Involvement of Transforming Growth Factor β and its Type 1 Receptor in the Development of Breast Cancer

Introduction of recent achievements of molecular biology into clinical practice contributes significantly to both prevention and early detection of breast cancer in women and development of new approaches, including genotyping, to screening for breast cancer. Screening for genetic polymorphisms in genes TGFβ1 and TGFβR1 reveals carriers of sporadic breast cancer. This provides an opportunity to reassess the role and place of preventive measures, also including surgical method, in the fight against breast cancer.     

BACE2 is Stored in Secretory Granules of Mouse and Rat Pancreatic β Cells

BACE2 is a protease homologous to BACE1 protein, an enzyme involved in the amyloid formation of Alzheimer disease (AD). However, despite the high homology between these two proteins, the biological role of BACE2 is still controversial, even though a few studies have suggested a pathogenetic role in sporadic inclusion-body myositis and hereditary inclusion-body myopathy, which are characterized by vacuolization of muscular fibers with intracellular deposits of proteins similar to those found in the brain of AD patients. Although BACE2 has also been identified in the pancreas, its function remains unknown and its specific localization in different pancreatic cell types has not been definitively ascertained. For these reasons, the authors have investigated the cellular and subcellular localization of BACE2 in normal rodent pancreases. BACE2 immunoreactivity was found in secretory granules of β cells, co-stored with insulin and IAPP, while it was lacking in the other endocrine and exocrine cell types. The presence of BACE2 in secretory granules of β cells suggests that it may play a role in diabetes-associated amyloidogenesis.     

Antigenic Regions on the β Chain of Human Chorionic Gonadotropin and Development of Hormone Specific Antibodies

Five peptides corresponding to four regions of the β chain of human chorionic gonadotropin (hCG), were synthesized, purified and characterized. The four regions studied were selected on the basis of sequence differences between the β chain of hCG (βhCG) and the β chains of related hormones. The peptides were found to bind rabbit and mouse anti-hCG antibodies as well as rabbit anti-β chain antibodies, but did not bind antibodies against the α chain or against other hormones. All the peptides, even in their free form, were able to elicit high titer antisera in both rabbits and mice. In all cases, anti-peptide antisera bound to the immunizing peptide as well as to the native hCG and the isolated β chain. These anti-peptide antisera did not bind to unrelated peptides, the α chain of hCG or to other hormones with very similar β chains such as human luteotropic hormone (hLH), ovine luteotropic hormone (oLH) and equine chorionic gonadotropin (eCG). Since the areas represented by these peptides elicit antibodies that are specific for human βhCG, they can formulate the basis for the development of discriminatory reagents for the β chain of hCG.     

Roles of estrogen receptor α and β in the regulation of proliferation in endometrial carcinoma

Endometrial carcinoma (EC), an estrogen-dependent gynecological malignancy, is prevalent worldwide. Estrogen receptor α (ERα) and estrogen receptor β (ERβ) are two main estrogen receptor isoforms, which mediate estrogen-induced proliferation in EC. However, the dynamic changes of ERα and ERβ subtype expression and their functions on proliferation in EC remain elusive. In this study, we aimed to investigate the expression of ERα and ERβ in para-tumor eutopic endometrium, endometrial atypical hyperplasia and EC by immunohistochemistry and then analyse their clinical significance. Subsequently, Ishikawa cells with ERα or ERβ knockdown by lentivirus transfection were used to explore the relationship between ERα/ERβ and cell proliferation, and preliminarily evaluate whether metformin’s inhibitory effect on estrogen-induced cell proliferation was mediated by ERα and ERβ. We found that the expression of ERα and ERβ were markedly changed in endometrial hyperplasia and EC compared with that in para-tumor eutopic endometrium and exhibited different expression trends. Through further analysis, we discovered that ERα presented higher expression in endometrial atypical hyperplasia and early stage of EC than that in para-tumor eutopic endometrium, while the expression of ERβ gradually decreased from para-tumor eutopic endometrium to EC. Additionally, the cell cycle-related protein, CyclinD1 was gradually increased but p21 decreased. Furthermore, knockdown of ERα and ERβ severally in Ishikawa cells either inhibited or promoted estrogen-induced cell proliferation through regulating CyclinD1 and p21 expression. Meanwhile, the inhibitory effect of metformin on estrogen-induced cell proliferation was respectively blunted or partly reversed by knockdown of ERα or ERβ. Altogether, ERα and ERβ have different expression patterns in the progression of EC either facilitating or suppressing cell proliferation through regulating the expression of CyclinD1 and p21 in EC cells, and may also mediate the inhibitory effect of metformin on estrogen-induced EC cells proliferation.     

Colocalization of Neurotensin and Corticotropin-Releasing Factor in Oval Nucleus of the Bed Nuclei of Stria Terminalis in the Rat

Ju et al first parcellated in detail the bed nuclei of stria terminalis according to its cyto-and chemoarchitecture. They found that the oval nucleus (OV) was rich in neurotensin (NT)-and corticotropin-releasing factor (CRF)like immunoreactivities. Using indirect immunohistochemical technique it has been shown in the present study that both neuropeptides show quite similar distribution pattern in OV of the Sprague-     

Abrogation of the Allelic Exclusion in a T Cell Receptor β Chain Gene Transgenic Mouse Strain

The expression of endogenous T cell receptor (TcR) β chains in a TcR β chain gene transgenic mouse (TGM) strain was examined. Unlike many other TGM strains reported, a considerable proportion of T cells from the thymus and spleen as well as organ cultured fetal thymus from our TGM express endogenous TCR β chains on their surface. Compatible with this was the elucidation of VDJ rearrangement of endogenous β chain genes by PCR. Three color flowcytometric analysis of thymus cell subpopulations revealed that the expression levels of both endogenous and transgenic TcR β genes are regulated in a maturational stage specific manner. Splenic T cells contained a several fold higher percentage of endogenous TcR β positive cells than thymus cells, suggesting a role of TcR on T cell peripherization. Vβ6 positive cells were deleted in the TGM carrying minor lymphocyte stimulating (Mls)-l^a antigen, indicating that the endogenous TcR β is functional in terms of transmiting a signal for clonal deletion.     

Innovation in Heart Preservation to Optimize the Sampling and Examination of Rodent Heart Valves Using the HistoGel™ Technique

Abstract

Certain classes of compounds have been linked to heart valve effects in humans. Preclinical screening therefore requires detailed and systematic examination of heart valves in laboratory animals, including rodents. However, inconsistencies in rodent heart valve sampling often are unavoidable because of inherent small heart size, thin valve-leaflets, and the various trimming/sectioning methods used. Therefore, we developed an innovative method of handling rodent hearts to allow for targeted sampling and examination of specific heart valves. At necropsy, rat hearts are quickly excised, flushed, and infused with 10% neutral buffered formalin (NBF). A hemostat is used to clamp the major vessels and the intact hearts are then submersed in 10% NBF for fixation. Three to six hours after formalin fixation, the hearts are infused with prewarmed (52°C) HistoGel?. The hearts are then submersed back into the 10% NBF for an additional 48 h. Fixed hearts are oriented in an acrylic heart matrix and consistently trimmed into three sections. The sections are processed and embedded face down in paraffin blocks. Using this technique, we are able to reliably trim, embed, section, and microscopically evaluate all four valves from the rat heart with minimal inconsistencies. (The J Histotechnol 32(2):64–68, 2009)

Submitted January 22, 2009; accepted April 15, 2009     

The Vesicle-containing Spines of the Dendrites in the Rat Hippocampus

The present article deals with the ultrastructure of the dendrites and their synapses in the rat hippocampus and the besicle-cotaining spines of the dendrites in the Ammon's Horn were first recorded.Using glutaraldehyde-osmic acid as fixatives for present work,the     

Plasma β amyloid and the risk of Alzheimer's disease in Down syndrome

Extracellular deposition of amyloid beta peptide (Aβ) has been implicated as a critical step in the pathogenesis of Alzheimer's disease (AD). In Down syndrome (DS), Alzheimer's disease is assumed to be caused by the triplication and overexpression of the gene for amyloid precursor protein (APP), located on chromosome 21. Plasma concentrations of Aβ1-40 and Aβ1-42 were determined in a population based study of 506 persons with DS, who were screened annually for dementia. We used Cox proportional hazards models to determine the risk of dementia. Demented persons with DS have a significantly higher plasma Aβ1-40 concentration than the nondemented (p = 0.05). Those with the highest concentrations of Aβ1-40 and Aβ1-42 have a higher risk to develop dementia. The risk to develop dementia during follow-up (mean 4.7 years) increased to 2.56 (95% confidence interval, 1.39-4.71) for Aβ1-42 and 2.16 (95% confidence interval, 1.14-4.10) for Aβ1-40. High plasma concentration of plasma Aβ1-40 and Aβ1-42 are determinants of the risk of dementia in persons with DS.     

Estrogen receptor-α in the raphe serotonergic and supramammillary area calretinin-containing neurons of the female rat

It is well established that estrogen affects hippocampal long-term potentiation and hippocampus-related memory processes. Furthermore, theta rhythm, in conjunction with long-term potentiation, influences memory and is regulated by subcortical structures, including the median raphe and supramammillary area. To test the validity of the hypothesis that the effects of estrogen on the hippocampus are mediated, at least partly, via these subcortical structures, it must first be determined whether the neurons of the median raphe and supramammillary area contain estrogen receptors. Light and electron microscopic double immunostaining for estrogen receptor-α plus serotonin and estrogen receptor-α plus calretinin on vibratome sections of the median raphe and supramammillary area, respectively, demonstrated that large populations of the median raphe serotonin and supramammillary area calretinin neurons exhibit estrogen receptor-immunoreactive nuclei. These observations indicate that circulating gonadal hormones can affect hippocampal electric activity indirectly, via those subcortical structures that are involved in theta rhythm regulation. Received: 1 February 1999 / Accepted: 17 May 1999     

Microglia Activated with the Toll-Like Receptor 9 Ligand CpG Attenuate Oligomeric Amyloid β Neurotoxicity in in Vitro and in Vivo Models of Alzheimer’s Disease

Soluble oligomeric amyloid β (oAβ) 1-42 causes synaptic dysfunction and neuronal injury in Alzheimer’s disease (AD). Although accumulation of microglia around senile plaques is a hallmark of AD pathology, the role of microglia in oAβ1-42 neurotoxicity is not fully understood. Here, we showed that oAβ but not fibrillar Aβ was neurotoxic, and microglia activated with unmethylated DNA CpG motif (CpG), a ligand for Toll-like receptor 9, attenuated oAβ1-42 neurotoxicity in primary neuron-microglia co-cultures. CpG enhanced microglial clearance of oAβ1-42 and induced higher levels of the antioxidant enzyme heme oxygenase-1 in microglia without producing neurotoxic molecules such as nitric oxide and glutamate. Among subclasses of CpGs, class B and class C activated microglia to promote neuroprotection. Moreover, intracerebroventricular administration of CpG ameliorated both the cognitive impairments induced by oAβ1-42 and the impairment of associative learning in Tg2576 mouse model of AD. We propose that CpG may be an effective therapeutic strategy for limiting oAβ1-42 neurotoxicity in AD.The senile plaque is a pathological hallmark of Alzheimer’s disease (AD). Fibrillar amyloid β (fAβ), a major component of senile plaques, induces tau hyperphosphorylation and neuronal dystrophy.^1,2 Soluble oligomeric Aβ (oAβ) has been reported to exhibit higher neurotoxicity than fAβ. Naturally secreted oAβ inhibits hippocampal long-term potentiation and disrupts synaptic plasticity in rats in vivo³. In addition, oAβ induces neuronal reactive oxygen species (ROS) through a mechanism requiring N-methyl-d-aspartate receptor activation.⁴ Exposure to oAβ induces rapid and massive neuronal death, while fAβ is required at higher concentrations and for longer incubations to cause neuronal dystrophy.⁵Microglia, macrophage-like cells in the central nervous system, cluster both in and around senile plaques and have been proposed to have pivotal roles in the pathogenesis of AD. Microglia activated with Aβ may be involved in the inflammatory component of AD.⁶ Both fAβ and oAβ stimulate microglial secretion of proinflammatory cytokines, chemokines, complement components, and free radicals.⁷ However, microglia also perform neuroprotective functions such as releasing neurotrophic factors⁸ and phagocytosing and degrading Aβ.^9,10Toll-like receptor (TLR) ligands enhance microglial phagocytosis of Aβ. Peptidoglycan, a TLR2 ligand, and unmethylated DNA CpG motifs (CpG), a TLR9 ligand, increase Aβ phagocytosis through the G protein-coupled formyl peptide receptor-like 2.^11,12 Similarly, the TLR4 ligand lipopolysaccharide increases phagocytosis through the CD14 receptor.¹³ However, microglia activated with TLR ligands also produce neurotoxic molecules such as proinflammatory cytokines, nitric oxide (NO), ROS, and peroxynitrite.¹⁴ In particular, lipopolysaccharide-activated microglia produce a large amount of glutamate, a neurotransmitter but also potent neurotoxin.¹⁵ Thus, factors that increase microglial clearance of oAβ without producing inflammatory mediators are candidates for the treatment of AD.Here, we investigated the role of microglia in neurotoxicity mediated by oAβ1-42. We found that microglia activated with a low dose of CpG attenuated the neurotoxic effects of oAβ1-42 without producing other neurotoxic molecules in vitro. Moreover, intracerebroventricular (ICV) administration of CpG ameliorated the cognitive impairment induced by ICV injection of oAβ1-42 and the impairment of associative learning in Tg2576 mouse model of AD.     

Development and Validation of Enzyme‐Linked Immunosorbent Assays for Quantification of Anti‐Methotrexate IgG and Fab in Mouse and Rat Plasma

Abstract

This laboratory is investigating the use of anti‐methotrexate IgG (AMI) and anti‐methotrexate Fab fragments (AMF) within an inverse targeting strategy that is designed to enhance the pharmacokinetic selectivity of intraperitoneal (i.p.) chemotherapy. The goal of this study was to develop enzyme‐linked immunosorbent assays (ELISAs) to determine concentrations of AMI and AMF in mouse and rat plasma. An antigen‐specific ELISA was developed for AMI and AMF in mouse and rat plasma. The assay was validated with respect to precision and accuracy by evaluating the recovery of AMI and AMF from mouse and rat plasma samples. Preliminary pharmacokinetic studies of AMI and AMF were performed in Sprague‐Dawley rats and Swiss Webster mice. The animals were instrumented with a jugular vein cannula and administered AMI or AMF, 15?mg?kg^?1 via the cannula. Plasma samples were taken at various time points and analyzed using the ELISA, and the observed concentration vs. time profiles were subjected to non‐compartmental pharmacokinetic analyses. Standard curves for the ELISAs were found to be linear over concentration ranges of 0–250 and 0–350?ng?mL^?1 for AMI and AMF, respectively. Intra‐assay and inter‐assay recovery of AMI and AMF from plasma samples were found to be within 15% of theoretical values. Preliminary pharmacokinetic investigations of AMI allowed estimation of AMI clearance to be 0.017?mL?kg^?1?min^?1 in the rat and 0.043?mL?kg^?1?min^?1 in the mouse. AMF clearance was estimated to be 0.038 and 1.93?mL?kg^?1?min^?1 in the mouse and rat, respectively. In conclusion, ELISAs have been developed and validated for quantitation of AMI and AMF in rat and mouse plasma. The assays will allow further investigations of AMI and AMF pharmacokinetics.     

Expression of estrogen receptors α and β in the trigeminal mesencephalic nucleus of adult women and men

Temporomandibular disorders are more prevalent in women than in men and phases of pain relate to the estrous cycle. Several studies described the location of estrogen receptors (ER) in the temporomandibular joint (TMJ), the masseteric muscles and cartilage, but it was unknown whether they are also expressed within the pseudounipolar neurons of the trigeminal mesencephalic nucleus, which receives direct sensory inputs from these structures. Therefore, we studied expression of ERα and ERβ protein in the trigeminal mesencephalic nucleus of ten human brains (five female/five male). Both receptors were uniformly expressed on neurons, but not other cell types within the target structure. Thus, sensory inputs from the TMJ and adjacent structures are likely to be modulated by estrogen at the level of the first sensory neuron which may underlie the well-known correlation of pain incidence and phases of the estrous cycle.     

Transforming growth factor β mRNA and protein expression in the ovary of the chicken embryo

The expression pattern of transforming growth factor beta (TGFβ) isoforms in chicken embryo gonads was studied at 6–10 days of incubation. TGFβ2 mRNA was expressed predominantly in the cortex of the left ovary from day 8 of incubation onwards. TGFβ3 mRNA was not detected at any of the stages studied. Similarly, immunofluorescence for the TGFβ protein revealed that at day 9 it was located throughout the cortex of the left ovary and in the medulla of both the left and right ovaries. The presence of phosphorylated Smad2 in the nuclei of these regions suggests that TGFβ signaling is most likely active at this developmental stage. Culturing the left ovary in a TGFβ1-supplemented medium induced a shift of cortical structures toward the medulla, suggesting a role for TGFβ in the morphogenesis of the female gonad in chickens.     

Differential Expression of the Five Somatostatin Receptor Subtypes in Human Benign and Malignant Insulinomas – Predominance of Receptor Subtype 4

Insulinomas constitute a subgroup of pancreatic endocrine tumors showing B cell differentiation and clinical symptoms related to inappropriate insulin secretion (WHO). Many endocrine tumors express somatostatin receptors (sstrs), which can be visualized by octreotide scintigraphy; however, about half of all insulinomas are reported to be negative. Previous immunohistochemical investigations with antibodies to sstr subtypes 1, 2, 3, and 5 have revealed differences in expression between various neuroendocrine tumors. In the present study, the immunoreactivity to all five human sstr was studied in ten benign and six malignant human insulinomas. Sstr₄ was the receptor subtype most frequently expressed in both benign and malignant tumors. A difference in the immunohistochemical sstr₅ expression pattern was seen between benign and malignant tumors: Three of the six malignant tumors, but none of the benign tumors, expressed sstr₅. The other receptor subtypes were expressed in low numbers with no difference between benign and malignant tumors. The finding of a strong expression of sstr₄ in both benign and malignant insulinomas suggests that this receptor subtype could be of importance for diagnostic and therapeutic use.