American Journal Of Pathology

Published by American Society for Investigative Pathology
Online ISSN: 1525-2191
Print ISSN: 0002-9440
Metaphase-based comparative genomic hybridization (CGH) has identified recurrent regions of gain on different chromosomes in bladder cancer, including 6p22. These regions may contain activated oncogenes important in disease progression. Using quantitative multiplex polymerase chain reaction (QM-PCR) to study DNA from 59 bladder tumors, we precisely mapped the focal region of genomic gain on 6p22. The marker STS-X64229 had copy number increases in 38 of 59 (64%) tumors and the flanking markers, RH122450 and A009N14, had copy number gains in 33 of 59 (56%) and 26 of 59 (45%) respectively. Contiguous gain was present for all three markers in 14 of 59 (24%) and for two (RH122450 and STS-X64229) in 25 of 59 (42%). The genomic distance between the markers flanking STS-X64229 is 0.5 megabases, defining the minimal region of gain on 6p22. Locus-specific interphase fluorescence in situ hybridization confirmed the increased copy numbers detected by QM-PCR. Current human genomic mapping data indicates that an oncogene, DEK, is centrally placed within this minimal region. Our findings demonstrate the power of QM-PCR to narrow the regions identified by CGH to facilitate identifying specific candidate oncogenes. This also represents the first study identifying DNA copy number increases for DEK in bladder cancer.
Angiogenesis in malignant melanoma (MM) was evaluated by comparing mean vessel number (MVN) in Spitz's nevi (SN), thick and thin MMs that metastasized, and thick and thin MMs with > or = 10-year survival. Vessels were identified with antibodies against factor VIII-related antigen (FVIII) and CD34 in 37 MMs (17 < or = 1.9 mm and 20 > or = 4.0 mm) with > or = 10-year follow-up and 10 SN from children (< or = 9 years old). Fields (x250) with the highest vessel density were counted by independent observers blinded to clinical outcome. There were no differences in MVN between SN versus MMs (P = 1.0), but the distribution of vessels was much more uniform in SN. Seven MM pairs (> or = 5.5 mm) and five pairs (< or = 0.75 mm) were matched by sex, age, site, stage, and primary treatment (paired t-test). In the pairs > or = 5.5 mm, there was no correlation with MVN with either metastasis or death (FVIII P = 0.98; CD34 P = 0.85). Among the thin paired lesions, high MVN (FVIII = 46, CD34 = 39) was significantly related not only to metastasis (FVIII P = 0.04, CD34 P = 0.03) but also to death (FVIII P = 0.04, CD34 P = 0.05). MVN does not separate SN versus MM nor predict outcome in thick (> or = 4.0 mm) MMs; however, high MVN (> or = 42 average) is predictive of metastasis and death in MMs < or = 0.75 mm. Larger matched studies are indicated to confirm this observation.
Ulex europaeus agglutinin 1 (UEA-1) peroxidase staining of vessels in histological sections of normal skin (A) and from the base of a melanoma (B). Vessels (arrows) are demonstrated by staining of the endothelial lining and are seen as circular or elliptical cross-sections. At the base of the melanoma, vessels are more plentiful than in norrnal skin, but are similar in size and shape in cross-section. The edge of the melanoma is marked by an interrupted line. 
The vascularity of 20 primary skin melanomas was assessed histologically. These cases were selected from patients with intermediate thickness melanomas (0.76-4.0 mm thick) treated surgically to provide two groups of ten patients. One group had no evidence of recurrence with a minimum follow-up of 9 years. The second group of ten patients developed locoregional or systemic metastasis under follow-up, and seven of these patients died of disseminated melanoma. Age, sex, Breslow's tumor thickness, and Clark's level of invasion were similar in the two groups. Vascular quantitation was carried out by image analysis after vascular definition by Ulex europaeus-I agglutinin staining. The percentage vascular area at the tumor base in the recurrence group was more than twice that in the recurrence-free group. This study suggests that increased vascularity at the tumor base may have prognostic significance in intermediate thickness melanomas.
Drug-induced liver injury is a major safety issue. It can cause severe disease and is a common cause of the withdrawal of drugs from the pharmaceutical market. Recent studies have identified the HLA-B(∗)57:01 allele as a risk factor for floxacillin (FLUX)-induced liver injury and have suggested a role for cytotoxic CD8(+) T cells in the pathomechanism of liver injury caused by FLUX. This study aimed to confirm the importance of FLUX-reacting cytotoxic lymphocytes in the pathomechanism of liver injury and to dissect the involved mechanisms of cytotoxicity. Cytotoxicity of FLUX-reacting T cells from 10 HLA-B(∗)57:01(+) healthy donors toward autologous target cells and HLA-B(∗)57:01-transduced hepatocytes was analyzed in vitro. Cytotoxicity of FLUX-reacting T cells was concentration-dependent and required concentrations in the range of peak serum levels after FLUX administration. Killing of target cells was mediated by different cytotoxic mechanisms. IHC staining of a liver biopsy from a patient with FLUX-induced liver injury revealed periportal inflammation and the infiltration of cytotoxic CD3(+) CD8(+) lymphocytes into the liver. The infiltration of cytotoxic lymphocytes into the liver of a patient with FLUX-induced liver injury demonstrates the importance of FLUX-reacting T cells in the underlying pathomechanism. Our findings emphasize the role of the adaptive immune system and especially of activated drug-reacting T cells in human leukocyte antigen-associated, drug-induced liver injury.
The pathogenesis of endometriosis, a disease widely believed to arise from an aberrant growth of endometrial tissue outside the uterus, is still unclear. We have previously observed that cytokine-stimulated endometrial cells of women with endometriosis secrete in vitro increased amounts of monocyte chemotactic protein-1 (MCP-1). This factor may be important in the recruitment and activation of peritoneal macrophages observed in endometriosis patients. The present study reports that, in the presence of the disease, such an up-regulation of MCP-1 expression arises in vivo and can be encountered in situ in the intrauterine endometrium. In women with endometriosis, MCP-1 expression was elevated in endometrial glands, both at the level of the protein (immunohistochemistry) and the mRNA (in situ hybridization). This was observed throughout the menstrual cycle and varied according to the stage of the disease. These findings strongly argue in favor of the presence of pathophysiological changes in the eutopic endometrium of patients with endometriosis and make plausible MCP-1 as a key effector cell mediator involved in the pathogenesis of the disease.
Histological assessment of fibrosis after a short course of CC14. Male or female Sprague Dawley rats received four doses of CC14 as described in Materials and Methods. At the time of sacrifice, a piece of liver was fixed in neutral buffered formalin, sectioned, and stained with sirius red14; representative areas are shown. Other samples from the same livers were analyzed for type I collagen mRNA (Figure 2). 
Effect of Safironil on Collagen Expression during Short-Course CC14 Administration 
Effect of Safironil on Hepatic Injury and Collagen Content After Long-Course CC14 
The perisinusoidal stellate cells of the liver in an injury milieu undergo activation, acquiring a myofibroblast-like phenotype. In this state, they are the principal source of collagen and related proteins in fibrosis. The present studies evaluate the mechanism of action of two novel antifibrotic compounds, HOE 077 and Safironil, which were designed as competitive inhibitors of collagen protein synthesis. Fibrosis was induced in rats by administration of carbon tetrachloride, and activation was monitored as the level of collagen I mRNA or smooth muscle alpha-actin. Both male and female rats were studied. Stellate cell activation, rather than collagen synthesis, proved to be the target of both HOE 077 and Safironil in the intact liver. In culture, the drugs not only prevented the activation of stellate cells but also accelerated their deactivation. They were no more effective in co-cultures containing hepatocytes than in pure stellate cell cultures, indicating that metabolic conversion of HOE 077 was not required. Interestingly, the response of cells from females was greater than that of male cells, leading to the conclusion that stellate activation is sexually dimorphic. This finding may be relevant to the observation that fibrosis in chronic viral hepatitis progresses less rapidly and that hepatocellular carcinoma is less frequent in females than in males.
The effects of 7 days of pretreatment with various inducers on the acute toxicity of either 5% vinyl chloride at 24 hours after onset of a 6-hour exposure or 0.02%/ 1,1dichloroethylene at 6 hours after onset of a 4-hour exposure. Control animals were pretreated with water. Photomicrographs show the effect of the inducers PBT, 1254, and HCB. Central vein toward the right, portal vein toward the left. (x 150) 
-Effect of MFOS Inducers on Liver Glutathione Content Following Exposure to Vinyl 
Midzonal parenchymal cells 2 hours after the onset of exposure to 0.02% DCE. Cell borders are retracted from sinusoidal walls at lower right and left center. Space formed contains cytoplasmic protrusions, material resembling fibrin and erythrocytes. Cytoplasm of retracted cells focally contain swollen mitochondria and clusters of small vacuoles in the regions of the golgi apparatus. Strands of rough endoplasmic reticulum are intact. Nuclei show striking segregation of chromatin towards the margins of the nuclear envelope. Bile canaliculi appear intact. Parenchymal 
Vinyl chloride, an occupational carcinogen, produces acute liver injury in rats pretreated with phenobarbital or Aroclor 1254. Injury appears related to morphologic changes in the endoplasmic reticulum. The degree of injury, as indicated by elevation of serum enzymes derived from the liver, correlates with the magnitude of induction of cytochrome P-450 and its reduction by NADPH. Hepatic injury following 1,1-dichloroethylene exposure differs strikingly from that caused by vinyl chloride and appears to involve plasma membranes, mitochondria, and chromatin and spares endoplasmic reticulum. Induction of cytochrome P-450 appears to protect against 1,1-dichloroethylene but not vinyl chloride.
Time course of acute hepatocellular cytorrhexis in livers of previously fasted rats during exposure to 1 ,1-dichloroethylene. Portal triads are at the left. Central veins are toward the right. At zero time "control" (A) portal cells seem slightly larger than centrolobular cells. Ergastoplasm (cytoplasmic clumps of rough endoplasmic reticulum) are prominent, and occasional larger, darker tetraploid nuclei are seen in parenchymal cells. One hour after onset of exposure (B) egastoplasm is still prominent and mitotic figures are numerous in parenchymal cells (arrows). By two hours (C) there are increased erythrocytes within midzonal and centrolobular hepatic cords, and occasional parenchymal cell nuclei in these areas show central rarefaction and displacement of their chromatin to the periphery (arrows). At 4 hours (D) centrolobular parenchyma (at righo is necrotic and hemorrhagic. (Immersion fixation in formaldehyde, x500) 
At 2 hours after the 
-Effects* of 200 ppm 1,1-DCE on Components of Liver Microsomes Time after onset of 1,1-DCE exposure 
Exposure of fasted rats to 200 ppm 1,1-dichloroethylene (1,1-DCE) for 1-4 hours resulted in striking aberrations in hepatic Na, K, Ca, and GSH levels which preceded and/or accompanied catastrophic histologic alterations of the liver. Na levels began to rise during the first hour, and preceded the morphologically apparent injury. Ca levels increased markedly and K levels declined between the second and fourth hour of exposure, and accompanied the catastrophic morphologic alterations. GSH levels were rapidly depleted but began to recover before the end of the exposure to 1,1-DCE. Functions of components of the mixed-function oxidase system of the liver endoplasmic reticulum were not appreciably affected early in the course of 1,1-DCE exposure; but after injury became massive, cytochrome P-450 and oxidative N-demethylase were deactivated. Thus effects on the functional components of the endoplasmic reticulum mixed-function oxidase system do not appear to be primary events in 1,1-DCE cytotoxicity. In contrast, there were progressive declines in mitochondrial K and marked imbalances in mitochondrial Na, Zn, and Mg preceding the massive influx of Ca into the cell, indicating that mitochondria are involved early in he evolution of injurious molecular events elicited by this potent hepatotoxin.
Canalicular and mitochondrial membranes were investigated as early foci of hepatocyte injury in fed and fasted male Sprague-Dawley rats given 50 mg of 1,1-dichloroethylene (DCE)/kg. Staining of the bile canaliculi localized enzymes, leucine aminopeptidase (LAP), and Mg++-dependent ATPase (Mg++-ATPase), was examined by histochemistry in frozen sections. Mitochondrial membrane enzymes, including succinate dehydrogenase, also were examined by histochemistry. Staining of two monoclonal antibodies, C-1 and 9-B1, whose binding is localized in the bile canalicular region, was examined by immunofluorescence in frozen sections. Fasted rats treated with DCE developed moderate liver damage by 4 hours as evidenced by increases in serum transaminase and bilirubin, whereas fed rats developed only slight cell damage. Centrolobular loss of immunocytochemical and histochemical canalicular staining, especially for C-1 and Mg++-ATPase, was evident as early as 1 hour after DCE and was striking by 2 hours in both fed and fasted rats. Decreases in mitochondrial enzymes were not evident histochemically in fed animals at any time after DCE and were found only at the later times in fasted animals given the toxin. Thus, DCE administration to fed rats provides a new model system of selective bile canaliculi injury.
Administration of 0.001% 1,2-dimethylhydrazine dihydrochloride, symmetrical, in the drinking water of 7-week-old randomly bred Swiss mice for the remainder of their lifetime induced blood vessel tumors and enhanced the incidence of lung neoplasms. Ninety-eight percent of the females and 92% of the males developed vascular lesions, whereas among the controls the incidence was 3% in the females and 1% in the males. In addition, the incidence of lung tumors rose from 12 to 44% in the females and from 10 to 24% in the males, as compared with the controls. The occurrence of the vascular tumors in order of decreasing frequency was as follows: muscle, pararenal, fat, liver, parametrial, paraepididymal tissues, etc. Gross, light and electron microscopic examinations of vascular lesions revealed the characteristic appearance of angiosarcomas. The type and extent of macroscopic and histologic involvements of the various tissues by the tumors are presented. The ultrastructural descriptions of hemorrhagic areas, vascular spaces, neoplastic endothelial cells, their cytoplasms and organelles are illustrated in detail.In conclusion, whereas hydrazine enhanced the development of lung tumors, when the dimethyl group was attached to it at symmetrical positions, it evoked vascular tumors. Thus, the present study provides evidence for the possible relationship between chemical structure and tumor induction at specific organ sites.
During inflammation, E- and P-selectins appear on activated endothelial cells to interact with leukocytes through sialyl-Lewis x and sialyl-Lewis a antigens (sLe(x/a)). These selectins can also interact with tumor cells in a sialyl-Lewis-dependent manner and for this reason, they are thought to play a key role in metastasis. Diverting the biosynthesis of sialyl-Lewis antigens toward nonadhesive structures is an attractive gene therapy for preventing the hematogenous metastatic spread of cancers. We have previously shown that transfection of alpha(1,2)-fucosyltransferase-I (FUT1) in Chinese hamster ovary (CHO) cells had a slight effect on the overall sialylation while the synthesis of sLE(x) was dramatically prevented. We herein delivered the gene of FUT1 by a human immunodeficiency virus-derived lentiviral vector to three human cancer cell lines including pancreatic (BxPC3), hepatic (HepG2), and colonic (HT-29) cancer cells. We found that on FUT1 transduction, all cells exhibited a dramatic decrease in sLe(x) synthesis with a concomitant increase in Le(y) and Le(b) expression, without any detectable effect on the level of cell surface sLe(a) antigens. In parallel, FUT1-transduced HT-29 and HepG2 cells, but not BxPC3 cells, failed to interact with E-selectin as assessed by E-selectin-binding assay or dynamic adhesion to activated endothelial cells. We show also that transduced FUT1 efficiently fucosylates the P-selectin ligand PSGL-1 without altering P-selectin binding. These results have important implications for understanding cell-specific reactions underlying the synthesis of selectin ligands in cancer cells and may provide a basis for the development of anti-metastatic gene therapy.
1,25-Dihydroxyvitamin D3 (1,25-[OH]2D3) was administered in doses of 1 (65 picomoles) or 5 (325 picomoles) units daily for 7 days to adult thyroparathyroidectomized rats fed a normal rodent diet. Both dose levels significantly increased serum calcium, decreased serum phosphorus, and increased urinary calcium and phosphorus concentrations. Numbers of osteoblasts were significantly increased in rats given 5 units 1,25-(OH)2D3. Ultrastructurally, these osteoblasts were larger and interpreted to be more active in bone matrix synthesis and mineralization, compared with osteoblasts in control rats. The number of osteoblasts in rats given 1 unit 1,25-(OH)2D3 was not increased, compared with controls, but they were morphologically similar to the osteoblasts in rats given 5 units 1,25-(OH)2D3. A granular electron-dense material, interpreted to be mineral, was present in the pericellular space of osteocytes in rats given 1 and 5 units 1,25-(OH)2D3. The size of osteocytes, organellar development, and contour of osteocytic lacunae were not affected by 1,25-(OH)2D3. The number of osteoclasts was not significantly elevated in 1,25-(OH)2D3-treated rats and their morphology was similar to that of osteoclasts in controls. It is concluded that in metaphyseal bone 1,25-(OH)2D3 increased the number and synthetic activity of osteoblasts without significantly enhancing osteoclasis or osteocytic osteolysis. This response was independent of parathyroid hormone and calcitonin in rats fed a normal diet.
Metabolic Parameters in Adult Dogs Following L-Thyroxine Administration for 60 Days* Interval (weeks)
The effects of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3), parathyroid hormone (PTH), and L-thyroxine (T4) on trabecular bone remodeling were evaluated by histomorphometric methods in adult female beagle dogs. Intravenous 1,25-(OH)2D3 (1.25 micrograms/day in equally divided doses) was administered intermittently for 6 days and withdrawn 14 days for three complete cycles. PTH was administered intravenously (2.5 U/kg/day) in divided doses 6 hours apart for 60 days. Thyroxine was given orally (1.0 mg/kg/day) in divided doses for a similar interval. Static and dynamic changes were evaluated using tetracycline and DCAF (2,4 BIS) N, N', Di (carboxymethyl) (amino methyl fluorescein) in vivo double labeling of bone from the iliac crest taken before treatment and after 60 days. The intermittent administration of 1,25-(OH)2D3 stimulated the bone resorption rate and depressed the formation rate. 1,25-(OH)2D3 increased trabecular resorption surfaces; osteoid surface, volume, and thickness; mineralization lag time; and osteoblast number but decreased the bone volume. Multiple small daily doses of PTH resulted in an overall negative balance in trabecular bone. This was associated with an increased trabecular surface-to-volume ratio, bone resorption and formation rates, active forming surfaces, osteoid volume and surface, life span of bone forming and resorbing sites, and the number of osteoclast nuclei. Thyroxine appeared to increase bone mass by enhancing the switch-over from the resorptive to the formative phase of remodeling. Coupling between osteoid apposition and mineralization was increased by recruiting more forming sites and prolonging their life span. Thyroxine increased bone resorption and formation rates, trabecular bone volume and balance, number of osteoclast nuclei, and life span of bone forming sites. The osteoid seam thickness and mineralization lag time were decreased. The present study demonstrated that 1,25-(OH)2D3, PTH, and thyroxine at the dose and schedule used, markedly altered stimulators of remodeling in trabecular bone of adult dogs.
The effects of intermittent low doses (1.25 mug daily, administered intravenously for 6 days and withdrawn for 14 days for 3 complete cycles) of 1,25-dihydroxycholecalciferol (1,25-[OH](2)D(3)) on cortical bone were determined and compared in ribs with steady state and regionally accelerated remodeling in adult intact female dogs. The bone changes were analyzed by dynamic bone histomorphometric methods, using tetracycline and DCAF (2,4 BIS) N, N' di (carboxymethyl) (amino methyl fluorescein) in vivo double labeling of bones before treatment and after 60 days of intermittent 1,25-(OH)(2)D(3) administration. Serum calcium and phosphorus levels increased during 1,25-(OH)(2)D(3) administration. Urinary hydroxyproline excretion increased during the first interval of 1,25-(OH)(2)D(3) administration but was not changed significantly during the last two intervals. In normal cortical bone (11th rib) following the administration of 1,25-(OH)(2)D(3) there was a marked decrease in the activation frequency, bone formation rate, osteoid seam thickness, seam circumference, and mean appositional rate. Although recruitment of new remodeling sites was decreased after 1,25-(OH)(2)D(3), previously existing remodeling units continued to completion. These effects resulted in a preponderance of mature osteons in normal cortical bone. The morphometric changes in cortical bone (9th rib) exposed to both 1,25-(OH)(2)D(3) and periosteal elevation were characterized by a marked increase in both the activation frequency and bone formation rate and associated with a decrease in the osteon formation time. Other morphometric parameters that were increased included radial closure rate, numbers of osteoid seams and resorption cavities, ratio of bone resorbing to forming sites, percentage labeled and circumference of osteoid seams, and total and cortical bone areas. The combined effect of periosteal elevation and 1,25-(OH)(2)D(3) were markedly different from those observed with 1,25-(OH)(2)D(3) alone. These findings suggest that the rapid bone turnover induced by tissue injury will mask or alter the effects of hormones on bone remodeling when studied over a relatively short period of time.
We have analyzed expression of 1,25-dihydroxyvitamin D(3) receptor (VDR) protein and mRNA in basal cell carcinomas (BCC) of human skin. VDR immunoreactivity in BCCs was compared with the staining pattern of the proliferation marker Ki-67 in the same tumors. Additionally, VDR staining was compared to staining pattern of apoptotic cells by terminal UTP nucleotide end labeling assay. Frozen sections of superficial type, nodular type, and fibrosing type BCCs were consistently immunoreactive for VDR (mAb 9A7gamma) with almost every tumor cell labeled (n = 15). In general, VDR staining was pronounced in peripheral tumor cells. VDR immunoreactivity was consistently stronger in tumor cells than in adjacent or unaffected epidermis. No visual correlation was found in BCCs comparing labeling patterns of Ki-67-positive or apoptotic cells and mAb 9A7gamma. VDR mRNA was increased in BCCs (n = 6) compared to normal human skin (n = 5), as revealed by reverse transcription-polymerase chain reaction analysis. Our findings indicate that VDR is strongly expressed in BCCs and may be involved in the growth regulation of this tumour, and VDR mRNA and protein are increased in BCCs as compared to normal human epidermis.
The objectives of this study were to evaluate the effects of vitamin D(3) (D(3)) and 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) on uremic bone disease independent of their action on the intestine. The histomorphology of tibial metaphyses in uremic (5/6 nephrectomized [5/6 Nx]) rats fed a low-calcium-low-phosphorus (LCLP) diet was compared with sham-operated (SO) rats fed an LCLP diet and 5/6 Nx rats fed an LCLP diet and given 15,000 IU D(3) or 5 units (135 ng) 1,25-(OH)(2)D(3) daily for 7 days. A marked osteomalacia characterized by an increased percentage of active and inactive trabecular osteoid surface and thickened growth plates developed in proximal tibial metaphyses in 5/6 Nx rats given the placebo, compared with SO rats. These bone changes were associated with relative hypophosphatemia, hypophosphaturia, and hypercalciuria in 5/6 Nx rats. In 5/6 Nx rats treated with D(3) or 1,25-(OH)(2)D(3) the growth plates had undergone mineralization and vascular invasion and were markedly reduced in thickness. Other parameters of osteomalacia in trabecular bone were not different from 5/6 Nx rats given the placebo. There was a significant decrease in osteoclasts per millimeter of trabecular surface perimeter in D(3)- and 1,25-(OH)(2)D(3)-treated rats. These bone changes were associated with hypercalcemia, hyperphosphatemia, and hyperphosphaturia, compared with 5/6 Nx rats given the placebo. It was concluded that in uremic rats fed the LCLP diet, shortterm treatment with either pharmacologic levels of D(3) or 1,25-(OH)(2)D(3) corrected only lesions in the growth plate. Osteoid seams were not reduced in treated rats, although the serum calcium-phosphorus product was elevated. The 5/6 Nx rat fed the LCLP diet appears to be a useful model for the rapid induction of uremic osteomalacia in adult animals.
Vitamin D deficiency is a major risk factor for central nervous system (CNS) demyelinating diseases including multiple sclerosis (MS) and its animal model, that of experimental autoimmune encephalomyelitis (EAE). Both vitamin D(3) and 1, 25-dihydroxyviatmin-D(3) (calcitriol) had beneficial effects in EAE/MS. However, the exact cause of vitamin D deficiency in EAE/MS is not clear. Previously, we documented that lovastatin (LOV) provides protection in EAE animals via inhibition of RhoA-ROCK signaling. Herein, we demonstrate that LOV prevents the lowering of circulating 25-hydroxyvitamin-D(3) and 1,25-dihydroxyviatmin-D(3) levels including 1,25-dihydroxyviatmin-D(3) levels in the peripheral lymphoid organs and CNS of treated EAE animals. These effects of LOV were attributed to enhanced expression of vitamin D synthesizing enzyme (1α-hydroxylase) in kidney and the CNS, with corresponding reduction of vitamin D catabolizing enzyme (24-hydorxylase) expression in the CNS of EAE animals via inhibition of RhoA-ROCK signaling. Ex vivo and in vitro studies established that autoreactive Th1/Th17 cells had higher expression of 24-hydroxylase than Th2/T regulatory cells, that was reverted by LOV or ROCK inhibitor. Interestingly, LOV-mediated regulation of vitamin D metabolism had improved vitamin D(3) efficacy to confer protection in EAE animals and that was ascribed to the LOV- and calcitriol-induced immunomodulatory synergy. Together, these data provide evidence that interfering with RhoA-ROCK signaling in autoreactive Th1/Th17 cells can improve vitamin D(3) efficacy in clinical trials of MS and related neurodegenerative disorders.
Thyroparathyroidectomized rats fed a low-calcium-normal-phosphorus diet were administered 1 or 5 units of 1,25-dihydroxyvitamin D3-(1,25-[OH]2D3) or placebo daily for 7 days. 1,25-(OH)2D3 elevated serum and urine calcium and decreased serum phosphorus. Rats given 1 unit of 1,25-(OH)2D3 had increased numbers of osteoclasts in metaphyseal trabeculae, Ultrastructurally, osteoclasts, osteoblasts, and osteocytes in rats given 1 unit of 1,25-(OH)2D3, were similar to those in rats given placebo. In rats given 5 units of 1,25-(OH)2D3, osteoclasis was markedly increased. Osteoblasts were more numerous and interpreted to be active in matrix production and mineralization. Lamellated electron-dense bodies were observed adjacent to the plasma membranes of less active osteoblasts and were interpreted to be modified matrix. Most osteocytes in rats given 5 units of 1,25-(OH)2D3 were indistinguishable from osteocytes in rats given placebo. However,the pericellular space of some osteocytes in rats given 5 units of 1,25-(OH)2D3 contained electron-dense granular deposits that were interpreted to be calcium phosphate. It is concluded that 1,25-(OH)2D3 is able to significantly elevate serum calcium independent of dietary calcium, parathyroid hormone, and calcitonin primarily by increasing ostoeclasis with minimal dependence on osteocytic osteolysis.
The α (1,3)-fucosyltransferases, types IV and VII (FUT4 and FUT7, respectively), are required for the synthesis of functional selectin-type leukocyte adhesion molecule ligands. The selectins and their ligands modulate leukocyte trafficking, and P-selectin and its ligand, P-selectin glycoprotein ligand-1, can modulate hemostasis and thrombosis. Regulation of thrombosis by FUT4 and/or FUT7 activity was examined in mouse models of carotid artery thrombosis and collagen/epinephrine-induced thromboembolism. Mice lacking both FUT4 and FUT7 (Fut(-/-) mice) had a shorter time to occlusive thrombus formation in the injured carotid artery and a higher mortality due to collagen/epinephrine-induced pulmonary thromboemboli. Mice lacking P-selectin or P-selectin glycoprotein ligand-1 did not have a prothrombotic phenotype. Whole blood platelet aggregation was enhanced, and plasma fibrinogen content, clot weight, and clot strength were increased in Fut(-/-) mice, and in vitro clot lysis was reduced compared with wild type. Fut4(-/-), but not Fut7(-/-), mice had increased pulmonary thromboembolism-induced mortality and decreased thromboemboli dissolution in vivo. These data show that FUT4 and FUT7 activity regulates thrombosis in a P-selectin- and P-selectin glycoprotein ligand-1-independent manner and suggest that FUT4 activity is important for thrombolysis.
Selectins mediate the initial adhesion of leukocytes to endothelial cells in many contexts of inflammation-dependent leukocyte recruitment. The glycans that contribute to P- and E-selectin counterreceptor activity arise through glycosylation reactions in which the terminal steps are catalyzed by alpha(1,3) fucosyltransferases (FTs). We examined how selectin ligand activities are controlled in eosinophils by characterizing FT expression profiles and regulatory mechanisms in eosinophils isolated from human blood. We found that FT-IV and FT-VII mRNAs were up-regulated by transforming growth factor-beta1, but the FT-IV transcript consistently predominated in eosinophils. To further define the physiological role of FT-IV and FT-VII in expression of eosinophil selectin ligand, we characterized models of dermal eosinophilia in FT-IV- and/or FT-VII-deficient mice in vivo. FT-IV deficiency yielded a significant decrease in eosinophil recruitment to the skin. Likewise, deficiency of FT-VII also yielded a decrease in eosinophil recruitment. Eosinophil recruitment that remained in the absence of FT-VII was further inhibited by blocking P- or E-selectin and was essentially absent in mice deficient in both enzymes. These observations indicate that FT-IV and FT-VII are both important contributors to selectin-dependent eosinophil recruitment to the skin and may represent therapeutic targets for treating diseases in which eosinophil recruitment contributes to pathophysiology.
Preterm birth (PTB) currently accounts for 13% of all births in the United States, with the leading cause of PTB being maternal infection. Endothelin-1, an extremely potent vasoconstrictor capable of increasing myometrial smooth muscle tone, has been shown to be up-regulated in the setting of infection in pregnancy, ultimately leading to PTB. In previous work, we have shown that infection-associated PTB is controlled in our murine model by using phospharamidon, an endothelin-converting enzyme-1 inhibitor; knocking down endothelin-converting enzyme-1 mRNA; or blocking the binding of endothelin-1 to the endothelin-A (ET(A)) receptor with either BQ-123 or with HJP-272, the 6-OH compound of our series of novel synthetic (ET(A)) receptor antagonists. In the current study, we show that HJP-272, a highly selective ET(A) receptor antagonist with an IC(50) of 70.1 nmol/L, binds in a noncompetitive manner to the ET(A) receptor. Additionally, we introduce n-propyl (HJP-286) and n-butyl (HJP-278) analogs of HJP-272. We find that the LD(50) of HJP-272, the analog in the series most effective in controlling preterm birth, is more than 20-fold higher than its therapeutic dose. Acute exposure to high doses of these compounds produces no histological changes in any organ, while chronic exposure produces only a rare hepatotoxic effect. These findings may be of clinical significance, as there is currently no FDA-approved therapy for women presenting with threatened preterm delivery.
We have previously demonstrated that hepatocyte proliferation induced by the mitogen 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) is independent of changes in cytokines, immediate early genes, and transcription factors that are considered to be necessary for regeneration of the liver after partial hepatectomy (PH) or necrosis. To further investigate the differences between mitogen-induced mouse hepatocyte proliferation and liver regeneration after PH, we have measured the expression of cyclin D1, cyclin D3, cyclin E, and cyclin A and of the cyclin-dependent kinases CDK2, CDK4, and CDK6. The involvement of the cyclin-dependent kinase inhibitors p21 and p27 and of the oncosuppressor gene p53 was also examined at different times after stimulation of hepatocyte proliferation. Results showed that a single administration of TCPOBOP caused a very rapid increase in the levels of cyclin D1, a G1 protein, when compared with two thirds PH (8 hours versus 30 hours). The early increase in cyclin D1 protein levels was associated with a faster onset of increased expression of S-phase-associated cyclin A (24 hours versus 36 hours with PH mice). Accordingly, measurement of bromodeoxyuridine (BrdU) incorporation revealed that, although approximately 8% of hepatocytes were BrdU-positive as early as 24 hours after TCPOBOP, no significant changes in BrdU incorporation were observed at the same time point after two thirds PH. The expression of other proteins involved in cell cycle control, such as cyclin-dependent kinases (CDK4, CDK2, CDK6), was also analyzed. Results showed that expression of CDK2 was induced much more rapidly in TCPOBOP-treated mice (2 hours) than in mice subjected to PH (36 hours). A different pattern of expression in the two models of hepatocyte proliferation, although less dramatic, was also observed for CDK4 and CDK6. Expression of the CDK inhibitors p21 and p27 and the oncosuppressor gene p53 variably increased after two thirds PH, whereas basically no change in protein levels was found in TCPOBOP-treated mice. The results demonstrate that profound differences in many cell cycle-regulatory proteins exist between direct hyperplasia and compensatory regeneration. Cyclin D1 induction is one of the earlier events in hepatocyte proliferation induced by the primary mitogen TCPOBOP and suggests that a direct effect of the mitogen on this cyclin may be responsible for the rapid onset of DNA synthesis observed in TCPOBOP-induced hyperplasia.
Cell-surface carbohydrate chains are known to contribute to cell migration, interactions, and proliferation, but their roles in skin wound healing have not been evaluated. We examined the biological roles of beta4-galactosylated carbohydrate chains in skin wound healing using mutant mice that lack beta-1,4-galactosyltransferase-I (beta4GalT-I), which is responsible for the biosynthesis of the type 2 chain in N-glycans and the core 2 branch in O-glycans. beta4GalT-I-deficient mice showed significantly delayed wound healing with reduced re-epithelialization, collagen synthesis, and angiogenesis, compared with control mice. Neutrophil and macrophage recruitment at wound sites was also impaired in these mice probably because of selectin-ligand deficiency. In accordance with the reduced leukocyte infiltration, the expression levels of macrophage-derived chemokines, transforming growth factor-beta1, and vascular endothelial growth factor were all reduced in beta4GalT-I(-/-) mice. These results demonstrate that beta4-galactosylated carbohydrate chains play a critical role in skin wound healing by mediating leukocyte infiltration and epidermal cell growth, which affects the production of chemokines and growth factors. This study introduces a suitable mouse model for investigating the molecular mechanisms of skin wound healing and is the first report showing that carbohydrate chains have a strong influence on skin wound healing.
Beta4 galactosylation of glycoproteins plays important roles in protein conformation, stability, transport, and clearance from the circulation. Recent studies have revealed that aberrant glycosylation causes various human diseases. Here we report that mice lacking beta-1,4-galactosyltransferase (beta4GalT)-I, which transfers galactose to the terminal N-acetylglucosamine of N- and O-linked glycans in a beta-1,4 linkage, spontaneously developed human immunoglobulin A nephropathy (IgAN)-like glomerular lesions with IgA deposition and expanded mesangial matrix. beta4GalT-I-deficient mice also showed high serum IgA levels with increased polymeric forms as in human IgAN. IgAN is the most common form of glomerulonephritis, and a significant proportion of patients progress to renal failure. However, pathological molecular mechanisms of IgAN are poorly understood. In humans, abnormal character of serum IgA, especially serum IgA1 with aberrant galactosylation and sialylation of O-glycans in its hinge region is thought to contribute to the pathogenesis of IgAN. Mouse IgA has N-glycans but not O-glycans, and beta4-galactosylation and sialylation of the N-glycans on the serum IgA from beta4GalT-I-deficient mice was completely absent. This is the first report demonstrating that genetic remodeling of protein glycosylation causes IgAN. We propose that carbohydrates of serum IgA are involved in the development of IgAN, whether the carbohydrates are O-glycans or N-glycans.
Administration of a hepatotoxic diet containing 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) induces biliary damage followed by hepatocyte injury, which is repaired through atypical ductular proliferation and oval cells and their subsequent differentiation to bile duct cells and hepatocytes. In this study, we examine whether excess β-catenin in transgenic (TG) mice would provide any reparative advantage in response to DDC. No differences in appearance or numbers of total A6-positive oval cells were observed after DDC administration. However, an increase in A6-positive "atypical hepatocytes" in the TG livers was observed after 14 and 28 days, coinciding with an increase in proliferating cell nuclear antigen-positive hepatocytes. Intriguingly, after chronic DDC administration for 150 days, a further increase in atypical hepatocytes was evident in TG mice, with higher numbers of proliferating cell nuclear antigen-positive hepatocytes exhibiting cytoplasmic/nuclear β-catenin and α-fetoprotein but not CK19, HNF1β, or Trop-2. Coincidently, we observed an improvement in intrahepatic cholestasis as seen by decreases in both serum bilirubin and alkaline phosphatase levels in TG mice, indicating an overall improvement in hepatic repair. TG mice exposed to DDC for 4 weeks followed by 2 days of normal chow showed decreases in alkaline phosphatase, atypical ductular proliferation, and periportal inflammation compared with wild-type animals, verifying improved biliary repair in TG livers. Thus, we report a potential role of β-catenin in liver repair, especially in enhancing the resolution of intrahepatic cholestasis after DDC injury.
In previous studies, we have shown that angiogenesis is often first noted in cutaneous malignant melanomas (CMMs) under 1.0 mm in thickness. Because angiogenesis may signal a more aggressive tumor phenotype, it is important to establish the circumstances associated with onset of angiogenesis. In the present study, we have quantified tumor vascularity in a series of CMMs under 1.0 mm in thickness and either associated with or lacking histologic regression. Microvessels were identified with the lectin Ulex europaeus agglutinin I and the vessels in five fields counted within an ocular grid (area 7.84 x 10(-2) mm2) at 400 x magnification. CMMs (mean 0.48 mm) with regression had greater microvessel counts (27.2 +/- 5.1) compared with CMMs (mean 0.61 mm) without regression (mean 20.1 +/- 7.9) (P < 0.01). However, of particular interest, CMMs in the radial growth phase only and associated with regression (mean 0.40 mm) had strikingly greater vascularity (mean 28.7 +/- 6.9) versus radial growth phase CMMs (mean 0.44 mm) lacking regression (mean 16.4 +/- 6.6) (P = 0.0013). CMMs in the vertical growth phase (mean 0.81 mm) without regression had slightly less vascularity (mean 24.4 +/- 7.3) compared with vertical growth phase CMMs with regression (mean microvessels 27.2 +/- 5.1) (P = 0.1878) but significantly greater microvessels versus radial growth phase CMMs without regression (P = 0.0213). These results suggest that the onset of angiogenesis in thin CMMs is related to at least two phenomena: 1) inflammatory regression and 2) development of the vertical growth phase.
Cell Numbers per Glomerular Cross Section after the First Injection of OX7 
Cell Numbers per Glomerular Cross Section after the Second Injection of OX7 
NO Generation by Macrophages Isolated from Control and Nephritic Rats after First Injection of OX7 
NO Generation by Macrophages from Control and Nephritic Rats after Second Injection of OX7 
-Glucuronidase Expression of Macrophages Isolated from Glomeruli of Rats Killed after the First Injection of OX7 and Healthy Controls 
Macrophages infiltrating glomeruli in telescoped nephrotoxic nephritis are programmed. The purpose of this study was to assess whether macrophages infiltrating glomeruli of rats with passively induced injury become similarly programmed, and to determine whether macrophage commitment is an early event. Glomerular macrophages isolated from rats with resolving and proliferative anti-Thy-1 nephritis were examined for nitric oxide (NO) generation and expression of lysosomal hydrolases. After a single injection of Thy-1 antibody the cells generated large amounts of NO that was attenuated ex vivo by transforming growth factor-beta and other anti-inflammatory cytokines. In contrast macrophages infiltrating glomeruli immediately after a second injection of Thy-1 antibody generated NO spontaneously and were unresponsive to alternative activation. beta-Glucuronidase expression was used as a second independent assay for macrophage activation and the results confirmed the observations made for NO. Furthermore, macrophages infiltrating the glomerulus after the second antibody injection exhibited a striking dichotomy in that 70% of the cells behave as programmed by interferon-gamma and 30% by transforming growth factor-beta. The results show that macrophage commitment occurs very early after monocyte migration and that infiltration itself does not invariably induce macrophage programming. It demonstrates that macrophages infiltrating inflamed glomeruli at the same time do not respond uniformly, but are capable of engaging different activation programs. This emphasizes the critical importance of the underlying disease process for macrophage functional development in an inflamed environment.
Chronic passive congestion (CPC) and centrilobular necrosis (CLN) are well recognized pathologic changes, but their exact relationship to different forms of cardiac dysfunction is uncertain. We reviewed clinical data and hepatic, renal, and adrenal morphology related to cardiac dysfunction in 1000 autopsy subjects at The Johns Hopkins Hospital whose hearts had been studied after postmortem arteriography and fixation in distention. Fourteen pathologic variables, including body and organ size, and microscopic changes graded on a semiquantitative scale, and 18 clinical variables including congestive heart failure, shock, and cardiovascular disease, were analyzed statistically. Distinct patterns of cardiac dysfunction emerged for the two spectra of hepatic morphologic change. Among patients with variable CPC, but slight or absent CLN, the amount of CPC was predicted in a multivariate analysis by severity of right-sided congestive heart failure. CPC severity correlated with cardiac weight and chamber enlargement (P less than 0.001). Among patients with variable CLN, but slight or absent CPC, CLN was predicted by profound hypotension and by renal failure. In addition, CLN, but not CPC, was significantly correlated with renal acute tubular necrosis (P less than 0.001) and adrenal cortical medullary junction necrosis (P less than 0.05), two lesions associated with shock. Among all 1000 patients CPC and CLN were highly significantly correlated (P less than 0.001). The results show that hepatic CPC arises from conditions producing elevated systemic venous pressure but that CLN arises from reduced systemic arterial pressure; and the presence of one potentiates the development of the other.
Immunosuppressive treatments of systemic lupus (SLE) remain associated with significant toxicities; hence, compounds with better toxicity profiles are needed. Dihydroorotate dehydrogenase (DHODH) inhibition with leflunomide has proven to be effective in autoimmune diseases including SLE, but leflunomide can cause a variety of side effects. We hypothesized that 4SC-101, a novel DHODH inhibitor with a more favorable toxicity profile, would be as effective as high-dose cyclophosphamide (CYC) in controlling experimental SLE of female MRL(Fas)lpr mice. Daily oral gavage of 30, 100, and 300 mg/kg 4SC-101 from 12 to 22 weeks of age was compared with either vehicle or CYC treatment (30 mg/kg/week, i.p.) in terms of efficacy and toxicity. Three hundred milligrams per kilogram 4SC-101 was as effective as CYC in depleting spleen autoreactive T cells, B cells, and plasma cells as well as the respective DNA and RNA serum autoantibodies. This was associated with a comparable amelioration of the renal, dermal, and pulmonary SLE manifestations of MRL(Fas)lpr mice. However, even the highest dose of 4SC-101 had no effect on bone marrow neutrophil counts, which were significantly reduced in CYC-treated mice. Together, the novel DHODH inhibitor 4SC-101 is as effective as high dose CYC in controlling SLE without causing myelosuppression. Hence, DHODH inhibition with 4SC-101 might be suitable to treat active SLE with fewer side effects than CYC.
Recent evidence has suggested an important role of miRNAs in liver biology and diseases, although the implication of miRNAs in cholangiocarcinoma remains to be defined further. This study was designed to examine the biological function and molecular mechanism of miR-101 in cholangiocarcinogenesis and tumor progression. In situ hybridization and quantitative RT-PCR were performed to determine the expression of miR-101 in human cholangiocarcinoma tissues and cell lines. Compared with noncancerous biliary epithelial cells, the expression of miR-101 is decreased in 43.5% of human cholangiocarcinoma specimens and in all three cholangiocarcinoma cell lines used in this study. Forced overexpression of miR-101 significantly inhibited cholangiocarcinoma growth in severe combined immunodeficiency mice. miR-101-overexpressed xenograft tumor tissues showed decreased capillary densities and decreased levels of vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2). The VEGF and COX-2 mRNAs were identified as the bona fide targets of miR-101 in cholangiocarcinoma cells by both computational analysis and experimental assays. miR-101 inhibits cholangiocarcinoma angiogenesis by direct targeting of VEGF mRNA 3'untranslated region and by repression of VEGF gene transcription through inhibition of COX-2. This study established a novel tumor-suppressor role of miR-101 in cholangiocarcinoma and it suggests the possibility of targeting miR-101 and related signaling pathways for future therapy.
It can be difficult to distinguish benign bile duct proliferations (BDPs) from well-differentiated metastatic peripancreatic adenocarcinomas on histological grounds alone. Most peripancreatic carcinomas harbor activating point mutations in codon 12 of the K-ras oncogene, suggesting that K-ras mutational status may provide a molecular basis for distinguishing BDPs from liver metastases. The ability of tests for mutations in codon 12 of K-ras to make this distinction was examined in a two-part study. In the first part we determined the K-ras mutational status of 56 liver lesions and 48 primary peripancreatic adenocarcinomas obtained from 48 patients. In the second part of this study an additional 45 liver lesions were studied. In the first 48 patients, activating point mutations in codon 12 of K-ras were detected in 28 (61%) of the 46 primary carcinomas, in 8 (100%) of 8 liver metastases, in 2 (6.5%) of 31 BDPs, and in none (0%) of 14 liver granulomas. Three BDPs and two primary carcinomas did not amplify. To further estimate the prevalence of K-ras mutations in BDPs we analyzed an additional series of 45 mostly incidental BDPs for K-ras mutations. Three (6.7%) of these 45 harbored K-ras mutations. These results suggest that K-ras mutations may be useful in distinguishing BDPs from metastases in the liver; however, there is some overlap in the mutational spectra of BDPs and pancreatic carcinomas.
Human neutrophils were isolated from peripheral blood by four methods: 1) Ficoll-Hypaque gradients and erythrocyte lysis, 2) plasma-Percoll gradients, 3) a "lipopolysaccharide (LPS)-free" method yielding 85% neutrophils, and 4) by centrifugation of cells prepared by Method 3 through a plasma-Percoll gradient to produce pure neutrophils. The use of the Ficoll-Hypaque method resulted in spontaneous change of cell shape, enhanced formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated release of superoxide anion, increased release of lysosomal enzymes upon subsequent FMLP stimulation, and reduced chemotactic responsiveness, by comparison with the other methods. These effects were not due to erythrocyte lysis by NH4C1 but were reproduced by exposure of neutrophils prepared by the "LPS-free" method or the use of plasma-Percoll gradients to 10-100 ng/ml LPS. Neutrophil change of shape and stimulated O-2 production were particularly sensitive markers of these effects. The effects of trace concentrations of LPS in the modulation of neutrophil function may have relevance to the pathophysiology of endotoxemia and its resultant tissue injury.
New Zealand white rabbits (10) were administered daily doses of nicotine (2.4 mg/kg/day) in their drinking water for 25 weeks. Nicotine-treated rabbits were compared with control rabbits (10) in terms of blood serum biochemistry and lipid profiles, blood cells counts, changes in aortic endothelial cell morphologic characteristic and distribution, and vessel wall permeability (Evans blue dye uptake). Fasting serum levels of glucose, triglycerides, total cholesterol, and LDL-cholesterol were elevated in nicotine-treated rabbits. No significant differences (nicotine vs control) were seen in leukocyte, erythrocyte and platelet counts, or hematocrit and hemoglobin. Control and nicotine-treated rabbit aortas showed similar focal areas of increased Evans blue dye uptake; staining was localized primarily to aortic arch areas. Endothelial cells (luminal surface) from non-Evans blue and Evans blue arch areas were examined by a combination of Häutchen preparation (silver-stained vessels) and scanning and transmission electron microscopy. Endothelial cells from nicotine-treated arch areas (Evans-blue-stained) showed extensive changes such as: increased cytoplasmic silver deposition, increased formation of microvilli, and numerous focal areas of "ruffled" endothelium (projections on cell surfaces). These data indicate that nicotine, administered orally to rabbits, has a demonstrable in vivo morphologic effect on endothelial cells in the aortic arch.
Polymerase chain reaction (PCR) amplification and product analysis for the detection of chromosomal translocations, such as the t(14;18), has traditionally been a two-step process. PCR product detection has generally entailed gel electrophoresis and/or hybridization or sequencing for confirmation of assay specificity. Using a microvolume fluorimeter integrated with a thermal cycler and a PCR-compatible double-stranded DNA (dsDNA) binding fluorescent dye (SYBR Green I), we investigated the feasibility of simultaneous thermal amplification and detection of MBR/JH translocation products by fluorescence melting curve analysis. We analyzed DNA from 30 cases of lymphoproliferative disorders comprising 19 cases of previously documented MBR/JH-positive follicle center lymphoma and 11 reactive lymphadenopathies. The samples were coded and analyzed blindly for the presence of MBR/JH translocations by fluorescence melting curve analysis. We also performed dilutional assays using the MBR/JH-positive cell line SUDHL-6. Multiplex PCR for MBR/JH and beta-globin was used to simultaneously assess sample adequacy. All (100%) of the 19 cases previously determined to be MBR/JH positive by conventional PCR analysis showed a characteristic sharp decrease in fluorescence at approximately 90 degrees C by melting curve analysis after amplification. Fluorescence melting peaks obtained by plotting the negative derivative of fluorescence over temperature (-dF/dT) versus temperature (T) showed melting temperatures (Tm) at 88.85+/-1.15 degrees C. In addition, multiplex assays using both MBR/JH and beta-globin primers yielded easily distinguishable fluorescence melting peaks at approximately 90 degrees C and 81.2 degrees C, respectively. Dilutional assays revealed that fluorescence melting curve analysis was more sensitive than conventional PCR and agarose gel electrophoresis with ultraviolet transillumination by as much as 100-fold. Simultaneous amplification and fluorescence melting curve analysis is a simple, reliable, and sensitive method for the detection of MBR/JH translocations. The feasibility of specific PCR product detection without electrophoresis or utilization of expensive fluorescently labeled probes makes this method attractive for routine molecular diagnostics.
Most colorectal cancers have loss of function mutations in the adenomatosis polyposis coli (APC) tumor suppressor gene. This leads to accumulation of beta-catenin, which together with the DNA binding protein TCF-4 functions as a transcriptional activator. Recently defined target genes are c-myc and cyclin D1, linking the APC gene defect to the capacity for autonomous proliferation of colon tumors. Here we report the identification of the matrix metalloproteinase MMP-7 as another target gene of beta-catenin/TCF-4. MMP-7 is overexpressed in 80% of human colorectal cancers and known to be an important factor for early tumor growth, with a potential function also for later progression steps, like invasion and metastasis. Our results explain the high percentage of MMP-7 overexpression in colon tumors. Moreover they indicate that defects in the APC tumor suppressor gene may also have an influence on later steps of colon tumor progression.
Risk factors for vulvar squamous cell carcinoma (SCC) are human papilloma virus (HPV) infections and lichen sclerosus (LS). The significance of monoclonal gamma-T-cell receptor (gamma-TCR) rearrangement in the lymphoid infiltrate of LS and the consequence for vulvar carcinogenesis is unknown. One hundred sixty-one biopsies of vulvar LS and SCC, with and without LS, were examined for monoclonal gamma-TCR rearrangement and HPV16 expression, and for the expression of B- and T-cell markers and fascin. Monoclonal gamma-TCR rearrangement was identified in 8 of 17 patients with LS and 11 of 21 patients with SCC arising in LS with only occasional HPV16 DNA detection. None of the 19 SCC without LS showed monoclonal gamma-TCR rearrangement, but 14 of 19 patients had strong HPV16 detection. The lichenoid infiltrate of LS with germline configuration consisted predominantly of T cells (CD8 > CD4), along with numerous B cells. However, in biopsies with monoclonally rearranged gamma-TCR, CD4-positive T cells dominated along with B cells and fascin-positive cells in the lichenoid infiltrate and in deeply located lymphocyte aggregates (LAs). These LAs additionally contained fascin-positive dendritic cells with only individual CD8, CD57, and granzyme-positive cells. LAs in biopsies with germline configuration demonstrated numerous T cells (CD8 >CD4), but only single peripheral B cells, CD57, and fascin-positive lymphocytes. Our data suggest that monoclonal gamma-TCR rearrangement is characteristic for and limited to LS and SCC arising in LS, raising the question for a LS-associated antigen. We interpret B cells, CD4-positive T cells, and fascin-expressing dendritic cells within LS as a cellular immune response to antigen or proliferating T-cell clones. The resulting local immune dysregulation in LS may provide a permissive environment for the development of a SCC.
SMCs stimulate collagen fibril assembly. In vitro collagen assembly was performed by incubating Texas Red-labeled, acid-solubilized collagen at 37 ° C in culture medium (M199 plus 10% fetal bovine serum, pH 7.4). Fibrils that formed and attached to the substrate were fixed in paraformaldehyde and imaged microscopically. a: Spontaneously assembled fibrils that formed and adhered to a fibronectin-coated coverslip after incubating 10 ␮ g/ml of Texas Red-Vitrogen collagen in cell-free culture media for 3 hours at 37 ° C. b: Fibrils are not evident when 2 ␮ g/ml of Texas Red-collagen is incubated with culture medium, indicating this concentration to be below the critical threshold for self-assembly under these conditions. c and d: Confocal microscopic images acquired 1 hour ( c ) and 3 hours ( d ) after addition of 1 ␮ g/ml of Texas Red-labeled Vitrogen collagen to human SMCs. Punctate accumulations of fluorescence are evident initially, followed by small fibrils on the surface of SMCs. e: Lower magnification image (nonconfocal) 24 hours after addition of soluble collagen to SMCs, showing extensive fibril formation. f: Cell-associated fibrils also formed after the addition of 1 ␮ g/ml of Texas Red-labeled rat-tail collagen to SMC cultures. Nuclei were counterstained with Hoechst 33258. Scale bars, 50 ␮ m. 
SMC-associated collagen assembly is mediated by ␣ 2 ␤ 1 integrin. Human SMCs were incubated for 48 hours with solubilized FITC-labeled collagen (2 ␮ g/ml) and either control IgG ( a ), an anti- ␣ 1 ␤ 1 integrin antibody (5E8D9, 20 ␮ g/ml) ( b ), or an anti- ␣ 2 ␤ 1 integrin antibody (BHA2.1, 20 ␮ g/ml) ( c ). Antibodies were added 30 minutes before addition of FITC-collagen monomers. Fibril assembly was also evaluated in cultures preincubated for 30 minutes with anti- ␣ 2 ␤ 1 integrin-stimulating antibody (JBS2, 1:50) and then incubated with Texas Red-labeled collagen (1 ␮ g/ml) for 6 hours. Compared to control conditions ( d ), the fibers in JBS2-treated cultures were more abundant and straighter ( e ). Washed cultures were fixed in paraformaldehyde and nuclei were stained with Hoechst 33258. Scale bars, 30 ␮ m. 
SMC-directed collagen assembly is dependent on the actin cytoskeleton. a: Collagen fibrils formed over the stretched tail of human SMCs ( arrow ) 6 hours after addition of soluble, Texas Red-labeled rat tail collagen (1 ␮ g/ml) to cultures (overlay of phase contrast with fluorescence image). b: Six hours after addition of soluble, FITC-labeled bovine skin collagen (2 ␮ g/ml) to cultures, early fibrils can be seen oriented parallel to the actin microfilament bundles, the latter stained using Texas Red-labeled phalloidin. c and d: Collagen assembly was evaluated in SMC cultures incubated for 24 hours with FITC-collagen and either DMSO ( c ) or cytochalasin D (10 ␮ mol/L) ( d ), both added 1 hour before addition of solubilized collagen. Nuclei were counterstained with Hoechst 33258. Scale bars, 30 ␮ m. 
SMC-directed collagen assembly is mediated by RhoA. Human arterial SMCs were infected with retrovirus containing empty vector (pLNCX, a), cDNA for dominant-negative RhoA (pLNCX-T19N RhoA, b), or cDNA for constitutively active RhoA (pLNCX-N63L RhoA, c). Transductants were selected by G418 treatment and then incubated with FITC-labeled collagen (2 g/ml) for 24 hours. Collagen fibrils are reduced on SMCs expressing dominant-negative RhoA and extensive on SMCs expressing constitutively active RhoA. Nuclei were counterstained with Hoechst 33258. Scale bars, 50 m.
LPA stimulates SMC-directed collagen assembly. SMCs were incubated for 12 hours with 2 ␮ g/ml of FITC-labeled soluble collagen and either vehicle ( a , b ) or LPA (20 ␮ mol/L) ( c , d ), added 30 minutes before addition of collagen. Cultures were fixed in 4% paraformaldehyde and nuclei stained with Hoechst 33258. Cultures were also stained with Texas Red-labeled phalloidin to show actin microfilament bundles ( b and d , control and LPA-treated, respectively). The Texas Red fluorescence images were acquired with identical exposure times. Scale bars, 50 ␮ m. 
Assembly of collagen into fibrils is widely studied as a spontaneous and entropy-driven process. To determine whether vascular smooth muscle cells (SMCs) impact the formation of collagen fibrils, we microscopically tracked the conversion of soluble to insoluble collagen in human SMC cultures, using fluorescent type I collagen at concentrations less than that which supported self-assembly. Collagen microaggregates were found to form on the cell surface, initially as punctate collections and then as an increasingly intricate network of fibrils. These fibrils displayed 67-nm periodicity and were found in membrane-delimited cellular invaginations. Fibril assembly was inhibited by an anti-alpha2beta1 integrin antibody and accelerated by an alpha2beta1 integrin antibody that stimulates a high-affinity binding state. Newly assembled collagen fibrils were also found to co-localize with newly assembled fibronectin fibrils. Moreover, inhibition of fibronectin assembly with an anti-alpha5beta1 integrin antibody completely inhibited collagen assembly. Collagen fibril formation was also linked to the cytoskeleton. Fibrils formed on the stretched tails of SMCs, ran parallel to actin microfilament bundles, and formed poorly on SMCs transduced with retrovirus containing cDNA for dominant-negative RhoA and robustly on SMCs expressing constitutively active RhoA. Lysophosphatidic acid, which activates RhoA and stimulates fibronectin assembly, stimulated collagen fibril formation, establishing for the first time that collagen polymerization can be regulated by soluble agonists of cell function. Thus, collagen fibril formation is under close cellular control and is dynamically integrated with fibronectin assembly, opening new possibilities for modifying collagen deposition.
Estrogen receptor (ER) action is modulated by posttranslational modifications. Although ERalpha phosphorylation correlates with patient outcome, ERbeta is similarly phosphorylated but its significance in breast cancer has not been addressed. We investigated whether ERbeta that is phosphorylated at serine 105 (S105-ERbeta) is expressed in breast cancer and assessed potential clinical implications of this phosphorylation. Following antibody validation, S105-ERbeta expression was studied in tissue microarrays comprising 108 tamoxifen-resistant and 351 tamoxifen-sensitive cases and analyzed against clinical data. S105-ERbeta regulation in vitro was assessed by Western blot, flow cytometry, and immunofluorescence. Nuclear S105-ERbeta was observed in breast carcinoma and was associated with better survival (Allred score > or =3), even in tamoxifen-resistant cases, and additionally correlated with ERbeta1 and ERbeta2 expression. Distinct S105-ERbeta nuclear speckles were seen in some higher grade tumors. S105-ERbeta levels increased in MCF-7 cells in response to 17beta-estradiol, the ERbeta-specific agonist diarylpropionitrile, and the partial ERbeta-agonist genistein. S105-ERbeta nuclear speckles were also seen in MCF-7 cells and markedly increased in size and number at 24 hours following 17beta-estradiol and, in particular diarylpropionitrile, treatment. These speckles were coexpressed with ERbeta1 and ERbeta2. Presence of S105-ERbeta in breast cancer and association with improved survival, even in endocrine resistant breast tumors suggest S105-ERbeta might be a useful additional prognostic marker in this disease.
It has been proposed that the skin is a functionally unique compartment of the immune system, although little direct evidence supporting this hypothesis has been presented. Here we show that lymphocyte populations at cutaneous sites can be differentiated from otherwise similar populations at noncutaneous sites by their preferential expression of an epitope defined by the MAb HECA-452. This MAb recognizes a predominantly 200-kd cell-surface glycoprotein present on about 16% of peripheral blood T cells, including both CD4+ and CD8+ T cells (17% and 11% HECA-452+, respectively), as well as TCR-delta-bearing T cells (32%+). Most thymocytes (99%) lacked HECA-452 antigen expression, and essentially all the HECA-452+ peripheral blood T cells were found in the adhesion molecule high, CD45R low putative memory cell subset, findings suggesting that HECA-452 expression develops peripherally as a consequence of antigenic stimulation. However, the HECA-452 antigen is not a conventional activation antigen because it was not upregulated with mitogen stimulation of peripheral blood T cells. Most significantly, among 54 diverse specimens of normal/reactive lymphoid tissues and sites of chronic inflammation, there was a clear association of lymphocyte HECA-452 expression and cutaneous location. In extracutaneous sites (n = 38) only about 5% of lymphocytes within the T-cell areas of these tissues expressed this antigen, whereas in inflammatory skin lesions (n = 16), 85% were HECA-452+. The association of HECA-452 expression and cutaneous location was also seen in a series of T-cell lymphomas. The malignant cells of 16 of 18 cases of epidermotropic (patch/plaque) stage mycosis fungoides were HECA-452+, as well as 2 of 7 nonmycosis fungoides peripheral T-cell lymphomas in skin. In contrast, this antigen was not expressed in thymic (lymphoblastic) lymphomas (n = 14), nonepidermotropic (tumor) stage mycosis fungoides (n = 5), and noncutaneous peripheral T-cell lymphomas (n = 15). Among lymphocytes, the preferential expression of the HECA-452 determinant by cutaneous T cells supports the hypothesis that the skin constitutes a immunologically unique lymphoid tissue and suggests that this molecule may play a role in either lymphocyte homing to skin or in lymphocyte interactions with the epidermis.
Galectin-3 is a beta-galactoside-binding protein which regulates many biological processes including cell adhesion, migration, cell growth, tumor progression, metastasis, and apoptosis. Although the exact function of galectin-3 in cancer development is unclear, galectin-3 expression is associated with neoplastic progression and metastatic potential. Since studies have suggested that tumor cell survival in microcirculation determines the metastatic outcome, we examined the effect of galectin-3 overexpression in human breast carcinoma cell survival using the liver ischemia/reperfusion metastasis model. While the majority of control cells died by hepatic ischemia/reoxygenation, nearly all of galectin-3 overexpressing cells survived. We showed that galectin-3 inhibits nitrogen free radical-mediated apoptosis, one of the major death pathways induced during hepatic ischemia/reperfusion. Galectin-3 inhibition of apoptosis involved protection of mitochondrial integrity, inhibition of cytochrome c release and caspase activation. Taking these results together with the previous observation that galectin-3 inhibits apoptosis induced by loss of cell adhesion, we propose that galectin-3 is a critical determinant for anchorage-independent and free radical-resistant cell survival during metastasis.
In this study in situ hybridization methods were used to examine biopsy samples from 13 adenocarcinomas of the colon for the presence of mRNA for the urokinase-type plasminogen activator (u-PA) and its specific cell-surface receptor (u-PAR). In all cases, u-PA mRNA was present in fibroblastlike cells in the stroma adjacent to the invasive tumor nodules. Urokinase-type plasminogen activator mRNA was not detected in the malignant cells. All specimens also contained u-PAR mRNA in cells located at the tumoral-stromal interface of invasive foci, but in contrast at least some of these cells were in all but one case identified as being of malignant origin. Stromal cells, probably tumor-infiltrating macrophages and neutrophils, also were positive in these areas. These results support the view that components of the plasminogen activation system may act to influence proteolytic events occurring at the interface between stroma and malignant cells in adenocarcinomas of the colon in humans.
Granulin (GRN, or progranulin) is a protein involved in wound repair, inflammation, and neoplasia. GRN has also been directly implicated in frontotemporal dementia and may contribute to Alzheimer's disease pathogenesis. However, GRN regulation expression is poorly understood. A high-throughput experimental microRNA assay showed that GRN is the strongest target for miR-107 in human H4 neuroglioma cells. miR-107 has been implicated in Alzheimer's disease pathogenesis, and sequence elements in the open reading frame-rather than the 3' untranslated region-of GRN mRNA are recognized by miR-107 and are highly conserved among vertebrate species. To better understand the mechanism of this interaction, FLAG-tagged Argonaute constructs were used following miR-107 transfection. GRN mRNA interacts preferentially with Argonaute 2. In vitro and in vivo studies indicate that regulation of GRN by miR-107 may be functionally important. Glucose supplementation in cultured cells that leads to increased miR-107 levels also results in decreased GRN expression, including changes in cell compartmentation and decreased secretion of GRN protein. This effect was eliminated following miR-107 transfection. We also tested a mouse model where miR-107 has been shown to be down-regulated. In brain tissue subjacent to 1.0 mm depth controlled cortical impact, surviving hippocampal neurons show decreased miR-107 with augmentation of neuronal GRN expression. These findings indicate that miR-107 contributes to GRN expression regulation with implications for brain disorders.
Galectin-3 is a member of a growing family of beta-galactoside-binding animal lectins. Previous studies have demonstrated a variety of biological activities for this protein in vitro, including activation of cells, modulation of cell adhesion, induction of pre-mRNA splicing, and regulation of apoptosis. To assist in fully elucidating the physiological and pathological functions of this protein, we have generated galectin-3-deficient (gal3(-/-)) mice by targeted interruption of the galectin-3 gene. Gal3(-/-) mice consistently developed fewer inflammatory cell infiltrations in the peritoneal cavities than the wild-type (gal3(+/+)) mice in response to thioglycollate broth treatment, mainly due to lower numbers of macrophages. Also, when compared to cells from gal3(+/+) mice, thioglycollate-elicited inflammatory cells from gal3(-/-) mice exhibited significantly lower levels of NF-kappaB response. In addition, dramatically different cell-spreading phenotypes were observed in cultured macrophages from the two genotypes. Whereas macrophages from gal3(+/+) mice exhibited well spread out morphology, those from gal3(-/-) mice were often spindle-shaped. Finally, we found that peritoneal macrophages from gal3(-/-) mice were more prone to undergo apoptosis than those from gal3(+/+) mice when treated with apoptotic stimuli, suggesting that expression of galectin-3 in inflammatory cells may lead to longer cell survival, thus prolonging inflammation. These results strongly support galectin-3 as a positive regulator of inflammatory responses in the peritoneal cavity.
Top-cited authors
Jeremy S Duffield
  • University of Washington Seattle
Michael P Lisanti
  • University of Salford
John Varga
  • Northwestern University
Ben Humphreys
  • Brigham and Women's Hospital
Bradley Hyman
  • Harvard University