Long-term outcomes for pediatric heart transplant recipients transitioning to adult care teams.

BACKGROUND: There are limited data evaluating the success of a structured transition plan specifically for pediatric heart transplant (HT) recipients following their transfer of care to an adult specialist. We sought to identify risk factors for poor adherence, graft failure, and mortality following the transfer of care to adult HT care teams. METHODS: We retrospectively reviewed all patients who underwent transition from the pediatric to adult HT program at our center between January 2011 and June 2021. Demographic characteristics, comorbid conditions, and psychosocial history were collected at the time of HT, the time of transition, and the most recent follow-up. Adverse events including mortality, graft rejection, infection, and renal function were also captured before and after the transition. RESULTS: Seventy-two patients were identified (54.1% male, 54.2% Caucasian). Mean age at the time of transition was 23 years after a median of 11.6 years in the pediatric program. The use of calcineurin inhibitors was associated with reduced mortality (HR .04, 95% CI .0-.6, p = .015), while prior psychiatric hospitalization (HR 45.3, 95% CI, 6.144-333.9, p = .0001) was associated with increased mortality following transition. Medication nonadherence and young age at the time of transition were markers for high-risk individuals prior to the transition of care. CONCLUSIONS: Transition of HT recipients from a pediatric program to an adult program occurs during a vulnerable time of emerging adulthood, and we have identified risk factors for mortality following transition. Development of a formalized transition plan with a large multidisciplinary team with focused attention on high-risk patients, including those with psychiatric comorbidities, may favorably influence outcomes.

Race and Socioeconomic Bias in Pediatric Cardiac Transplantation.

BACKGROUND: To date, no studies evaluated implicit bias among clinicians caring for children with advanced heart failure. OBJECTIVES: This study aims to evaluate implicit racial and socioeconomic bias among pediatric heart transplant clinicians. METHODS: A cross-sectional survey of transplant clinicians from the Pediatric Heart Transplant Society was conducted between June and August 2021. The survey consisted of demographic questions along with explicit and validated race and socioeconomic status (SES) implicit association tests (IATs). Implicit and explicit biases among survey group members were studied and associations were tested between implicit and explicit measures. RESULTS: Of 500 members, 91 (18.2%) individuals completed the race IAT and 70 (14%) completed the SES IAT. Race IAT scores indicated moderate levels of implicit bias (mean = 0.33, d = 0.76; P < 0.001; ie, preference for White individuals). SES IAT scores indicated strong implicit bias (mean = 0.52, d = 1.53; P < 0.001; ie, preference for people from upper SES). There were weak levels of explicit race and wealth bias. There was a strong level of explicit education bias (mean = 5.22, d = 1.19; P < 0.001; ie, preference for educated people). There were nonsignificant correlations between the race and the SES IAT and explicit measures (P > 0.05 for all). CONCLUSIONS: As observed across other health care disciplines, among a group of pediatric heart transplant clinicians, there is an implicit preference for individuals who are White and from higher SES, and an explicit preference for educated people. Future studies should evaluate how implicit biases affect clinician behavior and assess the impact of efforts to reduce such biases.

The physical and biochemical effects of pre-rigor high pressure processing of beef.

High pressure processing (HPP) of pre-rigor longissimus thoracis (strip loin) from prime and bull animals substantially decreased the shear force and improved consumer eating attributes of the final meat product. The improved tenderness in both prime and bull meat was associated with a lower myofibrillar fragmentation index and reduced calpain 1 activity which indicated the mechanism of tenderisation was different from that which occurred in chill aged meat. Light microscopy showed disruption to the fibre packing within the muscle and electron microscopy confirmed significant disruption of the Z discs and M lines and disappearance of the A lines. Thus, HPP is associated with a reduction in the structural integrity and strength of the sarcomeres. These effects were consistent in strip loins sourced from prime and bull stock. HPP also led to the movement of glycogen phosphorylase from the sarcoplasmic fraction to the insoluble myofibrillar fraction in all animals and this was associated with a higher pH at 24 h.

High pressure processing improves the tenderness and quality of hot-boned beef.

Strip loins from different grades of cattle were subjected to two levels of high pressure processing (HPP) within 1h of slaughter at a commercial meat processing plant and chilled for 1day before freezing. The physical and eating quality characteristics of longissimus thoracis (strip loin) steaks from HPP were compared to meat that was chill aged for 1 or 28days. HPP produced meat after 1day with 60% lower shear force and higher sensory eating quality scores than 1day chill aged meat. Extended chill storage for 28days produced steaks of similar tenderness to HPP meat. HPP also increased the ultimate pH and decreased the cooking loss. Chilled storage of the gluteus medius from prime cattle for 28days significantly improved the shear force by 18%, whilst HPP improved both the shear force by 43% and the sensory eating quality scores. HPP can produce high eating quality eye of rump medallions within 1day of slaughter.

A macrocyclic calpain inhibitor slows the development of inherited cortical cataracts in a sheep model.

PURPOSE: We used sheep with an autosomal dominant gene for cortical cataract as an animal model to evaluate novel macrocyclic calpain inhibitors with potential for the medical treatment of human cataract. METHODS: The macrocyclic aldehyde, CAT811, identified previously as a calpain inhibitor that prevents calcium-induced opacification in cultured sheep lenses, was tested for its ability to protect cytoskeletal proteins from calpain proteolysis. CAT811 and its alcohol analogue, CAT505, were formulated separately into ointments, and each was applied twice daily to the right eye of sheep with early cataracts for five months. Progress of cataracts in the sheep was determined by ophthalmologic examination and comparison with a matched sample of sheep treated similarly with ointment that did not contain the active ingredient. RESULTS: The novel macrocyclic aldehyde, CAT811, was able to inhibit calpain proteolysis of lens cytoskeletal proteins at micromolar concentrations. When applied topically to the eyes of sheep, CAT811 was able to slow cataract development by 27% in the initial three months of treatment (P < 0.05). Its alcohol analogue, CAT505, was not able to slow cataractogenesis significantly. CONCLUSIONS: The inherited sheep cataract provides a reproducible model of cortical cataract over a time scale of several months. The data reported here, using this model, demonstrated the potential of the macrocyclic calpain inhibitor, CAT811, to act as a therapeutic for treatment of cortical cataract.

Molecular modeling, synthesis, and biological evaluation of macrocyclic calpain inhibitors.

The design and elaboration of a series of macrocyclic templates that exhibit a propensity to adopt a beta-strand-like peptide-backbone conformation led to potent and selective inhibitors of calpain 2. Macrocycle 1 retarded calcium-induced opacification in an ovine-lens culture assay and is a lead compound for the development of a drug for cataract treatment. Cbz=carbobenzyloxy.

The involvement of calpains in opacification induced by Ca2+-overload in ovine lens culture.

OBJECTIVE: To investigate biochemical changes accompanying Ca(2+)-induced lens opacification and the possible role of calpain activation in opacification within an ovine lens culture system. METHODS: Sheep lenses were cultured in minimal media. Lens opacification was induced by exposure to the Ca(2+) ionophore, ionomycin, and graded by digital image analysis. Cell viability was estimated by the release of lactate dehydrogenase into the culture medium. Opaque lenses were fixed and stained for a microscopic view of the lens structural changes. Ionic changes in the lens were measured by atomic absorption spectroscopy. Calpain activation was determined by zymography on casein gels and proteolysis was investigated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2DE) and Western blotting. The calpain inhibitor, SJA6017, was used to investigate the involvement of calpains in lens opacification. RESULTS: Treatment of cultured ovine lenses with ionomycin increased total lens Ca(2+) concentration and caused the cortical region of the lens to become opaque. Addition of the Ca(2+) chelator, EGTA, inhibited the ionomycin-induced changes. Progress of opacification correlated with the death of lens cells and lens swelling in differentiating fiber cells. Autolysis of calpain 2, following ionomycin treatment, suggested activation of this protease. 2DE revealed that the ionomycin did not result in substantial proteolysis of the crystallins. However, Western blotting revealed significant breakdown of the cytoskeletal proteins, spectrin and vimentin. The pattern of the breakdown products was consistent with calpain proteolytic activity. SJA6017 retarded the cortical opacity induced by Ca(2+)-overload in the ovine lens. CONCLUSION: The ovine lens with Ca(2+)-induced opacification by ionomycin is associated with calpain activation and the subsequent proteolysis of cytoskeletal proteins. These events could be initial factors contributing to cell death and the loss of lens transparency which occurs in this ovine model of cataractogenesis. The ovine model supports the hypothesis that cytoskeletal proteins and Ca(2+) homeostasis play an important role in maintaining lens transparency.

Synthesis, biological evaluation and molecular modelling of N-heterocyclic dipeptide aldehydes as selective calpain inhibitors.

A series of N-heterocyclic dipeptide aldehydes 4-13 have been synthesised and evaluated as inhibitors of ovine calpain 1 (o-CAPN1) and ovine calpain 2 (o-CAPN2). 5-Formyl-pyrrole 9 (IC(50) values of 290 and 25nM against o-CAPN1 and o-CAPN2, respectively) was the most potent and selective o-CAPN2 inhibitor, displaying >11-fold selectivity. The amino acid sequences of o-CAPN1 and o-CAPN2 have been determined. Because of the lack of available structural information on the ovine calpains, in silico homology models of the active site cleft of o-CAPN1 and o-CAPN2 were developed based on human calpain 1 (h-CAPN1) X-ray crystal structure (PDB code 1ZCM). These models were used to rationalise the observed SAR for compounds 4-13 and the selectivity observed for 9. The o-CAPN2 selective inhibitor 9 (CAT0059) was assayed in an in vitro ovine lens culture system and shown to successfully protect the lens from calcium-induced opacification.

Evaluation of a novel calpain inhibitor as a treatment for cataract.

PURPOSE: The aim of this study is to evaluate the therapeutic potential of a newly synthesized calpain inhibitor, CAT0059, using a naturally occurring in vivo sheep cataract model. METHODS: The selectivity of CAT0059 was investigated by an in vitro protease assay. The efficacy of CAT0059 in preventing proteolysis of lens cytoskeletal proteins by calpain 2 was investigated using a lens-based cell-free method. The cytotoxicity and stability of CAT0059 in physiological conditions were examined using cultured sheep lenses. Protein binding of CAT0059 by ocular proteins was assessed and quantified by a modified high-performance liquid chromatography assay. CAT0059 was formulated in an eye drop solution and as an eye ointment. These were applied in vivo daily to one eye of the cataract lambs, over a 67- and 97-day trial period, respectively. The progression of cataracts in the treated and untreated eyes was assessed by an independent veterinary ophthalmologist using a slit-lamp microscope. RESULTS: In vitro assays revealed that CAT0059 was selective for cysteine proteases and also protected lens cytoskeletal proteins from degradation. CAT0059 was stable in physiological conditions and non-toxic to the lens. Only 15% of CAT0059 is bound to proteins in the aqueous humour but >90% bound to lens homogenate. The 67-day CAT0059 eye drop treatment was not effective in slowing the rate of cataract development. However, application of CAT0059 in an eye ointment initially slowed cataract development compared with the untreated eye. This effect was temporary. CONCLUSIONS: In vitro assays confirmed CAT0059 to be a potent calpain inhibitor. The two in vivo trials addressed the ability of CAT0059 to reach the lens and established its limitations as a therapeutic molecule for cataract treatment.

Investigation into the P3 binding domain of m-calpain using photoswitchable diazo- and triazene-dipeptide aldehydes: new anticataract agents.

The photoswitchable N-terminal diazo and triazene-dipeptide aldehydes 8a-d, 10a,b, and 17a,b present predominantly as the (E)-isomer, which purportedly binds deep in the S3 pocket of calpain. All compounds are potent inhibitors of m-calpain, with 8b being the most active (IC50 of 35 nM). The diazo-containing inhibitors 8a, 8c, and 10a were irradiated at 340 nm to give a photostationary state enriched in the (Z)-isomer, and in all cases, these were less active. The most water soluble triazene 17a (IC50 of 90 nM) retards calpain-induced cataract formation in lens culture.