Scalable production of tissue-like vascularized liver organoids from human PSCs.

The lack of physiological parity between 2D cell culture and in vivo culture has led to the development of more organotypic models, such as organoids. Organoid models have been developed for a number of tissues, including the liver. Current organoid protocols are characterized by a reliance on extracellular matrices (ECMs), patterning in 2D culture, costly growth factors and a lack of cellular diversity, structure, and organization. Current hepatic organoid models are generally simplistic and composed of hepatocytes or cholangiocytes, rendering them less physiologically relevant compared to native tissue. We have developed an approach that does not require 2D patterning, is ECM independent, and employs small molecules to mimic embryonic liver development that produces large quantities of liver-like organoids. Using single-cell RNA sequencing and immunofluorescence, we demonstrate a liver-like cellular repertoire, a higher order cellular complexity, presenting with vascular luminal structures, and a population of resident macrophages: Kupffer cells. The organoids exhibit key liver functions, including drug metabolism, serum protein production, urea synthesis and coagulation factor production, with preserved post-translational modifications such as N-glycosylation and functionality. The organoids can be transplanted and maintained long term in mice producing human albumin. The organoids exhibit a complex cellular repertoire reflective of the organ and have de novo vascularization and liver-like function. These characteristics are a prerequisite for many applications from cellular therapy, tissue engineering, drug toxicity assessment, and disease modeling to basic developmental biology.

"iPSC-derived liver organoids and inherited bleeding disorders: Potential and future perspectives".

The bleeding phenotype of hereditary coagulation disorders is caused by the low or undetectable activity of the proteins involved in hemostasis, due to a broad spectrum of genetic alterations. Most of the affected coagulation factors are produced in the liver. Therefore, two-dimensional (2D) cultures of primary human hepatocytes and recombinant overexpression of the factors in non-human cell lines have been primarily used to mimic disease pathogenesis and as a model for innovative therapeutic strategies. However, neither human nor animal cells fully represent the hepatocellular biology and do not harbor the exact genetic background of the patient. As a result, the inability of the current in vitro models in recapitulating the in vivo situation has limited the studies of these inherited coagulation disorders. Induced Pluripotent Stem Cell (iPSC) technology offers a possible solution to overcome these limitations by reprogramming patient somatic cells into an embryonic-like pluripotent state, thus giving the possibility of generating an unlimited number of liver cells needed for modeling or therapeutic purposes. By combining this potential and the recent advances in the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology, it allows for the generation of autologous and gene corrected liver cells in the form of three-dimensional (3D) liver organoids. The organoids recapitulate cellular composition and organization of the liver, providing a more physiological model to study the biology of coagulation proteins and modeling hereditary coagulation disorders. This advanced methodology can pave the way for the development of cell-based therapeutic approaches to treat inherited coagulation disorders. In this review we will explore the use of liver organoids as a state-of-the-art methodology for modeling coagulation factors disorders and the possibilities of using organoid technology to treat the disease.

Molecular Characterization of Two Homozygous Factor VII Variants Associated with Intracranial Bleeding.

Clinical parameters have been extensively studied in factor (F) VII deficiency, but the knowledge of molecular mechanisms of this disease is scarce. We report on three probands with intracranial bleeds at an early age, one of which had concomitant high titer of FVII inhibitor. The aim of the present study was to identify the causative mutations and to elucidate the underlying molecular mechanisms. All nine F7 exons were sequenced in the probands and the closest family members. A homozygous deletion in exon 1, leading to a frame shift and generation of a premature stop codon (p.C10Pfs*16), was found in proband 1. Probands 2 and 3 (siblings) were homozygous for a missense mutation in exon 8, resulting in a glycine (G) to arginine (R) substitution at amino acid 240 (p.G240R). All probands had severely reduced FVII activity (FVII:C < 1 IU/dL). Treatment consisted of recombinant FVIIa and/or plasma concentrate, and proband 1 developed a FVII inhibitor shortly after initiation of treatment. The FVII variants were overexpressed in mammalian cell lines. No FVII protein was produced in cells expressing the p.C10Pfs*16 variant, and the inhibitor development in proband 1 was likely linked to the complete absence of circulating FVII. Structural analysis suggested that the G to R substitution in FVII found in probands 2 and 3 would destabilize the protein structure, and cell studies demonstrated a defective intracellular transport and increased endoplasmic reticulum stress. The molecular mechanism underlying the p.G240R variant could be reduced secretion caused by protein destabilization and misfolding.

The effect of the chemical chaperone 4-phenylbutyrate on secretion and activity of the p.Q160R missense variant of coagulation factor FVII.

BACKGROUND: Congenital coagulation factor (F) VII deficiency is a rare bleeding disorder caused by mutations in the F7 gene. The missense factor FVII variant p.Q160R is the disease-causing mutation in all Norwegian FVII deficient patients and results in reduced biological activity and antigen levels of FVII in patient plasma. Previous in vitro studies on this variant demonstrated impaired intracellular trafficking and reduced secretion, possibly due to protein misfolding. The aim of the study was therefore to assess the impact of chemical chaperones on cellular processing and secretion of this variant using a cell model based on overexpression of the recombinant protein. RESULTS: Through screening of compounds, we identified 4-phenylbutyrate (4-PBA) to increase the secretion of recombinant (r) FVII-160R by ~ 2.5-fold. Additionally, treatment with 4-PBA resulted in a modest increase in specific biological activity. Intracellular localization studies revealed that upon treatment with 4-PBA, rFVII-160R was secreted through Golgi and Golgi reassembly-stacking protein (GRASP)-structures. CONCLUSIONS: The present study demonstrates that the chemical chaperone 4-PBA, restores intracellular trafficking and increases the secretion of a missense FVII variant with functional properties in the extrinsic coagulation pathway.

Activation of Endoplasmic Reticulum Stress and Unfolded Protein Response in Congenital Factor VII Deficiency.

Congenital factor (F) VII deficiency is a bleeding disorder caused by a heterogeneous pattern of mutations in the F7 gene. Protein misfolding due to mutations is a strong candidate mechanism to produce the highly represented type I FVII deficiency forms, characterized by a concomitant deficiency of FVII antigen and activity. Misfolded proteins can accumulate within the endoplasmic reticulum (ER) causing ER stress with subsequent activation of the unfolded protein response (UPR). So far, there are limited data on this important issue in FVII deficiency. In this study, we chose as candidate FVII model mutations, the p.Q160R, p.I289del and p.A354V-p.P464Hfs, which are all associated with severe to moderate type I FVII deficiency. In vitro expression of the recombinant (r) mutants rFVII-160R, rFVII-289del or rFVII-354V-464Hfs, which are characterized by either amino acid substitution, deletion, or by an extended carboxyl terminus, demonstrated inefficient secretion of the mutant proteins, probably caused by intracellular retention and association with ER chaperones. Both ER stress and UPR were activated following expression of all FVII mutants, with the highest response for rFVII-289del and rFVII-354V-464Hfs. These data unravel new knowledge on pathogenic mechanisms leading to FVII deficiency, and support the investigation of pharmaceutical modulators of ER stress and UPR as therapeutic agents.

Factor VII deficiency: Unveiling the cellular and molecular mechanisms underlying three model alterations of the enzyme catalytic domain.

Activated factor (F) VII is a vitamin K-dependent glycoprotein that initiates blood coagulation upon interaction with tissue factor. FVII deficiency is the most common of the rare congenital bleeding disorders. While the mutational pattern has been extensively characterized, the pathogenic molecular mechanisms of mutations, particularly at the intracellular level, have been poorly defined. Here, we aimed at elucidating the mechanisms underlying altered FVII biosynthesis in the presence of three mutation types in the catalytic domain: a missense change, a microdeletion and a frameshift/elongation, associated with severe or moderate to severe phenotypes. Using CHO-K1 cells transiently transfected with expression vectors containing the wild-type FVII cDNA (FVIIwt) or harboring the p.I289del, p.G420V or p.A354V-p.P464Hfs mutations, we found that the secretion of the FVII mutants was severely decreased compared to FVIIwt. The synthesis rate of the mutants was slower than the FVIIwt and delayed, and no degradation of the FVII mutants by proteasomes, lysosomes or cysteine proteases was observed. Confocal immunofluorescence microscopy studies showed that FVII variants were localized into the endoplasmic reticulum (ER) but were not detectable within the Golgi apparatus. These findings suggested that a common pathogenic mechanism, possibly a defective folding of the mutant proteins, was triggered by the FVII mutations. The misfolded state led to impaired trafficking of these proteins causing ER retention, which would explain the low to very low FVII plasma levels observed in patients carrying these mutations.

The chemical chaperone sodium 4-phenylbutyrate improves the secretion of the protein CA267T mutant in CHO-K1 cells trough the GRASP55 pathway.

Some inherited coagulation factor deficiencies are caused by intracellular retention or degradation of misfolded proteins, and chemical chaperones have been shown to reverse protein misfolding. The purpose of the present study was to investigate whether chemical chaperones may improve secretion of the protein CA267T (PCA267T) mutant in a cellular model. Using stably transfected Chinese hamster ovary cells (CHO-K1) expressing PCA267T we demonstrate that sodium 4-phenylbutyrate (PBA) increased the secretion of PCA267T by approximately 4-fold in comparison with untreated cells, and that this secretion seemed to follow an unconventional pathway via the Golgi reassembly stacking protein (GRASP55).

The nuclear hormone receptor PPARγ counteracts vascular calcification by inhibiting Wnt5a signalling in vascular smooth muscle cells.

Vascular calcification is a hallmark of advanced atherosclerosis. Here we show that deletion of the nuclear receptor PPARγ in vascular smooth muscle cells of low density lipoprotein receptor (LDLr)-deficient mice fed an atherogenic diet high in cholesterol, accelerates vascular calcification with chondrogenic metaplasia within the lesions. Vascular calcification in the absence of PPARγ requires expression of the transmembrane receptor LDLr-related protein-1 in vascular smooth muscle cells. LDLr-related protein-1 promotes a previously unknown Wnt5a-dependent prochondrogenic pathway. We show that PPARγ protects against vascular calcification by inducing the expression of secreted frizzled-related protein-2, which functions as a Wnt5a antagonist. Targeting this signalling pathway may have clinical implications in the context of common complications of atherosclerosis, including coronary artery calcification and valvular sclerosis.

Central nervous system paracoccidioidomycosis: case report and review.

Paracoccidioidomycosis is a systemic infection caused by a dimorphic fungus (Paracoccidioides brasiliensis). The most common lesions frequently occur in the bucopharinx mucosa. Other lesions occur in the adrenal glands, liver, bone, gastrointestinal tract, lungs and nervous system. We report here a case of neuroparacoccidioidomycosis. The patient was a 49 year-old male, who consulted due to neurological symptoms (cephalalgia, speech difficulty and one tonic clonic seizure with urinary incontinence) of eight months duration. Upon physical examination it was observed an emaciated male with nail clubbing, a skin ulcer with raised edges and a crusted bottom of 4 x 2 cm in diameter located in the right supraclavicular region and an ulcerated lesion in the left tonsil with edema. The rest of the physical examination reveled a discrete left side hemiparesis and pulmonary rales in the left hemitorax. The fungus was identified through direct examination of cerebrospinal fluid (CSF). The histopathology of suprarenal, lungs, brain and skin showed multiple paracoccidioidal granulomas. To the best of our knowledge, this is the third case reported in the literature. We review the literature on the pathogenesis and prevalence of neuroparacoccidioidomycosis.

Central nervous system paracoccidioidomycosis: case report and review

Paracoccidioidomycosis is a systemic infection caused by a dimorphic fungus (Paracoccidioides brasiliensis). The most common lesions frequently occur in the bucopharinx mucosa. Other lesions occur in the adrenal glands, liver, bone, gastrointestinal tract, lungs and nervous system. We report here a case of neuroparacoccidioidomycosis. The patient was a 49 year-old male, who consulted due to neurological symptoms (cephalalgia, speech difficulty and one tonic clonic seizure with urinary incontinence) of eight months duration. Upon physical examination it was observed an emaciated male with nail clubbing, a skin ulcer with raised edges and a crusted bottom of 4 × 2 cm in diameter located in the right supraclavicular region and an ulcerated lesion in the left tonsil with edema. The rest of the physical examination reveled a discrete left side hemiparesis and pulmonary rales in the left hemitorax. The fungus was identified through direct examination of cerebrospinal fluid (CSF). The histopathology of suprarenal, lungs, brain and skin showed multiple paracoccidioidal granulomas. To the best of our knowledge, this is the third case reported in the literature. We review the literature on the pathogenesis and prevalence of neuroparacoccidioidomycosis

Actividad de la telomerasa en leucocitos de sangre periférica de pacientes con hipertensión arterial esencial / Telomerase activity in peripheral blood leucocytes from patients with essential hypertension

FUNDAMENTO Y OBJETIVO: La activación primaria o secundaria de mecanismos inmunes, que lleva a una disfunción y/o proliferación de poblaciones celulares específicas, se ha implicado en la patogenia y/o complicaciones de la hipertensión arterial esencial. En vista de que la longitud de los telómeros determina el período de vida de una célula y que se han observado alteraciones replicativas en los leucocitos de sangre periférica de pacientes con hipertensión arterial esencial, investigamos la relación entre la actividad de la telomerasa en los leucocitos de sangre periférica, como un indicador del acortamiento telomérico, y la presencia de hipertensión arterial. PACIENTES Y MÉTODO: Determinamos la actividad de la telomerasa en leucocitos de sangre periférica aislados de controles normales, pacientes con hipertensión esencial controlada y no controlada. Los leucocios de sangre periférica se aislaron por gradiente de densidad, se lisaron y su ADN se amplificó por el método de reacción en cadena de la polimerasa (PCR). La actividad de la telomerasa se midió por un ELISA específico. RESULTADOS: El recuento de leucocitos fue significativamente mayor en los pacientes con hipertensión esencial en comparación con el de los individuos sanos (p < 0,05). La actividad de la telomerasa fue significativamente mayor en pacientes menores de 45 años con hipertensión no controlada, comparada con la de los individuos sanos y con los pacientes menores de 45 años con hipertensión controlada (p < 0,05). En este último grupo, la actividad de la telomerasa fue significativamente menor con respecto a los otros dos (p < 0,05). CONCLUSIONES: Nuestros resultados indican que existe una relación entre la actividad de la telomerasa en los leucocitos de sangre periférica, la proliferación de estos leucocitos y la presencia de hipertensión arterial esencial (AU)

[Mucormycosis. Report of 3 cases]. / Mucormicosis. Reporte de tres casos.

Mucormycosis is an acute and often fatal infection caused by a fungus of the Mucorales order of the Zygomycetes class. There are various clinical types, usually associated with an underlying disorder. The rhinocerebral mucormycosis is usually seen in diabetics, especially in ketoacidosis, but may also appear in healthy people. We report three cases of mucormycosis diagnosed since april 1987 through january 2001 at the Dr. Domingo Luciani Hospital, Caracas, Venezuela. Two of them had diabetes and one was apparently healthy. The most common clinical presentation of mucormycosis was the rhinocerebral infection, seen in the two diabetic patients, both of them had cavernous sinus thrombosis one with involvement of the carotid artery. The other patient with sinus involvement had no predisposing factors. All patients were treated with amphotericin B and two of them had surgical debridement of involved tissue. We emphasize the importance of an early clinical diagnosis and treatment with surgical debridement of infected tissue combined with amphotericin B.

Mucormicosis. Reporte de tres casos / Mucormycosis. Report of 3 cases

Mucormycosis is an acute and often fatal infection caused by a fungus of the Mucorales order of the Zygomycetes class. There are various clinical types, usually associated with an underlying disorder. The rhinocerebral mucormycosis is usually seen in diabetics, especially in ketoacidosis, but may also appear in healthy people. We report three cases of mucormycosis diagnosed since april 1987 through january 2001 at the Dr. Domingo Luciani Hospital, Caracas, Venezuela. Two of them had diabetes and one was apparently healthy. The most common clinical presentation of mucormycosis was the rhinocerebral infection, seen in the two diabetic patients, both of them had cavernous sinus thrombosis one with involvement of the carotid artery. The other patient with sinus involvement had no predisposing factors. All patients were treated with amphotericin B and two of them had surgical debridement of involved tissue. We emphasize the importance of an early clinical diagnosis and treatment with surgical debridement of infected tissue combined with amphotericin B.

Absceso esplénico: reporte de 5 casos y revisión de la literatura / Splenic abscess: report of 5 cases and review of the literature

El absceso esplénico es una enfermedad poco común a menudo de dificil diagnóstico y tratamiento. Estudios de autopsias han revelado una incidencia de 0.14 a 0.7 por ciento, sin embargo ésta se ha incrementado en los últimos años debido al avance y el uso de los procedimientos diagnósticos. Así como por la mayor frecuencia de estados de inmunosupresión en esta década. Se realizó un estudio retrospectivo de revisión de historias clínicas con diagnóstico de absceso esplenico en el período comprendido entre abril de 1987 y marzo del 2001, en el Hospital "Dr. Domingo Luciani" Caracas. Venezuela. Se encontraron 5 casos registrados con este diagnóstico, se obtuvieron datos como edad, género, motivo de consulta, enfermedades asociadas y tratamiento recibido. El motivo de consulta en todos los pacientes fue dolor abdominal y fiebre, y en tres de ellos se asoció, además, sintomatología respiratoria, lo cual coincide con lo reportado en la literatura en donde las manifestaciones clínicas iniciales de esta enfermedad son fiebre, dolor abdominal, en ocasiones acompañada de alteraciones en el exámen físico pulmonar. Enfermedades asociadas estuvieron presentes en los cinco pacientes, dos con hemoglobinopatías, uno con inmunodeficiencia común variable, uno con diabetes mellitus tipo 2, y el paciente restante con cardiopatía hipertensiva. En cuatro de los cinco pacientes el diagnóstico se corroboró por ultrasonografía. Se aisló en hemocultivos seriados Staphylococus Coagulasa Negativa en 3 pacientes y Staphylococus Aureos en un paciente. El tratamiento definitivo fue la esplenectomía junto con la antibioticoterapia, a excepción de un paciente con lesión única y de pequeño tamaño que sólo recibió tratamiento médico. En conclusión los abscesos son una infección infrecuente, generalmente en pacietes con factores o enfermedades predisponentes. Los métodos radiodiagnósticos son útiles para el reconocimiento de esta patología. El drenaje y la esplenectomía en unión con la antibioticoterapia indicada son cardinales en el tratamiento de esta afección.

Tuberculosis pulmonar y renal diagnósticadas por el método de reacción en cadena de la polimerasa (PCR) en sangre y orina / Diagnosis of kidney and lung tuberculosis by the polymerase chain reaction (PCR) in blood and urine

Se describe el caso de un paciente con TBC pulmonar diagnósticada clínicamente y por métodos radiológicos, con alta sospecha de TBC renal, no se identificó la micobacteria en la muestra de esputo ni de orina por los métodos tradicionales y se logró utilizando la reacción en cadena de la polimerasa (PCR) en muestras de esputo y sangre. Se enviaron al Laboratorio de Biología Molecular de Agentes Infecciosos en el Instituto de Medicina Experimental (IME) de la UCV, 2 muestras de sangre y 3 muestras de orina de la mañana para ser procesadas. Se aíslo DNA de las muestras clínicas utilizando los protocolos descritos por Brisson-Noel y col, y por Boom y col. Se logró amplificar un segmento de 245 pb (resultado positivo) a partir de DNA aislado de la muestra de sangre y de la tercera muestra de orina. Un desarrollo dramático en la detección directa e identificación del M.tuberculosis ha resultado de métodos que utilizan técnicas de amplificación de ácidos nucleicos como la PCR. Estas técnicas permiten la amplificación de secuencias específicas de ácidos nucleicos que posteriormente pueden ser detectados

Paracoccidioidomicosis: experiencia en el Hospital Dr. Domingo Luciani / Paracoccidioidomycosis: experence in the Hospital Dr. Domingo Luciani

Se realizó un estudio retrospectivo de revisión de historia clínicas con diagnóstico de paracoccidioidomicosis, en el Hospital Dr. Domingo Luciani, desde febrero de 1987 hasta enero del 2000. Se extrajeron los siguientes datos: edad, género, lugar de nacimiento, profesión, procedencia, motivo de consulta, tiempo transcurrido entre el inicio de los síntomas al momento de la consulta, enfermedades asociadas, estado nutricional, localización de las lesiones, tratamiento. Cada uno de estos parámetros fue asociado con la evolución de la enfermedad y se realizó una clasificación según la localización de las lesiones. Se encontraron treinta casos, veintiocho del género masculino, edad promedio de 48,8 años, 96,7 por ciento procedentes de la región central; el motivo de consulta más frecuente fue el momento de volumen en la región cervical. El estado nutricional fue determinado en 40 por ciento de los pacientes y el 15 por ciento tenía IMC<20. Treinta y seis por ciento de los pacientes tenía enfermedad asociada. La forma clínica más común fue la crónica multifocal del adulto. Los pacientes con la forma unifocal tegumentaria recibieron tratamiento con azoles evolucionando de manera satisfactoria. Aquellos con la forma clínica crónica multifocal fueron tratados con combinación de Anfotericina B y derivados imidazolínicos, no obteniéndose buenos resultados en 29,8 por ciento de los casos. Los síntomas neurológicos, como motivo de consulta y la forma crónica multifocal del adulto, con afección pulmonar, tuvieron peor pronóstico. Todos los pacientes con afección del sistema nervioso central fallecieron