Giovanna Angelini

Antigen-presenting dendritic cells provide the reducing extracellular microenvironment required for T lymphocyte activation

T lymphocytes are defective in cystine uptake and thus require exogenous thiols for activation and function. Here we show that monocyte-derived human dendritic cells (DCs) release cysteine in the extracellular space. Cysteine generation is increased by lipopolysaccharide and tumor necrosis factor α, and by contact with T cells specifically recognizing soluble or alloantigens. These stimuli also induce thioredoxin (TRX) accumulation in DCs. However, only the contact with antigen-specific T cells triggers TRX secretion by the antigen-presenting cells. Fewer extracellular thiols are recovered after DC–T cell interactions when cystine uptake or TRX activity are inhibited. In addition, glutamate (Glu) and anti-TRX-inactivating antibodies inhibit antigen-dependent T lymphocyte proliferation. These findings indicate that, during antigen presentation, DCs uptake cystine and release cysteine and TRX, thus providing a reducing microenvironment that facilitates immune response.

HLA-DP typing by DNA amplification and hybridization with specific oligonucleotides.

HLA-DP genotyping was performed using dot-blot analysis with synthetic oligonucleotide probes. Fourteen probes were designed based on the known sequence variations in the polymorphic segments of the DP beta second exon. Each probe was tested against genomic DNA amplified by the polymerase chain reaction, using DP beta-specific primers. A total of 45 HLA homozygous B-cell lines, selected from the Tenth International Histocompatability Workshop and pretyped for the known DP omega specificities, were analyzed. Different hybridization patterns were found for each DP omega specificity. The oligonucleotide hybridization performed on DP omega-negative B-cell lines gave a pattern distinct from those of known DP omega specificities, indicating the presence of novel DP allelic sequences. The use of sequence-specific oligonucleotides combined with DNA amplification allows a simple and reliable genotyping of DP antigens.

The thiol redox state of lymphoid organs is modified by immunization: Role of different immune cell populations†

Resting T lymphocytes can internalize reduced cysteine (Cys) but not cystine, the oxidized form of the amino acid that predominates extracellularly. In vitro studies have shown that DC provide Cys to T cells during antigen presentation, allowing their activation. Here, we show that increased thiol production is a hallmark of immune response in vivo. Indeed, the thiol content of LN increases dramatically after antigen injection. Non‐protein thiols co‐distribute with DC and are highly abundant in germinal centers. In agreement, activated but not resting B lymphocytes and macrophages release free thiols. Increased thiol release following activation requires thioredoxin and is paralleled by increased thioredoxin expression. The T zones of LN are consistently less stained, and both resting and activated T cells are unable to release thiols. Interestingly, the cystine/glutamate transporter x  c− is absent in resting T lymphocytes but is rapidly induced by TCR triggering in vitro, indicating that the release of T cells from the need of exogenous Cys occurs early after activation. These results indicate that a reducing microenvironment is essential to start the immune response but dispensable for its evolution, and support the emerging concept that extracellular redox is implicated in the control of crucial cellular functions.

Direct inhibition of hexokinase activity by metformin at least partially impairs glucose metabolism and tumor growth in experimental breast cancer

Emerging evidence suggests that metformin, a widely used anti-diabetic drug, may be useful in the prevention and treatment of different cancers. In the present study, we demonstrate that metformin directly inhibits the enzymatic function of hexokinase (HK) I and II in a cell line of triple-negative breast cancer (MDA-MB-231). The inhibition is selective for these isoforms, as documented by experiments with purified HK I and II as well as with cell lysates. Measurements of 18F-fluoro-deoxyglycose uptake document that it is dose- and time-dependent and powerful enough to virtually abolish glucose consumption despite unchanged availability of membrane glucose transporters. The profound energetic imbalance activates phosphorylation and is subsequently followed by cell death. More importantly, the “in vivo” relevance of this effect is confirmed by studies of orthotopic xenografts of MDA-MB-231 cells in athymic (nu/nu) mice. Administration of high drug doses after tumor development caused an evident tumor necrosis in a time as short as 48 h. On the other hand, 1 mo metformin treatment markedly reduced cancer glucose consumption and growth. Taken together, our results strongly suggest that HK inhibition contributes to metformin therapeutic and preventive potential in breast cancer.

The maturation potential of NK cell clones toward autologous dendritic cells correlates with HMGB1 secretion

Interaction of NK cells with autologous immature dendritic cells (iDCs) results in reciprocal activation. We have previously reported that NK cells trigger iDC to polarize and secrete IL‐18; in turn, DC‐activated NK cells secrete the nuclear protein/proinflammatory cytokine high mobility group box protein 1 (HMGB1), which induces DC maturation and prevents DC from lysis. However, activated NK cells can also kill iDC. To investigate whether effector and maturative properties may coexist or segregate in different NK subsets, human NK cell clones were generated and analyzed for their effects on iDC. We found that the ability of different NK cell clones to induce iDC maturation is unlinked to their phenotypic and cytolytic features but correlates with the relocation of HMGB1 from nucleus to cytoplasm. “Maturative” NK cell clones secrete HMGB1 spontaneously. It is interesting that secretion is strongly enhanced by engagement of the surface molecule NKp30 but only slightly induced by triggering of the activating NK receptor CD16. However, culturing freshly isolated NK cells for 1 week with low doses of anti‐CD16 triggers the relocation of HMGB1 from nucleus to cytoplasm and its spontaneous secretion, resulting in a stronger maturation potential of the NK cells. Together, our data indicate that NK cells comprise functionally different subsets, endowed with different capacities to secrete HMGB1 and to induce maturation of autologous iDC. Nonetheless, maturation properties can be modulated by different stimuli. This suggests that depending on the environmental stimuli, NK/iDC interaction can lead to different outcomes, thus influencing immune response.

Overexpression of the ATP binding cassette gene ABCA1 determines resistance to Curcumin in M14 melanoma cells

BackgroundCurcumin induces apoptosis in many cancer cells and it reduces xenograft growth and the formation of lung metastases in nude mice. Moreover, the plant derived polyphenol has been reported to be able to overcome drug resistance to classical chemotherapy. These features render the drug a promising candidate for tumor therapy especially for cancers known for their high rates concerning therapy resistance like melanoma.ResultsWe show here that the melanoma cell line M14 is resistant to Curcumin induced apoptosis, which correlates with the absence of any effect on NFκB signaling. We show that CXCL1 a chemokine that is down regulated in breast cancer cells by Curcumin in an NFκB dependant manner is expressed at variable levels in human melanomas. Yet in M14 cells, CXCL1 expression did not change upon Curcumin treatment. Following the hypothesis that Curcumin is rapidly removed from the resistant cells, we analyzed expression of known multi drug resistance genes and cellular transporters in M14 melanoma cells and in the Curcumin sensitive breast cancer cell line MDA-MB-231. ATP-binding cassette transporter ABCA1, a gene involved in the cellular lipid removal pathway is over-expressed in resistant M14 melanoma as compared to the sensitive MDA-MB-231 breast cancer cells. Gene silencing of ABCA1 by siRNA sensitizes M14 cells to the apoptotic effect of Curcumin most likely as a result of reduced basal levels of active NFκB. Moreover, ABCA1 silencing alone also induces apoptosis and reduces p65 expression.ConclusionResistance to Curcumin thus follows classical pathways and ABCA1 expression should be considered as response marker.

Mda-9/syntenin is expressed in uveal melanoma and correlates with metastatic progression.

Uveal melanoma is an aggressive cancer that metastasizes to the liver in about half of the patients, with a high lethality rate. Identification of patients at high risk of metastases may provide indication for a frequent follow-up for early detection of metastases and treatment. The analysis of the gene expression profiles of primary human uveal melanomas showed high expression of SDCBP gene (encoding for syndecan-binding protein-1 or mda-9/syntenin), which appeared higher in patients with recurrence, whereas expression of syndecans was lower and unrelated to progression. Moreover, we found that high expression of SDCBP gene was related to metastatic progression in two additional independent datasets of uveal melanoma patients. More importantly, immunohistochemistry showed that high expression of mda-9/syntenin protein in primary tumors was significantly related to metastatic recurrence in our cohort of patients. Mda-9/syntenin expression was confirmed by RT-PCR, immunofluorescence and immunohistochemistry in cultured uveal melanoma cells or primary tumors. Interestingly, mda-9/syntenin showed both cytoplasmic and nuclear localization in cell lines and in a fraction of patients, suggesting its possible involvement in nuclear functions. A pseudo-metastatic model of uveal melanoma to the liver was developed in NOD/SCID/IL2Rγ null mice and the study of mda-9/syntenin expression in primary and metastatic lesions revealed higher mda-9/syntenin in metastases. The inhibition of SDCBP expression by siRNA impaired the ability of uveal melanoma cells to migrate in a wound–healing assay. Moreover, silencing of SDCBP in mda-9/syntenin-high uveal melanoma cells inhibited the hepatocyte growth factor (HGF)-triggered invasion of matrigel membranes and inhibited the activation of FAK, AKT and Src. Conversely syntenin overexpression in mda-9/syntenin-low uveal melanoma cells mediated opposite effects. These results suggest that mda-9/syntenin is involved in uveal melanoma progression and that it warrants further investigation as a candidate molecular marker of metastases and a potential therapeutic target.

Frequency of DPB1 ∗0401 is significantly decreased in patients with allergic asthma in a Mulatto population

Allergic asthma (AA) is a multifactorial disease in which the IgE hyperresponsiveness to mite allergens is determinant for its pathogenesis and clinical picture. We have reported previously that IgE responsiveness to mite allergens in AA patients is linked to HLA and possibly controlled by a dominant suppression (Is) gene of that region. The present population study was done to detect alleles involved in the genetic control of mite IgE response that accompanies AA, using polymerase chain reaction and oligonucleotide DNA typing of DP locus. Instead of finding any significant positive association with AA, in this study we found that the allele DPB1*0401 is present mainly in the nonallergic control population and strikingly absent in patients (p less than 0.008), suggesting that this gene could confer resistance to AA and other atopic diseases. Our results add more evidence regarding the existence of Is genes in the HLA region involved in the control of IgE immune response to environmental allergens. Furthermore, they suggest that genes of HLA are important genetic components involved in the etiology of AA.

A prognostic multigene classifier for squamous cell carcinomas of the larynx.

Survival after diagnosis of laryngeal cancer has not improved over the last 20 years. Selection of patients for radio- and chemotherapy or surgery or follow-up strategies based on a prognostic classifier could improve survival without unduly extending radical surgery. We performed microarray gene expression analysis and developed a four-gene classifier for laryngeal cancer using Prediction Analysis of Microarray and leave-one-out cross validation. A four-gene classifier containing the non-coding gene H19, the histone HIST1H3F and the two small nucleolar RNAs, SNORA16A and SNORD14C was developed that assigns cases to low and high risk classes. The high risk class has a relative risk of 6.5 (CI=1.817-23.258, Fisher exact test p<0.0001). The maternally imprinted gene H19 is the top classifier gene.

Analysis of HLA DP, DQ, and DR allesles in adult Italian rheumatoid arthritis patients

We analyzed the distribution of DRB1, DQA1, DQB1, and DPB1 allelic variants in 48 rheumatoid arthritis (RA) patients, compared with 109 Italian random controls, using PCR amplification and hybridization with specific oligonucleotides. We confirm the previously reported increase of DR4 specificity, in comparison with healthy Italian individuals. In particular, we find a statistically significant positive association of DRB1*0401 and DRB1*0404 alleles with RA. However, when we compare the DR4+ groups, none of the DRB1*04 alleles is increased in the RA group. By sequence analysis, performed on 10 patients, we demonstrate that the DRB1*04 genes of RA show no difference from the DRB1*04 sequences previously published. From the molecular analysis of the other DRB1 polymorphic variants, we find a trend of positive association of DRB1*0101 in DR4-negative patients versus DR4-negative healthy controls and, in the group of DR4-negative and/or DR1-negative patients, a similar increase of DRB1*06. Also, we observe in RA patients a statistically significant increase of DQA1*0301 and DQB1*0302 accompanied by a significant decrease of DQA1*0201, DQA1*0501 and DQB1*0201. Finally, from the analysis of DPB1 gene, it can be assessed that the distribution of DPB1 alleles does not differ significantly between RA patients and healthy controls.