Safety and feasibility of the gene transfer of hemopexin for conditions with increased free heme.

Heme is a fundamental molecule for several biological processes, but when released in the extracellular space such as in hemolytic diseases, it can be toxic to cells and tissues. Hemopexin (HPX) is a circulating protein responsible for removing free heme from the circulation, whose levels can be severely depleted in conditions such as sickle cell diseases. Accordingly, increasing HPX levels represents an attractive strategy to mitigate the deleterious effects of heme in these conditions. Gene transfer of liver-produced proteins with adeno-associated virus (AAV) has been shown to be an effective and safety strategy in animal and human studies mainly in hemophilia. Here, we report the feasibility of increasing HPX levels using an AAV8 vector expressing human HPX (hHPX). C57Bl mice were injected with escalating doses of our vector, and expression was assessed by enzyme immunoassay (ELISA), Western blot, and quantitative polymerase chain reaction (qPCR). In addition, the biological activity of transgenic hHPX was confirmed using two different models of heme challenge consisting of serial heme injections or phenylhydrazine-induced hemolysis. Sustained expression of hHPX was confirmed for up to 26 weeks in plasma. Expression was dose-dependent and not associated with clinical signs of toxicity. hHPX levels were significantly reduced by heme infusions and phenylhydrazine-induced hemolysis. No clinical toxicity or laboratory signs of liver damage were observed in preliminary short-term heme challenge studies. Our results confirm that long-term expression of hHPX is feasible and safe in mice, even in the presence of heme overload. Additional studies are needed to explore the effect of transgenic HPX protein in animal models of chronic hemolysis.

Podoplanin and CLEC-2 levels in patients with COVID-19.

Introduction: Podoplanin (PDPN gene) and CLEC-2 are involved in inflammatory hemostasis and have also been related with the pathogenesis of thrombosis. Emerging evidence also suggest that podoplanin can exert protective effects in sepsis and in acute lung injury. In lungs, podoplanin is co-expressed with ACE2, which is the main entry receptor for SARS-CoV-2. Aim: To explore the role of podoplanin and CLEC-2 in COVID-19. Methods: Circulating levels of podoplanin and CLEC-2 were measured in 30 consecutive COVID-19 patients admitted due to hypoxia, and in 30 age- and sex-matched healthy individuals. Podoplanin expression in lungs from patients who died of COVID-19 was obtained from two independent public databases of single-cell RNAseq from which data from control lungs were also available. Results: Circulating podoplanin levels were lower in COVID-19, while no difference was observed in CLEC-2 levels. Podoplanin levels were significantly inversely correlated with markers of coagulation, fibrinolysis and innate immunity. scRNAseq data confirmed that PDPN is co-expressed with ACE2 in pneumocytes, and showed that PDPN expression is lower in this cell compartment in lungs from patients with COVID-19. Conclusion: Circulating levels of podoplanin are lower in COVID-19, and the magnitude of this reduction is correlated with hemostasis activation. We also demonstrate the downregulation of PDPN at the transcription level in pneumocytes. Together, our exploratory study questions whether an acquired podoplanin deficiency could be involved in the pathogenesis of acute lung injury in COVID-19, and warrant additional studies to confirm and refine these findings.

Association of Ang/Tie2 pathway mediators with endothelial barrier integrity and disease severity in COVID-19.

Endothelial barrier (EB) disruption contributes to acute lung injury in COVID-19, and levels of both VEGF-A and Ang-2, which are mediators of EB integrity, have been associated with COVID-19 severity. Here we explored the participation of additional mediators of barrier integrity in this process, as well as the potential of serum from COVID-19 patients to induce EB disruption in cell monolayers. In a cohort from a clinical trial consisting of thirty patients with COVID-19 that required hospital admission due to hypoxia we demonstrate that i) levels of soluble Tie2 were increase, and of soluble VE-cadherin were decreased when compared to healthy individuals; ii) sera from these patients induce barrier disruption in monolayers of endothelial cells; and iii) that the magnitude of this effect is proportional to disease severity and to circulating levels of VEGF-A and Ang-2. Our study confirms and extends previous findings on the pathogenesis of acute lung injury in COVID-19, reinforcing the concept that EB is a relevant component of this disease. Our results pave the way for future studies that can refine our understanding of the pathogenesis of acute lung injury in viral respiratory disorders, and contribute to the identification of new biomarkers and therapeutic targets for these conditions.

Association of heme-oxygenase 1, hemopexin, and heme levels with markers of disease severity in COVID-19.

Heme-oxygenase 1 (HO-1) is an enzyme with well-known anti-inflammatory and antioxidant properties, whose levels have been previously associated with disease severity in the context of sterile and infectious diseases. Moreover, the heme/HO-1 pathway has been associated with prothrombotic changes in other diseases. Accordingly, the potential of modulating HO-1 levels for the treatment of COVID-19 was extensively speculated during the COVID-19 pandemic, but very few actual data were generated. The aim of our study was to explore the association of HO-1, heme, and hemopexin (HPX) levels with COVID-19 severity and with markers of inflammation and coagulation activation. The study was conducted in 30 consecutive patients with COVID-19 admitted due to hypoxemia, and 30 healthy volunteers matched by sex, age, and geographic region. HO-1 and HPX levels were measured by enzyme immunoassay (ELISA) and heme levels were measured by a colorimetric method. A comprehensive panel of coagulation and fibrinolysis activation was also used. Patients with COVID-19 presented increased levels of HO-1 when compared to controls (5741 ± 2696 vs 1953 ± 612 pg/mL, respectively, P < 0.0001), as well as a trend toward increased levels of HPX (3.724 ± 0.880 vs 3.254 ± 1.022 mg/mL, respectively; P = 0.06). In addition, HO-1 and HPX levels reduced from admission to day + 4. HO-1 levels were associated with duration of intensive care unit stay and with several markers of coagulation activation. In conclusion, modulation of HO-1 could be associated with the prothrombotic state observed in COVID-19, and HO-1 could also represent a relevant biomarker for COVID-19. New independent studies are warranted to explore and expand these findings.

Changes in Heme Levels During Acute Vaso-occlusive Crisis in Sickle Cell Anemia.

OBJECTIVE/BACKGROUND: Sickle cell anemia (SCA) is associated with increased levels of extracellular heme, which is a key mediator of inflammation in this condition. Despite abundant evidence supporting this concept in cell and animal models, few studies addressed the association between heme levels and the development and severity of acute vasoocclusive crises (VOC) in humans. METHODS: A cross-sectional study was conducted in patients with acute VOC. Total extracellular heme levels were measured in both plasma and serum at admission and after convalescence, and correlated with other clinical and laboratory markers of SCA severity. RESULTS: A total of 28 episodes of VOC in 25 patients were included. Heme levels were similar between admission and convalescence, and correlated with the difference between pre and post hemoglobin, and SCA severity estimated by a composite score of clinical and laboratory markers. Heme levels were neither associated with VOC severity nor with markers of hemostasis activation, and were similar to those reported in an independent population of SCA patients at steady state. DISCUSSION: Acute VOC are not characterized by significant increases in total extracellular heme levels. Studies measuring the fraction of free extracellular heme unbound to proteins are warranted to further refine our understanding of the role of heme in acute VOC.

Contact and intrinsic coagulation pathways are activated and associated with adverse clinical outcomes in COVID-19.

Coagulation activation is a prominent feature of severe acute respiratory syndrome coronavirus 2 (COVID-19) infection. Activation of the contact system and intrinsic pathway has increasingly been implicated in the prothrombotic state observed in both sterile and infectious inflammatory conditions. We therefore sought to assess activation of the contact system and intrinsic pathway in individuals with COVID-19 infection. Baseline plasma levels of protease:serpin complexes indicative of activation of the contact and intrinsic pathways were measured in samples from inpatients with COVID-19 and healthy individuals. Cleaved kininogen, a surrogate for bradykinin release, was measured by enzyme-linked immunosorbent assay, and extrinsic pathway activation was assessed by microvesicle tissue factor-mediated factor Xa (FXa; MVTF) generation. Samples were collected within 24 hours of COVID-19 diagnosis. Thirty patients with COVID-19 and 30 age- and sex-matched controls were enrolled. Contact system and intrinsic pathway activation in COVID-19 was demonstrated by increased plasma levels of FXIIa:C1 esterase inhibitor (C1), kallikrein:C1, FXIa:C1, FXIa:α1-antitrypsin, and FIXa:antithrombin (AT). MVTF levels were also increased in patients with COVID-19. Because FIXa:AT levels were associated with both contact/intrinsic pathway complexes and MVTF, activation of FIX likely occurs through both contact/intrinsic and extrinsic pathways. Among the protease:serpin complexes measured, FIXa:AT complexes were uniquely associated with clinical indices of disease severity, specifically total length of hospitalization, length of intensive care unit stay, and extent of lung computed tomography changes. We conclude that the contact/intrinsic pathway may contribute to the pathogenesis of the prothrombotic state in COVID-19. Larger prospective studies are required to confirm whether FIXa:AT complexes are a clinically useful biomarker of adverse clinical outcomes.

HRT Atlas v1.0 database: redefining human and mouse housekeeping genes and candidate reference transcripts by mining massive RNA-seq datasets.

Housekeeping (HK) genes are constitutively expressed genes that are required for the maintenance of basic cellular functions. Despite their importance in the calibration of gene expression, as well as the understanding of many genomic and evolutionary features, important discrepancies have been observed in studies that previously identified these genes. Here, we present Housekeeping and Reference Transcript Atlas (HRT Atlas v1.0, www.housekeeping.unicamp.br) a web-based database which addresses some of the previously observed limitations in the identification of these genes, and offers a more accurate database of human and mouse HK genes and transcripts. The database was generated by mining massive human and mouse RNA-seq data sets, including 11 281 and 507 high-quality RNA-seq samples from 52 human non-disease tissues/cells and 14 healthy tissues/cells of C57BL/6 wild type mouse, respectively. User can visualize the expression and download lists of 2158 human HK transcripts from 2176 HK genes and 3024 mouse HK transcripts from 3277 mouse HK genes. HRT Atlas also offers the most stable and suitable tissue selective candidate reference transcripts for normalization of qPCR experiments. Specific primers and predicted modifiers of gene expression for some of these HK transcripts are also proposed. HRT Atlas has also been integrated with a regulatory elements resource from Epiregio server.

Endothelial Barrier Integrity Is Disrupted In Vitro by Heme and by Serum From Sickle Cell Disease Patients.

Free extracellular heme has been shown to activate several compartments of innate immunity, acting as a danger-associated molecular pattern (DAMP) in hemolytic diseases. Although localized endothelial barrier (EB) disruption is an important part of inflammation that allows circulating leukocytes to reach inflamed tissues, non-localized/deregulated disruption of the EB can lead to widespread microvascular hyperpermeability and secondary tissue damage. In mouse models of sickle cell disease (SCD), EB disruption has been associated with the development of a form of acute lung injury that closely resembles acute chest syndrome (ACS), and that can be elicited by acute heme infusion. Here we explored the effect of heme on EB integrity using human endothelial cell monolayers, in experimental conditions that include elements that more closely resemble in vivo conditions. EB integrity was assessed by electric cell-substrate impedance sensing in the presence of varying concentrations of heme and sera from SCD patients or healthy volunteers. Heme caused a dose-dependent decrease of the electrical resistance of cell monolayers, consistent with EB disruption, which was confirmed by staining of junction protein VE-cadherin. In addition, sera from SCD patients, but not from healthy volunteers, were also capable to induce EB disruption. Interestingly, these effects were not associated with total heme levels in serum. However, when heme was added to sera from SCD patients, but not from healthy volunteers, EB disruption could be elicited, and this effect was associated with hemopexin serum levels. Together our in vitro studies provide additional support to the concept of heme as a DAMP in hemolytic conditions.

Hypofibrinolysis induced by tranexamic acid does not influence inflammation and mortality in a polymicrobial sepsis model.

The biological relevance of fibrinolysis to the host response to sepsis is illustrated by pathogens such as S. pyogenes and Y. pestis, whose virulence factors are proteins that challenge the balance between pro- and anti-fibrinolytic factors of the host, and by the consistent finding of hypofibrinolysis in the early stages of sepsis. Whether this hypofibrinolytic response is beneficial or detrimental to the host, by containing the spread of pathogens while at the same time limiting the access of immune cell to infectious foci, is still a matter of debate. Tranexamic acid (TnxAc) is an antifibrinolytic agent that is being increasingly used to prevent and control bleeding in conditions such as elective orthopedic surgery, trauma, and post-partum-hemorrhage, which are frequently followed by infection and sepsis. Here we used a model of polymicrobial sepsis to evaluate whether hypofibrinolysis induced by TnxAc influenced survival, tissue injury and pathogen spread. Mice were treated with two doses of TnxAc bid for 48h, and then sepsis was induced by cecal ligation and puncture. Despite the induction of hypofibrinolysis by TnxAc, no difference could be observed in survival, tissue injury (measured by biochemical and histological parameters), cytokine levels or pathogen spread. Our results contribute with a new piece of data to the understanding of the complex interplay between fibrinolysis and innate immunity. While our results do not support the use of TnxAc in sepsis, they also address the thrombotic safety of TnxAc, a low cost and widely used agent to prevent bleeding.

Fratura radicular horizontal – relato de caso / Horizontal root fracture – case report

O objetivo do presente trabalho ‚ relatar um caso de fratura radicular horizontal tratada com Ca(OH) 2 e obturada com MTA. Paciente do sexo feminino, 20 anos, apresentou-se para tratamento queixando-se de dor e discreta mobilidade no dente 21. A paciente relatou ter sofrido uma queda há 2 meses e batido a boca. No exame clínico, observou-se que o dente 21 apresentava discreta mobilidade e alteração de cor, dor moderada na percussão vertical e horizontal, ausência de vitalidade pulpar, aspecto e coloração gengival normais, ausência de deslocamento da porção coronária e coroa do dente hígida. O exame radiográfico revelou fratura horizontal no terço médio desta raiz. Foi realizado o tratamento endodêntico do segmento radicular coronário … linha de fratura, sendo o canal radicular esvaziado, modelado e preenchido com Ca(OH) 2 a 1 mm aquém da linha de fratura. Este foi trocado com intervalos trimestrais até completar 1 ano e 3 meses. Com o auxílio de um microscópio clínico, foi realizado o preenchimento total do segmento radicular coronário … linha de fratura com MTA. Após 20 meses, o dente encontra-se assintomático e, radiograficamente, com presença de lâmina dura e ausência total de infecção. Concluiu-se que o MTA ‚ viável para obturação do remanescente coronário em um caso de fratura do terço médio radicular

The aim of this study was to report a case of horizontal root fracture treated with Ca(OH)2 and filled with MTA. The patient, a 20 year-old woman, presented for treatment complaining of pain and discrete mobility of tooth 21. The patient reported that she hit her mouth on an accidental fall 2 months before. During intra-oral examination it was observed that the tooth 21 presented discrete mobility and color alteration, moderate pain on vertical and horizontal percussion, negative response to vitality test, normal gingival aspect and coloring, no displacement of coronal portion, and the crown was healthy. Radiographic examination showed horizontal fracture in the middle third of this root. Access cavity and chemical-mechanical preparation were performed to 1 mm short of the fracture line, and Ca(OH) 2 was used for intra-canal dressing. Using a clinical microscope, the coronal portion of the canal was filled with MTA. After 20 months the tooth is asymptomatic; radiograph showed complete healing between the fragments with no infection. It was concluded that MTA filling can be an alternative to the routine treatments of horizontal root fracture in the middle third

Effects of light emitting diode (LED) therapy at 940 nm on inflammatory root resorption in rats.

The effects of LED therapy at 940 nm on periodontal healing, inflammatory cell infiltration, and root resorption were analyzed in an experimental model of orthodontic tooth movement in rats. Twenty-five male Wistar rats were allocated into four experimental groups: Control animals (Co, n = 5), Controls + LED therapy (CoLED, n = 6), animals submitted to orthodontic force (RR, n = 7) and submitted to orthodontic force + LED therapy (RRLED, n = 7). All procedures were approved by the Committee for Ethics in Animal Experimentation of the Universidade Estadual de Londrina (protocol CEEA 5/2010 37359). A force of 50 g was applied to the right upper molars of RR and RRLED groups. On days 2, 3, and 4 after orthodontic treatment, the CoLED and RRLED groups received LED irradiation (940 nm, 4 J/cm(2)). The animals were killed on day 7 for histological analysis. An increased number of root resorption lacunae was found only in the RR group (p < 0.05). The RR group also presented more osteoclasts (p < 0.005) and inflammatory cell infiltration (p < 0.005) than the control group. The RRLED group presented fewer osteoclasts (p < 0.005) and inflammatory cells (p < 0.005) in the periodontal ligament than the RR group. The CoLED and RRLED groups presented more periodontal fibroblasts (p < 0.005) than non-irradiated groups. RRLED presented more blood vessels (p < 0.01) in the periodontal ligament than the RR group. In conclusion, the results suggest that LED therapy improved periodontal tissue repair and decreased inflammation and root resorption after the application of orthodontic force.

Effects of light emitting diode (LED) therapy and cold water immersion therapy on exercise-induced muscle damage in rats.

The aim of this work is to analyze the effects of LED therapy at 940 nm or cold water immersion therapy (CWI) after an acute bout of exercise on markers of muscle damage and inflammation. Thirty-two male Wistar rats were allocated into four groups: animals kept at rest (control), exercised animals (E), exercised + CWI (CWI), and exercised + LED therapy (LED). The animals swam for 100 min, after which blood samples were collected for lactate analysis. Animals in the E group were returned to their cages without treatment, the CWI group was placed in cold water (10°C) for 10 min and the LED group received LED irradiation on both gastrocnemius muscles (4 J/cm(2) each). After 24 h, the animals were killed and the soleus muscles were submitted to histological analysis. Blood samples were used for hematological and CK analyses. The results demonstrated that the LED group presented fewer areas of muscle damage and inflammatory cell infiltration and lower levels of CK activity than the E group. Fewer areas of damaged muscle fiber were observed in the LED group than in CWI. CWI and LED did not reduce edema areas. Hematological analysis showed no significant effect of either treatment on leukocyte counts. The results suggest that LED therapy is more efficient than CWI in preventing muscle damage and local inflammation after exercise.

Effects of 940 nm light-emitting diode (led) on sciatic nerve regeneration in rats.

The objective of the present study was to evaluate the effect of 940 nm wavelength light emitting diode (LED) phototherapy on nerve regeneration in rats. Forty male Wistar rats weighing approximately 300 g each were divided into four groups: control (C); control submitted to LED phototherapy (CLed); Sciatic Nerve Lesion without LED phototherapy (L); Sciatic Nerve Lesion with LED phototherapy (LLed). The lesion was caused by crushing the right sciatic nerve. A dose of 4 J/cm(2) was used for ten consecutive days beginning on the first postoperative day. Groups C and L were submitted to the same procedure as the LLed group, but the equipment was turned off. The LED phototherapy with 940 nm wavelength reduced the areas of edema, the number of mononuclear cells present in the inflammatory infiltration, and increased functional recovery scores at 7, 14 and 21 days. The results suggest that the use of phototherapy at 940 nm after nerve damage improves morphofunctional recovery and nerve regeneration.

Efeitos biológicos da luz: aplicação de terapia de baixa potência empregando LEDs (Light Emitting Diode) na cicatrização da úlcera venosa: relato de caso / Biological effects of light: application of low power therapy using LEDs (Light Emitting Diode) on the healing of venous ulcers: a case report

O emprego de fontes de luz de baixa potência, como diodos emissores de luz-LEDs, pode propiciar um recurso terapêutico opcional aos convencionais ou ser utilizado em conjunto com estes, com a vantagem do baixo custo e comprovada eficiência no tratamento de úlceras e outras enfermidades. O estudo de caso foi realizado com um paciente do Ambulatório de Clinicas do HC/UEL que apresenta úlceras nos membros inferiores. A aplicação foi feita uma vez por semana, empregando LEDs com comprimento de onda de 628 nm na úlcera do membro inferior esquerdo e o direito foi utilizado como controle. A evolução foi medida por meio de registro fotográfico, medida da área e mensuração da dor. Foram realizadas 18 sessões. Os resultados apontaram mudanças nas características clínicas da lesão e a área cicatrizada foi 30% maior em relação à úlcera controle. Quanto à dor, a escala análoga visual variou de oito para zero no membro irradiado e de nove para dois no controle. Assim, pode-se concluir que a LED terapia é um recurso de eleição no tratamento da úlcera venosa, nos aspectos cicatriciais e redução da dor.

The use of low power light sources, such as diodes emitters of light – LEDs, provides an alternative and complementary therapeutic resource to the conventional ones, with the advantage of being cost-effective and reliable in the treatment of ulcers and other infirmities. This study was carried out on a patient from the HC/UEL Outpatient Clinic with ulcers on both lower limbs. LEDs were applied once a week, using a wave length of 628 nm on the left lower limb ulcer ,using the right limb as control. Evolution was measured through photographic registers, area size and pain assessment. Eighteen sessions were performed. Results showed that changes were observed on the clinical characteristics of the injury and the healed area was 30% larger when compared to the control. As for the pain assessment, the visual analogue scale varied from eight to zero on the limb radiated and from nine to two on the control. Thus, it can be concluded that the LED therapy is a resource of choice for treating venous ulcer, in both healing and pain reduction aspects.