Verification of the Usefulness of an Assessment and Risk Control Sheet that Promotes Management of Cancer Drug Therapy.

Background: Proper management of adverse events is crucial for the safe and effective implementation of anticancer drug treatment. Showa University Hospital uses our interview sheet (assessment and risk control [ARC] sheet) for the accurate evaluation of adverse events. On the day of anticancer drug treatment, a nurse conducts a face-to-face interview. As a feature of the ARC sheet, by separately describing the symptoms the day before treatment and the day of treatment and sharing the information on the medical record, it is possible to clearly determine the status of adverse events. In this study, we hypothesized that the usefulness and points for improvement of the ARC sheet would be clarified by using and evaluating a patient questionnaire. Methods: This study included 174 patients (144 at Showa University Hospital (Hatanodai Hospital) and 30 at Showa University Koto Toyosu Hospital (Toyosu Hospital) who underwent pre-examination interviews by nurses and received cancer chemotherapy at the outpatient center of Hatanodai and Toyosu Hospital. In the questionnaire survey, the ARC sheet's content and quality, respondents' satisfaction, structural strengths, and points for improvement were evaluated on a five-point scale. Results: The patient questionnaire received responses from 160 participants, including the ARC sheet use group (132 people) and the non-use group (28 people). Unlike the ARC sheet non-use group, the ARC sheet use group recognized that the sheet was useful to understand the adverse events of aphthous ulcers (p = 0.017) and dysgeusia (p = 0.006). In the satisfaction survey questionnaire, there was a high sense of security in the pre-examination interviews by nurses using the ARC sheet. Conclusions: The ARC sheet is considered an effective tool for comprehensively evaluating adverse events. Pre-examination interviews by nurses using ARC sheets accurately determined the adverse events experienced by patients with anxiety and tension due to confrontation with physicians.

Inhibition of osteoclast formation by 3-methylcholanthrene, a ligand for arylhydrocarbon receptor: suppression of osteoclast differentiation factor in osteogenic cells.

We investigated the effects of 3-methylcholanthrene (3MC), a ligand for arylhydrocarbon receptor (AhR), on osteoclastogenesis. Osteoclast-like cells, in cocultures with mouse spleen cells and clonal osteogenic stromal ST2 cells, are formed from spleen cells by a combination of the receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) produced by ST2 cells in response to 1alpha,25(OH)(2) Vitamin D(3). 3MC dose-dependently inhibited the formation of mono- and multinuclear osteoclast-like cells. However, 3MC did not inhibit the formation of osteoclast-like cells from mouse spleen cells which was supported by the exogenous soluble RANKL and M-CSF. 3MC did not affect the formation of an actin ring and pits on slices of dentine by osteoclast-like cells, both of which are typical indices of osteoclast activity. These results suggest that 3MC affects osteoclast-supporting cells such as ST2 cells but not osteoclast precursor cells and mature osteoclastic cells. When we measured the expression levels of RANKL mRNA in ST2 cells, 3MC dose-dependently decreased the level of this mRNA. However, 3MC did not affect levels of mRNAs for osteoprotegerin (OPG), M-CSF, and the receptor of 1alpha,25(OH)(2) Vitamin D(3) in ST2 cells. Furthermore, soluble RANKL was able to counteract the inhibitory effect of 3MC on the formation of osteoclast-like cells. Our findings indicate that 3MC inhibits osteoclastogenesis via the inhibition of RANKL expression in osteoblastic cells.

Treatment of myoblastic C2C12 cells with BMP-2 stimulates vitamin D-induced formation of osteoclasts.

The interaction of osteoclast precursors with osteoblasts and/or stromal cells is essential for the formation of mature osteoclasts and the resorption of bone. We found that myoblastic C2C12 cells induced the differentiation of mouse spleen cells into tartrate-resistant acid phosphatase-positive (TRAP-positive) multinucleated cells in the presence of 10(-7) M 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] and that C2C12 cells that had been treated with bone morphogenetic protein-2 (BMP-2) dose-dependently stimulated the formation of osteoclasts. The newly developed TRAP-positive multinucleated cells were capable of resorbing mineralized tissues. Treatment of C2C12 cells with BMP-2 for 24 h enhanced the subsequent expression in C2C12 cells of mRNA for the receptor activator of nuclear factor-kappaB ligand (RANKL) in the presence of 1alpha,25(OH)2D3. Since the formation of osteoclasts was inhibited dose-dependently by exogenous OPG, the expression of RANKL in response to BMP-2 appeared to be critical for the formation of osteoclasts. Our findings suggest that BMP-2 might play an important role in the differentiation of cells that support osteoclastogenesis.

Recovery process of arthritis induced by 6-sulfanilamidoindazole (6SAI) in rats.

6-Sulfanilamidoindazole (6SAI) is known to induce not only an acute arthritis but also serositis and arteritis which resemble those induced by some vasodilators in rats. In this study, the recovery process of ankle lesions was examined histopathologically for up to 12 weeks of recovery period in rats bearing arthritis induced by administration of 6SAI (500 mg/kg) for 2 weeks. At 2 weeks of 6SAI-treatment, exudative synovitis and exudative/edematous periarthritis with marked formation of granulation tissues and periosteal reactive bone formation were noted in the ankles, but no remarkable neutrophil infiltration was detected in those lesions. The ankle swelling induced by 6SAI diminished by 4 weeks of recovery period, and the elevated plasma fibrinogen levels were normalized by 2 weeks of recovery period. Although fibrosis and newly-formed periosteal bone were still observed after 2 weeks of recovery period, no inflammatory lesion was detected at that point. At 4 or 12 weeks of recovery periods, the ankles showed an almost normal appearance. These results indicate that 6SAI-induced arthritis is reversible in nature and does not develop into chronic phase.

Reciprocal control of expression of mRNAs for osteoclast differentiation factor and OPG in osteogenic stromal cells by genistein: evidence for the involvement of topoisomerase II in osteoclastogenesis.

Osteoclast-like cells, in cocultures with mouse spleen cells and clonal osteogenic stromal ST2 cells, are formed from spleen cells with monocyte/macrophage lineage in response to a combination of osteoclast differentiation factor (RANKL) and OPG, a decoy receptor for RANKL, produced by ST2 cells in response to 1alpha,25-dihydroxyvitamin D(3). Treatment of ST2 cells with the natural isoflavonoid genistein for 6 h before coculture with spleen cells inhibited the formation of tartrate-resistant acid phosphatase-positive osteoclast-like cells. When we measured levels of RANKL mRNA in ST2 cells, we found that genistein decreased the level of this mRNA. By contrast, the level of OPG mRNA was enhanced by genistein. Genistein is a specific inhibitor of topoisomerase II (topo II) and an inhibitor of protein tyrosine kinase, as well as being a potent phytoestrogen. To characterize the mode of action of genistein, we examined the effects of an inactive form of genistein (daidzein), 17beta-estradiol, inhibitors of topo II, and inhibitors of tyrosine kinases on the formation of tartrate-resistant acid phosphatase-positive osteoclast-like cells. Among the compounds tested, two inhibitors of topo II, amsacrine and etoposide, attenuated the formation of osteoclast-like cells via reciprocal regulation of the expression of mRNAs for RANKL and OPG in ST2 cells, acting similarly to genistein. Our findings indicate that genistein might inhibit the formation of osteoclast-like cells via inhibition of the activity of topo II, suggesting the novel possibility that topo II might play an important role in osteoclastogenesis.

Role of ascorbic acid in the osteoclast formation: induction of osteoclast differentiation factor with formation of the extracellular collagen matrix.

Osteoclasts are bone-resorbing multinucleated cells. Tartrate-resistant acid phosphatase-positive (TRAP-positive) mononuclear and multinucleated cells, which are osteoclast-like cells (OCLs), were formed as a result of the coculture of mouse bone marrow cells and clonal stromal ST2 cells in the presence of 1alpha,25-dihydroxy-vitamin D3. Removal of ascorbic acid from the culture medium prevented the formation of TRAP-positive OCLs. Addition of ascorbic acid to the medium formed TRAP-positive OCLs, and the effect of ascorbic acid was dose-dependent. When we examined the level of messenger RNA (mRNA) for osteoclast differentiation factor (RANKL/ODF) in ST2 cells, we found that ascorbic acid caused an approximately 5-fold increase in the level of this mRNA. The half-life of the mRNA was unaffected by ascorbic acid. To characterize the mechanism of action of ascorbic acid, we investigated the relationship between formation of TRAP-positive OCLs and formation of the collagen matrix. Inhibitors of the formation of collagen triple helices blocked both the formation of TRAP-positive OCLs and the expression of the mRNA for RANKL/ODF in response to ascorbic acid. Our findings suggest that ascorbic acid might be essential for osteoclastogenesis and might induce the formation of TRAP-positive OCLs via induction of the synthesis of RANKL/ODF that is somehow mediated by the extracellular matrix.

Primary effusion lymphoma with B-cell phenotype.

We describe here a case of primary effusion lymphoma that occurred in a 78-year-old woman. She was successively treated with prednisolone but died 15 months after the diagnosis of primary effusion lymphoma. The immunohistochemistry revealed the neoplastic cells to be CD19(+), CD20(+), CD21(+), Sm-Ig(+), and HLA-DR(+). This patient exhibited clonal IgH and clonal kappa light chain gene rearrangement, indicating a B-cell origin. The present case was distinguished from the majority cases of HHV-8-positive primary effusion lymphoma. Here we present clinical details of response to therapy in this case.

Nitric oxide accelerates the ascorbic acid-induced osteoblastic differentiation of mouse stromal ST2 cells by stimulating the production of prostaglandin E(2).

Nitric oxide (NO) promoted the differentiation of clonal stromal cells (ST2 cells) derived from mouse bone marrow to osteoblast-like cells. The level of expression of mRNA for osteocalcin, a marker of osteoblastic differentiation, and the formation of mineralized nodules, increased in ST2 cells treated with a donor of NO. We used the reverse transcriptase-polymerase chain reaction (RT-PCR) to identify the subtypes of NO synthase that were expressed in the ST2 cells and we detected the expression of an inducible NO synthase gene in response to tumor necrosis factor-alpha (TNF-alpha). In various types of cell, NO induces the synthesis of prostaglandin E(2) and cGMP, which are known as regulators of osteoblastic differentiation, by activating cyclooxygenases and soluble guanylate cyclase, respectively. Prostaglandin E(2) was generated in response to NO in ST2 cells, however, no synthesis of cGMP in response to NO was detected. Two inhibitors of cyclooxygenase-2, N-[4-nitro-2-phenoxyphenyl]-methanesulfonamide (nimesulide) and 1-(4-chlorobenzoyl)-5-methoxy-2-methylindole-3-acetic acid (indomethacin), inhibited the formation of mineralized nodules by ST2 cells. Our observations suggest that NO might promote osteoblastic differentiation of ST2 cells by stimulating the production of prostaglandin E(2).

Effects of nicotine on cultured cells suggest that it can influence the formation and resorption of bone.

The acute effects of nicotine [1-methyl-2-(3-pyridyl)pyrrolidine] on the formation and resorption of bone were examined in cultures of clonal rat calvarial osteogenic cells (ROB-C26) and clonal mouse calvarial preosteoblastic cells (MC3T3-E1), as well as in osteoclast-like cells formed during coculture of mouse bone marrow cells and clonal stromal cells from mouse bone marrow, ST2 cells, at concentrations that occur in the saliva of smokeless tobacco users. Nicotine stimulated the rate of deposition of Ca(2+) by ROB-C26 cells, as well as the alkaline phosphatase activity of these cells, in a dose-dependent manner. However, both activities decreased in MC3T3-E1 cells that had been exposed to nicotine. These results indicate that nicotine affected osteoblastic differentiation in osteoblast-like cells. By contrast, nicotine reduced, in a dose-dependent manner, the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) and the formation of pits on slices of dentine, both of which are typical characteristics of osteoclasts. Our results suggest that nicotine might have critical effects on bone metabolism.

Characterization of osteoblastic differentiation of stromal cell line ST2 that is induced by ascorbic acid.

The stromal cell line ST2, derived from mouse bone marrow, differentiated into osteoblast-like cells in response to ascorbic acid. Ascorbic acid induced alkaline phosphatase (ALPase) activity, the expression of mRNAs for proteins that are markers of osteoblastic differentiation, the deposition of calcium, and the formation of mineralized nodules by ST2 cells. We investigated the mechanism whereby ascorbic acid induced the differentiation of ST2 cells. Inhibitors of the formation of collagen triple helices completely blocked the effects of ascorbic acid on ST2 cells, an indication that matrix formation by type I collagen is essential for the induction of osteoblastic differentiation of ST2 cells by ascorbic acid. We furthermore examined the effects of bone morphogenetic proteins (BMPs) on the differentiation of ST2 cells induced by ascorbic acid. Ascorbic acid had no effect on the expression of mRNAs for BMP-4 and the BMP receptors. However, a soluble form of BMP receptor IA inhibited the induction of ALPase activity by ascorbic acid. These results suggest that ascorbic acid might promote the differentiation of ST2 cells into osteoblast-like cells by inducing the formation of a matrix of type I collagen, with subsequent activation of the signaling pathways that involve BMPs.

Ipriflavone down-regulates endothelin receptor levels during differentiation of rat calvarial osteoblast-like cells.

Ipriflavone (7-isopropoxy-3-phenyl-4H-1-benzopyran-4-one) is a synthetic flavonoid that has been shown to stimulate the activity of osteoblasts. We show here that ipriflavone also promotes the deposition of calcium and the formation of mineralized nodules by newborn rat calvarial osteoblast-like (ROB) cells as well as the activity of alkaline phosphatase. We reported previously that endothelin-1 inhibits the differentiation of ROB cells [Y. Hiruma et al. (1998) J. Cardiovasc. Pharmacol. 31, S521-S523]. Therefore, we examined the effects of ipriflavone on the expression of endothelin receptors in ROB cells by polymerase chain reaction-Southern blot analysis and in binding assays with 125I-labeled endothelin-1. Ipriflavone reduced levels of endothelin ETA receptors (to 48% of the control level) in ROB cells around day 7 in our standard cultures, while it had no apparent effect on the expression of the mRNA for the endothelin ETA receptor. By contrast, treatment with 10(-7) M endothelin-1 on days 6 through 9 alone suppressed mineralization by ROB cells. Ipriflavone also reduced the ability of endothelin-1 to inhibit mineralization by ROB cells. These results suggest that the acceleration of osteoblastic differentiation by ipriflavone might be due, at least in part, to a time-specific down-regulation of endothelin receptors.

[Epstein-Barr virus associated Richter's syndrome accompanied by interstitial pneumonia].

A 65-year-old man who had an 8-year history of chronic lymphocytic leukemia was admitted to our hospital on February 19, 1998 because of high fever, dry cough, and weight loss. Laboratory data on admission included serum lactate dehydrogenase at 980 IU/l, CRP at 21.8 mg/dl, and soluble interleukin-2 receptor at 7,280 U/ml. The results of serological tests for Epstein-Barr virus (EBV) antibodies were as follows: EBV capsid antigen IgG 1:2560, EBV early antigen IgG 1:640, and EBV nuclear antigens 1:20. Computed tomography revealed diffuse interstitial pneumonia in both lungs, hepatosplenomegaly with multiple nodules, and enlarged intra-abdominal lymph nodes. In addition, Gallium-67 scintigraphy demonstrated abnormal accumulations. Although the patient initially responded well to combination chemotherapy, he eventually deteriorated and died on November 2, 1988, despite salvage chemotherapy. Postmortem needle biopsy specimens from the liver and spleen revealed diffuse proliferation of polymorphic large lymphoma cells. The lymphoma cells were positive for L-26, latent membrane protein 1, and EBV nuclear antigen, but negative for UCHL-1 and CD3, 5, 10, and 30. In situ hybridization procedures disclosed the presence of EBV-encoded small RNA in lymphoma cells. These findings suggested the possibility of association with EBV infection in some cases of Richter's syndrome.

Correlation between induction of expression of biglycan and mineralization by C-type natriuretic peptide in osteoblastic cells.

We reported previously that C-type natriuretic peptide (CNP) promotes the differentiation and mineralization of osteoblastic cells [Am. J. Physiol. 270 (Cell Physiol. 39): C1311-C1318, 1996]. However, little information is available about the mechanism of action of CNP in differentiating osteoblastic cells. Using the technique known as differential display-polymerase chain reaction, we attempted to identify the mRNAs whose levels are regulated by CNP in mouse clonal preosteoblastic MC3T3-E1 cells. One species of mRNA whose level was increased by CNP was 99% homologous to the 3'-untranslated region of a mouse gene for biglycan (BGN), a small proteoglycan. BGN is known to be involved in bone formation by osteoblastic cells. Therefore, we investigated the relationship, during the formation of mineralized nodules, between CNP and BGN using calvarial osteoblast-like cells (ROB cells) from newborn rats, that are a good model for studies on bone formation in vitro. Northern blot analysis revealed that transcription of the mRNA for BGN was up-regulated by CNP in ROB cells on days 6 and 8, whereas no effect of CNP was observed on days 3 and 12. Brief treatment with 10(-7) M CNP on days 3 through 9 exclusively enhanced the deposition of calcium, a result that suggests that CNP might regulate the expression of mineralization-related genes and, probably, the gene for BGN during a specific time period.

Endothelins inhibit the mineralization of osteoblastic MC3T3-E1 cells through the A-type endothelin receptor.

We examined the effects of various endothelins on the mineralization of mouse clonal preosteoblastic MC3T3-E1 cells. MC3T3-E1 cells expressed mRNAs for endothelin (ET)-1 and the A-type receptor for ET (ETA). A pharmacological study also demonstrated the predominant expression of the ETA receptor. Northern blotting analysis revealed that ETs decreased the expression of mRNA for osteocalcin, which is a marker protein for the maturation of osteoblastic cells. ET-1 also decreased in the deposition of calcium by MC3T3-E1 cells in a dose-dependent manner and it had an inhibitory effect even at 10(-11) M. The rank order of potency of ETs was ET-1 = ET-2 > ET-3. Brief treatment with 10(-7) M ET-1 on days 6-8 alone suppressed mineralization. ET-1 enhanced the rate of production of inositol 1,4, 5-trisphosphate (IP3) in MC3T3-E1 cells, but it had no effect on the rate of production of cAMP. Taken together, our data indicate that ET-1 might inhibit the mineralization of osteoblastic cells via an interaction with the ETA receptor, with generation of IP3 as the intracellular signal.

Mutational analysis of a function of xeroderma pigmentosum group A (XPA) protein in strand-specific DNA repair.

To analyze the function of the xeroderma pigmentosum group A (XPA) protein in strand-specific DNA repair, we examined repair of UV-induced cyclobutane pyrimidine dimer (CPD) in transcribed and non-transcribed strands of the dihydrofolate reductase gene of xeroderma pigmentosum group A (XP-A) cell line (XP12ROSV) which was transfected with various types of mutant XPA cDNA. The transfectant overexpressing mutant XPA with a defect in the interaction with either ERCC1, replication protein A (RPA), or general transcription factor TFIIH, showed more or less decreased repair of CPD in each strand in parallel, while in the transfectant overexpressing R207G (Arg207to Gly) mutant XPA derived from XP129, a UV-resistant XP12ROSV revertant, the rate of CPD repair was almost normal in each strand. We also examined the dose responses of the XPA protein on CPD repair in each strand by the modulation of the expression levels of wild-type or R207G mutant XPA using an inducible expression system, LacSwitchtrade mark promoter. There were good correlations between the rate of CPD repair in each strand and the amount of XPA protein produced in these Lac cells. Our results indicate that the XPA protein is equally important for the CPD repair in both transcribed and non-transcribed strands and that the R207G mutation found in XP129 may not be responsible for a selective defect in CPD repair in the non-transcribed strand in XP129.

Effects of nitric oxide from exogenous nitric oxide donors on osteoblastic metabolism.

We examined the effects of nitric oxide (NO) on the differentiation and mineralization of newborn rat calvarial osteoblastic cells (ROB cells) using exogenous NO donors, sodium nitroprusside, 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamin e (NOC-7) and 2,2'-(hydroxynitrosoydrazino)bis-ethanamine (NOC-18). Sodium nitroprusside and NOC-7 dose-dependently enhanced the rate of production of intracellular cGMP in ROB cells and the rat clonal osteogenic cell line ROB-C26. We used NOC (NOC is the trade name for NO complex manufactured by Dojindo, Kumamoto, Japan) as an NO donor in our experiments because sodium nitroprusside exhibited a marked cytotoxicity. Northern blot analysis revealed that the level of mRNA for osteocalcin, one of the osteoblastic differentiation markers, was enhanced in the ROB cells, which was continuously treated by NOC-18. NOC-18, however, did not affect the level of mRNA for alkaline phosphatase and the activity of alkaline phosphatase. Both the number and the total area of mineralized nodules that are a model of in vitro bone formation were shown to be increased by 10(-5) M NOC-18. Our data suggest that NO might act as a local regulator of the metabolism of osteoblastic cells.

Endothelins inhibit mineralization of rat calvarial osteoblast-like cells.

We examined the effects of members of the endothelin (ET) family on mineralization of rat calvarial osteoblast-like cells. The accumulation of calcium in cells and cell layers was attenuated by ETs with the rank order of potency ET-1 = ET-2 > ET-3. We stained the mineralized nodules by von Kossa staining and measured the number and area of mineralized nodules. The inhibitory effects of ET-1 and ET-2 on the formation of mineralized nodules were stronger than those of ET-3. Our data suggest that ET-1 may inhibit the mineralization process of osteoblastic cells through the ETA receptor.

Inhibition of HIV-1 replication by targeting the Rev protein.

Human immunodeficiency virus type 1 (HIV-1) encodes two regulatory proteins, Tat and Rev. The Rev protein facilitates the transport of unspliced and singly-spliced RNA to the cytoplasm in infected host cells by binding to target RNA (Rev response element: RRE). A variety of approaches targeting Rev function, including gene therapy, have been developed that inhibit HIV-1 replication in cells cultured in vitro. This minireview summarizes the recent developments as well as our application of the Rev-binding element-based decoy approach using RNA-DNA chimera oligonucleotide modeling.

[A pathological study of cytomegalovirus infections in autopsied cases with adult T-cell leukemia].

In this study, cytomegalovirus (CMV) infection was found in eleven of 21 autopsied cases (52.4%) with adult T-cell leukemia (ATL). Seven cases (63.6%) revealed disseminated infection in more than three organs. The lungs were involved in all eleven cases (100%), adrenal glands in eight cases (72.7%), esophagus in four cases (36.4%), and stomach, small intestine and urinary bladder in three cases (27.3%). Histopathological findings suggested that lung involvement was the cause of death in five of the 11 cases, the small intestine were involved in two of the 3 cases, and the adrenal glands were involved in one of the 8 cases. In summary, CMV infection was found to be the main cause of death in five (45.5%) of the 11 ATL patients.

Decoy approach using RNA-DNA chimera oligonucleotides to inhibit the regulatory function of human immunodeficiency virus type 1 Rev protein.

Human immunodeficiency virus type 1 (HIV-1) encodes two regulatory proteins, Tat and Rev, that bind to target RNA sequences. These are the trans-activation responsive (TAR) RNA and the Rev-responsive element (RRE), respectively. The Rev protein shifts RNA synthesis to viral transcripts by binding to the RRE within the env gene. In the present study we prepared a RNA-DNA chimera consisting of 29 or 31 nucleotides to inhibit the Rev regulatory function by means of the decoy approach. The chimera oligonucleotides (anti-Rev oligonucleotides [AROs]) contained an RNA "bubble" structure (13 oligonucleotides; the Rev-binding element in RRE) that bound Rev with a high affinity in an in vitro assay. The controls were RNA-DNA chimera oligonucleotides (negative control oligonucleotides [NCOs]) similar to ARO, but without the bubble structure, that bound with considerably less affinity to Rev. When the inhibitory effects of these decoys on HIV-1 replication were examined, we found that AROs, but no NCOs, reduced more than 90% of the HIV-1 production generated by productively infected human T-cell lines. The production of primary HIV-1 isolates in healthy donor-derived peripheral blood mononuclear cells was also similarly inhibited by AROs. In addition, the induction of viral mRNAs and antigens in latently HIV-1-infected ACH-2 cells by tumor necrosis factor alpha was specifically inhibited by AROs, but not by NCOs. No apparent cytotoxicity was caused by either decoy. Thus, the use of a Rev-binding element-based decoy, the RNA-DNA chimera oligonucleotide, may represent a safer approach to gene therapy for reducing the virus load in HIV-1-infected individuals.