Alessandro Perrella

Binding of Hepatitis A Virus to Its Cellular Receptor 1 Inhibits T-Regulatory Cell Functions in Humans

BACKGROUND & AIMS CD4+ T-regulatory (Treg) cells suppress immune responses and control self-tolerance and immunity to pathogens, cancer, and alloantigens. Most pathogens activate Treg cells to minimize immune-mediated tissue damage and prevent clearance, which promotes chronic infections. However, hepatitis A virus (HAV) temporarily inhibits Treg-cell functions. We investigated whether the interaction of HAV with its cellular receptor 1 (HAVCR1), a T-cell co-stimulatory molecule, inhibits the function of Treg cells to control HAV infection. METHODS We studied the effects of HAV interaction with HAVCR1 on human T cells using binding, signal transduction, apoptosis, activation, suppression, cytokine production, and confocal microscopy analyses. Cytokines were analyzed in sera from 14 patients with HAV infection using bead arrays. RESULTS Human Treg cells constitutively express HAVCR1. Binding of HAV to HAVCR1 blocked phosphorylation of Akt, prevented activation of the T-cell receptor, and inhibited function of Treg cells. At the peak viremia, patients with acute HAV infection had no Treg-cell suppression function, produced low levels of transforming growth factor-β , which limited leukocyte recruitment and survival, and produced high levels of interleukin-22, which prevented liver damage. CONCLUSIONS Interaction between HAV and its receptor HAVCR1 inhibits Treg-cell function, resulting in an immune imbalance that allows viral expansion with limited hepatocellular damage during early stages of infection-a characteristic of HAV pathogenesis. The mechanism by which HAV is cleared in the absence of Treg-cell function could be used as a model to develop anticancer therapies, modulate autoimmune and allergic responses, and prevent transplant rejection.

Elevated CD4+/CD25+ T cell frequency and function during acute hepatitis C presage chronic evolution

Hepatitis C virus (HCV) determines an acute hepatitis evolving to persistent infection in 50–80% of patients.1 Different mechanisms have been proposed to explain disease evolution, including viral escape, failure of the T helper immune network, and host genetic factors.2 Among CD4+ T cell subsets, lymphocytes expressing constitutively CD25 (interleukin 2 receptor alpha chain), namely T regulatory cells (Treg), appear to play a critical role in controlling chronic evolution of HCV mediated liver diseases.3 Here we investigate the frequency and functional activity of CD3+/CD4+/CD25+ Tregs in acute HCV infection in relation to its evolution over time. The study was approved by the local ethics committee and included 16 consecutive patients with acute hepatitis C (AHC), 15 consecutive patients with acute hepatitis A (AHA), and …

Impaired function of CD4+/CD25+ T regulatory lymphocytes characterizes the self-limited hepatitis A virus infection

Background and Aim:  Hepatitis A virus (HAV) causes a transient illness leaving permanent protection against reinfection. Few data are available on the regulatory mechanisms involved in the CD4+ T helper activation. We aimed to investigate the frequency and function of CD3+/CD4+/CD25+ T cells with regulatory function (Tregs) during acute HAV infection.

Interleukin-10 and tumor necrosis factor-alpha : model of immunomodulation in multiple sclerosis

Abstract Objectives: The mechanisms involved in the pathogenesis of relapsing-remitting multiple sclerosis are still unclear. The aim of the present study was to evaluate both cerebrospinal fluid (CSF) CD4+ CD7+ T cells and peripheral blood (PB) interleukin-10 (IL-10) as well as tumor necrosis-alpha (TNF-α) levels in patients with definite multiple sclerosis of the relapsing-remitting type. Methods: To assess the above-mentioned cytokine levels we performed our test by the means of ELI-spot assay; the T-helper cell subset was assayed using flow cytometry. Results: PB IL-10 levels of multiple sclerosis (MS) patients in remission were significantly (p<0.001) higher than in MS patients in the active phase. There was significant and increased evidence of TNF-α levels only in the MS patients in the active phase. CD4+ CD7+ T cells, characterized by a preferential Th1-like cytokine profile, were detectable only in seven patients in the active phase without evidence of a statistical significance with respect to cytokine levels. Conclusion: The data indicate that the production of different cytokines characterized the expression of relapsing-remitting MS. The data also suggest that is it possible to control MS using the regulatory cytokine balance.

Immune system homeostasis during acute hepatitis C: Viral escape or T‐cell regulation?

We read with great interest the paper of Rahman et al. about the effects of antiviral therapy on the immune response during acute hepatitis C,1 and the conclusions of their investigation are fascinating. Hepatitis C virus (HCV) is characterized by a high mutational capacity,2 so a continuous antigenic stimulus is directed at T lymphocytes. The ability to mount a vigorous and multispecific Th1 response is the key for disease resolution at an early phase3; on the other hand, the presence of an increased Th2 network could be related to progression to chronic disease.4 Over the last years, many studies have shown the importance of antigen specific CD4 T cells to different epitopes in the natural history of HCV infection.5,6 To provide a wider analysis and to understand the possible modulation of Th2 to Th1 network in early phase of infection, Rahman et al. have assayed the HCV-specific T-cell response toward 600 overlapping HCV peptides and 6 proteins by an ex vivo enzyme-linked immunospot. The authors hypothesized that a sustained response to the antiviral treatment was not associated with a lasting enhancement of HCV-specific T-cell responsiveness in blood. Moreover, they suggest that the impaired proliferative response could represent a possible adverse effect of interferon . In view of these results, we have some questions. Is the absence of HCV-specific CD4 T-cell enhancement related to sustained treatment in the periphery or a demonstration of immune system disease resolution response? In fact, during an infection, due to the homeostasis rules, when the antigenic stimulation ends, the CD4 T-cell proliferation decreases.7 Thus, in the patients who had a sustained response and no viral load, the specific lymphocyte response could be suppressed probably and simply because it is no longer required. In contrast, when an antigen is not completely cleared, a T-cell immune response is still present. Indeed, this was the case in the study of 1 patient who developed a viral breakthrough while maintaining HCV-specific T-cell responses. Concerning possible effects of interferon on T-cell proliferation, the proposed schedule of the authors includes ribavirin; this drug also exhibits some important effects on T lymphocytes, stimulating the Th1 network.8 In light of such evidence, how can they explain the antiproliferative and myelosuppresive effects of interferon without considering a possible influence of ribavirin too? Moreover, an issue arises about the differences in HCV-specific T cells between the patients who resolve spontaneously and those who need treatment. Have the authors considered measuring possible inhibition mechanisms on HCV-specific T cells by means of other lymphocyte subsets, such as CD4 CD25 (Treg) in their patients? Probably, the subjects without spontaneous viral clearance and illness resolution may exhibit an increased Treg lymphocytes percentage with inhibition of CD4 CD25 T cells, responsible for the immune response. Finally, we think that the immune system homeostasis during acute HCV infection hides still-important data.

Infection Control in a Far Far Away Galaxy: New and Alternative Learning Tool from Popular Culture to Improve the Antimicrobial Stewardship.

Dear Editor, Infection control still represents one of the most important issue in clinical practice, particularly in critically-ill patients. Indeed, it has to be considered as a mandatory approach in clinical practice for all healthcare workers, nonetheless it is one of the most difficult to learn and to apply. A good and significant methodology to acquire a state of mind focused on infection control during clinical practice should start early during the training of healthcare workers and should be carefully managed, likely using all possible and available learning tools. It should also involve patients and their relatives, aiming to minimize possible infection spread. Previously case studies from popular culture have been proposed as teaching tool for medical student, but possibly also for all those individuals having relation with healthcare facilities. More recently Star Wars series has been used as learning tool to focus on pathophysiology of the lung according to one of the most important character of Star Wars series, Darth Vader. Indeed Star Wars and related imaginary world holds some other important learning suggestions to emphasize another important feature of the tragic story of this popular character, or else the infection control after severe skin burn in one of its most popular character.[1] Infection still remains a leading cause of morbidity and mortality following burns,[2] being related to poor wound healing and increased risk of scarring which may have a negative prognosis and functional impact on the patient.[2] Survival following burn improves with an early wound debridement surgery and skin replacement aiming to remove the source of wound infection and restore skin integrity with particular attention to aseptic condition.[3] Critical factors previously being correlated to increased risk of developing burn wound infection (BWI) include age, percentage of total body surface area (TBSA), presence of full-thickness burns, and presence of an inhalation injury that may contribute to tissue hypoxia reducing wound strength after healing and increasing the risk of infection.[4] Among these comorbidities, fungal infection may have an important role on prognosis if not treated with an accurate clinical approach.[5] In the specific case related to Star Wars series, Anakin Skywalker (the real name of Darth Vader before its evil change) during Star Wars Episode III, after a saber duel with his Jedi Master Obi-Wan Kenobi suffers traumatic amputations of both lower limbs and his left upper limb with burns, several wounds on the head and on the face with volcanic gas inhalation. All these events, according to reported literature data could clearly predispose to infections, with related increase in mortality.[4,6] Nevertheless, in the above mentioned episode, in a relatively few time, the protagonist is assisted with a medical capsule and shortly after the rescue, he is taken in a surgical operative room. In this aseptic environment a quickly wound debridement surgery and skin replacement is performed by medical robots and finally a whole-body suit is dressed, with possibly some healing property for the skin and lung injury from volcanic gas, with a final surgical and clinical success.[1] In the Episode IV of the movie series, 22 years later,we find Darth Vader (Anakin Skywalker) still dressing whole-body suit and most importantly still alive even if affected by chronic pulmonary disease. From a medical point of view, our villain not only may be considered an example of learning tool to underline some important aspect of acute and chronic respiratory failure,[6] but also an important model of the efficacy of anearly wound debridement surgery and skin replacement to minimize infection morbidities,with a long survival. Mainly, we think that there are two key factors in this “medical history”: The use of asterile medical capsule on the site ofthe traumatic event that could be considered as a correct aseptic approach (hand hygiene, appropriated devices use) to critically-ill patients after a traumatic event, Early surgical approach in aseptic and possibly hyperbaric chamber, to be considered as accurate and specific methodology to remove source of infection in aseptic conditions. Both these two elements strongly suggest that standard hygiene precautions and a rigorous specialist approaches are fundamental methodologies to minimize risk of the infection and to ensure good prognosis of the patients in a far far away galaxy as on the Earth. Despite it would seem controversial, the use of a such kind of approach to infection control training, may help healthcare workers to target both automatic associative learning processes and conscious decision making, in addition to ensuring good knowledge of all possible measure to prevent infections, as recently suggested.[6] In conclusion, Star Wars, as any other subjects from popular culture should be considered as a really valuable approach and an alternative as well as entertaining platform for a plenary discussion with medical students on one of the most important topic in current medical practice: Infection control.

P101 FK stimulation of hepatitis C virus-specific and nonspecific peripheral blood mononuclear cell of patients on waiting list to evaluate and tailor immunosuppresion before orthotopic liver transplantation

Introduction We previously found that FK did not induce a severe reduction of immune response compared to other clacineurin inhibitor (ILTS 2009 oral presentation). Aim We aimed to evaluate the in vitro effect of tacrolimus on hepatitis C virus (HCV)-specific immune response by ELISpot to establish early the possible effect of immunosuppression and so tailoring immunosuppressive schedule before OLTx to minimize or delay HCV recurrence. Method Twenty-five HCV+ve patients (20 male and 5 female) on waiting list were enrolled. Blood samples were taken and at least on 2–2.5×105 cells per well ELISpot was performed to evaluate IFN-specific response after coculturing with 1 ng/ml or 5 ng/ml of FK for 24 h. CMV and a specific stimulation with PMI-Ionomicin were used as control on HCV patients. Ten healthy donors were used as control. Results After 24 h of FK stimulation, PMA-Ion IFN-response was reduced in all patients (p<0.05) compared to basal values both with 1 ng/ml that 5 ng/ml while CMV- specific showed reduction only when cocultured with 5 ng/ml. Among patients on waiting list, we had that 8 out 25 had a major reduction of HCV and CMV specific response (p <0.05) while 17 out 25 had no statistical significant changes in their IFN response. Conclusion Based on these results and our previous data, ELISpot assay before OLTx may useful to establish the impact of immunosuppressive schedule. In those having a major reduction of HCV specific immune response after CNI stimulation (FK) a different drug should be used to minimize or delay the possible HCV recurrence. Further studies are required to establish in vitro effects.Abstract P101 Table 1 Results Results Patients parameters HCV CMV PMI-Ion HD-PMA-Ion Age (y, mean SD) 55, 3 55, 3 55, 3 35, 6 Male 20/25 20/25 20/25 7/10 Female 5/25 5/25 5/25 3/10 HCV-RNA (IU/mL mean SD) 310 232×103 n.d. n.d. n.d. Genotype 1a/1b 23/25 Non-1 2/25 Immunological parameters IFN-ELISpot (SFC) in 17/25 25/25 25/25 10/10 T0 without stimulation 65 25 192 103 225 100* 272 113* FK stimulation 1 ng/ml 58 19 176 98 176 88* 200 126* FK stimulation 5 ng/ml 52 50 128 42* 150 45* 133 23* IFN-ELISpot (SFC) in 8/25 pts T0 without stimulation 61 15* FK stimulation 1 ng/ml 41 7* FK stimulation 5 ng/ml 34 5* * p<0.05; p>0.05.

Immune response and liver transplantation

In an article published in the January 2005 issue of Liver Transplantation, Warle et al. provided an intriguing report on possible correlation between cytokine gene polymorphism and liver graft rejection.1 In brief, the authors suggest that interleukin IL-10 -1082 polymorphism could play a critical role in determining the human liver graft rejection. Two principal evaluations emerge from this study: first, the role of a preferential immune response related to liver transplant, and second, a possible involvement of immunoregulatory T cells such as CD4 CD25 T lymphocytes. A first key question is if a preferential cytokine network may play an absolute role in causing a liver graft rejection. In our previous paper, we have shown that a proinflammatory cytokine profile was related to liver acute rejection.2 Our study comprised a group of 14 patients with different causes of liver disease treated with the same immunosuppressive regimen of therapy and with the diagnosis of acute cellular rejection based on the presence of at least 2 of the following features: bile duct infiltration and damage, or venous endothelitis of the portal tracts; hepatic artery or portal vein occlusion was excluded by Doppler echography. In our patients we found a statistically significant increase (P 0.001) of IL-2 level in the liver with respect to peripheral blood. The determination was performed by enzyme linked immunosorbent assay test in day-3 culture supernatants collected by biopsy, processed into small clumps and simple suspensions, and incubated with anti-CD3.Our study showed that differences in the rate of acute rejection may be related to different factors such as different regimens and maintenance of therapy, type of initial cause of liver disease, and type of individual immune response. The individual immune response can be regulated by a specific activity of Treg cells. Recent evidence has implicated regulatory T cells in transplantation tolerance and rejection.3 Treg cells can suppress the immune system by several mechanisms.4 In particular, IL-10 and transforming growth factor 1 (TGF1) have been shown to be important mediators of Tregmediated suppression.5 IL-10 was originally described as a Th2 cytokine cell, yet IL-10 may also have the important role of feedback regulator to control the pathology associated with an overexuberant Th1 response. The source of IL-10 during acute rejection may be Th1, Th2, or regulatory T cells, on the basis of an individual background and pathogen. It is possible that the mechanisms of action of such T lymphocytes may be associated with different pathways related to differences in systemic vs. local inflammation. The molecular mechanism involved in regulation may depend on the level of inflammation accompanying an immune response.6 Finally, we think that the risk factor for acute rejection may not be related, in a stereotyped way, to a specific cytokine profile but, conversely, to individual Treg cells activated during liver transplant. Such cells can influence unrelated aspects of immune homeostasis.