Antiviral adoptive immunotherapy using antigen-specific T cells in allogeneic hematopoietic stem cell transplant recipients.

Adoptive immunotherapy using antiviral T cells (AVT) obtained from healthy donors is one of the advanced approaches considered as a breakthrough in the treatment of refractory and severe viral infections that often accompany primary immunodeficiencies or allogeneic hematopoietic stem cell transplantations. The review describes nearly 30 years of the development of AVT to human cytomegalovirus, Epstein-Barr virus, human adenovirus, and human polyomavirus BK. The review introduces the basic methodological approaches to their production and summarizes the results from clinical studies that tested the safety and efficiency of the procedures used. Recent studies indicate that the treatment of viral infections by frozen AVT stored in banks could become a commonly available therapeutic modality.

Genetic variability of cytomegalovirus glycoprotein O in hematopoietic stem cell transplant recipients.

UNLABELLED: Genetic variation of cytomegalovirus (CMV) strains can correlate with their pathogenicity for immunocompromised patients. Glycoprotein O (gO), together with glycoprotein L and glycoprotein H, mediate the fusion of the viral envelope with the cell membrane and promotes virus penetration, envelopment, and release. The variability of gO might play a role in CMV cell tropism. The goal was a retrospective analysis of gO variability in a cohort of hematopoietic stem cell transplant (HSCT) recipients to determine the distribution of gO genotypes and to investigate their impact on clinical outcome and manifestation of CMV infection. METHODS: In archived blood samples from 51 adult allogeneic HSCT recipients with active CMV infection, gO was analyzed by sequencing the N-terminal domain of the UL74 gene using the dye deoxy termination method. RESULTS: The gO1 and gO2 clades were most common (39% and 20%, respectively, and gO3 was associated with higher risk of symptomatic infection (P = 0.026 in multivariant analysis). Despite being associated with higher antigenemia levels (P = 0.02), gO4 had the best survival and lower rate of CMV recurrence. No significant differences were found in clinical manifestation and outcome of CMV disease between patients with various gO clades. Because CMV strains sharing an identical gO sequence differed in glycoprotein B genotypes, sequencing the N-terminal part of the gO gene does not seem to be optimal for the identification of strains. CONCLUSIONS: gO genotyping may contribute to the biological characterization of CMV strains in HSCT recipients.

[Genotyping of viral glycoprotein B (gB) in hematopoietic stem cell transplant recipients with active cytomegalovirus infection: analysis of the impact of gB genotypes on the patients' outcome]. / Genotypizace virového glykoproteinu B (gB) u príjemcu transplantátu kmenových bunek krvetvorby s aktivní infekcí cytomegalovirem--sledováni vlivu genotypu na prubeh infekce.

AIM OF THE STUDY: Genetic variation of CMV strains may correlate with their pathogenicity for immunocompromised patients. On the basis of sequence variation in the UL55 gene encoding the most abundant viral envelope glycoprotein gB, CMV can be classified into four major gB genotypes. The aim of the study was the analysis of the distribution of gB genotypes in a cohort of haematopoietic stem cell transplant (HSCT) recipients and of the correlation of genetic polymorphisms with clinical outcomes and manifestation of CMV infection. MATERIAL AND METHODS: Archived DNA isolates from consecutive blood samples of 53 adult allogeneic HSCT recipients with active CMV infection, transplanted in 2004-2005, were used for the genetic analysis. HCMV gB genotyping was performed by restriction fragment length polymorphism (RFLP) analysis and sequencing of the central variable region of UL55. The association of gB genotypes with selected clinical parameters was assessed by multivariate analysis after adjustment for graft donor type, HLA-matching and anti-thymocyte immunoglobulin (ATG) therapy. RESULTS: gB1, gB2, gB3, and gB4 genotypes were detected in 30%, 17%, 26% and 4% of the patients, respectively. An atypical gB genotype was found in one patient. Co-infection with two or more gB genotypes was revealed in 17% of the patients. The distribution of gB genotypes did not vary in time, despite the fact that the patients transplanted in 2005 had more severe CMV infection with higher viral loads in the blood than those transplanted in 2004. gB1 was associated with a lower viral load (p = 0.046) and a milder course of symptomatic CMV infection, but with a higher rate of acute graft versus host disease (OR 3.4; p = 0.067). Pancytopenia was less frequent in the patients infected with gB3 (OR 0.09; p = 0.075). In contrast, gB2-infected patients had a worse outcome of CMV infection with a higher rate of organ involvement and were less responsive to antiviral therapy (OR 6.65 and 0.18; p = 0.15 and 0.12, respectively). The prognostic impact of co-infection with two or more gB genotypes was not shown. CONCLUSIONS: gB genotype may have an impact on the course of CMV infection and its complications in HSCT recipients. Nevertheless, these results need to be tested on a larger group of patients in the context of genetic variability of other functionally important viral genes. The characterization of viral genetic factors determining CMV pathogenesis will be of relevance to the treatment of patients at high risk of CMV infection.

[Prevalence of glycoprotein B (gB) genotypes in the patients with high risk of symptomatic cytomegalovirus infection]. / Zastoupení ruzných genotypu glykoproteinu gB u pacientu s vysokým rizikem symptomatické infekce lidským cytomegalovirem (CMV).

OBJECTIVES: Based on genetic variability of the dominant envelope glycoprotein (gB), human cytomegalovirus is classified into four major genotypes. The aims were to determine the prevalence of particular gB genotypes in Czech CMV-infected patients and to compare three groups of the patients with high risk of symptomatic CMV infection, i.e., haematopoietic stem cell transplant (HSCT) recipients, HIV-positive persons and infants. MATERIAL AND METHODS: The study was performed on 134 archived CMV-positive DNA isolates from the patients tested in the National Reference Laboratory for Herpesviruses in 2004-2007. For genotyping, the variable part of gB was amplified and analysed using the restriction fragment length polymorphism (RFLP) method. RESULTS: The most frequently detected genotype was gB1 (33%), followed by gB2 (29%), gB3 (18%) and gB4 (7%). However, the distribution of gB genotypes varied between groups of high-risk patients: gB2 dominated in HIV-positives (55%, p = 0.004), while gB3 was most common in HSCT recipients (26%, p = 0.03) and gB4 was relatively more frequent in infants (20%, p = 0.03). In HSCT recipients, we found increased frequency of gB3 (26%, p = 0.03) and co-infection with two or more gB genotypes (17%, p = 0.016). CONCLUSIONS: The distribution of CMV gB genotypes from Czech CMV-infected patients is similar to that reported in other European countries or in the United States. Differences in the prevalence of CMV gB genotypes between groups of high-risk patients indicate variation in biological properties of particular gB genotypes, possibly resulting in distinct virulence, immunogenicity or epidemiological characteristics of circulating strains.

[Guidelines for the diagnosis and treatment of genital herpes in women]. / Doporucený postup pro diagnostiku a terapii genitálního herpesu u zen.

Presented are draft guidelines for genital herpes (GH) in women. The text is concerned with the incidence, clinical picture, diagnosis, prevention, prophylaxis and therapy of genital HSV infection. The aim is to provide valuable information on the approach to both initial and relapsing GH, with attention being paid to risk factors such as immunosuppression or pregnancy. Also mentioned are procedures to decrease the risk of vertical transmission of HIV infection and adequate measures to prevent disseminated neonatal herpes infection.

Prevalence of active infection with Chlamydia pneumoniae and human cytomegalovirus in patients with type II diabetes mellitus.

By promoting the inflammatory process in the arterial wall, Chlamydia pneumoniae (CPN) and human cytomegalovirus (CMV) participate in the pathogenesis of cardiovascular disease (CVD). Since patients with diabetes mellitus (DM) are at high risk of CVD, we studied markers of CMV and CPN infection in DM patients as possible predictors of cardiovascular complications. The seroprevalence rates of CMV in 44 DM patients and matched controls were 74 and 88%, respectively. Compared with controls, patients showed lower titers of IgG against CMV (p < 0.001) and higher titers of genus-specific IgA against CPN (p = 0.006). The titers of genus-specific IgG and prevalence rates of type-specific anti-CPN IgA, IgG or IgM were similar in both DM patients and controls. Serological markers of either active or recent CPN infection were detected in 54% of patients and 59% of controls. However, CPN DNA was not detected in the blood of any DM patient. CMV DNA was found in the blood of 1 (2.3%) patient. The results do not indicate an increased rate of CMV or CPN infection in patients with type II DM.

[A simultaneous detection of Chlamydia pneumoniae and Chlamydia trachomatis DNA by real-time PCR]. / Soucasný prukaz DNA Chlamydia pneumoniae a Chlamydia trachomatis pomoci real-time PCR.

STUDY OBJECTIVE: Design and validation of a real-time PCR assay for quantitative detection of C. pneumoniae and C. trachomatis in clinical specimens. MATERIAL AND METHODS: A part of the 16S RNA gene was selected as the target sequence for amplification. The reaction product was cloned in the bacterial plasmid and a recombinant calibrator and a combined internal standard were prepared, usable in three different PCR assays. Archived DNA isolates from various clinical specimens, DNA isolates from other infectious agents and control samples for DNA detection, CT (QCMD) and CPN (Instand), were used for validation. RESULTS: Reaction specificity and reproducibility were tested. The analytical sensitivity was set to 5-10 copies of the bacterial genome/reaction. As many as 105 DNA isolates from various types of clinical specimens (swabs, urine, peripheral blood and BAL fluid) were tested by real-time PCR and conventional PCR assays. The specimens that had not yielded concordant results were characterized by a third independent amplification test. The sensitivity and specificity of real-time PCR were 89% and 100%, respectively. The assay is suitable for both screening and diagnosis of Chlamydia.

Immunological profiles of patients with chronic myeloid leukaemia. I. State before the start of treatment.

In view of the increasing interest in the immunotherapy of CML it seems highly desirable to broaden the present knowledge on the immune reactivity of CML patients. A group of 24 patients and 24 healthy controls were studied for the total of 15 immunological parameters, including the prevalence of antibodies against human herpesviruses and papillomaviruses. To clearly discriminate between changes associated with the disease and those induced by the therapy, all patients were enrolled prior to the start of any anti-leukaemic therapy. Statistically significant differences between patients and controls were found in the levels of IgA, C4 component of complement, CRP and IL-6, the production of Th1 cytokines in stimulated CD3 cells and the E. coli stimulatory index. The analysis of the interrelationship between the results obtained in the individual patients presented some unexpected findings, such as the lack of correlation between the CRP and IL-6 levels. It will be the purpose of a follow-up to determine whether and how the immune status of the patients prior to the treatment correlates with their response to therapy and how the individual immunological profiles change in the course of the disease. These observations will be utilized in the future immunotherapeutic studies to constitute the vaccine- and placebo-treated groups.

Presence of herpes simplex virus (HSV) in peripheral leukocytes of patient who developed active HSV infection after bone marrow transplantation.

BACKGROUND: Despite of prophylactic antiviral therapy, latent HSV may be reactivated in bone marrow transplant (BMT) recipients and cause serious disease. Rapid diagnosis of HSV infection is needed to prompt institution of appropriate therapy. OBJECTIVES: We report a case of the allogenic BMT recipient, who developed ulcerative esophagitis which progressed to generalized HSV infection and graft versus host reaction (GVHR).We consider several diagnostic approaches to detection of active HSV infection in this patient. STUDY DESIGN: Polymerase chain reaction (PCR) was used to detect HSV DNA in esophageal biopsy specimens and peripheral leukocytes (PBL). Isolation of HSV in tissue culture was performed to prove infectious virus in swabs from mucocutaneous lesions or in PBL. RESULTS: Using PCR, HSV DNA was detected in peripheral leukocytes of the patient who had developed generalized HSV infection accompanied with hepatosplenomegaly and hepatitis. At that time, a fully infectious ACV-resistant HSV was isolated from his PBL. On the other hand, HSV DNA was not detected in PBL of other BMT-recipients with skin- or organ-localized infection. CONCLUSIONS: Presence of HSV-DNA in PBL of BMT recipients can signalize generalized HSV infection. Isolation of HSV from PBL by cocultivation with human fibroblasts can be used as an alternative diagnostic approach in these patients.

Seroprevalence of HHV 8 antibodies among the general population and HIV positive persons in the Czech Republic.

BACKGROUND: The seroprevalence rates of herpesvirus 8 (HHV 8) antibodies were determined for the general Czech population and HIV-positive individuals. OBJECTIVES: Six hundred and sixty six serum samples from the general Czech population and 129 serum samples from HIV-positive persons were tested for the presence of antibodies to the HHV 8 lytic and latent antigens. STUDY DESIGN: HHV 8 antibodies were detected by the indirect immunofluorescence test. RESULTS: In the general Czech population, only 2.4 and 0.3% of the serum samples tested positive for antibodies against the lytic and latent HHV 8 antigens, respectively. As many as 34.9 and 10.9% HIV positive individuals had antibodies to the HHV 8 antigens, respectively. Only three of them have developed Kaposi's sarcoma (KS) to date. At the time of KS diagnosis, the three patients had antibodies to both HHV 8 antigens. HIV-positive homo/bisexuals were at significantly higher risk of acquiring HHV 8 infection compared with HIV-positive heterosexuals. The increase in HHV 8 seroprevalence was associated with progression of the HIV infection from stage A to stage B. No correlation was found between the HHV 8 seroprevalence and CD 4+T-lymphocytes counts or the HIV viral load. CONCLUSIONS: Among the general Czech population, the HHV 8 seroprevalence is as low as in the West European countries. The mean HHV 8 seroprevalence rate in HIV-positive individuals was 34.9% and was comparable with those reported in other low seroprevalence countries.

Effect of high glucose concentrations on expression of ELAM-1, VCAM-1 and ICAM-1 in HUVEC with and without cytokine activation.

Diabetes mellitus is associated with an increased prevalence of endothelial dysfunction and development of atherosclerotic vascular diseases. We demonstrate here that hyperglycemia results in the expression of adhesion molecules on endothelial cells in vitro. Incubation of human umbilical vein endothelial cells (HUVEC) in a culture medium with 11.0 mM, 16.5 mM and 22.0 mM glucose concentrations induced the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelial-leukocyte adhesion molecule-1 (ELAM-1). This effect was detectable after 24 h incubation of HUVEC with a high glucose concentration. The effect of high glucose concentration on TNF-alpha induced expression of ELAM-1, VCAM-1 and ICAM-1 was negligible, if at all. These results show that even a short-term exposure of endothelial cells (ECs) to high glucose concentration leads to their activation associated with increased expression of adhesion molecules such as ELAM-1, VCAM-1 and ICAM-1.

Interaction of Borrelia burgdorferi sensu lato with Epstein-Barr virus in lymphoblastoid cells.

Since the possibility of interruption of latent EBV infection has been suggested by the induction of the lytic virus cycle with chemical substances, other viruses, and by immunosuppression, we hypothesized that the same effect might happen in B. burgdorferi sensu lato infection as happens in Lyme disease patients with positive serology for both agents. We have observed EBV replication in lymphoblastoid cells after superinfection with B. garinii and B. afzelii strains after 1 and 4 h of their interaction. We found that viral and borrelial antigens persisted in the lymphoblasts for 3 and 4 days. Morphological and functional transformation of both agents facilitate their transfer to daughter cells. Association with lymphoblasts and internalization of B. garinii by tube phagocytosis increased replication of viruses more successfully than B. afzelii and chemical inductors. Demonstration of such findings must be interpreted cautiously, but may prove a mixed borrelial and viral cause of severe neurological disease.

Effect of n-butyrate on superinfection of Raji lymphoblastoid cell line by Epstein-Barr virus.

The effect of n-butyrate on superinfectability of virus-nonproducer Raji cells by the P3HR-1 strain of Epstein-Barr virus (EBV) was investigated. n-Butyrate is known to be a potent inducer of virus antigen synthesis in virus-producer cell lines and of cell differentiation in virus nonproducers. The drug inhibited the growth of Raji cells but did not interfere markedly with cell viability. It induced a low rate of early antigen (EA) synthesis in about 1-2% of noninfected Raji cells. While the number of superinfectable cells remained relatively constant after treatment with butyrate, an increase in antigen positivity was noted in untreated cells. This relative decrease in sensitivity to superinfection in butyrate-treated Raji cells was more pronounced in cultures that had been treated with the drug for 48 or 72 hr as compared to those treated for 24 hr. A blocking of the treated cells in the certain cell-cycle phase and their drug-induced differentiation towards plasma cells might have been involved in the phenomenon described.

Typing of human herpesvirus-6 DNA by restriction endonuclease cleavage of the polymerase chain reaction product.

A 591 bp portion of the HindIII H (pZVH14) fragment of human herpesvirus-6 (HHV-6) strains GS (type A) and R-147 (type B) DNA was amplified by the polymerase chain reaction (PCR) using specific primers. While AluI cleaved the amplified DNA of both types of HHV-6, EcoRV cut the type A but not the type B DNA; vice versa, HaeIII cleaved the type B but not the type A DNA.

Effect of human cytomegalovirus and glucose on adhesion molecules expression in cultured human endothelial cells.

The aim this study was to investigate the effect of glucose on the induction of adhesion molecules by Human cytomegalovirus (HCMV) in endothelial cells in vitro. Primary cultures of human umbilical vein endothelial cells (HUVECs) pretreated with 16.5 mmol/l glucose for 24 hrs were infected with a HCMV strain with tropism for endothelial cells. Expression of adhesion nmolecules (ICAM-1, VCAM-1 and ELAM-1) was measured by flow cytometry. While high concentrations of glucoseperse activated the expression of all three adhesion molecules tested, HCMV induced the expression of ICAM-1 only. Moreover, it potentiated the expression of ICAM-1 in glucose-pretreated HUVECs, while it did not affect at all or slightly suppressed the glucose-activated expression of VCAM-1 and ELAM-1. The modulatory effect of glucose and HCMV on the expression of adhesion molecules in endothelial cells may be applied in increased vulnerability to patients with diabetes mellitus or atherosclerosis.

Epstein-Barr virus (EBV) antibodies in children with non-Hodgkin's lymphomas.

Antibody titres against Epstein-Barr virus (EBV) antigens in children suffering from non-Hodgkin's lymphoma (NHL) were determined. IgG antibody titres against the viral capsid antigen (VCA) and early antigen (EA) exceeded those found in healthy control subjects. On the other hand, antibody titres against EBV-determined nuclear antigen (EBNA complex) were generally lower than in the control group. The most striking phenomenon observed in the patient group was the frequent activation of latent virus infection as revealed by the periodical appearance of anti-EA and IgM class anti-VCA antibodies. Antibody titres against EBV antigens were generally lower among patients with progressing disease than in those with a more favourable course of the illness. The closest relation to EBV based on serological findings, was detected in lymphoblastic lymphomas of Burkitt-type histology, poorly differentiated lymphocytic lymphomas, and in lymphomas localized in the abdomen. The question whether EBV might be involved in a certain proportion of the cases examined is discussed and further approaches to elucidate this problem are suggested.

Antibody against synthetic peptide derived from Epstein-Barr virus-determined nuclear antigen 1 (EBNA-1) in child non-Hodgkin's lymphoma.

Antibody reactivity against a synthetic peptide derived from Epstein-Barr virus nuclear antigen 1 (EBNA-1) was determined in 56 cases of child non-Hodgkin's lymphoma and 31 controls. The patients were divided into subgroups based on tumour location and histology and the antibody responses in the various groups were compared. A significant increase in both IgG and IgM antipeptide titres was detected in patients with tumours localized in the abdomen. High IgG titres were also noted in Burkitt-type, lymphoblastic, and centroblastic lymphomas. On the other hand, low or nil IgG titres were found in unclassified malignant lymphomas, in four cases of centroblastic-centrocytic lymphoma and in lymphomas located in the mediastinum. Surprisingly, the occurrence of antipeptide IgM antibody was highest in those tumours, where IgG titres were low, i.e. in subjects with mediastinal tumours and in unclassified malignant lymphomas. However, with the exception of tumours localized in the abdomen and unclassified tumours, the IgM titres in positive individuals were low and comparable with titres found in a part of healthy controls.

Serological markers of Chlamydia pneumoniae, cytomegalovirus and Helicobacter pylori infection in diabetic and non-diabetic patients with unstable angina pectoris.

The possible role of inflammation in coronary artery disease (CAD) is being recognised, while markers of inflammation (e.g., CRP) and infection with Chlamydia pneumoniae (C. pneumoniae), cytomegalovirus (CMV) and Helicobacter pylori (H. pylori) have been proposed as risk factors for CAD. However, these associations require further evaluation. It is a known fact that diabetic patients suffer from impaired immune response to some pathogens and a high incidence of atherosclerosis. In this case-control study we investigated serological markers of infection with C. pneumoniae, CMV, and H. pylori in a group of 140 patients with unstable angina pectoris (UA), 52 of them having type 2 diabetes mellitus, and in a matched control group. Anamnestic (IgG) and acute infection (IgA) antibodies against the above agents were tested using ELISA or indirect immunofluorescence tests. In patients with UA we found a significantly higher seroprevalence and titres of IgG antibodies against C. pneumoniae (p = 0.04) and increased titres of IgG antibodies against CMV (p = 0.007). No differences were found in IgA antibody response to these pathogens. Antibody response to H. pylori was similar in both groups tested. In diabetic patients with UA, the frequency of group-common IgG antibodies against C. pneumoniae was higher than in the non-diabetic UA patients. The other serological markers studied were comparable in the patients with or without diabetes mellitus. Our findings confirmed association of C. pneumoniae and CMV with cardiovascular heart disease. Moreover, diabetes mellitus may predispose the patients to C. pneumoniae infection. However, serological markers observed do not indicate that destabilisation of angina pectoris is associated with acute C. pneumoniae or CMV infection. No relationship was found between UA and H. pylori infection.

[Signs of active infection with Chlamydia pneumoniae in patients with coronary heart disease]. / Sledování známek aktivní infekce Chlamydia pneumoniae u pacientu s ischemickou chorobou srdecní.

BACKGROUND: Possible relationship between Chlamydia pneumoniae (CPN) infection and atherosclerosis has been documented in many seroepidemiological, histological and biological studies. The objectives of the present study were to find out whether serological signs of active CPN infection in patients with coronary heart disease (CHD) are associated with the presence of bacterial DNA in peripheral blood and to correlate with clinical symptoms and to study the dynamics of the markers of CPN infection within a six-month follow-up. METHODS AND RESULTS: Seventy-one patients with acute CHD were enrolled in the study. They underwent clinical and biochemical tests and were screened for the presence of genus- and type-specific IgG, IgA and IgM antibodies against CPN at admission and then in 3- and 6-month intervals. CPN DNA was detected in peripheral blood using nested PCR. Serological markers of active CPN infection were found in 36 patients (51.4%) while bacterial DNA was detected in two patients only. Laboratory signs of active CPN infection did not correlate with either clinical symptoms or levels of biochemical markers. In most of the patients, titers of anti-CPN antibodies were stable throughout the follow-up. Increase in antibody titers was observed in 23% of patients and was associated with more frequent signs of unstable angina pectoris (p=0.06) but not with higher risk of myocardial infarction within 6 months after the acute episode of CHD. CONCLUSIONS: In patients with CHD, serological markers of active infection persist for a long time. Nevertheless, their association with the course of CHD or relapse risk was not proved. Bacterial DNA was rarely detected in peripheral blood of the patients. None of the currently available laboratory tests proved adequately effective for detection of ongoing or chronic CPN infection. This project was sponsored by grant IGA MZ CR NI/6811-3 and research plan of Natl. Inst.Publ.Health