Simultaneous inhibition of the constitutively activated nuclear factor kappaB and of the interleukin-6 pathways is necessary and sufficient to completely overcome apoptosis resistance of human U266 myeloma cells.

Elevated Nuclear Factor kappaB (NFkappaB) levels have been reported in multiple myeloma cells derived from patients relapsing after chemotherapy. In the search of an in vitro a model with molecular features similar to relapsing lesions, we focused our attention on an IL-6 autocrine human myeloma cell line (U266), characterized by apoptosis resistance due to upregulation of two constitutive signaling pathways: NFkappaB and STAT-3. NFkappaB activity was inhibited with proteasome inhibitory agents, such as PS-341 and Withaferin A, with an IKK inhibitor (Wedelolactone) or with the adenoviral vector HD IkappaBalphamut-IRES-EGFP encoding a mutant IkappaBalpha protein, resistant to proteasomal degradation. We observed that the NFkappaB intracellular dislocation at the beginning of the treatment affected therapeutic effectiveness of PS-341, Withaferin A and Wedelolactone; interestingly, the adenoviral vector was highly effective in inducing apopotosis even with NFkappaB being predominantly nuclear at the time of infection. We also observed that U266 treated with the Interleukin-6 antagonist Sant7 exhibited reduced STAT3 activity and preferential cytoplasmic NFkappaB location; moreover they became capable of undergoing apoptosis mainly from the G1 phase. Adenoviral vector treated U266 have NFkappaB localized completely in the cytoplasm and also showed downregulation of nuclear phospho STAT-3. Finally, combined targeting of NFkappaB and STAT3 signalling pathways was the most effective treatment in inducing apoptosis. These findings suggest that combined NFkappaB and STAT3 targeting warrants further investigations in other apoptosis resistant MM cell lines as well as in suitable MM animal models.

Bartonella quintana-induced apoptosis inhibition of human endothelial cells is associated with p38 and SAPK/JNK modulation and with stimulation of mitosis.

Previous studies demonstrated that live Bartonella quintana often induces angioproliferative lesions in humans. It modulates endothelial cell apoptotic and inflammatory patterns, thus inducing a very early overexpression of caspase 8 and Apaf-1 and increasing mRNA production of TNF-alpha, interleukin-8, and E-selectin. However, starting at 10 hours postinfection, the bacteria provoke antiapoptotic effects that induce an increase of bcl-2 gene transcription. To gain further insight into the cellular mechanisms that regulate apoptosis, survival and proliferation, we studied the modulation of mitogen-activated protein kinase (MAPK) and the activation state of cdc2 kinase, which regulates progression into mitosis. Confocal microscopy findings indicated a maximum rate of Bartonella entry into host cells between postinfection hours 6 and 10. Live bacteria caused substantially higher apoptosis of human umbilical vein endothelial cells-cryopreserved (HUVEC-C) than heat- and trypsin-inactivated microorganisms. During the first 6 hours postinfection, B. quintana triggered a peak of apoptosis, induced activation of p38 MAPK and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), with bacterial clusters appearing at the cellular surface of the HUVEC-C. However, at 8 to 24 hours postinfection, B. quintana was internalized and inhibited proapoptotic signals such as p38 MAPK and SAPK/JNK while inducing antiapoptotic signals. Indeed, expression of the bcl-2 gene and the increase of the bcl-2 kinase active form was concomitant to activation of mitosis, as shown by cdc2 protein activation. These data thus suggest that mechanisms that induce mitotic activity and inhibit apoptotic signals may contribute to the ability of B. quintana to cause vascular proliferation.

Changes in the prevalence of hepatitis C virus (HCV) genotype 4 in Calabria, Southern Italy.

The aim of this study was to assess changes in the prevalence of hepatitis C virus (HCV) genotypes, focusing on genotype 4, by surveying population of chronic hepatitis C patients within an area of Southern Italy. HCV-RNA was detected in serum using two commercial hepatitis C RNA PCR assays (Amplicor Roche Diagnostic System, and AmpliSensor HCV, Nuclear Laser Medicine). PCR products were analyzed for genotyping using a reverse hybridization of the amplified product by a line probe assay (INNO LIPA, Innogenetics). In our Institution we have previously observed, in a period of 18 months (January 1997-May 1998) an initial increase of the genotype 4 which appeared in 3.3% of HCV patients versus a percentage of 1.3%, during 1996. Later data obtained from 702 HCV-RNA positive patients, collected from June 1998 until December 1999 indicated a 3.7% of genotype 4. This percentage increased until to 4.7% in the most recent period studied (January 2000-February 2001). Drug addiction, blood transfusion and sporadically acquired infections represented the most frequent risk factors. In the Calabria region, genotype 1b, the most prevalent isolate (53.3%) and genotype 2a/2c (26.2%) were associated with older age, confirming our previous study. Genotype 4 was the fifth most prevalent genotype observed, just after 3a and 1a subtypes. Spread of genotype 4 in Calabria region is mostly associated to older age when compared to genotype 3a and 1a, but is statistically associated with a younger group of patients when compared with genotype 1b. In conclusion we demonstrated a fourfold increased prevalence of HCV genotype 4 during the last 5 years.