Elisabetta Ugolotti

High levels of PROM1 (CD133) transcript are a potential predictor of poor prognosis in medulloblastoma

The surface marker PROM1 is considered one of the most important markers of tumor-initiating cells, and its expression is believed to be an adverse prognostic factor in gliomas and in other malignancies. To date, to our knowledge, no specific studies of its expression in medulloblastoma series have been performed. The aims of our study were to evaluate the expression profile of the PROM1 gene in medulloblastoma and to assess its possible role as a prognostic factor. The PROM1 gene expression was evaluated by quantitative- polymerase chain reaction on 45 medulloblastoma samples by using specific dye-labeled probe systems. A significantly higher expression of PROM1 was found both in patients with poorer prognosis (P= .007) and in those with metastasis (P= .03). Kaplan-Meier analysis showed that both overall survival (OS) and progression-free survival (PFS) were shorter in patients with higher PROM1 mRNA levels than in patients with lower expression, even when the desmoplastic cases were excluded (P= .0004 and P= .002, for OS and PFS for all cases, respectively; P= .002 and P= .008 for OS and PFS for nondesmoplastic cases, respectively). Cox regression model demonstrated that PROM1 expression is an independent prognostic factor (hazard ratio, 4.56; P= .008). The result was validated on an independent cohort of 42 cases by microarray-based analysis (P= .019). This work suggests that high mRNA levels of PROM1 are associated with poor outcome in pediatric medulloblastoma. Furthermore, high PROM1 expression levels seem to increase the likelihood of metastases. Such results need to be confirmed in larger prospective series to possibly incorporate PROM1 gene expression into risk classification systems to be used in the clinical setting.

NK Cell Receptors and Their Interactions with MHC

MHC-specific Natural Killer inhibitory receptors display a conserved and fundamental function in the regulation of NK-mediated cytolysis. Their importance is substantiated by the fact that during speciation different molecular receptor structures have evolved to maintain inhibitory regulation of NK cells. The information gained during these last twenty years begins to be fruitfully used in the therapy of leukemias, but a lot has to be still done. In particular, we need to understand the role of activating KIR and their ligand(s), since their role in the course of different viral diseases is still intriguing.

CD8+ NK cells are predominant in chimpanzees, characterized by high NCR expression and cytokine production, and preserved in chronic HIV-1 infection

HIV‐1 infection in humans results in an early and progressive NK cell dysfunction and an accumulation of an “anergic” CD56− CD16+ NK subset, which is characterised by low natural cytotoxicity receptor expression and low cytokine producing capacity. In contrast to humans, chimpanzee NK cells do not display a distinguishable CD56bright and CD56dim subset but, as shown here, could be subdivided into functionally different CD8+ and CD8− subsets. The CD8+ NK cells expressed significantly higher levels of triggering receptors including NKp46 and, upon in vitro activation, produced more IFN‐γ, TNF‐α and CD107 than their CD8− counterparts. In addition, chimpanzee CD8− NK cells had relatively high levels of HLA‐DR expression, suggestive of an activated state. Killing inhibitory receptors were expressed only at low levels; however, upon in vitro stimulation, they were up‐regulated in CD8+ but not in CD8− NK cells and were functionally capable of inhibiting NKp30‐triggered killing. In contrast to HIV‐1‐infected humans, infected chimpanzees maintained their dominant CD8+ NK cell population, with high expression of natural cytotoxicity receptors.

Whole-genome sequencing as standard practice for the analysis of clonality in outbreaks of meticillin-resistant Staphylococcus aureus in a paediatric setting.

Meticillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of hospital-associated infections. This study investigated the potential use of whole-genome sequencing (WGS) for surveillance purposes by re-examining MRSA strains related to past outbreaks among hospitalized paediatric patients. WGS data ameliorated the genotypic profile previously obtained with Sanger sequencing and pulsed-field gel electrophoresis typing, and discriminated between strains that were related and unrelated to the outbreaks. This allowed strain clonality to be defined with a higher level of resolution than achieved previously. This study demonstrates the potential of WGS to trace hospital outbreaks, which may lead to WGS becoming standard practice in outbreak investigations.

Activating Killer Immunoglobulin Receptors and HLA-C: A successful combination providing HIV-1 control

Several studies demonstrated a relevant role of polymorphisms located within the HLA-B and -C loci and the Killer Immunoglobulin Receptors (KIRs) 3DL1 and 3DS1 in controlling HIV-1 replication. KIRs are regulatory receptors expressed at the surface of NK and CD8+ T-cells that specifically bind HLA-A and -B alleles belonging to the Bw4 supratype and all the -C alleles expressing the C1 or C2 supratype. We here disclose a novel signature associated with the Elite Controller but not with the long-term nonprogressor status concerning 2DS activating KIRs and HLA-C2 alleles insensitive to miRNA148a regulation. Overall, our findings support a crucial role of NK cells in the control of HIV-1 viremia.

Human leukocyte antigen–B (-Bw6/-Bw4 I80, T80) and human leukocyte antigen–C (-C1/-C2) subgrouping using pyrosequence analysis

Specific combinations of killer immunoglobulin-like receptors (KIR) and human leukocyte antigen (HLA) class I molecules characterized by a particular residue 80 are significantly associated with outcomes in different pathologic conditions, such as autoimmunity, pathogenic infection, cancer, and reproductive failure. Thus, a simplified method for HLA typing used in association with the analysis of KIR genotype (Kirotype) is of particular interest to extend the analysis of larger series. Here, we describe a quick and inexpensive method that allows use of pyrosequencing, a helpful subtyping of HLA class I molecules, into HLA-Bw6, -Bw4 I(80) or -Bw4 T(80), HLA-C1, or -C2 groups and HLA-A allotypes sharing Bw4+ epitope or the rare HLA-B allotypes displaying the C1 motif. In particular, this analysis is focused on the amino acids around residue 80, known to be relevant in defining the affinity of KIR/HLA interaction and in the functional effects. This method was demonstrated to have good sensitivity, specificity, and reproducibility of detection and it was validated using a panel of HLA-typed International Histocompatibility Workshop (IHW) cell lines and clinical isolates. Using an allele quantitative acquisition mode, the method permitted us to obtain an accurate sequencing as required in heterozygous and/or homozygous sample definition.

Troubleshooting Fine-Tuning Procedures for qPCR System Design

Quantitative real‐time PCR (qPCR) has been improved and optimized over the past decade for a wide range of applications. Design of primers and probes is one of the crucial steps to obtain high system efficiency of qPCR since design pitfalls influence negatively amplification performances. We report the results of some experiments. First, we demonstrate the utility of optimal primer design and concentration in PCR by constructing suboptimal primers, for instance with hairpin and primer–dimers secondarystructures, and quantifying the decrease in efficiency of amplification. Second, we show the adverse effects of the target sequence harboring stable secondary structures on the primer binding sites. Finally, we let see that the mere use of probe‐based detection is not enough to ensure robustness of qPCR data, because the eventual detrimental products generated by primers not well designed may influence in any case the PCR efficiency. J. Clin. Lab. Anal. 25:389–394, 2011.

Receptor modulation and functional activation of human CD34+Lin−‐derived immature NK cells in vitro by Mycobacterium bovis Bacillus Calmette‐Guerin (BCG)

It is not yet clear whether immature NK (iNK) cells are bystanders to or rather participate in immune responses to pathogens that may colocalize in areas of NK‐cell maturation such as bone marrow or lymph nodes. Mycobacteria, including Bacillus Calmette‐Guerin (BCG), have been shown to interact with peripheral NK cells and in vivo may colocalize in areas of iNK‐cell development. We studied infection with BCG of human cord blood CD34+Lin−‐derived cultures containing myelomonocytes and iNK cells in vitro. Increased iNK‐cell DNAM‐1 expression, transient natural cytotoxicity receptor modulation, and production of IFN‐γ were observed. Transcriptional receptor modulation was associated to BCG challenge, which determined increased iNK‐cell cytotoxic activity against tumor cell lines and also increased killing of immature dendritic cells (iDCs). No requirement for cell contact was recorded for BCG‐induced iNK‐cell activation, while cytokine production including IL‐18, IL‐10, GM‐CSF, and TGF‐β contributed to the observed effects. Thus, iNK cells are affected by mycobacteria in vitro and may contribute to shaping of adaptive mature innate responses through iDC‐iNK cross‐talk. In addition, iNK‐cell activation by BCG may represent a novel additional mechanism contributing to the effects observed upon BCG administration in vivo.

Stability and expression levels of HLA-C on the cell membrane modulate HIV-1 infectivity

ABSTRACT HLA-C expression is associated with a differential ability to control HIV-1 infection. Higher HLA-C levels may lead to better control of HIV-1 infection through both a higher efficiency of antigen presentation to cytotoxic T lymphocytes and the triggering of activating killer immunoglobulin-like receptors on NK cells, whereas lower levels may provide poor HIV-1 control and rapid progression to AIDS. We characterized the relative amounts of HLA-C heterotrimers (heavy chain/β2 microglobulin [β2m]/peptide) and HLA-C free heavy chains on peripheral blood mononuclear cells (PBMCs) from healthy blood donors harboring both alleles with stable or unstable binding to β2m/peptide. We analyzed the stability of HLA-C heterotrimers of different allotypes and the infectivity of HIV-1 virions produced by PBMCs with various allotypes. We observed significant differences in HLA-C heterotrimer stability and in expression levels. We found that R5 HIV-1 virions produced by PBMCs harboring unstable HLA-C alleles were more infectious than those produced by PBMCs carrying the stable variants. We propose that HIV-1 infectivity might depend both on the amounts of HLA-C molecules and on their stability as trimeric complex. According to this model, individuals with low-expression HLA-C alleles and unstable binding to β2m/peptide might have worse control of HIV-1 infection and an intrinsically higher capacity to support viral replication. IMPORTANCE Following HIV-1 infection, some people advance rapidly to AIDS while others have slow disease progression. HLA-C, a molecule involved in immunity, is a key determinant of HIV-1 control. Here we reveal how HLA-C variants contribute to the modulation of viral infectivity. HLA-C is present on the cell surface in two different conformations. The immunologically active conformation is part of a complex that includes β2 microglobulin/peptide; the other conformation is not bound to β2 microglobulin/peptide and can associate with HIV-1, increasing its infectivity. Individuals with HLA-C variants with a predominance of immunologically active conformations would display stronger immunity to HIV-1, reduced viral infectivity and effective control of HIV-1 infection, while subjects with HLA-C variants that easily dissociate from β2 microglobulin/peptide would have a reduced immunological response to HIV-1 and produce more infectious virions. This study provides new information that could be useful in the design of novel vaccine strategies and therapeutic approaches to HIV-1.

HLA-B and HLA-C Supratyping by Pyrosequencing ®

Usually, HLA typing has been performed either by serology-based typing incubating a panel of known anti-HLA antibodies with viable lymphocytes of unknown HLA type or by molecular typing including medium-resolution HLA typing by Sequence Specific Oligonucleotide Probes (SSOP) or high-resolution HLA typing by Sequence Based Typing (SBT). Traditionally, HLA antigens have been defined using serological techniques, but these methods have several disadvantages, such as low resolution, the requirement for viable cells, and cell surface expression of HLA molecules. HLA type screening methods are categorized as low, medium, and high resolution, and only sequencing-based typing methods provide the highest resolution and are considered the gold standard for HLA typing.Among the HLA SBT based-methods, the Pyrosequencing(®) technique is an extremely versatile and accurate real-time sequencing technique with some advantages compared to classic Sanger method.Here, we describe a quick and inexpensive method that allows through the use of Pyrosequencing subtyping of HLA class I molecules, into HLA-Bw6, -Bw4 I80, or -Bw4 T80 and HLA-C1, or -C2 groups. In particular, this analysis is focused on the amino acids around residue 80. This method demonstrated good sensitivity, specificity, and reproducibility. Using a quantitative allele acquisition mode, the method provides accurate sequence information required for the definition of heterozygous and/or homozygous samples.