Simultaneous determination of six antibiotics in human serum by high-performance liquid chromatography with UV detection.

Antibiotics are widely used in intensive care patients to treat severe infections. To avoid bacterial resistance or toxic side effects, the determination of serum concentration of ABs is advisable. Therefore, in this study, we developed and validated a simple and fast high-performance liquid chromatography method with UV detection for the simultaneous determination of four ß-lactam ABs (meropenem, imipenem, ceftazidime, and piperacillin) and two coadministered substances (cilastatin and tazobactam) in human serum. Sample preparation required a simple protein precipitation by methanol. The separation of the ABs occurred within a timeframe of 17 min. For this purpose, we used a Kinetex F5 column with a linear gradient of acetonitrile and phosphate buffer (pH 6.9). The UV detector recorded two separate chromatograms at 220 and 295 nm simultaneously. Validation has demonstrated that the method is linear, accurate, and precise within the clinically relevant range for each substance.

A mutation within the SH2 domain of slp-76 regulates the tissue distribution and cytokine production of iNKT cells in mice.

TCR ligation is critical for the selection, activation, and integrin expression of T lymphocytes. Here, we explored the role of the TCR adaptor protein slp-76 on iNKT-cell biology. Compared to B6 controls, slp-76(ace/ace) mice carrying a missense mutation (Thr428Ile) within the SH2-domain of slp-76 showed an increase in iNKT cells in the thymus and lymph nodes, but a decrease in iNKT cells in spleens and livers, along with reduced ADAP expression and cytokine response. A comparable reduction in iNKT cells was observed in the livers and spleens of ADAP-deficient mice. Like ADAP(-/-) iNKT cells, slp-76(ace/ace) iNKT cells were characterized by enhanced CD11b expression, correlating with an impaired induction of the TCR immediate-early gene Nur77 and a decreased adhesion to ICAM-1. Furthermore, CD11b-intrinsic effects inhibited cytokine release, concanavalin A-mediated inflammation, and iNKT-cell accumulation in the liver. Unlike B6 and ADAP(-/-) mice, the expression of the transcription factors Id3 and PLZF was reduced, whereas NP-1-expression was enhanced in slp-76(ace/ace) mice. Blockade of NP-1 decreased the recovery of iNKT cells from peripheral lymph nodes, identifying NP-1 as an iNKT-cell-specific adhesion factor. Thus, slp-76 contributes to the regulation of the tissue distribution, PLZF, and cytokine expression of iNKT cells via ADAP-dependent and -independent mechanisms.

Determination of meropenem levels in human serum by high-performance liquid chromatography with ultraviolet detection.

Meropenem is a ß-lactam broad-spectrum antibiotic and belongs to the subgroup of carbapenems. It is primarily used in intensive care units for intravenous treatment of severe infections. To avoid bacterial resistance or toxic side effects, the determination of serum meropenem concentration is highly advisable. A simple and fast method for the quantitative determination of meropenem in human serum using high-performance liquid chromatography with ultraviolet detection (HPLC/UV) was developed and validated. Meropenem was determined by an isocratic HPLC using a tris(hydroxymethyl)aminomethane buffer (pH 8.5; 15% methanol) as a mobile phase and UV detection at 300 nm, with a flow rate of 1.0 mL/min and an analysis time of 10 min. Chromatographic separation was performed on a Kinetex C18 column (5 µm, 150 × 4.6 mm). In order to remove undesired serum components, solid-phase extraction was used for sample preparation. Since meropenem is not stable in solution, sample and stock solution were stored at -80°C. After preparation, samples were stable at room temperature for at least 6 h. The calibration curve was linear from 3.5 to 200 mg/L with a correlation coefficient r2 of 0.999. The method is accurate with an intra- and inter-assay precision <18.5%.

Specific removal of C-reactive protein by apheresis in a porcine cardiac infarction model.

BACKGROUND: C-reactive protein (CRP) is a possible causative factor of the destructive processes observed during the weeks after myocardial infarction. METHODS: We developed a clinically relevant animal model including the removal of CRP from blood plasma utilizing a specific CRP adsorber and the visualization of the infarct scar in the living animal by cardiovascular magnetic resonance imaging as a tool to investigate the impact of CRP after acute myocardial infarction. RESULTS: We describe the facets of this model system and kinetics of clinical blood parameters like CRP and troponin. In addition, we demonstrate the potency of CRP apheresis reducing CRP levels by ~70% in the established treatment system. CONCLUSION: We showed for the first time that it is possible to conduct apheresis at the following 2 days after acute myocardial infarction in a porcine infarction model and to analyze the infarct by cardiovascular magnetic resonance imaging at day 1 and 14.

Comparison of the Serum Tumor Markers S100 and Melanoma-inhibitory Activity (MIA) in the Monitoring of Patients with Metastatic Melanoma Receiving Vaccination Immunotherapy with Dendritic Cells.

BACKGROUND: In patients with melanoma, early dissemination via lymphatic and hematogenous routes is frequently seen. Thus, besides clinical follow-up examination and imaging, reliable melanoma-specific serological tumor markers are needed. PATIENTS AND METHODS: We retrospectively compared two serum markers for melanoma, S100 and melanoma-inhibitory activity (MIA), for monitoring of patients with metastatic melanoma under either adjuvant or therapeutic vaccination immunotherapy with dendritic cells (DC). Serum was obtained from a total of 100 patients (28 patients in stage III and 72 patients in stage IV, according to the American Joint Committee on Cancer 2002) at regular intervals during therapy, accompanied by follow-up imaging. RESULTS: When relapse was detected, both markers often remained within normal range. In contrast, in patients with metastatic measurable disease receiving therapeutic and not adjuvant DC vaccination, an increase of both markers was a strong indicator for disease progression. When comparing both markers in the whole study population, MIA showed a superior sensitivity to detect disease progression. CONCLUSION: S100 and MIA are highly sensitive tumor markers for monitoring of patients with melanoma with current metastases, but less sensitive for monitoring of tumor-free patients. In the current study, MIA had a slightly superior sensitivity to detect progressive disease compared to S100 and seems to be more useful in monitoring of patients with metastatic melanoma receiving immunotherapy.

Efavirenz Has the Highest Anti-Proliferative Effect of Non-Nucleoside Reverse Transcriptase Inhibitors against Pancreatic Cancer Cells.

BACKGROUND: Cancer prevention and therapy in HIV-1-infected patients will play an important role in future. The non-nucleoside reverse transcriptase inhibitors (NNRTI) Efavirenz and Nevirapine are cytotoxic against cancer cells in vitro. As other NNRTIs have not been studied so far, all clinically used NNRTIs were tested and the in vitro toxic concentrations were compared to drug levels in patients to predict possible anti-cancer effects in vivo. METHODS: Cytotoxicity was studied by Annexin-V-APC/7AAD staining and flow cytometry in the pancreatic cancer cell lines BxPC-3 and Panc-1 and confirmed by colony formation assays. The 50% effective cytotoxic concentrations (EC50) were calculated and compared to the blood levels in our patients and published data. RESULTS: The in vitro EC50 of the different drugs in the BxPC-3 pancreatic cancer cells were: Efavirenz 31.5 µmol/l (= 9944 ng/ml), Nevirapine 239 µmol/l (= 63,786 ng/ml), Etravirine 89.0 µmol/l (= 38,740 ng/ml), Lersivirine 543 µmol/l (= 168,523 ng/ml), Delavirdine 171 µmol/l (= 78,072 ng/ml), Rilpivirine 24.4 µmol/l (= 8941 ng/ml). As Efavirenz and Rilpivirine had the highest cytotoxic potential and Nevirapine is frequently used in HIV-1 positive patients, the results of these three drugs were further studied in Panc-1 pancreatic cancer cells and confirmed with colony formation assays. 205 patient blood levels of Efavirenz, 127 of Rilpivirine and 31 of Nevirapine were analyzed. The mean blood level of Efavirenz was 3587 ng/ml (range 162-15,363 ng/ml), of Rilpivirine 144 ng/ml (range 0-572 ng/ml) and of Nevirapine 4955 ng/ml (range 1856-8697 ng/ml). Blood levels from our patients and from published data had comparable Efavirenz levels to the in vitro toxic EC50 in about 1 to 5% of all patients. CONCLUSION: All studied NNRTIs were toxic against cancer cells. A low percentage of patients taking Efavirenz reached in vitro cytotoxic blood levels. It can be speculated that in HIV-1 positive patients having high Efavirenz blood levels pancreatic cancer incidence might be reduced. Efavirenz might be a new option in the treatment of cancer.

The Passage of S100B from Brain to Blood Is Not Specifically Related to the Blood-Brain Barrier Integrity.

Following brain injury, S100B is released from damaged astrocytes but also yields repair mechanisms. We measured S100B in the cerebrospinal fluid (CSF) and serum (Cobas e411 electrochemiluminescence assay, Roche) longitudinally in a large cohort of patients treated with a ventricular drainage following traumatic brain injury (TBI) or subarachnoid hemorrhage (SAH). Statistical analysis was performed with SPSS software applying the Mann-Whitney rank sum test or chi-test where appropriate. S100B in CSF and serum was significantly increased following TBI (n = 71) and SAH (n = 185) for at least one week following injury. High S100B levels in CSF and serum were inconsistent associated with outcome. The passage of S100B from CSF to blood (100( *)serum(S100B)/CSF(S100B)) was significantly decreased although the albumin quotient suggested an "open" blood-CSF barrier. Events possibly interfering with the BBB did not affect the S100B passage (P = .591). In conclusion, we could not confirm S100B measurements to reliably predict outcome, and a compromised blood-CSF barrier did not affect the passage of S100B from CSF to serum.

Impaired cytotrophoblast cell-cell fusion is associated with reduced Syncytin and increased apoptosis in patients with placental dysfunction.

Preeclampsia (PE), Hemolysis Elevated Liver Enzymes and Low Platelets (HELLP)-syndrome, and intrauterine growth restriction (IUGR) are associated with abnormal placentation. In early pregnancy, placental cytotrophoblasts fuse and form multinuclear syncytiotrophoblasts. The envelope gene of the human endogenous retrovirus-W, Syncytin, is a key factor for mediating cell-cell fusion of cytotrophoblasts. This study investigated clinical parameters of PE and HELLP-associated IUGR and analyzed the cell-cell fusion index and beta-human chorionic gonadotropin (beta-hCG) secretion of cytotrophoblasts isolated and cultured from placentas of these patients. In addition, we performed absolute quantitation of Syncytin and determined the apoptosis rate in both cultured cytotrophoblasts and placental tissues. Cultured cytotrophoblasts from PE and HELLP-associated IUGR correlated with a pronounced lower cell-cell fusion index, 1.8- and 3.6-fold; less nuclei per syncytiotrophoblast, 1.4- and 2.0-fold; a significantly decreased beta-hCG secretion, 4.3- and 17.2-fold and a reduction of Syncytin gene expression, 8.1 (P = 0.019) and 222.7-fold (P = 0.011) compared with controls, respectively. In contrast, a significantly 2.3-fold higher apoptosis rate was observed in cultured PE/IUGR cytotrophoblasts (P = 0.043). Importantly, Syncytin gene expression in primary placental tissues of PE/IUGR was 5.4-fold lower (P = 0.047) and in HELLP/IUGR 10.6-fold lower (P = 0.019) along with a 1.8- and 1.9-fold significant increase in the apoptosis rate compared with controls, respectively. Low Syncytin expression in both cultured cytotrophoblasts and primary tissues from pathological placentas supports an intrinsic placenta-specific deregulation of cell-cell fusion in the formation of syncytiotrophoblasts leading to increased apoptosis. These processes could contribute to the development and severity of PE and HELLP-associated IUGR.