The International Consortium on Primary Graft Dysfunction: Redefining Clinical Risk Factors in the Contemporary Era of Heart Transplantation.

BACKGROUND: Primary graft dysfunction (PGD) is the leading cause of morbidity and mortality early after heart transplantation (HT). The International Consortium on PGD is a multicenter collaboration dedicated to identifying the clinical risk factors for PGD in the contemporary era of HT. The objectives of the current report were (1) to assess the incidence of severe PGD in an international cohort; (2) to evaluate the performance of the most strongly validated PGD risk tool, the RADIAL score, in a contemporary cohort; and (3) to redefine clinical risk factors for severe PGD in the current era of HT. METHODS: This is a retrospective, observational study of consecutive adult HT recipients between 2010 and 2020 in 10 centers in the United States, Canada and Europe. Patients with severe PGD were compared to those without severe PGD (comprising those with no, mild and moderate PGD). The RADIAL score was calculated for each transplant recipient. The discriminatory power of the RADIAL score was evaluated using receiver operating characteristic (ROC) analysis, and its calibration was assessed by plotting the percentage of PGD predicted vs that which was observed. To identify clinical risk factors associated with severe PGD, we performed multivariable mixed-effects logistic regression modeling to account for among-center variability. RESULTS: A total of 2746 patients have been enrolled in the registry to date, including 2015 (73.4%) from North America, and 731 (26.6%) from Europe; 215 participants (7.8%) met the criteria for severe PGD. There was an increase in the incidence of severe PGD over the study period (P value for trend by difference sign test = 0.004). The Kaplan-Meier estimate for 1-year survival was 75.7% (95% CI 69.4-80.9%) in patients with severe PGD as compared to 94.4% (95% CI 93.5-95.2%) in those without severe PGD (log-rank P value < 0.001). The RADIAL score performed poorly in our contemporary cohort and was not associated with severe PGD; it had an AUC of 0.53 (95% CI 0.48-0.58). In the multivariable regression model, acute preoperative dialysis (OR 2.41, 95% CI 1.31-4.43), durable left ventricular assist device support (OR 1.77, 95% CI 1.13-2.77), and total ischemic time (OR 1.20 for each additional hour, 95% CI 1.02-1.41) were associated with an increased risk of severe PGD. CONCLUSIONS: Our consortium has identified an increasing incidence of PGD in the modern transplant era. We identified contemporary risk factors for this early post-transplant complication, which confers a high mortality risk. These results may enable the identification of patients at high risk for developing severe PGD in order to inform peri-transplant donor and recipient management practices.

Direct T cell-tumour interaction triggers TH1 phenotype activation through the modification of the mesenchymal stromal cells transcriptional programme.

BACKGROUND: Mesenchymal stromal cells (MSCs) are heterogeneous cells with immunoregulatory and wound-healing properties. In cancer, they are known to be an essential part of the tumour microenvironment. However, their role in tumour growth and rejection remains unclear. To investigate this, we co-cultured human MSCs, tumour infiltrating lymphocytes (TIL), and melanoma cells to investigate the role of MSCs in the tumour environment. METHODS: Mesenchymal stromal cells were co-cultured with melanoma antigen-specific TIL that were stimulated either with HLA-A*0201(+) melanoma cells or with a corresponding clone that had lost HLA-A*0201 expression. RESULTS: Activated TIL induced profound pro-inflammatory gene expression signature in MSCs. Analysis of culture supernatant found that MSCs secreted pro-inflammatory cytokines, including TH1 cytokines that have been previously associated with immune-mediated antitumor responses. In addition, immunohistochemical analysis on selected markers revealed that the same activated MSCs secreted both the TH1 cytokine (interleukin-12) and indoleamine 2,3 dioxygenase (IDO), a classical immunosuppressive factor. CONCLUSIONS: This study reflected that the plasticity of MSCs is highly dependent upon microenvironment conditions. Tumour-activated TIL induced TH1 phenotype change in MSCs that is qualitatively similar to the previously described immunologic constant of rejection signature observed during immune-mediated, tissue-specific destruction. This response may be responsible for the in loco amplification of antigen-specific anti-cancer immune response.

In-situ gelling xyloglucan formulations as 3D artificial niche for adipose stem cell spheroids.

Three-dimensional spheroidal cell aggregates of adipose stem cells (SASCs) are a distinct upstream population of stem cells present in adipose tissue, with enhanced regeneration properties in vivo. The preservation of the 3D structure of the cells, from extraction to administration, can be a promising strategy to ensure optimal conditions for cell viability and maintenance of stemness potential. With this aim, an artificial niche was created by incorporating the spheroids into an injectable, in-situ gelling solution of partially degalactosylated xyloglucan (dXG) and an ad hoc formulated culture medium for the preservation of stem cell spheroid features. The evolution of the mechanical properties and the morphological structure of this artificial niche was investigated by small amplitude rheological analysis and scanning electron microscopy, respectively. Comparatively, systems produced with the same polymer and the typical culture medium (DMEM) used for adipose stem cell (ASC) growth in adherent cell culture conditions were also characterised. Cell viability of both SASCs and ASCs incorporated inside the hydrogel or seeded on top of the hydrogel were investigated as well as the preservation of SASC stemness conditions when embedded in the hydrogel.

An electronic patient-reported outcomes measurement system in paediatric orthopaedics.

PURPOSE: The purpose of the study was to evaluate the reliability, review differences and assess patient satisfaction of electronic patient-reported outcome measures (PROMs) compared with paper PROMs. METHODS: Participants between 12 and 19 years of age with a knee-related primary complaint were randomized into two groups. Group 1 completed paper PROMs followed by electronic, while Group 2 received the electronic followed by paper. PROMs included the Pediatric International Knee Documentation Committee (Pedi-IKDC), Hospital for Special Surgery (HSS) Pediatric Functional Activity Brief Scale (HSS Pedi-FABS), Tegner Activity Level Scale, Visual Analogue Scale (VAS), PedsQL Teen and a satisfaction survey. RESULTS: In all, 87 participants were enrolled with one excluded due to incomplete PROMs. Of the 86 participants, 54 were female and 32 were male with an average age of 14.3 years (12 to 18). A high degree of reliability was found when comparing the paper and electronic versions of the Pedi-IKDC (0.946; p < 0.001), HSS Pedi-FABS (0.923; p < 0.001), PedsQL Teen (0.894; p < 0.001), Tegner Activity Level Scale before injury (0.848; p < 0.001) and the Tegner Activity Level Scale after (0.930; p < 0.001). Differences were noted between the VAS scores, with paper scores being significantly higher than electronic (5.3 versus 4.6; p < 0.001). While not significant, a trend was noted in which electronic PROMs took, overall, less time than paper (10.0 mins versus 11.2 mins; p = 0.096).Of all participants, 69.8% preferred the electronic PROMs, 67.4% felt they were faster, 93.0% stated they would complete forms at home prior to appointments and 91.8% were not concerned about the safety/privacy of electronic forms. CONCLUSION: PROMs captured electronically were reliable when compared with paper. Electronic PROMs may be quicker, will not require manual scoring and are preferred by patients. LEVEL OF EVIDENCE: II.

Effects of inducible overexpression of DNp73alpha on cancer cell growth and response to treatment in vitro and in vivo.

The p73 gene has a complex regulation, which leads to the expression of different isoforms, often with opposite biological effects. We have generated in the human colocarcinoma cell line HCT116, expressing a wild-type p53, an inducible DNp73alpha expressing system. Two clones (HCT116/DN3 and HCT116/DN14), upon doxycycline addition, show a strong expression of DNp73alpha. In vitro the two DNp73alpha overexpressing clones grow at similar rate of the control transfected clone (HCT116/8a) and similarly respond to DNA damage. When injected in mice, HCT116/DN3, HCT116/DN14, and HCT116/8a cells grew similarly in the absence or presence of tetracycline. In HCT116/DN3 and HCT116/DN14 tumors, tetracycline induced a strong expression of DNp73alpha both as mRNA and protein. These results indicate that in this system the overexpression of the DNp73alpha does not induce a more aggressive phenotype and does not seem to be associated with a reduced response of the cells to treatment with anticancer agents.

Methodology to analyse small silicon samples by glow discharge mass spectrometry using a thin wafer mask.

Glow discharge mass spectrometry (GDMS) is widely used for trace element analysis of bulk solid samples. The geometry of the GD source limits the minimum size of the sample, which for the instrument used in this work (ThermoElementGD) is 20 mm in diameter. From time to time, there is the need to analyse smaller samples with this technique, and we present here a methodology to analyse samples of 9-20 mm diameter through the use of thin masks. Thin masks have been previously used mostly as secondary cathode for the analysis of non-conducting materials, with hole size smaller than the area of the glow discharge. The use of masks in this work includes the following customization:•The choice of highly-pure Si as mask material, to decrease the chance of interferences with the Si samples.•The use of a hole in the mask of the same size as the discharge area. This implies that the mask material is not sputtered, thus decreasing chances for contamination from the mask itself.

Gemcitabine increases systemic 5-fluorouracil exposure in advanced cancer patients.

A number of recent clinical trials testing the combination of 5-fluorouracil (5-FU) and gemcitabine in patients with advanced pancreatic adenocarcinoma have shown a significant clinical response rate, but also significant toxicity. As the two antimetabolites may interact at several biochemical levels along their pathways of activation, we investigated whether gemcitabine (GEM) affects 5-FU pharmacokinetics in cancer patients. Thus, we compared 5-FU pharmacokinetics in two groups of patients with various cancers who received the same schedule of 5-FU and folinic acid (FUFA), with or without GEM. There was a significant increase in systemic (5-FU) exposure and toxicity in the FUFA plus GEM group. Our finding may be useful in designing future studies of the combination in order to reduce the occurrence of side-effects and to maximise the antitumour activity.

Tumour-associated antigen (TAA)-specific cytotoxic T cell (CTL) response in vitro and in a mouse model, induced by TAA-plasmids delivered by influenza virosomes.

We investigated influenza virosomes as a TAA-gene delivery system for use in TAA-directed anti-cancer vaccine therapy. An engineered plasmid (GC90) expressing the parathyroid hormone-related peptide (PTH-rP), a protein secreted by prostate and lung carcinoma cells, was included in influenza virosomes (GC90V). The ability of GC90V to elicit a PTH-rP-specific cytotoxic T cell (CTL) response was demonstrated in BALB/c mice immunised with intranasal (i.n.) GC90V+/-adjuvant subcutaneous (s.c.) interleukin-2 (IL-2). A PTH-rP-specific CTL response with antitumour activity was also demonstrated in human peripheral blood mononuclear cells (PBMC) stimulated in vitro with GC90V infected autologous dendritic cells (DC). These results provide a rationale for investigating GC90V in clinical trials of anticancer vaccine therapy.

Recruitment of dendritic cells and enhanced antigen-specific immune reactivity in cancer patients treated with hr-GM-CSF (Molgramostim) and hr-IL-2. results from a phase Ib clinical trial.

Experimental findings suggest that granulocyte-monocyte-colony stimulating factor (GM-CSF) synergistically interacts with interleukin-2 (IL-2) in generating an efficient antigen-specific immune response. We evaluated the toxicity, antitumour activity and immunobiological effects of human recombinant (hr)-GM-CSF and hr-IL-2 in 25 cancer patients who subcutaneously (s.c.) received hr-GM-CSF 150 microg/day for 5 days, followed by hrIL-2 s.c. for 10 days and 15 days rest. Two of the most common side-effects were bone pain and fever. Of the 24 patients evaluable for response, 3 achieved partial remission, 13 experienced stable disease, and 8 progressed. Cytokine treatment increased the number of monocytes, dendritic cells (DC), and lymphocytes (memory T cells) in the peripheral blood and enhanced the antigen-specific immunoreactivity of these patients. Our results show that the hr-GM-CSF and hr-IL-2 combination is active and well tolerated. Its biological activity may support tumour associated antigen (TAA)-specific anticancer immunotherapy by increasing antigen presenting cell (APC) activity and T cell immune competence in vivo.

Striatal influence on ingestive behaviour in the cat.

In freely moving cats with chronically implanted electrodes an analysis was made of the effects on feeding behaviour of low-frequency long-duration stimulation of the caudate nucleus, the substantia nigra and the globus pallidus. In all 3 structures a significant reduction of food intake was observed and in the pallidus this reached the point of a complete block of feeding. The effects were always limited to the period of stimulation. At the end of stimulation the animals recovered and took in food quantities equal to those of controls. The results are interpreted on the basis of reciprocal connections between the basal ganglia and the hypothalamus; the role of the striatum on the selection of certain movements and its possible involvement in behaviour is also discussed.

Substantia nigra-mediated anticonvulsant action: a possible role of a dopaminergic component.

A number of neural pathways may mediate nigral control of epilepsy. According to the literature, a GABAergic nigrotectal pathway may be responsible for the control exerted by the substantia nigra on the diffusion of discharges toward spinal targets, while the nigrothalamic projection may transfer nigral influence on premotor neocortical epilepsy. Since there is probably an anatomical nigrohippocampal pathway arising from dopaminergic cells in the substantia nigra, we tested the effects of stimulating the substantia nigra pars compacta (SNpc) on focal hippocampal epilepsy induced by penicillin injection in the cat. The possibility of dopamine involvement was further tested by studying the effects of intraperitoneal injection of haloperidol, a dopamine receptor blocking agent on nigrohippocampal influences, while to verify the precise site of action, in other groups of cats, sulpiride and apomorphine (D-receptor antagonist and agonist, respectively) were locally administered in the dorsal hippocampus. Furthermore, modifications of hippocampal epileptiform EEG were studied in control conditions and following SNpc electrolytic lesions. Results showed a strong nigral suppressive effect on focal hippocampal epilepsy. Nigral stimulation induced a significant decrease in both frequency and amplitude of hippocampal spikes, which disappeared either about 10 min after i.p. injection of haloperidol 1 mg/kg or about 5 min after intrahippocampal administration of sulpiride, and did not return during a further hour or more of experimental observation. It should be emphasized that in the absence of nigral stimulation, both haloperidol and sulpiride did not modify hippocampal spike frequency. Apomorphine application to dorsal hippocampus induced a marked reduction of hippocampal epileptiform activity parallel to the effect observed during SNpc stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of desipramine and alprazolam on forced swimming behaviour of adult rats exposed to prenatal diazepam.

Pregnant rats were treated with a single daily s.c. injection of diazepam (2 mg/kg) over gestation days 14-20. This treatment led to a reduction in GABA receptor complex function since adult male offspring showed a strong decrease in electrographic hippocampal responses to alprazolam and a strongly increased response to picrotoxin after intra-locus coeruleus injection of the two compounds. No difference in immobility time in the forced swimming test and in spontaneous motor activity was observed between prenatally vehicle- and diazepam-exposed offspring. Conversely, prenatal exposure to diazepam potentiated the anti-immobility effect of subchronic desipramine (10 mg/kg i.p.) and made active a dose of desipramine (5 mg/kg i.p.) that was ineffective in prenatally vehicle-exposed rats. This effect was observed only in pretested rats. Prenatal exposure to diazepam blocked the anti-immobility effect of subchronic alprazolam (15 mg/kg i.p.) in both non-pretested and pretested rats. Spontaneous motor activity was strongly reduced in all groups. These findings suggest that a persistent reduction in GABA receptor complex function, induced by prenatal exposure to diazepam, does not alter the mobility of adult progeny in the forced swimming test, but it may have consequences when drugs acting on the GABA receptor complex are used.

Effects of substantia nigra and pallidum stimulation on hippocampal interictal activity in the cat.

In the present work the role played by substantia nigra pars compacta and globus pallidus pars interna on hippocampal bioelectrical activity is studied. Injections of sodium penicillin (i.v.) produce steady interictal spikes in the hippocampus. Substantia nigra stimulation induces regular theta rhythm and inhibits the spikes. Pallidal stimulation, on the contrary, appears to strongly enhance epileptiform activity, proceeding to generalized seizure activity. The results are discussed in the light of the interrelationships between basal ganglia and hippocampus, hypothesizing a putative feedback loop from striatal to limbic centers.

Striatal and septal influence on hippocampal theta and spikes in the cat.

The experiments studied the modulation exerted by the septum and the caudate nucleus on hippocampal activity in the cat. Injections (i.v.) of sodium penicillin were performed in order to obtain a steady interictal epileptic activity. Hippocampal slow rhythmic activity showed a marked decrease either in duration or in frequency following penicillin activation. Both septal and caudate electrical stimulation inhibited spike frequency through a theta eliciting mechanism. Caudate stimulation failed to determine any sort of effect after medial septum lesions. The importance of the septum as modulation station between basal ganglia and hippocampus is emphasized.

An electrophysiological study of habenular influence on hippocampus.

The action of lateral habenula (LH) stimulation on focal epileptiform activity in the hippocampus was studied. Local microinjection of sodium penicillin induced a steady interictal activity in the dorsal hippocampus. Low frequency electrical stimulation of the habenula caused a marked enhancement of spike activity in both frequency and amplitude. The effect was blocked by intraperitoneally injected methysergide. The facilitatory influence of the habenula on hippocampal activity might be due to a disinhibitory mechanism. The results are regarded as suggesting that the habenula may be a relay station between the basal ganglia and the hippocampal formation. LH as well as basal ganglia might modulate hippocampal excitability, exerting a control on the genesis and diffusion of abnormal activities.

Anticonvulsant activity of the noradrenergic locus coeruleus system: role of beta mediation.

Many experimental observations have demonstrated the modulatory role exerted by several neural structures and neurotransmitters on spontaneous and paroxysmal bioelectric activity of the hippocampus. Recently, the control exerted by locus coeruleus (LC) and its noradrenergic (NA) efferent pathway on different experimental models of epilepsy (e.g. cortical cobalt chronic epilepsy, amygdaloid and hippocampal kindling) was emphasised. On this basis, a series of experiments was performed to elucidate the functional role of LC-NA system on the hippocampal penicillin (PCN) focus and the type of adrenergic receptor involved. The experiments were carried out on 25 rats in which an epileptiform hippocampal focus was obtained through intrahippocampal PCN administration (100-200 I.U.). In these conditions, LC, ipsilateral to PCN hippocampal focus, was stimulated before and after intraperitoneal (i.p.) administration of a beta-adrenergic receptor antagonist propranolol (2 mg/kg). Results showed a significant reduction of hippocampal spiking frequency during LC stimulation; after i.p. propranolol injection, LC stimulation, at the same parameters, failed to induce any sort of modification of PCN hippocampal spiking frequency. Furthermore, intrahippocampal application of a beta-selective agonist 2-fluoro-noradrenaline (2-FNA) mimics the inhibitory effects of LC stimulation. All data suggest that the LC-NA system is able to induce a net reduction of hippocampal epileptiform focus and the inhibitory NA control involves the activation of adrenergic beta receptors.

Dopaminergic control of feline hippocampal epilepsy: a nigro hippocampal pathway.

Substantia nigra is a mesencephalic structure inserted along several circuits which appear to play a key role in epilepsy. In previous researches we postulated that substantia nigra pars compacta (SNpc) may be the site of a precise control of hippocampal epilepsy while substantia nigra pars reticulata (SNpr) may exert a modulation of both neocortical epilepsy and spreading of hyperactivity toward a motor target. In order to better understand mechanisms subserving nigral action in feline hippocampal epilepsy we electrically stimulated SNpc (dopaminergic), before and after sulpiride (dopamine receptor-antagonist) intravenous injection. Furthermore we compared hippocampal epileptiform activity prior to and after apomorphine (dopamine receptor-agonist) intrahippocampal injection as well as prior to and after SNpc electrolytic destruction. Results showed that SNpc is able to regulate hippocampal epilepsy. This effect is selectively antagonized by sulpiride while apomorphine exerts, synergically with SNpc stimulation, inhibitory effects. On the contrary SNpc lesions induces a significant enhancement of hippocampal epileptiform spikes. Experimental findings suggest that SNpc represents a strategic region for the control of hippocampal excitability and that this regulation appears to be dopaminergic in nature.

Striatonigral suppression of focal hippocampal epilepsy.

Both caudate nucleus (CN) and substantia nigra (SN) appear to be involved in the control of epileptogenic events. Previous investigations had demonstrated that both CN and SN stimulations are able to induce hippocampal theta (theta) rhythm and an inhibition of epileptiform spikes. Since the two structures are reciprocally linked by fibre pathways, experiments were carried out to test the possibility that CN influences the hippocampus via SN or vice versa. To this end, changes in penicillin-induced hippocampal spikes by CN or SN stimulation were studied before and after destruction of SN and CN respectively. Steady interictal activity was induced in the hippocampus of encéphale isolé cats by local injection of penicillin. Stimulations of both CN and SN induced statistically significant reduction of hippocampal spike frequency, and in some cases a clear and regular theta-rhythm. These effects were unchanged by the destruction of either CN or SN. The results add further information to the role played by the basal ganglia and SN in the control of epilepsy, and underline the possibility that caudate and nigral influences on the hippocampus are mediated by different pathways.

Modulation of paroxysmal activity in the hippocampus by caudate stimulation in the chronic cat.

Afterdischarges in the dorsal hippocampus (HADs) were studied in freely-moving cats with implanted electrodes following threshold stimulation of the mirror-image point on the contralateral side. Marked inhibition, similar so that seen in acute animals, was observed when the test stimulation was immediately preceded by a conditioning stimulus applied to the caudate nucleus. The inhibitory effect appeared to be larger in these chronic animals than in the acute preparations previously studied, probably because of the total absence of anaesthesia during the recording session. When the HAD is preceded by caudate stimulation, its duration can be graduated by the intensity of the hippocampal test stimulation. The results are discussed in terms of a possible modulation induced directly or indirectly by the caudate nucleus in the hippocampus, which reacts in a gradual manner to the excitatory volley.

Hippocampal seizures and striatal regulation: a possible functional pathway.

Experimental findings have suggested the possibility of a functional relationship between the basal ganglia and the hippocampus. Previous research has revealed a predominantly inhibitory action of the caudate nucleus (CN) and an excitatory effect of the globus pallidus (GP) on electrically induced hippocampal afterdischarges (HAD). The effects of electrolytic destruction of the CN on the threshold and duration of HAD has been studied in the 'encéphale isolé' cat. The threshold and duration of HAD was also studied following conditioning stimulation of the CN in animals in which the inner segment of the globus pallidus (GPi) and medial septal nucleus (MSN) had been destroyed. Following CN lesions, the hippocampal excitability threshold underwent a significant reduction, while the duration of HAD appeared to be increased. Following destruction of the GPi and MSN, the threshold and duration of HAD exhibited no change following conditioning stimulation of the CN. The results reveal a tonic inhibitory effect of the CN on the hippocampus and suggest that a strio-pallido-septal pathway is the anatomical substrate for the effect.