Oligoclonal expansion of cd8+ T-lymphocytes in cultured peripheral lymphocytes derived from asymptomatic HTLV-I carriers.

Peripheral blood mononulear cells (PBMC) derived from 10 asymptomatic human T lymphotropic virus type I (HTLV-I) carriers were cultured for a short term (10-14 days) in the absence of exogenous antigens. In 5 carriers, when compared with 5 HTLV-I non-carriers an apparent increase in the proportion of CD8+ DR+ cells was observed. The clonality of cultured lymphocytes was then examined by analyzing the usage of Vbeta families of T cell receptor genes. In three of the 5 carriers with an increased CD8+ population, two to four Vbeta genes were dominant in the CD8+ population but not in the CD4+ population. No dominance of Vbeta gene usage was observed in lymphocytes derived from the 5 noncarriers. The sequence of cDNA from Vbeta families which were especially dominant revealed their oligoclonal characteristics. These results were quite similar to our previous findings from HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients in whom the same oligoclonal CD8+ cells were expanded cytotoxic T lymphocyte clones for HTLV-I genome products. We posed the question of whether the dominance of TCR Vbeta usage in cultured PBMC was associated with the HTLV-I genome dose in the PBMC or with anti-HTLV-I antibody titers. The three carriers who showed an increased CD8+ population mentioned above all showed a rather high HTLV-I genome dose, which again was similar to HAM/TSP patients. These three carriers however, did not necessarily show high anti-HTLV-I antibody titers in contrast with HAM/TSP patients, who generally do.

[An autopsy case of AIDS with disseminated cytomegalovirus infection and neurological disturbance].

We report a case of acquired immunodeficiency syndrome (AIDS) complicated by disseminated CMV infection and neurological disturbance. A 21 years old male with hemophilia A was diagnosed as having AIDS in Feb. 1986 because of interstitial pneumonia and esophageal candidiasis. Since Jan. 1987 he had complained of hypesthesia in the legs. On Mar. 14 he was admitted due to diarrhea. The laboratory data revealed that WBC was 4,000/microliters including 29% of lymphocytes, 1.6% of OKT4+-, 71.6% of OKT8+-lymphocytes, T4/T8 ratio 0.02 and positive HIV antibody and HTLV-1 antibody. After the admission, sensory disturbance exacerbated to complicate paraplegia. He developed acute hepatitis associated with leukopenia, thrombocytopenia, pneumonia and melena, and eventually died on May 29. The autopsy findings disclosed CMV infection in the lungs, colons, and adrenal glands, suggesting that the primary cause of death was adrenal insufficiency. Degeneration of cerebro-spinal nerve cells and peripheral neuritis were thought to result from direct HIV infection to the nervous system.

Analysis of therapeutic effect in experimental chemoimmunotherapy for rat ascites tumor.

The lyophilized, squalene-treated Nocardia rubra cell wall skeleton (N-CWS) was confirmed to produce tumoricidal peritoneal macrophages resulting in inhibition of tumor growth when injected locally into the syngeneic ascites fibrosarcoma, AMC 60 in ACI/N rats. Furthermore, N-CWS was found to augment therapeutic effect when administered repeatedly after a single local injection of mitomycin-C (MMC). To analyze the effects, various in vitro cytolysis assays were performed using N-CWS-activated peritoneal macrophages. When tumor target cells were exposed in vitro to MMC, the resulting cytolysis in the presence of N-CWS-activated macrophages was similar to cytolysis of intact target cells. On the other hand, when N-CWS-activated macrophages were exposed to MMC, the tumoricidal activity was lost significantly, depending on exposure to MMC. When tumor target cells and N-CWS-activated macrophages were simultaneously exposed to MMC, tumor-cell cytolysis was strikingly depressed. In the final experiment, combined injection of MMC and N-CWS into the ascites tumor resulted in remarkable increases not only in peritoneal exudate cell number, but also in in vitro tumoricidal activity of peritoneal macrophages as compared to those induced by either agent alone. In addition, the production of tumoricidal macrophages by IP injection of MMC alone was also noticeable, as described previously. These results possibly indicate the involvement of macrophage activation in induction of therapeutic effect in chemoimmunotherapy.

Semiquantitative analysis of integrated genomes of human T-lymphotropic virus type I in asymptomatic virus carriers.

A semiquantitative estimation of human T-lymphotropic virus type I (HTLV-I) integration by peripheral blood mononuclear cells (PBMC) was performed. Genomic DNA samples derived from 134 HTLV-I carriers were subjected to 40 or 60 cycles of the polymerase chain reaction to amplify the pol region of HTLV-I. The HTLV-I genome was detected by dot hybridization using a 32P-labeled oligonucleotide probe for the pol region. The radioactivity of hybridized dot membranes was then counted with an RI Imaging System (Ambis Inc, San Diego, CA) and the HTLV-I genome dose was determined by comparison with standard curve for serially diluted HTLV-I genome-positive DNA. A wide range of variation of HTLV-I genome integration was observed. When the integrated genome dose was calculated as the number of HTLV-I copies per 100 PBMC, 7 carriers (5%) had more than 10 copies, 56 (42%) had 1 to 10 copies, 46 (34%) had 0.1 to 1 copy, and 24 (18%) had less than 0.1 copy. In one sample, the HTLV-I genome was undetectable, which may indicate that the integrated genome was present at less than 0.01 copies per 100 PBMC. Age- or sex-related variations in the distribution of individuals with different HTLV-I genome were rather limited. However, carriers with a high level of the HTLV-I genome were always more than 30 years old and were predominantly male (six of seven).

Relationship between the anti-HTLV-1 antibody level, the number of abnormal lymphocytes and the viral-genome dose in HTLV-1-infected individuals.

Two distinct diseases, adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), develop in a minor population of HTLV-1 carriers. We examined the relationship between the viral genome dose in the peripheral-blood mononuclear cells and the serological response in HTLV-1 carriers and patients with HAM/TSP. The antibody titer to HTLV-1 gag and env proteins, as well as the frequency of an antibody response to viral protein p40tax and the titer, increased with increasing viral genome dose. However, the number of abnormal lymphocytes was not directly related to the host viral load. Patients with HAM/TSP generally showed a higher genome dose than healthy carriers and also had higher antibody titers than healthy carriers with the same HTLV-1 load, supporting the existence of an augmented immune response in these patients. These findings suggest that the antibody titer to HTLV-1 genome products, and not the number of abnormal lymphocytes, intimately reflects the approximate viral load in HTLV-1-infected individuals.

Immune responses and serum levels of cytokines in adult T-cell leukemia patients and human T-cell leukemia virus type-I carriers.

To find predictive parameters for development and progression of adult T-cell leukemia (ATL) in human T-cell leukemia virus type-I (HTLV-I) carriers, we investigated cellular immune responses such as mitogenic responses and natural killer activity of the peripheral blood mononuclear cells (PBMC). And serum or plasma levels of cytokines, including tumor-necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and immunosuppressive acidic protein (IAP), were also measured in patients with ATL, healthy HTLV-I carriers and healthy HTLV-I non-carriers as controls. Results are as follows: (1) increased spontaneous proliferation and decreased mitogenic responses of PBMC already existed in HTLV-I carriers; (2) IAP was significantly higher in patients with acute/lymphoma type ATL than in those with chronic/smoldering type, HTLV-I carriers and HTLV-I non-carriers. These results suggest that spontaneous proliferation or mitogenic responses and IAP may be useful parameters for the development and progression of ATL from the carriers. Since HTLV-I carriers already have various grades of immunosuppression, we should seriously try to prevent further HTLV-I transmission.

In vitro tumor cell killing by peritoneal macrophages from mitomycin C-treated rats.

The local cellular response induced by intraperitoneal injection of mitomycin C was examined in terms of cell-mediated cytotoxicity for tumor cells. An in vitro cytolysis assay involving 125I-iododeoxyuridine-labeled tumor target cells revealed that treatment of normal ACI/N rats (200 g) with a single intraperitoneal injection of mitomycin C (50, 100, or 200 micrograms) induced tumoricidal macrophages in the peritoneal cavity. The tumoricidal activity was dependent on the dose of mitomycin C injected and it was detectable as early as 1 day after the intraperitoneal injection of mitomycin C. In addition to the increased tumoricidal activity, the functional activities of the peritoneal macrophages were found to be increased with respect both to uptake of 2-deoxy-D-glucose and to phagocytosis of latex beads. Additional experiments excluded the possibility that the tumor cell cytolysis was the result of direct cytotoxicity by mitomycin C that might have been incorporated in the peritoneal macrophages or of nutrient depletion in the medium during the cytolysis assay. Furthermore, endotoxin contamination of the mitomycin C, which might have produced the activated macrophages, was not detected. The mechanism by which mitomycin C injected intraperitoneally induced the tumoricidal macrophages locally remains uncertain; however, it is possible also in clinical situations.