Multicellular dynamics on structured surfaces: Stress concentration is a key to controlling complex microtissue morphology on engineered scaffolds.

Tissue engineers have utilised a variety of three-dimensional (3D) scaffolds for controlling multicellular dynamics and the resulting tissue microstructures. In particular, cutting-edge microfabrication technologies, such as 3D bioprinting, provide increasingly complex structures. However, unpredictable microtissue detachment from scaffolds, which ruins desired tissue structures, is becoming an evident problem. To overcome this issue, we elucidated the mechanism underlying collective cellular detachment by combining a new computational simulation method with quantitative tissue-culture experiments. We first quantified the stochastic processes of cellular detachment shown by vascular smooth muscle cells on model curved scaffolds and found that microtissue morphologies vary drastically depending on cell contractility, substrate curvature, and cell-substrate adhesion strength. To explore this mechanism, we developed a new particle-based model that explicitly describes stochastic processes of multicellular dynamics, such as adhesion, rupture, and large deformation of microtissues on structured surfaces. Computational simulations using the developed model successfully reproduced characteristic detachment processes observed in experiments. Crucially, simulations revealed that cellular contractility-induced stress is locally concentrated at the cell-substrate interface, subsequently inducing a catastrophic process of collective cellular detachment, which can be suppressed by modulating cell contractility, substrate curvature, and cell-substrate adhesion. These results show that the developed computational method is useful for predicting engineered tissue dynamics as a platform for prediction-guided scaffold design. STATEMENT OF SIGNIFICANCE: Microfabrication technologies aiming to control multicellular dynamics by engineering 3D scaffolds are attracting increasing attention for modelling in cell biology and regenerative medicine. However, obtaining microtissues with the desired 3D structures is made considerably more difficult by microtissue detachments from scaffolds. This study reveals a key mechanism behind this detachment by developing a novel computational method for simulating multicellular dynamics on designed scaffolds. This method enabled us to predict microtissue dynamics on structured surfaces, based on cell mechanics, substrate geometry, and cell-substrate interaction. This study provides a platform for the physics-based design of micro-engineered scaffolds and thus contributes to prediction-guided biomaterials design in the future.

Air-pressure-driven Separable Microdevice to Control the Anisotropic Curvature of Cell Culture Surface.

We report on a novel microdevice to tune the curvature of a cell-adhering surface by controlling the air-pressure and micro-slit. Human aortic smooth muscle cells were cultured on demi-cylindrical concaves formed on a microdevice. Their shape-adapting behavior could be tracked when the groove direction was changed to the orthogonal direction. This microdevice demonstrated live observation of cells responding to dynamic changes of the anisotropic curvature of the adhering surface and could serve as a new platform to pursue mechanobiology on curved surfaces.

[A case of relapsed neurosyphilis with progressive left hemiparesis].

A 52-year-old man presented with progressive dementia and left hemiparesis. He was treated for neurosyphilis at 44 years old in another hospital. An initial MRI revealed a widespread high-intensity area in the right temporal lobe on DWI. Findings on MRA were normal. He was treated initially with intravenous edaravone and glyceol, but neurological finding did not improved. Serological tests of serum and CSF demonstrated high titers of antibodies to Treponema pallidum. He was treated for relapsed neurosyphilis with daily penicillin G injections without improvement. Penicillin G was switched to erythromycin. After administration of erythromycin, neurological symptoms improved and MRI abnormality showed progression. This case could be considered as Lissauer form of general paresis because of left hemiparesis and MRI findings. Neurosyphilis should be considered in a case with revealing high density area in DWI.

[A Case of Laparoscopic Surgery Performed for Lynch Syndrome in a Patient Who Developed Cecal Cancer and Uterine Carcinosarcoma Synchronously].

A 51-year-old woman was referred to our hospitalfor treatment of endometrialcancer. She had 3 family members with colorectal cancer in the first degree. She was also diagnosed with advanced cecal cancer based on a preoperative examination. She underwent laparoscopic surgery, modified radical hysterectomy, and bilateral salpingo-oophorectomy for endometrial cancer, and ileocecal resection for cecal cancer simultaneously. Pathological examination of the uterine tumor revealed carcinosarcoma with carcinomatous and sarcomatous components. Since she fulfilled 4 of the revised Bethesda criteria, we suspected Lynch syndrome. Immunohistochemical analysis of mismatch repair proteins demonstrated the loss of MSH2/MSH6 expression in both cecalcancer and uterine carcinosarcoma tissues. Genetic testing by direct sequencing revealed a pathogenic germ line mutation of MSH2 in codon 2245 of exon 14, and she was definitively diagnosed with Lynch syndrome. Laparoscopic surgery is less invasive and would be useful for Lynch syndrome patients potentially requiring multiple surgeries or risk- reduction surgery.

Irreversible neurological defects in the lower extremities after haploidentical stem cell transplantation: possible association with nelarabine.

Severe peripheral neuropathy and myelopathy are rare complications after stem cell transplantation (SCT). In our institution, seven patients of precursor T lymphoblastic leukemia/lymphoma without the central nervous involvement who had been treated by nelarabine to control their diseases received SCT from HLA-haploidentical familial donor (HLA-haploidentical SCT) with the conditioning regimen including high-dose cytarabine (HDAC). Three of evaluable six patients developed irreversible paresthesia and muscle weakness in both lower extremities after neutrophil engraftment. The results of nerve conduction studies and short latency somatosensory evoked potentials suggested axonal neuropathy of both lower extremities in all three patients and myelopathy in two patients. Negative findings of PET-CT, and analyses of repeated cerebrospinal fluid samples and the bone marrow also indicated that tumor involvement was improbable. In all three patients, the symptoms worsened or persisted despite administration of corticosteroid and intravenous immunoglobulin. The high frequency of the neurological symptoms in our patients previously treated by nelarabine strongly suggested the association of the nelarabine use. Furthermore, the HLA-haploidentical SCT setting and the use of a potentially neurotoxic agent, HDAC might augment the neurotoxicity of nelarabine. It may be desirable that HLA-haploidentical SCT candidates avoid receiving nelarabine.

[A case of intravascular malignant lymphoma with initial progressive non-specific neurological symptoms].

A 79-year-old woman was admitted to a nearby hospital for seven days due to low-grade fever, loss of appetite and general fatigue. She was diagnosed with normal condition and discharged. She was admitted to our hospital one week later with disturbed consciousness. Laboratory findings upon admission revealed anemia, elevated alanine amino transferase, elevated total birirubin and thrombocytopenia. Abdominal CT demonstrated multiple low intensity lesions in the liver. Enhanced brain CT revealed multiple lesions with increased signal intensity lesions in the white matter and cortex. The value of soluble IL-2 receptor antibody was 16,000U/ml. Intravascular lymphoma was suspected because of brain CT finding and IL-2 receptor antibody titer. Methylprednisolone pulse therapy was started considering her age and general condition, but she was died thirteen days after admission. Postmorten examination revealed widespread intravascular aggregation of malignant lymphoma cells in the liver, spleen, bone marrow, bladder, ovary and stomach indicating a diagnosis of an Asian variant of intravascular large B cell lymphoma (AIVL). Neurological abnormalities are not usually associated with AIVL, but this patient had rare AIVL presenting with initial progressive nonspecific neurological symptoms.

Production of scFv-displaying BmNPV in silkworm larvae and its efficient purification.

Baculovirus-display technology utilizing the gp64 envelope protein has been developed. A simple and efficient process to separate the virus from the majority of the protein contaminants may be needed for the future demand of pure and functional baculovirus vectors ideal for vaccine- and gene-delivery applications. In the present study, using Bombyx mori (silkworm) larvae as a host, scFv (single-chain variable fragment)-surface displaying recombinant baculovirus production and its purification from silkworm larval haemolymph by SEC (size-exclusion chromatography) were demonstrated. The amounts of scFv were 4-8 µg/ml in the haemolymph. The scFv-gp64 fusion protein was confirmed to be incorporated into the cell membrane and the BmNPV (B. mori nucleopolyhedrovirus) surface by immunofluorescence microscopy and Western blotting. rBmNPV (recombinant BmNPV) was purified to higher purity by SEC using Sephacryl S-1000 column chromatography than by sucrose-density-gradient centrifugation. The recovery of purified rBmNPV was 22.2%, and the virus purity in the SEC fraction was increased 269-fold compared with its purity in haemolymph. Judging from the results of ELISA, approx. 0.9% of the total baculovirus-particle proteins were occupied by scFv on their surface. A BmNPV-based silkworm-larval system is suitable for large-scale production of baculovirus-surface-displayed proteins or peptides in comparison with a cell-culture system. The present study will be useful for future BmNPV-application studies for gene delivery and vaccine trials.

Enhanced gene expression in insect cells and silkworm larva by modified polyhedrin promoter using repeated Burst sequence and very late transcriptional factor-1.

The Burst of expression from polyhedrin (polh) promoter during very late phase of baculovirus infection requires a sequence located between TAAG and the translation initiation site, typically referred to as burst sequence (BS). The expression of polh promoter is stimulated by specifically binding of very late transcriptional factor 1 (VLF-1) to BS. In order to enhance the production of recombinant proteins the polh promoter was modified via a multiple BS bacmid system in which the number of BSs was increased. Compared to an expression from a normal polh promoter, ß-glucuronidase (GUS) activity in High Five insect cells was three times higher with a modified polh promoter containing two BSs. Using a modified polh promoter that contains nine BSs in silkworm expression system, ß1-3-N-acetylglucosaminyltransferase 2 (ß3GnT2) activity per larva was 6.8-fold higher than control. Furthermore, the co-expression of modified promoters along with VLF-1-enhanced ß3GnT activity. Thus, an increased optimal number of BS and its co-expression with VLF-1 leads to the production of higher level of gene expression in insect cells and silkworm larvae. This new modified promoter engineered in the current study is the strongest promoter for overexpressing foreign proteins in an eukaryotic cell and system, thus leading a progress in baculovirus-insect cell and silkworm biotechnology.

Improved secretion of molecular chaperone-assisted human IgG in silkworm, and no alterations in their N-linked glycan structures.

Human 29IJ6 IgG was expressed in silkworm using a Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid system. The mean amounts of 296IJ6 IgG produced in larval hemolymph and whole pupae were 30.1 microg/larva and 78.0 microg/pupa, respectively. The use of molecular chaperones including calreticulin (CRT), calnexin (CNX), and immunoglobulin heavy chain binding protein (BiP, GRP78) improved the production of 296IJ6 IgG secretion in the larvae fivefold. The total yield of recombinant 29IJ6 IgG was 239 microg/mL when coexpressed with CRT. However, the overexpression of molecular chaperones had negative effects on secretion. The N-linked glycans of secreted 296IJ6 IgG in silkworm hemolymph were dominated by paucimannose structures. Small amounts of GlcNAc residues linked to the Manalpha1,3 branch were detected. When molecular chaperones were coexpressed, the compositions of N-linked glycans in the IgG1 produced were unchanged compared with those produced without them. This suggests that N-glycosylation is controlled by a regulatory function in the Golgi apparatus even though the post-translational modification of 296IJ6 IgG was assisted by the coexpression of molecular chaperones. Therefore, if the glycosylation pathways that coexpress N-acetylglucosaminyltransferase, galactosyltransferase, and sialyltransferase could be improved, silkworm larvae might prove a useful system for producing human antibodies.

Comparison of the N-linked glycosylation of human beta1,3-N-acetylglucosaminyltransferase 2 expressed in insect cells and silkworm larvae.

N-Glycosylation of human beta1,3N-acetylglucosaminyltransferase 2 (beta3GnT2) is essential for its biological function. beta3GnT2 fused to GFP(uv) (GFP(uv)-beta3GnT2) was produced by non-virus expression systems in stably transformed insect cells and silkworm larvae using a recombinant BmNPV bacmid, and purified for analysis of N-glycosylation. The N-glycan structure of beta3GnT2 was identified by glycoamidase A digestion, labeling with 2-aminopyridine (PA), and HPLC mapping. The paucimannosidic N-glycan structure (73.2%) was predominant in stably transformed Trichoplusia ni cells. In contrast, N-glycan with Gal (21.3%) and GlcNAc (16.2%) terminal residues linked to Manalpha(1,3) branch were detected on beta3GnT2 expressed in silkworm larvae. The presence of terminal Gal and bisecting GlcNAc residues such as Galbeta1, 4GlcNAcbeta1, 2Manalpha1,3(GlcNAcbeta1,4)(Manalpha1,6)Manbeta1, 4GlcNAc is not typical structure for lepidopteran insect N-glycosylation. Although allergenic alpha1,3-fucose residues have been found in T. ni cells, only alpha1,6-fucose residues were attached to the beta3GnT2 glycan in silkworm larvae. Therefore, silkworm larvae might be a useful host for producing human glycoproteins.

Human single-chain antibody expression in the hemolymph and fat body of silkworm larvae and pupae using BmNPV bacmids.

Seven sets of recombinant expression vectors were constructed for the expression of the human anti-bovine serum albumin (BSA) single-chain Fv fragment (scFv) 13CG2 fused to the C terminus of GFP(uv) or bombyxin (bx) signal peptide in the hemolymph and fat body of silkworm larvae and pupae, using cysteine protease- (BmNPV-CP(-)), and cysteine protease- and chitinase-deficient (BmNPV-CP(-)Chi(-)) bacmids. When BmNPV-CP(-) or BmNPV-CP(-)Chi(-) bacmids were used, 16.9-18.9 mg/l (11.6-15.0 microg/larva) of scFv was expressed at 6 d.p.i., whereas wild-type BmNPV bacmid expressed only 4.4 mg/l, probably because of proteolytic degradation of the protein. The scFv yield in silkworm pupae was only 0.67-1.0 microg/pupa, which was 5.4% of that in the hemolymph of silkworm larvae. The bx signal peptide enabled the secretion of scFv into the hemolymph. Without the signal sequence, the fusion protein accumulated in the fat body and lost its biological function. The removal of GFP(uv) significantly increased the scFv yield in the hemolymph of silkworms to 188.4 mg/l (132.4 mg/larva), which was ten times higher than that of the fusion protein.

Human IgG1 expression in silkworm larval hemolymph using BmNPV bacmids and its N-linked glycan structure.

A Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid expressing heavy and light chains of human 29IJ6 IgG was constructed and used to secrete recombinant antibody into silkworm larval hemolymph. Fifth instar silkworm larvae were reared and injected into the dorsum of the larvae with recombinant cysteine protease- and chitinase-deficient BmNPV (BmNPV-CP(-)-Chi(-)) bacmid/29IJ6 IgG and harvested after approximately 6 days. The total yield of recombinant 29IJ6 IgG was 36 microg/larvae, which is equivalent to 8 mg/kg of larvae. The recombinant antibody was purified to homogeneity using a HiTrap rProtein A FF column with a purification yield of 83.1%. The purified protein was identified by Western blot and ELISA experiments. The N-linked glycan structure of the purified protein was determined by the HPLC mapping method. The N-glycans of the 29IJ6 IgG glycoprotein produced in, and secreted by the silkworm larvae were composed exclusively of two kinds of paucimannose-type oligosaccharides, Manalpha1-6Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc and Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc.

Hypertensive brainstem encephalopathy without parieto-occipital lesion--two case reports.

Two patients presented with malignant hypertension associated with encephalopathy predominantly manifesting as brainstem lesion. T(2)-weighted and fluid-attenuated inversion recovery magnetic resonance (MR) imaging revealed diffuse hyperintense areas in the pons and scattered lesions in the cerebellum, basal ganglia, and cerebral subcortex without parieto-occipital lesions. Diffusion-weighted MR imaging demonstrated these lesions as normal intensity, indicating vasogenic edema. These lesions resolved rapidly once hypertension was controlled. Review of clinical findings for 14 other patients with hypertensive brainstem encephalopathy without parieto-occipital lesions suggested that anterior circulation structures supplied by the carotid artery are frequently involved in such patients.

Macrophage colony-stimulating factor inhibits tumor necrosis factor production and prolongs skin graft survival.

BACKGROUND: Despite the availability of a variety of immunosuppressive agents, acute rejection and infection after organ transplantation remain serious problems. METHODS AND RESULTS: We examined the effect of macrophage colony-stimulating factor (M-CSF) on the production of tumor necrosis factor (TNF) in a Bacille de Calmette Guérin-lipopolysaccharide-challenged mouse model. Both serial and repeated injections of M-CSF inhibited TNF production in a dose-dependent manner. Electrophoretic mobility shift assay showed that M-CSF-induced inhibition of TNF production was a result of suppression of nuclear factor-kappaB. High-dose M-CSF significantly prolonged skin graft survival in mice with orthotopic transplantation compared with the control and low-dose M-CSF groups. The combined administration of low-dose M-CSF and cyclosporine also significantly prolonged graft survival compared with the control and low-dose single agent-treated groups. CONCLUSIONS: Our results indicate that M-CSF at a high dose is a potent inhibitor of cytokine production and can potentially be used as an immunosuppressive agent for allograft rejection.