Chemokines, chemokine receptors and adhesion molecules on different human endothelia: discriminating the tissue-specific functions that affect leucocyte migration.

The selective accumulation of different leucocyte populations during inflammation is regulated by adhesion molecules and chemokines expressed by vascular endothelium. This study examined how chemokine production and the expression of adhesion molecules and chemokine receptors vary between endothelia from different vascular beds. Human saphenous vein endothelium was compared with lung and dermal microvascular endothelia and with umbilical vein endothelium and a bone-marrow endothelial cell line. All endothelia produced CCL2 and CXCL8 constitutively, whereas CXCL10 and CCL5 were only secreted after tumour necrosis factor (TNF)-alpha or interferon (IFN)-gamma stimulation. In combination with TNF-alpha, IFN-gamma suppressed CXCL8 but enhanced CCL5 and CXCL10, whereas transforming growth factor (TGF)-beta reduced secretion of all chemokines. Basal chemokine secretion was higher from umbilical vein than other endothelial cells. Chemokine receptors, CXCR1, CXCR3 and CCR3, were present on all endothelia but highest on saphenous vein. CCR4, CCR5, CCR6, CXCR2, CXCR4 and CXCR5 were also detected at variable levels on different endothelia. The variation between endothelia in chemokine secretion was much greater than the variations in adhesion molecules, both on resting cells and following cytokine stimulation. These results indicate that it is the tissue-specific variations in endothelial chemokine secretion rather than variations in adhesion molecules that can explain the different patterns of inflammation and leucocyte traffic seen in non-lymphoid tissues.

Brain engraftment of autologous macrophages transduced with a lentiviral flap vector: an approach to complement brain dysfunctions.

Transplantation of ex vivo gene-corrected autologous cells represents an attractive therapeutic approach for brain diseases. Among the cells of the central nervous system, brain macrophages are promising candidates due to their role in tissue homeostasis and their implication in several neurological diseases. Up to now, gene transfer into macrophages has proven difficult by most currently available gene delivery methods. We describe herein, an efficient transduction of rat bone marrow-derived and brain macrophages with an HIV-1-derived vector containing a central DNA flap and encoding the GFP reporter gene (TRIP-DeltaU3-GFP). In primary cultures of macrophages our results show that more than 90% of the cells were transduced by the TRIP vector and that GFP expression remained stable for 1 month without cytopathic effect. In vivo, transplants of transduced macrophages into the striatum of adult rats exhibited long-term expression of GFP up to 3 months. Transduced macrophages were observed around the brain injection site and exhibited the brain macrophage/microglia phenotype. There was no significant sign of astrogliosis around the graft. These results confirm the potential of lentiviral vectors for efficient and stable ex vivo transduction of macrophages. Moreover, transduced autologous macrophages appear as a valuable vehicle for long-term and localized gene expression into the brain.

Expression and detection of macrophage-tropic HIV-1 gp120 in the brain using conformation-dependent antibodies.

HIV-1 envelope proteins gp120 and gp41 are likely to play a role in the pathogenesis of HIV-associated neurocognitive disorders. While detection of gp120 in HIV-infected cell cultures is easy, it has not yet been possible to identify gp120 in human or animal brains in situ. The difficulty in detecting gp120 could be due to low expression levels of the protein, to the shedding of gp120 from infected macrophages/microglia, or to the use of inappropriate gp-specific antibodies. We addressed these questions by analyzing the subcellular localization, oligomeric structure, and shedding behavior of gp120 from a macrophage-tropic, CCR5-dependent primary isolate, BX08, expressed by a Semliki Forest virus replicon (SFVenvBX08) in vitro. We used the same SFV system injected in vivo into the rat brain in an attempt to detect gp120 in situ. Our results show that gp120/41 is expressed as monomers, dimers, and trimers in cell culture. Immunocytochemical analysis revealed that intracytoplasmic gp120 can be recognized by an anti-V3 antibody, whereas gp120 at the plasma membrane is detected exclusively by a conformation-dependent antibody. In the rat brain, the SFV vector allows gene expression in neurons from day 3 to day 9 after injection without any apparent brain damage nor reactive astrogliosis. In SFVenvBX08-infected neurons only conformation-dependent antibodies allowed gp120 labeling. These results suggest that previous difficulties in detecting gp120 in brain tissues may be due to the use of antibodies which were unable to recognize gp120 at the plasma membrane.

Multifactorial drug-resistance phenomenon in acute leukemias: impact of P170-MDR1, LRP56 protein, glutathione-transferases and metallothionein systems on clinical outcome.

The multidrug resistance phenomenon can be observed in cases which do not express the P170 protein and these cases are suspected as having activated different resistance phenomena. Four phenomena were studied at the time of diagnosis in a series of 35 lymphoblastic and 25 myeloblastic acute (de novo) leukemias, by an immunocytochemical method. Two energetic drug transport processes were investigated: the classical MDR/P170 and the P110/LRP56 proteins, and two physiological detoxifying activities such as the glutathione transferases (GST alpha, mu, pi) and the metallothioneins (Mts). The results demonstrate that these phenomena are independent but their synergic activity can increase their impact on the outcome. P110/LRP56 positive cases demonstrated 48.8% complete remission (CR) rate compared to 71.4% for negative tests. When P170 and P110 were both positive or negative, the CR rates were 27.3% and 81.8% respectively (p = 0.0120), and survival curves were also different (p = 0.030). The CR rate in AML or ALL is weakly affected by GST pi, alpha or mu but relapses are more frequently observed for Positive-GST pi ALL (p = 0.0658). Patients with both P170 and GST pi positive reactions had a 53.3% CR rate compared to 78.9% for both negative reactions. Survival curves for these two groups were different. The CR rate in AMl was 100% for Mts positive and 43.7% for negative cases (p = 0.050), however the median survival was totally different for these two groups (p = 0.046). CR rates were 26.6% for patients who were P170 positive and Mts negative compared to 100% for P170 negative and Mts positive (p = 0.038) patients. Survival curves were also different (p = 0.0510). We conclude that these four mechanisms induce an independent drug resistance but their synergic action increase their impact on the outcome. The metallothioneins seem to have a major impact on the drug resistance phenomenon and its effect should be investigated with high priority, in the light of these results.

P-glycoprotein (P-170) and CD34 expression in adult acute myeloid leukemia (AML).

We investigated the prognosis value of CD34 and P-170 expression in blast cells of adult patients affected by de novo acute myeloid leukemia (AML). CD34 antigen was analyzed by indirect immunofluorescence (IFI) and alkaline phosphatase-labeled streptavidin biotine (AP-LSAB) in 62 patients (median age: 51 years). P-170 expression was determined by AP-LSAB in 51 cases using JSB1 and C219 monoclonal antibodies. All patients were treated with conventional chemotherapy induction regimen. Follow-up was from 6 to 79 months. Complete remission (CR) rate was not statistically different between CD34+ and CD34- patients (67 vs. 84%, p = 0.2). The duration of CR and survival were not influenced by CD34 expression. Karyotype abnormalities were more frequent among MDR+ patients (65 vs. 21%, p < 0.01). CR rate was statistically lower in MDR+ patients as compared to MDR- patients (63 vs. 96%, p = 0.01). Median disease-free survival (DFS) was shorter for MDR+ patients but the difference was not significant (5 vs. 10 months, p = 0.09). Patients who were positive for both parameters CD34 and P-170, had a poor prognosis with a 50 vs. 100% CR rate for CD34/P-170 negative patients, (p = 0.002), a lower median DFS (3 vs. 12 months, p = 0.01) and overall survival (OS) (3 vs. 14.5 months, p = 0.01). Results of cytogenetic analysis did not influence CR rate but the relapse rate was higher, although not significant, for the patients with unfavorable karyotype (63 vs. 33%). The seven CD34+/MDR+ patients with poor prognosis karyotype had a statistically lower CR rate, median DFS and OS than the 7 CD34-/MDR- patients with normal or favorable karyotype (CR: 29% vs. 100%, p = 0.02), (DFS: 3 vs. > 12 months, p = 0.01), (OS: 4 vs. > 12 months, p = 0.02). Our data indicate that P-170 but not CD34 expression is predictive for a lower CR rate. The identification of a bad prognosis subgroup of CD34+/MDR+ AML patients (and especially those with poor prognosis karyotype) has to be confirmed on larger series using uniform methodology.