Decreased functional T lymphocyte-mediated cytokine responses in patients with chemotherapy-induced neutropenia.

OBJECTIVES: The degree of immunosuppression in patients with haematological malignancies treated with chemotherapy is routinely measured as number of circulating cells (preferable neutrophils) in peripheral blood. A parallel decline in the number of T cells is expected, but a possible alteration in their functionality has been less well explored. The ability of T cells to secrete more than one cytokine simultaneously is known to indicate protective immunity. The aim of this study was to determine whether the function of circulating T cells is altered in patients with chemotherapy-induced neutropenia. DESIGN, SETTING AND SUBJECTS: In this cross-sectional study, we used the FluoroSpot assay to investigate the proportion of T cells secreting either interferon-γ or interleukin-2, or both cytokines simultaneously, after anti-CD3 stimulation. Peripheral blood mononuclear cells from 53 adult patients with chemotherapy-induced neutropenia and 20 healthy individuals were investigated. RESULTS: There were significantly fewer T cells secreting interferon-γ in patients with neutropenia compared with healthy control subjects (P = 0.02), but the difference was greatest for dual cytokine-secreting T cells (P = 0.001). Furthermore, the amount of secreted cytokine per T cell appeared to be reduced in patients, compared with control subjects. CONCLUSION: Our results suggest that the functionality of T cells is altered in patients with haematological malignancies with chemotherapy-induced neutropenia. In parallel with a decline in T cell count, this may further increase the risk of severe infections.

Antibody-dependent cellular cytotoxicity to clinical isolates of HIV-1 and SIVcpz: comparison of human and chimpanzees.

OBJECTIVE: To investigate differences in the antibody-dependent cellular cytotoxicity (ADCC) responses between HIV-1-infected humans and chimpanzees. DESIGN: The breadth of the ADCC responses in the two populations were tested against autologous and heterologous HIV-1 and SIVcpz clinical isolates as well as against reference isolates. METHODS: ADCC was tested in a 51Cr-release assay using human peripheral blood mononuclear cells as effector cells and infected Jurkat/Tat-cells as target cells. RESULTS: The majority of sera from chronically HIV-1-infected humans and chimpanzees had ADCC responses to HIV-1LAI. Interestingly, vaccinated chimpanzees with a low virus load during the immediate post-challenge period had low ADCC responses 3 years after challenge. In contrast, when ADCC activity to clinical isolates was evaluated, HIV-1-infected chimpanzees had more frequent heterologous (broader) responses than HIV-1-infected humans. ADCC was also tested in consecutive serum samples from two patients and two chimpanzees against autologous isolates, but was only detected to a low degree in one of the animals, although heterologous ADCC was demonstrated in all cases. The naturally infected (SIVcpz) chimpanzee did not have detectable heterologous or autologous ADCC responses. CONCLUSIONS: HIV-1-infected chimpanzees had broader ADCC reactivity to heterologous HIV-1 clinical isolates than the HIV-1-infected humans. These findings are consistent with subtle differences in host-virus relationships of these two species.

Seroprevalence of viral childhood infections in Eritrea.

BACKGROUND: The seroprevalence of viral childhood infections in Africa has not been thoroughly investigated. The relatively recently discovered human parvovirus B19 (B19) and human herpesvirus 6 (HHV-6) have received particularly little attention. OBJECTIVE: To investigate the seroprevalence of viral childhood infections in different Eritrean populations and to define groups at high risk for infection. STUDY DESIGN: Five population groups in Eritrea have been examined to define the prevalence of specific antibodies to several childhood viruses. The study population of more than 400 persons consisted of children, pregnant women, female sex workers and members of a secluded tribe called Rashaida. RESULTS: All groups showed a high prevalence of antibodies to measles and HHV-6 (> 85%). For rubella, the seroprevalence was very high in all adult groups (93-99%) except the Rashaida group (71%). The mumps prevalence was surprisingly low in the Rashaida group (29%) compared to 46-85% in the other adults. Late encounter of mumps and rubella was also observed among the Rashaidas. The pattern of antibodies to B19 showed a higher seroprevalence in all groups (56-91%) compared to what has been reported from the western world. CONCLUSION: The findings represent what might be expected in an unvaccinated population. The exception was the Rashaidas, which had low seroprevalences and late encounter of mumps and rubella. This is of importance because it makes this tribe vulnerable to these infections, which are associated with complications when acquired in adult age. Also noteworthy is the high frequency of antibodies to HHV-6 and particularly B19 in all groups, indicative of an early encounter of both these viruses.

Seroprevalence of human herpes virus 8 in different Eritrean population groups.

BACKGROUND: Human herpesvirus 8 (HHV-8) is associated with Kaposi's sarcoma. In the US and Europe, HHV-8 is believed to be mainly sexually transmitted, but reports from some African countries suggest non-sexual transmission. OBJECTIVES: To find out more about HHV-8 seroprevalence and transmission in Eastern Africa. STUDY DESIGN: In this study, 411 serum samples from different population groups in Eritrea (children, pregnant women, female sex workers and members of the isolated Rashaida tribe) were examined for HHV-8 antibodies with an immunofluorescence assay detecting antibodies to latent and lytic HHV-8 antigens. RESULTS: Antibodies to HHV-8 latent antigen were found in 0-2% of Eritrean children, 5% of pregnant women, 8% of female sex workers and 26% of Rashaidas, respectively. No correlation was found between detectable HHV-8 antibodies and seropositivity to HIV or herpes simplex 2. CONCLUSIONS: These results suggest that HHV-8 infection is relatively common in Eritrea and that viral transmission occurs predominantly through non-sexual route in this region.

[Parvovirus B19 infection--an insidious chameleon]. / Parvovirus B19-infektion--en lömsk kameleont.

Parvovirus B19 is a common source of infection with a seroprevalence of 60-70 per cent in the adult population. The most common manifestation is erythema infectiosum ('fifth disease'), with exanthem, fever and upper airway symptoms in children. The infection can give rise to a multifacetted clinical picture and is probably underdiagnosed, particularly in risk groups (individuals with haemolytic anaemia or immunosuppression, and fetuses). Serological diagnosis can now be complemented with the demonstration of viral DNA using the PCR (polymerase chain reaction) test in various body fluids, or tissue biopsy. Recent years have witnessed manifest increase in clinical knowledge of parvovirus B19-associated complications, and their diagnosis and treatment.

Cytokine responses in acute and persistent human parvovirus B19 infection.

The aim of this study was to characterize the proinflammatory and T helper (Th)1/Th2 cytokine responses during acute parvovirus B19 (B19) infection and determine whether an imbalance of the Th1/Th2 cytokine pattern is related to persistent B19 infection. Cytokines were quantified by multiplex beads immunoassay in serum from B19-infected patients and controls. The cytokine responses were correlated with B19 serology, quantitative B19 DNA levels and clinical symptoms. In addition to a proinflammatory response, elevated levels of the Th1 type of cytokines interleukin (IL)-2, IL-12 and IL-15 were evident at time of the initial peak of B19 viral load in a few patients during acute infection. This pattern was seen in the absence of an interferon (IFN)-gamma response. During follow-up (20-130 weeks post-acute infection) some of these patients had a sustained Th1 cytokine response. The Th1 cytokine response correlated with the previously identified sustained CD8+ T cell response and viraemia. A cross-sectional study on patients with persistent B19 infection showed no apparent imbalance of their cytokine pattern, except for an elevated level of IFN-gamma response. No general immunodeficiency was diagnosed as an explanation for the viral persistence in this later group. Neither the acutely infected nor the persistently infected patients demonstrated a Th2 cytokine response. In conclusion, the acutely infected patients demonstrated a sustained Th1 cytokine response whereas the persistently infected patients did not exhibit an apparent imbalance of their cytokine pattern except for an elevated IFN-gamma response.

Clinical aspects of parvovirus B19 infection.

Parvovirus B19 is a significant human pathogen that causes a wide spectrum of clinical complications ranging from mild, self-limiting erythema infectiosum in immunocompetent children to lethal cytopenias in immunocompromised patients and intrauterine foetal death in primary infected pregnant women. The infection may also be persistent and can mimic or trigger autoimmune inflammatory disorders. Another important clinical aspect to consider is the risk of infection through B19-contaminated blood products. Recent advances in diagnosis and pathogenesis, new insights in the cellular immune response and newly discovered genotypes of human parvoviruses form a platform for the development of modern therapeutic and prophylactic alternatives.

A highly restricted T-cell receptor dominates the CD8+ T-cell response to parvovirus B19 infection in HLA-A*2402-positive individuals.

Six of seven HLA-A*2402-positive individuals with acute parvovirus B19 infections made vigorous CD8-positive cytotoxic T-cell (CTL) responses to the viral epitope FYTPLADQF. All responders showed highly focused T-cell receptor (TCR) usage, using almost exclusively BV5.1. The BV5.1 TCR dominated the acute response, was maintained over time, and was also used by a remotely infected individual. Nine CTL clones and two oligoclonal lines obtained from three unrelated individuals used BV5.1, BJ2.1, and a conserved TCR CDR3 of nine amino acids. This commonly recognized epitope is likely important in long-term protective immunity and should be included in vaccine design.

Evaluation of serological assays for identification of parvovirus B19 immunoglobulin M.

Three different enzyme immunoassays (EIAs) (Parvoscan-B19, IBL parvovirus B19, and IDEIA parvovirus B19) and one immunofluorescence assay (Biotrin Parvo B19 IFA) were evaluated for detection of parvovirus B19 immunoglobulin M (IgM) antibodies in 203 clinical serum samples. An IgM antibody capture radioimmunoassay was used as a reference test. Serum specimens obtained from patients with clinical symptoms suggestive of parvovirus B19 infections were used to evaluate the sensitivities of the assays, which were shown to be comparable for the Biotrin IFA and IDEIA (97%) and lower for the other two EIAs (90%). In order to test the specificity of the assays, clinical serum samples with IgM antibodies against other viruses were examined, as well as sera with rheumatoid factor activity and sera from healthy pregnant women. The specificities of B19 IgM antibody detection were 96% for the Biotrin IFA, 96% for IDEIA, 90% for Parvoscan, and 88% for the IBL assay. These results show that all four assays can be recommended for diagnostic purposes, although false-positive results may be seen with other acute viral infections, healthy pregnant women, and rheumatoid factor-positive samples.

Persistent B19 parvovirus infection in pediatric malignancies.

BACKGROUND AND PROCEDURE: The frequency and clinical importance of parvovirus B19 infection were studied in children investigated or treated for various malignancies and cytopenias. RESULTS: B19 infection was thus demonstrated in six out of 53 unselected children with malignancies by bone marrow examination, using the B19, DNA-specific, polymerase chain reaction (PCR). Examinations using the PCR in serum samples were equally or less sensitive than in bone marrow samples. One of the children had a persistent B19 infection during maintenance therapy for acute lymphoblastic leukemia. She developed a prolonged and severe cytopenia, and the clinical signs included facial rash, chills, high undulating fever, and pharyngitis. She also seroconverted and became B19 IgM-antibody positive during the study period. CONCLUSIONS: Parvovirus B19 infection was detected in 10% of the children and was either asymptomatic or was associated with severe and prolonged cytopenia. Bone marrow examinations are recommended for the detection of B19 DNA in immunosuppressed children.

No association between human parvovirus B19 and testicular germ cell cancer.

The incidence of testicular germ cell cancer, which is the most common cancer among young male adults, is increasing. The aetiology remains unknown, although a virus has been proposed. A previous study has shown a high prevalence of human parvovirus B19 (B19) DNA in the testes of patients with testicular germ cell tumours (85%) and suggested that B19 may play a role in tumour development. To address this question of causality, seroreactivity to B19 was studied among cases (n=80) and controls (n=241) using serum samples drawn before the onset of disease, in addition to an elucidation of the frequency of virus DNA in a retrospectively collected 2-year testicular carcinoma series. No association was found between B19 seropositivity and the risk of testicular cancer (odds ratio=1.03; 95% confidence interval=0.60-1.77) nor was there any dose-response relation (P for trend=0.53). This study did, however, confirm the observation that B19 DNA can be detected in testicular carcinoma tissue, as 4 of 24 cases were found to be positive, while no B19 DNA could be detected in the control cases. It is speculated that this finding may be due to susceptibility of the carcinoma cells to B19 virus owing to high-level expression of the viral receptor glycosphingolipid (Gb4) and possible other putative cellular factors resulting in a localized persistence initiated after the development of cancer.

Mapping of B-cell epitopes on human parvovirus B19 non-structural and structural proteins.

In the present study, the immune reactivity to Parvovirus B19 (B19) proteins and variations in antigenic reactivity in different clinical manifestations were investigated. Sera from healthy B19 IgG positive individuals were evaluated for antibody reactivity against linear peptides. Three antigenic regions (amino acid number 191-206, 271-286, 371-386) on the B19 non-structural (NS) protein 1 were identified. The highest seroreactivity against these peptides was found against amino acid number 271-286. Seroreactivity in this group of individuals was also investigated against peptides representing selected neutralising regions of the B19 capsid proteins viral protein (VP) 1/VP2. The antigenic NS1 and VP1/VP2 regions, thus defined, were further mapped by seroreactivity against peptides containing specific deletions. The frequencies of seroreactivity against the NS1 and VP1/VP2 peptides in healthy B19 IgG positive individuals were similar to those in HIV-seropositive and persistently B19 infected patients, except that the latter group showed a lower reactivity to the C-terminal end of VP1/VP2. The identification of antigenic regions and corresponding seroreactivity in asymptomatic and persistently B19-infected patients is important for the understanding of B19-pathogenesis and for the development of B19 vaccine candidates.

Parvovirus B19 infection: association with third-trimester intrauterine fetal death.

OBJECTIVE: To identify the presence of parvovirus B19 infection as a possible cause of fetal loss in the third trimester. DESIGN: Prospective study of women experiencing third-trimester intrauterine fetal death (IUFD). SETTING: All cases of IUFD at Danderyd Hospital from 1992 to 1998. POPULATION: Ninety-three women with IUFD in 33,759 deliveries (0.3%). METHODS: Detection of B19 DNA by polymerase chain reaction (PCR) in placental and fetal tissue. Placental pathology and B19-specific immunohistochemistry. Maternal serology in consecutive samples. RESULTS: Among 93 cases of IUFD, seven (7.5%) had detectable B19 DNA in freshly-frozen placental tissue. The detection of B19 DNA in these tissues was confirmed by detection of B19 DNA in six separately stored paraffin-embedded placental tissues. No other explanations for the fetal deaths were found. None of the women had experienced any clinical signs of infection prior to fetal demise. None of the seven fetuses were hydropic. Histopathologic examination of the placentas revealed only minor abnormalities. Serology on maternal samples at birth revealed delayed or absent B19 IgG responses in five of seven cases. Two women were B19 IgG seropositive at the time of delivery but had unusual infection patterns; persistent viraemia for at least five months before birth in one case and likely persistence or re-infection by B19 in the other. CONCLUSION: In our study, 7.5% of IUFDs in the third trimester may have been caused by parvovirus B19 infection, without signs of fetal hydrops. This finding indicates that B19 PCR should be included in the routine investigation of IUFD.

Clinical and laboratory findings in immunocompetent patients with persistent parvovirus B19 DNA in bone marrow.

The clinical relevance of parvovirus B19 DNA persistence in bone marrow was examined in 10 immunocompetent individuals undergoing examinations for unexplained fever, arthralgia or chronic leukopenia. Common causes of these symptoms had been ruled out and bone marrow aspiration was indicated at this stage of investigation. In addition to morphological analysis of the bone marrow, a test for B19 DNA was performed with 2 nested PCRs. Five of these 10 selected patients had detectable B19 DNA in their bone marrow, whereas no viraemia was observed. Additional bone marrow samples were collected at least 6 months after the first sample from the B19 DNA-positive patients, of whom 3 were found to be still positive. Indeed, 2 of the patients have been positive for more than 5 y of follow-up. Sera from all patients with persistent B19 DNA in bone marrow could neutralize the virus. One patient responded to treatment with immunoglobulin but later relapsed. No other cause of the symptoms was found, despite extensive investigations, and at least some of the prolonged disease manifestations may be due to parvovirus B19.

Prevalence of parvovirus B19 DNA in bone marrow of patients with haematological disorders.

Patients with haematological disorders (n = 100) were examined for prevalence of parvovirus B19 DNA in the bone marrow and serum, irrespective of B19-related symptoms. B19 DNA was studied using 2 nested PCRs and the serum samples were further analysed with B19-specific IgG, IgM and avidity as well as seroreactivity against linear and conformational epitopes of the B19 VP2 antigen. The latter assays specify whether the IgG antibody response represents acute or past B19 infection. B19 DNA was detected in 4 of the 100 bone marrow samples, whereas all the serum samples were B19 DNA negative. None of the 4 B19 DNA positive patients had symptoms typical of B19 infection and serology showed past infection. Furthermore, 2 were still B19 DNA positive in bone marrow more than 1 y after the first sample indicating virus persistence. The seroprevalence for B19 IgG was 59% and 2 patients were B19 IgM positive. Thus, presence of B19 DNA in bone marrow from patients with haematological disorders is not a general finding in seropositive patients. B19 DNA can persist in bone marrow, but in our material this finding showed no clear correlation with symptomatic B19 infection.

Recombinant parvovirus B19 empty capsids inhibit fetal hematopoietic colony formation in vitro.

Erythroid lineage cells are target cells for human parvovirus B19, and a natural infection often results in transient anemia. To determine whether recombinant B19 capsid proteins (VP1/VP2) also inhibit human hematopoietic progenitor growth, a model system was set up. The B19 capsids were inoculated into primary cultures of hematopoietic stem cells derived from human fetal liver, resulting in a 70-95% reduction of BFU-E (burst-forming unit erythroid cells) as compared with the medium control. A similar effect was seen in human hematopoietic stem cell cultures derived from cord blood and adult bone marrow. Preincubation of the B19 capsids with either a monoclonal antibody to the virus or with B19 IgG positive human sera reduced the inhibitory effect. Furthermore, the inhibitory effect could be reduced by preincubating the target cells with a monoclonal antibody to the cellular receptor for the virus, the P antigen. These findings thus show that the inhibition of colony formation of human hematopoietic stem cells can occur in the absence of parvovirus B19 nonstructural proteins. We speculate that B19 capsid could provide a possible strategy to downregulate indigenous hematopoiesis in fetal stem cell transplantations.

Frequency of human parvovirus B19 infection in intrauterine fetal death.

BACKGROUND: Parvovirus B19 is known to cause fetal death in the second trimester, mainly in combination with hydrops fetalis. However, the frequency of parvovirus-B19-associated non-hydropic fetal loss in the late second and third trimester has not been thoroughly investigated. We aimed to investigate the frequency of parvovirus B19 infection in unselected cases of intrauterine fetal death and to assess the sensitivity of different diagnostic procedures. METHODS: Of 14147 deliveries in three hospitals in the major Stockholm area of Sweden, all cases of intrauterine fetal death (>22 gestational weeks) that occurred between January, 1998, and May, 1999 (n=47), referred cases of miscarriage (<22 gestational weeks, n=37), and induced abortions (n=29), were included in the study. Placental and fetal tissues were examined by means of parvovirus-B19-specific PCR, histopathology, and immunohistochemistry. Placental tissues from 53 normal pregnancies at term were also examined. FINDINGS: Significantly more cases of intrauterine fetal death were positive for parvovirus B19 DNA (seven [15%]) than were normal pregnancies at term (zero, p=0.049). Furthermore, parvovirus B19 DNA was found in two (5%) of the miscarriages but not in any of the cases of induced abortion. Only three of nine DNA-positive cases had parvovirus-B19-associated inclusions and stained positive for viral proteins. All but one of the DNA-positive cases of intrauterine fetal death were non-hydropic. INTERPRETATION: The presence of parvovirus B19 DNA in cases of late second-trimester and third-trimester fetal death is common, and most are non-hydropic. The sensitivity of conventional diagnostic procedures for intrauterine fetal death could be greatly improved by addition of parvovirus B19 PCR.

[Parvovirus B19 infection--an incidious chameleon]. / Parvovirus B19-infektion--en lömsk kameleont.

Parvovirus B19 is a common source of infection with a seroprevalence of 60-70 per cent in the adult population. The most common manifestation is erythema infectiosum ("fifth disease"), with exanthem, fever and upper airway symptoms in children. The infection can give rise to a multifaceted clinical picture and is probably underdiagnosed, particularly in risk groups (individuals with haemolytic anaemia or immunosuppression, and fetuses). Serological diagnosis can now be complemented with the demonstration of viral DNA using the PCR (polymerase chain reaction) test in various body fluids, or tissue biopsy. Recent years have witnessed manifest increase in clinical knowledge of parvovirus B19-associated complications, and their diagnosis and treatment.

Direct ex vivo measurement of CD8(+) T-lymphocyte responses to human parvovirus B19.

Parvovirus B19 is a common human pathogen which can cause severe syndromes, including aplastic anemia and fetal hydrops. The mapping of the first parvovirus B19-derived CD8(+) T-lymphocyte epitope is described. This HLA-B35-restricted peptide derives from the nonstructural (NS1) protein and is strongly immunogenic in B19 virus-seropositive donors.