Fluorescence-activated Cell Analysis Research Articles

Abstract 1271: PMEPA1 acts as a switch to modulate cooperative cellular invasion and drive NSCLC tumor progression

Abstract Metastatic disease drives cancer patient mortality. One primary mode of metastasis is collective invasion, whereby cohesive groups of cells invade into the adjacent stroma while maintaining cell-cell contacts. Importantly, these cellular packs harbor genetically and phenotypically heterogeneous subpopulations that cooperate to drive invasion. To deconstruct how phenotypic cellular heterogeneity facilitates distinct molecular profiles within a single tumor, we established the technique Spatiotemporal Cellular and Genomic Analysis (SaGA). SaGA is an image-guided approach that optically highlights phenotypically defined cell(s) for live fluorescence-activated cell sorting and analysis. To elucidate the transcriptional heterogeneity across invasive packs within a 3D physiologically-relevant microenvironment, we combined SaGA with single cell RNA sequencing for isolation and analysis of actively invading cells. This novel single cell transcriptomics approach reveals that - during cellular invasion into the microenvironment - the follower subpopulation supplies TGFβ to heterogeneously stimulate both autocrine canonical and paracrine noncanonical signaling pathways. This cooperative exchange facilitates a symbiotic relationship between tumor subpopulations in non-small cell lung carcinoma both in vitro and in vivo. In vitro, we determine that PMEPA1 protein levels mitigate TGFβ downstream effector signaling within heterogeneous tumor cells to modulate JAG1 expression and activity. In vivo, we show that a reduction in JAG1 levels decreases primary tumor cell invasion and progression. We mechanistically define a TGFβ-PMEPA1-JAG1 signaling axis across leader and follower subpopulations critical for tumor cell invasion and cancer progression. Together, these data highlight the importance of cellular heterogeneity and subpopulation communication required for tumor progression. Citation Format: Tala O. Khatib, Brian A. Pedro, Christina M. Knippler, Veronika Y. Matsuk, Sarah F. Webster, Sergei Bombin, Bhakti Dwivedi, Jeanne Kowalski, Henry R. Johnston, Janna K. Mouw, Adam I. Marcus. PMEPA1 acts as a switch to modulate cooperative cellular invasion and drive NSCLC tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1271.

Carbohydrate ligand engagement with CD11b enhances differentiation of tumor-associated myeloid cells for immunotherapy of solid cancers

BackgroundEfforts to modulate the function of tumor-associated myeloid cell are underway to overcome the challenges in immunotherapy and find a cure. One potential therapeutic target is integrin CD11b, which can...

Local immune cell infiltration in cutaneous acute graft versus host disease.

BackgroundHematopoietic stem cell transplant is a crucial intervention to definitively treat many hematopoietic malignancies, but it carries great risks of morbidity and mortality often associated with graft-versus-host disease (GVHD). Acute and chronic GVHD are distinct entities, defined by a combination of historical, clinical, and pathologic data, but both are generally thought to stem from self-propagating aberrantly activated immune cells inflicting end organ damage, with the potential to cause significant illness or even death. Event-free survival rates after hematopoietic stem cell transplant continue to improve each year, but GVHD remains a major hurdle in improving the efficacy and safety of transplant.ObjectiveRecent studies demonstrating tissue-specific immune effector phenotypes underscore the need for a deeper understanding of the cellular and molecular pathways driving the destruction of target tissues in patients with acute GVHD.MethodsSamples were collected from lesional and unaffected skin in five patients with acute cutaneous GHVD. Fresh tissue was processed for fluorescence-activated cell sorting and analysis of macrophages and lymphocytes.ResultsThe percentage of lymphocytes and macrophages as a representation of total cells varied among patients and was not always consistent between lesional and unaffected sites. The heterogeneity in immune cell profiling observed in patients in this study could reflect the diverse demographics, conditioning, and transplant conditions of each individual.ConclusionThis study provides initial insight into the underlying molecular mechanisms of cutaneous GVHD progression and paves the way for additional studies to examine the cellular and molecular landscape in greater detail.

Elevated Exogenous Pyruvate Potentiates Mesodermal Differentiation through Metabolic Modulation and AMPK/mTOR Pathway in Human Embryonic Stem Cells.

SummaryPyruvate is a key metabolite in glycolysis and the tricarboxylic acid (TCA) cycle. Exogenous pyruvate modulates metabolism, provides cellular protection, and is essential for the maintenance of human preimplantation embryos and human embryonic stem cells (hESCs). However, little is known about how pyruvate contributes to cell-fate determination during epiblast stage. In this study, we used hESCs as a model to demonstrate that elevated exogenous pyruvate shifts metabolic balance toward oxidative phosphorylation in both maintenance and differentiation conditions. During differentiation, pyruvate potentiates mesoderm and endoderm lineage specification. Pyruvate production and its mitochondrial metabolism are required in BMP4-induced mesoderm differentiation. However, the TCA-cycle metabolites do not have the same effect as pyruvate on differentiation. Further study shows that pyruvate increases AMP/ATP ratio, activates AMPK, and modulates the mTOR pathway to enhance mesoderm differentiation. This study reveals that exogenous pyruvate not only controls metabolism but also modulates signaling pathways in hESC differentiation.

Bacterial Cell Surface Display of a Multifunctional Cellulolytic Enzyme Screened from a Bovine Rumen Metagenomic Resource.

A cell surface display system for heterologous expression of the multifunctional cellulase, CelEx-BR12, in Escherichia coli was developed using truncated E. coli outer membrane protein C (OmpC) as an anchor motif. Cell surface expression of CelEx-BR12 cellulase in E. coli harboring OmpC-fused CelEx-BR12, designated MC4100 (pTOCBR12), was confirmed by fluorescence-activated cell sorting and analysis of outer membrane fractions by western blotting, which verified the expected molecular mass of OmpC-fused CelEx-BR12 (~72 kDa). Functional evidence for exocellulase activity was provided by enzymatic assays of whole cells and outer membrane protein fractions from E. coli MC4100 (pTOCBR12). The stability of E. coli MC4100 (pTOCBR12) cellulase activity was tested by carrying out repeated reaction cycles, which demonstrated the reusability of recombinant cells. Finally, we showed that recombinant E. coli cells displaying the CelEx-BR12 enzyme on the cell surface were capable of growth using carboxymethyl cellulose as the sole carbon source.

A Transgenic Prox1-Cre-tdTomato Reporter Mouse for Lymphatic Vessel Research

The lymphatic vascular system plays an active role in immune cell trafficking, inflammation and cancer spread. In order to provide an in vivo tool to improve our understanding of lymphatic vessel function in physiological and pathological conditions, we generated and characterized a tdTomato reporter mouse and crossed it with a mouse line expressing Cre recombinase under the control of the lymphatic specific promoter Prox1 in an inducible fashion. We found that the tdTomato fluorescent signal recapitulates the expression pattern of Prox1 in lymphatic vessels and other known Prox1-expressing organs. Importantly, tdTomato co-localized with the lymphatic markers Prox1, LYVE-1 and podoplanin as assessed by whole-mount immunofluorescence and FACS analysis. The tdTomato reporter was brighter than a previously established red fluorescent reporter line. We confirmed the applicability of this animal model to intravital microscopy of dendritic cell migration into and within lymphatic vessels, and to fluorescence-activated single cell analysis of lymphatic endothelial cells. Additionally, we were able to describe the early morphological changes of the lymphatic vasculature upon induction of skin inflammation. The Prox1-Cre-tdTomato reporter mouse thus shows great potential for lymphatic research.

Synaptic mitochondria: a brain mitochondria cluster with a specific proteome.

Neurons communicate with each other through synapse, a compartment metabolically isolated from the cell body. Mitochondria are concentrated in presynaptic terminals by active transport to provide energy supply for information transfer. Mitochondrial composition in the synapse may be different than in the cell body as some examples have demonstrated altered mitochondrial composition with cell type and cellular function in the muscle, heart and liver. Therefore, we posed the question whether protein composition of synaptic mitochondria reflects its specific functions. The determined protein difference pattern was in accordance with known functional specialties of high demand synaptic mitochondria. The data also suggest specifically tuned metabolic fluxes for energy production by means of interaction with glial cells surrounding the synapse. These findings provide possible mechanisms for dynamically adapting synaptic mitochondrial output to actual demand. In turn, an increased vulnerability of synaptic mitochondria to oxidative stress is implied by the data. This is important from theoretical but potentially also from therapeutic aspects. Mitochondria are known to be affected in some neurodegenerative and psychiatric disorders, and proteins with elevated level in synaptic mitochondria, e.g. C1qbp represent targets for future drug development, by which synaptic and non-synaptic mitochondria can be differentially affected.

Transient Brown Adipocyte-Like Cells Derive from Peripheral Nerve Progenitors in Response to Bone Morphogenetic Protein 2

Perineurial-associated brown adipocyte-like cells were rapidly generated during bone morphogenetic protein 2 (BMP2)-induced sciatic nerve remodeling in the mouse. Two days after intramuscular injection of transduced mouse fibroblast cells expressing BMP2 into wild-type mice, there was replication of beta-3 adrenergic receptor(+) (ADRB3(+)) cells within the sciatic nerve perineurium. Fluorescence-activated cell sorting and analysis of cells isolated from these nerves confirmed ADRB3(+) cell expansion and their expression of the neural migration marker HNK1. Similar analysis performed 4 days after BMP2 delivery revealed a significant decrease in ADRB3(+) cells from isolated sciatic nerves, with their concurrent appearance within the adjacent soft tissue, suggesting migration away from the nerve. These soft tissue-derived cells also expressed the brown adipose marker uncoupling protein 1 (UCP1). Quantification of ADRB3-specific RNA in total hind limb tissue revealed a 3-fold increase 2 days after delivery of BMP2, followed by a 70-fold increase in UCP1-specific RNA after 3 days. Expression levels then rapidly returned to baseline by 4 days. Interestingly, these ADRB3(+) UCP1(+) cells also expressed the neural guidance factor reelin. Reelin(+) cells demonstrated distinct patterns within the injected muscle, concentrated toward the area of BMP2 release. Blocking mast cell degranulation-induced nerve remodeling resulted in the complete abrogation of UCP1-specific RNA and protein expression within the hind limbs following BMP2 injection. The data collectively suggest that local BMP2 administration initiates a cascade of events leading to the expansion, migration, and differentiation of progenitors from the peripheral nerve perineurium to brown adipose-like cells in the mouse, a necessary prerequisite for associated nerve remodeling.

Identification of fibrocytes from mesenchymal stem cells in keloid tissue: a potential source of abnormal fibroblasts in keloid scarring

Abnormal fibroblasts have been implicated in keloid formation, a benign but fibroproliferative skin disorder. However, the exact source of these cells remains unknown. Fibrocytes are considered to be hybrid mesenchymal/hematopoietic cells, having been identified in various fibrotic disorders as the precursors of fibroblasts. Therefore, we hypothesized that a population of fibrocytes is present in keloid tissue as opposed to the mesenchymal stem cells (MSCs). We compared the proportion of MSCs in keloid versus bone marrow-derived cells and compared fibrocytes in keloid as opposed to normal scar tissue. We also investigated the propagation of fibrocytes in serum-supplemented versus serum-free media. Using multicolor fluorescence-activated cell analysis, we found distinct populations of MSCs (CD34(-)CD73(+)CD90(+)CD105(+)) that were different from CD45RO(+)25F9(+)MRP8/14(+) fibrocytes present in keloid tissue, while very few fibrocytes were observed in normal scar tissue. The proportion of keloid-derived cells in serum-free or serum-supplemented cultures expressing these fibrocyte markers was greater than from cultures derived from normal scar tissue (p<0.05). We found that the proliferation of CD45(+)/Col1(+) keloid cells in vitro was greater in serum-free media compared to serum-supplemented media. This is the first study to have identified fibrocytes isolated from keloid tissue and normal scars, utilizing a specific set of markers for fibrocytes. This finding may aid our understanding of the origin of abnormal fibroblasts identified in keloid scarring. The identification of fibrocyte populations distinct from MSCs in keloid scar tissue could lead to novel targets for therapeutic intervention, treatment and prevention of recurrence.

Establishment of a docetaxel-induced human breast carcinoma drug-resistant cell line MCF7/docetaxel and its biological characteristics

Objective To establish a docetaxel-induced human breast carcinoma drug-resistant cell line MCF7/Docetaxel and study its biological characteristics. Methods The drug-resistant cell line MCF7/Docetaxel was established in culture by exposing MCF7 parent cells to moderate concentration of docetaxel over a period of 11 months. The resistance index to docetaxel was determined by methyl thiazol tetrazolium (MTT) assay. Cell growth curve was drawed and the doubling time was accounted. Flow cytometry (FCM) was performed to determine cell cycle. Fluorescence activated cell analysis (FACS) was employed to determine the concentration of fluorescence dye of rhodamine 123 ( Rh123) in the cells for the evaluation of drug-absorptive and expelling capacity. Results The resistant index of MCF7/Docetaxel was 16. 2. When MCF7/Docetaxel was stored with docetaxel at - 196℃ for 3 months and then went back to the normal environment, its characteristic of anti-tumor drug resistance was still maintained. But after being cultured in RPMI 1640 without docetaxel for a period of 1 month, MCF7/Docetaxel showed a decrease in the resistant index. As compared with the parent cells, MCF7/Docetaxel exhibited a prolonged doubling time [(39. 89 ±0. 43 vs 27. 51 ±0. 55 h,P＜0. 05] and lower growth rate. The percentages of cells in G2 and M phases were significantly increased in MCF7/Docetaxel in comparison with those in MCF7 [(39. 50 ± 3. 83) % vs (23. 59 ± 2. 51) % , P ＜ 0.05]. In soft agar growth test, MCF7/Docetaxel cells formed more cell clones than MCF7 cells (67% vs 23% ,P ＜0. 05). Finally, the concentration of Rh123 in MCF7/Docetaxel was lower than that in MCF7. Conclusion The newly established stable cell line MCF7/Docetaxel possesses typical drug-resistant characteristics and its drug-resistant phenotype is stable. Key words: Docetaxel; Drug resistance; Breast carcinoma

Optimization of a cAMP response element signal pathway reporter system

A sensitive cAMP response element (CRE) reporter system is essential for studying the cAMP/protein kinase A/cAMP response element binding protein signal pathway. Here we have tested a few CRE promoters and found one with high sensitivity to external stimuli. Using this optimal CRE promoter and the enhanced green fluorescent protein as the reporter, we have established a CRE reporter cell line. This cell line can be used to study the signal pathway by fluorescent microscope, fluorescence-activated cell analysis and luciferase assay. This cell line's sensitivity to forskolin, using the technique of fluorescence-activated cell sorting, was increased to approximately seven times that of its parental HEK 293 cell line, which is currently the most commonly used cell line in the field for the signal pathway study. Therefore, this newly created cell line is potentially useful for studying the signal pathway's modulators, which generally have weaker effect than its mediators. Our research has also established a general procedure for optimizing transcription-based reporter cell lines, which might be useful in performing the same task when studying many other transcription-based signal pathways.

New cholesterol-specific antibodies remodel HIV-1 target cells’ surface and inhibit their in vitro virus production

The importance of membrane rafts in HIV-1 infection is still in the focus of interest. Here, we report that new monoclonal anticholesterol IgG antibodies (ACHAs), recognizing clustered membrane cholesterol (e.g., in lipid rafts), rearrange the lateral molecular organization of HIV-1 receptors and coreceptors in the plasma membrane of HIV-1 permissive human T-cells and macrophages. This remodeling is accompanied with a substantial inhibition of their infection and HIV-1 production in vitro. ACHAs promote the association of CXCR4 with both CD4 and lipid rafts, consistent with the decreased lateral mobility of CXCR4, while Fab fragments of ACHAs do not show these effects. ACHAs do not directly mask the extracellular domains of either CD4 or CXCR4 nor do they affect CXCR4 internalization. No significant inhibition of HIV production is seen when the virus is preincubated with the antibodies prior to infection. Thus, we propose that the observed inhibition is mainly due to the membrane remodeling induced by cholesterol-specific antibodies on the target cells. This, in turn, may prevent the proper spatio-temporal juxtaposition of HIV-1 glycoproteins with CD4 and chemokine receptors, thus negatively interfering with virus attachment/entry.

Increased levels of circulating endothelial progenitor cells in subjects with moderate to severe chronic periodontitis

Emerging evidence shows that periodontal disease is associated with endothelial dysfunction. The purpose of this study was to determine the association between chronic periodontitis (CP) and circulating endothelial progenitor cells (EPC). Eighty-six non-smoking subjects (36 males and 50 females, aged 35-80 years) were recruited, including 23 subjects with no or mild CP and 63 subjects with moderate to severe CP. The levels of circulating EPC were quantitatively determined by fluorescence-activated cell analysis, including CD34+/kinase insert domain-containing receptor (KDR)+ (more mature EPC) and CD133+/KDR+ (less mature EPC). Periodontal conditions, the intima-media thickness of carotid arteries and circulating biomarkers were examined. Subjects with moderate to severe CP exhibited an increased risk of high EPC count, compared with those with no or mild CP: CD34+/KDR+ EPC [odds ratio (OR)=9.5, 95% confidence interval (95% CI) 1.5-61.0, p=0.018; CD133+/KDR+ EPC, OR=4.6, 95% CI 1.1-19.5, p=0.039]. C-reactive protein was significantly associated with high CD34+/KDR+ EPC count and age was inversely related with high EPC count. Age, gender and CD34+/KDR+ EPC were independent variables of increased carotid intima-media thickness (p<0.05). This study shows for the first time that moderate to severe CP is associated with an increased level of circulating EPC.

Therapierefraktäre Hauttuberkulose nach Methylprednisolon-Therapie

Systemic lupus erythematodes was suspected in a 55-year-old patient with arthralgias, secondary Raynaud's syndrome with acral necroses and increased antinuclear antibodies. Treatment with methylprednisolone was started but despite initial improvement of symptoms, the fever, weight loss and nocturnal sweating continued and the patient developed a progressive erythema on his left thigh. Skin biopsy revealed Mycobacterium tuberculosis. Drug sensitivity tests of the initial culture isolate from the skin biopsy indicated response to all tested first-line drugs. The initial chest-x-ray showed only posttubercular lesions in both upper lobes. Computed tomography revealed a progressive infiltration in the right upper lobe and multiple disseminated lesions in both lungs. Mycobacteria were cultured from sputum. A fluorescence-activated cell analysis of peripheral leucocytes showed a decreased total T-cell-count of 173/microl. Tuberculostatic treatment with five first-line drugs led to a temporary improvement of the patient's general condition and of the inflammatory signs. But the initially small cutaneous erythema expanded and developed into a large abscess, which could not be controlled even with surgery. Multiple necrotic lesions then developed rapidly in different areas of the skin without any response to various tuberculostatic drugs. Despite of several months of treatment the patient died of septic multi-organ-failure. A possible explanation for the treatment-failure may have been the state of low immunity, either as a result of an underlying disease or after the methylprednisolone therapy. An alternative reason could be the insufficient penetration of the antibiotics into the necrotic abscesses.

Proliferation, differentiation, and cytokine secretion of human umbilical cord blood–derived mononuclear cells in vitro

Human umbilical cord blood (hUCB)-derived mononuclear cells were previously shown to exert therapeutic effects in a number of animal models of nervous system impairment. However, the mechanisms underlying the structural and functional improvements are still unclear. As cell replacement seems to be a rare or absent event in vivo, we suggest secondary mechanisms, by which the therapeutic effect of transplanted mononuclear cells might be mediated. We investigated the potential of hUCB-derived mononuclear cells in vitro to proliferate, differentiate, and to secrete factors possibly beneficial for the host brain tissue in vivo. Using a succession of distinct culture media, mononuclear cells were stimulated by growth factor combinations, e.g., epidermal growth factor (EGF)/fibroblast growth factor-2 (FGF-2) or nerve growth factor (NGF)/retinoic acid (RA). Expression of hematological and neural marker proteins was investigated by immunoblotting, immunocytochemistry, and fluorescence-activated cell analysis. Secretion of proteins was assayed using a human cytokine antibody array, and quantified via enzyme-linked immunosorbent assay. Mononuclear cells were shown to undergo proliferation in the presence of EGF/FGF-2. When cells were cultured in NGF/RA-containing medium, neuronal and glial marker proteins were expressed, indicating differentiation. In the presence of either growth factor combination, cells in vitro secrete interleukins, growth factors, and chemotactic proteins. Although capable of incipient differentiation, cytokine secretion of hUCB-derived mononuclear cells envisages the potential of an indirect effect in vivo. Most factors detected in conditioned medium are renowned for their anti-inflammatory, neuroprotective, angiogenic, or chemotactic actions, thus, providing the means for a therapeutic outcome mediated by secondary effects.

Genetic control of NKT cell numbers maps to major diabetes and lupus loci.

Natural killer T cells are an immunoregulatory population of lymphocytes that plays a critical role in controlling the adaptive immune system and contributes to the regulation of autoimmune responses. We have previously reported deficiencies in the numbers and function of NKT cells in the nonobese diabetic (NOD) mouse strain, a well-validated model of type 1 diabetes and systemic lupus erythematosus. In this study, we report the results of a genetic linkage analysis of the genes controlling NKT cell numbers in a first backcross (BC1) from C57BL/6 to NOD.Nkrp1(b) mice. The numbers of thymic NKT cells of 320 BC1 mice were determined by fluorescence-activated cell analysis using anti-TCR Ab and CD1/alpha-galactosylceramide tetramer. Tail DNA of 138 female BC1 mice was analyzed for PCR product length polymorphisms at 181 simple sequence repeats, providing greater than 90% coverage of the autosomal genome with an average marker separation of 8 cM. Two loci exhibiting significant linkage to NKT cell numbers were identified; the most significant (Nkt1) was on distal chromosome 1, in the same region as the NOD mouse lupus susceptibility gene Babs2/Bana3. The second most significant locus (Nkt2) mapped to the same region as Idd13, a NOD-derived diabetes susceptibility gene on chromosome 2.

Increased lactotrophs despite decreased somatotrophs in the dwarf (dw/dw) rat: a defect in the regulation of lactotroph/somatotroph cell fate?

The dwarf (dw/dw) rat differs from all other rodent models of GH deficiency in that its pituitary prolactin (PRL) content is normal or even increased. We have now studied this throughout postnatal development, using a combination of immunocytochemistry, RIA and fluorescence-activated cell sorting (FACS) and analysis. Compared with normal Albino Swiss (AS) rats, adult dw/dw rats showed a profound reduction in pituitary GH content accompanied by increased PRL content, significantly so in females (AS vs dw/dw; P<0.01). Somatotroph hypoplasia was evident in the adult dw/dw rats, with most GH(+ve) cells showing weak immunostaining, whereas many more strongly stained PRL cells were evident in pituitary sections from dw/dw rats. Facs analysis confirmed both somatotroph hypoplasia and relative lactotroph hyperplasia in dw/dw rats at all ages studied (9-144 days); the difference in somatotrophs increased with age whereas the difference in lactotrophs declined with age. At 9 days, the percentage of lactotrophs was 10-fold higher in dw/dw rats than in AS rats. Young dw/dw rats also had a higher proportion of mammosomatotrophs than AS rats, although this difference disappeared as the mammosomatotroph proportions increased with age in both strains. GHRH released GH from both dw/dw and as cells maintained in culture for 5 days. The sensitivity to GHRH and the amount of GH released was lower in the dw/dw cultures, mostly explained by their fewer GH cells and lower initial GH content. GHRH increased cAMP in as but not in dw/dw cultures, even when these were greatly enriched for dw/dw somatotrophs by FACS sorting prior to culture. These results suggest that GHRH-induced cAMP stimulation is required for trophic effects on GH synthesis and somatotroph proliferation, but is not required for GHRH-stimulated GH release. The inverse changes in somatotroph and lactotroph numbers suggest that the dw/dw mutation disturbs the mechanism that specifies and retains appropriate numbers of somatotrophs in their differentiated state, and results in a higher proportion of the remaining cells progressing to lactotrophs. The dw/dw phenotype is thus not confined to somatotrophs.

Phagocytosis by Emigrated, Intra-Abdominal Neutrophils Is Depressed during Human Secondary Peritonitis

The phagocytic function of neutrophils is a crucial element in host defense against invading microorganisms. Patients with diffuse peritonitis depend on adequate reactivity of neutrophils, in particular locally in the peritoneal cavity as well as in the circulation. This study examined phagocytosis as well as numerical expression of Fcγ I–III (CD16, CD32, CD64) and complement receptors (CD18, CD35) of emigrated, intra-abdominal and circulating neutrophils during human secondary peritonitis using fluorescence-activated cell analysis. Optimally opsonized E. coli bacteria were used independently of the well-known low level of opsonic molecules during peritonitis. Compared with controls (abdominal surgery without peritonitis), the percentage of emigrated neutrophils which engulfed E. coli bacteria was significantly depressed until 48 h after diagnosis of, and surgery for, peritonitis. When patients with complicated peritonitis (septic shock, multiple organ failure) were compared with patients without complications, phagocytosis was even more depressed in patients with complications. Numerical expression of CD64 (Fcγ RI) and CD35 (CR1) increased significantly on emigrated polymorphonuclear leukocytes (PMNs) during peritonitis when compared to controls. There was no difference in CD18 and CD32 (Fcγ RII) expression between the two groups. Numerical expression of CD16 (Fcγ RIII) on emigrated PMNs decreased significantly in peritonitis. This was more pronounced in patients with complicated peritonitis. We conclude that there is a long-lasting depression of phagocytosis by emigrated PMNs during peritonitis, independent of the opsonic activity. Our data suggest that decreased phagocytosis might be correlated to the profound drop in CD16 on these cells.

Use of VSV-G pseudotyped retroviral vectors to target murine osteoprogenitor cells.

Marrow stromal cells (MSC) and neonatal calvarial cells have the potential to differentiate and express markers of mature osteoblasts. Furthermore, MSCs can generate multiple differentiated connective tissue phenotypes. These properties and their ability to be expanded ex vivo make them good models for ex vivo gene therapy. In this study we examined the ability of vesicular stomatitis virus (VSV-G) pseudotyped retroviral vectors to transduce osteoprogenitor cells derived from bone marrow and from neonatal calvaria. Retrovectors encoding either beta-galactosidase or green fluorescent protein (eGFP) were used for transduction of primary murine marrow stromal and primary neonatal calvarial cell cultures. High infection efficiency was demonstrated by fluorescence-activated cell analysis when GFP was used as a marker or by estimating the number of beta-galactosidase-positive cells. Expression of markers of differentiated bone cells, including Col1a1, bone sialoprotein, and osteocalcin mRNA and alkaline phosphatase activity was not impaired by retroviral transduction. Our data suggest that VSV-G pseudotypes retroviral vectors are suitable for introducing genes into osteoprogenitor cells without affecting osteoprogenitor lineage progression.

Monocytes coexpress endothelial and macrophagocytic lineage markers and form cord-like structures in Matrigel under angiogenic conditions.

It has been shown that circulating human non-adherent CD34+ cells coexpressing vascular endothelial growth factor (VEGF)-R2 and AC133 have the capacity to differentiate into adherent mature endothelial cells. However, prior studies have demonstrated that a much bigger subset of primary adherent mononuclear cells can also form endothelial progenitor cells (EPC). To determine the origin of the latter cell population we tested the hypothesis: do monocytes as a firmly adherent and plastic cell type have the potential to differentiate into an endothelial phenotype. CD34-/CD14+ monocytes were isolated from human peripheral blood by adherence separation and magnetic bead selection (purity >90%) and cultured on fibronectin-coated plastic dishes (medium containing VEGF 10 ng/ml, basic fibroblast growth factor (bFGF) 2 ng/ml, insulin like growth factor (IGF-1) 1 ng/ml, 20% fetal calf serum). After 2 weeks of culture, using fluorescence activated cell analysis we observed a new expression of the endothelial markers von Willebrand factor (vWf), VE-cadherin (VE) and ec-NOS in 45.2, 12.4 and 9.8% of the cells, respectively. The proportion of cells expressing these markers further increased after 4 weeks (94.2, 89.7 and 58.8% of these cells, respectively). The proportion of CD45 expressing cells remained unchanged during this period. However, after 14 days the specific macrophage antigen CD68 was newly expressed in 62% of the analysed cells with a further increase to 90% after 28 days of culture. In three-dimensional gel (Matrigel the formation of cord- and tubular-like structures was observed. The present data indicate that under angiogenic stimulation macrophages develop an endothelial phenotype with expression of specific surface markers and even form cord- and tubular-like structures in vitro suggesting that this cell population may be recruited for vasculogenesis.