Sexual Dimorphism in Alternative Metabolic Pathways of L-Arginine in Circulating Leukocytes in Young People with Type 1 Diabetes Mellitus.

Background: Sexual dimorphism in specific biochemical pathways and immune response, underlies the heterogeneity of type 1 diabetes mellitus (T1DM) and affects the outcome of immunotherapy. Arginase and nitric oxide (NO) synthase (NOS) metabolize L-arginine and play opposite roles in the immune response and autoimmune processes.Objective: We hypothesized that the above mentioned enzymes can be involved in sex and age differences in T1DM and its treatment. Based on this, the enzymes have been studied in peripheral blood leukocytes (PBL) and plasma of young people with T1DM.Methods: Patients were recruited from Muratsan University Hospital (Yerevan, Armenia) and were divided into groups: girls and boys by age, from children to adolescents and adolescents/young adults with recent-onset T1DM (RO-T1DM) (0.1-1 years) and long-term T1DM (LT-T1DM) (1.6-9.9 years). Arginase activity was assessed by L-arginine-dependent production of L-ornithine, and the NOS activity was assessed by NO/nitrite production. Glycemic control was assessed using hemoglobin A1c test. Plasma HbA1c concentration below 7.5% (median (range) 6.7 [6.2-7.5]) was taken as good glycemic control (+) and above 7.5% (median (range) 10.5 [7.6-13]) as poor glycemic control (-). Healthy volunteers with corresponding sex and age were used as the control group.Results: All the patients with RO-T1DM, with poor glycemic control, had increased arginase activity in the cytoplasm (cARG) and mitochondria (mARG) in PBL. In girls with RO-T1DM, with good glycemic control, the subcellular arginase activity decreased, and normalized in LT-T1DM, regardless of age. In contrast, boys from both age groups showed high arginase activity, regardless of glycemic control and duration of T1DM along with insulin therapy. At the same time, a significant decrease in the subcellular production of bioavailable NO was observed in children/preadolescents, regardless of glycemic control and duration of diabetes. In adolescents/young adult boys with RO-T1DM, with (-), the subcellular production of NO decreased significantly, and with LT-T1DM, the decrease was attenuated, but even with (+) remained lower than in healthy people. In contrast, in the group of same age girls with RO-T1DM, NO production increased above normal in both cellular compartments, while with LT-T1DM it normalized in the cytoplasm. In adolescents/young adults with LT-T1DM, NO production in PBL mitochondria decreased by almost a half, regardless of glycemic control and gender. Changes in the metabolic pathways of L-arginine in plasma differed and were less substantial than in the PBL cellular compartments in T1DM.Conclusions: Glycemic status and duration of T1DM along with insulin therapy affect the activity of arginase and NOS-dependent production of bioavailable NO in the cytoplasm and mitochondria in PBL of young patients with T1DM, depending on sex and age. Arginase and NOS can directly affect the processes occurring in the pancreas and the outcome of therapy through infiltrated leukocytes. Obtained data can be useful for understanding the heterogeneity of T1DM and using it to develop available criteria for assessing the severity and treatment of autoimmune diabetes.

Application of a glycinated bile acid biomarker for diagnosis and assessment of response to treatment in Niemann-pick disease type C1.

Niemann-Pick disease type C (NPC) is a neurodegenerative disease in which mutation of NPC1 or NPC2 gene leads to lysosomal accumulation of unesterified cholesterol and sphingolipids. Diagnosis of NPC disease is challenging due to non-specific early symptoms. Biomarker and genetic tests are used as first-line diagnostic tests for NPC. In this study, we developed a plasma test based on N-(3ß,5α,6ß-trihydroxy-cholan-24-oyl)glycine (TCG) that was markedly increased in the plasma of human NPC1 subjects. The test showed sensitivity of 0.9945 and specificity of 0.9982 to differentiate individuals with NPC1 from NPC1 carriers and controls. Compared to other commonly used biomarkers, cholestane-3ß,5α,6ß-triol (C-triol) and N-palmitoyl-O-phosphocholine (PPCS, also referred to as lysoSM-509), TCG was equally sensitive for identifying NPC1 but more specific. Unlike C-triol and PPCS, TCG showed excellent stability and no spurious generation of marker in the sample preparation or aging of samples. TCG was also elevated in lysosomal acid lipase deficiency (LALD) and acid sphingomyelinase deficiency (ASMD). Plasma TCG was significantly reduced after intravenous (IV) 2-hydroxypropyl-ß-cyclodextrin (HPßCD) treatment. These results demonstrate that plasma TCG was superior to C-triol and PPCS as NPC1 diagnostic biomarker and was able to evaluate the peripheral treatment efficacy of IV HPßCD treatment.

Immunogenicity, efficacy, safety, and mechanism of action of epitope vaccine (Lu AF20513) for Alzheimer's disease: prelude to a clinical trial.

The Alzheimer's disease (AD) process is understood to involve the accumulation of amyloid plaques and tau tangles in the brain. However, attempts at targeting the main culprits, neurotoxic Aß peptides, have thus far proven unsuccessful for improving cognitive function. Recent clinical trials with passively administrated anti-Aß antibodies failed to slow cognitive decline in mild to moderate AD patients, but suggest that an immunotherapeutic approach could be effective in patients with mild AD. Using an AD mouse model (Tg2576), we tested the immunogenicity (cellular and humoral immune responses) and efficacy (AD-like pathology) of clinical grade Lu AF20513 vaccine. We found that Lu AF20513 induces robust "non-self" T-cell responses and the production of anti-Aß antibodies that reduce AD-like pathology in the brains of Tg2576 mice without inducing microglial activation and enhancing astrocytosis or cerebral amyloid angiopathy. A single immunization with Lu AF20513 induced strong humoral immunity in mice with preexisting memory T-helper cells. In addition, Lu AF20513 induced strong humoral responses in guinea pigs and monkeys. These data support the translation of Lu AF20513 to the clinical setting with the aims of: (1) inducing therapeutically potent anti-Aß antibody responses in patients with mild AD, particularly if they have memory T-helper cells generated after immunizations with conventional tetanus toxoid vaccine, and (2) preventing pathological autoreactive T-cell responses.

Self-concept in children with Down syndrome.

Self-concept is a critical indicator of quality of life but few studies have examined this subject in children with Down syndrome (DS). In this study, we propose a novel methodology to assess the self-concept of children with DS by analyzing their responses towards two dolls, one with a "typically developing" (TD) appearance and one with the phenotypic features of DS. Fifty-four children with DS participated in play sessions with both dolls and were then interviewed to assess doll preference, resemblance, and attribution of positive qualities. We observed that children with DS: (i) exhibited a preference for the TD doll regardless of age, gender, IQ or self-awareness; (ii) attributed more positive qualities to the TD doll than the DS doll; and (iii) believed that they resembled the TD doll, rather than the more phenotypically accurate representation of themselves. Older participants were more likely to exhibit self-recognition by this technique. These findings contribute to current understandings of how people with DS view themselves and their disability.

Delivery of a DNA vaccine for Alzheimer's disease by electroporation versus gene gun generates potent and similar immune responses.

BACKGROUND: Induction of a humoral response against amyloid-ß peptide may be beneficial for Alzheimer's disease (AD) patients and may alleviate the onset and progression of AD. DNA-based vaccination provides a unique alternative method of immunization for treatment and prevention of AD. Currently, the two major delivery methods used for enhancing DNA uptake and immune responses to DNA vaccines in humans are electroporation (EP) and gene gun (GG). OBJECTIVE: The goal of this translational study was to evaluate the efficacy of an AD DNA epitope vaccine (DepVac) delivered intramuscularly by EP or intradermally by GG. METHODS: Humoral and cellular immune responses to immunization with DepVac were evaluated by ELISA and ELISPOT, respectively. Functional activity of the antibodies was also assessed. RESULTS: EP- and GG-mediated immunizations with DepVac induced similar anti-amyloid-ß (Aß) antibody and T cell responses. Anti-Aß antibodies bound to amyloid plaques in AD brain tissue and to toxic forms of Aß(42) peptide. CONCLUSION: Both delivery methods are effective at promoting potent antibodies specific for Aß.

The immunological potency and therapeutic potential of a prototype dual vaccine against influenza and Alzheimer's disease.

BACKGROUND: Numerous pre-clinical studies and clinical trials demonstrated that induction of antibodies to the ß-amyloid peptide of 42 residues (Aß42) elicits therapeutic effects in Alzheimer's disease (AD). However, an active vaccination strategy based on full length Aß42 is currently hampered by elicitation of T cell pathological autoreactivity. We attempt to improve vaccine efficacy by creating a novel chimeric flu vaccine expressing the small immunodominant B cell epitope of Aß42. We hypothesized that in elderly people with pre-existing memory Th cells specific to influenza this dual vaccine will simultaneously boost anti-influenza immunity and induce production of therapeutically active anti-Aß antibodies. METHODS: Plasmid-based reverse genetics system was used for the rescue of recombinant influenza virus containing immunodominant B cell epitopes of Aß42 (Aß1-7/10). RESULTS: Two chimeric flu viruses expressing either 7 or 10 aa of Aß42 (flu-Aß1-7 or flu-Aß1-10) were generated and tested in mice as conventional inactivated vaccines. We demonstrated that this dual vaccine induced therapeutically potent anti-Aß antibodies and anti-influenza antibodies in mice. CONCLUSION: We suggest that this strategy might be beneficial for treatment of AD patients as well as for prevention of development of AD pathology in pre-symptomatic individuals while concurrently boosting immunity against influenza.

Cancer-testis antigen, BORIS based vaccine delivered by dendritic cells is extremely effective against a very aggressive and highly metastatic mouse mammary carcinoma.

Here, we analyze for the first time the immunological and therapeutic efficacy of a dendritic cell (DC) vaccine based on a cancer-testis antigen, Brother of regulator of imprinted sites (BORIS), an epigenetically acting tumor-promoting transcription factor. Vaccination of mice with DC loaded with truncated form of BORIS (DC/mBORIS) after 4T1 mammary tumor implantation induced strong anti-cancer immunity, inhibited tumor growth (18.75% of mice remained tumor-free), and dramatically lowered the number of spontaneous clonogenic metastases (50% of mice remained metastases-free). Higher numbers of immune effector CD4 and CD8 T cells infiltrated the tumors of vaccinated mice vs. control animals. Vaccination significantly decreased the number of myeloid-derived suppressor cells (MDSCs) infiltrating the tumor sites, but not MDSCs in the spleens of vaccinated animals. These data suggest that DC-based mBORIS vaccination strategies have significant anti-tumor activity in a therapeutic setting and will be more effective when combined with agents to attenuate tumor-associated immune suppression.

Abnormally increased carotid intima media-thickness and elasticity in patients with Morquio A disease.

BACKGROUND: Cardiovascular disease frequently causes morbidity and mortality in mucopolysaccharidoses (MPS); however, cardiovascular anatomy and dysfunction in MPS IVA (Morquio A disease) is not well described. Consequently, the study aimed to compare carotid artery structure and elasticity of MPS IVA patients with other MPS patients and healthy control subjects, and quantitate frequency of MPS IVA cardiac structural and functional abnormalities. METHODS: Prospective, multi-center echocardiogram and carotid ultrasound evaluations of 12 Morquio A patients were compared with other MPS and healthy control subjects. Average differences between groups were adjusted for age, sex, and height with robust variance estimation for confidence intervals and P-values. RESULTS: Morquio A patients demonstrated significantly higher (P < 0.001) adjusted carotid intima-media thickness (cIMT), mean (SD) of 0.56 mm (0.03) compared to control subjects, 0.44 mm (0.04). The Morquio A cohort had significantly greater adjusted carotid elasticity (carotid cross-sectional compliance + 43%, P < 0.001; carotid incremental elastic modulus - 33%, P = 0.003) than control subjects and other MPS patients. Aortic root dilatation was noted in 56% of the Morquio A cohort, which also had highly prevalent mitral (73%) and aortic (82%) valve thickening, though hemodynamically significant valve dysfunction was less frequent (9%). CONCLUSIONS: Increased carotid elasticity in Morquio A patients is an unexpected contrast to the reduced elasticity observed in other MPS. These Morquio A cIMT findings corroborate MPS IVA arterial post-mortem reports and are consistent with cIMT of other MPS. Aortic root dilatation in Morquio A indicates arterial elastin dysfunction, but their carotid hyperelasticity indicates other vascular intima/media components, such as proteoglycans, may also influence artery function. Studying MPS I and IVA model systems may uniquely illuminate the function of glycosaminoglycan-bearing proteoglycans in arterial health.

Alzheimer's disease peptide epitope vaccine reduces insoluble but not soluble/oligomeric Abeta species in amyloid precursor protein transgenic mice.

Active vaccination of elderly Alzheimer's disease (AD) patients with fibrillar amyloid-beta peptide (Abeta42), even in the presence of a potent Th1 adjuvant, induced generally low titers of antibodies in a small fraction (approximately 20% responders) of those that received the AN-1792 vaccine. To improve the immunogenicity and reduce the likelihood of inducing adverse autoreactive T-cells specific for Abeta42, we previously tested in wild-type mice an alternative approach for active immunization: an epitope vaccine that selectively initiate B cell responses toward an immunogenic self-epitope of Abeta in the absence of anti-Abeta T cell responses. Here, we describe a second generation epitope vaccine composed of two copies of Abeta(1-11) fused with the promiscuous nonself T cell epitope, PADRE (pan human leukocyte antigen DR-binding peptide) that completely eliminates the autoreactive T cell responses and induces humoral immune responses in amyloid precursor protein transgenic 2576 mice with pre-existing AD-like pathology. Based on the titers of anti-Abeta(1-11) antibody experimental mice were divided into low, moderate and high responders, and for the first time we report a positive correlation between the concentration of anti-Abeta(1-11) antibody and a reduction of insoluble, cerebral Abeta plaques. The reduction of insoluble Abeta deposition was not associated with adverse events, such as CNS T cell or macrophage infiltration or microhemorrhages. Surprisingly, vaccination did not alter the levels of soluble Abeta. Alternatively, early protective immunization before substantial neuropathology, neuronal loss and cognitive deficits have become firmly established may be more beneficial and safer for potential patients, especially if they can be identified in a preclinical stage by the development of antecedent biomarkers of AD.

Mannan-Abeta28 conjugate prevents Abeta-plaque deposition, but increases microhemorrhages in the brains of vaccinated Tg2576 (APPsw) mice.

BACKGROUND: New pre-clinical trials in AD mouse models may help to develop novel immunogen-adjuvant configurations with the potential to avoid the adverse responses that occurred during the clinical trials with AN-1792 vaccine formulation. Recently, we have pursued an alternative immunization strategy that replaces QS21 the Th1 type adjuvant used in the AN-1792 clinical trial with a molecular adjuvant, mannan that can promote a Th2-polarized immune response through interactions with mannose-binding and CD35/CD21 receptors of the innate immune system. Previously we established that immunization of wild-type mice with mannan-Abeta28 conjugate promoted Th2-mediated humoral and cellular immune responses. In the current study, we tested the efficacy of this vaccine configuration in amyloid precursor protein (APP) transgenic mice (Tg2576). METHODS: Mannan was purified, activated and chemically conjugated to Abeta28 peptide. Humoral immune responses induced by the immunization of mice with mannan-Abeta28 conjugate were analyzed using a standard ELISA. Abeta42 and Abeta40 amyloid burden, cerebral amyloid angiopathy (CAA), astrocytosis, and microgliosis in the brain of immunized and control mice were detected using immunohistochemistry. Additionally, cored plaques and cerebral vascular microhemorrhages in the brains of vaccinated mice were detected by standard histochemistry. RESULTS: Immunizations with low doses of mannan-Abeta28 induced potent and long-lasting anti-Abeta humoral responses in Tg2576 mice. Even 11 months after the last injection, the immunized mice were still producing low levels of anti-Abeta antibodies, predominantly of the IgG1 isotype, indicative of a Th2 immune response. Vaccination with mannan-Abeta28 prevented Abeta plaque deposition, but unexpectedly increased the level of microhemorrhages in the brains of aged immunized mice compared to two groups of control animals of the same age either injected with molecular adjuvant fused with an irrelevant antigen, BSA (mannan-BSA) or non-immunized mice. Of note, mice immunized with mannan-Abeta28 showed a trend toward elevated levels of CAA in the neocortex and in the leptomeninges compared to that in mice of both control groups. CONCLUSION: Mannan conjugated to Abeta28 provided sufficient adjuvant activity to induce potent anti-Abeta antibodies in APP transgenic mice, which have been shown to be hyporesponsive to immunization with Abeta self-antigen. However, in old Tg2576 mice there were increased levels of cerebral microhemorrhages in mannan-Abeta28 immunized mice. This effect was likely unrelated to the anti-mannan antibodies induced by the immunoconjugate, because control mice immunized with mannan-BSA also induced antibodies specific to mannan, but did not have increased levels of cerebral microhemorrhages compared with non-immunized mice. Whether these anti-mannan antibodies increased the permeability of the blood brain barrier thus allowing elevated levels of anti-Abeta antibodies entry into cerebral perivascular or brain parenchymal spaces and contributed to the increased incidence of microhemorrhages remains to be investigated in the future studies.

DNA epitope vaccine containing complement component C3d enhances anti-amyloid-beta antibody production and polarizes the immune response towards a Th2 phenotype.

We have engineered a DNA epitope vaccine that expresses 3 self-B cell epitopes of Abeta(42) (3Abeta(1-11)), a non-self T helper (Th) cell epitope (PADRE), and 3 copies of C3d (3C3d), a component of complement as a molecular adjuvant, designed to safely reduce CNS Abeta. Immunization of mice with 3Abeta(1-11)-PADRE epitope vaccine alone generated only moderate levels of anti-Abeta antibodies and a pro-inflammatory T helper (Th1 phenotype) cellular immune response. However, the addition of 3C3d to the vaccine construct significantly augmented the anti-Abeta humoral immune response and, importantly, shifted the cellular immune response towards the potentially safer anti-inflammatory Th2 phenotype.

Immunostimulant adjuvant patch enhances humoral and cellular immune responses to DNA immunization.

The focus of this report is on the development of an improved DNA immunization protocol, which takes advantage of the strengths of DNA immunization, as well as those associated with adjuvant delivered by transcutaneous immunostimulatory (IS) patches. Because transcutaneous delivery of adjuvants to the skin at the vaccination site has been shown to amplify the immune response to protein antigens, we hypothesized that the same IS patch when placed on the skin at the site of DNA injection could further enhance the immune response to a DNA influenza vaccine. We have combined an influenza DNA vaccine, hemagglutinin fused with three copies of complement C3d, to enhance uptake and antigen presentation, with an IS patch containing heat-labile enterotoxin from Escherichia coli. Coadministration of a potent adjuvant in IS patches placed on the skin at the site of DNA vaccination dramatically amplifies anti-influenza antibody immune response. Supplementing DNA vaccines with IS patches may be a particularly valuable strategy because DNA vaccines can be rapidly modified in response to mutations in pathogens, and individuals with compromised immune systems such as transplant patients and the elderly will benefit from the enhanced antibody response induced by the IS patches.

Case Report: Ursodeoxycholic acid treatment in Niemann-Pick disease type C; clinical experience in four cases.

In this case series, we demonstrate that Ursodeoxycholic acid (UDCA) improves liver dysfunction in Niemann-Pick type C (NPC) and may restore a suppressed cytochrome p450 system. NPC disease is a progressive neurodegenerative lysosomal storage disease caused by mutations in either the NPC1 or NPC2 genes. Liver disease is a common feature presenting either acutely as cholestatic jaundice in the neonatal period, or in later life as elevated liver enzymes indicative of liver dysfunction. Recently, an imbalance in bile acid synthesis in a mouse model of NPC disease was linked to suppression of the P450 detoxification system and was corrected by UDCA treatment. UDCA (3α, 7ß-dihydroxy-5ß-cholanic acid), a hydrophilic bile acid, is used to treat various cholestatic disorders. In this report we summarise the findings from four independent cases of NPC, three with abnormal liver enzyme levels at baseline, that were subsequently treated with UDCA. The patients differed in age and clinical features, they all tolerated the drug well, and in those with abnormal liver function, there were significant improvements in their liver enzyme parameters.

Abeta-immunotherapy for Alzheimer's disease using mannan-amyloid-Beta peptide immunoconjugates.

In Alzheimer's disease (AD) the accumulation of pathological forms of the beta-amyloid (Abeta) peptide are believed to be causal factors in the neurodegeneration that results in the loss of cognitive function in patients. Anti-Abeta antibodies have been shown to reduce Abeta levels in transgenic mouse models of AD and in AN-1792 clinical trial on AD patients; however, the clinical trial was halted when some patients developed meningoencephalitis. Theories on the cause of the adverse events include proinflammatory "primed patients," a Th1-inducing adjuvant, and Abeta autoreactive T cells. New immunotherapy approaches are being developed to eliminate these putative risk factors. Mannan, which is recognized by pattern recognition receptors of the innate immune system, can be utilized as a molecular adjuvant to promote a Th2-mediated immune response to conjugated B cell epitopes. The N-terminus of Abeta was conjugated to mannan, and used to immunize mice with low concentrations of immunoconjugate, without a conventional adjuvant. Mannan induced a significant and highly polarized toward Th2 phenotype anti-Abeta antibody response not only in BALB/c, but also in B6SJL F1 mice. New preclinical trials in AD mouse models may help to develop novel immunogen-adjuvant configurations with the potential to avoid the adverse immune response that occurred in the first clinical trial.

Refinement of a DNA based Alzheimer's disease epitope vaccine in rabbits.

We previously demonstrated that our second-generation DNA-based Alzheimer disease (AD) epitope vaccine comprising three copies of a short amyloid-ß (Aß) B cell epitope, Aß 11 fused with the foreign promiscuous Th epitope, PADRE (p3Aß 11-PADRE) was immunogenic in mice. However, since DNA vaccines exhibit poor immunogenicity in large animals and humans, in this study, we sought to improve the immunogenicity of p3Aß 11-PADRE by modifying this vaccine to express protein 3Aß 11-PADRE with a free N-terminal aspartic acid fused with eight additional promiscuous Th epitopes. Generated pN-3Aß 11-PADRE-Thep vaccine has been designated as AV-1955. We also delivered this vaccine using the TriGrid electroporation system to improve the efficiency of DNA transfection. This third-generation DNA epitope vaccine was evaluated for immunogenicity in rabbits in comparison to the parent construct p3Aß 11-PADRE. AV-1955 vaccination induced significantly stronger humoral immune responses in rabbits compared with p3Aß 11-PADRE vaccine. Anti-Aß 11 antibodies recognized all forms of human ß-amyloid peptide (monomers, oligomers and fibrils), bound to amyloid plaques in brain sections from an AD case and reduced oligomer- and fibril-mediated cytotoxicity ex vivo. These findings suggest that AV-1955 could represent an effective DNA epitope vaccine for AD therapy, pending safety and efficacy studies that are currently being conducted in Rhesus monkeys.

Down syndrome and dementia: seizures and cognitive decline.

The objective of this study was to determine the association of seizures and cognitive decline in adults with Down syndrome (DS) and Alzheimer's-type dementia. A retrospective data analysis was carried out following a controlled study of antioxidant supplementation for dementia in DS. Observations were made at baseline and every 6 months for 2 years. Seizure history was obtained from study records. The primary outcome measures comprised the performance-based Severe Impairment Battery (SIB) and Brief Praxis Test (BPT). Secondary outcome measures comprised the informant-based Dementia Questionnaire for Mentally Retarded Persons and Vineland Adaptive Behavior Scales. Because a large proportion of patients with seizures had such severe cognitive decline as to become untestable on the performance measures, time to "first inability to test" was measured. Adjustments were made for the potentially confounding co-variates of age, gender, APOE4 status, baseline cognitive impairment, years since dementia onset at baseline, and treatment assignment. The estimated odds ratio for the time to "first inability to test" on SIB comparing those with seizures to those without is 11.02 (95% CI: 1.59, 76.27), a ratio that is significantly different from 1 (p = 0.015). Similarly, we estimated an odds ratio of 9.02 (95% CI: 1.90, 42.85) on BPT, a ratio also significantly different than 1 (p = 0.006). Results from a secondary analysis of the informant measures showed significant decline related to seizures. We conclude that there is a strong association of seizures with cognitive decline in demented individuals with DS. Prospective studies exploring this relationship in DS are indicated.

Low concentrations of anti-Aß antibodies generated in Tg2576 mice by DNA epitope vaccine fused with 3C3d molecular adjuvant do not affect AD pathology.

It has been demonstrated that an active vaccination strategy with protein- or DNA-based epitope vaccines composed of the immunodominant self B cell epitope of amyloid-ß42 (Aß42) and a non-self T helper (Th) cell epitope is an immunotherapeutic approach to preventing or treating Alzheimer's disease (AD). As a DNA-based epitope vaccine, we used a plasmid encoding three copies of Aß(1-11) and Th cell epitope, PADRE (p3Aß(1-11)-PADRE). We have previously reported that three copies of component of complement C3d (3C3d) acts as a molecular adjuvant significantly enhancing immune responses in wild-type mice of the H2(b) haplotype immunized with p3Aß(1-11)-PADRE. Here, we tested the efficacy of p3Aß(1-11)-PADRE and the same vaccine fused with 3C3d (p3Aß(1-11)-PADRE-3C3d) in a transgenic (Tg) mouse model of AD (Tg2576) of the H2(bxs) immune haplotype. The overall responses to both vaccines were very weak in Tg2576 mice despite the fact that the 3C3d molecular adjuvant significantly enhanced the anti-Aß response to 3Aß(1-11)-PADRE. Importantly, generation of low antibody responses was associated with the strain of amyloid precursor protein Tg mice rather than with a molecular adjuvant, as a p3Aß(1-11)-PADRE-3C3d vaccine induced significantly higher antibody production in another AD mouse model, 3xTg-AD of the H2(b) haplotype. Finally, this study demonstrated that low concentrations of antibodies generated by both DNA vaccines were not sufficient for the reduction of Aß pathology in the brains of vaccinated Tg2576 animals, confirming previous reports from preclinical studies and the AN-1792 clinical trials, which concluded that the concentration of anti-Aß antibodies may be essential for the reduction of AD pathology.

Detection of the active components of calf thymus nuclear proteins (TNP), histones that are binding with high affinity to HIV-1 envelope proteins and CD4 molecules.

VGV-1, a clinical-grade formulation of bovine thymus nuclear protein (TNP) has been demonstrated to possess anti-viral activity in HIV-1 patients in five clinical trials, one of which was placebo controlled double-blinded. However, to date molecular mechanisms remain to be identified. Using surface plasmon resonance we observed TNP components bind with high affinity to HIV-1 proteins involved in viral entry, gp41 and pg120, as well as the T cell HIV-1 receptor CD4. To identify protein components of TNP, gel electrophoresis was performed followed by tandem mass spectrometry (MS/MS). Searching of bovine protein databases revealed the presence of numerous histones. Further analysis of TNP by immunoaffinity chromatography using gp120 and CD4 molecules as targets followed by gel electrophoresis and MS/MS analysis confirmed these data, demonstrating that H1.1, H2B, H4, and H2A histones are the active component of TNP that bind to HIV envelop glycoprotein and its receptor. To conclusively demonstrate binding of histones to target proteins, we repeated the surface plasmon resonance experiments using commercially available bovine histones and demonstrated high-affinity interaction of histones with gp120, and CD4. The binding of histone proteins to CD4, as well as viral molecules has profound implications for basic understanding of immune functions as well as a possible mechanism of VGV-1 activity in AIDS patients.

Reducing AD-like pathology in 3xTg-AD mouse model by DNA epitope vaccine - a novel immunotherapeutic strategy.

BACKGROUND: The development of a safe and effective AD vaccine requires a delicate balance between providing an adequate anti-Abeta antibody response sufficient to provide therapeutic benefit, while eliminating an adverse T cell-mediated proinflammatory autoimmune response. To achieve this goal we have designed a prototype chemokine-based DNA epitope vaccine expressing a fusion protein that consists of 3 copies of the self-B cell epitope of Abeta(42) (Abeta(1-11)) , a non-self T helper cell epitope (PADRE), and macrophage-derived chemokine (MDC/CCL22) as a molecular adjuvant to promote a strong anti-inflammatory Th2 phenotype. METHODS AND FINDINGS: We generated pMDC-3Abeta(1-11)-PADRE construct and immunized 3xTg-AD mouse model starting at age of 3-4 months old. We demonstrated that prophylactic immunizations with the DNA epitope vaccine generated a robust Th2 immune response that induced high titers of anti-Abeta antibody, which in turn inhibited accumulation of Abeta pathology in the brains of older mice. Importantly, vaccination reduced glial activation and prevented the development of behavioral deficits in aged animals without increasing the incidence of microhemorrhages. CONCLUSIONS: Data from this transitional pre-clinical study suggest that our DNA epitope vaccine could be used as a safe and effective strategy for AD therapy. Future safety and immunology studies in large animals with the goal to achieve effective humoral immunity without adverse effects should help to translate this study to human clinical trials.

Anti-A beta 1-11 antibody binds to different beta-amyloid species, inhibits fibril formation, and disaggregates preformed fibrils but not the most toxic oligomers.

Different strategies proposed as therapy for Alzheimer disease (AD) have aimed to reduce the level of toxic forms of A beta peptide in the brain. Here, we directly analyze the therapeutic utility of the polyclonal anti-A beta(1-11) antibody induced in 3xTg-AD mice vaccinated with the second generation prototype epitope vaccine. Substoichiometric concentrations of purified anti-A beta(1-11) antibody prevented aggregation of A beta(42) and induced disaggregation of preformed A beta(42) fibrils down to nonfilamentous and nontoxic species. Anti-A beta(1-11) antibody delayed A beta(42) oligomer formation but ultimately appeared to stabilize nonfibrillar conformations, including oligomer-like assemblies. The reduced oligomer-mediated cytotoxicity observed upon preincubation of A beta oligomers with the anti-A beta(1-11) antibody in the absence of oligomer disaggregation suggests a possible oligomer rearrangement in the presence of the antibody. These in vitro observations suggest that preventive vaccination may protect from AD or may delay the onset of the disease, whereas therapeutic vaccination cannot disrupt the toxic oligomers and may only minimally alleviate preexisting AD pathology.