Factors Affecting the Evaluation of Collagen Deposition and Fibrosis In Vitro.

Immune responses to biomedical implants, wound healing, and diseased tissues often involve collagen deposition by fibroblasts and other stromal cells. Dysregulated collagen deposition can lead to complications, such as biomaterial fibrosis, cardiac fibrosis, desmoplasia, liver fibrosis, and pulmonary fibrosis, which can ultimately result in losses of organ function or failure of biomedical implants. Current in vitro methods to induce collagen deposition include growing the cells under macromolecular crowding conditions or on fibronectin-coated surfaces. However, the majority of these methods have been demonstrated with a single cell line, and the combined impacts of culture conditions and postculture processing on collagen deposition have not been explored in detail. In this work, the effects of macromolecular crowding versus fibronectin coating, fixation with methanol versus fixation with paraformaldehyde, and use of plastic substrates versus glass substrates were evaluated using the WI-38 human lung fibroblast cell line. Fibronectin coating was found to provide enhanced collagen deposition under macromolecular crowding conditions, while a higher plating density led to improved collagen I deposition compared with macromolecular crowding. Collagen deposition was found to be more apparent on plastic substrates than on glass substrates. The effects of primary cells versus cell lines, and mouse cells versus human cells, were evaluated using WI-38 cells, primary human lung fibroblasts, primary human dermal fibroblasts, primary mouse lung fibroblasts, primary mouse dermal fibroblasts, and the L929 mouse fibroblast cell line. Cell lines exhibited enhanced collagen I deposition compared with primary cells. Furthermore, collagen deposition was quantified with picrosirius red staining, and plate-based drug screening through picrosirius red staining of decellularized extracellular matrices was demonstrated. The results of this study provide detailed conditions under which collagen deposition can be induced in vitro in multiple cell types, with applications including material development, development of potential antifibrotic therapies, and mechanistic investigation of disease pathways. Impact Statement This study demonstrated the effects of cell type, biological conditions, fixative, culture substrate, and staining method on in vitro collagen deposition and visualization. Further the utility of plate-based picrosirius red staining of decellularized extracellular matrices for drug screening through collagen quantification was demonstrated. These results should provide clarity and a path forward for researchers who aim to conduct in vitro experiments on collagen deposition.

Dynamics of SARS-CoV-2 Seroprevalence in a Large US population Over a Period of 12 Months.

Due to a combination of asymptomatic or undiagnosed infections, the proportion of the United States population infected with SARS-CoV-2 was unclear from the beginning of the pandemic. We previously established a platform to screen for SARS-CoV-2 positivity across a representative proportion of the US population, from which we reported that almost 17 million Americans were estimated to have had undocumented infections in the Spring of 2020. Since then, vaccine rollout and prevalence of different SARS-CoV-2 variants have further altered seropositivity trends within the United States population. To explore the longitudinal impacts of the pandemic and vaccine responses on seropositivity, we re-enrolled participants from our baseline study in a 6- and 12- month follow-up study to develop a longitudinal antibody profile capable of representing seropositivity within the United States during a critical period just prior to and during the initiation of vaccine rollout. Initial measurements showed that, since July 2020, seropositivity elevated within this population from 4.8% at baseline to 36.2% and 89.3% at 6 and 12 months, respectively. We also evaluated nucleocapsid seropositivity and compared to spike seropositivity to identify trends in infection versus vaccination relative to baseline. These data serve as a window into a critical timeframe within the COVID-19 pandemic response and serve as a resource that could be used in subsequent respiratory illness outbreaks.

Development of a High-Color Flow Cytometry Panel for Immunologic Analysis of Tissue Injury and Reconstruction in a Rat Model.

The rat model is an important resource in biomedical research due to its similarities to the human immune system and its use for functional studies. However, because of the preponderance of mouse models in foundational and mechanistic immunological studies, there is a relative lack of diverse, commercially available flow cytometry antibodies for immunological profiling in the rat model. Available antibodies are often conjugated to common fluorophores with similar peak emission wavelengths, making them hard to distinguish on conventional flow cytometers and restricting more comprehensive immune analysis. This can become a limitation when designing immunological studies in rat injury models to investigate the immune response to tissue injury. In addition, this lack of available antibodies limits the number of studies that can be done on the immune populations in lymphoid organs in other research areas. To address this critical unmet need, we designed a spectral flow cytometry panel for rat models. Spectral cytometry distinguishes between different fluorophores by capturing their full emission spectra instead of their peak emission wavelengths. This flow cytometry panel includes 24 distinct immune cell markers to analyze the innate and adaptive immune response. Importantly, this panel identifies different immune phenotypes, including tolerogenic, Type 1, and Type 2 immune responses. We show that this panel can identify unique immune populations and phenotypes in a rat muscle trauma model. We further validated that the panel can identify distinct adaptive and innate immune populations and their unique phenotypes in lymphoid organs. This panel expands the scope of previous rat panels providing a tool for scientists to examine the immune system in homeostasis and injury while pairing mechanistic immunological studies with functional studies.

Age-dependent characterization of carotid and cerebral artery geometries in a transgenic mouse model of sickle cell anemia using ultrasound and microcomputed tomography.

To define morphological changes in carotid and cerebral arteries in sickle cell transgenic mice (SS) as they age, a combination of ultrasound and microcomputed tomography of plastinated arteries was used to quantify arterial dimensions and changes in mice 4, 12, and 24 weeks of age. 12-week SS mice had significantly larger common carotid artery diameters than AS mice, which continued through to the extracranial and intracranial portions of the internal carotid artery (ICA). There were also side specific differences in diameters between the left and right vessels. Significant ICA tapering along its length occurred by 12- and 24-weeks in SS mice, decreasing by as much as 70%. Significant narrowing along the length was also measured in SS anterior cerebral arteries at 12- and 24-weeks, but not AS. Collectively, these findings indicate that sickle cell anemia induces arterial remodeling in 12- and 24-weeks old mice. Catalog of measurements are also provided for the common carotid, internal carotid, anterior cerebral, and middle cerebral arteries for AS and SS genotypes, as a reference for other investigators using mathematical and computational models of age-dependent arterial complications caused by sickle cell anemia.

Microbial indicators and molecular markers used to differentiate the source of faecal pollution in the Bogotá River (Colombia).

Intestinal pathogenic microorganisms are introduced into the water by means of faecal contamination, thus creating a threat to public health and to the environment. Detecting these contaminants has been difficult due to such an analysis being costly and time-intensive; as an alternative, microbiological indicators have been used for this purpose, although they cannot differentiate between human or animal sources of contamination because these indicators are part of the digestive tracts of both. To identify the sources of faecal pollution, the use of chemical, microbiological and molecular markers has been proposed. Currently available markers present some geographical specificity. The aim of this study was to select microbial and molecular markers that could be used to differentiate the sources of faecal pollution in the Bogotá River and to use them as tools for the evaluation and identification of the origin of discharges and for quality control of the water. In addition to existing microbial source markers, a phage host strain (PZ8) that differentiates porcine contamination was isolated from porcine intestinal content. The strain was identified biochemically and genotypically as Bacteroides. The use of this strain as a microbial source tracking indicator was evaluated in bovine and porcine slaughterhouse wastewaters, raw municipal wastewaters and the Bogotá River. The results obtained indicate that the selected microbial and molecular markers enable the determination of the source of faecal contamination in the Bogotá River by using different algorithms to develop prediction models.

Epidemiology of Spotted Fever Group Rickettsioses and Acute Undifferentiated Febrile Illness in Villeta, Colombia.

Etiology of acute undifferentiated febrile syndrome (AUFS) is often unknown, leading to inaccurate diagnosis and treatment. Villeta town has been identified as an endemic area for spotted fever group (SFG) rickettsioses but little is known about possible amplifier hosts and other Rickettsia species different from Rickettsia rickettsii. Besides, few studies have approached other AUFS etiologies in the region. We investigated the role of dengue, leptospirosis, rickettsioses, human anaplasmosis, and Q fever as possible causes of AUFS in patients from Villeta. Sera specimens and ticks from animals as well as ticks from vegetation were studied for the presence of different Rickettsia spp. Among 104 sera from patients with AUFS, 16.4%, 24.0%, and 2.9% patients seroconverted to dengue, Leptospira, and SFG Rickettsia, respectively, with a case of probable coinfection or cross-reaction with Anaplasma phagocytophilum. None of the samples were reactive for Coxiella burnetii. Sera samples from 74 horses, 118 dogs, and 62 bovines were collected and showed 33.8%, 14.4%, and 50.0% of seroprevalence for SFG Rickettsia, respectively. A total of 1,287 ixodid ticks were collected from animals/vegetation and processed in pools for polymerase chain reaction. Among them, 1.7% was positive for Rickettsia genes, and Rickettsia amblyommii, R. rickettsii, and Rickettsia spp. were found. These results confirm the circulation of dengue, different SFG Rickettsia species and the relevance of other etiologies like leptospirosis and human anaplasmosis. Further studies must identify different epidemiological variables to establish proper surveillance and control programs.

The characterization of trans-pecos copperhead (Agkistrodon contortrix pictigaster) venom and isolation of two new dimeric disintegrins.

The vast amounts of toxins within the venom of snakes, while known to cause medical emergencies, display various biological functions. Trans-pecos copperhead (Agkistrodon contortrix pictigaster) crude venom separated by cation-exchange chromatography showed several fractions with fibrinolytic, hemorrhagic, gelatinase and platelet activities. Venom fractions 1, 2, 4, 5, and 12-17 contained fibrinolytic activity. Venom fractions 1, 2, 5 and 12-14 had hemorrhagic activity. Fractions 1, 2, 12, 13 and 17 contained gelatinase activity. Reverse-Phase C18 High Performance Liquid Chromatography was also used to purify and isolated disintegrins from this venom. Anti-platelet aggregation activity of the C18 fractions collected and performed on whole human blood showed that they inhibited platelet aggregation in presence of several agonists. Results from both SDS-PAGE and N-terminal sequencing determined that pictistatin 1 obtained from the Trans-Pecos copperhead venom was a dimeric disintegrin, and pictistatin 2 was a heterodimeric disintegrin. The molecules with anti-platelet activity could be considered in the development of more effective drugs, for numerous blood-related diseases such as stroke, heart attacks, thrombosis, and other medical conditions. In this study, we are presenting the first report of the purification, isolation, and partial characterization of two new dimeric disintegrins isolated from the venom of trans-pecos copperhead.

Biological and biochemical characterization of venom from the broad-banded copperhead (Agkistrodon contortrix laticinctus): isolation of two new dimeric disintegrins.

Disintegrins represent a family of effective cell-cell and cell-matrix inhibitors by binding to integrin receptors. Integrins are heterodimeric, transmembrane receptors that are the bridges for these cell interactions. Disintegrins have been shown to have many therapeutic implications for the treatment of strokes, heart attacks, and cancer. Two novel heterodimeric disintegrins were isolated from the venom of the broad-banded copperhead (Agkistrodon contortrix laticinctus). Crude venom separated by cation-exchange chromatography resulted in several fractions possessing hemorrhagic, fibrinolytic, gelatinase, and platelet activities. Venom fractions 2-3 and 17-19 showed fibrinolytic activity. Fractions 2-6, 8-11, and 16-21 had hemorrhagic activity. Gelatinase activity was found in fractions 3, 11, and 19. The isolation of laticinstatins 1 and 2 was accomplished by fractionating crude venom using reverse phase chromatography. Data from both SDS-PAGE and N-terminal sequencing determined that laticinstatins 1 and 2 were heterodimeric disintegrins, and both were assayed for their ability to inhibit platelet aggregation in human whole blood. Future functional evaluation of snake venom disintegrins shows considerable promise for elucidating the biochemical mechanisms of integrin-ligand interactions that will allow the development of adequate medications for hemostatic pathologies such as thrombosis, stroke, and cerebral and cardiac accidents. In this study, we are presenting the first report of the purification, and partial characterization of two new dimeric disintegrins isolated from the venom of broad-banded copperhead snakes.

Conductas de salud y factores de riesgo en la salud sexual y reproductiva de una población universitaria / Health behaviors and risk factors in sexual and reproductive health of a university population

Este estudio, iniciado en el año 2008, tuvo como objetivo describir los factores de riesgo y las conductas de la salud sexual y reproductiva de una población universitaria. Se realizó un estudio observacional de corte transversal, con una muestra conformada por 491 estudiantes de una universidad de Bogotá. Los resultados muestran que el género predominante fue el femenino (63,1%,), con una edad promedio de 20,4±2,6 años; el 80% inició su actividad sexual y la edad entre los 15 y los 18 años. El método anticonceptivo más usado es el preservativo y el 37,9% tiene conocimientos escasos sobre anticoncepción. La prevalencia de infecciones de transmisión sexual es baja, pero sólo la mitad han recibido tratamiento. El 10% de la población se ha realizado la prueba para el virus de inmunodeficiencia humana. El 10% de la población ha presentado embarazos, de los cuales el 75,3% no han sido planeados y el 47,4% han terminado en aborto, de los cuales el 80% han sido inducidos. Hay cifras altas de antecedentes familiares de cáncer. El 84% de los hombres no se realizan el auto examen de testículo. En las mujeres, el 56% se realiza auto examen de seno. La prevalencia de citologías es baja (38%), no todas han reclamado el resultado y aquellas que presentaron anormalidades (78%) no recibieron tratamiento. El 86,9% de los estudiantes desean que la universidad desarrolle el programa de salud sexual y reproductiva; el medio preferido es la consulta personalizada y la pagina web con asesoría en línea. El análisis de los resultados permite anotar que los jóvenes requieren programas de promoción de la salud que conlleven a que éstos asuman su sexualidad con autonomía, responsabilidad e información adecuada, en el marco de sus derechos sexuales y reproductivos.