Regional infusion of a class C TLR9 agonist enhances liver tumor microenvironment reprogramming and MDSC reduction to improve responsiveness to systemic checkpoint inhibition.

Myeloid-derived suppressor cells (MDSCs) expand in response to malignancy and suppress responsiveness to immunotherapy, including checkpoint inhibitors (CPIs). Within the liver, MDSCs have unique immunosuppressive features. While TLR9 agonists have shown promising activities in enhancing CPI responsiveness in superficial tumors amenable to direct needle injection, clinical success for liver tumors with TLR9 agonists has been limited by delivery challenges. Here, we report that regional intravascular infusion of ODN2395 into mice with liver metastasis (LM) partially eliminated liver MDSCs and reprogrammed residual MDSC. TLR9 agonist regional infusion also induced an increase in the M1/M2 macrophage ratio. Enhanced TLR9 signaling was demonstrated by an increased activation of in NFκB (pP65) and production of IL6 compared with systemic infusion. Further, PBMC-derived human MDSCs express TLR9, and treatment with class C TLR9 agonists (ODN2395 and SD101) reduced the expansion of MDSC population. TLR9 stimulation induced MDSC apoptosis and increased the M1/M2 macrophage ratio. Regional TLR9 agonist infusion along with systemic anti-PD-1 therapy improved control of LM. With effective delivery, TLR9 agonists have the potential to favorably reprogram the liver TME through reduction of MDSCs and favorable macrophage polarization, which may improve responsiveness to systemic CPI therapy.

The Origin and Diversification of the Hyperdiverse Flora in the Chocó Biogeographic Region.

Extremely high levels of plant diversity in the American tropics are derived from multiple interactions between biotic and abiotic factors. Previous studies have focused on macro-evolutionary dynamics of the Tropical Andes, Amazonia, and Brazil's Cerrado and Atlantic forests during the last decade. Yet, other equally important Neotropical biodiversity hotspots have been severely neglected. This is particularly true for the Chocó region on the north-western coast of South and Central America. This geologically complex region is Earth's ninth most biodiverse hotspot, hosting approximately 3% of all known plant species. Here, we test Gentry's [1982a,b] hypothesis of a northern Andean-Central American Pleistocene origin of the Chocoan flora using phylogenetic reconstructions of representative plant lineages in the American tropics. We show that plant diversity in the Chocó is derived mostly from Andean immigrants. Contributions from more distant biogeographical areas also exist but are fewer. We also identify a strong floristic connection between the Chocó and Central America, revealed by multiple migrations into the Chocó during the last 5 Ma. The dated phylogenetic reconstructions suggest a Plio-Pleistocene onset of the extant Chocó flora. Taken together, these results support to a limited extend Gentry's hypothesis of a Pleistocene origin and of a compound assembly of the Chocoan biodiversity hotspot. Strong Central American-Chocoan floristic affinity may be partly explained by the accretion of a land mass derived from the Caribbean plate to north-western South America. Additional densely sampled phylogenies of Chocoan lineages also well represented across the Neotropics could enlighten the role of land mass movements through time in the assembly of floras in Neotropical biodiversity hotspots.

Alteration of marrow cell gene expression, protein production, and engraftment into lung by lung-derived microvesicles: a novel mechanism for phenotype modulation.

Numerous animal studies have demonstrated that adult marrow-derived cells can contribute to the cellular component of the lung. Lung injury is a major variable in this process; however, the mechanism remains unknown. We hypothesize that injured lung is capable of inducing epigenetic modifications of marrow cells, influencing them to assume phenotypic characteristics of lung cells. We report that under certain conditions, radiation-injured lung induced expression of pulmonary epithelial cell-specific genes and prosurfactant B protein in cocultured whole bone marrow cells separated by a cell-impermeable membrane. Lung-conditioned media had a similar effect on cocultured whole bone marrow cells and was found to contain pulmonary epithelial cell-specific RNA-filled microvesicles that entered whole bone marrow cells in culture. Also, whole bone marrow cells cocultured with lung had a greater propensity to produce type II pneumocytes after transplantation into irradiated mice. These findings demonstrate alterations of marrow cell phenotype by lung-derived microvesicles and suggest a novel mechanism for marrow cell-directed repair of injured tissue.

Bone marrow production of lung cells: the impact of G-CSF, cardiotoxin, graded doses of irradiation, and subpopulation phenotype.

OBJECTIVE: Previous studies have demonstrated the production of various types of lung cells from marrow cells under diverse experimental conditions. Our aim was to identify some of the variables that influence conversion in the lung. METHODS: In separate experiments, mice received various doses of total-body irradiation followed by transplantation with whole bone marrow or various subpopulations of marrow cells (Lin(-/+), c-kit(-/+), Sca-1(-/+)) from GFP(+) (C57BL/6-TgN[ACTbEGFP]1Osb) mice. Some were given intramuscular cardiotoxin and/or mobilized with granulocyte colony-stimulating factor (G-CSF). RESULTS: The production of pulmonary epithelial cells from engrafted bone marrow was established utilizing green fluorescent protein (GFP) antibody labeling to rule out autofluorescence and deconvolution microscopy to establish the colocaliztion of GFP and cytokeratin and the absence of CD45 in lung samples after transplantation. More donor-derived lung cells (GFP(+)/CD45(-)) were seen with increasing doses of radiation (5.43% of all lung cells, 1200 cGy). In the 900-cGy group, 61.43% of GFP(+)/CD45(-) cells were also cytokeratin(+). Mobilization further increased GFP(+)/CD45(-) cells to 7.88% in radiation-injured mice. Up to 1.67% of lung cells were GFP(+)/CD45(-) in radiation-injured mice transplanted with Lin(-), c-kit(+), or Sca-1(+) marrow cells. Lin(+), c-kit(-), and Sca-1(-) subpopulations did not significantly engraft the lung. CONCLUSIONS: We have established that marrow cells are capable of producing pulmonary epithelial cells and identified radiation dose and G-CSF mobilization as variables influencing the production of lung cells from marrow cells. Furthermore, the putative lung cell-producing marrow cell has the phenotype of a hematopoietic stem cell.

hRad9 rapidly binds DNA containing double-strand breaks and is required for damage-dependent topoisomerase II beta binding protein 1 focus formation.

Checkpoint proteins protect the genomic integrity of a cell, repeatedly impaired by DNA damage and normal cellular processes, such as replication. Checkpoint proteins hRad9, hRad1, and hHus1 form a heterotrimeric complex that is thought to act as a genomic surveyor of DNA damage. We show here that, when DNA double-strand breaks (DSBs) are specifically generated in a subnuclear area, hRad9 is rapidly retained at the damaged DNA, within 2 min of damage induction. Rapid localization of hRad9 to regions of DNA containing DSBs is most efficient during replication. Furthermore, hRad9 colocalizes with the phosphorylated form of damage-response protein H2AX (gamma H2AX) after DNA damage. This localization is independent of the damage repair kinase ataxia telangiectasia-mutated kinase (ATM), because hRad9/gamma H2AX colocalization still occurs in ATM(-/-) fibroblasts. Secondly, hRad9 interacts with replication and checkpoint protein topoisomerase II beta binding protein 1 (TopBP1) before and after DNA damage, and this interaction is dependent on the COOH-terminal 17 amino acids of hRad9. Overexpression of a COOH-terminally deleted form of hRad9 abolishes the colocalization of TopBP1 to gamma H2AX, ablating TopBP1 but not gamma H2AX foci formation. The loss of TopBP1 containing foci, but not of gamma H2AX containing foci, indicates that hRad9 is required for TopBP1 focus formation after damage, but is not required for gamma H2AX formation at DSBs. These results are consistent with a model in which the hRad9/hHus1/hRad1 complex acts as a checkpoint sensor during S phase by rapidly localizing to sites of DNA damage and transducing checkpoint responses by facilitating proper localization of downstream checkpoint proteins, including TopBP1.

The stem cell continuum.

Hematopoietic stem cells have been felt to exist in a hierarchical structure with a relatively fixed phenotype at each stage of differentiation. Recent studies on the phenotype of the marrow hematopoietic stem cell indicate that it is not a fixed entity, but rather that it fluctuates and shows marked heterogeneity. Past studies have shown that stem cell engraftment characteristics, adhesion protein, and gene expression varies with the phase of the cell cycle. More recently, we demonstrated that progenitor numbers and differentiation potential also vary reversibly during one cytokine-induced cell cycle transit. We have also shown high levels of conversion of marrow cells to skeletal muscle and lung cells, indicating a different level of plasticity. Recently, we demonstrated that homing to lung and conversion to lung cells in a mouse transplant model also fluctuates reversibly with cell cycle transit. This could be considered plasticity squared. These data indicate that marrow stem cells are regulated on a continuum related to the cell cycle both as to hematopoietic and to nonhematopoietic differentiation.

Robust conversion of marrow cells to skeletal muscle with formation of marrow-derived muscle cell colonies: a multifactorial process.

OBJECTIVE: Murine marrow cells are capable of repopulating skeletal muscle fibers. A point of concern has been the "robustness" of such conversions. We have investigated the impact of type of cell delivery, muscle injury, nature of delivered cell, and stem cell mobilizations on marrow-to-muscle conversion. METHODS: We transplanted green fluorescence protein (GFP)-transgenic marrow into irradiated C57BL/6 mice and then injured anterior tibialis muscle by cardiotoxin. One month after injury, sections were analyzed by standard and deconvolutional microscopy for expression of muscle and hematopoietic markers. RESULTS: Irradiation was essential to conversion, although whether by injury or induction of chimerism is not clear. Cardiotoxin- and, to a lesser extent, PBS-injected muscles showed significant number of GFP(+) muscle fibers, while uninjected muscles showed only rare GFP(+) cells. Marrow conversion to muscle was increased by two cycles of G-CSF mobilization and to a lesser extent by G-CSF and steel or GM-CSF. Transplantation of female GFP to male C57BL/6 and GFP to ROSA26 mice showed fusion of donor cells to recipient muscle. High numbers of donor-derived muscle colonies and up to 12% GFP(+) muscle cells were seen after mobilization or direct injection. These levels of donor muscle chimerism approach levels that could be clinically significant in developing strategies for the treatment of muscular dystrophies. CONCLUSION: In summary, the conversion of marrow to skeletal muscle cells is based on cell fusion and is critically dependent on injury. This conversion is also numerically significant and increases with mobilization.

Implementing an evidence-based metabolic syndrome prevention and treatment program utilizing group visits.

PURPOSE: To develop and implement a pilot program designed as a shared medical group visit targeting metabolic syndrome prevention in two ethnically diverse patient populations. DATA SOURCES: The Cooperative Health Care Clinics (CHCC) module was utilized for group sessions to focus on interactive discussions following the L.E.A.R.N. format in order to encourage healthy lifestyle changes. Participants completed a pre- and postknowledge base test that encompassed information on healthy lifestyle changes in addition to disease processes associated with metabolic syndrome. Each didactic session was evaluated using mean ± standard deviation for the knowledge tests. Analysis of variance was used in determining body mass index (BMI) and weight measured at weeks 1, 5, and 10. Participants completed a 5-point Likert scale satisfaction survey on week 10. Independent means t-test compared clinic results along with the Satterthwaite approximate t-test because of unequal sample sizes to evaluate differences in means. CONCLUSIONS: There were no significant statistical differences in mean weight or BMI on weeks 1 and 5. However, on week 10, there was a statistically significant difference for waist circumference in both clinics (p= .0466). Knowledge base improved in both clinics with a premean (0 score = 87 ± 18) and postmean (0 score = 93 ± 14). Both sites received high scores for patient satisfaction. IMPLICATIONS FOR PRACTICE: The shared medical group visits program implemented in both clinic sites demonstrated that this is an effective model in which to provide intensive patient education, foster peer support, and facilitate health-related behavioral changes. Peer support, self-management, and continuity appear to be important factors in behavior change and improved knowledge.

Cytokines inducing bone marrow SCA+ cells migration into pancreatic islet and conversion into insulin-positive cells in vivo.

We hypothesize that specific bone marrow lineages and cytokine treatment may facilitate bone marrow migration into islets, leading to a conversion into insulin producing cells in vivo. In this study we focused on identifying which bone marrow subpopulations and cytokine treatments play a role in bone marrow supporting islet function in vivo by evaluating whether bone marrow is capable of migrating into islets as well as converting into insulin positive cells. We approached this aim by utilizing several bone marrow lineages and cytokine-treated bone marrow from green fluorescent protein (GFP) positive bone marrow donors. Sorted lineages of Mac-1(+), Mac-1(-), Sca(+), Sca(-), Sca(-)/Mac-1(+) and Sca(+)/Mac-1(-) from GFP positive mice were transplanted to irradiated C57BL6 GFP negative mice. Bone marrow from transgenic human ubiquitin C promoter GFP (uGFP, with strong signal) C57BL6 mice was transplanted into GFP negative C57BL6 recipients. After eight weeks, migration of GFP positive donor' bone marrow to the recipient's pancreatic islets was evaluated as the percentage of positive GFP islets/total islets. The results show that the most effective migration comes from the Sca(+)/Mac(-) lineage and these cells, treated with cytokines for 48 hours, were found to have converted into insulin positive cells in pancreatic islets in vivo. This study suggests that bone marrow lineage positive cells and cytokine treatments are critical factors in determining whether bone marrow is able to migrate and form insulin producing cells in vivo. The mechanisms causing this facilitation as well as bone marrow converting to pancreatic beta cells still need to be investigated.

Haematopoietic stem cells participate in muscle regeneration.

It has previously been shown that bone marrow cells contribute to skeletal muscle regeneration, but the nature of marrow cell(s) involved in this process is unknown. We used an immunocompetent and an immunocompromised model of bone marrow transplantation to characterize the type of marrow cells participating regenerating skeletal muscle fibres. Animals were transplanted with different populations of marrow cells from Green Fluorescent Protein (GFP) transgenic mice and the presence of GFP(+) muscle fibres were evaluated in the cardiotoxin-injured tibialis anterior muscles. GFP(+) muscle fibres were found mostly in animals that received either CD45(-), lineage(-), c-Kit(+), Sca-1(+) or Flk-2(+) populations of marrow cells, suggesting that haematopoietic stem cells (HSC) rather than mesenchymal cells or more differentiated haematopoietic cells are responsible for the formation of GFP(+) muscle fibres. Mac-1 positive population of marrow cells was also associated with the emergence of GFP(+) skeletal muscle fibres. However, most of this activity was limited to either Mac-1(+) Sca(+) or Mac-1(+)c-Kit(+) cells with long-term haematopoietic repopulation capabilities, indicating a stem cell phenotype for these cells. Experiments in the immunocompromised transplant model showed that participation of HSC in the skeletal muscle fibre formation could occur without haematopoietic chimerism.

Regulation of lens aldose reductase activity by nitric oxide.

To examine the regulation of aldose reductase (AR) activity by nitric oxide (NO) in human lens epithelial cells (HLEC), cultured rat lens, and normal and diabetic rat lens, we have incubated HLEC or cultured rat lenses with 1 mm of the NO donors S-nitroso-N-acetylpenicillamine (SNAP) or S-nitrosoglutathione (GSNO), and the AR activity and sorbitol content were measured. Non-diabetic and diabetic (treated with streptozotocin 65 mg kg(-1) body wt, i.p.) rats were injected with the nitric oxide synthase (NOS) inhibitor, L-NAME (50 mg kg(-1) body wt day(-1), x 10 days i.p.) or NOS substrate, L-arginine (200 mg kg(-1) body wt day(-1), x 10 days i.p.). In a separate group of rats, a nitroglycerin (NG)-patch that releases 200 ng min(-1) NO was applied to the dorsal neck region. After 10 days of treatment, the lenses were removed and their AR activity and sorbitol content were measured. Incubation of HLEC with SNAP or GSNO reduced AR activity. A similar reduction in AR activity and sorbitol accumulation was observed when diabetic and non-diabetic rat lenses were cultured in the presence of SNAP and GSNO. Total protein-SSG in diabetic lens was lower compared to normal lens. Treatment of diabetic and non-diabetic rats with L-NAME enhanced AR activity and sorbitol accumulation, whereas NG patch and L-arginine significantly decreased AR activity and sorbitol accumulation in diabetic lenses compared to non-diabetic. Increased S-glutathiolation of AR was observed in the presence of SNAP. These results suggest that decreased glutathiolation of cellular proteins in diabetic rat lens compared to non-diabetic lens is related to decreased NO availability in diabetic rats which would decrease GSNO. Restoring the NO levels in diabetic animals increases glutathiolation of cellular proteins, inhibits AR activity and prevents sorbitol accumulation. Exogenous delivery of NO may represent a potentially useful strategy for preventing or delaying diabetic cataractogenesis and the development of other diabetic complications.

Critical variables in the conversion of marrow cells to skeletal muscle.

We have studied conversion of marrow cells to skeletal muscle in cardiotoxin-injured anterior tibialis muscle in a green fluorescent protein (GFP) to C57BL/6 transplantation model and ascertained that total body irradiation (TBI) with establishment of chimerism is a critical factor. Local irradiation has little effect in lower doses and was detrimental at higher doses. Whole body (1000 cGy) with shielding of the leg or a combination of 500 cGy TBI and 500 cGy local radiations was found to give the best results. In non-obese diabetic-severe combined immunodeficient (NOD-SCID) recipients, we were able to show that conversion could occur without radiation, albeit at relatively lower levels. Within 3 days of cardiotoxin injury, GFP-positive mononuclear cells were seen in the muscle, and within 2 weeks GFP-positive muscle fibers were identified. Conversion rates were increased by increasing donor-cell dose. Timing of the cardiotoxin injury relative to the transplantation was critical. These studies show that variables in transplantation and injury are critical features of marrow-to-muscle conversions. Irradiation primarily effects conversion by promoting chimerism. These data may explain the differences in the literature for the frequency of marrow-to-skeletal muscle conversion and can set a platform for future models and perhaps clinical protocols.

Homing and conversion of murine hematopoietic stem cells to lung.

The hematopoietic stem cell population, lineage negative-Sca positive (HSC), displays a homing defect into bone marrow (BM) after 48-h exposure to interleukin (IL)-3, IL-6, IL-11, and steel factor [J. Hematother. Stem Cell Res. 11 (2002) 913]. Cytokine treatment of murine marrow leads to reversible alterations in adhesion protein expression, which may explain the changes in homing. We evaluated 3 h homing to nonhematopoietic organs of marrow cells exposed to cytokines for 0, 18, 24, 40 and 48 h. HSC cells from C57BL/6J mice were cultured and labeled with the cytoplasmic fluorescent dye CFSE. We found homed events from uncultured cells in spleen, liver and lung, but no events were seen in duodenum or anterior tibialis muscle. Culture in cytokines led to decreased homing to marrow at 24 and 48 h with parallel changes in spleen homing. There was little variability of homing to liver, however the number, of homed events in lung was markedly increased when 24-h cultured cells were assessed. This was approximately a 10-fold increase compared to the 0 h time point (flow cytometry). Homing was determined by evaluation of frozen section (8 microm) by fluorescent microscopy for spleen, liver, duodenum, anterior tibialis and lung. Data were confirmed by flow cytometry from each organ including marrow. These data indicate the presence of a lung homing "hotspot" at 24 h of cytokine culture; this is a time when the stem progenitors cells are in mid S-phase. Altogether these data suggest that homing of marrow cell to nonmarrow organs may fluctuate with cell cycle transit and that there is a lung homing hotspot in mid-S.

Stem cell plasticity: an overview.

The capacity of adult bone marrow cells to convert to cells of other tissues, referred to by many as stem cell plasticity, was the focus of the meeting in Providence entitled "Challenges in the Era of Stem Cell Plasticity". The meeting provided a showcase for the many impressive positive results on tissue restoration including the capacity of purified marrow stem cells to restore heart, skin, and liver function in impaired mice or humans. This area of research has become a center of controversy, although it is not clear why. Calls for clonality, robustness, and function have been shown to be erroneous or premature. A call for clonality (which has been shown nicely in one study) is meaningless on a predefined stem cell population which is intrinsically heterogeneous, as they all are. Robustness means nothing; it all depends on the details of the situation. Function on an organ level is, of course, the goal of many investigators and should not be raised as a limiting consideration. Lastly, fusion has been highlighted as undermining studies with adult stem cells. It, of course, does not. Fusion is simply a means to a final goal, which occurs in certain settings of marrow conversions (transdifferentiation) and not in others. We hypothesize that the conversion phenomena may, in fact, be due to one or several marrow stem cells with broad differentiation potential which can be expressed when the cell is placed in an environment with the appropriate inductive signals. Furthermore, initial events may be relatively rare and significant conversion numbers may be obtained with massive or ongoing selection. Fusion appears in an initial mechanism in some cases and not in others. Overall, the therapeutic potential of adult marrow stem cells is very intriguing, and successful use therapeutically will probably depend on definition of the most appropriate transplant model and tissue injury.