Synthesis and immunological activity of a branched peptide carrying the T-cell epitope of gp43, the major exocellular antigen of Paracoccidioides brasiliensis

The 43 kDa glycoprotein (gp43) of Paracoccidioides brasiliensis is the major diagnostic antigen of paracoccidioidomycosis (PCM), a prevalent fungal infection in South America. A 15-mer sequence from gp43, denominated P10, induced T-CD4+ T helper 1 cellular immune responses in mice of three different haplotypes and protected against intratracheal challenge by a virulent isolate of P. brasiliensis. In an attempt to improve delivery of P10, a promiscuous antigen also presented by human leucocyte antigen-DR alleles, aiming at immunotherapy, we synthesized a multiple antigen peptide with the protective T-cell epitope expressed in a tetravalent 13-mer analog of P10 (M10). M10 induced specific lymph node cell proliferation in mice preimmunized with peptides in complete Freund's adjuvant (CFA). In addition, M10 immunization without CFA significantly protected intratracheally infected mice. We conclude that M10 is a candidate for an anti-PCM vaccine. In this report we describe: (1) the synthesis of M10; (2) the induction of M10-elicited T-cell response and (3) in vivo protection of mice immunized with M10 and challenged by a virulent strain of P. brasiliensis.     

Significance of PBMC response of rheumatic fever patients and control individuals to immunodominant streptococcal M5 protein epitopes

β-hemolytic streptococcal infection in developing countries still causes thousands of cases of Rheumatic Fever (RF). Molecular mimicry between streptococcal M protein (strep M) and heart components has been proposed as the triggering factor leading to autoimmunity in individuals with genetic susceptibility, which is linked to different HLA-DR alleles in different populations. In our hands, RF was significantly associated to HLA-DR7/53. Previous work in our lab has shown that heart-infiltrating T cells that simultaneously recognize strep M and heart proteins. Further, such T cells predominantly recognized the 81-103 strep M5 epitope. In this work, we analysed the proliferative response of peripheral blood mononuclear cells of 99 RF patients and 40 normal controls. Eighty-nine of the RF patients were HLA-typed. As among heart-infiltrating T cells, the 81-103 strep M5 protein epitope is the most frequently recognized epitope among RF PBMC (35.4%), against a 7.5% frequency of proliferation among normal controls (p=0.0018, chi square). However, the 81-103 epitope was as frequently recognized by HLA-DR7,53 positive as by negative individuals (45.2% vs 54.8%, respectively). Taken together, the results suggest that the 81-103 strep M5 epitope may be the immunodominant epitope, “promiscuously” recognized by T cells in a genetically diverse population. The demonstration that molecular mimicry is targeted to a discrete immunodominant “promiscuous” epitope in strep M5 may allow the development of a safe anti-streptococcal synthetic vaccine devoid of such epitopes.     

Genetic load caused by variation in the amount of rDNA in a wasp

Extensive variation in the size of the short (heterochromatic) arm of chromosome 14 was found in the wasp Trypoxylon (Trypargilum) albitarse. Ten different variants were differentiated by size and C-banding pattern. Fluorescent in-situ hybridization (FISH) revealed that ribosomal DNA in this species is clustered in the darkly C-banded parts of the heterochromatic short arm of chromosome 14. On this basis, we got an indirect estimate of the amount of rDNA from the area of these dark C-bands. The significant absence in males of the three chromosome variants with lower amounts of rDNA indicates that these three variants are lethal in this sex, and suggests the existence of a threshold marking the minimum amount of rDNA which is tolerable in haploidy. This implies about 4% genetic load in the population caused by variation in rDNA amount. This revised version was published online in July 2006 with corrections to the Cover Date.     

Short-term pharmacologically induced growth study of ontogenetic allometry of oxygen uptake in children

BACKGROUND: A range of allometric coefficients have been proposed in describing the maximal oxygen uptake (VO2max): body mass relation in children using weight-bearing ergometry. However, a wide deviation in the allometric coefficients for VO2max may be apparent when selected pediatric cohorts are studied in conjunction with clinical intervention for growth abnormalities. AIM: The purpose of this study was to determine the allometric coefficients for VO2max after short-term pharmacologically induced growth in pre- and early pubescent children. SUBJECTS AND METHODS: The treatment group consisted of nine subjects with non-growth hormone (GH)-deficient short stature and one with GH-deficient short stature (mean age: 13.7+/-1.7 years). Ten pre- and early pubescent children matched for age, height, weight, VO2max and body mass index (BMI) were controls. The treatment group were evaluated before (Pre-GH) and after (Post-GH) 4 months of subcutaneous GH therapy (0.05 mgkg(-1)day(-1) x 6 days week(-1)). RESULTS: The mean ontogenetic coefficient for the treatment group was 1.50+/-0.20 and for the control group was 0.77+/-0.34. The mean allometric coefficient for body mass relative to VO2max was significantly higher in the treatment group compared with the control group (p<0.05). Height, weight, fat free mass (FFM), VO2max indexed to body mass (mLkg(-1)min(-1)) and FFM (mLkgFFM(-1)min(-1)) increased (p<0.05) with GH therapy. GH therapy also increased insulin-like growth factor-I (IGF-I) and served as a biochemical marker of GH therapy (p<0.05). The control group had no significant differences in all the variables tested (p<0.05). CONCLUSION: The scaling for oxygen uptake (VO2) for body mass varies with GH treatment and the increase in VO2max that commonly occurs in conjunction with physical growth in the pre-and early pubescent individual may be linked to an increase in FFM and linear size.     

Solubilization and immunological identification of presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in the rat hippocampus

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors have been identified mostly as postsynaptic receptors mediating fast glutamatergic synaptic transmission. However, neurochemical studies based on the modulation of neurotransmitter release have suggested the existence of presynaptic AMPA receptors. We have used a recently described technique that allows a high-purity fractionation of the pre- and postsynaptic proteins of synaptic junctions to evaluate the distribution of the different AMPA receptor subunits in rat hippocampal synapses. Surprisingly, we found very high levels of GluR1- and GluR2/3-like immunoreactivity in the presynaptic fraction, but also in the postsynaptic and extrasynaptic fractions. GluR4-like immunoreactivity was much less abundant but was still detected, predominantly in the postsynaptic fraction. This methodology appears to be far more sensitive than the classical immunogold electron microscopy to determine the localization of synaptic receptors.     

Characterization of Paracoccidioides brasiliensis isolates by random amplified polymorphic DNA analysis.

We initially used 25 different random primers in order to test their ability to generate random amplified polymorphic DNA fragments from the dimorphic human pathogenic fungus Paracoccidioides brasiliensis. From the tested primers we chose five to distinguish between seven isolates of this microorganism. The DNA amplification patterns allowed clear differentiation of the seven isolates into two distinct groups with only 35% genomic identity. One of these groups contained two subgroups with 81% genetic similarity. The random amplified polymorphic DNA analysis method proved to be a good tool for analyzing and comparing different genomes of P. brasiliensis isolates.     

Metacyclogenesis is a major determinant of Leishmania promastigote virulence and attenuation.

The in vivo virulence patterns of promastigote populations defined on the basis of agglutination by the lectin peanut agglutinin (PNA) were studied for various cloned lines of Leishmania major. Promastigotes derived from logarithmic-phase cultures, which were routinely 100% agglutinated at 100 micrograms of PNA per ml, were relatively avirulent for BALB/c mice. The relative virulence of stationary-phase promastigotes appeared to be attributable to the proportion of nonagglutinable (PNA-) promastigotes contained within these populations. Purification of PNA- organisms from stationary cultures provided for each clone the most virulent inoculum, supporting the view that this change in lectin binding accurately reflects the development of infective metacyclic stage promastigotes. By studying this marker, we found that there was considerable variation in the degree to which different strains and clones underwent metacyclogenesis during growth. Examination of a reportedly avirulent L. major clone revealed that metacyclogenesis was unusually delayed and inefficient for this clone, but that those PNA- promastigotes which could be recovered from late-stationary-phase cultures were virulent for BALB/c mice. The loss of virulence associated with frequent subculture could also be attributed to a drastic diminution in metacyclogenesis potential over time. A clone which yielded over 90% PNA- promastigotes during growth within passage 1 generated fewer than 10% PNA- promastigotes during growth by passage 94. Subcloning of late-passage attenuated promastigotes yielded a clone for which no PNA- promastigotes could be generated during growth, and an infective population could not be derived from this clone. Thus, metacyclogenesis does not appear to be stable for even cloned lines of Leishmania promastigotes, and virulence comparisons between different strains and clones can be meaningfully made only if the metacyclic populations contained within the respective inocula are determined.     

Haptic selective attention by foot and by hand

Nonvisual perceptions of a wielded object's spatial properties are based on the quantities expressing the object's mass distribution, quantities that are invariant during the wielding. The mechanoreceptors underlying the kind of haptic perception involved in wielding – referred to as effortful, kinesthetic, or dynamic touch – are those embedded in the muscles, tendons, and ligaments. The present experiment's focus was the selectivity of this muscle-based form of haptic perception. For an occluded rod grasped by the hand at some intermediate position along its length, participants can attend to and report selectively the rod's full length, its partial lengths (fore or aft of the hand), and the position of the grip. The present experiment evaluated whether participants could similarly attend selectively when wielding by foot. For a given rod attached to and wielded by foot or attached to (i.e. grasped) and wielded by hand, participants reported (by magnitude production) the rod's whole length or fractional length leftward of the point of attachment. On measures of mean perceived length, accuracy, and reliability, the degree of differentiation of partial from full extent achieved by means of the foot matched that achieved by means of the hand. Despite their neural, anatomical, and experiential differences, the lower and upper limbs seem to abide by the same principles of selective muscle-based perception and seem to express this perceptual function with equal facility.     

Mycobacteria and human autoimmune disease: direct evidence of cross-reactivity between human lactoferrin and the 65-kilodalton protein of tubercle and leprosy bacilli.

We document here by Western immunoblotting and immunogold ultracytochemistry that polyclonal antibodies against human lactoferrin (Lf) bind to tubercle and leprosy bacilli. In situ immunogold labeling of Mycobacterium leprae (present in armadillo liver and in human skin) and of Mycobacterium tuberculosis indicated that receptors for anti-Lf antibodies were present both on the cytoplasm and on the envelope of the bacilli. We found by immunoblotting that the 65-kDa heat shock protein is the major component of M. leprae and M. tuberculosis that is responsible for the binding of the anti-Lf probe. Furthermore, we show that anti-Lf immunoglobulin G eluted from the nitrocellulose-transferred mycobacterial 65-kDa protein band did bind back to Lf. Ultracytochemistry of biopsy samples of human lepromas showed that dead or severely damaged M. leprae was strongly marked by the anti-Lf antibodies; a similar pattern of immunogold marking was observed on M. leprae when antibodies against the 65-kDa mycobacterial protein were used. Our results offer direct evidence that the 65-kDa protein of leprosy and tubercle bacilli is recognized with specificity by antibodies against the human protein Lf. The Lf-65-kDa protein antigenic cross-reactivity may contribute to the formation of autoantibodies and immune complexes as well as to other autoimmune events that are frequent in tuberculosis and leprosy. Our immunocytochemical data also suggest that the cross-reactivity may persist for some time after the death of mycobacteria in infected hosts.