RESUMO
Genetically manipulated organisms with dysfunction of specific tissues are crucial for the study of various biological applications and mechanisms. However, the bioengineering of model organisms with tissue-specific dysfunction has not progressed because the challenges of expression of proteins, such as cytotoxins, in living cells of individual organisms need to be overcome first. Here, we report the establishment of a transgenic silkworm (Bombyx mori) with posterior silk glands (PSGs) that was designed to express the cabbage butterfly (Pieris rapae) cytotoxin pierisin-1A (P1A). P1A, a homolog of the apoptosis inducer pierisin-1, had relatively lower DNA ADP ribosyltransferase activity than pierisin-1; it also induced the repression of certain protein synthesis when expressed in B. mori-derived cultured cells. The transgene-derived P1A domain harboring enzymatic activity was successfully expressed in the transgenic silkworm PSGs. The glands showed no apoptosis-related morphological changes; however, an abnormal appearance was evident. The introduced truncated P1A resulted in the dysfunction of PSGs in that they failed to produce the silk protein fibroin. Cocoons generated by the silkworms solely consisted of the glue-like glycoprotein sericin, from which soluble sericin could be prepared to form hydrogels. Embryonic stem cells could be maintained on the hydrogels in an undifferentiated state and proliferated through stimulation by the cytokines introduced into the hydrogels. Thus, bioengineering with targeted P1A expression successfully produced silkworms with a biologically useful trait that has significant application potential.
Assuntos
ADP Ribose Transferases , Animais Geneticamente Modificados , Bombyx , Citotoxinas , Glândulas Exócrinas/metabolismo , Hidrogéis/farmacologia , Proteínas de Insetos , Células-Tronco Embrionárias Murinas/metabolismo , Sericinas , ADP Ribose Transferases/biossíntese , ADP Ribose Transferases/genética , ADP Ribose Transferases/farmacologia , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Bombyx/genética , Bombyx/metabolismo , Citocinas/biossíntese , Citotoxinas/biossíntese , Citotoxinas/genética , Citotoxinas/farmacologia , Proteínas de Insetos/biossíntese , Proteínas de Insetos/genética , Proteínas de Insetos/farmacologia , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Sericinas/biossíntese , Sericinas/genética , Sericinas/farmacologiaRESUMO
BACKGROUND: Herpes zoster (HZ) is caused by reactivation of a latent varicella zoster virus (VZV). The potential to develop HZ increases with age due to waning of memory cell-mediated immunity (CMI), mainly the CD4 response. Therefore, VZV-CD4-memory T cells (CD4-M) count in blood could serve as a barometer for HZ protection. However, direct quantification of these cells is known to be difficult because they are few in number in the blood. We thus developed a method to measure the proliferation level of CD4-M cells responding to VZV antigen in whole blood culture. METHODS: Blood samples were collected from 32 children (2-15â¯years old) with or without a history of varicella infection, 18 young adults (28-45â¯years old), and 80 elderly (50-86â¯years old) with a history of varicella infection. The elderly group was vaccinated, and blood samples were taken 2â¯months and 1â¯year after VZV vaccination. Then, 1â¯mL of blood was mixed with VZV, diluted 1/10 in medium, and cultured. CD4-M cells were identified and measured by flow cytometry. RESULTS: There was distinct proliferation of CD3+CD4highCD45RA-RO+ (CD4high-M) cells specific to VZV antigen at day 9. The majority of CD4high-M cells had the effector memory phenotype CCR7- and was granzyme B-positive. CD4high-M cells were detected in blood culture from varicella-immune but not varicella-non-immune children. Meanwhile, a higher level of CD4high-M proliferation was observed in young adults than in the elderly. The CD4high-M proliferation level was boosted 2â¯months after VZV vaccination and maintained for at least 1â¯year in the elderly. CONCLUSION: Quantifying VZV responder CD4high -M cell proliferation is a convenient way to measure VZV CMI using small blood volumes. Our method can be applied to measure VZV vaccine-induced CMI in the elderly. Clinical study registry numbers: (www.clinicaltrials.jp) 173532 and 183985.
Assuntos
Vacina contra Herpes Zoster/uso terapêutico , Herpes Zoster/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Hemocultura , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células/fisiologia , Feminino , Citometria de Fluxo , Humanos , Imunidade Celular/imunologia , Imunidade Celular/fisiologia , Masculino , Pessoa de Meia-Idade , Vacinação/métodos , Vacinas Atenuadas/uso terapêuticoRESUMO
Since thrombotic complications, such as superficial thrombophlebitis and subsequent skin pigmentation, are common after sclerotherapy, we conducted a study to evaluate whether combining sclerotherapy with ligation of varicose veins minimizes complications and what timing for sclerotherapy would be most beneficial-accompanying surgery or several weeks postsurgery. Surgical intervention and compression sclerotherapy were performed consecutively on 111 limbs (group A), and sclerotherapy was performed 28 days after surgical intervention on 87 limbs (group B). The volume of sclerosant used and the frequency of complications (thrombus formation and pigmentation) were analyzed. Plasma levels of thrombin-antithrombin III complex (TAT) and D-dimer (DD), as markers for activation of coagulation, were compared. In group A, the total volume of sclerosant used in patients with complications was significantly higher than that in patients without complications. The frequency of thrombus formation and of pigmentation was significantly lower (p <0.01) in group B (10% and 18%, respectively) than in group A (21% and 37%, respectively). The plasma levels of TAT 7 days after treatment were significantly lower in group B (3.4 +/- 1.2 mg/L) than in group A (4.9 +/- 1.1 mg/L). Performing compression sclerotherapy 28 days after surgical intervention is effective for reducing complications and a good alternative for patients with an underlying hypercoagulable state.
Assuntos
Peptídeo Hidrolases/sangue , Escleroterapia/métodos , Varizes/terapia , Procedimentos Cirúrgicos Vasculares/métodos , Adulto , Idoso , Antitrombina III , Bandagens , Biomarcadores , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Humanos , Ligadura , Masculino , Pessoa de Meia-Idade , Fatores de Tempo , Resultado do Tratamento , Varizes/imunologiaRESUMO
BACKGROUND: Since platelet function is known to play a major role in arterial thrombosis, we investigated postsurgery alterations in platelet function that might predispose patients with upper gastrointestinal malignancy to postoperative thrombotic complications. SUBJECTS AND METHODS: Shear-induced platelet aggregation (SIPA) in platelet-rich plasma was measured in 23 patients who elected to undergo abdominal surgery. Measurement was done by cone-plate viscometer under low shear stress (12 dyn/cm(2)), a physiological condition, and under high shear stress (108 dyn/cm(2)), a pathological condition that simulates in vivo conditions such as those in stenotic arteries. Platelet microparticle (PMP) formation was analyzed by flow cytometry. Plasma von Willebrand factor (vWF) was also measured. RESULTS: SIPA under high shear stress was significantly enhanced from 44.0 +/- 13.4% preoperatively to 69.5 +/- 15.8% on postoperative day (POD) 1, and it returned to preoperative levels on POD 14. PMP formation under high shear stress was enhanced before surgery (140.8 +/- 38.7%) compared to that under a static condition, and the enhancement was further augmented on POD 1 (219.1 +/- 49.3%). The enhancement of SIPA and PMP formation had no association with disease stage. vWF levels increased significantly on POD 1. Exogenous vWF augmented SIPA and PMP formation under high shear stress, and this augmentation was inhibited by anti-vWF antibody. CONCLUSIONS: Because PMPs are highly procoagulant, increased SIPA and PMP formation induced by surgical intervention possibly contribute to thrombotic complications. Blockage of platelet interaction with vWF may prevent arterial thrombus formation perioperatively.