Outcomes after fingolimod to alemtuzumab treatment shift in relapsing–remitting MS patients: a multicentre cohort study

Journal of Neurology - A high reactivation of multiple sclerosis (MS) was reported in patients treated with alemtuzumab after fingolimod. We aimed to understand whether this shift enhanced the risk...     

In vitro use of autologous dendritic cells improves detection of T cell responses to hepatitis B virus (HBV) antigens

T lymphocyte responses to hepatitis B virus (HBV) core antigen (HBcAg) are vigorous and easily detectable in vitro during recovery from acute hepatitis B but significantly weaker in patients with chronic HBV infection. In contrast, T cell responses to hepatitis B surface antigen (HBsAg) are almost undetectable during infection and even in a substantial fraction of subjects receiving vaccination with HBsAg. The aim of this study was to investigate whether the use of dendritic cells (DCs) in an in vitro assay could increase the detection of HBV‐specific T cells in these conditions. Autologous monocyte‐derived DCs, compared to direct HBsAg addition to the cultures, increased the stimulation of HBs‐ specific T cells. These were detected in 73% of healthy subjects who had recently received hepatitis B vaccine and in 43% of patients recovering from acute hepatitis B. Likewise, proliferation in response to DC‐presented HBcAg was detected in both CD4⁺ and CD8⁺ T cells from the majority of chronic hepatitis B patients. A longitudinal evaluation of HBc‐specific T cell responses during and after a 1‐year treatment with pegylated interferon (IFN)‐α showed that HBc‐specific CD4⁺ T cell responses had no correlation with sustained virus suppression whereas CD8⁺ T cell responses were more frequently detected in patients able to control HBV replication after therapy interruption. The use of autologous DCs as antigen‐presenting cells appears applicable to clinically relevant in vitro evaluation of T cell responses, particularly in those conditions characterized by low frequency of circulating antigen‐specific cells and suboptimal in vivo activation. J. Med. Virol. 81:332–339, 2009. © 2008 Wiley‐Liss, Inc.     

Detection of KRAS mutations in colorectal cancer with Fast COLD-PCR

Patients with metastatic colorectal carcinoma (mCRC) carrying activating mutations of the KRAS gene do not benefit from treatment with anti-epidermal growth factor receptor (EGFR) monoclonal antibodies. Therefore, KRAS mutation testing of mCRC patients is mandatory in the clinical setting for the choice of the most appropriate therapy. Co-amplification-at-lower denaturation-temperature PCR (COLD-PCR) is a novel modification of the conventional PCR method that selectively amplifies minority alleles from a mixture of wild-type and mutant sequences irrespective of the mutation type or position within the sequence. In this study, we compared the sensitivity of a COLD-PCR method with conventional PCR/sequencing and the real-time PCR-based Therascreen kit to detect KRAS mutations. By using dilutions of KRAS mutant DNA in wild-type DNA from colon cancer cell lines with known KRAS status, we found that Fast COLD-PCR is more sensitive than the conventional PCR method, showing a sensitivity of 2.5% in detecting G>A and G>T mutations. The detection of G>C transversions was not improved by either Fast COLD-PCR or Full COLD-PCR. We next analyzed by COLD-PCR, conventional PCR and Therascreen 52 formalin-fixed paraffin-embedded samples from mCRC patients. Among 36 samples with >30% tumor cells, 8 samples were negative by conventional PCR, Therascreen and Fast COLD-PCR; 20 mutations identified by conventional PCR were confirmed by Therascreen and Fast COLD-PCR; 8 cases undetermined by conventional PCR were all confirmed to carry KRAS G>A or G>T mutations by using either Therascreen or Fast COLD-PCR. Conventional PCR was able to detect only 2 KRAS mutations among 16 samples with <30% tumor cells (12.5%), whereas Therascreen and Fast COLD-PCR identified 6 mutants (37.5%). These data suggest that Fast COLD-PCR has a higher clinical sensitivity as compared with conventional PCR in detecting G>C to A>T changes in the KRAS gene, which represent >90% of the mutations of this oncogene in CRC.     

AZD3409 inhibits the growth of breast cancer cells with intrinsic resistance to the EGFR tyrosine kinase inhibitor gefitinib

AKT and MAPK signaling are involved in the resistance of breast cancer cells to the EGFR tyrosine kinase inhibitor gefitinib. RAS proteins are upstream mediators that transfer messages from surface receptors to intracellular signal transducers including MAPK and AKT pathways. AZD3409 is a novel prenyl inhibitor that has shown activity against both farnesyl transferase and geranylgeranyl transferase in isolated enzyme studies. We explored the activity of AZD3409 on breast cancer cell lines with high (SK-Br-3), intermediate (MDA-MB-361) or low (MDA-MB-468) sensitivity to gefitinib. We found that AZD3409 inhibits the growth of breast cancer cells in a dose-dependent manner, with the MDA-MB-468 and MDA-MB-361 cell lines showing higher sensitivity as compared with SK-Br-3 cells. Treatment with AZD3409 produced a significant reduction in the levels of activation of AKT in the three cell lines. AZD3409 also induced an increase in the expression of p27kip-1 and of hypophosphorylated forms of pRb2 in MDA-MB-468 cells that was associated with accumulation of cells in G0/G1 and the appearance of a sub-G1 peak suggestive of apoptosis. In contrast, AZD3409 produced a G2 arrest associated with reduced expression of pRb2 in MDA-MB-361 cells. A synergistic anti-tumor effect was observed when MDA-MB-468 or MDA-MB-361 cells were treated with both AZD3409 and gefitinib, whereas this combination was only additive in SK-Br-3 cells. However, treatment of breast cancer cells with AZD3409 and gefitinib did not produce a more significant blockade of AKT signaling as compared with gefitinib alone. These data suggest that AZD3409 might be active in gefitinib-resistant breast carcinoma.     

Metastases to the human mandible

Two clinical cases are reported of mandibular swelling caused by metastatic jaw neoplasms. They analyse. The clinical characteristics of the swelling are analysed and all laboratory and radiographic tests performed are examined. It is concluded that metastatic localisations in the maxillofacial bone are very rare, but that the mandible is certainly the most frequent site. The authors also highlight the fact that the presence of metastatic lesions in the jaw may be the first symptom of the neoplastic disease.     

Unraveling the active conformation of urotensin II

Urotensin II (U-II) is a disulfide-bridged undecapeptide recently identified as the ligand of an orphan G-protein-coupled receptor. Human U-II (H-Glu-Thr-Pro-Asp-cyclo[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) has been described as the most potent vasoconstrictor compound identified to date.With the aim of elucidating the active conformation of hU-II, we have performed a spectroscopic analysis of hU-II minimal active fragment hU-II(4-11) in different environmental conditions. The analysis indicated that hU-II(4-11) was highly structured in the anisotropic membrane mimetic SDS solution, showing a type II' beta-turn structure, which is almost unprecedented for L-amino acid peptides. Micelle bound structure of hU-II(4-11) was then compared with those of four synthetic analogues recently synthesized in our lab, bearing modified Cys residues at position 5 and/or position 10 and characterized by different levels of agonist activity. The structures of the active compounds were found to be very similar to that of hU-II(4-11), while a barely active compound does not show any propensity to beta-turn formation. Furthermore, distances among putative pharmacophoric points in the structures of the active compounds obtained in SDS solution are in good agreement with those found in a recently described non-peptide agonist of the hU-II receptor. A type II' beta-turn structure was already found for the somatostatin analogue octreotide. On the basis of the similarity of the primary and 3D structures of U-II and somatostatin analogues and on the basis of the sequence homology between the GPR14/UT-II receptor and members of the somatostatin receptor family, a common evolutionary pathway for the signal transmission system activated by these peptide can be hypothesized.