Antileishmanial activity in Israeli plants.

Leishmaniasis is a vector-borne disease caused by flagellated protozoan parasites of the genus Leishmania, which affects both humans and other mammals. Most of the available drugs against the disease are toxic and parasite resistance to some of the drugs has already developed. In the present study, the leishmanicidal activities of methanolic extracts of some Israeli plants have been evaluated in vitro, against the free-living promastigotes and intracellular amastigotes of Leishmania major. Of the 41 extracts examined, those of two plants (Nuphar lutea>Withania somnifera) were highly effective (with a maximum inhibitory effect of >50%), those of three other species (Pteris vittata>Smyrnium olusatrum>Trifolium clypeatum) were moderately effective (25%-50%) and another four extracts (Erodium malacoides>Hyparrhenia hirta>Thymelaea hirsuta>Pulicaria crispa) showed a marginal effect (15%-22%) against the parasites. Extracts of nine plant species therefore showed antileishmanial activity but only the extract of N. lutea, used at 1.25 microg/ml, eliminated all the intracellular parasites within 3 days of treatment, with no detectable toxicity to the host macrophages. The mean (S.D.) values recorded for the median inhibitory concentrations of this extract (IC50) against the promastigotes [2.0 (0.12) microg/ml] and amastigotes [0.65 (0.023) microg/ml] and the median lethal concentration (LD50) against macrophages [2.1 (0.096) microg/ml] were encouraging, giving a therapeutic selectivity index [LD50/IC50 for amastigotes)] of 3.23. The extract of N. lutea was, in fact, generally as effective as the paromomycin that was used as the 'gold standard' drug. These results indicate that N. lutea and probably also Withania somnifera might be potential sources of clinically useful, antileishmanial compounds.

Nuphar lutea: in vitro anti-leishmanial activity against Leishmania major promastigotes and amastigotes.

UNLABELLED: Several anti-leishmanial drugs of choice are of plant origin. Many of the available drugs against the disease are toxic and in certain cases parasite drug resistance is developed. The development of new compounds is urgently required. AIMS OF THE STUDY: To determine the leishmanicidal activity of the Nuphar lutea plant extract against Leishmania major in vitro. MATERIALS AND METHODS: The leishmanicidal activity of methanolic plant extract against L. major free living promastigotes and intracellular amastigotes was evaluated, using microscopic examinations and the enzymatic XTT assay. RESULTS: Methanolic extract of N. lutea was highly effective against both Leishmania promastigotes and L. amastigotes (IC(50)=2+/-0.12 microg/ml; ID(50)=0.65+/-0.02 3 microg/ml; LD(50)=2.1+/-0.096 microg/ml, STI=3.23). The extract at 1.25 microg/ml totally eliminated the intracellular parasites within 3 days of treatment. Also, a synergistic anti-leishmanial activity was demonstrated with N. lutea extract combined with the anti-leishmanial drug--paromomycin. The partially purified N. lutea active component was found to be a thermo-stable alkaloid(s) with no electrical charge and is resistant to boiling and to methanol, dichloromethane and xylene treatment. CONCLUSIONS: The present study suggests that N. lutea might be a potential source of anti-leishmanial compounds.

Alpha 2 macroglobulin activity in rats infected with Typanosoma lewisi and treated with cyclophosphamide and its effect on the malignancy of the disease.

BACKGROUND & OBJECTIVES: Trypanosoma lewisi is a common, flagellated parasite of the rat. Our previous study showed that rabbits injected with serum collected from rats infected with Trypanosoma lewisi and treated with cyclophosphamide (CyI) produced high levels of antibodies against a new protein in the CyI rat serum. RESULTS: In the present study, this protein was characterised as alpha2 macroglobulin (alpha2M) and the kinetics of its production and its influence on the malignancy of the disease were determined. In rats infected with T. lewisi, alpha2M was first demonstrated and peaked on the second day post-infection (972 microg/ml) and then reduced gradually, reaching a level of 32 microg/ml on the eighth day post-infection. However, in the CyI rats the level of alpha2M was gradually increased as the disease progressed, reaching a level of 890 microg/ml on the eighth day post-infection. Injection of both crude and purified alpha2M into rats infected with T. lewisi led to increased parasitaemia. INTERPRETATION & CONCLUSION: The present study suggests that increased levels of alpha2M in the CyI rats contribute to the malignancy of the disease.

Pathological developments mediated by cyclophosphamide in rats infected with Trypanosoma lewisi.

Trypanosoma lewisi is an obligatory, flagellated parasite of the rat. Despite the fact that naturally the rats overcome the disease, a lethal infection can be induced by the administration of an immunosuppressive agent, i.e. cyclophosphamide (Cy). In the Cy treated infected rats (CyI) the severity of the trypanosome infection was demonstrated in the internal organs, in the following order: lungs>liver>heart>spleen>kidney. The parasites were not detected in the brain. The accumulation of the parasites in the lungs led to the development of hemorrhagic inflammatory foci. The rupture of blood vessels was accompanied by lymphocyte infiltrations into the damaged tissues and multiple foci of edema around the blood vessels. In most cases the lungs were dark brown in color due to intra-alveolar hemorrhages. The spleen of the CyI rats showed general deformation of the tissue's architecture, migration of macrophages and cell depletion due to the Cy action. The liver showed inflammatory hemorrhagic foci associated with massive destruction of the parenchyma. In spite of the heavy parasitemia (>50%) developed in the CyI rats the brain remained free of parasites, which might explain the non-virulent character of this parasite compared to the African trypanosomes.

The flow-cytometry-based evaluation of cellular immunity in cases of cutaneous leishmaniasis and healthy controls from the endemic area in southern Israel.

Only limited data are available on the early immunological events associated with human cutaneous leishmaniasis (CL). In this study, peripheral-blood mononuclear cells were obtained from 66 individuals (34 patients with cutaneous lesions and 32 apparently healthy controls) who had each spent no more than 3 months in the endemic region of Qetzioth, in southern Israel. These cells' responses to Leishmania major antigen were then explored, by the flow-cytometry-based evaluation of blast transformation (BT). The lymphocytes from 17 (50%) of the patients but only one (3%) of the controls displayed BT. When, in an ELISA, most (52) of the subjects were checked for anti-L. major antibodies, none of the 22 controls investigated but 19 (63%) of the 30 patients were found seropositive. Although 14 (47%) of the 30 patients who were checked for antibodies were BT-positive, the seropositive patients were not significantly more or less likely to be BT-positive than the seronegative patients (P<0.919). These data indicate that, in CL, the hosts' cellular and humoral responses develop independently within the first 3 months post-infection, but further investigation is required to confirm this hypothesis.

Case report: clinical and serological evaluation of echinococcosis of the spine.

A 53-year-old male with a severe destructive lesion of the L4 vertebral body caused by an Echinococcus granulosus hydatid cyst was studied. He underwent surgery twice, and was treated continuously with albendazole, followed by albendazole combined with praziquantel. Specific anti-echinococcal immunoglobulin (Ig) G, IgG4, and IgE activities before and after surgery were further determined. In spite of long-term chemotherapeutic treatment combined with surgery, no eradication of the disease was achieved.

Prevalence of cystic echinococcosis among Muslim and Jewish populations in southern Israel.

Following a review of records at the Soroka University Medical Center (SUMC) showing a recent increase in the annual incidence of hydatidosis among the Bedouin population of southern Israel, a seroepidemiological survey was conducted. A total of 1439 blood samples were collected from Bedouins and Jews living in the Negev area in southern Israel. An enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G, indirect haemagglutination (IHA) and immunoelectrophoresis (IEP) were used to detect anti-Echinococcus granulosus antibodies. The seroprevalence in the Bedouin group was 0.68% (7/1026), including two children under the age of 10. Among Jews, a seroprevalence of 0.5% was recorded in patients over 60 years of age only. A high rate of infection was shown in goats and sheep slaughtered in abattoirs in Bedouin localities. The results indicate that echinococcosis is a common disease in the Muslim communities of southern Israel.

Interleukin 1alpha activity of peritoneal and bone marrow macrophages infected with Leishmania major and Leishmania donovani in vitro.

In this study, the pattern of interleukin-1alpha (IL-1alpha) production by both peritoneal (PM) and bone marrow macrophages (BMM) from resistant (C3H/HeJ) and susceptible (BALB/c) mice was investigated, using a bioassay and an IL-1alpha-specific ELISA kit. PM from normal uninfected mice showed either an initial high (C3H/HeJ) or a neglected (BALB/c) level of IL-1alpha activity, respectively, probably due to thioglycollate stimulation. Infection with Leishmania major induced only a marginal effect on IL-1 production by both cells. Normal, uninfected and unstimulated BMM from both mice did not produce IL-1alpha over a 7-day period of cultivation in vitro. Upon stimulation with either lipopolysaccharide (LPS) (BALB/c) or concanavalin A (Con A) (C3H/HeJ), both cell types produced IL-1alpha that peaked within the first 12-24 h following stimulation. BMM from C3H/HeJ and BALB/c mice failed to produce IL-1alpha when infected in vitro with L. major or L. donovani promastigotes. However, infection with these two parasites did not interfere with the capability of the host cell to produce IL-1alpha when stimulated with LPS or Con A. The level of IL-1alpha production was independent of the degree of parasitization of the macrophages. Similar results were observed with IL-1beta and IL-6 production by BMM, even though their levels were generally slightly higher than those obtained with IL-1alpha.

Giardia lamblia carriage in Israeli Bedouin infants: risk factors and consequences.

Giardiasis is a common protozoan infection, with varying clinical manifestations. We investigated the associations between Giardia lamblia carriage and environmental, family, illness, and growth characteristics. Bedouin infants (n=234) were followed from birth to age 18-23 months. At monthly home visits, stool samples were obtained, history of illness was determined, and an environmental assessment was done. The comparisons presented are between 4 groups defined by length of carriage of G. lamblia. Study children had a mean+/-SD of 4.1+/-2.9 diarrhea episodes. No illness, environmental, or family characteristics were associated with length of carriage. Significant differences were found in weight-for-age and weight-for-height z scores between the never-positive-for-G. lamblia group and all other carriage groups combined. Faltering growth was shown to be subsequent to G. lamblia infection rather than preceding it. Our findings confirm that G. lamblia carriage is not associated with diarrhea. However, the effect on growth deserves further investigation.

Persistent diarrhea in a cohort of Israeli Bedouin infants: role of enteric pathogens and family and environmental factors.

This study examined the role of enteric pathogens and infant, family, and household characteristics in persistent diarrhea. Bedouin infants from southern Israel were followed from birth to age 18-23 months. During monthly home visits, stool samples were obtained, and feeding practices and history of diarrhea were determined, and at age 3 months, an environmental assessment was done. Diarrhea surveillance was either via a network covering all community health care facilities or via weekly interviews with the mother. None of the enteric pathogens examined, including Cryptosporidium parvum and enteroaggregative Escherichia coli, were associated with persistent diarrhea. In multivariate analyses, age at first diarrheal illness and maternal age and maternal education were independently and significantly associated with the risk of persistent diarrhea. These data suggest that persistent diarrhea is a clinical entity that may be related less to a specific enteric pathogen and more to the health experiences of children and their home environment.

An epidemiological survey of Echinococcus granulosus and other helminths in animal populations in northern Israel.

In a survey carried out during the period May 1995 to November 1996, in communities of various ethnic groups in northern Israel, 206 dogs were examined for Echinococcus granulosus and other intestinal helminth parasites by arecoline hydrobromide purges and the coproantigen-ELISA. The arecoline test was performed close to the owners' homes, using plastic sheets secured to the ground. From 56 dogs examined in the Muslim town of Tamra, six (10.7%) were found to be infected with E. granulosus. Four of them also had a mixed infection of Taenia hydatigena and Dipylidium caninum (two dogs), and the remaining two dogs were infected with either D. caninum or Taenia pisiformis. An additional 18 dogs were infected with either T. pisiformis (eight dogs), D. Caninum (seven dogs), or T. hydatigena (three dogs). Two of these dogs harboured mixed infections whereas the remaining 32 dogs were free of helminths. In the Jewish villages, none of the 150 dogs examined were infected with E. granulosus, although 26 (17.3%) were infected with D. caninum, four (2.7%) with Ancylostoma spp. and one (0.7%) with Toxocara canis. Only one of the 22 stray dogs and none of the 15 jackals examined were infected with E. granulosus. However, 21 (95.4%) of the dogs and 12 (80%) of the jackals harboured helminth infections, including: D. caninum (16 dogs and seven jackals), Ancylostoma spp. (five jackals), T. hydatigena (three dogs), and T. canis (one dog). Approximately 18% of the dogs and 33% of the jackals showed mixed infections with two or more of the above helminths. In the abattoirs, 52 (5.9%) of the 874 sheep and 33 (5.3%) of the 616 goats from 17 herds slaughtered in the Muslim and Druze villages were found to be infected with E. granulosus, compared with a 0% infection rate observed in 93 sheep from two herds in Jewish villages.

Natural history of Giardia lamblia and Cryptosporidium infections in a cohort of Israeli Bedouin infants: a study of a population in transition.

The natural history of Giardia lamblia and Cryptosporidium infections were determined in a cohort of 164 Bedouin children, from a population not previously studied, which is in transition from nomadism to a settled life style. Stools were sampled monthly from birth to two years of age and at all diarrhea episodes. The risk of infection with G. lamblia and Cryptosporidium infection by age two was 91.5% and 48.8%, respectively. Cryptosporidium prevalence was 3-4% at all ages, whereas G. lamblia prevalence was > 30% after age one. Giardia lamblia and Cryptosporidium asymptomatic detection rates were high, 28.5% and 1.6%, respectively. Detection of G. lamblia was higher in diarrhea episode samples obtained before six months of age, but after that age and overall, the detection was lower than in nondiarrhea samples (odds ratio [OR] = 0.8, 95% confidence interval [CI] = 0.7-0.9, P < 0.05). Detection rates of C. parvum were higher in episode-related samples in all age groups (OR = 2.8, 95% CI = 1.9-4.2, P < 0.05) and infections in boys were more frequently symptomatic than in girls. While G. lamblia does not appear to be a consistent pathogen in this population where it is hyperendemic, Cryptosporidium has been shown to be an important cause of diarrhea in young children in the community.

Cellular and humoral immune responses of hydatidosis patients to Echinococcus granulosus purified antigens.

The cellular and humoral immune responses of 41 hydatidosis patients, 12 healthy uninfected individuals, and seven patients with other parasitic diseases were determined using serologic and lymphoproliferative assays (LPAs), respectively. Echinococcal antigens were obtained by gel filtration of crude hydatid cyst fluid (HCF) on a Sephadex G-200 column. The fractions contained either a mixture of antigens A plus B or antigen B alone that was further enriched by boiling. All the hydatidosis patients responded positively by LPA to either crude or purified echinococcal antigens: 95% of them responded to either crude HCF, or a mixture of antigens A plus B and 83% to antigen B alone. The degree of the response to crude HCF (mean stimulation index [SI] = 75.3) was higher than that of purified antigens (SI = 39.1 for a mixture of antigens A plus B and SI = 36.9 for antigen B alone). No positive LPA response was obtained with the control groups. Serologic examinations showed that 78% of cases were positive by immunoblot, 73% by indirect hemagglutination, and 46% by immunoelectrophoresis. No correlation between the degree of cellular and humoral responsiveness to both crude and purified echinococcal antigens was observed. Nine of the 41 patients examined who were serologically negative also developed a high lymphoproliferative (LP) response to either crude or purified echinococcal antigens. The LP response remained positive over a long period after successful treatment. No relationship was observed between the results of treatment and the LP response. The present study indicates that the LPA could be used as an additional tool for the diagnosis of hydatid disease, particularly in seronegative cases, although it is unsuitable for effective monitoring treatment/surgery.

Echinococcus granulosus: a seroepidemiological survey in northern Israel using an enzyme-linked immunosorbent assay.

Following an intensive health education programme, 8651 finger-prick blood samples, 4122 from a predominantly adult group attending a primary care clinic and 4529 from schoolchildren, were collected in Tamra, northern Israel. An enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) was used to detect anti-Echinococcus granulosus antibodies, using both crude and purified antigens. The seroprevalence in the adult group was 0.48% (20/4122); optical density values were 0.1-0.14 in 10 subjects, 0.15-0.19 in 9, and > or = 0.2 in one; prevalences did not differ significantly between males and females or among age groups. Twenty-six of the schoolchildren (0.57%) were seropositive, 23 with optical densities of 0.1-0.14, one of 0.15-0.19, and 2 > or = 0.2. A high correlation was observed between ELISA positivity and both positivity in the arc 5 immunoelectrophoresis test and the presence of a high titre in the indirect immunofluorescence assay. Cross reactivity was observed with sera from schistosomiasis and ancylostomiasis patients, using both crude and purified echinococcal antigens. The results indicated that the IgG ELISA, using both crude and purified antigens, was very useful for seroepidemiological screening for echinococcosis, and that this condition is an emerging disease in northern Israel.

Evaluation of an enzyme immunoassay for the detection of Cryptosporidium spp. in stool specimens from infants and young children in field studies.

Diagnosis of Cryptosporidium is made by the identification of oocysts in stool specimens. Screening in field studies relies mainly on acid-fast staining followed by microscopic examination. The more sensitive immunofluorescent antibody (IFA) staining method is time-consuming, may involve technical difficulties, and is extremely costly as a screening procedure in field studies. We evaluated the diagnostic utility of a commercially available enzyme immunoassay (EIA), which detects Cryptosporidium-specific antigen, in 204 unprocessed stool specimens obtained from patients less than three years of age from a field study in southern Israel. When compared with the routine screening procedure applied in this field study (screening by acid-fast staining and microscopy after concentration, and confirmation of positive results by IFA), both the sensitivity and specificity were 98%. Of 139 specimens negative by microscopy, 13 (9.3%) were positive by the EIA. Eleven of these were confirmed by inhibition with antibody to Cryptosporidia-specific antigen. The EIA is an important tool for identifying Cryptosporidium in fecal specimens in field studies since it is sensitive, specific, simple to use, and unaffected by the presence of a preservative.

Suppression of antigen-specific T cell responses by Leishmania major excreted factor: inhibition of activation signals linked to the T cell antigen receptor and interleukin 2 receptor.

An excreted factor (EF) derived from culture medium of Leishmania major was found to suppress ConA-induced polyclonal activation of mouse T cells. To further dissect the effect of EF on cell-mediated immune responses, we used in vivo primed antigen-specific murine lymph node cells. EF inhibited the proliferative response of keyhole limpet hemocyanin (KLH) or ovalbumin (OA)-primed T cells upon in vitro challenge with the antigen. In addition, it suppressed the induction of interleukin 2 receptor (IL2-R) alpha following mitogen stimulation of unprimed T cells or antigen challenge of KLH-primed T cells. Thus, EF affects early events in signal transduction that follow the T cell antigen receptor (TCR) triggering. To test whether EF may interfere with more remote events in the activation process of T cells, we used IL2-R positive T cells and tested their response to IL2 in the presence of EF. We found that EF inhibited also IL2-dependent T cell proliferation in a dose-dependent manner. The data suggest, therefore, that the locus of inhibitory effect of EF is at both the early and late stages of T cell activation and apparently involves two different signal transduction pathway linked to the receptors for the antigen and IL2.

Three- to 7-year follow-up after albendazole treatment of 68 patients with cystic echinococcosis (hydatid disease).

Sixty-eight cystic echinococcosis patients were each treated with 800 mg albendazole/day in four 28-day courses separated by 14-day drug-free intervals. Follow-up for 3-7 years showed that this treatment alone eradicated the cysts in many patients; in most of the remainder, disease progression stopped. Of the 59 patients with liver cyst(s), 41% were cured (all cysts disappeared), 17% showed marked improvement (> 25% contraction, complete consolidation), 24% limited improvement and 15% no change. Of the 12 patients with lung cyst(s), 72% were cured, 9% showed limited improvement and 18% showed no change (18%). No patient worsened but a recurrence occurred in two patients at about 56 months. The times taken to detect an initial cyst change and to detect cure were longer than is generally expected; initial cyst changes and cure could only be detected after 1 year in 12% and 69% of patients with liver cysts and 22% and 37% of those with lung cysts, respectively.