The clinical implications of reduced viral fitness

Viral fitness, defined as the extent of viral adaptation to the host environment, arises from tissue tropism, immune system evasion, drug resistance, and viral replication capacity. The fitness of wild-type and drug-resistant HIV-1 varies widely, associating with plasma viremia, CD4+ T-cell count, and clinical progression. HIV-1 fitness may be measured in competitive culture assays, single cycle assays, or single cycle assays based on a subgenomic fragment of HIV-1, which has been standardized as the replication capacity assay (pol RC). During virologic failure of antiretroviral therapy, CD4 T-cell counts remain elevated while pol RC declines and remains durably lower because of drug-selected changes in the gag and pol genes. CD4 T-cell sparing also is observed among patients without evidence of drug resistance who carry a low pol RC virus. Reduced HIV-1 replication capacity and virulence may occur because of drug resistance or viral escape from host immune responses.     

Neuropeptides in migraine and cluster headache

The cerebral circulation is invested by a rich network of neuropeptide Y (NPY) and noradrenaline containing sympathetic nerve fibers in arteries, arterioles and veins. However, the nerve supply of vasoactive intestinal peptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP) containing fibers is sparse. While noradrenaline and NPY cause vasoconstriction, VIP, SP and CGRP are potent vasodilators. Stimulation of the trigeminal ganglion in cat and man elicits release of SP and CGRP. Subjects with spontaneous attacks of migraine show release of CGRP in parallel with headache. Cluster headache patients have release of CGRP and VIP during bouts. Treatment with sumatriptan aborts headache in migraine and cluster headache as well as the concomitant peptide release.     

Confirmation of hepatitis C infection: a comparison of five immunoblot assays

BACKGROUND: Recently, new immunoblot assays for the detection of antibodies to hepatitis C virus (HCV) became available. STUDY DESIGN AND METHODS: The performance of five confirmatory anti-HCV immunoblot assays was studied with samples with known HCV antibody and HCV RNA status. The assays were a third-generation strip recombinant immunoblot assay (RIBA-3, Chiron Corp., Emeryville, CA), a second-generation HCV blot (DB-2 blot, Diagnostic Biotechnology, Singapore), the Wellcozyme HCV Western blot (Murex blot, Murex Diagnostics, Dartford, UK), an immunodot HCV assay (Matrix, Abbott Laboratories, Chicago, IL), and the third-generation HCV line immunoassay (Liatek-III, Organon Teknika, Boxtel, The Netherlands). RESULTS: Sensitivity on samples from 48 HCV RNA-positive, second-generation RIBA (RIBA-2)-positive persons and specificity on samples from 31 low-risk donors was 96 percent or better for all assays. The sensitivity on 31 HCV RNA-positive, RIBA-2- indeterminate samples was as follows: Liatek-III, 94 percent; RIBA-3, 90 percent; Murex blot, 61 percent; Matrix, 55 percent; and DB-2 blot, 39 percent. In testing 39 HCV RNA-negative, RIBA-2-indeterminate donor samples, the percentage found to be negative was Liatek-III, 77 percent; RIBA-3, 67 percent; Murex blot, 49 percent; DB-2 blot, 33 percent; and Matrix, 15 percent. The order of sensitivity on four HCV seroconversion series was (from high to low): RIBA-3, Liatek-III, DB-2 blot, Murex blot, and Matrix; the differences were small. CONCLUSION: Detection of HCV antibodies was not refined by the addition of new HCV antigens (NS5, E2/NS1), but by improved classical antigens (core, NS3, NS4). Replacement of the commonly used RIBA-2 will resolve the status of a high proportion of RIBA-2-indeterminate samples.     

Effect of dipyridamole therapy on myocardial ischemia in patients with stable angina pectoris receiving concurrent anti-ischemic therapy.

The effects of oral dipyridamole on exercise performance and anginal symptoms were evaluated in 15 men with stable angina pectoris. In a double-blind, randomized, crossover design, patients received 75 mg of dipyridamole or placebo every 8 hours for 2 weeks in addition to their previously prescribed cardiac medications. Graded exercise tolerance testing was performed twice before randomization, at the end of each treatment period, and after single-blind placebo washout. When compared with baseline tests, the time to onset of 0.1 mV ST-segment depression was similar between dipyridamole and placebo treatments (316 +/- 89 vs 345 +/- 102 seconds, respectively, p = not significant). No significant differences existed between treatments in the peak systolic blood pressure-heart rate product or in the duration of exercise. Angina pectoris occurred during all 3 baseline exercise tests in 7 of the 15 subjects; the time to onset of angina was unchanged by either treatment. Analysis of symptom diaries conducted in 13 patients revealed no significant alteration in reported anginal symptoms during dipyridamole treatment compared with placebo treatment (0.6 +/- 0.9 vs 0.3 +/- 0.4 episodes per week). Ambulatory electrocardiographic monitoring in 12 patients revealed few episodes of ischemia during daily activities with no alteration in frequency of episodes during treatment periods. Plasma concentrations of dipyridamole did not correspond with the outcomes of exercise testing. It is concluded that chronic oral dipyridamole therapy given in its usual clinical dose does not adversely affect exercise performance, daily anginal episodes or ambulatory ischemia in patients receiving concurrent anti-ischemic medication.     

Sustained CD4+ T cell response after virologic failure of protease inhibitor-based regimens in patients with human immunodeficiency virus infection

The relationship between plasma human immunodeficiency virus (HIV) RNA levels and peripheral CD4+ T cell counts was examined in 380 HIV-infected adults receiving long-term protease inhibitor therapy. Patients experiencing virologic failure (persistent HIV RNA >500 copies RNA/mL) generally had CD4+ T cell counts that remained greater than pretherapy baseline levels, at least through 96 weeks of follow-up. The CD4+ T cell response was directly and independently related to degree of viral suppression below the pretreatment baseline. For any given HIV RNA level measured 12 weeks after virologic failure, subsequent CD4+ T cell decline was slower in patients receiving a protease inhibitor-based regimen than in a historical control group of untreated patients. These observations suggest that transient or partial declines in plasma HIV RNA levels can have sustained effects on CD4+ T cell levels.     

重组犬钩虫分泌蛋白抗血清的免疫反应性

目的　分析重组犬钩虫分泌蛋白抗血清与钩虫不同种、期抗原的免疫反应性。　方法　用微型垂直电泳槽进行 SDS- PAGE,以低分子量标准蛋白作参照。EL IB试验 :以重组犬钩虫分泌蛋白 - 1(Ac- r Asp- 1)或重组犬钩虫分泌蛋白 - 2 (Ac- r Asp- 2 )免疫鼠血清作第一抗体 ,羊抗鼠 Ig G- HRP作第二抗体 ,用 Western blotting发光底物试剂反应 ,全自动摄影 ,按照底片中显示带的位置测出相应分子量。　结果与结论　 Ac- r Asp- 1组分为 45k Da,其免疫血清能识别犬钩虫第 期幼虫 (Ac- L3)抗原和 Ac- r Asp- 1,不与十二指肠钩虫成虫 (Ad- A)、十二指肠钩虫第 期幼虫 (Ad- L3)、美洲钩虫成虫 (Na- A)、犬钩虫成虫 (Ac- A)、巴西日圆线虫成虫 (Nb- A)抗原和 Ac- r Asp- 2起反应 ;Ac- r Asp- 2组分为 2 4k Da,其免疫血清能识别 Ad- A、Ad- L3、Na- A、Ac- A、Ac- L3抗原和 Ac- r Asp- 2 ,不与Nb- A抗原和 Ac- r Asp- 1起反应。     

Development of a marrow transplant regimen for acute leukemia using targeted hematopoietic irradiation delivered by 131I-labeled anti-CD45 antibody, combined with cyclophosphamide and total body irradiation

In an attempt to decrease the relapse rate after bone marrow transplantation (BMT) for advanced acute leukemia, we initiated studies using 131I-labeled anti-CD45 antibody (BC8) to deliver radiation specifically to hematopoietic tissues, followed by a standard transplant preparative regimen. Biodistribution studies were performed in 23 patients using 0.5 mg/kg trace 131I-labeled BC8 antibody. The BC8 antibody was cleared rapidly from plasma with an initial disappearance half-time of 1.5 +/- 0.2 hours, presumably reflecting rapid antigen- specific binding. The mean radiation absorbed doses (cGy/mCi131I administered) were as follows: marrow, 7.1 +/- 0.8; spleen, 10.8 +/- 1.4; liver, 2.7 +/- 0.2; lungs, 2.1 +/- 0.1; kidneys, 0.7 +/- 0.1; and total body, 0.4 +/- 0.03. Patients with acute myelogenous leukemia (AML) in relapse had a higher marrow dose (11.4 cGy/mCi) than those in remission (5.2 cGy/mCi; P = .001) because of higher uptake and longer retention of radionuclide in marrow. Twenty patients were treated with a dose of 131I estimated to deliver 3.5 Gy (level 1) to 7 Gy (level 3) to liver, with marrow doses of 4 to 30 Gy and spleen doses of 7 to 60 Gy, followed by 120 mg/kg cyclophosphamide (CY) and 12 Gy total body irradiation (TBI). Nine of 13 patients with AML or refractory anemia with excess blasts (RAEB) and two of seven with acute lymphocytic leukemia (ALL) are alive disease-free at 8 to 41 months (median, 17 months) after BMT. Toxicity has not been measurably greater than that of CY/TBI alone, and the maximum tolerated dose has not been reached. This study demonstrates that with the use of 131I-BC8 substantially greater doses of radiation can be delivered to hematopoietic tissues as compared with liver, lung, or kidney, which may improve the efficacy of marrow transplantation.     

The growth factor requirements of STRO-1-positive human bone marrow stromal precursors under serum-deprived conditions in vitro

Factors that regulate the growth and development of primitive bone marrow stromal cell precursors are not well defined. We have examined 25 purified recombinant growth factors for their ability to initiate and support clonogenic growth of fibroblast colony-forming cells (CFU- F) from adult human bone marrow. Assays were performed using bone marrow mononuclear cells (BMMNC) enriched in CFU-F by magnetic- activated cell sorting (MACS) using the monoclonal antibody (MoAb) STRO- 1. A serum-deprived assay was developed to avoid components of fetal calf serum (FCS) that may mask or otherwise modify the response of CFU- F to exogenously added factors. L-ascorbate and the glucocorticoid dexamethasone were found to be essential for CFU-F colony development under serum-deprived conditions. Importantly, clonogenic growth of CFU- F in this culture system was absolutely dependent on an exogenous source of growth factor. Platelet-derived growth factor-BB (PDGF) and epidermal growth factor (EGF) demonstrated the greatest ability to support colony growth. Colony formation was dose-dependent, with half- maximal colony numbers at approximately 0.2 ng/mL for either factor and plateau numbers at concentrations in excess of 1.0 ng/mL. Simultaneous addition of PDGF and EGF had no effect on the number of colonies initiated but resulted in dose-dependent increases in mean colony diameter that were significant (P < or = .05) when compared with the effect of either factor alone or with the size of colonies elicited in control cultures by 20% FCS. Fluorescence-activated cell sorting (FACS) of BMMNC using MoAbs to the alpha chain of the PDGF receptor and to the EGF receptor in combination with the Moab STRO-1 demonstrated constitutive expression of both receptors by greater than 90% on CFU-F. Receptors for insulin-like growth factor-1 (IGF-1) and nerve growth factor (NGF) were also detected on STRO-1+ CFU-F, but in vitro both IGF- 1 and NGF did not support colony growth. This report demonstrates the development of a simple, reproducible, and stringent culture system for the growth and assay of stromal precursors under serum-deprived conditions and represents an important prerequisite for future studies of the role of growth factors in the regulation of stromal cell proliferation, differentiation, and development.     

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.