Continuous multi-membrane chromatography of large viral particles.

Continuous multi-column chromatography (CMCC) has been successfully implemented to address biopharmaceutical biomolecule instability, to improve process efficiency, and to reduce facility footprint and capital cost. This paper explores the implementation of a continuous multi-membrane chromatography (CMMC) process, using four membrane units, for a large viral particle in just few weeks. CMMC improves the efficiency of the chromatography step by enabling higher loads with smaller membranes for multiple cycles of column use and enables steady-state continuous bioprocessing. The separation performance of CMMC was compared to a conventional batch chromatographic capture step used at full manufacturing scale. The product step yield was 80% using CMMC versus 65% in batch mode while increasing slightly the relative purity. Furthermore, the total amount of membrane area required for the CMMC approach was approximately 10% of the area needed for batch operation, while realizing similar processing times. Since CMMC uses smaller membrane sizes, it can take advantage of the high flow rates achievable for membrane chromatography that are not typically possible at larger membrane scales due to skid flow rate limitations. As such, CMMC offers the potential for more efficient and cost-effective purification trains.

Design and In Situ Validation of Low-Cost and Easy to Apply Anti-Biofouling Techniques for Oceanographic Continuous Monitoring with Optical Instruments.

Biofouling is the major factor that limits long-term monitoring studies with automated optical instruments. Protection of the sensing areas, surfaces, and structural housing of the sensors must be considered to deliver reliable data without the need for cleaning or maintenance. In this work, we present the design and field validation of different techniques for biofouling protection based on different housing materials, biocides, and transparent coatings. Six optical turbidity probes were built using polylactic acid (PLA), acrylonitrile butadiene styrene (ABS), PLA with copper filament, ABS coated with PDMS, ABS coated with epoxy and ABS assembled with a system for in situ chlorine production. The probes were deployed in the sea for 48 days and their anti-biofouling efficiency was evaluated using the results of the field experiment, visual inspections, and calibration signal loss after the tests. The PLA and ABS were used as samplers without fouling protection. The probe with chlorine production outperformed the other techniques, providing reliable data during the in situ experiment. The copper probe had lower performance but still retarded the biological growth. The techniques based on transparent coatings, epoxy, and PDMS did not prevent biofilm formation and suffered mostly from micro-biofouling.

Central memory T cells are the most effective precursors of resident memory T cells in human skin.

The circulating precursor cells that give rise to human resident memory T cells (TRM) are poorly characterized. We used an in vitro differentiation system and human skin-grafted mice to study TRM generation from circulating human memory T cell subsets. In vitro TRM differentiation was associated with functional changes, including enhanced IL-17A production and FOXP3 expression in CD4+ T cells and granzyme B production in CD8+ T cells, changes that mirrored the phenotype of T cells in healthy human skin. Effector memory T cells (TEM) had the highest conversion rate to TRM in vitro and in vivo, but central memory T cells (TCM) persisted longer in the circulation, entered the skin in larger numbers, and generated increased numbers of TRM. In summary, TCM are highly efficient precursors of human skin TRM, a feature that may underlie their known association with effective long-term immunity.

Presence of Skin Tissue-Resident Memory T Cells in Human Nonmalignant and Premalignant Melanocytic Skin Lesions and in Melanoma.

ABSTRACT: The infiltration of tissue-resident memory (TRM) cells in melanoma correlates with improved survival, suggesting an important role for TRM cells in immunity against melanoma. However, little is known about the presence of TRM cells in nonmalignant and premalignant melanocytic lesions. This study aimed to evaluate the presence of TRM cells in human skin melanocytic lesions, representing the spectrum from healthy skin to metastatic melanoma. FFPE sections from healthy skin, sun-exposed skin, benign nevi, lentigo maligna (LM), primary LM melanoma, and primary cutaneous and metastatic melanoma were analyzed by immunohistochemistry. The number of infiltrating cells expressing TRM-associated markers, CD3, CD4, CD8, CD69, CD103, and CD49a, was quantified by digital analyses. Multiplex immunofluorescence was performed to analyze coexpression of TRM cell markers. More T cells and CD69+ cells were found in melanoma lesions, as compared with healthy skin and nevi. CD103+ and CD49a+ cell numbers did not significantly differ. More importantly, no differences were seen in expression of all markers between healthy skin and benign nevi. Similar results, except for CD69, were observed in LM melanoma, as compared with LM and sun-exposed skin. Interestingly, multiplex immunofluorescence showed that nevi tissues have comparable CD103+ T cell numbers with healthy skin but comprise more CD103+ CD8+ cells. Expression of TRM cell markers is significantly increased in melanoma, as compared with nonmalignant skin. Our data also show that TRM cells are not abundantly present already in premalignant tissues. Further studies on the specificity of TRM cells for melanocyte/melanoma antigens may reveal their significance in cancer immunosurveillance.

Underwater Energy Harvesting to Extend Operation Time of Submersible Sensors.

A linear electromagnetic energy harvesting device for underwater applications, fabricated with a simple manufacturing process, was developed to operate with movement frequencies from 0.1 to 0.4 Hz. The generator has two coils, and the effect of the combination of the two coils was investigated. The experimental study has shown that the energy capture system was able to supply energy to several ocean sensors, producing 7.77 mJ per second with wave movements at 0.4 Hz. This study shows that this energy is enough to restore the energy used by the battery or the capacitor and continue supplying energy to the sensors used in the experimental work. For an ocean wave frequency of 0.4 Hz, the generator can supply power to 8 sensors or 48 sensors, depending on the energy consumed and its optimization.

Labeling and tracking of immune cells in ex vivo human skin.

Human skin harbors various immune cells that are crucial for the control of injury and infection. However, the current understanding of immune cell function within viable human skin tissue is limited. We developed an ex vivo imaging approach in which fresh skin biopsies are mounted and then labeled with nanobodies or antibodies against cell surface markers on tissue-resident memory CD8+ T cells, other immune cells of interest, or extracellular tissue components. Subsequent longitudinal imaging allows one to describe the dynamic behavior of human skin-resident cells in situ. In addition, this strategy can be used to study immune cell function in murine skin. The ability to follow the spatiotemporal behavior of CD8+ T cells and other immune cells in skin, including their response to immune stimuli, provides a platform to investigate physiological immune cell behavior and immune cell behavior in skin diseases. The mounting, staining and imaging of skin samples requires ~1.5 d, and subsequent tracking analysis requires a minimum of 1 d. The optional production of fluorescently labeled nanobodies takes ~5 d.

Peripheral host T cells survive hematopoietic stem cell transplantation and promote graft-versus-host disease.

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in hematopoietic stem cell transplantation (HSCT). Donor T cells are key mediators in pathogenesis, but a contribution from host T cells has not been explored, as conditioning regimens are believed to deplete host T cells. To evaluate a potential role for host T cells in GVHD, the origin of skin and blood T cells was assessed prospectively in patients after HSCT in the absence of GVHD. While blood contained primarily donor-derived T cells, most T cells in the skin were host derived. We next examined patient skin, colon, and blood during acute GVHD. Host T cells were present in all skin and colon acute GVHD specimens studied, yet were largely absent in blood. We observed acute skin GVHD in the presence of 100% host T cells. Analysis demonstrated that a subset of host T cells in peripheral tissues were proliferating (Ki67+) and producing the proinflammatory cytokines IFN-γ and IL-17 in situ. Comparatively, the majority of antigen-presenting cells (APCs) in tissue in acute GVHD were donor derived, and donor-derived APCs were observed directly adjacent to host T cells. A humanized mouse model demonstrated that host skin-resident T cells could be activated by donor monocytes to generate a GVHD-like dermatitis. Thus, host tissue-resident T cells may play a previously unappreciated pathogenic role in acute GVHD.

Author Correction: Tissue patrol by resident memory CD8+ T cells in human skin.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Maturation and Phenotypic Heterogeneity of Human CD4+ Regulatory T Cells From Birth to Adulthood and After Allogeneic Stem Cell Transplantation.

CD4+ Regulatory T cells (Treg) play a critical role in maintaining immune homeostasis. Various Treg subsets have been identified, however the heterogeneity of Treg subpopulations during development remains uncharacterized. Using mass cytometry we obtained single cell data on expression of 35 functional markers to examine the heterogeneity of Treg cells at birth and in adults. Unsupervised clustering algorithms FlowSOM and ACCENSE were used to quantify Treg heterogeneity. As expected, Treg in umbilical cord blood were predominately naïve while Treg in adult blood were predominately central memory and effector memory cells. Although umbilical cord blood Treg are mostly naïve cells, we observed multiple phenotypic Treg subsets in cord blood. Nevertheless, peripheral blood in adults contained higher percentages of Treg and the heterogeneity of Treg was significantly increased in adults. We also studied Treg heterogeneity throughout a 2-year period after allogeneic hematopoietic stem cell transplantation (alloHSCT) and in patients with chronic graft-versus-host disease (cGVHD). Treg heterogeneity recovered rapidly after alloHSCT and gradually increased in the first two years post-transplant. However, patients with cGVHD had significantly fewer distinct Treg subpopulations, proposing a correlation between a disrupted Treg heterogeneity and cGVHD. Our study is the first to compare human Treg heterogeneity at birth, in healthy adults and in patients after alloHSCT with and without cGVHD. This approach to characterize Treg heterogeneity based on expression of a large panel of functional markers may enable future studies to identify specific Treg defects that contribute to immune dysfunction.

Eficácia da técnica de breath stacking na função respiratória em mulheres submetidas a cirurgia bariátrica / Effectiveness of the breath-stacking technique in the respiratory function of women undergoing bariatric surgery / Eficacia de la técnica de breath stacking sobre la función respiratoria en mujeres sometidas a cirugía bariátrica

Enquadramento: A cirurgia abdominal alta está associada a alta incidência de complicações pulmonares. A técnica breath stacking (BS) perspetiva-se como recurso com potencial terapêutico para a enfermagem de reabilitação. Objetivo: Avaliar a eficácia da técnica BS na melhoria da função respiratória. Metodologia: Estudo quasi-experimental com 36 mulheres submetidas a cirurgia bariátrica, distribuídas pelos grupos de controlo (GC) e intervenção (GI). Foram avaliadas no pré e pós-operatório as medidas capacidade vital forçada (CVF), volume expiratório forçado no primeiro segundo (VEF1), pressão inspiratória máxima (PIm), pressão expiratória máxima (PEm), saturação periférica de oxigénio (SpO2) e frequência respiratória (FR). Foi aplicada a técnica BS no GI no pré e pós-operatório. Recorreu-se à análise estatística descritiva e inferencial. Resultados: Observaram-se diferenças estatisticamente significativas pós-operatórias, entre o GC e o GI, na CVF ( -20,29 vs. -13,60), VEF1 (-23,05 vs. -13,38), PIm (-22,96 vs. -14,93), PEm (-14,10 vs -10,32) e FR (12,29 vs 6,45). Conclusão: A técnica de BS permitiu melhorar a função respiratória do GI e reverter as alterações previsíveis no pós-operatório de cirurgia bariátrica.

Background: Upper abdominal surgery is associated with a high incidence of pulmonary complications. The breath-stacking (BS) technique is considered a resource with therapeutic potential for rehabilitation nursing. Objective: To assess the effectiveness of the BS technique in improving the respiratory function. Methodology: Quasi-experimental study with 36 women undergoing bariatric surgery, distributed into a control group (CG) and an intervention group (IG). The forced vital capacity (FVC), the forced expiratory volume in one second (FEV1), the maximal inspiratory pressure (MIP), the maximal expiratory pressure (MEP), the peripheral oxygen saturation (SpO2), and the respiratory rate (RR) were evaluated in the pre and postoperative periods. The BS technique was applied to the IG in the pre and postoperative periods. Both descriptive and inferential statistics were used. Results: Statistically significant postoperative differences were observed between the CG and the IG in FVC (-20.29 vs. -13.60), FEV1 (-23.05 vs. -13.38), MIP (-22.96 vs. -14.93), MEP (-14.10 vs. -10.32), and RF (12.29 vs. 6.45). Conclusion: The BS technique improved the respiratory function of the IG and reversed the predictable changes in the postoperative period of bariatric surgery.

Marco contextual: La cirugía abdominal alta se asocia con una elevada incidencia de complicaciones pulmonares. La técnica de breath stacking (BS) se considera un recurso con potencial terapéutico para la enfermería de rehabilitación. Objetivo: Evaluar la efectividad de la técnica de BS para mejorar la función respiratoria. Metodología: Estudio cuasi-experimental con 36 mujeres sometidas a cirugía bariátrica, distribuidas entre el grupo de control (GC) y el de intervención (GI). Las mediciones de la capacidad vital forzada (CVF), el volumen espiratorio forzado en el primer segundo (VEF1), la presión inspiratoria máxima (PIm), la presión espiratoria máxima (PEm), la saturación periférica de oxigeno (SpO2) y la frecuencia respiratoria (FR) se evaluaron GI que en el GC antes y después de la cirugía. La técnica BS se aplicó en el GI antes y después de la cirugía. Se utilizó un análisis estadístico descriptivo e inferencial. Resultados: Se observaron diferencias posoperatorias estadísticamente significativas entre el GC y el GI en la CVF (-20,29 frente a -13,60), VEF1 (-23,05 frente a -13,38), PIm (-22,96 frente a -14,93), PEm (-14,10 frente a -10,32) y FR (12,29 frente a 6,45). Conclusión: La técnica BS permitió mejorar la función respiratoria del GI y revertir los cambios predecibles en el posoperatorio de cirugía bariátrica.

Erratum: Martins, M.S., et al. Wideband and Wide Beam Polyvinylidene Difluoride (PVDF) Acoustic Transducer for Broadband Underwater. Sensors 2019, 19, 3991.

The authors wish to make the following erratum to this paper [...].

Wideband and Wide Beam Polyvinylidene Difluoride (PVDF) Acoustic Transducer for Broadband Underwater Communications.

The advances in wireless communications are still very limited when intended to be used on Underwater Communication Systems mainly due to the adverse proprieties of the submarine channel to the acoustic and radio frequency (RF) waves propagation. This work describes the development and characterization of a polyvinylidene difluoride ultrasound transducer to be used as an emitter in underwater wireless communications. The transducer has a beam up to 10° × 70° degrees and a usable frequency band up to 1 MHz. The transducer was designed using Finite Elements Methods and compared with real measurements. Pool trials show a transmitting voltage response (TVR) of approximately 150 dB re µPa/V@1 m from 750 kHz to 1 MHz. Sea trials were carried in Ria Formosa, Faro (Portugal) over a 15 m source-receiver communication link. All the signals were successfully detected by cross-correlation using 10 chirp signals between 10 to 900 kHz.

Skin-resident memory T cells as a potential new therapeutic target in vitiligo and melanoma.

Tissue-resident memory T (TRM ) cells are abundant in the memory T cell pool and remain resident in peripheral tissues, such as the skin, where they act as alarm sensors or cytotoxic killers. TRM cells persist long after the pathogen is eliminated and can respond rapidly upon reinfection with the same antigen. When aberrantly activated, skin-located TRM cells have a profound role in various skin disorders, including vitiligo and melanoma. Autoreactive TRM cells are present in human lesional vitiligo skin and mouse models of vitiligo, which suggests that targeting these cells could be effective as a durable treatment strategy for vitiligo. Furthermore, emerging evidence indicates that induction of melanoma-reactive TRM cells is needed to achieve effective protection against tumor growth. This review highlights seminal reports about skin-resident T cells, focusing mainly on their role in the context of vitiligo and melanoma, as well as their potential as therapeutic targets in both diseases.

Tissue patrol by resident memory CD8+ T cells in human skin.

Emerging data show that tissue-resident memory T (TRM) cells play an important protective role at murine and human barrier sites. TRM cells in the epidermis of mouse skin patrol their surroundings and rapidly respond when antigens are encountered. However, whether a similar migratory behavior is performed by human TRM cells is unclear, as technology to longitudinally follow them in situ has been lacking. To address this issue, we developed an ex vivo culture system to label and track T cells in fresh skin samples. We validated this system by comparing in vivo and ex vivo properties of murine TRM cells. Using nanobody labeling, we subsequently demonstrated in human ex vivo skin that CD8+ TRM cells migrated through the papillary dermis and the epidermis, below sessile Langerhans cells. Collectively, this work allows the dynamic study of resident immune cells in human skin and provides evidence of tissue patrol by human CD8+ TRM cells.

Separation of Nucleic Acids Using Single- and Multimodal Chromatography.

The needs for purified nucleic acids for preparative and analytical applications have increased constantly, demanding for the development of new and more efficient methods for their recovery and isolation. DNA molecules harbour some intrinsic chemical properties that render them suitable for chromatographic separations. These include a negatively charged phosphate backbone as well as a hydrophobic character originating mainly from the major groove of DNA which exposes the base pairs on the surface of the molecule. In addition, single stranded DNA often allows for a free exposure of the hydrophobic aromatic bases. In this review, multimodal chromatography (MMC) has been evaluated as an alternative tool for complex separations of nucleic acids. MMC embraces more than one kind of interaction between the chromatographic ligand and the target molecules. These resins have often proved superior to conventional single-mode chromatographic materials for DNA isolation, including, e.g., the purification of plasmid DNA from crude cell lysates and for the preparation of DNA fragments before or after a polymerase chain reaction (PCR).

Phase I/II Trial of a Combination of Anti-CD3/CD7 Immunotoxins for Steroid-Refractory Acute Graft-versus-Host Disease.

Effective therapies for treating patients with steroid-refractory acute graft-versus-host-disease (SR-aGVHD), particularly strategies that reduce the duration of immunosuppression following remission, are urgently needed. The investigated immunotoxin combination consists of a mixture of anti-CD3 and anti-CD7 antibodies separately conjugated to recombinant ricin A (CD3/CD7-IT), which induces in vivo depletion of T cells and natural killer (NK) cells and suppresses T cell receptor activation. We conducted a phase I/II trial to examine the safety and efficacy of CD3/CD7-IT in 20 patients with SR-aGVHD; 17 of these patients (85%) had severe SR-aGVHD, and all 20 patients had visceral organ involvement, including 18 (90%) with gastrointestinal (GI) involvement and 5 (25%) with liver involvement. A validated 2-biomarker algorithm classified the majority of patients (11 of 20) as high risk. On day 28 after the start of CD3/CD7-IT therapy, the overall response rate was 60% (12 of 20), with 10 patients (50%) achieving a complete response. The 6-month overall survival rate was 60% (12 of 20), including 64% (7 of 11) classified as high risk by biomarkers. The 1-week course of treatment with CD3/CD7-IT caused profound but transient depletion of T cells and NK cells, followed by rapid recovery of the immune system with a diverse TCR Vß repertoire, and preservation of Epstein-Barr virus- and cytomegalovirus-specific T cell clones. Furthermore, our results indicate that CD3/CD7-IT appeared to be safe and well tolerated, with a relatively low prevalence of manageable and reversible adverse events, primarily worsening of hypoalbuminemia, microangiopathy, and thrombocytopenia. These encouraging results suggest that CD3/CD7-IT may improve patient outcomes in patients with SR-aGVHD.

A primary role for human central memory cells in tissue immunosurveillance.

Central memory T cells (TCM) patrol lymph nodes, providing central immunosurveillance against known pathogens, but have not been described as conducting primary tissue immunosurveillance. We analyzed the expression of tissue-homing addressins in human TCM vs effector memory T cells (TEM) from the same donors. In humans, the majority of human TCM were tropic for either skin or gut, and the overall tissue tropism of TCM was comparable to that of TEM TCM were present in healthy, noninflamed human skin, lung, colon, and cervix, suggesting a role for TCM in the primary immunosurveillance of peripheral tissues. TCM also had potent effector functions; 80% of CD8+ TCM produced TC1/TC2/TC17/TC22 cytokines. TCM injected into human skin-grafted mice migrated into skin and induced inflammatory eruptions comparable to TEM-injected mice. In summary, human TCM express peripheral tissue-homing receptors at levels similar to their effector memory counterparts, are found in healthy human tissues, have impressive effector functions, and can act alone to induce skin inflammation in human engrafted mice. Our studies support a novel role for human TCM in primary immunosurveillance of peripheral tissues and highlight the important role of this long-lived cell type in tissue-based immune responses.