Rapid Immunomagnetic Negative Enrichment of Neutrophil Granulocytes from Murine Bone Marrow for Functional Studies In Vitro and In Vivo

Polymorphonuclear neutrophils (PMN) mediate early immunity to infection but can also cause host damage if their effector functions are not controlled. Their lack or dysfunction is associated with severe health problems and thus the analysis of PMN physiology is a central issue. One prerequisite for PMN analysis is the availability of purified cells from primary organs. While human PMN are easily isolated from peripheral blood, this approach is less suitable for mice due to limited availability of blood. Instead, bone marrow (BM) is an easily available reservoir of murine PMN, but methods to obtain pure cells from BM are limited. We have developed a novel protocol allowing the isolation of highly pure untouched PMN from murine BM by negative immunomagnetic isolation using a complex antibody cocktail. The protocol is simple and fast (∼1 h), has a high yield (5–10*106 PMN per animal) and provides a purity of cells equivalent to positive selection (>80%). Most importantly, cells obtained by this method are non-activated and remain fully functional in vitro or after adoptive transfer into recipient animals. This method should thus greatly facilitate the study of primary murine PMN in vitro and in vivo

Cdc45 Limits Replicon Usage from a Low Density of preRCs in Mammalian Cells

Little is known about mammalian preRC stoichiometry, the number of preRCs on chromosomes, and how this relates to replicon size and usage. We show here that, on average, each 100-kb of the mammalian genome contains a preRC composed of approximately one ORC hexamer, 4–5 MCM hexamers, and 2 Cdc6. Relative to these subunits, ∼0.35 total molecules of the pre-Initiation Complex factor Cdc45 are present. Thus, based on ORC availability, somatic cells contain ∼70,000 preRCs of this average total stoichiometry, although subunits may not be juxtaposed with each other. Except for ORC, the chromatin-bound complement of preRC subunits is even lower. Cdc45 is present at very low levels relative to the preRC subunits, but is highly stable, and the same limited number of stable Cdc45 molecules are present from the beginning of S-phase to its completion. Efforts to artificially increase Cdc45 levels through ectopic expression block cell growth. However, microinjection of excess purified Cdc45 into S-phase nuclei activates additional replication foci by three-fold, indicating that Cdc45 functions to activate dormant preRCs and is rate-limiting for somatic replicon usage. Paradoxically, although Cdc45 colocalizes in vivo with some MCM sites and is rate-limiting for DNA replication to occur, neither Cdc45 nor MCMs colocalize with active replication sites. Embryonic metazoan chromatin consists of small replicons that are used efficiently via an excess of preRC subunits. In contrast, somatic mammalian cells contain a low density of preRCs, each containing only a few MCMs that compete for limiting amounts of Cdc45. This provides a molecular explanation why, relative to embryonic replicon dynamics, somatic replicons are, on average, larger and origin efficiency tends to be lower. The stable, continuous, and rate-limiting nature of Cdc45 suggests that Cdc45 contributes to the staggering of replicon usage throughout S-phase, and that replicon activation requires reutilization of existing Cdc45 during S-phase

The Transmembrane Adaptor Protein SIT Inhibits TCR- Mediated Signaling

Transmembrane adaptor proteins (TRAPs) organize signaling complexes at the plasma membrane, and thus function as critical linkers and integrators of signaling cascades downstream of antigen receptors. We have previously shown that the transmembrane adaptor protein SIT regulates the threshold for thymocyte selection. Moreover, T cells from SIT-deficient mice are hyperresponsive to CD3 stimulation and undergo enhanced lymphopenia-induced homeostatic proliferation, thus indicating that SIT inhibits TCR-mediated signaling. Here, we have further addressed how SIT regulates signaling cascades in T cells. We demonstrate that the loss of SIT enhances TCR-mediated Akt activation and increased phosphorylation/ inactivation of Foxo1, a transcription factor of the Forkhead family that inhibits cell cycle progression and regulates T-cell homeostasis. We have also shown that CD4 + T cells from SIT-deficient mice display increased CD69 and CD40L expression indicating an altered activation status. Additional biochemical analyses further revealed that suppression of SIT expression by RNAi in human T cells resulted in an enhanced proximal TCR signaling. In summary, the data identify SIT as an importan

Incidence and Risk Factors of Pneumonia in Individuals With Acute Spinal Cord Injury: A Multi-national, Multi-center, Prospective Cohort Study

Objective: To describe the occurrence of pneumonia in individuals with acute spinal cord injury (SCI) and identify its key predictors. Design: Multi-centric, longitudinal cohort study. Setting: 10 specialized SCI rehabilitation units in Europe and Australia. Participants: Eligible were 902 men and women with acute SCI, aged 18 years or older, with cervical or thoracic lesions and not dependent on 24-hour mechanical ventilation; 503 participated in the study (N=503). Interventions: Not applicable. Main Outcome Measures: We assessed demographics and lesion related parameters at study entry, and any pneumonia events throughout inpatient rehabilitation. Respiratory function, decubitus, and urinary tract infections were assessed at 1, 3, and 6 months post injury as well as at discharge from inpatient rehabilitation. Time to event (pneumonia) analyses were done using the Kaplan-Meier method, and potential predictors for pneumonia were analyzed with multivariable survival models. Results: Five hundred three patients with SCI were included, with 70 experiencing at least 1 pneumonia event. 11 participants experienced 2 or more events during inpatient rehabilitation. Most events occurred very early after injury, with a median of 6 days. Pneumonia risk was associated with tetraplegia (hazard ratio [HR]=1.78; 95% confidence interval [CI] 1.00-3.17) and traumatic etiology (HR=3.75; 95% CI 1.30-10.8) American Spinal Injury Impairment Scale (AIS) A (HR=5.30; 95% CI 2.28-12.31), B (HR=4.38; 95% CI 1.77-10.83), or C (HR=4.09; 95% CI 1.71-9.81) lesions. For every 10 cmH 2O increase in inspiratory muscle strength, pneumonia risk was reduced by 13% (HR=0.87; 95% CI 0.78-0.97)

Incidence and Risk Factors of Pneumonia in Individuals With Acute Spinal Cord Injury:A Multi-national, Multi-center, Prospective Cohort Study

Objective: To describe the occurrence of pneumonia in individuals with acute spinal cord injury (SCI) and identify its key predictors. Design: Multi-centric, longitudinal cohort study. Setting: 10 specialized SCI rehabilitation units in Europe and Australia. Participants: Eligible were 902 men and women with acute SCI, aged 18 years or older, with cervical or thoracic lesions and not dependent on 24-hour mechanical ventilation; 503 participated in the study (N=503). Interventions: Not applicable. Main Outcome Measures: We assessed demographics and lesion related parameters at study entry, and any pneumonia events throughout inpatient rehabilitation. Respiratory function, decubitus, and urinary tract infections were assessed at 1, 3, and 6 months post injury as well as at discharge from inpatient rehabilitation. Time to event (pneumonia) analyses were done using the Kaplan-Meier method, and potential predictors for pneumonia were analyzed with multivariable survival models. Results: Five hundred three patients with SCI were included, with 70 experiencing at least 1 pneumonia event. 11 participants experienced 2 or more events during inpatient rehabilitation. Most events occurred very early after injury, with a median of 6 days. Pneumonia risk was associated with tetraplegia (hazard ratio [HR]=1.78; 95% confidence interval [CI] 1.00-3.17) and traumatic etiology (HR=3.75; 95% CI 1.30-10.8) American Spinal Injury Impairment Scale (AIS) A (HR=5.30; 95% CI 2.28-12.31), B (HR=4.38; 95% CI 1.77-10.83), or C (HR=4.09; 95% CI 1.71-9.81) lesions. For every 10 cmH2O increase in inspiratory muscle strength, pneumonia risk was reduced by 13% (HR=0.87; 95% CI 0.78-0.97). Conclusion: Pneumonia is a major complication after SCI with the highest incidence very early after injury. Individuals with traumatic or AIS A, B, or C tetraplegia are at highest risk for pneumonia.</p

Incidence and Risk Factors of Pneumonia in Individuals With Acute Spinal Cord Injury:A Multi-national, Multi-center, Prospective Cohort Study

Objective: To describe the occurrence of pneumonia in individuals with acute spinal cord injury (SCI) and identify its key predictors. Design: Multi-centric, longitudinal cohort study. Setting: 10 specialized SCI rehabilitation units in Europe and Australia. Participants: Eligible were 902 men and women with acute SCI, aged 18 years or older, with cervical or thoracic lesions and not dependent on 24-hour mechanical ventilation; 503 participated in the study (N=503). Interventions: Not applicable. Main Outcome Measures: We assessed demographics and lesion related parameters at study entry, and any pneumonia events throughout inpatient rehabilitation. Respiratory function, decubitus, and urinary tract infections were assessed at 1, 3, and 6 months post injury as well as at discharge from inpatient rehabilitation. Time to event (pneumonia) analyses were done using the Kaplan-Meier method, and potential predictors for pneumonia were analyzed with multivariable survival models. Results: Five hundred three patients with SCI were included, with 70 experiencing at least 1 pneumonia event. 11 participants experienced 2 or more events during inpatient rehabilitation. Most events occurred very early after injury, with a median of 6 days. Pneumonia risk was associated with tetraplegia (hazard ratio [HR]=1.78; 95% confidence interval [CI] 1.00-3.17) and traumatic etiology (HR=3.75; 95% CI 1.30-10.8) American Spinal Injury Impairment Scale (AIS) A (HR=5.30; 95% CI 2.28-12.31), B (HR=4.38; 95% CI 1.77-10.83), or C (HR=4.09; 95% CI 1.71-9.81) lesions. For every 10 cmH2O increase in inspiratory muscle strength, pneumonia risk was reduced by 13% (HR=0.87; 95% CI 0.78-0.97). Conclusion: Pneumonia is a major complication after SCI with the highest incidence very early after injury. Individuals with traumatic or AIS A, B, or C tetraplegia are at highest risk for pneumonia.</p

Incidence and Risk Factors of Pneumonia in Individuals With Acute Spinal Cord Injury: A Multi-national, Multi-center, Prospective Cohort Study

OBJECTIVE To describe the occurrence of pneumonia in individuals with acute spinal cord injury (SCI) and identify its key predictors. DESIGN Multi-centric, longitudinal cohort study. SETTING 10 specialized SCI rehabilitation units in Europe and Australia. PARTICIPANTS Eligible were 902 men and women with acute SCI, aged 18 years or older, with cervical or thoracic lesions and not dependent on 24-hour mechanical ventilation; 503 participated in the study (N=503). INTERVENTIONS Not applicable. MAIN OUTCOME MEASURES We assessed demographics and lesion related parameters at study entry, and any pneumonia events throughout inpatient rehabilitation. Respiratory function, decubitus, and urinary tract infections were assessed at 1, 3, and 6 months post injury as well as at discharge from inpatient rehabilitation. Time to event (pneumonia) analyses were done using the Kaplan-Meier method, and potential predictors for pneumonia were analyzed with multivariable survival models. RESULTS Five hundred three patients with SCI were included, with 70 experiencing at least 1 pneumonia event. 11 participants experienced 2 or more events during inpatient rehabilitation. Most events occurred very early after injury, with a median of 6 days. Pneumonia risk was associated with tetraplegia (hazard ratio [HR]=1.78; 95% confidence interval [CI] 1.00-3.17) and traumatic etiology (HR=3.75; 95% CI 1.30-10.8) American Spinal Injury Impairment Scale (AIS) A (HR=5.30; 95% CI 2.28-12.31), B (HR=4.38; 95% CI 1.77-10.83), or C (HR=4.09; 95% CI 1.71-9.81) lesions. For every 10 cmH$_{2}$O increase in inspiratory muscle strength, pneumonia risk was reduced by 13% (HR=0.87; 95% CI 0.78-0.97). CONCLUSION Pneumonia is a major complication after SCI with the highest incidence very early after injury. Individuals with traumatic or AIS A, B, or C tetraplegia are at highest risk for pneumonia

Analysis of activation markers in SIT^−/− mice.

<p>A) Splenic CD4⁺ T cells from 1 year-old SIT-deficient and wild-type mice were analyzed for CD69 expression. Numbers within histograms indicate the percentage of cells. One representative profile per group is shown. B) Comparison of CD40L expression on CD4⁺ T cells isolated from SIT-deficient (empty histogram) and wild-type (filled histogram) mice. Mean fluorescent intensity (MFI) from one representative mouse per group is indicated. C) Comparison of CD5 expression on CD4⁺ T cells isolated from SIT-deficient (empty histogram) and wild-type (filled histogram) mice. Bar graphs in A), B), and C) represent statistical analyses of CD69, CD40L, and CD5 expression, respectively. Data derive from at least 5 mice per group. Statistical significance * p<0.05 and ***p<0.0001.</p