Prophylactic antineoplastic activity of Toxoplasma gondii RH derived antigen against ehrlich solid carcinoma with evidence of shared antigens by comparative immunoblotting.

BACKGROUND: With cancer cases escalation, an urgent request to develop novel combating strategies arise. Pathogen-based cancer-immunotherapy is getting more consideration. Autoclaved parasitic antigens seem promising candidates, taking steadily their first steps. Our aim was to examine the prophylactic antineoplastic activity of autoclaved Toxoplasma vaccine (ATV) and to test for the shared antigen theory between Toxoplasma gondii and cancer cells. METHODS: Mice were immunized with ATV followed by Ehrlich solid carcinoma (ESC) inoculation. Tumor weight, volume, histopathology, and immunohistochemistry for CD8+ T cells, Treg cells and VEGF were assessed. In addition, the proposed shared antigen theory between parasites and cancer was also verified using SDS-PAGE and immunoblotting. RESULTS: Results revealed powerful prophylactic activity of ATV with 13.3% inhibition of ESC incidence, significant reduction in tumor weight and volume in ATV vaccinated mice. Immunologically, significantly higher CD8+T cells and lower FOXP3+ Treg cells surrounded and infiltrated ESC in ATV immunized mice with higher CD8+T/Treg cells ratio and significant antiangiogenic effect. Moreover, SDS-PAGE and immunoblotting showed four shared bands between Ehrlich carcinoma and ATV of approximate molecular weights 60, 26, 22 and 12.5 KDa. CONCLUSION: Exclusively, we demonstrated a prophylactic antineoplastic activity of autoclaved Toxoplasma vaccine against ESC. Moreover, to the best of our knowledge this is the first report highlighting the existence of cross-reactive antigens between Toxoplasma gondi parasite and cancer cells of Ehrlich carcinoma.

Toxoplasma gondii-derived antigen modifies tumor microenvironment of Ehrlich solid carcinoma murine model and enhances immunotherapeutic activity of cyclophosphamide.

Pathogen-based cancer vaccine is a promising immunotherapeutic weapon to stimulate cancer immunosuppressive state. Toxoplasma gondii is a potent immunostimulant, and low-dose infection was linked to cancer resistance. Our goal was to evaluate the therapeutic antineoplastic activity of autoclaved Toxoplasma vaccine (ATV) against Ehrlich solid carcinoma (ESC) in mice in reference to and in combination with low-dose cyclophosphamide (CP), a cancer immunomodulator. Mice inoculation with ESC was followed by applying different treatment modalities including ATV, CP, and CP/ATV. We evaluated the impact of the different treatments on liver enzymes and pathology, tumor weight, volume, and histopathological changes. Using immunohistochemistry, we evaluated CD8+ T cell, FOXP3+ Treg, CD8+/Treg outside and inside ESC, and angiogenesis. Results showed significant tumor weights and volumes reduction with all treatments with 13.3% inhibition of tumor development upon combined CP/ATV use. Significant necrosis and fibrosis were noted in ESC by all treatments with improved hepatic functions versus non-treated control. Although ATV was almost equivalent to CP in tumor gross and histopathology, it promoted an immunostimulatory activity with significant Treg cells depletion outside ESC and CD8+ T cells infiltration inside ESC with higher CD8+ T/Treg ratio inside ESC superior to CP. Combined with CP, ATV exhibited significant synergistic immunotherapeutic and antiangiogenic action compared to either treatment alone with significant Kupffer cells hyperplasia and hypertrophy. Exclusively, therapeutic antineoplastic and antiangiogenic activity of ATV against ESC was verified that boosted CP immunomodulatory action which highlights a novel biological cancer immunotherapeutic vaccine candidate.

Effect of alginate nanoparticles on the immunogenicity of excretory-secretory antigens against acute toxoplasmosis in murine model.

Efficacy of alginate nanoparticles (Alg-NPs) as vaccine delivery for the excretory-secretory antigens (ESAs) against the virulent strain of Toxoplasma gondii was evaluated. Swiss albino mice were intraperitoneally immunized with three doses of either in vivo and in vitro-prepared ESA vaccines, 20 µg each, at 2-week intervals, then were challenged with 2500 tachyzoites of the RH HXGPRT (-) Toxoplasma gondii strain, four weeks later. Mice mortality, tachyzoite number in both peritoneal fluid and impression smear, and viability, ultrastructural tachyzoite changes, measuring immunological markers, and histopathological changes of both liver and spleen were studied, in comparison to alum adjuvanted ESAs and infected control subgroups. The in vivo-prepared Alg-NPs loaded ESAs vaccinated subgroups induced significant reduction in mice mortality, tachyzoite count in both peritoneal fluid and impression smears and viability. Scanning electron microscopy revealed tachyzoites deformities with multiple irregularities, while transmission electron microscopy showed tachyzoites distortion, disrupted plasma membranes, loss of nuclear integrities, and absence of dense granules with extensive vacuolations. A statistically significant increase in the level of both IFN-γ and anti-Toxoplasma IgG was noted. Histopathological results recorded amelioration of the pathological changes induced by Toxoplasma infection in both liver and spleen, with scanty parasites. Therefore, Alg-NPs proved its effectiveness in enhancing the ESAs antigencity, and recommended to test its potentiality as drugs carrier for anti-Toxoplasma agents to enhance their therapeutic efficacy.

Auramine-phenol vs. modified Kinyoun's acid-fast stains for detection of coccidia parasites.

OBJECTIVE: Auramine-phenol stain was compared with Kinyoun's acid-fast stain to detect coccidia parasites in fecal samples from immunocompromised patients. The comparison was based on the number of detected cases, sensitivity, specificity, time required for the procedure, ease of use, interpretation, and cost. METHODS: A total of 112 fecal specimens were examined by conventional methods: Direct wet saline smear, iodine smear, and formol ether sedimentation technique. Duplicate smears of the fecal concentrates were stained by both procedures. RESULTS: Kinyoun's and auramine-phenol stains detected 22 and 24 positive coccidia specimens respectively. The control group (27 immunocompetent relatives) showed a high incidence of Giardia lamblia infection. Kinyoun smears were difficult to interpret, while auramine smears were extremely easy to read, thus requiring less time. Artifacts were readily recognizable. The cost of auramine-phenol reagents was much higher than Kinyoun's acid-fast stain. CONCLUSION: Although the overall ranking of both staining techniques was high, the auramine-phenol stain was a more desirable test despite its higher cost.

Evaluation of enzyme immunoassay techniques for diagnosis of the most common intestinal protozoa in fecal samples.

OBJECTIVE: This study was designed to evaluate the antigen capture enzyme immunoassays (EIAs) Triage parasite panel and TechLab Entamoeba histolytica II in detecting Giardia intestinalis, Cryptosporidium sp, and Entamoeba histolytica in fecal samples in comparison to microscopy, and in differentiating Entamoeba histolytica from Entamoeba dispar. METHODS: The Triage EIA was evaluated using 100 stool specimens that were tested by standard ova and parasite examination, including staining with both trichrome and modified acid-fast stains. Differentiation between E. histolytica and E. dispar was performed using TechLab. RESULTS: Microscopic examination revealed that 19% of the samples were positive for Giardia, 4% for Cryptosporidium, and 1% for E. histolytica/E. dispar, and other parasites were found in 5%. By Triage, 23% of the samples were infected with Giardia, 5% with Cryptosporidium, and 2% with E. histolytica/E. dispar. Triage showed a sensitivity of 100% and specificity of 91.5%. The TechLab assay was negative for both samples diagnosed as E. histolytica/E. dispar by Triage, which suggested that they were E. dispar. Both tests showed no cross-reactivity with other intestinal protozoa. CONCLUSION: These results indicate that antigen detection by EIA has the potential to become a valuable tool, capable of making stool diagnostics more effective.

Use of pooled sodium acetate acetic acid formalin-preserved fecal specimens for the detection of intestinal parasites.

This study aimed at comparing detection of intestinal parasites from single unpreserved stool sample vs. sodium acetate acetic acid formalin (SAF)-preserved pooled samples, and stained with chlorazol black dye in routine practice. Unpreserved samples were collected from 120 patients and represented as Group I. Other three SAF-preserved samples were collected from the same patients over a 6-day period and represented as Groups IIa, IIb, and IIc. The latter groups were equally subdivided into two subgroups. The first subgroup of each of the three samples was examined individually, whereas the second subgroup of each were pooled and examined as a single specimen. All groups were examined by the routine diagnostic techniques; however, in group II when the diagnosis was uncertain, the chlorazol black dye staining procedure was carried out. Results demonstrated that out of 74 patients who continued the study, 12 cases (16%) were positive in group I, compared with 29 (39%) in the subgroups examined individually, and 27 (36%) in the pooled subgroups. Therefore, pooling of preserved fecal samples is an efficient and economical procedure for the detection of parasites. Furthermore, the chlorazol black dye was simple and effective in detecting the nuclear details of different parasites.

Evaluation of the co-agglutination test in diagnosis of experimental microsporidiosis.

Microsporidiosis is an emerging and opportunistic infection associated with wide range of clinical syndromes in humans. Confirmation of the presence of microsporidia in different samples is laborious, costly and often difficult. The present study was designed to evaluate the utility of the Co-agglutination test (Co-A test) for detection of urinary, fecal and circulating microsporidial antigens in experimentally infected mice. One hundred and twenty male Swiss albino mice were divided into non infected control and infected experimental groups which were further subdivided into two equal subgroups; immunosuppressed and immunocompetent. Microsporidial spores were isolated from human stools and identified to be Encephalitozoon intestinalis by the molecular methods. They were used to infect each subgroup of mice, then their urine, stools and sera were collected at the 1st, 3rd, 5th, 7th and 9th days post-infection (PI). Co-A test, using prepared hyperimmune serum, was used to detect antigens in all samples collected. The cross reactivity of microsporidial hyperimmune sera with antigens of Cyclospora cyatenensis and Cryptosporidium parvum was investigated by Co-A test. The results showed that Co-A test was effective in detecting microsporidial antigen in stool of immunosuppressed infected mice from the 1st day PI, and in urine and serum from the 3rd day PI till the end of the study. In the immunocompetent subgroup, Co-A test detected microsporidial antigens in stool, serum and urine of mice from the 1st day, 3rd day and the 5th day PI, respectively till the end of the study, without cross reactivity with C. cyatenensis or C. parvum in both subgroups. Co-A test proved to be simple and suitable tool for detecting microsporidial antigen in different specimens and did not need sophisticated equipment. It is very practical under field or rural conditions and in poorly equipped clinical laboratories.

The utility of direct agglutination (DAT) and fast agglutination screening (FAST) tests in serodiagnosis of experimental microsporidiosis.

The present study was designed to evaluate the efficiency of two serodiagnostic tests; the direct agglutination test (DAT) and the fast agglutination screening test (FAST) in the diagnosis of Microsporidia in experimentally infected mice and to differentiate between different species of the parasite. The swiss albino mice were divided into non infected control and infected experimental groups which were further subdivided into ten subgroups. Ten samples of microsporidial spores were isolated from ten human stools and each one was used to infect each subgroup of mice. Stool and sera were collected weekly from each subgroup from the 1st to the 4th week post infection (PI). DAT & FAST tests, using antigen prepared from the different species of microsporidial spores were used to detect antibodies in sera of different mice subgroups. The cross reactivity of microsporidial spores with the antibodies of Cyclospora cyatenensis and Cryptosporidium parvum was investigated by DAT & FAST. The results proved that DAT & FAST were effective in detecting microsporidial antibodies in sera of experimentally infected mice from the 2nd week PI till the end of the study, without cross reactivity with C. cyatenensis or C. parvum. They failed to differentiate between different Microspoiridia species used but, they gave good interpretation and they were specific and sensitive, and did not need sophisticated equipments.

Effect of solar disinfection on viability of intestinal protozoa in drinking water.

The effect of solar disinfection on the viability of intestinal protozoa Giardia lamblia, Microsporidia sp., Cryptosporidium parvum, Cyclospora cyatenensis and Entamoeba histolytica in drinking water was studied as compared to chlorine disinfection. The protozoa were collected from stool samples, to infect to the distilled water. Chlorinated water samples were prepared at concentration of 4 ppm, and the parasites were incubated overnight at room temperature with the treated water. Sun treatment was applied for 2 exposures (6 & 24 hrs), in summer and winter. Sun treated water samples were put in tubes and exposed to sun. The 2 disinfection methods were tested in plastic and glass test tubes. Parasites viability was assessed by viability assay using trypan blue stain (0.4%), and bioassay infectivity tests in experimentally laboratory bred mice. Results proved that all parasites' viability was not affected by chlorine, following solar disinfection treatment, parasites became dark blue in colour and deformed by trypan blue stain. High parasites death was recorded for all parasites except Microsporidia sp. Bioassay infectivity test showed a statistically significant reduction in mean number of all parasites in intestinal sections compared to controls. The best results were tubes exposure to sun for 24 hrs in summer, where G. lamblia, C. parvum and C. cyatenensis were inactivated or absence in intestinal sections. No statistically significant difference was between the use of plastic and glass tubes, either in chlorine or sun treated parasites. So, solar disinfection proved a simple, cheap and effective means for improving water for human use, particularly in developing countries.

Immunization against trichinellosis using microwaved larvae of Trichinela spiralis.

The present study evaluated the effect of microwave irradiation on infective larvae of Trichinella spiralis (T. spiralis) by the ultrastructure changes of the microwaved larvae (ML) using scanning electron microscope (SEM). The ML tested the ability to immunize mice against a challenge infection with T. spiralis. For the optimal dose and the best route of immunization inducing protection against challenge infection, two doses were used; 300 & 600 ML as one or two-dose regimen, each dose was given orally and intraperitoneally (IP). SEM revealed tegumental damage of the ML in the form of distortion, loss of normal fold pattern and depressions or papillae protruded from their outer surface. After administration of the ML (orally or IP) to the non-infected control mice, neither adults nor larvae were detected in the intestines or muscles respectively. This indicated loss of larvae infectivity after exposure to the microwave irradiation. Also, a significant protection against challenge infection with T. spiralis was demonstrated in experimental mice immunized by ML, orally or IP. This was assessed by a statistically significant decrease in adult and muscle larval count, compared with the non-immunized infected control. Complete protection against both adults and larvae (100%) was achieved by IP injection of two doses of 600 ML, two weeks apart. The results suggested the feasible application of the microwave irradiation on meat for its decontamination from T. spiralis larvae. Such a method might be a promising a prophylaxis vaccine against trichinellosis in animals and/or humans.

The use of ELISA and immunohistochemistry techniques for detection of Toxoplasma gondii antigen in tissues of experimentally infected mice.

The capability of double antibody sandwich enzyme-linked immunosorbent assay (ELISA) for detecting antigens of Toxoplasma gondii (T. gondii) in different mice tissue specimens was evaluated in comparison to the immunohistochemistry (IHC) technique. Results proved that tissue antigens were detectable in liver, kidney and mesenteric lymph node (LN) specimens by both methods from the second day of infection, with statistically significant increase in its amount in all organs throughout the period of the study. Using ELISA technique, the highest antigen level was recorded on the second day (0.120+/-0.0015) and the fourth day (0.147+/-0.0034) of infection in LN specimens, while, the liver showed the highest antigen level at the sixth day post infection (PI)(0.165+/-0.0066). On the other hand, using the IHC technique, the highest number of tachyzoites was recorded in LN sections in all studied durations, the second, the fourth and the sixth days PI (1.1+/-0.875, 1.6+/-1.173 & 3.1+/-1.370 respectively). Thus, sandwich ELISA technique might offer a valuable aid for rapid diagnosis of acute toxoplasmosis in human tissues, and it has proved to be more accurate than IHC technique, since its results was coincided with the pathogenesis of the disease.

Effect of Schistosoma mansoni infection on physiological gastrointestinal transit and contractility.

The work aimed to study the effect of Schistosoma mansoni (S. mansoni) on gastrointestinal transit and contractility of the colonic muscles of two subgroups of experimental mice, infected by 50 & 200 cercaria/mouse respevtively, at 8th & 12th week postinfection (PI). In addition, the histopathologic changes in the colon, and the immunological changes of the host were studied at different durations. At 8th weeks PI, in both subgroups, gastrointestinal transit was statistically significant decreased, in concurrent with statistically significant increase in the colonic muscle contractility compared to the controls. The colon was inflamed as shown by mucosal inflammatory infiltrates, with large size and number of schistosomal granulomas. The serum antigen was absent, while the serum antibody was detectable at low titre. At 12th weeks PI, there was a more statistically significant decrease in gastro-intestinal transit, and increase in the colonic muscle contractility. The colon was still inflamed, but the granulomas were reduced in size and in number, with increase in the fibrocytes density. These alterations coincided with absence of serum antigen and increase in the antibody titre. All changes were more pronounced in the 2nd group of mice (200 ceraria/mouse) than the 1st one (50 cercaria/mouse). So, intestinal schistosomiasis is associated with great structural, functional and immunological changes, related to the time course and the infection intensity which may be involved in the pathogenesis and clinical manifestations of the disease.

Experimental trichinosis: parasitological, electrophysiological and biochemical studies.

Trichinosis is a parasitic infection affecting the gut and the muscles causing mild gastrointestinal symptoms followed by periorbital oedema, muscle pains, fever and eosinophilia. The infection evokes functional disturbances in physiological effector systems. Furthermore, several biochemical changes are associated with the infection. Therefore, this work was carried out to study the electrophysiological changes in intestine, striated and cardiac muscles by electromyography (EMG) and to assess the biochemical changes through measurement of serum cholinesterase and intestinal myeloperoxidase activity (MPO) in both light and heavy infected experimental animals by Trichinella spiralis (T. spiralis). Electrophysiological results showed increased contractility of the smooth muscle layers of the intestine only early in the infection, whereas both striated and cardiac muscles showed increase in the contractility with the progress of infection in both light and heavy infection. Significant myocardial dysfunction in the form of bradycardia, in addition to major histopathological changes in the heart occurred from the beginning of the infection and increased till the end of the study. Biochemical study showed gradual increase in serum cholinesterase, while, the intestinal MPO showed increase only in the early stage of the infection. It was noticed that all changes were more pronounced in the heavily infected group than the lightly infected one.