The histopathological comparison on the destruction of the periodontal tissue between normal junctional epithelium and long junctional epithelium.

BACKGROUND AND OBJECTIVE: The barrier function of long junctional epithelium is thought to be important after periodontal initial therapy and periodontal surgery. Although the difference between long junctional epithelium and normal junctional epithelium regarding their resistance to destruction of periodontal tissue has been investigated, the mechanism still remains unclear. Using our rat experimental periodontitis model in which loss of attachment and resorption of alveolar bone is induced by the formation of immune complexes, we investigated the resistance of periodontal tissue containing long junctional epithelium and normal junctional epithelium to destruction. MATERIAL AND METHODS: Rats were divided into four groups. In the immunized long junctional epithelium (I-LJE) group, rats were immunized with lipopolysaccharide (LPS), and curettage and root planing procedures were performed on the palatal gingiva of the maxillary first molars to obtain reattachment by long junctional epithelium. In the immunized normal junctional epithelium (I-JE) group, rats were immunized without curettage and root planing procedures. In the nonimmunized long junctional epithelium (nI-LJE) group, rats were not immunized but curettage and root-planing procedures were performed. In the control group, neither immunization nor curettage and root-planing was performed. In all rats, periodontal inflammation was induced by topical application of LPS into the palatal gingival sulcus of maxillary first molars. The rats were killed at baseline and after the third and fifth applications of LPS. Attachment loss and the number of inflammatory cells and osteoclasts in the four groups were compared histopathologically and histometrically. RESULTS: After the third application of LPS in the I-LJE group, attachment loss showed a greater increase than in control and nI-LJE groups, and inflammatory cell infiltration and osteoclasts were increased more than in the other groups. After the fifth application of LPS, attachment loss was greater and there was a higher degree of inflammatory cell infiltration in nI-LJE and I-LJE groups than in control and I-JE groups. CONCLUSION: Our findings suggest that the destruction of periodontal tissue is increased in tissue containing long junctional epithelium compared with normal junctional epithelium and that the immunized condition accelerates the destruction by forming immune complexes.

Green tea extract inhibits the onset of periodontal destruction in rat experimental periodontitis.

BACKGROUND AND OBJECTIVE: Green tea extract exerts a variety of biological effects, including anti-inflammatory activities. However, there has been no report on the effect of green tea extract on loss of attachment, which is an important characteristic of periodontitis. Here, we examined the inhibitory effects of green tea extract on the onset of periodontitis in a rat model. MATERIAL AND METHODS: Rats were immunized intraperitoneally with Escherichia coli lipopolysaccharide (LPS). The LPS group (n = 12) received a topical application of LPS onto the palatal gingival sulcus every 24 h. The green tea extract group (n = 12) received a topical application of LPS mixed with green tea extract, sunphenon BG, every 24 h. The phosphate-buffered saline (PBS) group (n = 6) received a topical application of PBS every 24 h. The levels of anti-LPS immunoglobulin G (IgG) in serum were determined using ELISA. Rats in the LPS and green tea extract groups were killed after the 10th and 20th applications. Rats in the PBS group were killed after the 20th application. Loss of attachment, level of alveolar bone and inflammatory cell infiltration were investigated histopathologically and histometrically. RANKL-positive cells and the formation of immune complexes were evaluated immunohistologically. RESULTS: There was no significant difference in the serum levels of anti-LPS IgG between the LPS group and the green tea extract group. In contrast, loss of attachment, level of alveolar bone, inflammatory cell infiltration and RANKL expression in the green tea extract group were significantly decreased compared with those in the LPS group. CONCLUSION: These findings demonstrate that green tea extract suppresses the onset of loss of attachment and alveolar bone resorption in a rat model of experimental periodontitis.

Occlusal trauma accelerates attachment loss at the onset of experimental periodontitis in rats.

BACKGROUND AND OBJECTIVE: Occlusal trauma is an important factor that influences the progression of periodontitis, but it is unclear whether occlusal trauma influences periodontal destruction at the onset of periodontitis. We established an experimental periodontitis model with both site-specific loss of attachment and alveolar bone resorption. The purpose of the present study was to investigate the effects of occlusal trauma on periodontal destruction, particularly loss of attachment, at the onset of experimental periodontitis. MATERIAL AND METHODS: Sixty rats were used in the present study. Forty-eight rats immunized with lipopolysaccharide (LPS) intraperitoneally were divided into four groups. In the trauma (T) group, occlusal trauma was induced by placing an excessively high metal wire in the occlusal surface of the mandibular right first molar. In the inflammation (I) group, periodontal inflammation was induced by topical application of LPS into the palatal gingival sulcus of maxillary right first molars. In the trauma + inflammation (T+I) group, both trauma and periodontal inflammation were simultaneously induced. The PBS group was administered phosphate-buffered saline only. Another 12 nonimmunized rats (the n-(T+I) group) were treated as described for the T+I group. All rats were killed after 5 or 10 d, and their maxillary first molars with surrounding tissues were observed histopathologically. Loss of attachment and osteoclasts on the alveolar bone crest were investigated histopathologically. To detect immune complexes, immunohistological staining for C1qB was performed. Collagen fibers were also observed using the picrosirius red-polarization method. RESULTS: There were significant increases in loss of attachment and in the number of osteoclasts in the T+I group compared with the other groups. Moreover, widespread distribution of immune complexes was observed in the T + I group, and collagen fibers oriented from the root surface to the alveolar bone crest had partially disappeared in the T, T+I and n-(T+I) groups. CONCLUSION: When inflammation was combined with occlusal trauma, immune complexes were confirmed in more expanding areas than in the area of the I group without occlusal trauma, and loss of attachment at the onset of experimental periodontitis was increased. Damage of collagen fibers by occlusal trauma may elevate the permeability of the antigen through the tissue and result in expansion of the area of immune-complex formation and accelerating inflammatory reaction. The periodontal tissue destruction was thus greater in the T+I group than in the I group.

Gram-positive bacteria as an antigen topically applied into gingival sulcus of immunized rat accelerates periodontal destruction.

BACKGROUND AND OBJECTIVE: Periodontitis is generally accepted to relate to gram-negative bacteria, and the host defense system influences its onset and progression. However, little is known about the relation between gram-positive bacteria and periodontitis. In this study, we topically applied gram-positive and gram-negative bacterial suspensions to the gingival sulcus in rats after immunization, and then histopathologically examined their influence on periodontal destruction. MATERIALS AND METHODS: Rats previously immunized with heat-treated and sonicated Staphylococcus aureus or Aggregatibacter actinomycetemcomitans were used as immunized groups. The non-immunized group received only sterile phosphate-buffered saline. In each animal, S. aureus or A. actinomycetemcomitans suspension was applied topically to the palatal gingival sulcus of first molars every 24 h for 10 d. Blood samples were collected and the serum level of anti-S. aureus or anti-A. actinomycetemcomitans immunoglobulin G (IgG) antibodies was determined by enzyme-linked immunosorbent assay. The first molar regions were resected and observed histopathologically. Osteoclasts were stained with tartrate-resistant acid phosphatase (TRAP). The formation of immune complexes was confirmed by immunohistological staining of C1qB. RESULTS: Serum levels of anti-S. aureus and anti-A. actinomycetemcomitans IgG antibodies in the immunized groups were significantly higher than those in the non-immunized groups were. The loss of attachment, increase in apical migration of the junctional epithelium, and decreases in alveolar bone level and number of TRAP-positive multinuclear cells in each immunized group were significantly greater than in each non-immunized group. The presence of C1qB was observed in the junctional epithelium and adjacent connective tissue in the immunized groups. CONCLUSIONS: Heat-treated and sonicated S. aureus and A. actinomycetemcomitans induced attachment loss in rats immunized with their suspensions. Our results suggest that not only gram-negative but also gram-positive bacteria are able to induce periodontal destruction.

Topical application of lipopolysaccharide into gingival sulcus promotes periodontal destruction in rats immunized with lipopolysaccharide.

BACKGROUND AND OBJECTIVE: The causes of periodontitis are bacteria and the host immune system, but the role of the immune system in the onset and progression of periodontal disease is still unclear. Our previous report showed that the formation of an immune complex in the gingival sulcus induces periodontal destruction. This study was carried out to investigate how the immune system, particularly immunization, is involved in periodontal destruction. MATERIAL AND METHODS: Animals immunized intraperitoneally with lipopolysaccharide (LPS) were used as the immunized group. The nonimmunized group received only phosphate-buffered saline. LPS was applied daily onto the palatal gingival sulcus in both groups 1 d after the booster injection. Serum levels of anti-LPS IgG were determined. Loss of attachment and the level of alveolar bone were histopathologically and histometrically investigated. RANKL-bearing cells and the expression of C1qB were immunohistologically evaluated. RESULTS: The serum levels of anti-LPS IgG were elevated in the early experimental period in the immunized group. There were significant increases in loss of attachment, level of alveolar bone and the number of RANKL-bearing cells in the immunized group. C1qB was observed in the junctional epithelium and adjacent connective tissue. The nonimmunized group showed similar findings at and after the time when the serum level of anti-LPS IgG was elevated. CONCLUSION: Topical application of LPS as an antigen induced periodontal destruction when the serum level of anti-LPS IgG was elevated in rats immunized with LPS. The presence of C1qB suggests that the formation of immune complexes is involved in this destruction.

The formation of immune complexes is involved in the acute phase of periodontal destruction in rats.

BACKGROUND AND OBJECTIVE: Loss of clinical attachment and alveolar bone destruction are major symptoms of periodontitis, caused by not only the destructive effect of periodontopathic bacteria but also the overactive response of the host immune system against periodontal pathogens. The details of the participation of the immune system in the onset and progression of periodontitis are unclear. In this study, we attempted to determine whether the host immune system, and in particular the formation of immune complexes, is involved in the periodontal destruction. MATERIAL AND METHODS: We applied ovalbumin or lipopolysaccharide (LPS) as antigens and their specific immunoglobulin G (IgG) antibodies purified from rat serum to rat gingival sulcus alternately. Loss of attachment, alveolar bone destruction and the numbers of inflammatory cells infiltrating the periodontal tissue and osteoclasts on the alveolar bone surface were investigated histometrically. The formation of immune complex was confirmed by immunohistological staining of complement C1qB. RESULTS: Loss of attachment and the presence of C1qB were observed histopathologically in both experimental groups. The group that had been treated with LPS and anti-LPS IgG showed greater loss of attachment. The number of inflammatory cells in the periodontal tissue was increased in both experimental groups, while osteoclasts at the alveolar bone crest were observed only in the group that had been treated with LPS and anti-LPS IgG. CONCLUSION: In the present study, we showed that the formation of immune complex appears to be involved in the acute phase of periodontal destruction and that the biological activity of antigens is also important.

Proteomic inventory of myocardial proteins from patients with chronic Chagas' cardiomyopathy.

Chronic Chagas' disease cardiomyopathy (CCC) is an often fatal outcome of Trypanosoma cruzi infection, with a poorer prognosis than other cardiomyopathies. CCC is refractory to heart failure treatments, and is the major indication of heart transplantation in Latin America. A diffuse myocarditis, plus intense myocardial hypertrophy, damage and fibrosis, in the presence of very few T. cruzi forms, are the histopathological hallmarks of CCC. To gain a better understanding of the pathophysiology of CCC, we analyzed the protein profile in the affected CCC myocardium. Homogenates from left ventricular myocardial samples of end-stage CCC hearts explanted during heart transplantation were subjected to two-dimensional electrophoresis with Coomassie blue staining; protein identification was performed by MALDI-ToF mass spectrometry and peptide mass fingerprinting. The identification of selected proteins was confirmed by immunoblotting. We demonstrated that 246 proteins matched in gels from two CCC patients. They corresponded to 112 distinct proteins. Along with structural/contractile and metabolism proteins, we also identified proteins involved in apoptosis (caspase 8, caspase 2), immune system (T cell receptor ss chain, granzyme A, HLA class I) and stress processes (heat shock proteins, superoxide dismutases, and other oxidative stress proteins). Proteins involved in cell signaling and transcriptional factors were also identified. The identification of caspases and oxidative stress proteins suggests the occurrence of active apoptosis and significant oxidative stress in CCC myocardium. These results generated an inventory of myocardial proteins in CCC that should contribute to the generation of hypothesis-driven experiments designed on the basis of the classes of proteins identified here.

Proteomic inventory of myocardial proteins from patients with chronic Chagas' cardiomyopathy

Chronic Chagas' disease cardiomyopathy (CCC) is an often fatal outcome of Trypanosoma cruzi infection, with a poorer prognosis than other cardiomyopathies. CCC is refractory to heart failure treatments, and is the major indication of heart transplantation in Latin America. A diffuse myocarditis, plus intense myocardial hypertrophy, damage and fibrosis, in the presence of very few T. cruzi forms, are the histopathological hallmarks of CCC. To gain a better understanding of the pathophysiology of CCC, we analyzed the protein profile in the affected CCC myocardium. Homogenates from left ventricular myocardial samples of end-stage CCC hearts explanted during heart transplantation were subjected to two-dimensional electrophoresis with Coomassie blue staining; protein identification was performed by MALDI-ToF mass spectrometry and peptide mass fingerprinting. The identification of selected proteins was confirmed by immunoblotting. We demonstrated that 246 proteins matched in gels from two CCC patients. They corresponded to 112 distinct proteins. Along with structural/contractile and metabolism proteins, we also identified proteins involved in apoptosis (caspase 8, caspase 2), immune system (T cell receptor ß chain, granzyme A, HLA class I) and stress processes (heat shock proteins, superoxide dismutases, and other oxidative stress proteins). Proteins involved in cell signaling and transcriptional factors were also identified. The identification of caspases and oxidative stress proteins suggests the occurrence of active apoptosis and significant oxidative stress in CCC myocardium. These results generated an inventory of myocardial proteins in CCC that should contribute to the generation of hypothesis-driven experiments designed on the basis of the classes of proteins identified here.

Inhibition of root caries progression by an antibacterial adhesive.

A dentin primer containing the antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB) has been shown to penetrate and kill the bacteria in artificially demineralized dentin. We hypothesized that an experimental adhesive system, which incorporates the MDPB-containing primer, would be effective in inhibiting the progression of root caries in vitro. Artificial caries lesions were prepared by either an acid-gel or a Streptococcus mutans culture technique on the roots of extracted human teeth. The progression of these lesions after the application of the experimental or proprietary adhesive system was examined. Further demineralization was completely prevented by the experimental adhesive system, while lesions managed with the proprietary materials showed limited ability to inhibit further demineralization. We conclude that the experimental adhesive system can inhibit the progression of root-surface caries in vitro, through a combination of its antimicrobial activity and sealing of the demineralized dentin.

Evaluation of the automatic fluorescent image analyzer, Image Titer, for quantitative analysis of antinuclear antibodies.

By making comparisons with the usual manual method, we evaluated an automatic fluorescent image analyzer (Image Titer, Tripath Imaging, Burlington NC), the software for which was developed to simplify measuring indirect immunofluorescent antinuclear antibodies (FANAs). In this new system, images of the stained sample are displayed, and it measures the FANA titer and staining pattern using only 1 slide per subject and does not required the staining of a series of diluted samples as does the manual method. This system showed good reproducibility and linearity for 4 types of control serum samples (with homogeneous, speckled, discrete speckled, and nucleolar staining patterns). In 132 serum samples, consistency between the methods was 100% for the FANA staining pattern and 93.9% for the FANA titer. The Image Titer system detected each pattern in samples with 2 mixed patterns. This system should partly reduce labor and lead to results with minimum differences among individuals, including newly trained persons.

The challenges and rewards of institutional collaboration in distance education.

Faculty from five nursing schools created an innovative RN to BSN program, which is offered via distance education technology. The development of a distance education academic program leading to a degree involved working to overcome many barriers; however, this strategy for delivering an academic degree has a promising future.

Developing collaborative partnerships in continuing nursing education: walking the talk.

The purpose of this article is to describe and analyze a reciprocal partnership developed among four university-based continuing education providers in Wisconsin. Members of this partnership collaborated in developing a statewide program to address the educational needs of nurses moving from acute or long-term care into community nursing. Components of the reciprocal partnership model were applied in developing this program. The partners identified that, despite the initial time investment, the collaborative approach was an advantage. The collaborative approach enhanced the quality of the program developed and reached a wider audience. Continuing nursing educators may want to consider partnership arrangements for program development and implementation.

A long term follow-up of a randomized trial comparing interferon-alpha with busulfan for chronic myelogenous leukemia. The Kouseisho Leukemia Study Group.

To evaluate the long-term effectiveness of interferon-alpha (IFN-alpha) therapy in patients with chronic myelogenous leukemia (CML) in chronic phase, we examined the updated outcomes of 159 patients who had been enrolled between 1988 and 1991 into a randomized trial comparing IFN-alpha with busulfan. At a median follow-up of 73 months, the median survival was 71 months in the IFN-alpha group and 55 months in the busulfan group (P=0.0563), and the median time of remaining in chronic phase was 58 months in the IFN-alpha group and 39 months in the busulfan group (P=0.4676). Landmark analysis showed a significant advantage in survival (P=0.009) and duration of chronic phase (P=0.0001) in patients with any cytogenetic response among the IFN-alpha group. About half patients were discontinued IFN-alpha administration in spite of cytogenetic response in this study. It appears that continuation of IFN-alpha might possibly confer a survival advantage. Pretreatment factors associated with cytogenetic response included high hemoglobin level, low percentage of peripheral basophils and low leukocyte counts. Multivariate analysis identified lower percentage of bone marrow basophilia (P=0.007) for survival advantage. If a group with a very good prognosis is predicted by a new prognostic model, it might be an option to wait for bone marrow transplantation.

A descriptive study of continuing nursing education programs in academic settings.

This article describes survey results from 24 collegiate continuing nursing education programs in the United States. The purpose was to collect baseline data regarding the role of the continuing education (CE) administrator, the characteristics of financial information, and the organizational structure of the department. The findings showed that roles and responsibilities of the directors in continuing nursing education include both administrative activities and faculty responsibilities. The trend toward decreasing enrollments of CE registrants will make it imperative for the CE administrator to explore creative programming directions, configure an affordable staffing arrangement within the organization, and generate new sources of program revenue.

[Clinical study of itraconazole for systemic fungal infections complicated with hematological malignancies--multicenter cooperative study. West-Japan Systemic Fungal Infection Study Group].

Itraconazole, an oral antifungal agent, was evaluated for the clinical efficacy and safety in patients documented or suspected of systemic fungal infection (SFI) complicated with hematological malignancies. The data was also evaluated according to serological tests and fungal culture. Out of a total of 79 patients, the clinical efficacy of itraconazole was evaluated in 52 patients, comprising 4 patients with proven SFI, 33 with clinical SFI, and 15 with suspected SFI. The overall efficacy was 67.3% (35/52), in which 25.0% in the established SFI group (1/4), 78.8% in the clinical SFI group (26.33), and 53.3% in the suspected SFI (8/15). When assessed according to the severity of SFI, the effectiveness rates were 75.0% in the mild group (15/20), 76.2% in the moderate group (16/21), and 36.4% in the severe group (4/11), resulting in significantly higher rates in the mild and the moderate groups than in the severe group (p = 0.0479). Out of the 10 patients who were switched from the previous antifungals, 7 patients were judged as showing marked or good responses. All of these patients were switched from intravenous fluconazole therapy. With respect to the safety of itraconazole, among 79 patients eligible for safety evaluation, adverse events such as hepatic dysfunction, increase in GOT and/or GPT, nausea and vomiting, and chest pain were observed in 6 patients (7.6%), and itraconazole may have been associated with them. As a conclusion, itraconazole proved to be effective and safe on SFI developed in patients with various hematological malignancies.

Association between frequency of amino acid changes in core region of hepatitis B virus (HBV) and the presence of precore mutation in Japanese HBV carriers.

To elucidate the relationship between the frequency of core mutations and precore mutation of hepatitis B virus (HBV) in Japanese HBV carriers, we investigated the nucleotide sequence of the precore/core region of HBV in 26 Japanese HBV carriers [15 who were HBe antigen-negative (HBeAg-) and 11 who were HBeAg-positive (HBeAg+)]. The number of amino acid changes (5.9 +/- 3.8) in the core region of HBV in HBeAg-carriers was significantly greater than that in the HBeAg+ carriers (1.5 +/- 1.0; P < 0.005). The precore stop codon mutation was found in 93.3% of HBeAg-negative HBV carriers, while no precore mutation was found in the HBeAg-positive HBV carriers, suggesting that the frequency of core mutations may be associated with the presence of the precore stop codon mutation. However, there was no significant difference in the frequency of amino acid changes among HBeAg-HBV carriers. The mean number of core amino acid changes of liver cirrhosis patients, chronic active hepatitis patients, chronic persistent hepatitis patients, and asymptomatic carriers were 2.7 +/- 1.5, 6.0 +/- 2.2, 4.7 +/- 1.2, and 8.4 +/- 5.3, respectively. We detected hot spots for core mutations, which showed characteristic localizations and specific substitutions: Gly-87, Leu-97, and Thr-130 were detected exclusively in patients with chronic liver disease with or without HBeAg. To address further the relationship between frequency of core mutations and the presence of the precore stop codon mutation, we investigated the precore/core nucleotide sequence serially along with seroconversion in three patients with chronic hepatitis B in whom the hepatitis either became inactive or remained active after the seroconversion. Emergence of the precore stop codon mutation and a significant increase in core amino-acid changes after seroconversion were noted in all three patients. Our results suggest a close association between the frequency of core amino acid changes and the presence of the precore stop codon mutation; some characteristic core mutations may be associated with the clinical course of chronic hepatitis B in Japanese patients.

Pyridoxine refractory X-linked sideroblastic anemia caused by a point mutation in the erythroid 5-aminolevulinate synthase gene.

To elucidate how pyridoxine-refractory X-linked sideroblastic anemia (XLSA) develops, we analyzed the erythroid-specific 5-aminolevulinate synthase (ALAS-E) gene of a patient with the anemia. The activity and amount of the enzyme in bone marrow cells of the patient were found to be approximately 5% of the normal control. We identified a point mutation, which introduces an amino acid substitution from Asp 190 to Val. In transient transfection analyses using quail fibroblasts, accumulation of aberrantly processed proteins, the sizes of which were larger than that of mature ALAS-E, was found in mitochondria. The proteins were reproducibly detected in assays combining in vitro transcription/translation of ALAS-E precursor and import of the precursor into isolated mouse mitochondria. These results suggest that the mutation causing pyridoxine-refractory XLSA affects the processing of the ALAS-E precursor, thus provoking instability of the ALAS-E protein.

Molecular and genetic analysis of two patients with Bernard-Soulier syndrome--identification of new mutations in glycoprotein Ib alpha gene.

We investigated two unrelated patients with Bernard-Soulier syndrome (BSS) by performing molecular and genetic analysis. A flow cytometric and immunoblotting analysis showed GP Ib alpha to be absent from the platelet membrane of both patients. Other glycoproteins that formed GP Ib/IX/V complex were present on the platelets, but in decreased amounts. Therefore, GP Ib alpha gene from both cases was sequenced after PCR amplification and subcloning. We identified a homozygous mutation of a dinucleotide deletion within the TGTG repeat at cDNA number 972 to 975 in GP Ib alpha gene from Case 1. In Case 2, compound heterozygosity was demonstrated in GP Ib alpha gene; an insertion of a single base (T) at cDNA number 1,418 in one allele, and a deletion of a single base (A) within the 7-adenine repeat at cDNA number 1,438 to 1,444 in another allele. The three new mutations in both patients appeared to cause a frameshift, which created a new termination codon shortly thereafter, and thus lead to a GP Ib alpha deficiency on the platelet membrane. Truncated mutant proteins could be detected in the plasma and platelets of Case 2, but not of Case 1. According to these findings, it is thus supposed that the properties and conformation of additional COOH-terminal peptides, which were supposedly synthesized as results of the mutations, may have an important role on the processing of mutant GP Ib alpha in megakaryocytes and platelets.

Audiographics teleconferencing. A method of distance learning.

Teleconferencing is considered an alternative to traditional educational programming and can be accomplished through various methods. In this article, the authors explain audiographics, a form of teleconferencing that combines audio conferencing with a personal computer-based visual conferencing system. The authors discuss the system, the planning process, and the implementation and evaluation of audiographics during an 8-week continuing education course.

Oligoclonal accumulation of T cells in peripheral blood from patients with idiopathic thrombocytopenic purpura.

To determine whether clonal T cells accumulate in idiopathic thrombocytopenic purpura (ITP), we performed single-strand conformation polymorphism (SSCP) analysis to detect T-cell receptor (TCR) beta-chain usage of peripheral T cells. We detected significantly more oligoclonal T cells (15.5 +/- 8.9 bands representative for clonal T-cell expansions) in peripheral blood from ITP patients than from healthy donors (2.8 +/- 2.6 bands). Frequently used V beta genes in these accumulated T cells in ITP were V beta 3, 6, 10, 13.1 and 14. To determine whether these bands were derived from clonal T cells, presumably in a preactivated state, we established some T-cell clones (expressing CD4 and TCR V beta 6. 13.1. or 14) by nonspecific stimulation from patients peripheral mononuclear cells, and examined their clonotypes. Clonal identities for three out of seven clones tested were confirmed using SSCP analyses to compare the migration of their beta-chain complementarity determining region 3 (CDR3) cDNAs, expanded by polymerase chain reaction (PCR) with those from peripheral blood. Therefore, distinctive T-cell clones accumulated in the periphery in ITP and they may be related to the autoimmune-mediated destruction of platelets.