The revised Tokuhashi score; analysis of parameters and assessment of its accuracy in determining survival in patients afflicted with spinal metastasis.

PURPOSE: To determine the significance of each parameter of the revised Tokuhashi score and identify which is associated with survival. BACKGROUND: Spinal metastases are common and can be a challenging medical issue. Treatment options depend on patients' prognosis. Many scoring systems in the literature help estimate prognosis, such as the Tokuhashi, revised Tokuhashi, and Tomita scoring systems. METHODS: A retrospective review of all patients from 2003 to 2012 treated for spinal metastases in one center was conducted. Imaging, pathology, and charts were reviewed to determine the modified Tokuhashi scores. Scores were then compared to the actual documented survival. Univariate and multiple regression analyses were used to assess the importance of each individual parameter and survival time. Linear regression was used to determine the relationship between the Tokuhashi score and weighted Tokuhashi score with survival time. RESULTS: A total of 126 patients were reviewed. All parameters in the revised Tokuhashi score were significantly associated with survival time except for primary site using univariate analysis. Only the number of spinal metastases and metastasis to major organs showed statistical significance when multiple variable analysis was used. CONCLUSION: A number of spinal metastases and metastasis to major organs were the most important predictors of actual survival. Modification to the score based on population characteristics would help better identify patients with spinal metastases that can benefit from surgery.

Soluble human leucocyte antigen-G molecules in peripheral blood haematopoietic stem cell transplantation: a specific role to prevent acute graft-versus-host disease and a link with regulatory T cells.

Haematopoietic stem cell transplantation is often complicated by the life-threatening graft-versus-host disease (GVHD) which consists of an allogeneic reaction of the graft cells against the host organs. The aim of this study was to investigate the putative involvement of soluble human leucocyte antigen (sHLA) class I molecules, and particularly sHLA-G molecules, in the occurrence and/or prevention of acute GVHD (aGVHD) in allogeneic peripheral blood stem cell (PSC) transplantation. Whole sHLA class I molecules seem to be involved in aGVHD pathogenesis because detection of a high concentration of these molecules in the first month post allograft is correlated with aGVHD occurrence. Conversely, a high level of sHLA-G molecules before and after allograft could indicate good prognosis in PSC allograft transplantation. sHLA-G molecules seem to be involved in aGVHD prevention, not only because they are enriched in plasma of patients without aGVHD, but also because: (i) a positive correlation has been found between sHLA-G level and CD4+ CD25+ CD152+ natural regulatory T cell (T(reg)) frequency in the blood of transplanted patients; and (ii) the presence of CD4+ CD25+ CD152+ natural T(reg) is correlated with increased sHLA-G expression in in vitro mixed leucocyte reaction cultures. Altogether, these results support the immunomodulatory function of sHLA-G molecules that might create a regulatory network together with the natural T(reg) to foster the induction of a tolerogenic environment and improve PSC transplantation favourable outcome.

Clinical spectrum of gammadelta+ T cell LGL leukemia: analysis of 20 cases.

We report on the clinico-biological characteristics of 20 cases of gammadelta T cell large granular lymphocyte (LGL) leukemia. All the data were compared to that of 196 cases with alphabeta T cell subtype, which represents the majority of T cell LGL leukemias. Clinical findings were quite similar in the two groups regarding age, sex ratio, recurrent infections, and association with auto-immune diseases especially rheumatoid arthritis. Gammadelta LGL predominantly expressed a CD3+/CD4-/CD8+/CD16+/CD57+ phenotype, in 50% of cases. Clinical outcome was favorable for these patients with overall survival of 85% at 3 years. Fifty percent of gammadelta patients required treatment and the response to therapy was estimated at 55%. gammadelta and alphabeta T cell LGL leukemia harbor a very similar clinico-biological behavior and represent part of an antigen-driven T cell lymphoproliferation.

Elevated levels of soluble non-classical major histocompatibility class I molecule human leucocyte antigen (HLA)-G in the blood of HIV-infected patients with or without visceral leishmaniasis.

The non-classical class I major histocompatibility complex molecules human leucocyte antigen (HLA)-G have been shown to play a role in HIV persistence, but no data are available on the expression of the soluble forms HLA-G5 and sHLA-G1 in HIV-infected patients with and without opportunistic infections. The soluble HLA-G isoform was measured with an enzyme-linked immunosorbent assay (ELISA) method in plasma from 94 subjects: 31 HIV-1-seropositive, 17 with visceral leishmaniasis (VL), seven with both VL and HIV-1 infection and 39 healthy HIV-seronegative subjects. Between groups, the frequency of sHLA-G positivity was statistically different: 81% of HIV-infected patients were positive, as were 57% of HIV-Leishmania infantum co-infected patients, 35% of HIV-seronegative patients with VL and 3% of healthy controls. Levels of the soluble forms of the immunomodulatory molecules HLA-G are elevated during HIV infection. In HIV-Leishmania co-infected patients, sHLA-G secretion could contribute to the tolerogenic environment and to Leishmania immune evasion.

[Flow cytometry: application for the diagnosis and the follow-up of hematological malignancies]. / La cytométrie en flux: intérêt dans le diagnostic phénotypique et le suivi des hémopathies malignes.

Diagnosis of haematological malignancies is based on multiparametric analysis such as morphology, phenotype and genotype studies. Some entities are only defined by one of these approach. Flow-cytometry (FCM) is useful to determined the normal counterpart of the tumoral process and its differentiation status within the involved lineage. Furthermore, FCM is able to detect clonality in B or T proliferations and criteria for malignancies such as abnormal phenotype. Finally it also specifies prognosis criterias. Among the different haematological malignancies, acute lymphoblastic leukaemia (ALL) can be diagnosed using FCM, whereas acute myeloblastic leukaemia diagnosis is only confirmed by this methodology, which could moreover determine prognosis factors. A scoring system (EGIL) determine the normal counterpart of tumoral cells using a panel of different markers. Immunophenotyping is also useful in chronic lymphoproliferative disorders, such as chronic lymphocytic leukaemia (CLL) by using a similar scoring system (so-called Matutes scoring). Since FCM is able to detect simultaneously numerous cell markers it could be more accurate than immunohistochemistry for the diagnosis of follicular lymphoma, mantle cell lymphoma or hairy cell leukaemia. Finally, during treatment follow-up, minimal residual disease characterised by the detection of rare specific events, may be examined using FCM, in some situations.

[Computer navigation in dorsal instrumentation of the cervical spine--an in vitro study]. / Computernavigation bei der dorsalen Instrumentierung der HWS--eine In-vitro-Studie.

Transarticular C1/2 screws are widely used in posterior cervical spine instrumentation. Pedicle screws in the cervical spine remain uncommon until now. In view of improved biomechanical stability compared to lateral mass screws, pedicle screws could be used, especially for patients with poor bone quality or defects in the anterior column. Nevertheless, there are potential risks of iatrogenic damage to the spinal cord, nerve roots, or the vertebral artery related to both techniques of posterior cervical spine instrumentation. Therefore, the aim of this study was to evaluate whether C1/2 transarticular screws as well as transpedicular screws in C3 and C4 can be applied safely and with high accuracy using a computer-assisted surgery (CAS) system. C1/2 transarticular screws as well as transpedicular screws in the cervical spine can be applied safely and with high accuracy using a CAS system in vitro. Therefore, this technique may be used in the clinical setup due to improved accuracy and reduced radiation dose for the patient and medical staff. Nevertheless, to prevent iatrogenic damage, users should be aware of known sources of possible errors that cause inaccuracies. Small pedicles with a diameter below 4.0 mm may not be suitable for pedicle screws.

Interference during the use of an electromagnetic tracking system under OR conditions.

Many computer-assisted surgery applications use electromagnetic tracking devices and several sources of interference may reduce the accuracy of this type of system in clinical situations. This study aims to quantify interference sources in an operating room (OR) and determine if their impact on the tracking system is excessive for applications requiring millimetric accuracy. Electromagnetic noise levels were measured in a controlled environment and compared with measurements in an OR. Errors generated by this noise remained below the 0.15mm RMS level. OR equipment was also brought in proximity to the electromagnetic receivers and the errors generated by the ensuing interference were measured. Ferromagnetic and electrical devices can produce large interference (translation errors up to 8.4mm RMS and rotation up to 166 degrees ). However, these devices can be identified and placed at sufficient distances to decrease the magnitude of their interference. In conclusion, in the absence of significant ferromagnetic or electromagnetic distortion caused by equipment often present in an OR, this electromagnetic tracking system provides valid relative measurements with millimetric accuracy to computer-assisted surgical applications. This distortion can be reduced by maximizing the distances to the interfering OR equipment and integrating noise-reducing algorithms in associated software.

High multidrug resistance protein activity in acute myeloid leukaemias is associated with poor response to chemotherapy and reduced patient survival.

Multidrug resistance protein (MRP) activity was investigated in 44 newly diagnosed acute myeloid leukaemia (AML) patients using a functional assay based on efflux of carboxy-2',7'-dichlorofluorescein, an anionic dye handled by both MRP1 and MRP2. Elevated MRP transport was detected in 29% of cases, but was not significantly correlated with sex, age, white blood cell count at diagnosis or karyotype. In contrast, it was associated with secondary AML (P = 0.002), CD34 positivity (P = 0.041) and P-glycoprotein activity (P = 0.01). There was a lower rate of complete remission in MRP-positive patients versus MRP-negative patients (23% versus 81%; P = 0.001); overall survival was also better for MRP-negative patients (P = 0.004). These data indicate a probable role for MRP activity in the clinical outcome of AML.

Lung macrophages and dendritic cells express HLA-G molecules in pulmonary diseases.

HLA-G is selectively expressed in extravillous trophoblast of human placenta, which does not express classical HLA-A and -B molecules. Several studies report the role of HLA-G as a molecule involved in immune tolerance. By interacting with NK and T cells inhibitory receptors, HLA-G may downregulate their cytotoxicity functions. To appreciate the biologic and clinical relevance of HLA-G expression in lung diseases, HLA class I and HLA-G expression were analyzed in a panel of 36 ex vivo neoplastic tissues and 8 non-neoplastic lung tissues. Immunohistochemical analysis was performed using a pan-HLA class I antibody (W6/32) and three different specific anti-HLA-G antibodies (87G, MEMG/9 and 4H84). These findings demonstrated that HLA-G products were not expressed in pulmonary structural cells. However, HLA-G molecules were detected in activated macrophages and dendritic cells infiltrating lung carcinomas (33%) and nontumoral pulmonary diseases (25%). HLA-G expression was not correlated with classical HLA alterations. No statistical correlation was found between HLA-G expression and clinical or biologic parameters except high tumor size. The expression of HLA-G in myelo-monocytic cells infiltrating lung pathologic tissues could alter antigenic presentation and contribute to decrease immune response efficiency, subsequently favoring the progression of tumoral or inflammatory processes.

Differential expression of the efflux pumps P-glycoprotein and multidrug resistance-associated protein in human monocyte-derived dendritic cells.

P-glycoprotein (P-gp), an ATP-binding cassette (ABC) drug efflux pump, has been recently shown to play an important role in the physiology of Langherans cells, a subtype of dendritic cells (DC) found in the skin. The present study was designed to investigate expression and activity of P-gp and of multidrug resistance-associated protein (MRP), another ABC efflux pump sharing numerous substrates with P-gp, in human monocyte-derived DC. Immunolabeling experiments and dye efflux assays indicated that such cells displayed elevated levels of MRP activity and expression when compared to those present in parental monocytes. Generation of DC from monocytes in the presence of the MRP inhibitor indomethacin did not, however, alter the capacity of DC to stimulate allogeneic T cells proliferation in mixed lymphocyte reaction. In addition, indomethacin did not inhibit the up-regulation of the CD1a, a marker occurring during the differentiation of monocytes into DC. In contrast to that of MRP, functional expression of P-gp was not detected in monocyte-derived DC. Such antigen presenting cells that constitute a promising tool for antitumor vaccinal therapy therefore display differential expression of the efflux pumps P-gp and MRP.

Multidrug resistance protein (MRP) activity in normal mature leukocytes and CD34-positive hematopoietic cells from peripheral blood.

Multidrug resistance proteins (MRPs) such as MRP1, MRP2 and MRP3 are membrane efflux pumps involved in multidrug resistance and handling organic anions. In the present study, MRP activity was investigated in normal mature leucocytes and CD34-positive hematopoietic cells from peripheral blood using the flow cytometric carboxy-2',7'-dichlorofluorescein (CF) efflux assay. Basal and similar cellular exports of CF, an anionic fluorescent dye substrate for MRP1 and MRP2 transporters, were evidenced in lymphocytes whatever their subsets (CD3, CD4, CD8, CD20 and CD56 cells), in CD14 monocytes and in CD15 granulocytes whereas higher CF efflux was found in CD34 cells. Such outwardly-directed transports of CF were inhibited by known blockers of MRP function such as probenecid whereas the P-glycoprotein modulator verapamil did not alter the retention of the dye in the blood leukocytes. Peripheral mature blood leukocytes were moreover found to express MRP1 mRNAs and MRP1 protein as assessed by Northern-blot and Western-blot analyses, whereas MRP2 and MRP3 transcripts were not present or only at very low levels. Mature leukocytes therefore display basal constitutive MRP-related transport activity regardless of cell lineage and likely related to MRP1 expression whereas higher MRP-related efflux can be detected in peripheral CD34 hematopoietic cells.

Computer-assisted surgery in posterior instrumentation of the cervical spine: an in-vitro feasibility study.

Transarticular C1/2 screws are widely used in posterior cervical spine instrumentation. The use of pedicle screws in the cervical spine remains uncommon. Due to superior biomechanical stability compared to lateral mass screws, pedicle screws can be used, especially for patients with poor bone quality or defects in the anterior column. Nevertheless there are potential risks of iatrogenic damage to the spinal cord, nerve roots or the vertebral artery associated with both posterior cervical spine instrumentation techniques. Therefore, the aim of this study was to evaluate whether C1/2 transarticular screws as well as transpedicular screws in C3 and C4 can be applied safely and with high accuracy using a computer-assisted surgery (CAS) system. We used 13 human cadaver C0-C5 spine segments. We installed 1.4-mm Kirschner wires transarticular in C 1/2, using a specially designed guide, and drilled 2.5-mm pedicle holes in C3 and C4 with the assistance of the CAS system. Hole positions were evaluated by palpation, CT and dissection. Forty-eight (92%) of the 52 drilled pedicles were correctly positioned after palpation, imaging and dissection. The vertebral artery was not injured in any specimen. All of the 26 C1/2 Kirschner wires were placed properly after imaging and dissection evaluations. No injury to vascular or bony structures was observed. C /2 transarticular screws as well as transpedicular screws in the cervical spine can be applied safely and with high accuracy using a CAS system in vitro. Therefore, this technique may be used in a clinical setting, as it offers improved accuracy and reduced radiation dose for the patient and the medical staff. Nevertheless, users should take note of known sources of possible faults causing inaccuracies in order to prevent iatrogenic damage. Small pedicles, with a diameter of less than 4.0 mm, may not be suitable for pedicle screws.

Comparative results between conventional and computer-assisted pedicle screw installation in the thoracic, lumbar, and sacral spine.

STUDY DESIGN: A comparative study on the position of pedicle screws in patients treated surgically with and without computer assistance. OBJECTIVES: To evaluate the accuracy of computer-assisted pedicle screw installation, and to evaluate its clinical benefit as compared with conventional pedicle screw installation techniques. SUMMARY OF BACKGROUND DATA: In vitro and clinical studies have documented a significant rate of misplaced screws in the thoracolumbar area. Neurologic complications are recognized problems caused by screw misplacement. METHODS: Patients treated surgically with computer assistance were compared with a historical control group of patients treated surgically with conventional techniques in the same hospital and by the same surgical team. All screw positions were measured with a postoperative magnetic resonance tomography, and cortical effractions were categorized in 2-mm increments. Patients' charts also were reviewed to assess individual neurologic outcomes. RESULTS: The control cohort was composed of 100 patients, with 544 screws from T5 to S1. The computer-assisted cohort was composed of 50 patients, with 294 screws from T2 to S1. In the control cohort, 461 of 544 screws (85%) were found completely within their pedicles as compared with 278 of 294 screws (95%) correctly placed in the computer-assisted group (P < 0.0001). All 16 screws incorrectly placed with computer assistance were found 0.1 mm to 2 mm from the pedicle cortex. In the control cohort, 68 screws were found 0.1 mm to 2 mm, 10 screws 2.1 mm to 4 mm, and 5 screws more than 4 mm from the pedicle cortex. Seven patients in the control cohort were surgically retreated because of postoperative neurologic deficits, whereas no patients in the computer-assisted group were surgically retreated. CONCLUSIONS: Computer assistance can decrease the incidence of incorrectly positioned pedicle screws.

HLA-DR-mediated apoptosis susceptibility discriminates differentiation stages of dendritic/monocytic APC.

Professional APC are characterized by their ability to present peptide via HLA class II in the presence of costimulatory molecules (CD40, CD80, and CD86). The efficiency of Ag presentation can be classed as follows: mature dendritic cells (DC) are most efficient, immature DC and macrophages are intermediate, and monocytes are considered poor APC. There is a large body of evidence demonstrating that HLA-DR transmits signals in the APC. In this study, we have addressed the question of the outcome of HLA-DR signals on APC of the monocyte/DC lineages throughout their differentiation from immature to mature APC. DC were generated from both monocytes and CD34+ cells of the same individual, macrophages were differentiated from monocytes. Immunophenotypical analysis clearly distinguished these populations. HLA-DR-mediated signals led to marked apoptosis in mature DC of either CD34 or monocytic origin. Significantly less apoptosis was observed in immature DC of either origin. Nonetheless, even immature DC were more susceptible to HLA-DR-mediated apoptosis than macrophages, whereas monocytes were resistant to HLA-DR-mediated apoptosis. The mechanism of HLA-DR-mediated apoptosis was independent of caspase activation. Taken together, these data lead to the notion that signals generated via HLA-DR lead to the demise of mature professional APC, thereby providing a means of limiting the immune response.

Modulation of HLA-G antigens expression in myelomonocytic cells.

As trophoblast cells and macrophages share cellular characteristics, we investigated the expression of HLA-G antigens during the myelomonocytic differentiation. Analyses with the 87G and 16G1 monoclonal antibodies demonstrated that HLA-G was not expressed in peripheral blood monocytes, in in vitro differentiated dendritic cells and macrophages, and in resident mononuclear phagocytes infiltrating healthy tissues. Conversely, activated macrophages and dendritic cells localized in tumoral biopsies of some lung carcinomas expressed HLA-G antigens. Induction of HLA-G expression at the cell surface of the monohistiocytic cell line U 937 with different cytokines strongly suggests that cytokines secreted during inflammation may be involved in this specific upregulation. Bronchoalveolar macrophages collected from patients suffering from acute HCMV pneumonitis also expressed HLA-G molecules. In vitro, we thus demonstrated that HLA-G antigens are produced during viral reactivation in the macrophages generated after allogeneic stimulation of HCMV latently infected monocytes. Our data suggest that inflammatory processes in lung tissues, like tumoral transformation and HCMV acute infection, are likely to induce HLA-G molecules in infiltrating macrophages and dendritic cells. The expression of molecules capable of downregulating both the innate and adoptive immunity could be a mechanism that helps tumoral and HCMV infected cells to escape immune response.

HLA-DR inhibits granulocytic differentiation without inducing apoptosis of CD34 cells.

Hematopoietic progenitors express HLA-DR molecules. However the significance of HLA-class II molecules on CD34+ cells remains unknown. The primary role of HLA-class-II molecules is antigen presentation although a second role, that of signal transduction, has been established in B cells. The role of HLA-DR in hematopoiesis was examined by determining the ability of CD34+ progenitor cells to differentiate to "Colony Forming Unit Granulocyte-Macrophage" (CFU-GM) and "Burst Forming Unit Erythrocyte" (BFU-E) in the presence of anti-HLA-DR monoclonal antibody. We observed a reduction in the number of CFU-GM which was due in part to down regulation of granulocyte rather than monocyte differentiation. These observations suggest that HLA-DR signals can regulate myelopoiesis. We point out especially the role of the HLA-DR molecule in the switch of CFU-GM between granulocyte or monocyte lineages. Although HLA-DR mediated apoptosis has been described in mature B lymphocytes apoptosis of CD34+ cells was excluded as a mechanism.

Differential expression of major histocompatibility complex class Ia, Ib, and II molecules on monocytes-derived dendritic and macrophagic cells.

Blood monocyte derived antigen presenting cells (APC) such as dendritic cells and macrophages are considered as major promising tools for antitumoral immunotherapy. In order to contribute to their phenotype characterization, we have precisely investigated their levels of expression of MHC class Ia, Ib (HLA-G) and II molecules using mainly flow cytometry quantification assays. APC were generated from monocytes cultured for 7 days in the presence of GM-CSF and IL-4 or M-CSF. These cells, which exhibited known morphological and immunological features of dendritic cells and macrophages respectively, were evidenced to display high expression of MHC class Ia and class II antigens in comparison to that found in monocytes. Dendritic cells and macrophages thus expressed 2-fold more and 4-fold more MHC class Ia molecules and 5-fold and 3-fold more MHC class II DR molecules than parental monocytes. In addition, expression of MHC class II DP and DQ molecules, not or only barely detected in monocytes, was clearly demonstrated in the two kinds of APC. In contrast, monocytes, dendritic cells and macrophages failed to express MHC class Ib HLA-G antigen. The up-regulation in monocyte-derived APC of MHC class Ia and II molecules mediating the presentation of antigen peptides to lymphocytes fully supports the interest of such APC in antitumoral immunotherapy.

Use of the anionic dye carboxy-2',7'-dichlorofluorescein for sensitive flow cytometric detection of multidrug resistance-associated protein activity.

Multidrug resistance-associated protein (MRP) and P-glycoprotein are drug efflux pumps conferring multidrug resistance to tumor cells and sharing numerous substrates. In order to determine a flow cytometric assay allowing to analyse MRP activity in cancerous cells in a sensitive and specific manner, cellular accumulation and efflux of the anionic fluorescent dye carboxy-2',7'-dichlorofluorescein (CDF) were studied by flow cytometry using mainly MRP-overexpressing lung GLC4/Sb30 cells and parental GLC4 cells. GLC4/Sb30 cells were found to display reduced accumulation and enhanced efflux of the dye when compared to their parental counterparts. Probenecid, a well known blocker of MRP, strongly enhanced CDF accumulation in GLC4/Sb30 cells through inhibiting efflux of the dye; it also increased CDF levels in GLC4 cells, although to a lesser extent, which may likely be linked to the low, but detectable, expression of MRP in these cells. Comparison of CDF retention with that of calcein demonstrated that the former dye was the most efficiently effluxed by GLC4/Sb30 cells. In contrast to MRP overexpression, that of P-glycoprotein was not found to alter cellular CDF labelling whereas it strongly impaired calcein staining. These results indicate that CDF is a substrate for MRP, but not for P-gp, which may likely be useful for sensitive and specific flow cytometric determination of MRP activity in clinical samples.

HLA-G protein expression is not induced during malignant transformation.

To evaluate the biological relevance of HLA-G expression during tumoral transformation, we analyzed its expression in different malignant cells and immune effector cells infiltrating solid tumors. Our analysis of 33 tumor cell lines and 53 tumoral biopsies demonstrated that: i) six tumor cell lines display HLA-G transcription with differential alternative splicing patterns and only Jeg3 choriocarcinoma and MCF-7 breast adenocarcinoma cell lines express HLA-G translated products; and ii) HLA-G antigens are not expressed in malignantly transformed cells derived from lung (n=18), liver (n=5), colon (n=5), breast (n=10), kidney (n=5), ovary (n=5), and larynx (n=5) tissues ex vivo. The healthy tissues surrounding these tumor tissues do not express HLA-G molecules either. On the other hand, surprisingly, HLA-G products were detected in activated macrophages and dendritic cells localized in tumoral biopsies of 5 out of 18 different lung carcinomas. No HLA-G labelling was observed in resident mononuclear phagocytes of surrounding healthy tissues. Our observations clearly demonstrate that HLA-G is not a marker of malignant cells but appears as a gene expressed in tumor-associated macrophages and dendritic cells, preferentially in those recruited in lung carcinomas. Our findings suggest that specific environmental factors around lung tumors could be involved in the induction of HLA-G protein expression.