Effect of Transcranial Low-Level Light Therapy vs Sham Therapy Among Patients With Moderate Traumatic Brain Injury: A Randomized Clinical Trial.

Importance: Preclinical studies have shown that transcranial near-infrared low-level light therapy (LLLT) administered after traumatic brain injury (TBI) confers a neuroprotective response. Objectives: To assess the feasibility and safety of LLLT administered acutely after a moderate TBI and the neuroreactivity to LLLT through quantitative magnetic resonance imaging metrics and neurocognitive assessment. Design, Setting, and Participants: A randomized, single-center, prospective, double-blind, placebo-controlled parallel-group trial was conducted from November 27, 2015, through July 11, 2019. Participants included 68 men and women with acute, nonpenetrating, moderate TBI who were randomized to LLLT or sham treatment. Analysis of the response-evaluable population was conducted. Interventions: Transcranial LLLT was administered using a custom-built helmet starting within 72 hours after the trauma. Magnetic resonance imaging was performed in the acute (within 72 hours), early subacute (2-3 weeks), and late subacute (approximately 3 months) stages of recovery. Clinical assessments were performed concomitantly and at 6 months via the Rivermead Post-Concussion Questionnaire (RPQ), a 16-item questionnaire with each item assessed on a 5-point scale ranging from 0 (no problem) to 4 (severe problem). Main Outcomes and Measures: The number of participants to successfully and safely complete LLLT without any adverse events within the first 7 days after the therapy was the primary outcome measure. Secondary outcomes were the differential effect of LLLT on MR brain diffusion parameters and RPQ scores compared with the sham group. Results: Of the 68 patients who were randomized (33 to LLLT and 35 to sham therapy), 28 completed at least 1 LLLT session. No adverse events referable to LLLT were reported. Forty-three patients (22 men [51.2%]; mean [SD] age, 50.49 [17.44] years]) completed the study with at least 1 magnetic resonance imaging scan: 19 individuals in the LLLT group and 24 in the sham treatment group. Radial diffusivity (RD), mean diffusivity (MD), and fractional anisotropy (FA) showed significant time and treatment interaction at 3-month time point (RD: 0.013; 95% CI, 0.006 to 0.019; P < .001; MD: 0.008; 95% CI, 0.001 to 0.015; P = .03; FA: -0.018; 95% CI, -0.026 to -0.010; P < .001).The LLLT group had lower RPQ scores, but this effect did not reach statistical significance (time effect P = .39, treatment effect P = .61, and time × treatment effect P = .91). Conclusions and Relevance: In this randomized clinical trial, LLLT was feasible in all patients and did not exhibit any adverse events. Light therapy altered multiple diffusion tensor parameters in a statistically significant manner in the late subacute stage. This study provides the first human evidence to date that light therapy engages neural substrates that play a role in the pathophysiologic factors of moderate TBI and also suggests diffusion imaging as the biomarker of therapeutic response. Trial Registration: ClinicalTrials.gov Identifier: NCT02233413.

Wide-Field Functional Microscopy of Peripheral Nerve Injury and Regeneration.

Severe peripheral nerve injuries often result in partial repair and lifelong disabilities in patients. New surgical techniques and better graft tissues are being studied to accelerate regeneration and improve functional recovery. Currently, limited tools are available to provide in vivo monitoring of changes in nerve physiology such as myelination and vascularization, and this has impeded the development of new therapeutic options. We have developed a wide-field and label-free functional microscopy platform based on angiographic and vectorial birefringence methods in optical coherence tomography (OCT). By incorporating the directionality of the birefringence, which was neglected in the previously reported polarization-sensitive OCT techniques for nerve imaging, vectorial birefringence contrast reveals internal nerve microanatomy and allows for quantification of local myelination with superior sensitivity. Advanced OCT angiography is applied in parallel to image the three-dimensional vascular networks within the nerve over wide-fields. Furthermore, by combining vectorial birefringence and angiography, intraneural vessels can be discriminated from those of the surrounding tissues. The technique is used to provide longitudinal imaging of myelination and revascularization in the rodent sciatic nerve model, i.e. imaged at certain sequential time-points during regeneration. The animals were exposed to either crush or transection injuries, and in the case of transection, were repaired using an autologous nerve graft or acellular nerve allograft. Such label-free functional imaging by the platform can provide new insights into the mechanisms that limit regeneration and functional recovery, and may ultimately provide intraoperative assessment in human subjects.

Resolvin D2 Limits Secondary Tissue Necrosis After Burn Wounds in Rats.

Secondary burn necrosis is the expansion and deepening of the original burn injury several days after injury. Limiting the extent of secondary burn necrosis may improve outcomes. In this study, we examined the ability of the lipid mediator of inflammation-resolution resolvin D2 (RvD2) and chromatin-lysing enzyme (DNase) to reduce secondary burn necrosis. Male Wistar rats were injured using a brass comb with 4 prongs heated in boiling water. This method created 2 parallel rows of 4 rectangular burned areas separated by 3 unburned interspaces. Starting at 2 hours after the burn injury, rats received either 25 ng/kg RvD2 intravenously daily for 7 days or 200 U/kg DNase every 12 hours for 3 days. We documented the necrosis around the initial wounds by digital photography. We used laser Doppler to assess the total blood flux in the burn area. We evaluated the functionality of the capillary network in the interspaces by optical coherence tomography angiography. We performed histological examination of wound skin tissue samples collected at 14 days postburn. We found that the interspace areas were preserved and had higher blood flow in the RvD2-treated group, while the burn areas expanded into the interspace areas, which were confluent by 7 days postburn, in the control-untreated group. We found a larger monocyte-to-neutrophil ratio in the RvD2-treated group compared with the DNase-treated and control groups (P < .05). Overall, RvD2 suppresses secondary necrosis and starts regeneration, highlighting the role of inflammation resolution as a potential therapeutic target in burn care.

Physiological Translocation of Lactic Acid Bacteria during Pregnancy Contributes to the Composition of the Milk Microbiota in Mice.

The human milk microbiota is a complex and diverse ecosystem that seems to play a relevant role in the mother-to-infant transmission of microorganisms during early life. Bacteria present in human milk may arise from different sources, and recent studies suggest that at least some of them may be originally present in the maternal digestive tract and may reach the mammary gland through an endogenous route during pregnancy and lactation. The objective of this work was to elucidate whether some lactic acid bacteria are able to translocate and colonize the mammary gland and milk. For this purpose, two lactic acid bacteria strains (Lactococcus lactis MG1614 and Lactobacillus salivarius PS2) were transformed with a plasmid containing the lux genes; subsequently, the transformed strains were orally administered to pregnant mice. The murine model allowed the visualization, isolation, and Polymerase Chain Reaction (PCR)-detection of the transformed bacteria in different body locations, including mammary tissue and milk, reinforcing the hypothesis that physiological translocation of maternal bacteria during pregnancy and lactation may contribute to the composition of the mammary and milk microbiota.

Prostaglandins induce early growth response 1 transcription factor mediated microsomal prostaglandin E2 synthase up-regulation for colorectal cancer progression.

Cyclooxygenase2 (COX2) has been associated with cell growth, invasiveness, tumor progression and metastasis of colorectal carcinomas. However, the downstream prostaglandin (PG)-PG receptor pathway involved in these effects is poorly characterized.We studied the PG-pathway in gene expression databases and we found that PTGS2 (prostaglandin G/H synthase and cyclooxygenase) and PTGES (prostaglandin E synthase) are co-expressed in human colorectal tumors. Moreover, we detected that COX2 and microsomal Prostaglandin E2 synthase 1 (mPGES1) proteins are both up-regulated in colorectal human tumor biopsies.Using colon carcinoma cell cultures we found that COX2 overexpression significantly increased mPGES1 mRNA and protein. This up-regulation was due to an increase in early growth response 1 (EGR1) levels and its transcriptional activity. EGR1 was induced by COX2-generated PGF2α. A PGF2α receptor antagonist, or EGR1 silencing, inhibited the mPGES1 induction by COX2 overexpression. Moreover, using immunodeficient mice, we also demonstrated that both COX2- and mPGES1-overexpressing carcinoma cells were more efficient forming tumors.Our results describe for the first time the molecular pathway correlating PTGS2 and PTGES in colon cancer progression. We demonstrated that in this pathway mPGES1 is induced by COX2 overexpression, via autocrine PGs release, likely PGF2α, through an EGR1-dependent mechanism. This signaling provides a molecular explanation to PTGS2 and PTGES association and contribute to colon cancer advance, pointing out novel potential therapeutic targets in this oncological context.

Complex differential variance algorithm for optical coherence tomography angiography.

We describe a complex differential variance (CDV) algorithm for optical coherence tomography based angiography. The algorithm exploits both the intensity and phase changes of optical coherence tomography (OCT) signals from flowing blood to achieve high vascular contrast, and also intrinsically reject undesirable phase signals originating from small displacement axial bulk tissue motion and instrument synchronization errors. We present this algorithm within a broader discussion of the properties of OCT signal dynamics. The performance of the algorithm is compared against two other existing algorithms using both phantom measurements and in vivo data. We show that the algorithm provides better contrast for a given number of measurements and equivalent spatial averaging.

Monte Carlo modeling of angiographic optical coherence tomography.

Optical coherence tomography (OCT) provides both structural and angiographic imaging modes. Because of its unique capabilities, OCT-based angiography has been increasingly adopted into small animal and human subject imaging. To support the development of the signal and image processing algorithms on which OCT-based angiography depends, we describe here a Monte Carlo-based model of the imaging approach. The model supports arbitrary three-dimensional vascular network geometries and incorporates methods to simulate OCT signal temporal decorrelation. With this model, it will be easier to compare the performance of existing and new angiographic signal processing algorithms, and to quantify the accuracy of vascular segmentation algorithms. The quantitative analysis of key algorithms within OCT-based angiography may, in turn, simplify the selection of algorithms in instrument design and accelerate the pace of new algorithm development.

In Vivo Fluorescent Labeling of Tumor Cells with the HaloTag® Technology.

Many fluorescent sensors are currently available for in vitro bio-physiological microscopic imaging. The ability to label cells in living animals with these fluorescent sensors would help translate some of these assays into in vivo applications. To achieve this goal, the first step is to establish a method for selectively labeling target cells with exogenous fluorophores. Here we tested whether the HaloTag® protein tagging system provides specific labeling of xenograft tumors in living animals. After systemic delivery of fluorophore-conjugated ligands, we performed whole animal planar fluorescent imaging to determine uptake in tag-expressing HCT116 xenografts. Our results demonstrate that HaloTag ligands containing red or near-infrared fluorophores have enhanced tumor uptake and are suitable for non-invasive in vivo imaging. Our proof-of-concept results establish feasibility for using HaloTag technology for bio-physiological imaging in living animals.

Angiotensin-converting enzyme inhibition prevents the release of monocytes from their splenic reservoir in mice with myocardial infarction.

RATIONALE: Monocytes recruited to ischemic myocardium originate from a reservoir in the spleen, and the release from their splenic niche relies on angiotensin (Ang) II signaling. OBJECTIVE: Because monocytes are centrally involved in tissue repair after ischemia, we hypothesized that early angiotensin-converting enzyme (ACE) inhibitor therapy impacts healing after myocardial infarction partly via effects on monocyte traffic. METHODS AND RESULTS: In a mouse model of permanent coronary ligation, enalapril arrested the release of monocytes from the splenic reservoir and consequently reduced their recruitment into the healing infarct by 45%, as quantified by flow cytometry of digested infarcts. Time-lapse intravital microscopy revealed that enalapril reduces monocyte motility in the spleen. In vitro migration assays and Western blotting showed that this was caused by reduced signaling through the Ang II type 1 receptor. We then studied the long-term consequences of blocked splenic monocyte release in atherosclerotic apolipoprotein (apo)E(-/-) mice, in which infarct healing is impaired because of excessive inflammation in the cardiac wound. Enalapril improved histologic healing biomarkers and reduced inflammation in infarcts measured by FMT-CT (fluorescence molecular tomography in conjunction with x-ray computed tomography) of proteolytic activity. ACE inhibition improved MRI-derived ejection fraction by 14% on day 21, despite initially comparable infarct size. In apoE(-/-) mice, ischemia/reperfusion injury resulted in larger infarct size and enhanced monocyte recruitment and was reversible by enalapril treatment. Splenectomy reproduced antiinflammatory effects of enalapril. CONCLUSION: This study suggests that benefits of early ACE inhibition after myocardial infarction can partially be attributed to its potent antiinflammatory impact on the splenic monocyte reservoir.

CD69 limits the severity of cardiomyopathy after autoimmune myocarditis.

BACKGROUND: Experimental autoimmune myocarditis (EAM), a mouse model of post-infectious cardiomyopathy, reflects mechanisms of inflammatory cardiomyopathy in humans. EAM is characterized by an infiltration of inflammatory cells into the myocardium that can be followed by myocyte fibrosis, edema, and necrosis, leading to ventricular wall dysfunction and heart failure. Different data indicate that CD69 exerts an important immunoregulatory effect in vivo. However, the possible role of CD69 in autoimmune myocarditis has not been studied. METHODS AND RESULTS: We have explored the role of the leukocyte regulatory molecule CD69 in the inflammation that leads to cardiac dysfunction after myocardial injury in EAM. We have found that after induction of EAM, the draining lymph nodes from CD69-deficient mice developed an exacerbated Th17 inflammatory response, resulting in increases in the numbers of infiltrating leukocytes in the myocardium. In the chronic phase of EAM, transthoracic echocardiography revealed a significantly reduced left ventricular fractional shortening and a decreased ejection fraction in CD69-deficient mice, indicative of an impaired cardiac contractility. This condition was accompanied by a greater extent of myocardial fibrosis, an elevated number of sinus pauses on ECG, and an enhanced ratio of heart weight to body weight in CD69-/- mice. Moreover, both bone marrow transplantation and adoptive transfer of Th17 cells isolated from immunized CD69-/- mice with EAM into naive wild-type recipients reproduced the severity of the disease, demonstrating that CD69 exerts its function within the lymphocyte compartment. CONCLUSION: Our findings indicate that CD69 negatively regulates heart-specific Th17 responses, cardiac inflammation, and heart failure progression in EAM.

Role of Trypanosoma cruzi autoreactive T cells in the generation of cardiac pathology.

Chagas disease, caused by Trypanosoma cruzi, affects several million people in Central and South America. About 30% of chronic patients develop cardiomyopathy probably caused by parasite persistence and/or autoimmunity. While several cross-reactive antibodies generated during mammal T. cruzi infection have been described, very few cross-reactive T cells have been identified. We performed adoptive transfer experiments of T cells isolated from chronically infected mice. The results showed the generation of cardiac pathology in the absence of parasites. We also transferred cross-reactive SAPA-specific T cells and observed unspecific alterations in heart repolarization, cardiac inflammatory infiltration, and tissue damage.

Immune suppression in advanced chronic fascioliasis: an experimental study in a rat model.

Chronicity and Th2 immune responses are features of helminth infections in humans. The liver fluke promotes its own survival through several strategies to down-regulate the immune response of the host during the early phase of infection. However, there is no evidence that this modulation occurs much later. The immune response in advanced chronic fascioliasis was analyzed in an experimental rat model at 20 weeks after infection. Cytokine quantification in infected rat serum revealed basal levels. The predominant immunoglobulin (Ig) isotype was IgG1. Flow cytometry analysis of T cell (CD3(+), CD4(+), and CD8a(+)), B cell (CD45R(+)), and macrophage (CD11b(+)) populations in spleens showed no significant differences between infected and control rats. Mononuclear cell proliferation in the spleen in response to T and B mitogens was strongly inhibited in infected versus control rats. During early chronic infection, there is a predominance of a Th2 response, which decreases in advanced chronic infection characterized by a persistent immune suppression.

Coexpression of virulence and fosfomycin susceptibility in Listeria: molecular basis of an antimicrobial in vitro-in vivo paradox.

Discrepancies between resistance in vitro and therapeutic efficacy in vivo are generally attributed to failure of laboratory susceptibility tests to reflect an antibiotic's pharmacokinetic or pharmacodynamic properties. We show here that this phenomenon can result from differential in vitro-in vivo expression of bacterial determinants of antibiotic susceptibility. We found that an in vivo-induced virulence factor, Hpt, also mediates uptake of fosfomycin in Listeria monocytogenes. These bacteria therefore seem resistant to fosfomycin in vitro, although they are in fact susceptible to the antibiotic during infection.

Hpt, a bacterial homolog of the microsomal glucose- 6-phosphate translocase, mediates rapid intracellular proliferation in Listeria.

Efficient replication in vivo is essential for a microparasite to colonize its host and the understanding of the molecular mechanisms by which microbial pathogens grow within host tissues can lead to the discovery of novel therapies to treat infection. Here we present evidence that the foodborne bacterial pathogen Listeria monocytogenes, a facultative intracellular parasite, exploits hexose phosphates (HP) from the host cell as a source of carbon and energy to fuel fast intracellular growth. HP uptake is mediated by Hpt, a bacterial homolog of the mammalian translocase that transports glucose-6-phosphate from the cytosol into the endoplasmic reticulum in the final step of gluconeogenesis and glycogenolysis. Expression of the Hpt permease is tightly controlled by the central virulence regulator PrfA, which upon entry into host cells induces a set of virulence factors required for listerial intracellular parasitism. Loss of Hpt resulted in impaired listerial intracytosolic proliferation and attenuated virulence in mice. Hpt is the first virulence factor to be identified as specifically involved in the replication phase of a facultative intracellular pathogen. It is also a clear example of how adaptation to intracellular parasitism by microbial pathogens involves mimicry of physiological mechanisms of their eukaryotic host cells.