Femoral Access-Site Complications with Tenecteplase versus Alteplase before Mechanical Thrombectomy for Large-Vessel-Occlusion Stroke.

BACKGROUND AND PURPOSE: IV thrombolysis with alteplase before mechanical thrombectomy for emergent large-vessel-occlusion stroke is associated with access-site bleeding complications. However, the incidence of femoral access-site complications with tenecteplase before mechanical thrombectomy requires exploration. Here, femoral access-site complications with tenecteplase versus alteplase before mechanical thrombectomy for large-vessel-occlusion stroke were compared. MATERIALS AND METHODS: All patients receiving IV thrombolytics before mechanical thrombectomy for large-vessel-occlusion stroke who presented from January 2020 to August 2022 were reviewed. In May 2021, our health care system switched from alteplase to tenecteplase as the primary thrombolytic for all patients with stroke, facilitating the comparison of alteplase-versus-tenecteplase femoral access-site complication rates. Major (requiring surgery) and minor (managed conservatively) access-site complications were assessed. RESULTS: One hundred thirty-nine patients underwent transfemoral mechanical thrombectomy for large-vessel-occlusion stroke, of whom 46/139 (33.1%) received tenecteplase and 93/139 (66.9%) received alteplase. In all cases (n = 139), an 8F sheath was inserted without sonographic guidance, and vascular closure was obtained with an Angio-Seal. Baseline demographics, concomitant antithrombotic medications, and periprocedural coagulation lab findings were similar between groups. The incidence of conservatively managed groin hematomas (2.2% versus 4.3%), delayed access-site oozing requiring manual compression (6.5% versus 2.2%), and arterial occlusion requiring surgery (2.2% versus 1.1%) was similar between the tenecteplase and alteplase groups, respectively (P = not significant). No dissection, arteriovenous fistula, or retroperitoneal hematoma was observed. CONCLUSIONS: Tenecteplase compared with alteplase before mechanical thrombectomy for large-vessel-occlusion stroke is not associated with an alteration in femoral access-site complication rates.

Harnessing alveolar macrophages for sustained mucosal T-cell recall confers long-term protection to mice against lethal influenza challenge without clinical disease.

Vaccines that induce T cells, which recognize conserved viral proteins, could confer universal protection against seasonal and pandemic influenza strains. An effective vaccine should generate sufficient mucosal T cells to ensure rapid viral control before clinical disease. However, T cells may also cause lung injury in influenza, so this approach carries inherent risks. Here we describe intranasal immunization of mice with a lentiviral vector expressing influenza nucleoprotein (NP), together with an NFκB activator, which transduces over 75% of alveolar macrophages (AM). This strategy recalls and expands NP-specific CD8+ T cells in the lung and airway of mice that have been immunized subcutaneously, or previously exposed to influenza. Granzyme B-high, lung-resident T-cell populations persist for at least 4 months and can control a lethal influenza challenge without harmful cytokine responses, weight loss, or lung injury. These data demonstrate that AM can be harnessed as effective antigen-presenting cells for influenza vaccination.

Characterization of HIV-1 vectors with gammaretrovirus envelope glycoproteins produced from stable packaging cells.

We have recently described a novel, stable human immunodeficiency virus type 1 (HIV-1) vector packaging system, STAR. High-titre HIV-1 vectors bearing gammaretrovirus envelopes (Env) are continuously produced from STAR cells. Here we compare the properties of such vectors, with the amphotropic murine leukaemia virus (MLV-A) Env, a modified gibbon ape leukaemia virus (GALV) Env and two modified versions of the cat endogenous retrovirus RD114 Env, produced from STAR cells, to transiently produced HIV-1 vectors with vesicular stomatitis virus G protein (VSV-G). Our results indicate that gammaretrovirus pseudotypes from STAR cells are relatively stable at 37 degrees C and are resistant to inactivation by freeze/thaw cycling or incubation with human sera. HIV-1(VSV-G) was, however, sensitive to freeze/thaw when harvested in serum-free media and was readily inactivated in human sera. Furthermore, the titre of 'gamma-retrovirus' pseudotypes, but not HIV-1(VSV-G), could be increased by the use of a combination of polybrene and spinoculation. All pseudotypes could be efficiently concentrated, but soluble gammaretrovirus Env could act as an inhibitor of infection.

Gene transduction efficiency in cells of different species by HIV and EIAV vectors.

The ability of human immunodeficiency virus (HIV)- and equine infectious anaemia virus (EIAV)-based vectors to transduce cell lines from a range of species was compared. Both vectors carried the vesicular stomatitis virus G (VSV-G) envelope protein and encoded an enhanced green fluorescent protein (eGFP) gene driven by a human cytomegalovirus (CMV) early promoter. Immunostaining for viral core proteins and VSV-G was used to demonstrate that the HIV and EIAV vector preparations contained similar numbers of virus particles. Various cell lines were transduced with these vectors and the transduction efficiency was estimated by measuring eGFP expression. Efficient transduction by both vectors was observed in human, hamster, pig, horse, cat and dog cell lines, although EIAV vector was about 10-fold less efficient in human, hamster and pig cells normalised to the total number of viral particles. This could be partly explained by the lower RNA genome levels per particle for EIAV as measured by real-time RT-PCR. Rodent cells appeared to be transduced inefficiently with both vectors, but when the CMV promoter was substituted with the EF1alpha promoter in the HIV vectors, the expression level increased leading to an increase in the measurable level of transduction.

The JNK phosphatase M3/6 is inhibited by protein-damaging stress.

Cells respond to stresses such as osmotic shock and heat shock by activating stress-activated protein kinases (SAPKs), including c-Jun N-terminal kinase (JNK) [1]. Activation of JNK requires phosphorylation of threonine and tyrosine residues in the TPY activation loop motif [2, 3] and can be reversed by the removal of either phosphate group. Numerous JNK phosphatases including dual-specificity phosphatases [4, 5], have been identified. Many stimuli activate JNK by increasing its rate of phosphorylation; however, JNK dephosphorylation is inhibited in cells after heat shock [6], suggesting that a JNK phosphatase(s) is inactivated. M3/6 is a dual-specificity phosphatase selective for JNK [7, 8]. We have previously expressed M3/6 in the mouse bone marrow cell line BAF3 in order to show that JNK activation by IL-3 is necessary for cell survival and proliferation [9]. Here we report that M3/6 dissociates from JNK and appears in an insoluble fraction after heat shock. These data identify M3/6 as a JNK phosphatase that is inactivated by heat shock and provide a molecular mechanism for the activation of JNK by heat shock.

Role of Bax in apoptosis of IL-3-dependent cells.

IL-3 removal was reported to induce membrane association of the apoptotic effector Bax. This report demonstrates that IL-3-dependent cells from Bax-null mice failed to activate caspases after IL-3 removal and survived in an 10-fold lower concentration of IL-3. As IL-3 removal also down-regulates expression of Bcl-X, we examined the relationship between Bcl-X decrease and Bax membrane association. IL-3 removal from BAF-3 cells, followed by sorting caspase-active and caspase-inactive populations, showed that both expressed similar levels of Bcl-X. Inhibition of IL-3 signalling via PI-3 kinase and MEK1/2 resulted in cells with minimal Bcl-X, which remained viable with soluble Bax. However BAF-3-derived cells, which maintained Bcl-X expression without IL-3, also remained viable with soluble Bax on IL-3 removal. Therefore a decrease in Bcl-X is necessary, though not sufficient, for Bax membrane association on IL-3 removal. In contrast, treatment of BAF-3 cells with hydroxyurea induced apoptosis in the absence of a Bcl-X decrease. Furthermore, IL-3-dependent cells from Bax-null mice activated caspases after hydroxyurea treatment and show the same sensitivity to a variety of cytotoxic drugs. Thus, apoptosis after IL-3 removal requires a decrease in Bcl-X and Bax membrane association, whereas that induced by cytotoxic drugs does not.

The role of p53 in death of IL-3-dependent cells in response to cytotoxic drugs.

This report examines the cytotoxicity of chemotherapeutic agents to primary bone marrow-derived IL-3-dependent cells. Such cells derived from p53-null mice were resistant to almost 100-fold higher concentrations of the inhibitors of deoxyribonucleotide synthesis FUdR, methotrexate and hydroxyurea than cells with wild-type p53. In contrast, the cytotoxicity of the DNA damaging agents X-irradiation, cisplatin or bleomycin was p53-independent. The topoisomerase II inhibitor etoposide induced p53-dependent death, which suggests that DNA damage may not be its primary mechanism of cytotoxicity in this cell type. An IL-3-dependent cell line which expresses wild-type p53 was used to demonstrate that the ability of cytotoxic drugs to increase p53 expression level does not control their ability to induce p53-dependent loss of clonigenicity. Finally, comparison with a p53-null IL-3-dependent cell line was used to show that absence of p53 delays the rate of entry into apoptosis following treatment with either DNA damaging agents or inhibitors of deoxyribonucleotide synthesis. This distinguishes short-term effects of p53 on rate of entry into apoptosis from its role in controlling ultimate cell survival. Oncogene (2000) 19, 3556 - 3559

Improved safety and titre of murine leukaemia virus (MLV)-based retroviral vectors.

Many retroviral vectors based on murine leukaemia virus (MLV) contain the first 420 nucleotides of the gag gene, as this was reported to increase vector titre by increasing the efficiency of RNA packaging. In this study, deletion of this gag sequence from its original location did not decrease the titre of two retroviral vectors, pBabe puro and MFG-S-. The two vectors could be improved by replacing the gag sequence with a CTE from Mason-Pfizer monkey virus (MPMV). This substitution improved vector titre, while eliminating a region of homology between vector and packaging constructs. Gene Therapy (2000) 7, 300-305.

Interferon-alpha (IFN-alpha) stimulates anti-melanoma cytotoxic T lymphocyte (CTL) generation in mixed lymphocyte tumour cultures (MLTC).

IFN-alpha administration after primary tumour resection improves the survival of melanoma patients at high risk of relapse. To investigate whether this response might be due to stimulation of anti-tumour immunity, the effect of IFN-alpha on anti-melanoma CTL generation in MLTC was measured. IFN-alpha increased both allogeneic and autologous anti-melanoma CTL generation from peripheral blood lymphocytes stimulated with irradiated primary melanoma cultures. IFN-alpha up-regulated MHC class I expression on primary melanoma cultures, whereas IFN-gamma up-regulated both MHC class I and II expression. However, the effect of IFN-alpha on anti-melanoma CTL generation was often more potent than that of IFN-gamma, equalling the effect of the optimal combination of IL-2 and IL-12. Pre-treatment of primary melanoma cultures with IFN-gamma was sufficient for CTL generation in MLTC, whereas IFN-alpha needed to be present during the MLTC. While direct anti-proliferative effects of IFN-alpha on some tumour cells have been described, IFN-alpha did not inhibit proliferation of primary melanoma cultures. These results suggest that the clinical effects of IFN-alpha in melanoma patients may be immune-mediated.

Efficient gene delivery to quiescent interleukin-2 (IL-2)-dependent cells by murine leukemia virus-derived vectors harboring IL-2 chimeric envelope glycoproteins.

Interleukin-2 (IL-2) is a cytokine that induces the proliferation of certain IL-2 receptor expressing quiescent cells. Human IL-2 was fused to the amino-terminus of amphotropic murine leukemia virus (MLV) envelope glycoproteins. Retroviral vectors were pseudotyped with both the IL-2 chimeric envelope and the wild-type amphotropic MLV envelope. The chimeric IL-2 glycoproteins were incorporated on retroviral vectors and the IL-2-displaying vector particles could bind specifically to cell surface IL-2 receptors. In addition, the IL-2-displaying vectors could infect proliferating cells through amphotropic receptors irrespective of whether the cells expressed the IL-2 receptor. IL-2-displaying vector particles could also transiently stimulate the cell cycle entry and proliferation of several IL-2-dependent cell lines. Finally, retroviral vectors displaying IL-2 could efficiently transduce G0/G1-arrested cells expressing the IL-2 receptor at a 34-fold higher efficiency compared with vectors with unmodified envelopes. This new strategy, whereby C-type retroviral vector particles display a ligand that activates the cell cycle of the target cells at the time of virus entry, may represent an alternative to lentivirus-derived retroviral vectors for the infection of quiescent cells. In addition, upon infection of an heterogeneous population of nonproliferating cells, MLV-retroviral vectors that display cytokines/growth factors will allow the transgene of interest to be integrated specifically in quiescent cells expressing the corresponding cytokine/growth factor receptor.

Gene therapy with autologous, interleukin 2-secreting tumor cells in patients with malignant melanoma.

We vaccinated metastatic melanoma patients with irradiated, autologous melanoma cells genetically engineered to secrete interleukin 2 (IL-2) to investigate whether an anti-tumor immune response would be induced. Melanoma cell cultures were established from surgical specimens and were engineered to secrete IL-2 by infection with recombinant retrovirus. Twelve patients were vaccinated subcutaneously one, two, or three times with approximately 10(7) irradiated, autologous, IL-2-secreting tumor cells. Treatment was well tolerated, with local reactions at 11 of 24 injection sites and minor systemic symptoms of fever and headache after 6 injections. One patient developed anti-tumor DTH after the first vaccination and showed an increased response after the second vaccination. Anti-autologous tumor CTLs could be detected prevaccination in the peripheral blood of seven patients and their activity increased after vaccination in four patients. No UICC-defined clinical responses were seen, but three patients had stable disease for 7-15 months, one of whom has not yet progressed (15+ months). Thus, patient vaccination with autologous, genetically engineered tumor cells is feasible and safe. Anti-tumor DTH and CTLs can be induced in some patients with such a vaccine.

Cationic liposomes enhance the rate of transduction by a recombinant retroviral vector in vitro and in vivo.

Cationic liposomes enhanced the rate of transduction of target cells with retroviral vectors. The greatest effect was seen with the formulation DC-Chol/DOPE, which gave a 20-fold increase in initial transduction rate. This allowed an efficiency of transduction after brief exposure of target cells to virus plus liposome that could be achieved only after extensive exposure to virus alone. Enhancement with DC-Chol/DOPE was optimal when stable virion-liposome complexes were preformed. The transduction rate for complexed virus, as for virus used alone or with the polycation Polybrene, showed first-order dependence on virus concentration. Cationic liposomes, but not Polybrene, were able to mediate envelope-independent transduction, but optimal efficiency required envelope-receptor interaction. When virus complexed with DC-Chol/DOPE was used to transduce human mesothelioma xenografts, transduction was enhanced four- to fivefold compared to that for virus alone. Since the efficacy of gene therapy is dependent on the number of cells modified, which is in turn dependent upon the balance between transduction and biological clearance of the vector, the ability of cationic liposomes to form stable complexes with retroviral vectors and enhance their rate of infection is likely to be important for in vivo application.

Induction of apoptosis by valinomycin: mitochondrial permeability transition causes intracellular acidification.

In order to determine whether disruption of mitochondrial function could trigger apoptosis in murine haematopoietic cells, we used the potassium ionophore valinomycin. Valinomycin induces apoptosis in the murine pre-B cell line BAF3, which cannot be inhibited by interleukin-3 addition or Bcl-2 over-expression. Valinomycin triggers rapid loss of mitochondrial membrane potential. This precedes cytoplasmic acidification, which leads to cysteine-active-site protease activation, DNA fragmentation and cell death. Bongkrekic acid, an inhibitor of the mitochondrial permeability transition, prevents acidification and subsequent induction of apoptosis by valinomycin.

Sarcoma cells engineered to secrete IFN-gamma or IL-2 acquire sensitization to immune cell killing via different mechanisms.

The murine fibrosarcoma FS29 can be more efficiently killed by syngeneic lymphocytes when it has been engineered to secrete either interferon gamma (IFN-gamma), or interleukin 2 (IL-2). The mechanisms by which the two cytokines enhance target sensitivity differ. Supernatant from IFN-gamma-secreting cells can enhance the sensitivity of unmodified cells. The enhanced sensitivity correlates with MHC upregulation observed on both the IFN-gamma-secreting and supernatant-treated cells. In contrast, supernatant from IL-2-secreting cells does not affect the sensitivity of unmodified cells. IL-2 can be detected, by a bioassay, bound to the extracellular matrix of the secreting tumour cells.

Overexpression of a heterologous thymidine kinase delays apoptosis induced by factor deprivation and inhibitors of deoxynucleotide metabolism.

Perturbing deoxyribonucleoside triphosphate (dNTP) metabolism with inhibitors of the de novo synthesis of dNTP causes apoptosis in the interleukin-3 (IL-3)-dependent pre-B cell line BAF3. Under these conditions apoptosis is prevented when deoxyribonucleosides for dNTP synthesis are supplied in the culture medium. On the other hand, removal of IL-3 from cultures of BAF3 cells resulted in down-regulation of thymidine kinase activity, rapid imbalance in dNTP levels, and apoptosis. In this study we show that overexpression of a heterologous thymidine kinase, herpes simplex virus thymidine kinase (TK), in BAF3 cells protects these cells from apoptosis induced by either inhibitors of dNTP synthesis or IL-3 deprivation. This protection against apoptosis is abrogated by 9-(4-hydroxybutyl)-N2-phenylguanine, a specific inhibitor of herpes simplex virus-1 TK. These results suggest that deoxyribonucleoside kinases, particularly TK, may be important in the regulation of apoptosis in hemopoietic cells.

p53 is not required for regulation of apoptosis or radioprotection by interleukin-3.

Primary interleukin-3 (IL-3)-dependent mast cell cultures from bone marrow of p53-null mice and littermate controls were established. Both p53-null and wild-type cells entered apoptosis on IL-3 removal, showing that p53 is not required for entry into apoptosis after factor deprivation. After X-irradiation, a lower proportion of the p53-null than wild-type cells underwent G2 arrest, but their radiosensitivity was similar. An IL-3-dependent cell line expressing wild-type p53 was used to show that cells die at a fixed time after X-irradiation rather than from a specific cell cycle point.

The bystander effect of the nitroreductase/CB1954 enzyme/prodrug system is due to a cell-permeable metabolite.

The bystander effect is an important part of tumor kill using gene-directed enzyme prodrug therapy (GDEPT). Recently, we have described a novel enzyme prodrug system using bacterial nitroreductase and the prodrug CB1954 (NTR/CB1954). We demonstrate here the presence of a cell-permeable cytotoxic activity in the conditioned growth medium of nitroreductase (NTR)-transduced cells treated with CB1954 and show that its appearance corresponds to the appearance of two metabolites of CB1954 previously identified (Friedlos et al., 1992). The degree of bystander effect and the degree of transferred cytotoxicity correlates with the level of NTR enzyme expression. Two other prodrugs for NTR show little bystander killing and do not produce detectable cell permeable metabolites. The elucidation of the mechanism of the bystander effect may allow the more effective use of NTR/CB1954.

Intracellular acidification induces apoptosis by stimulating ICE-like protease activity.

ICE-like protease activation and DNA fragmentation are preceded by a decrease in intracellular pH (pHi) during apoptosis in the IL-3 dependent cell line BAF3. Acidification occurs after 7 hours in cells deprived of IL-3 and after 4 hours when cells are treated with etoposide, close to the time of detection of ICE-like protease activity. Increasing extracellular pH reduces ICE-like protease activation and DNA fragmentation. Bcl-2 over-expression both delays acidification and inhibits ICE-like protease activation. Generation of a rapid intracellular pH decrease, using the ionophore nigericin, induces ICE-like protease activation and apoptosis. ZVAD, a cell permeable inhibitor of ICE-like proteases, does not affect acidification but inhibits apoptosis induced by IL-3 removal or nigericin treatment. These data suggest that intracellular acidification triggers apoptosis by directly or indirectly activating ICE-like proteases.

Irradiated NC adenocarcinoma cells transduced with both B7.1 and interleukin-2 induce CD4+-mediated rejection of established tumors.

Previous studies have shown that expression of the immune co-stimulator B7.1 reduces the tumorigenicity of some, but not all, malignant cell lines. However, B7.1-expressing tumor cells are not very effective in inducing the rejection of established tumors. This may in part be due to induction of anergy in the potentially reactive T cells. Previous studies have shown that IL-2 can reverse the anergic state both in vitro and in vivo. Therefore, we have examined the effect of retrovirus-mediated delivery and expression of murine B7.1 and interleukin-2 on tumor formation and rejection of established MHC class I+/II- NC adenocarcinomas. Neither the expression of B7.1 nor IL-2 alone had a significant effect on NC tumorigenicity. In contrast, combined expression of B7.1 and IL-2 substantially decreased the tumorigenicity of these cells in the immunecompetent syngeneic hosts. T-cell depletion studies show this to be dependent primarily on the activation of CD4+ cells. Furthermore, distant subcutaneous injection of irradiated NC/IL-2/B7.1 can induce, much more effectively than NC/B7.1 or NC/IL-2, the rejection of small NC tumors, and prevent the recurrence of large surgically resected tumors. Together, these results suggest that tumor cells genetically modified to express B7.1 and IL-2 can induce the immune-mediated rejection of established class II- tumors by a mechanism involving CD4+ cells.

In bone marrow derived Baf-3 cells, inhibition of apoptosis by IL-3 is mediated by two independent pathways.

The inhibition of cell death by growth factors plays a key role in the maintenance of the haematopoietic system homeostasis. However the mechanisms involved in this inhibition are still poorly understood. In order to determine if inhibition of apoptosis by growth factors is dependent only on the expression of survival genes, we have studied that process in the bone marrow derived IL-3 dependent cell line Baf-3. We show that, following IL-3 starvation, mRNA and protein levels of Bcl-X but not Bcl-2 decrease rapidly preceeding the onset of death. The death of IL-3 starved cells is asynchronous, starting between 6 to 8 h with 50% death being reached after 10 to 12 h. At any time point, apoptosis can be rapidly inhibited by growth factor re-addition. This has allowed us to determine that the inhibition of apoptosis by growth factor takes place at two levels. The first one, which we have called short term inhibition, is independent of mRNA and protein synthesis i.e. it takes place in the absence of survival gene neosynthesis and can be demonstrated during the first 6 h following growth factor re-addition. The second one corresponds to long-term survival-more than 24 h survival-and is strongly correlated with the induction of Bcl-X but not Bcl-2 gene expression. This induction of Bcl-X by IL-3 is shown to be dependent on MAP-kinase activation.