Biology subjects

Salama, A.

Publications and source records attributed to Salama, A..

6 recordsLinked to original sources

bioRxiv2024

Three-step scalable production of extracellular vesicles from pancreatic beta cells in stirred tank bioreactors promotes cell maturation and release of ectosomes with preserved immunomodulatory properties

Small extracellular vesicles (sEV) released by healthy beta cells are promising candidates for diabetes therapy thanks to their aptitude to modulate inflammation, to induce or maintain pancreatic function and to prevent pathogenic mechanisms. To advance the clinical development of therapeutics, there is a crucial need for scalable production methods. Stirred tank bioreactors (STR) are widely used in the industry due to their ability to provide homogeneous gas and nutrient supply, online monitoring, and efficient scale up. Anchorage- dependent cells can be cultured in STR on microcarriers or as spheroids, but may experience shear stress, which can affect sEV phenotype and function. Using pancreatic beta cells, this study identifies critical cell culturing parameters, including culture mode (monolayer vs. spheroids), medium formulation (with or without serum, glucose control), and process parameters (stirring, duration, cell density). The findings show that small spheroid culture promotes beta cell maturation without decreasing the yield of sEV per cell, despite a reduced cell surface exchange area. However, stirring increased expression of cellular stress markers and decreased cell viability. Set up of a three-step bioprocess allowed to maximize cell viability and sEV yields at high cell density over short production duration. sEV produced under these conditions maintained high purity, membrane integrity, and the aptitude to reduce T- lymphocyte proliferation and IFN-{gamma} cytokine secretion in a mixed lymphocyte reaction. Flow cytometry analysis revealed lower CD63/CD81 ratios in STR, indicating enhanced ectosome production. Switch from high glucose expansion to low glucose production medium further allowed to direct sorting of the antigen insulin into beta-sEV. This study demonstrates the feasibility of producing functional sEV from mature beta cells cultured as small spheroids, suitable for upscale. Production of sEV in STR may be particularly beneficial for ectosome- enriched compound loading for therapeutic applications. Graphical abstractRational development of a scalable bioprocess to control extracellular vesicles production & function. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/611247v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@6eaefeorg.highwire.dtl.DTLVardef@a2da3borg.highwire.dtl.DTLVardef@1a58f35org.highwire.dtl.DTLVardef@5cfd1d_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LISmall 3D-spheroid culture promotes cell maturation C_LIO_LIStirring induces cellular stress responses & promotes ectosome release from the cytoplasmic membrane C_LIO_LIDesign of experiment efficiently enhances cell viability and EV yield with preserved immune-modulatory properties C_LIO_LIGlucose-starvation during the production phase directs insulin-sorting into extracellular vesicles C_LI

bioengineering↗

bioRxiv2022

LIS1, a glyco-humanized swine polyclonal anti-lymphocyte globulin, as a novel induction treatment in solid organ transplantation

Anti-thymocyte/lymphocyte globulins (ATGs/ALGs) are immunosuppressive drugs used in induction therapies to prevent acute rejection in solid organ transplantation. Because of animal origin, ATGs/ALGs contain highly immunogenic carbohydrate xenoantigens eliciting antibodies that are associated with subclinical inflammatory events possibly impacting long-term graft survival. Their strong and long-lasting lymphodepleting activity also increases the risk for infections. To circumvent these drawbacks, LIS1 has been engineered as a glyco-humanized polyclonal antibody obtained by immunizing genetically modified pigs knocked out for major xeno-antigens Gal and Neu5Gc. It is Fc-silenced in humans and differs from other ATGs/ALGs by its mechanism of action excluding antibody-dependent cell-mediated cytotoxicity and being restricted to complement and phagocyte-mediated cytotoxicity, apoptosis and antigen masking, resulting in profound inhibition of T-cell alloreactivity in mixed-leucocyte-reactions. Preclinical evaluation in macaques showed that LIS1 impacted CD4+, CD8+ effector T cells but not T-reg, B cells or myeloid cells. Compared with rabbit ATG, LIS1 induced transient depletion of target T cells in the peripheral blood but was equivalent in preventing allograft rejection in a skin allograft model. The novel therapeutic modality of LIS1 might present advantages in induction treatment after organ transplantation by shortening the T-cell depletion period while maintaining adequate immunosuppression and reducing immunogenicity.

immunology↗

bioRxiv2022

Effect of psilocybin on marble-burying in ICR mice: Role of 5-HT1A receptors and implications for the treatment of obsessive-compulsive disorder

BackgroundPreliminary clinical findings, supported by preclinical studies employing behavioral paradigms such as marble-burying, suggest that psilocybin may be effective in treating obsessive-compulsive disorder. AimsTo explore 1) the role of 5-HT2A and 5-HT1A receptors in the effect of psilocybin on marble-burying; 2) the effect of staggered versus bolus psilocybin administration and persistence of the effect; 3) the effect of the 5-HT1A partial agonist, buspirone, on marble-burying and the head-twitch response (HTR) induced by psilocybin, a rodent correlate of psychedelic effects. MethodsMale ICR mice were administered psilocybin 4.4 mg/kg, escitalopram 5 mg/kg, 8-OH-DPAT 2 mg/kg, M100907 2 mg/kg, buspirone 5 mg/kg, WAY100635 2 mg/kg or combinations, intraperitoneally, and were tested on the MBT. HTR was examined in a magnetometer-based assay. Results1) Psilocybin and escitalopram significantly reduced marble-burying. The effect of psilocybin was not attenuated by the 5-HT2A antagonist, M100907. The 5-HT1A agonist, 8-OH-DPAT reduced marble-burying as did the 5-HT1A partial agonist, buspirone. The effect of 8-OH-DPAT was additive to that of psilocybin but that of buspirone was not. The 5-HT1A antagonist, WAY100635, attenuated the effect of 8-OH-DPAT and buspirone but not the effect of psilocybin. 2) Psilocybin injections over 3.5 hours had no effect on marble-burying and the effect of bolus injection was not persistent. 3) Co-administration of buspirone with psilocybin blocked its effect on HTR ConclusionsNeither 5-HT2A nor 5-HT1A receptors are pivotally implicated in the effect of psilocybin on marble-burying. Co-administration with buspirone may block the psychedelic effects of psilocybin without impeding its anti-obsessional effects.

pharmacology and toxicology↗

bioRxiv2022

IL-34 deficiency impairs FOXP3+ Treg function and increases susceptibility to autoimmunity

Immune homeostasis requires fully functional Tregs with a stable phenotype to control autoimmunity. Although IL-34 is a cytokine first described as mainly involved in monocyte cell survival and differentiation, we recently described its expression by CD8+ Tregs in a rat model of transplantation tolerance and by activated FOXP3+ CD4+ and CD8+ Tregs in human healthy individuals. However, its role in autoimmunity and potential in human diseases remain to be determined. Here we report that the absence of expression of IL-34 in Il34-/- rodents leads to an unstable phenotype, with production of multiple auto-antibodies, exacerbated under inflammatory conditions with increased susceptibility to DSS- and TNBS-colitis in deficient animals. Moreover, we strikingly revealed the inability of Il34-/- CD4+ Tregs to protect Il2rg-/- rats from a wasting disease induced by transfer of pathogenic cells, in contrast to Il34+/+ CD4+ Tregs. We also showed that IL-34 treatment delayed EAE in mice as well as GVHD and human skin allograft rejection in immune humanized immunodeficient NSG mice. Finally, we show that presence of IL-34 in the serum is associated with a longer rejection-free period in kidney transplanted patients. Altogether, our data emphasize on the crucial necessity of IL-34 for immune homeostasis and for CD4+ Tregs suppressive function. Our data also shows the therapeutic potential of IL-34 in human transplantation and auto-immunity.

immunology↗

bioRxiv2020

High neutralizing potency of swine glyco-humanized polyclonal antibodies against SARS-CoV-2

Perfusion of convalescent plasma (CP) has demonstrated a potential to improve the pneumonia induced by SARS-CoV-2, but procurement and standardization of CP are barriers to its wide usage. Many monoclonal antibodies (mAbs) have been developed but appear insufficient to neutralize SARS-CoV-2 unless two or three of them are being combined. Therefore, heterologous polyclonal antibodies of animal origin, that have been used for decades to fight against infectious agents might represent a highly efficient alternative to the use of CP or mAbs in COVID-19 by targeting multiple antigen epitopes. However, conventional heterologous polyclonal antibodies trigger human natural xenogeneic antibody responses particularly directed against animal-type carbohydrate epitopes, mainly the N-glycolyl form of the neuraminic acid (Neu5Gc) and the Gal 1,3-galactose (Gal), ultimately forming immune complexes and potentially leading to serum sickness or allergy. To circumvent these drawbacks, we engineered animals lacking the genes coding for the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) and 1,3-galactosyl-transferase (GGTA1) enzymes to produce glyco-humanized polyclonal antibodies (GH-pAb) lacking Neu5Gc and -Gal epitopes. We found that pig IgG Fc domains fail to interact with human Fc receptors and thereby should confer the safety advantage to avoiding macrophage dependent exacerbated inflammatory responses, a drawback possibly associated with antibody responses against SARS-CoV-2 or to avoiding a possible antibody-dependent enhancement (ADE). Therefore, we immunized CMAH/GGTA1 double knockout (DKO) pigs with the SARS-CoV-2 spike receptor-binding domain (RBD) to elicit neutralizing antibodies. Animals rapidly developed a hyperimmune response with anti-SARS-CoV-2 end-titers binding dilutions over one to a million and end-titers neutralizing dilutions of 1:10,000. The IgG fraction purified and formulated following clinical Good Manufacturing Practices, named XAV-19, neutralized Spike/angiotensin converting enzyme-2 (ACE-2) interaction at a concentration < 1g/mL and inhibited infection of human cells by SARS-CoV-2 in cytopathic assays. These data and the accumulating safety advantages of using glyco-humanized swine antibodies in humans warranted clinical assessment of XAV-19 to fight against COVID-19.

immunology↗

bioRxiv2019

Prenatal heat stress effects on gestation and postnatal behavior in kid goats

Consequences of heat stress during pregnancy can affect the normal development of the offspring. In the present experiment, 30 Murciano-Granadina dairy goats (41.8 {+/-} 5.7 kg) were exposed to 2 thermal environments varying in temperature-humidity index (THI) from 12 days before mating to 45 days of gestation. The environmental conditions were: gestation thermal-neutral (GTN; THI = 71 {+/-} 3); and gestation heat stress (GHS; THI = 85 {+/-} 3). At 27 {+/-} 4 days old, GTN-born female kids (n = 16) and GHS-born ones (n = 10) were subjected to 2 tests: arena test (AT) and novel object test (NOT), the latter was repeated at 3 months of age. Additionally, 8 months after birth, a subset of growing goats (n = 8) coming from GTN and GHS (16.8 {+/-} 3.4 kg BW) were exposed consecutively to 2 environmental conditions: a basal thermal-neutral period (THI = 72 {+/-} 3) for 7 days, and a heat-stress period (THI = 87 {+/-} 2) for 21 days. In both periods, feeding behavior, resting behavior, other active behaviors (exploring, grooming), thermally-associated behaviors and posture were recorded. The gestation length was shortened by 3 days in GHS goats. In the AT, GHS kids showed a lower number of sniffs (P < 0.01) compared to GTN. In the NOT, GHS kids also tended to show a lower number of sniffs (P = 0.09). During heat exposure, GTN and GHS growing goats spent more time resting as well as exhibited more heat-stress related behaviors such as panting and drinking (P < 0.001); however, no differences were observed between both groups. In conclusion, heat stress during the first third of pregnancy shortened gestation length and influenced the exploratory behavior of the kids in the early life without impact on the behavior during the adulthood when exposed to heat stress.

animal behavior and cognition↗