Influence of process parameters in the ASES process on particle properties of budesonide for pulmonary delivery.

Budesonide was micronized by precipitation in supercritical carbon dioxide by using the aerosol solvent extraction system at various process conditions. The process is characterised by spraying an organic solution of budesonide into the supercritical fluid (SCF), precipitation of the drug in the SCF and extraction and recovery of the solvent. The micronized budesonide particles were characterized physico-chemically for their morphology, crystallinity, size distribution and for their aerodynamic behaviour. The particle size distribution of the powder products was similar, regardless of the process conditions used for the crystallization. Also, the morphology of the particles did not differ between the batches. However, the aerodynamic properties of the precipitated batches were significantly different between the batches produced and as compared to a jet-milled budesonide powder. In conclusion, the process conditions in the SCF precipitation may influence the aerodynamic properties, although other physico-chemical parameters appear to be similar.     

Prediction of individual clinical outcome in MCI by means of genetic assessment and (18)F-FDG PET.

Patients with mild cognitive impairment (MCI) represent a risk population for progressing to dementia of the Alzheimer type (DAT). However, clinical criteria do not ensure reliable individual prognosis in these patients. The objective of this longitudinal, prospective study was to examine the value of (18)F-FDG PET of cerebral glucose metabolism and of genetic susceptibility, as defined by an APOEepsilon4-positive genotype, with regard to the early diagnosis of DAT in patients with MCI. METHODS: In 30 patients with the diagnosis of MCI (16 female, 14 male; age, 70 +/- 8 y), baseline and follow-up examinations (mean observation period, 16 mo) were performed. In all patients, the APOE genotype was assessed and cerebral glucose metabolism was evaluated at baseline using cranial (18)F-FDG PET. Individual PET data were screened for findings suggestive of Alzheimer's disease (AD), with the help of an automated computer program. After stereotactical normalization of the PET images, this program performs an observer-independent statistical comparison with an age-matched reference database (n = 22). RESULTS: In 43% of all MCI subjects, a PET scan suggestive of AD pathology according to our predefined criteria was observed at baseline (PET+); 57% of all MCI patients were carriers of the APOE epsilon4 allele (e4+). In 40% of all patients, progression of symptoms within the observation period justified the clinical diagnosis of probable DAT at the time of follow-up reevaluation. Statistical evaluation revealed the best results for PET with regard to early diagnosis of DAT in MCI patients (sensitivity, 92%; specificity, 89%). Classification according to the APOE genotype was significantly less successful (sensitivity, 75%; specificity, 56%). However, a combination of both diagnostic tests allowed early diagnosis with either very high specificity (PET+ AND e4+: sensitivity, 67%; specificity, 100%) or very high sensitivity (PET+ OR e4+: sensitivity, 100%; specificity, 44%). CONCLUSION: (18)F-FDG PET of cerebral glucose metabolism is a valuable diagnostic tool for the prediction of clinical outcome in individual MCI patients. Results are superior to the exclusive assessment of the APOE genotype. A combination of both functional imaging and genotyping may allow an early high-risk or low-risk stratification of patients with either very high sensitivity or very high specificity. This may be valuable, for example, for patient selection in scientific studies.     

Selective targeted delivery of TNFalpha to tumor blood vessels

We sought to enhance the selective toxicity of tumor necrosis factor alpha (TNFalpha) to permit its systemic use in cancer therapy. Because ligand-targeted therapeutics have proven successful in improving the selective toxicity of drugs, we prepared a fusion protein (L19mTNFalpha) composed of mouse TNFalpha and a high-affinity antibody fragment (L19 scFv) to the extradomain B (ED-B) domain of fibronectin, a marker of angiogenesis. L19mTNFalpha was expressed in mammalian cells, purified, and characterized. L19mTNFalpha was an immunoreactive and biologically active homotrimer. Radiolabeled L19mTNFalpha selectively targeted tumor neovasculature in tumor-bearing mice, where it accumulated selectively and persistently (tumor-to-blood ratio of the percentage of injected dose per gram [%ID/g] of 700, 48 hours from injection). L19mTNFalpha showed a greater anticancer therapeutic activity than both mTNFalpha and TN11mTNFalpha, a control fusion protein in which an antibody fragment, irrelevant in the tumor model used, substituted for L19. This activity was further dramatically enhanced by its combination with melphalan or the recently reported fusion protein L19-IL2. In conclusion, L19mTNFalpha allows concentrating therapeutically active doses of TNFalpha at the tumor level, thus opening new possibilities for the systemic use of TNFalpha in cancer therapy.     

Antiphospholipid antibodies in pediatric and adult patients with rheumatic disease are associated with parvovirus B19 infection

OBJECTIVE: To show a possible association between parvovirus B19 infection and the presence of antiphospholipid antibodies (aPL) in patients with rheumatic diseases. METHODS: Serum samples obtained from 88 children with various forms of juvenile rheumatic disease and from 40 adults with systemic lupus erythematosus, the antiphospholipid syndrome, or other rheumatic disease, who had previously been tested and shown to be positive for IgG aPL, were analyzed for the presence of B19 DNA, for antibodies against the B19 viral proteins VP1, VP2, and NS1, and for IgG aPL (anticardiolipin, anti-beta(2)-glycoprotein I, and antiphosphatidylserine). As controls, serum samples obtained from 135 children with noninflammatory bone diseases or growth retardation were also analyzed. RESULTS: Twenty-four (27%) of the 88 children with rheumatic diseases had detectable amounts of IgG aPL. Fourteen (58%) of these 24 IgG aPL-positive patients showed IgG against VP1/VP2 and viral genomes, indicating the presence of acute (2 patients) or persistent (12 patients) infection. Past parvovirus B19 infection was identified in 7 (29%) of 24 IgG aPL-positive children, as indicated by VP1/VP2-specific IgG in the absence of viral DNA. Three (12%) of 24 IgG aPL-positive children had not been infected with B19. Sixty-nine (51%) of 135 control children displayed VP1/VP2-specific IgG. Three (2%) of these 135 children were IgG aPL positive (2 children had past parvovirus B19 infection, and 1 was negative for parvovirus B19). Analysis of the parvovirus B19 status of 40 adult IgG aPL-positive patients showed that 33 (83%) were anti-IgG VP1/VP2-positive, and viral DNA was detected in 11 patients (28%). Ten of these 11 viremic patients were in the subgroup of 28 IgG aPL-positive SLE patients. CONCLUSION: Antiphospholipid antibodies are preferentially found in serum of children with juvenile idiopathic arthritis who have been previously infected with parvovirus B19 and have established, persistent infection. Adult patients with IgG aPL positivity have a high incidence of persistent parvovirus B19 infection. We conclude that parvovirus B19 might be directly involved in the elicitation of autoimmune reactions partly mediated by aPL.     

Near-infrared spectroscopic imaging of the whole hand: A new tool to assess tissue perfusion and peripheral microcirculation in scleroderma

Objectives

Systemic sclerosis (SSc) causes functional and structural microcirculatory dysfunction, affecting also distal extremities. Optical Near-InfraRed Spectroscopy (NIRS) of blood HbO₂ saturation (stO₂) is able to evaluate O₂ delivery/consumption balance in the explored tissue. The NIRS-sensitive camera non-invasively detects stO₂ values in superficial tissues, automatically generating 2D-imaging maps in real time. We aimed at testing whether NIRS hand imaging may evaluate peripheral microcirculatory dysfunction and its spatial heterogeneity in SSc patients compared to controls.

Activation of the small GTPases, rac and cdc42, after ligation of the platelet PAR-1 receptor

Stimulation of platelet PAR-1 receptors results in the rapid (10 to 30 seconds) and extensive (30% to 40% of total) guanosine triphosphate (GTP) charging of endogenous platelet rac, previously identified as a possible key intermediate in the signal pathway between PAR-1 and actin filament barbed-end uncapping, leading to actin assembly. During PAR-1-mediated platelet activation, rac distributes from the cell interior to the cell periphery, and this reorganization is resistant to the inhibition of PI-3-kinase activity. Rac, in resting or activated platelets, is Triton X-100 soluble, suggesting that it does not form tight complexes with actin cytoskeletal proteins, though its retention in octyl-glucoside-treated platelets and ultrastructural observations of activated platelets implies that rac binds to plasma membranes, where it can interact with phosphoinositide kinases implicated in actin assembly reactions. PAR-1 stimulation also rapidly and extensively activates cdc42, though, in contrast to rac, some cdc42 associates with the actin cytoskeleton in resting platelets, and the bound fraction increases during stimulation. The differences in subcellular distribution and previous evidence showing quantitatively divergent effects of rac and cdc42 on actin nucleation in permeabilized platelets indicate different signaling roles for these GTPases.     

Cortical Thickness Changes Associated with Photoparoxysmal Response

Photoparoxysmal response (PPR) is an EEG trait of spike and spike-wave discharges in response to photic stimulation that is closely linked to idiopathic generalized epilepsy (IGE). In our previous studies we showed that PPR is associated with functional alterations in the occipital and frontal cortices. The aim of the present study was to determine structural changes associated with PPR. For this purpose we analysed the cortical thickness as derived from T1 MRI images in PPR-positive-subjects (n = 12; 15.5 ± 8.6 years; 4 males), PPR-positive-IGE-patients (n = 12; 14.9 ± 2.7 years; 4 males) and compared these groups with a group of PPR-negative-healthy-controls (HC, n = 17; 15.3 ± 3.6 years; 6 males). Our results revealed an increase of cortical thickness in the occipital, frontal and parietal cortices bilaterally in PPR-positive-subjects in comparison to HC. Moreover PPR-positive-subjects presented a significant decrease of cortical thickness in the temporal cortex in the same group contrast. IGE patients exhibited lower cortical thickness in the temporal lobe bilaterally and in the right paracentral region in comparison to PPR-positive-subjects. Our study demonstrates structural changes in the occipital lobe, frontoparietal regions and temporal lobe, which also show functional changes associated with PPR. Patients with epilepsy present changes in the temporal lobe and supplementary motor area.