Evidence for humoral and cellular reactivity against keratin and thyroglobulin in HTLV-I infected rabbits.

Human T cell leukemia virus type I (HTLV-I) infection was initially associated with T cell leukemia and a progressive neurologic disease but has since been linked to an increasing number of autoimmune disorders, including Sjogren's syndrome, uveitis, and polyarthritis. A survey of serum samples from a rabbit model of HTLV-I infection revealed that all had antibodies against keratin and thyroglobulin. Sera from several infected rabbits also reacted with collagen, while antibody reactions with other autoantigens tested, including DNA, were rare and sporadic. In addition to antibodies, cellular reactivity to keratin, but not thyroglobulin, was demonstrated by cellular proliferation in presence of IL-2 and keratin. Expanded cell cultures were positive for T cell activation markers and CD8. Association of the auto-reactivity with HTLV-I infection rather than random anti-cellular responses was supported by the fact that no antikeratin or antithyroglobulin was seen in uninfected controls, including that inoculated with uninfected lymphocytes. Finding autoantibodies in rabbits infected using naked HTLV-I DNA clones provided further assurance that infection induced the autoimmune reactions detected.

Functional characterization of a lysosomal sorting motif in the cytoplasmic tail of HLA-DObeta.

HLA-DO is an intracellular non-classical class II major histocompatibility complex molecule expressed in the endocytic pathway of B lymphocytes, which regulates the loading of antigenic peptides onto classical class II molecules such as HLA-DR. The activity of HLA-DO is mediated through its interaction with the peptide editor HLA-DM. Here, our results demonstrate that although HLA-DO is absolutely dependent on its association with DM to egress the endoplasmic reticulum, the cytoplasmic portion of its beta chain encodes a functional lysosomal sorting signal. By confocal microscopy and flow cytometry analysis, we show that reporter transmembrane molecules fused to the cytoplasmic tail of HLA-DObeta accumulated in Lamp-1(+) vesicles of transfected HeLa cells. Mutagenesis of a leucine-leucine motif abrogated lysosomal accumulation and resulted in cell surface redistribution of reporter molecules. Finally, we show that mutation of the di-leucine sequence in DObeta did not alter its lysosomal sorting when associated with DM molecules. Taken together, these results demonstrate that lysosomal expression of the DO-DM complex is mediated primarily by the tyrosine-based motif of HLA-DM and suggest that the DObeta-encoded motif is involved in the fine-tuning of the intracellular sorting.

Cellular distribution of a mixed MHC class II heterodimer between DRalpha and a chimeric DObeta chain.

Human MHC class II antigens include HLA-DR, -DQ, and -DP molecules that present antigens to CD4+ T cells, as well as the non-classical molecules HLA-DM and -DO. HLA-DM promotes peptide binding to class II molecules in endocytic compartments and HLA-DO, which is physically associated with HLA-DM in B lymphocytes, regulates HLA-DM function. Antibodies specific for the DObeta chain were obtained by immunization of mice with a heterodimer consisting of a chimeric DObeta chain (DR/DObeta), containing 18 N-terminal residues of DRbeta, paired with the DRalpha chain and isolated from transfected murine fibroblasts. The specificity of this serum for the DObeta chain and the lysosomal expression of the HLA-DO protein was confirmed using mutant human B cell lines lacking DR or DO molecules. The lysosomal localization of HLA-DO in human B cells contrasts with the cell surface expression of the mixed pair in transfected murine fibroblasts and raises questions concerning the role of the putative targeting motifs in HLA-DO. Transfection of the chimeric DR/DObeta chain along with DRalpha into human epithelial HeLa cells resulted in high levels of expression of the mixed isotypic pair at the surface of transfectants as well as in lysosomes. The same pattern was observed in HeLa cells transfected with the DObeta chimera and a DRa chain lacking the cytoplasmic tail. Taken together, these results suggest that functional sorting motifs exist in the DObeta chain but that the tight compartmentalization of HLA-DO observed inside B lymphocytes is controlled by the HLA-DOalpha chain and HLA-DM.

Conserved structural features between HLA-DO beta and -DR beta.

HLA-DO is a non-classical MHC class II molecule presumed to play a specialized role in the antigen processing pathway. We have modeled the HLA-DO beta-chain and found its overall structure compatible with the one of DR beta. Functional studies further highlighted the similarity between these beta-chains of the class II family of proteins. Indeed, a mixed heterodimer composed of the DR alpha and a chimeric DO beta-chains presented bacterial superantigens to T cells and was shown to interact with CD4. The implications of such structural conservation for the in vivo functions of HLA-DO are discussed.

A comparative multicentre trial of spinal needles for caesarean section.

We studied 681 patients in a randomised, multicentre, double-blind, parallel group trial designed to assess the incidence of headache following spinal anaesthesia for Caesarean section using four different pencil point spinal needles. The needles used were: Whitacre 25G (n = 170), Polymedic 25G (n = 170), Sprotte 24G (n = 173) and Polymedic 24G (n = 168). The incidence of all headaches prior to discharge was 11.1%. Only five headaches (0.75%) were severe with features of post dural puncture headache (PDPH) and required an epidural blood patch: Whitacre 25G = 0, Polymedic 25G = 1 (0.6%), Sprotte 24G = 2 (1.2%), Polymedic 24G = 2 (1.2%). There was no statistically significant difference between the four groups for PDPH. We conclude that all four needles studied performed satisfactorily and comparably.

The reinforced laryngeal mask airway for dento-alveolar surgery.

We have evaluated the reinforced laryngeal mask airway (LMA) for use during dento-alveolar surgery in 100 ASA I and II day-case patients allocated randomly to receive either a nasotracheal tube or reinforced LMA. We recorded ease of airway insertion, airway complications, quality of recovery and replies to a 24-h postoperative questionnaire. In addition, a fibreoptic assessment was made of laryngotracheal soiling, and the effect of head movement and the position of the reinforced LMA. There were no significant differences in difficulty in airway positioning or perioperative oxygen desaturation. Nineteen patients in the nasotracheal tube group had epistaxis (P = 0.001) and laryngotracheal soiling occurred in three of these patients. Two reinforced LMA were dislodged on moving into the operating theatre and in a further five patients in this group there was partial airway obstruction (compared with none in the nasotracheal tube group; P = 0.018) which was caused by downward pressure on the mandible by the surgeon. There were no differences in postoperative complications. No surgeon reported poor access to the operating field. Overall the reinforced LMA provided satisfactory conditions for this surgery but vigilance of the airway was required, especially at the time of extraction.

HLA-DRB and -DBQ typing by PCR amplification using sequence-specific primers (PCR-SSP): assessment after 1 year of routine use by three laboratories.

Using sequence-specific amplifications, a practical and fast technique for DRB and DQB typing has been developed. The primers are chosen in order to amplify groups of alleles corresponding to the same serological specificity. In a second step, precise allelic determination is obtained by studying the restriction fragment length polymorphism of the PCR products. The experience of three laboratories using this technique in the context of organ or bone marrow transplantation is reported.

Localization of the conformational alteration of MHC molecules induced by the association of mouse class I heavy chain with a xenogeneic beta 2-microglobulin.

Changes in the antigenicity of major histocompatibility complex (MHC) class I molecules resulting from the association of bovine beta 2-microglobulin (beta 2-m) with mouse class I heavy chains were investigated. Mice (H-2b) were immunized with syngeneic Concanavalin A (Con A) blasts induced in the presence of fetal calf serum (FCS) in conditions allowing exchange between mouse and bovine beta 2-microglobulin (beta 2-m). Spleen cells from hyperimmunized mice were fused with myeloma cells and two monoclonal antibodies which required for their reactivity the presence of FCS have been further studied. One of them (CAB 297) recognized a determinant of bovine beta 2-m which is present on free molecules in solution as well as when they are associated with either mouse or bovine class I heavy chains. In contrast, the second monoclonal antibody (CBB 70) did not react with free bovine beta 2-m molecules, nor with beta 2-m associated with bovine class I heavy chains. It did react with cells of some H-2 haplotypes (b, f, p and r) but only when their class I heavy chains are associated with bovine or with human beta 2-m. Therefore, expression of the CBB 70 defined antigenic determinant requires both xenogeneic beta 2-m and class I heavy chain of a given H-2 molecule. In order to precisely localize the antigenic determinant defined by this monoclonal antibody and therefore the region altered by the association of class I heavy chain with xenogeneic beta 2-m, we made use of exon shuffled class I molecules. The results indicate that changes induced by the association of bovine beta 2-m with H-2 class I heavy chain affect the conformation of the alpha 2 domain. These studies illustrate that MHC class I molecules exhibit a considerable conformational flexibility which could influence their ability to bind and present various peptides to the T-cell receptor.

Oral fluids prior to day surgery. The effect of shortening the pre-operative fluid fast on postoperative morbidity.

One hundred day surgical patients undergoing first trimester termination of pregnancy were randomly allocated to receive either 150 ml of clear fluid 1.5-2 hours before anaesthesia or to remain fasted from midnight the night before. Patients were anaesthetised using a total intravenous technique which consisted of propofol and alfentanil. No adverse intra-operative events were noted in either group. There were no significant differences in immediate recovery time, or pain, nausea and headache scores at 30 or 120 minutes following recovery. The fasted group had less pain (p less than 0.05) at 60 minutes after recovery than the fluid group, although the mean pain scores in both groups were low. Eighty two per cent of the patients returned questionnaires about pain, nausea and headache scores on arriving home, and at 12 and 24 hours after surgery. There were no significant differences between the two groups. In conclusion, pain, nausea and headache scores are low following total intravenous anaesthesia with propofol and alfentanil for termination of pregnancy and these were unaffected by the administration of 150 ml of clear fluid given approximately 1.5 hours pre-operatively.

H-2 influence on the production of real but not of apparent H-2-specific antibodies induced by the association of syngeneic class I heavy chains with bovine beta 2-microglobulin.

Immunogenic properties of class I molecules resulting from the association of mouse class I heavy chains with a xenogeneic beta 2-microglobulin (beta 2-m) were investigated by studying the antibody response of mice of injections to syngeneic Con A lymphoblasts, induced in conditions allowing the replacement of endogenous beta 2-m by exogenously added bovine beta 2-m provided by fetal calf serum (FCS-Con A blasts). Lymphocytotoxic antibodies were regularly produced and according to their specificities they could be divided into two types: antibodies whose reactivity was (1) dependent on and (2) independent of the presence of bovine beta 2-m on target cells. Although both types displayed an H-2 dependent polymorphic reaction pattern, only antibodies recognizing class I molecules without bovine beta 2-m can be considered as real H-2-specific antibodies. The others are only apparent H-2-specific antibodies: their polymorphic reaction pattern is dependent both on the presence of bovine beta 2-m on the surface of target cells and on their H-2 haplotype. A comparison of the antibody response of mice with various H-2 haplotypes to injections of syngeneic FCS-Con A blasts showed no significant difference in the induction of bovine beta 2-m-dependent antibodies (apparent H-2-specific) among the mice from all strains tested (H-2b, H-2p, H-2q, and H-2s). Unexpectedly, for most strains more than 60% of the immunized mice produced also beta 2-m-independent antibodies (real H-2-specific), with the exception of H-2q mice, in which only 30% of sera were positive. The real H-2-specific antibody response is of two types: some mice (H-2p and H-2s) produced antibodies only reactive with allogeneic target cells whereas others (H-2b and H-2q) produced in addition antibodies that were reactive with syngeneic cells. Thus H-2 appears to play an important role in the induction and specificity of the lymphocytotoxic H-2-specific antibodies induced upon immunization with cells expressing syngeneic class I heavy chains associated with bovine beta 2-m.

Cerebrovascular response to intracarotid infusion of 5-hydroxytryptophan and L-DOPA in cats.

Intracarotid administration of 5-hydroxytryptophan (10 mg/kg) results in a slight vasoconstriction of cerebral vessels. L-DOPA (20 mg/kg) administered by the same route enhances cerebral blood flow. The effect on cerebral blood flow of L-DOPA (20 mg/kg) following intracarotid 5-hydroxytryptophan (10 mg/kg) administration shows a potentiation of cerebral blood flow response in non-pretreated as well as in nialamide- and reserpine-pretreated cats.