[Effects of celastrol on autophagy and endoplasmic reticulum stress-mediated apoptosis in a mouse model of nonalcoholic fatty liver disease].

Objective: To investigate the effect of celastrol (CEL) on autophagy and endoplasmic reticulum stress-mediated apoptosis in a mouse model of nonalcoholic fatty liver disease (NAFLD). Methods: Eighteen male C57BL/6J mice were randomly divided into normal control (NC, n=6), high-fat diet (HFD, n=6) and celastrol group (HFD+CEL, n=6). The normal control group was fed with regular diet, and the high-fat diet and celastrol group were fed with high-fat diet for 12 weeks. After successful modeling, celastrol group were injected with 100 µg⋅kg-1⋅d-1 celastrol intraperitoneally for 4 weeks, and NC and HFD group were injected intraperitoneally with the same doses of normal saline. Serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) were measured in mouse after 4-weeks of intervention. HE and Oil Red O staining were used to observe the pathomorphological changes and lipid droplet deposition in mouse liver, and the findings were scored according to NAFLD activity score (NAS). Western blot was used to detect the expression levels of liver microtubule associated protein 1 light chain 3 (LC3), P62, glucose-regulated protein 78 (GRP78), protein kinase R-like endoplasmic reticulum kinase (PERK), phosphorylated PERK (p-PERK), activated transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), cleaved Caspase-3(cleaved caspase-3), B-cell lymphoma-2 (Bcl-2) and Bcl-2 related X protein (Bax).TUNEL staining was used to observe the apoptosis of hepatocytes. One-way analysis of variance was used for the intergroup comparison. Results: Serum levels of ALT (68.71±8.57) U/L, AST (209.63±28.64) U/L, TG (0.97±0.14) mmol/L, TC (4.12±0.64) mmol/L, and LDL -C (0.40±0.06) mmol/L were lower in celastrol group mouse than HFD group [(110.19±10.79) U/L, (399.72±73.47) U/L, (1.44±0.13) mmol/L, (5.65±0.54) mmol /L, (0.61±0.07) mmol/L] (P<0.05); while the serum HDL-C level (1.29±0.17) mmol/L was higher in celastrol than HFD group (0.72±0.13) mmol/L (P<0.05). HE and Oil Red O staining showed that lipid deposition and intralobular inflammation were apparent in the liver tissue of HFD group mouse, and the NAS score was significantly increased, while the hepatocyte steatosis and intralobular inflammation were alleviated after celastrol intervention, and the NAS score was decreased significantly (P<0.05). Compared with HFD group, the ratio of LC3II/I was significantly increased in the liver of celastrol group mouse, and the P62 was significantly decreased (P<0.05). Meanwhile, the expression level of GRP78, p-PERK/PERK , ATF4, and CHOP was significantly lower in celastrol than HFD group (P<0.05). In addition, the expressions of cleaved caspase-3 and Bax were significantly lower in celastrol than HFD group, and the expression of Bcl-2 was significantly increased (P<0.05). At the same time, the apoptosis rate of hepatocytes was also significantly lower in celastrol than HFD group (P<0.05). Conclusion: Celastrol can effectively alleviate the lipid deposition, protect hepatocytes and delay the progression of non-alcoholic fatty liver disease in mouse liver with non-alcoholic fatty liver disease. In addition, its mechanism of action may be related to the induction of autophagy, inhibition of endoplasmic reticulum stress PERK/ATF4/CHOP pathway and its mediated apoptosis.

[Fluid resuscitation strategy and efficacy evaluation in shock stage in severely burned children with different burn areas in different age groups].

Objective: To explore the fluid resuscitation strategy in shock stage in severely burned children with different burn areas in different age groups, and to evaluate the curative effect. Methods: A retrospective cohort study was conducted. From January 2015 to June 2020, 235 children with severe and above burns who met the inclusion criteria were hospitalized in the First Affiliated Hospital of Nanchang University, including 150 males and 85 females, aged 3 months to 12 years. After admission, it was planned to rehydrate the children with electrolyte, colloid, and water according to the domestic rehydration formula for pediatric burn shock, and the rehydration volume and speed were adjusted according to the children's mental state, peripheral circulation, heart rate, blood pressure, and urine output, etc. The actual input volume and planned input volume of electrolyte, colloid, water, and total fluid of all the children were recorded during the 8 hours since fluid replacement and the first and second 24 hours after injury. According to urine output during the 8 hours since fluid replacement, all the children were divided into satisfactory urine output maintenance group (119 cases) with urine output ≥1 mL·kg-1·h-1 and unsatisfactory urine output maintenance group (116 cases) with urine output <1 mL·kg-1·h-1, and the electrolyte coefficient, colloid coefficient, and water coefficient of the children were calculated during the 8 hours since fluid replacement. According to the total burn area, children aged <3 years (155 cases) and 3-12 years (80 cases) were divided into 15%-25% total body surface area (TBSA) group and >25%TBSA group, respectively. The electrolyte coefficient, colloid coefficient, water coefficient, and urine output of the children were calculated or counted during the first and second 24 hours after injury, and the non-invasive monitoring indicators of body temperature, heart rate, respiratory rate, and percutaneous arterial oxygen saturation and efficacy indicators of hematocrit, platelet count, hemoglobin, albumin, creatinine, and alanine aminotransferase (ALT) of the children were recorded 48 hours after injury. The prognosis and outcome indicators of all the children during the treatment were counted, including complications, cure, improvement and discharge, automatic discharge, and death. Data were statistically analyzed with independent sample or paired sample t test, Mann-Whitney U test, chi-square test, and Fisher's exact probability test. Results: During the 8 hours since fluid replacement, the actual input volume of electrolyte of all the children was significantly more than the planned input volume, and the actual input volumes of colloid, water, and total fluid were significantly less than the planned input volumes (Z=13.094, 5.096, 13.256, 7.742, P<0.01). During the first and second 24 hours after injury, the actual input volumes of electrolyte of all the children were significantly more than the planned input volumes, and the actual input volumes of water and total fluid were significantly less than the planned input volumes (Z=13.288, -13.252, 3.867, 13.183, -13.191, 10.091, P<0.01), while the actual input volumes of colloid were close to the planned input volumes (P>0.05). During the 8 hours since fluid replacement, compared with those in unsatisfactory urine output maintenance group, there was no significant change in electrolyte coefficient or colloid coefficient of children in satisfactory urine output maintenance group (P>0.05), while the water coefficient was significantly increased (Z=2.574, P<0.05). Among children <3 years old, compared with those in >25%TBSA group, the electrolyte coefficient and water coefficient of children were significantly increased and the urine output of children was significantly decreased in 15%-25%TBSA group during the first and second 24 hours after injury (Z=-3.867, -6.993, -3.417, -5.396, -5.062, 1.503, P<0.05 or P<0.01), while the colloid coefficient did not change significantly (P>0.05); the levels of efficacy indicators of hematocrit, platelet count, and hemoglobin at 48 h after injury were significantly increased, while ALT level was significantly decreased (Z=-2.720, -3.099, -2.063, -2.481, P<0.05 or P<0.01); the levels of the rest of the efficacy indicators and non-invasive monitoring indicators at 48 h after injury did not change significantly (P>0.05). Among children aged 3-12 years, compared with those in >25%TBSA group, the electrolyte coefficient and water coefficient of children in 15%-25%TBSA group were significantly increased during the first and second 24 hours after injury, the colloid coefficient during the second 24 h was significantly decreased (Z=-2.042, -4.884, -2.297, -3.448, -2.480, P<0.05 or P<0.01), while the colloid coefficient during the first 24 hours after injury, urine output during the first and second 24 hours after injury, and the non-invasive monitoring indicators and efficacy indicators at 48 hours after injury did not change significantly (P>0.05). Complications occurred in 17 children during the treatment. Among the 235 children, 211 cases were cured, accounting for 89.79%, 5 cases were improved and discharged, accounting for 2.13%, 16 cases were discharged automatically, accounting for 6.81%, and 3 cases died, accounting for 1.28%. Conclusions: The electrolyte volume in early fluid resuscitation in severely burned children exceeding the volume calculated by the formula can obtain a good therapeutic effect. Among children <3 years old, the volume of fluid resuscitation should be appropriately increased in children with extremely severe burns compared with children with severe burns during fluid resuscitation; among children aged 3-12 years, the colloid volume should be appropriately increased in children with extremely severe burns compared with children with severe burns during fluid resuscitation; non-invasive monitoring indicators can be used to monitor hemodynamics and guide fluid resuscitation in severely burned children.

[Role of 14-3-3σgene in the regulation of endotoxin/lipopolysaccharide-induced inflammatory responses in human pulmonary epithelial cells].

Objective: To explore the mechanism of 14-3-3σgene in regulating inflammatory response of human pulmonary epithelial cells induced by endotoxin/lipopolysaccharide (LPS). Methods: (1) Cells of human normal pulmonary epithelial cell line BEAS-2B cultured in logarithmic growth period were collected and divided into control group and PCMV6-14-3-3σgroup using the random number table, with 3 wells in each group. Cells in control group were transfected with empty plasmid, and cells in PCMV6-14-3-3σgroup were transfected with PCMV6-14-3-3σplasmid. The protein expression of 14-3-3σin cell was detected by Western blotting at 48 hours after transfection. (2) Cells of human normal pulmonary epithelial cell line BEAS-2B cultured in logarithmic growth period were collected and divided into control group, PCMV6-14-3-3σgroup, PCMV6-14-3-3σ+ LPS group, and LPS group using the random number table, with 3 wells in each group. Cells in control group were transfected with empty plasmid for 42 hours. Cells in PCMV6-14-3-3σgroup were transfected with PCMV6-14-3-3σplasmid for 42 hours. Cells in PCMV6-14-3-3σ+ LPS group were stimulated with 1 µg/mL LPS (the same final mass concentration below) for 6 hours after being transfected with PCMV6-14-3-3σplasmid for 42 hours. Cells in LPS group were stimulated by LPS for 6 hours. The protein expressions of Bax and B-cell lymphoma-2 (Bcl-2) were detected by Western blotting, and the ratio of Bax to Bcl-2 was calculated. Apoptotic rate was detected by flow cytometry. The mRNA expressions of tumor necrosis factor alpha (TNF-α) and interleukin 1beta (IL-1ß) in cells were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction technique. Content of TNF-α and IL-1ß in cell culture supernatant was detected by enzyme-linked immunosorbent assay. Data were statistically analyzed with t test, one-way analysis of variance, and least significant difference test. Results: (1) At 48 hours after transfection, the protein expression of 14-3-3σin cells of PCMV6-14-3-3σgroup (1.05±0.03) was significantly higher than that in control group (0.78±0.04, t=5.41, P<0.01). (2) Compared with those in control group, the ratio of Bax to Bcl-2, apoptotic rate, mRNA expressions of TNF-α and IL-1ß, and content of TNF-α and IL-1ß in cell supernatant in PCMV6-14-3-3σgroup showed no significant difference (P>0.05); the above-mentioned indexes of cells in LPS group were significantly higher or increased (P<0.01). Compared with those in LPS group, the above-mentioned indexes of cells in PCMV6-14-3-3σ+ LPS group were significantly lower or decreased (P<0.01). Conclusions: 14-3-3σis a key factor in regulating apoptosis. It can alleviate the LPS-induced inflammatory responses by regulating the ratio of apoptotic regulators Bax to Bcl-2 and inhibiting apoptosis of human pulmonary epithelial cells.

[Analysis of differential gene expressions of inflammatory and repair-related factors in chronic refractory wounds in clinic].

Objective: To compare the tissue morphology and gene expressions of inflammatory and repair-related factors in chronic refractory wound tissue including pressure ulcers and diabetic feet. Methods: During August 2016 to September 2017, 10 samples of prepuce were collected after circumcision of 10 urological patients [all male, aged (38±4) years old] admitted in the First Affiliated Hospital of Nanchang University and included in normal skin group, samples of tissue around the edge of wounds with blood supply were collected from 9 heat or electric burn patients [6 male patients, 3 female patients, aged (51±8) years old], 13 pressure ulcer patients [9 male patients, 4 female patients, aged (51±14) years old] and 10 diabetic foot patients [8 male patients, 2 female patients, aged (61±10) years old] during the operations. The samples were divided into burn wound group (9 samples), pressure ulcer group (13 samples), and diabetic foot group (10 samples). Ten slices were taken from pressure ulcer group and diabetic foot group respectively, and 5 slices in each group were used to observe the tissue morphology and expressions of Ki67 and CD31 of wounds respectively with immunofluorescence method. Ten samples from normal skin group, 9 samples from burn wound group, 13 samples from pressure ulcer group, and 10 samples from diabetic foot group were collected for analysis of mRNA expressions of vascular endothelial growth factor 192 (VEGF192), transforming growth factor ß (TGF-ß), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) , interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor α (TNF-α) by real time fluorescent quantitative reverse transcription polymerase chain reaction. Data were processed with Mann-Whitney U test and Kruskal-Wallis rank-sum test. Results: (1) The expression level of Ki67 in diabetic foot group (390±100) was higher than that of pressure ulcer group (182±14, Z=-2.611, P<0.01). (2) Although there were a large number of vascular endothelial cells (CD31 positive cells) in wounds of diabetic foot group, their distribution was disordered and failed to form intact lumen. There were less vascular endothelial cells in wounds of pressure ulcer group than those of diabetic foot group, but the complete lumen was formed. (3) The mRNA expression levels of VEGF192 in wounds of burn wound group, pressure ulcer group, and diabetic foot group were significantly lower than the level in normal skin group (H=13.72, 30.50, 15.20, P<0.05 or P<0.01), and the level was the lowest in pressure ulcer group. The mRNA expression level of VEGF192 in wounds of pressure ulcer group was significantly lower than that of diabetic foot group (H=15.30, P<0.01). Compared with that of normal skin group, the mRNA expression level of TGF-ß in wounds of burn wound group showed no significant difference (H=-9.50, P>0.05), while the mRNA expression levels of TGF-ß in wounds of pressure ulcer group and diabetic foot group were significantly decreased (H=18.04, 14.50, P<0.01). The mRNA expression level of TGF-ß in wounds of pressure ulcer group was similar to that of diabetic foot group (H=3.54, P>0.05). (4) Compared with those of normal skin group, the mRNA expression levels of VCAM-1 in wounds of burn wound group and pressure ulcer group were significantly increased (H=-22.50, -11.50, P<0.05 or P<0.01), and there was no significant difference in the mRNA expression level of VCAM-1 in wounds of diabetic foot group (H=10.00, P>0.05); the mRNA expression level of ICAM-1 in wounds of burn wound group showed no significant difference (H=-9.50, P>0.05), and the levels of ICAM-1 in wounds of pressure ulcer group and diabetic foot group were significantly decreased (H=16.50, 16.50, P<0.01). The mRNA expression level of VCAM-1 in wounds of pressure ulcer group was significantly higher than that of diabetic foot group (H=-21.50, P<0.01), the mRNA expression level of ICAM-1 in wounds of pressure ulcer group was similar to that of diabetic foot group (H=0, P>0.05). (5) Compared with those of normal skin group, except for the mRNA expression level of IL-1ß in wounds of diabetic foot group showed no significant difference (H=-10.00, P>0.05), the mRNA expression levels of IL-1ß in wounds of burn wound group and pressure ulcer group were significantly increased (H=-32.50, -21.50, P<0.01); the mRNA expression levels of IL-6 were significantly increased in wounds of burn wound group, pressure ulcer group, and diabetic foot group (H=-17.50, -30.50, -11.80, P<0.05 or P<0.01); except for the mRNA expression level of TNF-α in wounds of burn wound group showed no significant difference (H=-9.50, P>0.05), the mRNA expression levels of TNF-α in wounds of pressure ulcer group and diabetic foot group were significantly decreased (H=18.04, 14.50, P<0.01). The mRNA expression levels of IL-1ß and TNF-α in wounds of pressure ulcer group were significantly lower than those of burn wound group (H=11.00, 27.54, P<0.05 or P<0.01), while the mRNA expression level of IL-6 was significantly higher (H=-13.00, P<0.05). The mRNA expression levels of IL-1ß and TNF-α in wounds of diabetic foot group were significantly lower than those of burn wound group (H=22.50, 24.00, P<0.01), while the mRNA expression level of IL-6 showed no significant difference (H=5.70, P>0.05). Conclusions: The phenotypes of diabetic foot and pressure ulcer vary from the expressions levels of proliferating cell nuclear antigen and blood vessels forming ability to the expression levels of growth factors, cell adhesion factors, and inflammatory cytokines.

[Effects of aerosol inhalation of recombinant human keratinocyte growth factor 2 on the lung tissue of rabbits with severe smoke inhalation injury].

Objective: To investigate the effect of recombinant human keratinocyte growth factor 2 (rhKGF-2) on lung tissue of rabbits with severe smoke inhalation injury. Methods: A total of 120 New Zealand rabbits were divided into 5 groups by random number table after being inflicted with severe smoke inhalation injury, with 24 rats in each group. Rabbits in the simple injury group inhaled air, while rabbits in the injury+phosphate buffer solution (PBS) group inhaled 5 mL PBS once daily for 7 d. Rabbits in injury+1 mg/kg rhKGF-2 group, injury+2 mg/kg rhKGF-2 group, and injury+5 mg/kg rhKGF-2 group received aerosol inhalation of 1 mg/kg, 2 mg/kg, and 5 mg/kg rhKGF-2 (all dissolved in 5 mL PBS) once daily for 7 d, respectively. On treatment day 1, 3, 5, and 7, blood samples were taken from the ear central artery of 6 rabbits in each group. After the blood was taken, the rabbits were sacrificed, and the tracheal carina tissue and lung were collected. Blood pH value, arterial oxygen partial pressure (PaO(2)), arterial blood carbon dioxide pressure (PaCO(2)), and bicarbonate ion were detected by handheld blood analyzer. The expressions of pulmonary surfactant-associated protein A (SP-A) and vascular endothelial growth factor (VEGF) in lung tissue were detected by Western blotting. Pathomorphology of lung tissue and trachea was observed by hematoxylin-eosin staining. Data were processed with analysis of variance of two-way factorial design and Tukey test. Results: (1) Compared with those in simple injury group, the blood pH values of rabbits in the latter groups on treatment day 1-7 had no obvious change (P>0.05). The PaO(2) of rabbits in injury+2 mg/kg rhKGF-2 group on treatment day 5 and 7 were (75.0±2.4) and (71.0±4.5) mmHg (1 mmHg=0.133 kPa), respectively, which were significantly higher than (62.0±6.8) and (63.0±3.0) mmHg in simple injury group (q=4.265, 8.202, P<0.05 or P<0.01). The PaO(2) of rabbits in injury+5 mg/kg rhKGF-2 group on treatment day 7 was (82.0±4.9) mmHg, which was significantly higher than that in simple injury group (q=6.234, P<0.01). Compared with that in simple injury group, the PaCO(2) of rabbits in injury+2 mg/kg rhKGF-2 group on treatment day 3 was significantly decreased (q=4.876, P<0.01) and significantly increased on treatment day 5 (q=5.562, P<0.01); the PaCO(2) of rabbits in injury+5 mg/kg rhKGF-2 group was significantly increased on treatment day 5 and 7 (q=5.013, 4.601, P<0.05 or P<0.01). Compared with that in simple injury group, the serum bicarbonate ion of rabbits in injury+1 mg/kg rhKGF-2 group on treatment day 7 was significantly increased (q=5.142, P<0.01); the serum bicarbonate ion of rabbits in injury+2 mg/kg rhKGF-2 group on treatment day 5 and 7 were significantly increased (q=4.830, 6.934, P<0.01); the serum bicarbonate ion of rabbits in injury+5 mg/kg rhKGF-2 group on treatment day 5 were significantly increased (q=3.973, P<0.05). (2) The expressions of SP-A in lung tissue of rabbits in simple injury group and injury+PBS group in each treatment time point were close (P>0.05). The expressions of SP-A in lung tissue of rabbits in injury+2 mg/kg rhKGF-2 group and injury+5 mg/kg rhKGF-2 group on treatment day 3 were 0.091±0.007 and 0.101±0.009, respectively, significantly higher than 0.069±0.009 in simple injury group (q=10.800, 13.580, P<0.01). The expressions of SP-A in lung tissue of rabbits in injury+1 mg/kg rhKGF-2 group, injury+2 mg/kg rhKGF-2 group, and injury+5 mg/kg rhKGF-2 group on treatment day 5 and 7 were 0.127±0.008, 0.132±0.006, 0.194±0.006, 0.152±0.017, 0.166±0.004, 0.240±0.008, significantly higher than 0.092±0.003 and 0.108±0.005 in simple injury group (q=6.789, 12.340, 17.900, 9.875, 31.480, 40.740, P<0.01). (3) On treatment day 1 and 5, there was no significant difference in the expression of VEGF in lung tissue of rabbits among the 5 groups (P>0.05). Compared with those in simple injury group, the expressions of VEGF in lung tissue of rabbits in injury+2 mg/kg rhKGF-2 group on treatment day 3 and 7 were significantly increased (q=4.243, 8.000, P<0.05 or P<0.01), and the expression of VEGF in lung tissue of rabbits in injury+5 mg/kg rhKGF-2 group on treatment day 7 was significantly increased (q=20.720, P<0.01). (4) On treatment day 1, the injury of rabbits in each group was similar, with a large number of neutrophils infiltrated and abscess formed in the alveolar and interstitial tissue, thickened alveolar septum, some collapsed alveolar and atelectasis; large area of tracheal mucosa was degenerated and necrotic, with a large amount of inflammatory exudates blocking in the cavity. On treatment day 3, the inflammation of lung tissue and trachea in each group were improved, but the inflammation in simple injury group and injury+PBS group was also serious. On treatment day 5, the inflammation in lung tissue and trachea of rabbits in injury+2 mg/kg rhKGF-2 group and injury+5 mg/kg rhKGF-2 group were improved much obviously than those in the other groups. On treatment day 7, the inflammation in lung tissue of rabbits in injury+5 mg/kg rhKGF-2 group alleviated obviously than those in the other groups, most alveoli had no obvious exudative fluid, the alveolar cavity was intact and clear, the local alveolar dilated like a cyst, and the alveolar septum thinning; the improvement of inflammation of trachea was more obvious than the other groups, the tracheal mucosa tended to be more complete, and few neutrophils were infiltrated in the endotracheal cavity. Conclusions: Atomization inhalation of rhKGF-2 can improve the PaO(2) level of rabbits with severe smoke inhalation injury, reduce airway inflammation, increase the expression of SP-A and VEGF in lung tissue, thus promoting the repair of lung tissue.

Functional cloning of Src-like adapter protein-2 (SLAP-2), a novel inhibitor of antigen receptor signaling.

In an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expressed in hematopoietic cells. Overexpression of SLAP-2 in B and T cell lines specifically impaired antigen receptor-mediated signaling events, including CD69 surface marker upregulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homology domains, but lacks a tyrosine kinase domain. In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl. Deletion of the COOH terminus of SLAP-2 blocked function and abrogated its association with Cbl. Mutation of the putative myristoylation site of SLAP-2 compromised its inhibitory activity and impaired its localization to the membrane compartment. Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.

Impaired NFATc translocation and failure of Th2 development in Itk-deficient CD4+ T cells.

Naive Itk-deficient CD4+ T cells were unable to establish stable IL-4 production, even when primed in Th2-inducing conditions. In contrast, IFNgamma production was little affected. Failure to express IL-4 occurred even among cells that had gone through multiple cell divisions and was associated with a delay in the kinetics and magnitude of NFATc nuclear localization. IL-4 production was restored genetically by retroviral reconstitution of Itk or biochemically by augmenting the calcium flux with ionomycin. In vivo, Itk-deficient mice were unable to establish functional Th2 cells. Development of protective Th1 cells was unimpeded. These data define a nonredundant role for Itk in modulating signals from the TCR/CD28 pathways that are specific for the establishment of stable IL-4 but not IFNgamma expression.

Impaired cytokine signaling in mice lacking the IL-1 receptor-associated kinase.

Stimulation of the type 1 IL-1R (IL-1R1) and the IL-18R by their cognate ligands induces recruitment of the IL-1R-associated kinase (IRAK). Activation of IRAK leads in turn to nuclear translocation of NF-kappaB, which directs expression of innate and adaptive immune response genes. To study IRAK function in cytokine signaling, we generated cells and mice lacking the IRAK protein. IRAK-deficient fibroblasts show diminished activation of NF-kappaB when stimulated with IL-1. Immune effector cells without IRAK exhibit a defective IFN-gamma response to costimulation with IL-18. Furthermore, mice lacking the Irak gene demonstrate an attenuated response to injected IL-1. Deletion of Irak, however, does not affect the ability of mice to develop delayed-type hypersensitivity or clear infection with the intracellular parasite, Listeria monocytogenes. These results demonstrate that although IRAK participates in IL-1 and IL-18 signal transduction, residual cytokine responsiveness operates through an IRAK-independent pathway.

Requirement for Tec kinases Rlk and Itk in T cell receptor signaling and immunity.

T cell receptor (TCR) signaling requires activation of Zap-70 and Src family tyrosine kinases, but requirements for other tyrosine kinases are less clear. Combined deletion in mice of two Tec kinases, Rlk and Itk, caused marked defects in TCR responses including proliferation, cytokine production, and apoptosis in vitro and adaptive immune responses to Toxoplasma gondii in vivo. Molecular events immediately downstream from the TCR were intact in rlk-/-itk-/- cells, but intermediate events including inositol trisphosphate production, calcium mobilization, and mitogen-activated protein kinase activation were impaired, establishing Tec kinases as critical regulators of TCR signaling required for phospholipase C-gamma activation.

Antiviral immune responses in Itk-deficient mice.

Mice lacking Itk, a T-cell-specific protein tyrosine kinase, have reduced numbers of T cells and reduced responses to allogeneic major histocompatibility molecules. This study analyzed antiviral immune responses in mice deficient for Itk. Primary cytotoxic T-lymphocyte (CTL) responses were analyzed after infection with lymphocytic choriomeningitis virus (LCMV), vaccinia virus (VV), and vesicular stomatitis virus (VSV). Ex vivo CTL activity was consistently reduced by a factor of two to six for the different viruses. CTL responses after restimulation in vitro were similarly reduced unless exogenous cytokines were added. In the presence of interleukin-2 or concanavalin A supernatant, Itk-deficient and control mice responded similarly. Interestingly, while LCMV was completely eliminated by day 8 in both Itk-deficient and control mice, VV cleared from itk-/- mice with delayed kinetics. Antibody responses were evaluated after VSV infection. Both the T-cell-independent neutralizing immunoglobulin M (IgM) and the T-cell-dependent IgG responses were similar in Itk-deficient and control mice. Taken together, the results show that CTL responses are reduced in the absence of Itk whereas antiviral B-cell responses are not affected.

Itk negatively regulates induction of T cell proliferation by CD28 costimulation.

CD28 is a cell surface molecule that mediates a costimulatory signal crucial for T cell proliferation and lymphokine production. The signal transduction mechanisms of CD28 are not well understood. Itk, a nonreceptor protein tyrosine kinase specifically expressed in T cells and mast cells, has been implicated in the CD28 signaling pathway because of reports that it becomes phosphorylated on tyrosines and associates with CD28 upon cross-linking of the cell surface molecule. To determine whether Itk plays a functional role in CD28 signaling, we compared T cells from Itk-deficient mice and control mice for their responses to CD28 costimulation. T cells defective in Itk were found to be fully competent to respond to costimulation. Whereas the CD3-mediated proliferative response was severely compromised in the absence of Itk, the calcineurin-independent CD28-mediated response was significantly elevated when compared with cells from control animals. The augmented proliferation was not due to increased production of interleukin-2. The results suggest that Itk has distinct roles in the CD3 versus the CD28 signaling pathways. By negatively regulating the amplitude of signaling upon CD28 costimulation, Itk may provide a means for modulating the outcome of T cell activation during development and during antigen-driven immune responses.

Itk and Fyn make independent contributions to T cell activation.

Itk is a member of the Btk/Tec/Itk family of nonreceptor protein tyrosine kinases (PTKs), and has been implicated in T cell antigen receptor (TCR) signal transduction. Lck and Fyn are the Src-family nonreceptor PTKs that are involved in TCR signaling. To address the question of how these members of different families of PTKs functionally contribute to T cell development and to T cell activation, mice deficient for both Itk and either Lck or Fyn were generated. The Itk/Lck doubly deficient mice exhibited a phenotype similar to that of Lck-deficient mice. The phenotype of the Itk/Fyn doubly deficient mice was similar to that of Itk deficient mice. However the Itk/Fyn doubly deficient mice exhibited a more severe defect in TCR-induced proliferation of thymocytes and peripheral T cells than did mice deficient in either kinase alone. These data support the notion that Itk and Fyn both make independent contributions to TCR-induced T cell activation.

Altered T cell receptor signaling and disrupted T cell development in mice lacking Itk.

Itk is a T cell protein tyrosine kinase (PTK) that, along with Btk and Tec, belongs to a family of cytoplasmic PTKs with N-terminal pleckstrin homology domains. Btk plays a critical role in B lymphocyte development. To determine whether Itk has an analogous role in T lymphocytes, we used gene targeting to prepare mice lacking expression of Itk. Such animals had decreased numbers of mature thymocytes, an effect most clearly observed in mice expressing T cell receptor (TCR) transgenes. Mature T cells from Itk-deficient mice had reduced proliferative responses to allogeneic MHC stimulation and to anti-TCR cross-linking, but responded normally to stimulation with phorbol ester plus ionomycin or with IL-2. These results provide genetic evidence that Itk is involved in T cell development and also suggest that Itk has an important role in proximal events in TCR-mediated signaling pathways.

The yeast MUD2 protein: an interaction with PRP11 defines a bridge between commitment complexes and U2 snRNP addition.

In characterizing a series of yeast (Saccharomyces cerevisiae) mutants synthetic lethal with U1 RNA, we have identified a yeast gene (MUD2) with sequence similarity to the well-studied metazoan splicing factor U2AF65. The biochemical characterization indicates that the MUD2 gene product (MUD2P) contacts pre-mRNA directly and is a component of the pre-mRNA-U1 snRNP complex (commitment complex) that forms during early spliceosome assembly in yeast extracts. Unlike U1 snRNP itself, the association of MUD2P with pre-mRNA is dependent on a proper yeast branchpoint sequence. Genetic experiments indicate that MUD2P affects U2 snRNP addition. Moreover, experiments in the two-hybrid system show that PRP11P, a recently identified component of U2 snRNP, can interact directly with MUD2P. The experiments identify a specific inter-snRNP protein-protein contact that occurs during spliceosome assembly and more generally support substantial functional similarity between U2AF65 and MUD2P.

U1 small nuclear ribonucleoprotein particle-protein interactions are revealed in Saccharomyces cerevisiae by in vivo competition assays.

Two highly conserved regions of the 586-nucleotide yeast (Saccharomyces cerevisiae) U1 small nuclear RNA (snRNA) can be mutated or deleted with little or no effect on growth rate: the universally conserved loop II (corresponding to the metazoan A loop) and the yeast core region (X. Liao, L. Kretzner, B. Séraphin, and M. Rosbash, Genes Dev. 4:1766-1774, 1990). To examine the contribution of these regions to U1 small nuclear ribonucleoprotein particle (snRNP) activity, a competitor U1 gene, encoding a nonfunctional U1 snRNA molecule, was introduced into a number of strains carrying a U1 snRNA gene with loop II or yeast core mutations. The presence of the nonfunctional U1 gene lowered the growth rate of these mutant strains but not wild-type strains, consistent with the notion that mutant U1 RNAs are less active than wild-type U1 snRNAs. A detailed analysis of the U1 snRNA levels and half-lives in a number of merodiploid strains suggests that these mutant U1 snRNAs interact with U1 snRNP proteins less well than do their wild-type counterparts. Competition for protein factors during snRNP assembly could account for a number of previous observations in both yeast and mammalian cells.

An enhancer screen identifies a gene that encodes the yeast U1 snRNP A protein: implications for snRNP protein function in pre-mRNA splicing.

In an enhancer screen for yeast mutants that may interact with U1 small nuclear RNA (snRNA), we identified a gene that encodes the apparent yeast homolog of the well-studied human U1A protein. Both in vitro and in vivo, the absence of the protein has a dramatic effect on the activity of U1 snRNP containing the mutant U1 snRNA used in the screen. Surprisingly, the U1A gene is inessential in a wild-type U1 RNA background, as growth rate and the splicing of endogenous pre-mRNA transcripts are normal in these strains that lack the U1A protein. Even in vitro, the absence of the protein has little effect on splicing. On the basis of these observations, we suggest that a principal role of the U1A protein is to help fold or maintain U1 RNA in an active configuration.

Requirements for U2 snRNP addition to yeast pre-mRNA.

The in vitro spliceosome assembly pathway is conserved between yeast and mammals as U1 and U2 snRNPs associate with the pre-mRNA prior to U5 and U4/U6 snRNPs. In yeast, U1 snRNP-pre-mRNA complexes are the first splicing complexes visualized on native gels, and association with U1 snRNP apparently commits pre-mRNA to the spliceosome assembly pathway. The current study addresses U2 snRNP addition to commitment complexes. We show that commitment complex formation is relatively slow and does not require ATP, whereas U2 snRNP adds to the U1 snRNP complexes in a reaction that is relatively fast and requires ATP or hydrolyzable ATP analogs. In vitro spliceosome assembly was assayed in extracts derived from strains containing several U1 sRNA mutations. The results were consistent with a critical role for U1 snRNP in early complex formation. A mutation that disrupts the base-pairing between the 5' end of U1 snRNA and the 5' splice site allows some U2 snRNP addition to bypass the ATP requirement, suggesting that ATP may be used to destabilize certain U1 snRNP:pre-mRNA interactions to allow subsequent U2 snRNP addition.