Freeze-drying process monitoring using a cold plasma ionization device.

A cold plasma ionization device has been designed to monitor freeze-drying processes in situ by monitoring lyophilization chamber moisture content. This plasma device, which consists of a probe that can be mounted directly on the lyophilization chamber, depends upon the ionization of nitrogen and water molecules using a radiofrequency generator and spectrometric signal collection. The study performed on this probe shows that it is steam sterilizable, simple to integrate, reproducible, and sensitive. The limitations include suitable positioning in the lyophilization chamber, calibration, and signal integration. Sensitivity was evaluated in relation to the quantity of vials and the probe positioning, and correlation with existing methods, such as microbalance, was established. These tests verified signal reproducibility through three freeze-drying cycles. Scaling-up studies demonstrated a similar product signature for the same product using pilot-scale and larger-scale equipment. On an industrial scale, the method efficiently monitored the freeze-drying cycle, but in a larger industrial freeze-dryer the signal was slightly modified. This was mainly due to the positioning of the plasma device, in relation to the vapor flow pathway, which is not necessarily homogeneous within the freeze-drying chamber. The plasma tool is a relevant method for monitoring freeze-drying processes and may in the future allow the verification of current thermodynamic freeze-drying models. This plasma technique may ultimately represent a process analytical technology (PAT) approach for the freeze-drying process.

Early hepatic cytokine mRNA expression in experimental rat fasciolosis.

We studied the development of the cellular response, particularly with respect to Th1 and Th2 cytokine mRNA levels, in rat liver during the first 14 days of experimental infection with Fasciola hepatica. We analysed the panel of cytokines involved in initiation of the inflammatory and immune response. The levels of various mRNAs, particularly those primarily associated with the acute inflammatory response, and those commonly associated with T-cell proliferation and differentiation, were assessed by reverse transcription-polymerase chain reaction (RT-PCR) in liver samples. We also investigated the immune and inflammatory mediators balance in the liver, draining lymph node and spleen, by RT-competitive PCR quantification of mRNA levels for IL-4, IL-10 and IFN-gamma. Our data provide the first evidence that, in the early phase of infection, the inflammatory response in the liver of infected animals is transiently depressed or delayed. A Th0 profile was initially observed in the liver and hepatic lymph node, which developed into a Th2 profile 2 weeks after infection in the liver only. In the spleen, cytokine down-regulation was initiated and maintained during this period, suggesting that the parasite acts differently locally and in the periphery.

Comparison of hepatic and renal drug-metabolising enzyme activities in sheep given single or two-fold challenge infections with Fasciola hepatica.

The activity of drug-metabolising enzymes was compared in liver and kidneys of adult sheep given single or two-fold fluke infection. Fascioliasis was induced by oral administration of 200 metacercariae of Fasciola hepatica to female sheep either 10 or 20 weeks (mono-infections) or 10 and 20 weeks (bi-infection) before killing. The parasitic pathology was ascertained at autopsy and by clinical observation of animals. In the liver of both mono- and bi-infected animals, significant decreases (P<0.05) (17-44%) were observed in the microsomal content of cytochrome P450 and in the two measured P450-dependent monooxygenase activities, benzphetamine and ethylmorphine N-demethylations. Moreover, Western blot analysis of microsomes demonstrated a decrease in the expression of cytochrome P4503A subfamily correlative with that of its presumed corresponding activity ethylmorphine N-demethylase. By contrast, the conjugation of chloro-dinitrobenzene to glutathione remained unchanged in liver cytosolic fractions prepared from all these animals. In kidneys, a significant decrease (P<0.05) (30%) in microsomal cytochrome P450 level of 10-week mono-infected sheep was observed whereas there was no change in the other groups of animals. The inflammatory origin and the consequences in terms of pathology and animal productivity of the fascioliasis-induced decreases in tissue-oxidative drug metabolism are discussed, particularly in the case of adult sheep suffering repetitive infections.

Fasciola hepatica: influence of gender and liver biotransformations on flukicide treatment efficacy of rats infested and cured with either clorsulon/ivermectin or triclabendazole.

Two fasciolicide preparations have been compared in 130 rats experimentally infected with Fasciola hepatica. Parasitological, immunological, and biochemical parameters have been followed to monitor the efficacy of the treatments. While Fascinex (triclabendazole) efficiently cured both male and female rats when administered as soon as 4 weeks postinfection, treatment with Ivomec-D (clorsulon + ivermectin) displayed a low efficacy on either male or female rats at this time point (54 and 0%, respectively). Moreover, when administered 8 weeks postinfection, the Ivomec-D treatment proved highly efficient on male rats while it displayed little effect on the female population (100 and 53%, respectively). This unexpected result has been related to an overexpression of a P4503A isoform that is observed only in females that have been treated with Ivomec-D. The influence of this P4503A cytochrome on drug metabolism and the need for the incorporation of both genders in clinical trials are discussed.

Use of an enzyme-linked lectinsorbent assay to monitor the shift in polysaccharide composition in bacterial biofilms.

An enzyme-linked lectinsorbent assay (ELLA) was developed for quantification and characterization of extracellular polysaccharides produced by 1- and 4-day biofilms of 10 bacterial strains isolated from food industry premises. Peroxidase-labeled concanavalin A (ConA) and wheat germ agglutinin (WGA) were used, as they specifically bind to saccharide residues most frequently encountered in biofilms matrices: D-glucose or D-mannose for ConA and N-acetyl-D-glucosamine or N-acetylneuraminic acid for WGA. The ELLA applied to 1- and 4-day biofilms colonizing wells of microtiter plates was able to detect that for Stenotrophomonas maltophilia and to a lesser extent Staphylococcus sciuri, the increase in production of exopolysaccharides over time was not the same for sugars binding with ConA and those binding with WGA. Differences in extracellular polysaccharides produced were observed among strains belonging to the same species. These results demonstrate that ELLA is a useful tool not only for rapid characterization of biofilm extracellular polysaccharides but also, in studies of individual strains, for detection of changes over time in the proportion of the exopolysaccharidic component within the polymeric matrix.

Local hepatic immune response in rats during primary infection with Fasciola hepatica.

The distribution of lymphocyte subpopulations (TCD4+, TCD8+, TCD43+ and Ig+ cells), macrophages and eosinophils were analysed in the inflammatory infiltrates associated with hepatic lesions and in hepatic lymph nodes (HLN) from rats experimentally infected with F. hepatica and necropsied 1, 2, 3, 4, 6 and 8 week post infection (WPI). We also investigated the fixation of immunoglobulin isotypes on migrating flukes in the liver. As early as 1WPI, portal tract areas surrounding migratory tunnels were infiltrated with immune and inflammatory cells. The dominant cells were eosinophils and to lesser extent, macrophages and lymphocytes (TCD4+, TCD8+ and B). Most of the inflammatory and immune cells reached the posterior part of flukes, whereas in front of the parasites these cells were fewer in number. Except for eosinophils, no immune cells penetrated through granuloma consisting of hepatic necrotic cells. As early as 1WPI, IgM could be detected in the liver, and to a lesser extent IgA, IgG2a and IgG2b. At 2WPI, IgE and IgG1 began being detected. IgG2c was detectable at 3WPI. In HLN, we observed numerous microscopic follicles in the cortical zone with proliferation of germinal centres and medullary cords. The protective role of infiltrating cell populations and immunoglobulin isotypes and possible mechanisms of immune evasion by the parasite are discussed.

New Caledonia: Solidarite SIDA - telephone hot-line.

PIP: This article traces the experience of the first 2 years of managing the Solidarite SIDA toll-free telephone hotline service, including the services offered, the training needed for those answering the calls, statistics on those using the service, and the types of questions asked. SIDA was established in May 1996 as a support network. The goals of Solidarite SIDA have been encouraging contacts and the exchange of information; defending and promoting the rights and dignity of the people living with HIV and AIDS, their families, and friends; and sharing experiences with official organizations and other associations. The SOS AIDS telephone hotline was opened on December 1, 1996, to provide a sympathetic ear and to give information, advice and moral support to callers and to document the problems. Overall, it is noted that majority who utilize the services are young Melanesians who require information most often related to HIV transmission and its preventative measures. It is also indicated that the free phone is a source of advice and support with regard to worries connected to sexuality. In view of this, more advertising is necessary and documentation of calls should be continued in order to provide statistics to official institutions and other associations who work in HIV and AIDS. The expansion of the program is projected.^ieng

Mimotopes of polyreactive anti-DNA antibodies identified using phage-display peptide libraries.

Three monoclonal IgG2a anti-DNA polyreactive autoantibodies, derived from lupus-prone mice (NZB x NZW)F1, were studied by surface plasmon resonance (BIAcore) analysis using three different synthetic double-stranded (ds) oligonucleotides of 25, 30, and 25 base pairs (bp). These monoclonal antibodies (mAb) exhibited dissociation rate constants (k(off)), ranging from 0.0001 (mAb F14.6 and F4.1) to 0.01/s (mAb J20.8) and k(on) ranging from 2 x 10(5) to 2 x 10(6) /M/s. The screening of a constrained random peptide library displayed on M13 bacteriophages on these mAb allowed the determination of the specific consensus motifs (mimotopes) for mAb F14.6 and J20.8, but not for mAb F4.1. No cross-reaction was observed between F14.6- and J20.8-specific peptides (and vice versa). Binding of all phages selected on F14.6 was inhibited with 700 ng/ml soluble DNA. The binding of one group of peptides selected on J20.8 was inhibited by 400 ng/ml soluble DNA, of a second group by 2500 ng/ml, while binding of a third group could not be inhibited. The determined consensus sequences do not match with known sequences. Peptides specific for F14.6 share negative charges and aromatic rings that may mimic a DNA backbone, while peptides selected with J20.8 do not bear any negative charge, implying a different kind of molecular recognition, for example hydrogen or salt bonds. The peptides selected on J20.8 also bind serum antibodies from human patients with systemic lupus erythematosus. In addition, BALB/c mice immunized with some of the selected phages exhibit high serum titers of IgG3 anti-dsDNA antibodies, further supporting the hypothesis that peptide epitopes may mimic an oligonucleotide structure.

A FIV epitope defined by a phage peptide library screened with a monoclonal anti-FIV antibody.

Phage peptide libraries constitute powerful tools for the mapping of epitopes recognized by monoclonal antibodies. We report here the characterization of an antibody directed against a 20-residue peptide derived from the surface glycoprotein of the feline immunodeficiency virus. The isolation of the WRPDF consensus sequence from a phage display library defined the exact epitope recognized by the mAb. Compared with known immunogenic peptides of the FIV envelope, it corresponds to the most immunodominant peptide found in the whole molecule. Kinetic data describing the antibody-peptide interactions were obtained by surface plasmon resonance. The antibody binds the peptide with a KD in the nanomolar range.

Comparison of serological tests for the diagnosis of feline immunodeficiency virus infection of cats.

A new enzyme-linked-immunosorbent assay (ELISA) for the detection of antibodies to feline immunodeficiency virus was compared with previously described ELISAs. Serum samples from 184 infected or uninfected cats were tested using a whole virus lysate kit and ELISAs based on recognition of one of two synthetic peptides (P237 and P253) localized in the transmembrane domain of the viral envelope. The whole virus lysate commercial kit led to the detection of 6% false positive and 4.3% false negative sera. The ELISA based on peptide P253 gave no false positive result and failed to detect only one serum that was subsequently shown to be positive by radio-immunoprecipitation assay. A sandwich-ELISA test using Galanthus nivalis agglutinin, a lectin that specifically binds terminal mannose groups of the envelope proteins was used as a confirmatory test for equivocal results with peptide ELISA and gave similar results. This study indicates that recognition of P253 could serve as a sensitive and specific test for the diagnosis of seropositivity to feline immunodeficiency virus, and moreover that the Galanthus nivalis ELISA could be useful in equivocal cases as a confirmatory test.

[Resection of lung neoplasm in patients aged 70 years and over. Apropos of a series of 193 resections]. / L'exérèse du néoplasme bronchique chez les sujets de 70 ans et plus. A propos d'une série de 193 exérèses.

Lung cancer in the elderly is an active cancer. Due to the poor tolerance of radiotherapy and chemotherapy, and the ever increasing life expectancy of patients over the age of 70 years, surgery remains the treatment of choice for these lesions. We report a series of 193 patients over the age of 70 years, the high postoperative mortality of 19% during the first month was counterbalanced by a 5-year survival of more than 20%. The criteria of operability must be rigorous and based on objective elements, especially lung function and the patient's good psychological state.

[The first- and second-arch syndrome. A treatment analysis and plan]. / Syndrome du premier et du deuxième arc. Analyse et plan de traitement.

There is a very great lesion staging in these syndromes, and a great number of procedures have been described, according to the age and severity of the lesions. The initial assessment is therefore important, since it allows classifying these malformations according to their severity in key sectors. Scheduling the treatment in time must take account of: growth, the necessity to perform successive operations in one region, since no operation must hinder a subsequent one. It is essential to determine the optimum time for bone reconstruction. An early treatment with a conventional bone graft, without any intrinsic growth potential, condemns the reconstructed region to immobility, therefore to a progressive degradation of the result in time and to successive corrections. Early reconstruction is justified only if the available reconstruction means allow the reconstructed area to grow, either naturally or with the aid of orthopaedic stimulation. Some means seem to be available to date. A choice must therefore be made between: delayed morphological surgery, early functional surgery. Considering the extent of the means implemented, this is reserved for severe cases. The other great problem is the reconstruction of the auricle, which most often requires a series of operations scheduled over 2 years. The middle ear may not be operated if the lesions are unilateral. The other malformations, including macrostomia, muscular and neural abnormalities, involvement of the eyelids, sometimes require correction, which must fit in a repair schedule that must be established as soon as possible and must take account of the predictable procedures in order to prevent them from hindering each other.

[Osteosarcomas of the mandible]. / Les ostéosarcomes de la mandibule.

On the base of 3 cases of mandibular osteosarcoma, the current role of surgery is specified. The most often used procedure is Rosen's protocol, which includes: preoperative chemotherapy, radical surgery with reconstruction, and postoperative chemotherapy. The key element is the pathological study of the surgical specimen. This will determine the response of the tumor to preoperative chemotherapy and, by comparing the histology of the initial biopsy specimen to that of the surgical specimen, will be used as a basis to select the postoperative chemotherapy. For this type of tumor, chemotherapy has completely substituted for radiation therapy, which is no longer used in the initial treatment. Prophylactic lung radiation therapy has also been given up due to the risks of pulmonary fibrosis entailed by the effective doses. Surgery still has a role of choice in the therapeutic schedule, even if it cannot be contemplated as the sole treatment. Considering the usually young age of the patients, we currently think that reconstruction must be immediate and must resort to all the techniques likely to produce the best possible functional and morphological result.

[Bone substitutes and infection in maxillofacial surgery]. / Substituts osseux et infection en chirurgie maxillo-faciale.

Bony substitutes are in fashion in maxillofacial surgery. They are used to fill bony cavities, in bony reconstruction to give shape-lines and re-create area of support or to fill bony defects. Then their use is frequently crowned by success in general surgery, it is not the same thing in maxillofacial surgery because of the usual impossibility to fulfil a requirement to biomaterial utilisation: the watertight. Furthermore, they don't have yet, for the most part, an essential quality: malleability. In our experience, their use are frequently disappointing because of postoperative infections and we stay faithful to the autograft bone. However it is highly probable that these biomaterials will take an importance more and more considerable when these problems will be overcame.

[A case of cellular blue nevus with bone and meningeal invasion]. / A propos d'un cas de naevus bleu cellulaire avec envahissement osseux et méningé.

The blue naevus is an essentially benign tumour. However, the cellular variety may rarely undergo malignant transformation. The authors report an unusual case of blue naevus in terms of size and evolution. This tumour which involved the occipitovertebral and parotid regions infiltrated underlying tissues and organs, in particular, the occipital bone, the dura mater and the parotid gland. The clinical, histological and histogenic problems associated with the blue naevus are discussed. There are only scattered reports of tumours of a similar nature in the literature and for this reason we felt that this case merited being reported.

[Surgical problems arising from ethmoid adenocarcinomas]. / Problèmes chirurgicaux posés par les adénocarcinomes de l'ethmoïde.

Adenocarcinomas are the most frequent ethmoid tumors. The only curative treatment at present is surgery. Potential extensions of these tumors require a wide exeresis. This is best ensured with an approach that is cranial and of the Cairns type at first, then in some cases facial and patterned on the Moure-Sebileau course of approach. This procedure demands the immediate reconstruction of the 3 levels involved: the dura mater, the bone and the mucosa of the nasal fossae. We reconstruct the dura mater with a pericranial flap, the bone with bone grafts, and the mucosa with a flap from the galea.

[Pulmonary metastasis of a dermatofibrosarcoma]. / Métastase pulmonaire d'un dermatofibrosarcome.

Progressive and recurrent dermatofibrosarcoma, described by Darier and Ferrand in 1924, is a fibrous skin tumour with essentially local malignancy. The authors report a case with pulmonary metastasis, a rare event as only 13 cases of visceral metastases have been reported in the literature. The clinical course of this case was favourable (follow-up of 5 years), in contrast with the usually pejorative nature of metastatic disease (death after several months to one year following the discovery of the first metastasis).

[Evaluation of the use of free flaps of the anterior iliac crest. Review of 49 cases]. / Bilan d'utilisation du lambeau libre de crête iliaque antérieure. A propos de 49 observations.

Because of the septic environment, the reconstruction of extensive losses of substance of the mandible has always raised numerous problems, especially as in a certain number of cases, concomitant radiotherapy represents an additional difficulty. Free flaps offer an important contribution. Up until now, we have essentially used the anterior iliac crest flap vascularised by the deep iliac circumflex pedicle. After 55 flaps of this type, the authors present an analysis of their results and try to analyse the causes for the failures. On the basis of their experience, they consider: that it is preferable to perform the bone and soft tissue repair by means of two different procedures (or at least by means of two different pedicles); that it is preferable, as far as possible, to perform immediate reconstructions rather than deferred reconstructions, especially in the case of malignant tumours.