Association study by genetic clustering detects multiple inflammatory response loci in non-inbred mice

We tested the possibility to map loci affecting the acute inflammatory response (AIR) in an (AIRmax AIRmin) F2 intercrossmouse population derived from non-inbred parents, by association analysis in the absence of pedigree information. Using 1064 autosomal single nucleotide polymorphisms (SNPs), we clustered the intercross population into 12 groups of genetically related individuals. Association analysis adjusted for genetic clusters allowed to identify two loci, inflammatory response modulator 1 (Irm1) on chromosome 7 previously detected by genetic linkage analysis in the F2 mice, and a new locus onchromosome 5 (Irm2), linked to the number of infiltrating cells in subcutaneous inflammatory exudates (Irm1: P»6.3 10 7; Irm2: P»8.2 10 5) and interleukin 1 beta (IL-1b) production (Irm1: P»1.9 10 16; Irm2: P»1.1 10 6). Use of a polygenic model based on additive effects of the rare alleles of 15 or 18 SNPs associated at suggestive genome-wide statistical threshold(Po3.4 10 3) with the number of infiltrating cells or IL-1b production, respectively, allowed prediction of the inflammatory response of progenitor AIR mice. Our findings suggest the usefulness of association analysis in combination with genetic clustering to map loci affecting complex phenotypes in non-inbred animal species.

A new model of outbred genetically selected mice which present a strong acute inflammatory response in the absence of complement component C5.

OBJECTIVE: Mice selected for a strong (AIRmax) or weak (AIRmin) acute inflammatory response present different susceptibilities to bacterial infections, autoimmune diseases and carcinogenesis. Variations in these phenotypes have been also detected in AIRmax and AIRmin mice rendered homozygous for Slc11a1 resistant (R) and susceptible (S) alleles. Our aim was to investigate if the phenotypic differences observed in these mice was related to the complement system. MATERIAL: AIRmax and AIRmin mice and AIRmax and AIRmin groups homozygous for the resistance (R) or susceptibility (S) alleles of the solute carrier family 11a1 member (Slc11a1) gene, formerly designated Nramp-1. METHODS AND RESULTS: While no difference in complement activity was detected in sera from AIRmax and AIRmin strains, all sera from AIRmax Slc11a1 resistant mice (AIRmax(RR)) presented no complement-dependent hemolytic activity. Furthermore, C5 was not found in their sera by immunodiffusion and, polymerase chain reaction and DNA sequencing of its gene demonstrated that AIRmax(RR) mice are homozygous for the C5 deficient (D) mutation previously described in A/J. Therefore, the C5D allele was fixed in homozygosis in AIRmax(RR) line. CONCLUSIONS: The AIRmax(RR) line is a new experimental mouse model in which a strong inflammatory response can be triggered in vivo in the absence of C5.

Maximal inflammatory response benefits syngeneic skin graft acceptance.

OBJECTIVE AND DESIGN: We investigated the influence of acute inflammation in skin isograft acceptance. METHODS: Two mouse lines selected for maximal (AIRMAX) or minimal inflammatory response (AIRMIN) were transplanted with syngeneic skin. Cellular infiltrates and cytokine production were measured 1, 3, 7 or 14 days post-transplantation. The percentage of CD4+CD25+Foxp3+ cells in the lymph nodes was also evaluated. RESULTS: Grafts were totally accepted in 100% of AIRMAX and in 26% of AIRMIN mice. In the latter, partial acceptance was observed in 74% of the animals. Emigrated cells were basically PMN and were enhanced in AIRMAX transplants. IL-10 production by graft infiltrating cells showed no interline differences. IFN-gamma was increased in AIRMIN grafts at day 14 and lower percentages of CD4+CD25+Foxp3+ cells in the lymph nodes were observed in these mice. CONCLUSIONS: Our data suggest that differences in graft acceptance might be due to a lack of appropriate regulation of the inflammatory response in AIRMIN mice compromising the self/non-self recognition.

Hypoglycemic activity of Ailanthus excelsa leaves in normal and streptozotocin-induced diabetic rats.

The hypoglycemic activity of a 70% methanol extract from the leaves of Ailanthus excelsa Roxb. (Simaroubaceae) was studied in normal, transiently hyperglycemic and streptozotocin (STZ)-induced diabetic rats. Oral administration of the extract at doses of 14, 70 and 350 mg/kg body weight caused no significant changes in fasting blood glucose levels of normal rats. In an oral glucose tolerance test, the extract produced a significant decrease in glycemia 90 min after the glucose pulse. Daily administration of A. excelsa extract for 60 days produced a significant hypoglycemic effect in diabetic animals. In addition, this treatment improved the altered renal function observed in diabetic control rats. This study suggests that Ailanthus leaf extract could be potentially useful for post-prandial hyperglycemia treatment.

Slc11a1 (formerly NRAMP1) gene modulates both acute inflammatory reactions and pristane-induced arthritis in mice.

Mice selected for the maximum acute inflammatory reaction (AIRmax) are highly susceptible to pristane-induced arthritis (PIA), whereas mice selected for the minimum response (AIRmin) are resistant. These lines show distinct patterns of leukocyte infiltration and R and S allele frequency disequilibrium of the solute carrier family 11a member 1 (Slc11a1) gene. In order to study the interactions of the Slc11a1 R and S alleles with the inflammation modulating Quantitative Trait Loci (QTL) during PIA development, homozygous AIRmax(RR), AIRmax(SS), AIRmin(RR) and AIRmin(SS) lines were produced by genotype-assisted breedings. These mice received two intraperitoneal injections of 0.5 ml pristane at 60-day intervals, and the subsequent development of arthritis was assessed for 210 days. Cytokine-secreting cell profiles were investigated using enzyme-linked immunospot. Arthritis incidence in AIRmax(RR) mice reached 29%, whereas PIA incidence in AIRmax(SS) mice was 70% by day 180. AIRmin(RR) mice were resistant, whereas 13.3% of AIRmin(SS) mice became arthritic. The presence of the defective S allele also increased arthritis severity, although acute inflammation was higher in mice bearing the R allele. A predominant Th0/Th2-type response in Slc11a1(SS) mice was observed. These results indicate that Slc11a1 is a strong candidate for the QTL modulating acute inflammation and for PIA.

Involvement of antibody production quantitative trait loci in the susceptibility to pristane-induced arthritis in the mouse.

Mice obtained by bidirectional selective breeding for high (HIII) or low (LIII) antibody (Ab) production are resistant or extremely susceptible to pristane-induced arthritis (PIA), respectively. Several quantitative trait loci regulating Ab production (Ab QTL) have been mapped in these lines, which were used to investigate the influence of these Ab QTL in PIA. Parental HIII and LIII mice and their F1 and F2 intercrosses were injected twice with pristane, and arthritis was observed for 200 days. In LIII mice PIA was more severe and incidence was 100% at day 105, while F1 and F2 mice showed intermediate values. HIII mice were totally resistant. Microsatellite polymorphisms of Ab QTL were analysed and D3Mit100 alleles cosegregated significantly with PIA incidence, severity and onset in F2 intercross mice, while the other four markers showed suggestive values. Results indicate colocalization of QTL for Ab production and PIA susceptibility. Moreover, the different cytokine and IgG isotype profiles observed in HIII and LIII lines after PIA induction are useful to candidate genes endowed with the regulation of the Ab production and arthritis phenotypes.

Ordered mesoporous silica SBA-15 A new effective adjuvant to induce antibody response

The applicability of SBA-15 mesostructure as an adjuvant and evaluation of its efficiency to induce antibody response, was discussed. It was observed that better encapsulation of biomolecules of variable shape and size can be achieved using a antigen to SBA-15 weight ratio of 1: 2.5. Efficient antibody generation could be achieved because SBA-15 was able to attract antigens effectively due to its high surface area and proper mesopore size. The results show that SBA-15 and related silica mesostructures are promising nanosystems for vaccine delivery.

Genetic potential for an acute inflammatory response in IgA glomerulonephritis in mice

Mice selected on the basis of an acute inflammatory response (AIR) can provide information about the immunopathological mechanisms of glomerulonephritis. We studied the differences between mice selected for a maximal AIR (AIRmax that attract more polymorphonuclear cells to the site of injury) or a minimal AIR (AIRmin that attract more mononuclear cells) in an experimental model of IgA nephropathy in order to investigate the effect of genetic background on glomerular disease progression and the participation of the monocyte chemoattractant protein-1 (MCP-1) chemokine. IgA nephropathy was induced by intraperitoneal ovalbumin injection and bile duct ligation in AIRmax and AIRmin mice. Histological changes, urinary protein/creatinine ratio, serum IgA levels, immunofluorescence for IgA, IgG and complement C3 fraction, immunohistochemistry for macrophages and MCP-1, and MCP-1 levels in macerated kidney were determined. Mesangial IgA deposition was seen only in AIRmin mice, which presented more renal lesions. Increased serum IgA levels (1.5 ± 0.4 vs 0.3 ± 0.1 mg/mL, P < 0.001), high glomerular MCP-1 expression and decreased monocyte/macrophage infiltration in the interstitial area (0.3 ± 0.3 vs 1.1 ± 0.9 macrophages/field, P < 0.05) were detected in AIRmin mice compared to AIRmax mice. No glomerular monocyte/macrophage infiltration was detected in either strain. In spite of the absence of IgA deposition, AIRmax mice presented discrete or absent mesangial proliferation. The study showed that there are differences between mice selected for AIRmax and AIRmin with respect to serum IgA levels, histological damage and MCP-1 chemokine production after ovalbumin injection in combination with bile duct ligation.

Genetic potential for an acute inflammatory response in IgA glomerulonephritis in mice.

Mice selected on the basis of an acute inflammatory response (AIR) can provide information about the immunopathological mechanisms of glomerulonephritis. We studied the differences between mice selected for a maximal AIR (AIRmax that attract more polymorphonuclear cells to the site of injury) or a minimal AIR (AIRmin that attract more mononuclear cells) in an experimental model of IgA nephropathy in order to investigate the effect of genetic background on glomerular disease progression and the participation of the monocyte chemoattractant protein-1 (MCP-1) chemokine. IgA nephropathy was induced by intraperitoneal ovalbumin injection and bile duct ligation in AIRmax and AIRmin mice. Histological changes, urinary protein/creatinine ratio, serum IgA levels, immunofluorescence for IgA, IgG and complement C3 fraction, immunohistochemistry for macrophages and MCP-1, and MCP-1 levels in macerated kidney were determined. Mesangial IgA deposition was seen only in AIRmin mice, which presented more renal lesions. Increased serum IgA levels (1.5 +/- 0.4 vs 0.3 +/- 0.1 mg/mL, P < 0.001), high glomerular MCP-1 expression and decreased monocyte/macrophage infiltration in the interstitial area (0.3 +/- 0.3 vs 1.1 +/- 0.9 macrophages/field, P < 0.05) were detected in AIRmin mice compared to AIRmax mice. No glomerular monocyte/macrophage infiltration was detected in either strain. In spite of the absence of IgA deposition, AIRmax mice presented discrete or absent mesangial proliferation. The study showed that there are differences between mice selected for AIRmax and AIRmin with respect to serum IgA levels, histological damage and MCP-1 chemokine production after ovalbumin injection in combination with bile duct ligation.

Pristane-induced arthritis in mice selected for maximal or minimal acute inflammatory reaction.

The role of inflammatory and specific immune responses in pristane-induced arthritis (PIA) was investigated in mouse lines produced by bi-directional selective breedings for maximal (AIRmax) or minimal (AIRmin) acute inflammatory reaction, comparing the outcome of PIA and the humoral and cellular response to hsp65. Symptoms of arthritis were detected in 50 % AIRmax mice 120 days after pristane injection, reaching a maximal incidence of 65 %, whereas only 7 % of AIRmin mice developed arthritis within an observation period of 200 days. The production of IgG antibody against hsp65 was found to be similar on both lines, although the IgG1 isotype was predominant in AIRmax, and IgG2a in AIRmin line. In vitro T cell proliferation to hsp65 was similar in the two lines, however, ELISPOT assays carried out soon after pristane treatment, demonstrated higher numbers of IL-6-, TNF-alpha- and IL-4-secreting cells in the spleen of AIRmax than in AIRmin mice, while higher numbers of IFN-gamma-producing cells were found in AIRmin mice. These results suggest a major participation of acute inflammatory mechanisms in the susceptibility to PIA. The genetic background which determines high or low AIR favors a Th2-like response in susceptible AIRmax and Th1-like response in resistant AIRmin mice at the initial phase of arthritis induction.

Effect of the bacillus of Calmette-Guérin, Propionibacter acnes and avridine as immunomodulators in antirabies vaccination of mice using the Fuenzalida-Palacios mouse brain vaccine.

Using the laboratory mice, Fuenzalida-Palacios mouse brain human rabies vaccine was administered in groups of animals previously inoculated with rabies virus and then submitted to treatments with the immunomodulators onco-BCG, avridine and Propionibacterium acnes. Humoral and cellular immune responses were evaluated through the macrophage inhibition factor (MIF), intra-pad inoculation (IPI) and serum neutralization (SN) tests and by the detection of gamma-interferon (IFN-gamma). The IPI test was not effective in detecting the response of delayed-type hypersensitivity, contrary to MIF, which showed the immune cellular response. Higher levels of IFN-gamma were observed in the groups of mice vaccinated and treated with avridine and P. acnes. Although immunomodulating activities have been detected, the use of adjuvants with the Fuenzalida-Palacios type vaccine in mice did not reveal any encouraging results.

Effect of bacillus of Calmette-Guérin, avridine and Propionibacterium acnes as immunomodulators on rabies in mice.

The cellular and humoral immune responses of mice inoculated with rabies virus and treated with the Bacillus of Calmette-Guérin, Avridine and Propionibacterium acnes were evaluated in this paper. There was a higher percentage of surviving mice in groups submitted to P. acnes treatment. Lower levels of interferon-gamma (IFN-gamma) were found in infected mice. The intra-pad inoculation test (IPI) was not effective to detect cellular immune response, contrary to the results found in MIF reaction. The survival of mice did not present correlation with the levels of antirabies serum neutralizing (SN) antibodies titers, IFN-gamma concentration and MIF response.

Low antibody responsiveness is found to be associated with resistance to chemical skin tumorigenesis in several lines of Biozzi mice.

High and low antibody responder lines of mice from Selections I, III and G were assayed for two-step skin tumorigenesis using a protocol consisting in initiation with 7,12-dimethylbenz[a]anthracene (DMBA) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Concordant results were obtained in the three selections: low antibody responder mice were shown to be significantly more resistant to tumor induction than the high responder counterparts. The difference was observed for all parameters: kinetics and percentages of tumor incidence and tumor multiplicity. The three bidirectional selective breeding experiments differed in several respects namely, the origin of the foundation populations, the antigens and immunization protocols used during the selection, as well as the breeding unit environments. Therefore, the consistent results relative to tumorigenesis strongly suggest that some of the alleles relevant to multispecific 'low' antibody production could contribute to the resistance to cutaneous chemical tumorigenesis.

Effect of bacillus of Calmette-Guerin, avridine and Propionibacterium acnes as immunomodulators on rabies in mice

Avaliou-se a resposta imune celular e humoral de camundongos inoculados com virus rabico de rua e submetidos aos imunomoduladores Onco-BCG, avridina e Propionibacterium acnes. Os animais submetidos ao tratamento com P. acnes apresentaram um maior percentual de sobrevivencia quando comparados aos dos demais tratamentos. Foram observados menores niveis de IFN-gama nos animais infectados, sugerindo imunossupressao viral. O teste do Coxim Plantar nao foi eficaz para a deteccao da resposta de hipersensibilidade retardada na metodologia utilizada, contrariamente ao MIF. A sobrevivencia dos animais nao apresentou correlacao com os niveis de anticorpos soroneutralizantes, concentracao de IFN-gama e resposta ao MIF

Innate resistance to infection by intracellular bacterial pathogens differs in mice selected for maximal or minimal acute inflammatory response.

The intensity of nonspecific immune reaction and the host resistance to facultative intracellular pathogens are found to be associated in lines of mice selected for maximal (AIRmax) or minimal (AIRmin) acute inflammatory reactivity. AIRmax are more resistant than AIRmin mice to Salmonella typhimurium and Listeria monocytogenes infection, the differences between lines in LD50 being > 1000 and 100 times, respectively. This difference was shown to be related to the initial bacterial containment at the infectious focus, and to the control of bacterial multiplication in the spleen during the 1st week after s. c. inoculation of the bacteria. Specific immune responses were not deeply affected by the selective process: antibody production and delayed-type hypersensitivity were both of similar intensity in AIRmax and AIRmin mice. The differential susceptibility to infection seems independent of the Nramp-1 locus polymorphism; therefore, these two lines represent a powerful model for investigating the role of other genetic loci regulating the nonspecific immunity effectors in the course of infectious diseases.

Effect of vaccination and the immunomodulators "bacillus of Calmette-Guérin, avridine and Propionibacterium acnes" on rabies in mice.

Responses of vaccination and treatment to immunomodulators against rabies in mice were evaluated through macrophage inhibition factor (MIF), intra-pad inoculation (IPI) and serum neutralization (SN) tests and by the detection of gamma-interferon (IFN-gamma). Onco-BCG, Avridine and Propionibacterium acnes were administered to groups of mice. Higher survival rates were found in animals treated with P. acnes. Lower levels of IFN-gamma were observed in the groups of infected and vaccinated mice. The IPI was not effective on detecting the response of delayed-type hypersensitivity. Vaccine induced in the infected animals a more intense response to MIF reaction.

Innate resistance to infection by intracellular bacterial pathogens differs in mice selected for maximal or minimal acute inflammatory response

The intensity of nonspecific immune reaction and the host resistance to facultative intracellular pathogens are found to be associated in lines of mice selected for maximal (AIRmax) or minimal (AIRmin) acute inflammatory reactivity. AIRmax are more resistant than AIRmin mice to Salmonella typhimurium and Listeria monocytogenes infection, the differences between lines in LD50 being > 1000 and 100 times, respectively. This difference was shown to be related to the initial bacterial containment at the infectious focus, and to the control of bacterial multiplication in the spleen during the 1st week after s. c. inoculation of the bacteria. Specific immune responses were not deeply affected by the selective process: antibody production and delayed-type hypersensitivity were both of similar intensity in AIRmax and AIRmin mice. The differential susceptibility to infection seems independent of the Nramp-1 locus polymorphism; therefore, these two lines represent a powerful model for investigating the role of other genetic loci regulating the nonspecific immunity effectors in the course of infectious diseases.

Mortality in elderly patients with acute renal failure.

In a retrospective study, we identified 55 elderly patients with acute renal failure (ARF) admitted to our hospital during an 8-year period from 1985 to 1993. Information about the etiology, complications, laboratory data, and treatment course were obtained from the clinical history. Of the 200 patients with ARF admitted to the hospital during this period, 28% were patients more than 60 years old (41 male and 14 female) with an average age of 68.5 +/- 7 years. The main causes of ARF were sepsis, volume depletion, low cardiac output, arterial hypotension, nephrotoxicity by antibiotics, and obstructive uropathy. The global mortality of elderly patients with ARF was 53%. The mortality rate of the different types of the ARF were: prerenal 35%, intrinsic 64% (oliguric 76%, nonoliguric 50%), and postrenal 40%. Mortality as a result of sepsis occurred in 18 patients (62%), by cardiovascular disease in 4 patients (13%), by acute respiratory failure in 2 patients (7%), and by other causes in 5 patients (18%). In the cases of sepsis, Pseudomonas was detected in 7 cases (39%), Escherichia coli in 2 cases (11%), Gram-negative nonspecific in 3 cases (17%), Klebsiella in 1 case (5%), and in 5 cases (16%), the hemoculture was negative. The patient survival rate was 47% (26 of 55 patients). Of these patients, 19 recovered their normal renal function (73%), but 7 patients remained with renal failure (27%). In conclusion, the global mortality in the elderly patients without considering the types of ARF was 53%. The oliguric form had the highest mortality rate with 76%. The main causes for mortality were sepsis with 62%, cardiovascular disease with 13%, and other causes 18%.

Reduced humoral immunity in uremic mice genetically selected for high antibody response.

Biozzi's Selection IV-A mice, genetically selected for 25 generations for high and low antibody response to sheep red blood cells (SRBC), 2-3 months old, were made uremic by subtotal nephrectomy and characterized for antibody production against the selection antigen. T cell activity was evaluated in vitro by lymphocyte proliferation and interleukin 2 (IL 2) production in response to the super antigen staphylococcal enterotoxin B (SEB). Total and IgM antibody titers (log2) were similar in uremic and non-uremic low responder mice (total antibody: 4.0 +/- 0.6 vs 3.6 +/- 0.6; IgG: 3.0 +/- 0.7 vs 2.4 +/- 0.4), while uremic high responders presented with non-uremic animals (total antibody: 10.8 +/- 1.6 vs 13.0 +/- 0.2; IgG: 10.3 +/- 1.5 vs 11.7 +/- 0.3). T cell proliferation and IL 2 production were similar in uremic and non-uremic groups after SEB stimulation. The results indicate a genetic effect on sensitivity to humoral immune response modulation by uremic status, with deficient antibody production despite a normal T cell proliferative response to mitogen stimulation in short-duration renal failure in high responder mice.

Reduced humoral immunity in uremic mice genetically selected for high antibody response

Biozzi's Selection IV-A mice, genetically selected for 25 generations for high and low antibody response to sheep red blood cells (SRBC), 2-3 months old, were made uremic by subtotal nephrectomy and characterized for antibody production against the selection antigen. T cell activity was evaluated in vitro by lymphocyte proliferation and interleukin 2 (IL 2) production in response to the superantigen staphylococcal enterotoxin B (SEB). Total and IgM antibody titers (log2) were similar in uremic and non-uremic low responder mice (total antobody: 4.0 +/- 0.6 vs 3.6 +/- 0.6; IgG: 3.0 +/- 0.7 vs 2.4 +/- 0,4), while uremic high responders presented a blunted humoral immune response to SRBC when compared with non-uremic animals (total antibody: 10.8 +/- 1.6 vs 13.0 +/- 0.2; IgG: 10.3 +/- 1.5 vs 11.7 +/- 0.3). T cell proliferation and IL 2 production were similar in uremic and ...