Meylan P R, Bürgisser P, Weyrich-Suter C, Spertini F
Institute of Microbiology, CHUV, Lausanne, Switzerland.
J Acquir Immune Defic Syndr Hum Retrovirol. 1996 Sep;13(1):39-47. doi: 10.1097/00042560-199609000-00007.
We wished to establish the feasibility of fine needle aspiration of lymph nodes as a noninvasive method for measuring subsets of immune cells and viral load in HIV-infected patients. Twenty-five patients (CD4+ T cell range 4-760/microl, median 362) were selected. Lymph node aspiration was attempted in 21 patients. Lymph node cells (LNC), ranging from 6 x 10(3) to 2 x 10(6) (median 6 x 10(5)) were obtained in 17 subjects, and compared with peripheral blood mononuclear cells (PBMC) obtained simultaneously. Immunophenotype could be determined by flow cytometry in 9 patients. Mean percent of CD4+ CD3+ T cells in LNC and PBMC and 23.2 and 14.6. Mean percent of CD8+ CD3+ T cells in LNC and PBMC was 23.1 and 45.0, respectively. Therefore, CD4+/CD8+ ratios were much higher in LNC (mean +/- SD: 1.06 +/- 0.31) than in PBMC (0.35 +/- 0.13). The amount of HIV DNA (11 patients) and RNA (8 patients) was determined in the plasma, LNC, and PBMC by competitive reverse transcriptase-polymerase chain reaction (RT-PCR). The number of copies of viral DNA/10(5) cells was higher in LNC than in PBMC (LNC/PBMC ratio ranger: 0.54-25, median 3.4). The number of copies of unspliced viral RNA/10(5) cells was much higher in LNC than in PBMC (LNC/PBMC ratio range 65-1,159, median 435). The plasma RNA copy number, a measure of circulating cell-free virus, was correlated with the RNA copy number in PBMC, but not in LNC. A RT-PCR system specific for spliced transcripts was also used to assess the level of transcripts independent of genomic RNA. This assay also detected more signal in LNC than in PBMC. The level of spliced transcripts in LNC and PBMC correlated with the amount of full-length RNA detected by competitive PCR. A semiquantitative coculture assay with lymphoblasts from healthy donors was used to assess the infectivity of LNC as compared with PBMC in 14 patients. The minimum number of LNC necessary to cause a positive coculture ranged from 10(3) to > 10(5) (median 10(4)); the corresponding number for PBMC ranged from 10(3) to > 10(6) (median 5 x 10(5)). In most patients selected for palpable lymph nodes, LNC could be obtained by fine needle aspiration, thus allowing noninvasive monitoring of viral burden in lymphoid tissue. The present study also suggests that both T-cell subsets and viral load differ in the blood and lymphoid tissue, which raises the question of whether the study of the lymphoid tissue would yield better prognostic markers of disease course.
我们希望确定细针穿刺淋巴结作为一种非侵入性方法来测量HIV感染患者免疫细胞亚群和病毒载量的可行性。选取了25例患者（CD4 + T细胞范围为4 - 760/微升，中位数为362）。对21例患者尝试进行淋巴结穿刺。在17例受试者中获取了6×10³至2×10⁶（中位数为6×10⁵）个淋巴结细胞（LNC），并与同时获取的外周血单个核细胞（PBMC）进行比较。9例患者可通过流式细胞术确定免疫表型。LNC和PBMC中CD4 + CD3 + T细胞的平均百分比分别为23.2和14.6。LNC和PBMC中CD8 + CD3 + T细胞的平均百分比分别为23.1和45.0。因此，LNC中的CD4⁺/CD8⁺比值（平均值±标准差：1.06±0.31）远高于PBMC中的比值（0.35±0.13）。通过竞争性逆转录 - 聚合酶链反应（RT - PCR）测定了11例患者血浆、LNC和PBMC中的HIV DNA量以及8例患者中的RNA量。每10⁵个细胞中病毒DNA的拷贝数在LNC中高于PBMC（LNC/PBMC比值范围：0.54 - 25，中位数为3.4）。每10⁵个细胞中未剪接病毒RNA的拷贝数在LNC中远高于PBMC（LNC/PBMC比值范围65 - 1159，中位数为435）。血浆RNA拷贝数作为循环游离病毒的指标，与PBMC中的RNA拷贝数相关，但与LNC中的RNA拷贝数无关。还使用了一种针对剪接转录本的特异性RT - PCR系统来评估独立于基因组RNA的转录本水平。该检测在LNC中检测到的信号也比在PBMC中更多。LNC和PBMC中剪接转录本的水平与竞争性PCR检测到的全长RNA量相关。使用来自健康供体的淋巴细胞进行半定量共培养试验，以评估14例患者中LNC与PBMC相比的感染性。导致共培养呈阳性所需的LNC的最小数量范围为10³至>10⁵（中位数为10⁴）；PBMC的相应数量范围为10³至>10⁶（中位数为5×10⁵）。在大多数因可触及淋巴结而入选的患者中，可通过细针穿刺获取LNC，从而实现对淋巴组织中病毒载量进行非侵入性监测。本研究还表明，血液和淋巴组织中的T细胞亚群和病毒载量均存在差异，这就提出了一个问题，即对淋巴组织的研究是否会产生更好的疾病进程预后标志物。
