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Correction for 'Rapid fabrication of functionalised poly(dimethylsiloxane) microwells for cell aggregate formation' by A. Forget et al., Biomater. Sci., 2017, 5, 828-836.

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Cell aggregates reproduce many features of the natural architecture of functional tissues, and have therefore become an important in vitro model of tissue function. In this study, we present an efficient and rapid method for the fabrication of site specific functionalised poly(dimethylsiloxane) (PDMS) microwell arrays that promote the formation of insulin-producing beta cell (MIN6) aggregates. Microwells were prepared using an ice templating technique whereby aqueous droplets were frozen on a surface and PDMS was cast on top to form a replica. By employing an aqueous alkali hydroxide solution, we demonstrate exclusive etching and functionalisation of the microwell inner surface, thereby allowing the selective absorption of biological factors within the microwells. Additionally, by manipulating surface wettability of the substrate through plasma polymer coating, the shape and profile of the microwells could be tailored. Microwells coated with antifouling Pluronic 123, bovine serum albumin, collagen type IV or insulin growth factor 2 were employed to investigate the formation and stability of MIN6 aggregates in microwells of different shapes. MIN6 aggregates formed with this technique retained insulin expression. These results demonstrate the potential of this platform for the rapid screening of biological factors influencing the formation and response of insulin-producing cell aggregates without the need for expensive micromachining techniques.

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Islet transplantation, the only curative therapy for type I diabetes, requires isolation of the graft in highly specialized facilities for its later dispatch to remote transplantation centres. During transport and culture, many valuable cells are lost due to several factors such as mechanical stress, islet aggregation and dissociation. Here, we evaluate a porous microwell array sheet made of natural collagen type I extracellular matrix (ECM) protein as a novel islet culture substrate. This culture platform can be coated with IGF-2, a growth factor favorable for islet survival, and allows segregation of the islets within the porous microwell sheet, preventing aggregation. This design shows promising results for improving human pancreatic islets viability and function during culture and could form a novel paradigm for the transport of islets between isolation and transplantation centres.

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In this study, the antibacterial efficacy of NO-releasing porous silicon nanoparticles (pSiNPs) is reported. NO-releasing pSiNPs were produced via the conjugation of S-nitrosothiol (SNO) and S-nitrosoglutathione (GSNO) donors to the nanoparticle surfaces. The release of the conjugated NO caused by the decomposition of the conjugated SNO and GSNO was boosted in the presence of ascorbic acid. The released NO was bactericidal to Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli), and eliminated bacterial growth within 2 h of incubation without compromising the viability of mammalian cells. These results demonstrate the advantages of NO-releasing pSiNPs for antibacterial applications, for example, in chronic wound treatment.

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In this study, thermally hydrocarbonised porous silicon nanoparticles (THCpSiNPs) capped with polyethylenimine (PEI) were fabricated, and their potential for small interfering RNA (siRNA) delivery was investigated in an in vitro glioblastoma model. PEI coating following siRNA loading enhanced the sustained release of siRNA, and suppressed burst release effects. The positively-charged surface improved the internalisation of the nanoparticles across the cell membrane. THCpSiNP-mediated siRNA delivery reduced mRNA expression of the MRP1 gene, linked to the resistence of glioblastoma to chemotherapy, by 63% and reduced MRP1-protein levels by 70%. MRP1 siRNA loaded nanoparticles did not induce cytotoxicity in glioblastoma cells, but markedly reduced cell proliferation. In summary, the results demonstrated that non-cytotoxic cationic THCpSiNPs are promising vehicles for therapeutic siRNA delivery.

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The human G1m1 allotype comprises two amino acids, D12 and L14, in the CH3 domain of IGHG1. Although the G1m1 allotype is prevalent in human populations, ~40% of Caucasiods are homozygous for the nG1m1 allotype corresponding to E12 and M14. Peptides derived from the G1m1 region were tested for their ability to induce CD4+ T-cell proliferative responses in vitro. A peptide immediately downstream from the G1m1 sequence was recognized by CD4+ T cells in a large percentage of donors (peptide CH315-29). CD4+ T-cell proliferative responses to CH315-29 were found at an increased frequency in nG1m1 homozygous donors. Homozygous nG1m1 donors possessing the HLA-DRB1*07 allele displayed the highest magnitudes of proliferation. CD4+ T cells from donors homozygous for nG1m1 proliferated to G1m1-carrying Fc-fragment proteins, whereas CD4+ T cells from G1m1 homozygous donors did not. The G1m1 sequence creates an enzymatic cleavage site for asparaginyl endopeptidase in vitro. Proteolytic activity at D12 may allow the presentation of the CH315-29 peptide, which in turn may result in the establishment of tolerance to this peptide in G1m1-positive donors. Homozygous nG1m1 patients may be more likely to develop CD4+ T-cell-mediated immune responses to therapeutic antibodies carrying the G1m1 allotype.

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During puberty, pregnancy, lactation and post-lactation, breast tissue undergoes extensive remodelling and the disruption of these events can lead to cancer. In vitro studies of mammary tissue and its malignant transformation regularly employ mammary epithelial cells cultivated on matrigel or floating collagen rafts. In these cultures, mammary epithelial cells assemble into three-dimensional structures resembling in vivo acini. We present a novel technique for generating functional mammary constructs without the use of matrix substitutes.

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Human CD4(+) T-cell epitopes were identified in interferon-beta (IFN-beta)-1b. A prominent peptide epitope region was found that induced a proliferative response in 16% of all donors tested. Responses corresponded to the presence of the HLA-DR2 haplotype. Responsive donors expressing the HLA-DQ6 allele showed an increased level of proliferation to the epitope as compared to peptide-responsive HLA-DQ6 negative donors. A similar result was found for HLA-DR15-expressing donors. PBMC from donors expressing HLA-DR15 were more likely to proliferate in response to IFN-beta in a whole-protein in vitro assay than donors who did not carry this haplotype. It is striking that the common DQ6 allele HLA-DQB1(*)0602 is found in linkage disequilibrium with HLA-DRB1(*)1501, and this combination defines the HLA genotype associated with the development of multiple sclerosis. The HLA association between a response to IFN-beta and MS might explain the prevalence of neutralizing antibody development, and may underlie the etiology of the disease.

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OBJECTIVES: To characterize the bilateral lower-extremity kinematics and kinetics associated with squatting exercise after anterior cruciate ligament (ACL) reconstruction. DESIGN: We evaluated bilaterally sagittal plane kinematics and kinetics of the ankle, knee, and hip joints during submaximal squatting exercise in rehabilitating patients after ACL reconstruction. Comparisons were performed between involved and noninvolved limbs, and regression models were created to examine the relations between the bilateral kinetic differences and time postsurgery. SETTING: A motion analysis laboratory. PARTICIPANTS: Eight adults (27.9+/-6.8y) with unilateral ACL reconstruction (postsurgical time, 30+/-12wk). INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Sagittal plane ankle, knee, and hip peak net moments of force, maximum joint excursion angles, and peak vertical ground reaction forces. RESULTS: Peak vertical ground reaction forces did not differ between limbs. The peak knee extensor moment generated during the submaximal squatting exercise was 25.5% greater in the noninvolved limb than in the involved limb (P=.003). The peak ankle plantarflexor moment did not differ between limbs (P=.85); however, there was a trend toward a greater hip extensor moment in the involved limb (P=.06). The ratio of the peak hip extensor moment to the peak knee extensor moment was 46.5% greater in the involved limb (P=.02). Only the peak dorsiflexion angle differed between limbs (P=.02). None of the linear models examining the relations between differences in the involved limb and noninvolved limb kinetics, and postsurgical time, were statistically significant. CONCLUSIONS: Patients performing the squat exercise, within 1 year of ACL reconstructive surgery, used 2 strategies for generating the joint torques required to perform the movement: (1) in the noninvolved limb, patients used a strategy that equally distributed the muscular effort between the hip and knee extensors, and (2) in the involved limb, patients used a strategy that increased the muscular effort at the hip and reduced the effort at the knee. These intra- and interlimb motor-programming alterations (ie, substitution strategies) could potentially slow or limit rehabilitation, and induce strength and performance deficits.

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OBJECTIVE: To investigate labour-associated changes in: 1. the myometrial contractile response to arginine vasopressin compared with oxytocin in vitro 2. fetal production of arginine vasopressin and 3. myometrial vasopressin V1a receptor mRNA. DESIGN: The contractile response to vasopressin (compared with oxytocin) was investigated in paired myometrial strips in vitro. Blood was taken from the umbilical artery and vein at delivery and arginine vasopressin measured by radio-immunoassay. V1a receptor mRNA was determined by in situ hybridisation. RESULTS: Myometrium was more sensitive to arginine vasopressin than oxytocin (P<0.05 for frequency, amplitude and activity integral in paired strips) after, but not before labour. There was a marked umbilical arteriovenous difference in arginine vasopressin concentration at delivery suggesting fetal production which was not influenced by labour. Myometrial vasopressin V1a receptor mRNA was not increased after the onset of labour. CONCLUSIONS: The human uterus is extremely sensitive to arginine vasopressin in vitro. Arginine vasopressin is produced by the fetus but fetal formation is not increased during labour.

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OBJECTIVE: B-1a, B-1b, and B-2 cells represent the three B-cell subsets in mice. Previous studies have demonstrated that peritoneal B-1a cell development is absent, or nearly so, from adult bone marrow transfers into irradiated adult hosts. The majority of these studies have been performed under a limited set of conditions with irradiated host mice. Here we examined that under a variety of conditions, peritoneal B-1a cells can develop in significant numbers from adult bone marrow transfers into severe combined immunodeficient (SCID) and recombination activation gene 2(-) (RAG-2(-)) mice. MATERIALS AND METHODS: Adult bone marrow was transferred into various strains of irradiated and nonirradiated adult immunodeficient RAG-2(-) and SCID mice. Peritoneal B-cell engraftment was examined by fluorescein-activated cell sorting analysis and unpaired t-tests were used to determine significant differences of B-cell engraftment among the various conditions of cell transfer. RESULTS: The level of B-1a cell engraftment was variously affected by the type of host immunodeficiency, the combination of donor and host strains, and the time allowed for engraftment. Irradiation of SCID, but not RAG-2(-), host mice inhibited B-1a-cell engraftment. Additionally, decreasing the number of bone marrow progenitor cells transferred was not found to preferentially affect B-1a cell development in irradiated RAG-2(-) hosts. CONCLUSION: In the context of these strains, we conclude that adult murine bone marrow contains progenitors that have the capacity to reconstitute peritoneal B-1a cell populations to donor levels.

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The engineering of protein therapeutics to improve their stability, their efficacy, or to create "humanized" versions introduces changes to the amino acid sequence that are potential T-cell epitopes. Until now, there has been no available assay to detect primary T-cell responses to novel epitopes in humans. Currently available in vitro protocols for epitope determination rely on peripheral blood lymphocytes from environmentally exposed or disease-bearing donors. This severely limits the opportunity to confirm T-cell epitopes in novel proteins, because exposed donors are not available to novel or engineered proteins. Other methods for determining T-cell epitopes are either computer-modeled predictions based on potential binding to HLA molecules or the identification of peptides presented by HLA molecules removed from the surface of tumor cells or protein-pulsed antigen-presenting cells. Because HLA binding is necessary, but not sufficient, for T-cell responses, these methods must be validated by in vitro presentation assays. The authors describe a dendritic cell-based assay that identifies CD4+ T-cell epitopes in novel proteins using unexposed donors. Predicted T-cell epitopes in the protein of interest were confirmed using cells from two verified exposed donors. The major CD4+ T-cell epitope of the novel protein examined in this study associated with the expression of HLA DRb1*15. This assay reflects de novo priming in vitro, and it accurately identifies primary T-cell epitopes. This assay is a powerful tool for determining relevant immunostimulatory T-cell epitopes for all types of immunoregulatory applications.

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We present fundamental studies examining the design of a phase/Doppler laser light-scattering system applicable to on-line measurements of small-diameter (<15 mum) fibers during fiberglass manufacturing. We first discuss off-line diameter measurement techniques currently used in the fiberglass industry and outline the limitations and problems associated with these methods. For the phase/Doppler design study we have developed a theoretical computer model for the response of the measurement system to cylindrical fibers, which is based on electromagnetic scattering theory. The model, valid for arbitrary fiber diameters and hardware configurations, generates simulated detector output as a function of time for a finite absorbing, cylindrical fiber oriented perpendicular to the two incident laser beams. Results of experimental measurements are presented, confirming predictions of the theoretical model. Parametric studies have also been conducted using the computer model to identify experimental arrangements that provide linear phase-diameter relationships for small-diameter fibers, within the measurement constraints imposed by the fiberglass production environment. The effect of variations in optical properties of the glass as well as fiber orientation effects are discussed. Through this research we have identified phase/Doppler arrangements that we expect to have future applications in the fiberglass industry for on-line diameter monitoring and process control.

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Human immunoglobulin transgenic mice provide a method of obtaining human monoclonal antibodies (Mabs) using conventional hybridoma technology. We describe a novel strain of human immunoglobulin transgenic mice and the use of this strain to generate multiple high-avidity human sequence IgG kappa Mabs directed against a human antigen. The light chain transgene is derived in part from a yeast artificial chromosome clone that includes nearly half of the germline human V kappa region. In addition, the heavy-chain transgene encodes both human mu and human gamma 1 constant regions, the latter of which is expressed via intratransgene class switching. We have used these animals to isolate human IgG kappa Mabs that are specific for the human T-cell marker CD4, have high binding avidities, and are immunosuppressive in vitro. The human Mab-secreting hybridomas display properties similar to those of wild-type mice including stability, growth, and secretion levels. Mabs with four distinct specificities were derived from a single transgenic mouse, consistent with an extensive diversity in the primary repertoire encoded by the transgenes.

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We have introduced human germline-configuration heavy and kappa light chain minilocus transgenes into mice that have been engineered so that their endogenous heavy and kappa light chain loci are inactive. The two human transgenes are inserted by pronuclear microinjection, while the two endogenous mouse genes are disrupted by homologous recombination in embryonic stem cells. The resulting animals contain four unlinked genetic modifications and must rely on the introduced transgenes for the development of the B-cell lineage and for the generation of an antibody repertoire. The heavy chain transgene includes both the human mu and the human gamma 1 constant region gene segments, as well as upstream switch region sequences. Although mouse B cells and human B cells exhibit species-specific differences in the induction of gamma isotype expression, the transgenic mouse B cells appear to undergo regulated switching to human gamma 1 both in vivo and in vitro. This observation defines a subset of the heavy chain constant region that is sufficient for class switching, and implies that the human gamma 1 switch region includes a core of sequence that is functionally homologous to those cis-acting regulatory elements that direct mouse gamma switching.

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Human sequence monoclonal antibodies, which in theory combine high specificity with low immunogenicity, represent a class of potential therapeutic agents. But nearly 20 years after Köhler and Milstein first developed methods for obtaining mouse antibodies, no comparable technology exists for reliably obtaining high-affinity human antibodies directed against selected targets. Thus, rodent antibodies, and in vitro modified derivatives of rodent antibodies, are still being used and tested in the clinic. The rodent system has certain clear advantages; mice are easy to immunize, are not tolerant to most human antigens, and their B cells form stable hybridoma cell lines. To exploit these advantages, we have developed transgenic mice that express human IgM, IgG and Ig kappa in the absence of mouse IgM or Ig kappa. We report here that these mice contain human sequence transgenes that undergo V(D)J joining, heavy-chain class switching, and somatic mutation to generate a repertoire of human sequence immunoglobulins. They are also homozygous for targeted mutations that disrupt V(D)J rearrangement at the endogenous heavy- and kappa light-chain loci. We have immunized the mice with human proteins and isolated hybridomas secreting human IgG kappa antigen-specific antibodies.

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