Human B-cell ontogeny in humanized NOD/SCID γc(null) mice generates a diverse yet auto/poly- and HIV-1-reactive antibody repertoire.

Characterization of the human antibody (Ab) repertoire in mouse models of the human immune system is essential to establish their relevance in translational studies. Single human B cells were sorted from bone marrow and periphery of humanized NOD/SCID γc(null) (hNSG) mice at 8-10 months post engraftment with human cord blood-derived CD34(+) stem cells. Human IG variable heavy (V(H)) and kappa (V(κ)) genes were amplified, cognate V(H)-V(κ) gene-pairs assembled as single-chain variable fragment-Fc Abs (scFvFcs) and functional studies were performed. Although overall distribution of V(H) genes approximated the normal human Ab repertoire, analysis of the V(H)-third complementarity-determining regions in the mature B-cell subset demonstrated an increase in length and positive charges, suggesting autoimmune characteristics. Additionally, >70% of V(κ) sequences utilized V(κ)4-1, a germline gene associated with autoimmunity. The mature B-cell subset-derived scFvFcs displayed the highest frequency of autoreactivity and polyspecificity, suggesting defects in checkpoint control mechanisms. Furthermore, these scFvFcs demonstrated binding to recombinant HIV envelope corroborating previous observations of poly/autoreactivity in anti-HIVgp140 Abs. These data lend support to the hypothesis that anti-HIV broadly neutralizing antibodies may be derived from auto/polyspecific Abs that escaped immune elimination and that the hNSG mouse could provide a new experimental platform for studying the origin of anti-HIV-neutralizing Ab responses.

Drinking habits and risk of altered liver enzymes in the general population of a rural area in Southern Italy.

PURPOSE: To assess the overall drinking habits (amount and duration of alcohol intake, as well as type of alcoholic drinks consumed) and their potential for alteration of liver enzymes in a random sample of the general population aged > or =18 years of a rural area in Southern Italy. MATERIALS AND METHODS: Of the 4000 subjects selected, 3306 (82.7%) agreed to take part in the study. Of these, 41% were teetotallers (54.4% females, 26.1% males; p<0.01). A very small proportion of subjects reported > or =4 drinks/day (11.9% males, 0.8% females; p<0.01). RESULTS: Increased aspartate aminotransferase and/or alanine aminotransferase values were observed in 148 (4.5%) subjects. Hepatitis C virus positivity alone, excessive body mass index alone and alcohol intake alone were observed in 28.6, 23.8 and 18.4% of cases, respectively. After exclusion of subjects with chronic viral hepatitis infections (hepatitis B virus and/or hepatitis C virus) and adjustment for the confounding effect of age (>50 years) and body mass index (> or =25) by multiple logistic regression analysis, subjects who reported consuming >4 drinks/day were 2.4-fold (95%CI=1.1-5.2) more likely than teetotallers to have altered liver enzyme values; subjects reporting intake below this threshold were not at risk of alterations in aspartate aminotransferase/alanine aminotransferase (OR 1.4; 95%CI=0.7-2.6). CONCLUSIONS: These findings indicate that only a small proportion of the rural population studied (particularly females) can be considered as alcohol misusers. Moreover, a mild alcohol intake (< or =4 drinks/day) is not associated with alterations in aspartate aminotransferase/alanine aminotransferase levels in the absence of other factors such as hepatitis viruses and impaired body mass index.

Frequency and distribution of megakaryoblasts in FAB subtypes of ANLL: an immunocytochemical ultrastructural study.

BACKGROUND: Identification of early megakaryocyte (MK) precursors is difficult and conflicting. The prevalence of leukemias with a megakaryocytic component may be underestimated when conventional techniques are utilized. The recent development of immuno-ultrastructural procedures for simultaneous detection of platelet peroxidase reaction and specific platelet membrane antigens has increased our capability of recognizing blast cells of the megakaryocytic lineage. METHODS: In this study the occurrence and distribution of megakaryoblastic in the FAB subtypes of adult "de novo" acute myeloid leukemias was employed to detect megakaryocytic precursors. RESULTS: A megakaryoblastic proliferation was demonstrated in 8/42 cases (19%), with a much higher percentage than previously reported and an unusual distribution in the M2 subtype. CONCLUSIONS: These findings indicate that megakaryocytic involvement may be found rather frequently when an immunocytochemical ultrastructural method is applied to the characterization of leukemias.

Morphologic, immunologic and cytogenetic studies in acute myeloid leukemia following occupational exposure to pesticides and organic solvents.

In order to analyze the correlation between environmental exposure and the clinicopathological picture in acute myeloid leukemia (AML), cytogenetic, cyto-immunologic and clinical studies were performed in 70 newly diagnosed AML patients, 30 of which were anamnestically exposed to pesticides (21 cases) or to organic solvents (9 cases). Clonal chromosome aberrations, with involvement of chromosome 5 and/or 7 were more frequently encountered among exposed patients. While the classical t(15;17), t(8;21) and t(9;11) were detected more frequently among non-exposed patients, other recurring chromosome changes in the exposed group were: rearrangements leading to total or partial monosomy 17p (5 cases), structural aberrations involving the band 16q22 (4 cases), trisomy 11q (2 cases), breaks involving bands 6p23, 7p14, 11q13 (2 cases each). Cytologically, trilineage myelodysplasia was observed in 21 exposed patients, whereas morphologic aberrations of the non-blast cell population were confined to a minority of cells in most patients non-exposed. Immunologic studies revealed positivity for the CD34 stem cell marker in 80% exposed patients vs 22% in the non-exposed group. Conventional chemotherapy achieved complete remission in 3/21 patients exposed and in 16/32 patients non-exposed. Median survival was 2 months in the former group and 9 months in the latter group. These findings show that AML following occupational exposure to pesticides and organic solvents may represent a distinct cytogenetic and clinicopathological entity.

A new chromosomal breakpoint in Ph positive, bcr negative chronic myelogenous leukemia. Report of a case.

We report a new case of Ph positive chronic myeloid leukemia (CML) without the classical rearrangement in Mbcr. By Southern blot analysis the molecular breakpoint was mapped 3 to 8 kb upstream of Mbcr. This region has not been shown to be rearranged in any other described case of CML. We did not detect any specific abnormal BCR-ABL transcript even with the use of the very sensitive RNA-PCR technique.

Non-radioactive in situ hybridization for the detection and monitoring of trisomy 12 in B-cell chronic lymphocytic leukaemia.

Non-radioactive in situ hybridization (NISH) with a chromosome 12-specific alpha satellite probe was performed on 20 patients with chronic lymphocytic leukaemia (CLL) with normal karyotype (15 cases) or with inadequate mitotic yield (5 cases) from mitogen-stimulated cultures. All patients had over 70% lymphocytes coexpressing the CD5/CD23 antigens. While less than 1% interphase nuclei showed three fluorescent spots in 16/20 patients, evidence of trisomy 12 in 15-25% interphase cells was detected in four patients. According to the FAB classification the diagnosis in these patients was typical B-CLL, stage III (Rai's staging system) in one case, CLL/PLL, stage II and III in two cases, PLL, stage III in one case. In order to confirm these results, NISH was repeated after 1 month in one patient and after 2 years in three patients. All patients had been treated with chemotherapy in the period between the two NISH experiments. In all cases a 1.8-3-fold increase of percentage of trisomic interphase cells was detected. These findings suggest that in B-CLL clones with trisomy 12 may have proliferative advantage over clonal B-lymphocyte without +12 and, possibly, that they may be more resistant to chemotherapy. We conclude that NISH is a sensitive technique allowing for the detection and monitoring of trisomy 12 in a fraction of B-CLL patients with normal karyotype or with no analysable mitoses despite employment of polyclonal B-cell mitogens.

[Cytofluorometric study of myeloperoxidase (MPO) expression in neutrophilic granulocytes of subjects with primary or secondary MPO deficiency: analysis of the histographic antigen distribution using the Kolmogorov-Smirnov mathematical model]. / Studio citofluorimetrico della espressione della mieloperossidasi (MPO) nei granulociti neutrofili dei soggetti con deficit primitivo e secondario di MPO: analisi della distribuzione istografica degli antigeni secondo il modello matematico di Kolmogorov-Smirnov.

Neutrophil granulocytes from 12 subjects with primitive myeloperoxidase (MPO) deficiency (6 totally deficient) and 16 patients with secondary partial MPO deficiency were tested using two different anti-MPO monoclonal antibodies (MoAbs), in combination with a flow cytometer. Results demonstrated three different patterns of immunoreactivity with the MPO protein:i) a bright MPO antigenic expression, typical of patients with secondary MPO deficiency (comparable with that observed in the control group); ii) a medium MPO antigenic expression, typical of subjects with hereditary partial MPO deficiency; and iii) a dim MPO antigenic expression, characteristic of individuals with hereditary total MPO deficiency. No significant differences in granulocyte MPO reactivity were demonstrated for the two MoAbs. Furthermore, in two individuals with complete primitive deficiency, the single histogram analysis of MPO fluorescence seemed to show that only 38% (case 1) and 44% (case 2) of neutrophil were reactive with the MoAbs anti-MPO: the use of multiple histogram analysis in combination with Kolmogorov-Smirnov statistics allowed us to demonstrate that all the cells express a low density of MPO antigen. These data suggest that patients with primary MPO deficiency have different amount of MPO antigens in the neutrophils, and the levels of MPO fluorescence seem to decline concurrently with enzyme activity, thereby suggesting the presence of a diminished MPO production. On the contrary, in most cases of acquired MPO deficit, the reduced cytochemical activity contrasts with normal antigenic reactivity: this might be the result of the presence of an inactive enzyme.