Anionic Complex with Efficient Expression and Good Safety Profile for mRNA Delivery.

We previously found that a complex comprising plasmid DNA (pDNA), polyethylenimine (PEI), and γ-polyglutamic acid (γ-PGA) had high transgene efficiency without cytotoxicity in vitro and in vivo. However, messenger RNA (mRNA) remains an attractive alternative to pDNA. In this study, we developed a safe and effective delivery system for mRNA to prevent its degradation and efficiently deliver it into target cells. Various cationic and anionic complexes were produced containing PEI, γ-PGA, and an mRNA encoding firefly luciferase. Their physicochemical properties and cytotoxicities were analyzed and the in vitro and in vivo protein expression were determined. The cationic mRNA/PEI complex showed high in vitro protein expression with strong cytotoxicity. The anionic complex was constructed as mRNA/PEI8/γ-PGA12 complex with a theoretical charge ratio of 1:8:12 based on the phosphate groups of the mRNA, nitrogen groups of PEI, and carboxylate groups of γ-PGA. It was stable and showed high in vitro protein expression without cytotoxicity. After intravenous administration of mRNA/PEI8/γ-PGA12 complex to mice, high protein expression was observed in the spleen and liver and slight expression was observed in the lung over 24 h. Thus, the newly constructed mRNA/PEI8/γ-PGA12 complex provides a safe and effective strategy for the delivery of mRNA.

Excellent Islet Yields after 18-h Porcine Pancreas Preservation by Ductal Injection, Pancreas Preservation with MK Solution, Bottle Purification, and Islet Purification Using Iodixanol with UW Solution and Iodixanol with MK Solution.

Successful islet isolation is the key to successful islet transplantation. Our group recently modified the islet isolation protocol to include pancreatic ductal injection of the preservation solution, pancreas storage in modified extracellular-type trehalose-containing Kyoto (MK) solution, and use of an iodixanol-based purification solution and bottle purification. In this study, we applied these methods to porcine islet isolation after 18-h pancreas preservation and compared two solutions with different compositions in bottle purification. Islet yield before purification was 651,661 ± 157,719 islet equivalents (IE) and 5576 ± 1538 IE/g pancreas weight. An IU solution was made by adding iodixanol to University of Wisconsin solution and an IK solution was made by adding iodixanol to MK solution. The efficacy of the two solutions for islet isolation was compared. There were no significant differences between the two purification methods with regard to islet yield, survival rate, purity, score, or stimulation index. These results indicate that our isolation protocol produces efficient islet yields from prolonged cold-stored pancreas and that IU and IK solutions are equally useful for islet purification.

A Novel Preservation Solution Containing a JNK Inhibitory Peptide Efficiently Improves Islet Yield for Porcine Islet Isolation.

BACKGROUND: For islet transplantation, pancreas preservation in University of Wisconsin (UW) solution is associated with disadvantages, such as collagenase inhibition, resulting in poor islet yield and islets with poor viability. In this study, we evaluated a novel preservation solution, the extracellular-type c-Jun N-terminal kinase (JNK) inhibitor-containing (EJ) solution. METHODS: The EJ solution has high sodium-low potassium composition with low viscosity compared to UW solution. Moreover, EJ solution contains a recently developed JNK inhibitor from our laboratory. RESULTS: We first compared the performance of EJ solution with that of UW solution. Islet yield before and after purification was significantly higher in the EJ group than in the UW group. Second, we compared the performance of EJ solution with that of EJ solution without the JNK inhibitor (EJ-J solution). After pancreas preservation in EJ solution, JNK activity was maintained at a relatively low level during islet isolation. Islet yield before and after purification was significantly higher in the EJ group than in the EJ-J group. After islet transplantation into streptozotocin-induced diabetic mice, blood glucose levels reached the normoglycemic range in 61.5% and 7.7% of diabetic mice in the EJ and EJ-J groups, respectively. Moreover, EJ solution exhibited reduced inhibition of collagenase digestion compared with UW solution. CONCLUSIONS: Advantages of EJ solution over UW solution were inhibition of JNK activity and reduced collagenase inhibition. EJ solution may therefore be more suitable for islet isolation than UW solution.

Modified cell-permeable JNK inhibitors efficiently prevents islet apoptosis and improves the outcome of islet transplantation.

We previously reported that treatment with a JNK inhibitory peptide (11R-JNKI) prevents islet apoptosis and enhances the islet function in vivo. In the present study, we explored more efficient JNK inhibitors. The inhibition of the JNK activity by five types of deletion peptides in 11R-JNKI was investigated. One of the peptides, 8R-sJNKI(-9), significantly prevented JNK activation. At a concentration of 1 µM, 8R-sJNKI(-9) inhibited JNK activity similarly to 10 µM 11R-JNKI and the inhibition of the JNK activity by 10 µM 8R-sJNKI(-9) was significantly greater than that by 10 µM 11R-JNK. To evaluate the effects of 8R-sJNKI(-9), porcine islets were cultured with 1 µM of 8R-sJNKI(-9) or 8R-mutant sJNKI(-9) (8R-mJNKI(-9)). After 1 day of culture, the numbers of islets in the 8R-sJNKI(-9)-treated group was significantly higher than that in the 8R-mJNKI(-9)-treated group. After islet transplantation, the blood glucose levels reached the normoglycemic range in 58.3% of streptozotocin-induced diabetic mice in the 8R-sJNKI(-9) group and 0% of the mice in the 8R-mJNKI(-9)-treated group. These data suggest that 8R-sJNKI(-9) inhibits islet apoptosis and improves islet function.

Evaluation of Islet Purification Methods for Making a Continuous Density Gradient and Loading Tissue.

Islet purification is one of the most important steps of islet isolation for pancreatic islet transplantation. We previously reported that a purification method using large plastic bottles effectively achieved a high yield of islets from porcine pancreas. In this study, we evaluated the methods for making a continuous density gradient and loading tissue. One method involved loading digested tissue on top of a continuous density gradient (top loading). The other method involved mixing digested tissue with low-density solution and then making a continuous gradient (mixed loading). There were no significant differences between the 2 purification methods in terms of the islet yield, rate of viability or purity, score, or in the stimulation index after purification. Furthermore, there were no marked differences in the attainability or suitability of posttransplantation normoglycemia. Our study shows the equivalency of these 2 methods of islet purification.

A Comparison of Pancreatic Islet Purification using Iodixanol with University of Wisconsin Solution and with Na-Lactobionate and Histidine Solution.

Purification of pancreatic islets is an important step in islet isolation for islet transplantation. In this study, to investigate how a solution composed mainly of Na-lactobionate and histidine (HL) influences the purification of islets, iodixanol was added to a purified solution for porcine islet isolation. A solution (IU) made by adding iodixanol to University of Wisconsin solution and a solution (IHL) made by adding iodixanol to HL solution were used to evaluate the islet isolation performance. We noted no significant differences between the two purification methods with regard to the islet yield, survival rate or purity, score, or stimulation index. These results show that IHL solution is as useful as IU solution for islet purification.

Comparison of Tissue Loading Before and After the Creation of a Continuous Density Gradient in Porcine Islet Purification.

The purification step is one of the most important and difficult procedures in islet isolation for pancreatic islet transplantation. We previously reported that a purification method using large plastic bottles effectively achieved a high yield of islets from the porcine pancreas. In this study, we evaluated the impact of the timing of tissue loading on porcine islet purification using large plastic bottles. One method involved loading digested tissue after creating a continuous density gradient (tissue after gradient [TAG]). The other method involved loading digested tissue before creating a continuous density gradient (tissue before gradient [TBG]). There were no significant differences between TAG and TBG in terms of the islet yield, rates of viability and purity, score, and in the stimulation index after purification. Furthermore, there were no marked differences in the attainability or suitability of post-transplantation normoglycemia. Our study shows the equivalency of these two methods of islet purification.

Critical roles of a dendritic cell subset expressing a chemokine receptor, XCR1.

Dendritic cells (DCs) consist of various subsets that play crucial roles in linking innate and adaptive immunity. In the murine spleen, CD8α(+) DCs exhibit a propensity to ingest dying/dead cells, produce proinflammatory cytokines, and cross-present Ags to generate CD8(+) T cell responses. To track and ablate CD8α(+) DCs in vivo, we generated XCR1-venus and XCR1-DTRvenus mice, in which genes for a fluorescent protein, venus, and a fusion protein consisting of diphtheria toxin receptor and venus were knocked into the gene locus of a chemokine receptor, XCR1, which is highly expressed in CD8α(+) DCs. In both mice, venus(+) cells were detected in the majority of CD8α(+) DCs, but they were not detected in any other cells, including splenic macrophages. Venus(+)CD8α(+) DCs were superior to venus(-)CD8α(+) DCs with regard to their cytokine-producing ability in response to TLR stimuli. In other tissues, venus(+) cells were found primarily in lymph node (LN)-resident CD8α(+), LN migratory and peripheral CD103(+) DCs, which are closely related to splenic CD8α(+) DCs, although some thymic CD8α(-)CD11b(-) and LN CD103(-)CD11b(-) DCs were also venus(+). In response to dsRNAs, diphtheria toxin-treated XCR1-DTR mice showed impaired CD8(+) T cell responses, with retained cytokine and augmented CD4(+) T cell responses. Furthermore, Listeria monocytogenes infection and anti-L. monocytogenes CD8(+) T cell responses were defective in diphtheria toxin-treated XCR1-DTRvenus mice. Thus, XCR1-expressing DCs were required for dsRNA- or bacteria-induced CD8(+) T cell responses. XCR1-venus and XCR1-DTRvenus mice should be useful for elucidating the functions and behavior of XCR1-expressing DCs, including CD8α(+) and CD103(+) DCs, in lymphoid and peripheral tissues.

Cytokine secretion from human monocytes potentiated by P-selectin-mediated cell adhesion.

BACKGROUND/AIM: P-selectin is a carbohydrate-recognizing cell adhesion molecule expressed on activated platelets and endothelial cells. It plays a crucial role in the recruitment of leukocytes to inflammatory and hemorrhagic sites. Cell adhesion mediated by P-selectin induces leukocyte activation, such as the generation of reactive oxygen species and the expression of blood coagulation factors. We assessed how P-selectin-mediated cell adhesion affects cytokine secretion from monocytes. METHODS: Human peripheral blood monocytes were cultured in a plate that had been coated with P-selectin purified from human platelets, and cytokines released in the culture supernatant from monocytes were determined by ELISA. RESULTS: The secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-8, IL-12 and macrophage inflammatory protein-1ß increased 3- to 10-fold in response to P-selectin compared with unstimulated monocytes. We next examined the effects of cytokine treatment of monocytes on their susceptibility to P-selectin. The secretion of TNF-α from monocytes in response to P-selectin was increased when monocytes were preincubated with granulocyte/macrophage colony-stimulating factor, monocyte chemotactic protein-1 or interferon-γ (IFN-γ); IFN-γ was the most effective in potentiating TNF-α secretion from monocytes. CONCLUSION: These results suggest that the interaction of monocytes with P-selectin plays an important role not only in their trafficking but also in the regulation of cytokine production by these cells.

Conservation of a chemokine system, XCR1 and its ligand, XCL1, between human and mice.

Understanding dendritic cell (DC) subset functions should lead to the development of novel types of vaccine. Here we characterized expression of XC chemokine receptor 1 (XCR1) and its ligand, XCL1. Murine XCR1 was the only chemokine receptor selectively expressed in CD8alpha(+) conventional DCs. XCL1 was constitutively expressed in NK cells, which contribute to serum XCL1 levels. NK and CD8(+) T cells increased XCL1 production upon activation. These expression patterns were conserved in human blood cells, including the BDCA3(+) DC subset. Thus, in human and mice, certain DC subsets should be chemotactic towards NK or activated CD8(+) T cells through XCR1.

Staphylococcal superantigen-like protein 5 inhibits matrix metalloproteinase 9 from human neutrophils.

Staphylococcal superantigen-like proteins (SSLs) constitute a family of exoproteins exhibiting structural similarities to superantigens and enterotoxins but no superantigenic activity. In this article, we present evidence that SSL5 specifically binds to matrix metalloproteinase 9 (MMP-9) and inhibits its enzymatic activity. When human neutrophil cell lysate was applied to recombinant His-tagged SSL5 conjugated to Sepharose, the bound fraction gave a major band of approximately 100 kDa in SDS-polyacrylamide gel electrophoresis. This protein was identified as the proform of MMP-9 (proMMP-9) by peptide mass fingerprinting analysis. The recombinant SSL5-Sepharose also bound to proMMP-9 secreted by interleukin 8 (IL-8)-stimulated neutrophils and HT1080 fibrosarcoma cells. Surface plasmon resonance analysis revealed that recombinant SSL5 bound to proMMP-9 with rather high affinity (dissociation constant [K(D)] = 1.9 nM). Recombinant SSL5 was found to effectively inhibit MMP-9-catalyzed hydrolysis of gelatin and a synthetic fluorogenic peptide in a noncompetitive manner (K(i) = 0.097 nM), as assessed by zymography and the fluorescence quenching method. Finally, the transmigration of neutrophils across Matrigel basement membranes in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP) was suppressed by the presence of recombinant SSL5. We discuss possible roles that SSL5 may play in immune evasion of staphylococci by inhibiting MMP and interfering with leukocyte trafficking.

Staphylococcal superantigen-like protein 10 (SSL10) binds to human immunoglobulin G (IgG) and inhibits complement activation via the classical pathway.

Staphylococcal superantigen-like (SSL) proteins are a family of exoproteins that share structural similarity with staphylococcal superantigens but exhibit no superantigenic activity. It was previously reported that two members (SSL5 and SSL7) bound to serum components and cell adhesion molecules involved in host immune response; however, the other family members have not been functionally characterized. In this study, we attempted to isolate SSL10-binding proteins from human serum and found that recombinant His-tagged SSL10 bound two major polypeptides of approximately 50 and approximately 25 kDa after affinity purification and SDS-polyacrylamide gel electrophoresis. These polypeptides were identified as heavy and light chains of human IgG by peptide mass fingerprinting analysis. The specific interaction between recombinant SSL10 and human IgG was confirmed by far Western blot analysis using immobilized SSL10 and pull-down analysis using SSL10-conjugated Sepharose. Surface plasmon resonance analysis revealed that the dissociation equilibrium constant for the interaction between human IgG and recombinant SSL10 was estimated to be 220 nM. We also found that recombinant SSL10 inhibited the binding of complement component C1q to IgG-Sepharose and hemolysis of IgG-sensitized sheep erythrocytes via the classical complement activation pathway. These results suggest that SSL10 may play a role in the evasion of Staphylococcus aureus from the host immune system via interfering complement activation.

[A case of multiple sclerosis with bilateral useless hand syndrome as a main clinical feature].

A 41-year-old, right-handed man was admitted to our hospital on September 12, 2002, due to progressive clumsiness in both hands. The patient had been diagnosed as having multiple sclerosis three years prior to admission. He noticed difficulty in manipulating objects three months before admission. Cervical T2-weighted MRI showed a high signal intensity at the level of C3-4 which was enhanced on T1-weighted image with gadolinium. On admission, neurological examinations revealed impairment of dexterity, deep sensory disturbance, and astereognosis in both hands. The clumsiness of complex finger movements was predominant on the left side, and was exaggerated with the eyes closed in association with pseudoathetosis. After steroid therapy, his clumsy hands improved gradually. This type of clumsiness in multiple sclerosis had been described as useless hand syndrome by Oppenheim. In accordance with our case, useless hand syndrome has been reported to arise from high cervical (C2-4) lesions, mainly involving the posterior cord ipsilateral to the clumsy hand. Although the majority of reported cases with useless hand syndrome had other neurological complications, such as hemiparesis, tetraparesis, and truncal ataxia, our patient exhibited a pure form of useless hand syndrome. In addition, useless hand syndrome is usually unilateral, and bilateral useless hand syndrome is very rare. Clumsiness of fine finger movements with astereognosis in our patient is similar to numb clumsy hands or limb-kinetic apraxia due to cervical spondylosis or postcentral gyrus lesion, respectively. This indicates an important role of the high cervical posterior cord in conveying a kinesthetic sense necessary to guide fine finger movements. It should be kept in mind that high cervical lesions in multiple sclerosis causes clumsy hands mimicking limb-kinetic apraxia.

[A case of pure autonomic failure (PAF) with sleep apnea syndrome (SAS) and successful treatment of dysautonomia with nasal continuous positive airway pressure (CPAP)].

A 72-year-old man was admitted to our hospital due to dysuria and frequent syncope. The patient had been well until the age of 70 years, when he began with these symptoms and neurogenic bladder was diagnosed in the other hospital. On admission, neurological examinations revealed no abnormal findings except blepharoptosis, anisocoria and orthostatic hypotension. Frequent apnea was evident during sleep. Autonomic function tests showed mainly sympathetic postganglionic dysfunction. Brain magnetic resonance imaging showed lacunar infarctions without cerebello-pontine atrophy or abnormal signals of the basal ganglia. We diagnosed pure autonomic failure (PAF) with sleep apnea syndrome (SAS). After starting nasal continuous positive airway pressure (CPAP) for SAS, his sneezing and sleep apnea drastically improved. Interestingly, CPAP also decreased the severity of orthostatic hypotension and syncope. Ambulatory blood pressure monitoring (ABPM) showed remarkable improvement in diurnal fluctuation of blood pressure after CPAP therapy. Although SAS is frequently associated with Shy-Drager syndrome but not with PAF, patients with PAF had been reported to have degenerative changes in the central nervous system overlapping with Shy-Drager syndrome or Lewy body disease. This case raised the possibility that nasal CPAP may be useful for orthostatic hypotension as well as SAS in neurodegenerative diseases.

Anterior cerebral artery territory infarctions presenting with ascending tetraparesis.

We describe a patient with ascending tetraparesis following stroke. The patient presented initially with spastic paraparesis which acutely evolved to tetraparesis with abulia. Magnetic resonance imaging revealed acute infarctions in the bilateral medial frontal regions but not in the brainstem or spinal cord. Multiple infarctions in the anterior cerebral artery territory appeared to originate from artery to artery embolism. The present case provides distinct clinical features of anterior cerebral artery syndrome which mimic myelopathy or brainstem lesions.