Mass cytometry reveals cladribine-induced resets among innate lymphoid cells in multiple sclerosis.

Here we present a comprehensive mass cytometry analysis of peripheral innate lymphoid cell (ILC) subsets in relapsing/remitting MS (RRMS) patients prior to and after onset of cladribine tablets (CladT). ILC analysis was conducted on CyTOF data from peripheral blood mononuclear cells (PBMC) of MS patients before, 2 and 6 months after onset of CladT, and non-MS controls. Dimensionality reduction was used for immunophenotyping ILC subsets. CladT reduced all ILC subsets, except for CD56bright NK cells and ILC2. Furthermore, CD38+ NK cell and CCR6+ ILC3 were excluded from CladT-induced immune cell reductions. Post-CladT replenishment by immature ILC was noted by increased CD5+ ILC1 proportions at 2 months, and boosted CD38-CD56bright NK cell numbers at 6 months. CladT induce immune cell depletion among ILC but exclude CD56bright NK cells and ILC2 subsets, as well as CD38+ NK cell and CCR6+ ILC3 immunophenotypes. Post-CladT ILC expansions indicate ILC reconstitution towards a more tolerant immune system phenotype.

A Novel Computerized Cognitive Stress Test to Detect Mild Cognitive Impairment.

BACKGROUND: The Loewenstein Acevedo Scales of Semantic Interference and Learning (LASSI-L) is a novel and increasingly employed instrument that has outperformed widely used cognitive measures as an early correlate of elevated brain amyloid and neurodegeneration in prodromal Alzheimer's Disease (AD). The LASSI-L has distinguished those with amnestic mild cognitive impairment (aMCI) and high amyloid load from aMCI attributable to other non-AD conditions. The authors designed and implemented a web-based brief computerized version of the instrument, the LASSI-BC, to improve standardized administration, facilitate scoring accuracy, real-time data entry, and increase the accessibility of the measure. OBJECTIVE: The psychometric properties and clinical utility of the brief computerized version of the LASSI-L was evaluated, together with its ability to differentiate older adults who are cognitively normal (CN) from those with amnestic Mild Cognitive Impairment (aMCI). METHODS: After undergoing a comprehensive uniform clinical and neuropsychological evaluation using traditional measures, older adults were classified as cognitively normal or diagnosed with aMCI. All participants were administered the LASSI-BC, a computerized version of the LASSI-L. Test-retest and discriminant validity was assessed for each LASSI-BC subscale. RESULTS: LASSI-BC subscales demonstrated high test-retest reliability, and discriminant validity was attained. CONCLUSIONS: The LASSI-BC, a brief computerized version of the LASSI-L is a valid and useful cognitive tool for the detection of aMCI among older adults.

Stable thrombus formation on irradiated microvascular endothelial cells under pulsatile flow: Pre-testing annexin V-thrombin conjugate for treatment of brain arteriovenous malformations.

BACKGROUND: Our goal is to develop a vascular targeting treatment for brain arteriovenous malformations (AVMs). Externalized phosphatidylserine has been established as a potential biomarker on the endothelium of irradiated AVM blood vessels. We hypothesize that phosphatidylserine could be selectively targeted after AVM radiosurgery with a ligand-directed vascular targeting agent to achieve localized thrombosis and rapid occlusion of pathological AVM vessels. OBJECTIVE: The study aim was to establish an in vitro parallel-plate flow chamber to test the efficacy of a pro-thrombotic conjugate targeting phosphatidylserine. METHODS: Conjugate was prepared by Lys-Lys cross-linking of thrombin with the phosphatidylserine-targeting ligand, annexin V. Cerebral microvascular endothelial cells were irradiated (5, 15, and 25 Gy) and after 1 or 3 days assembled in a parallel-plate flow chamber containing whole human blood and conjugate (1.25 or 2.5 µg/mL). Confocal microscopy was used to assess thrombus formation after flow via binding and aggregation of fluorescently-labelled platelets and fibrinogen. RESULTS AND CONCLUSIONS: The annexin V-thrombin conjugate induced rapid thrombosis (fibrin deposition) on irradiated endothelial cells under shear stress in the parallel-plate flow device. Unconjugated, non-targeting thrombin did not induce fibrin deposition. A synergistic interaction between radiation and conjugate dose was observed. Thrombosis was greatest at the highest combined doses of radiation (25 Gy) and conjugate (2.5 µg/mL). The parallel-plate flow system provides a rapid method to pre-test pro-thrombotic vascular targeting agents. These findings validate the translation of the annexin V-thrombin conjugate to pre-clinical studies.

Brain endothelial cells increase the proliferation of Plasmodium falciparum through production of soluble factors.

We here describe the novel finding that brain endothelial cells in vitro can stimulate the growth of Plasmodium falciparum through the production of low molecular weight growth factors. By using a conditioned medium approach, we show that the brain endothelial cells continued to release these factors over time. If this mirrors the in vivo situation, these growth factors potentially would provide an advantage, in terms of enhanced growth, for sequestered parasitised red blood cells in the brain microvasculature. We observed this phenomenon with brain endothelial cells from several sources as well as a second P. falciparum strain. The characteristics of the growth factors included: <3 kDa molecular weight, heat stable, and in part chloroform soluble. Future efforts should be directed at identifying these growth factors, since blocking their production or actions might be of benefit for reducing parasite load and, hence, malaria pathology.

Is routine endocrine evaluation necessary after paediatric traumatic brain injury?

BACKGROUND: Traumatic brain injury (TBI) is a common event in childhood. It is a recognised cause of hypopituitarism both in adult and paediatric patients. Routine endocrine evaluation has been proposed for adult TBI-survivors; nevertheless, incongruous data have been reported in children. AIM: The goal of this study was to describe the prevalence of pituitary dysfunction after TBI in a cohort of children. MATERIAL/SUBJECTS AND METHODS: This is a cross-sectional study comprising retrospective medical record review and prospective testing. Children with brain injury discharged from the Paediatric Intensive Care Unit from year 2004 to 2009 were recruited. Height and weight were recorded, systemic examination was performed and baseline pituitary function tests were undertaken. Provocative tests were performed only if abnormal basal levels were detected. RESULTS: Thirty-six patients were collected; the mean age at assessment was 7.2 years and the mean interval since injury 3.3 years. All patients had skull fracture or intracranial haemorrhage; 36.6 % of them had moderate to severe TBI. No abnormalities were found on examination. Low serum IGF 1 levels were detected in four patients and two patients had low serum cortisol levels with inappropriately normal plasma ACTH concentrations. No evidence of pituitary dysfunction was observed in these patients after clinical follow-up, repeated baseline hormone levels or dynamic function tests. CONCLUSIONS: No endocrine sequelae have been detected in this population. The routine endocrine evaluation in children with mild to moderate TBI might not be justified, according to our findings.

Single-cell clones of liver cancer stem cells have the potential of differentiating into different types of tumor cells.

Cancer stem cells (CSCs) are believed to be a promising target for cancer therapy because these cells are responsible for tumor development, maintenance and chemotherapy resistance. Finding out the critical factors regulating CSC fate is the key for target therapy of CSCs. Just as normal stem cells are regulated by their microenvironment (niche), CSCs are also regulated by cells in the tumor microenvironment. However, whether various tumor microenvironments can induce CSCs to differentiate into different cancer cells is not clear. Here, we show that single-cell-cloned CSCs, accidentally obtained from a human liver cancer microvascular endothelial cells, express classic stem cell markers, genes associated with self-renewal and pluripotent factors and possess colony-forming ability in vitro and the ability of serial transplantation in vivo. The single-cell-cloned CSCs treated with the different tumor cell/tissue-derived conditioned culture medium, which is a mimic of carcinoma microenvironment, could differentiate into corresponding tumor cells and express specific markers of the respective type of tumor cells at the gene, protein and cell levels, respectively. Interestingly, this multilineage differentiation potential of single-cell-cloned liver CSCs sharply declined after the specific knockdown of octamer-binding transcription factor 4 (Oct4) alone, even though they were under the same induction conditions (carcinoma microenvironments). These data support the hypothesis that single-cell-cloned liver CSCs have the potential of differentiating into different types of tumor cells, and the tumor microenvironment does play a crucial role in deciding differentiation directions. Simultaneously, Oct4 in CSCs is indispensable in this process. These factors are promising targets for liver CSC-specific therapy.

Microparticles and their emerging role in cancer multidrug resistance.

Drug resistance is a major obstacle to the successful treatment of cancer as tumor cells either fail to reduce in size following chemotherapy or the cancer recurs after an initial response. The phenomenon of multidrug resistance (MDR) is particularly problematic as it involves the simultaneous resistance to numerous chemotherapeutics of different classes. MDR is predominantly attributed to the overexpression of efflux transporters such as P-glycoprotein (P-gp) and the Multidrug Resistance-Associated Protein 1 (MRP1). P-gp and MRP1 are members of the ATP Binding Cassette (ABC) superfamily of transporters and are capable of effluxing many chemotherapeutics out of cancer cells, allowing them to survive the toxic insult. Numerous strategies have been developed over the years to circumvent MDR. Of these, the discovery and implementation of P-gp and MRP1 inhibitors have been most extensively studied. However, these inhibitors have not been able to be used clinically. While research continues in this area, it must also be acknowledged that other avenues must be explored. Recently, the novel 'non-genetic' acquisition of P-gp-mediated MDR by microparticles (MPs) has been reported. MPs are vesicles 0.1-1µm in diameter that are released via plasma membrane blebbing. They are important mediators of inflammation, coagulation and vascular homeostasis. In addition to surface P-gp protein, MPs also carry various nucleic acid species as cargo. This 'non-genetic' intercellular transfer provides an alternative pathway for the cellular acquisition and dissemination of traits and implicates MPs as important mediators in the spread of MDR and provides a novel pathway for the circumvention of MDR.

Resistance to anti-tuberculosis medications in the Horn of Africa.

We performed drug susceptibility testing among chronic patients hospitalised in the tuberculosis (TB) referral centre of the capital of Djibouti. Among 36 patients tested, 27 had multidrug-resistant TB and four had extensively drug-resistant TB. National guidelines must be revised urgently and the administration of second-line TB medications strictly controlled.

Membrane microparticles mediate transfer of P-glycoprotein to drug sensitive cancer cells.

Multidrug resistance (MDR), a significant impediment to the successful treatment of cancer clinically, has been attributed to the overexpression of P-glycoprotein (P-gp), a plasma membrane multidrug efflux transporter. P-gp maintains sublethal intracellular drug concentrations by virtue of its drug efflux capacity. The cellular regulation of P-gp expression is currently known to occur at either pre- or post-transcriptional levels. In this study, we identify a 'non-genetic' mechanism whereby microparticles (MPs) serve as vectors in the acquisition and spread of MDR. MPs isolated from drug-resistant cancer cells (VLB(100)) were co-cultured with drug sensitive cells (CCRF-CEM) over a 4 h period to allow for MP binding and P-gp transfer. Presence of P-gp on MPs was established using flow cytometry (FCM) and western blotting. Whole-cell drug accumulation assays using rhodamine 123 and daunorubicin (DNR) were carried out to validate the transfer of functional P-gp after co-culture. We establish that MPs shed in vitro from drug-resistant cancer cells incorporate cell surface P-gp from their donor cells, effectively bind to drug-sensitive recipient cells and transfer functional P-gp to the latter. These findings serve to substantially advance our understanding of the molecular basis for the emergence of MDR in cancer clinically and lead to new treatment strategies which target and inhibit MP mediated transfer of P-gp during the course of treatment.

Clinical presentation, haematological indices and management of children with severe and uncomplicated malaria in Douala, Cameroon.

This study carried out from January to June 2007, was undertaken to describe the clinical presentation of childhood malaria in Douala, a meso-endemic area as far as malaria transmission is concerned. One hundred and seventy eight children were enrolled after informed consent of their parents. The sample characteristics were recorded and clinical as well as preliminary laboratory investigations were performed. Thirty eight children coming for vaccination and counselling was targeted to serve as control. According to the results obtained, cerebral malaria (CM) seems to be associated with young age, whilst Malaria anaemia (MA) was predominant among older children. Hyperpyrexia and hyperparasitaemia were high among CM patients and 11.1% of them died, however, no neurological squeal was noticed immediately after discharge on those who survived. Haemoglobin and glycaemia were low on MA and CM patients; these groups had low percentage in bed nets utilization as well. These results suggest that the clinical presentation of the disease differ with the geographic location and malaria disease features varies according to the severity. Such studies could contribute to the management of the disease.

Reproductibilidad interobservador de la Escala AVD Alzheimer, entre las disciplinas de psicología, fisioterapia y terapia ocupacional / Interobserver reliability on the Alzheimer's ADL Scale, in the health areas of psychology, physiotherapy and occupational therapy

Fundamentos: el objetivo del presente estudio fue realizar la reproductibilidad interobservador de la Escala AVD Alzheimer entre tres profesionales (uno del área de la "psicología", otro del área de la "fisioterapia" y otro del área de la "terapia ocupacional"), para adaptarla a otras disciplinas sanitarias (fisioterapia y terapia ocupacional).Métodos: se utilizó un diseño longitudinal prospectivo durante 6 meses aproximadamente, con un grupo de 50 sujetos (cuidadores de enfermos diagnosticados de enfermedad de Alzheimer), provenientes del Centro Residencial Putxet (Barcelona), Centro de Día "Cuidem la Memòria" (Barcelona), Residencia Pedrell (Barcelona), Residencia Campoamor (Barcelona) y Residencia Bon Estar (Premiá de Mar). La Escala AVD Alzheimer fue administrada por tres profesionales (uno del área de la "psicología", otro del área de "fisioterapia" y otro del área de la "terapia ocupacional"). Dado que se comparaban profesiones distintas, permitiría que se cumpliera el objetivo: la multidisciplinariedad e interdisciplinariedad. Resultados: las puntuaciones de las kappas son de aceptables a buenas, dado que la gran mayoría de ellas se encuentran por encima de 0,7 (p<0.01). La Escala AVD Alzheimer, presenta las características métricas necesarias en cuanto a su fiabilidad interobservador entre las áreas sanitarias de "psicología", "fisioterapia" y "terapia ocupacional". Conclusiones: todo y con las diferencias existentes entre las disciplinas sanitarias de "psicología", "fisioterapia" y "terapia ocupacional", se observa que los resultados son óptimos. La Escala AVD Alzheimer puede ser administrada por las disciplinas "fisioterapia" y "terapia ocupacional", con buenos resultados (AU)

[Pathogenesis of cerebral malaria: facts and hypotheses]. / Pathogenèse du neuropaludisme: faits et hypothèses.

Cerebral malaria (CM) is one of the most serious complications of Plasmodium falciparum infection. It is characterized by sequestration of parasitized red blood cells (PRBC) in cerebral capillaries and venules. Although the exact cause of CM remains unclear, current evidence has clearly implicated metabolic disturbances and host immune responses. Studies on mouse CM models suggest the involvement of host cells and in particular platelets. These results led us to study the role of platelets in human CM. Our findings demonstrated that significantly greater accumulation of platelets occurred in capillaries and venules of Malawian patients who died from CM than from other diseases. We also assessed the role of platelets in cytoadherence of PRBCs using PRBC adhering only on CD36, platelets and endothelial cells (EC) constitutively devoid of CD36. Cultures using the three components showed that platelets played a role in inducing cytoadherence of PRBC on EC via a cellular bridging resistant to physiological flow conditions. Having established the link between platelets and sequestration, the next step will be to examine the link between platelets and CM. A combination of approaches from different disciplines will be needed to gain further insight into the mechanisms underlying the complications of malaria.

Pathogenesis of cerebral malaria: recent experimental data and possible applications for humans.

Malaria still is a major public health problem, partly because the pathogenesis of its major complication, cerebral malaria, remains incompletely understood. Experimental models represent useful tools to better understand the mechanisms of this syndrome. Here, data generated by several models are reviewed both in vivo and in vitro; we propose that some pathogenic mechanisms, drawn from data obtained from experiments in a mouse model, may be instrumental in humans. In particular, tumor necrosis factor (TNF) receptor 2 is involved in this syndrome, implying that the transmembrane form of TNF may be more important than the soluble form of the cytokine. It has also been shown that in addition to differences in immune responsiveness between genetically resistant and susceptible mice, there are marked differences at the level of the target cell of the lesion, namely, the brain endothelial cell. In murine cerebral malaria, a paradoxical role of platelets has been proposed. Indeed, platelets appear to be pathogenic rather than protective in inflammatory conditions because they can potentiate the deleterious effects of TNF. More recently, it has been shown that interactions among platelets, leukocytes, and endothelial cells have phenotypic and functional consequences for the endothelial cells. A better understanding of these complex interactions leading to vascular injury will help improve the outcome of cerebral malaria.

The spanish version of the paediatric asthma quality of life questionnaire (PAQLQ): metric characteristics and equivalence with the original version.

UNLABELLED: The Paediatric Asthma Quality of Life Questionnaire (PAQLQ), developed by Juniper et al., assesses the impact of asthma on children's daily life. It contains 23 items, covering three dimensions: symptoms, activities limitation and emotional function. AIMS: To develop an equivalent Spanish version of the PAQLQ, and to assess its measurement characteristics. METHODS: The forward and back-translation method was used for the adaptation. A longitudinal study (assessments at the 1st and 5th weeks), with patients from the emergency and outpatient departments of three Spanish hospitals, was designed to test the properties of the new adapted version. At each visit, a trained interviewer administered the PAQLQ, a Global Index of Change and a General Health Perception scale. The Peak Expiratory Flow Rate (PEFR) was also recorded daily, together with symptoms, during the prior week. RESULTS: Ninety-nine patients (66.7% males, 42.4% suffering an exacerbation, mean age of 11.3 years) with the following types of asthma were evaluated: mild intermittent (31.3%), mild persistent (36.4%), moderate persistent (29.3%) and severe persistent (3%). At the 1st visit, the mean pre-bronchodilator %PEFR was 87.3%. The Cronbach's alpha ranged from 0.86 to 0.95. As expected, correlations between the PAQLQ scores, and the Asthma Control Score (0.53-0.67), the General Health Perception (0.34-0.55), and the %PEFR (0.44-0.55) were moderate. The PAQLQ scores remained unchanged in stable patients while increased significantly in those showing improvements. CONCLUSIONS: After a standard cross-cultural adaptation process, the Spanish version of the PAQLQ has shown to be equivalent to the original, with similar internal consistency reliability, validity and sensitivity to clinical changes.

Diagnostic value of procalcitonin, interleukin-6, and interleukin-8 in critically ill patients admitted with suspected sepsis.

To assess the diagnostic value of procalcitonin (PCT), interleukin (IL)-6, IL-8, and standard measurements in identifying critically ill patients with sepsis, we performed prospective measurements in 78 consecutive patients admitted with acute systemic inflammatory response syndrome (SIRS) and suspected infection. We estimated the relevance of the different parameters by using multivariable regression modeling, likelihood-ratio tests, and area under the receiver operating characteristic curves (AUC). The final diagnosis was SIRS in 18 patients, sepsis in 14, severe sepsis in 21, and septic shock in 25. PCT yielded the highest discriminative value, with an AUC of 0.92 (CI, 0.85 to 1.0), followed by IL-6 (0.75; CI, 0.63 to 0.87), and IL-8 (0.71; CI, 0.59 to 0.83; p < 0.001). At a cutoff of 1.1 ng/ml, PCT yielded a sensitivity of 97% and a specificity of 78% to differentiate patients with SIRS from those with sepsis-related conditions. Median PCT concentrations on admission (ng/ ml, range) were 0.6 (0 to 5.3) for SIRS; 3.5 (0.4 to 6.7) for sepsis; 6.2 (2.2 to 85) for severe sepsis; and 21.3 (1.2 to 654) for septic shock (p < 0.001). The addition of PCT to a model based solely on standard indicators improved the predictive power of detecting sepsis (likelihood ratio test; p = 0.001) and increased the AUC value for the routine value-based model from 0.77 (CI, 0.64 to 0.89) to 0.94 (CI, 0.89 to 0.99; p = 0.002). In contrast, no additive effect was seen for IL-6 (p = 0.56) or IL-8 (p = 0.14). Elevated PCT concentrations appear to be a promising indicator of sepsis in newly admitted, critically ill patients capable of complementing clinical signs and routine laboratory parameters suggestive of severe infection.

Lectin-deficient TNF mutants display comparable anti-tumour but reduced pro-metastatic potential as compared to the wild-type molecule.

In this study, we characterised the anti-tumour as well as the pro-metastatic activities of TNF mutants deficient in their lectin-like activity.1619 We report that, despite reduced systemic toxicity as compared to wild-type (wt) mTNF, a (T104A) and a (T104A-E106A-E109A) mTNF mutant (triple mTNF) retained most of their necrotic and tumouristatic activities, as measured in a CFS-1 fibrosarcoma and a B16BL6 melanoma tumour model, respectively. These mutants also conserved their anti-angiogenic activity, as measured in an in vitro endothelial morphogenesis assay.26 In contrast, the pro-metastatic activity of the T104A and the triple mTNF mutants in the CFS-1 fibrosarcoma and the 3LL-R Lewis lung carcinoma tumour model was significantly lower than that of the wt molecule. These results thus indicate that the lectin-like domain of TNF is not implicated in its necrotic, tumouristatic and anti-angiogenic activities, but that it can contribute to the pro-metastatic effect of the cytokine. In conclusion, in view of their reduced systemic toxicity and pro-metastatic capacity, but their retained anti-tumour activities, lectin-deficient TNF mutants might prove to be therapeutically interesting alternatives to wt TNF.

Irreducible lateral dislocation of patella due to an intact retinaculum.

Lateral dislocation of the patella is a common injury that can be treated successfully nonoperatively. The authors present a patient with lateral dislocation of the patella in which the patella rotated 90 degrees about its vertical axis. The retinaculum had been avulsed from the anterior aspect of the patella but was otherwise intact. Because the intact retinaculum prohibited closed reduction, open reduction was performed. The patient has returned to full, unrestricted activities.

Evaluation of the acute cervical spine: a management algorithm.

BACKGROUND: Safe, efficient, and cost-effective evaluation of the spine is the goal in the trauma setting. At our Level I trauma facility, the trauma service, emergency medicine, radiology, anesthesia, and the spine service combined individual concerns into one agreed-upon clearance protocol. Here, we present the effectiveness of a new cervical spine clearance protocol. METHODS: A retrospective review was initiated of all trauma patients evaluated in a Level I trauma center the year before and after implementation of a new cervical spine protocol to determine the incidence of missed cervical injuries. An additional 6 months were reviewed to detect any missed injuries late in the study period. RESULTS: During the 2-year study period, 4,460 patients presented to the emergency room with some form of cervical spine precautions. Blunt trauma comprised 90% of the study population. According to the protocol, approximately 45% required further cervical radiographs after presentation. In the preprotocol year, 77 of 2,217 (3.4%) patients were diagnosed with cervical spine injuries, 16 of 77 (21%) with multiple level of injuries, and 25 of 77 (32%) with neurologic compromise. Three of 2,217 patients had missed cervical spine injuries on their initial evaluations. In the postprotocol year, 84 of 2,243 (3.4%) patients had cervical injuries, 25 of 84 (30%) with multiple levels of injuries and 28 of 84 (28%) with neurologic compromise. No patient evaluated during the protocol year was missed. All statistics between the two groups were not significant. CONCLUSION: The current protocol by risk stratifying patients on presentation is effective in assessing patients for cervical spine injuries.