Roberto P. Stock

Secretion by Trypanosoma cruzi of a peptidyl-prolyl cis-trans isomerase involved in cell infection.

Macrophage infectivity potentiators are membrane proteins described as virulence factors in bacterial intracellular parasites, such as Legionella and Chlamydia. These factors share amino acid homology to eukaryotic peptidyl‐prolyl cis‐trans isomerases that are inhibited by FK506, an inhibitor of signal transduction in mammalian cells with potent immunosuppressor activity. We report here the characterization of a protein released into the culture medium by the infective stage of the protozoan intracellular parasite Trypanosoma cruzi. The protein possesses a peptidyl‐prolyl cis‐trans isomerase activity that is inhibited by FK506 and its non‐immunosuppressing derivative L‐685,818. The corresponding gene presents sequence homology with bacterial macrophage infectivity potentiators. The addition of the protein, produced heterologously in Escherichia coli, to cultures of trypomastigotes and simian epithelial or HeLa cells enhances invasion of the mammalian cells by the parasites. Antibodies raised in mice against the T.cruzi isomerase greatly reduce infectivity. A similar reduction of infectivity is obtained by addition to the cultures of FK506 and L‐685,818. We concluded that the T.cruzi isomerase is involved in cell invasion.

Bringing antivenoms to Sub-Saharan Africa

To reduce unacceptably high death rates from snakebite envenomation, sub-Saharan Africa must adopt not only a new generation of multivalent biotech antivenoms, but also an infrastructure to deliver them.

Inhibition of gene expression in Entamoeba histolytica with antisense peptide nucleic acid oligomers.

Peptide nucleic acids (PNAs) may be a potent tool for gene function studies in medically important parasitic organisms, especially those that have not before been accessible to molecular genetic knockout approaches. One such organism is Entamoeba histolytica, the causative agent of amebiasis, which infects about 500 million people and is the cause of clinical disease in over 40 million each year, mainly in the tropical and subtropical world. We used PNA antisense oligomers to inhibit expression of an episomally expressed gene (neomycin phosphorotransferase, NPT) and a chromosomal gene (EhErd2, a homolog of Erd2, a marker of the Golgi system in eukaryotic cells) in axenically cultured trophozoites of E. histolytica. Measurement of NPT enzyme activity and EhErd2 protein levels, as well as measurement of cellular proliferation, revealed specific decreases in expression of the target genes, and concomitant inhibition of cell growth, in trophozoites treated with micromolar concentrations of unmodified antisense PNA oligomers.

Molecular characterization and transcription of the histone H2B gene from the protozoan parasite Trypanosoma cruzi

The structure, genomic organization and transcription of the gene encoding histone H2B in the protozoan parasite Trypanosoma cruzi have been studied. This gene consists of a 746‐nucleotlde unit, tandemly repeated at least 18 times in each of two clusters. DNA probes corresponding to histones H2B and H3 hybridized to different chromosomes revealing that the genes coding for these two histones are not physically linked in the genome of T. cruzi. The primary transcription product of the H2B gene is processed by trans‐splicing and polyadenylation. Inhibition of DNA synthesis with aphidicolin resulted in the reduction of histone H2B mRNA to undetectable levels in about two hours, suggesting that its abundance is regulated throughout the cell cycle as it occurs in other eukaryotes. in addition, a concomitant inhibition of translation by cycloheximide reverted this effect indicating that de novo protein synthesis is required for RNA instability. Histone mRNA abundance was dependent on the life‐cycle stage of T. cruzi: abundant in amastigotes and epimastigotes, the dividing forms in the host cell and the insect vector, respectively, white undetected in trypomastigotes, the parasites non‐dividing life stage.

Paraspecific neutralization of the venom of African species of cobra by an equine antiserum against Naja melanoleuca: a comparative study.

Venoms of snakes belonging to the same Genera tend to share biochemical, toxinological and antigenic characteristics. Accordingly, paraspecific neutralization of venom lethality by experimental antisera and commercial antivenoms has been reported. We studied the spectrum of neutralization of lethality of an experimental monovalent equine antiserum against the strongly neurotoxic African forest cobra (Naja melanoleuca) when tested against venoms of most species of African Naja, both neuro and cytotoxic as described by some authors. We report a comparison of the median lethal doses (LD50) of the venoms and the paraspecific median effective doses (ED50) of the antiserum calculated using three methods: Spearman-Kärber and Probit (currently recommended by the World Health Organization), and non-linear regression. An ample--but not complete--spectrum of paraspecific neutralization of lethality was observed against both spitting and non-spitting species of African Naja with a clearly more efficient neutralization of the more potent venoms, the implications of which are discussed. The median lethal and effective doses calculated by the three methods are remarkably consistent and may warrant consideration of non-linear regression methods for the calculation of venom lethality and antivenom potency by venom/antivenom researchers and producers.

Methodology of clinical studies dealing with the treatment of envenomation

A total of 142 clinical studies have been devoted to the treatment of envenomations, of which 115 address snake bites, 20 scorpion stings, and 8 other animals (one addresses both snake and spider envenomation). Antivenom use was studied in 118, of which 82 addressed efficacy, 43 evaluated safety, 23 studied dosage and 8 explored other issues. Besides anecdotal clinical reports, three classes of clinical studies are distinguished: (a) observational clinical studies (55 of the total) which analyze series of cases, (b) comparative clinical studies (36) which compare therapeutic products or treatment regimens without a gold standard for comparison and (c) randomized clinical trials (RCT, 51). The goals, methods and constraints of design of RCT are determined by whether explanatory (analytical) or pragmatic considerations are prioritized. Explanation-oriented RCT rely on strict group comparability before and during treatment, in order to ensure the internal validity of the study. The pragmatically-oriented RCT aims at establishing the superiority of a treatment over another, the goal being to maximize the external validity of the trial (that is, its application in current practice). We found that all clinical studies of treatment of envenomation lean markedly toward the explanatory end and suggest that, given some particularities of envenomation as a medical condition, a more pragmatic approach may be of value, particularly under the conditions prevalent for clinical studies in developing nations.

Sphingomyelinase D Activity in Model Membranes: Structural Effects of in situ Generation of Ceramide-1-Phosphate

The toxicity of Loxosceles spider venom has been attributed to a rare enzyme, sphingomyelinase D, which transforms sphingomyelin to ceramide-1-phosphate. The bases of its inflammatory and dermonecrotic activity, however, remain unclear. In this work the effects of ceramide-1-phosphate on model membranes were studied both by in situ generation of this lipid using a recombinant sphingomyelinase D from the spider Loxosceles laeta and by pre-mixing it with sphingomyelin and cholesterol. The systems of choice were large unilamellar vesicles for bulk studies (enzyme kinetics, fluorescence spectroscopy and dynamic light scattering) and giant unilamellar vesicles for fluorescence microscopy examination using a variety of fluorescent probes. The influence of membrane lateral structure on the kinetics of enzyme activity and the consequences of enzyme activity on the structure of target membranes containing sphingomyelin were examined. The findings indicate that: 1) ceramide-1-phosphate (particularly lauroyl ceramide-1-phosphate) can be incorporated into sphingomyelin bilayers in a concentration-dependent manner and generates coexistence of liquid disordered/solid ordered domains, 2) the activity of sphingomyelinase D is clearly influenced by the supramolecular organization of its substrate in membranes and, 3) in situ ceramide-1-phosphate generation by enzymatic activity profoundly alters the lateral structure and morphology of the target membranes.

Cellular organization and appearance of differentiated structures in developing stages of the parasitic platyhelminth Echinococcus granulosus

Echinococcus granulosus is the causative agent of hydatidosis, a major zoonoses that affects humans and herbivorous domestic animals. The disease is caused by the pressure exerted on viscera by hydatid cysts that are formed upon ingestion of E. granulosus eggs excreted by canine. Protoscoleces, larval forms infective to canine, develop asynchronously and clonally from the germinal layer (GL) of hydatid cysts. In this report, we describe the cellular organization and the appearance of differentiated structures both in nascent buds and developed protoscoleces attached to the GL. Early protoscolex morphogenesis is a highly complex and dynamic process starting from the constitution of a foramen in the early bud, around which nuclei are distributed mainly at the lateral and apical regions. Similarly, distribution of nuclei in mature protoscoleces is not homogenous but underlies three cellular territories: the suckers, the rostellar pad, and the body, that surrounds the foramen. Several nuclei are associated to calcareous corpuscles (Cc), differentiated structures that are absent in the earlier bud stages. The number of nuclei is similar from the grown, elongated bud stage to the mature protoscolex attached to the GL, strongly suggesting that there is no significant cellular proliferation during final protoscolex development. The amount of DNA per nucleus is in the same range to the one described for most other platyhelminthes. Our results point to a sequential series of events involving cell proliferation, spatial cell organization, and differentiation, starting in early buds at the GL of fertile hydatid cysts leading to mature protoscoleces infective to canine.

Myotoxicity and nephrotoxicity by Micrurus venoms in experimental envenomation.

Micrurus venoms are essentially neurotoxic but other activities, such as myotoxicity, may be apparent under experimental conditions. Although this myotoxicity has been occasionally reported, there are no studies addressing it systematically across the genus, particularly in its relationship to other systemic manifestations such as renal impairment. The lethal potency of Micrurus fulvius, Micrurus nigrocinctus, Micrurus surinamensis, Micrurus altirostris, Micrurus balyocoriphus and Micrurus pyrrhocryptus venoms determined by us were in the range described for the genus and all venoms exhibited phospholipase activity, albeit at significantly different levels. Intramuscular venom injection caused variable local inflammation-edema; myotoxicity (as determined by plasma creatine kinase levels and histopathology) was apparent only in those venoms with highest phospholipase activity, namely M. fulvius, M. nigrocinctus and M. pyrrhocryptus. Kidneys of animals injected with these strongly myotoxic venoms showed lesions consisting in extensive tubular necrosis with nuclear fragmentation, destruction of the brush border, rupture of basal membrane and epithelial exfoliation of tubular cells, granular cast and thickening of tubules. The histological characteristics of the lesions suggest an important role for indirect glomerular damage by myoglobin deposits. Phospholipase and myotoxic activities did not correlate significantly to the lethal potency; renal lesions were, however, evident only in those venoms that caused extensive muscular damage. Although kidney lesions have not been described in clinical cases of Micrurus envenomation, the potential for nephrotoxicity of some of these venoms should be considered in the overall toxicological picture, at least in experimental conditions.

Neutralization of Vipera and Macrovipera venoms by two experimental polyvalent antisera: A study of paraspecificity

We conducted an extensive study of neutralization of lethality of 11 species and one subspecies of snakes of the genus Vipera, and of five species of Macrovipera, by two experimental equine antisera. One antiserum was a trivalent preparation raised against the venoms of Vipera aspis aspis, Vipera berus berus and Vipera ammodytes ammodytes; the other was a pentavalent preparation that also included venoms of Vipera (now Montivipera) xanthina and Macrovipera lebetina obtusa. We measured specific neutralization of lethality against all venoms included in the immunization schemes, and paraspecific neutralization against the venoms of Vipera ammodytes montandoni, Vipera (Montivipera) bornmuelleri, Vipera latastei, Vipera (Mo.) latifii, Vipera (Mo.) lotievi, Vipera (Daboia) palaestinae, Vipera (Mo.) raddei and Vipera seoanei, as well as against Macrovipera (D.) deserti, Macrovipera lebetina cernovi, Macrovipera lebetina turanica and Macrovipera schweitzeri. We found an important degree of paraspecific protection within each genera (omitting recent reclassification) that was quite independent of both the lethal potency of the venoms and their geographic origin. This information may be of use to clinicians charged with the treatment of Vipera or Macrovipera envenomations with non-specific antivenoms.