Tirofiban and activated protein C synergistically inhibit the Instant Blood Mediated Inflammatory Reaction (IBMIR) from allogeneic islet cells exposure to human blood.

Instant blood mediated inflammatory reaction (IBMIR) occurs when islets are exposed to blood and manifests clinically as portal vein thrombosis and graft failure. The aim of this study was to determine the impact of recombinant human activated protein C (rhAPC) and platelet inhibition on IBMIR in order to develop a better targeted treatment for this condition. Five thousand human islet cell equivalents (IEQ) were mixed in a PVC loop system with 7 mL of ABO compatible human blood and incubated with rhAPC, either alone or in combination with tirofiban. Admixing human islets and blood caused rapid clot formation, consumption of platelets, leukocytes, fibrinogen, coagulation factors and raised d-dimers. Islets were encased in a fibrin and platelet clot heavily infiltrated with neutrophils. Tirofiban monotherapy was ineffective, whereas rhAPC monotherapy prevented IBMIR in a dose-dependent manner, preserving islet integrity while maintaining platelet and leukocyte counts, fibrinogen and coagulation factor levels, and reducing d-dimer formation. The combination of tirofiban and low-dose rhAPC inhibited IBMIR synergistically with an efficacy equal to high dose rhAPC. Tirofiban and rhAPC worked synergistically to preserve islets, suggesting that co-inhibition of the platelet and coagulation pathways' contribution to thrombin generation is required for the optimal anti-IBMIR effect.

Single-Cell RNA Sequencing Reveals Novel Markers of Male Pituitary Stem Cells and Hormone-Producing Cell Types.

Transcription factors and signaling pathways that regulate stem cells and specialized hormone-producing cells in the pituitary gland have been the subject of intense study and have yielded a mechanistic understanding of pituitary organogenesis and disease. However, the regulation of stem cell proliferation and differentiation, the heterogeneity among specialized hormone-producing cells, and the role of nonendocrine cells in the gland remain important, unanswered questions. Recent advances in single-cell RNA sequencing (scRNAseq) technologies provide new avenues to address these questions. We performed scRNAseq on ∼13,663 cells pooled from six whole pituitary glands of 7-week-old C57BL/6 male mice. We identified pituitary endocrine and stem cells in silico, as well as other support cell types such as endothelia, connective tissue, and red and white blood cells. Differential gene expression analyses identify known and novel markers of pituitary endocrine and stem cell populations. We demonstrate the value of scRNAseq by in vivo validation of a novel gonadotrope-enriched marker, Foxp2. We present novel scRNAseq data of in vivo pituitary tissue, including data from agnostic clustering algorithms that suggest the presence of a somatotrope subpopulation enriched in sterol/cholesterol synthesis genes. Additionally, we show that incomplete transcriptome annotation can cause false negatives on some scRNAseq platforms that only generate 3' transcript end sequences, and we use in vivo data to recover reads of the pituitary transcription factor Prop1. Ultimately, scRNAseq technologies represent a significant opportunity to address long-standing questions regarding the development and function of the different populations of the pituitary gland throughout life.

Genetics of Combined Pituitary Hormone Deficiency: Roadmap into the Genome Era.

The genetic basis for combined pituitary hormone deficiency (CPHD) is complex, involving 30 genes in a variety of syndromic and nonsyndromic presentations. Molecular diagnosis of this disorder is valuable for predicting disease progression, avoiding unnecessary surgery, and family planning. We expect that the application of high throughput sequencing will uncover additional contributing genes and eventually become a valuable tool for molecular diagnosis. For example, in the last 3 years, six new genes have been implicated in CPHD using whole-exome sequencing. In this review, we present a historical perspective on gene discovery for CPHD and predict approaches that may facilitate future gene identification projects conducted by clinicians and basic scientists. Guidelines for systematic reporting of genetic variants and assigning causality are emerging. We apply these guidelines retrospectively to reports of the genetic basis of CPHD and summarize modes of inheritance and penetrance for each of the known genes. In recent years, there have been great improvements in databases of genetic information for diverse populations. Some issues remain that make molecular diagnosis challenging in some cases. These include the inherent genetic complexity of this disorder, technical challenges like uneven coverage, differing results from variant calling and interpretation pipelines, the number of tolerated genetic alterations, and imperfect methods for predicting pathogenicity. We discuss approaches for future research in the genetics of CPHD.

Histopathological and immunohistological analyses in IgA deficient lymphoplasia (aly/aly) mouse.

The aly/aly mouse has a severe immunodeficiency, because it lacks peripheral lymph nodes as well as IgA and IgG immunoglobulin synthesis. In the present study, we performed histopathological and immunohistological examinations to clarify histological disorders of various immune organs in these mice. Carbon CH40 injections into the apex of the tongue confirmed the absence of submandibular lymph nodes in aly/aly mice. The thymus had a poorly constructed cortex and medulla, and the number of lymphoid follicles was clearly decreased in the spleen. No IgG- or IgA- producing cells were found in any immune organs, including the mucosal immune sites, though several IgM -producing cells were identified. Other characteristic findings included perivascular lymphocytes accumulation in the salivary glands, lungs, liver and pancreas, which caused tissues damage. These results demonstrated that the various lymphoid tissues disorders and organ-specific lymphocyte infiltration cause immuno-deficiency in the aly/aly mouse.

[Evaluation of the chemotherapy of a transplantable bladder tumour strain (NM-B-1) by two anti-cancer drugs in combination with a pressor agent in nude mice].

The anti-cancer effects of either Cis-diammine (Glycolato) platinum II (CDGP-II), a novel platinum derivative, or ifosfamide (IFX) on bladder carcinoma strain NM-B-1 inoculated in nude mice were examined. These chemotherapeutic agents were compared with each other in terms of their inhibitory effect on tumor growth, their histology, and the concentration of each agent in the blood, tumor tissue, liver and kidneys. (1) The tumor growth was inhibited by CDGP-II in all three different dosage groups (p < 0.05), but not by IFX (p > 0.05). These results were also confirmed by histological examination. (2) The amount of CDGP-II (30 mg/kg) in the tumor tissue increased in a time-dependent manner, while in the blood plasma and kidney tissue, it decreased. Total value (TV) and active metabolite (AML) of IFX (500 mg/kg) were examined. TV in the tumor tissue decreased in a time-dependent manner. AML could not be detected. There was no change of AML in the blood plasma, liver, and kidneys. (3) Transmigration of single loading of CDGP-II or IFX with Angiotensin II (AT-II) from blood into the tissue was examined. CDGP-II in the blood plasma, tumor, liver and kidneys increased in a time-dependent manner. After single administration of IFX, TV decreased in the blood plasma, tumor, liver and kidneys, while AML increased in the blood and kidneys (p < 0.01). (4) The results suggest that CDGP-II may have an anti-tumor effect of NM-B-1. However, the effect can not be enhanced under the influence of a pressor chemotherapeutic agent.(ABSTRACT TRUNCATED AT 250 WORDS)

Ontogeny of the murine Ig joining chain gene and protein.

We used Northern blot analysis in order to investigate the ontogeny of the murine joining (J)-chain gene. No J-chain expression was detected in embryonic tissues, including liver, spleen and intestine, but an expression of mu heavy chain was detected in foetal liver at day 17. J-chain expression was detected in the spleen at day 9 and in the intestine at day 15 after birth. Western blot analysis was carried out in order to compare the protein levels of J and mu heavy chains in serum from day 8 to day 24 after birth, using antihuman J chain and antimouse mu chain antibodies. Although mu chain protein could be detected in serum from day 8, J-chain protein was detectable only at day 24. These results suggest that the expression of J chain is a later event than the mu chain in the mouse, which thus differs in embryogenesis from humans.