Adult stem cell activity in naked mole rats for long-term tissue maintenance – Nature.com

Ethics

This study involved undertaking animal procedures in four different countries: U.K, USA, Austria, and the Republic of South Africa. Animal procedures were carried out in accordance with Home Office, UK regulations and the Animals (Scientific Procedures) Act, 1986 of UK, the Institutional Animal Care and Use Committee (IACUC) of USA, Act 7, 1991 of South Africa, and the Directive 2010/63/EU of the European Parliament.

Normal human colonoscopy samples were collected under the research tissue bank ethics 16/YH/0247 supported by NIHR Biomedical Research Centre, Oxford, U.K. and under the London Dulwich Research Ethics Committee (reference number 15/LO/1998). Written informed consent was obtained from all participants undergoing routine bowel cancer or IBD screening. All samples were anonymized.

Wild-caught mice (F1) were acquired from a founder population trapped in lower Austria and Vienna (2016) and housed at the Konrad Lorenz Institute of Ethology, University of Vienna, Austria. All C57BL/6J mice used in this study were purchased from Charles River (Kent, UK) or the Jackson Laboratory (USA) and housed at Biomedical Services Unit in John Radcliffe Hospital, Oxford, UK or at Rutgers University Animal Facility in Newark, New Jersey, USA. Mice were housed in individually ventilated cages under specific pathogen-free conditions and maintained at 1923C temperature with 45-65% relative humidity, in an alternating 12-h light/12-h dark cycles and fed with food and water ad libitum.

Naked mole rats (NMRs) were housed at the Animal Facility of the Department of Zoology and Entomology, University of Pretoria. The NMRs were kept in tunnel systems consisting of several Perspex chambers containing wood shavings as nestling material. The NMR room was maintained at temperatures ranging between 2932C, with relative humidity around 40-60%. NMRs were fed chopped fresh fruits and vegetables (apple, sweet potato, cucumber, and capsicum) daily ad libitum along with weekly supplement of ProNutro (Bokomo). Since NMRs obtain all their necessary water from food sources, no drinking water was provided to the animals. All scientific procedures on NMRs were conducted under ethics approval (NAS046-19 and NAS289-2020) by the Animal Ethics Committee, University of Pretoria. In addition, DAFF section 20 approval was granted (SDAH-Epi-20111909592).

For all analyses, both male and female mice, NMRs, and humans were included in the study.

15mg/mL solution of BrdU (5-bromo-2-deoxyuridine, Abcam, ab142567) and 12.3mg/mL solution of EdU (5-ethynyl-2-deoxyuridine, Merck, 900584) were prepared in sterile 1 PBS (Gibco, 10010023) and filtered through a 0.2m strainer. Using a 27-gauge needle and 1mL syringe, 100mg per kg bodyweight BrdU and 82.14mg per kg bodyweight EdU were administered intraperitoneally. Animals were checked regularly for signs of discomfort (hunched back, shivering, low mobility) after the injection.

For cumulative labelling protocol using BrdU, the first injection in naked mole rats was administered between 14:00 to 15:00. Subsequent BrdU injections were given every 8h for a duration of 5 days and intestinal tissues were collected every 8h after the first injection. In C57BL/6J mice, the first BrdU injection was also given between 14:00 to 15:00, with further injections given every 6h for a total of 2.25 days. Mouse intestinal tissues were collected 1h after each injection. The rationale for the frequency and total number of injections in the two species is discussed in Supplementary Note1.

Dextran sulphate sodium (DSS) salt (Merck, 42867) was dissolved in sterile ddH2O to prepare 0 to 8.75% (w/V) solution. Using a 2mL syringe fitted with a plastic feeding tube (Prime Bioscience, FTP-20-38), 50mL per kg bodyweight of DSS solution in NMRs or 12mL per kg bodyweight in mice was administered orally at specific intervals for 3 days. Body mass was monitored daily and stool samples collected while animals were also checked for signs of discomfort (e.g. hunched back, shivering, low mobility) every 3h.

After sacrificing the animals by approved procedures, the intestine was immediately isolated from the abdominal cavity and fatty tissue was removed. The small intestine was then divided into three equal sections: SB1 (duodenum), SB2 (jejunum) and SB3 (ileum). All three parts of the small intestine and colon were then flushed with 1 PBS (Phosphate Buffered Saline) solution using a P1000 pipette to clean all the faecal material. Each tissue section was then cut open longitudinally using a gut cutting device86 and the edges pinned down onto a 3MM filter paper such that the luminal side was facing upward. The tissue was then fixed in 10% neutral buffered formalin overnight at room temperature. The following day fixed intestinal tissues were rolled using the Swiss-rolling technique87 and stored in 70% ethanol at 4C. Next, formalin-fixed Swiss-rolls were dehydrated through increasing concentrations of ethanol, cleared through xylene, and embedded in paraffin. The paraffin blocks were sectioned at 4m thickness using a microtome (Anglia Scientific).

Tissue sections on SuperFrost Plus slides (VWR, 6310108) were deparaffinized by submerging slides in xylene (2 times, 10min each) and rehydrated in 100% ethanol (2 times, 5min each), 95% ethanol (2min), 70% ethanol (2min), 50% ethanol (2min), and distilled water (5min). Sections were then stained with Harris Haematoxylin (Merck, HHS32) for 2min 45s followed by washing in running tap water for 5min. Next, slides were dipped in 95% ethanol ten times before sections were counter-stained with Eosin solution (Merck, 117081) for 3min. This was followed by tissue sections being dehydrated in 95% ethanol (15s) and 100% ethanol (2 times, 15s each), dipped in xylene (2 times, 5min each), and finally coverslipped using DPX Mountant (Merck, 06522).

Tissue sections on SuperFrost Plus slides (VWR, 6310108) were first deparaffinized with xylene (2 times, 5min each). They were rehydrated in 100%, 90%, 70% ethanol (5min each) and tap water (2min), dipped in 3% acetic acid solution (3min) before staining with Alcian blue 8GX (Merck, A5268) solution (pH 2.5) for 30min. Tissue sections were then washed (5min) in running tap water and counterstained (5min) with Nuclear Fast Red (Merck, N3020). After 1min wash in running tap water again, tissue sections were dehydrated in ethanol, dipped in xylene and finally coverslipped using DPX Mountant (Merck, 06522).

To preserve the mucus layer of the colonic epithelium, contact with any aqueous solution was avoided after the excision of the intestinal tissue. Without removing the faecal matter, several segments of the colon were cut using a scalpel and fixed overnight at room temperature in methacran/Carnoys solution which was composed of 60% methanol, 30% chloroform, and 10% glacial acetic acid. On the second day, fixed tissues were processed in 100% methanol (2 times, 30min each), 100% ethanol (3 times, 60min each) and xylene (2 times, 60min each). Processed tissues were embedded in paraffin and 4m thick sections cut and stained with Alcian blue as described above. Stained tissues were photomicrographed at 60 magnification on an Olympus BX51 brightfield microscope. For both NMRs and mice, 30 independent measurements of the mucus layer were taken from 3 animals using the measure tool in Fiji package88.

Tissue sections on SuperFrost Plus slides (VWR, 6310108) were deparaffinized in xylene (2 times, 5min each) and rehydrated in 100%, 90%, 70% ethanol (5min each) and distilled water (5min). A hydrophobic barrier was drawn around the tissue sections using a PAP pen (Vector Lab, H-4000) before incubating in the AB solution (AP Staining kit, SystemBio, AP100B-1) for 20min at room temperature in the dark. All sections were then washed in 1 PBS (5min, on a shaker), counterstained with Nuclear Fast Red (5min), washed in running tap water (1min), dehydrated in ethanol, dipped in xylene and finally coverslipped with DPX Mountant (Merck, 06522).

4m thick formalin-fixed paraffin-embedded (FFPE) sections were cut using a microtome and dried overnight on SuperFrost Plus slides (VWR, 6310108). Tissue sections were baked at 60C for 1h the next day, deparaffinized in 3 rounds of xylene (5min each) and rehydrated in 100%, 90%, 70% ethanol and distilled H2O (5min each). Endogenous peroxidase activity was quenched by incubating sections in 3% H2O2 (Merck, 8222871000) for 20min. A heat mediated antigen retrieval was performed by boiling sections in 10mM sodium citrate buffer (pH 6.0) for 10min which was followed by 20min of cooling down in the same solution. This was followed by incubating the tissue sections in 1 PBSTX (0.1% Triton X) for 10min. All sections were then blocked for 1h at room temperature using 5% serum which matched the species of the secondary antibody. Next, primary antibodies were diluted in antibody diluent (1% BSA dissolved in 1 PBS) which was applied to the tissue sections and incubated overnight at 4C. The primary antibodies used in this study were Chromogranin A (Abcam, ab15160) at 1:2000 and BrdU (Abcam, ab6326) at 1:500. It is noteworthy that in our BrdU staining, we did not use HCl-mediated DNA denaturation and only performed heat-mediated antigen retrieval (98-100C) which has been shown to produce a brighter signal than acid hydrolysis89. After 3 rounds of washes (5min each) with 1 PBST (0.1% Tween20 in 1 PBS), tissue sections were then incubated for 1h at room temperature with biotinylated secondary antibodies diluted at 1:300. For our study specifically, we used goat anti-rabbit IgG (Vector Laboratories, BA-1000) and goat anti-rat IgG (Abcam, ab207997). To detect the biotinylated target, we used the Avidin/Biotinylated enzyme Complex (ABC) kit (Vector Laboratories, PK-6101) and developed the signal using the DAB (3,3-diaminobenzidine) solution (R&D systems, 4800-30-07). The tissue sections were then counterstained with Harris Haematoxylin (Merck, HHS32) for 5s, dehydrated in 70%, 90% and 100% ethanol for 15s each, dipped in xylene and coverslipped using DPX Mountant (Merck, 06522).

Species-specific RNAscope probes from ACD Bio-techne were used to detect Lgr5 mRNA expression in NMR (584631), mouse (312171) and human (311021) intestinal tissues. We used the RNAscope Multiplex Fluorescent Detection Kit v2 (ACD Bio-techne, 323110) and followed the instructions of the manufacturer (document number 323100-USM, ACD Bio-techne) to detect Lgr5 mRNA targets at a single cell level in FFPE tissue sections mounted on SuperFrost Plus slides (VWR, 6310108).

To enable multiplexing of mRNA and proteins, we adapted the manufacturers instructions (document number 323100-USM, ACD Bio-techne) for RNAscope Multiplex Fluorescent Detection Kit v2 (ACD Bio-techne, 323110) to exclude the step involving protease treatment. Once the mRNA signal was developed, we proceeded to detect proteins by first washing tissue sections (2 times, 2min each) in 1 TBST (0.1% Tween20 in 1 Tris-buffered saline). This was followed by blocking for 1h at room temperature with 10% serum which matched the species of the secondary antibodies. Multiple primary antibodies (diluted in 1% BSA in 1 TBS) were then applied to the tissue sections and incubated overnight at 4C. The dilutions of various primary antibodies used in our study were 1:500 for EpCAM (Abcam, ab71916), 1:500 for Ki67 (Cell Signaling, 12202), 1:200 for p27Kip1 (Cell Signaling, 3686 and 2552), 1:500 for BrdU (Abcam, ab6326) and 1:2000 for PHH3-S28 (Abcam, ab32388). Following primary antibody incubation, the next day we washed the sections thrice in 1 TBST (5min each) before incubating them with fluorophore-linked secondary antibodies (at 1:500 dilution) for 1h at room temperature. Fluorescent secondary antibodies used in our study included goat anti-rabbit Alexa 488 (Invitrogen, A11008), goat anti-rat Alexa 488 (Invitrogen, A11006), goat anti-rabbit Alexa 555 (Invitrogen, A21428) and goat anti-rabbit Alexa 633 (Invitrogen, A21070). Following the secondary antibody incubation, tissue sections were washed three times in 1 TBST (5min each) and counterstained with DAPI (Invitrogen, D1306) for 15min at room temperature before mounting with coverslips (VWR, 631-0138) using Diamond Antifade Mountant (Invitrogen, P36961).

Click-iT Plus TUNEL Assay Kit (Invitrogen, C10617) was used following the manufacturers instructions to detect apoptotic cells FFPE tissue sections.

EdU-Click 488 kit (Base Click, BCK-EdU488-1) was used according to the instructions provided by the manufacturer to detect EdU-positive cells in FFPE tissue sections.

Plasma BrdU concentration was determined following the protocol described by Barker et al.90. In brief, 100L naked mole rat blood was collected by a tail vein puncture after 8hand 16h of BrdU injection. The blood was mixed with heparin to stop clotting and centrifuged at 13,000g for 15min to separate all blood cells. Plasma was collected from the top layer and stored at 80C.

HEK293T cells (ATCC, CRL-3216) were cultured in high-glucose DMEM (Merck, D6546) containing 10% FBS (Gibco, 10270), 1 Penicillin-Streptomycin (Merck, P4333-100ML), and 2mM l-glutamine (Gibco, 25030-024) at 37C with 5% CO2. Cells were plated on a 13mm sterile glass coverslip precoated with poly l-lysine (VWR, 631-0149) in a 24-well plate (Starlab, CC7682-7524) and cultured overnight. The media was replaced with 500L fresh culture media containing 10L plasma or standard BrdU solution (3, 10, 20, 30, 40, 50g/ml) and incubated at 37C for 4h. Cells were then washed with 1 PBS and fixed in 4% paraformaldehyde for 20min at room temperature. Fixed cells were kept in 1 PBS at 4C before proceeding to immunocytochemical detection of BrdU.

Fixed cells on coverslips in 24 well plates were incubated with 3% H2O2 for 10min at room temperature. After washing with 1 PBS, cells were incubated in 2N HCl for 1h at room temperature to denature DNA strands. Fixed cells were then incubated in 0.1M Borate buffer (pH 8.5) for 30min at room temperature and in 1 PBSTX (0.1% Triton X) for 10min. Cells were blocked with 5% goat serum for 1h at room temperature and incubated with rat anti-BrdU primary antibody (Abcam, ab6326, 1:2000) overnight at 4C. The next day, cells were washed three times in 1 PBST and incubated with goat anti-rat-biotin-linked secondary antibody (Abcam, ab207997, 1:400) for 1h at room temperature. The biotinylated signal was developed using the ABC Kit (Vectastain, PK-6101) following the manufacturers instructions and detected with DAB solution (R&D systems, 4800-30-07). Gills No. 3 Haematoxylin (Merck, GHS316-500ML) was used for counterstaining and cells on the coverslips were mounted on glass slides using Aquatex mounting agent (Merck, 108562).

Intestinal tissue was washed with PBS, cut open longitudinally and laid flat on a glass slide with the luminal side facing upward. The small intestinal villi were scrapped off the flat tissue by a glass slide and collected in cold 1 PBS. The remaining tissue containing crypts was chopped into <2mm pieces using a scalpel, washed three times with ice-cold 1 PBS and incubated in chelation medium (2mM EDTA in 1 PBS without Ca2+ and Mg2+, Gibco 10010023) for 40min with agitation at 4C. The digested tissue was shaken vigorously for 30s in 1 PBS to release crypts and villi. To separate out crypts and villi of the small intestine, the solution was passed through a 100m cell strainer. The isolated crypts in the flow through were pelleted and transferred to RLT Buffer (Qiagen, 79216). RNeasy microkit (Qiagen, 74004) was used for RNA extraction. Extracted RNAs were incubated with DNase1 (ThermoFisher, EN0521) at 37C for 30min, followed by a 10min incubation with EDTA at 65C. High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814) was used to generate complementary DNA from total RNA. Quantitative real-time-PCR (qRT-PCR) was performed on LightCycler96 (Roche) with mouse and naked mole rat Gapdh used as an endogenous control. The IDs of Taqman Gene expression assays (Applied Biosystems) used in this study are Gapdh (Mm99999915_g1, Hg05064520_gH), Muc2 (Mm01276681_m1, Hg05250665_g1), Synaptophysin (Mm00436850_m1, Hg05249763_m1), and Aldolase B (Mm00523293_m1, Hg05103981_m1). The 2-Ct method was used to calculate the relative gene expression levels.

Brightfield images of tissue sections were captured using an Olympus BX51 microscope coupled with an Olympus DP70 camera system using DP controller software. Villi were imaged using 10 objective while crypts were imaged with 20 (for colon) or 60 (for small intestine) objective lens. Histopathological scoring in this study was performed based on the digital images obtained on Hamamatsu (Nanozoomer HT) scanner at 40 magnification.

To quantify cell numbers in crypt-villous structures from brightfield images, cell counter plugin of Fiji software was used. The dimensions of crypt-villous structure were calculated using the measure tool in Fiji.

Fluorescent images of intestinal crypts were acquired from 4m thick tissue sections with a Plan Apochromat 63 or 100 1.4 oil objective on a Zeiss LSM 780 upright or inverted confocal microscope. Images were acquired in Zen SP7 FP3 (black) software using 405nm, 488nm, 561nm, and 633nm laser lines in sequential tracks. Z-stacks of 6-12 optical sections with 50% overlap between subsequent planes were captured within the span of a single cell at 0.3m z-distance, 0.087m pixel dimension, and 12-bit depth.

For generating the RGB images used in the figures (Figs.1a, b, 2ad, 3d, 4d, 7a, d, Supplementary Figs.14, 5d, e, 11b), the original.czi raw files were imported into Fiji software package and a maximum intensity z-projection was created from the stacks. Using the split channel option of Fiji, the multicolour fluorescent images were separated into individual channels (DAPI, Alexa 488, Cy3, Alexa 633). The maximum and minimum displayed pixel values of individual channels were adjusted across the entire image set including in negative controls (i.e. linear adjustment) to correct for autofluorescence that had been introduced in the image stacks during acquisition. Then, using merge channel option in Fiji, two/more channels were combined to create a composite image (Lgr5/Ki67 or LGR5/KI67, Lgr5/EpCAM or LGR5/EPCAM, Lgr5/p27 or LGR5/P27, Lgr5/BrdU, Lgr5/pHH3 or LGR5/PHH3) while keeping the individual channels intact. Finally, all the individual and composite images were converted into RGB color type and saved in TIFF format. These images (TIFF) were compiled in Adobe Illustrator 2020 software to produce the panels presented in the figures.

Z-stack images were processed in batch mode of Fiji package. Firstly, a maximum intensity projection was created to generate a 2D image from the stacks. Next, each channel of the image was separated, and maximum and minimum displayed pixel values were adjusted across the entire image set including negative controls. To quantify the number of rodent Lgr5 or human LGR5 mRNA expressed in a single cell, all the ISH dots were manually counted within the cell periphery demarcated by EpCAM staining. As the Lgr5 or LGR5 signal was captured using confocal microscopy at a resolution of 237nm, overlapping/merged Lgr5 or LGR5 mRNA signal dots were rarely observed. To calculate the distribution of Lgr5+ or LGR5+ cells relative to other cells along the crypt axis, the cell present at the crypt apex was assigned position 0 and we counted cells on each side of this cell to acquire datapoints in our quantifications. Any cell containing more than three Lgr5 or LGR5 mRNA puncta was considered positive for Lgr5 or LGR5 expression (Lgr5+ or LGR5+).

We observed significant variation in autofluorescence levels between mouse, human and NMR intestinal tissues, with mouse tissue emitting the most and naked mole rats the least. This variation necessitated adjusting the laser powers of the confocal microscope during image acquisition so that maximal image contrast was achieved while also reducing the autofluorescence signals. The maximum and minimum displayed pixel values of individual channels were adjusted across the entire image set (i.e. linear adjustment), including in negative controls, to correct for autofluorescence. These adjustments resulted in varying intensities for specific signals in the three species and, therefore, we took a binary approach for the quantification of the antibody-based signals. The presence of any specific signal in the target compartment inside a cell was considered positive regardless of the staining intensity.

We determined the length of the cell cycle (TT) and S-phase (TS) in CBC cells (Lgr5+CBC) of naked mole rats by counting the fraction of BrdU-labelled Lgr5+CBC cells after successive pulsing over 5 days in NMRs and 2.25 days in mice. As the CBC cells (Lgr5+CBC) cells are on average asynchronously and asymmetrically dividing45, the labelling index (LI) which provides the ratio of labelled cells to the total population (LI=Lgr5+CBCBrdU+/Lgr5+CBC) at any given time (t) can be modelled by Eq.1 below where TT is the total cell division time33.

$${{{{{rm{LI}}}}}}= (1/{{{{{rm{T}}}}}}_{{{{{rm{T}}}}}}){{{{{rm{X}}}}}}t+({{{{{rm{T}}}}}}_{{{{{rm{S}}}}}}/{{{{{rm{T}}}}}}_{{{{{rm{T}}}}}}),{{{{{rm{for}}}}}},{t}{{{{{rm{le }}}}}}{{{{{{rm{T}}}}}}}_{{{{{{rm{T}}}}}}}-{{{{{{rm{T}}}}}}}_{{{{{{rm{S}}}}}}}\ {{{{{rm{LI}}}}}}= 1,{{{{{rm{for}}}}}},t > {{{{{{rm{T}}}}}}}_{{{{{{rm{T}}}}}}}-{{{{{{rm{T}}}}}}}_{{{{{{rm{S}}}}}}}$$

(1)

Equation1 assumes that there are no or only very few stem cells (based on p27 negativity in NMR and mouse Lgr5+CBC cells) that remain quiescent for the duration of the BrdU experiment. The lfit tool in STATA was used to calculate the least square fit of the data by considering the time points before LI reached saturation. We derived TT from the slope of the regression (TT=1/slope). When t=0, LI0=TS/TT which is the y-intercept of the graph. Thus, the duration of S-phase (TS) was estimated from the y-intercept of the regression line.

For human LGR5+CBC cells, we assumed KI67 is undetectable at G1/S transition and detected in the S to M phases of the cell cycle46. We determined the fraction of LGR5+CBC cells that expressed KI67 and calculated the length of S, G2 and M-phase (T(S, G2, M)) using Eq.2:

$${{{{{{rm{T}}}}}}_{{({{{{{rm{S}}}}}},{{{{{rm{G}}}}}}2,{{{{{rm{M}}}}}})}}}{{{{{rm{KI}}}}}}67^{+}={{{{{{{rm{T}}}}}}}_{{{{{rm{T}}}}}}^{({{{{{{rm{Ref}}}}}}},31)}}{{{{{rm{X}}}}}},{{{{{{rm{LGR}}}}}}5}^{+{{{{{rm{CBC}}}}}}}{{{{{rm{KI}}}}}}67^{+}/{{LGR}5}^{+{{{{{rm{CBC}}}}}}}$$

(2)

The time in mitosis (TM) was calculated after quantifying the fraction of rodent (mouse or NMR) Lgr5+CBC or human LGR5+CBC cells positive for phospho-histone H3 using Eq.3:

$${{{{{{rm{T}}}}}}{{{{{rm{M}}}}}}}^{{{{{{rm{Ki}}}}}}67+}={{{{{{{rm{T}}}}}}}_{{{{{{rm{T}}}}}}}}^{({{{{{rm{linear}}}}}},{{{{{rm{regression}}}}}})}{{{{{rm{X}}}}}},{{Lgr}5}^{+{{{{{rm{CBC}}}}}}}{{{{{rm{pHH}}}}}}3+({{{{{rm{Ser}}}}}}28)/{{Lgr}5}^{+{{{{{rm{CBC}}}}}}}$$

(3)

or

$${{{{{{{rm{T}}}}}}}_{{{{{{rm{M}}}}}}}}^{{{{{{rm{KI}}}}}}67+}={{{{{{{rm{T}}}}}}}_{{{{{{rm{T}}}}}}}}{({{{{{rm{ref}}}}}}31)}{{{{{rm{X}}}}}},{{LGR}5}^{+{{{{{rm{CBC}}}}}}}{{{{{{rm{PHH}}}}}}3}^{+}({{{{{rm{Ser}}}}}}28)/{{LGR}5}^{+{{{{{rm{CBC}}}}}}}$$

Using TS estimated by Ishikawa et al.31 previously, the length of G2-phase (TG2) was calculated using Eq.4:

$${{{{{{{rm{T}}}}}}}_{{{{{{rm{G}}}}}}2}}^{{{{{{rm{KI}}}}}}67+}={{{{{{rm{T}}}}}}}_{({{{{{rm{S}}}}}},{{{{{rm{G}}}}}}2,{{{{{rm{M}}}}}})}{{{{{{rm{KI}}}}}}67}^{+}-left({{{{{{{rm{T}}}}}}}_{{{{{{rm{S}}}}}}}}^{{{{{{rm{KI}}}}}}67+}+{{{{{{{rm{T}}}}}}}_{{{{{{rm{M}}}}}}}}^{{{{{{rm{KI}}}}}}67+}right)$$

(4)

After quantifying the fraction of LGR5+CBC cells expressing P27, we calculated the time spent in G0 and G1 (T(G1, G0)P27+) using Eq.5:

$${{{{{{{rm{T}}}}}}}_{({{{{{rm{G}}}}}}1,{{{{{rm{G}}}}}}0)}}^{{{{{{rm{P}}}}}}27+}={{{{{{{rm{T}}}}}}}_{{{{{{rm{T}}}}}}}}{({{{{{rm{ref}}}}}}31)}{{{{{rm{X}}}}}},{{LGR}5}^{+{{{{{rm{CBC}}}}}}}{{{{{rm{P}}}}}}27+/{{LGR}5}^{+{{{{{rm{CBC}}}}}}}$$

(5)

We took the fraction of LGR5+P27+ cells in G0 phase (QF) from Ishikawa et al. 31 to calculate the length of G0 in human LGR5+CBC cells using Eq.6:

$${{{{{{{rm{T}}}}}}}_{{{{{{rm{G}}}}}}0}}^{{{{{{rm{P}}}}}}27+}={{{{{{rm{QF}}}}}}}{({{{{{rm{ref}}}}}}31)}{{{{{rm{X}}}}}},{{{{{{{rm{T}}}}}}}_{(G1,G0)}}^{{{{{{rm{P}}}}}}27+}$$

(6)

Finally, using Eq.7, we quantified the time human colonic LGR5+CBC cells spend in G1 (TG1):

$${{{{{{rm{T}}}}}}}_{{{{{{rm{T}}}}}}}={{{{{{{rm{T}}}}}}}_{{{{{{rm{G}}}}}}0}}^{{{{{{rm{P}}}}}}27+}+{{{{{{{rm{T}}}}}}}_{{{{{{rm{G}}}}}}1}}^{{{{{{rm{P}}}}}}27+}+{{{{{{{rm{T}}}}}}}_{{{{{{rm{S}}}}}}}}^{{{{{{rm{KI}}}}}}67+}+{{{{{{{rm{T}}}}}}}_{{{{{{rm{G}}}}}}2}}^{{{{{{rm{KI}}}}}}67+}+{{{{{{{rm{T}}}}}}}_{{{{{{rm{M}}}}}}}}^{{{{{{rm{KI}}}}}}67+}$$

(7)

In NMR and mouse, Lgr5+CBC cells are negative for p27 such that TG0=0. For these species, we derived the combined length of time spent in G1 and G2 (TG1+TG2) from Eq.7.

Using the length of TS from cumulative BrdU labelling in Lgr5+CBC cells and assuming no change in TS in Lgr5+ cells located at different positions within the crypt31, we measured the total cell division time (TT) of Lgr5+above crypt base cells using Eq.1 by measuring the labelling index (LI) at a single time point (t) after pulsing animals with BrdU in vivo. More specifically, in C57BL/6 mice (n=3 animals, 4 months old), we administered BrdU once and analysed intestinal tissue at t=0.5h. In NMRs (n=3 animals, 6-24 months-old), we pulsed the animals with BrdU every 8h and analysed the intestine after t=1 day.

We used Microsoft Excel (v16.77.1) for inputting raw data after collection. All statistical tests and graphs displayed in this paper were generated using StataMP 14.1. Details of statistical tests performed are described in figure legends. P-values are generated by conducting two-tailed t-tests, F-test and Wilcoxon rank sum test as indicated in each figure legend. No blinding and randomization were performed during the analysis.

All the figures presented in this manuscript were prepared using Adobe Illustrator 2020 (version 24.1). Vector line arts shown in Figs.1c, d, 3a, h, 4a, h, 6a, Supplementary Figs.5d, e, and 9a, b were created using the curvature tool of Adobe Illustrator.

Further information on research design is available in theNature Portfolio Reporting Summary linked to this article.

Read the original here:
Adult stem cell activity in naked mole rats for long-term tissue maintenance - Nature.com

NHS England NHS to offer stem cell transplants to cure life-limiting … – NHS England

Patients with severe inherited blood disorders will be offered stem cell transplants on the NHS that could cure their condition and help avoid life-long blood transfusions.

Thalassaemia is a rare disorder that affects the production of haemoglobin in the blood, leading to severe anaemia and debilitating tiredness, with patients needing to have blood transfusions every two to four weeks to survive which has a major impact on their quality of life.

Now, curative stem cell transplants will be funded by the NHS for the first time for adult patients with thalassaemia following new guidance.

The procedure called allogeneic haematopoietic stem cell transplant (Allo-HSCT) involves replacing the bone marrow stem cells of a patient with ones from a matched sibling donor. Stem cells are given to the patient via an intravenous infusion which helps to re-establish healthy blood cell production.

Previously, this type of treatment was offered only to children because of the potential risk of complications among adults.

But now, following advances in transplant treatment such as better medications to manage a patient before, during and after transplantation, new guidance from NHS Englands Clinical Priorities Advisory Group has recommended the treatment be made available for the first time to eligible thalassaemia patients over the age of 18.

It is estimated that there are more than 600 adults across the UK who have transfusion-dependent thalassaemia a severe form of the condition, which was previously fatal in childhood, many of whom could be eligible for the stem cell transplant. Overall, there are 2281 people with thalassaemia registered on the National Haemoglobinopathy Registry in the UK, including 1332 over the age of 18.

Thalassaemia is more prevalent amongst Southern European, Middle Eastern, South American, Caribbean, Asian and South East Asian communities in the UK, and providing access to this treatment will play a role in reducing health inequalities for these populations.

Professor Sir Stephen Powis, NHS England Medical Director, said: Expanding the availability of stem cell transplants to adults living with thalassaemia is another vital step forward to help change the lives of those living with this deeply debilitating condition.

Thalassaemia can be an incredibly painful condition with difficult symptoms for patients as well as the impact on their heart, liver and bones, and its fantastic that offering this evidence-based curative stem cell treatment can now offer new hope to help significantly improve patients quality of life.

Sonal Mistry, 38, from Birmingham was diagnosed with thalassaemia as a baby and received a stem cell transplant in 1991, when she was five years old. Sonal had received regular blood transfusions to manage her condition as a young child but was cured when she received the donor cells from her younger sister, Krishna.

After initially suffering complications from the transplant, including permanent scarring, Sonal now lives a healthy life, free of disease, only requiring annual blood tests to check in on her condition.

Sonal said: I havent been on any medication since I was 15 years old.

All my test results came back as normal, and I now live a healthy life. Im married and work as a stem cell scientist. Its my way of giving back, by doing for someone else what somebody did for me.

As well as working as a stem cell scientist, Sonal previously worked in a hospital that specialises in treating patients with thalassaemia and sickle cell disease, giving her firsthand experience of the benefits of her treatment.

She said: Ive met a lot of patients with serious heart and liver complications from their condition.

If it wasnt for the transplant, Id probably still be having blood transfusions and regular hospital appointments.

Im so grateful to be on the other side of my journey, and its so nice to now be helping other people. Todays news is really exciting, and I hope that for adults offered transplants, it will be life changing for them, like it was for me.

Kirthana Balachandran, a 20-year-old medical student from West London, was diagnosed with thalassaemia when she was three months old. Although she previously had the potential to receive a stem cell transplant as a child and teenager, unfortunately no matched donors were found, meaning Kirthana still requires blood transfusions every three weeks and medication to manage her condition.

Kirthana said: My condition affects me a lot.

Sometimes I have muscle pain, back pain and I can even feel breathless or have palpitations when I walk uphill. It just depends on your haemoglobin level. When its low, I feel so tired and it can affect me at the most inconvenient times, like when I have exams. But when my levels are good, I dont really feel any symptoms at all.

For Kirthana, the possibility of a stem cell donation in adulthood gives her an option to cure her condition, that previously wouldnt have been available to her.

She said: When I was younger, we looked for a donor from my family, but no one was a match and the possibility just faded away. With todays news, if we were able to find a donor, that would be an amazing possibility, as it could really change my life. I would never need to go for a blood transfusion again for my condition and I wouldnt have to worry about the side effects from transfusions or my health in future either.

Its amazing that theyve made this treatment option available for adults, because it means we can still have a chance of finding a donor, even at an older age.

Romaine Maharaj, UK Thalassaemia Society Executive Director, said: We celebrate the long-awaited approval of Allo-HSCT for adults with transfusion dependent thalassaemia. This remarkable milestone offers hope to adults with donor matches who were previously excluded from accessing a curative option.

While it is a huge step in the right direction and a monumental win for thalassaemia, we also eagerly await the much-needed approval for gene therapies. Having both curative options available will grant more patients the chance to live transfusion-independent lives, enhancing both their quality of life and life expectancy.

Earlier this year, the NHS became the first healthcare system in the world to provide blood group genotyping for people with thalassaemia and sickle cell disease. This is a detailed DNA analysis of each patients blood group to match more accurately those in need of transfusions to donated blood.

The landmark new programme, delivered in partnership by NHS England and NHS Blood and Transplant, will help ensure patients receive the best treatment for them, reducing the risk/impact of reactions to donor blood and the development of antibodies that attack the donor blood cells.

View post:
NHS England NHS to offer stem cell transplants to cure life-limiting ... - NHS England

Mechanisms of Wharton’s Jelly-derived MSCs in enhancing … – Nature.com

Preparation and culture of human WJ-MSCs

This study was approved by the Institutional Review Board of Asan Medical Center (No. 20150303), and the WJ-MSCs were provided by the Stem Cell Center, Asan Institute for Life Sciences, Seoul, Korea. All experiments were performed in accordance with relevant guidelines and regulations. Informed consent from the mothers was obtained for the use of umbilical cords. Umbilical cords were cut into 0.31.0cm pieces without blood vessels. The matrix was minced and transferred to culture dishes in minimal essential medium supplemented with 10% fetal bovine serum and an antibioticantimycotic mixture at 37C in a 5% CO2 incubator in vitro as described previously45. When the cells reached 80% confluency, they were replated at a 1:3 split ratio.

This study complied with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of Asan Medical Center and Ulsan University College of Medicine (No. 2017-12-127), and all the following methods were performed in accordance with the relevant guidelines and regulations. After isoflurane induction, rats were euthanized by CO2 inhalation. Sciatic nerve segments (10mm in length) were harvested from male SpragueDawley (SD) rats (78weeks old, weight 250350g) (Orient Bio Inc., Seongnam, Korea). To prepare ANGs, sciatic nerve pieces were decellularized using a series of detergents as described by Shin et al.2. Briefly, the nerves were treated with detergents, including aprotinin, CHAPS, and DNase and RNase solutions. Then, the decellularized segments were washed several times with phosphate-buffered saline (PBS) to remove residual reagents and stored in PBS at 4C until use. All solutions were autoclaved or filter-sterilized before use.

Seventy-four adult male SD rats were randomly assigned to two groups: WJ-MSCs group (which was implanted with WJ-MSCs-laden ANG; n=37) and control group (which was implanted with ANG only; n=37). After anesthetization, the left sciatic nerve of rats was exposed and transected, and 10mm of the nerve was removed. The 10-mm piece of WJ-MSCs-laden ANG or ANG was sutured using 90 nylon (Ethicon, Somerville, NY) under a microscope.

Seven rats were selected from each group, and their ankle angles at the toeoff phase were measured at 4, 8, 12, and 16weeks postoperatively to evaluate serial functional recovery. A walking track (length 1m, width 10cm, and height 10cm) was built for this test. During the test, video was acquired with a digital camera (Canon SX730HS, Canon, Tokyo, Japan) at a distance of 1m and calibrated to prevent optical distortion. Records were repeated until three satisfactory trials were obtained per rat. The ankle angle at the toeoff phase was measured at maximal plantar flexion in the experimental lateral ankle joint. After the foot and leg segments were manually identified in the video frames, the ankle angles at the toeoff phase were displayed in degrees.

All rats were anesthetized at 16weeks postoperatively, and the maximum isometric tetanic force was measured. The sciatic nerve was fully exposed through previous operation incision, and another skin incision was made anterior to the ankle to expose and transect the tibialis anterior (TA) tendon distally. The TA tendon was connected to a force transducer using a custom clamp with the knee and ankle joints immobilized to a platform. A bipolar stimulator (Grass S88, Grass Instrument Corp, Quincy, MA) was used to generate stimulus and processed on a computer using LabVIEW software (National Instruments, Austin, TX). All contractions were performed at supramaximal voltage to ensure maximal activation of all TA motor units. The strength of muscles was standardized as a percentage of the value from the contralateral side.

Sciatic nerve axonal regeneration in each group was directly examined using toluidine blue staining at 16weeks postoperatively. The implanted sciatic nerves were harvested by including the distal sites, and 2.5% glutaraldehyde solution was used for fixation. The harvested nerves were further fixed in 1% osmium tetroxide, dehydrated in ethanol, and embedded in EPON resin (Miller-Stephenson Chemical Co., Sylmar, CA, USA). Cross-sections (1m thick) were stained with toluidine blue to visualize myelin with light microscopy. Digital images of nonoverlapping fields were taken at 400magnification using unbiased random sampling. The total number of myelinated axons was calculated using ImageJ software (National Institutes of Health, Bethesda, MD).

RT-qPCR was performed to evaluate the mRNA expression levels of factors related to peripheral nerve regeneration. First, macrophage markers (CD206 and interleukin 10 [IL10]) were assessed to investigate the immunomodulatory effects of WJ-MSCs. Second, NGF, BDNF, and VEGF were assessed to analyze the paracrine effects of WJ-MSCs on ANGs. Third, SC markers (S100 and MBP) were assessed to confirm the recruitment of SCs by WJ-MSCs in ANGs. All experiments were performed using nerve grafts harvested from five rats in each group at 3, 7, and 14days postoperatively. Gene expressions were analyzed as described previously5. Total RNA was isolated from ANGs or WJ-MSCs-laden ANGs using TRIzol (Thermo Fisher Scientific). Approximately 1g of total RNA was used for cDNA synthesis using a first-strand cDNA synthesis kit. Quantitative estimation of mRNA expression was conducted using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems/Thermo Fisher Scientific). All experiments were performed in triplicates and independently repeated more than three times. The following primers were used: CD206 (forward, TTA CTT TAA GGG GGC GTG TG; reverse, AGT TGG TTG GGG AGT GTC AG), IL10 (forward, CTC CAC CTG GCA AAC AAA AT; reverse, CTG CCT AGC CCA CAA AGA AG), NGF (forward, ACT CGG CTC CTT TGA GTT GA; reverse, CCC GTC CTA CAG AAG CAG AG), BDNF (forward, GAA GGT GAG GAA AGC AGC AC; reverse, TGC ACA GTC ATC TGG AAA GC), VEGF (forward, TGC TTC CTA GTG GGC TCT GT; reverse, CAC ACA TAC ACT CCG GCA TC), S100 (forward, GAA TTG GGG CAG AGA AAT GA; reverse, GGC TTG AGC TTC TTG GAA TG), MBP (forward, AAT GTT TCA GGG CAC CGT AG; reverse, AAA AAC CAG CCA GCT GAG AA), and GAPDH (forward, ATG GTG AAG GTC CCT GTG AAC G; reverse, CTT GCC GTG GGT AGA GTC AT). The comparative Ct method (2Ct)46 was used to analyze the relative amount of gene expression.

The protein levels of CD206, CD68, NGF, BDNF, VEGF, and S100 were evaluated using immunofluorescence staining at 3, 7, and 14days postoperatively. CD68 (a total marker of all macrophages) was analyzed to identify and quantify macrophages. In addition, double-staining for human nuclei and S100 was performed only in the WJ-MSCs group at 3 and 7days postoperatively to investigate the differentiation potential of WJ-MSCs in ANGs. All experiments were performed using nerve grafts harvested from five rats in each group.

The grafts were snap-frozen in liquid nitrogen with frozen section compound (Leica Biosystems, Wetzlar, Germany) as described previously47. Samples were cut into 6-m-thick cross-sections using a Cryo-Star HM560 freezing microtome (Thermo Fisher Scientific). After fixation, sections were permeabilized and then blocked with 10% normal goat serum. Thereafter, they were incubated overnight with the primary antibody at 4C. The following antibodies were used: anti-CD206 (ab64693; Abcam, Cambridge, UK), anti-CD68 (ab201340; Abcam), anti-NGF (ab6199; Abcam), anti-BDNF (ab108319; Abcam), anti-VEGF (ab1316), anti-S100b (ab52642; Abcam), and anti-hNuclei (MAB1281; Millipore, Burlington, MA, USA). Secondary antibodies, anti-rabbit Alexa 555 (A32732, Invitrogen), and anti-mouse Alexa 546 (Invitrogen A11003), were applied for 1h at room temperature in the dark. Finally, 4,6-diamidino-2-phenylindole was used to counterstain nuclei. Analysis of staining was performed using an LSM-810 confocal microscope (Zeiss, Oberkochen, Germany) at 100magnification.

All experiments were repeated three times or more. Values are presented as meanstandard error of the mean (SEM). Statistical significance was considered at p<0.05. All statistical analyses were performed using Students t-test in IBM SPSS Statistics for Windows v. 27.0 (IBM, USA).

Go here to read the rest:
Mechanisms of Wharton's Jelly-derived MSCs in enhancing ... - Nature.com

Is Sight Care Legit? Review How SightCare Ingredients Work Before … – Bellevue Reporter

It is common knowledge today that your eye health suffers indiscriminately because of increased exposure to blue light emitted from electronic gadgets. Every day we are on our mobile phones or laptops, scrolling through either work or social media. This makes our eyesight more vulnerable and affects night vision and cognitive function.

You can rely on a supplement that claims to reverse poor eye health. Our team has identified the Sight Care eye health formula as one of the best ways to attain clear vision.

Sight Care can prove to be your go-to supplement if you spend your days in front of devices that emit blue light. The Sight Care formula comprises natural ingredients including minerals, herbs, roots, and plants. These ingredients are backed by science to produce impressive results and carry multiple health benefits for the human body.

It is a powerful supplement that helps you attain optimal eye health naturally. The formula of Sight Care has been developed from the work of a Nobel Prize winner who found a way to reverse age-related macular degeneration. This scientific vision breakthrough does not just improve visual acuity, it takes care of your overall health.

In this Sight Care review, we will explore all the details, big and small, about this supplement to help you make an informed choice about its purchase. But first, let us go through its summary below.

Category:

Dietary Supplement

Product Form:

Capsule

Serving Quantity:

60 capsules

Dosage Guideline:

Consume 2 capsules daily

Key Ingredients:

Astaxanthin, Quercetin, N-Acetyl-L-Cysteine, Zeaxanthin, Lutein, L-Lysine, Eyebright, Bilberry Fruit Extract, etc.

Product Characteristics:

Price:

$69 for one bottle (Official Website)

Key Benefits:

Bonus Products:

Yes

Money-Back Guarantee:

180-money-back guarantee

Sight Care is one of the best natural eye health supplements that is resplendent with features to benefit users immensely. This supplement can help you enjoy healthy vision without affecting other functions of your body. With the help of Sight Care capsules, you can notice significant improvements in your overall eye health. Lets go through the highlights of Sight Care below.

Made only using natural ingredients, Sight Care is a wonder for those people who easily catch side effects from artificial elements.

Sight Care is a peoples favorite because we found so many positive reviews about this supplement online.

The Sight Care eye health supplement works mainly to provide you with 20/20 vision and top-notch visual acuity so that you can differentiate between objects from far away.

If your night vision is suffering, you can use the exclusively designed formula of Sight Care which is scientifically backed to support optimal vision.

According to the Sight Care official website, this supplement is manufactured in FDA-registered and GMP-certified facilities in the USA.

The ingredients added in Sight Care are tested by third-party labs to protect you against side effects.

Get started today and see the difference SightCare can make >>>

The makers of Sight Care made a very important revelation through their research about the eye health of humans. According to them, the main reason behind poor eyesight is the low levels of adult repair stem cells after a certain age. Every person has these cells in abundance at a young age.

When your eyes suffer damage in your 20s or 30s, these cells transform themselves into healthy eye cells to keep your vision immaculate. According to the work of a Nobel Prize winner, these cells do not have any assigned role and make up for any damaged cells in the body.

So, as you grow older, you already use up most of these cells and as a result, develop age-related macular degeneration and poor night vision.

The Sight Care formula has been specially designed to boost the levels of adult repair stem cells using natural ingredients like astaxanthin, eyebright, quercetin, lutein, and zeaxanthin. It is one of the only natural eye health supplements to take this approach to improve your vision. As a result, Sight Care is popular among people who have increased exposure to blue light.

Sight Care ingredients work together to influence the growth of these cells so that you can experience healthy eyesight. These ingredients tell your body to produce brand-new stem cells to improve visual acuity and support clear vision. Several studies have proven the efficacy of Sight Care ingredients in improving stem cell potency.

This helps protect eye health and prevent age-related macular disease in old age. Sight Care is filled with nutrients that fight free radicals which in turn helps support eye health. This supplement improves blood circulation to the eye cells which allows the restoration of good vision at a cellular level. SightCare can protect you from blue light that is emitted from electronic devices.

The formula of SightCare is rich in antioxidants that support your cognitive function. By boosting blood flow in the body, Sight Care detoxifies your body and supports high energy levels. It can also help you adhere to a healthy lifestyle which helps promote your liver health. All in all, SightCare can prove highly beneficial for your overall well-being.

The formula of SightCare is rich in antioxidants that support your cognitive function. The connection between the eye and brain health is astonishing. When your brain receives proper nourishment, it prepares a foundation for better ocular health. Sight Care provides holistic support for superior brain health which paves the path for clear vision and better eye well-being.

Sight Care uses natural ingredients rich in amino acids which act as the building blocks for your brain cells. This tones down vision problems and helps improve visual acuity. Once your brain health becomes optimal, the eye tissues become stronger to support better vision.

One of the very few natural eye health supplements to support brain health and fight age-related eye complications, Sight Care improves your decision-making skills.

Sight Care Is On Sale Now For A Limited Time!

Sight Care supplements are brimming with ingredients that are very nourishing for your body. These capsules carry multiple health benefits for the human body. They can improve the well-being of your eyes, brain, liver, and immune system. Lets take a detailed look at the benefits of Sight Care.

This supplement supports healthy eyesight by boosting blood circulation in the body. It can help you in maintaining eye health so that you dont develop poor eyesight. This eye health formula can nourish your eye cells with better blood flow to offer improved eye health. It helps greatly in maintaining healthy vision and tackling vision problems.

Apart from promoting eye health, Sight Care also helps improve your brain health with the help of a nutrient-rich formulation. If you have poor eye health, it might be because of slow cognitive function and improper blood flow to the brain. Sight Care capsules help you attain optimal brain health to lead a healthy lifestyle.

The SightCare eye health supplement helps you maintain healthy eyesight by improving your visual acuity and supporting 20/20 vision. These capsules improve night vision and promote clear vision at all times to reduce the chances of vision impairment.

Sight Care supports a healthy inflammatory response to support optimal vision. It contains anti-inflammatory agents that support healthy vision in users.

This vision health supplement is well-formulated to improve your night vision and enhance visual acuity. SightCare helps protect eye health at nighttime using natural ingredients that boost healthy blood flow. It can help in maintaining night vision so that you can go out without anyones help.

The formula of the Sight Care vision supplement is rich in nutrients that are capable of boosting your energy levels. This vision health supplement can do more than just promote healthy vision so that you enjoy your life and lead a healthy lifestyle.

When you consume Sight Care supplements along with a balanced diet, it detoxifies your body and improves your liver health.

The natural ingredients present in the formula of this vision health supplement can nourish your body deeply. This supplement can improve your immunity and support brain function to support better overall health. You can notice a vast improvement in your general well-being after taking this dietary supplement daily.

To enjoy the benefits of SightCare, click here to order your supply now!

The natural ingredients in SightCare are backed by science. The ingredients, which include lutein and zeaxanthin, have been studied for their potential to improve overall eye health and help protect the eyes from certain conditions such as age-related macular degeneration (AMD). Below is an overview of some of these ingredients:

Astaxanthin consists of a long chain of conjugated double bonds with various functional groups attached, resulting in a highly complex structure. This compound is primarily found in certain types of microalgae, as well as in the marine animals that consume these algae.

It is able to neutralize harmful free radicals that are generated by exposure to ultraviolet (UV) radiation, environmental pollutants, and other sources of oxidative stress. By doing so, astaxanthin helps to protect the delicate tissues of the eye, including the retina, from damage caused by these free radicals, which can contribute to the development of age-related eye conditions such as macular degeneration and cataracts.

Furthermore, astaxanthin has been found to enhance blood flow to the retina and improve microcirculation within the eye. This can have a positive impact on visual acuity by ensuring an adequate supply of oxygen and nutrients to the cells of the retina, which are responsible for capturing light and transmitting visual signals to the brain.

N-Acetyl-L-Cysteine (NAC) is a powerful antioxidant that is added to Sight Care pills to promote eye health and boost night vision. This compound works by supporting the production of glutathione, a potent antioxidant that helps protect the eyes from oxidative stress and damage.

A clinical trial conducted by researchers at the University of Melbourne examined the effects of NAC supplementation on healthy individuals with impaired night vision. The study involved 52 participants who were randomly assigned to receive either NAC or a placebo for a period of three months. The participants night vision was assessed using a device that measured their ability to detect low levels of light.

The results of the study showed that the group receiving NAC supplementation experienced a significant improvement in night vision compared to the placebo group. Specifically, the participants who took NAC had a 45% improvement in their ability to detect low levels of light, while the placebo group showed no significant improvement.

Click here to claim your discount!

Eyebright, also known by its botanical name Euphrasia officinalis, is a small flowering plant that belongs to the Orobanchaceae family. It is native to Europe and has been used for centuries as a herbal remedy for various eye conditions. Eyebright gets its name from the belief that its flowers resemble an eye, with a dark spot at the center resembling the pupil.

From a scientific perspective, Eyebright contains several bioactive compounds, such as flavonoids, iridoid glycosides, and tannins, which contribute to its beneficial effects on mental sharpness and eye health.

Moreover, iridoid glycosides found in Eyebright possess anti-inflammatory properties that can benefit both mental sharpness and eye health.

By inhibiting inflammatory pathways, iridoid glycosides in Eyebright help to reduce inflammation in the brain, thereby supporting mental clarity and cognitive performance.

Bilberry fruit, known for its vibrant blue color and small size, resembles a blueberry but with a slightly darker hue. This small, power-packed berry has been used for centuries to alleviate signs of eye cell inflammation and boost overall vision.

A study involved 36 participants with early signs of age-related macular degeneration (AMD), a condition characterized by the deterioration of the central part of the retina. The participants were divided into two groups, with one group receiving bilberry extract and the other group receiving a placebo.

The study reported a 63% improvement in visual acuity and a 42% increase in macular pigment density in the bilberry group, compared to the placebo group.

Lutein and zeaxanthin act as natural antioxidants, which means they have the ability to neutralize harmful free radicals in the eye. A clinical trial conducted by the Age-Related Eye Disease Study 2 (AREDS2) Research Group demonstrated the effectiveness of these carotenoids in reducing the risk of advanced age-related macular degeneration (AMD).

The study showed that a daily supplement containing lutein (10 mg) and zeaxanthin (2 mg) significantly reduced the progression to advanced AMD by 18% in high-risk individuals.

A study published in the Journal of Optometry investigated the effects of lutein and zeaxanthin supplementation on visual function in healthy young individuals. The participants who received a supplement containing lutein (10 mg) and zeaxanthin (2 mg) for six months experienced a significant improvement in contrast sensitivity and glare recovery compared to the placebo group.

Contrast sensitivity refers to the ability to distinguish objects of similar brightness, while glare recovery is the ability to regain clear vision after exposure to bright lights.

Dont miss out on the benefits that Sight Care can provide order now!

SightCare is an amazing eye health supplement that can improve your visual acuity and prevent the onset of age-related eye diseases in older adults. There are many Sight Care reviews online where people have left admiring comments about this supplement. You will find this Sight Care review particularly insightful.

I was looking for a supplement to combat vision impairment and I came across SightCare. After taking it for a short time, I noticed a stark difference in my ocular health and I was impressed beyond words. As time went by, my eyesight improved and I have a perfect 20/20 vision now.

Sight Care is a pretty safe eye health supplement because it does not contain GMOs, gluten, dairy, stimulants, or chemical compounds in its formulation. The usage of 100% natural ingredients in Sight Care makes it safe for everyday intake for enhanced visual clarity.

Moreover, SightCare is manufactured in GMP-certified and FDA-registered facilities in the USA and tested for purity and quality. There are also no cases of side effects according to multiple positive Sight Care reviews.

There are 60 capsules in every bottle of the Sight Care supplement. When we looked through the official website of this supplement, we found that you need to take two petite Sight Care capsules every day to deal with vision problems and maintain healthy eyesight.

The Sight Care supplement makes use of blue light-obstructing ingredients that work efficiently to help maintain optimal eye health in users. This supplement can prevent age-related vision problems by blocking damage from blue light emitted from electronic devices. This blue light can cause age-related macular degeneration and degrade your eyesight.

According to the official website of Sight Care, this supplement helps in maintaining optimal eye health by reversing the damage caused by blue light.

You can find SightCare on its official website. Every bottle of this vision health supplement contains 30 servings and is available for $69. You can also buy this supplement for a lower price if you buy it in bulk.

If you want crystal clear vision, you must order the 3-bottle pack of SightCare for $177 ($59/per bottle). But, if you want improved overall eye health in the long run, you must order the 6-bottle pack of Sight Care for $294 ($49/per bottle).

The makers of Sight Care offer a whopping 180-day money-back guarantee on every order of this supplement from its official website. Every person is eligible for this 180-day money-back guarantee with no exceptions.

If you dont get any relief from age-related eye diseases, you can avail of this 180-day money-back guarantee to claim your refund. We should warn you that this 180-day money-back guarantee is only valid on the official website of Sight Care.

The Sight Care vision supplement comes with a bonus book on every three-bottle and six-bottle purchase from its official website. This bonus book is The Truth About Vision and it helps you to promote vision health naturally. You also get access to a private VIP Client Area where you can gain resources, guides, and videos about improving eye health.

Sight Care is one of the many natural eye health supplements available on the market. There are other supplements or eye vitamins that claim to enhance vision and tackle age-related vision diseases. We compared SightCare with these supplements to identify its strengths and weaknesses.

Divine Vision 12 is a well-known vision supplement that promises to promote optimal eye health in daily users. This supplement contains natural ingredients like bilberry and grape seed which are also found in the formula of Sight Care. Together with other ingredients, they improve vision and support optimal brain health.

This supplement blocks out the damaging blue light from digital devices to protect you against age-related macular disease. However, the benefits of Divine Vision 12 do not encapsulate your overall health. Unlike it, SightCare boosts your brain health, liver health, and immune health. Furthermore, it works to enhance adult repair stem cells which is scientifically proven to improve vision.

Click Here to Get SightCare At Discounted Price!!!

Eyesight Max is another vision health supplement that claims to support healthy eye function in users. The working of Eyesight Max is different from Sight Care. According to the makers of Eyesight Max, the main reason behind vision loss is PM 2.5 particles which are dangerous pollutants found everywhere in the atmosphere.

These particles damage your brain health which in turn worsens your vision. When these particles enter your blood circulation, your eyes suffer extensive damage. To support healthy vision, Eyesight Max forms a natural immune barrier. It helps cleanse your body of toxins to reduce the risk of developing age-related macular degeneration.

Both natural eye health supplements, Eyesight Max and SightCare enhance visual acuity and promote your overall well-being. However, there are no bonus books available with Eyesight Max. But, you get The Truth About Vision with Sight Care to improve your eyesight.

The last eye health supplement that we will compare Sight Care with is ReVision. It is a natural supplement that contains vitamins and minerals to boost blood flow to your eyes and improve your ocular health. Although it contains some of the same ingredients as SightCare, there is nothing special about the working of the ReVision formula.

Sight Care, on the other hand, aims to boost the levels of adult repair stem cells which are the newly discovered cause of vision loss in older adults. Low levels of these cells can lead to age-related macular disease and worsen your visual clarity. The formula of SightCare helps in promoting your vision well-being to block out the damage caused by blue light.

This supplement also provides other health benefits like improved energy levels and liver health. It also helps in promoting healthy brain function and improving eye-brain coordination.

Order SightCare Right Here At The Best Prices!!

Ensuring optimal vision goes beyond routine eye check-ups; its intrinsically linked to what we fuel our bodies with. A balanced diet rich in essential nutrients significantly contributes to maintaining healthy eyes.

Vitamin A takes the spotlight, playing a pivotal role in preventing night blindness and promoting overall eye health. Leafy greens, carrots, and sweet potatoes are rich sources. Equally crucial are antioxidants like lutein and zeaxanthin found in kale, spinach, and eggs, safeguarding against age-related macular degeneration.

Omega-3 fatty acids, found in fish like salmon and flaxseeds, nurture the eyes cell membranes. Zinc, prevalent in meat and dairy, supports the health of the retina. Antioxidant-rich fruits like berries add another layer of defense.

In essence, a diet encompassing a spectrum of colorful fruits, vegetables, and nutrient-dense proteins ensures a holistic approach to eye health, fortifying your vision for the long haul.

Recognizing signs of deteriorating eye health is paramount for timely intervention. Regular self-assessment can be a powerful tool. If you notice persistent blurriness, difficulty seeing in low light, or sudden changes in vision, its a clarion call to schedule an eye examination.

Go here to see the original:
Is Sight Care Legit? Review How SightCare Ingredients Work Before ... - Bellevue Reporter

Comparison of fludarabine/melphalan (FluMel) with fludarabine … – Nature.com

Granot N, Storb R. History of hematopoietic cell transplantation: challenges and progress. Haematologica. 2020;105:271629.

Article PubMed PubMed Central Google Scholar

Spyridonidis A, Labopin M, Savani BN, Niittyvuopio R, Blaise D, Craddock C, et al. Redefining and measuring transplant conditioning intensity in current era: a study in acute myeloid leukemia patients. Bone Marrow Transpl. 2020;55:111425.

Article CAS Google Scholar

Ciurea SO, Kongtim P, Varma A, Rondon G, Chen J, Srour S, et al. Is there an optimal conditioning for older patients with AML receiving allogeneic hematopoietic cell transplantation? Blood. 2020;135:44952.

Article PubMed PubMed Central Google Scholar

Eapen M, Brazauskas R, Hemmer M, Perez WS, Steinert P, Horowitz MM, et al. Hematopoietic cell transplant for acute myeloid leukemia and myelodysplastic syndrome: conditioning regimen intensity. Blood Adv. 2018;2:2095103.

Article PubMed PubMed Central CAS Google Scholar

Baron F, Labopin M, Peniket A, Jindra P, Afanasyev B, Sanz MA, et al. Reduced-intensity conditioning with fludarabine and busulfan versus fludarabine and melphalan for patients with acute myeloid leukemia: a report from the Acute Leukemia Working Party of the European Group for Blood and Marrow Transplantation. Cancer. 2015;121:104855.

Article PubMed CAS Google Scholar

Oran B, Ahn KW, Fretham C, Beitinjaneh A, Bashey A, Pawarode A, et al. Fludarabine and melphalan compared with reduced doses of busulfan and fludarabine improve transplantation outcomes in older patients with myelodysplastic syndromes. Transpl Cell Ther. 2021;27:921.e1921.e10.

Article Google Scholar

Duque-Afonso J, Finke J, Labopin M, Craddock C, Protheroe R, Kottaridis P, et al. Comparison of fludarabine-melphalan and fludarabine-treosulfan as conditioning prior to allogeneic hematopoietic cell transplantation-a registry study on behalf of the EBMT acute leukemia working party. Bone Marrow Transpl. 2022;57:126976.

Article CAS Google Scholar

Wsch R, Reisser S, Hahn J, Bertz H, Engelhardt M, Kunzmann R, et al. Rapid achievement of complete donor chimerism and low regimen-related toxicity after reduced conditioning with fludarabine, carmustine, melphalan and allogeneic transplantation. Bone Marrow Transpl. 2000;26:24350.

Article Google Scholar

Bertz H, Potthoff K, Finke J. Allogeneic stem-cell transplantation from related and unrelated donors in older patients with myeloid leukemia. J Clin Oncol. 2003;21:14804.

Article PubMed Google Scholar

Spyridonidis A, Bertz H, Ihorst G, Grllich C, Finke J. Hematopoietic cell transplantation from unrelated donors as an effective therapy for older patients (> or = 60 years) with active myeloid malignancies. Blood. 2005;105:41478.

Article PubMed CAS Google Scholar

Marks R, Potthoff K, Hahn J, Ihorst G, Bertz H, Spyridonidis A, et al. Reduced-toxicity conditioning with fludarabine, BCNU, and melphalan in allogeneic hematopoietic cell transplantation: particular activity against advanced hematologic malignancies. Blood. 2008;112:41525.

Article PubMed CAS Google Scholar

Ciurea SO, Saliba R, Rondon G, Pesoa S, Cano P, Fernandez-Vina M, et al. Reduced-intensity conditioning using fludarabine, melphalan and thiotepa for adult patients undergoing haploidentical SCT. Bone Marrow Transpl. 2010;45:42936.

Article CAS Google Scholar

Ciurea SO, Saliba RM, Hamerschlak N, Karduss Aurueta AJ, Bassett R, Fernandez-Vina M, et al. Fludarabine, melphalan, thiotepa and anti-thymocyte globulin conditioning for unrelated cord blood transplant. Leuk Lymphoma. 2012;53:9016.

Article PubMed PubMed Central CAS Google Scholar

Majolino I, Davoli M, Carnevalli E, Locasciulli A, Di Bartolomeo P, Scim R, et al. Reduced intensity conditioning with thiotepa, fludarabine, and melphalan is effective in advanced multiple myeloma. Leuk Lymphoma. 2007;48:75966.

Article PubMed CAS Google Scholar

Duque-Afonso J, Ihorst G, Waterhouse M, Zeiser R, Wsch R, Bertz H, et al. Impact of lung function on bronchiolitis obliterans syndrome and outcome after allogeneic hematopoietic cell transplantation with reduced-intensity conditioning. Biol Blood Marrow Transpl. 2018;24:227784.

Article Google Scholar

Duque-Afonso J, Ihorst G, Waterhouse M, Zeiser R, Wsch R, Bertz H, et al. Comparison of reduced-toxicity conditioning protocols using fludarabine, melphalan combined with thiotepa or carmustine in allogeneic hematopoietic cell transplantation. Bone Marrow Transpl. 2021;56:11020.

Article CAS Google Scholar

Kanate AS, Nagler A, Savani B. Summary of scientific and statistical methods, study endpoints and definitions for observational and registry-based studies in hematopoietic cell transplantation. Clin Hematol Int. 2019;2:24.

Article PubMed PubMed Central Google Scholar

Nagler A, Shimoni A Conditioning. In: Carreras E, Dufour C, Mohty M, Krger N, editors. The EBMT Handbook: Hematopoietic Stem Cell Transplantation and Cellular Therapies 2019. 7th edition. Cham (CH): Springer. Chapter 13.

Passweg JR, Baldomero H, Chabannon C, Basak GW, de la Cmara R, Corbacioglu S, et al. Hematopoietic cell transplantation and cellular therapy survey of the EBMT: monitoring of activities and trends over 30 years. Bone Marrow Transplant. 2021;56:165164.

Article PubMed PubMed Central Google Scholar

Saraceni F, Scortechini I, Fiorentini A, Dubbini MV, Mancini G, Federici I, et al. Conditioning regimens for frail patients with acute leukemia undergoing allogeneic stem cell transplant: how to strike gently. Clin Hematol Int. 2021;3:15360.

Article PubMed PubMed Central Google Scholar

Rambaldi A, Grassi A, Masciulli A, Boschini C, Mic MC, Busca A, et al. Busulfan plus cyclophosphamide versus busulfan plus fludarabine as a preparative regimen for allogeneic haemopoietic stem-cell transplantation in patients with acute myeloid leukaemia: an open-label, multicentre, randomised, phase 3 trial. Lancet Oncol. 2015;16:152536.

Article PubMed CAS Google Scholar

Russell JA, Tran HT, Quinlan D, Chaudhry A, Duggan P, Brown C, et al. Once-daily intravenous busulfan given with fludarabine as conditioning for allogeneic stem cell transplantation: study of pharmacokinetics and early clinical outcomes. Biol Blood Marrow Transpl. 2002;8:46876.

Article CAS Google Scholar

de Lima M, Couriel D, Thall PF, Wang X, Madden T, Jones R, et al. Once-daily intravenous busulfan and fludarabine: clinical and pharmacokinetic results of a myeloablative, reduced-toxicity conditioning regimen for allogeneic stem cell transplantation in AML and MDS. Blood. 2004;104:85764.

Article PubMed Google Scholar

Beelen DW, Stelljes M, Remnyi P, Wagner-Drouet EM, Dreger P, Bethge W, et al. Treosulfan compared with reduced-intensity busulfan improves allogeneic hematopoietic cell transplantation outcomes of older acute myeloid leukemia and myelodysplastic syndrome patients: final analysis of a prospective randomized trial. Am J Hematol. 2022;97:102334.

Article PubMed CAS Google Scholar

Shimoni A, Hardan I, Shem-Tov N, Rand A, Herscovici C, Yerushalmi R, et al. Comparison between two fludarabine-based reduced-intensity conditioning regimens before allogeneic hematopoietic stem-cell transplantation: fludarabine/melphalan is associated with higher incidence of acute graft-versus-host disease and non-relapse mortality and lower incidence of relapse than fludarabine/busulfan. Leukemia. 2007;21:210916.

Article PubMed CAS Google Scholar

Bernardo ME, Zecca M, Piras E, Vacca A, Giorgiani G, Cugno C, et al. Treosulfan-based conditioning regimen for allogeneic haematopoietic stem cell transplantation in patients with thalassaemia major. Br J Haematol. 2008;143:54851.

Article PubMed Google Scholar

Bernardo ME, Piras E, Vacca A, Giorgiani G, Zecca M, Bertaina A, et al. Allogeneic hematopoietic stem cell transplantation in thalassemia major: results of a reduced-toxicity conditioning regimen based on the use of treosulfan. Blood. 2012;120:4736.

Article PubMed CAS Google Scholar

Finke J, Schmoor C, Stelljes M, Burchert A, Dreger P, Hegenbart U, et al. Thiotepa-fludarabine-treosulfan conditioning for 2nd allogeneic HCT from an alternative unrelated donor for patients with AML: a prospective multicenter phase II trial. Bone Marrow Transpl. 2022;57:166470.

Article CAS Google Scholar

Sanz J, Boluda JC, Martn C, Gonzlez M, Ferr C, Serrano D, et al. Single-unit umbilical cord blood transplantation from unrelated donors in patients with hematological malignancy using busulfan, thiotepa, fludarabine and ATG as myeloablative conditioning regimen. Bone Marrow Transpl. 2012;47:128793.

Article CAS Google Scholar

Di Bartolomeo P, Santarone S, De Angelis G, Picardi A, Cudillo L, Cerretti R, et al. Haploidentical, unmanipulated, G-CSF-primed bone marrow transplantation for patients with high-risk hematologic malignancies. Blood. 2013;121:84957.

Article PubMed Google Scholar

Raiola AM, Dominietto A, Ghiso A, Di Grazia C, Lamparelli T, Gualandi F, et al. Unmanipulated haploidentical bone marrow transplantation and posttransplantation cyclophosphamide for hematologic malignancies after myeloablative conditioning. Biol Blood Marrow Transpl. 2013;19:11722.

Article CAS Google Scholar

Sora F, Grazia CD, Chiusolo P, Raiola AM, Bregante S, Mordini N. et al. Allogeneic hemopoietic stem cell transplants in patients with Acute Myeloid Leukemia (AML) prepared with Busulfan and Fludarabine (BUFLU) or Thiotepa, Busulfan, and Fludarabine (TBF): a retrospective study. Biol Blood Marrow Transpl. 2020;26:698703.

Article CAS Google Scholar

Saraceni F, Labopin M, Hamladji RM, Mufti G, Soci G, Shimoni A, et al. Acute leukemia working party of the European society for Blood and Marrow Transplantation (EBMT). Thiotepa-busulfan-fludarabine compared to busulfan-fludarabine for sibling and unrelated donor transplant in acute myeloid leukemia in first remission. Oncotarget. 2017;9:337993.

Article PubMed PubMed Central Google Scholar

Craddock C, Jackson A, Loke J, Siddique S, Hodgkinson A, Mason J, et al. Augmented reduced-intensity regimen does not improve postallogeneic transplant outcomes in acute myeloid leukemia. J Clin Oncol. 2021;39:76878.

Article PubMed CAS Google Scholar

Marsh RA, Lane A, Mehta PA, Neumeier L, Jodele S, Davies SM, et al. Alemtuzumab levels impact acute GVHD, mixed chimerism, and lymphocyte recovery following alemtuzumab, fludarabine, and melphalan RIC HCT. Blood. 2016;127:50312.

Article PubMed CAS Google Scholar

Bertz H, Spyridonidis A, Wsch R, Grllich C, Egger M, Finke J. A novel GVHD-prophylaxis with low-dose alemtuzumab in allogeneic sibling or unrelated donor hematopoetic cell transplantation: the feasibility of deescalation. Biol Blood Marrow Transpl. 2009;15:156370.

Article CAS Google Scholar

Read this article:
Comparison of fludarabine/melphalan (FluMel) with fludarabine ... - Nature.com

T-Cell Malignancies Reported in Patients Who Received BCMA … – OncLive

The FDA has received reports of T-cell malignancies in patients who have been treated with CD19- or BCMA-directed autologous CAR T-cell immunotherapies.1 These T-cell malignancies include CAR-positive lymphoma and were reported from postmarketing adverse effect (AE) data sources and clinical trials in patients who received several CAR T-cell products in the class.

The FDA has determined that the risk of T-cell malignancies is apparent in all currently approved CD19- and BCMA-directed genetically modified autologous CAR T-cell immunotherapies, including:

The FDA emphasizes that although the benefits of these products in their indicated uses continue to outweigh their potential risks, it is investigating the identified risk of T-cell malignancy, which may have serious outcomes, such as hospitalization and death.1 The agency is evaluating whether regulatory action pertaining to this risk is necessary.

All gene therapy products with integrating lentiviral or retroviral vectors, including CD19- and BCMA-directed genetically modified autologous T-cell immunotherapies, are labeled with a United States prescribing information class warning for the development of secondary malignancies. The initial FDA approvals of these products included postmarketing requirements per Section 505(o) of the Federal Food, Drug, and Cosmetic Act for investigators to conduct 15-year observational follow-up safety studies for the purpose of assessing the long-term safety profiles of these products and the risk of developing secondary malignancies after treatment.

Patients being treated with these CD19- and BCMA-directed genetically modified autologous T-cell immunotherapies should receive life-long monitoring for the development of new malignancies. If a patient develops a new malignancy after receiving treatment with these products, they should contact the respective products manufacturer to report the incident and receive instructions regarding the collection of patient samples that will be tested for the presence of the CAR transgene.

Suspected AEs, including T-cell malignancies, related to these products can be reported to the FDA at 1-800-FDA-1088 or http://www.fda.gov/medwatch.

Health care providers, clinical investigators, caregivers, and patients with questions about these products can contact the FDAs Center for Biologics Evaluation and Research atocod@fda.hhs.gov.

See the original post:
T-Cell Malignancies Reported in Patients Who Received BCMA ... - OncLive

NOT-AR-23-022: Request for Information on Themes for the NIAMS … – National Institutes of Health (.gov)

Request for Information on Themes for the NIAMS Strategic Plan for Fiscal Years 2025-2029

The National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) supports research into the causes, treatment, and prevention of arthritis and musculoskeletal and skin diseases; the training of basic and clinical scientists to carry out this research; and the dissemination of information on research progress in these diseases. NIAMS is updating its Strategic Plan to help guide the research, training, and information dissemination programs it supports between fiscal years 2025 through 2029. The new Plan will focus on cross-cutting thematic research opportunities that position the Institute to make a difference in the lives of all Americans.Because public input is a crucial step in this effort, the Institute issued a Request for Information (NOT-AR-22-023) and hosted a meeting attended by approximately 160 researchers, patient representatives, and staff from other Federal entities to gain insight into topics that could be included in the new Strategic Plan.

Through this Request for Information, NIAMS invites feedback from researchers in academia and industry, health care professionals, patient advocates and health advocacy organizations, scientific or professional organizations, Federal agencies, and other interested members of the public on the Institutes distillation of the input received to date. Professional societies and patient organizations are strongly encouraged to submit a single response that reflects the views of their entire membership.

Please provide your perspective on the following potential cross-cutting themes, examples, and bold aspirations. NIAMS is particularly interested in suggestions for additional or alternative:

Examples:

Bold Aspirations:

Examples:

Bold Aspiration:

Note: Efforts to identify and reduce health disparities and provide all Americans with equitable access to clinical and epidemiologic studies and healthcare should be considered for NIAMS-funded research projects whenever possible.

Examples:

Bold Aspirations:

Examples:

Bold Aspiration:

Note: Consistent with the note under Health disparities and health equity, studies of lifestyle factors and environmental exposures should include efforts to identify and reduce health disparities and provide all Americans with equitable access to clinical and epidemiologic studies and healthcare whenever possible.

Examples:

Bold Aspiration:

Note: Consistent with the note under Health disparities and health equity, clinical and epidemiologic research should include efforts to identify and reduce health disparities and provide all Americans with equitable access to clinical and epidemiologic studies and healthcare whenever possible.

Examples:

Bold Aspiration:

Examples:

Bold Aspiration:

Note: Training and workforce efforts are essential for the pursuit of all cross-cutting thematic research areas in the new NIAMS Strategic Plan.

Examples:

Bold Aspiration:

Examples:

Bold Aspirations:

Responses to this RFI must be submitted electronically at https://rfi.grants.nih.gov/?s=654a7bc81e7ccb6f7d03d792.

Responses must be received by Monday, January 1, 2024.

Responses to this RFI are voluntary. Do not include any proprietary, classified, confidential, trade secret, or sensitive information in your response. The responses will be reviewed by NIAMS staff, leadership, and Advisory Council members. Individual feedback will not be provided to any respondent. NIAMS will use the information submitted in response to this RFI at its discretion and will not provide comments to any respondents submission. Respondents are advised that the Government is under no obligation to acknowledge receipt of the information received or provide feedback to respondents with respect to any information submitted.The Government reserves the right to use any submitted information on public NIH websites, in reports, in summaries of the state of the science, in any possible resultant solicitation(s), grant(s), or cooperative agreement(s), or in the development of future funding opportunity announcements.

This RFI is for information and planning purposes only and shall not be construed as a solicitation, grant, or cooperative agreement, or as an obligation on the part of the Federal Government, the NIH, or individual NIH Institutes and Centers to provide support for any ideas identified in response to it. The Government will not pay for the preparation of any information submitted or for the Governments use of such information. No basis for claims against the U.S. Government shall arise as a result of a response to this request for information or from the Governments use of such information.

We look forward to your input and hope that you will share this RFI document with your colleagues.

More:
NOT-AR-23-022: Request for Information on Themes for the NIAMS ... - National Institutes of Health (.gov)

Fish-like genetic program used to turn human retinal cells into neurons – EurekAlert

image:

Overall, our study provides a proof-of-principle that human glia can be reprogrammed to cells that are capable of making new neurons," said Thomas Reh, PhD, University of Washington, USA. "This opens up an entirely new way to repair the retina in people that have lost neurons to disease or trauma."

Credit: Thomas Reh, PhD

Loss of neurons in in the retina due to trauma or disease leads to vision impairment or blindness, a process which is irreversible in humans. Interestingly, some animals like fish have the built-in ability to regenerate retinal neurons by turning another retinal cell type called Muller glia into neurons. This conversion does not happen spontaneously in humans and other mammals, but new research by Thomas Reh, Juliette Wohlschlegel, and colleagues at the University of Washington, USA, published in the journalStem Cell Reports, shows that human Muller glia can be coaxed into changing identity in the laboratory, which could serve as a potential source of new neurons to treat vision loss.

"Overall, our study provides a proof-of-principle that human glia can be reprogrammed to cells that are capable of making new neurons,said Thomas Reh, PhD, University of Washington."This opens up an entirely new way to repair the retina in people that have lost neurons to disease or trauma."

Muller glia are supportive cells in the retina which help photoreceptors and other retinal neurons to function properly. In some species like fish and birds, Muller glia turn into immature retinal cells upon injury and subsequently generate new retinal neurons. By contrast, Muller glia in the mammalian retina react to injury with scar formation and inflammation without making new neurons. This difference in behavior is based on different genetic programs being activated in fish versus mammalian Muller glia after injury. Artificial activation of a fish-like genetic program can turn mouse Muller glia into retinal neurons according to prior research. However, up until now, it has not been known if the same strategy can be used to convert human Muller glia into neurons. To answer this question, the researchers genetically modified human Muller glia in the lab to switch on neurons-specific genetic programs, as it naturally happens in fish. Indeed, within a week, the genetically modified cells adopted a neuron-like characteristics resembling immature retinal neurons. These findings suggest that human Muller glia can be coaxed into neurons and may serve as a resource to generate new neurons in patients retinas in the future. Of note, Muller glia in this study were derived from immature Muller glia and it remains to be seen if similar approaches can transform adult human Muller glia into neurons, and to what efficiency.

Stem Cell Reports

ASCL1 induces neurogenesis in human Muller glia

30-Nov-2023

Disclaimer: AAAS and EurekAlert! are not responsible for the accuracy of news releases posted to EurekAlert! by contributing institutions or for the use of any information through the EurekAlert system.

Continued here:
Fish-like genetic program used to turn human retinal cells into neurons - EurekAlert

Dr Hurwitz on Ongoing Investigations of the Use of CAR T-Cell … – OncLive

Michael Hurwitz, MD, PhD, associate professor, internal medicine (medical oncology), Yale School of Medicine, discusses the ongoing investigation into the use of CAR T-cell therapies in patients with solid tumors, such as kidney cancers.

Hurwitz begins by stating that considerations surrounding the use of CAR T-cell therapy in solid tumors, such as renal cell carcinoma (RCC), have been uncertain. The phase 1 COBALT-RCC trial (NCT04438083), which investigated CTX130 allogeneic CRISPR/Cas9engineered CAR T-cell therapy in patients with advanced clear cell RCC, is currently inactive. However, a new agent with similar attributes to the CAR T-cell product investigated in COBALT-RCC is under development and may improve upon the outcomes seen in COBALT-RCC, Hurwitz begins.

Another trial, the phase 1 TRAVERSE trial (NCT04696731), is ongoing at some sites, he explains. This trial involves off-the-shelf CAR T-cell therapy, Hurwitz explains. These modified CAR T cells are engineered to evade the recipient's immune response and eliminate the need for personalized CAR T-cell production, offering a faster turnaround that is crucial for individuals with advanced solid tumors, Hurwitz explains.Traditionally, introducing foreign T cells into the body triggers immune responses, which are addressed by removing human leukocyte antigens, so the body does not recognize the T cells as foreign. In these modified CAR T cells, the endogenous T-cell receptors are also removed, ensuring these cells do not perceive the body as foreign, he expands.

Along with the FDA approvals of CAR T cells for patients with hematologic malignancies, their application in solid tumors is evolving, Hurwitz emphasizes. Ongoing preclinical research aims to engineer safe, specific, and effective CAR T cells, he states.

These innovations with CAR T-cell therapy promise highly targeted, safe cancer treatments for patients with solid tumors, Hurwitz continues. Looking forward, the possibility of synergies between CAR T-cell therapy and other treatments looms, he notes. Although the timing of integrating CAR T-cell therapies into the solid tumor treatment armamentarium is uncertain, combining these products with other agents offers a glimpse into a future where cancer treatment is more effective and personalized.In essence, technological advances in cancer therapy are just beginning to unfold, Hurwitz adds. The future promises innovations and a convergence of technologies to reshape cancer treatment, ushering in an era of hope and healing, he concludes.

Go here to read the rest:
Dr Hurwitz on Ongoing Investigations of the Use of CAR T-Cell ... - OncLive

Gene-editing therapy by Vertex and CRISPR poised for FDA approval – The Boston Globe

Its kind of surreal, said Tornyenu, 22, who grew up in Bethlehem, Pa., and participated in a clinical trial at Childrens Hospital of Philadelphia. Im, like, wait, I dont have sickle cell anymore.

The life-changing drug, developed by Boston-based Vertex and its Swiss partner CRISPR Therapeutics, is expected to be approved by the Food and Drug Administration by Friday for people with severe cases of the disease. Called Casgevy, it would usher in a new era not only for those with sickle cell but also for medicine: The drug would be the first gene-editing therapy authorized by US regulators, and uses a tool called CRISPR.

The likely approval Casgevy was cleared by British regulators last month raises both the promise of cures for diseases as well as the ethical concerns that come with the power to manipulate the building blocks of human life. With an expected price tag in the seven figures, it also touches on issues of equity in medicine.

Sickle cell primarily afflicts people of African descent. Research on the disease languished for decades, which many experts blame on structural racism, particularly in funding.

For Tornyenu, Casgevy has meant an end to the searing pain crises that caused her to miss a week of classes every month as a high school senior. After getting the treatment, she took the spring semester off from Cornell to recover from the debilitating effects of chemotherapy that made room in her blood marrow for gene-edited cells.

But now she no longer dreads the arrival of cold weather, which would often induce excruciating pain in her hips and legs. A senior at Cornell, she has a job lined up as a consultant at PricewaterhouseCoopers in Boston after graduation.

Im very hopeful [Casgevy] will be approved, she said, because I dont know what I would have done otherwise.

The gene-editing method that became known as CRISPR was first reported in a landmark 2012 paper by American biochemist Jennifer Doudna of the University of California, Berkeley, and French microbiologist Emmanuelle Charpentier of the Max Planck Institute for Infection Biology. They would share the 2020 Nobel Prize in chemistry for their work on the tool.

Sickle cell was an obvious choice for scientists to tackle with CRISPR. It was the first human disorder understood on a molecular level, its underpinnings explained in a landmark 1949 paper written by the future two-time Nobel laureate Linus Pauling. Yet progress against the disease was slow for decades afterward.

Sickle cell affects hemoglobin, the oxygen-carrying protein in red blood cells. It causes the round, flexible blood cells to deform into a sickle shape and stick to vessel walls. That deprives tissues of oxygen, causing crushing pain that can often only be relieved with opioids and blood transfusions.

Sickle cell can also lead to strokes, damage organs, and cause early death. A 2019 study in JAMA Network Open estimated the life expectancy of adults with sickle cell in the US is 54 years, about 20 years shorter than the general population.

In a clinical trial, Casgevy demonstrated remarkable results. The medicine completely relieved 29 of 30 sickle cell patients of debilitating episodes of pain for at least one year among trial participants who were followed for at least 18 months, according to Vertex. The patients received a one-time intravenous infusion of edited stem cells that flipped a genetic switch to restore their blood cells ability to carry oxygen throughout their bodies.

This is what a potential cure looks like, said Dr. Stephan Grupp, chief of the Cellular Therapy and Transplant Section at Childrens Hospital of Philadelphia. He was the principal investigator at the trial site where Tornyenu got Casgevy and was paid by Vertex to help organize the study at locations across the US.

About 100,000 Americans, most of them Black or Hispanic, are believed to have sickle cell. The Vertex-CRISPR treatment was geared for those with severe and repeated pain crises, roughly 20,000 people in the US. As of 2021, almost 8 million people around the world live with sickle cell, according to the Institute for Health Metrics and Evaluation at the University of Washington in Seattle.

The FDA has approved four medicines for the disorder, but none has been remotely as effective as Casgevy, which is expected to cost more than $1 million for a one-time infusion in the US, according to experts. (No price has been announced in the United Kingdom.) Sickle cell can be cured with a bone-marrow transplant, but few patients have compatible donors.

Patients are already inquiring about Casgevy, said Dr. Sharl Azar, a hematologist at Massachusetts General Hospital and medical director of its Comprehensive Sickle Cell Disease Treatment Center. He said he is eager to see whether the FDA clears it, how broad the approval would be, and whether Medicare and Medicaid would cover it.

Theres a lot of unknowns that were looking forward to working out in the coming months, he said. But I think everyone, from patients to providers, recognizes that this is a big deal.

Rahman Oladigbolu, a 52-year-old Harvard-educated filmmaker in Brockton, is among local patients interested in Casgevy. He has had six joints his hips, shoulders, and knees surgically replaced since 2000 because of damage from sickle cell. He walks with a cane at times, often gets lightheaded, and takes opioids to relieve persistent pain.

When Oladigbolu was growing up in Nigeria, his grandmother would take him to traditional medicine men and medicine women who prescribed herbs and potions, some of which they rubbed into his aching limbs after cutting him with a razor blade. He moved to the US when he was 28 and currently takes a sickle cell drug called crizanlizumab, which reduces his pain but doesnt eliminate it.

Pursuing a cure has been like a side job all my life, said Oladigbolu, who receives treatment at Boston Medical Center.

CRISPR-based treatments will likely be approved for other disorders in the coming years, experts say, although its hard to predict for what and when. Researchers, including scientists at multiple biotech companies and hospitals in Massachusetts, are studying the potential of gene editing for a variety of diseases, from ALS to forms of cancer.

There will be other gene-editing therapies, certainly, but each disease is different, said Dr. Stuart Orkin, a researcher at Dana-Farber Cancer Institute and Boston Childrens Hospital who in 2008 helped identify the gene that Casgevy snips to treat sickle cell. For some diseases, its not clear what to edit. People will argue about whats the right target. Each one is a special case.

Gene editing has also raised ethical concerns. In 2018, a Chinese scientist, He Jiankui, was widely condemned when he announced that he used CRISPR to edit DNA in human embryos to try to make them immune to HIV. The experiment sparked fears that He had opened the door to creating so-called designer babies children whose genetic makeup is altered to produce desired traits.

Dr. George Q. Daley, dean of Harvard Medical School, was among those who said Hes experiment raised the specter of a Brave New World of eugenics. Casgevy, he said recently, is completely different. The modifications it makes to DNA only helps sickle cell patients and cannot be passed on to their children.

Daleys bigger worry concerns access to Casgevy. While wealthy countries like the US have hospitals and doctors capable of preparing patients for the treatment and administering it, he said, millions of people with sickle cell in sub-Saharan Africa dont have those options.

This is a triumph of modern biomedicine, he said. The major ethical concerns now are issues of cost and equitable distribution.

Casgevy isnt the only gene-based medicine on the horizon for sickle cell. The Somerville biotech Bluebird Bio hopes the FDA approves a so-called gene therapy, lovo-cel, by Dec. 20. It also proved remarkably effective in clinical trials.

Unlike Casgevy, which cuts a gene, lovo-cel adds a modified gene into a patients DNA to enable blood cells to deliver oxygen. The FDA has approved at least eight gene therapies for mostly rare diseases since 2017.

Both Casgevy and lovo-cel are expected to be breathtakingly expensive. That has renewed questions about whether the health care system can afford such cutting-edge medicines.

Still, the Institute for Clinical and Economic Review, or ICER, an independent Boston-based drug-pricing watchdog, estimates that either drug could cost nearly $2 million and be worth it, given the cumulative costs of treating sickle cell over a lifetime and the benefits the new approaches would bring to patients and families.

Casgevy, which was called exa-cel in clinical trials, works by editing a patients bone marrow stem cells to make high levels of fetal hemoglobin the healthy, oxygen-carrying form of hemoglobin produced during fetal development that is replaced by adult hemoglobin soon after birth.

Unlike adult hemoglobin, fetal hemoglobin resists forming a crescent shape in sickle cell patients, and scientists have long searched for a way to restart it. The researchers behind Casgevy solved the problem by editing a gene called BCL11A, which regulates fetal hemoglobin.

The treatment involves multiple steps over several months. Patients must donate stem cells to be modified at a laboratory. Then donors have to undergo a grueling regimen of chemotherapy to make room in their bone marrow for the genetically altered cells. Finally, the patients get the cells back through a single infusion.

Dr. David Altshuler, Vertexs chief scientific officer, acknowledged that the gene-editing treatment is extremely complex and resource intensive. He said Vertex is researching the possibility of developing a pill that could do what Casgevy does without gene editing. (Vertexs business partner for Casgevy, CRISPR Therapeutics, is based in Zug, Switzerland, but has most of its workforce in Boston.)

FDA officials have raised concerns about the possibility that Casgevy could inadvertently change patients DNA beyond the targeted disease so-called off-target editing. Dr. Daniel E. Bauer, a staff physician at Dana-Farber Cancer Institute and Boston Childrens Hospital, told an FDA advisory panel on Oct. 31 that Casgevy contains hundreds of millions of edited cells and one could undoubtedly go off target and cause leukemia. But he described the risk as modest given the benefits of the treatment.

Altshuler said recently that there is no evidence to date of off-target editing, but it is important to be humble and to continue to follow patients. Vertex and CRISPR have pledged to follow trial participants for 15 years to make sure they stay healthy.

Tornyenu, the Cornell student, says she considers Casgevy a miracle and would celebrate Dec. 8 every year if the FDA approves the drug that day.

For lack of a better term, she said, its a big FU to sickle cell.

Jonathan Saltzman can be reached at jonathan.saltzman@globe.com.

Read this article:
Gene-editing therapy by Vertex and CRISPR poised for FDA approval - The Boston Globe