Tumor cell invasion of model 3-dimensional matrices: demonstration of migratory pathways, collagen disruption, and intercellular cooperation.

We report a novel 3-dimensional model for visualizing tumor cell migration across a nylon mesh-supported gelatin matrix. To visualize migration across these model barriers, cell proteolytic activity of the pericellular matrix was detected using Bodipy-BSA (fluorescent upon proteolysis) and DQ collagen (fluorescent upon collagenase activity). For 3-dimensional image reconstruction, multiple optical images at sequential z axis positions were deconvoluted by computer analysis. Specificity was indicated using well-known inhibitors. Using these fluorescent proteolysis markers and imaging methods, we have directly demonstrated proteolytic and collagenolytic activity during tumor cell invasion. Moreover, it is possible to visualize migratory pathways followed by tumor cells during matrix invasion. Using cells of differing invasive potentials (uPAR-negative T-47D wild-type and uPAR-positive T-47D A2--1 cells), we show that the presence of the T-47D-A2--1 cells facilitates the entry of T-47D wild-type cells into the matrix. In some cases, wild-type cells follow T-47D A2--1 cells into the matrix whereas other T-47D-wild-type cells appear to enter without the direct intervention of T-47D A2--1 cells. Thus, we have developed a new 3-dimensional model of tumor cell invasion, demonstrated protein and collagen disruption, mapped the pathways followed by tumor cells during migration through an extracellular matrix, and illustrated cross-talk among tumor cell populations during invasion.

Apoptotic cells of an epithelial cell line, AsPC-1, release monocyte chemotactic S19 ribosomal protein dimer.

A pancreatic carcinoma cell line, AsPC-1, underwent apoptosis in vitro when heat-treated for 60 min at 43 degrees C. Apoptotic AsPC-1 cells liberated a monocyte chemotactic factor into the culture supernatant 24 to 30 h after the heat-treatment. This factor was immunologically identified as the cross-linked homodimer of S19 ribosomal protein (RP S19), since the majority of the chemotactic activity was absorbed by both anti--RP S19 rabbit antibodies and an anti--isopeptide bond monoclonal antibody immobilized on agarose beads. Intracellular transglutaminase activity increased during the apoptotic process, reaching the peak strength between 18 and 24 h after the heat-treatment. A recombinant RP S19 acquired the monocyte chemotactic capacity when incubated with the apoptotic cell extract obtained at the 18th hour. The chemotactic activity acquirement as well as the transglutaminase activity were blocked by treatment of the extract with anti--type II transglutaminase rabbit antibodies. When the recombinant RP S19 was treated with an authentic type II transglutaminase, the dimerization of RP S19 concomitant with the generation of the monocyte chemotactic activity was observed. Peptide-map analyses involving amino acid sequencing demonstrated that the inter-molecular isopeptide bond was heterogeneous: Gln12 or Gln137 and Lys29 or Lys122 were cross-linked. Site-directed mutagenic analysis indicated that the cross-linking of Gln137, but not other residues such as Gln12, Lys29, and Lys122, was essential for expression of the chemotactic activity.

Enamel proteins and extracellular matrix molecules are co-localized in the pseudocystic stromal space of adenomatoid odontogenic tumor.

In order to examine the functional differentiation of tumor cells of adenomatoid odontogenic tumor (AOT) as ameloblasts and to determine the participation of the extracellular matrix (ECM) in the formation of its characteristic histologic architecture, tissue samples from five cases of adenomatoid odontogenic tumor were examined by immunohistochemical staining for enamel proteins and ECM molecules. Amelogenin, enamelin, laminin, heparan sulfate proteoglycan, fibronectin, collagen type IV and type V were immunolocalized within the luminal space and along the inner rim of duct-like structures. Eosinophilic hyaline droplets within the whorled or rosette masses of tumor cells showed basically the same staining pattern as the luminal contents. High columnar tumor cells that formed duct-like structures were immunopositive for amelogenin, while the staining intensity decreased with flattening of the cells, which was a result of luminal growth. The findings suggest that the constituent cells of duct-like structures are differentiated once to ameloblasts but fail to mature further due instead to increased production of ECM molecules and due to their retention in the lumina. It is possible to regard these special structures in AOT as stromal pseudocysts.

[Clinical usefulness of half-dose gadolinium-enhanced MR angiography of portal venous system (MR portography)--combined use with conventional dynamic MR imaging].

The clinical usefulness of half-dose Gadolinium-enhanced MR portography combined with the conventional full-dose dynamic MR imaging was evaluated on normal volunteers and patients. Half-dose MR portography was performed immediately following conventional full-dose Gd-enhanced dynamic MR imaging. The visualization of the extrahepatic portal system was either good or excellent in most cases excluding the splenic vein which was poor in 20.7%. That of the right and left main portal branches was also excellent. As for segmental branches, the visualization was poor of the left lobe. The comparison with digital subtraction angiography showed that the visualization of the right and left main portal branches was mostly equal. That of the splenic vein alone was worse on MR portography. We conclude that half-dose Gadolinium-enhanced MR portography is useful for the overall assessment of the portal venous system and can be added to the conventional dynamic MR imaging.

Acquired immune response as a consequence of the macrophage-dependent apoptotic cell clearance and role of the monocyte chemotactic S19 ribosomal protein dimer in this connection.

A connection between the apoptotic cell clearance system and the acquired immune system was studied in vivo. When fluorescence-labeled apoptotic HL-60 cells were inoculated into footpads of guinea pigs and rabbits, monocyte/ macrophage infiltration rapidly occurred and subsequently the apoptotic cells as well as the macrophages disappeared from the lesion by 48 hours without any macroscopical signs of inflammation. Inversely, the fluorescent cell debris, which had been engulfed by the macrophages, appeared and chronologically increased in the draining lymphatics and the popliteal lymph nodes by 48 hours. Subsequently, proliferation of T and B lymphocytes in the popliteal lymph nodes was observed. Secondary inoculation of HL-60 cells in the flank skin of guinea pigs on day 3 after the initial inoculation induced an acute immunologic dermatitis with erythema, edema, vascular permeability enhancement, and polymorphonuclear leukocyte infiltration. In vitro characterizations demonstrated the presence of compliment dependent cytotoxic IgM antibody against HL-60 cells in their sera. The infiltration of monocytes/macrophages at the apoptotic cell injection site and the subsequent production of the anti-HL-60 cell IgM antibodies were significantly suppressed by in situ injections of anti-S19 ribosomal protein rabbit antibodies. These results indicated that the serial events with the rapid apoptotic cell clearance by macrophages, the macrophage migration to lymph nodes, and the antigen presentation to T lymphocytes by the macrophages acquire immunity against apoptotic cells. It was also indicated that the S19 ribosomal protein dimer was the major chemotactic factor in the initial monocyte/macrophage infiltration to apoptotic cells.

Clinical evaluation of irreversible data compression for computed radiography of the chest.

Efficient data compression is essential for practical daily operation of computed radiography (CR) systems. In this study the clinical applicability of type III irreversible high data compression using an FCR 9501 chest unit (Fuji Photo Film, Tokyo, Japan) was evaluated. Sixty-eight normal and 93 various abnormal cases, with an additional 15 cases of lung cancers with solitary lung nodules, were selected from the file. A pair of hard copies of original images and images reconstructed using type III compression was made for each case. Six radiologists evaluated the image quality by visual rating and receiver operating characteristic (ROC) curve analysis. For all five anatomic regions of normal cases, "original equal to compressed" was the most common response, followed by "original significantly better than compressed." When abnormal cases were evaluated for diagnostic information, there was no significant difference between the compressed and original images. ROC curve analysis on lung nodules with lung cancer showed no significant difference between the two. Compressed CR images using the type III irreversible technique are clinically applicable and acceptable despite slight degradation of image quality.

[Computer-aided diagnosis in the detection of simulated clustered microcalcifications on mammography].

The usefulness of a computer-aided diagnosis (CAD) using a mathematical morphology (Tophat method) for the detection of simulated clustered microcalcifications on mammography (MMG) was evaluated with an acrylic phantom as well as a specially made phantom breast. The sites of microcalcifications indicated by CAD were printed as dots on the films. With the acrylic phantom, CAD detected microcalcifications as small as 0.177 mm, but with the specially made phantom breast the sensitivity decreased from 100% for microcalcifications of 0.250 mm to 25% for those of 0.210 mm. The detectability of microcalcifications was influenced by the contrast with background opacity. A receiver-operating-characteristic (ROC) study performed on 80 images of the phantom breast by 8 radiologists showed no significant difference between the results with and without CAD assistance. However, when the observers were divided into two groups according to the results of interpretation without CAD assistance, the detectability of microcalcifications with CAD significantly improved in the group with the poorer performance. The detection of microcalcifications was influenced by contrast with the surrounding substance in the phantom study. The usefulness of this CAD method presented for the detection of microcalcifications on MMG seems to be limited for inexperienced observers, but it may have potential to be widely applicable with further refinement of the technique.

A monocyte chemotactic factor, S19 ribosomal protein dimer, in phagocytic clearance of apoptotic cells.

HL-60 cells derived from a human promyelocytic leukemia underwent apoptosis by heat treatment. When the heat-treated HL-60 cells were injected into guinea pig skin, monocyte/macrophage infiltration was observed 24 or 36 hours later, and the apoptotic cells were phagocytically cleared by 48 hours after their injection. The infiltration and clearance patterns were quite different from those observed in injection of necrotic or boil-fixed HL-60 cells. The apoptotic cells released a monocyte chemotactic factor in vitro 24 hours after the heat treatment. The chemotactic factor generated was identified as the cross-linked homodimer of S19 ribosomal protein by its immunologic and physicochemical properties. A serine protease that inactivates the monocyte chemotactic factor was also released from the apoptotic cells 30 hours after the heat treatment. A super infusion of this protease into the skin where the apoptotic cells had been injected diminished the number of infiltrated monocytes. The present results indicate an important role of the S19 ribosomal protein dimer in the phagocytic clearance of apoptotic cells.

Cerebral venous angiomas associated with hemorrhagic lesions. Their MRI manifestations.

Using two 1.5-T magnetic resonance (MR) scanners, 82 venous angiomas (VAs) were imaged. There were 13 (16%) VAs associated with hemorrhagic lesions. Ten of the 13 hemorrhagic lesions were considered hematomas in the subacute or chronic stage, caused by VAs or coexisting cavernous hemangiomas. Two of the 13 were subacute intracerebral hematomas; the remaining one was a sequela of a hemorrhagic venous infarct. After analysis of our data, it was concluded that infratentorial VAs and deeply draining supratentorial VAs in relatively young adults, especially females, are relatively frequently associated with intracerebral hemorrhagic lesions. MR imaging proved useful for diagnosing VAs and associated hemorrhagic lesions.

Role of milk whey in the transmission of human cytomegalovirus infection by breast milk.

Breast-fed infants are susceptible to human cytomegalovirus (HCMV) infection via breast milk. In our previous study, HCMV was isolated more frequently from breast milk at later than one month after delivery than from colostrum or early breast milk. To clarify the role of milk cells and whey in vertical infection by breast feeding, we separated breast milk into milk cells and whey and examined each fraction for the presence of HCMV. We collected breast milk from mothers who breast-fed their infants (aged from 3 days to 2 months). The breast milk was centrifuged and separated into the middle layer (layer of milk whey) and the pellet (containing milk cells). We attempted to isolate HCMV from whey and to detect HCMV immediate early (IE) DNA in both milk whey and cells. HCMV was isolated from 7 out of 35 (20.0%) whey samples and HCMV IE DNA was detected from 15 out of 35 (42.9%) whey and/or milk cells. Detection rates of HCMV IE DNA in the whey layer and milk cells were 39.1% (25 out of 64) and 17.2% (11 out of 64), respectively. HCMV IE DNA was not detected in colostrum, but was detected in breast milk samples one month after delivery. Therefore, cell-free HCMV shed into milk whey may have a more important role in vertical infection by breast milk than cell-associated HCMV in the milk.

[Effects of anti-Bacteroides gingivalis (Bg), and anti-Actinobacillus actinomycetemcomitans (Aa) antibodies on Bg and Aa].

The purpose of this study was to determine the effects of anti-Bacteroides gingivalis (Bg) and anti-Actinobacillus actinomycetemcomitans (Aa) antibodies on Bg and Aa respectively. Bg and Aa were resistant to complement-mediated bactericidal activity, and killing by PMN. However a significant reduction in CFU of Bg and Aa was found when these bacteria were incubated with the antibodies and not agitated. This would suggest that the antibody was capable of aggregating or phagocytizing, but not killing, these bacteria when complement and PMN were added. Moreover the antibodies had no effect on bacterial proliferation. Anti Aa antibody also had no effects on killing of Aa or PMN infiltration in experimentally established periodontal pockets in dogs.

The polypeptide of a human calicivirus.

Viral particles morphologically resembling animals caliciviruses in the faeces of a patient with acute gastroenteritis were purified, radiolabeled with [125I], and analyzed by SDS-PAGE. A single major structural protein with a mol. mass 62,000 daltons was identified by immunoprecipitation technique. The finding is consistent with human calicivirus-like particles associated with gastroenteritis being a member of the family Caliciviridae.

Prevalence of antibody to human calicivirus in general population of northern Japan.

Serum specimens from children and adults living in Saporo, Japan, were tested for antibody against human calicivirus by immune electron microscopy (IEM), using virus-rich faecal extracts as the source of antigen. Of 83 serum specimens tested, 49 (59%) were positive for calicivirus antibody. Age-related prevalence of antibody to calicivirus was as follows: 23% (3/13) in the 0-5-month-old group, 30% (6/20) in the 6-23-month-old group, 65% (13/20) in the 2-5-year-old group, and 90% in school children (18/20) and adults (9/10). As for IEM antibody ratings scored from 0 to 4, almost all positive sera from older infants and preschool children scored 3 to 4. Antibody scores were rather more scattered in school children. The results indicated that caliciviral infection is prevalent in younger children in this part of Japan.

Fecal shedding of virus in relation to the days of illness in infantile gastroenteritis due to calicivirus.

Fecal shedding of virus in relation to the days of illness was studied by electron microscopic examinations of stool specimens collected during two consecutive outbreaks of gastroenteritis associated with calicivirus in an orphanage in the city of Sapporo, Japan. Of 61 stool specimens examined, 29 (48%) were found to contain typical calicivirus particles. Although caliciviruses were found in none of the seven stools obtained by chance before the onset of illness, they were found in 18 (95%) of 19 stool specimens collected within four days after the onset of illness. Seven (50%) of 14 specimens collected during the next five days were virus-positive, and the viruses were rarely detected in the stools collected thereafter. Thus correlation between viral shedding and the days of illness was clearly demonstrated. This finding should provide additional evidence for the etiologic role of calicivirus in acute infantile gastroenteritis.

Small round virus-like particles associated with acute gastroenteritis in Japanese children.

Nine patients with acute gastroenteritis shed small round virus-like particles in the faeces, and seven of them developed serum antibody against those particles as judged by immune electron microscopy. The small round virus-like particle was measured at 29-32 nm in diameter and showed a fine spiky structure on the surface. The buoyant density of the small round virus-like particles was determined to be 1.37-1.40 gm/cm3 in cesium chloride density gradient. The small round virus-like particle differed morphologically from astrovirus and calicivirus, and was antigenically dissimilar to Norwalk agent and W agent. These observations suggest that the small round virus-like particle is one of the new gastroenteritis viruses or may be a new serotype of them. Attempts to cultivate the small round virus-like particles were unsuccesful.

Recurrent attack of rotavirus gastroenteritis after adenovirus-induced diarrhoea.

A 14-month-old baby boy suffered a second attack of rotavirus gastroenteritis within one month of the initial one. The second attack followed a diarrhoeal episode associated with adenovirus. Gastrointestinal symptoms in the second attack were more severe than those of the first. The adenovirus-associated enteritis was mild compared with the illness in both episodes of rotavirus infection.