Experience in the perioperative management of patients with hip fracture in southwestern Colombia. Retrospective cohort study

Introduction Hip fracture is one of the main causes of morbidity and mortality among the elderly population. In Colombia there is a shortage of scientific literature on the perioperative management of this population of patients. Objective To describe the perioperative management of hip fracture patients at a tertiary university hospital in Cali, Colombia. Methods An observational study was conducted with relational scope of a historical cohort of patients with hip fracture who underwent surgical management between January 2018 and June 2022. A non-probability sampling method was used and contingency tables were designed aimed at describing the relationship between the patients' characteristics and the postoperative outcomes. Results 235 patients were included, of which 57 % were males. The mean age was 79 years and 49.8 % were classified as ASA III or higher. Spinal anesthesia was the most commonly used technique in 63.4 % of the cases. The most frequent outcomes were delirium in 17.9 %, and acute kidney failure in 6.8 %. 83.4 % of the patients underwent surgery within 48 hours of admission to the emergency department and intra-hospital mortality was 2.6 %. Conclusions The results of this study provide relevant information to identify opportunities for improvement and their implementation, such as the reduction in the time elapsed until surgical management and the development of care protocols in the region.

Introducción: La fractura de cadera es una de las principales causas de morbimortalidad en la población adulta mayor. En Colombia hay un déficit en la literatura científica acerca del manejo perioperatorio de esta población de pacientes. Objetivo: Describir el manejo perioperatorio de pacientes con fractura de cadera en un hospital universitario de alto nivel de complejidad de Cali, Colombia. Métodos: Se realizó un estudio observacional con alcance relacional de una cohorte histórica de pacientes con fractura de cadera, que recibieron manejo quirúrgico entre enero de 2018 y junio de 2022. Se hizo un muestreo no probabilístico y se diseñaron tablas de contingencia con el propósito de describir relaciones entre las características y los desenlaces posoperatorios. Resultados: Se incluyeron 235 pacientes de los cuales el 57 % fueron hombres. La mediana de edad fue de 79 años y el 49,8 % tenían una clasificación ASA III o mayor. La anestesia espinal fue la técnica más utilizada en el 63,4 %. Los desenlaces más frecuentes fueron el delirio en el 17,9 %, y la falla renal aguda en el 6,8 %. El 83,4 % de los pacientes fueron intervenidos quirúrgicamente antes de las 48 horas desde el ingreso a urgencias y la mortalidad intrahospitalaria fue del 2,6 %. Conclusiones: Los resultados de este estudio brindan información relevante que permite la implementación de oportunidades de mejora como disminución en los tiempos hasta el manejo quirúrgico y el desarrollo de protocolos de atención en la región.

Postoperative Outcomes of Analgesic Management with Erector Spine Plane Block at T5 Level in Pediatric Patients Undergoing Cardiac Surgery with Sternotomy: A Cohort Study.

Introduction: There is limited evidence on the impact of erector spinae plane block (ESPB) as part of multimodal analgesia in pediatric population undergoing cardiac surgery. Methods: A retrospective cohort study was conducted in patients under 18 years of age, who underwent cardiac surgery Risk Adjusted classification for Congenital Heart Surgery (RACHS-1) ≤3 by sternotomy. The study aims to evaluate the effect of ESPB as part of multimodal analgesia in pediatric patients undergoing cardiac surgery compared to conventional analgesia (CA) on relevant clinical outcomes: length of hospital stay, length of ICU stay, opioid consumption, time to extubation, mortality, and postoperative complications. The participants included were treated in a reference hospital in Colombia from July 2019 to June 2022. Results: Eighty participants were included, 40 in the ESPB group and 40 in the CA group. There was a significant decrease (Log rank test p = 0.007) in days to length of hospital stay in ESPB group (median 6.5 days (IQR: 4-11)) compared to the CA group (median 10.5 days (IQR: 6-25)). Likewise, there was a higher probability of discharge from the ICU in the ESPB group (HR 1.71 (95% CI: 1.05-2.79)). The ESPB group had lower opioid consumption (p < 0.05). There were no differences in time to extubation, mortality, and postoperative complications. Conclusion: ESPB as part of multimodal analgesia in pediatric patients undergoing cardiac surgery is feasible and associated with shorter hospital length of stay, faster ICU discharge and lower opioid consumption.

Frequency and function of circulating regulatory T-cells in biliary atresia.

PURPOSE: Although the impairment of regulatory T-cells (Tregs) has been shown in the liver or portal area of biliary atresia (BA) the frequency and function of circulating Tregs in BA patients is poorly understood. We aimed to investigate the frequency and function of circulating Tregs in BA patients. METHODS: Peripheral blood mononuclear cells were collected from 25 BA patients and 24 controls. Treg frequency was measured by flow cytometry; function was determined by T-cell proliferation assay. We also assessed the association between Treg frequency/function and clinical parameters in BA cases. RESULTS: There was no significant difference between the two groups in both frequency (BA: 3.4%; control: 3.2%; p = 0.97) and function (BA: 22.0%; control: 7.5%; p = 0.23) of Tregs. We further focused on 13 preoperative BA patients and 14 age-matched controls. Neither Treg frequency nor function were significantly different (frequency: BA: 4.6%; control: 3.4%; p = 0.38, function: BA: 2.7%; control: 7.6%; p = 0.89). There was no association between Treg frequency/function and clinical parameters. CONCLUSION: Neither the frequency nor function of circulating Tregs was affected in BA patients, suggesting the negative role of circulating Tregs in the pathogenesis of BA. Further investigation of local Treg profiles is warranted.

Safety of neuraxial anesthesia in patients twin pregnancy and twin-to-twin transfusion syndrome taken to laser photocoagulation. Retrospective cohort study / Seguridad de la anestesia neuroaxial en mujeres con embarazo gemelar y síndrome de transfusión feto-fetal, sometidas a fotocoagulación láser. Estudio de cohorte retrospectiva

Objective: To describe the clinical characteristics and the frequency of maternal, fetal and neonatal complications in accordance with the neuraxial anesthesia (NA) technique in women with twin-totwin transfusion syndrome (TTTS) treated with laser photocoagulation. Materials and Methods: Descriptive retrospective cohort study of pregnant patients with TTTS treated with laser photocoagulation under NA at Fundación Valle del Lili, Cali (Colombia), between 2013-2017. Patients with Quintero stage V TTTS were excluded. The protocol was approved by the institutional ethics committee. Results: Of the participants, 32 met the inclusion and exclusion criteria. The study population consisted of young, multiparous women. Urgent interventions were performed in 87.5% of cases; 43.7% were Quintero stage III and epidural anesthesia was used in 56.2% of the women. Maternal hemodynamic variables were similar, in accordance with the timing of surgery and the neuraxial technique used. Sustained hypotension occurred in 65.6% of the pregnant women and 9.3% developed pulmonary edema. Pre-term delivery occurred in 65.6% of the patients and 18.7% had premature rupture of membranes. There were 14 fetal demises and five neonatal deaths. There were no cases of maternal mortality. Conclusions: In patients with TTTS requiring laser photocoagulation, the use of epidural, spinal or combined anesthesia is likely associated with similar maternal hemodynamic variables at the time of surgery. Practitioners providing care to these pregnant women must be aware of the frequent occurrence of maternal, fetal and neonatal complications. Prospective studies to assess the safety and effectiveness of the different neuraxial anesthesia techniques in patients with TTTS are required.

Objetivo: describir las características clínicas y la frecuencia de complicaciones maternas, fetales y neonatales, según técnica de anestesia neuroaxial (AN) en mujeres con síndrome de transfusión feto-fetal (STFF) tratadas con fotocoagulación láser (FL). Materiales y métodos: estudio de cohorte retrospectiva descriptivo. Se incluyeron gestantes con STFF tratadas con FL y AN en la Fundación Valle del Lili, Cali (Colombia) entre 2013-2017. Se excluyeron pacientes con STFF estadio-V de Quintero. Se usó estadística descriptiva. El protocolo fue aprobado por el Comité de Ética de la institución. Resultados: 32 participantes cumplieron con los criterios de inclusión y de exclusión. La población estuvo constituida por mujeres jóvenes, multíparas. En el 87,5% de los casos se realizó intervención de urgencia. El 43,7% presentaba el estadio-III de Quintero y en el 56,2 % de las gestantes se utilizó anestesia epidural. Las variables hemodinámicas maternas exhibieron un comportamiento similar, acorde al momento de la cirugía y la técnica neuoraxial implementada. El 65,6 % de las gestantes presentó hipotensión sostenida y el 9,3 % desarrolló edema pulmonar. El 65,6 % de las pacientes experimentó parto pretérmino y el 18,7 % ruptura prematura de membranas. Se registraron 14 muertes fetales y cinco neonatales. No se registraron casos de mortalidad materna. Conclusiones: en pacientes con STFF que requieren FL, el uso de la anestesia epidural, espinal o combinada probablemente se asocia con un comportamiento similar al de las variables hemodinámicas maternas, durante los momentos de la cirugía. Los profesionales que brindan atención a estas gestantes deben estar alerta ante la frecuente aparición de complicaciones maternas, fetales y neonatales. Se requieren estudios prospectivos que evalúen la seguridad y la efectividad de las diferentes técnicas de anestesia neuroaxial en pacientes con STFF.

Seguridad de la anestesia neuroaxial en mujeres con embarazo gemelar y síndrome de transfusión feto-fetal, sometidas a fotocoagulación láser. Estudio de cohorte retrospectiva / Safety of neuraxial anesthesia in patients twin pregnancy and twin-to-twin transfusion syndrome taken to laser photocoagulation. Retrospective cohort study

Objetivo: describir las características clínicas y la frecuencia de complicaciones maternas, fetales y neonatales, según técnica de anestesia neuroaxial (AN) en mujeres con síndrome de transfusión feto-fetal (STFF) tratadas con fotocoagulación láser (FL). Materiales y métodos: estudio de cohorte retrospectiva descriptivo. Se incluyeron gestantes con STFF tratadas con FL y AN en la Fundación Valle del Lili, Cali (Colombia) entre 2013-2017. Se excluyeron pacientes con STFF estadio-V de Quintero. Se usó estadística descriptiva. El protocolo fue aprobado por el Comité de Ética de la institución. Resultados: 32 participantes cumplieron con los criterios de inclusión y de exclusión. La población estuvo constituida por mujeres jóvenes, multíparas. En el 87,5% de los casos se realizó intervención de urgencia. El 43,7% presentaba el estadio-III de Quintero y en el 56,2 % de las gestantes se utilizó anestesia epidural. Las variables hemodinámicas maternas exhibieron un comportamiento similar, acorde al momento de la cirugía y la técnica neuoraxial implementada. El 65,6 % de las gestantes presentó hipotensión sostenida y el 9,3 % desarrolló edema pulmonar. El 65,6 % de las pacientes experimentó parto pretérmino y el 18,7 % ruptura prematura de membranas. Se registraron 14 muertes fetales y cinco neonatales. No se registraron casos de mortalidad materna. Conclusiones: en pacientes con STFF que requieren FL, el uso de la anestesia epidural, espinal o combinada probablemente se asocia con un comportamiento similar al de las variables hemodinámicas maternas, durante los momentos de la cirugía. Los profesionales que brindan atención a estas gestantes deben estar alerta ante la frecuente aparición de complicaciones maternas, fetales y neonatales. Se requieren estudios prospectivos que evalúen la seguridad y la efectividad de las diferentes técnicas de anestesia neuroaxial en pacientes con STFF.

Objective: To describe the clinical characteristics and the frequency of maternal, fetal and neonatal complications in accordance with the neuraxial anesthesia (NA) technique in women with twin-to- twin transfusion syndrome (TTTS) treated with laser photocoagulation. Materials and Methods: Descriptive retrospective cohort study of pregnant patients with TTTS treated with laser photocoagulation under NA at Fundación Valle del Lili, Cali (Colombia), between 2013-2017. Patients with Quintero stage VTTTS were excluded. The protocol was approved by the institutional ethics committee. Results: Of the participants, 32 met the inclusion and exclusion criteria. The study population consisted of young, multiparous women. Urgent interventions were performed in 87.5% of cases; 43.7% were Quintero stage III and epidural anesthesia was used in 56.2% of the women. Maternal hemodynamic variables were similar, in accordance with the timing of surgery and the neuraxial technique used. Sustained hypotension occurred in 65.6% of the pregnant women and 9.3% developed pulmonary edema. Pre-term delivery occurred in 65.6% of the patients and 18.7% had premature rupture of membranes. There were 14 fetal demises and five neonatal deaths. There were no cases of maternal mortality. Conclusions: In patients with TTTS requiring laser photocoagulation, the use of epidural, spinal or combined anesthesia is likely associated with similar maternal hemodynamic variables at the time of surgery. Practitioners providing care to these pregnant women must be aware of the frequent occurrence of maternal, fetal and neonatal complications. Prospective studies to assess the safety and effectiveness of the different neuraxial anesthesia techniques in patients with TTTS are required.

Genetic background-dependent abnormalities of the enteric nervous system and intestinal function in Kif26a-deficient mice.

The Kif26a protein-coding gene has been identified as a negative regulator of the GDNF-Ret signaling pathway in enteric neurons. The aim of this study was to investigate the influence of genetic background on the phenotype of Kif26a-deficient (KO, -/-) mice. KO mice with both C57BL/6 and BALB/c genetic backgrounds were established. Survival rates and megacolon development were compared between these two strains of KO mice. Functional bowel assessments and enteric neuron histopathology were performed in the deficient mice. KO mice with the BALB/c genetic background survived more than 400 days without evidence of megacolon, while all C57BL/6 KO mice developed megacolon and died within 30 days. Local enteric neuron hyperplasia in the colon and functional bowel abnormalities were observed in BALB/c KO mice. These results indicated that megacolon and enteric neuron hyperplasia in KO mice are influenced by the genetic background. BALB/c KO mice may represent a viable model for functional gastrointestinal diseases such as chronic constipation, facilitating studies on the underlying mechanisms and providing a foundation for the development of treatments.

Establishment and analysis of a mouse model that regulates sex-related differences in liver drug metabolism.

The adult liver performs many metabolic functions for maintaining homeostasis. There are several sex differences in liver function and disease pathogenesis. One important function of the liver is drug metabolism, where cytochrome p450s (CYPs) in hepatocytes are the main enzymes involved. The toxicity of various drugs and chemicals differs with sex due to differences in hepatocytic CYP expression. However, the molecular mechanism regulating sex-related differences in drug metabolism remains unknown. In this study, we identified transcriptional regulator B-cell lymphoma 6 (Bcl6) as an important factor in sex-biased differential CYP expression. Microarray analysis of livers derived from liver-specific Bcl6-knockout mice showed that Bcl6 is required for sex-biased CYP expression patterns in the liver. Additionally, quantitative PCR analysis revealed that hepatocytic expression of male-biased genes, such as Cyp2d9, Cyp2u1, Cyp4a12a/12b, and Cyp7b1, in liver-specific Bcl6-knockout male mice significantly decreased to levels similar to those observed in wild-type female mice. Conversely, hepatocytic expression of female-biased genes, such as Cyp2a4/2a5, Cyp2b9, Cyp3a41, and Cyp17a1, significantly increased in liver-specific Bcl6-knockout male mice. Deletion of Bcl6 caused female-like expression of CYPs in male livers. These results suggest that Bcl6 is a key regulator of sex-related differential regulation of drug metabolism. Moreover, serum sex hormone levels and fertility did not change in liver-specific, Bcl6-knockout mice. Hepatocytic Bcl6 regulates sex-related differential CYP expression in the liver without changing the sex of the whole body. Thus, this mouse model is useful for analyzing liver-specific sex-dependent regulation of drug metabolism and pathogenesis.

Allergic TH2 Response Governed by B-Cell Lymphoma 6 Function in Naturally Occurring Memory Phenotype CD4+ T Cells.

Transcriptional repressor B-cell lymphoma 6 (Bcl6) appears to regulate TH2 immune responses in allergies, but its precise role is unclear. We previously reported that Bcl6 suppressed IL-4 production in naïve CD4+ T cell-derived memory TH2 cells. To investigate Bcl6 function in allergic responses in naturally occurring memory phenotype CD4+ T (MPT) cells and their derived TH2 (MPTH2) cells, Bcl6-manipulated mice, highly conserved intron enhancer (hcIE)-deficient mice, and reporter mice for conserved noncoding sequence 2 (CNS2) 3' distal enhancer region were used to elucidate Bcl6 function in MPT cells. The molecular mechanisms of Bcl6-mediated TH2 cytokine gene regulation were elucidated using cellular and molecular approaches. Bcl6 function in MPT cells was determined using adoptive transfer to naïve mice, which were assessed for allergic airway inflammation. Bcl6 suppressed IL-4 production in MPT and MPTH2 cells by suppressing CNS2 enhancer activity. Bcl6 downregulated Il4 expression in MPTH2 cells, but not MPT cells, by suppressing hcIE activity. The inhibitory functions of Bcl6 in MPT and MPTH2 cells attenuated allergic responses. Bcl6 is a critical regulator of IL-4 production by MPT and MPTH2 cells in TH2 immune responses related to the pathogenesis of allergies.

Development of chronic allergic responses by dampening Bcl6-mediated suppressor activity in memory T helper 2 cells.

Mice deficient in the transcriptional repressor B-cell CLL/lymphoma 6 (Bcl6) exhibit similar T helper 2 (TH2) immune responses as patients with allergic diseases. However, the molecular mechanisms underlying Bcl6-directed regulation of TH2 cytokine genes remain unclear. We identified multiple Bcl6/STAT binding sites (BSs) in TH2 cytokine gene loci. We found that Bcl6 is modestly associated with the BSs, and it had no significant effect on cytokine production in newly differentiated TH2 cells. Contrarily, in memory TH2 (mTH2) cells derived from adaptively transferred TH2 effectors, Bcl6 outcompeted STAT5 for binding to TH2 cytokine gene loci, particularly Interleukin4 (Il4) loci, and attenuated GATA binding protein 3 (GATA3) binding to highly conserved intron enhancer regions in mTH2 cells. Bcl6 suppressed cytokine production epigenetically in mTH2 cells to negatively tune histone acetylation at TH2 cytokine gene loci, including Il4 loci. In addition, IL-33, a pro-TH2 cytokine, diminished Bcl6's association with loci to which GATA3 recruitment was inversely augmented, resulting in altered IL-4, but not IL-5 and IL-13, production in mTH2 cells but no altered production in newly differentiated TH2 cells. Use of a murine asthma model that generates high levels of pro-TH2 cytokines, such as IL-33, suggested that the suppressive function of Bcl6 in mTH2 cells is abolished in severe asthma. These findings indicate a role of the interaction between TH2-promoting factors and Bcl6 in promoting appropriate IL-4 production in mTH2 cells and suggest that chronic allergic diseases involve the TH2-promoting factor-mediated functional breakdown of Bcl6, resulting in allergy exacerbation.

Systemic and Local Cytokine Profile in Biliary Atresia.

Purpose Systemic and local immune environments in human biliary atresia (BA) were analyzed. Methods Plasma concentrations of 19 inflammatory components in 16 preoperative BA patients (median age, 51 days), 13 normal controls (NCs) (44 days), and 15 cholestatic controls (CC) (23 days) were measured using flow cytometry, and compared according to post-Kasai outcomes in BA patients. Hepatic mRNA levels of representative helper T (Th) cell cytokines and forkhead box protein 3 (FoxP3) quantified by real-time reverse transcription polymerase chain reaction were compared between BA and non-BA. Results No significant differences were observed between BA and control in serum Th1, Th2, or macrophage markers, while soluble cellular adhesion molecule (CAM) levels were significantly higher in BA (p < 0.0001 for NC; p = 0.0003 for CC). No statistically significant difference was discovered between BA with favorable and unfavorable prognosis. Hepatic mRNA levels of interferon-gamma or interleukin-4 showed no significant differences between BA and non-BA, while FoxP3 was significantly higher in BA (p = 0.01). Conclusions A skewed bias toward specific Th-oriented immunity was not demonstrated in either the systemic or local environment in the early stage of human BA, with patient prognoses not necessarily revealed by preoperative plasma inflammatory component levels. CAM and regulatory T cell roles and functions warrant further investigation.

Induced Expression of Cancer Stem Cell Markers ALDH1A3 and Sox-2 in Hierarchical Reconstitution of Apoptosis-resistant Human Breast Cancer Cells.

We established an experimental system that can induce p53-dependent apoptosis by doxycycline treatment to analyze characteristics of the apoptosis-resistant cancer cell subpopulation in the human breast cancer cell line HCC1937. Expression patterns of the stem cell markers, ALDH1A3 and Sox-2, the luminal differentiation marker, GATA3 and the proliferation index marker, Ki-67 were analyzed using immunostaining and fluorescence-activated cell sorting (FACS). After doxycycline treatment, the number of viable cells was gradually decreased over seven days in a time-dependent manner due to p53-induced apoptosis; however, the number of smaller-sized ALDH1A3+ cells assessed by immunostaining increased sharply after 1 day of doxycycline treatment, suggesting their apoptosis-resistant nature. The expression of ALDH1A3 was also detected in 78% of small-sized Ki-67+ proliferating progenitor cells, followed by the transient expression of GATA3, which presumably indicated the ability to differentiate into luminal progenitor cells. Although 42.2-58.5% of residual cells were positive for both ALDH1A3 and GATA3, their expression patterns exhibited an inverse correlation. The expression pattern of another stem cell marker, Sox-2, was similar, but more drastically altered after p53 induction compared with ALDH1A3. These findings may aid in understanding the hierarchical responses of cancer stem cells to therapeutic stresses.

Role of Bcl-3 in the development of follicular helper T cells and in the pathogenesis of rheumatoid arthritis.

OBJECTIVE: We have previously shown that expression of the Bcl-3 gene, a member of the IκB family, is down-regulated in CD4+ T cells from patients with rheumatoid arthritis (RA) following tocilizumab therapy. The objective of this study was to examine the role of Bcl-3 in the pathogenesis of RA. METHODS: DNA microarray analysis was used to compare the signal intensity of Bcl-3 in CD4+ T cells from untreated RA patients and healthy controls. We examined the roles of interleukin-6 (IL-6)/STAT-3 signaling in the induction of Bcl-3. In addition, we analyzed the gene expression profiles of Bcl-3-transduced CD4+ T cells by RNA sequencing. The effects of enforced expression as well as gene silencing of Bcl-3 on the development of follicular helper T (Tfh) cells were evaluated. Finally, we examined correlations between the signal intensities of Bcl-3 and Tfh cell-related genes in CD4+ T cells from untreated RA patients. RESULTS: Bcl-3 levels were significantly higher in RA patients than in healthy controls. IL-6 induced Bcl-3 expression in CD4+ T cells in a STAT-3-dependent manner. Transcriptome analysis revealed that the expression of Bcl-6, a master regulator of Tfh cell differentiation, was significantly up-regulated by the enforced Bcl-3 expression. The enforced Bcl-3 expression increased, but Bcl-3 silencing decreased, the numbers of IL-21-producing Tfh-like cells. Bcl-3 levels in CD4+ T cells from RA patients correlated positively with the levels of Tfh cell-related genes CXCR5, inducible costimulator, and achaete-scute homolog 2. CONCLUSION: Bcl-3 is involved in the development of Tfh cells and the pathogenesis of RA, presumably by inducing IL-21 production.

DA-Raf-dependent inhibition of the Ras-ERK signaling pathway in type 2 alveolar epithelial cells controls alveolar formation.

Alveolar formation is coupled to the spatiotemporally regulated differentiation of alveolar myofibroblasts (AMYFs), which contribute to the morphological changes of interalveolar walls. Although the Ras-ERK signaling pathway is one of the key regulators for alveolar formation in developing lungs, the intrinsic molecular and cellular mechanisms underlying its role remain largely unknown. By analyzing the Ras-ERK signaling pathway during postnatal development of lungs, we have identified a critical role of DA-Raf1 (DA-Raf)-a dominant-negative antagonist for the Ras-ERK signaling pathway-in alveolar formation. DA-Raf-deficient mice displayed alveolar dysgenesis as a result of the blockade of AMYF differentiation. DA-Raf is predominantly expressed in type 2 alveolar epithelial cells (AEC2s) in developing lungs, and DA-Raf-dependent MEK1/2 inhibition in AEC2s suppresses expression of tissue inhibitor of matalloprotienase 4 (TIMP4), which prevents a subsequent proteolytic cascade matrix metalloproteinase (MMP)14-MMP2. Furthermore, MMP14-MMP2 proteolytic cascade regulates AMYF differentiation and alveolar formation. Therefore, DA-Raf-dependent inhibition of the Ras-ERK signaling pathway in AEC2s is required for alveolar formation via triggering MMP2 activation followed by AMYF differentiation. These findings reveal a pivotal role of the Ras-ERK signaling pathway in the dynamic regulation of alveolar development.

Lack of both α2-antiplasmin and plasminogen activator inhibitor type-1 induces high IgE production.

AIMS: We investigated the pathophysiological changes in mice lacking α2-antiplasmin (α2-AP) and plasminogen activator inhibitor type-1 (PAI-1) genes, and elucidated the involvement of these inhibitors for fibrinolysis in immune response. MAIN METHODS: The pathophysiological changes induced by a lack of both α2-AP and PAI-1 were investigated using double knockout (KO) mice. The lung, liver, kidney and spleen tissues from α2-AP/PAI-1-double KO mice were compared with those from wild-type (WT) mice. Furthermore, the bone marrow cells from α2-AP/PAI-1-double KO mice were transplanted into 10-Gy X ray irradiated WT mice, and then the effects of the transplantation were studied. KEY FINDINGS: Plasma IgE levels in the α2-AP/PAI-1-double KO mice increased with age and exceeded 1000 ng/mL after 6 months of age. The plasma cells that produced IgE were detected in perivascular assembled lymphocytes. In the α2-AP/PAI-1-double KO mice, perivascular lymphocyte infiltration was observed in the lung, liver, and kidneys and peribronchial lymphocyte infiltration was present in the lung. When the bone marrow cells from α2-AP/PAI-1-double KO mice were transplanted into 10-Gy X ray irradiated WT mice, the phenotypes of the recipients were similar to those of α2-AP/PAI-1-double KO mice. SIGNIFICANCE: The simultaneous expression of both the α2-AP and PAI-1 genes contributes to the maintenance of immunological functions that are related to IgE. Moreover, it is suggested that both α2-AP and PAI-1 are involved in the recruitment of lymphocytes in the peripheral tissues.

ADAR1 protein induces adenosine-targeted DNA mutations in senescent Bcl6 gene-deficient cells.

Somatic mutations accumulate in senescent cells. Bcl6, which functions as a transcriptional repressor, has been identified as a potent inhibitor of cell senescence, but a role of Bcl6 in the accumulation of somatic mutations has remained unclear. Ig class-switch recombination simultaneously induces somatic mutations in an IgM class-switch (Ig-Sµ) region of IgG B cells. Surprisingly, mutations were detected in the Ig-Sµ region of Bcl6-deficient IgM B cells without class-switch recombination, and these mutations were mainly generated by conversion of adenosine to guanosine, suggesting a novel DNA mutator in the B cells. The ADAR1 (adenosine deaminase acting on RNA1) gene was overexpressed in Bcl6-deficient cells, and its promoter analysis revealed that ADAR1 is a molecular target of Bcl6. Exogenous ADAR1 induced adenosine-targeted DNA mutations in IgM B cells from ADAR1-transgenic mice and in wild-type mouse embryonic fibroblasts (MEFs). These mutations accumulated in senescent MEFs accompanied with endogenous ADAR1 expression, and the frequency in senescent Bcl6-deficient MEFs was higher than senescent wild-type MEFs. Thus, Bcl6 protects senescent cells from accumulation of adenosine-targeted DNA mutations induced by ADAR1.

CD40 amplifies Fas-mediated apoptosis: a mechanism contributing to emphysema.

Excessive apoptosis and prolonged inflammation of alveolar cells are associated with the pathogenesis of pulmonary emphysema. We aimed to determine whether CD40 affects alveolar epithelial cells and endothelial cells, with regard to evoking apoptosis and inflammation. Mice were repeatedly treated with agonistic-anti CD40 antibody (Ab), with or without agonistic-anti Fas Ab, and evaluated for apoptosis and inflammation in lungs. Human pulmonary microvascular endothelial cells and alveolar epithelial cells were treated with agonistic anti-CD40 Ab and/or anti-Fas Ab to see their direct effect on apoptosis and secretion of proinflammatory molecules in vitro. Furthermore, plasma soluble CD40 ligand (sCD40L) level was evaluated in patients with chronic obstructive pulmonary disease (COPD). In mice, inhaling agonistic anti-CD40 Ab induced moderate alveolar enlargement. CD40 stimulation, in combination with anti-Fas Ab, induced significant emphysematous changes and increased alveolar cell apoptosis. CD40 stimulation also enhanced IFN-γ-mediated emphysematous changes, not via apoptosis induction, but via inflammation with lymphocyte accumulation. In vitro, Fas-mediated apoptosis was enhanced by CD40 stimulation and IFN-γ in endothelial cells and by CD40 stimulation in epithelial cells. CD40 stimulation induced secretion of CCR5 ligands in endothelial cells, enhanced with IFN-γ. Plasma sCD40L levels were significantly increased in patients with COPD, inversely correlating to the percentage of forced expiratory volume in 1 s and positively correlating to low attenuation area score by CT scan, regardless of smoking history. Collectively CD40 plays a contributing role in the development of pulmonary emphysema by sensitizing Fas-mediated apoptosis in alveolar cells and increasing the secretion of proinflammatory chemokines.

CXCR4 expression on activated B cells is downregulated by CD63 and IL-21.

CXCR4 expression is critical for localization of centroblasts in the dark zone of germinal centers (GCs), and centrocytes downregulate CXCR4 and thus leave the dark zone to reside in the light zone. However, mechanisms governing CXCR4 downregulation on centrocytes are not known. In this study, we show that the amount of intracellular CXCR4 in centroblasts was similar to that in centrocytes, suggesting differential control of CXCR4 protein expression in these GC B cells. Restimulation of activated B cells with IL-21, which is a major cytokine produced by T follicular helper cells, accelerated CXCR4 internalization by inducing endocytosis-related GRK6 expression. Although CXCR4 expression was downregulated on GC B cells by IL-21 stimulation, CXCR4(low) centrocytes developed in the spleens of IL-21R-deficient mice, suggesting other mechanisms for downregulation. The level of CD63 (which recruits CXCR4 to late endosome in CD4 T cells) in centrocytes was more than that in centroblasts and was strikingly elevated in activated Bcl6-deficient B cells. Bcl6, a transcriptional repressor, was detected on the chromatin of the CD63 gene in resting B cells, therefore CD63 is a molecular target of Bcl6. Downregulation of CD63 mRNA in activated Bcl6-deficient B cells by small interfering RNA upregulated CXCR4 expression on the B cells. Furthermore, addition of Bcl6 inhibitor to activated B cell cultures increased CD63 mRNA expression in (and downregulated CXCR4 expression on) those activated B cells. Thus, CXCR4 can be downregulated on activated B cells by IL-21-induced endocytosis and CD63-mediated endosomal recruitment, and these mechanisms may contribute to downregulation of CXCR4 on centrocytes.

Bcl6 is required for the development of mouse CD4+ and CD8α+ dendritic cells.

Th2-type inflammation spontaneously shown in Bcl6-knockout (KO) mice is mainly caused by bone marrow (BM)-derived nonlymphoid cells. However, the function of dendritic cells (DCs) in Bcl6-KO mice has not been reported. We show in this article that the numbers of CD4(+) conventional DCs (cDCs) and CD8α(+) cDCs, but not of plasmacytoid DCs, were markedly reduced in the spleen of Bcl6-KO mice. Generation of cDCs from DC progenitors in BM cells was perturbed in the spleen of irradiated wild-type (WT) mice transferred with Bcl6-KO BM cells, indicating an intrinsic effect of Bcl6 in cDC precursors. Although cDC precursors were developed in a Bcl6-KO BM culture with Fms-like tyrosine kinase 3 ligand, the cDC precursors were more apoptotic than WT ones. Also p53, one of the molecular targets of Bcl6, was overexpressed in the precursors. The addition of a p53 inhibitor to Bcl6-KO BM culture protected apoptosis, suggesting that Bcl6 is required by cDC precursors for survival by controlling p53 expression. Furthermore, large numbers of T1/ST2(+) Th2 cells were naturally developed in the spleen of Bcl6-KO mice. Th2 skewing was accelerated in the culture of WT CD4 T cells stimulated with Ags and LPS-activated Bcl6-KO BM-derived DCs, which produced more IL-6 and less IL-12 than did WT DCs; the addition of anti-IL-6 Abs to the culture partially abrogated the Th2 skewing. These results suggest that Bcl6 is required in cDC precursors for survival and in activated DCs for modulating the cytokine profile.

The Blimp1-Bcl6 axis is critical to regulate osteoclast differentiation and bone homeostasis.

Controlling osteoclastogenesis is critical to maintain physiological bone homeostasis and prevent skeletal disorders. Although signaling activating nuclear factor of activated T cells 1 (NFATc1), a transcription factor essential for osteoclastogenesis, has been intensively investigated, factors antagonistic to NFATc1 in osteoclasts have not been characterized. Here, we describe a novel pathway that maintains bone homeostasis via two transcriptional repressors, B cell lymphoma 6 (Bcl6) and B lymphocyte-induced maturation protein-1 (Blimp1). We show that Bcl6 directly targets 'osteoclastic' molecules such as NFATc1, cathepsin K, and dendritic cell-specific transmembrane protein (DC-STAMP), all of which are targets of NFATc1. Bcl6-overexpression inhibited osteoclastogenesis in vitro, whereas Bcl6-deficient mice showed accelerated osteoclast differentiation and severe osteoporosis. We report that Bcl6 is a direct target of Blimp1 and that mice lacking Blimp1 in osteoclasts exhibit osteopetrosis caused by impaired osteoclastogenesis resulting from Bcl6 up-regulation. Indeed, mice doubly mutant in Blimp1 and Bcl6 in osteoclasts exhibited decreased bone mass with increased osteoclastogenesis relative to osteoclast-specific Blimp1-deficient mice. These results reveal a Blimp1-Bcl6-osteoclastic molecule axis, which critically regulates bone homeostasis by controlling osteoclastogenesis and may provide a molecular basis for novel therapeutic strategies.

A kelch family protein Nd1-L functions as a metastasis suppressor in cancer cells via Rho family proteins mediated mechanism.

The BTB-kelch protein Nd1-L acts as an actin cytoskeleton stabilizer expressed ubiquitously in mouse tissues. We examined the effect of Nd1-L on cancer cell invasion and metastasis. Over-expression of Nd1-L in murine colon carcinoma cell line Colon 26 and murine melanoma cell line B16 resulted in suppression of pulmonary and liver metastasis after inoculation of these cells to syngeneric mice and in increased survival in an animal model. On the other hand, knock down of Nd1-L by RNA interference promoted metastasis ability of these cells. Increased expression of Nd1-L inhibited migration and Matrigel invasion capacity of cancer cell lines in vitro. Thus, Nd1-L expression inversely correlated with invasive and metastasis capacity of cancer cells. Furthermore, increased expression of Nd1-L in NIH3T3 cells inhibited growth factor induced activation of Rho family small GTPases such as Rho, Rac and cdc42. These results suggest that Nd1-L is involved in invasion and metastasis of cancer cells by regulating the actin cytoskeleton and Rho family proteins.