Development of recombinant human granulocyte colony-stimulating factor (nartograstim) production process in Escherichia coli compatible with industrial scale and with no antibiotics in the culture medium.

The granulocyte colony-stimulating factor (G-CSF) is a hematopoietic cytokine that has important clinical applications for treating neutropenia. Nartograstim is a recombinant variant of human G-CSF. Nartograstim has been produced in Escherichia coli as inclusion bodies (IB) and presents higher stability and biological activity than the wild type of human G-CSF because of its mutations. We developed a production process of nartograstim in a 10-L bioreactor using auto-induction or chemically defined medium. After cell lysis, centrifugation, IB washing, and IB solubilization, the following three refolding methods were evaluated: diafiltration, dialysis, and direct dilution in two refolding buffers. Western blot and SDS-PAGE confirmed the identity of 18.8-kDa bands as nartograstim in both cultures. The auto-induction medium produced 1.17 g/L and chemically defined medium produced 0.95 g/L. The dilution method yielded the highest percentage of refolding (99%). After refolding, many contaminant proteins precipitated during pH adjustment to 5.2, increasing purity from 50 to 78%. After applying the supernatant to cation exchange chromatography (CEC), nartograstim recovery was low and the purity was 87%. However, when the refolding solution was applied to anion exchange chromatography followed by CEC, 91%-98% purity and 2.2% recovery were obtained. The purification process described in this work can be used to obtain nartograstim with high purity, structural integrity, and the expected biological activity. KEY POINTS: • Few papers report the final recovery of the purification process from inclusion bodies. • The process developed led to high purity and reasonable recovery compared to literature. • Nartograstim biological activity was demonstrated in mice using a neutropenia model.

Development of recombinant human granulocyte colony-stimulating factor (nartograstim) production process in Escherichia coli compatible with industrial scale and with no antibiotics in the culture medium

The granulocyte colony-stimulating factor (G-CSF) is a hematopoietic cytokine that has important clinical applications for treating neutropenia. Nartograstim is a recombinant variant of human G-CSF. Nartograstim has been produced in Escherichia coli as inclusion bodies (IB) and presents higher stability and biological activity than the wild type of human G-CSF because of its mutations. We developed a production process of nartograstim in a 10-L bioreactor using auto-induction or chemically defined medium. After cell lysis, centrifugation, IB washing, and IB solubilization, the following three refolding methods were evaluated: diafiltration, dialysis, and direct dilution in two refolding buffers. Western blot and SDS-PAGE confirmed the identity of 18.8-kDa bands as nartograstim in both cultures. The auto-induction medium produced 1.17 g/L and chemically defined medium produced 0.95 g/L. The dilution method yielded the highest percentage of refolding (99%). After refolding, many contaminant proteins precipitated during pH adjustment to 5.2, increasing purity from 50 to 78%. After applying the supernatant to cation exchange chromatography (CEC), nartograstim recovery was low and the purity was 87%. However, when the refolding solution was applied to anion exchange chromatography followed by CEC, 91%-98% purity and 2.2% recovery were obtained. The purification process described in this work can be used to obtain nartograstim with high purity, structural integrity, and the expected biological activity.

Local and Systemic STAT3 and p65 NF-KappaB Expression as Progression Markers and Functional Targets for Patients With Cervical Cancer.

Cervical cancer, which main etiologic factor is Human Papillomavirus (HPV) infection, continues to be a burden for public health systems in developing countries. Our laboratory has been working with the hypothesis that signals generated in the tumor microenvironment can modulate local and systemic immune responses. In this context, it would be reasonable to think that tumors create pro-tumoral bias in immune cells, even before they are recruited to the tumor microenvironment. To understand if and how signaling started in the tumor microenvironment can influence cells within the tumor and systemically, we investigated the expression of key proteins in signaling pathways important for cell proliferation, viability, immune responses and tolerance. Besides, we used detection of specific phosphorylated residues, which are indicative of activation for Akt, CREB, p65 NFκB, and STAT3. Our findings included the observation of a significant STAT3 expression increase and p65 NFκB decrease in circulating leukocytes in correlation with lesion grade. In light of those observations, we started investigating the result of the inhibition of STAT3 in a tumor experimental model. STAT3 inhibition impaired tumor growth, increased anti-tumor T cell responses and decreased the accumulation of myeloid cells in the spleen. The concomitant inhibition of NFκB partially reversed these effects. This study indicates that STAT3 and NFκB are involved in immunomodulatory tumor effects and STAT3 inhibition could be considered as therapy for patients with cervical cancer.

NFAT1 Regulates Ly6Chi Monocyte Recruitment to the CNS and Plays an Essential Role in Resistance to Toxoplasma gondii Infection.

Monocytes play key roles in the maintenance of homeostasis and in the control of the infection. Monocytes are recruited from the bone marrow to inflammatory sites and are essential for antimicrobial activity to limit tissue damage and promote adaptive T cell responses. Here, we investigated the role of Nuclear Factor of Activated T cells 1 (NFAT1) in the regulation of Ly6Chi inflammatory monocyte recruitment to the CNS upon T. gondii infection. We show that NFAT-1-deficient monocytes are unable to migrate to the CNS of T. gondii-infected mice. Moreover, NFAT1-/- mice are highly susceptible to chronic T. gondii infection due to a failure to control parasite replication in the CNS. The inhibition of Ly6Chi inflammatory monocyte recruitment to the CNS severely blocked CXCL10 production and consequently the migration of IFN-γ-producing CD4+ T cells. Moreover, the transfer of Ly6Chi monocytes to infected NFAT1-/- mice favored CD4+ T cell migration to the CNS and resulted in the inhibition of parasite replication and host defense. Together, these results demonstrated for the first time the contribution of NFAT1 to the regulation of Ly6Chi monocyte recruitment to the CNS and to resistance during chronic T. gondii infection.