Analysis of T cell receptor V(beta) gene expression and clonality in bronchoalveolar fluid lymphocytes from a patient with chronic eosinophilic pneumonitis.

Chronic eosinophilic pneumonitis (CEP) is characterized by longstanding respiratory symptoms accompanied by a massive pulmonary eosinophil infiltration. T lymphocytes in bronchoalveolar lavage (BAL) from patients with chronic eosinophilic pneumonitis are considered to recognize unknown antigens. To analyze the pathogenesis of CEP, we examined the T cell receptor (TCR) repertoire and T cell clonotype of BAL lymphocytes and peripheral blood lymphocytes (PBLs) in a 66-year-old woman patient with CEP. The expression of TCR BV gene was analyzed by the family PCR method using specific primers for 20 TCR BV genes and BC gene. The clonotype of BAL and peripheral T cells was examined by the PCR-single-strand conformation polymorphism (SSCP) method. Functional sequences of some T cell clones were also carried out. A TCR repertoire of BAL T cells was heterogeneous as well as PBLs. However, SSCP analysis showed that distinct T cell clonotypes were detected in BAL T cells, TCR BV3, BV4, BV6, BV8, BV9, BV14, and BV18-positive T cell clones especially, expanded clonally in BAL from the patient. Sequencing analysis showed that GVD, LGG, RDXS, and SSG amino acid sequence motif were found in the CDR3 in lung-specific T cells. BAL-specific T cell clones accumulated in the patient with CEP. Thus, we can conclude that BAL T cells are induced by the antigen-driven stimulation and these cells might play a crucial role in the generation of CEP.

Umbilical cord blood T lymphocytes are induced to apoptosis after being allo-primed in vitro.

In this study, the immunity of umbilical cord blood (UCB) T lymphocytes against allo-antigens was investigated by a standard MLC. No significant difference, between the UCB T cells or peripheral blood (PB) mature T cells, was observed in the primary responses (stimulation index (SI), 51.8 +/- 14.8 and 46.5 +/- 15.0, respectively). In contrast, in the secondary response, the SI obtained with the CD4 T cells from UCB decreased dramatically (16.3 +/- 6.4), while it increased with the CD4 T cells from PB (118.5 +/- 21.7). UCB (CD4 and CD8) T cells separately showed much higher frequencies of apoptosis after a primary allo-priming, compared with PB CD4 and CD8 T cells (CD4, UCB 30.5% vs PB 0.8%; CD8, UCB 32% vs PB 1.3%). The higher apoptotic level of the UCB CD4 T cells was confirmed by a second, ELISA-based, Tunel assay (OD values, UCB CD4 1.93 +/- 0.31 vs PB CD4 0.59 0.9; P < 0.01). Those apoptotic steps were not attributed to the amount of cytokine (IL-2, 4 and IFN-gamma) production, which was found to be similar in both cases. In conclusion, UCB lymphocytes are much more likely to be induced to apoptosis by allo-priming than adult lymphocytes. This supports their possible, successful engraftment across barriers of HLA incompatibility.

T-cell clonal change after allo-kidney transplantation in humans.

Whether T cells circulating peripherally express changes at a clonal level after renal transplantation is uncertain. To clarify this issue, we analyzed T-cell clonality of peripheral blood lymphocytes (PBLs) in 12 renal transplant recipients by a novel polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) method that can discriminate T-cell clones with different T-cell receptor (TCR) Vbeta motifs. The PCR-SSCP study showed that after transplantation, only a few distinct T-cell clonotypes accumulated in the absence of clinical episodes, irrespective of the compatibility of HLA antigens. In contrast, various T-cell clones appeared in cases of acute rejection (AR) and infection. These subsided immediately after the AR was resolved; however, they remained long after the resolution of the infection. In a case of AR followed by an infectious episode, distinct T-cell clones appeared concomitantly with each episode. Several of them disappeared or remained thereafter. In one case, significant numbers of accumulating bands were observed by in-vitro stimulation by mixed lymphocyte reaction (MLR); several were identical to those found in vivo. However, some of those that did not appear in vitro were apparent in vivo. In conclusion, the appearance of T-cell clonotypes at a peripheral level indicates the existence of immunologically activated T-cell clones, which were significantly affected by immunosuppressive therapy. It was also determined that the T-cell immune system is much more complicated in vivo than in vitro.

[Bilateral hilar and mediastinal lymphadenopathy accomporying pulmonary infiltration with eosinophilia].

A 46-year-old man had had an occasional dry cough in the early morning since about the age of 20, but had received no treatment. He had been taking an antirheumatic drug for 2 years for rheumatoid arthritis. The patient complained of fever and dry coughing that began in the middle of November 1995, and he was treated for acute bronchitis. His condition did not improve, and he was admitted to the hospital in early December. Wheezing and rhonchi were heard in both lung fields. His white blood cell count was 19,000/mm3, and the eosinophil percent age was 48%. A chest CT scan revealed macular lesions with an increased density in both lung fields, and markedly swollen mediastinal and hilar lymph nodes. Analysis of alveolar lavage fluid revealed an increased number of cells (total) and eosinophilia (37%), and examination of a transbronchial lung biopsy specimen indicated infiltration with eosinophils and lymphocytes. Our diagnosis was eosinophilic pneumonia. The patient's condition improved soon after the start of pulse therapy with steroids. Bilateral swelling of mediastinal and hilar lymph nodes is rare in patients who have pulmonary in filtration with eosinophilia (the PIE syndrome).

Association between HLA-DPB1 matching and 1-year rejection-free graft survival in high-risk corneal transplantation.

We analyzed the effect of matching for HLA class II alleles on corneal graft outcome in a single-center, retrospective study from January 1991 through April 1996. The study involved 81 transplant recipients at high and low risk of corneal graft rejection, who were typed by the polymerase chain reaction-restriction fragment length polymorphism method and who completed at least 1-year of follow-up. The DRB1, DQB1, and DPB1 alleles were analyzed together and transplant recipients were subdivided into groups with matching (one to four alleles matched in the high risk or one to five alleles matched in the low risk) and without matching (no allele matched) for HLA class II. A significantly higher rate of 1-year rejection-free graft survival was revealed in high-risk transplant recipients with matching, compared with those without matching (P=0.0238). We have shown that matching for at least one HLA class II allele was actually beneficial in high-risk transplants. An analysis of matching for each allele separately, detected that only HLA-DPB1 matching was significantly associated with a higher rate of 1-year rejection-free graft survival in high-risk transplant recipients with matching (one or two alleles matched) compared with those without matching (no allele matched) (P=0.0139). In particular, matching for one DPB1 allele was significantly beneficial compared with no matching (P=0.0140). There was no significant effect of HLA-DRB1 and -DQB1 matching (P=0.3177 and P=0.2878, respectively). Furthermore, a strong association between DPB1 matching and 1-year rejection-free graft survival was observed in DRB1-incompatible high-risk transplant recipients (P=0.0308). Nevertheless, no significant effect of DPB1 matching was detected in DQB1-incompatible transplant recipients. Our findings indicate that HLA class II DNA typing is clinically relevant for corneal transplant recipients and that especially HLA-DPB1 matching has a beneficial effect in high-risk corneal transplantation.

Xenogeneic iso-skin graft and mixed lymphocyte reaction studies using HLA-DP transgenic mice.

To elucidate the cellular responses against xeno-MHC antigens, in vitro mixed lymphocyte culture (MLC) and in vivo skin grafting (SG) studies were conducted using HLA-DP transgenic mice (B6-DP mice). Xenogenic iso-(B6-DP to B6 mice) MLC showed positive but much lower responses compared to allo-MLC responses. Nevertheless, B6-DP skin grafts were rejected in a similar time course as allo-skin grafts. To examine mechanisms underlining skin graft rejection, both in vitro cytotoxic lymphocyte (CTL) responses and delayed-type hypersensitivity (DTH) reactions were tested. The studies showed that DTH but not CTL reactions were involved for the graft rejection. SG was again conducted after the administration of anti-CD4 and/or CD8 monoclonal antibody (mAb). Mice treated with both CD4 and CD8 mAb accepted B6-DP SG for as long as up to 60 days and those treated with either CD4 or CD8 mAb alone rejected skin grafts on its own most of the time (75% in anti-CD4 mAb treated mice, 88.9% in anti-CD8 mAb treated mice), which suggests that the strict T cell restrictions for xeno-DP antigens do not exist. Even in these finally rejected cases, longer median survival time and final rejection time were observed, and in the other mice (25% in anti-CD4 mAb treated, and 11.1% in anti-CD8 mAb treated mice), graft acceptance was found. Therefore, it was suggested that the immunological reactions leading to the graft rejection occurs most efficiently when both T cell subsets are present. The above results indicate that xenogeneic HLA-DP antigens could act as significant transplantation antigens equivalent to alloantigens despite their lower stimulative activity in vitro, and also support the interpretation that DP antigens act like a minor histocompatibility antigen beyond the difference of species. Monomorphic anti-HLA class II antibodies were detected in recipients' sera as early as 2 weeks and even at 6 months, indicating that xeno-MHC antigens are prone to be memorized to B cells. It was concluded that HLA transgenic mice are useful for the investigation of cellular responses across xeno-MHC barriers.

Stable clonal expansion of T cells induced by bone marrow transplantation.

The immune mechanisms of T cells regeneration after bone marrow transplantation (BMT) and the factors maintaining allogeneic marrow graft in the host are still unknown. To pursue this issue, we analyzed T-cell clonality of peripheral blood lymphocytes (PBLs) in BMT recipients, using reverse transcription polymerase chain reaction with T-cell receptor (TCR) V beta gene segment-specific primers and single-strand conformation polymorphism. PBLs from patients and donors showed a heterogeneous T-cell population with oligoclonal accumulations of CD8+ T cells. When PBLs were cultured in HLA-matched mixed lymphocytes reaction in vitro, no distinct clonal expansion was observed. However, after BMT, oligoclonal expansions were induced in the recipients in vivo, without a restriction of TCR V beta gene usage. Although part of the expansion was transient, the majority was repeatedly detected even several months later. Our results suggested that certain in vivo mechanisms maintain a stable clonal expansion of distinct T cells in marrow recipients. We also found in a single patient with graft-versus-host disease a replacement of expanded clones by other clones during follow-up. Diminishing numbers of accumulation clones were found in long-term marrow recipients, indicating a general tendency for clonal expansion to subside progressively. Considered together, our data suggest the involvement of clonally expanded T cells in lymphoid regeneration and in acute and chronic immune responses after BMT.

[Usefulness of the DNA-HLA class II typing in corneal transplantation].

DNA-HLA (human leukocyte antigen) typing was performed for ocular tissues in order to determine the usefulness of the method in corneal transplantation. Each type of ocular tissue was dissected from eye bank eyes (n = 3). DNA was extracted, and HLA-DRB1, DQB1, and DPB1 genes were amplified using PCR (polymerase chain reaction) method. DNA can be extracted and amplified from each ocular tissue except for the crystalline lens, but DNA from iris and choroid can be amplified only after diluting the samples 10 to 100 times. HLA class II antigens were successfully determined in these ocular tissues by the RFLP (restriction fragment length polymorphism) method. The method was applied to the corneal tissues of donor and recipient used for the corneal transplantation (n = 7). HLA class II antigens can be determined in both the donor and recipient corneal samples, and the results of recipients' HLA typing were the same as those determined using the blood samples. These results indicate that the DNA-HLA typing can be used for ocular tissues. Since DNA-HLA typing is more accurate than the conventional serological typing, the method is promising for the study of HLA class II typing in corneal transplantation.

Evaluation of prognoses of patients with gastric cancer by the responsiveness of peripheral blood lymphocyte to OK432 in vitro.

The response of peripheral blood lymphocytes to the streptococcal preparation OK432 was examined in vitro in 193 gastric cancer patients. When the patients were divided by stimulation index (SI) into two groups, SI > or = 20 and SI < 20, the response rate of SI > or = 20 group of stage IV was 37.5%, and higher than those of the other stages (p < 0.05). In the relationship between SI and the survival period in stage III, the SI > or = 20 group showed longer survival than the SI < 20 group (p < 0.05). There was no statistically significant difference in survival between SI > or = 20 and SI < 20 in stage IV, but no member of the SI < 20 group survived more than 2 years, whereas the five-year survival of the SI > or = 20 group was 38.9%. In the overall survival of stage III and IV, the SI > or = 20 group showed longer survival than the SI < 20 group (p = 0.001). The lymphoproliferative responses to OK432 decreased in very advanced gastric cancer patients and this might lead to poor prognosis.

Studies on transfer of varicella-zoster-virus specific T-cell immunity from bone marrow donor to recipient.

The transfer of antigen-specific cellular immunity in human bone marrow transplantation (BMT) was studied in 49 donor-recipient pairs, using a varicella-zoster-virus (VZV) specific lymphoproliferative response (LPR) assay. Posttransplant VZV-LPR could be serially measured in 31 long-term surviving recipients. VZV-specific T-cell immunity was detected in the early posttransplant period in 4 of 16 recipients who were, and whose donors were, immune to VZV before BMT, but two of those positive responses diminished in the first 100 days posttransplant. No positive response was detected in the immediate posttransplant period when either only the recipient or the donor was immune to VZV pretransplant. Herpes zoster or chickenpox developed in the recipients depending on a history of pretransplant VZV infection when the VZV-LPR became negative, and recovery from VZV infection was always followed by quick conversion of VZV-LPR. Long-lasting positive VZV-LPR was observed in the two recipients who experienced VZV infection in the immediate pretransplant period and received marrow graft from an immune donor. Our results indicate that a simple or direct transfer of VZV-specific cellular immunity from a marrow donor to a recipient cannot be expected in usual clinical bone marrow transplantation and that there might be a collaboration or recruitment of immune responses involving both donor and recipient that permits the VZV-LPR to remain positive posttransplant.

[Clinical evaluation of the metal-based denture using superplastic titanium alloy for upper edentulous patients].

The purpose of this study was to evaluate clinically superplastic titanium alloy for maxillary complete denture. The subjects that were the 30 patients with upper edentulous ridges were selected from Tsurumi University Dental Hospital. 17 patients were maxillary and mandibular edentulous, the other patients were only maxillary edentulous. Each of metal base was made of 90 Ti-6 Al-4 V alloy by Sankin Dental Co.. The portion of a resin was used of PMMA resin with 4-META (4-methacryloxyethyl trimellitic anhydride). At the time of inserting the denture and one month after, the dentures were clinically observed about the retentive force and the adhesive condition on finish line. At the same time, the patients were examined oral mucose and inquired of the feeling relating to wear new denture. Furthermore each weight of new dentures was compared with each of old dentures. The results were as following: 1. The width and depth of the palate were measured previously on posterior palatal sealed area. The mean value of the depth was 8.8 +/- 1.8 mm and the mean value of the width was 44.9 +/- 2.7 mm. 2. The retentive force of new dentures was clinically adequate enough to achievement of function without two cases. 3. The adhesive condition on the finish line was adequate in all cases. 4. The mean weight value of the new denture was 20.1 +/- 3.9 g. 5. 2 of 30 patients were finded the slightly redness without pain on the middle palatal mucose. 6. The feelings of patients relating to wear were as following: 1) Lighter than old denture. 2) More extensive tongue space.(ABSTRACT TRUNCATED AT 250 WORDS)