Balance of inflammatory cytokines related to severity and mortality of murine sepsis.

We tested the hypothesis that, during sepsis, the balance of pro- and anti-inflammatory cytokines is related to severity and survival. Cecal ligation and puncture (CLP) with a large (18-gauge)-, intermediate (21-gauge)-, or small (26-gauge)-diameter needle, or sham laparotomy, was performed on outbred CD-1 mice. Concentrations of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and the anti-inflammatory cytokine IL-10 were measured (by enzyme-linked immunosorbent assay) in serum, peritoneal lavage fluid, and liver and lung samples at 4, 8, 24, 48, and 96 h. As the diameter of the CLP needle decreased, the mortality rate decreased (at 48 h: large, 80%; intermediate, 40%; small, 20%; P < 0.05), the TNF-alpha and IL-6 concentrations decreased, and the time-to-peak TNF-alpha expression increased. In contrast, IL-10 concentration increased compared with baseline (serum at 24 h: large, 2.3-fold +/- 1.6-fold; intermediate, 2.0-fold +/- 0.5-fold; small, 49.9-fold +/- 8.3-fold; P < 0.05). Administration of IL-10 (5 microg, intraperitoneal) prior to CLP decreased mortality (P < 0.001). Administration of polyclonal anti-IL-10 serum prior to CLP (0.5 ml intraperitoneal) had the opposite effect and increased mortality (P < 0.001) and TNF-alpha, IL-6, and TNF-alpha mRNA expression compared with controls. Thus, severe sepsis is associated with a largely unopposed inflammatory response, and a largely unopposed inflammatory response (with anti-IL-10) results in severe sepsis and death. Less severe sepsis is associated with greater anti-inflammatory mediator expression, and greater anti-inflammatory mediator expression (with IL-10) results in less severe sepsis. Thus, the balance of inflammatory mediators is related to the severity and mortality of murine sepsis.     

Evidence for major alterations in the thymocyte subpopulations in murine models of autoimmune diseases

Thymocytes can be divided into four major subpopulations: CD4+CD8+ (double-positive), CD4-CD8- (double-negative), CD4+CD8- (CD4+) and CD4-CD8+ (CD8+) cells. Recent studies have shown that T-cell development in the thymus progresses as: CD4-CD8(-)----CD4+CD8(+)----CD4+ or CD8+ cells. In the present study we investigated these and other subpopulations of thymocytes in autoimmune MRL(-)+/+, MRL-lpr/lpr, C57BL/6-lpr/lpr, BXSB and NZB mice before (1-month old) and after (4-6-months old) the onset of lymphadenopathy and autoimmune disease. All the autoimmune strains at one month of age and other H-2, sex and age-matched controls (C3H, DBA/2, and C57BL/6) demonstrated normal proportions of thymocyte subsets with approximately 75% double-positive cells, 5-7% double-negative cells, 11-15% CD4+ cells and 3-5% CD8+ cells. By 4-6 months of age, MRL(-)+/+ mice demonstrated a moderate increase in double-negative cells (approximately 13%) and a decrease in double-positive cells (approximately 46%). Interestingly, in the presence of the lpr gene, as seen in MRL-lpr/lpr mice, the double-negative cells increased to approximately 47% and the double-positive cells decreased to approximately 16%. In contrast, 4-6-month-old C57BL/6-lpr/lpr mice failed to demonstrate any alterations in the thymocyte subsets thereby suggesting that background genes, in addition to the lpr gene, played a role in the thymocyte differentiation. BXSB male mice with severe lymphadenopathy behaved very similarly to MRL-lpr/lpr mice, inasmuch as their thymus contained approximately 48% double-negative cells and only approximately 8% double-positive cells. In contrast to MRL-lpr/lpr and BXSB strains, NZB mice at 6 or 10 months of age had normal composition of thymocyte subsets. In MRL and BXSB animals, although there was a significant increase in CD4+ cells (approximately 23-33%), due to a consequent increase in CD8+ cells (approximately 11%), the ratio of CD4+:CD8+ cells remained 2-3:1, similar to that seen in normal mice. Furthermore, using the J11d marker expressed by the majority of the double-negative and all double-positive thymocytes but not by mature functional T cells, we confirmed the above findings and demonstrated further that MRL-lpr/lpr mice at 4-6 months of age had an increased percentage of J11d- double-negative cells and a decrease in J11d+ double-negative cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunopathologic effects of tumor necrosis factor alpha in murine mycobacterial infection are dose dependent

In experimental mycobacterial infection, tumor necrosis factor alpha (TNF-alpha) is required for control of bacillary growth and the protective granulomatous response, but may cause immunopathology. To directly examine the positive and detrimental effects of this cytokine, a murine model was used in which different amounts of TNF-alpha were delivered to the site of infection. Mice with a disruption in the TNF-alpha gene (TNF-KO) or wild-type mice were infected with low or high doses of recombinant Mycobacterium bovis BCG that secreted murine TNF-alpha (BCG-TNF). Infection of TNF-KO mice with BCG containing the vector (BCG-vector) at a low dose led to increased bacillary load in all organs and an extensive granulomatous response in the lungs and spleen. The mice succumbed to the infection by approximately 40 days. However, when TNF-KO mice were infected with low doses of BCG-TNF, bacillary growth was controlled, granulomas were small and well differentiated, the spleen was not enlarged, and the mice survived. Infection with high inocula of BCG-TNF resulted in bacterial clearance, but was accompanied by severe inflammation in the lungs and spleen and earlier death compared to the results from the mice infected with high inocula of BCG-vector. Wild-type mice controlled infection with either recombinant strain, but showed decreased survival following high-dose BCG-TNF infection. The effects of TNF-alpha required signaling through an intact receptor, since the differential effects were not observed when TNF-alpha receptor-deficient mice were infected. The results suggest that the relative amount of TNF-alpha at the site of infection determines whether the cytokine is protective or destructive.     

The disconnect between animal models of sepsis and human sepsis

Frequently used experimental models of sepsis include cecal ligation and puncture, ascending colon stent peritonitis, and the i.p. or i.v. injection of bacteria or bacterial products (such as LPS). Many of these models mimic the pathophysiology of human sepsis. However, identification of mediators in animals, the blockade of which has been protective, has not translated into clinical efficacy in septic humans. We describe the shortcomings of the animal models and reasons why effective therapy for human sepsis cannot be derived readily from promising findings in animal sepsis.     

Stage-specific alterations in murine B lymphopoiesis with age

Although it is reported that B lymphopoiesis declines with age,the precursor stage(s) affected and the age of onset are ambiguous.Each progressive phase of B cell differentiation has distinctrequirements; therefore, precise identificatIon of the stage(s)that decline would yield insight into the age-related mechanismsaffecting humoral immunity. We analysed the composition of Blineage cells of mice 1, 4, 12 and 24 months of age using flowcytometry. Numbers of prepro-B and pro-B cells were unchanged,and a profound decrease occurred only in the numbers of pre-Bcells. This decrease occurred in two distinct phases: between1 and 4 months and between 12 and 24 months. Notably, the numbersof newly formed B cells did not decline in parallel, suggestingthat mechanisms are established to overcome the deficiency ofpre-B cells. Since the age-related changes are limited to thepre-B cell stage, we hypothesized that the impairment acts atthe pro-B to pre-B transition. We therefore evaluated whetherthe pro-B cells or the supporting stromal cells, which are necessaryfor normal progression of this stage, changed with age. Theabiiity of pro-B cells to proliferate in the presence of stromalcells was reduced by 24 months of age, as was the ability ofthe stromal cells to support pro-B cell proliferation. In contrast,the ability to mature into IgM⁺ cells was unchanged. Thus, strategiesthat supplement the stromal environment may enhance B lymphopoiesisin aged animals.     

Advances in sepsis research derived from animal models

Inflammation is the basic process by which tissues of the body respond to infection. Activation of the immune system normally leads to removal of microbial pathogens, and after resolution of the inflammation immune homeostasis is restored. This controlled process, however, can be disturbed resulting in disease. Therefore, many studies using infection models have investigated the participating immune mechanisms aiming at possible therapeutic interventions. Defined model substances such as bacterial lipopolysaccharide (endotoxin) have been used to mimic bacterial infections and analyze their immune stimulating functions. A complex network of molecular mechanisms involved in the recognition and activation processes of bacterial infections and their regulation has developed from these studies. More complex infection models will now help to interpret earlier observations leading to the design of relevant new infection models.     

Strain dependence in murine models of monoarthritis

Objective and design: This study explores the inflammatory response in various murine strains. Utilising several approaches, monoarthritis was induced in the knee, providing an inflammatory model relevant to arthritis. Methods: Acute (carrageenan/kaolin; C/K) or chronic inflammatory models (Freund’s complete adjuvant; FCA) or antigen-induced arthritis (AIA), were induced by peri- and/or intra-articular injection. Results: C/K elicited an acute inflammatory response in various strains of mice, with significant (P < 0.005) phenotypic variation. FCA induction provided a chronic inflammatory response. The magnitude of the response in both acute and chronic models was strain dependent, with BalbC exhibiting the most resistance to swelling in all models. AIA produced only an acute response in three strains tested. Conclusions: The data presented, demonstrating variation in the magnitude of acute and chronic inflammatory responses in different mice strains, allows informed selection of appropriate strains and models for future experimental studies. Received 28 March 2007; returned for revision 1 June 2007; accepted by J. Di Battista 27 June 2007     

Detrimental role of CC chemokine receptor 4 in murine polymicrobial sepsis

CC chemokine receptor 4 (CCR4) and its two ligands, CCL17 and CCL22, are critically involved in different immune processes. In models of lipopolysaccharide-induced shock, CCR4-deficient (CCR4^−/−) mice showed improved survival rates associated with attenuated proinflammatory cytokine release. Using CCR4^−/− mice with a C57BL/6 background, this study describes for the first time the role of CCR4 in a murine model of polymicrobial abdominal sepsis, the colon ascendens stent peritonitis (CASP). CASP-induced sepsis led to a massive downregulation of CCR4 in lymphoid and nonlymphoid tissues, whereas the expression of CCL17 and CCL22 was independent of the presence of CCR4. After CASP, CCR4^−/− animals showed a strongly enhanced bacterial clearance in several organs but not in the peritoneal lavage fluid and the blood. In addition, significantly reduced levels of proinflammatory cytokines/chemokines were measured in organ supernatants as well as in the sera of CCR4^−/− mice. CCR4 deficiency consequently resulted in an attenuated severity of systemic sepsis and a strongly improved survival rate after CASP or CASP with intervention. Thus, our data provide clear evidence that CCR4 plays a strictly detrimental role in the course of polymicrobial sepsis.     

Dendrite and dendritic spine alterations in Alzheimer models

Synaptic damage and loss are factors that affect the degree of dementia experienced in Alzheimer disease (AD) patients. Multicolor DiOlistic labeling of the hippocampus has been undertaken which allows the full dendritic arbor of targeted neurons to be imaged. Using this labeling technique the neuronal morphology of two transgenic mouse lines (J20 and APP/PS1) expressing mutant forms of the Amyloid Precursor Protein (APP), at various ages, have been visualized and compared to Wild Type (WT) littermate controls. Swollen bulbous dystrophic neurites with loss of spines were apparent in the transgenic animals. Upon quantification, statistically significant reductions in the number of spines and total dendrite area was observed in both transgenic mouse lines at 11 months of age. Similar morphological abnormalities were seen in human AD hippocampal tissue both qualitatively and quantitatively. Immunohistochemistry and DiOlistic labeling was combined so that Abeta plaques were imaged in relation to the dendritic trees. No preferential localization of these abnormal dystrophic neurites was seen in regions with plaques. DiI labeled reative astrocytes were often apparent in close proximity to A beta plaques.     

Functional alterations of murine peritoneal macrophages during pregnancy.

We have studied some functional characteristics of murine peritoneal macrophages (MOp) related to hormonal changes produced during pregnancy. Maximal expression of Ia antigen and release of interleukin 1 (IL1) were observed during the first week of pregnancy (implantation), when the highest peak of estradiol was produced. Both Ia antigen expression and IL1 levels progressively decreased as gestation advanced. Inversely, MOp capability to phagocyte and reduce nitro blue tetrazolium (NBT) was diminished at the beginning of pregnancy but returned to normal values in the last week. Sexual steroid levels (estradiol and progesterone) were inversely related, and the release of prostaglandin E2 (PGE2) by MOp decreased when progesterone levels increased. Although PGE2 production had no evident relation with Ia antigen expression and IL1 activity during the first and second weeks of pregnancy, the increment in PGE2 values and percentages of NBT+ cells could indicate a different stage of macrophage activation. These results suggest a possible hormonal regulation of macrophage functionality.     

Flow microfluorometry analysis of alterations in T-lymphocyte subsets during murine listeriosis

C57BL/6 mice were infected intravenously with 6 X 10(3) Listeria monocytogenes organisms. As early as day 3 of infection, there was a marked reduction in the number of lymphocytes recovered from the peripheral blood, bone marrow, and thymuses of infected animals. Concomitantly, there was an increase in the number of splenic lymphocytes. By day 14, both the total and differential cell counts were similar in both infected and normal animals. Flow microfluorometric studies comparing the Thy-1.2, Lyt-1, Lyt-2, and surface immunoglobulin (SIg) phenotypes of lymphocytes from normal and infected mice were performed. Between days 3 and 5, there was a decrease in the percentage of Thy-1.2+ cells in the spleens of L. monocytogenes-infected animals. Conversely, the percentages of Lyt-1+, Lyt-2+, and SIg+ cells remained constant. At day 7 of infection, the percentage of Thy-1.2+ splenocytes was within normal limits, and at day 10, the percentage of Thy-1.2+ cells was elevated slightly. The absolute numbers of Thy-1.2+ cells were comparable in both infected and normal animals at early stages (days 3 to 5) of L. monocytogenes infection, but there was a marked elevation of Thy-1.2+ splenocytes at days 7 to 14 of infection. Lyt-1+, Lyt-2+, and SIg+ splenocytes increased in absolute numbers as early as day 3 of infection and were still elevated at day 14. Adrenalectomy before infection had no effect on the results obtained, suggesting that these changes were not mediated by endogenous steroids.     

The role of cow milk allergy in increasing the severity of atopic dermatitis

Previous studies have shown that up to 33% of children with atopic dermatitis have experienced food hypersensitivity and among different kinds of food allergens Cow Milk (CM) has almost always been one of the most common food allergens in children. The aim of this study is to evaluate the cow milk allergy (CMA) as an increasing factor of severity of atopic dermatitis. One hundred and nineteen children (between 1.5 months and 12 years of age) with atopic dermatitis in the sense of Hanifin and Rajka's criteria entered this study and the severity of atopic dermatitis was identified via the SCORAD index. In order to make the diagnosis of cow milk allergy, a careful history, and a familial history of allergy was taken and the results of skin prick test (SPT) with CM and 4 other food allergen extracts, Radioallergosorbent test (RAST) with CM allergens and a food challenge test with cow milk (fresh or dried) were used. Also a total serum IgE determination and an eosinophil count (with a stool exam) were accomplished. The clinical manifestations of atopic dermatitis in patients was started from their first day of life up to 10 years of age. The family history in 83% of the patients was positive. Positive skin prick test and RAST with CM allergens were positive in 37.9% and 29.3% of cases respectively and the response to challenge test with cow milk was positive in 35 out of 40 patients and in total 44.5% had CMA according to a positive history of cow milk allergy and a positive outcome of the IgE tests (SPT and/or RAST) or a positive challenge test with CM allergens. The results showed that the most common food allergens in patients with atopic dermatitis are certainly cow milk allergens (44.5%) whereas other food allergens are tomato (29.41%), egg (28.57%), nuts (9.24%) and wheat (3.36%) according to the skin prick test. The mean total serum IgE was 307.11 +/- 6.56 IU/ml (range = 6-5000) in children with CMA and 81.04 +/- 5.97 IU/ml (range = 1-5000) in children without CMA while the mean eosinophil count was 569.52 +/- 3.02 count/ml (range = 67-8500) and 314.22 +/- 2.94 count/ml (range = 5-5000) respectively. The mean severity of atopic dermatitis according to the SCORAD index was 60.76 in children with CMA and 44.29 in children without CMA. The severity of atopic dermatitis in patients with CMA was significantly higher than patients without CMA (p < 0.0001). Also the mean total serum IgE and mean eosionophil counts in children with CMA were significantly higher than in children without CMA (P < 0.01 and p < 0.0001, respectively). It shows the important role of CM allergen proteins in the induction and in increasing the severity of AD in children.     

Immunopathologic studies of cutaneous lupus erythematosus

The studies, as outlined above, strongly suggest that there may be several pathophysiologic mechanisms resulting in the development of cutaneous lupus lesions. It appears that all lupus lesions are associated predominantly with a T-cell infiltrate. Based upon the studies of the neonatal lupus infants, it has been hypothesized that the U1RNP and Ro(SS-A) autoantibodies of maternal origin play a direct pathologic role in the genesis of the annular polycyclic SCLE lesions and this may be mediated by antibody-dependent cellular cytotoxicity mechanisms in which the antibody binds to the respective antigen present on the keratinocyte plasma membrane and the effector cells are T cells derived from the infants. Other studies, using direct immunofluorescence techniques, have demonstrated an association of cutaneous lupus lesions occurring in the presence of immunoglobulin and complement at the dermal/epidermal junction (positive lupus band test) in which the neoantigen of the complement membrane attack complex (C5b-C9) is detected. These data have been interpreted as indicating that immunoglobulin and complement, perhaps in the form of immune complexes, may play a role in the pathogenesis of some cutaneous lupus lesions. Additional studies have determined that there is a substantial number of lupus patients with cutaneous disease, without antinuclear antibodies, who fail to demonstrate the deposition of immunoglobulin and complement at the dermal/epidermal junction. Furthermore, other studies have indicated that ultraviolet light is capable of inducing lesions in lupus patients that histologically are identical to those of cutaneous lupus erythematosus but that failed to demonstrate the deposition of the immunoglobulin and complement components. Since discoid lupus lesions demonstrate a preponderance of T cells, it has been proposed that some of these lesions are the direct result of a T-cell cytotoxic event. However, the nature of the autoantigens responsible for this putative T cell-mediated cytotoxic response is unknown at the present time. The role of ultraviolet light in the genesis of the cutaneous lupus lesions appears to involve, within the epidermis, the generation of autoantigen macromolecules which then react with autoantibodies or specific T cells of the lupus host.     

Role of T cells in a murine model of Escherichia coli sepsis

To study the role of T cells in gram-negative sepsis, we developed a mouse model in which i.v. injection of Escherichia coli results in severe systemic illness, with high mortality rates after day 5. A large proportion of both CD4(+) and CD8(+) T cells are activated within 1 day after infection, as evidenced by up-regulation of CD69 and down-regulation of CD62L. Even more surprisingly, T cell-deficient mice exhibit markedly decreased disease severity compared to WT mice, indicating a pathogenic role of T cells. Mice lacking IFN-gamma also show diminished disease, and exhibit reduced T cell activation. Therefore, the pathogenic role of T cells may be mediated by IFN-gamma. Both T cell- and IFN-gamma-deficient mice have reduced serum IL-6 levels compared to WT mice, suggesting that T cells may stimulate innate immune responses, resulting in enhancement of disease. These data indicate an important role for T cells in a mouse model of E. coli sepsis, and reveal an unexpected early and pathogenic T cell response to this bacterial infection.     

Immunopathologic aspects of local tuberculous tissue reactions

The importance of immunologic factors in the local defense against myobacterial disease derives from different mechanisms of action. These may be characterized as follows: mycobacteria act as antigens or antigenic fragments to cause local sensitization, activation, and proliferation of T-lymphocytes leading to the formation of giant cells of Langhans' type. Antibodies play a role only as opsonines and are not directly responsible for the destruction of the invading organisms. Lymphokines on the other hand activate connective tissue cells and thereby contribute to the walling of infection and the development of scar tissue.     

Age-associated alterations in CXCL1 chemokine expression by murine B cells

Background  

The CXCL1 chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (KC), have been shown to play a role in a number of pathophysiological disease states including endotoxin-induced inflammation and bacterial meningitis. While the expression of these chemokines has been identified in a variety of cell types in the mouse, little is known about their expression with murine B-lymphocytes.     

Glutamatergic alterations and mitochondrial impairment in a murine model of Alzheimer disease

Deficits in glutamate neurotransmission and mitochondrial functions were detected in the frontal cortex (FC) and hippopcampus (HIPP) of aged 3×Tg-Alzheimer's disease (AD) mice, compared with their wild type littermates (non-Tg). In particular, basal levels of glutamate and vesicular glutamate transporter 1 (VGLUT1) expression were reduced in both areas. Cortical glutamate release responded to K(+) stimulation, whereas no peak release was observed in the HIPP of mutant mice. Synaptosomal-associated protein 25 (SNAP-25), glutamate/aspartate transporter (GLAST), glutamate transporter 1 (GLT1) and excitatory amino acid carrier 1 (EAAC1) were reduced in HIPP homogenates, where the adenosine triphosphate (ATP) content was lower. In contrast, glutamate transporter 1 and glial fibrillary acidic protein (GFAP) were found to be higher in the frontal cortex. The respiration rates of complex-I, II, IV, and the membrane potential were reduced in cortical mitochondria, where unaltered proton leak, F(0)F(1)-ATPase activity and ATP content, with increased hydrogen peroxide production (H(2)O(2)), were also observed. In contrast, complex-I respiration rate was significantly increased in hippocampal mitochondria, together with increased proton leak and H(2)O(2) production. Moreover, loss of complex-IV and F(0)F(1)-ATPase activities were observed. These data suggest that impairments of mitochondrial bioenergetics might sustain the failure in the energy-requiring glutamatergic transmission.     

Immunopathologic aspects of woodchuck hepatitis.

The natural history of infection with woodchuck hepatitis virus (WHV) has been studied in a colony of 38 Marmota monax. Besides serologic assessment for WHV markers, light-microscopic findings of 61 liver biopsies were correlated with the results of immunofluorescence analysis for nucleocapsid (WHcAg) and surface (WHsAg) antigens. Twenty-four chronic WHsAg carriers all featured signs of continuous viral replication. Two major immunomorphologic patterns were observed in their livers: 1) portal hepatitis in which WHcAg accumulated in the cytoplasm and WHsAg was associated with the hepatocyte membrane and 2) periportal hepatitis in which WHcAg shifted toward nuclear localization and WHsAg became mostly intracytoplasmic. Progression from portal to periportal hepatitis, observed in 7 woodchucks, appeared to be induced by a partial recovery of specific immune reactivity to WHV, insufficient, however, to interrupt WHV replication. Deposits of WHsAg and immunoglobulins were present in the kidney and spleen of animals with severe hepatitis.