Human immune responses to dengue viruses

Dengue fever (DF) and dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS) are major public health problems in many areas of the world. We are analyzing the human immune responses to dengue viruses, in order to understand the mechanism of recovery from dengue virus infections and the pathogenesis of DHF/DSS. Human natural killer (NK) cells lyse dengue virus-infected cells to a greater degree than uninfected cells. Antibodies to dengue viruses augment the lysis of dengue virus-infected cells by NK cells. Dengue virus-infected monocytes produce high levels of interferon alpha (IFN alpha). DR+ lymphocytes also produce high levels of IFN alpha after contact with dengue virus-infected monocytes. The IFN alpha produced protects uninfected monocytes from dengue virus infection. These results suggest that NK cells and IFN alpha may play an important role in controlling primary dengue virus infection. Dengue virus-specific CD4+CD8(-)T lymphocytes and CD4(-)CD8+T lymphocytes are present in the peripheral blood mononuclear cell population from donors who were infected with dengue virus. Most of CD4+T lymphocytes are dengue serotype-crossreactive. They lyse dengue virus-infected autologous cells in an HLA class II-restricted fashion, and produce interferon gamma (IFN gamma). IFN gamma augments dengue virus infection of monocytic cells in the presence of antidengue virus antibodies by increasing the number of Fc gamma receptors. Dengue virus-specific CD8+T lymphocytes lyse dengue virus-infected autologous cells in an HLA class I-restricted fashion. These CD8+T lymphocytes are also dengue serotype-crossreactive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitation of T lymphocyte subsets helps to distinguish dengue hemorrhagic fever from classic dengue fever during the acute febrile stage

Activation of immunoregulatory T lymphocyte subsets has been observed in dengue viral infection, being more evident in dengue hemorrhagic fever (DHF) than in classical dengue fever (DF). There are, however, as yet no well-defined host markers to determine which patients with dengue viral infection will develop severe complications during the acute febrile stage of the disease. A study was performed to compare the cellular immune status in DHF, DF and non-dengue viral infections (NDF) in order to determine the value of these parameters in distinguishing DHF from classic DF and other viral infections during the acute febrile stage of the disease. This study involved 109 febrile patients admitted because of suspected DHF. Fifty patients were serologically confirmed cases of dengue infection, of which 25 had grade 1 or 2 DHF. There was a reduction in total T (CD3), CD4 and CD8 cells in DHF and demonstrated that a low level of CD3, CD4, CD8 and CD5 cells discriminated DHF from DF patients during the febrile stage of the illness. In contrast, B (CD19) cells and natural killer (NK) cells did not appear to be discriminatory in this study. Receiver operating characteristic (ROC) curve analysis showed that a combination of CD3 cell of < or = 45% and CD5 cell of < or = 55% was the best marker to identify DHF patients (sensitivity = 84% and specificity = 52% for CD3 cell of < or = 45%; sensitivity = 92% and specificity = 71% for CD5 cell of < or = 55%). CD4 cell of < or = 25% and CD8 cell < or = 30% were equally good in discriminating DHF from DF patients. On the other hand, the ROC curves indicated no clear difference between the immunoregulatory cell counts in DF from NDF Lymphopenia, atypical lymphocytosis and thrombocytopenia were significantly more evident in dengue compared to non-dengue infection but did not appear to be discriminatory among DHF and DF patients. The reduction in CD3, CD4, CD8, CD5 cells correlated with the degree of thrombocytopenia in DHF (p < 0.05) which suggests that these cells probably participate in a common pathogenetic mechanism.     

Demonstration of dengue antibody complexes on the surface of platelets from patients with dengue hemorrhagic fever.

By the direct immunofluorescent technic, dengue antigen, human immunoglobulins, and beta 1C globulin were detectable on the surfaces of platelet suspensions from 48% of patients with dengue hemorrhagic fever. The percentages of positive-staining platelets were not related to the severity of thrombocytopenia, which was marked on the day after the patient developed shock or subsidence of fever. It is suggested that an immunologic mechanism is one of the factors associated with the thrombocytopenia caused by increased platelet destruction.     

登革病毒感染者外周血T淋巴细胞亚群数量的变化及其临床意义

目的观察登革热患者外周血T淋巴细胞亚群数量变化的特点并探讨其临床意义。方法用流式细胞仪检测不同发病时间71例登革热患者外周血中总T淋巴细胞(CD3+)、CD4+T淋巴细胞和CD8+T淋巴细胞的数量,并将急性期患者、恢复期患者及正常人T淋巴细胞及其亚群数量进行比较,组间数据比较采用t检验。结果与正常对照组相比,急性期登革热患者T细胞和CD4+T细胞都显著减少(P<0.01)、CD4+T/CD8+T比值显著降低(P<0.01),恢复期登革热患者上述指标没有显著性差异(P>0.05);与急性期相比,恢复期登革热患者CD4+T细胞显著升高(P<0.01);急性期、恢复期登革热患者和正常对照组CD8+T细胞数量没有显著性差异(P>0.05)。登革热患者T淋巴细胞亚群数量和CD4+T/CD8+T比值随着发病时间的增加而逐渐上升。结论登革热患者急性期细胞免疫异常,通过治疗可以逐渐恢复。T淋巴细胞亚群的检测可作为监测登革热患者免疫功能的一个指标,对于病人的治疗和预后有一定的意义。细胞免疫异常可能在登革热的发病中起重要作用。     

Neonatal dengue infection: report of dengue fever in a 1-day-old infant

A male infant was admitted because of fever. He was born at 37-weeks' gestation. His mother had experienced acute febrile illness with headache and myalgia. Her illness persisted with onset of active labor pain on day 5, which prompted cesarean section; postoperatively, the hematocrit decreased, requiring transfusion. The infant was well until fever developed at 16 hours after birth. There were petichiae on his face and trunk and the liver was enlarged. Fever subsided on day 5 without evidence of plasma leakage or severe hemorrhage. He made an uneventful recovery after 8 days of illness. Leukopenia and thrombocytopenia were present in the mother and infant. Both were diagnosed as dengue fever. Dengue type 1 was recovered from the infant by polymerase chain reaction. The dengue enzyme-linked immunoassay showed secondary infection in the mother and primary infection in the infant. In dengue-endemic areas, clinicians should be alert to dengue fever/dengue hemorrhagic fever in pregnant women presenting with acute febrile illness, and be prepared for proper management.     

The pattern and nature of the lymphocyte population response in dengue hemorrhagic fever.

Eighty-eight specimens consisting of lymphocytes separated from peripheral blood samples from 76 patients with dengue hemorrhagic fever (DHF) were studied. The results revealed a significant increase in numbers of atypical lymphocytes and B lymphocytes, and a decrease in T lymphocytes by comparison with normal controls. Anti-T lymphocyte antibody was also detected in the serum of all patients with DHF. The greatest increments and decrements of the above were noted on the day of shock of subsidence of fever.     

Parturient and perinatal dengue hemorrhagic fever

A Thai woman in the shock stage of dengue hemorrhagic fever delivered a healthy infant by normal delivery. She had high, prolonged fever for five days, hepatomegaly, thrombocytopenia and a right pleural effusion. The serology performed later established a diagnosis of secondary dengue infection. She had no serious complications except for postpartum anemia. The newborn became febrile at the 48th hour of life. He had fever for 2 days, hepatomegaly, thrombocytopenia and a right pleural effusion. Dengue virus type 1 was detected from his serum by polymerase chain reaction (PCR). Although he developed prolonged, marked thrombocytopenia (12 days), his illness was uncomplicated. He recovered uneventfully and was discharged with his mother. This report was one of dengue vertical transmission with dengue hemorrhagic fever in humans.     

Defective immunoregulation in primary biliary cirrhosis: CD4+, Leu-8+ T cells have abnormal activation and suppressor function in vitro

To determine whether abnormalities of lymphocyte function in primary biliary cirrhosis are due to altered function of immunoregulatory T cell subpopulations, phenotypic and functional characteristics of CD4+ T cells were examined. The proportion of CD4+ T cells expressing the Leu-8 and CD45R antigens was normal in patients with primary biliary cirrhosis. The capacity of CD4+, Leu-8- T cells to provide helper function for pokeweed mitogen-stimulated immunoglobulin synthesis by B cells in vitro was similar in patients and controls. However, in contrast to normal individuals and patients with other liver diseases, CD4+, Leu-8+ T cells from six of 10 patients with primary biliary cirrhosis did not suppress, but enhanced immunoglobulin synthesis. Whereas treatment of CD4+ T cells from normal individuals with anti-Leu-8 monoclonal antibody enhanced their suppressor function, similar treatment of CD4+ T cells from patients with primary biliary cirrhosis did not increase their suppressor function. To determine whether the abnormal regulatory function of CD4+, Leu-8+ T cells was due to a defect of cell activation, the proliferative response of CD4+ T cell subpopulations to mitogenic stimulation was examined. The proliferative responses of CD4+, Leu-8- T cells from patients with primary biliary cirrhosis and controls were similar, but the proliferative responses of CD4+, Leu-8+ T cells from patients with primary biliary cirrhosis were lower than those of control cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early CD69 expression on peripheral blood lymphocytes from children with dengue hemorrhagic fever.

Recent reports have demonstrated immune activation in dengue hemorrhagic fever (DHF) by cytokine and soluble receptor detection in blood. The goal of this study was to determine which cell types are activated and likely to be responsible for cytokine production. Whole blood specimens from 51 Thai children presenting within 72 h of fever onset and with detectable plasma dengue viral RNA were studied by flow cytometry. Absolute CD4 T cell, CD8 T cell, NK cell, and gammadelta T cell counts were decreased in children with DHF compared with those with dengue fever (DF) early in the course of illness. The percent of cells expressing CD69 was increased on CD8 T cells and NK cells in children who developed DHF more than in those with DF. These data directly demonstrate that cellular immune activation is present early in acute dengue and is related to disease severity.     

Cellular immune activation in children with acute dengue virus infections is modulated by apoptosis

Apoptosis is an important modulator of cellular immune responses during systemic viral infections. Peripheral-blood mononuclear cell (PBMC) apoptosis and plasma soluble levels of CD95, a mediator of apoptosis, were determined in sequential samples from children participating in a prospective study of dengue virus (DV) infections. During the period of defervescence, levels of PBMC apoptosis were higher in children developing dengue hemorrhagic fever (DHF), the most severe form of illness, than in those with dengue fever (DF) and other, nondengue, febrile illnesses. CD8(+) T lymphocytes made up approximately half of the peak circulating apoptotic PBMCs in DHF and DF. Maximum plasma levels of soluble CD95 were also higher in children with DHF than in those with DF. The level of PBMC apoptosis correlated with dengue disease severity. Apoptosis appears to be involved in modulation of the innate and adaptive immune responses to DV infection and is likely involved in the evolution of immune responses in other viral hemorrhagic fevers.     

Peripheral blood T,B, and NK cells in relation to histological hepatitis activity and fibrosis stage in chronic hepatitis C

BACKGROUND/AIMS: Many data on the pathogenesis of chronic hepatitis C have pointed to host's immune system disorders and a high variety of virus. However, there are no known criteria that could prognose the course of chronic hepatitis C infection. The analysis of T and B lymphocyte subpopulations in the peripheral blood was undertaken in patients with chronic hepatitis C of more than 6 months of duration. METHODOLOGY: Fluorescein isothiocyanate or phycoerythryne conjugated monoclonal antibodies for CD3+, CD4+, CD8+, CD19+, CD3++ HLA DR+, CD16++ CD56+ were used. The correlation between histological hepatitis activity and fibrosis (according Scheuer's scale) and the distribution of lymphocytes in the peripheral blood was sought. RESULTS: All patients with chronic hepatitis showed statistically significant increase in active lymphocytes CD3++ HLA DR+ and CD16++ CD56+ NK cells in peripheral blood. We observed the correlation between these cells and histological hepatitis activity and fibrosis. There was no correlation between the value of CD3+ and CD8+ cells and the stage of liver failure. In the early stage of chronic hepatitis C we noted decrease CD4+ cells with increase B cells CD19+. CD4+/CD8+ ratio was maintained as slightly decreased in chronic hepatitis C in favor of lymphocytes CD8+. CONCLUSIONS: The results show the correlation between peripheral blood value of activated T cell (HLA DR+) and NK cells with histological activity and fibrosis in chronic hepatitis C. Lymphocyte T (CD4+, CD8+) and B (CD19+) did not correlate with grade and stage of hepatitis C.     

Classification of dengue illness based on readily available laboratory data

The aim of this study was to examine retrospective dengue-illness classification using only clinical laboratory data, without relying on X-ray, ultrasound, or percent hemoconcentration. We analyzed data from a study of children who presented with acute febrile illness to two hospitals in Thailand. Multivariable logistic regression models were used to distinguish: (1) dengue hemorrhagic fever (DHF) versus dengue fever (DF), (2) DHF versus DF + other febrile illness (OFI), (3) dengue versus OFI, and (4) severe dengue versus non-severe dengue + OFI. Data from the second hospital served as a validation set. There were 1,227 patients in the analysis. The sensitivity of the models ranged from 89.2% (dengue versus OFI) to 79.6% (DHF versus DF). The models showed high sensitivity in the validation dataset. These models could be used to calculate a probability and classify patients based on readily available clinical laboratory data, and they will need to be validated in other dengue-endemic regions.     

Resolution of primary murine listeriosis and acquired resistance to lethal secondary infection can be mediated predominantly by Thy-1+ CD4- CD8- cells.

Mice depleted of CD4+, CD8+, Thy-1+ CD4- CD8-, or all three types of cells by appropriate monoclonal antibody treatment were tested for their ability to express resistance against a primary and secondary Listeria monocytogenes infection. It was found that mice depleted of CD8+, CD4+, or both of these subsets of T cells remained capable of controlling and then resolving both a primary infection and a secondary infection lethal for normal animals. In contrast, depleting mice of a population of Thy-1+ CD8- CD4- cells that remain after CD4+ and CD8+ T cells had been removed, rendered them incapable of resolving a sublethal primary infection or of expressing acquired immunity to a lethal secondary infection. The results point to the possibilities that Thy1+ CD4- CD8- alpha beta T cells, Thy1+ CD4- CD8- gamma delta T cells, Thy-1+ NK cells, or all three types of these host cells are involved in anti-L. monocytogenes resistance.     

Early immune activation in acute dengue illness is related to development of plasma leakage and disease severity

T lymphocyte activation and increased cytokine levels have been described in retrospective studies of children presenting with dengue hemorrhagic fever (DHF). Serial plasma samples obtained in a prospective study of Thai children presenting with <72 h of fever were studied. Plasma levels of 80-kDa soluble tumor necrosis factor receptors (sTNFRs) were higher in children who developed DHF than in those with dengue fever (DF) or other nondengue febrile illnesses (OFIs) and were correlated with the degree of subsequent plasma leakage. Soluble CD8 and soluble interleukin-2 receptor levels were also elevated in children with DHF compared with those with DF. Interferon-gamma and sTNFR 60-kDa levels were higher in children with dengue than in those with OFIs. TNF-alpha was detectable more often in DHF than in DF or OFIs (P<.05). These results support the hypothesis that immune activation contributes to the pathogenesis of DHF. Further studies evaluating the predictive value of sTNFR80 for DHF are warranted.     

大强度运动对大鼠脾脏淋巴细胞、巨噬细胞、T淋巴细胞亚群的影响

目的探讨大强度运动对大鼠脾脏淋巴细胞、巨噬细胞、T淋巴细胞亚的影响。方法40只SD大鼠按体重随机分为安静组(A组,n=10)、运动组(B组,n=30),在最后一次力竭运动后,将B组大鼠分为运动后即刻组(B 1组,n=10)、运动后12 h组(B 2组,n=10),运动后24 h组(B 3组,n=10)。A组不运动,B组大鼠进行为期9 w的大强度训练,末次训练后,A组、B 1组摘取脾脏,制作脾细胞悬液,噻唑蓝(MTT)法分别测T淋巴细胞增殖能力、B淋巴细胞增殖能力,流式细胞仪测CD4^+、CD8^+细胞数。中性红比色法测巨噬细胞吞噬能力。B 2组、B 3组分别在运动后12 h、运动后24 h按上述方法测试。结果与A组相比,B 1组T淋巴细胞增殖、B淋巴细胞增殖、CD4^+、CD8^+、CD4^+/CD8^+、巨噬细胞吞噬能力显著降低,B 2组T淋巴细胞增殖、B淋巴细胞增殖、CD4^+、CD4^+/CD8^+显著降低,巨噬细胞吞噬能力差异无统计学意义,B 3组T淋巴细胞、B淋巴细胞增殖能力、CD4^+、CD8^+、CD4^+/CD8^+、巨噬细胞吞噬能力差异无统计学意义;与B 1相比,B 2组、B 3组T淋巴细胞增殖、B淋巴细胞增殖、CD4^+、CD8^+、CD4^+/CD8^+、巨噬细胞吞噬能力显著升高;与B 2相比,B 3组T淋巴细胞增殖、B淋巴细胞增殖、CD4^+均显著升高,CD8^+、CD4^+/CD8^+、巨噬细胞吞噬能力无显著差异。结论大强度运动后,大鼠脾脏、B淋巴细胞、T淋巴增殖能力降低、巨噬细胞吞噬能力降低、CD3^+、CD4^+、CD8^+、CD4^+/CD8^+降低,恢复期间逐步上升,其中CD8^+、CD4^+/CD8^+、巨噬细胞吞噬能力在恢复期12 h基本达到正常状态,T淋巴细胞增殖能力、B淋巴细胞增殖能力、CD4^+在恢复期24 h达到正常状态。     

Evidence that maternal dengue antibodies are important in the development of dengue hemorrhagic fever in infants

To establish the role of maternal dengue-specific antibodies in the development of dengue hemorrhagic fever and dengue shock syndrome caused by dengue 2 virus in infants, we examined sera from mothers of infants and toddlers with dengue hemorrhagic fever or dengue shock syndrome and mothers of infants with pyrexia of unknown origin. The mean titers of hemagglutination inhibition, neutralization, and infection-enhancing activities against dengue 2 virus were not statistically different among the three groups. However, among infants who developed dengue hemorrhagic fever/dengue shock syndrome there was a strong correlation between the mothers' dengue 2 neutralizing titers and infant age at the time of onset of severe illness, where no such correlation was found among the other two groups. Furthermore, the actual age at which dengue hemorrhagic fever/dengue shock syndrome occurred in each infant correlated with the age at which maximum enhancing activity for dengue 2 infection in mononuclear phagocytes was predicted. This critical time for the occurrence of dengue hemorrhagic fever/dengue shock syndrome was observed to be approximately 2 months after the time calculated for maternal dengue 2 neutralizing antibodies to degrade below a protective level. In addition, sera of mothers of infants with dengue hemorrhagic fever/dengue shock syndrome enhanced dengue 2 virus infection to a slightly greater degree than did sera from mothers of infants with pyrexia of unknown origin and toddlers with dengue hemorrhagic fever/dengue shock syndrome. These data are consistent with the hypothesis that maternal dengue antibodies play a dual role by first protecting and later increasing the risk of development of dengue hemorrhagic fever/dengue shock syndrome in infants who become infected by dengue 2 virus.     

Case report: Dengue hemorrhagic fever with encephalopathy in an adult

Encephalopathy in dengue hemorrhagic fever is a very rare condition and usually occurrs in the febrile stage. We report a 29-year-old woman, who presented with acute fever, thrombocytopenia and positive IgM antibodies for dengue virus. On the fourth hospital day, the fever subsided and she developed a confusional stage. CT scan and MRI of the brain were within normal limits. Electroencephalography (EEG) revealed generalized theta waves. Cerebrospinal fluid was normal. She was treated with supportive treatment. Five days later, she was fully recovered without any neurological deficits. This is a first case of encephalopathy in dengue hemorrhagic fever that developed after the fever subsided.     

Studies on natural killer cells in chronic myeloid leukemia patients in remission.

In our earlier studies, the natural killer (NK) cell mediated cytotoxicity was found to be impaired in 55% of chronic myeloid leukemia (CML) patients in remission (low NK responders), while the rest exhibited normal range of cytotoxicity (normal NK responders). In the present investigations probable factors contributing to the impaired NK activity of low NK responder CML patients have been analyzed. Peripheral blood lymphocytes (PBL) from both low and normal NK responder CML patients possessed normal percentages of HNK-1+, CD3+ and CD8+ cells. The proportion of HNK-1+ cells and CD8+ cells in low density fractions (LDF) and high density fractions (HDF) respectively of Percoll separated PBL from low and normal NK responder CML patients and healthy donors was comparable. However, the NK activity of LDF cells of low NK responder CML patients was much lower. Also, HDF cells from low NK responder CML patients exhibited a regulatory effect on NK cytotoxicity of PBL from healthy donors. Therefore, it is likely that the presence of suppressor cells and perhaps an intrinsic inability of HNK-1+ cells may together contribute to the impaired NK cytotoxicity of low NK responder CML patients.     

Changes in intraepithelial lymphocyte subpopulations in coeliac disease and enteropathy associated T cell lymphoma (malignant histiocytosis of the intestine).

Studies of the morphologic and phenotypic diversity of intraepithelial T cells in human small intestine have shown them to be heterogeneous, yet distinct from most extra intestinal T cells. In this study sequential immunoenzymatic staining was used to define new intraepithelial lymphocyte subpopulations in man. In normal human jejunum approximately 6% of the intraepithelial T cells expressing CD3 (an antigen associated with the T cell receptor) do not express the T cell subset antigens CD4 or CD8. Approximately 20% of CD7+ cells (T cells and null cells) do not express CD4 or CD8 and 14% of the CD7+ cells do not express CD3 and are therefore not T cells. The CD7+, CD3+/-, CD4-, CD8- population is concentrated in the tips of the villi. In coeliac disease, the ratios of the subsets change significantly. The percentage of CD3+, 4-, 8- cells increases to 28%, the proportion of CD7+, 4-, 8- cells remains unchanged and the CD7+, CD3- (non-T cell) population is reduced to 1.4% of the CD7+ cells. In contrast, in patients with villous atrophy of uncertain aetiology, all CD4-, CD8- lymphocyte subsets are decreased compared with normal biopsies. Finally, in enteropathy associated T cell lymphoma (malignant histiocytosis of the intestine) in which the 'uninvolved mucosa' is histologically similar to untreated coeliac disease, the changes in the intraepithelial T cell sub-sets are indistinguishable from those in coeliac disease, suggesting that the lymphoma is a complication of coeliac disease.     

In vitro inhibition of normal human hematopoiesis by marrow CD3+, CD8+, HLA-DR+, HNK1+ lymphocytes

We previously demonstrated that after allogeneic bone marrow transplantation (BMT) a subset of CD8, HNK1, and DR-positive T lymphocytes are able to inhibit CFU-GM and BFU-E growth with an HLA-DR restriction. In this study we investigated whether these cells, present in normal marrow in low concentration (less than 1%), play the same role. HNK1-positive sorted marrow cells forming rosettes (E+C) were able to inhibit BFU-E and CFU-GM growth when added back to the marrow E-C at a ratio of 1:10 (HNK1+ E+C/E-C) in a range from 40% to 60%. This inhibitory effect was also detected for a cellular ratio of 1:100, which is the normal marrow value for this subset of T cell. HNK1+ DR+-sorted E+C after double-immunofluorescent labeling also showed the same inhibitory activity as the HNK1+ E+C, whereas the negative fraction including all the other E+C had no detectable inhibitory activity. CD3 and CD8 antigens were also present on the membrane of these cells, as demonstrated in two cases by double-immunofluorescent labeling performed with anti-CD3 or anti-CD8 monoclonal antibodies (MoAbs) and HNK1 MoAb, respectively, and subsequent cell sorting. Blocking experiments, performed by adding in culture anti-CD4 and anti-CD8 MoAbs to HNK1+ T cells showed that only the last MoAb was able to prevent inhibition of hematopoietic colony growth. These results confirmed that one subset of CD3+, CD8+, HNK1+, and DR+ T cells was responsible for in vitro inhibition of normal hematopoiesis. In addition, this inhibition was genetically restricted to HLA-class II antigens, since in three co-culture experiments with unrelated bone marrow cells inhibition occurred only when cells with one haplo-identical HLA-DR antigen was added back to the culture. Indeed, this effect was really HLA-DR restricted, since in blocking experiments with different anti-HLA class II MoAbs (anti-DR, anti-DP, and anti-DQ MoAbs) only an anti-HLA-DR MoAb was able to prevent the colony growth inhibition by CD3+ HNK1+, or CD8+ HNK1+ E+C. In conclusion, the CD3+, HNK1+, CD8+, DR+ cells may be the T-cell subset able to inhibit normal hematopoiesis with an HLA-DR restriction.