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Irradiated human peripheral blood lymphoid cells undergo apoptosis and progressively exhibit typical changes in light scatter and plasma membrane integrity that can be easily tracked by flow cytometry. Using this model, we assessed the capacity of a newly developed fluorochrome, Apostain, in identifying early apoptosis in unfixed samples. This probe is a plasma membrane permeant DNA dye that can be conveniently excited at 488 nm and has an emission wavelength >650 nm. To identify apoptotic cells, Apostain relies on the transient changes of chromatin texture that allow to accommodate more of a DNA dye occurring in early apoptosis. As early as 4 h after irradiation, a proportion of cells showed an enhanced Apostain uptake. Consistent with their initial apoptotic nature, these cells had a still integer plasma membrane, as assessed by ethidium bromide, and unaltered light scatter. With time, cells showing the enhanced Apostain uptake started to bind dimly Annexin-V and, later, reduced their forward scatter. After 18 h from irradiation, cells exhibiting a reduced forward scatter exhibited a bright staining with Annexin-V with a concomitant reduction in Apostain uptake, reflecting the gross chromatin disruption characterising the endpoint of apoptosis.

Fifty uniformly treated adult AML patients were analyzed with respect to pre-treatment and post-induction risk factors. Forty-two patients achieving complete hematological remission were assessed for minimal residual disease (MRD) by WT1 gene expression; 34 by flow-cytometry (flow-MRD). Patients who were flow-MRD negative had a better 3-year disease-free (DFS; 79.5% vs. 27.3%; p=.032) compared with patients who were still positive after induction. Interestingly, DFS of flow-MRD positive patients was not related to the amount of flow-detected clone population (≥ or

Introduction With standard intensive induction regimens, up to 80% of Acute Myeloid leukemia (AML) patients can achieve complete remission (CR). Several evidences demonstrated that the persistence of detectable disease (MRD) assessed with highly sensitive techniques such as Multicolor-Flow-Cytometry (MFC) and PCR based molecular analysis, retains a prognostic value among patients achieving morphological remission (Walter RB, 2015; Araki D et al, 2016, Zhou Y et al, 2016). The aim of the present study was to retrospectively evaluate the prognostic impact of MRD in a cohort of uniformly treated AML patients. One hundred and ten consecutive AML patients who had been treated in our center between January 2004 and December 2014 were retrospectively analyzed. All patients had received a fludarabine-containing induction (FLAI-5) and received second cycle and further consolidation therapy according to our published strategy (Guolo F, 2016). Median age was 47 years (range 17-61). Median follow up was 59 months. Patients features are summarized in Table I. MRD assessment was performed through 4-colour MFC analysis (MFC-MRD)and through WT1-gene expression analysis, as previously described (Guolo F, 2016). Three different MRD time-points (TP)were considered: TP1, after induction I; TP2, after induction II; TP3, after consolidation therapy for patients who did not undergo HSCT and at HSCT for patients who underwent HSCT. Relapse-free survival (RFS) was calculated from the time of diagnosis until last follow-up or documented leukemic relapse. CR rate after 1st and 2ndinduction was 82.7 and 85.5%, respectively, whereas 30 and 60 days mortality was 6.4% and 8.2%, respectively. Overall, patients showed MRD reduction from TP1 to TP2. Detailed MRD negativity rates are provided in table II. MRD clearance probability was significantly influenced only by ELN risk group and Karyotype (p In the whole cohort, 2 years RFS was 62.4% (median not reached). RFS duration was significantly influenced by secondary disease (p Overall survival (OS) analysis led to similar results. However, at TP1, when performing MFC-MRD evaluation with a "Leukemia-associated immunophenotype" (LAP) approach, the choice of the deeper 0.025% cut-off for positivity identified patients at lower risk of relapse (2-year RFS of 88.9%) whereas patients with MFC MRD >0.1% had an high probability of relapse, with a 2-year RFS of only 21.5%. Moreover, the combination of MFC and WT1- MRD assessment identified three subgroups of patients with significantly different outcome (2-years OS 100, 64.4 and 46.7%, respectively, for MFC-neg/WT1 neg, MFC-pos/WT1 neg, MFC-pos/WT1 pos), as we previously described (Marani C, 2013). Consolidation chemotherapy with High Dose Ara-C was able to improve prognosis and increase MRD TP3 negativity rate only in patients not scheduled for HSCT in 1st CR and only if at least 2cycles were administered. The positive impact was higher if MFC MRD at TP2 was Conclusions Our data clearly show that the quantification of MFC-MRD assessment at different time-points during treatment retains a strong prognostic impact in AML and can improve patients-risk stratification. In our experience the established threshold of 0.1% to define MFC negativity is confirmed as the most useful in post induction program setting. However, performing a MFC-MRD evaluation with LAP approach, choosing a deeper cut-off value can discriminate patients with a significantly lower risk of relapse. Prospective randomized trials evaluating the prognostic impact of MRD-driven therapeutic decisions are strongly needed. Disclosures Gobbi: Takeda: Consultancy; Janssen: Consultancy, Honoraria; Gilead: Honoraria; Celgene: Consultancy; Roche: Honoraria; Novartis: Consultancy, Research Funding; Mundipharma: Consultancy, Research Funding.

BACKGROUND AND AIMS Allogeneic bone marrow transplantation (BMT) offers the greatest chance of cure for high-risk acute myeloid leukemia (AML) patients. Persistence of disease or high levels of pre BMT minimal residual disease (MRD) have been reported to predict relapse risk after BMT. Multicolor flow cytometry (MFC) is the most common tool to evaluate MRD, whereas WT1 gene expression is the most widely applicable and standardized molecular MRD marker in AML. The aim of this study was to analyze the role of pre-BMT combined MFC-molecular MRD assessment as predictor for the post-transplant relapse risk. MATERIALS AND METHODS We retrospectively analyzed the outcome of 224 consecutive AML patients receiving allo-BMT in 1st or 2nd CR. Pre-BMT marrow samples were analysed for WT1 expression and MFC as MRD evaluation. Median age at transplant was 44 years. Disease phase was CR1 in 161 (72%) and CR2 in 63 patients (28%). BMT conditioning wasmyeloablative in 163 (73%), whereas 61 patients (27%) received reduced intensity conditioning. Stem cell source was HLA-identical sibling in 79 (35%),haploidentical in 59 (27%) and alternative donor in 86 (38%).Median follow-up was 64 months (95% CI 50.3 - 77.3 months). Cumulative Incidence of Relapse (CI of relapse) at various time-points was calculated in competing risk analysis by takingin account non relapse mortality as competing event. Overall Survival (OS) was calculated from the time of BMT until death by any cause or last follow-up. A positive MFC MRD was defined by the presence of no less than 25 clustered leukemic cells/105 total events (threshold of 2.5x10-4 residual leukemic cells) at four-color flow-cytometry. WT1 copy number/Abl copy number 500x104 was used as cut-off value for abnormal WT1 expression. RESULTS Relapse occurred in 62 patients (27.7%). Three-year estimate of CI of relapse was 25.9% (median not reached). The CI of relapse was significantly affected by occurrence of acute GVHD (lower for grade ≥2, p Specifically, patients with double positive molecular and MFC MRD had a very poor outcome, with a 3-year CI of relapse of 51.9% (Figure 1). On the contrary, patients with negative combined MRD had a very low CI of relapse, regardless of the conditioning regimen received, whereas a trend toward benefit frommyeloablativeconditioning was observed in patients with MFC positive/WT1 negative MRD and double positive patients. Patients with negative MFC-MRD undergoing BMT in 1st or 2nd CR had a very similar outcome (3-year CI of relapse 8.3 and 5.6%, respectively). Similarly, MFC positive/WT1 negative MRD patients had a 3-year CI of relapse of 17.3 and 18.% for CR1 and CR2 patients, respectively. Patients who were MRD positive by both methods undergoing BMT in CR1 or CR2 had a similar, very high CI of relapse (Figure 2). MRD evaluation was also a strong predictor of long term survival: 3- years OS was 73.5% for MFC negative and 36.7% for double WT1 and MFC MRD positive patients, respectively, (p CONCLUSIONS Pre transplant MRD evaluation by combined molecular and MFC assessment on bone marrow samples is a reliable predictor of relapse risk. Patients with both negative pre-BMT MRD markers have a significantly lower CI of relapse, while patients with both positive MRD markers display an higher risk of relapse. Given the high prognostic value, combined molecular/MFC pre-transplant MRD status may be taken in account when considering the most appropriate stem cell source or conditioning for each patient. Furthermore, identifying patient in morphological CR at BMT but with a very high risk of relapse, such as double positive patients, could open the way to apply pre-emptive therapeutic strategies to prevent AML relapse, from donor lymphocyte infusion to other innovative approaches. Figure 1. CI of Relapse and Overall Survival according to risk group Figure 1. CI of Relapse and Overall Survival according to risk group Figure 2. a: OS according to MRD in patients transplanted in CR1 b: OS according to MRD in patients transplanted in CR2 Figure 2. a: OS according to MRD in patients transplanted in CR1. / b: OS according to MRD in patients transplanted in CR2 Disclosures Gobbi: Janssen: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; Celgene: Consultancy; Roche: Honoraria; Mundipharma: Consultancy, Research Funding; Takeda: Consultancy; Gilead: Honoraria.

In addition to HIV infection, several acquired immunodeficiencies lead to depletion of CD4 lymphocytes. These include immunosuppression resulting from high dose cancer chemotherapy or induced to control graft rejection, as well as in autoimmune diseases. The consequence of this depletion is an increased susceptibility to opportunistic infections or the inability to control primary infection in the case of HIV infection. In all instances a full or partial immunoreconstitution is desirable. In order to monitor the cellular immune state of a patient, rational information cannot be simply derived from phenotypic quantification of T lymphocytes. Instead loss or recovery of CD4 cells should be monitored by defining the specificity, the function and the clonality of the relevant cell population. Several methods are now available for this type of investigation. Here we describe an approach for the definition of clonal heterogeneity of antigen specific CD4 lymphocytes, a parameter that may help monitor loss or reconstitution in acquired immunodeficiencies. As examples of antigen specific CD4 T cell responses we focused on Pneumocystis carinii and on cytomegalovirus, as prototypic opportunistic pathogens which are responsible for severe infections in AIDS and in other immunosuppressive conditions which arise for instance following transplantation. Specific CD4 T cell lines were generated from normal controls and from seropositives in order to select antigen specific lymphocytes. The cells were subsequently analyzed for clonal diversity according to TCR BV gene family usage and according to TCR CDR3 size heterogeneity (spectratyping).

The 'dangerous liaison' between CD4 and gp120 that offers the first entry opportunity to HIV may also provoke perturbations of the immune control of the host with far-reaching immunopathological consequences. We wondered whether a mechanism of intermolecular help (T help across the gap of a non-covalent bond, in contrast to the intramolecular help of carrier to hapten) could break self-tolerance and be the cause of the frequent anti-CD4 autoantibodies found in AIDS patients. To determine whether this hypothesis deserves further testing, we designed a series of in vitro and in vivo experiments of increasing complexity, focused on the presentation of gp120 to specific T cells by antigen presenting cells (APC) exposed to the envelope protein in the form of non-covalent complexes. Bi-molecular complexes were constructed by allowing gp120 or gp160 to bind specific human mAbs. Tri-molecular complexes were constructed by introducing CD4 as an intermediate ligand between gp120 and mouse mAbs specific for CD4. In all cases the use of complexes did enhance the immunogenic capacity of substimulatory doses of gp120 or gp160 by facilitating uptake by APC via Fc receptor and consequent presentation to specific human T cell clones. Finally, help for the production in vivo of anti-CD4 antibodies was obtained from T lymphocytes specific for gp120 when CD4-primed memory B cells were pulsed with CD4 complexed with gp120, thus demonstrating in the mouse the entire cycle of intermolecular help via non-covalent interaction, and setting the stage for future experiments on self-tolerance breakage in a human molecular context.

Surrogate markers for monitoring immuno-virological discordant responders, in addition to plasma viral load and CD4 cells, are still lacking. We assessed the diagnostic utility of CD38 expression on CD8 T cell assay, alone or in association with lymphocyte proliferation to mycotic antigens, in evaluating antiretroviral response. 28 vertically HIV-infected youths, 21 HAART- and seven 2 nucleotide reverse transcriptase inhibitors-treated, were enrolled in a retrospective study. Responders (57.1%) and non-responders (42.9%) to stable antiretroviral therapy for a minimum of 6 months, on the basis of viral load and CD4 T cells, comprehensively evaluated by CD38 expression on CD8 T lymphocytes [measured as CD38 antibody bound per CD8 T cell (CD38 ABC) and %CD38+ of total CD8 T cells (%CD38/CD8)] and lymphocyte proliferation to P. jiroveci, C. albicans, C. neoformans, A. fumigatus at a single time point after treatment, were selected. CD38 expressionor =2401 CD38 ABC andor =85% CD38/CD8 cut-off points, accurately discriminates responders versus non-responders, both measures resulting in 75.0% (CI 42.8-94.5) sensitivity (identification of non-responder) and 93.8% (CI 69.8-99.8) specificity (identification of responder), when considered as single assays. The association 'or =2401 CD38 ABC oror =85% CD38/CD8' improved sensitivity to 83.3% (CI 51.6-97.9), while the association '2401 CD38ABC (or85% CD38/CD8) and lymphoproliferative response positive toor =2 tested organisms' improved specificity to 100% (CI 79.4-100). In conclusions, CD38 expression and mycotic antigen-specific T-cell proliferation may be used as additional parameters to existing criteria to evaluate antiretroviral response in immuno-virological discordant patients.

Activation of a galactosidase-specific murine T hybridoma clone and of a human tetanus toxoid-specific T clone by antigen-presenting cells (APC) was used to evaluate the regulatory function of antibodies complexed with the relevant antigen. Complexed antigen, in fact, is taken up with high efficiency thanks to Fc receptors borne by APC. Antibody/antigen ratio in the complexes proved to be a critical parameter in enhancing antigen presentation. Complexes in moderate antibody excess provided optimal T cell activation independently of the physical state of the complexes (precipitated by a second antibody or solubilized by complement). Complexes in extreme antibody excess, on the contrary, did not yield T cell activation although taken up by APC efficiently. The effect of antibodies at extreme excess was observed with substimulatory dose of antigen (loss of potentiation) and with optimal dose of antigen (loss of stimulation). An excess of specific polyclonal antibodies hampers proteolytic degradation of antigen in vitro, supporting the view that a similar mechanism may operate within the APC that have internalized immune complexes in extreme antibody excess. The possibility that immune complex forming in extreme antibody excess may turn off the T cell response is proposed as a regulatory mechanism.

Germinal centres are areas of B lymphocytes proliferation that appear in primary lymphoid follicles after immunization. The results summarized here implicate these structures in the establishment of immunological memory for antibody production. It appears that after primary immunization antigen-antibody-complement complexes become trapped on the membrane of specialized dendritic cells in lymphoid follicles, and these complexes provide the stimulus for germinal centre formation. In support of this, immunization with preformed antigen-antibody complexes, rather than with antigen, leads to the earlier appearance of germinal centres and memory cells, and also accelerates the selective triggering of precursors capable of producing high affinity antibodies.

Background Mesenchymal stromal cells are multipotent cells considered to be of great promise for use in regenerative medicine. However, the cell dose may be a critical factor in many clinical conditions and the yield resulting from the ex vivo expansion of mesenchymal stromal cells derived from bone marrow may be insufficient. Thus, alternative sources of mesenchymal stromal cells need to be explored. In this study, mesenchymal stromal cells were successfully isolated from second trimester amniotic fluid and analyzed for chromosomal stability to validate their safety for potential utilization as a cell therapy product.Design and Methods Mesenchymal stromal cells were expanded up to the sixth passage starting from amniotic fluid using different culture conditions to optimize large-scale production.Results The highest number of mesenchymal stromal cells derived from amniotic fluid was reached at a low plating density; in these conditions the expansion of mesenchymal stromal cells from amniotic fluid was significantly greater than that of adult bone marrow-derived mesenchymal stromal cells. Mesenchymal stromal cells from amniotic fluid represent a relatively homogeneous population of immature cells with immunosuppressive properties and extensive proliferative potential. Despite their high proliferative capacity in culture, we did not observe any karyotypic abnormalities or transformation potential in vitro nor any tumorigenic effect in vivo.Conclusions Fetal mesenchymal stromal cells can be extensively expanded from amniotic fluid, showing no karyotypic abnormalities or transformation potential in vitro and no tumorigenic effect in vivo. They represent a relatively homogeneous population of immature mesenchymal stromal cells with long telomeres, immunosuppressive properties and extensive proliferative potential. Our results indicate that amniotic fluid represents a rich source of mesenchymal stromal cells suitable for banking to be used when large amounts of cells are required.

Objective Endothelial progenitor cells (EPCs) are involved in neovessel formation. So far, therapeutic angiogenesis is hampered by the low frequency and limited proliferative potential of these cells isolated from peripheral blood. Recently, it has been shown that cord blood–derived EPCs (CB EPCs) can be ex vivo expanded on a clinical scale. In this study, we evaluated the expansion potential of CB EPCs together with their phenotypic, functional, and chromosomal stability over time. Materials and Methods Flow cytometry, in vitro tube formation, and proliferation assays were performed to characterize CB EPC–derived cells. Chromosomal stability was evaluated by karyotype analysis. In vitro and in vivo tumorigenicity was evaluated by soft agar assay and injection into nonobese diabetic/severe combined immunodeficient mice, respectively. Results We showed that CB EPC–derived cells displayed phenotypic and functional features of EPCs, although a process of maturation was observed over time. Although we confirmed that CB EPCs have a greater expansion potential compared to peripheral blood EPCS, we observed a high incidence of cytogenetic alterations (71%) in the expanded endothelial cell population, even at early times of culture. In two cases, spontaneous transformation in vitro was documented, but none of the samples tested showed tumorigenic potential in vivo. Conversely, no karyotype alterations have been observed on peripheral blood EPCs–derived cells. Conclusions We confirm that CB represents a good source for clinical ex vivo expansion of EPCs. However, because of high frequency of karyotype alterations, these cells cannot be considered free of risk in clinical application.

Tumor growth is allowed by its ability to escape immune system surveillance. An important role in determining tumor evasion from immune control might be played by tumor-infiltrating regulatory lymphocytes. This study was aimed at characterizing phenotype and function of CD8+CD28− T regulatory cells infiltrating human cancer. Lymphocytes infiltrating primitive tumor lesion and/or satellite lymph node from a series of 42 human cancers were phenotypically studied and functionally analyzed by suppressor assays. The unprecedented observation was made that CD8+CD28− T regulatory lymphocytes are almost constantly present and functional in human tumors, being able to inhibit both T cell proliferation and cytotoxicity. CD4+CD25+ T regulatory lymphocytes associate with CD8+CD28− T regulatory cells so that the immunosuppressive activity of tumor-infiltrating regulatory T cell subsets, altogether considered, may become predominant. The infiltration of regulatory T cells seems tumor related, being present in metastatic but not in metastasis-free satellite lymph nodes; it likely depends on both in situ generation (via cytokine production) and recruitment from the periphery (via chemokine secretion). Collectively, these results have pathogenic relevance and implication for immunotherapy of cancer.

Background This study analyzed morbidity, mortality, and quality of life after aortic valve replacement with mechanical and biologic prostheses in octogenarian patients. Methods A retrospective analysis was performed in 345 consecutive patients, mean age of 82 ± 2 years (range, 80 to 92), who had aortic valve replacement from May 1991 to April 2005. A bioprosthesis (group I) was used in 200 patients (58%), and 145 (42%) received a mechanical prosthesis (group II). Associated cardiac procedures were done in 211 patients (61%), of which 71% were coronary artery bypass grafting. Patients had symptomatic aortic stenosis (84.3%) or associated aortic insufficiency; 88% were in New York Heart Association (NYHA) class III or IV. The mean preoperative aortic valve gradient was 62 ± 16 mm Hg (range, 25 to 122 mm Hg). The mean left ventricular ejection fraction was good (0.52 ± 0.12); 30 patients (8.7%) had an ejection fraction of less than 0.30. Results The in-hospital mortality rate was 7.5% (26 patients); 17 (8.5%) in group I and 9 (6.2%) in group II ( p = 0.536) Significant predictors of operative mortality were preoperative renal insufficiency (blood creatinine > 2.00 mg/mL) and need for urgent operation. Mean follow-up, complete at 100%, was 40 ± 33 months (range, 1 to 176 months). Long-term follow-up, using Kaplan-Meier analysis, showed an overall survival of 61% at 5 years and 21% at 10 years; survival by type of prosthesis was significantly higher with mechanical prostheses (log-rank p = 0.03). Freedom from cerebrovascular events (thromboembolic/hemorrhagic) at 5 and 10 years was 89% and 62% in the mechanical group and 92% and 77% in the biologic group ( p = 0.76). Postoperative NYHA functional class was I or II in 96% of patients. Quality-of-life scores were excellent considering the age of the patients. No differences were found between the two groups. Conclusions Surgical treatment for symptomatic aortic stenosis in octogenarians has an acceptable operative risk with excellent long-term results and good quality of life. In this cohort, survival rate is slightly but significantly higher with mechanical prostheses.

BACKGROUND AND AIMS The prognosis of Philadelphia positive (Ph+) acute lymphoblastic leukemia (ALL) patients has improved since the introduction of tyrosine kinase inhibitors (TKI). The inclusion of TKIs in standard ALL protocols allows a great increase in complete molecular responses, but at the price of non negligible toxicities and high rates of toxic deaths. On the other and TKI monotherapy as induction treatment allows to rapidly achieve complete hematologic remission (CR) but only a minority of patients achieve a complete molecular response with high risk of relapse. On the other hand, In the last years we tested a combination of Fludarabine, Cytarabine, Daunoxome (FLAD) with or without TKIs (mainly Dasatinib) as salvage regimen in relapsed-refractory ALL, with acceptable toxicity and good efficacy. We decided to apply the same schedule in newly diagnosed Ph+ ALL as consolidation treatment after a two months TKI (Dasatinib) monotherapy induction on a minimal residual disease condition. MATERIALS AND METHODS FLAD regimen consisted with a three-days administration of Fludarabine 30 mg/sqm followed four hours later by Cytarabine 2000 mg/sqm and Daunoxome 100 mg/sqm. TKI were suspended during chemotherapy administration and were re-administrated starting from day 5. G-CSF was given to all patients from day 4 to complete hematological recovery. FLAD was administrated for up to two cycles; all patients with available donor proceeded to allogeneic bone marrow transplantation (allo-BMT) after FLAD. Minimal residual disease (MRD) was evaluated in all patients after each FLAD either by RQ-PCR for VDJ rearrangements, multicolor flow cytometry (MFC) and RQ-PCR for BCR/Abl. Ten Ph+ ALL have been treated with FLAD + TKIs from January 2008 to December 2014: six patients received FLAD as salvage regimen, two of them in post allo-BMT setting, whereas four patients were treated frontline, after hematological CR was obtained with Dasatinib + steroids induction. All frontline patients proceeded to allo-BMT after two FLAD. Median age for frontline patients was 50 years (range 29-58), median follow-up was 20 months. RESULTS As salvage regimen, 5/6 patients achieved hematological CR after FLAD, with three patients achieving also MFC MRD negativity and clearance of VDJ and BCR/Abl transcript. All patients who did not receive subsequent BMT relapsed, whereas of the two transplanted patients one is still in CR after a follow-up of 38 months. In the frontline setting, all patients received 70 days induction of Dasatinib + Steroids and achieved CR with complete hematological recovery. BCR/Abl transcript could be detected in all patients on BM samples on day 33 and on day 70 (Fig. 1), two patientshad MFC MRD positivity both on day 33 and on day 70, whereas two patients achieved MFC MRD negativity on day 33. FLAD was very well tolerated, with negligible non hematological toxicity, with a median duration of ANC Two patients achieved BCR/Abl negativity after first FLAD. All patients achieved molecular complete response after the second course (Fig. 1). No patient experienced relapse, whereas one patient died in CR on day +289 after allo-BMT due to myocardial viral infection. CONCLUSIONS FLAD has a very good efficacy in adult Ph+ ALL, with an acceptable toxicity profile. Deep responses have been observed in relapsed patients, and all newly diagnosed patients who received FLAD as consolidation regimen had achieved molecular CR before allo-BMT. Achieving complete hematological response with Dasatinib + steroids allowed us to safely administer two FLAD courses. Figure 1. BCR/abl on bone marrow samples at different timepoints for each of the four patients receiving FLAD as consolidation therapy Figure 1. BCR/abl on bone marrow samples at different timepoints for each of the four patients receiving FLAD as consolidation therapy Disclosures Off Label Use: Use of liposomal daunorubicin in the treatment of ALL.

Recent works demonstrated the presence of a multipotent epithelial cell population in the bulge region of adult human hair follicles. These cells can be cultured in vitro, thus leading to the preparation of dermal-epidermal substitutes which are applicable in the treatment of burns and ulcers. We evaluated the main marker expression in cells obtained from stripped human hair follicles. A pool of hair follicles were incubated at 37 degrees C and 5% CO(2) in a growth medium. The cells were then labelled with antibodies (anti-CD34, anti-CD38, anti-CD45, anti-CD90, anti-CD133, anti-CD146) and analysed by cytometry. We also used hair follicles for immunohistochemical studies, employing antibodies such as CD34, Actin Smooth Muscle, Filaggrin, Desmin, Vimentin, Glial Fibrillary Acidic Protein, Ki-67, PanCytokeratin, CK15, CK19. The cytometry results revealed that a part of bulge cells were CD34+ (1-2%). CD34+ population comprises both large, CD45-, CD133-, CD146- cells and small, CD45+, CD133+, CD146+ cells. Thus, a part of CD34+ cells present a mature endothelial marker (CD146). An expression of the proliferation marker Ki-67 and the stem cell marker CD34 is present in the follicle bulge region. In conclusion, we observed that the stripped hair follicle has the same multipotent cell population as adult and fetal scalp hair follicles.

Our genetic system for expression of heterologous proteins on the surface of the Gram-positive bacterium Streptococcus gordonii was used to express a human T-helper epitope of HIV-1 envelope glycoprotein gp120. In previous work on the naive repertoire of human T-helper cells, it was shown that a 15-amino acid synthetic peptide of the HIV-1 gp120 sequence contained an immunodominant T-helper epitope. Synthetic DNA coding for this peptide was cloned in frame within the gene for the streptococcal surface protein M6, and the gene fusion was integrated by transformation into the chromosome of S. gordonii. The expected M6-gp120 fusion protein was found to be expressed on the surface of the recombinant streptococci. To test whether the T epitope could be recognized by T cells when expressed on the bacterial surface within the context of M6, recombinant bacteria were used as antigen in proliferation assays to stimulate the 15-amino acid-specific human T-helper clone, in the presence of autologous antigen-presenting cells. Bacteria expressing the T epitope were efficiently recognized by the T cells in culture. In proliferation assays, 10(6)-10(7) bacteria induced responses comparable to those obtained by standard amounts of synthetic peptide (0.02-0.2 micrograms). Recombinant S. gordonii, a candidate for a live vaccine vector, appeared suitable for delivering T epitopes to the immune system.

Cholesterol embolism is often an unrecognized complication of some cardiac and vascular procedures (i.e. coronarography, angioplasty, aortocoronary bypass, abdominal aortic aneurysmectomy) and of therapies affecting coagulation (thrombolysis, anticoagulation). The degree of pain associated with ischaemic and necrotic lesions secondary to cholesterol embolism involving the lower limbs is disproportionate to the extension of tissue involvement. Spinal cord stimulation (SCS) has been recognized as effective in relief of pain of ischaemic and neuropathic nature, although its mechanism of action is still not completely clear. The authors are unaware of previous reports of peripheral cholesterol embolism treated by SCS. Two case reports of inferior limb ischaemia secondary to cholesterol embolism in patients who had undergone cardiac invasive procedures. Temporary surgical implantation of SCS devices, which were removed after 4 to 6 weeks. Pain relief was achieved within 1 to 4 hours of surgical procedure. Any analgesic medications could be immediately discontinued. Pain control was effective and normal daily activities were rapidly regained. Ischaemic lesions healed within 4 to 6 weeks of SCS. Pain control is the most critical aspect of the management of peripheral cholesterol embolism without visceral organ involvement. SCS provided effective pain relief in the reported cases and its established ability to improve peripheral microcirculation allowed rapid resolution of necrotic lesions. Temporary SCS should be considered in the management of painful necrotic skin lesions secondary to iatrogenic cholesterol embolism.

Background: Allogeneic bone marrow transplantation (BMT) offers the greatest chance of cure for most patients affected by acute myeloid leukemia (AML). Persistence of disease or high levels of pre BMT minimal residual disease (MRD) have been reported to predict disease relapse after BMT. WT1 expression levels and multicolor flow cytometry (MFC) are widely used as markers of MRD. We recently reported that combined evaluation of MRD by WT1 and MFC after induction therapy can predict relapse risk in AML patients. Aims: The aim of the present study was to apply the same MRD assessment in pre BMT setting to evaluate its reliability in predicting relapse. Methods: We retrospectively analyzed BMT outcome of 66 AML patients with both WT1-based and MFC-based MRD evaluation on bone marrow samples before transplant. Median age at transplant was 44 years. Forty-one were transplanted in first and twenty-five in second or subsequent complete remission. Induction therapies included fludarabine-containing regimens or standard ara-C and daunorubicin schedule (3+7). Median follow-up was 44 months (range 0-119 months); pre-transplantation evaluations were performed at a median of one month before transplant (range 1-3). Disease-free survival (DFS) was calculated from the time of transplantation until last follow-up or documented leukemic relapse. Overall survival was calculated from the time of transplantation to the last follow-up or death for any cause. All causes of death not directly due to relapse or progression of leukemia were considered as non-relapse mortality. A positive MFC MRD was defined by the presence of no less than 25 clustered leukemic cells /105 total events (threshold of 2.5x10-4 residual leukemic cells) at four-color flow-cytometry. Real-time PCR for WT1 was performed on DNA Engine 2 (Opticon®, MJResearch®). WT1 copy number/Abl copy number 500x104 was used as cut-off value for high WT1 expression. Results: Twenty-five relapses (37.9%) were observed. Median DFS was 31 months. Our analysis shows that the probability of relapse was significantly influenced only by disease status (first or subsequent CR) and MRD status at transplantation. Specifically, MFC-MRD was the strongest predictor of longer disease free survival (p Conclusion: pre BMT MRD evaluation by WT1 and MFC on bone marrow samples is a reliable tool to predict relapse risk. Patients with negative pre-BMT MRD have a significantly longer DFS and OS, while MRD positive patients by both methods display a higher risk of relapse. Patients at higher risk of poor outcome should undergo a more stringent program of post BMT evaluations, in order to detect disease relapse earlier and might be candidate for pre-emptive therapeutic interventions aimed at delaying or avoiding AML reoccurrence. Disclosures No relevant conflicts of interest to declare.

The aim of the paper is to evaluate retrospectively the series of patients affected by colon polyps and colon cancer to purpose a planned follow-up for patients who underwent colon polypectomy.Out of 198 patients affected by colon carcinoma, the authors describe 95 cases of previous, synchronous or metachronous polyps, with a global prevalence of 47.9%, and respectively of 13.6%, 16.6%, and 17.6%.The evolutive sequence between colon polyp and carcinoma is shown through both clinical experience and experimental tests. The epidemiologic curve of adenoma rate precedes by five years the curve of carcinoma, and such is the average period of time for the transformation of an adenoma into invasive carcinoma. In clinical practice, it is frequent to note synchronous or metachronous polyps with respect to colon carcinoma. It is also possible to note carcinoma in patients with previous polypectomies.On the basis of the retrospective evaluation of the clinical cases, and reviewing international literature, the authors suggest their diagnostic-therapeutic and endoscopic follow-up protocol for patients affected by colon neoformations (Fig. 1).

The enumeration of absolute levels of cells and their subsets in clinical samples is of primary importance in human immunodeficiency virus (HIV)+ individuals (CD4+ T- lymphocyte enumeration), in patients who are candidates for autotransplantation (CD34+ hematopoietic progenitor cells), and in evaluating leukoreduced blood products (residual white blood cells). These measurements share a number of technical options, namely, single- or multiple-color cell staining and logical gating strategies. These can be accomplished using single- or dual-platform counting technologies employing cytometric methods. Dual-platform counting technologies couple the percentage of positive cell subsets obtained by cytometry and the absolute cell count obtained by automated hematology analyzers to derive the absolute value of such subsets. Despite having many conceptual and technical limitations, this approach is traditionally considered as the reference method for absolute cell count enumeration. As a result, the development of single-platform technologies has recently attracted attention with several different technical approaches now being readily available. These single-platform approaches have less sources of variability. A number of reports clearly demonstrate that they provide better coefficients of variation (CVs) in multicenter studies and a lower chance to generate aberrant results. These methods are therefore candidates for the new gold standard for absolute cell assessments. The currently available technical options are discussed in this review together with the results of some cross-comparative studies. Each analytical system has its own specific requirements as far as the dispensing precision steps are concerned. The importance of precision reverse pipetting is emphasized. Issues still under development include the establishment of the critical error ranges, which are different in each test setting, and the applicability of simplified low-cost techniques to be used in countries with limited resources.

Abdominal aortic aneurysm and cholelithiasis are two common diseases in the elderly population. The prevalence of abdominal aortic aneurysms ranges between 1.8 and 6.6% in autoptic series and it's estimated that 2.5% of the over sixty year old population is affected. Carcinoma of the gallbladder is the most common malignant tumor of the biliary tract and in the United States is the fifth most frequent digestive tract malignancy; it's incidence ranges between 2 to 10 cases of 100,000 persons/year. No adequate guidelines are now available to assist the surgeon, in the case of concomitant gallbladder disease and abdominal aortic aneurysm. In this paper the management of abdominal aortic aneurysm in a patient with gallbladder disease is discussed in order to assist the surgeon deciding whether to perform concomitant aneurysm resection and cholecystectomy. In 162 aneurysmectomies (1987-1997) 18 (11.11%) patients underwent combined aneurysmectomy and cholecystectomy operation. The patients ranged in age from 49 to 88 years (average 69 years). In two cases the anatomo-histological specimen examinations (twelve sections) demonstrated a gallbladder carcinoma. The overall mortality rate was 5.56% either for aneurysmectomy alone or for combined therapy. In case of abdominal aortic aneurysm and concomitant gallbladder disease, in choosing simple endoaneurysmectomy, the surgeon has to consider the risk of early and late complications of leaving a diseased gallbladder in place. In case of concomitant performance of both operations, the risks of a possible septic graft contamination must be considered. We believe that the patient may be best served by performing the vascular and nonvascular procedures in the same operation. In this paper a new proof, till now never considered in the international literature, is presented to support our opinion: the possibility of concomitant unknown cancer or precancerous lesions in a lithiasic gallbladder. Diagnosis of these lesions is, indeed, not easy to perform in the preoperative phase and is often a postoperative anatomo-histological detection.

Apoptosis has been indicated as a mechanism of T cell depletion in HIV-infected subjects and useful in monitoring disease progression. We investigated for the presence of apoptotic T lymphocytes in 130 HIV subjects in various stages of disease by the newly developed cell permeant DNA dye Apostain. Blood was collected in EDTA, lysed in buffered ammonium chloride, fixed in freshly prepared 1% paraformaldehyde and stored in aliquots at -80 degrees C. Samples were thawed and double stained with FITC conjugated-CD3 monoclonal antibody and Apostain. Flow cytometry was then performed and T cells gated on a CD3 versus side scatter dot plot. Normal samples treated in the same manner served to establish the boundary separating non-apoptotic from apoptotic cells. There was no statistically significant association between the proportion of subjects with detectable apoptotic cells and CDC clinical categories A, B and C at the time of admission to the study, although a trend toward a lower apoptotic rate in category A (A= 29%, B=40% and C=41%) was noticed. Conversely, CDC T cell categories 2 and 3 contained significantly higher proportions of Apostain positive patients (1=6%, 2=32% and 3=49%, P=0.072, by chi(2) test). Most importantly, Apostain test identified subjects at risk of disease progression during a 3.5-7 months follow-up in CDC category B and 2 (P=0.008 and P=0.0003, by Fisher's exact test, respectively). A similar, albeit not statistically significant trend was observed also in the other categories. Not requiring extensive manipulation of fresh samples nor cumbersome culture techniques, Apostain test appears suitable for identifying HIV subjects at higher risk of disease progression in clinical settings.

The enumeration of absolute levels of cells and their subsets in clinical samples is of primary importance in human immunodeficiency virus (HIV)+ individuals (CD4+ T- lymphocyte enumeration), in patients who are candidates for autotransplantation (CD34+ hematopoietic progenitor cells), and in evaluating leukoreduced blood products (residual white blood cells). These measurements share a number of technical options, namely, single- or multiple-color cell staining and logical gating strategies. These can be accomplished using single- or dual-platform counting technologies employing cytometric methods, Dual-platform counting technologies couple the percentage of positive cell subsets obtained by cytometry and the absolute cell count obtained by automated hematology analyzers to derive the absolute value of such subsets. Despite having many conceptual and technical limitations, this approach is traditionally considered as the reference method for absolute cell count enumeration. As a result, the development of single-platform technologies has recently attracted attention with several different technical approaches now being readily available. These single-platform approaches have less sources of variability. A number of reports clearly demonstrate that they provide better coefficients of variation (CVs) in multicenter studies and a lower chance to generate aberrant results. These methods are therefore candidates for the new gold standard for absolute cell assessments. The currently available technical options are discussed in this review together with the results of some cross-comparative studies, Each analytical system has its own specific requirements as far as the dispensing precision steps are concerned. The importance of precision reverse pipetting is emphasized, Issues still under development include the establishment of the critical error ranges, which are different in each test setting, and the applicability of simplified low-cost techniques to be used in countries with limited resources. Cytometry (Comm. Clin. Cytometry) 42:327-346, 2000. (C) 2000 Wiley-Liss, Inc.

Aortic cross-clamping is the cornerstone of abdominal aortic aneurysm surgery. The transient ischemia of the inferior hemisoma, and mainly of the large bowel, is then a current condition, usually well tolerated. At the time of vascular clamps removal, the ischemia-reperfusion syndrome may take place, and evolution toward multiple organ failure is an actual risk. The large bowel has a crucial role in the sequence of events causing ischemia-reperfusion syndrome, even when intestinal ischemia is not evident during aneurysmectomy. In this paper, current concepts of ischemia-reperfusion syndrome are reviewed, and the role of the colon after abdominal aortic cross-clamping for aneurysmectomy is focused. Principles of prevention of MOF from ischemia-reperfusion syndrome are pointed out.

The fine specificity of the cellular immune response to Candida albicans (i.e., recognition of different antigenic components) between normal controls and human immunodeficiency virus-infected patients in various stages of disease was compared. C. albicans-specific T cells, enriched by antigen stimulation and interleukin-2 expansion, were challenged with antigenic fractions of different molecular weight obtained by SDS-gel fractionation of C. albicans extracts in the presence of autologous mononuclear cells as antigen-presenting cells. Proliferative responses showed similar patterns of reactivity between controls and category A and B seropositive subjects. Category C patients with concurrent C. albicans infections did not give rise to C. albicans-specific T cell lines, confirming the T cell defect. Patients without clinically evident C. albicans infection had a low but broad reactivity pattern of C. albicans-specific T cells. These results suggest that depletion of C. albicans-specific T cells, independent of their fine specificity, occurs along with disease progression.

Antigen derived peptides bound on MHC class II molecules on presenting cells stimulate specific CD4 lymphocytes that are in a naive state if antigen is given for the first time, or in a memory state if antigen has been previously encountered. In order to compare clonal heterogeneity of the human CD4+ T helper repertoire in primary vs. recall responses, we have generated T cell lines in vitro by repeated stimulation of peripheral lymphocytes with primary or with recall antigens. Clonal heterogeneity was broad in the case of recall response to tetanus toxoid or PPD, with a high frequency of specific precursors (> 100 cells/10(6) lymphocytes). In contrast, T cell lines responsive to primary antigens (HIV gp120 or HIV p66) were oligoclonal as defined by TCR V beta gene usage and by spectratyping, and the precursor frequency was low (< 2 cells/10(6) lymphocytes). Primary T cell lines generated from blood samples drawn at different times from the same donor showed that clones with identical TCR CDR3 region coding sequences were expanded, suggesting that in these individuals a large progeny derived from one single precursor is present, even though a previous encounter with the antigen was not documented. Assuming an even in vivo distribution of such cells, the presence of one precursor every 10(6) CD4 lymphocytes (within the CD4 T repertoire that comprises roughly 10(11) CD4 T cells) indicates that approximately 10(5) identical T cells from the same clonal precursor account for the primary response against the model antigens we have studied.

Human T helper cells specific for mycobacterial antigens have been extensively investigated. Differences have been detected according to antigen specificity and to fine epitope specificity. In this work we have analyzed two additional parameters that allow discrimination among antigen specific T helper cells: requirement for certain types of antigen presenting cells (APC) and requirement for protease-sensitive antigen processing pathways. We used T cell clones from peripheral blood or from pleural exudates, and specific for different antigenic fractions of M. tuberculosis. APC were autologous peripheral blood mononuclear cells, adherent monocytes, adherent pleural monocytes, EBV transformed B lymphocytes and dendritic cells. Seven clones out of twelve were stimulated by all APC irrespective of their specificity, whereas other clones had more selective requirements. When protease inhibitors were used during antigen pulsing of APC, the production of certain epitopes, and thus T cell activation, was impaired with six clones out of sixteen. These results demonstrate that the human T helper repertoire specific for mycobacterial antigens is highly diverse also according to APC populations needed for presentation and to processing mechanisms required for production of the relevant T epitopes.

The International Society of Analytical Cytology (ISAC) Biohazard Working Group presents guidelines for sorting of unfixed cells, including known biohazardous samples, using jet-in-air, deflected-droplet cell sorters. There is a risk that personnel operating these instruments could become exposed to droplets and aerosols containing biological agents present in the samples. The following guidelines can aid in the prevention of exposures of laboratory personnel to pathogens contained in the sort samples. The document provides biosafety recommendations for sample handling, operator training and protection, laboratory facility design, and instrument setup and maintenance. In addition, it describes in detail methods for assessment of instrument aerosol containment. Recommendations provided here may also help laboratories to obtain institutional and/or regulatory agency approval for sorting of unfixed and known biohazardous samples.

Summary Antigen-specific T helper cells play an important role in retroviral infections. Indeed, they provide help for B-cell activation and antibody production and for clonal expansion of cytolytic lymphocytes. Therefore, we used retrovirus-specific human T helper clones in order to define modes of antigen presentation, antigen-presenting cells and the molecular context of Th epitopes that could be exploited in the design of immunogens aimed at optimizing the Th cell response. In particular, we describe several mechanisms of receptor-mediated antigen uptake that enhance the stimulation of human T-cell clones specific for HIV and HTLV-1 antigens; we report on the differential recognition of Th epitopes depending on the molecular-viral context; we show that dendritic cells are the most efficient presenting cells and are essential for the induction of in vitro primary Th cell responses; and finally, we propose that Th cells specific for internal, conserved antigens of HIV such as reverse transcriptase, may be candidates for intrastructural help resulting in induction of envelope specific antibodies.

CD4+ T cell lines and clones specific for human immunodeficiency virus (HIV) antigens have been generated from peripheral lymphocytes of naive individuals by priming with the envelope protein gp120, the enzyme reverse transcriptase (p66), and their synthetic peptides. T cells were tested for proliferation to proteins, to peptides, and to HIV virions. Different patterns of reaction were identified. T cells primed in vitro with the whole antigen responded to the protein, but recognition of overlapping peptides occurred with a fraction of the lines or clones. The virus was recognized by some, but not all, of the gp120- and p66-specific T cells, with an efficiency 2 logs higher than the recombinant soluble proteins on a molar basis. One T cell line specific for gp120 responded to virions presented by B cells, but not by monocytes. In contrast, T cells induced with peptides did not always respond to the proteins. Generation of T cell lines from naive individuals may be an in vitro model for T cell immunization, and the response patterns may have implications for the design of vaccines aimed at inducing a T helper response. In fact our in vitro data suggest that (a) immunization with peptides does not always induce T cells recognizing the whole protein, (b) immunization with proteins does not always induce T cells recognizing the protein in the context of the HIV virus, and (c) recognition of gp120 in the context of HIV may be dictated by the type of presenting cells.