Rosa Di Noto

Novel Synthetic, Salt-Resistant Analogs of Human Beta-Defensins 1 and 3 Endowed with Enhanced Antimicrobial Activity

ABSTRACT Human beta-defensins (hBDs) are antimicrobial peptides of human innate immunity. The antibacterial activities of hBDs 1, 2, and 4 but not the activity of hBD3 are impaired by high salt levels. We have designed and synthesized seven novel hBD analogs, constituted by different domains of hBD1 (which is constitutively expressed in humans) and of hBD3 (which is induced by microorganisms and inflammatory factors in humans), that would maintain and potentially increase the wild-type antimicrobial activities and be salt resistant. We have compared the antibacterial, antiviral, and chemotactic activities of the analogs with those of hBD1 and hBD3. We show that the hBD1 internal region and the hBD3 C-terminal region are critical for antibacterial activity also at high salt concentrations, whereas deletion of the N-terminal region of hBD3 results in an increase in antibacterial activity. All analogs inhibited herpes simplex virus; antiviral activity was enhanced by the hBD1 internal region and the hBD3 C-terminal region. Wild-type and analog peptides were chemotactic for granulocytes and monocytes, irrespective of the salt concentrations. These new peptides may have therapeutic potential.

Combined CD133/CD44 expression as a prognostic indicator of disease-free survival in patients with colorectal cancer.

HYPOTHESIS Because of some inconsistencies in the traditional model of human colorectal carcinogenesis, the cancer stem cell (CSC) model was recently proposed, in which tumor results from neoplastic transformation of stem cells, which become CSCs. Identification of CSCs by expression of surface antigens remains a critical issue because no biomarker has been shown to be completely reliable. CD133 and CD44 are commonly used as CSC markers, and correlation of their expression with colorectal cancer (CRC) clinicopathological features and outcomes may be useful. DESIGN Pilot study. SETTING University hospital. PATIENTS Thirty-six consecutive patients with CRC. CD133 and CD44 expression (alone or combined) was determined in nontumor cells and in tumor cells by flow cytometry, which identified viable cells only. MAIN OUTCOME MEASURES Correlation of CD133 and CD44 expression with each other, with other prognostic indicators, and with disease-free survival. RESULTS CD133 and CD44 expression was significantly higher in tumor cells than in nontumor cells, and expression of one did not necessarily correlate with expression of the other. CD133 or CD44 expression alone was variable, while combined CD133/CD44 expression identified a small subset of cells positive for CRC. CD133 or CD44 overexpression was not associated with CRC recurrence; only high frequencies of CD133(+)/CD44(+) cells were a strong indicator of worse disease-free survival and an independent risk factor for CRC recurrence. CONCLUSION Evaluation of combined CD133/CD44 expression could be useful to identify putative colorectal CSCs and tumors with a poor prognosis.

CD66c is a novel marker for colorectal cancer stem cell isolation, and its silencing halts tumor growth in vivo

Despite the well recognized expression of the cell surface markers cluster of differentiation 44 (homing cell adhesion molecule) and CD133 (Prominin 1) on human colorectal cancer stem cells (CCSCs), these molecules do not appear to be effective targets for stem cell‐directed therapies. Because the surface marker CD66c (also known as carcinoembryonic antigen‐related cell adhesion molecule 6) has demonstrated promise as a therapeutic target in pancreatic malignancy, the authors evaluated its potential as a target for stem cell‐directed treatment of colorectal cancer.

CD200/OX2, a cell surface molecule with immuno-regulatory function, is consistently expressed on hairy cell leukaemia neoplastic cells

CD200 (formerly called OX2) is a transmembrane glycoprotein with immunosuppressive functions. It is expressed on normal B-lymphocytes, T-lymphocytes, dendritic cells and several solid tissues (Kawasaki & Farrar, 2008). CD200 receptor expression is limited to myeloid leucocytes and a subset of Tlymphocytes (Kawasaki & Farrar, 2008). In mouse systems, the binding of CD200 to its receptor (i) decreases the production of T-helper cell type 1 (Th1)-like cytokines, such as interleukin (IL)-2 and interferon c, (ii) increases the release of Th2-like cytokines, such as IL-10 and IL-4 (Gorczynski, 2001) and (iii) promotes the in vitro differentiation of T cells toward CD4CD25Foxp3 Treg lymphocytes (Gorczynski et al, 2008). CD200 is constantly overexpressed on chronic lymphocytic leukaemia (CLL) cells (McWhirter et al, 2006). The addition of CLL cells to mixed lymphocyte reactions causes an immunological shift from a Th1-like response to a Th2-like response, confirming that CD200 plays an important role in controlling T-cytotoxic immune response (McWhirter et al, 2006). Starting from these data, we extended the investigation of CD200 expression to another B-chronic lymphoproliferative disorder i.e. hairy cell leukaemia (HCL). Hairy cell leukaemia is a distinct disease entity in the World Health Organization (WHO) classification, displaying unique clinico-pathological and biological features (Tiacci et al, 2006). As hairy cells display a specific immunophenotype, multicolour flow cytometry is currently the best tool for HCL diagnosis. A total of 10 specimens (six peripheral blood samples and four bone marrow aspirates), collected from 10 patients with newly diagnosed HCL, were studied. As normal controls we analysed 10 peripheral blood specimens and two bone marrow aspirates from 12 healthy donors. An aliquot (50 ll) of each sample was incubated at 4 C for 30 min in the presence of appropriate amounts of monoclonal antibodies. The mixtures were then diluted 1:20 in ammonium chloride lysing solution, incubated at room temperature for 10 min and finally washed prior to flow cytometric analysis with the FACSCanto II flow cytometer (Becton Dickinson, San Jose, CA, USA). The following antigens were analysed: CD200, SmIg-kappa, SmIg-lambda, CD45, CD19, CD5, CD23, CD20, CD22, CD103, CD11c, CD25, CD43, CD10, CD3, CD56 and CD81. Hairy cells were gated as CD45CD19 ‘monocytoid cells’ (i.e. cells with light scatter features typical of monocytes). In addition, in the majority of cases, we also were able to perform a full immunological gate on CD45 CD19CD103CD11c cells. With regard to the normal controls included in our study, B-lymphocytes were simply gated as CD45CD19 cells. In all specimens cell doublets and debris were excluded from our analysis by forward-scatter versus side-scatter dotplot examination. To set the cut-off point to distinguish between CD200 negative and positive cells, we used the ‘Fluorescence Minus One’ technique as described by Perfetto et al (2004). A single case was arbitrarily judged CD200 positive when the percentage of positive cells (PPC) was higher than 30%. All HCL samples were CD200 positive with PPC and median fluorescence intensity (MFI) median values of 99 (25th–75th percentile 92–99) and 3016 (25th–75th percentile 1382–5430), respectively. Although CD200 was positive in 12 out of 12 normal controls, the PPC and MFI median values were of 71 (25th–75th percentile 64–83) and 582 (25th–75th percentile 406–725), respectively. Differences in PPCs and MFIs between HCLs and normal controls were statistically significant (Mann–Whitney U, two-tailed testing, P < 0Æ0001). Data regarding MFI analysis are shown in Fig 1. Whereas normal controls showed weak CD200 fluorescence intensity with a bimodal distribution, HCL samples showed bright CD200 expression in a homogeneous pattern (Fig 2). This is the first documented direct evidence of CD200 overexpression in HCL. As described above, CD200 promotes Th2-like cytokines synthesis. IL-4 and IL-10 are reported to reduce anti-tumour cytotoxic T cell response (McWhirter

Protein cross-talk in CD133+ colon cancer cells indicates activation of the Wnt pathway and upregulation of SRp20 that is potentially involved in tumorigenicity

The cancer stem cell (CSC) theory represents a breakthrough in cancer research. We characterized the protein pattern of CSCs to identify specific intracellular pathways in this subpopulation of tumor cells. We studied colon CSCs using two different colon cancer cell lines: CaCo‐2 and HCT‐116. Putative CSCs were separated from non‐CSCs by flow cytometry using CD133 as stemness marker. Total protein extracts of CD133+ cells were then compared to protein extracts of CD133– cells by 2D DIGE. The protein spots differentially expressed in the two subpopulations of cells were analyzed by mass spectrometry. Bioinformatics analysis of the identified proteins indicated alteration of two main processes: energy metabolism and the Wnt pathway. Interestingly, we observed upregulation of the splicing factor SRp20, a newly identified target gene of the Wnt/β‐catenin pathway, and we demonstrated a direct cause–effect relationship between Wnt pathway activation and the increased SRp20 expression. Our results also show that SRp20 influences cell proliferation, which suggests it plays a role in the tumorigenicity of CD133+ cells. In conclusion, activation of the Wnt pathway in CD133+ cells and upregulation of SRp20, which is implicated in tumorigenesis, raises the possibility of a sequential series of molecular events occurring in connection with this process.

Extended flow cytometry characterization of normal bone marrow progenitor cells by simultaneous detection of aldehyde dehydrogenase and early hematopoietic antigens: implication for erythroid differentiation studies.

BackgroundAldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood, as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow, detailed characterization of ALDH+ cells has been addressed by one single study (Gentry et al, 2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens, with particular attention to the expression of ALDH on erythroid precursors. To this aim, we used three kinds of approach: i) multidimensional analytical flow cytometry, detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells, followed by in vitro induction of erythroid differentiation; iii) detection of ALDH+ cellular subsets in bone marrow from pure red cell aplasia patients.ResultsIn normal bone marrow, we identified three populations of cells, namely ALDH+CD34+, ALDH-CD34+ and ALDH+CD34- (median percentages were 0.52, 0.53 and 0.57, respectively). As compared to ALDH-CD34+ cells, ALDH+CD34+ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells, with brighter expression of CD117 and CD133, accompanied by lower display of CD38 and CD45RA. Of interest, ALDH+CD34- population disclosed a straightforward erythroid commitment, on the basis of three orders of evidences. First of all, ALDH+CD34- cells showed a CD71bright, CD105+, CD45- phenotype. Secondly, induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally, ALDH+CD34- precursors were not detectable in patients with pure red cell aplasia (PRCA).ConclusionOur study, comparing surface antigen expression of ALDH+/CD34+, ALDH-/CD34+ and ALDH+/CD34- progenitor cell subsets in human bone marrow, clearly indicated that ALDH+CD34- cells are mainly committed towards erythropoiesis. To the best of our knowledge this finding is new and could be useful for basic studies about normal erythropoietic differentiation as well as for enabling the employment of ALDH as a red cell marker in polychromatic flow cytometry characterization of bone marrow from patients with aplastic anemia and myelodysplasia.

Culture Conditions Allow Selection of Different Mesenchymal Progenitors from Adult Mouse Bone Marrow

The use of adult stem cells in tissue engineering approaches will benefit from the establishment of culture conditions that allow the expansion and maintenance of cells with stem cell-like activity and high differentiation potential. In the field of adult stem cells, bone marrow stromal cells (BMSCs) are promising candidates. In the present study, we define, for the first time, conditions for optimizing the yields of cultures enriched for specific progenitors of bone marrow. Using four distinct culture conditions, supernatants from culture of bone fragments, marrow stroma cell line MS-5, embryonic fibroblast cell line NIH3T3, and a cocktail of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), we isolated four different sub-populations of murine BMSCs (mBMSCs). These cells express a well-known marker of undifferentiated embryonic stem cells (Nanog) and show interesting features in immunophenotype, self-renewal ability, and differentiation potency. In particular, using NIH3T3 conditioned medium, we obtained cells that showed impairment in osteogenic and chondrogenic differentiation while retaining high adipogenic potential during passages. Our results indicate that the choice of the medium used for isolation and expansion of mBMSCs is important for enriching the culture of desired specific progenitors.

Chimeric Beta-Defensin Analogs, Including the Novel 3NI Analog, Display Salt-Resistant Antimicrobial Activity and Lack Toxicity in Human Epithelial Cell Lines

ABSTRACT Human beta-defensins (hBDs) are crucial peptides for the innate immune response and are thus prime candidates as therapeutic agents directed against infective diseases. Based on the properties of wild-type hBD1 and hBD3 and of previously synthesized analogs (1C, 3I, and 3N), we have designed a new analog, 3NI, and investigated its potential as an antimicrobial drug. Specifically, we evaluated the antimicrobial activities of 3NI versus those of hBD1, hBD3, 1C, 3I, and 3N. Our results show that 3NI exerted greater antibacterial activity against Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis than did hBD1 and hBD3, even with elevated salt concentrations. Moreover, its antiviral activity against herpes simplex virus 1 was greater than that of hBD1 and similar to that of hBD3. Subsequently, we investigated the cytotoxic effects of all peptides in three human epithelial carcinoma cell lines: A549 from lung, CaCo-2 from colon, and Capan-1 from pancreas. None of the analogs significantly reduced cell viability versus wild-type hBD1 and hBD3. They did not induce genotoxicity or cause an increase in the number of apoptotic cells. Using confocal microscopy, we also investigated the localization of the peptides during their incubation with epithelial cells and found that they were distributed on the cell surface, from which they were internalized. Finally, we show that hBD1 and hBD3 are characterized by high resistance to serum degradation. In conclusion, the new analog 3NI seems to be a promising anti-infective agent, particularly given its high salt resistance—a feature that is relevant in diseases such as cystic fibrosis.

Complete remission in acute myeloid leukaemia with t(8;21) following treatment with G‐CSF: flow cytometric analysis of in vivo and in vitro effects on cell maturation

A 75‐year‐old patient diagnosed as having acute myeloid leukaemia with t(8;21) received G‐CSF alone as induction therapy. Complete remission was achieved following 2 weeks of treatment. Flow cytometric analysis, performed by CD45 technique modified by the introduction of preliminary gating with LDS‐751, confirmed the disappearance of blast cells along with myeloid maturation. Finally, in vitro studies demonstrated that G‐CSF, as compared to other differentiation inducers, was able to induce a striking effect toward neutrophilic differentiation.

Immunophenotypic analysis enables the correct prediction of t(8;21) in acute myeloid leukaemia.

Immunophenotypic findings from 14 patients affected by acute myeloid leukaemia (AML) with t(8;21) were compared to those obtained from 79 AML patients with normal or other aberrant karyotypes. Classic lineage markers, adhesion molecules, surface enzymes, stem‐cell‐ related antigens and HLA‐DR were investigated. Following evaluation by the Mann‐Whitney test, we found that t(8;21) AMLs showed a significantly higher expression of CD19, CD34, CD56, CD45RA and CD54. Conversely, blasts from patients in the control group significantly expressed higher levels of CD45R0, CD33, CD36, CD11b and CD14. In order to split the data at the best cut‐off point to achieve the most homogenous subset with regard to cytogenetic pattern, i.e. t(8;21) or not, the CART (Classification and Regression Trees) method was applied. In the univariate analysis by CART, statistically significant differences were found when CD19 was dichotomized at 10%, CD34 at 37%, CD45RA at 84%, CD54 at 21%, CD56 at 12% , CD36 at 14%, CD45R0 at 25%, CD11b at 18% and CD14 at 12%. Once cut‐off points were established by CART, we applied the logistic regression model to establish which combination of two or more antigens was most predictive for t(8;21). The combination CD19‐CD34 at the cut‐off points indicated above correctly classified 92/93 cases (98.9%). The addition of any other antigen combination to the CD19/CD34 model failed to improve the level of prediction. We conclude that AML with t(8;21) displays an exclusive immunophenotype that is highly predictive of the cytogenetic pattern.