Fused Cells between Human-Adipose-Derived Mesenchymal Stem Cells and Monocytes Keep Stemness Properties and Acquire High Mobility.

Human-adipose-derived mesenchymal stem cells (hADMSCs) are multipotent stem cells which have become of great interest in stem-cell therapy due to their less invasive isolation. However, they have limited migration and short lifespans. Therefore, understanding the mechanisms by which these cells could migrate is of critical importance for regenerative medicine. Methods: Looking for novel alternatives, herein, hADMSCs were isolated from adipose tissue and co-cultured with human monocytes ex vivo. Results: A new fused hybrid entity, a foam hybrid cell (FHC), which was CD90+CD14+, resulted from this co-culture and was observed to have enhanced motility, proliferation, immunomodulation properties, and maintained stemness features. Conclusions: Our study demonstrates the generation of a new hybrid cellular population that could provide migration advantages to MSCs, while at the same time maintaining stemness properties.

Modulation of endothelial function by Toll like receptors.

Endothelial cells (EC) are able to actively control vascular permeability, coagulation, blood pressure and angiogenesis. Most recently, a role for endothelial cells in the immune response has been described. Therefore, the endothelium has a dual role controlling homeostasis but also being the first line for host defence and tissue damage repair thanks to its ability to mount an inflammatory response. Endothelial cells have been shown to express pattern-recognition receptors (PRR) including Toll-like receptors (TLR) that are activated in response to stimuli within the bloodstream including pathogens and damage signals. TLRs are strategic mediators of the immune response in endothelial cells but they also regulate the angiogenic process critical for tissue repair. Nevertheless, endothelial activation and angiogenesis can contribute to some pathologies. Thus, inappropriate endothelial activation, also known as endothelial dysfunction, through TLRs contributes to tissue damage during autoimmune and inflammatory diseases such as atherosclerosis, hypertension, ischemia and diabetes associated cardiovascular diseases. Also TLR induced angiogenesis is required for the growth of some tumors, atherosclerosis and rheumatoid arthritis, among others. In this review we discuss the importance of various TLRs in modulating the activation of endothelial cells and their importance in immunity to infection and vascular disease as well as their potential as therapeutic targets.

Construction of a Mycoviral Infectious Clone for Reverse Genetics in Botrytis cinerea.

The most important advances in our understanding of the viral life cycle, such as genome replication, packaging, transmission, and host interactions, have been made via the development of viral infectious full-length clones. Here, we describe the detailed protocols for the construction of an infectious clone derived from Botrytis virus F (BVF), a mycoflexivirus infecting the plant pathogenic fungus Botrytis cinerea, the determination of the complete sequence of the cloned mycovirus, the preparation of fungal protoplasts, and the transfection of protoplasts using transcripts derived from the BVF infectious clone.

Usefulness of a low-cost simulation model for teaching internal manual aortic compression. A survey-based mannequin evaluation.

OBJECTIVE: To evaluate the users' opinion on internal manual aortic compression (IMAC) training, using a low-cost simulation model. METHODS: An educational strategy was designed to teach IMAC, which included: (1) guided reading of educational material and viewing an explanatory video of IMAC; (2) an introductory lecture with the anatomical considerations, documentation of the cessation of femoral arterial flow during IMAC, and real clinical cases in which this procedure was used; and (3) simulated practice of IMAC with a new low-cost manikin. The educational strategy was applied during three postpartum hemorrhage workshops in three Latin American countries and the opinions of the participants were measured with a survey. RESULTS: Almost all of the participants in the IMAC workshop, including the simulation with the low-cost mannikin, highlighted the usefulness of the strategy (scores of 4/5 and 5/5 on the Likert scale) and would recommend it to colleagues. CONCLUSION: We present a low-cost simulation model for IMAC as the basis of an educational strategy perceived as very useful by most participants. The execution of this strategy in other populations and its impact on postpartum hemorrhage management should be evaluated in further studies.

Complete genome sequence of hepatitis E virus from rabbits in the United States.

Hepatitis E virus (HEV) is a single-strand positive-sense RNA virus in the family Hepeviridae. The disease caused by HEV, hepatitis E, is an important public health problem in developing countries of Asia and Africa and is also endemic in many industrialized countries, including the United States. HEV has been identified from several other animal species in addition to humans, including the pig, chicken, mongoose, deer, rabbit, ferret, bat, and fish. Here we report the complete genome sequence of the first strain of HEV from rabbits in the United States. Sequence and phylogenetic analyses revealed that the U.S. rabbit HEV is a distant member of the zoonotic genotype 3 HEV, thus raising a concern for potential zoonotic human infection. A unique 90-nucleotide insertion within the X domain of the ORF1 was identified in the rabbit HEV, and this insertion may play a role in the species tropism of HEV.

Rescue of a genotype 4 human hepatitis E virus from cloned cDNA and characterization of intergenotypic chimeric viruses in cultured human liver cells and in pigs.

Hepatitis E virus (HEV) is an important but extremely understudied human pathogen. Genotypes 1 and 2 are restricted to humans, whereas genotypes 3 and 4 are zoonotic, infecting both humans and pigs. This report describes, for the first time, the successful rescue of infectious HEV in vitro and in vivo from cloned cDNA of a genotype 4 human HEV (strain TW6196E). The complete genomic sequence of the TW6196E virus was determined and a full-length cDNA clone (pHEV-4TW) was assembled. Capped RNA transcripts from the pHEV-4TW clone were replication competent in Huh7 cells and infectious in HepG2/C3A cells. Pigs inoculated intrahepatically with capped RNA transcripts from pHEV-4TW developed an active infection, as evidenced by faecal virus shedding and seroconversion, indicating the successful rescue of infectious genotype 4 HEV and cross-species infection of pigs by a genotype 4 human HEV. To demonstrate the utility of the genotype 4 HEV infectious clone and to evaluate the potential viral determinant(s) for species tropism, four intergenotypic chimeric clones were constructed by swapping various genomic regions between genotypes 1 and 4, and genotypes 1 and 3. All four chimeric clones were replication competent in Huh7 cells, but only the two chimeras with sequences swapped between genotypes 1 and 4 human HEVs produced viruses capable of infecting HepG2/C3A cells. None of the four chimeras was able to establish a robust infection in pigs. The availability of a genotype 4 HEV infectious clone affords an opportunity to delineate the molecular mechanisms of HEV cross-species infection in the future.

Cross-species infection of pigs with a novel rabbit, but not rat, strain of hepatitis E virus isolated in the United States.

Hepatitis E virus (HEV) is an important human pathogen. In addition to humans, HEV has also been identified in pig, chicken, mongoose, deer, rat, rabbit and fish. There are four recognized and two putative genotypes of mammalian HEV. Genotypes 1 and 2 are restricted to humans, while genotypes 3 and 4 are zoonotic. The recently identified rabbit HEV is a distant member of genotype 3. Here, we first expressed and purified the recombinant capsid protein of rabbit HEV and showed that the capsid protein of rabbit HEV cross-reacted with antibodies raised against avian, rat, swine and human HEV. Conversely, we showed that antibodies against rabbit HEV cross-reacted with capsid proteins derived from chicken, rat, swine and human HEV. Since pigs are the natural host of genotype 3 HEV, we then determined if rabbit HEV infects pigs. Twenty pigs were divided into five groups of four each and intravenously inoculated with PBS, US rabbit HEV, Chinese rabbit HEV, US rat HEV and swine HEV, respectively. Results showed that only half of the pigs inoculated with rabbit HEV had low levels of viraemia and faecal virus shedding, indicative of active but not robust HEV infection. Infection of pigs by rabbit HEV was further verified by transmission of the virus recovered from pig faeces to naïve rabbits. Pigs inoculated with rat HEV showed no evidence of infection. Preliminary results suggest that rabbit HEV is antigenically related to other HEV strains and infects pigs and that rat HEV failed to infect pigs.

Mutational analysis of the hypervariable region of hepatitis e virus reveals its involvement in the efficiency of viral RNA replication.

The RNA genome of the hepatitis E virus (HEV) contains a hypervariable region (HVR) in ORF1 that tolerates small deletions with respect to infectivity. To further investigate the role of the HVR in HEV replication, we constructed a panel of mutants with overlapping deletions in the N-terminal, central, and C-terminal regions of the HVR by using a genotype 1 human HEV luciferase replicon and analyzed the effects of deletions on viral RNA replication in Huh7 cells. We found that the replication levels of the HVR deletion mutants were markedly reduced in Huh7 cells, suggesting a role of the HVR in viral replication efficiency. To further verify the results, we constructed HVR deletion mutants by using a genetically divergent, nonmammalian avian HEV, and similar effects on viral replication efficiency were observed when the avian HEV mutants were tested in LMH cells. Furthermore, the impact of complete HVR deletion on virus infectivity was tested in chickens, using an avian HEV mutant with a complete HVR deletion. Although the deletion mutant was still replication competent in LMH cells, the complete HVR deletion resulted in a loss of avian HEV infectivity in chickens. Since the HVR exhibits extensive variations in sequence and length among different HEV genotypes, we further examined the interchangeability of HVRs and demonstrated that HVR sequences are functionally exchangeable between HEV genotypes with regard to viral replication and infectivity in vitro, although genotype-specific HVR differences in replication efficiency were observed. The results showed that although the HVR tolerates small deletions with regard to infectivity, it may interact with viral and host factors to modulate the efficiency of HEV replication.

Three amino acid mutations (F51L, T59A, and S390L) in the capsid protein of the hepatitis E virus collectively contribute to virus attenuation.

Hepatitis E virus (HEV) is an important but extremely understudied human pathogen, and the mechanisms of HEV replication and pathogenesis are largely unknown. We previously identified an attenuated genotype 3 HEV mutant (pSHEV-1) containing three unique amino acid mutations (F51L, T59A, and S390L) in the capsid protein. To determine the role of each of these mutations, we constructed three HEV single mutants (rF51L, rT59A, and rS390L) which were all found to be replication competent in Huh7 liver cells. To determine the pathogenicities of the mutants, we utilized the specific-pathogen-free (SPF) pig model for HEV and a unique inoculation procedure that bypasses the need for propagating infectious HEV in vitro. A total of 60 pigs were intrahepatically inoculated, via an ultrasound-guided technique, with in vitro-transcribed full-length capped RNA transcripts from the infectious clones of each single mutant, the pSHEV-1 triple mutant, wild-type pSHEV-3, or phosphate-buffered saline (PBS) buffer (n = 10). The results showed that the F51L mutation partially contributed to virus attenuation, whereas the T59A and S390L mutations resulted in more drastic attenuation of HEV in pigs, as evidenced by a significantly lower incidence of viremia, a delayed appearance and shorter duration of fecal virus shedding and viremia, and lower viral loads in liver, bile, and intestinal content collected at three different necropsy times. The results indicate that the three mutations in the capsid protein collectively contribute to HEV attenuation. This study has important implications for developing a modified live-attenuated vaccine against HEV.

Construction and Characterization of a Botrytis Virus F Infectious Clone.

Botrytis virus F (BVF) is a positive-sense, single-stranded RNA (+ssRNA) virus within the Gammaflexiviridae family of the plant-pathogenic fungus Botrytis cinerea. In this study, the complete sequence of a BVF strain isolated from B. cinerea collected from grapevine fields in Spain was analyzed. This virus, in this work BVF-V448, has a genome of 6827 nt in length, excluding the poly(A) tail, with two open reading frames encoding an RNA dependent RNA polymerase (RdRP) and a coat protein (CP). The 5'- and 3'-terminal regions of the genome were determined by rapid amplification of cDNA ends (RACE). Furthermore, a yet undetected subgenomic RNA species in BVF-V448 was identified, indicating that the CP is expressed via 3' coterminal subgenomic RNAs (sgRNAs). We also report the successful construction of the first BVF full-length cDNA clone and synthesized in vitro RNA transcripts using the T7 polymerase, which could efficiently transfect two different strains of B. cinerea, B05.10 and Pi258.9. The levels of growth in culture and virulence on plants of BVF-V448 transfected strains were comparable to BVF-free strains. The infectious clones generated in this work provide a useful tool for the future development of an efficient BVF foreign gene expression vector and a virus-induced gene silencing (VIGS) vector as a biological agent for the control of B. cinerea.

Overcoming tumor resistance mechanisms in CAR-NK cell therapy.

Despite the impressive results of autologous CAR-T cell therapy in refractory B lymphoproliferative diseases, CAR-NK immunotherapy emerges as a safer, faster, and cost-effective approach with no signs of severe toxicities as described for CAR-T cells. Permanently scrutinized for its efficacy, recent promising data in CAR-NK clinical trials point out the achievement of deep, high-quality responses, thus confirming its potential clinical use. Although CAR-NK cell therapy is not significantly affected by the loss or downregulation of its CAR tumor target, as in the case of CAR-T cell, a plethora of common additional tumor intrinsic or extrinsic mechanisms that could also disable NK cell function have been described. Therefore, considering lessons learned from CAR-T cell therapy, the emergence of CAR-NK cell therapy resistance can also be envisioned. In this review we highlight the processes that could be involved in its development, focusing on cytokine addiction and potential fratricide during manufacturing, poor tumor trafficking, exhaustion within the tumor microenvironment (TME), and NK cell short in vivo persistence on account of the limited expansion, replicative senescence, and rejection by patient's immune system after lymphodepletion recovery. Finally, we outline new actively explored alternatives to overcome these resistance mechanisms, with a special emphasis on CRISPR/Cas9 mediated genetic engineering approaches, a promising platform to optimize CAR-NK cell function to eradicate refractory cancers.

Colorectal Cancer Stem Cells Fuse with Monocytes to Form Tumour Hybrid Cells with the Ability to Migrate and Evade the Immune System.

BACKGROUND: The cancer cell fusion theory could be one of the best explanations for the metastasis from primary tumours. METHODS: Herein, we co-cultured colorectal cancer (CRC) stem cells with human monocytes and analysed the properties of the generated tumour hybrid cells (THCs). The presence of THCs in the bloodstream together with samples from primary and metastatic lesions and their clinical correlations were evaluated in CRC patients and were detected by both FACS and immunofluorescence methods. Additionally, the role of SIGLEC5 as an immune evasion molecule in colorectal cancer was evaluated. RESULTS: Our data demonstrated the generation of THCs after the in vitro co-culture of CRC stem cells and monocytes. These cells, defined as CD45+CD14+EpCAM+, showed enhanced migratory and proliferative abilities. The THC-specific cell surface signature allows identification in matched primary tumour tissues and metastases as well as in the bloodstream from patients with CRC, thus functioning as a biomarker. Moreover, SIG-LEC5 expression on in vitro generated THCs has shown to be involved in the mechanism for immune evasion. Additionally, sSIGLEC5 levels correlated with THC numbers in the prospective cohort of patients. CONCLUSIONS: Our results indicate the generation of a hybrid entity after the in vitro co-culture between CRC stem cells and human monocytes. Moreover, THC numbers present in patients are related to both prognosis and the later spread of metastases in CRC patients.

Hepatitis E virus in rabbits, Virginia, USA.

We identified hepatitis E virus (HEV) in rabbits in Virginia, USA. HEV RNA was detected in 14 (16%) of 85 serum samples and 13 (15%) of 85 fecal samples. Antibodies against HEV were detected in 31 (36%) of 85 serum samples. Sequence analyses showed that HEV from rabbits is closely related to genotype 3.

Zn-Mg and Zn-Cu alloys for stenting applications: From nanoscale mechanical characterization to in vitro degradation and biocompatibility.

In the recent decades, zinc (Zn) and its alloys have been drawing attention as promising candidates for bioresorbable cardiovascular stents due to its degradation rate more suitable than magnesium (Mg) and iron (Fe) alloys. However, its mechanical properties need to be improved in order to meet the criteria for vascular stents. This work investigates the mechanical properties, biodegradability and biocompatibility of Zn-Mg and Zn-Cu alloys in order to determine a proper alloy composition for optimal stent performance. Nanoindentation measurements are performed to characterize the mechanical properties at the nanoscale as a function of the Zn microstructure variations induced by alloying. The biodegradation mechanisms are discussed and correlated to microstructure, mechanical performance and bacterial/cell response. Addition of Mg or Cu alloying elements refined the microstructure of Zn and enhanced yield strength (YS) and ultimate tensile strength (UTS) proportional to the volume fraction of secondary phases. Zn-1Mg showed the higher YS and UTS and better performance in terms of degradation stability in Hanks' solution. Zn-Cu alloys presented an antibacterial effect for S. aureus controlled by diffusion mechanisms and by contact. Biocompatibility was dependent on the degradation rate and the nature of the corrosion products.

Chagas disease reactivation in rheumatologic patients: association with immunosuppressive therapy and humoral response.

Evidence for Chagas disease reactivation (CDR) in rheumatologic patients under rheumatologic treatments (RTs) is scarce. To screen and follow-up patients with rheumatic diseases and concomitant Chagas disease under RT to detect CDR and to describe a possible relationship between CDR and specific RT. An observational, longitudinal, prospective, consecutive study was carried out between 2018 and 2020. Included patients were evaluated during the follow-up for clinical and laboratorial manifestations of CDR. Direct blood parasitological examination (Strout method) and polymerase chain reaction (PCR) were employed to diagnose CDR. The dynamic of anti-T. cruzi-specific antibodies was also assessed by IHA and ELISA (total IgG and Anti-SAPA). Fifty-one patients were included (86% women). Rheumatoid arthritis was the predominant disease (57%). Classic DMARDs (86.3%) and corticosteroids (61%) were the most frequent RT. CDR was developed in 6 patients (11.7%), exhibiting both positive Strout and PCR. Symptomatic reactivation of CD (fever, asthenia, arthralgias, myalgias) occurred in two patients who had previously been diagnosed with it. Regardless of the different RT, all patients who experienced CDR had previously received more than ≥ 20 mg/day of prednisone equivalent. Despite immunosuppression, patients with CDR exhibited increased levels of specific anti-T. cruzi and anti-SAPA antibodies, which decreased after anti-parasitic treatment. CDR is possible in rheumatologic patients, especially after receiving high doses of corticosteroids. Since CDR symptoms may mimic rheumatic disease activity, monitoring of Chagas disease is highly recommended before, during and after immunosuppression. Key Points • Chagas disease reactivation (CDR) in the context of rheumatological treatment was associated to high doses of corticosteroids. • CDR was associated with an increase in anti-T. cruzi antibodies despite the immunosuppressive treatment. • Suspecting and anticipating CDR is mandatory in this patient population to diagnose and treat it.

NKG2D-CAR-transduced natural killer cells efficiently target multiple myeloma.

CAR-T-cell therapy against MM currently shows promising results, but usually with serious toxicities. CAR-NK cells may exert less toxicity when redirected against resistant myeloma cells. CARs can be designed through the use of receptors, such as NKG2D, which recognizes a wide range of ligands to provide broad target specificity. Here, we test this approach by analyzing the antitumor activity of activated and expanded NK cells (NKAE) and CD45RA- T cells from MM patients that were engineered to express an NKG2D-based CAR. NKAE cells were cultured with irradiated Clone9.mbIL21 cells. Then, cells were transduced with an NKG2D-4-1BB-CD3z-CAR. CAR-NKAE cells exhibited no evidence of genetic abnormalities. Although memory T cells were more stably transduced, CAR-NKAE cells exhibited greater in vitro cytotoxicity against MM cells, while showing minimal activity against healthy cells. In vivo, CAR-NKAE cells mediated highly efficient abrogation of MM growth, and 25% of the treated mice remained disease free. Overall, these results demonstrate that it is feasible to modify autologous NKAE cells from MM patients to safely express a NKG2D-CAR. Additionally, autologous CAR-NKAE cells display enhanced antimyeloma activity demonstrating that they could be an effective strategy against MM supporting the development of NKG2D-CAR-NK-cell therapy for MM.

Brain manifestations secondary to auricular myxoma.

Cardiac myxoma is the most common benign tumor of the heart. In most cases, it is in the left atrium, which can generate neurological embolization; that can manifest as an ischemic event, aneurysm formation and less frequently as brain metastases. This is a case report of a 56-year-old male patient with cerebral embolisms secondary to a left cardiac myxoma. In these patients, the role of neurologic imaging is to detect the firsts complications and avoid secondary complications.

Bi-layered silane-TiO2/collagen coating to control biodegradation and biointegration of Mg alloys.

Magnesium alloys have shown high potential as biodegradable implants for bone repair applications. However, their fast degradation in physiological media demands tuning their corrosion rate to accompany the natural tissue healing processes. Here, a new bi-layered silane-TiO2/collagen coating efficient in stabilizing and biofunctionalizing the surface of AZ31 and ZE41 Mg alloys is presented. Corrosion tests performed in cell culture medium over 7 weeks showed that the bi-layered coating promotes the formation of a stable layer of Mg(OH)2/MgCO3/CaCO3 that provides effective protection to the alloys at advanced immersion stages. The intrinsic reactivity of each alloy plus formation of transitory calcium phosphate phases, resulted in distinct corrosion behavior in the short term. Cell experiments showed that the bi-layered coating improved osteoblasts and fibroblasts proliferation compared to bare and silane-TiO2-coated alloys. Different responses in terms of cell adhesion could be related to the intrinsic corrosion rate of each alloy and some toxicity from the alloying elements. The results evidenced the complex interplay between alloy nature, coating-alloy combination and cell type. The silane-TiO2/collagen coating showed to be a promising strategy to improve cell response and viability and to control degradation rate of Mg alloys in the long term.