Respiratory epithelial adenomatoid hamartomas of the sinonasal tract: A histopathological analysis of 50 patients.

Respiratory epithelial adenomatoid hamartoma (REAH) is a benign lesion of the nasal cavity and paranasal sinuses. Here, we report the clinicopathological characteristics of REAH identified in 2065 cases with nasal/paranasal polypoid lesions treated with endoscopic sinus surgery (ESS) at our hospital from 2008 to 2021. Cases including the olfactory area were reviewed and 50 patients of REAH were identified pathologically (50/2065, 2.4%). The average age was 58.9 years old and the male/female ratio was 45/5. Grossly, REAH showed a whitish surface and elastic firm consistency. The histopathological characteristics included proliferation of small to medium-sized glands composed of ciliated respiratory epithelium containing goblet cells; thickening of the basement membrane compared to that for inverted papilloma (9.6 ± 2.4 vs. 1.3 ± 1.6 µm, p < 0.001); and no intra-epithelial neutrophilic infiltration. Among the REAH cases, 81% were associated with sinonasal inflammatory polyps. Many olfactory cleft polyps were REAH (38/98, 39%). The rate of REAH found in ESS in the last 7 years was higher than that in the first 7 years (3.17% vs. 1.62%, p = 0.032). Our results in Japanese patients are similar to those found in other countries, including male predominance. REAH is relatively common and that 39% of polyps taken from olfactory clefts are REAH.

Altered cytokine profiles in relapsed paucibacillary leprosy: a case report.

BACKGROUND: Individuals with relapses of leprosy should be monitored carefully, however, with respect to paucibacillary (PB) leprosy, it is sometimes difficult to make a definitive diagnosis of relapse, because the bacillary index is often negative. To evaluate the usefulness of cytokine profiling in a patient with relapsed PB leprosy who tested negative for anti-phenolic glycolipid-I antibodies, we analyzed the Mycobacterium leprae protein-induced cytokine expression in peripheral blood mononuclear cells of the patient. CASE PRESENTATION: An 89-year-old-male relapsed PB patient, first treated for leprosy over 50 years prior, was examined. In April 2012, he noticed three skin lesions consisting of annular erythema in the thighs. Slit skin smear tests were negative, and skin biopsies revealed a pathology of indeterminate-to-borderline tuberculoid leprosy. He received 600 mg of rifampicin once per month and 75 mg of dapsone daily for 12 months. The annular erythemas disappeared after starting treatment. Before treatment, and 6 and 12 months after starting treatment, the Th1/Th2 cytokine profiles in the supernatant of mononuclear cells from the patient before and after stimulation with Mycobacterium leprae soluble protein (MLS) were examined using a Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II. The CBA Enhanced Sensitivity Flex Set system was applied to detect small amounts of cytokines in the serum just before treatment and one year before relapse. In the culture supernatant, just before treatment, increases in IFN-γ level and the IFN-γ/IL-10 ratio and a decreased IL-6 level were observed without stimulation. Upon stimulation with MLS, just before treatment, both the IFN-γ and TNF levels increased markedly, and twelve months after starting treatment, the IFN-γ and TNF levels decreased greatly. In the serum, just before treatment, increases in IFN-γ and TNF levels and the IFN-γ/IL-10 ratio were evident compared with those measured one year before relapse. CONCLUSIONS: Cytokine profiling using culture supernatants and serum samples may be useful for the diagnosis of relapsed PB leprosy.

[Review of WHO Expert Committee on Leprosy 8th report, --comparison to 7th report].

In 2012 the WHO Expert Committee on Leprosy published its 8th report, 14 years after the publication of its 7th report in 1998. This report, the first since the leprosy reduction goal was met in 2000, highlights key points such as improvements in the quality of various services available to patients and the efforts of individuals and societies, in addition to medical progress in diagnosis and treatment. This review will mainly describe the changes made since the 7th report. Some of the main modifications are the deletion of single lesion paucibacillary type, elongated treatment of patients with high bacterial indices, the introduction of promising new drugs, and a shift from reducing the statistical number of patients to a new target for disability prevention.

Heterotopic salivary duct inclusions in Warthin tumor - A cryptic histological finding involved in its pathogenesis.

Warthin tumor (WT) is the second most common benign parotid gland tumor after pleomorphic adenoma. WT is characterized by cystic and papillary proliferation of a two-layered oncocytic epithelium supported by lymphoid tissue. Heterotopic salivary duct inclusions (SDIs) are frequently observed in lymph nodes (LNs) of WT (SDI/LNs), and are thought to be the origin of WT. If this is true, SDIs should also persist in the lymphoid tissue of WT itself (SDI/WT), as a missing link between SDIs and WTs, but studies of this issue are limited. From 2008-2023, 138 WT cases were surgically excised at our hospital. SDI/LNs and SDI/WTs were histologically examined. Of 100 WT cases with LNs, SDI/LNs were observed in 67 cases (67 %). SDI/WTs were detected in 114 of 138 cases (82.6 %), including 107 of 127 smokers (84.3 %) and 7 of 11 never-smokers (63.6 %). SDI/WTs were located mainly in the subcapsular lymphoid tissue and often surrounded by a fibrous coat resembling salivary excretory ducts. This study revealed a high incidence of SDIs in WT itself, strongly supporting the theory that WT develops from heterotopic salivary ducts.

[Guidelines for the treatment of Hansen's disease in Japan (third edition)].

ad hoc committee of Japanese Leprosy Association recommends revised standard treatment protocol of leprosy in Japan, which is a modification of World Health Organization's multidrug therapy (WHO/MDT, 2010). For paucibacillary (PB) leprosy, 6 months treatment by rifampicin and dapsone (MDT/PB) is enough. However, for high bacterial load multibacillary (MB) leprosy, 12 months treatment seems insufficient. Thus, (A) For MB with bacterial index (BI) > 3 before treatment, 2 years treatment by rifampicin, dapsone and clofazimine (MDT/MB) is necessary. When BI becomes negative and active lesion is lost within 2 years, no maintenance therapy is necessary. When BI is still positive, one year of MDT/MB is added (3 years in total), followed by maintenance therapy by dapsone and clofazimine until BI negativity and loss of active lesions. (B) For MB with BI < 3 or fresh MB (less than 6 months after the onset of the disease) with BI > 3, 1 year treatment by MDT/MB is necessary. When BI becomes negative and active lesion is lost within one year, no maintenance therapy is necessary. When BI is still positive or active lesion is remaining, additional therapy with MDT/MB for one more year is recommended. Brief summary of diagnosis, purpose of therapy, character of drugs, and prevention of deformity is also described.

PGL-III, a Rare Intermediate of Mycobacterium leprae Phenolic Glycolipid Biosynthesis, Is a Potent Mincle Ligand.

Although leprosy (Hansen's disease) is one of the oldest known diseases, the pathogenicity of Mycobacterium leprae (M. leprae) remains enigmatic. Indeed, the cell wall components responsible for the immune response against M. leprae are as yet largely unidentified. We reveal here phenolic glycolipid-III (PGL-III) as an M. leprae-specific ligand for the immune receptor Mincle. PGL-III is a scarcely present trisaccharide intermediate in the biosynthetic pathway to PGL-I, an abundant and characteristic M. leprae glycolipid. Using activity-based purification, we identified PGL-III as a Mincle ligand that is more potent than the well-known M. tuberculosis trehalose dimycolate. The cocrystal structure of Mincle and a synthetic PGL-III analogue revealed a unique recognition mode, implying that it can engage multiple Mincle molecules. In Mincle-deficient mice infected with M. leprae, increased bacterial burden with gross pathologies were observed. These results show that PGL-III is a noncanonical ligand recognized by Mincle, triggering protective immunity.

A novel anti-MUC1 antibody against the MUC1 cytoplasmic tail domain: use in sensitive identification of poorly differentiated cells in adenocarcinoma of the stomach.

BACKGROUND: Isolated cancer cells of non-solid type poorly differentiated adenocarcinoma (por2) or signet-ring cell carcinoma (sig) are frequently seen in scirrhous gastric cancers with a very poor prognosis. These cells are often scattered in granulation tissue or desmoplastic fibrotic tissue and tend to be overlooked in routine pathological examination. We aimed to raise a novel antibody that can identify the isolated cancer cells easily. METHODS: Because the MUC1 cytoplasmic tail domain (CTD) has many biological roles including tumor progression and cell adhesion disturbance and is expected to be expressed in isolated cancer cells, we raised a novel monoclonal antibody (MAb) MUC1-014E against an intracellular nonrepeating 19-amino-acid sequence (RYVPPSSTDRSPYEKVSAG: N-1217-1235-C) of the MUC1 CTD, using a synthetic peptide including the 7-amino-acid epitope (STDRSPY: N-1223-1229-C). RESULTS: In the immunohistochemical staining of 107 gastrectomy specimens including 48 por2 and 31 sig lesions, the MAb MUC1-014E showed high rates of positive staining (≥5% of carcinoma cells stained) for por2 (100%) and sig (97%), and of the highest intensity staining (4+, ≥75% of carcinoma cells stained) for por2 (100%) and sig (90%). In the 89 biopsy specimens including 82 por2 and 38 sig lesions, the MAb MUC1-014E showed high rates of positive staining for por2 (100%) and sig (100%) and of 4+ staining for por2 (87%) and sig (84%). All the rates were significantly higher than those with cytokeratins (AE1/AE3 or CAM5.2). CONCLUSIONS: The MAb MUC1-014E is very useful for accurate detection of isolated cancer cells in scirrhous gastric cancers.

Therapeutic efficacy of rifalazil (KRM-1648) in a M. ulcerans-induced Buruli ulcer mouse model.

Buruli ulcer (BU) is a skin disease caused by Mycobacterium ulcerans infection that requires long-term antibiotic treatment and/or surgical excision. In this study, we investigated the therapeutic efficacy of the rifamycin derivative, rifalazil (RLZ) (also known as KRM-1648), in an advanced M. ulcerans infection model. Six-week-old female BALB/c mice were infected with 3.25 x 104 colony-forming units (CFU) of M. ulcerans subcutaneously into the bilateral hind footpads. At 33 days post-infection, when the footpads exhibited significant redness and swelling, mice were treated orally with 5 or 10 mg/kg of RLZ for up to 15 weeks. Mice were followed for an additional 15 weeks following treatment cessation. Untreated mice exhibited a progressive increase in footpad redness, swelling, and erosion over time, and all untreated mice reached to endpoint within 5-8 weeks post-bacterial injection. In the RLZ-treated mice, footpad redness and swelling and general condition improved or completely healed, and no recurrence occurred following treatment cessation. After 3 weeks of treatment, the CFU counts from the footpads of recovered RLZ-treated mice showed a 104 decrease compared with those of untreated mice. We observed a further reduction in CFU counts to the detection limit following 6 to 15 weeks of treatment, which did not increase 15 weeks after discontinuing the treatment. Histopathologically, bacteria in the treated mice became fragmented one week after RLZ-treatment. At the final point of the experiment, all the treated mice (5mg/kg/day; n = 6, 10mg/kg/day; n = 7) survived and had no signs of M. ulcerans infection. These results indicate that the rifamycin analogue, RLZ, is efficacious in the treatment of an advanced M. ulcerans infection mouse model.

Genome-wide screening identified SEC61A1 as an essential factor for mycolactone-dependent apoptosis in human premonocytic THP-1 cells.

Buruli ulcer is a chronic skin disease caused by a toxic lipid mycolactone produced by Mycobacterium ulcerans, which induces local skin tissue destruction and analgesia. However, the cytotoxicity pathway induced by mycolactone remains largely unknown. Here we investigated the mycolactone-induced cell death pathway by screening host factors using a genome-scale lenti-CRISPR mutagenesis assay in human premonocytic THP-1 cells. As a result, 884 genes were identified as candidates causing mycolactone-induced cell death, among which SEC61A1, the α-subunit of the Sec61 translocon complex, was the highest scoring. CRISPR/Cas9 genome editing of SEC61A1 in THP-1 cells suppressed mycolactone-induced endoplasmic reticulum stress, especially eIF2α phosphorylation, and caspase-dependent apoptosis. Although previous studies have reported that mycolactone targets SEC61A1 based on mutation screening and structural analysis in several cell lines, we have reconfirmed that SEC61A1 is a mycolactone target by genome-wide screening in THP-1 cells. These results shed light on the cytotoxicity of mycolactone and suggest that the inhibition of mycolactone activity or SEC61A1 downstream cascades will be a novel therapeutic modality to eliminate the harmful effects of mycolactone in addition to the 8-week antibiotic regimen of rifampicin and clarithromycin.

DNA methylation and histone H3-K9 modifications contribute to MUC17 expression.

MUC17 glycoprotein is a membrane-associated mucin that is mainly expressed in the digestive tract. It has been suggested that MUC17 expression is correlated with the malignancy potential of pancreatic ductal adenocarcinomas (PDACs). In the present study, we provided the first report of the MUC17 gene expression through epigenetic regulation such as promoter methylation, histone modification and microRNA (miRNA) expression. Near the transcriptional start site, the DNA methylation level of MUC17-negative cancer cell lines (e.g. PANC1) was high, whereas that of MUC17-positive cells (e.g. AsPC-1) was low. Histone H3-K9 (H3-K9) modification status was also closely related to MUC17 expression. Our results indicate that DNA methylation and histone H3-K9 modification in the 5' flanking region play a critical role in MUC17 expression. Furthermore, the hypomethylation status was observed in patients with PDAC. This indicates that the hypomethylation status in the MUC17 promoter could be a novel epigenetic marker for the diagnosis of PDAC. In addition, the result of miRNA microarray analysis showed that five potential miRNA candidates existed. It is also possible that the MUC17 might be post-transcriptionally regulated by miRNA targeting to the 3'-untranslated region of its mRNA. These understandings of the epigenetic changes of MUC17 may be of importance for the diagnosis of carcinogenic risk and the prediction of outcomes for cancer patients.

Mucins in human neoplasms: clinical pathology, gene expression and diagnostic application.

Mucins are high molecular weight glycoproteins that play important roles in carcinogenesis and tumor invasion. Our immunohistochemical studies demonstrated that MUC1 or MUC4 expression is related to the aggressive behavior and poor outcome of human neoplasms. MUC2 is expressed in indolent pancreatobiliary neoplasms, but these tumors sometimes show invasive growth with MUC1 expression in invasive areas. MUC5AC shows de novo high expression in many types of precancerous lesions of pancreatobiliary cancers and is an effective marker for early detection of the neoplasms. The combination of MUC1, MUC2, MUC4 and MUC5AC expression may be useful for early detection and evaluation of the potential for malignancy of pancreatobiliary neoplasms. Regarding the mechanism of mucin expression, we have recently reported that expression of the mucin genes is regulated epigenetically in cancer cell lines, using quantitative MassARRAY analysis, methylation-specific polymerase chain reaction analysis and chromatin immunoprecipitation analysis, with confirmation by the treatment with 5-aza-2'-deoxycytidine and trichostatin A. We have also developed a monoclonal antibody against the MUC1 cytoplasmic tail domain, which has many biological roles. Based on all of the above findings, we suggest that translational research into mucin gene expression mechanisms, including epigenetics, may provide new tools for early and accurate detection of human neoplasms.

Acute respiratory distress syndrome as an initial presentation of hemophagocytic lymphohistiocytosis after induction therapy for acute myeloid leukemia.

A 7-month-old girl with acute myeloid leukemia (AML) developed acute respiratory distress syndrome (ARDS) during the pancytopenic period after induction chemotherapy. Respiratory failure did not improve despite intensive treatments. Eventually, hemophagocytic lymphohistiocytosis (HLH) was diagnosed based on hemophagocytosis in bone marrow, and high soluble interleukin-2 receptor (sIL-2R) and ferritin levels. Even after cyclosporin A was started against HLH, she did not recover. Autopsy showed macrophage proliferation in bone marrow and lymph nodes. HLH should be considered, even in the pancytopenic period after chemotherapy, when patients develop ARDS that does not respond to supportive therapies.

[Role of mycolactone in the nerve damage of Buruli ulcer (Mycobacterium ulcerans infection)].

Buruli ulcer is a skin disease caused by Mycobacterium ulcerans (M. ulcerans). In this review, we introduce our recent studies and other important works. Lesions of Buruli ulcer are usually painless, despite the extensive tissue necrosis. We have reported that mice inoculated with M ulcerans show nerve degeneration and absence of pain, but the mechanism evoking the nerve damage have not been clarified. In order to define whether mycolactone, a toxic lipid produced by M. ulcerans, can induce nerve damages, we have injected mycolactone A/B to BALB/c mouse footpads. Mycolactone induced footpad swelling, and sensory test showed hyperesthesia on day 7 and 14, recovery on day 21, and hypoesthesia on days 28 and 42. Histologically, nerve bundles showed hemorrhage, neutrophilic infiltration, and loss of Schwann cell nuclei on days 7 and 14. Semithin section studies revealed vacuolar change of Schwann cells started on day 14, which subsided by day 42, but myelinated fiber density remained low. This study suggests that mycolactone directly damages nerves and is responsible for the absence of pain characteristic of Buruli ulcer. In the human lesions, presence of neuritis is reported (Rondini S, 2006), and murine studies showed "autoamputation" (Addo P, 2005). In order to prevent the serious deformities evoked by Buruli ulcer, further studies are necessary.

[Japanese guideline on thalidomide usage in the management of erythema nodosum leprosum].

Treatment of erythema nodosum leprosum (ENL, type 2 reaction) using thalidomide provides effective alternative choice to steroid therapy. Yet, the Japanese National Health Insurance approves thalidomide prescription only for the treatment of multiple myeloma under the Thalidomide Education and Risk Management System (TERMS). Benefit of thalidomide therapy for patients with ENL is already an established fact based on various reports from other countries, but limited experiences and standards in Japan have hindered application of the medication to our patients. This led us to compose a local guideline. Based on and following the TERMS, we suggest starting thalidomide from 50-100 mg/day and then onwards adjusting the dose according to the symptoms of each patient, not to exceed the maximum recommended dose of 300 mg/day, for the treatment of ENL.

Expression of mucin antigens (MUC1 and MUC16) as a prognostic factor for mucinous adenocarcinoma of the uterine cervix.

AIM: Only limited data are available regarding mucin antigen expression in adenocarcinoma of the uterine cervix. METHODS: Fifty-two patients with mucinous adenocarcinoma of the endocervical type were enrolled for making clear implication of the mucin antigens. The expression of mucin antigens in hysterectomized specimens were determined immunohistochemically. Clinicopathological characteristics and prognosis included International Federation of Gynecology and Obstetrics (FIGO) stage, histological grade, nodal and ovarian metastases, overall and disease-free survivals. The relationships of the expression of mucin antigens to various variables were investigated using uni- and multivariate analyses. RESULTS: The majority of mucinous adenocarcinoma showed overexpression of MUC1 and MUC16. Overexpression of both MUC1 and MUC16 was associated with lower survival rates. Particularly, overexpression of MUC1 was associated with a lower disease-free survival rate and lymph node metastasis. However, absence of expression of MUC1 and/or MUC16 was associated with longer overall and disease-free survival. In cases classified as FIGO stage Ib (n = 35), after adjusting for patient age at diagnosis and stratifying by histological grade, MUC1 and/or MUC 16 overexpression was an independent prognostic factor for overall survival (hazard ratio [HR] = 6.16, 95% confidence interval [CI] = 1.01-118.5, P < 0.05). After stratifying by ovarian metastasis, MUC1 and/or MUC 16 overexpression was also an independent prognostic factor for disease-free survival (HR = 5.50, 95% CI = 0.82-87.4, P < 0.05). CONCLUSION: The expressions of MUC1 and/or MUC16 may be available as independent prognostic factors for the endocervical type of mucinous adenocarcinoma.

Expression profiles of MUC1, MUC2, and MUC4 mucins in human neoplasms and their relationship with biological behavior.

Mucins are high molecular weight glycoproteins that play important roles in carcinogenesis or tumor invasion. To clarify the relationship of the expression patterns of mucins in human neoplasms with their biological behavior, we examined the expression profiles of MUC1, MUC2, and MUC4 mucins in various human neoplasms using immunohistochemistry and in situ hybridization, and compared them with clinicopathologic factors including outcome of the patients. MUC1 or MUC4 expression is related with the aggressive behavior of human neoplasms and a poor outcome of the patients. In contrast, MUC2 expression tends to be related with the indolent behavior of human neoplasms and a favorable outcome of the patients, although indolent pancreatobiliary neoplasms sometimes show invasive growth with MUC1 expression in the invasive areas. The expression of MUC2 mucin in indolent pancreatobiliary neoplasms coincided with expression of MUC2 mRNA. Our recent studies to clarify the MUC2 gene regulation mechanism disclosed that DNA methylation and histone modification in the 5' flanking region of the MUC2 promoter may play an important role. Further studies of the epigenetics also in MUC1 and MUC4 gene expression may be needed to understand the relationship between the expression of mucins in human neoplasms with their biological behavior.

Mycolactone is responsible for the painlessness of Mycobacterium ulcerans infection (buruli ulcer) in a murine study.

Buruli ulcer is a chronic skin disease caused by Mycobacterium ulcerans, which produces a toxic lipid mycolactone. Despite the extensive necrosis and tissue damage, the lesions are painless. This absence of pain prevents patients from seeking early treatment and, as a result, many patients experience severe sequelae, including limb amputation. We have reported that mice inoculated with M. ulcerans show loss of pain sensation and nerve degeneration. However, the molecules responsible for the nerve damage have not been identified. In order to clarify whether mycolactone alone can induce nerve damage, mycolactone A/B was injected to footpads of BALB/c mice. A total of 100 microg of mycolactone induced footpad swelling, redness, and erosion. The von Frey sensory test showed hyperesthesia on day 7, recovery on day 21, and hypoesthesia on day 28. Histologically, the footpads showed epidermal erosion, moderate stromal edema, and moderate neutrophilic infiltration up to day 14, which gradually resolved. Nerve bundles showed intraneural hemorrhage, neutrophilic infiltration, and loss of Schwann cell nuclei on days 7 and 14. Ultrastructurally, vacuolar change of myelin started on day 14 and gradually subsided by day 42, but the density of myelinated fibers remained low. This study demonstrated that initial hyperesthesia is followed by sensory recovery and final hypoesthesia. Our present study suggests that mycolactone directly damages nerves and is responsible for the absence of pain characteristic of Buruli ulcer. Furthermore, mice injected with 200 microg of mycolactone showed pulmonary hemorrhage. This is the first study to demonstrate the systemic effects of mycolactone.

Quantitative evaluation of maxillary bone deformation by computed tomography in patients with leprosy.

BACKGROUND: Facial deformation as a sequela of leprosy is caused not only by a saddle nose but also by regression of the maxilla, as well documented in paleopathological observations of excavated skeletal remains of patients with leprosy. However, maxillary changes in living patients have been evaluated only by the subjective visual grading. Here, we attempted to evaluate maxillary bone deformation in patients with leprosy using three-dimensional computed tomography (3D-CT). METHODS: Three-dimensional images centered on the maxilla were reconstructed using multiplanar reconstruction methods in former patients with leprosy (n = 10) and control subjects (n = 5); the anterior-posterior length of the maxilla (MA-P) was then measured. The difference between the MA-P of the patients and those of controls was evaluated after compensating for individual skull size. These findings were also compared with those from previous paleopathological studies. FINDINGS: Three former patients with lepromatous leprosy showed marked atrophy of the maxilla at the prosthion (-8.6, -11.1 and -17.9 mm) which corresponded with the visual appearance of the maxillary deformity, and these results were consistent with paleopathological findings of excavated skeletal remains. Additionally, the precise bone defects of the maxilla could be individually calculated for accurate reconstructive surgery. INTERPRETATION: We have successfully illustrated maxillary bone deformities in living patients with leprosy. This study also confirmed the maxillary regression described in paleopathological studies.

Naturally occurring a loss of a giant plasmid from Mycobacterium ulcerans subsp. shinshuense makes it non-pathogenic.

Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU), a WHO-defined neglected tropical disease. All Japanese BU causative isolates have shown distinct differences from the prototype and are categorized as M. ulcerans subspecies shinshuense. During repeated sub-culture, we found that some M. shinshuense colonies were non-pigmented whereas others were pigmented. Whole genome sequence analysis revealed that non-pigmented colonies did not harbor a giant plasmid, which encodes elements needed for mycolactone toxin biosynthesis. Moreover, mycolactone was not detected in sterile filtrates of non-pigmented colonies. Mice inoculated with suspensions of pigmented colonies died within 5 weeks whereas those infected with suspensions of non-pigmented colonies had significantly prolonged survival (>8 weeks). This study suggests that mycolactone is a critical M. shinshuense virulence factor and that the lack of a mycolactone-producing giant plasmid makes the strain non-pathogenic. We made an avirulent mycolactone-deletion mutant strain directly from the virulent original.