High prevalence of Microsporidium infection in HIV-infected patients.

Sixty-four patients infected with human immunodeficiency virus (HIV) participated in a study to determine opportunistic enteric pathogens and compare them with the patients' clinical status. The most frequently found pathogens were microsporidium (81.2%), Cryptosporidium parvum (20.3%), Candida albicans (12.5%) and Blastocystis hominis (10.9%). Less frequently found pathogens were Giardia intestinalis (6.2%), Cyclospora (4.7%), Opishorchis viverrini ova (3.1%), Strongyloides stercoralis larvae (3.1%) and hookworm ova (1.6%). The presence of enteric pathogens was not significantly associated with sex, length of HIV seropositivity and diarrheal symptoms. A high prevalence of microsporidium, based on microscopic examination, was found in Thai HIV-infected patients. This confirms the importance of microsporidium in HIV-infected/AIDS patients and the necessity for stool evaluation in all HIV-infected patients.

A murine model of allergy caused by American cockroach (CR), Periplaneta americana.

An animal model resembling the human immuno-pathological features of CR allergy is needed for CR allergy research, e.g., measuring allergenicity of novel allergens, testing immunotherapeutic efficacies of drugs and vaccines. In this study we develop a murine model of American CR, P. americana allergy. BALB/c mice, 6 weeks old, were individually intraperitoneally injected with three doses (days 0, 7 and 14) of alum adjuvanted-crude extract of P. americana. On days 21 and 23, they were given crude CR extract in PBS intranasally (10 microl) and aerosolically (10 ml) via an air-pressure nebulizer, respectively. Mice received alum alone and PBS instead of the CR extract served as non-allergenic controls. All mice were bled twenty four hours after the nebulization and sacrificed. Their serum samples, broncho-alveolar lavage fluids (BALF), and lung tissues were collected. BALF of all allergen-treated mice had marked cellular infiltration notably neutrophils, eosinophils and lymphocytes. The average total cell count in BALF of the allergenic mice was 1.9 x 10(5) cells/ml which out-numbered those of the non-allergenic controls (8 x 10(4) cells/ml). The eosinophil infiltration was pronounced in lungs of the allergen-treated mice. Specific serum IgE to the CR extract elevated in serum samples of all allergen treated mice and nil in the sera of the controls. None of the mice showed detectable level of IgG2a to the CR extract. RT-PCR revealed that all allergen-treated mice had marked increase of IL-13, IL-4 and TNF-alpha gene expressions, slight increase of IL-5 gene expression, and absence of detectable IFN-gamma gene expression in comparison to the non-allergenic controls. None of the allergen-treated mice and 50% of the non-allergenic controls had IL-12 gene expression as detected by RT- PCR. One allergen treated-mouse (25%) had subpar level of the IL-18 gene expression compared to the controls. Results of the quantitative real-time PCR conformed to those of the RT-PCR. A murine model of P. americana resembling human allergic manifestations was successfully developed.

Murine monoclonal antibodies neutralizing the cytotoxic activity of diphtheria toxin.

In this study, murine monoclonal antibodies that specifically bound to the A and B subunits of diphtheria toxin (DT) were produced by conventional hybridoma technology using the spleens of BALB/c mice immunized with diphtheria DTP vaccine and CRM197. Monoclonal antibodies specific to the A subunit, i.e. clone AC5, as well as those specific to the B subunit, i.e. clone BB7, could neutralize the DT-mediated cytotoxicity to Vero cells in microcultures. The DT neutralizing mechanisms have yet to be determined. The MAbBB7 is hypothesized to either interfere with the DT receptor binding or with the pore forming function of the T domain of the B subunit. The MAbAC5 could neutralize the DT mediated cytotoxicity when mixed with the DT before adding to the Vero cell culture thus suggesting that the antibody interfered with the translocation of the A subunit. The A subunit-antibody complex might be too large to pass through the membrane channel formed by the T domain and thus prevent the accessibility of the A subunit to the cytosolic target. It is also possible that the MAb AC5 blocked the enzymatic active site of the enzyme catalytic subunit. While further experiments are needed to localize the epitopes of the two MAbs on the holo-DT in order to reveal the DT neutralizing mechanisms, both MAbs in their humanized forms have a high potential as human therapeutic antibodies for diphtheria.

Immunohistochemical study of acute and chronic toxoplasmosis in experimentally infected mice.

Acute and chronic Toxoplasma infections were evaluated in mice using stage specific antibodies and immunocytochemistry. Mice with acute toxoplasmosis were less active, had erectile body hair and seldom took food or water resulting in weight loss. All mice died within 7 days post-inoculation. The immunohistochemical technique enhanced visualization of parasites allowing their distribution to be accurately followed. Following intraperitoneal infection, tachyzoites were initially identified on the surface of the liver and spleen. There was a rapid increase in the number of tachyzoites associated with invasion from the surrounding connective tissue into the organs with formation of inflammatory lesions in the liver. The focal inflammatory lesions showed increasing numbers of tachyzoites with the period post-inoculation. Similar increases in tachyzoites were observed for the spleen. In contrast, only a few individual tachyzoites were seen in the brain at the final time point. In chronic infections, the mice were asymptomatic but tissue cysts containing large numbers of bradyzoites were observed in all brains with the average number of 295 tissue cysts per half brain and the average cystic size of 46.02 +/- 5.08 microm. By histology and immunostaining, the tissue cysts were readily identifiable along with a mild inflammatory cell infiltration into the meninges and perivascular cuffing. Double immunocytochemical labelling confirmed the exclusive presence of tachyzoites during the acute phase and bradyzoites during the chronic phase.

OmpL1 DNA vaccine cross-protects against heterologous Leptospira spp. challenge.

Available leptospirosis vaccines made up of inactivated bacteria or their membrane components elicit immunity which is serovar specific and unsatisfactory immunological memory. A vaccine that protects across Leptospira serogroups/serovars, i.e. broad spectrum, and induces long-lasting memory is needed for both human and veterinary uses. In this study, a plasmid DNA vaccine was constructed from cloning gene encoding a transmembrane porin protein, OmpL1, of pathogenic Leptospira interrogans, serogroup Icterohaemorrhagiae, serovar Copenhageni into a mammalian expression vector pcDNA3.1(+). The protective efficacy of the ompL1-pcDNA3.1(+) plasmid DNA vaccine was studied by immunizing hamsters intramuscularly with three doses of the vaccine (100 microg per dose) at two week intervals. The empty pcDNA3.1(+) and PBS were used as mock as negative vaccine controls, respectively. All animals were challenged with the heterologous Leptospira interrogans, serogroup Pomona, serovar Pomona (10 LD50), at one week after the last vaccine booster. The ompL1-pcDNA3.1(+) plasmid DNA vaccine rescued some vaccinated animals from the lethal challenge and delayed death time, reduced morbidity, e.g. fever, and/or the numbers of Leptospira in the tissues of the vaccinated animals. While the results are encouraging, further studies are needed to optimize the immunization schedule, vaccine dosage and formulation in order to maximize the efficacy of the vaccine.

Traffic of antibody-secreting cells after immunization with a liposome-associated, CpG-ODN-adjuvanted oral cholera vaccine.

An oral cholera vaccine made up of heat-treated recombinant cholera toxin (rCT), V. cholerae lipopolysaccharide (LPS), and recombinant toxin-co-regulated pili subunit A (rTcpA), entrapped in liposomes in the presence of unmethylated bacterial CpG-DNA (ODN#1826) was used to orally immunize a group of eight week old rats. A booster dose was given 14 days later. Control rats received placebo (vaccine diluent). The kinetics of the immune response were investigated by enumerating the antigen specific-antibody secreting cells (ASC) in the blood circulation and intestinal lamina propria using the ELISPOT assay and a histo-immunofluorescence assay (IFA), respectively. ASC of all antigenic specificities were detected in the blood of the vaccinated rats as early as two days after the booster dose. The numbers of LPS-ASC and TcpA-ASC in the blood were at their peak at day 3 post booster while the number of CT-ASC was highest at day 4 after the booster immunization. At day 13 post immunization, no ASC were detected in the blood. A several fold increase in the number of ASC of all antigenic specificities in the lamina propria above the background numbers of the control animals were found in all vaccinated rats at days 6 and 13 post booster (earlier and later time points were not studied). Vibriocidal antibody and specific antibodies to CT, LPS and TcpA were detected in 57.1% and 52.4%, 14.3%, and 19.0% of the orally vaccinated rats, respectively. The data indicated that rats orally primed with the vaccine could produce a rapid anamnestic response after re-exposure to the V. cholerae antigens. Thus, a single dose of the vaccine is expected to elicit a similar anamnestic immune response in people from cholera endemic areas who have been naturally primed to V. cholerae antigens, while two doses at a 14 day interval should be adequate for a traveler to a disease endemicarea.

A quantitative ultrastructural study of the liver and the spleen in fatal falciparum malaria.

We performed a retrospective study of 25 patients who died of severe falciparum malaria in Thailand and Vietnam using electron microscopy. The aims of the study were: to determine if there was any significant association between parasitized red blood cells (PRBC) sequestered in liver and spleen and particular pre-mortem clinical complications, and to compare the degree of parasite load between the liver and spleen within the same patients. PRBC sequestrations in each organ were compared with the pre-mortem parasitemia, to calculate the sequestration index (S.I.). The S.I. showed that the degree of PRBC sequestration in the spleen was higher than the liver (S.I. median = 3.13, 0.87, respectively) (p < 0.05). The results of quantitative ultrastructural study showed a significantly high parasite load in the liver of patients with jaundice, hepatomegaly and liver enzyme elevation (p < 0.05). We found a significant correlation between PRBC sequestration in the liver and a high serum bilirubin level, a high aspartate aminotransferase (AST) level and an increase in the size of the liver (Spearman's correlation coefficient = 0.688, 0.572, 0.736, respectively). Furthermore, a higher parasite load was found in the liver of patients with acute renal failure (ARF) compared to patients without ARF (p < 0.05). These findings suggest that PRBC sequestration in the liver is quantitatively associated with pre-mortem hepatic dysfunction and renal impairment. There was no significant difference between splenomegaly and PRBC sequestration. The size of a palpable spleen was not correlated with parasite load in the spleen. When ultrastructural features were compared between the two reticuloendothelial organs, we found that the spleen had more PRBC and phagocytes than the liver. The spleen of non-cerebral malaria (NCM) patients had more phagocytes than cerebral malaria (CM) patients. This observation reveals that the spleen plays a major role in malaria parasite clearance, and is associated with host defence mechanisms against malaria.

Ultrastructural changes of pancreatic islets microcirculation in nonobese diabetic (NOD) mice.

The objective of this study was to investigate the ultrastructural changes of vascular pancreatic islets using a transmission electron microscopic technique. The major ultrastructural changes of microvessel in NOD mice are indicated by the swelling and vacuolization of the endothelial cell. Swollen cells are the first noticeable lesion of the cell response in reversible degeneration that is caused by the failure of homeostatic control. Loss in endothelial cell homeostasis is primarily a marker of endothelial dysfunction that plays a key role in the pathogenesis of diabetic vascular disease by losing the control of vascular tone. Diabetes also associates with an increased generation of oxygen-derived free radicals that may impair vasodilatation through the inactivation of vasodilators. In conclusion, consistent with a hypothesis that loss of the modulatory role of the endothelium may be a critical and initiating factor in the development of diabetic vascular disease, the ultrastructural changes in this study may indicate the first sign of endothelial dysfunction. This dysfunction correlates to the relationship between diabetes and reversible lesions of vessels in NOD mice, making for a better understanding of the pathophysiology of diabetic vascular disease to set the stage for further investigation to restore endothelial dysfunction in diabetes.

CpG DNA, liposome and refined antigen oral cholera vaccine.

An oral cholera vaccine made up of three Vibrio cholerae antigens, i.e. lipopolysaccharide (LPS), recombinant toxin co-regulated pili (rTcpA) and heat-treated cholera toxin (H-CT) has been developed in six different formulations. Eight-week-old Wistar rats were divided into nine groups and immunized as follows: the first group received the oral vaccine 1 consisting of the three antigens (LPS, rTcpA and H-CT) associated with a liposome (L) and bacterial CpG-DNA (ODN#1826). The rats of groups 2 and 3 received oral vaccines 2 and 3 consisting of the liposome-associated three antigens with and without non-bacterial CpG-DNA (ODN#1982), respectively. Rats of groups 4 received oral vaccine 4 consisting of the three antigens mixed with the ODN#1826, similar to vaccine 1, but without liposome. Rats of groups 5 and 6 received oral vaccines 5 and 6 consisting of the three antigens with and without ODN#1982, respectively, similar to vaccines 2 and 3, but without liposome. Rats of groups 7, 8 and 9 received oral placebos, namely liposomes (L), ODN#1826 (CpG), and vaccine diluent, i.e. 5% NaHCO3 solution, respectively. All vaccines were given in three doses at 14-day intervals. It was found that the combination of liposome and ODN#1826 in vaccine 1 evoked the highest immune response to V. cholerae antigen compared to other vaccine formulations and placebos, as measured by the appearance of antigen-specific antibody-producing cells in the intestinal lamina propria. The immunogenicity according to the magnitude of the immune response was: V1>V2=V3>V4>V5=V6>V7=V8=V9. The results of this study indicate that CpG-DNA and liposome are effective mucosal adjuvants for an oral cholera vaccine prepared from refined V. cholerae antigens and their combination seems to be synergistic. The potential role of liposome as a vaccine delivery vehicle has been confirmed.

Pathologic study of acute toxoplasmosis in experimental animals.

We studied the pathology of acute toxoplasmosis in experimental mice inoculated with RH strain tachyzoites of Toxoplasma gondii. All died from severe disseminated toxoplasmosis involving the liver, spleen and pancreas. Pathological features of acute toxoplasmosis in susceptible mice could be regarded as an excellent model for acute reactivation of Toxoplasma in the immunosuppressed host.

Specific monoclonal antibodies to Strongyloides stercoralis: a potential diagnostic reagent for strongyloidiasis.

In this study, specific hybridomas secreting monoclonal antibodies (MAb) to antigen of Strongyloides stercoralis filariform larvae were produced. Specific epitopes targeted by the MAb were protein in nature and located in situ in the internal content of the filariform larvae of the parasite but not in the esophagus. The MAb reacted to the homologous antigen in an indirect ELISA but did not reveal any reaction to the SDS-PAGE separated-homologous antigen in a Western blot analysis (WB) suggesting a conformational epitope specificity. The MAb were of IgG1 isotype which is the isotype known to have high affinity to this epitope so they were used in a dot-ELISA to detect the antigen of the parasite. The assay could detect the epitopes in 78 ng or more of the crude filariform larval extract but did not reveal any positive result when applied to detect antigen in stool samples of parasitologically confirmed strongyloidiasis patients. The negative antigen test results can be explained as follows. Either the MAb were filariform stage-specific and thus did not recognize the rhabditiform larval antigen mainly contained in the patient's stool or the amounts of antigen in the stool samples were too small and/or unevenly dispersed. In the latter instance, the MAb developed in this study would have a diagnostic potential if used in an immunological test design where more volume of fresh stool sample could be accommodated in the test, e.g. a sandwich plate ELISA.