[Practical implementation and usefulness of guidelines for the management of syncope: a professional practice study]. / Application pratique et intérêt des recommandations de prise en charge des syncopes : évaluation de pratiques professionnelles.

AIMS: To assess the practical implementation of international guidelines and their impact on syncope management in a 500-bed general hospital. PATIENTS AND METHODS: Three groups of 63 consecutive patients admitted for syncope to the emergency care unit (ECU) were studied: group 1, before the guidelines delivered to the practitioners, group 2 immediately after the diffusion of guidelines and group 3, one year later. The study evaluates the mean duration of stay (MDS) and the relevance of the diagnostic strategy. RESULTS: In group 1 compared to group 2, MDS were respectively 6.8±5.5 and 5.4±2.8 days (P=0.07) and the unexplained syncope number respectively 22% and 24% (P=0.8). The search of orthostatic hypotension became more systematic (13% versus 86% in group 1 and 2 respectively, P<0.001). The agreement (kappa coefficient) between initial and final diagnostic increased in 0.34 to 0.44. One year later MDS in group 3 was 7.1±4.7 days (P=0.8 versus group 1 and P=0.015 versus group 2) with only 6.3% systematic search for orthostatic hypotension (P<0.001). CONCLUSIONS: Guidelines optimize the syncope management in the ECU and the agreement between the emergency and discharge diagnostic without change of unexplained syncope and. MDS tend to be shorter when guidelines are actively implemented. Nevertheless, the positive impact of guidelines implementation is of limited duration.

RT-PCR Quantitation of HSP60 mRNA Expression.

Heat shock protein 60 (HSP60, HSPD1) is a "chaperonin" that facilitates folding of nascent proteins into proper conformations (1). It is thought to play a critical role in the assembly, folding, and transport of proteins in the mitochondria. HSP60 also interacts with nascent cellular proteins to prevent their denaturation under heat stress (2). The HSP60 gene sequence is known and is highly conserved (3). Expression of the HSP60 gene has been associated with cisplatin resistance in several preclinical model systems and in ovarian carcinoma patients (1-6); we quantitated HSP60 mRNA expression in preclinical human ovarian and bladder carcinoma models.

Expression of melanoma inhibitory activity in melanoma and nonmelanoma tissue specimens.

Melanoma inhibitory activity (MIA) is a small soluble protein secreted by malignant melanoma cells and chondrocytes. Prior studies suggested that MIA expression was relatively tissue-specific, making it a potentially useful marker for melanoma. The current investigations sought to more clearly define the range of tumor/tissue-types where MIA is expressed, compared with expression of 4 other potential melanoma marker genes (tyrosinase melanoma antigen recognized by T cells [MART-1/MelanA], gp100, and melanoma growth-stimulatory activity [MGSA/Gro alpha]). Expression of these genes was assayed by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry in 23 melanoma tumor specimens and in 25 additional nonmelanoma or nonmalignant specimens. MIA, tyrosinase, and MGSA were expressed in most melanoma specimens. Specificity was highest for MART-1, followed by MIA and tyrosinase. Increasing the number of cycles of amplification from 35 to 40 increased sensitivity but decreased specificity of most markers, though MIA was relatively robust. MIA mRNA was also detected in carcinomas of the colon, ovary, kidney, and head/neck, as well as in normal laryngeal epithelium. Although MIA discriminated melanoma from nonmelanoma at least as well as tyrosinase, no single mRNA marker had accuracy greater than 71%, raising potential concern about application of these particular mRNA markers to the minimal disease setting. HUM PATHOL 31:1381-1388.

Relationship between heat shock protein 60 (HSP60) mRNA expression and resistance to platinum analogues in human ovarian and bladder carcinoma cell lines.

Heat shock protein 60 (HSP60) participates in protein assembly, folding and transport. Increased HSP60 mRNA expression is associated with cisplatin resistance in some in vitro models and with shorter survival among ovarian cancer patients. HSP60 mRNA expression was quantitated in three independent model systems by reverse-transcription PCR (A2780 human ovarian carcinoma cell line and sublines selected for cisplatin or oxaliplatin resistance in vitro and also in the UCRU-BL13 human bladder carcinoma cell line and a cisplatin-resistant subline). Increased HSP60 mRNA expression was observed in all resistant sublines (range 2.5-15 fold), correlated with relative resistance to cisplatin and oxaliplatin. No differences in HSP60 gene copy number were apparent in resistant sublines. These data provide further evidence of a strong association between in vitro resistance to platinum compounds and increased HSP60 mRNA expression.

Altered glutathione metabolism in oxaliplatin resistant ovarian carcinoma cells.

Elevation of glutathione (GSH) is commonly observed in cellular resistance to a number of anticancer agents. Most frequently reported change in GSH metabolism that is associated with the elevated GSH levels is increased mRNA expression and activity of gamma-glutamyl cysteine synthetase (gamma GCS), the first enzyme of the GSH biosynthetic pathway. We have isolated sublines of the A2780 ovarian carcinoma cell line (C10 and C25) that are 8- and 12-fold resistant to oxaliplatin by repeatedly exposing the cells to increasing concentrations of the platinum agent. The GSH levels in C10 and C25 cell sublines are 3.1- and 3.8-fold higher than the parent A2780 cell line. The mRNA levels and activities for gamma GCS and that for gamma-glutamyl transpeptidase (gamma GT), the GSH salvage pathway enzyme, were measured in these cells. The mRNA for gamma GT and gamma GCS were measured by RT-PCR, with quantitation of the PCR product by HPLC; mRNA levels are expressed as ratios to beta-actin mRNA, used as an endogenous standard. GSH and gamma GCS activity were measured by HPLC assays and gamma GT activity by a colorimetric assay. The increase in GSH in C10 and C25 was associated with an elevation in gamma GT mRNA (2.5- and 8-fold) and gamma GT activity (2.7- and 2.8-fold). No changes were observed in gamma GCS mRNA levels or activity. The data indicate that alterations in GSH metabolism leading to elevations in cellular GSH in A2780 ovarian carcinoma cells selected for low levels of resistance to oxaliplatin are mediated by gamma GT, the "salvage' pathway, rather than an increase in GSH biosynthesis.

A one step PCR procedure for analysis of tumor specific T lymphocyte responses.

In an effort to develop optimal conditions for analysis of tumor specific T lymphocyte responses, we have studied the effect of changes in the concentration of oligonucleotide primers on the synthesis of TCR cDNAs in one step PCR procedure using Vbeta10 gene subfamily as a model. It was found that synthesis of the TCR cDNAs increases in a roughly linear fashion at primer concentrations between 0.005-0.05 muM. Evaluation of the use of low concentration (0.005 muM) of primers showed these conditions to be adequate for the analysis of TCR Vbeta subfamilies in the spleen of BALB/c mice, but not in the peritoneal exudate cells (PECs), the latter requiring ten-fold higher concentrations of the variable region primers to compensate for the overall low frequency of T lymphocytes in the PECs in comparison to the spleen. Use of these optimal conditions to detect L1210 tumor specific T lymphocyte responses showed that, in the immunized mice, L1210 specific T lymphocyte responses are detectable in the PECs, but not in the spleen cells from these mice. Thus, upon i.p. immunization of DBA/2 mice with irradiated L1210 lymphoma cells, followed by analysis of the PECs by RT/PCR, three TCR Vbeta subfamilies, including Vbeta8.2, Vbeta15 and Vbeta16 were found to contain specific major TCR cDNA bands. The approach described here is very efficient, as it uses a small amount of the 32P isotope (0.5 muCi) followed by direct analysis of the PCR products on a denaturing acrylamide/urea gel. Furthermore, data is also presented that shows that quantitative differences in the levels of individual TCR cDNAs in a particular Vbeta subfamily are preserved during PCR amplification.

Induction of gamma-glutamyl transpeptidase mRNA by platinum complexes in a human ovarian carcinoma cell line.

Elevation of glutathione (GSH) is widely observed in cellular resistance to platinum agents. Our previous studies have shown that sublines of human ovarian carcinoma cell line A2780, which exhibited low levels of resistance to oxaliplatin, showed elevated steady state levels of mRNA and activity of gamma-glutamyl transpeptidase (gamma-GT, EC 2.3.2.2), but not of gamma-glutamylcysteine synthetase (gamma-GCS, EC 6.3.2.2) [El-akawi et al., Cancer Lett. 105:5-14; 1966]. To understand this phenomenon better, we have studied the effect of single exposures of oxaliplatin or cisplatin on the mRNA expression of gamma-GT and gamma-GCS in A2780 cells. The mRNAs of gamma-GT and gamma-GCS were measured by reverse transcriptase PCR, with quantitation of the PCR product by HPLC; mRNA levels are expressed as ratios to beta-actin mRNA, used as an endogenous standard. GSH was measured by HPLC. The gamma-GT activity was measured by a colorimetric assay. Single exposures of cells to oxaliplatin induced a time- and concentration-dependent increase in the mRNA of gamma-GT, but not of gamma-GCS. Cisplatin also induced an elevation in gamma-GT mRNA, but to a lower degree. The gamma-GT enzyme activity increased corresponding to the elevation in mRNA expression. The gamma-GT-induced cells showed an increase in cellular GSH when incubated in medium containing GSH. The data suggest that a) single, brief exposures to pharmacologically relevant concentrations of platinum complexes induce elevation in mRNA of gamma-GT, b) elevation in gamma-GT mRNA translates into elevated gamma-GT activity and increase in GSH salvage, and c) the degree of induction of gamma-GT mRNA differs between platinum complexes.

Allelic differences in the VHOx-1 gene explain the absence of a B cell clonal dominance in the primary response of C57BL/6 mice to phthalate.

A highly conserved Id (CRIXmp-1) associated with the murine (BALB/c) humoral immune response to the hapten phthalate (Xmp) is conspicuously absent in C57BL/6 mice. The absence of this Id in C57BL/6 mice is shown here to be due to the absence of the appropriate VH gene (VHOx-1) usage in the Xmp response. To determine whether the failure to utilize this VH was due to an active suppression or to the lack of the requisite VH gene in the available repertoire, VHOx-1 gene-specific primers were used to amplify the germ-line VHOx-1 gene from genomic DNA from BALB/c and C57BL/6 mice. The germ-line coding sequence of the C57BL/6 allele of the VHOx-1 gene is 99% similar to the germ-line coding sequence of the BALB/c allele. Amplification of cDNA made from splenic RNA from C57BL/6 mice confirmed that this gene is expressed. There are four nucleotide differences that lead to three amino acid changes in the predicted protein sequence. Each change is either in or immediately adjacent to a complementarity-determining region (CDR). Two of these changes are unique to the C57BL/6 allele and are not shared with CRIXmp-1-expressing strains. These two changes are predicted to alter the Xmp binding capabilities of the C57BL/6 allelic form of this VH gene, thereby explaining the absence of the Xmp-1 clonotype, which is dominant in the primary Xmp immune response of most other strains of mice.

Alternatively spliced MHC class I mRNAs show specific deletion of sequences encoding the extracellular polymorphic domains.

Analysis of structural diversity at the 5' end of H-2 class I mRNAs showed that a small fraction of Kd mRNA from L1210 lymphoma (approximately 10%) or from various normal tissue (2-3%) of DBA/2 mice carries a precise deletion of the sequences encoding the second extracellular domain. Nucleotide sequence of the coding region of the second domain-lacking Kd mRNA was found to be identical to the known sequence of the Kd gene from DBA/2 liver with the exception of the above deletion and a single nucleotide silent substitution at position 150, suggesting that the novel Kd RNA is a product of the functional Kd gene. In addition, various normal tissues that are known to express varying levels of Kd antigen did not show changes in the expression levels of the second domain-lacking Kd mRNA thus ruling out the possibility that synthesis of this RNA is coupled to control the expression levels of the canonical Kd mRNA, hence the Kd antigen. Analysis by polymerase chain reaction showed that all normal tissues including the testis and sperm express this RNA. Preliminary analysis of the Kb mRNA from the spleen and thymus of C57BL/6 mouse also showed the presence of second domain-lacking Kb mRNA in these mice. Furthermore, preliminary structural analysis of the Dd and Ld mRNAs has revealed additional polymorphic extracellular domain-lacking mRNA species including a first domain-lacking Dd mRNA and two Ld mRNAs that lack sequences encoding either the first extracellular domain or the second extracellular domain respectively. These results together show that H-2 mRNAs lacking sequences that specify individual extracellular polymorphic domains are a frequent feature of the structural diversity at the 5' ends of these mRNAs. Potential significance of these domain-lacking H-2 mRNAs is discussed, particularly with regard to the function of the putative encoded peptides as targets of natural killer cells.

Tumorigenicity of interleukin-2 (IL-2)-cDNA-transfected L1210 lymphoma and its in vivo variants is modulated by changes in IL-2 expression; potential therapeutic implications.

To study parameters that affect the tumorigenicity of L1210 lymphoma we have analyzed the structure of MHC class I antigens of this tumor. In addition this tumor was transfected with interleukin-2 (IL-2) cDNA in order to determine the effects of high concentrations of IL-2 within the tumor environment. The nucleotide sequence of the class I Kd, Dd and Ld mRNAs from this tumor showed that the encoded amino acid sequence of the corresponding antigens is normal, thus suggesting that the tumorigenicity of L1210 lymphoma is not due to defective antigen presentation to tumor-specific cytotoxic T cells. In contrast, induction of IL-2 expression by cDNA transfection led to loss of tumorigenicity of the IL-2-secreting tumor cells. However, a fraction of long-term-surviving mice developed progressively growing variant tumors that showed substantial decrease or loss of IL-2 expression. These results suggest that IL-2 secretion by tumors is suicidal but, because of tumor heterogeneity, IL-2-loss-variant tumors may arise that are able to escape the immune defenses of the host. The observed consistent loss of IL-2 expression in variant tumors implies that specific targeting of large quantities of IL-2 to tumor cells may be a valuable approach to immunotherapy of cancer. In addition we find that under specific gamma ray irradiation IL-2-secreting tumor cells lose their ability to multiply yet continue to secrete IL-2 at levels equivalent to those secreted by unirradiated cells. Such IL-2-secreting irradiated tumor cells were found to be superior immunogens in comparison to the irradiated parental tumor cells, suggesting their use as tumor vaccines.

Identification of an alternatively spliced Kd and the Qa-6d mRNAs by using amplified cDNA.

We have employed the primer chain reaction method for direct sequencing of H-2 mRNAs. This approach is highly sensitive and permits quantitation and sequencing of the canonical as well as alternatively spliced mRNAs that may be expressed at 5-10% level in comparison to the major H-2 species. Using this technique, we have identified a novel species of alternatively spliced Kd mRNA expressed in L1210 lymphoma and in the spleen and liver of DBA/2 mice. Similarly, we found a previously described alternatively spliced species of H-2Dd mRNA to be expressed in L1210 lymphoma and have determined the sequence of the cytoplasmic domain of Ld mRNA. In addition, we have identified a Class I MHC transcript presumably encoded by a gene allelic to Q6 gene of BALB/c mice.

Selective elimination of idiotype-binding cells in vivo by a drug-idiotype conjugate demonstrates the functional significance of these cells in immune regulation.

A receptor-specific cytotoxic drug delivery system has been used to eliminate idiotype-binding cells in vivo to ascertain the possible functional significance of these cells in regulating the humoral immune response to dextran. Protein M104E, a mouse myeloma protein that binds dextran, expresses a private idiotope that is present on a significant proportion of the normal dextran-specific antibody repertoire. Immunocompetent cells that bind and internalize M104E idiotype-bearing molecules were eliminated by the intravenous administration of a single dose of cytosine arabinonucleoside conjugated to purified M104E protein. The administration of this cytotoxic drug-idiotype conjugate had a profound effect upon the expression of the M104E idiotype in euthymic but not in athymic BALB/c mice following immunization with dextran. In euthymic mice, the depletion of the idiotype-binding cells resulted in a marked elevation in the level of M104E idiotype present in the immune sera. Moreover, treated but not control mice developed idiotype-positive molecules that did not bind dextran. These results demonstrate the functional significance of idiotype-binding cells in the regulation of individual clonotypes during an immune response.

Antigen-specific drug-targeting used to manipulate an immune response in vivo.

The administration of dextran-conjugated cytosine arabinonucleoside (araC) to BALB/c mice at various times prior to but not subsequent to immunization with native dextran renders mice unresponsive to this thymic-independent antigen. These results demonstrate that the primary immune response to an antigen can be selectively and efficiently suppressed or eliminated in vivo by the delivery of a single dose of an appropriate antigen-cytotoxic drug conjugate. Evidence presented here indicates that the dextran-araC conjugate (toxogen) acts directly and selectively upon unprimed dextran-specific antibody-forming cell precursors, presumably by binding to their receptors and subsequent internalization of the resultant receptor-toxogen complexes. The resistance of antigen-primed mice to the cytotoxic effect of the toxogen could result from the failure of dextran-primed cells to reexpress antigen-specific receptors, from an alternative processing of the toxogen, or from the inability of the antigen-primed cells to internalize a second round of receptor-ligand complexes. We also determined that B cells responding to thymic-dependent antigens were not affected by the prior exposure to a toxogen. The inability to eliminate or suppress the primary response to a thymic-dependent antigen via the administration of a cytotoxic drug-antigen conjugate distinguishes the thymic-independent set of B cells from the thymic-dependent B-cell repertoire. The difference between these two B-cell compartments could be due either to differences in the amount of ligand bound to receptors or to differences in the trafficking patterns of receptor-ligand complexes within each cell type.

Antibody response of BALB/c mice to dextran B1355S: alterations in the expression of an idiotype associated with the depletion of idiotype-binding cells.

In this study, we established that BALB/c mice recognize and respond to the idiotype (M104E IdI) of a major dextran-specific clonotype within the BALB/c mouse repertoire. This idiotype recognition is established by demonstrating the presence of idiotype-binding cells and by the production of antibodies specific for the private M104E idiotype. To determine whether or not the idiotype-recognizing cells play a regulatory role during an immune response to dextran, the idiotype-binding cells were selectively removed either by panning or by radiation-induced killing. Two significant effects are observed when the depleted spleen cells are immunized with dextran. First, there is a substantial increase in the proportion of anti-dextran antibodies that are M104E IdI+. The second effect of the idiotype-specific cell depletion is the production of significant amounts of M104E IdI+ immunoglobulin molecules which do not bind dextran. The depletion experiments produced no alteration in the concentration of anti-dextran antibodies found in the serum or in the number of dextran-specific PFC in the spleen. The data indicate that idiotype-reactive cells can play a role in regulating the level of individual clonotype expression (i.e., the M104E clonotype), but that an alternative mechanism must exist for regulating the absolute amount of anti-dextran antibody produced after immunization.