Anakinra Protects Against Serum Deprivation-Induced Inflammation and Functional Derangement in Islets Isolated From Nonhuman Primates.

We investigated whether serum deprivation induces islet amyloid polypeptide (IAPP) oligomer accumulation and/or a proinflammatory response and, if so, whether the addition of interleukin (IL)-1 receptor antagonist to the culture medium can relieve the proinflammatory response during serum-deprived culture of nonhuman primate (NHP) islets. After culture in medium with and without Ana under serum-deprived culture conditions, IAPP oligomer/amyloid accumulation, in vitro viability, islet function, cytokine secretion, and posttransplantation outcome in streptozotocin-induced diabetic nude mice were determined in islets isolated from heterozygote human IAPP transgenic (hIAPP+/- ) mice and/or NHP islets. Serum deprivation induced accumulation of IAPP oligomer, but not amyloid, in NHP islets. Anakinra (Ana) protected islets from the serum deprivation-induced impairment of in vitro viability and glucose-stimulated insulin secretion and attenuated serum deprivation-induced caspase-1 activation, transcription, and secretion of IL-1ß, IL-6, and tumor necrosis factor-α in hIAPP+/- mice and NHP islets. Supplementation of medium with Ana during serum-deprived culture also improved posttransplantation in vivo outcomes of NHP islets. In conclusion, serum deprivation induced accumulation of IAPP oligomers and proinflammatory responses in cultured isolated islets. Supplementation of the culture medium with Ana attenuated the functional impairment and proinflammatory responses induced by serum deprivation in ex vivo culture of NHP islets.

Improved revascularization of islet grafts using an angiogenic monocyte subpopulation derived from spheroid culture of bone marrow mononuclear cells.

The spheroid culture method is an effective strategy for ex vivo expansion of an autologous therapeutic cell population. We investigated if cotransplantation of bone marrow-derived spheroids (BM-spheroid) formed using 3D culture of BM-derived mononuclear cells (BM-MNCs) could improve the posttransplant outcome of islet grafts using a mouse syngeneic marginal mass renal subcapsular islet transplantation model. Using green fluorescent protein transgenic (GFP-Tg) mice, the role of the BM-spheroids and the contribution of vessels derived from donors and recipients in grafted areas were assessed by immunohistochemistry. Compared to fresh BM-MNCs and nonspheroid remnant cells (BM-nonspheroid), the BM-spheroids, mainly composed of CXCR4(+) CD14(+) myeloid cells, showed higher angiogenic capacity, such as in vitro self-formed vessel structures; increased expression of angiogenic and chemoattractive factors; and incorporation into new vessel formation in basement membrane matrix plugs. BM-spheroid cotransplantation with islets improved the posttransplant outcomes in terms of glucose tolerance, serum insulin level, and diabetes reversal rate when compared with cotransplantation of BM-nonspheroids. Immunohistochemistry revealed that cotransplantation of the BM-spheroids increased vessel density, area of grafted endocrine and non-endocrine tissue, and ß cell proliferation. In conclusion, cotransplantation of islets and BM-spheroids improved islet function through facilitation of revascularization and an increase in cell proliferation and islet cell mass.

Life-threatening methemoglobinemia after unintentional ingestion of antifreeze admixtures containing sodium nitrite in the construction sites.

CONTEXT: Construction workers are exposed to a wide variety of health hazards such as poisoning at the construction sites. Various forms of poisoning incidents in construction workers have been reported. However, studies on methemoglobinemia caused by unintentional ingestion of antifreeze admixtures containing sodium nitrite at the construction sites have not been reported yet. OBJECTIVE: The aim of this study was to evaluate life-threatening methemoglobinemia after unintentional ingestion of antifreeze admixtures containing sodium nitrite at the construction sites and describe similar incidents involving ingestion of antifreeze admixtures in Korea. MATERIALS AND METHODS: Retrospective observational case series study on patients with methemoglobinemia after unintentional ingestion of antifreeze admixtures containing sodium nitrite admitted to the emergency department (ED) from January 1, 2010 to December 31, 2012 and cases reported to the Korea Occupational Safety and Health Agency (KOSHA) was performed. Results. Six victims were admitted to our ED. They had methemoglobin levels ranging from 32.4% to 71.5% and all of them recovered after receiving one (2 mg/kg) or two doses infusion of methylene blue. From the data of the KOSHA, six incidents that caused 27 victims were identified. Of 27 victims, five were included in the ED cases. For all incidents, antifreeze admixtures were not contained in their original containers and all new containers did not have a new label. All workers mistook antifreeze admixtures for water. Among the 28 victims included in this study, four died. CONCLUSION: Unintentional ingestion of antifreeze admixtures containing sodium nitrite at the construction sites can cause life-threatening methemoglobinemia. There is a need to store and label potentially hazardous materials properly to avoid unintentional ingestion at the construction sites.

A case of severe methemoglobinemia caused by hair dye poisoning.

CONTEXT: Hair dyes are widely used and very popular xenobiotics. Most of these products contain paraphenylenediamine (PPD) that can cause methemoglobinemia. We here report a case of severe methemoglobinemia that we treated using large amounts of methylene blue. CASE DETAILS: A 30-year-old man visited a regional hospital with cyanosis. He was congenitally blind and had autism. For several weeks, he had mistaken hair dye for toothpaste. When he arrived at a regional hospital, he was drowsy with cyanosis and his initial serum methemoglobin (MetHb) level was 59.5%. After being treated with 2 mg/kg methylene blue (1 mg/kg × 2 administrations), he was transferred to a tertiary university hospital. Upon presentation at the Emergency Department in the tertiary hospital, his MetHb level was found to be 49.4% and his oxygen saturation was 80%. He was then admitted to the intensive care unit. After treatment with 4 mg/kg methylene blue (1 mg/kg × 4 administrations), he successfully recovered. DISCUSSION: Because PPD can result in serious methemoglobinemia, clinicians should test it in cyanotic patients who have been exposed to hair dye for an extended period.

Co-transplantation of bone marrow-derived endothelial progenitor cells improves revascularization and organization in islet grafts.

Bone marrow-derived early endothelial progenitor cells (BM-EPCs) are a clinical tool for enhancing revascularization. However, the therapeutic efficacy of co-transplantation of BM-EPC with islets has not been investigated. In this study, marginal mass islets were co-transplanted with or without BM-EPCs under the kidney capsules of syngeneic streptozotocin-induced diabetic mice. Using green fluorescent protein transgenic (GFP-Tg) mice as BM-EPC and islet donors or recipients, the role of EPCs in revascularization was assessed for graft morphology, vascular density and fate of EPCs by immunohistochemistry. Islet-EPC co-transplantation improved the outcome of islet transplantation as measured by glucose tolerance, serum insulin level and diabetes reversal rate, compared with transplantation of islets alone. Between groups, the morphology of islet grafts showed significant differences in size and composition of grafted endocrine tissues. Significantly more vessel density derived from donors and recipients was detected with islet-EPC co-transplantation. Abundant GFP-Tg mice-derived BM-EPCs (GFP-EPCs) were observed in or around islet grafts and incorporated into CD31-positive capillaries. Remaining GFP-EPCs expressed VEGF. In conclusion, co-transplantation of islets with BM-EPCs could improve the outcome of marginal mass islet transplantation by promoting revascularization and preserving islet morphology.

Intracardiac thrombus formation induced by carbon monoxide poisoning.

INTRODUCTION: Carbon monoxide (CO) is one of the leading causes of poisoning; it inhibits oxygen delivery, subsequently causing ischemic changes and ultimately death by multiorgan failure. Furthermore, thromboembolic episodes due to CO poisoning have been reported. However, intracardiac thrombus formation following exposure to CO has been very rarely described. Here, a case of right atrial large thrombus formation after CO poisoning is presented. CASE PRESENTATION: A previously healthy 24-year-old woman was referred for CO poisoning. She has attempted suicide, and her initial mental status was drowsy with focal memory loss. Her initial CO fraction was 16%, and initial laboratory data showed creatinine kinase-myocardial bound of 90.6 ng/mL (upper limit 5 ng/mL) and troponin I of 1.899 ng/mL (upper limit 1.5 ng/mL). A transthoracic echocardiography was performed 24 h after the accident, revealing a 30 15 mm nodular echogenic mass in the right atrium. Anticoagulation with low-molecular-weight heparin was started along with hyperbaric oxygen therapy. After 7 days of heparinization, the large thrombus in right atrium had resolved. CONCLUSION: This report describes an intracardiac thrombus formation induced by CO poisoning. Because intracardiac thrombus can result in pulmonary embolism and cerebral embolic infarction, its consideration following CO poisoning is important.

Counting small hypointense spots confounds the quantification of functional islet mass based on islet MRI.

Iron-containing fragmented islets or free iron released from dying cells could confound the interpretation of MRI of iron nanoparticle-labeled islets. Exclusion of small hypointense spots could be a useful strategy to avoid such artifact. We investigated whether this strategy could improve the estimation of functioning islet mass after islet transplantation. Using a rat syngeneic intraportal islet transplantation model, we quantitatively assessed the relationships between total area, number of hypointense spots on MRI that belong to each size quartile and glycemic control of the recipients. The total area of hypointense spots on MRI was greater in the recipients that achieved diabetes reversal (p = 0.002), whereas the total number of hypointense spots was not different (p = 0.757). Exclusion of small hypointense spots improved the association between the number of hypointense spots and the blood glucose level of the recipients (p < 0.001). Ex-vivo imaging and histologic study confirmed that some small hypointense spots represent the phagocytosed free iron. Exclusion of small hypointense spots improved the quantification of the functional islet mass based on islet MRI. This would be a useful principle in the development of an algorithm to estimate functioning islet mass based on islet MRI.

Simultaneous sternothoracic cardiopulmonary resuscitation: a new method of cardiopulmonary resuscitation.

No existing device for cardiopulmonary resuscitation (CPR) is designed to exploit both the "cardiac pump" and the "thoracic pump" effect simultaneously. The purpose of this study was to measure the haemodynamic effect of a new simultaneous sternothoracic cardiopulmonary resuscitation (SST-CPR) device that could compress the sternum and constrict the thoracic cavity simultaneously in a canine cardiac arrest model. After 4 min of ventricular fibrillation, 24 mongrel dogs were randomized to receive standard CPR (n=12) or SST-CPR (n=12). SST-CPR generated a new pattern of the aortic pressure curve presumed to be the result of both sternal compression and thoracic constriction. SST-CPR resulted in significantly higher mean arterial pressure than standard CPR (68.9+/-16.1 vs. 30.5+/-10.0 mmHg, P<0.01). SST-CPR generated higher coronary perfusion pressure than standard CPR (47.0+/-11.4 vs. 17.3+/-8.9 mmHg, P<0.01). End tidal CO(2) tension was also higher during SST-CPR than standard CPR (11.6+/-6.1 vs. 2.17+/-3.3 mmHg, P<0.01). In this preliminary animal model study, simultaneous sternothoracic cardiopulmonary resuscitation generated better haemodynamic effects than standard, closed chest cardiopulmonary resuscitation.

Inhibition of fungal appressorium formation by pepper (Capsicum annuum) esterase.

A pepper esterase gene (PepEST) that is highly expressed during an incompatible interaction between pepper (Capsicum annuum) and the anthracnose fungus Colletotrichum gloeosporioides has been previously cloned. Glutathione-S-transferase-tagged recombinant PepEST protein expressed in Escherichia coli showed substrate specificity for p-nitrophenyl esters. Inoculation of compatible unripe pepper fruits with C. gloeosporioides spores amended with the recombinant protein did not cause anthracnose symptoms on the fruit. The recombinant protein has no fungicidal activity, but it significantly inhibits appressorium formation of the anthracnose fungus in a dose-dependent manner. An esterase from porcine liver also inhibited appressorium formation, and the recombinant protein inhibited appressorium formation in the rice blast fungus, Magnaporthe grisea. Inhibition of appressorium formation in M. grisea by the recombinant protein was reversible by treatment with cyclic AMP (cAMP) or 1,16-hexadecanediol. The results suggest that the recombinant protein regulates appressorium formation by modulating the cAMP-dependent signaling pathway in this fungus. Taken together, the PepEST esterase activity can inhibit appressorium formation of C. gloeosporioides, which may result in protection of the unripe fruit against the fungus.

Coexpression of a defensin gene and a thionin-like via different signal transduction pathways in pepper and Colletotrichum gloeosporioides interactions.

The anthracnose fungus, Colletotrichum gloeosporioides, interacts incompatibly with the ripe fruit of pepper (Capsicum annuum). It interacts compatibly with the unripe-mature fruit. We isolated a defensin gene, jl-l, and a thionin-like gene, PepThi, expressed in the incompatible interaction by using an mRNA differential display method. Both genes were developmentally regulated during fruit ripening, organ-specifically regulated, and differentially induced during the compatible and incompatible interactions. Expression of the PepThi gene was rapidly induced in the incompatible-ripe fruit upon fungal infection. The fungus-inducible PepThi gene is highly inducible only in the unripe fruit by salicylic acid. In both ripe and unripe fruit, it was induced by wounding, but not by jasmonic acid. Expression of the jl-l gene is enhanced by jasmonic acid in the unripe fruit but suppressed in the ripe fruit. These results suggest that both small and cysteine-rich protein genes are induced via different signal transduction pathways during fruit ripening to protect the reproductive organs against biotic and abiotic stresses.

Isolation and characterization of a cDNA encoding the cysteine proteinase inhibitor, induced upon flower maturation in carnation using suppression subtractive hybridization.

The suppression subtractive hybridization (SSH) method was used to isolate differentially expressed genes during carnation flower maturation. Five cDNA clones, designated as carnation flower maturation-induced (CFMI), were verified as flower maturation-induced cDNAs. Sequence analysis of five CFMI (CFMI-5, CFMI-6, CFMI-7, CFMI-9, and CFMI-10) clones revealed that one of the clones, CFMI-5, showed high sequence similarity to the cysteine proteinase inhibitor gene, predicted to be involved in flower maturation. The full length cDNA clone CFMI-5 was 531 nucleotides (nts) long and consisted of an open reading frame of 294 nucleotides, encoding a 98 amino acid protein, 12 nucleotides of 5'-untranslated region and 3'-untranslated region (225 nts) with a poly(A)+ tail. The predicted CFMI-5 amino acid sequence had a conserved sequence Gln-Val-Val-Ala-Gly, which corresponds to the active site of proteinase inhibition. Northern blot analysis revealed tissue-specific expression of CFMI-5 transcripts, as the transcripts were expressed preferentially in petals and styles. A PCR-based cDNA subtraction method, termed suppression subtractive hybridization, was identified as a rapid method to screen differentially expressed genes in a short time.

Cymbidium hybrida dihydroflavonol 4-reductase does not efficiently reduce dihydrokaempferol to produce orange pelargonidin-type anthocyanins.

Some angiosperms are limited to a range of possible flower colors. This limitation can be due to the lack of an anthocyanin biosynthetic gene or to the substrate specificity of a key anthocyanin biosynthetic enzyme, dihydroflavonol 4-reductase (DFR). Cymbidium hybrida orchid flowers primarily produce cyanidin-type (pink to red) anthocyanins and lack the pelargonidin-type (orange to brick-red) anthocyanins. To investigate the underlying molecular mechanism of this flower color range, we cloned a Cymbidium DFR gene and transformed it into a DFR- petunia line. We found that the Cymbidium DFR did not efficiently reduce dihydrokaempferol (DHK), which is an essential step for pelargonidin production. Phylogenetic analysis of a number of DFR sequences indicate that the inability to catalyze DHK reduction has occurred at least twice during angiosperm evolution. Our results indicate that developing a pelargonidin-type orange flower color in Cymbidium may require the transformation of a DFR gene that can efficiently catalyze DHK reduction.

A cytochrome P450 gene is differentially expressed in compatible and incompatible interactions between pepper (Capsicum annuum) and the anthracnose fungus, Colletotrichum gloeosporioides.

The anthracnose fungus, Colletotrichum gloeosporioides, was previously shown to have an incompatible interaction with ripe-red fruit of pepper (Capsicum annuum). However, the fungus had a compatible interaction with unripe-mature-green fruit. Using mRNA differential display, we isolated and characterized a PepCYP gene expressed in the incompatible interaction. The PepCYP gene encodes a protein homologous to cytochrome P450 proteins containing a heme-binding domain. The expression level of PepCYP is higher in the incompatible interaction than in the compatible interaction, and then remains elevated in the incompatible interaction. In the compatible interaction, the expression of PepCYP is transient. The induction of PepCYP gene is up-regulated by wounding or jasmonic acid treatment during ripening. Analysis of PepCYP expression by in situ hybridization shows that the accumulation of PepCYP mRNA is localized in the epidermal cell layers, but not in the cortical cell layers. An examination of transverse sections of the fruits inoculated with the fungus shows that the fungus invades and colonizes the epidermal cell layers of the unripe fruit at 24 and 72 h after inoculation, respectively, but not those of the ripe fruit. These results suggest that the PepCYP gene product plays a role in the defense mechanism when the fungus invades and colonizes the epidermal cells of fruits in the incompatible interaction during the early fungal infection process.

Induction of early flowering in Cymbidium niveo-marginatum Mak in vitro.

Many orchids take several years to flower. We have been able to induce early flowering in the temperate orchid Cymbidium niveo-marginatum Mak in vitro. The combined treatment of cytokinin (6-benzylaminopurine), restricted nitrogen supply with phosphorus enrichment, and root excision (pruning) induced transition of the Cymbidium shoot from a vegetative to a reproductive stage. Nearly 100% of the plants flowered within 90 days only when the combined treatment was applied. When root excision and/or 6-benzylaminopurine were omitted from the combined treatments, flower induction was significantly reduced. The auxin transport inhibitor, 2,3,5-triiodobenzoic acid prevented flowering of Cymbidium in vitro, although auxin (α-naphthaleneacetic acid) itself did not induce flowering. Gibberellic acid markedly delayed flowering in C. niveo-marginatum even when the flower-promoting treatment was applied. Paclobutrazol, an anti-gibberellin agent, totally blocked the inductive effects of either cytokinin or pruning. These observations suggest that concerted actions of auxin, cytokinin, and gibberellin, as well as nutrient concentration and putative promoting/suppressing agents, determine the timing of Cymbidium orchid transition from the vegetative to reproductive stage.

Nonalcoholic steatohepatitis: Mayo Clinic experiences with a hitherto unnamed disease.

Nonalcoholic steatohepatitis is a poorly understood and hitherto unnamed liver disease that histologically mimics alcoholic hepatitis and that also may progress to cirrhosis. Described here are findings in 20 patients with nonalcoholic steatohepatitis of unknown cause. The biopsy specimens were characterized by the presence of striking fatty changes with evidence of lobular hepatitis, focal necroses with mixed inflammatory infiltrates, and, in most instances, Mallory bodies; Evidence of fibrosis was found in most specimens, and cirrhosis was diagnosed in biopsy tissue from three patients. The disease was more common in women. Most patients were moderately obese, and many had obesity-associated diseases, such as diabetes mellitus and cholelithiasis. Presence of hepatomegaly and mild abnormalities of liver function were common clinical findings. Currently, we know of no effective therapy.