Giardia Alters Commensal Microbial Diversity throughout the Murine Gut.

Giardia lamblia is the most frequently identified protozoan cause of intestinal infection. Over 200 million people are estimated to have acute or chronic giardiasis, with infection rates approaching 90% in areas where Giardia is endemic. Despite its significance in global health, the mechanisms of pathogenesis associated with giardiasis remain unclear, as the parasite neither produces a known toxin nor induces a robust inflammatory response. Giardia colonization and proliferation in the small intestine of the host may, however, disrupt the ecological homeostasis of gastrointestinal commensal microbes and contribute to diarrheal disease associated with giardiasis. To evaluate the impact of Giardia infection on the host microbiota, we used culture-independent methods to quantify shifts in the diversity of commensal microbes throughout the gastrointestinal tract in mice infected with Giardia We discovered that Giardia's colonization of the small intestine causes a systemic dysbiosis of aerobic and anaerobic commensal bacteria. Specifically, Giardia colonization is typified by both expansions in aerobic Proteobacteria and decreases in anaerobic Firmicutes and Melainabacteria in the murine foregut and hindgut. Based on these shifts, we created a quantitative index of murine Giardia-induced microbial dysbiosis. This index increased at all gut regions during the duration of infection, including both the proximal small intestine and the colon. Giardiasis could be an ecological disease, and the observed dysbiosis may be mediated directly via the parasite's unique anaerobic fermentative metabolism or indirectly via parasite induction of gut inflammation. This systemic alteration of murine gut commensal diversity may be the cause or the consequence of inflammatory and metabolic changes throughout the gut. Shifts in the commensal microbiota may explain observed variations in giardiasis between hosts with respect to host pathology, degree of parasite colonization, infection initiation, and eventual clearance.

Effect of tumor necrosis factor alpha on insulin-dependent diabetes mellitus in NOD mice. I. The early development of autoimmunity and the diabetogenic process.

Tumor necrosis factor (TNF) alpha is a cytokine that has potent immune regulatory functions. To assess the potential role of this cytokine in the early development of autoimmunity, we investigated the effect of TNF on the development of insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice, a spontaneous murine model for autoimmune, insulin-dependent type I diabetes. Treatment of newborn female NOD mice with TNF every other day for 3 wk, led to an earlier onset of disease (10 versus 15 wk of age in control mice) and 100% incidence before 20 wk of age (compared to 45% at 20 wk of age in control phosphate-buffered saline treated female mice). In contrast, administration of an anti-TNF monoclonal antibody, TN3.19.12, resulted in complete prevention of IDDM. In vitro proliferation assays demonstrated that mice treated with TNF developed an increased T cell response to a panel of beta cell autoantigens, whereas anti-TNF treatment resulted in unresponsiveness to the autoantigens. In addition, autoantibody responses to the panel of beta cell antigens paralleled the T cell responses. The effects mediated by TNF appear to be highly age dependent. Treatment of animals either from birth or from 2 wk of age had a similar effect. However, if treatment was initiated at 4 wk of age, TNF delayed disease onset. These data suggest that TNF has a critical role in the early development of autoimmunity towards beta-islet cells.

The abundance of sterile transcripts in Giardia lamblia.

The protozoan parasite Giardia lamblia synthesizes a diverse and surprisingly abundant array of sterile transcripts unable to code for proteins. Random sampling of cDNAs from two evolutionarily divergent Giardia strains indicates that approximately 20% of cDNAs in the libraries represent polyadenylated sterile transcripts. RNase protection analysis and northern blot hybridization of three sterile transcript loci demonstrated that both the sterile transcript and a complementary mRNA were made in each case, further categorizing these sterile transcripts as antisense transcripts. Investigation of the genomic loci for these same three sterile antisense transcripts showed typical transcription units for the sense transcripts, but still failed to reveal a usable open reading frame for the sterile antisense transcripts. 5'-RACE mapped the transcription start site for one of the sterile antisense transcripts to an AT-rich region, as is typical for GIARDIA: It is unclear whether these sterile transcripts represent errors in transcription or whether they have regulatory functions within the cell, although preliminary investigations failed to reveal evidence for a role in developmental gene regulation. In either case, the presence of such a large pool of sterile antisense transcripts is dramatic evidence of the unusual molecular machinery of the early diverging protist G.lamblia.

Reduction in diabetes incidence in an I-Ag7 transgenic nonobese diabetic mouse line.

Susceptibility to IDDM is strongly associated with major histocompatibility complex (MHC) class II genotypes. Nonobese diabetic (NOD) mice develop a similar autoimmune diabetes and have a unique MHC class II I-A allele that is required for the development of diabetes. A number of groups have shown that the introduction of resistant MHC class II alleles as transgenes into the NOD mouse protects from diabetes. We made control transgenic NOD mice, expressing their own I-Abetag7 molecule as a transgene. One of two lines of these mice showed a reduced incidence of diabetes, without any change in T-cell proliferative response to a number of diabetes autoantigens or any change in insulitis severity. This line developed a subtle decrease in the percentage of splenic B-cells that progressed with age. This defect was not associated with any other phenotypic abnormalities. Our findings suggest that assessment of splenic B-cell number is necessary in interpretation of the effects of MHC class II transgenes on the development of diabetes in the NOD mouse.

Prevention of diabetes in NOD mice by a mutated I-Ab transgene.

Susceptibility to the human autoimmune disease IDDM is strongly associated with those haplotypes of the major histocompatibility complex (MHC) carrying DQB1 alleles that do not encode aspartic acid at codon 57. Similarly, in a spontaneous animal model of this disease, the NOD mouse, the genes of the MHC play an important role in the development of diabetes. The DQB1 homolog in NOD mice, I-Ab(g7), encodes a histidine at codon 56 and a serine at codon 57, while all other known I-Ab alleles encode proline and aspartic acid, respectively, at these positions. We therefore mutated the NOD I-Ab allele to encode proline at position 56 and aspartic acid at position 57 and introduced this allele onto the NOD genetic background to study the effect of these substitutions on susceptibility to diabetes. No transgenic mice developed diabetes by 8 months of age, and transgenic mice had markedly reduced lymphocytic infiltration in the pancreas compared with nontransgenic littermates. Furthermore, splenocytes from transgenic mice failed to proliferate or secrete gamma-interferon in response to a panel of beta-cell autoantigens, although the mice did produce beta-cell specific antibodies. Interestingly, the proportion of IgG1 and IgE relative to IgG2a comprising these autoantibodies was much greater in transgenic mice compared with nontransgenic control mice. Finally, T-cells from transgenic mice inhibited the adoptive transfer of diabetes to irradiated recipients. This inhibition was partially reversed by treatment of the recipients with a combination of anti-interleukin (IL)-4 and anti-IL-10 monoclonal antibodies. Thus, a transgenic class II MHC allele encoding aspartic acid at B57 prevents diabetes, in part, by promoting the production of IL-4 and IL-10, which interfere with the effector phase of the diabetic process.

Episomal and integrated maintenance of foreign DNA in Giardia lamblia.

Giardia lamblia is an early diverging eukaryote which causes gastrointestinal disease throughout the world. Different subgroups of Giardia have been defined based on several biochemical and genetic criteria. We have developed a method for stably introducing DNA into the nuclei of the parasite using puromycin acetyltransferase (pac) as a dominant selectable marker. Transfected circular DNAs were maintained as episomes in the isolate WB, a representative of one Giardia subgroup. When input DNAs were linearized, integration was observed to occur by homologous recombination producing gene replacements in this isolate. In isolate GS, which represents a different subgroup, both linear and circular transfected DNAs were integrated into the genome by homologous recombination. In GS, linear DNA again produced gene replacements, while circular DNA produced duplicative integration events. The failure of GS to replicate episomes may reflect differences in the structure or recognition of DNA replication origins between these subgroups. A plasmid shuttle vector was also developed for expression of other genes in Giardia lamblia. Utilizing the green fluorescent protein as a reporter gene in the WB isolate, we show that gene expression from this vector correlated with plasmid copy number over a range of two orders of magnitude. Together these tools should greatly enhance our ability to study both the basic biology and the pathogenesis of this ubiquitous parasite.

Initiator and upstream elements in the alpha2-tubulin promoter of Giardia lamblia.

Giardia lamblia, one of the earliest diverging eukaryotes and a major cause of diarrhea world-wide, has unusually short intergenic regions, raising questions concerning its regulation of gene expression. We have approached this issue through examination of the alpha2-tubulin promoter and in particular investigated the function of an AT-rich element surrounding the transcription start site. Its placement and the ability of this sequence to direct transcription initiation in the absence of any other promoter elements is similar to the initiator element in higher eukaryotes. However, the sequence diversity of extremely short (8-10 bp) initiator elements is surprising, as is their ability to independently direct substantial levels of transcription. We also identified a large AT-rich element located between -64 and -29 bp upstream of the transcriptional start site and show using both deletions and site-specific mutations of this region that sequences between -60 and the start of transcription are important for promoter strength; interestingly this AT-rich sequence is not highly conserved among different Giardia promoters. These data suggest that while the overall structure of the core promoter has been conserved throughout eukaryotic evolution, significant variation and flexibility is allowed in element consensus sequences and roles in transcription. In particular, the short and diverse sequences that function in transcription initiation in Giardia suggest the potential for relaxed transcriptional regulation.

Tau in aluminum-induced neurofibrillary tangles.

Aluminum is a neurotoxin and in susceptible species induces a neurofibrillary pathology characterized by argentophilic masses in neuronal perikarya and in axonal spheroids. These inclusions are known to contain neurofilament proteins. Using immunocytochemistry and immunoblotting, we demonstrate that tau is a component of these aluminum-induced neurofibrillary tangles (Al-NFTs) in rabbits. Double-label immunocytochemistry experiments reveal co-localization of phosphorylated neurofilaments (using SMI31) and tau (using tau-1, tau-5, AT8, and PHF-1) in the perikaryal Al-NFTs. Non-phosphorylated tau (detected using tau-1) occupies a smaller area of the Al-NFT than the total pool of tau proteins (detected using tau-5). The area of total tau and non-phosphorylated tau immunolabeling in the Al-NFT increases as the size of the Al-NFT (i.e., the proportion of cell area occupied by the Al-NFT) increases. The proportion of cell area (outside of the Al-NFT) occupied by tau (as indicated by tau-5) decreases as the area of tau in the Al-NFT increases and as the size of the Al-NFT in the cell increases. Immunoblotting experiments demonstrate 1) the specificity of the tau antibody labeling and verify a lack of cross-reactivity of the tau-5 antibody to neurofilament proteins in rabbit tissue; and 2) no alterations in the levels of tau resulting from aluminum-treatment. These data suggest that as the size of the Al-NFT in a cell increases there is less tau in the neuronal perikarya. Therefore, there may be less tau in the perikarya available to perform normal functions such as microtubule polymerization and stabilization. Tau and neurofilament proteins are perturbed in a number of neurodegenerative disorders such as Alzheimer's disease, diffuse Lewy body disease, and Parkinson's disease. Aluminum-induced neurofibrillary pathology may provide a model to study perturbation in tau and neurofilaments, their phosphorylation and deposition into pathological inclusions.

Mortality and morbidity associated with the distribution of monthly welfare payments.

OBJECTIVE: The impact of major social policy decisions on community health is rarely considered or analyzed. This article describes the association of major community and health resource use in relation to the distribution of monthly welfare payments. METHODS: A descriptive, retrospective study was performed using existing accessible databases in the city of Vancouver, British Columbia (BC), and St. Paul's Hospital, a tertiary care, downtown institution. The mean numbers of admissions or responses per week and per day related to the monthly welfare check issue day in 1993 were collected from the following health agencies: the BC Ambulance Service, the Vancouver Fire Department, the BC Coroner's Office, the Vancouver Detox Center, the Vancouver City Police Jail for public drunkenness, and St. Paul's Hospital ED. RESULTS: Comparison of weekly events for non-payweeks vs the week starting on welfare payday (mean +/- SD) are; St. Paul's ED, 949 +/- 51 vs 993 +/- 81 (p = 0.10); Detox Center observation admissions, 29 +/- 5.6 vs 40 +/- 7.3 (p < 0.001); Vancouver Fire Department medical responses, 453 +/- 44 vs 527 +/- 45 (p < 0.001); BC Ambulance Service responses, 3,338 +/- 101 vs 3,634 +/- 85 (p < 0.001); and coroner-reported deaths, 8.8 +/- 3.0 vs 13.6 +/- 2.6 (p < 0.0001). CONCLUSIONS: As measured in multiple independent databases, there is a significant increase in morbidity and mortality in the week after the distribution of monthly welfare paychecks.

Aluminum-induced neuropathology: transient changes in microtubule-associated proteins.

In susceptible species, aluminum induces cytoskeletal changes in which neurofilaments accumulate in neuronal cell bodies and proximal axonal enlargements. To determine if microtubule-associated proteins (MAPs) are altered in this model, we examined the spinal cords of aluminum- and saline-treated control rabbits at several time points after treatment. Transient decreases in tau and MAP2 immunoreactivity in neurons in aluminum-intoxicated rabbits were demonstrated with immunocytochemistry. An antibody directed against Alzheimer's disease paired helical filaments labeled neurons in aluminum-treated rabbits but not controls. MAP5 immunoreactivity in the cell body cytoplasm was displaced by aluminum-induced tangles. The transient decreases in MAP2 and tau immunoreactivity did not reflect alterations in protein levels measured using immunoblotting. The transient antigenic changes in tau and MAP2 may reflect conformational changes in these cytoskeletal proteins. Aluminum-induced pathology provides a model for studying perturbations in MAPs and neurofilament proteins that are characteristic of many human neurodegenerative diseases such as Alzheimer's disease, diffuse Lewy body disease, Parkinson's disease, and amyotrophic lateral sclerosis.

Participatory aspects in the qualitative research design of phase II of the ethnocultural communities facing AIDS study.

This paper describes the steps taken in generating and implementing a qualitative research design for Phase II of the Ethnocultural Communities Facing AIDS Study. Theoretically framed by the macro-level, sociocultural model of health behaviour developed by Kleinman, the methodological procedures are an adaptation of Scrimshaw's Rapid Assessment Procedures (RAP) and a participatory approach involving stakeholders from each ethnocultural community. Qualitative data-on behaviours conducive to HIV transmission in six ethnocultural communities in Canada-were elicited using a combination of key communicator interviews, focus groups, and participant observation techniques. Data were analyzed using systematic content analysis techniques. Inter-rater reliability checks and procedures of triangulation demonstrated the validity of evidence generated. A commitment to research partnership with community persons, and an accountability loop that provided assurances of how the data would be scientifically represented, were critical elements in the process of design construction.

Overview of the Canadian study on the determinants of ethnoculturally specific behaviours related to HIV/AIDS.

Canada's population is composed of heterogenous ethnocultural communities. There is a need for information and educational initiatives on HIV and AIDS directed specifically at these communities. For such interventions to be effective we must determine the existing personal and sociocultural factors related to HIV transmission. There has been little such research in Canada. In this supplement we report on various aspects of a study conducted between May 1992 and December 1994 to determine the factors related to HIV transmission in several ethnocultural communities. This paper describes some innovative aspects of the project: the conceptual framework, the community participatory model, the use of a multi-method research design, and the ongoing communication strategy. The combination of these elements makes the study unique. The value of the study lies not only in the information obtained but also in the model it provides for future research in other settings.

HIV/AIDS in the context of culture: selection of ethnocultural communities for study in Canada.

This article reports on the methodology used to select six ethnocultural communities invited to participate in subsequent phases of the project on HIV/AIDS in the context of culture in Canada. Selection was based on quantitative data on demography, qualitative assessment of ethnocultural cohesion; and quantitative data and qualitative data of exposure to risk for sexually transmitted disease. A principle of partnership insured that the final selection was completed by interaction between the investigators and the National Advisory Committee representing ethnocultural communities in Canada. The six communities asked to participate in Phase II of the study were: in Montreal, the Latin American and the Arabic-speaking communities; in Toronto, the English-speaking Caribbean communities and communities from the Horn of Africa; in Vancouver, the Chinese and the South Asian communities. The results are significant for the future both of research on ethnicity in Canada and of control of HIV and AIDS.

Many voices--sociocultural results of the ethnocultural communities facing AIDS study in Canada.

This paper presents the results of Phase II of the Ethnocultural Communities Facing AIDS Study, the sociocultural investigation of factors contributing to risk behaviour associated with HIV/AIDS in six ethnocultural communities in Canada in three urban sites. In Vancouver, the South Asian and Chinese communities were studied, the Horn of Africa and English-speaking Caribbean communities in Toronto and the Latin American and Arabic-speaking communities in Montreal. Results demonstrated that there are common elements across these ethnocultural communities that increase the risk for HIV transmission. HIV/AIDS awareness and prevention in ethnocultural communities must address sociocultural differences, particularly sex role differences between men and women in terms of power within relationships to negotiate for safer sexual practices.

Understanding use of condoms among Canadian ethnocultural communities: methods and main findings of the survey.

The aim of this study was to understand the intention to use a condom for each instance of sexual intercourse with a new partner in three of Canada's non-dominant ethnocultural communities: Latin American (N = 346), English-speaking Caribbean (N = 358), and South Asian (N = 355). All respondents were recruited from multiple ethnocultural venues using predetermined sampling frames and quotas for each community. Anonymous questionnaires assessing culturally specific theoretical constructs were completed. This paper presents the methodology and the main findings. The high quality of the results of this study demonstrate the advantage of establishing strong partnerships with members of communities being studied.

PIP: During April-May 1994, in Canada, 346 members of the Latin American community aged 18-49, 358 members of the English-speaking Caribbean community aged 16-49, and 355 members of the South Asian community aged 18-45 completed a questionnaire developed by the Research Group on Psychosocial Aspects of Health Behavior at Laval University in Quebec. This study aimed to identify the variables influencing intention to use a condom for each instance of sexual intercourse with a new partner in three nondominant ethnocultural communities. 81% of Latin Americans, 75% of the Caribbeans, and 71% of the South Asians intended to use a condom for each instance of sexual intercourse with a new partner during the next 3 months. Yet, among people who had had sex with a new partner in the last year, only 30% of Latin Americans, 28% of the Caribbeans, and 47% of South Asians always used a condom. Significant predictors of intent to use a condom in each instance of sexual intercourse with a new partner were personal normative belief (a measure of the personal feelings of moral obligation or responsibility to use or refuse to use a condom), perceived behavioral control, and role beliefs (p .0001). For the Latin American community, these three constructs explained 70.7% of the variance (partial R2 = 51.1% for personal normative belief, 10% for role beliefs, and 4.1% for perceived behavioral control). For the English-speaking Caribbean community, they explained 51% of the variance (partial R2 = 35.7% for personal normative belief, 11.9% for perceived behavioral control, and 3.5% for role beliefs). For the South Asian community, they accounted for 76% of the variance (partial R2 = 63.5% for perceived behavioral control, 9.4% for personal normative belief, and 3.1% for role beliefs). These findings serve as a basis for recommendations for the prevention of HIV transmission among the participating ethnocultural communities.

Canadian ethnocultural communities facing AIDS: overview and summary of survey results from phase III.

This paper describes the survey results reporting demographic profiles, behaviours, opinions, beliefs, attitudes, and intentions related to condom use for three Canadian ethnocultural communities (Latin American, English-speaking Caribbean and South Asian) participating in the Ethnocultural Communities Facing AIDS Study. Specific recommendations are presented for HIV-prevention programming based on the research results.

Regulation of intestinal epithelial cell cytoskeletal remodeling by cellular immunity following gut infection.

Gut infections often lead to epithelial cell damage followed by a healing response. We examined changes in the epithelial cell cytoskeleton and the involvement of host adaptive immunity in these events using an in vivo model of parasitic infection. We found that both ezrin and villin, key components of the actin cytoskeleton comprising the brush border (BB) of intestinal epithelial cells (IECs), underwent significant post-translational changes following gut infection and during the recovery phase of gut infection. Intriguingly, using mice lacking either CD4(+) or CD8(+) T-cell responses, we demonstrated that the mechanisms by which ezrin and villin are regulated in response to infection are different. Both ezrin and villin undergo proteolysis during the recovery phase of infection. Cleavage of ezrin requires CD4(+) but not CD8(+) T cells, whereas cleavage of villin requires both CD4(+) and CD8(+) T-cell responses. Both proteins were also regulated by phosphorylation; reduced levels of phosphorylated ezrin and increased levels of villin phosphorylation were observed at the peak of infection and correlated with reduced BB enzyme activity. Finally, we show that infection also leads to enhanced proliferation of IECs in this model. Cytoskeletal remodeling in IECs can have critical roles in the immunopathology and healing responses observed during many infectious and non-infectious intestinal conditions. These data indicate that cellular immune responses can be significant drivers of these processes.

Tumour necrosis factor alpha contributes to protection against Giardia lamblia infection in mice.

Giardia lamblia is a ubiquitous parasite that causes diarrhoea. Effective control of Giardia infections in mice has been shown to involve IgA, T cells, mast cells and IL-6. We now show that Tumour necrosis factor alpha (TNFalpha) also plays an important role in the early control of giardiasis. Mice treated with neutralizing anti-TNFalpha antibodies or genetically deficient in TNFalpha were infected with the G. lamblia clone GS/(M)-H7. In both cases, mice lacking TNFalpha had much higher parasite numbers than controls during the first 2 weeks of infections. However, anti-parasite IgA levels, mast cell responses, and IL-4 and IL-6 mRNA levels do not appear significantly altered in the absence of TNFalpha. In addition, we show that mice infected with G. lamblia exhibit increased intestinal permeability, similar to human Giardia infection, and that this increase occurs in both wild-type and TNFalpha deficient mice. We conclude that TNFalpha is essential for host resistance to G. lamblia infection, and that it does not exert its effects through mechanisms previously implicated in control of this parasite.