A Case of Lemierre's Syndrome Caused by Streptococcus Milleri Group.

A 64-year-old woman was transported to the emergency room with a headache and fever. She presented with a right ocular protrusion, hyperemia, and tenderness in the neck. Contrast-enhanced MRI of the head showed a high DWI signal in the bilateral sphenoid sinuses and contrast defects along the bilateral internal jugular and superior ophthalmic veins. Blood and CSF cultures revealed Streptococcus milleri group. Surgery was performed for Lemierre's syndrome secondary to sphenoid sinusitis. The patient was treated with antibiotics and anticoagulant therapy, but a duodenal ulcer and brain abscess thereafter developed. However, multidisciplinary endoscopic and surgical treatment saved her life.

Case report of a 28-year-old male with the rapid progression of steroid-resistant central nervous system vasculitis diagnosed by a brain biopsy.

A 28-year-old Japanese male without a significant past medical history presented with new-onset generalized clonic seizure and headache. A brain MRI revealed multiple enhanced lesions on both cerebral hemispheres. Laboratory exams showed no evidence of systemic inflammation or auto-immune antibodies such as ANCAs. Despite four courses of high-dose methylprednisolone pulse therapy and five treatments with plasmapheresis, his symptoms worsened and the MRI lesions progressed rapidly. During these treatments, we performed a targeted brain biopsy, that revealed histological findings consistent with a predominant angiitis of parenchymal and subdural small vessels. He was provided with diagnosis of central nervous system vasculitis (CNSV). Subsequent cyclophosphamide pulse therapy enabled a progressive successful improvement of his symptoms. While diagnostic methods for CNSV remain controversial, histological findings are thought to be more useful in obtaining a more definitive diagnosis than findings in image studies, such as MRI and angiography. We suggest that a brain biopsy should be considered during the early period of cases with suspected CNSV and rapid clinical deterioration. We also detected human herpesvirus 7 (HHV-7) using PCR technology in brain biopsy specimens, however the relationship between CNSV and HHV-7 infection is unknow.

Reactive oxygen species-induced actin glutathionylation controls actin dynamics in neutrophils.

The regulation of actin dynamics is pivotal for cellular processes such as cell adhesion, migration, and phagocytosis and thus is crucial for neutrophils to fulfill their roles in innate immunity. Many factors have been implicated in signal-induced actin polymerization, but the essential nature of the potential negative modulators are still poorly understood. Here we report that NADPH oxidase-dependent physiologically generated reactive oxygen species (ROS) negatively regulate actin polymerization in stimulated neutrophils via driving reversible actin glutathionylation. Disruption of glutaredoxin 1 (Grx1), an enzyme that catalyzes actin deglutathionylation, increased actin glutathionylation, attenuated actin polymerization, and consequently impaired neutrophil polarization, chemotaxis, adhesion, and phagocytosis. Consistently, Grx1-deficient murine neutrophils showed impaired in vivo recruitment to sites of inflammation and reduced bactericidal capability. Together, these results present a physiological role for glutaredoxin and ROS- induced reversible actin glutathionylation in regulation of actin dynamics in neutrophils.

Catheter displacement into inferior epigastric vein causing local phlebitis and cellulitis.

Catheter insertion for intravenous hyperalimentation is a commonly and widely used clinical technique. When compared with the incidence of complications associated with insertions into the internal jugular vein or the subclavian vein, complications associated with insertions into the femoral vein are less frequent. In this paper, we describe a very rare complication of femoral vein catheter insertion-namely, catheter displacement into the inferior epigastric vein.

Intracranial transplantation of monocyte-derived multipotential cells enhances recovery after ischemic stroke in rats.

Cell transplantation has emerged as a potential therapy to reduce the neurological deficits caused by ischemic stroke. We previously reported a primitive cell population, monocyte-derived multipotential cells (MOMCs), which can differentiate into mesenchymal, neuronal, and endothelial lineages. In this study, MOMCs and macrophages were prepared from rat peripheral blood and transplanted intracranially into the ischemic core of syngeneic rats that had undergone a left middle cerebral artery occlusion procedure. Neurological deficits, as evaluated by the corner test, were less severe in the MOMC-transplanted rats than in macrophage-transplanted or mock-treated rats. Histological evaluations revealed that the number of microvessels that had formed in the ischemic boundary area by 4 weeks after transplantation was significantly greater in the MOMC-transplanted rats than in the control groups. The blood vessel formation was preceded by the appearance of round CD31(+) cells, which we confirmed were derived from the transplanted MOMCs. Small numbers of bloodvessels incorporating MOMC-derived endothelial cells expressing a mature endothelial marker RECA-1 were detected at 4 weeks after transplantation. In addition, MOMCs expressed a series of angiogenic factors, including vascular endothelial growth factor, angiopoetin-1, and placenta growth factor (PlGF). These findings provide evidence that the intracranial delivery of MOMCs enhances functional recovery by promoting neovascularization in a rat model for ischemic stroke.

Reactive oxygen species as signaling molecules in neutrophil chemotaxis.

Neutrophil chemotaxis is a critical component in innate immunity. Recently, using a small-molecule functional screening, we identified NADPHoxidase- dependent Reactive Oxygen Species (ROS) as key regulators of neutrophil chemotactic migration. Neutrophils depleted of ROS form more frequent multiple pseudopodia and lost their directionality as they migrate up a chemoattractant concentration gradient. Here, we further studied the role of ROS in neutrophil chemotaxis and found that multiple pseudopodia formation induced by NADPH inhibitor diphenyleneiodonium chloride (DPI) was more prominent in relatively shallow chemoattractant gradient. It was reported that, in shallow chemoattractant gradients, new pseudopods are usually generated when existing ones bifurcate. Directional sensing is mediated by maintaining the most accurate existing pseudopod, and destroying pseudopods facing the wrong direction by actin depolymerization. We propose that NADPH-mediated ROS production may be critical for disruption of misoriented pseudopods in chemotaxing neutrophils. Thus, inhibition of ROS production will lead to formation of multiple pseudopodia.

Natural product Celastrol destabilizes tubulin heterodimer and facilitates mitotic cell death triggered by microtubule-targeting anti-cancer drugs.

BACKGROUND: Microtubule drugs are effective anti-cancer agents, primarily due to their ability to induce mitotic arrest and subsequent cell death. However, some cancer cells are intrinsically resistant or acquire a resistance. Lack of apoptosis following mitotic arrest is thought to contribute to drug resistance that limits the efficacy of the microtubule-targeting anti-cancer drugs. Genetic or pharmacological agents that selectively facilitate the apoptosis of mitotic arrested cells present opportunities to strengthen the therapeutic efficacy. METHODOLOGY AND PRINCIPAL FINDINGS: We report a natural product Celastrol targets tubulin and facilitates mitotic cell death caused by microtubule drugs. First, in a small molecule screening effort, we identify Celastrol as an inhibitor of neutrophil chemotaxis. Subsequent time-lapse imaging analyses reveal that inhibition of microtubule-mediated cellular processes, including cell migration and mitotic chromosome alignment, is the earliest events affected by Celastrol. Disorganization, not depolymerization, of mitotic spindles appears responsible for mitotic defects. Celastrol directly affects the biochemical properties of tubulin heterodimer in vitro and reduces its protein level in vivo. At the cellular level, Celastrol induces a synergistic apoptosis when combined with conventional microtubule-targeting drugs and manifests an efficacy toward Taxol-resistant cancer cells. Finally, by time-lapse imaging and tracking of microtubule drug-treated cells, we show that Celastrol preferentially induces apoptosis of mitotic arrested cells in a caspase-dependent manner. This selective effect is not due to inhibition of general cell survival pathways or mitotic kinases that have been shown to enhance microtubule drug-induced cell death. CONCLUSIONS AND SIGNIFICANCE: We provide evidence for new cellular pathways that, when perturbed, selectively induce the apoptosis of mitotic arrested cancer cells, identifying a potential new strategy to enhance the therapeutic efficacy of conventional microtubule-targeting anti-cancer drugs.

Integrin-independent role of CalDAG-GEFI in neutrophil chemotaxis.

Chemotaxis and integrin activation are essential processes for neutrophil transmigration in response to injury. CalDAG-GEFI plays a key role in the activation of beta1, beta2, and beta3 integrins in platelets and neutrophils by exchanging a GDP for a GTP on Rap1. Here, we explored the role of CalDAG-GEFI and Rap1b in integrin-independent neutrophil chemotaxis. In a transwell assay, CalDAG-GEFI-/- neutrophils had a 46% reduction in transmigration compared with WT in response to a low concentration of LTB4. Visualization of migrating neutrophils in the presence of 10 mM EDTA revealed that CalDAG-GEFI-/- neutrophils had abnormal chemotactic behavior compared with WT neutrophils, including reduced speed and directionality. Interestingly, Rap1b-/- neutrophils had a similar phenotype in this assay, suggesting that CalDAG-GEFI may be acting through Rap1b. We investigated whether the deficit in integrin-independent chemotaxis in CalDAG-GEFI-/- neutrophils could be explained by defective cytoskeleton rearrangement. Indeed, we found that CalDAG-GEFI-/- neutrophils had reduced formation of F-actin pseudopodia after LTB4 stimulation, suggesting that they have a defect in polarization. Overall, our studies show that CalDAG-GEFI helps regulate neutrophil chemotaxis, independent of its established role in integrin activation, through a mechanism that involves actin cytoskeleton and cellular polarization.

Small-molecule screen identifies reactive oxygen species as key regulators of neutrophil chemotaxis.

Neutrophil chemotaxis plays an essential role in innate immunity, but the underlying cellular mechanism is still not fully characterized. Here, using a small-molecule functional screening, we identified NADPH oxidase-dependent reactive oxygen species as key regulators of neutrophil chemotactic migration. Neutrophils with pharmacologically inhibited oxidase, or isolated from chronic granulomatous disease (CGD) patients and mice, formed more frequent multiple pseudopodia and lost their directionality as they migrated up a chemoattractant concentration gradient. Knocking down NADPH oxidase in differentiated neutrophil-like HL60 cells also led to defective chemotaxis. Consistent with the in vitro results, adoptively transferred CGD murine neutrophils showed impaired in vivo recruitment to sites of inflammation. Together, these results present a physiological role for reactive oxygen species in regulating neutrophil functions and shed light on the pathogenesis of CGD.

Progressive multifocal leukoencephalopathy developed in incomplete Heerfordt syndrome, a rare manifestation of sarcoidosis, without steroid therapy responding to cidofovir.

Progressive multifocal leukoencephalopathy (PML) is a severe demyelinating disease of the central nervous system caused by the JC virus; the mortality rate is high and it is usually refractory to treatment. In non-HIV patients, PML occurs as a late consequence of hematologic malignancies or during prolonged immunosuppression for transplantation or autoimmune disease. We describe a 34-year-old PML patient with incomplete Heerfordt syndrome, a rare type of sarcoidosis, who had not received any immunosuppressants, including steroids, at the onset and who was clinically and radiologically responsive to the antiviral drug cidofovir.

Focal adhesion kinase regulates pathogen-killing capability and life span of neutrophils via mediating both adhesion-dependent and -independent cellular signals.

Various neutrophil functions such as phagocytosis, superoxide production, and survival are regulated by integrin signaling. Despite the essential role of focal adhesion kinase (FAK) in mediating this signaling pathway, its exact function in neutrophils is ill defined. In this study, we investigated the role of FAK in neutrophils using a myeloid-specific conditional FAK knockout mouse. As reported in many other cell types, FAK is required for regulation of focal adhesion dynamics when neutrophils adhere to fibronectin or ICAM-1. Adhesion on VCAM-1-coated surfaces and chemotaxis after adhesion were not altered in FAK null neutrophils. In addition, we observed significant reduction in NADPH oxidase-mediated superoxide production and complement-mediated phagocytosis in FAK null neutrophils. As a result, these neutrophils displayed decreased pathogen killing capability both in vitro and in vivo in a mouse peritonitis model. In adherent cells, the defects associated with FAK deficiency are likely due to suppression of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) signaling and chemoattractant-elicited calcium signaling. Disruption of FAK also reduced chemoattractant-elicited superoxide production in suspended neutrophils in the absence of cell adhesion. This may be solely caused by suppression of PtdIns(3,4,5)P3 signaling in these cells, because the fMLP-elicited calcium signal was not altered. Consistent with decreased PtdIns(3,4,5)P3/Akt signaling in FAK null neutrophils, we also observed accelerated spontaneous death in these cells. Taken together, our results revealed previously unrecognized roles of FAK in neutrophil function and provided a potential therapeutic target for treatment of a variety of infectious and inflammatory diseases.

Identification of biomarkers associated with migraine with aura.

The diagnosis of migraine can sometimes be difficult because some patients do not fulfill the International Headache Society's criteria for migraine. Hence, an accurate and reliable diagnostic marker for migraine is required. In this study, lymphocytes were used to establish Epstein-Barr virus (EBV)-immortalized lymphoblast cell lines, which were then analyzed using a differential cRNA microarray analysis. The gene expression results were validated using real-time polymerase chain reaction. Gene expression profiling identified 15 genes as being differentially expressed in lymphoblasts originating from patients diagnosed as having migraine with aura (MA). One-fifth of these genes were associated with cytoskeletal proteins. The expressions of seven genes increased significantly by more than 50% of the value in the controls, while the expressions of eight genes decreased significantly by more than 50% of the value in the controls. We also verified that the expression of alpha-fodrin, which was 1 of the 15 genes that were differentially expressed in lymphoblasts originating from patients with MA, increased after cortical spreading depression in an animal model. Thus, alpha-fodrin might play an important role in the pathophysiology of migraine, possibly serving as a migraine biomarker.

Cancer cell-derived clusterin modulates the phosphatidylinositol 3'-kinase-Akt pathway through attenuation of insulin-like growth factor 1 during serum deprivation.

Cancer cells in their respective microenvironments must endure various growth-constraining stresses. Under these conditions, the cancer cell-derived factors are thought to modulate the signaling pathways between cell growth and dormancy. Here, we describe a cancer cell-derived regulatory system that modulates the phosphatidylinositol 3'-kinase (PI3K)-Akt pathway under serum deprivation stress. Through the use of biochemical purification, we reveal that cancer cell-secreted insulin-like growth factor 1 (IGF-1) and clusterin, an extracellular stress protein, constitute this regulatory system. We show that secreted clusterin associates with IGF-1 and inhibits its binding to the IGF-1 receptor and hence negatively regulates the PI3K-Akt pathway during serum deprivation. This inhibitory function of clusterin appears to prefer IGF-1, as it fails to exert any effects on epidermal growth factor signaling. We demonstrate furthermore that the constitutive activation of oncogenic signaling downstream of IGF-1 confers insensitivity to the inhibitory effects of clusterin. Thus, the interplay between cancer cell-derived clusterin and IGF-1 may dictate the outcome of cell growth and dormancy during tumorigenic progression.

Inositol trisphosphate 3-kinase B (InsP3KB) as a physiological modulator of myelopoiesis.

Inositol trisphosphate 3-kinase B (InsP3KB) belongs to a family of kinases that convert inositol 1,4,5-trisphosphate (Ins(1,4,5)P3 or IP3) to inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). Previous studies have shown that disruption of InsP3KB leads to impaired T cell and B cell development as well as hyperactivation of neutrophils. Here, we demonstrate that InsP3KB is also a physiological modulator of myelopoiesis. The InsP3KB gene is expressed in all hematopoietic stem/progenitor cell populations. In InsP3KB null mice, the bone marrow granulocyte monocyte progenitor (GMP) population was expanded, and GMP cells proliferated significantly faster. Consequently, neutrophil production in the bone marrow was enhanced, and the peripheral blood neutrophil count was also substantially elevated in these mice. These effects might be due to enhancement of PtdIns(3,4,5)P3/Akt signaling in the InsP3KB null cells. Phosphorylation of cell cycle-inhibitory protein p21(cip1), one of the downstream targets of Akt, was augmented, which can lead to the suppression of the cell cycle-inhibitory effect of p21.

Inflammatory myopathy with anti-Golgi antibody and anti-SS-A/Ro antibody.

We report a 74-year-old woman with anit-Golgi antibody and anti-SS-A/Ro antibody who contracted inflammatory myopahy presenting 'ALS-like' symptoms. We identified anti-Golgi antibody directly using confocal microscopy and successfully treated her with steroid. This report suggests that there is a new categorized subgroup of inflammatory myopathy with these specific antibodies and the pattern of autoantibody in these patients indicates the specific clinical course and treatment strategy.

Inositol 1,3,4,5-tetrakisphosphate negatively regulates phosphatidylinositol-3,4,5- trisphosphate signaling in neutrophils.

Many neutrophil functions are regulated by phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) that mediates protein membrane translocation via binding to pleckstrin homolog (PH) domains within target proteins. Here we show that inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), a cytosolic small molecule, bound the same PH domain of target proteins and competed for binding to PtdIns(3,4,5)P3. In neutrophils, chemoattractant stimulation triggered rapid elevation in Ins(1,3,4,5)P4 concentration. Depletion of Ins(1,3,4,5)P4 by deleting the gene encoding InsP3KB, which converts Ins(1,4,5)P3 to Ins(1,3,4,5)P4, enhanced membrane translocation of the PtdIns(3,4,5)P3-specific PH domain. This led to enhanced sensitivity to chemoattractant stimulation, elevated superoxide production, and enhanced neutrophil recruitment to inflamed peritoneal cavity. On the contrary, augmentation of intracellular Ins(1,3,4,5)P4 concentration blocked PH domain-mediated membrane translocation of target proteins and dramatically decreased the sensitivity of neutrophils to chemoattractant stimulation. These findings establish a role for Ins(1,3,4,5)P4 in cellular signal transduction pathways and provide another mechanism for modulating PtdIns(3,4,5)P3 signaling in neutrophils.

RNAi screen identifies UBE2D3 as a mediator of all-trans retinoic acid-induced cell growth arrest in human acute promyelocytic NB4 cells.

All-trans retinoic acid (ATRA) has been widely used in differentiation therapy for acute promyelocytic leukemia (APL). ATRA binds to retinoic acid receptor (RAR) and triggers the formation of the transcription coactivator complex, which leads to changes in gene expression, APL cell-cycle arrest and differentiation, and clinical remission. The mechanisms responsible for ATRA's beneficial effects are still ill-defined. Here, we conducted a large-scale, unbiased short hairpin RNA (shRNA) screen aiming to identify mediators of ATRA-induced differentiation and growth arrest of APL cells. Twenty-six proteins were identified. They cover a wide range of cellular functions, including gene expression, intracellular signaling, cell death control, stress responses, and metabolic regulation, indicating the complexity of ATRA-induced cell growth control and differentiation in APL. One of these proteins, the ubiquitin-conjugating enzyme UBE2D3, is up-regulated in ATRA-treated acute promyelocytic NB4 cells. UBE2D3 is physically associated with cyclin D1 and mediates ATRA-induced cyclin D1 degradation. Knocking down UBE2D3 by RNA interference (RNAi) leads to blockage of ATRA-induced cyclin D1 degradation and cell-cycle arrest. Thus, our results highlight the involvement of the ubiquitin-mediated proteolysis pathway in ATRA-induced cell-cycle arrest and provide a novel strategy for modulating ATRA-elicited cellular effects.