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CD1 glycoproteins are a class of antigen presenting molecules that bind and present non-peptidic antigens (lipids and glycolipids) for immune recognition. CD1 polymorphisms, although limited, could have a critical role in antimicrobial, anticancer, and autoimmune responses and disease susceptibility. Ethnic differences and interactions between genetic and environmental factors make it attractive the study of these molecules in autoimmune inflammatory disorders, such as celiac disease (CD), in which a strong genetic predisposition (HLA-DQ2/DQ8) and pressure of environmental factors have a central role. CD1A, CD1D and CD1E polymorphisms in exon 2 were assessed in patients from Morocco affected by CD, using direct sequencing analysis, in order to investigate possible associations with the disease in a North African population. Differences in genotype and haplotype distribution of CD1E between celiac patients and controls were found: in particular, an increase of CD1E*02/02 homozygous (OR 2.93, CI 1.30-6.59, p = 0.007) and CD1A*02-E*02 estimated haplotypes in CD, compared with controls. Frequencies of CD1A and CD1D genotypes/alleles were not different between groups. CD1E*02/02, previously suggested as a potential immune protective genotype to malaria susceptibility, could be an additional gene involved in celiac risk in this geographic area.

Celiac disease (CD) is a chronic inflammatory enteropathy caused by gluten (protein from wheat, rye and, barley) in genetically predisposed individuals carrying the HLA-DQ2/HLA-DQ8 genotype. This pathology has a multifactorial etiology in which HLA genes, the microbiome, gluten and, other environmental factors are involved in the development of the disease. Its pathogenesis involves both innate and adaptive immunity as well as upregulation of IL-15. The objective of this review is to examine the results of current studies on genetic and environmental variables to better understand the pathogenesis of this enteropathy. The complex etiology of celiac disease makes our understanding of the pathogenesis of the disease incomplete, and a better knowledge of the many genetic and environmental components would help us better understand the pathophysiology of celiac disease.

Background Human leukocyte antigen (HLA)-G is a non-classical HLA molecule with immunomodulant and immunosuppressive functions, involved in transplantation tolerance. HLA-G14bp ins/del polymorphism in exon 8 has been associated with allograft rejection and kidney transplant outcome, with controversial results. We investigated associations of HLA-G14bp ins/del polymorphism on onset of some of the main post-transplant risk factors, like excess body weight, lipid abnormalities, increased fasting plasma glucose. Polymorphisms of cytokines with both immunosuppressive and metabolic effects were also assessed for comparisons and associated analysis. Methods The present study involved kidney transplant recipients (n = 173) in which body mass index, cholesterol, triglycerides, fasting plasma glucose were registered in the first years after transplantation and analyzed in association with genotypes. Presence of hypertension and smoking habits, demographic, transplant-related and therapeutic data of patients were also recorded. Polymerase chain reaction, sequence-specific primer amplification and Taqman allelic discrimination techniques were used for genotyping of HLA-G14bp ins/del, interleukin (IL)-10(−1082G > A,-819 T > C,–592A > C), transforming growth factor-β(+ 869 T > C,+915C > G), IL-6(−174G > C), tumor necrosis factor-α(−308G > A) and IL-18(−137G > C,-607C > A). Effects of genotypes on clinical markers at each time point (pre-transplant and 1 to 5 years after transplant) were analyzed using a repeated-measures general linear model analysis; adjustment for potential confounders was performed. Results Results showed that HLA-G14bp ins/ins was significantly associated with obesity, in particular after transplantation (3 years, p = 0.002, OR = 4.48, 95% CI:1.76–11.41). Post-transplant body mass index was significantly increased in HLA-G14bp ins/ins carriers (3 and 4 years, p = 0.033 and p = 0.044); effects of HLA-G14bp genotypes on post-transplant BMI were confirmed by using repeated-measures analysis and after controlling for confounding variables. Cytokine genotypes did not associate with the examined factors. Conclusions The study of transplanted patients allowed to evidence a potential relationship between post-transplant weight gain and HLA-G14bp ins/del polymorphism, previously involved in rejection for its immunosuppressive/tolerogenic activity. This novel association could widen the knowledge of the role and functions of HLA-G molecules in diseases and transplantation.

Tesi di laurea magistrale: POLIMORFISMI GENETICI ED APLOTIPI DELLA REGIONE 3'UTR DEL GENE HLA-G NEL TRAPIANTO DI RENE. La maggiore sfida per il paziente trapiantato è il mantenimento di una stabile funzione renale a lungo termine in assenza di reazioni di rigetto dell'organo, cercando di prevenire le principali complicanze (metaboliche e cardiovascolari, infezioni, neoplasie) associate alla terapia immunosoppressiva. L'insorgenza di disordini metabolici, quali diabete, dislipidemie ed obesità post trapianto (che costituiscono fattori di rischio per aumentata morbidità e mortalità nel lungo termine), oltre ad essere indotta dal trattamento farmacologico, può essere favorita da fattori genetici di predisposizione. Le conseguenze dell'insorgenza di alterazioni metaboliche sulla prognosi del trapianto di rene, a breve e lungo termine, sono oggetto di studio e di crescente interesse. Nella popolazione generale, circa il 40-70% della variabilità interindividuale dell'indice di massa corporea (BMI) è stato attribuito a fattori genetici e sono stati individuati numerosi geni che, con differenti meccanismi, influenzano i valori di BMI e lo sviluppo di obesità. Più recentemente, l'attenzione si è concentrata sull'associazione tra obesità, diabete ed infiammazione di basso grado, con l'individuazione di polimorfismi genetici di molecole coinvolte nell'immunità/infiammazione. Human leukocyte antigen (HLA)-G è una molecola HLA non classica con funzioni immunomodulanti ed immunosuppressive e coinvolta nei meccanismi di tolleranza nei trapianti d'organo. Il polimorfismo costituito da una inserzione/delezione di 14bp nell'esone 8, HLA-G14bpins/del, è uno dei polimorfismi più studiati in varie condizioni patologiche; nei trapianti d'organo è stato principalmente analizzato in associazione con l'insorgenza di complicanze immunologiche, quali rigetto ed infezioni post-trapianto. Fino ad ora, nel campo dei trapianti, l'interesse per questa molecola è stato prevalentemente di tipo immunologico, tuttavia alcuni studi ne stanno ipotizzando un coinvolgimento in altri ambiti fisiopatologici. Nel presente lavoro sperimentale è stata effettuata un'analisi preliminare dei polimorfismi della regione regolatrice 3'UTR del gene HLA-G, per valutarne eventuali associazioni genotipiche e/o aplotipiche con l'insorgenza di fattori di rischio post-trapianto, quali obesità, diabete e rigetto cronico. Lo studio è stato condotto presso l'Istituto di Farmacologia Traslazionale del Consiglio Nazionale delle Ricerche (CNR - IFT), sede dell'Aquila. L'analisi dei polimorfismi della regione 3'UTR dell'HLA-G è stata effettuata mediante sequenziamento diretto. I risultati hanno evidenziato associazioni significative per alcuni genotipi (+3142G/G) ed una differente distribuzione degli aplotipi della regione 3'UTR tra riceventi obesi e non obesi. I risultati confermano l'ipotesi di un ruolo funzionale di HLA-G nella regolazione metabolica, fino ad ora solo marginalmente evidenziata in altre condizioni patologiche, ed indicano l'opportunità di approfondire lo studio di nuovi meccanismi fisiopatologici della molecola HLA-G, di potenziale interesse in varie condizioni cliniche.

Il trapianto renale rappresenta la migliore opzione terapeutica per i pazienti con insufficienza renale cronica. Tuttavia, Per migliorare l'evoluzione del trapianto e la sopravvivenza del paziente è necessario il controllo delle alterazioni metaboliche e nutrizionali associate al trapianto stesso. L'aumento di peso dopo il trapianto è estremamente comune. Negli ultimi anni, attraverso studi di associazione genome-wide (Genome-Wide Association Study, GWAS) sono state identificate diverse varianti genetiche associate all'obesità e allo sviluppo di eventuali comorbidità. Scopo del seguente elaborato è stato quello di ricercare in letteratura eventuali lavori che abbiano analizzato, in pazienti sottoposti a trapianto di rene, varianti genetiche potenzialmente coinvolte nell'insorgenza di obesità e disturbi metabolici post trapianto. Poiché i polimorfismi a singolo nucleotide trasmettono effetti di piccola entità e i punteggi di rischio genetico hanno un valore predittivo relativamente basso, finora non soddisfano i criteri per diventare biomarcatori adatti per la medicina di precisione. Ad oggi, per prevenire l'insorgenza di obesità nei pazienti riceventi il trapianto di rene, vengono applicate delle strategie generali di management post-trapianto che prevedono cambiamenti comportamentali e dello stile di vita. Tuttavia, il trapianto può rappresentare un modello di studio e prevenzione del rischio cardiovascolare e degli effetti di componenti genetiche eventualmente coinvolte in tale rischio. Una buona gestione dei rischi su base genetica potrebbe indirizzare trattamenti personalizzati e migliorare, in futuro, l'outcome a lungo termine.

CD1 molecules are specialized in capturing and presenting lipids and glycolipids to distinct subsets of T and NKT cells. Glycolipid presentation could play a significant role in the immune response against microbial infections. There are five closely linked CD1 genes in humans, named CD1A, B, C, D, and E, which all show a limited polymorphism. In this study, exon 2 polymorphisms of CD1A, CD1D and CD1E were investigated and allele, genotype and haplotype frequencies of these loci were reported in a Moroccan population. A comparison with allele, genotype and haplotype frequencies observed in other geographic areas was also performed. Results confirmed the presence of ethnic differences in CD1 polymorphism, mainly in CD1D (in this population two additional CD1D variant alleles, CD1D ∗ 03 and CD1D ∗ 04, were described) and E genes. These data could be useful to evaluate a possible pathogenetic role of CD1 in diseases. Increasing the knowledge in this field may offer possibilities for the development of new immunotherapeutic approaches.

The fat mass and obesity-associated (FTO) gene is one of the most important obesity susceptibility genes. Some FTO gene polymorphisms have been associated with obesity, diabetes, and hypertension, all conditions for which, after transplant, there is increased susceptibility, due to effects of immunosuppressive regimens. To evaluate whether FTO could be a candidate for targeted preventive intervention in the transplant setting, we investigated whether the common genetic variation, FTO rs9939609T>A, could affect weight gain and risk of cardiovascular complications in kidney transplantation. Methods In 198 kidney transplant recipients, FTO rs9939609 was investigated in association with body mass index (BMI)/obesity and with other clinical markers of posttransplant risk, then monitored up to 5 years after transplantation. Genotyping was performed using an allelic discrimination method on a real-time polymerase chain (PCR) system. Associations were analyzed using the chi-square test; differences between genotypes were examined with analysis of variance or Kruskal-Wallis test; tests for repeated measures and a general linear model analysis controlling for age and gender were also utilized. Results Allele and genotype frequencies of FTO rs9939609 in recipients (T/T, 29.8%; T/A, 49.0%; A/A, 21.2%; A, 45.7%; T, 54.3%) reflect those present in healthy Caucasian populations. In the face of pre-/posttransplant differences in total cholesterol, triglycerides, or fasting glucose, results did not show significant changes in these factors among genotypes either before or after transplantation. Conclusion This study highlights a lack of association of FTO rs9939609T>A genotypes and posttransplant weight gain, plasma lipids, and fasting blood glucose in kidney transplantation.

A novel MICA allele, MICA*078, has been identified during HLA/MICA high resolution typing of Moroccan patients with celiac disease. MICA*078 shows an uncommon variation at a highly conserved nucleotide position (nt 493, G -> A), resulting in one amino acid change at codon 142 (V -> I) of MICA gene (compared to MICA*002:01), located in the alpha 2-domain, in which V142 is the common residue. (C) 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Genetic and environmental factors are responsible for differences in the prevalence of some diseases across countries. Human leukocyte antigen (HLA) allele frequencies in North African populations show some differences in their distribution compared to Europeans, Mediterraneans, and sub-Saharans, and some specific alleles and haplotypes could be clinically relevant. Celiac disease (CD) has been fast increasing in prevalence in North Africa; but few immunogenetic data are available for this area, in which a high prevalence of the disease has been described. In this report, we assess and discuss results of HLA class II (HLA-DQA1/DQB1/DRB1) typing in Moroccan patients with CD and compare them with a control population from Morocco--genetically well characterized--and with other North African, Mediterranean, and European populations. The classical HLA-DQ associations were confirmed in Moroccans with CD. The high frequency of DQ2.5 homozygosity (45.2%) found in Moroccans with CD was noteworthy as compared with other populations (23%-32%). The genetic risk gradient for CD, identified by previous studies, has been confirmed in Moroccans with some differences, mainly concerning DQ8 genotypes. This study provides the immunogenetic framework of CD in Moroccans and confirms the need to learn more about associations with additional HLA and non-HLA genetic factors.

La malattia di Alzheimer (Alzheimer's Disease, AD) è la più comune forma di demenza. Lo scopo del presente studio è stato verificare il valore predittivo del polimorfismo della gliossalasi 1 (rs1049346) nei confronti della malattia di Alzheimer nell'uomo, per la quale non sono presenti lavori in letteratura. È ormai noto ed accettato che la patogenesi della malattia di Alzheimer è in parte correlata al potenziamento del processo glicativo, coinvolto nello sviluppo dei principali biomarkers della patologia (placche senili, grovigli neurofibrillari e atrofia corticale). Inoltre, l'allele ?4 del gene APOE ha una elevata capacità di interazione con i prodotti finali (Advanced Glycation End products, AGEs). La glicazione può essere favorita sia da fattori esogeni (dieta) che endogeni (alterazioni degli equilibri con i sistemi di disintossicazione). Il metilgliossale, in particolare, è tra i più efficienti induttori della glicazione ed agisce direttamente sia sulle proteine (albumina del siero, collagene, emoglobina e proteine del cristallino) e sia sui nucleotidi (deossiguanosina). I residui di arginina, in particolare, sono i siti di elezione per l'interazione con le proteine e la successiva formazione degli idroimidazoloni metilgliossale-derivati (MG-derived hydroimidazolones, MG-Hs). Nel 90% dei casi, l'isoforma prodotta è la MG-H1. I livelli serici di MG-H1, pertanto, rappresentano un ottimo indicatore delle quantità di metilgliossale e dei livelli di glicazione presenti nell'organismo. La funzione di contenimento dei danni da un eccesso di metilgliossale è svolta dal sistema della gliossalasi, il quale interviene prima che vengano aggrediti i residui di arginina, prevenendo l'alterazione delle proteine e la formazione degli addotti. Il processo di disintossicazione coinvolge nell'ordine, 2 enzimi: la gliossalasi 1 e la gliossalasi 2. La gliossalasi 1 è un enzima glutatione-dipendente ed agisce sull'emitioacetale che quest'ultimo forma con il metilgliossale, restituendo il prodotto intermedio S-D-lattoilglutatione. Successivamente, la gliossalasi 2 catalizza l'ultima reazione, liberando il prodotto finale D-lattato ed fornendo nuovamente al sistema il glutatione necessario al ciclo successivo, configurando in tal modo un pathway autoalimentantesi. Questo sistema, tuttavia, può andare incontro ad un calo della funzionalità. Ad esempio, con il normale processo di invecchiamento, la quantità di glutatione disponibile diminuisce progressivamente, con conseguente rallentamento dell'attività della gliossalasi 1 ed innalzamento dei livelli di metilgliossale circolante. Da un punto di vista prettamente genetico, alcuni di polimorfismi della gliossalasi 1 sono associati a variazioni dell'attività enzimatica e all'efficienza della via stessa. In particolare, l'allele T per i polimorfismi rs1130534 e rs1049346 è significativamente correlato con il calo della funzionalità enzimatica. Muovendo da queste premesse, lo scopo della presente ricerca è stato lo studio delle distribuzioni alleliche e genotipiche del polimorfismo rs1049346 in pazienti con malattia di Alzheimer e verificare se questo possa essere considerato come un fattore di esposizione alla malattia. Secondariamente, data la relazione esistente tra livelli di metilgliossale, glicazione e neurodegenerazioni, sono stati analizzati i livelli serici di MG-H1.

Summary Two novel CD1D alleles were identified in unrelated individuals from Morocco. They differ each from the common CD1D*01 allele by one nucleotide substitution in exon 2 resulting in one amino acid change in the G-ALPHA1-LIKE domain. According to the IMGT unique numbering for G domain, CD1D*03 has one nucleotide transition c136 > t in codon 46, with an arginine-to-cysteine amino acid change (R46 > C) in the D-STRAND, whereas CD1D*04 has one transition c98 > t in codon 33, with a threonine-to-methionine amino acid change (T33 > M) in the C-STRAND. This suggests that CD1D is more polymorphic than previously assumed.

The MICA gene encodes a family of nonclassical major histocompatibility complex class I molecules. Data on MICA polymorphism in different populations are still limited. In the present study, MICA allele frequencies (af) were assessed in 82 unrelated healthy individuals from a Moroccan Berber population named Metalsa (ME) by means of sequence-based typing of exons 2, 3, 4, and 5. In consideration of the linkage disequilibrium existing between MICA and human leukocyte antigen (HLA) class I alleles, MICA/HLA-B, MICA/HLA-Cw, and MICA/HLA-A haplotype frequencies (hf) were estimated. A wide allelic distribution including 16 different MICA alleles was found in ME. The most common MICA alleles were MICA*00801 (af = 0.268), *004 (0.232), *00902 (0.140), *00901 (0.085), and *00901 (0.073). The most common MICA/HLA-B haplotypes were MICA*004-B*4403 and MICA*009-B*50 (hf = 0.113 for both these haplotypes). Some known MICA and HLA-B associations were confirmed in this population. Noteworthy was the high frequency of MICA*009 (af = 0.226); the high frequency of B*50 found in ME (af = 0.114) permitted us to evidence the associations of MICA*00902 with B*5001 (hf = 0.068) or *5002 (hf = 0.045), whereas MICA*00901 was mainly associated with B*5101 (hf = 0.038), which corresponds to the previously described association MICA*009/A6-HLA-B*51. This study extends the previous knowledge on MICA polymorphism to a North African white population and may have implications for disease associations and transplantation.

We describe an additional HLA-Cw*02 variant, HLA-Cw*0208, which has been identified in a renal transplant recipient of Caucasian origin (Italy). After performing preliminary serological typing, we analyzed exons 2 and 3 of the HLA-C locus polymorphism by cloning the amplified DNA and using a sequence-based typing method. The new allele differs from Cw*020202 by one nucleotide substitution at nucleotide 61 (G-->A) of exon 2, which translates to a difference of one amino acid at residue 21 (His-->Arg) of the HLA-C heavy chain. We propose that Cw*0208 was generated by a random point mutation in codon 21 from the Cw*020202 allele, or through gene conversion of Cw*020202 with another allele, probably the Cw*1205 and Cw*1602 alleles.

This report presents a novel allele, HLA-B*4427, which was identified in a bone marrow donor of Caucasian origin, and a confirmatory sequence (B*44022). Sequence analysis revealed the new allele differs from B*44021 by a single nucleotide exchange at position 668 (C-->T), which is located in exon 4. At the protein level, it is the only B*44 variant to produce an Ala in place of a Val at codon 199. Its structure suggests that it may have originated from a point mutation in B*44021 or by gene conversion with a variety of HLA-B alleles. Cloning and sequencing of the allele B*44022 revealed a sequence identical to B*44021 and B*44 exon 4, with the codon GTC (Val) in position 199.

In order to study the role of interleukin-1beta (IL-1beta) in homeostasis, hypoxia and recovery of neuronal cells, we studied the expression and release of tumor necrosis factor-alpha (TNF-alpha) and nerve growth factor (NGF), in relation to the presence or absence of this cytokine in culture medium. Moreover, we evaluated cell mortality in the same conditions. For this aim, we used untreated and IL-1beta pre-immunoneutralized hippocampal neuronal cultures exposed to mild hypoxic stress and left to reoxygenate. Semiquantitative reverse-transciptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) determined gene expression and protein levels. Mild hypoxic stress provokes a decrease in both the expression and release of TNF-alpha and NGF. IL-1beta neutralization results in an inversion of this pattern since treated hypoxic cultures exhibited an increase of both expression and release of NGF. In pretreated hypoxic cells the increased expression of TNF-alpha was not followed by a rise in release. Reoxygenation reversed the observed effects in both cultures and the levels of cytokine expression and release were approaching control values. Our data show that in physiological conditions IL-1beta may have a neuroprotective action through positive modulation of NGF. Contrary to that, in presence of insult, IL-1beta may have an opposite role, since neutralization provoked an increase of expression and release of NGF. In addition, we demonstrated that neuronal cells are biochemically capable, not only of maintaining and recovering the homeostasis, but also of activating the appropriate response to insult. IL-1beta may have a pivotal role in this mechanism through the modulation of NGF and to a lesser degree of TNF-alpha.

Severe combined immunodeficiency (SCID) represents a group of rare, sometimes fatal, congenital disorders in which there is a combined absence of T-lymphocyte and B-lymphocyte function. Children with SCID die within two years of age, if untreated. The effective treatment for SCID is a hematopoietic stem cell transplantation (HSCT). It has been repeatedly described that in peripheral blood of infants with SCID maternal T cells can be found. Here we report a case of blood chimerism in a one-year-old boy with SCID.

B*3812 is a novel allele identified in our laboratory during the typing procedure for hematopoietic cell transplantation purpose. The name B*3812 has been officially assigned by the WHO Nomenclature Committee in November 2005.

The most common monogenic form of obesity is caused by mutations in the gene encoding the melanocortin-4 receptor (MC4R). We have screened the MC4R coding sequence in 291 patients of a Dutch outpatient pediatric obesity clinic. We analyzed the minimal promoter region of the gene in a random subgroup of 217 children. Our aims were (i) to determine the frequency of MC4R mutations in a cohort of Dutch clinically obese children and (ii) to search for mutations in the promoter of the gene. Eleven MC4R coding variants were detected. Five children had mutations that have been shown to affect receptor function by other research groups (p.Y35X, p.I251fs, p.G231S). These children did not have earlier onset of obesity or higher BMI-SDS than the remainder of the cohort. One child had a novel nonsynonymous coding mutation (p.L304F). This variant showed a markedly decreased cell surface expression in in vitro experiments and is thus expected to be pathogenic. We detected 12 variants in the MC4R flanking regions. Five of these were not previously described (c.-1101C>T, c.-705A>T, c.-461A>G, c.-312T>C, c.-213A>G). We investigated these mutations by family studies and a bioinformatic approach. We conclude that rare heterozygous mutations in the coding sequence of MC4R account for some severe obesity cases in the Dutch population. These patients are difficult to recognize in a clinical setting. We generated a list of all MC4R variants that were described in the literature so far, which can aid the interpretation of mutations found in a diagnostic setting.

The aim of this study was to provide genetic and anthropological information on the Chaouya (CH), an Arabic-speaking population living in West Morocco, Atlantic coast (Settat). In 98 unrelated healthy CH volunteers, we first investigated the human leukocyte antigen (HLA) class I and II allele polymorphisms using a sequence-based typing method and examined haplotypes and relatedness of this group to other African and Mediterranean populations. The study showed the close relatedness with Tunisian population and other North Africans, together with a strong influence of various immigrations, mainly Spaniards, French, and Portuguese, as expected. Nevertheless, analysis of class II allele frequencies (afs) showed that Oromo and Amhara Ethiopian groups cluster together with the Berbers and other North Africans, confirming the relationship between these populations (Afro-Asiatic linguistic group, Hamites). South and sub-Saharan Africans cluster separately at a great distance from CH, except the sub-Saharan Bantu population from Congo Kinshasa, which shows a relatively close genetic relationship ascribable to the effect of a diversifying selection. On the other hand, considering HLA class I afs analyses, it was noteworthy that CH grouped together with sub-Saharans, showing a close genetic distance mainly with Ugandas and Kenians Luo.

Acknowledgments: This research was supported in part by funding from the C.N.R., P.F. “Biotecnologie”. We wish to acknowledge and thank Dr. Maria Teresa Vicentini, Dr. Lorena Pompucci and Dr. Anna Aureli for their support with HLA typing. Abstract: This communication describes a new DRB1*11 allele identified in a familial group of Caucasian individuals from central Italy. The new sequence has been officially named DRB1*11272 by the World Health Organization (WHO) Nomenclature Committee (February 2000). It encodes an HLA antigen serologically recognized as DR11. The sequence variation of this new allele was localized to codon 77 of exon 2. It differs at position 230 from DRB1*11011 allele (A replacing C), and at position 231 from DRB1*1127 (C replacing T). At the amino acid level, the DRB1*11272 mutation is silent with respect to the DRB*1127 phenotype, coding for a synonymous asparagine.

Alterations in the immune response play an important role in the pathogenesis of atopic dermatitis (AD). We evaluated the role of Th1/Th2 cytokine imbalance in paediatric AD and its progression towards other allergic manifestations. The levels of total interleukin (IL)-12 (p70+p40), IL-12 p70 and soluble CD30 (sCD30) were measured in paediatric patients affected by AD and in age-matched, non-atopic subjects (controls). The serum levels of total IL-12 and sCD30 were higher in patients than in controls (p=0.003 and p=0.053, respectively). Total IL-12 and sCD30 were also particularly increased in patients with severe disease. Serum levels of total IL-12 were negatively correlated with patient age (p=0.001): they were significantly higher in patients younger than 30 months, as compared to age-matched controls and to older patients and controls. Total IL-12 and sCD30 production were not significantly correlated with disease outcomes (atopic march) or with a family history of atopy. Our data show that total IL-12 levels are strongly associated with Th2 activation and severe disease in children with AD. The increased production of total IL-12 at an early age might indicate a different immune modulation and suggests the possibility of new therapeutic approaches for allergic diseases, in particular AD, early in childhood.

The high prevalence of nasopharyngeal cancer (NPC) in Southern Asia and Mediterranean Northern Africa suggests genetic predisposition among other factors. While Human Leukocyte Antigen (HLA) haplotypes have been conclusively associated with NPC predisposition in Asians, Northern African Maghrebians have been less intensely studied. However, low resolution serological methods identified weak positive associations with HLA-B5, B13 and B18 and a negative with HLA-B14. Using sequence based typing (SBT), we performed a direct comparison of HLA class I frequencies in a cohort of 136 Tunisian patients with NPC matched for gender, age and geographical residence to 148 normal Tunisians. The bimodal age distribution of NPC in Maghrebians was also taken into account. HLA frequencies in normal Tunisians were also compared with those of Northern Moroccan Berbers (ME) to evaluate whether the Tunisian population in this study could be considered representative of other Maghrebian populations. HLA-B14 and -Cw08 were negatively associated with NPC (odd ratio = 0.09 and 0.18 respectively, Fisher p2-value = 0.0001 and = 0.003). Moreover, positive associations were observed for HLA-B-18, -B51 (split of -B5) and -B57 (p2-value < 0.025 in all) confirming previous findings in Maghrebs. The HLA-B14/Cw*08 haplotype frequency (HF) was 0.007 in NPC patients compared to 0.057 in both Tunisian (OR = 0.12; p2-value = 0.001) and Moroccan controls. This study confirms several previous associations noted by serologic typing between HLA class I alleles and the prevalence of NPC in Maghrebians populations. In addition, we identified a putative haplotype rare in Tunisian patients with NPC that may serve as a genetic marker for further susceptibility studies.

B*3812 is a novel allele identified in our laboratory during the typing procedure for hematopoietic cell transplantation purpose. The name B*3812 has been officially assigned by the WHO Nomenclature Committee in November 2005.

A novel HLA-A*02 allele was detected in a Caucasian patient from central Italy, requiring a hematopoietic cell transplantation. Direct sequencing identified a variation in one nucleotide position, which was confirmed by cloning. The name A*027401 was officially assigned by the WHO Nomenclature Committee in November 2004 (AY796418, AY796419, AY796420, and HWS10002684). A*027401 differs from A*02010101 by a single G to A substitution at nucleotide position 595 in exon 3. The new variant would lead to a nonsynonymous nucleotide change (GGG to AGG) at codon 175, resulting in a basic Arg in the α-helix of the α2-domain, in place of a non-polar Gly. The presence of an uncommon variation at a highly conserved nucleotide position could have implications in unrelated hematopoietic cell transplantation.

Class I human leukocyte antigen (HLA) polymorphism was examined in a Berber population from North Morocco, named Metalsa (ME). All data were obtained at high-resolution level, using sequence-based typing. The most frequent alleles were: HLA-A*0201 and A*0101; HLA-B*44 (B*4403 and B*4402); B*0801 and the B*50 allele group (B*5001 and B*5002); HLA-Cw*0602; and Cw*07 group (Cw*070101, Cw*070102, Cw*0702, Cw*0704, and Cw*0706), and Cw*040101. The novel HLA-B*570302 allele was identified. It differs at position 486 and position 855 from B*570301, resulting in synonymous Thr and Val. The analysis also evidenced some alleles common in Africans (A*3402, A*6802, A*7401, B*1503, B*4102, B*4202, B*7801, B*5802, Cw*1701, and Cw*1703) and some uncommon alleles (A*3004, B*2702, B*2703, B*5001,02, B*3503, and Cw*0706). The predominant HLA-A-Cw-B-DRB1-extended haplotypes in ME population were A*0101-Cw*0501-B*4402-DRB1*0402, A*240201-Cw*0701-B*0801-DRB1*030101, A*2301-Cw*040101-B*4403-DRB1*040501, A*0201-Cw*040101-B*4403-DRB1*1302, and A*3002-Cw*0602-B*5002-DRB1*0406. This study demonstrates a strong relatedness of ME to other Moroccan and North African populations, some characteristics of sub-Saharan Africans and evidenced the influence of various immigrations during centuries. Nevertheless, this study highlights some unique genetic traits of the ME population compared to other ethnic groups within Morocco, which could be of great interest for clinical aims, transplantation, and diseases.

We analysed the specific effects of IL-1beta immunoneutralization on the expression of IL-6 in different pure cultures of neurones and glia after both experimental subliminal hypoxia and recovery. Whereas the IL-1beta-deprivation signal induced a decrease in IL-6 expression and release of normoxic neurones, it provoked an increase in IL-6 protein in hypoxic neurones. Moreover, the direct correlation between IL-1beta and IL-6, observed in normal and recovering neuronal cultures, was reversed in hypoxic conditions. These reversals were not observed in glial cells, in which IL-1beta immunosuppression led to a decrease in IL-6 under all conditions considered. In conclusion, the IL-1beta modulates IL-6 in different ways according to the ambient physiological or pathological conditions, and also acts via different mechanisms, depending on the cellular phenotype.

To examine the genetic diversity in Morocco, the polymorphism at the HLA-DRB1 locus was investigated in two populations: the Metalsa group consisting of Berbers from north Morocco (who speak the Tarifit language and live in the Nador area), and the Chaouya group who are Arabic-speaking people from west Morocco (Atlantic coast) living in the Settat area. The DRB1 alleles of 197 healthy unrelated individuals were identified by direct DNA sequencing of exon 2 using fluorescently-labeled primers. A total of 28 and 29 alleles at DRB1 locus were identified in the Metalsa and Chaouya groups, respectively. The most frequent alleles in the Metalsa group are DRB1∗03011 (20.2%), DRB1∗0701 (12.12%), and DRB1∗1302 (11.11%). In the Chaouya group, DRB1∗0701 (16.33%), DRB1∗15011 (12.76%), and DRB1∗03011 (11.73%) are most common. Each population exhibits some specific variants and some uncommon alleles. The frequency of the DRB1∗03011 allele differs significantly between the two populations ( p = 0.0311). The DRB1 frequency distributions in the two groups suggest the effects of balancing selection. The interpopulation analysis highlighted a strong relatedness, based on genetic distances, between the two Moroccan groups and the other north Africans (the Moroccans from El Jadida area, Moroccan Souss Berbers, Algerians, and Tunisians), and to a lesser extent with the Iberians, French, and Ethiopians.

The expression of cytokine mRNAs in tumor tissue, normal mucosa, and peripheral blood mononuclear cells (PBMCs) was studied in 12 patients with colorectal cancer undergoing surgical resection, to characterize local immune conditions. mRNA transcripts for interleukin (IL)-1 beta, IL-2, IL-2-R(p55), IL-4, IL-5, IL-6, and IL-10 were detected using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. IL-6 mRNA was expressed in tumor tissue in 83% of the cases but only in one case in normal mucosa (p < 0.001); serum levels of IL-6 did not show any correlation with IL-6 mRNA; IL-1 beta transcripts were present in all tumor tissue samples; no IL-4 expression was detected; IL-2 mRNA was only present in two tumors; IL-2R(p55) mRNA was found in 58% of tumors but not in normal mucosae (p = 0.005). The expression of IL-10 suggests that it does not play a central role in colorectal cancer immunosuppression, and cytokine expression in PBMCs indicates a different and independent activation. This study suggests a pattern of expression of inflammatory cytokines in the tumor microenvironment, probably produced by infiltrating immune cells. The absence of the specific immune-activating cytokines, IL-2 and IL-4, could indicate an impairment of the anticancer immune response; IL-2R results confirm the dysregulation of the IL-2/IL-2R activation pathway. These findings may lead to a better understanding of the role of cytokines and especially IL-6 at the tumor site and hence their importance in developing an effective immunotherapy.

The mechanisms for the recruitment of immune cells to sites of inflammation have not been fully elucidated. In order to understand the mechanisms of IL-8 a potent chemotactic and activating chemokine, during immune response, we examined in colon cancer patients and controls, serum levels of IL-8, IL7, IL-6, IL-4, TNF-α, IFN-γ IL-2, sIL-2R: IL-8 production in supernatants of PBMC with and without activation agents: PBMC phenotype (CD3, CD4, CD8, CD16, CD56, CD57, CD25): PBMC proliferative responses to IL-2, IL-4, anti-CD3. Our results show that IL-8 serum levels in patients (p) were higher than in controls (c) (n.p = 65, n.c = 51 P = 0.007) and this was significant only at stage II (n.p = 25 n.c = 51 P = 0.01) and III (n.p = 8 n.c = 51 P = 0.03). It is of great interest to note that our preliminary data of IL-8 production from: PBMC (n.p = 14 n.c = 4 P = 0.0002), plus PHA (n.p = 15 n.c. = 3 P = 0.008), plus anti-CD3 (n.p = 3 n.c = 3 P = 0.03) show that levels in patients are significantly lower than in controls. Correlation between IL-8 serum levels and other serum cyokines evidenced no significant values, but at stage I (n.p = 9 P

Immunological evaluations on cancer patients provide informations to understand the disregulations which allow the tumor to locate and progress in the host. In colon cancer patients and controls we examined the serum levels of various cytokines and the proliferative response of PBMC to IL-2, IL-4, anti-CD3mAb. Our results show that in patients there was a significant serum increase of IFNγ, IL-4, IL-6, whereas IL-2 significantly decreases. This situation is indicative for the functional presence of inflammatory CD4 + TH2 cells. In patients it is possible to note a negative correlation between the proliferative response to IL-2 and stage of the disease ( * P = 0.0024), which changes into positive when anti-CD3 is added ( * P = 0.007) (indicative for a functional increase of CD4 + TH2 cells with the disease progression). Moreover in patients the proliferative response to IL-2 + anti-CD3 was comparable to controls (§ P = 0.42), whereas in the former there wasa significant reduction after IL-4 addition (§ P = 0.005). This effect of IL-4 on IL-2 + anti-CD3 starts at stage II, whereas at stage I the response is still comparable to controls and there is a greater response to IL-2 alone (§ P = 0.0035). Analysing our data, it seems that the start of the immunological response in patients is of inflammatory type and it degenerates because IL-4, which raises with the disease progression, interferes with IL-2 mechanisms. Serum levels patients compared to controls * stage correlations § IFNγ n = 73 P = 0.003 n = 43 n = 63 r = 0.19 P = 0.14 § IL-4 n = 24 P n = 33 n = 24 r = 0.89 P ° IL-6 n = 56 P = 0.0001 n = 33 n = 55 r = 0.21 P = 0.12 ° TNFα n = 56 P = 0.1 n = 33 n = 51 r = 0.35 P = 0.014 § IL-2 n = 42 P n = 27 n = 41 r = −0.56 p = 0.0004 * Spearman rank correlations § Student's t-test ° Mann-Whitney test

The current research has still not clarified why serum levels of sIL-2R increase in cancer patients. In order to establish whether this could be an independent event or there are correlated mechanisms, in colon cancer patients and controls we examined the level of this soluble receptor in correlation with phenotype of PBMC; serum levels of IL-6, TNF-α, IL-4, IFN-γ, IL-2; changes of HLA-I and HLAII antigen expression on tumor tissue respect to normal mucosa; PBMC proliferative response to IL-2, IL-4, anti-CD3. Correlations with the disease progression were evaluated. Our results show that sIL-2R level of patients (p) was higher than in controls (c) (n.p = 72, n.c = 79 P P = 0.0001), IFN-γ (n.p = 73, n.c = 43 p = 0.003), IL-4 (n.p = 24, n.c = 33 P P P = 0.033). Moreover, from the evaluation of HLA antigens it is possible to note that when the expression of HLAI is the same in tumor tissue and normal mucosa (situation more often present at stage I: 66.7%), sIL-2R of patients was higher than controls (P = 0.018). With regard to the correlations with the proliferative response, merely a positive association with IL-2 plus anti-CD3 was determined (n.p = 29 P = 0.013). So, from our overall results, as IL-4 is produced by CD4 + TH2 cells and the anti-CD3 addition to IL-2 is well-known to be discriminant for the activation of these cells, it seems that the increase of sIL-2R in serum of cancer patients is an event dependent on CD4 + TH2 cells. This hypothesis is confirmed by the fact that when HLAI antigen expression was lower than normal, the HLAII antigens was higher, supporting an inflammatory situation.