Mesenchymal Stem Cells Reconditioned in Their Own Serum Exhibit Augmented Therapeutic Properties in the Setting of Acute Respiratory Distress Syndrome.

Mesenchymal stem cells (MSCs) are a promising form of therapy for acute respiratory distress syndrome (ARDS). The objective of this study was twofold: (a) to characterize cytokine expression in serum from ARDS subjects receiving MSCs and (b) to determine MSC function following "preconditioning" with ARDS serum. In phase I, serum from three cohorts of animals (uninjured [no ARDS, n = 4], injured untreated [n = 5], and injured treated with approximately 6 million per kilogram MSCs [n = 7]) was analyzed for expression of inflammatory mediators. In phase II, the functional properties of bone marrow porcine MSCs were assessed following "preconditioning" with serum from the three cohorts. In phase III, the findings from the previous phases were validated using human bone marrow MSCs (hBM-MSCs) and lipopolysaccharide (LPS). Serum from injured treated animals had significantly lower levels of interferon-γ and significantly higher levels of interleukin (IL)-1 receptor antagonist (IL-1RA) and IL-6. Similarly, upon exposure to the injured treated serum ex vivo, the MSCs secreted higher levels of IL-1RA and IL-10, dampened the secretion of proinflammatory cytokines, exhibited upregulation of toll-like receptor 4 (TLR-4) and vascular endothelial growth factor (VEGF) genes, and triggered a strong immunomodulatory response via prostaglandin E2 (PGE2 ). hBM-MSCs demonstrated a similar augmented therapeutic function following reconditioning in a LPS milieu. Administration of MSCs modulated the inflammatory milieu following ARDS. Exposure to ARDS serum ex vivo paralleled the trends seen in vivo, which appear to be mediated, in part, through TLR-4 and VEGF and PGE2 . Reconditioning MSCs in their own serum potentiates their immunotherapeutic function, a technique that can be used in clinical applications. Stem Cells Translational Medicine 2019;8:1092-1106.

Bench-to-bedside optimization of mesenchymal stem cell isolation, processing, and expansion for in vivo administration.

Aim: In this study, we aimed at identifying the optimal conditions for isolation, processing and expansion of mesenchymal stem cells (MSCs). Methods: Porcine bone marrow was obtained from either small- or large-volume bone marrow aspirate (BMA). Next, three BMA processing methods were compared. Finally, the best condition was selected from various culture parameters, including basal media, supplementation and seeding density. Results: Our results demonstrate that a small-volume BMA and direct plating yields significantly higher concentration of MSCs. Basal media supplementation with 10% platelet lysate and seeding density of 1000 cells/cm2 can generate large numbers of multipotent MSCs with augmented function and low population doublings. Conclusion: This work provides guidance for preparation of robust MSCs for future clinical trials.

Short-term physiological hypoxia potentiates the therapeutic function of mesenchymal stem cells.

BACKGROUND: In the bone marrow, MSCs reside in a hypoxic milieu (1-5% O2) that is thought to preserve their multipotent state. Typically, in vitro expansion of MSCs is performed under normoxia (~ 21% O2), a process that has been shown to impair their function. Here, we evaluated the characteristics and function of MSCs cultured under hypoxia and hypothesized that, when compared to normoxia, dedicated hypoxia will augment the functional characteristics of MSCs. METHODS: Human and porcine bone marrow MSCs were obtained from fresh mononuclear cells. The first study evaluated MSC function following both long-term (10 days) and short-term (48 h) hypoxia (1% O2) culture. In our second study, we evaluated the functional characteristics of MSC cultured under short-term 2% and 5% hypoxia. MSCs were evaluated for their metabolic activity, proliferation, viability, clonogenicity, gene expression, and secretory capacity. RESULTS: In long-term culture, common MSC surface marker expression (CD44 and CD105) dropped under hypoxia. Additionally, in long-term culture, MSCs proliferated significantly slower and provided lower yields under hypoxia. Conversely, in short-term culture, MSCs proliferated significantly faster under hypoxia. In both long-term and short-term cultures, MSC metabolic activity was significantly higher under hypoxia. Furthermore, MSCs cultured under hypoxia had upregulated expression of VEGF with concomitant downregulation of HMGB1 and the apoptotic genes BCL-2 and CASP3. Finally, in both hypoxia cultures, the pro-inflammatory cytokine, IL-8, was suppressed, while levels of the anti-inflammatories, IL-1ra and GM-CSF, were elevated in short-term hypoxia only. CONCLUSIONS: In this study, we demonstrate that hypoxia augments the therapeutic characteristics of both porcine and human MSCs. Yet, short-term 2% hypoxia offers the greatest benefit overall, exemplified by the increase in proliferation, self-renewing capacity, and modulation of key genes and the inflammatory milieu as compared to normoxia. These data are important for generating robust MSCs with augmented function for clinical applications.

The effect of acute respiratory distress syndrome on bone marrow-derived mesenchymal stem cells.

BACKGROUND: It is known that, following a physiological insult, bone marrow-derived mesenchymal stem cells (MSCs) mobilize and home to the site of injury. However, the effect of injury on the function of endogenous MSCs is unknown. In this study, MSCs harvested from the bone marrow of swine with or without acute respiratory distress syndrome (ARDS) were assessed for their characteristics and therapeutic function. METHODS: MSCs were harvested from three groups of anesthetized and mechanically ventilated swine (n = 3 in each group): 1) no ARDS ('Uninjured' group); 2) ARDS induced via smoke inhalation and 40% burn and treated with inhaled epinephrine ('Injured Treated' group); and 3) ARDS without treatment ('Injured Untreated' group). Cellular evaluation of the three groups included: flow cytometry for MSC markers; colony forming unit-fibroblast (CFU-F) assay; proliferative and metabolic capacity; gene expression using quantitative real-time polymerase chain reaction (qRT-PCR); and a lipopolysaccharide (LPS) challenge, with or without coculture with mononuclear cells (MNCs), for evaluation of their protein secretion profile using Multiplex. Statistical analysis was performed using one- or two-way analysis of variance (ANOVA) with a Tukey's post-test; a p-value less than 0.05 was considered statistically significant. RESULTS: Cells from all groups exhibited nearly 100% expression of MSC surface markers and retained their multidifferentiation capacity. However, the MSCs from the 'Injured Untreated' group generated a significantly higher number of colonies compared with the other two groups (p < 0.0001), indicative of increased clonogenic capacity following ARDS. Following an LPS challenge, the MSCs from the 'Injured Untreated' group exhibited a significant reduction in their proliferative capacity (p = 0.0002), significant downregulation in the expression of high-mobility group box 1 (HMGB1; p < 0.001), Toll-like receptor (TLR)-4 (p < 0.01), and vascular endothelial growth factor (VEGF; p < 0.05) genes, and significantly diminished secretory capacity for the inflammatory mediators interleukin (IL)-6 (p < 0.0001), IL-8 (p < 0.05), and tumor necrosis factor (TNF)-α (p < 0.05) compared with the 'Uninjured' group. CONCLUSIONS: The results suggest that, following ARDS, there is an increase in the clonogenic capacity of MSCs to increase the available stem cell pool in vivo. However, MSCs harvested from subjects with ARDS seem to exhibit a diminished capacity to proliferate, express regenerative signals, and secrete pro/anti-inflammatory mediators.

A model of recovery from inhalation injury and cutaneous burn in ambulatory swine.

Inhalation injury commonly accompanies thermal injury, increasing the likelihood of mortality and multiple organ dysfunction (MOD). Large animal models have given important insight into the pathophysiology of this injury; however recapitulating late MOD has remained difficult. The current report describes experiments using a smoke inhalation and burn model, with follow-up of ambulatory swine for 14days with bronchoscopy, CT scanning, and bronchoalveolar lavage fluid (BALF)/blood collection. Clinically, animals cleared airway damage in the first several days after-injury. This was mirrored with erythematous airways on day 2 after-injury, which resolved by the end of the experiment, as did parenchymal damage seen on CT. An initial rise in the protein content of BALF immediately after-injury was followed by a dramatic increase in the concentration of leukocytes. Circulating neutrophils increased while lymphocytes decreased; both correlated with cell counts in BALF. IL8 levels in BALF increased 30-fold and remained elevated throughout the experiment. IL1ra increased circulation immediately after-injury, and afterwards in BALF. Other cytokines (TNFα, IL12) transiently increased in BALF (and decreased in circulation) on day 2. Taken together, these results display a remarkable capability for the lungs to recover in the absence of intubation, with further evidence of the role of cytokines such as IL8 and IL1ra. The possible exacerbating effects of clinical practices such as ventilation and bronchoscopies should be considered.

Eliminating tuberculosis one neighborhood at a time.

OBJECTIVES: We evaluated a strategy for preventing tuberculosis (TB) in communities most affected by it. METHODS: In 1996, we mapped reported TB cases (1985-1995) and positive tuberculin skin test (TST) reactors (1993-1995) in Smith County, Texas. We delineated the 2 largest, densest clusters, identifying 2 highest-incidence neighborhoods (180 square blocks, 3153 residents). After extensive community preparation, trained health care workers went door-to-door offering TST to all residents unless contraindicated. TST-positive individuals were escorted to a mobile clinic for radiography, clinical evaluation, and isoniazid preventive treatment (IPT) as indicated. To assess long-term impact, we mapped all TB cases in Smith County during the equivalent time period after the project. RESULTS: Of 2258 eligible individuals, 1291 (57.1%) were tested, 229 (17.7%) were TST positive, and 147 were treated. From 1996 to 2006, there were no TB cases in either project neighborhood, in contrast with the preintervention decade and the continued occurrence of TB in the rest of Smith County. CONCLUSIONS: Targeting high-incidence neighborhoods for active, community-based screening and IPT may hasten TB elimination in the United States.

[Eliminating tuberculosis one neighborhood at a time]. / Eliminación de la tuberculosis, un vecindario por vez.

OBJECTIVES: We evaluated a strategy for preventing tuberculosis (TB) in communities most affected by it. METHODS: In 1996, we mapped reported TB cases (1985-1995) and positive tuberculin skin test (TST) reactors (1993-1995) in Smith County, Texas. We delineated the 2 largest, densest clusters, identifying 2 highest-incidence neighborhoods (180 square blocks, 3153 residents). After extensive community preparation, trained health care workers went door-to-door offering TST to all residents unless contraindicated. TST-positive individuals were escorted to a mobile clinic for radiography, clinical evaluation, and isoniazid preventive treatment (IPT) as indicated. To assess long-term impact, we mapped all TB cases in Smith County during the equivalent time period after the project. RESULTS: Of 2258 eligible individuals, 1291 (57.1%) were tested, 229 (17.7%) were TST positive, and 147 were treated. From 1996 to 2006, there were no TB cases in either project neighborhood, in contrast with the preintervention decade and the continued occurrence of TB in the rest of Smith County. CONCLUSIONS: Targeting high-incidence neighborhoods for active, community-based screening and IPT may hasten TB elimination in the United States.

Eliminación de la tuberculosis, un vecindario por vez / Eliminating tuberculosis one neighborhood at a time

OBJETIVOS: Evaluamos una estrategia para la prevención de la tuberculosis en las comunidades más afectadas por esta enfermedad. MÉTODOS: En 1996, trazamos un mapa de los casos de tuberculosis notificados (1985-1995) y de las personas con reacción positiva a la prueba de la tuberculina (1993-1995) en el condado de Smith, Texas, Estados Unidos de América. Definimos los dos conglomerados de mayor tamaño y densidad, identificando los dos vecindarios con mayor incidencia (180 manzanas y 3 153 residentes). Tras una preparación intensiva de la comunidad, personal sanitario capacitado ofreció a todos los residentes, de vivienda en vivienda, la posibilidad de hacerse la prueba de la tuberculina, a menos que estuviera contraindicado. A las personas con resultados positivos en esta prueba se las acompañó a un consultorio móvil para realizarles ahí radiografías, una evaluación clínica y, según fuera pertinente, proceder con el tratamiento preventivo con isoniazida. Para evaluar las repercusiones a largo plazo, trazamos un mapa de todos los casos de tuberculosis que se registraron en el condado de Smith durante el período equivalente después del proyecto. RESULTADOS: De las 2 258 personas que cumplían los requisitos para participar, 1 291 (57,1%) se sometieron a la prueba de la tuberculina, 229 (17,7%) presentaron resultados positivos en dicha prueba y 147 fueron tratadas. De 1996 al 2006, no se registró ningún caso de tuberculosis en ninguno de los vecindarios del proyecto, a diferencia de lo ocurrido en el decenio anterior a la intervención y en el resto del condado de Smith, donde aparecieron continuamente casos de tuberculosis. CONCLUSIONES: Dirigirse a los vecindarios con una incidencia alta para realizar el tamizaje activo en la comunidad y aplicar tratamiento preventivo con isoniazida puede acelerar la eliminación de la tuberculosis en los Estados Unidos.

OBJECTIVES: We evaluated a strategy for preventing tuberculosis (TB) in communities most affected by it. METHODS: In 1996, we mapped reported TB cases (1985-1995) and positive tuberculin skin test (TST) reactors (1993-1995) in Smith County, Texas. We delineated the 2 largest, densest clusters, identifying 2 highest-incidence neighborhoods (180 square blocks, 3153 residents). After extensive community preparation, trained health care workers went door-to-door offering TST to all residents unless contraindicated. TST-positive individuals were escorted to a mobile clinic for radiography, clinical evaluation, and isoniazid preventive treatment (IPT) as indicated. To assess long-term impact, we mapped all TB cases in Smith County during the equivalent time period after the project. RESULTS: Of 2258 eligible individuals, 1291 (57.1%) were tested, 229 (17.7%) were TST positive, and 147 were treated. From 1996 to 2006, there were no TB cases in either project neighborhood, in contrast with the preintervention decade and the continued occurrence of TB in the rest of Smith County. CONCLUSIONS: Targeting high-incidence neighborhoods for active, community-based screening and IPT may hasten TB elimination in the United States.

Eliminating tuberculosis one neighborhood at a time.

OBJECTIVES: We evaluated a strategy for preventing tuberculosis (TB) in communities most affected by it. METHODS: In 1996, we mapped reported TB cases (1985-1995) and positive tuberculin skin test (TST) reactors (1993-1995) in Smith County, Texas. We delineated the 2 largest, densest clusters, identifying 2 highest-incidence neighborhoods (180 square blocks, 3153 residents). After extensive community preparation, trained health care workers went door-to-door offering TST to all residents unless contraindicated. TST-positive individuals were escorted to a mobile clinic for radiography, clinical evaluation, and isoniazid preventive treatment (IPT) as indicated. To assess long-term impact, we mapped all TB cases in Smith County during the equivalent time period after the project. RESULTS: Of 2258 eligible individuals, 1291 (57.1%) were tested, 229 (17.7%) were TST positive, and 147 were treated. From 1996 to 2006, there were no TB cases in either project neighborhood, in contrast with the preintervention decade and the continued occurrence of TB in the rest of Smith County. CONCLUSIONS: Targeting high-incidence neighborhoods for active, community-based screening and IPT may hasten TB elimination in the United States.

Wood bark smoke induces lung and pleural plasminogen activator inhibitor 1 and stabilizes its mRNA in porcine lung cells.

Although aberrant fibrinolysis and plasminogen activator inhibitor 1 (PAI-1) are implicated in acute lung injury, the role of this serpin in the pathogenesis of wood bark smoke (WBS)-induced acute lung injury (SIALI) and its regulation in resident lung cells after exposure to smoke are unclear. A total of 22 mechanically ventilated pigs were included in this study. Immunohistochemical analyses were used to assess fibrin and PAI-1 in the lungs of pigs with SIALI in situ. Plasminogen activator inhibitor 1 was measured in bronchoalveolar lavage fluids by Western blotting. Induction of PAI-1 was determined at the protein and mRNA levels by Western and polymerase chain reaction analyses in primary porcine alveolar type II cells, fibroblasts, and pleural mesothelial cells. Plasminogen activator inhibitor 1 mRNA stability was determined by transcription chase studies. Gel shift analyses were used to characterize the mechanism regulating PAI-1 mRNA stability. Smoke-induced ALI induced PAI-1, with prominent extravascular fibrin deposition in large and small airways as well as alveolar and subpleural compartments. In pleural mesothelial cells, lung fibroblasts, and alveolar type II cells, PAI-1 mRNA was stabilized by WBS extract and contributed to induction of PAI-1. The mechanism involves dissociation of a novel 6-phospho-d-gluconate-NADP oxidoreductase-like PAI-1 mRNA binding protein from PAI-1 mRNA. Exposure to WBS induces prominent airway and mesothelial expression of PAI-1, associated with florid distribution of fibrin in SIALI in vivo Wood bark smoke components induce PAI-1 in vitro in part by stabilization of PAI-1 mRNA, a newly recognized pathway that may promote extravascular fibrin deposition and lung dysfunction in SIALI.

Lower interbreath interval complexity is associated with extubation failure in mechanically ventilated patients during spontaneous breathing trials.

OBJECTIVE: To determine whether lower complexity of interbreath interval as measured with nonlinear analysis techniques will identify patients who fail to separate from mechanical ventilation after 30-minute spontaneous breathing trials (SBTs). METHODS: Respiratory waveforms from SBT of patients in surgical or burn intensive care units were recorded for later analysis. The decision to extubate was made by attending physician. Extubated patients were observed for 48 hours; during this time, reintubation or noninvasive positive pressure ventilation was considered as a failure. Analysis of waveform data by software was performed post hoc. Sample entropy (SampEn) and other nonlinear measures were 48 hours of extubation. RESULTS: Thirty-two patients (24 burn, 8 trauma/surgical admissions; mean age, 40.2 +/- 16.9 years; 26 men and 6 women) who were intubated >24 hours were extubated after SBT. Twenty-four patients were successfully separated from mechanical ventilation and eight failed. Age, gender, and mechanism of injury did not influence outcome. SampEn calculated for the two groups presented in this study was different with the cohort that failed extubation having a lower mean value (1.35 +/- 0.39 vs. 1.87 +/- 0.27; p < 0.001). Other nonlinear metrics were moved in concert with SampEn. The stationarity in the respiratory signal was not different between groups. CONCLUSION: In intubated patients, the interbreath interval in those who were successfully separated from mechanical ventilation was more irregular than those who failed, as measured by nonlinear techniques. When available at bedside, these metrics may be useful markers of pulmonary health and assist in clinical decision making.

Solution structure and backbone dynamics of the TGFbeta type II receptor extracellular domain.

Isoforms of transforming growth factor beta (TGFbeta) are 25 kDa homodimeric polypeptides that signal by binding and bringing together two related, functionally distinct cell surface receptors designated as TbetaR1 and TbetaR2. Here, we report the solution structure of the 13.8 kDa extracellular domain of human TbetaR2 (ecTbetaR2) as calculated from N(N)-H(N), C(alpha)-H(alpha), and C(alpha)-C(O) residual dipolar coupling restraints in conjunction with NOE distance, dihedral angle, and scalar coupling restraints. Comparison of the free ecTbetaR2 solution structure with the TGFbeta3-bound ecTbetaR2 crystal structure reveals backbone conformations that superimpose with RMSDs of 1.0 A over the regions of regular secondary structure and 1.4 A overall. The differences in structure fall mainly in loop regions that are either poorly defined by the available NMR data or are involved in crystal contacts. The noted similarities between the NMR structure of the free form and the crystal structure of the TGFbeta-bound form are also consistent with the close correspondence, 0.16 A RMSD for regions of secondary structure and 0.51 A RMSD overall, for the crystal structure of free ecTbetaR2 as compared to the crystal structure of TGFbeta3-bound ecTbetaR2. Despite the apparent similarities between the free and the bound forms, there appears to be small but significant differences in structure involving the interfacial contact region of the receptor. Measurements of backbone (15)N relaxation times and interpretation of these by the model-free formalism with axial diffusional anisotropy further reveal significant ms to micros time scale motions centered about two of the conserved disulfide bonds and in several residues that comprise the TGFbeta binding surface. Together, these observations indicate that binding likely occurs through a mechanism with a small component of induced fit character, whereby flexibility within the receptor facilitates the transition to the TGFbeta-bound state.