Gregorio Costa

NK cells provide helper signal for CD8+ T cells by inducing the expression of membrane-bound IL-15 on DCs

NK cell recognition of cells that do not express or express low amounts of MHC class I molecules results not only in direct killing of target cells but also in the generation of specific T cell responses consequent to the induction of dendritic cell (DC) activation. While IL-12 production by NK cell-activated DCs is generally thought to play a critical role, a similar DC-mediated NK cell help has been reported also in IL-12-knockout mice. Here, we show that human NK cells can induce on DC surface membrane, via IFN-gamma secretion, the expression of high levels of IL-15. Remarkably, we show that DC expression of this membrane-bound form of IL-15, which is only partially associated with IL-15R molecules, is essential to promote specific CD8(+) T lymphocyte response in the absence of DC-derived IL-12.

Seroma fluid subsequent to axillary lymph node dissection for breast cancer derives from an accumulation of afferent lymph

Seroma is a frequent complication of breast cancer surgery, the etiology of which remains indefinite. It represents a subcutaneous accumulation of fluid frequently reported after surgical procedures such as axillary lymph node dissection. Despite previous studies have associated seroma fluid to an inflammatory exudate, the surgical removal of draining lymph nodes may indicate that seroma might not represent a mere exudate but rather an accrual of lymph drained from tributary tissues. To verify this hypothesis, seromas were collected at different intervals of time in patients operated upon for axillary lymph node removal. Fluids were analyzed in details by flow cytometry and biochemical assays for their cellular content and for their molecular features and relevant cytokine content. Lymphocytes and other peculiar blood mononuclear cells were present, while erythrocytes, platelets and granulocytes were absent or extremely rare. The protein concentration resulted lower (median 64%) than in peripheral blood. However, specific proteins related to locoregional tissues resulted highly concentrated (e.g. up to 500% for ferritin and 300% for lactate deydrogenase and exclusive presence of interleukin-6) whereas all enzymes and proteins synthesized in the liver or other organs (e.g. alkaline phosphatase, ALT, gammaGT, prealbumin, transferrin, ceruloplasmin, C3 and C4, alpha2 macroglobulin from liver; apolipoproteins from liver and gut; amylase and lipase from pancreas) were represented in reduced concentrations, thus ruling out that seroma proteins derive directly from blood serum. As a whole, this comprehensive cytological and molecular analysis provided evidences that seroma is constituted by serum ultrafiltrated-derived extracellular fluid of regions located upstream of removed lymph nodes. This fluid is then enriched by proteins and cells collected in the drained regions. Remarkably, seroma fluids collected in the same patient at different time points (up to 50 days following surgery) displayed similar biochemical features, clearly indicating that fluid composition was not significantly affected by post-surgical locoregional flogosis. Finally, the period of seroma formation indicates that lymph accumulates in the axillary region during the interval of time needed for afferent lymphatic vessels to re-anastomose with the efferent ducts. Therefore, seroma fluid represents a font of biological material suitable for investigating the biology of breast cancer, healing tissues and lymph.

Membrane Transfer from Tumor Cells Overcomes Deficient Phagocytic Ability of Plasmacytoid Dendritic Cells for the Acquisition and Presentation of Tumor Antigens

The potential contribution of plasmacytoid dendritic cells (pDCs) in the presentation of tumor cell Ags remains unclear, and some controversies exist with regard to the ability of pDCs to phagocytose cell-derived particulate Ags and cross-present them to MHC class I–restricted T lymphocytes. In this study, we show that human pDCs, although inefficient in the internalization of cell membrane fragments by phagocytosis, can efficiently acquire membrane patches and associated molecules from cancer cells of different histotypes. The transfer of membrane patches to pDCs occurred in a very short time and required cell-to-cell contact. Membrane transfer also included intact HLA complexes, and the acquired Ags could be efficiently recognized on pDCs by tumor-specific CD8+ T cells. Remarkably, pDCs isolated from human colon cancer tissues displayed a strong surface expression of epithelial cell adhesion molecule, indicating that the exchange of exogenous Ags between pDCs and tumor cells also can occur in vivo. These data demonstrate that pDCs are well suited to acquire membrane patches from contiguous tumor cells by a cell-to-cell contact–dependent mechanism that closely resembles “trogocytosis.” This phenomenon may allow pDCs to proficiently present tumor cell–derived Ags, despite limited properties of endophagocytosis.

Characterization of Human Afferent Lymph Dendritic Cells from Seroma Fluids

Dendritic cells (DCs) migrate from peripheral tissues to secondary lymphoid organs (SLOs) through the afferent lymph. Owing to limitations in investigating human lymph, DCs flowing in afferent lymph have not been properly characterized in humans until now. In this study, DCs present in seroma, an accrual of human afferent lymph occurring after lymph node surgical dissection, were isolated and analyzed in detail. Two main DC subsets were identified in seroma that corresponded to the migratory DC subsets present in lymph nodes, that is, CD14+ and CD1a+. The latter also included CD1abright Langerhans cells. The two DC subsets appeared to share the same monocytic precursor and to be developmentally related; both of them spontaneously released high levels of TGF-β and displayed similar T cell–activating and –polarizing properties. In contrast, they differed in the expression of surface molecules, including TLRs; in their phagocytic activity; and in the expression of proteins involved in Ag processing and presentation. It is worth noting that although both subsets were detected in seroma in the postsurgical inflammatory phase, only CD1a+ DCs migrated via afferent lymph under steady-state conditions. In conclusion, the high numbers of DCs contained in seroma fluids allowed a proper characterization of human DCs migrating via afferent lymph, revealing a continuous stream of DCs from peripheral regions toward SLOs under normal conditions. Moreover, we showed that, in inflammatory conditions, distinct subsets of DCs can migrate to SLOs via afferent lymph.

Intralesional sonographically guided injections of lymphokine-activated killer cells and recombinant interleukin-2 for the treatment of liver tumors: a pilot study.

Primary and metastatic liver cancers have a poor prognosis. At present, sonographically guided alcohol injection results in a partial reduction of cancer masses even if severe toxic effects (including pain and bleeding) are always present. For these reasons, a pilot study was started to evaluate the feasibility of an intralesional adoptive immunotherapeutic approach, using lymphokine-activated killer (LAK) cells and recombinant interleukin-2 (rIL-2). Nine patients (one with primary hepatocarcinoma and eight with liver metastases) entered the study. Four cycles of weekly injections of LAK cells (ranging from 2 to 9 x 10(8)) and 10(6) IU rIL-2 were performed percutaneously under ultrasonic guidance. In the same period, 3 x 10(6) IU rIL-2/day, for 24 days, was injected subcutaneously. All patients but one completed the therapy. Side effects were limited to grade 1-2 fever and were mostly related to rIL-2 subcutaneous injections. No patients complained of having pain during intralesional therapy. Two complete responses were detected. One partial response, four stable diseases, and one progressive disease were observed. One patient was not evaluable. These preliminary results suggest that sonographically guided intralesional adoptive immunotherapy of liver tumors is feasible, safe, and could offer promising therapeutic advantages in cancers for which conventional treatment is generally unsatisfactory.

Divergent signaling pathways regulate IL-12 production induced by different species of Lactobacilli in human dendritic cells.

Recent studies have indicated that different strains of Lactobacilli differ in their ability to regulate IL-12 production by dendritic cells (DCs), as some strains are stronger inducer of IL-12 while other are not and can even inhibit IL-12 production stimulated by IL-12-inducer Lactobacilli. In this report we demonstrate that Lactobacillus reuteri 5289, as previously described for other strains of L. reuteri, can inhibit DC production of IL-12 induced by Lactobacilllus acidophilus NCFM. Remarkably, L. reuteri 5289 was able to inhibit IL-12 production induced not only by Lactobacilli, as so far reported, but also by bacteria of different genera, including pathogens. We investigated in human DCs the signal transduction pathways involved in the inhibition of IL-12 production induced by L. reuteri 5289, showing that this potential anti-inflammatory activity, which is also accompanied by an elevated IL-10 production, is associated to a prolonged phosphorilation of ERK1/2 MAP kinase pathway. Improved understanding of the immune regulatory mechanisms exerted by Lactobacilli is crucial for a more precise employment of these commensal bacteria as probiotics in human immune-mediated pathologies, such as allergies or inflammatory bowel diseases.

Curcumin ameliorates the in vitro efficacy of carfilzomib in human multiple myeloma U266 cells targeting p53 and NF-κB pathways

Multiple myeloma (MM) is a malignant B-cell neoplasm with accumulation of malignant plasma cells in bone marrow. Pharmacological therapy improves response frequency even if with various associated toxicities. Herein, we investigated if combination of curcumin with carfilzomib (CFZ) can induce a better cytotoxic effect on in vitro cultured U266 cells. Cell viability data showed that curcumin significantly ameliorates CFZ cytotoxic effect. Furthermore, curcumin alone did not affect proteasome at the tested dose, confirming the involvement of different mechanisms in the observed effects. U266 cells exposure to curcumin or CFZ increased reactive species (RS) levels, although their production did not appear further potentiated following drugs combination. Interestingly, NF-κB nuclear accumulation was reduced by treatment with CFZ or curcumin, and was more deeply decreased in cells treated with CFZ-curcumin combinations, very likely due to the different mechanisms through which they target NF-κB. Our results confirmed the induction of p53/p21 axis and G0/G1 cell cycle arrest in anticancer activities of both drugs, an effect more pronounced for the CFZ-curcumin tested combinations. Furthermore, curcumin addition enhanced CFZ proapoptotic effect. These findings evidence that curcumin can ameliorate CFZ efficacy, and lead us to hypothesize that this effect might be useful to optimize CFZ therapy in MM patients.

Micropapillary Carcinoma of the Breast with Necrosis-like Cell Death: A Case Report

A primary invasive micropapillary carcinoma of the breast in a 46-year-old woman is reported. Histologically, it was composed predominantly of papillary tumor cell clusters without fibrovascular cores, surrounded by a clear space. Tumor cells were positive for cytokeratin (CK) 7, estrogen receptor (ER), and progesterone receptor (PR), but negative for p53, CK 20, CD34, c-Erb-B2, CK5, epidermal growth factor receptor (EGFR), vimentin, and c-kit. MUC1 expression was found at the reversed apical membrane of neoplastic cell clusters. Accordingly, electron microscopy showed the lack of basement membrane and presence of microvilli at the basal surface of the tumor cells. Moreover, ultrastructural examination revealed single tumor cell death characterized by patchy condensations of chromatin throughout the nucleus. These nuclear alterations were associated with the occurrence of empty cytoplasmic vacuoles, conferring a necrosis-like phenotype to this cell death. Alternative programmed cell deaths are reviewed and their morphologic distinction is discussed.

Morphological changes of follicular cell basal borders and basement membranes in benign and malignant nodular lesions of the thyroid gland: an ultrastructural study.

Microfollicular nodular lesions of the thyroid gland may represent a differential diagnosis problem. Firstly, nodular areas of follicular hyperplasia have to be distinguished from follicular adenomas. On the other hand, nodular microfollicular areas exhibiting large pale nuclei, occasionally found in hyperplastic nodules and follicular adenomas, must be discriminated from latent papillary carcinomas with predominant follicular architecture. The diagnosis of follicular carcinoma still requires the detection of vascular and/or capsular microinvasion. A more refined study was planned to search for additional descriptors useful for diagnosis The authors report the results of an ultrastructural investigation carried out on 220 thyroid nodular lesions and 50 specimens of macroscopically nonnodular glands. An infolding arrangements of the thyreocyte basal border(TBB) and follicular basement membrane (FBM) was demonstrated in 50/50 nonnodular thyroid tissue specimens and 53/67 (79.1%) hyperplastic nodular lesions (p < .005). A linear arrangement of the TBB and FBM was found in 85/121(70.2%) follicular adenomas and in 32/32 differentiated carcinomas (p < .001). In the last group, 12/32 (37.5%) cases showed focal discontinuities of FBM. In conclusion, the benign thyroid nodules show a prevalently infolding arrangements of TBBs, whereas the majority of proliferative lesions display a linear morphology. In absence of an infiltrating pattern there is no morphological evidence of discriminating potentially malignant vs. benign lesions. The linear distribution of TBBs and FBMs places the case in a group of borderline lesions that involve a more careful postsurgery investigation.