Impact of Long-Term Cryopreservation on Blood Immune Cell Markers in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Implications for Biomarker Discovery.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex neuroimmune disorder characterized by numerous symptoms of unknown etiology. The ME/CFS immune markers reported so far have failed to generate a clinical consensus, perhaps partly due to the limitations of biospecimen biobanking. To address this issue, we performed a comparative analysis of the impact of long-term biobanking on previously identified immune markers and also explored additional potential immune markers linked to infection in ME/CFS. A correlation analysis of marker cryostability across immune cell subsets based on flow cytometry immunophenotyping of fresh blood and frozen PBMC samples collected from individuals with ME/CFS (n = 18) and matched healthy controls (n = 18) was performed. The functionality of biobanked samples was assessed on the basis of cytokine production assay after stimulation of frozen PBMCs. T cell markers defining Treg subsets and the expression of surface glycoprotein CD56 in T cells and the frequency of the effector CD8 T cells, together with CD57 expression in NK cells, appeared unaltered by biobanking. By contrast, NK cell markers CD25 and CD69 were notably increased, and NKp46 expression markedly reduced, by long-term cryopreservation and thawing. Further exploration of Treg and NK cell subsets failed to identify significant differences between ME/CFS patients and healthy controls in terms of biobanked PBMCs. Our findings show that some of the previously identified immune markers in T and NK cell subsets become unstable after cell biobanking, thus limiting their use in further immunophenotyping studies for ME/CFS. These data are potentially relevant for future multisite intervention studies and cooperative projects for biomarker discovery using ME/CFS biobanked samples. Further studies are needed to develop novel tools for the assessment of biomarker stability in cryopreserved immune cells from people with ME/CFS.

Screening NK-, B- and T-cell phenotype and function in patients suffering from Chronic Fatigue Syndrome.

BACKGROUND: Chronic Fatigue Syndrome (CFS) is a debilitating neuro-immune disorder of unknown etiology diagnosed by an array of clinical manifestations. Although several immunological abnormalities have been described in CFS, their heterogeneity has limited diagnostic applicability. METHODS: Immunological features of CFS were screened in 22 CFS diagnosed individuals fulfilling Fukuda criteria and 30 control healthy individuals. Peripheral blood T, B and NK cell function and phenotype were analyzed by flow cytometry in both groups. RESULTS: CFS diagnosed individuals showed similar absolute numbers of T, B and NK cells, with minor differences in the percentage of CD4+ and CD8+ T cells. B cells showed similar subset frequencies and proliferative responses between groups. Conversely, significant differences were observed in T cell subsets. CFS individuals showed increased levels of T regulatory cells (CD25+/FOXP3+) CD4 T cells, and lower proliferative responses in vitro and in vivo. Moreover, CD8 T cells from the CFS group showed significantly lower activation and frequency of effector memory cells. No clear signs of T-cell immunosenescence were observed. NK cells from CFS individuals displayed higher expression of NKp46 and CD69 but lower expression of CD25 in all NK subsets defined. Overall, T cell and NK cell features clearly clustered CFS individuals. CONCLUSIONS: Our findings suggest that alterations in T-cell phenotype and proliferative response along with the specific signature of NK cell phenotype may be useful to identify CFS individuals. The striking down modulation of T cell mediated immunity may help to understand intercurrent viral infections in CFS.

The electric field induced by light can explain cellular responses to electromagnetic energy: a hypothesis of mechanism.

When cells are irradiated with visible and near-infrared wavelengths a variety of stimulatory effects are observed in their metabolism. To explain the observed light effects, researchers try to identify the chromophores that are involved in the processes. However, the mechanism of light absorption by a chromophore does not explain many of the experimental observations and therefore the primary mechanism for cellular light responses remains unproven. In addition to the ability of photons to produce electronic excitation in chromophores, light induces a wave-like alternating electric field in a medium that is able to interact with polar structures and produce dipole transitions. These dipole transitions are analyzed in the present article at different cellular and biochemical levels, leading to the proposal that the primary mechanism for the observed light effects is related to the light-induced electric field.

Modification of the intrinsic fluorescence and the biochemical behavior of ATP after irradiation with visible and near-infrared laser light.

In this work, the effects of visible (655 nm) and near-infrared (830 nm) light on ATP in solution were examined. The addition of irradiated ATP to the hexokinase reaction caused significant differences in the reaction rates and in the Michaelis-Menten kinetic parameters, k(m) and v(max). Irradiated ATP cleavage by hexokinase occurred in less time. Changes were wavelength and dose dependent. Excitation of ATP with a 260 nm wavelength ultraviolet light induced a fluorescence emission that was decreased when Mg2+ was added due to ion binding of the phosphates, which are the structures that modify the fluorescence produced by the adenine dipoles. The irradiation of this ATP.Mg2+ solution using 655 and 830 nm light increased the fluorescence by a possible displacement of Mg2+ from the phosphates. In conclusion, visible and near-infrared light modifies the biochemical behavior of ATP in the hexokinase reaction and the fluorescence intensity of the molecule thus altering the Mg2+ binding strength to the oxygen atoms in the phosphate group.

Neurotransmitter release changes induced by low power 830 nm diode laser irradiation on the neuromuscular junctions of the mouse.

BACKGROUND AND OBJECTIVE: Treating patients with a Gallium-Aluminum-Arsenide (GaAlAs) infrared (IR) diode laser reduces muscle spasm and increases mobility in the muscles. The effect of low intensity laser irradiation on nerve function, growth, and repair mechanisms is a contentious area of research. We have addressed one aspect of this controversy by systematically examining the influence of 830 nm laser radiation on neurotransmitter release in neuromuscular junctions (NMJ) of the mouse diaphragm. STUDY DESIGN/MATERIALS AND METHODS: Thirty adult mice were studied. Diode laser GaAlAs 830 nm (4 and 12 J/cm2) was used. Neurotransmitter release was studied by conventional intracellular recording techniques on curarized muscles or high magnesium media. The quantal content, amplitude, and latency of the end-plate potentials (EPPs) were analyzed. Frequency and amplitude were evaluated for the miniature end-plate potentials (MEPPs). Facilitation of the neurotransmitter release was also evaluated by paired pulse stimulation. RESULTS AND CONCLUSIONS: The irradiated (12 J/cm2) muscles showed a significant reduction in quantal content (P = 0.01) and EPP amplitude (P = 0.04), but the latency, spontaneous transmitter release (MEPPs) and paired pulse facilitation did not change. No alterations were observed in NMJ irradiated with 4 J/cm2. We conclude that 830 nm diode laser irradiation (at a dose of 12 J/cm2) can affect the evoked neurotransmitter release in the mouse motor endplates.

Effect of low power 655 nm diode laser irradiation on the neuromuscular junctions of the mouse diaphragm.

BACKGROUND AND OBJECTIVES: Low level laser therapy (LLLT) in specific wavelengths and fluence maintains the electrophysiological activity of injured peripheral nerve in rats, preventing scar formation (at injury site) as well as degenerative changes in the corresponding motor neurons of the spinal cord, thus accelerating regeneration of the injured nerve. We studied the effect of LLLT on the neurotransmitter release in neuromuscular junctions of the mouse diaphragm. STUDY DESIGN/MATERIALS AND METHODS: Thirty-nine diaphragm muscles were studied. LLLT with GaAlAs 655 nm (1-12 J/cm(2)) was used. Neurotransmitter release was studied by conventional intracellular recording techniques on curarised or high magnesium media. Quantal content, amplitude, latency and rise time were analysed for end-plate potentials (EPPs). Frequency and amplitude were evaluated for the miniature end-plate potentials (MEPPs). Short-term plasticity of the neurotransmitter release (fast facilitation) was also evaluated by paired pulse stimulation. RESULTS AND CONCLUSIONS: This study showed that LLLT (655 nm) in these doses has no detectable physiological effect on the motor end-plate neurotransmitter release in mice.

Effect of low-power GaAlAs laser (660 nm) on bone structure and cell activity: an experimental animal study.

Low-level laser therapy (LLLT) is increasingly being used in the regeneration of soft tissue. In the regeneration of hard tissue, it has already been shown that the biomodulation effect of lasers repairs bones more quickly. We studied the activity in bone cells after LLLT close to the site of the bone injury. The femurs of 48 rats were perforated (24 in the irradiated group and 24 in the control group) and the irradiated group was treated with a GaAlAs laser of 660 nm, 10 J/cm2 of radiant exposure on the 2nd, 4th, 6th and 8th days after surgery (DAS). We carried out histomorphometry analysis of the bone. We found that activity was higher in the irradiated group than in the control group: (a) bone volume at 5 DAS (p=0.035); (b) osteoblast surface at 15 DAS (p=0.0002); (c) mineral apposition rate at 15 and 25 DAS (p=0.0008 and 0.006); (d) osteoclast surface at 5 DAS and 25 DAS (p=0.049 and p=0.0028); and (e) eroded surface ( p=0.0032). We concluded that LLLT increases the activity in bone cells (resorption and formation) around the site of the repair without changing the bone structure.

Pulsed and scanned carbon dioxide laser resurfacing 2 years after treatment: comparison by means of scanning electron microscopy.

Studies have reported short-term and long-term (1-year) findings for laser skin resurfacing. Two of the most popular systems used for this procedure, the continuous-wave Sharplan 40C SilkTouch system and the pulsed Coherent 5000C UltraPulse system with a computer pattern generator, were previously compared for a range of follow-up times up to 1 year, using light microscopy and transmission electron microscopy. This study analyzed the 2-year morphological differences using scanning electron microscopy. Tissue samples were obtained from 10 patients (age range, 50 to 72 years; skin types II and III) who had undergone laser resurfacing 2 years previously. One half of the face of each patient had been treated with the continuous-wave system and the other half with the pulsed system. The samples were subjected to scanning electron microscopy. On the continuous-wave-treated side, significantly better dermal collagen organization was observed at 2 years, with plump-appearing fibers that were closely knit to form a compact structure. On the side treated with the pulsed system, the collagen fibers in the papillary dermis were more loosely arranged and appeared drier. In both the continuous-wave-treated and pulsed-treated areas, the epidermis appeared healthy and exhibited some signs of age-related deterioration, with slightly flatter plaques and somewhat more flaking keratin on the pulsed-treated side. Probably because of the greater degree of residual thermal damage associated with the continuous-wave system, at 2 years after treatment there was more prolific synthesis and better orientation of collagen fibers, which were maintained for longer times, compared with the pulsed-treated specimens.