Novel coronavirus SARS-CoV-2 (Covid-19) dynamics inside the human body.

A knowledge-based cybernetic framework model representing the dynamics of SARS-CoV-2 inside the human body has been studied analytically and in silico to explore the pathophysiologic regulations. The following modeling methodology was developed as a platform to introduce a predictive tool supporting a therapeutic approach to Covid-19 disease. A time-dependent nonlinear system of ordinary differential equations model was constructed involving type-I cells, type-II cells, SARS-CoV-2 virus, inflammatory mediators, interleukins along with host pulmonary gas exchange rate, thermostat control, and mean pressure difference. This formalism introduced about 17 unknown parameters. Estimating these unknown parameters requires a mathematical association with the in vivo sparse data and the dynamic sensitivities of the model. The cybernetic model can simulate a dynamic response to the reduced pulmonary alveolar gas exchange rate, thermostat control, and mean pressure difference under a very critical condition based on equilibrium (steady state) values of the inflammatory mediators and system parameters. In silico analysis of the current cybernetical approach with system dynamical modeling can provide an intellectual framework to help experimentalists identify more active therapeutic approaches.

2'-Fluoro-Pyrimidine-Modified RNA Aptamers Specific for Lipopolysaccharide Binding Protein (LBP).

Lipopolysaccaride binding protein (LBP), a glycosylated acute phase protein, plays an important role in the pathophysiology of sepsis. LBP binds with high affinity to the lipid part of bacterial lipopolysaccaride (LPS). Inhibition of the LPS-LBP interaction or blockage of LBP-mediated transfer of LPS monomers to CD14 may be therapeutical strategies to prevent septic shock. LBP is also of interest as a biomarker to identify septic patients at high risk for death, as LBP levels are elevated during early stages of severe sepsis. As a first step toward such potential applications, we isolated aptamers specific for murine LBP (mLBP) by in vitro selection from a library containing a 60-nucleotide randomized region. Modified RNA pools were transcribed in the presence of 2'-fluoro-modified pyrimidine nucleotides to stabilize transcripts against nuclease degradation. As verified for one aptamer experimentally, the selected aptamers adopt a "three-helix junction" architecture, presenting single-stranded 7-nt (5'-YGCTTCY) or 6-nt (5'-RTTTCY) consensus sequences in their core. The best binder (aptamer A011; Kd of 270 nM for binding to mLBP), characterized in more detail by structure probing and boundary analysis, was demonstrated to bind with high specificity to murine LBP.

Lipocalin-2 promotes m1 macrophages polarization in a mouse cardiac ischaemia-reperfusion injury model.

Ischaemia-reperfusion (IR) injury is a major issue in cardiac transplantation. Inflammatory processes play a major role in myocardial IR injury. Lipocalin-2 (Lcn2), which is also known as neutrophil gelatinase-associated lipocalin, has multiple functions that include the regulation of cell death/survival, cell migration/invasion, cell differentiation and iron delivery. In our study, the hearts of C57BL/6 mice were flushed with and stored in cold Bretschneider solution for 8 h and then transplanted into a syngeneic recipient. We found that Lcn2 neutralization decreased the recruitment of neutrophils and macrophages. Troponin T (TnT) production, 24 h after myocardial IR injury, was reduced through anti-Lcn2 antibody administration. The cardiac output at 60 mmHg of afterload pressure was significantly increased in hearts administrated with anti-Lcn2 antibody administration (anti-Lcn-2: 58.9 ± 5.62 ml/min; control: 25.8 ± 4.1 ml/min; P < 0.05). Anti-Lcn2 antibody treatment suppressed M1 marker (IL-12, IL-23 and iNOS) expression but increased M2 marker (IL-10, Arg1 and Mrc1) expression. Furthermore, in our vitro and vivo experiments, we found that anti-Lcn2 antibody treatment failed to induce M1-related gene expression in response to LPS and that Lcn2 neutralization enhanced the expression of M2-related genes following IL-4 treatment. In conclusion, Lcn2 promotes M1 polarization, and Lcn2 neutralization attenuates cardiac IR injury.

Inhibiting metastatic breast cancer cell migration via the synergy of targeted, pH-triggered siRNA delivery and chemokine axis blockade.

Because breast cancer patient survival inversely correlates with metastasis, we engineered vehicles to inhibit both the C-X-C chemokine receptor type 4 (CXCR4) and lipocalin-2 (Lcn2) mediated migratory pathways. pH-responsive liposomes were designed to protect and trigger the release of Lcn2 siRNA. Liposomes were modified with anti-CXCR4 antibodies to target metastatic breast cancer (MBC) cells and block migration along the CXCR4-CXCL12 axis. This synergistic approach--coupling the CXCR4 axis blockade with Lcn2 silencing--significantly reduced migration in triple-negative human breast cancer cells (88% for MDA-MB-436 and 92% for MDA-MB-231). The results suggested that drug delivery vehicles engineered to attack multiple migratory pathways may effectively slow progression of MBC.

Liver transplantation and inflammation: is lipopolysaccharide binding protein the link?

BACKGROUND: Lipopolysaccharide (LPS) binding protein (LBP) is an acute phase protein, which upregulated in response to surgical interventions. LBP plays an important role in modulating LPS-induced inflammatory response. We investigated the expression of LBP and the translocation of LPS in rat models of hepatic ischemia reperfusion injury and liver transplantation (LTx). We also elucidated the effect of LBP on the inflammatory response. METHODS: In this study, cold ischemia (CI), warm ischemia/reperfusion (WI/R), and LTx models were used to model relevant physiologic situations during LTx. Serum and effluent protein levels as well as hepatic-mRNA and protein levels of LBP were examined. LBP released into the effluent during CI was used in a macrophage-LPS-stimulation assay to investigate the role of LBP in modulating the LPS-induced inflammatory response. Blocking experiments using an LBP-inhibitory peptide were performed to confirm the relevance of LPS/LBP for the induction of the inflammatory response. Impairment of the intestinal barrier and translocation of LPS into the liver was visualized by immunohistochemistry. Induction of tumor necrosis factor-alpha (TNF-α) mRNA expression in the liver was taken as indicator of the inflammatory response. RESULTS: Upregulation of LBP in serum and/or liver tissue was observed after WI/R, CI and LTx, respectively. The LBP released during CI enhanced the LPS induced inflammatory response in vitro as indicated by an induction of TNF-α. On the other hand, blocking LBP using LBP inhibitory peptide, suppressed the induction of TNF-α in vitro markedly. After LTx, elevated serum LBP levels were associated with post-operative LPS translocation and production of inflammatory cytokines. CONCLUSIONS: Our findings suggest that translocation of LPS occurs after LTx and that LBP is mediating the LPS-induced inflammatory response after LTx. Blocking LBP using LBP-inhibitory peptide might represent a novel strategy to reduce the I/R-induced inflammatory response.

Identification of lipopolysaccharide-binding peptide regions within HMGB1 and their effects on subclinical endotoxemia in a mouse model.

Lipopolysaccharide (LPS) triggers deleterious systemic inflammatory responses when released into the circulation. LPS-binding protein (LBP) in the serum plays an important role in modifying LPS toxicity by facilitating its interaction with LPS signaling receptors, which are expressed on the surface of LPS-responsive cells. We have previously demonstrated that high mobility group box 1 (HMGB1) can bind to and transfer LPS, consequently increasing LPS-induced TNF-α production in human peripheral blood mononuclear cells (PBMCs). We report here on the identification of two LPS-binding domains within HMGB1. Furthermore, using 12 synthetic HMGB1 peptides, we define the LPS-binding regions within each domain. Among them, synthetic peptides HPep1 and HPep6, which are located in the A and B box domains of HMGB1, bind to the polysaccharide and lipid A moieties of LPS respectively. Both HPep1 and HPep6 peptides inhibited binding of LPS to LBP and HMGB1, LBP-mediated LPS transfer to CD14, and cellular uptake of LPS in RAW264.7 cells. These peptides also inhibited LPS-induced TNF-α release in human PBMCs and induced lower levels of TNF-α in the serum in a subclinical endotoxemia mouse model. These results indicate that HMGB1 has two LPS-binding peptide regions that can be utilized to design anti-sepsis or LPS-neutralizing therapeutics.

Klebsiella pneumoniae yersiniabactin promotes respiratory tract infection through evasion of lipocalin 2.

Klebsiella pneumoniae is a pathogen of increasing concern because of multidrug resistance, especially due to K. pneumoniae carbapenemases (KPCs). K. pneumoniae must acquire iron to replicate, and it utilizes iron-scavenging siderophores, such as enterobactin (Ent). The innate immune protein lipocalin 2 (Lcn2) is able to specifically bind Ent and disrupt iron acquisition. To determine whether K. pneumoniae must produce Lcn2-resistant siderophores to cause disease, we examined siderophore production by clinical isolates (n = 129) from respiratory, urine, blood, and stool samples and by defined siderophore mutants through genotyping and liquid chromatography-mass spectrometry. Three categories of K. pneumoniae isolates were identified: enterobactin positive (Ent(+)) (81%), enterobactin and yersiniabactin positive (Ent(+) Ybt(+)) (17%), and enterobactin and salmochelin (glycosylated Ent) positive (Ent(+) gly-Ent(+)) with or without Ybt (2%). Ent(+) Ybt(+) strains were significantly overrepresented among respiratory tract isolates (P = 0.0068) and ß-lactam-resistant isolates (P = 0.0019), including the epidemic KPC-producing clone multilocus sequence type 258 (ST258). In ex vivo growth assays, gly-Ent but not Ybt allowed evasion of Lcn2 in human serum, whereas siderophores were dispensable for growth in human urine. In a murine pneumonia model, an Ent(+) strain was an opportunistic pathogen that was completely inhibited by Lcn2 but caused severe, disseminated disease in Lcn2(-/-) mice. In contrast, an Ent(+) Ybt(+) strain was a frank respiratory pathogen, causing pneumonia despite Lcn2. However, Lcn2 retained partial protection against disseminated disease. In summary, Ybt is a virulence factor that is prevalent among KPC-producing K. pneumoniae isolates and promotes respiratory tract infections through evasion of Lcn2.

A Randomized Double-Masked Phase 2a Trial to Evaluate Activity and Safety of Topical Ocular Reproxalap, a Novel RASP Inhibitor, in Dry Eye Disease.

Purpose: To determine whether reproxalap, a novel reactive aldehyde species (RASP) inhibitor, is safe and effective for the treatment of the signs and symptoms of dry eye disease (DED). Methods: In a randomized double-masked parallel-group Phase 2a trial of 3 topical ocular reproxalap formulations (0.1% ophthalmic solution, 0.5% ophthalmic solution, and 0.5% lipid ophthalmic solution), 51 patients with DED were randomly assigned 1:1:1 at a single US site. Eyes were treated bilaterally 4 times daily for 28 days, and standard DED signs and symptoms were assessed at baseline and after 7 and 28 days of dosing. Tear RASP levels were assessed at baseline and at day 28. Results: The effect of treatment on DED signs and symptoms was similar across the treatment arms, and pooled data from the 28-day treatment period demonstrated significant improvement from baseline in Symptom Assessment in Dry Eye Disease score (P = 0.003), Ocular Discomfort Scale score (P < 0.0001), Ocular Discomfort Score and 4-Symptom Questionnaire overall score (P = 0.0004), Schirmer's test (P = 0.008), tear osmolarity (P = 0.003), and lissamine green total staining score (P = 0.002). Improvements in DED symptoms were evident within 1 week of therapy, and effect sizes generally approached or exceeded 0.5. No significant changes in safety measures were observed. Conclusion: The results suggest that the novel RASP inhibitor reproxalap has the potential to mitigate the signs and symptoms of DED, and may represent a new, rapidly and broadly active treatment approach for DED (NCT03162783).

Lipocalin-2: pro- or anti-apoptotic?

Survival and apoptosis signaling pathways are altered concomitantly in response to numerous endogenous and exogenous stressors. The lipocalin family of small soluble proteins has been implicated in modulating apoptosis. However, the overall effect of these proteins has been variable, showing both pro- and anti-apoptotic activities. The goal of this minireview is to summarize the studies on lipocalins and apoptosis and consider what roles lipocalin-2 may play in cell death and survival.

Healing of incisional wounds in the embryonic chick wing bud: characterization of the actin purse-string and demonstration of a requirement for Rho activation.

Small skin wounds in the chick embryo do not heal by lamellipodial crawling of cells at the wound edge as a skin wound does in the adult, but rather by contraction of an actin purse-string that rapidly assembles in the front row of epidermal cells (Martin, P., and J. Lewis. 1992. Nature (Lond.). 360:179-183). To observe the early time course of actin purse-string assembly and to characterize other cytoskeletal components of the contractile machinery, we have followed the healing of incisional or slash wounds on the dorsum of the chick wing; these wounds take only seconds to create and heal within approximately 6 h. Healing of the epithelium depends on a combination of purse-string contraction and zipper-like closure of the gap between the cut edges of the epithelium. Confocal laser scanning microscope studies show that actin initially aligns into a cable at the wound margin in the basal layer of the epidermis within approximately 2 min of wounding. Coincident with actin cable assembly, we see localization of cadherins into clusters at the wound margin, presumably marking the sites where segments of the cable in adjacent cells are linked via adherens junctions. A few minutes later we also see localization of myosin II at the wound margin, as expected if myosin is being recruited into the cable to generate a contractile force for wound healing. At the time of wounding, cells at the wound edge become transiently leaky, allowing us to load them with reagents that block the function of two small GTPases, Rho and Rac, which recently have been shown to play key roles in reorganiztion of the actin cytoskeleton in tissue-culture cells (Hall, A. 1994. Annu. Rev. Cell Biol. 10:31-54). Loading wound edge epidermal cells with C3 transferase, a bacterial exoenzyme that inactivates endogenous Rho, prevents assembly of an actin cable and causes a failure of healing. No such effects are seen with N17rac, a dominant inhibitory mutant Rac protein. These findings support the view that in this system the actin cable is required for healing-both the purse-string contraction and the zipping up-and that Rho is required for formation of the actin cable.

Identification of new accessible tumor antigens in human colon cancer by ex vivo protein biotinylation and comparative mass spectrometry analysis.

One of the most promising new strategies for the development of efficacious cancer therapies relies on the targeted delivery of biopharmaceutical to the tumor environment by the use of selective and specific antibodies. The identification of accessible perivascular proteins selectively overexpressed in cancer tissue may facilitate the development of antibody-based biopharmaceutical administration. This approach is potentially highly selective and specific, combining the presence of tumor biomarkers readily accessible from the blood vessels and the high rate of angiogenesis characteristic of cancer tissues. We performed ex vivo perfusions of surgically resected human colon cancer using a reactive ester derivative of biotin, thus achieving a selective covalent modification of accessible proteins in vascular structures and stroma. After extraction and purification, biotinylated proteins were digested and the resulting peptides submitted to a comparative mass spectrometry-based proteomic analysis, revealing quantitative differences between normal and cancer colon. Sixty-seven of the total 367 proteins identified were found to be preferentially expressed at the tumor site. We generated human monoclonal antibodies against 2 potential tumor targets, NGAL and GW112, and we proved their selective expression in cancer colon and not or barely in healthy tissues. This article presents the first proteomic analysis of human colorectal cancer structures readily accessible from the tumor vasculature, revealing the overexpression of novel tumor antigens which may serve as selective targets for antibody-based imaging and therapeutic biomolecular strategies.

Antioxidants modulate the IL-6 induced inhibition of negative acute-phase protein secretion in HepG2 cells.

Despite increasing evidence on the potential of dietary antioxidants in modulating the etiology of certain chronic diseases such as cancer and cardiovascular diseases, little is known about their beneficial role in acute-phase responses and inflammatory diseases. From this viewpoint the aim of this study was to investigate the effect of selected dietary antioxidants in modulating the secretion of negative acute-phase proteins caused by interleukin-6 (IL-6) in HepG2 cells. Cells were first stimulated with a fixed dose of IL-6 for 24 h then incubated for a further 8 h with varying concentrations of eight antioxidants, alpha-lipoic acid (LA), (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCG), alpha-tocopherol (TOC), ascorbic acid (AA) and N-acetylcysteine (NAC). The culture supernatants were assayed for transthyretin (TTR) and retinol binding protein (RBP) using ELISA. The data revealed that IL-6 significantly reduced TTR and RBP secretion compared with the basal production. All tested antioxidants attenuate the reduction in TTR and RPB levels. The strongest effects were achieved with the highest concentration of each antioxidant. The order of effect were LA > EGCG > ECG > TOC > EGC > EC > NAC > AA. In conclusion, these results provide evidence that the dietary antioxidants can play a fundamental role in inflammatory processes.

Up-regulation of miR-138 inhibits hypoxia-induced cardiomyocyte apoptosis via down-regulating lipocalin-2 expression.

Hypoxia-induced cardiomyocyte apoptosis contributes significantly to the development of numerous cardiac diseases, such as ischemic heart disease, heart failure, etc. Promoting cell survival by inhibiting apoptosis is one of the available strategies to attenuate cardiac dysfunction caused by cardiomyocyte loss. Previous studies have been demonstrated that miR-138 and lipocalin-2 (Lcn2) play important roles in cardiomyocyte apoptosis and survival. We presently determined whether Lcn2 is a target gene of miR-138 involved in hypoxia-induced cardiomyocyte apoptosis. Firstly, mimics of miR-138 were transfected into HL-1 cells to investigate its effect on cell apoptosis. Using 3-(4,5-dimethyl-thiazol-2-y1) 2,5-diphenyl tetrazolium bromide (MTT) and Annexin V-FITC/PI flow cytometer assays, over-expression of miR-138 significantly enhanced the cell growth and significantly attenuated the cell apoptosis in hypoxic conditions. Dual-luciferase reporter gene and western blot results confirmed Lcn2 was a direct target of miR-138. Then, the recombinant plasmid, pcDNA3.1/Lcn2 was transfected into the HL-1 cells that over-expressed miR-138. We further observed that the over-expression of Lcn2 diminished the protection of miR-138 over-expression from hypoxia-induced cell survival and apoptosis. In conclusion, our study demonstrated that up-regulation of miR-138 inhibits the hypoxia-induced cardiomyocyte apoptosis via down-regulating the pro-apoptotic gene expression of Lcn2.

ICAM-1-Targeted, Lcn2 siRNA-Encapsulating Liposomes are Potent Anti-angiogenic Agents for Triple Negative Breast Cancer.

Lipocalin 2 (Lcn2) is a promising therapeutic target as well as a potential diagnostic biomarker for breast cancer. It has been previously shown to promote breast cancer progression by inducing the epithelial to mesenchymal transition in breast cancer cells as well as by enhancing angiogenesis. Lcn2 levels in urine and tissue samples of breast cancer patients has also been correlated with breast cancer status and poor patient prognosis. In this study, we have engineered a novel liposomal small interfering RNA (siRNA) delivery system to target triple negative breast cancer (TNBC) via a recently identified molecular target, intercellular adhesion molecule-1 (ICAM-1). This ICAM-1-targeted, Lcn2 siRNA- encapsulating liposome (ICAM-Lcn2-LP) binds human TNBC MDA-MB-231cells significantly stronger than non-neoplastic MCF-10A cells. Efficient Lcn2 knockdown by ICAM-Lcn2-LPs led to a significant reduction in the production of vascular endothelial growth factor (VEGF) from MDA-MB-231 cells, which, in turn, led to reduced angiogenesis both in vitro and in vivo. Angiogenesis (neovascularization) is a requirement for solid tumor growth and progression, and its inhibition is an important therapeutic strategy for human cancers. Our results indicate that a tumor-specific strategy such as the TNBC-targeted, anti-angiogenic therapeutic approach developed here, may be clinically useful in inhibiting TNBC progression.

Inhibition of constitutively active Stat3 suppresses proliferation and induces apoptosis in glioblastoma multiforme cells.

Glioblastoma multiforme (GBM), the most common and malignant central nervous system tumor in humans, is highly proliferative and resistant to apoptosis. Stat3, a latent transcription factor being activated by aberrant cytokine or growth factor signaling, acts as a suppressor of apoptosis in a number of cancer cells. Here we report that GBM tumors and cell lines contain high levels of constitutively activated Stat3 when compared with normal human astrocytes, white matter, and normal tissue adjacent to tumor. The persistent activation of Stat3 is in part, attributable to an autocrine action of interleukin-6 in the GBM cell line U251. Janus kinase inhibitor AG490 inhibits Stat3 activation with a concomitant reduction in steady-state levels of Bcl-X(L), Bcl-2 and Mcl-1 proteins and induces apoptosis in U251 cells as revealed by Poly (ADP-ribose) polymerase cleavage and Annexin-V staining. Expression of a dominant negative mutant Stat3 protein or treatment with AG490 markedly reduces the proliferation of U251 cells by inhibiting the constitutive activation of Stat3. These results provide evidence that constitutive activation of Stat3 contributes to the pathogenesis of glioblastoma by promoting both proliferation and survival of GBM cells. Therefore, targeting Stat3 signaling may provide a potential therapeutic intervention for GBM.

Differential involvement of the actin cytoskeleton in differentiation and mitogenesis of thyroid cells: inactivation of Rho proteins contributes to cyclic adenosine monophosphate-dependent gene expression but prevents mitogenesis.

In thyroid epithelial cells, TSH via cAMP induces a rounding up of the cells associated with actin stress fiber disruption, expression of differentiation genes and cell cycle progression. Here we have evaluated the role of small G proteins of the Rho family and their impact on the actin cytoskeleton in these different processes in primary cultures of canine thyrocytes. TSH and forskolin, but not growth factors, rapidly inactivated RhoA, Rac1, and Cdc42, as assayed by detection of GTP-bound forms. Using toxins that inactivate Rho proteins (toxin B, C3 exoenzyme) or activate them [cytotoxic necrotizing factor 1 (CNF1)], in comparison with disruption of the actin cytoskeleton by dihydrocytochalasin B (DCB) or latrunculin, two unexpected conclusions were reached: 1) inactivation of Rho proteins by cAMP, by disorganizing actin microfilaments and inducing cell retraction, could be necessary and sufficient to mediate at least part of the cAMP-dependent induction of thyroglobulin and thyroid oxidases, but only partly necessary for the induction of Na(+)/I(-) symporter and thyroperoxidase; 2) as indicated by the effect of their inhibition by toxin B and C3, some residual activity of Rho proteins could be required for the induction by cAMP-dependent or -independent mitogenic cascades of DNA synthesis and retinoblastoma protein (pRb) phosphorylation, through mechanisms targeting the activity, but not the stimulated assembly, of cyclin D3-cyclin-dependent kinase 4 complexes. However, at variance with current concepts mostly derived from fibroblast models, DNA synthesis induction and cyclin D3-cyclin-dependent kinase 4 activation were resistant to actin depolymerization by dihydrocytochalasin B in canine thyrocytes, which provides a first such example in a normal adherent cell.

Protein-bound polysaccharide isolated from basidiomycetes inhibits endotoxin-induced activation by blocking lipopolysaccharide-binding protein and CD14 functions.

The protein-bound polysaccharide isolated from basidiomycetes (PSK) is a biological response modifier capable of exhibiting various biological activities, such as antitumor and antimicrobial effects. In the present study, we found that PSK suppressed interleukin (IL)-6 production in murine peritoneal macrophages stimulated with endotoxic lipopolysaccharide (LPS) and its synthetic lipid A (compound 506). Nitric oxide production and p38 mitogen-associated protein kinase phosphorylation induced in a murine macrophage cell line, J774-A1, by LPS and compound 506 were also inhibited by PSK. Further, PSK distinctly suppressed nuclear factor-kappaB activation in Ba/F3 cells expressing mouse Toll-like receptor 4 and MD-2, following stimulation with LPS and compound 506, however, not with Taxol. These PSK-induced inhibitory activities were caused by inhibition of the physical associations of LPS with LPS-binding protein (LBP) and CD14. PSK also protected mice from LPS-induced lethality, presumably by down-regulating IL-6 and tumor necrosis factor-alpha concentrations in serum. These findings indicate that PSK, which also has an ability to regulate LBP/CD14 functions, may be useful for clinical control of endotoxic sepsis.

Drug-loaded red blood cell-mediated clearance of HIV-1 macrophage reservoir by selective inhibition of STAT1 expression.

Current highly active antiretroviral therapy (HAART) cannot eliminate HIV-1 from infected persons, mainly because of the existence of refractory viral reservoir(s). Beyond latently-infected CD4+-T lymphocytes, macrophages (M/M) are important persistent reservoirs for HIV in vivo, that represent a major obstacle to HIV-1 eradication. Therefore, a rational therapeutic approach directed to the selective elimination of long-living HIV-infected M/M may be relevant in the therapy of HIV infection. Here we report that HIV-1 chronic infection of human macrophages results in the marked increase of expression and phosphorylation of STAT1, a protein involved in the regulation of many functions such as cell growth, differentiation, and maintenance of cellular homeostasis, thereby providing a new molecular target for drug development. A single and brief exposure to 9-(beta-D-arabinofuranosyl)-2-fluoroadenine 5'-monophosphate (FaraAMP, Fludarabine), a potent antileukemic nucleoside analog active against STAT1 expressing cells, selectively kills macrophage cultures infected by HIV-1 without affecting uninfected macrophages. Furthermore, encapsulation of Fludarabine into autologous erythrocytes (RBC) and targeting to macrophages through a single-18 h treatment with drug-loaded RBC, not only abolishes the Fludarabine-mediated toxic effect on non-phagocytic cells, but also enhances the selective killing of HIV-infected macrophages. As a final result, a potent (>98%) and long-lasting (at least 4 weeks without rebound) inhibition of virus release from drug-loaded RBC-treated chronically-infected macrophages was achieved. Taken together, the evidence of HIV-1-induced increase of STAT1, and the availability of a selective drug targeting system, may prove useful in the design of new pharmacological treatments to clear the HIV-1 macrophage reservoir.

Balancing Innate Immunity and Inflammatory State via Modulation of Neutrophil Function: A Novel Strategy to Fight Sepsis.

Sepsis and SIRS (systemic inflammatory response syndrome) belong to a severe disease complex characterized by infection and/or a whole-body inflammatory state. There is a growing body of evidence that neutrophils are actively involved in sepsis and are responsible for both release of cytokines and phagocytosis of pathogens. The neutrophil level is mainly regulated by G-CSF, a cytokine and drug, which is widely used in the septic patient with neutropenia. This review will briefly summarize the role of neutrophils and the therapeutic effect of G-CSF in sepsis. We further suggest that targeting neutrophil function to modulate the balance between innate immunity and inflammatory injury could be a worthwhile therapeutic strategy for sepsis.

ADP-ribosylation of Rho enhances actin polymerization-coupled shape oscillations in human neutrophils.

Stimulated neutrophils exhibit coordinated sinusoidal oscillations in filamentous actin content and cellular shape. We investigated the effect of inhibition of the small G protein Rho on neutrophil actin polymerization, shape changes and oscillations using a genetically engineered toxin that enters cells and selectively ADP-ribosylates endogenous Rho. This treatment increased the amplitudes and frequencies of shape oscillations and duration of the oscillating transient. However, it had no effect on the initial actin polymerization and shape changes induced by N-formyl-Met-Leu-Phe. Regulation of these oscillations may be important for the control of neutrophil motility.