Expression of Protein Markers in Spermatogenic and Supporting Sertoli Cells Affected by High Abdominal Temperature in Cryptorchidism Model Mice.

Cryptorchidism is a congenital abnormality resulting in increased rates of infertility and testicular cancer. We used cryptorchidism model mice that presented with the translocation of the left testis from the scrotum to the abdominal cavity. Mice underwent the surgical procedure of the left testis at day 0 and were sacrificed at days 3, 5, 7, 14, 21, and 28 post-operatively. The weight of the left cryptorchid testis decreased significantly at days 21 and 28. The morphological changes were observed after 5 days and showed detached spermatogenic cells and abnormal formation of acrosome at day 5, multinucleated giant cells at day 7, and atrophy of seminiferous tubules at days 21 and 28. The high abdominal temperature disrupted the normal expression of cell adhesion molecule-1, Nectin-2, and Nectin-3 which are essential for spermatogenesis. In addition, the pattern and alignment of acetylated tubulin in cryptorchid testes were also changed at days 5, 7, 14, 21, and 28. Ultrastructure of cryptorchid testes revealed giant cells that had been formed by spermatogonia, spermatocytes, and round and elongating spermatids. The study's findings reveal that cryptorchidism's duration is linked to abnormal changes in the testis, impacting protein marker expression in spermatogenic and Sertoli cells. These changes stem from the induction of high abdominal temperature.

Quantitative evaluation of spermatogenesis by fluorescent histochemistry.

Identifying the types of spermatogenic cells that compose seminiferous tubules, as well as qualitative confirmation of the presence or absence of disorders, has been regarded as crucial in spermatogenesis. Sperm count and fertilizing capacity, both of which depend on the quality as well as quantity of spermatogenesis, are factors critical to fertilization. However, the quantitative assessment of spermatogenesis is not commonly practiced. Spermatogenesis has species-specific stages; when the specific stage in the seminiferous tubules is precisely determined, the types of spermatogenic cells in each stage can be spontaneously identified. Thereafter, a unique marker is used to classify the cells observed in each stage. Quantitative assessment of spermatogenesis has the potential to detect inapparent spermatogenesis disorders or numerically indicate the degree of the disorder. To this end, a histochemical approach using unique markers is indispensable for the quantitative assessment of spermatogenesis. Future developments in techniques to measure cell populations using computer software will further facilitate the establishment of quantitative assessment of spermatogenesis as a standard analysis method that can contribute significantly to advance our understanding of spermatogenesis.

Nitric oxide facilitates the targeting Kupffer cells of a nano-antioxidant for the treatment of NASH.

Kupffer cells are a key source of reactive oxygen species (ROS) and are implicated in the development of steatohepatitis and fibrosis in nonalcoholic steatohepatitis (NASH). We recently developed a polythiolated and mannosylated human serum albumin (SH-Man-HSA), a nano-antioxidant that targets Kupffer cells, in which the mannosyl units on albumin allows their specific uptake by Kupffer cells via the mannose receptor C type 1 (MRC1), and in which the polythiolation confers antioxidant activity. The aim of this study was to investigate the therapeutic potential of SH-Man-HSA in NASH model mice. In livers from mice and/or patients with NASH, we observed a reduced blood flow in the liver lobes and the down-regulation in MRC1 expression in Kupffer cells, and SH-Man-HSA alone failed to improve the pathological phenotype in NASH. However, the administration of a nitric oxide (NO) donor restored hepatic blood flow and increased the expression of the mannose receptor C type 2 (MRC2) instead of MRC1. Consequently, treatment with a combination of SH-Man-HSA and an NO donor improved oxidative stress-associated pathology. Finally, we developed a hybrid type of nano-antioxidant (SNO-Man-HSA) via the S-nitrosation of SH-Man-HSA. This nanomedicine efficiently delivered both NO and thiol groups to the liver, with a hepatoprotective effect that was comparable to the combination therapy of SH-Man-HSA and an NO donor. These findings suggest that SNO-Man-HSA has the potential for functioning as a novel nano-therapy for the treatment of NASH.

Oral Coadministration of Zn-Insulin with d-Form Small Intestine-Permeable Cyclic Peptide Enhances Its Blood Glucose-Lowering Effect in Mice.

Oral delivery of insulin remains a challenge owing to its poor permeability across the small intestine and enzymatic digestion in the gastrointestinal tract. In a previous study, we identified a small intestine-permeable cyclic peptide, C-DNPGNET-C (C-C disulfide bond, cyclic DNP peptide), which facilitated the permeation of macromolecules. Here, we showed that intraintestinal and oral coadministration of insulin with the cyclic DNP derivative significantly reduced blood glucose levels by increasing the portal plasma insulin concentration following permeation across the small intestine of mice. We also found that protecting the cyclic DNP derivative from enzymatic digestion in the small intestine of mice using d-amino acids and by the cyclization of DNP peptide was essential to enhance cyclic DNP derivative-induced insulin absorption across the small intestine. Furthermore, intraintestinal and oral coadministration of insulin hexamer stabilized by zinc ions (Zn-insulin) with cyclic D-DNP derivative was more effective in facilitating insulin absorption and inducing hypoglycemic effects in mice than the coadministration of insulin with the cyclic D-DNP derivative. Moreover, Zn-insulin was more resistant to degradation in the small intestine of mice compared to insulin. Intraintestinal and oral coadministration of Zn-insulin with cyclic DNP derivative also reduced blood glucose levels in a streptozotocin-induced diabetes mellitus mouse model. A single intraintestinal administration of the cyclic D-DNP derivative did not induce any cytotoxicity, either locally in the small intestine or systemically. In summary, we demonstrated that coadministration of Zn-insulin with cyclic D-DNP derivative could enhance oral insulin absorption across the small intestine in mice.

Development of Kupffer cell targeting type-I interferon for the treatment of hepatitis via inducing anti-inflammatory and immunomodulatory actions.

Because of its multifaceted anti-inflammatory and immunomodulatory effects, delivering type-I interferon to Kupffer cells has the potential to function as a novel type of therapy for the treatment of various types of hepatitis. We report herein on the preparation of a Kupffer cell targeting type-I interferon, an albumin-IFNα2b fusion protein that contains highly mannosylated N-linked oligosaccharide chains, Man-HSA(D494N)-IFNα2b, attached by combining albumin fusion technology and site-directed mutagenesis. The presence of this unique oligosaccharide permits the protein to be efficiently, rapidly and preferentially distributed to Kupffer cells. Likewise IFNα2b, Man-HSA(D494N)-IFNα2b caused a significant induction in the mRNA levels of IL-10, IL-1Ra, PD-L1 in RAW264.7 cells and mouse isolated Kupffer cells, and these inductions were largely inhibited by blocking the interferon receptor. These data indicate that Man-HSA(D494N)-IFNα2b retained the biological activities of type-I interferon. Man-HSA(D494N)-IFNα2b significantly inhibited liver injury in Concanavalin A (Con-A)-induced hepatitis model mice, and consequently improved their survival rate. Moreover, the post-administration of Man-HSA(D494N)-IFNα2b at 2 h after the Con-A challenge also exerted hepato-protective effects. In conclusion, this proof-of-concept study demonstrates the therapeutic effectiveness and utility of Kupffer cell targeting type-I interferon against hepatitis via its anti-inflammatory and immunomodulatory actions.

Origin of IgM(+)IgG(+) lymphocytes in the bursa of Fabricius.

IgM(+)IgG(+) B cells were detected, by immunofluorescence staining of single cells, in the bursa of Fabricius after hatching. To study the role of maternal IgG (MIgG) in this emergence of IgM(+)IgG(+) B cells, MIgG-free chicks were established from surgically bursectomized hens. Deprivation of MIgG in chicks completely prevented the appearance of IgM(+)IgG(+) B cells in the bursa 1 week after hatching. However, introduction of fluorescein-isothiocyanate-labeled MIgG to MIgG-free chick embryos on day 18 of incubation retrieved IgM(+)IgG(+) B cells in the bursa 1 week after hatching. Thus, IgM(+)IgG(+) B cells are induced by the binding of MIgG to IgM(+) B cells in the bursa after hatching. Nevertheless, no binding of MIgG to IgM(+) B cells was observed in the bursa of chick embryos in which B-cell proliferation and differentiation were independent of external antigens (Ags). Additionally, the binding of MIgG to IgM(+) B cells after hatching was prevented by the isolation of the bursa from environmental stimuli by bursal duct ligation. Therefore, Ag stimulation from the external environment to the bursa is indispensable for the binding of MIgG to IgM(+) B cells in the bursa. Taken together, the data demonstrate that IgM(+)IgG(+) B cells are generated by Ag-dependent binding of MIgG to IgM(+) B cells in the bursa after hatching.

New insight into the origin of IgG-bearing cells in the bursa of Fabricius.

The bursa of Fabricius is a primary lymphoid organ for B-cell development and gut-associated lymphoid tissue. After hatching, IgG-containing cells with reticular branches are found in the medulla of bursal follicles on frozen sections stained with anti-Cγ antibody, and IgM(+)IgG(+) B cells are detected in single-cell suspension of the bursa. IgG-containing cells in the medulla do not biosynthesize IgG and are composed of aggregated maternal IgG and environmental antigens. Then, those cells in the medulla are acknowledged as follicular dendritic cells retaining immune complexes. Also, it is presumed that IgM(+)IgG(+) B cells are generated by the attachment of immune complexes to IgM(+) bursal B cells because IgM(+)IgG(+) B cells are induced by antigen-dependent attachment of maternal IgG. Therefore, it is reasonable to suppose that immune complexes exert further B-cell differentiation in the medulla.

Immune complexes of E. coli antigens and maternal IgG in the bursa of Fabricius.

The bursa of Fabricius of the chicken is known to be both a primary lymphoid organ and a secondary lymphoid tissue. Bursal follicles are equipped with antigen-trapping follicle-associated epithelium. However, bioactive antigens such as protein and bacteria have not been detected in the bursal parenchyma. By immunoperoxidase staining with a polyspecific antibody (Ab) against Escherichia coli, we detected aggregated E. coli antigens in the medulla of bursal follicles after hatching. The distribution of aggregated E. coli antigens is restricted to the medulla of bursal follicles. The antigens are not found in the spleen or the parenchyma of the caecal tonsil. The bursa is thus a trapping site for E. coli antigens from the external environment. Furthermore, two-color immunostaining clarified that these antigens form immune complexes with maternal IgG (MIgG) and are retained by reticular cells. Additionally, immune complexes in the bursa were shown to induce the rapid development of serum IgM Ab for indigenous E. coli. Our results suggest that immune complexes of MIgG and environmental antigens in the medulla of bursal follicles exert positive effects on B-cell differentiation in the bursa in situ.

The origin of IgG-containing cells in the bursa of Fabricius.

The bursa of Fabricius of the chicken is known as a primary lymphoid organ for B-cell development. Morphologically, the origin of IgG-containing cells in the bursa has not been clear until now, because abundant maternal IgG (MIgG) is transported to the chick embryo and distributed to the bursal tissue around hatching. Thus, it has been difficult to find out whether these cells themselves biosynthesize IgG or if they acquire MIgG via attachment to their surface. Our present study employing in situ hybridization clarified that IgG-containing cells in the medulla of bursal follicles did not biosynthesize IgG. To study the role of MIgG in the development of those IgG-containing cells, MIgG-free chicks were established from surgically bursectomized hen (SBx-hen). We found that, on the one hand, deprivation of MIgG from chicks completely inhibited the development of IgG-containing cells in the medulla after hatching. On the other hand, administration of MIgG to MIgG-free chicks recovered the emergence of those cells. In addition, we observed that those cells did not bear a B-cell marker and possessed dendrites with aggregated IgG. These results demonstrate that IgG-containing cells in the medulla are reticular cells that capture aggregated MIgG. Moreover, we show that the isolation of the bursa from environmental stimuli by bursal duct ligation (BDL) suppressed the development of IgG-containing cells after hatching. Thus, it is implied that environmental stimulations play a key role in MIgG aggregations and dendritic distributions of aggregated MIgG in the medulla after hatching.