Effect of silicon-doped calcium phosphate cement on angiogenesis based on controlled macrophage polarization.

Vascularization is an important early indicator of osteogenesis involving biomaterials. Bone repair and new bone formation are associated with extensive neovascularization. Silicon-based biomaterials have attracted widespread attention due to their rapid vascularization. Although calcium phosphate cement (CPC) is a mature substitute for bone, the application of CPC is limited by its slow degradation and insufficient promotion of neovascularization. Calcium silicate (CS) has been shown to stimulate vascular endothelial proliferation. Thus, CS may be added to CPC (CPC-CS) to improve the biocompatibility and neovascularization of CPC. In the early phase of bone repair (the inflammatory phase), macrophages accumulate around the biomaterial and exert both anti- and pro-inflammatory effects. However, the effect of CPC-CS on macrophage polarization is not known, and it is not clear whether the effect on neovascularization is mediated through macrophage polarization. In the present study, we explored whether silicon-mediated macrophage polarization contributes to vascularization by evaluating the CPC-CS-mediated changes in the immuno-environment under different silicate ion contents both in vivo and in vitro. We found that the silicon released from CPC-CS can promote macrophage polarization into the M2 phenotype and rapid endothelial neovascularization during bone repair. Dramatic neovascularization and osteogenesis were observed in mouse calvarial bone defects implanted with CPC-CS containing 60% CS. These findings suggest that CPC-CS is a novel biomaterial that can modulate immune response, promote endothelial proliferation, and facilitate neovascularization and osteogenesis. Thus, CPC-CS shows potential as a bone substitute material.

[Value of blood routine indexes and their ratios in judging the prognosis of adult patients with extensive burns].

Objective: To investigate the value of neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and blood platelet count (BPC) in judging the prognosis of adult patients with extensive burns. Methods: From January 2012 to December 2018, 99 adult patients with extensive burns who met the inclusion criteria were admitted to Union Hospital of Fujian Medical University, including 76 males and 23 females, aged 18 to 75 (43±13) years. According to the prognosis, the patients were divided into survival group of 79 cases and death group of 20 cases. Their clinical data were retrospectively analyzed by the method of case-control study. The gender, age, total burn area, inhalation injury, use of mechanical ventilation and white blood cell count, neutrophil count, lymphocyte count, and BPC on post injury day (PID) 1, 3, and 7 were collected, and the NLR, PLR, difference value of BPC on PID 3 and PID 1 (ΔBPC3), difference value of NLR on PID 3 and PID 1 (ΔNLR3), difference value of PLR on PID 3 and PID 1 (ΔPLR3), difference value of BPC on PID 7 and PID 1 (ΔBPC7), difference value of NLR on PID 7 and PID 1 (ΔNLR7), difference value of PLR on PID 7 and PID 1 (ΔPLR7) of patients in the two groups were calculated. Data were statistically analyzed with Mann-Whitney U test, independent sample t test, chi-square test to screen the death-related factors of patients. Binary classification single factor and multifactor logistic regression analysis were used to analyze the death-related factors of patients. The receiver's operating characteristic (ROC) curve of the independent risk factor of death of patients predicting the prognosis of adult patients with extensive burns was drawn, and the area under the curve, the optimal threshold and its sensitivity and specificity were calculated. Results: (1) There were statistically significant differences in total burn area and use of mechanical ventilation of patients between the two groups (Z=-2.615, χ(2)=7.282, P<0.01). (2) On PID 1, there was statistically significant difference in NLR of patients between the two groups (Z=-2.414, P<0.05). On PID 3, there were statistically significant differences in BPC and ΔNLR3 of patients between the two groups (Z=-2.048, -2.780, P<0.05 or P<0.01). On PID 7, there were statistically significant differences in lymphocyte count, BPC, NLR, and ΔNLR7 of patients between the two groups (Z=-2.248, -2.231, -2.641, -3.669, P<0.05 or P<0.01). (3) Binary classification single factor logistic regression analysis showed that the total burn area, mechanical ventilation, BPC and NLR on PID 7, and ΔNLR7 were related to death of patients (odds ratio=1.038, 0.193, 0.990, 1.086, 1.105, 95% confidence interval=1.010-1.067, 0.062-0.598, 0.982-0.998, 1.012-1.165, 1.037-1.178, P<0.05 or P<0.01). Binary classification multifactor logistic regression analysis showed that ΔNLR7 was the independent risk factor of death of adult patients with extensive burns (odds ratio=1.090, 95% confidence interval=1.008-1.178, P<0.05). (4) The optimal threshold of ROC curve of ΔNLR7 for predicting the prognostic death of 97 adult patients with extensive burns was -0.073 4. The sensitivity under the optimal threshold was 65.0%, and the specificity was 78.5%. The area under the ROC curve was 0.776 (95% confidence interval=0.650-0.882, P<0.01). Conclusions: Dynamic monitoring of NLR and BPC is of great significance to assist in judging the prognosis of adult patients with extensive burns. ΔNLR7 is an independent predictor of death in adult patients with extensive burns, while PLR can not predict the death of adult patients with extensive burns.

[Effects of glutamine on skeletal muscle membrane repair in severely burned mice and the functional mechanism].

Objective: To observe how glutamine affect the skeletal muscle membrane repair in severely burned mice through promoting the mitsugumin 53 (MG53) dimerization in skeletal muscle and to explore its functional mechanism. Methods: (1) Animal experiments. A total of 179 BALB/c male mice aged 6 to 8 weeks were divided into sham injury group (n=43), burn group (n=73) and burn+ glutamine group (n=63) according to the random number table (the same grouping method below). Mice in sham injury group were sham injured on the back, and mice in burn group and burn+ glutamine group were inflicted with 30% total body surface area full-thickness scald (hereinafter referred to as burn) on the back. Mice in burn+ glutamine group were intragastrically administered with glutamine (1 mg/kg), and the other two groups were given the same amount of amino acid solution once per day for 14 days. On post burn hour 12, 10 mice from burn group were taken for preparation of burn serum, which is used in the following cell experiments. Blood samples were collected from the hearts to prepare serum from 10 mice in sham injury group immediately after burn and from 10 mice in burn group and burn+ glutamine group on post burn day (PBD) 5, 10, and 14, respectively. And then the whole gastrocnemius muscle was harvested after the mice were sacrificed. On PBD 10, the whole flexor brevis digitorum was harvested from 6 mice in the 3 groups respectively after the mice were sacrificed. On PBD 5, 10, and 14, the whole gastrocnemius muscle tissue was harvested from another 9 mice in the 3 groups respectively after the mice were sacrificed. The mass of the whole gastrocnemius muscle of mice was weighed. The total protein content of gastrocnemius muscle of mice was detected by coomassie brilliant blue method. The repair function of myolemma of flexor brevis digitorum of mice was detected by two-photon laser fiber membrane perforating. The serum content of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) of mice was determined with radioimmunoassay. The expressions of MG53 dimer and monomer in gastrocnemius of mice were determined with non-reductive electrophoresis-Western blotting. The protein expressions of endoplasmic reticulum stress sign proteins CCAAT/enhancer binding protein homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) in gastrocnemius of mice were determined with Western blotting. (2) Cell experiments. Mice skeletal muscle precursor cells C2C12 were cultured in vitro, and cells of the second passage were selected for the experiments. The cells were divided into normal control group, burn serum group, and burn serum+ glutamine group, with 3 dishes in each group and 1×10(3) cells in each dish. Cells in normal control group were cultured with 1 mL Dulbecco's modified Eagle medium (DMEM) with fetal bovine serum of volume fraction 10%, cells in burn serum group were cultured with 1 mL DMEM with burn serum of volume fraction 10%, and cells in burn serum+ glutamine group were cultured with 1 mL DMEM with burn serum of volume fraction 10% and 4 µL glutamine with a final molar concentration of 8 mmol/L. After 24 hours of culturing, the repair function of myocyte membrane after differentiation of skeletal muscle precursor cells in mice was detected with the same method before. Another cells were grouped and cultured as before, with 3 wells in each group and 1×10(5) cells in each well. After 24 hours of culturing, the expressions of MG53 dimer and monomer and endoplasmic reticulum stress marker proteins in the cells were detected as before. Data were processed with analysis of variance of factorial design, one-way analysis of variance, least significant difference t test, and Student Newman Keuls test. Results: Animal experiments. (1) Compared with those in sham injury group, the mass and total protein content of gastrocnemius muscle of mice in burn group were significantly decreased on PBD 5, 10, and 14 (P<0.05). Compared with those in burn group, the mass and total protein content of gastrocnemius muscle of mice in burn+ glutamine group were significantly increased on PBD 5, 10, and 14 (P<0.05). (2) Compared with that in sham injury group (0.9±0.4), the fluorescence intensity of FM1-43 in myofiber of mice in burn group (7.8±0.4) was significantly increased on PBD 10 (t=7.75, P<0.05). Compared with that in burn group, the fluorescence intensity of FM1-43 in myofiber of mice in burn+ glutamine group (4.0±0.4) was significantly decreased on PBD 10 (t=-4.31, P<0.05). (3) Compared with that in sham injury group, the serum content of TNF-α and IL-6 of mice in burn group was significantly increased on PBD 5, 10, and 14 (P<0.05). Compared with that in burn group, the serum content of TNF-α and IL-6 of mice in burn+ glutamine group was significantly decreased on PBD 5, 10, and 14 (P<0.05). (4) Compared with 56.97±2.82, 44.89±4.72, 42.46±1.06, 14.26±0.99, 62.36±2.74, and 29.45±0.84 in sham injury group, the expressions of MG53 dimer and monomer in gastrocnemius of mice were significantly decreased in burn group on PBD 5, 10, and 14 (6.16±0.25, 26.09±1.22, 28.86±1.53, 5.63±0.25, 26.74±0.79, 4.41±0.52, P<0.05). Compared with those in burn group, the expression of MG53 dimer of gastrocnemius of mice in burn+ glutamine group was significantly increased on PBD 10 and 14 (36.79±1.44, 43.96±1.62), and the expression of MG53 monomer of gastrocnemius muscle of mice in burn+ glutamine group was significantly increased on PBD 14 (13.16±2.17, P<0.05). Compared with those in sham injury group, the protein expressions of CHOP and GRP78 in gastrocnemius muscle of mice in burn group were significantly elevated on PBD 5, 10, and 14 (P<0.05). Compared with those in burn group, the protein expressions of CHOP and GRP78 in gastrocnemius of mice in burn+ glutamine group were significantly reduced on PBD 5, 10 (P<0.05). Cell experiments. (1) Compared with that in normal control group (1.76±0.25), the fluorescence intensity of FM1-43 in cells in burn serum group (9.46±1.22) was significantly increased after 24 hours of culturing (t=12.28, P<0.05). Compared with that in burn serum group, the fluorescence intensity of FM1-43 in cells in burn serum+ glutamine group (4.71±0.45) was significantly decreased after 24 hours of culturing (t=-7.59, P<0.05). (2) The expressions of MG53 monomer of cells were similar in normal control group, burn serum group, and burn+ glutamine group after 24 hours of culturing (P>0.05). Compared with 58.5±1.8 in normal control group, the expression of MG53 dimer of cells in burn serum group was significantly decreased after 24 hours of culturing (14.1±1.4, P<0.05). Compared with that in burn serum group, the expression of MG53 dimer of cells in burn serum+ glutamine group was significantly increased after 24 hours of culturing (30.9±0.6, P<0.05). Compared with those in normal control group, the protein expressions of CHOP and GRP78 of cells were significantly elevated in burn serum group after 24 hours of culturing (P<0.05). Compared with those in burn serum group, the protein expressions of CHOP and GRP78 of cells were significantly reduced in burn serum+ glutamine group after 24 hours of culturing (P<0.05). Conclusions: Glutamine can promote MG53 dimerization by alleviating endoplasmic reticulum stress in severely burned mice. Thus it can accelerate skeletal muscle membrane repair, reduce the local inflammatory reaction of skeletal muscle and consumption of skeletal muscle.

[Effects of different doses of dopamine on organ function of rats at early stage of severe scald].

Objective: To explore the effects of different doses of dopamine on organ function of rats at early stage of severe scald. Methods: Thirty-two male Wistar rats aged 8 to 12 weeks were divided into sham injury (SI) group, simple resuscitation (SR) group, small dose (SD) group, and moderate dose (MD) group according to the random number table, with 8 rats in each group. After rats in the 4 groups were performed cardiac catheterization, rats in group SI were sham injured on the back by immersing in 37 ℃ warm water for 18 s, and rats in the other 3 groups were inflicted with 30% total body surface area (TBSA) full-thickness scald on the back by immersing in 97 ℃ hot water for 18 s. Rats in group SI were not treated after the injury, while rats in the other 3 groups were performed fluid resuscitation for 24 h through jugular vein catheter with micro syringe pump according to the Parkland formula. They were given 4.0 mL·kg(-1)·% TBSA(-1) normal saline during the first 24 h, of which they were given half of the total amount for the first 8 h, and they were given half of the total amount for the second and third 8 h. Rats in group SR were infused normal saline only, while rats in group SD and group MD were infused normal saline+ 1.25 µg·kg(-1)·min(-1)dopamine and normal saline+ 6.00 µg·kg(-1)·min(-1) dopamine respectively. Volume of 0.5 mL venous blood of all rats were taken through the cardiac catheter with serum separated at post injury hour (PIH) 1, 3, 6, 12, and 24. Serum content of cardiac troponin I (cTnI) was determined by enzyme-linked immunosorbent assay; serum content of diamine oxidase (DAO) was detected by ultraviolet spectrophotometer; serum content of ß(2)-microglobulin (ß(2)-MG) was determined by latex-enhanced immunoturbidimetric assay; serum content of total bile acid (TBA) was determined by enzyme colorimetry; serum content of lactic acid, malondialdehyde, and myeloperoxidase (MPO) was determined by ultraviolet spectrophotometer. Data were processed with analysis of variance for repeated measurement, one-way analysis of variance, least significant difference test, and Bonferroni correction. Results: (1) At PIH 1, 3, 6, 12, and 24, serum content of cTnI of rats in group SR, group SD, and group MD [(2.69±0.19), (3.04±0.19), (4.96±0.25), (6.88±0.28), (4.75±0.31) µg/L, (2.70±0.14), (3.08±0.13), (5.06±0.19), (7.11±0.21), (4.89±0.16) µg/L, (2.18±0.14), (2.54±0.09), (3.97±0.14), (5.46±0.34), (3.32±0.33) µg/L] were higher than that in group SI [(1.70±0.08), (1.70±0.08), (1.69±0.11), (1.69±0.08), (1.70±0.08) µg/L, P<0.05], serum content of cTnI of rats in group SR and group SD was similar (P>0.05), and serum content of cTnI of rats in group MD was lower than that in group SR and group SD (P<0.05). (2) At PIH 1 to 24, serum content of DAO of rats in group SR, group SD, and group MD was higher than that in group SI (P<0.05), serum content of DAO of rats in group SR and group MD was similar (P>0.05), and serum content of DAO of rats in group SD was lower than that in group SR and group MD (P<0.05). (3) At PIH 1 to 24, serum content of ß(2)-MG of rats in group SR, group SD, and group MD was higher than that in group SI (P<0.05), serum content of ß(2)-MG of rats in group SR and group MD was similar (P>0.05), and serum content of ß(2)-MG of rats in group SD was lower than that in group SR and group MD (P<0.05). (4) At PIH 1 to 24, serum content of TBA of rats in group SR, group SD, and group MD was similar (P>0.05) and higher than that in group SI (P<0.05). (5) At PIH 1 to 24, serum content of lactic acid of rats in group SR, group SD, and group MD was higher than that in group SI (P<0.05), serum content of lactic acid of rats in group SR and group MD was similar (P>0.05), and serum content of lactic acid of rats in group SD was lower than that in group SR and group MD (P<0.05). (6) At PIH 1 to 24, serum content of malondialdehyde and MPO of rats in group SR, group SD, and group MD was higher than that in group SI (P<0.05), serum content of malondialdehyde and MPO of rats in group SR and group MD was similar (P>0.05), and serum content of malondialdehyde and MPO of rats in group SD was significantly lower than that in group SR and group MD (P<0.05). Conclusions: With effective liquid recovery, dopamine of MD can improve early cardiac function of rats with severe scald, while dopamine of SD can alleviate tissue ischemia and hypoxia, reduce oxygen free radical damage in internal organs, and improve functions of intestine and kidney.

Association of killer cell immunoglobulin-like receptors with pulmonary tuberculosis in Chinese Han.

Killer cell immunoglobulin-like receptors (KIRs) are involved in the pathogenesis of a variety of diseases. However, whether KIR polymorphism is associated with susceptibility to pulmonary tuberculosis was unknown. We examined a possible association of KIR polymorphism with susceptibility to pulmonary tuberculosis in Chinese Han. We analyzed 15 KIR genes in 109 pulmonary tuberculosis patients and 110 healthy controls using sequence-specific primer PCR analysis of genomic DNA. We found that the frequencies of KIR2DS1, 2DS3 and 3DS1 were significantly higher in patients than in the control group. In addition, the number of subjects carrying more than two activating KIR genes in the patient group was significantly higher than in the control group. The gene cluster containing KIR3DS1-2DL5-2DS1-2DS5 was also significantly more frequent in the patient group. In conclusion, KIR genes 2DS1, 2DS3 and 3DS1 appear to be associated with resistance to pulmonary tuberculosis in the Chinese Han population. KIR genes apparently have a role in resistance to pulmonary tuberculosis.

A protective role for periostin and TGF-ß in IgE-mediated allergy and airway hyperresponsiveness.

BACKGROUND: The pathophysiology of asthma involves allergic inflammation and remodelling in the airway and airway hyperresponsiveness (AHR) to cholinergic stimuli, but many details of the specific underlying cellular and molecular mechanisms remain unknown. Periostin is a matricellular protein with roles in tissue repair following injury in both the skin and heart. It has recently been shown to be up-regulated in the airway epithelium of asthmatics and to increase active TGF-ß. Though one might expect periostin to play a deleterious role in asthma pathogenesis, to date its biological role in the airway is unknown. OBJECTIVE: To determine the effect of periostin deficiency on airway responses to inhaled allergen. METHODS: In vivo measures of airway responsiveness, inflammation, and remodelling were made in periostin deficient mice and wild-type controls following repeated intranasal challenge with Aspergillus fumigatus antigen. In vitro studies of the effects of epithelial cell-derived periostin on murine T cells were also performed. RESULTS: Surprisingly, compared with wild-type controls, periostin deficient mice developed increased AHR and serum IgE levels following allergen challenge without differences in two outcomes of airway remodelling (mucus metaplasia and peribronchial fibrosis). These changes were associated with decreased expression of TGF-ß1 and Foxp3 in the lungs of periostin deficient mice. Airway epithelial cell-derived periostin-induced conversion of CD4(+) CD25(-) cells into CD25(+) , Foxp3(+) T cells in vitro in a TGF-ß dependent manner. CONCLUSIONS AND CLINICAL RELEVANCE: Allergen-induced increases in serum IgE and bronchial hyperresponsiveness are exaggerated in periostin deficient mice challenged with inhaled aeroallergen. The mechanism of periostin's effect as a brake on allergen-induced responses may involve augmentation of TGF-ß-induced T regulatory cell differentiation.

Deletion of a coordinate regulator of type 2 cytokine expression in mice.

Mechanisms that underlie the patterning of cytokine expression in T helper (T(H)) cell subsets remain incompletely defined. An evolutionarily conserved approximately 400-bp noncoding sequence in the intergenic region between the genes Il4 and Il13, designated conserved noncoding sequence 1 (CNS-1), was deleted in mice. The capacity to develop T(H)2 cells was compromised in vitro and in vivo in the absence of CNS-1. Despite the profound effect in T cells, mast cells from CNS-1(-/-) mice maintained their capacity to produce interleukin 4. A T cell-specific element critical for the optimal expression of type 2 cytokines may represent the evolution of a regulatory sequence exploited by adaptive immunity.

Faithful expression of the human 5q31 cytokine cluster in transgenic mice.

Interleukins -4, -5, and -13, cardinal cytokines produced by Th2 cells, are coordinately expressed and clustered in 150-kb syntenic regions on mouse chromosome 11 and human chromosome 5q31. We analyzed two sets of human yeast artificial chromosome transgenic mice that contained the 5q31 cytokines to assess whether conserved sequences required for their coordinate and cell-specific regulation are contained within the cytokine cluster itself. Human IL-4, IL-13, and IL-5 were expressed under Th2, but not Th1, conditions in vitro. Each of these cytokines was produced during infection with Nippostrongylus brasiliensis, a Th2-inducing stimulus, and human IL-4 was generated after activation of NK T cells in vivo. Consistently fewer cells produced the endogenous mouse cytokines in transgenic than in control mice, suggesting competition for stable expression between the mouse and human genes. These data imply the existence of both conserved trans-activating factors and cis-regulatory elements that underlie the coordinate expression and lineage specificity of the type 2 cytokine genes in lymphocytes.

Identification of a coordinate regulator of interleukins 4, 13, and 5 by cross-species sequence comparisons.

Long-range regulatory elements are difficult to discover experimentally; however, they tend to be conserved among mammals, suggesting that cross-species sequence comparisons should identify them. To search for regulatory sequences, we examined about 1 megabase of orthologous human and mouse sequences for conserved noncoding elements with greater than or equal to 70% identity over at least 100 base pairs. Ninety noncoding sequences meeting these criteria were discovered, and the analysis of 15 of these elements found that about 70% were conserved across mammals. Characterization of the largest element in yeast artificial chromosome transgenic mice revealed it to be a coordinate regulator of three genes, interleukin-4, interleukin-13, and interleukin-5, spread over 120 kilobases.

Functional screening of an asthma QTL in YAC transgenic mice.

Many quantitative trait loci (QTLs) contributing to genetically complex conditions have been discovered, but few causative genes have been identified. This is mainly due to the large size of QTLs and the subtle connection between genotype and quantitative phenotype associated with these conditions. Transgenic mice have been successfully used to analyse well-characterized genes suspected of contributing to quantitative traits. Although this approach is powerful for examining one gene at a time, it can be impractical for surveying the large genomic intervals containing many genes that are typically associated with QTLs. To screen for genes contributing to an asthma QTL mapped to human chromosome 5q3 (refs 6,7), we characterized a panel of large-insert 5q31 transgenics based on studies demonstrating that altering gene dosage frequently affects quantitative phenotypes normally influenced by that gene. This panel of human YAC transgenics, propagating a 1-Mb interval of chromosome 5q31 containing 6 cytokine genes and 17 partially characterized genes, was screened for quantitative changes in several asthma-associated phenotypes. Multiple independent transgenic lines with altered IgE response to antigen treatment shared a 180-kb region containing 5 genes, including those encoding human interleukin 4 (IL4) and interleukin 13 (IL13 ), which induce IgE class switching in B cells. Further analysis of these mice and mice transgenic for mouse Il4 and Il13 demonstrated that moderate changes in Il4 and Il13 expression affect asthma-associated phenotypes in vivo. This functional screen of large-insert transgenics enabled us to identify genes that influence the QTL phenotype in vivo.

Genetic regulation of commitment to interleukin 4 production by a CD4(+) T cell-intrinsic mechanism.

The dysregulated expression of interleukin 4 (IL-4) can have deleterious effects on the outcome of infectious and allergic diseases. Despite this, the mechanisms by which naive T cells commit to IL-4 expression during differentiation into mature effector cells remain incompletely defined. As compared to cells from most strains of mice, activated CD4(+) T cells from BALB mice show a bias towards IL-4 production and T helper 2 commitment in vitro and in vivo. Here, we show that this bias arises not from an increase in the amount of IL-4 produced per cell, but rather from an increase in the proportion of CD4(+) T cells that commit to IL-4 expression. This strain-specific difference in commitment was independent of signals mediated via the IL-4 receptor and hence occurred upstream of potential autoregulatory effects of IL-4. Segregation analysis of the phenotype in an experimental backcross cohort implicated a polymorphic locus on chromosome 16. Consistent with a role in differentiation, expression of the phenotype was CD4(+) T cell intrinsic and was evident as early as 16 h after the activation of naive T cells. Probabilistic gene activation is proposed as a T cell-intrinsic mechanism capable of modulating the proportion of naive T cells that commit to IL-4 production.

Interferon gamma derived from CD4(+) T cells is sufficient to mediate T helper cell type 1 development.

Interferon gamma (IFN-gamma) has been implicated in T helper type 1 (Th1) cell development through its ability to optimize interleukin 12 (IL-12) production from macrophages and IL-12 receptor expression on activated T cells. Various systems have suggested a role for IFN-gamma derived from the innate immune system, particularly natural killer (NK) cells, in mediating Th1 differentiation in vivo. We tested this requirement by reconstituting T cell and IFN-gamma doubly deficient mice with wild-type CD4(+) T cells and challenging the mice with pathogens that elicited either minimal or robust IL-12 in vivo (Leishmania major or Listeria monocytogenes, respectively). Th1 cells developed under both conditions, and this was unaffected by the presence or absence of IFN-gamma in non-T cells. Reconstitution with IFN-gamma-deficient CD4(+) T cells could not reestablish control over L. major, even in the presence of IFN-gamma from the NK compartment. These data demonstrate that activated T cells can maintain responsiveness to IL-12 through elaboration of endogenous IFN-gamma without requirement for an exogenous source of this cytokine.

Distribution of protein kinase C isoforms after infection of macrophages with Leishmania major.

We characterized the effects of Leishmania infection on activation-induced translocation of protein kinase C (PKC) isoforms in murine bone marrow-derived macrophages. Although PKC-dependent gene expression was attenuated by infection, the distribution and translocation of PKC isoforms were unaffected. However, subsequent dissociation from membranes was substantially delayed for some isoforms, particularly PKCbeta.

Leishmania major: targeting IL-4 in successful immunomodulation of murine infection.

Protection against Leishmania major infection among inbred strains of mice is dependent upon successful expansion and activation of type 1 CD4+ effector (Th1) cells, a process that is aberrant in highly susceptible BALB strains. We sought to establish whether vaccination strategies using whole parasite lysates or a characterized immunodominant antigen, the Leishmania homolog of mammalian receptor for activated protein kinase C (LACK), would be capable of protecting subsequently infected BALB mice if given within a cytokine milieu capable of biasing the immune response toward Th1 cells. When given with neutralizing antibody to IL-4, but not when given alone, subcutaneously administered soluble Leishmania antigens mediated substantial protection to BALB/c mice against subsequent infection with parasites as assessed by size of the local lesion, enhanced Th1-type immune responses, and decreased parasite burdens. Similarly, when given with recombinant IL-12, LACK conferred substantial protection to cohorts of BALB.B, BALB/c, and BALB.K mice that was associated with reduction in serum IgE levels, consistent with effects on IL-4 production. Thus altering the cytokine milieu during administration of vaccine antigens by neutralizing IL-4 induced powerful Th1 recall responses during infection that were capable of mediating substantial levels of protection.

Expression of smooth muscle myosin isoforms in urinary bladder smooth muscle during hypertrophy and regression.

BACKGROUND: Partial ligation of the urinary out-flow tract of rabbit bladder induces hypertrophy of the smooth muscle layer in the bladder wall, and it is reversible by the removal of the ligature. The expression of smooth muscle myosin heavy chain isoforms SM1 and SM2 after hypertrophy and the regression of hypertrophy (reversal) was investigated at the translational and transcriptional levels using this experimental model. DESIGN: The contractile activity of smooth muscle strips derived from normal, hypertrophied, and reversal bladders was measured using electrical stimulation. Expression of SM1 and SM2 in normal, hypertrophied, and reversal muscle tissue was characterized using SDS-PAGE, reverse transcriptase-PCR (RT-PCR), and RNase protection assay. RESULTS: Smooth muscle strips from hypertrophied urinary bladder revealed a decrease in both force and rate of force generation in response to field stimulation. These alterations in contractility were reversed by removal of the obstruction. The altered function in bladder hypertrophy was also associated with changes in translation and transcription of the smooth muscle heavy chain isoforms SM1 and SM2. Upon regression of the hypertrophy by removal of the obstruction, the relative ratio of myosin heavy chain SM2:SM1 returned to nearly normal values. Analyses by RT-PCR showed a decrease in the mRNA transcript for SM2 in hypertrophied bladder muscle; and, on reversal of the hypertrophy, the SM2 mRNA level returned to that of normal bladder. These data suggest that the obstruction-induced hypertrophy activates a down-regulating mechanism for the expression of myosin SM2 heavy chain. CONCLUSIONS: Obstruction-induced alteration in the contractile characteristics of the urinary bladder smooth muscle is associated with changes in the expression of smooth muscle myosin heavy chains at both the protein and mRNA levels. The contractile function and the myosin heavy chain expression return to normal after regression of the smooth muscle hypertrophy on removal of the obstruction.

Expression cloning of a protective Leishmania antigen.

Parasite-specific CD4+ T cells have been shown to transfer protection against Leishmania major in susceptible BALB/c mice. An epitope-tagged expression library was used to identify the antigen recognized by a protective CD4+ T cell clone. The expression library allowed recombinant proteins made in bacteria to be captured by macrophages for presentation to T cells restricted to major histocompatibility complex class II. A conserved 36-kilodalton member of the tryptophan-aspartic acid repeat family of proteins was identified that was expressed in both stages of the parasite life cycle. A 24-kilodalton portion of this antigen protected susceptible mice when administered as a vaccine with interleukin-12 before infection.

Interferon gamma-independent effects of interleukin 12 administered during acute or established infection due to Leishmania major.

Interleukin 12 (IL-12) is a powerful stimulus for the growth of activated T and natural killer cells, their generation of interferon gamma (IFN-gamma), and the differentiation of T helper type 1 (Th1) effector cells from naive precursors in vitro. These activities are consistent with the capacity of exogenous IL-12 to heal otherwise susceptible BALB/c mice infected with the intramacrophage parasite Leishmania major. Using this characterized model of CD4 cell subset differentiation, we examined the immunologic effects of IL-12 administered either at the time of infection, when naive T cells are primed, or after 14 days of infection, by which time CD4+ subset differentiation has occurred. Given with the inoculation of parasites, IL-12 induced IFN-gamma and IL-10 and markedly suppressed IL-4. Effects on IL-10 and IL-4 were comparable in mice with homozygous disruption of the IFN-gamma gene (IFN-gamma 0/0), and suppression of IL-4 was unchanged by administration of neutralizing anti-IL-10 antibody. Induction of IFN-gamma and IL-10 mRNA by IL-12 also occurred in infected SCID mice. Given after day 14 of infection, however, IL-12 not only induced IFN-gamma and IL-10 but also induced IL-4 in normal and IFN-gamma 0/0 mice. These data demonstrate direct effects of IL-12 independent of IFN-gamma, IL-10, and IL-4 and demonstrate that the ineffectiveness of IL-12 administered following infection with L. major correlates with resistance of differentiated Th2 cells to the IL-4-suppressing activity of IL-12.

CD4+ effector cells default to the Th2 pathway in interferon gamma-deficient mice infected with Leishmania major.

Mice with homologous disruption of the interferon gamma (IFN-gamma) gene on the C57BL/6 background were infected with Leishmania major and the immune response assessed. In contrast to wild-type or heterozygous knockout mice, deficient animals were unable to restrict growth of the parasite and suffered lethal infection over 6-8 wk. Although wild-type and heterozygous littermates developed CD4+ cells that contained transcripts for IFN-gamma and lymphotoxin, typical of T helper type 1 (Th1) cells, the knockout mice developed CD4+ cells that contained transcripts for interleukin 4 (IL-4), IL-5, and IL-13, typical of Th2 cells. ELISPOT assays confirmed the reciprocal patterns of IFN-gamma or IL-4 production by T cells in similar frequencies in the respective groups of mice, and antibody analysis confirmed the presence of Th2-mediated isotype switching in the knockout mice. These data suggest that CD4+ T cells that normally respond to antigens by differentiation to Th1 cells default to the Th2 pathway in the absence of endogenous IFN-gamma.

Leishmania promastigotes evade interleukin 12 (IL-12) induction by macrophages and stimulate a broad range of cytokines from CD4+ T cells during initiation of infection.

Leishmania major are intramacrophage parasites whose eradication requires the induction of T helper 1 (Th1) effector cells capable of activating macrophages to a microbicidal state. Interleukin 12 (IL-12) has been recently identified as a macrophage-derived cytokine capable of mediating Th1 effector cell development, and of markedly enhancing interferon gamma (IFN-gamma) production by T cells and natural killer cells. Infection of macrophages in vitro by promastigotes of L. major caused no induction of IL-12 p40 transcripts, whereas stimulation using heat-killed Listeria or bacterial lipopolysaccharide induced readily detectable IL-12 mRNA. Using a competitor construct to quantitate a number of transcripts, a kinetic analysis of cytokine induction during the first few days of infection by L. major was performed. All strains of mice examined, including susceptible BALB/c and resistant C57BL/6, B10.D2, and C3H/HeN, had the appearance of a CD4+ population in the draining lymph nodes that contained transcripts for IL-2, IL-4, and IFN-gamma (and in some cases, IL-10) that peaked 4 d after infection. In resistant mice, the transcripts for IL-2, IL-4, and IL-10 were subsequently downregulated, whereas in susceptible BALB/c mice, these transcripts were only slightly decreased, and IL-4 continued to be reexpressed at high levels. IL-12 transcripts were first detected in vivo by 7 d after infection, consistent with induction by intracellular amastigotes. Challenge of macrophages in vitro confirmed that amastigotes, in contrast to promastigotes, induced IL-12 p40 mRNA. Reexamination of the cytokine mRNA at 4 d revealed expression of IL-13 in all strains analyzed, suggesting that IL-2 and IL-13 may mediate the IL-12-independent production of IFN-gamma during the first days after infection. Leishmania have evolved to avoid inducing IL-12 from host macrophages during transmission from the insect vector, and cause a striking induction of mRNAs for IL-2, IL-4, IL-10, and IL-13 in CD4+ T cells. Each of these activities may favor survival of the organism.

Identification of the ligand-binding regions in the macrophage colony-stimulating factor receptor extracellular domain.

The c-fms gene encodes the receptor for the macrophage colony-stimulating factor (M-CSF), and its extracellular domain consists of five immunoglobulin-like subdomains. To identify which of the five immunoglobulin-like regions are involved in ligand binding, we polymerase chain reaction-cloned five segments of the extracellular domain of the murine c-fms gene, each starting with the normal initiation codon and containing successive additions of the immunoglobulin-like subdomains. These protein segments are designated A, B, C, D, and E and contain, from the N-terminal end, either one, two, three, four, or all five immunoglobulin-like subdomains, respectively. Each segment was expressed as a secreted soluble protein from a baculovirus expression vector in Sf9 insect cells. In addition, segments A, B, C, and E were produced as soluble alkaline phosphatase fusion proteins, as was a segment containing only the fourth and fifth immunoglobulin domains. These segments of the Fms extracellular domain were used to assess M-CSF binding by competition radioimmunoassays, plate binding immunoassays, and immunoprecipitation analyses. The results indicated that the first two N-terminal immunoglobulin-like domains did not interact with M-CSF but, in combination with the third immunoglobulin-like domain, provided high-affinity M-CSF binding. The fourth and fifth immunoglobulin-like domains near the cell membrane did not exhibit M-CSF binding and may inhibit interaction of M-CSF with the first three immunoglobulin domains. These results suggest that the three N-terminal immunoglobulin-like domains constitute the high-affinity M-CSF binding region and that the fourth and fifth immunoglobulin-like domains may perform functions other than ligand binding.