Tumor suppressor TNFAIP3 (A20) is frequently deleted in Sézary syndrome.

Despite recent therapeutic improvements, the prognosis for patients suffering from Sézary syndrome (SS), a disseminated form of cutaneous T-cell lymphomas, is still poor. We identified bi- and monoallelic deletions of the tumor necrosis factor-α-induced protein 3 gene (TNFAIP3; A20) in a high proportion of SS patients as well as biallelic A20 deletion in the SS-derived cell line SeAx. Furthermore, we demonstrate that inhibition of A20 activates the NF-κB pathway thereby increasing the proliferation of normal T lymphocytes. On the other hand, the reconstitution of A20 expression slowed down the cell cycle in SeAx cells. Recently A20 inactivation has been reported in various B-cell lymphomas. In this study, we show that A20 is also a putative tumor suppressor in the T-cell malignancy-SS.

Molecular and serological characterization of hepatitis B virus genotype A and D infected blood donors in Poland.

Hepatitis B virus (HBV) genotypes have distinct geographical distributions and influence severity of clinical outcome and response to antiviral therapies. HBV polymorphism in HBV surface antigen (HBsAg) positive first time blood donors from Poland was examined. HBV serological markers and HBV DNA were tested in 170 samples. Whole genome (n = 53) or specific region sequences: pre-S/S and basic core promoter/precore (BCP/PC) region (91 and 154 samples, respectively) were phylogenetically analyzed. The median age of infected donors was 21 years. Anti-HBs, anti-HBe and hepatitis B e antigen were detected in 5%, 92.4% and 10.5% of tested donors, respectively. The HBV DNA load ranged between unquantifiable and 3.1 x 10(10) IU/mL (median: 4.10 x 10(3) IU/mL). Genotypes A2 (81.2%) and D (18.8%) co-circulated. Phylogenetic analyses revealed differences between the genotypes. Viral load and level of HBsAg tended to be lower in genotype D. The median HBsAg/HBV DNA ratio expressed in IU/mL was one for both genotypes, but very low or very high ratios appeared more frequent in genotype D infections. Higher amino acid variability in the surface proteins (median: 4%vs 1.5%; P = 0.01) and in the major hydrophilic region was observed in genotype D (P = 0.01). BCP/PC region analysis revealed the double mutation 1762T/1764A in 49/125 (39.2%) genotype A2 and 6/29 (20.7%) genotype D strains (P = 0.08). Mutations in PC and BCP regions correlated neither with HBsAg nor HBV DNA levels. HBV genotype A2 is dominant in HBsAg positive donors in Poland. Minority genotype D strains are significantly more substituted than genotype A2 strains potentially affecting the course of infection.

Pure red cell aplasia due to parvovirus B19 infection transmitted probably through hematopoietic stem cell transplantation.

Human parvovirus B19 is a very common infectious pathogen in humans. In healthy subjects, B19 infection is the cause of a self-limiting subclinical erythroid aplasia, followed by rash or arthralgia. In immunocompromised patients B19 can cause chronic anemia. This report presents the case of a 19-year-old male who developed severe anemia shortly after successful allogeneic hematopoietic stem cell transplantation. His marrow showed selective erythroid aplasia, and real-time polymerase chain reaction assay confirmed parvovirus B19 infection. Despite repeated high-dose immunoglobulin treatment, he remained anemic. His hematological status markedly improved after cessation of immunosuppression. Retrospective examination of the donor's blood suggests that hematopoietic stem cells could be the source of infection.

Inhibition of BCL11B expression leads to apoptosis of malignant but not normal mature T cells.

The B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B gene (BCL11B) encodes a Krüppel-like zinc-finger protein, which plays a crucial role in thymopoiesis and has been associated with hematopoietic malignancies. It was hypothesized that BCL11B may act as a tumor-suppressor gene, but its precise function has not yet been elucidated. Here, we demonstrate that the survival of human T-cell leukemia and lymphoma cell lines is critically dependent on Bcl11b. Suppression of Bcl11b by RNA interference selectively induced apoptosis in transformed T cells whereas normal mature T cells remained unaffected. The apoptosis was effected by simultaneous activation of death receptor-mediated and intrinsic apoptotic pathways, most likely as a result of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) upregulation and suppression of the Bcl-xL antiapoptotic protein. Our data indicate an antiapoptotic function of Bcl11b. The resistance of normal mature T lymphocytes to Bcl11b suppression-induced apoptosis and restricted expression pattern make it an attractive therapeutic target in T-cell malignancies.

Preliminary results of HBV DNA testing of Polish haemophilia patients--lack of occult HBV infection.

Identification of hepatitis B virus (HBV) infection in the absence of surface antigen (HbsAg) became possible with the introduction of HBV DNA detection methods. Such occult HBV infection was diagnosed recently in about half of the Japanese HBsAg-negative haemophilia patients. The aim of our study was to assess the prevalence of occult HBV infection in Polish severe haemophilia population on the sample of 115 haemophilia A and B patients (mean age 34.9 +/- 10.9) treated with non-virus inactivated clotting factor preparations before 1995. HBV DNA was detected in nine HBsAg-positive patients (7.8%). The mean HBV DNA load was 72,800 IU mL(-1) (250-400,000 IU mL(-1)). Hepatitis C virus (HCV) RNA was found in six out of nine HBV-positive patients. In conclusion, HBV DNA was identified only in HBsAg-positive patients. Unlike in Japan, the frequency of occult HBV infection in Polish haemophilia population seems extremely rare or absent.

Disruption of the BCL11B gene through inv(14)(q11.2q32.31) results in the expression of BCL11B-TRDC fusion transcripts and is associated with the absence of wild-type BCL11B transcripts in T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is associated with chromosomal aberrations characterized by juxtaposition of proto-oncogenes to T-cell receptor gene loci (TCR), resulting in the deregulated transcription of these proto-oncogenes. Here, we describe the molecular characterization of a novel chromosomal aberration, inv(14)(q11.2q32.31), in a T-ALL sample, involving the recently described BCL11B gene and the TCRD locus. The inversion joined the 5' part of BCL11B, including exons 1-3, to the TRDD3 gene segment of the TCRD locus, whereas the reciprocal breakpoint fused the TRDV1 gene segment to the fourth exon of BCL11B. The TRDV1-BCL11B joining region was 1344 bp long and contained fragments derived from 20q11.22, 3p21.33 and from 11p12, indicating the complex character of this aberration. A strong expression of in-frame transcripts with truncated BCL11B and TCRD constant region (TRDC) were observed, but in contrast to normal T cells and other T-ALL samples, no wild-type BCL11B transcripts were detected in the T-ALL sample. Screening of 37 other T-ALLs revealed one additional case with expression of the BCL11B-TRDC fusion transcript. As BCL11B appears to play a key role in T-cell differentiation, BCL11B disruption and disturbed expression may contribute to the development of T-cell malignancies in man.

The hepatitis C virus genotype and subtype frequency in hepatitis C virus RNA-positive, hepatitis C virus antibody-negative blood donors identified in the nucleic acid test screening program in Poland.

BACKGROUND: Since 2002, blood donors in Poland have been tested not only for hepatitis C virus antibodies (anti-HCV) but also for HCV RNA or HCV core antigen. This screening program identifies asymptomatic, recently infected individuals with no anti-HCV (in the "window period"). The aim of this study was to compare HCV genotype and subtype distribution in window-period (wp) donors, anti-HCV-positive donors, and chronic hepatitis C (CHC) patients. STUDY DESIGN AND METHODS: A total of 2.37 million donors were investigated for HCV RNA, and 340,000 for HCV core antigen. HCV genotypes and subtypes were investigated in 50 HCV RNA-positive, anti-HCV-negative donors; in 70 anti-HCV-positive donors; and in 170 CHC patients. Re-questioning of wp donors for probable risk factors was introduced. RESULTS: HCV RNA was detected in 50 donors of 2.71 million (1:54,200) anti-HCV-negative blood donations. Of these 50 donors, 36 percent exhibited Subtype 1b, whereas Subtypes 3a and 4c/d were identified in 40 and 14 percent, respectively. In anti-HCV-positive donors and CHC patients, the frequency of Subtype 1b was significantly higher (75.7 and 85.3%, respectively); in both groups the lower frequency of Subtypes 3a (14.3 and 10.6%, respectively) and 4c/d (4.3 and 1.2%, respectively) was found. The probable source of infection was identified in 9 wp donors. CONCLUSIONS: The frequency of wp donors is 18.5 per 1 million. The unexpected high frequency of Genotype 4 and Subtype 3a and the low frequency of Subtype 1b was observed in wp donors compared to anti-HCV-positive individuals. Additional epidemiologic questioning introduced after HCV RNA detection may help to identify infection source.

Loss of tumorigenicity of murine colon carcinoma MC38/0 cell line after transduction with a retroviral vector carrying murine IL-12 genes.

Cells of transplantable MC38 colon carcinoma of C57BL/6 mice were adapted to growth in vitro as the MC38/0 cell line. Along the establishing process, MC38/0 cells preserved their tumorigenicity. After transduction with a retroviral vector carrying murine interleukin 12 (mIL-12) genes and further selection, stable MC38/IL-12 transductant cells were obtained. These cells produced IL-12 (approx. 2500 ng/ml/5x10(5) cells/48 h) as evaluated in the optimized bioassay. After subcutaneous inoculation into syngeneic mice, the IL-12-modified cells demonstrated reduced tumorigenicity as compared to parental MC38/0 cells. Mice that rejected the MC38/IL-12 tumour became protected against subsequent challenge with MC38/0 cells. The obtained data indicate that the IL-12-transduced murine colon carcinoma cells could be used both as a model tumour for the study of mechanisms of anticancer immunity and/or as an adjuvant to cancer vaccines.

Polymorphism of the TT virus and its frequency in Polish blood donors.

OBJECTIVE: To determine, in Polish blood donors, the frequency of TT virus (TTV) using different primers and the sequence diversity of TTV genotypes. MATERIALS AND METHODS: Two-hundred blood donors were studied. TTV DNA was detected by the polymerase chain reaction (PCR) using primers for the coding (ORF1) and non-coding (NC) regions. Twenty isolates were genotyped by sequencing the ORF1 fragment. RESULTS: TTV DNA was detected in 78% of donors using NC primers and in 10% using ORF1 primers. The frequency of TTV DNA detection by NC primers was observed to increase with donor age, whereas the frequency of detection by ORF primers did not differ between various age-groups. The nucleotide sequence homology of Polish TTV isolates ranged from 59 to 99%. Three genotypes (1b, 2b and 2c) were identified. CONCLUSIONS: The frequency of TTV detection depends on the primers used for the PCR. Using the NC primers the virus is detected in the majority of donors, whereas the ORF1 primers strongly underestimate the prevalence of TTV. The frequency of TTV DNA increases with age. Polish TTV isolates are highly polymorphic and are classified as 1b, 2b and 2c.

Genetically modified dendritic cells--a new, promising cancer treatment strategy?

Dendritic cells (DCs), the most potent antigen-presenting cells (APCs), were discovered almost 30 years ago. Due to the priming of antigen-specific immune responses mediated by CD4+ and CD8+ lymphocytes, DCs are crucial for the induction of adaptive immunity against cancer. Therefore, vaccination of cancer patients with DCs presenting tumour-associated antigens (TAAs) have been believed to be a promising anticancer strategy. Multiple clinical trials have been carried out in order to evaluate the safety and efficacy of cancer vaccines based on antigen-pulsed DCs. However, pulsing of DCs with particular peptides has several disadvantages: i) short-time duration of antigen-major histocompatability complex (MHC) complexes, ii) a requirement for matching defined peptides with MHC complexes and iii) exclusive presentation of single antigen epitopes. Application of gene transfer technologies in the field of DC-based vaccines made possible the development of novel, anticancer immunisation strategies. In several animal models, DCs modified with genes encoding TAA or immunostimulatory proteins have been shown to be effective in the induction of antitumour immune responses. Based on these encouraging results, a first clinical trial of prostate cancer patients vaccinated with gene modified DCs has recently been initiated. In this article, methods used for genetic modification of DCs and anticancer vaccination strategies based on genetically modified DCs are reviewed.

Recent developments in retroviral gene delivery systems.

Since the first gene therapy clinical trial carried out by Rosenberg et al. in 1990 [1], recombinant retroviral vectors are still the most popular gene delivery tools for gene therapy purposes. According to the databases of the Journal of Gene Medicine [101] 35% of gene therapy protocols employ retroviruses, other vectors such as adenoviral vectors are used in 27%, pox viral vectors in 6%, adeno-associated viral vectors in 2% and herpes simplex based vectors in 0.5% of clinical trials. It has become clear that the early retroviral vectors based on simple retroviruses (e.g., murine leukaemia viruses (MLV) are characterised by relatively low viral titre, low transduction efficiency and difficulties in in vivo administration. On the other hand, lentiviral vectors (complex retroviruses) can be grown to a high titre, can stably transduce non-dividing cells and can effectively deliver genes to human progenitor cells (CD34+). This article aims to summarise the first year of the 21st century's developments in the retroviral gene delivery system.

Analysis of hepatitis G virus infection markers in blood donors and patients with hepatitis.

The incidence and clinical significance of hepatitis G virus (HGV) is still not fully known. The aim of our study was to assess the frequency of HGV RNA and antibody to HGV E2 protein (anti-E2) in Polish blood donors and patients with hepatitis, and to compare the sequence of HGV clones with those reported by others. Two-hundred and nineteen blood donors and 83 patients with hepatitis were studied. HGV was detected in 3.2% and anti-E2 in 24.2% of blood donors and in 26.5% and 8.4% of patients with hepatitis, respectively. HGV was detected as a co-infection with HCV in four of 18 patients with chronic hepatitis, in four of 16 patients with acute hepatitis and in one of six patients with fulminant liver failure (FLF), and as a co-infection with HBV in one of six patients with FLF and in three of 10 patients with chronic hepatitis. In non-A-C hepatitis, eight of 23 patients with acute hepatitis and one of four patients with FLF were positive for HGV but all 10 patients with chronic cryptogenic hepatitis were negative. In the follow-up studies of patients with HGV alone, a correlation with viraemia and clinical symptoms was observed in two patients, but in three others HGV RNA was detected in spite of clinical resolution. Two HGV clones were sequenced, and the sequence of the HGV helicase region of the HGV isolates from donor and patient were homologous to those described by others. Hence, the frequency of HGV RNA in blood donors is similar to that obtained in other countries but the anti-E2 (marker of a past infection) frequency is higher. The incidence of HGV RNA and anti-E2 in hepatitis patients suggests that HGV plays a role in liver pathology, but careful analysis of individual cases does not confirm this.

Anti-HCV RIBA/LiaTek reactivity and HCV genotype in EIA-negative patients with viremia.

Individuals infected with hepatitis C virus (HCV) usually produce anti-HCV antibodies detectable by enzyme immunoassay (EIA); however, in certain viremic cases this antibody does not appear. To investigate whether anti-HCV in these cases is detectable by Western blot (WB), 38 HCV RNA positive/anti-HCV EIA-negative sera were tested by RIBA 3.0 or LiaTek III. The HCV genotypes (INNO-LiPA) were analyzed to determine whether the variance in these genotypes can be the reason for the late, weak antibody production or its absence. As the control group, 282 EIA-positive/HCV RNA-positive patients were examined. A single band reactivity of various intensities by RIBA or LiaTek was observed in 16/38 EIA negative sera. Positive results with NS3 were detected in 4 sera and weak positive (+/-) with core, NS3, and NS5 in 5, 6, and 1 sera, respectively. In 3 cases with anti-NS3, the seroreversion was observed in follow-up. The distribution of genotypes in anti-HCV-negative versus anti-HCV-positive groups was: 1b alone, 50.0% vs. 78.0%; 3a alone, 13.2% vs. 15.6%; and mixed (1b+3a), 36.8% vs. 5.0%, respectively. The follow-up studies showed that viremia was lost spontaneously in 12/35 patients. In some patients infected with two genotypes, the spontaneous loss of the 3a genotype was observed. The study showed that WB tests are useful for serological confirmation of HCV infection in some EIA negative/HCV RNA-positive patients but, because seroreversion may occur, sequential sera samples should be tested. No unusual HCV genotype was detected in anti-HCV-negative/HCV RNA-positive cases, but the frequency of mixed infection with the 1b+3a genotypes in this group was found to be higher than that in anti-HCV-positive hepatitis patients.

[Diagnosis of human parvovirus B19 infection in nonimmune hydrops fetalis]. / Diagnostyka zakazenia ludzkim parwowirusem B19 w nieimmunologicznym obrzeku plodu.

Parvovirus B19 (PV B19) infection was investigated in 29 pregnant women with fetal hydrops, after exclusion of feto-maternal incompatibility within red blood cell antigens, TORCH infections, feto-maternal hemorrhage and genetics reasons. The active viral infection was detected in 9 women (31%) by PCR amplification of DNA B19; in 2 of them IgM and IgG, in 1 IgM and in 4 IgG antibodies were also present. In 6 women (20%) IgG antibodies were only found, but not IgM and DNA B19, which confirmed infection in the past. In addition in 9 cases DNA B19 was evaluated in the fetal blood. The results in the mothers and their fetuses were concordant (4 positive, 5 negative). Our conclusion is that in nonimmune hydrops fetalis, PV B19 infection should be based on the viral DNA evaluation in the blood of mother (or fetus). IgM antibodies, in time of fetal disorders, might not be detected.

Thymus-directed immunotoxicity of airborne dust particles from Upper Silesia (Poland) under acute extrapulmonary studies in mice.

Industrial air pollutants from Upper Silesia, Poland, contain over 250 polycyclic and heterocyclic aromatic hydrocarbons and heavy metals, including mutagenic and carcinogenic chemicals that have been shown to form DNA adducts. Over 4 million habitants of Silesia are permanently exposed to the industrial pollution by pulmonary and dermal routes and by contaminated food and water. These chemicals, when examined separately in animals models, were proven immunotoxic. We studied the extrapulmonary immunotoxic potential of a typical mixture of Silesian filter-suspended matter from a selected area, over a specific season and time period. Early changes in the immune system were analyzed in BALB/c mice exposed ip to acute doses of 20-330 mg dust mixture/kg body weight (0.06-1.0 LD50). No major changes were noted for weight and the cellularity of spleen, liver and kidneys. However, dramatic decrease in thymus weight index and thymocyte cell count were noted as early as 24-72 h postexposure, which correlated with almost complete depletion of immature, double-positive CD4+CD8+ thymocytes. Changes in spleen were less profound; however, increased depletion of B cells over T cells was noted at high doses of the suspended matter. Exposure to the airborne dust also decreased cytokine production by spleen cells, such as interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). Overall, a single exposure to Silesian dust, even at the relatively low 0.06 LD50 dose, affected lymphokine production, suppressed B-cell proliferative response, and depleted thymuses of immature, double-positive CD4+CD8+ cells. A chemical synergism is suspected. To our knowledge, none of the known components of Silesian suspended matter, when examined as a single chemical, was shown to exert such a profound biological effect.