Actinobacillus actinomycetemcomitans is a facultative gram-negative microorganism which has been implicated as an etiologic agent in localized juvenile periodontitis and in subacute bacterial endocarditis and abscesses. Although resistant to serum bactericidal action and to oxidant injury mediated by superoxide anion (O2-) and hydrogen peroxide (H2O2), this organism is sensitive to killing by the myeloperoxidase-hydrogen peroxide-chloride system (K.T. Miyasaki, M.E. Wilson, and R.J. Genco, Infect. Immun. 53:161-165, 1986). In this study, we examined the sensitivity of A. actinomycetemcomitans to killing by intact neutrophils under aerobic conditions, under anaerobic conditions, and under aerobic conditions in the presence of the heme-protein inhibitor sodium cyanide. Intact neutrophils killed opsonized A. actinomycetemcomitans under aerobic and anaerobic conditions, and the kinetics of these reactions indicated that both oxidative and nonoxidative mechanisms were operative. Oxidative mechanisms contributed significantly, and most of the killing attributable to oxidative mechanisms was inhibited by sodium cyanide, which suggested that the myeloperoxidase-hydrogen peroxide-chloride system participated in the oxidative process. We conclude that human neutrophils are capable of killing A. actinomycetemcomitans by both oxygen-dependent and oxygen-independent pathways, and that most oxygen-dependent killing requires myeloperoxidase activity.
Ninety percent of the 500,000 annual new cases of visceral leishmaniasis occur in India/Bangladesh/Nepal, Sudan and Brazil. Importantly, 80-90% of human infections are sub-clinical or asymptomatic, usually associated with strong cell-mediated immunity. Understanding the environmental and genetic risk factors that determine why two people with the same exposure to infection differ in susceptibility could provide important leads for improved therapies. Recent research using candidate gene association analysis and genome-wide linkage studies (GWLS) in collections of families from Sudan, Brazil and India have identified a number of genes/regions related both to environmental risk factors (e.g. iron), as well as genes that determine type 1 versus type 2 cellular immune responses. However, until now all of the allelic association studies carried out have been underpowered to find genes of small effect sizes (odds ratios or OR<2), and GWLS using multicase pedigrees have only been powered to find single major genes, or at best oligogenic control. The accumulation of large DNA banks from India and Brazil now makes it possible to undertake genome-wide asscociation studies (GWAS), which are ongoing as part of phase two of the Wellcome Trust Case Control Consortium. Data from this analysis should seed research into novel genes and mechanisms that influence susceptibility to visceral leishmaniasis.
leishmaniasis; candidate genes; genome scans
HIV has become increasingly prevalent in the Northeast region of Brazil where Leishmania infantum chagasi is endemic, and concurrent AIDS and visceral leishmaniasis (VL) has emerged. In this study, persons with HIV/AIDS and VL (n = 17) had a mean age of 37.3 years (range 29–53 years) compared with 12.5 years (1–80 years) for persons with VL alone (n = 2836). Males accounted for 88% of cases versus 65%. The mean CD4 count and antileishmanial antibody titre were lower and recurrence of VL and death were more likely with co-infection. Considering the prevalences of L.i. chagasi and HIV in the region, this may herald the emergence of an important public health problem.
Visceral leishmaniasis; HIV; AIDS; Leishmania chagasi infection; Brazil
Psychological stress may impair premenopausal ovarian function and contribute to risk for chronic disease. Soy isoflavones may also influence ovarian function and affect health. Here, we report the effects of a psychological stressor (subordinate social status) and dietary soy on reproductive function and related health indices in female monkeys. We hypothesized that reproductive compromise and adverse health outcomes would be induced in subordinate when compared with dominant monkeys and be mitigated by exposure to soy.
Subjects were 95 adult cynomolgus monkeys (Macaca fascicularis) housed in social groups of five or six. Animals consumed a soy-free, animal protein-based diet during an 8-month Baseline phase and then, during a 32-month Treatment phase, consumed either the baseline diet or an identical diet that substituted high-isoflavone soy protein for animal protein.
Across more than 1200 menstrual cycles, subordinate monkeys consistently exhibited ovarian impairment [increased cycle length (P < 0.02) and variability (P < 0.02) and reduced levels of progesterone (P < 0.04) and estradiol (P < 0.04)]. Subordinate status was confirmed behaviorally and was associated with elevated cortisol (P < 0.04) and relative osteopenia (P < 0.05). Consumption of the soy diet had no significant effects.
(i) Psychological stress adversely affects ovarian function and related health indices in a well-accepted animal model of women's health; (ii) Similar effects may extend to women experiencing reproductive impairment of psychogenic origin; (iii) soy protein and isoflavones neither exacerbate nor mitigate the effects of an adverse psychosocial environment; and (iv) this study was limited by an inability to investigate the genetic and developmental determinants of social status.
functional hypothalamic anovulatory syndrome; stress; reproduction; diet
Visceral leishmaniasis (VL) in northeast Brazil is a disease caused by infection with the protozoan Leishmania chagasi. Infection leads to variable clinical outcomes ranging from asymptomatic infection to potentially fatal disease. Prior studies suggest the genetic background of the host contributes to the development of different outcomes after infection, although it is not known if ancestral background itself influences outcomes. VL is endemic in peri-urban areas about the city of Natal in northeast Brazil. The population of northeast Brazil is a mixture of distinct racial and ethnic groups. We hypothesized that some sub-populations may be more susceptible than others to develop different clinical outcomes after L. chagasi infection. Using microsatellite markers, we examined whether admixture of the population as a whole, or markers likely inherited from a distinct ethnic background, differed between individuals with VL, individuals with an asymptomatic infection, or individuals with no infection. There was no apparent significant difference in overall population admixture proportions among the three clinical phenotype groups. However, one marker on Chr. 22 displayed evidence of excess ancestry from putative ancestral populations among different clinical phenotypes, suggesting this region may contains genes determining the course of L. chagasi infection.
leishmaniasis; Brazil; admixture; STRUCTURE; susceptibility; variation
Aims: To compare optotype acuities and re-operation rates in children corrected with a contact lens (CL) compared with an intraocular lens (IOL) following unilateral cataract extraction during infancy in a non-randomised, retrospective case series.
Methods: 25 infants with a unilateral congenital cataract underwent cataract surgery with (IOL group, n = 12) or without (CL group, n = 13) IOL implantation when <7 months of age. Optotype acuities were assessed in 19 of these children at a mean age of 4.3 years (range 3.3–5.5 years). The number of re-operations were assessed in 21 children.
Results: The visual acuity results were similar in the two treatment groups (p = 0.99); however, two of the four (50%) children in the IOL group compared with two of the seven (28%) children in the CL group undergoing surgery during the first 6 weeks of life had 20/40 or better visual acuity. The children in the IOL group had more re-operations than the children in the CL group (mean 1.1 v 0.36). Most of the re-operations in the IOL group were membranectomies performed during the first year of life (median 8.0 months) whereas all of the re-operations in the CL group were the implantation of a secondary IOL later in childhood (mean 2.2 years).
Conclusion: Optotype acuities were similar for the children corrected with a CL compared with IOL, while the children in the IOL group underwent more re-operations .
cataract; amblyopia; intraocular lens; contact lens; infants
Paediatric cataract blindness presents an enormous problem to developing countries in terms of human morbidity, economic loss, and social burden. Managing cataracts in children remains a challenge: treatment is often difficult, tedious, and requires a dedicated team effort. To assure the best long term outcome for cataract blind children, appropriate paediatric surgical techniques need to be defined and adopted by ophthalmic surgeons of developing countries. The high cost of operative equipment and the uneven world distribution of ophthalmologists, paediatricians, and anaesthetists create unique challenges. This review focuses on issues related to paediatric cataract management that are appropriate and suitable for ophthalmic surgeons in the developing world. Practical guidelines and recommendations have also been provided for ophthalmic surgeons and health planners dealing with childhood cataract management in the developing world.
paediatric cataract surgery; intraocular lens; developing world
Sera of localized juvenile periodontitis (LJP) patients colonized by Actinobacillus actinomycetemcomitans serotype b often contain markedly elevated levels of immunoglobulin G (IgG) antibodies to serospecific determinants in the O polysaccharide of lipopolysaccharide (LPS), as well as to outer membrane proteins of this species. IgG antibodies in LJP sera are known to opsonize A. actinomycetemcomitans for subsequent phagocytosis and killing by human neutrophils. The objective of this study was to determine whether outer membrane proteins or serospecific determinants in LPS are the primary target for opsonic IgG antibodies in LJP sera. An A. actinomycetemcomitans serotype b O-polysaccharide affinity column was constructed and subsequently used to purify LPS-specific IgG antibodies from LJP serum. The affinity-purified anti-LPS IgG antibodies were enriched in content of IgG2 (66.2%, compared with 37.0% in the total IgG fraction) and were immunospecific for A. actinomycetemcomitans serotype b LPS. In an opsonophagocytic assay using neutrophils from donors who were homozygous for the H131 allotype of Fcy receptor IIa (CD32), it was found that LPS-specific IgG antibodies exhibited substantially greater opsonic activity toward A. actinomycetemcomitans serotype b than an LJP IgG fraction that was depleted of LPS-reactive antibodies but contained antibodies against outer membrane proteins of this species. The results of this study indicate that serospecific determinants in the O polysaccharide of A. actinomycetemcomitans serotype b are a principal target for opsonic antibodies in sera of LJP subjects.
The objective of this study was to determine if a relationship exists between antibody reactive with the Actinobacillus actinomycetemcomitans serotype b lipopolysaccharide (LPS) and severity of periodontal disease in generalized early-onset periodontitis (G-EOP). The concentration of antibody reactive with A. actinomycetemcomitans serotype b LPS was determined for 102 G-EOP subjects. Analysis of the relationship between antibody reactive with A. actinomycetemcomitans serotype b LPS and measures of periodontal attachment loss indicated that the patients with the highest concentrations of antibody reactive with A. actinomycetemcomitans serotype b LPS had significantly less attachment loss. These high-responder subjects also had anti-A. actinomycetemcomitans serotype b LPS with significantly higher relative avidity. The results suggest that antibody reactive with A. actinomycetemcomitans serotype b LPS is protective in G-EOP patients.
The ability of the protozoan Leishmania chagasi to infect a vertebrate host depends on its ability to survive intracellularly in a mammalian macrophage. Novel patterns of gene expression are probably important for conversion from the extracellular promastigote to the obligate intracellular amastigote parasite form. We found that the human macrophage-like cell line U937 provided an in vitro model of phagocytosis of L. chagasi promastigotes and intracellular conversion to amastigotes, allowing examination of parasite protein and RNA expression. The Leishmania surface protease gp63 assumed three isoforms during stage conversion, and a 64-kDa form of gp63 not present in promastigotes became the most prominent form in amastigotes. gp63 RNAs derived from the three different classes of msp genes (mspS, mspL, and mspC) were also differentially expressed. Infectious promastigotes contained mRNAs from mspS and mspC genes, whereas converting parasites expressed only mspL and mspC mRNAs. Sequence analysis of clones from an amastigote cDNA library confirmed the presence of gp63 mRNAs only from mspL and mspC class genes in tissue-derived amastigotes. Finally, 24 h after phagocytosis, there was a transient increase in the level of hsp70 and hsp90 proteins that subsequently decreased to baseline; this increase was not due to heat shock alone. We conclude that a unique pattern of selected L. chagasi proteins and RNAs is induced following phagocytosis by macrophages.
We previously reported that the serotype b antigen of Actinobacillus actinomycetemcomitans is a constituent of the polysaccharide region of lipopolysaccharide (LPS) and contains significant amount of the neutral sugars rhamnose and fucose (M. Wilson and R. Schifferle, Infect. Immun. 59:1544-1551, 1991). In the present study, we determined the structure of the O antigen of A. actinomycetemcomitans Y4 (serotype b) LPS. Aqueous phase LPS was obtained from a phenol-water extract of A. actinomycetemcomitans Y4. This material was found to react with rabbit polyclonal antiserum to serotype b but not with antisera specific for other A. actinomycetemcomitans serotypes. Analyses revealed that the O polysaccharide of Y4 LPS consists of a polymer of trisaccharide repeating units composed of D-Fuc, AL-Rha, and D-GalNAc residues. An identical structure was obtained for the O polysaccharide of LPS from A. actinomycetemcomitans JP2, another serotype b strain. These results indicate that the serotype b antigen of A. actinomycetemcomitans is defined by a trisaccharide repeating unit present in the O polysaccharide of LPS.
Cellular immune mechanisms resulting in gamma interferon production are critical for protection against visceral leishmaniasis. Antigens stimulating T-cell responses are likely present in the intracellular amastigote form of the parasite, since this is the form found in a mammalian host. To identify T-cell antigens of Leishmania chagasi, the parasite causing South American visceral leishmaniasis, we used a double antibody-T-cell technique to screen an amastigote cDNA library. One cDNA selected (Lcr1) encodes an antigen that stimulated proliferation of splenic T lymphocytes from infected mice that were either resistant (C3H.HeJ) or susceptible (BALB/c) to L. chagasi infection. The Lcr1 cDNA contains four highly divergent 201-bp repeats homologous to the 204-bp repeat of a Trypanosoma cruzi flagellar antigen gene. Results are consistent with a single copy of the Lcr1 gene producing an mRNA of > 10 kb and a protein of > 200 kDa. Recombinant Lcr1, cloned adjacent to polyhistidine and purified on a nickel affinity column, stimulated gamma interferon but not interleukin-4 (IL-4), IL-5, or IL-10 secretion by T-cell-enriched splenocytes from either susceptible or resistant mice during L. chagasi infection. Immunization with Lcr1 partially protected BALB/c mice against challenge with L. chagasi, indicating the utility of the double screening approach in selecting relevant T-cell antigens.
Travel is a potent force in the emergence of disease. Migration of humans has been the pathway for disseminating infectious diseases throughout recorded history and will continue to shape the emergence, frequency, and spread of infections in geographic areas and populations. The current volume, speed, and reach of travel are unprecedented. The consequences of travel extend beyond the traveler to the population visited and the ecosystem. When they travel, humans carry their genetic makeup, immunologic sequelae of past infections, cultural preferences, customs, and behavioral patterns. Microbes, animals, and other biologic life also accompany them. Today's massive movement of humans and materials sets the stage for mixing diverse genetic pools at rates and in combinations previously unknown. Concomitant changes in the environment, climate, technology, land use, human behavior, and demographics converge to favor the emergence of infectious diseases caused by a broad range of organisms in humans, as well as in plants and animals.
Sera from patients with localized juvenile periodontitis (LJP) often contain markedly elevated levels of immunoglobulin G2 (IgG2) antibodies reactive to cell envelope constituents of Actinobacillus actinomycetemcomitans. The objective of this study was to determine if these IgG2 antibodies are capable of supporting phagocytosis and killing of A. actinomycetemcomitans by human neutrophils. Polyclonal IgG2 antibodies were prepared from high-titer LJP serum by affinity chromatography, yielding a preparation which was > 99% subclass restricted and retained immunoreactivity to A. actinomycetemcomitans antigens. Affinity-purified IgG2 antibodies were evaluated by an in vitro opsonophagocytic assay that employed neutrophils obtained from donors who were homozygous for the H131 allotype of Fc gamma receptor type IIa (CD32), which efficiently binds human IgG2 antibodies. Affinity-purified IgG2 antibodies from LJP serum but not from sera of periodontally healthy individuals promoted phagocytosis and killing of A. actinomycetemcomitans. The expression of IgG2-dependent opsonic activity required the presence of complement. Incubation of A. actinomycetemcomitans with neutrophils in the presence of an optimal concentration of LJP IgG2 (50 micrograms/ml) and 5% hypogammaglobulinemic serum (as a complement source) resulted in a > 1 log10 reduction in bacterial viability within 30 min. The opsonic activity of IgG2 antibodies was found to be comparable to that observed with affinity-purified IgG1 antibodies. Moreover, IgG1 antibodies interacted synergistically with IgG2 antibodies in promoting opsonophagocytosis of A. actinomycetemcomitans. The results of this study indicate that LJP serum contains IgG2 antibodies which, when employed in conjunction with neutrophils that express Fc gamma receptors capable of recognizing this subclass, are opsonic for A. actinomycetemcomitans.
The cell envelope of Actinobacillus actinomycetemcomitans includes a number of outer membrane proteins (OMPs) which appear to be important targets for immunoglobulin G (IgG) antibodies in sera from localized juvenile periodontitis (LJP) patients. In this study, we examined the subclass distribution of IgG antibodies reactive to the 16.6- and 29-kDa OMPs of A. actinomycetemcomitans in sera from LJP patients and periodontally healthy individuals. Antibody responses were determined in a quantitative enzyme-linked immunosorbent assay that employed human IgG subclass-restricted monoclonal antibodies. High-titer LJP sera (93% black; geometric mean titer, 32,673) were found to contain significantly elevated levels of IgG1, IgG2, and IgG3 antibodies to the 29-kDa OMP of A. actinomycetemcomitans, compared with those of low-titer LJP sera (mean titer, 1,421) and sera from periodontally healthy, race-matched control subjects. The concentration of IgG2 antibody to this protein was greater than or equal to the corresponding IgG1 concentration in 7 of 14 high-titer sera, although mean IgG1 and IgG2 concentrations were not significantly different. The concentrations of IgG1 and IgG2 antibodies to the 16.6-kDa protein were also significantly elevated in LJP sera, although of considerably lesser magnitude than that observed for the 29-kDa protein. The IgG2 response to the 29-kDa protein could not be attributed to the presence of IgG2 antibodies to lipopolysaccharide contaminants or to Fc-binding activity, nor does this molecule appear to be a glycoprotein. Hence, LJP subjects produce IgG2 antibodies, as well as IgG1 and IgG3 antibodies, directed to at least one of the major OMPs of A. actinomycetemcomitans.
The aim of the present study was to determine the effect of the antibiotic cefpodoxime on the gram-negative periodontopathic microorganism Actinobacillus actinomycetemcomitans and its interaction with elements of the host immune system. Growth of A. actinomycetemcomitans in subinhibitory concentrations of cefpodoxime induced morphological changes in the bacteria, causing the organisms to grow as filaments rather than coccobacilli. Growth in cefpodoxime did not render these bacteria susceptible to killing by serum, nor did it abrogate the requirement for serum opsonins to support the bactericidal activity of neutrophils. Cefpodoxime enhanced the susceptibility of A. actinomycetemcomitans to the bactericidal activity of neutrophils. In the presence of suitable opsonins, neutrophils were able to kill four times as many cefpodoxime-induced A. actinomycetemcomitans filaments as untreated A. actinomycetemcomitans CFU. This effect was due to antibiotic actions on the bacterium and not on the neutrophil. At inhibitory concentrations, the bactericidal activities of cefpodoxime and neutrophils were additive, and cefpodoxime did not interfere with the normal functioning of the neutrophils. Concomitant with these morphological and functional changes, the expression of two outer membrane proteins (66 and 29 kDa) and one inner membrane protein (57 kDa) was decreased in A. actinomycetemcomitans grown in cefpodoxime. The concentration range over which cefpodoxime is effective against A. actinomycetemcomitans in vivo may be extended by the ability of subinhibitory concentrations to enhance the susceptibility of this organism to host immune defenses.
We compared the sensitivities of oral and nonoral isolates of Actinobacillus actinomycetemcomitans, Haemophilus segnis, H. aphrophilus, and H. paraphrophilus to the bactericidal action of reagent hydrogen peroxide (H2O2). Susceptibility to a range of H2O2 concentrations (10(-6) to 10(-3) M) was assessed by incubating bacterial suspensions for 1 h at 37 degrees C in the presence of H2O2 and plating on chocolate agar to determine the concentration of H2O2 that would produce a 50% reduction in CFU (50% lethal dose). As a group, A. actinomycetemcomitans was more resistant to H2O2 than the oral haemophili, and H. aphrophilus was much more sensitive than all other organisms tested. The range of 50% lethal dose values for A. actinomycetemcomitans was between 8.5 X 10(-5) and 10(-3) M H2O2 or above. In contrast, H. aphrophilus exhibited 50% lethal dose values from below 1 X 10(-6) to 3.4 X 10(-4) M H2O2. The resistance of A. actinomycetemcomitans to H2O2 may be sufficient to protect these organisms from direct H2O2-mediated killing by host phagocytes.
Gentamicin- and methicillin-resistant strains of Staphylococcus aureus have been isolated from Spring 1979 to the present from many hospitals in New York City. A large proportion of the strains were resistant to the majority of antistaphylococcal antibiotics. The ratio of multiply resistant strains was highest among tetracycline-resistant strains. There were significant differences in phage susceptibility patterns and the resistance spectrum of strains isolated at different hospitals, whereas strains isolated at the same hospital often showed a marked degree of similarity. This suggests multiple origins of gentamicin- and methicillin-resistant strains isolated in New York City.
Ten anaesthetists were asked to make judgments on fitness for elective operation on data derived from 200 patients. The extent of their agreement was measured using a kappa statistic, and clusters of anaesthetists who agreed well with each other were identified. Using an alternative technique, the "true" fitness category of each patient was estimated using a maximum likelihood method which estimated the error involved in making judgments on limited amounts of information. It was possible to compare the performance of each anaesthetist against the consensus and to measure deviation on an "optimism--pessimism" continuum. A simple questionnaire predicted fitness for operation by all 10 anaesthetists in 96% of cases.
At the onset of infection, Leishmania promastigotes are phagocytized by mammalian macrophages. They must survive despite exposure to toxic oxidants such as hydrogen peroxide (H2O2) and superoxide (.O2-) generated during phagocytosis. We investigated the effects of these oxidants on Leishmania chagasi promastigotes and promastigote mechanisms for oxidant resistance. According to spin trapping and electron paramagnetic resonance spectrometry, .O2- could be generated by exposure of promastigotes to the redox-cycling compound menadione. Incubation in either menadione or H2O2 caused a concentration-dependent loss of promastigote viability. However, incubation in sublethal concentrations of H2O2 or menadione caused a stress response in promastigotes. This oxidant-induced response was associated with an increase in the amount of heat shock protein hsp70. Induction of a stress response by exposure of promastigotes either to heat shock or to sublethal oxidants (H2O2 or menadione) caused promastigotes to become more resistant to H2O2 toxicity. Sublethal menadione also caused promastigotes to become more virulent in a BALB/c mouse model of leishmaniasis. We previously correlated H2O2 cytotoxicity for promastigotes with the formation of hydroxyl radical (.OH) from H2O2. However, according to electron paramagnetic resonance spectrometry, the increase in H2O2 resistance after exposure to sublethal oxidants was not associated with diminished generation (i.e., scavenging) of .OH. These data suggest that there is a cross-protective stress response that occurs after exposure of L. chagasi promastigotes to heat shock or to sublethal H2O2 or .O2-, exposures that also occur during natural infection. This response results in increased resistance to H2O2 toxicity and increased virulence for a mammalian host.