Four-color flow cytometric analysis of peripheral blood donor cell chimerism

Passenger leukocytes have been demonstrated to play significant roles in initiating and also regulating immune reactions after organ transplantation. Reliable techniques to detect donor leukocytes in recipients after organ transplantation are essential to analyze the role, function, and behavior of these leukocytes. In this report we describe a simple, reliable method to detect donor cells with low frequencies using peripheral blood samples. Detection of small numbers of major histocompatibility complex (MHC) mismatched cells was first studied using four-color flow cytometry in artificially created cell mixtures. By selecting the CD45(+) population and simultaneous staining with several leukocyte lineage markers (CD3, CD4, CD8, CD56, and CD19), MHC-mismatched leukocytes were consistently detected in cell suspensions prepared from directly stained whole blood samples with a threshold sensitivity as low as 0.1%-0.2%. When the fresh peripheral blood mononuclear cells were separated by conventional Ficoll gradient purification, similar, but slightly lower levels of donor cells were detected. Blood samples obtained 1-5 months after liver, kidney, and intestine transplants revealed that the kind of organ allograft influenced levels and lineage pattern of the circulating donor cells. This procedure provided a simple and reliable method in determining early chimerism in transplant recipients. However, the detection of MHC-mismatched leukocytes of all lineages was much lower when frozen peripheral blood mononuclear cells were used.     

Malignant granular cell tumor of the lateral femoral cutaneous nerve: report of a case with cytogenetic analysis

Malignant granular cell tumors (MGCTs) are rare neoplasms of uncertain histogenesis. We report a case of MGCT involving a peripheral nerve with peritoneal and omental dissemination in which cytogenetic findings are available. Our results show that MGCTs share some cytogenetic abnormalities with malignant peripheral nerve sheath tumors (MPNSTs), supporting the hypothesis that they may represent histogenetically related lesions.     

Epidemiological and Environmental Investigations of Legionella pneumophila Infection in Cattle and Case Report of Fatal Pneumonia in a Calf

A fatal pneumonia due to Legionella pneumophila was diagnosed in a young calf reared in a dairy herd located in northern Italy. Clinical symptoms consisted of watery diarrhea, hyperthermia, anorexia, and severe dyspnea. The pathological and histological findings were very similar to those observed in human legionellosis. Legionella pneumophila serogroup 1 (SG1) and SG10 were isolated from the calf’s lung, and L. pneumophila SG1 was isolated from the calf’s liver. L. pneumophila SG1 was also demonstrated in the lung tissue by immunofluorescence and immunohistochemical examinations. Nine of 10 L. pneumophila SG1 isolates belonged to the Olda subtype, and 1 belonged to the Camperdown subtype. A very low prevalence of antibodies to Legionella was detected in cows and calves reared in the same herd. Cultures of aqueous sediment of an old electric water heater which supplied hot water for the feeding of the calves yielded L. pneumophila SG1. Four of the colonies tested belonged to the Olda subtype. Ten clinical and four environmental isolates were examined for the presence of plasmids. Nine of them were also examined by pulsed-field gel electrophoresis assay, and the same patterns were found for L. pneumophila SG1 Olda strains isolated from the calf and from the electric heater. This is the first report of a documented case of a naturally occurring Legionella pneumonia in an animal. Cattle probably act as accidental hosts for legionellae, much the same as humans.     

Dpc4 is expressed in virtually all primary and metastatic pancreatic endocrine carcinomas

DPC4/Smad4 is inactivated in about 50% of pancreatic ductal cancers. It has been recently reported that this gene is also inactivated in neoplasms arising from pancreatic islet cells, a phenomenon suggested to be related to similar progressions of malignancy found in common ductal cancers. To evaluate this possibility, we analysed 20 metastases of pancreatic endocrine carcinomas and their corresponding primary lesion for inactivation of DPC4 using immunohistochemical staining. In fact, immunohistochemical labelling has been shown to correlate with DPC4 gene status with high sensitivity and specificity. The cancers included 18 nonfunctioning tumours, one gastrinoma and one ViPoma all with liver, nodal and/or adrenal metastases. Seventeen were well-differentiated and three poorly differentiated endocrine carcinomas. Dpc4 expression was absent in only one primary well-differentiated endocrine cancer and its liver metastasis, while all the remaining 19 primary tumours and their metastases stained positive for the protein. All positively staining cases showed diffuse cytoplasmic and nuclear staining in virtually all neoplastic cells. Our data suggest that DPC4 is only rarely involved in pancreatic endocrine tumourigenesis and give further weight to the hypothesis that tumours arising from pancreatic exocrine and endocrine epithelia are genetically distinct.     

Clinical,immunological, and molecular analysis in a large cohort of patients with X-linked agammaglobulinemia: an Italian multicenter study

A questionnaire-based retrospective clinical and immunological survey was conducted in 73 males with a definite diagnosis of X-linked agammaglobulinemia based on BTK sequence analysis. Forty-four were sporadic and 29 familial cases. At December 2000, the patients' ages ranged from 2 to 33 years; mean age at diagnosis and mean duration of follow-up were 3.5 and 10 years respectively. After the mid-1980s all but 2 were on intravenous immunoglobulin (IVIG) substitution therapy, with residual IgG >500 mg/dl in 94% of the patients at the time of enrollment. Respiratory infections were the most frequent manifestation both prior to diagnosis and over follow-up. Chronic lung disease (CLD) was present in 24 patients, in 15 already at diagnosis and in 9 more by 2000. The cumulative risk to present at diagnosis with CLD increased from 0.17 to 0.40 and 0.78 when the diagnosis was made at the ages of 5, 10, and 15 years respectively. For the 9 patients who developed CLD during follow-up, the duration of follow-up, rather than age at diagnosis; previous administration of intramuscular immunoglobulin; and residual IgG levels had a significant effect on the development of CLD. Chronic sinusitis was present in 35 patients (48%), in 15 already at diagnosis and in 20 by 2000. Sistemic infections such as sepsis and meningitis/meningoencephalitis decreased over follow-up, probably due to optimal protection provided by high circulating IgG levels reached with IVIG.     

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.     

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.     

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.     

Analysis of the α/β T-cell receptor repertoire by competitive and quantitative family-specific PCR with exogenous standards and high resolution fluorescence based CDR3 size imaging

The characterization of the human T-cell receptor (TCR) repertoire in various physiological and pathological conditions has become an important tool in studies of the immune response. Therefore, a number of PCR based strategies for the semiquantitative analysis of the TCR repertoire have been described. Family specific amplification of TCR cDNA has been employed in a number of studies often with contradictory results. We have developed a strategy utilizing exogenous standards with homologous primer binding sites for the quantitative analysis of the α/β T-cell receptor repertoire. This system allows the detection of even minute differences in T-cell populations based on quantitative PCR (Q-PCR) and competitive PCR (C-PCR). Results presented here demonstrate that expansions of T-cell subsets as defined by the specificity of the variable gene segments can be readily monitored when exceeding 1% of the total repertoire. In addition, the proposed method reveals direct information of CDR3 size heterogeneity and can be used to estimate the T-cell repertoire complexity and monitor clonal expansions. We discuss variables such as cell number and experimental conditions influencing accuracy and reproducibility of the analyses. We have used this protocol based on non-radioactive techniques for characterization of the fine specificity of the T-cell repertoire in peripheral and organ-infiltrating T-lymphocytes. The analyses revealed information about polyclonal or clonal expansion of T-cells in vivo and in vitro following various stimuli such as superantigenic stimulation of T-cell subsets as well as antigen-driven shaping of the α/β T-cell repertoire in autoimmune and infectious diseases.