Eduardo Chuluyan

Local and Systemic Cellular Immunity in Early Renal Artery Atherosclerosis

BACKGROUND AND OBJECTIVES Modern imaging techniques have increased the incidental detection of renal atherosclerotic disease (RAD). Because immune activation may hasten RAD progression, identifying cellular immune markers might provide clues to clinical activity. In this study, cellular immune markers were assessed in early RAD. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Immune cell markers in peripheral blood of two groups of hypertensive patients with normal carotid and coronary arteries were evaluated: 28 patients had incidental RAD and 22 patients had normal renal arteries; 21 renal arteries obtained at necropsy from individuals with history of hypertension and tissue evidence of RAD were examined and matched with 21 individuals with normal renal arteries. Cell subpopulations were measured by flow cytometry in peripheral blood and direct cell count, respectively, using T and dendritic cells monoclonal antibodies. RESULTS Peripheral blood of RAD patients showed increased numbers of cells expressing CD3, CD4, CD83, and CD86. CD4 to CD8 ratio was 8.3 ± 1.4 (RAD) to 3.4 ± 0.9 (normal; P<0.001). No differences were found in CD25, CD8, and S100 among groups. Postmortem samples from RAD showed increased CD3+, CD4+, CD86+, and S100+ cells, whereas CD25+ and CD8+ were unmodified between groups. CD4+ to CD8+ ratio was higher in the RAD(PM) group. CONCLUSIONS These results are consistent with an increased expression of immune cell markers in early RAD. Additional studies will explore if they may potentially turn into treatment targets to prevent disease progression.

Cross-Talk between CD31 and the Signaling Lymphocytic Activation Molecule—Associated Protein during Interferon-γ Production against Mycobacterium tuberculosis

Effective host defense against tuberculosis requires Th1 cytokine responses. We studied the regulation of interferon (IFN)- gamma production during tuberculosis by investigating the role of CD31, a receptor that attenuates T cell receptor signals. After antigen stimulation, CD3(+)CD31(+) blood lymphocytes decreased in healthy donors and in tuberculosis patients with robust Th1 responses to Mycobacterium tuberculosis and IFN- gamma was secreted only by CD31(-) T cells. In contrast, in patients with weak Th1 cytokine responses to M. tuberculosis, the level of CD3(+)CD31(+) lymphocytes was increased and IFN- gamma production was low. Furthermore, the inverse relationship between CD31 expression and IFN- gamma production was in contrast to signaling lymphocytic activation molecule (SLAM) expression, an IFN- gamma inducer in tuberculosis. Interestingly, CD31 bound to SLAM-associated protein (SAP), an IFN- gamma inhibitor in tuberculosis, and when CD31 and SAP were coexpressed in lymphocytes, their association inhibited the IFN- gamma response to M. tuberculosis. Thus, CD31, when binding to SAP, interferes with Th1 responses, suggesting that CD31 has a key regulatory role in the signaling pathway(s) leading to the IFN- gamma response to M. tuberculosis.

Secretory Leukocyte Protease Inhibitor (SLPI) expression downregulates E-cadherin, induces β-catenin re-localisation and triggers apoptosis-related events in breast cancer cells

Epithelial cadherin (E‐cadherin) is involved in cell–cell adhesion through its extracellular domain, whereas the intracellular domain interacts with adaptor proteins, i.e. β‐catenin, links E‐cadherin to the actin cytoskeleton and participates in signal transduction events. E‐cadherin protects mammary epithelial cells from apoptosis and its loss during tumour progression has been documented. Secretory Leukocyte Protease Inhibitor (SLPI) has anti‐ and pro‐tumourigenic activities but its role in breast cancer has not been fully elucidated. Notwithstanding its relevance, how SLPI affects E‐cadherin in breast cancer is still unknown. This study evaluated the effect of SLPI upon E‐cadherin/β‐catenin expression and apoptosis‐related markers in murine (F3II) and human (MCF‐7) breast tumour cells either treated with exogenous recombinant human SLPI (rhSLPI) or stably transfected with a plasmid encoding its sequence.

Mood, Th-1/Th-2 cytokine profile, and autonomic activity in older adults with acute/decompensated heart failure: preliminary observations.

In order to assess the relationships among mood, peripheral autonomic output and circulating immunoinflammatory mediators in older individuals with decompensated heart failure (CHF), 20 consecutive patients (78±7 years, 35% women) admitted to the coronary care unit with a clinical diagnosis of acute/decompensated CHF of coronary origin were examined. Mood was evaluated by the 21-item Hamilton Depression Scale (HAM-D). Four patients met the criteria for major depression. Heart rate variability (HRV) analysis and the levels of tumour necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-2, IL-4, IL-6 and IL-10 were measured within 24–72 h of admission. A significant positive relationship between score in HAM-D and serum IL-6 levels was detected with a similar trend as far as IL-2 levels. Circulating IL-2 levels were strongly associated with the HRV L/H quotient, an index of increased sympathetic and/or decreased parasympathetic thoracic activity. A negative correlation between vagal activity (as assessed by HRV) and IL-4 occurred. Neither TNF-α nor IL-10 were detectable in this group of elderly patients. The results add to the concept that mood and autonomic unbalance are associated with increased systemic inflammation in old patients with decompensated CHF, a potential mechanism for mood-related worsened prognosis of heart failure at an advanced age.

Diabetic retinopathy: could the alpha-1 antitrypsin be a therapeutic option?

Diabetic retinopathy is one of the most important causes of blindness. The underlying mechanisms of this disease include inflammatory changes and remodeling processes of the extracellular-matrix (ECM) leading to pericyte and vascular endothelial cell damage that affects the retinal circulation. In turn, this causes hypoxia leading to release of vascular endothelial growth factor (VEGF) to induce the angiogenesis process. Alpha-1 antitrypsin (AAT) is the most important circulating inhibitor of serine proteases (SERPIN). Its targets include elastase, plasmin, thrombin, trypsin, chymotrypsin, proteinase 3 (PR-3) and plasminogen activator (PAI). AAT modulates the effect of protease-activated receptors (PARs) during inflammatory responses. Plasma levels of AAT can increase 4-fold during acute inflammation then is so-called acute phase protein (APPs). Individuals with low serum levels of AAT could develop disease in lung, liver and pancreas. AAT is involved in extracellular matrix remodeling and inflammation, particularly migration and chemotaxis of neutrophils. It can also suppress nitric oxide (NO) by nitric oxide sintase (NOS) inhibition. AAT binds their targets in an irreversible way resulting in product degradation. The aim of this review is to focus on the points of contact between multiple factors involved in diabetic retinopathy and AAT resembling pleiotropic effects that might be beneficial.

Polyomavirus BK-Associated Nephropathy After Kidney Transplantation: A Single-Center Retrospective Analysis

Polyomavirus BK-associated nephropathy is cause of kidney transplant morbidity and graft failure. Reported prevalence can vary significantly between centers, probably depending on the immunosuppressive regime used, averaging 5% and the reported incidence of allograft failure ranges from 15 to 50% of affected individuals. Aim To evaluate the evolution of BK virus nephropathy in our transplant center Materials: A descriptive, retrospective study of the clinical evolution of BK virus nephropathy in adult kidney transplant patients from 10 / 2011-10 / 2016. The diagnostic suspicion was through the technique of screening and / or renal dysfunction and confirm by kidney biopsy. Screening was performed by Polymerasa Chain Reaction for BKV(+ greater than 10 4 copies / ml) at 2-6-9-12-month and then annually and after the treatment episode due to graft rejection. Patients with a diagnosis of BK nephropathy we decided 1- Reduction of immunosuppression (suspension of mycophenolate by leflunomide and tacrolimus reduction (maintaining dosages average 5ng / ml )or sirolimus, corticoids and leflunomide), and following with 2-Evaluation of renal function,3- Viral load, 4-antiHLA antibodies and 5-Renal biopsy. Half of the patients also received IVIG 400mg / kg / day for 5 days as a treatment. Results 18/774 renal tx patients had BK nephropathy, at 553 ± 355 days of transplant. 6 for screening and the rest for renal dysfunction. Age 42 ± 12y, 50% women, 14 LD, 4 DD. 6/18 presented previous rejections 2 Mixed 4 cellular Kidney. Biopsies showed Stage I Nephropathy 8 patients (44.4%), S II 9 patients (50%) and S III 1 patient (5.6%). Average basal creatinine (n = 18) 2.5 ± 0.18, 6 months (n = 16) 3.1 ± 1.6, 12 m (n = 15) 2.7 ± 0.8, 24 m (n = 10) 2.62 ± 0.8, 36m (n = 6) 2.9 ± 0.2. 2 patients presented by biopsy, cellular rejection at 3 months of the diagnosis, treated with steroid pulses. No development of de novo antiHLA DSA was observed in this group of patients. The negativization of the viral load BKV was at 3 months in 5 patients and the rest at 6 months. Only 12 renal biopsies were performed(X 12m), presenting an increase in interstitial infiltration and tubulitis in 2 patients). Loss of grafts in 3 patients (16.6%), 2/18 at 4 months and 1/18 at 10 months. There were no deaths. Conclusions A low incidence (2,34%) of BK virus nephropathy is observed in this population. There were no differences in the evolution of patients treated with / without IVIG (pNS) especially in the negativization of viral load. Higher percentage of living donors (77%) with BK nephropathy due probably immunosuppression charge and / or unidentified serology of BK virus in donors.

Antibody Monitoring in Pancreas Transplantation: When should we do protocol biopsies?

Introduction In kidney transplantation de novo donor-specific HLA antibodies (DSA) have been found to correlate to poor graft survival and Consensus Guidelines recommend a protocol biopsy. In pancreas transplantation DSA had also been associated with poor graft outcomes however there are no recommendations on protocol biopsies. In June, 2013 a screening protocol was initiated for detection of anti-HLA antibodies (Abs) by Luminex. Testing was performed on patients presenting with dysfunction and by protocol at 0, 3, 6, 12 months and yearly for new transplants and yearly for previous transplants. Patients with DSA Abs and patients with high MFI non-DSA Abs were considered for protocol biopsy of both organs. Results During the study period 144 pancreas transplant recipients were screened for anti-HLA antibodies. 106 patients had initially negative antibodies and 20 among them produced de novo antibodies. 38 had positive antibodies at the initial testing. 86 patients had negative antibodies though the study period. Among them 10 presented dysfunction and biopsy proven rejection of either organ during follow-up, being 8 acute cellular rejections (ACR) and 2 antibody mediated rejections (AMR). There were 20 patients with initially negative Abs that presented de novo Abs during follow up. Seven of these were found to have positive Abs when presenting with rejection (all non-DSA Abs). The other 13 cases were detected by screening. Among these, there were 7 non-DSA and 6 DSA cases. No biopsies were performed in the non-DSA cases with 1 presenting rejection at 3 years of follow-up. All 6 patients with DSA Abs had protocol biopsies with 2 of them showing subclinical kidney AMR. We found 38 patients with positive Abs at the initial testing. 10 of them were detected when presenting with dysfunction and all were biopsied. Among these, 5 had DSA and 5 non-DSA Abs and all but one patient had rejection. The other 28 cases were detected by screening. In this group, 18 patients had non-DSA Abs and 8 of them were biopsied with negative results and remained rejection free. The last 10 cases had DSA Abs initially. Six of them were biopsied and had subclinical rejection on the kidney (3), the pancreas (1) or both (2). Of the remaining four patients without biopsy, 2 presented kidney humoral rejection during at 6 and 12 months of follow-up and 2 remained rejection free. Discussion Our protocol detected 40% of patients with positive anti-HLA Abs (26% at the initial testing and 14% de novo during follow-up) with 36% of them being DSA abs. In non-dysfunctioned patients with non-DSA abs protocol biopsies were negative in all biopsied patients and only 4% of them presented rejection during follow-up. In contrast, in non-dysfunctioned patients with DSA abs protocol biopsies found subclinical rejection in 66% of them and half the non-biopsied patients presented rejection during follow-up. We conclude that patients with DSA abs may benefit from protocol biopsies of both grafts.

α-lipoic acid reduces postreperfusion syndrome in human liver transplantation - a pilot study

A double‐blind randomized controlled trial was performed to compare the safety and efficacy of α‐lipoic acid (ALA) in liver transplantation (LT). The grafts were randomized to receive ALA or placebo before the cold ischemia time. Furthermore, patients transplanted with the ALA‐perfused graft received 600 mg of intravenous ALA, while patients with the nonperfused graft received the placebo just before graft reperfusion. Hepatic biopsy was performed 2 h postreperfusion. Blood samples were collected before, during and 1 and 2 days after reperfusion. Quantitative polymerase chain reaction (qPCR) analysis was performed on biopsies to assess genes involved in the response to hypoxia, apoptosis, cell growth, survival and proliferation, cytokine production and tissue damage protection. Nine of 40 patients developed postreperfusion syndrome (PRS), but seven of them belonged to the control group. There was a decrease in PHD2 and an increase in alpha subunit of hypoxia‐inducible factor‐1 (HIF‐1α) and baculoviral IAP repeat containing 2 (Birc2) transcript levels in the biopsies from the ALA‐treated versus the control group of patients. Additionally, plasma levels of alarmins were lower in ALA‐treated patients than control patients, which suggests that ALA‐treated grafts are less inflammatory than untreated grafts. These results showed that ALA is safe for use in LT, induces gene changes that protect against hypoxia and oxidative stress and reduces the appearance of PRS.

Cementoin–SLPI fusion protein binds to human monocytes and epithelial cells and shows higher biological activity than SLPI

Secretory Leukocyte Proteinase Inhibitor (SLPI) is an antiinflammatory peptide that blocks the activity of serine proteases, primarily the neutrophil elastase. In an attempt to direct the activity of SLPI on inflamed sites, a chimera consisting of the transglutaminase II substrate domain of trappin 2 (cementoin), and the mature SLPI protein was constructed. Cell attachment and biological activity were compared between SLPI and this chimera. By using whole cell ELISA, fluorescence microscopy and flow cytometry assays we observed that the cementoin-SLPI fusion protein (FP) but not SLPI attached to a human lung epithelial cell line and monocytes. A maximum attachment was achieved 15 min after FP was added to the cell cultures. In an elastase activity assay, we observed that FP retained its antiprotease activity and that at equimolar amount of proteins, FP was more efficient than SLPI in the inhibition. Both, FP and SLPI inhibits IL-2-induced lymphocyte proliferation, however, lower amounts of FP were required to achieve this inhibition. Furthermore, FP binds to mycobacteria and maintained the bactericidal activity observed for SLPI. Overall, these results show that this new chimera is able to attach to the cell surfaces retaining and improving some biological activities described for SLPI.

Alpha Lipoic Acid: A Therapeutic Strategy that Tend to Limit the Action of Free Radicals in Transplantation

Organ replacement is an option to mitigate irreversible organ damage. This procedure has achieved a considerable degree of acceptance. However, several factors significantly limit its effectiveness. Among them, the initial inflammatory graft reaction due to ischemia-reperfusion injury (IRI) has a fundamental influence on the short and long term organ function. The reactive oxygen species (ROS) produced during the IRI actively participates in these adverse events. Therapeutic strategies that tend to limit the action of free radicals could result in beneficial effects in transplantation outcome. Accordingly, the anti-oxidant α-lipoic acid (ALA) have been proved to be protective in several animal experimental models and humans. In a clinical trial, ALA was found to decrease hepatic IRI after hepatic occlusion and resection. Furthermore, the treatment of cadaveric donor and recipient with ALA had a protective effect in the short-term outcome in simultaneous kidney and pancreas transplanted patients. These studies support ALA as a drug to mitigate the damage caused by IRI and reinforce the knowledge about the deleterious consequences of ROS on graft injury in transplantation. The goal of this review is to overview the current knowledge about ROS in transplantation and the use of ALA to mitigate it.