Cloning and expression of a Leishmania donovani gene instructed by a peptide isolated from major histocompatibility complex class II molecules of infected macrophages.

The studies reported here describe the isolation of peptides from MHC class II molecules of murine macrophages infected with Leishmania donovani, and the use of the derived peptide sequences to rescue the pathogen peptide donor protein. The isolation of the peptides was carried out by comparing the RP HPLC profile of peptides extracted from infected macrophages with the peptides extracted from noninfected cells. Several distinct HPLC peaks unique to infected macrophages were sequenced. One of the peptides that was not homologous to any known protein was used to instruct the designing of an oligonucleotide sense primer that was used in combination with an oligo dT nucleotide (anti-sense primer) to amplify by PCR a DNA fragment from L. donovani cDNA. The amplified DNA fragment was cloned and used as a probe to screen a L. donovani cDNA library. The cloned gene (Ld peptide gene) has an open reading frame of 525 bp and has no homology with any known protein/gene sequence. Northern blot analyses indicated that the Ld peptide/gene is broadly distributed and expressed among species of the Leishmania genus, in both the amastigote and promastigote life cycle forms. Using the pGEX 2T vector, the gene was expressed and the relationship of the purified recombinant protein with L. donovani was confirmed using both antibody and T cell responses from immunized or infected animals. The gene encodes a 23-kD molecule (Ldp 23) associated with the cell surface of L. donovani promastigotes. In addition, T cells purified from the lymph nodes of BALB/c mice immunized with L. donovani or infected with L. major, and from CBA/J mice infected with L. amazonensis were stimulated to proliferate by the recombinant Ldp 23 and produced high levels of IFN-gamma and no IL 4. This observation suggests that the Ldp 23 is an interesting parasite molecule for the studies concerning the host/parasite interaction because the Th1 pattern of cytokine response that it induces is correlated with resistance to Leishmania infections. These results clearly point to an alternative strategy for the purification of proteins useful for the development of both vaccines and immunological diagnostic tools not only against leishmaniasis but also for other diseases caused by intracellular pathogens.

Ancylostoma caninum anticoagulant peptide-5: immunolocalization and in vitro neutralization of a major hookworm anti-thrombotic.

Hookworm infection is a major cause of gastrointestinal blood loss and iron deficiency anemia in the developing world. Recently two major anticoagulant serine protease inhibitors have been identified and cloned from adult Ancylostoma caninum hookworms. One of these, A. caninum anticoagulant peptide 5 (AcAP5), is a potent and specific inhibitor of human coagulation factor Xa. A polyclonal IgG has been purified from rabbits immunized with recombinant AcAP5 using affinity chromatography. Using immunohistochemistry, the polyclonal alpha-rAcAP5 IgG localized to the cephalic or amphidial glands, confirming previous biochemical studies that had identified this secretory gland as the primary source of anticoagulant activity in the adult worm. This polyclonal IgG also neutralized the inhibitory activity of recombinant and native AcAP using a single stage chromogenic assay of coagulation factor Xa activity. In addition, the polyclonal IgG also neutralized the anticoagulant activity of native and recombinant AcAP5 as measured by the activated partial thromboplastin time clotting assay. Importantly, this neutralizing activity is species specific, as the polyclonal IgG failed to neutralize the anticoagulant activity of A. ceylanicum. Taken together, these data suggest that the hookworm anticoagulant AcAP5 represents a viable target for future immunization strategies aimed at inhibiting the ability of the adult hookworm to feed on blood in vivo.

Heterogeneity of tachykinin-like immunoreactive peptides in rat spinal cord and dorsal root ganglia.

The concentrations of tachykinins in rat spinal cord and dorsal root ganglia (DRGs) were measured using a combination of high performance liquid chromatography (HPLC) and radioimmunoassays (RIAs). Substance P-like immunoreactivity (SPLI) was found to be significantly higher than either substance K-like immunoreactivity (SKLI) or neuromedin K-like immunoreactivity (NMKLI) in both tissues. In the spinal cord, the concentration of SKLI was comparable to that of NMKLI. In DRGs, NMKLI is present at concentrations much lower than those of SKLI or SPLI. In addition to immunoreactive components co-eluting with the three mammalian tachykinins SP, SK and NMK, analyses using reverse-phase HPLC revealed an immunoreactive peak co-eluting with the C-terminal octapeptide of SK (SK3-10), and a yet to be identified peak eluting before SK. This study also demonstrates the use of a novel and highly specific RIA for NMK to measure NMKLI without the need of reverse-phase HPLC.

A novel radioimmunoassay for neuromedin K. I. Absence of neuromedin K-like immunoreactivity in guinea pig ileum and urinary bladder. II. Heterogeneity of tachykinins in guinea pig tissues.

A novel and highly specific radioimmunoassay for the tachykinin peptide neuromedin K (NMK, also known as neurokinin beta, neurokinin B) has been developed and used to determine the distribution of this peptide in extracts of guinea pig tissues. In addition to immunoreactive components coeluting with the 3 mammalian tachykinins, substance P (SP), substance K (SK) and NMK, analyses using reverse-phase HPLC revealed immunoreactive peaks coeluting with the C-terminal octapeptide of SK (SK-(3-10], an N-terminally extended form of SK (gamma-preprotachykinin-(72-92)amide), and a yet unidentified peak eluting before NMK in the extracts of guinea pig brain and spinal cord. In contrast to the other tachykinins, SP and SK, which were present in high concentrations in extracts of all peripheral and central tissues examined, NMK-like immunoreactivity was detected only in extracts of central tissues. NMK-like immunoreactivity was not detected in extracts of terminal ileum and urinary bladder.

[Anticardiolipin antibodies and primary antiphospholipid syndrome of the central nervous system: report of 10 clinical cases]. / Anticuerpos anticardiolipinas y síndrome antifosfolípidos primario del sistema nervioso central: comunicación de 10 casos clínicos.

An association between anti-phospholipid antibodies and different disorders of the central nervous system has been described recently. We used an ELISA technique and detected anti-cardiolipin antibodies of the IgG or IgM variety in a series of 10 patients: 4 had occlusive stroke, 2 brain hemorrhage, 2 chorea, 1 a Sneddon syndrome and 1 vascular cephalea. The diagnosis of primary anti-phospholipid syndrome was suggested after ruling out systemic lupus in all patients.

[Multidisciplinary study of cerebrovascular disorders. II. Cardiovascular profile of occlusive vascular disorders]. / Estudio multidisciplinario de los accidentes vasculares encefálicos. II--Perfil cardiovascular de los accidentes vasculares oclusivos.

Ischemic stroke constitute a mayor cause of morbidity and mortality in the adult population, particularly in the elderly. Heart disease may predispose to ischemic stroke, especially in the presence of transient or permanent precipitating factors such as atrial fibrillation. To elucidate the role of heart disease in predisposing to ischemic stroke we studied the clinical and non invasive cardiac profile (EKG, 2D-Echo, Holter) of 186 consecutive patients, 91 of them embolic (GI) and 96 non embolic (lacunar, atherothrombotic, others) (GII), as determined by brain CT scan and thorough clinical evaluation. Age and male/female ratio were significantly different (71 + 13 vs 65 + 12 years, 40/60 vs 65/35, p < 0.003). Hypertension was equally common in both groups (38 and 40%). Patients in GI had higher prevalence of valvular heart disease (23 vs 1%), atrial fibrillation (67 vs 10%), 2D Echo left atrial enlargement (45 vs 16%) and supraventricular ectopy in Holter (59 vs 32%) p < 0,001. By contrast absence of heart disease (45 vs 19%), ST-T changes in EKG (28 vs 14%), left ventricular hypertrophy in 2D Echo (28 vs 9%) and ventricular ectopy in Holter (54 vs 23%) were more prevalent in GII patients, p < 0.001. Multiple stepwise logistic regression analysis showed age > 70 years (relative risk (RR) 1.67), valvular heart disease (RR 2.25), chronic AF (RR 2.44) and paroxysmal AF (RR 1.89) were significant independent predictors of embolic stroke, whereas the presence of left ventricular hypertrophy in 2D-Echo (RR 0.76) and frequent ventricular premature beats in Holter (RR 0.47) were predictors of occlusive non embolic stroke. Thus, the clinical and non invasive cardiac profile of embolic and non embolic ischemic stroke is significantly different, which is relevant to preventive strategies.

Taspine is the cicatrizant principle in Sangre de Grado extracted from Croton lechleri.

Sangre de Grado extract used by Peruvian natives as a cicatrizant agent, was collected from trees of the species Croton lechleri growing in the Peruvian jungle. The Sangre de Grado was found to contain one alkaloid identified as taspine and which was shown to be the active cicatrizant principle by an in vivo test in mice. This alkaloid exhibited a dose-related cicatrizant effect and an ED50 of 0.375 mg/kg. Experiments with taspine hydrochloride in order to study its mechanism of action in cell culture systems showed that the alkaloid was non-toxic to human foreskin fibroblasts at concentrations below 150 ng/ml and that it had no effect on cell proliferation. On the other hand, taspine hydrochloride was found to increase the migration of human foreskin fibroblasts. This effect on the migration of fibroblasts is probably the mechanism by which Sangre de Grado and taspine hydrochloride accelerate the wound healing process. Using the two-stage mouse skin carcinogenesis system, we have been able to show that neither Sangre de Grado nor taspine hydrochloride had carcinogenic or tumour promoter activity after 17 months of treatment.