Aggregatibacter actinomycetemcomitans induces detachment and death of human gingival epithelial cells and fibroblasts via elastase release following leukotoxin‐dependent neutrophil lysis

Aggregatibacter actinomycetemcomitans is considered to be associated with periodontitis. Leukotoxin (LtxA), which destroys leukocytes in humans, is one of this bacterium's major virulence factors. Amounts of neutrophil elastase (NE), which is normally localized in the cytoplasm of neutrophils, are reportedly increased in the saliva of patients with periodontitis. However, the mechanism by which NE is released from human neutrophils and the role of NE in periodontitis is unclear. In the present study, it was hypothesized that LtxA induces NE release from human neutrophils, which subsequently causes the breakdown of periodontal tissues. LtxA‐treatment did not induce significant cytotoxicity against human gingival epithelial cells (HGECs) or human gingival fibroblasts (HGFs). However, it did induce significant cytotoxicity against human neutrophils, leading to NE release. Furthermore, NE and the supernatant from LtxA‐treated human neutrophils induced detachment and death of HGECs and HGFs, these effects being inhibited by administration of an NE inhibitor, sivelestat. The present results suggest that LtxA mediates human neutrophil lysis and induces the subsequent release of NE, which eventually results in detachment and death of HGECs and HGFs. Thus, LtxA‐induced release of NE could cause breakdown of periodontal tissue and thereby exacerbate periodontitis.     

Postglacial environmental change of the Pacific Ocean off the coasts of central Japan

Downcore changes in microfossil assemblages and oxygen isotope ratios in three piston cores recovered from the Northwestern Pacific, off central Japan, show that the subtropical Kuroshio front was located to the south of C-4 core site (Lat. 33° N) during the last glacial. The front then advanced northward, passing over the C-4 site and the C-6 site (34.6° N) at about 13 ka and 10 ka, respectively, and reached the C-1 core site (36° N) at about 7 ka. After 5.5 ka it retreated to the area between the C-1 and C-6 sites. A brief but significant cold event, the readvance of the cold Oyashio Current, is recognized between 11 and 10 ka in the two northern cores, but the current did not reach the southern C-4 site. A contemporaneous cold event is known in the North Atlantic, and the cooling was probably a global phenomenon likely to be associated with lowering of sea level. Contamination of isotopically light water is apparent between 14 and 11 ka in the marked change in isotopic composition of benthic foraminifers. Oxygen isotope ratios of planktonic foraminifers show that prior to the advance of the Kuroshio front, the surface water at these core sites was isotopically lighter than the Kuroshio water at that time.     

Functional and phenotypic analyses of interleukin 2-activated tumor-infiltrating lymphocytes

The tumor-infiltrating lymphocytes (TILs) were cultured with interleukin 2 (IL-2) to induce the activated killer cells possessing autologous tumor-killing activity, and analysed their cell surface phenotypes and assessed anti-tumor killing activity. Furthermore, the activated TILs were transferred into 7 patients adoptively resulting in complete remission in a patient with pancreatic cancer and partial remission in another patient with gastric cancer.The cytotoxic activities of activated TILs at 3 weeks-incubation was 72 ± 15, 42 ± 26, 27 ± 21 and 25 ± 15% against K562, Daudi, KATO-III and autologous tumor, respectively. The negative selection method, indicated that the killer cells recognizing autologous tumor cells consisted of CD4- or CD8-positive T lymphocytes and CD16- or CD56-positive natural killer cells. The activated TILs could not only lyse cultured tumor cell lines, but also autologous tumor cells.     

Fetus-in-fetu: report of a case

A 5 month-old female was brought to our clinic because of diarrhea and abdominal distension. A plain radiograph demonstrated a mass with a vertebral column in the right upper quadrant of the abdomen. At operation a mass was found to be retroperitoneal, well encapsulated, and connected to the abdominal aorta of the host by two small vessels; no other connections and adhesions were seen between the mass and the host. The ovaries, uterus, and other pelvic and abdominal viscera of the host were normal. The mass was diagnosed as a fetus-in-fetu. The fetus-in-fetu, encapsulated with an amniotic capsule, was covered with skin and had a top with long hair, two protuberances, an amniotic hernial sac, upper limbs with syndactylic fingers, a gluteal region, and lower limbs with polysyndactylic toes. A brain mass and a spinal cord were identified in the cranial cavity and the vertebral canal. Several spinal ganglia and a nerve plexus were found. A noselike structure, upper lip, maxillalike bone with teeth, tonguelike structure, intestines, ribs, bones of the extremities, and skeletal muscles were also identified. A cloacalike cyst was observed to have an opening in the external female genitalia. Microscopically, a small number of motor neurons were seen in the brain mass and the anterior horn of the spinal cord. In the spinal ganglia, ganglion cells were differentiated. The submucosal and myenteric plexuses were seen in the intestinal wall. Well-differentiated muscle fibers were often accompanied with myelinated nerve fibers. Hematopoiesis was observed in the cranial bone marrow. The presence of the sex chromatin was confirmed in the nuclei of motor neurons and polymorphonuclear leukocytes. Thus, the present fetus-in-fetu, which was connected to the abdominal aorta of the host by two vessels, was a monozygotic twin which developed within its own amniotic cavity.     

Alterations in migrating cranial neural crest cells in embryos of mice fed retinoic acid

Alterations in migrating neural crest cells induced by all-trans retinoic acid (RA) were studied morphologically and immunohistochemically in the cranial portion of 8-day-old mouse embryos which were derived from dams given 60, 40 or 0 mg kg of RA and killed 2 to 8 h later. Additionally, the embryos exposed to 4 mg/kg of actinomycin D (AD) on day 8 of gestation for 5 h were examined similarly. Light microscopy revealed that RA was cytotoxic and caused the appearance of pleomorphic nuclei, extra-large nucleoli and cytoplasmic budding which replaced lamellipodia and spike-like projections. Electron microscopy revealed pleomorphic nuclei containing nucleoli with major granular portions frequently surrounded with heterochromatin, monosomes, and phagosomes. A monosomal distribution pattern was different from that seen in the neural crest cells exposed to AD. The latter showed incomplete polyribosomal dispersion with fewer nucleolar components. Fewer neural crest cells with choline acetyltransferase-like immunoreactivity were detected in RA- and AD-exposed embryos than in the controls. These findings suggest that excess RA inhibits acetylcholine synthesis of the migrating neural crest cells, in a manner different from AD, and that it enhances phagocytosis. These phenomena modify the characteristics of neural crest cells resulting in craniofacial malformations.     

Relationship between cytosolic Ca2+ concentration and amylase release in rat parotid acinar cells following muscarinic stimulation.

Carbachol (CCh), a muscarinic-cholinergic agonist, increased both cytosolic free calcium concentration ([Ca2+]i) and amylase release in rat parotid acinar cells or acini in a dose-dependent manner. Treatment of acinar cells with the intracellular Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), or the intracellular Ca2+ chelator, 1,2-bis(O-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (BAPTA), strongly attenuated the increases in [Ca2+]i evoked by CCh, but amylase release from acini was not significantly suppressed by the treatment with TMB-8 or BAPTA. Low concentrations (0.02-0.5 microM) of ionomycin, a Ca2+ ionophore, caused increases in [Ca2+]i comparable to those induced by CCh, but the same concentrations had only a little effect on amylase release. The protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated amylase release in quantities similar to those induced by CCh, although TPA alone did not cause any change in [Ca2+]i. Combined addition of TPA and ionomycin potentiated only modestly amylase release stimulated by TPA alone. Staurosporine, a protein kinase C-inhibitor, similarly inhibited both the CCh- and TPA-induced amylase release. These results suggest that an increase in [Ca2+]i elicited by CCh does not play an essential role for inducing amylase release in rat parotid acini. Amylase release by muscarinic stimulation may be mediated mainly by activation of protein kinase C.     

A new regulatory element that augments the Tax-dependent enhancer of human T-cell leukemia virus type 1 and cloning of cDNAs encoding its binding proteins.

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) trans activates the 21-bp enhancer of HTLV-1. A sequence of more than two copies of the 21-bp enhancer is efficiently activated by Tax, but one copy is not activated extensively. Another sequence (TRE-2, positions -163 to -117) adjacent to the 21-bp enhancer in the long terminal repeat of HTLV-1 can enhance a single copy of the 21-bp enhancer activity in trans activation by Tax. This sequence contains motifs related to the Ets- and NF-kappa B-binding sequences, but mutations at these sites indicated that neither is responsive to cooperation with the 21-bp enhancer. A deletion mutation of TRE-2 identified 25 bases at positions -158 to -134 (TRE-2S) as an essential sequence, and TRE-2S was sufficient to give maximum cooperation with one copy of the 21-bp enhancer in trans activation by Tax protein. Using TRE-2S as a probe, we screened a cDNA library of HUT102 cells by the Southwestern (DNA-protein) procedure and isolated two cDNA clones, THP-1 and -2. These two clones encode TRE-2S-binding proteins, and they differ by only an extra 17 amino acids in THP-2. Both THP proteins contain five zinc finger motifs which are strikingly similar to those of the GLI family, an amplified gene product in glyoma cells. The binding site of THP-1 and -2 was GAACCACCCA in TRE-2S, which is highly homologous to the GLI-binding site. These results suggest that binding of THP to TRE-2S may be involved in cooperation with one copy of the 21-bp enhancer in responding to Tax trans activation.