Background: Type 1 diabetes (T1D) results in autoreactive T cells chronically destroying pancreatic islets. This often results in irreplaceable loss of insulin-producing beta cells. To reverse course, a combinatorial strategy of employing glucose-responsive insulin restoration coupled with inhibiting autoreactive immune responses is required. Methods: Non-obese diabetic mice received a single intraperitoneal implantation of a novel biomaterial co-seeded with insulin-producing islets and T regulatory cells (Tregs). Controls included biomaterial seeded solely with islets, or biomaterial only groups. Mice were interrogated for changes in inflammation and diabetes progression via blood glucose monitoring, multiplex serum cytokine profiling, flow cytometry and immunohistochemistry assessments. Results: Islet and Tregs co-seeded biomaterial recipients had increased longevity, insulin secretion, and normoglycemia through 180 days post-implantation compared to controls. Serum profile revealed reduced TNFα, IFNγ, IL-1β and increased IL-10, insulin, C-Peptide, PP and PPY in recipients receiving co-seeded biomaterial. Evaluation of the resected co-seeded biomaterial revealed reduced infiltrating autoreactive CD8 + and CD4 + T cells concomitant with sustained presence of Foxp3 + Tregs; further analysis revealed that the few infiltrated resident effector CD4+ or CD8+ T cells were anergic, as measured by low levels of IFNγ and Granzyme-B upon stimulation when compared to controls. Interestingly, studies also revealed increased Tregs in the pancreas. However, there was no restoration of the pancreas beta cell compartment, suggesting normoglycemia and production of insulin levels were largely supported by the implanted co-seeded biomaterial. Conclusion These studies show the efficacy of a combinatorial approach seeding Tregs with pancreatic islets in a novel self-assembling organoid for reversing T1D.

Background: Type 1 diabetes (T1D) results in autoreactive T cells chronically destroying pancreatic islets. This often results in irreplaceable loss of insulin-producing beta cells. To reverse course, a combinatorial strategy of employing glucose-responsive insulin restoration coupled with inhibiting autoreactive immune responses is required. Methods: Non-obese diabetic mice received a single intraperitoneal implantation of a novel biomaterial co-seeded with insulin-producing islets and T regulatory cells (Tregs). Controls included biomaterial seeded solely with islets, or biomaterial only groups. Mice were interrogated for changes in inflammation and diabetes progression via blood glucose monitoring, multiplex serum cytokine profiling, flow cytometry and immunohistochemistry assessments. Results: Islet and Tregs co-seeded biomaterial recipients had increased longevity, insulin secretion, and normoglycemia through 180 days post-implantation compared to controls. Serum profile revealed reduced TNFα, IFNγ, IL-1β and increased IL-10, insulin, C-Peptide, PP and PPY in recipients receiving co-seeded biomaterial. Evaluation of the resected co-seeded biomaterial revealed reduced infiltrating autoreactive CD8 + and CD4 + T cells concomitant with sustained presence of Foxp3 + Tregs; further analysis revealed that the few infiltrated resident effector CD4+ or CD8+ T cells were anergic, as measured by low levels of IFNγ and Granzyme-B upon stimulation when compared to controls. Interestingly, studies also revealed increased Tregs in the pancreas. However, there was no restoration of the pancreas beta cell compartment, suggesting normoglycemia and production of insulin levels were largely supported by the implanted co-seeded biomaterial. Conclusion These studies show the efficacy of a combinatorial approach seeding Tregs with pancreatic islets in a novel self-assembling organoid for reversing T1D.

Multiple morphological abnormalities of the flagella (MMAF) represent a severe form of sperm defects leading to asthenozoospermia and male infertility. In this study, we identified a novel homozygous splicing mutation (c.871-4 ACA>A) in the adenylate kinase 7 (AK7) gene by whole-exome sequencing in infertile individuals. Spermatozoa from affected individuals exhibited typical MMAF characteristics, including coiled, bent, short, absent, and irregular flagella. Transmission electron microscopy analysis showed disorganized axonemal structure and abnormal mitochondrial sheets in sperm flagella. Immunofluorescence staining confirmed the absence of AK7 protein from the patients' spermatozoa, validating the pathogenic nature of the mutation. This study provides direct evidence linking the AK7 gene to MMAF-associated asthenozoospermia in humans, expanding the mutational spectrum of AK7 and enhancing our understanding of the genetic basis of male infertility.
Humans ; Male ; Sperm Tail/ultrastructure* ; Homozygote ; Consanguinity ; Asthenozoospermia/pathology* ; Infertility, Male/genetics* ; Mutation ; Pakistan ; Adenylate Kinase/genetics* ; Adult ; Pedigree ; RNA Splicing ; Exome Sequencing ; Spermatozoa

Male infertility can result from impaired sperm motility caused by multiple morphological abnormalities of the flagella (MMAF). Distinct projections encircling the central microtubules of the spermatozoal axoneme play pivotal roles in flagellar bending and spermatozoal movement. Mammalian sperm-associated antigen 17 ( SPAG17 ) encodes a conserved axonemal protein of cilia and flagella, forming part of the C1a projection of the central apparatus, with functions related to ciliary/flagellar motility, skeletal growth, and male fertility. This study investigated two novel homozygous SPAG17 mutations (M1: NM_206996.2, c.829+1G>T, p.Asp212_Glu276del; and M2: c.2120del, p.Leu707*) identified in four infertile patients from two consanguineous Pakistani families. These patients displayed the MMAF phenotype confirmed by Papanicolaou staining and scanning electron microscopy assays of spermatozoa. Quantitative real-time polymerase chain reaction (PCR) of patients' spermatozoa also revealed a significant decrease in SPAG17 mRNA expression, and immunofluorescence staining showed the absence of SPAG17 protein signals along the flagella. However, no apparent ciliary-related symptoms or skeletal malformations were observed in the chest X-rays of any of the patients. Transmission electron microscopy of axoneme cross-sections from the patients showed incomplete C1a projection and a higher frequency of missing microtubule doublets 1 and 9 compared with those from fertile controls. Immunofluorescence staining and Western blot analyses of spermatogenesis-associated protein 17 (SPATA17), a component of the C1a projection, and sperm-associated antigen 6 (SPAG6), a marker of the spring layer, revealed disrupted expression of both proteins in the patients' spermatozoa. Altogether, these findings demonstrated that SPAG17 maintains the integrity of spermatozoal flagellar axoneme, expanding the phenotypic spectrum of SPAG17 mutations in humans.
Humans ; Male ; Infertility, Male/pathology* ; Sperm Tail/ultrastructure* ; Homozygote ; Microtubule-Associated Proteins/genetics* ; Axoneme/genetics* ; Spermatozoa/ultrastructure* ; Adult ; Mutation ; Sperm Motility/genetics* ; Pedigree ; Microtubules ; Microtubule Proteins/genetics*

The syndrome of multiple morphological abnormalities of the sperm flagella (MMAF) is one of the most serious kinds of sperm defects, leading to asthenoteratozoospermia and male infertility. In this study, we use whole-exome sequencing to identify genetic factors that account for male infertility in a patient born from a consanguineous Pakistani couple. A homozygous frameshift mutation (c.1399_1402del; p.Gln468ArgfsTer2) in axonemal dynein light chain domain containing 1 ( AXDND1 ) was identified in the patient. Sanger sequencing data showed that the mutation was cosegregated recessively with male infertility in this family. Papanicolaou staining and scanning electron microscopy analysis of the sperm revealed severely abnormal flagellar morphology in the patient. Immunofluorescence and western blot showed undetectable AXDND1 expression in the sperm of the patient. Transmission electron microscopy analysis showed disorganized sperm axonemal structure in the patient, particularly missing the central pair of microtubules. Immunofluorescence staining showed the absence of sperm-associated antigen 6 (SPAG6) and dynein axonemal light intermediate chain 1 (DNALI1) signals in the sperm flagella of the patient. These findings indicate that AXDND1 is essential for the organization of flagellar axoneme and provide direct evidence that AXDND1 is a MMAF gene in humans, thus expanding the phenotypic spectrum of AXDND1 frameshift mutations.
Humans ; Male ; Sperm Tail/ultrastructure* ; Frameshift Mutation ; Infertility, Male/pathology* ; Pakistan ; Pedigree ; Consanguinity ; Axonemal Dyneins/genetics* ; Adult ; Spermatozoa ; Exome Sequencing

Background: Type 1 diabetes (T1D) results in autoreactive T cells chronically destroying pancreatic islets. This often results in irreplaceable loss of insulin-producing beta cells. To reverse course, a combinatorial strategy of employing glucose-responsive insulin restoration coupled with inhibiting autoreactive immune responses is required. Methods: Non-obese diabetic mice received a single intraperitoneal implantation of a novel biomaterial co-seeded with insulin-producing islets and T regulatory cells (Tregs). Controls included biomaterial seeded solely with islets, or biomaterial only groups. Mice were interrogated for changes in inflammation and diabetes progression via blood glucose monitoring, multiplex serum cytokine profiling, flow cytometry and immunohistochemistry assessments. Results: Islet and Tregs co-seeded biomaterial recipients had increased longevity, insulin secretion, and normoglycemia through 180 days post-implantation compared to controls. Serum profile revealed reduced TNFα, IFNγ, IL-1β and increased IL-10, insulin, C-Peptide, PP and PPY in recipients receiving co-seeded biomaterial. Evaluation of the resected co-seeded biomaterial revealed reduced infiltrating autoreactive CD8 + and CD4 + T cells concomitant with sustained presence of Foxp3 + Tregs; further analysis revealed that the few infiltrated resident effector CD4+ or CD8+ T cells were anergic, as measured by low levels of IFNγ and Granzyme-B upon stimulation when compared to controls. Interestingly, studies also revealed increased Tregs in the pancreas. However, there was no restoration of the pancreas beta cell compartment, suggesting normoglycemia and production of insulin levels were largely supported by the implanted co-seeded biomaterial. Conclusion These studies show the efficacy of a combinatorial approach seeding Tregs with pancreatic islets in a novel self-assembling organoid for reversing T1D.

Background: Type 1 diabetes (T1D) results in autoreactive T cells chronically destroying pancreatic islets. This often results in irreplaceable loss of insulin-producing beta cells. To reverse course, a combinatorial strategy of employing glucose-responsive insulin restoration coupled with inhibiting autoreactive immune responses is required. Methods: Non-obese diabetic mice received a single intraperitoneal implantation of a novel biomaterial co-seeded with insulin-producing islets and T regulatory cells (Tregs). Controls included biomaterial seeded solely with islets, or biomaterial only groups. Mice were interrogated for changes in inflammation and diabetes progression via blood glucose monitoring, multiplex serum cytokine profiling, flow cytometry and immunohistochemistry assessments. Results: Islet and Tregs co-seeded biomaterial recipients had increased longevity, insulin secretion, and normoglycemia through 180 days post-implantation compared to controls. Serum profile revealed reduced TNFα, IFNγ, IL-1β and increased IL-10, insulin, C-Peptide, PP and PPY in recipients receiving co-seeded biomaterial. Evaluation of the resected co-seeded biomaterial revealed reduced infiltrating autoreactive CD8 + and CD4 + T cells concomitant with sustained presence of Foxp3 + Tregs; further analysis revealed that the few infiltrated resident effector CD4+ or CD8+ T cells were anergic, as measured by low levels of IFNγ and Granzyme-B upon stimulation when compared to controls. Interestingly, studies also revealed increased Tregs in the pancreas. However, there was no restoration of the pancreas beta cell compartment, suggesting normoglycemia and production of insulin levels were largely supported by the implanted co-seeded biomaterial. Conclusion These studies show the efficacy of a combinatorial approach seeding Tregs with pancreatic islets in a novel self-assembling organoid for reversing T1D.