Chiara Quadrelli

Characterization of Specific Immune Responses to Different Aspergillus Antigens during the Course of Invasive Aspergillosis in Hematologic Patients

Several studies in mouse model of invasive aspergillosis (IA) and in healthy donors have shown that different Aspergillus antigens may stimulate different adaptive immune responses. However, the occurrence of Aspergillus-specific T cells have not yet been reported in patients with the disease. In patients with IA, we have investigated during the infection: a) whether and how specific T-cell responses to different Aspergillus antigens occur and develop; b) which antigens elicit the highest frequencies of protective immune responses and, c) whether such protective T cells could be expanded ex-vivo. Forty hematologic patients have been studied, including 22 patients with IA and 18 controls. Specific T cells producing IL-10, IFN-γ, IL-4 and IL-17A have been characterized through enzyme linked immunospot and cytokine secretion assays on 88 peripheral blood (PB) samples, by using the following recombinant antigens: GEL1p, CRF1p, PEP1p, SOD1p, α1–3glucan, β1–3glucan, galactomannan. Specific T cells were expanded through short term culture. Aspergillus-specific T cells producing non-protective interleukin-10 (IL-10) and protective interferon-gamma (IFN-γ) have been detected to all the antigens only in IA patients. Lower numbers of specific T cells producing IL-4 and IL-17A have also been shown. Protective T cells targeted predominantly Aspergillus cell wall antigens, tended to increase during the IA course and to be associated with a better clinical outcome. Aspergillus-specific T cells could be successfully generated from the PB of 8 out of 8 patients with IA and included cytotoxic subsets able to lyse Aspergillus hyphae. Aspergillus specific T-cell responses contribute to the clearance of the pathogen in immunosuppressed patients with IA and Aspergillus cell wall antigens are those mainly targeted by protective immune responses. Cytotoxic specific T cells can be expanded from immunosuppressed patients even during the infection by using the above mentioned antigens. These findings may be exploited for immunotherapeutic purposes in patients with IA.

Changes in the immune responses against human herpesvirus-8 in the disease course of posttransplant kaposi sarcoma

In nine patients with posttransplant Kaposi sarcoma (KS) T-cell responses to human herpesvirus (HHV)-8 latent and lytic antigens, as detected by enzyme-linked-immunospot (Elispot) assay, were absent at disease onset. Virus-specific T-cell responses were detected in six renal recipients at remission after a reduction of calcineurin inhibitors (CIs), and in two HHV-8 seropositive renal recipients without KS. In two liver recipients undergoing switch from CIs to sirolimus (SRL), normalization of the T-cell repertoire and recovery of both HHV-8-specific effector and memory T lymphocytes were associated with complete KS remission. In a renal recipient undergoing SRL conversion, the early recovery of HHV-8-specific effector but not of memory T lymphocytes, was associated only with partial remission. Neither rejection nor changes in graft function were observed after SRL conversion. HHV-8-specific T-cell responses are required to achieve posttransplant KS remission, and may be restored under SRL, while maintaining effective immunosuppression.

Mucorales -specific T cells emerge in the course of invasive mucormycosis and may be used as a surrogate diagnostic marker in high-risk patients

Mucorales-specific T cells were investigated in 28 hematologic patients during the course of their treatment. Three developed proven invasive mucormycosis (IM), 17 had infections of known origin but other than IM, and 8 never had fever during the period of observation. Mucorales-specific T cells could be detected only in patients with IM, both at diagnosis and throughout the entire course of the IM, but neither before nor for long after resolution of the infection. Such T cells predominantly produced IL-4, IFN-γ, IL-10, and to a lesser extent IL-17 and belonged to either CD4(+) or CD8(+) subsets. The specific T cells that produced IFN-γ were able to directly induce damage to Mucorales hyphae. None of the 25 patients without IM had Mucorales-specific T cells. Specific T cells contribute to human immune responses against fungi of the order Mucorales and could be evaluated as a surrogate diagnostic marker of IM.

Emergence of BCR-ABL–specific cytotoxic T cells in the bone marrow of patients with Ph + acute lymphoblastic leukemia during long-term imatinib mesylate treatment

Imatinib mesylate has been demonstrated to allow the emergence of T cells directed against chronic myeloid leukemia cells. A total of 10 Philadelphia chromosome-positive acute lymphoblastic leukemia patients receiving high-dose imatinib mesylate maintenance underwent long-term immunological monitoring (range, 2-65 months) of (p190)BCR-ABL-specific T cells in the bone marrow and peripheral blood. (p190)BCR-ABL-specific T lymphocytes were detected in all patients, more frequently in bone marrow than in peripheral blood samples (67% vs 25%, P < .01) and resulted significantly associated with lower minimal residual disease values (P < .001), whereas absent at leukemia relapse. Specific T cells were mainly effector memory CD8(+) and CD4(+) T cells, producing interferon-gamma, tumor necrosis factor-alpha, and interleukin-2 (median percentage of positive cells: 3.34, 3.04, and 3.58, respectively). Cytotoxic subsets able to lyse BCR-ABL-positive leukemia blasts also were detectable. Whether these autologous (p190)BCR-ABL-specific T cells may be detectable under other tyrosine-kinase inhibitors, expanded ex vivo, and exploited for immunotherapy remains to be addressed.

Diagnosis of invasive aspergillosis by tracking Aspergillus-specific T cells in hematologic patients with pulmonary infiltrates.

Invasive aspergillosis (IA) is a leading cause of infection-related mortality in hematologic patients, with death rate ranging from 50 to 90%.1 The reason for this extremely poor outcome is because of the difficulties in a timely and undoubted diagnosis, which still relies on a very high degree of suspicion. The current diagnostic tools are limited by invasiveness, slowness, relative insensitiveness, lack of standardization and unpredictable kinetics.2 The most widely studied test, Galctomannan antigenemia (GM), has been demonstrated to be highly variable in performance, with sensitivity ranging between 29 and 100%, and is affected by several factors related either to the fungus or to the host.2 Furthermore, the detection of Aspergillus DNA through polymerase chain reaction (PCR) is still hampered by the difficulties in understanding the fungal DNA release and kinetics other than by technical barriers.2 For all these reasons, establishing an early diagnosis of IA remains a challenge.1, 2

Parvoviruses in Blood Donors and Transplant Patients, Italy

To the Editor: Parvoviruses (PARV) 4 and 5 are 2 genotypes of a novel human parvovirus, with 92% nucleotide identity, identified in the plasma sample of a patient screened for acute HIV infection and in samples of manufactured plasma pools (1,2). Recently, PARV4 and PARV5 were identified in blood samples from 3 of 26 cadavers from the United Kingdom, all of whom were positive for hepatitis C virus RNA and had a history of intravenous drug use (3). PARV4/5 were also found in bone marrow (BM) and lymphoid tissues from 17 of 24 HIV-positive cadavers from Scotland (4) and in BM aspirates from 16 of 35 Italian patients with AIDS (5). Little or no information is available about the epidemiology and clinical correlates of infection with these novel viruses. To provide insights into their pathogenic potential in vivo, we assessed the frequency of PARV4/5 viremia in healthy patients, transplant patients, and those with suspected viral disease. We performed a retrospective molecular study for the presence of PARV4/5 sequences in 4 groups of 417 Italian HIV-negative persons. Group 1 consisted of 100 blood donors recruited from the Transfusion Centre of Modena (northern Italy); group 2, 84 patients with hematologic diseases showing clinical signs of viral etiology but negative results for the most common viruses (herpesviruses, adenovirus, hepatitis virus, and coxsackie virus). For both of these groups, DNA was extracted for analysis from serum specimens and peripheral blood mononuclear cells (PBMCs). Groups 3 and 4 comprised recipients of kidney and allogeneic BM/peripheral blood stem cell (PBSC) transplants, for which DNA was extracted from serum specimens collected at 6 and 12 months, respectively, after transplantation. The nested PCR method was used to amplify a shared sequence of PARV4 and its variant PARV5 and was specific for the open reading frame 1. First step PCR was performed as previously described (2) with a sensitivity of 1–10 copies, on 1 μg PBMC DNA and on one fifth of DNA extracted from 0.25 mL of serum. Primers for second round PCR were PV4NS1Fn2 (5′-GTTGATGGYCCTGTGGTTAG-3′) and PV4NS1Rn2 (5′-CCTTTCATATTCAGTTCCTGTTCAC-3′). All positive results were confirmed by direct sequencing. We found 3 positive case-patients, including 2 renal transplant recipients and 1 patient with a suspected viral disease; none of the blood donors tested positive on single-round PCR. On nested PCR, 1 blood donor had positive results; the positivity rate did not increase in the other groups (Table). In the first 2 groups, PARV4/5 sequences were detected only in the serum samples, not in the PBMCs collected at the same time. These sequences suggest that PBMCs are not a major site of viral replication. Similar to B19 infection, which is rarely reactivated in the setting of BM/PBSC transplantation (6,7), none of the BM/PBSC transplant patients were PARV4/5 positive. The detection of PARV4/5 sequences in the serum collected at 12 months after transplantation was not associated with the occurrence of any symptoms in the 2 renal recipients. Of note, the available serum samples collected from both recipients before transplantation, and at 6 and 24 months after transplantation, were PARV4/5 negative, which suggests that asymptomatic PARV4/5 infection may transiently occur after solid organ transplantation or may be acquired throughout transfusion or transplantation. Similarly, the rate of B19 infection in solid organ transplant recipients is low (1.4%–1.8%), and most B19 DNA–positive patients remain asymptomatic (8,9). Table Analysis of 417 patients tested for parvoviruses 4/5 by PCR* PARV4/5 sequences were detected in the serum collected from 1 patient affected with Wegener granulomatosis. This patient was under long-term treatment with steroids, concomitant with the development of a clinical syndrome for which a viral cause was suspected, including fever, severe anemia, a histologic-examination–proven postinfectious glomerulonephritis, and eryhtroid hypoplasia, with dsyserythropoiesis and dismegakaryopoiesis on BM examination. Serologic and molecular tests for the most common viruses, including B19, were negative and the patient died of multiple organ failure 1 month later. Single-cell PCR performed on the DNA extracted from isolated BM erythroid and myeloid progenitors in the formalin-fixed, paraffin-embedded BM tissue biopsy specimens, collected 2 days before death and at autopsy, were PARV4/5 negative. While the PARV4/5 viremia, in the absence of other known viral agents, suggests a possible contribution of this novel parvovirus to the patient’s clinical syndrome, the absence of the virus in the BM cells suggests that its in vivo tropism may markedly differ from that of B19. In conclusion, although the frequency of PARV4/5 viremia is very low in the general Italian population, it is slightly higher in certain subgroups of iatrogenically immunosuppressed patients and it is not clear to which extent immunosuppression enhances viral reactivation and/or primary infection. Failure to detect PARV4/5 DNA in all but 4 study patients does not necessarily indicate a rarity of past viral exposure or infection in transplant patients or indeed in the general population. Further studies are needed to confirm a possible pathogenic role of PARV4/5 infection,

Indirect antitumor effects of mammalian target of rapamycin inhibitors against Kaposi sarcoma in transplant patients.

of urine leakage around the bladder. Radiological imaging demonstrated leakage at the vesico-ureteral anastomosis. After insertion of a percutaneous nephrostomy catheter and later a double J-stent, the patient recovered and the anastomosis healed. As illustrated in Figure 1, serum creatinine values increased during the urine leakage, whereas the serum cystatin C concentration and the cystatin C-based estimeated GFR (eGFR) (5) indicated normal renal function. Creatinine clearance calculated from 24-hr urine was even lower than the Schwartz eGFR during urinary leakage (6).

Mucorales-Specific T Cells in Patients with Hematologic Malignancies

Background Invasive mucormycosis (IM) is an emerging life-threatening fungal infection. It is difficult to obtain a definite diagnosis and to initiate timely intervention. Mucorales-specific T cells occur during the course of IM and are involved in the clearance of the infection. We have evaluated the feasibility of detecting Mucorales-specific T cells in hematological patients at risk for IM, and have correlated the detection of such cells with the clinical conditions of the patients. Methods and Findings By using an enzyme linked immunospot assay, the presence of Mucorales-specific T cells in peripheral blood (PB) samples has been investigated at three time points during high-dose chemotherapy for hematologic malignancies. Mucorales-specific T cells producing interferon-γ, interleukin-10 and interleukin-4 were analysed in order to detect a correlation between the immune response and the clinical picture. Twenty-one (10.3%) of 204 patients, accounting for 32 (5.3%) of 598 PB samples, tested positive for Mucorales-specific T cells. Two groups could be identified. Group 1, including 15 patients without signs or symptoms of invasive fungal diseases (IFD), showed a predominance of Mucorales-specific T cells producing interferon-gamma. Group 2 included 6 patients with a clinical picture consistent with invasive fungal disease (IFD): 2 cases of proven IM and 4 cases of possible IFD. The proven patients had significantly higher number of Mucorales-specific T cells producing interleukin-10 and interleukin-4 and higher rates of positive samples by using derived diagnostic cut-offs when compared with the 15 patients without IFD. Conclusions Mucorales-specific T cells can be detected and monitored in patients with hematologic malignancies at risk for IM. Mucorales-specific T cells polarized to the production of T helper type 2 cytokines are associated with proven IM and may be evaluated as a surrogate diagnostic marker for IM.

Assessment of Aspergillus-Specific T Cells for Diagnosis of Invasive Aspergillosis in a Leukemic Child with Liver Lesions Mimicking Hepatosplenic Candidiasis

ABSTRACT A child with acute myeloid leukemia presented with multiple liver lesions mimicking hepatosplenic candidiasis during the neutropenic phase following the induction chemotherapy. All the available diagnostic tools showed repeatedly negative results, including galactomannan. An enzyme-linked immunospot (ELISPOT) assay showed a high number of Aspergillus-specific T cells producing interleukin-10 [TH2(IL-10)] and a low number of Aspergillus-specific T cells producing gamma interferon [TH1(IFN-γ)], revealing invasive aspergillosis (IA) before the confirmatory biopsy. A progressive skewing from the predominance of TH2(IL-10) to a predominance of TH1(IFN-γ) was observed close to the complete resolution of the infection and foreshadowed the outcome. The ELISPOT assay holds promise for diagnosing pediatric IA.

May the indirect effects of CIHHV-6 in transplant patients be exerted through the reactivation of the viral replicative machinery?

We read with great interest the article by Lee et al. (1) about the clinical significance of the human herpesvirus 6 integrated in host chromosome (CIHHV-6) in liver transplant patients. The authors show that CIHHV-6 may be associated with higher rates of allograft rejection and opportunistic infections, previously attributed only to active HHV-6 infection, possibly as a result of viral immunomodulation (1, 2). Unfortunately, no data have so far been reported on how such an immunomodulatory activity may be exerted, in particular whether it may be mediated by the activation of the replicative machinery of the integrated genome. Of note, it has recently been demonstrated that CIHHV-6 may actively replicate upon chemical stimuli and infect permissive cells, at least in vitro (3). We would like to discuss the issue of the pathogenicity of CIHHV-6 by adding new experimental data and by extending the clinical follow-up of a previously reported patient with the variant A CIHHV-6, undergoing an allogeneic stem-cell transplantation (allo-SCT) from an unrelated donor for a high-risk myelodysplastic syndrome (4). At day 275 posttransplant, the patient underwent a bone marrow (BM) biopsy because of severe leukopenia and increasing HHV-6A DNA plasma values. Graft-versus-host disease manifestations were absent and either peripheral or BM blood donor chimerisms resulted almost 100% (Fig. 1A). The reduction of HHV-6 loads would be expected with improving donor chimerism in alloSCT patients, with such a CIHHV-6 donor/recipient combination (5). The biopsy demonstrated a reduced BM cellularity without signs of myelodysplasia. After antiviral treatment, leukocytes recovered and HHV-6 loads reduced. A second similar episode was recorded at day 342 and again resolved with the administration of antiviral therapy. Long-term BM cultures (LTBMCs) were performed with the BM-derived cells from the patient, either alone (LTBMC1) or further allogeneically stimulated with BM-derived cells from another histoincompatible subject (LTBMC2) (6). Either the human leukocyte antigen-unrelated SCT donor or the histoincompatible subject was HHV-6B seropositive. After 48 hr, nonadherent cells have been removed, and immunohistochemical analysis with a mouse monoclonal antibody against the HHV-6A early protein (gp41/38) (Advanced Biotechnologies) was performed, as described (4), on ethanolfixed cells either at days 10, 30, and 50 of LTBMC1 or at days 17, 32, and 57 of LTBMC2. The gp41/38 antibody highlighted cytoplasmic reactivity in some BM adherent cells either at day 10 of LTBMC1 or at day 32 of LTBMC2, confirming their productive infection with HHV-6A (Fig. 1B, C). Unfortunately, the same analysis on formalin-fixed paraffin-embedded BM biopsy specimens resulted negative, possibly due to the antiviral treatment, reducing HHV-6 gene expression, in vivo. These results demonstrate that CIHHV-6 may produce proteins, at least in ex vivo culture, possibly as a result of the allogeneic reaction between the patient’s cells and those of either his human leukocyte antigen-unrelated SCT donor or the histoincompatible subject. It has been reported that human cytomegalovirus, a beta herpesvirus closely related to HHV-6, may reactivate from latency in peripheral blood mononuclear cells after allogeneic stimulation (6). In our case, the possibility that the protein expression comes from a superinfecting episomal copy is very unlikely because (a) HHV-6A latency is unlikely to involve the formation of viral episomes (3) and (b) either the SCT donor or the histoincompatible subject was latently infected with the HHV-6B. These findings cast doubt on a possible CIHHV-6 reactivation as culprit in the patient’s two episodes of leukopenia with increased HHV-6 loads, at day 263 and 342 posttransplant, respectively, also when considering that both episodes resolved after the administration of antivirals. In conclusion, Lee’s, Arbuckle’s, and our data challenge the belief that CIHHV-6 could be an inert genome and only represents a confounding element in the diagnosis of HHV-6 active infection (1, 3, 7). Alternatively, as it may be supposed that HHV-6 may exert its pathogenic potential through either full replication or the sole expression of viral proteins, we believe that transplant clinicians should be alerted to the possibility of HHV-6-associated diseases/indirect effects in their CIHHV-6 patients and also consider the administration of antivirals in those patients with signs and symptoms consistent with viral infection, when all the other causes have been extensively and repeatedly ruled out. The observation by Lee et al. (1) that the only CIHHV-6 patient undergoing valganciclovir prophylaxis developed neither rejection nor opportunistic infections may spur investigations on whether the administration of antiviral prophylaxis with an HHV-6 active agent may reduce the occurrence of HHV-6 indirect effects in patients with CIHHV-6. Further in vivo studies are needed to clearly define the replicative potential of CIHHV-6.