Safety of DNA and modified vaccinia virus Ankara vaccines against liver-stage P. falciparum malaria in non-immune volunteers.

A series of phase I clinical studies were conducted to evaluate the safety of plasmid DNA and modified vaccinia virus Ankara malaria vaccines. The vaccines each encoded a polyepitope string fused to whole Plasmodium falciparum TRAP antigen. Forty-three healthy adult volunteers received the vaccines alone or in DNA/MVA prime-boost combinations. The DNA vaccine was administered either intramuscularly by needle or intradermally by a needleless delivery device. The MVA vaccine was administered intradermally by needle. The vaccines were well-tolerated by all three routes and in various DNA/MVA immunisation regimes. There were no severe or serious adverse events.

In vivo antigen challenge in celiac disease identifies a single transglutaminase-modified peptide as the dominant A-gliadin T-cell epitope.

Celiac disease (CD) is an increasingly diagnosed enteropathy (prevalence, 1:200-1:300) that is induced by dietary exposure to wheat gliadins (as well as related proteins in rye and barley) and is strongly associated with HLA-DQ2 (alpha1*0501, beta1*0201), which is present in over 90% of CD patients. Because a variety of gliadin peptides have been identified as epitopes for gliadin-specific T-cell clones and as bioactive sequences in feeding studies and in ex vivo CD intestinal biopsy challenge, it has been unclear whether a 'dominant' T-cell epitope is associated with CD. Here, we used fresh peripheral blood lymphocytes from individual subjects undergoing short-term antigen challenge and tissue transglutaminase-treated, overlapping synthetic peptides spanning A-gliadin to demonstrate a transient, disease-specific, DQ2-restricted, CD4 T-cell response to a single dominant epitope. Optimal gamma interferon release in an ELISPOT assay was elicited by a 17-amino-acid peptide corresponding to the partially deamidated peptide of A-gliadin amino acids 57-73 (Q65E). Consistent with earlier reports indicating that host tissue transglutaminase modification of gliadin enhances gliadin-specific CD T-cell responses, tissue transglutaminase specifically deamidated Q65 in the peptide of A-gliadin amino acids 56-75. Discovery of this dominant epitope may allow development of antigen-specific immunotherapy for CD.

DNA-based vaccines for malaria: a heterologous prime-boost immunisation strategy.

A generic approach to inducing high level CD8+ T cell responses would be of value for prophylactic and therapeutic immunisation against several infectious diseases. However, it has been very difficult to achieve such immune responses using available vaccination strategies. Malaria is one of several diseases against which a new generation of better CD8+ T cell-inducing vaccines might be useful and is unusual in that it allows assessment of vaccine efficacy in small numbers of volunteers in carefully controlled challenge studies. Here we review the identification of a heterologous prime-boost regime using DNA priming and recombinant modified vaccinia Ankara (MVA) boosting that induces high level T cell responses in both mice and non-human primates. Clinical trials to determine whether this prime-boost approach is immunogenic in humans are in progress.

Gene gun intradermal DNA immunization followed by boosting with modified vaccinia virus Ankara: enhanced CD8+ T cell immunogenicity and protective efficacy in the influenza and malaria models.

In influenza and malaria, CD8+ T cells play an important role in protective immunity in mice. An immunization strategy consisting of DNA priming followed by boosting with recombinant modified vaccinia virus Ankara (MVA) induces complete protection, associated with high levels of CD8+ T cells, against Plasmodium berghei sporozoite challenge in mice. Intradermal delivery of DNA with a gene gun requires smaller amounts of DNA than intramuscular injection, in order to induce similar levels of immune responses. The present study compares both routes for the induction of specific CD8+ T cell responses and protection using different prime-boost immunization regimes in the influenza and the malaria models. In the DNA/MVA regime, equally high CD8+ T cell responses and levels of protection are achieved using ten times less DNA when delivered with a gene gun compared to intramuscular injection.

Induction of CD8+ T cells using heterologous prime-boost immunisation strategies.

One of the current challenges in vaccine design is the development of antigen delivery systems or vaccination strategies that induce high protective levels of CD8+ T cells. These cells are crucial for protection against certain tumours and intracellular pathogens such as the liver-stage parasite of malaria. A liver-stage malaria vaccine should therefore include CD8+ T-cell-inducing components. This review provides an overview of prime-boost immunisation strategies that result in protective CD8+ T-cell responses against malaria with an emphasis on work from our laboratory. Possible mechanisms explaining why heterologous prime-boost strategies, in particular boosting with replication-impaired recombinant poxviruses, are so effective are discussed.

Intradermal DNA immunization of mice against influenza A virus using the novel PowderJect system.

The PowderJect system, a device that uses compressed helium gas to accelerate microscopic particles into the skin, was used as a delivery system for DNA vaccines to elicit a virus-specific cytotoxic T cell response (CTL) in mice. Transient expression of beta-galactosidase (beta-Gal) was observed in the epidermis when gold particles coated with beta-Gal expression plasmid were delivered to mouse skin with the device. When DNA encoding the nucleoprotein gene (NP) of influenza A virus was used to coat gold particles, a strong and specific anti-NP CTL response was elicited by immunizations with nanogram amounts of the NP DNA vector. This study shows the potential for application of the PowderJect system to intradermal delivery of DNA in order to elicit an immune response.

Amylin (islet amyloid polypeptide) inhibition of insulin release in the perfused rat pancreas: implication of the adenylate cyclase/cAMP system.

Amylin inhibits glucose-induced insulin secretion in the rat pancreas. To study the mechanism by which amylin acts on the B-cell, we have investigated, in the perfused rat pancreas, the effect of synthetic rat amylin (75 pM) on insulin release elicited by secretagogues acting on the B-cell via the adenylate cyclase/cAMP system, i.e., glucagon (10 nM), gastric inhibitory polypeptide (GIP, 1 nM), forskolin (1 microM) and isobutylmethylxanthine (IBMX, 75 microM). In addition, we examined the effect of amylin on GIP-induced insulin release in pancreata from rats pretreated with pertussis toxin, an agent which inactivates certain Gi proteins coupled to adenylate cyclase. Amylin inhibited the insulin response to glucagon (approx. 70%), GIP (approx. 90%), IBMX (approx. 75%) as well as the early phase of forskolin-induced insulin output (approx. 74%). However, amylin failed to modify GIP-induced insulin release in pancreata obtained from pertussis toxin pretreated rats. These results would indicate that the inhibitory effect of amylin on insulin secretion could be, at least in part, attributed to its interfering with the adenylate cyclase/cAMP system. Furthermore, prevention of the inhibitory effect of amylin on GIP-induced insulin output by pertussis toxin pretreatment, supports the concept that amylin can inhibit insulin release via a pertussis toxin-sensitive Gi protein coupled to the adenylate cyclase system.

Determination of the molecular mass of the native beta-cell sulfonylurea receptor.

In the present study we have determined the molecular mass of the beta-cell sulfonylurea receptor in its native form by two different experimental approaches; gel filtration chromatography and radiation inactivation analysis. We first confirmed that the denatured photolabelled MIN6 beta-cell receptor had a molecular size of 141 +/- 2 kDa (mean +/- S.E., n = 8). Under non-denaturing conditions, using gel filtration chromatography, apparent molecular masses of 166 +/- 1 kDa (mean +/- S.E., n = 3) and 182 +/- 5 kDa (mean +/- S.E., n = 4) were determined for the photoaffinity-labelled and unlabelled sulfonylurea receptor, respectively. We conclude that in the solubilized state the receptor exists as a monomer. Radiation inactivation analysis indicated that the receptor has a target size of 250 +/- 30 kDa (mean +/- S.E., n = 7). This value for the molecular mass is larger than that obtained from SDS-PAGE following photolabelling of the receptor (141 kDa) suggesting that the beta-cell sulfonylurea receptor is composed of more than one subunit in the native membrane.

Reversal of the inhibitory effects of calcitonin gene-related peptide (CGRP) and amylin on insulin secretion by the 8-37 fragment of human CGRP.

The 8-37 fragment of human calcitonin gene-related peptide [(8-37)hCGRP] antagonizes the effects of calcitonin gene-related peptide (CGRP) and amylin in a number of tissues. We have studied the influence of (8-37)hCGRP on the effects of both CGRP and amylin on insulin secretion. In the perfused rat pancreas, homologous CGRP and amylin, at 75 pM, exerted comparable inhibitory effects on the insulin response to 9 mM glucose (ca. 70%; P < 0.025). These effects were antagonized by (8-37)hCGRP (1 microM). Our results suggest that CGRP and amylin act on the B-cell, at least in part, through a common receptor.

Amylin inhibits glucose-induced insulin secretion in a dose-dependent manner. Study in the perfused rat pancreas.

Islet amyloid polypeptide (IAPP), also called amylin, has been localized in the B-cell secretory granule and is co-secreted with insulin. We have investigated the effect of synthetic amidated rat amylin on the insulin release evoked by 9 mM glucose in the isolated, perfused rat pancreas. Amylin, in a range of 75 nM-75 pM, significantly inhibited this insulin response in a dose-dependent manner. The correlation between the logarithm of amylin concentrations and the percentages of inhibition was highly significant (r = 0.98, P < 0.01). The lowest effective amylin concentration tested (75 pM) is within the range of amylin levels reported for the effluent of the perfused rat pancreas. Finally, pre-infusion of the rat pancreas with a high amylin concentration (75 nM) did not alter the insulin response to glucose, thus excluding a toxic effect of amylin on the B-cell. These observations support the concept that amylin plays a role in the control of insulin secretion.

Effects of rat pancreatic polypeptide on islet-cell secretion in the perfused rat pancreas.

Pancreatic polypeptide (PP) secretory cells are abundant in the islets of Langerhans. Results concerning the effects of exogenous PP on islet-cell secretion are controversial. This might be due in part to species specificity, given that most reports refer to studies performed using PP of bovine, porcine, or human origin in a heterologous animal model. Thus, we have investigated the influence of synthetic rat PP (80 nmol/L) on unstimulated insulin, glucagon, and somatostatin release, and on the responses of these hormones to glucose (11 mmol/L) and to arginine (3.5 mmol/L) in a homologous animal model, the perfused rat pancreas. Infusion of rat PP (rPP) reduced unstimulated insulin release by 35% (P = .03), and the insulin responses to glucose by 65% (P = .029) and to arginine by 50% (P = .026), without modifying glucagon output. rPP did not affect somatostatin secretion, either in unstimulated conditions or in the presence of 11 mmol/L glucose. However, it induced a clear-cut increase in somatostatin release during 3.5 mmol/L arginine infusion. Our observation that rPP inhibited insulin secretion without affecting glucagon and somatostatin output points to a direct effect of PP on B-cell function. However, during aminogenic priming of the D cell, the inhibition of insulin output induced by rPP was accompanied by an increase in somatostatin release. Thus, in this circumstance, it might be considered that the blocking effect of PP on B-cell secretion could be, at least in part, mediated by a D-cell paracrine effect.

Homologous pancreastatin inhibits insulin secretion without affecting glucagon and somatostatin release in the perfused rat pancreas.

The identification of pancreastatin in pancreatic extracts prompted the investigation of its effects on islet cell function. However, in most of the investigations to date, pig pancreastatin was tested in heterologous species. Since there is great interspecies variability in the amino acid sequence of pancreastatin, we have investigated the influence of rat pancreastatin on insulin, glucagon and somatostatin secretion in a homologous animal model, namely the perfused rat pancreas. During 5.5 mM glucose infusion, pancreastatin (40 nM) inhibited insulin secretion (ca. 40%, P less than 0.025) as well as the insulin responses to 10 mM arginine (ca. 50%, P less than 0.025) and to 1 nM vasoactive intestinal polypeptide (ca. 50%; P less than 0.05). Pancreastatin failed to significantly modify glucagon or somatostatin release under any of the above experimental conditions. In addition, a lower pancreastatin concentration (15.7 nM) markedly suppressed the insulin release evoked by 11 mM glucose (ca. 85%, P less than 0.05). Our present observations reinforce the concept that pancreastatin is an effective inhibitor of insulin secretion, influencing the B-cell function directly and not through an A-cell or D-cell paracrine effect.

Inhibition of insulin release by amylin is not mediated by changes in somatostatin output.

We have investigated the effect of a high concentration (750 nM) of synthetic amidated rat amylin on unstimulated somatostatin and insulin secretion as well as on the response of these hormones to arginine. Amylin consistently reduced insulin output but it did not significantly modify somatostatin release. These findings indicate that the inhibitory effect of amylin on insulin secretion is not mediated by a D-cell paracrine effect.

Inhibitory effect of rat amylin on the insulin responses to glucose and arginine in the perfused rat pancreas.

Amylin, a 37-amino acid polypeptide, is the main component of amyloid deposits in the islets of Langerhans, and has been identified in the B-cell secretory granules. We have investigated the effect of rat amylin on the insulin and glucagon release by the isolated, perfused rat pancreas. Amylin infusion at 750 nM, markedly reduced unstimulated insulin release (ca. 50%, P less than 0.025), whereas it did not modify glucagon output. At the same concentration, amylin also blocked the insulin response to 9 mM glucose (ca. 80%, P less than 0.025) without affecting the suppressor effect of glucose on glucagon release. The inhibitory effect of amylin on glucose-induced insulin secretion was confirmed by lowering the amylin concentration (500 nM) and increasing the glucose stimulus (11 mM); again, no effect of amylin on glucagon release was observed. Finally, amylin, at 500 nM, reduced the insulin response to 3.5 mM arginine (ca. 40%, P less than 0.025) without modifying the secretion of glucagon elicited by this amino acid. It can be concluded that, in the rat pancreas, the inhibitory effect of homologous amylin on unstimulated insulin secretion, as well as on the insulin responses to metabolic substrates (glucose and arginine), favours the concept of this novel peptide as a potential diabetogenic agent.

Inhibition of insulin and somatostatin secretion and stimulation of glucagon release by homologous galanin in perfused rat pancreas.

Results of studies on the effects of exogenous galanin on islet cell secretion are controversial. Until recently, only pig galanin has been available, and structural dissimilarities among the galanin molecules of different species might have contributed to discrepancies among the study results. Thus, we investigated the influence of synthetic rat galanin (50 nM) on unstimulated insulin, glucagon, and somatostatin release and on the responses of these hormones to arginine (10 mM), glucose (16.6 mM), and vasoactive intestinal polypeptide (VIP; 1 nM) in a homologous animal model, the perfused rat pancreas. In addition, the effect of an equimolar concentration of pig galanin on arginine-induced islet cell secretion was examined. Infusion of rat galanin reduced unstimulated insulin release (approximately 60%, P less than 0.01) and the insulin responses to arginine (approximately 30%, P less than 0.025), glucose (100%, P less than 0.01), and VIP (approximately 80%, P less than 0.025). Galanin also inhibited unstimulated somatostatin secretion (approximately 15%, P less than 0.05) and virtually abolished the somatostatin output evoked by arginine, glucose, and VIP. Conversely, rat galanin increased unstimulated glucagon output (approximately 20%, P less than 0.05), potentiated the glucagon response to arginine (approximately 50%, P less than 0.05) and VIP (approximately 90%, P less than 0.05), and counteracted the suppressor effect of glucose on alpha-cell secretion. Pig galanin inhibited the insulin output elicited by arginine (approximately 45%, P less than 0.05) but did not affect the somatostatin and glucagon responses to the aminogenic stimulus. In conclusion, the opposite effects of galanin on insulin and glucagon secretion favor the concept of galanin as a diabetogenic agent. Galanin also behaves as a potent inhibitor of somatostatin release. Finally, the importance of using homologous galanin to study the biological activity of this peptide must be emphasized.

An evaluation based theorem prover.

A noninductive method for mechanical theorem proving is presented, which deals with a recursive class of theorems involving iterative functions and predicates. The method is based on the symbolic evaluation of the formula to be proved and requires no inductive step. Induction is avoided since a metatheorem is proved which establishes the conditions on the evaluation of any formula which are sufficient to assure that the formula actually holds. The proof of a supposed theorem consists in evaluating the formula and checking the conditions. The method applies to assertions that involve element-by-element checking of typed homogeneous sequences which are hierarchically constructed out of the primitive type consisting of the truth values. The sequences can be computed by means of iterative and ``accumulator'' functions. The paper includes the definition of a simple typed iterative language in which both predicates and functions are expressed. The language precisely defines the scope of the proof method. The method proves a wide variety of theorems about iterative functions on sequences, including that which states that REVERSE is its own inverse, and that it can be inversely distributed on APPEND, that FLATTEN can be distributed on APPEND and that each element of any sequence is a MEMBER of the sequence itself. Although the method is not complete, it does provide the basis for an extremely efficient tool to be used in a complete mechanical theorem prover.