Two cases of BRAF-mutated, bulbar conjunctival melanoma, and review of the published literature.

We describe two patients with BRAF-mutated melanoma of the epithelioid cell type arising from primary acquired melanosis with severe atypia of the right bulbar conjunctiva. Patient 1 was a 71-year-old Japanese man. After adjuvant cryotherapy and enucleation of the right eyeball, therapy with vemurafenib was administered for a distant metastasis to a lumbar vertebra, accompanied by erythema multiforme and two keratinous tumours. The patient died due to metastases to the liver and multiple vertebrae, despite therapy with nivolumab and combination therapy with dabrafenib plus trametinib. Patient 2 was a 72-year-old Japanese man. After adjuvant cryotherapy, periodic mitomycin C eye drops, and excision of the superficial portion of the right parotid gland and the dissection of cervical lymph nodes, he was treated with adjuvant combination therapy with dabrafenib plus trametinib. Dermatologists should be familiar with BRAF-mutated conjunctival melanoma, which is usually located on the bulbar conjunctiva and associated with more frequent distant metastasis.

Granulocyte and monocyte apheresis can control juvenile generalized pustular psoriasis with mutation of IL36RN.

Patients with deficiency of interleukin-36 receptor antagonist (DITRA), due to mutation of IL36RN, exhibit psoriatic phenotypes, typically generalized pustular psoriasis (GPP). We report a paediatric patient with DITRA, whose cutaneous lesions varied from psoriasis vulgaris in infancy to annular pustular psoriasis with acute exacerbation to GPP at 13 years of age. Conventional systemic treatments for GPP, which include oral retinoids, ciclosporin and methotrexate, are controversial in paediatric cases, because of their adverse effects and uncertain long-term consequences. Granulocyte monocyte apheresis, a process associated with few adverse events, promptly controlled the GPP of our paediatric patient, and has potential as a suitable alternative treatment for paediatric patients with DITRA.

Induction of lymphokine-activated killer-like cells by cancer chemotherapy.

Natural cell-mediated cytotoxicity against NK-resistant target tumor cells was found in the peripheral blood of tumor-bearing patients approximately 1 mo after combined chemotherapy. The recognition specificity of these effector cells was broad and had no restriction. From the experiments of negative selection with mAbs and complements, these newly developed killer cells after chemotherapy were thought to be LAK-like cells. Contribution of these LAK-like cells to the mechanism of action of anticancer drugs remains to be clarified.

Inactivation of hepatitis A virus by heat and high hydrostatic pressure: variation among laboratory strains.

BACKGROUND AND OBJECTIVES: Hepatitis A virus (HAV) transmission via contaminated blood products has been reported. Cell-adapted HAV strains are generally used to confirm virus inactivation in manufacturing blood products, but the strains may differ in their sensitivity to inactivation treatment. To select an appropriate cell-adapted HAV strain for virus validation, we compared the inactivation efficiency among four strains under two different physical inactivation treatments: heat and high hydrostatic pressure. MATERIALS AND METHODS: The cell-adapted HAV strains used here were KRM238, KRM003 (subgenotype IIIB), KRM031 (IA), and TKM005 (IB). The strains were treated at 60 degrees C for up to 10 h or under high hydrostatic pressure (up to 420 MPa). The reduction in HAV infectivity was measured by an immunofocus-staining method. RESULTS: The heat treatment at 60 degrees C for 10 h reduced HAV infectivity in the range of 3 to 5 log(10) among the strains; KRM238 and TKM005 were harder to inactivate than the other two. The high hydrostatic pressure treatment at 420 MPa also reduced infectivity in the range of 3 to 5 log(10) among the strains, and KRM031 was easier to inactivate than the other strains. CONCLUSION: Heat treatment and high hydrostatic pressure treatment revealed differences in inactivation efficiencies among cell-adapted HAV strains, and each strain reacted differently depending on the treatment. KRM238 may be the best candidate for virus validation to ensure the safety of blood products against viral contamination, as it is harder to inactivate and it replicates better in cell culture than the other strains.

Adiponectin plays a protective role in caerulein-induced acute pancreatitis in mice fed a high-fat diet.

BACKGROUND: Obesity is a risk factor for acute pancreatitis (AP), but the molecular mechanism remains unclear. Adiponectin, an adipose tissue-derived secretory factor, has anti-inflammatory properties in addition to various biological functions, and its plasma concentrations are reduced in obese subjects. However, the role of adiponectin in AP has not been investigated. AIM: To determine the effects of adiponectin on AP. METHODS: We investigated the effects of adiponectin on experimental AP by using adiponectin-knockout (APN-KO) mice and adenovirus-mediated adiponectin over-expression. AP was induced by 10 hourly intraperitoneal injections of low-dose caerulein (10 microg/kg) after 2 week feeding of normal chow or a high-fat diet (HFD) in wild-type (WT) and APN-KO mice. We evaluated the severity of AP biochemically and morphologically. RESULTS: Low-dose caerulein treatment did not induce pancreatic damage in either WT or APN-KO mice under normal chow feeding. APN-KO mice, but not WT mice, fed a HFD and then treated with caerulein developed pancreatic damage and inflammation, accompanied by increased macrophage/neutrophil infiltration and upregulation of pro-inflammatory mediators such as tumour necrosis factor alpha in the pancreas. Adenovirus-mediated over-expression of adiponectin attenuated the severity of HFD/caerulein-induced AP in APN-KO mice. CONCLUSIONS: Adiponectin plays a protective role in caerulein-induced AP in HFD-fed mice.

Antitumor activities of newly synthesized 5-carbamoyl-1 H-imidazol-4yl 1-adamantanecarboxylate and 5-carbamoyl-1H-imidazol-4yl piperonylate.

In synthetic studies on the chemical modification of the nucleoside antibiotic bredinin, two new derivatives, 5-carbamoyl-1H-imidazol-4-yl 1-adamantanecarboxylate and 5-carbamoyl-1H-imidazol-4-yl piperonylate, were found to possess a potent antitumor activity in several experimental tumor systems, even though bredinin itself shows only in vitro cytotoxicity and thus lacks therapeutic effectiveness. These two derivatives of bredinin exhibited antitumor activity against a wide variety of tumors, including leukemias L1210 and P388, Lewis lung carcinoma, B16 melanoma, Colon 26 and 38 adenocarcinomas. Ehrlich carcinoma, and Sarcoma 180. It is noteworthy that these agents showed good therapeutic effects not only against ascitic types of tumors but also against a number of slow-growing solid tumor lines, particularly the ascitic and solid forms of Ehrlich carcinoma. At their optimal doses, both compounds effected a complete cure of all or most of the mice treated. Although the mechanisms of action of these compounds remain unknown, they are able to suppress in vivo tumor growth, presumably by being slowly anabolized in vivo to an active form and inhibiting purine de novo synthesis as bredinin does.

Mutations of the bak gene in human gastric and colorectal cancers.

The Bcl-2 homologue Bak is a potent inducer of apoptosis. We performed PCR-based single-strand conformational polymorphism and sequencing analysis of the entire coding region of the bak gene (exons 2-6) in 24 primary gastric cancers (6 early-stage and 18 advanced-stage cancers) and 20 primary colorectal cancers (6 early-stage and 14 advanced-stage cancers). The data herein demonstrate, for the first time, the mutation of the bak gene in gastric and colorectal cancers. Missense bak gene mutations were observed in 3 of 24 (12.5%) gastric cancers and 2 of 20 (10.0%) colorectal cancers. Sequence alterations without amino acid alteration were observed 1 of 24 (4.2%) gastric cancers and 2 of 20 (10.0%) colorectal cancers. Mutations in the bak gene were observed only in advanced-stage gastrointestinal cancers but not in early-stage cancers. Our observations suggest that mutations in this gene predispose bearers to the development of gastrointestinal malignancies in at least a subset of the cases.

Modulation of apoptosis by endogenous Bcl-xL expression in MKN-45 human gastric cancer cells.

This study was designed to clarify the role of endogenous Bcl-xL expression in modulating apoptosis of malignant cells. Administration of bcl-x-antisense oligonucleotides decreased Bcl-xL protein levels in the MKN-45 human gastric cancer cell line. The decrease in Bcl-xL protein content resulted in increased cell death induced by serum deprivation or Fas-antibody administration. Flow cytometric analysis revealed that the increased apoptotic cell death was more prominent in bcl-x-antisense-treated cells as compared to control cells, bcl-x-sense-treated cells, or bcl-x-nonsense-treated cells. To inhibit the effect of intrinsic Bcl-xL protein, we overexpressed Bak, which binds Bcl-xL and inhibits the anti-apoptotic effect of Bcl-xL, by transfection into MKN-45 cells. Bak-overexpressing cells showed increased apoptotic cell death induced by Fas-antibody when compared to parent cells and MKN-neo-transfected cells. Bak-overexpressing cells also showed greater sensitization to 5-fluorouracil and cisplatin than parent cells and MKN-neo-transfected cells. In conclusion, we demonstrated that administration of bcl-x-antisense oligonucleotides or overexpression of Bak protein induces sensitization to apoptosis in MKN-45 gastric cancer cells, suggesting that endogenous Bcl-xL expression in cancer cells is an important modulator of apoptosis.

Plasticity of neurohypophysial terminals with increased hormonal release during dehydration: ultrastructural and biochemical analyses.

Arginine vasopressin- (AVP) and oxytocin- (OXT) secreting magnocellular neurons undergo gross structural changes with chronic physiological stimulation. Here, we investigated subcellular aspects of plasticity in rat neurohypophysial terminals during dehydration. Ultrastructural analyses demonstrated that chronic dehydration by 2% NaCl drinking for 7 days significantly decreased the numbers of neurosecretory granules and microvesicles but not the numbers of mitochondria. Moreover, in dehydrated rats, terminals making neurovascular contacts enlarged, whereas terminals in apposition to astrocytes, i.e., neuroglial contacts, became smaller. Western blot analyses demonstrated significant decreases in the levels of F3 and Thy-1 together with those of AVP- and OXT-neurophysin, but the levels of synaptophysin, SNAP-25, and GAP-43 were unchanged. Both F3 and Thy-1 were recovered in the buffer-insoluble pellet, and phosphatidyl inositol-specific phospholipase C treatment released both molecules from the crude membrane fraction, indicating that they are attached to terminal membranes by glycosylphosphatidyl inositol anchors. Confocal microscopic observations demonstrated that F3 colocalized with Thy-1 in the same terminals of magnocellular neurons. In contrast, the level of calretinin, a Ca(2+) binding protein was significantly increased with chronic dehydration. Thus, the present results suggest that enhancement of neurovascular contacts results from rearrangement of terminal-astrocyte and terminal-vessel contacts rather than enlargement or sprouting of magnocellular terminals themselves. The down-regulation of F3 and Thy-1 may contribute to enhancement of neurovascular contacts that accompany increased peptide release during dehydration.

Expression of the IgLON cell adhesion molecules Kilon and OBCAM in hypothalamic magnocellular neurons.

The vasopressin (AVP) and oxytocin (OXT) magnocellular neurons in the hypothalamic supraoptic (SON) and paraventricular nuclei (PVN) display reversible structural plasticity of neurons and glial cells under different conditions of neuropeptide secretion. In the present study, we investigated the expression of two immunoglobulin superfamily (IgSF) proteins, Kilon and OBCAM, in the magnocellular neurons by using monoclonal antibodies. Anti-Kilon antibody reacted specifically with the bacterially expressed recombinant Kilon but not with the recombinant OBCAM, and similarly anti-OBCAM antibody specifically recognized the recombinant OBCAM. Western blotting analysis revealed the specific expression of Kilon and OBCAM in the SON homogenates. Although Kilon and OBCAM of the SON homogenates were present as the insoluble form, most Kilon was present in the Triton-insoluble fraction, and OBCAM was localized mainly in the Triton-soluble fraction. Immunocytochemistry revealed Kilon and OBCAM immunoreactivity in the magnocellular neurons of the SON and PVN of the rat hypothalamus compared with outside of the SON and PVN in the hypothalamus. The double-labeling study with confocal microscopy further demonstrated that Kilon immunoreactivity was observed mainly in the dendrites of AVP-secreting neurons and also occasionally OXT-secreting neurons. However, OBCAM immunoreactivity was exclusively seen in the dendrites of AVP-secreting magnocellular neurons. Chronic physiological stimulation by 2% NaCl had no effect on the expression levels of either IgLON protein in the SON. Our study thus demonstrated specific expression of Kilon and OBCAM in the hypothalamic magnocellular neurons, particularly in dendrites, suggesting that they confer on magnocellular neurons the ability to rearrange dendritic connectivity.

Responses of anterior hypothalamic-preoptic thermosensitive neurons to substance P and capsaicin.

The effects of capsaicin and substance P, applied locally, on the activity of thermosensitive and thermally-insensitive neurons in the anterior hypothalamic-preoptic area were studied with the use of multibarrelled microelectrodes in the urethane-anesthetized rat. In a total of 34 thermosensitive neurons (21 warm-units and 13 cold-units) in the anterior hypothalamic-preoptic area, only 7 units responded to substance P and 27 units (79.4%) were not affected. In contrast, 23 of 57 (40.4%) thermally-insensitive neurons responded to substance P. Of 74 thermosensitive and thermally-insensitive neurons tested with both substance P and capsaicin, only 7 units (9.5%) showed the same direction of change in activity in response to the application of both drugs. The majority of the neurons responded to only one of the drugs (32 units, 43.2%) or responded to substance P and capsaicin in opposite directions (9 units, 12.2%). A substance P antagonist, (D-Pro2, D-Trp7,9)-substance P, blocked the substance P-induced excitatory responses in 5 of 6 neurons tested in the anterior hypothalamic-preoptic area, but not the capsaicin-induced excitatory or inhibitory responses (n = 5). It is unlikely that substance P in the anterior hypothalamic-preoptic area participates in the hypothermic effects of capsaicin, observed after local injection.

Responses of anterior hypothalamic-preoptic thermosensitive neurons to thyrotropin releasing hormone and cyclo(His-Pro).

Effects of local application of thyrotropin releasing hormone (TRH) and its metabolite, histidyl-proline diketopiperazine [cyclo (His-Pro)], on the activity of thermosensitive and thermally-insensitive neurons of the anterior hypothalamic-preoptic area were investigated in urethane-anesthetized rats. Microelectrophoretic application of TRH changed the activity of 126 of 206 neurons tested. Thyrotropin releasing hormone predominantly decreased the activity of warm-sensitive neurons and increased the activity of cold-sensitive neurons. Since it has been generally assumed that warm-sensitive and cold-sensitive neurons in the anterior hypothalamic-preoptic area mediate heat and cold defence responses, respectively, the present results are consistent with previous findings showing hyperthermia after injection of TRH into the hypothalamus in the rat. Cyclo (His-Pro) affected the activity of 59 of 153 neurons tested. In addition, cyclo (His-Pro) did not preferentially affect warm- or cold-sensitive neurons. These results indicate that the previously-determined hypothermic effect of cyclo (His-Pro) cannot be explained by its effects on thermosensitive neurons in the anterior hypothalamic-preoptic area.

GABA(B) receptor-mediated presynaptic inhibition of glutamatergic and GABAergic transmission in the basolateral amygdala.

The information processing at central synapses is mediated not only by homosynaptic transmission with direct synaptic connections but also by heterosynaptic interactions between distinct synaptic inputs. Using rat brain slices and whole-cell recordings this study aimed to examine the roles of GABA(B) receptors in synaptic interactions in the basolateral amygdala (BLA), a critical brain structure related to fear and anxiety. Stimulation in the BLA produced non-NMDA type glutamate receptor antagonist-sensitive excitatory postsynaptic currents (EPSCs) and bicuculline-sensitive inhibitory postsynaptic currents (IPSCs) in the BLA neurons. The GABA(B) receptor agonist baclofen markedly inhibited both EPSCs and IPSCs in a concentration-dependent manner, and the baclofen-induced inhibition was selectively abolished by the GABA(B) receptor antagonist CGP55845A. The paired-pulse ratio of EPSC and IPSC amplitude was increased by baclofen. The effect of baclofen was mimicked by lowering the external Ca2+ concentration but not by glutamate- and GABA(A)-receptor antagonists. The frequency but not the mean amplitude of miniature EPSCs and IPSCs was decreased by baclofen. The findings suggest that activation of GABA(B) receptors by baclofen reduces the strength of excitatory and inhibitory transmission in the BLA by a presynaptic mechanism. Repetitive conditioning stimulation applied to GABAergic synaptic inputs exerted an inhibitory action on glutamatergic excitatory transmission, and the stimulation-induced inhibition was abolished by CGP55845A. Furthermore, the paired-pulse ratio of EPSCs was increased during the stimulation-induced inhibition. The results in this study provide evidence that synaptic activation of GABA(B) heteroreceptors elicits presynaptic inhibition of glutamatergic excitatory transmission in the BLA.

Responses of anterior hypothalamic-preoptic thermosensitive neurons to locally applied capsaicin.

The effects of local application of capsaicin on the activity of single thermosensitive neurons in the anterior hypothalamic-preoptic area were studied in the urethane-anesthetized rat. Local injection of capsaicin through a cannula to the vicinity of the neurons increased the activity in 15 of 28 warm-units, decreased the activity in 2 of 4 cold-units and had no effect on 5 of 10 thermally-insensitive units. Electrophoretic application of capsaicin with the use of multibarrelled microelectrodes excited 16 of 27 warm-units, inhibited 12 of 17 cold-units and had no effect on 35 of 60 thermally-insensitive units. Progressive decreases in the responsiveness of the neurons to both capsaicin and the hypothalamic temperature were observed with repeated applications of capsaicin. Many neurons ceased firing after showing excitatory or inhibitory responses to single or repeated applications of capsaicin either by local injection or electrophoretic application. The results may explain the acute thermolytic response, as well as the subsequent decrease in responsiveness to the injection of capsaicin into the anterior hypothalamic-preoptic area, on the basis of changes in the activity of thermosensitive neurons in the anterior hypothalamic-preoptic area.

Expression of high levels of tubulin and microtubule-associated protein 2d in the neurohypophysial astrocytes of adult rat.

The hypothalamo-neurohypophysial system, containing arginine vasopressin and oxytocin, is well known to show reversible morphological reorganization for both neurons and glial cells during chronic physiological stimulation. To determine the molecular background for these morphological changes, we investigated the expression of tubulin and microtubule-associated protein (MAP) 2d in the neurohypophysial astrocytes, pituicytes of adult rats by using reverse transcription-polymerase chain reaction, western blot, and immunohistochemistry. The mRNA of MAP2d was expressed at higher levels than that of MAP2c in the neurohypophysis, cerebral cortex, and cerebellum. In contrast, predominant expression of mRNA of MAP2c was detected in the olfactory bulb. Western blot analysis showed the presence of MAP2d in the neurohypophysis, however the amount was below the detection level in the cerebral cortex and cerebellum. A double labeling study using a confocal laser scanning microscope showed intense tubulin immunoreactivity in the glial fibrillary acidic protein (GFAP)-positive pituicytes of the intact neurohypophysis. Almost no tubulin immunoreactivity was observed in the astrocytes of the intact cerebral cortex, cerebellum, and supraoptic nucleus, in contrast to strong tubulin immunoreactivity in neuronal dendrites and somata. Interestingly, intense tubulin immunoreactivity was also observed in the GFAP-positive reactive astrocytes in the immediate vicinity of the artificial lesion of the cerebral cortex. Electron microscopic observation further demonstrated the presence of a lot of microtubules in the pituicytes of intact rats.The present results demonstrate that pituicytes in the adult rat neurohypophysis expresses high levels of tubulin and MAP2d compared with normal brain astrocytes, and suggest that the ability of astrocytic morphological alteration may be at least partly ascribed to this high expression of microtubule proteins.

Microtubule-associated protein-2 in the hypothalamo-neurohypophysial system: low-molecular-weight microtubule-associated protein-2 in pituitary astrocytes.

Microtubule-associated protein-2 is the most abundant microtubule-associated protein in the brain and is responsible for morphogenesis and maintenance of the nervous system. In the present experiments, we have examined the localization of microtubule-associated protein-2 in the hypothalamo-neurohypophysial system of the rat using western blots and immunohistochemistry. Two monoclonal antibodies against microtubule-associated protein-2, antibody C and AP20, were used: antibody C recognizes both the high- and low-molecular-weight isoforms of microtubule-associated protein-2; antibody AP20 specifically detects high-molecular-weight microtubule-associated protein-2 only. Western blot analysis revealed expression of high-molecular-weight microtubule-associated protein-2 in the whole brain, hippocampus and whole hypothalamus. While the supraoptic nucleus expressed only high-molecular-weight microtubule-associated protein-2, the adult posterior pituitary predominantly expressed low-molecular-weight microtubule-associated protein-2, which was also seen in the embryonic whole brain. Light microscopic immunohistochemistry revealed that both antibody C and AP20 intensely stained dendrites of the dendritic and somatic zones in the supraoptic nucleus. Double labeling with antibodies against microtubule-associated protein-2 and oxytocin (or vasopressin) demonstrated that microtubule-associated protein-2 was localized in dendrites of magnocellular neurons in the supraoptic nucleus. In the posterior pituitary, however, antibody C stained fine processes and cell bodies of astrocytes, which were identified by an antibody against glial fibrillary acidic protein. Antibody AP20 also stained fine processes of some astrocytes in the posterior pituitary, but the intensity of immunoreactivity with antibody AP20 was weaker than that with antibody C. This result suggests that microtubule-associated protein-2 in astrocytes of the posterior pituitary is predominantly of the low-molecular-weight type. Moreover, western blots revealed low-molecular-weight microtubule-associated protein-2 of the posterior pituitary at a molecular weight slightly higher than embryonically expressed low-molecular-weight microtubule-associated protein-2, indicating that low-molecular-weight microtubule-associated protein-2 in the posterior pituitary is possibly the isoform microtubule-associated protein-2d. The present results demonstrate that astrocytes in the posterior pituitary of adult rats still retain the ability to express the immature variant of microtubule-associated protein-2, low-molecular-weight microtubule-associated protein-2, and its expression is probably linked to structural plasticity.