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Biomedical subjects

A Crisanti

Publications and source records attributed to A Crisanti.

At least 73 records · Page 4Linked to original sources
PubMed1993

Members of a trypsin gene family in Anopheles gambiae are induced in the gut by blood meal.

Serine proteases are among the enzymes that play a crucial role during the digestion of the blood meal in the gut of mosquitoes. The identification of the corresponding genes would have important implications for the control of mosquitoes and mosquito-borne diseases. Analysis of the genomic organization of these genes may lead to the isolation of a gut-specific, inducible promoter for the expression of anti-parasitic agents in transgenic mosquitoes. Moreover, specific inhibitors could be designed on the basis of the structural properties of the enzymes. We report here on the identification of a trypsin gene family in Anopheles gambiae, the mosquito vector of malaria in Africa. Mosquito trypsin-related sequences were amplified by PCR using as template cDNA derived from RNA of blood fed mosquitoes. Cloning of the PCR product revealed two distinct sequences. Corresponding full-length cDNA clones were obtained and sequenced. Antryp1 and Antryp2 code for proteins of 274 and 277 amino acids respectively, showing 75% homology at the amino acid level. The deduced amino acid sequences clearly identify them as trypsins. Five additional trypsin sequences were found in overlapping genomic clones. The genes identified are tightly clustered within 11 kb and sequencing indicates that no introns are present. Northern and PCR analysis indicated that the transcription of both Antryp1 and Antryp2 is induced by blood feeding. Moreover, the Antryp1 protein was detected among the proteins of a midgut lysate of blood fed mosquitoes using antisera against recombinant Antryp1. In addition, the recombinant polypeptides derived from Antryp1 and Antryp2 expressed in Escherichia coli showed a strong proteolytic activity against different sets of blood proteins. We conclude that the products of Antryp1 and Antryp2 play an important role in the breakdown of the proteins during the digestion of the blood meal in the mosquito gut.

Amino Acid Sequence↗
PubMed1993

A method for collecting large quantities of Cryptosporidium parasites.

The intestinal mucoso may be regarded as a potential and abundant source of Cryptosporidium parvurn parasites from which all developmental stages might be collected. If intracellular stages could be recovered from the brush border, many of the limitations concerned with the use of oocysts and in vitro cultures may be overcome. Hans-Michael Muller, Lorella Ranucci, Edoordo Pozio and Andrea Crisonti discuss here how this can be done.

Journal Article↗
PubMed1993

Characterization and immunolocalization of a Cryptosporidium protein containing repeated amino acid motifs.

The oocyst wall is one of the components that permits cryptosporidia both to survive in the environment and to retain infectivity. With the aim of identifying Cryptosporidium proteins specifically expressed at the oocyst stage, we screened lambda gt11 genomic libraries of Cryptosporidium parvum with both an oocyst antiserum and a specific genetic probe. We isolated, from distinct libraries, two overlapping clones containing an open reading frame encoding a 1,252-amino-acid polypeptide. The analysis of the deduced amino acid sequence revealed unusually high contents of cysteine, proline, and histidine. The sequence was also characterized by two distinct amino acid motifs, each repeated several times. The DNA sequences coding for the amino acid repeats showed a high frequency of synonymous mutations, a result suggesting that the repeated motifs may be functionally and/or structurally important to the parasite. Antisera and monoclonal antibodies developed against a recombinant polypeptide encompassing the first 786 amino acids revealed that the corresponding protein in C. parvum had an apparent molecular weight of 190,000. Moreover, confocal microscopy analysis with immunofluorescence indicated that the protein was localized on the oocyst wall as a uniform stain and within the oocyst itself as bright granules in close association with the residual body.

Amino Acid Sequence↗
PubMed1993

Analysis of the human antibody response to thrombospondin-related anonymous protein of Plasmodium falciparum.

Thrombospondin-related anonymous protein (TRAP) of the malaria parasite Plasmodium falciparum shares two sequence motifs with other proteins which possess adhesive properties. Recently, findings indicate that TRAP is an antigen which contributes to antisporozoite immunity. We have cloned and expressed the TRAP coding sequences in Escherichia coli to investigate the human humoral immune response against this protein in a region of malaria endemicity of West Africa characterized by a seasonal transmission. Our results show that antibodies against TRAP are present in infected individuals. The anti-TRAP antibodies were analyzed in both a longitudinal and a prospective study. The longitudinal analysis shows seasonal fluctuations of the levels of specific antibodies as well as age-dependent quantitative differences. The immune response is long-lived in most of the adults and some of the older children but short-lived in young children. More importantly, the prospective analysis suggests that the presence of anti-TRAP antibodies in older children before the beginning of malaria transmission correlates with the subsequent control of parasite densities.

Adolescent↗
PubMed1993

Thrombospondin related anonymous protein (TRAP) of Plasmodium falciparum in parasite-host cell interactions.

Thrombospondin related anonymous protein (TRAP) of Plasmodium falciparum is characterized by the presence of an amino acid motif based on the sequence Trp-Ser-Pro-Cys-Ser-Val-Thr-Cys-Gly (WSPCSVTCG) that is found in a growing family of proteins. The sequence WSPCSVTCG is considered to confer sulpho-galactosyl-cerebroside (sulphatide) binding properties to antistasin, TSP, CS protein and properdin. The observation that TRAP is localized both on the micronemes and on the surface of P. falciparum sporozoites would suggest a role played by TRAP, and its putative sulphated glycoconjugates binding motif, in the recognition and/or entry of hepatocytes by the sporozoite. Our results indicated that TRAP constructs, expressed in E. coli, bind to sulpho-galactosyl-cerebrosides (sulphatides) and to the surface of HepG2 cells using the conserved amino acid motif WSPCSVTCG. Antisera raised against TRAP constructs inhibited sporozoite invasion of HepG2 cells thus suggesting, thus, that TRAP may be one of the parasite-encoded molecules implicated in the sporozoite invasion of hepatocytes. Moreover, the possibility that TRAP antibodies may be relevant in malaria immunity is supported by the results obtained in a prospective study conducted in a malaria endemic area. In adolescents, the presence of TRAP antibodies, before malaria transmission, correlated positively with the control of parasite density.

Adolescent↗
PubMed1993

Temporal and spatial expression of serine protease genes in Anopheles gambiae.

Serine proteases play a crucial role during the digestion of the blood meal in the mosquito gut. The isolation and the analysis of the genomic organisation of the corresponding genes may lead to the characterization of gut-specific, inducible promoters, suitable for the expression of anti-parasitic agents in the gut of transgenic mosquitoes. We report here on the identification of a trypsin and a chymotrypsin gene family of Anopheles gambiae. Following a blood meal, the transcription of all members of the two identified gene clusters, seven trypsin genes (Antryp1-7) and two chymotrypsin genes (Anchym1-2), is induced. Recombinant Antryp1 and Antryp2, expressed in E. coli, were both active in vitro against blood proteins. Moreover, mouse sera raised against Antryp1, Anchym1 and Anchym2 recognized the corresponding proteases among the proteins of a lysate prepared from dissected guts of An. gambiae mosquitoes.

Animals↗
PubMed1991

Human antibody response to the major merozoite surface antigen of Plasmodium falciparum is strain specific and short-lived.

The precursor of the major merozoite antigen of Plasmodium falciparum, gp190, is considered a candidate for inclusion in a malaria vaccine. This protein, which consists of conserved, dimorphic, and polymorphic sequences, is very immunogenic in humans. In a longitudinal study carried out with 94 inhabitants of a rural community in Mali, West Africa, we show that in this endemic area naturally acquired gp190-specific antibodies are predominantly directed against the dimorphic parts of one of the main alleles of gp190. The presence of antibodies against these dimorphic regions correlates with the prevalence of the corresponding antigen in the infecting parasite population. Moreover, qualitative as well as quantitative differences were found in the time course of the humoral immune response to the dimorphic regions in adults and children, who differ in their susceptibility to malaria infection.

Adolescent↗
PubMed1991

The human immune response against the major merozoite surface antigen of Plasmodium falciparum.

The major surface antigen of the merozoite (MMSA) is very immunogenic in humans and it is considered a candidate for developing a malaria vaccine. This protein consists of conserved, dimorphic and polymorphic sequences that might differ in their ability to induce immunity. Epidemiological studies were undertaken in two different endemic areas of West Africa with the aim to identify the sequences within the protein that are the target of the humoral and cellular immune responses. Recombinant polypeptides expressed in E. coli, covering the conserved, the dimorphic and polymorphic regions, were used to evaluate the reactivity of sera and of Peripheral Blood Mononuclear Cells (PBMC) from inhabitants of rural communities exposed to P. falciparum transmission. The analysis of the humoral immune response against the MMSA showed that both qualitative and quantitative differences exist among groups of individuals with different susceptibility to P. falciparum infection. Furthermore, an association between intensity of transmission and antibody reactivity against the dimorphic regions was observed in individuals living in a malaria endemic area. The proliferative response of the PBMC was in most cases very low, however, several T cell clones could be established. The dimorphic region of MMSA was shown to contain T cell epitopes together with sequences most frequently recognized by human sera.

Adult↗
PubMed1990

Polymorphism of the TRAP gene of Plasmodium falciparum.

Natural sequence variation of the thrombospondin related anonymous protein (TRAP) gene of Plasmodium falciparum has been investigated by DNA analysis following the polymerase chain reaction amplification, and this shows the gene to be highly polymorphic. The region containing the sequence motif Trp-Ser-Pro-Cys-Ser-Val-Thr-Cys-Gly (WSPCSVTCG), common to TRAP, the circumsporozoite protein, properdin, and thrombospondin, was invariant. Elsewhere in the molecule, over 50 amino acid substitutions are described including the insertion of an in-frame, small-variable tandemly repeating motif between amino acid residues 352 and 353. Only one silent mutation was observed. Most nucleotide changes that occur in the first two codon positions result in conservative amino acid changes. Restriction fragment length polymorphism (RFLP) analysis was used to examine inheritance of TRAP in a cross between the HB3 and 3D7 clones of P. falciparum. Out of nine progeny examined, four possessed the HB3 gene and five the 3D7 gene. The TRAP gene hybridized to chromosome 13. Previous work has shown that a subtelomeric region of chromosome 13 from the 3D7 parent (marked by the HRP-III gene) was favoured strongly in this cross. The TRAP gene, however, is over 1 Mb away from this subtelomeric region and exhibits no such linkage because of chromosome crossovers. Five geographically separate isolates shared the same TRAP sequence as well as the same variant of the Th2R/Th3R region from the circumsporozoite protein. The correlation between independent markers in these isolates suggests that they have a common provenance.

Amino Acid Sequence↗
PubMed1990

The T cell reactivity against the major merozoite protein of Plasmodium falciparum.

We have undertaken a systematic search for T cell epitopes within the sequence of the major merozoite surface antigen (GP190) of Plasmodium falciparum. Recombinant polypeptides expressed in E. coli were used to evaluate the reactivity of peripheral blood mononuclear cells (PBMC) from both inhabitants of a rural community of West Africa exposed to P. falciparum transmission and from German patients with diagnosis of acute malaria. Although the proliferative response of the PBMC was in most cases very low, several T cell clones could be established. Deletion analysis of each gp190-derived polypeptide allowed the identification of six different T cell epitopes. Epitopes could be mapped within the dimorphic region of gp190, which also contains the sequences most frequently recognized by sera from adult individuals living in endemic areas.

Adult↗
PubMed1990

Ultrastructural localization of the putative precursors of the A4 amyloid protein associated with Alzheimer's disease.

Any explanation of the causes of Alzheimer's disease and of its unique cerebral pathologic features must take into account the distribution and ultrastructural localization of the pre-A4 amyloid proteins in tissues and organs. The authors have analyzed the expression of the pre-A4 amyloid proteins in several tissues by immunogold electron microscopy and by immunofluorescence. For this purpose, they have used a mouse monoclonal antibody and a guinea pig antiserum raised against two synthetic peptides corresponding to two different sequences common to all the full-length forms of the A4 amyloid precursors. They observed a tissue-specific distribution of the secreted and the transmembrane form of the precursors. The authors could determine that the secreted form is generated in vivo within the cytoplasm. In the salivary glands and in the adenohypophysis, all the immunoreactivity is associated with the process of secretion, whereas in the muscle, a staining pattern compatible with the presence of the pre-A4 amyloid proteins in the sarcoplasmic reticulum has been observed. This difference in the localization may reflect tissue-specific processing pathways and suggests that posttranslational modifications such as proteolytic removal of the transmembrane and cytoplasmic domains contribute to the structural and thus functional diversity of the A4 amyloid precursors.

Alzheimer Disease↗
PubMed1989

A new tool for the serodiagnosis of acute Plasmodium falciparum malaria in individuals with primary infection.

We have developed an ELISA which detects, with high specificity, antibodies against a major surface protein of P. falciparum merozoites which is a processing product of the precursor glycoprotein gp190. This assay can be used in the diagnosis of acute malaria in individuals with primary infection. Two partial sequences of gp190 were expressed in E. coli as beta-galactosidase (beta-Gal) fusion proteins. The same sequences fused to chloramphenicol acetyltransferase (CAT) or mouse dihydrofolate reductase (DHFR) react with high frequency when sera of acute malaria patients are analyzed in immunoblots. Antibodies from such sera crosslink, via their antigen binding sites, the beta-Gal fusions to the corresponding CAT or DHFR fusions adsorbed to a solid phase as demonstrated by the captured beta-Gal activity. The assay is highly specific, shows extremely low cut off values and should therefore be widely applicable.

Animals↗
PubMed1989

Steroid sensitivity of thymocyte subpopulations during intrathymic differentiation. Effects of 17 beta-estradiol and dexamethasone on subsets expressing T cell antigen receptor or IL-2 receptor.

We have studied the effects of the steroid hormones, 17 beta-estradiol and dexamethasone, on the relative proportion of thymocyte expression of CD4 (L3T4), CD8 (Ly-2), TCR and IL-2R, used to identify different stages of thymocyte differentiation. After short-term in vivo steroid treatment, a significant decrease in the number and proportion of the CD4+/CD8+, double positive subpopulation was observed in parallel with a proportional increase in the percentage of the CD4+/CD8- single positive, of the CD4-/CD8-, double negative and, to a lesser extent, of the CD8+/CD4- subsets. Either steroid treatment increased the proportion of cells expressing either the epsilon-chain of the CD3 complex and/or the beta-chain of the TCR (beta-TCR) (TCR+/CD3+) and the 55 kDa protein of the IL-2R (IL-2R+), related to the increase of CD4+ SP thymocytes and of DN cells, respectively. Furthermore, the increased proportion of CD3+ cells could also be partially related to the increase of both the CD4+ and DN subsets. A decrease of the effect on either DN/IL-2R+ cells or CD4+ SP cells was selectively observed after long-term treatments with 17 beta-estradiol or DEX, respectively. It is concluded that after short-term administration, different steroid hormones mediate a similar selective depletion of DP, TCR-/CD3-, IL2R- cells presumably in an intermediate stage of differentiation. However, either steroid effects evolve differently in long-term treatment schedules, resulting in different effects on early (DN/IL2R+) and late (SP/IL2R-) steps of thymocyte differentiation.

Animals↗
PubMed1989

Development of the human immune response against the major surface protein (gp190) of Plasmodium falciparum.

The 190-kilodalton glycoprotein (gp190) of Plasmodium falciparum, the precursor of the major surface proteins of merozoites, is considered a promising candidate for a blood stage malaria vaccine. DNA sequences specific for the gp190 of the two isolates K1 and MAD20 were subcloned and expressed in Escherichia coli. The panel of fusion proteins obtained represents about 80% of the polymorphic sequences observed so far within various isolates of P. falciparum. Sera from individuals living in a malaria-endemic area of West Africa were tested in immunoblots against the gp190 fusion proteins, and antibody reactivity was mapped to defined regions of the gp190. Depending on the age of the individual and on the presence of parasites in the blood, distinct regions of gp190 were differentially recognized by the respective antibodies. Similarly, the analysis of sera from German patients with acute malaria revealed a distinct pattern. When grouped according to age and to parasitemia, the reactivity of the sera of people living in malaria-endemic areas may indicate a correlation between certain gp190 regions and protective immune response.

Animals↗