Immunomodulatory properties of porcine, bone marrow-derived multipotent mesenchymal stromal cells and comparison with their human counterpart.

Thanks to their immunonodulatory properties, multipotent mesenchymal stromal cells (MSCs) are a promising strategy for preventing/reducing the risk of graft rejection after hematopoietic cell and solid organ transplantation. We have previously demonstrated that porcine MSCs (pMSCs) can be isolated from bone marrow and display similar morphology and differentiative capacity as compared to human MSC (hMSCs). In this study, we investigated the in vitro immunomodulatory properties (namely the ability to suppress lymphocyte proliferation in response to phytohemagglutinin and the cytokine production in the culture supernatants) of pMSCs from six Large White 6-month old piglets. Similarly to hMSCs, pMSCs reduced the phytohemagglutinin-induced lymphocyte proliferation. High levels of IL-6 were found in culture supernatants, whereas IL-10 and TGF-ß were not detectable. In conclusion, ex vivo expanded pMSCs share selected biological/functional properties with hMSCs. pMSCs may be used in in vivo models to investigate novel approaches of prevention of graft rejection in solid organ transplantation.

Phenotypic and genotypic alterations characterize patients bearing plasma cell dyscrasias with a high M-component.

As at present only a long-term follow-up can fully determine whether monoclonal gammapathies of undetermined significance (MGUS) will evolve into multiple myeloma (MM), this study attempted to identify other variables connected with the amount of monoclonal component (MC), generally considered as the most reliable marker of malignant evolution. Thirty-four MGUS subjects showing a high MC (> or = 15.0 g/l) but without clinical evidence of MM (MGUS group b), were characterized for their phenotypic and genotypic profile by comparing them either with 40 MM patients or with 24 subjects affected by a benign form of monoclonal gammapathy (MGUS group a) according to the standard criteria. In addition to the usual laboratory markers, the levels of expression of a panel of CD membrane subsets were measured on B and T lymphocytes. Also, the serum level of the p53 mutant protein and the structural alterations of the c-myc oncogene were evaluated. The results show that for MGUS group b patients, an increased M-protein was accompanied by significantly increased levels of peripheral blood CD3+ T cells and oncogenetic aberrations in c-myc. Since a high serum MC level seems to indicate a greater likelihood of malignant transformation for MGUS patients, these findings suggest that this relationship may be a result of the concomitant alterations observed at a phenotypic and genotypic level. Such alterations may be potentially useful as surrogate markers for the transition of benign to malignant (MM) plasma cell dyscrasia.

Autoimmune thrombocytopenic purpura in pregnancy: maternal risk factors predictive of neonatal thrombocytopenia.

Pregnancy in ATP women is not unusual. The problem of this association concerns the possibility of disease transmission to the fetus due to the crossing of maternal antiplatelet antibodies through the placenta. Maternal risk factors predictive of neonatal thrombocytopenia, can be identified as follows: severe thrombocytopenia, previous splenectomy, high titre of PA-IgG and/or SPB-IgG. In 63 pregnancies in ATP patients, we have evaluated whether the above maternal risk factors, considered in the third trimester, can provide useful criteria for the prediction of neonatal thrombocytopenia. In the third trimester, the distribution of maternal risk factors was as follows: 0 in 7 cases, 1 in 27 cases, 2 in 15 cases, 3 in 12 cases, 4 in 2 cases. From a statistical evaluation, the neonatal platelet values and the maternal risk factors seem inversely correlated (r -0.437; p = 0.0005). In particular, neonatal and maternal platelet count correlated positively (r = 0.249; p = 0.025); moreover, neonatal platelet count correlated negatively with Splenectomy (r = -0.209; p = 0.05), PA-IgG (r = -0.401; p < 0.0005) and SPB-IgG (r = -0.338; p < 0.005). We tried to apply a multiple regression model for all the above parameters which appears statistically significant (p = 0.001); the variability was about 30%. This regression model could be validated if applied to a larger number of cases, and it could represent an alternative to the invasive methods used for the diagnosis of neonatal thrombocytopenia.

Detection of Helicobacter pylori carriers by discriminant analysis of urea and pH levels in gastric juices.

An alternative approach to the problems inherent in current methods for detecting Helicobacter pylori carriers--that of being generally time-consuming, expensive, and not sufficiently sensitive--was devised by using the urea concentration and pH levels of gastric juices. A linear discriminant analysis of these variables, measured in 54 patients submitted to digestive endoscopy for gastritis, provided a mathematical formula for assigning the subjects (previously classified by other standard methods) to groups of either positive or negative H pylori carriers. The results obtained showed a correct classification in 52 out of 54 cases with only one false negative and one false positive case.

Incidence of chromosomes 1 and 17 aneusomy in breast cancer and adjacent tissue: an interphase cytogenetic study.

BACKGROUND: Characterization of the biopathologic events underlying the early steps of breast carcinogenesis may have a dramatic impact on reducing breast cancer mortality. Genes involved in breast tumorigenesis are localized on chromosomes 1 and 17, and numeric aberrations of these chromosomes have been correlated with breast cancer tumorigenesis and progression. According to the field cancerization hypothesis, specific chromosome aberrations may be present in breast cancer and in normal-appearing adjacent tissue. The latter changes reflect the genomic damage that follows longterm carcinogenic exposure and precede the morphologically detectable neoplastic transformation. We hypothesize that detection of these aberrations in benign breast epithelium may provide a tool for molecular risk assessment. STUDY DESIGN: Using fluorescence in situ hybridization with centromere-specific probes, we determined the status of chromosomes 1 and 17 in fresh imprints of 28 samples of primary tumors and 54 samples of their surrounding uninvolved parenchyma taken from patients undergoing operations for breast carcinoma. Ten contralateral breast biopsy specimens collected from patients with previous breast carcinoma were also evaluated as a surrogate of a high-risk group to rule out the hypothesis that chromosomal aneusomy in tumor-adjacent tissue could be related to a paracrine effect of the primary tumor. Ten samples of benign breast tissue taken from patients at low risk were used as controls to define tolerance limits for aneusomy definition. RESULTS: Using threshold values of 40% of signal loss and 13% of signal gain to define chromosome aneusomy (ie, mean + 3 SDs of the control group signals), we found the following: 1) almost all primary breast tumors were aneusomic for chromosomes 1 and 17; 2) primary breast tumor and adjacent uninvolved parenchyma shared the same pattern of chromosomes 1 and 17 aneusomy in 66.7% of patients; and 3) chromosomes 1 and 17 aneusomies in contralateral benign breast samples from high-risk patients were not different from those in primary breast tumor or adjacent tissue samples. CONCLUSIONS: These results suggest that chromosomes 1 and 17 aneusomy may represent an intermediate biomarker of breast tumorigenesis potentially useful to detect patients at high risk of breast carcinoma who may benefit from preventive interventions.

Specificity of antinuclear and antiphospholipid antibodies in sera of patients with autoimmune lymphoproliferative disease (ALD).

OBJECTIVE: A human lymphoproliferative syndrome characterized by a defect of the Fas-mediated apoptosis pathway in the absence of a fas gene mutation (Autoimmune Lymphoproliferative Disease) has recently been described and characterized by autoimmune phenomena. The aim of this study was to investigate the presence of antinuclear and antiphospholipid antibodies and to define their specificity in 5 pediatric patients with this syndrome. METHODS: Antinuclear antibodies were investigated by Western Blot and IIF performed under standard as well as apoptotic conditions. The fine specificity of antiphospholipid antibodies was dissected by an ELISA for anti-beta 2-glycoprotein I, anti-prothrombin, anti-annexin V and anti-protein S antibodies, and by immunostaining on thin layer chromatography plates for antiphospholipid molecule antibodies. RESULTS: This study showed that the autoantibodies found in these patients targeted a broad spectrum of nuclear antigens which undergo redistribution from the nucleus to the cytoplasm and plasma membrane during the course of the apoptotic process. This reactivity does not comprise known specificities such as anti-extractable nuclear antigens or anti-dsDNA. Antiphospholipid antibodies were also found in these sera. A further characterization of the antiphospholipid antibodies showed the presence of a heterogeneous response with antibodies directed to negatively-charged phospholipids and antibodies targeting coagulation-related proteins (beta 2-GPI, prothrombin, annexin V) which are considered relevant antigens in the antiphospholipid syndrome. CONCLUSIONS: These results suggest that lack of tolerance due to a defect of Fas-mediated apoptosis allows the survival of B and T clones involved in the antinuclear and antiphospholipid immune responses.

Fibrinolysis components as prognostic markers in breast cancer and colorectal carcinoma.

The impact of prognostic markers on disease-free and overall survival reflects their relative role in tumour biology. Breast cancer and colon carcinoma can be taken as examples to demonstrate the clinical and biological relevance of 'new markers' of neoplastic disease. In breast cancer, receptor-bound urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 seem to play an important role in the dissolution of the surrounding tissue and the formation of tumour stroma. These processes are prerequisites for invasion and metastasis. The study of 'classical' and 'new' prognostic factors showed that uPA and PAI-1 content of breast cancer tissue are strong and independent prognostic factors. Also in colorectal cancer the prognostic relevance of plasminogen activators and inhibitors was analysed. In particular, low tissue plasminogen activator (tPA) levels, as antigen or as activity, high uPA: tPA antigen ratio in corresponding normal mucosa, high levels of uPA-related antigen and activity and of PAI-2 antigen in neoplastic tissue, and high uPA (neoplastic mucosa): tPA (normal mucosa) ratio, were all parameters associated with a poor overall survival. In conclusion, all these observations show the clinical importance of plasminogen activators and inhibitors at tissue levels with respect to cancer development and survival of patients affected by breast carcinoma or colorectal neoplasia. These new prognostic markers will also permit a better patient selection for a possible adjuvant treatment.

Predictive value of cell kinetics in endometrial adenocarcinoma.

Flow cytometric DNA content and proliferative kinetic markers, S-phase fraction (SPF) and thymidine labeling index (TLI), were evaluated in 68 patients with endometrial carcinoma. A high rate of aneuploid tumors was detected (48.4%); median values of SPF and TLI were 6.4 and 6.2, respectively. No significant relationship emerged between ploidy status and proliferative markers in respect to clinical and pathological variables. Aneuploid tumors had a higher recurrence rate than diploid tumors (21.8% vs 9.6%), but the difference was not statistically significant. According to the median value of both kinetic markers, the study population was divided into low and high-risk, where DFS was 100% and 71.4%, respectively (p = 0.05). Furthermore, high-TLI tumors (> 6.2) had a significantly worse DFS (75.4%) than low-TLI (100%) only among patients assigned to Stage I of the disease, regardless of other pathological variables. At multivariate analysis myometrial invasion resulted as an independent and significant factor. Flow cytometric ploidy analysis was useless as a predictive biological parameter and did not add any further prognostic information to the pathologic variables. SPF and TLI values could indicate a subset of women with unexpected poor outcome in a group of patients generally considered at low-risk, i.e. Stage I. If further investigation confirms these data, it could prove useful for therapeutic planning in endometrial cancer patients. At the present time, pathological and clinical factors are still the most reliable predictive parameters.

Detection of C-myb genetic alterations and mutant p53 serum protein in patients with benign and malignant colon lesions.

C-myb structural alterations were analysed by Southern blot hybridization in 55 adenomatous polyps and 21 adenocarcinomas of the colon. Gene amplification was observed in 8 cases (14.5%) and c-myb rearrangements in 3 cases (5.4%) of the preneoplastic lesions analysed. A higher percentage of c-myb abnormalities (23.8%) was shown by malignant tumors. As far as mutant p53 protein is concerned, it was detected both in sera of adenoma and adenocarcinoma patients, though at different levels. No statistically significant correlations were found between c-myb or p53 abnormalities and clinico-pathological variables.

TAG-72 expression and its role in the biological evaluation of human colorectal cancer.

Immunohistochemical studies showed that TAG-72 is expressed in more than 80% of colorectal carcinomas, but is rarely expressed in normal epithelium and benign diseases. TAG-72 can also be found in the body fluids of patients with adenocarcinomas, and its direct measurement can be used in conjunction with immunocytochemical analysis to help in discriminating benign from malignant effusions. The evaluation of TAG-72 in serum of colorectal carcinoma patients showed a sensitivity of approximately 40%, comparable to that of the widely used CEA. TAG-72 serum levels correlate with the stage of disease, suggesting its utility in discriminating between early-stage versus late-stage colon carcinoma. Longitudinal studies demonstrated that TAG-72 serum levels may be used as a predictive marker of recurrences. Moreover, the simultaneous measurement of TAG-72 and CEA serum markers improves the monitoring of recurrent disease. Therefore, these data suggest that TAG-72 is a well suitable marker for colorectal cancer.

Behaviour of several 'progression markers' during the HIV-Ab seroconversion period. Comparison with later stages.

Two acute phase reactants, four cytokines, five soluble factors and lymphocyte subpopulations have been simultaneously evaluated in 16 subjects before and closely after the HIV-Ab seroconversion time. The same variables have also been determined in 50 HIV-Ab-negative high risk subjects, in 36 CDC II-III and in 30 CDC IV patients, utilizing a mixed longitudinal epidemiological model. The results show significant variations of few parameters in the early phases (increase: sCD8, beta-2-Microglobulin, sIL-2R, sCD23, Neopterin, IFN-alpha; decrease: CD4+ lymphocytes). In the course of the disease, many others parameters progressively increase (IFN-tau, IL-4, IL-6, acid-alpha 1-glycoprotein, alpha 1-antitrypsin) or decrease (B- and T-lymphocytes). Ferritin, in particular, highly increases only in CDC IV stage. These data may be useful to monitor patients during the entire course of their disease and to suggest the time elapsed from seroconversion.

Autoimmune thrombocytopenic purpura: maternal and fetal disease.

We have followed up 63 pregnancies in women with autoimmune thrombocytopenic purpura (ATP). Of these, 15 were previously splenectomized. The characteristics of the sample can be summed up as follows: average age 27 years (17-41); platelets at the beginning of pregnancy, mean 129.5 x 10(9)/l (range 16-488); platelets at delivery, mean 133 x 10(9)/l (range 8-477); PA-IgG at delivery, mean 320 ng IgG/10(7) platelets (range 10-1000); SPB-IgG at delivery, mean 262 ng IgG/10(7) platelets (range 10-1000). There were 30 spontaneous deliveries and 33 cesarean sections. Forty-two newborns had a platelet count within the normal range while nine had a platelet count less than or equal to 150 x 10(9)/l, while six had less than or equal to 100 x 10(9)/l and a further six less than or equal to 50 x 10(9)/l. The aim of this study is the evaluation of maternal risk and of possible feto-neonatal thrombocytopenia at birth. In this regard, the following parameters were considered: previous maternal splenectomy; the platelet count at the beginning of pregnancy; the platelet count and the titres of PA-IgG and SPB-IgG at delivery. Preliminary statistical evaluation of these parameters enabled us to identify a risk score. From this it was possible to obtain an optimum management of the final stage of pregnancy regarding the therapeutic approach and the timing of delivery.

DNA aberrations in urinary bladder cancer detected by flow cytometry and FISH: prognostic implications.

We evaluated the genetic changes in bladder cancer biopsy by fluorescence in situ hybridization (FISH) and related them to stage and grade of the tumor, ploidy (FCM) and clinical outcome, to determine a simple method to identify tumors with a poorer prognosis. Using FISH the numerical aberrations of chromosomes 1, 7, 9, 17 in tumor's imprints of 70 patients with transitional cell cancer (TCC) were determined. First of all, the data demonstrated that the sensitivity of FISH in detecting quantitative DNA aberrations exceeds FCM's sensitivity. The frequency of chromosome 1 and 9 aberrations did not show significant differences in diploid and aneuploid tumors in different stage and grade. On the contrary, the chromosome 7 and 17 aneusomy showed greater differences between pT1 and pT2-3 tumors (p<0.032 and p<0.0006, respectively) than between stage pTa and pT1. In our investigation, an increasing number of aberrations was observed in all chromosomes examined in tumors of patients who afterwards underwent cystectomy and/or had recurrent tumors. These results suggest that chromosome 7 and 17 aneusomy could be predictive of adverse outcome in a subgroup of patients with superficial tumors at presentation.

[IL-6, p53 and proto-oncogene c-myc play different roles as biological markers of plasma cell dyscrasias]. / IL-6, p53 e il proto-oncogene c-myc esercitano ruoli diversi come marcatori biologici di discrasie plasmacellulari.

PURPOSE: To determine the role of serum levels of IL-6 and p53 mutant protein as well as of c-myc proto-oncogene alterations: a) in discriminating between benign (MGUS) and malignant Plasma cell dyscrasias (Multiple and Microsecreting Myeloma, Plasmocytoma); b) in monitoring the clinical course of malignant forms of this disease. PATIENTS AND METHODS: Eighty-eight patients affected by Plasma cell dyscrasias (58 MGUS, 24 MM and 6 PLC) entered this study. Using commercially available ELISA kits, serum levels of IL-6 and p53 have been determined in all the patients. In addition, a selected group of patients (n = 30) was also analyzed for structural c-myc gene alterations by Southern blot technique. RESULTS: The results show that, conversely from p53 protein, IL-6 and c-myc gene may represent useful diagnostic markers for discriminating benign from malignant forms of Plasma cell dyscrasia. On the contrary, preliminary findings of the same work indicate a potential role for the mutant p53 protein in monitoring the response to chemotherapy of patients affected by MM or PLM. CONCLUSIONS: Overall, these data suggest that the combined use of IL-6, p53 and c-myc may provide a new approach for a more rational management of Plasma cell dyscrasia patients.

MTHFR C677T mutation, factor II G20210A mutation and factor V Leiden as risks factor for youth retinal vein occlusion.

OBJECTIVE: To determine whether methylene tetrahydrofolate reductase (MTHFR) C677T mutation, factor II G20210A mutation and factor V Leiden are risk factors for retinal vein occlusion (RVO) in patients under fifty years of age. METHODS: Comparison of 29 patients, under 50 years old of age, as affected RVO and 62 age matched normal controls. Plasma MTHFR C677T genotype, Factor II G20210A genotype, Factor V Leiden genotype, S protein level, C protein level, APCR presence (Actived Protein C Resistance), homocysteine level and Beta-thromboglobulin level were determined. RESULTS: Seventeen RVO patients and twenty-one controls were heterozygous for the MTHFR C677T mutation. Three RVO patients and twenty-three controls were homozygous for the MTHFR C677T mutation. Three RVO patients and two controls were heterozygous for the factor II G20210A mutation. One control was heterozygous for the factor V Leiden. CONCLUSIONS: This study fails to demonstrate that these mutations are risk factors for RVO in patients under fifty years of age.

Isolation and ex vivo expansion of bone marrow-derived porcine mesenchymal stromal cells: potential for application in an experimental model of solid organ transplantation in large animals.

Pharmacological aspecific immunosuppression, despite being widely used in solid organ transplantation recipients, is unable to completely prevent allograft rejection. It promotes the occurrence of sometimes life-threatening infections. Due to their immunosuppressive and anti- inflammatory properties, there is great interest in the therapeutic use of bone marrow (BM)-derived mesenchymal stromal cells (MSC). Large animal models play a crucial role to investigate the biological and functional properties of MSCs as novel cellular therapy. In the current study we sought to isolate expand ex vivo, and phenotypically characterize MSC derived from BM of 4 Large White 6-month-old piglets. Porcine MSC (pMSC) were characterized for their in vitro differentiation capacity. pMSC were successfully isolated from all BM samples. They showed spindle-shaped morphology and a stable doubling time on culture. They were positive for CD90, CD29, CD105, and negative for CD45 and CD11b. Furthermore, they differentiated, upon specific in vitro conditions toward adipogenic and osteogenic lineages. The optimization of methods for the isolation and characterization of pMSC may be useful to elucidate their biological and functional properties. The anatomy and physiology of the pig, which is similar to humans, make this animal model more attractive than small animals to test the safety and efficacy of MSC in the context of solid organ transplantation.