The role of Bryostatin and PMA (Phorbole Myristate Acetate) in enhancing ZFN’s Anti-HIV effects

Purpose: Investigate the efficacy of protein kinase activators in enhancing the activity of ZFN and maintain the average of CD4/CD8 in the treatment of AIDS by using protein kinase activators like bryostatin and PMA. Materials and methods: Balb/C mice were infected with hiv-1ADA (tCID50 102×5 per mouse), A week later, ZFN was then injected with a concentration of 3,100ng, PMA with a dose 100ng per animal and Bryostatin 40 μg/kg, intraperitoneally. And Antiviral treatment was continued for seven weeks, using oral tenofovir at a dose of 4.5mg, emtricitabine at a dose of 3mg, and efavirenz at a dose of 18mg, daily At the end of the study, blood samples were withdrawn from the retro-orbital mouse eye and CD4/CD8 was measured by flow cytometry. Results: The pathophysiological changes decreased in the group treated with ZFN compared to the control infected group with significant differences, but there are no significant differences between and the groups treated with ZFN+Bry (HIV+ Z +B), ZFN +PMA (HIV+ Z +PMA) and ART group with CD4/CD8 ratio in these compared to the infected irradiated control group (HIV). Conclusion: Bryostatin and PMA cannot enhance the effect of ZFN in treating HIV infection.

Rab10-Positive Tubular Structures Represent a Novel Endocytic Pathway That Diverges From Canonical Macropinocytosis in RAW264 Macrophages

Using the optogenetic photo-manipulation of photoactivatable (PA)-Rac1, remarkable cell surface ruffling and the formation of a macropinocytic cup (premacropinosome) could be induced in the region of RAW264 macrophages irradiated with blue light due to the activation of PA-Rac1. However, the completion of macropinosome formation did not occur until Rac1 was deactivated by the removal of the light stimulus. Following PA-Rac1 deactivation, some premacropinosomes closed into intracellular macropinosomes, whereas many others transformed into long Rab10-positive tubules without forming typical macropinosomes. These Rab10-positive tubules moved centripetally towards the perinuclear Golgi region along microtubules. Surprisingly, these Rab10-positive tubules did not contain any endosome/lysosome compartment markers, such as Rab5, Rab7, or LAMP1, suggesting that the Rab10-positive tubules were not part of the degradation pathway for lysosomes. These Rab10-positive tubules were distinct from recycling endosomal compartments, which are labeled with Rab4, Rab11, or SNX1. These findings suggested that these Rab10-positive tubules may be a part of non-degradative endocytic pathway that has never been known. The formation of Rab10-positive tubules from premacropinosomes was also observed in control and phorbol myristate acetate (PMA)-stimulated macrophages, although their frequencies were low. Interestingly, the formation of Rab10-positive premacropinosomes and tubules was not inhibited by phosphoinositide 3-kinase (PI3K) inhibitors, while the classical macropinosome formation requires PI3K activity. Thus, this study provides evidence to support the existence of Rab10-positive tubules as a novel endocytic pathway that diverges from canonical macropinocytosis.

Sustained Surface ICAM-1 Expression and Transient PDGF-B Production by Phorbol Myristate Acetate-Activated THP-1 Cells Harboring Blau Syndrome-Associated NOD2 Mutations

Objectives: Blau syndrome is a distinct class of autoinflammatory syndrome presenting with early-onset systemic granulomatosis. Blau syndrome-causing NOD2 mutations located in the central nucleotide-oligomerization domain induce ligand-independent basal NF-κB activation in an in vitro reporter assay. However, the precise role of this signaling on granuloma formation has not yet been clarified. Methods: Blau syndrome-causing NOD2 mutations were introduced into human monocytic THP-1 cells, and their morphological and molecular changes from parental cells were analyzed. Identified molecules with altered expression were examined in the patient’s lesional skin by immunostaining. Results: Although the production of proinflammatory cytokines was not altered without stimulation, mutant NOD2-expressing THP-1 cells attached persistently to the culture plate after stimulation with phorbol myristate acetate. Sustained surface ICAM-1 expression was observed in association with this phenomenon, but neither persistent ICAM-1 mRNA expression nor impaired ADAM17 mRNA expression was revealed. However, the transient induction of PDGF-B mRNA expression was specifically observed in stimulated THP-1 derivatives. In the granulomatous skin lesion of a Blau syndrome patient, ICAM-1 and PDGF-B were positively immunostained in NOD2-expressing giant cells. Conclusions: Sustained surface ICAM-1 expression and transient PDGF-B production by newly differentiating macrophages harboring mutant NOD2 might play a role in granuloma formation in Blau syndrome.

Rab10-positive tubular structures represent a novel endocytic pathway that diverges from canonical macropinocytosis in RAW264 macrophages

Using the optogenetic photo-manipulation of photoactivatable (PA)-Rac1, remarkable cell surface ruffling and the formation of a macropinocytic cup (premacropinosome) could be induced in the region of RAW264 macrophages irradiated with blue light due to the activation of PA-Rac1. However, the completion of macropinosome formation did not occur until Rac1 was deactivated by the removal of the light stimulus. Following PA-Rac1 deactivation, some premacropinosomes closed into intracellular macropinosomes, whereas many others transformed into long Rab10-positive tubules without forming typical macropinosomes. These Rab10-positive tubules moved centripetally towards the Golgi region along microtubules. Surprisingly, these Rab10-positive tubules did not contain any endosome/lysosome compartment markers, such as Rab5, Rab7, or LAMP1, suggesting that the Rab10-positive tubules were not part of the degradation pathway for lysosomes. These Rab10-positive tubules were distinct from recycling endosomal compartments, which are labeled with Rab4, Rab11, or SNX1. These findings suggested that these Rab10-positive tubules belonged to a novel, non-degradative, endocytic pathway. The formation of Rab10-positive tubules from premacropinosomes was also observed in control and phorbol myristate acetate (PMA)-stimulated macrophages, although their frequencies were low. Interestingly, the formation of Rab10-positive premacropinosomes and tubules was not inhibited by phosphoinositide 3-kinase (PI3K) inhibitors, while the classical macropinosome formation requires PI3K activity. Thus, this study provides evidence to support the existence of Rab10-positive tubules as a novel, non-degradative, endocytic pathway that diverges from canonical macropinocytosis.

Abstract 369: Infarct Macrophage Secretome Factors Regulate Neutrophil Degranulation

Following myocardial infarction (MI), the left ventricle undergoes wound healing that begins withan intense inflammatory response to recruit leukocytes to the infarcted region. Infiltratedneutrophils degranulate, releasing a series of proteases including matrix metalloproteinase(MMP)-9 into the extracellular space. Macrophages also infiltrate into the infarct region, and wehypothesized that the macrophage may secrete factors that regulate neutrophil degranulation.Stimulating bone marrow derived neutrophils from C57BL/6J mice (3-6 month old male) withphorbol myristate acetate (PMA) induced degranulation, as evidenced by release of MMP-9.Co-stimulation with MI day 1 macrophage secretome (10% by volume) reduced PMA-induceddegranulation of MMP-9 by 8-fold (p<0.0014). Transcriptomic analysis of day 1 MI macrophagesrevealed galectin-3, vimentin, fibronectin, and murinoglobulin-1 were highly expressed.Examination of the day 1 MI macrophage secretome also showed high expression of vimentinand fibronectin. To further unmask signaling connections, neutrophils were co-stimulated withPMA and day 1 MI macrophage secretome, along with blocking antibodies for the 4 proteinsindividually. Galectin-3 inhibition promoted the macrophage secretome effect, whilemurinoglobulin-1 inhibition reversed the macrophage secretome effect. Neutrophil stimulationwith recombinant galectin-3 showed no additive effect with PMA on MMP-9 release, suggestingthat galectin-3 works through the protein kinase C signaling pathway to induce neutrophildegranulation. Neither Vimentin inhibition nor stimulation had an effect on neutrophildegranulation Overall, our results uncovered a role for galectin-3 and fibronectin in inducingneutrophil degranulation while murinoglobulin-1 may be a potent inhibitor of degranulation. Theday 1 MI macrophage therefore secretes both factors that promote as well as factors thattemper neutrophil degranulation. The balance of these factors determines the net effect of themacrophage secretome on neutrophil degranulation.

Dietary antioxidants significantly reduced phorbol myristate acetate induced oxidative stress of peripheral blood mononuclear cells of patients with rheumatoid arthritis

Background: Rheumatoid arthritis (RA) is an auto immune disease responsible for maximum human morbidity in modern life whereas oxidative stress is the ultimate potential biomarker for determining disease activity in patients with RA. Objective: The present study scientifically validated the effectiveness of antioxidant commonly present in different food supplement as to neutralize the free radicals mediated oxidativestress in isolated peripheral blood mononuclear lymphocytes (PBML) of patients with RA. Methods: The study population included the patients with Rheumatoid arthritis, RA (n =15) who fulfilled the American College of Rheumatology criteria for RA. Peripheral blood was collected and isolated mononuclear lymphocyte cells (PBML) were pretreated with phorbol myristate acetate (PMS) and furthermore incubated with different concentration of Naringenin, β carotene and N-acetyl cysteine (NAC) in an ex vivo condition. Resultant cell lysate was used for further studies for determination of other oxidative biomarkers. Increase of superoxide and nitric oxide production was observed when PBML were treated PMS. Results: Importantly, the increased oxidative stress was effectively decreased by the selected plant derived compounds β-carotene and naringenin. Conclusion: The study scientifically evaluated the efficacy of the molecules validated by one-way ANOVA followed by Dunnett’s post hoc test of significance. Collectively, our results indicate that both β carotene and naringenin may be a promising non-toxic food supplement in attenuating the oxidative stress associated pathology in RA, meriting further pharmacological studies on other inflammatory cells like neutrophils.