Soft Imaging System Research Articles

Regulatory effects of arachidonate 5-lipoxygenase on hepatic microsomal TG transfer protein activity and VLDL-triglyceride and apoB secretion in obese mice

As 5-lipoxygenase (5-LO) is an emerging target in obesity and insulin resistance, we have investigated whether this arachidonate pathway is also implicated in the progression of obesity-related fatty liver disease. Our results show that 5-LO activity and 5-LO-derived product levels are significantly elevated in the liver of obese ob/ob mice with respect to wild-type controls. Treatment of ob/ob mice with a selective 5-LO inhibitor exerted a remarkable protection from hepatic steatosis as revealed by decreased oil red-O staining and reduced hepatic triglyceride (TG) concentrations. In addition, 5-LO inhibition in ob/ob mice downregulated genes involved in hepatic fatty acid uptake (i.e., L-FABP and FAT/CD36) and normalized peroxisome proliferator-activated receptor alpha (PPARalpha) and acyl-CoA oxidase expression, whereas the expression of lipogenic genes [i.e., fatty acid synthase (FASN) and SREBP-1c] remained unaltered. Furthermore, 5-LO inhibition restored hepatic microsomal TG transfer protein (MTP) activity in parallel with a stimulation of hepatic VLDL-TG and apoB secretion in ob/ob mice. Consistent with these findings, 5-LO products directly inhibited MTP activity and triggered cytosolic TG accumulation in CC-1 cells, a murine hepatocyte cell line. Taken together, these findings identify a novel steatogenic role for 5-LO in the liver through mechanisms involving the regulation of hepatic MTP activity and VLDL-TG and apoB secretion.

Tumor Recovery by Angiogenic Switch from Sprouting to Intussusceptive Angiogenesis after Treatment with PTK787/ZK222584 or Ionizing Radiation

Inhibitors of angiogenesis and radiation induce compensatory changes in the tumor vasculature both during and after treatment cessation. To assess the responses to irradiation and vascular endothelial growth factor-receptor tyrosine kinase inhibition (by the vascular endothelial growth factor tyrosine kinase inhibitor PTK787/ZK222854), mammary carcinoma allografts were investigated by vascular casting; electron, light, and confocal microscopy; and immunoblotting. Irradiation and anti-angiogenic therapy had similar effects on the tumor vasculature. Both treatments reduced tumor vascularization, particularly in the tumor medulla. After cessation of therapy, the tumor vasculature expanded predominantly by intussusception with a plexus composed of enlarged sinusoidal-like vessels containing multiple transluminal tissue pillars. Tumor revascularization originated from preserved alpha-smooth muscle actin-positive vessels in the tumor cortex. Quantification revealed that recovery was characterized by an angiogenic switch from sprouting to intussusception. Up-regulated alpha-smooth muscle actin-expression during recovery reflected the recruitment of alpha-smooth muscle actin-positive cells for intussusception as part of the angio-adaptive mechanism. Tumor recovery was associated with a dramatic decrease (by 30% to 40%) in the intratumoral microvascular density, probably as a result of intussusceptive pruning and, surprisingly, with only a minimal reduction of the total microvascular (exchange) area. Therefore, the vascular supply to the tumor was not severely compromised, as demonstrated by hypoxia-inducible factor-1alpha expression. Both irradiation and anti-angiogenic therapy cause a switch from sprouting to intussusceptive angiogenesis, representing an escape mechanism and accounting for the development of resistance, as well as rapid recovery, after cessation of therapy.

Formation of stromal collagen fibrils and proteoglycans in the developing zebrafish cornea

Collagen fibrils and proteoglycans are the main components of the corneal extracellular matrix and corneal transparency depends crucially on their proper arrangement. In the present study, we investigated the formation of collagen fibrils and proteoglycans in the developing cornea of the zebrafish, a model organism used to study vertebrate embryonic development and genetic disease. We employed thin-section electron microscopy to investigate the ultrastructure of the zebrafish cornea at different developmental stages. The layering of the zebrafish cornea into an epithelium, a Bowman's layer, stroma and endothelium was observed starting at 72 hr post-fertilization. At this stage, the stroma contained orthogonally arranged collagen fibrils and small proteoglycans. The density of proteoglycans increased gradually throughout subsequent development of the cornea. In the stroma of 2-week-old larvae, the collagen fibrils were organized into thin lamellae and were separated by very large, randomly distributed proteoglycans. At 4 weeks, a regular arrangement of proteoglycans in relation to the collagen fibrils was observed for the first time and the lamellae were also thickened. The present study, for the first time, provides ultrastructural details of collagen fibril and proteoglycan development in the zebrafish cornea. Furthermore, it directly correlates the collagen fibril and proteoglycan composition of the zebrafish cornea with that of the human cornea. The similarities between the two species suggest that the zebrafish could serve as a model for investigating the genetics of human corneal development and diseases.

Ultrastructural features of hepatocytes in cultured Eurasian perch (Perca fluviatilisL.) as affected by nutritional and husbandry conditions

A wide range of factors can be attributed to the syndrome of fatty liver observed in some cultured fish species. The objective of the study was therefore to quantify different hepatocyte ultrastructural features as potentially influenced by twelve nutritional and husbandry factors, in order to discriminate the most influent factors in Eurasian perch (Perca fluviatilis), a typical carnivorous temperate fish species. Twenty-four groups of juveniles (initial weight 57.6 (SD 14.4) g) were intensively reared for 116 d and fed sixteen different isoproteic diets. The distribution of the experimental treatments was based on a multivariate fractional factorial design (L(24) 2(12)) with either high (+1) or low (-1) level of each of the following factors: diet (lipid and protein sources, lipid content, astaxanthin enrichment), feeding level, daily and weekly distribution frequency, fish density, initial weight heterogeneity, temperature, photoperiod, and light spectrum. Liver lipid droplets, glycogen, mitochondria and rough endoplasmic reticulum (RER) were semi-quantified and analysed by a soft imaging system using transmission electronic microscopy photographs. Important variability of hepatocyte ultrastructural features was observed. The present study confirms that the rearing temperature, through its influences in the general metabolic activity, seems to be the main factor modifying mainly lipid droplet accumulation and RER development. However, factors that could be pooled under the designation of factors leading to food accessibility and lipid and protein quality intensify or compensate the effect of temperature.

Sortilin-related Receptor with A-type Repeats (SORLA) Affects the Amyloid Precursor Protein-dependent Stimulation of ERK Signaling and Adult Neurogenesis

Sortilin-related receptor with A-type repeats (SORLA) is a sorting receptor that impairs processing of amyloid precursor protein (APP) to soluble (s) APP and to the amyloid beta-peptide in cultured neurons and is poorly expressed in patients with Alzheimer disease (AD). Here, we evaluated the consequences of Sorla gene defects on brain anatomy and function using mouse models of receptor deficiency. In line with a protective role for SORLA in APP metabolism, lack of the receptor results in increased amyloidogenic processing of endogenous APP and in aggravated plaque deposition when introduced into PDAPP mice expressing mutant human APP. Surprisingly, increased levels of sAPP caused by receptor deficiency correlate with pro-found stimulation of neuronal ERK signaling and with enhanced neurogenesis, providing in vivo support for neurotrophic functions of sAPP. Our data document a role for SORLA not only in control of plaque burden but also in APP-dependent neuronal signaling and suggest a molecular explanation for increased neurogenesis observed in some AD patients.

Primary cutaneous carcinosarcoma: case report with expanded immunohistochemical analysis

An 83‐year‐old woman presented with a nodular, eroded tumor on the skin between the nose and the upper lip of 18 months’ duration. There were no palpable lymph nodes and no infiltrates on chest radiography. Complete surgical excision showed a tumor measuring 65 × 40 × 30 mm. On histopathologic examination, it was composed of typical basal cell carcinoma (BCC) nodules and large sheets of oval or short spindle cells (Fig. 1a), with vesicular nuclei, distinct nucleoli, moderate pleomorphism, and pronounced mitotic activity (more than 40 mitoses/10 high‐power fields). In parts abutting the upper lip, BCC nodules were found in the muscle layer, but the small salivary glands were uninvolved (Fig. 1b). Immunohistochemical analysis (Table 1) revealed a cytokeratin (CK)‐positive, Ber‐EP4‐positive, and vimentin‐negative BCC component (Fig. 2a), and a vimentin‐positive, CK‐negative sarcomatous component (Fig. 2b). In addition, mesenchymal tumor components were focally positive for smooth muscle actin (SMA). The BCC component showed irregular reaction with CK7, which stained some lobules and parts of individual nests (Fig. 2c). The final diagnosis was primary cutaneous carcinosarcoma. At the last visit, 3 months after operation, no signs of recurrence or metastatic spread were observed. Additional immunohistochemical analyses showed preserved membranous β‐catenin staining in the BCC component, without nuclear reaction in the mesenchymal component. The labeling index (LI) is expressed as the percentage of positive cells, and is calculated from the number of positive tumor cells divided by the total number of tumor cells counted (minimum 300 cells) in the areas with most pronounced immunopositivity. Counting was performed on images taken from microscopic high‐power fields with an Olympus DP70 digital camera (Olympus Corporation, Tokyo, Japan). The program analySYS (Soft Imaging System, Munster, Germany) was used, with the screen grid and the manual touch‐count method. The LI values of Ki‐67 and telomerase reverse transcriptase (hTERT) were higher in the sarcomatous component. hTERT displayed enhanced nucleolar localization and diffuse staining of mitotic cells. Histone deacetylase 1 (HDAC1) was expressed in a smaller percentage of cells than HDAC2, with a higher LI in the sarcomatous component. HDAC2 was the only marker analyzed that stained more cells in the BCC component (Fig. 2d).(a) Basal cell carcinoma (BCC) nodules and diffuse proliferation of sarcomatous cells. (b) Deep peripheral parts of the tumor show BCC nodules infiltrating into the muscle layer, but the small salivary glands are uninvolved. Hematoxylin and eosin; original magnification: (a) ×100; (b) ×40image Immunohistochemical staining in carcinosarcoma Antigen Source Epithelial (BCC) component Mesenchymal component CK (AE1/AE3) Dako* + – EA (Ber‐EP4) Dako + – CK7 Dako + (variable) – EMA Dako – – CEA Dako – – S‐100 Dako – – HMB‐45 Dako – – Vimentin Dako – + Factor VIII‐related Ag Dako – – CD34 Dako – – CD68 Dako – – CD99 Dako – – LCA Dako – – α‐SMA Dako – + (focal) Desmin Dako – – β‐Catenin Santa Cruz† + (membranous) – Ki‐67 (LI) Dako 10% 32% hTERT (LI) Novocastra‡ 22% 41% HDAC1 (LI) Santa Cruz 16% 40% HDAC2 (LI) Santa Cruz 76% 37% CEA, carcinoembryonic antigen; CK, cytokeratin; EA, epithelial antigen; EMA, epithelial membrane antigen; HDAC, histone deacetylase; hTERT, telomerase reverse transcriptase; LCA, leukocyte common antigen; LI, labeling index; SMA, smooth muscle actin. Glostrup, Denmark. Santa Cruz, CA, USA. Newcastle, UK. (a) Cytokeratin (CK) stain reveals epithelial (basal cell carcinoma, BCC) nodules, whereas the large sheet of sarcomatous cells (top left) is unstained. (b) Vimentin‐positive sarcomatous component and negative BCC nodules. (c) Variations of CK7 staining between BCC nodules and inside individual nodules. (d) Histone deacetylase 2 (HDAC2) expression is higher in the BCC component (right) than in the sarcomatous component. Streptavidin–biotin; original magnification: (a) ×12.5; (b, c) ×100; (d) ×400image

Lipid mapping in human dystrophic muscle by cluster-time-of-flight secondary ion mass spectrometry imaging

Human striated muscle samples, from male control and Duchenne muscular dystrophy-affected children, were subjected to cluster-time-of-flight secondary ion mass spectrometry (cluster-ToF-SIMS) imaging using a 25 keV Bi(3)(+) liquid metal ion gun under static SIMS conditions. Spectra and ion density maps, or secondary ion images, were acquired in both positive and negative ion mode over several areas of 500 x 500 microm(2) (image resolution, 256 x 256 pixels). Characteristic distributions of various lipids were observed. Vitamin E and phosphatidylinositols were found to concentrate within the cells, whereas intact phosphocholines accumulated over the most damaged areas of the dystrophic muscles, together with cholesterol and sphingomyelin species. Fatty acyl chain composition varied depending on the region, allowing estimation of the local damage extent.

Abstract No. 154: Success of a Flat Panel C-Arm with Soft Tissue Imaging and Needle Guidance System for Interventional Procedures

Abstract No. 154: Success of a Flat Panel C-Arm with Soft Tissue Imaging and Needle Guidance System for Interventional Procedures

Thickness of glomerular and tubular basement membranes in preclinical and clinical stages of diabetic nephropathy

Aims:This study aimed to elucidate the early renal changes in diabetes mellitus (DM) with and without clinical symptoms related to renal damage.Methods:Renal biopsy was studied in 25 patients (14 with microalbuminuria and 11 with albuminuria) both by light and electron microscopies (LM and EM, respectively) for renal changes and morphometry was performed to study glomerular and tubular basement membranes (GBM and TBM, respectively) width using a Soft Imaging System GmBH (analysis 3).Results:A significant increase was noted in the mean GBM and TBM thickness in both the preclinical and clinical groups compared to the control group. The changes in the TBM were noted to be predominant in both preclinical and clinical patients.Conclusions:This study indicates the importance of morphometric evaluation of the GBM and TBM width in the elucidation of early renal damage in diabetic nephropathy, especially in the absence of LM changes. The significance of identification of early renal changes using morphometric techniques for better management of these patients requires further studies.

Excess Adhesive Flash Upon Bracket Placement

To determine if there is any significant difference in excess adhesive flash (EAF) with the use of APC PLUS adhesive coated appliance system and Transbond XT and if other variables such as side of the mouth, time taken to bond the brackets, the age of the orthodontic specialist, or the years of clinical experience affected the amount of EAF. Twenty orthodontic specialists bonded 20 preadjusted straight wire brackets (Victory, 3M Unitek) on a typodont with a Class I crowded malocclusion in a split-mouth design, with one-half of the brackets bonded with APC PLUS adhesive system and the other half with Transbond XT. The teeth were sputter coated with gold and an image was taken at 32x magnification in a scanning electron microscope (Philips XL30). The resultant tagged image file (TIF) was opened in AnalySIS Pro 3.1 (Soft Imaging System, Munich, Germany) and the area of EAF was measured. t-Tests demonstrated that: (1) there was no statistically significant difference in EAF between two adhesives when bonding in different quadrants of dentition; and (2) there was no statistically significant difference in EAF between two adhesives overall. Analysis of variance models demonstrated there were no statistically significant differences found regarding the age of the orthodontic specialists, years of clinical experience, and time in performing the bonding. The addition of a coloring agent to assist in the visualization of the excess adhesive does not reduce the amount of excessive adhesive around orthodontic brackets.

Blockade of VEGFR3-signalling specifically inhibits lymphangiogenesis in inflammatory corneal neovascularisation

Inflammatory corneal hem- and lymphangiogenesis occurring both prior to as well as after penetrating keratoplasty significantly increase the risk for subsequent immune rejections. The purpose of this study was to analyze whether the blocking anti-VEGFR3 antibody mF4-31C1 is able to inhibit the outgrowth of pathologic new lymphatic vessels in a mouse model of suture-induced, inflammatory corneal neovascularisation, and whether this antibody specifically inhibits lymphangiogenesis without affecting hemangiogenesis. Three interrupted 11-0 nylon sutures were placed into the corneal stroma of BALB/c mice (6 weeks old) and left in place for 7 days to induce neovascularisation. The treatment group (n = 9) received the anti-VEGFR3 antibody mF4-31C1 intraperitoneally on the day of surgery and 3 days later (0.5 mg/mouse). Control mice received an equal amount of control IgG solution. For immunohistochemistry, corneal flat mounts were stained with LYVE-1 as a specific lymphatic vascular endothelial marker and with CD31 as panendothelial marker. Morphometry was performed with the image analysis software analySIS;B (Soft Imaging Systems GmbH, Münster, Germany). To improve the objectivity and precision of the morphometrical analysis, we established a modified method using grey filter sampling on monochromatic pictures. The mF4-31C1 antibody-treated mice displayed nearly complete inhibition of lymphangiogenesis compared with IgG controls (p < 0.006). In contrast, there was no significant inhibitory effect observed with respect to blood vessel growth (p > 0.05). Inflammatory corneal lymphangiogenesis seems to depend on VEGFR3-signalling. By blocking this receptor the ingrowths of lymphatic vessels can be inhibited almost completely, and specifically without affecting hemangiogenesis. This may open new treatment options to promote (corneal) graft survival without affecting hemangiogenesis.

Quantification of Neuroretinal Rim Loss Using Digital Planimetry in Long-term Follow-up of Normals and Patients With Ocular Hypertension

The purpose of this study was to investigate if digital planimetry is appropriate for quantification of neuroretinal rim loss in patients with ocular hypertension (OHT) and if there is an age-related neuroretinal rim loss in normals. Fifty-six patients with OHT without optic disc change, 13 patients with OHT and conversion to early glaucoma during follow-up and 42 age-matched controls were recruited from the Erlangen Glaucoma Registry. Annually, all patients underwent complete ophthalmologic examination including detailed diagnostic testing concerning glaucoma. Gold standard for morphologic evaluation of the optic nerve head was the semiquantitative 2-dimensional-method described by Jonas. Optic disc images from baseline and after 5 or 10 years follow-up were used for digital planimetry. Optic disc area and cup area were measured using commercial software: Soft Imaging System analysis. The investigator was masked for diagnosis and time point of examination. Mean neuroretinal rim loss was 0.36% per year in controls, 0.54% per year in patients with OHT without progressive disease, and 0.95% per year in OHT and conversion. Neuroretinal rim loss was highest in the group of OHT with conversion to early glaucoma during follow-up. In the control group we detected a very low mean neuroretinal rim loss during 10-year follow-up. In ocular hypertensive patients without progressive disease mean neuroretinal rim loss was approximately twice compared with normals.

Inhibition of Inflammatory Lymphangiogenesis by Integrin α5 Blockade

The interaction between endothelial cells and extracellular matrix proteins plays an important role in (hem)angiogenesis. Integrins are able to mediate the outgrowth of newly formed blood vessels. In contrast, the role of integrins in lymphangiogenesis, ie, the outgrowth of new from pre-existing lymphatic vessels, has so far been unclear. Here, expression and functional relevance of integrins on lymphatic endothelium in vivo was investigated using the mouse model of combined inflammatory corneal hemangiogenesis and lymphangiogenesis. Immunohistochemistry revealed novel expression of both integrin alpha5 and alphav on both resting and activated lymphatic vessels in vivo. Integrin alpha5-inhibiting small molecules significantly blocked the outgrowth of new lymphatic vessels into the cornea in a dose-dependent manner. The outgrowth of blood vessels was less significantly affected by this treatment, thus allowing for selective inhibition of lymphangiogenesis at lower dosages. Combined inhibition of integrin alpha5 and alphav using inhibiting molecules did not significantly increase the anti-lymphangiogenic effect in vivo, thus suggesting an important functional role of integrin alpha5 in lymphangiogenesis. In summary, our findings demonstrate novel expression of specific integrins on growing lymphatic endothelial cells in vivo and reveal their functional role during lymphangiogenesis. This opens new treatment options for selective inhibition of lymphangiogenesis, eg, in oncology and transplant immunology.

Connective tissue alterations in women with pelvic organ prolapse and urinary incontinence

Alterations in collagen synthesis and metabolism have previously been reported in patients with pelvic organ prolapse (POP) and/or urodynamic stress incontinence (USI). Since urinary incontinence does not always associate with POP, the objective of this study was to examine connective tissues from patients with USI plus POP, and patients with prolapse only. Biopsies from the uterosacral ligaments were obtained during the operation from POP patients (n =28), and from continent women (control group, n =12) who underwent surgery for other benign reasons. POP patients were classified following urodynamic tests and symptom questionnaire with respect to the presence (n =14) or absence (n =14) of USI. N-terminal propeptides of collagen (PINP and PIIINP), TGF-beta and leptin were measured in plasma. Hydroxyproline and glycosaminoglycan (GAGs) concentrations and total hexosaminidase activity were measured in tissue samples. Histological sections were prepared using Masson's trichrome technique, and digitised solutions were used for imaging provided by Soft Imaging System GmBh. Statistical evaluations were made by the Kruskal-Wallis test. A significant decrease in hydroxyproline content was found in USI+POP women in comparison to controls (p<0.05). In contrast, histopathological examination revealed an increased density of collagen in USI+POP patients. Hexosaminidase activity was decreased in both groups with POP, but no change in the amount of GAGs was observed. Markers of collagen synthesis (PINP, PIIINP), and factors related to the collagen synthesis (TGF-beta, leptin) remained unaltered. Our biochemical and morphological findings suggest a different organisation of collagen fibres in tissues of patients with USI+POP, when compared with both the controls and the POP patients.

Formation of Hyaluronan- and Versican-rich Pericellular Matrix by Prostate Cancer Cells Promotes Cell Motility

Previous studies have demonstrated that high levels of hyaluronan (HA) and the chondroitin sulfate proteoglycan, versican in the peritumoral stroma are associated with metastatic spread of clinical prostate cancer. In vitro integration of HA and versican into a pericellular sheath is a prerequisite for proliferation and migration of vascular smooth muscle cells. In this study, a particle exclusion assay was used to determine whether human prostate cancer cell lines are capable of assembling a pericellular sheath following treatment with versican-containing medium and whether formation of a pericellular sheath modulated cell motility. PC3 and DU145, but not LNCaP cells formed prominent polarized pericellular sheaths following treatment with prostate fibroblast-conditioned medium. The capacity to assemble a pericellular sheath correlated with the ability to express membranous HA receptor, CD44. HA and versican histochemical staining were observed surrounding PC3 and DU145 cells following treatment with prostatic fibroblast-conditioned medium. The dependence on HA for integrity of the pericellular sheath was demonstrated by its removal following treatment with hyaluronidase. Purified versican or conditioned medium from Chinese hamster ovary K1 cells overexpressing versican V1, but not conditioned medium from parental cells, promoted pericellular sheath formation and motility of PC3 cells. Using time lapse microscopy, motile PC3 cells treated with versican but not non-motile cells exhibited a polar pericellular sheath. Polar pericellular sheath was particularly evident at the trailing edge but was excluded from the leading edge of PC3 cells. These studies indicate that prostate cancer cells recruit stromal components to remodel their pericellular environment and promote their motility.

Overexpression of Rapsyn in Rat Muscle Increases Acetylcholine Receptor Levels in Chronic Experimental Autoimmune Myasthenia Gravis

The primary autoantigen in myasthenia gravis, the acetylcholine receptor (AChR), is clustered and anchored in the postsynaptic membrane of the neuromuscular junction by rapsyn. Previously, we found that overexpression of rapsyn by cDNA transfection protects AChRs in rat muscles from antibody-mediated loss in passive transfer experimental autoimmune myasthenia gravis (EAMG). Here, we determined whether rapsyn overexpression can reduce or even reverse AChR loss in muscles that are already damaged by chronic EAMG, which mimics the human disease. Active immunization against purified AChR was performed in female Lewis rats. Rapsyn overexpression resulted in an increase in total muscle membrane AChR levels, with some AChR at neuromuscular junctions but much of it in extrasynaptic membrane regions. At the ultrastructural level, most endplates in rapsyn-treated chronic EAMG muscles showed increased damage to the postsynaptic membrane. Although rapsyn overexpression stabilized AChRs in intact or mildly damaged endplates, the rapsyn-induced increase of membrane AChR enhanced autoantibody binding and membrane damage in severe ongoing disease. Thus, these results show the complexity of synaptic stabilization of AChR during the autoantibody attack. They also indicate that the expression of receptor-associated proteins may determine the severity of autoimmune diseases caused by anti-receptor antibodies.

Mutant Presenilin 1 Alters Synaptic Transmission in Cultured Hippocampal Neurons

Mutations in presenilins are the major cause of familial Alzheimer disease, but the precise pathogenic mechanism by which presenilin (PS) mutations cause synaptic dysfunction leading to memory loss and neurodegeneration remains unclear. Using autaptic hippocampal cultures from transgenic mice expressing human PS1 with the A246E mutation, we demonstrate that mutant PS1 significantly depressed the amplitude of evoked alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate receptor-mediated synaptic currents. Analysis of the spontaneous miniature synaptic activity revealed a lower frequency of miniature currents but normal miniature amplitude. Both alterations could be rescued by the application of a gamma-secretase blocker. On the other hand, the application of synthetic soluble Abeta42 in wild-type neurons induced the PS1 mutant phenotype on synaptic strength. Together, these findings strongly suggest that the expression of mutant PS1 in cultured neurons depresses synaptic transmission by causing a physical reduction in the number of synapses. This hypothesis is consistent with morphometic and semiquantitative immunohistochemical analysis, revealing a decrease in synaptophysin-positive puncta in PS1 mutant hippocampal neurons.

Reduction of Amyloid Angiopathy and Aβ Plaque Burden after Enriched Housing in TgCRND8 Mice: Involvement of Multiple Pathways

Reduction of Amyloid Angiopathy and Aβ Plaque Burden after Enriched Housing in TgCRND8 Mice: Involvement of Multiple Pathways

Species-Specific Fibroblasts Required for Triggering Invasiveness of Partially Transformed Oral Keratinocytes

This study tests the hypothesis that invasion of partially transformed keratinocytes is initiated by diffusible, proinvasive signals provided by species-specific fibroblasts. In vitro organotypic cultures of neoplastic human oral mucosa were constructed by growing a partially transformed, nontumorigenic keratinocytic cell line isolated from a dysplastic human oral lesion (DOK-ECACC94122104) on top of various types of connective tissue equivalents. Cultured tissues were analyzed by histomorphometry (depth and area of invasion: Dinv, Ainv) and immunohistochemistry. Presence of human fibroblasts in the matrix induced a local invasion of DOK (Dinv = 95.6 +/- 7.1 microm, Ainv = 45.8 +/- 3.5%). Minimal invasion (P < 0.05) was observed when DOK grew on simple collagen matrix (Dinv = 14.1 +/- 2.1 microm, Ainv = 3.7 +/- 0.8%) or matrices containing fibroblasts from mouse (Dinv = 11.5 +/- 4.0 microm, Ainv = 4.3 +/- 1.0%) or rat (Dinv = 15.6 +/- 1.2 microm, Ainv = 6.1 +/- 0.5%). In these cultures, local invasion could be induced by the presence of human fibroblasts in a bottom layer of the collagen matrix (P < 0.05) or by conditioned medium from organotypic cultures of DOK on human fibroblast-containing matrix (P < 0.05) but not by conditioned medium from human fibroblast monocultures (P > 0.05). Deposition of human collagen IV was observed at epithelial-matrix interface only when DOK behaved invasively. In conclusion, invasion of partially transformed oral keratinocytes was triggered by keratinocyte-induced fibroblast-derived diffusible factor(s) in a species-specific manner and associated with de novo synthesis of collagen IV.

Alcohol Abuse Enhances Neuroinflammation and Impairs Immune Responses in an Animal Model of Human Immunodeficiency Virus-1 Encephalitis

Neuroinflammatory disorders (including human immunodeficiency virus-1 encephalitis, HIVE) are associated with oxidative stress and inflammatory brain injury, and excessive alcohol use can exacerbate tissue damage. Using a murine model of HIVE, we investigated the effects of alcohol abuse on the clearance of virus-infected macrophages and neuroinflammation. Severe combined immunodeficient mice were reconstituted with human lymphocytes, and encephalitis was induced by intracranial injection of HIV-1-infected monocyte-derived macrophages (HIV-1(+) MDM). Animals were fed an ethanol-containing diet beginning 2 weeks before lymphocyte engraftment and for the entire duration of the experiment. Lymphocyte engraftment was not altered by ethanol exposure. Alcohol-mediated immunosuppression in ethanol-fed mice was manifested by a significant decrease in CD8(+)/interferon-gamma(+) T lymphocytes, a fivefold increase in viremia, and diminished expression of immunoproteasomes in the spleen. Although both groups showed similar amounts of CD8(+) T-lymphocyte infiltration in brain areas containing HIV-1(+) MDMs, ethanol-fed mice featured double the amounts of HIV-1(+) MDMs in the brain compared to controls. Ethanol-exposed mice demonstrated higher microglial reaction and enhanced oxidative stress. Alcohol exposure impaired immune responses (increased viremia, decreased immunoproteasome levels, and prevented efficient elimination of HIV-1(+) MDMs) and enhanced neuroinflammation in HIVE mice. Thus, alcohol abuse could be a co-factor in progression of HIV-1 infection of the brain.