Knockout of apolipoprotein A-I decreases parenchymal and vascular ß-amyloid pathology in the Tg2576 mouse model of Alzheimer's disease.

AIMS: Apolipoprotein A-I (apoA-I), the principal apolipoprotein associated with high-density lipoproteins in the periphery, is also found at high concentrations in the cerebrospinal fluid. Previous studies have reported either no impact or vascular-specific effects of apoA-I knockout (KO) on ß-amyloid (Aß) pathology. However, the putative mechanism(s) by which apoA-I may influence Aß deposition is unknown. METHODS: We evaluated the effect of apoA-I deletion on Aß pathology, Aß production and clearance from the brain in the Tg2576 mouse model of Alzheimer's disease (AD). RESULTS: Contrary to previous reports, deletion of the APOA1 gene significantly reduced concentrations of insoluble Aß40 and Aß42 and reduced plaque load in both the parenchyma and blood vessels of apoA-I KO × Tg2576 mice compared to Tg2576 animals. This was not due to decreased Aß production or alterations in Aß species. Levels of soluble clusterin/apoJ were significantly higher in neurons of apoA-I KO mice compared to both wildtype (WT) and apoA-I KO × Tg2576 mice. In addition, clearance of Aß along intramural periarterial drainage pathways was significantly higher in apoA-I KO mice compared to WT animals. CONCLUSION: These data suggest that deletion of apoA-I is associated with increased clearance of Aß and reduced parenchymal and vascular Aß pathology in the Tg2576 model. These results suggest that peripheral dyslipidaemia can modulate the expression of apolipoproteins in the brain and may influence Aß clearance and aggregation in AD.

Systemic lupus erythematosus in Europe at the change of the millennium: lessons from the "Euro-Lupus Project".

The "Euro-Lupus Cohort" is composed by 1000 patients with systemic lupus erythematosus (SLE) that have been followed prospectively since 1991. These patients have been gathered by a European consortium--the "Euro-Lupus Project Group". This consortium was originated as part of the network promoted by the "European Working Party on SLE", a working group created in 1990 in order to promote research in Europe on the different problems related to this disease. The "Euro-Lupus Cohort" provides an updated information on the SLE morbidity and mortality characteristics in the present decade as well as defines several clinical and immunological prognostic factors.

Expression of ATP7B in normal human liver.

ATP7B is a copper transporting P-type ATPase, also known as Wilson disease protein, which plays a key role in copper distribution inside cells. Recent experimental data in cell culture have shown that ATP7B putatively serves a dual function in hepatocytes: when localized to the Golgi apparatus, it has a biosynthetic role, delivering copper atoms to apoceruloplasmin; when the hepatocytes are under copper stress, ATP7B translocates to the biliary pole to transport excess copper out of the cell and into the bile canaliculus for subsequent excretion from the body via the bile. The above data on ATP7B localization have been mainly obtained in tumor cell systems in vitro. The aim of the present work was to assess the presence and localization of the Wilson disease protein in the human liver. We tested immunoreactivity for ATP7B in 10 human liver biopsies, in which no significant pathological lesion was found using a polyclonal antiserum specific for ATP7B. In the normal liver, immunoreactivity for ATP7B was observed in hepatocytes and in biliary cells. In the hepatocytes, immunoreactivity for ATP7B was observed close to the plasma membrane, both at the sinusoidal and at the biliary pole. In the biliary cells, ATP7B was localized close to the cell membrane, mainly concentrated at the basal pole of the cells. The data suggest that, in human liver, ATP7B is localized to the plasma membrane of both hepatocytes and biliary epithelial cells.

Unrelated donor stem cell transplantation in adult patients with thalassemia.

Allogeneic SCT remains the only potential cure for patients with thalassemia. However, most BMT candidates lack a suitable family donor and require an unrelated donor (UD). We evaluated whether BMT using UDs in high-risk adult thalassemia patients can offer a probability of cure comparable to that reported employing an HLA-compatible sibling as donor. A total of 27 adult thalassemia patients (15 males and 12 females, median age 22 years) underwent BMT from a UD selected by high-resolution HLA molecular typing. The conditioning regimen consisted of Busulphan (BU, 14 mg/kg) plus Cyclophosphamide (CY, 120 or 160 mg/kg) in 12 cases and BU (14 mg/kg), Thiotepa (10 mg/kg) and CY (120-160 mg/kg) in the remaining 15 cases. Cyclosporine-A and short-term Methotrexate were used for graft-versus-host disease (GVHD) prophylaxis. In all, 19 patients (70%) are alive and transfusion-independent after a median follow-up of 43 months (range 16-137). A total of 10 patients (37%) developed grade II-IV acute GVHD and six (27%) chronic GVHD. Eight patients (30%) died from transplant-related causes. UD-BMT can cure more than two-thirds of adult thalassemia patients, and is a particularly attractive option for patients who are not compliant with conventional treatment.

Association of HLA-DRB3*0202 and serum IgG antibodies to Chlamydia pneumoniae with essential hypertension in a highly homogeneous population from Majorca (Balearic Islands, Spain).

Separate studies investigating the relationship of essential hypertension (EH) with the HLA system and with Chlamydia pneumoniae (C. pneumoniae) infection have given conflicting results. Our aim was to clarify these relationships and determine whether the HLA system and C. pneumoniae infection interact with respect to the risk for EH. An association study (110 essential hypertensives and 107 controls) was conducted in a highly homogeneous population in the Balearic Island of Majorca (Spain). Molecular typing of HLA-B and HLA-DRB and quantification of serum levels of IgG antibodies to C. pneumoniae (sIgGa-Cp) were determined. Student's t-test, chi(2)-statistics, logistic regression analysis, and general linear model ANOVA were used for statistical analysis. The results showed that EH was related with HLA-DRB3*0202 in the whole study population, and with levels of sIgGa-Cp>63.5 BU/ml in the group of individuals with sIgGa-Cp>30 BU/ml (OR (95% CI) adjusted for obesity, familial history of EH and diabetes=2.06 (1.07-3.97), P=0.03, and =4.60 (1.06-19.90), P=0.04, respectively). The association between EH and sIgGa-Cp was observed in the DRB3*0202(+) individuals, but not in the DRB3*0202(-) subgroup (OR (95% CI)=11.14 (1.92-64.54), P=0.004, and =0.98 (0.22-4.43), P=0.64, respectively (P of the Mantel-Haenszel test for homogeneity of OR=0.06)). In our population, EH was positively associated with HLA-DRB3*0202 and with high levels of sIgGa-Cp. Moreover, a significant interaction of DRB3*0202 on the effect of sIgGa-Cp was observed, as the association of EH with these antibodies depended on the presence of DRB*0202.

A psoriasis vulgaris protective gene maps close to the HLA-C locus on the EH18.2-extended haplotype.

We determined the molecular haplotypes of the HLA-A, HLA-C and HLA-B loci and the MHC class I-B-related (MIB) microsatellite in 179 unrelated psoriatic patients (72 familial cases) and in 120 controls. The HLA-A*3002-Cw*0501-B*1801-MIB1 haplotype showed a strong negative association with psoriasis vulgaris (PV) and in particular with familial PV, revealing the presence of a PV-protective gene. Analysis of association and linkage disequilibrium of the single alleles and the various two-three-four-locus segments of this haplotype indicated the presence of a protective gene telomeric to the HLA-C locus. This finding was confirmed in 13 informative multiplex PV families, in which at least one parent carried the EH18.2 haplotype. In two families, an affected sibling presented HLA-A/C recombination on the EH18.2 haplotype. A study of 12 polymorphic microsatellites in all members of the informative families, 145 PV patients, 120 controls and 32 EH18.2 homozygous healthy individuals demonstrated that the protection conferred by the EH18.2 haplotype lies within a 170 kb interval between the C143 and C244 loci, most probably in a 60 kb segment between the C132 and C244 loci.

Psoriasis is associated with a SNP haplotype of the corneodesmosin gene (CDSN).

A psoriasis susceptibility locus has been mapped to the HLA region in the proximity of the HLA-C locus. This critical region also contains the CDSN gene coding for the corneodesmosin protein. In a case-control association study of psoriasis in the Sardinian population, we analyzed the allele distribution of eight intragenic SNPs (positions 619, 767, 1215, 1118, 1236, 1243, 1331, 1593) of the CDSN gene and the six haplotypes that are coded by these SNPs. Our study showed that these CDSN haplotypes are very stable and well-conserved in the Sardinian population. The CDSN2 haplotype was found to be associated with susceptibility to psoriasis. The association did not depend upon any one of the intragenic SNPs taken separately. At the HLA-C locus, the Cw6 and Cw7 alleles were dragged along by linkage disequilibrium with the CDSN2 haplotype and only revealed a trend towards association with the disease. Therefore, the intragenic SNPs of the CDSN gene and the HLA-Cw6 and Cw7 alleles are not directly involved in susceptibility to psoriasis. However, the strong association of the CDSN2 haplotype suggests a possible role for the CDSN gene and its chromosome region in susceptibility to psoriasis.

Unrelated bone marrow transplantation in thalassemia. The experience of the Italian Bone Marrow Transplant Group (GITMO).

BACKGROUND AND OBJECTIVES: Allogeneic bone marrow transplantation (BMT) is a widely accepted therapeutic approach in homozygous beta-thalassemia. However, the majority of patients do not have a genotypically identical donor within the family. This prompted us to conduct a pilot study to investigate the feasibility of matched unrelated bone marrow transplantation in thalassemia. The major drawback was the high risk of immunologic and transplant-related complications, mainly graft-versus-host disease (GvHD) and graft failure. DESIGN AND METHODS: Our aim was to reduce this risk through careful selection of donor/recipient pairs. HLA haplotypes that show a high linkage disequilibrium among their class I, class II and class III alleles are considered extended or ancestral haplotypes. RESULTS: These haplotypes are conserved and can be shared by apparently unrelated individuals. Our study shows that matching for these haplotypes significantly improves the outcome of unrelated bone marrow transplantation in thalassemia. In fact, results were comparable to those obtained in transplants using HLA-identifical family donors. INTERPRETATION AND CONCLUSIONS: Better results were obtained in patients with lesser iron overload and when the donor shared an identity for the DPB1 alleles.

Molecular variation of HLA class I genes in the Corsican population: approach to its origin.

Three human leucocyte antigen (HLA) class I loci (HLA-A, -B and -Cw) were typed for the first time at the DNA level in the Corsican population. This analysis was performed on 100 individuals of Corsican origin living in central Corsica (46 individuals) and in south-western Corsica (54 individuals). The genetic structure of these two subpopulations was analysed on the basis of the molecular polymorphisms of the HLA-A, -B and -Cw genes. A phylogenetic and a haplotypic analysis were performed. The genotypic analysis did not detect genetic differentiation. Examination of the allelic and haplotypic frequencies did, however, reveal some significant differences between the two zones. Similarities with the Sardinian population were found, and were particularly evident in the south-western sample. However, Corsica has probably been subject to greater West European influence, which can be seen in the Corte sample (Haute Corse).

Lessons from the "Euro-Lupus Cohort".

The "Euro-Lupus Cohort" is composed by 1,000 patients with systemic lupus erythematosus (SLE) that have been followed prospectively since 1991. These patients have been gathered by a European consortium - the "Euro-Lupus Project Group". This consortium was originated as part of the network promoted by the "European Working Party on SLE", a working group created in 1990 in order to promote research in Europe on the different problems related to this disease. The "Euro-Lupus Cohort" provides an updated information on the SLE morbidity and mortality characteristics in the present decade as well as defines several clinical and immunological prognostic factors.

A novel approach to search for identity by descent in small samples of patients and controls from the same mendelian breeding unit: a pilot study on myopia.

Autosomal dominant high myopia, a genetic disorder already mapped to region 18p11.31, is common in Carloforte (Sardinia, Italy), an isolated village of 8,000 inhabitants descending from a founder group of 300 in the early 1700s. Fifteen myopic propositi and 36 normal controls were selected for not having ancestors in common at least up to the grandparental generation, although still descendants of the original founders. All subjects were genotyped for 14 markers located on autosome 18 at a resolution of about 10 cM. Allelic distributions were found to be similar at all tested loci in propositi and controls, except for the candidate marker D18S63 known to segregate in close linkage association with high myopia. In particular, the frequency of allele 85 among the propositi was almost double that of the controls (Fisher's exact test, p = 0.037). The association is more striking when the frequency of the genotype 85/85 in the two groups is compared (Fisher's exact test, p = 0.005). This conclusion was further evaluated through a bootstrap analysis by computing the overall probability of the observed data under the null hypothesis (i.e. no difference between the two groups in frequency distributions for the chromosome 18 markers). Again, marker D18S63 was found to have a sample probability lower than 0.004, which is significant at the 0.05 level after correcting for simultaneous testing of multiple loci. The study demonstrates the efficiency of our novel strategy to detect identity by descent (IBD) in small numbers of patients and controls when they are both part of well-defined Mendelian breeding units (MBUs). The iterative application of our strategy in separate MBUs is expected to become the method of choice to evaluate the ever-growing number of reported associations between candidate genes and multifactorial traits and diseases.

The role of heterocellular hereditary persistence of fetal haemoglobin in beta(0)-thalassaemia intermedia.

Beta(0)-thalassaemia intermedia (beta(0)-TI) describes patients who lack beta-globin synthesis yet manifest a non-transfusion-dependent form of beta-thalassaemia. Co-inheritance of alpha-thalassaemia, certain variants of the beta-like globin gene cluster and elevated fetal haemoglobin (HbF) production are all associated with beta(0)-TI. However, the mild phenotypes of many beta(0)-TI patients are unexplained. Genetically determined HbF levels in beta-thalassaemia are difficult to assess because erythrocytes containing HbF (F cells) preferentially survive over erythrocytes without HbF. To evaluate the importance of genetically elevated HbF in beta-thalassaemia, F-cell levels of 19 TI patients' relatives were compared with relatives of transfusion-dependent beta-thalassaemia major patients and those of beta-globin genotype-matched controls. The beta-globin and alpha-globin genotypes, as well as their Ggamma promoter were also examined. Using this approach, in all but one patient the mild phenotype was attributable to either alpha-globin genotype, gamma-globin promoter polymorphism or inherited elevated F-cell levels. The findings of this study establish the F-cell levels required to modify the degree of disease severity significantly and demonstrate that F-cell level is a crucial parameter in the understanding of phenotypic variation in beta-thalassaemia.

West Mediterranean islands (Corsica, Balearic islands, Sardinia) and the Basque population: contribution of HLA class I molecular markers to their evolutionary history.

The genetic structure of Balearic islands (Corsica and Sardinia), situated on the same trans-Mediterranean maritime routes and having very similar histories, were compared and their position among the neighbouring Caucasian populations was inferred. For this purpose, three HLA loci (HLA-A, -B and -Cw) were typed at the DNA level in these populations and the allelic and haplotypic frequencies were estimated. Because previous studies have shown common genetic features in the Sardinians and Basques, HLA-Cw molecular typing was also performed in a sample of French Basques in order to establish the haplotypic structure of this population for a more accurate comparison with the three others. By its allelic composition, the Corsican population has an intermediate position between the two other islander populations. Its close relationship with the Sardinian population, however, was clearly revealed by the phylogenetic analysis which also suggests a proximity with eastern Mediterranean peoples, whereas the Balearic islands are more narrowly related to Spain and western Europe. Peculiarities were observed in the distributions of some common haplotypes in the populations of the islands that confirm the results of the phylogenetic analysis and could be related to their history. Noteworthy is the presence of the HLA-A30-Cw*0501-B18 haplotype at frequencies approximately 2% in Corsica and the Balearic islands, yet the estimated frequencies of this haplotype are much lower than in the Sardinian and Basque populations.

A randomized trial comparing the introduction of ritonavir or indinavir in 1251 nucleoside-experienced patients with advanced HIV infection.

ISS-IP1, a multicenter, randomized, 48-week open trial, was designed to compare the introduction of ritonavir or indinavir in patients with previous nucleoside experience and CD4+ cell counts below 50/mm3. Concomitant antiretroviral treatment with nucleoside analogs was allowed. Primary efficacy measures were survival and time to a new AIDS-defining event or death, analyzed through the whole period of observation by the intention-to-treat approach. Primary toxicity measures were time to treatment discontinuation and adverse events, grade at least 3/serious, analyzed by an on-treatment approach. Evaluation-of efficacy also included CD4+ cell and RNA response. The trial enrolled 1251 patients in 5 months. At baseline, mean CD4+ cell count was about 20 cells/mm3 and mean HIV RNA copy number was 4.9 log10/ml in both groups. Overall, 402 patients in the ritonavir group and 250 patients in the indinavir group permanently discontinued the assigned treatment (relative risk, 1.96; 95% CI, 1.68-2.30; p = 0.0001), with most of this difference dependent on a higher number of discontinuation for adverse events in the ritonavir group. After a mean follow-up of 307 days (ritonavir, 304; indinavir, 309), 124 deaths (ritonavir, 61; indinavir, 63; relative risk, 0.96; 95% CI, 0.67-1.36; p = 0.80) and 330 new AIDS-defining events (ritonavir, 170; indinavir, 160; relative risk, 1.05; 95% CI, 0.85-1.31; p = 0.60) were observed. CD4+ cell counts increased in both groups in patients still receiving treatment, with about 100 cells gained by week 24 and 150 cells gained by week 48. Body weight also increased over time in both groups. Analysis of RNA response showed a decrease of 1.5 log10 or higher in both treatment groups. Overall, 400 patients in the ritonavir group and 338 patients in the indinavir group developed at least one grade 3/serious new adverse event during follow-up (relative risk, 1.48; 95% CI, 1.28-1.72; p = 0.0001). Favorable CD4+ cell and RNA responses at 24 and 48 weeks were observed in both groups of patients remaining on treatment. Indinavir showed slightly better effects in sustaining RNA, CD4+ cell, and body weight responses. Ritonavir and indinavir results were comparable in terms of clinical outcome (survival and AIDS-defining events).

Identifying human herpesvirus 8 infection: performance characteristics of serologic assays.

Epidemiologic studies of infection with the oncogenic human herpesvirus 8 (HHV-8) depend on serologic methods to diagnose infection. However, optimal strategies for identifying HHV-8 infection remain undefined. We therefore evaluated four enzyme-linked immunoassays (EIAs) and one immunofluorescence assay (IFA) using sera from 87 individuals with the prototype HHV-8 disease, Kaposi's sarcoma (KS), and 210 participants in a hemophilia study (who were presumed not to be infected with HHV-8). Assays performed reasonably well in distinguishing between infected and uninfected persons, with receiver operator curve areas between 0.86 and 0.96. Nonetheless, IFA had only 86% sensitivity and 88% specificity, and no EIA simultaneously had sensitivity and specificity above 90% for any of the optical density (OD) cutpoints used to define seropositivity. Some assays were markedly less sensitive with diluted KS sera, suggesting that they poorly identify low-titer antibodies present in asymptomatic infection. We also developed a classification tree that categorized individuals as seropositive if they had OD > 2.00 on recombinant K8.1 protein EIA or if they had both K8.1 OD between 0.51 and 2.00 and positive IFA results; this strategy had between 80% and 90% sensitivity and 95% and 100% specificity. Overall, assays performed adequately for use in most epidemiologic investigations, but wider applications will require improved tests.

Familial hypercholesterolemia study in Sardinia using 6 LDLR polymorphic markers based on PCR.

Twenty-two Sardinian families with multiple cases of hypercholesterolemia were investigated with six polymorphic markers of the low-density lipoprotein receptor (LDLR) gene that could be quickly analyzed by PCR-based methods. Five single nucleotide polymorphisms (SNP) in exons 8, 10, 13, 15, and 18 and a microsatellite marker flanking the 3' end of the LDLR gene were used to define the haplotypes at the LDLR locus for familial hypercholesterolemia (FH) diagnosis within families. No significant differences were observed between the allele frequencies of the normal and mutant chromosomes. In two families, hypercholesterolemia did not cosegregate with the LDLR locus. In the remaining 20 FH chromosomes, seven different haplotypes were identified. The same haplotypes were found with a similar frequency among the 61 normal chromosomes. Other five haplotypes were characteristic only of normal chromosomes. These data provide no evidence for a gene founder effect in the Sardinian population and, instead, highlight a pattern of genetic heterogeneity comparable with that found in mainland European populations. The replacement of the restriction fragment length polymorphisms currently used in the genetic analysis of FH with PCR-based markers proved to be a simple and less time-consuming method and did not reduce informativity in the molecular analysis of FH families.

Population genomics in Sardinia: a novel approach to hunt for genomic combinations underlying complex traits and diseases.

The availability of highly polymorphic markers permits testing whether complex traits and diseases result from genomic interactions between nonallelic normal variants at separate loci. Such variants may be identified by deviations from the expected distributions of alleles at a high number of polymorphic loci, when individuals with the phenotype of interest are compared to normal controls of the same breeding unit, provided that both groups share the same remote ancestry and had no ancestors in common for the last three to four generations. The circumstances needed for such studies are ideally met on the island of Sardinia. The recurrent finding of the same type of association in separate breeding units between the phenotype of interest and a given genotype should allow a distinction between true genetic identity by descent and randomly occurring identities, as these will be obviously different in separate breeding units. The availability of several breeding units located in sharply different ecological environments will permit assessment of the role of nature/nurture factors in the degree of manifestation of each newly discovered genotype/phenotype association. A pilot study to evaluate the proposed strategy has been carried out in the Sardinian village of Carloforte, a community of about 8,000 individuals who have remained genetically homogeneous. Fifty-five control samples have been genotyped with six tetranucleotide microsatellites and with a subset of the 400 markers contained in the ABI PRISM linkage mapping panel, version 2. The allele frequencies for these microsatellite markers have been determined for these 55 individuals and compared to those from a random sampling of subsets of these 55 persons. For the six tetranucleotide microsatellites, a subset of as few as 20 people displayed the same allele frequency distributions as observed with the original 55 unrelated individuals. In conclusion, when samples are chosen from the same breeding unit, the number of individuals sufficient to draw the genomic profile of an isolated population can be relatively small. Likewise, the number of probands with the phenotype of interest can be even smaller when they are ascertained with the same genealogical criteria as the normal controls. By comparing the genomic profile of the probands to a fraction of the control samples within each of several separate breeding units of common remote ancestry, the search for genotype/phenotype association for mono- and multifactorial traits and diseases should be simplified and yield unequivocal results.

HLA-DMA alleles are possible new markers of rheumatoid arthritis: study of a Corsican group.

The HLA-DMA gene, along with the HLA-DMB gene, encodes the not classical class II molecule. This molecule catalyzes the class-II-associated invariant-chain peptide (CLIP)-antigen peptide exchange in classical class II molecule peptide-binding groove. As such, the DM heterodimer is an antigen presentation regulator and may be linked to immune system deficiencies such as those observed in autoimmune diseases. The study of DMA gene polymorphism seems be a reasonable approach to provide an answer to this question. Thanks to PCR-derived methods, the relationship between DMA gene polymorphism and rheumatoid arthritis (RA) was demonstrated in the present study. The DMA*0101 allele was observed to confer a significant predisposition to RA while the DMA*0102 allele significantly protected from this disease. Polymorphism experiments with the HLA-DRB1 gene revealed that this relationship between DMA polymorphism and RA is not a consequence of a linkage disequilibrium with the HLA-DRB1 alleles implicated in this pathology. The study of the DMA gene could therefore prove to be very useful in the early diagnosis of RA.