Extracellular nucleotides are agonists at the family of receptors known as the P2 receptors, and in keratinocytes the P2Y2 subtype is known to elevate the intracellular free calcium concentration ...(Cai) and stimulate proliferation. In this study, we have investigated the presence of other functional members of the P2Y subgroup in both normal human keratinocytes and the HaCaT cell line. Using reverse transcription polymerase chain reaction, the expression of mRNA for P2Y1, P2Y2, P2Y4, and P2Y6 receptors was demonstrated in HaCaT cells and differentiated and undifferentiated normal human keratinocytes. Cai was monitored in response to a panel of P2Y receptor agonists. To couple mobilized Cai to a downstream cellular response, cell proliferation was also addressed. In both cell types, adenosine 5′-triphosphate and uridine 5′-triphosphate induced Cai transients of approximately equal duration, magnitude, and shape, confirming the presence of functional P2Y2 receptors. In HaCaT cells, additional characteristic responses were observed in a subpopulation of cells; adenosine 5′-triphosphate failed to elevate Cai in some cells responding to uridine 5′-triphosphate, indicating the presence of P2Y4 receptors, whereas the P2Y1-specific agonist 2-methylthio-5′-adenosine diphosphate was, again, only effective in a small subpopulation. Uridine 5′-diphosphate was ineffective, indicating the absence of functional P2Y6 receptors. Adenosine 5′-triphosphate and uridine 5′-triphosphate equally promoted cell growth in normal human keratinocytes in comparison with the control. In HaCaT cells, adenosine 5′-triphosphate, uridine 5′-triphosphate, and adenosine 5′-diphosphate significantly increased proliferation in comparison to the controls, with a 30% higher response to uridine 5′-triphosphate than with adenosine 5′-triphosphate. These data demonstrate that multiple P2Y receptors (P2Y1, P2Y2, and P2Y4 subtypes) are differentially involved in the regulation of proliferation in human keratinocytes and therefore may be important in wound healing.
•Fundamental diseases are rare diseases that help elucidate mechanisms of common disorders.•Alkaptonuria (AKU), an iconic Mendelian disease, is a prototypic fundamental disease.•Studying AKU has ...identified new disease mechanisms in osteoarthritis including the exposed collagen hypothesis.•HDMPs, identified in AKU and then OA, constitute a newly discovered mechanism of joint destruction.
“Fundamental diseases” is a term introduced by the charity Findacure to describe rare genetic disorders that are gateways to understanding common conditions and human physiology. The concept that rare diseases have important lessons for biomedical science has been recognised by some of the great figures in the history of medical research, including Harvey, Bateson and Garrod. Here we describe some of the recently discovered lessons from the study of the iconic genetic disease alkaptonuria (AKU), which have shed new light on understanding the pathogenesis of osteoarthritis. In AKU, ochronotic pigment is deposited in cartilage when collagen fibrils become susceptible to attack by homogentisic acid (HGA). When HGA binds to collagen, cartilage matrix becomes stiffened, resulting in the aberrant transmission of loading to underlying subchondral bone. Aberrant loading leads to the formation of pathophysiological structures including trabecular excrescences and high density mineralised protrusions (HDMPs). These structures initially identified in AKU have subsequently been found in more common osteoarthritis and appear to play a role in joint destruction in both diseases.
Alkaptonuria (AKU) is a rare, inherited disorder of tyrosine metabolism, where patients are unable to breakdown homogentisic acid (HGA), which increases systemically over time. It presents with a ...clinical triad of features; HGA in urine, ochronosis of collagenous tissues, and the subsequent ochronotic arthritis of these tissues. In recent years the advance in the understanding of the disease and the potential treatment of the disorder looks promising with the data on the efficacy of nitisinone. However, there are limited methods for the detection and monitoring of ochronosis in vivo, or for treatment monitoring.
The study aim was to test the hypothesis that Raman spectra would identify a distinct chemical fingerprint for the non-ochronotic, compared to ochronotic cartilage.
Ochronotic and non-ochronotic cartilage from human hips and ears were analysed using Raman spectroscopy.
Non-ochronotic cartilage spectra were similar and reproducible and typical of normal articular cartilage. Conversely, the ochronotic cartilage samples were highly fluorescent and displayed limited or no discernible Raman peaks in the spectra, in stark contrast to their non-ochronotic pairs. Interestingly, a novel peak was observed associated with the polymer of HGA in the ochronotic cartilage that was confirmed by analysis of pigment derived from synthetic HGA.
This technique reveals novel data on the chemical differences in ochronotic compared with non-ochronotic cartilage, these differences are detectable by a technique that is already generating in vivo data and demonstrates the first possible procedure to monitor the progression of ochronosis in tissues of patients with AKU.
Abstract
Alkaptonuria is an inherited disease caused by homogentisate 1,2-dioxygenase (HGD) deficiency. Circulating homogentisic acid (HGA) is elevated and deposits in connective tissues as ...ochronotic pigment. In this study, we aimed to define developmental and adult HGD tissue expression and determine the location and amount of gene activity required to lower circulating HGA and rescue the alkaptonuria phenotype.
We generated an alkaptonuria mouse model using a knockout-first design for the disruption of the HGD gene. Hgd tm1a −/− mice showed elevated HGA and ochronosis in adulthood. LacZ staining driven by the endogenous HGD promoter was localised to only liver parenchymal cells and kidney proximal tubules in adulthood, commencing at E12.5 and E15.5 respectively. Following removal of the gene trap cassette to obtain a normal mouse with a floxed 6th HGD exon, a double transgenic was then created with Mx1-Cre which conditionally deleted HGD in liver in a dose dependent manner. 20% of HGD mRNA remaining in liver did not rescue the disease, suggesting that we need more than 20% of liver HGD to correct the disease in gene therapy.
Kidney HGD activity which remained intact reduced urinary HGA, most likely by increased absorption, but did not reduce plasma HGA nor did it prevent ochronosis. In addition, downstream metabolites of exogenous 13C6-HGA, were detected in heterozygous plasma, revealing that hepatocytes take up and metabolise HGA.
This novel alkaptonuria mouse model demonstrated the importance of targeting liver for therapeutic intervention, supported by our observation that hepatocytes take up and metabolise HGA.
Dialogic Reading Towson, Jacqueline A.; Gallagher, Peggy A.; Bingham, Gary E.
Journal of early intervention,
12/2016, Letnik:
38, Številka:
4
Journal Article
Recenzirano
Dialogic reading is an evidence-based practice for preschool children who are typically developing or at-risk; yet there is limited research to evaluate if it has similar positive effects on the ...language and preliteracy skills of children with disabilities. This quasi-experimental study examined the effects of dialogic reading, with the incorporation of pause time, on the language and preliteracy skills of 42 preschool children with disabilities. Following random assignment of students at the classroom level, participants were equally distributed into an intervention (n = 21) and a comparison group (n = 21). Children received either dialogic reading or typical storybook reading for 10 to 15 min per day, 3 days per week, for 6 weeks. Children in the intervention group scored significantly higher on receptive and expressive near-transfer vocabulary assessments. This occurred both for words that were specifically targeted during dialogic reading, and for additional vocabulary words in the storybook.
Highlights • Studying rare syndromes helps elucidate disease mechanisms of more common disorders. • Lessons from rare bone diseases have contributed to the development of osteoanabolic therapies. • ...Osteoarthritis (OA) is a common disease with currently no effective therapies. • Investigation of rare cartilage syndromes is identifying new targets in OA. • HDMPs identified in AKU and then OA constitute a new mechanism of joint destruction.
Altered activity of specific enzymes in phenylalanine‐tyrosine (phe‐tyr) metabolism results in incomplete breakdown of various metabolite substrates in this pathway. Increased biofluid concentration ...and tissue accumulation of the phe‐tyr pathway metabolite homogentisic acid (HGA) is central to pathophysiology in the inherited disorder alkaptonuria (AKU). Accumulation of metabolites upstream of HGA, including tyrosine, occurs in patients on nitisinone, a licenced drug for AKU and hereditary tyrosinaemia type 1, which inhibits the enzyme responsible for HGA production. The aim of this study was to investigate the phe‐tyr metabolite content of key biofluids and tissues in AKU mice on and off nitisinone to gain new insights into the biodistribution of metabolites in these altered metabolic states. The data show for the first time that HGA is present in bile in AKU (mean ±SD = 1003±410 μmol/L; nitisinone‐treated AKU mean ±SD = 45±23 μmol/L). Biliary tyrosine, 3(4‐hydroxyphenyl)pyruvic acid (HPPA) and 3(4‐hydroxyphenyl)lactic acid (HPLA) are also increased on nitisinone. Urine was confirmed as the dominant elimination route of HGA in untreated AKU, but with indication of biliary excretion. These data provide new insights into pathways of phe‐tyr metabolite biodistribution and metabolism, showing for the first time that hepatobiliary excretion contributes to the total pool of metabolites in this pathway. Our data suggest that biliary elimination of organic acids and other metabolites may play an underappreciated role in disorders of metabolism. We propose that our finding of approximately 3.8 times greater urinary HGA excretion in AKU mice compared with patients is one reason for the lack of extensive tissue ochronosis in the AKU mouse model.
Undignifying institutions Seedhouse, D; Gallagher, A
Journal of medical ethics,
12/2002, Letnik:
28, Številka:
6
Journal Article
Recenzirano
Odprti dostop
Declarations of the importance of dignity in health care are commonplace in codes of practice and other mission statements, yet these documents never clarify dignity’s meaning. Their vague ...aspirations are compared to comments from staff and patients about opportunities for and barriers against the promotion of dignity in elderly care institutions. These suggest that while nurses and health care assistants have an intuitive understanding of dignity, they either do not or cannot always bring it about in practice. Thus, despite stated intentions to promote dignity, it appears that the circumstances of at least some elderly care institutions cause patients to experience avoidable indignities. Such institutions are “undignifying institutions” because they fail to acknowledge dignity’s basic components, focus excessively on quantifiable priorities, and have insufficient resources available to assure consistently dignifying care. As a partial solution, we argue that health workers should be taught to understand and specify the components of dignity, which will better prepare them to challenge undignifying practices and to recognise opportunities for dignity promotion.
PDF
