Lactococcus lactis as an Interleukin Delivery System for Prophylaxis and Treatment of Inflammatory and Autoimmune Diseases.

Target delivery of therapeutic agents with anti-inflammatory properties using probiotics as delivery and recombinant protein expression vehicles is a promising approach for the prevention and treatment of many diseases, such as cancer and intestinal immune disorders. Lactococcus lactis, a Lactic Acid Bacteria (LAB) widely used in the dairy industry, is one of the most important microorganisms with GRAS status for human consumption, for which biotechnological tools have already been developed to express and deliver recombinant biomolecules with anti-inflammatory properties. Cytokines, for  example, are immune system communication molecules present at virtually all levels of the immune response. They are essential in cellular and humoral processes, such as hampering inflammation or adjuvating in the adaptive immune response, making them good candidates for therapeutic approaches. This review discusses the advances in the development of new therapies and prophylactic approaches using LAB to deliver/express cytokines for the treatment of inflammatory and autoimmune diseases in the future.

Biological characterization of the UW402, UW473, ONS-76 and DAOY pediatric medulloblastoma cell lines.

Medulloblastoma (MB) is the most common malignant brain tumor in children. Recent advances in molecular technologies allowed to classify MB in 4 major molecular subgroups: WNT, SHH, Group 3 and Group 4. In cancer research, cancer cell lines are important for examining and manipulating molecular and cellular process. However, it is important to know the characteristics of each cancer cell line prior to use, because there are some differences among them, even if they originate from the same cancer type. This study aimed to evaluate the similarities and differences among four human medulloblastoma cell lines, UW402, UW473, DAOY and ONS-76. The medulloblastoma cell lines were analyzed for (1) cell morphology, (2) immunophenotyping by flow cytometry for some specifics surface proteins, (3) expression level of adhesion molecules by RT-qPCR, (4) proliferative potential, (5) cell migration, and (6) in vivo tumorigenic potential. It was observed a relationship between cell growth and CDH1 (E-chaderin) adhesion molecule expression and all MB cell lines showed higher levels of CDH2 (N-chaderin) when compared to other adhesion molecule. ONS-76 showed higher gene expression of CDH5 (VE-chaderin) and higher percentage of CD144/VE-chaderin positive cells when compared to other MB cell lines. All MB cell lines showed low percentage of CD34, CD45, CD31, CD133 positive cells and high percentage of CD44, CD105, CD106 and CD29 positive cells. The DAOY cell line showed the highest migration potential, the ONS-76 cell line showed the highest proliferative potential and only DAOY and ONS-76 cell lines showed tumorigenic potential in vivo. MB cell lines showed functional and molecular differences among them, which it should be considered by the researchers in choosing the most suitable cellular model according to the study proposal.

Adipogenic differentiation of murine bone marrow mesenchymal stem cells induced by visible light via photo- induced biomodulation.

BACKGROUND: Bone marrow mesenchymal stem cells (BM-MSCs) are undifferentiated cells that can proliferate and differentiate into specialized cells for tissue self-repair. Low-level laser (LLL) can induce biomodulatory effects such as cellular proliferation, differentiation, and migration. We investigated the biomodulatory effects of the photoactive compound chloroaluminum phthalocyanine nanoemulsion (AlClPc/NE) on the adipogenic differentiation of BM-MSCs, when combined with LLL (AlClPc/NE-LLL). METHODS: The BM-MSCs used in this work were isolated from green fluorescent protein-positive (GFP+) C57BL6 mice. Cells were first treated with AlClPc/NE, a well-designed photoactive nano-drug and were then subjected to in vitro expansion, morphological and immunophenotypic characterization, and cellular cytotoxicity analysis. Subsequently, BM-MSCs were induced to differentiate into adipocytes by photo-induced biomodulation with AlClPc/NE-LLL. RESULTS: Our results showed that the isolated cell population was consistent with murine BM-MSCs. The cellular cytotoxicity analysis revealed that the optimal nanoemulsion dose to induce BM-MSC biomodulation was 5.0 µmol/L. Twenty-four hours following treatment with AlClPc/NE, BM-MSC were subjected to visible light irradiation of 20 mJ/cm2 at 670 nm. Six days after photo-induced biomodulation, cells maintained high GFP expression level, and expressed detectable mRNA levels of adipogenic genes (lipoprotein lipase and PPARγ); formation of lipid vacuoles was observed, and the cells did not show any tumorigenic potential in vivo. CONCLUSIONS: Our results indicated that photo-induced biomodulation via visible light using AlClPc/NE and LLL can induce adipogenic differentiation of murine BM-MSCs. Therefore, cell therapy with BM-MSCs and photo-induced biomodulation may contribute to the development of new therapeutic strategies that are faster and more effective than traditional methods to trigger MSC differentiation.

Functional analysis of HOXA10 and HOXB4 in human medulloblastoma cell lines.

Medulloblastoma (MB) is a malignant childhood brain tumor which at molecular level is classified into at least four major subtypes: WNT, SHH, group C and group D differing in response to treatment. Previous studies have associated changes in expression levels and activation of certain HOX genes with MB development. In the present study, we investigate the role of HOX genes in two attributes acquired by tumor cells: migration and proliferation potential, as well as, in vivo tumorigenic potential. We analyzed UW402, UW473, DAOY and ONS-76 human pediatric MB cell lines and cerebellum primary cultures. Two-color microarray-based gene expression analysis was used to identify differentially expressed HOX genes. Among the various HOX genes significantly overexpressed in DAOY and ONS-76 cell lines compared to UW402 and UW473 cell lines, HOXA10 and HOXB4 were selected for further analysis. The expression levels of these HOX genes were validated by real-time PCR. A mouse model was used to study the effect of the HOXA10 and HOXB4 genes on the in vivo tumorigenic potential and the in vitro proliferative and migration potential of MB cell lines. Our results show that the inhibition of HOXA10 in DAOY cell line led to increased in vitro cell migration while in vitro cell proliferation or in vivo tumorigenic potential were unaffected. We also observed that induced expression of HOXB4 in the UW473 cell line significantly reduced in vitro cell proliferation and migration capability of UW473 cells with no effect on the in vivo tumorigenicity. This suggests that HOXA10 plays a role in migration events and the HOXB4 gene is involved in proliferation and migration processes of medulloblastoma cells, however, it appears that these genes are not essential for the tumorigenic process of these cells.

The F309S mutation increases factor VIII secretion in human cell line

OBJECTIVES: The capacity of a human cell line to secrete recombinant factor VIII with a F309S point mutation was investigated, as was the effect of the addition of chemical chaperones (betaine and sodium-4-phenylbutyrate) on the secretion of factor VIII. METHODS: This work used a vector with a F309S mutation in the A1 domain to investigate FVIII production in the HEK 293 human cell line. Factor VIII activity was measured by chromogenic assay. Furthermore, the effects of chemical drugs on the culture were evaluated. RESULTS: The addition of the F309S mutation to a previously described FVIII variant increased FVIII secretion by 4.5 fold. Moreover, the addition of betaine or sodium-4-phenylbutyrate increased the secretion rate of FVIIIÄB proteins in HEK 293 cells, but the same effect was not seen for FVIIIÄB-F309S indicating that all the recombinant protein produced had been efficiently secreted. CONCLUSION: Bioengineering factor VIII expressed in human cells may lead to an efficient production of recombinant factor VIII and contribute toward low-cost coagulation factor replacement therapy for hemophilia A. FVIII-F309S produced in human cells can be effective in vivo.

The F309S mutation increases factor VIII secretion in human cell line.

OBJECTIVES: The capacity of a human cell line to secrete recombinant factor VIII with a F309S point mutation was investigated, as was the effect of the addition of chemical chaperones (betaine and sodium-4-phenylbutyrate) on the secretion of factor VIII. METHODS: This work used a vector with a F309S mutation in the A1 domain to investigate FVIII production in the HEK 293 human cell line. Factor VIII activity was measured by chromogenic assay. Furthermore, the effects of chemical drugs on the culture were evaluated. RESULTS: The addition of the F309S mutation to a previously described FVIII variant increased FVIII secretion by 4.5 fold. Moreover, the addition of betaine or sodium-4-phenylbutyrate increased the secretion rate of FVIIIΔB proteins in HEK 293 cells, but the same effect was not seen for FVIIIΔB-F309S indicating that all the recombinant protein produced had been efficiently secreted. CONCLUSION: Bioengineering factor VIII expressed in human cells may lead to an efficient production of recombinant factor VIII and contribute toward low-cost coagulation factor replacement therapy for hemophilia A. FVIII-F309S produced in human cells can be effective in vivo.

Significant differences in integration sites of Moloney murine leukemia virus/Moloney murine sarcoma virus retroviral vector carrying recombinant coagulation factor IX in two human cell lines.

Ligation-mediated-PCR was performed followed by the mapping of 177 and 150 integration sites from HepG2 and Hek293 transduced with chimera vector carrying recombinant human Factor IX (rhFIX) cDNA, respectively. The sequences were analyzed for chromosome preference, CpG, transcription start site (TSS), repetitive elements, fragile sites and target genes. In HepG2, rhFIX was had an increased preference for chromosomes 6 and 17; the median distance to the nearest CpG islands was 15,240 base pairs and 37 % of the integrations occurred in RefSeq genes. In Hek293, rhFIX had an increased preference for chromosome 5; the median distance to the nearest CpG islands was 209,100 base pairs and 74 % of the integrations occurred in RefSeq genes. The integrations in both cell lines were distant from the TSS. The integration patterns associated with this vector are different in each cell line.

Murine leukemia virus-derived retroviral vector has differential integration patterns in human cell lines used to produce recombinant factor VIII.

OBJECTIVE: Nowadays recombinant factor VIII is produced in murine cells including in Chinese hamster ovary (CHO) and baby hamster kidney cells (BHK). Previous studies, using the murine leukemia virus-derived retroviral vector pMFG-FVIII-P140K, modified two recombinant human cell lines, HepG2 and Hek293 to produce recombinant factor VIII. In order to characterize these cells, the present study aimed to analyze the integration pattern of retroviral vector pMFG-FVIII-P140K. METHODS: This study used ligation-mediated polymerase chain reaction to locate the site of viral vector integration by sequencing polymerase chain reaction products. The sequences were compared to genomic databases to characterize respective clones. RESULTS: The retroviral vector presented different and non-random profiles of integration between cells lines. A preference of integration for chromosomes 19, 17 and 11 was observed for HepG2FVIIIdB/P140K and chromosome 9 for Hek293FVIIIdB/P140K. In genomic regions such as CpG islands and transcription factor binding sites, there was no difference in the integration profiles for both cell lines. Integration in intronic regions of encoding protein genes (RefSeq genes) was also observed in both cell lines. Twenty percent of integrations occurred at fragile sites in the genome of the HepG2 cell line and 17% in Hek293. CONCLUSION: The results suggest that the cell type can affect the profile of chromosomal integration of the retroviral vector used; these differences may interfere in the level of expression of recombinant proteins.

Murine leukemia virus-derived retroviral vector has differential integration patterns in human cell lines used to produce recombinant factor VIII

OBJECTIVE: Nowadays recombinant factor VIII is produced in murine cells including in Chinese hamster ovary (CHO) and baby hamster kidney cells (BHK). Previous studies, using the murine leukemia virus-derived retroviral vector pMFG-FVIII-P140K, modified two recombinant human cell lines, HepG2 and Hek293 to produce recombinant factor VIII. In order to characterize these cells, the present study aimed to analyze the integration pattern of retroviral vector pMFG-FVIII-P140K. METHODS: This study used ligation-mediated polymerase chain reaction to locate the site of viral vector integration by sequencing polymerase chain reaction products. The sequences were compared to genomic databases to characterize respective clones. RESULTS: The retroviral vector presented different and non-random profiles of integration between cells lines. A preference of integration for chromosomes 19, 17 and 11 was observed for HepG2FVIIIdB/P140K and chromosome 9 for Hek293FVIIIdB/P140K. In genomic regions such as CpG islands and transcription factor binding sites, there was no difference in the integration profiles for both cell lines. Integration in intronic regions of encoding protein genes (RefSeq genes) was also observed in both cell lines. Twenty percent of integrations occurred at fragile sites in the genome of the HepG2 cell line and 17% in Hek293. CONCLUSION: The results suggest that the cell type can affect the profile of chromosomal integration of the retroviral vector used; these differences may interfere in the level of expression of recombinant proteins. .

Differential expression of AURKA and AURKB genes in bone marrow stromal mesenchymal cells of myelodysplastic syndrome: correlation with G-banding analysis and FISH.

It has been demonstrated that genomic alterations of cells in the hematopoietic microenvironment could induce myelodysplastic syndromes (MDS) with ineffective hematopoiesis and dysmorphic hematopoietic cells, and subsequent transformation to acute myeloid leukemia. This investigation is the first attempt to correlate the gene expression profile of AURKA and AURKB in a cytogenetically stratified population of mesenchymal stem cells (MSCs) from MDS patients. We found that AURKA messenger RNA was expressed at significantly higher levels in MSCs even with normal/altered karyotype when compared with hematopoietic cells and healthy donors. In addition, we found that the presence of chromosomal abnormalities (mainly aneuploidy) in hematopoietic cells/MSCs was also associated with higher levels of AURKA. Different from previous investigations, our findings, regarding AURKA expression support the hypothesis that the presence of chromosomal abnormalities in MSCs from MDS is not a consequence of the method used for chromosome preparation. They may reflect the genomic instability present in the bone marrow microenvironment of MDS patients. This information is also supported by differences observed in the growth kinetics between MSCs from healthy donors (normal karyotype) and from MDS patients with abnormal karyotype. In summary, our results may not be considered evidence that MDS and MSCs are originated from a single neoplastic clone. In fact, both cells (hematopoietic and MSCs) may probably be altered in response to damage-inducing factors, and the presence of genomic abnormalities in MSCs suggests that an unstable bone marrow microenvironment may facilitate the expansion of MDS/leukemic cells.

SK-HEP cells and lentiviral vector for production of human recombinant factor VIII.

Hemophilia A is caused by a deficiency in coagulation factor VIII. Recombinant factor VIII can be used as an alternative although it is unavailable for most patients. Here, we describe the production of a human recombinant B-domain-deleted FVIII (rBDDFVIII) by the human cell line SK-HEP-1, modified by a lentiviral vector rBDDFVIII was produced by recombinant SK-HEP cells (rSK-HEP) at 1.5-2.1 IU/10(6) in 24 h. The recombinant factor had increased in vitro stability when compared to commercial pdFVIII. The functionality of rBDDFVIII was shown by its biological activity and by tail-clip challenge in hemophilia A mice. The rSK-HEP cells grew in a scalable system and produced active rBDDFVIII, indicating that this platform production can be optimized to meet the commercial production scale needs.

Human hepatic stellate cell line (LX-2) exhibits characteristics of bone marrow-derived mesenchymal stem cells.

The LX-2 cell line has characteristics of hepatic stellate cells (HSCs), which are considered pericytes of the hepatic microcirculatory system. Recent studies have suggested that HSCs might have mesenchymal origin. We have performed an extensive characterization of the LX-2 cells and have compared their features with those of mesenchymal cells. Our data show that LX-2 cells have a phenotype resembling activated HSCs as well as bone marrow-derived mesenchymal stem cells (BM-MSCs). Our immunophenotypic analysis showed that LX-2 cells are positive for activated HSC markers (αSMA, GFAP, nestin and CD271) and classical mesenchymal makers (CD105, CD44, CD29, CD13, CD90, HLA class-I, CD73, CD49e, CD166 and CD146) but negative for the endothelial marker CD31 and endothelial progenitor cell marker CD133 as well as hematopoietic markers (CD45 and CD34). LX-2 cells also express the same transcripts found in immortalized and primary BM-MSCs (vimentin, annexin 5, collagen 1A, NG2 and CD140b), although at different levels. We show that LX-2 cells are capable to differentiate into multilineage mesenchymal cells in vitro and can stimulate new blood vessel formation in vivo. LX-2 cells appear not to possess tumorigenic potential. Thus, the LX-2 cell line behaves as a multipotent cell line with similarity to BM-MSCs. This line should be useful for further studies to elucidate liver regeneration mechanisms and be the foundation for development of hepatic cell-based therapies.

Association between Knops blood group polymorphisms and susceptibility to malaria in an endemic area of the Brazilian Amazon.

Complement receptor 1 (CR1) gene polymorphisms that are associated with Knops blood group antigens may influence the binding of Plasmodium parasites to erythrocytes, thereby affecting susceptibility to malaria. The aim of this study was to evaluate the genotype and allele and haplotype frequencies of single-nucleotide polymorphisms (SNPs) of Knops blood group antigens and examine their association with susceptibility to malaria in an endemic area of Brazil. One hundred and twenty-six individuals from the Brazilian Amazon were studied. The CR1-genomic fragment was amplified by PCR and six SNPs and haplotypes were identified after DNA sequence analysis. Allele and haplotype frequencies revealed that the Kn(b) allele and H8 haplotype were possibly associated with susceptibility to Plasmodium falciparum. The odds ratios were reasonably high, suggesting a potentially important association between two Knops blood antigens (Kn(b) and KAM(+)) that confer susceptibility to P. falciparum in individuals from the Brazilian Amazon.

Association between Knops blood group polymorphisms and susceptibility to malaria in an endemic area of the Brazilian Amazon

Complement receptor 1 (CR1) gene polymorphisms that are associated with Knops blood group antigens may influence the binding of Plasmodium parasites to erythrocytes, thereby affecting susceptibility to malaria. The aim of this study was to evaluate the genotype and allele and haplotype frequencies of single-nucleotide polymorphisms (SNPs) of Knops blood group antigens and examine their association with susceptibility to malaria in an endemic area of Brazil. One hundred and twenty-six individuals from the Brazilian Amazon were studied. The CR1-genomic fragment was amplified by PCR and six SNPs and haplotypes were identified after DNA sequence analysis. Allele and haplotype frequencies revealed that the Kn b allele and H8 haplotype were possibly associated with susceptibility to Plasmodium falciparum. The odds ratios were reasonably high, suggesting a potentially important association between two Knops blood antigens (Kn b and KAM+) that confer susceptibility to P. falciparum in individuals from the Brazilian Amazon.

Effects of high-dose chemotherapy on bone marrow multipotent mesenchymal stromal cells isolated from lymphoma patients.

OBJECTIVE: High-dose chemotherapy (HDCT) followed by autologous stem cell transplantation is a widely applied treatment for hematological and autoimmune diseases. Little is known about the effects of this therapy on multipotent mesenchymal stromal cells (MSCs). We aimed to characterize, morphologically and functionally, MSCs isolated from bone marrow aspirates of patients after HDCT. MATERIALS AND METHODS: We studied 12 consecutive lymphoma patients submitted to BEAM conditioning regimen followed by autologous stem cell transplantation 28 to 1836 days before the sample collection. Thirteen normal donors were used as control. MSCs were isolated by adherence to plastic and expanded ex vivo by culture in flasks containing alpha-minimum essential medium plus 15% fetal bovine serum. RESULTS: The cell population isolated showed a typical MSC morphology, immunophenotype, and differentiation capacity into adipogenic, osteogenic, and chondrogenic lineages. The MSCs obtained from patients with Hodgkin's disease and non-Hodgkin's lymphoma showed decreased fibroblastoid colony-forming unit count (p = 0.023) and increased doubling time (p = 0.031) related to the control group. The total cell expansion of MSCs from normal subjects was marginally superior to the patient group (p = 0.064). There were no differences in gene expression profile, MSCs plasticity, or hematopoiesis support capability between control and patient group. CONCLUSIONS: Results suggest that HDCT applied to lymphoma patients damaged MSCs, which was demonstrated by their reduced clonogenic potential, doubling time, and cell expansion rates when compared to controls.

Mechanisms involved in the therapeutic properties of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have been described as being able to give rise to several quite different mesenchymal cell phenotypes. However, the ability to differentiate is not the only characteristic that makes these cells attractive for therapeutic purposes. The secretion of a broad range of bioactive molecules by MSCs, such as growth factors, cytokines and chemokines, constitutes their most biologically significant role under injury conditions. Understanding this intricate secretory activity as well as the properties of MSCs in vivo is central to harnessing their clinical potential. Herein, we identify some of the molecules involved in the paracrine effects of MSCs with a perspective that these cells intrinsically belong to a perivascular niche in vivo, and discuss how this knowledge could be advantageously used in clinical applications.

The chimeric cytokine Hyper-IL-6 enhances the efficiency of lentiviral gene transfer in hepatocytes both in vitro and in vivo.

Lentiviral vectors have been used for gene transfer into the liver but their ability to efficiently transduce quiescent hepatocytes remains controversial. Lentivirus-mediated gene transfer is more efficient in cycling cells. We determine the effect of H-IL6 in the lentiviral transduction. The lentiviral vector was used to transduce HepG2 cells and mice liver cells, previously treated with H-IL6. The highest transduction level was observed in HepG2 cells treated with 30 ng/mL H-IL6 and in the mice that received 4 microg H-IL6. Our results suggest that H-IL6 is an inducer of lentiviral gene transfer into the liver cells without any toxicity.

Knops blood group haplotypes among distinct Brazilian populations.

BACKGROUND: The Knops blood group system consists of antigens encoded by exon 29 of complement receptor 1 (CR1) gene. To better elucidate the complexity of Knops group system, the frequency of six single-nucleotide polymorphisms (SNPs) in three Brazilian populations is determined. STUDY DESIGN AND METHODS: A total of 118 individuals descendant from Europe, Asia, and Africa were studied. The genomic fragment of CR1 was amplified by polymerase chain reaction, and the SNPs and haplotypes were determined after DNA sequence analysis. RESULTS: Among the six polymorphisms characterized, one of them was described for the first time. The analysis of allele frequency showed that these six SNPs did not differ between the European and Asian groups. The African group presented a higher frequency of alleles McC(b), Sl2, and KAM+. The six polymorphisms gave origin to 12 haplotypes that were defined for the first time. The haplotypes 1 (4646A, Kn(a), McC(a), Sl1, Sl4, KAM+), 2 (4646A, Kn(a), McC(a), Sl1, KAM-), and 3 (4646A, Kn(a), McC(a), Sl2, Sl4, KAM-) are the most frequent in all populations. The H2 presents similar frequency in all populations; however, whereas the H1 presented a higher prevalence in the European and Asian groups, in the African group H3 was present in a higher prevalence. CONCLUSIONS: In this study, a new SNP substituting serine for asparagine at amino acid 1540 was identified. Moreover 12 haplotypes were identified. The differences in haplotype frequencies strongly suggest that the H1 and H2 might be the ancestral one while the H3 may have originated in Africa and may have fixed there by positive selection.

[Cloning and transmembrane glycoprotein expression of the retrovirus HTLV-1 in mammals' cells]. / Clonagem e expressão da glicoproteína transmembrana do retrovírus HTLV-1 em células de mamíferos.

The retrovirus HTLV-1 is the etiological agent of the adult T-cell leukemia and HTLV-1 associated myelopathy/tropical spastic paraparesis. The proviral genome has 9,032 base pairs, showing regulatory and structural genes. The env gene encodes for the transmembrane glycoprotein gp 21. The development of methodologies for heterologous protein expression, as well as the acquisition of a cellular line that constituently expresses the recombinant, were the main goals of this work. The DNA fragment that encodes for gp 21 was amplified by nested-PCR and cloned into a pCR2.1-TOPO vector. After which, a sub-cloning was realized using the expressing vector pcDNA3.1+. The transfection of mammalian cells HEK 293 was performed transitorily and permanently. Production of the recombinant gp 21 was confirmed by flux cytometry experiments and the cell line producing protein will be used in immunogenicity assays.

Clonagem e expressão da glicoproteína transmembrana do retrovírus HTLV-1 em células de mamíferos / Cloning and transmembrane glycoprotein expression of the retrovirus HTLV-1 in mammals' cells

O retrovírus linfotrópico de células T humanas tipo 1 é o agente etiológico da leucemia das células T do adulto e da paraparesia espástica tropical/mielopatia associada ao HTLV-1. O genoma proviral é composto por 9.032 pares de bases, contendo genes estruturais e regulatórios. A glicoproteína transmembrana gp 21 é codificada pelo gene estrutural env. O desenvolvimento de metodologias para a expressão heteróloga de proteínas, assim como a obtencão de uma linhagem celular que expresse a gp 21 recombinante constitutivamente são os principais objetivos do trabalho. O fragmento codificante da gp 21 foi amplificado por Nested-PCR e clonado no vetor pCR2.1-TOPO. Posteriormente, foi realizada a subclonagem no vetor de expressão pcDNA3.1+. A transfeccão da linhagem celular de mamíferos HEK 293 foi realizada de maneira transitória e permanente. A producão da gp 21 recombinante foi confirmada por citometria de fluxo e a linhagem celular produtora será utilizada em ensaios de imunogenicidade.