Nutritional management in renal transplant recipients: A transplant team opportunity to improve graft survival.

AIMS: The nutritional management of renal transplant recipients (RTR) represents a complex problem either because the recovery of renal function is not complete and for the appearance of "unavoidable" metabolic side effects of immunosuppressive drugs. Nevertheless, it remains a neglected problem, whereas an appropriate dietary intervention could favorably affect graft survival. DATA SYNTHESIS: Renal transplantation is associated with steroids and calcineurin inhibitors administration, liberalization of diet after dialysis restrictions, and patients' better quality of life. These factors predispose, from the first months after surgery, to body weight gain, enhanced post transplant diabetes, hyperlipidemia, metabolic syndrome, with negative consequences on graft outcome. Unfortunately, specific guidelines about this topic and nutritional counseling are scarce; moreover, beyond the low adherence of patients to any dietary plan, there is a dangerous underestimation of the problem by physicians, sometimes with inadequate interventions. A prompt and specific nutritional management of RTR can help prevent or minimize these metabolic alterations, mostly when associated with careful and repeated counseling. CONCLUSIONS: A correct nutritional management, possibly tailored to enhance patients' motivation and adherence, represents the best preventive maneuver to increase patients' life and probably improve graft survival, at no cost and with no side effects.

Oscillatory mTOR inhibition and Treg increase in kidney transplantation.

Intracellular metabolic pathways dependent upon the mammalian target of rapamycin (mTOR) play a key role in immune-tolerance control. In this study, we focused on long-term mTOR-dependent immune-modulating effects in kidney transplant recipients undergoing conversion from calcineurin inhibitors (CNI) to mTOR inhibitors (everolimus) in a 1-year follow-up. The conversion to everolimus is associated with a decrease of neutrophils and of CD8(+) T cells. In addition, we observed a reduced production of interferon (IFN)-γ by CD8(+) T cells and of interleukin (IL)-17 by CD4(+) T lymphocytes. An increase in CD4(+) CD25(+) forkhead box protein 3 (FoxP3)(+) [regulatory T cell [(Treg)] numbers was also seen. Treg increase correlated with a higher proliferation rate of this regulatory subpopulation when compared with the CD4(+) FoxP3(-) effector counterpart. Basal phosphorylation level of S6 kinase, a major mTOR-dependent molecular target, was substantially maintained in patients treated with everolimus. Moreover, oscillations in serum concentration of everolimus were associated with changes in basal and activation-dependent S6 kinase phosphorylation of CD4(+) and CD8(+) T cells. Indeed, T cell receptor (TCR) triggering was observed to induce significantly higher S6 kinase phosphorylation in the presence of lower everolimus serum concentrations. These results unveil the complex mTOR-dependent immune-metabolic network leading to long-term immune-modulation and might have relevance for novel therapeutic settings in kidney transplants.

Efficacy of subcutaneous epoetin-zeta on anemia in renal transplant recipients: a single-center experience.

BACKGROUND: Persistent or "de novo" anemia (plasma hemoglobin<11 g/dL) may complicate the graft outcome in a significant number of renal transplant recipients. We describe a single-center experience with epoetin-zeta (EPO-Z), the biosimilar form for epoetin-alfa. METHODS: Twenty patients were included in the study, 10 in treatment with different erythropoiesis-stimulating agents (ESA) and shifted to EPO-Z (shift group) and 10 who started EPO-Z treatment for anemia (naive group). All the patients had stable renal function and normal values of main inflammation markers and were prospectively followed up for 12 months. Iron supplements were administered during the study, as needed. RESULTS: In the shift group, mean plasma hemoglobin levels>11 g/dL were maintained for the entire 1-year follow-up period, with average EPO-Z doses 3.4% higher than the corresponding doses of previous ESA; in the naive group, the target value was reached between the first and third months and remained stable throughout the study. Mean corpuscular volume did not vary in either group. No change was observed in glomerular filtration rate, nor in proteinuria or in main laboratory data. No drug-related side effect was reported. CONCLUSIONS: EPO-Z may be considered a valid alternative to different ESAs in renal transplant recipients, with an interesting pharmaco-economic profile, considering its lower cost.

T-cell subpopulations express a different pattern of dopaminergic markers in intra- and extra-thymic compartments.

An involvement of dopamine in regulation of the immune function has been assessed and dopaminergic system has been found widely represented in thymus. Nevertheless detail on the characterization of dopaminergic system in assisting thymocytes development and lymphocytes mature physiology are still lacking. The present study was designed to characterize dopamine plasma membrane transporter (DAT), vesicular dopamine transporters (VMAT)-1 and -2, and dopamine D1-like and D2-like receptors in rat thymocytes, splenocytes and peripheral blood mononuclear cells. Western blot and RT-PCR analyses, performed on these cells, showed an expression of dopamine transporters and receptors during thymocyte development (when of CD4 and CD8 markers are differently expressed). Furthermore FACS analysis, indicates that DAT and dopamine D1-like receptors are expressed at high levels in thymocytes, splenocytes, and peripheral lymphocytes. The percentage of CD4+ CD8+ (double-positive) thymocytes expressing dopaminergic markers was significantly higher compared to the percentage of double-negative ones. The percentage of CD8+ single positive cells expressing dopaminergic markers was significantly higher than that of CD4+ cells. The results suggest that the dopaminergic system plays a role in the thymus microenvironment during T-cell development. The more pronounced expression of dopaminergic markers in CD8+ subsets suggests that dopamine plays a role in development of cytotoxic T-cells. Our findings indicate dopaminergic system to have a role during the maturation and selection of lymphocytes, and support its involvement in the active phases of immune response.

Effects of switching from agalsidase Beta to agalsidase alfa in 10 patients with anderson-fabry disease.

Anderson-Fabry disease (AFD) is a multiorgan X-linked lysosomal storage disease that particularly affects the heart, kidneys, and cerebrovascular system. Current treatment is enzyme replacement therapy (ERT) with agalsidase beta (Fabrazyme(®), Genzyme Corporation, Cambridge, MA, USA) or agalsidase alfa (Replagal(®), Shire Human Genetic Therapies AB, Lund, Sweden). It was recommended that patients switch to agalsidase alfa due to a manufacturing shortage of agalsidase beta beginning in June 2009. This study assessed the effect of switching to agalsidase alfa on clinical outcomes in patients with AFD previously treated with agalsidase beta. Ten patients (seven male, three female) with genetically confirmed AFD and at least 48 months' continuous data collected during treatment with agalsidase beta 1 mg/kg every other week were switched to agalsidase alfa 0.2 mg/kg every other week for at least 20 months, with prospective clinical evaluations every 6 months. Pre-switch data was collected retrospectively from patient charts. Cardiac functional parameters were assessed using magnetic resonance imaging. Results showed that renal function was normal (estimated glomerular filtration rate ≥90 mL/min/1.73 m(2)) in 8 of 10 patients prior to agalsidase alfa and generally remained stable after the switch. Cardiac mass decreased significantly (p < 0.05 vs pre-ERT) after agalsidase beta and remained unchanged after switching to agalsidase alfa. Symptoms of pain and health status scores did not deteriorate during agalsidase alfa therapy. Adverse events were mostly mild and infusion related. In conclusion, switching to agalsidase alfa was relatively well tolerated and associated with stable clinical status and preserved renal and cardiac function.

Non pigmented melanocytic nevus of the oral cavity: a case report with emphasis on the surgical excision procedures.

We report a case of a 37-year-old caucasian woman presenting a 1 cm pinkish nodular asymptomatic lesion of the hard palate, slowly growing in the last years. The lesion underwent to biopsy. Histological analysis showed the nevus tissue layered under a continuous squamous epithelium. The stroma contained nests of medium-sized round cells, with regular monomorphous nuclei. The nevus cells were immunohistochemically positive for S-100 protein, while melanin, visualized by Masson-Fontana silver staining, was absent. Therefore a diagnosis of non pigmented melanocytic nevus was formulated. Because of its rarity and to avoid any risk of malignant transformation, a surgical treatment with wide excision was chosen; the surgical wound was previously covered with a membrane of fibrin and autologous platelets, and subsequently sutured, resulting in a total heal. This procedure seems to be the most reliable to approach melanocytic lesions of the oral cavity. Clinical diagnosis of non-pigmented nevi, either flat or protruding, is difficult, because the nevus shows a pinkish colour that is indistinguishable from that of the surrounding mucosa. Moreover, attention is required when similar clinical evidence occurs, because the localization inside the oral cavity may offer several problems of differential diagnosis.

Analysis of nerve supply pattern in human lymphatic vessels of young and old men.

BACKGROUND: The present work deals with innervation patterns along collector lymphatic vessels from cervical, mesenteric, and femoral regions, and lymph capillaries in young and elderly subjects. METHODS AND RESULTS: Morphological and morphometric analysis of nerve fibers along lymph vessels was performed by immunohistochemistry for PGP 9.5, NPY, TH, ChAT, VIP, SP, and dopamine. Nerves containing NPY and TH were frequent, whereas immunoreactivity for ChAT and VIP were few. SP-positive fibers were widely distributed in the medial and endothelial layers. Dopamine neurotransmitters were observed in a few short nerve fibers. A more diffuse presence of nerve fibers in mesenteric and femoral lymph vessels, compared to cervical ones, was detected. In lymph capillary vessels, a few nerve fibers positive for neuropeptides and neurotransmitters were detected, whereas no dopamine and VIP immunoreactive fibers were detected. A wide reduction of all specific nerve fibers analyzed was detected in lymph vessels from elderly subjects. CONCLUSIONS: The presence on lymph vessels of sympathetic and parasympathetic nerve systems can be declared. The differences observed in lymphatic vessel innervation patterns may note the involvement in lymph flow regulation, calling attention in aging, when nerve fibers reduction may cause functional default of lymph vessels.

Neuropeptides of human thymus in normal and pathological conditions.

Human thymus of healthy subjects and patients affected by thymoma-associated Myastenia Gravis were studied in order to visualize and compare the morphological distributive pattern of four neuropeptides: vasoactive intestinal peptide, substance P, neuropeptide Y, and neurotensin. Based on our observations, we formulated hypotheses on their relations in neuro-immunomodulation under physiological and pathophysiological conditions. Immuno-histochemical staining for neuropeptides was performed and morphological and morphometrical analyses were conducted on healthy and diseased thymus. In normal thymus, a specific distributive pattern was observed for the several neuropeptide-positive nerves in different thymus lobular zones. In particular substance P-positive fibers were observed in subcapsular zone, specifically located into parenchyma, where they represent the almost total amount of fibers; neurotensin-positive fibers were observed primarily located in parenchyma than perivascular site of several thymus lobular zones, and more abundant the cortico-medullary and medullary zones. Instead VIP- and NPY-positive fibers were widely distributed in perivascular and parenchymal sites of several thymus lobular zones. In thymoma, the distribution of neuropeptide-positive fibers was quantitatively reduced, while cells immunopositive to VIP and substance P were quantitatively increased and dispersed. Observation of the perivascular and parenchymal distribution of the analyzed neuropeptides suggests evidence that a regulatory function is performed by nerves and cells that secrete neuropeptide into the thymus. The alteration of neuropeptide patterns in thymoma suggests that these neurotransmitters play a role in autoimmune diseases such as Myastenia Gravis.

RETRACTED: Intrinsic innervation and dopaminergic markers after experimental denervation in rat thymus.

The aim of this study was to examine rat thymus innervation using denervation techniques and to explore the related micro-anatomical localization of dopamine, D1, D2 receptors and dopamine membrane transporter (DAT). In the thymus subcapsular region, the parenchymal cholinergic fibers belong exclusively to phrenic nerve branching. No somatic phrenic nerve branching was detected in any other analysed thymus lobule regions. In rats subjected to sympathetic or parasympathetic ablation, it was observed that catecholaminergic and cholinergic nerve fibers respectively contributed to forming plexuses along vessel walls. In the subcapsular and septal region, no parenchymal nerve branching, belonging to sympathetic or parasympathetic nervous system was noted. Instead, in the deep cortical region, cortico-medullary junction (CM-j) and medulla, catecholaminergic and cholinergic nerve fibers were detected along the vessels and parenchyma. Dopamine and dopamine receptors were widely diffused in the lobular cortico-medullary junction region and in the medulla, where the final steps of thymocyte maturation and their trafficking take place. No variation in dopamine and DAT immune reaction was observed following total or partial parasympathectomy or phrenic nerve cutting. After chemical or surgical sympathectomy however, neither dopamine nor DAT immune reaction was noted again. Instead, D1 and D2 dopamine receptor expression was not affected by thymus denervation. In rats subjected to specific denervation, it was observed the direct intraparenchymal branching of the phrenic nerve and sympathetic and parasympathetic fibers into thymus parenchyma along vessels. These findings on the dopaminergic system highlight the importance of neurotransmitter receptor expression in the homeostasis of neuroimmune modulation.

Serum anti-p53 antibodies as a diagnostic tumor marker: observations in patients with malignant and premalignant oral cavity lesions.

AIM: To determine whether serum anti-p53 antibody (p53-Abs) positivity in patients with oral carcinoma corresponds with tumor localization, histological grade, stage, and recurrence. METHODS: The study population was divided into three groups: controls; patients with a premalignant lesion; and patients with oral squamous cell carcinoma (SCC). The third group was composed of patients attending outpatient services for pathological diagnosis or for follow-up monitoring only. The cancer patients had undergone resective surgery in local anesthesia. Serum p53-Abs levels were measured using a commercial enzyme-linked immunosorbent assay (ELISA) and monitored over a 3-year follow-up period. RESULTS: Controls and patients with premalignant lesions did not test positive for p53-Abs at ELISA testing. Patients with a malignant lesion tested positive at initial diagnosis when a high histopathological grade lesion was present or localized to the posterior region of the oral cavity. Postoperative serum p53-Abs levels gradually declined until complete seronegativity. Patients with a recurrent tumor tested positive for p53-Abs. CONCLUSION: Seropositivity for p53-Abs may be associated with histopathological tumor grade, localization, and recurrence. The findings suggest that serum p53-Abs analysis is a useful diagnostic marker for oral SCC.

Overstressed mechanical stretching activates survival and apoptotic signals in fibroblasts.

The interest of scientists in the effects of mechanical stresses on cells is growing, in order to reproduce and understand cell behaviour in an environment closely reproducing physiological conditions. There have been many studies showing that mechanical stimulations are involved in regulating the proliferation, apoptosis and synthesis of proteins and cell morphology. In this study, we have considered the effects of a 20% stretching mechanical stress on MRC5 lung fibroblast cells in order to verify the role of survival/apoptotic pathways. As a survival pathway, the activation of Akt has been studied in association with pro-apoptotic or anti-apoptotic signals such as the Bax/Bcl-2 ratio and cleavage of caspases 3 and 9. Findings have shown the effects of overstressed cellular stretching to be a balance of a cause-and-effect reaction between survival and apoptosis.

Effects of voriconazole on tacrolimus metabolism in a kidney transplant recipient.

Infection occurs frequently in the organ transplant recipients during the post-transplant period because of immunosuppression. Therefore, prophylactic antimicrobial agents are often used. The azole antifungals, widely prescribed prophylactically, are known to have many drug-drug interactions. This report presents a case of drug-drug interaction between voriconazole and tacrolimus in a kidney transplant recipient. Voriconazole treatment led to a dramatic increase in tacrolimus concentration that required its discontinuation in spite of the manufacturer's guidelines that recommend a reduction of tacrolimus dosage by one-third. The present drug-drug interaction can be attributed to a strong inhibitory effect on cytochrome P450-3A4 activity by voriconazole. When voriconazole and tacrolimus are coadministered, close monitoring of tacrolimus blood levels is recommended as the rule-of-thumb reduction of tacrolimus dose by one-third may not be satisfactory.

Long-term assessment of plasma lipids in transplant recipients treated with tacrolimus in relation to fatty liver.

Immunosuppression has improved graft and recipient survival in transplantation but is associated with possible adverse effects including cardiovascular diseases. The impact of tacrolimus on the lipidic profile has been debated for several years. Twenty-nine kidney transplant recipients on tacrolimus treatment were monitored for six years, and multiple laboratory parameters investigating the lipid asset, as well as glucose profile, were carried out. Tacrolimus has been responsible for significant changes in plasma lipid concentrations only for the first six months, but not for the remaining time of observation. Similarly, in the same periods, glycemic imbalance was highlighted. The liver enzyme activity showed a modest derangement during the tacrolimus treatment, suggesting the presence of lipid accumulation in the liver. Fatty liver reversed in the long term follow-up. Tacrolimus, although it is not a completely safe option in the first months of the immunosuppressive protocols in organ transplanted recipients, still retains a certain role in the long-term post-transplantation immunosuppressive approach with high cardiovascular risks.

Quantification of sirolimus and everolimus by immunoassay techniques: test specificity and cross-reactivity evaluation.

The possible cross-reactivity of immunoassays with structurally-related drugs was investigated. Innofluor Certican (FPIA) calibrators were measured by using IMx Sirolimus assay (MEIA) and MEIA Sirolimus calibrators were analysed by using FPIA Certican assay. Drug concentrations were measured in 95 and 100 samples from renal transplanted patients (RTP) on sirolimus or everolimus treatment by using immunoassays and LC/ESI-MSMS. A high cross-reactivity was found both for MEIA and FPIA. High correlation degrees, confirmed by the Bland-Altman and the Eksborg tests, were found between drug concentrations measured in real samples by both immunoassays (r = 0.909 and r = 0.970, respectively). LC/ESI-MSMS analysis of samples containing sirolimus showed no positivity for everolimus. Similarly, samples from patients on treatment with everolimus resulted negative as far as regards sirolimus. MEIA and FPIA could be considered mutually reliable and accurate alternatives for the specific-drug immunoassay. It should be noticed that in patients switching from one drug to the other unreal overestimation of the blood levels of the current administered immunosuppressant can occur.

Stability of sirolimus and everolimus measured by immunoassay techniques in whole blood samples from kidney transplant patients.

The measurement of blood concentration of immunosuppressive drugs is strongly recommended because of the narrow therapeutic range. An important aspect in the therapeutic monitoring of a drug is its possible degradation. This paper is aimed at investigating the stability of two widely-used immunosuppressants, sirolimus and everolimus. Short (storage at 30 degrees C for 3 or 7 days) and long term (storage at -20 degrees C for 0-90 days with a single freeze-thaw cycle) stability of sirolimus and everolimus in whole blood samples from kidney transplant patients were examined by using MEIA and FPIA. Sirolimus and everolimus samples stored at 30 degrees C in light for up to a week showed a decrement in concentration of 5.2 percent and 6.1 percent, respectively. Our findings on long term stability for both sirolimus and everolimus highlight the possibility of storing samples at -20 degrees C for up to 90 days, without the need to use lower storage temperatures. The results have important implications for patients living far from laboratories where drug concentration is measured or when the storing of blood samples is needed for pharmacokinetic studies.

GABAA receptors expression pattern in rat brain following low pressure distension of the stomach.

It is known that gastric mechanoreceptor stimuli are widely integrated into neuronal circuits that involve visceral nuclei of hindbrain as well as several central brain areas. GABAergic neurons are widely represented in hindbrain nuclei controlling gastric motor functions, but limited information is available specifically about GABA(A)-responding neurons in brain visceral areas. The present investigation was designed to determine the central sensory neuronal pathways and their GABA(A)-alpha1 and -alpha3 receptor presenting neurons that respond to gastric mechanoreceptor stimulation within the entire rat brain. Low pressure gastric distension was used to deliver physiological mechanical stimuli in anesthetized rats, and different protocols of gastric distension were performed to mimic different stimulation patterns with and without sectioning vagal and/or splanchnic afferent nerves. Mapping of activated neurons was investigated using double colorimetric immunohistochemistry for GABA(A)-alpha1 or -alpha3 subunits and c-Fos. Following stomach distension, neurons expressing GABA(A) receptors with alpha1 or alpha3 subunits were detected. Low frequency gastric distension induced c-Fos expression in nucleus tractus solitarii (NTS) only, whereas in the high frequency gastric distension c-Fos positive nuclei were found in lateral reticular nucleus and in NTS in addition to some forebrain areas. In contrast, during the tonic-rapid gastric distension the neuronal activation was found in hindbrain, midbrain and forebrain areas. Moreover different protocols of gastric stimulation activated diverse patterns of neurons presenting GABA(A)-alpha1 or -alpha3 receptors within responding brain nuclei, which may indicate a probable functional significance of differential expression of GABA(A)-responding neurons. The same protocol of gastric distension performed in vagotomized rats has confirmed the primary role of the vagus in the response of activation of gastric brain areas, whereas neuronal input of splanchnic origins was shown to play an important role in modulating the mechanogastric response of brain areas.

Cell behaviour on phospholipids-coated surfaces.

Effective integration of orthopedic biomaterials requires the rapid formation of the inorganic mineral phase during the first hours of implantation and the subsequent adhesion and proliferation of the osteoblasts. It has recently been demonstrated that phosphatidylserine-rich phospholipid coatings can induce a fast mineralisation of titanium implant surfaces on incubation in simulated body fluids. The aim of this work was to investigate the biocompatibility of these coatings in terms of cytotoxicity and ability to support osteoblast adhesion and activity. Cytotoxicity and cell adhesion to uncoated titanium, calcified phospholipid-coated titanium and HA-coated titanium was assessed using fibroblasts and osteoblast-like cells. The synthesis of type I collagen by osteoblast-like cells cultured on the calcified-phospholipid coatings was also comparable to that observed for osteoblast-like cells cultured on the titanium and HA-Ti surfaces.The results suggest that the fast mineralization of the phospholipid matrix, obtained in vitro by its pre-treatment in a SBF, exposes the cells to an environment similar to that present in the bone during its natural formation that allow cells to adhere, proliferate and produce proteins fundamental for bone growth. The biocompatibility of these phospholipid-based coatings, in combination with their ability to initiate rapid mineralisation, provides a promising material that could in vivo create bone cell interactions and bone integration.