A Case Series and Literature Review of Isavuconazole Use in Pediatric Patients with Hemato-oncologic Diseases and Hematopoietic Stem Cell Transplantation.

We analyzed the use of isavuconazole (ISA) as treatment or prophylaxis for invasive fungal disease (IFD) in children with hemato-oncologic diseases. A multicentric retrospective analysis was performed among centers belonging to the Italian Association for Pediatric Hematology and Oncology (AIEOP). Pharmacokinetic (PK) monitoring was applied by a high-performance liquid chromatography-tandem mass spectrometry (HLPC-MS/MS) assay. Twenty-nine patients were studied: 10 during chemotherapy and 19 after allogeneic hematopoietic stem cell transplantation (HSCT). The patients consisted of 20 males and 9 females with a median age of 14.5 years (age range, 3 to 18 years) and a median body weight of 47 kg (body weight range, 15 to 80 kg). ISA was used as prophylaxis in 5 patients and as treatment in 24 cases (20 after therapeutic failure, 4 as first-line therapy). According to European Organization for Research and Treatment of Cancer (EORTC) criteria, we registered 5 patients with proven IFD, 9 patients with probable IFD, and 10 patients with possible IFD. Patients with a body weight of <30 kg received half the ISA dose; the others received ISA on the adult schedule (a 200-mg loading dose every 8 h on days 1 and 2 and a 200-mg/day maintenance dose); for all but 10 patients, the route of administration switched from the intravenous route to the oral route during treatment. ISA was administered for a median of 75.5 days (range, 6 to 523 days). The overall response rate was 70.8%; 12 patients with IFD achieved complete remission, 5 achieved partial remission, 5 achieved progression, and 3 achieved stable IFD. No breakthrough infections were registered. PK monitoring of 17 patients revealed a median ISA steady-state trough concentration of 4.91 mg/liter (range, 2.15 to 8.54 mg/liter) and a concentration/dose (in kilograms) ratio of 1.13 (range, 0.47 to 3.42). Determination of the 12-h PK profile was performed in 6 cases. The median area under the concentration-time curve from 0 to 12 h was 153.16 mg·h/liter (range, 86.31 to 169.45 mg·h/liter). Common Terminology Criteria for Adverse Events grade 1 to 3 toxicity (increased transaminase and/or creatinine levels) was observed in 6 patients, with no drug-drug interactions being seen in patients receiving immunosuppressants. Isavuconazole may be useful and safe in children with hemato-oncologic diseases, even in the HSCT setting. Prospective studies are warranted.

New insights into HLA-G mediated tolerance.

Human Leukocyte Antigen G (HLA-G) is a nonclassical HLA class I molecule with well-characterized immunomodulatory activities. HLA-G was first described as a regulatory molecule that allows the fetus to elude the maternal immune response. In the last decade it has become evident that HLA-G is involved in modulating both innate and adaptive immune responses, in maintaining tolerance in autoimmune and inflammatory diseases and after transplantation, and in promoting immune escape in cancer and infectious diseases. HLA-G exerts its modulatory/regulatory functions directly by interacting with specific inhibitory receptors. The expression of HLA-G is finely tuned by genetic variations in the noncoding region of the locus. The recent discovery of dendritic cells-10 (DC-10) as naturally occurring HLA-G-expressing dendritic cells opens new perspectives in the identification of the molecular and cellular mechanisms underlying HLA-G-mediated tolerance. An overview on the HLA-G-mediated inhibition of innate and adaptive immune cells, on the genetic influence on HLA-G expression, and on HLA-G-expressing DC-10 is presented. Moreover, we discuss the central and critical role of DC-10 in the HLA-G-mediated tolerance.

Transplant tolerance to pancreatic islets is initiated in the graft and sustained in the spleen.

The immune system is comprised of several CD4(+) T regulatory (Treg) cell types, of which two, the Foxp3(+) Treg and T regulatory type 1 (Tr1) cells, have frequently been associated with transplant tolerance. However, whether and how these two Treg-cell types synergize to promote allograft tolerance remains unknown. We previously developed a mouse model of allogeneic transplantation in which a specific immunomodulatory treatment leads to transplant tolerance through both Foxp3(+) Treg and Tr1 cells. Here, we show that Foxp3(+) Treg cells exert their regulatory function within the allograft and initiate engraftment locally and in a non-antigen (Ag) specific manner. Whereas CD4(+) CD25(-) T cells, which contain Tr1 cells, act from the spleen and are key to the maintenance of long-term tolerance. Importantly, the role of Foxp3(+) Treg and Tr1 cells is not redundant once they are simultaneously expanded/induced in the same host. Moreover, our data show that long-term tolerance induced by Foxp3(+) Treg-cell transfer is sustained by splenic Tr1 cells and functionally moves from the allograft to the spleen.

Genotypes and haplotypes in the 3' untranslated region of the HLA-G gene and their association with clinical outcome of hematopoietic stem cell transplantation for beta-thalassemia.

Polymorphisms in the 3' untranslated region (3'UTR) of HLA-G, an important player in immunological tolerance, could be involved in post-transcriptional expression control, and their association with different clinical immune-related conditions including autoimmunity and transplantation is of mounting interest. Most studies have focused on a 14 base pair (bp) insertion/deletion (ins/del), while additional single-nucleotide polymorphisms (SNPs) in the HLA-G 3'UTR have been described but not extensively investigated for their clinical relevance. Here we have comparatively studied the association between 3'UTR haplotypes of HLA-G, or the 14 bp ins/del, with clinical outcome of HLA-identical sibling hematopoietic stem cell transplantation (HSCT) in 147 Middle Eastern beta-thalassemia patients. Sequence based typing of 3'UTR HLA-G polymorphisms in the patients and in 102 healthy Italian blood donors showed strong linkage disequilibrium between the 14 bp ins/del and five 3'UTR SNPs, which together could be arranged into eight distinct haplotypes based on expectation-maximization studies, with four predominant haplotypes (UTRs1-4). After HSCT, we found a moderate though not significant association between the presence of UTR-2 in double dose and protection from acute graft versus host disease (hazard ratio (HR) 0.45, 95% confidence intervals (CI): 0.14-1.45; P = 0.18), an effect that was also seen when the corresponding 14 bp ins/ins genotype was considered alone (HR 0.42, 95% CI: 0.16-1.06; P = 0.07). No association was found with rejection or survival. Taken together, our data show that there is no apparent added value of considering entire 3'UTR HLA-G haplotypes for risk prediction after allogeneic HSCT for beta-thalassemia.

Dendritic cells in networks of immunological tolerance.

Dendritic cells (DC) represent pacemakers of the immune system because they play a major role as antigen-presenting cells in inducing adaptive immune responses on the one hand and are critically involved in promoting and maintaining immunological tolerance on the other. The latter function is mediated by specialized subsets of DC, named tolerogenic DC, as well as by DC activated or differentiated in the presence of specific biological or chemical agents. Suppression by tolerogenic DC is primarily mediated via the induction of regulatory T (Tr) cells. In the present review, we will focus on human tolerogenic DC with the aim to: (1) describe subsets of human tolerogenic DC; (2) define the modes of in vitro induction of myeloid tolerogenic DC and their ability to induce Tr cells; (3) elucidate the role of tolerogenic DC in orchestrating tolerance induction in vivo; and (4) envisage the use of tolerogenic DC as therapeutic tool to trigger immunoregulatory mechanisms.

A peptide-binding motif for I-A(g7), the class II major histocompatibility complex (MHC) molecule of NOD and Biozzi AB/H mice.

The class II major histocompatibility complex molecule I-A(g7) is strongly linked to the development of spontaneous insulin-dependent diabetes mellitus (IDDM) in non obese diabetic mice and to the induction of experimental allergic encephalomyelitis in Biozzi AB/H mice. Structurally, it resembles the HLA-DQ molecules associated with human IDDM, in having a non-Asp residue at position 57 in its beta chain. To identify the requirements for peptide binding to I-A(g7) and thereby potentially pathogenic T cell epitopes, we analyzed a known I-A(g7)-restricted T cell epitope, hen egg white lysozyme (HEL) amino acids 9-27. NH2- and COOH-terminal truncations demonstrated that the minimal epitope for activation of the T cell hybridoma 2D12.1 was M12-R21 and the minimum sequence for direct binding to purified I-A(g7) M12-Y20/K13-R21. Alanine (A) scanning revealed two primary anchors for binding at relative positions (p) 6 (L) and 9 (Y) in the HEL epitope. The critical role of both anchors was demonstrated by incorporating L and Y in poly(A) backbones at the same relative positions as in the HEL epitope. Well-tolerated, weakly tolerated, and nontolerated residues were identified by analyzing the binding of peptides containing multiple substitutions at individual positions. Optimally, p6 was a large, hydrophobic residue (L, I, V, M), whereas p9 was aromatic and hydrophobic (Y or F) or positively charged (K, R). Specific residues were not tolerated at these and some other positions. A motif for binding to I-A(g7) deduced from analysis of the model HEL epitope was present in 27/30 (90%) of peptides reported to be I-A(g7)-restricted T cell epitopes or eluted from I-A(g7). Scanning a set of overlapping peptides encompassing human proinsulin revealed the motif in 6/6 good binders (sensitivity = 100%) and 4/13 weak or non-binders (specificity = 70%). This motif should facilitate identification of autoantigenic epitopes relevant to the pathogenesis and immunotherapy of IDDM.

Impact of HLA-G polymorphism on the outcome of allogeneic hematopoietic stem cell transplantation for metastatic renal cell carcinoma.

Renal cell carcinoma (RCC) is particularly sensitive to immune intervention. HLA-G, a non-classical HLA class I molecule with immunomodulatory properties, has been studied with regard to outcome after hematopoietic stem cell transplantation (HSCT), in particular the 14 bp insertion/deletion polymorphism in the 3' untranslated region. Here we analyzed n=56 patients affected by metastatic RCC who received an allogeneic HSCT between 1998 and 2006 in Milano, Marseille, Clermont-Ferrand and Stockholm. The 14 bp polymorphism was analyzed in correlation with overall survival (OS), PFS, acute and chronic GvHD. With a median follow-up of 13 years, a trend towards better outcome was observed when homozygosity for the 14bp-del allele was present: multivariate hazard ratio was 0.50 (95% confidence interval (CI): 0.23-1.13; P=0.10) and 0.57 (95% CI: 0.26-1.26; P=0.17) for OS and PFS, respectively, when 14bp-del/del was compared with 14bp-ins/X. Further exploratory analysis revealed a significant association between T/C at p3003 and improved OS (P=0.05) and PFS (P=0.006) compared with T/T. To our knowledge this is the first study on HLA-G and outcome after HSCT for a solid malignancy. After a coordinated multicenter study, we found that the more tolerogenic polymorphisms (14bp-del/del) is associated with better PFS and OS. The finding on p3003 deserves further investigation.

Safety of arylsulfatase A overexpression for gene therapy of metachromatic leukodystrophy.

Successful gene therapy approaches for metachromatic leukodystrophy (MLD), based either on hematopoietic stem/progenitor cells (HSPCs) or direct central nervous system (CNS) gene transfer, highlighted a requirement for high levels of arylsulfatase A (ARSA) expression to achieve correction of disease manifestations in the mouse model. Full assessment of the safety of ARSA expression above physiological levels thus represents a prerequisite for clinical translation of these approaches. Here, using lentiviral vectors (LVs), we generated two relevant models for the stringent evaluation of the consequences of ARSA overexpression in transduced cells. We first demonstrated that ARSA overexpression in human HSPCs does not affect their clonogenic and multilineage differentiation capacities in clonogenic assays and in a neonatal hematochimeric mouse model. Further, we studied ARSA overexpression in all body tissues by generating transgenic mice overexpressing the ARSA enzyme by LV up to 15-fold above the normal range and carrying multiple copies of LV in their genome. Characterization of these mice demonstrated the safety of ARSA overexpression in two main gene therapy targets, HSPCs and neurons, with maintenance of the complex functions of the hematopoietic and nervous system in the presence of supraphysiological enzyme levels. The activity of other sulfatases dependent on the same common activator, sulfatase-modifying factor-1 (SUMF1), was tested in ARSA-overexpressing HSPCs and in transgenic mice, excluding the occurrence of saturation phenomena. Overall, these data indicate that from the perspective of clinical translation, therapeutic levels of ARSA overexpression can be safely achieved. Further, they demonstrate an experimental platform for the preclinical assessment of the safety of new gene therapy approaches.

White-coat hypertension: a selection bias? Harvest Study Investigators. Hypertension and Ambulatory Recording Venetia Study.

BACKGROUND: Results of several studies have shown that subjects with white-coat hypertension (WCH) have more target-organ damage than do normotensive controls with similar ambulatory blood pressures. OBJECTIVE: To investigate whether this is due to a selection bias. SETTING: Seventeen hypertension clinics in northeast Italy. MAIN OUTCOME MEASURES: Echocardiographic data in relation to WCH status. PATIENTS AND METHODS: Mild hypertensive subjects from the HARVEST (n = 565) who underwent two ambulatory blood pressure monitorings 3 months apart and M-mode echocardiography, and 95 normotensive control subjects. RESULTS: From first ambulatory monitoring, 90 hypertensive subjects were classified as having WCH (mean daytime blood pressure < 130/80 mmHg). Their 24 h blood pressure was similar to that of the normotensive subjects, but their left ventricular mass index was greater. From second ambulatory monitoring, only 38 of the 90 subjects still had WCH, whereas 24 h blood pressure in the other 52 had risen beyond the limit of WCH. Left ventricular mass index (89.2 +/- 2.4 g/m2), wall thickness (18.1 +/- 0.3 mm), and relative wall thickness (0.359 +/- 0.006%) of the 38 subjects with WCH at both recordings were still greater than those of the normotensive subjects (82.4 +/- 1.5 g/m2, P = 0.02; 17.2 +/- 0.2 mm, P = 0.002; and 0.337 +/- 0.004%, P = 0.025) and similar to those of the 52 subjects who no longer had WCH (88.5 +/- 2.0 g/m2, 18.7 +/- 0.2 mm, and 0.375 +/- 0.005%, all NS). CONCLUSIONS: Owing to regression toward the mean, over 50% of the subjects with WCH could no longer be classified as such from repeated ambulatory monitoring, indicating that the current diagnosis of WCH is subject to selection bias. Cardiac remodeling was present also in the subjects confirmed to have WCH by repeated blood pressure recording, suggesting that the effect of WCH has an actual impact on target organs.

Prevalence and clinical correlates of microalbuminuria in stage I hypertension. Results from the Hypertension and Ambulatory Recording Venetia Study (HARVEST Study).

The objective of the present study was to examine the association between albumin excretion rate (AER) and office and ambulatory blood pressures (BP), and other recognized cardiovascular risk factors in stage I hypertension. The study was carried out in 870 never-treated 18- to 45-year-old hypertensives (628 men, 242 women). Office and ambulatory BP, 24-h urinary collection for AER assessment, and echocardiographic left ventricular mass (n = 587) were obtained. AER was similar in men and women (12.3 v 12.5 mg/24 h) and was unrelated to age and body mass index. In 85.2% of the subjects, AER was < 16 mg/24 h, in 8.3% it was between 16 and 29 mg/24 h (borderline microalbuminuria), and in 6.1% it was >or= 30 mg/24 h (overt microalbuminuria). Office systolic BP was not different in the three groups, whereas 24-h systolic BP was higher in the subjects with microalbuminuria than in those with normal AER (P < .0001) and was similar in the two microalbuminuric groups. Office and 24-h diastolic BPs were higher in the subjects with overt microalbuminuria than in those with normal AER. Left ventricular mass was correlated to systolic (P < .0001) and diastolic (P = .01) 24-h BP, but was unrelated to AER. Family history for hypertension, smoking, coffee and alcohol intake, and physical activity habits did not influence AER. In a logistic regression analysis, 24-h systolic BP emerged as the only determinant of microalbuminuria (P < .0001). In conclusion, these results indicate that borderline levels of microalbuminuria may also be clinically relevant in stage I hypertension. Overweight and lifestyle factors do not appear to influence AER in these patients. Finally, the lack of correlation between AER and left ventricular mass suggests that renal and cardiac involvement do not occur in a parallel fashion in the initial phase of hypertension.

Secretory defects induced by immunosuppressive agents on human pancreatic beta-cells.

Despite the considerable interest for islet and pancreas transplantation, remarkably little is known about the direct effects of immunosuppressive drugs on human beta-cell function. We measured different insulin secretory parameters and insulin gene expression of human islets cultured for 5 days in the presence of mycophenolate mofetil (MMF), cyclosporin A (CsA), tacrolimus (FK506) or a mixture of 3 cytokines. Basal insulin release after exposure to cytokines and FK506 was significantly higher than in control islets. Responsiveness to an acute glucose stimulus did not differ significantly between control and treated islets. However, absolute incremental insulin responses (delta-AUCs) of islets exposed to cytokines or FK506 were significantly higher compared to islets exposed to CsA or MMF, mainly because of the higher basal release. Indeed, maximal over basal release (stimulation index, SI) tended to be lower in islets exposed to FK506 than in control islets. Insulin gene expression was significantly reduced only in islets exposed to CsA. FK506 was, among those tested, the immunosuppressive drug that most profoundly altered the normal insulin secretory pattern of human beta-cells, whereas CsA was the only inhibiting insulin gene expression. Although the abnormalities induced by the immunosoppressive drugs utilized in this study were modest, these in vitro data are consistent with the reported in vivo diabetogenicity of CsA and FK506 and point to MMF as the ideal immunosuppressive agent from a pancreatic beta-cell point of view.

Serotonin S2 receptors blockage and generalized anxiety disorders. A double-blind study on ritanserin and lorazepam.

Serotonin (5-HT) and 5-HT receptors are involved in mood disturbances, such as anxiety and depression. Ritanserin is a new substance with highly selective blocking activity on S2 receptors for 5-HT in the central nervous system. Ritanserin, (20 mg daily) and lorazepam (5 mg daily) were administered to 24 patients suffering from generalized anxiety disorders (DSM III), in a double-blind fashion for six weeks. The results obtained showed comparable improvement in almost all patients with both drugs. Future studies should pay particular attention to psychosomatic disturbances, depressed mood and dysthymic-like disorders, in which ritanserin seems to be more efficacious, according to the best responding items of the general anxiety check list used.

Monoclonal antibodies against recombinant human growth hormone as probes to study immune function.

Monoclonal antibodies were raised against human recombinant growth hormone (rhGH) and those that did not cross-react with other human recombinant proteins like prolactin (PRL), interleukin 2 (IL-2), insulin, or bovine pituitary growth hormone were selected. The selected hybridoma supernatants were studied for their ability to influence T lymphocyte proliferation when induced either by a mitogen, such as phytohemagglutinin (PHA), or by alloantigen. All supernatants inhibited proliferation. Three MAbs were then purified by several passages on antimouse IgG (or IgM)-agarose columns, and characterized. These MAbs recognized three different epitopes, as revealed by competition study, although their inhibitory effect on PHA-induced T cell proliferation was quite similar. The data demonstrate that the MAbs were not cytolytic, that they did not interfere with the PHA binding to T cell membranes, and, as revealed by FACS analysis, did not bind to the membrane. Finally, these MAbs immunoprecipitated a 44-kDa molecule from PHA-activated T cell-concentrated supernatants. These data indicate that the MAbs recognized a soluble factor that plays a central role in T cell proliferation and that is probably the immune growth hormone.

Study of the structure of troponin-I by measuring the relative reactivities of lysines with acetic anhydride.

A competitive labeling method that measures the relative reactivity of lysines was used to study the structure of troponin-I. Troponin-I was acetylated free and complexed with troponin-C and troponin-T in the native state with [3H]acetic anhydride. The [3H]troponin-I was combined with [14C]troponin-I that had been acetylated in 6 M guanidine HCl and completely chemically labeled. Peptides containing labeled lysines were isolated following digestion with trypsin and Staphylococcus aureus protease and identified in the published sequence. The 3H/14C ratio of these peptides was used as a measure of the relative reactivity of the lysines. Troponin-I contains 24 lysines; we have identified 23 of these in 16 peptides. When troponin-I is labeled in a native complex, the lysines in the region from residues 40 to 98 are influenced: five become relatively less reactive (40, 65, 70, 78, and 90) and three become relatively more reactive (84, 87), and 98). All of these changes except Lys 70 can be seen when troponin-I binds to troponin-T. Lys 70 is reduced in reactivity when it binds to troponin-C. The lysines that appear to be important in binding of troponin-I to troponin-T are influenced by the binding of Ca2+ to troponin-C in the native troponin complex (in the presence of 2 mM MgCl2), suggesting for the first time that the troponin-IT interaction is affected by Ca2+.

The frequency of high avidity T cells determines the hierarchy of determinant spreading.

Autoimmunity often spreads in a predefined pattern during the progression of T cell-mediated autoimmune diseases. This progression has been well described in animal models and in man, but the basis for this phenomenon is little understood. To gain insight into the factors that determine this spreading hierarchy, we characterized the binding affinity of a panel of beta cell-autoantigenic peptides to I-Ag7, as well as the precursor frequency, functional avidity, and phenotype of the T cells that recognize these peptides in type 1 diabetes-prone nonobese diabetic mice. We observed that autoimmunity gradually spreads from a beta cell determinant, which had the largest precursor pool of high avidity T cells, to beta cell determinants with progressively smaller and lower avidity T cell precursor pools. This correlation between the sequential development of spontaneous T cell autoimmunity and the frequency and avidity of autoantigen-reactive T cells suggests that the extent to which T cells were negatively selected by the self-determinants is the key factor determining the spreading hierarchy.