Restriction in the repertoire of detectable autoantibodies in polyclonal B cell activations and the mimicry of autoantigens by idiotopes

Despite the existence of erythrocyte-autoreactive B cells in normal animals, erythrocyte-autoantibodies could not be detected during polyclonal B-cell activation (PBA) both in patients with visceral leishmaniasis and in bacterial lipopolysacharide (LPS) - injected mice. The failure to detect these autoantibodies in mice with PBA di not seem to be due to suppressor-cell activity, since (1) transfer of spleen cells from LPS-treated mice to naive recipients did not affect the erythrocyte-autoantibody response elicited by subsequent injections of rat erythrocytes and (2) low doses of X-radiation did no lead to erythrocyte-autoantibody detection in LPS-treated mice. The possibility that the detection of erytrocyte-autoantibodies could be affected by autoantibodies with idiotopes mimicring erythrocyte epitopes, the synthesis of which would also be triggerred in PBA, is discussed. Indirect evidence for the existence in normal animal of an expanded lymphocyte population with DNP-binding. Ia-mimicring antigen receptors is presented.

Inputs to the hormonal control of egg development in Rhodnius prolixus

This paper re-examines existing data on the environmental inputs governing egg production in Rhodnius prolixus. Feeding has a direct effect on egg production such that the product of the unfed weight of the female times the weight of the blood meal is a good predictor of the number of eggs produced. Mating modifies this input, so that mated females produce more eggs. Egg production is governed by the corpus allatum, and indirect evidence suggests that the number of eggs producted by a female is a function of the length of time that juvenile hormone is secreted by the corpus allatum. The input which determines the times at which the corpus allatum is switched off originates in the stretch induced by the amount of the meal remaining in the crop, modified by the matedness status of the female. The precise nature of the sensors detecting stretch is not yet clear, but the integrity of the dorsal aorta is essential to the transmission of the information. These data are related to the survival strategy for Rhodnius.

Metabolic and respiratory effects of infused sodium acetate in healthy human subjects.

The metabolic and respiratory effects of intravenous 0.5 M sodium acetate (at a rate of 2.5 mmol/min during 120 min) were studied in nine normal human subjects. O2 consumption (VO2) and CO2 production (VCO2) were measured continuously by open-circuit indirect calorimetry. VO2 increased from 251 +/- 9 to 281 +/- 9 ml/min (P < 0.001), energy expenditure increased from 4.95 +/- 0.17 kJ/min baseline to 5.58 +/- 0.16 kJ/min (P < 0.001), and VCO2 decreased nonsignificantly (211 +/- 7 ml/min vs. 202 +/- 7 ml/min, NS). The extrapulmonary CO2 loss (i.e., bicarbonate generation and excretion) was estimated at 48 +/- 5 ml/min. This observation is consistent with 1 mol of bicarbonate generated from 1 mol of acetate metabolized. Alveolar ventilation decreased from 3.5 +/- 0.2 l/min basal to 3.1 +/- 0.2 l/min (P < 0.001). The minute ventilation (VE) to VO2 ratio decreased from 22.9 +/- 1.3 to 17.6 +/- 0.9 l/l (P < 0.005), arterial PO2 decreased from 93.2 +/- 1.9 to 78.7 +/- 1.6 mmHg (P < 0.0001), arterial PCO2 increased from 39.2 +/- 0.7 to 42.1 +/- 1.1 mmHg (P < 0.0001), pH from 7.40 +/- 0.005 to 7.50 +/- 0.007 (P < 0.005), and arterial bicarbonate concentration from 24.2 +/- 0.7 to 32.9 +/- 1.1 (P < 0.0001). These observations indicate that sodium acetate infusion results in substantial extrapulmonary CO2 loss, which leads to a relative decrease of total and alveolar ventilation.

In vivo and in vitro effects of somatostatin and insulin on glucagon release in a human glucagonoma.

Inhibition of pancreatic glucagon secretion has been reported to be mediated by glucose, insulin and somatostatin. As no human pancreatic alpha-cell lines are available to study in vitro the relative importance of insulin and glucose in the control of pancreatic glucagon release, we investigated a patient presenting with a malignant glucagonoma who underwent surgical resection of the tumour. Functional somatostatin receptors were present as octreotide administration decreased basal glucagon and insulin secretion by 52 and 74%, respectively. The removed tumour was immunohistochemically positive for glucagon, chromogranin A and pancreatic polypeptide but negative for insulin, gastrin and somatostatin. The glucagonoma cells were also isolated and cultured in vitro. Incubation experiments revealed that change from high (10 mM) to low (1 mM) glucose concentration was unable to stimulate glucagon secretion. A dose-dependent inhibition of glucagon release by insulin was however, observed at low glucose concentration. These findings demonstrate that insulin could inhibit glucagon secretion in vitro in the absence of elevated glucose concentrations. These data suggest, as observed in vivo and in vitro in several animal studies, that glucopenia-induced glucagon secretion in humans is not mediated by a direct effect of low glucose on alpha-cells but possibly by a reduction of insulin-mediated alpha-cell suppression and/or an indirect neuronal stimulation of glucagon release.

Correlation of wear in vivo and six laboratory wear methods.

OBJECTIVE: We examined the correlation between clinical wear rates of restorative materials and enamel (TRAC Research Foundation, Provo, USA) and the results of six laboratory test methods (ACTA, Alabama (generalized, localized), Ivoclar (vertical, volumetric), Munich, OHSU (abrasion, attrition), Zurich). METHODS: Individual clinical wear data were available from clinical trials that were conducted by TRAC Research Foundation (formerly CRA) together with general practitioners. For each of the n=28 materials (21 composite resins for intra-coronal restorations [20 direct and 1 indirect], 5 resin materials for crowns, 1 amalgam, enamel) a minimum of 30 restorations had been placed in posterior teeth, mainly molars. The recall intervals were up to 5 years with the majority of materials (n=27) being monitored, however, only for up to 2 years. For the laboratory data, the databases MEDLINE and IADR abstracts were searched for wear data on materials which were also clinically tested by TRAC Research Foundation. Only those data for which the same test parameters (e.g. number of cycles, loading force, type of antagonist) had been published were included in the study. A different quantity of data was available for each laboratory method: Ivoclar (n=22), Zurich (n=20), Alabama (n=17), OHSU and ACTA (n=12), Munich (n=7). The clinical results were summed up in an index and a linear mixed model was fitted to the log wear measurements including the following factors: material, time (0.5, 1, 2 and 3 years), tooth (premolar/molar) and gender (male/female) as fixed effects, and patient as random effect. Relative ranks were created for each material and method; the same was performed with the clinical results. RESULTS: The mean age of the subjects was 40 (±12) years. The materials had been mostly applied in molars (81%) and 95% of the intracoronal restorations were Class II restorations. The mean number of individual wear data per material was 25 (range 14-42). The mean coefficient of variation of clinical wear data was 53%. The only significant correlation was reached by OHSU (abrasion) with a Spearman r of 0.86 (p=0.001). Zurich, ACTA, Alabama generalized wear and Ivoclar (volume) had correlation coefficients between 0.3 and 0.4. For Zurich, Alabama generalized wear and Munich, the correlation coefficient improved if only composites for direct use were taken into consideration. The combination of different laboratory methods did not significantly improve the correlation. SIGNIFICANCE: The clinical wear of composite resins is mainly dependent on differences between patients and less on the differences between materials. Laboratory methods to test conventional resins for wear are therefore less important, especially since most of them do not reflect the clinical wear.

Photodynamic therapy : a pathway for enhanced delivery of liposomal doxorubicin to tumors

Abstract Part I : Background : Isolated lung perfusion (ILP) was designed for the treatment of loco-regional malignancies of the lung. In contrast to intravenous (IV) drug application, ILP allows for a selective administration of cytostatic agents such as doxorubicin to the lung while sparing non-affected tissues. However, the clinical results with ILP were disappointing. Doxorubicinbased ILP on sarcoma rodent lungs suggested high overall doxorubicin concentrations within the perfused lung but a poor penetration of the cytostatic agent into tumors. The same holds true for liposomal-encapsulated macromolecular doxorubicin (LiporubicinTM) In specific conditions, low-dose photodynamic therapy (PDT) can enhance the distribution of macromolecules across the endothelial bamer in solid tumors. It was recently postulated that tumor neovessels were more responsive to PDT than the normal vasculature. We therefore hypothesized that Visudyne®-mediated PDT could selectively increase liposomal doxorubicin (LiporubicinTM) uptake in sarcoma tumors to rodent lungs during intravenous (IV) drug administration and isolated lung perfusion (ILP). Material and Methods : A sarcoma tumor was generated in the left lung of Fisher rats by subpleural injection of a sarcoma cell ,suspension via thoracotomy. Ten days later, LiporubicinTM is administered IV or by single pass antegrade ILP, with or without Visudyne® -mediated low-dose PDT pre-treatment of the sarcoma bearing lung. The drug concentration and distribution were assessed separately in tumors and lung tissues by high pressure liquid chromatography (HPLC) and fluorescence microscopy (FNI~, respectively. Results : PDT pretreatment before IV LiporubicinTM administration resulted in a significantly higher tumor drug uptake and tumor to lung drug ratio compared to IV drug injection alone without affecting the blood flow and drug distribution in the lung. PDT pre-treatment before LiporubicinTM-based ILP also resulted in a higher tumor drug uptake and a higher tumor to lung drug ratio compared to ILP alone, however, these differences were not significant due to a heterogeneous blood flow drug distribution during ILP which was further accentuated by PDT. Conclusions : Low-dose Visudyne®-mediated PDT pre-treatment has the potential to selectively enhance liposomal encapsulated doxorubicin uptake in tumors but not in normal lung tissue after IV drug application in a rat model of sarcoma tumors to the lung which opens new perspectives for the treatment of superficially spreading chemoresistant tumors of the chest cavity such as mesothelioma or malignant effusion. However, the impact of PDT on macromolecular drug uptake during ILP is limited since its therapeutic advantage is circumvented by ILP-induced heterogeneicity of blood flow and drug distribution Abstract Part II Background : Photodynamic therapy (PDT) with Visudyne® acts by direct cellular phototoxicity and/or by an indirect vascular-mediated effect. Here, we demonstrate that the vessel integrity interruption by PDT can promote the extravasation of a macromolecular agent in normal tissue. To obtain extravasation in normal tissue PDT conditions were one order of magnitude more intensive than the ones in tissue containing neovessels reported in the literature. Material and Methods : Fluorescein isothiocyanate dextran (FITC-D, 2000kDa), a macromolecular agent, was intravenously injected 10 minutes before (LKO group, n=14) or 2 hours (LK2 group, n=16) after Visudyne® mediated PDT in nude mice bearing a dorsal skin fold chamber. Control animals had no PDT (CTRL group, n=8). The extravasation of FITC-D from blood vessels in striated muscle tissue was observed in both groups in real-time for up to 2500 seconds after injection. We also monitored PDT-induced leukocyte rolling in-vivo and assessed, by histology, the corresponding inflammatory reaction score in the dorsal skin fold chambers. Results : In all animals, at the applied PDT conditions, FITC-D extravasation was significantly enhanced in the PDT treated areas as compared to the surrounding non-treated areas (p<0.0001). There was no FITC-D leakage in the control animals. Animals from the LKO group had significantly less FITC-D extravasation than those from the LK2 group (p = 0.0002). In the LKO group FITC-D leakage correlated significantly with the inflammation (p < 0.001). Conclusions: At the selected conditions, Visudyne-mediated PDT promotes vascular leakage and FITC-D extravasation into the interstitial space of normal tissue. The intensity of vascular leakage depends on the time interval between PDT and FITC-D injection. This concept could be used to locally modulate the delivery of macromolecules in vivo. Résumé : La perfusion cytostatique isolée du poumon permet une administration sélective des agents cytostatiques sans implication de la circulation systémique avec une forte accumulation au niveau du poumon mais une faible pénétration dans les tumeurs. La thérapie photodynamique (PDT) qui consiste en l'application d'un sensibilisateur activé par lumière laser non- thermique d'une longueur d'onde définie permet dans certaines conditions, une augmentation de la pénétration des agents cytostatiques macromoléculaires à travers la barrière endothéliale tumorale. Nous avons exploré cet avantage thérapeutique de la PDT dans un modèle expérimental afin d'augmenter d'une manière sélective la pénétration tumorale de la doxorubicin pegylée, liposomal- encapsulée macromoléculaire (Liporubicin). Une tumeur sarcomateuse a été générée au niveau du poumon de rongeur suivie d'administration de Liporubicin, soit par voie intraveineuse soit par perfusion isolée du poumon (ILP). Une partie des animaux ont reçus un prétraitement de la tumeur et du poumon sous jacent par PDT avec Visudyne comme photosensibilisateur. Les résultats ont démontrés que la PDT permet, sous certaines conditions, une augmentation sélective de Liporubicin dans les tumeurs mais pas dans le parenchyme pulmonaire sous jacent. Après administration intraveineuse de Liporubicin et prétraitement par PDT, l'accumulation dans les tumeurs était significative par rapport au poumon, et aux tumeurs sans PDT. Le même phénomène est observé après ILP du poumon. Cependant, les différences avec ou sans PDT n'étaient pas significatives lié à und distribution hétérogène de Liporubicin dans le poumon perfusé après ILP. Dans une deuxième partie de l'expérimentation, nous avons exploré la microscopie intra-vitale pour déterminer l'extravasion des substances macromoléculaires (FITS) à travers la barrière endothéliale avec ou sans Visudyne-PDT au niveau des chambres dorsales des souris nues. Les résultats montrent qu'après PDT, l'extravasion de FITS a été augmentée de manière significative par rapport au tissu non traité. L'intensité de l'extravasion de FITS dépendait également de l'intervalle entre PDT et injection de FITS. En conclusion, les expérimentations montrent que la PDT est capable, sous certaines conditions, d'augmenter de manière significative l'extravasion des macromolécules à travers la barrière endothéliale et leur accumulation dans des tumeurs mais pas dans le parenchyme pulmonaire. Ces résultats permettent une nouvelle perspective de traitement pour des tumeurs superficielles intrathoraciques chimio-résistent comme l'épanchement pleural malin ou le mésothéliome pleural.

Evaluation of Chagas' disease transmission through breast-feeding

On hundred milk or colostrum samples from 78 mothers with chronic Chagas' disease were parasitologically studied for Trypanosoma cruzi infection by means of direct examination and inoculation of mice. The mice were submitted to direct bllod examination three times a week. At the end of 45 days, xenodiagnosis and indirect immunofluorescent test (IFAT) for T. cruzi antibodies were carried out in the animals. No parasitized sample was observed even though five mothers had parasitemia at milk collection. In addition, 97 breast-fed children of chronic chagasic mothers, born free of infection, were tested for IgG antibodies to T. cruzi using IFAT. No case of T. cruzi infection was detected. The authors conclude that breast-feeding should not be avoided for children for chronic chagasic women. However, as these mothers had intermittent parasitemia, they should avoid nursing when there is nipple bleeding.

Effects of hyperglycemia on glucose metabolism before and after oral glucose ingestion in normal men.

The plasma glucose excursion may influence the metabolic responses after oral glucose ingestion. Although previous studies addressed the effects of hyperglycemia in conditions of hyperinsulinemia, it has not been evaluated whether the route of glucose administration (oral vs. intravenous) plays a role. Our aim was to determine the effects of moderately controlled hyperglycemia on glucose metabolism before and after oral glucose ingestion. Eight normal men underwent two oral glucose clamps at 6 and 10 mmol/l plasma glucose. Glucose turnover and cycling rates were measured by infusion of [2H7]glucose. The oral glucose load was labeled by D-[6,6-2H2]glucose to monitor exogenous glucose appearance, and respiratory exchanges were measured by indirect calorimetry. Sixty percent of the oral glucose load appeared in the systemic circulation during both the 6 and 10 mmol/l plasma glucose tests, although less endogenous glucose appeared during the 10 mmol/l tests before glucose ingestion (P &lt; 0.05). This inhibitory effect of hyperglycemia was not detectable after oral glucose ingestion, although glucose utilization was increased (+28%, P &lt; 0.05) due to increased nonoxidative glucose disposal [10 vs. 6 mmol/l: +20%, not significant (NS) before oral glucose ingestion; +40%, P &lt; 0.05 after oral glucose ingestion]. Glucose cycling rates were increased by hyperglycemia (+13% before oral glucose ingestion, P &lt; 0.001; +31% after oral glucose ingestion, P &lt; 0.05) and oral glucose ingestion during both the 6 (+10%, P &lt; 0.05) and 10 mmol/l (+26%, P &lt; 0.005) tests. A moderate hyperglycemia inhibits endogenous glucose production and contributes to glucose tolerance by enhancing nonoxidative glucose disposal. Hyperglycemia and oral glucose ingestion both stimulate glucose cycling.

Jatobal virus antigenic characterization by ELISA and neutralization test using EIA as indicator, on tissue culture

A virus antigenic characterization methodology using an indirect method of antibody detection ELISA with virus-infected cultured cells as antigen and a micro virus neutralisation test using EIA (NT-EIA) as an aid to reading were used for antigenic characterization of Jatobal (BeAn 423380). Jatobal virus was characterized as a Bunyaviridae, Bunyavirus genus, Simbu serogroup virus. ELISA using infected cultured cells as antigen is a sensitive and reliable method for identification of viruses and has many advantages over conventional antibody capture ELISA's and other tests: it eliminates solid phase coating with virus and laborious antigen preparation; it permits screening of large numbers of virus antisera faster and more easily than by CF, HAI, or plaque reduction NT. ELISA and NT using EIA as an aid to reading can be applicable to viruses which do not produce cytopathogenic effect. Both techniques are applicable to identification of viruses which grow in mosquito cells.

Detection of viral infection by immunofluorescence in formalin-fixed tissues, pretreated with trypsin

The presence of viral antigen in sections from formalin-fixed and paraffin-embedded human tissues was demonstrated by trypsin digestion followed by direct or indirect immunofluorescence. The specimens may be used for retrospective diagnosis. The immunofluorescence technique has to be adapted to the suspected virus infection on the basis of previous histopathology study. Variations of trypsin concentration time and temperature of incubation, expose different viral antigens and have to be previously tested for each unknown system. For measles virus detection in lung a stronger digestion has to be applied as compared to adenovirus or respiratory disease viruses in the same tisue. Flavivirus in liver tissue needs a weaker digestion. The reproducibility of the method makes it useful as a routine technique in diagnosis of virus infection.

Development and current use of parenteral nutrition in critical care - an opinion paper.

Critically ill patients depend on artificial nutrition for the maintenance of their metabolic functions and lean body mass, as well as for limiting underfeeding-related complications. Current guidelines recommend enteral nutrition (EN), possibly within the first 48 hours, as the best way to provide the nutrients and prevent infections. EN may be difficult to realize or may be contraindicated in some patients, such as those presenting anatomic intestinal continuity problems or splanchnic ischemia. A series of contradictory trials regarding the best route and timing for feeding have left the medical community with great uncertainty regarding the place of parenteral nutrition (PN) in critically ill patients. Many of the deleterious effects attributed to PN result from inadequate indications, or from overfeeding. The latter is due firstly to the easier delivery of nutrients by PN compared with EN increasing the risk of overfeeding, and secondly to the use of approximate energy targets, generally based on predictive equations: these equations are static and inaccurate in about 70% of patients. Such high uncertainty about requirements compromises attempts at conducting nutrition trials without indirect calorimetry support because the results cannot be trusted; indeed, both underfeeding and overfeeding are equally deleterious. An individualized therapy is required. A pragmatic approach to feeding is proposed: at first to attempt EN whenever and as early as possible, then to use indirect calorimetry if available, and to monitor delivery and response to feeding, and finally to consider the option of combining EN with PN in case of insufficient EN from day 4 onwards.

Differential regulations of AQP4 and Kir4.1 by triamcinolone acetonide and dexamethasone in the healthy and inflamed retina.

PURPOSE: Glucocorticoids are used to treat macular edema, although the mechanisms underlying this effect remain largely unknown. The authors have evaluated in the normal and endotoxin-induced uveitis (EIU) rats, the effects of dexamethasone (dex) and triamcinolone acetonide (TA) on potassium channel Kir4.1 and aquaporin-4 (AQP4), the two main retinal Müller glial (RMG) channels controlling retinal fluid movement. METHODS: Clinical as well as relatively low doses of dex and TA were injected in the vitreous of normal rats to evaluate their influence on Kir4.1 and AQP4 expression 24 hours later. The dose-dependent effects of the two glucocorticoids were investigated using rat neuroretinal organotypic cultures. EIU was induced by footpad lipopolysaccharide injection, without or with 100 nM intraocular dex or TA. Glucocorticoid receptor and channel expression levels were measured by quantitative PCR, Western blot, and immunohistochemistry. RESULTS: The authors found that dex and TA exert distinct and specific channel regulations at 24 hours after intravitreous injection. Dex selectively upregulated Kir4.1 (not AQP4) in healthy and inflamed retinas, whereas TA induced AQP4 (not Kir4.1) downregulation in normal retina and upregulation in EIU. The lower concentration (100 nM) efficiently regulated the channels. Moreover, in EIU, an inflammatory condition, the glucocorticoid receptor was downregulated in the retina, which was prevented by intravitreous injections of the low concentration of dex or TA. CONCLUSIONS: The results show that dex and TA are far from being equivalent to modulate RMG channels. Furthermore, the authors suggest that low doses of glucocorticoids may have antiedematous effects on the retina with reduced toxicity.

Effects of infused amino acids on glucose production and utilization in healthy human subjects.

Amino acids have been reported to increase endogenous glucose production in normal human subjects during hyperinsulinemia: however, controversy exists as to whether insulin-mediated glucose disposal is inhibited under these conditions. The effect of an amino acid infusion on glucose oxidation rate has so far not been determined. Substrate oxidation rates, endogenous glucose production, and [13C]glucose synthesis from [13C]bicarbonate were measured in six normal human subjects during sequential infusions of exogenous glucose and exogenous glucose with (n = 5) or without (n = 5) exogenous amino acids. Amino acids increased endogenous glucose production by 84% and [13C]glucose synthesis by 235%. Glucose oxidation estimated from indirect calorimetry decreased slightly after amino acids, but glucose oxidation estimated from [13C]glucose-13CO2 data was increased by 14%. It is concluded that gluconeogenesis is the major pathway of amino acid degradation. During amino acid administration, indirect calorimetry underestimates the true rate of glucose oxidation, whereas glucose oxidation calculated from the 13C enrichment of expired CO2 during [U-13C]glucose infusion does not. A slight stimulation of glucose oxidation during amino acid infusion, concomitant with an increased plasma insulin concentration, indicates that amino acids do not inhibit glucose oxidation.

An outbreak of american cutaneous leishmaniasis (Leishmania braziliensis braziliensis) in a periurban area of Rio de Janeiro city, Brazil: clinical and epidemiological studies

From July 1984 to September 1986, 105 cases of American cutaneous leishmaniasis were studied in a locality closely situated to an urbanized area of the city of Rio de Janeiro, Brazil. Settement in this area was established at least 20 years ago but the first cases were noted six months prior to the beginning of this study. Cases were almost exlusively cutaneous and ulcerated, with one to six months of evolution. Montenegro's skin tests were positive in all cases and anti-Leishmania antibodies were detected by indirect immunofluorescence test in 74.3% of the patients. Parasites were demonstrated in 69.5% of cases. Domestic animals were easily found infected; 32% of the examined dogs and 30.8% of the examined equines were positive to the presence of Leishmania in cutaneous ulcerated lesions. Parasite isolates from human, dog andequines were immunologically characterized and identified as L. b. braziliensis. 73,0% of the sandfly population were Lutzomyia intermedia mainly caught on human baits and on domestic animals. Our observations suggest that this is an area of recent established L. b. braziliensis infection and that transmission probably occurs indoors or outdoors close to the houses.

Patterns of serologic response to human immunodeficiency virus type 1 (HIV-1) in brazilians with different clinical forms of HIV infection

In order to investigate the IgG HIV-1 antibodies rectivity to structural components of the virus, 85 sera from infected Brazilians, comprising the total spectrum of HIV infection, were analysed by Western blot assay. The sera were confirmed as being positive to HIV with enzyme linked immuno assay (ELISA) and indirect immunofluorescence (IIF). Although the sera from patients reacted less intensively to the gag polypeptide of 55KDa, no distinctive antigen reaction patterns were observed between sera patients with different clinical forms. Because of the higher frequency of reactivity to the gag p24 in AIDS patients, the patterns of anti-HIV IgG responses are similar to those observed in their African counterparts.