Complement binding by two developmental stages of Leishmania major promastigotes varying in expression of a surface lipophosphoglycan.

The binding of complement by two developmentally distinct stages of Leishmania major has been studied. Noninfective log phase growth (LOG) promastigotes (serum sensitive) activate complement with deposition of covalently bound C3b onto the surface of the parasite. Infective, peanut agglutinin (PNA-) metacyclic stage promastigotes (serum resistant) also bear mainly C3b after incubation in serum, but a major portion of deposited C3 is present as a 110 X 10(3) mol wt C3 fragment. Whereas deposition of C3b on LOG promastigotes is mediated through the alternative pathway. PNA- parasites are unable to activate the alternative pathway in nonimmune serum. C3 is released from the parasite surface by proteolytic cleavage, at a rate which is nearly threefold greater for LOG than for PNA- promastigotes. Immunoprecipitation experiments show that the developmentally regulated lipophosphoglycan is a major C3 acceptor on both LOG and PNA- parasites. These experiments, which are the first to compare the form and processing of complement on infective and noninfective promastigotes of Leishmania, provide a framework for further definition of the differential C3 receptor-dependent uptake and survival of these parasites within mononuclear phagocytes.

Risk factors associated with congenital defects that alter hearing or vision in children born in the city of Bogotá between 2002 and 2016.

INTRODUCTION: Congenital defects affecting the auditory and visual capacity of newborns represent a public health problem as they result in substantial disability, directly impacting the quality of life of newborns and their families. OBJECTIVE: To evaluate risk factors associated with congenital defects that alter hearing or vision in newborns in the city of Bogotá between 2002 and 2016. METHOD: Data from the Bogotá Birth Defects Surveillance and Follow-up Program was used, which consolidated data regarding 167 ECLAMC study (Estudio Colaborativo Latino Americano de Malformaciones Congénitas, in spanish) variables in a case-control design to identify risk factors for birth defects after parents provided signed informed consent. Cases were defined as any newborn (alive or stillborn) with a weight greater than 500 g with any visual or hearing abnormality. Controls were defined as newborn in the same hospital and month with no birth defects. Groups were formed according to the case presentation as follows: isolated eye anomaly, isolated ear anomaly, polymalformative, syndromic, and teratogenic. RESULTS: In total, 402,657 births were reviewed, of which 968 cases had some congenital defects that alter hearing or vision. An association was found between the presence of defects and prematurity, as well as between syndromic cases and increasing maternal age. When comparing cases and controls with the risk of having a birth defect, multiparity had an odds ratio (OR) of 1.47 (95% CI: 1.27-1.71), acute respiratory infection had an OR of 2.41 (95% CI: 1.04-5.58), low maternal education level had an OR of 1.34 (95% CI:1.10-1.62), low paternal education had an OR of 1.42, (95% CI:1.17-1.73), manual labor in the maternal occupation had an OR of 1.31 (95% CI:1.03-1.67), and a history of congenital anomalies in the family had an OR of 1.55 (95% CI:1.19-2.00). CONCLUSION: This research allowed the identification of epidemiological data and significant risk factors for congenital defects that alter hearing or vision in the population of Bogotá.

Internal capsule stroke in the common marmoset.

White matter (WM) impairment and motor deficit after stroke are directly related. However, WM injury mechanisms and their relation to motor disturbances are still poorly understood. In humans, the anterior choroidal artery (AChA) irrigates the internal capsule (IC), and stroke to this region can induce isolated motor impairment. The goal of this study was to analyze whether AChA occlusion can injure the IC in the marmoset monkey. The vascular distribution of the marmoset brain was examined by colored latex perfusion and revealed high resemblance to the human brain anatomy. Next, a new approach to electrocoagulate the AChA was developed and chronic experiments showed infarction compromising the IC on magnetic resonance imaging (MRI) scanning (day 4) and histology (day 11). Behavioral analysis was performed using a neurologic score previously developed and our own scoring method. Marmosets showed a decreased score that was still evident at day 10 after AChA electrocoagulation. We developed a new approach able to induce damage to the marmoset IC that may be useful for the detailed study of WM impairment and behavioral changes after stroke in the nonhuman primate.

Sustained improvement in growth velocity and recovery from suboptimal growth hormone (GH) secretion after treatment with human pituitary GH-releasing hormone-(1-44)-NH2.

The sustained effect of human pancreatic GH-releasing hormone [hpGHRH-(1-44)-NH2] on growth rate and GH secretory patterns was studied in 14 patients (10 males and 4 females; aged 10-16 yr; all Tanner stage I or II). Nine children had inadequate spontaneous GH secretion (ISGHS), while 5 had classic GH deficiency. Seven of 9 patients with ISGHS and 1 of 5 patients with GH deficiency were given 2 sc injections/day of 5 micrograms/kg GHRH for 2-3 months; the others received 5 pulses of GHRH (5 micrograms/kg BW.pulse) for 6 nights a week for 2-13 months, given every 3 h. Six of the nine ISGHS patients increased their growth velocity in response to GHRH therapy. These same six patients maintained an increased growth velocity for up to 24 months after GHRH was discontinued. The remaining three ISGHS patients did not show a significant growth response to GHRH administration. Neither a temporary nor a sustained growth response was correlated with spontaneous overnight GH secretion in these patients. In contrast, three of five classical GH deficiency patients exhibited increased growth velocity while undergoing GHRH therapy, but growth returned to preintervention rates upon discontinuation of treatment. The other two of the five classic GH deficiency patients failed to demonstrate any growth response to GHRH treatment. The increased growth velocity that was sustained for long intervals even after discontinuation of GHRH in ISGHS patients may indicate restoration of normal regulation of the hypothalamic-pituitary GH secretion axis.

Partial characterization and staphylocidal activity of thrombin-induced platelet microbicidal protein.

Thrombin-induced platelet microbicidal protein (PMP) is considered to play an important role in preventing an important role in preventing streptococcal endocarditis. However, the structural features and functions of PMPs have not been well characterized, and their antibacterial spectra against other common endocarditis pathogens, such as the staphylococci, are not known. Thrombin stimulation of washed rabbit platelets (10(8)/ml) yielded a PMP-rich preparation with a specific activity of approximately 25 U/mg of protein as determined by Bacillus subtilis bioassay. Twenty-eight clinical and laboratory Staphylococcus aureus isolates, exposed to a standardized PMP preparation (100 U/ml for 2 h at 37 degrees C), exhibited a Poisson-distributed heterogeneity to the bactericidal action of PMP, with approximately one-third designated as PMP resistant. Gel filtration chromatography (Sephadex G-50) identified the bioactive moiety within PMP preparations to be in the major protein elution peak; sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) presumptively identified PMP as a low-molecular-weight (MW) (8,500) protein present only in such bioactive protein peaks. Both the bioactivity of PMP preparations and the low-MW protein band were removable by specific anionic membranes (e.g., cellulose-acetate/nitrate), as well as by a variety of anionic resins, further corroborating the suspected cationic charge of PMP. In addition, both PMP bioactivity and the low-MW protein band were recoverable by 1.5 M NaCl elution of the anionic membrane filters post-PMP adsorptive removal. Adsorption of bioactive PMP preparations by highly PMP-susceptible B. subtilis (10(8) CFU/ml, 30 min) resulted in a near-complete loss of residual bioactivity; in contrast, adsorption of bioactive PMP preparations with less PMP-susceptible S. aureus strains failed to reduce bioactivity. Significant lysozyme contamination of PMP-rich preparations was ruled out by determination of differences between bioactive PMP preparations and exogenous lysozyme as regards (i) relative heat stabilities; (ii) differential bactericidal activity versus B. subtilis and Micrococcus luteus; and (iii) SDS-PAGE protein profiles. These data show that the bioactive PMP protein moiety is of low MW, is heat stable, is probably cationic (similar to leukocyte-derived defensins), and possesses potent bactericidal activity against a significant percentage of S. aureus isolates.

Nonimmune binding of human immunoglobulin A to type II group B streptococci.

The binding of 125I-labeled human myeloma immunoglobulin A (IgA) to four type II strains and one nontypable strain of group B streptococci was measured after streptococcal chains were broken by brief sonication. Some IgA binding was observed with all strains, but specific binding (binding that was inhibited by excess unlabeled IgA, was dose dependent, and was saturable) occurred only with those strains possessing the trypsin-sensitive beta component of the c protein. Similar amounts of binding were observed with myeloma IgA and IgA1 purified from normal serum. Specific binding was more rapid at 25 degrees C than at 0 or 37 degrees C and reached a plateau in 6 to 8 h. Binding was drastically reduced (85 to 90%) when streptococci had been heated (90 degrees C for 1 h). Most radioactivity bound to group B streptococci could be displaced (greater than 60% in 3 days) by the addition of excess unlabeled IgA. The binding capacity of one strain (10(8) streptococci in 1 ml of buffer) was saturated by approximately 24 micrograms of IgA. When transformed for Scatchard analysis, these data indicated that there was a specific binding capacity of 16,000 molecules of monomeric serum IgA per single streptococcal cell. The dissociation constant for IgA binding was 19.3 nM. Since enzyme-linked immunosorbent assay studies showed that the myeloma IgA used for the studies described above was IgA1, our quantitative data apply only to the binding of this subclass to group B streptococci. However, an enzyme-linked immunosorbent-filtration assay showed that both IgA1 and IgA2 bound to a type II group B streptococcus bearing the c protein.

Binding and release of C3 from Leishmania donovani promastigotes during incubation in normal human serum.

We have examined the nature and extent of C3 deposition on Leishmania donovani, strain 1S, clone 2D, promastigotes. Total molecules of C3 bound/parasite after 60 min was similar for parasites incubated in normal human serum, normal human serum adsorbed to remove natural antibody, or either serum source chelated with Mg-EGTA to limit activation to the alternative pathway. A comparison of parasites grown to early, mid, late-log or stationary phases revealed no difference in the extent and kinetics of C3 binding. C3 bound covalently to the parasite primarily through a hydroxylamine resistant (putatively amide) linkage. Of the bound C3, 75% was present as hemolytically inactive iC3b. Nearly 50% of the bound C3 was spontaneously released within 30 min at 37 degrees C. This spontaneous release was due to an unusual proteolytic cleavage event that released C3 from the C3 acceptor on the parasite surface. These results define and characterize the unusual features of C3 binding to L. donovani promastigotes during incubation in serum.

Complement binding on serum-sensitive and serum-resistant transformants of Neisseria gonorrhoeae: effect of presensitization with a non-bactericidal monoclonal antibody.

The binding of C3 and C9 on serum sensitive (FA635) and serum resistant (FA638) transformants of serum sensitive Neisseria gonorrhoeae strain F62 was examined. Previous studies showed that these transformants have Protein IAs which are minimally different by proteinase K cleavage and primary structural and peptide mapping and bear LPS which vary slightly on SDS-PAGE. Binding of C3 and C9 on FA635 exceeded binding on FA638 in NHS and in adsorbed NHS. Monoclonal antibody 4G5, which binds to PI on FA638 but not FA635, increases C9 binding on FA638 to levels 3-3.5 fold greater than on FA635 but does not result in killing. The majority of additional 125IC9 deposited on FA638 following presensitization with 4G5 is released from the bacterial surface by trypsin. These results extend our earlier results with N. gonorrhoeae by showing that, although PI monoclonals can lead to substantial deposition of non-bactericidal C5b-9, this C5b-9 is not fully inserted into the gonococcal outer membrane.

Serum resistance of metacyclic stage Leishmania major promastigotes is due to release of C5b-9.

The mechanism of serum resistance for infective promastigotes of Leishmania major was investigated. Prior results suggested that the mechanism of resistance was mediated at a step after C3 deposition. Equivalent amounts of C3b were deposited on serum-susceptible, noninfective promastigotes harvested from log stage cultures (LOG) and on C-resistant, infective, metacyclic promastigotes (MP) purified from stationary stage cultures. Whereas binding of C9 to LOG was stable during incubation in serum, C9 binding to MP was minimal and unstable, because molecules bound initially to MP were released with continued incubation. Failure to bind C9 was not a result of inability to activate C; the kinetics of C3, C6, and C9 consumption were similar for LOG and MP. Deposition of C5b-7 on MP was stable, indicating that the initial steps in terminal complex formation were intact. Instead, the majority of C5b-9 formed on MP was spontaneously released into the serum as SC5b-9. Residual C5b-9 on MP was released with 1 M NaCl. These data show that developmental modification of the promastigote membrane during transition from a noninfective to an infective stage blocks insertion of lytic C5b-9 into the promastigote membrane.