Expansion of functional human mucosal-associated invariant T cells via reprogramming to pluripotency and redifferentiation.

Mucosal-associated invariant T (MAIT) cells play an important physiological role in host pathogen defense and may also be involved in inflammatory disorders and multiple sclerosis. The rarity and inefficient expansion of these cells have hampered detailed analysis and application. Here, we report an induced pluripotent stem cell (iPSC)-based reprogramming approach for the expansion of functional MAIT cells. We found that human MAIT cells can be reprogrammed into iPSCs using a Sendai virus harboring standard reprogramming factors. Under T cell-permissive conditions, these iPSCs efficiently redifferentiate into MAIT-like lymphocytes expressing the T cell receptor Vα7.2, CD161, and interleukin-18 receptor chain α. Upon incubation with bacteria-fed monocytes, the derived MAIT cells show enhanced production of a broad range of cytokines. Following adoptive transfer into immunocompromised mice, these cells migrate to the bone marrow, liver, spleen, and intestine and protect against Mycobacterium abscessus. Our findings pave the way for further functional analysis of MAIT cells and determination of their therapeutic potential.

Dermal mast cells reduce progressive tissue necrosis caused by subcutaneous infection with Streptococcus pyogenes in mice.

A single subcutaneous (s.c.) infection with 1×10(7) c.f.u. GAS472, a group A streptococcus (GAS) serotype M1 strain isolated from the blood of a patient suffering from streptococcal toxic shock syndrome, led to severe damage of striated muscle layers in the feet of mast cell (MC)-deficient WBB6F(1)-Kit(W)/Kit(W-v) (W/W(v)) mice 72 h after infection. In contrast, no damage was recognized in striated muscle layers in the feet of the control WBB6F(1)-Kit(+/+) (+/+) mice 72 h after infection. In addition, adoptively transferred MCs reduced progressive tissue necrosis of the feet of W/W(v) mice after infection. However, there was no significant difference in the mortality rates between the W/W(v) and +/+ mice, or between the human CD46-expressing transgenic (Tg) mouse bone marrow-derived cultured MC-reconstituted W/W(v) and non-Tg mouse bone marrow-derived cultured MC-reconstituted W/W(v) mice after infection. Consequently, although MCs can help to reduce the severity of necrosis of the feet caused by s.c. infection with GAS472, such reduction of tissue necrosis scarcely improves the mortality rates of these mice. Moreover, human CD46 does not play a crucial role in the MC-mediated innate immune defence against GAS infection.

CD46 transgenic mouse model of necrotizing fasciitis caused by Streptococcus pyogenes infection.

We developed a human CD46-expressing transgenic (Tg) mouse model of subcutaneous (s.c.) infection into both hind footpads with clinically isolated 11 group A streptococcus (GAS) serotype M1 strains. When the severity levels of foot lesions at 72 h and the mortality rates by 336 h were compared after s.c. infection with 1x10(7) CFU of each GAS strain, the GAS472 strain, isolated from the blood of a patient suffering from streptococcal toxic shock syndrome (STSS), induced the highest severity levels and mortality rates. GAS472 led to a 100% mortality rate in CD46 Tg mice after only 168 h postinfection through the supervention of severe necrotizing fasciitis (NF) of the feet. In contrast, GAS472 led to a 10% mortality rate in non-Tg mice through the supervention of partial necrotizing cutaneous lesions of the feet. The footpad skin sections of CD46 Tg mice showed hemorrhaging and necrotic striated muscle layers in the dermis, along with the exfoliation of epidermis with intracellular edema until 48 h after s.c. infection with GAS472. Thereafter, the bacteria proliferated, reaching a 90-fold or 7-fold increase in the livers of CD46 Tg mice or non-Tg mice, respectively, for 24 h between 48 and 72 h after s.c. infection with GAS472. As a result, the infected CD46 Tg mice appeared to suffer severe liver injuries. These findings suggest that human CD46 enhanced the progression of NF in the feet and the exponential growth of bacteria in deep tissues, leading to death.

Evaluation of antibiotic therapy for eradication of "Candidatus Helicobacter heilmannii".

Triple-agent therapy with lansoprazole (15 mg/kg)-clarithromycin (50 mg/kg)-amoxicillin (50 mg/kg) twice daily for 7 days fully cleared "Candidatus Helicobacter heilmannii" from infected mouse stomachs. Moreover, gastric mucosa-associated lymphoid tissue lymphoma-like lesions in the stomach nearly disappeared in the treated mice 4 months after the therapy.

Comparative efficacies of different antibiotic treatments to eradicate nontypeable Haemophilus influenzae infection.

BACKGROUND: Nonencapsulated and nontypeable Haemophilus influenzae (NTHi) is a major cause of human respiratory tract infections. Some strains of NTHi can cause invasive diseases such as septicemia and meningitis, even if H. influenzae is not generally considered to be an intracellular pathogen. There have been very few reports about the therapeutic efficacy of antibiotics against respiratory tract infection caused by NTHi in mice because it is difficult for H. influenzae to infect mice. Therefore, we evaluated the efficacy of antibiotics against NTHi in both a cell culture model and a mouse model of infection. METHODS: We used six strains of NTHi isolated from adult patients with chronic otitis media, namely three beta-lactamase-negative ampicillin (AMP)-resistant (BLNAR) strains and three beta-lactamase-negative AMP-susceptible (BLNAS) strains, to evaluate the efficacy of AMP, cefcapene (CFPN), levofloxacin (LVX), clarithromycin (CLR), and azithromycin (AZM) in both a cell culture infection model and a mouse infection model. In the cell culture infection model, strains that invade A549 human alveolar epithelial cells were treated with each antibiotic (1 mug/ml). In the mouse infection model, female C57BL/6 mice were intraperitoneally injected with cyclophosphamide (200 mg/kg) three days before intranasal infection with 1 x 109 colony-forming units (CFU) of NTHi and on the day of infection. After infection, the mice were orally administered each antibiotic three times daily for three days, except for AZM, which was administered once daily for three days, at a dose of 100 mg/kg/day. RESULTS: In the cell culture infection model, it was found that two BLNAR strains were able to enter the cell monolayers by the process of macropinocytosis, and treatment with LVX yielded good bactericidal activity against both strains inside the cells. In the mouse infection model, no bacteria were detected by means of plating the lung homogenates of LVX-treated mice at day 4 after infection, while more than 105 CFU of bacteria per tissue sample were detected in nontreated mice. CONCLUSION: Our findings show the outcome and rich benefits of fluoroquinolone treatment of respiratory infections caused by either invasive or noninvasive BLNAR strains of NTHi.

Microcirculatory alteration in low-grade gastric mucosa-associated lymphoma by Helicobacter heilmannii infection: its relation to vascular endothelial growth factor and cyclooxygenase-2.

BACKGROUND: There are clinical reports that Helicobacter heilmannii, as well as Helicobacter pylori, has been clinically reported to cause gastric low-grade mucosa-associated lymphoid tissue-type (MALT) lymphoma, although its precise mechanism remains to be clarified. Thus, the present study was undertaken to elucidate the alteration of the microcirculatory structure and the relation to angiogenetic factors in mice infected with H. heilmannii for 3 and 6 months. METHODS: Immunohistochemical studies have been performed by FITC-dextran intra-aortic infusion or CD31, vascular endothelial growth factor-A, cyclooxygenase 2 antibodies using our recently established model of gastric mucosa-associated lymphoid tissue-type gastric B-cell lymphoma in C57BL/6 mice. RESULTS: Increased microcirculatory network was recognized surrounding the MALT lymphoma tissues by both the FITC-dextran infusion method and CD31 immunoreactivity. Vascular endothelial growth factor-A immunoreactivity was recognized within the lymphoma tissues as well as in the marginal area, while cyclooxygenase-2 immunoreactivity was localized in the area surrounding the MALT lymphoma tissues. CONCLUSION: Increased microvascular network as well as enhanced VEGF-A immunoreactivity was shown to be related to expansion of the MALT lymphoma formed by Helicobacter heilmannii infection.

Expressed Salmonella antigens within macrophages enhance the proliferation of CD4+ and CD8+ T lymphocytes by means of bystander dendritic cells.

ATP-dependent Lon protease-deficient Salmonella enterica serovar Typhimurium (strain CS2022) appeared to invade successfully the mesenteric lymph nodes (MLN) and Peyer's patches (PP) of BALB/c mice and appeared to be easily eradicated by the host after oral immunization. As detected by flow cytometry, the population of major histocompatibility complex class I (MHC-I)-expressing macrophages and dendritic cells (DCs) was increased in the PP of mice immunized with CS2022 on day 6 after immunization. Thereafter, the population of splenic surface CD69(+) T lymphocytes prepared from mice immunized with CS2022 6 weeks prior to measurement increased as a result of the administration of the extracellular vesicles of RAW264.7 macrophage-like cells derived by Salmonella challenge. In addition, the proliferation of CD8(+) and even of CD4(+)T cells isolated from mouse spleens immunized with CS2022 was enhanced after cocultivation with naive DCs in the presence of the extracellular vesicles. These findings indicate that the extracellular vesicles prepared from the Salmonella-challenged macrophages carried salmonellae antigens to bystander DCs, thereby stimulating T-cell responses. Therefore, as antigen presentation after phagocytosis should be a central process in the T-cell activation that occurs in response to Salmonella infection, an oral immunization with CS2022 sufficiently induces T cell-mediated immunity in mice.

An oral Salmonella vaccine promotes the down-regulation of cell surface Toll-like receptor 4 (TLR4) and TLR2 expression in mice.

A single oral immunization with the Lon-protease-deficient Salmonella enterica serovar Typhimurium (strain CS2022) induced protective immunity in mice against a subcutaneous challenge with virulent Listeria monocytogenes as well as virulent Salmonella serovar Typhimurium. The populations of cell surface Toll-like receptor 4 (TLR4) and TLR2 on peritoneal macrophages decreased at week 6 after immunization. This population decrease was not reversed after a challenge with either Salmonella or Listeria. These results suggest that oral immunization with CS2022 induced immune tolerance correlated with the down-regulation of cell surface TLR expression. This down-regulation may in part account for the development of cross-protection against a Listeria challenge by immunization with CS2022.

Use of QuantiFERON-TB Gold to investigate tuberculosis contacts in a high school.

BACKGROUND AND OBJECTIVE: QuantiFERON-TB Gold (QFT-G) was employed in a contact investigation in a high school to evaluate its performance in adolescents. METHODS: Students of the same school grade as the index case were screened with tuberculin skin test (TST) and CXR examination as an initial contact investigation. QFT-G was performed for students demonstrating a positive TST (erythema larger than 30 mm). RESULTS: Of 349 students whose TST was completed, 95 had positive TST responses, although the distribution of TST responses was similar for both high and low exposure groups. In contrast, only four of the 88 TST-positive students tested with QFT-G were positive by this test, and three of these were from the high exposure group. Chemoprophylaxis was provided to only those four QFT-G-positive students. Follow up of the 91 students who were TST-positive, but QFT-G-negative (or not tested), for more than 3.5 years revealed that none have developed active tuberculosis. CONCLUSIONS: QFT-G appears more specific than TST as contacts with positive TST and negative QFT-G responses were not offered prophylaxis and none developed tuberculosis during 3.5 years of follow up. The replacement of TST with QFT-G, or perhaps combined use of TST and QFT-G, may be more useful in diagnosing true infection and thus reducing the number of subjects indicated for chemoprophylaxis.

"Candidatus Helicobacter heilmannii" from a cynomolgus monkey induces gastric mucosa-associated lymphoid tissue lymphomas in C57BL/6 mice.

Both Helicobacter pylori and "Candidatus Helicobacter heilmannii" infections are associated with peptic ulcers, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue (MALT) lymphomas. However, good animal models of H. pylori clinical diseases are rare. In this study, we aimed to establish an animal model of "Candidatus Helicobacter heilmannii" gastric MALT lymphoma. We used a urease-positive gastric mucosal and mucus homogenate from a cynomolgus monkey maintained in C57BL/6 mouse stomachs. The bacterium in the homogenate was identified as "Candidatus Helicobacter heilmannii" based on a DNA sequence analysis of the 16S rRNA and urease genes. Mucosal and mucus homogenates were used to inoculate C57BL/6 mice, which were then examined for 24 months. We observed a gradual increase in the surface area of protrusive lesions in almost all infected C57BL/6 mouse fundic stomachs 6 months after infection. Light microscopic observations revealed an accumulation of B lymphocytes along with destruction of glandular elements and the presence of lymphoepithelial lesions consistent with low-grade MALT lymphomas. Electron microscopic observation revealed numerous "Candidatus Helicobacter heilmannii" bacilli in the fundic glandular lumen, the intracellular canaliculi, and the cytoplasm of intact cells, as well as damaged parietal cells. In conclusion, "Candidatus Helicobacter heilmannii" induced gastric MALT lymphomas in almost 100% of infected C57BL/6 mice after a 6-month period associated with the destruction of parietal cells.

Screening for tuberculosis infection using whole-blood interferon-gamma and Mantoux testing among Japanese healthcare workers.

OBJECTIVE: To examine the hypothesis that results of the QuantiFERON-TB Gold assay (QFT-G), a whole-blood test for detection of tuberculosis infection, are more significantly related to known risk factors for tuberculosis infection in healthcare workers (HCWs) who have received bacille Calmette-Guerin vaccine than are results of the Mantoux tuberculin skin test (TST). DESIGN: All HCWs (approximately 510) from a 370-bed general hospital in Tokyo where patients with and patients without tuberculosis are treated were invited to participate in the study. All study participants completed a questionnaire about their Mycobacterium tuberculosis infection risk factors as HCWs at the general hospital. They were then tested for LTBI by means of the QFT-G, followed by the TST. Statistical analyses were performed to compare results of each test with M. tuberculosis infection risk factors (age, length of employment in the healthcare industry, history of working with tuberculosis-positive patients in a tuberculosis ward or in the outpatient department of the hospital's tuberculosis clinic for more than 1 year, chest radiograph evidence of healed tuberculosis, history of performing bronchoscope procedures, and job classification), and for TST-positive HCWs, to compare the QFT-G result with the TST induration diameter. RESULTS: A total of 332 HCWs (95% of whom had been vaccinated with BCG) participated in the study, and 33 had positive QFT-G results, suggesting a prevalence of LTBI of 9.9%. Of 304 HCWs who underwent TST, 283 (93.1%) had an induration diameter of 10 mm or more. Multiple logistic regression analysis revealed that positive QFT-G results were significantly associated with age and with a history of working in a tuberculosis ward or an outpatient department of a tuberculosis clinic. TST results were not correlated with any of the tuberculosis infection risk factors we evaluated. CONCLUSIONS: Positive QFT-G results were closely associated with the presence of risk factors for LTBI in a hospital setting, suggesting that the QFT-G can detect LTBI in a population composed predominantly of BCG vaccinees. Because most HCWs worldwide have been vaccinated with BCG, the QFT-G offers a significant improvement over the TST in tuberculosis screening programs and minimizes unwarranted use of tuberculosis prophylaxis.

Evaluation of the Lon-deficient Salmonella strain as an oral vaccine candidate.

We evaluated the efficacy of CS2022 (the Lon protease-deficient mutant strain of Salmonella enterica serovar Typhimurium) as a candidate live oral vaccine strain against subsequent oral challenge with a virulent strain administered to BALB/c and C57BL/6 mice. CS2022 persistently resided in the spleen, mesenteric lymph nodes, Peyer's patches, and cecum of both strains of mice after a single oral inoculation with 1 x 10(8) colony-forming units. Finally, CS2022 almost disappeared from each tissue sample by week 12 in BALB/c mice, whereas CS2022 still resided in each tissue type at week 12 after inoculation of C57BL/6 mice. A significant increase in the serovar Typhimurium lipopolysaccharide-specific secretory immunoglobulin A (s-IgA), as measured for one of the mucosal immune responses, was detected in bile and intestinal samples of both strains of immunized mice at week 4 after immunization. In addition, the expression of gamma interferon mRNA in the spleens of both strains of immunized mice, especially those of C57BL/6 mice, was significantly increased at week 4 after immunization and was boosted during the following 5 days after the challenge was administered to the mice. Furthermore, peritoneal macrophages isolated from immunized mice at week 4 after immunization exhibited an increase in intracellular killing activity against both virulent and avirulent Salmonella. The present results suggested that salmonellae-specific s-IgA on the mucosal surfaces induced by immunization with CS2022 generally prevented mice from succumbing to an oral challenge with a virulent strain. Simultaneously, CS2022 promoted the protective immunity associated with macrophages in both strains of mice.

[Detection of tuberculosis infection using a whole blood interferon gamma assay in a contact investigation--evaluation using quantiFERon TB-2G].

OBJECTIVE: The purpose of this study was to evaluate the usefulness of a novel method of detecting tuberculosis infection, QuantiFERON TB-2G (QFT), in a large scale contact investigation when an outbreak of mass tuberculosis infection was suspected. SUBJECTS AND METHODS: The index case was a health-care worker who worked in a maternity hospital. The investigated contacts were categorized as follow according to the grade of closeness of contact; the "very close" contact group (11 subjects), the "close" contact group (33 subjects), and the "non-close" contact group (3,791 subjects). For the former two groups, tuberculin skin test (TST), chest X-ray examination and QFT were conducted. For the last contact group, TST and chest X-ray examination were conducted only to subjects who aged less than 29 years old, while only chest X-ray examination was conducted to those aged 30 years or older. The QFT test, i.e., a whole blood interferon-gamma assay using Mycobacterium tuberculosis-specific antigens, was performed to the "very close" and "close" contacts, and for strong tuberculin reactors among the "non-close" contacts. RESULTS: The number of infected subjects in the "very close" contact group, the "close" contact group, and the "non-close" contact group were 7, 7, and 277, respectively, based on TST results. On the other hand, the number of infected subjects in each group were 3, 2, and 5, respectively, based on the QFT test. CONCLUSION: If the indication of chemoprophylaxis was determined based on TST test, this case would have been regarded a large tuberculosis outbreak. However, the use of the QFT test greatly reduced the number of the infected persons, so that the possibility of such massive TB outbreak was denied. Thus, the use of QFT, with which TB infection could be detected more accurately, seems to be very beneficial in contact investigations.

Characterization of M. Tuberculosis-derived IL-12-inducing material by alveolar macrophages.

We have investigated the substance derived from Mycobacterium tuberculosis (Mtb) that induces interleukin (IL)-12 production by alveolar macrophages (AMs) in vitro. The cytosol fraction of live Mtb H37Rv induced IL-12 production by AMs in a dose-dependent manner. The addition of interferon-gamma (IFN-gamma) augmented IL-12 production. IL-12-inducing activity by AMs (termed as surely active keeping rescue antigen, SAKRA) was purified by gel filtration and ion exchange column chromatography, and the molecular weight of SAKRA was estimated by gel filtration to be more than 700 kDa. SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting of SAKRA using rabbit anti-SAKRA antibody suggested that SAKRA is composed with several low molecular weight proteins. Amino acids sequence analysis of several bands after SDS-PAGE suggested that SAKRA is a part of ribosomes. RT-PCR showed that SAKRA induced not only expression of IL-12 p40 mRNA, but expression of tumor necrosis factor (TNF)-alpha and inducible nitric oxide synthase (iNOS) mRNA at least 6 h after stimulation, suggesting that SAKRA activates the bactericidal activity of macrophages. To investigate the potential use of SAKRA as a vaccine against tuberculosis, SAKRA was administered to BALB/c mouse that had been immunized with BCG for 18 months, and mouse were infected with Mtb H37Rv via a respiratory route. Replication of Mtb in lungs and spleens was examined 6 weeks after infection. Administration of SAKRA to BCG-vaccinated mice significantly reduced the numbers of Mtb in lungs and spleens as compared with BCG-vaccinated control mice. Taken together, these results suggest that SAKRA is one of the Mtb-derived immunomodulatory substances which induce IL-12 production during infection and also increases mycobactericidal activities of macrophages, and that SAKRA may be a promising new vaccine candidate against tuberculosis.

[Basic characteristics of a novel diagnostic method (QuantiFERON TB-2G) for latent tuberculosis infection with the use of Mycobacterium tuberculosis-specific antigens, ESAT-6 and CFP-10].

PURPOSES: To determine the optimum cut-off level of a newly developed method for diagnosing tuberculosis infection based on whole-blood interferon-gamma measurement, and to study the basic characteristics of the method. STUDY SUBJECTS: 1) A total of 220 young, healthy individuals having no apparent exposure to tuberculosis infection, most of whom have had a vaccination with BCG vaccine. 2) One hundred eighteen tuberculosis patients who were diagnosed by positive Mycobacterium tuberculosis on culture. 3) A group of 75 youngsters exposed to an infectious tuberculosis patient and who showed a strong tuberculin reaction (with erythema diameter of 30 mm or more). METHOD: Whole-blood specimens of donors were stimulated with antigens, i.e., ESAT-6 and CFP-10, and then cultured. Plasma concentrations of interferon-gamma discharged were then determined with QuantiFERON-CMI. Correlation between interferon-gamma concentrations in response to ESAT-6 and CFP-10, and their correlation with Mantoux test results were analyzed for various categories of donors. The Receiver Operating Characteristics analysis was performed considering the loss due to misclassification. [ RESULTS AND DISCUSSION: The optimum cut-off level was determined as 0.35 IU/ml for both ESAT-6 and CFP-10. This gave the test a sensitivity of 89.0% and specificity of 98.1% in detecting tuberculosis infection. The correlation of interferon-gamma response with tuberculin tests among BCG-vaccinated individuals was low, which suggested that the test was not influenced by previous BCG vaccination. The low correlation between ESAT-6 and CFP-10 tests suggested that the simultaneous use of the two tests was beneficial. As in the case of clinical tests in general, the cut-off should be set at a lower level when the test is applied to high prevalence situation and vice versa.

[Usefulness of a novel diagnostic method of tuberculosis infection, QuantiFERON TB-2G, in an outbreak of tuberculosis].

OBJECTIVE: The purpose of this study was to evaluate QuantiFERON TB-2G (QFT), a novel method of detecting tuberculosis infection among contacts of a tuberculosis patient by determining the whole-blood interferongamma response to the specific antigens. SUBJECTS AND METHODS: A teacher of a college who had been coughing for the preceding two months was diagnosed with smear-positive tuberculosis. About 270 students of the college were considered to have been exposed to tuberculosis infection, of whom 73 were in closer contact with the index case because they participated in a one-week group excursion attended by the teacher. Two of the contact students developed active tuberculosis shortly thereafter. Tuberculin tests were conducted to almost all students, and QFT was performed for only those with tuberculin reactions having erythema diameters of 30 mm or larger. RESULTS: Tuberculin tests of students, all of whom had been vaccinated with BCG at least once, revealed that the distribution of the close contact group was slightly shifted to right (larger side) than those with less close contacts. The QFT positive rate for close contacts was 45.5%, while that for less close contacts was only 7.1%, which obviously indicates that QFT is hardly affected by the tuberculin allergy due to past BCG vaccination. The distribution of interferon-gamma measurements (log-transformed) of the close contacts showed typical bimodality, one mode representing the infected, another the non-infected. This was not clear for the less close contacts. The correlation of interferon-gamma measurements (log-transformed) with tuberculin reaction erythema size was weak, if not non-significant. CONCLUSION: It was concluded that QFT was a useful method for diagnosing tuberculosis infection and was unaffected by the BCG-caused tuberculin allergy. In the case of the outbreak mentioned above, QFT greatly reduced the indication of chemoprophylaxis, from 28% of all the contacts solely based on tuberculin test to only 7%. Although there remains some problems to be overcome for QFT to be widely used with high confidence, this technology will provide a high possibility for wider and more accurate indication of chemoprophylaxis and will be one of the essential tools of tuberculosis control of the 21st century in Japan.