A new monoclonal antibody (CAL2) detects CALRETICULIN mutations in formalin-fixed and paraffin-embedded bone marrow biopsies.

Recent advances in the diagnostic of myeloproliferative neoplasms (MPNs) discovered CALRETICULIN (CALR) mutations as a major driver in these disorders. In contrast to JAK2 mutations being mainly associated with polycythaemia vera, CALR mutations are only associated with primary myelofibrosis (PMF) and essential thrombocythaemia (ET). CALR mutations are present in the majority of PMF and ET patients lacking JAK2 and MPL mutations. As these CALR mutations are absent from reactive bone marrow (BM) lesions their presence indicates ET or PMF. So far these mutations are detectable only by molecular assays. Their molecular detection is cumbersome because of the great CALR mutation heterogeneity. Therefore, the availability of a simple assay would be of great help. All CALR mutations reported lead to a frameshift generating a new 36 amino-acid C-terminus. We generated a monoclonal antibody (CAL2) to this C-neoterminus by immunizing mice with a representative peptide and compared its performance with Sanger sequencing data in 173 MPNs and other BM diseases. There was a 100% correlation between the molecular and the CAL2 immunohistochemical (IHC) assays. Thus, the detection of CALR mutations by the CAL2 IHC is a specific, sensitive, rapid, simple and low-cost method.

[Pathogenicity of Mycobacterium kansasii]. / Pathogenität von Mycobacterium kansasii.

BACKGROUND: In a recent prospective study on pulmonary infections with non-tuberculous mycobacteria (NTM) led by the WATL group, disease rates in patients with M. kansasii infection were found to be 100 %. In the present study we re-evaluated the pathogenicity of M. kansasii infections in a large lung diseases treatment center in Berlin (Lungenklinik Heckeshorn). METHODS: All patients in whose respiratory specimen cultures M. kansasii was detected between January 2003 and June 2013 were included. The 2007 ATS diagnostic criteria were applied to differentiate disease from asymptomatic infection. The strains were further investigated by sequencing of the 16S-23S rDNA internal transcribed spacer (ITS) region. RESULTS: We evaluated 43 consecutive cases. Complete patient data were available in 38 cases. In one patient, no culture results were obtained, in 37 patients M. kansasii was isolated and patient data could be retrieved. In 25/37 patients (68 %) clinical disease was present so that a specific treatment was initiated (underlying diseases were COPD in 8/25 (32 %), bronchiectasis in 5/25 (20 %), TB scar or scar due to prior chest surgery in 3/25 (12 %) and alcohol abuse in 4/25 (16 %)). Twelve out of 37 patients (32 %) were found to be colonized or asymptomatically infected (underlying diseases were COPD in 7/12 (58 %), bronchiectasis in 3/12 (25 %) and TB scar or scar due to prior chest surgery in 3/12 (25 %)). Sequencing results identified 30 strains as genotype I, and 2 strains as genotype II. In 22/30 cases (73 %) genotype I was considered pathogenic. CONCLUSIONS: In our cohort, we could not confirm the high M. kansasii pathogenicity of 100 % found in a previous multi-center study; we therefore support the clinical and semiquantitative microbiologic diagnostic criteria also for infection with M. kansasii.

Chromogenic in situ hybridisation (CISH) is a powerful method to detect ALK-positive non-small cell lung carcinomas.

AIM: We assessed the potential of a chromogenic in situ hybridisation (CISH) assay in comparison with quantitative reverse transcription (RT)-PCR (qPCR) to detect anaplastic lymphoma kinase (ALK) break apart-positive lung carcinomas. METHODS: Dual-colour CISH using a break apart probe for the ALK gene on 2p23 was performed with 181 formalin-fixed, paraffin-embedded tissue and agar block sections from 175 cases of non-small cell lung carcinomas (NSCLC). Stained slides were analysed with a standard bright-field microscope at 1000× magnification by counting signals from 60 non-overlapping nuclei from three different tumour areas. Samples with ≥15% of positive nuclei were judged as ALK break apart-positive. All samples were simultaneously analysed by qPCR for EML4-ALK to validate CISH results, and positive samples were subject to Sanger sequencing. RESULTS: CISH was successful with 173 of 181 hybridised samples (96%), and seven ALK break apart-positive cases were detected. CISH signals were specific and distinct for both colours. All positive cases were confirmed by qPCR and Sanger sequencing, and concordance between CISH and qPCR was 100%. Nearly all samples (9/10) which failed by qPCR were accessible to CISH analysis. CONCLUSIONS: CISH is a very reliable, convenient and inexpensive method to detect ALK-positive NSCLC. CISH success rate is comparably high as with qPCR, and it detects all ALK break apart events in a single assay. It is of special value when RNA quality is poor, or when small biopsies with a very limited amount of tumour cells have to be analysed.

The epidemiology of atypical chronic lung disease in extremely low birth weight infants.

OBJECTIVE: To determine the incidence and associated complications of atypical chronic lung disease (ACLD) in extremely low birth weight infants. STUDY DESIGN: All infants born at Johns Hopkins Hospital between 1996 and 2001, with birthweight <1000 g, gestational age <31 weeks, no major anomalies or genetic syndromes, and living at least 21 days were eligible for inclusion. Data pertaining to demographics, hospital course, diagnosis of atypical chronic lung disease, patterns of surfactant use, complications of prematurity and severity of lung disease were collected. RESULT: Using inclusion criteria, 215 eligible infants were identified, of which 185 had hospital charts available for review. Twenty-eight infants (15%) met the criteria for atypical chronic lung disease. Of the remaining 157 infants, 57 patients met the criteria for mild bronchopulmonary dysplasia (BPD) (supplemental oxygen requirement at 28 days of life), 38 patients had moderate/severe BPD (supplemental oxygen requirement at both 28 days of life and 36 weeks post-menstrual age), and 38 infants did not have chronic lung disease. Infants with ACLD had much higher rates of sepsis (46%) and pneumothorax (18%) than infants in the comparison groups. CONCLUSION: Infants with respiratory distress syndrome in the first week of life, which initially resolves are still at risk for an atypical form of chronic lung disease. The prolonged respiratory support they require as a result of this type of lung disease increases their risk for complications of prematurity, which may outlast their lung disease. We speculate that inflammation secondary to infection acquired shortly after birth may be an important step in the pathogenesis of ACLD.

Multiple unit gastroretentive drug delivery systems: a new preparation method for low density microparticles.

The aim of this study was to develop a new preparation method for low density foam-based, floating microparticles and to demonstrate the systems' performance in vitro. Major advantages of the novel preparation technique include: (i) short processing times, (ii) no exposure of the ingredients to high temperatures, (iii) the possibility to avoid toxic organic solvents, and (iv) high encapsulation efficiencies close to 100%. Floating microparticles consisting of polypropylene foam powder, model drug [chlorpheniramine maleate (CPM), diltiazem HCl, theophylline or verapamil HCl] and polymer [Eudragit RS or polymethyl methacrylate (PMMA)] were prepared by soaking the microporous foam carrier with an organic solution of drug and polymer and subsequent drying. The effects of various formulation and processing parameters on the resulting in vitro floating behaviour, internal and external particle morphology, drug loading, in vitro drug release and physical state of the incorporated drug were studied. Good in vitro floating behaviour was observed in most cases and a broad variety of drug release patterns could be achieved by varying the drug loading and type of polymer. Interestingly, PMMA-based microparticles showed incomplete drug release with verapamil HCl. This restriction could be overcome by forming the free base of the drug prior to microparticle preparation. In contrast to the salt, the free base acted as a plasticizer for PMMA, resulting in sufficiently high diffusion coefficients and, consequently, complete drug release. The low density microparticles were compressed into rapidly disintegrating tablets in order to provide an administrable oral dosage form.

Floating matrix tablets based on low density foam powder: effects of formulation and processing parameters on drug release.

The aim of this study was to develop and physicochemically characterize single unit, floating controlled drug delivery systems consisting of (i). polypropylene foam powder, (ii). matrix-forming polymer(s), (iii). drug, and (iv). filler (optional). The highly porous foam powder provided low density and, thus, excellent in vitro floating behavior of the tablets. All foam powder-containing tablets remained floating for at least 8 h in 0.1 N HCl at 37 degrees C. Different types of matrix-forming polymers were studied: hydroxypropyl methylcellulose (HPMC), polyacrylates, sodium alginate, corn starch, carrageenan, gum guar and gum arabic. The tablets eroded upon contact with the release medium, and the relative importance of drug diffusion, polymer swelling and tablet erosion for the resulting release patterns varied significantly with the type of matrix former. The release rate could effectively be modified by varying the "matrix-forming polymer/foam powder" ratio, the initial drug loading, the tablet geometry (radius and height), the type of matrix-forming polymer, the use of polymer blends and the addition of water-soluble or water-insoluble fillers (such as lactose or microcrystalline cellulose). The floating behavior of the low density drug delivery systems could successfully be combined with accurate control of the drug release patterns.

Floating microparticles based on low density foam powder.

The aim of this study was to develop a novel multiparticulate gastroretentive drug delivery system and to demonstrate its performance in vitro. Floating microparticles consisting of (i) polypropylene foam powder; (ii) verapamil HCl as model drug; and (iii) Eudragit RS, ethylcellulose (EC) or polymethyl methacrylate (PMMA) as polymers were prepared with an O/W solvent evaporation method. The effect of various formulation and processing parameters on the internal and external particle morphology, drug loading, in vitro floating behavior, in vitro drug release kinetics, particle size distribution and physical state of the incorporated drug was studied. The microparticles were irregular in shape and highly porous. The drug encapsulation efficiency was high and almost independent of the theoretical loading. Encapsulation efficiencies close to 100% could be achieved by varying either the ratio 'amount of ingredients: volume of the organic phase' or the relative amount of polymer. In all cases, good in vitro floating behavior was observed. The release rate increased with increasing drug loading and with decreasing polymer amounts. The type of polymer significantly affected the drug release rate, which increased in the following rank order: PMMA

Understanding and predicting drug delivery from hydrophilic matrix tablets using the "sequential layer" model.

PURPOSE: The objectives of this work were (i) to study and understand the physicochemical phenomena which are involved in the swelling and drug release from hydrophilic matrix tablets using the "sequential layer" model, and (ii) to predict the effect of the initial radius height and size of the tablets on the resulting drug release profiles. METHODS: Tablets were prepared by direct compression, using hydroxypropyl methylcellulose (HPMC) grades with different average molecular weights as matrix-forming polymers. The in vitro release of chlorpheniramine maleate, propranolol HCl, acetaminophen, theophylline and diclofenac sodium was studied in phosphate buffer (pH 7.4) and 0.1 M HCl, respectively. The initial drug loading varied from 1 to 70%, while the radius and height of the tablets varied from 1 to 8 mm. RESULTS: The "sequential layer" model considers water and drug diffusion with non-constant diffusivities and moving boundary conditions, non-homogeneous polymer swelling, drug dissolution, and polymer dissolution. We showed that this model was able to predict the resulting drug release kinetics accurately in all cases. CONCLUSIONS: The "sequential layer" model can be used to elucidate the swelling and drug release behavior from hydrophilic matrix tablets and to simulate the effect of the device geometry on the drug release patterns. Hence, it can facilitate the development of new pharmaceutical products.

Bimodal drug release achieved with multi-layer matrix tablets: transport mechanisms and device design.

The aim of this study was to develop new multi-layer matrix tablets to achieve bimodal drug release profiles (fast release/slow release/fast release). Hydroxypropyl methylcellulose acetate succinate (HPMCAS, type MF) was chosen as a matrix former, because it is water-insoluble at low, and water-soluble at high pH values. Studies focused on the elucidation of the drug release mechanisms from HPMCAS-MF:drug tablets. In 0.1 N HCl the resulting release kinetics can be described using Fick's second law of diffusion, taking into account axial and radial mass transfer in cylindrical geometry. As the diffusion coefficients are found to be constant and the boundary conditions to be stationary, these systems are purely drug diffusion-controlled. In contrast, the dominating mass transport phenomena in phosphate buffer pH 7.4 are more complex. Due to polymer dissolution the resulting matrix structure is time-variant, leading to increasing drug diffusion coefficients and decreasing tablet dimensions, and thus moving boundary conditions. Drug release is affected by water imbibition, drug diffusion and polymer dissolution and is faster compared to 0.1 N HCl. With knowledge of these underlying release mechanisms, multi-layer matrix tablets were developed to achieve bimodal drug release. HPMCAS-MF:drug mixtures were used as tablet cores. As expected, changing the release medium from 0.1 N HCl to phosphate buffer pH 7. 4 after 2 h, lead to a significant increase in drug release. The abruptness of this rate change could be enhanced by adding two drug-free HPMCAS-MF barrier layers (one on each side) to the system. The addition of a fourth, drug-containing and fast disintegrating initial dose layer yielded the desired bimodal drug release patterns. The process and formulation parameters affecting the resulting release rates were investigated using theophylline and acetaminophen as model drugs.

pH-independent release of a weakly basic drug from water-insoluble and -soluble matrix tablets.

Weakly basic drugs or salts thereof demonstrate pH-dependent solubility. The resulting release from conventional matrix tablets decreases with increasing pH-milieu of the gastrointestinal tract. The aim of this study was to overcome this problem and to achieve pH-independent drug release. Two different polymers were used as matrix formers, the water-insoluble and almost unswellable ethylcellulose (EC), and the water-soluble and highly swellable hydroxypropyl methylcellulose (HPMC). Two different approaches to solve the problem of pH-dependent release of weakly basic drugs are demonstrated in this paper. The first one is based on the addition of hydroxypropyl methylcellulose acetate succinate (HPMCAS, an enteric polymer), the second one on the addition of organic acids such as fumaric, succinic or adipic acid to the drug-polymer system. The first approach failed to achieve pH-independent drug release, whereas the addition of organic acids to both matrix formers was found to maintain low pH values within the tablets during drug release in phosphate buffer (pH 6.8 or 7.4). Thus, the micro-environmental conditions for the dissolution and diffusion of the weakly basic drug were almost kept constant. The release of verapamil hydrochloride from tablets composed of ethylcellulose or HPMC and organic acids was found to be pH-independent.

Novel diagnostic algorithm for identification of mycobacteria using genus-specific amplification of the 16S-23S rRNA gene spacer and restriction endonucleases.

A novel genus-specific PCR for mycobacteria with simple identification to the species level by restriction fragment length polymorphism (RFLP) was established using the 16S-23S ribosomal RNA gene (rDNA) spacer as a target. Panspecificity of primers was demonstrated on the genus level by testing 811 bacterial strains (122 species in 37 genera from 286 reference strains and 525 clinical isolates). All mycobacterial isolates (678 strains among 48 defined species and 5 indeterminate taxons) were amplified by the new primers. Among nonmycobacterial isolates, only Gordonia terrae was amplified. The RFLP scheme devised involves estimation of variable PCR product sizes together with HaeIII and CfoI restriction analysis. It yielded 58 HaeIII patterns, of which 49 (84%) were unique on the species level. Hence, HaeIII digestion together with CfoI results was sufficient for correct identification of 39 of 54 mycobacterial taxons and one of three or four of seven RFLP genotypes found in Mycobacterium intracellulare and Mycobacterium kansasii, respectively. Following a clearly laid out diagnostic algorithm, the remaining unidentified organisms fell into five clusters of closely related species (i.e., the Mycobacterium avium complex or Mycobacterium chelonae-Mycobacterium abscessus) that were successfully separated using additional enzymes (TaqI, MspI, DdeI, or AvaII). Thus, next to slowly growing mycobacteria, all rapidly growing species studied, including M. abscessus, M. chelonae, Mycobacterium farcinogenes, Mycobacterium fortuitum, Mycobacterium peregrinum, and Mycobacterium senegalense (with a very high 16S rDNA sequence similarity) were correctly identified. A high intraspecies sequence stability and the good discriminative power of patterns indicate that this method is very suitable for rapid and cost-effective identification of a wide variety of mycobacterial species without the need for sequencing. Phylogenetically, spacer sequence data stand in good agreement with 16S rDNA sequencing results, as was shown by including strains with unsettled taxonomy. Since this approach recognized significant subspecific genotypes while identification of a broad spectrum of mycobacteria rested on identification of one specific RFLP pattern within a species, this method can be used by both reference (or research) and routine laboratories.