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RG6102 is a bispecific 2+1 monoclonal antibody (mAb) under development for the treatment of Alz- heimer’s disease (AD). It combines the anti-amyloid beta antibody gantenerumab with a transferrin receptor 1-binding “Brain Shuttle” module, enabling active receptor-mediated transport across the blood–brain barrier.In preclinical studies, RG6102 has shown superior distribution, target engagement and amyloid plaque clearance compared with gantenerumab. In a human Phase Ia study, there was a markedly increased cerebrospinal fluid (CSF)/plasma ratio for RG6102 compared with typical mAbs.Brainshuttle AD is a 28-week, randomised, global, multicentre, double-blind, placebo-controlled, parallel- group Phase Ib/IIa study evaluating the safety, tolerability, immunogenicity and pharmacokinetics/phar- macodynamics (PK/PD) of RG6102. Multiple-ascending intravenous doses of RG6102 are administered every 4 weeks to patients with prodromal or mild-to-moderate AD. The study consists of a screening period, a double-blind treatment period and a safety follow-up period.The primary objective is to evaluate the safety and tolerability of RG6102. Secondary outcome measures include the change from baseline in brain amyloid load, plasma and CSF concentration of RG6102 and incidence of anti-drug antibodies to RG6102. Exploratory endpoints include the clinical effect of multiple doses of RG6102 on clinical outcome measures and on various PD biomarkers.Recruitment for Brainshuttle AD is currently ongoing.

Singing is one of the most used forms of language for the child and is present in all cultures. The musicality at the base of singing causes measurable biological responses in the singing adult and in the listening child. This article summarizes the most recent scientific evidence on the effects of adult singing (parent or caregiver) during the first years of life of a child. Singing reduces stress, promotes adult-child relationship and the child’s attention skills. Finally, the knowledge of singing’s positive effects emphasizes the importance of this knowledge for the pediatrician in informing families of this useful practice for supporting child development.

We tested the possibility to prepare a hyperproteic and hyperenergetic supplementary food for malnutrition rehabilitation in children starting from available ingredients in popular markets in Sierra Leone. Twelve residents in Paediatrics from University of Parma, Italy, prepared in a hospital near the capital Freetown with modest technology a mixture of peanut flour, palm oil, milk powder, sugar and vitamins to which they gave the name of "Parma pap". Three hundred and thirty-two malnourished children (mean age 14±6.3 months) who were receiving Feeding Program Supplementations (FPS), were enrolled in the study: 177 participants received randomly FSP portions only (Group 1), and 159 participants were treated with FSP regimen plus a supplement of "Parma pap" (Group 2). Outcomes of the study were computed as WHZ-score increment (Δ value) by subtracting the discharge WHZ-score from the admission WHZ-score. The best Δ-WHZ-scores (gt;+4) were recorded among participants of Group 2 (64%) rather than in Group 1 (21%; p=0.040). The children receiving FSP portions plus "Parma pap" recovered faster (5.54 week on average) than those treated with FSP regimen only (8.16 on average). The percentage of children who did not recover was higher in Group 1 (25.3%) than in Group 2 (; 13%; p=0.05). A slight positive correlation has been found between WHZ-scores at admission and at the end of the study (r=0.19; p=0.045). During the experience in Sierra Leone we have had the chance to give "Parma pap" to twenty one malnourished children admitted to Xaverian Mission in Makeni, northern Sierra Leone, not taking other supplementary food. Sixteen of these children recovered in 4.9 week on average and five in 6 to 8 weeks. Mean Δ-WHZ-scores ranged between + 1 and + 5. The data from the present study suggest that "Parma pap" could be an effective additional food to FPS regimen in malnutrition recovering. Further researches are needed on the contrary to prove if "Parma pap" could be defined as a veritable ready to use therapeutic food, although this characteristic seems already to result from the experience in Makeni Mission.

The major histocompatibility complex (MHC) comprises a set of genes that are essential to immunity and surveillance against neoplastic transformation. MHC antigens not only regulate antitumor immune responses in experimental animal models but also directly correlate with survival and prognosis of patients with various types of cancers. Effective recognition of tumor cells by effector T cells may be affected by the genotype and the extent of expression of human leukocyte antigen (HLA)-peptide complexes. Therefore, MHC antigens may serve as potential biomarkers for prognosis and allow selection of cancer patients for specific therapy. We describe PCR-based method to determine the HLA genotype in healthy individuals and patients using blood and tumor tissue as DNA source.

BACKGROUND AND AIM: Colon crypts are characterized by a hierarchy of cells distributed along the crypt axis. Aim of this paper was to develop an in vitro system for separation of epithelial cell subsets in different maturation stages from normal human colon. METHODOLOGY AND MAJOR FINDINGS: Dissociated colonic epithelial cells were stained with PKH26, which allows identification of distinct populations based on their proliferation rate, and cultured in vitro in the absence of serum. The cytofluorimetric expression of CK20, Msi-1 and Lgr5 was studied. The mRNA levels of several stemness-associated genes were also compared in cultured cell populations and in three colon crypt populations isolated by microdissection. A PKH(pos) population survived in culture and formed spheroids; this population included subsets with slow (PKH(high)) and rapid (PKH(low)) replicative rates. Molecular analysis revealed higher mRNA levels of both Msi-1 and Lgr-5 in PKH(high) cells; by cytofluorimetric analysis, Msi-1(+)/Lgr5(+) cells were only found within PKH(high) cells, whereas Msi-1(+)/Lgr5(-) cells were also observed in the PKH(low) population. As judged by qRT-PCR analysis, the expression of several stemness-associated markers (Bmi-1, EphB2, EpCAM, ALDH1) was highly enriched in Msi-1(+)/Lgr5(+) cells. While CK20 expression was mainly found in PKH(low) and PKH(neg) cells, a small PKH(high) subset co-expressed both CK20 and Msi-1, but not Lgr5; cells with these properties also expressed Mucin, and could be identified in vivo in colon crypts. These results mirrored those found in cells isolated from different crypt portions by microdissection, and based on proliferation rates and marker expression they allowed to define several subsets at different maturation stages: PKH(high)/Lgr5(+)/Msi-1(+)/CK20(-), PKH(high)/Lgr5(-)/Msi-1(+)/CK20(+), PKH(low)/Lgr5(-)/Msi-1(+)/Ck20(-), and PKH(low)/Lgr5(-)/Msi-1(-)/CK20(+) cells. CONCLUSIONS: Our data show the possibility of deriving in vitro, without any selection strategy, several distinct cell subsets of human colon epithelial cells, which recapitulate the phenotypic and molecular profile of cells in a discrete crypt location.

ObjectivePremature pubarche (PP) is the most frequent sign of nonclassic congenital adrenal hyperplasia (NCCAH) due to 21-hydroxylase deficiency in childhood. The aim of this study was to assess the relationship between the CYP21A2 genotype and baseline and ACTH-stimulated 17-hydroxyprogesterone (17-OHP) and cortisol serum levels in patients presenting with PP.Patients and methodsA total of 152 Italian children with PP were studied. Baseline and ACTH-stimulated 17-OHP and cortisol serum levels were measured and CYP21A2 gene was genotyped in all subjects.ResultsBaseline and ACTH-stimulated serum 17-OHP levels were significantly higher in NCCAH patients than in both heterozygotes and children with idiopathic PP (IPP). Of the patient population, four NCCAH patients (7.3%) exhibited baseline 17-OHP values CYP21A2 mutations, 41.8% were compound heterozygotes for one mild and one severe CYP21A2 gene mutations, and 3.6% had two severe CYP21A2 gene mutations.ConclusionIn children with PP, baseline 17-OHP levels are not useful to rule out the diagnosis of NCCAH, which is accomplished by means of ACTH testing only. The different percentages of severe and mild CYP21A2 gene mutations found in PP children compared with adult NCCAH patients is an indirect evidence that the enzyme defect is under-diagnosed in childhood, and it might not lead to the development of hyperandrogenic symptoms in adulthood. Stress-dose glucocorticoids should be considered in patients with suboptimal cortisol response to ACTH stimulation.

HLA abnormalities on tumour cells for immune escape have been widely described. In addition, cellular components of the tumour microenvironment, in particular myeloid derived suppressor cells (MDSC) and alternatively activated M2 tumour-associated macrophages (TAMs), are involved in tumour promotion, progression, angiogenesis and suppression of anti-tumour immunity. However, the role of HLA in these activities is poorly understood. This review details MHC class I characteristics and describes MHC class I receptors functions. This analysis established the basis for a reflection about the crosstalk among the tumour cells, the TAMs and the cells mediating an immune response. The tumour cells and TAMs exploit MHC class I molecules to modulate the surrounding immune cells. HLA A, B, C and G molecules down-regulate the macrophage myeloid activation through the interaction with the inhibitory LILRB receptors. HLA A, B, C are able to engage inhibitory KIR receptors negatively regulating the Natural Killer and cytotoxic T lymphocytes function while HLA-G induces the secretion of pro-angiogenic cytokines and chemokine thanks to an activator KIR receptor expressed by a minority of peripheral NK cells. The open conformer of classical MHC-I is able to interact with LILRA receptors described as being associated to the Th2-type cytokine response, triggering a condition for the M2 like TAM polarization. In addition, HLA-E antigens on the surface of the TAMs bind the inhibitory receptor CD94/NKG2A expressed by a subset of NK cells and activated cytotoxic T lymphocytes protecting from the cytolysis. Furthermore MHC class II expression by antigen presenting cells is finely regulated by factors provided with immunological capacities. Tumour-associated macrophages show an epigenetically controlled down-regulation of the MHC class II expression induced by the decoy receptor DcR3, a member of the TNFR, which further enhances the M2-like polarization. BAT3, a positive regulator of MHC class II expression in normal macrophages, seems to be secreted by TAMs, consequently lacking its intracellular function, it looks like acting as an immunosuppressive factor. In conclusion HLA could cover a considerable role in tumour-development orchestrated by tumour-associated macrophages.

We updated a cohort study with the aim of evaluating if an up-front Rituximab supplemented high-dose sequential chemotherapy (R-HDS), supported by autologous bone marrow transplantation (ASCT) can induce log-term remissions in Mantle Cell Lymphoma (MCL), with an acceptable toxic profile. From 1997 to 2005, 77 consecutive MCL patients, 1 IPI risk factors, while >1 extranodal sites prevailed in Group 1 and a poor ECOG-PS in Group 2. After 2–3 cycles of either doxorubicin- or cisplatin-containing chemotherapy, Group 1 received R-HDS including: HD-cyclophosphamide (CTX) 7 gr/sqm and HD-Ara-C (2 g/sqm every 12 hours for 6 days), followed by HDS HD-melphalan (180 mg/sqm) and/or HD-mitoxantrone plus melphalan (60 and 180 mg/sqm) and ASCT. Rituximab (375 mg /sqm) was given for a total of 6 doses, twice after HD-CTX and HD-Ara-C, as in vivo purging before CD34+ cells harvest and twice after ASCT. Sixty-nine patients (90%) in Group 1 completed the planned program and a median number of 6.3 x 10^6 cells CD34+/kg were transplanted. In Group 2, 85% of patients received the planned chemotherapy. The CR rate was 86% in Group 1 and 35% in Group 2 and the treatment-related mortality was 1.3% and 0.8%, respectively. Moreover, in Group 1, 4 patient developed sMDS, and 4 died of solid tumors (n=3), or septic shock (n=1). After a median follow-up of 50 months (range 3–111) in Group 1 and 37 months (range 1–141) in Group 2, the rate of 1st CCR was 65% and 14%, respectively. The 5-year projected OS, EFS and DFS were 74%, 61% and 70%, in Group 1 and 31%, 14% and 25%, in Group 2, respectively. Cox multivariate analysis failed to identify within potential prognostic markers factors predictive for OS and EFS. In conclusion, R-HDS induces long-term remissions with a manageable toxicity in patients with newly diagnosed MCL. Studies comparing this program with other intensive regimens are needed for ascertaining the role of R-HDS in the treatment of this otherwise incurable disease.

We showed recently that the dismal outcome of MCL might be improved in 28 young patients (pts) (60 years with an age-adapted R-HDS. We report the outcome of 54 young pts (group 1), including the first 28 cases and that of 19 elderly pts (group 2). The majority of both groups had an advanced stage and bone marrow infiltration, while in elderly pts prevail B symptoms and > 1 extranodal site. One third of cases had >2 adverse prognostic features according to IPI. After 2–3 cycles of either doxorubicin- or cisplatin-containing chemotherapy, group 1 received standard R-HDS including: HD-cyclophosphamide (CTX) 7 gr/sqm and HD-Ara-C (2 g/sqm every 12 hours for 6 days). Following HDS chemotherapy a conditioning program with HD-melphalan (180 mg/sqm) and/or HD-mitoxantrone plus melphalan (60 and 180 mg/sqm) with ASCT was planned. Rituximab (375 mg /sqm) was given for a total of 6 doses, twice after HD-CTX and HD-Ara-C, as in vivo purging before CD34+ cells harvest and twice after ASCT. Elderly patients received an age-adapted R-HDS: HD-CTX (3–4 gr/sqm) and HD-Ara-C (1–1.5 g/sqm every 12 hours for 3–5 days), followed by HD-melphalan (120 mg/sqm) and HD-mitoxantrone plus melphalan (40 and 120 mg/sqm). 35 pts (65%) in group 1 and 9 (47%) in group 2 completed the planned program and a median number of 7.6 and 6.8 x 10^6 cells CD34+/kg were transplanted. After ASCT the CR rate was 88% in young and 95% in elderly patients. One young pt (2%) died during treatment, 1 developed sMDS and 5 died of late toxicity including a case of lung carcinoma. Among elderly pts only one died tardily because of cardiac disease. With a median follow-up of 48 months (range 8–101) in group 1 and 25 months (range 9–68) in group 2, the 5-year estimated OS, EFS and DFS were 77%, 60% and 71%, in group 1 and 55%, 53% and not yet achieved in group 2. The Cox multivariate analysis failed to identify within potential prognostic markers factors predictive for OS and EFS. We conclude that R-HDS is an effective regimen for the induction of complete remissions in pts with newly diagnosed MCL. The manageable toxicity of the program in elderly pts proved that an age-adjusted R-HDS regimen can be safely applied to this age subgroup still producing long-term remissions.

B-cell malignancies represent a potential target for anti-cancer vaccination programs due to the expression of tumor-specific antigens. Although immunization with tumor-derived idiotype protein is a frequently used procedure, vaccination with DCs loaded with killed tumor cells may activate response to a much wider range of antigens, without requiring prior molecular identification of such determinants. Furthermore, such DC-based vaccines could be available to all patients, irrespective of the HLA type. To evaluate the safety and tolerability of this approach, 18 patients with measurable relapse/refractory follicular (FCL; n= 12) and lymphoplasmocytoid (n= 6) lymphoma have been enrolled in a phase I study. Median prior number of treatment regimens was 2 (range 1–5) comprising 4 patients treated with high-dose chemotherapy supported by autologous stem cell transplantation. The vaccination was started after at least 6-months from the last chemotherapy treatment. All patients were evaluable for toxicity and 16/18 patients for efficacy with a median follow-up of 12.5 months (range 3–29 months). Each patient received 4 intradermal/subcutaneous injections at 2-weekly intervals of 50x10e6 tumor-loaded DCs. Immature DCs were generated by 5-days culture of autologous monocytes in the presence of IL-4 and GM-CSF. After selection by immunomagnetic technique, autologous CD19+ tumor cells, harvested from lymph nodes (n= 12) and/or peripheral blood (n= 6), were heat shocked and then irradiated by UVC. DCs were loaded for 48 hrs with killed tumor cells and then, to induce their maturation, were cultured for 12 hrs in the presence of TNF-alfa. Overall, vaccinations were well tolerated and no autoimmune reactions were observed. Mild erythema in the site of injection developed in the majority of patients (12/18), but only in 2 cases induration and extended erythema was observed. Six of 16 (37.5%) evaluable patients had objective responses. Two patients had partial responses (PR). One is still in PR and the other had a PR lasting 7 months. Four patients had complete remission (CR). Two patients are still in CR and the other 2 patients had a mean CR duration of 14.5 months. The remaining 10 patients had stable disease (n=5) or progressive disease (n=5). The overall monitoring of immune responses is ongoing. However, in one patient in PR, we evaluated the frequency of anti autologous tumor-specific T cells, by ELISPOT assay for IFN-gamma, on pathologic lymph nodes harvested before and after 2 months from the last vaccination. A significant increase of specific T-cell frequency was observed in the post-vaccination lymph node, compared to the tissue sample taken before vaccination. Moreover, evaluation of CD8+ T cell maturation markers, by analysis for CCR7 and CD45RA expression, indicated a shift of tumor-infiltrating T cells towards memory and effector stages in the lymph-node isolated after vaccination. In conclusion, injection of DCs loaded with killed tumor cells is a well-tolerated procedure achieving clinical and immunological responses also in the presence of significant tumor burden. However, further strategies, following DC-vaccination, are needed to ensure durable immune and clinical responses.