The amoeboid migration of monocytes in confining channels requires the local remodeling of the cortical actin cytoskeleton by cofilin-1.

Within the bloodstream, monocytes must traverse the microvasculature to prevent leukostasis, which is the entrapment of monocytes within the confines of the microvasculature. Using the model cell line, THP-1, and VCAM-1 coated channels to simulate the microvasculature surface, we demonstrate that monocytes predominantly adopt an amoeboid phenotype, which is characterized by the formation of blebs. As opposed to cortical actin flow in leader blebs, cell movement is correlated with myosin contraction at the cell rear. It was previously documented that cofilin-1 promotes cortical actin turnover at leader bleb necks in melanoma cells. In monocytes, our data suggest that cofilin-1 promotes the local upregulation of myosin contractility through actin cytoskeleton remodeling. In support of this concept, cofilin-1 is found to localize to a single cell edge. Moreover, the widespread upregulation of myosin contractility was found to inhibit migration. Thus, monocytes within the microvasculature may avoid entrapment by adopting an amoeboid mode of migration.

The amoeboid migration of monocytes in confining channels requires the local remodeling of the cortical actin cytoskeleton by cofilin-1.

Within the bloodstream, monocytes must traverse the microvasculature to prevent leukostasis, which is the entrapment of monocytes within the confines of the microvasculature. Using the model cell line, THP-1, and VCAM-1 coated channels to simulate the microvasculature surface, we demonstrate that monocytes predominantly adopt an amoeboid phenotype, which is characterized by the formation of blebs. As opposed to cortical actin flow in leader blebs, cell movement is correlated with myosin contraction at the cell rear. It was previously documented that cofilin-1 promotes cortical actin turnover at leader bleb necks in melanoma cells. In monocytes, our data suggest that cofilin-1 promotes the local upregulation of myosin contractility through actin cytoskeleton remodeling. In support of this concept, cofilin-1 is found to localize to a single cell edge. Moreover, the widespread upregulation of myosin contractility was found to inhibit migration. Thus, monocytes within the microvasculature may avoid entrapment by adopting an amoeboid mode of migration.

The amoeboid migration of monocytes in confining channels requires the local remodeling of the cortical actin cytoskeleton by cofilin-1.

Within the bloodstream, monocytes must traverse the microvasculature to prevent leukostasis, which is the entrapment of monocytes within the confines of the microvasculature. Using the model cell line, THP-1, and VCAM-1 coated channels to simulate the microvasculature, we demonstrate that monocytes predominantly adopt an amoeboid phenotype, which is characterized by the formation of blebs. As opposed to cortical actin flow in leader blebs, cell movement is correlated with myosin contraction at the cell rear. Previously, we documented that cofilin-1 promotes cortical actin turnover at leader bleb necks in melanoma cells. In monocytes, our data suggest that cofilin-1 promotes the local upregulation of myosin contractility through actin cytoskeleton remodeling. In support of this concept, cofilin-1 is found to localize to a single cell edge. Moreover, the widespread upregulation of myosin contractility was found to inhibit migration. Thus, monocytes within the microvasculature may avoid entrapment by adopting an amoeboid mode of migration.

PKC-ε regulates vesicle delivery and focal exocytosis for efficient IgG-mediated phagocytosis.

Protein kinase C (PKC)-ε is required for membrane addition during IgG-mediated phagocytosis, but its role in this process is ill defined. Here, we performed high-resolution imaging, which reveals that PKC-ε exits the Golgi and enters phagosomes on vesicles that then fuse. TNF and PKC-ε colocalize at the Golgi and on vesicles that enter the phagosome. Loss of PKC-ε and TNF delivery upon nocodazole treatment confirmed vesicular transport on microtubules. That TNF+ vesicles were not delivered in macrophages from PKC-ε null mice, or upon dissociation of the Golgi-associated pool of PKC-ε, implies that Golgi-tethered PKC-ε is a driver of Golgi-to-phagosome trafficking. Finally, we established that the regulatory domain of PKC-ε is sufficient for delivery of TNF+ vesicles to the phagosome. These studies reveal a novel role for PKC-ε in focal exocytosis - its regulatory domain drives Golgi-derived vesicles to the phagosome, whereas catalytic activity is required for their fusion. This is one of the first examples of a PKC requirement for vesicular trafficking and describes a novel function for a PKC regulatory domain. This article has an associated First Person interview with the first author of the paper.

Protein Kinase C-epsilon in Membrane Delivery during Phagocytosis.

During phagocytosis, internal membranes are recruited to the site of pathogen binding and fuse with the plasma membrane, providing the membrane needed for pseudopod extension and target uptake. The mechanism by which vesicles destined for the phagosome are generated, targeted, and fuse is unknown. We established that Golgi-associated protein kinase C-epsilon (PKC-ε) is necessary for the addition of membrane during FcyR-mediated phagocytosis. PKC-ε is tethered to the Golgi through interactions between its' regulatory domain and the Golgi lipids PI4P and diacylglycerol; disruption of these interactions prevents PKC-ε concentration at phagosomes and decreases phagocytosis. The accumulated evidence suggests that PKC-ε orchestrates vesicle formation at the Golgi by a mechanism requiring lipid binding but not enzymatic activity. This review discusses how PKC-ε might mediate vesicle formation at the level of budding and fission. Specifically, we discuss PKC-ε binding partners, the formation of lipid subdomains to generate membrane curvature, and PKC-ε mediated links to the actin and microtubule cytoskeleton to provide tension for vesicle fission. Assimilating information from several model systems, we propose a model for PKC-ε mediated vesicle formation for exocytosis during phagocytosis that may be applicable to other processes that require directed membrane delivery and fusion.

Golgi-Associated Protein Kinase C-ε Is Delivered to Phagocytic Cups: Role of Phosphatidylinositol 4-Phosphate.

Protein kinase C-ε (PKC-ε) at phagocytic cups mediates the membrane fusion necessary for efficient IgG-mediated phagocytosis. The C1B and pseudosubstrate (εPS) domains are necessary and sufficient for this concentration. C1B binds diacylglycerol; the docking partner for εPS is unknown. Liposome assays revealed that the εPS binds phosphatidylinositol 4-phosphate (PI4P) and PI(3,5)P2 Wortmannin, but not LY294002, inhibits PKC-ε concentration at cups and significantly reduces the rate of phagocytosis. As Wortmannin inhibits PI4 kinase, we hypothesized that PI4P mediates the PKC-ε concentration at cups and the rate of phagocytosis. PKC-ε colocalizes with the trans-Golgi network (TGN) PI4P reporter, P4M, suggesting it is tethered at the TGN. Real-time imaging of GFP-PKC-ε-expressing macrophages revealed a loss of Golgi-associated PKC-ε during phagocytosis, consistent with a Golgi-to-phagosome translocation. Treatment with PIK93, a PI4 kinase inhibitor, reduces PKC-ε at both the TGN and the cup, decreases phagocytosis, and prevents the increase in capacitance that accompanies membrane fusion. Finally, expression of the Golgi-directed PI4P phosphatase, hSac1-K2A, recapitulates the PIK93 phenotype, confirming that Golgi-associated PI4P is critical for efficient phagocytosis. Together these data are consistent with a model in which PKC-ε is tethered to the TGN via an εPS-PI4P interaction. The TGN-associated pool of PKC-ε concentrates at the phagocytic cup where it mediates the membrane fusion necessary for phagocytosis. The novelty of these data lies in the demonstration that εPS binds PI4P and PI(3,5)P2 and that PI4P is necessary for PKC-ε localization at the TGN, its translocation to the phagocytic cup, and the membrane fusion required for efficient Fc [γ] receptor-mediated phagocytosis.

Attomole quantification and global profile of RNA modifications: Epitranscriptome of human neural stem cells.

Exploration of the epitranscriptome requires the development of highly sensitive and accurate technologies in order to elucidate the contributions of the more than 100 RNA modifications to cell processes. A highly sensitive and accurate ultra-high performance liquid chromatography-tandem mass spectrometry method was developed to simultaneously detect and quantify 28 modified and four major nucleosides in less than 20 min. Absolute concentrations were calculated using extinction coefficients of each of the RNA modifications studied. A comprehensive RNA modifications database of UV profiles and extinction coefficient is reported within a 2.3-5.2 % relative standard deviation. Excellent linearity was observed 0.99227-0.99999 and limit of detection values ranged from 63.75 attomoles to 1.21 femtomoles. The analytical performance was evaluated by analyzing RNA modifications from 100 ng of RNA from human pluripotent stem cell-derived neural cells. Modifications were detected at concentrations four orders of magnitude lower than the corresponding parental nucleosides, and as low as 23.01 femtograms, 64.09 attomoles. Direct and global quantitative analysis of RNA modifications are among the advantages of this new approach.

A novel method using blinatumomab for efficient, clinical-grade expansion of polyclonal T cells for adoptive immunotherapy.

Current treatment of chronic lymphocytic leukemia (CLL) patients often results in life-threatening immunosuppression. Furthermore, CLL is still an incurable disease due to the persistence of residual leukemic cells. These patients may therefore benefit from immunotherapy approaches aimed at immunoreconstitution and/or the elimination of residual disease following chemotherapy. For these purposes, we designed a simple GMP-compliant protocol for ex vivo expansion of normal T cells from CLL patients' peripheral blood for adoptive therapy, using bispecific Ab blinatumomab (CD3 × CD19), acting both as T cell stimulator and CLL depletion agent, and human rIL-2. Starting from only 10 ml CLL peripheral blood, a mean 515 × 10(6) CD3(+) T cells were expanded in 3 wk. The resulting blinatumomab-expanded T cells (BET) were polyclonal CD4(+) and CD8(+) and mostly effector and central memory cells. The Th1 subset was slightly prevalent over Th2, whereas Th17 and T regulatory cells were <1%. CMV-specific clones were detected in equivalent proportion before and after expansion. Interestingly, BET cells had normalized expression of the synapse inhibitors CD272 and CD279 compared with starting T cells and were cytotoxic against CD19(+) targets in presence of blinatumomab in vitro. In support of their functional capacity, we observed that BET, in combination with blinatumomab, had significant therapeutic activity in a systemic human diffuse large B lymphoma model in NOD-SCID mice. We propose BET as a therapeutic tool for immunoreconstitution of heavily immunosuppressed CLL patients and, in combination with bispecific Ab, as antitumor immunotherapy.

Altered insulin receptor signalling and ß-cell cycle dynamics in type 2 diabetes mellitus.

Insulin resistance, reduced ß-cell mass, and hyperglucagonemia are consistent features in type 2 diabetes mellitus (T2DM). We used pancreas and islets from humans with T2DM to examine the regulation of insulin signaling and cell-cycle control of islet cells. We observed reduced ß-cell mass and increased α-cell mass in the Type 2 diabetic pancreas. Confocal microscopy, real-time PCR and western blotting analyses revealed increased expression of PCNA and down-regulation of p27-Kip1 and altered expression of insulin receptors, insulin receptor substrate-2 and phosphorylated BAD. To investigate the mechanisms underlying these findings, we examined a mouse model of insulin resistance in ß-cells--which also exhibits reduced ß-cell mass, the ß-cell-specific insulin receptor knockout (ßIRKO). Freshly isolated islets and ß-cell lines derived from ßIRKO mice exhibited poor cell-cycle progression, nuclear restriction of FoxO1 and reduced expression of cell-cycle proteins favoring growth arrest. Re-expression of insulin receptors in ßIRKO ß-cells reversed the defects and promoted cell cycle progression and proliferation implying a role for insulin-signaling in ß-cell growth. These data provide evidence that human ß- and α-cells can enter the cell-cycle, but proliferation of ß-cells in T2DM fails due to G1-to-S phase arrest secondary to defective insulin signaling. Activation of insulin signaling, FoxO1 and proteins in ß-cell-cycle progression are attractive therapeutic targets to enhance ß-cell regeneration in the treatment of T2DM.

The surface density of the glutamate transporter EAAC1 is controlled by interactions with PDZK1 and AP2 adaptor complexes.

The glutamate transporter excitatory amino acid carrier (EAAC1/EAAT3) mediates the absorption of dicarboxylic amino acids in epithelial cells as well as the uptake of glutamate from the synaptic cleft. Its cell-surface density is regulated by interaction with accessory proteins which remain to be identified. We detected a consensus sequence for interaction with post-synaptic density-95/Discs large/Zonula occludens (PDZ) proteins (-SQF) and a tyrosine-based internalization signal (-YVNG-) in the C-terminus of EAAC1, and investigated their role in the transporter localization. We demonstrated that PDZ interactions are required for the efficient delivery to and the retention in the plasma membrane of EAAC1 and we identified PDZK1/NHERF3 (Na+/H+-exchanger regulatory factor 3) as a novel EAAC1 interacting protein. Expression of PDZK1 in Madin-Darby canine kidney (MDCK) cells tethered EAAC1 to filopodia and increased its surface activity. Removal of the PDZ-target motif promoted the EAAC1 binding to α-adaptin and clathrin and the transporter internalization in endocytic/degradative compartments. This defect was largely prevented by hypertonic treatment or overexpression of the dominant-negative µ2-W421A-subunit of AP-2 clathrin-adaptor. The rate of transporter endocytosis was attenuated following tyrosine mutagenesis in the internalization signal, thus indicating that this motif can regulate the transporter endocytosis. We suggest that EAAC1 density is controlled by balanced interactions with PDZK1 and adaptor protein 2 (AP2): the former promotes the transporter expression at the cell surface, and the latter mediates its constitutive endocytosis.

LIN7 mediates the recruitment of IRSp53 to tight junctions.

In this study, we examined the role of the L27 [(LIN2-LIN7) domain] and PDZ domain (domain previously found in PSD95-DlgA-ZO-1) for protein-protein interaction of the scaffold protein LIN7 in tight junction (TJ) assembly in Madin-Darby canine kidney (MDCK) cells and found that the stable expression of a LIN7 mutant lacking the L27 domain (DeltaL27 mutant) acts as a dominant interfering protein by inhibiting TJ localization of endogenous LIN7. The loss of LIN7 did not alter the localization of the PALS1 (protein associated with LIN7) partner of the L27 domain but prevented TJ localization of the insulin receptor substrate p53 (IRSp53), a partner of the PDZ domain of LIN7. The function of both L27 and PDZ domains of LIN7 in IRSp53 localization to TJs has been further demonstrated by reducing the expression of LIN7 (LIN7 small hairpin RNA experiments) and by expression of IRSp53 deleted of its motif for PDZ interaction (IRSp53Delta5) or fused to the L27 domain of LIN7 (L27-IRSp53Delta5). Cell lines with decreased localization of LIN7 and IRSp53 to TJs showed defects during assembly of TJs and cyst polarization and failed to activate Rac1, a member of the Rho guanosine triphosphatases family crucially involved in actin organization and orientation of apicobasal polarity. These data therefore indicate that LIN7-IRSp53 association plays a role during assembly of functional TJs and surface polarization in epithelial cells.

Dose-intensity temozolomide after concurrent chemoradiotherapy in operated high-grade gliomas.

PURPOSE: We performed a new phase II trial enrolling patients with newly diagnosed high-grade glioma (HGG) to test the efficacy of a weekly alternating temozolomide (TMZ) schedule after surgery and concomitant chemoradiotherapy. METHODS: From January 2005 to January 2007, 34 patients (21 men, 13 women; age range 30-70, mean age 53) were enrolled. There were 32 glioblastoma multiforme and two anaplastic astrocytoma. Each patient after surgery received standard concurrent chemoradiotherapy. After a 4-week break, patients were then to receive 12 cycles of 1-week-on/1-week-off TMZ, with 75 mg/m(2) for the first cycle, 100 mg/m(2) for the second, 125 mg/m(2) for the third, and 150 mg/m(2) from the fourth to the 12th. Hematological toxicity was monitored every week during concomitant chemoradiotherapy and then every 4 weeks. RESULTS: After 12 months from the end of radiotherapy, the overall survival (OS) rate was 59% (20/38), distributed as follows: 60% (18/30) for recursive partitioning analysis (RPA) class 4 patients and 33% (1/3) for RPA class 6 patients; the only RPA class 1 patient was alive and disease free at the time of writing. Median OS was 13 months [95% confidence interval (CI) 11.02-14.98 months]. Hematological toxicity was seen in six patients (18%): grade 1 neutropenia in four, grade 2 thrombocytopenia in one, and grade 4 thrombocytopenia plus grade 1 neutropenia in one. There was one case of opportunistic infection (Pneumocystis carinii pneumonitis). CONCLUSION: The toxicity of the TMZ dose-dense regimen was very low. Results seem to be encouraging for RPA lower classes (patients with good prognostic factors).

Secondary azoospermia after vasovasostomy.

OBJECTIVES: To determine the frequency of secondary azoospermia after microsurgical vasovasostomy and to determine what factors increase the risk of its occurrence. METHODS: We performed a retrospective review of three surgeons' experience. Patency was defined as the presence of sperm in at least one postoperative semen sample. Transient patency was defined as azoospermia or no motile sperm after previous documentation of motile sperm. RESULTS: A total of 242 patients underwent 245 procedures (233 bilateral, 12 unilateral). The mean patient age was 39.2 +/- 0.4 years (range 24 to 56), and the mean obstructive interval was 8.7 +/- 0.3 years (range 0.25 to 24). The patency rates were 91% (224 of 245, sperm present in the semen) and 86% (208 of 245, motile sperm present in the semen). The transient patency rates were 5.3% (13 of 245, presence of sperm, with subsequent azoospermia) and 2.9% (7 of 241, motile sperm in the semen, with subsequent azoospermia) at a follow-up of 9.0 +/- 0.7 months (range 1 to 60). Transient patency occurred at a mean of 9.7 +/- 2.3 months. A greater risk of transient patency was observed with unilateral cases than bilateral cases (3 of 12, 25% versus 10 of 233, 4.3%; P = 0.0196, Fisher's exact test), and the obstructive interval was shorter for patent than for transient patent cases (Mann-Whitney U test, P = 0.0458). CONCLUSIONS: The results of our study demonstrated that secondary azoospermia after vasovasostomy is rare. It is more common in unilateral cases and the obstructive interval for transiently patent cases is longer. Sperm cryopreservation, when motile sperm appear in the semen postoperatively, can circumvent the problem of secondary azoospermia, but most men will not need the frozen sperm.

Pregnancy outcomes after vasectomy reversal for female partners 35 years old or older.

PURPOSE: We review the outcomes after vasectomy reversal for couples with female partners 35 years old or older. MATERIALS AND METHODS: A retrospective review of experience at 2 institutions was performed. Patency was defined as the presence of motile sperm. Patients with less than 6 months of followup were excluded from the patency rate analysis unless they had sperm in the semen. Similarly, patients with less than 12 months of followup or no ongoing interest in establishing conception were excluded from the pregnancy rate analysis unless they had established a pregnancy or they were azoospermic with sufficient followup. RESULTS: A total of 46 men with partners 35 years old or older underwent vasectomy reversal at 2 institutions. Mean partner age was 37 +/- 2 years, and median obstructive interval was 10 years. Bilateral vasovasostomy was performed in 43 men, unilateral vasovasostomy in 2 and vasovasostomy/vasoepididymostomy in 1. Of the 46 men 27 had followup semen analyses with a patency rate of 81% (22). Transient patency occurred in 2 cases (7%). Pregnancy occurred in 35% of the couples (14 of 40 patients) with sufficient followup. The ongoing/live delivery rate was 33% (13 of 40 cases). The pregnancy and ongoing/delivery rates were 46% (12 of 26 patients) and 46% (12 of 26) for female partners 35 to 39 years old, and 14% (2 of 14) and 7% (1 of 14) for female partners older than 40, respectively. CONCLUSIONS: Vasectomy reversal offers reasonable chance for success when the female partner is 35 years old or older. The chance for success is similar to that of a single cycle of in vitro fertilization with intracytoplasmic sperm injection. These couples should not be eliminated from consideration for reversal simply because the female partner is 35 years old or older.

Outcomes for vasovasostomy with bilateral intravasal azoospermia.

We conducted an evaluation of outcomes for microsurgical vasectomy reversal in which sperm are absent from the vas fluid in order to determine a threshold obstructive interval when vasoepididymostomy (VE) may be indicated. Vasectomy reversal was performed for 32 patients with intravasal azoospermia: 25 received bilateral vasovasostomy (VV), 1 had a bilateral VV, 5 underwent VV/VE, and 1 had bilateral VE. Overall, the patency rate was 50% (14 of 28). Five pregnancies (20%) and 3 live births (12%) occurred in 25 patients with sufficient follow-up. One pregnancy was electively terminated and the other is ongoing, for an ongoing or delivered rate of 16%. The patency rate for VV (either bilateral or unilateral) was 55% (12 of 22). Median obstructive interval was 7 years in patent and 15 years in nonpatent cases, respectively, (P =.0027). Sperm were not observed after VV in any case n which the obstructive interval was greater than 11 years. If VV was limited to obstructive intervals of 11 years or less, then the patency rate was 80% (12 of 15) and the pregnancy rate was 38% (5 of 13). The patency rate for bilateral VV was 67% (8 of 12) if clear fluid was observed on at least one side. We conclude that VE is not required in every case of intravasal azoospermia, but it could improve success rates in this setting. Based on our experience, VE may be indicated for intravasal azoospermia if the obstructive interval is more than 11 years.

Outcomes for vasectomy reversal performed after obstructive intervals of at least 10 years.

OBJECTIVES: To determine the outcomes for vasectomy reversal performed after at least 10 years of obstruction. METHODS: We performed a retrospective review of three surgeons' experience with microsurgical vasectomy reversal for obstructive intervals of at least 10 years. RESULTS: The overall pregnancy rate was 37%. The patency/pregnancy rate for an obstructive interval of 10 to 15, 16 to 19, and 20 or more years was 74%/40%, 87%/36%, and 75%/27%, respectively. The overall ongoing/delivered rate was 35%. The ongoing/delivered rates equaled the pregnancy rates, except in the 16 to 19-year group, for which the ongoing/delivered rate was 27%. Assuming a live delivery rate per cycle of 25% for intracytoplasmic sperm injection (ICSI), the delivery rate for vasectomy reversal would not be exceeded until an obstructive interval of at least 20 years. Assuming a live delivery rate of 28.6% per cycle for ICSI with obstructive azoospermia, the delivery rate for vasectomy reversal would not be exceeded until an obstructive interval of at least 15 years. CONCLUSIONS: Even after prolonged obstructive intervals, vasectomy reversal offers better or comparable success rates to ICSI. For each center, depending on their success rates, a threshold obstructive interval exists at which ICSI surpasses vasectomy reversal. Depending on their wishes, couples who have an obstructive interval that exceeds this threshold may be better served by ICSI. As with all infertile couples, close collaboration between the urologists and gynecologists is essential to provide the most appropriate care.