[Advances in supercooling liver preservation].

Liver transplantation is currently the only effective treatment for end-stage liver disease. The preservation of donor liver before transplantation is important. But both traditional static cold storage and machine perfusion are limited by the preservation time, so that the allotment space of donor liver is limited, which inevitably leads to the abandonment of part of donor liver.At present, to find a preservation technology that not only guarantees the quality of donor liver but also has a longer effective preservation time is the direction of joint efforts of all clinicians. Supercooling liver preservation(SLP) to find a preservation technology that not only guarantees the quality of donor liver but also has a longer effective preservation time is the direction of joint efforts of all clinicians. SLP, a new method based on using cryoprotectants to keep donor liver under -6 ℃ and recovering the graft with subnormothermic machine perfusion that enables long-term transplantation survival following 4 days of liver preservation, made a revolutionary breakthrough in the field of liver preservation, carved out a new field for the research of liver preservation. This article reviews the latest experimental research progress of SLP in the field of liver transplantation.

Effects of Mycoplasma pneumoniae infection on airway neurokinin-1 receptor expression in BALB/c mice.

The aim of this study was to establish a BALB/c mouse model of Mycoplasma pneumoniae (MP) infection and to explore the expression of neurokinin-1 receptor (NK1-R) in the trachea and lung tissue and changes in its relative content at different time points (on the 3rd, 7th, 14th, 21st, and 30th days after infection) in MP-infected BALB/c mice. Immunohistochemistry and Western blot analysis were performed to determine NK1-R expression in the trachea and lung tissue and changes in relative content in MP-infected BALB/c mice. After MP infection, the expression of NK1-R on the surfaces of upper tracheal and bronchial epithelial cells, submucosa, and alveolar epithelial cells, as well as around the smooth muscle, was upregulated more significantly in the infection group than in the control group (P < 0.05); NK1-R protein expression was enhanced on the 3rd, 7th, 14th, 21st, and 30th days after infection compared with that of the control group (P < 0.05). NK1-R expression in the trachea, bronchus, and lung tissue increased in MP-infected BALB/c mice, which may explain why wheezing occurs after MP infection.

Kidney transplantation from cardiac death donors with terminal acute renal failure.

OBJECTIVES: Kidney transplantation from donation after cardiac death (DCD) donors with terminal acute renal failure (ARF) is not widely accepted due to concern about the organ quality. Here we report our initial clinical outcomes of kidney transplantation from DCD donors with ARF. MATERIALS AND METHODS: The results of 29 kidney transplants from ARF DCD donors were compared with those of 60 kidney transplants from non-ARF DCD donors performed at our center from August 2011 to March 2013. RESULTS: There was no difference in the incidence of delayed graft function and acute rejection between ARF and non-ARF kidneys (27.6% vs 16.7%, 10.3% vs 8.3%, respectively). Estimated glomerular filtration rate at 12 months was similar between ARF and non-ARF kidneys. With a mean follow-up of 18 months (range 7 to 26 months), actual patient and graft survival rates for ARF DCD recipients were 100% and 96.6%, respectively, which were similar to those of the control group of kidney transplants from non-ARF kidneys (98.3% and 95.0%). CONCLUSIONS: Kidneys from DCD donors with terminal ARF have excellent short-term outcomes and may represent another potential method to safely expand the donor pool.

Complete eradication of hepatomas using an oncolytic adenovirus containing AFP promoter controlling E1A and an E1B deletion to drive IL-24 expression.

Interleukin (IL)-24, a promising therapeutic gene, has been widely used for Cancer Targeting Gene-Viro-Therapy (CTGVT). In this study, IL-24 was inserted into an oncolytic adenovirus in which the E1A gene is driven by an enhanced, short α-fetoprotein (AFP) promoter and the E1B gene is completely deleted to form Ad.enAFP-E1A-ΔE1B-IL-24. This construct has a potent antitumor effect on liver cancer cell lines in vitro, but little or no effect on normal cell lines, such as L-02 and QSG-7701. In vivo, the complete elimination of Huh-7 liver cancer in nude mice with Ad.enAFP-E1A-ΔE1B-IL-24 intratumor injection was observed. The design of Ad.enAFP-E1A-ΔE1B-IL-24 and its potent antitumor effect on liver cancer have not been published previously. The mechanism of the potent antitumor effect of Ad.enAFP-E1A-ΔE1B-IL-24 is due to the upregulation of GADD34 and intrinsic and extrinsic apoptotic signaling.

Tumour necrosis factor-like weak inducer of apoptosis (TWEAK), an important mediator of endothelial inflammation, is associated with the pathogenesis of Henoch-Schonlein purpura.

Tumour necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumour necrosis factor (TNF) family, has been implicated as a proinflammatory cytokine in many types of autoimmune and infectious diseases. However, information about TWEAK in dermatological diseases is limited. Herein, we investigated the role of TWEAK in patients with Henoch-Schonlein purpura (HSP) and the ability of TWEAK on chemokine production in the human dermal microvascular endothelial cell line (HMEC-1). Serum TWEAK levels in patients with HSP, together with patients with psoriasis vulgaris (PV) and atopic dermatitis (AD), were detected by enzyme-linked immunosorbent assay (ELISA). HMEC-1 cells were treated with TWEAK at concentrations ranging from 1 ng/ml to 100 ng/ml. Serum levels of TWEAK were elevated in patients with HSP in the acute stage but not in patients with PV or AD. Moreover, TWEAK levels were correlated with the severity of HSP. TWEAK markedly induced CCL5 and CXCL8 production at both mRNA and protein levels in HMEC-1 cells. In addition, TWEAK-stimulated HMEC-1 supernatant enhanced HL-60 or human acute monocytic leukaemia cell line (THP-1) cell migration. Finally, Western blot data revealed that TWEAK can induce rapid phosphorylation of inhibitor of κB-α (IκBα) in HMEC-1 cells. In conclusion, we show that serum levels of TWEAK were elevated in patients with acute stage HSP. TWEAK may act as a regulator of nuclear factor-κB (NF-κB) activation and chemokine production in human dermal microvascular endothelial cells, thus promoting leucocyte migration in cutaneous vasculitis.

The suppressive effect of resveratrol on protein kinase C theta in peripheral blood T lymphocytes in a rat liver transplantation model.

UNLABELLED: Our aim was to study the effect of resveratrol on the expressions of protein kinase C isotypes (PKC alpha, theta) in peripheral blood lymphocytes and on the expression of IkappaB kinase-beta (IKK beta) in lymphocytes in allografts in a rat liver transplantation model. METHODS: Orthotopic liver transplantations (OLT) were performed from Sprague Dawley rats to Wistar rats. The recipients were divided into two groups after OLT. In the RES group, resveratrol was given intraperitoneally once a day (100 mg kg(-1)) after OLT, whereas in the control group vehicle buffer was given. The expressions of PKC alpha, theta in peripheral blood lymphocytes, expression of IKK beta in lymphocytes in allograft, and survival periods were compared between the groups. RESULTS: The mean survival period after OLT in the RES group was significantly longer than that in control group (P < .05). On posttransplant day 7, the expression of PKC theta in peripheral blood lymphocytes in the RES group was significantly decreased compared with that in the control group (P < .05), whereas there was no obvious difference in the expressions of PKC alpha between the two groups (P > .05), and the positive rate of IKK beta protein in lymphocytes in allografts in RES group was significantly decreased compared with that in the control group (P < .05). CONCLUSION: Resveratrol showed an immunosuppressive effect on lymphocytes for allograft rejection in the rat. Down-regulation of the expression of PKC theta in peripheral blood lymphocytes may be part of the mechanism.

Secondary degeneration reduced by inosine after spinal cord injury in rats.

STUDY DESIGN: Assessment of the potential protective effects of inosine on an animal model of spinal cord injury. OBJECTIVES: Our previous studies have demonstrated that inosine can directly protect neurons in vitro from zinc-induced injury and axotomized retinal ganglion cells of rats in vivo. This investigation was carried out to examine the possible protective effects of inosine on spinal cord secondary degeneration. SETTING: Institute of Neurosciences, The Fourth Military Medical University, Xi'an, China. METHODS: Compressive spinal cord injury (95-g load for 1 min) model was established in rats, and inosine was administrated beginning at different time points (2, 12, or 24 h) after spinal cord injury. RESULTS: Using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) technique and hematoxylin and eosin staining, our study demonstrated that administration of inosine as late as 12 h after injury significantly reduced the total volume of spinal cord degenerative areas and the number of apoptotic cells 3 days following the trauma. CONCLUSION: Inosine can significantly reduce the spread of secondary degeneration and the cell death following spinal cord injury in adult rats. These findings may find a clinical application in the treatment of acute spinal cord injury.

Astrocytes in injured adult rat spinal cord may acquire the potential of neural stem cells.

It has been well documented that in adult rats astrocytes in the subventricular zone and subgranular layer of the dentate gyrus are neural stem cells. Elsewhere in the CNS astrocytes are not generally recognized as stem cells. Here we describe nestin expression in a population of astrocytes in the spinal cord of adult rat following cord injury. In either hemitransectioned or longitudinally cut spinal cord, there was widespread nestin expression in astrocytes of both the gray and white matters. Isolation of the lateral part of the spinal cord from the central canal region, where stem cells may reside, could not block the appearance of nestin-immunoreactive astrocytes in the lateral cord, and none of them showed Fast DiI labeling after the central canal ependyma had been labeled by the dye, indicating that the nestin-immunoreactive astrocytes can evolve locally in the lateral cord. They were found to be undergoing a process of de-differentiation. Culture of the nestin-immunoreactive astrocytes of the lateral cord generated neurospheres, the cells of which had the ability of self-renewal, and were able to differentiate into neurons, astrocytes, or oligodendrocytes. Taken together, the results indicate that the astrocytes in injured adult rat spinal cord may acquire the potential of neural stem cells.

Phosphorylation of extracellular signal-regulated kinases 1/2 is predominantly enhanced in the microglia of the rat spinal cord following dorsal root transection.

The present study was initiated to investigate the role of extracellular signal-regulated kinases (ERK) 1/2 signaling pathway in the early response of spinal cord and associated dorsal root ganglion (DRG) to rhizotomy by using Western blotting and immunohistochemical techniques in a rat model of L3 and L4 dorsal root transection. The results showed that there were a considerable amount of total and phosphorylated ERK 1/2 protein in both spinal cord and DRG in normal animals killed under pentobarbital anesthesia. The total ERK 1/2 distributed in both glia and neurons, while phosphorylated ERK 1/2 dominantly existed in the latter in the gray matter of spinal cord, as demonstrated with double immunofluorescent staining. Twenty-four and forty-eight hours after axotomy, the phosphorylation level of ERK 1/2 in the operation side of dorsal spinal cord was much higher than that in the contralateral side, while the total ERK 1/2 level seemed unchanged. The increased expression of Fos protein was also seen in the dorsal spinal cord at lesion side twelve and twenty-four hours after axotomy. Double fluorescent staining proved that the phosphorylated ERK 1/2 positive cells in the ipsilateral dorsal spinal cord after axotomy predominantly were microglia and small portion was oligodendrocytes, whereas the Fos expression was mainly in neurons. In normal DRG, most neurons, especially the medium and small-sized ones, and the satellite cells contained total ERK 1/2-like immunoreactivity, whereas only a small portion of neurons and satellite cells contained phosphorylated ERK 1/2. After unilateral dorsal rhizotomy, there were no detectable changes for the phosphorylation of ERK 1/2 in either neurons or satellite cells in DRG.Collectively, the present results suggest that both ERK and Fos signal pathways involve the cellular activation in the spinal cord following dorsal rhizotomy, with ERK mainly in microglia and Fos in neurons. The increase of phosphorylation of ERK 1/2 in microglia of spinal cord after rhizotomy implicates that ERK signaling pathway involves intracellular activity of microglia responding to the experimental injury.

Aplastic anaemia associated with parvovirus B19 infection.

Parvovirus B19 involvement was investigated in 30 children with severe aplastic anaemia. Active or recent parvovirus B19 infection, as shown by B19 DNA viraemia, positive B19 specific IgM antibodies, or both, was diagnosed in six patients. There were no other plausible causes. We suggest that parvovirus B19 infection might be associated with severe aplastic anaemia.

Increase in cytochrome oxidase activity in regenerating nerve fibers of hemitransected spinal cord in the rat.

We explored the possibility of cytochrome oxidase (CO) involvement in spinal cord regeneration in adult rats. The spinal cord was hemitransected at T9. After one month's survival, the animals were deeply anesthetized and perfused. The spinal cord segments including the lesion site were removed and sectioned horizontally for CO histochemistry. Under light microscope, a substantial number of CO-reactive nerve fibers and boutons were identified in the lateral funiculus adjacent to the lesion site. Under electron microscope, moderately to highly CO-reactive mitochondria could be seen within nerve fibers and boutons. Synaptic contacts were identified among them. The increase in CO activity in nerve fibers and boutons may indicate their high-energy demand for synaptic and spontaneous activity following spinal cord hemisection.

[Transplantation of cultured human keratinocyte on collagen sponge].

OBJECTIVE: To investigate the skin regeneration using cultured human keratinocytes with collagen sponge transplanted into thickness wound of nude mice. METHODS: Human foreskin from foreskin ectomy procedures was detached with 0.5% Dispase II. Epidermis sheets were separated from dermis and digested with 0.05% Trypsin into single cell suspension. Keratinocytes were cultured and seeded into collagen sponge during logarithmic growth phase. After 3 days, the keratinocytes-collagen sponge were grafted on full thickness wound of nude mice, compared with simple collagen sponge without keratinocytes. The histological, immunohistochemical examination and electron microscopy were detected. RESULTS: After the epidermal substitute was grafted onto wound, the human keratinocytes were able to further proliferate and differentiate and develop into new epithelia. Compared with the control group, the wound healed earlier and contracted less, epithelia matured earlier, and the collagen fiber was less beneath epithelia. CONCLUSION: Keratinocytes can grow on collagen sponge and migrate onto wound to develop into stratified epithelia and inhibit wound contract. The keratinocyte graft can be used to repair skin defect.

Activity of p44/42 MAP kinase in the caudal subnucleus of trigeminal spinal nucleus is increased following perioral noxious stimulation in the mouse.

The extracellular signal-regulated protein kinase-1 and -2 (ERK1 and ERK2), also referred to as the p44/42 mitogen-activated protein kinase (p44/42 MAP kinase), plays an essential role in neuronal signal transduction, but its function involved in nociceptive response has not been deeply studied yet. Here we report immunohistochemical evidence that p44/42 MAPK might be critical in nociceptive response. We found that after formalin was injected into the perioral skin of the upper lip of mice, the number of activated p44/42 MAPK-like immunoreactive neurons was significantly increased in the laminae I and II of the caudal subnucleus of the trigeminal spinal nucleus (Sp5C). The positive neurons and fibers were mostly concentrated in the middle portion of Sp5C dorsoventrally, where the afferent fibers innervating the skin of the upper lip are terminated. The reactive products were localized in perikarya, dendrites, nuclei, and diffusely in the neuropil. The present result suggests that p44/42 MAPK may be important in the transmission and modulation of noxious information in Sp5C.

[Tissue engineering of skin].

OBJECTIVE: To review the recent progresses on tissue engineering of skin. METHODS: Recent original articles about tissue engineering of skin were extensively reviewed, which focused on the progresses and major problems concerning the epidermal substitutes, dermal substitutes, cultured-epidermal composite skin graft. RESULTS: Most investigators had come to conclusion that the optimal skin substitute should provide for immediate reconstruction of both the lost epidermis and dermis. The research was mainly focused on how to transplant epidermal cells immediately, preserve their activity and function, and develop the extracellular matrix which could effectively accelerate the function of transplanted cells, induce vascular growth from the wound bed, could be biodegradable, no toxicity and no danger of carrying pathogen. CONCLUSION: The major research trends of tissue engineering of skin should be focused on the study of immediate transplantation of epidermal cells, accelerate wound healing and developing extracellular matrix of dermis.

Prefabrication of bilaminar-epithelialized composite flap with tissue expander and cultured keratinocytes.

This study investigated the feasibility of prefabrication of a bilaminar-epithelialized flap by using a tissue expander and cultured keratinocytes, for reconstruction of perforate defects in the oral cavity and upper aerodigestive tract. In each of six rats, a 10-ml volume expander was implanted under the inferior epigastric flap and a thin silicon catheter was introduced into periexpander space. Seven days after implantation, 10 x 10(6) cultured keratinocytes, isolated from inbred donor rats, were suspended in fibrin glue and injected into the periexpander space through the catheter (n = 4 of 6). The expansion was started immediately after cell inoculation and lasted at least 3 weeks at the speed of 2 to 3 ml every 5 to 7 days. At the end of expansion, the periexpander space was opened and the capsule around the tissue expander was found to be covered completely with a neoepithelium. Thus, a bilaminar-epithelialized flap based on femoral vessels was elevated and successfully transferred to cover the excisional perforate defect in the oral cavity with the neoepithelial side as inner lining. All flaps treated with 10 x 10(6) cultured keratinocytes survived with complete wound healing during a 1-week follow-up (n = 4 of 6). Both macroscopic and histologic findings demonstrated that a bilaminar-epithelialized composite flap can be fabricated by using a tissue expander and keratinocyte-fibrin glue suspension.

[The study of new bio-membrane-bred epidermal cell grafts].

OBJECTIVE: The human epidermal cells were bred on a kind of bio-membrane, the bio-brane, in engineering a kind of new epidermal substitute, the bio-membrane bred cell graft. METHODS: Fresh and frozen grafts of biomembrane bred epidermal cells were transplanted into the full-thickness wounds of nude mice and those received simple Bio-brane were served as control. The wounds of the two groups were observed daily and biopsy was taken on the 3, 5, 7, 10, 21 and 35 days respectively. RESULTS: Epidermal cells could be cultured in vitro on the bio-membrane reaching the sub-saturated state of 60 to 70 percents. The bio-membrane after being grafted the epidermal cells continued to proliferate and differentiate to form a layer of new epidermis. There was no difference between the fresh and the frozen bio-membranes. CONCLUSION: Bio-membrane bred with epidermal cells could be a kind of ideal epidermal substitute.

[Continuous delivery of epidermal growth factor to wounds in vivo by genetically modified fibroblasts transfected with a novel chimeric construct].

OBJECTIVE: This paper aims to explore the new method of continuous delivery of epidermal growth factor to wounds by transfected fibroblasts to promote wound repair. METHODS: It was constructed a novel chimeric expression plasmid in which the biologically active portion of the human epidermal growth factor (EGF) gene was fused in-frame to the human granulocyte colony-stimulating factor signal sequence. RESULTS: Clonally selected human fibroblasts transfected with this construct could secrete biologically active EGF. After the transplantation of irradiated gene-transfected fibroblasts suspended in fibrin glue to murine full-thickness wounds, EGF could be demonstrated for at least seven days in the wounds, slowly decreasing from initially 470 ng/L to 140 ng/L in 7 days. No EGF was found in the wound at 14 days. CONCLUSION: A single application of irradiated EGF gene transfected fibroblasts to wounds can continuously deliver the transgene in vivo and can be used to administer drugs to the wound bed during the crucial first seven days of wound-healing.