Neurological phenotype of Potocki–Lupski syndrome

Potocki–Lupski syndrome is a condition mainly characterized by infantile hypotonia, developmental delay/intellectual disability (DD/ID), and congenital anomalies, caused by duplications of the 17p11.2 region, encompassing RAI1 gene. Its clinical presentation is extremely variable, especially for what concerns the cognitive level and the behavioral phenotype. Such aspects, as well as the dysmorphic/malformative ones, have been covered by previous studies; otherwise neurological features have never been systematically described. In order to delineate the neurological phenotype of Potocki–Lupski Syndrome, we collect an 8‐patients cohort. Developmental milestones are delayed and a mild to moderate cognitive impairment is present in all patients, variably associated with features of autism spectrum disorder, behavioral disturb, and sleep disturb. Hypotonia appears a less frequent finding than what previously reported, while motor clumsiness/coordination impairment is frequent. EGG registration demonstrated a common pattern with excess of diffuse rhythmic activity in sleep phases or while the patient is falling asleep. Brain MRI did not reveal common anomalies, although unspecific white matter changes may be present. We discuss such findings and compare them to literature data, offering an overview on the neurological and cognitive‐behavioral presentation of the syndrome.     

Micronuclei frequencies in hospital workers occupationally exposed to low levels of ionizing radiation: influence of smoking status and other factors

In the context of a medical surveillance program aimed at preventing cancer risk from exposure to ionizing radiation, we investigated chromosomal damage in peripheral lymphocytes from 37 hospital workers exposed to low levels of ionizing radiation and 37 controls. The micronuleus (MN) assay was used as a biomarker of genetic damage. The influence of confounding factors like smoking status, age and gender was investigated by multiple regression analysis. The results indicated that, overall, MN frequency was higher in exposed workers than in controls, although the difference was not statistically significant. Interestingly, smoking status significantly raised MN frequency among the exposed workers but not among controls. This suggests that smoking can influence chromosomal damage induced in humans by ionizing radiation. Among both exposed workers and controls, MN frequency was found to increase with age. Female gender influenced the increase in MN frequency in the exposed group. Our results suggest that the effect of cigarette smoking should be carefully factored into genetic monitoring studies assessing the risks associated with low level radiation exposure.     

ALK Expression Defines a Distinct Group of T/Null Lymphomas (“ALK Lymphomas”) with a Wide Morphological Spectrum

The t(2;5)(p23;q35) translocation associated with CD30-positive anaplastic large cell lymphoma results in the production of a NPM-ALK chimeric protein, consisting of the N-terminal portion of the NPM protein joined to the entire cytoplasmic domain of the neural receptor tyrosine kinase ALK. The ALK gene products were identified in paraffin sections by using a new anti-ALK (cytoplasmic portion) monoclonal antibody (ALKc) that tends to react more strongly than a previously described ALK1 antibody with the nuclei of ALK-expressing tumor cells after microwave heating in 1 mmol/L ethylenediaminetetraacetic acid buffer, pH 8.0. The ALKc monoclonal antibody reacted selectively with 60% of anaplastic large cell lymphoma cases (60 of 100), which occurred mainly in the first three decades of life and consistently displayed a T/null phenotype. This group of ALK-positive tumors showed a wide morphological spectrum including cases with features of anaplastic large cell lymphoma “common” type (75%), “lymphohistiocytic” (10%), “small cell” (8.3%), “giant cell” (3.3%), and “Hodgkin’s like” (3.3%). CD30-positive large anaplastic cells expressing the ALK protein both in the cytoplasm and nucleus represented the dominant tumor population in the common, Hodgkin’s-like and giant cell types, but they were present at a smaller percentage (often with a perivascular distribution) also in cases with lymphohistiocytic and small cell features. In this study, the ALKc antibody also allowed us to identify small neoplastic cells (usually CD30 negative) with nucleus-restricted ALK positivity that were, by definition, more evident in the small cell variant but were also found in cases with lymphohistiocytic, common, and “Hodgkin’s-like” features. These findings, which have not been previously emphasized, strongly suggest that the neoplastic lesion (the NPM-ALK gene) must be present both in the large anaplastic and small tumor cells, and that ALK-positive lymphomas lie on a spectrum, their position being defined by the ratio of small to large neoplastic cells. Notably, about 15% of all ALK-positive lymphomas (usually of the common or giant cell variant) showed a cytoplasm-restricted ALK positivity, which suggests that the ALK gene may have fused with a partner(s) other than NPM. From a diagnostic point of view, detection of the ALK protein was useful in distinguishing anaplastic large cell lymphoma cases of lymphohistiocytic and small cell variants from reactive conditions and other peripheral T-cell lymphoma subtypes, as well as for detecting a small number of tumor cells in lymphohemopoietic tissues. In conclusion, ALK positivity appears to define a clinicopathological entity with a T/null phenotype (“ALK lymphomas”), but one that shows a wider spectrum of morphological patterns than has been appreciated in the past.     

PG-M1: A New Monoclonal Antibody Directed against a Fixative-Resistant Epitope on the Macrophage-Restricted Form of the CD68 Molecule

A new anti-macrophage monoclonal antibody (PG-M1) was produced by immunizing BALB/c mice with fresh spleen cells from a patient with Gaucher's disease. PG-M1 reacts strongly with a fixative-resistant epitope of an intracytoplasmic molecule, selectively expressed by virtually all macrophages of the human body. Although attempts to immunoprecipitate the molecule recognized by PG-M1 have failed so far, the reactivity of the antibody with COS-1 and WOP cells transfected with a human complementary DNA clone encoding for the CD68 antigen suggests that PG-M1 is a new member of the CD68 cluster. However, unlike other CD68 antibodies (KP1, EBM11, etc.), which react with both macrophages and myeloid cells, PG-M1 detects a fixative-resistant epitope on the macrophage-restricted form of the CD68 antigen. In 957 routinely fixed, paraffin-embedded samples, PG-M1 showed a more restricted reactivity with elements of the monocyte/macrophage lineage than the previously described monoclonal antibodies MAC-387 (anti-calgranulins), KP1 (CD68) and Ki-M1P. Among hematological malignancies, PG-M1 only labels acute leukemias of M4 and M5 type and rare examples of malignant histiocytosis/true histiocytic sarcoma. In contrast, acute leukemias of the M1, M2, M3, M6, M7, and L1-L3 types, non-Hodgkin's lymphomas, and Hodgkin and Reed-Sternberg cells of Hodgkin's disease are consistently PG-M1-negative. In the daily diagnostic practice, PG-M1 seems to be particularly valuable for the diagnosis of myelomonocytic or monocytic leukemia and neoplasms of true histiocytic origin in routine paraffin sections.     

Comparative Evaluation of Ligation-Mediated PCR and Spoligotyping as Screening Methods for Genotyping of Mycobacterium tuberculosis Strains

Spoligotyping has been suggested as a screening test in multistep genotyping of Mycobacterium tuberculosis strains. Relying on restriction fragment length polymorphism (RFLP) analysis with IS6110 (IS6110 RFLP analysis) as a "gold standard," we performed a comparative evaluation of spoligotyping and ligation-mediated PCR (LMPCR), a recently described PCR-based typing method, as rapid screening tests for fingerprinting of 158 M. tuberculosis strains collected in Verona, Italy. LMPCR seemed to be comparable to spoligotyping in terms both of feasibility with rapidly extracted DNA and of generation of software-analyzable images. Moreover, LMPCR grouped considerably fewer strains than spoligotyping (38 versus 67%) and was found to reduce the cluster overestimation rate (26.3 versus 58%) and to give a better discriminatory index (0.992 versus 0.970) compared to spoligotyping. In our geographical region, where there was no evidence of clustered strains carrying fewer than six IS6110 copies, LMPCR was found to be more discriminatory than spoligotyping. We also evaluated two models of three-step typing strategies, involving the use of spoligotyping and LMPCR as screening methods and IS6110 RFLP analysis as a further supporting test. LMPCR proved to be a more effective first-step test than spoligotyping, significantly reducing the need for subtyping. LMPCR should be considered an alternative to spoligotyping as a rapid screening method for M. tuberculosis fingerprinting, particularly in areas with a low prevalence of M. tuberculosis strains carrying few copies of IS6110.     

In vitro and in vivo modulation of MDR1/P-glycoprotein in HIV-infected patients administered highly active antiretroviral therapy and liposomal doxorubicin

P-glycoprotein (P-gp) transports a wide range of structurally unrelated drugs, such as HIV protease inhibitors (PIs) and cytotoxic compounds such as anthracyclines. Because modification of P-gp phenotype and function is an important underlying mechanism of drug interactions, the current study was conducted in order to evaluate whether highly active antiretroviral therapy (HAART), HIV plasma viral load (VL), or cancer chemotherapy may induce in vivo changes of P-gp phenotype in peripheral blood mononuclear cells (PBMCs) from HIV-infected treatment-naive and -experienced subjects at different stages of HIV infection and/or disease, including patients with HIV-associated Kaposi sarcoma (KS). Our results show that neither HAART nor HIV VL, nor the stage of HIV infection and/or disease, significantly alter P-gp expression on PBMCs. In particular, surface P-gp expression is expressed at low levels by T-cell subsets, B cells, and NK cells, whereas almost all monocytes are double positive and these results are not modified by HIV PI-containing regimens. By contrast, a significant phenotype modification is detected in PBMCs from AIDS/KS patients after challenge with the liposomal formulation of the anthracycline doxorubicin (L-DOX) with the higher expression reached 24 hours after the end of the drug infusion. In addition, accumulation of L-DOX is unaffected by P-gp-mediated drug efflux as documented by in vitro experiments, in sharp contrast to the kinetic of free DOX, based on HIV PI blockade experiments. Finally, P-gp expression was found in KS spindle cells from HIV-infected treatment-naive AIDS/KS patients. We conclude that P-gp phenotype in PBMCs and specific subsets is not altered by HAART and/or HIV, whereas a significant increase is induced by specific anticancer drugs such as L-DOX. Moreover, HIV PIs possess an inhibitory effect on P-gp function that may improve DOX sensitivity in KS spindle cells.     

Stimulation of NaCl reabsorption by antidiuretic hormone in the cortical thick ascending limb of Henle's loop of the mouse

The effect of antidiuretic hormone (ADH) on transepithelial Na⁺, Cl^–, Ca²⁺ and Mg²⁺ net fluxes (J_Na, J_Cl, J_Mg, J_Ca) was investigated in isolated perfused cortical thick ascending limb segments (cTAL) of the mouse nephron, using the microperfusion technique and the electron microprobe analysis to determine the ionic composition of the collected tubular fluid. Simultaneously, the transepithelial potential difference (PD_te) and the transepithelial resistance (R_te) were recorded. Prior to the flux measurements cTAL segments were perfused for one hour. During this equilibration period PD_te decreased significantly from +19.9±1.6 to +14.9±1.l mV and R_te increased from 30.6±3.5 cm² to 38.8±2.4 cm² (n=7), reflecting a decline in NaCl transport. After ADH was added to the bath solution at 10^–10 mol.l^–1, PD_te increased from +14.4±1.1 to +18.0±1.5 mV, accompanied by a rise in J_Na and J_Cl from 205±11 to 273±19 and from 216±12 to 283±21 pmol.min^–1.mm^–1 (n=7), respectively. J_Ca and J_Mg also increased from 0.81±0.07 to 1.50±0.12 and from 0.43±0.11 to 0.76±0.08 pmol.min^–1.mm^–1 (n=7), respectively. All these effects were fully reversible after withdrawal of the hormone. In conclusion our data indicate that ADH stimulates divalent cation transport and NaCl transport in the cortical thick ascending limb of Henle's loop of the mouse.     

Replacement of Candida albicans with C. dubliniensis in human immunodeficiency virus-infected patients with oropharyngeal candidiasis treated with fluconazole

Candida dubliniensis is an opportunistic yeast that has been increasingly implicated in oropharyngeal candidiasis (OPC) in human immunodeficiency virus (HIV)-infected patients but may be underreported due to its similarity with Candida albicans. Although most C. dubliniensis isolates are susceptible to fluconazole, the inducibility of azole resistance in vitro has been reported. Thus, the use of fluconazole prophylaxis in the treatment of these patients may have contributed to the increasing rates of isolation of C. dubliniensis. In this study, yeast strains were collected from the oral cavities of HIV-infected patients enrolled in a longitudinal study of OPC. Patients received fluconazole for the suppression or treatment of OPC, and isolates collected at both study entry and end of study were chosen for analysis. Samples were plated on CHROMagar Candida medium for initial isolation and further identified by Southern blot analysis with the species-specific probes Ca3 (for C. albicans) and Cd25 (for C. dubliniensis). Fluconazole MICs were determined by using NCCLS methods. At study entry, susceptible C. albicans isolates were recovered from oral samples in 42 patients who were followed longitudinally (1 to 36 months). C. albicans strains from 12 of these patients developed fluconazole resistance (fluconazole MIC, >/=64 micro g/ml). C. dubliniensis was not detected at end of study in any of these patients. Of the remaining 30 patients, eight (27%) demonstrated a replacement of C. albicans by C. dubliniensis when a comparison of isolates obtained at baseline and those from the last culture was done. For the 22 of these 30 patients in whom no switch in species was detected, the fluconazole MICs for initial and end-of-study C. albicans isolates ranged from 0.125 to 2.0 micro g/ml. For the eight patients in whom a switch to C. dubliniensis was detected, the fluconazole MICs for C. dubliniensis isolates at end of study ranged from 0.25 to 64 micro g/ml: the fluconazole MICs for isolates from six patients were 0.25 to 2.0 micro g/ml and those for the other two were 32 and 64 micro g/ml, respectively. In conclusion, a considerable number of patients initially infected with C. albicans strains that failed to develop fluconazole resistance demonstrated a switch to C. dubliniensis. C. dubliniensis in this setting may be underestimated due to lack of identification and may occur due to the impact of fluconazole on the ecology of oral yeast species.     

Giant ragweed specific immunotherapy is not effective in a proportion of patients sensitized to short ragweed: analysis of the allergenic differences between short and giant ragweed

BACKGROUND: Short ragweed and giant ragweed pollen allergens are considered largely cross-reactive, and it is generally believed that 1 species is sufficient for skin testing and immunotherapy. However, in the area north of Milan (a zone widely invaded only by short ragweed), about 50% of patients submitted to injection specific immunotherapy with giant ragweed showed little or no clinical response, but showed an excellent outcome if they were shifted to short ragweed specific immunotherapy. OBJECTIVE: To investigate allergenic differences between short and giant ragweed. METHODS: IgE reactivity to short ragweed of sera from 16 patients allergic to ragweed was assessed by immunoblot before and after absorption with short and giant ragweed. Moreover, 41 ragweed-monosensitive patients underwent skin prick test with both ragweed species. RESULTS: In several cases, preabsorption of sera with giant ragweed extract was unable to inhibit IgE reactivity fully against both a 43-kd allergen and other allergens at different molecular weights in short ragweed. On skin prick test, short ragweed induced larger wheals than giant ragweed in the majority of patients, and 6 of 41 (15%) patients were strongly short ragweed-positive but giant ragweed-negative. The immunoblot with the serum from 1 of these subjects showed a strong IgE reactivity to short ragweed at about 43 kd in the absence of any reactivity to giant ragweed. CONCLUSION: Short and giant ragweed are not allergenically equivalent. Allergenic differences involve both the major allergens Amb a 1-2/Amb t 1-2 and some minor allergens. In patients allergic to ragweed, both diagnosis in vivo and immunotherapy should always be performed by using the ragweed species present in that specific geographic area.     

Characterization of Sicilian strains of spotted fever group rickettsiae by using monoclonal antibodies.

Twenty-two hybridomas producing anti-Rickettsia conorii monoclonal antibodies were obtained by nine fusion experiments. The strain chosen for immunization of mice was MAVI, an R. conorii strain isolated from a Sicilian patient with Boutonneuse fever. When tested for immunoglobulin isotype by an indirect immunofluorescence (IIF) assay, 46.6% of supernatants from the 22 hybridomas were immunoglobulin M. The supernatants were tested in the IIF assay for binding to the MAVI strain and four spotted fever group rickettsia strains isolated from Sicilian ticks (two virulent and two nonpathogenic when inoculated intraperitoneally in male guinea pigs). Only five of the supernatants showed a positive IIF result on all tested strains, although they produced different titers to the various strains, possibly an indication that they recognized an antigen common to spotted fever group rickettsiae. Immunodominant epitopes for humans were determined by using patient sera to analyze inhibition of binding to the MAVI strain. Although a limited number of serum samples were screened, a high percentage of Boutonneuse fever patients produced antibodies recognizing the same epitopes as were recognized by the mouse monoclonal antibodies. A striking heterogeneity was found both in the expression of mouse-recognized epitopes on the five rickettsial strains and in the serum antibody responses of Boutonneuse fever patients to these epitopes.