The human G1m1 allotype associates with CD4+ T-cell responsiveness to a highly conserved IgG1 constant region peptide and confers an asparaginyl endopeptidase cleavage site.

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.

CD28-B7 interactions allow the induction of CD8+ cytotoxic T lymphocytes in the absence of exogenous help.

The activation requirements for the generation of CD8+ cytotoxic T cells (CTL) are poorly understood. Here we demonstrate that in the absence of exogenous help, a CD28-B7 interaction is necessary and sufficient for generation of class I major histocompatibility complex-specific CTL. Costimulation is required only during the inductive phase of the response, and not during the effector phase. Transfection of the CD28 counter receptor, B7, into nonstimulatory P815 cells confers the ability to elicit P815-specific CTL, and this response can be inhibited by anti-CD28 Fab or by the chimeric B7-binding protein CTLA4Ig. Anti-CD28 monoclonal antibody (mAb) can provide a costimulatory signal to CD8+ T cells when the costimulatory capacity of splenic stimulators is destroyed by chemical fixation. CD28-mediated signaling provokes the release of interleukin 2 (IL-2) from the CD8+ CTL precursors, as anti-CD28 mAb could be substituted for by the addition of IL-2, and an anti-IL-2 mAb can block the generation of anti-CD28-induced CTL. CD4+ cells are not involved in the costimulatory response in the systems examined. We conclude that CD8+ T cell activation requires two signals: an antigen-specific signal mediated by the T cell receptor, and an additional antigen nonspecific signal provided via a CD28-B7 interaction.

High-avidity human IgG kappa monoclonal antibodies from a novel strain of minilocus transgenic mice.

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.

Rapid fabrication of functionalised poly(dimethylsiloxane) microwells for cell aggregate formation.

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.

Correction: Rapid fabrication of functionalised poly(dimethylsiloxane) microwells for cell aggregate formation.

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

IGF-2 coated porous collagen microwells for the culture of pancreatic islets.

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.

Antibacterial properties of nitric oxide-releasing porous silicon nanoparticles.

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.

Peritoneal B-cell development depends on strain, radiation, and time.

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.

Small interfering RNA delivery by polyethylenimine-functionalised porous silicon nanoparticles.

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.

MHC restriction of T-cell proliferative responses in Xenopus.

The MHC restriction of Xenopus allogeneic MHC- and antigen-specific T-cell proliferative responses was assessed. Xenopus MHC-specific monoclonal antibodies that recognize class I and class II molecules were tested for inhibitory effects on the generation of secondary T-cell proliferative responses. Antigen-specific T-cell lines were inhibited by anti-class II but not anti-class I monoclonal antibodies. Secondary alloantigen-specific proliferative responses also demonstrated MHC class II restriction. Allogeneic MHC- and antigen-specific T-cell lines demonstrated differential sensitivity to anti-class II monoclonal antibodies directed at discrete class II epitopes. These results indicate that Xenopus T cells interact with antigen-presenting cells similarly to mammals, and directly confirm previous data indicating that MHC class II restriction of proliferative responses is present in amphibians.

Immune system activation associated with a naturally occurring infection in Xenopus laevis.

Immune system activation correlated with a naturally occurring infection has been found in the South African clawed frog Xenopus laevis. The microorganism thought to be the cause of this infection is coccobacilloid and approximately 1 micron in diameter. Since this microorganism does not grow on conventional bacterial media and it has been observed intracellularly, it may be an obligate intracellular bacterium. It has been found in Xenopus peripheral blood and in highly vascularized organs such as the spleen and liver. Splenomegaly is the only pathology thus far described for infected frogs; infection is not associated with increased morbidity or mortality. This infection has been found in all outbred frogs examined in shipments from one South African and three separate North American vendors, and has been transmitted to animals bred and raised in our laboratory. This infection has a profound effect on the immune system of Xenopus. Significant numbers of splenocytes from infected individuals exhibit morphology commonly associated with activated T lymphocytes. There is constitutive production of T-cell growth factor (TCGF) and both IgM and IgY. Freshly harvested splenocytes from infected animals proliferate in response to a TCGF-containing supernatant, indicating that they express receptors for TCGF, a trait exclusively exhibited by activated lymphocytes. These splenocytes also show an increase in the activation marker recognized by the monoclonal antibody FJ17.

Class switching in human immunoglobulin transgenic mice.

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.

Effect of magnesium deficiency and food restriction on the immune response in young mice.

Similar batches of five-week-old C57 Bl/6 mice were given either a magnesium-deficient diet (4mg Mg/100 g), or a control diet (40 mg/100 g). Control diet intake was either ad libitum or reduced. After immunization with SRBC (sheep red blood cells), the immune response was studied by estimating the number of spleen AFC (antibody-forming cells) capable of lysing SRBC, and by a cytoadhesion test to determine the number of RFC (rosette forming cells). Limitation of the control diet slowed the growth rate in mice. Whenever food intake was reduced from 4g/day to 2.9 or 2.6g/day, the AFC response intensified but the RFC response remained similar. Food limitation might therefore mainly affect immature IgM producing cells with a high dividing rate. Magnesium deficiency produced a drastic fall in the primary and secondary immune responses, as measured by the number of spleen AFC. The number RFC was also much lower in the spleen of deficient animals. Consequently, the spleen immune system is deeply affected by this deficiency.

Aromatic amines (serotonin and histamine) and magnesium deficiency in the rat.

Rats were made deficient by giving a 4 mg Mg/100 g diet. The control diet content was 40 mg Mg/100 g. Serotonin injected to magnesium deficient and control rats was metabolized at the same rate in both groups and the urinary derivatives were similar. Serotonin catabolism can occur in liver as shown by isolated hepatocyte technique. Hyperemia of the ears and dermatosis appeared in magnesium deficient rats, while histaminemia rised. This allergy-like crisis was delayed and milder, when the food intake was reduced. The evolution of histaminemia was studied in parallel with the different white blood cells, during three periods of magnesium deficiency. An important rise of total white blood cells, specially poly morphonuclear and eosinophil cells was observed. The peak for eosinophils occured before the histaminemia and basophils peaks. Basophils cells found only in magnesium deficient group were partly degranualted.

Testing for antibiotic residues in milk.

Milk from dairy farms in England and Wales has been tested regularly for antibiotic residues since 1965. The sensitivity of the test organism was 0.02 iu/ml penicillin or equivalent until the change to 0.01 iu/ml in January 1986. In 1984-85, 99.6 per cent of the 2,000,000 milk samples tested passed the test and there was an average of 695 failures per month. From 7500 on-farm investigations over the two years 1983-85 the most frequent reasons suggested by farmers for their test failures were not withholding milk for the full withdrawal period (19.3 to 16.5 per cent) and accidental transfer of milk (16.3 to 16.7 per cent). Lactating and dry cow intramammary antibiotic preparations were held responsible for rather over 50 per cent and 25 per cent respectively of the failures in both years.