Nicotinamide phosphoribosyl transferase in mammary gland epithelial cells is required for nicotinamide mononucleotide production in mouse milk.

Tissue-specific deficiency of nicotinamide phosphoribosyl transferase (NAMPT), the rate-limiting enzyme of the nicotinamide adenine dinucleotide (NAD+)-salvage pathway, causes a decrease of NAD+ in the tissue, resulting in functional abnormalities. The NAD+-salvage pathway is drastically activated in the mammary gland during lactation, but the significance of this has not been established. To investigate the impact of NAD+ perturbation in the mammary gland, we generated two new lines of mammary gland epithelial-cell-specific Nampt-knockout mice (MGKO). LC-MS/MS analyses confirmed that the levels of NAD+ and its precursor nicotinamide mononucleotide (NMN) were significantly increased in lactating mammary glands. We found that murine milk contained a remarkably high level of NMN. MGKO exhibited a significant decrease in tissue NAD+ and milk NMN levels in the mammary gland during lactation periods. Despite the decline in NAD+ levels, the mammary glands of MGKO appeared to develop normally. Transcriptome analysis revealed that the gene profiles of MGKO were indistinguishable from those of their wild-type counterparts, except for Nampt. Although the NMN levels in milk from MGKO were decreased, the metabolomic profile of milk was otherwise unaltered. The mammary gland also contains adipocytes, but adipocyte-specific deficiency of Nampt did not affect mammary gland NAD+ metabolism or mammary gland development. These results demonstrate that the NAD+ -salvage pathway is activated in mammary epithelial cells during lactation and suggest that this activation is required for production of milk NMN rather than mammary gland development. Our MGKO mice could be a suitable model for exploring the potential roles of NMN in milk.

Hepatocyte ß-Klotho regulates lipid homeostasis but not body weight in mice.

ß-Klotho (ß-Kl), a transmembrane protein expressed in the liver, pancreas, adipose tissues, and brain, is essential for feedback suppression of hepatic bile acid synthesis. Because bile acid is a key regulator of lipid and energy metabolism, we hypothesized potential and tissue-specific roles of ß-Kl in regulating plasma lipid levels and body weight. By crossing ß-kl(-/-) mice with newly developed hepatocyte-specific ß-kl transgenic (Tg) mice, we generated mice expressing ß-kl solely in hepatocytes (ß-kl(-/-)/Tg). Gene expression, metabolomic, and in vivo flux analyses consistently revealed that plasma level of cholesterol, which is over-excreted into feces as bile acids in ß-kl(-/-), is maintained in ß-kl(-/-) mice by enhanced de novo cholesterogenesis. No compensatory increase in lipogenesis was observed, despite markedly decreased plasma triglyceride. Along with enhanced bile acid synthesis, these lipid dysregulations in ß-kl(-/-) were completely reversed in ß-kl(-/-)/Tg mice. In contrast, reduced body weight and resistance to diet-induced obesity in ß-kl(-/-) mice were not reversed by hepatocyte-specific restoration of ß-Kl expression. We conclude that ß-Kl in hepatocytes is necessary and sufficient for lipid homeostasis, whereas nonhepatic ß-Kl regulates energy metabolism. We further demonstrate that in a condition with excessive cholesterol disposal, a robust compensatory mechanism maintains cholesterol levels but not triglyceride levels in mice.

[THE MINIMUM NUMBER OF SPUTUM SMEAR SAMPLES NEEDED FOR THE DIAGNOSIS OF TUBERCULOSIS].

[Objective] To determine whether three sputum examinations with fluorescent staining are necessary to diag- nose tuberculosis (TB) in our hospital. [Patients] From April 2005 to December 2012, 379 TB patients were admitted and received anti-TB therapy in our hospital. [Methods] A retrospective study was conducted to assess the positivity rates of sputum smears based on three exami- nations. The positivity rate of first sputum smear and the cumulative smear-positive rates in the second and third were determined. Then, we also determined difference of positivity rates in sputum properties, sampling procedures and cavity formation. [Results] Of the 379 patients who met the screening criteria, 300 tested positive based on the first sputum smear (79.2%). The positivity rate of the first sputum smears was higher in the purulent sputum group than in the mucous sputum group (91.2% vs. 72.3%). Cavity formation, and sputum extraction procedures were not related to the positivity rate of the first sputum smears. In the mucous sputum group, the cumulative smear-positive rate in the second test significantly rose, but did not rise in the third test. [Conclusions] Three sputum smear examinations were necessary in patients who submitted mucous sputum samples. It is important to get purulent sputum.

[[CASE OF TUBERCULOSIS COMPLICATIONS DURING PREGNANCY LEADING TO AN INFECTION OUTBREAK].]

A 31-year-old woman developed a constant cough during the 8th week of pregnancy and was diagnosed with bronchial asthma. She was prescribed prednisolone and inhaled corticosteroids. At 28 weeks of pregnancy, she showed worsening weight loss, fever, night sweats, hoarseness, and coughs. At 31 weeks of pregnancy, a scatter shadow and cavitary lesions were detected on the chest radiograph. Acid- fast bacilli smear test and tuberculosis (TB) polymerase chain reaction tests yielded positive results (G-8), and she was diagnosed with TB. Contact tracing and screening indicated 3 patients with TB onset and 18 patients with latent TB infec- tion attributed to the initial patient, who infected a total of 36 people. In the present case, physicians were reluctant to order a chest radiograph for fear of harming the fetus and did not order sputum or interferon gamma release (IGRA) assay tests either. The diagnosis was delayed by 152 days, which was considered as a factor that caused the outbreak. The diag- nosis of TB in a pregnant patient may be very challenging because symptoms may initially be ascribed to the pregnan- cy, and delayed diagnosis and treatment of military TB can lead to the death of the mother and fetus. Consequently, to ensure early diagnosis and treatment, chest radiography and sputum and IGRA tests are recommended for pregnant women who have TB symptoms or are at high risk for TB.

[AN OUTBREAK OF PULMONARY TUBERCULOSIS DUE TO DEFINITE EXOGENOUS REINFECTION AMONG ELDERLY INDIVIDUALS IN WELFARE FACILITIES].

PURPOSE: We report an outbreak of 64 cases of tuberculosis (TB) that spread in a welfare facility for elderly individuals. OBJECTIVE AND METHODS: First, 64 TB patients who had contact with the source patient were screened at our hospital. We examined the time course up to the discovery of symptoms and analyzed the results for variable numbers of tandem repeats (VNTR) and the drug susceptibility tests. Second, we performed chest computed tomography to examine lesions due to a previous TB infection. RESULT: The source patient had recurrent aspiration pneumonia. The delay in doctor consultation was considered day 0, and the delay of diagnosis was 267 days. On examining the contacts, we found that 29 patients had TB while 35 had a latent TB infection. Results of the VNTR and the drug susceptibility tests showed that all the patients who developed TB had the same pattern as that of the source patient. Chest computed tomography showed lesions due to a previous TB infection in 8 patients. CONCLUSION: Based on the results of the VNTR and drug susceptibility tests, we concluded that the outbreak was due to an exogenous infection from the same source. All 8 patients who showed lesions due to a previous TB infection were aged > 81 years, and TB in these patients was found to be due to exogenous re-infection.

Feto-maternal cholesterol transport regulated by ß-Klotho-FGF15 axis is essential for fetal growth.

ß-Klotho (ß-KL) is indispensable to regulate lipid, glucose, and energy metabolism in adult animals. ß-KL is highly expressed in the yolk sac, but its role in the developmental stages has not been established. We hypothesized that ß-KL is required for metabolic regulation in the embryo and aimed to clarify the role of ß-KL during development. Here, we show that ß-KL regulates feto-maternal cholesterol transport through the yolk sac by mediating FGF 15 signaling, and also that impairment of the ß-KL-FGF15 axis causes fetal growth restriction (FGR). Embryos of ß- kl knockout (ß-kl-/-) mice were morphologically normal but exhibited FGR before placental maturation. The body weight of ß-kl-/- mice remained lower after birth. ß-KL deletion reduced cholesterol supply from the maternal blood and led to lipid shortage in the embryos. These phenotypes were similar to those of embryos lacking FGF15, indicating that ß-KL-FGF15 axis is essential for growth and lipid regulation in the embryonic stages. Our findings suggest that lipid abnormalities in early gestation provoke FGR, leading to reduced body size in later life.

Hemolytic Anemia Occurring 14 Years after Ascending Aortic Replacement for Acute Type A Aortic Dissection Due to Aortic Pseudoaneurysm Derived from Anastomotic Leakage: A Case Report.

A 64-year-old male patient who presented with symptoms indicative of hemolytic anemia was referred to our hospital. After obtaining the patient's history, it was found that hemolysis occurred 14 years after he underwent ascending aortic replacement for acute type A aortic dissection. Enhanced computed tomography revealed an aortic pseudoaneurysm at the proximal anastomosis, which was thought to be the cause of hemolysis. Furthermore, aortic valve regurgitation and dilatation of the sinus of Valsalva were also found on a transthoracic echocardiogram. Therefore, the Bentall procedure was performed. During the surgery, aortic pseudoaneurysm formation and vascular graft stenosis were observed. The postoperative course was uneventful, and hemolysis diminished soon after the surgery.

Clinical outcome of intraoperative hemodialysis using a hemoconcentrator during cardiopulmonary bypass for dialysis-dependent patients.

OBJECTIVES: The basic materials and structure of a hemoconcentrator incorporated into cardiopulmonary bypass (CPB) circuits are similar to those of hemodialyzers. Gravity drainage hemodiafiltration (GHDF) is an easy-to-use intraoperative renal replacement therapy (RRT) that utilizes a hemoconcentrator. This study aimed to verify whether GHDF can correct electrolyte imbalance and remove uremic toxins in dialysis-dependent patients and to evaluate the clinical outcomes of GHDF by comparing it with a conventional method of dilutional ultrafiltration (DUF). METHODS: This study retrospectively compared perioperative clinical values of 41 dialysis-dependent patients (21 patients with GHDF and 20 patients with DUF) who underwent open-heart surgery. Changes in serum parameters before and after passing through the hemoconcentrator were also compared. RESULTS: Compared to DUF, GHDF significantly lowered potassium, blood urea nitrogen, and creatinine levels at the outflow of the hemoconcentrator. Less catecholamine was needed to wean CPB in GHDF than in DUF. The P/F ratio (arterial blood oxygen pressure/inhaled oxygen concentration) at the end of surgery was significantly higher in GHDF than in DUF (450.8 ± 149.7 vs. 279.3 ± 153.5; p < 0.001). Postoperative intubation time was shorter in GHDF than in DUF (8.3 ± 5.9 vs. 18.7 ± 16.1 h; p = 0.006). The major morbidity and mortality rates were comparable in both groups. CONCLUSIONS: GHDF removed both potassium and uremic toxins more efficiently than DUF in dialysis-dependent patients. Less catecholamine was needed to wean CPB using GHDF. It improved the immediate postoperative respiratory function and enabled earlier extubation. GHDF is a novel and effective option for intraoperative RRT in dialysis-dependent patients undergoing open-heart surgery.

[Clinical analysis of non-tuberculous mycobacteriosis cases complicated with pulmonary aspergillosis].

OBJECTIVES: To clarify the clinical features of nontuberculous mycobacteriosis (NTM) complicated with chronic pulmonary aspergillosis (CPA), we analyzed 257 cases diagnosed with newly developed NTM during the last 12 years in our hospital. RESULTS: Fifty-six per cent of the patients were females. Ten cases (3.9%) of them were complicated with CPA in their clinical course. Mean age at the diagnosis of CPA was 65.5 years, and 8 of 10 cases were males. The average period from the diagnosis of NTM to CPA was almost 7 years. Six NTM cases were classified as the cavitary type and 4 as the nodular-bronchiectasis type. At the time of the diagnosis of CPA, NTM bacilli were isolated in 5 cases, but in the other 5 bacilli were not detected. Radiologically it was found that in many cases the infiltrative shadow had increased and the cavity wall had thickened. Antifungal drug administration was effective in 67% of the cases. In particular, in cases with progressive infiltrative shadows, the antifungal drug was effective in 83% of patients. DISCUSSION: For the clinical management of NTM, careful attention to the complication of CPA is required.

[Pulmonary tuberculosis with atypical radiological findings in a patient with chronic obstructive pulmonary disease].

A 77-year-old-man who had been treated for chronic obstructive pulmonary disease (COPD) was referred to our hospital for further examination of a chest X-ray abnormality. The chest X-ray showed consolidation in the right upper and middle lung field. Chest computed tomography showed an airspace consolidation extending subpleurally and nonsegmentally without nodular lesions. The tentative diagnosis was cryptogenic organizing pneumonia. However, bronchoalveolar lavage fluid was positive for acid-fast bacilli on smear and also positive for tuberculosis PCR, leading to a diagnosis of tuberculous pneumonia. Tuberculous pneumonia in COPD patients can be non-segmental and mimic organizing pneumonia.

Thermococcus sp. KS-1 PPIase as a fusion partner improving soluble production of aromatic amino acid decarboxylase.

Peptidyl-prolyl cis-trans isomerase (PPIase, EC 5.2.1.8) catalyzes the racemization reaction of proline residues on a polypeptide chain. This enzyme is also known to function as a molecular chaperon to stabilize protein conformation during the folding process. In this study, we noted FK506 binding protein (FKBP)-type PPIase from a hyperthemophilic archaeon Thermococcus sp. strain KS-1 (PPIase KS-1) to improve the solubility of Pseudomonas putida aromatic amino acid decarboxylase (AADC) that is an indispensable enzyme for fermentative production of plant isoquinoline alkaloids. AADC fused N-terminally with the PPIase KS-1 (PPIase KS-1-AADC), which was synthesized utilizing Escherichia coli host, showed improved solubility and, consequently, the cell-free extract from the recombinant strain exhibited 2.6- to 3.4-fold elevated AADC activity than that from the control strain that expressed the AADC gene without PPIase KS-1. On the other hand, its thermostability was slightly decreased by fusing PPIase KS-1. The recombinant E. coli cells expressing the PPIase KS-1-AADC gene produced dopamine and phenylethylamine from L-dopa and phenylalanine by two- and threefold faster, respectively, as compared with the control strain. We further demonstrated that the efficacy of PPIase KS-1-AADC in solubility and activity enhancement was a little but obviously higher than that of AADC fused N-terminally with NusA protein, which has been assumed to be the most effective protein solubilizer. These results suggest that PPIase KS-1 can be used as one of the best choices for producing heterologous proteins as active forms in E. coli.

[The changes of hospitalization in our tuberculous ward after new discharge criteria].

PURPOSE: After new criteria for discharge from a tuberculosis ward were introduced, we studied changes in the length of hospitalization before and after adoption of these criteria. METHODS: We evaluated monthly data on hospitalization in our tuberculosis ward between April 2003 and September 2008. RESULTS: Although the number of complicated patients increased, hospitalization decreased after the change in discharge criteria. CONCLUSION: After adoption of the new discharge crite-ria, the length of hospitalization in our tuberculosis ward decreased.

[A case of multidrug-resistant tuberculosis who acquired additional resistance to ethambutol before the result of the initial drug sensitivity test was reported].

On December 6, 2008, a 52-year-old man presented to a clinic with chronic cough, sputum, and chest discomfort, which had lasted since mid-November. Since the chest radiograph showed a small cavity with small nodules and granular shadows, he was referred to another hospital. On TB-PCR, the gastric juice was positive. Therefore, on December 16, 2008, treatment for pulmonary tuberculosis was initiated with isoniazid, rifampicin, ethambutol, and pyrazinamide. However, on February 4, 2009, a drug susceptibility test revealed that the bacilli were resistant to isoniazid and rifampicin. Therefore, he was referred to our hospital. At that time, he had no symptoms and his sputum smear was negative. We performed a right upper lobectomy. The smear result of the surgical specimen was heavily positive (equivalent to Gaffky 6), and the drug susceptibility test showed resistance to ethambutol in addition to isoniazid and rifampicin. After surgery, we treated him with pyrazinamide, streptomycin, para-aminosalicylate, ethionamide, and levofloxacin. We report this case of multidrug-resistant tuberculosis without past treatment who acquired additional resistance to ethambutol during the first 2 months of chemotherapy. When treating multidrug-resistant tuberculosis, very careful consideration of susceptibility to other drugs is warranted.

[A case of miliary tuberculosis and esophageal perforation secondary to tuberculous mediastinal lymphadenitis].

An 80-year-old woman was admitted to a local hospital following transient disturbance of consciousness after a fall. High intermittent fever developed after hospitalization and she was diagnosed as having mediastinal abscess with esophageal perforation. She underwent mediastinal drainage and surgical repair of the esophagus. Acid-fast bacilli were detected in her sputum. Chest CT scanning showed a diffuse granular shadow. Then she was diagnosed as having miliary tuberculosis and treated with combination of INH, RFP, EB, and PZA. However, five days after treatment was initiated, fever and skin eruption appeared and treatment has to be stopped after one month. Then she was referred to our hospital. We gradually increased the dosages of INH and RFP, which resulted in pyrexia. Therefore, we changed EB to SM. Fever subsided and we were able to administer the full dose of drugs from the beginning of January 2007. Thereafter, the patient improved gradually. However, she died in February 2007. At autopsy, we identified tuberculous mediastinal lymphadenitis, inflammatory granuloma under the esophageal mucosa and miliary tuberculosis. We report this case as a rare case of miliary tuberculosis and esophageal perforation secondary to tuberculous mediastinal lymphadenitis.

[A case of axillary lymph node tuberculosis with paradoxical worsening in breast lesion].

A 80-year-old woman was admitted to our hospital because of left axillary swelling. Needle biopsy specimen showed negative results on a smear for acid-fast bacilli and PCR. The histological findings showed epithelioid cell granuloma with caseous necrosis. QFT TB-2G showed positivity of 1.9 IU/ml in ESAT-6. We diagnosed tuberculous lymphadenopathy and administered antituberculous drugs. After 3 weeks of treatment, in spite of the regression of lymphadenopathy, mammary swelling had progressed. We performed a biopsy of the mammary lesion, but did not detect any abnormal findings. As the mammary lesion had regressed by the continued treatment of antituberculous drugs, we thought the mammary swelling was paradoxical worsening.

Aortic sarcoma mimicking a mycotic aneurysm in the thoracoabdominal aorta.

Aortic sarcoma is a rare primary tumor with dismal prognosis. Here, we report a case involving a 74-year-old female patient with aortic sarcoma masquerading as a mycotic aneurysm in the thoracoabdominal aorta. She underwent aortic resection with Dacron prosthetic graft replacement because of rapid growth. The postoperative pathological findings of the resected specimen confirmed the diagnosis of aortic mural sarcoma, which was an unexpected result based on repeat computed tomography angiography performed within 2 months preoperatively. The preoperative diagnosis of aortic sarcoma is often difficult because of its rarity, and this case demonstrates some of the diagnostic pitfalls.

[A case of Mycobacterium intracellulare pleurisy without active lung lesion].

Pleural effusion without occurrence of active pulmonary lesion due to nontuberculous mycobacteria is extremely rare. We report a case of Mycobacterium intracellulare pleurisy in an 84-year-old woman. The patient was admitted to a nearby hospital because of dyspnea. Massive right pleural effusion was observed on chest roentgenogram. Bacteriological examinations, smear and culture of the sputum or pleural effusion were negative. First we thought pleurisy was caused by M. tuberculosis as pleural effusion showed predominant lymphocyte count and high adenosine deaminase level. However, M. intracellulare was identified by the polymerase chain reaction method from pleural effusion. Based on clinical findings and laboratory data, we suspected pleurisy was due to M. intracellulare infection. Clarithromycin, kanamycin, rifampicin and ethambutol were administered. After four months of treatment pleural effusion disappeared without accompanying the active pulmonary lesion. Therefore, we diagnosed this case as pleurisy without pulmonary lesion due to M. intracellulare.

[A case of multi-drug resistant pulmonary tuberculosis after administration of standard anti-tuberculosis treatment for two times].

We reported a case in which multi-drug resistant tuberculosis was recognized after two courses of anti-tuberculosis treatment. A 41-year-old woman who had received two courses of anti-tuberculosis treatment for pulmonary tuberculosis was admitted to our hospital due to productive cough, high fever and positive sputum smear showing acid fast bacillus. In the past treatment, drug susceptibility was unknown because of culture-negative TB. Chest radiograph showed atelectasis of the right upper lobe. The pathological examination of surgically resected lung specimen revealed that atelectasis was formed by a granulation tissue with caseous necrosis progressed to the bronchus wall. We examined cultures three times using both solid and liquid media. Liquid culture of the first time specimen was positive for Mycobacterium tuberculosis after six weeks and multi-drug resistant tuberculosis was recognized on drug susceptibility test. Thereafter she was treated with KM, LVFX, PZA and PAS, and maintained sputum smear negative for 7 months after treatment. Physicians must consider possibility of MDR-TB despite findings showing smear-positive and culture-negative TB.

[Quantiferon TB-2G among nurses with a history of working in our tuberculosis ward].

PURPOSE: To investigate the infection rate of tuberculosis among nurses with a history of working in our hospital's tuberculosis ward (TW). METHODS: We measured interferon gamma levels in 50 nurses who had worked in our TW, and evaluated the infection rate among these nurses before (I) and after (II) the use of our hospital's tuberculosis infection control manual. RESULTS: The infection rate including probable infection was 6/50 (12.0%) in all TW nurses. The infection rate in the group I was 17.6%, but that in group II was 0%. CONCLUSION: Our hospital's tuberculosis infection control manual was effective for decreasing the infection rate, despite a high rate of infection in group I.