Rational design of alcoholic fermentation targeting extracellular carbon.

Breeding yeast strains for industrial alcoholic fermentation requires laborious screening due to the lack of in vivo modification strategies. Here we show that quiescence-specific cell wall thickening via synthesis of a major component, 1,3-ß-glucan, critically antagonizes cellular fermentation ability by sequestering the available cytoplasmic carbon sources. This study provides insights into glycolytic control and reports an effective and reliable rational fermentation design.

Effect of physical restraint on glucose tolerance in cynomolgus monkeys.

BACKGROUND: Physiologic stress has been demonstrated to impair glucose tolerance. METHODS: Glucose tolerance tests were performed using six cynomolgus monkeys. RESULTS: Chair-restrained subjects elicited higher elevations of plasma glucose and cortisol compared with squeezing device-restrained subjects. CONCLUSIONS: The responses to a glucose challenge are altered by different restraint procedures.

Establishment of a novel experimental model of human angiosarcoma and a VEGF-targeting therapeutic experiment.

BACKGROUND: Angiosarcoma is one of the most life-threatening neoplasms with strong resistance to conventional chemotherapy/radiotherapy; consequently, alternative therapeutic agents are urgently required. One factor in delaying the therapy development is the limitation of experimental models. OBJECTIVE: We established a novel experimental angiosarcoma model. METHODS: From surgically resected tissue, human AS cell line was established. Using xenograft of AS cell line, we performed therapeutic experiments with the anti-human VEGF Ab or the receptor tyrosine kinase inhibitor. RESULTS: First we generated an angiosarcoma cell line, HAMON (human angiosarcoma, monoclonal), which expresses CD31 and produces tumors in immunodeficient mice. HAMON expresses VEGFR2 and that exogenous VEGF leads to HAMON proliferation in vitro. Anti-human VEGF Ab bevacizumab treatment failed to suppress HAMON proliferation in vitro and in vivo. Furthermore, the receptor tyrosine kinase inhibitor sunitinib did not suppress HAMON proliferation in vitro. Similarly, in in vivo therapeutic experiments, even high doses of sunitinib failed to inhibit tumor growth. Finally, we checked whether compensatory activation of VEGF signaling occurred after sunitinib addition. VEGF protein secretion, VEGF mRNA synthesis and VEGFR2 phosphorylation all were unaffected in HAMON after sunitinib treatment. CONCLUSION: A novel in vitro and in vivo experimental model of human angiosarcoma has been successfully established. With this model, we were able to perform therapeutic experiments. In addition, our angiosarcoma cell line, HAMON, is quite useful for identifying key molecules in angiosarcoma.

Stevens-Johnson syndrome/toxic epidermal necrolysis mouse model generated by using PBMCs and the skin of patients.

BACKGROUND: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening cutaneous reactions caused by drugs or infections and exhibiting widespread epidermal necrosis. Currently, there is no animal model that reproduces SJS/TEN symptoms. OBJECTIVE: We sought to develop a novel mouse model of SJS/TEN by using PBMCs and skin from patients who had recovered from SJS/TEN. METHODS: For our mouse model, patients' PBMCs were injected intravenously into immunocompromised NOD/Shi-scid, IL-2Rγ(null) (NOG) mice, followed by oral administration of a causative drug. Subsequently, to replace human skin, unaffected skin specimens obtained from patients who had recovered from SJS/TEN were grafted onto NOG mice, after which patient-derived PBMCs and the causative drug were applied. RESULTS: Mice injected with PBMCs from patients with SJS/TEN and given the causative drug showed marked conjunctival congestion and numerous cell death of conjunctival epithelium, whereas there were no symptoms in mice injected with PBMCs from patients with ordinary drug skin reactions. CD8(+) T lymphocyte-depleted PBMCs from patients with SJS/TEN did not elicit these symptoms. In addition, skin-grafted mice showed darkening of the skin-grafted areas. Cleaved caspase-3 staining showed that dead keratinocytes were more numerous in the skin-grafted mice than in the healthy control animals. CONCLUSION: We have established a novel human-oriented SJS/TEN mouse model and proved the importance of CD8(+) T lymphocytes in SJS/TEN pathogenesis. The mouse model promises to promote diagnostic and therapeutic approaches.

Rapid immunochromatographic test for serum granulysin is useful for the prediction of Stevens-Johnson syndrome and toxic epidermal necrolysis.

BACKGROUND: Life-threatening adverse drug reactions such as Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) sometimes start with clinical features of ordinary drug-induced skin reactions (ODSRs) and it may be difficult to make a correct diagnosis before severe mucocutaneous erosions occur. We have reported that serum granulysin levels are elevated (cut off: 10 ng/mL) in patients with SJS/TEN before generalized blisters form. OBJECTIVE: We sought to develop a rapid detection system for elevated serum granulysin to predict the progression from ODSRs. METHODS: Serum samples from 5 patients with SJS/TEN at 2 to 4 days before mucocutaneous erosions formed were analyzed. Sera from 24 patients with ODSRs and 31 healthy volunteers were also investigated as control subjects. We developed a rapid immunochromatographic assay for the detection of high levels of serum granulysin using two different antigranulysin monoclonal antibodies. RESULTS: The immunochromatographic test showed positive results for 4 of 5 patients with SJS/TEN but only one patient of 24 with ODSRs. The results correlated closely with those of enzyme-linked immunosorbent assays. LIMITATIONS: The validation of the long-time stability in this test strip has not been investigated. CONCLUSION: This novel test enables the prediction of SJS/TEN occurrence in patients even when only features of ODSRs are noted clinically.

DNA vaccination against macrophage migration inhibitory factor improves atopic dermatitis in murine models.

BACKGROUND: Atopic dermatitis (AD) is a common chronic inflammatory skin disease. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that has been implicated in the pathogenesis of AD. Recently, we developed a novel DNA vaccine that generates neutralizing endogenous anti-MIF antibodies. OBJECTIVE: This study explores the preventive and therapeutic effects of this MIF-DNA vaccine in mouse models of AD. METHODS: Two different AD model mice (DS-Nh and NC/Nga) received MIF-DNA vaccination to analyze preventive and therapeutic effects, as assessed by clinical skin scores, histologic findings, and serum IgE levels. RESULTS: In murine models of AD, MIF-DNA vaccination prevented the occurrence of the AD skin phenotype. Furthermore, administration of MIF-DNA vaccine to mice that had already developed AD produced a rapid improvement in AD skin manifestation. There were reduced histologic signs of inflammation and lower serum IgE levels in treated mice compared with those seen in control animals. Finally, passive transfer of IgG from MIF-DNA vaccinated mice to AD mice also produced a significant therapeutic effect. These results demonstrate that MIF-DNA vaccination not only prevents the development of AD but also improves the symptoms of pre-existing AD. CONCLUSION: Taken together, the induction of an anti-MIF autoantibody response using MIF-DNA vaccination appears to be a useful approach in the treatment of AD.

Effects of tributyltin on the development of atopic dermatitis-like eruptions in DS-Nh mice.

BACKGROUND: In the last few decades, numerous chemical compounds have been produced as a result of industrial development. At the same time, the number of atopic dermatitis (AD) patients has been increasing. It has been reported that tributyltin (TBT) compounds have effects not only on the reproductive system but also on the immune system. OBJECTIVE: To investigate whether TBT has an effect on AD, we fed a diet containing TBT to DS-Nh mice, which spontaneously developed dermatitis under conventional conditions. METHODS: DS-Nh mice fed TBT or a control diet were examined for skin changes, number of Staphylococcus aureus on the skin and serum IgE levels. To determine Th1/Th2 cytokine production by lymphocytes, lymphocytes of DS-Nh mice fed TBT and of controls were cultured with staphylococcal enterotoxin B and cytokine levels in the supernatants were measured by ELISA. We observed not only spontaneous dermatitis but also dermatitis induced by sensitization with 2,4,6-trinitrochlorobenzene (TNCB). RESULTS AND CONCLUSION: The AD-like lesions induced by TNCB sensitization were more severe in the mice fed TBT than in those fed the control diet. A greater increase in S. aureus on the skin was observed in the mice fed TBT than in the mice fed the control diet. A decrease in IFN-gamma production and an increase in IL-5 and IL-13 production were observed in the mice fed the TBT diet and treated with TNCB. These findings suggest that the increase in S. aureus and the enhancement of Th2 response induced by TBT exacerbate the AD-like lesions in mice treated with TNCB.