Single-cell clones of liver cancer stem cells have the potential of differentiating into different types of tumor cells.

Cancer stem cells (CSCs) are believed to be a promising target for cancer therapy because these cells are responsible for tumor development, maintenance and chemotherapy resistance. Finding out the critical factors regulating CSC fate is the key for target therapy of CSCs. Just as normal stem cells are regulated by their microenvironment (niche), CSCs are also regulated by cells in the tumor microenvironment. However, whether various tumor microenvironments can induce CSCs to differentiate into different cancer cells is not clear. Here, we show that single-cell-cloned CSCs, accidentally obtained from a human liver cancer microvascular endothelial cells, express classic stem cell markers, genes associated with self-renewal and pluripotent factors and possess colony-forming ability in vitro and the ability of serial transplantation in vivo. The single-cell-cloned CSCs treated with the different tumor cell/tissue-derived conditioned culture medium, which is a mimic of carcinoma microenvironment, could differentiate into corresponding tumor cells and express specific markers of the respective type of tumor cells at the gene, protein and cell levels, respectively. Interestingly, this multilineage differentiation potential of single-cell-cloned liver CSCs sharply declined after the specific knockdown of octamer-binding transcription factor 4 (Oct4) alone, even though they were under the same induction conditions (carcinoma microenvironments). These data support the hypothesis that single-cell-cloned liver CSCs have the potential of differentiating into different types of tumor cells, and the tumor microenvironment does play a crucial role in deciding differentiation directions. Simultaneously, Oct4 in CSCs is indispensable in this process. These factors are promising targets for liver CSC-specific therapy.

Microparticles and their emerging role in cancer multidrug resistance.

Drug resistance is a major obstacle to the successful treatment of cancer as tumor cells either fail to reduce in size following chemotherapy or the cancer recurs after an initial response. The phenomenon of multidrug resistance (MDR) is particularly problematic as it involves the simultaneous resistance to numerous chemotherapeutics of different classes. MDR is predominantly attributed to the overexpression of efflux transporters such as P-glycoprotein (P-gp) and the Multidrug Resistance-Associated Protein 1 (MRP1). P-gp and MRP1 are members of the ATP Binding Cassette (ABC) superfamily of transporters and are capable of effluxing many chemotherapeutics out of cancer cells, allowing them to survive the toxic insult. Numerous strategies have been developed over the years to circumvent MDR. Of these, the discovery and implementation of P-gp and MRP1 inhibitors have been most extensively studied. However, these inhibitors have not been able to be used clinically. While research continues in this area, it must also be acknowledged that other avenues must be explored. Recently, the novel 'non-genetic' acquisition of P-gp-mediated MDR by microparticles (MPs) has been reported. MPs are vesicles 0.1-1µm in diameter that are released via plasma membrane blebbing. They are important mediators of inflammation, coagulation and vascular homeostasis. In addition to surface P-gp protein, MPs also carry various nucleic acid species as cargo. This 'non-genetic' intercellular transfer provides an alternative pathway for the cellular acquisition and dissemination of traits and implicates MPs as important mediators in the spread of MDR and provides a novel pathway for the circumvention of MDR.

Membrane microparticles mediate transfer of P-glycoprotein to drug sensitive cancer cells.

Multidrug resistance (MDR), a significant impediment to the successful treatment of cancer clinically, has been attributed to the overexpression of P-glycoprotein (P-gp), a plasma membrane multidrug efflux transporter. P-gp maintains sublethal intracellular drug concentrations by virtue of its drug efflux capacity. The cellular regulation of P-gp expression is currently known to occur at either pre- or post-transcriptional levels. In this study, we identify a 'non-genetic' mechanism whereby microparticles (MPs) serve as vectors in the acquisition and spread of MDR. MPs isolated from drug-resistant cancer cells (VLB(100)) were co-cultured with drug sensitive cells (CCRF-CEM) over a 4 h period to allow for MP binding and P-gp transfer. Presence of P-gp on MPs was established using flow cytometry (FCM) and western blotting. Whole-cell drug accumulation assays using rhodamine 123 and daunorubicin (DNR) were carried out to validate the transfer of functional P-gp after co-culture. We establish that MPs shed in vitro from drug-resistant cancer cells incorporate cell surface P-gp from their donor cells, effectively bind to drug-sensitive recipient cells and transfer functional P-gp to the latter. These findings serve to substantially advance our understanding of the molecular basis for the emergence of MDR in cancer clinically and lead to new treatment strategies which target and inhibit MP mediated transfer of P-gp during the course of treatment.

Pathogenesis of cerebral malaria: recent experimental data and possible applications for humans.

Malaria still is a major public health problem, partly because the pathogenesis of its major complication, cerebral malaria, remains incompletely understood. Experimental models represent useful tools to better understand the mechanisms of this syndrome. Here, data generated by several models are reviewed both in vivo and in vitro; we propose that some pathogenic mechanisms, drawn from data obtained from experiments in a mouse model, may be instrumental in humans. In particular, tumor necrosis factor (TNF) receptor 2 is involved in this syndrome, implying that the transmembrane form of TNF may be more important than the soluble form of the cytokine. It has also been shown that in addition to differences in immune responsiveness between genetically resistant and susceptible mice, there are marked differences at the level of the target cell of the lesion, namely, the brain endothelial cell. In murine cerebral malaria, a paradoxical role of platelets has been proposed. Indeed, platelets appear to be pathogenic rather than protective in inflammatory conditions because they can potentiate the deleterious effects of TNF. More recently, it has been shown that interactions among platelets, leukocytes, and endothelial cells have phenotypic and functional consequences for the endothelial cells. A better understanding of these complex interactions leading to vascular injury will help improve the outcome of cerebral malaria.

Diagnostic value of procalcitonin, interleukin-6, and interleukin-8 in critically ill patients admitted with suspected sepsis.

To assess the diagnostic value of procalcitonin (PCT), interleukin (IL)-6, IL-8, and standard measurements in identifying critically ill patients with sepsis, we performed prospective measurements in 78 consecutive patients admitted with acute systemic inflammatory response syndrome (SIRS) and suspected infection. We estimated the relevance of the different parameters by using multivariable regression modeling, likelihood-ratio tests, and area under the receiver operating characteristic curves (AUC). The final diagnosis was SIRS in 18 patients, sepsis in 14, severe sepsis in 21, and septic shock in 25. PCT yielded the highest discriminative value, with an AUC of 0.92 (CI, 0.85 to 1.0), followed by IL-6 (0.75; CI, 0.63 to 0.87), and IL-8 (0.71; CI, 0.59 to 0.83; p < 0.001). At a cutoff of 1.1 ng/ml, PCT yielded a sensitivity of 97% and a specificity of 78% to differentiate patients with SIRS from those with sepsis-related conditions. Median PCT concentrations on admission (ng/ ml, range) were 0.6 (0 to 5.3) for SIRS; 3.5 (0.4 to 6.7) for sepsis; 6.2 (2.2 to 85) for severe sepsis; and 21.3 (1.2 to 654) for septic shock (p < 0.001). The addition of PCT to a model based solely on standard indicators improved the predictive power of detecting sepsis (likelihood ratio test; p = 0.001) and increased the AUC value for the routine value-based model from 0.77 (CI, 0.64 to 0.89) to 0.94 (CI, 0.89 to 0.99; p = 0.002). In contrast, no additive effect was seen for IL-6 (p = 0.56) or IL-8 (p = 0.14). Elevated PCT concentrations appear to be a promising indicator of sepsis in newly admitted, critically ill patients capable of complementing clinical signs and routine laboratory parameters suggestive of severe infection.

Lectin-deficient TNF mutants display comparable anti-tumour but reduced pro-metastatic potential as compared to the wild-type molecule.

In this study, we characterised the anti-tumour as well as the pro-metastatic activities of TNF mutants deficient in their lectin-like activity.1619 We report that, despite reduced systemic toxicity as compared to wild-type (wt) mTNF, a (T104A) and a (T104A-E106A-E109A) mTNF mutant (triple mTNF) retained most of their necrotic and tumouristatic activities, as measured in a CFS-1 fibrosarcoma and a B16BL6 melanoma tumour model, respectively. These mutants also conserved their anti-angiogenic activity, as measured in an in vitro endothelial morphogenesis assay.26 In contrast, the pro-metastatic activity of the T104A and the triple mTNF mutants in the CFS-1 fibrosarcoma and the 3LL-R Lewis lung carcinoma tumour model was significantly lower than that of the wt molecule. These results thus indicate that the lectin-like domain of TNF is not implicated in its necrotic, tumouristatic and anti-angiogenic activities, but that it can contribute to the pro-metastatic effect of the cytokine. In conclusion, in view of their reduced systemic toxicity and pro-metastatic capacity, but their retained anti-tumour activities, lectin-deficient TNF mutants might prove to be therapeutically interesting alternatives to wt TNF.

Delayed mortality and attenuated thrombocytopenia associated with severe malaria in urokinase- and urokinase receptor-deficient mice.

We explored the role of urokinase and tissue-type plasminogen activators (uPA and tPA), as well as the uPA receptor (uPAR; CD87) in mouse severe malaria (SM), using genetically deficient (-/-) mice. The mortality resulting from Plasmodium berghei ANKA infection was delayed in uPA(-/-) and uPAR(-/-) mice but was similar to that of the wild type (+/+) in tPA(-/-) mice. Parasitemia levels were similar in uPA(-/-), uPAR(-/-), and +/+ mice. Production of tumor necrosis factor, as judged from the plasma level and the mRNA levels in brain and lung, was markedly increased by infection in both +/+ and uPAR(-/-) mice. Breakdown of the blood-brain barrier, as evidenced by the leakage of Evans Blue, was similar in +/+ and uPAR(-/-) mice. SM was associated with a profound thrombocytopenia, which was attenuated in uPA(-/-) and uPAR(-/-) mice. Administration of aprotinin, a plasmin antagonist, also delayed mortality and attenuated thrombocytopenia. Platelet trapping in cerebral venules or alveolar capillaries was evident in +/+ mice but absent in uPAR(-/-) mice. In contrast, macrophage sequestration in cerebral venules or alveolar capillaries was evident in both +/+ and uPAR(-/-) mice. Polymorphonuclear leukocyte sequestration in alveolar capillaries was similar in +/+ and uPAR(-/-) mice. These results demonstrate that the uPAR deficiency attenuates the severity of SM, probably by its important role in platelet kinetics and trapping. These results therefore suggest that platelet sequestration contributes to the pathogenesis of SM.

Differences in levels of soluble E-selectin and VCAM-1 in malignant versus non malignant Mediterranean spotted fever.

In the present study, we investigated the plasma levels of soluble adhesion molecules E-selectin, P-selectin, intercellular adhesion molecule- (ICAM- ) and vascular cell adhesion molecule-1(VCAM-1) in 24 patients with Mediterranean spotted fever (MSF), 6 of whom with a malignant form. Measurements were performed on blood samples collected before treatment (T1), then twice during treatment (T2 and T3). Before treatment, MSF patients taken as a whole presented elevated levels of sICAM-1 and sVCAM-1 and normal levels of sE-selectin and sP-selectin compared to healthy controls. We found that sICAM-1 was elevated both in mild and malignant MSF. sE-selectin and sVCAM-1 were elevated only in patients with the malignant form and allowed to discriminate the two clinical subgroups. Their levels decreased after treatment with sE-selectin reaching control values at T2 whereas sVCAM-1 remained higher over the course of the malignant form. In patients with mild MSF, sP-selectin steadily increased after treatment, whereas it did not present any modification at any of the two sampling times in patients with the malignant form. Raised plasma levels of sE-selectin and sVCAM-1 reflect endothelial activation in malignant rickettsial disease and may be sufficiently early markers to influence the therapeutic decision.

The lectin-like domain of tumor necrosis factor-alpha increases membrane conductance in microvascular endothelial cells and peritoneal macrophages.

Herein, we show that TNF exerts a pH-dependent increase in membrane conductance in primary lung microvascular endothelial cells and peritoneal macrophages. This effect was TNF receptor-independent, since it also occurred in cells isolated from mice deficient in both types of TNF receptors. A TNF mutant in which the three amino acids critical for the lectin-like activity were replaced by an alanine did not show any significant effect on membrane conductance. Moreover, a synthetic 17-amino acid peptide of TNF, which was previously shown to exert lectin-like activity, also increased the ion permeability in these cells. The amiloride sensitivity of the observed activity suggests a binding of TNF to an endogenous ion channel rather than channel formation by TNF itself. This may have important implications in mechanisms of TNF-mediated vascular pathology.

Interferon-beta inhibits activated leukocyte migration through human brain microvascular endothelial cell monolayer.

Perivascular leukocyte infiltration into the central nervous system is characteristic of multiple sclerosis (MS) pathology. Interferon-beta (IFN-beta) has shown efficacy in the treatment of patients with MS, but the relevant mechanisms remain incompletely understood. In this study the effects of IFN-beta on leukocyte transendothelial migration were investigated using cells relevant to MS pathogenesis, namely human brain microvascular endothelial cells (HB-MVEC). Activated, but not resting leukocytes exhibited a high transendothelial migration capacity. HB-MVEC prestimulated with tumor necrosis factor (TNF) and IFN-gamma significantly promoted leukocyte transendothelial migration. IFN-beta inhibited the activated leukocyte transendothelial migration on TNF/IFN-gamma-activated HB-MVEC in a dose-dependent manner. A matrix metalloproteinase (MMP) inhibitor and monoclonal antibodies to lymphocyte function antigen-1 (LFA-1) or intercellular adhesion molecule-1 (ICAM-1), but not to very late antigen-4 or to vascular cell adhesion molecule-1 significantly inhibited the transendothelial migration of stimulated leukocytes, suggesting that this phenomenon involves the LFA-1/ICAM-1 interaction and MMP. However IFN-beta did not interfere with the binding of leukocytes to HB-MVEC unless IFN-beta was preincubated with leukocytes or added to HB-MVEC at the time of stimulation. Furthermore IFN-beta did not modulate the expression of adhesion molecules on either stimulated leukocytes or activated HB-MVEC, but partially reduced TNF and interleukin-1 production from stimulated leukocytes during coculture with HB-MVEC. Interestingly, in the presence of IFN-beta, a significant down-regulation of MMP-9 release from stimulated leukocytes was found, especially for the activated form of MMP-9. These results indicate that inhibition of leukocyte transendothelial migration is an important mechanism accounting for the beneficial effects of IFN-beta in the treatment MS patients.

In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant.

Microparticles (MPs) resulting from vesiculation of platelets and other blood cells have been extensively documented in vitro and have been found in increased numbers in several vascular diseases, but little is known about MPs of endothelial origin. The aim of this study was to analyze morphological, immunological, and functional characteristics of MPs derived from human umbilical vein endothelial cells (HUVECs) stimulated by TNF, and to investigate whether these MPs are detectable in healthy individuals and in patients with a prothrombotic coagulation abnormality. Electron microscopy evidenced bleb formation on the membrane of TNF-stimulated HUVECs, leading to increased numbers of MPs released in the supernatant. These endothelial microparticles (EMPs) expressed the same antigenic determinants as the corresponding cell surface, both in resting and activated conditions. MPs derived from TNF-stimulated cells induced coagulation in vitro, via a tissue factor/factor VII-dependent pathway. The expression of E-selectin, ICAM-1, alphavbeta3, and PECAM-1 suggests that MPs have an adhesion potential in addition to their procoagulant activity. In patients, labeling with alphavbeta3 was selected to discriminate EMPs from those of other origins. We provide evidence that endothelial-derived MPs are detectable in normal human blood and are increased in patients with a coagulation abnormality characterized by the presence of lupus anticoagulant. Thus, MPs can be induced by TNF in vitro, and may participate in vivo in the dissemination of proadhesive and procoagulant activities in thrombotic disorders.

Acute systemic reaction and lung alterations induced by an antiplatelet integrin gpIIb/IIIa antibody in mice.

Shock is frequently accompanied by thrombocytopenia. To investigate the pathogenic role of platelets in shock, we examined the in vivo effects of monoclonal antibodies (MoAbs) against mouse platelet membrane proteins. Injection of the platelet-specific MoAb MWReg30 to the fibrinogen receptor (gpIIb/IIIa) rendered mice severely hypothermic within minutes. Isotype-matched control antibodies, even if they also recognized platelet surface antigens, did not induce comparable signs. MWReg30 induced early signs of acute lung injury with increased cellularity in the lung interstitium and rapid engorgement of alveolar septal vessels. Despite this in vivo activity, MWReg30 inhibited rather than stimulated platelet aggregation in vitro. MWReg30-binding to platelets led to phosphorylation of gpIIIa, but did not induce morphological signs of platelet activation. The MWReg30-induced reaction was abolished after treatment with MoAbs 2.4G2 to FcgammaRII/III and was absent in FcgammaRIII-deficient mice, clearly demonstrating the requirement for FcgammaRIII on involved leukocytes. Simultaneous administration of tumor necrosis factor exacerbated, whereas a tolerizing regimen of tumor necrosis factor or bacterial lipopolysaccharide completely prevented the reaction. These data suggest that platelet surface-deposited MWReg30-immune complexes lead to an acute Fc-mediated reaction with pulmonary congestion and life-threatening potential that could serve as an in vivo model of acute lung injury.

Differential reactivity of brain microvascular endothelial cells to TNF reflects the genetic susceptibility to cerebral malaria.

Upon infection with Plasmodium berghei ANKA (PbA), various inbred strains of mice exhibit different susceptibility to the development of cerebral malaria (CM). Tumor necrosis factor-alpha (TNF) and interferon-gamma (IFN-gamma) have been shown to be crucial mediators in the pathogenesis of this neurovascular complication. Brain microvascular endothelial cells (MVEC) represent an important target of both cytokines. In the present study, we show that brain MVEC purified from CM-susceptible (CM-S) CBA/J mice and CM-resistant (CM-R) BALB/c mice exhibit a different sensitivity to TNF. CBA/J brain MVEC displayed a higher capacity to produce IL-6 and to up-regulate intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in response to TNF than BALB/c brain MVEC. In contrast, no difference was found in the induction of E-selectin after TNF challenge. CM-S brain MVEC were also significantly more sensitive to TNF-induced lysis. This differential reactivity to TNF was further substantiated by comparing TNF receptor expression on CM-S and CM-R brain MVEC. Although the constitutive expression of TNF receptors was comparable on cells from the two origins, TNF induced an up-regulation of both p55 and p75 TNF receptors in CM-S, but not in CM-R brain MVEC. A similar regulation was found at the level of TNF receptor mRNA, but not for receptor shedding. Although a protein kinase C inhibitor blocked the response to TNF in both the brain MVEC, an inhibitor of protein kinase A selectively abolished the response to TNF in CM-R, but not CM-S brain MVEC, suggesting a differential protein kinase involvement in TNF-induced activation of CM-S and CM-R brain MVEC. These results indicate that brain MVEC purified from CM-S and CM-R mice exhibit distinctive sensitivity to TNF This difference may be partly due to a differential regulation of TNF receptors and via distinct protein kinase pathways.

Both TNF receptors are required for direct TNF-mediated cytotoxicity in microvascular endothelial cells.

The conditions under which tumor necrosis factor-alpha (TNF) induces apoptosis in primary microvascular endothelial cells (MVEC) were investigated. In the absence of sensitizing agents, TNF induced apoptosis after 3 days of incubation in confluent MVEC. In contrast, upon addition of the transcriptional inhibitor actinomycin D (Act. D), confluence was no longer required and apoptosis occurred already after 16 h. To assess the role of either TNF receptor (TNFR) type in apoptosis, MVEC isolated from mice genetically deficient in TNFR1 (Tnfr1o mice) or TNFR2 (Tnfr2o mice) were incubated with TNF in the presence or absence of Act. D. Under sensitized conditions, Tnfr2o MVEC were lysed like controls, whereas Tnfr1o MVEC were completely resistant, indicating an exclusive role for TNFR1. In contrast, in the absence of Act. D, confluent monolayers of wild-type cells were lysed by TNF, but both Tnfr1o and Tnfr2o MVEC were resistant to TNF-mediated toxicity, indicating a requirement for both TNFR types. Overexpression of the anti-apoptotic protein bcl-xL in MVEC led to a protection against the direct, but not the sensitized cytotoxicity of TNF. In conclusion, in pathophysiologically relevant conditions, both TNFR appear to be required for TNF-induced apoptosis in MVEC.

Multiple forms of angiostatin induce apoptosis in endothelial cells.

Angiostatin is a circulating inhibitor of angiogenesis generated by proteolytic cleavage of plasminogen. In this study we have used recombinant human and murine angiostatins (kringles 1-4) as well as native human angiostatin (prepared by elastase digestion of plasminogen [kringles 1-3] or by plasmin autocatalysis in the presence of a free sulfhydryl donor [kringles 1-4]). We report that angiostatin reduces endothelial cell number in a 4-day proliferation assay without affecting cell cycle progression into S-phase (as determined by bromodeoxyuridine labeling). This suggested that the reduction in cell number in the proliferation assay might in part be due to cytotoxicity. This was confirmed by the observation that ethidium homodimer incorporation (a measure of plasma membrane integrity) into endothelial cells was increased by angiostatin in a manner similar to that seen with tumor necrosis factor- (TNF-) and transforming growth factor-beta1 (TGF-beta1), both of which induce apoptosis in endothelial cells. In contrast to TNF- and TGF-beta1, angiostatin did not induce cytotoxicity in human MRC-5 fibroblast, rat smooth muscle, canine MDCK epithelial, or murine B16-F10 melanoma cell lines. Angiostatin-induced apoptosis was confirmed by endothelial cell nuclear acridine orange incorporation as well as by annexin V and TUNEL staining. These in vitro findings point to endothelial cell apoptosis as a mechanism for the antiangiogenic effect of angiostatin in vivo.

An improved method for isolation of microvascular endothelial cells from normal and inflamed human lung.

Microvascular endothelial cells (MVEC), which differ from large vessel endothelial cells, have been isolated successfully from lungs of various species, including man. However, contamination by nonendothelial cells remains a major problem in spite of several technical improvements. In view of the organ specificity of MVEC, endothelial cells should be derived from the tissue involved in the diseases one wishes to study. Therefore, to investigate some of the immunopathological mechanisms leading to acute respiratory distress syndrome (ARDS), we have attempted to isolate lung MVEC from patients undergoing thoracic surgery for lung carcinoma and patients dying of ARDS. The method described here includes four main steps: (1) full digestion of pulmonary tissue with trypsin and collagenase, (2) aggregation of MVEC induced by human plasma, (3) Percoll density centrifugation, and (4) selection and transfer of MVEC after local digestion with trypsin/EDTA under light microscopy. Normal and ARDS-derived lung MVEC purified by this technique presented contact inhibition (i.e., grew in monolayer), and expressed classical endothelial markers, including von Willebrand factor (vWF), platelet endothelial cell adhesion molecule 1(PECAM-1, CD31), and transcripts for the angiotensin converting enzyme (ACE). The cells also formed capillarylike structures, took up high levels of acetylated low-density lipoprotein (Ac-LDL), and exhibited ELAM-1 inducibility in response to TNF. Contaminant cells, such as fibroblasts, smooth muscle cells, or pericytes, were easily recognized on the basis of morphology and were eliminated by selection of plasma-aggregated cells under light microscopy. The technique presented here allows one to study the specific involvement and contribution of pulmonary endothelium in various lung diseases.

Demonstration of anti-disease immunity to Plasmodium vivax malaria in Sri Lanka using a quantitative method to assess clinical disease.

Clinical immunity to malaria was studied by quantifying the intensity of symptoms as well as by measurement of several hematologic indicators of pathology (the erythrocyte sedimentation rate [ESR], serum bilirubin, reticulocyte count, plasma tumor necrosis factor-alpha [TNF-alpha], and blood glucose levels) in 39 Plasmodium vivax malaria patients exposed to endemic malaria in southern Sri Lanka, and for comparison in 43 nonimmune patients who were residents of nonmalarious regions of the country. The intensity of 11 symptoms was scored numerically in all patients using a questionnaire. This clinical score was validated by introducing internal controls to the questionnaire, and by correlating it with the underlying pathology. Both the intensity of clinical disease as well as the degree of underlying pathology were found to be significantly lower in endemic area patients (mean clinical score = 8.8, median ESR = 8 mm) compared with the nonendemic area patients (mean clinical score = 19.0, median ESR 31.5 mm). Endemic area patients also had lower parasite densities (mean = 0.06%) than those from the nonendemic area (0.12%) (P < 0.05). However, at any parasite density, both clinical disease and pathology were significantly less in the endemic area patients (P < 0.001, for both clinical score and ESR), indicating that the clinical immunity seen in the endemic area patients was a true tolerance of parasites. Although plasma TNF-alpha levels were elevated in both groups of patients, they were significantly higher in the nonendemic area patients than in patients from the endemic area (P < 0.01). Furthermore, at comparable levels of plasma TNF-alpha, nonendemic area patients had both a higher intensity of clinical disease and an underlying pathology than those from the endemic area, suggesting that if TNF-alpha is indeed a mediator of clinical disease, the endemic area patients may be tolerant to its effects. Hypoglycemia was not observed in any of these P. vivax patients despite some with high levels of plasma TNF-alpha.

Inhibition of leukocyte adherence and transendothelial migration in cultured human liver vascular endothelial cells by prostaglandin E1.

Primary graft dysfunction is a major complication of orthotopic liver transplantation, and hepatic ischemic reperfusion injury is considered to be its major determinant cause. Although oxygen free radicals play an important role, leukocytes, cytokines, and adhesion molecules also contribute to hepatic ischemic reperfusion injury. Prostaglandin E1 (PGE1) has been shown to protect against impairment and dysfunction of transplanted livers in various experimental models as well as in clinical liver transplantation. In this study, the role of PGE1 on leukocyte adherence and transendothelial migration was investigated in cultured human liver vascular endothelial cells (HLVEC). Our results indicated that stimulated, but not resting, leukocytes exhibited high adhesion and transmigration capacity. HLVEC incubated with tumor necrosis factor (TNF) promoted leukocyte adherence and transendothelial migration. PGE1 inhibited leukocyte adherence to HLVEC when it was preincubated with either HLVEC or leukocytes. Moreover, PGE1 also suppressed stimulated leukocyte transendothelial migration in a dose-dependent manner. The inhibitory activity of PGE1 was further investigated on both HLVEC and leukocytes with attention to adhesion molecules. On HLVEC, PGE1 down-regulated TNF-induced expression of endothelial cell leukocyte adhesion molecule 1 and vascular adhesion molecule 1, but not intercellular adhesion molecule 1. On leukocytes, PGE1 inhibited expression of CD11a/CD18 and membrane-bound TNF on PHA-stimulated leukocytes. PGE1 also suppressed TNF release from the stimulated leukocytes. These results indicated that inhibition of leukocyte adherence and transendothelial migration is one of the mechanisms by which PGE1 protects liver grafts.

Adhesion molecules in HIV-related and idiopathic polymyositis: immunohistochemical studies.

Idiopathic polymyositis (IPM) and HIV polymyositis (HIV-PM) are considered to be related autoimmune diseases whose target is skeletal muscle. They have been associated to a T cell-mediated and MHC-I-restricted cytotoxic phenomenon, but both etiology and physiopathology remain incompletely understood. Their histological hallmarks are mononuclear leukocyte infiltrates as well as necrosis, degeneration, and regeneration of muscle fibers. In the present study, we have investigated the immunohistochemical expression of cell adhesion molecules, cytokines, and leukocyte surface antigens in biopsies of HIV-PM and IPM patients. The aim was to better define factors involved in lymphocyte recruitment and in inflammatory changes seen in PM. Notable upregulation of ICAM-1 and TNF-alpha was detected on capillary and venular endothelia and on inflammatory cells, whereas no significant VCAM-1 and ELAM-1 expression was present. LFA-1, the main ICAM-1 counter-receptor, was found to be highly expressed on lymphocytes and monocytes, especially at the vicinity of damaged fibers. The majority of infiltrating cells were CD8+CD45 RO-T cells, which are thought to have memory capacities. These findings suggest that in IPM and HIV-PM, enhanced ICAM-1 and LFA-1 expression possibly induced by TNF-alpha, may regulate the homing process of selected lymphocyte clones in muscle tissue. Lymphocyte proliferation and differentiation into memory subsets may further potentiate tissue-restricted homing capabilities.