Female-specific neuroprotection against transient brain ischemia observed in mice devoid of prion protein is abolished by ectopic expression of prion protein-like protein.

This study was designed to examine the function of cellular prion protein and prion protein-like protein/Doppel, in transient ischemia-related neuronal death in the hippocampus. Two different lines of mice devoid of cellular prion protein, Zrch I Prnp(0/0) and Ngsk Prnp(0/0), were used. The former lacks cellular prion protein whereas the latter ectopically expresses prion protein-like protein/Doppel in the brain in the absence of cellular prion protein. Mice were subjected to 10 min-occlusion of the bilateral common carotid arteries with recovery for 14 days. Less than 10% of the pyramidal neurons in the CA1 subfield were degenerated in male and female wild-type mice. In contrast, more than half of the neurons were lost in male Zrch I Prnp(0/0) and Ngsk Prnp(0/0) mice. Such severe neuronal loss was also observed in female Ngsk Prnp(0/0) mice. However, female Zrch I Prnp(0/0) mice showed mild neuronal loss similar to wild-type mice. Flunarizine, a T- and L-type Ca(2+)-channel antagonist, significantly reduced the neuronal loss in female but not in male Ngsk Prnp(0/0) mice. These results indicate that loss of cellular prion protein renders hippocampal neurons susceptible to ischemic insult specifically in male but not female mice and the ectopic expression of prion protein-like protein/Doppel aggravates the ischemic neuronal death in female prion protein-null mice probably via overloading of Ca(2+)-dependent signaling.

HTLV-1 proviruses encoding non-functional TAX in adult T-cell leukemia.

Adult T-cell leukemia (ATL) is associated with prior infection with human T-cell leukemia virus type 1 (HTLV-1). TAX, the major transactivator of HTLV-1, has been implicated in the immortalization of infected T-cells, but molecular mechanisms of in vivo malignant cell transformation induced by HTLV-1 remain unclear. To investigate the role of TAX in the monoclonal proliferation of ATL cells, we determined the nucleotide sequence of tax DNA clones obtained from 6 ATL patients and analysed the biological function of their products. We found that ATL cells from 2 of these patients possessed tax with a nonsense or frame-shift mutation resulting in the premature termination of its protein product, which was no longer functional. This strongly argued against an indispensable role of TAX for the maintenance of ATL cells in vivo. On the other hand, the frequency of nucleotide substitutions found in non-functional tax DNA clones from these patients was significantly lower than those in functional tax DNA clones from the others, suggesting a role for TAX in the genome instability of infected cells. Although mismatch repair defects in the microsatellite markers, including those in hMSH3, hMSH6, BAX, TGF-beta RII, and E2F4 genes, were infrequent, we found an increase in the number of CAG repeats of the E2F4 microsatellite marker in 1 patient. These findings indicate that while TAX may be a necessary prerequisite for malignant transformation of infected cells, it is not essential for the maintenance of ATL cells in vivo.

PrP fragment 106-126 is toxic to cerebral endothelial cells expressing PrP(C).

A hydrophobic, fibrillogenic peptide fragment of human prion protein (PrP106-126) had in vitro toxicity to neurons expressing cellular prion protein (PrP(C)). In this study, we proved that primary cultures of mouse cerebral endothelial cells (MCEC) express PrP(C). Incubation of MCEC with PrP106-126 (25-200 microM) caused a dose-dependent toxicity assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase release, bis-benzimide staining for nuclear morphology, and trypan blue exclusion test. Pentosan polysulphate (50-100 microg/ml), a drug effective in scrapie prophylaxis, dose-dependently attenuated the injury. MCEC cultures from mice homogenous for the disrupted PrP gene were resistant to the toxicity of PrP106-126. In conclusion, cerebral endothelium expressing PrP(C) may be directly damaged during spongiform encephalopathies.

Pentosan polysulfate regulates scavenger receptor-mediated, but not fluid-phase, endocytosis in immortalized cerebral endothelial cells.

1. Effects of pentosan polysulfate (PPS) and the structurally related sulfated polyanions dextran sulfate, fucoidan, and heparin on the scavenger receptor-mediated and fluidphase endocytosis in GP8 immortalized rat brain endothelial cells were investigated. 2. Using 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarboxyamine perchlorate-labeled acetylated low-density lipoprotein (DiI-AcLDL), we found a binding site with high affinity and low binding capacity, and another one with low affinity and high binding capacity. Increasing ligand concentrations could not saturate DiI-AcLDL uptake. DiI-AcLDL uptake, but not binding, was sensitive to pretreatment with filipin, an inhibitor of caveola formation. 3. PPS (20-200 microg/ml) significantly reduced the binding of DiI-AcLDL after coincubation for 3 hr, though this effect was less expressed after 18 hr. Among other polyanions, only fucoidan decreased the DiI-AcLDL binding after 3 hr, whereas dextran sulfate significantly increased it after 18 hr. PPS treatment induced an increase in DiI-AcLDL uptake, whereas other polysulfated compounds caused a significant reduction. 4. Fluid-phase endocytosis determined by the accumulation of Lucifer yellow was concentration and time dependent in GP8 cells. Coincubation with PPS or other sulfated polyanions could not significantly alter the rate of Lucifer yellow uptake. 5. In conclusion. PPS decreased the binding and increased the uptake of DiI-AcLDL in cerebral endothelial cells, an effect not mimicked by the other polyanions investigated.

Physiological expression of the gene for PrP-like protein, PrPLP/Dpl, by brain endothelial cells and its ectopic expression in neurons of PrP-deficient mice ataxic due to Purkinje cell degeneration.

Recently, a novel gene encoding a prion protein (PrP)-like glycoprotein, PrPLP/Dpl, was identified as being expressed ectopically by neurons of the ataxic PrP-deficient (PRNP(-/-)) mouse lines exhibiting Purkinje cell degeneration. In adult wild-type mice, PrPLP/Dpl mRNA was physiologically expressed at a high level by testis and heart, but was barely detectable in brain. However, transient expression of PrPLP/Dpl mRNA was detectable by Northern blotting in the brain of neonatal wild-type mice, showing maximal expression around 1 week after birth. In situ hybridization paired with immunohistochemistry using anti-factor VIII serum identified brain endothelial cells as expressing the transcripts. Moreover, in the neonatal wild-type mice, the PrPLP/Dpl mRNA colocalized with factor VIII immunoreactivities in spleen and was detectable on capillaries in lamina propria mucosa of gut. These findings suggested a role of PrPLP/Dpl in angiogenesis, in particular blood-brain barrier maturation in the central nervous system. Even in the ataxic Ngsk PRNP(-/-) mice, the physiological regulation of PrPLP/Dpl mRNA expression in brain endothelial cells was still preserved. This strongly supports the argument that the ectopic expression of PrPLP/Dpl in neurons, but not deregulation of its physiological expression in endothelial cells, is involved in the neuronal degeneration in ataxic PRNP(-/-) mice.

Prion infection impairs the cellular response to oxidative stress.

The molecular mechanism of neurodegeneration in transmissible spongiform encephalopathies remains uncertain. In this study, it was demonstrated that prion-infected hypothalamic neuronal GT1 cells displayed a higher sensitivity to induced oxidative stress over noninfected cells. In addition, the infected cells presented an increased lipid peroxidation and signs of apoptosis associated with a dramatic reduction in the activities of the glutathione-dependent and superoxide dismutase antioxidant systems. This study indicates for the first time that prion infection results in an alteration of the molecular mechanisms promoting cellular resistance to reactive oxygen species. This finding is vital for future therapeutic approaches in transmissible spongiform encephalopathies and the understanding of the function of the prion protein.

Identification of genes associated with the progression of adult T cell leukemia (ATL).

Patients with adult T-cell leukemia/lymphoma (ATL) exhibit a variety of clinical features, and this disease is therefore clinically subclassified into acute, lymphomatous, chronic, and smoldering types. Acute ATL is a typical leukemic form of ATL with rapid progression, and chronic ATL is a less aggressive clinical form allowing long-term survival even without chemotherapy. In the present study, we used fresh peripheral blood mononuclear cells (PBMC) from both types of ATL patients to identify molecules that may contribute to the difference between acute and chronic ATL. Isolated mRNAs expressed differentially between the two types of ATL include a T-cell differentiation antigen (MAL), a lymphoid-specific member of the G-protein-coupled receptor family (EBI-1 / CCR7), a novel human homologue to a subunit (MNLL) of the bovine ubiquinone oxidoreductase complex, and a human fibrinogen-like protein (hpT49). We found that the former three are upregulated in acute ATL and the last is down-regulated in both chronic and acute ATL. We speculate that dysregulation of the genes may account for the malignant features of ATL cells, in terms of growth, energy metabolism, and motility.

Comparative molecular analysis of HTLV-I proviral DNA in HTLV-I infected members of a family with a discordant HTLV-I-associated myelopathy in monozygotic twins.

In order to elucidate the underlying mechanisms of a discordant case with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in monozygotic twins, we investigated HTLV-I tax sequences of 10 - 18 polymerase chain reaction-based clones each derived from peripheral blood mononuclear cells of the twins as well as their infected mother and an elder brother who also suffered from HAM/TSP. Sequence comparison revealed that three of the infected individuals including a twin with HAM/TSP shared the consensus tax sequence identical to the reference, ATK-1, but that of another healthy twin was different at five nucleotide positions including three nonsynonymous changes from ATK-1. This finding strongly suggested that different HTLV-I strains infected the monozygotic twins and the difference in infected proviral sequences determined the discordant clinical outcomes. Transfection and subsequent reporter assays failed to show a significant difference in transactivation activity on HTLV-I LTR and NF-kappaB elements between the products of the two sequences. Two HAM/TSP patients (a twin and elder brother) among three members infected with the ATK-1 type virus shared a paternal HLA allele which was absent in the healthy individual (mother). Genetic analysis of sequence variation in the tax sequences of the discordant twins showed that the Dn/Ds ratio was high in the healthy twin but low in the twin with HAM/TSP, implying the presence of more intense selection forces in the carrier. Our findings strongly suggested that a particular combination of HTLV-I strains with an HLA genotype would be a risk for HAM/TSP.

Up-regulation of human T lymphotropic virus type 1 (HTLV-1) tax/rex mRNA in infected lung tissues.

HTLV-1 has been implicated in certain pulmonary diseases. We previously demonstrated that expression of HTLV-1 tax/rex mRNA, encoding the transcriptional transactivator Tax, was closely associated with infiltration of activated T lymphocytes into lung tissue. To explore mechanisms of tax/rex expression in the lung, tax/rex mRNA expression and proviral DNA load were compared between peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage cells (BALC) from four patients with HTLV-1-associated myelopathy (HAM/TSP) and 13 carriers with various pulmonary symptoms. Semiquantitative detection of tax/rex mRNA strongly suggested that the lung was a preferential site for its expression. Proviral DNA loads in non-HAM/TSP carriers were variable but correlated well between PBMC and BALC in each individual, and revealed no relationship with tax/rex mRNA expression. In contrast, both cell groups from four HAM/TSP patients expressed detectable tax/rex mRNA accompanied by high proviral DNA load. The ratio of tax/rex mRNA expression to proviral DNA load was higher in BALC than in PBMC in three of four carriers and in three of four HAM/TSP patients, suggesting up-regulation of tax/rex mRNA in infected lung tissue. To analyse differences in distribution of HTLV-1 quasispecies between the two tissues, phylogenetic analysis was performed for sequence sets of the proviral tax open reading frame (ORF: 1059 bp) derived from PBMC and BALC of two infected individuals. Sequences derived from the two tissues distributed similarly to branches of phylogenetic trees, and there was no evidence of selective distribution of certain quasispecies in the lung. Our results suggest the presence of tissue-specific conditions that activate viral expression in infected cells in the lung. Constitutive exposure of this tissue to foreign antigens leading to up-regulation of basal viral promoter activity is likely to be one such mechanism.

High frequency of postnatal transmission of TT virus in infancy.

DNA of TT virus (TTV), a novel human circovirus, was tested for in 116 mother-infant pairs who had participated in the adult T-cell leukemia prevention program (APP) in Nagasaki, Japan, and refrained from breast-feeding. By polymerase chain reaction with Okamoto's seminested primers, 36 of the 115 (31%) mothers were positive. At the age of 6-8 months, 7 of 29 (24%) and 6 of 72 (8%) infants born to infected and uninfected mothers were positive, respectively (P = 0.047; RR, 2.90). Maternal TTV DNA load did not correlate with infantile infections. Since 99 of 100 (99%) cord blood samples were negative and all the mothers refrained from breast-feeding, the infantile TTV transmission would not be intrauterine or milk-borne. Between 6-8 and 12-21 months of age, 4 of 12 (33%) and 5 of 22 (23%) children born to infected and uninfected mothers turned positive, respectively (NS). At 12-21 months of age, 8 of 21 (38%) and 12 of 32 (38%) children born to infected and uninfected mothers were positive, respectively (NS). These results indicate that the TTV infection prevails in children at a frequency comparative to that in their mothers within the first 2 years of life, regardless of the maternal TTV status.

Evaluation of adult T-cell leukemia/lymphoma incidence and its impact on non-Hodgkin lymphoma incidence in southwestern Japan.

The incidence of adult T-cell leukemia/lymphoma (ATL) and its impact on that of total non-Hodgkin lymphoma (NHL) were evaluated in Nagasaki, an area in southwestern Japan where human T-cell lymphotropic virus type I (HTLV-I) is endemic. The first study area comprised 4 towns located on the K Islands, which had a population of 26,870 in 1990. The overall HTLV-I seroprevalence estimated from the serologic survey of 18,485 subjects was 16.2%. By using the data from the Nagasaki Prefectural Cancer Registry (NPCR) and reviewing clinical and laboratory information, we identified 40 cases of ATL and 35 cases of other NHL diagnosed between 1985 and 1995. The crude annual incidence of ATL among 100,000 HTLV-I carriers aged 30 or older was estimated at 137.7 for men and 57.4 for women, with a significant sex difference after adjustment for age (rate ratio = 2.50, 95% confidence interval 1.32-4.73). The cumulative risk from 30 to 79 years of age was estimated at approximately 6.6% for men and 2.1% for women. Among the entire population, ATL accounted for 51 to 59% of the total NHL incidence, showing the strong impact of HTLV-I infection. The second study area comprised the whole of Nagasaki Prefecture (total population in 1990 = 1.56 million). Between 1985 and 1995, 989 cases of ATL and 1,745 cases of other NHL were registered in the NPCR. The world age-standardized annual incidence rate of ATL per 100,000 persons aged 30 or older was estimated at 10.5 for men and 6.0 for women, which accounted for approximately 37 to 41% of the total NHL incidence.

Low copy numbers of human T-cell lymphotropic virus type I (HTLV-I) tax-like DNA detected in the salivary gland of seronegative patients with Sjögren's syndrome in an HTLV-I endemic area.

To evaluate the hypothesis, proposed in previous reports from HTLV-I non-endemic areas, that HTLV-I is involved in a significant proportion, about a quarter, of Sjögren's syndrome patients who lack serum antibodies to the virus, we examined for the presence or absence of HTLV-I in DNA samples isolated from salivary gland tissues of 17 seronegative as well as 7 seropositive patients with Sjögren's syndrome in Nagasaki, Japan, where the virus is highly endemic. The nested two-step polymerase chain reaction (PCR), with a sensitivity capable of detecting a single DNA molecule, failed to amplify the HTLV-I tax sequence from DNA of 14 of the 17 seronegative patients. The tax was only amplifiable from the tissue DNA of the remaining three seronegative patients. The detection rate, 3/17 (18%), was, unexpectedly, less than those previously reported from the HTLV-I non-endemic areas. Moreover, in contrast to high viral loads (10(-1) to 10(-3) per cell) in the salivary gland of the seropositive patients, a semiquantitative PCR revealed that the copy number of the HTLV-I tax in the gland tissue of these seronegative patients was very low, 10(-5) per cell. This level is unlikely to be sufficient to promote an inflammatory reaction in the tissue. Our findings might argue against the involvement of "prototype" HTLV-I in the pathogenesis of Sjögren's syndrome in seronegative patients.

Deleted HTLV-1 provirus in cord-blood samples of babies born to HTLV-1-carrier mothers.

We screened 596 cord-blood samples by nested short PCRs for the gag and pX regions of HTLV-1 which are capable of detecting a single copy. The samples were derived from 449 and 147 babies born to seropositive and seronegative mothers respectively. Of these, 20 samples were positive in at least one of the PCRs: 9 (45%) were positive in both PCRs, but 10 and 1 samples in either the pX or the gag PCR respectively. These samples were tested further in nested long PCRs directed for gag-pX, gag-pol and pol-pX regions capable of detecting 8, 1 and 2 copies respectively. Of 9 dually positive samples, 7 (77%) showed the predicted 6.2-kbp band in the gag-pX PCR; only 2 of them had the predicted band alone; 7 samples had discrete bands shorter than the predicted size. In the gag-pol PCR, all 9 samples showed the predicted 2.2-kbp band alone. In the pol-pX PCR, 819 samples showed the predicted 4.2-kbp band, including one with an additional 2.1-kbp band, and the last a 1.0-kbp band alone. Thus, all of the dually positive samples had proviruses harboring gag, pol and pX priming sites. In contrast, none of the 11 singly positive samples showed the predicted band in the gag-pX PCR: 5 had no visible band, and the other 6 had shorter bands only. None of these 11 samples showed any positive signal in either gag-pol or pol-pX PCR. Our results suggest that HTLV-1 proviruses in the cord blood are frequently defective.

Cultured skin fibroblasts isolated from mice devoid of the prion protein gene express major heat shock proteins in response to heat stress.

Recent evidence has suggested that molecular chaperones participate in the conformational change between the normal cellular prion protein (PrPC) and its scrapie isoform (PrPSc). To study a role of PrPC in the regulation of expression of heat shock proteins (HSPs), a group of molecular chaperones, heat-induced expression of major HSPs (HSP105, HSP90alpha, HSP72, HSC70, HSP60, and HSP25) was investigated in cultured skin fibroblasts isolated from the mice homogeneous for a disrupted PrP gene (PrP-/- mice) by Western blot analysis and immunocytochemistry. Two lines of fibroblasts were established and designated SFK derived from the PrP-/- mice and SFH derived from the PrP+/+ mice, respectively. In both SFK and SFH cells, HSP105, HSP72, and HSP25 were expressed at low levels under unstressed conditions but they were induced markedly following exposure to heat stress (43 degreesC/20 min) at 3-72 h postrecovery. In both cell types, HSC70 and HSP60 were expressed at high levels under unstressed conditions and their levels remained unchanged after heat shock treatment. HSP90alpha was undetectable in both cell types under any conditions examined. The pattern of expression, induction, and subcellular location of HSP105, HSP72, HSC70, HSP60, and HSP25 was not significantly different between SFK and SFH cells under unstressed and heat-stressed conditions. Furthermore, the levels of constitutive expression of HSP105, HSC70, HSP60, and HSP25 were similar between the brain tissues isolated from the PrP-/- and PrP+/+ mice. These results indicate that HSP induction is not affected by either the existence or the absence of PrPC in the cells.

Primary prevention of HTLV-1 in Japan.

The Nagasaki Prefecture, Japan (population: 1.5 million), is one of the hot endemic foci of Human T-lymphotropic virus type 1 (HTLV-1). Prevalence of HTLV-1 carriers are approximately 10% in the age group over 40 years old (40,000 individuals), approximately 10 times of the national average. Annual registry of adult T-cell leukemia (ATL) in the Prefecture is approximately 60 cases (estimated incidence: 100 cases), or a half percent of total deaths. A effective measure to control the endemic cycle of HTLV-1 has been imperative, since practical ways to prevent or control ATL are not available. A prefecture wide intervention at Nagasaki by refrain from breast-feeding blocked approximately 80% of mother-to-child transmission of HTLV-1.

Integration patterns of HTLV-I provirus in relation to the clinical course of ATL: frequent clonal change at crisis from indolent disease.

We examined human T-lymphotropic virus type I (HTLV-I) DNA integration in 68 patients with adult T-cell leukemia/ lymphoma (ATL) by Southern blotting using EcoRI, which does not cut within the 9 kb of the genome and probes for pX and gag-pol region of HTLV-I. We detected defective proviral integration as a monoclonal band of various sizes with the pX but not with the gag-pol probe, or a monoclonal band of less than 9 kb with the pX probe, in 20 patients (29.4%). These were designated defective (D) type. With both probes, a single band greater than 9 kb was detected in 34 (50.0%), designated complete (C) type, and two or more bands greater than 9 kb, were designated multiple (M) type, in 14 (20.6%). Advanced age, a high LDH value, and hypercalcemia were more frequent in D type patients. The median survival time (MST) was 6.8, 24.4, and 33.3 months, for D, C, and M types, respectively (log rank P = .006). Among 52 sequentially examined patients, the HTLV-I integration patterns changed in 4 (7.5%). In three of these four, the rearrangements of the T-cell receptor (TCR)b gene concomitantly changed, suggesting the appearance of a new ATL clone. Another patient had the same rearrangement of the TCRb gene, indicating clonal evolution. The HTLV-I integration pattern changed at crisis from indolent to aggressive ATL in three patients. These findings suggested that the HTLV-I integration patterns have clinical implications in ATL pathophysiology. In contrast to the clonal evolution characteristic of the multistep carcinogenesis of most human malignancies, the frequent clonal change of ATL at crisis is a peculiar phenomenon, probably reflecting the emergence of multiple premalignant clones in viral leukemogenesis as suggested in Epstein-Barr virus associated lymphomagenesis in the immunocompromised host.

High seroprevalence of anti-HTLV-I antibody in rheumatoid arthritis.

OBJECTIVE: To investigate the association between human T lymphotropic virus type I (HTLV-I) infection and rheumatoid arthritis (RA) in Nagasaki, an area highly endemic for HTLV-I infection. METHODS: Sera from 113 female patients with RA and 19,796 female blood donors were screened for anti-HTLV-I antibodies with a gelatin particle agglutination kit and confirmed using an immunoblotting kits. RESULTS: The age-adjusted summary odds ratio of HTLV-I infection among RA patients, as compared with blood donors, was 2.8 (95% confidence interval [95% CI] 1.8-4.6). The etiologic fraction, i.e., the proportion of RA in the study population that is attributable to HTLV-I infection, was estimated to be 13.2% (95% CI 5.1-21.2). There was no significant difference in the clinical and laboratory findings between HTLV-I- infected and HTLV-I-uninfected RA patients. CONCLUSION: These epidemiologic findings support the idea that HTLV-I infection is a risk factor for RA, and suggest that approximately 13 % of the cases of RA in females living in Nagasaki are associated with HTLV-I infection.

Activity of Fgr protein-tyrosine kinase is reduced in neutrophils of patients with myelodysplastic syndromes and chronic myelogenous leukemia.

The Fgr protein-tyrosine kinase, p55(c-fgr), is specifically expressed and functions in cells of myelomonocytic lineages. We examined levels of expression and enzymatic activity of p55(c-fgr) peripheral blood neutrophils of patients with myelodysplastic syndromes (MDS) and chronic myelogenous leukemia (CML) by comparison with those of normal individuals. While neutrophils of eight normal subjects gave uniform results, the specific enzymatic activity of p55(c-fgr), a ratio of the total kinase activity versus the protein level was reduced in seven out of eight patients with MDS and all of five patients with CML. The specific kinase activity of p55(c-fgr) correlated significantly with the activity of neutrophil alkaline phosphatase (NAP) which has been considered to be a marker of neutrophil maturity (r=0.568, P<0.01). The reduced activity of this tyrosine kinase was considered to be a biological parameter for immaturity and to reflect dysfunction of neutrophils of patients with MDS and with CML.

[HTLV-I infection in primary Sjögren's syndrome--epidemiological, clinical and virological studies].

The HTLV-I seroprevalence rate among the patients with Sjögren's syndrome (SjS, 23.0%) was significantly higher than that among blood donors (3.4%). The age-adjusted summary odds ratio of HTLV-I infection among SjS patients as compared with blood donors was 3.1. The etiologic fraction, i.e., the proportion of SjS in the study population that are attributable to HTLV-I infection, was estimated to be 17.6%. Titers of serum antibodies to HTLV-I in the seropositive SjS patients were significantly higher than those among healthy carriers. IgM class antibodies were commonly detected in sera of SjS patients. Salivary IgA class antibodies were common among seropositive SjS patients, but not in HAM patients or in healthy subjects. The findings strongly suggest that HTLV-I is involved in the pathogenesis of the disease in a subset of patients with SjS in endemic areas.