Natural and engineered chemokine (C-X-C motif) receptor 4 agonists prevent acute respiratory distress syndrome after lung ischemia-reperfusion injury and hemorrhage.

We compared therapeutic properties of natural and engineered chemokine (C-X-C motif) receptor 4 (CXCR4) agonists in a rat acute respiratory distress syndrome (ARDS) model utilizing the PaO2/FiO2-ratio as a clinically relevant primary outcome criterion. Ventilated rats underwent unilateral lung ischemia from t = 0-70 min plus hemorrhage to a mean arterial blood pressure (MAP) of 30 mmHg from t = 40-70 min, followed by reperfusion/fluid resuscitation until t = 300 min. Natural CXCR4 agonists (CXCL12, ubiquitin) and engineered CXCL12 variants (CXCL121, CXCL22, CXCL12K27A/R41A/R47A, CXCL12 (3-68)) were administered within 5 min of fluid resuscitation. Animals treated with vehicle or CXCL12 (3-68) reached criteria for mild and moderate ARDS between t = 90-120 min and t = 120-180 min, respectively, and remained in moderate ARDS until t = 300 min. Ubiquitin, CXCL12, CXCL121 and CXCL122 prevented ARDS development. Potencies of CXCL12/CXCL121/CXCL122 were higher than the potency of ubiquitin. CXCL12K27A/R41A/R47A was inefficacious. CXCL121 > CXCL12 stabilized MAP and reduced fluid requirements. CXCR4 agonists at doses that preserved lung function reduced histological injury of the post-ischemic lung and reduced mortality from 55 to 9%. Our findings suggest that CXCR4 protein agonists prevent development of ARDS and reduce mortality in a rat model, and that development of new engineered protein therapeutics with improved pharmacological properties for ARDS is possible.

MODERATION OF QUANTITATIVE CHANGES OF REGENERATING ERYTHROPOIETIC CELLS BY EXTRACELLULAR UBIQUITIN.

Hematological disorders caused by radiation remain the most challenging problem of the last decades. Environmental radiation, as well as medical application of radiation causes serious problems especially from the point of view of blood formation and passage of blood functional cells. Bone marrow emptying followed by the rise of immature cells in the bloodstream is the main pathology that must be eliminated. The importance of the issue is well recognized by all investigators. Opening of agents for regulation of spontaneous regeneration of hematopoietic cell lines is of prime importance in cancer treatment. Ubiquitin is a globular protein of eukaryotic cells. It is involved in regulation of cell cycle. Recently we studied the influence of extracellular ubiquitin on regeneration of leukopoiesis. Interestingly what is its effect on erythropoietic cell lines? Erythrocytes are more resistant to irradiation, than nucleic cells. Their depletion-elevation picks during blood regeneration clearly reveal low sharpness. Nevertheless, depletion of erythropoietic cells if not treated, may cause short- and long-term side effects. We studied the influence of intraperitoneally injected ubiquitin on the process of spontaneous regeneration of erythropoietic cell lines of bone marrow (BM) and peripheral blood (PB) after irradiation in mice. The source of radiation was 137Cs with dose rate 1Gy/min., the duration of exposure 3 min and 5 min. Nonlinear white mice of 22±2 gr. were used for experiment. Animals were divided into five groups (6 animals in each group): the first control group of intact mice; the second test group of mice irradiated by the sublethal dose of 3Gy; the third test group of mice irradiated by 3Gy intraperitoneally injected by ubiquitin by the dose of 100µg/ml at the 72 hour point after irradiation; the fourth test group of mice irradiated by the dose of LD50 5Gy; the fifth test group of mice irradiated by 5Gy intraperitoneally injected by ubiquitin at the 72 hour point after irradiation. PB and BM samples from the control group and the test groups of mice have been taken every 24 hours after irradiation during 7 days. Microscopy and statistical methods have been implicated for calculation of cell count of PB and BM. Analyzing the results we concluded that Ubiquitin prevents depletion-elevation picks of erythrocytes and erythroblasts regardless of the severity of damage caused by different doses of radiation indicating normalization of the regeneration process after irradiation. The study is important for opening new therapeutic agents for normalization of regeneration hematopoiesis after irradiation.

Pharmacological targeting of chemokine (C-X-C motif) receptor 4 in porcine polytrauma and hemorrhage models.

BACKGROUND: Recent evidence suggests that chemokine receptor CXCR4 regulates vascular α1-adrenergic receptor function and that the noncognate CXCR4 agonist ubiquitin has therapeutic potential after trauma/hemorrhage. Pharmacologic properties of ubiquitin in large animal trauma models, however, are poorly characterized. Thus, the aims of the present study were to determine the effects of CXCR4 modulation on resuscitation requirements after polytrauma, to assess whether ubiquitin influences survival times after lethal polytrauma-hemorrhage, and to characterize its dose-effect profile in porcine models. METHODS: Anesthetized pigs underwent polytrauma (PT, femur fractures/lung contusion) alone (Series 1) or PT/hemorrhage (PT/H) to a mean arterial blood pressure of 30 mmHg with subsequent fluid resuscitation (Series 2 and 3) or 40% blood volume hemorrhage within 15 minutes followed by 2.5% blood volume hemorrhage every 15 minutes without fluid resuscitation (Series 4). In Series 1, ubiquitin (175 and 350 nmol/kg), AMD3100 (CXCR4 antagonist, 350 nmol/kg), or vehicle treatment 60 minutes after PT was performed. In Series 2, ubiquitin (175, 875, and 1,750 nmol/kg) or vehicle treatment 60 minutes after PT/H was performed. In Series 3, ubiquitin (175 and 875 nmol/kg) or vehicle treatment at 60 and 180 minutes after PT/H was performed. In Series 4, ubiquitin (875 nmol/kg) or vehicle treatment 30 minutes after hemorrhage was performed. RESULTS: In Series 1, resuscitation fluid requirements were significantly reduced by 40% with 350-nmol/kg ubiquitin and increased by 25% with AMD3100. In Series 2, median survival time was 190 minutes with vehicle, 260 minutes with 175-nmol/kg ubiquitin, and longer than 420 minutes with 875-nmol/kg and 1,750-nmol/kg ubiquitin (p < 0.05 vs. vehicle). In Series 3, median survival time was 288 minutes with vehicle and 336 minutes and longer than 420 minutes (p < 0.05 vs. vehicle) with 175-nmol/kg and 875-nmol/kg ubiquitin, respectively. In Series 4, median survival time was 147.5 minutes and 150 minutes with vehicle and ubiquitin, respectively (p > 0.05). CONCLUSION: These findings further suggest CXCR4 as a drug target after PT/H. Ubiquitin treatment reduces resuscitation fluid requirements and provides survival benefits after PT/H. The pharmacological effects of ubiquitin treatment occur dose dependently.

A Toxoplasma gondii vaccine encoding multistage antigens in conjunction with ubiquitin confers protective immunity to BALB/c mice against parasite infection.

BACKGROUND: Toxoplasma gondii is a widely prevalent intracellular parasite which infects almost all warm-blooded animals including humans and causes serious zoonotic toxoplasmosis. DNA vaccines have proved effective in the protection against parasites. However, the problems of weak immunity and inefficient delivery of DNA vaccine remain major issues. Therefore, comprehensive antigens derived from all stages of the parasite, effective adjuvants and delivery systems should be considered in the vaccine construction. METHODS: SAG3101-144,ROP18347-396, MIC6288-347, GRA7182-224, MAG158-125, BAG1156-211 and SPA142-200, derived from antigens in tachyzoite, bradyzoite and sporozoite stages of T. gondii were screened based on CD8(+) T cell epitope binding affinity to HLA and H-2. We constructed a recombinant DNA vaccine and an adenovirus vaccine encoding multi-stage antigen of T. gondii linked to ubiquitin molecules and vaccinated BALB/c mice with different strategies. Antibodies, cytokines, splenocytes proliferation, as well as the percentage of CD4(+) and CD8(+) T cells in immunized mouse were analyzed by the Enzyme-Linked Immunosorbent Assays (ELISA), Flow Cytometry (FCM). Protective efficacy was evaluated by challenging immunized mice with type I and type II parasite. RESULTS: Our results indicated that the DNA vaccine had the advantage of inducing a stronger humoral response, whereas the adenovirus-vectored vaccine effectively improved the cellular immune response. Priming with DNA vaccine and boosting with adenovirus-vectored vaccine induced Th1-type immune responses with highest levels of IgG2a and secretion of cytokines IL-2 and IFN-γ. Effective protection against type I and type II parasite with an increase in survival rate and a decrease in brain cyst burden was achieved in immunized mice. CONCLUSIONS: Priming vaccination with DNA vaccine and boosting with the recombinant adenovirus vaccine encoding ubiquitin conjugated multi-stage antigens of T. gondii was proved to be a potential strategy against the infection of type I and type II parasite.

A novel system enhancing the endosomal escapes of peptides promotes Bak BH3 peptide inducing apoptosis in lung cancer A549 cells.

Therapeutic peptides have been proven useful for treating various diseases. However, it is difficult for therapeutic peptides to reach their target sites with an effective concentration due to inefficient intracellular delivery. Although Tat transduction peptide is a promising tool to deliver therapeutic peptides into cells, the entrapment within endosomes and the nuclear localization of Tat transduction peptide severely limited the biological effects of Tat-linked cargos. In this study, we designed a novel peptide delivering system, Tat-INF7-ubiquitin (TIU), which consisted of Tat transduction peptide, endosomal escape enhancer peptide INF7, and ubiquitin protein. We found that the TIU system was able to efficiently deliver the mCherry fluorescent proteins and the apoptosis-inducing Bak BH3 peptide into the cytosol. The Bak BH3 peptide transported into the cells by the TIU system increased the apoptotic rate to 46.15 ± 4.86% (p < 0.001) in A549 cells, while Tat-BH3 could result in only 20.45 ± 2.89%. These results demonstrated that the TIU system could enhance the effects of therapeutic peptides by facilitating the transmembrane delivery of peptides into the cells and the escape of target proteins from the endosome efficiently.

Ubiquitin in combination with alcohol stimulates proliferative activity of hepatocytes.

There are several factors involved in aetiology of Alcoholic Liver Disease. The one of the most important role in regulation of intracellular transformations belongs to ubiquitin proteasome system that is comparably well studied, but less is known about the functions of extracellular ubiquitin. The goal of presented work is to study the effect of extracellular ubiquitin on partially hepatectomized alcoholic liver regeneration. Experiments were carried out on white female rats. Proliferative activity of hepatocytes was determined by colchicine mitotic index and immunohistochemical staining on Ki67. We have shown that in vivo injected extracellular ubiquitin stimulates proliferative activity of hepatocytes in partially hepatectomized alcoholic liver whereas in non-alcoholic rats hepatocytes proliferative activity was decreased. The main attraction of the experiment is the combination of alcohol and ubiquitin, as without alcohol ubiquitin inhibits proliferative activity of hepatocytes and blood cells. Further investigation of its action will provide new insights into possible pathway of ubiquitilation involved in stimulation of proliferative activity in alcoholic liver.

Giant hollow M(n)L(2n) spherical complexes: structure, functionalisation and applications.

Drawing inspiration from the self-assembly of hollow spherical virus capsids and protein cages found in nature, a family of roughly spherical coordination polyhedra with general formula MnL2n was designed and several members of the series have been synthesised. These spherical complexes are self-assembled upon reaction of bent bis(pyridine) ligands with Pd(2+) ions. The introduction of functional side chains into the ligands is straightforward, making the synthesis of both exo- and endohedrally functionalised spherical complexes possible. Accumulation of a high density of functional groups at the periphery of the spherical framework results in an enhancement of the weak interactions used in biomolecular recognition processes and the strong and selective interaction of the complex with a variety of substrates. Discrete and well-defined environments are generated within the spherical framework by functionalisation of the interior of the complex. These environments can be used for the selective encapsulation of guest molecules, including species as diverse as simple metal ions, fluoroalkanes and fullerenes. The well-defined cavity of the spherical complexes can also be exploited for the synthesis of precisely size-controlled nanoparticles and polymers. Most recently, a protein was successfully enclosed within a hollow self-assembled spherical complex, with a long-term view towards the control of protein functions for the development of new applications.

In vivo investigation of extracellular ubiquitin effect on liver histoarchitectonics.

Ubiquitin is a small regulatory protein of living cells. It affects almost all cellular processes. Investigation of ubiquitin is very important for discovering the new therapeutics for different heavy human diseases. The main goal of presented work was to reveal effect of extracellular ubiquitin on liver histoarchitectonic of mature and immature white rats. The obtained data indicate the inefficiency of used doses--200 µg/ml and 500 µg/ml--of extracellular ubiquitin single and multiple in vivo injections to evident changes of histoarchitectonics of immature and mature white rat liver. Further investigation of in vivo injection effects on histoarchitectonics of intact and damaged liver and hepatocyte cell cycle regulation by immunological assays seems to be actual and interesting.

Ubiquitin conjugation of open reading frame F DNA vaccine leads to enhanced cell-mediated immune response and induces protection against both antimony-susceptible and -resistant strains of Leishmania donovani.

Resistance of Leishmania donovani to sodium antimony gluconate has become a critical issue in the current, prolonged epidemic in India. Hence, there is an urgent need for a vaccine that is protective against both antimony-susceptible and -resistant strains of L. donovani. The multigene LD1 locus located on chromosome 35 of Leishmania is amplified in approximately 15% of the isolates examined. The open reading frame F (ORFF), a potential vaccine candidate against visceral leishmaniasis, is part of the multigene LD1 locus. ORFF was expressed as a chimeric conjugate of ubiquitin to elicit an Ag-specific cell-mediated immune response. Analysis of the cellular immune responses of ubiquitin-conjugated ORFF (UBQ-ORFF) DNA-immunized, uninfected BALB/c mice demonstrated that the vaccine induced enhanced IFN-gamma-producing CD4(+) and CD8(+) T cells compared with nonubiquitinated ORFF DNA vaccine. Higher levels of IL-12 and IFN-gamma and the low levels of IL-4 and IL-10 further indicated that the immune responses with UBQ-ORFF were mediated toward the Th1 rather than Th2 type. Infection of immunized mice with either the antimony-susceptible (AG83) or -resistant (GE1F8R) L. donovani strain showed that UBQ-ORFF DNA vaccine induced higher protection when compared with ORFF DNA. UBQ-ORFF DNA-immunized and -infected mice showed a significant increase in IL-12 and IFN-gamma and significant down-regulation of IL-10. High levels of production of nitrite and superoxide, two macrophage-derived oxidants that are critical in controlling Leishmania infection, were observed in protected mice. The feasibility of using ubiquitinated-conjugated ORFF DNA vaccine as a promising immune enhancer for vaccination against both antimony-susceptible and -resistant strains of L. donovani is reported.

Prolongation of skin graft survival by exogenous ubiquitin.

Recently, it was shown that exogenous ubiquitin has anti-inflammatory actions in vivo and that the ubiquitin-decapeptide 50-59 has immunosuppressive effects similar to cyclosporine. Immunosuppressive effects of the native ubiquitin molecule and its therapeutic potential in transplantation are unknown. We tested the hypothesis that ubiquitin inhibits alloreactivity and increases allograft survival in a murine model of skin transplantation in fully mismatched strain combinations (C3H/HEJ-DBA2). Ubiquitin dose-dependently inhibited mixed leukocyte reaction in C3H/HEJ splenocytes in vitro. Intraperitoneal ubiquitin administration (25 microg/h for 14 days) was well-tolerated, dose-dependently increased ubiquitin serum concentrations and median allograft survival from 10 days (with albumin; control) to 17 days in DBA2 mice (survival ratio: 1.7, 95% confidence interval: 1.266-2.134; P=0.0005). The in vivo effects in this study combined with our previous work strongly indicate that ubiquitin is a potent immune modulator with broad therapeutic potential. Ubiquitin treatment could be a novel strategy to improve immunosuppressive regimens in transplantation.

Ubiquitin reduces fluid shifts after traumatic brain injury.

BACKGROUND: Ubiquitin has well-described intracellular properties. Recent data also suggest pleiotropic effects of extracellular ubiquitin, including induction of apoptosis, regulation of immune functions, and therapeutic potential during fluid resuscitation from severe trauma. However, the actions of exogenous ubiquitin after traumatic brain injury (TBI) are unknown. METHODS: Series 1: Thirty-five minutes after TBI and hemorrhage, 1.5 mg ubiquitin/kg (n = 5) or albumin (n = 5) intravenous was followed by fluid resuscitation to maintain mean arterial and cerebral perfusion pressure. Series 2: Ubiquitin (n = 5) or vehicle (n = 6) was administered after TBI only. Ubiquitin was measured with enzyme-linked immunosorbent assay in serum, urine (series 1), and cerebrospinal fluid (series 2) for 300 minutes. RESULTS: Series 1: After intravenous bolus, serum ubiquitin peaked at t = 45 minutes with a half-life of 54 minutes. Recovery in urine was 10%. With albumin versus ubiquitin, 85% more resuscitation fluid was required to stabilize systemic and cerebral hemodynamics (P < .05 for t = 150 to 300 minutes), but hematocrit was similar. With albumin there were progressive increases in intracranial pressure, peak inspiratory pressure, and decreases in oxygenation. All were significantly attenuated by ubiquitin (all P < .05 vs albumin). Series 2: Intravenous ubiquitin altered cerebrospinal fluid ubiquitin with an increased time to peak (t = 88 +/- 13 min vs 45 +/- 7 min, P < .05) and area under the concentration-time curve (82 +/- 22 vs 23 +/- 11 microg/min(1)/mL(-1), P < .05). CONCLUSIONS: After TBI, intravenous ubiquitin crossed the blood-brain barrier and significantly reduced third spacing of fluid into the brain and lung during resuscitation.

Effects of exogenous ubiquitin in lethal endotoxemia.

BACKGROUND: Recent data indicated a potential role for extracellular ubiquitin in hematopoiesis and inflammation. The biological significance and therapeutic potential of these findings in vivo are unknown. Based on its in vitro abilities to inhibit endotoxin-stimulated tumor necrosis factor alpha (TNFalpha) production, we hypothesized that exogenous ubiquitin has salutary effects on sequelae caused by endotoxin in vivo. METHODS: Anesthetized and mechanically ventilated swine were infused with endotoxin for 3 hours. Ubiquitin was administered intravenously either 15 minutes before or 45 minutes after the endotoxin infusion was started. Albumin was administered to a control group. An additional control group received only ubiquitin. Ex vivo endotoxin evoked TNFalpha production was measured using a whole blood assay. Ubiquitin and TNFalpha concentrations were determined by enzyme-linked immunosorbent assay. RESULTS: Ubiquitin reduced mortality (P <.05), prevented development of pulmonary failure (P <.05), reduced fluid requirements (P <.05), and diminished erythema and edema formation. Ubiquitin pretreatment was more effective than treatment 45 minutes after an endotoxin infusion was started. In vivo ubiquitin administration alone inhibited ex vivo endotoxin-evoked TNFalpha secretion, but had no effect on TNFalpha serum levels after endotoxin infusion. CONCLUSION: In vivo ubiquitin administration has salutary actions during lethal endotoxemia and inhibits ex vivo whole blood TNFalpha production upon endotoxin stimulation. The clinical appearance after ubiquitin treatment in endotoxemia indicates the endothelium as another potential target cell population for interactions with ubiquitin. A novel therapeutic approach to a broad variety of diseases, in which endotoxin triggers immune activation, is suggested.