Autoimmune Lymphoproliferative Syndrome (ALPS) Disease and ALPS Phenotype: Are They Two Distinct Entities?

Autoimmune lymphoproliferative syndrome (ALPS) is an inherited disorder of lymphocyte homeostasis classically due to mutation of FAS, FASL, and CASP10 genes (ALPS-FAS/CASP10). Despite recent progress, about one-third of ALPS patients does not carry classical mutations and still remains gene orphan (ALPS-U, undetermined genetic defects). The aims of the present study were to compare the clinical and immunological features of ALPS-FAS/CASP10 versus those of ALPS-U affected subjects and to deepen the genetic characteristics of this latter group. Demographical, anamnestic, biochemical data were retrieved from medical record of 46 ALPS subjects. An enlarged panel of genes (next-generation sequencing) was applied to the ALPS-U group. ALPS-U subjects showed a more complex phenotype if compared to ALPS-FAS/CASP10 group, characterized by multiorgan involvement (P = 0.001) and positivity of autoimmune markers (P = 0.02). Multilineage cytopenia was present in both groups without differences with the exception of lymphocytopenia and autoimmune neutropenia that were more frequent in ALPS-U than in the ALPS-FAS/CASP10 group (P = 0.01 and P = 0.04). First- and second-line treatments were able to control the symptoms in 100% of the ALPS-FAS/CASP10 patients, while 63% of ALPS-U needed >2 lines of treatment and remission in some cases was obtained only after target therapy. In the ALPS-U group, we found in 14 of 28 (50%) patients 19 variants; of these, 4 of 19 (21%) were known as pathogenic and 8 of 19 (42%) as likely pathogenic. A characteristic flow cytometry panel including CD3CD4-CD8-+TCRαß+, CD3+CD25+/CD3HLADR+, TCR αß+ B220+, and CD19+CD27+ identified the ALPS-FAS/CASP10 group. ALPS-U seems to represent a distinct entity from ALPS-FAS/CASP10; this is relevant for management and tailored treatments whenever available.

E2F3 gene expression is a potential negative prognostic marker for localised and MYCN not-amplified neuroblastoma: Results of in silico analysis of 786 samples.

BACKGROUND: Neuroblastoma (NB) is an enigmatic childhood malignancy characterised by a wide range of clinical behaviour. Many potential oncogenes for NB have recently been identified. Among them, E2 transcription factor 3 (E2F3) expression was associated with a poor survival in 134 stage 4S patients, but evidence for other stage groups remains poorly investigated. METHODS: We have analysed the expression of E2F3 gene from a database of 786 NB samples. Overall and event-free survivals (EFS) were assessed by the Kaplan-Meier method, splitting the data on the median and tertile expression values. The Cox model was applied to control for the confounding by stage, age and MYCN amplification. Validation was performed by an in silico analysis of an independent cohort of 283 NB patients. Furthermore, an immunofluorescence analysis on 48 formalin-fixed, paraffin-embedded NB specimens was also performed. RESULTS: E2F3 overexpression was associated with a poor survival (EFS = 84%, 95% CI: 79%-95%, for low expression levels; EFS = 62%, 95% CI: 56%-68% for middle levels; EFS = 30%, 95% CI: 24%-36%, for high levels, p < .001). This association was confirmed in multivariable analysis and was more evident in patients with MYCN not-amplified and localised stages. Immunofluorescence results and the validation on an independent cohort of NB primary samples confirmed these findings. CONCLUSIONS: E2F3 is a new potential prognostic marker in NB with favourable characteristics at diagnosis. Further studies are needed to elucidate the potential role of E2F3 in NB oncogenesis and progression, in order to identify new targets for therapeutic interventions.

Late-onset and long-lasting autoimmune neutropenia: an analysis from the Italian Neutropenia Registry.

Primary autoimmune neutropenia (pAN) is typified by onset in early infancy and a mild/moderate phenotype that resolves within 3 years of diagnosis. In contrast, secondary AN is classically an adult disease associated with malignancy, autoimmunity, immunodeficiency, viral infection, or drugs. This study describes a cohort of 79 children from the Italian Registry who, although resembling pAN, did not fully match the criteria for pAN because neutropenia either appeared after age 5 years (LO-Np) or lasted longer than 3 years (LL-Np). These 2 categories compared with classical pAN showed a far inferior rate of resolution (P < .001), lower severity of neutropenia (P = .03), leukopenia (P < .001), lymphopenia (P < .001) with low B+ (P = .001), increased need of granulocyte colony-stimulating factor (P = .04), and increased frequency of autoimmunity over the disease course (P < .001). A paired comparison between LO-Np and LL-Np suggested that LO-Np had a lower rate of resolution (P < .001) and lower white blood cell (P < .001) and lymphocyte (P < .001) values, higher occurrence of apthae (P = .008), and a stronger association with autoimmune diseases/markers (P = .001) than LL-Np, thus suggesting a more pronounced autoimmune signature for LO-Np. A next-generation sequencing panel applied in a small subgroup of LO-Np and LL-Np patients identified variants related to immune dysregulations. Overall, these findings indicate that there are important differences among pAN LL-Np and LO-Np. Forms rising after 3 years of age, with low tendency to resolution, require tight monitoring and extensive immune investigations aimed to early identify underlying immunologic disease.

Reliability and diagnostic use of a test for the search of the hepatitis C virus Ag (AgHCV).

BACKGROUND/AIMS: The detection of serum HCV nucleocapsid (core) antigen, besides being a valid alternative, by virtue of its low cost, to direct analysis of the virus in everyday transfusion practice, also aims to be employed in monitoring patients subjected to antiviral therapy. The verification of strict correlation between the two tests is the presupposition for such use. METHODOLOGY: In a group of 112 HCV-positive subjects, we assessed blood transaminases, viremia (by PCR), and the circulating core antigen (by ELISA). RESULTS: Only 16 out of 112 patients were AgHCV-negative, with viremia levels in the 10(2) to 10(4) range; 96 patients were HCV-positive, as indicated both by viremia and by Ag detection (1.9 to 292.4pg/mL). Sensitivity of the ELISA test corresponds to 3.6x10(4) IU/mL of viral load. There is an evident aggregation of results in groups according to antigenemia classes and the corresponding viremia levels: <10pg/mL--10(4) IU/mL (6/96); up to 100pg/mL--10(5) IU/mL (55/96); 100-200pg/mL--10(6) IU/mL (31/96); and more than 200pg/mL--10(7) IU/mL (4/96). CONCLUSIONS: AgHCV is correlated with elevation of ALT and high or medium-high viral loads. It can discriminate between ongoing and previous infection and is suitable for monitoring the pharmacological therapy in the presence of sufficiently high viral loads and for evaluating the onset of medium-long-term relapses. Neither the genotypes nor pharmacological therapy appear to affect the comparison of viremia with antigenemia. Moreover, this analysis is cheaper as compared with molecular techniques.

IgV gene intraclonal diversification and clonal evolution in B-cell chronic lymphocytic leukaemia.

Intraclonal diversification of immunoglobulin (Ig) variable (V) genes was evaluated in leukaemic cells from a B-cell chronic lymphocytic leukaemia (B-CLL) case over a 2-year period at four time points. Intraclonal heterogeneity was analysed by sequencing 305 molecular clones derived from polymerase chain reaction amplification of B-CLL cell IgV heavy (H) and light (C) chain gene rearrangements. Sequences were compared with evaluating intraclonal variation and the nature of somatic mutations. Although IgV intraclonal variation was detected at all time points, its level decreased with time and a parallel emergence of two more represented V(H)DJ(H) clones was observed. They differed by nine nucleotide substitutions one of which only caused a conservative replacement aminoacid change. In addition, one V(L)J(L) rearrangement became more represented over time. Analyses of somatic mutations suggest antigen selection and impairment of negative selection of neoplastic cells. In addition, a genealogical tree representing a model of clonal evolution of the neoplastic cells was created. It is of note that, during the period of study, the patient showed clinical progression of disease. We conclude that antigen stimulation and somatic hypermutation may participate in disease progression through the selection and expansion of neoplastic subclone(s).

[Cardiac surgery in octogenarians: a six-year follow-up with a multidimensional intervention]. / Vantaggi di un approccio multidimensionale personalizzato al paziente anziano sottoposto a chirurgia cardiaca: risultati a breve e lungo termine su 193 pazienti.

BACKGROUND: Elderly subjects frequently experience a decline in function following hospitalization and surgery. Specific changes in the provision of acute hospital care can improve the ability of acutely ill older patients to perform activities of daily living at the time of discharge and the quality of life. The aim of this study was to investigate outcomes of older (age > or =80 years) cardiac surgery patients managed with multicomponent intervention. METHODS: Between 1998 and 2004, we studied records of 193 octogenarian patients who underwent cardiac surgery and were treated with a multicomponent intervention that included: specially designed environment, patient-centered care, planning for patient discharge at home, and an interdisciplinary approach that incorporates in- and out-of-hospital health professionals. RESULTS: Mean follow-up was 26.4 months and 100% complete. Mean age of patients was 82.3 +/- 2 years. Eighty-nine patients had myocardial revascularization (CABG), 40 aortic valve replacement (AVR), 34 AVR + CABG, 8 mitral valve replacement (MVR), 11 MVR + CABG and 11 other interventions. Rates of hospital death, major complications and prolonged stay (> 14 days) were as follows: CABG 4 (4.4%), 3 (3.3%), 6 (6.4%); AVR 1 (2.5%), 3 (7.5%), 2 (5%); AVR + CABG 1 (2.9%), 2 (5.8%), 4 (11.7%); MVR 0 (0%), 0 (0%), 1 (12.5%); MVR + CABG 2 (18.1%), 2 (18.1%), 3 (27.2%). Multivariate predictors of hospital deaths were NYHA class, cardiopulmonary bypass and cross-clamping time, urgent procedure and ischemic mitral valve procedures. The actuarial 6-year survival was as follows: CABG 91%,AVR 92.5%, AVR + CABG 88.2%, MVR + CABG 81.8%. Total survival rate, free from rehospitalization and redo surgery, was 89.7, 69.8 and 99% respectively. Multivariate predictors of late death were urgent procedure and ischemic mitral valve procedures. At follow-up NYHA classification had improved a median of two classes. Global patients' satisfaction was excellent in 76.7% of survivors; 95.7% were autonomous, 40.5% live at home, 64% had a light-moderate physical activity, and 70% of patients had good social relationships and quality of life. Medical therapy was reduced in 29.3% and level of anxiety improved in 76%. CONCLUSIONS: An interdisciplinary approach and multicomponent intervention with an appropriate postoperative care, provides beneficial effects on outcome in geriatric cardiac surgery patients.

CD1d is expressed on B-chronic lymphocytic leukemia cells and mediates alpha-galactosylceramide presentation to natural killer T lymphocytes.

Generation of immune responses against B cell chronic lymphocytic leukemia (B-CLL) has been the aim of several studies that have demonstrated a poor antigen presenting ability of B-CLL cells and an inconsistent emergence of T cells capable of killing efficiently the leukemic cells. CD1d is a restriction element structurally related to the major histocompatibility complex (MHC) and capable of presenting lipid antigens to CD1d-restricted T cells (also defined as natural killer-T [NKT] cells). The synthetic lipid alpha-galactosylceramide (alpha-GalCer) has been characterized as a potent stimulator of CD1d-restricted T cells. We have investigated the expression of CD1d on B-CLL cells. CD1d was detected by flow cytometric analyses on leukemic cells of all B-CLL cases studied (n = 38) and was expressed at higher density on cells carrying unmutated immunoglobulin variable region (IgV) genes. In addition, CD1d on B-CLL cells mediated the presentation of alpha-GalCer to CD1d-restricted T cells, which in turn induced B-CLL cell death. At variance with another study (Metelitsa et al., Leukemia 2003;17:1068-77), no correlation between expression levels of CD1d and susceptibility to NKT cell lysis was observed. Proliferation and production of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) by CD1d-restricted T cells, in the presence of B-CLL cells loaded with alpha-GalCer, were also observed. Our study demonstrates that B-CLL cells express a monomorphic restriction element that is functionally capable of antigen presentation and can be useful to design novel B-CLL immunotherapies.

Role of amniotic membrane transplantation for conjunctival reconstruction in ocular-cicatricial pemphigoid.

PURPOSE: To evaluate the role and the effectiveness over time of amniotic membrane transplantation (AMT) as a first-step procedure to treat conjunctival reconstruction in late-stage ocular-cicatricial pemphigoid (OCP). DESIGN: Prospective interventional noncomparative case series. PARTICIPANTS: Nine eyes (9 patients) with advanced OCP. METHODS: Preoperatively, the ocular surface conditions were evaluated by immunohistochemistry of conjunctival biopsy and impression cytology specimens. The amniotic membrane was obtained during cesarean section from women who were 39 weeks pregnant and seronegative for human immunodeficiency virus, hepatitis B and C, and syphilis; it was processed, histologically tested, and stored at -80 degrees C. After scar tissue was removed, the preserved amniotic membrane was placed over the cornea, the bulbar, and tarsal conjunctiva, and was secured with 8-0 Vicryl sutures to the conjunctival edges and the deep fornices with double-armed 6-0 silk sutures. In 2 cases a double layer of amniotic membrane was transplanted. All patients received immunosuppressive systemic therapy and preservative-free tear substitutes and steroids topically for at least 6 months. During follow-up (average, 48 weeks; range, 28-96 weeks), a new standardized method was used to evaluate the fornix depth, and impression cytology testing was performed and conjunctival inflammation recorded and used as parameters for monitoring disease activity. MAIN OUTCOME MEASURES: Symblepharon, increased inferior fornix depth, presence of conjunctival goblet cells, and the degree of conjunctival inflammation. RESULTS: The conjunctival surface was free from symblepharon in all subjects for the first 16 weeks. At the week 28 examination, a small area of symblepharon was present in four eyes (44.4%). The depth of the fornix was significantly (P < 0.0001, analysis of variance) improved at weeks 4, 16, and 28. The normal conjunctival epithelium with goblet cells was restored in 6 of 9 eyes (66.7%) at the week 4 examination and in 4 eyes (44.4%) at the week 28 examination. Conjunctival inflammation was clinically but not statistically reduced. The visual acuity improved in 5 subjects. CONCLUSIONS: AMT can be a first-step procedure for ocular surface reconstruction in OCP, but its effectiveness deteriorates slightly over time.

Systemic linoleic and gamma-linolenic acid therapy in dry eye syndrome with an inflammatory component.

PURPOSE: To evaluate the efficacy and anti-inflammatory activity of systemic linoleic (LA) and gamma-linolenic acid (GLA), which decrease chronic inflammation in rheumatoid arthritis, on the ocular surface of patients with keratoconjunctivitis sicca. METHODS: In a randomized clinical trial, 26 patients with aqueous-deficient keratoconjunctivitis sicca were consecutively selected from patients presenting to Department of Neurosciences, Ophthalmology and Genetics, University of Genoa. The diagnosis was based on dry eye symptom survey score, Schirmer-1 test values, positive vital staining with lissamine green, and fluorescein break-up time (FBUT). All patients had ocular surface inflammation based on HLA-DR expression, a major histocompatibility class II antigen, on epithelial bulbar conjunctiva samples. The subjects were randomly divided into two groups of 13 patients each. The study group received tablets containing LA (28.5 mg) and GLA (15 mg) twice daily for 45 days and used tears; the control group received a tear substitute and a placebo tablet for 45 days. RESULTS: Statistically significant changes in symptoms (p < 0.005), lissamine green staining (p < 0.005), and ocular surface inflammation (p < 0.05) occurred in the study group compared with controls. HLA-DR expression varied from 58.5 +/- 14.1% positive conjunctival cells to 41.3 +/- 18.9% in the treated group and from 61.4 +/- 21.9% to 58.0 +/- 13.3% in the controls. No statistically significant difference between groups was found for FBUT and the Schirmer-1 test. CONCLUSIONS: Therapy with LA and GLA and tear substitutes reduces ocular surface inflammation and improves dry eye symptoms. Long-term studies are needed to confirm the role of this new therapy for keratoconjunctivitis sicca.