Prehospital Ground and Helicopter-Based Extracorporeal Cardiopulmonary Resuscitation (ECPR) Reduce Barriers to ECPR: A GIS Model.

INTRODUCTION: Evidence suggests that Extracorporeal Cardiopulmonary Resuscitation (ECPR) can improve survival rates for nontraumatic out-of-hospital cardiac arrest (OHCA). However, when ECPR is indicated over 50% of potential candidates are unable to qualify in the current hospital-based system due to geographic limitations. This study employs a Geographic Information System (GIS) model to estimate the number of ECPR eligible patients within the United States in the current hospital-based system, a prehospital ECPR ground-based system, and a prehospital ECPR Helicopter Emergency Medical Services (HEMS)-based system. METHODS: We constructed a GIS model to estimate ground and helicopter transport times. Time-dependent rates of ECPR eligibility were derived from the Resuscitation Outcome Consortium (ROC) database, while the Cardiac Arrest Registry to Enhance Survival (CARES) registry determined the number of OHCA patients meeting ECPR criteria within designated transportation times. Emergency Medical Services (EMS) response time, ECPR candidacy determination time, and on-scene time were modeled based on data from the EROCA trial. The combined model was used to estimate the total ECPR eligibility in each system. RESULTS: The CARES registry recorded 736,066 OHCA patients from 2013 to 2021. After applying clinical criteria, 24,661 (3.4%) ECPR-indicated OHCA were identified. When considering overall ECPR eligibility within 45 min from OHCA to initiation, only 11.76% of OHCA where ECPR was indicated were eligible in the current hospital-based system. The prehospital ECPR HEMS-based system exhibited a four-fold increase in ECPR eligibility (49.3%), while the prehospital ground-based system showed a more than two-fold increase (28.4%). CONCLUSIONS: The study demonstrates a two-fold increase in ECPR eligibility for a prehospital ECPR ground-based system and a four-fold increase for a prehospital ECPR HEMS-based system compared to the current hospital-based ECPR system. This novel GIS model can inform future ECPR implementation strategies, optimizing systems of care.

Eligibility of out-of-hospital cardiac arrest patients for extracorporeal cardiopulmonary resuscitation in the United States: A geographic information system model.

BACKGROUND: Recent evidence suggest that extracorporeal cardiopulmonary resuscitation (ECPR) may improve survival rates for nontraumatic out-of-hospital cardiac arrest (OHCA). Eligibility criteria for ECPR are often based on patient age, clinical variables, and facility capabilities. Expanding access to ECPR across the U.S. requires a better understanding of how these factors interact with transport time to ECPR centers. METHODS: We constructed a Geographic Information System (GIS) model to estimate the number of ECPR candidates in the U.S. We utilized a Resuscitation Outcome Consortium (ROC) database to model time-dependent rates of ECPR eligibility and the Cardiac Arrest Registry to Enhance Survival (CARES) registry to determine the total number of OHCA patients who meet pre-specified ECPR criteria within designated transportation times. The combined model was used to estimate the total number of ECPR candidates. RESULTS: There were 588,203 OHCA patients in the CARES registry from 2013 to 2020. After applying clinical eligibility criteria, 22,104 (3.76%) OHCA patients were deemed eligible for ECPR. The rate of ROSC increased with longer resuscitation time, which resulted in fewer ECPR candidates. The proportion of OHCA patients eligible for ECPR increased with older age cutoffs. Only 1.68% (9,889/588,203) of OHCA patients in the U.S. were eligible for ECPR based on a 45-minute transportation time to an ECMO-ready center model. CONCLUSIONS: Less than 2% of OHCA patients are eligible for ECPR in the U.S. GIS models can identify the impact of clinical criteria, transportation time, and hospital capabilities on ECPR eligibility to inform future implementation strategies.

Melanocortin 1 receptor genotype: an important determinant of the damage response of melanocytes to ultraviolet radiation.

The melanocortin 1 receptor gene is a main determinant of human pigmentation, and a melanoma susceptibility gene, because its variants that are strongly associated with red hair color increase melanoma risk. To test experimentally the association between melanocortin 1 receptor genotype and melanoma susceptibility, we compared the responses of primary human melanocyte cultures naturally expressing different melanocortin 1 receptor variants to α-melanocortin and ultraviolet radiation. We found that expression of 2 red hair variants abolished the response to α-melanocortin and its photoprotective effects, evidenced by lack of functional coupling of the receptor, and absence of reduction in ultraviolet radiation-induced hydrogen peroxide generation or enhancement of repair of DNA photoproducts, respectively. These variants had different heterozygous effects on receptor function. Microarray data confirmed the observed differences in responses of melanocytes with functional vs. nonfunctional receptor to α-melanocortin and ultraviolet radiation, and identified DNA repair and antioxidant genes that are modulated by α-melanocortin. Our findings highlight the molecular mechanisms by which the melanocortin 1 receptor genotype controls genomic stability of and the mutagenic effect of ultraviolet radiation on human melanocytes.

Melanocytes derived from patients with Hermansky-Pudlak Syndrome types 1, 2, and 3 have distinct defects in cargo trafficking.

Hermansky-Pudlak Syndrome (HPS) is a genetically heterogeneous disorder in which mutations in one of several genes interrupts biogenesis of melanosomes, platelet dense bodies, and lysosomes. Affected patients have oculocutaneous albinism, a bleeding diathesis, and sometimes develop granulomatous colitis or pulmonary fibrosis. In order to assess the role of HPS genes in melanosome biogenesis, melanocytes cultured from patients with HPS subtypes 1, 2, or 3 were assessed for the localization of various melanocyte proteins. Tyrosinase, Tyrp1, and Dct/Tyrp2 were atypically and distinctly expressed in HPS-1 and HPS-3 melanocytes, whereas only tyrosinase showed an atypical distribution in HPS-2 melanocytes. The HPS1 and AP3B1 (i.e., HPS-2) gene products showed no expression in HPS-1 and HPS-2 melanocytes, respectively, whereas HPS-3 melanocytes exhibited normal expression for both proteins. In normal human melanocytes, the HPS1 protein was expressed as an approximately 80 kDa molecule with both granular and reticular intracellular profiles. In HPS-1, lysosome associated membrane protein 1 (LAMP1), and LAMP3 were localized to abnormal large granules; in HPS-2, all LAMPs exhibited a normal granular expression; and in HPS-3, LAMP1, and LAMP3 exhibited a distinct less granular and more floccular pattern. In contrast, the expressions of Rab 27, transferrin, and cKit were unaffected in all three HPS genotypes. These data demonstrate that the three initially identified subtypes of human HPS exhibit distinct defects in the trafficking of various melanocyte-specific proteins.

Melanocytic galectin-3 is associated with tyrosinase-related protein-1 and pigment biosynthesis.

Galectin-3 is a family member of the carbohydrate-binding proteins widely expressed by many cell types and exhibits multiple cellular functions. We demonstrate that melanocytes express galectin-3, which is predominantly localized to the cell body peripherally along the Golgi zone. Downregulation of galectin-3 in human melanocytes using short hairpin RNA technology resulted in the reduction of both melanin synthesis and expression/activity of tyrosinase-related protein-1 (Tyrp-1). In the cell body, galectin-3 colocalizes with melanosome-destined cargo, specifically tyrosinase and Tyrp-1. We studied melanocytes cultured from patients with forms of Hermansky-Pudlak syndrome (HPS) containing defects in trafficking steps governed by biogenesis of lysosome-related organelle complex-2 (BLOC-2) (HPS-5), BLOC-3 (HPS-1), and adaptin-3 (HPS-2). We found that galectin-3 expression mimicked the defective expression of the tyrosinase cargo in dendrites of HPS-5 melanocytes, but it was not altered in HPS-1 or HPS-2 melanocytes. In addition, galectin-3 colocalized predominantly with the HPS-5 component of BLOC-2 in normal human melanocytes. These data indicate that galectin-3 is a regulatory component in melanin synthesis affecting the expression of Tyrp-1.

Preferential secretion of inducible HSP70 by vitiligo melanocytes under stress.

Inducible HSP70 (HSP70i) chaperones peptides from stressed cells, protecting them from apoptosis. Upon extracellular release, HSP70i serves an adjuvant function, enhancing immune responses to bound peptides. We questioned whether HSP70i differentially protects control and vitiligo melanocytes from stress and subsequent immune responses. We compared expression of HSP70i in skin samples, evaluated the viability of primary vitiligo and control melanocytes exposed to bleaching phenols, and measured secreted HSP70i. We determined whether HSP70i traffics to melanosomes to contact immunogenic proteins by cell fractionation, western blotting, electron microscopy, and confocal microscopy. Viability of vitiligo and control melanocytes was equally affected under stress. However, vitiligo melanocytes secreted increased amounts of HSP70i in response to MBEH, corroborating with aberrant HSP70i expression in patient skin. Intracellular HSP70i colocalized with melanosomes, and more so in response to MBEH in vitiligo melanocytes. Thus, whereas either agent is cytotoxic to melanocytes, MBEH preferentially induces immune responses to melanocytes.

Hydrolytic enzymes of the interfollicular epidermis differ in expression and correlate with the phenotypic difference observed between light and dark skin.

Degradation of melanosomes in light skin (LS, i.e. phototype I/II) appears to occur more rapidly than dark skin (DS, i.e. phototype IV/V). Hydrolytic enzymes known to reside and be expressed in a differential pattern within the interfollicular epidermis are implicated in playing a role in epidermal differentiation and potentially melanosome degradation. The aim of this present study was to evaluate the differential expression of hydrolytic enzymes that may correlate with physiological and phenotypic differences seen between DS and LS. Expression of six hydrolytic enzymes was confirmed by microarray analysis of the suprabasal epidermis from LS and DS. Specific lysosomal hydrolases identified by microarray analysis were analyzed by indirect immunofluorescence (IIF) and immunoblot analysis. Immunogold electron microscopy (IEM) was completed to visualize cellular expression of the hydrolytic enzyme cathepsin L2 (Cath L2) and biochemical assay was performed to ascertain Cath L2 activity. Immunoblotting of light and dark epidermal lysates demonstrated that of the six enzymes initially analyzed, both prostatic acid phosphatase (ACPP) and Cath L2 were reproducibly upregulated in DS and LS, respectively. IIF and IEM analyses of Cath L2 in tissue confirmed this differential expression. Biochemical analysis of Cath L2 in light and dark epidermal lysates displays increased activity of Cath L2 in LS. The results of this study confirm differential expression of ACPP and Cath L2 in DS and LS at gene and protein level. Additionally, Cath L2 displays increased activity in LS-derived epidermal lysates. This study indentified two acid hydrolases that may play a role in melanosome degradation and pigment processing.

Epidermal keratinocytes from light vs. dark skin exhibit differential degradation of melanosomes.

Modification of skin complexion coloration has traditionally been accomplished by interruption or attenuation of melanogenesis and/or melanosome transfer. Post-transfer modification of pigmented melanosomes provides an attractive and distinct avenue of modulating skin pigmentation. The processing of melanosomes during keratinocyte (KC) terminal differentiation and the degradative variability observed between light and dark skin (LS and DS) remains enigmatic. To evaluate this, we developed a model system to investigate the loss of fluorescently labeled and isolated melanosomes by cultured human KCs. The extent of melanosome loss has been qualitatively assessed using transmission electron microscopy and indirect immunofluorescence with confocal microscopy, and quantitatively assessed using flow cytometry analysis. Results show that melanosomes are incorporated into the cytoplasm of both light and dark keratinocytes (LKCs and DKCs) and trafficked to a perinuclear region. Within 48 hours, confocal microscopy images suggest that LKCs display accelerated melanosome loss. This time-dependent decrease in carboxyfluorescein diacetate (CFDA) fluorescence was then quantitatively analyzed using flow cytometry. Consistent with the results of the confocal analysis, over a 48-hour time frame, LKCs appear to lose melanosomes more efficiently than DKCs. These experiments show that melanosomes are more rapidly lost in KCs derived from LS as opposed to DS.

Monobenzyl ether of hydroquinone and 4-tertiary butyl phenol activate markedly different physiological responses in melanocytes: relevance to skin depigmentation.

Monobenzyl ether of hydroquinone (MBEH) is a Food and Drug Administration approved drug used for depigmentation therapy of advanced vitiligo. Here, the working mechanism of MBEH is explored in comparison to 4-tertiary butyl phenol (4-TBP), a known causative agent for occupational vitiligo mediating apoptotic melanocytic death. Cytotoxic experiments reveal that similar to 4-TBP, MBEH induces specific melanocyte death. To compare death pathways initiated by 4-TBP and MBEH, classical apoptotic hallmarks were evaluated in treated melanocytes. MBEH induced cell death without activating the caspase cascade or DNA fragmentation, showing that the death pathway is non-apoptotic. Release of High Mobility Group Box-1 protein by MBEH-treated melanocytes and ultrastructural features further confirmed a necrotic death pathway mediated by MBEH. A negative correlation between MBEH-induced cell death and cellular melanin content supports a cytoprotective role for melanin. Moreover, MBEH exposure upregulated the levels of melanogenic enzymes in cultured melanocytes and skin explants, whereas 4-TBP reduced the expression of the same. In summary, exposure to MBEH or 4-TBP has profoundly different consequences for melanocyte physiology and activates different death pathways. As the mode of cell death defines the nature of the immune response that follows, these findings help to explain the relative efficacy of these agents in mediating depigmentation.

Improper trafficking of melanocyte-specific proteins in Hermansky-Pudlak syndrome type-5.

Hermansky-Pudlak syndrome (HPS) is a disorder of lysosome-related organelle biogenesis resulting in melanosome dysfunction and absent platelet dense bodies. HPS patients have oculocutaneous albinism, bruising, and bleeding. HPS-5 results from deficiency of the HPS5 protein, a component of the biogenesis of lysosome-related organelles complex-2 (BLOC-2). HPS5 has an unknown function and lacks homology to known proteins. We performed ultrastructural studies of HPS-5 melanocytes revealing predominantly early-stage melanosomes with many small 3,4(OH)2-phenylalanine-positive vesicles throughout the cell body and dendrites. These findings resemble the distinct ultrastructural features of HPS-3 melanocytes; HPS3 is also a BLOC-2 component. Immunofluorescence and immunoEM studies showed decreased TYRP1 labeling in the dendrites of HPS-5 melanocytes, and the overall abundance of TYRP1 was reduced. No substantial differences were observed in the distribution or abundance of Pmel17 in HPS-5 melanocytes. In normal melanocytes, endogenous tyrosinase colocalized with Pmel17 and TYRP1 in the perinuclear area and dendritic tips; this was much reduced in HPS-5 melanocytes, particularly in the tips. We conclude that early stage melanosome formation and Pmel17 trafficking are preserved in HPS5-deficient cells. Tyrosinase and TYRP1 are mistrafficked, however, and fail to be efficiently delivered to melanosomes of HPS-5 melanocytes.

Membranous complexes characteristic of melanocytes derived from patients with Hermansky-Pudlak syndrome type 1 are macroautophagosomal entities of the lysosomal compartment.

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder resulting from mutations in a family of genes required for efficient transport of lysosomal-related proteins from the trans-Golgi network to a target organelle. To date, there are several genetically distinct forms of HPS. Many forms of HPS exhibit aberrant trafficking of melanosome-targeted proteins resulting in incomplete melanosome biogenesis responsible for oculocutaneous albinism observed in patients. In HPS-1, melanosome-targeted proteins are localized to characteristic membranous complexes, which have morphologic similarities to macroautophagosomes. In this report, we evaluated the hypothesis that HPS-1-specific membranous complexes comprise a component of the lysosomal compartment of melanocytes. Using indirect immunofluorescence, an increase in co-localization of misrouted tyrosinase with cathepsin-L, a lysosomal cysteine protease, occurred in HPS-1 melanocytes. In addition, ribophorin II, an integral endoplasmic reticulum protein that is also a component of macroautophagosomes, and LC3, a specific marker of macrophagosomes, demonstrated localization to membranous complexes in HPS-1 melanocytes. At the electron microscopic level, the membranous complexes exhibited acid phosphatase activity and localization of exogenously supplied horseradish peroxidase (HRP)-conjugated gold particles, indicating incorporation of lysosomal and endosomal components to membranous complexes, respectively. These results confirm that membranous complexes of HPS-1 melanocytes are macroautophagosomal representatives of the lysosomal compartment.

Melanocyte-specific proteins are aberrantly trafficked in melanocytes of Hermansky-Pudlak syndrome-type 3.

Hermansky-Pudlak Syndrome-type 3 (HPS-3) is a relatively mild subtype of HPS with minimal cutaneous and ocular depigmentation. The HPS-3 gene encodes a novel protein of unknown function with a predicted molecular weight of 114 kd. To assess the role of the HPS3 protein in melanization, cultured melanocytes developed from HPS-3 patients were evaluated biochemically and histologically for activity and localization of melanocyte-specific proteins. Endogenous tyrosinase activity of HPS-3 melanocytes was substantial, but tyrosinase activity and melanin synthesis was suppressed in intact melanocytes. However, the level of suppression, as well as extent to which up-regulation by isobutylmethylxanthine and cholera toxin was muted, was less that in HPS-1 melanocytes. Ultrastructurally, HPS-3 melanocytes contained morphologically normal melanosomes, predominantly of stage I and II with minimal stage III and few stage IV melanosomes. Dihydroxyphenylalanine (DOPA) histochemistry demonstrated an increase in melanization of melanosomes. Unique to HPS-3 melanocytes were numerous DOPA-positive 50-nm vesicles and tubular elements present throughout the cell body and dendrites. Tyrosinase, tyrosinase-related protein-1 (Tyrp1), dopachrome tautomerase (Dct), and LAMP1 and 3 localization in HPS-3 melanocytes, as evaluated by immunocytochemistry and confocal microscopy, demonstrated a fine, floccular distribution in contrast to the coarse, granular distribution characteristic of control melanocytes. The localization profile of other proteins expressed by melanocytes (ie, Silver/Pmel17, Melan-A/MART-1, LAMP2, Rab 27, transferrin, c-kit, adaptin-3, and the HPS1 protein) appeared normal. These results suggest that a specific subset of melanocyte proteins are aberrantly trafficked throughout the HPS-3 melanocyte and may be responsible for the reduction in melanin synthesis.

Effective inhibition of melanosome transfer to keratinocytes by lectins and niacinamide is reversible.

Skin pigmentation results in part from the transfer of melanized melanosomes synthesized by melanocytes to neighboring keratinocytes. Plasma membrane lectins and their glycoconjugates expressed by these epidermal cells are critical molecules involved in this transfer process. In addition, the derivative of vitamin B(3), niacinamide, can inhibit melanosome transfer and induce skin lightening. We investigated the effects of these molecules on the viability of melanocytes and keratinocytes and on the reversibility of melanosome-transfer inhibition induced by these agents using an in vitro melanocyte-keratinocyte coculture model system. While lectins and neoglycoproteins could induce apoptosis in a dose-dependent manner to melanocytes or keratinocytes in monoculture, similar dosages of the lectins, as opposed to neoglycoproteins, did not induce apoptosis to either cell type when treated in coculture. The dosages of lectins and niacinamide not affecting cell viability produced an inhibitory effect on melanosome transfer, when used either alone or together in cocultures of melanocytes-keratinocytes. Cocultures treated with lectins or niacinamide resumed normal melanosome transfer in 3 days after removal of the inhibitor, while cocultures treated with a combination of lectins and niacinamide demonstrated a lag in this recovery. Subsequently, we assessed the effect of niacinamide on facial hyperpigmented spots using a vehicle-controlled, split-faced design human clinical trial. Topical application of niacinamide resulted in a dose-dependent and reversible reduction in hyperpigmented lesions. These results suggest that lectins and niacinamide at concentrations that do not affect cell viability are reversible inhibitors of melanosome transfer.