Trend towards reduction of transfusion reactions using prestorage leukocyte-reduced and pooled whole blood-derived platelets and cost savings compared with poststorage whole blood-derived random platelets as evidenced by real-time hemovigilance.

BACKGROUND: Due to chemotherapy-induced neutropenia or hematologic malignancies, immunocompromised cancer patients may have higher incidence of febrile nonhemolytic transfusion reactions compared with the general population and frequently require platelet transfusions. This quality improvement project compared the safety of transfusion using prestorage leukocyte-reduced and pooled whole blood-derived platelets (Acrodose/WBD) with conventionally produced poststorage WBD platelets (RDP) using an active hemovigilance system. METHODS: Every patient receiving a blood product at the hospital was virtually monitored in real time by trained nurses from a remote hemovigilance unit. These nurses monitor a digital dashboard, which populates a watch list of patients from the time blood product administration is initiated until 12 hours posttransfusion. Over the course of 6 months, 371 patients receiving 792 RDP transfusions and 423 patients receiving 780 Acrodose/WBD platelets transfusions were monitored for transfusion reactions. RESULTS: We identified 26 transfusion reactions in RDP but only 12 transfusion reactions in the Acrodose/WBD platelet group. CONCLUSION: Acrodose platelet transfusion was associated with fewer transfusion reactions, which resulted in significant cost savings.

Technical Note: Use of a Novel Bacterial Culture Sampling Device for Pooled Whole Blood Platelets.

OBJECTIVE: Transfusion associated sepsis is a serious risk after platelet transfusion. Although platelet culture can be performed to avoid such risk, culture results are often available after transfusion due to the 4-hour shelf-life after pooling. To decrease such risk, we implemented a needleless closed system device to culture for contamination before pooling and release for transfusion. METHODS: We customized a needleless device to permit sterile sampling of whole blood platelets without retrograde or cross-contamination. Then aliquots of platelets were injected into culture media for detection of aerobic organisms and cultured for 24 hours but released for transfusion after 12 hours of negative culture. RESULTS: In a period of two years, we used this device in 5,741 whole blood derived pooled platelets and only 24 units tested positive (0.4%) but none of initial positive was later confirmed. There were 11 Staphylococcus and 9 Bacillus species identified. All but one of the positive units were discarded; there was no clinical impact in the patient who received the positive unit. CONCLUSION: This device allows for sampling of whole blood derived platelets before pooling, warranting a transfusion of a culture negative unit after 12 hours of negative culture, consequently reducing transfusion of bacterially contaminated whole blood derived pooled platelets.

Digitally enabled hemovigilance allows real time response to transfusion reactions.

BACKGROUND: Transfusion carries a risk of transfusion reaction that is often underdiagnosed due to reliance on passive reporting. The study investigated the utility of digital methods to identify potential transfusion reactions, thus allowing real-time intervention for affected patients. METHOD: The hemovigilance unit monitored 3856 patients receiving 43,515 transfusions under the hemovigilance program. Retrospective comparison data included 298,498 transfusions. Transfusion medicine physicians designed and validated algorithms in the electronic health record that analyze discrete data, such as vital sign changes, to assign a risk score during each transfusion. Dedicated hemovigilance nurses remotely monitor all patients and perform real-time chart reviews prioritized by risk score. When a reaction is suspected, a hemovigilance trained licensed clinician responds to manage the patient and ensure data collection. Board-certified transfusion medicine physicians reviewed data and classified transfusion reactions under various categories according to the Centers for Disease Control hemovigilance definitions. RESULTS: Transfusion medicine physicians diagnosed 564 transfusion reactions (1.3% of transfusions)-a 524% increase compared to the previous passive reporting. The rapid response provider reached the bedside on average at 12.4 min demonstrating logistic feasibility. While febrile reactions were most diagnosed, recognition of transfusion-associated circulatory overload demonstrated the greatest relative increase. Auditing and education programs further enhanced transfusion reaction awareness. DISCUSSION: The model of digitally-enabled expert real-time review of clinical data that prompts rapid response improved recognition of transfusion reactions. This approach could be applied to other patient deterioration events such as early identification of sepsis.

Oncology Patients Who Develop Transfusion-Associated Circulatory Overload: An Observational Study.

BACKGROUND: Transfusion-associated circulatory overload (TACO) is a largely preventable transfusion complication that results in significant morbidity and mortality. Cancers, related treatments, and comorbidities are among the factors that can predispose patients to TACO, but currently there are limited data on this topic in the literature. METHODS: We collected data retrospectively from the electronic health records of 93 adult patients with cancer who met Centers for Disease Control and Prevention (CDC) criteria for TACO from July 1, 2019, through October 31, 2020. The parameters we studied included demographics, comorbidities, treatment modalities, transfusion practices, and outcomes. We summarized data by means and ranges for continuous variables, and proportions for categorical variables. RESULTS: During the study period, the incidence of TACO among oncology patients was 0.84 per 1000 transfusions (95% CI, 0.68-1.02), representing 6.6% of all reactions. This percentage is high, compared with 1%-6% among other populations. Unique characteristics such as hematology malignancy (75.3%), receipt of cardiotoxic chemotherapy (87.1%), pneumonia (57.0%), preexisting oxygen use (59.1%), dyspnea (62.4%), hypertension (55.9%), renal insufficiency (46.2%), daily use of corticosteroids (43.0%), daily use of diuretics (40.9%), daily use of beta-blockers (36.6%), and elevated NT-proBNP (33.3%) were frequently observed in these group of oncology patients. CONCLUSIONS: Our study indicates that oncology patients have unique factors that may lead to diagnosis of TACO. Developing appropriate guidelines that apply to oncology patients, in addition to those set forth by the CDC, should be considered. Implementation by ordering healthcare providers of a tools that can predict TACO can help in early recognition and mitigation of TACO.

Pancreatic hamartoma in a premature Trisomy 18 female.

Pancreatic hamartomas are extremely rare tumors in adults and even more so in children. They are lesions characterized by acinar, islet and ductal components found in varying proportions and in a disorganized pattern. We report a case of a premature female with trisomy 18 diagnosed by amniocentesis. The newborn was delivered by cesarean section at thirty-three weeks of gestation and expired within one hour of birth. Postmortem examination exhibited numerous features associated with Trisomy 18 including lanugo on the torso and arms, micrognathia, microstomia, left low-set ear with small flat pinna, closed ear canal, clenched fists with overlapping fingers, rocker-bottom feet, narrow pelvis, large right diaphragmatic hernia and left pulmonary hypoplasia. Microscopic examination of the pancreas revealed an area, 1.2 cm in greatest dimension, with branching ducts and cysts lined by cuboidal epithelium intermingled within primitive mesenchymal proliferation and exocrine glands. The cysts measured up to 0.2 cm and were surrounded by a collarette of proliferating spindle cells as highlighted by Masson's trichrome stain. A diagnosis of pancreatic hamartoma was rendered. A total of thirty-four cases of pancreatic hamartomas have been reported in the literature including twenty-seven in adults, five in children and two in newborns. Our case may be the third pancreatic hamartoma reported in association with Trisomy 18. We recommend that careful examination of the pancreas be performed in individuals with Trisomy 18 to further characterize this lesion as one of the possible abnormal findings associated with this syndrome.

Pancreatic hamartoma in a premature Trisomy 18 female

Pancreatic hamartomas are extremely rare tumors in adults and even more so in children. They are lesions characterized by acinar, islet and ductal components found in varying proportions and in a disorganized pattern. We report a case of a premature female with trisomy 18 diagnosed by amniocentesis. The newborn was delivered by cesarean section at thirty-three weeks of gestation and expired within one hour of birth. Postmortem examination exhibited numerous features associated with Trisomy 18 including lanugo on the torso and arms, micrognathia, microstomia, left low-set ear with small flat pinna, closed ear canal, clenched fists with overlapping fingers, rocker-bottom feet, narrow pelvis, large right diaphragmatic hernia and left pulmonary hypoplasia. Microscopic examination of the pancreas revealed an area, 1.2 cm in greatest dimension, with branching ducts and cysts lined by cuboidal epithelium intermingled within primitive mesenchymal proliferation and exocrine glands. The cysts measured up to 0.2 cm and were surrounded by a collarette of proliferating spindle cells as highlighted by Masson's trichrome stain. A diagnosis of pancreatic hamartoma was rendered. A total of thirty-four cases of pancreatic hamartomas have been reported in the literature including twenty-seven in adults, five in children and two in newborns. Our case may be the third pancreatic hamartoma reported in association with Trisomy 18. We recommend that careful examination of the pancreas be performed in individuals with Trisomy 18 to further characterize this lesion as one of the possible abnormal findings associated with this syndrome.

Bilateral renal dysplasia, nephroblastomatosis, and bronchial stenosis. A new syndrome?

Bilateral nephroblastomatosis (NB) is an uncommon renal anomaly characterized by multiple confluent nephrogenic rests scattered through both kidneys, with only a limited number of cases reported in the medical literature. Some of these children may have associated either Perlman or Beckwith-Wiedemann syndrome and others do not demonstrate syndromic features. We report a full-term boy with anteverted nose, bilateral bronchial stenosis due to lack of cartilage, bilateral obstructive renal dysplasia and NB with glomeruloid features. The infant had visceromegaly, but neither gigantism nor hemihypertrophy. Immunohistochemistry for PAX2 (Paired box gene-2) and WT-1 (Wilms Tumor 1) were strongly positive in the areas of NB. GLEPP-1 (Glomerular Epithelial Protein) did not stain the areas of NB with a glomeruloid appearance, but was positive in the renal glomeruli as expected. We found neither associated bronchial stenosis nor the histology of NB resembling giant glomeruli in any of the reported cases of NB.

Sphingomyelinase-like phosphodiesterase 3b expression levels determine podocyte injury phenotypes in glomerular disease.

Diabetic kidney disease (DKD) is the most common cause of ESRD in the United States. Podocyte injury is an important feature of DKD that is likely to be caused by circulating factors other than glucose. Soluble urokinase plasminogen activator receptor (suPAR) is a circulating factor found to be elevated in the serum of patients with FSGS and causes podocyte αVß3 integrin-dependent migration in vitro. Furthermore, αVß3 integrin activation occurs in association with decreased podocyte-specific expression of acid sphingomyelinase-like phosphodiesterase 3b (SMPDL3b) in kidney biopsy specimens from patients with FSGS. However, whether suPAR-dependent αVß3 integrin activation occurs in diseases other than FSGS and whether there is a direct link between circulating suPAR levels and SMPDL3b expression in podocytes remain to be established. Our data indicate that serum suPAR levels are also elevated in patients with DKD. However, unlike in FSGS, SMPDL3b expression was increased in glomeruli from patients with DKD and DKD sera-treated human podocytes, where it prevented αVß3 integrin activation by its interaction with suPAR and led to increased RhoA activity, rendering podocytes more susceptible to apoptosis. In vivo, inhibition of acid sphingomyelinase reduced proteinuria in experimental DKD but not FSGS, indicating that SMPDL3b expression levels determined the podocyte injury phenotype. These observations suggest that SMPDL3b may be an important modulator of podocyte function by shifting suPAR-mediated podocyte injury from a migratory phenotype to an apoptotic phenotype and that it represents a novel therapeutic glomerular disease target.

Coordination of hypothalamic and pituitary T3 production regulates TSH expression.

Type II deiodinase (D2) activates thyroid hormone by converting thyroxine (T4) to 3,5,3'-triiodothyronine (T3). This allows plasma T4 to signal a negative feedback loop that inhibits production of thyrotropin-releasing hormone (TRH) in the mediobasal hypothalamus (MBH) and thyroid-stimulating hormone (TSH) in the pituitary. To determine the relative contributions of these D2 pathways in the feedback loop, we developed 2 mouse strains with pituitary- and astrocyte-specific D2 knockdown (pit-D2 KO and astro-D2 KO mice, respectively). The pit-D2 KO mice had normal serum T3 and were systemically euthyroid, but exhibited an approximately 3-fold elevation in serum TSH levels and a 40% reduction in biological activity. This was the result of elevated serum T4 that increased D2-mediated T3 production in the MBH, thus decreasing Trh mRNA. That tanycytes, not astrocytes, are the cells within the MBH that mediate T4-to-T3 conversion was defined by studies using the astro-D2 KO mice. Despite near-complete loss of brain D2, tanycyte D2 was preserved in astro-D2 KO mice at levels that were sufficient to maintain both the T4-dependent negative feedback loop and thyroid economy. Taken together, these data demonstrated that the hypothalamic-thyroid axis is wired to maintain normal plasma T3 levels, which is achieved through coordination of T4-to-T3 conversion between thyrotrophs and tanycytes.

Disruption of thyroid hormone activation in type 2 deiodinase knockout mice causes obesity with glucose intolerance and liver steatosis only at thermoneutrality.

OBJECTIVE: Thyroid hormone accelerates energy expenditure; thus, hypothyroidism is intuitively associated with obesity. However, studies failed to establish such a connection. In brown adipose tissue (BAT), thyroid hormone activation via type 2 deiodinase (D2) is necessary for adaptive thermogenesis, such that mice lacking D2 (D2KO) exhibit an impaired thermogenic response to cold. Here we investigate whether the impaired thermogenesis of D2KO mice increases their susceptibility to obesity when placed on a high-fat diet. RESEARCH DESIGN AND METHODS: To test this, D2KO mice were admitted to a comprehensive monitoring system acclimatized to room temperature (22°C) or thermoneutrality (30°C) and kept either on chow or high-fat diet for 60 days. RESULTS: At 22°C, D2KO mice preferentially oxidize fat, have a similar sensitivity to diet-induced obesity, and are supertolerant to glucose. However, when thermal stress is eliminated at thermoneutrality (30°C), an opposite phenotype is encountered, one that includes obesity, glucose intolerance, and exacerbated hepatic steatosis. We suggest that a compensatory increase in BAT sympathetic activation of the D2KO mice masks metabolic repercussions that they would otherwise exhibit. CONCLUSIONS: Thus, upon minimization of thermal stress, high-fat feeding reveals the defective capacity of D2KO mice for diet-induced thermogenesis, provoking a paradigm shift in the understanding of the role of the thyroid hormone in metabolism.

KISS1R intracellular trafficking and degradation: effect of the Arg386Pro disease-associated mutation.

The goal of this study was to investigate how the Arg386Pro mutation prolongs KiSS-1 receptor (KISS1R) responsiveness to kisspeptin, contributing to human central precocious puberty. Confocal imaging showed colocalization of wild-type (WT) KISS1R with a membrane marker, which persisted for up to 5 h of stimulation. Conversely, no colocalization with a lysosome marker was detected. Also, overnight treatment with a lysosome inhibitor did not affect WT KISS1R protein, whereas overnight treatment with a proteasome inhibitor increased protein levels by 24-fold. WT and Arg386Pro KISS1R showed time-dependent internalization upon stimulation. However, both receptors were recycled back to the membrane. The Arg386Pro mutation did not affect the relative distribution of KISS1R in membrane and internalized fractions when compared to WT KISS1R for up to 120 min of stimulation, demonstrating that this mutation does not affect KISS1R trafficking rate. Nonetheless, total Arg386Pro KISS1R was substantially increased compared with WT after 120 min of kisspeptin stimulation. This net increase was eliminated by blockade of detection of recycled receptors, demonstrating that recycled receptors account for the increased responsiveness of this mutant to kisspeptin. We therefore conclude the following: 1) WT KISS1R is degraded by proteasomes rather than lysosomes; 2) WT and Arg386Pro KISS1R are internalized upon stimulation, but most of the internalized receptors are recycled back to the membrane rather than degraded; 3) the Arg386Pro mutation does not affect the rate of KISS1R trafficking--instead, it prolongs responsiveness to kisspeptin by decreasing KISS1R degradation, resulting in the net increase on mutant receptor recycled back to the plasma membrane.

Matrix metalloproteinase 1: role in sarcoma biology.

In carcinomas stromal cells participate in cancer progression by producing proteases such as MMPs. The expression MMP1 is a prognostic factor in human chondrosarcoma, however the role in tumor progression is unknown. Laser capture microdissection and In Situ hybridization were used to determine cellular origin of MMP1 in human sarcomas. A xenogenic model of tumor progression was then used and mice were divided in two groups: each harboring either the control or a stably MMP1 silenced cell line. Animals were sacrificed; the neovascularization, primary tumor volumes, and metastatic burden were assessed. LCM and RNA-ISH analysis revealed MMP1 expression was predominantly localized to the tumor cells in all samples of sarcoma (p = 0.05). The percentage lung metastatic volume at 5 weeks (p = 0.08) and number of spontaneous deaths secondary to systemic tumor burden were lower in MMP1 silenced cell bearing mice. Interestingly, this group also demonstrated a larger primary tumor size (p<0.04) and increased angiogenesis (p<0.01). These findings were found to be consistent when experiment was repeated using a second independent MMP1 silencing sequence. Prior clinical trials employing MMP1 inhibitors failed because of a poor understanding of the role of MMPs in tumor progression. The current findings indicating tumor cell production of MMP1 by sarcoma cells is novel and highlights the fundamental differences in MMP biology between carcinomas and sarcomas. The results also emphasize the complex roles of MMP in tumor progression of sarcomas. Not only does metastasis seem to be affected by MMP1 silencing, but also local tumor growth and angiogenesis are affected inversely.

MicroRNA signature of the human developing pancreas.

BACKGROUND: MicroRNAs are non-coding RNAs that regulate gene expression including differentiation and development by either inhibiting translation or inducing target degradation. The aim of this study is to determine the microRNA expression signature during human pancreatic development and to identify potential microRNA gene targets calculating correlations between the signature microRNAs and their corresponding mRNA targets, predicted by bioinformatics, in genome-wide RNA microarray study. RESULTS: The microRNA signature of human fetal pancreatic samples 10-22 weeks of gestational age (wga), was obtained by PCR-based high throughput screening with Taqman Low Density Arrays. This method led to identification of 212 microRNAs. The microRNAs were classified in 3 groups: Group number I contains 4 microRNAs with the increasing profile; II, 35 microRNAs with decreasing profile and III with 173 microRNAs, which remain unchanged. We calculated Pearson correlations between the expression profile of microRNAs and target mRNAs, predicted by TargetScan 5.1 and miRBase algorithms, using genome-wide mRNA expression data. Group I correlated with the decreasing expression of 142 target mRNAs and Group II with the increasing expression of 876 target mRNAs. Most microRNAs correlate with multiple targets, just as mRNAs are targeted by multiple microRNAs. Among the identified targets are the genes and transcription factors known to play an essential role in pancreatic development. CONCLUSIONS: We have determined specific groups of microRNAs in human fetal pancreas that change the degree of their expression throughout the development. A negative correlative analysis suggests an intertwined network of microRNAs and mRNAs collaborating with each other. This study provides information leading to potential two-way level of combinatorial control regulating gene expression through microRNAs targeting multiple mRNAs and, conversely, target mRNAs regulated in parallel by other microRNAs as well. This study may further the understanding of gene expression regulation in the human developing pancreas.

Absence of thyroid hormone activation during development underlies a permanent defect in adaptive thermogenesis.

Type 2 deiodinase (D2), which is highly expressed in brown adipose tissue (BAT), is an enzyme that amplifies thyroid hormone signaling in individual cells. Mice with inactivation of the D2 pathway (D2KO) exhibit dramatically impaired thermogenesis in BAT, leading to hypothermia during cold exposure and a greater susceptibility to diet-induced obesity. This was interpreted as a result of defective acute activation of BAT D2. Here we report that the adult D2KO BAT has a permanent thermogenic defect that stems from impaired embryonic BAT development. D2KO embryos have normal serum T3 but due to lack of D2-generated T3 in BAT, this tissue exhibits decreased expression of genes defining BAT identity [i.e. UCP1, PGC-1alpha and Dio2 (nonfunctional)], which results in impaired differentiation and oxidative capacity. Coinciding with a reduction of these T3-responsive genes, there is oxidative stress that in a cell model of brown adipogenesis can be linked to decreased insulin signaling and decreased adipogenesis. This discovery highlights the importance of deiodinase-controlled thyroid hormone signaling in BAT development, where it has important metabolic repercussions for energy homeostasis in adulthood.

ATP-gated P2X3 receptors constitute a positive autocrine signal for insulin release in the human pancreatic beta cell.

Extracellular ATP has been proposed as a paracrine signal in rodent islets, but it is unclear what role ATP plays in human islets. We now show the presence of an ATP signaling pathway that enhances the human beta cell's sensitivity and responsiveness to glucose fluctuations. By using in situ hybridization, RT-PCR, immunohistochemistry, and Western blotting as well as recordings of cytoplasmic-free Ca(2+) concentration, [Ca(2+)](i), and hormone release in vitro, we show that human beta cells express ionotropic ATP receptors of the P2X(3) type and that activation of these receptors by ATP coreleased with insulin amplifies glucose-induced insulin secretion. Released ATP activates P2X(3) receptors in the beta-cell plasma membrane, resulting in increased [Ca(2+)](i) and enhanced insulin secretion. Therefore, in human islets, released ATP forms a positive autocrine feedback loop that sensitizes the beta cell's secretory machinery. This may explain how the human pancreatic beta cell can respond so effectively to relatively modest changes in glucose concentration under physiological conditions in vivo.

Endocrine cell clustering during human pancreas development.

The development of efficient, reproducible protocols for directed in vitro differentiation of human embryonic stem (hES) cells into insulin-producing beta cells will benefit greatly from increased knowledge regarding the spatiotemporal expression profile of key instructive factors involved in human endocrine cell generation. Human fetal pancreases 7 to 21 weeks of gestational age, were collected following consent immediately after pregnancy termination and processed for immunostaining, in situ hybridization, and real-time RT-PCR expression analyses. Islet-like structures appear from approximately week 12 and, unlike the mixed architecture observed in adult islets, fetal islets are initially formed predominantly by aggregated insulin- or glucagon-expressing cells. The period studied (7-22 weeks) coincides with a decrease in the proliferation and an increase in the differentiation of the progenitor cells, the initiation of NGN3 expression, and the appearance of differentiated endocrine cells. The present study provides a detailed characterization of islet formation and expression profiles of key intrinsic and extrinsic factors during human pancreas development. This information is beneficial for the development of efficient protocols that will allow guided in vitro differentiation of hES cells into insulin-producing cells.

MicroRNA miR-7 is preferentially expressed in endocrine cells of the developing and adult human pancreas.

MicroRNAs (miRNA) are small non-coding RNAs that inhibit gene expression through binding to complementary messenger RNA sequences. miRNAs have been predicted to target genes important for pancreas development, proper endocrine cell function and metabolism. We previously described that miRNA-7 (miR-7) was the most abundant and differentially expressed islet miRNA, with 200-fold higher expression in mature human islets than in acinar tissue. Here we have analyzed the temporal and spatial expression of miR-7 in human fetal pancreas from 8 to 22 weeks of gestational age (wga). Human fetal (8-22wga) and adult pancreases were processed for immunohistochemistry, in situ hybridization, and quantitative RT-PCR of miRNA and mRNA. miR-7 was expressed in the human developing pancreas from around 9wga and reached its maximum expression levels between 14 and 18wga, coinciding with the exponential increase of the pancreatic endocrine hormones. Throughout development miR-7 expression was preferentially localized to endocrine cells and its expression persisted in the adult pancreas. The present study provides a detailed analysis of the spatiotemporal expression of miR-7 in developing human pancreas. The specific localization of miR-7 expression to fetal and adult endocrine cells indicates a potential role for miR-7 in endocrine cell differentiation and/or function. Future functional studies of a potential role for miR-7 function in islet cell differentiation and physiology are likely to identify novel targets for the treatment of diabetes and will lead to the development of improved protocols for generating insulin-producing cells for cell replacement therapy.

Retinoic acid promotes the generation of pancreatic endocrine progenitor cells and their further differentiation into beta-cells.

The identification of secreted factors that can selectively stimulate the generation of insulin producing beta-cells from stem and/or progenitor cells represent a significant step in the development of stem cell-based beta-cell replacement therapy. By elucidating the molecular mechanisms that regulate the generation of beta-cells during normal pancreatic development such putative factors may be identified. In the mouse, beta-cells increase markedly in numbers from embryonic day (e) 14.5 and onwards, but the extra-cellular signal(s) that promotes the selective generation of beta-cells at these stages remains to be identified. Here we show that the retinoic acid (RA) synthesizing enzyme Raldh1 is expressed in developing mouse and human pancreas at stages when beta-cells are generated. We also provide evidence that RA induces the generation of Ngn3(+) endocrine progenitor cells and stimulates their further differentiation into beta-cells by activating a program of cell differentiation that recapitulates the normal temporal program of beta-cell differentiation.