Eosinophils Contribute to Oral Tolerance via Induction of RORγt-Positive Antigen-Presenting Cells and RORγt-Positive Regulatory T Cells.

Oral tolerance has been defined as the specific suppression of immune responses to an antigen by prior oral administration of the antigen. It has been thought to serve to suppress food allergy. Previous studies have shown that dendritic cells (DCs) and regulatory T cells (Tregs) are involved in the induction of oral tolerance. However, the detailed mechanisms of Treg induction in oral tolerance remain largely unknown. Eosinophils have been recognized as effector cells in allergic diseases, but in recent years, the diverse functions of tissue-resident eosinophils have been reported. Eosinophils in the intestine have been reported to induce Tregs by releasing TGF-ß, but the role of eosinophils in oral tolerance is still controversial. In this study, we analyzed the roles of eosinophils in oral tolerance using eosinophil-deficient ΔdblGATA mice (mice lacking a high-affinity GATA-binding site in the GATA1 promoter). ΔdblGATA mice showed impaired antigen-induced oral tolerance compared to wild-type mice. The induction of RORγt+ Tregs in mesenteric lymph nodes (MLNs) by oral tolerance induction was impaired in ΔdblGATA mice compared to wild-type mice. An increase in RORγt+ antigen-presenting cells (APCs), which are involved in RORγt+ Treg differentiation, in the intestine and MLNs was not seen in ΔdblGATA mice. Notably, the expansion of group 3 innate lymphoid cells (ILC3s), a subset of RORγt+ APCs, by oral tolerance induction was seen in wild-type mice but not ΔdblGATA mice. These results suggest that eosinophils are crucial in the induction of oral tolerance, possibly via the induction of RORγt+ APCs and RORγt+ Tregs.

Type 1 Cryoglobulinemic Vasculitis Due to Monoclonal Gammopathy of Undetermined Significance Successfully Treated by Bortezomib Plus Dexamethasone.

Cryoglobulins are immunoglobulins that precipitate in cold conditions. Type I cryoglobulinemic vasculitis is associated with hematological malignancies. We herein report a case of steroid-resistant type 1 cryoglobulinemic vasculitis associated with monoclonal gammopathy of undetermined significance (MGUS) in a 47-year-old woman. By immunofixation of cryoglobulin, we found that the main component of cryoglobulin was the M protein due to MGUS, so treatment of MGUS was needed. Bortezomib+dexamethasone therapy resulted in a rapid decrease in cryoglobulin and improvement in the symptoms of cryoglobulinemic vasculitis. In refractory type I cryoglobulinemic vasculitis, treatment of the underlying gammaglobulinopathy should be considered.

Polymerization Properties of n-Butyl Cyanoacrylate Mixed with Ethiodized Oil in the Lymphatic System: In Vivo Investigation in a Rabbit Model.

PURPOSE: To evaluate the polymerization properties of a mixture of n-butyl cyanoacrylate (nBCA) and ethiodized oil in the lymphatic system using an animal model. MATERIALS AND METHODS: Nineteen male Japanese White rabbits underwent 28 lymphatic embolization procedures under fluoroscopic guidance using manually injected mixtures of nBCA and ethiodized oil at ratios of 1:2 (nBCA density of 33%), 1:4 (20%), 1:6 (14%), and 1:8 (11%) via the popliteal lymph node. The time required for polymerization and the distance traveled by the mixture were evaluated and compared among the groups using the Kruskal-Wallis test. Histopathologic intergroup comparisons and time-course changes were also evaluated using embolized lymph nodes. RESULTS: Among 23 successful procedures, the mean polymerization times were 14 ± 3, 88 ± 93, 331 ± 292, and 932 seconds ± 540 and the mean distances traveled were 13 ± 10, 31 ± 44, 85 ± 89, and 108 mm ± 35 in the 33% (n = 5), 20% (n = 6), 14% (n = 6), and 11% (n = 6) groups, respectively. The 11% group demonstrated a significantly longer polymerization time than the 33%, 20%, and 14% groups and distance traveled than the 33% group. Pathologically, the embolized lymph nodes showed inflammatory changes and massive necrosis regardless of the nBCA density. CONCLUSIONS: Polymerization times and distances traveled were increased when nBCA was diluted with increasing quantitites of ethiodized oil in this rabbit model of lymphatic embolization. These relationships should be considered when dilution is prescribed for clinical use.

Calcineurin inhibitor inhibits tolerance induction by suppressing terminal exhaustion of donor T cells after allo-HCT.

Calcineurin inhibitor-based graft-versus-host disease (GVHD) prophylaxis is standard in allogeneic hematopoietic stem cell transplantation (HCT) but fails to induce long-term tolerance without chronic GVHD (cGVHD) in a considerable number of patients. In this study, we addressed this long-standing question in mouse models of HCT. After HCT, alloreactive donor T cells rapidly differentiated into PD-1+ TIGIT+ terminally exhausted T cells (terminal Tex). GVHD prophylaxis with cyclosporine (CSP) suppressed donor T-cell expression of TOX, a master regulator to promote differentiation of transitory exhausted T cells (transitory Tex), expressing both inhibitory receptors and effector molecules, into terminal Tex, and inhibited tolerance induction. Adoptive transfer of transitory Tex, but not terminal Tex, into secondary recipients developed cGVHD. Transitory Tex maintained alloreactivity and thus PD-1 blockade restored graft-versus-leukemia (GVL) activity of transitory Tex and not terminal Tex. In conclusion, CSP inhibits tolerance induction by suppressing the terminal exhaustion of donor T cells, while maintaining GVL effects to suppress leukemia relapse.

Subcutaneous Panniculitis-like T-cell Lymphoma Lacking Subcutaneous Tumor Mimicking Adult-onset Still's Disease.

We herein report a case of subcutaneous panniculitis-like T-cell lymphoma (SPTCL) resembling adult-onset Still's disease (AOSD). A 40-year-old woman presented with a fever, erythema, and painful subcutaneous nodules on the trunk. Laboratory data and a bone marrow analysis showed hemophagocytic syndrome. Although AOSD was suspected, based on a histopathological evaluation of the erythema, she was diagnosed with SPTCL. She was refractory to combination chemotherapy but achieved durable remission with cyclosporine monotherapy. Genetic testing revealed a homozygous HAVCR2 c.245A>G variant (rs184868814) that had caused NLRP3 inflammasome activation. SPTCL and AOSD share a pathogenesis in terms of NLRP3 inflammasome activation, so the clinical phenotype of SPTCL reasonably mimics AOSD.

Evaluation of the correlation between multiple organ calcification on CT and disease severity in patients with TAFRO syndrome.

PURPOSE: The purpose of this study was to investigate the incidence of multiple organ calcification and the correlation between multiple organ calcification and clinical severity in patients with thrombocytopenia, anasarca, fever, reticulin fibrosis, renal dysfunction, and organomegaly (TAFRO) syndrome. METHODS: We retrospectively identified 13 patients with TAFRO syndrome who were treated at our hospital between February 2019 and March 2021. Computed tomography (CT) images of TAFRO patients, which were acquired at admission and one month after admission, were evaluated. Additionally, clinical and laboratory data related to organ calcification and severity classification of TAFRO syndrome were investigated. The correlation between the presence of organ calcification on CT and TAFRO syndrome-severity classification was evaluated. RESULTS: One month after admission, calcification of the myocardium, adrenal glands, gallbladder wall, pancreas, kidney, skeletal muscle, and skin were observed in 38%, 46%, 15%, 15%, 15%, 23%, and 15% of the thirteen patients, respectively. The occurrence rate of calcifications in the myocardium, adrenal glands, and skeletal muscle was significantly higher in patients with a grade 4 or higher clinical severity than in those with a level up to grade 3 (p = 0.001, p = 0.005, and p = 0.035, respectively). CONCLUSIONS: Our results suggest that the higher the clinical severity in patients with TAFRO syndrome, the higher is the frequency of calcification in the myocardium, adrenal glands, and skeletal muscle; therefore, the assessment of these organ calcifications on CT images may be useful in predicting the severity of TAFRO syndrome.

Reactive granulopoiesis depends on T-cell production of IL-17A and neutropenia-associated alteration of gut microbiota.

Granulopoiesis in the bone marrow adjusts cellular output as demand for neutrophils changes. Reactive granulopoiesis is induced by profound neutropenia, but its mechanism remains to be clarified. We herein explored its mechanisms using mouse models of syngeneic hematopoietic stem cell transplantation (SCT) and 5-fluorouracil-induced neutropenia. After SCT, T cell production of IL-17A was up-regulated. Neutrophil recovery was significantly delayed in IL-17A-deficient or T cell-deficient RAG1-/- mice, and adoptive transfer of wild-type (WT) T cells facilitated neutrophil engraftment. Gut decontamination with oral antibiotics suppressed T cell production of IL-17A and impaired neutrophil recovery. Transplantation of fecal microbiota collected from neutropenic, not naive, mice promoted neutrophil recovery in these mice, suggesting that neutropenia-associated microbiota had a potential to stimulate reactive granulopoiesis. Our study uncovered a cross talk between gut microbiota and neutropenia after SCT and chemotherapy.

Gilteritinib enhances graft-versus-leukemia effects against FLT3-ITD mutant leukemia after allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-SCT) is a potentially curative therapy for FLT3 internal tandem duplication mutant (FLT3-ITD+) acute myeloid leukemia, but relapse rate is high. A recent study showed that sorafenib, a first generation FLT3 and multikinase inhibitor, enhanced graft-versus-leukemia (GVL) effects against FLT3-ITD+ leukemia via interleukin-15 (IL-15) production. However, it remains to be clarified whether this effect could be mediated by selective FLT3 inhibition. We investigated whether gilteritinib, a selective FLT3 inhibitor, could enhance GVL effects against FLT3-ITD transfected Ba/F3 leukemia (Ba/F3-FLT3-ITD) in mice. Oral administration of gilteritinib from day +5 to +14 after allo-SCT reduced expression of the co-inhibitory receptors PD-1 and TIGIT on donor CD8+ T cells and enhanced IL-15 expression in Ba/F3-FLT3-ITD. Bioluminescent imaging using luciferase-transfected Ba/F3-FLT3-ITD demonstrated that gilteritinib significantly suppressed leukemia expansion after allo-SCT, whereas it did not impact the morbidity or mortality of graft-versus-host disease (GVHD), resulting in significant improvement of overall survival. In conclusion, short-term administration of gilteritinib after allo-SCT enhanced GVL effects against FLT3-ITD+ leukemia without exacerbating GVHD.

Flavonoids in the flowers of Primula ×polyantha Mill. and Primula primulina (Spreng.) H. Hara (Primulaceae).

Two undescribed anthocyanins and two undescribed flavonols were isolated from the flowers of Primula ×polyantha Mill., along with five known anthocyanins and four known flavonols. The two undescribed anthocyanins and the two undescribed flavonols were determined to be hirsutidin 3-O-ß-galactopyranoside-5-O-ß-glucopyranoside, 7-O-methyl-petunidin 3-O-ß-galactopyranoside-5-O-ß-glucopyranoside, quercetin 3-O-ß-[(6""-acetylglucopyranosyl)-(1 â†’ 2)-ß-glucopyranosyl-(1 â†’ 6)-ß-glucopyranoside], and kaempferol 3-O-ß-[(6""-acetylglucopyranosyl)-(1 â†’ 2)-ß-glucopyranosyl-(1 â†’ 6)-ß-glucopyranoside] using chemical and spectroscopic methods. They were also found in the flowers of the Himalayan wild species, Primula primulina (Spreng.) H. Hara except for quercetin 3-O-ß-[(6""-acetylglucopyranosyl)-(1 â†’ 2)-ß-glucopyranosyl-(1 â†’ 6)-ß-glucopyranoside]. The flower color variations of P. ×polyantha cultivars, reflected by the hue values (b*/a*) of the colors, were due to the glycosidic patterns in the anthocyanins and their concentrations in the petals. Moreover, in the P. ×polyantha cultivars with violet-blue flowers, both the intermolecular copigmentation occurs between hirsutidin 3-O-ß-galactopyranoside-5-O-ß-glucopyranoside and another flavonol, quercetin 3-O-ß-glucopyranosyl-(1 â†’ 2)-ß-glucopyranosyl-(1 â†’ 6)-ß-glucopyranoside. Moreover, the flower color variation was affected by the pH value.

Intestinal goblet cells protect against GVHD after allogeneic stem cell transplantation via Lypd8.

Graft-versus-host disease (GVHD) and infection are major obstacles to successful allogeneic hematopoietic stem cell transplantation (HSCT). Intestinal goblet cells form the mucus layers, which spatially segregate gut microbiota from host tissues. Although it is well known that goblet cell loss is one of the histologic features of GVHD, effects of their loss in pathophysiology of GVHD remain to be elucidated. In mouse models of allogeneic HSCT, goblet cells in the colon were significantly reduced, resulting in disruption of the inner mucus layer of the colon and increased bacterial translocation into colonic mucosa. Pretransplant administration of interleukin-25 (IL-25), a growth factor for goblet cells, protected goblet cells against GVHD, prevented bacterial translocation, reduced plasma concentrations of interferon-γ (IFN-γ) and IL-6, and ameliorated GVHD. The protective role of IL-25 was dependent on Lypd8, an antimicrobial molecule produced by enterocytes in the colon that suppresses motility of flagellated bacteria. In clinical colon biopsies, low numbers of goblet cells were significantly associated with severe intestinal GVHD, increased transplant-related mortality, and poor survival after HSCT. Goblet cell loss is associated with poor transplant outcome, and administration of IL-25 represents an adjunct therapeutic strategy for GVHD by protecting goblet cells.

Vogt-Koyanagi-Harada disease-like uveitis following nivolumab administration treated with steroid pulse therapy: a case report.

BACKGROUND: Immune checkpoint inhibitors can cause various adverse effects. Recently it has been shown that Vogt-Koyanagi-Harada (VKH) disease-like uveitis can occur in patients treated with nivolumab. CASE PRESENTATION: A 69-year-old man developed bilateral panuveitis after nivolumab treatment for recurrent hypopharyngeal cancer. Slit lamp examination revealed bilateral granulomatous keratic precipitates, anterior chamber cells and partial synechiae. Fundus examination revealed bilateral optic disc edema and diffuse serous retinal detachment. His human leukocyte antigen (HLA) typing showed HLA-DRB1*04:05 allele. A lumbar puncture did not demonstrate pleocytosis. Bilateral sub-tenon injections of triamcinolone acetonide were initiated. As his panuveitis did not regress completely, steroid pulse therapy was administered. That therapy led to the resolution of his serous retinal detachment and to rapid improvement in his vision. Following this, we treated him with 50 mg/day of prednisolone for 1 week and then reduced it by 5 mg every week. No bilateral uveitis relapse had occurred by his 3-month follow-up; however, he subsequently died because of his cancer. CONCLUSION: To our knowledge, this is the first report of a patient with NVKH who underwent a lumbar puncture. Unlike VKH, our case did not show meningismus or pleocytosis. NVKH may, therefore, have a different etiology from VKH. In cases of NVKH with posterior uveitis, steroid pulse therapy may be considered as a treatment option, as it is in VKH.

Multiple liver metastases of pulmonary carcinoid successfully treated by two-stage hepatectomy combined with embolization of portal vein branches: Report of a case.

INTRODUCTION: The prognosis of atypical pulmonary carcinoid with liver metastases is extremely poor, and patients with multiple liver metastases are often treated using non-surgical therapies. We report a case with multiple liver metastases from atypical pulmonary carcinoid that was successfully treated using two-stage hepatectomy combined with embolization of portal vein branches. PRESENTATION OF CASE: A 48-year-old man was referred to our department after multiple liver tumors were detected in both liver lobes on computed tomography. He had undergone right upper lobectomy of the lung for atypical pulmonary carcinoid (T2a, N0, M0; Stage IB) 2 years previously. Positron emission tomography-computed tomography showed no extrahepatic tumor manifestations. The tumors were located in segment 2, 3, 5/8 and the right hepatic vein drainage area. We planned complete resection of metastases in a two-stage hepatectomy. The first stage comprised concomitant left lateral segmentectomy, partial hepatectomy of segment 5/8 and portal vein embolization of the posterior segmental branches. The second stage comprised resection of the right hepatic vein drainage area, performed 21 days after the first surgery. Histopathological diagnosis was liver metastases of atypical pulmonary carcinoid. Postoperative bile leak developed, which was treated with endoscopic retrograde biliary drainage and percutaneous bile leak drainage. He has been followed for 24 months postoperatively without tumor recurrence. DISCUSSION: Two-stage hepatectomy may represent an option for bilobar multiple liver metastases from atypical pulmonary carcinoid. CONCLUSION: We successfully treated a patient with multiple liver metastases of atypical pulmonary carcinoid using a two-stage hepatectomy combined with portal vein embolization of the posterior segmental branches.

Myasthenia gravis after allogeneic bone marrow transplantation: A case report and literature review.

A 52-year-old man with acute myeloid leukemia underwent allogeneic hematopoietic stem cell transplantation and developed extensive chronic graft-versus-host disease and myasthenia gravis (MG), which became involved with oculobulbar and proximal upper and lower limb weakness in 677days. In the literature, we identified 24 cases where MG developed after allo-SCT. Graft-versus-host disease development and male recipients of female donors might be prone to the development of posttransplant MG (odds ratio, 3.75).

[Thrombotic microangiopathy developing subsequent to tocilizumab therapy in a patient with TAFRO syndrome].

We report a case of a 60-year-old male who presented with fever and anasarca as well as hepatosplenomegaly, general lymphadenopathy, and disseminated intravascular coagulation (DIC), and was, therefore, admitted to our hospital. In addition, the patient suffered from respiratory failure and renal dysfunction and had pleural effusion and ascites. The pathological diagnosis from lymph node biopsy suggested multicentric Castleman's disease of the plasma cell type; however, the presence of high IL-6 levels, myelofibrosis, thrombocytopenia, anasarca, renal dysfunction, and hepatosplenomegaly led to a definitive diagnosis of TAFRO syndrome. Tocilizumab was administered on day 15 of disease diagnosis, resulting in the improvement in DIC but not other symptoms. As schizocytes were detected in the peripheral blood, he also experienced disturbance of consciousness and thrombotic microangiopathy (TMA) was considered. Following plasma exchange (PE) and continuous hemodiafiltration (CHDF), his symptoms temporarily improved. However, his condition worsened again, and he eventually died on day 33. Pathological autopsy revealed that although the lymph nodes were not enlarged, he had organomegaly, gastrointestinal and omental hemorrhage, and acute necrotizing pancreatitis. Since TMA developed after the administration of tocilizumab, the possibility of drug-induced secondary TMA cannot be ruled out.

Efficacy and prognosis of antiviral therapy on hepatitis C following treatment of lymphoma in HCV-positive diffuse large-cell lymphoma.

The purpose of this study is to study the usefulness of post-remission antiviral therapy in cases of HCV-RNA-positive diffuse large-cell lymphoma. Antiviral therapy against HCV was performed after remission using CHOP or CHOP-like chemotherapy in combination with rituximab in five successive cases of HCV-RNA-positive diffuse large-cell lymphoma. The control groups consisted of a group of HCV-RNA-positive diffuse large-cell lymphoma cases prior to this trial (control 1), and a group of cases that tested negative for HIV, HCV, and HBV (control 2). All the cases were in remission at the time of initial treatment. There were no significant differences between the three groups in terms of age, sex, treatment, stage, or International Prognosis Index (IPI). When HCV antiviral therapy was performed after treatment for diffuse large-cell lymphoma, we observed no recurrence or deaths, and the 2-year overall survival and progression-free survival rates were significantly greater than those in the control 1 group (P = 0.0246). It is possible that a better prognosis can be achieved by performing HCV antiviral therapy after achieving remission in cases of HCV-RNA-positive diffuse large-cell lymphoma through the use of R-CHOP or similar treatments.

Evaluation of Secure Computation in a Distributed Healthcare Setting.

Issues related to ensuring patient privacy and data ownership in clinical repositories prevent the growth of translational research. Previous studies have used an aggregator agent to obscure clinical repositories from the data user, and to ensure the privacy of output using statistical disclosure control. However, there remain several issues that must be considered. One such issue is that a data breach may occur when multiple nodes conspire. Another is that the agent may eavesdrop on or leak a user's queries and their results. We have implemented a secure computing method so that the data used by each party can be kept confidential even if all of the other parties conspire to crack the data. We deployed our implementation at three geographically distributed nodes connected to a high-speed layer two network. The performance of our method, with respect to processing times, suggests suitability for practical use.

Determination of Singlet Oxygen and Electron Transfer Mediated Mechanisms of Photosensitized Protein Damage by Phosphorus(V)porphyrins.

The mechanism of photosensitized protein damage byphosphorus(V) tetraphenylporphyrin derivatives (P(V)TPPs) wasquantitatively clarified. P(V)TPPs bound to human serum albumin(HSA), a water-soluble protein, and damaged its tryptophan residueduring photoirradiation. P(V)TPPs photosensitized singlet oxygen ((1)O(2))generation, and the contribution of (1)O(2) to HSA damage was confirmedby the inhibitory effect of sodium azide, a (1)O(2) quencher. However,sodium azide could not completely inhibit HSA damage, suggesting thecontribution of an electron transfer mechanism to HSA damage. Thedecrement in the fluorescence lifetime of P(V)TPPs by HSA supportedthe electron transfer mechanism. The contribution of these processes could be determined by the kinetic analysis of the effect ofsodium azide on the photosensitized protein damage by P(V)TPPs.

[Applicability of PCR methods for detection of shrimp and crab in processed food].

According to Japanese food allergen labeling regulations, an ELISA screening test is used for detection of crustacean proteins in food and a shrimp/crab-PCR confirmation test is used to confirm a positive ELISA screening test and to exclude false positives. Forty-six kinds of processed foods labeled as containing shrimp/crab were subjected to ELISA screening test and PCR confirmation test and the usefulness of the shrimp/crab-PCR was evaluated. Twenty-seven of the 46 samples contained total crustacean protein levels of 10 ppm or more in the ELISA screening test. All of the samples were positive in the shrimp/crab-PCR confirmation test. The results of the confirmation test were consistent with the declaration in the list of ingredients and with the results of the ELISA screening test. The shrimp/crab-PCR confirmation test was demonstrated to be applicable to various kinds of foods, including powder, extract, seasoning paste, prepared frozen food, snack food, retort food and canned food.

APOLLON protein promotes early mitotic CYCLIN A degradation independent of the spindle assembly checkpoint.

In the mammalian cell cycle, both CYCLIN A and CYCLIN B are required for entry into mitosis, and their elimination is also essential to complete the process. During mitosis, CYCLIN A and CYCLIN B are ubiquitylated by the anaphase-promoting complex/cyclosome (APC/C) and then subjected to proteasomal degradation. However, CYCLIN A, but not CYCLIN B, begins to be degraded in the prometaphase when APC/C is inactivated by the spindle assembly checkpoint (SAC). Here, we show that APOLLON (also known as BRUCE or BIRC6) plays a role in SAC-independent degradation of CYCLIN A in early mitosis. APPOLON interacts with CYCLIN A that is not associated with cyclin-dependent kinases. APPOLON also interacts with APC/C, and it facilitates CYCLIN A ubiquitylation. In APPOLON-deficient cells, mitotic degradation of CYCLIN A is delayed, and the total, but not the cyclin-dependent kinase-bound, CYCLIN A level was increased. We propose APPOLON to be a novel regulator of mitotic CYCLIN A degradation independent of SAC.

Disjunct distribution of chloroplast DNA haplotypes in the understory perennial Veratrum album ssp. oxysepalum (Melanthiaceae) in Japan as a result of ancient introgression.

• The Quaternary climatic changes resulted in range shifts of species, providing chances for hybridization. However, the genetic signatures of such ancient introgression have rarely been reported. To investigate such signatures, we performed a phylogeographical study on the perennial plant Veratrum album ssp. oxysepalum, which may have hybridized long ago with another congeneric species, V. stamineum. • Sequence variations in chloroplast DNA (cpDNA) were examined in 43 populations in Japan and adjacent areas. Phylogenetic analyses of different cpDNA haplotypes were conducted on the basis of cpDNA and nuclear ribosomal internal transcribed spacer (nrITS) variations. • In the Japanese archipelago, two major groups of haplotypes were detected, one of which was distributed in a disjunct pattern. The major haplotype, occupying the central part of the species' distribution, formed a monophyletic group with V. stamineum in phylogenetic trees on the basis of cpDNA variation, although the two species did not form a monophyletic group in phylogenetic trees on the basis of nrITS variation. • Historical hybridization between V. album ssp. oxysepalum and V. stamineum in refugia during the Quaternary climatic oscillations, and the resulting chloroplast capture of V. stamineum by V. album ssp. oxysepalum, are most probably responsible for the disjunct distribution of cpDNA in V. album ssp. oxysepalum.