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 disease. A 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% (12 mol/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.

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  µ mol/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  ].