Lentivirus-induced 'Smart' dendritic cells: Pharmacodynamics and GMP-compliant production for immunotherapy against TRP2-positive melanoma.

Monocyte-derived conventional dendritic cells (ConvDCs) loaded with melanoma antigens showed modest responses in clinical trials. Efficacy studies were hampered by difficulties in ConvDC manufacturing and low potency. Overcoming these issues, we demonstrated higher potency of lentiviral vector (LV)-programmed DCs. Monocytes were directly induced to self-differentiate into DCs (SmartDC-TRP2) upon transduction with a tricistronic LV encoding for cytokines (granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4)) and a melanoma antigen (tyrosinase-related protein 2 (TRP2)). Here, SmartDC-TRP2 generated with monocytes from five advanced melanoma patients were tested in autologous DC:T cell stimulation assays, validating the activation of functional TRP2-specific cytotoxic T lymphocytes (CTLs) for all patients. We described methods compliant to good manufacturing practices (GMP) to produce LV and SmartDC-TRP2. Feasibility of monocyte transduction in a bag system and cryopreservation following a 24-h standard operating procedure were achieved. After thawing, 50% of the initial monocyte input was recovered and SmartDC-TRP2 self-differentiated in vitro, showing uniform expression of DC markers, detectable LV copies and a polyclonal LV integration pattern not biased to oncogenic loci. GMP-grade SmartDC-TRP2 expanded TRP2-specific autologous CTLs in vitro. These results demonstrated a simpler GMP-compliant method of manufacturing an effective individualized DC vaccine. Such DC vaccine, when in combination with checkpoint inhibition therapies, might provide higher specificity against melanoma.

Sorafenib and pegylated interferon-α2b in advanced metastatic melanoma: a multicenter phase II DeCOG trial.

BACKGROUND: The combination of sorafenib, a multikinase inhibitor, and pegylated interferon-α2b (Peg-IFN-α2b) could potentially lead to an improved antitumoral response. Previously, combinations of interferon and sorafenib have been used in renal cell cancer. PATIENTS AND METHODS: Patients with stage IV metastatic melanoma and no previous systemic therapies apart from adjuvant immunotherapy received Peg-IFN-α2b 3 µg/kg once per week, and sorafenib 400-mg b.i.d. for a minimum of 8 weeks. The primary study end point was disease control rate (DCR). RESULTS: Between February 2008 and February 2009, 55 patients were enrolled with a median age of 64 years (20-85). At 8 weeks, 2 patients (3.6%) had a partial response (PR) and 14 patients a stable disease (25.5%), for a DCR of 29.1% in the intention-to-treat (ITT) population. The median progression-free survival in the ITT population was 2.47 months (95% confidence interval 1.22-3.72 months). The toxicity of sorafenib and Peg-IFN-α2b combination was characterized by mainly hematological side-effects, including one treatment-related bleeding complication with a fatal outcome. Other grade 3/4 toxic effects were fatigue and flu-like symptoms. CONCLUSION: The combination of sorafenib and Peg-IFN-α2b showed modest clinical activity and some serious side-effects including fatal bleeding complications.

[Cutaneous side effects of medical tumor therapy]. / Kutane Nebenwirkungen der medikamentösen Tumortherapie.

Agents used in medical tumor therapies can cause peculiar cutaneous side effects. In particular the newly developed targeted therapies are associated with specific cutaneous side effects, such as a papulopustular exanthems associated with EGFR inhibitors and epithelial skin cancers associated with inhibitors of mutated BRAF. In this review the clinical pictures as well as pathogenetic and therapeutic aspects of the hand-foot-syndrome, papulopustular exanthems, epithelial skin cancers, paronychia and changes of hair and nails as side effects of medical tumor therapies are summarized. Knowledge of these side effects is important to avoid interruption or termination of the tumor therapy, in particular since some of the cutaneous reactions have been shown to correlate with the therapeutic benefit of the tumor therapy.

Does further clean-up reduce the matrix enhancement effect in gas chromatographic analysis of pesticide residues in food?

Sample extracts of apples, peas, green beans, oranges, raspberries, clementines, carrots, and wheat obtained using the Food and Drug Administration (acetone extraction) and Canadian Pest Management Regulatory Agency (acetonitrile extraction) multiresidue methods for pesticides were subjected to clean-up using different solid-phase extraction (SPE) cartridges in an attempt to reduce or eliminate the matrix enhancement effect. The matrix enhancement effect is related to the blocking of active sites on the injector liner by matrix components, thereby increasing signal in the presence of matrix versus standards in solvent in which the pesticides themselves interact with the active sites. Graphitized carbon black (GCB) was often used in combination with various anion-exchange SPE cartridges. The extracts were then spiked with organophosphorus insecticides. These process standards were then compared to standards in acetone of the same concentration using gas chromatography with flame photometric detection or ion trap mass spectrometric detection. Sample matrix enhancement varied from little to no effect for some pesticides (e.g. chlorpyrifos, malathion) to >200% in the case of certain susceptible pesticides. The GCB removed color components but showed little effect in reducing matrix enhancement by itself. The anion-exchange cartridges in combination with GCB or not, substantially reduced the matrix enhancement effect but did not eliminate it.

Chromatographic methods of analysis of antibiotics in milk.

The widespread use of antibiotics in dairy cattle management may result in the presence of antibiotic residues in milk. While rapid screening tests are commonly used to detect the presence of antibiotics in milk, more accurate chromatographic methods are required by government regulatory agencies to identify and confirm the identity and quantity of antibiotic present. This paper review recent developments in the chromatographic determination of antibiotic residues in milk.

Determination of organochlorine and organophosphorus pesticide residues in eggs using a solid phase extraction cleanup.

A multiresidue solid phase extraction (SPE) method for the isolation and subsequent gas chromatographic determination of nonpolar organochlorine and polar organophosphorus pesticide residues in eggs is described. The method uses an acetonitrile extraction followed by an SPE cleanup using graphitized carbon black and aminopropyl SPE columns. Organophosphorus pesticides are determined by gas chromatography with flame photometric detection. After further cleanup of the extract using Florisil SPE columns, organochlorine pesticides are determined by gas chromatography with electron capture detection. Studies were performed using eggs containing both fortified and incurred pesticide residues. The average recoveries were 86-108% for 8 fortified organochlorine pesticide residues and 61-149% for 28 fortified organophosphorus pesticide residues.

Stability of tetracycline antibiotics in raw milk under laboratory storage conditions.

Raw milk samples collected from bulk milk tankers may be screened for the presence of tetracycline antibiotics using rapid screening tests. If tetracycline residues are detected, the milk may be shipped to a laboratory for high-pressure liquid chromatography (HPLC) analysis. Because the milk may be shipped on ice blocks, it is important to know whether tetracycline residues are stable at that temperature and for how long. Control raw milk samples fortified with 50 ppb each chlortetracycline, demeclocycline, methacycline hydrochloride, minocycline, oxytetracycline, and tetracycline were incubated at 4 degrees C or 25 degrees C, then analyzed using a metal chelate affinity chromatography extraction and HPLC. No loss of tetracycline was observed after 48 h of storage at 4 degrees C or 24 h at 25 degrees C. Losses ranging from 4 to 13% and 0 to 18% were noted after 72 h at 4 degrees C and 48 h at 25 degrees C, respectively.

Screening of nonfatty fish for organochlorine pesticide residues by solid-phase extraction cleanup: interlaboratory study.

Six U.S. Food and Drug Administration laboratories participated in an Interlaboratory trial of a solid-phase extraction cleanup method for determination of pesticides in nonfatty seafood products. The participants analyzed control and fortified (about 0.050 ppm lindane, heptachlor epoxide, p,p'-DDE, and endrin) croaker and flounder samples and a sea trout sample containing incurred p,p'-DDE residues. Mean recoveries of the fortified residues from the fish ranged from 89.1 to 107.8%. The within-laboratory coefficients of variation (CVs) ranged from 4.2 to 8.5%, and the among-laboratory CVs ranged from 10.9 to 26.5%. The 6 laboratories reported a mean value of 0.040 ppm p,p'-DDE in a fish sample which contained incurred residues. The same value (0.040 ppm) was obtained by using official methodology. The within-laboratory CVs ranged from 3.5 to 18.3%, and the among-laboratory CV was 17.3%.

Multiresidue determination of abamectin, doramectin, ivermectin, and moxidectin in milk using liquid chromatography and fluorescence detection.

Abamectin, doramectin, ivermectin, and moxidectin are macrocyclic lactones derived from soil dwelling actinomycetes, and are very effective against nematode, insect, and arthropod infestations. These compounds, known as endectocides, have been approved for use in beef cattle in the United States; however, they are currently not approved for use in dairy cattle. Abamectin, doramectin, ivermectin, and moxidectin residues were isolated from milk by a series of liquid-liquid extraction steps, derivatized with trifluoroacetic anhydride, and determined by liquid chromatography with fluorescence detection. Recovery studies were performed in 2 laboratories. Recoveries of > 80% (1-30 ng/mL) were achieved for all 4 compounds.

Determination of organochlorine pesticide and polychlorinated biphenyl residues in fatty fish by tandem solid-phase extraction cleanup.

A rapid, multiresidue solid-phase extraction (SPE) technique for determination of organochlorine pesticide and polychlorinated biphenyl (PCB) residues in nonfatty fish was modified for use with fatty fish. In the modified procedures, samples are extracted with acetonitrile, and the extract is cleaned up with both C18 and Florisil SPE columns. Residues are determined by gas chromatography with electron capture detection. The original method was modified for use with fatty fish by reducing the amount of tissue extracted and by using an improved Florisil SPE cleanup. Recovery data are presented for 24 fortified organochlorine pesticide residues (0.12 ppm) and 3 fortified PCB residues (0.80 ppm) from flounder, bluefish, and shad samples, which contained 0.8, 5.4, and 22.6% fat, respectively. For the 3 types of fish, recoveries of 23 of 24 fortified organochlorine pesticide residues ranged from 55 to 129%, and recoveries of 3 fortified PCB residues ranged from 55 to 104%. There were no significant differences in recovery based on fish species and/or fat content for the majority of residues studied. This SPE method and the official AOAC method yielded comparable results for fish containing incurred organochlorine residues.

Florisil solid-phase extraction cartridges for cleanup of organochlorine pesticide residues in foods.

Florisil solid-phase extraction cartridges were evaluated for cleanup of organochlorine pesticide residues in food extracts. Elution patterns and recoveries were determined for 24 organochlorine pesticides. A range of elution solvents was evaluated. A 2% ethyl ether-petroleum ether eluant optimized overall recoveries while minimizing interferences from coextractants.

Screening procedure for organochlorine and organophosphorus pesticide residues in eggs using a solid-phase extraction cleanup and gas chromatographic detection.

A solid-phase extraction (SPE) screening procedure for the isolation and gas chromatographic (GC) determination of organochlorine and organophosphorus pesticide residues in eggs is described. Eggs are extracted with acetonitrile. The extract is subjected to a cleanup on tandem C18 and Florisil SPE columns. Organochlorine and organophosphorus pesticide residues are determined by GC with electron capture and flame photometric detection, respectively. Because the injected extracts are free from matrix interferences, the amount of residue present is easy to calculate. The average recoveries of 9 spiked organochlorine pesticide residues (0.01-1.0 ppm) ranged from 80.9 to 91.1%. The average recoveries of 7 spiked organophosphorus pesticide residues (0.02-0.50 ppm) ranged from 80.3 to 89.5%. The SPE method results in a 90% reduction in organic solvent consumption and an 85% reduction in hazardous waste production compared with the AOAC methodology.

Levels of satisfaction with current chronic constipation treatment options in Europe - an internet survey.

BACKGROUND: Data on treatment satisfaction in European men and women with chronic constipation are limited. AIM: To assess satisfaction with current treatment among European men and women with chronic constipation. METHODS: An internet-based survey was conducted in 2009 in 10 European countries: Austria, Germany, France, Ireland, Italy, Spain, Switzerland, the UK, Belgium and the Netherlands. Participants had self-reported chronic constipation (<3 bowel movements/week and ≥1 symptoms for ≥6 months of: pain during defecation; lumpy/hard faeces; and feeling of incomplete evacuation). Demographic data and disease history were collected. For participants using laxatives, drug name/class, satisfaction with treatment and interest in other treatments were collected. RESULTS: Of the 1941 participants screened, 1355 had chronic constipation and met the inclusion criteria (chronic constipation population). The majority of the chronic constipation population who disclosed their sex (n = 811) were women (82%). Sixty-eight per cent of respondents (n = 855/1255) reported using laxatives, with the proportion of laxative users differing between subsets. Twenty-eight per cent (n = 225/793) were (very) satisfied with their treatment, whereas 44% (n = 345/793) were neutral and 28% (n = 223/793) (very) dissatisfied. There was no relationship between type of laxative and degree of (dis)satisfaction. Interest in other treatments was high with 83% (n = 686/827) of respondents 'absolutely' or 'probably' interested. Respondents dissatisfied with their treatment were more likely to be interested in other treatments. CONCLUSIONS: Laxative-use is common for chronic constipation. In this large survey, 28% of participants were dissatisfied with their treatment, with the majority interested in other treatments.

[Fluorescein angiography findings in intermediate uveitis]. / Fluoreszenzangiographische Befunde bei intermediärer Uveitis.

The present paper describes the results of angiographic examinations of 48 eyes (29 patients) with intermediate uveitis. More than 50% of the cases displayed pathologic changes of the retinal blood vessels, such as increased fluorescein staining of the vessel walls and leakages of the retinal veins or venules, respectively. Some 20% of the eyes manifested cystoid macular edema and/or edema of the optic disk which had gone undetected by ophthalmoscopy. These findings suggest that vascular changes may play a role in the pathogenesis of intermediate uveitis. The question as to whether this disease might be caused rather by retinal vasculitis than by uveitis is discussed.

Retinal vasculitis with multifocal retinochoroiditis.

We found a multifocal retinochoroiditis in 16 patients (13 female, 3 male). Patients also showed vitreous cells, in some cases pronounced retinal vasculitis, cystoid macular edema and papilledema. The patients ranged in age from 62 to 77 years. The anterior segment was involved in 13 cases. One patient demonstrated subretinal neovascularisation in both eyes. Fluorescein angiograms showed leakage from the retinal vessels, papilledema, macular edema and subretinal neovascularisation. A convincing classification of the described changes within any known disease entity was impossible. The findings most closely resembled a disease described by Dreyer and Gass in 1984 as 'multifocal choroiditis and panuveitis'.

[Retinal vasculitis with multifocal chorioretinitis]. / Vasculitis retinae mit multifokaler Retinochorioiditis.

Multifocal retinochoroiditis was diagnosed in 16 patients (13 female, 3 male). The patients also had cells in the vitreous, and in some cases pronounced retinal vasculitis, cystoid macular edema and papilledema. The patients' ages ranged from 62 to 77 years. There was anterior segment involvement in 13 cases. One patient had subretinal neovascularization in both eyes. Fluorescein angiograms revealed leaks from the retinal vessels, papilledema, and macular edema. So far, the authors have not succeeded in classifying the changes described as belonging to a known disease entity. The findings most closely resembled a disease described by Dreyer and Gass in 1984 as "multifocal choroiditis and panuveitis". Similar changes were seen by Tiedemann in 1987, in patients with Epstein-Barr viral antibodies.

The effect of storage at 4 degrees C on the stability of ampicillin residues in raw milk.

Raw milk samples collected from tanker trucks are routinely screened for beta-lactam antibiotic drug residues using rapid screening tests. If drug residues are detected, the milk may be shipped on ice blocks to a laboratory for further analysis. A study was conducted to determine the stability of ampicillin in raw milk stored at +4 degrees C in order to predict if shipping the milk would result in the degradation of ampicillin residues. Milk samples were spiked with 20 ppb ampicillin, stored at +4 degrees C and -70 degrees C for 1-6 days, and then analysed by HPLC with fluorescence detection. No loss of ampicillin residues was found in milk stored at +4 degrees C for 1-6 days.