Efficient expression of bioactive murine IL12 as a self-processing P2A polypeptide driven by inflammation-regulated promoters in tumor cell lines.

Interleukin 12 (IL12) is a heterodimeric proinflammatory cytokine that has shown promise as an anticancer agent. However, despite encouraging results in animal models, clinical trials involving IL12 have been unsuccessful due to toxic side effects associated with its systemic administration, prompting investigation into new delivery methods to confine IL12 expression to the tumor environment. In this study we used the self-cleaving property of the 2A peptide to express both codon-optimized murine IL12 subunits (muIL12opt) as a self-processing polypeptide (muIL12opt-P2A). We cloned muIL12opt-P2A driven by different inflammation-induced lentiviral expression systems to transduce murine tumor cell lines commonly employed in syngeneic tumor models. We confirmed the inducibility of these systems in vitro and in vivo and demonstrated the successful expression of both IL12 subunits and the release of bioactive IL12 upon proinflammatory stimulation in vitro. Therefore, IL12 release driven by these inflammation-regulated expression systems might be useful not only to address the impact of IL12 expression in the tumor environment but also to achieve a local IL12 release controlled by the inflammation state of the tumor, thus avoiding toxic side effects associated with systemic administration.

Prognostic irrelevance of HLA-G in B-cell chronic lymphocytic leukemia.

In the last few years, it has been suggested that the involvement of human leukocyte antigen-G (HLA-G) in several tumoral processes and its likely participation as a factor of immune tolerance in malignant cells. Recently, positive HLA-G surface expression has been associated with a poor prognosis in a small group of patients with B-cell chronic lymphocytic leukemia (B-CLL), a lymphoproliferative disorder characterized by a heterogeneous clinical course. In the present work, 169 patients suffering from B-CLL were analyzed for the expression of HLA-G by flow cytometry in order to verify its prognostic value in a larger cohort. We observed a low expression of this molecule on leukemic B cells and no significant relation to clinical data or progression-free survival time, indicating that this molecule is not as good immunologic prognostic marker for B-CLL as suggested.

Interleukin-10 promoter polymorphisms in patients with systemic lupus erythematosus from the Canary Islands.

The purpose of this study was to examine whether several allelic variants in the polymorphic interleukin (IL)-10 promoter region were related with an increased risk of developing systemic lupus erythematosus (SLE) in Spanish patients from Canary Islands. Microsatellites (MS) at positions -4000 and -1200 (IL10R and IL10G, respectively) and single nucleotide polymorphisms (SNPs) (MS) at positions -1082G/A, -819C/T and -592C/A of the IL-10 promoter were analysed in patients with SLE and healthy controls from Canary Islands (Spain). We found that SNPs but not MS were associated with SLE. The GCC haplotype frequency was significantly higher in SLE patients (0.43) than in healthy donors (0.33) [P = 0.02; OR = 1.50 (95% CI = 1.06-2.14)], whereas the ACC haplotype was less represented in patients (0.28 vs. 0.37) [P = 0.02; OR = 0.64 (95% CI = 0.44-0.92)]. To assess the functional role of genotypes, serum IL-10 levels from patients and controls were quantified by ELISA. Also, the lipopolysaccharide-induced IL-10 secretion by monocytes from healthy controls was evaluated in vitro. Serum IL-10 levels were higher in patients [median (interquartile range) = 2.8 pg/mL (1.8-4.2)] than in controls [0.9 pg/mL (0-3.5)] (P = 0.02), but no association was observed between serum IL-10 levels or lipopolysaccharide-induced IL-10 secretion and the IL-10 promoter haplotypes. These data suggest that the IL-10 promoter haplotype that produces higher levels of cytokine is associated with SLE in patients from Canary Islands.

A sustained activation of PI3K/NF-kappaB pathway is critical for the survival of chronic lymphocytic leukemia B cells.

The progressive rise of mature CD5+ B lymphocytes, despite the low proportion of proliferating cells, has led to the notion that B cell chronic lymphocytic leukemia (B-CLL) is primarily related to defective apoptosis. The microenvironment likely plays a prominent role because the malignant cells progressively accumulate in vivo, whereas they rapidly undergo spontaneous apoptosis when cultured in vitro. To assess microenvironment-mediated survival signals, B-CLL cells were cultured with a murine fibroblast cell line, Ltk-, with and without an agonistic antibody to CD40. Spontaneous apoptosis was associated with the loss of Akt and NF-kappaB activities. Interactions with fibroblasts sustained a basal level of Akt and NF-kappaB activities, which was dependent on phosphatidylinositol-3 kinase (PI3K). Constitutive activity of the PI3K pathway in B-CLL cells when cultured with fibroblasts prevented the downregulation of the prosurvival Bcl-2 family protein Bcl-xL and the caspase inhibitor proteins FLIPL and XIAP, and consequently caspase-3 activation and apoptosis. CD40 crosslinking in B-CLL cells did not further prevent murine fibroblasts-mediated apoptosis but induced cell proliferation, which was associated with an increase of Akt and NF-kappaB activation compared with cells cultured with fibroblasts alone. The PI3K pathway seems to play a pivotal role in B-CLL cell survival and growth.