Plasminogen binding and activation by Mycoplasma fermentans

The binding of plasminogen to Mycoplasma fermentans was studied by an immunoblot analysis and by a binding assay using iodine-labeled plasminogen. The binding of 125I-labeled plasminogen was inhibited by unlabeled plasminogen, lysine, and lysine analog epsilon-aminocaproic acid. Partial inhibition was obtained by a plasminogen fragment containing kringles 1 to 3 whereas almost no inhibition was observed with a fragment containing kringle 4. Scatchard analysis revealed a dual-phase interaction, one with a dissociation constant (kd) of 0.5 microM and the second with a kd of 7.5 microM. The estimated numbers of plasminogen molecules bound were calculated to be 110 and 790 per cell, respectively. Autoradiograms of ligand blots containing M. fermentans membrane proteins incubated with 125I-labeled plasminogen identified two plasminogen-binding proteins of about 32 and 55 kDa. The binding of plasminogen to M. fermentans enhances the activation of plasminogen to plasmin by the urokinase-type plasminogen activator (uPA), as monitored by measuring the breakdown of chromogenic substrate S-2251. Enhancement was more pronounced with the low-molecular-weight and the single-chain uPA variants, known to have low plasminogen activator activities. The binding of plasminogen also promotes the invasion of HeLa cells by M. fermentans. Invasion was more pronounced in the presence of uPA, suggesting that the ability of the organism to invade host cells stems not only from its potential to bind plasminogen but also from the activation of plasminogen to plasmin.     

Urokinase mediates fibrinolysis in the pulmonary microvasculature

The role of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in fibrinolysis remains unsettled. The contribution of uPA may depend on the vascular location, the physical properties of the clot, and its impact on tissue function. To study the contribution of urokinase within the pulmonary microvasculature, a model of pulmonary microembolism in the mouse was developed. Iodine 125 ((125)I)-labeled fibrin microparticles injected intravenously through the tail vein lodged preferentially in the lung, distributing homogeneously throughout the lobes. Clearance of (125)I-microemboli in wild type mice was rapid and essentially complete by 5 hours. In contrast, uPA(-/-) and tissue-type plasminogen activator tPA(-/-) mice, but not uPAR(-/-) mice, showed a marked impairment in pulmonary fibrinolysis throughout the experimental period. The phenotype in the uPA(-/-) mouse was rescued completely by infusion of single chain uPA (scuPA). The increment in clot lysis was 4-fold greater in uPA(-/-) mice infused with the same concentration of scuPA complexed with soluble recombinant uPAR. These data indicate that uPA contributes to endogenous fibrinolysis in the pulmonary vasculature to the same extent as tPA in this model system. Binding of scuPA to its receptor promotes fibrinolytic activity in vivo as well as in vitro. The physical properties of fibrin clots, including size, age, and cellular composition, as well as heterogeneity in endothelial cell function, may modify the participation of uPA in endogenous fibrinolysis. (Blood. 2000;96:1820-1826)     

Inflammation and dephosphorylation of the tight junction protein occludin in an experimental model of multiple sclerosis

Multiple sclerosis (MS) is a disease of the CNS in which inflammation, demyelination and neurodegeneration contribute to its initiation and progression. A frequently employed model of MS is experimental autoimmune encephalomyelitis (EAE). Here, to gain new insights into the disease process, an analysis of proteins in extracts of lumbar spinal cord from naïve and EAE rats was undertaken. The data mainly confirm that inflammation and blood–brain barrier (BBB) breakdown are the major hallmarks of disease in this model. Given their importance in the BBB, junctional proteins were further investigated. Occludin, a protein localizing to tight junctions in brain endothelial cells, showed strikingly increased migration in EAE when analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). This increased migration was mimicked by in vitro phosphatase treatment, implying its dephosphorylation in EAE. Occludin dephosphorylation coincided with the onset of inflammation, slightly preceding visible signs of disease, and was just prior to apparent changes in BBB permeability. These findings suggest occludin is a target for signaling processes in EAE, perhaps regulating the response of the BBB to the inflammatory environment as seen in MS.     

Early infection in post-autologous hematopoietic stem cell transplant patients: Princess Noorah Oncology Center experience

Objectives:To assess local epidemiology and risk factors for bacterial, fungal, and viral infections among the autologous bone marrow transplant population.Methods:This study is a retrospective correlational cohort design comprising 150 adult patients who underwent autologous transplants at Princess Noorah Oncology Center, Jeddah, Saudi Arabia between 2014 and 2020.Results:The study findings indicate that bacterial infection prevalence differed significantly across the different disease status pre-salvage as patients with the relapsed disease were more likely to have bacterial infections. The median of engraftment days differed significantly between those who had a bacterial infection and those who did not. Interestingly, previous pneumonia infection had a positive relationship with the number of hospital stays.Conclusions:Bacterial infections are the dominant type of infection among the autologous patient population. The research reflects authentic practice and reports unique characteristics of autologous transplant patients in terms of the prevalence and types of infection these patients experience.     

The alpha-defensins stimulate proteoglycan-dependent catabolism of low-density lipoprotein by vascular cells: a new class of inflammatory apolipoprotein and a possible contributor to atherogenesis

Inflammation may contribute to the pathogenesis of atherosclerosis. On the basis of previous reports that human atherosclerotic lesions contain alpha-defensins, a class of cationic proteins released by activated neutrophils, the study was designed to ask whether defensins modulate the binding and catabolism of low-density lipoprotein (LDL) by human vascular cells. The results of the study demonstrated that defensin stimulated the binding of (125)I-LDL to cultured human umbilical vein endothelial cells, smooth muscle cells, and fibroblasts approximately 5-fold in a dose-dependent and saturable manner. Defensin and LDL formed stable complexes in solution and on cell surfaces. Stimulation of LDL binding by defensin was not inhibited by antibodies against the LDL-receptor (LDL-R), or by recombinant receptor-associated protein, which blocks binding of ligands to the alpha(2)-macroglobulin receptor/LDL-R-related protein and other LDL-R family members. Furthermore, defensin stimulated the binding, endocytosis, and degradation of LDL by fibroblasts lacking LDL-R. Stimulation of LDL degradation by defensin was inhibited approximately 75% by low concentrations of heparin (0.2 units/mL) and was similarly reduced in CHO cells lacking heparan-sulfate-containing proteoglycans. The effect of defensin was substantially increased in cells overexpressing the core protein of the syndecan-1 heparan sulfate proteoglycan. The alpha-defensins released from activated neutrophils may provide a link between inflammation and atherosclerosis by changing the pattern of LDL catabolism from LDL-R to the less efficient LDL-R-independent, proteoglycan-dependent pathway. (Blood. 2000;96:1393-1398)     

The fibrinolytic system attenuates vascular tone: effects of tissue plasminogen activator (tPA) and aminocaproic acid on renal microcirculation

1. The renal medulla is a major source of plasminogen activators (PA), recently shown to induce vasodilation in vitro. Treatment with PA inhibitors has been associated with renal dysfunction, suggesting compromised renal microvasculature. We investigated the impact of the PA inhibitor epsilon amino-caproic acid (EACA) upon vascular tone in vitro, and studied the effect of both tPA and EACA upon intrarenal hemodynamics in vivo. 2. In vitro experiments were carried out in isolated aortic rings and with cultured vascular smooth muscle cells. Studies of renal microcirculation and morphology were conducted in anesthetized Sprague-Dawley rats. 3. In isolated aortic rings, EACA (but not the other inhibitors of the fibrinolytic system PAI-1 or alpha-2 antiplasmin) reduced the half-maximal effective concentration of phenylephrine (PE) required to induce contraction (from 32 nm in control solution to 2 and 0.1 nm at EACA concentrations of 1 and 10 microm, respectively). Using reteplase (retavase) in the same model, we also provide evidence that the vasoactivity of tPA is in part kringle-dependent. In cultured vascular smooth muscle cells, Ca(2+) internalization following PE was enhanced by EACA, and retarded by tPA. 4. In anesthetized rats, EACA (150 mg x kg(-1)) did not affect systemic blood pressure, total renal or cortical blood flow. However, the outer medullary blood flow declined 12+/-2% below the baseline (P<0.03). By contrast, tPA (2 mg x kg(-1)), transiently increased outer medullary blood flow by 8+/-5% (P<0.02). Fibrin microthrombi were not found within the renal microvasculature in EACA-treated animals. 5. In conclusion, both fibrinolytic and antifibrinolytic agents modulate medullary renal blood flow with reciprocal effects of vasodilation (PA) and vasoconstriction (EACA). In vitro studies suggest that these hemodynamic responses are related to direct modulation of the vascular tone.     

Oral cancer in Saudi Arabia: the role of alqat and alshammah

The purpose of this retrospective clinical study is to look into the etiological role of alshammah and alqat consumption in the causation of squamous cell carcinoma of the head and neck (SCHN) in Arabia. Alshammah and alqat are traditional forms of chewable tobacco very commonly used in southern Saudi Arabia and Yemem. In a 2 year period 64 patients with histologically proven SCHN were found. Most of them were originally from southern Saudi Arabia and Yemen. Of these 64 patients 52 (81%) admitted alshammah consumption. Of these 52 patients 22 admitted alqat consumption in addition. Thirty-eight (59%) had oral cancer, while the rest had pharyngeal and laryngeal cancer. All the 38 patients with oral cancer either used alshammah alone (16 patients) or used both alshammah and alqat (22 patients). The median duration of alshammah consumption was 15 years and that of alqat was 12 years. Alcohol was not consumed among the group and only few were cigarette smokers. Most patients presented in the late stage of the disease. The evidence implicating alshammah and alqat in the causation of SCHN is only clinically based. Further controlled prospective clinical and experimental studies are needed to confirm the theory.     

tPA-S481A prevents neurotoxicity of endogenous tPA in traumatic brain injury

Traumatic brain injury (TBI) is associated with loss of autoregulation due to impaired responsiveness to cerebrovascular dilator stimuli, which leads to cerebral hypoperfusion and neuronal impairment or death. Upregulation of tissue plasminogen activator (tPA) post-TBI exacerbates loss of cerebral autoregulation and NMDA-receptor-mediated impairment of cerebral hemodynamics, and enhances excitotoxic neuronal death. However, the relationship between NMDA-receptor activation, loss of autoregulation, and neurological dysfunction is unclear. Here, we evaluated the potential therapeutic efficacy of a catalytically inactive tPA variant, tPA S481A, that acts by competing with wild-type tPA for binding, cleavage, and activation of NMDA receptors. Lateral fluid percussion brain injury was produced in anesthetized piglets. Pial artery reactivity was measured via a closed cranial window, and cerebrospinal fluid (CSF) extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) was quantified by enzyme-linked immunosorbent assay (ELISA). tPA-S481A prevented impairment of cerebral autoregulation and reduced histopathologic changes after TBI by inhibiting upregulation of the ERK isoform of MAPK. Treatment with this tPA variant provides a novel approach for limiting neuronal toxicity caused by untoward NMDA-receptor activation mediated by increased tPA and glutamate following TBI.     

Polymerase chain reaction pool screening used to compare prevalence of infective black flies in two onchocerciasis foci in northern Sudan

Onchocerciasis remains an important debilitating disease in many areas of Africa, including Sudan. The status of infection transmission in 2007 was assessed in the vectors of two disease foci in Sudan: Abu Hamed in northern Sudan, which has received at least 10 years of annual treatment and Galabat focus in eastern Sudan, where only minor, largely undocumented treatment activity has occurred. Assessment of more than 30,000 black flies for Onchocerca volvulus infectious stage L3 larvae by using an O-150 polymerase chain reaction protocol showed that black fly infectivity rates were 0.84 (95% confidence interval = 0.0497-1.88) per 10,000 flies for Abu Hamed and 6.9 (95% confidence interval = 1.1-16.4) infective flies per 10,000 for Galabat. These results provide entomologic evidence for suppressed Onchocerca volvulus transmission in the Abu Hamed focus and a moderate transmission rate of the parasite in the Galabat focus.