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Biomedical subjects

M M Rodrigues

Publications and source records attributed to M M Rodrigues.

At least 37 records · Page 2Linked to original sources
PubMed1998

Malaria vaccine: roadblocks and possible solutions.

Malaria remains the most prevalent and devastating parasitic disease worldwide. Vaccination is considered to be an approach that will complement other strategies for prevention and control of the disease in the future. In the last 10 years, intense studies aimed at the development of a malaria vaccine have provided important knowledge of the nature of the host immunological mechanisms of protection and their respective target antigens. It became well established that protective immune responses can be generated against the distinct stages of Plasmodium. However, in general, protective immune responses are directed at stage-specific antigens. The elucidation of the primary structure of these antigens made possible the generation of synthetic and recombinant proteins that are being extensively used in experimental immunizations against the infection. Today, several epitopes of limited polymorphism have been described and protective immunity can be generated by immunization with them. These epitopes are being tested as primary candidates for a subunit vaccine against malaria. Here we critically review the major roadblocks for the development of a malaria vaccine and provide some insight on how these problems are being solved.

Animals↗
PubMed1997

Temperature differences for trans-glycosylation and hydrolysis reaction reveal an acceptor binding site in the catalytic mechanism of Trypanosoma cruzi trans-sialidase.

Trypanosoma cruzi, the agent of Chagas disease, expresses on its surface a trans-sialidase that catalyzes preferentially the transference of alpha-2,3-linked sialic acid to acceptors containing terminal beta-galactosyl residues, instead of the typical hydrolysis reaction, found in most sialidases. The trans-sialidase is responsible for the acquisition of the host sialic acid by this protozoan parasite, which does not synthesize sialic acids. Here, we have studied some kinetic properties of a recombinant trans-sialidase expressed in Escherichia coli. We found that it has sequential-type kinetics for the transferase reaction, as shown for the parasite-derived enzyme. The rates of sialic acid transfer to water (hydrolysis), and to beta-galactosyl residues have a unique behavior with respect to the reaction temperature. While the hydrolysis rate of sialyllactose increases continuously up to 35 degrees C, the temperature for the maximal rate of trans-glycosylation depends on the acceptor concentration. At low acceptor concentrations the rate of trans-glycosylation is maximal at 13 degrees C and independent of the amount of sialic acid donors. With increasing acceptor concentrations, maximal rates of trans-glycosylation are shifted to higher temperatures. This finding is explained by an 8-fold increase in the Km for the acceptor from 13 degrees C to 33 degrees C. Differences in hydrolysis and transfer rates were also obtained by using 4-methylumbelliferyl-N-acetyl-neuraminic acid. However, its hydrolysis rate is much higher than the rate of transference to lactose, suggesting that a long-lived enzyme-sialosyl intermediate is not formed. In addition, lactose does not increase the rate of methyl-umbelliferone release at any temperature, indicating that the rate limiting step is the aglycon release. Based on these results we propose that transglycosylation in T. cruzi sialidase is favored by the existence of a binding site for beta-galactosyl residues, which accepts the new glycosidic bond as sialic acid is released from the donor. With increasing temperature the affinity for the acceptor decreases, resulting in a concomitant increase in the rate of transfer to water, which, in turn, can be suppressed by increasing the acceptor concentration.

Animals↗
PubMed1997

Acquired immune responses to the N- and C-terminal regions of Plasmodium vivax merozoite surface protein 1 in individuals exposed to malaria.

In this study, we evaluated the naturally acquired immune response to Plasmodium vivax merozoite surface protein 1 (PvMSP1) in individuals with recent clinical episodes of malaria from the state of Para, Brazil. Ten recombinant proteins representing the first 682 amino acids (aa) of the N-terminal region and one representing the final 111 aa of the C-terminal region were expressed in Escherichia coli as glutathione S-transferase fusion proteins. Both of these regions have been suggested as candidates for development of a vaccine against Plasmodium sp. The total frequencies of individuals with antibodies and cellular immune responses to PvMSP1 were high (83.8 and 75%, respectively). The recombinant proteins representing the N- and C-terminal regions were recognized by 51.4 and 64.1% of sera, respectively. The frequency of responders to the C-terminal region increased according to the number of previous malaria episodes, reaching 83.3% after four episodes. Cellular immune response was measured by in vitro proliferation and gamma interferon production. Peripheral blood mononuclear cells of 75 and 47.2% of individuals proliferated in response to stimulation by the N- and C-terminal regions, respectively. Also, we found that one protein representing the N terminus and a second representing the C terminus of PvMSP1 stimulated 54.5% of individuals to secrete gamma interferon. We concluded that PvMSP1 is immunogenic to a large proportion of individuals exposed to malaria. Our results also suggested that the C-terminal region of PvMSP1 containing the two epidermal growth factor-like domains is particularly immunogenic to antibodies and T cells during natural infection in humans.

Adolescent↗
PubMed1997

Passive transfer of a monoclonal antibody specific for a sialic acid-dependent epitope on the surface of Trypanosoma cruzi trypomastigotes reduces infection in mice.

Trypanosoma cruzi, the parasite that causes Chagas' disease, proliferates in the cytosol of mammalian cells. When the trypomastigote forms exit the infected cell, they become extensively sialylated because the parasite contains an enzyme called trans-sialidase. This enzyme efficiently catalyzes the transfer of bound sialic acid residues from host glycoconjugates to acceptors containing terminal beta-galactosyl residues on the parasite surface. The sialic acid acceptors are developmentally regulated mucin-like glycoproteins that are extremely abundant on the trypomastigote surface. In the present study, we determined whether passive transfer of monoclonal antibodies specific for sialic acid acceptors could reduce the acute infection induced by T. cruzi in a highly susceptible mouse strain. We found that passive transfer to naive mice of an immunoglobulin G1 monoclonal antibody directed to a sialylated epitope of these mucin-like glycoproteins significantly decreased parasitemia and the number of tissue parasites as measured by a DNA probe specific for T. cruzi. Upon challenge with trypomastigotes, mice which received this antibody also had a significant increase in survival. A statistically significant reduction in parasitemia could be accomplished with relatively small doses of immunoglobulin, and Fab fragments alone could not mediate protective immunity. The precise mechanism of parasite elimination is unknown; however, this monoclonal antibody does not lyse trypomastigotes in vitro in the presence of human complement or mouse spleen cells.

Animals↗
PubMed1996

Accumulation of HDL apolipoproteins accompanies abnormal cholesterol accumulation in Schnyder's corneal dystrophy.

Schnyder's corneal dystrophy is an autosomal dominant disorder that results in clouding of the central cornea and premature development of peripheral arcus in the cornea. Previous studies showed that abnormal lipid accumulation is the basis for the corneal clouding. We examined whether apolipoproteins are involved in this disorder and characterized the lipid accumulation in the central portion of corneas removed from patients with Schnyder's dystrophy. Our findings show that cholesterol and phospholipid contents increased greater than 10-fold and 5-fold, respectively, in affected compared with normal corneas. In addition, the percentage of cholesterol that was unesterified (63% versus 50%) and the molar ratio of unesterified cholesterol to phospholipid (1.5 versus 0.5) were higher in affected compared with normal corneas. Large multilamellar vesicles and electron-dense granules (100 to 300 nm in diameter) as well as cholesterol crystals accumulated in the extracellular matrix of affected corneas. Immunohistochemical analysis showed that apolipoprotein constituents of HDL (apoA-I, apoA-II, and apoE), but not apoB, a marker of LDL, accumulated in the affected cornea. Western blot analysis confirmed the increased amounts of these HDL apolipoproteins in affected corneas and showed that the apparent molecular weights of the apolipoproteins were normal. Our findings show for the first time that HDL apolipoproteins accumulate in the corneas of patients with Schnyder's corneal dystrophy. Thus, this disorder influences the metabolism of HDL in the corneas of these patients.

Apolipoprotein A-I↗
PubMed1995

Quantification of antigen specific CD8+ T cells using an ELISPOT assay.

An ELISPOT assay to detect and determine the number of antigen specific CD8+ T cells was standardized using cloned murine CD8+ T cells specific for the epitope SYVPSAEQI of a rodent malaria antigen. This assay is based on the detection of IFN-gamma secretion by single cells after their stimulation with antigen. The interferon secretion is visualized as spots revealed by using enzyme labeled anti-IFN-gamma monoclonal antibodies. Using known numbers of cloned murine CD8+ T cells it was determined that the assay detects 80-95% of these CD8+ T cells. The optimal culture conditions for the stimulation of the CD8+ T cells were determined and the antigen concentration, number of antigen presenting cells and supplement of growth factors required to perform the assay were defined. This ELISPOT assay can be performed with spleen cells from immunized mice, and provide the precise number of antigen specific CD8+ T cells present in mixed lymphocyte populations. This method is more sensitive than the chromium-51 release assay, and much simpler than the conventional precursor frequency analysis, providing the number of antigen specific CD8+ T cells in 36-48 h.

Amino Acid Sequence↗
PubMed1994

Effect of local diets with added sucrose on glycemic profiles of healthy and diabetic Brazilian subjects.

OBJECTIVE: We set out to determine the daily glycemic profile of healthy and non-insulin-dependent diabetes mellitus (NIDDM) persons and to test the hypothesis that small amounts of sucrose added to NIDDM meals would not change their responses. METHODS: Thirteen NIDDM and six healthy volunteers participated in the study. They initially consumed a diet similar to their home diet and later a diabetic hospital dietary regimen, with and without 30 g/day sugar replacing equivalent food energy. The hospital diet included their usual foods: bread, milk, rice, beans, meat, vegetables and fruits at breakfast, lunch and dinner. To follow their glycemic profile we drew several blood samples during a 22-hour period. RESULTS: The day-long plasma glucose profile of the NIDDM and healthy subjects showed similar patterns, increasing after the meals and returning later to baseline levels. The extra amount of sucrose consumed did not change the daily glycemic profile or the calculated glycemic area under the 22-hour glycemic curves. These results call attention to the importance of the 22-hour glycemic profile compared to other shorter glycemic indexes. CONCLUSION: The 22-hour profile has obvious advantage for planning day-long diabetic diets, taking in consideration local daily foods and usual eating habits. Maintenance of a small and traditional amount of food, e.g., sucrose, without harmful effects to the subjects, is another advantage of this proposition. It improves patient compliance as well as social daily life.

Adult↗
PubMed1994

[Calculation of recommendations regarding protein intake: their application to preschool, school students and adults taking Brazilian foods].

The recommendations for protein consumption depend on the essential amino acid and total nitrogen content of a diet, and food digestibility. International recommendations are based on egg or milk proteins. However, populations eat different food mixtures. Brazilians use rice and beans as their main protein food source. This study presents different Brazilian diets, with variable amount of rice and beans. The results show that for each diet there is a different amount of protein recommended. Pre-school children, for example, must receive from 1.15 to 1.77 g/protein/day, depending on the mixture of their dietary protein intake. Besides the diet protein's quality and quantity, the total food intake and presence of other essential nutrients, such as iron, calcium and vitamin C has also to be considered. The correct protein recommendation with respect to a diet or a mixture of food, should take into consideration: digestibility, total nitrogen, essential amino acids, presence of others nutrients and weight of food consumed.

Adult↗
PubMed1994

Intrasplenic immunization with infected hepatocytes: a mouse model for studying protective immunity against malaria pre-erythrocytic stage.

Malaria liver forms are the target of antibody or T-cell-mediated immune mechanisms induced by previous or subsequent developmental stages of the parasite. The potential for vaccine development of antigens expressed exclusively in the liver stages has not been fully explored partly because of the lack of an experimental animal model. Here we show that protective immunity against sporozoite-induced infection with Plasmodium yoelii and P. berghei can be obtained by intrasplenic injection of a small number of liver stages of the parasites. The serum of the protected animals did not contain antibodies against sporozoites, liver or blood stage malaria parasites. Protective immunity was abolished by depletion of either CD4+ or CD8+ T cells from the vaccinated mice before challenge.

Animals↗
PubMed1993

Gelsolin immunoreactivity in corneal amyloid, wound healing, and macular and granular dystrophies.

Immunohistologic studies of tissue sections obtained from patients with type 1 or type 2 lattice corneal dystrophy, polymorphic amyloid degeneration, or gelatinous amyloid degeneration were performed by using a monoclonal antibody raised to a chymotryptic fragment inclusive of the carboxy-terminal half of plasma gelsolin, and also with a series of polyclonal antibodies specific for synthetic peptides corresponding to immunogenic epitopes of gelsolin. These epitopes are parts of sequences at the amino- and carboxy-terminal ends of gelsolin, as well as adjacent to and inclusive of the codon 187 mutant 7-11 kD fragment that has been shown to be the subunit protein of amyloid fibrils occurring systemically in patients affected by Finnish type familial amyloidosis. These antibodies were also tested on tissue sections obtained from patients with granular and macular corneal dystrophy, corneal wounds, and normal control corneas. Specificity of staining was established by absorption with gelsolin purified from plasma, or the appropriate synthetic peptide. Gelsolin immunoreactivity was detected in the conjunctival and skin amyloid in familial amyloidosis by using familial amyloid (Finnish type) antibody. In other types of corneal amyloid, including lattice dystrophy type 1, immunoreactivity with gelsolin and synthetic peptides was observed adjacent to the deposits, but rarely within them. In macular dystrophy, variable staining of the deposits could result from the association of subunit proteins with glycosaminoglycans.

Amyloidosis↗
PubMed1993

Spontaneous ulcerative keratitis in immunocompromised patients.

We studied the occurrence of ulcerative keratitis in five eyes of four patients who were examined at the University of Maryland Hospital ophthalmology clinic over a 12-month period. All were young women who were intravenous drug abusers, with no known predisposing factors for ulcerative keratitis. Two patients had acquired immunodeficiency syndrome (AIDS), one was human immunodeficiency virus (HIV)-positive, and the fourth refused HIV testing. One had a corneoscleral limbus to corneoscleral limbus keratitis; three had inferiorly located corneal ulcers (bilateral in one patient with AIDS). Corneal cultures disclosed Capnocytophaga species in the corneoscleral limbus to corneoscleral limbus keratitis. The remaining ulcers were polymicrobial; cultures of three grew Candida albicans, cultures of two grew alpha-hemolytic streptococci, cultures of two grew Staphylococcus aureus, and culture of one grew Pseudomonas aeruginosa. Treatment with topical fortified antibiotics and antifungal agents resulted in complete healing in all four inferiorly located ulcers. The corneal ulcer became perforated and the eye was eviscerated. Histopathologic analysis of the eviscerated specimen disclosed acute keratitis with necrosis and no microorganisms.

Acquired Immunodeficiency Syndrome↗
PubMed1993

A guinea-pig model of corneal jellyfish envenomations.

Corneal jellyfish stings are painful, self-limited injuries which usually produce conjunctival hyperemia and chemosis, corneal edema, and mild iridocyclitis with resolution in 48 hr. We have developed a guinea-pig model of corneal jellyfish stings in order to study pathogenesis of human injury. Guinea-pig eyes were exposed to sea nettle (Chrysaora quinquecirrha) venom in vivo in the following ways: contact with live tentacles, intracameral injection of crude venom, and intracorneal injection of crude jellyfish venom. Slit lamp examination and histologic sections of the eye performed at intervals up to 8 days after exposure to the venom demonstrated conjunctival hyperemia and chemosis, corneal stromal inflammatory edema, anterior chamber inflammation, and opacities on the anterior capsule of the lens. Nematocysts adherent to the corneal epithelium were noted in eyes exposed to live tentacles.

Animals↗
PubMed1993

Dacryocystitis associated with malignant lymphoma of the lacrimal sac.

BACKGROUND: Tumors of the lacrimal sac are unusual, and lymphomas of the lacrimal sac are quite rare. Four patients with a history of well-differentiated, small cell lymphoma or chronic lymphocytic leukemia presented with either acute or chronic dacryocystitis and epiphora due to lymphomatous infiltration of the lacrimal sac. METHODS: All four patients underwent dacryocystorhinostomy with lacrimal sac biopsy. All tissues underwent complete histopathologic evaluation including immunohistochemical studies for cell surface markers and, in addition, were compared with previous biopsies performed for the initial diagnosis of lymphoma. FINDINGS: All biopsies demonstrated small cell well-differentiated lymphoma on histologic and immunofluorescent examination. No patient demonstrated orbital involvement on computed tomography. One patient had previously diagnosed chronic lymphocytic leukemia and one patient demonstrated diffuse lymph node involvement on postoperative systemic evaluation. All four patients underwent additional chemotherapy. No recurrence of dacryocystitis or epiphora occurred. CONCLUSION: Four patients presented with dacryocystitis secondary to lacrimal sac lymphoma. Lymphomatous lacrimal sac infiltration is an unusual cause of dacryocystitis. Biopsy of the lacrimal sac plays a diagnostically important role in dacryocystorhinostomy even in the absence of obvious tumorous involvement of the lacrimal sac mucosa.

Acute Disease↗
PubMed1993

Laser photocoagulation of experimental corneal stromal vascularization. Efficacy and histopathology.

BACKGROUND: Conventional treatment of corneal stromal vascularization is often inadequate. The authors developed a rabbit model of corneal stromal vascularization, treated it with laser photocoagulation, and then studied the histopathology. METHODS: A reproducible model of corneal stromal vascularization was developed in albino rabbits by injecting sodium hydroxide into the corneal stroma. Corneal stromal vascularization was produced in both eyes of 13 rabbits, and treated after stabilization at 5 weeks with 577-nm yellow dye laser in 1 eye of each rabbit. Seven rabbits were followed for 6 months with corneal angiography and photography, and the corneal stromal vascularization quantified with a grid. The other 6 rabbits were killed at 1, 4, 8, 24, 48 hours, and 6 days after laser photocoagulation and examined by light and transmission electron microscopy. RESULTS: Stable corneal stromal vascularization was observed in the anterior and midstroma for at least 6 months in the model. Laser photocoagulation reduced corneal stromal vascularization significantly compared with the controls (P < or = 0.05), resulting in 40.7% +/- 5.0%, 45.3% +/- 3.3%, and 34.9% +/- 5.2% (mean +/- standard error of the mean) reduction at 2, 4, and 6 months, respectively. Maximum inflammatory cell infiltrates were detected at 8 hours after laser photocoagulation, which diminished markedly at 6 days. The stroma of unlasered eyes showed no inflammatory cells and considerably more patent blood vessels than the lasered eyes. In the lasered eyes, transmission electron microscopy showed damaged vascular endothelial cells, extravasated erythrocytes, haphazardly arranged collagen fibrils, thrombus formation, and ghost vessels in the stroma. No damage was observed in the deep corneal stroma or endothelium in the lasered eyes. CONCLUSION: Laser photocoagulation is effective in reducing corneal stromal vascularization in this model for at least 6 months. It does not damage the deeper stroma or endothelium.

Animals↗
PubMed1993

Retinoblastoma. Interphotoreceptor retinoid binding protein mRNA analysis by polymerase chain reaction.

The authors used the polymerase chain reaction (PCR) to detect the mRNA for interphotoreceptor retinoid-binding protein (IRBP/RBP3), a photoreceptor specific protein, in small samples. They carried out these experiments to assess the feasibility of applying this technique to small tumor samples. Surgically excised tumor samples from four enucleations were analyzed. Messenger RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction was followed by phenol-chloroform purification, reverse-transcription and amplification. The primers used were 5' TGATGACTCTGTCAGTG 3' in exon 3 (sense) and 5' TTGTGCTGGAGCATCTC 3' in exon 4 (antisense). Controls included an IRBP cDNA pIRBP 20-700 and RNA from normal human retina. All samples amplified the same size band if detected. Three tumor samples contained IRBP mRNA as indicated by amplified 234 bp band. These three samples showed a high IRBP protein level by slot blot and RNA for IRBP detected by northern blot. Hematoxylin-eosin staining of one of these samples revealed a well differentiated tumor with numerous Flexner-Wintersteiner rosettes. In the fourth tumor, a poorly differentiated neoplasm, no IRBP mRNA was detected. The authors' results showed a qualitative variation of IRBP mRNA levels, usually related to the histologic differentiation, with IRBP expressed in well differentiated tumors as well as in the normal human retina in contrast to a poorly differentiated tumor with no detectable IRBP. The feasibility of the reverse transcriptase-PCR (RT-PCR) technique to detect IRBP mRNA in small retinoblastoma tumors, was demonstrated.

Base Sequence↗