achieve sensitivity 91%, specificity 95%, PPV 89%, and NPV 96%. Time to perform screening examinations averaged 3.2 minutes per patient. Three patients had common carotid lesions not identified on the limited internal carotid screening examinations. CONCLUSIONS: Screening carotid examinations are a rapid, reliable, and relatively inexpensive method for detection of patients with asymptomatic internal carotid artery stenosis. Limited screening examinations should be developed in each vascular laboratory and utilized in high-risk patients.     

Coagulative interstitial laser-induced thermotherapy of benign prostatic hyperplasia: online imaging with a T2-weighted fast spin-echo MR sequence--experience in six patients

PURPOSE: To determine if hypointense lesions clearly outline on T2-weighted fast spin-echo (SE) magnetic resonance (MR) images obtained during coagulative interstitial laser-induced thermotherapy (LITT) of a prostate with benign hyperplasia. MATERIALS AND METHODS: In six patients with benign prostatic hyperplasia (BPH), 12 LITT treatments were followed online with repetitive axial T2-weighted fast SE imaging (repetition time, 3,700 msec; echo time, 138 msec; acquisition time, 19 seconds). Development, time course, correlation with interstitial tissue temperature, and diameters of hypointense lesions around the laser diffusor tip were investigated. Lesion diameters on T2-weighted images acquired during LITT were compared with diameters of final lesions on T2-weighted images and unperfused lesions on enhanced T1-weighted SE images obtained at the end of therapy. RESULTS: Hypointense lesions developed within 20-40 seconds of LITT. Average correlation coefficients between interstitial temperature development and signal intensity development were 0.92 during LITT and 0.90 after LITT. Regression slopes were significantly steeper during LITT (0.67% signal intensity change per degree Celsius) than after LITT (0.47% per degree Celsius; P = .038). Lesions remained visible after LITT for all procedures. Average maximum diameters of lesions were 1-3 mm larger during LITT than after LITT (P = .0006-.019). CONCLUSION: Repetitive T2-weighted fast SE MR imaging during interstitial coagulative LITT of BPH demonstrates the development of permanent hypointense prostate lesions. However, posttherapeutic lesion diameters tend to be overestimated during LITT.     

Recombinant Human Erythropoietin and Hemoglobin Concentration at Operation and during the Postoperative Period: Reduced Need for Blood Transfusions in Patients Undergoing Colorectal Surgery—Prospective Double-blind Placebo-controlled Study

p < 0.05). On postoperative days 3 and 7 the values were 7.2 (5.3–8.2) and 7.5 (5.4–9.4) mmol/L, respectively, in the erythropoietin group compared to 6.7 (5.2–7.8) and 6.9 (5.1–8.6) mmol/L in the placebo group ( p < 0.01). At discharge the hemoglobin concentration was 7.8 (5.9–8.8) mmol/L in the erythropoietin group and 7.2 (5.4–8.6) mmol/L in the placebo group ( p < 0.002). The blood loss during operation was similar in the two groups. In the erythropoietin group the median value was 280 ml (range 25–2000 ml), with the lower and upper quartiles 150 and 500 ml, respectively. In the placebo group the blood loss was median 300 ml (range 50–1800 ml), with the lower and upper quartiles 200 and 750 ml, respectively. The number of blood transfusions given was significantly lower in the erythropoietin group, with a mean of 0.3 (range 0–6) units compared to 1.6 (0–9) units in the control group ( p < 0.05). In conclusion, the hemoglobin concentration at the time of surgery and during the week following surgery was significantly higher in the group of patients receiving r-HuEPO perioperatively compared to the placebo group together with a significant lower use of blood transfusions in the r-HuEPO group. However, the clinical implications of these findings has yet to be proven.RID=" ID=" <E5>Correspondence to:</E5> N. Qvist, M.D., D.Sci.     

Biological monitoring of isocyanates and related amines

Summary Five men were exposed to toluene diisocyanate (TDI) atmospheres for 7.5 h. The TDI atmospheres were generated by a gas-phase permeation method, and the exposures were performed in an 8-m3 stainless-steel test chamber. The mean air concentration of TDI was ca. 40 g/m³, which corresponds to the threshold limit value (TLV) of Sweden. The inhaled doses of 2,4- and 2,6-TDI were ca. 120 g. TDI in the test chamber air was determined by an HPLC method using the 9-(N-methyl-aminomethyl)-anthracene reagent and by a continuous-monitoring filter-tape instrument. After hydrolysis of plasma and urine, the related amines, 2,4- and 2,6-toluenediamine 2,4-, and 2,6-TDA), were determined as pentafluoropropionic anhydride (PFPA) derivatives by capillary gas-chromatography using selected ion monitoring (SIM) in the electron-impact mode. The urinary elimination of the TDAs showed a possible biphasic pattern, with rapid first phases for 2,4-TDA (mean t _1/2 for the concentration in urine, 1.9 h) and for 2,6-TDA (mean t _1/2 for the concentration in urine, 1.6 h). The cumulative amount of 2,4-TDA excreted in urine within 28 h ranged from 8% to 14% of the estimated dose of 2,4-TDI, and the cumulative amount of 2,6-TDA in urine ranged from 14% to 18% of the 2,6-TDI dose. The average urinary level of 2,4-TDA was 5 g/l in the 6 to 8-h sample (range 2.8–9.6 g/l), and the corresponding value for 2,6-TDA was 8.6 g/l (range, 5.6–16.6 g/l). Biological monitoring of exposure to 2,4- and 2,6-TDI by analysis of 2,4- and 2,6-TDA in urine is feasible.     

Biological monitoring of isocyanates and related amines

Summary Two men were exposed to toluene diisocyanate (TDI) atmospheres at three different air concentrations (ca. 25, 50 and 70g/m³) . The TDI atmospheres were generated by a gas-phase permeation method, and the exposures were performed in an 8-m³ stainless-steel test chamber. The effective exposure period was 4h. The isomeric composition of the air in the test chamber was 30% 2,4-TDI and 70% 2,6-TDI. The concentration of TDI in air of the test chamber was determined by an HPLC method using the 9-(N-methyl-amino-methyl)-anthracene reagent and by a continuous-monitoring filter-tape instrument. Following the hydrolysis of plasma and urine, the related amines, 2,4-toluenediamine (2,4-TDA) and 2,6-toluenediamine (2,6-TDA), were determined as pentafluoropropionic anhydride (PFPA) derivatives by capillary gas chromatography using selected ion monitoring (SIM) in the electron-impact mode. In plasma, 2,4- and 2,6-TDA showed a rapid-phase elimination half-time of ca. 2–5 h, and that for the slow phase was > 6 days. A connection was observed between concentrations of 2,4- and 2,6-TDI in air and the levels of 2,4- and 2,6-TDA in plasma. The cumulated amount of 2,4-TDA excreted in the urine over 24 h was ca. 15%–19% of the estimated inhaled dose of 2,4-TDI, and that of 2,6-TDA was ca. 17%–23% of the inhaled dose of 2,6-TDI. A connection was found between the cumulated (24-h) urinary excretion of 2,4- and 2,6-TDA and the air concentration of 2,4- and 2,6-TDI in the test chamber. A connection was also observed between the rate of urinary excretion of 2,4- and 2,6-TDA over the last 2h of exposure and the air concentration of 2,4- and 2,6-TDI in the test chamber. Biological monitoring of exposure to monomeric 2,4- and 2,6-TDI by the analysis of 2,4- and 2,6-TDA in biological media is feasible. A method based on 24-h urine sampling and determination levels of 2,4- and 2,6-TDA in hydrolysed urine is recommended. However, exposure to TDI is often associated with aerosols containing polymeric TDI, and we do not know whether analysis of TDA in urine can also be used as a marker of exposure to TDI prepolymers.     

Multiple-dose pharmacokinetics of epirubicin at four different dose levels: studies in patients with metastatic breast cancer

Summary Pharmacokinetic analysis of epirubicin and its metabolites epirubicinol and 7-deoxy-13-dihydro-epirubicinol aglycone during the first and the fourth courses of treatment was performed in 78 patients with metastatic breast cancer. The patients were treated every 3 weeks with epirubicin given as 10-min i.v. infusions at four different dose levels: 40, 60, 90 and 135 mg/m². In most cases (76 of 78 cases), plasma concentration-time curves fitted to a three-compartmental pharmacokinetic model. The terminal half-life of epirubicin was independent of dose and duration of treatment. Large interindividual differences were demonstrated (meant _1/2, 21.6±7.9 h; range, 10.6–69 h;n=110). In two subjects, extremely long half-lives and high serum bilirubin concentrations indicated impaired liver function. No correlation was found between the half-life and levels of liver alanine aminotransferase (ALAT) or serum creatinine. The metabolite epirubicinol appeared quickly after epirubicin administration and its half-lives were shorter than that of the parent compound (meant _1/2, 18.1±4.8 h; range, 8.2–38.4 h;n=105).Formation of the aglycone metabolite was delayed and the half-life of this metabolite was shorter than that of epirubicin (meant _1/2, 13±4.6 h; range, 2.7–29 h;n=104). The AUC of epirubicin and the total AUC (drug and metabolites) were linearly proportional to the dose, with the former value constituting two-thirds of the latter. A correlation was found between AUC and the plasma concentration of epirubicin at two time points (2 and 24 h after administration). The proposed model was AUC=9.44×c ₂+62.5×c ₂₄+157.7 (r=0.953).This work was supported by the Lundbeck Foundation, the Michaelsen Foundation and Farmitalia Carlo Erba Ltd.     

Circadian variations in serum eosinophil cationic protein, and serum and urine eosinophil protein X

Biochemical evaluation of inflammation may be a useful adjunct to measures of pulmonary function and symptoms in children with asthma. However, little data have been provided to validate the markers in children. The aim of the present study was to assess circadian variations in serum eosinophil cationic protein (ECP), and serum and urine eosinophil protein X (EPX) in children. Five girls and two boys aged 10–14 years were studied. The first sample of urine consisted of urine collected from 24.00 hours the night before until 08.00 hours on the morning of the day of investigation. Thereafter urine was collected at 4-h intervals until 24.00 hours and in another 8-h interval from 24.00 to 08.00 hours. Blood samples for assessment of serum ECP and serum EPX were collected every 2 h during the 24 h. Statistically significant circadian variations in serum ECP (F=3.2, p=0.002), serum EPX (F=3.1, p=0.002) and in urine EPX/creatinine (F=5.4, p=0.003) were detected. The concentrations were higher during the night compared to daytime. Peak levels of serum ECP (mean [± SEM]) were found at 06.00 hours (16.3 [5.3] µg/l), trough levels at 08.00 hours (3.9 [0.7] µg/l) (p=0.01). Peak levels of serum EPX were seen at 06.00 (43.7 [9.5] µg/l) with trough levels at 12.00 hours (22.0 [3.5] µg/l) (p=0.01). Peak levels of urine EPX/creatinine occurred in urine collected from 24.00 to 08.00 hours (90.0 [27.7] µg/mmol), trough levels in the 16.00–20.00 hours sample (29.7 [8.9] µg/mmol) (p=0.02). Serum ECP, serum EPX and urine EPX exhibit a circadian variation in children with nocturnal and early morning peak levels. To avoid confounding influence from circadian variations in ECP and EPX in clinical studies blood or urine should be sampled at consistent times.