PINK1 deficiency in β-cells increases basal insulin secretion and improves glucose tolerance in mice

The Parkinson''s disease (PD) gene, PARK6, encodes the PTEN-induced putative kinase 1 (PINK1) mitochondrial kinase, which provides protection against oxidative stress-induced apoptosis. Given the link between glucose metabolism, mitochondrial function and insulin secretion in β-cells, and the reported association of PD with type 2 diabetes, we investigated the response of PINK1-deficient β-cells to glucose stimuli to determine whether loss of PINK1 affected their function. We find that loss of PINK1 significantly impairs the ability of mouse pancreatic β-cells (MIN6 cells) and primary intact islets to take up glucose. This was accompanied by higher basal levels of intracellular calcium leading to increased basal levels of insulin secretion under low glucose conditions. Finally, we investigated the effect of PINK1 deficiency in vivo and find that PINK1 knockout mice have improved glucose tolerance. For the first time, these combined results demonstrate that loss of PINK1 function appears to disrupt glucose-sensing leading to enhanced insulin release, which is uncoupled from glucose uptake, and suggest a key role for PINK1 in β-cell function.     

TGF-β1 induces an age-dependent inflammation of nerve ganglia and fibroplasia in the prostate gland stroma of a novel transgenic mouse

TGF-β1 is overexpressed in wound repair and in most proliferative disorders including benign prostatic hyperplasia and prostate cancer. The stromal microenvironment at these sites is reactive and typified by altered phenotype, matrix deposition, inflammatory responses, and alterations in nerve density and biology. TGF-β1 is known to modulate several stromal responses; however there are few transgenic models to study its integrated biology. To address the actions of TGF-β1 in prostate disorders, we targeted expression of an epitope tagged and constitutively active TGF-β1 via the enhanced probasin promoter to the murine prostate gland epithelium. Transgenic mice developed age-dependent lesions leading to severe, yet focal attenuation of epithelium, and a discontinuous basal lamina. These changes were associated with elevated fibroplasia and frequency of collagenous micronodules in collapsed acini, along with an induced inflammation in nerve ganglia and small vessels. Elevated recruitment of CD115+ myeloid cells but not mature macrophages was observed in nerve ganglia, also in an age-dependent manner. Similar phenotypic changes were observed using a human prostate epithelium tissue recombination xenograft model, where epithelial cells engineered to overexpress TGF-β1 induced fibrosis and altered matrix deposition concurrent with inflammation in the stromal compartment. Together, these data suggest that elevated TGF-β1 expression induces a fibroplasia stromal response associated with breach of epithelial wall structure and inflammatory involvement of nerve ganglia and vessels. The novel findings of ganglia and vessel inflammation associated with formation of collagenous micronodules in collapsed acini is important as each of these are observed in human prostate carcinoma and may play a role in disease progression.     

Matrix metalloproteinase-8 regulates transforming growth factor-β1 levels in mouse tongue wounds and fibroblasts in vitro

Matrix metalloproteinase-8 (MMP-8)-deficient mice (Mmp8-/-) exhibit delayed dermal wound healing, but also partly contradicting results have been reported. Using the Mmp8-/- mice we investigated the role of MMP-8 in acute wound healing of the mobile tongue, and analyzed the function of tongue fibroblasts in vitro. Interestingly, in the early phase the tongue wounds of Mmp8-/- mice healed faster than those of wild type (wt) mice resulting in significant difference in wound widths (P=0.001, 6–24 h). The Mmp8-/- wounds showed no change in myeloperoxidase positive myeloid cell count, but the level of transforming growth factor (TGF)-β1 was significantly increased (P=0.007) compared to the wt tongues. Fibroblasts cultured from wt tongues expressed MMP-8 and TGF-β1. However, higher TGF-β1 levels were detected in Mmp8-/- fibroblasts, and MMP-8 treatment decreased phosphorylated Smad-2 levels and α-smooth muscle actin expression in these fibroblasts suggesting reduced TGF-β1 signaling. Consistently, a degradation of recombinant TGF-β1 by MMP-8 decreased its ability to activate the signaling cascade in fibroblasts. Moreover, collagen gels with Mmp8-/- fibroblasts reduced more in size. We conclude that MMP-8 regulates tongue wound contraction rate and TGF-β1 levels. In vitro analyses suggest that MMP-8 may also play a role in regulating TGF-β1 signaling of stromal fibroblasts.     

The relationship between predicted peptide–MHC class II affinity and T-cell activation in a HLA-DRβ1*0401 transgenic mouse model

The HLA-DRB1*0401 MHC class II molecule (DR4) is genetically associated with rheumatoid arthritis. It has been proposed that this MHC class II molecule participates in disease pathogenesis by presenting arthritogenic endogenous or exogenous peptides to CD4⁺ T cells, leading to their activation and resulting in an inflammatory response within the synovium. In order to better understand DR4 restricted T cell activation, we analyzed the candidate arthritogenic antigens type II collagen, human aggrecan, and the hepatitis B surface antigen for T-cell epitopes using a predictive model for determining peptide–DR4 affinity. We also applied this model to determine whether cross-reactive T-cell epitopes can be predicted based on known MHC–peptide–TCR interactions. Using the HLA-DR4-IE transgenic mouse, we showed that both T-cell proliferation and Th1 cytokine production (IFN-γ) correlate with the predicted affinity of a peptide for DR4. In addition, we provide evidence that TCR recognition of a peptide–DR4 complex is highly specific in that similar antigenic peptide sequences, containing identical amino acids at TCR contact positions, do not activate the same population of T cells.     

Conditional overexpression of TGFβ1 promotes pulmonary inflammation,apoptosis and mortality via TGFβR2 in the developing mouse lung

Background

Earlier studies have reported that transforming growth factor beta 1(TGFβ1) is a critical mediator of hyperoxia-induced acute lung injury (HALI) in developing lungs, leading to impaired alveolarization and a pulmonary phenotype of bronchopulmonary dysplasia (BPD). However, the mechanisms responsible for the TGFβ1-induced inflammatory signals that lead to cell death and abnormal alveolarization are poorly understood. We hypothesized that TGFβ1 signaling via TGFβR2 is necessary for the pathogenesis of the BPD pulmonary phenotype resulting from HALI.

Methods

We utilized lung epithelial cell-specific TGFβ1 overexpressing transgenic and TGFβR2 null mutant mice to evaluate the effects on neonatal mortality as well as pulmonary inflammation and apoptosis in developing lungs. Lung morphometry was performed to determine the impaired alveolarization and multicolor flow cytometry studies were performed to detect inflammatory macrophages and monocytes in lungs. Apoptotic cell death was measured with TUNEL assay, immunohistochemistry and western blotting and protein expression of angiogenic mediators were also analyzed.

Results

Our data reveals that increased TGFβ1 expression in newborn mice lungs leads to increased mortality, macrophage and immature monocyte infiltration, apoptotic cell death specifically in Type II alveolar epithelial cells (AECs), impaired alveolarization, and dysregulated angiogenic molecular markers.

Conclusions

Our study has demonstrated the potential role of inhibition of TGFβ1 signaling via TGFβR2 for improved survival, reduced inflammation and apoptosis that may provide insights for the development of potential therapeutic strategies targeted against HALI and BPD.     

C/EBP-α, involvement of a novel transcription factor in leptin-induced VCAM-1 production in mouse chondrocytes

Leptin and vascular cell adhesion molecules-1 (VCAM-1) are two important mediators in obesity-related osteoarthritis, while the molecular mechanism linking leptin to VCAM-1 production is still obscure. Here we show that leptin upregulates VCAM-1 mRNA and protein levels in a time- and dose-dependent manner. Mechanistically, leptin induces VCAM-1 promoter activity by increasing the expression of C/EBP-α and facilitating its binding to a newly identified element in the VCAM-1 gene. Gain or loss of function studies reveal a regulatory role of C/EBP-α on VCAM-1 expression. Finally, elevated plasma leptin level correlates to increased C/EBP-α and VCAM-1 production in chondrocytes from obese mice.     

Adaptive response to mutagenesis and its molecular basis in a human T-cell leukemia line primed with a low dose ofγ-rays

The effect was studied of a low dose of-ray preexposure on the frequency and molecular spectrum of radiation-induced mutations at the hprt locus in a human T-cell leukemia line. When the cells were preexposed to 0.01 Gy of-rays, the yield of mutations induced by a subsequent 2-Gy challenge dose was reduced by 60%, compared with the 2 Gy of irradiation alone. The data of Southern blot analysis showed that 47% of the mutants induced by 2 Gy in the cells without low-dose preexposure were of the deletion or rearranged mutations type. In contrast, in the low-dose radioadapted cells the proportion of this type of 2-Gy-induced mutations decreased to 28%. This is close to the control level (22%) of spontaneous mutations. Our results confirm that a low dose of-ray preexposure leads to a decreased susceptibility to gene deletions and rearrangements after high-dose irradiation.     

TGF-β1 and high levels of glucose do not increase insulin cell proportions in the avian embryonic pancreas

TGF-β1 is thought to decrease the proportion of embryonic pancreatic β-cells with respect to α-cells, whereas glucose is thought to enhance β-cell proportions in rats. However, chick pancreatic cells may respond in a dissimilar way to glucose. Thus, the effect of TGF-β1 on the proportion of β-cells in embryonic chick dorsal pancreatic buds (DPBs) in vitro was tested with short-term exposure to high levels of glucose. Five-day-old chick DPBs were cultured on growth factor-reduced Matrigel, which contains reduced levels of growth factors including TGF-β1, and a variety of culture media with and without high levels of glucose. TGF-β1 reduced the proportion of β-cells, as expected. A similar decrease in the proportion of β-cells occurred in the presence of high levels of glucose.     

Chemically mimicked hypoxia modulates gene expression and protein levels of the sodium calcium exchanger in HEK 293 cell line via HIF-1α

Up to now a little is known about the effect of hypoxia on the sodium calcium exchanger type 1 (NCX1) expression and function. Therefore, we studied how dimethyloxallyl glycine (DMOG), an activator and stabilizer of the hypoxia-inducible factor (HIF)-1α, could affect expression of the NCX1 in HEK 293 cell line. We also tried to determine whether this activation can result in the induction of apoptosis in HEK 293 cells. We have found that DMOG treatment for 3 hours significantly increased gene expression and also protein levels of the NCX1. This increase was accompanied by a decrease in intracellular pH. Wash-out of DMOG did not result in reduction of the NCX1 mRNA and protein to original - control levels, although pH returned to physiological values. Using luciferase reporter assay we observed increase in the NCX1 promoter activity after DMOG treatment and using wild-type mouse embryonic fibroblast (MEF)-HIF-1(+/+) and HIF-1-deficient MEF-HIF-1(-/-) cells we have clearly shown that in the promoter region, HIF-1α is involved in DMOG induced upregulation of the NCX1. Moreover, we also showed that an increase in the NCX1 mRNA due to the apoptosis induction is not regulated by HIF-1α.     

Reduced basal and novelty-induced levels of activity-regulated cytoskeleton associated protein (Arc) and c-Fos mRNA in the cerebral cortex and hippocampus of APPswe/PS1ΔE9 transgenic mice

Activity-regulated cytoskeletal-associated protein (Arc) and c-Fos are immediate early gene (IEG) products induced by novelty in the hippocampus and involved in the consolidation of synaptic plasticity and long-term memory. We investigated whether induction of arc and c-fos after exposure to a novel open field environment was compromised in different neocortical areas and the hippocampal formation in APP/PS1ΔE9 transgenic mice characterized by pronounced accumulation and deposition of beta amyloid (Aβ). Notably, the basal level of Arc and c-fos mRNA in the neocortex was significantly lower in APP/PS1ΔE9 compared to wild-type mice. Novelty exposure induced an increase in Arc and c-Fos mRNA in the medial prefrontal cortex (mPFC), parietal cortex, and hippocampal formation in both APP/PS1ΔE9 transgenic and wild-type mice. However, novelty-induced IEG expression did not reach the same levels in APP/PS1ΔE9 as in the wild-type mice. In contrast, synaptophysin levels did not differ between mutant and wild type mice, suggesting that the observed effect was not due to a general decrease in the number of presynapses. These data suggest a reduction in basal and novelty-induced neuronal activity in a transgenic mouse model of Alzheimer’s disease, which is most pronounced in cortical regions, indicating that a decreased functional response in IEG expression could be partly responsible for the cognitive deficits observed in patients with Alzheimer’s disease.     

Association of T869C gene polymorphism of transforming growth factor-β1 with low protein levels and anthropometric indices in osteopenia/osteoporosis postmenopausal Thai women

Osteoporosis is the most common metabolic bone disease; it is an important health problem among postmenopausal women. We evaluated the association of three polymorphisms, T869C, C-509T and G915C, of the TGF-β1 gene with bone mineral density (BMD) serum TGF-β1 levels in 278 postmenopausal female osteopenia/osteoporosis subjects and 95 postmenopausal female control subjects. Serum TGF-β1 levels were significantly lower in osteopenia/osteoporosis subjects than in control subjects. Serum TGF-β1 levels of the CT+CC (T869C) genotype group were significantly lower in osteopenia/osteoporosis subjects than in control subjects (11.3 vs 15.8 ng/mL). There was a significant difference in the CT+CC (T869C) genotype frequencies between the osteopenia/osteoporosis and control subjects (74.18 vs 60.22%; OR = 1.90, 95%CI = 1.16-3.12). In the age group of more than 50 years, subjects with the TC+CC genotype of T869C polymorphism had significantly increased risk of osteopenic/ osteoporotic bones at L1 (OR = 2.36, 95%CI = 1.37-4.07), L2 (OR = 1.71, 95%CI = 1.01-2.90), L3 (OR = 2.21, 95%CI = 1.23-3.98), L4 (OR = 1.74, 95%CI = 1.00-3.03) and the femoral neck (OR = 1.80, 95%CI = 1.04-3.12). The CT+CC genotype of the T869C polymorphism of the TGF-β1 gene was found to be associated with lower serum TGF-β1 in osteopenia/osteoporosis subjects and increased risk of osteopenic and osteoporotic fracture at L1-4, femoral neck and total hip in postmenopausal Thai women. Logistic regression analysis showed that T869C polymorphism is a significant risk factor for osteopenia/ osteoporosis. We concluded that T869C polymorphism of the TGF-β1 gene has an impact on decreased serum TGF-β1 levels and influences susceptibility to osteopenia/osteoporosis in Thai women.     

Specific stimulation of basal lamina heparan sulfate proteoglycan in mouse uterine epithelium by matrigel and by transforming growth factor-β1

Summary The basal lamina of differentiated epithelium normally turns over only slowly unless stimulated by tissue repair and growth. We show here that one mechanism of this stimulation, as modeled by basal lamina proteoglycan synthesis, may be the release of basal lamina-bound transforming growth factor (TGF-β). A large heparan sulfate proteoglycan (HSPG, 0.2K _av on Sepharose CL-4B) that was extractable from mouse uterine epithelium with 4M guanidine-HCl or 1M KCl was recognized by a specific monoclonal antibody to the basal lamina HSPG, perlecan. This HSPG was metabolically inactive with respect to [³⁵S]-sulfate labeling in pieces of whole uterus during 4 h of culture, but it was labeled in isolated cells under the same conditions, provided that the cells had been cultured at least 6 to 12 h before labeling. The rate of labeling was then constant during at least 4 days in culture in serum-containing medium. Cultures on Matrigel showed an enhanced [³⁵S]-sulfate labeling specifically in the 0.2K _av HSPG fraction. Partial stimulation was obtained with a serum-free medium extract of Matrigel, which fractionated on Sephadex G-50 in two components; a major one >30 kDa and the other at about 15 to 25 kDa. The specific stimulation was mimicked by the addition of 10 ng/ml of TGF-β1, but there was no specific stimulation by basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulinlike growth factor-1 (IGF-1), or interleukin-1 (IL-1). TGF-β1 was identified as a 12.5 kDa monomer in thiol-reduced Matrigel and Matrigel extracts by polyclonal blocking antibodies on transblots following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Failure of excess amounts of these antibodies to block Matrigel-stimulated basal lamina HSPG synthesis indicates that TGF-β1 may be only one component of Matrigel that is important in stimulating basal lamina HSPG synthesis in culture. We suggest that in vivo TGF-β1 is bound to macromolecular components of mouse uterine epithelial basal lamina, where it may be sequestered until microenvironmental changes make it available to promote basal lamina HSPG synthesis.     

Vitreous TIMP-1 levels associate with neovascularization and TGF-β2 levels but not with fibrosis in the clinical course of proliferative diabetic retinopathy

In proliferative diabetic retinopathy (PDR), vascular endothelial growth factor (VEGF) and CCN2 (connective tissue growth factor; CTGF) cause blindness by neovascularization and subsequent fibrosis. This angio-fibrotic switch is associated with a shift in the balance between vitreous levels of CCN2 and VEGF in the eye. Here, we investigated the possible involvement of other important mediators of fibrosis, tissue inhibitor of metalloproteinases (TIMP)-1 and transforming growth factor (TGF)-β2, and of the matrix metalloproteinases (MMP)-2 and MMP-9, in the natural course of PDR. TIMP-1, activated TGF-β2, CCN2 and VEGF levels were measured by ELISA in 78 vitreous samples of patients with PDR (n = 28), diabetic patients without PDR (n = 24), and patients with the diabetes-unrelated retinal conditions macular hole (n = 10) or macular pucker (n = 16), and were related to MMP-2 and MMP-9 activity on zymograms and to clinical data, including degree of intra-ocular neovascularization and fibrosis. TIMP-1, CCN2 and VEGF levels, but not activated TGF-β2 levels, were significantly increased in the vitreous of diabetic patients, with the highest levels in PDR patients. CCN2 and the CCN2/VEGF ratio were the strongest predictors of degree of fibrosis. In diabetic patients with or without PDR, activated TGF-β2 levels correlated with TIMP-1 levels, whereas in PDR patients, TIMP-1 levels, MMP-2 and proMMP-9 were associated with degree of neovascularization, like VEGF levels, but not with fibrosis. We confirm here our previous findings that retinal fibrosis in PDR patients is significantly correlated with vitreous CCN2 levels and the CCN2/VEGF ratio. In contrast, TIMP-1, MMP-2 and MMP-9 appear to have a role in the angiogenic phase rather than in the fibrotic phase of PDR.     

Hydrogen sulfide improves spatial memory impairment and decreases production of Aβ in APP/PS1 transgenic mice

Alzheimer’s disease (AD) is defined both by its progressive cognitive deterioration and hallmark increase in neuronal Aβ plaque formation. However, many of the underlying neurobiological facets of this disease are still being elucidated. Previous research has demonstrated that production of neuronal hydrogen sulfide (H₂S) is significantly decreased in patients with AD. Moreover, systemic plasma H₂S levels are negatively correlated with its severity. However, how a decrease in H₂S production might be correlated with either the etiology or pathophysiology of AD remains unknown. To better understand the role of H₂S in AD, we examined both levels of H₂S and the expression and activity H₂S-synthesizing enzyme (cystathionine beta synthase or CBS) in an APP/PS1 transgenic mouse line at 3, 6, 9 and 12 months. After intraperitoneal (i.p.) administration of an H₂S donor (NaHS) into APP/PS1 mice, application of exogenous H₂S resulted in improved spatial learning and memory acquisition in APP/PS1 mice. H₂S administration also led to significant decrease in extracellular levels of Aβ40 and Aβ42, the expression of BACE1 and PS1, and a significant increase of ADAM17 expression. Similarly, an increase in non-amyloidogenic C83 fragment generation and a decrease in amyloidogenic C99 fragment generation were also observed. Thus, NaHS application resulted in a shift from the plaque-forming beta pathway to the non-plaque forming alpha pathway of APP cleavage in 6 and 12 month APP/PS1 mice. These results indicate the importance of H₂S to AD severity and that administration of exogenous H₂S can promote a non-amyloidogenic processing of APP.     

Visualization and phenotyping of proinflammatory antigen-specific T cells during collagen-induced arthritis in a mouse with a fixed collagen type II-specific transgenic T-cell receptor β-chain

Introduction  

The Vβ12-transgenic mouse was previously generated to investigate the role of antigen-specific T cells in collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis. This mouse expresses a transgenic collagen type II (CII)-specific T-cell receptor (TCR) β-chain and consequently displays an increased immunity to CII and increased susceptibility to CIA. However, while the transgenic Vβ12 chain recombines with endogenous α-chains, the frequency and distribution of CII-specific T cells in the Vβ12-transgenic mouse has not been determined. The aim of the present report was to establish a system enabling identification of CII-specific T cells in the Vβ12-transgenic mouse in order to determine to what extent the transgenic expression of the CII-specific β-chain would skew the response towards the immunodominant galactosylated T-cell epitope and to use this system to monitor these cells throughout development of CIA.     

Nr5a1-Cre-mediated Tspo conditional knockout mice with low growth rate and prediabetes symptoms – A mouse model of stress diabetes

Translocator protein (TSPO) is a high-affinity cholesterol- and drug-binding mitochondrial protein. Nuclear receptor subfamily 5 group A member 1 or steroidogenic factor 1 (Nr5a1)-Cre mice were previously used to generate steroidogenic cell-specific Tspo gene conditional knockout (cKO) mice. TSPO-depleted homozygotes showed no response to adrenocorticotropic hormone (ACTH) in stimulating adrenal cortex corticosterone production but showed increased epinephrine synthesis in the medulla. No other phenotype was observed under normal growth conditions. During these studies, we noted that pairing two cKO mice resulted in the generation of small pups. These pups showed low growth rate at weaning, which has been linked to the development of type 2 diabetes (T2D) in adulthood. Experimental verification of T2D symptoms via blood testing of the adult mice, including glycated hemoglobin and insulin C-peptide measurements, showed that these Tspo cKO mice exhibited sustained hyperglycemia, a sign of prediabetes, likely due to the augmentation of hepatic glucose production mediated by the increased epinephrine. We also observed increased expression of the S100a8 gene, which is upregulated after chronic glucose stimulation. Taken together, the observed prediabetes phenotype and lack of response to ACTH indicate that Tspo cKO mice (Nr5a1-Cre^+/?, Tspo^fl/fl) could provide a useful model to study the link between diabetes and stress.     

TGF-β1 influence on TNF-α production and sTNF-Rs shedding in a coculture of colon carcinoma cell spheroids with normal cells

In this study, the levels of TNF-α and its soluble receptors sTNF-Rp55 and sTNF-Rp75 were analyzed in cocultures of human colon carcinoma cell spheroids prepared from different grades of tumors with normal human colon epithelium, myofibroblast, and endothelial cell monolayers. Additionally, the influence of exogenously added rhTGF-β1 (2 ng/ml) on the TNF-α and sTNF-Rs levels was tested. Direct interactions of colon carcinoma spheroids with normal cells caused decreases in TNF-α levels and normal cell-dependent changes in sTNF-Rs amounts as compared to normal cells cultured alone. The addition of rhTGF-β1 to the cocultures caused a significant increase in TNF-α levels with a simultaneous decrease in the amounts of both sTNF-Rs. During direct interactions of colon carcinoma cells with normal tissue, paracrine effects are very important. We showed that TGF-β1 acts synergistically with TNF-α and significantly limits sTNF-Rs shedding. Therefore, TNF-Rs bound to cellular membranes, but not their soluble forms, play an important role in tumor/normal cell interactions. TGF-β1 and sTNF-Rs, in turn, may be valuable factors in colon cancer development and metastasis.     

Chronic intranasal treatment with an anti-Aβ(30-42) scFv antibody ameliorates amyloid pathology in a transgenic mouse model of Alzheimer's disease

Amyloid-beta peptide (Aβ)-directed active and passive immunization therapeutic strategies reduce brain levels of Aβ, decrease the severity of beta-amyloid plaque pathology and reverse cognitive deficits in mouse models of Alzheimer's disease (AD). As an alternative approach to passive immunization with full IgG molecules, single-chain variable fragment (scFv) antibodies can modulate or neutralize Aβ-related neurotoxicity and inhibit its aggregation in vitro. In this study, we characterized a scFv derived from a full IgG antibody raised against the C-terminus of Aβ, and studied its passage into the brains of APP transgenic mice, as well as its potential to reduce Aβ-related pathology. We found that the scFv entered the brain after intranasal application, and that it bound to beta-amyloid plaques in the cortex and hippocampus of APP transgenic mice. Moreover, the scFv inhibited Aβ fibril formation and Aβ-mediated neurotoxicity in vitro. In a preventative therapeutic approach chronic intranasal treatment with scFv reduced congophilic amyloid angiopathy (CAA) and beta-amyloid plaque numbers in the cortex of APPswe/PS1dE9 mice. This reduction of CAA and plaque pathology was associated with a redistribution of brain Aβ from the insoluble fraction to the soluble peptide pool. Due to their lack of the effector domain of full IgG, scFv may represent an alternative tool for the treatment of Aβ-related pathology without triggering Fc-mediated effector functions. Additionally, our observations support the possibility that Aβ-directed immunotherapy can reduce Aβ deposition in brain vessels in transgenic mice.