Potency testing of mesenchymal stromal cell growth expanded in human platelet lysate from different human tissues.

BACKGROUND: Mesenchymal stromal cells (MSCs) have been largely investigated, in the past decade, as potential therapeutic strategies for various acute and chronic pathological conditions. MSCs isolated from different sources, such as bone marrow (BM), umbilical cord tissue (UCT) and adipose tissue (AT), share many biological features, although they may show some differences on cumulative yield, proliferative ability and differentiation potential. The standardization of MSCs growth and their functional amplification is a mandatory objective of cell therapies. The aim of this study was to evaluate the cumulative yield and the ex vivo amplification potential of MSCs obtained from various sources and different subjects, using defined culture conditions with a standardized platelet lysate (PL) as growth stimulus. METHODS: MSCs isolated from BM, UCT and AT and expanded in human PL were compared in terms of cumulative yield and growth potential per gram of starting tissue. MSCs morphology, phenotype, differentiation potential, and immunomodulatory properties were also investigated to evaluate their biological characteristics. RESULTS: The use of standardized PL-based culture conditions resulted in a very low variability of MSC growth. Our data showed that AT has the greater capacity to generate MSC per gram of initial tissue, compared to BM and UCT. However, UCT-MSCs replicated faster than AT-MSCs and BM-MSCs, revealing a greater proliferation capacity of this source irrespective of its lower MSC yield. All MSCs exhibited the typical MSC phenotype and the ability to differentiate into all mesodermal lineages, while BM-MSCs showed the most prominent immunosuppressive effect in vitro. CONCLUSIONS: The adoption of standardized culture conditions may help researchers and clinicians to reveal particular characteristics and inter-individual variability of MSCs sourced from different tissues. These data will be beneficial to set the standards for tissue collection and MSCs clinical-scale expansion both for cell banking and for cell-based therapy settings.

Culture of human cell lines by a pathogen-inactivated human platelet lysate.

Alternatives to the use of fetal bovine serum (FBS) have been investigated to ensure xeno-free growth condition. In this study we evaluated the efficacy of human platelet lysate (PL) as a substitute of FBS for the in vitro culture of some human cell lines. PL was obtained by pools of pathogen inactivated human donor platelet (PLT) concentrates. Human leukemia cell lines (KG-1, K562, JURKAT, HL-60) and epithelial tumor cell lines (HeLa and MCF-7) were cultured with either FBS or PL. Changes in cell proliferation, viability, morphology, surface markers and cell cycle were evaluated for each cell line. Functional characteristics were analysed by drug sensitivity test and cytotoxicity assay. Our results demonstrated that PL can support growth and expansion of all cell lines, although the cells cultured in presence of PL experienced a less massive proliferation compared to those grown with FBS. We found a comparable percentage of viable specific marker-expressing cells in both conditions, confirming lineage fidelity in all cultures. Functionality assays showed that cells in both FBS- and PL-supported cultures maintained their normal responsiveness to adriamycin and NK cell-mediated lysis. Our findings indicate that PL is a feasible serum substitute for supporting growth and propagation of haematopoietic and epithelial cell lines with many advantages from a perspective of process standardization, ethicality and product safety.

Counting of leukocytes in samples from G-CSF mobilized donors, leukapheresis products, and cord blood: the performances of an analyzer with dedicated profiles.

INTRODUCTION: Accurate white blood cell counting (WBC) and differential count by blood analyzers could allow a more informative characterization of granulocyte colony-stimulating factor (G-CSF) mobilized blood (MB), leukapheresis products (LP), and cord blood (CB). However, reliable counting by a blood cell analyzer in this setting is a major challenge owing to quali-quantitative abnormalities of blood cells. METHODS: We evaluated the performances of the analyzer Pentra DX 120 by Horiba ABX working with dedicated cell-gating profiles, which generate three-part differential counts in samples obtained from donors' MB, LP, and CB. The results of the analyzer were compared to counts obtained by flow cytometry and manual counts, the latter performed for reference validation and in the case of discrepant results between study and reference counts. RESULTS: Pentra DX 120 generated highly correlated counts (R > 0.91 in all cases) to those obtained by flow cytometry in all samples (MB, LP, and CB) with high degree of count accuracy in most cases and referred to WBC absolute count and differential count including lymphocytes (LYM) %, monocytes (MON) %, and polymorphonuclear leukocytes (PMN) %. Accuracy, judged by the difference between study and reference counts and expressed as percentage of reference count, ranged from 0.8% to 8.6%, and sporadic loss of accuracy occurred for MON % only in no more than 10% of CB samples. CONCLUSION: The ABX Pentra DX 120 provided accurate WBC count and differential count during MB, LP, and CB analyses and allowed a better characterization of donors' hematologic status and graft composition.

Evaluation of the analytical performances of a portable, 18-parameter hemometric system using capillary blood samples for blood donor enrolment.

BACKGROUND AND OBJECTIVES: Blood donor enrolment process is frequently based on the sole capillary haemoglobin (Hb) evaluation while platelet donors by apheresis also requires platelet (Plt) count. The 'sole Hb' approach prevents a complete donor evaluation and does not allow Plt donor enrolment. To extend blood counts before donations, we evaluated the performances of a multiparametric counter using capillary blood. MATERIALS AND METHODS: The ABX Micros 60 (Micros 60) blood analyzer was employed on capillary blood and compared with venous counts by a reference counter (Coulter AcT 5diff) in a first series of 416 donors and in a second series of 136, after a 3-month period of routine use of this study counter. An average of 50 microl of capillary blood was collected whose 10 microl had been aspirated by Micros 60. RESULTS: High correlations were found between capillary counts using Micros 60 and venous counts using the reference counter. Mean Plt counts differed of 37 x 10(9)/l less for capillary approach in the first series of comparisons, but decreased to 10 x 10(9)/l less in the second series due to a greater expertise of operators in capillary sampling. All other parameters were accurate and never reached clinical relevance albeit they showed statistically significant differences. CONCLUSION: Data on Micros 60 demonstrated that capillary predonation counts may represent a feasible and effective approach to realize an accurate enrolment process of blood and Plt donors.

Hepatitis B virus blood screening: impact of nucleic amplification technology testing implementation on identifying hepatitis B surface antigen non-reactive window period and chronic infections.

BACKGROUND: Despite improvements in hepatitis B surface antigen (HBsAg) test sensitivity, post-transfusion hepatitis B virus (HBV) infection still occurs because HBsAg is undetectable during the early window phase (WP) of the infection, in the convalescence core window phase of the infection, or in serologically silent chronic hepatitis or in mutant forms of HBV. HBV-DNA screening using high sensitivity nucleic amplification technology (NAT) assays has recently been introduced to reduce the residual risk of transmission of HBV by transfusion of blood components. MATERIALS: Over 1 year 75 063 donations were individually screened for HBV-DNA by the Ultrio Procleix assay on the Tigris platform. The donations were collected in the Latium region, an area of the central Italy, and they accounted for the 40% of the total blood units collected in this area per year. The initial reactive samples were re-tested and confirmed by the discriminatory HBV assay. Additional HBV serological markers were also performed. Suspected WP infections were followed-up to monitor the development of the immune response. All HBV-DNA-positive donors were called back to check up their infectious status. RESULTS: The results of testing the 75 063 donations are: 33 donations HBsAg positive, 31 out of them HBV-DNA-positive and two HBV-DNA negative; 22 donations HBsAg-negative but HBV-DNA positive with low viral load. Six of the 22 were found to be consistently HBV-DNA reactive whereas the remaining 16 donations showed inconsistent results on multiple NAT retesting. One WP infection was confirmed by the follow-up of the donor for 3 months following the index blood donation. CONCLUSIONS: In the donor population of the Latium region, NAT screening has revealed a higher than expected number of donors who were HBsAg non-reactive but HBV-DNA-positive with three donors showing HBV-DNA as the only marker of infection. The adoption of genome screening has increased the safety of the blood supply and has also contributed to the protection of donor health by identifying either WP or clinically silent infections.

ADAM-HCV, a new-concept diagnostic assay for antibodies to hepatitis C virus in serum.

We screened phage libraries using sera from noninfected individuals and patients infected by hepatitis C virus (HCV). By applying different selection and maturation strategies, we identified a wide collection of efficient phage-borne ligands for HCV-specific antibodies. The selected ligands retained their antigenic properties when expressed as multimeric synthetic peptides. Peptides that mimic several immunodominant epitopes of the virus were used to develop a novel type of diagnostic assay which efficiently detects antibodies to HCV in serum. This type of analysis provides a conclusive diagnosis for many patients identified as indeterminate according to presently available serological assays.

Human immunodeficiency virus-1 specific and natural cellular immunity in HIV seronegative subjects with multiple sexual exposures to virus.

The probability of HIV infection by sexual contact, although it varies greatly, appears to be lower than that of infection by other routes of exposure. The aim of this study was to evaluate immunological determinants involved in protection against HIV infection in subjects with multiple and repeated sexual exposures to the virus. Twenty-two subjects were studied for CD8+ cell anti-HIV suppression activity and serum neutralizing activity against the HIV strain of their own partners, beta-chemokine production, and natural killer cell activity. CD8+ cell anti-HIV activity and neutralizing activity of sera were found in 13 (76%) and 12 (70.5%) out of 17 HIV-1 negative subjects, respectively. Six individuals had a relevant immune response against HIV: three subjects with a high CD8+ cell antiviral suppression activity and three individuals with sera neutralizing activity titer >1:10. These last three subjects had the highest beta-chemokine levels, a very prolonged period of multiple sexual intercourse (>6 years) and a seropositive partner with a high viral load. A partial reduction of neutralizing activity titer was observed when pre-incubating the sera with anti-beta-chemokine neutralizing antibodies. A spontaneous natural killer cell activity was suppressed in the majority of HIV-1 negative subjects with sexual exposure in comparison with normal individuals. The protection from sexual HIV transmission appears to be the result of a network of different humoral and cellular factors.

Prevalence of TT viral DNA in italian blood donors with and without elevated serum ALT levels: molecular characterization of viral DNA isolates.

BACKGROUND AND OBJECTIVE: A novel non-enveloped DNA virus, called TT virus (TTV), has been reported to be associated with post-transfusion hepatitis of unknown etiology. Although its clinical role still remains obscure, its presence in blood donations might cause problems. It, therefore, appeared of interest to investigate TTV prevalence in voluntary blood donors. DESIGN AND METHODS: A total of 595 Italian blood donors with and without elevated serum alanine aminotransferase (ALT) levels were tested by polymerase chain reaction using two sets of semi-nested primers that amplify the well-known region in the N22 clone. The amplified products were then sequenced to assess the genotype by phylogenetic and restriction fragment length polymorphism analyses. RESULTS: The prevalence of TTV in blood donors was 5+/-1.9% (25 out of 500) with a 95% confidence limit. A similar prevalence was found in 95 selected blood donors with increased ALT levels. A viral load of 10(3)-10(4) viral DNA molecules/mL was found, thus indicating a rather narrow range of variability. A phylogenetic tree built up on the basis of 210 base sequences of ORF1 allowed isolates to be classified into 2 groups corresponding, at least, to two of the putatives TTV genotypes, group 1 and group 2 of Okamoto's classification. A similar classification was also obtained by site restriction enzyme analysis. INTERPRETATION AND CONCLUSIONS: The results show that TTV infection is present among Italian blood donors. No significant difference in prevalence of TTV infection was found between patients with normal and increased ALT, making the association between TTV infection and human hepatitis questionable.

Relationship of blood transfusion, post-operative infections and immunoreactivity in patients undergoing surgery for gastrointestinal cancer.

BACKGROUND AND OBJECTIVE: The immunosuppression induced by perioperative blood transfusion (BT) and its effect on the incidence of post-surgical infectious complications remains controversial. In this study, the relationship between BT and postoperative infections was investigated in 136 gastrointestinal cancer patients submitted to curative surgery. METHODS: Clinical and laboratory variables, data on postoperative infections, infection risk factors and types of transfusion were analyzed. Immune function was evaluated in 76 patients and compared before and after surgery. RESULTS: The overall postoperative infection rate was 28% for the transfused and 4.6% for the untransfused patients. The univariate analysis of investigated variables indicated that BT, progressive cancer stage, duration of surgery, drains, all had significant association with infection. The multiple logistic regression analysis confirmed BT (p = 0.0028) and advanced cancer stage (p < 0.001) as significant risk factors for the postoperative infections. The results of immunological tests showed no significant differences between transfused and untransfused patient groups, after surgery. Comparing pre- and postoperative data from individual patients, an impairment of natural killer (NK) activity was observed in all patients regardless of their transfusional status; the synthesis of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) was also decreased respectively in the untransfused and in the transfused patients. INTERPRETATION AND CONCLUSIONS: These results indicate that other factors, beside BT, can induce immunosuppressive effects in these patients and thus increase their susceptibility to postoperative infections.

Plasma viremia titration and RNA quantitation in ICD-p24 negative HIV type-1-infected patients.

Quantitative culture of human immunodeficiency virus (HIV) was performed on 202 plasma samples obtained from asymptomatic and early symptomatic HIV-1 infected patients (mean CD4+ count: 186/mm3) before antiretroviral therapy was started. HIV could be isolated from 84% of the plasma samples (titers ranging from 10(0) to 10(2.75) TCID50/ml). Immune complex dissociated p24 antigen (ICD-p24) was detected in 66% of the samples. Only 23 samples (11%) were negative for both ICD-p24 as well as HIV culture. Discordant results were obtained in 55 samples, and 45 samples negative for ICD-p24 were positive for HIV culture. A significant proportion (42%) of patients that were negative for ICD-p24 belonged to a very advanced group with very low CD4+ cell count. However, almost 90% of these ICD-p24 negative samples were positive for HIV plasma viremia, stressing the value of this virological marker in patients with low CD4+ cell count and without any detectable ICD-p24 antigenemia. HIV-1 RNA was detected in all ICD-p24 negative plasma samples tested by the branched DNA (bDNA) assay. A very good correlation was found between high RNA copy number and HIV plasma isolation in samples obtained from patients with low CD4+ cell count, suggesting that HIV-1 RNA quantitation may also reflect viral infectivity of plasma.

Recombinant interleukin-2 treatment before and after autologous stem cell transplantation in hematologic malignancies: clinical and immunologic effects.

Autologous bone marrow transplantation (ABMT) for hematologic malignancies is associated with a high relapse rate. Interleukin-2 (IL-2) administration is a therapy that may prevent relapse if used when the tumor burden is minimal. In this study we administered recombinant IL-2 (rIL-2) therapy to 12 patients affected by hematologic malignancies either before or after autologous stem cell transplantation (ASCT). rIL-2 was given by a 6 day continuous intravenous infusion with escalating doses, up to 18 x 10(6)/m2/day, depending on patient tolerance. The functional immune responses of the patients were assessed as natural killer (NK) and lymphokine-activated killer (LAK) cytotoxic activities and in vitro interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) synthesis. During rIL-2 treatment, the expected side effects occurred; only 3 patients, who showed severe cardiovascular toxicity, required suspension of the treatment. All toxicities reversed after the end of the therapy. Immunologic monitoring was carried out the day before starting rIL-2 infusion and then repeated on days 3, 7, and 14 after rIL-2 was discontinued. Following every rIL-2 course, a pronounced increase in CD3+, CD8+, CD56+ cells was found, with a peak value on day 3. The NK and LAK activities showed a significant increase on day 3 (p < 0.001) over pretherapy values; the increase lasted until day 14, although the difference at later time points was not significant. Before transplant the synthesis of both IFN-gamma and TNF-alpha decreased following rIL-2 therapy, whereas higher levels of these lymphokines were found after posttransplant rIL-2 courses.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoresponsiveness of cancer patients: effect of blood transfusion and immune reactivity of tumor infiltrating lymphocytes.

The immunoreactivity of cancer patients submitted to surgery and perioperatively transfused was investigated. Peripheral blood mononuclear cells (PBMC) and tumor infiltrating lymphocytes (TIL) were tested for the natural killer (NK) cytotoxic activity, for the in vitro synthesis of interleukin-2 (IL-2), interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and prostaglandin E2 (PGE2). The serum levels and the production of PGE2 by PBMC were significantly higher in patients than in controls, whereas no significant differences in the tested immunological variables emerged between the two groups of subjects. Instead, TIL produced significant larger amounts of spontaneous PGE2 (p < 0.001) and significant lower amounts of IFN-gamma (p < 0.001) and TNF-alpha (p < 0.001) than autologous PBMC, suggesting an involvement of PGE2 in the impairment of the host immunoreactivity at the tumor site. To evaluate the immunomodulating effect of blood transfusion, the patients were reexamined 8 to 20 days after surgery. No differences were found in the NK cytotoxic activity, lymphokine synthesis, serum levels, and production of PGE2 between transfused and untransfused patients. These results do not support the hypothesis that blood transfusions negatively affect the immune response of neoplastic patients.

[Analysis of growth, phenotype and cytotoxic activity of tumor infiltrating lymphocytes expanded in vitro with interleukin-2 and interleukin-4: preliminary results]. / Analisi della crescita, del fenotipo e dell'attività citotossica dei linfociti infiltranti i tumori espansi in vitro con interleuchina 2 ed interleuchina 4: risultati preliminari.

Tumor infiltrating lymphocytes (TIL) play an important role in the host immune response to cancer. When these cells are reinfused into cancer patients after in vitro expansion with lymphokines such as interleukin 2 (rIL-2), they often induce regression of tumor metastases. We obtained TIL of enzymatic digestion of 7 human solid tumors and then cultured them with rIL-2 and interleukin 4 (IL-4) at different concentrations for about 36 days. Immunophenotypic analysis was performed at the end of the second and fourth week; cytotoxic activity against autologous and heterologous targets was assessed on the 30th day of culture. The best lymphocytic growth was observed when we used rIL-2 and IL-4 for the first two weeks of culturing and then continued with rIL-2 alone. CD3 and CD56 cells formed the majority of TIL in all cultures. In 4 cases CD4 cells predominated at the initial stage of culturing, with CD8 cells gradually increasing and finally inverting the CD4/CD8 ratio. Autologous cytotoxicity (3/4 cases) appeared to be better in those patients in whom the CD4/CD8 ratio was inverted. These data enable identification of the combination of lymphokines that will will best provide expansion of live TIL active against tumoral cells. This procedure must be followed before in vivo reinfusion of expanded lymphocytes is carried out.

Effects of blood transfusion on the immune responsiveness and survival of cancer patients: a prospective study.

To evaluate whether blood transfusion exerts an adverse influence on cancer evolution, a prospective clinical and immunologic investigation was carried out on 58 surgical patients with gastric or colorectal adenocarcinoma. None had had previous transfusion; 35 received perioperative transfusion. Among preoperative variables, only red cell count and hemoglobin concentration were significantly reduced in the patients transfused at operation. Other clinical characteristics and immunologic functions (except interferon-gamma release) did not differ significantly from those of untransfused patients. The survival rate of transfused patients, although shorter, was not significantly different from that of untransfused patients. Immunologic tests done after surgery on 30 patients (17 transfused and 13 untransfused) did not show significant differences in the two groups. Significant increases in interleukin-2-stimulated production and immunoglobulin M synthesis were observed in transfused patients after surgery. Patients transfused perioperatively with more than 3 units of blood had some evidence of decreased immune function, but differences were not significant. While shorter survival and some immunologic changes may correlate with the number of transfusions, more patients must be studied to determine whether this relationship will be confirmed.

Investigation on the humoral immune response in patients with gastro-intestinal cancer.

The humoral immune reactivity was studied in patients affected by gastro-intestinal cancer. The number of peripheral B lymphocytes, the concentration of serum immunoglobulins (Ig) and of C3 complement factor and the frequency of circulating immuno complexes (CIC) did not significantly differ between patients and age-matched controls, while the C4 factor level was significantly increased. The frequency of serum monoclonal components (M-components) was higher in the patients than in the elderly subjects. Moreover the results concerning the "in vitro" functional response of patient B lymphocytes showed a significant decrease of the proliferative responses to Staphylococcus Aureus Cowan I (SAC) and a significant increase of the IgG and IgM synthesis by peripheral blood mononuclear cells (PBMC) in unstimulated and pokeweed mitogen (PWM) stimulated cultures. The meaning of these results, taken together with those reported by others, is discussed.

Functional analysis of lymphocytes from two patients affected by common variable immunodeficiency (CVI).

Two patients affected by severe hypogammaglobulinemia classified as CVI were studied. Both patients showed an increase in peripheral T cells and a normal or elevated number of surface immunoglobulin-bearing cells (sIg+); the T cell subsets showed a decrease of CD4 and an increment of CD8 cells with an inversion of the CD4/CD8 ratio. Patient peripheral blood mononuclear cells (PBMC) did not proliferate after Staphylococcus aureus Cowan I (SAC) activation. Moreover, patient PBMC were not able to differentiate into plaque - forming cells (PFC) either spontaneously or after pokeweed mitogen (PWM) stimulation. The immunoglobulin synthesis from patient PBMC stimulated in vitro by PWM was very little as compared to controls. When isolated patient B cells were cultured in the presence of exogenous B cell growth factor (BCGF) and BCGF plus anti-mu and anti-delta antibodies, no proliferation was observed. Taken together the results concerning B cell function of our CVI patients indicate the presence of an intrinsic defect of B cells. These cells are normal in number, but they are not able to leave the resting state, enter the activation state, proliferate and differentiate into Ig secreting cells. Moreover the alteration of the T cell subset proportions seems to suggest an impaired cooperation between B and T cells.

Effects of the allogeneic stimulation on B cell function in thalassaemic polytransfused patients.

The immunological abnormalities of polytransfused patients affected by beta-thalassaemia major have been investigated in relation to B cell function. The spontaneous in vitro production of Ig by patient peripheral mononuclear cells was not modified, while the pokeweed mitogen induced IgM synthesis was significantly reduced. However, by comparing the splenectomized and non-splenectomized patients, this alteration proved to be present only in the splenectomized group. The proliferation of patient peripheral B cells in vitro stimulated with B cell growth factor alone was not significantly enhanced in respect to the controls. These results indicate that in vivo activation state of patient B cells is not different from that of the controls. The stimulation of peripheral B cell with anti-mu or anti-alpha antibodies and anti-mu and anti-alpha antibodies plus B cells growth factor induced a significant increase in proliferative responses of B cells from the splenectomized patients. Taken together, these findings suggest that circulating B cells of thalassaemic polytransfused patients are not hyperactivated. The splenectomy can account for the immunological changes observed in our thalassaemic sample as compared to control group.

Immunoenzymatic determination of immunoglobulins secreted by human peripheral blood lymphocytes in pokeweed mitogen and lipo-polysaccharide stimulated cultures.

Immunoglobulins (Ig) production by peripheral blood mononuclear cells (PBMC) from 27 healthy subjects at the 6th and 12th day of in vitro stimulation by pokeweed mitogen (PWM) or bacterial lipo-polysaccharide (LPS) was investigated. Total IgG, IgA, IgM in the culture supernatants were measured by an enzyme linked immunosorbent assay (ELISA). The average values of Ig production (in ng per ml of culture supernatants) under PWM and LPS stimulation after 12 days of culture are respectively: 3397 and 2557 for IgG, 512 and 374 for IgA, 3172 and 1439 for IgM. The use of PWM and LPS mitogens for stimulating Ig secreting cells may provide insights into the nature of the interacting cells on immune responses and into the pathogenesis of different diseases.