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Large clonal expansions of peripheral CD8+ T cells carrying receptors for single epitopes of CMV and EBV are common in the elderly and may be associated with an immune risk phenotype predicting mortality. Here we show that the frequency of CD8+ T cells expressing the inhibitory killer cell lectin-like receptor G1 (KLRG1), a marker of cells unable to undergo further clonal expansion, was markedly elevated in CD8+ T cells from old donors. Moreover, tetramer staining revealed that the elevated frequency of CMV-specific CD8+ T cells in the elderly was due to an accumulation of cells bearing this dominant negative receptor. The fraction of CMV-specific T cells able to secrete interferon-gamma after specific antigenic stimulation was significantly lower in the elderly than in the young, although the total number of functional cells was comparable. Therefore, the majority of the clonally expanded virus-specific CD8+ cells in the elderly was dysfunctional. Thus, T cell responses are altered in the aged by an accumulation of replicatively senescent dysfunctional T cells carrying receptors for persistent herpes viruses. The presence of clonal expansions of such virus-specific cells may shrink the available repertoire for other antigens and contribute to the increased incidence of infectious disease in the elderly.

PCR-based preemptive therapy with ganciclovir has been shown to reduce the incidence of CMV disease after BMT. Failures of this treatment strategy are CMV disease and secondary non-viral infections. Eighty-six consecutive patients at high risk for CMV disease who received PCR-based preemptive therapy with ganciclovir were assessed for treatment failures and possible risk factors. Ganciclovir was initiated in 57 of 86 patients (66%). Only 28 of 86 (32%) patients received 4 or more weeks of ganciclovir. Recurrence of CMV infection after successful treatment was more frequent among recipients of a BMT from an unrelated compared to a sibling donor (P = 0.004). Three (3.5%) patients developed non-fatal early onset CMV disease and seven of 68 (10.3 %) late onset CMV disease (>100 days post transplant). Risk factors for late onset CMV disease were cGVHD (P = 0.0017) and duration of prior antiviral therapy >4 weeks (P = 0. 0073). The incidence of secondary non-viral infections was 28% with the duration of antiviral treatment being a significant risk factor for secondary bacterial (P = 0.0045) and invasive fungal infections (P = 0.006). Thus, PCR-based preemptive treatment with ganciclovir reduces early onset CMV disease, but the duration of antiviral therapy prior to day +100 is a significant risk factor for late onset CMV disease as well as secondary non-viral infections.

Cytomegalovirus (CMV) disease is associated with a high mortality in recipients of an allogeneic stem cell transplant. Apparent differences in biological behaviour have been noted among clinical CMV isolates. By amplifying specific functionally relevant regions of the CMV genome [immediate early (IE) exon 3, glycoprotein B (gB)], a possible association of strain variation and clinical symptoms of infection was analysed in 24 patients. A high number of genome mutations of the IE exon 3 region could be documented translating into amino acid changes of viral isolated of 8 out of 15 patients with symptomatic and 2 out of 9 patients with asymptomatic CMV infection. Identical IE mutations and gB types were observed in isolates from two different sites in 6 patients. gB strain 2 was found to be associated with symptomatic CMV infection (P = 0.03). Thus, apart from host factors viral factors might influence the virus-host interaction in severely immunosuppressed patients.

Thirty patients undergoing allogeneic BMT were screened post-transplant together with their marrow donors for CMV-specific T cell proliferation and the occurrence of CMV disease. Twenty-one of these patients received a marrow transplant from an HLA-matched sibling donor, and nine from an HLA-matched unrelated donor. All these patients were either CMV seropositive and/or had received a transplant from a CMV-seropositive donor. Patients were monitored for CMV-viraemia until day +100 post-BMT by PCR and virus culture, and thereafter by virus culture only when clinically indicated. The proliferative T cell response was investigated at regular monthly intervals beginning on day +30. A proliferative response to HCMV (median, day +123) was documented in these patients between day +37 and +730 post-BMT. None of the patients with a documented CMV-specific T cell proliferation on day 120 (n = 17) developed CMV disease in the later post-transplant period, but of the patients lacking CMV-specific proliferation (n = 13), 30.8% developed CMV disease after day 120. Thus, patients lacking a CMV-specific T-helper cell response might benefit from sensitive screening for CMV infection and pre-emptive therapy after day +100.

Sensitive screening for cytomegalovirus (CMV) by polymerase chain reaction (PCR) following autologous bone marrow or peripheral blood progenitor cell transplantation has not been evaluated. In a three-center study, 98 autograft transplant recipients were prospectively screened for CMV infection by PCR and culture techniques. At a median of 20 days (range, 3-28) after transplantation, 21 (39.6%) of 53 CMV-seronegative patients were PCR positive for CMV, and at a median of 17 days (range, 7-84) after transplantation, 19 (42.2%) of 45 CMV-seropositive patients were PCR positive for CMV. Low-level DNAemia (1-10 fg CMV DNA/mL blood) occurred for 1 week in 31 patients but was never associated with CMV disease. Of 9 patients who presented with at least two consecutive positive PCR results, 1 developed CMV pneumonia. No patients died because of CMV disease. Screening for CMV infection by PCR had a negative predictive value of 100% (as also observed after allogeneic transplantation), but its positive predictive value was significantly lower.

Recent findings indicate that the kinetics of B-cell reconstitution after marrow transplantation mimic normal ontogeny. The early B-cell repertoire during ontogeny is characterized by a high degree of autoreactivity and interconnectivity. Therefore, in a prospective analysis, 95 consecutive recipients of an allogeneic marrow transplant were screened for the occurrence of various autoantibodies and 47 of these 95 were also screened for monoclonal gammopathies. None of the patients developed antibodies specific for systemic autoimmune disorders. In contrast, a high prevalence of natural antibodies (79/95) was found early post-transplant, with 58 of these 79 patients developing two or more autoantibodies. According to multiple regression, the mean number of natural antibodies (95% confidence limits in parentheses) depends significantly (P = 0.006) on the status of CMV infection: 0.9 (0.4; 1.6) CMV-negative: 2.0 (1.0; 3.3) asymptomatic CMV infection; 3.1 (1.7; 5.0) CMV disease. Sex, age, underlying disease, conditioning therapy, acute graft-versus-host disease and CMV serology of donor and recipient pretransplant did not affect the number of natural autoantibodies. Monoclonal gammopathies were detected in 12/47 patients with a predominance of the IgG-kappa subtype. All these 12 patients suffered from a viral infection (CMV, n = 11: influenza strain A, n = 1). The high degree of self-reactivity post-transplant further supports the hypothesis that B-cell reconstitution mimics ontogeny. Moreover, these data indicate nonspecific polyclonal, CMV-mediated, presumably T-cell independent B-cell stimulation and disturbed T-cell regulatory function following allogeneic BMT.

Culture-based preemptive therapy with ganciclovir was shown to reduce the incidence of cytomegalovirus (CMV) disease after bone marrow transplantation (BMT). Culture techniques did not detect CMV in 12% to 13% of patients before the onset of CMV disease. In a prospective study, 71 patients either received preemptive therapy based on polymerase chain reaction (PCR) technique (37 patients) or on culture assays (34 patients). In both groups, therapy was continued until clinical signs disappeared and PCR negativity was documented. Twenty-two patients in the PCR group and 15 patients in the culture group received antiviral therapy. PCR allowed detection of the virus (median day, +32 v day +49; P = .006) and introduction of antiviral therapy (median day, +44 v day +54; P = .02) earlier than did culture assays. The incidences of CMV disease (2 of 37 v 8 of 34 in PCR group v culture group, respectively; P = .02) and CMV-associated mortality (0 of 37 v 5 of 34 in PCR group v culture group, respectively; P = .02) were decreased, and the duration of ganciclovir therapy (P < .001) was shorter in the PCR-monitored group. Incidence and median duration of severe neutropenia (less than 500/microL) were lower in the PCR group (two v eight episodes, P = .02; median duration, 1.5 v 5 days, P = .04), as was the incidence of nonviral infections during/after antiviral therapy (2 of 37 v 9 of 34; P = .012). Thus, preemptive therapy based on more sensitive detection methods such as the PCR assay reduces the incidence of CMV disease and CMV-related mortality. Additionally, stopping and withholding antiviral therapy in a PCR-negative patient is safe and allows reduction of the duration and side effects of antiviral therapy.

In a retrospective analysis lung biopsy specimens obtained postmortem from 30 consecutive allogeneic bone marrow transplant recipients who had died of either either interstitial pneumonitis (IP; 18/30 patients) or various other causes (12/30 patients) were studied for the local presence of human cytomegalovirus (HCMV) by culture, in situ hybridization, polymerase chain reaction (PCR) and immunohistochemistry for HCMV proteins. All patients suffering from IP were found to be HCMV positive in the lung biopsy. PCR revealed the highest sensitivity for HCMV detection in lung biopsies, but in 15/18 PCR-positive samples local HCMV infection could be confirmed by at least one additional technique. All the lung biopsies obtained from the 12 patients without IP were negative for HCMV by all techniques applied, except one with a weak HCMV-DNA signal in the PCR assay. The severity of the clinical, as well as histological and immunohistological alterations in the lung did not correlate with the amount of HCMV-DNA or the number of HCMV-positive cells detected in the biopsy. An increase of HLA-class II antigen and of ICAM-1 expression on the alveolar epithelium, as well as presence of activated CD8+ or CD4+ lymphocytes infiltrating only HCMV-positive lung biopsies revealed T cell-mediated immune reactions to be involved in the pathogenesis of IP. Since all analyzed patients presented with severe acute or extensive chronic graft-versus-host disease (GvHD), but only those with pulmonary HCMV infection developed IP, dissemination of HCMV appears to be the primary requirement for the initiation of IP. GvHD, however, may interfere with normal control of subsequent antiviral immune response and, thus, provoke the immunopathology of IP.

Sixty-three recipients of an allogeneic marrow transplant were screened for the occurrence of cytomegalovirus (CMV) infection and clinical parameters possibly predicting the development of CMV disease in a retrospective study. Blood and urine samples obtained from these patients were screened weekly after bone marrow transplantation (BMT) for the presence of CMV by polymerase chain reaction (PCR) and virus culture technique. Forty-six of the 63 patients studied were found to be CMV-positive by PCR technique in blood and urine samples at a median of 29 days after BMT. In 33 of these 46 patients, CMV could be cultured from urine samples and 16 of the 46 had culture-positive viremia. Twenty-eight of these 46 PCR-positive patients developed CMV disease. Whereas PCR assays showed an optimal negative predictive value and sensitivity for the development of CMV disease, their positive predictive value was 61% and could not be remarkably increased when culture-proven viruria (64%) and viremia (69%) were considered. Acute graft-versus-host disease (GVHD) grade 2 to 4 (P < .05), but not underlying disease, conditioning therapy, or GVHD prophylaxis, was associated with CMV infection. On day +49, a remarkable decrease (P < .001) in the lymphocyte count, as well as in the absolute number of CD4+, CD8+, and CD56+ lymphocytes, occurred only among the patients who later developed CMV disease. The decrease of all of these cell counts, but predominantly the CD4+ T cells, to less than 100/microL on day +49 after BMT showed a very high positive predictive value (100%) for the development of CMV disease in patients with PCR-proven viremia. Persisting CD4 lymphopenia after antiviral therapy was only observed in patients who finally died of CMV disease. Thus, immunophenotyping of the patients after BMT in addition to a highly sensitive virus detection assay might help to identify patients at high risk to develop CMV disease and indicate the need for additional adoptive immunotherapy.

HCMV infection diagnosed by the highly sensitive polymerase chain reaction (PCR) technology in blood, urine and skin biopsies of patients after bone marrow transplantation (BMT) correlated with the reconstitution of peripheral blood lymphocytes and dermal immunohistological alterations to evaluate the interaction of viral infection with the recovery of the immune system, as well as with the induction or aggravation of graft-versus-host disease (GVHD). In a prospective study 73% of 63 patients showed viremia at a median time of 25 days after BMT. Only 44% of these cases that also presented with a higher frequency of acute GVHD symptoms developed HCMB disease later on. In the skin, similar immunohistological alternations, as well as frequent primary local HCMV infection before the development of cutaneous signs of GVHD, was found, suggesting the direct involvement of anti-HCMV immune responses in the induction of GVHD-associated organ lesions.

Human cytomegalovirus (HCMV) has been suspected to participate in the pathogenesis of IgA nephropathy (IgAN). However, with regard to the presence of HCMV in the renal tissue of IgAN, conflicting results have been reported using a variety of different techniques. Renal biopsies of 29 patients with IgAN, of 7 with focal segmental glomerulosclerosis (FSGS) and of 11 normal kidneys were analyzed for the presence of HCMV-DNA using the polymerase chain reaction. HCMV-DNA was detected by hybridization with digoxigenin-labelled probes in 14 of 19 analyzed frozen renal biopsies from patients with IgAN. However, only 1 of 17 renal biopsies of IgAN embedded in paraffin was positive for HCMV-DNA. Furthermore, HCMV-DNA was detected in 4 of 18 frozen normal kidneys but in none of the tissues from patients with FSGS. The present results provide further evidence of an association between the presence of HCMV in renal tissue and IgAN.

A sensitive and specific method to monitor suppression of cytomegalovirus (CMV) replication is essential in patients treated with ganciclovir after allogeneic bone-marrow transplantation. In this study, antiviral therapy of eighteen episodes of symptomatic CMV infection in 15 such patients were followed up clinically and by virus culture and polymerase chain reaction (PCR). Clinical improvement, culture, and PCR were assessed for their ability to predict the efficacy of ganciclovir therapy in each patient. In eleven successfully treated episodes of CMV disease (disappearance of symptoms and improvement in biochemical variables) clinical improvement was associated with an effective suppression of virus replication as shown by negative culture and PCR assays of blood and urine specimens obtained after antiviral therapy. 1 patient who did not improve clinically when receiving antiviral therapy remained both culture positive and PCR positive for CMV. 6 patients with early relapse of CMV disease or who died after an initial clinical improvement were PCR positive but culture negative after termination of therapy. Demonstration of CMV in blood and urine by PCR after stopping antiviral therapy (even when culture is negative) points to incomplete suppression of virus replication. The findings show that PCR is a better predictor of the efficacy of antiviral therapy than are culture or clinical assessment.

Twenty-eight patients undergoing bone marrow transplantation (BMT) were followed-up at weekly intervals from day -10 to discharge from hospital after BMT for human cytomegalovirus (HCMV) infection using polymerase chain reaction (PCR), slot-blot hybridization, and conventional virus culture. High specificity of the PCR assay applied could be shown by failure to amplify DNA extracted from a wide range of other viruses frequently infecting marrow transplant recipients. The PCR technique allowed us to diagnose viremia and viruria in 20 (83%) of 24 seropositive patients after BMT, whereas culture assays showed 16 (67%) of 24 of these patients to be viruric and 9 (37%) of 24 cases to be viremic. Slot-blot hybridization showed a frequency of viruria and viremia in 12 (50%) of 24 seropositive patients. By application of PCR techniques, HCMV detection could be achieved even in the very early posttransplant period. HCMV was detected in five patients even before the onset of clinical symptoms of acute graft-versus-host disease. Analysis by PCR techniques of 33 organ biopsy specimens from patients after BMT showed the presence of HCMV in 13 of 14 liver samples obtained from patients with HCMV viremia; three liver specimens from patients without viremia were negative by all the techniques applied. HCMV could also be demonstrated in postmortem lung biopsy specimens from all patients (n = 10) with interstitial pneumonia.

A total of 317 different clinical samples obtained from patients following bone marrow transplantation and 56 blood and urine samples from seronegative control persons were screened for the presence of human cytomegalovirus (CMV) using virus culture and slot-blot hybridization. Immunohistochemical techniques using monoclonal antibodies to various viral antigens and in situ hybridization techniques were also utilised for detection of CMV in tissue samples. In cell suspensions of blood, bone marrow and effusions, and in liver biopsies, CMV DNA could be demonstrated more often by slot-blot and in situ hybridization techniques than by virus culture or immunostaining of viral antigens. For detection of CMV in lung biopsies and other clinical samples containing mainly cell-free virus, such as urine, bronchial lavage and throat washings, virus culture was found to be at least as sensitive as the hybridization techniques. Immunostaining proved to be a fast and sensitive technique for detection of CMV in tissues and may thus provide additional information about viral replication and clinical relevance of the virus infection.

Hybridization techniques (slot-blot and in-situ hybridization assays) and immunostaining using murine monoclonal antibodies directed against different proteins of the human cytomegalovirus (HCMV) were compared for their sensitivity and specificity for detection of HCMV. A model system with HCMV infected human embryonic lung fibroblasts and lung biopsy specimens obtained from patients with culture positive HCMV interstitial pneumonia were used for evaluation of these techniques. The hybridization techniques were found to provide an improved sensitivity compared to immunostaining. Additionally a good correlation was found between the virus dose determined by TCID50 and the amount of viral DNA detected by slot-blot hybridization and by the number of autoradiographic silver grains per 100 cells per 2 weeks exposure time detected in the infected fibroblasts by in-situ hybridization. Thus, at least in the model system quantification of the virus was achieved by hybridization assays.

Bone marrow transplantation was performed between IV/82 and X/85 in 64 patients with acute leukemia (n = 36), chronic myelogenous leukemia (CML; n = 13), severe aplastic anemia (n = 12), and neuroblastoma stage IV (n = 3). Of these patients 57 received allogeneic marrow from HLA-ABCDR identical, MLC-negative sibling donors. Six transplants were performed with syngenic marrow and one with autologous marrow. Of the 64 patients 48 survived 40-1,250 days after transplantation, resulting in a survival rate (SR) of 75% and a survival probability (SP) of 71%. Of the 36 patients suffering from acute leukemia (SR = 64%, SP = 51%), patients with acute myelogenous leukemia (AML) in first complete remission (n = 11; SR = 81%, SP = 76%), as well as patients with acute lymphatic leukemia (ALL) in 1st to 4th complete remission at the time of transplantation (n = 14; SR = 81%, SP = 76%) show a favorable prognosis. A poor survival rate was seen for patients with AML when transplanted in second or partial remission (1/5; SR = 20%), as well as for patients suffering from ALL and transplanted during relapse or partial remission (1/6; SR = 16%). Of 13 patients suffering from CML 12 survived the transplantation free of relapse (SR = 93%, SP = 92%), and one patient died from varicella zoster pneumonia. Of the transplanted patients with severe aplastic anemia, 12 of 13 are surviving with complete hematologic reconstitution; one patient, however, died on day 10 from a sepsis. In our patient group, the SR as well as the SP has been improved through changes in the irradiation protocol concomitant with prophylactic application of anti-CMV hypergammaglobulin, as well as through additional oral medication of Azyklovir. The 41 patients (BMT No. 7-47) with total body irradiation at one time show an SR of 44% and an SP of 41%. The following 46 patients (BMT No. 48-93) have reached an SR of 83% and an SP of 74% under the regimen of fractionated total body irradiation, plus prophylaxis with anti-CMV hypergammaglobulin and Azyklovir. Within this group, no fatal CMV pneumonia was encountered as opposed to six patients lost from CMV pneumonia in the first group.