Molgramostim (rHuGM-CSF)

Background/Target.  Growth factor treatment could be beneficial in both inflammatory bowel disease and experimental colitis. The aim of this study was to investigate the effect of colony stimulating factor (CSF) and recombinant human (rHu) granulocyte stimulating factor (GSF) on experimental colitis in rats. methods  . Experimental colitis was induced in 62 male Wistar rats divided into 9 groups using 2,4,6-trinitrobenzenesulfonic acid (TNBS). Group 1:   Ten rats with colitis without treatment (control group). Euthanasia after 15 days. Group 2:   Ten animals with colitis without treatment (control group). Euthanasia after 30 days. Group 3:   Six animals with colitis. Immediate treatment with CSF. Euthanasia after 19 days. Group 4:   Six animals with colitis. Treatment began 7 days after colitis induction. The animals were kept for 19 days. Group 5:   Six animals with colitis. Treatment began 2 weeks after induction of colitis. 

Group 6:   Six animals with colitis, same as in group 3. Treatment with GSF. Group 7:   Six animals with colitis, the same as in group 4. Treatment with GSF. Group 8.   Six animals with colitis same as in Group 5. Treatment with GSF. Group 9:   Six animals with colitis. Immediate treatment with prednisolone. Euthanasia after 15 days. Results  . CSF and GSF administration significantly improved histological score (□  )  and reduced malondialdehyde content (  □ ) in   all animals compared to control groups. CSF was superior to GSF and prednisolone. Conclusion .Administration of both CSF and GSF could significantly improve histological score and oxidative stress in experimental colitis in rats.

 introduction

Inflammatory bowel disease (IBD) is a chronic and relapsing inflammatory disease of the gastrointestinal tract with two main entities, Crohn’s disease (CD) and ulcerative colitis (UC). The primary therapeutic goals are to improve quality of life by inducing and maintaining remission, predicting, preventing, and treating complications, restoring nutritional deficiencies, and altering the natural history of the diseaseA dysfunctional innate immune system could be instrumental in the pathogenesis of CD. Therefore, treatments aimed at overcoming congenital immunodeficiencies could represent valuable therapeutic alternatives.

Recombinant Human Granulocyte-Macrophage Colony Stimulating Factor [GM-CSF (rHu GM-CSF)] [Molgramostim (Mielogen)] is a human protein produced by a strain of Escherichia coli containing a plasmid encoding the human GM-CSF gene encoded. Cells expressing receptors for GM-CSF include neutrophils, monocytes, macrophages and antigen presenting cells. These receptors are also produced by specialized intestinal epithelial cells, including Paneth cells and CD4 T lymphocyte cells. Molgramostim stimulates the formation of macrophage colonies, which are essential for the process of hematopoiesis and the function of mature myeloid cells.In addition, molgramostim increases white blood cell count and functions, increases antimicrobial phagocytosis, and promotes oxidative metabolism, functions associated with the host’s innate immunological defense mechanisms.

Lenograstim is the glycosylated recombinant form of human granulocyte colony-stimulating factor with effects similar to molgramostim  . Lenograstim is a valuable adjunct capable of minimizing the hematological toxicity of myelosuppressive chemotherapy in patients with malignancies .

Of the currently used animal models, 2,4,6-trinitrobenzenesulfonic acid ( TNBS )-induced colitis resembles human IBD in its various histological features, including infiltration of the colonic mucosa by neutrophils and macrophages and increased production of inflammatory mediators. The immune-stimulating functions of growth factors could potentially be beneficial in patients with IBD.

Finally, malondialdehyde (MDA) is one of the end products of lipid peroxidation in cells. An increase in free radicals during an inflammatory process such as colitis causes an overproduction of MDA. For this reason, MDA levels are often used as a marker for oxidative stress.

The aim of this study was to examine the influence of the two growth factors; Granulocyte colony-stimulating factor (G-CSF,   Lenograstim)  and recombinant human granulocyte-macrophage colony-stimulating factor (rHu GM-CSF,   Molgramostim, Mielogen)  ] as potential therapeutics in experimental colitis in rats.

 MATERIALS AND METHODS

experimental animals

Sixty-two male Wistar rats weighing more than 250  g were used. The animals were housed in the laboratory for at least 10 days prior to the experiment according to international regulations. The experimental procedures described below have been approved by the Animal Care Committee under European Union Law and Greek Law 160, A-64, May 1991 and are designed to minimize pain and discomfort to the animals. All animals were euthanized by barbiturate overdose (150  mg/kg pentobarbital sodium). Choosing different time points at which euthanasia was performed allowed us to examine clinical worsening or remission of colitis over different time intervals.

 Induction of experimental colitis

Distal colitis was induced by intracolonial installation of 25  mg TNBS dissolved in 0.25  ml 50% ethanol. The solution was injected into the colon 8 cm proximal to the anus using a PE-50 needle. To ensure that the TNBS-ethanol solution was not immediately excreted from the rat, the cannula  was left in place for 15 s prior to its removal.

groups

The animals were divided into 9 groups. The first two groups consisted of ten rats each (control groups, total of 20 animals). All other groups consisted of 6 rats each (42 animals in total). The number of animals used per group was based on a similar estimate to those used in other relevant studies in the literature. These numbers were the highest possible for the available budget to allow safe conclusions. In other words, we recruited the highest possible number of mice for the given budget; We also internally allocated a specific number of mice per group and compared this to other studies with a similar focus to ensure that reasonable study power was achieved within cost constraints.

Prednisolone was chosen as a positive control group.

The nine groups were as follows  : Group 1:   Ten rats with TNBS colitis without treatment (control group 1). Euthanasia was performed after 15 days. Group 2:   Ten animals with TNBS colitis, again without treatment (control group 2). Euthanasia was performed after 30 days. Group 3:   Six animals with TNBS colitis. Immediate treatment with liquor. Euthanasia was performed after 19 days. Group 4:   Six animals with TNBS colitis. CSF treatment began 7 days after colitis induction.

Euthanasia was performed after 19 days. Group 5:   Six animals with TNBS colitis. CSF treatment began 14 days after colitis induction. Euthanasia was performed after 19 days. Group 6:   Six animals with TNBS colitis, immediate treatment with GSF. Euthanasia was performed after 19 days.  Group 7:   Six animals with TNBS colitis, treatment with GSF started 7 days after colitis induction. Euthanasia was performed after 19 days.  Group 8.   Six animals with TNBS colitis, treatment with GSF started 14 days after colitis induction. Euthanasia was performed after 19 days  . Group 9:  Six animals with TNBS colitis. Immediate treatment with prednisolone and euthanasia after 15 days.

pathology

The resected tissue samples were fixed in 10% buffered formaldehyde and each sample was completely embedded in 3-4 paraffin blocks depending on the size. Histological sections from each block were cut at 4  μm  thickness and stained with hematoxylin-eosin [  8  ]. First, the sections were blindly examined separately by two pathologists (CB and AN) and a finally agreed histological diagnosis was made. Results were evaluated using the Geboes [  9 ] Histological score based on the following features: normal histology, acute ulcers/erosions/chronic ulcers, neutrophilic activity, crypt distortion, and chronic inflammation of the lamina propria. The above features were diagnostic of induced colitis. Based on this score, the colonic mucosa was characterized as normal (grade 0), colitis in remission (grades 1 and 2), and active colitis (grades 3-5). The histological features of healing ulcers with complete or incomplete re-epithelialization, crypt loss and lamina propria/submucosa fibrosis or crypt regeneration were evaluated as signs of remission.

Tissue Malondialdehyde Estimate

The malondialdehyde(MDA) measurement was based on the reaction of a chromogenic reagent,   N  -methyl-2-phenylindole (MPI), with MDA at 45°C. Reagents used included Reagent MPI   10.3  mmol/L   N  -Methyl-2-phenylindole in acetonitrile, MDA standard   10  mmol/L 1,1,3,3-Tetramethoxypropane in 20  mmol/L Tris-HCl, 500  mmol/L butylated hydroxytoluene, in acetonitrile, 20  mmol/L Tris buffer pH 7.4, 0.9% NaCl, 37% (12mol/L) HCl, methanol, HPLC grade, acetonitrile and HPLC grade. Before the procedure, three volumes of MPI reagent were diluted with one volume of 100% methanol. Tissue samples were rinsed with ice-cold isotonic saline prior to homogenization, which was performed using Tris buffer 20  mmol/L pH 7.4 and an ULTRA-TURRAX (IKA-Labortechnik) blender. One milliliter of buffer was used for 0.1  g of tissue.

Molgramostim (rHuGM-CSF)

rHuGM-CSF Amyotop 300µg/vial 249 EUR

PRE-GMP rHu GM-CSF, Molgramostim-Leukoma

04-RHUGM-CSF-7A10 Gentaur Genprice 300 µg 385 EUR

Filgrastim (rHuG-CSF)

rHuG-CSF Amyotop 300µg/vial 225 EUR

Mouse GM-CSF ELISA Kit, 96 tests, Quantitative

MGM-CSF-100 Alpha Diagnostics 1 kit 482 EUR

Human GM-CSF ELISA Kit, 96 tests, Quantitative

HGM-CSF-100 Alpha Diagnostics 1 kit 482 EUR

CSF/ Rat CSF ELISA Kit

ELA-E1111r Lifescience Market 96 Tests 886 EUR

G-CSF

E21-002 EnoGene 10ug 343 EUR

GM-CSF

E21-003 EnoGene 10ug 343 EUR

M-CSF

E21-417 EnoGene 10ug 343 EUR

GM-CSF

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CSF Tubes

213361-(V100) ImmunoStep 100 tubes 1688 EUR

CSF Tubes

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CSF Tubes

213361-(V6) ImmunoStep 6 tubes 186.5 EUR

CSF Tubes

abx290032-100tubes Abbexa 100 tubes 1957 EUR

CSF Tubes

abx290032-25tubes Abbexa 25 tubes 662 EUR

CSF Tubes

abx290032-6tubes Abbexa 6 tubes 314 EUR

Recombinant Mouse M-CSF/CSF-1 Protein

RP01216 Abclonal 10 μg 202 EUR

Recombinant human M-CSF/CSF-1 Protein

RP01221 Abclonal 10 μg 230 EUR

G-CSF/ Rat G- CSF ELISA Kit

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G-CSF, Human

HY-P7015 MedChemExpress 100ug 946 EUR

GM-CSF, Human

HY-P7016A MedChemExpress 50ug 441 EUR

M-CSF, Human

HY-P7050 MedChemExpress 50ug 612 EUR

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GM-CSF, Mouse

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M-CSF, Rat

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M-CSF/CSF1

E21-B34 EnoGene 10ug 343 EUR

GM-CSF/CSF2

E21-C79 EnoGene 10ug 343 EUR

His-GM-CSF

E13-010-1 EnoGene 10μg 291 EUR

His-GM-CSF

E13-010-2 EnoGene 50μg 700 EUR

His-G-CSF

E13-011-1 EnoGene 10μg 291 EUR

His-G-CSF

E13-011-2 EnoGene 50μg 700 EUR

G - CSF Human

PR27066 Neuromics 2 ug 344 EUR

G - CSF Mouse

PR27067 Neuromics 2 ug 191 EUR

GM - CSF Human

PR27068 Neuromics 2 ug 191 EUR

GM - CSF Rat

PR27071 Neuromics 2 ug 148 EUR

GM - CSF Sf9

PR27072 Neuromics 2 ug 148 EUR

GM - CSF Mouse

PR27074 Neuromics 2 ug 191 EUR

M-CSF Human

PR27079 Neuromics 2 ug 148 EUR

rHu G-CSF

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rHu G-CSF

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rHu G-CSF

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rHu G-CSF

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rHu GM-CSF

AK9707-0005 Akron Biotech 5µg Ask for price

rHu GM-CSF

AK9707-0020 Akron Biotech 20µg Ask for price

rHu GM-CSF

AK9707-0100 Akron Biotech 100µg Ask for price

rHu GM-CSF

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G-CSF, Human

LF-P0270 Abfrontier 10 ug 270 EUR

G-CSF, Human

LF-P0270A Abfrontier 100 ug 1811 EUR

GM-CSF, Human

LF-P0278 Abfrontier 10 ug 270 EUR

GM-CSF, Human

LF-P0278A Abfrontier 100 ug 1811 EUR

anti-G-CSF

LF-PA0229 Abfrontier 100 ul 334 EUR

GM-CSF antibody

70-GR30 Fitzgerald 50 ug 298 EUR

M-CSF antibody

70R-CR018 Fitzgerald 100 ug 381 EUR

GM-CSF antibody

70R-GR026 Fitzgerald 50 ug 273 EUR

M-CSF antibody

70R-MG004 Fitzgerald 50 ug 273 EUR

GM-CSF protein

30R-AG014 Fitzgerald 5 ug 133 EUR

GM-CSF protein

30R-AG016 Fitzgerald 50 ug 529 EUR

GM-CSF protein

30R-AG022 Fitzgerald 20 ug 273 EUR

M-CSF protein

30R-AM004 Fitzgerald 50 ug 691 EUR

M-CSF protein

30R-AM005 Fitzgerald 10 ug 273 EUR

M-CSF protein

30R-AM069 Fitzgerald 10 ug 273 EUR

anti-GM-CSF

YF-PA11125 Abfrontier 100 ug 403 EUR

anti-G-CSF

YF-PA11129 Abfrontier 50 ul 363 EUR

anti-G-CSF

YF-PA11130 Abfrontier 50 ug 363 EUR

anti-G-CSF

YF-PA11131 Abfrontier 100 ul 403 EUR

anti-G-CSF

YF-PA11132 Abfrontier 100 ug 403 EUR

G-CSF Antibody

5094-100 Biovision 316 EUR

G-CSF Antibody

5094-30T Biovision 146 EUR

G-CSF Antibody

5094R-100 Biovision 338 EUR

G-CSF Antibody

5094R-30T Biovision 146 EUR

GM-CSF Antibody

5100-100 Biovision 316 EUR

GM-CSF Antibody

5100-30T Biovision 146 EUR

GM-CSF Antibody

5101-100 Biovision 316 EUR

GM-CSF Antibody

5101-30T Biovision 146 EUR

M-CSF Antibody

5237-100 Biovision 316 EUR

M-CSF Antibody

5237-30T Biovision 146 EUR

M-CSF Antibody

5238-100 Biovision 316 EUR

M-CSF Antibody

5238-30T Biovision 146 EUR

G-CSF-R/ Rat G- CSF- R ELISA Kit

ELA-E2217r Lifescience Market 96 Tests 886 EUR

G-CSF ab/ Rat G- CSF ab ELISA Kit

ELA-E1717r Lifescience Market 96 Tests 886 EUR

Recombinant Human M-CSF/ CSF-1 Protein, Untagged, E.coli-100ug

QP10351-ec-100ug EnQuireBio 100ug 1396 EUR

Recombinant Human M-CSF/ CSF-1 Protein, Untagged, E.coli-10ug

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Recombinant Human M-CSF/ CSF-1 Protein, Untagged, E.coli-250ug

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Recombinant Human M-CSF/ CSF-1 Protein, Untagged, E.coli-2ug

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Recombinant Mouse M-CSF/ CSF-1 Protein, Untagged, E.coli-100ug

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Recombinant Mouse M-CSF/ CSF-1 Protein, Untagged, E.coli-10ug

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Recombinant Mouse M-CSF/ CSF-1 Protein, Untagged, E.coli-250ug

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Recombinant Mouse M-CSF/ CSF-1 Protein, Untagged, E.coli-2ug

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Recombinant Human M-CSF/ CSF-1 Protein, Untagged, E.coli-1mg

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Recombinant Human M-CSF/ CSF-1 Protein, Untagged, E.coli-500ug

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Recombinant Mouse M-CSF/ CSF-1 Protein, Untagged, E.coli-1mg

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Recombinant Rat M-CSF/ CSF-1 Protein, Untagged, E.coli-100ug

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Recombinant Rat M-CSF/ CSF-1 Protein, Untagged, E.coli-10ug

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Recombinant Rat M-CSF/ CSF-1 Protein, Untagged, E.coli-1mg

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Recombinant Rat M-CSF/ CSF-1 Protein, Untagged, E.coli-2ug

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Recombinant Mouse M-CSF/ CSF-1 Protein, His, E.coli-100ug

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Ten milliliters of 500  mmol/LBHT were added to 1  ml of tissue homogenate to prevent sample oxidation. The homogenate was centrifuged at 3000 rpm at 4°C for 10  min. Then 0.2  ml of sample (plasma or tissue homogenate supernatant) and 0.65  ml of diluted MPI reagent were added to a polypropylene microcentrifuge tube. The mixture was vortexed and then 0.15  mL of 12  M HCl was added. The tubes were  incubated at 45°C for 60 min and centrifuged at 6000 rpm for 15 min  . Then 0.8  ml of the supernatant was  measured at 586 nm. MDA standards for the standard curve were obtained by diluting the 10th mm TMOP stock solution. The final concentrations were 2.08, 4.16, 8.33, 12.5 and 16.66  µmol/L and the test procedure was followed as for the samples. The absorbance was 0.059, 0.124, 0.264, 0.4, and 0.545, respectively [  10  ].

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