In vitro and in vivo effects of insulin-producing cells generated by xeno-antigen free 3D culture with RCP piece.

To establish widespread cell therapy for type 1 diabetes mellitus, we aimed to develop an effective protocol for generating insulin-producing cells (IPCs) from adipose-derived stem cells (ADSCs). We established a 3D culture using a human recombinant peptide (RCP) petaloid µ-piece with xeno-antigen free reagents. Briefly, we employed our two-step protocol to differentiate ADSCs in 96-well dishes and cultured cells in xeno-antigen free reagents with 0.1 mg/mL RCP µ-piece for 7 days (step 1), followed by addition of histone deacetylase inhibitor for 14 days (step 2). Generated IPCs were strongly stained with dithizone, anti-insulin antibody at day 21, and microstructures resembling insulin secretory granules were detected by electron microscopy. Glucose stimulation index (maximum value, 4.9) and MAFA mRNA expression were significantly higher in 3D cultured cells compared with conventionally cultured cells (P < 0.01 and P < 0.05, respectively). The hyperglycaemic state of streptozotocin-induced diabetic nude mice converted to normoglycaemic state around 14 days after transplantation of 96 IPCs under kidney capsule or intra-mesentery. Histological evaluation revealed that insulin and C-peptide positive structures existed at day 120. Our established xeno-antigen free and RCP petaloid µ-piece 3D culture method for generating IPCs may be suitable for clinical application, due to the proven effectiveness in vitro and in vivo.

Choice of xenogenic-free expansion media significantly influences the myogenic differentiation potential of human bone marrow-derived mesenchymal stromal cells.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) have great potential for use in cell-based therapies for restoration of structure and function of many tissue types including smooth muscle. METHODS: We compared proliferation, immunophenotype, differentiation capability and gene expression of bone marrow-derived MSCs expanded in different media containing human serum, plasma and platelet lysate in combination with commonly used protocols for myogenic, osteogenic, chondrogenic and adipogenic differentiation. Moreover, we developed a xenogenic-free protocol for myogenic differentiation of MSCs. RESULTS: Expansion of MSCs in media complemented with serum, serum + platelet lysate or plasma + platelet lysate were multipotent because they differentiated toward four mesenchymal (myogenic, osteogenic, chondrogenic, adipogenic) lineages. Addition of platelet lysate to expansion media increased the proliferation of MSCs and their expression of CD146. Incubation of MSCs in medium containing human serum or plasma plus 5% human platelet lysate in combination with smooth muscle cell (SMC)-inducing growth factors TGFß1, PDGF and ascorbic acid induced high expression of ACTA2, TAGLN, CNN1 and/or MYH11 contractile SMC markers. Osteogenic, adipogenic and chondrogenic differentiations served as controls. DISCUSSION: Our study provides novel data on the myogenic differentiation potential of human MSCs toward the SMC lineage using different xenogenic-free cell culture expansion media in combination with distinct differentiation medium compositions. We show that the choice of expansion medium significantly influences the differentiation potential of human MSCs toward the smooth muscle cell, as well as osteogenic, adipogenic and chondrogenic lineages. These results can aid in designing studies using MSCs for tissue-specific therapeutic applications.

Strategies and implications for prime-boost vaccination to generate memory CD8 T cells.

Generating a large population of memory CD8 T cells is an appealing goal for vaccine design against a variety of human diseases. Indeed, experimental models have demonstrated that the overall number of memory CD8 T cells present at the time of infection correlates strongly with the ability to confer host protection against a range of different pathogens. Currently, the most conceivable approach to rapidly generate a large population of memory CD8 T cells is through the use of prime-boost vaccination. In addition, recent experimental findings have uncovered important principles that govern both the rate and magnitude of memory CD8 T cell formation. Thus, this has resulted in novel prime-boost vaccination strategies that could potentially be used in humans to generate protective populations of memory CD8 T cells.

In vivo neutralization of naturally existing antibodies against linear alpha(1,3)-galactosidic carbohydrate epitopes by multivalent antigen presentation: a solution for the first hurdle of pig-to-human xenotransplantation.

Pig-to-human xenotransplantation of islet cells or of vascularized organs would offer a welcome treatment alternative for the ever-increasing number of patients with end-stage organ failure who are waiting for a suitable allograph. The main hurdle are preexisting antibodies, most of which are specific for 'Linear-B', carbohydrate epitopes terminated by the unbranched Gal-alpha(1,3)Gal disaccharide. These antibodies are responsible for the 'hyper-acute rejection' of the xenograft by complement mediated hemorrhage. For depletion of such antibodies we have developed an artificial injectable antigen, a glycopolymer (GAS914) with a charge neutral poly-lysine backbone (degree of polymerization n = 1000) and 25% of its side chains coupled to Linear-B-trisaccharide. With an average molecular weight of 400 to 500 kD, presenting 250 trisaccharide epitopes per molecule, this multivalent array binds anti-alphaGal antibodies with at least three orders of magnitude higher avidity on a per-saccharide basis than the monomeric epitope. In vivo experiments with non-human primates documented that rather low doses--1 to 5 mg/kg of GAS914 injected i.v.--efficiently reduce the load of anti-Linear-B antibodies quickly by at least 80%. This treatment can be repeated without any sensitization to GAS914. Interestingly, although the antibody levels start raising 12 h after injection, they do not reach pretreatment levels. The polymer is degraded and excreted within hours, with a minute fraction remaining in lymphoid tissue of anti-alphaGal producing animals only, probably binding to and inhibiting antibody-producing B-cells. The results of pig-to-non-human primate xenotransplantations established GAS914 as a relevant therapeutic option for pig-to-human transplantations as well. The synthesis of GAS914 was successfully scaled up to kg amounts needed for first clinical studies. Key was the use of galactosyl transferases and UDP-galactose for the synthesis of the trisaccharide.

N-glycolylneuraminic acid xenoantigen contamination of human embryonic and mesenchymal stem cells is substantially reversible.

Human embryonic and mesenchymal stem cell therapies may offer significant benefit to a large number of patients. Recently, however, human embryonic stem cell lines cultured on mouse feeder cells were reported to be contaminated by the xeno-carbohydrate N-glycolylneuraminic acid (Neu5Gc) and considered potentially unfit for human therapy. To determine the extent of the problem of Neu5Gc contamination for the development of stem cell therapies, we investigated whether it also occurs in cells cultured on human feeder cells and in mesenchymal stem cells, what are the sources of contamination, and whether the contamination is reversible. We found that N-glycolylneuraminic acid was present in embryonic stem cells cultured on human feeder cells, correlating with the presence of Neu5Gc in components of the commercial serum replacement culture medium. Similar contamination occurred in mesenchymal stem cells cultured in the presence of fetal bovine serum. The results suggest that the Neu5Gc is present in both glycoprotein and lipid-linked glycans, as detected by mass spectrometric analysis and monoclonal antibody staining, respectively. Significantly, the contamination was largely reversible in the progeny of both cell types, suggesting that decontaminated cells may be derived from existing stem cell lines. Although major complications have not been reported in the clinical trials with mesenchymal stem cells exposed to fetal bovine serum, the immunogenic contamination may potentially be reflected in the viability and efficacy of the transplanted cells and thus bias the published results. Definition of safe culture conditions for stem cells is essential for future development of cellular therapies.

Estimation of interaction between human keratinocytes and xenogenic collagen in vitro.

This paper describes the interaction observed between human keratinocytes and xenogenic collagen in vitro modified by HCl. Human keratinocytes were cultivated for 3-10 days, on modified and control support. Their growth, morphology and interaction with support were analyzed. It was found that on both control and experimental (modified) collagen cells proliferated in a similar way. Within 3-10 days, the culture became multilayered and mature and differentiation of cells was visible. Using electron microscope elements of basal membrane interacting with support were seen. On modified support processes of cells penetrating the support are occasionally seen. By use of the immunofluorescent, cytochemical techniques was found the presence of: BP-180 (antigen), beta(4) integrin, laminin 5 and collagen IV, VII, VIIc. On the modified support the above listed elements appeared between 3 and 7 days of culture, whereas on the control between 7th and 10th days. On 10th day of culture, the presence of elements of basal membranes became less evident. Results give some hope for using xenogenic, modified collagen as support of keratinocytes culture in process of human skin engineering.

Xenogeneic DNA immunization in melanoma models for minimal residual disease.

INTRODUCTION: DNA immunization with xenogeneic genes encoding homologous antigens protects mice against tumor challenge with syngeneic melanoma in a lung metastasis model. The effect of xenogeneic human TRP-2 (hTRP2) DNA immunization on disease confined to an orthotopic site, the skin, and in a model of minimal residual disease that is relevant to a setting of adjuvant therapy for micrometastatic cancer is reported. METHODS: Immunization and tumor challenge with B16F10LM3 melanoma were performed in C57BL/6 mice and in mice genetically deficient in MHC class I or II molecules. A melanoma variant of B16 with a predilection for lung metastasis was selected and used to challenge C57BL/6 mice. Tumor challenge in the footpad with the B16 variant was followed by local tumor growth and lung metastasis. The tumor-bearing distal extremities were surgically resected and mice were randomized to receive hTRP2 DNA immunization or no treatment. Approximately 3-5 weeks after surgical resection, lungs were harvested and metastases counted. RESULTS: Xenogeneic DNA immunization with hTRP2 prevented tumor growth in the skin by a mechanism requiring CD4(+) and CD8(+) T cells but did not inhibit the growth of established tumors. Adjuvant immunization with hTRP2 DNA after resection significantly reduced lung metastases and decreased local recurrence rates after surgical resection. CONCLUSIONS: Xenogeneic DNA immunization with hTRP2 was effective in protecting mice from intradermal tumor challenge. Immunization prevented local recurrence and the development of metastases in a mouse model of minimal residual disease, supporting a role for DNA immunization against melanosomal antigens as an adjuvant to surgery in high-risk primary melanomas.

Evaluation of intradermal sensitivity test in patients submitted to heterologous serotherapy

This review deals with heterologous sera produced used in Brazil. The authors studied 64 patients. Of these, 35 had been attacked by domestic and wild animals and received antirabies serum; 20 had been bitten by venomous animals (snakes and scorpions) and were treated with specific antivenoms; and 9 had traumas and received antitetanic serum. All these patients were submitted to the intradermal sensitivity test before, and 30 days after receiving heterologous serotherapy. The following results obtained by the authors agree with those in literature: - The intradermal test using undiluted heterologous serum produced a more acute reaction than that using heterologous serum diluted 1:10; - The larger the volume of serum, the larger was the wheal directly after inoculation; - The antigenic concentration influenced the final local reaction; - The reading 30 minutes after inoculation was always higher than that 15 min after; - No systemic reaction was observed during the tests; - The use of promethazine as a prophylactic medication did not protect against the early reactions; - Tests performed 30 days after serotherapy showed a higher reactivity, probably due to sensitization; - The intradermal sensitivity test did not allow the authors to predict early reactions. After these observations, the authors do not recommend the intradermal sensitivity test for patients submitted to heterologous serotherapy. They, however, strongly recommend a careful observation during the infusion and clinical follow up in the first 24 hours.

[A comparative study of the nonhistone chromosomal proteins (pp23) from rat and bull kidneys]. / Sravnitel'noe izuchenie khromosomnykh negistonovykh belkov (pp23) iz pochek krysy i byka.

The bovine nonhistone protein pp23 differs from that of rat origin both immunologically (precipitation, specific interaction with monoclonal antibodies to rat pp23) and functionally (capability to stimulate expression of hetero-organic antigens on the membrane of intact hepatocytes cultured in suspension). These differences, however, are not recognized in the experiments on stimulation of DNA synthesis in Swiss 3T3 cell resting culture, when a more general cell function (proliferation) is dealt with, rather than a narrow specific one, such as synthesis of tumor-associated hetero-organic antigen of kidney origin.

Immunological memory in mice. 3. Memory to heterologous erythrocytes in both T cell and B cell populations and requirement for T cells in expression of B cell memory. Evidence using immunoglobulin allotype and mouse alloantigen theta markers with congenic mice.

Using anti-allotype sera and AKR anti thetaC3H sera, a requirement for two cell types has been demonstrated in the adoptive secondary response of mice to heterologous erythrocytes. The cell types have been designated B cells [precursors of plaque-forming cells (PFC)] and T cells (thymus-influenced cells, not providing precursors of detectable PFC). The in vivo indirect PFC response of spleen cells from primed mice is markedly reduced by in vitro treatment of the cells with a mixture of anti-theta serum and guinea pig serum (Anti theta + GPS). This B cell response is fully restored to control levels by thymus cells from normal mice which do not themselves provide precursors of indirect PFC. Thus memory is carried by the B cell lineage but the expression of this memory is dependent on the presence of a cell population which is sensitive to Anti theta + GPS and which is replaced functionally by unprimed T cells. When assayed for T cell activity, thoracic duct cells from specifically primed mice are better than cells from nonspecifically primed mice in restoring the B cell response of spleen cells from immunized mice. Moreover, the T cell activity of a reconstitutive cell population from primed mice is reduced by incubation with Anti theta + GPS. We conclude that memory to heterologous erythrocyte antigens is carried by the T cell lineage as well as the B cell lineage even though unprimed T cells are sufficient for expression of B cell memory.