Reduction of postendodontic pain after one-visit root canal treatment using three irrigating regimens with different temperature.

OBJECTIVE: The aim of this clinical trial was to assess whether controlled irrigation with three different irrigation regimens with different temperature would result in reduction of post-endodontic pain after one-visit root canal treatment (RCT). MATERIALS AND METHODS: A total of 240 (129 females and 111 male) aged 18 - 65 years were referred and integrated in this clinical trial, All patients presented with a vital maxillary or mandibular molar, premolar or front teeth designated for conventional root canal treatment for prosthetic reasons detected with only vital pulps. All canals were cleaned and shaped with Reciproc instruments, and were used with a micro motor (VDW, Munich Germany). Final irrigation was done with cold (4°C, 2.5°C, and room temperature) 17% EDTA and 10 mL of cold saline solution. RESULTS: A total of 240 of 279 patients (129 females and 111 male) aged 18 - 65 years were referred and integrated in this clinical trial, whereas 29 were rejected as not completing the requirements needed. All patients presented with a vital maxillary or mandibular molar, premolar, or front teeth designated for intentional endodontic RCT for prosthetic reasons. No statistically significant difference (P > 0.05) among the groups was found regarding degree or duration of pain. There was no statistically significant difference (P > 0.05) among the 4oC and 2.5oC groups. CONCLUSION: The approach in both selecting the patients participating in the study and analyzing the data in this randomized clinical trial allows us to conclude that cryotherapy is an aid of clinical procedures to clean and shape the canals to reduce the occurrence of postendodontic pain and the need for medication in patients presenting with a diagnosis of vital pulp.

Short report: Prevalence of fecal Encephalitozoon sp. spores among hospitalized patients in Nepal.

During the months of June and July 1998, stool samples from 341 hospitalized patients (mean age, 32.7 +/- 16.3 years; range, 1-86 years) from Kathmandu, Nepal, were screened for the prevalence of Encephalitozoon sp. by use of anti-Encephalitozoon sp. monoclonal antibody 3B6-based immunofluorescence assay. The cross-sectional study revealed the presence of Encephalitozoon spores in 0.6% (2 of 341) patients. By use of direct microscopic examination, 27% (93 of 341) of patients were diagnosed with various gastrointestinal pathogens, among which Ascaris lumbricoides and Ancylostoma duodenale were the most commonly found, with prevalence rates of 8.8% (30 of 341) and 7.6% (26 of 341), respectively. To our knowledge, this is the first study to report the presence of Encephalitozoon sp. among humans in Nepal.

Differential modulation of murine cellular immune responses by salivary gland extract of Aedes aegypti.

The ability of salivary gland extract (SGE) of Aedes aegypti to modulate cellular immune responses was investigated in a mouse model. Cytokine production was induced in naive and antigen-primed murine (BALB/c) spleen cells in vitro by stimulation with the T cell mitogen concanavalin A or the T cell-dependent antigen ovalbumin (OVA), respectively. Inclusion of Ae. aegypti SGE in in vitro culture with naive cells caused significant suppression of the cytokines interleukin-2 (IL-2) and interferon gamma in culture supernatants, while levels of other cytokines (IL-4 and IL-5) were unaffected by SGE. In contrast, SGE did not affect cytokine production by antigen-activated cells derived from OVA-primed mice. To determine whether SGE could inhibit the responsiveness of cells to exogenous cytokine stimuli, optimized quantities of lymphocyte growth factor cytokines IL-2 and IL-4 were added to SGE-treated spleen cells and the degree of cellular proliferation was determined. Cellular proliferation in response to IL-2 was markedly suppressed by prior exposure of cells to SGE, while the proliferative response to IL-4 was also affected by SGE but to a lesser extent. These results confirm that mosquito SGE can modulate host immune responses, and suggest that in Ae. aegypti modulation is directed primarily against cytokines associated with type 1 lymphocyte responses. The mode of immunomodulation and the possible relevance of these results to vector-borne disease research are discussed.

Cryptosporidium parvum in children with diarrhea in Mexico.

Using an anti-oocyst wall monoclonal antibody-based immunofluorescence assay, the presence of Cryptosporidium parvum was evaluated in children with diarrhea from rural areas (selected from a door-to-door community survey) and from urban areas (patients attending hospitals) in the State of Puebla, Mexico. Prevalences of 9.4% in a rural population (n = 85) and 29.6% in a hospital-based urban population (n = 81). There was no consistent correlation between water source and other environmental data and the presence of C. parvum in stools. It is concluded that C. parvum may be an important pathogen associated with diarrhea in this Mexican state.

Modulation of murine immunological responses by salivary gland extract of Simulium vittatum (Diptera: Simuliidae).

The influence of Simulium vittatum Zetterstedt salivary gland extract on several immunological mechanisms was investigated in murine model hosts (laboratory mice). These mechanisms included the expression of major histocompatibility complex class II cell surface molecules, the in vitro mitogen responsiveness of lymphoid cells, and the antibody responses to heterologous foreign antigens (sheep erythrocytes). Experiments were designed to determine the influence of salivary gland extract following in vivo inoculation or in vitro inclusion in cell culture. In vivo inoculation of salivary gland extract reduced the percentage of Ia+ cells in spleen cell populations, although this difference was ameliorated by a 2 d in vitro culture period, regardless of whether salivary gland extract was included in culture. Salivary gland extract had no effect on Ia expression by cells derived from regional lymph nodes or the skin (epidermis). In vivo inoculation with salivary gland extract did not affect the responsiveness of splenic lymphocytes to mitogens, whereas in vitro exposure to salivary gland extract reduced both T and B cell mitogenesis. Finally, antibody responses to sheep erythrocytes were enhanced if salivary gland extract was included as a coinoculant, although this was expressed only at the systemic level regardless of the route of antigen delivery. In light of these results, immunomodulatory functions of black fly saliva are postulated; they are operative at different levels on different subcompartments of the immune system, possibly via cytokine modulation.

Antibody responses of BALB/c mice to salivary antigens of hematophagous black flies (Diptera: Simuliidae).

The humoral antibody responses to salivary antigens of Simulium vittatum Zetterstedt were investigated in a BALB/c mouse laboratory model. Production of antisera was stimulated by intraperitoneal immunization with salivary gland extract or by feeding flies directly on depilated mice. Antibody responses in these two groups of mice were compared by western blotting, thus characterizing "true" salivary immunogens present in salivary gland extract. Immunized mice developed IgG, IgM, and IgE antibodies which recognized several salivary gland components, ranging in molecular weight between 26 and 67 kDa. Sera from bitten mice recognized fewer antigens, indicating that some components of the salivary gland extract were poorly immunogenic or absent from the saliva secreted during blood feeding. Antisera raised against S. vittatum also were used to identify cross-reactive immunogens and allergens in salivary gland extracts from other New World simuliids (Simulium argus Williston, S. metallicum Bellardi, and S. ochraceum Walker). SDS-PAGE protein profiles indicated a high degree of similarity between salivary gland extract of S. vittatum and S. argus, and several cross-reacting antigens were identified by western blotting. In contrast, protein profiles of S. ochraceum and S. metallicum differed from the former species, both qualitatively and quantitatively. Antisera demonstrated a low degree of cross-reactivity against salivary gland extract of S. ochraceum, whereas no cross-reactivity was detected against S. metallicum. These observations were confirmed using a monoclonal antibody raised against S. vittatum salivary gland extract (designated SVSG.1.F10), which showed cross-reactivity against S. argus but failed to recognize salivary gland components of either S. ochraceum or S. metallicum.

Differential intra-epithelial lymphocyte phenotypes following Cryptosporidium parvum challenge in susceptible and resistant athymic strains of mice.

The lack of immunocompetent laboratory animal models has limited our understanding of functional immune responses to Cryptosporidium parvum infection, but such responses have been studied in susceptible laboratory rodents with genetic, acquired, or induced immunodeficiencies. We previously observed that athymic C57BL/6J nude mice inoculated with C. parvum oocysts had lower or absent fecal oocyst excretion when compared to inoculated athymic BALB/cJ nude mice. This discrepancy prompted us to explore potential differences in intestinal immune responses in both strains. Prior to and after C. parvum challenge, BALB/cJ nude and C57BL/6J nude mice did not differ in either spleen cell numbers or in parasite-specific proliferation. However, both strains of mice exhibited a significant increase in intra-epithelial lymphocyte (IEL) numbers prior to and following C. parvum inoculation when compared to uninoculated controls (P<0.05). Prior to challenge, C57BL/6J nude mice had a higher percentage of both CD8+ and CD8+ gammadelta+ IEL than BALB/cJ nude mice. Following challenge, resistant C57BL/6J nude mice had a higher percentage of gammadelta+, CD4+, and CD8+ gammadelta+ IEL than uninoculated C57BL/6J nude mice and than susceptible BALB/cJ nude mice (P<0.05). Conversely, inoculated C57BL/6J nude mice had a significantly lower percentage of alphabeta+ IEL than inoculated BALB/cJ nude mice (P<0.05). We conclude that gammadelta+, CD4+, and/or CD8+ gammadelta+ IEL may influence responses to cryptosporidiosis in athymic murine models, and that the increased percentage of alphabeta+ IEL in susceptible BALB/cJ nude mice could reflect a preferential expression during chronic C. parvum infection and/or might downregulate local protective responses.

Adoptive transfer of immunity with intraepithelial lymphocytes in Cryptosporidium parvum-infected severe combined immunodeficient mice.

BACKGROUND: Intestinal infections with the protozoan parasite Cryptosporidium parvum are prevalent in both immunocompetent and immunocompromised hosts. Although C parvum is an important cause of outbreaks and opportunistic infections worldwide, little is known about protective mucosal immune responses. This is in part because animal models of infection are limited to those with genetic or induced immunodeficiencies. METHOD: In this report, we isolated immune (primed) or nonimmune (unprimed) intraepithelial lymphocytes (IEL) from BALB/cJ mouse intestines, adoptively transferred them into C parvum-infected severe combined immunodeficient (SCID) mice, and evaluated infection and cell phenotype responses. RESULTS: Control SCID mice that received no IEL shed large numbers of oocysts throughout the experimental period (day 18 to day 72). Transfer of primed IEL significantly reduced fecal oocyst shedding in recipient SCID mice compared with SCID mice that received unprimed IEL or no IEL. SCID mice transferred with unprimed IEL shed variable numbers of fecal oocysts that increased and decreased in bursts until day 57 after infection. SCID mice transferred with primed IEL exhibited significantly higher proportions of T-cell receptor (TCR) alphabeta+, CD8+, and CD8alphabeta+ EL compared with inoculated SCID mice that received unprimed or no IEL. CONCLUSION: We conclude that primed IEL from immunocompetent mice may influence protective mucosal response against cryptosporidiosis when transferred into SCID mice. In addition, the increased percentage of TCR alphabeta+, CD8+, CD8alphabeta+ IEL in recipient SCID mice may reflect mucosal cell populations involved in these responses during chronic C parvum infection.

Influence of immunizing dose and presence or absence of adult worms on the development of resistance to Nematospiroides dubius challenge infections of mice.

H-2 congenic strains expressing resistant (H-2q, H-2f) or susceptible (H-2k) haplotypes were compared for their ability to resist challenge infection with N. dubius following a 6- or 14-day ivermectin-abbreviated immunizing infection. B10.BR mice (H-2k) were considerably more resistant to infection when the priming interval was shortened from 14 to 6 days. B10.Q (H-2q) and B10.M (H-2f) mice resisted challenge regardless of which immunization regimen was used. The influence of parasite numbers on the response to challenge was studied by comparing infections in resistant DBA/1 (H-2q) and susceptible CBA/J (H-2k) mice that differ at both H-2 and non-H-2 genes. DBA/1 mice, immunized with 50 or 150 L3 of N. dubius for 14 days, resisted challenge, whereas mice receiving 300 worms did not. In contrast, CBA/J mice failed to resist challenge at all priming doses tested. When the immunizing infection was shortened from 14 to 6 days, DBA/1 mice resisted challenge regardless of priming dose and CBA/J mice resisted challenge only when the highest dose of 300 worms was used for priming. The data suggest that susceptible strains of mice may be preferentially immunosuppressed, particularly at low infective doses, and that suppression is associated with adult worms present in the lumen of the small intestine.

In vivo and in vitro egg production by Nematospiroides dubius during primary and challenge infections in resistant and susceptible strains of mice.

Experiments were conducted to examine adult worm burdens, fecal egg output, and in vitro fecundity of Nematospiroides dubius in resistant LAF1 and susceptible CBA mice 12, 15, 18, and 21 days following primary and challenge infections. A strong correlation was obtained on the number of eggs produced by worms cultured in vitro and the egg production as assessed by fecal egg count. Worm counts, fecal egg counts, and in vitro fecundity were similar on all days studied following a primary infection in both mouse strains. However, after challenge infection, LAF1 mice showed lower worm burdens, fecal egg output, and in vitro egg production when compared to CBA mice. Although the egg production of surviving female worms from immune LAF1 mice was decreased, it never fell below a threshold of 100 eggs/day. The reduced fecundity may be a manifestation of a general anti-worm response rather than responses directed specifically at worm reproduction.

Increased proportion of antigen-specific antibody-producing hybridomas following an in vitro immunization with in vivo immunized mouse spleen cells.

Development of murine monoclonal antibodies to weakly immunogenic antigens was accomplished by combining both in vivo and in vitro immunizations. Following immunization of mice with Treponema hyodysenteriae outer membrane antigens, Manduca sexta apolipoproteins, and Drosophila melanogaster DNA polymerase, respectively, a significant increase in percentage of antibody-producing hybrids were identified when immune spleens were subjected to an in vitro immunization prior to fusion with SP2/0 myeloma cells. The hybrids developed, produced Abs to a T. hyodysenteriae 14 Kd carbohydrate, M. sexta apolipoproteins I, II, and III, and D. melanogaster DNA polymerase. The use of both in vivo and in vitro immunizations may increase the likelihood of generating monoclonal antibodies to weakly immunogenic antigens.

Role of CD4+ TH1- and TH2-cell-secreted cytokines in cryptosporidiosis.

Cryptosporidium parvum is now recognized as an important gastrointestinal pathogen around the world. Unfortunately, control measures for cryptosporidiosis are currently not well defined and very little is known about the immunological events relevant to the control of infection. Cytokine depletion experiments were conducted by injecting adult BALB/c mice with monoclonal antibodies directed to IL-2, IL-4, and IL-5, prior and during C. parvum infection. Faecal oocyst excretion and intestinal parasite number were monitored from day 4 to day 31 post-infection. Mice injected with monoclonal antibodies to IL-5 or IL-4/IL-5, but not IL-4 alone, harboured significantly higher numbers of both intestinal parasites and excreted oocysts. It is concluded that IL-5 complemented with IL-4 appears to be an important TH2-dependent mechanism for controlling cryptosporidiosis. Nevertheless, we suggest that cytokines secreted by both TH1 and TH2 cells may operate in concert in controlling cryptosporidiosis, triggering different functional mechanisms in a dynamic and simultaneous up- and down-regulatory fashion.

Cryptosporidium infections in inbred strains of mice.

Cryptosporidium, a protozoan parasite of man and animals, is an important etiological agent of diarrhea throughout the world, particularly in children and immunocompromised individuals such as AIDS patients. Unfortunately, because of the lack of both in vivo laboratory models and reliable in vitro parasite culture systems, virtually nothing is known about the immunological events occurring during disease. In order to identify reliable animal models for infection, we studied C. parvum infections in 19 different strains of mice representing 12 H-2 haplotypes: A/J, AKR/J, B10.D2/J, B10.M/J, C3H/HeJ, C57BL/65, C57BL/6J-bgJ, CBA/NJ, DBA/1J, DBA/2J, HRS/J, HTG/J, NZB/B1NJ, NZW/J, P/J, RIII/J, SJL/J, SWR/J, and WB/ReJ, and in one gerbil: Meriones unguiculatus. Fecal samples and histological sections of the intestine taken on day 7 post-Cryptosporidium inoculation indicated that only the beige mouse (C57BL/6J-bgJ) harbored significant numbers of parasites compared to the other strains. The numbers of parasites harbored in these NK cell-deficient beige mice were, however, considerably lower than those seen in neonatal mice. Adult inbred mouse strains susceptible to Cryptosporidium infections are discussed.

Heligmosomoides polygyrus: a model for chronic gastrointestinal helminthiasis.

Establishment of chronic infections and strain-dependent variation in resistance to challenge infections are well-known features of the relationship between mice and the intestinal nematode parasite Heligmosomoides polygyrus. Here, Fernando Monroy and Javier Enriquez examine host responses, immunogenic and nonimmunogenic antigens of the parasite, and parasite immune evasion strategies in this useful laboratory mouse model of nematode parasitism of mammals.

Role of immunoglobulin A monoclonal antibodies against P23 in controlling murine Cryptosporidium parvum infection.

Cryptosporidium parvum is an important diarrhea-causing protozoan parasite of immunocompetent and immunocompromised hosts. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections with bacteria, viruses, and parasites, but little is known about the role of IgA in the control of C. parvum infection. We assessed the role of IgA during C. parvum infection in neonatal mice. IgA-secreting hybridomas were developed by using Peyer's patch lymphocytes from BALB/c mice which had been orally inoculated with viable C. parvum oocysts. Six monoclonal antibodies (MAbs) were selected for further study based on indirect immunofluorescence assay reactivity with sporozoite and merozoite pellicles and the antigen (Ag) deposited on glass substrate by gliding sporozoites. Each MAb was secreted in dimeric form and recognized a 23-kDa sporozoite Ag in Western immunoblots. The Ag recognized comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with P23, a previously defined neutralization-sensitive zoite pellicle Ag. MAbs were evaluated for prophylactic or therapeutic efficacy against C. parvum, singly and in combinations, in neonatal BALB/c mice. A combination of two MAbs given prophylactically prior to and 12 h following oocyst challenge reduced the number of intestinal parasites scored histologically by 21.1% compared to the numbers in mice given an isotype-matched control MAb (P < 0.01). Individual MAbs given therapeutically in nine doses over a 96-h period following oocyst challenge increased efficacy against C. parvum infection. Four MAbs given therapeutically each reduced intestinal infection 34.4 to 42.2% compared to isotype-matched control MAb-treated mice (P < 0.05). One MAb reduced infection 63.3 and 72. 7% in replicate experiments compared to isotype-matched control MAb-treated mice (P < 0.0001). We conclude that IgA MAbs directed to neutralization-sensitive P23 epitopes may have utility in passive immunization against murine C. parvum infection.

Nematospiroides dubius: genetic control of immunity to infections of mice.

Inbred strains of mice differ in their susceptibility and resistance to challenge infections with Nematospiroides dubius. In our studies, F1 hybrid mice from resistant SJL and susceptible CBA parents were resistant to N. dubius challenge infections. Only 22% of backcrosses to SJL were susceptible while backcrosses to CBA had a wide range of susceptibility. Male mice were more susceptible than female mice. In another experiment, inbred strains of mice were compared in their ability to resist N. dubius challenge infection: SJL and A.SW (H-2s) mice became resistant after one immunizing infection, A, A/He (both H-2a), as well as BALB/c and DBA/2 (both H-2d) mice became resistant after two immunizing infections, while C57BL/6 (H-2b), C3H/He, CBA, and AKR (H-2k) mice remained susceptible. The resistance to reinfections was characterized by reduction of worm burdens between Days 6 and 14 postinfection. It was concluded that (1) resistance to N. dubius challenge infections is inherited in a dominant fashion and that multiple genes may influence such response, which in turn might be modulated by the Y chromosome; (2) both MHC and non-MHC genes may influence, in conjunction with the number of exposures to parasite antigens, the resistance to challenge infections.

Presence of non-Fab IgE binding molecules in the intestinal nematode parasite of mice Heligmosomoides polygyrus.

Unsuccessful attempts to identify serum parasite-specific immunoglobulin E (IgE) responses in mice following infections with the intestinal nematode parasite Heligmosomoides polygyrus prompted us to explore the possibility that IgE bound within the parasite antigen could account for the false-positive results observed. A live-worm ELISA was developed. Following incubation, irrelevant IgE monoclonal antibody to DNP, IgE present in normal mouse serum, as well as IgE in immune serum were independently identified within live adult worms in this H. polygyrus-modified ELISA. It was concluded that in addition to parasite-specific IgE binding to H. polygyrus, the parasite may attract both parasite-specific and non-parasite-specific IgE via non-Fab IgE-binding molecules.

Modulation of murine cellular immune responses and cytokines by salivary gland extract of the black fly Simulium vittatum.

Salivary gland extract (SGE) of the blood-feeding black fly Simulium vittatum is known to modulate immunological responses. In the present study, the ability of S. vittatum SGE to modulate responses during heterologous antigenic challenge was investigated in a murine model, with particular emphasis on characterizing the patterns of cytokine response. Mice were injected repeatedly with SGE or saline (sham), then challenged with the T dependent antigen ovalbumin (OVA) to generate antigen-specific lymphoblasts. Spleen cells from OVA-primed mice were then co-cultured with OVA in vitro to stimulate cytokine secretion. Cells from mice that had been injected with SGE prior to OVA challenge produced lower levels of interleukins 5 and 10 (IL-5 and IL-10) in in vitro culture, when stimulated with OVA, compared to mice that had been sham-injected with saline. Levels of IFN-gamma, IL-2 and IL-4 did not differ significantly between SGE- and saline-injected groups. Mice injected repeatedly with SGE prior to OVA challenge had fewer circulating eosinophils than sham-injected mice, while other leukocyte levels were unaffected by SGE. Prior exposure to SGE did not affect levels of serum IgE or IgA significantly. The effect of SGE on the ability of murine spleen cells to respond in vitro to the recombinant cytokines IL-2 and IL-4 was also investigated. Naive spleen cells pre-incubated with SGE proliferated less in response to both IL-2 and IL-4 in in vitro culture than cells pre-incubated with saline as a control.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of an anti-exospore monoclonal antibody on microsporidial development in vitro.

In this study we evaluated the effects of the anti-microsporidial exospore monoclonal antibody 3B6, recognizing 3 Encephalitozoon species, Encephalitozoon intestinalis (Syn. Septata intestinalis), Encephalitozoon cuniculi, and Encephalitozoon hellem on microsporidial growth in vitro. Pre-treatment of spores for 24 h with mAb 3B6 resulted in 21-29% fewer infected host cells 4 days after inoculation of the cultures compared to cultures pre-treated with medium or an irrelevant isotype control mAb (P < 0.001). Fewer intracellular spores (1.2 +/- 0.2) in infected cells were found when mAb 3B6 was present in cultures compared to cultures with medium alone (4.3 +/- 0.8) or an irrelevant isotype control mAb (4.2 +/- 0.9; P < 0.001). This decrease appeared not to be dependent on time of exposure, mAb concentration, or presence of complement. It is concluded that antibodies, particularly those directed to potential neutralizing-sensitive epitopes on spores, may have a role in the control of microsporidial growth in vitro.