In vivo imaging of translocator protein, a marker of activated microglia, in alcohol dependence.

Neuroinflammation may be a critical component of the neurobiology of alcohol use disorders, yet the exact nature of this relationship is not well understood. This work compared the brain and peripheral immune profile of alcohol-dependent subjects and controls. Brain levels of 18-kDa translocator protein (TSPO), a marker of microglial activation and neuroinflammation, were measured with [11C]PBR28 positron emission tomography imaging in 15 healthy controls and 15 alcohol-dependent subjects. Alcohol-dependent subjects were imaged 1-4 days (n=14) or 24 days (n=1) after their last drink. Linear mixed modeling of partial-volume-corrected [11C]PBR28 data revealed a main effect of alcohol dependence (P=0.034), corresponding to 10% lower TSPO levels in alcohol-dependent subjects. Within this group, exploratory analyses found a negative association of TSPO levels in the hippocampus and striatum with alcohol dependence severity (P<0.035). Peripheral immune response was assessed in a subset of subjects by measuring cytokine expression from monocytes cultured both in the presence and absence of lipopolysaccharide. Peripheral monocyte response to lipopolysaccharide stimulation was lower in alcohol-dependent subjects compared with controls for the proinflammatory cytokines interleukin-6 and interleukin-8. Thus, alcohol-dependent individuals exhibited less activated microglia in the brain and a blunted peripheral proinflammatory response compared with controls. These findings suggest a role for pharmaceuticals tuning the neuroimmune system as therapeutics for alcohol dependence.

A unique antibody gene signature is prevalent in the central nervous system of patients with multiple sclerosis.

B cells isolated from the CSF of patients with multiple sclerosis (MS) have a unique accumulation of somatic hypermutation within the B cell receptor, termed the antibody gene signature (AGS). The focus of this study was to investigate whether the AGS could also be detected in MS brain tissue. Genetic analysis of B cells isolated from post-mortem CNS tissue samples from four MS brains demonstrated that signature enriched B cells are present at the site of tissue injury as well as in the circulating CSF.

Anti-myelin antibodies modulate clinical expression of childhood multiple sclerosis.

Anti-myelin basic protein (MBP) antibodies in pediatric-onset MS and controls were characterized. Serum samples were obtained from 94 children with MS and 106 controls. Paired CSF and serum were obtained from 25 children with MS at time of their initial episode of acute demyelinating syndrome (ADS). Complementary assays were applied across samples to evaluate the presence, and the physical binding properties, of anti-MBP antibodies. While the prevalence and titers of serum anti-MBP antibodies against both immature and mature forms of MBP were similar in children with MS and in controls, binding characteristics and formal Surface Plasmon Resonance (SPR) studies indicated surprisingly high binding affinities of all pediatric anti-MBP antibodies. Serum levels of anti-MBP antibodies correlated significantly with their CSF levels, and their presence in children with MS was associated with significantly increased risk of an acute disseminated encephalomyelitis-like initial clinical presentation. While antibodies to both immature and mature forms of MBP can be present as part of the normal pediatric humoral repertoire, these anti-myelin antibodies are of surprisingly high affinity, can access the CNS during inflammation, and have the capacity to modulate disease expression. Our findings identify an immune mechanism that could contribute to the observed heterogeneity in spectrum of clinical presentations in early-onset MS.

Plasma cells in muscle in inclusion body myositis and polymyositis.

BACKGROUND: Previous immunohistochemical studies of muscle from patients with inclusion body myositis and polymyositis found many more T cells than B cells, suggesting a role for intramuscular cell-mediated immune mechanisms rather than humoral mechanisms. METHODS: Microarray studies were performed on muscle biopsy specimens from 40 patients with inclusion body myositis (IBM; n = 23), polymyositis (PM; n = 6), and without neuromuscular disease (n = 11). Reverse transcription PCR of selected immunoglobulin gene transcripts was performed on two patient samples. Qualitative immunohistochemical studies for B-cell lineage cell surface markers were performed on 28 muscle specimens and quantitative studies performed on a subset of 19 untreated patients with IBM or PM. CD138+ cells were isolated from muscle using laser capture microdissection, and immunoglobulin transcripts were PCR amplified to determine the presence or absence of immunoglobulin gene rearrangements unique to the B-cell lineage. RESULTS: Immunoglobulin gene transcripts accounted for 59% in IBM and 33% in PM of the most stringently defined highest differentially expressed muscle transcripts compared with normal. Plasma cells, terminally differentiated B cells expressing CD138 but not CD19 or CD20, are present in IBM and PM muscle in numbers several times higher than B cells. CONCLUSIONS: There are differentiated B cells in the form of CD138+ plasma cells within the muscle of patients with inclusion body myositis and polymyositis. The principle of linked recognition of B-cell activation predicts several strategies for autoantigen discovery that could not otherwise be pursued through the study of the infiltrating T-cell population alone.

Immunological memory: contribution of memory B cells expressing costimulatory molecules in the resting state.

Traditionally, emphasis has been placed on the roles of Th cells in generating and amplifying both cellular and humoral memory responses. Little is known about the potential contributions of B cell subsets to immunological memory. Resting memory B cells have generally been regarded as poor APC, attributed in part to the relative paucity of costimulatory molecules identified on their surface. We describe a novel subpopulation of human memory B cells that express CD80 in their resting state, are poised to secrete particularly large amounts of class switched Igs, and can efficiently present Ag to and activate T cells. This functionally distinct B cell subset may represent an important mechanism by which quiescent human B cells can initiate and propagate rapid and vigorous immune memory responses. Finally, these studies extend recent observations in the murine system and highlight the phenotypic and functional diversity that exists within the human B cell memory compartment.

The rearranged V(H) domain of a physiologically selected anti-single-stranded DNA antibody as a precursor for formation of IgM and IgG antibodies to diverse antigens.

It has been proposed that autoreactivity of modest affinity contributes to positive selection of a preimmunization B cell repertoire, whereas high-affinity autoreactivity leads to negative selection. This hypothesis predicts that a B cell producing a physiologically selected unmutated ssDNA-binding Ab should be a precursor of cells that respond to diverse exogenous Ags. To test this prediction, we prepared transgenic mice bearing the rearranged V(H) domain of an IgM Ab from a nonautoimmune mouse immunized with a DNA-protein complex, poly(dC)-methylated BSA. The Ab, dC1, binds both poly(dC) and ssDNA. It is encoded by V(H) and V(L) gene segments with no mutations, suggesting that the producing cell may have been selected before and activated during immunization. The dC1V(H) transgene was targeted to the IgH locus. In heterozygous mice, on a nonautoimmune C57BL/6 background, the transgene allotype was expressed on B cell surfaces and in serum Ig, but about one-third of B cells expressed the endogenous allele instead. Total serum Ig concentrations were normal and included both transgene- and endogenous gene-coded IgM and IgG. The transgene V(H) D(H)J(H) was expressed in splenic IgM cDNA with few or no mutations, and in IgG cDNA with multiple mutations. The transgene allotype was also expressed in Abs formed on immunization with thyroglobulin, pneumococcal polysaccharide, and ssDNA-methylated BSA. Consistent with the hypothesis, cells with a rearranged autoreactive V(H) domain selected for reactivity with a form of ssDNA did serve as precursors for cells producing IgM and IgG Abs to diverse Ags.

Dynamics of spheroid self-assembly in liquid-overlay culture of DU 145 human prostate cancer cells.

The in vitro self-assembly of multicellular spheroids generates highly organized structures in which the three-dimensional structure and differentiated function frequently mimic that of in vivo tissues. This has led to their use in such diverse applications as tissue regeneration and drug therapy. Using Smoluchowski-like rate equations, herein we present a model of the self-aggregation of DU 145 human prostate carcinoma cells in liquid-overlay culture to elucidate some of the physical parameters affecting homotypic aggregation in attachment-dependent cells. Experimental results indicate that self-aggregation in our system is divided into three distinct phases: a transient reorganization of initial cell clusters, an active aggregation characterized by constant rate coefficients, and a ripening phase of established spheroid growth. In contrast to the diffusion-controlled aggregation previously observed for attachment-independent cells, the model suggests that active aggregation in our system is reaction-controlled. The rate equations accurately predict the aggregation kinetics of spheroids containing up to 30 cells and are dominated by spheroid adhesive potential with lesser contributions from the radius of influence. The adhesion probability increases with spheroid size so that spheroid-spheroid adhesions are a minimum of 2.5 times more likely than those of cell-cell, possibly due to the upregulation of extracellular matrix proteins and cell-adhesion molecules. The radius of influence is at least 1.5 to 3 times greater than expected for spherical geometry as a result of ellipsoidal shape and possible chemotactic or Fröhlich interactions. Brownian-type behavior was noted for spheroids larger than 30 microm in diameter, but smaller aggregates were more motile by as much as a factor of 10 for single cells. The model may improve spheroid fidelity for existing applications of spheroids and form the basis of a simple assay for quantitatively evaluating cellular metastatic potential as well as therapies that seek to alter this potential.

The neuroimmunology of multiple sclerosis: possible roles of T and B lymphocytes in immunopathogenesis.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system white matter. The association of the disease with MHC genes, the inflammatory white matter infiltrates, similarities with animal models, and the observation that MS can be treated with immunomodulatory and immunosuppressive therapies support the hypothesis that autoimmunity plays a major role in the disease pathology. Evidence supports activated CD4+ myelin-reactive T cells as major mediators of the disease. In addition, a renewed interest in the possible contribution of B cells to MS immunopathology has been sparked by nonhuman primate and MS pathological studies. This review focuses on the immunopathology of MS, outlining the hypothetical steps of tolerance breakdown and the molecules that play a role in the migration of autoreactive cells to the CNS. Particular focus is given to autoreactive T cells and cytokines as well as B cells and autoantibodies and their role in CNS pathogenesis in MS.

Recognition of DNA by VH and Fv domains of an IgG anti-poly(dC) antibody with a singly mutated VH domain.

Secondary antigen stimulation usually produces IgG antibodies with hypermutated V segments. Studying a strong secondary response to the polynucleotide antigen poly(dC), however, we found a highly selective IgG antibody (mAb dC7) with only one mutation (a conservative Leu to Ileu substitution) throughout the whole VH domain. To investigate the roles of VH and VL domains in selective binding by this mAb, we prepared its VH, VL and single-chain Fv (scFv) fragments. A bacterial expression system produced soluble monomeric V region proteins. CD spectra confirmed that they had the beta-secondary structure expected for Ig domains. Both the scFv and VH fragments bound to single-stranded non-protonated poly(dC) and to ssDNA but not to protonated, more structured poly(dC) or dsDNA. The VL domain alone did not bind to nucleic acids, but VL association modified the VH binding, giving the scFv a 10-fold higher affinity than the VH for poly(dC) and greatly increasing the cytosine-dependent selectivity. Non-ionic interactions were prominent in the Fv reaction with a (dC)( n) sequence. Ionic interactions were revealed in Fv cross-reactions with ssDNA, and were more prominent in binding of either poly(dC) or ssDNA by VH alone, consistent with the lesser base selectivity of the VH. Thus, the Fv and VH alone bind to a single antigen, poly(dC), but mechanistic differences result from additional subsites in the Fv. Generation of a selective IgG with very few CDR mutations in either VH or VL, which was accompanied by IgM antibodies with unmutated V regions, also suggests that nucleic acid binding activity is a property of the B cell repertoire even before immunization.

Levels of spinal cord injury and predictors of neurologic recovery.

A comprehensive physical examination of the patient with acute spinal cord injury is essential in determining the initial level of injury and is the most accurate method of prognosticating neurologic recovery. Understanding neurologic recovery helps predict ultimate functional capability and needs, and helps evaluate the effectiveness of pharmacologic and therapeutic interventions.

Characterization of aggregation and protein expression of bovine corneal endothelial cells as microcarrier cultures in a rotating-wall vessel.

Rotating-wall vessels are beneficial to tissue engineering in that the reconstituted tissue formed in these low-shear bioreactors undergoes extensive three-dimensional growth and differentiation. In the present study, bovine corneal endothelial (BCE) cells were grown in a high-aspect rotating-wall vessel (HARV) attached to collagen-coated Cytodex-3 beads as a representative monolayer culture to investigate factors during HARV cultivation which affect three-dimensional growth and protein expression. A collagen type I substratum in T-flask control cultures increased cell density of BCE cells at confluence by 40% and altered the expression of select proteins (43, 50 and 210 kDa). The low-shear environment in the HARV facilitated cell bridging between microcarrier beads to form aggregates containing upwards of 23 beads each, but it did not promote multilayer growth. A kinetic model of microcarrier aggregation was developed which indicates that the rate of aggregation between a single bead and an aggregate was nearly 10 times faster than between two aggregate and 60 times faster than between two single beads. These differences reflect changes in collision frequency and cell bridge formation. HARV cultivation altered the expression of cellular proteins (43 and 70 kDa) and matrix proteins (50, 73, 89 and 210 kDa) relative to controls perhaps due to hypoxia, fluid flow or distortion of cell shape. In addition to the insight that this work has provided into rotating-wall vessels, it could be useful in modeling aggregation in other cell systems, propagating human corneal endothelial cells for eye surgery and examining the response of endothelial cells to reduced shear.

Duplex ultrasound screening for deep vein thrombosis in spinal cord injured patients at rehabilitation admission.

OBJECTIVE: To determine the rate of deep vein thrombosis (DVT) newly diagnosed by duplex ultrasound in patients with acute spinal cord injury (SCI) at admission for rehabilitation. DESIGN: Retrospective case-control study. SETTING: Independent specialized spinal cord rehabilitation hospital. PATIENTS: Data were collected from records of 189 SCI patients admitted for rehabilitation over a 1-year period who underwent a duplex scan and were not admitted with a known diagnosis of DVT. MAIN OUTCOME MEASURES: A DVT newly diagnosed by duplex ultrasound at rehabilitation admission. RESULTS: Twenty-two patients (11.6%) had a newly diagnosed DVT at time of admission. Chi-square analysis found no statistically significant relationship between level of injury (tetraplegia vs paraplegia), motor complete (ASIA A and B) versus incomplete status (ASIA C and D), or cause of SCI (traumatic vs nontraumatic injury) in determining a positive or negative duplex result (chi2 = 1.709, p = .191; chi2 = 1.314, p = .252; chi2 = 3.155, p = .076; respectively). Prophylaxis for DVT decreased the risk of developing a DVT: 4.1% of patients administered prophylaxis as compared to 16.4% of patients not given prophylaxis (chi2 = 6.558, p = .01). Only 38.6% of patients transferred to rehabilitation were undergoing DVT prophylaxis. CONCLUSIONS: The prevalence of DVT in acute SCI patients at admission to rehabilitation is significant. A duplex ultrasound is an important noninvasive technique to screen patients with acute and subacute SCI for DVT on admission to the rehabilitation setting regardless of the completeness, level, or cause of the patients' injury.

Predictors of dysphagia after spinal cord injury.

OBJECTIVE: To quantify the incidence of swallowing deficits (dysphagia) and to identify factors that predict risk for dysphagia in the rehabilitation setting following acute traumatic spinal cord injury. DESIGN: Retrospective case-control study. SETTING: Freestanding rehabilitation hospital. PATIENTS: Data were collected on 187 patients with acute traumatic spinal cord injury admitted for rehabilitation over a 4-year period who underwent a swallowing screen, in which 42 underwent a videofluoroscopic swallowing study (VFSS). MAIN OUTCOME MEASURES: VFSS was performed on patients with suspected swallowing problems. Possible antecedents of dysphagia were recorded from the medical record including previous history of spine surgery, surgical approach and technique, tracheostomy and ventilator status, neurologic level of injury, ASIA Impairment Classification, orthosis, etiology of injury, age, and gender. RESULTS: On admission to rehabilitation 22.5% (n = 42) of spinal cord injury patients had symptoms suggesting dysphagia. In 73.8% (n = 31) of these cases, testing confirmed dysphagia (aspiration or requiring a modified diet), while VFSS ruled out dysphagia in 26.2% (n = 11) cases. Logistic regression and other analyses revealed three significant predictors of risk for dysphagia: age (p < .028), tracheostomy and mechanical ventilation (p < .001), and spinal surgery via an anterior cervical approach (p < .016). Other variables analyzed had no relation or at best a slight relation to dysphagia. Tracheostomy at admission was the strongest predictor of dysphagia. The combination of tracheostomy at rehabilitation admission and anterior surgical approach had an extremely high rate of dysphagia (48%). CONCLUSION: Swallowing abnormalities are present in a significant percentage of patients presenting to rehabilitation with acute traumatic cervical spinal cord injury. Patients with a tracheostomy appear to have a substantially increased risk of development of dysphagia, although other factors are also relevant. Risk of dysphagia should be evaluated to decrease the potential for morbidity related to swallowing abnormalities.

Three-dimensional cultures of prostatic cells: tissue models for the development of novel anti-cancer therapies.

This review addresses the application of three-dimensional cultures of prostatic cells to the development of novel anti-cancer therapies. A variety of therapeutic agents to combat prostate cancer are currently under development. These include cytotoxins, differentiation agents and, more recently, genetically modified tumor vaccines. Three-dimensional cultures of prostatic cells are increasingly used in preclinical research in the design of new therapies and in the development of delivery strategies for these treatments. These tissue-like structures more realistically model the structural architecture and differentiated function of the human prostate than a cellular monolayer. In doing so, three-dimensional cultures produce an in vivo-like response to therapeutic agents. Advances in tissue engineering have improved the variety, fidelity and quantity of these prostate models. To date, they have been applied to estimate the dose of new drug therapies, evaluate drug penetration into solid tumors, assess the effectiveness of drug combinations, and develop tumor vaccines.

Treatment of acute deep vein thrombosis in spinal cord injured patients with enoxaparin: a cost analysis.

Enoxaparin, a low molecular weight heparin, has been demonstrated to be effective in the prophylaxis and treatment of deep vein thrombosis (DVT) in the general population. This study presents an analysis of the costs of subcutaneous (SQ) enoxaparin compared with intravenous (IV) heparin. Data were gathered on six spinal cord injured (SCI) patients in an acute freestanding rehabilitation center who were initially treated with SQ enoxaparin (n=3) or IV heparin (n=3) for a proximal DVT. No patients in either group developed further clinical complications. Comparison of the total costs of each treatment was performed, including the direct cost of the drug, as well as some of the costs of administration. Although the cost of enoxaparin per unit dose is higher, the total costs of enoxaparin are slightly lower, because its labor and administration costs are less. Subcutaneous enoxaparin is a safe, cost-effective, and less labor-intensive treatment, and can be of substantial benefit in the treatment of DVT in SCI patients in the rehabilitation setting.

Predicting neurologic recovery in traumatic cervical spinal cord injury.

OBJECTIVE: Traumatic spinal cord injury (SCI) affects 8,000 to 10,000 individuals per year in the United States. One of the most difficult tasks confronting the clinician is the discussion of neurologic recovery and prognosis with the patient and/or family. Our objective is to provide a guide for practitioners to accurately predict neurologic outcome in acute traumatic cervical SCI (tetraplegia). DATA SOURCE: Published reports obtained through MEDLINE search, texts, and studies presented at national conferences. STUDY SELECTION: Peer reviewed studies, in English language, that discussed prognosis after traumatic SCI. CONCLUSION: A comprehensive physical examination of the acute SCI patient is essential in determining the initial level and classification of the injury and is the most accurate method to predict neurologic recovery. Other diagnostic tests, including somatosensory evoked potentials, magnetic resonance imaging, and transcranial magnetic stimulation, may be helpful in further determining outcome when used in association with the clinical examination. The understanding of neurologic recovery should help predict ultimate functional capability and potential needs.

Bowel care practices in chronic spinal cord injury patients.

OBJECTIVE: To determine current characteristics of bowel care practices of chronic spinal cord injury (SCI) patients. DESIGN: Prospective interview and examination of 100 SCI patients injured for more than 1 year. SETTING: Freestanding rehabilitation outpatient SCI center. PARTICIPANTS: One hundred chronic SCI patients. RESULTS: The following bowel program characteristics were found: alternate-day programs were most common; most subjects performed their programs in the morning; and tetraplegic subjects performed their programs less often, used suppositories more often, required greater assistance, and took longer to complete their programs. CONCLUSIONS: Bowel dysfunction in chronic SCI need not be associated with complications in the majority of cases.

Anemia and serum protein deficiencies in patients with traumatic spinal cord injury.

This study was conducted to further investigate the natural history of the anemia, hypoproteinemia and hypoalbuminemia commonly observed in patients with traumatic spinal cord injury (SCI). Blood samples were taken from 46 traumatic SCI patients at the time of initial admission to an acute rehabilitation hospital and again approximately one year later during a routine follow-up appointment. At initial admission, 65 percent of patients were anemic (hemoglobin < 13.0 g/dl), 86.7 percent were hypoalbuminemic (albumin < 3.2 g/dl) and 48.9 percent were hypoproteinemic (total protein < 6.5 g/dl). Deficiencies were most commonly observed in tetraplegics and in patients with complete injuries. At the time of follow-up, 6.8 percent of patients were anemic, 2.2 percent demonstrated abnormally low serum protein concentrations and a significant (p = 0.01) decrease in the incidence of each deficiency was observed. Our findings suggest that anemia and decreased serum protein concentrations, while commonly observed in the acute SCI population, are much less frequently encountered in the more chronically injured. If noted in the chronic SCI patient, these deficiencies should alert clinicians to the likelihood of a concurrent process.

Cultivation of fall armyworm ovary cells in simulated microgravity.

A methodology is presented to culture Fall Armyworm Ovary cells in simulated micrograviy using a novel bioreactor developed by NASA, the High-Aspect Ratio Vessel. In this vessel, the growth and metabolic profile for these insect cells were profoundly different than those obtained in shaker-flask culture. Specifically, stationary phase in the NASA vessel was extended from 24 h to at least 7 d while cell concentration and viability remained in excess of 1 x 10(7) viable cells/ml and 90%, respectively. Measurements of glucose utilization, lactate production, ammonia production, and pH change indicate that simulated microgravity had a twofold effect on cell metabolism. Fewer nutrients were consumed and fewer wastes were produced in stationary phase by as much as a factor of 4 over that achieved in shaker culture. Those nutrients that were consumed in the NASA vessel were directed along different metabolic pathways as evidenced by an extreme shift in glucose utilization from consumption to production in lag phase and a decrease in yield coefficients by one half in stationary phase. These changes reflect a reduction in hydrodynamic forces from over 1 dyne/cm2 in shaker culture to under 0.5 dyne/cm2 in the NASA vessel. These results suggest that cultivation of insect cells in simulated microgravity may reduce production costs of cell-derived biologicals by extending production time and reducing medium requirements.