Two cases of elevated tryptase in abdominal aortic aneurysm.

INTRODUCTION: From the literature, patients with a history of anaphylaxis to hymenoptera venom and positive specific IgE have shown a correlation between elevated tryptase levels and two clinical situations: systemic mastocytosis and an increased risk of reactions to venom immunotherapy or hymenoptera sting. Other clinical scenarios could explain elevated tryptase levels. MATERIAL AND METHODS: A 67 year old male (P1) and a 77 year old male (P2) were evaluated for previous severe anaphylaxis to hymenoptera sting. They underwent standard diagnostic work-up for hymenoptera venom allergy. Having found elevated tryptase levels, these were followed by a bone marrow biopsy to rule out systemic mastocytosis. RESULTS: P1: specific IgE and skin tests were positive for Vespula species; tryptase 52.8 ng/ml; P2: specific IgE and skin tests were positive for Vespa cabro and tryptase 153 ng/ml. Bone marrow biopsy results were negative for mastocytosis. We carried out magnetic resonance imaging, in P1 to better characterize the severe osteoporosis and in P2 because during physical examination a pulsating mass had been identified in the mesogastrium, and an aneurysm of the abdominal aorta which required surgical intervention in both patients was detected. Eight months after surgery, tryptase levels had diminished significantly (P1: 11.6 ng/ml and P2: 14.5 ng/ml). DISCUSSION: The elevated tryptase levels were correlated to abdominal aneurysm in both patients. In fact, post-surgery tryptase levels dramatically decreased. These two cases demonstrate that high tryptase levels in subjects with a history of hymenoptera venom anaphylaxis can be associated to undiagnosed aneurysmatic disease.

Hexamethylene bisacetamide inhibits malignant phenotype in T-ALL cell lines.

T acute lymphoblastic leukemia cell lines treated with hexamethylene bisacetamide (HMBA) undergo a delay in cell cycle progression and increase susceptibility to apoptosis, although they never overcome the differentiation block. In accordance with changes in cell cycle and apoptosis, transitory p53 pathway activation commonly occurs. Bcl-2 inhibition further favours the pro-apoptotic effect of HMBA. Notch1 expression is down regulated by reduction of its transcription level. Accordingly, Notch1 protein and transcriptional activity were affected. Even if HMBA generally reduces Notch1 level in T acute lymphoblastic leukemia (T-ALL) cell lines, this does not commonly influence the biological response; in fact all the analysed cell lines, except CEM cells, display no biological effect following DAPT-induced Notch inhibition.

Efficacy of single dose pegfilgrastim in enhancing the mobilization of CD34+ peripheral blood stem cells in aggressive lymphoma patients treated with cisplatin-aracytin-containing regimens.

Systematic data on the ability of pegfilgrastim to mobilize stem cells after chemotherapy are scarce. We evaluated the efficacy of a single 6 mg dose of pegfilgrastim for mobilizing peripheral blood stem cells (PBSC) in aggressive lymphoma patients. Between July 2004 and October 2005, 17 aggressive non-Hodgkin's lymphoma and 11 poor-risk Hodgkin's lymphoma were treated with cycles containing cisplatin-aracytin. At the end of chemotherapy, the patients received 6 mg of pegfilgrastim. Duration of grade 4 neutropenia, adverse events, time to neutrophil recovery, peak and harvest of CD34+ cells were recorded. Twenty-seven out of 28 patients harvested a median of 17.3 x 10(6)/CD34+ cells (range 2.5-28.9) after a median of 9 days (range 8-12 days), with a single apheresis procedure in 25 cases. All patients had grade 3-4 neutropenia, median duration 3 days. The only adverse event was mild bone pain. To date, 13 patients have been autografted with a median of 15.4 x 10(6) CD34+ pegfilgrastim-mobilized cells per kg (range 2.5-28.9) with rapid and sustained engraftment. Mobilization, harvesting and autografting of pegfilgrastim-mobilized PBC can be successfully achieved in pretreated patients with aggressive lymphoma.

Successful CD34+ cell mobilization by intermediate-dose Ara-C in chronic lymphocytic leukemia patients treated with sequential fludarabine and Campath-1H.

Chronic lymphocytic leukemia (CLL) cells could be undetectable by flow cytometry or polymerase chain reaction after sequential treatment with fludarabine and Campath-1H. Concern has been raised regarding the ability to mobilize sufficient peripheral blood progenitor cells (PBPCs) for autografting after purine analogues, and there are few data about PBPC collection after Campath-1H. In all, 16 CLL patients responding to sequential chemo-immunotherapy entered the study. In 10, mobilization regimen consisted of granulocyte colony-stimulating factor (G-CSF) 5-10 microg/kg/die. Patients failing mobilization or not achieving the target of 2.5 x 10(6) CD34+ cells/kg underwent a second attempt using intermediate-dose (ID) Ara-C, 800 mg/m(2) every 12 h for six doses+G-CSF. PBPC collection after G-CSF alone was successful in two out of 10 patients. An adequate number of CD34+ cells were collected after ID Ara-C+G-CSF in eight patients failing the mobilization with G-CSF alone and in five out of six who did not receive G-CSF before. Greater yields of PBPCs were collected with Ara-C+G-CSF compared with G-CSF alone (13.8 vs 3.3). The extrahematologic toxicity was manageable. In conclusion, PBPC collection is feasible in CLL patients treated with sequential therapy including fludarabine and Campath-1H. Excellent yields were obtained in 92.8% of patients primed with ID Ara-C+G-CSF.

The "vanishing counting bead" phenomenon: effect on absolute CD34+ cell counting in phosphate-buffered saline-diluted leukapheresis samples.

BACKGROUND: Using a single-platform protocol to count absolute CD34+ hematopoietic precursor cell (HPC) levels with different reference microbeads, we recorded occasionally artifactually high CD34+ HPC counts in some leukapheresis bags, whereas dual-platform calculations were always consistent. Abnormal countings were observed only when phosphate-buffered saline (PBS)-diluted leukapheresis samples were vortexed before analysis. A large series of blood samples analyzed similarly for CD34+ and CD4+ absolute counts did not show any sample or vortexing effect. With the volumetric absolute counting cytometer Partec-PAS, lower counts were also observed when different reference beads were vortexed before the instrument checking procedures. The counting abnormality was caused by a drop in microbead concentration (the "vanishing bead phenomenon"). This phenomenon reduced the total and relative bead event number in experimental and routine samples and in calibration procedures. This altered the bead denominator used to calculate absolute CD34+ HPC levels and it also reduced the concentration of standard calibration beads. METHODS: Using the Partec-PAS to measure volumetrically the actual bead concentration, we studied the vanishing bead phenomenon. Different types of counting and reference microbeads were resuspended in media with or without proteins or cells. Replicates were submitted either to gentle manual mixing or to vortexing before counting. RESULTS: Vortex agitation almost invariably induced the vanishing bead phenomenon when beads were resuspended in saline media or when an insufficient protein concentration was present, such as in diluted leukapheresis samples. Different bead types showed various degrees of sensitivity to vortexing. The bead disappearance was not caused by bubble formation or disruption. The addition of small amounts of protein completely prevented the vanishing bead phenomenon. The causative effect of the electrostatic charging of tube induced by vortexing is hypothesized. CONCLUSIONS: Sample suspensions containing counting beads for single-platform analysis must be resuspended in media with protein supplements to prevent the vanishing bead phenomenon and to ensure accurate counting.

Cytofluorometric methods for assessing absolute numbers of cell subsets in blood. European Working Group on Clinical Cell Analysis.

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.

Demonstration of antibodies to human T-lymphotropic retrovirus type III in lymphoadenopathy syndrome patients and in individuals at risk for acquired immune deficiency syndrome (AIDS) in Italy.

An epidemiologic survey of the distribution of lymphoadenopathy syndrome in six Italian cities and its correlation with human T-lymphotropic retrovirus III (HTLV-III) is reported. Serum samples of nine patients with acquired immune deficiency syndrome (AIDS) were tested, 180 from patients with lymphoadenopathy and 349 from individuals belonging to groups such as homosexuals, drug addicts, hemophiliacs, and polytransfused considered at increased risk for AIDS. Prevalence of HTLV-III antibodies was 78% in AIDS patients and 61% in 180 patients with lymphoadenopathy syndrome (variation among drug abusers by city from 51% in Cagliari to 87% in Rome). The percentage of positive sera in individuals at risk for AIDS or lymphoadenopathy ranged from 0% in polytransfused to 8.5% in homosexuals, 14% in drug addicts, and 19.5% in hemophiliacs. No positive sera were found among 660 healthy individuals including relatives of patients with AIDS or lymphoadenopathy or in 342 patients suffering from immunologic or infectious diseases. These results strongly suggest that HTLV-III is the causative agent of AIDS and lymphoadenopathy. Since none of the healthy subjects were positive while a substantial percentage of people at risk for AIDS showed antibodies to HTLV-III, it may be presumed that this infection is also prevalent in the Italian population group in which AIDS and lymphoadenopathy are most likely to develop.

Immunological responses of patients with recurrent herpes genitalis.

Immunological responses of patients with recurrent herpes genitalis (RHG) were assayed in comparison to those of patients affected by recurrent herpes labialis (RHL) and to those of normal seropositive and seronegative controls. Total T cells were normal while a significant (p less than 0.001) reduction of cells with helper phenotype was found only in people with RHG, who were also lacking specific cell-mediated immunity (CMI) to herpes simplex virus (HSV) antigen. HSV-type 2 was demonstrated in their active lesions. Number of human NK-1 positive (HNK-1 +) cells was increased in RHG showing an inverse correlation with depressed natural killer (NK) activity. These abnormalities were found irrespective or not if tests were carried out during the active phase of the disease or during quiescence and were confirmed during the 2-year follow-up period to which patients were submitted. Similar abnormalities were found in RHL only during the active phase and reversed to normal during quiescence. Our data indicate the presence of specific cellular and NK unresponsiveness in subjects with severe and long-lasting herpes genitalis.

Epidemiological aspects of HTLV-III infection in Italy.

An epidemiological survey on the presence of serum antibodies to human T lymphotropic retrovirus III (HTLV-III) is reported. The study was carried out on people living in large as well as small cities in different Italian regions. Serum samples of 21 patients with AIDS, 402 with ARCS, and 920 from individuals at risk for these diseases were analyzed. The percentage of positive sera varied from 81% in AIDS, to 100% -33.3% in ARC according to the different geographical areas and/or the various categories of people with ARC. The percentage of positive sera in individuals at risk for AIDS or ARC varied from 11.9% in homosexuals, to 21% in drug abusers and 23.1% in haemophiliacs. No positive sera were observed among 660 normal individuals, relatives of patients with AIDS or ARC and in 114 patients suffering from immunological or infectious disease and among people at risk living in small cities. Sera were also negative in patients with classical Kaposi's sarcoma or T-cell chronic lymphocytic leukemia. Since none of the 660 unselected normal adults were positive while a significant percentage of people at risk for AIDS showed antibodies to HTLV-III, we may presume that this infection is prevalent in the Italian categories in which AIDS and ARC are most likely to develop.

Immunological and virological investigation in patients with lymphoadenopathy syndrome and in a population at risk for acquired immunodeficiency syndrome (AIDS), with particular focus on the detection of antibodies to human T-lymphotropic retroviruses (HTLV III).

Thirteen patients affected with unexplained lymphoadenopathy, fever, weight loss, and diarrhea (lymphoadenopathy syndrome; LAS) were evaluated for the possible appearance of acquired immunodeficiency syndrome (AIDS) and for immunological and virological characterization. The patients belonged to categories of individuals at risk for AIDS and were homosexual and/or drug abusers or hemophiliacs. Lymph node biopsy showed the histological picture of a follicular hyperplasia. The study of cell-mediated immunity (CMI), humoral immune response, and natural killer (NK) activity demonstrated a significant decrease in T cells with the helper/inducer phenotype (OKT4+ cells) and a relatively increased number of lymphocytes with the suppressor/cytotoxic phenotype (OKT8+ cells). NK activity was significantly lower than in normal controls. The in vitro response to polyclonal activators (phytohemagglutinin; PHA) and the cutaneous responsiveness to recall skin tests were impaired, whereas immunoglobulin production was increased, mainly in the IgG fraction. Virological studies showed high serum antibody titers to cytomegalovirus (CMV) but a lack of specific CMI as assayed by the leukocyte migration inhibition test (LMIT). CMV was also isolated from the urine specimen of one patient. The antibody pattern to Epstein-Barr virus (EBV) showed the uncommon contemporary presence of both Epstein-Barr nuclear antigen (EBNA) and early antigen (EA) antibodies. Antibodies to human T-lymphotropic retroviruses (HTLV III) were positive in 10 patients and the virus was isolated in 3 of them. In some patients the presence of serum antibodies to HTLV III was not associated with an impairment of the immune function. A group of individuals at risk for AIDS without LAS was also evaluated for the presence of HTLV III antibodies; the percentage of positive sera was 11.4.(ABSTRACT TRUNCATED AT 250 WORDS)

A preliminary report on the immunological responses after oral vaccine (Ty 21a).

Humoral and cell-mediated immune responses, specific for lipopolysaccharide (LPS), were evaluated before and after oral immunization with the Ty 21a strain of Salmonella typhi in a group of healthy volunteers. No rise in seric and foecal antibody titres, detected by the ELISA technique, was seen after vaccination. On the contrary, we were able to demonstrate the development of specific cell-mediated immunity, as assayed by the leukocyte migration inhibition test, 21 days after vaccination. This conversion was still present 40 days after the last dose of vaccine. We believe that the Ty 21a strain of S. typhi, recently proposed for immunization against typhoid fever and based on the use of live micro-organisms, is effective due to its ability to induce specific cell-mediated immunity.