Recombinant polio-rhinovirus immunotherapy for recurrent paediatric high-grade glioma: a phase 1b trial.

BACKGROUND: Outcomes of recurrent paediatric high-grade glioma are poor, with a median overall survival of less than 6 months. Viral immunotherapy, such as the polio-rhinovirus chimera lerapolturev, is a novel approach for treatment of recurrent paediatric high-grade glioma and has shown promise in adults with recurrent glioblastoma. The poliovirus receptor CD155 is ubiquitously expressed in malignant paediatric brain tumours and is a treatment target in paediatric high-grade glioma. We aimed to assess the safety of lerapolturev when administered as a single dose intracerebrally by convection enhanced delivery in children and young people with recurrent WHO grade 3 or grade 4 glioma, and to assess overall survival in these patients. METHODS: This phase 1b trial was done at the Duke University Medical Center (Durham, NC, USA). Patients aged 4-21 years with recurrent high-grade malignant glioma (anaplastic astrocytoma, glioblastoma, anaplastic oligoastrocytoma, anaplastic oligodendroglioma, or anaplastic pleomorphic xanthoastrocytoma) or anaplastic ependymoma, atypical teratoid rhabdoid tumour, or medulloblastoma with infusible disease were eligible for this study. A catheter was tunnelled beneath the scalp for a distance of at least 5 cm to aid in prevention of infection. The next day, lerapolturev at a dose of 5 × 107 median tissue culture infectious dose in 3 mL infusate loaded in a syringe was administered via a pump at a rate of 0·5 mL per h as a one-time dose. The infusion time was approximately 6·5 h to compensate for volume of the tubing. The primary endpoint was the proportion of patients with unacceptable toxic effects during the 14-day period after lerapolturev treatment. The study is registered with ClinicalTrials.gov, NCT03043391. FINDINGS: Between Dec 5, 2017, and May 12, 2021, 12 patients (11 unique patients) were enrolled in the trial. Eight patients were treated with lerapolturev. The median patient age was 16·5 years (IQR 11·0-18·0), five (63%) of eight patients were male and three (38%) were female, and six (75%) of eight patients were White and two (25%) were Black or African American. The median number of previous chemotherapeutic regimens was 3·50 (IQR 1·25-5·00). Six of eight patients had 26 treatment-related adverse events attributable to lerapolturev. There were no irreversible (ie, persisted longer than 2 weeks) treatment-related grade 4 adverse events or deaths. Treatment-related grade 3 adverse events included headaches in two patients and seizure in one patient. Four patients received low-dose bevacizumab on-study for treatment-related peritumoural inflammation or oedema, diagnosed by both clinical symptoms plus fluid-attenuated inversion recovery MRI. The median overall survival was 4·1 months (95% CI 1·2-10·1). One patient remains alive after 22 months. INTERPRETATION: Convection enhanced delivery of lerapolturev is safe enough in the treatment of recurrent paediatric high-grade glioma to proceed to the next phase of trial. FUNDING: Solving Kids Cancer, B+ Foundation, Musella Foundation, and National Institutes of Health.

Central nervous system monoamine metabolite response to alcohol exposure is associated with future alcohol intake in a nonhuman primate model (Macaca mulatta).

It is widely held that the central monoamine neurotransmitters modulate alcohol intake. Few studies, however, directly assess the relationship between baseline and alcohol-induced monoamine turnover, as well as the change from baseline, as predictors of alcohol intake. Using a nonhuman primate model, this study investigates baseline, alcohol-induced and alcohol-induced change in monoamine activity and their relationship with alcohol intake. Alcohol-naïve, adolescent rhesus macaques (Macaca mulatta, N = 114) were administered a standardized intravenous bolus of alcohol solution (16.8%, v/v) on two occasions, approximately 1 month apart. One month prior to and 1 h following each alcohol infusion, cisternal cerebrospinal fluid (CSF) was obtained and assayed for monoamine metabolite concentrations. Approximately 6-7 months later, subjects were allowed unfettered access to an aspartame-sweetened alcohol solution (8.4%, v/v) for 1 h/day, 5 days/week, over 5-7 weeks. Results showed strong positive correlations between baseline and post-infusion CSF monoamine metabolite concentrations, indicating a trait-like response. Low baseline and post-infusion serotonin and dopamine metabolite concentrations and a smaller change in serotonin and dopamine metabolites from one infusion to the next were associated with higher alcohol intake. Low baseline and post-infusion norepinephrine metabolite concentrations predicted high alcohol intake, but unlike the other monoamines, a greater change in norepinephrine metabolite concentrations from one infusion to the next was associated with higher alcohol intake. These findings suggest that individual differences in naturally occurring and alcohol-induced monoamine activity, as well as the change between exposures, are important modulators of initial alcohol consumption and may play a role in the risk for excessive alcohol intake.

Alginate/κ-Carrageenan-Based Edible Films Incorporated with Clove Essential Oil: Physico-Chemical Characterization and Antioxidant-Antimicrobial Activity.

This study aimed to enhance the properties of CaCl2 crosslinked sodium alginate/k-carrageenan (SA/KC) incorporated with clove essential oil (CEO). An evaluation of the modification effects on physicochemical, morphological, antioxidant, and antibacterial properties was performed. The properties were observed at various SA/KC ratios (10/0 to 1.5/1), CEO (1.5% to 3%), and CaCl2 (0% to 2%). The surface morphology was improved by addition of KC and CaCl2. The Fourier transform infrared (FTIR) result showed insignificant alteration of film chemical structure. The X-ray diffraction (XRD) result confirmed the increased crystallinity index of the film by CaCl2 addition. On physicochemical properties, a higher proportion of SA/KC showed the declined tensile strength, meanwhile both elongation at break and water solubility were increased. The incorporated CEO film reduced both tensile strength and water solubility; however, the elongation at break was significantly increased. The presence of Ca2+ ions remarkably increased the tensile strength despite decreased water solubility. Overall, the addition of KC and CaCl2 helped in repairing the mechanical properties and flexibility. CEO incorporation showed the effectiveness of profiling the antioxidant and antimicrobial activity indicated by high 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity up to 90.32% and inhibition zone of E. coli growth up to 113.14 mm2.

Very low mutation burden is a feature of inflamed recurrent glioblastomas responsive to cancer immunotherapy.

Several immunotherapy clinical trials in recurrent glioblastoma have reported long-term survival benefits in 10-20% of patients. Here we perform genomic analysis of tumor tissue from recurrent WHO grade IV glioblastoma patients acquired prior to immunotherapy intervention. We report that very low tumor mutation burden is associated with longer survival after recombinant polio virotherapy or after immune checkpoint blockade in recurrent glioblastoma patients. A relationship between tumor mutation burden and survival is not observed in cohorts of immunotherapy naïve newly diagnosed or recurrent glioblastoma patients. Transcriptomic analyses reveal an inverse relationship between tumor mutation burden and enrichment of inflammatory gene signatures in cohorts of recurrent, but not newly diagnosed glioblastoma tumors, implying that a relationship between tumor mutation burden and tumor-intrinsic inflammation evolves upon recurrence.

Correlation of whole-bone marrow dual-time-point 18F-FDG, as measured by a CT-based method of PET/CT quantification, with response to treatment in newly diagnosed multiple myeloma patients.

The practical application of dual-time-point-imaging (DTPI) technique still remains controversial. One of the issues is that current parameters of DTPI quantification suffer from some deficiencies, mainly limited sampling of the diseased sites by confining measurements to specific locations. We aimed to examine the correlation between the percent change from early to delayed scans in whole-bone marrow (WBM) 18F-FDG uptake, as measured by a CT-based method of PET/CT quantification, and response to treatment in multiple myeloma (MM) patients. Pre-treatment 18F-FDG-PET/CT scans of 36 newly diagnosed MM patients were collected in a prospective study at 1 h and 3 h post tracer injection (NCT02187731). A threshold algorithm based on bone Hounsfield units on CT was applied to segment and quantify WBM 18F-FDG uptake. Patients were separated into two treatment groups: high-dose therapy with autologous stem cell transplant (HDT) and non-high dose therapy (non-HDT). The International Response Criteria for MM patients was used to determine each patient's response to treatment. In the HDT group, WBM 18F-FDG uptake increased significantly in patients that had a poor response to treatment, from a median of 1.31 (IQR: 1.13-1.64) at 1 h to a median of 1.85 (1.45-2.10) at 3 h. The median percent change was 37.77% (IQR: 23.47-46.4), with a range of 6.10-50.73 (P = 0.003). However, no significant change in uptake was observed in patients with a complete response (P = 0.24). The same trend was observed for the non-HDT group. WBM uptake of 18F-FDG assessed with dual-time-point imaging may have a role in predicting treatment response in MM.

Comparison of 18F-sodium fluoride uptake in the whole bone, pelvis, and femoral neck of multiple myeloma patients before and after high-dose therapy and conventional-dose chemotherapy.

AIM: To compare the effects of high-dose therapy (HDT consisting of high-dose chemotherapy followed by autologous stem cell transplantation) and conventional-dose chemotherapy (non-HDT) on the uptake of 18F-sodium fluoride (NaF) in the whole bone, pelvis, and femoral neck of multiple myeloma (MM) patients. METHOD: The data of 19 MM patients who received HDT (61.5 (SD 5.6) years) and 11 MM patients who received conventional-dose chemotherapy (70.9 (SD 7.2) years) were collected in a prospective study. NaF PET/CT imaging was performed at baseline, and 8 weeks and 2 weeks after treatment for the HDT group and the non-HDT group, respectively. A CT-based algorithm was applied to segment the bones, and the global mean SUV (GSUVmean) of the whole bone and pelvis was calculated (OsiriX MD v.9.0, Pixmeo SARL; Bernex, Switzerland). In addition, regions of interest for the whole, medial, and lateral femoral neck were delineated bilaterally. Whole bone and pelvis measurements were replicated by two observers. RESULTS: The average GSUVmean in the whole bone and pelvis of the patients who underwent HDT significantly decreased from before to after treatment (- 16.27%, p = 0.02 and - 16.54%, p = 0.01, respectively). A significant decrease in the whole and lateral femoral neck was also observed bilaterally in the HDT group. No significant decrease in average GSUVmean was observed in the non-HDT group. A high level of inter-observer reliability was found in intra-class correlation (ICC for pre-treatment whole bone 0.983, post-treatment whole bone 0.989, pre-treatment whole pelvis 0.998, post-treatment whole pelvis 0.996). CONCLUSION: NaF uptake significantly decreased after treatment in patients who received high-dose therapy. A high level of agreement was observed between two operators for whole bone and pelvis measurements.

Sacroiliac Joint Asymmetry Regarding Inflammation and Bone Turnover: Assessment by FDG and NaF PET/CT.

OBJECTIVES: This study was undertaken to determine the role of computed tomography (CT)-based methodology to segment the SI joint and quantify the metabolic activity using positron emission tomography (PET). We measured tracer uptake in the right and left SI joints independently to look for differences between the two sides. Further, we correlated tracer uptake with BMI and studied the inter-observer variation with regard to estimated tracer uptake in the SI joints. METHODS: In this retrospective study, a total of 103 subjects (48 females, 55 males) from the CAMONA study database collected 2012-2016 at Odense University Hospital in Denmark were included. Mean age was 48±14.59 years, mean BMI was 26.68±4.31 kg/m2. The SI joints were segmented on fused PET/CT images using a 3D growing algorithm with adjustable upper and lower Hounsfield Units (HU) thresholds. The metabolic activities on the two sides were correlated with BMI. RESULTS: For FDG, we found a higher average SUVmean on the right side (right: 1.3±0.33, left: 1.13±0.30; <0.0001). Similarly, for NaF, the uptake was higher on the right side (right: 5.9±1.29, left: 4.27±1.23; <0.0001). Positive correlations were present between BMI and FDG uptake (P<0.01) as well as NaF uptake (P<0.01). CONCLUSION: The PET-based molecular imaging probes along with the CT-based segmentation techniques revealed a significant difference in the metabolic activity between the two SI joints with higher inflammation and reactive bone formation on the right side. FDG and NaF uptakes correlated significantly and positively with BMI.

Evolving Role of PET-Based Novel Quantitative Techniques in the Management of Hematological Malignancies.

"The role of 18F-fluorodeoxyglucose PET/computed tomography in hematological malignancies continues to expand in disease diagnosis, staging, and management. A key advantage of PET over other imaging modalities is its ability to quantify tracer uptake, which can be used to determine degree of disease activity. Although tracer uptake with PET is conventionally measured in focal lesions, novel quantitative techniques are being investigated that set objective protocols and produce robust parameters that represent total disease activity portrayed by PET. This article discusses recent advances in PET quantification that can improve reliability and accuracy of characterizing hematological malignancies."

Efficacy of 18F-FDG and 18F-NaF PET/CT imaging: A novel semi-quantitative assessment of the effects of age and obesity on hip joint inflammation and bone degeneration.

OBJECTIVE: Osteoarthritis (OA) is characterized by synovial tissue inflammation and underlying bone degeneration in the joints. Aging and obesity are among the major risk factors. This study evaluated the effects of aging and body mass index (BMI) on hip joint inflammation and bone degeneration using fluorine-18-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) and fluorine-18 sodium fluoride (18F-NaF) PET/CT imaging, respectively. SUBJECTS AND METHODS: In this retrospective study, a total of 116 subjects (58 males and 58 females) who had undergone both 18F-FDG and 18F-NaF PET/CT imaging were analyzed. The mean age of these subjects was 48.6±14.5 with an age range of 21-75 years. Fluorine-18-FDG and 18F-NaF PET/CT imaging was conducted 180min and 90min (respectively) after intravenous administration of the appropriate tracer. The hip joint was segmented on fused PET/CT images using OsiriX MD v.9.5 (DICOM viewer and image-analysis program, Pixmeo SARL; Bernex, Switzerland). The region of interest (ROI) for the hip joint was indicated by using a 3D-growing region algorithm with upper/lower Hounsfield Units (HU) followed by a morphological closing algorithm. The metabolic activity for the left and right side of the joint was measured and correlated with age and BMI. RESULTS: Fluorine-18-FDG uptake in the hip was 0.83±0.22 (right side: 0.83±0.23, left side: 0.83±0.22, P=0.82). Fluorine-18-NaF uptake in the hip was 3.20±1.07 (right side: 3.25±1.14, left side: 3.15±1.04, P=0.02). Body mass index positively correlated with both 18F-FDG (r=0.29, P=0.001) and NaF (r=0.26, P=0.005) uptake. No significant correlation was seen between age and either 18F-FDG (r=0.12, P=0.19) or 18F-NaF (r=0.03, P=0.78) uptake. CONCLUSION: Body mass index had a significant impact on 18F-FDG and 18F-NaF uptake, whereas age had no correlation with either tracer uptake. Obesity increases the mechanical forces applied on weight-bearing joints such as the hip. Body mass index was related to increased joint inflammation and bone degeneration. These findings further support the studies explaining the role of adipose tissue in promoting OA.

CT-based tissue segmentation to assess knee joint inflammation and reactive bone formation assessed by 18F-FDG and 18F-NaF PET/CT: Effects of age and BMI.

OBJECTIVE: This study was conducted to determine the role of computed tomography (CT)-based segmentation methodology to semi-quantify the degree of inflammation and reactive bone formation in the knee joints by fluorine-18-fluorodeoxyglucose (18F-FDG) and 18F-sodium fluoride positron emission tomography/CT (18F-NaF PET/CT) imaging, respectively. Furthermore, we assessed the impact of aging and body mass index (BMI) on these biological responses. SUBJECTS AND METHODS: In this retrospective study, we examined a total of 97 subjects who had undergone both 18F-FDG and 18F-NaF PET/CT scanning. The mean age was 49.3±14.9 (21-75) and the mean BMI was 26.7±4.3 (17.7-42.0). Whole joint compartments and osseous compartments were segmented on fused PET/CT images using a 3D-growing algorithm with an adjustable upper/lower Hounsfield Units (HU) thresholds and manual tools. The metabolic activity and volume of each compartment was measured, values from the osseous compartment were subtracted from the whole joint to get the volume and metabolic activity of the soft tissue. The metabolic activity was correlated with age and BMI. RESULTS: Fluorine-18-FDG uptake in the soft tissues surrounding the joint was 0.35±0.07 while 0.19±0.04 in the osseous structures (P<0.0001). Aging positively correlated with 18F-FDG uptake in the soft tissue (r=0.37, P=0.0001). Body mass index positively correlated with 18F-FDG uptake in the soft tissue (r=0.53, P<0.0001), osseous compartment (r=0.58, P<0.0001) and 18F-NaF uptake in the joint (r=0.37, P=0.0001). A positive association was noted between the degree of new bone formation and the inflammatory reaction (P<0.01). CONCLUSION: The PET-based molecular imaging probes along with the CT-based segmentation techniques revealed an association between aging and the inflammatory activity of the soft tissue compartment. Similarly, a positive correlation was noted between BMI and inflammation and reactive bone f ormation of the knee joint compartments.

Recurrent Glioblastoma Treated with Recombinant Poliovirus.

BACKGROUND: The prognosis of patients with recurrent World Health Organization (WHO) grade IV malignant glioma is dismal, and there is currently no effective therapy. We conducted a dose-finding and toxicity study in this population of patients, evaluating convection-enhanced, intratumoral delivery of the recombinant nonpathogenic polio-rhinovirus chimera (PVSRIPO). PVSRIPO recognizes the poliovirus receptor CD155, which is widely expressed in neoplastic cells of solid tumors and in major components of the tumor microenvironment. METHODS: We enrolled consecutive adult patients who had recurrent supratentorial WHO grade IV malignant glioma, confirmed on histopathological testing, with measurable disease (contrast-enhancing tumor of ≥1 cm and ≤5.5 cm in the greatest dimension). The study evaluated seven doses, ranging between 107 and 1010 50% tissue-culture infectious doses (TCID50), first in a dose-escalation phase and then in a dose-expansion phase. RESULTS: From May 2012 through May 2017, a total of 61 patients were enrolled and received a dose of PVSRIPO. Dose level -1 (5.0×107 TCID50) was identified as the phase 2 dose. One dose-limiting toxic effect was observed; a patient in whom dose level 5 (1010 TCID50) was administered had a grade 4 intracranial hemorrhage immediately after the catheter was removed. To mitigate locoregional inflammation of the infused tumor with prolonged glucocorticoid use, dose level 5 was deescalated to reach the phase 2 dose. In the dose-expansion phase, 19% of the patients had a PVSRIPO-related adverse event of grade 3 or higher. Overall survival among the patients who received PVSRIPO reached a plateau of 21% (95% confidence interval, 11 to 33) at 24 months that was sustained at 36 months. CONCLUSIONS: Intratumoral infusion of PVSRIPO in patients with recurrent WHO grade IV malignant glioma confirmed the absence of neurovirulent potential. The survival rate among patients who received PVSRIPO immunotherapy was higher at 24 and 36 months than the rate among historical controls. (Funded by the Brain Tumor Research Charity and others; ClinicalTrials.gov number, NCT01491893 .).

Association and expression of the antigen-processing gene PSMB8, coding for low-molecular-mass protease 7, with vitiligo in North India: case-control study.

BACKGROUND: Vitiligo is a multifactorial, autoimmune, depigmenting disorder of the skin where aberrant presentation of autoantigens may have a role. OBJECTIVES: To study the association of two antigen-processing genes, PSMB8 and PSMB9, with vitiligo. METHODS: In total 1320 cases of vitiligo (1050 generalized and 270 localized) and 752 healthy controls were studied for the PSMB9 exon 3 G/A single-nucleotide polymorphism (SNP), PSMB8 exon 2 C/A SNP and PSMB8 intron 6 G/T SNP at site 37 360 using polymerase chain reaction (PCR)-restriction fragment length polymorphism. Real-time PCR was used for transcriptional expression of PSMB8 and cytokines. Expression of ubiquitinated proteins and phosphorylated-p38 (P-p38) was studied by Western blotting. RESULTS: Significant increases in PSMB8 exon 2 allele A (P < 2.07 × 10-6 , odds ratio 1·93) and genotypes AA (P < 1.03 × 10-6 , odds ratio 2·51) and AC (P < 1.29 × 10-6 , odds ratio 1·63) were observed in patients with vitiligo. Interferon-γ stimulation induced lower expression of PSMB8 in peripheral blood mononuclear cells of cases compared with controls, suggesting impaired antigen processing, which was confirmed by accumulation of ubiquitinated proteins in both lesional and nonlesional skin of patients with vitiligo. Expression of proinflammatory cytokines - interleukin (IL)-6, IL-1ß and IL-8 - was higher in the lesional skin. P-p38 expression was variable but correlated with the amount of ubiquitinated proteins in the lesional and nonlesional skin, suggesting that the inflammatory cytokine responses in lesional skin could be a result of both P-p38-dependent and -independent pathways. CONCLUSIONS: The PSMB8 exon 2 SNP is significantly associated with vitiligo. Accumulation of ubiquitinated proteins in skin of cases of vitiligo suggests their aberrant processing, which may promote the development of the disease.

Quantification of chondroitin sulfate and dermatan sulfate in danaparoid sodium by (1)H NMR spectroscopy and PLS regression.

Danaparoid sodium (the active pharmaceutical ingredient in Orgaran; Merck Sharp and Dohme) is a biopolymeric non-heparin drug used as anticoagulant and antithrombotic agent approved for the prophylaxis of post-operative deep-vein thrombosis, which may lead to pulmonary embolism in patients undergoing, e.g., elective hip replacement surgery. It consists of a mixture of three glycosaminoglycans (GAGs): heparan sulfate (HS), dermatan sulfate (DS), and chondroitin sulfate (CS). Currently, the CS and DS content are quantified by means of a time-consuming enzymatic method. In this paper the use of (1)H NMR in combination with multivariate regression (partial least-squares, PLS) is proposed as a new method. In order to evaluate the proposed method, a series of danaparoid sodium samples were analyzed and the results were compared with those obtained by the enzymatic method (reference method). The results showed that the proposed (1)H NMR method is a good alternative for analysis of CS and DS in danaparoid sodium. Accuracy of ±0.7% (w/w) and ±1.1% (w/w) for CS and DS was obtained by the (1)H NMR method and accuracy of ±1.0% (w/w) and ±1.3% (w/w) by the enzymatic method. Furthermore, the use of (1)H NMR in combination with PLS results in a fast quantification. The analysis time is reduced to 35 min per sample instead of 60 h for a maximum of 16 samples.

Detection of antibody to the foot-and-mouth disease virus (FMDV) non-structural polyprotein 3ABC in sheep by ELISA.

The specificity and sensitivity of an ELISA for detecting IgG to the 3ABC non-structural protein of foot-and-mouth disease (FMD) virus was evaluated in FMD naive, aerosol-infected, aerosol plus direct contact infected and field-exposed sheep. All 12 sheep that were experimentally infected without prior vaccination seroconverted in the test, although fewer field sera from FMD-exposed sheep were scored seropositive compared to test results for structural protein antibodies. The 3ABC test specificity was 98 or 100% according to whether sera reacting in the doubtful range were scored as positive or negative. The test was then used to investigate the antibody response of sheep vaccinated against FMD and exposed to the virus by an aerosol challenge 4-14 days later. The response of individual animals varied. Whether immunised with high or low doses of vaccine, the development of 3ABC antibody was most likely in sheep from which live virus was recovered at or beyond 9 days post-challenge. Non-structural responses were also more frequent in animals from which multiple incidences of live FMD virus isolation (perhaps more indicative of true virus replication) were demonstrated.

Emergency vaccination of sheep against foot-and-mouth disease: protection against disease and reduction in contact transmission.

The ability of several emergency FMD vaccine formulations to elicit early protective immunity in sheep was examined. All vaccine formulations were shown to protect sheep against airborne challenge with homologous FMDV within 4 days of vaccination. Protection was associated in part with the induction of serum antibody responses but was also demonstrated in the absence of any detectable antibody response at the time of challenge. Aqueous Al(OH)3/saponin vaccine formulations and oil emulsion vaccines based on Montanide ISA 206 adjuvant reduced virus replication and the numbers of animals subclinically infected up to 28 days post-challenge, when compared with non-vaccinated animals, consequently limiting transmission of the disease or infection to in-contact susceptible animals.

The Drosophila melanogaster-related angiotensin-I-converting enzymes Acer and Ance--distinct enzymic characteristics and alternative expression during pupal development.

Drosophila melanogaster express two distinct angiotensin-I-converting enzymes (ACEs) called Ance and Acer, which display a high level of primary structure similarity. We have expressed Acer in the yeast Pichia pastoris and purified the recombinant enzyme with a view to developing biochemical tools to distinguish between Acer and Ance. Purified Acer and Ance expressed in yeast were used to raise anti-Acer Ig and anti-Ance Ig that specifically cross-reacted with the respective enzyme on immunoblotting, but did not act as specific inhibitors. Acer cleaves the C-terminal dipeptides from benzoylglycyl-histidyl-leucine and [Leu5]enkephalin, and Acer and Ance are both able to act as endopeptidases, releasing the C-terminal dipeptideamide from [Leu5]enkephalinamide. However, Acer hydrolyses this substrate at a slightly faster rate than [Leu5]enkephalin, whereas Ance hydrolyses the peptide with a free C-terminus with a kcat 15-fold higher than [Leu5]enkephalinamide. In addition, Acer did not cleave angiotensin I. In contrast, Ance hydrolysed 25% of this substrate at an 8-fold lower enzyme concentration. Furthermore, Acer did not hydrolyse the synthetic substrates Phe-Ser-Pro-Arg-Leu-Gly-Arg-Arg and Phe-Ser-Pro-Arg-Leu-Gly-Lys-Arg, two partially processed putative locustamyotropin precursors, under conditions where Ance produced 82% substrate hydrolysis. Acer was inhibited by captopril, trandolaprilat and enalaprilat, with apparent Ki values in the nanomolar range, whereas lisinopril and fosinoprilat were less potent. We show that the two Drosophila ACEs are alternatively expressed in stages P1 (white puparium)-P15 (eclosion) of pupal development; Ance is expressed predominantly during stages P4-P7, whereas the ACE activity expressed during stages P9-P12 is mainly due to Acer suggesting different roles for the two enzymes during pupal development.

Emergency vaccination of pigs against foot-and-mouth disease: protection against disease and reduction in contact transmission.

The protective ability of two novel oil-based FMD vaccines in pigs was examined. Both vaccine formulations were shown to protect pigs against airborne challenge with homologous FMDV within four days of vaccination, but not at two and three days post-vaccination. Protection was associated with the induction of variable and low titre serum antibody responses. A transmission study showed that protective immunisation resulted in reduced virus excretion. Vaccination at seven days, but not at four days, prior to challenge prevented contact transmission of FMD. The two formulations tested in this study have the favourable characteristics of low viscosity, low reactivity and high potency emergency FMD vaccines for use in strategic vaccination campaigns to assist the control of outbreaks of FMD.