There is a current biodefense interest in protection against anthrax. Here, we developed a new generation of stable and effective anthrax vaccine. We studied the immune response elicited by recombinant protective antigen (rPA) delivered intranasally with a novel mucosal adjuvant, a mast cell activator compound 48/80 (C48/80). The vaccine formulation was prepared in a powder form by spray-freeze-drying (SFD) under optimized conditions to produce particles with a target size of D(50) = 25 μm, suitable for delivery to the rabbit nasal cavity. Physicochemical properties of the powder vaccines were characterized to assess their delivery and storage potential. Structural stability of rPA was confirmed by circular dichroism and attenuated total reflectance-Fourier transform infrared spectroscopy, whereas functional stability of rPA and C48/80 was monitored by cell-based assays. Animal study was performed using a unit-dose powder device for direct nasal application. Results showed that C48/80 provided effective mucosal adjuvant activity in rabbits. Freshly prepared SFD powder vaccine formulations or powders stored for over 2 years at room temperature elicited significantly elevated serum PA-specific and lethal toxin neutralization antibody titers that were comparable to that induced by intramuscular immunization with rPA. Nasal delivery of this vaccine formulation may be a viable alternative to the currently licensed vaccine or an attractive vaccine platform for other mucosally transmitted diseases.

Pharmaceutical spray-freeze drying (SFD) includes a heterogeneous set of technologies with primary applications in apparent solubility enhancement, pulmonary drug delivery, intradermal ballistic administration and delivery of vaccines to the nasal mucosa. The methods comprise of three steps: droplet generation, freezing and sublimation drying, which can be matched to the requirements given by the dosage form and route of administration. The objectives, various methods and physicochemical and pharmacological outcomes have been reviewed with a scope including related fields of science and technology.

OBJECTIVE

To compare the relative cost effectiveness of salmeterol (50 microg)/ fluticasone propionate (100 microg) with that of oral montelukast (10mg) as initial maintenance therapy in patients with persistent asthma uncontrolled on short-acting beta2-agonist therapy alone.

METHODS

A cost-effectiveness analysis was performed based on effectiveness and resource utilisation data that was prospectively collected from a randomised, double-blind, double-dummy, 12-week trial.

METHODS

Patients (>15 years of age) who had asthma for at least 6 months. Effectiveness measurements in this analysis included improvement in forced expiratory volume in 1 second (FEV(1)) and symptom-free days (SFDs). Cost of asthma drug treatment as well as costs related to an asthma exacerbation were used in the cost analysis. The study assumed a payer's perspective. All costs are in 2001 US dollars.

RESULTS

Of the 423 patients eligible for the study, 211 were randomised to salmeterol/fluticasone propionate and 212 to montelukast. Treatment with salmeterol/fluticasone propionate resulted in a significantly higher proportion of patients who achieved a 12% increase in FEV(1) (successful treatment) [salmeterol/fluticasone propionate: 71% vs montelukast: 39%; p < 0.001] and percentage of SFDs (salmeterol/fluticasone propionate: 46.8% vs montelukast: 21.5%; p < 0.001) compared with montelukast. The mean daily costs per successfully treated patient were lower in the salmeterol/fluticasone propionate group (US dollars 5.03, 95% CI US dollars 4.61 to US dollars 5.50) compared with the montelukast group (US dollars 8.25, 95% CI US dollars 6.98 to US dollars 9.93). Furthermore, per patient mean daily cost per SFD was lower with salmeterol/fluticasone propionate (US dollars 7.63, 95% CI US dollars 6.90 to US dollars 8.50) compared with montelukast (US dollars 14.89, 95% CI US dollars 12.36 to US dollars 17.98). Incremental cost-effectiveness ratios (ICERs) showed that the additional costs to achieve these benefits with salmeterol/fluticasone propionate were minimal. With regards to improvement in lung function, the ICER was US dollars 1.33 (95% CI US dollars 0.80 to US dollars 2.02) and with regards to SFD, the ICER was US dollars 1.69 (95% CI US dollars 1.01 to US dollars 2.48). Sensitivity analysis demonstrated the stability of the results over a range of assumptions.

CONCLUSIONS

From a third-party payer perspective, this analysis shows that based on increased efficacy and only a slight increase in cost, twice-daily treatment with salmeterol/fluticasone propionate is more cost effective than once-daily treatment with montelukast as initial maintenance therapy for persistent asthma. This finding complements the results of the clinical analyses indicating that treatment of both inflammation and bronchoconstriction with products such as salmeterol/ fluticasone propionate may be more cost effective as initial maintenance asthma therapy than the use of leukotriene modifiers such as montelukast.

Snake fungal disease (SFD) is an emerging skin infection of wild snakes in eastern North America. The fungus Ophidiomyces ophiodiicola is frequently associated with the skin lesions that are characteristic of SFD, but a causal relationship between the fungus and the disease has not been established. We experimentally infected captive-bred corn snakes (Pantherophis guttatus) in the laboratory with pure cultures of O. ophiodiicola. All snakes in the infected group (n = 8) developed gross and microscopic lesions identical to those observed in wild snakes with SFD; snakes in the control group (n = 7) did not develop skin infections. Furthermore, the same strain of O. ophiodiicola used to inoculate snakes was recovered from lesions of all animals in the infected group, but no fungi were isolated from individuals in the control group. Monitoring progression of lesions throughout the experiment captured a range of presentations of SFD that have been described in wild snakes. The host response to the infection included marked recruitment of granulocytes to sites of fungal invasion, increased frequency of molting, and abnormal behaviors, such as anorexia and resting in conspicuous areas of enclosures. While these responses may help snakes to fight infection, they could also impact host fitness and may contribute to mortality in wild snakes with chronic O. ophiodiicola infection. This work provides a basis for understanding the pathogenicity of O. ophiodiicola and the ecology of SFD by using a model system that incorporates a host species that is easy to procure and maintain in the laboratory.

OBJECTIVE

Skin infections in snakes, referred to as snake fungal disease (SFD), have been reported with increasing frequency in wild snakes in the eastern United States. While most of these infections are associated with the fungus Ophidiomyces ophiodiicola, there has been no conclusive evidence to implicate this fungus as a primary pathogen. Furthermore, it is not understood why the infections affect different host populations differently. Our experiment demonstrates that O. ophiodiicola is the causative agent of SFD and can elicit pathological changes that likely impact fitness of wild snakes. This information, and the laboratory model we describe, will be essential in addressing unresolved questions regarding disease ecology and outcomes of O. ophiodiicola infection and helping to conserve snake populations threatened by the disease. The SFD model of infection also offers utility for exploring larger concepts related to comparative fungal virulence, host response, and host-pathogen evolution.

Mitochondrial transcription factor A (TFAM), a nucleus-encoded protein, regulates the initiation of transcription and replication of mitochondrial DNA (mtDNA). Decreased expression of nuclear-encoded mitochondrial genes has been associated with onset of obesity in mice. Therefore, we hypothesized genetic variants in TFAM gene influence mitochondrial biogenesis consequently affecting body fat deposition and energy metabolism. In the present study, both cDNA (2259 bp) and genomic DNA (16,666 bp) sequences were generated for the bovine TFAM gene using a combination of in silico cloning with targeted region PCR amplification. Alignment of both cDNA and genomic sequences led to the determination of genomic organization and characterization of the promoter region of the bovine TFAM gene. Two closely linked A/C and C/T single nucleotide polymorphisms (SNPs) were found in the bovine TFAM promoter and then genotyped on 237 Wagyu x Limousin F(2) animals with recorded phenotypes for marbling and subcutaneous fat depth (SFD). Statistical analysis demonstrated that both SNPs and their haplotypes were associated with marbling (P=0.0153 for A/C, P=0.0026 for C/T, and P=0.0004 for haplotype) and SFD (P=0.0200 for A/C, P=0.0039 for C/T, and P=0.0029 for haplotype), respectively. A search for transcriptional regulatory elements using MatInspector indicated that both SNPs lead to a gain/loss of six putative-binding sites for transcription factors relevant to fat deposition and energy metabolism. Our results suggest for the first time that TFAM gene plays an important role in lipid metabolism and may be a strong candidate gene for obesity in mammals.

METHODS

We conducted a multinational pharmacoeconomic evaluation comparing the immediate release form of a new class of serotonin norepinephrine reuptake inhibitor (SNRI), venlafaxine IR to the selective serotonin reuptake inhibitors (SSRIs) and the tricyclic antidepressants (TCAs) in the treatment of acute major depressive disorder (MDD) in 10 countries (Germany, Italy, Netherlands, Poland, Spain, Sweden, Switzerland, United Kingdom, United States, and Venezuela). We designed a decision analytic model assessing the acute phase of MDD treatment within a 6-month time horizon. Six decision tree models were customized with country-specific estimates from a clinical management analysis, meta-analytic rates from two published meta-analyses, and a resource valuation of treatment costs representing the inpatient and outpatient settings within each country. The meta-analyses provided the clinical rates of success defined as a 50% reduction in depression scores on the Hamilton Depression Scale (HAM-D) or the Montgomery-Asberg Depression Rating Scale (MADRS). Treatment regimen costs were determined from standard lists, fee schedules, and communication with local health economists in each country. The meta-analytic rates were applied to the decision analytic model to calculate the expected cost and expected outcomes for each antidepressant comparator. Cost-effectiveness was determined using the expected values for both a successful outcome, and a composite measure of outcome termed symptom-free days. A policy analysis was conducted to examine the health system budget impact in each country of increasing the utilization of the most effective antidepressant found in our study.

RESULTS

Initiating treatment of MDD with venlafaxine IR yielded a lower expected cost compared to the SSRIs and TCAs in all countries except Poland in the inpatient setting, and Italy and Poland within the outpatient settings. The weighted average expected cost per patient varied from US$632 (Poland) to US$5647 (US) in the six-month acute phase treatment of MDD. The estimated total budgetary impact for each 1% of venlafaxine utilization, assuming a population of one million MDD patients, ranged from US$1600 (Italy) to US$29,049 (US).

CONCLUSIONS

Within the inpatient and outpatient treatment settings, venlafaxine IR was a more cost-effective treatment of MDD compared to the SSRIs and TCAs. Additionally, the results of this investigation indicate that increased utilization of venlafaxine in most settings across Europe and the Americas will have favorable impact on health care payer budgets. ADR, adverse drug reaction; CMA, clinical management analysis; ECT, electroconvulsive therapy; HAM-D, Hamilton Depression Scale; MADRS, Montgomery-Asberg depression rating scale; MDD, major depressive disorder; SFD, symptom-free day; SNRI, serotonin-norepinephrine reuptake inhibitor; SSRI, selective serotonin reuptake inhibitor; TCA, tricyclic antidepressant; WHO, world health organization.

Sorsby's fundus dystrophy (SFD) is an autosomal dominant degenerative disease of the macula caused by mutations in the tissue inhibitor of metalloproteinase-3 (TIMP-3) gene. Choroidal neovascularization is a hallmark of this disease, which closely resembles the exudative form of age-related macular degeneration. However, the mechanism by which TIMP-3 mutations induce the disease phenotype in SFD remains unknown. To address this question we established human retinal pigment epithelial cell lines expressing wild type or S156C (Ser(156) changed to cysteine) mutant TIMP-3. S156C TIMP-3 had reduced matrix metalloproteinase (MMP) inhibitory activity in retinal pigment epithelial cells and resulted in increased secretion and activation of gelatinase A and B. The conditioned medium from these cells induced angiogenesis in "in vivo" chick chorioallantoic membrane assays that could be reversed with recombinant wild type TIMP-3. Our data indicate that the choroidal neovascularization in SFD may be a result of increased MMP activity, which could lead to the stimulation of angiogenesis. These results also suggest the potential therapeutic use of TIMP-3 or synthetic MMP inhibitors in this disease.

Sorsby's fundus dystrophy (SFD) is a rare autosomal dominant disorder that results in degeneration of the macular region of the retina, with onset usually in the fourth to fifth decade of life. It leads to the rapid loss of central vision, often followed by further loss of peripheral vision. SFD shares several pathological features commonly found in the 'wet' or exudative form of age-related macular degeneration (AMD), the most common cause of blindness in the elderly in developed countries. These phenotypic similarities have led to SFD being proposed as an acceptable genetic model for AMD. Whereas AMD appears to have a complex aetiology, with both genetic and environmental factors playing a role, SFD has been shown to be a single-gene disorder, linked to mutations in exon 5 of the tissue inhibitor of metalloproteinases 3 (TIMP3) gene on chromosome 22q12-q13. This review confines itself to a discussion of the known biochemical properties of the wild-type and SFD TIMP3 proteins and attempts to relate these to the pathology encountered in SFD patients. We also discuss briefly how, despite the lack of inherited mutations in the structural gene, the TIMP3 protein might play a role in the onset and progression of AMD.

The objectives of this study were to produce nanostructured protein microparticles with the spray freezing into liquid (SFL) cryogenic process and to demonstrate a smaller degree of protein denaturation and aggregation than observed in spray freeze drying (SFD). Nanostructured microparticles were formed by atomization of an aqueous buffer solution containing bovine serum albumin (BSA) with and without excipients beneath the surface of a cryogenic liquid. Lyophilization was used to sublime the water in the frozen particles. The resulting BSA dry powder was characterized by size exclusion chromatography, Fourier-transform infrared spectroscopy, scanning electron microscopy (SEM), light scattering, and specific surface area analysis. SEM revealed highly porous microparticle with features smaller than 500 nm. The specific surface area of the BSA microparticles ranged from 19.2 to 97.7 m(2)/g as a function of the total protein and excipient content in the aqueous feed solution. SFL produced less denaturation and aggregation of protein monomer than SFD, despite the extremely high surface areas in both processes. The intense atomization and ultra-rapid freezing in the SFL process lead to nanostructured BSA microparticles with high surface areas. Protein denaturation and aggregation are reduced in SFL relative to SFD. The more rapid freezing in SFL lowers the time for proteins to aggregate or diffuse to water-air and water-ice interfaces where they may be denatured.

Following the observation by Brown et al. (Am J Physiol 1997; 272: C937-49) that primary rat adipocytes in culture secrete gelatinase A (MMP-2), we have evaluated gelatinase expression in adipose tissue with the use of mouse models of obesity. Wild-type mice were kept on a standard fat diet (SFD) or on a high fat diet (42% fat, HFD) and- genetically obese db/db mice were kept on SFD; gonadal and subcutaneous fat pads were removed and analysed ex vivo. These studies revealed that: 1) the HFD induced adipocyte hypertrophy; 2) after 32 weeks, significantly higher levels of 70 kDa (p <0.05) and 65 kDa proMMP-2 (p <0.01) were observed in extracts of gonadal fat pads of mice on HFD; 3) the contribution of active MMP-2 to the total level was comparable in SFD and HFD groups (20 to 30%); and 4) gelatinase B (MMP-9) was not consistently detected. These findings were confirmed by gelatin zymography and by mRNA determination using competitive RT-PCR. The presence of MMP-2 in the adipose tissue was confirmed immunologically and its localization in adipocytes revealed by immunogold electron microscopy. The potential functional role of MMP-2 in adipose tissue remains to be determined.

Lipid-polymer hybrid nanoparticles - polymeric nanoparticles enveloped by lipid layers - have emerged as a potent therapeutic nano-carrier alternative to liposomes and polymeric nanoparticles. Herein we perform comparative studies of employing spray drying (SD) and spray freeze drying (SFD) to produce inhalable dry-powder form of drug-loaded lipid-polymer hybrid nanoparticles. Poly(lactic-co-glycolic acid), lecithin, and levofloxacin are employed as the polymer, lipid, and drug models, respectively. The hybrid nanoparticles are transformed into micro-scale nanoparticle aggregates (or nano-aggregates) via SD and SFD, where the effects of (1) different excipients (i.e. mannitol, polyvinyl alcohol (PVA), and leucine), and (2) nanoparticle to excipient ratio on nano-aggregate characteristics (e.g. size, flowability, aqueous reconstitution, aerosolization efficiency) are examined. In both methods, PVA is found more effective than mannitol for aqueous reconstitution, whereas hydrophobic leucineis needed to achieve effective aerosolization as it reduces nano-aggregate agglomeration. Using PVA, both methods are equally capable of producing nano-aggregates having size, density, flowability, yield and reconstitutibility in the range ideal for inhaled delivery. Nevertheless, nano-aggregates produced by SFD are superior to SD in terms of their aerosolization efficiency manifested in the higher emitted dose and fine particle fraction with lower mass median aerodynamic diameter.

OBJECTIVE

A comparative study of the spinopelvic sagittal alignment in patients with lumbar disc degeneration or herniation (LDD/LDH) in normal population was designed to analyse the role of sagittal anatomical parameter (pelvic incidence, PI) and positional parameters in the pathogenesis and development of the disease. Several comparative studies of these patients with asymptomatic controls have been done. However, in previous studies without lumbar MRI, a certain number of asymptomatic LDD patients should have been included in the control group and then impacted on the results.

METHODS

Based on MRI findings, we divided 60 LDD or LDH patients and 110 asymptomatic volunteers into the normal group (NG) and the degeneration group (DG), which was further subdivided into the symptomatic (SDG) and asymptomatic (ADG) subgroups according to patients' symptoms. Standing full spine radiographs were used to measure sagittal parameters, including PI, sacral slope (SS), pelvic tilt (PT), lumbar lordosis (LL), thoracic kyphosis (TK), sagittal vertical axis (SVA), and sacrum-bicoxofemoral distance (SFD).

RESULTS

The PI, SS and LL in DG were significantly lower than NG, while the SVA and SFD were significantly greater (P < 0.05). PI correlated well with the SS and LL in all subjects. However, the trend lines of SS or LL over PI were downward in DG. PI was similar in SDG and ADG (P = 0.716) but SS and LL were significantly lower and SVA was significantly greater (P < 0.05).

CONCLUSIONS

PI may play a predisposing role in the pathogenesis of lumbar disc degenerative diseases. The secondary structural and compensatory factors would lead to a straighter spine after disc degenerative change.

Sorsby's fundus dystrophy (SFD) has been mapped to a genetic interval of 8 cM between loci D22S275 and D22S278. A total of 15 families, unrelated on the basis of genealogy and expressing the SFD phenotype were identified from a large data base of genetic eye disease families originating from diverse parts of the British Isles. The identification of the same Ser181Cys mutation cosegregating with disease in each family led us to consider the hypothesis of a founder effect being present. In all families studied, the same relatively infrequent allele (occurring in just 11% of the control group) was associated with disease at marker locus D22S280. A highly significant disease-associated haplotype, spanning across 3 cM of the SFD locus, was conserved in 11 of the 15 families (68% of all affected chromosomes); a further extended haplotype spanning up to 7 cM, was identified in 5 families (27% of SFD-associated chromosomes) and possibly represents the ancestral haplotype. This haplotype analysis has refined the TIMP3 gene localization to a 1- to 3-cM interval between marker loci D22S273 and D22S281 and provides strong evidence for a single mutational event being responsible for the majority of SFD identified in the British Isles.

Nanofibrillated cellulose (NFC) aerogels were prepared by spray freeze-drying (SFD). Their structural, mechanical and thermal insulation properties were compared to those of NFC aerogels prepared by conventional freeze-drying (CFD). The purpose of this investigation is to develop superinsulating bioaerogels by reducing their pore size. Severe reduction of the aerogel pore size and skeleton architecture were observed by SEM, aerogels prepared by SFD method show a fibril skeleton morphology, which defines a mesoporous structure. BET analyses confirm the appearance of a new organization structure with pores of nanometric sizes. As a consequence, the thermal insulation properties were significantly improved for SFD materials compared to CFD aerogel, reaching values of thermal conductivity as low as 0.018W/(mK). Moreover, NFC aerogels have a thermal conductivity below that of air in ambient conditions, making them one of the best cellulose based thermal superinsulating material.

Genetic and ecological factors may interact in their effects on fitness. Such interactions are thus to be expected between inbreeding and exposure of a population to a toxicant. The magnitude of inbreeding depression is thought to increase in stressful environments. To test this hypothesis, we investigated the combined effects of environmental conditions and inbreeding on fitness in the self-fertile snail Lymnaea stagnalis, using a stress gradient (0-2) applied to a 100 isolated and paired lineages: laboratory control (0), outdoor microcosm control (1) and pesticide exposure under outdoor microcosm conditions (2). Outdoor stress conditions were maintained for 28 days prior to measurements of fitness traits (fecundity, hatching success, and size at hatching) under laboratory conditions, so that delayed environmental effects could be estimated. Under laboratory control conditions, we found significant initial family level heterogeneity for most measured traits, including physiological performances as assessed through energetic biomarkers. Whatever the environmental conditions, inbreeding depression was very low for progeny performances. Negative values of self-fertilization depression (SFD) were obtained. Unexpectedly, SFD showed a negative relationship with the assumed stress intensity, reflecting a higher sensitivity under pairing than under selfing, mostly due to parental fecundity. This suggests that stressful conditions may favour selfing. Stress intensity increased the distribution limits of both depression indices, suggesting that changes in fitness are less predictable in a population under stress. Implications of such findings for environmental risk assessment of pesticides are discussed.

Sorsby's fundus dystrophy (SFD) is an autosomal dominant macular dystrophy which is developed usually in the third or fourth decade of life, and is characterized by central visual loss and nyctalopia due to fundus changes of exudative or atrophic macular lesions. Its functional prognosis is usually poor because of disciform macular scars and peripheral chorioretinal atrophies. To date, five different mutations in the tissue inhibitor of the metalloproteinases-3 (TIMP3) gene have been identified in families of a wide geographic origin, all of which are missense mutations that cause replacement by cysteine of conserved amino acids in the C-terminus of exon 5 of TIMP3. We have studied two Japanese families with SFD, the first report from the Eastern world, and identified a novel 3' splice site mutation in the TIMP3 gene, namely a single base insertion at the intron 4/exon 5 junction which converts the consensus sequence CAG to CAAG in the splice acceptor site. In addition, our patients displayed a distinctive clinical expression in that they developed macular dystrophies at an approximately 30-year later age of onset and preserved functional vision until later life with essentially uninvolved peripheral retina. The present findings may provide some insight into the genotype-phenotype relationship in SFD.

OBJECTIVE

Ion channel mechanism of cardiac electrophysiological effects of Shenfu Decoction (SFD, Ginseng and Aconiti Praeparatae Decoction), a traditional Chinese medicine (TCM) prescription, and its ingredient bases were investigated in guinea pigs.

METHODS

After administration of an injection made from SFD (Shenfu Injection, SFI), the indexes of transmembrane action potential (TAP) in vivo and sodium channels in isolated ventricular myocyte were assayed by suspended microelectrodes and patch clamp techniques respectively, and ingredients of SFD were compared with.

RESULTS

After administration of SFI, the action potential amplitude (APA) and maximum velocity (V(max)) of TAP decreased. In the presence of either SFI or Fuzi active ingredient (FZAI)(5, 10, and 15%), not any other ingredient, the density of voltage-dependent sodium current (I(Na)) decreased significantly, while the inhibition ratio of SFI was larger. EC(50) of SFI was less than the one of FZAI, and SFI displayed effects on I(Na) in wider voltage scope than FZAI in current-voltage curve. Both SFI and FZAI shifted the steady-state inactivation curve of sodium channels to the left, and the recovery curve to the right.

CONCLUSIONS

The results indicated that the cardiac electrophysiological effects of SFI were exerted by blocking sodium channels, and FZAI contributed to such effects most but inferior to SFI, which justified its use in anti-arrhythmia, myocardial protection, etc.

The expression of hTERT gene, encoding the catalytic subunit of telomerase, is a feature of most cancer cells. Changes in the chromatin environment of its promoter and binding of transcriptional factors have been reported in differentiating cells when its transcription is repressed. However, it is not clear whether these changes are directly involved in this repression or only linked to differentiation. In a maturation-resistant acute promyelocytic leukemia (APL) cell line (NB4-LR1), we have previously identified a new pathway of retinoid-induced hTERT repression independent of differentiation. Using a variant of this cell line (NB4-LR1(SFD)), which resists to this repression, we show that although distinct patterns of histone modifications and transcription factor binding at the proximal domain of hTERT gene promoter could concur to modulate its expression, this region is not sufficient to the on/off switch of hTERT by retinoids. DNA methylation analysis of the hTERT promoter led to the identification of two distinct functional domains, a proximal one, fully unmethylated in both cell lines, and a distal one, significantly methylated in NB4-LR1(SFD) cells, whose methylation was further re-enforced by retinoid treatment. Interestingly, we showed that the binding to this distal domain of a known hTERT repressor, WT1, was defective only in NB4-LR1(SFD) cells. We propose that epigenetic modifications targeting this distal region could modulate the binding of hTERT repressors and account either for hTERT reactivation and resistance to retinoid-induced hTERT downregulation.

This work investigates the use of spray freeze-drying (SFD) to produce protein loaded particles suitable for epidermal delivery. In the first part of the study, the effects of formulation and process conditions on particle properties are examined. Aqueous solutions of trehalose produce SFD particles in the size range 20-80 microm, with a smooth, textured surface, but having high internal porosity. The latter was visualized using SEM and a novel particle embedding and sectioning technique. Use of an annealing step during the freeze-drying cycle caused the particles to shrink, reducing hereby porosity and also the measured rate of moisture uptake into these amorphous particles. SFD pure mannitol was approximately 40% amorphous, but not hygroscopic. Incorporation of dextran 37,500 into a combined amorphous trehalose/mannitol formulation led to increased particle shrinkage and lower particle porosity on annealing. The model protein trypsinogen lost approximately 15% activity during SFD of solutions containing 50 mg/mL protein, but was only marginally aggregated (1.4%). It is suggested that trypsinogen forms an irreversible partially unfolded state or molten globule on SFD/rehydration. The pure protein was also partially inactivated without aggregation during atomization into air. Surprisingly, neither activity loss nor aggregation were detected on atomization of the protein solution into liquid nitrogen. Quench-freezing of small droplets may reverse the partial unfolding of trypsinogen occurring on atomization into air. The origin of the trypsinogen inactivation during SFD must therefore be the subsequent freeze-drying step of this multistep process. Isolated freeze drying of trypsinogen produces strong aggregation and equivalent inactivation. This result suggests that trypsinogen behaves differently during freeze drying from frozen droplets and from bulk solution in a vial. In the former case the protein forms an irreversible partially unfolded state, whereas in the latter case aggregates are formed. Trypsinogen inactivation during SFD could be completely prevented by the presence of trehalose in the formulation. Electron Spectroscopy for Chemical Analysis (ESCA) showed a high surface excess of the protein in the SFD particles, which was reduced on inclusion of Polysorbate 80, but not trehalose. Taken together, these results help to elucidate the complex destabilization behavior of trypsinogen during SFD.

OBJECTIVE

Patients with somatoform disorders (SFD) are likely to overutilize healthcare services. This study investigates (a) whether extraordinarily high medical costs can be predicted from patient characteristics or psychopathology, and (b) whether high-utilizing patients respond differently to cognitive-behavioral treatment.

METHODS

We compared 42 SFD high utilizers with 53 SFD average utilizers and 29 patients suffering from other than SFD mental disorders. High utilization was defined by healthcare expenditures of>> or = 2500 euros during the past 2 years. Costs were computed from medical and billing records of health insurance companies. Somatization distress, hypochondriasis, depression, dysfunctional cognitions related to bodily symptoms, general psychopathology, personality profiles, and psychosocial disabilities were assessed before treatment.

RESULTS

High utilizers had higher levels of self- and observer-rated illness behavior, self-perceived bodily weakness, and psychosocial disabilities. Although they did not report more somatization symptoms, their subjective symptom distress was higher. There were no differences between high and average utilizers concerning general psychopathology, DSM-IV comorbidity, and personality profiles. Treatment improvements were similar.

CONCLUSIONS

High- and average-utilizing somatizers represent distinguishable subgroups. The results emphasize the importance of mechanisms specifically related to SFD and may enhance the early detection of patients who are likely to develop overutilization.

OBJECTIVE

To evaluate the physical growth and sexual maturation of children born with low birth weight (< 2000 g). (LBW).

METHODS

Longitudinal follow up.

METHODS

Hospital born urban cohort.

METHODS

Weight, height, head circumference and pubertal changes were recorded till 14 years at specified intervals in 252 LBW and 176 control (term neonates with birth weights>> or = 2500 g) children. Effect of prematurity and fetal growth retardation (SFD) was studied in 79 preterm appropriate for gestation and 45 term SFD children.

RESULTS

LBW boys significantly lagged behind their controls for all physical growth parameters till 14 years, while the LBW girls had a physical growth comparable to controls after 11 years. Preterms had comparable weight, height and head circumference with their controls after 11 years. The SFDs, however, remained significantly handicapped in their overall physical growth even at 14 years. In comparison to controls, menarche occured 6 months earlier in preterms and 12 months earlier in SFD girls. However, there was no change in the sequence of pubertal changes in either preterms or SFDs.

CONCLUSIONS

Fetal growth retardation has a lasting adverse effect on later physical growth, while most preterms catch up with their peers by adolescence.

Spray-freeze drying (SFD) is an attractive technique to prepare highly porous dry powders for inhalation. However, there have been few reports of its application to dry powder inhalers (DPIs). Therefore, in this study, we prepared dry plasmid DNA (pDNA) powders with different molecular ratios of chitosan to pDNA (N/P ratios) by SFD. All the pDNA powders were spherical and highly porous, with particles approximately 20-40microm in geometric diameter. The morphology changed little with the alteration of the N/P ratio. On electrophoresis, a band of linear pDNA was detected in the preparation without chitosan, suggesting the destabilization of pDNA through SFD. However, the addition of chitosan protected pDNA from destabilization. Moreover, the pDNA powders were evaluated for pulmonary gene transfection efficiency using an in vivo dual imaging technique for gene DPIs developed previously. Maximum gene expression was observed at 9-12h following pulmonary administration of the powders into mice. The powder with the N/P ratio of 10 had the highest gene transfection efficiency. A higher affinity of chitosan for pDNA and a smaller (approximately 100nm) pDNA/chitosan complex (N/Pf10) were found at pH 6.5 (in lung) than at pH 7.4 (in physiological conditions), suggesting that the effective compaction of pDNA by chitosan at the N/P ratio of 10 at pH 6.5 contributes to the gene transfection efficiency in the lung. These results suggest inhalable dry pDNA powders with chitosan prepared by SFD to be a suitable formulation for pulmonary gene therapy.

OBJECTIVE

To investigate the association of reticular pseudodrusen (RPD) with Sorsby fundus dystrophy (SFD).

METHODS

Prospective, monocenter, cross-sectional case series.

METHODS

Sixteen patients of 4 unrelated families with SFD caused by mutations in TIMP3.

METHODS

All subjects underwent multimodal imaging including near-infrared (NIR) reflectance and fundus autofluorescence with a confocal scanning laser ophthalmoscope and spectral-domain optical coherence tomography (SD OCT).

METHODS

Prevalence, topographic distribution, and phenotype of RPD.

RESULTS

Mean age of the investigated patients was 56.8 years (range, 23-78 years). Reticular pseudodrusen were identified frequently in SFD patients in the sixth decade of life (5 of 7 [71%]) and were absent in younger (n = 3) or older (n = 6) patients. They were most abundant in the superior quadrant and spared the foveal region. Reticular pseudodrusen appeared as yellowish round to oval (dot subtype; n = 5) or confluent, wriggled (ribbon subtype; n = 3) lesions, sometimes forming irregular networks. Reticular pseudodrusen were hyporeflective on NIR reflectance and hypofluorescent on fundus autofluorescence imaging. They appeared as subretinal deposits on SD OCT imaging. Other lesions, such as peripheral pseudodrusen and soft drusen, were present less frequently.

CONCLUSIONS

Reticular pseudodrusen are a frequent finding in patients with SFD. Although SFD patients with RPD are younger, distribution and phenotype of RPD are similar to those observed in patients with age-related macular degeneration. The association of RPD with SFD implicates a role of Bruch's membrane, the Bruch's membrane-retinal pigment epithelium interface, or both in the pathogenesis of RPD.

The potential of aerosol phage therapy for treating lung infections has been demonstrated in animal models and clinical studies. This work compared the performance of two dry powder formation techniques, spray freeze drying (SFD) and spray drying (SD), in producing inhalable phage powders.

A Pseudomonas podoviridae phage, PEV2, was incorporated into multi-component formulation systems consisting of trehalose, mannitol and L-leucine (F1 = 60:20:20 and F2 = 40:40:20). The phage titer loss after the SFD and SD processes and in vitro aerosol performance of the produced powders were assessed.

A significant titer loss (~2 log) was noted for droplet generation using an ultrasonic nozzle employed in the SFD method, but the conventional two-fluid nozzle used in the SD method was less destructive for the phage (~0.75 log loss). The phage were more vulnerable during the evaporative drying process (~0.75 log further loss) compared with the freeze drying step, which caused negligible phage loss. In vitro aerosol performance showed that the SFD powders (~80% phage recovery) provided better phage protection than the SD powders (~20% phage recovery) during the aerosolization process. Despite this, higher total lung doses were obtained for the SD formulations (SD-F1 = 13.1 ± 1.7 × 10(4) pfu and SD-F2 = 11.0 ± 1.4 × 10(4) pfu) than from their counterpart SFD formulations (SFD-F1 = 8.3 ± 1.8 × 10(4) pfu and SFD-F2 = 2.1 ± 0.3 × 10(4) pfu).

Overall, the SD method caused less phage reduction during the powder formation process and the resulted powders achieved better aerosol performance for PEV2.