Investigating the candidacy of LPS-based glycoconjugates to prevent invasive meningococcal disease: conjugates based on core oligosaccharides.

In this study we have prepared glycoconjugates with core oligosaccharides (OS) from the lipopolysaccharide (LPS) of Neisseria meningitidis, thus avoiding the neo-epitopes of the deacylated lipid A region of the derived LPS molecule identified in our previous studies. A comprehensive investigation was performed with glycoconjugates prepared from the most extended to the most truncated core OS still maintaining the conserved inner core epitope. As previously, we have established reproducible bactericidal killing of the homologous antigen elaborating strain, but a failure to kill wild-type strains. In these studies it was evident that the linker molecules used in the conjugation methodologies were dominating the immune response. However, when galE core OS based conjugates were prepared without utilizing linkers, via direct reductive amination, we failed to generate an immune response to even the homologous antigen. We also identified that immunisation with the galE antigen via linker methodologies provoked an immune response that was dependent upon key residues of the conserved inner core OS structure, whereas the immune responses to lgtB and lgtA antigens did not involve the inner core OS. This comprehensive study has, despite our best efforts, cast significant doubt as to the utility of the conserved inner core region of the meningococcal LPS as a potential vaccine antigen.

A combined approach to assess the potential coverage of a multicomponent protein-based vaccine.

Meningococcal disease caused by Neisseria meningitidis serogroup B is a public health concern even in developed countries. Despite glycoconjugate vaccines against the other invasive serogroups (A, C, W135, Y) are already available and successfully introduced in many countries, no vaccine is currently in use for prevention of serogroup B meningitis. A protein based, multicomponent vaccine (4CMenB) has been developed and proposed for prevention of invasive serogroup B meningococcal disease (MenB). This novel vaccine has been tested in clinical trials and shown to be well tolerated and immunogenic, inducing bactericidal antibodies in infants, adolescents and adults. The high level of genetic and antigenic variability observed in MenB clinical isolates, requires a suitable method to assess the ability of the 4CMenB vaccine to cover genetically diverse menigococcal strains and to estimate the potential public health impact. To this purpose the Meningococcal Antigen Typing System (MATS) has been developed and recently described. This method provides a quick and reproducible tool to estimate the level of expression and immunoreactivity of each of the vaccine antigens, in any meningococcal isolate, and it is related to the likelihood that the isolate will be killed by sera from immunized subjects. A multi-laboratory study involving several European countries, demonstrates that the 4CMenB has the potential to protect against a significant proportion of menB strains circulating in Europe.

Photoactivation enhances the mitochondrial toxicity of the cationic rhodacyanine MKT-077.

In this study, the mitochondrial phototoxicity of the cationic rhodacyanine MKT-077 was investigated by comparing its effects on the inhibition of mitochondrial respiration and the structural integrity of mitochondrial DNA (mtDNA) in the presence and absence of added high-intensity visible light (7.5 J/cm2). Results indicate that photoirradiation significantly enhances the mitochondrial toxicity of MKT-077 at both the biochemical and DNA levels. For example, the concentration of MKT-077 required to achieve one-half maximal inhibition of ADP-stimulated respiration was observed to be 6-fold lower in the presence versus absence of high-intensity light (one-half maximal inhibition at 2.5 versus 15 microg MKT-077/ mg, respectively). In addition, photoirradiation produced a 25-fold increase in inhibition of succinate-cytochrome c reductase activity by MKT-077 (one-half maximal inhibition at 2 versus 50 microg MKT-077/ml, +/-light, respectively) and a 6-fold increase in inhibition of cytochrome oxidase activity (one-half maximal inhibition at 5 versus 30 microg MKT-077/ml, +/-light, respectively). Furthermore, the combination of 25 microg/ml MKT-077 and 7.5 J/cm2 visible light caused significant degradation of mtDNA in isolated rat liver mitochondria, whereas the same concentration of dye in the absence of light had only a modest effect on mtDNA. Evaluation of light-induced MKT-077 lipid peroxidation in mitochondrial membrane fragments by the thiobarbituric acid test and by measurement of nonrespiratory-linked oxygen uptake suggests that mitochondrial phototoxicity by MKT-077 may be the result of lipid peroxidation via reactive oxygen species. These results have important implications with regard to the potential use of MKT-077 in photochemotherapy.

Selective damage to carcinoma mitochondria by the rhodacyanine MKT-077.

We investigated the mitochondrial toxicity of the lipophilic cation, MKT-077, and the role of mitochondria in selective malignant cell killing by this compound by examining the effect of MKT-077 on mitochondrial structure and function in treated cells and in isolated organelles. Results of this study demonstrate changes in mitochondrial ultrastructure that are induced by MKT-077 treatment in carcinoma cells but not in similarly treated normal epithelial cells. In addition, MKT-077 was found to inhibit respiratory activity in isolated intact mitochondria and electron transport activity in freeze-thawed mitochondrial membrane fragments in a dose-dependent manner. The concentration of MKT-077 necessary to obtain half-maximal inhibition of ADP-stimulated respiration was approximately 4-fold greater in mitochondria isolated from cells of the normal epithelial cell line, CV-1 (15 micrograms MKT-077/mg protein), as compared to the human colon carcinoma cell line, CX-1 (4 micrograms MKT-077/mg protein). Further, the data show a selective loss of mitochondrial DNA in CX-1 and CRL1420 cells (carcinoma) but not CV-1 cells (normal epithelial) treated with 3 microgram/ml MKT-077 for up to 3 days. Under the same conditions, nuclear DNA was unaffected in all three cell lines. The sensitivity of the cell lines tested to mitochondrial damage by MKT-077 correlates well with their sensitivity to cytotoxicity by MKT-077. These results demonstrate selective mitochondrial damage by MKT-077 at the cellular, biochemical, and molecular levels and suggest that selective effects on mitochondrial structure and function may provide a basis for the selective malignant cell killing exhibited by this compound.

[Results and considerations regarding auditory neonatal screening based on the use of transient evoked otoacoustic emission]. / Risultati e considerazioni a proposito di uno screening audiologico neonatale basato sull'impiego delle otoemissioni acustiche evocate transienti.

The authors present a 5-year clinical experience in audiological screening performed at the neonatal center of the Policlinico of Perugia, Italy. The study was performed using an IL088 Otodynamics unit produced by Bray & Kemp. A total of 1328 newborns (2656 ears) were tested on the 4th day of life and during spontaneous sleep. None of the children had any audiological risk factors. The test was repeated one month later for all subjects who lacked Transient Evoked Otoacoustic Emissions and in many cases ABR testing was performed by 3 months of age. The authors present the undoubted advantages of the present method which include the fact that it is a) easy to perform, b) non invasive, c) sensitive and d) effective. They then discuss the main problems which arose during the course of the screening and advance some solutions. For the most part these problems involved the high number of false positives (13.1%) and the high percentage of subjects who were lost to subsequent controls (approximately 6% of the total population). The number of false positives could be reduced by using a linear acoustic stimulation (rather than the non-linear stimulation which is the default parameter for the machine). Such a linear stimulation can improve the signal-to-noise ratio, thus making it possible to adopt a reproducibility index lower than the 70% presently used (however, this brings with it the risk of including a certain number of false negatives). Finally, they discuss the possibility of only retesting those subjects with bilateral lack of TEOAEs, thus reducing the number of check-ups to be performed a month later.

[Products of acoustic distortion in the examination of deafness in an infant]. / Les produits de distorsion acoustique dans le dépistage de la surdité du jeune enfant.

Distortion-product otoacoustic emissions (DPOAEs) are otoacoustic emissions evoked by two pure tones. The greatest advantage of DPOAEs is their frequency specificity with respect to the eliciting bitonal stimuli. The purpose of this study was to compare DPOAEs in two populations. This paper reports input/output DPOAEs functions and DPOAE-audiograms for audiometric frequencies of f2 between 696 and 6006 Hz in a normal neonate population and in an adult control group. Fifteen healthy fullterm newborns (29 ears) and 8 normal-hearing adults (16 ears) participated as subjects. Results at medium frequencies indicate that the maximum amplitudes of the DPOAEs were generated by neonates, the detection threshold was better and the dynamic range was greater than in adults, making them potentially valid for studying cochlear functioning in infants.

[Non-ketotic hypoglycemia caused by carnitine palmitoyl transferase 1 deficiency]. / Ipoglicemia non chetotica da deficit di carnitina palmitoil trasferasi 1.

A 9-year-old girl was referred to our hospital after recurrent episodes of hypoglycemia, altered consciousness and persistent vomiting without acetonemia or myopathic symptoms. Other pertinent laboratory data included elevated BUN, hyperammonemia and very low levels of triglycerides with elevated free fatty acids. The patient was born from unaffected but related parents (second cousins) and the illness was previously diagnosed as Reye encephalopathy. Recurrence of similar attacks suggested an underlying metabolic disorder. Several syndromes of impaired FFA beta oxidation were taken into account and discarded successively after laboratory investigations: systemic carnitine deficiency, Medium and Long Chain Acyl-CoA Dehydrogenase deficiency and Multiple Acyl CoA Dehydrogenation deficiency (Glutaric aciduria, Ethylmalonic-adipic aciduria and riboflavin-responsive multiple acyl CoA dehydrogenation deficiency). Urinary and hematic gas-chromatography and Mass-Spectrometry show no abnormality in Medium Chain fatty acids and in C6-C10 dicarboxylic acids. Carnitine plasma concentrations (both total and free) were above normal levels while in urine acetyl carnitine was low in respect to longer acyclic radicals. Among metabolic defects located at the level of hepatic fatty acid oxidation, only Carnitine Transferase deficiency can explain this peculiar mosaic of data (precursors of the blocked reaction are elevated in blood whereas lack of the metabolites derived uniquely from this reaction explains all the clinical manifestations).

[Use of a personal computer in contrast evaluation in magnetic resonance tomography]. / Impiego del personal computer nella valutazione del contrasto in tomografia a risonanza magnetica (RMT).

Contrast dependency in MR Tomography from several intrinsic and extrinsic parameters makes this technique versatile and sensitive; at the same time it makes more complex the image contrast evaluation and the optimized sequence choice. The approach to this problem from the viewpoint both of training and practical application becomes easier using the proposed personal computer program.

[Preliminary evaluation of the clinical use of a system for intraoperative radiotherapy with electrons (IORT)]. / Valutazioni preliminari all'impiego clinico di un sistema dedicato per radioterapia intraoperatoria con elettroni (IORT).

The preliminary evaluation of the dosimetric characteristics of a dedicated intraoperative electron beam therapy system is described (Bionix Corporation). The linear accelerator is the Varian 2100 C with electron beam energies of 6, 9, 12, 16 e 20 MeV. The isodose distribution for each of 15 available applicators (5 plane and 10 beveled) has been studied with a water phantom. The problems of the homogeneity of dose distribution, scattered and leakage radiation outside the applicators and X-ray contamination of the electron beams have been addressed, at the nominal 20 MeV energy for exemplification. The accurate knowledge of such physical and dosimetric properties are necessary for the implementation of the IORT techniques in terms of choice of the applicator size and electron beam energy for tumor coverage and dose sparing of critical organs.

[Physico-technical aspects of total skin irradiation with high-intensity-dose electrons]. / Aspetti fisico-tecnici nell'irradiazione cutanea totale con elettroni in alta intensità di dose.

The authors have addressed the physical and technical problems involved in the total skin irradiation with a linear accelerator Varian Clinac 2100 C expressly preset for this modality with electrons of 6 MeV nominal energy and dose rate of about 2500 UM/min at the isocenter. The six dual field technique has been implemented and the effect of interposed degrading filters has been evaluated with film dosimetry on an anthropomorphic phantom. It was verified that the total skin electron irradiation expressly offered by the manufacturer as an established treatment option on the Clinac 2100 C with high performance ionizing chambers and high dose rate makes the all procedure safer and simpler.

[The Monte Carlo method and parallel estimation in the drawing up of radiosurgery treatment plans]. / Metodo Monte Carlo e calcolo parallelo nella stesura dei piani di trattamento radiochirurgici.

PURPOSE: We investigated the practical application of a calculation algorithm based on the Monte Carlo method to stereotactic radiosurgery treatment planning. In radiosurgery, high dose gradients and the lack of electronic disequilibrium make high resolution matrices and high computing power and speed necessary to obtain accurate dose distribution. To date, the main obstacle to the wider-spread use of the Monte Carlo method has been the huge computing time necessary to obtain a dose distribution on current hardware. MATERIAL AND METHODS: In this project, developed within the ESPRIT program, funded by the European Union, a Parsytec CC (Cognitive Computing) computer was used with 9 processors (Power PC 604, 133 Mhz, RAM 64 Mb) with IBM AIX/EPX OS and availability for Fortran parallel codes compilation, connected to a PC for data input, results rendering, and dose distribution calculation with a conventional algorithm for comparison with the Monte Carlo code (an EGS4 user code). The module named Rapt Region Extractor performs data compression with an octree method without decreasing resolution, for RAM and computing time requirements to remain acceptable. A model of the 6 MV photon beam from Clinac 2100C Varian linear accelerator was devised, based on incident photon energy spectrum and, for each collimator dimension, on bidimensional dose distribution orthogonal to beam direction measured at SSd = SAD = 100 cm. RESULTS: Parallelization was carried out on event numbers, allowing a simulation speed to number of processor ratio close to unity. A new random number generator was used, capable of correctly running on the parallel architecture. The simulation procedure includes: 1) CT acquisition in DICOM 3.0 format, Analyze or with scanner; 2) Target delineation, treatment arc definition. 3) Dose calculation, with both conventional and Monte Carlo methods. 4) Dose distribution rendering on every transverse, sagittal or coronal planes overlapped in color wash on anatomical representation. Comparison between conventional and Monte Carlo algorithms were carried out on an anthropomorphic phantom and 10 real patients, with 2.5 mm anatomical resolution and standard deviation never exceeding 2%. A simulation with 10,000,000 events and 1% maximum variance can be run in 43'. When PTV is an homogeneous areas the differences between the two methods are around 5%, while when PTV is localized in dishomogeneous areas discrepancies reach 20% in the bone. CONCLUSIONS: In conclusion, the feasibility of direct simulation with the Monte Carlo method in radiosurgery has been demonstrated within time and hardware costs compatible with clinical practice.

[Verification of 3 computerized systems for drafting a plan for treatment with electrons]. / Verifica di tre sistemi computerizzati per la stesura dei piani di trattamento con elettroni.

The electron beam treatment planning systems, with the availability of more and more developed hardware for complex calculation algorithms, need quality controls of accuracy and comparability of treatment plans from different systems. In this paper three systems have been evaluated; their similar computation algorithms (pencil beam) have been used but in some conditions (beam reconstruction, oblique incidence and dishomogeneity) different results have been drawn. The basic data (PDD, dose profile, etc.) were the same for every system, therefore it has been observed that different results are mainly due to differences in: a) input of parameter values, b) calculation algorithm approximations, c) image acquisitions and elaboration.

A novel mimetic antigen eliciting protective antibody to Neisseria meningitidis.

Molecular mimetic Ags are of considerable interest as vaccine candidates. Yet there are few examples of mimetic Ags that elicit protective Ab against a pathogen, and the functional activity of anti-mimetic Abs has not been studied in detail. As part of the Neisseria meningitidis serogroup B genome sequencing project, a large number of novel proteins were identified. Herein, we provide evidence that genome-derived Ag 33 (GNA33), a lipoprotein with homology to Escherichia coli murein transglycosylase, elicits protective Ab to meningococci as a result of mimicking an epitope on loop 4 of porin A (PorA) in strains with serosubtype P1.2. Epitope mapping of a bactericidal anti-GNA33 mAb using overlapping peptides shows that the mAb recognizes peptides from GNA33 and PorA that share a QTP sequence that is necessary but not sufficient for binding. By flow cytometry, mouse antisera prepared against rGNA33 and the anti-GNA33 mAb bind as well as an anti-PorA P1.2 mAb to the surface of eight of nine N. meningitidis serogroup B strains tested with the P1.2 serosubtype. Anti-GNA33 Abs also are bactericidal for most P1.2 strains and, for susceptible strains, the activity of an anti-GNA33 mAb is similar to that of an anticapsular mAb but less active than an anti-P1.2 mAb. Anti-GNA Abs also confer passive protection against bacteremia in infant rats challenged with P1.2 strains. Thus, GNA33 represents one of the most effective immunogenic mimetics yet described. These results demonstrate that molecular mimetics have potential as meningococcal vaccine candidates.