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Biomedical subjects

A Malaspina

Publications and source records attributed to A Malaspina.

At least 19 recordsLinked to original sources
PubMed2002

SOD1 activity and protective factors in familial ALS patients with L84F SOD1 mutation.

METHOD: The activity and amount of SOD1 in erythrocyte lysates and the plasma amino acid content were evaluated in four familial ALS patients bearing the L84F SOD1 mutation (fALS), in an asymptomatic family member with the mutation (L84F(5)), in sporadic ALS patients (sALS) and controls. Three of the fALS patients and the L84F(5) subject were tested once a year for three consecutive years. RESULTS: At the first evaluation SOD1 activity was similar in controls, sALS and fALS; the amount of SOD1 protein was lower (P < 0.01) in fALS. In the subsequent 2 years, 34% and 52% decrease of SOD1 activity was recorded in fALS patients. The plasma amino acid pattern did not differ between controls and sALS, whereas fALS patients displayed high levels of plasma aspartate and glutamate. Aspartate was in the normal range but glutamate was still elevated in the subsequent evaluations. The L84F(5) subject had remarkably low levels of aspartate, glutamate and branched-chain amino acids. CONCLUSIONS: The method of measuring mutant SOD1 amount is indirect but the results are indicative of a reduction of mutant SOD1 taking place during fast-worsening phases of the disease. Since the disease onset of fALS patients is 42.8 +/- 11.3 years and the L84F(5) family member is asymptomatic at the age of 66, low levels of excitotoxic and branched-chain amino acids in plasma may constitute a protective factor against disease development.

Amino Acids↗
PubMed2001

Epitope mapping using the X-ray crystallographic structure of complement receptor type 2 (CR2)/CD21: identification of a highly inhibitory monoclonal antibody that directly recognizes the CR2-C3d interface.

Complement receptor type 2 (CR2)/CD21 is a B lymphocyte cell membrane C3d/iC3b receptor that plays a central role in the immune response. Human CR2 is also the receptor for the EBV viral membrane glycoprotein gp350/220. Both C3d and gp350/220 bind CR2 within the first two of 15-16 repetitive domains that have been designated short consensus/complement repeats. Many mAbs react with human CR2; however, only one currently available mAb is known to block both C3d/iC3b and gp350/220 binding. We have used a recombinant form of human CR2 containing the short consensus/complement repeat 1-2 ligand-binding fragment to immunize Cr2(-/-) mice. Following fusion, we identified and further characterized four new anti-CR2 mAbs that recognize this fragment. Three of these inhibited binding of CR2 to C3d and gp350/220 in different forms. We have determined the relative inhibitory ability of the four mAbs to block ligand binding, and we have used overlapping peptide-based approaches to identify linear epitopes recognized by the inhibitory mAbs. Placement of these epitopes on the recently solved crystal structure of the CR2-C3d complex reveals that each inhibitory mAb recognizes a site either within or adjacent to the CR2-C3d contact site. One new mAb, designated 171, blocks CR2 receptor-ligand interactions with the greatest efficiency and recognizes a portion of the C3d contact site on CR2. Thus, we have created an anti-human CR2 mAb that blocks the C3d ligand by direct contact with its interaction site, and we have provided confirmatory evidence that the C3d binding site seen in its crystal structure exists in solution.

Animals↗
PubMed2001

HIV-1 induces phenotypic and functional perturbations of B cells in chronically infected individuals.

A number of perturbations of B cells has been described in the setting of HIV infection; however, most remain poorly understood. To directly address the effect of HIV replication on B cell function, we investigated the capacity of B cells isolated from HIV-infected patients to respond to a variety of stimuli before and after reduction of viremia by effective antiretroviral therapy. B cells taken from patients with high levels of plasma viremia were defective in their proliferative responses to various stimuli. Viremia was also associated with the appearance of a subpopulation of B cells that expressed reduced levels of CD21. After fractionation into CD21(high)- and CD21(low)-expressing B cells, the CD21(low) fraction showed dramatically reduced proliferation in response to B cell stimuli and enhanced secretion of immunoglobulins when compared with the CD21(high) fraction. Electron microscopic analysis of each fraction revealed cells with plasmacytoid features in the CD21(low) B cell population but not in the CD21(high) fraction. These results indicate that HIV viremia induces the appearance of a subset of B cells whose function is impaired and which may be responsible for the hypergammaglobulinemia associated with HIV disease.

Antigens, CD19↗
PubMed2001

Differential expression of 14 genes in amyotrophic lateral sclerosis spinal cord detected using gridded cDNA arrays.

In order to obtain insight into the aetiology and pathogenesis of amyotrophic lateral sclerosis (ALS), high-density gene discovery arrays (GDA human version 1.2) containing 18 400 non-redundant EST cDNAs pooled from different tissue libraries have been used to monitor gene expression in lumbar spinal cord from ALS cases compared with controls. Quantitative filter analysis revealed differential expression of cDNAs normalized to internal standards. These candidates have been further investigated and their expression in spinal cord characterized in a panel of ALS and control subjects. Significant differential expression was obtained for 14 genes, 13 being elevated (up to six-fold) and one decreased (by 80%) in ALS. Amongst those elevated in ALS were thioredoxin and glial fibrilary acid protein, which have already been shown to be up-regulated in ALS, thus supporting the reliability of this approach. The other differentially regulated transcripts confirmed in the expression studies represent potential candidates in ALS pathogenesis being involved in antioxidant systems, neuroinflammation, the regulation of motor neurone function, lipid metabolism, protease inhibition and protection against apoptosis. The use of the GDA system has greatly facilitated the screening and retrieval of sequence information and has generated useful information on the cascade of molecular events occurring in ALS and potentially may highlight new candidates playing a role in the aetiology and progression of this disease.

Adolescent↗
PubMed2000

B cells of HIV-1-infected patients bind virions through CD21-complement interactions and transmit infectious virus to activated T cells.

The impact of HIV-associated immunopathogenesis on B cells has been largely associated with indirect consequences of viral replication. This study demonstrates that HIV interacts directly with B cells in both lymphoid tissues and peripheral blood. B cells isolated from lymph node and peripheral blood mononuclear cells (PBMCs) of 4 and 23 chronically infected patients, respectively, demonstrated similar capacities to pass virus to activated HIV-negative PBMCs when compared with CD4(+) cells from the same patients. However, in contrast to T cells, virus associated with B cells was surface bound, as shown by its sensitivity to pronase and the staining pattern revealed by in situ amplification of HIV-1 RNA. Cell sorting and ligand displacing approaches established that CD21 was the HIV-binding receptor on B cells, and that this association was mediated through complement-opsonized virus. These B cells were also found to express significantly lower levels of CD21 compared with HIV-negative individuals, suggesting a direct perturbing effect of HIV on B cells. These findings suggest that B cells, although they themselves are not readily infected by HIV, are similar to follicular dendritic cells in their capacity to serve as extracellular reservoirs for HIV-1. Furthermore, B cells possess the added capability of circulating in peripheral blood and migrating through tissues where they can potentially interact with and pass virus to T cells.

Acquired Immunodeficiency Syndrome↗
PubMed2000

Migraine with aura and white matter abnormalities: Notch3 mutation.

The authors report on an Italian family with eight affected members who show autosomal dominant migraine with prolonged visual, sensory, motor, and aphasic aura. These symptoms are associated with white matter abnormalities on brain MRI. All living affected members carry a Notch3 mutation (Arg153Cys) previously reported in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). White matter abnormalities occur in a variable percentage of the general migraine population; CADASIL should be suspected in migraineurs with prolonged atypical aura and white matter abnormalities.

Brain↗
PubMed2000

A 14-3-3 mRNA is up-regulated in amyotrophic lateral sclerosis spinal cord.

We have recently isolated a 2.2-kb cDNA clone (1C5) from a human spinal cord cDNA library with partial identity to the 14-3-3 protein mRNA encoding the theta protein (YWHAQ). 14-3-3 protein transcripts are highly expressed in large projection neurones of the hippocampus, cerebellum, and spinal cord and have been found to be significantly up-regulated in rat motor neurones following hypoglossal nerve axotomy. In this study we investigated whether the 1C5 transcript (YWHAQ) isolated from spinal cord was involved in amyotrophic lateral sclerosis (ALS). We found a significant up-regulation of 1C5 (YWHAQ) in lumbar spinal cord from patients with sporadic ALS compared with controls, with the highest levels of expression being found in individuals with predominant lower motor neurone involvement. A 6-bp tandem repeat in the 5'-untranslated region of the gene was found to be polymorphic, but no significant association with disease was found following genomic analysis of this region. The localisation of 1C5 (YWHAQ) to chromosome 2 was determined and coincides with that reported for clone HS1 (EMBL accession no. X57347). These results show the marked up-regulation of the 14-3-3 isoform (YWHAQ) in ALS spinal cord and indicate the involvement of a potential 14-3-3-mediated survival pathway in the pathogenesis of ALS.

14-3-3 Proteins↗
PubMed2000

Characterization of trinucleotide- and tandem repeat-containing transcripts obtained from human spinal cord cDNA library by high-density filter hybridization.

In order to identify trinucleotide- and tandem repeat-containing transcripts in human spinal cord, hybridization of a high-density spinal cord cDNA library filter was carried out using a radioactively labeled degenerate oligonucleotide designed to detect different trinucleotide repeats including those known to occur in disease-associated expansions, in a single step. The sequence analysis of the trinucleotide repeat-containing transcripts (TNRTs) revealed 23 known mammalian genes with trinucleotide repeat-containing regions (TNRs), some of which were not previously reported to contain TNRs, and 18 cDNA clones with no or insignificant sequence homology to known genes. Amongst the known genes detected was the fragile X gene (FMR-1) containing (CGG)30. Other genes containing extended TNRs of 9 to 21 repeats were calcium-dependent protease, ATBF1-A, ferritin H chain, and the G protein Gsalpha2. Ten sequences containing perfect TNRs and two sequences containing perfect tandem repeats (derived from 11 TNRTs) were further analyzed for allelic variation using primers flanking the TNR, and five were shown to exhibit two to five alleles per TNR. These transcripts were further investigated for their chromosomal localization where unknown or only partially characterized. The transcripts that were polymorphic in the TNR region were ATBF1-A (a homeodomain protein), clone 390013 on chromosome Xp11, a member of the family of the 14.3.3 protein kinase C regulators, a human translation initiation factor (an isolog of the yeast Suilisol gene 1), and a novel sequence (TR21). Only the first two transcripts showed the presence of rare expanded alleles. Characterization of polymorphic TNRs in novel and even known genes expressed in human spinal cord is likely to help in the identification of new candidates for genes involved in neurodegenerative disorders.

Chromosome Mapping↗
PubMed1999

Identification and molecular characterization of NKp30, a novel triggering receptor involved in natural cytotoxicity mediated by human natural killer cells.

Two major receptors involved in human natural cytotoxicity, NKp46 and NKp44, have recently been identified. However, experimental evidence suggested the existence of additional such receptor(s). In this study, by the generation of monoclonal antibodies (mAbs), we identified NKp30, a novel 30-kD triggering receptor selectively expressed by all resting and activated human natural killer (NK) cells. Although mAb-mediated cross-linking of NKp30 induces strong NK cell activation, mAb-mediated masking inhibits the NK cytotoxicity against normal or tumor target cells. NKp30 cooperates with NKp46 and/or NKp44 in the induction of NK-mediated cytotoxicity against the majority of target cells, whereas it represents the major triggering receptor in the killing of certain tumors. This novel receptor is associated with CD3zeta chains that become tyrosine phosphorylated upon sodium pervanadate treatment of NK cells. Molecular cloning of NKp30 cDNA revealed a member of the immunoglobulin superfamily, characterized by a single V-type domain and a charged residue in the transmembrane portion. Moreover, we show that NKp30 is encoded by the previously identified 1C7 gene, for which the function and the cellular distribution of the putative product were not identified in previous studies.

Animals↗
PubMed1999

Clinical characteristics of SOD1 gene mutations in UK families with ALS.

Five to ten percent of patients with ALS have a family history of the disease, inheritance is usually autosomal dominant. Mutations of the SOD1 gene were first identified in a proportion of families with ALS by Rosen et al. The SOD1 gene encodes the enzyme copper zinc superoxide dismutase. Patients were studied from throughout the UK, where more than one individual in the family had ALS. Clinical history and examination of the individual and family were obtained, and DNA extracted from leukocytes of whole blood samples. Mutations were identified by standard sequencing methods. To date, 12 different mutations of SOD1 have been identified in 17 different families, representing around 20% of all ALS families studied. The mutations were mainly single base substitutions - H48Q, G72S, G93R, G93V, E100G, D101N, D101G, G108V, I113T, D125H, I149T - and also an insertion mutation - 132insTT - leading to a premature stop codon. The mutations were present in exons 2-5. We did not identify mutations in exon 1, although these have been identified by others in different patient samples. We have identified SOD1 mutations in around 20% of UK families with ALS studied. This is similar to that reported in other populations. Mutations have now been identified in all exons of SOD1. The individual mutations do not precisely predict disease severity, and generally it is difficult to give a specific prognosis based on the individuals' SOD1 mutations. We continue to investigate the possible pathogenic mechanisms of the SOD1 mutations. We have studied the neuropathology in patients with SOD1 mutations. We are also performing linkage studies to identify the genes involved in the 80% of families where an SOD1 mutation has not been identified.

Adult↗
PubMed1999

Clustering of ALS patients in central Italy due to the occurrence of the L84F SOD1 gene mutation.

OBJECTIVE: To study three new apparently unrelated Italian families with ALS and several sporadic ALS patients living in the same rural area. BACKGROUND: One Italian family with ALS carrying a superoxide dismutase 1 (SOD1) gene mutation (G41S) and no regional ALS clustering has been reported in Italy. METHODS: Genetic analysis was performed by automated and manual sequencing of the SOD1 gene in 13 family members and in 6 of 10 unrelated patients with sporadic cases of ALS living in the same area. The authors also determined SOD1 activity in erythrocytes and lymphocytes. RESULTS: The three families included a total of 28 affected members distributed over six generations. Despite a wide variability in age at onset and disease duration, the clinical pattern is uniform, with onset in the lower limbs, ascending progression, and predominant lower motor neuron involvement in all subjects. Generational anticipation is evident in the last two generations. All familial ALS patients and one of the six sporadic patients carry the same L84F missense point mutation in exon 4 of the SOD1 gene. SOD1 enzyme activity and SOD1 protein levels were not decreased significantly in the L84F patients. CONCLUSION: The ALS patients carrying the L84F mutation derive from a common ancestor. This mutation is responsible for ALS clustering in the area. The L84F mutation does not modify SOD1-specific activity.

Adult↗
PubMed1999

NKp44, a triggering receptor involved in tumor cell lysis by activated human natural killer cells, is a novel member of the immunoglobulin superfamily.

Surface receptors involved in natural killer (NK) cell triggering during the process of tumor cell lysis have recently been identified. Of these receptors, NKp44 is selectively expressed by IL-2- activated NK cells and may contribute to the increased efficiency of activated NK cells to mediate tumor cell lysis. Here we describe the molecular cloning of NKp44. Analysis of the cloned cDNA indicated that NKp44 is a novel transmembrane glycoprotein belonging to the Immunoglobulin superfamily characterized by a single extracellular V-type domain. The charged amino acid lysine in the transmembrane region may be involved in the association of NKp44 with the signal transducing molecule killer activating receptor-associated polypeptide (KARAP)/DAP12. These molecules were found to be crucial for the surface expression of NKp44. In agreement with data of NKp44 surface expression, the NKp44 transcripts were strictly confined to activated NK cells and to a minor subset of TCR-gamma/delta+ T lymphocytes. Unlike genes coding for other receptors involved in NK cell triggering or inhibition, the NKp44 gene is on human chromosome 6.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Heterogeneous distribution of amyotrophic lateral sclerosis patients with SOD-1 gene mutations: preliminary data on an Italian survey.

We report the absence of superoxide dismutase (SOD-1) gene mutations in 30 patients with amyotrophic lateral sclerosis (ALS) including individuals with a confirmed family history of ALS (familial ALS/FALS), ALS with an unclear family history (UFALS) and sporadic ALS (SALS). Single strand conformation polymorphism (SSCP) and sequence analysis of the 5 SOD-1 gene exons were undertaken to improve the accuracy of the mutation detection. Our preliminary data appear to diverge from the results of studies by other groups using different populations. We discuss the possible reasons for this disparity and the apparent heterogeneous distribution of ALS with SOD-1 gene mutations among different ethnic groups.

Adult↗
PubMed1999

CD40-Mediated induction of CD4 and CXCR4 on B lymphocytes correlates with restricted susceptibility to human immunodeficiency virus type 1 infection: potential role of B lymphocytes as a viral reservoir.

Human immunodeficiency virus type 1 (HIV-1) replicates primarily in lymphoid tissues where it has ready access to activated immune competent cells. We used one of the major pathways of immune activation, namely, CD40-CD40L interactions, to study the infectability of B lymphocytes isolated from peripheral blood mononuclear cells. Highly enriched populations of B lymphocytes generated in the presence of interleukin-4 and oligomeric soluble CD40L upregulated costimulatory and activation markers, as well as HIV-1 receptors CD4 and CXCR4, but not CCR5. By using single-round competent luciferase viruses complemented with either amphotropic or HIV-derived envelopes, we found a direct correlation between upregulation of HIV-1 receptors and the susceptibility of the B lymphocytes to infection with dual-tropic and T-tropic strains of HIV-1; in contrast, cells were resistant to M-tropic strains of HIV-1. HIV-1 envelope-mediated infection was completely abolished with either an anti-CD4 monoclonal antibody or a peptide known to directly block CXCR4 usage and partially blocked with stromal cell-derived factor 1, all of which had no effect on the entry of virus pseudotyped with amphotropic envelope. Full virus replication kinetics confirmed that infection depends on CXCR4 usage. Furthermore, productive cycles of virus replication occurred rapidly yet under most conditions, without the appearance of syncytia. Thus, an activated immunological environment may induce the expression of HIV-1 receptors on B lymphocytes, priming them for infection with selective strains of HIV-1 and allowing them to serve as a potential viral reservoir.

B-Lymphocytes↗
PubMed1998

Molecular cloning of NKp46: a novel member of the immunoglobulin superfamily involved in triggering of natural cytotoxicity.

NKp46 has been shown to represent a novel, natural killer (NK) cell-specific surface molecule, involved in human NK cell activation. In this study, we further analyzed the role of NKp46 in natural cytotoxicity against different tumor target cells. We provide direct evidence that NKp46 represents a major activating receptor involved in the recognition and lysis of both human and murine tumor cells. Although NKp46 may cooperate with other activating receptors (including the recently identified NKp44 molecule) in the induction of NK-mediated lysis of human tumor cells, it may represent the only human NK receptor involved in recognition of murine target cells. Molecular cloning of the cDNA encoding the NKp46 molecule revealed a novel member of the immunoglobulin (Ig) superfamily, characterized by two C2-type Ig-like domains in the extracellular portion. The transmembrane region contains the positively charged amino acid Arg, which is possibly involved in stabilizing the association with CD3zeta chain. The cytoplasmic portion, spanning 30 amino acids, does not contain immunoreceptor tyrosine-based activating motifs. Analysis of a panel of human/hamster somatic cell hybrids revealed segregation of the NKp46 gene on human chromosome 19. Assessment of the NKp46 mRNA expression in different tissues and cell types unambiguously confirmed the strict NK cell specificity of the NKp46 molecule. Remarkably, in line with the ability of NKp46 to recognize ligand(s) on murine target cells, the cDNA encoding NKp46 was found to be homologous to a cDNA expressed in murine spleen. In conclusion, this study reports the first characterization of the molecular structure of a NK-specific receptor involved in the mechanism of NK cell activation during natural cytotoxicity.

Amino Acid Sequence↗
PubMed1998

Isolation and characterization of trinucleotide repeat containing partial transcripts in human spinal cord.

We report the isolation of 41 partial transcripts containing trinucleotide repeats (TNRs) (CAG/CCG or CAA) from human spinal cord using a polymerase chain reaction-based method. The sequence analysis and database search at the nucleic acid and protein level revealed several classes of TNR-containing partial transcripts (TNRPTs). The TNRPTs included 16 known genes and 10 contained or partially overlapped with 13 expressed sequence tags (ESTs), some of which are known to contain TNRs and others which have previously not been shown to contain these repeats (e.g., clone 54 with homology for the homeobox protein HOX-A5). A further 15 partial transcripts showed no homologies in the databases and therefore may be unique. The validity of this approach is supported by the detection of nervous system-specific genes (e.g., glial fibrillary acid protein) and genes known to show trinucleotide expansions in disease (e.g., AAD10 associated with spino cerebellar ataxia type 2). This method provides a simple approach for the isolation of TNRPTs, from which full-length transcripts can be obtained and the discovery of TNR-containing genes may be facilitated. TNRPTs can also be used to study quantitative gene expression at the transcriptional level, to construct TNR-enriched cDNA libraries, and to make larger contigs from ESTs.

Base Sequence↗