Interindividual conservation of T-cell receptor beta chain variable regions by minor histocompatibility antigen-specific HLA-A*0201- restricted cytotoxic T-cell clones

Minor histocompatibility antigens (mHags) are involved in the induction of graft-versus-host disease (GVHD) after HLA-identical bone marrow transplantation. Previously, we isolated a series of HLA-A*0201- restricted cytotoxic T-cell (CTL) clones specific for the same mHag HA- 1 from peripheral blood of three unrelated patients who were suffering from GVHD. We have now analyzed the composition of the T-cell receptor (TCR) V regions of 12 of these mHag HA-1-specific HLA-A*0201-restricted CTL clones by DNA sequencing of the alpha and beta chains. Of these 12 clones, derived from three unrelated individuals, five independent TCR alpha V- and beta V-region sequences were established. The TCR alpha chains were composed of varying TCR alpha V and TCR alpha J genes with no obvious similarities in structure in the N regions. However, the TCR beta chains all used the TCR beta V6S9 gene segment, and showed remarkable similarities within the N-D-N regions; ie, three independent beta-chain sequences (originating from donors Ha and Gy) shared a leucine/valine amino acid pair, whereas the other two (originating from donors Ha and Wi) shared a serine/threonine pair, all at positions 99 and 100 of the TCR beta V region. In conclusion, the TCR analysis of HA- 1 mHag-specific CTL clones has shown that the HA-1 mHag/HLA-A*0201 complex selects for highly similar TCR beta V regions.     

Engraftment of bone marrow cells into normal unprepared hosts: effects of 5-fluorouracil and cell cycle status

Bone marrow from animals treated with 5-fluorouracil (5FU) competes equally with normal marrow when assessed in vivo in an irradiated mouse, but shows markedly defective engraftment when transplanted into noncytoablated hosts. Using Southern Blot analysis and a Y-chromosome specific probe, we determined the level of engraftment of male donor cells in the bone marrow, spleen, and thymus of unprepared female hosts. We have confirmed the defective engraftment of marrow harvested 6 days after 5FU (FU-6) and transplanted into unprepared hosts and shown that this defect is transient; by 35 days after 5FU (FU-35), engraftment has returned to levels seen with normal marrow. FU-6 marrow represents an actively cycling population of stem cells, and we hypothesize that the cycle status of the stem cell may relate to its capacity to engraft in the nonirradiated host. Accordingly, we have evaluated the cycle status of engrafting normal and FU-6 marrow into normal hosts using an in vivo hydroxyurea technique. We have shown that those cells engrafting from normal marrow and over 70% of the cells engrafting from FU-6 marrow were quiescent, demonstrating no killing with hydroxyurea. We have also used fluorescent in situ hybridization (FISH) analysis with a Y-chromosome probe and demonstrated that normal and post-5FU engraftment patterns in peripheral blood were similar to those seen in bone marrow, spleen, and thymus. Altogether these data indicate that cells engrafting in normal, unprepared hosts are dormant, and the defect that occurs after 5FU is concomitant with the induction of these cells to transit the cell cycle.     

The significance of HLA-DRB1 matching on clinical outcome after HLA-A, B, DR identical unrelated donor marrow transplantation

Despite matching for serologically defined HLA-A, B, DR antigens, acute graft-versus-host disease (GVHD) is a major complication contributing to increased morbidity and mortality in patients who undergo marrow transplantation from unrelated donors. The extent to which unrecognized mismatching for alleles that encode DR1-DR18 contribute to the increased risk of acute GVHD and overall survival is unknown. We analyzed 364 patients and their HLA-A, B, DR serologically matched donors to determine whether molecular typing of DRB1 alleles can allow more accurate donor/recipient matching and thereby improve clinical outcome after marrow transplantation. DRB1 alleles were typed by sequence-specific oligonucleotide probe hybridization methods. Selected alleles were confirmed by DNA sequencing. Of the 364 pairs, 305 were matched and 59 were mismatched for DRB1. The probability of moderate to severe acute GVHD was .48 for the matched and .70 for the mismatched patients. Compared with mismatched patients, the estimated relative risk (RR) of GVHD for matched patients was .58 (95% confidence interval [CI], .40 to .85). DRB1 matching decreased the risk of transplant- related mortality (RR, .66; 95% CI, .44 to .97) and was associated with decreased overall mortality (RR, .71; 95% CI, .51 to 1.0). Therefore, matching DRB1 alleles of the donor and recipient decreases the risk of acute GVHD and improves survival after unrelated marrow transplantation. These results indicate that prospective matching of patients and donors for DRB1 alleles is warranted.     

Unrelated donor marrow transplantation in children

Eighty-eight children 0.5 to 17 years of age (median, 9 years of age) received an unrelated donor marrow transplant for treatment of chronic myeloid leukemia (CML; n = 16), acute lymphoblastic leukemia (ALL) in first or second remission (n = 15) or more advanced stage (n = 28), acute myeloid leukemia (AML; n = 13), or other hematologic diseases (n = 16) between June 1985 and April 1993. All patients were conditioned with cyclophosphamide and total body irradiation and received a combination of methotrexate and cyclosporine as graft-versus-host disease (GVHD) prophylaxis. Fourty-six patients received transplants from HLA-identical donors and 42 patients received transplants from donors who were minor-mismatched at one HLA-A or B or D/DRB1 locus. The Kaplan-Meier estimates of disease-free survival and relapse were 75% and 0% for patients with CML, 47% and 20% for ALL in first or second remission, 10% and 60% for ALL in relapse or third remission, 46% and 46% for AML in first remission (n = 1) or more advanced disease (n = 12), and 29% and 69% for other diseases. HLA disparity was not significantly associated with lower disease-free survival, but the results suggest more relapses in HLA-matched recipients and there was significantly more transplant-related mortality in mismatched recipients (51% v 24%, P = .04). Most deaths were due to infections associated with acuteor chronic GVHD and occurred within the first 2 years after transplantation. Granulocyte engraftment occurred in all evaluable patients. Sixty-three percent of HLA-matched and 57% of HLA- mismatched recipients were discharged home disease-free at a median of 98 and 103 days, respectively, after transplantation (P = not significant [NS]). The incidence of grades II-IV acute GVHD was 83% in HLA-matched and 98% in HLA-mismatched recipients (P = .009). The incidence of chronic GVHD was 60% in HLA-matched and 69% in HLA- mismatched recipients (P = NS). One or multiple late adverse events such as cataracts, osteonecrosis of the hip or knee, restrictive or obstructive pulmonary disease, and hypothyroidism have occurred in 11 of 33 (33%) surviving patients. Immunosuppression was discontinued in 58% of surviving patients, including all 12 patients surviving more than 3.2 years, all of whom have a Lansky or Karnofsky score of 100%.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chemokine expression and leucocyte infiltration in Sjogren's syndrome

OBJECTIVE: To investigate the expression and source of chemokines in minor salivary gland biopsies (MSGs) in patients with Sjogren's syndrome (SS). METHODS: Immunohistochemical analysis was used to determine the pattern of chemokine expression in MSGs from patients with (n=6) and without (n=5) SS, as well as to examine the phenotype of both resident and infiltrating cells expressing chemokines. RESULTS: Significant differences in the number of infiltrating mononuclear (MN) cells in patients with and without SS were noted. Ductal epithelial cells of SS biopsies expressed significantly increased levels of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, interleukin-8 (IL-8) and RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted). Biopsies from patients with SS showed that MIP-1beta was expressed by 51% of infiltrating cells, while 41% expressed MIP-1alpha, whereas 22 and 7% expressed RANTES and IL-8, respectively. CONCLUSION: Chemokines expressed by ductal epithelial cells may attract circulating leucocytes, in particular CD4+ T cells, towards the site of inflammation, thereby orchestrating the influx of MN cells characteristically seen in MSGs in SS. Chemokines may be induced directly by a putative triggering agent for SS, or secondary to the release of pro-inflammatory cytokines produced by epithelial cells. These findings further implicate epithelial cells as playing a major role in the pathogenesis of SS and implicate chemokines in the leucocyte recruitment in this setting.      

Association of HbA1c levels with vascular complications and death in patients with type 2 diabetes: evidence of glycaemic thresholds

Aims/hypothesis

There is conflicting evidence regarding appropriate glycaemic targets for patients with type 2 diabetes. Here, we investigate the relationship between HbA_1c and the risks of vascular complications and death in such patients.

Methods

Eleven thousand one hundred and forty patients were randomised to intensive or standard glucose control in the Action in Diabetes and Vascular disease: Preterax and Diamicron Modified Release Controlled Evaluation (ADVANCE) trial. Glycaemic exposure was assessed as the mean of HbA_1c measurements during follow-up and prior to the first event. Adjusted risks for each HbA_1c decile were estimated using Cox models. Possible differences in the association between HbA_1c and risks at different levels of HbA_1c were explored using linear spline models.

Results

There was a non-linear relationship between mean HbA_1c during follow-up and the risks of macrovascular events, microvascular events and death. Within the range of HbA_1c studied (5.5?C10.5%), there was evidence of ??thresholds??, such that below HbA_1c levels of 7.0% for macrovascular events and death, and 6.5% for microvascular events, there was no significant change in risks (all p?>?0.8). Above these thresholds, the risks increased significantly: every 1% higher HbA_1c level was associated with a 38% higher risk of a macrovascular event, a 40% higher risk of a microvascular event and a 38% higher risk of death (all p?Conclusions/interpretation In patients with type 2 diabetes, HbA_1c levels were associated with lower risks of macrovascular events and death down to a threshold of 7.0% and microvascular events down to a threshold of 6.5%. There was no evidence of lower risks below these levels but neither was there clear evidence of harm.

Trial Registration:

ClinicalTrial.gov NCT00145925

Funding:

Servier and the National Health and Medical Research Council of Australia (project grant ID 211086 and programme grant IDs 358395 and 571281)     

Risk of infective endocarditis in mitral valve prolapse with and without precordial systolic murmurs

The risk of infective endocarditis (IE) associated with a systolic murmur in patients with mitral valve prolapse (MVP) was investigated in a case-control study. The case group comprised all patients with MVP (n = 19) from a series of 136 consecutive adult admissions for IE. Three matched control subjects were chosen for each case from a series of 144 MVP patients without IE. Seventeen of the 19 cases (89%) had documented evidence of systolic murmurs existing before the IE episode; systolic murmurs were documented in 25 of the 57 control subjects (47%). The data indicate a significant increase in the risk of IE in MVP patients with a systolic murmur (p less than 0.01). The absolute probability of IE developing in a patient with MVP and a murmur was estimated to be approximately 1 in 1,400 per year; this was 35 times greater than the probability in a patient with MVP without a murmur. The results suggest that by restricting prophylaxis to MVP patients with a systolic murmur, cover would be provided for almost 90% of those with MVP in whom IE would be likely to develop.     

Long-term engraftment of normal and post-5-fluorouracil murine marrow into normal nonmyeloablated mice [see comments]

We report the successful long-term engraftment of normal male donor bone marrow (BM) transfused into noncytoablated female mice, challenging the assumption that "niches" need to be created for marrow to engraft. We have used chromosomal banding and Southern blot analysis to identify transplanted male marrow cells, and shown the long-term stability of the chimeric marrows. Balb/C, BDF1, or CBA-J female hosts (no irradiation) received for 5 consecutive days 40 x 10(6) male cells (per day) of the same strain, and repopulation patterns were observed. Parallel studies were performed using tibia/femur equivalents of normal marrow or marrow from Balb/C mice pretreated 6 days previously with 150 mg/kg 5-fluorouracil (5-FU). Chromosome banding techniques showed that 5% to 46% of marrow cells were male 3 to 9 months posttransplant with normal donor marrow. Southern blot analysis, using the pY2 probe, showed continued engraftment at 21 to 25 months posttransplant, ranging from 15% to 42% male engrafted cells in marrow. Normal donor male marrow engrafted significantly better than 5-FU-pretreated male marrow as shown 1 to 12 months posttransplant in non-cytoablated female recipients. Percentages of male engrafted cells in BM ranged from 23% to 78% for recipients of normal donor marrow and from 0.1% to 39% for recipients of 5-FU marrow. Mean engraftment for 6 mice receiving normal marrow was 38%, whereas that for 6 mice receiving post-5-FU marrow was 8%, as assayed 1 to 3 months posttransplant. At 10 to 12 months, mean engraftment for the normal donor group was 46%, compared with 16% for the 5-FU group. The patterns of engraftment with normal and 5-FU marrow were similar for spleen and thymus. These results show that long-term chimerism can be established after transplantation of normal donor marrow to normal nonirradiated host mice and indicate that marrow spaces do not have to be created for successful engraftment. They suggest that transplanted marrow competes equally with host marrow for marrow space. Finally, these data show that post-5-FU Balb/C male marrow is markedly inferior in the repopulation of Balb/C female host marrow, spleen, and thymus, and suggest that this population of cells may not be the ideal population for gene transfer studies.     

中国心力衰竭流行病学调查及其患病率

目的　了解我国成年人慢性心力衰竭 (心衰 )的患病率和分布特征。方法　中国心血管健康多中心合作研究应用四阶段整群随机抽样方法 ,在全国 1 0个省市 (南方和北方各 5个省市 )抽取具有代表性的样本 ,年龄在 35～ 74岁之间 ,城市和农村各半 ,男、女人数均衡。统计不同年龄组、不同性别和不同地区人群的心衰患病率。结果　共抽样调查 35～ 74岁城乡居民 1 5 51 8人 ,心衰患病率为0 9% ;其中男性为 0 7% ,女性为 1 0 % ,女性患病率高于男性 (P <0 0 5)。 35～ 44岁、45～ 54岁、55～64岁、65～ 74岁年龄组的心衰患病率分别为 0 4%、1 0 %、1 3 %和 1 3 % ;随着年龄增高 ,心衰的患病率显著上升 (P <0 0 1 )。我国北方地区心衰患病率为 1 4% ,南方地区心衰患病率为 0 5 % ,北方明显高于南方 (P <0 0 1 ) ;城市人群心衰患病率为 1 1 % ,农村人群心衰患病率为 0 8% ,城市人群心衰患病率高于农村。结论　心衰正在成为我国心血管病领域的重要公共卫生问题