Acquired immune deficiency in Haitians: opportunistic infections in previously healthy Haitian immigrants

We describe acquired immune deficiency manifested by opportunistic infections in 10 previously healthy heterosexual Haitian men. The opportunistic pathogens included Toxoplasma gondii (in four patients), Cryptococcus neoformans (in one), Pneumocystis carinii (in four patients), and Candida albicans (in three). Six of the patients also had Mycobacterium tuberculosis. Immunologic studies of three patients showed a decrease in the numbers and activity of helper T cells, with normal or increased populations of suppressor T cells. Serologic markers for previous infections from hepatitis A, cytomegalovirus, and herpes simplex virus were detected in several patients. Six of the patients died despite specific antimicrobial therapy. The clinical and immunologic findings in these 10 Haitians are similar to those reported in drug addicts and homosexuals with the acquired immune-deficiency syndrome.     

Expression in Escherichia coli of a dominant immunogen of Trypanosoma cruzi recognized by human chagasic sera.

A genomic clone expressing a Trypanosoma cruzi antigen in Escherichia coli was identified using human chagasic sera. Chagasic antibodies affinity purified on extracts of this clone recognized a high-molecular-weight protein expressed in all developmental stages of the parasite life cycle, as well as in various T. cruzi strains. The antigen is associated with the cytoskeleton of the parasite and localizes along the attachment region between the flagellum and the cell body. Antibodies to the recombinant antigen were detected in the sera of 115 chagasic patients from different endemic regions, but not in sera of patients with leishmaniasis, T. rangeli infection, or other parasitic diseases. Our data suggest that the presence of antibodies to this antigen may be specifically associated with Chagas' disease.     

Mapping of B-Cell Epitopes in a Trypanosoma cruzi Immunodominant Antigen Expressed in Natural Infections

Tc40 is an immunodominant antigen present in natural Trypanosoma cruzi infections. This immunogen was thoroughly mapped by using overlapping amino acid sequences identified by gene cloning and chemical peptide synthesis. To map continuous epitopes of the Tc40 antigen, an epitope expression library was constructed and screened with sera from human chagasic patients. A major, linear B-cell epitope spanning residues 403 to 426 (PAKAAAPPAA) was identified in the central domain of Tc40. A synthetic peptide spanning this region reacted strongly with 89.8% of the serum samples from T. cruzi-infected individuals. This indicates that the main antigenic site is defined by the linear sequence of the peptide rather than a conformation-dependent structure. The major B-cell epitope of Tc40 shares a high degree of sequence identity with T. cruzi ribosomal and RNA binding proteins, suggesting the existence of cross-reactivity among these molecules.     

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.     

A dengue-2 Envelope fragment inserted within the structure of the P64k meningococcal protein carrier enables a functional immune response against the virus in mice

A gene fragment encoding for the amino acids (aa) 286-426 from the dengue Envelope (E) protein was expressed in Escherichia coli as two forms of fusion proteins. In one case, the E fragment was fused to the first 45 aa of the P64k protein from Neisseria meningitidis (PD2) while, in the other, it was inserted within the lipoil-binding domain of the aforementioned bacterial protein (PD3). PD2 was obtained as insoluble form within the cytoplasm of the bacteria while PD3 was distributed equally as soluble and insoluble forms. The insoluble forms of each protein as well as the soluble fraction of PD3 were semipurified to test the antigenicity and the immunogenicity in mice. The forms containing the entire P64k protein exhibited the highest recognition with different polyclonal and monoclonal antibodies. Consequently, the neutralizing antibodies elicited by the recombinant proteins were higher in the case of PD3 forms than with PD2, independently of the solubility status. In addition, mice inoculated with the semipurified insoluble form of PD3 were partially protected against lethal challenge with dengue-2 virus, administered by intracerebral inoculation. The results suggested the folding and carrier capacity of the P64k protein over the E fragment, converting PD3 as an attractive vaccine candidate against dengue-2 virus.     

Interleukin-12 mediates resistance to Trypanosoma cruzi in mice and is produced by murine macrophages in response to live trypomastigotes.

Host resistance to infection by Trypanosoma cruzi is dependent on both natural and acquired immune responses. During the first week of infection in mice, NK cell-derived gamma interferon (IFN-gamma) is involved in controlling intracellular parasite replication, mainly through the induction of NO biosynthesis by activated macrophages. Interleukin-12 (IL-12) has been shown to be a powerful cytokine in inducing IFN-gamma synthesis by NK cells, as well as in mediating resistance to different intracellular protozoa. We have therefore studied the ability of T. cruzi to elicit IL-12 synthesis by macrophages and the role of this cytokine in controlling parasite replication during acute infection in mice. Our results show that macrophages cultured in the presence of live trypomastigote forms (but not epimastigotes) release IL-12 that can induce IFN-gamma production by normal spleen cells. IL-12 was detected in as little as 12 h after the addition of the trypomastigotes, and the level of IL-12 peaked at 48 h after the initial macrophage-parasite incubation. The addition of anti-IL-12 monoclonal antibody to macrophage-trypomastigote supernatants dose-dependently inhibited IFN-gamma production by naive splenocytes. Finally, the in vivo role of IL-12 in resistance to infection by T. cruzi was analyzed. Mice treated with anti-IL-12 monoclonal antibody had significantly increased parasitemia and mortality in comparison with those of control infected mice treated with control antibody. Together, these results suggest that macrophage-derived IL-12 plays a major role in controlling the parasitemia in T. cruzi-infected mice and that the animal's resistance during the acute phase of infection may, at least in part, be a consequence of postinfection levels of IL-12.     

Induction of ribonucleotide reductase activity in cells infected with African swine fever virus.

Infection of Vero cells with African swine fever virus (ASFV) resulted in a marked increase in ribonucleotide reductase activity. The induction of ribonucleotide reductase was detected early after infection and was proportional to the multiplicity of infection. Inhibition of viral DNA replication did not affect the induction of the enzyme. Several characteristics could distinguish the virus-induced from the normal cell enzyme. ASFV-induced ribonucleotide reductase was inhibited by magnesium, was more strongly inhibited by hydroxyurea, and had a fourfold lower Km. The virus-induced enzyme was inhibited by deoxyribonucleoside triphosphates and by ATP. The isolation of hydroxyurea-resistant ASFV mutants provided genetic evidence for the viral origin of the induced ribonucleotide reductase. The resistance to hydroxyurea was due to a threefold overproduction of ribonucleotide reductase, as compared to enzyme induction by wild-type ASFV. Hydroxyurea had similar effect in vitro on ribonucleotide reductases induced by wild-type or mutant virus. The gene for the small subunit of the viral enzyme was mapped within a 2.3-kb fragment by hybridization with an oligonucleotide probe designed from a conserved aminoacid sequence of eukaryotic and viral ribonucleotide reductases.     

Presynaptic dopamine D1 receptors attenuate excitatory and inhibitory limbic inputs to the shell region of the rat nucleus accumbens studied in vitro.

1. Intracellular recordings were made from the shell region of the nucleus accumbens in an in vitro slice preparation. The mean resting membrane potential, input resistance, and action potential amplitude of these neurons were -76 +/- 1 mV, 87 +/- 5 M omega and 94 +/- 2 mV (N = 108), respectively. A sample of these neurons (N = 18) was identified as medium spiny neurons with the use of the biocytin-avidin labeling technique. 2. Electrical stimulation of the fornix, subcortical fibers, or neuropil within the nucleus accumbens shell itself elicited a depolarizing postsynaptic potential (PSP). Dopamine (10-100 microM) attenuated PSPs elicited by stimulation of all of these sites. In a paired-pulse stimulation protocol, dopamine was observed to enhance the facilitation of the test response with respect to the conditioning response. 3. The suppressive effect of dopamine was mimicked by the D1 receptor agonist SKF 82958 (10-30 microM), whereas the D2 receptor agonist quinpirole (10-30 microM) was ineffective. The action of dopamine was antagonized by the D1 receptor antagonist Sch 23390 (10-30 microM), but not by the D2 receptor antagonist sulpiride (10-50 microM) or various adrenergic receptor antagonists. 4. The PSP was usually composed of an excitatory postsynaptic potential (EPSP)-inhibitory postsynaptic potential (IPSP) sequence. Dopamine equally attenuated the excitatory and inhibitory component of the synaptic response. The attenuation of both EPSP and IPSP did not depend on membrane potential. 5. Dopamine effects on the resting membrane potential and input resistance were variable and did not correlate with changes in the PSP. Two further indications were found in favor of a presynaptic locus of dopaminergic modulation. First, the time course of the PSP was not altered during dopamine application. Second, dopamine did not attenuate depolarizations induced by bath-applied L-glutamate. In extracellular recordings, it was found that dopamine reduced the population spike but not the presynaptic fiber volley. 6. These findings strongly indicate that dopaminergic modulation of synaptic responses in neurons located in the accumbens shell region is mediated by presynaptic D1 receptors. Notably, dopamine does not exert a purely inhibitory effect on synaptic excitability in the nucleus accumbens, because it suppresses both the excitatory and inhibitory component of the synaptic response.     

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.