Modeling the coating layer thickness in a pharmaceutical coating process.

Although mechanistic numerical simulations can offer great insights into a process, they are limited with respect to resolved process time. While statistical models provide long-term predictability, determining the underlying probability distributions is often challenging. In this work, detailed CFD-DEM simulations of a pharmaceutical Wurster coating process for microspheres are used to evaluate the input parameters for a novel Monte-Carlo simulation approach. The combined strengths of both modeling approaches make it possible to predict the coating mass and thickness distributions over the entire process time. It was observed that smaller beads receive a thicker coating layer since they pass the spray zone closer to the nozzle. Moreover, it was established that, in contrast to the airflow rate, the spray rate has a great impact on the inter-particle coating variability. A stochastic model was developed to investigate the relative contribution of coating layer variability and fill weight variability to the product non-uniformity in a capsule filling process of Multiple Unit Pellet Systems (MUPS).

Evaluation of some compression aids in tableting of roller compacted swellable core drug layer.

Swellable core technology (SCT) represents a broadly applicable oral osmotic drug delivery platform for the controlled release of drugs. SCT tablets control drug delivery by using osmosis to regulate the influx of water into the tablet's core. The tablet consists of two layers; drug layer and sweller layer, with a semi-permeable membrane coating and delivery port located in the drug layer side of the tablet. The key component of SCT formulations is polyethylene oxide (PEO), which is typically wet granulated with organic solvents to prevent rapid gel hydration observed during contact with aqueous environments. However, the use of organic solvents has their own environmental and cost considerations which make this form of processing undesirable. To overcome this issue, dry granulation can be employed. However, PEO is a very plastic material and problems may be encountered during the tableting process, when work hardening occurs upon double compression. The addition of compression aids to the drug layer will help to increase the roll force when generating ribbons - reducing fines and segregation potential - while also reducing work hardening effects which impact tablet friability. The five compression aids used in this study were microcrystalline cellulose (MCC), xylitol, di-calcium phosphate (anhydrous), lactose monohydrate and starch. The work undertaken here studies the compression properties of the drug layer blends with different levels of the five compression aids as part of the formulation. Roller compaction properties are also varied to provide granules with differing solid fractions. The results of this study indicate that addition of microcrystalline cellulose in the formulation in levels between 10% and 30% significantly improve the tablet hardness at lower tablet compression forces. Further work is required to investigate the impact on dissolution.

Specificity grouping of the accessory gene regulator quorum-sensing system of Staphylococcus epidermidis is linked to infection.

Staphylococcus epidermidis represents the most frequent pathogen involved in nosocomial infections and infections of indwelling medical devices. The strain-to-strain variation of the gene encoding the quorum-sensing pheromone of S. epidermidis as well as the correlation between specificity groups and origin from infection were determined. The pro-pheromone gene was highly conserved and showed infrequent, non-synonymous, single-nucleotide polymorphisms that led to conservative amino acid exchanges only. Importantly, one specificity group was significantly more frequent among strains isolated from infection. The finding that quorum-sensing specificity groups are linked to infection demonstrates the relevance of quorum-sensing for virulence in this critical human pathogen and contributes to the scientific basis needed for the development of quorum-sensing-targeting drugs.

Neutrophils as early immunologic effectors in hemorrhage- or endotoxemia-induced acute lung injury.

Acute lung injury is characterized by accumulation of neutrophils in the lungs, accompanied by the development of interstitial edema and an intense inflammatory response. To assess the role of neutrophils as early immune effectors in hemorrhage- or endotoxemia-induced lung injury, mice were made neutropenic with cyclophosphamide or anti-neutrophil antibodies. Endotoxemia- or hemorrhage-induced lung edema was significantly reduced in neutropenic animals. Activation of the transcriptional regulatory factor nuclear factor-kappaB after hemorrhage or endotoxemia was diminished in the lungs of neutropenic mice compared with nonneutropenic controls. Hemorrhage or endotoxemia was followed by increases in pulmonary mRNA and protein levels for interleukin-1beta (IL-1beta), macrophage inflammatory protein-2 (MIP-2), and tumor necrosis factor-alpha (TNF-alpha). Endotoxin-induced increases in proinflammatory cytokine expression were greater than those found after hemorrhage. The amounts of mRNA or protein for IL-1beta, MIP-2, and TNF-alpha were significantly lower after hemorrhage in the lungs of neutropenic versus nonneutropenic mice. Neutropenia was associated with significant reductions in IL-1beta and MIP-2 but not in TNF-alpha expression in the lungs after endotoxemia. These experiments show that neutrophils play a central role in initiating acute inflammatory responses and causing injury in the lungs after hemorrhage or endotoxemia.

Cloning and preliminary characterization of a novel cuticular antigen from the filarial parasite Dirofilaria immitis.

We have described here the cloning and partial characterization of a cDNA encoding a cuticular antigen of Dirofilaria immitis. A 48-h third-stage larval D. immitis cDNA library was immunoscreened with sera raised in mice against third-stage larval cuticles (mouse anti-L3 cuticle antisera). A strongly immunoreactive clone (L3MC4) was isolated. Sequence analysis of L3MC4 showed that it was a partial length cDNA. The missing 5' end of the clone was amplified by PCR from D. immitis adult female first-strand cDNA using the nematode 22-base splice leader sequence and a L3MC4-specific antisense primer. The composite cDNA sequence comprised 616 bases (nDiL3MC4) encoding a full-length protein of 146 amino acids (DiL3MC4). GenBank analysis showed that DiL3MC4 shared some homology to an unknown C. elegans gene product (31%) at the amino acid level. However, there were no related filarial expressed sequence tags in the current GenBank database. Antibodies to recombinant DiL3MC4 (rDiL3MC4) identified a 19-kDa native antigen in the adults and in the L3 and L4 larval stages of D. immitis. In addition, the antibodies bound to the cortical layers of the L3 cuticle, as revealed by immuno-gold electron microscopy. The native protein was not detected in larval and adult excretory-secretory products. Immunoblot analysis showed that serum from a rabbit that was repeatedly injected with a small number of D. immitis third stage larvae reacted with rDiL3MC4. Thus, DiL3MC4 is a novel cuticular antigen of a filarial parasite.

HMG-1 as a mediator of acute lung inflammation.

Acute inflammatory lung injury is often a delayed complication of critical illness and is associated with increased mortality. High mobility group-1 (HMG-1) protein, in addition to its role as a transcriptional regulatory factor, has recently been identified as a late mediator of endotoxin lethality. In the present studies, HMG-1 given intratracheally produced acute inflammatory injury to the lungs, with neutrophil accumulation, the development of lung edema, and increased pulmonary production of IL-1beta, TNF-alpha, and macrophage-inflammatory protein-2. In endotoxin-induced acute lung inflammation, administration of anti-HMG-1 Abs either before or after endotoxin exposure decreased the migration of neutrophils to the lungs as well as lung edema. These protective effects of anti-HMG-1 were specific, because pulmonary levels of IL-1beta, TNF-alpha, or macrophage-inflammatory protein-2 were not decreased after therapy with anti-HMG-1. Together, these findings indicate that HMG-1 is a distal mediator of acute inflammatory lung injury.

Removal of hydrogen peroxide by a 1-cysteine peroxiredoxin enzyme of the filarial parasite Dirofilaria immitis.

Prior studies have shown that filarial nematodes can effectively metabolize hydrogen peroxide in excess of that generated by activated host cells. However, the mechanisms of H2O2 removal by the filarial parasites are unclear. Herein we report the results of studies carried out on the biochemical activity and on immunolocalization of a recombinant peroxiredoxin (Prx) enzyme from the dog filarial parasite Dirofilaria immitis. A full-length cDNA encoding a 1-Cys Prx enzyme from the dog heartworm D. immitis was expressed in Escherichia coli as a recombinant polyhistidine fusion protein (rDiPrx-1). rDiPrx-1 was capable of reducing H2O2 in the presence of dithiothreitol. The apparent kinetic constants determined for DiPrx-1 using H2O2 as a substrate were a Michaelis constant (Km) of 16.28 mM and a maximal velocity (Vmax) of 16 micromol/min(-1). Consistent with the enzyme activity, D. immitis adult worms could detoxify exogenously added H2O2 in vitro. Antibodies to rDiPrx-1 identified a 27-kDa native antigen in parasite extracts and larval and adult excretory-secretory products. The antibodies were used to localize the native antigen to the lateral hypodermal chords of both male and female worms by immunohistochemistry. In addition, labeling was seen in the afibrillar muscle cells in male worms and in some areas of the uterine wall in female worms. Thus, DiPrx-1 is the first parasite Prx to be shown to detoxify exogenously added H2O2 in an in vitro system.

Identification of an asparagine amidohydrolase from the filarial parasite Dirofilaria immitis.

The nematode cuticle is a complex extracellular structure which is secreted by an underlying syncytium of hypodermal cells. Recent studies have demonstrated that the cuticle of parasitic nematodes is a dynamic structure with important absorptive, secretory, and enzymatic activities. In addition, the cuticle serves as a protective barrier against the host. A 48-h third stage larval Dirofilaria immitis cDNA library was immunoscreened with sera raised against larval cuticles. One clone, L3MC4 that reacted strongly with the anti-cuticle antisera was sequenced. The composite cDNA sequence comprises 2073 bp coding for a full-length protein of 590 amino acids. GenBank analysis showed that DiAsp had significant similarity to a Caenorhabditis elegans gene-product (54% identity) and to other asparaginases at the amino acid level. Escherichia coli-expressed recombinant DiAsp (rDiAsp) catalysed the hydrolysis of asparagine to aspartate and ammonia. Antibodies raised against D. immitis larval cuticles reacted with rDiAsp in immunoblots. This is the first report of identification of a cDNA clone encoding an asparaginase enzyme from a parasitic nematode.

Molecular characterization of a calcium-binding protein from the filarial parasite Dirofilaria immitis.

A full length D. immitis cDNA (nDiCal) encoding a protein with significant similarity to the calreticulin protein family was isolated from a 6-day fourth-stage larval cDNA expression library by immunoscreening, using serum from a rabbit immunized by repeated injection of small numbers of third-stage larvae. nDiCal is 1538 bp long and contains the 21 bp nematode splice leader sequence SL1 at the 5' end. nDiCal encodes for a protein (pDiCal) with a predicted molecular mass of 46 kDa. pDiCal sequence analysis revealed similarities with calreticulin, a protein that typically resides in the endoplasmic reticulum. pDiCal possesses three consensus sequences of the calreticulin family of proteins: a neutral N-terminal region with a putative signal sequence; a proline- and tryptophan-rich P region; and a highly acidic C-terminal region. A 45Ca2+-overlay assay showed that recombinant pDiCal (rDiCal) is a Ca2+-binding protein. Antibodies to rDiCal identified a 56 kDa native antigen in all developmental stages including the excretory-secretory products derived from larvae and adult worms. Localization studies demonstrated the ubiquitous presence of pDiCal with intense expression in the hypodermis and syncitial muscle cells in both male and female adult worms. Labeling was also seen in the developing embryos within the uterus of the female worms. Sera from immune as well as chronically-infected microfilaremic dogs contained antibodies that bind rDiCal. In addition, immunoblot analysis showed that serum from a rabbit immunized with L3 cuticles reacted with rDiCal.

Finnish En(a-) propositus with anti-EnaFS and anti-EnaFR: in vitro and in vivo characteristics.

A third Finnish En(a-) individual (ERP) with alloanti-Ena has been identified. Subsequent family studies revealed that ERP is distantly related to the 2 previous Finnish En(a-) propositi. Serologically, ERP's erythrocytes were found to be M-N-'N'+S-s+U+En(a-)Wr(a-b-) and were sialic acid deficient. SDS-PAGE studies confirmed that the red cell membranes lacked the MN-sialoglycoprotein, glycophorin-A. ERP's serum contained an IgG antibody which demonstrated two separable specificities, a ficin-sensitive specificity (anti-EnFS) and a ficin-resistant specificity (anti-EnFR), which reacted by the indirect antiglobulin technique. The antibodies were probably pregnancy induced, as ERP had never been transfused. A 51Cr-labelled red cell survival study showed that the antibodies were capable of causing significant destruction of incompatible red cells.

Survival after spinal cord trauma. A life table analysis.

The records accruing from the care of spinal cord injury patients in hospitals of Veterans Administration (VA) make available a unique opportunity to study survival rates of a large group. This study analyzes the survival experience of patients whose initial treatment in a VA hospital for trauma to the spinal cord occurred between Oct 1, 1955, and Sept 30, 1965. Life table methodology enabled survival rates to be calculated for various intervals after injury and allowed for maximum use of each patient's experience. Age at injury, level of lesion, and extent of paralysis were all found to be important factors in survival. High mortality occurs in the first three months regardless of age at injury or level of lesion. Of those paraplegic and quadriplegic patients who survived the first three months after injury, the ten-year survival rates are quite similar, 86% and 80%, respectively.