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Biomedical subjects

P Hervé

Publications and source records attributed to P Hervé.

At least 19 recordsLinked to original sources
PubMed1999

Vascular endothelium viability and function after total cardiopulmonary bypass in neonatal piglets.

Endothelium dysfunction with severe pulmonary hypertension may occur after total cardiopulmonary bypass (CPB) in infants as a result of a widespread inflammatory response. The aim of this study was to separate out the effects of lung ischemia-reperfusion from membrane oxygenator-induced activation of leukocytes on the function and viability of the pulmonary and systemic endothelia in neonatal piglets submitted to 90-min total CPB followed by 60-min reperfusion or in sham animals. Hemodynamics, gas exchange, endothelial-dependent relaxation in pulmonary and femoral arteries, and lung and skeletal muscle myeloperoxidase activity were assessed before, during, and after CPB, i.e., after reperfusion. Pulmonary and aortic endothelial cells and circulating leukocytes were harvested to assess reperfusion-induced changes in endothelial cells' viability and proliferation, and leukocyte-endothelial cell adhesion and cytotoxicity. Gas exchange worsened after reperfusion with pulmonary hypertension, increase in lung but not skeletal myeloperoxidase, and reduction of endothelial-dependent relaxation in pulmonary but not femoral arteries. After reperfusion, viabilities of pulmonary and aortic endothelial cells were reduced to 50%, endothelial cell growths were faster in pulmonary arteries than aorta, and leukocyte-pulmonary endothelial cell adhesion and cytotoxicity increased. These results suggest that in total CPB lung ischemia-reperfusion aggravates the inflammatory response and predisposes the lung endothelium to leukocyte-mediated injury.

Animals↗
PubMed1998

Gene therapy for oxidant injury-related diseases: adenovirus-mediated transfer of superoxide dismutase and catalase cDNAs protects against hyperoxia but not against ischemia-reperfusion lung injury.

Hyperoxia and ischemia-reperfusion cause profound lung cellular damage mediated, in part, by generation of oxygen radicals. We hypothesized that gene therapy can be used to overcome oxidant injury by augmenting intracellular antioxidant enzymes. Adult rats were injected intratracheally with an adenovirus (Ad) vector encoding human superoxide dismutase (CuZn-SOD) or catalase cDNA, a mixture of both Ad vectors, or a control Ad vector containing no exogenous gene. Expression of human catalase and CuZn-SOD was demonstrated 3 days later in distal lung epithelial cells and alveolar macrophages, using ELISA and immunochemistry. After exposure to 100% O2 for 62 hr, survival was greater in rats injected with the catalase and/or SOD Ad vectors than in control rats. Ischemia-reperfusion injury was evaluated in the isolated perfused lung model. Overexpression of SOD worsened ischemia-reperfusion injury. Interestingly, concomitant overexpression of catalase prevented this adverse effect, but did not protect against ischemia-reperfusion injury. We conclude that Ad-mediated transfer to lungs of both catalase and SOD cDNAs protects from pulmonary O2 toxicity. Absence of protection against ischemia-reperfusion using intratracheal Ad injections may be related to the lack of endothelial protection, despite epithelial expression of catalase and SOD.

Adenoviridae↗
PubMed1998

In vivo alloreactive potential of ex vivo-expanded primary T lymphocytes.

BACKGROUND: We are presently investigating the therapeutic potential of herpes simplex-thymidine kinase-expressing donor T cells in the setting of a T cell-depleted allogeneic bone marrow transplantation. The generation, expansion, and selection of the gene-modified T cells require a 12-day ex vivo culture period in high-dose interleukin (IL)-2 that could significantly alter their in vivo alloreactivity. METHODS: We evaluated the alloreactive potential of such cultured cells in a murine allogeneic bone marrow transplantation model. RESULTS: The present studies demonstrate that ex vivo-expanded cultured T cells are capable of strong alloreactivity as evidenced by the occurrence of lethal acute graft-versus-host disease (GVHD). However, GVHD mortality after administration of the cultured T cells occurred later than after the administration of a same number of fresh T cells. Similar kinetics of GVHD-induced mortality between cultured and fresh T cells required a 10-fold increase in the number of cultured T cells, indicating a reduced alloreactive potential of these cells. The addition of a 2-day "resting" period in low-dose IL-2 resulted in T cells with enhanced alloreactive potential identical to the alloreactivity observed with fresh T cells. CONCLUSION: Ex vivo IL-2-expanded T cells are capable of significant in vivo alloreactivity. However, an increase in the number of cultured T cells administered or the introduction of a short resting culture period prior to infusion is necessary in order to achieve in vivo alloreactivity identical to the alloreactivity observed with fresh T cells.

Acute Disease↗
PubMed1998

Retrovirus-mediated transfer of the herpes simplex type I thymidine kinase gene in alloreactive T lymphocytes.

We have demonstrated in previous studies that retrovirus-mediated transfer of the herpes simplex thymidine kinase (HS-tk) and neomycin phosphotransferase (neo) genes in CD3/IL-2 stimulated primary T lymphocytes followed by G418 selection resulted in T cells retaining both interleukin-2 (IL-2) and alloresponsiveness and specifically inhibited by ganciclovir (GCV). A clinical trial examining the therapeutic potential of such gene-modified donor T cells after allogeneic bone marrow transplantation is presently underway. In the present study, we have investigated the feasibility and consequences of replacing polyclonal stimulation of T cells by an allogeneic stimulation prior to retrovirus-mediated gene transfer. Exposure of allostimulated primary donor T lymphocytes to retrovirus-containing supernatant resulted in T cells resistant to G418 while maintaining a strong, GCV-sensitive, allogeneic response when subsequently restimulated with the initial allogeneic cells. Control nontransduced cells identically stimulated exhibited a weaker, GCV-insensitive, allogeneic proliferative response. The transduced T cells were also capable of GCV-sensitive alloreactivity when exposed to third-party cells with, however, a lower proliferative response than that seen with the allogeneic cells used for stimulation at the time of transduction. Importantly, this difference in the proliferative responses was not observed with control nontransduced cells identically stimulated. A similar response pattern was observed with respect to pre-cytotoxic T lymphocyte (CTL) frequencies. Overall, retrovirus-mediated gene transfer after an allogeneic stimulation can lead to efficient transduction and the pattern of alloreactivity of the HS-tk-expressing cells is consistent with the preferential transduction of alloantigen-specific dividing T cells. Such an approach could be used to generate cells both strongly alloreactive and GCV-sensitive for in vivo therapeutic use.

Antimetabolites↗
PubMed1998

Differential effects of cyclosporin A on the alloreactivity of fresh and ex vivo-expanded T lymphocytes.

GVHD remains a major source of morbidity and mortality after non-T cell-depleted BMT. The use of donor T lymphocytes expressing a suicide gene could lead to specific immunomodulation after BMT. We are currently evaluating such an approach in a phase I clinical study. A 12-day ex vivo expansion is required to generate gene-modified donor T lymphocytes. CsA is commonly used for GVHD prophylaxis. We analyzed, in a murine GVHD model, the effects of CsA administration on the alloreactivity of fresh or ex vivo-expanded T cells. Variable amounts of fresh or ex vivo-expanded T cells were administered in conjunction with a marrow graft to lethally irradiated allogeneic mice. As expected, a protective effect of CsA with a delayed GVHD-related mortality (P < 0.01 vs saline treatment) was observed in mice receiving fresh splenocytes. However, CsA treatment had no effect (P = NS) in mice experiencing lethal GVHD induced by ex vivo-expanded T cells whether or not the T cells had been 'rested' in low-dose IL-2 prior to in vivo administration. In agreement with the in vivo findings, CsA also inhibited the in vitro proliferation of alloreactive fresh T cells while having no significant inhibitory effect on the alloreactivity of ex vivo-expanded T lymphocytes. Overall, we demonstrate that the alloreactivity of ex vivo-expanded T lymphocytes is not sensitive to CsA and that this differential effect of CsA is not related to the alloreactive potential of the infused T cells. These findings could be highly relevant when considering allogeneic T cell therapy approaches.

Animals↗
PubMed1998

A closed culture system for the ex vivo transduction and expansion of human T lymphocytes.

A phase I clinical trial is currently being performed at our institution, with the aim of evaluating the feasibility and toxicity related to the administration of herpes simplex thymidine kinase gene-expressing human primary T lymphocytes following allogeneic hematopoietic stem cell transplantation. The need for safe and standardized preparation conditions for gene-modified cells is crucial. We describe the closed culture system used in the current trial for ex vivo retroviral-mediated gene transfer and transduced cell selection. Cell handling is performed in closed systems using a sterile connection device that avoids opening the culture system. Cell numbers during the production process increased from 93 +/- 16 on day 0 to 440 +/- 92 x 10(6) on day 12 (7.2 +/- 1.4-fold increase) (n = 11). Transduction efficiency before and after G418 resistance-based selection was 13.5 +/- 3.8% and 90.0 +/- 1.4%, respectively. Safety and efficacy testing included a search for replication-competent retrovirus, endotoxins, Mycoplasma, and bacterial contamination (n = 0/9), PCR-DNA, % CD3+ cells (91 +/- 2%), and viability after thawing (82 +/- 3%). Effective working time from day 0 to day 12 is approximately 20 h. The closed system we developed allows for safe and reproducible ex vivo preparation of gene-modified primary T lymphocytes for clinical use.

Cell Culture Techniques↗
PubMed1998

Retroviral-mediated marker gene transfer in hematopoiesis-supportive marrow stromal cells.

A Moloney-derived retrovirus containing both LacZ and NeoR genes (G1BgSVNa from Genetic Therapy, Inc.), was used to transduce human and murine bone marrow stromal cells. Different kinds of stromal cells that were able to support hematopoiesis were transduced by incubation for 24 h in the presence of virus-containing supernatant. Semiconfluent layers of MRC-5 (human, myofibroblastic, fetal, pulmonary) and MS-5 (murine, myofibroblastic, medullary) cells were successfully transduced after one 24-h incubation, as demonstrated by G418 resistance and Escherichia coli beta-galactosidase staining. In contrast, human stromal cells, purified from primary confluent layers grown for 3-4 weeks, could not be transduced. However, stromal cells generated after 10-12 days in culture from Stro-1+ and 1B10+ stromal precursors were successfully transduced in the presence of basic fibroblast growth factor. Transduced stromal cells maintained a myofibroblastic phenotype, although with a decreased number of alpha-SM actin-positive microfilaments in MS-5 cells. The ability to support the generation of stroma-adherent colony-forming cells from cocultured cord blood CD34+ cells after 4 weeks in culture was similar before and after transduction and G418 selection. In conclusion, human primary stromal precursors can be efficiently transduced, and the stromal cell phenotype and function are not significantly altered after retroviral-mediated transfer of marker genes.

Animals↗
PubMed1998

Antibodies to human adhesion molecules and von Willebrand factor: in vitro cross-species reactivity in the xenotransplantation setting.

Endothelial cell activation is thought to play an important role in xenograft rejection through cell retraction and expression of pro-coagulant and pro-inflammatory factors. Identification of antibodies recognizing porcine endothelial molecules would be useful to study and manipulate the inflammatory response to a xenograft. The aim of this study was to investigate the cross-reactivity of antibodies directed against human adhesion molecules and von Willebrand factor (vWF). Binding of monoclonal antibodies (mAbs) directed against human CD3 1, CD44, CD49, CD54, CD62E, CD102, and CD106 was evaluated on resting and activated endothelial cells from human and pig by flow cytometry. Among 30 antibodies tested, 4 were shown to react with pig cells. Two of them, directed against human CD62E (E-selectin) and rabbit CD106 (VCAM-1) reacted strongly with activated and/or resting pig cells, whereas two others, directed to human CD31 (PECAM) and CD44 (H-CAM), bound weakly to pig cells. In addition, we analyzed the cross-reactivity of five polyclonal or monoclonal antibodies to human or pig vWF with human, baboon, rhesus, pig, and rat vWF. Binding of antibodies was tested by ELISA by using platelet lysates as source of vWF from the different species. Four anti-human or porcine vWF antibodies exhibited a broad reactivity with vWF from all species, whereas one anti-human vWF antibody was specific for primate vWF. In this study, we identified a small number of cross-reacting antibodies that may prove useful to study in vitro and in vivo xenogeneic responses. However, the weak antibody cross-reactivity observed with most porcine molecules points out the necessity of producing species-specific antibodies to study the immune response to xenografts or for use as specific immunosuppressive therapeutic reagents.

Animals↗
PubMed1998

Pulmonary vascular disorders in portal hypertension.

The wide spectrum of pulmonary vascular disorders in liver disease and portal hypertension ranges from the hepatopulmonary syndrome characterized by intrapulmonary vascular dilatations, to pulmonary hypertension (portopulmonary hypertension), in which pulmonary vascular resistance is elevated. Since hepatopulmonary syndrome and portopulmonary hypertension have been reported in patients with nonhepatic portal hypertension, the common factor that determines their development must be portal hypertension. The clinical presentations are very different, with gas exchange impairment in the hepatopulmonary syndrome and haemodynamic failure in portopulmonary hypertension. The severity of hepatopulmonary syndrome seems to parallel the severity of liver failure, whereas no simple relationship has been identified between hepatic impairment and the severity of portopulmonary hypertension. Resolution of hepatopulmonary syndrome is common after liver transplantation, which has an uncertain effect in portopulmonary hypertension. The pathophysiology of both syndromes may involve vasoactive mediators and angiogenic factors.

Hemodynamics↗
PubMed1998

Primary pulmonary hypertension associated with the use of fenfluramine derivatives.

Fenfluramine derivatives (Fds) are a well-established risk factor for primary pulmonary hypertension (PPH). We compared 62 Fd-PPH patients (61 women) evaluated in our center between 1986 and 1997 with 125 sex-matched PPH patients nonexposed to Fd referred during the same period (control PPH). In the Fd-PPH group, 33 patients (53%) used dexfenfluramine alone, 7 patients (11%) used fenfluramine alone, and 5 patients (8%) used both drugs. In 17 cases (27%), Fd use was associated with that of amphetamines. Most of the exposed patients used Fd for at least 3 months (81%). The interval between the onset of dyspnea and that of drug intake was 49+/-68 months (27 days to 23 years). At the time of diagnosis, Fd-PPH and control PPH were similar in terms of New York Heart Association functional class and symptoms. The two groups significantly differed only in terms of age (50+/-12 vs 40+/-14 years) and body mass index (28+/-6 vs 23+/-4). The two groups displayed similar severe baseline hemodynamics (total pulmonary vascular resistance: 32+/-12 vs 31+/-12 IU/m2), but the percentage of responders to acute vasodilator testing was higher in control PPH (27% vs 10%, p < 0.01). As a result, more patients were treated with oral vasodilators in the control PPH group (36% vs 16%, p < 0.01) and long-term epoprostenol infusion was more frequently used in the Fd-PPH group (52% vs 31%, p < 0.01). Overall survival was similar in the two groups with a 3-year survival rate of 50%.

Adult↗
PubMed1998

Reverse ventilation-perfusion mismatch in lung cancer suggests intrapulmonary functional shunting.

We report on a patient with squamous cell cancer of the left lung who was first considered ineligible for surgery because of severe hypoxemia. A ventilation-perfusion scan showed "reverse" ventilation-perfusion mismatch, with 20% of the total lung perfusion going to the left lung, which showed no ventilation with radioactive aerosols. This pattern suggested that the hypoxemia was due to intrapulmonary functional shunting and could therefore be improved by surgical resection of the tumor. Balloon occlusion of the left pulmonary artery resulted in an immediate rise in PaO2, indicating a right-to-left intrapulmonary shunt. After left pneumonectomy, PaO2 levels were normal. This patient provides an example of dysregulation of the pulmonary hypoxic vasoconstriction response in a non-small cell lung cancer. Lung cancer patients with severe hypoxemia should undergo ventilation-perfusion scanning to look for reverse ventilation-perfusion mismatch suggestive of intrapulmonary functional shunting.

Aerosols↗
PubMed1997

Inhaled nitric oxide attenuates reperfusion injury in non-heartbeating-donor lung transplantation. Paris-Sud University Lung Transplantation Group.

BACKGROUND: Non-heartbeating-donor (NHBD) lung transplantation could help reduce the current organ shortage. Polymorphonuclear neutrophil (PMN) activation plays a pivotal role in ischemia-reperfusion injury (I-R), and can be inhibited by nitric oxide (NO). We hypothesized that inhaled NO might be beneficial in NHBD lung transplantation. METHODS: The effect of inhaled NO on PMNs was studied by measuring in vivo PMN lung sequestration (myeloperoxidase activity) and adhesion of recipient circulating PMNs to cultured pulmonary artery endothelial cells (PAECs) in vitro. Pigs were randomly assigned to an NO or a control group (n=9 each). In the NO group, cadavers and recipients were ventilated with oxygen and 30 parts per million of NO. After 3 hr of postmortem in situ warm ischemia and 2 hr of cold ischemia, left allotransplantation was performed. The right pulmonary artery was ligated, and hemodynamic and gas exchange data were recorded hourly for 9 hr. Recipient PMN adherence to tumor necrosis factor-alpha- and calcium ionophore-stimulated PAECs was measured before and after reperfusion, and lung PMN sequestration was determined after death. RESULTS: NO-treated animals exhibited lowered pulmonary vascular resistance (P<0.01), as well as improved oxygenation (P<0.01) and survival (P<0.05). Adhesion of PMNs to PAECs was inhibited in the NO group before (P<0.001) and after reperfusion (P<0.0001). Lung PMN sequestration was reduced by NO (P<0.05). CONCLUSIONS: Inhaled NO attenuates I-R injury after NHBD lung transplantation. This is likely due to the prevention of I-R-induced pulmonary vasoconstriction and to the direct effect on peripheral blood PMN adhesion to endothelium, which results in reduced sequestration and tissue injury.

Administration, Inhalation↗