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Objectives To assess the humoral and cell-mediated response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) elicited by the mRNA BNT162b2 vaccine in SARS-CoV-2-experienced and -naive subjects against a reference strain and SARS-CoV-2 variants. Methods The humoral response (including neutralizing antibodies) and T-cell-mediated response elicited by BNT162b2 vaccine in 145 healthcare workers (both naive and positive for previous SARS-CoV-2 infection) were evaluated. In a subset of subjects, the effect of SARS-CoV-2 variants on antibody level and cell-mediated response was also investigated. Results Overall, 125/127 naive subjects (98.4%) developed both neutralizing antibodies and specific T cells after the second dose of vaccine. Moreover, the antibody and T-cell responses were effective against viral variants since SARS-CoV-2 NT Abs were still detectable in 55/68 (80.9%) and 25/29 (86.2%) naive subjects when sera were challenged against β and δ variants, respectively. T-cell response was less affected, with no significant difference in the frequency of responders (p 0.369). Of note, two doses of vaccine were able to elicit sustained neutralizing antibody activity against all the SARS-CoV-2 variants tested in SARS-CoV-2-experienced subjects. Conclusions BNT162b2 vaccine elicited a sustained humoral and cell-mediated response in immunocompetent subjects after two-dose administration of the vaccine, and the response seemed to be less affected by SARS-CoV-2 variants, the only exceptions being the β and δ variants. Increased immunogenicity, also against SARS-CoV-2 variant strains, was observed in SARS-CoV-2-experienced subjects. These results suggest that triple exposure to SARS-CoV-2 antigens might be proposed as valuable strategy for vaccination campaigns.

Letermovir (LTV), recently approved for the prophylaxis of human cytomegalovirus (HCMV) reactivation after hematopoietic stem cell transplantation (HSCT), has been shown to decrease the rate of infection in the first months post-transplantation. The aim of this study was to evaluate the impact of LTV prophylaxis on immune reconstitution and late-onset infection. We studied HCMV infection and HCMV-specific T cell reconstitution in 2 matched groups of HSCT recipients, those treated with LTV prophylaxis (n = 30; LTV group) and those receiving preemptive therapy (n = 31; PET group). We analyzed the rates of graft-versus-host disease (GVHD), neutropenia, baseline disease recurrence, and overall survival in the 2 groups. Clinically significant infections necessitating preemptive therapy showed a similar rate in the 2 groups (PET: 21 of 31 [68%]; LTV: 17 of 30 [57%]; P = .434) but occurred significantly later (after prophylaxis discontinuation) in the LTV group. There was no between-group difference in peak HCMV DNAemia level (P = .232). HCMV-specific T cell recovery was delayed by approximately 100 days in the LTV group. HCMV-specific CD4 and CD8 T cell counts were significantly lower in the LTV group at days 120 to 360 and days 90 to 120, respectively. A lower rate of chronic GVHD (P = .024) was seen in the LTV group. Time to engraftment, rate of disease relapse, and 1-year survival were not different between the 2 groups, whereas trends toward a lower rate of neutropenia (P = .124) and a higher rate of acute GVHD grade III-IV (P = .103) were observed in the LTV group. Because LTV prophylaxis delays HCMV infection and HCMV-specific immune reconstitution, immunologic and virologic monitoring should be implemented after discontinuation of prophylaxis. The potential effect of LTV prophylaxis in reducing chronic GVHD should be evaluated in prospective studies.

The development and persistence of SARS-CoV-2-specific immune response in immunocompetent (IC) and immunocompromised patients is crucial for long-term protection. Immune response to SARS-CoV-2 infection was analysed in 57 IC and 15 solid organ transplanted (TX) patients. Antibody responses were determined by ELISA and neutralization assay. T-cell response was determined by stimulation with peptide pools of the Spike, Envelope, Membrane, and Nucleocapsid proteins with a 20-h Activation Induced Marker (AIM) and 7-day lymphoproliferative assays. Antibody response was detected at similar levels in IC and TX patients. Anti-Spike IgG, IgA and neutralizing antibodies persisted for at least one year, while anti-Nucleocapsid IgG declined earlier. Patients with pneumonia developed higher antibody levels than patients with mild symptoms. Similarly, both rapid and proliferative T-cell responses were detected within the first two months after infection at comparable levels in IC and TX patients, and were higher in patients with pneumonia. T-cell response persisted for at least one year in both IC and TX patients. Spike, Membrane, and Nucleocapsid proteins elicited the major CD4+ and CD8+ T-cell responses, whereas the T-cell response to Envelope protein was negligible. After SARS-CoV-2 infection, antibody and T-cell responses develop rapidly and persist over time in both immunocompetent and transplanted patients.

BNT162b2 vaccine was introduced in Italy on 27th December 2020 and healthcare workers were rapidly vaccinated. In this study, we demonstrated that one vaccine dose was sufficient for eliciting a sustained humoral and cell-mediated response in SARS-CoV-2 experienced healthcare workers but had a lower effect in SARS-CoV-2 naïve subjects. However, 98% naïve subjects developed both neutralizing antibodies and Spike-specific T-cells after the second dose. Moreover, the antibody and T-cell responses were effective against viral variants since a partial reduction in antibody response was observed only against the South-African variant in SARS-CoV-2 naïve individuals, while the T-cell response was less affected.

Human cytomegalovirus (HCMV) infection is one of the major causes of mortality and morbidity after allo-hematopoietic stem cell transplantation (HSCT). Antiviral therapies are associated with toxicity and high economic burden. The aim of this retrospective study was to identify allo-HSCT HCMV-seropositive recipients at low risk of clinically significant HCMV infection who could avoid antiviral therapies. Sixty adult patients who underwent allo-HSCT were clustered in two groups: i) 22 (37%) spontaneously controlling HCMV reactivation (Controllers); ii) 38 (63%) developing clinically significant HCMV infection and receiving pre-emptive therapy (Non-Controllers). We analyzed several patient baseline characteristics, total/HCMV-specific CD4+ and CD8+ T-cell counts and their cytokine production (IFNγ, TNFα, IL2). Controllers presented a higher number of total/HCMV-specific CD4+ and CD8+ T-cells (P=0.001 and P=0.017 for total CD4+ and CD8+ T-cells respectively; P

α1-Acid glycoprotein (AGP) is an acute-phase protein (APP) that modulates immune responses, probably – at least in humans – owing to the modification of its glycosylation pattern. On this perspective, feline AGP can be a useful comparative model, as it has different concentrations in cats susceptible or resistant to some disease. As a preliminary approach to the study of feline AGP (fAGP) we have purified this protein from feline serum by HPLC using human AGP (hAGP) as a model. Immunoblotting with a polyclonal antibody against fAGP and with a monoclonal antibody against hAGP was performed on serum from healthy cats, from cats exposed to feline coronavirus (FCoV) infection and from cats with purulent inflammations, such as feline infectious peritonitis (FIP), feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV). Immunohistochemistry on tissues from healthy cats and from cats with different diseases (FIP, FIV, FeLV, locally extensive inflammation) was also performed with the same antibodies. Both hAGP and fAGP have been purified to homogenity as determined by SDS-PAGE. fAGP did not react with the anti-hAGP antibody which, in contrast, detected in feline serum a low MW protein that we called fAGP-related protein (fAGPrP). This protein was underexpressed in cats with FeLV and FIP. Both fAGP and fAGPrP were immunohistochemically detected in plasma and hepatocytes with a stronger intensity in cats with FIP and some inflammatory conditions. Moreover, fAGPrP was detected in the cytoplasm of tissue cells, most likely identifiable with plasma cells. These cells were rarely detectable in cats with FIV and FeLV, and numerous in cats with FIP and with locally extensive inflammation. In conclusion, purified fAGP has physicochemical characteristics similar to those of hAGP, but does not cross-react with anti-hAGP antibodies. In contrast, the anti-hAGP detected an AGP-related protein whose blood concentration and tissue distribution was not related to that of fAGP. Moreover, both fAGP and fAGPrP were differently expressed in cats with pathologic conditions compared to controls. Further study of these proteins by analysing their structural characteristics is required.

Purpose The potential association of human cytomegalovirus (HCMV) infection and acute rejection with the occurrence of chronic lung allograft dysfunction (CLAD) was analysed in lung transplant recipients not receiving anti-HCMV prophylaxis, but followed with a pre-emptive strategy. Methods We analysed 129 lung transplant recipients (Feb 2004 -feb 2018). HCMV infection in blood was monitored by HCMV DNA (101 patients) or pp65-antigenemia (28 patients, with retrospective confirmation of HCMV DNA), and in broncoalveolar lavage (BAL) by HCMV DNA. Protocol cut-off values for pre-emptive treatment initiation with Gancyclovir (GCV) or valganciclovir (VGCV) were; 100 pp65-positive /200,000 WBC or 300,000 HCMV DNA copies/ml blood, 100.000 HCMV DNA copies/ml BAL. Results HCMV infection was detected in 121 (94%) patients: 52 (40%) patients remained asymptomatic and resolved without treatment (peak HCMV DNA: 2,930, [ 100.000,10,000-100,000 or Conclusion By means of this retrospective analysis we can state that high levels of HCMV in blood, besides the occurrence of acute rejection, are associated with higher risk of CLAD occurrence. Although we cannot prove a causal relationship between HCMV load in blood and CLAD, on this basis cut-off for pre-emptive treatment must be lowered in order to decrease a potential risk for CLAD occurrence.

Purpose Clinical characteristics of SARS-CoV-2 infection and virus-specific humoral and cellular response were analyzed in 4 heart (HR) and 3 lung (LR) Tx recipients in standard triple immunosuppressive regimen. Methods SARS-CoV-2 infection was diagnosed by real-time PCR on naso-pharingeal swabs (NPS). T-cell response to structural antigens Spike (S), Envelope (E), Membrane (M) and Nucleocapsid (N) was evaluated by PBMC stimulation with overlapping peptides spanning the entire viral proteins and subsequent detection of cell activation markers CD137 and CD25. Serum IgG antibody to S and N, and IgA antibody to S were determined by ELISA. Results Three patients developed SARS-CoV-2 infection early (3 years) after Tx. One HR was asymptomatic, one LR presented only gastrointestinal symptoms, and five patients developed dyspnea with radiologic signs of interstitial pneumonia (in one HR ICU admittance was necessary. All patients recovered from SARS-CoV-2 infection, with viral clearance from NPS within 3 weeks. However, two HR (one early and one late HR) died at 6 and 4 months after infection because of multi-organ failure and sudden death. Both deaths were considered as unrelated to SARS-CoV2 infection. Patients who had no lung involvement did not develop specific antibody response, while all the other five patients developed IgG and IgA antibodies to S, and IgG antibody to N, within 2 months after infection. All the symptomatic patients developed a detectable CD4+ T-cell response to two or more antigens. Four patients were subsequently examined >3 months after infection, showing the persistence of IgG and IgA antibodies to S and a decline of IgG antibody to N, while CD4+ T-cell response to N was maintained. Timing of Tx did not affect the occurrence of virus specific immunity. Conclusion Although this series is small, the data indicate that immunosuppression does not prevent the development of a specific humoral and cellular anti-SARS-CoV-2 response, more likely in patients who have experienced clinically relevant pneumonia. These preliminary data encourage the maintenance of regular follow-up to monitor the persistence of immune response and the potential occurrence of SARS-CoV-2-related sequelae in heart and lung recipients.

Human cytomegalovirus (HCMV)-specific T-cell response in kidney transplant recipients (KTR) helps to identify patients at risk for severe infection. To assess the T-cell response, this study compared our in-house developed reference test, based on T-cell (both CD4+ and CD8+) stimulation by autologous HCMV-infected dendritic cells (iDC) and subsequent detection by cytokine flow cytometry (CFC-iDC), with the Quanti-FERON-CMV (QF-CMV) assay. Fifty-three HCMV-seropositive KTR were enrolled. At the DNAemia peak, 33 (62%) had low viral load (LVL,3x105 DNA copies/mL) self-resolving infection, 19 (36%) high viral load (HVL,3x105 DNA copies/mL) infection treated with antivirals, and one LVL patient (2%) tissue-invasive disease alone. Both assays showed a delayed recovery of HCMV-specific T-cell immunity in HVL vs LVL patients. Immune reconstitution kinetics did not significantly differ between the two assays in HVL patients. QF-CMV and CFC-iDC showed comparable sensitivities, but QF-CMV had a lower (although not significantly) specificity. Indeed, 7/19 HVL patients (37%) were erroneously considered protected from severe infection by QF-CMV, whereas CFC-iDC misidentified only 3/19 (16%) patients as protected. Although our reference test takes longer to complete, it appears slightly better at predicting patients at risk for severe HCMV infection. Moreover, QF-CMV may provide false negative results with some HLA types.

Immune correlates of protection against human cytomegalovirus (HCMV) infection are still debated. This study aimed to investigate which arm of the immune response plays a major role in protection against HCMV infection in kidney transplant recipients (n = 40) and heart transplant recipients (n = 12). Overall, patients were divided into 2 groups: one including 37 patients with low viral load (LVL), and the other including 15 patients with high viral load (HVL). All LVL patients resolved the infection spontaneously, whereas HVL patients were all treated with one or more courses of antivirals. In HVL patients, viral DNAemia, which was more than 100 times higher than LVL, appeared and peaked at significantly earlier times, but disappeared much later than in LVL patients. During a 1-year follow-up, all LVL patients had levels of HCMV-specific CD4+ (and CD8+ ) T cells significantly higher than HVL patients. On the contrary, titers of neutralizing antibodies and enzyme-linked immunosorbent assay-IgG antibodies to gB, gHgLgO, and pentamer gHgLpUL128L were overlapping in the 2 patient groups. In conclusion, while a valid HCMV-specific T-cell response was detected in more than 90% of LVL patients, >90% of HVL patients lacked an adequate T-cell response. Antibody responses did not appear to be associated directly or indirectly with protection.

Human cytomegalovirus (HCMV) is still the most common viral infection in solid-organ transplant recipients (SOTR). Our study aimed to identify the predictive values of the T-cell response able to protect from HCMV disease, according to different assays. Viral DNA was determined by real-time PCR. The T-cell immune response to HCMV infection was investigated in SOTR according to the following assays and stimuli: cytokine flow cytometry (CFC) after peripheral blood mononuclear cell (PBMC) stimulation with autologous HCMV-infected dendritic cells (iDC) vs three ELISPOT assays using PBMCs stimulated with: 1. HCMV-infected cell lysate (iCL); 2. a pool of 34 epitopic peptides (PP) from different HCMV proteins; 3. a commercial pp65 peptide pool (CPM). ELISPOT results were normalized to T-cell counts. Overall, 51 SOTR were enrolled: 29 (57%) had low viral load (LVL) self-resolving infections, 19 (37%) high viral load (HVL) infections treated with antiviral drugs, and 3 (6%) tissue-invasive disease (TID). At DNAemia peak, ROC analysis showed that CFC-iDC CD4+ and the ELISPOT-iCL assays yielded overlapping area under the curve (AUC) results. The time needed to reconstitute protective T-cell immunity in SOTR with HVL infections was significantly longer with each assay compared to LVL infections. Using the CFC-iDC assay as a reference test (requiring 7 days to complete), the 24h ELISPOT-iCL assay provides similar results in terms of protection prediction from HCMV infection.

Introduction HCMV infection is a significant viral complication in HSCTR, causing either a subclinical infection or a severe disease (systemic syndrome and localized tissue invasive disease (TID)). Pre-emptive therapy used for the management of HCMV infection and prevention of disease requires the monitoring of HCMV load in blood to start antiviral therapy after detection of a viral load threshold associated to the risk for HCMV severe infection and disease. In previous studies we observed that patients reconstituting both CD4+ and CD8+ HCMV-specific T cells are generally protected from HCMV disease. Thus, determination of specific T cells may discriminate patients lacking immunity and requiring stricter virological monitoring (negative T-cell response) from those early recovering T-cell immunity and thus not requiring antiviral interventions (positive T-cell response). Aims The aim of our work is to assess the possibility to manage HCMV infection by combined virological and immunological monitoring. The primary endpoint of this study is to compare the frequency of HCMV disease between two groups of HSCTR: in the first one, pre-emptive therapy was started according to combined virological/immunological methods; the second group consisted of an historical cohort of patients monitored with HCMV-DNA as the only guiding parameter for pre-emptive therapy. Methods We analyzed adult patients receiving allogeneic HSCT: the recipients, the donor or both had to be HCMV seropositive. Virological follow-up (HCMV-DNA determination in whole blood) was performed weekly for the first 3 months, then monthly during the first year. Immunological follow-up was performed at 1, 2, 3, 4, 6, 8, 10 and 12 months. Peripheral blood HCMV-specific T cells were determined by two assays: i) iCL (infected Cell Lysate)-ELISPOT, considering as "protective" immunity a blood count of specific T-cell ≥ 0.1/µL ii) iDC-CFC (infected Dendritic Cells-Cytokine Flow Cytometry), considering as "protective" immunity a blood count of CD4+ T-cell ≥ 1/µL and CD8+ T cells ≥ 3/µL. According to study, within the first 3 months following transplantation pre-emptive antiviral therapy was given to all patients presenting a viral load ≥30.000 copies/mL blood (severe infection). Beyond 3 months following transplantation, patients with negative T-cell response continued receiving a pre-emptive antiviral therapy as reported above. In patients with positive T-cell response confirmed by both assays ("protected" patients), antiviral therapy was deferred even if viral load would reach ≥30.000 copies/mL blood. Results We performed an analysis of 36 patients with a follow-up ≥4 months from HSCT (median follow-up 245 days, 120-397; see Table 1 for patients' characteristics). Among them, 32 HCMV-seropositive HSCTR developed HCMV infection whereas 4 HCMV-seronegative patient receiving HSCT from seropositive donors did not (nor developed specific immunity). Among the 32 HSCTR with HCMV reactivation, 13 (41%) resolved spontaneously HCMV reactivation, whereas 19 (59%) had a severe infection (11 [34%] controlled the infection after a single course of Valganciclovir therapy and the remaining 8 [25%] required multiple courses for relapsing episodes). Thirty days after HSCT, levels of total and HCMV-specific CD4+ and CD8+ T cells were significantly higher in the 13 patients spontaneously resolving the infection than in the 19 patients with severe infection. The two immunological assays were concordant in detecting time to protective immune reconstitution (median time: 177 vs 183 days for iDC and iCL-ELISPOT, respectively). HCMV-specific immune reconstitution was significantly delayed in patients with multiple infection episodes (median time: 250 days post-transplantation) vs patients with a single episode of severe infection (187 days) and patients with self-resolving infection (89 days). No patient developed HCMV severe infection after HCMV-specific immune reconstitution. Currently, no patient developed HCMV disease. Conclusion These results suggest that combined virological/immunological monitoring can be useful to identify patients not requiring strict virological controls and antiviral interventions. Disclosures No relevant conflicts of interest to declare.

INTRODUCTION: Human cytomegalovirus (HCMV) infection is a significant cause of mortality and morbidity after hemopoietic stem cell transplantation (HSCT). Pre-emptive therapy with Ganciclovir (GCV) reduces the risk for HCMV disease but may be associated with significant toxicity. Letermovir (LTV) has been recently approved for prophylaxis of HCMV infection in HCMV-seropositive patients. AIM: to compare costs associated with GCV preemptive therapy or LTV prophylaxis within 100 days after HSCT. METHODS: A retrospective analysis of GCV preemptive therapy-related costs was performed on 63 HCMV-seropositive patients receiving allogeneic HSCT. For comparison, we estimated LTV prophylaxis-related costs according to dosage and duration reported in the phase 3 trial (Marty et al., NEJM 2017). According to definitions adopted in the LTV prophylaxis study, patients were considered at high risk for HCMV-disease if having one of the following: a mismatched (related or unrelated) or a haploidentical donor; umbilical cord blood as the stem-cell source; ex vivo T-cell-depleted grafts; and grade ≥2 GVHD. Clinically significant HCMV infection was defined as HCMV disease (i.e. HCMV end-organ infection in association with clinical symptoms) or HCMV DNAemia leading to pre-emptive treatment (i.e. ≥ 30.000 copies/mL in blood in the GCV-preemptive group; ≥ 150 copies/mL or ≥ 300 copies/mL in plasma for high-risk or low-risk patients in the LTV-prophylaxis study). All diagnostic and therapeutic procedures performed within 100 days post-HSCT were recorded. Costs associated to HCMV antiviral therapy and to the management HCMV infection (including also, but not limited to, diagnostic procedures, transfusions and hospitalizations) were calculated. RESULTS Study populations Baseline characteristics of the GCV-preemptive group in comparison with the LTV-prophylaxis group are shown in Table 1. A higher proportion of patients at high risk for HCMV disease has been observed in the preemptive group (49.2% vs 32.4%). HCMV infection and disease GCV-preemptive group: 39 patients (62%) developed a clinically significant HCMV infection and in all cases pre-emptive therapy resolved the infection. Drug switch due to GCV-induced neutropenia was not necessary in any case. Gastrointestinal HCMV disease occurred beyond day 100: in 2 patients (3.2%) occurred within 24 weeks post-HSCT and in additional 7 patients later on (median time: 314, range 144-387 days after HSCT). LTV-prophylaxis group: clinically significant HCMV infection occurred in 57/325 (17.5%) patients with 5 (1.5%) developing gastrointestinal HCMV disease within 24 weeks after HSCT. Data for HCMV disease beyond week 24 are not reported. Costs analysis GCV-preemptive group: 5 mg/kg GCV has been administered i.v. twice daily. One vial of GCV 500 mg is purchased at a cost of 57.67 €. The average duration of GCV therapy was 16.9 days and the average cost was 77 €/day, i.e. 1301.67 €/patient. Six patients (10%) were hospitalized for the management of HCMV infection, for a total cost of 22,428 € (356 €/patient). Thus, the overall cost was 1657.67 €/patient (78% directly related to GCV-therapy and 22% related to the healthcare management costs). LTV-prophylaxis group: 240 mg/day of oral LTV were administered for of 100 days. Twenty-eight tablets of LTV cost of 13,863.36 €. Thus the total cost for LTV administration is 49,512 €/patient (or 24,756 € for patients receiving cyclosporine). We did not consider costs associated to HCMV reactivation. The costs for HCMV management with LTV prophylaxis appears to be ≥15 times superior to the costs for GCV preemptive therapy CONCLUSIONS: LTV prophylaxis is effective in reducing the risk for HCMV disease. However, the costs are superior to those for GCV-preemptive therapy (also considering hospitalization costs). Prospective randomized studies are needed to compare directly different outcomes in HSCT recipients receiving LTV prophylaxis vs GCV preemptive therapy (such as overall costs, indirect effects of subclinical HCMV infection, overall patient's survival). Nevertheless, the identification of patients at lower risk for HCMV infection (thus not requiring prophylaxis), as well as a personalized administration of LTV prophylaxis on the basis of the individual time to immune reconstitution (thus tailoring duration of prophylaxis), will be of great help for sparing costs in Health Systems with limited resources. Disclosures No relevant conflicts of interest to declare.

Autoimmunity to heat shock protein 60 (HSP60) has been related to atherosclerosis. Chlamydia pneumoniae (CP), the most studied infectious agent implicated in promoting atherosclerosis, produces a form of HSP60, which can induce an autoimmune response, due to high antigenic homology with human HSP60 (hHSP60). In this study, we evaluated the correlations among anti-hHSP60 antibodies, CP infection, and cardiovascular disease (CVD) in a high-risk population, such as patients undergoing hemodialysis (HD). Thirty-two patients (67.9 ± 13.9 years; male/female, 23:9) on regular HD were enrolled. Global absolute cardiovascular risk (GCR) was assessed using the Italian CUORE Project’s risk charts, which evaluate age, gender, smoking habits, diabetes, systolic blood pressure, and serum cholesterol. The occurrence of cardiovascular events during a 24-month follow-up was recorded. Seropositivity to CP and the presence of anti-hHSP60 antibodies were tested by specific enzyme-linked immunosorbent assays. Inflammation was assessed by measurement of C-reactive protein (CRP) serum levels. Fifteen healthy sex and age-matched (61.9 ± 9.5 years; male/female, 11:4) subjects were the control group. Fifteen of 32 patients resulted seropositive for CP. CP + patients were older than CP−, while they did not differ for GCR, CRP, and dialytic parameters. CVD incidence was significantly higher in CP+ (9 CP+ vs 2 CP−, p

The comparative long-term kinetics of human cytomegalovirus (HCMV) load and HCMV-specific antibody responses in the immunocompetent and immunocompromised solid-organ transplanted host during primary HCMV infection was investigated. In total, 40 immunocompetent subjects and 17 transplanted patients were examined for viral load as well as for IgG antibody responses to HCMV glycoproteins gH/gL/pUL128L, gH/gL and gB, and neutralizing antibodies in ARPE-19 epithelial cells and human fibroblasts. In parallel, the CD4(+) and CD8(+) HCMV-specific T-cell responses were determined by cytokine flow cytometry. Transplanted patients reached significantly higher viral DNA peaks, which persisted longer than in immunocompetent subjects. The ELISA-IgG responses to the pentamer, gH/gL and gB were significantly higher in primary infections of the immunocompetent until six months after onset, with the two antibody levels then overlapping from six to 12 months. Antibody levels neutralizing infection of epithelial cells were significantly higher in transplanted patients after six months, persisting for up to a year after transplantation. This trend was not observed for antibodies neutralizing infection of human fibroblasts, which showed higher titres in the immunocompetent over the entire one-year follow-up. In conclusion, in immunocompromised patients the viral load peak was much higher, while the neutralizing antibody response exceeded that detected in the immunocompetent host starting six months after onset of follow-up, often concomitantly with a lack of specific CD4(+) T cells. In this setting, the elevated antibody response occurred in the presence of differentiated follicular helper T cells in the blood, which decreased in number as did antibody titres upon reappearance of HCMV-specific CD4(+) T cells.

In solid-organ transplant recipients (SOTR) the protective role of human cytomegalovirus (HCMV)-specific CD4+, CD8+ and γδ T-cells vs. HCMV reactivation requires better definition. The aim of this study was to investigate the relevant role of HCMV-specific CD4+, CD8+ and γδ T-cells in different clinical presentations during the post-transplant period. Thirty-nine SOTR underwent virologic and immunologic follow-up for about 1 year after transplantation. Viral load was determined by real-time PCR, while immunologic monitoring was performed by measuring HCMV-specific CD4+ and CD8+ T cells (following stimulation with autologous HCMV-infected dendritic cells) and γδ T-cells by flow cytometry. Seven patients had no infection and 14 had a controlled infection, while both groups maintained CD4+ T-cell numbers above the established cut-off (0.4 cell/µL blood). Of the remaining patients, 9 controlled the infection temporarily in the presence of HCMV-specific CD8+ only, until CD4+ T-cell appearance; while 9 had to be treated preemptively due to a viral load greater than the established cut-off (3×10(5) DNA copies/mL blood) in the absence of specific CD4+ T-cells. Polyfunctional CD8+ T-cells as well as Vδ2- γδ T-cells were not associated with control of infection. In conclusion, in the absence of HCMV-specific CD4+ T-cells, no long-term protection is conferred to SOTR by either HCMV-specific CD8+ T-cells alone or Vδ2- γδ T-cell expansion.

Following primary human cytomegalovirus (HCMV) infection, both humoral and T-cell-mediated immune responses develop in immunocompetent subjects. However, while antibodies may be measured by different methodologies, the T-cell-mediated response remains to be analyzed in its polyfunctional aspects, in view of defining (following different stimuli) the optimal assay to monitor the HCMV-specific T-cell response in HCMV-seropositive subjects.In a group of 30 HCMV-seropositive adults, T-cell response revealed by the HCMV-infected dendritic cell (iDC) stimulus was compared with those given by the HCMV-infected cell lysate (iCL), and by a 34-peptide pool (PP).All HCMV-seropositive subjects showed presence of both HCMV-specific CD4(+) and CD8(+) T-cells in peripheral blood following iDC stimulation. One subject did not respond to PP. As compared to iDC, the number of HCMV-specific stimulated T-cells/μl blood was slightly lower for iCL (P = 0.195) and significantly lower for PP (P = 0.001). Polyfunctional analysis of the T-cell response indicated that the lower number of CD4(+) T-cells stimulated by iCL was due to the bifunctional (IFN-γ(+) TNF-α(+)) and CD40L-negative T-cell reduction, while the reduction in specific PP-stimulated CD8(+) T-cells was attributable to the reduction in tri-(IFN-γ(+) TNF-α(+) IL2(+)), bi-(IFN-γ(+) TNF-α(+)) and mono-(IFN-γ(+)) functional T-cells. In addition, 15/30 (50 %) subjects showed a CD4(+) cross-response to PP, and 11/30 (37 %) a CD8(+) cross-response to iCL.HCMV-specific stimulus given by iDC is not significantly different from that of iCL on CD4(+) and is significantly superior to that of PP on CD8+ T-cells. However, iCL may contribute significantly to CD8(+), and PP to CD4(+) T-cell stimulation.

It is debated whether human cytomegalovirus (HCMV) infection/disease of the pulmonary compartment in lung transplant recipients (LTRs) may be controlled by the HCMV-specific systemic T-cell response or requires a local (lung) T-cell response. Systemic and local HCMV loads were investigated in parallel by real-time PCR in 20 LTRs. T-cell responses were measured by intracellular cytokine staining of HCMV-specific IFN-? + CD4+ and CD8+ T-cells in PBMC, and by enzyme-linked immunospot (ELISpot) assay in lung (BAL) mononuclear cells. Patients were grouped at time of peak of infection based on viral load in blood and BAL. Immunological testing results showed that five patients with no HCMV infection (either local or systemic) had both local and systemic T-cell responses; four patients with systemic infection had no systemic T-cell response; five patients with both systemic and lung infection had neither local nor systemic T-cell responses; and six patients with lung infection had no local and a partial (only CD8+ in the absence of CD4+) systemic T-cell response. These results indicate that local immunity is associated with resolution of lung infection. Systemic T-cell response alone is not sufficient to provide lung protection from HCMV infection.

CD40 and its ligand (CD40L) regulate several cellular functions, including T and B-cell activation. The soluble form of CD40 (sCD40) antagonizes CD40/CD40L interaction. Patients undergoing hemodialysis (HD) present elevated sCD40 serum levels, which underlying molecular mechanisms are unknown. We studied sCD40 serum and urinary levels, CD40 membrane and gene expression and membrane shedding in HD, uremic not-HD patients (UR) and healthy subjects (N). We found that in HD sCD40 serum levels were higher than UR and N, being significantly elevated in anuric patients, and that sCD40 correlated to renal function in UR subjects, who presented also a reduced sCD40 urinary excretion rate. HD and UR presented reduced CD40 membrane and gene expression. The concentration of TNF-α converting enzyme (TACE), responsible for CD40 cleavage was not different between HD and N. Therefore the reduced renal clearance is the main cause of elevated sCD40 levels in HD. This finding could have relevant clinical implications.

MSC (mesenchymal stromal cells) can differentiate into renal adult cells, and have anti-inflammatory and immune-modulating activity. In the present study, we investigated whether MSC have protective/reparative effects in anti-Thy1 disease, an Ab (antibody)-induced mesangiolysis resulting in mesangioproliferative nephritis. We studied five groups of rats: (i) rats injected with anti-Thy1.1 Ab on day 0 (group A); (ii) rats injected with anti-Thy1.1 Ab on day 0+MSC on day 3 (group B); (iii) rats injected with anti-Thy1.1 Ab on day 0+mesangial cells on day 3 (group C); (iv) rats injected with saline on day 0+MSC on day 3 (group D); and (v) rats injected with saline on day 0 (group E). Rats were killed on days 1, 3, 7 and 14. MSC prevented the increase in serum creatinine, proteinuria, glomerular monocyte influx and glomerular histopathological injury. Furthermore, MSC suppressed the release of IL-6 (interleukin-6) and TGF-β (transforming growth factor-β), modulated glomerular PDGF-β (platelet-derived growth factor-β), and reset the scatter factors and their receptors, potentiating HGF (hepatocyte growth factor)/Met and inactivating MSP (macrophage-stimulating protein)/Ron (receptor origin nantaise). Few MSC were found in the kidney. These results indicate that MSC improve anti-Thy 1 disease not by replacing injured cells, but by preventing cytokine-driven inflammation and modulating PDGF-β and the scatter factors, i.e. systems that regulate movement and proliferation of monocytes and mesangial cells.

Chondrosarcomas represent 20% of all primary bone sarcomas, and many studies have attempted to unravel molecular targets for future development of new therapies. The aim of this study was to investigate the expression/activation of PDGFRα, PDGFRβ and KIT receptor tyrosine kinases (RTKs) as potential therapeutic targets in conventional central primary chondrosarcomas (CCS). The expression of PDGFRα, PDGFRβ and KIT RTKs was detected in 16 CCSs using immunohistochemistry (IHC), and their level of expression and activation status were analysed by immunoprecipitation and western blot experiments. PDGFRα, PDGFRβ and KIT cDNAs were screened to verify the presence of activating mutations and the presence of the cognate ligands was analysed by means of RT-PCR. RTK gene amplification was further studied by means of fluorescence in situ hybridization (FISH) analysis. The immunophenotyping and biochemical analyses showed that the CCSs co-expressed PDGFRα and PDGFRβ, with the latter showing definitively greater protein expression and phosphorylation levels. PDGFRβ was expressed but not activated in control healthy joint cartilage, in line with no PDGFB detection. Conversely, the KIT gene product did not seem to play a relevant role. These findings, in the absence of activating mutations or an abnormal genomic profile and the presence of PDGFA and PDGFB expression, are consistent with an autocrine/paracrine loop activation of the corresponding receptors. The CCS gene profile described here offers a rationale for the use of RTK inhibitors alone or in combination with chemotherapy, and supports further investigation of RTKs and their downstream signals. Copyright © 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

BACKGROUND Gastrointestinal stromal tumors (GISTs) are noncomplex sarcomas that often are due to c-kit-activating and platelet-derived growth factor receptor α gene (PDGFRα)-activating mutations and perturbations of their related signaling pathways. Molecular and cytogenetic findings have indicated correlations between tumor progression and high-risk GISTs with c-kit mutations, the overexpression of genes such as ezrin, and losses at 9p. In particular, it was reported recently that malignant GISTs showed alterations in the p16INK4a gene located at the 9p21 locus. METHODS To assess the involvement of p14ARF and p15INK4b in addition to p16INK4a in GISTs, the authors undertook a molecular and cytogenetic study of the 9p21 locus. A series of 22 pre-Gleevec era, cryopreserved, high-risk GISTs that were characterized well in terms of KIT and PDGFRα receptors were investigated for mRNA expression, homozygous deletions, mutations, and promoter methylation of locus 9p21, in some instances complemented by fluorescent in situ hybridization studies. RESULTS The results indicated the loss of p16INK4a mRNA expression in 41% of the GISTs, mainly due to the homozygous deletion of both the p16INK4a gene and the p14ARF gene (24%). No mutations were found, and promoter methylation (detected by means of methylation-specific polymerase chain reaction analysis in 27% of tumors) was restricted mainly to the p15INK4b gene (20%). It is noteworthy that, in all of the methylated GISTs, the epigenetic promoter alteration was coupled with mRNA expression. CONCLUSIONS Alterations in the 9p21 locus were found cumulatively in 54% of the tumors in the current series and were represented mainly by the loss of tumor suppressor gene expression. The p16INK4a deletion, which always was coupled with p14ARF gene loss, seemed to be the most common 9p21 inactivation mechanism. Cancer 2005. © 2005 American Cancer Society.

Age-related structural and neurochemical changes occurring in the central nervous system have been related to changes in some rhythmometric parameters. In spite of their clinical importance, only a few studies have investigated the modifications over time of serum electrolytes in senescence. The aim of our study was to evaluate the circadian pattern of serum potassium, chloride, sodium, calcium and phosphorus in 30 clinically healthy elderly subjects, with no cognitive impairment, and to compare the findings with those given by 24 healthy young controls. The subjects were synchronized as regards their daily activities, sleeping/waking habits, time/quality of meals and dietary electrolyte intake. After an overnight fast, samples were taken beginning at 08.00 and every 4 h thereafter until 20.00, and every 2 h from 20.00 to 04.00. Both the young and the elderly subjects exhibited statistically significant circadian rhythms for all serum electrolytes considered. Our findings suggest that circadian organization of serum electrolytes is maintained in physiological aging, even though it should be noted that sodium and phosphorus acrophases differed significantly in the two experimental groups.

We read with great interest the paper by Gruden et al.[1] recently published in Journal of Internal Medicine.The authors reported a significant inverse correlationbetween diabetic micro- and macroangiopathy andserum levels of anti-Heat Shock Protein 70 (HSP70)antibodies in a large cohort of type 1 diabetic patients.HSP70 levels resulted higher in healthy subjects com-pared with diabetic patients, while no significant dif-ference was found in anti-HPS60 levels betweenpatients and controls. In a longitudinal study, weinvestigated whether anti-HSP60 antibodies are asso-ciated with cardiovascular risk in adult patients onhaemodialysis (HD). We evaluated anti-HSP60 serumlevels in 33 nondiabetic patients (66.5 ± 10.4 years,m⁄f: 17⁄16) on regular HD. Then, we correlated thetiter of the antibodies to cardiovascular risk factors,inflammation and incidence of cardiovascular events,during a 24-month follow-up. Cardiovascular risk fac-tors considered were: smoking habit, diabetes, systolicblood pressure and serum cholesterol. Inflammationwas assessed by high sensitivity C-Reactive Protein(hs-CRP) serum levels. Thirty healthy sex-agematched subjects (63.3 ± 7.7 years, m⁄f: 15⁄15) werethe controls. Anti-HSP60 serum levels were measuredby ELISA that detected IgG, IgA and IgM isotypes(Stressgen Biotechnologies Corporation,Victoria, BC,Canada). Anti-HSP60 were present in all analysedsubjects; there was no significant difference in anti-body levels between patients and controls(41.84 ± 34.78 vs. 38.47 ± 11.34 lgmL

The relative contribution of human cytomegalovirus (HMCV)-specific CD4(+) and CD8(+) T cells to the control of HCMV infection in hematopoietic stem cell transplant (HSCT) recipients is still controversial. HCMV reactivation and HCMV-specific CD4(+) and CD8(+) T cell reconstitution were monitored for 1 year in 63 HCMV-seropositive patients receiving HSCT. HCMV reactivation was detected in all but 2 patients. In 20 of 63 (31.7%) patients (group 1) HCMV infection resolved spontaneously, whereas 32 of 63 (50.8%) patients (group 2) controlled the infection after a single short-course of pre-emptive therapy and the remaining 9 (14.3%) patients (group 3) suffered from relapsing episodes of HCMV infection, requiring multiple courses of antiviral therapy. The kinetics and magnitude of HCMV-specific CD8(+) T cell reconstitution were comparable among the 3 groups, but HCMV-specific CD4(+) T cells were lower in number in patients requiring antiviral treatment. HCMV-seronegative donors, as well as unrelated donors (receiving antithymocyte globulin) and acute graft-versus-host disease (GVHD) were associated with both delayed HCMV-specific CD4(+) T cell reconstitution and severity of infection. Conversely, these risk factors had no impact on HCMV-specific CD8(+) T cells. Eight patients with previous GVHD suffered from HCMV gastrointestinal disease, although in the presence of HCMV-specific CD4(+) and CD8(+) systemic immunity and undetectable HCMV DNA in blood. Reconstitution of systemic HCMV-specific CD4(+) T cell immunity is required for control of HCMV reactivation in adult HSCT recipients, but it may not be sufficient to prevent late-onset organ localization in patients with GVHD. HCMV-specific CD8(+) T cells contribute to control of HCMV infection, but only after HCMV-specific CD4(+) T cell reconstitution.