Comparison between three adjuvants for a vaccine against canine leishmaniasis: In vitro evaluation of macrophage killing ability.

The aim of this study was to evaluate, in terms of dog macrophage killing ability in vitro, a vaccine based on Leishmania infantum promastigote soluble antigen (LSA) formulated with three different adjuvants (BCG, AdjuPrime, MPL/TDM/CWS). A significant increase of the macrophage killing ability was observed in dogs vaccinated with LSA+MPL/TDM/CWS after 1 month from vaccination. A similar increase of macrophage parasitocidal ability was present only after 5 months in dogs vaccinated with LSA+BCG or LSA+AdjuPrime. In all dogs the augmented killing percentage was still present after 12 months from vaccination. Therefore, in particular LSA+MPL/TDM/CWS vaccine seems promising for further studies in dogs.

Cytokine expression in dogs with natural Leishmania infantum infection.

The aim of this study was to evaluate cytokine expression in 22 Leishmania infantum naturally infected dogs, in order to correlate this parameter with the clinical status of infected animals. After 4 and 8 months from the first diagnosis of Leishmania infection, clinical and laboratory examination of dogs was performed and peripheral blood mononuclear cells (PBMC) were isolated. The cytokine profile was analysed in terms of IFN-gamma, IL-4, IL-10 and TNF-alpha mRNA expression in cultured PBMC by a semi-quantitative reverse transcriptase-PCR. Thirteen out of 22 Leishmania-infected dogs remained asymptomatic in the follow-up, while 9 showed clinical signs of leishmaniasis. IL-4, IL-10, TNF-alpha and IFN-gamma mRNA levels were not significantly different in asymptomatic compared to symptomatic animals 4 months from the diagnosis of Leishmania infection, but were significantly higher in symptomatic versus asymptomatic dogs after 8 months from diagnosis. In addition, IL-4, IL-10 and TNF-alpha mRNA levels significantly increased only in symptomatic dogs at 8 months, in comparison to the levels found at 4 months. These results show a mixed Th1 and Th2 cytokine response in Leishmania-infected dogs, with higher cytokine expression in dogs with manifest clinical disease, during the second follow-up after 8 months from the first diagnosis of infection.

Acute gastric anisakiasis: an Italian experience.

Anisakidosis is a parasitic disease of the human gastrointestinal tract caused by ingestion of larvae of marine nematodes such as Anisakis spp. or, rarely, Pseudoterranova spp., present in raw or undercooked fish. We report the first series of gastric Anisakis infection (anisakiasis) from a single centre in Italy. In our department, we observed 3 cases, all in women who were urgently hospitalized following intense epigastric pain and vomiting, developed after the ingestion of raw fish. The patients underwent urgent gastroscopy within a few hours. In each, a worm was extracted from the gastric mucosa by means of biopsy forceps. This was followed by prompt clinical improvement. The worm was identified by its macroscopic and microscopic characteristics as an Anisakis spp. larva (L3). In 2 cases, laboratory tests revealed marked leukocytosis and eosinophilia in the peripheral blood 3-4 days after ingestion of the raw fish. The diagnosis of anisakiasis can be made by endoscopy, radiology and abdominal ultrasound, but is often made only at surgery. In the gastric form of the disease, urgent gastroscopy has both a diagnostic and a therapeutic role, because the worm can be removed by means of biopsy forceps.

Giardia cysts and Cryptosporidium oocysts in membrane-filtered municipal wastewater used for irrigation.

A wastewater tertiary treatment system based on membrane ultrafiltration and fed with secondary-treated municipal wastewater was evaluated for its Giardia cyst and Cryptosporidium oocyst removal efficiency. Giardia duodenalis (assemblages A and B) and Cryptosporidium parvum were identified in feed water but were found in filtered water only during occasional failure of the filtration system.

Waterborne transmission of Giardia and Cryptosporidium.

Giardia and Cryptosporidium spp. are parasitic protozoa which are frequent etiologic agents of waterborne diseases. This lecture will summarize the main biological and environmental factors involved in the potential risk for waterborne transmission of giardiosis and cryptosporidiosis, which have caused many outbreaks in different geographical areas. In particular, the current epidemiological situation of these parasitoses in Italy will be analysed, on the basis of research carried out on humans and on the environment. Finally, current methods for evaluating the presence of Giardia cysts and Cryptosporidium oocysts in water and new methods for cyst/oocyst removal from drinking water and wastewater will be examined.

Intestinal parasites in healthy subjects in Albania.

The purpose of this study was to evaluate the prevalence of intestinal parasites in 277 healthy subjects in the city of Mamuras (Albania, South Eastern Europe) and the correlation between parasitic infections and possible risk factors. Faecal samples collected with sodium-acetate-formalin fixative were concentrated by formalin ethylacetate sedimentation and examined as wet mounts, permanent stains and by anti-Giardia/Cryptosporidium fluorescent antibodies. Data concerning age, sex, level of education, availability of piped water, number of people living in the same house, and residence in rural or urban area were collected for each subject. Statistical analysis was performed by chi-square test and regression logistic analysis. The overall prevalence of intestinal parasites was 183/277 (66.06%). In particular, pathogenic protozoa or helminths were found in 67 subjects (24.18%), including Trichuris trichiura in 34 (12.27%), Giardia duodenalis in 31 (11.19%), Hymenolepis nana in 5 (1.8%), Ascaris lumbricoides in 3 (1.08%). A significant correlation was observed only between parasite colonization and older age and between Trichuris trichiura infection and residence in rural areas.

Rapid immunochromatographic test for serodiagnosis of canine leishmaniasis.

An rK39 immunochromatographic test and immunofluorescent-antibody test (IFAT) for serodiagnosis of canine leishmaniasis were evaluated. The two tests showed correlation for all but one of the sera obtained from 68 dogs confirmed as leishmaniasis cases and 40 dogs (22 healthy dogs and 18 dogs with other diseases) from areas where the disease is not endemic. Specificity was 100% for both tests, while sensitivity was 97% for the rapid test and 99% for IFAT.

Reduced expression of the chemokine receptor CCR1 in human macrophages and U-937 cells in vitro infected with Leishmania infantum.

Chemokines exert their actions through G-proteinlinked receptors, which are expressed to variable extents by different cell types. In accordance with the chemokine classification, these receptors are designated as CXC, CC, XC, and CX(3)C, followed by R and a number. The purpose of this investigation was to evaluate CCR1 expression in human peripheral blood-derived macrophages and the human monocytic U-937 cell line. Cells in vitro were infected with live Leishmania infantum promastigotes (zymodeme MON1); cell lysates were then subjected to SDS-PAGE and immunoblotting, by using an anti-CCR1 affinity purified polyclonal antibody. The expression of the CCR1 gene was analyzed by RT-PCR, using specific human primers. The results of both immunoblotting and RT-PCR showed that CCR1 expression in Leishmania-infected cells was lower than in uninfected control cells. These results indicate that Leishmania infantum infection causes a down-regulation of the CCR1 gene and protein expression, suggesting that reduced phagocyte recruitment at the inflammation sites could favor parasite progression and the spread of Leishmania infection.

Evidences for iNOS expression and nitric oxide production in the human macrophages.

Nitric oxide (NO) is a pleiotropic mediator of numerous biological processes, including smooth muscle relaxation, neurotransmission and defence against pathogens. In addition, NO is involved in the pathogenesis and control of inflammation, tumors, autoimmunity, and infectious and chronic degenerative diseases. NO, a highly reactive radical, is produced from L-arginine and oxygen by the enzyme NO synthase (NOS). Three NOS isoforms have been identified: two distinct NOS isoforms are constitutively expressed in cells, whereas a third isoform, inducible NOS (iNOS), is transcribed in response to specific stimuli. In particular, iNOS is responsible for the discontinuous synthesis of high amounts of NO and was originally characterized in murine macrophages after exposure to cytokines and/or microbial products. A wide range of microorganisms is sensibly inhibited in its development by NO, like fungi, bacteria, protozoa and viruses. Although NO production and its antimicrobial effect appear well established in rodent macrophages, the existence of L-arginine pathway in human mononuclear phagocytes has long been disputed. Recently, evidences showing the iNOS activity and NO production in other animal models, including humans, are now emerging, even if the NO induction has been more difficult to demonstrate. The present observations provide evidence for the occurrence of iNOS protein expression and NO production in human macrophages cultured in vitro.

Macrophage chemotactic protein-1 and macrophage inflammatory protein-1 alpha induce nitric oxide release and enhance parasite killing in Leishmania infantum-infected human macrophages.

Chemokines are a group of structurally defined small proteins that act as chemoattractants for leukocytes and are involved in many different biological activities, including leukocyte activation for antimicrobial mechanisms. We studied the effect of the chemokines monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1 alpha on nitric oxide release and parasitocidal ability of peripheral blood-derived human macrophages in vitro infected with Leishmania infantum, zymodeme MON1. In infected human macrophages, treatment with MCP-1 or MIP-1 alpha significantly enhanced nitric oxide production and leishmanicidal ability, compared with untreated cells, to the same levels induced by interferon-gamma. Both nitric oxide release and parasitocidal ability of macrophages were significantly reduced by addition of L- N(G)monomethylarginine ( L-NMMA), which is a competitive inhibitor of the L-arginine nitric oxide pathway. These data suggest that MCP-1 and MIP-1 alpha mediate macrophage activation for nitric oxide release and subsequent parasite clearance, and thus may play a role in the containment of Leishmania infection.

Unusual presentation of leishmaniasis as an adrenal cystic mass.

An unusual presentation of leishmaniasis that occurred in an Italian immunocompetent woman is described. The patient had a long history of coagulopathy due to factor VIII deficiency and pain in the right lumbar region. Computed axial tomography demonstrated a cystic mass in the right adrenal gland. Histological examination of the surgically removed cyst showed the presence of histiocytes containing Leishmania amastigotes. Serodiagnosis for leishmaniasis performed through immunofluorescent antibody testing and the rK39 enzyme immunoassay was positive, whereas a bone marrow aspirate did not reveal any parasite. The patient was not treated for leishmaniasis and recovered well after surgery. Serological testing was still positive 2 years after surgery, but clinical follow-up did not reveal the signs typical of visceral leishmaniasis.

Evaluation of a rapid immunochromatographic test for serodiagnosis of visceral leishmaniasis.

The purpose of this study was to compare the performance of a rapid immunochromatographic dipstick test for the qualitative detection of circulating antibodies to the leishmanial recombinant antigen K39 with that of a classical immunofluorescent antibody test for serodiagnosis of visceral leishmaniasis. Sera from 143 Italian subjects, including 69 patients with clinically suspected visceral leishmaniasis, 23 patients with hypergammaglobulinemia and 51 healthy controls, were tested. The immunochromatographic test was performed according to the manufacturer's instructions, using antigen-impregnated nitrocellulose paper strips. The immunofluorescent antibody test was performed according to an established method, using promastigotes of Leishmania infantum zymodeme Montpellier 1 as antigen. In 11 patients, diagnosis of active Leishmania infection was established by microscopic examination of biopsy samples and/or clinical response to meglumine antimoniate. Results of the two tests correlated for all but two sera examined. In two patients, one with proven infectious mononucleosis and one with bacterial pneumonia, the immunofluorescent antibody test was positive and the dipstick test was negative. In the restricted sample of patients in whom a definitive diagnosis was established, the immunochromatographic test was positive in 11 of 11 patients with confirmed Leishmania infection and negative in 103 of 103 subjects who either had other documented diseases or were healthy controls, showing 100% sensitivity and 100% specificity.

Inducible nitric oxide synthase expression in Leishmania-infected dog macrophages.

Nitric oxide (NO) production by the inducible NO synthase (iNOS or NOS2) represents one of the main microbicidal mechanisms of murine macrophages, but its role in other animal models is poorly investigated. Therefore, the aim of this work was to evaluate NOS2 expression in dog macrophages infected with Leishmania infantum. Macrophages obtained from peripheral blood of healthy dogs were activated with recombinant human interferon (rhIFN)-gamma and bacterial lipopolysaccharide (LPS) and then infected with L. infantum promastigotes. zymodeme MONI. For the immunofluorescence assay fixed macrophages were incubated with polyclonal rabbit anti-NOS2 and then with rhodamine F(ab')2 goat anti-rabbit IgG. For immunoblotting, cell lysates were submitted to SDS-PAGE and blots were incubated with polyclonal rabbit anti-NOS2 and then with horseradish peroxidase-conjugated goat anti-rabbit IgG. Results demonstrated that L. infantum-infected cells, after stimulation with rhIFN-gamma and LPS, displayed high levels of fluorescence for the NOS2 in their cytoplasm, unlike unstimulated uninfected macrophages. In western blotting, polyclonal anti-NOS2 reacted specifically with a protein band corresponding to 130 kDa. The signal produced in Leishmania-infected cells stimulated with rhIFN-gamma and LPS was higher than that produced in Leishmania-infected unstimulated cells. No band was detected in cellular lysates from uninfected unstimulated cells. These results indicate that dog macrophages can express NOS2, and suggest a role for IFN-gamma and LPS in NOS2 induction also in this animal model.

Nitric oxide production by macrophages of dogs vaccinated with killed Leishmania infantum promastigotes.

Human visceral leishmaniosis is endemic in Southern Italy, where the dog is the main reservoir of viscerotropic strains of Leishmania infantum. The release of nitric oxide (NO) by interferon (IFN)-gamma-activated macrophages is an important leishmanicidal mechanism in several animal species. In this work NO production, phagocytosis and killing capacity of monocyte-derived dog macrophages were evaluated in vitro before and after administration of a vaccine composed of killed Leishmania infantum promastigotes. Moreover, IFN-gamma content was measured in concanavalin A-activated dog peripheral blood mononuclear cell (PBMC) supernatants employed for macrophage stimulation. Phagocytosis, killing capacity and NO production by canine macrophages increased significantly 1 month after vaccine administration, and the increase also persisted 5 months later. In addition, the amount of IFN-gamma in PBMC supernatants was significantly higher after vaccination. Overall, our results suggest the usefulness of evaluating the in vivo protective role of this promastigote preparation in dogs.

Nitric oxide production by Leishmania-infected macrophages and modulation by cytokines and prostaglandins.

Nitric oxide (NO) produced by an inducible nitric oxide synthase (iNOS or NOS2) plays a major microbicidal role in murine macrophages and its importance is now emerging also in the dog and human models. In dogs we demonstrated that macrophages in vitro infected with Leishmania infantum produced NO, after stimulation with cytokine-enriched peripheral blood mononuclear cell supernatants. In addition, parasite killing was reduced by the NOS inhibitor L-NG monomethylarginine. On the contrary, canine blood monocytes before macrophage differentiation did not release NO, and their leishmanicidal activity was instead correlated with superoxide anion and interferon (IFN)-gamma production. In human macrophage cultures, after infection with Leishmania infantum, we showed both iNOS expression by immunofluorescence and western blotting and NO release by the Griess reaction for nitrites. Various cytokines and prostaglandins can differently modulate NO synthesis. In our experiments, stimulation by recombinant human IFN-gamma and bacterial lipopolysaccharide greatly enhanced iNOS expression and NO production in human macrophages. In addition, the prostaglandin E2 increased NO release in activated, Leishmania-infected human macrophages. These results are interesting in the light of a possible immunological or pharmacological regulation of NO synthesis and microbicidal functions of macrophages.

Nitric oxide production by Leishmania-infected macrophages and modulation by prostaglandin E2.

Nitric oxide (NO), produced by the nitric oxide synthase (iNOS) enzyme, is the most-important molecule responsible for the killing of Leishmania parasites by macrophages. In previous work we have demonstrated that, after activation with recombinant human interferon-gamma and/or bacterial lipopolysaccharide, human macrophages infected with Leishmania infantum are able to produce nitric oxide and to express nitric oxide synthase. The arachidonate derivative prostaglandin E2 has been shown to modulate various macrophage activities, and in particular nitric oxide production, sometimes with opposite effects, related to experimental conditions. In this work we have evaluated nitric oxide release and parasite killing by peripheral blood-derived L. infantum-infected human macrophages in vitro stimulated with lipopolysaccharide and simultaneously treated with prostaglandin E2. Experiments were also performed in the presence of the nitric oxide synthase inhibitor L-NGmonomethylarginine (L-NMMA) and of the cyclooxygenase inhibitor indomethacin. Nitric oxide release in supernatants of macrophage cultures was measured by the Griess reaction for nitrites. Parasite killing was microscopically evaluated by fluorescent dyes. Results demonstrated that macrophages stimulated with lipopolysaccharide and treated with prostaglandin E2 exhibited increased nitric oxide production and parasite killing, which were significantly reduced by either L-NMMA or indomethacin. In indomethacin-treated macrophages, nitric oxide production and leishmanicidal ability were partially restored by the addition of exogenous prostaglandin E2. Taken together, these results indicate that prostaglandin E2 may be involved in nitric oxide production, and possibly in the host-protective immune response against Leishmania. Moreover, the demonstration of a stimulatory role of prostaglandin E2 on nitric oxide production induced by intracellular pathogens in humans is interesting in the light of a possible pharmacological regulation of nitric oxide by modulation of prostaglandin E2 synthesis.

Effect of antiretroviral therapy on cryptosporidiosis and microsporidiosis in patients infected with human immunodeficiency virus type 1.

To better understand whether potent antiretroviral therapies can modify the natural history of HIV-1-associated microsporidiosis and cryptosporidiosis, the response to antimicrobial treatment of these opportunistic infections was evaluated in patients with or without antiretroviral treatment. Fifty patients with diarrhoea, all positive for Cryptosporidium parvum or Enterocytozoon bieneusi, were included in the study. Retrospective data were collected concerning demographics, clinical and microbiological characteristics of the parasitic infection, antiretroviral therapy and prophylaxis against opportunistic infections. Faecal samples were prepared using the Richie formalin-ethyl acetate method and stained using the modified Ziehl-Neelsen method for detection of Cryptosporidium parvum and Isospora belli, the modified trichrome and calcofluor white technique for detection of Enterocytozoon spp., and iodine for detection of ova, cysts or vegetative forms. Diarrhoea was defined as an abnormal increase in stool liquidity, an abnormal increase in stool frequency and a daily stool weight of more than 250 g for a period of at least 4 days. Patients treated with double antiretroviral therapy or protease inhibitors demonstrated an excellent response and a sustained therapeutic effect after follow-up (range, 5-36 months). The relapse of cryptosporidiosis in two patients who discontinued antiretroviral therapy suggests that the infection might remain in a latent stage. The resolution of the diarrhoea seems to be related to an increased CD4+ cell count rather than to the viral load. In conclusion, these data strongly support the hypothesis that combination antiretroviral therapy is able to greatly modify the course of cryptosporidiosis and microsporidiosis in patients infected with HIV-1.

Interactions between Leishmania parasites and host cells.

Several species of protozoa belonging to the genus Leishmania are pathogenic for humans, causing visceral and cutaneous diseases. They are transmitted by phlebotomine sandflies as flagellated promastigotes to mammals hosts, where they live as aflagellated amastigotes mainly within macrophages. Studies performed on mice infected with Leishmania major demonstrated that host defence against this infection depends on the interleukin-12-driven expansion of the T helper 1 cell subset, with production of cytokines such as interferon-gamma, which activate macrophages for parasite killing through the release of nitric oxide. The parasitocidal role of this radical is now emerging also in the human and canine model. Healing or progression of the infection is related to the genetic and immune status of the host, and to the virulence of different species and strains of Leishmania. The parasite survival ultimately depends on the ability to evade the host immune response by several mechanisms. Among them, inhibition of the signal transduction pathway of the host cells is particularly important. In fact, promastigotes inhibit protein kinase C activation, cause Ca++ influx into the host cell and decrease the levels of myristoylated alanine-rich C kinase substrate-related proteins, which are substrates for PKC. In addition, Leishmania infection blocks IFN-gamma-induced tyrosine kinase phosphorylation, with consequent impairment of signalling for IL-12 and nitric oxide production. Finally, Leishmania activates protein phosphotyrosine phosphatases, which down-regulate mitogen-activated protein kinase signalling and c-fos and nitric oxide synthase expression. New pharmacological applications, including protein tyrosine phosphatase and protein farnesyltransferase inhibitors, are being evaluated against leishmaniosis in vitro and in vivo in the murine model.