Use of subepithelial connective tissue graft as a biological barrier: a human clinical and histologic case report.

The aim of the present study was to develop a method to study the healing process after gingival grafting and to observe the histologic results after use of the modified edentulous ridge expansion technique. A 47-year-old nonsmoking woman with a noncontributory past medical history affected by edentulism associated with a horizontal alveolar ridge defect was referred to the authors for surgical correction of the deficit to improve implant support and the final esthetics of an implant-borne prosthesis. At the 4-month follow-up visit, a biopsy was performed by a punch technique in the same sites of healing abutment connection. The tissue was elevated from the attached gingival. Clinically, the grafted tissues seemed to be attached to the bone surfaces. The histologic findings revealed dense grafted tissues, providing long-term stability to the area. No ligament or bone, characteristic for periodontal regeneration, were observed. The presence of thick attached keratinized tissue around implants may constitute a protective factor against marginal inflammation or trauma.

Knockdown resistance mutations (kdr) and insecticide susceptibility to DDT and pyrethroids in Anopheles gambiae from Equatorial Guinea.

OBJECTIVES: To determine the frequency of knockdown resistance (kdr) mutations in the malaria vector Anopheles gambiae s.s. from continental Equatorial Guinea; and to relate kdr genotypes with susceptibility to DDT and pyrethroid insecticides in this vector. METHODS: Female mosquitoes were collected in two villages, Miyobo and Ngonamanga, of mainland Equatorial Guinea. Insecticide susceptibility tests were performed following WHO procedures. Anopheles gambiae complex specimens were identified to species and molecular form by PCR. Genotyping of the kdr locus was performed by allele-specific PCR and direct sequencing in a subset of samples. RESULTS: Both M and S molecular forms of A. gambiae were found in Ngonamanga whereas only the S-form was identified in Miyobo. The two kdr mutations were detected in S-form samples of both villages, with a higher frequency of the kdr-e (Leu-1014-Ser) allele (Miyobo: 16%; Ngonamanga: 40%). The kdr-w (Leu-1014-Phe) mutation was also detected in 3% of the M-form. All individuals tested for pyrethroids were susceptible. A mortality rate of 86% was obtained for DDT. An overall kdr allele frequency (i.e. kdr-e + kdr-w) of 22% was detected in DDT resistant individuals, whereas susceptible individuals had a kdr frequency of 6%. CONCLUSION: The co-occurrence of both kdr mutations and reduced susceptibility to DDT found in A. gambiae highlights the importance of implementing efficient surveillance of insecticide resistance in Equatorial Guinea.

Studies on antimalarial drug susceptibility in Colombia, in relation to Pfmdr1 and Pfcrt.

In Colombia, Plasmodium resistance to antimalarials such as chloroquine and antifolates is a serious problem. As a result, the national Colombian health authorities are monitoring the efficacy of alternative drugs and schemes. The study of genetic polymorphisms related with drug resistance is required in the region. In vitro responses to chloroquine, quinine, mefloquine, amodiaquine, desethylamodiaquine, artesunate and dihydroartesunate were carried out by HRP ELISA. SNP analysis in Pfcrt and Pfmdr1 genes was performed by PCR-RFLP in 77 samples from the North West region of Colombia. In vitro resistance to chloroquine was high (74%), followed by mefloquine (30%) and desethylamodiaquine (30%). A positive correlation between the IC(50) of paired drugs was also detected. The allele Pfmdr1 N86 (wild) was present in 100% of the samples and 1246Y (mutant) in 92%. However, their presence did not correlate with in vitro drug resistance. Presence of the mutations K76T and N75E in Pfcrt was confirmed in all samples. Analysis of 4 codons (72, 74, 75 and 76) in pfcrt confirmed the presence of the haplotypes CMET in 91% and SMET in 9% of the samples.

A primer-introduced restriction analysis-polymerase chain reaction method to detect knockdown resistance mutations in Anopheles gambiae.

In the major malaria vector Anopheles gambiae Giles, two point mutations at the voltage-gated sodium channel have been associated with knockdown resistance (kdr) to DDT and pyrethroid insecticides. Simple allele-specific polymerase chain reaction (PCR) assays to detect these single-nucleotide polymorphisms are prone to lack of specificity and therefore alternative techniques have been proposed. However, these may not be easily implemented in many laboratories from malaria endemic regions. Here, we describe a primer-introduced restriction analysis (PIRA)-PCR method to detect kdr mutations in An. gambiae. This method unambiguously identified all six genotypes for the kdr locus in a sample of 113 field-collected mosquitoes for which kdr genotypes had been confirmed by DNA sequencing. Co-occurrence of both kdr alleles was found in sites from Equatorial Guinea and Gabon and the L1014F mutation was detected in M-form individuals from Angola. The PIRA-PCR proved to be a reliable, robust, and simpler alternative for the detection of kdr mutations in this malaria vector.

The three-dimensional reconstruction of the jaw with "bone slat technique" in conjunction with third molar removal.

BACKGROUND: The purpose of this study was to report the outcome of the management of both horizontal and vertical defects of alveolar crest using the bone slat technique approach in conjunction with third molar removal prior to implant placement in the aesthetic area. METHODS: We present a 20-year-old female patient who lost a maxillary lateral incisor. The objective of treatment was to replace the lateral incisor with an implant-supported crown restoration without interfering with the integrity and topography of the adjacent gingival tissues. Because the future implant site showed horizontal and vertical bone defect the Authors decided to perform bone regeneration. The need for such bone augmentation in the younger patient often coincides with the timing for third molar removal. By combining third molar extraction with bone harvest and alveolar grafting, the patient undergoes only one surgical approach. The bone height (9.5 mm) and width (5.7 mm) were measured at the point of interest (tooth 12) both before and after implant placement in the reconstructed panoramic and parasagittal views by Cone Beam Computed Tomography (CBCT) scan. RESULTS: The final results demonstrated an increase in length of 5 mm after bone slat technique (from 9.5 mm to 13.5 mm) and an increase in width of 1 mm (from 5.7 mm to 6.7 mm). ISQ measurements were recorded at the time of implant placement (the mean was: 68.5) and immediately after individualized screw-retained provisional crown (the mean was: 77). CONCLUSIONS: This technique is reliable and aesthetic and functional results appear to be stable and respect this requisite: simple and fast graft harvesting and low risk of morbidity especially in conjunction with third molar removal.

Effect of antibodies on the expression of Plasmodium falciparum circumsporozoite protein gene.

Antibodies are known to play an important role in the control of malaria infection. However, they can modulate parasite development enhancing infection. The effect of anti-Plasmodium antibodies on the expression of circumsporozoite protein gene (csp) was investigated. Plasmodium falciparum 3D7 in vitro cultures were submitted to: i) anti- circumsporozoite protein monoclonal antibody (anti-CSP-mAb) [1microg/ml, 0.1microg/ml, 0.01microg/ml and 0.001microg/ml] and ii) purified IgG Fab fragment from a pool of malaria patients [1mg/ml and 1microg/ml]; and compared to control cultures. After 24h the number of ring infected erythrocytes was determined in order to calculate invasion efficacy. At 48h culture supernatant was collected, and the amount of circumsporozoite protein determined by ELISA, parasitaemia was calculated and cells were processed for RNA preparation. Expression of csp gene was quantified using Real time RT-PCR. There was an increase in parasite growth when treated with lower anti-CSP-mAb concentration, which was associated with lower csp expression, while 1mug/ml anti-CSP-mAb treatment presented a growth inhibitory effect accompanied by high csp expression.

Effect of chloroquine on the expression of genes involved in the mosquito immune response to Plasmodium infection.

Chloroquine has been described to increase Plasmodium infectivity to the mosquito vector and is known to affect the vertebrate host immune response including during malarial infection. Although knowledge of the mosquito immune response has recently improved, nothing is known about the impact of chloroquine on mosquito immunity. In order to characterize the influence of chloroquine on the mosquito immune system, we have analyzed the effect of chloroquine on Anopheles gambiae (i) serine proteases and (ii) antimicrobial peptide gene expression, in uninfected and Plasmodium berghei infected mosquitoes, using real-time PCR. We have demonstrated for the first time that mosquitoes fed on chloroquine-treated mice showed a significant down regulation of some immune-related genes. This effect was independent of midgut bacterial burden. These results suggest that chloroquine might act on the Anopheles serine proteases cascade, interfering with signal transduction pathways and at a transcriptional activation level.

High prevalence of natural antibodies against Plasmodium falciparum 83-kilodalton apical membrane antigen (PF83/AMA-1) as detected by capture-enzyme-linked immunosorbent assay using full-length baculovirus recombinant PF83/AMA-1.

The 83-kilodalton (kD) apical membrane antigen of Plasmodium falciparum (PF83/AMA-1) is a potential asexual blood stage vaccine component. This antigen has been expressed as a full-length, nonfusion, recombinant baculovirus protein (PF83-7G8-1) using the authentic predicted signal peptide for appropriate postsynthetic routing. When purified by a novel high-performance, ion exchange chromatography (HPIEC) method, PF83-7G8-1 induced polyclonal antibodies in rats that immunoprecipitated both 83- and 66-kD forms of PF83/AMA-1 from 35S-methionine metabolically labeled parasite extracts. Using HPIEC-purified PF83-7G8-1 in combination with a rat monoclonal antibody against the highly conserved carboxy-terminal (CT) region of PF83/AMA-1, we developed a CT-capture-enzyme-linked immunosorbent assay to measure naturally acquired responses against the entire PF83/AMA-1 molecule. Analysis of populations from villages in Guinea-Bissau and in an area of high malarial transmission in Senegal demonstrated a very high prevalence (94-100%) of naturally acquired serum IgG responses to PF83/AMA-1. Analysis of these natural responses showed that PF83/AMA-1 may be a well-recognized asexual parasite antigen. A statistically significant age-related change in antibody levels to PF83/AMA-1 was observed in Guinea-Bissau. No such correlation was observed in the Senegalese population, although an age-related antibody response was seen for total parasite antigen. No significant correlation was observed between PF83/AMA-1 responses and the parameters of parasite load and malaria-related fever.

Quantitation of antisporozoite immunoglobulins in the hemolymph of Anopheles stephensi after bloodfeeding.

Passage of rat antibodies induced by Plasmodium falciparum circumsporozoite protein (anti-CS IgG) from the bloodmeal into the hemocoel of uninfected Anopheles stephensi mosquitoes was quantified using enzyme-linked immunosorbent assay (ELISA) techniques. Anti-CS IgG were present in hemolymph immediately upon cessation of mosquito feeding. Titers peaked at 3 hr post-ingestion then declined steadily, becoming negligible at 18 hr. Substantial titers were present in the bloodmeal at 24 hr post-ingestion. By 48 hr, anti-CS IgG in both mosquito hemolymph and bloodmeal were virtually absent. Estimated quantities of anti-CS IgG in the hemolymph at 3 hr post-ingestion were 905-958 ng/ml, representing approximately 0.5% of that present in the host serum. Rat IgG subclasses 1, 2a, and 2b passed into hemolymph more readily than did IgM and possibly IgG2c. Hemolymph volume of unengorged mosquitoes (0.53 microliters) increased after a bloodmeal (0.73 microliters at 3 hr post-ingestion), suggesting that anti-CS IgG may move into the hemocoel in an aqueous solution.

Prevalence of the dihydrofolate reductase Asn-108 mutation as the basis for pyrimethamine-resistant falciparum malaria in the Brazilian Amazon.

Pyrimethamine resistance in cultivated laboratory isolates of Plasmodium falciparum is linked to the dihydrofolate reductase mutation Asn-108, a mutation that acts by interrupting drug binding within the active site of the enzyme. To determine the prevalence of this mutation in endemic regions harboring pyrimethamine-resistant malaria, we used a mutation-specific polymerase chain reaction assay to survey P. falciparum strains from a wide section of the Brazilian Amazon. Mutations were identified directly from blood samples without intervening steps of in vitro cultivation. Of 42 samples collected from four states in Brazil, 38 (90%) contained the Asn-108 codon AAC that confers pyrimethamine resistance, four samples contained only the wild-type Ser-108 codon AGC, and none contained the Thr-108 codon ACC found in cycloguanil-resistant pyrimethamine-sensitive strains. These findings indicate that a very high incidence of the Asn-108 DHFR mutation is responsible for pyrimethamine resistance in the Amazon, and they are consistent with recent failure rates reported for Fansidar (pyrimethamine-sulfadoxine). We suggest that limited use of proguanil be evaluated as an alternative to pyrimethamine.

Plasmodium falciparum: administration of anti-sporozoite antibodies during sporogony results in production of sporozoites which are not neutralized by human anti-circumsporozoite protein vaccine sera.

Five days after receiving a Plasmodium falciparum NF54 infectious blood meal, Anopheles stephensi mosquitoes were fed rat anti-P, falciparum sporozoite or rabbit anti-R32tet32 antibodies. Sporozoites isolated from salivary glands were tested by the inhibition of sporozoite invasion (ISI) assay using monoclonal antibody (Mab) 2A10 to P. falciparum circumsporozoite protein or sera from human volunteers immunized with P. falciparum R32tet32 or (NANP)3-TT vaccines. Whereas sporozoites from control mosquitoes were neutralized by Mab 2A10 and vaccine sera, only the Mab and not the vaccine sera neutralized sporozoites from immune-fed mosquitoes. The implications of these results in vaccine design and the impact on transmission are discussed.

The importance of sensitive detection of malaria parasites in the human and insect hosts in epidemiological studies, as shown by the analysis of field samples from Guinea Bissau.

A method based on the polymerase chain reaction (PCR) for highly sensitive detection and identification of human malaria parasites was applied to blood and mosquito samples obtained from a village in Guinea Bissau. The prevalence of parasites in the human population was shown to be greatly underestimated by microscopical examination. In particular, a high incidence of Plasmodium malariae and P. ovale parasites was revealed only by the PCR assay. Preliminary evidence was obtained to show that the distribution of P. malariae infections within the village was non-random. This was supported by analysis of the parasite species infecting the mosquito vector. The implication of these results for the design and interpretation of epidemiological surveys is discussed.

Activity of human volunteer sera to candidate Plasmodium falciparum circumsporozoite protein vaccines in the inhibition of sporozoite invasion assay of human hepatoma cells and hepatocytes.

Sera from human volunteers immunized with either synthetic peptide (NANP)3-TT or recombinant protein R32tet32 Plasmodium falciparum CS vaccines were tested in the inhibition of sporozoite invasion (ISI) assays using human hepatoma (HepG2-A16) cells or primary human hepatocytes. Sera or purified immunoglobulin (Ig) from volunteers who were completely protected against P. falciparum sporozoite challenge had higher ISI activity than sera from non-protected volunteers, or the highest titre endemic serum. However, Ig from protected and non-protected volunteers did not block sporozoite invasion of human hepatocytes, suggesting that P. falciparum sporozoites invade hepatocytes by mechanisms which differ from those concerned with invasion of HepG2-A16 cells.

Genotyping of Plasmodium falciparum infections by PCR: a comparative multicentre study.

Genetic diversity of malaria parasites represents a major issue in understanding several aspects of malaria infection and disease. Genotyping of Plasmodium falciparum infections with polymerase chain reaction (PCR)-based methods has therefore been introduced in epidemiological studies. Polymorphic regions of the msp1, msp2 and glurp genes are the most frequently used markers for genotyping, but methods may differ. A multicentre study was therefore conducted to evaluate the comparability of results from different laboratories when the same samples were analysed. Analyses of laboratory-cloned lines revealed high specificity but varying sensitivity. Detection of low-density clones was hampered in multiclonal infections. Analyses of isolates from Tanzania and Papua New Guinea revealed similar positivity rates with the same allelic types identified. The number of alleles detected per isolate, however, varied systematically between the laboratories especially at high parasite densities. When the analyses were repeated within the laboratories, high agreement was found in getting positive or negative results but with a random variation in the number of alleles detected. The msp2 locus appeared to be the most informative single marker for analyses of multiplicity of infection. Genotyping by PCR is a powerful tool for studies on genetic diversity of P. falciparum but this study has revealed limitations in comparing results on multiplicity of infection derived from different laboratories and emphasizes the need for highly standardized laboratory protocols.

The palpal ratio method compared with PCR to distinguish between Anopheles gambiae s.s. and A. melas from Guinea Bissau, West Africa.

We compared the palpal ratio method with the polymerase chain reaction (PCR) to distinguish between Anopheles gambiae s.s. and A. melas. At the end of the rainy season of 1995, female mosquitoes were collected indoors in the Antula area of Bissau, Guinea Bissau. A subsample of 354 mosquitoes were identified first with PCR and then with the palpal ratio method (study A). Subsequently, another 195 mosquitoes were identified first with the palpal ratio method and then with PCR (study B). The highest percentage (100%, n = 16) of correctly identified A. melas was obtained at the palpal ratio cut-off point of 0.83. However, at this point 4.0% (14/347) and 11.3% (21/186) of the A. gambiae were erroneously identified as A. melas in study A and B, respectively. This suggests that the palpal ratio method is not sufficiently reliable to distinguish between A. gambiae and A. melas from the Bissau area.

Non-immune patients in the Democratic Republic of São Tomé e Principe reveal a high level of transmission of P. ovale and P. vivax despite low frequency in immune patients.

Malaria parasite species circulating in immune inhabitants of the Democratic Republic of São Tomé e Principe (DRSTP) and those acquired by non-immune travellers returning from this country have been compared. Using sensitive detection and species identification by PCR, Plasmodium parasites were found in the blood of 16 of the 43 travellers, who reported during the first 8 months of 1995 to a clinical diagnosis laboratory in Lisbon. Plasmodium vivax was found in four (25%) and Plasmodium ovale in ten (63%) of these patients. The observed prevalence of these two species in infected patients of DRSTP during 1995/1996 was <2%.

Malaria in São Tomé and Príncipe: parasite prevalences and vector densities.

A cross-sectional survey was carried out in 16 localities on the island of São Tomé and three on the island of Príncipe, at the end of the rainy season of 1997, to determine malaria prevalence and vector densities. Blood samples from 664 inhabitants of all ages were examined by optical microscopy (OM) and PCR. Mosquito collections were made by outdoor landing captures from 21:00-23:00 h. Great differences were found between OM and PCR readings. OM had a sensitivity of 66%, a specificity of 79% and failed to reveal any mixed-infections. Overall prevalence, determined by PCR, was higher in São Tomé (53%) than in Príncipe (35%). It was highest in children below 16 years-old. All four human Plasmodium species occurred in São Tomé but P. ovale was not detected in Príncipe. The human population was largely asymptomatic. Bednet users had lower prevalence than did non-users. The FOREST form of Anopheles gambiae s.s., identified by PCR and cytogenetics, was the only vector on the islands. The sporozoite rate in São Tomé, assessed by ELISA, was 0.5%. Parasite prevalence and vector densities were positively correlated in São Tomé, where malaria transmission must occur predominantly in the more populated coastal areas.

Dogs as a favored host choice of Anopheles gambiae sensu stricto (Diptera: Culicidae) of São Tomé West Africa.

The host source and human blood index (HBI) of an exophilic population of the "forest" cytoform of Anopheles gambiae Giles sensu stricto, from a peri-urban area of the island of São Tomé were assessed. Blood meals of 434 An. gambiae females from all-night indoor light-trap collections, 193 from indoor and 422 from outdoor resting collections, were determined by ELISA. Significant differences were found in the HBI estimates from insects collected indoors (0.93) and outdoors (0.27). Blood-fed insects collected resting outdoors provided the most representative sample for host determination. Dogs were the predominant hosts, followed by humans and pigs. Of all human feeds, it was estimated that 81.5% were taken inside houses. The low HBI of 0.27 for the An. gambiae population explains the low sporozoite rate and the meso-endemicity of malaria in the island.

Refractoriness of Callithrix penicillata red blood cells to Plasmodium falciparum in vivo and in vitro.

Attempts to infect the New World marmot Callithrix penicillata with Plasmodium falciparum were unsuccessful. Attempts were also made to infect red blood cells of C. penicillata and Saimiri sciureus with P. falciparum in vitro, and these too were unsuccessful due to a high rate of hemolysis produced by apparently adverse culture conditions. It is concluded that modifications to the existing culture conditions will need to be made before successful parasitemia can be induced in vitro in simian erythrocytes.

Metabolism of primaquine by liver homogenate fractions. Evidence for monoamine oxidase and cytochrome P450 involvement in the oxidative deamination of primaquine to carboxyprimaquine.

The role of monoamine oxidase (MAO) and cytochrome P450 (P450) in the oxidative deamination of primaquine by rat liver fractions was studied. Rat liver fractions including liver homogenate, mitochondria, microsomes and 100,000 g supematant fractions were prepared from a pool of rat livers and characterised using benzylamine as a probe for MAO activity and N,N-dimethylbenzamide as a probe for P450 N-dealkylation activity. Incubation of all fractions with primaquine yielded carboxyprimaquine as the only metabolite detectable by HPLC. The mitochondrial fraction, which contained MAO activity but not P450 activity, presented the highest Vmax/K(M) value for the formation of carboxyprimaquine (8.5 x 10(-6) dm3mg(-1)h(-1). A substantially lower Vmax/K(M) value (1.3 x 10(-6) dm3mg(-1)h(-1)) was obtained in the microsomal fraction, which contained P450 but not MAO activity. The liver homogenate fraction presented a similar value (1.8 x 10(-6) dm3mg(-1)h(-1), though it contained both enzyme systems. Incubations of all the fractions that presented MAO activity, in presence of the MAO inhibitor pargiline, resulted in a marked inhibition of primaquine oxidation. P450 inhibitor SKF 525-A effectively inhibited primaquine metabolism in the microsomal fraction but inhibition in the liver homogenate was less effective. The results are consistent with an important role for MAO in primaquine biotransformation, though clearly metabolism by P450 has a contribution role.