An algorithm for the sedation of patients with obstructive sleep apnea by non-anesthesiologists.

OBJECTIVE: Patients with obstructive sleep apnea (OSA) are at risk for adverse events when moderate sedation is administered by nurse protocols (NAMS) under the guidance of non-anesthesiologists. An algorithm was applied for the appropriate section of patients to receive NAMS and the application of continuous positive airway pressure (CPAP). METHODS: An algorithm was developed for patients with OSA who were scheduled for gastroenterology, radiology, and cardiology procedures using NAMS. Those with normal airways and without contraindications for NAMS were classified as CPAP-independent (CPAP-I; not routinely used) or CPAP-dependent (CPAP-D; always used). CPAP machines were brought in by CPAP-D patients or supplied by the hospital and set at a patient's routine setting or 10 cm H2O if not known. CPAP-D patients for procedures for which CPAP could not be applied were done under anesthesia care. We retrospectively examined this program for the 2008-2018 period. RESULTS: Since the inception of this protocol in 2008, 803 patients with OSA safely underwent procedures using either personal CPAP or CPAP provided by the hospital. CONCLUSIONS: Patients with OSA can safely have NAMS for procedures when CPAP is applied based on a protocol that considers airway evaluation, the procedure, and whether there is dependence upon CPAP.

Evaluation of novel highly specific antibodies to cancer testis antigen Centrin-1 for radioimmunoimaging and radioimmunotherapy of pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) accounts for >90% of pancreatic malignancies, and has median survival of <6 months. There is an urgent need for diagnostic and therapeutic options for PDAC. Centrin1 (CETN1) is a novel member of Cancer/Testis Antigens, with a 25-fold increase of CETN1 gene expression in PDX from PDAC patients. The absence of selective anti-CETN1 antibodies is hampering CETN1 use for diagnosis and therapy. Here we report the generation of highly specific for CETN1 antibodies and their evaluation for radioimmunoimaging and radioimmunotherapy (RIT) of experimental PDAC. METHODS: The antibodies to CETN1 were generated via mice immunization with immunogenic peptide distinguishing CETN1 from CETN2. Patient tumor microarrays were used to evaluate the binding of the immune serum to PDAC versus normal pancreas. The antibodies were tested for their preferential binding to CETN1 over CETN2 by ELISA. Mice bearing PDAC MiaPaCa2 xenografts were imaged with microSPECT/CT and treated with 213 Bi- and 177 Lu-labeled antibodies to CETN1. RESULTS: Immune serum bind to 50% PDAC cases on patient tumor microarrays with no specific binding to normal pancreas. Antibodies demonstrated preferential binding to CETN1 versus CETN2. Antibody 69-11 localized to PDAC xenografts in mice in vivo and ex vivo. RIT of PDAC xenografts with 213 Bi-labeled antibodies was effective, safe, and CETN1-specific. CONCLUSIONS: The results demonstrate the ability of these novel antibodies to detect CETN1 both in vitro and in vivo; as well, the RIT treatment of experimental PDAC when radiolabeled with 213 Bi is highly efficient and safe. Further evaluation of these novel reagents for diagnosis and treatment of PDAC is warranted.

Survival and redox activity of Friedmanniomyces endolithicus, an Antarctic endemic black meristematic fungus, after gamma rays exposure.

Despite living organisms are not exposed to acute ionizing radiation under natural conditions, some exhibit a high radiation resistance. Understanding this phenomenon is important for assessing the impact of radiation-related accidents, occupational exposures and space missions. In this context, in this study we analyzed the effect of gamma rays on the Antarctic cryptoendolithic melanized fungus Friedmanniomyces endolithicus CCFEE 5208 and demonstrated its resistance to acute doses of gamma radiation (up to 400 Gy), accompanied by increase in metabolic activity.

Morphological changes in melanized and non-melanized Cryptococcus neoformans cells post exposure to sparsely and densely ionizing radiation demonstrate protective effect of melanin.

There is a need for novel and effective prophylactic treatments and radioprotective materials to protect civilians and military personnel from ionizing radiation in contaminated environments. Melanin, a naturally occurring, ubiquitous pigment, has been shown to confer radioresistance, acting as a potential radioprotective agent. We have demonstrated that melanized Cryptococcus neoformans (CN) cells had improved survival post ionizing irradiation than non-melanized ones. The goal of this study was to identify morphological changes in melanized and non-melanized CN cells following irradiation with densely-ionizing deuterons and alpha particles relative to sparsely-ionizing gamma radiation. We observed significant differences between the melanized and non-melanized CN cellular ultrastructure following irradiation. Melanized CN cells were relatively resistant to mid and max-dose levels of alpha particles and deuterons irradiation. Following irradiation the capsule was stripped, but the cell wall was intact and structural integrity was maintained. At the maximum dose, cytoplasmic vacuolization, and mitochondrial swelling started to occur. In contrast, the non-melanized CN strain was sensitive to the mid-dose radiation. Non-melanized cells presented two morphologies: small condensed, and swollen, lacking structural integrity. This morphological investigation provides the first direct evidence of the radioprotective properties of melanin in CN cells subjected to high RBE and high LET ionizing radiation.

The effect of protracted X-ray exposure on cell survival and metabolic activity of fast and slow growing fungi capable of melanogenesis.

The aim of this study was to analyse how protracted exposure to X-rays delivered at low dose rates of 0.0032-0.052 kGy h-1 affects the survival and metabolic activity of two microfungi capable of melanogenesis: fast-growing Cryptococcus neoformans (CN) and slow-growing Cryomyces antarcticus (CA). Melanized CN and CA cells survived the protracted exposure better than non-melanized ones, which was consistent with previous reports on the radioprotective role of melanin in these fungi after high dose rate exposures. The survival data were described by the linear quadratic dose response model. The XTT metabolic profiles were practically identical for melanized CN and CA with activity dose-dependent increasing: no changes in the activity of the non-melanized CN and CA were recorded by this assay. In contrast, the MTT assay, which measures the intracellular energy-related processes, recorded an increase in activity of non-melanized CN and CA cells, but not in their melanized counterparts. This could reflect intensive repair processes initiated by the non-melanized cells post exposure. This study suggests that differences in radiation responses between melanized and non-melanized fungal cells occur over a wide range of radiation dose rates.

Melanin, Radiation, and Energy Transduction in Fungi.

Melanin pigments are found in many diverse fungal species, where they serve a variety of functions that promote fitness and cell survival. Melanotic fungi inhabit some of the most extreme habitats on earth such as the damaged nuclear reactor at Chernobyl and the highlands of Antarctica, both of which are high-radiation environments. Melanotic fungi migrate toward radioactive sources, which appear to enhance their growth. This phenomenon, combined with the known capacities of melanin to absorb a broad spectrum of electromagnetic radiation and transduce this radiation into other forms of energy, raises the possibility that melanin also functions in harvesting such energy for biological usage. The ability of melanotic fungi to harness electromagnetic radiation for physiological processes has enormous implications for biological energy flows in the biosphere and for exobiology, since it provides new mechanisms for survival in extraterrestrial conditions. Whereas some features of the way melanin-related energy transduction works can be discerned by linking various observations and circumstantial data, the mechanistic details remain to be discovered.

Melanin is effective in protecting fast and slow growing fungi from various types of ionizing radiation.

Melanin is a ubiquitous pigment with unique physicochemical properties. The resistance of melanized fungi to cosmic and terrestrial ionizing radiation suggests that melanin also plays a pivotal role in radioprotection. In this study, we compared the effects of densely-ionizing deuterons and sparsely-ionizing X-rays on two microscopic fungi capable of melanogenesis. We utilized the fast-growing pathogenic basiodiomycete forming an induced DOPA-melanin, Cryptococcus neoformans (CN); and the slow-growing environmental rock-inhabiting ascomycete synthesizing a constitutive DHN-melanin, Cryomyces antarcticus (CA); melanized and non-melanized counterparts were compared. CA was more resistant to deuterons than CN, and similar resistance was observed for X-rays. Melanin afforded protection against high-dose (1.5 kGy) deuterons for both CN and CA (p-values < 10-4 ). For X-rays (0.3 kGy), melanin protected CA (p-values < 10-4 ) and probably CN. Deuterons increased XTT activity in melanized strains of both species, while the activity in non-melanized cells remained stable or decreased. For ATP levels the reverse occurred: it decreased in melanized strains, but not in non-melanized ones, after deuteron exposure. For both XTT and ATP, which reflect the metabolic activity of the cells, larger and more statistically-significant differences as a function of melanization status occurred in CN. Our data show, for the first time, that melanin protected both fast-growing and slow-growing fungi from high doses of deuterons under physiological conditions. These observations may give clues for creating melanin-based radioprotectors.

A fully human antibody to gp41 selectively eliminates HIV-infected cells that transmigrated across a model human blood brain barrier.

OBJECTIVE: Many HIV patients on combined antiretroviral therapy exhibit HIV-associated neurocognitive disorders because the brain becomes a viral reservoir. There is a need for therapeutics that can enter the central nervous system (CNS) and eradicate the virus. DESIGN: Radiolabeled human mAb 2556 to HIV gp41 selectively kills HIV-infected cells in vivo and in vitro. Here we tested the ability of 213Bi-2556 to cross a tissue culture model of the human blood brain barrier and kill HIV-infected peripheral blood mononuclear cells (PBMCs) and monocytes on the CNS side of the barrier. METHODS: 2556 mAb isoelectric point was determined with isoelectric focusing. The ability of radiolabeled 2556 to penetrate through the barrier was studied by adding it to the upper chamber of the barriers and its penetration into the CNS side was followed for 5 h. To assess the ability of Bi-2556 to kill the HIV-infected cells on the CNS side of barrier, the HIV-infected and uninfected PBMCs and monocytes were allowed to transmigrate across the barriers overnight followed by application of Bi-2556 or control mAb Bi-1418 to the top of the barrier. Killing of cells was measured by TUNEL and Trypan blue assays. The barriers were examined by confocal microscopy for overt damage. RESULTS: The isoelectric point of Bi-2556 was 9.6 enabling its penetration through the barrier by transcytosis. Bi-2556 killed significantly more transmigrated HIV-infected cells in comparison to Bi-1418 and uninfected cells. No overt damage to barriers was observed. CONCLUSION: We demonstrated that Bi-2556 mAb crossed an in-vitro human blood brain barrier and specifically killed transmigrated HIV-infected PBMCs and monocytes without overt damage to the barrier.

Effects of radiation type and delivery mode on a radioresistant eukaryote Cryptococcus neoformans.

INTRODUCTION: Most research on radioresistant fungi, particularly on human pathogens such as Cryptococcus neoformans, involves sparsely-ionizing radiation. Consequently, fungal responses to densely-ionizing radiation, which can be harnessed to treat life-threatening fungal infections, remain incompletely understood. METHODS: We addressed this issue by quantifying and comparing the effects of densely-ionizing α-particles (delivered either by external beam or by (213)Bi-labeled monoclonal antibodies), and sparsely-ionizing (137)Cs γ-rays, on Cryptococcus neoformans. RESULTS: The best-fit linear-quadratic parameters for clonogenic survival were the following: α = 0.24 × 10(-2) Gy(-1) for γ-rays and 1.07 × 10(-2) Gy(-1) for external-beam α-particles, and ß = 1.44 × 10(-5) Gy(-2) for both radiation types. Fungal cell killing by radiolabeled antibodies was consistent with predictions based on the α-particle dose to the cell nucleus and the linear-quadratic parameters for external-beam α-particles. The estimated RBE (for α-particles vs. γ-rays) at low doses was 4.47 for the initial portion of the α-particle track, and 7.66 for the Bragg peak. Non-radiological antibody effects accounted for up to 23% of cell death. CONCLUSIONS: These results quantify the degree of C. neoformans resistance to densely-ionizing radiations, and show how this resistance can be overcome with fungus-specific radiolabeled antibodies.

Treatment of experimental pancreatic cancer with 213-Bismuth-labeled chimeric antibody to single-strand DNA.

BACKGROUND: Novel approaches to treatment of pancreatic cancer (PCa) are urgently needed. A chimeric monoclonal antibody (mAb) chTNT3 binds to single-strand DNA (ssDNA) and RNA released from the non-viable cells in fast growing tumors. Here the authors investigated whether radioimmunotherapy (RIT) using chTNT3 mAb radiolabeled with 213-Bismuth ((213)Bi) could be effective in treatment of experimental PCa. METHODS: Two human PCa cell lines, Panc1 and MiaPaCa-2, were used for in vitro experiments. The xenografts in mice were established using MiaPaCa-2 cells. Therapy compared (213)Bi-chTNT3 (700 µCi) to gemcitabine or cisplatin, untreated controls and 'cold' chTNT3. RESULTS: RIT abrogated the tumors growth while tumors in control groups grew aggressively. Chemotherapy was less effective than RIT and toxic to mice while RIT did not have any side effects. CONCLUSIONS: RIT with (213)Bi-chTNT3 was safe and effective in the treatment of experimental PCa in comparison with chemotherapy. This makes α-RIT targeting ssDNA a promising modality for further development.

Mathematical modeling predicts enhanced growth of X-ray irradiated pigmented fungi.

Ionizing radiation is known for its cytotoxic and mutagenic properties. However, recent evidence suggests that chronic sub-lethal irradiation stimulates the growth of melanin-pigmented (melanized) fungi, supporting the hypothesis that interactions between melanin and ionizing photons generate energy useful for fungal growth, and/or regulate growth-promoting genes. There are no quantitative models of how fungal proliferation is affected by ionizing photon energy, dose rate, and presence versus absence of melanin on the same genetic background. Here we present such a model, which we test using experimental data on melanin-modulated radiation-induced proliferation enhancement in the fungus Cryptococcus neoformans, exposed to two different peak energies (150 and 320 kVp) over a wide range of X-ray dose rates. Our analysis demonstrates that radiation-induced proliferation enhancement in C. neoformans behaves as a binary "on/off" phenomenon, which is triggered by dose rates <0.002 mGy/h, and stays in the "on" position. A competing dose rate-dependent growth inhibition becomes apparent at dose rates >5000 mGy/h. Proliferation enhancement of irradiated cells compared with unirradiated controls occurs at both X-ray peak energies, but its magnitude is modulated by X-ray peak energy and cell melanization. At dose rates <5000 mGy/h, both melanized and non-melanized cells exposed to 150 kVp X-rays, and non-melanized cells exposed to 320 kVp X-rays, all exhibit the same proliferation enhancement: on average, chronic irradiation stimulates each founder cell to produce 100 (95% CI: 83, 116) extra descendants over 48 hours. Interactions between melanin and 320 kVp X-rays result in a significant (2-tailed p-value = 4.8 × 10(-5)) additional increase in the number of radiation-induced descendants per founder cell: by 55 (95% CI: 29, 81). These results show that both melanin-dependent and melanin-independent mechanisms are involved in radiation-induced fungal growth enhancement, and implicate direct and/or indirect interactions of melanin with high energy ionizing photons as an important pro-proliferative factor.

Radioimmunotherapy of Cryptococcus neoformans spares bystander mammalian cells.

AIM: Previously, we showed that radioimmunotherapy (RIT) for cryptococcal infections using radioactively labeled antibodies recognizing the cryptococcal capsule reduced fungal burden and prolonged survival of mice infected with Cryptococcus neoformans. Here, we investigate the effects of RIT on bystander mammalian cells. MATERIALS & METHODS: Heat-killed C. neoformans bound to anticapsular antibodies, unlabeled or labeled with the ß-emitter rhenium-188 (16.9-h half-life) or the α-emitter bismuth-213 (46-min half-life), was incubated with macrophage-like J774.16 cells or epithelial-like Chinese hamster ovary cells. Lactate dehydrogenase activity, crystal violet uptake, reduction of tetrazolium dye (2,3)-bis-(2-methoxy-4-nitro-5-sulfenyl)-(2H)-terazolium-5-carboxanilide and nitric oxide production were measured. RESULTS: The J774.16 and Chinese hamster ovary cells maintained membrane integrity, viability and metabolic activity following exposure to radiolabeled C. neoformans. CONCLUSION: RIT of C. neoformans is a selective therapy with minimal effects on host cells and these results are consistent with observations that RIT-treated mice with cryptococcal infection lacked RIT-related pathological changes in lungs and brain tissues.

Complement-dependent cytotoxicity of an antibody to melanin in radioimmunotherapy of metastatic melanoma.

AIM: Novel treatments for metastatic melanoma are urgently needed. MATERIALS & METHODS: We developed radioimmunotherapy of metastatic melanoma using 6D2 monoclonal antibody (mAb) to melanin with encouraging therapeutic results, preclinically and in patients. RESULTS: We observed tumor suppression with the unlabeled 6D2 mAb and investigated its tumoricidal mechanisms. In melanoma tumor-bearing mice, we detected more complement-C3 deposition in the tumors from 188-rhenium-labeled 6D2 mAb-treated mice when compared with untreated controls. 6D2 and isotype-control mAb TEPC caused suppression of tumor growth in A2058 melanoma tumor-bearing mice. Tumors of mice treated with the unlabeled 6D2 mAb were infiltrated with more lymphocytes compared with controls. In vitro antibody-dependent cell-mediated cytotoxicity did not contribute to the tumor-suppressive effect of 6D2 mAb, while 6D2 mAb demonstrated a strong effect on initiating complement-dependent cytotoxicity. CONCLUSION: We concluded that 6D2 mAb mediated complement-dependent cytotoxicity, resulting in killing of the tumor cells and suppression of tumor growth. These observations will help to improve the treatment protocols of radioimmunotherapy, as well as immunotherapy.

Compton scattering by internal shields based on melanin-containing mushrooms provides protection of gastrointestinal tract from ionizing radiation.

There is a need for radioprotectors that protect normal tissues from ionizing radiation in patients receiving high doses of radiation and during nuclear emergencies. We investigated the possibility of creating an efficient oral radioprotector based on the natural pigment melanin that would act as an internal shield and protect the tissues via Compton scattering followed by free radical scavenging. CD-1 mice were fed melanin-containing black edible mushrooms Auricularia auricila-judae before 9 Gy total body irradiation. The location of the mushrooms in the body before irradiation was determined by in vivo fluorescent imaging. Black mushrooms protected 80% of mice from the lethal dose, while control mice or those given melanin-devoid mushrooms died from gastrointestinal syndrome. The crypts of mice given black mushrooms showed less apoptosis and more cell division than those in control mice, and their white blood cell and platelet counts were restored at 45 days to preradiation levels. The role of melanin in radioprotection was proven by the fact that mice given white mushrooms supplemented with melanin survived at the same rate as mice given black mushrooms. The ability of melanin-containing mushrooms to provide remarkable protection against radiation suggests that they could be developed into oral radioprotectors.

Treatment of early and established Cryptococcus neoformans infection with radiolabeled antibodies in immunocompetent mice.

We investigated the utility of radioimmunotherapy (RIT) in early and established cryptococcal infection in immunocompetent mice. RIT with (213)Bi-18B7 antibody completely eliminated fungus from mouse lungs and brains for early infection, while (188)Re-18B7 significantly reduced CFU in the lungs or both lungs and brains during early and established infection, respectively. The results point to the independence of RIT of the immune status of the host, which is encouraging for translation of this strategy into the clinic.

Protection of melanized Cryptococcus neoformans from lethal dose gamma irradiation involves changes in melanin's chemical structure and paramagnetism.

Certain fungi thrive in highly radioactive environments including the defunct Chernobyl nuclear reactor. Cryptococcus neoformans (C. neoformans), which uses L-3,4-dihydroxyphenylalanine (L-DOPA) to produce melanin, was used here to investigate how gamma radiation under aqueous aerobic conditions affects the properties of melanin, with the aim of gaining insight into its radioprotective role. Exposure of melanized fungal cell in aqueous suspensions to doses of γ-radiation capable of killing 50 to 80% of the cells did not lead to a detectable loss of melanin integrity according to EPR spectra of melanin radicals. Moreover, upon UV-visible (Xe-lamp) illumination of melanized cells, the increase in radical population was unchanged after γ-irradiation. Gamma-irradiation of frozen cell suspensions and storage of samples for several days at 77 K however, produced melanin modification noted by a reduced radical population and reduced photoresponse. More direct evidence for structural modification of melanin came from the detection of soluble products with absorbance maxima near 260 nm in supernatants collected after γ-irradiation of cells and cell-free melanin. These products, which include thiobarbituric acid (TBA)-reactive aldehydes, were also generated by Fenton reagent treatment of cells and cell-free melanin. In an assay of melanin integrity based on the metal (Bi(+3)) binding capacity of cells, no detectable loss in binding was detected after γ-irradiation. Our results show that melanin in C. neoformans cells is susceptible to some damage by hydroxyl radical formed in lethal radioactive aqueous environments and serves a protective role in melanized fungi that involves sacrificial breakdown.

The effects of gamma radiation, UV and visible light on ATP levels in yeast cells depend on cellular melanization.

Previously we have shown that growth of melanized fungi is stimulated by low levels of gamma radiation. The goal of this study was to examine the effects of visible light, UV light, and gamma radiation on the energy level (ATP concentration) in melanized Cryptococcus neoformans cells. Melanized C. neoformans cells as well as non-melanized controls were subjected to visible, UV or gamma radiation, and ATP was quantified by measuring the amount of light emitted by the ATP-dependent reaction of luciferase with luciferin. We found that all three forms of radiation led to a reduction in the ATP levels in melanized C. neoformans cells. This points to a universal melanin-related mechanism underlying observation of ATP decrease in irradiated melanized cells. In contrast, in non-melanized cells visible light led to increase in ATP levels; gamma radiation did not cause any changes while UV exposure resulted in some ATP decrease, however, much less pronounced than in melanized cells.

Radioimmunotherapy is more effective than antifungal treatment in experimental cryptococcal infection.

Radioimmunotherapy (RIT) prolongs the survival of mice infected with Cryptococcus neoformans. To compare the efficacy of RIT with that of amphotericin B, we infected AJ/Cr mice intravenously with either nonmelanized or melanized C. neoformans cells. Infected mice were either left untreated or treated 24 h after infection with (213)Bi-18B7 antibody, amphotericin B, or both. Melanization before infection did not increase resistance of C. neoformans to RIT in vivo. (213)Bi-18B7 treatment almost completely eliminated colony-forming units from the lung and brain, whereas amphotericin B did not decrease the number of colony-forming units. We conclude that RIT is more effective than amphotericin B against systemic infection with C. neoformans.

Physico-chemical evaluation of rationally designed melanins as novel nature-inspired radioprotectors.

BACKGROUND: Melanin, a high-molecular weight pigment that is ubiquitous in nature, protects melanized microorganisms against high doses of ionizing radiation. However, the physics of melanin interaction with ionizing radiation is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We rationally designed melanins from either 5-S-cysteinyl-DOPA, L-cysteine/L-DOPA, or L-DOPA with diverse structures as shown by elemental analysis and HPLC. Sulfur-containing melanins had higher predicted attenuation coefficients than non-sulfur-containing melanins. All synthetic melanins displayed strong electron paramagnetic resonance (2.14.10(18), 7.09.10(18), and 9.05.10(17) spins/g, respectively), with sulfur-containing melanins demonstrating more complex spectra and higher numbers of stable free radicals. There was no change in the quality or quantity of the stable free radicals after high-dose (30,000 cGy), high-energy ((137)Cs, 661.6 keV) irradiation, indicating a high degree of radical stability as well as a robust resistance to the ionizing effects of gamma irradiation. The rationally designed melanins protected mammalian cells against ionizing radiation of different energies. CONCLUSIONS/SIGNIFICANCE: We propose that due to melanin's numerous aromatic oligomers containing multiple pi-electron system, a generated Compton recoil electron gradually loses energy while passing through the pigment, until its energy is sufficiently low that it can be trapped by stable free radicals present in the pigment. Controlled dissipation of high-energy recoil electrons by melanin prevents secondary ionizations and the generation of damaging free radical species.

Radioimmunotherapy is effective against high-inoculum Cryptococcus neoformans infection in mice and does not select for radiation-resistant cryptococcal cells.

We investigated the utility of radioimmunotherapy (RIT) in the treatment of experimental cryptococcal infection with high-inoculum and the possibility of RIT treatment selecting for fungal cells with radiation-resistant phenotypes. RIT reduced mortality in high-burden infections, and we found no evidence for the development of radiation-resistant cells.