A gene expression map of host immune response in human brucellosis.

Brucellosis is a common zoonotic disease caused by intracellular pathogens of the genus Brucella. Brucella infects macrophages and evades clearance mechanisms, thus resulting in chronic parasitism. Herein, we studied the molecular changes that take place in human brucellosis both in vitro and ex vivo. RNA sequencing was performed in primary human macrophages (Mφ) and polymorphonuclear neutrophils (PMNs) infected with a clinical strain of Brucella spp. We observed a downregulation in the expression of genes involved in host response, such as TNF signaling, IL-1ß production, and phagosome formation in Mφ, and phosphatidylinositol signaling and TNF signaling in PMNs, being in line with the ability of the pathogen to survive within phagocytes. Further transcriptomic analysis of isolated peripheral blood mononuclear cells (PBMCs) and PMNs from patients with acute brucellosis before treatment initiation and after successful treatment revealed a positive correlation of the molecular signature of active disease with pathways associated with response to interferons (IFN). We identified 24 common genes that were significantly altered in both PMNs and PBMCs, including genes involved in IFN signaling that were downregulated after treatment in both cell populations, and IL1R1 that was upregulated. The concentration of several inflammatory mediators was measured in the serum of these patients, and levels of IFN-γ, IL-1ß and IL-6 were found significantly increased before the treatment of acute brucellosis. An independent cohort of patients with chronic brucellosis also revealed increased levels of IFN-γ during relapse compared to remissions. Taken together, this study provides for the first time an in-depth analysis of the transcriptomic alterations that take place in human phagocytes upon infection, and in peripheral blood immune populations during active disease.

Expression of heat shock proteins in brain tumors.

AIM: Heat shock proteins (HSP) are an evolutionary conserved family of proteins that serve as molecular chaperones, preventing the formation of nonspecific protein aggregates and assisting proteins in the acquisition of their native structures. Furthermore, HSPs have anti-apoptotic properties and have been found to be elevated in many human cancers; their overexpression has been associated with poor survival and response to therapy. In the present study we assessed the HSP expression in brain tumors. MATERIAL AND METHODS: Simultaneous detection of HSP27, HSP40, HSP60, HSP70, HSP90a, total Akt and phospho- Akt in 19 brain tumor specimens was performed using the multiplex bead array assay. RESULTS: There was expression of HSP27 (pSer82), HSP27 (pSer15), HSP40, HSP60, HSP70, HSP90a, total-Akt and phospho- Akt in both gliomas and meningiomas. Significantly higher levels of HSP70 and a trend towards higher levels of HSP40 were found in meningiomas compared to gliomas. There was a significant correlation between HSP27 (pSer82) and HSP27 (pSer15) expression and between HSP90a and both total-AKT and phospho- AKT. A significant correlation between HSP27 and total-AKT was observed. CONCLUSION: Since HSPss are an attractive target for anticancer therapy, further studies are needed in order to better assess their relationship with tumor aggressiveness and patient prognosis.

Expression of heat shock proteins in medulloblastoma.

OBJECT: Medulloblastoma (MB) is the most common malignant brain tumor in children. Heat shock proteins (HSPs) comprise a superfamily of proteins that serve as molecular chaperones and are overexpressed in a wide range of human cancers. The purpose of the present study was to investigate the expression of HSP27 (pSer(82)), HSP27 (pSer(15)), HSP40, HSP60, HSP70, HSP90-α, Akt, and phospho-Akt by multiplex bead array assay of MBs. The results of HSP and Akt expression were correlated with MB subtype; immunohistochemical expression of Ki-67 index, bcl-2, and p53; and patients' prognosis. METHODS: The authors retrospectively evaluated 25 children with MB who underwent surgery. Immunohistochemical analysis of Ki-67, p53, and bcl-2 expression was performed in all cases. By using multiplex bead array assay, a simultaneous detection of HSP27 (pSer(82)), HSP27 (pSer(15)), HSP40, HSP60, HSP70, HSP90-α, Akt, and phospho-Akt was performed. RESULTS: Medulloblastoma with extensive nodularity had significantly lower HSP27 (pSer(15)) expression (p = 0.039) but significantly higher HSP60 expression (p = 0.021) than classic MB. Large-cell MB had significantly higher HSP70 expression (p = 0.028) than classic MB. No significant difference was found between HSP27 (pSer(82)), HSP40, HSP90-α, Akt, or phospho-Akt expression and MB subtype. Large-cell MBs had significantly higher Ki-67 index compared with classic MBs (p = 0.033). When analyzing all MBs, there was a significant negative correlation between HSP27 (pSer(15)) and Ki-67 index (r = -0.475, p = 0.016); a significant positive correlation between HSP70 expression and Ki-67 index (r = 0.407, p = 0.043); and a significant positive correlation between HSP70 expression and bcl-2 index (r = 0.491, p = 0.023). Patients with large-cell MB had a worse survival than those with classic MB, but the difference did not reach statistical significance (p = 0.076). CONCLUSIONS: A substantial expression of several HSPs in MB was observed. Given that HSPs represent an attractive strategy for anticancer therapy, further studies, involving larger series of patients, are obviously necessary to clarify the relationship of HSPs with tumor aggressiveness and prognosis.

Cisplatin-induced apoptosis in HL-60 human promyelocytic leukemia cells: differential expression of BCL2 and novel apoptosis-related gene BCL2L12.

The anti-apoptotic molecule BCL2 delays cell-cycle entry from quiescence. We have recently cloned a new member of the BCL2 family of apoptosis-related genes, BCL2L12. In the present study, the expression of BCL2 and BCL2L12 genes during cisplatin-induced apoptosis in HL-60 leukemic cells was investigated. The kinetics of apoptosis induction and cell toxicity were evaluated by DNA laddering and the MTT method, respectively. BCL2 and BCL2L12 expression was analyzed by RT-PCR using gene-specific primers. The ratio of apoptotic cells increased with increasing concentrations of cisplatin and exposure time of cell culture to the drug. Gradual, time-dependent downregulation of BCL2 gene was observed during cisplatin treatment. Up-regulation of BCL2L12 was observed 3 h (no DNA fragmentation) and 6 h (initiation of the DNA fragmentation) after treatment with cisplatin, followed by a decrease of its expression after 12-h continuous treatment with the drug. It is known that the main anti-carcinogenic effect of cisplatin is due to the induction of cell apoptosis. Present results indicate that downregulation of the BCL2 and upregulation of the BCL2L12 gene may be the underlying mechanisms.