Strategic discussion on funding and access to therapies targeting rare diseases in Spain: an expert consensus paper.

BACKGROUND: In recent years, significant advances have been made in the field of rare diseases (RDs). However, there is a large number of RDs without specific treatment and half of these treatments have public funding in Spain. The aim of the FINEERR project was to carry out a multidisciplinary strategic discussion on the challenge of funding and access to RD-targeted drugs in Spain, in order to agree on specific proposals for medium-term improvement and hence support decision-making in the Spanish National Healthcare System (SNHS). RESULTS: The FINEERR Project was organized around a CORE Advisory Committee, which provided an overview, agreed on the design and scope of the project, and selected the members within each of four working groups (WG). Overall, 40 experts discussed and reached a consensus on different relevant aspects, such as conditioning factors for initial funding and access, evaluation and access to RD-targeted therapies, funding of these therapies, and implementation of a new funding and access model. From these meetings, 50 proposals were defined and classified by their level of relevance according to the experts. A descriptive analysis of responses was performed for each proposal. Thereafter, experts completed another questionnaire where they ranked the 25 most relevant proposals according to their level of feasibility of being implemented in the SNHS. The most relevant and feasible proposals were to improve: process of referral of patients with RDs, control over monitoring mechanisms, and communication between healthcare professionals and patients. CONCLUSIONS: The FINEERR project may provide a starting point for stakeholders involved in the process of funding and access to RD-targeted therapies in Spain to provide the necessary resources and implement measures to improve both the quality of life and life expectancy of patients with RDs.

Production, purification and characterization of serraticin A, a novel cold-active antimicrobial produced by Serratia proteamaculans 136.

AIM: This study focuses on the production, purification and characterization of serraticin A, a novel cold-active antimicrobial produced by Serratia proteamaculans 136. METHODS AND RESULTS: A Ser. proteamaculans strain producing a novel cold-active antimicrobial was isolated from Isla de los Estados, Argentina. Antimicrobial production was optimized in a BIOFLO 101 bioreactor under batch culture mode, with temperature, pH and dissolved oxygen controlled conditions. A purification protocol was developed including activated charcoal adsorption, solid-phase C18 extraction (SPE) and semi-preparative HPLC. The molecular weight was determined by LC/QTOF/MS/MS mass analysis. CONCLUSIONS: Serratia proteamaculans 136 produces a cold-active low molecular bacteriocin-like compound named serraticin A. In this work, it has been laboratory-scale produced, purified and partially characterized. Cross-immunity test revealed that serraticin A is very different from other well-known microcins assayed, with a wide inhibitory spectrum, showing an interesting biotechnology potential to be applied as a control agent against pathogenic bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study is the first report of a cold-active compound with antimicrobial activity from Ser. proteamaculans. The work also highlights that cold environments could be a suitable source of micro-organisms with ability to produce cold-active biomolecules of biotechnological interest.

Dynamic testing in columns for soil heavy metal removal for a car park SUDS.

The increase in urban runoff brought about by a rise in impermeable surfaces has triggered the alteration and pollution of many aquatic systems. The overall goal of this research was to design a 'Sustainable Urban Drainage System' (SUDS) for the retention of heavy metals from a car park consisting of mixing autochthonous soil (70%) with sand (30%) to improve the hydrological conductivity and adsorption capacity. To quantify the retention of metals we characterize the adsorption kinetics and isotherms of the soil mixture and perform dynamic experiments. The proposed methodology allowed us to work out the amount of heavy metal retention by the adsorbent and the retention mechanisms. The retention capacity of the adsorbent mixture was as follows: Cr3+ ≈ Cu2+ ≫ Zn2+ > Ni2+ > Cd2+. Chromium and copper ions were mainly retained by precipitation, whereas zinc, nickel and cadmium were retained by ionic exchange with calcium ions that saturate the soil colloids. The soil mixture buffered pH was found to change when fed with an acid solution of metallic ions.

The effect of moisture regimes on the anaerobic degradation of municipal solid waste from Metepec (México).

The State of México, situated in central México, has a population of about 14 million, distributed in approximately 125 counties. Solid waste management represents a serious and ongoing pressure to local authorities. The final disposal site ("El Socavón") does not comply with minimum environmental requirements as no liners or leachate management infrastructure are available. Consequently, leachate composition or the effects of rain water input on municipal solid waste degradation are largely unknown. The aim of this work was to monitor the anaerobic degradation of municipal solid waste (MSW), simulating the water addition due to rainfall, under two different moisture content regimes (70% and 80% humidity). The study was carried out using bioreactors in both laboratory and pilot scales. The variation of organic matter and pH was followed in the solid matrix of the MSW. The leachate produced was used to estimate the field capacity of the MSW and to determine the pH, COD, BOD and heavy metals. Some leachate parameters were found to be within permitted limits, but further research is needed in order to analyze the leachate from lower layers of the disposal site ("El Socavón").

Thermal diffusivity measurement in thin metallic filaments using the mirage method with multiple probe beams and a digital camera.

Photothermal beam deflection is a well-established technique for measuring thermal diffusivity. In this technique, a pump laser beam generates temperature variations on the surface of the sample to be studied. These variations transfer heat to the surrounding medium, which may be air or any other fluid. The medium in turn experiences a change in the refractive index, which will be proportional to the temperature field on the sample surface when the distance to this surface is small. A probe laser beam will suffer a deflection due to the refractive index periodical changes, which is usually monitored by means of a quadrant photodetector or a similar device aided by lock-in amplification. A linear relationship that arises in this technique is that given by the phase lag of the thermal wave as a function of the distance to a punctual heat source when unidimensional heat diffusion can be guaranteed. This relationship is useful in the calculation of the sample's thermal diffusivity, which can be obtained straightforwardly by the so-called slope method, if the pump beam modulation frequency is well-known. The measurement procedure requires the experimenter to displace the probe beam at a given distance from the heat source, measure the phase lag at that offset, and repeat this for as many points as desired. This process can be quite lengthy in dependence of the number points. In this paper, we propose a detection scheme, which overcomes this limitation and simplifies the experimental setup using a digital camera that substitutes all detection hardware utilizing motion detection techniques and software digital signal lock-in post-processing. In this work, the method is demonstrated using thin metallic filaments as samples.

Fluvastatin as co-medication in heart transplant recipients with elevated creatine-kinase.

BACKGROUND: Statins reduce the risk for transplant vasculopathy and mortality among heart transplant recipients. Interactions between commonly used statins (ie, simvastatin, atorvastatin) and immunosuppressant drugs lead to side effects or withdrawal of statin therapy. Fluvastatin shows fewer interactions with the immunosuppressant agents because it is not metabolized via the cytochrome P-450 3A4 pathway like most immunosuppressants, steroids, or other statins. This study investigated the impact of a switch from other statins to fluvastatin in heart transplant recipients who revealed elevated creatine-kinase levels. METHODS: A total of 23 heart transplant patients with elevated creatine-kinase levels were included in this study. Statins were replaced with an equal dosage of fluvastatin. We measured on the day of replacement as well as there after at 3, 6, 9, and 12 months creatine-kinase, lipid status, ALT, AST, and creatinine levels. RESULTS: After 6 months creatine-kinase showed a significant reduction of 25% (P < .05) and after 9 months of 38% (P < .05). The HDL-cholesterol levels were significantly reduced at 6 months (8%; P < .05) and 9 months (23%; P < .05). At 3 months, triglyceride levels were significantly elevated (18%; P < .05). No differences were observed in ALT, AST, creatinine, total, and LDL-cholesterol at any time. CONCLUSION: A conversion from commonly used statins to fluvastatin in heart transplant patients with elevated creatine-kinase was safe, leading to a significant reduction in creatine-kinase levels. Except for an initial raise in triglycerides and a lowering of HDL-cholesterol, no changes in lipid status were seen. This conversion might help to maintain lipid-lowering therapy in transplant recipients who show side effects using conventional statins.

Targeting of HIV gp120 by oligonucleotide-photosensitizer conjugates. Light-induced damages.

Some guanine-rich oligonucleotides inhibit HIV infectivity through interaction with the gp120 glycoprotein. Besides, photoinactivation of viruses attracts attention for blood decontamination. The feasibility of targeting a red light-absorbing chlorin-type photosensitizer to gp120 through covalent coupling with 8-mer phosphodiester oligodeoxynucleotides is investigated. Some conjugates inhibit binding of antibodies directed to gp120. Inhibition is significantly increased upon red light activation. The activity of the conjugates correlates with their ability to self-associate, a process strongly favored by the propensity of the hydrophobic chlorin moiety to dimerize. Thus, the photosensitizer moiety both promotes structures with a higher affinity for gp120 and, upon light activation, can induce site-directed damages to the protein.

Late seroconversion of C virus markers in hemodialysis patients.

We examined the prevalence of the IgG C virus C 100-3 antibody (anti-HCV) in a group of 43 patients on hemodialysis in our center during three periods: A: 1988; B: 1989; C: 1990. During period A, the anti-HCV prevalence was 30% (13 of 43 patients), these patients being regarded as chronic carriers of these antibodies. In period B, two patients displayed seroconversion, and another seven during period C, all of whom had tested negative during period A. These patients were considered acute. During the three years under study, all of the patients shared the same hemodialysis monitors. High ALT values were found in four of nine acute patients (44.4%) and nine of thirteen (69.23%) of the chronic patients. In 10 anti-HCV patients, hypertransaminemia continued long-term (> 6 months). Two patients had been given contaminated blood, four were multi-transfused (> 14 transfusions), two less than 4 units, and one had never received a transfusion. The period between the initial high and/or maximum ALT and the determination of HCV Ac was up to 10 to 11 months in three patients. These findings indicate the lack of sensitivity of ALTs as a diagnostic tool for HCV, the possible late C 100-3 seroconversion, which makes it necessary to carry out periodic serological checks in hemodialysis patients and the decisive role transfusions in HCV transmission, without excluding other possible intra-dialysis contagion sources.

Follow-up of a human accidental infection by Leishmania (Viannia) braziliensis using conventional immunologic techniques and polymerase chain reaction.

The results of a two-year follow-up study of an accidental human infection with Leishmania (Viannia) braziliensis are presented. Western blots, indirect immunofluorescence, and intradermal reaction assays were not able to detect the infection before week 18 postinfection, whereas using the polymerase chain reaction (PCR) on blood samples, parasite DNA was detected at week 8. All test results, except the intradermal reaction followed the cure process and 12 weeks after the last chemotherapy injection (week 36 postinfection), all were negative. However, a year after cure, the PCR became positive again and has remained so for two years. To determine in which blood cell fraction the parasite was evident, enriched lymphocyte and monocyte fractions were assayed by the PCR. These results are discussed in the context of persistent infections.

Leishmania colombiensis in Venezuela.

Flagellate parasites isolated in Venezuela from bone marrow aspirates of a human (MHOM/VE/70/Chuao) and a dog (MCAN/VE/72/Talisman2) were subsequently identified by isozyme analysis as Leishmania colombiensis. Data are presented describing genetic similarity among Panama, Colombia, and Venezuela populations of this species.

Construction of an integrative shuttle vector for Zymomonas mobilis.

An integrative shuttle vector, pZMOCP1, was constructed by ligating EcoRV digests of the plasmid cloning vector pBluescript and pZMP1, a cryptic plasmid of Zymomonas mobilis PROIMI A1. The 7.2-kb plasmid pZMOCP1 replicated in Escherichia coli and could also be transferred from this host by electroporation to Z. mobilis ATCC 29191. The transformants were selected by ampicillin resistance. The integrative characteristic was detected by hybridization in situ. The vector was stably maintained in Z. mobilis after 200 generations without selective pressure.

Chlorin-type photosensitizers photochemically derived from vinyl porphyrins.

The reaction of singlet oxygen with four vinyl-substituted dicarboxylic porphyrins, vinyldeuteroporphyrin (VD), ethylvinyldeuteroporphyrin (EVD), hydroxyethylvinyldeuteroporphyrin (HVD) and protoporphyrin (PP) in organic solutions is investigated. The main products, the "hydroxyaldehyde" chlorin-type derivatives, are formed with a concentration-dependent photochemical quantum yield that reaches a maximum of 7.4 +/- 1.6 x 10(-3). However, owing to the high turnover of singlet-oxygen production, these chlorin-type compounds are easily prepared photochemically with a chemical yield of 70% and little side product formation. In chemical ionization mass spectrometry, these compounds display an unusual fragmentation with a loss of 16 mass units. This is attributed to the loss of the oxygen bound to the saturated carbon of the modified pyrrole unit. All these compounds sensitize the formation of singlet oxygen with a yield around 0.8. They interact with singlet oxygen with rate constants of 5 x 10(6)-9 x 10(6) M-1 s-1, lower than those measured for vinyl porphyrins. These data are likely to help in the characterization of photoproducts of vinyl porphyrins relevant to photodynamic therapy (PP, HVD). As exemplified with VD and EVD, they also point out the reaction of singlet oxygen as an efficient route to chlorin-type photosensitizers.

In vitro uptake of dicarboxylic porphyrins by human atheroma. Kinetic and analytical studies.

Human atheromatous aorta segments as well as presumably disease-free control aorta were obtained at autopsy. They were incubated with solutions of various purified dicarboxylic porphyrins including hematoporphyrin (HP) and hydroxyethylvinyldeuteroporphyrin (HVD), and with solutions of Photofrin. Selective labelling of the atheroma was shown by macroscopic and microscopic observations of the characteristic porphyrin fluorescence associated with the atheromatous plaques. The time dependence of the uptake, monitored by absorption spectrophotometry or by high performance liquid chromatography, was inferred from the disappearance of the porphyrins in the incubation medium. Significant binding was observed in the absence of albumin or serum proteins. The uptake of HP was less than that of the more hydrophobic compounds HVD or Photofrin when these porphyrins were used alone. The presence of albumin or serum drastically reduces atheroma labelling. Some competition between HP and HVD for binding sites is also seen. The present results do indicate that hydrophobic porphyrins have an intrinsic affinity for atheroma and that they can be taken up through passive processes. Taking into account previous data on animal models (Photochem. Photobiol. (1989), 731-737), it appears however that, in vivo, interactions with proteins and pharmacokinetics will primarily determine plaque labelling.

Antibody response and antigen-specific gamma-interferon profiles of vaccinated and unvaccinated pregnant sheep experimentally infected with Brucella melitensis.

It is well known that the immune response in sheep against Brucella melitensis is subject to individual variation, depending on diverse factors. It bears asking whether these factors (e.g. clinical disease, active infection, state of previous immunity), when affecting a group, can cause variation in the performance of different diagnostic tests. To clarify some of the circumstances in which this immune response can vary, we examine the immune-response profile of sheep protected against the clinical disease by prior vaccination with strain Rev. 1 in comparison with the profile of unprotected females showing the classical brucellosis symptoms. An experimental infection was provoked at midpregnancy under controlled conditions of both non-vaccinated (n=7) and previously Rev.1-vaccinated ewes (n=5). Their immune response was monitored from 7 to 9 weeks before abortion or normal birth to 30 weeks afterwards. Antibody response was assessed by classical tests (Rose Bengal test, complement fixation test (CFT)) in comparison with other diagnostic tests (indirect ELISA (iELISA), competitive ELISA (cELISA), fluorescence polarization assay (FPA), immunocapture test (ICT)). In addition, the cell-mediated immune response was indirectly evaluated by the in vitro antigen-specific release of gamma-interferon. The antibody levels and antigen-specific gamma-IFN profile of the non-vaccinated ewes having the disease and excreting the pathogen was notably high and differed significantly (P<0.05 or P<0.01) from those of vaccinated ewes that neither contracted brucellosis nor excreted the pathogen. In general, all the tests detect the infection in the non-vaccinated ewes with substantial effectiveness. It can be concluded that the high levels of circulating antibodies and of antigen-specific gamma-IFN are related to active Brucella infection. Similarly, the state of protection against the disease, but not necessarily against infection, due to a previous immunization with the Rev. 1 vaccination, appears to be responsible for a low level of detectable immune response. Nevertheless, the design of the study limits conclusions to pregnant ewes and cannot be extrapolated to non-pregnant ewes or rams. Likewise, the study provides no information on animals which are carriers of B. melitensis.

Photodynamic inactivation of cell-free HIV strains by a red-absorbing chlorin-type photosensitizer.

We have investigated the photodynamic activity of a new chlorin-type photosensitizer on a reference human immunodeficiency virus type 1 (HIV-1) strain, two wild-type HIV-1 isolates and two drug-resistant HIV-1 isolates. This chlorin was highly effective for the inactivation of free viruses, as assessed by two different quantitative cell culture assays. In the absence of blood components, all the HIV strains, including wild-type and drug-resistant mutant isolates, were totally inactivated using 30 micrograms ml-1 of chlorin and 0.75 J cm-2 of 661 nm light. Successful killing of HIV-1 strains in either plasma or whole blood was also obtained by increasing the chlorin concentration moderately. Our results demonstrate the antiviral efficiency of this chlorin, suggesting the potential application of dye-sensitized photoirradiation to decontaminate blood products.

Simultaneous serological evidence of Actinobacillus pleuropneumoniae, PRRS, Aujeszky's disease and influenza viruses in Spanish finishing pigs.

A total of 198 pigs with tachypnoea and temperature >/= 40 degrees C were selected on a Spanish finishing unit, and their sera were examined for antibodies to Actinobacillus pleuropneumoniae (App), porcine reproductive and respiratory syndrome virus (PRRSV), Aujeszky' disease virus (ADV), and swine influenza virus (SIV). Eighty-nine point nine per cent of the pigs were seropositive to App, 88.6 per cent to PRRS, 73.0 per cent to ADV, and 30.6 per cent to SIV. Thirty-one pigs (15.6 per cent) were seropositive for App, PRRSV, ADV and SIV, and only one (0.5 per cent) was seronegative for all. Statistical association was assessed for dual infections but it was not found in any case (P > 0.05). Other parameters (dyspnoea, nasal discharge and coughing) were also recorded, and no significant associations between them and the presence of antibodies against any of the four infections was found.

Effects of trap design and CO2 presentation on the measurement of adult mosquito abundance using Centers for Disease Control-style miniature light traps.

Centers for Disease Control miniature light traps augmented with CO2 provide an effective method of monitoring Culex abundance and may provide a useful supplement to New Jersey light traps used by the California Mosquito Surveillance Program. To assist in standardizing sampling protocols, the present research compared the catch of adult mosquitoes collected using 4 trap designs and 3 CO2 presentation methods. When augmented with dry ice, the Arbovirus Field Station (AFS) trap (consisting of a 3-in. fan mounted into a white polyvinyl chloride pipe and operated without a light source or rain shield) collected as many or more Culex females than similar traps purchased from John W. Hock and American Biophysics, or a trap with a 4.25-in. 2-bladed fan constructed by the Orange County Vector Control District (similar to the Encephalitis Virus Surveillance model distributed by Bioquip). Few blooded or gravid females and males were collected, indicating that CO2 released from the dry ice and not light probably was the primary attractant. Catch of Culex tarsalis females in traps baited with CO2 released at 0.5-1.5 liters/min from gas cylinders was significantly greater than in traps baited with dry ice, even though the CO2 release rates from the dry ice at dusk probably were comparable to that released from the cylinders and averaged 0.4-0).5 liters/min for the night. Traps baited with 0.5 liters/ min of CO2 gas released in 15 3- or 2-sec bursts per hour collected the fewest mosquitoes. In all experiments, trap location effects were significant and accounted for as much variability in catch size as trap design or CO2 presentation. Sampling efficiency of all trap designs or CO2 presentations were consistent over time, space, and different levels of mosquito abundance.

Comparative effectiveness of three adult mosquito sampling methods in habitats representative of four different biomes of California.

The effectiveness of New Jersey (NJ) light, dry ice baited, and gravid female traps for collecting adult mosquitoes was compared at representative habitats in the Coachella, San Joaquin, and Sacramento valleys and the Los Angeles basin of California. The NJ light traps effectively sampled Anopheles freeborni, Culex tarsalis, Psorophora columbiae, and several Aedes when abundance was high in rural areas with minimal competitive illumination. Dry ice-baited encephalitis virus surveillance or CDC style traps collected significantly more females of most species at most localities than did NJ light traps, regardless of background illumination. The Cummings modification of the Reiter gravid female trap baited with a bulrush (Schoenoplectus) infusion was the best method for collecting Culex pipiens complex females in most habitats. In the Los Angeles basin, gravid traps baited with bulrush infusion collected, on average, 4.5 times more Culex quinquefasciatus females than did traps baited with the Reiter infusion. The bulrush infusion in combination with the Cummings trap design seemed to provide resting site cues and collected males as well as empty and bloodfed females. Mosquito surveillance programs in California should include the systematic operation of dry ice-baited and gravid female traps to improve surveillance sensitivity for selected species in appropriate habitats.

Isolation and characterization of solventogenic, cellulase-free xylanolytic Clostridia from cow rumen.

Hemicellulolytic obligate anaerobes capable of converting a range of agricultural substrates and monomeric sugars of hemicellulose to solvents and acids were isolated from cow rumen fluid. The strains were identified as 5 strains of C. beijerinckii (3 different genotypes), 3 strains of C. acetobutylicum (2 different genotypes), 1 strain of C. butyricum and 3 strains of C. bifermentans (2 different genotypes). All of them produced cellulase-free xylanase activity. C. acetobutylicum LU1 produced solvent, a high of 5.1 gl(-1) and C.bifermentans LU1 produced xylanase activity a high of 4.2 IU ml(-1).

Surgery with computerized virtual reality for the automatic detection of tumors.

We present a novel and highly accurate system based on informatics engineering capable of automatic detection of tumors directly in the operating field. The system can identify the outlines of the tumor, determine whether it is malignant or not, detect lymphadenopathy and determine whether nodes are metastasized or not. The highly elaborate system, based on artificial vision, has been used in 30 gastric and 5 pancreatic neoplasms, among other tumor types. Images of the surgical field were recorded with a video camera connected to a computer, which was operated by the engineer. Questions asked by the surgeon during the procedure were processed immediately and sent to the virtual reality helmet worn by the surgeon, to the TV monitor in the operating room, or to both. The system is based on purely physical and mathematical processes that work reliably; in this sense it is free from errors and is self-consistent, operator errors or hardware failure excepted. In all cases tested here the system correctly identified the tumor as benign or malignant, revealed the extension of the tumor, and detected lymph node metastases. In every case these results were confirmed by histological examination.