Giuseppe A. Palumbo

Mutation and Haplotype Studies of Familial Mediterranean Fever Reveal New Ancestral Relationships and Evidence for a High Carrier Frequency with Reduced Penetrance in the Ashkenazi Jewish Population

Familial Mediterranean fever (FMF) is a recessive disorder characterized by episodes of fever with serositis or synovitis. The FMF gene (MEFV) was cloned recently, and four missense mutations were identified. Here we present data from non-Ashkenazi Jewish and Arab patients in whom we had not originally found mutations and from a new, more ethnically diverse panel. Among 90 symptomatic mutation-positive individuals, 11 mutations accounted for 79% of carrier chromosomes. Of the two mutations that are novel, one alters the same residue (680) as a previously known mutation, and the other (P369S) is located in exon 3. Consistent with another recent report, the E148Q mutation was observed in patients of several ethnicities and on multiple microsatellite haplotypes, but haplotype data indicate an ancestral relationships between non-Jewish Italian and Ashkenazi Jewish patients with FMF and other affected populations. Among approximately 200 anonymous Ashkenazi Jewish DNA samples, the MEFV carrier frequency was 21%, with E148Q the most common mutation. Several lines of evidence indicate reduced penetrance among Ashkenazi Jews, especially for E148Q, P369S, and K695R. Nevertheless, E148Q helps account for recessive inheritance in an Ashkenazi family previously reported as an unusual case of dominantly inherited FMF. The presence of three frequent MEFV mutations in multiple Mediterranean populations strongly suggests a heterozygote advantage in this geographic region.

M2 Macrophages Phagocytose Rituximab-Opsonized Leukemic Targets More Efficiently than M1 Cells In Vitro

Because macrophages have been implicated as major players in the mechanism of action of rituximab, we have investigated the factors that modulate their tumor cell killing potential. Human macrophages, differentiated in vitro from peripheral blood monocytes, were used in binding and phagocytosis assays using B-chronic lymphocytic leukemia or lymphoma target cells opsonized with rituximab. Phagocytosis was maximal at 0.1 μg/ml rituximab and was not significantly affected by CD20 expression levels or by CD16A polymorphism at position 158 (Val/Phe). The role of FcγRs was demonstrated by complete inhibition of phagocytosis by excess human Igs. Because macrophages can be differentiated to M1- or M2-type cells with either GM-CSF or M-CSF, respectively, and can be classically activated by proinflammatory stimuli (IFN-γ/LPS) or undergo alternative activation with cytokines such as IL-4 or IL-10, we have analyzed the effect of these different polarization programs on the phagocytosis mediated by rituximab. Macrophages differentiated in presence of M-CSF showed a 2- to 3-fold greater phagocytic capacity compared with GM-CSF-induced cells. Furthermore, addition of IL-10 significantly increased, whereas IL-4 decreased phagocytosis by both M-CSF- and GM-CSF-differentiated macrophages. LPS/IFN-γ had little effect. Expression of CD16, CD32, and CD64 in different macrophage populations correlated with phagocytic activity. Interestingly, several B lymphoma cell lines were observed to secrete 400-1300 pg/ml IL-10 in vitro, and coculture of human macrophages with lymphoma conditioned medium increased significantly their phagocytic capacity. Our data suggest that cytokines secreted by lymphoma cells can favor alternate activation of macrophages with a high phagocytic capacity toward rituximab-opsonized targets.

Genotype-Phenotype Relationship in Human ATP6i-Dependent Autosomal Recessive Osteopetrosis

Autosomal-recessive osteopetrosis is a severe genetic disease caused by osteoclast failure. Approximately 50% of the patients harbor mutations of the ATP6i gene, encoding for the osteoclast-specific a3 subunit of V-ATPase. We found inactivating ATP6i mutations in four patients, and three of these were novel. Patients shared macrocephaly, growth retardation and optic nerve alteration, osteosclerotic and endobone patterns, and high alkaline phosphatase and parathyroid hormone levels. Bone biopsies revealed primary spongiosa lined with active osteoblasts and high numbers of tartrate-resistant acid phosphatase (TRAP)-positive, a3 subunit-negative, morphologically unremarkable osteoclasts, some of which located in shallow Howship lacunae. Scarce hematopoietic cells and abundant fibrous tissue containing TRAP-positive putative osteoclast precursors were noted. In vitro osteoclasts were a3-negative, morphologically normal, with prominent clear zones and actin rings, and TRAP activity more elevated than in control patients. Podosomes, alphaVbeta3 receptor, c-Src, and PYK2 were unremarkable. Consistent with the finding in the bone biopsies, these cells excavated pits faintly stained with toluidine blue, indicating inefficient bone resorption. Bone marrow transplantation was successful in all patients, and posttransplant osteoclasts showed rescue of a3 subunit immunoreactivity.

CD200 expression may help in differential diagnosis between mantle cell lymphoma and B-cell chronic lymphocytic leukemia

Chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma (MCL) share many features and their differential diagnosis may be challenging, especially when a leukemic picture alone is present. Monoclonal antibody panels are often useful, with CD23 being the most reliable. However, MCL diagnosis should be confirmed by immunohistochemical cyclin D1 detection, sometimes with equivocal or even negative results. Other cytofluorimetric, cytogenetics or molecular techniques are reliable but not widely available. B-CLL leukemic cells express CD200, a membrane glycoprotein belonging to the immunoglobulin superfamily. We investigated its expression on fresh neoplastic cells of 93 patients with a CD5+ lymphoproliferative disease (79 selected B-CLL and 14 MCL in leukemic phase). Although these data cannot be generalized, all B-CLL samples we examined were positive, with CD200 present on the vast majority of the cells while, in MCL patients, CD200 was expressed by a small minority of CD5+ cells in three subjects and totally absent in the remaining 11. We then examined CD200 expression on paraffin-embedded lymphoid tissues and bone marrow (BM) trephine biopsies from 23 B-CLL and 44 MCL patients. Again, all B-CLL cells were CD200+ both in lymph nodes and in BM while all MCL cells were negative. Adding CD200 in routine panels could be of diagnostic utility in excluding MCL diagnosis.

Clinical and endocrine effects of finasteride, a 5α-reductase inhibitor, in women with idiopathic hirsutism

OBJECTIVE To evaluate the effects of long-term administration of finasteride on hirsutism score, basal gonadotropin, and androgen secretion in women with idiopathic hirsutism. DESIGN Randomized single-blinded study. PATIENTS Eighteen patients with moderate-severe hirsutism were recruited for the study. INTERVENTIONS Nine hirsute patients received 7.5 mg/d oral finasteride for a period of 9 months whereas the other nine were treated with placebo. Hirsutism score, serum basal gonadotropin, androgens, estrogen, and sex hormone-binding globulin (SHBG) levels were evaluated in all patients before treatment and every 3 months during treatment. RESULTS After 6 and 9 months of treatment, the hirsutism score improved significantly in the patients receiving finasteride, whereas no significant modifications were observed in patients treated with placebo. The side effects observed were headache and depression of modest entity during the 1st month of treatments, whereas libido did not change. Serum levels of LH, FSH, androstenedione, unbound T, DHEAS, E2, 17 alpha-hydroxyprogesterone, and SHBG did not change during therapy. Hirsute patients treated with finasteride exhibited a marked decrease of dihydrotestosterone and a significant increase of T serum levels from the 3rd and 6th months of treatment, respectively. CONCLUSION Finasteride decreased the hirsutism score of patients affected by idiopathic hirsutism with few side effects during treatment. No modification of libido was observed.

Ex vivo expansion of mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) are adult multipotent cells that can be isolated from several human tissues. MSCs represent a novel and attractive tool in strategies of cellular therapy. For in vivo use, MSCs have to be ex vivo expanded in order to reach the numbers suitable for their clinical application. Despite being efficacious, the use of fetal calf serum for MSC ex vivo expansion for clinical purposes raises concerns related to immunization and transmission of zoonoses; the standardization of expansion methods, possibly devoid of animal components, such as those based on platelet lysate, are discussed in this paper. Moreover, this review focuses on the search of novel markers for the prospective identification/isolation of MSCs and on the potential risks connected with ex vivo expansion of MSCs, in particular that of their malignant transformation. Available tests to study the genetic stability of ex vivo expanded MSCs are also analyzed.

Angiogenesis in Chronic Myeloproliferative Diseases

Increased angiogenic activity has been demonstrated in myelofibrosis with myeloid metaplasia (MMM), chronic myeloid leukemia (CML), and essential thrombocythemia (ET) by both bone marrow microvessel density evaluation and measurement of circulating angiogenic factors. MMM is probably the disease with the more pronounced angiogenesis among myeloproliferative disorders but the significance of this finding remains speculative since the angiogenic activity is not correlated with any of the clinical and laboratory features of the disease. Circulating serum levels of angiogenic factors such as vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were found increased in MMM, CML and ET but the frequent thrombocytosis that accompanies these diseases could limit the interpretation of these data since platelets and megakaryocytes may be considered a major source at least for VEGF. However, CML patients treated with interferon were found to have lower VEGF and HGF levels than untreated or hydroxyurea-treated patients, thus suggesting a possible antiangiogenic mechanism of this drug. In addition, preliminary experiences with the antiangiogenic drug thalidomide have shown therapeutic activity in some myeloproliferative disorders.

Negative depletion of α/β+ T cells and of CD19+ B lymphocytes: A novel frontier to optimize the effect of innate immunity in HLA-mismatched hematopoietic stem cell transplantation

In recent years, infusion of T-cell depleted hematopoietic stem cells from an HLA-haploidentical relative has been shown to represent a suitable and effective, alternative option in patients in need of an allograft who lack an HLA-identical relative. In particular, this type of allograft is associated with the enormous advantage of offering an immediate transplant treatment to virtually all pediatric patients without an HLA-matched donor, whether related or unrelated, or a suitable umbilical cord blood unit. Several studies have shown that in patients given a T-cell depleted transplant relevant part of the anti-leukemia effect is mediated by alloreactive (i.e. KIR/HLA mismatched) Natural Killer cells originated from donor hematopoietic stem cells. After infusion of positively selected hematopoietic stem cell, fully functioning Natural Killer cells emerge in the recipient peripheral blood, persisting over time, only several weeks after the allograft. We have developed a new method of T-cell depletion (based on the physical elimination of mature T cells carrying α and β chains of the T-cell receptor), which permits to maintain mature donor-derived alloreactive Natural Killer cells and γδ(+) T cells in the graft. We, thus, started a formal study in children with hematological disorders aimed at evaluating the safety and efficacy of this approach. Preliminary results on 60 children transplanted so far after this type of graft manipulation are particularly promising.

13q14 Deletion size and number of deleted cells both influence prognosis in chronic lymphocytic leukemia

Deletion at 13q14 is detected by fluorescence in situ hybridization (FISH) in about 50% of chronic lymphocytic leukemia (CLL). Although CLL with 13q deletion as the sole cytogenetic abnormality (del13q‐only) usually have good prognosis, more aggressive clinical courses are documented for del13q‐only CLL carrying higher percentages of 13q deleted nuclei. Moreover, deletion at 13q of different sizes have been described, whose prognostic significance is still unknown. In a multi‐institutional cohort of 342 del13q‐only cases and in a consecutive unselected cohort of 265 CLL, we investigated the prognostic significance of 13q deletion, using the 13q FISH probes locus‐specific identifier (LSI)‐D13S319 and LSI‐RB1 that detect the DLEU2/MIR15A/MIR16‐1 and RB1 loci, respectively. Results indicated that both percentage of deleted nuclei and presence of larger deletions involving the RB1 locus cooperated to refine the prognosis of del13q‐only cases. In particular, CLL carrying <70% of 13q deleted nuclei with deletions not comprising the RB1 locus were characterized by particularly long time‐to‐treatment. Conversely, CLL with 13q deletion in <70% of nuclei but involving the RB1 locus, or CLL carrying 13q deletion in ≥70% of nuclei, with or without RB1 deletions, collectively experienced shorter time‐to‐treatment. A revised flowchart for the prognostic FISH assessment of del13q‐only CLL, implying the usage of both 13q probes, is proposed.

Multidrug resistance mechanisms in chronic lymphocytic leukaemia

Summary. We evaluated the presence of P‐glycoprotein (P‐gp)‐170, multidrug resistance protein (MRP), lung resistance protein (LRP)‐56 and Bcl‐2 in CD19‐positive cells from 100 cases of chronic lymphocytic leukaemia (CLL). P‐gp‐170 was found in 73% of the CLL cases with no significant difference regarding stage or previous treatment. LRP‐56 protein was homogeneously distributed with no differences for stage or treatment. MRP protein was detected at a low level of expression in 49·4% of CLL patients with no differences for stage or treatment. Bcl‐2 protein was expressed at a high level in all CLL patients and higher levels were found in the advanced stage. This leads us to conclude that P‐gp, MRP, LRP‐56 and Bcl‐2 are frequently expressed in CLL. P‐gp, MRP and LRP are not correlated to stage or previous treatment. Bcl‐2 is higher in advanced‐stage patients. The clinical and biological significance of these zMDR mechanisms in CLL remains to be fully explained.