Bidirectional Changes in Anisotropy Are Associated with Outcomes in Mild Traumatic Brain Injury.

BACKGROUND AND PURPOSE: Mild traumatic brain injury results in a heterogeneous constellation of deficits and symptoms that persist in a subset of patients. This prospective longitudinal study identifies early diffusion tensor imaging biomarkers of mild traumatic brain injury that significantly relate to outcomes at 1 year following injury. MATERIALS AND METHODS: DTI was performed on 39 subjects with mild traumatic brain injury within 16 days of injury and 40 controls; 26 subjects with mild traumatic brain injury returned for follow-up at 1 year. We identified subject-specific regions of abnormally high and low fractional anisotropy and calculated mean fractional anisotropy, axial diffusivity, radial diffusivity, and mean diffusivity across all white matter voxels brain-wide and each of several white matter regions. Assessment of cognitive performance and symptom burden was performed at 1 year. RESULTS: Significant associations of brain-wide DTI measures and outcomes included the following: mean radial diffusivity and mean diffusivity with memory; and mean fractional anisotropy, radial diffusivity, and mean diffusivity with health-related quality of life. Significant differences in outcomes were found between subjects with and without abnormally high fractional anisotropy for the following white matter regions and outcome measures: left frontal lobe and left temporal lobe with attention at 1 year, left and right cerebelli with somatic postconcussion symptoms at 1 year, and right thalamus with emotional postconcussion symptoms at 1 year. CONCLUSIONS: Individualized assessment of DTI abnormalities significantly relates to long-term outcomes in mild traumatic brain injury. Abnormally high fractional anisotropy is significantly associated with better outcomes and might represent an imaging correlate of postinjury compensatory processes.

Possible mechanisms of diarrheal side effects associated with the use of a novel chemotherapeutic agent, flavopiridol.

The novel cyclin-dependent kinase inhibitor flavopiridol has recently completed Phase I trials for the treatment of refractory neoplasms. The dose-limiting toxicity observed with this agent was severe diarrhea. Because the compound otherwise showed promise, the present study sought to determine possible mechanisms underlying the diarrheal side effects. Flavopiridol was tested for its ability to modify chloride secretory responses of the human colonic epithelial cell line, T84. Studies were conducted in vitro in modified Ussing chambers. High concentrations of flavopiridol (10(-4) M), above those likely to be clinically relevant, had a direct stimulatory effect on chloride secretion, probably ascribable to an increase in cyclic AMP. Lower, clinically relevant concentrations of flavopiridol (10(-6) M) had no effect on chloride secretion by themselves but potentiated responses to the calcium-dependent secretagogue, carbachol. The drug also potentiated responses to thapsigargin and taurodeoxycholate and reversed the inhibitory effects of carbachol and epidermal growth factor on calcium-dependent chloride secretion. Pretreatment with the cyclic AMP-dependent secretagogue, forskolin, potentiated responses to flavopiridol, but not vice versa. Thus, diarrheal side effects induced by flavopiridol are likely multifactorial in origin and may involve interactions with endogenous secretagogues such as acetylcholine and bile acids. A better understanding of the diarrhea induced by flavopiridol should allow optimization of therapy with this otherwise promising drug and/or the development of related agents with improved toxicity profiles.

Comparison of transformation mechanisms of Haemophilus parainfluenzae and Haemophilus influenzae.

Transformation pathways in two closely related bacterial species, Haemophilus parainfluenzae and Haemophilus influenzae, were studied. Both organisms rapidly take up transforming DNA within minutes into specialized membranous structures on the cell surface (transformasomes). DNA within transformasomes is in a protected state, inaccessible to external DNase or internal restriction and modification enzymes. However, the subsequent processing of donor DNA differs in these two organisms. In H. influenzae, linear DNA immediately undergoes degradation from one end at a constant rate, leaving a lower-molecular-weight intermediate in the transformasome. The end undergoing degradation is searching for homologous regions of the chromosome. Once pairing is initiated, the remaining lower-molecular-weight DNA exits from the transformasome, and a single strand undergoes efficient integration. In contrast, in H. parainfluenzae little degradation of donor DNA is observed, with the majority remaining intact within the transformasomes after 1 h. Thus, whereas only 10% of donor DNA molecules leave the protected state after 1 h, portions of each molecule appear to become quantitatively integrated.

Directional transport and integration of donor DNA in Haemophilus influenzae transformation.

DNA transport and integration in Haemophilus influenzae transformation was studied with a plasmid clone of homologous DNA (pCML6). Our results indicate that: (i) donor DNA enters specialized membranous extensions on the cell surface, which we have termed "transformasomes"; (ii) linear DNA undergoes degradation upon exiting transformasomes; and (iii) DNA without a free end remains within transformasomes and is not degraded. By comparing the fate of label from uniformly labeled versus middle-labeled DNA, it appears that donor DNA undergoes degradation from an end prior to recombining with the chromosome. Using donor DNA with covalently closed termini (hairpin ends) prevents efficient exit from transformasomes. When one hairpin is removed, exit of donor DNA is shown to be directional from the free end, with preferential homologous integration of the 3' strand from that end.

Transformasomes: specialized membranous structures that protect DNA during Haemophilus transformation.

The mechanism by which Haemophilus protects donor DNA from cellular restriction and degradative enzymes during transformation is unclear. In this report, we demonstrate that donor DNA enters Haemophilus influenzae through specialized membranous extensions, which we have termed "transformasomes." DNA within transformasomes is in a protected state--resistant to external DNase and cellular restriction enzymes, although remaining unmodified and double-stranded. The ability of donor DNA to exit from transformasomes is dependent on its topological conformation. Circular DNA remains intact within transformasomes, while linear DNA rapidly exits and undergoes homologous recombination. Protected donor DNA can be preferentially removed from the surface of competent cells by extraction with organic solvents. Structurally intact transformasomes containing donor DNA could be partitioned into the organic layer and can be further purified by density centrifugation.

Possible mechanism for donor DNA binding and transport in Haemophilus.

Morphological differences were observed in competent and noncompetent Haemophilus parainfluenzae and Haemophilus influenzae when thin sections of these cells were examined by electron microscopy. The membranous extensions present on the surface of competent H. parainfluenzae cells disappeared on treatment with transforming DNA, while vacuole-like structures appeared in the periplasm. Noncompetent cells had 1/5th as many extensions on their surface as competent cells, and no vacuoles were observed after treatment with homologous DNA. Competent cells treated with radiolabeled DNA were disrupted and the clarified lysate was centrifuged on CsCl density gradients. Material having a density of 1.34 g/ml was found to contain the majority of the DNase-resistant radioactive DNA recovered from the bacteria and was shown by electron microscopy to be composed of membrane vesicles. The polypeptide composition of this dense membrane fraction was similar to that of H. parainfluenzae outer membrane.

Occurrence of pili on and adhesive properties of Haemophilus parainfluenzae.

Of 20 clinical isolates of Haemophilus parainfluenzae, 13 produced a mannose-resistant hemagglutinin that agglutinated erythrocytes from chickens, horses, rabbits, and sheep. Examination with the electron microscope showed that only strain HR-885 was pilate. Grown in static liquid culture, the 12 hemagglutinating, nonpilate isolates formed small, tightly packed clumps, whereas strain HR-885 formed large, loosely packed clumps. However, seven isolates did not produce a hemagglutinin, did not clump, and were nonpilate.