Successful xenografting of cryopreserved primary pancreatic cancers

In order to assess the suitability of cryopreserved neoplastic tissues for xenografting into nude (nu/nu) mice, we compared the take rate in 28 samples of pancreatic ductal carcinoma. Eleven fresh samples were implanted in nu/nu mice, and 17 were frozen in cryopreserving solution and implanted at a later time. All samples were examined for the presence of neoplastic tissue in cryostat sections. A total of 15 tumors grew in the animals; five from the freshly implanted samples and ten from those cryopreserved. Ten xenografted tumors were characterized for alterations in p53, K-ras, and p16 genes, which were found in six, eight, and nine cases, respectively. Our results demonstrate that the take rate for xenografting is comparable between cryopreserved and fresh tissue samples. The procedure allows for the exchange of tumor material between institutions and permits the establishment of centralized facilities for the storage of an array of different primary tumor samples suitable for the production of in vivo models of cancers.     

S-100 protein in white preadipocytes: An lmmunoelectronmicroscopic study

Differentiation of adipocytes from their precursor cells (preadipocytes) is an important problem in the study of the pathogenesis of obesity. Unfortunately, among the immature stages of adipocytes, only relatively differentiated forms can be identified by their fine structure; because early preadipocytes cannot be distinguished from fibroblasts solely on the basis of their morphology, it is impossible to assess the size of the preadipocyte population. S-100 protein has been identified in various mammalian tissues and recently mature adipocytes have been shown to be positive for this protein. Because fibroblasts are negative for S-100 protein, the present study tested the S-100 immunoreactivity of preadipocytes by the peroxidase-antiperoxidase (PAP) preembedding method at the ultrastructural level both in vivo and in culture. Mature adipocytes and early preadipocytes, including fibroblast-like cells devoid of lipid droplets, were positive both in vivo and in culture. Endothelial cells and pericytes were negative; but flattened, lipid-free, fibroblast-like cells surrounding the pericytes were positive. True fibroblasts both in vivo and in culture were negative. Therefore, S-100 protein can be a useful biochemical marker in distinguishing fibroblasts from early preadipocytes.     

Tumor cell purging by ex vivo expansion of hemopoietic stem cells from breast cancer patients combined with targeting ErbB receptors.

Tumor cell contamination might induce relapse after autologous transplantation in breast cancer patients. We used an ex vivo purging strategy to decrease the number of contaminating breast tumor cells in leukaphereses without altering the engraftment potential of the hemopoietic progenitor cells. This method is based on immunoselection of CD34+ cells derived from mobilized peripheral blood of patients with metastatic breast cancer and expansion in the presence of flt3 ligand, stem cell factor, interleukin 6, and thrombopoietin. Tumor contamination before and after culture was monitored by mammaglobin messenger RNA amplification by quantitative polymerase chain reaction. We analyzed both adherent and suspended cells obtained after 2 weeks of culture. Hemopoietic progenitors were increased among suspended cells. In this fraction, tumor cell contamination was decreased, whereas it increased within the adherent cell fraction. Experimental models using CD34+ cells from healthy donors spiked with breast cancer cells were also constructed to investigate whether treatment with anti-ErbB-receptor drugs could further reduce the tumor load without affecting the clonogenic potential of hemopoietic cells. For this purpose, we successfully assayed trastuzumab, a monoclonal antibody against ErbB-2, and gefitinib, an epidermal growth factor receptor tyrosine kinase receptor inhibitor. These results suggest that positively selected CD34+ cells from cancer patients contain tumor cells and that ex vivo expansion can reduce the tumor load of the suspended fraction. Target-based agents against ErbB-2, epidermal growth factor receptor, or both--such as trastuzumab or gefitinib--might increase the efficiency of purging.     

Exhaled breath condensate cysteinyl leukotrienes are increased in children with exercise-induced bronchoconstriction

BACKGROUND: It is recognized that airway inflammation has a central role in the pathogenesis of asthma, but how it relates to exercise-induced bronchoconstriction (EIB) is not completely understood. OBJECTIVE: The aim of our study was to investigate the relationship between EIB and baseline concentrations of cysteinyl leukotrienes (Cys-LTs) and other inflammatory markers in exhaled breath condensate (EBC). METHODS: EBC was collected, and the fraction of exhaled nitric oxide (FE NO ) was measured in a group of 19 asthmatic children, after which they performed a treadmill exercise test. Fourteen healthy children were enrolled as control subjects. RESULTS: The asthmatic children were divided into the EIB group (decrease in FEV 1 , > or =12%) and the non-EIB group. The EBC was analyzed for the presence of Cys-LTs, leukotriene B 4 , and ammonia. Asthmatic patients with EIB (mean FEV 1 decrease, 23% +/- 3%) had higher Cys-LT concentrations than either asthmatic patients without EIB or control subjects (42.2 pg/mL [median] vs 11.7 pg/mL and 5.8 pg/mL; P < .05 and P < .001, respectively). Ammonia concentrations were lower in both the EIB and non-EIB groups than in control subjects (253.2 microM and 334.6 microM vs 798.4 microM; P < .01 and P < .05, respectively). No difference in EBC leukotriene B 4 levels was found among the 3 groups. Both asthmatic groups had higher FE NO levels than control subjects ( P < .001). EBC Cys-LT ( P < .01; r = 0.7) and FE NO ( P < .05; r = 0.5) values both correlated significantly with the postexercise FEV 1 decrease. CONCLUSION: this study shows that EBC Cys-LT values are higher in asthmatic children with EIB and correlate with the decrease in FEV 1 after exercise. These findings suggest that the pathways of both Cys-LT and nitric oxide are involved in the pathogenesis of EIB.     

Fascin,an actin-bundling protein,modulates colonic epithelial cell invasiveness and differentiation in vitro

In epithelial tissue, cell-matrix and cell-cell adhesive interactions have important roles in the normal organization and stabilization of the cell layer. The malignant conversion of epithelial cells involves alterations in the expression and function of these adhesion systems that enable a switch to a migratory phenotype in tumor invasion and metastasis. Fascin is an actin-crosslinking protein that is found in the core actin bundles of cell-surface spikes and projections that are implicated in cell motility. We demonstrate that fascin is not detectable in normal colonic epithelium, but is dramatically up-regulated in colorectal adenocarcinoma. To test the hypothesis that fascin could participate in tumor invasive behavior, we developed a cell culture model to examine the effect of fascin expression on the adhesive interactions, invasiveness, and differentiation of colonic epithelial cells. We report marked effects on the organization of cell-surface protrusions, actin cytoskeleton, and focal adhesions in the absence of alterations in the protein levels of the major components of these structures. These effects correlate with alterations in cell movements on two-dimensional matrix, and increased invasiveness in three-dimensional matrix. The cells also show increased proliferation and decreased capacity for normal glandular differentiation in collagen gels. We propose that up-regulation of fascin, by promoting the formation of protrusive, actin-based, cell-motility structures, could be a significant component in the acquisition of invasive phenotype in colonic carcinoma.     

Tuning of innate immunity and polarized responses by decoy receptors

After the identification of the interleukin (IL)-1 type II receptor as the prototype, decoy receptors have been identified for a number of members of the IL-1/IL-18, TNF, IL-10 and IL-13 receptor families. Moreover, the silent receptor D6 is a promiscuous decoy and scavenger receptor of inflammatory chemokines. The IL-1 decoy receptor is regulated by pro- and anti-inflammatory signals and its levels may serve as a readout of the activation of anti-inflammatory pathways, for instance by glucocorticoid hormones. Decoy receptors represent a strategy to tune inflammatory and polarized adaptive responses.     

Effector and regulatory events during natural killer–dendritic cell interactions

Summary:  The different cell types of the innate immune system can interact with each other and influence the quality and strength of an immune response. The cross talk between natural killer (NK) cells and myeloid dendritic cells (DCs) leads to NK cell activation and DC maturation. Activated NK cells are capable of killing DCs that fail to undergo proper maturation ('DC editing'). Encounters between NK cells and DCs occur in both inflamed peripheral tissues and lymph nodes, where both cell types are recruited by chemokines released in the early phases of inflammatory responses. Different NK cell subsets (CD56^brightCD16⁻ versus CD56⁺CD16⁺) differ in their homing capabilities. In particular, CD56^brightCD16⁻ NK cells largely predominate the lymph nodes. In addition, these two subsets display major functional differences in their cytolytic activity, cytokine production, and ability to undergo proliferation. NK cell functions are also greatly influenced by the presence of polarizing cytokines such as interleukin (IL)-12 and IL-4. The cytokine microenvironment reflects the presence of different cell types that secrete such cytokines in response to microbial products acting on different Toll-like receptors (TLRs). Moreover, NK cells themselves can respond directly to microbial products by means of TLR3 and TLR9. Thus, it appears that the final outcome of a response to microbial infection may greatly vary as a result of the interactions occurring between different pathogen-derived products and different cell types of the innate immunity system. These interactions also determine the quality and strength of the subsequent adaptive responses. Remarkably, NK cells appear to play a key role in this complex network.     

Screening of HIV-1 isolates by reverse heteroduplex mobility assay and identification of non-B subtypes in Italy

OBJECTIVE: The increasing prevalence of HIV-1 transmission through heterosexual contacts and the growing number of immigrants from non-Western countries, where non-B subtypes and recombinant forms are prevalent, suggest the possible emergence in Italy of a new epidemic wave of HIV-1 non-B subtypes as well as recombinant forms. METHODS: The distribution of HIV-1 subtypes has been evaluated in 63 seropositive individuals residing in Italy, most of whom were infected through a sexual route during the last 5 years. A modified heteroduplex mobility assay (HMA) strategy, reverse HMA (rHMA), has been developed in our laboratory, allowing rapid identification of divergent-from-B-subtype isolates, which have been subsequently characterized by detailed molecular and phylogenetic analyses. RESULTS: Five samples show, on rHMA, an electrophoretic pattern compatible with a non-B subtype classification. Their phylogenetic analysis, performed on both env and gag regions, confirms the rHMA subtyping prediction, given that 3 samples fall into the "A-family" subtype and 2 into the G subtype. The 5 non-B-subtype HIV-1 isolates have been identified among 23 variants (prevalence, 21.74%) isolated during the 2000 to 2001 period in heterosexuals. In parallel, B-subtype isolates show high levels of intrasubtype nucleotide divergence, compatible with a constant HIV-1 molecular diversification. CONCLUSION: The Italian HIV-1 epidemic is still mostly attributable to the B subtype, which shows an increasing nucleotide heterogeneity. Heterosexual transmission and the interracial blending, however, are slowly introducing novel HIV-1 subtypes, and the data indicate that rHMA represents a powerful tool for HIV-1 biomolecular screening in epidemics characterized by a mono-/dual-subtype predominance.     

Inhibition of hepatitis C virus nonstructural protein, helicase activity, and viral replication by a recombinant human antibody clone

Hepatitis C virus (HCV) nonstructural protein 3 (NS3), with its protease, helicase, and NTPase enzymatic activities, plays a crucial role in viral replication, and therefore represents an ideal target for the development of anti-viral agents. We have developed a recombinant human antibody (Fab) that reacts with the helicase domain of HCV NS3. The affinity-purified Fab antibody completely inhibited the helicase activity of HCV NS3 at equimolar concentration. To evaluate the effect of the Fab on HCV replication, the clone encoding the Fab gene was put into an expression vector, which converts Fab into a complete IgG1 antibody. Using a DNA-based transfection model, we demonstrated that intracellular expression of this antibody resulted in significant reduction of HCV-negative strand RNA synthesis. Intracellular expression of this antibody into either a stable cell line replicating subgenomic RNA, or a transient full-length HCV replication model, reduced both HCV RNA and viral protein expression. These results support the use of recombinant antibody fragments to inhibit NS3 enzyme as a novel, feasible, and effective approach for inhibiting HCV replication.     

Congenital afibrinogenaemia caused by uniparental isodisomy of chromosome 4 containing a novel 15-kb deletion involving fibrinogen Aalpha-chain gene

Among rare inherited deficiencies of coagulation factors, congenital afibrinogenaemia is characterised by the lack of fibrinogen in plasma. In the last few years, several genetic defects underlying afibrinogenaemia (mostly point mutations) have been described in the fibrinogen gene cluster. In this study, the molecular basis responsible for afibrinogenaemia in a Thai proband was defined. Point mutation screening was accomplished by directly sequencing the three fibrinogen genes. The impossibility to amplify fibrinogen Aalpha-chain gene (FGA) exons 5 and 6 suggested the presence of a homozygous deletion. A specific long-range PCR assay enabled the identification of a novel 15-kb deletion, representing the largest afibrinogenaemia-causing deletion described so far. Direct sequencing of the deletion junction allowed mapping of the breakpoints in FGA intron 4 and in the intergenic region between Aalpha- and Bbeta-chain genes. Since the mutation was inherited only from the mother and nonpaternity was ruled out, a maternal uniparental disomy (UPD) was hypothesised. UPD test, carried out with markers covering the whole chromosome 4, revealed that maternal isodisomy was responsible for homozygosity of the 15-kb deletion in the proband. The apparently normal phenotype of the proband, except for afibrinogenaemia, suggests that UPD for chromosome 4 is clinically silent. This represents the first case of a documented complete isodisomy of chromosome 4 causing the phenotypic expression of a recessive disorder. In silico analyses of the regions surrounding the breakpoints suggested that the 15-kb deletion might have originated from an inappropriate repair of a double-strand break by the nonhomologous end joining mechanism.