Clonality of cold agglutinins in patients with hemolytic anemia: an analysis by high-resolution two-dimensional gel electrophoresis.

High-resolution two-dimensional gel electrophoresis (2-DGE) was used to analyse plasma samples and partially purified cold agglutinins (CA) obtained from two selected patients. Both presented an acute hemolytic anemia with CA of high thermal amplitude, normal immunoglobulin levels, no detectable paraproteinemia, and no clinical evidence of a malignant B-cell disorder. The electrophoretograms of their plasma showed evident alternations of the "normal" protein profile, which were directly related to hemolysis (absence of the spots of haptoglobin and in one case of those of hemopexin), but no monoclonal gammopathy. The electrophoretograms of their purified CA revealed two clearly different spot patterns respectively corresponding to a monoclonal IgM and to polyclonal IgM. These results show that the clonality of CA associated with hemolytic anemia can be easily determined by 2-DGE. This technique may be very useful to discriminate chronic cold agglutinin disease in the early phase from "parainfectious" CA.     

Measurement of post-operative pain and narcotic administration in infants using a new clinical scoring system

We developed a clinical neurologic and behavioral scoring system composed of 10 items to measure the post-operative pain levels in infants: (1) sleep during preceeding hour, (2) facial expression of pain, (3) quality of cry, (4) spontaneous motor activity, (5) Spontaneous excitability, (6) flexion of fingers and toes, (7) sucking, (8) global evaluation of tone, (9) consolability and (10) sociability. Using this system, a group of infants ranging from one to seven months in age and undergoing minor surgical procedures was studied. The infants were randomly assigned to two groups: Group I received Fentanyl intravenously (3 g/kg) prior to surgery, and Group II received a placebo. The infants then were studied post-operatively in the recovery room at 30, 60, 90 and 120 min intervals. Over the entire post-operative observation period, 54% of the infants in Group I had satisfactory analgesia compared to 18% in Group II. There were no significant differences in Group I and Group II in oxygenation, carbon dioxide elimination, blood pressure, heart rate or temperature.     

Distinct Leishmania braziliensis isolates induce different paces of chemokine expression patterns

Inflammatory events during Leishmania braziliensis infection in mice were investigated. Large lesions were directly correlated with the inflammatory reaction but not with parasite burden. Different L. braziliensis strains induce different paces of chemokine expression patterns, leading to diverse cell recruitment and differential inflammatory responses.     

Lutzomyia longipalpis salivary gland homogenate impairs cytokine production and costimulatory molecule expression on human monocytes and dendritic cells

In this report, we describe an investigation of the effects of Lutzomyia longipalpis sand fly salivary gland homogenates (SGH) on cytokine production and expression of costimulatory molecules on human monocytes, macrophages (Ms), and dendritic cells (DCs). SGH of L. longipalpis induced an increase in interleukin-6 (IL-6), IL-8 and IL-12p40 production but a decrease in tumor necrosis factor alpha and IL-10 production by lipopolysaccharida (LPS)-stimulated monocytes. We also examined the expression of costimulatory molecules on the surface of monocytes, Ms, and DCs. Whereas SGH affected the expression of these molecules on monocytes and Ms, it had little effect on these molecules on DCs. However, when DCs were generated from human monocytes in the presence of SGH, SGH inhibited the expression of costimulatory molecules. In addition, a decrease in the maturation of DCs induced by CD40L was observed in the presence of SGH. Finally, preincubating SGH with human sera containing anti-SGH-specific antibodies abolished the effects of SGH on cytokine production by LPS-stimulated monocytes.     

The in vitro cytotoxicity of ascorbate depends on the culture medium used to perform the assay and involves hydrogen peroxide

Reports about the effects of ascorbate (vitamin C) on cultured cells are confusing and conflicting. Some authors show inhibition of cell death by ascorbate, whereas others demonstrate that ascorbate is cytotoxic. In this report, using three different cell types and two different culture media (Dulbecco's modified Eagle's medium and RPMI 1640), we show that the toxicity of ascorbate is due to ascorbate-mediated production of H2O2, to an extent that varies with the medium used to culture the cells. For example, 1 mM ascorbate generates 161 +/- 39 microM H2O2 in Dulbecco's modified Eagle's medium and induces apoptosis in 50% of HL60 cells, whereas in RPMI 1640 only 83 +/- 17 microM H2O2 is produced and no apoptosis is detected. Apoptosis is prevented by catalase, and direct addition of H2O2 at the above concentration to the cells has similar effects to ascorbate. These results show that ascorbate itself is not toxic to the cell lines used and that effects of ascorbate in vivo cannot be predicted from studies on cultured cells. The ability of ascorbate to interact with different cell culture media to produce H2O2 at different rates could account for many or all of the conflicting results obtained using ascorbate in cultured cell assays.     

Characterization of wild-type recombinant Bet v 1a as a candidate vaccine against birch pollen allergy

BACKGROUND: We describe the production in Escherichia coli as a recombinant protein of clinical grade wild-type Bet v 1a (rBet v 1a), to be used as a candidate vaccine against birch pollen allergy. METHODS: This recombinant protein was purified by hydrophobic interaction and ion exchange chromatography and characterized by SDS-PAGE, immunoprint and circular dichroism in parallel with natural Bet v 1 (nBet v 1) purified from a birch pollen extract. We also compared rBet v 1 and nBet v 1 for their capacity to induce histamine release from basophils and to stimulate T lymphocyte proliferation. RESULTS: rBet v 1a appears in SDS-PAGE as an 18-kDa monomeric protein, whereas purified nBet v 1 comprises a mixture of isoforms (resolving as three distinct bands and six spots after 1-dimensional and 2-dimensional electrophoresis, respectively). Both recombinant and natural purified Bet v 1 molecules are recognized by IgE from birch pollen-allergic patients as well as anti-Bet v 1 murine monoclonal antibodies, suggesting that the recombinant protein is correctly folded in a native configuration. Circular dichroism analysis confirmed that the two Bet v 1 molecules exhibit similar 3-dimensional structures, even if rBet v 1a appears more compact and stable in thermodenaturation/renaturation experiments. Both rBet v 1 and nBet v 1 induce the degranulation of sensitized basophils and proliferation of Bet v 1-specific T lymphocytes in a similar manner. CONCLUSIONS: On the basis of these structural and biological properties, rBet v 1a is a valid candidate vaccine against birch pollen allergy, currently evaluated in humans.     

Ocular counterrolling induced by centrifugation during orbital space flight

During the 1998 Neurolab mission (STS-90), four astronauts were exposed to interaural centripetal accelerations (Gy centrifugation) of 0.5g and 1g during rotation on a centrifuge, both on Earth and during orbital space flight. Subjects were oriented either left-ear out or right-ear out, facing or back to motion. Binocular eye movements were measured in three dimensions using a video technique. On Earth, tangential centrifugation that produces 1g of interaural linear acceleration combines with gravity to tilt the gravitoinertial acceleration (GIA) vector 45° in the roll plane relative to the head vertical, generating a summed vector of 1.4g. Before flight, this elicited mean ocular counterrolling (OCR) of 5.7°. Due to the relative absence of gravity during flight, there was no linear acceleration along the dorsoventral axis of the head. As a result, during in-flight centrifugation, gravitoinertial acceleration was strictly aligned with the centripetal acceleration along the interaural axis. There was a small but significant decrease (mean 10%) in the magnitude of OCR in space (5.1°). The magnitude of OCR during postflight 1g centrifugation was not significantly different from preflight OCR (5.9°). Findings were similar for 0.5g centrifugation, but the OCR magnitude was approximately 60% of that induced by centrifugation at 1g. OCR during pre- and postflight static tilt was not significantly different and was always less than OCR elicited by centrifugation on Earth for an equivalent interaural linear acceleration. In contrast, there was no difference between the OCR generated by in-flight centrifugation and by static tilt on Earth at equivalent interaural linear accelerations. These data support the following conclusions: (1) OCR is generated predominantly in response to interaural linear acceleration; (2) the increased OCR during centrifugation on Earth is a response to the head dorsoventral 1g linear acceleration component, which was absent in microgravity. The dorsoventral linear acceleration could have activated either the otoliths or body-tilt receptors that responded to the larger GIA magnitude (1.4g), to generate the increased OCR during centrifugation on Earth. A striking finding was that magnitude of OCR was maintained throughout and after flight. This is in contrast to most previous postflight OCR studies, which have generally registered decreases in OCR. We postulate that intermittent exposure to artificial gravity, in the form of the centripetal acceleration experienced during centrifugation, acted as a countermeasure to deconditioning of this otolith-ocular orienting reflex during the 16-day mission. Electronic Publication