Efficient targeting of NY-ESO-1 tumor antigen to human cDC1s by lymphotactin results in cross-presentation and antigen-specific T cell expansion.

BACKGROUND: Type 1 conventional dendritic cells (cDC1s) are characterized by their ability to induce potent CD8+ T cell responses. In efforts to generate novel vaccination strategies, notably against cancer, human cDC1s emerge as an ideal target to deliver antigens. cDC1s uniquely express XCR1, a seven transmembrane G protein-coupled receptor. Due to its restricted expression and endocytic nature, XCR1 represents an attractive receptor to mediate antigen-delivery to human cDC1s. METHODS: To explore tumor antigen delivery to human cDC1s, we used an engineered version of XCR1-binding lymphotactin (XCL1), XCL1(CC3). Site-specific sortase-mediated transpeptidation was performed to conjugate XCL1(CC3) to an analog of the HLA-A*02:01 epitope of the cancer testis antigen New York Esophageal Squamous Cell Carcinoma-1 (NY-ESO-1). While poor epitope solubility prevented isolation of stable XCL1-antigen conjugates, incorporation of a single polyethylene glycol (PEG) chain upstream of the epitope-containing peptide enabled generation of soluble XCL1(CC3)-antigen fusion constructs. Binding and chemotactic characteristics of the XCL1-antigen conjugate, as well as its ability to induce antigen-specific CD8+ T cell activation by cDC1s, was assessed. RESULTS: PEGylated XCL1(CC3)-antigen conjugates retained binding to XCR1, and induced cDC1 chemoattraction in vitro. The model epitope was efficiently cross-presented by human cDC1s to activate NY-ESO-1-specific CD8+ T cells. Importantly, vaccine activity was increased by targeting XCR1 at the surface of cDC1s. CONCLUSION: Our results present a novel strategy for the generation of targeted vaccines fused to insoluble antigens. Moreover, our data emphasize the potential of targeting XCR1 at the surface of primary human cDC1s to induce potent CD8+ T cell responses.

Potential of a polyherbal drug to prevent antimicrobial resistance in bacteria to antibiotics.

Persistence of antibacterial drugs for prolonged period in milk increases the probability of antimicrobial resistance progress. Ceftizoxime was found to be excreted in milk for a prolonged period in goats, cows and buffaloes following intravenous injection of ceftriaxone and ceftizoxime. A single dose of ceftriaxone was administered intravenously in healthy control goats (group I) and a single oral dose of the commercial mammary protective polyherbal drug (1.9 gm) was given one hour prior to intravenous ceftriaxone injection in healthy (group II) and induced mastitic (group III) goats to evaluate milk disposition of ceftizoxime following single intravenous dosing of ceftriaxone at 42.25 mg kg-1.Ceftriaxone/ceftizoxime was analyzed by HPLC. The t1/2α and t1/2ß values were 14.755 ± 2.733 and 149.079 ± 18.565 hour, respectively indicating prolonged persistence of ceftizoxime in milk. The polyherbal drug increased the milk concentration at later hours and hastened the excretion of ceftizoxime from milk compared to control group. Ceftriaxone could not be detected in milk. The study suggested that adjunct single or repeated therapy of  the polyherbal drug may cause non persistence of ceftriaxone and shorter persistence of ceftizoxime in milk.

Direct regulation of IL-2 by curcumin.

Interleukin-2 (IL-2) is a crucial growth factor for both regulatory and effector T cells. Thus, IL-2 plays a critical role in the stimulation and suppression of immune responses. Recently, anti-IL-2 antibodies (Abs) have been shown to possess strong IL-2 modulatory activities by affecting the interaction between IL-2 and IL-2 receptors. In this study, we screened an herbal library to identify a compound that regulates IL-2, which resulted in the identification of curcumin as a direct binder and inhibitor of IL-2. Curcumin is a phytochemical with well-known anti-cancer properties. In this study, curcumin mimicked or altered the binding pattern of anti-IL-2 Abs against IL-2 and remarkably inhibited the interaction of recombinant IL-2 with the IL-2 receptor α, CD25. Interestingly, curcumin neutralized the biological activities of IL-2 both in vitro and in vivo. In this report, we elucidated the unsolved mechanism of the anti-cancer effect of curcumin by identifying IL-2 as a direct molecular target. Curcumin, as a small molecule IL-2 modulator, has the potential to be used to treat IL-2 related pathologic conditions.

Safety and patient response as indicated by biomarker changes to binding immunoglobulin protein in the phase I/IIA RAGULA clinical trial in rheumatoid arthritis.

OBJECTIVES: Binding immunoglobulin protein (BiP) is a human endoplasmic reticulum-resident stress protein. In pre-clinical studies it has anti-inflammatory properties due to the induction of regulatory cells. This randomized placebo-controlled, dose ascending double blind phase I/IIA trial of BiP in patients with active RA, who had failed accepted therapies, had the primary objective of safety. Potential efficacy was measured by DAS28-ESR and changes in biomarkers. METHODS: Twenty-four patients with active RA who had failed one or more DMARDs were sequentially assigned to three groups each of eight patients randomly allocated to receive placebo (two patients) or BiP (six patients), 1, 5 or 15 mg. Patients received a single i.v. infusion over 1 h and were observed as inpatients overnight. A 12-week follow-up for clinical, rheumatological and laboratory assessments for safety, efficacy (DAS28-ESR) and biomarker analysis was performed. RESULTS: No infusion reactions or serious adverse drug reactions were noted. Adverse events were evenly distributed between placebo and BiP groups with no BiP-related toxicities. Haematological, renal and metabolic parameters showed no drug-related toxicities. Remission was only achieved by patients in the 5 and 15 mg groups, and not patients who received placebo or 1 mg BiP. Good DAS28-ESR responses were achieved in all treatment groups. The BiP responding patients showed significantly lower serum concentrations of CRP, 2 weeks post-infusion compared with pre-infusion levels, and of VEGF and IL-8 from the placebo group. CONCLUSION: BiP (⩽15 mg) is safe in patients with active RA. Some patients had clinical and biological improvements in RA activity. BiP merits further study. TRIAL REGISTRATION: ISRCTN registry, http://isrctn.com, ISRCTN22288225 and EudraCT, https://eudract.ema.europa.eu, 2011-005831-19.

Low-dose amphotericin B and murine dialyzable spleen extracts protect against systemic candida infection in mice.

Candida albicans causes opportunistic systemic infections with high mortality (30%-50%). Despite significant nephrotoxicity, amphotericin (AmB) is still used for the treatment of this serious fungal infection. Therefore, alternative treatments are urgently needed. Dialyzable leukocyte extracts have been used successfully to treat patients with mucocutaneous candidiasis, but their effectiveness in systemic candidiasis has not been evaluated. In this study, low-dose AmB (0.1 mg/kg) plus 10 pg of murine dialyzable spleen extracts (mDSE) were tested in a systemic candidiasis mouse model. Survival, tissue fungal burden, kidney damage, kidney cytokines, and serum levels of IL-6 and hepcidin were evaluated. Our results showed that the combined treatment of low-dose AmB plus mDSE improved survival and reduced kidney fungal burden and histopathology; these effects correlated with increased kidney concentration of IFN- γ and TGF- ß 1, decreased levels of TNF- α , IL-6, and IL-10, as well as high levels of systemic IL-6 and hepcidin. Low-dose AmB and mDSE synergized to clear the infectious agent and reduced tissue damage, confirming the efficacy of a low dose of AmB, which might decrease the risk of drug toxicity. Further studies are necessary to explore these findings and its implications in future therapeutic approaches.

Platelet-derived growth factor CC-mediated neuroprotection against HIV Tat involves TRPC-mediated inactivation of GSK 3beta.

Platelet-derived growth factor-CC (PDGF-CC) is the third member of the PDGF family, and has been implicated both in embryogenesis and development of the CNS. The biological function of this isoform however, remains largely unexplored in the context of HIV-associated dementia (HAD). In the present study, we demonstrate that exposure of human neuroblastoma cells SH-SY5Y to HIV transactivator protein Tat resulted in decreased intrinsic expression of PDGF-CC as evidenced by RT-PCR and western blot assays. Reciprocally, pretreatment of SH-SY5Y cells with PDGF-CC abrogated Tat-mediated neurotoxicity by mitigating apoptosis and neurite & MAP-2 loss. Using pharmacological and loss of function approaches we identified the role of phosphatidylinositol 3-kinase (PI3K)/Akt signaling in PDGF-CC-mediated neuroprotection. We report herein a novel role about the involvement of transient receptor potential canonical (TRPC) channel 1 in modulation of calcium transients in PDGF-CC-mediated neuroprotection. Furthermore we also demonstrated PDGF-CC-mediated inactivation of the downstream mediator--glycogen synthase kinase 3ß (GSK3ß) evidenced by its phosphorylation at Ser-9. This was further validated by gain and loss of function studies using cells transfected with either the wild type or mutant GSK3ß constructs. Intriguingly, pretreatment of cells with either the PI3K inhibitor or TRPC blocker resulted in failure of PDGF-CC to inactivate GSK3ß, thereby suggesting the intersection of PI3K and TRPC signaling at GSK3ß. Taken together our findings lead to the suggestion that PDGF-CC could be developed as a therapeutic target to reverse Tat-mediated neurotoxicity with implications for HAD.

Modulation of immunogenicity and immunoprotection of mucosal vaccine against coxsackievirus B3 by optimizing the coadministration mode of lymphotactin adjuvant.

Induction of potent mucosal immune response is a goal of current vaccine strategies against mucus-infectious pathogens such as Coxsackievirus B3 type (CVB3). We previously showed that administration of lymphotactin (LTN) as an adjuvant could enhance the specific immune responses against a mucosal gene vaccine, chitosan-pVP1, against CVB3. To optimize the coadministration mode of the mucosal adjuvant, we compared the mucosal immune responses induced by chitosan-DNA vaccine with different combinations of the target VP1 antigen gene and the adjuvant LTN gene. The two genes were either cloned in separate vectors or coexpressed as a fusion or bicistron protein in the same vector before encapsulation in chitosan nanoparticles. Four doses of various adjuvant-combined chitosan-DNA were intranasally administrated to mice before challenge with CVB3. The results indicated that chitosan-formulated pVP1-LTN fusion plasmid exhibited very weak improvement of CVB3-specific immune responses. Although the bicistronic coexpression of LTN with VP1 was expected to be powerful, this combination had enhanced effects on serum IgG and systemic T cell immune responses, but not on mucosal T cell immunity. Coimmunization with VP1 and LTN as separate chitosan-DNA formulation remarkably enhanced antibody and T cell immune responses both in systemic and mucosal immune compartments, leading to the most desirable preventive effect on viral myocarditis. Taken together, how the adjuvant is combined with the target antigen has a strong influence on the mucosal immune responses induced by mucosal DNA vaccines.

Reduced tumorigenesis of EG7 after interleukin-10 gene transfer and enhanced efficacy in combination with intratumorally injection of adenovirus-mediated lymphotactin and the underlying mechanism.

Although interleukin-10 (IL-10) is commonly regarded as an immunosuppressive cytokine, a wealth of evidence is accumulating that IL-10 also possesses some immunostimulating antitumor properties. Previous studies demonstrated that forced expression of the IL-10 gene in tumor cells could unexpectedly produce antitumor effects. In this study, we explored the tumorigenesis of EG7 cells transduced with IL-10 gene. In vivo, IL-10 gene transfer reduced tumorigenic capacity of EG7 cells and prolonged survival of the EG7 tumor-bearing mice. It was found that the cytotoxicities of cytotoxic T lymphocytes (CTL) and natural killer cells (NK cells) were enhanced. Assessment of the immune status of the animals showed prevalence of a systemic and tumor-specific Th2 response (high levels of IL-4 and IL-10). To improve the therapeutic efficacy, we combined with intratumoral injection of adenovirus-mediated lymphotactin (Ad-Lptn) into the overestablished EG7 tumor model. More significant inhibition of tumor growth were observed in EG7 tumor-bearing mice that received combined treatment with IL-10 and Lptn gene than those of mice treated with IL-10 or Lptn gene alone. The highest NK cells and CTL activity was induced in the combined therapy group, increasing the production of IL-2 and interferon-γ (IFN-γ) significantly but decreasing the expression of immune suppressive cells (CD4(+)Foxp3(+) Treg cells and Gr1(+)CD11b(+) MDSCs). The necrosis of tumor cells was markedly observed in the tumor tissues, accompanying with strongest expression of Mig (monokine induced by interferon-gamma) and IP-10 (interferon-inducible protein 10), weakest expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases-2 (MMP-2). In vivo, depletion analysis demonstrated that CD8(+) T cells and NK cells were the predominant effector cell subset responsible for the antitumor effect of IL-10 or Lptn gene. These findings may provide a potential strategy to improve the antitumor efficacy of IL-10 and Lptn.

A parenteral DNA vaccine protects against pneumonic plague.

The chemokine, lymphotactin (LTN), was tested as a molecular adjuvant using bicistronic DNA vaccines encoding the protective Yersinia capsular (F1) antigen and virulence antigen (V-Ag) as a F1-V fusion protein. The LTN-encoding F1-V or V-Ag vaccines were given by the intranasal (i.n.) or intramuscular (i.m.) routes, and although serum IgG and mucosal IgA antibodies (Abs) were induced, F1-Ag boosts were required for robust anti-F1-Ag Abs. Optimal efficacy against pneumonic plague was obtained in mice i.m.-, not i.n.-immunized with these DNA vaccines. These vaccines stimulated elevated Ag-specific Ab-forming cells and mixed Th cell responses, with Th17 cells markedly enhanced by i.m. immunization. These results show that LTN can be used as a molecular adjuvant to enhance protective immunity against plague.

Recombinant Fv-Hsp70 protein mediates neuroprotection after focal cerebral ischemia in rats.

BACKGROUND AND PURPOSE: This study investigated the effects of intravenous recombinant Fv-Hsp70 protein on infarction volume and behavior after experimental ischemic stroke. METHODS: Focal cerebral ischemia was produced by occluding the middle cerebral artery using the intraluminal suture technique. Rats subjected to 2 hours of focal ischemia were allowed to survive 24 hours. At 2(1/4) hours and 3 hours after onset of ischemia, Fv-Hsp70 recombinant protein (0.5 mg/kg) or saline was injected through the tail vein. Sensorimotor function and infarction volume were assessed at 24 hours after ischemia. RESULTS: Administration of Fv-Hsp70 after focal cerebral ischemia significantly decreased infarct volume by 68% and significantly improved sensorimotor function compared with the saline-treated control group. Western blots showed Fv-Hsp70 in ischemic but not in control brain; and Fv-Hsp70 suppressed endogenous Hsp70. CONCLUSIONS: Fv-Hsp70 protected the ischemic brain in this experimental stroke model.

Delivering cytokines at tumor site: The immunocytokine-conjugated anti-EDB-fibronectin antibody case.

Although considerable efforts have been made in the discovery of new agents for cancer treatment, several promising therapeutics cannot be applied systemically because of their severe side effects. This is the case for various recombinant pro-inflammatory cytokines that, despite their potent anti-cancer activity, can not find their way to clinical exploitation due to their devastating toxicity shown during dose escalation to therapeutically active concentrations. To circumvent these problems, an elegant and efficient way to accumulate therapeutic agents at the tumor site, thus reducing systemic side effects, is their conjugation to tumor-specific antibodies. Here, we review preclinical data about immunocytokines conjugated to a promising single-chain human antibody that selectively targets tumor-associated stroma and blood vessels by binding with high affinity and specificity to the extra domain-B (EDB) of fibronectin.

Targeted deletion of CCR2 impairs deep vein thombosis resolution in a mouse model.

CCR2 is required for monocyte recruitment in many inflammatory processes, as well as conferring Th1 lymphokine responses. Deep vein thrombosis (DVT) resolution represents a specific inflammatory response whereby the thrombus must be dissolved for restoration of blood flow. Using a stasis model of DVT in the mouse, we investigated the role of CCR2 on DVT resolution. Genetic deletion of CCR2 (CCR2-/-) was associated with larger thrombi at early and later time points, increased thrombus collagen, fewer thrombus monocytes (F4/80), and significantly impaired neovascularization. IL-2 and IFN-gamma were significantly reduced in early CCR2-/- thrombi, whereas MCP-1 was significantly increased, and Th2 lymphokines were unaffected. Supplementation of CCR2-/- mice with IFN-gamma normalized early thrombus resolution without increasing monocyte influx. Neither Ab depletion of IFN-gamma nor genetic deletion of IFN-gamma impaired early DVT resolution. Early fibrinolysis was not impaired in CCR2-/- mice, but a significant reduction in both matrix metalloproteinase (MMP)-2 and MMP-9 activity was observed. However, only MMP-9 activity was restored with administration of IFN-gamma. We conclude that an early CCR2-dependent Th1 lymphokine response predominates in normal DVT resolution, mediates this in part by MMP-9 activation, but is not solely dependent on IFN-gamma.

Dose-dependent neovascularization-promoting effect of adenovirus vector CI-1023 in a rat hindlimb ischaemic model.

CI-1023 (AdGVVEGF121.10) is a replication-deficient adenovirus vector (complete E1a-, partial E1b-, partial E3-) delivering human vascular endothelial growth factor-121 gene. Previous studies from this group have established that CI-1023 can successfully transfer human vascular endothelial growth factor-121 gene resulting in local tissue expression of vascular endothelial growth factor protein. The purpose of this study was to evaluate neovascularization-promoting potency and efficacy of CI-1023 in a wide dose range. In a rat hindlimb ischaemic model, we measured neovascularization-promoting effect of CI-1023 using three end-points: post mortem angiography, immuno-histochemistry and Laser Doppler scanning of tissue blood perfusion. Neovascularization-promoting activity of CI-1023 over the dose range of 4 x 10(6) pu-4 x 10(10) pu was evaluated. Our data demonstrated an obvious dose-dependent effect between 4 x 10(6) pu-4 x 10(8) pu. The neovascularizing effect is somewhat plateaued at the levels between 4 x 10(8) pu and 4 x 10(10) pu. We conclude CI-1023 is a potent neovascularization-promoting compound, with a dose-dependent effect between 4 x 10(6) pu-4 x 10(8) pu in the rat hindlimb ischaemic model.

Histologic and immunohistochemical characterization of tumor and inflammatory infiltrates in oral squamous cell carcinomas treated with local multikine immunotherapy: the macrophage at the front line.

Squamous cell carcinomas of the head and neck (SCCHN) are excellent candidates for local immunotherapy owing to their accessibility and their infiltration by mononuclear cells that are susceptible to immunomodulation. A response rate of 25-60% has been reported for treatment with natural IL-2 or a mixture of natural lymphokines. In the present study, biopsies and posttreatment excision specimens from nine patients with operable SCCHN treated systemically with a variety of immunomodulators and locally with natural lymphokines (multikine, CelSci) were analyzed in an attempt to correlate clinical response to histopathological and immunohistochemical changes. Formalin-fixed, paraffin-embedded tissues were stained with antibodies against lymphocytes (CD45, CD3, CD4, CD8, CD20), macrophages (CD68) including dendritic cells (S-100), markers for lymphocyte activation (CD30, HLA-DR), natural killer cells (CD56 and CD57), beta-2-microglobulin and keratin. One patient showed a complete response to treatment and two a partial response. Tumor size was significantly smaller after therapy. Clinical and pathological regression were more prominent in the smaller tumors. Numerous macrophages, both mononucleated and multinucleated, were present along the tumor-stroma interface in the posttreatment specimens of seven patients, most prominently in the three patients with tumor regression. The increase in the number of CD68+ and S-100+ macrophages after treatment was statistically significant. Lymphocytic infiltrates, which showed some increase following treatment, were composed of a mixture of T and B lymphocytes, the former mostly in contact with the tumor and the latter placed more peripherally. CD8+ lymphocytes extended into the tumors, whereas CD4+ lymphocytes showed minimal extension. Intensity of beta-2-microglobulin staining in tumors was significantly higher following therapy and associated with a better outcome. The marked increase in macrophages following treatment may indicate that the macrophage plays a major role in tumor recognition, destruction and clearance. An increase in the number of macrophages in a posttreatment specimen may indicate immunoresponsiveness.

Topical VEGF enhances healing of thoracic aortic anastomosis for coarctation in a rabbit model.

BACKGROUND: Recurrent stenosis after extended end-to-end anastomosis for aortic coarctation is the primary indication for further interventions in children. Tension because of the extended resection and local arterial wall hypoxia are possible pathogenetic mechanisms. We hypothesized that (1) tension interferes with healing and (2) that vascular endothelial growth factor (VEGF), a hypoxia sensitive angiogenic inducer, may enhance healing of the vascular anastomosis. METHODS AND RESULTS: In a model of coarctation repair, rabbits underwent thoracic aortic end-to-end anastomosis after transection (no-tension; n=15), resection of an aortic ring (tension; n=14) or resection and topical VEGF treatment (0.75 microg VEGF165; tension+VEGF; n=14). Gross and histologic characteristics of the aortic wall were assessed at 1 week, 1 and 2 months. In the tension only group at 1 month, the severity of vascular remodeling was increased with fibrosis and calcification compared with controls. At 2 months, this group also revealed more luminal stenosis (29% versus 19%; P<0.001). Exogenous VEGF resulted in significantly less fibrosis, calcification and chondroid metaplasia at 1 month (P<0.05) and luminal area was only reduced 3% at 2 months (P<0.001 versus tension group). CONCLUSIONS: In a rabbit model of coarctation repair, the addition of tension on the vascular anastomosis resulted in poor healing and luminal stenosis. Topical VEGF maintained luminal integrity by decreasing fibrosis and calcification. These findings suggest that topical VEGF may be a promising new strategy to enhance healing and improve the outcome of vascular anastomoses for coarctation of the aorta.

Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease: a phase II randomized, double-blind, controlled study of adenoviral delivery of vascular endothelial growth factor 121 in patients with disabling intermittent claudication.

BACKGROUND: "Therapeutic angiogenesis" seeks to improve perfusion by the growth of new blood vessels. The Regional Angiogenesis with Vascular Endothelial growth factor (RAVE) trial is the first major randomized study of adenoviral vascular endothelial growth factor (VEGF) gene transfer for the treatment of peripheral artery disease (PAD). METHODS AND RESULTS: This phase 2, double-blind, placebo-controlled study was designed to test the efficacy and safety of intramuscular delivery of AdVEGF121, a replication-deficient adenovirus encoding the 121-amino-acid isoform of vascular endothelial growth factor, to the lower extremities of subjects with unilateral PAD. In all, 105 subjects with unilateral exercise-limiting intermittent claudication during 2 qualifying treadmill tests, with peak walking time (PWT) between 1 to 10 minutes, were stratified on the basis of diabetic status and randomized to low-dose (4x10(9) PU) AdVEGF121, high-dose (4x10(10) PU) AdVEGF121, or placebo, administered as 20 intramuscular injections to the index leg in a single session. The primary efficacy end point, change in PWT (DeltaPWT) at 12 weeks, did not differ between the placebo (1.8+/-3.2 minutes), low-dose (1.6+/-1.9 minutes), and high-dose (1.5+/-3.1 minutes) groups. Secondary measures, including DeltaPWT, ankle-brachial index, claudication onset time, and quality-of-life measures (SF-36 and Walking Impairment Questionnaire), were also similar among groups at 12 and 26 weeks. AdVEGF121 administration was associated with increased peripheral edema. CONCLUSIONS: A single unilateral intramuscular administration of AdVEGF121 was not associated with improved exercise performance or quality of life in this study. This study does not support local delivery of single-dose VEGF121 as a treatment strategy in patients with unilateral PAD.

Behavioural and anatomical effects of systemically administered leukemia inhibitory factor in the SOD1(G93A G1H) mouse model of familial amyotrophic lateral sclerosis.

We investigated the anatomical and behavioural effects of daily intraperitoneal injection of 25 microg/kg of LIF in the SOD1(G93A G1H) mouse model of familial ALS. We found some subtle beneficial behavioural changes in LIF treated mice. These included later onset of clinical disease in females as determined by clinical scoring; better grip strength in males; and delayed development of motor impairment in males as determined by the rotarod test. However, we found no significant rescue of motoneurons or prolongation of survival as a result of this systemic dose of LIF in these mice.

Exogenous leukaemia inhibitory factor enhances nerve regeneration after late secondary repair using a bioartificial nerve conduit.

The clinical outcome of peripheral nerve injuries remains disappointing, even in the ideal situation of a primary repair performed with optimal microsurgical techniques. Primary repair is appropriate for only about 85% of injuries, and outcome is worse following secondary nerve repair, partly owing to the reduced regenerative potential of chronically axotomised neurons. Leukaemia inhibitory factor (LIF) is a gp-130 neurocytokine that is thought to act as an 'injury factor', triggering the early-injury phenotype within neurons and potentially boosting their regenerative potential after secondary nerve repair. At 2-4 months after sciatic nerve axotomy in the rat, 1 cm gaps were repaired using either nerve isografts or poly-3-hydroxybutyrate conduits containing a calcium alginate and fibronectin hydrogel. Regeneration was determined by quantitative immunohistochemistry 6 weeks after repair, and the effect of incorporating recombinant LIF (100 ng/ml) into the conduits was assessed. LIF increased the regeneration distance in repairs performed after both 2 months (69%, P=0.019) and 4 months (123%, P=0.021), and was statistically comparable to nerve graft. The total area of axonal immunostaining increased by 21% (P>0.05) and 63% (P>0.05), respectively. Percentage immunostaining area was not increased in the 2 months group, but increased by 93% in the repairs performed 4 months after axotomy. Exogenous LIF, therefore, has a potential role in promoting peripheral nerve regeneration after secondary repair, and can be effectively delivered within poly-3-hydroxybutyrate bioartificial conduits used for nerve repair.

Adenoviral-mediated gene transfer of vascular endothelial growth factor in critical limb ischemia: safety results from a phase I trial.

Critical limb ischemia (CLI) is typified by rest pain and/or tissue necrosis secondary to advanced peripheral arterial disease (PAD) and is characterized by diminution in limb perfusion at rest. We tested the safety of an angiogenic strategy with CI-1023 (Ad(GV)VEGF121.10), a replication-deficient adenovirus encoding human vascular endothelial growth factor isoform 121 in patients with CLI as part of a phase I trial. Fifteen subjects >35 years of age with CLI and angiographic disease involving the infra-inguinal vessels underwent intramuscular injection of CI-1023 (4 x 10(8) to 4 x 10(10) particle units, n = 13) or placebo (n = 2). All of the patients tolerated the injection well and there were no serious complications related to the procedure. Transient edema was noted in one patient. A total of 79 adverse events were reported over the course of one year. One death (day 136) and one malignancy (day 332) occurred in the CI-1023 group. CI-1023 appears to be well tolerated and safe for single-dose administration in patients with critical limb ischemia due to PAD. Further studies are needed to determine the efficacy of this form of therapeutic angiogenesis.