An immune-molecular hypothesis supporting infectious aetiopathogenesis of Kawasaki disease in children.

The competitive binding between CpG-ODN (single-stranded DNA from pathogens) and HLA-B and HLA-A ligands for the inhibitory Killer Immunoglobulin-like Receptors (KIR)3DL1/2 may lead to possible hypo-sensing of pathogens and ineffective clearance. We observed an overabundance of HLA ligands for inhibitory KIR with three domains in KD subjects.

Decreased interferon-γ production by NK cells from KIR haplotype B carriers in hepatitis C virus infection.

BACKGROUND AND AIMS: Different population genetics studies showed that interactions between killer-cell immunoglobulin-like receptors (KIR) and HLA play a role in viral disease outcome, but functional correlates are missing. Building upon our previous work pointing to a regulatory role for KIR3DL1/DS1 in hepatitis C virus (HCV) infection, we analysed whether its expression may affect natural killer (NK) cell function in the presence or absence of its principal ligand HLA-Bw4 in KIR haplotype A and B carriers, which are characterized by a different representation of activating and inhibitory KIRs. METHODS: We performed KIR and HLA class I genotypic analysis in 54 healthy donors (HD) and 50 HCV+ subjects and examined NK cell cytokine secretion and degranulation in the context of KIR3DL1-HLA-Bw4 match stratified by KIR haplotype. RESULTS: KIR3DL1-HLA-Bw4 match induced functional NK cell modulation, reflected by reduced interferon (IFN)γ production in haplotype B HCV+ patients compared to HD. This functional impairment could be ascribed to the KIR3DS1 negative HCV-infected patient population, whose NK cells also showed a significantly decreased proportion of KIR3DL1. Haplotype A HCV-infected patients showed increased NK cell degranulation compared with HD in the absence of KIR-HLA-Bw4 match and this activity was associated with increased phosphorylation of signal transducer and activator of transcription (STAT) 1. CONCLUSIONS: Our data show that NK cells from HCV+ patients have an unbalanced ability to produce IFNγ and to kill target cells in haplotype A and B carriers, suggesting the existence of complex functional differences governed by KIR-HLA interaction, particularly on KIR3DL1 expressing NK cells.

HLA-DQ genetics in children with celiac disease: a meta-analysis suggesting a two-step genetic screening procedure starting with HLA-DQ ß chains.

BackgroundSpecific HLA-DQ genes have been recognized as necessary - but not sufficient - factors for the occurrence of Celiac Disease (CD). Through a meta-analysis, evaluating the distribution of CD-related HLA genotypes in children, we aimed at providing insights for a potential widened screening strategy.MethodsAfter a systematic search on the association between class II HLA genes and CD in children, 46 publications were obtained and assessed for eligibility. A total of 13 eligible studies were submitted to data extraction and analysis (10 case-control studies and 3 cohort studies). Case-control studies collectively enrolled 740 CD patients and 943 controls.ResultsIn the population-stratified analysis, the following alleles conferred a significantly increased risk for CD: HLA-DQB1*02 (odds ratio [OR]=10.28) and HLA-DQB1*03:02 (OR=2.24). By drafting a risk gradient to develop CD according to HLA genetic background, the highest risk is confirmed to exist for DQ2/DQ2 homozygous subjects, regardless of the ethnicities (OR=5.4). Actually, the genotype DQ2/ß2 showed basically the same risk (OR=5.3). Indeed, no differences have been found in CD risk between DQ2/ß2 and DQ2/DQ2, as well as between DQ8/ß2 and DQ2/DQ8, and between ß2/DQX and DQ2/X.ConclusionThe HLA-DQB1*02:01 allele is present in more than 90% CD children. In the perspective of a widened pediatric population screening for CD, a double-step process might be suggested: HLA-DQB1*02:01 might be investigated first and, only if this result is positive, children might be candidate for a prospective serologic screening, as a second step.

SJS/TEN overlap associated with lomefloxacin: case report and molecular typing studies.

BACKGROUND: Stevens-Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN) may develop in susceptible patients after administration of different drugs. Only mild cutaneous reactions have been related to lomefloxacin. A correlation between human leucocyte antigen (HLA) and cutaneous adverse reaction has been identified. CASE REPORT: Twenty-four hours after intake of lomefloxacin, a 30-year-old Caucasian woman developed a severe skin reaction with symptoms suggesting SJS/TEN. The fast onset reaction worsened with skin blisters and 20% body surface area skin detachment within 48 h. Burn unit admittance was required; corticosteroids and human immunoglobulins were administered. Complete recovery occurred within 3 months, except for epidermal discoloration. Molecular studies showed a peculiar profile characterized by HLA class I genotype rich of ligands for natural killer cell immunoglobulin-like receptors (KIR) and HLA class II haplotype, HLA-DRB1*03:01,DQB1*02:01, prone to autoimmunity. CONCLUSION: While the HLA profile approaches our case to other well-documented drug-induced SJS/TEN, KIR involvement still remains puzzling.

DQA1*0102 DQB1*0602 haplotype distinguishes coeliac disease and its complications from gluten unrelated enteropathies.

BACKGROUND: Duodenal villous atrophy is due not only to coeliac disease and its complications but also to other rare enteropathies unrelated to gluten consumption, defined as noncoeliac enteropathies. The diagnosis of noncoeliac enteropathies remains challenging, and HLA typing has been widely used to exclude coeliac disease if DQ2 and DQ8 alleles are absent. However, the frequency of the various HLA alleles in noncoeliac enteropathies is still unknown. AIMS: To describe the HLA genetic profile of patients affected by noncoeliac enteropathies who have been evaluated at our centres between 2000 and 2021, and to investigate the diagnostic role of HLA typing. METHODS: Genomic DNA was collected from 44 Italian and 19 British adult patients with noncoeliac enteropathies. Patient genotypes were compared with those of healthy Italian and British populations obtained from HLA bone marrow donors' banks. In addition, genotypes were also compared with those of patients with coeliac disease and complicated coeliac disease. RESULTS: Both in the Italian and in the British group, the DQA1*0102 DQB1*0602 haplotype and related alleles occurred significantly more frequently in patients with noncoeliac enteropathies compared to coeliac disease and complicated coeliac disease. CONCLUSIONS: Together with negative HLA-DQ2 and DQ8 haplotypes, the DQA1*0102 DQB1*0602 haplotype can be used to guide the differential diagnosis between coeliac disease and noncoeliac enteropathies.

In vitro evaluation of graft-versus-graft alloreactivity as a tool to identify the predominant cord blood unit before double cord blood transplantation.

The transplantation of two cord blood (CB) units obtained from unrelated donors (double CBT) is an effective strategy for adult patients with hematologic malignancies. Sustained hematopoiesis after double CBT is usually derived from a single donor, and only a few transplantation recipients displaying a stable mixed donor-donor chimerism have been reported. We investigated the mechanisms underlying single-donor predominance in double CBT by studying in vitro the role of the graft-versus-graft cell-mediated immune effect in two-way mixed-lymphocyte culture, along with the contribution of differential hematopoietic progenitor (HP) potency in HP mixed cultures. Results for the two-way mixed-lymphocyte culture showed that despite the weak and variable alloantigen-specific cytotoxic potential displayed by CB mononuclear cells, an immune-mediated dominance for one of the two CB units was detected in the majority of experiments. Alloantigen-induced cytotoxic activity was directed toward both CB-HP and phytohemagglutinin (PHA)-activated T lymphoblastoid cells. The CB unit with the higher fold expansion of CD34(+) cells in single-expansion culture was prevalent in the HP mixed-expansion culture, as shown by DNA chimerism evaluation. Based on these data, we hypothesize that the dominant CB unit is able to develop prevalent cytotoxic activity toward activated lymphocytes of the other CB unit, thereby preventing them from exerting alloantigen-specific cytotoxic potential against both activated lymphocytes and HPs of the dominant unit. In accordance with this hypothesis, we propose the evaluation of alloantigen-induced cytotoxic activity generated in two-way mixed-lymphocyte culture and directed toward PHA-activated T lymphoblastoid cells as a tool to identify the potentially predominant CB unit before double CBT.

Cytokine-Induced Memory-Like NK Cells with High Reactivity against Acute Leukemia Blasts and Solid Tumor Cells Suitable for Adoptive Immunotherapy Approaches.

The limited efficacy of Natural Killer (NK) cell-based immunotherapy results in part from the suboptimal expansion and persistence of the infused cells. Recent reports suggest that the generation of NK cells with memory-like properties upon in vitro activation with defined cytokines might be an effective way of ensuring long-lasting NK cell function in vivo. Here, we demonstrate that activation with IL-12, IL-15 and IL-18 followed by a one-week culture with optimal doses of Interleukin (IL-2) and IL-15 generates substantial numbers of memory-like NK cells able to persist for at least three weeks when injected into NOD scid gamma (NSG) mice. This approach induces haploidentical donor-derived memory-like NK cells that are highly lytic against patients' myeloid or lymphoid leukemia blasts, independent of the presence of alloreactive cell populations in the donor and with negligible reactivity against patients' non-malignant cells. Memory-like NK cells able to lyse autologous tumor cells can also be generated from patients with solid malignancies. The anti-tumor activity of allogenic and autologous memory-like NK cells is significantly greater than that displayed by NK cells stimulated overnight with IL-2, supporting their potential therapeutic value both in patients affected by high-risk acute leukemia after haploidentical hematopoietic stem cell transplantation and in patients with advanced solid malignancies.

The HLA alleles DRB1*13 and DQB1*06 are associated to Whipple's disease.

BACKGROUND & AIMS: Whipple's disease is a systemic, chronic, relapsing disorder caused by a combination of environmental (Tropheryma whipplei) and unknown host factors. Because it is a rare disease, the association between HLA type and Whipple's disease has been studied in only small numbers of patients; these studies have led to conflicting results. We aimed to investigate whether disease phenotype and outcome are associated with HLA type in 122 patients with Whipple's disease. METHODS: Genomic DNA was collected from 103 German, 11 Italian, and 8 Austrian patients with Whipple's disease, along with 62 healthy Austrian workers exposed to T whipplei (14 stool samples contained the bacterium). HLA class I and II alleles were identified by polymerase chain reaction analysis. Patient genotypes were compared with those of healthy German and Austrian populations; data for Italian controls were obtained from the Pavia HLA bone marrow donors' bank. RESULTS: HLA-DRB1*13 and DQB1*06 alleles occurred significantly more frequently in patients with Whipple's disease but not in healthy individuals who had been exposed to T Whipplei. The cumulative odds ratios for disease were 2.23 for the DRB1*13 allele (P < .0001) and 2.25 for the DQB1*06 allele (P < .0001). CONCLUSIONS: DRB1*13 and DQB1*06 alleles were found to be risk factors in the largest HLA study ever performed in patients with Whipple's disease.

Reduced Efficacy of Biological Drugs in Psoriatic Patients with HLA-A Bw4-80I KIR Ligands.

BACKGROUND: Biological drugs (biologics) are a highly effective therapy for the moderate to severe form of psoriasis, an immune-mediated dermatosis with a strong immunogenetic component. The interaction between human leukocyte antigen (HLA) class I ligands and killer immunoglobulin-like receptors (KIR) has a functional significance in the education of natural killer (NK) cells, and can thus influence the response to biologics. OBJECTIVE: In this study, we investigated the impact of HLA-A and -B KIR ligands in the response to biologics in a cohort of psoriatic patients. METHODS: Eighty-five patients with moderate to severe psoriasis treated with biologics (adalimumab, etanercept, infliximab, ustekinumab and secukinumab) were enrolled in the study. Clinical response was evaluated as patients attaining 50%, 75% or 90% reduction in the Psoriasis Area and Severity Index (PASI) (PASI 50, 75 or 90, respectively) over 6 months' follow-up. Poor response was defined as PASI 50, and in this case patients shifted to treatment with a different biologic. Fifty-two patients (61.2%) showed excellent response (PASI 90) to the first biologic, while 33 patients (38.8%), needed two or more biologics before reaching an excellent response (PASI 90) and were considered difficult to treat. RESULTS: Only HLA-A Bw4-80I ligands were associated with the response to biologics; in particular, they were linked with reduced response both at univariable analysis (odds ratio [OR] 3.11, 95% confidence interval [CI] 1.19-8.07; p = 0.019) and multivariable analysis (OR 5.02, 95% CI 1.40-17.97; p = 0.013). CONCLUSION: We suggest that the HLA-A Bw4-80I epitope could be a marker of reduced responsiveness to biologics. The possible reason for this is an increase of tumour necrosis factor (TNF)-α and the silencing of NK cells through the predominant interaction with the KIR3DL/S pair. HLA-KIR affinities might lead to a more efficient way to prescribe biologics.

Comparative Study of Salivary, Duodenal, and Fecal Microbiota Composition Across Adult Celiac Disease.

BACKGROUND: Growing evidence suggests that an altered microbiota composition contributes to the pathogenesis and clinical features in celiac disease (CD). We performed a comparative analysis of the gut microbiota in adulthood CD to evaluate whether: (i) dysbiosis anticipates mucosal lesions, (ii) gluten-free diet restores eubiosis, (iii) refractory CD has a peculiar microbial signature, and (iv) salivary and fecal communities overlap the mucosal one. METHODS: This is a cross-sectional study where a total of 52 CD patients, including 13 active CD, 29 treated CD, 4 refractory CD, and 6 potential CD, were enrolled in a tertiary center together with 31 controls. A 16S rRNA-based amplicon metagenomics approach was applied to determine the microbiota structure and composition of salivary, duodenal mucosa, and stool samples, followed by appropriate bioinformatic analyses. RESULTS: A reduction of both α- and ß-diversity in CD, already evident in the potential form and achieving nadir in refractory CD, was evident. Taxonomically, mucosa displayed a significant abundance of Proteobacteria and an expansion of Neisseria, especially in active patients, while treated celiacs showed an intermediate profile between active disease and controls. The saliva community mirrored the mucosal one better than stool. CONCLUSION: Expansion of pathobiontic species anticipates villous atrophy and achieves the maximal divergence from controls in refractory CD. Gluten-free diet results in incomplete recovery. The overlapping results between mucosal and salivary samples indicate the use of saliva as a diagnostic fluid.

[Genotyping in patients affected by HLA-related diseases. App development for diagnostic support.] / Tipizzazione genetica nelle patologie HLA-correlate. Sviluppo di una app per il supporto diagnostico.

HLA typing requests for association studies of immune-mediated diseases are often redundant and inadequate. We designed a series of meta-analyses to evaluate the accuracy of typing and distribution of HLA alleles predisposing to diseases, aiming at developing an app that can help doctors in choosing the most suitable molecular analysis. The first study was on celiac disease (CD) and HLA-DQ in children. We searched all english articles published in the main bibliographic databases up to May 2016. The search strategy has been developed using controlled terms (e.g. MeSH) and free terms. We identified 1885 articles. 1334 abstracts were examined. 46 manuscripts were evaluated, and 13 studies were included in the meta-analysis (740 CD and 943 controls). The risk of developing CD in children with allelic variants encoding the HLA-DQ2.5 and/or HLA-DQ8 molecules has been confirmed. The greatest CD risk resides in carriers of two DQ2.5 molecules, i.e. subjects homozygous for the DQB1*02:01 and DQA1*05 alleles (OR=5.4, 95 % CI=4.1-6.8) compared to any other DQ genotype. Carriers of two DQB1*02:01 (chain ß2) alleles and one DQA1*05 (chain α5) allele have the same risk (p=0.8089) of DQ2.5 homozygotes (OR=5.3%, 95 CI=4,1 to 6.5). We found no differences between DQ8/ß2 and DQ2.5/DQ8, nor between ß2/DQX and DQ2.5/X. We suggest a two-step process: first typing the DQB1*02:01 allele and, in case of a negative result, full typing of HLA-DQ.

Luminex® xMAP® technology is an effective strategy for high-definition human leukocyte antigen typing of cord blood units prior to listing.

INTRODUCTION: Allele-level donor-recipient match at HLA-A, HLA-B, HLA-C and HLA-DRB1 loci impacts the outcome after cord blood transplantation for hematologic malignancies and modifies the strategy of donor selection. High definition of both class I and II HLA loci at time of listing is a way to improve the attractiveness of cord blood bank inventories, reducing the time for donor search and procurement and simplifying donor choice, in particular, for patients of non-European heritage. METHODS: In 2014, Luminex® xMAP® technology was introduced in our laboratory practice and was applied to cord blood units typing. In this study, we evaluated the impact of this strategy in comparison with the platform in use until 2013, relying on LiPA reverse polymerase chain reaction-sequence-specific oligonucleotide (revPCR-SSO) plus polymerase chain reaction-sequence-specific primer (PCR-SSP). RESULTS: In 2014, the time for testing was shorter (141 vs 181 days on average), the number of test repetitions was lower (in particular for HLA-A locus, p = 0.026), and the cost reduced (240.7 vs 395.6 euros per unit on average) compared to 2013, demonstrating that Luminex xMAP technology is superior to the previous approach. CONCLUSION: Luminex xMAP platform has useful application in cord blood banking programs, to achieve high-definition HLA typing of cord blood units at the time of banking in a quick, accurate, and cost-effective manner.

Pro-inflammatory variants of DRB1 and RAGE genes are associated with susceptibility to pediatric type 1 diabetes: a new hypothesis on the adaptive role of autoimmunity.

Functional polymorphisms of two MHC genes (DRB1 and RAGE) were analysed in Italian pediatric patients with Type 1 diabetes and in a control group. The diabetic condition is related positively to the positive electric charge of the pocket 4 of pro-inflammatory DRB1 alleles (R = 0.5072, P = 0.0001) and negatively to at least one anti-inflammatory RAGE allele (R = -0.2200, P = 0.0106). The association DRB1-disease decreases from high risk positively charged alleles to low risk negatively ones. A multiple regression model including the effect of electric charges at positions 70 and 74 of the DRB1 explains more than 31% of the variability of our data. The addition of the RAGE dependent variables does not increase the significance of the model. Our results confirm that the interaction between a negatively charged amino acid of insulin autoantigenic peptides and a positively charged DRB1 is the key event triggering the autoimmune process. The linkage disequilibrium between RAGE and DRB1 is the main cause of the association between the variants of RAGE and the initial outcome of the disease. However, since RAGE ligands increase during the disease progression, the observed association suggests that the proinflammatory RAGE and DRB1 polymorphisms synergize to activate the immune response which leads to the complications of diabetes. These evidences support a new hypothesis that considers the largely unexplored role of the MHC genes in genetic adaptation to a variable environment and in the maintenance of the metabolic biodiversity. A mechanism based on the maternal immunization against the negatively charged autoantigens, such as the insulin peptide B9-23, and on the fetal-maternal interaction might transform the physiological adaptation into adaptive changes of the genetic population structure. According to the "thrifty-genotype" hypothesis, "thrifty DRB alleles" with a positive charge are responsible for the susceptibility to diabetes and for an efficient storage of caloric intake in arctic climates with scarce food availability while "non-thrifty DRB alleles" with a negative or neutral charge are advantaged in tropical climates with constant food supply.

HLA-DRB1 alleles and juvenile idiopathic arthritis: Diagnostic clues emerging from a meta-analysis.

Juvenile Idiopathic Arthritis (JIA) is characterized with a variable pattern of articular involvement and systemic symptoms and, thus, it has been classified in several subtypes. Genetic predisposition to JIA is mainly due to HLA class II molecules (HLA-DRB1, HLA-DPB1), although HLA class I molecules and non-HLA genes have been implicated, too. Here, we carried out a meta-analysis including selected studies designed to assess HLA genetic background of JIA patients, compared to healthy controls; particularly, we focused our attention on HLA-DRB1. In summary, our meta-analysis showed four main findings regarding HLA-DRB1 locus as a genetic factor of JIA: i) HLA-DRB1*08 is a strong factor predisposing to JIA, both for oligo-articular and poly-articular forms (oJIA>pJIA); ii) HLA-DRB1*01 and HLA-DRB1*04 may be involved in the genetic predisposition of Rheumatoid Factor (RF) positive forms of JIA; iii) HLA-DRB1*11 was confirmed to be predisposing to oligo-articular JIA; iv) HLA-DRB1*04 was confirmed to have a role in systemic JIA. Importantly, RF positivity seems to select the JIA clinical subset with the strongest immunogenetic similarities with adult rheumatoid arthritis.

The Immunosignature of Mother/Fetus Couples in Gestational Diabetes Mellitus: Role of HLA-G 14 bp ins/del and PAPP-A A/C Polymorphisms in the Uterine Inflammatory Milieu.

We enrolled 151 healthy mother/newborn couples and 26 with gestational diabetes mellitus (GDM). HLA-G and PAPP-A plasma levels were measured by ELISA at first and second trimesters, at delivery, and in cord blood. HLA-G 14 bp ins/del and PAPP-A A/C polymorphisms were genotyped. HLA-G del/del and PAPP-A C/C genotypes were more frequent among GDM mothers than controls. We observed a genetic epistasis between the two polymorphisms: the HLA-G del/del and PAPP-A C/C combination was carried by 8% of GDM mothers and 1.3% of controls (OR = 9.5, 95% CI = 0.8-109, p = 0.07). GDM mothers showed increased sHLA-G levels compared to controls (p = 0.004), and those carrying the HLA-G del/del genotype produced more sHLA-G at the second trimester and at delivery (p = 0.014). A genetic pressure by fetal genotype on maternal sHLA-G production was observed in GDM mothers with heterozygous HLA-G del/ins newborns (p = 0.02). Babies born to GDM mothers showed higher sHLA-G concentrations compared to those born to healthy mothers, and those carrying HLA-G del/del showed the highest sHLA-G levels (p = 0.013). PAPP-A amounts significantly increased along pregnancy (p < 0.001), but the median levels at the first and second trimesters were significantly lower in GDM (p = 0.03). Our findings first suggest an involvement of HLA-G and PAPP-A gene-protein interaction in GDM and highlight a possible contribution of the fetus in balancing maternal inflammation.

Excess of activating killer cell immunoglobulin­like receptors and lack of HLA-Bw4 ligands: a two­edged weapon in chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is an inflammatory disease of unknown aetiology. Researchers have proposed infectious, neurological and immunological causes of this syndrome. Recently, the xenotropic murine leukemia virus-related virus was detected in 67% of patients with CFS in a US study. This observation is in agreement with one ascertained aspect of the disease: a decreased efficiency in NK cell lytic activity in CFS patients. Here, we analyzed the genomic polymorphism of killer cell immunoglobulin-like receptors (KIRs) and their HLA class I cognate ligands in patients with certified CFS. An excess of KIR3DS1 was found in CFS patients with respect to controls, as well as an increased frequency of the genotype missing KIR2DS5. Forty-four CFS patients and 50 controls also underwent genomic typing for the HLA-ligands. In the patients, a great proportion of KIR3DL1 and KIR3DS1 receptors were found to be missing their HLA-Bw4Ile80 binding motif. We hypothesize that an excess of KIR3DS1, combined with an excess of ligand-free KIR3DL1 and KIR3DS1 receptors, may hamper the clearance of a pathogen via NK cells, thus favouring the chronicity of the infection.

Specific polymorphisms of cytokine genes are associated with different risks to develop single-system or multi-system childhood Langerhans cell histiocytosis.

Cytokines and chemokines determine mobilisation of Langerhans cells and their dysregulation is implicated in the pathogenesis of Langerhans cell histiocytosis (LCH). Twenty point mutations of 12 different cytokine genes were studied in 41 Italian children, 15 with single-system (SS) and 26 with multi-system disease. The allele and genotype distributions of interleukin-4 (IL-4) and interferon-gamma (IFNgamma) were significantly different in patients vs. 140 controls (P = 0.007, and P = 0.018). Older children with single-system disease shared the 'anti-inflammatory profile' determined by the intermediate producer genotype IFNgamma +874A/T (P = 0.029) and the high-producer genotypes IL-4 -590C/T and T/T (P = 0.029). Our findings suggest that specific cytokine gene variants affect susceptibility to LCH and its clinical heterogeneity.

No evidence of autoimmunity in 6-year-old children immunized at birth with recombinant hepatitis B vaccine.

OBJECTIVES: Taking into account that genetic predisposition, marked by human leukocyte antigen (HLA) class I and II genes, augments the probability of developing an autoimmune disorder after a triggering vaccination, as largely debated, we investigated the frequency of autoantibody production after recombinant hepatitis B vaccine (rHBv) in 6-year-old children immunized at birth to evaluate an association between autoimmune disorders and hepatitis B virus vaccination. METHODS: We investigated the presence of autoantibodies in 210 6-year-old children who were immunized at birth with rHBv: 200 showed anti-hepatitis B surface antigen concentrations > or =10 mUI/mL at seroconversion (responders), and 10 were nonresponders. Data were compared with those obtained in 109 unvaccinated children. All participants were screened for the presence of antinuclear antibodies (ANAs), anti-DNA, antimitochondrial, anti-liver/kidney microsomal, antireticulin, anti-smooth muscle (SMA), and antiribosomal antibodies. All participants were also screened for the presence of antithyroid antibodies, such as antithyroglobulin and antiperoxidase, and for antibodies found in type 1 diabetes, such as tyrosine phosphatase (IA-2A) and glutamic acid decarboxylase (GADA). HLA typing was extended to all 10 nonresponders. RESULTS: Autoantibodies were found in 16 of the 200 responders: ANAs were found in 12 (6%), smooth muscle antibodies were found in 4 (2.0%), and antireticulin antibodies and endomysial antibodies were found in 1 girl with ANAs. Antithyroid antibodies, IA-2A, and GADA were not present in any of the participants. No significant difference was found in the frequency of autoantibodies between vaccinated and control children. Three of the 10 nonresponder children were SMA-positive (30% vs 2% of responders); they also carried the supratype HLA-C4AQ0,DRB1*0301,DQB1*02. A family history for autoimmune disorders was present in 3 (18%; 95% confidence interval [CI]: 4.0%-45.6%) of the 16 responder infants with autoantibodies, in 15 (8.4%; 95% CI: 4.6%-13.1%) of responder children without autoantibodies, and in 1 (10%) of the 10 nonsreponder children. CONCLUSIONS: From our data, vaccination with rHBv given during the neonatal period does not seem to increase autoantibody production in a 6-year-old children. Autoantibodies, referred to as natural autoantibodies, can be found in healthy participants, but their significance is unclear. These autoantibodies often cross-react with bacteria or tumor antigens, suggesting their importance in innate immunity. It has been demonstrated in an animal model that self-antigen can promote B-cell accumulation, and that a significant proportion of natural autoantibodies is the product of this self-antigen- dependent process. Consequently, it has been speculated that self-antigens play a positive role in recruiting B cells as a part of innate immunity, but this process carries a potential risk for unregulated growth. Spreading of the immune response is a common theme in organ-specific and systemis autoimmune diseases, and this could be initiated by exogenous agents, in genetically susceptible hosts, owing to molecular mimicry of natural antigen. Moreover, 3 (18%) of the 16 children who had autoantibodies had a family history of autoimmume diseases. Thus, it is apparent that susceptibility to autoimmunity is determined by genetic factors rather than by vaccine challenge. Among all the children considered, only 1 girl (0.5%) developed celiac disease, reflecting the prevalence described in the literature. GADA and IA-2A were not found in our children; this observation is in agreement with data showing that type 1 diabetes risk may not be altered by vaccinations administered during childhood. On the contrary, a high frequency (30%) of autoantibodies, in particular SMA, was observed in the nonresponder children. The 3 SMA-positive children carried the HLA-C4Q0,DRB1*0301,DQB1*02 haplotype, a well-known predisposing factor for autoimmune disorders. On the other hand, the presence of autoantibodies to smooth muscle is known to be common in hepatitis B infection, and, it has been shown that cross-reactive immunity targeting homologous self-protein may partly account for autoantibody production. Although hepatitis B vaccination given during the neonatal period does not increase autoantibody production in 6-year-old immunized children, we deem useful a more prolonged follow-up for these nonresponder children carrying certain HLA haplotypes (such as C4AQ0,DRB1*0301,DQB1*02), particularly because most autoimmune diseases do not develop until later in life.