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Equivalent Gene Expression Profiles between Glatopa™ and Copaxone®.

D'Alessandro JS, Duffner J, Pradines J, Capila I, Garofalo K, Kaundinya G, Greenberg BM, Kantor D, Ganguly TC - PLoS ONE (2015)

Bottom Line: Comparative analysis was also performed on sets of transcripts relevant to T-cell biology and antigen presentation, among others that are known to be modulated by glatiramer acetate.No statistically significant differences were observed between Copaxone and Glatopa in the expression levels (magnitude and direction) of these glatiramer acetate-regulated genes.In conclusion, multiple methods consistently supported equivalent gene expression profiles between Copaxone and Glatopa.

View Article: PubMed Central - PubMed

Affiliation: Momenta Pharmaceuticals, Inc., Cambridge, MA, United States of America.

ABSTRACT
Glatopa™ is a generic glatiramer acetate recently approved for the treatment of patients with relapsing forms of multiple sclerosis. Gene expression profiling was performed as a means to evaluate equivalence of Glatopa and Copaxone®. Microarray analysis containing 39,429 unique probes across the entire genome was performed in murine glatiramer acetate--responsive Th2-polarized T cells, a test system highly relevant to the biology of glatiramer acetate. A closely related but nonequivalent glatiramoid molecule was used as a control to establish assay sensitivity. Multiple probe-level (Student's t-test) and sample-level (principal component analysis, multidimensional scaling, and hierarchical clustering) statistical analyses were utilized to look for differences in gene expression induced by the test articles. The analyses were conducted across all genes measured, as well as across a subset of genes that were shown to be modulated by Copaxone. The following observations were made across multiple statistical analyses: the expression of numerous genes was significantly changed by treatment with Copaxone when compared against media-only control; gene expression profiles induced by Copaxone and Glatopa were not significantly different; and gene expression profiles induced by Copaxone and the nonequivalent glatiramoid were significantly different, underscoring the sensitivity of the test system and the multiple analysis methods. Comparative analysis was also performed on sets of transcripts relevant to T-cell biology and antigen presentation, among others that are known to be modulated by glatiramer acetate. No statistically significant differences were observed between Copaxone and Glatopa in the expression levels (magnitude and direction) of these glatiramer acetate-regulated genes. In conclusion, multiple methods consistently supported equivalent gene expression profiles between Copaxone and Glatopa.

No MeSH data available.


Related in: MedlinePlus

T-helper cell pathway diagram.Transcripts measured in the current study are shown in the diagram as nodes and are colored based on the differences observed when cells are stimulated with Copaxone in comparison with cell culture media alone (P < 1e-3 [Student’s t-test]; red for increase, green for decrease). Molecules with P > 1e-3 are shown in gray. Genes refer to the human ortholog. Human HLA-DMA, HLA-DMB, and HLA-DQB1 represent murine H2-Dma, H2-Dmb2, and H2-Ab1, respectively. Blue arrows show the flow of activation by major Th1- and Th2-influencing molecules in the pathway. For example, APCs produce IL-4, which binds to and activates the IL-4 receptor, leading to the phosphorylation and activation of the transcription factor STAT6. Red and green arrows show the expected transcriptional outcomes of Th1 and Th2 polarization; red and green arrows indicate that activation will cause the transcript to increase and decrease, respectively. Expected transcriptional outcomes are based on reports in the literature on Th1/Th2 T-cell polarization and on studies conducted with GA [3,4,8,10,17,29–34]. Gray lines indicate members of a group. APCs, antigen-presenting cells; CXCR1, chemokine (C-X-C motif) receptor 1; CXCR3, chemokine (C-X-C motif) receptor 3; CXCR5, chemokine (C-X-C motif) receptor 5; CXCL10, chemokine (C-X-C motif) ligand 10; GA, glatiramer acetate; HLADMA, major histocompatibility complex class II, DM alpha; HLADMB, major histocompatibility complex class II, DM beta; HLADQB1, major histocompatibility complex class II, DQ beta 1; ICOSLG, inducible T-cell costimulatory ligand; IL-4, interleukin-4; MHC class II, major histocompatibility complex class II; LTB, lymphotoxin beta (tumor necrosis factor superfamily, member 3); MYD, myeloid differentiation primary response protein; S100A10, S100 calcium-binding protein A10; STAT, signal transducer and activator of transcription; TCR, T-cell receptor; Th, T-helper.
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pone.0140299.g006: T-helper cell pathway diagram.Transcripts measured in the current study are shown in the diagram as nodes and are colored based on the differences observed when cells are stimulated with Copaxone in comparison with cell culture media alone (P < 1e-3 [Student’s t-test]; red for increase, green for decrease). Molecules with P > 1e-3 are shown in gray. Genes refer to the human ortholog. Human HLA-DMA, HLA-DMB, and HLA-DQB1 represent murine H2-Dma, H2-Dmb2, and H2-Ab1, respectively. Blue arrows show the flow of activation by major Th1- and Th2-influencing molecules in the pathway. For example, APCs produce IL-4, which binds to and activates the IL-4 receptor, leading to the phosphorylation and activation of the transcription factor STAT6. Red and green arrows show the expected transcriptional outcomes of Th1 and Th2 polarization; red and green arrows indicate that activation will cause the transcript to increase and decrease, respectively. Expected transcriptional outcomes are based on reports in the literature on Th1/Th2 T-cell polarization and on studies conducted with GA [3,4,8,10,17,29–34]. Gray lines indicate members of a group. APCs, antigen-presenting cells; CXCR1, chemokine (C-X-C motif) receptor 1; CXCR3, chemokine (C-X-C motif) receptor 3; CXCR5, chemokine (C-X-C motif) receptor 5; CXCL10, chemokine (C-X-C motif) ligand 10; GA, glatiramer acetate; HLADMA, major histocompatibility complex class II, DM alpha; HLADMB, major histocompatibility complex class II, DM beta; HLADQB1, major histocompatibility complex class II, DQ beta 1; ICOSLG, inducible T-cell costimulatory ligand; IL-4, interleukin-4; MHC class II, major histocompatibility complex class II; LTB, lymphotoxin beta (tumor necrosis factor superfamily, member 3); MYD, myeloid differentiation primary response protein; S100A10, S100 calcium-binding protein A10; STAT, signal transducer and activator of transcription; TCR, T-cell receptor; Th, T-helper.

Mentions: T-helper cell differentiation can be induced by T-cell receptor stimulation by APCs. In our study, APCs (naive splenocytes) were mixed with T cells derived from GA-immunized mouse lymph nodes to provide T-cell receptor stimulation. APCs process the GA copolymer, load major histocompatibility complex II (MHC II) receptors with peptide fragments, and present these peptides to T cells. In the context of T-cell stimulation and differentiation, several cytokines can induce T-cell polarization toward a Th2 or a Th1 phenotype. Fig 6 shows a pathway diagram of Th1 and Th2 T-cell polarization, created using literature surrounding T-helper cell differentiation and studies with GA stimulation of T-cell populations.[3,4,8,10,17,29–34]. T-helper cells differentiate toward a Th2 phenotype and produce a Th2 response in the presence of the cytokine IL-4 and T-cell receptor stimulation by MHC II molecules. Th2 cells themselves also produce IL-4. In our study, we observed increased IL-4 expression when cells were stimulated with GA. IL-4 signals through its cognate IL-4 receptor, activating the JAK/STAT pathway, specifically STAT6. STAT6 is regulated by phosphorylation in the cytoplasm, then translocates to the nucleus to induce activation of another transcription factor, GATA3. Consistent with posttranslational regulation by the phosphorylation of STAT6 and GATA3, no changes were observed in gene expression of these signaling molecules. These transcription factors, however, can induce and inhibit an array of genes further downstream of the signaling cascade. As expected, STAT6/GATA3 induced increases in expression of the Th2 cytokines IL-3, IL-4, IL-5, and IL-13; the MHC II family genes; and IL-4R. Similarly, decreases were observed in the expression of other STAT6/GATA3-regulated genes, such as S100A10, ICOSLG, and LTB. This subset of genes can be considered GA treatment-specific markers for the Th2-polarized T-cell test system, and our observation of changes in the gene expression of these markers is consistent with the literature regarding Th2 cells and the purported mechanisms of GA on immune cells [3,4,7,8,10,17,29–34].


Equivalent Gene Expression Profiles between Glatopa™ and Copaxone®.

D'Alessandro JS, Duffner J, Pradines J, Capila I, Garofalo K, Kaundinya G, Greenberg BM, Kantor D, Ganguly TC - PLoS ONE (2015)

T-helper cell pathway diagram.Transcripts measured in the current study are shown in the diagram as nodes and are colored based on the differences observed when cells are stimulated with Copaxone in comparison with cell culture media alone (P < 1e-3 [Student’s t-test]; red for increase, green for decrease). Molecules with P > 1e-3 are shown in gray. Genes refer to the human ortholog. Human HLA-DMA, HLA-DMB, and HLA-DQB1 represent murine H2-Dma, H2-Dmb2, and H2-Ab1, respectively. Blue arrows show the flow of activation by major Th1- and Th2-influencing molecules in the pathway. For example, APCs produce IL-4, which binds to and activates the IL-4 receptor, leading to the phosphorylation and activation of the transcription factor STAT6. Red and green arrows show the expected transcriptional outcomes of Th1 and Th2 polarization; red and green arrows indicate that activation will cause the transcript to increase and decrease, respectively. Expected transcriptional outcomes are based on reports in the literature on Th1/Th2 T-cell polarization and on studies conducted with GA [3,4,8,10,17,29–34]. Gray lines indicate members of a group. APCs, antigen-presenting cells; CXCR1, chemokine (C-X-C motif) receptor 1; CXCR3, chemokine (C-X-C motif) receptor 3; CXCR5, chemokine (C-X-C motif) receptor 5; CXCL10, chemokine (C-X-C motif) ligand 10; GA, glatiramer acetate; HLADMA, major histocompatibility complex class II, DM alpha; HLADMB, major histocompatibility complex class II, DM beta; HLADQB1, major histocompatibility complex class II, DQ beta 1; ICOSLG, inducible T-cell costimulatory ligand; IL-4, interleukin-4; MHC class II, major histocompatibility complex class II; LTB, lymphotoxin beta (tumor necrosis factor superfamily, member 3); MYD, myeloid differentiation primary response protein; S100A10, S100 calcium-binding protein A10; STAT, signal transducer and activator of transcription; TCR, T-cell receptor; Th, T-helper.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608686&req=5

pone.0140299.g006: T-helper cell pathway diagram.Transcripts measured in the current study are shown in the diagram as nodes and are colored based on the differences observed when cells are stimulated with Copaxone in comparison with cell culture media alone (P < 1e-3 [Student’s t-test]; red for increase, green for decrease). Molecules with P > 1e-3 are shown in gray. Genes refer to the human ortholog. Human HLA-DMA, HLA-DMB, and HLA-DQB1 represent murine H2-Dma, H2-Dmb2, and H2-Ab1, respectively. Blue arrows show the flow of activation by major Th1- and Th2-influencing molecules in the pathway. For example, APCs produce IL-4, which binds to and activates the IL-4 receptor, leading to the phosphorylation and activation of the transcription factor STAT6. Red and green arrows show the expected transcriptional outcomes of Th1 and Th2 polarization; red and green arrows indicate that activation will cause the transcript to increase and decrease, respectively. Expected transcriptional outcomes are based on reports in the literature on Th1/Th2 T-cell polarization and on studies conducted with GA [3,4,8,10,17,29–34]. Gray lines indicate members of a group. APCs, antigen-presenting cells; CXCR1, chemokine (C-X-C motif) receptor 1; CXCR3, chemokine (C-X-C motif) receptor 3; CXCR5, chemokine (C-X-C motif) receptor 5; CXCL10, chemokine (C-X-C motif) ligand 10; GA, glatiramer acetate; HLADMA, major histocompatibility complex class II, DM alpha; HLADMB, major histocompatibility complex class II, DM beta; HLADQB1, major histocompatibility complex class II, DQ beta 1; ICOSLG, inducible T-cell costimulatory ligand; IL-4, interleukin-4; MHC class II, major histocompatibility complex class II; LTB, lymphotoxin beta (tumor necrosis factor superfamily, member 3); MYD, myeloid differentiation primary response protein; S100A10, S100 calcium-binding protein A10; STAT, signal transducer and activator of transcription; TCR, T-cell receptor; Th, T-helper.
Mentions: T-helper cell differentiation can be induced by T-cell receptor stimulation by APCs. In our study, APCs (naive splenocytes) were mixed with T cells derived from GA-immunized mouse lymph nodes to provide T-cell receptor stimulation. APCs process the GA copolymer, load major histocompatibility complex II (MHC II) receptors with peptide fragments, and present these peptides to T cells. In the context of T-cell stimulation and differentiation, several cytokines can induce T-cell polarization toward a Th2 or a Th1 phenotype. Fig 6 shows a pathway diagram of Th1 and Th2 T-cell polarization, created using literature surrounding T-helper cell differentiation and studies with GA stimulation of T-cell populations.[3,4,8,10,17,29–34]. T-helper cells differentiate toward a Th2 phenotype and produce a Th2 response in the presence of the cytokine IL-4 and T-cell receptor stimulation by MHC II molecules. Th2 cells themselves also produce IL-4. In our study, we observed increased IL-4 expression when cells were stimulated with GA. IL-4 signals through its cognate IL-4 receptor, activating the JAK/STAT pathway, specifically STAT6. STAT6 is regulated by phosphorylation in the cytoplasm, then translocates to the nucleus to induce activation of another transcription factor, GATA3. Consistent with posttranslational regulation by the phosphorylation of STAT6 and GATA3, no changes were observed in gene expression of these signaling molecules. These transcription factors, however, can induce and inhibit an array of genes further downstream of the signaling cascade. As expected, STAT6/GATA3 induced increases in expression of the Th2 cytokines IL-3, IL-4, IL-5, and IL-13; the MHC II family genes; and IL-4R. Similarly, decreases were observed in the expression of other STAT6/GATA3-regulated genes, such as S100A10, ICOSLG, and LTB. This subset of genes can be considered GA treatment-specific markers for the Th2-polarized T-cell test system, and our observation of changes in the gene expression of these markers is consistent with the literature regarding Th2 cells and the purported mechanisms of GA on immune cells [3,4,7,8,10,17,29–34].

Bottom Line: Comparative analysis was also performed on sets of transcripts relevant to T-cell biology and antigen presentation, among others that are known to be modulated by glatiramer acetate.No statistically significant differences were observed between Copaxone and Glatopa in the expression levels (magnitude and direction) of these glatiramer acetate-regulated genes.In conclusion, multiple methods consistently supported equivalent gene expression profiles between Copaxone and Glatopa.

View Article: PubMed Central - PubMed

Affiliation: Momenta Pharmaceuticals, Inc., Cambridge, MA, United States of America.

ABSTRACT
Glatopa™ is a generic glatiramer acetate recently approved for the treatment of patients with relapsing forms of multiple sclerosis. Gene expression profiling was performed as a means to evaluate equivalence of Glatopa and Copaxone®. Microarray analysis containing 39,429 unique probes across the entire genome was performed in murine glatiramer acetate--responsive Th2-polarized T cells, a test system highly relevant to the biology of glatiramer acetate. A closely related but nonequivalent glatiramoid molecule was used as a control to establish assay sensitivity. Multiple probe-level (Student's t-test) and sample-level (principal component analysis, multidimensional scaling, and hierarchical clustering) statistical analyses were utilized to look for differences in gene expression induced by the test articles. The analyses were conducted across all genes measured, as well as across a subset of genes that were shown to be modulated by Copaxone. The following observations were made across multiple statistical analyses: the expression of numerous genes was significantly changed by treatment with Copaxone when compared against media-only control; gene expression profiles induced by Copaxone and Glatopa were not significantly different; and gene expression profiles induced by Copaxone and the nonequivalent glatiramoid were significantly different, underscoring the sensitivity of the test system and the multiple analysis methods. Comparative analysis was also performed on sets of transcripts relevant to T-cell biology and antigen presentation, among others that are known to be modulated by glatiramer acetate. No statistically significant differences were observed between Copaxone and Glatopa in the expression levels (magnitude and direction) of these glatiramer acetate-regulated genes. In conclusion, multiple methods consistently supported equivalent gene expression profiles between Copaxone and Glatopa.

No MeSH data available.


Related in: MedlinePlus