Milk Contamination by Mycobacterium tuberculosis Complex, Implications for Public Health in Amazonas, Brazil.

ABSTRACT: In Brazil, contamination of raw milk with Mycobacterium tuberculosis complex (MTC) has been reported in several states. The highest rate of consumption of raw milk and its derivatives in Brazil occurs in Amazonas. This state also has the highest prevalence of tuberculosis in both humans and livestock. We assessed the contamination of cow's milk and buffalo's milk with MTC in Amazonas, focusing on Mycobacterium bovis, the species most commonly found in cattle and buffalo. In 2019, 250 samples of raw milk (91 from cattle, 159 from buffalo) were collected before processing from three milk plants in the state of Amazonas. The samples were placed into 21 pools and analyzed using shotgun metagenomic sequencing and taxonomic classification with Kraken 2 and MegaBLAST. To confirm the identity of mycobacterial species found, BLASTN was used to identify specific genomic positions in the TbD1 and RD1 regions and flanking RD4 region. MTC genetic material was identified in all pools of raw milk. Genetic material consistent with M. bovis was identified in seven pools of raw milk (1 from cattle, 6 from buffalo). Buffalo's milk had significantly higher MTC reads than did cow's milk. The common practice of consumption of raw milk and its derivatives in Amazonas presents a risk to public health. Urgent measures to prevent transmission of foodborne tuberculosis are needed in the Amazon region. Greater efforts and resources also should be directed toward elimination of bovine tuberculosis in cattle and buffalo herds in Amazonas and the rest of Brazil.

Phase II study of irinotecan combined with carboplatin in previously untreated small-cell lung cancer.

To determine the efficacy and toxicity of irinotecan combined with carboplatin, we conducted a phase II trial. Eligibility criteria were: chemotherapy-naïve, small-cell lung cancer (SCLC), good performance status (PS: 0-2), age

Phase I study of irinotecan and cisplatin with concurrent split-course radiotherapy in limited-disease small-cell lung cancer.

We conducted a phase I study of irinotecan (CPT-11) and cisplatin with concurrent split-course radiotherapy in limited-disease small-cell lung cancer (LD-SCLC). This study aimed to determine the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of this therapy. Four chemotherapy cycles of CPT-11 (days 1, 8 and 15) and cisplatin (day 1) were repeated every 28 days. Radiotherapy of 2 Gy/day commenced on day 2 of each chemotherapy cycle with 20 Gy administered from the first to the third cycles (a total of 60 Gy). 17 patients were enrolled at three dose levels (CPT-11/cisplatin: 40/60, 50/60 and 60/60 mg/m(2)), and 16 were evaluable for toxicity and outcome. 2 of 4 patients at 60/60 mg/m(2) refused continuation of therapy because of general fatigue, and the relative dose intensity of CPT-11 at 50/60 mg/m(2) was approximately 50%. These levels were considered as the MTD. Tumour responses included four complete responses (CR), 11 partial responses (PR) and one no change (NC), and the overall response rate was 93.8% (95% confidence interval: (CI) 71.7-98.9%). This combined modality is tolerable, and CPT-11/cisplatin of 40/60 mg/m(2) in this modality is recommended for phase II study.

Phase I and pharmacokinetic study of paclitaxel and irinotecan for patients with advanced non-small cell lung cancer.

We conducted a phase I study of paclitaxel and irinotecan (CPT-11) in advanced non-small cell lung cancer (NSCLC). This study aimed to determine the maximum tolerated doses (MTD). The pharmacokinetics of CPT-11 and its major active metabolite, SN-38, were also analysed. Patients received paclitaxel (day 1) followed by CPT-11 (days 1, 8 and 15), in a 4-week cycle, and paclitaxel and CPT-11 were escalated from 120 and 40 mg/m(2), respectively. 28 patients were enrolled, who were evaluated for toxicity. 2 of 6 patients at 210 mg/m(2) paclitaxel and 50 mg/m(2) CPT-11, and 2 of 4 at 180 and 60 mg/m(2) developed dose-limiting toxicity (DLT) (neutropenia, fever, neurotoxicity and diarrhoea). The area under the plasma concentration-time curve (AUC) of CPT-11 on day 1 was significantly higher than that on days 8 or 15 at each dose level (P=0.002). The AUC of SN-38 on day 1 was significantly increased using paclitaxel doses >or=150 mg/m(2). A preceding paclitaxel administration changed the pharmacokinetics of CPT-11 and SN-38. However, the toxicity was tolerable. Paclitaxel 180 mg/m(2) and CPT-11 50 mg/m(2) were the recommended doses for further phase II study of this combination.

Phase I study of irinotecan and cisplatin with concurrent split-course radiotherapy in unresectable and locally advanced non-small cell lung cancer.

We conducted a phase I study of irinotecan (CPT-11) and cisplatin with concurrent split-course radiotherapy in locally advanced stage III non-small cell lung cancer (NSCLC). This study aimed to determine the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of this therapy. Two chemotherapy cycles of CPT-11 (days 1, 8 and 15) and cisplatin (day 1) were repeated with a 28-day interval. Radiotherapy of 2 Gy/day commenced on day 2 of each chemotherapy cycle, with 24 Gy and 36 Gy administered for the first and second cycle, respectively. 24 eligible patients were enrolled at five dose levels (CPT-11/cisplatin: 40/60, 50/60, 60/60, 60/70 and 60/80 mg/m(2)), and 23 patients were evaluated for toxicity and clinical outcome. Only 1 patient experienced a DLT with neutropenia and diarrhoea at 60/60 mg/m(2). Dose escalation was limited to 60/80 mg/m(2) which was the recommended dose for CPT-11/cisplatin alone in NSCLC. Tumour responses included one complete response (CR), 15 partial response (PR), and 7 no change (NC), and the overall response rate was 69.6% (95% confidence interval (CI) 47.1-86.8%). This combined modality is tolerable, and CPT-11/cisplatin of 60/80 mg/m(2) in this modality is recommended for phase II study.

Phase I study of second-line chemotherapy with docetaxel and carboplatin in advanced non-small-cell lung cancer.

PURPOSE: Docetaxel and carboplatin have a broad spectrum of antitumor activity. We conducted a phase I study of docetaxel and carboplatin as second-line chemotherapy in previously treated non-small-cell lung cancer (NSCLC). This study aimed to determine the maximum tolerated dose (MTD) and the dose-limiting toxicities in this second-line combination chemotherapy. METHODS: Patients with advanced NSCLC were treated with escalating docetaxel doses in combination with a fixed-target area under the concentration-time curve (AUC) of 5 mg min/ml of carboplatin on day 1 of a 3-4-week cycle. The carboplatin dose was determined by multiplying the AUC by the clearance predicted using the Chatelut formula. The docetaxel dose was escalated from 40 mg/m2 to the MTD by 10 mg/m2 increments. RESULTS: A total of 16 patients previously treated with anticancer drugs were enrolled through three dose levels (40, 50 and 60 mg/m2 of docetaxel). All patients were assessable for toxicity and response. The MTD was docetaxel 60 mg/m2 with a carboplatin target AUC of 5 mg min/ml, and the dose-limiting toxicities in two of four patients were neutropenia and thrombocytopenia. Overall, neutropenia and thrombocytopenia of grade 3/4 occurred in eight patients (50%) and three patients (19%), respectively. Four patients (25%) and two patients (13%) experienced both grade 1 diarrhea and dermatitis, respectively. Allergic reactions, fluid retention, pneumonitis, neurotoxicity and mucositis were not observed. Of 16 patients, 5 showed an objective response (response rate 31%; 95% CI 14-56%). CONCLUSIONS: The combination of docetaxel and carboplatin is a feasible and well-tolerated second-line chemotherapy regimen in the treatment of NSCLC. Docetaxel 50 mg/m2 under the carboplatin target AUC of 5 mg x min/ml using the Chatelut formula was the recommended dose for phase II study.

Phase I study of irinotecan combined with carboplatin in previously untreated solid cancers.

Irinotecan (CPT-11) and carboplatin have broad anti-tumor activities. We conducted a Phase I study of CPT-11 combined with carboplatin in previously untreated solid cancers, especially advanced lung cancer. The aim of the study was to determine the maximum tolerated dose (MTD) and the dose-limiting toxicities in this regimen. In addition, we prospectively evaluated the Chatelut formula for predicting carboplatin clearance. Patients with advanced cancer were treated with CPT-11 (days 1, 8, and 15) and carboplatin (day 1) of a fixed-target area under the concentration-time curve (AUC) of 5 mg x min/ml. Carboplatin dose was determined by multiplying the AUC by the clearance predicted using the Chatelut formula. The CPT-11 dose was escalated from 40 mg/m2 to the MTD by 10 mg/m2. A total of 27 patients, 26 lung cancer patients and 1 colon cancer patient, were enrolled in this study. Dose-limiting leukoneutropenia, thrombocytopenia, and diarrhea, including one treatment-related death, were observed at 60 mg/m2 CPT-11, indicating that this level was the MTD. In 11 patients, the actual AUCs of carboplatin almost achieved the target AUC of 5. Fifteen (60%) of 25 evaluable patients showed an objective response, with an 85% response rate [11 of 13 patients (complete response, 31%; partial response, 54%)] in small cell lung cancers and a 36% response rate (4 of 11 patients) in non-small cell lung cancers. Neutropenia, thrombocytopenia, and diarrhea were the dose-limiting toxicities in this regimen. CPT-11 (50 mg/m2) under the carboplatin target AUC of 5 using the Chatelut formula was the recommended dose for further Phase II study, and this regimen seems to be active for small cell lung cancer.

Massive adrenal hemorrhage secondary to metastasis of lung cancer.

Hemorrhagic adrenal metastasis from lung cancer is extremely rare, although adrenal involvement is common in widely disseminated cancer. We report a case of massive adrenal hemorrhage secondary to metastasis of lung cancer. A 47-year-old female was treated by left upper lobectomy and mediastinal lymph node resection for an adenocarcinoma with intrapulmonary metastasis in the left upper lobe. Eight months later, she presented with right flank and back pain, and abdominal ultrasonography and computed tomography showed a right solitary adrenal tumor with massive hemorrhage. The tumor was not resectable and partially responded to chemotherapy. A massive adrenal hemorrhage, secondary to metastasis of lung cancer, presents with nonspecific clinical signs and symptoms. In lung cancer patients with an acute flank or back pain, hemorrhagic adrenal metastasis should be considered in the differential diagnosis.

Multidrug resistance-associated protein gene expression and drug sensitivity in human lung cancer cells.

Multidrug resistance-associated protein (MRP) mRNA expression and drug sensitivity in lung cancer cells were examined, and the effects of verapamil, a modulating agent for MRP, on drug sensitivity were also tested. Nine cell lines expressed various levels of MRP gene expression but not the MDR1 gene. The levels were higher in non-small cell carcinoma cells (NSCLC) than in small cell carcinoma cells (SCLC). Clear correlations between the MRP gene level and the sensitivity to etoposide (VP-16) and doxorubicin (Dox) were observed except for one cell line which highly expressed DNA topoisomerase II. Positive correlations between the MRP gene levels in three cell lines and the modulation effects of verapamil in VP-16, Dox, and vincristine were observed. The present results indicate that MRP probably confers intrinsic multidrug resistance in NSCLC rather than in SCLC.

Gene mutation analysis and quantitation of DNA topoisomerase I in previously untreated non-small cell lung carcinomas.

To elucidate whether gene alterations of topoisomerase I (topo I) exist in untreated non-small cell lung carcinomas (NSCLC), polymerase chain reaction-single strand conformation polymorphism analysis was performed in forty-four NSCLC tissue samples. Gene alterations of topo I were sought in three regions, near codons 361 and 363, 533, and 722 and 729, where point mutations have been found in resistant tumor cell lines selected by chronic camptothecin exposure. In addition, nuclear topo I contents were determined by immunoblotting. No mobility shifts were observed compared to the pattern observed in a normal control at any of the three regions in any sample, whereas topo I levels showed an approximately 12-fold variation. The variation is remarkably large compared to those seen in previous in vitro and in vivo studies. The results suggest that mutations of topo I may not contribute to intrinsic resistance of NSCLC to camptothecins, but low topo I levels may account, at least in part, for the resistance.

The clinical role of MDR1 gene expression in human lung cancer.

Human P-glycoprotein (Pgp) encoded by the MDR1 gene confers multidrug resistance to cancer cells. The clinical role of MDR1/Pgp in lung cancer is not fully understood. A total of 87 lung cancer surgical tissue samples, including previously untreated 84 non-small-cell (NSCLC) and three small-cell lung carcinoma (SCLC), were analyzed for levels of MDR1 mRNA determined by Northern blotting and compared with MDR1-positive cell lines. Fifteen percent (13/87) of the tumors were positive for the MDR1 gene, but the level was low in all samples except in one adenocarcinoma which expressed a high level of MDR1. The gene expression in these tumors did not relate with any pathologic factors such as histologic type, pathologic stage and tumor size. The SCLC and only one of the 14 MDR1-negative NSCLC responded to adjuvant chemotherapy after surgery. The present results indicate that the MDR1 gene is not associated in NSCLC with tumor progression and drug resistance.

A novel quinoline derivative, MS-209, overcomes drug resistance of human lung cancer cells expressing the multidrug resistance-associated protein (MRP) gene.

PURPOSE AND METHODS: MS-209 is a newly synthesized quinoline compound used orally to overcome human P-glycoprotein (Pgp)-mediated multidrug resistance (MDR). The multidrug resistance-associated protein (MRP) gene is thought to play an important role in MDR in lung cancer. To investigate whether MS-209 could also overcome MRP-mediated MDR, we examined the effect of the compound using a cytotoxicity assay on MDR1 gene-negative drug-selected MDR and wildtype lung cancer cells with various levels of MRP gene expression. The effects of MS-209 were compared with those of verapamil (VER) and cyclosporin A (CsA). The level of MRP gene expression in the cells was evaluated semiquantitatively by RT-PCR. For vincristine (VCR), intracellular accumulation of [3H]-VCR was measured with or without MS-209. RESULTS: In MDR UMCC-1/VP small-cell lung carcinoma cell line, 5 microM of MS-209 and VER enhanced the cytotoxicity of etoposide, doxorubicin (DOX) and VCR more than twofold, and completely reversed the resistance to VCR. The mean reversing effects of MS-209 on DOX and VCR were significantly stronger than those of VER and CsA. In wildtype non-small-cell lung carcinoma cells, the effects of MS-209 were almost equal to those of VER and CsA. The effect of these three agents correlated with the level of MRP gene expression. The MS-209-induced increase in intracellular accumulation of VCR was proportional to the level of MRP gene expression in these cells. CONCLUSION: Our results indicate that MS-209 is a potentially useful drug that can overcome MRP-mediated intrinsic and acquired MDR in human lung cancer.

The clinical implications of glutathione S-transferase-pi expression in lung cancer.

We performed immunohistochemical staining of glutathione S-transferase-pi (GST-pi) in 97 lung cancer specimens. In untreated patients, 86% (48/56) of NSCLC and none (0/8) of SCLC stained for GST-pi, and all squamous cell carcinomas were positive (28/28). The proportion of positive NSCLC with preoperative chemotherapy was similar to that of the untreated NSCLC, but the proportion of treated SCLC positive for GST-pi (14/18) was significantly higher than untreated tumors. GST-pi in NSCLC may be a tumor marker rather than being involved in drug resistance, and GST-pi-induction by chemotherapy may relate to acquired resistance in SCLC.

Bronchial artery aneurysm as a cause of atelectasis.

A 54-year-old nonsmoker female developed atelectasis of the anterior basal segment of the right lower lobe. A non-pulsating endobronchial tumor was observed bronchoscopically obstructing the right basal bronchus. The tumor was confirmed on arteriography to be a saccular aneurysm of the right bronchial artery. The aneurysm was treated with bronchial artery embolization. Bronchial artery aneurysm, without a predisposing disease, is quite rare, but should be considered as an etiological factor of atelectasis.

Enhanced expression of metallothionein in human non-small-cell lung carcinomas following chemotherapy.

Metallothioneins (MTs) are induced by various stimuli and probably confer drug resistance in tumor cells in vitro. To investigate whether MT expression in lung cancer is induced by chemotherapy, ninety-seven surgical specimens from patients who had or not received chemotherapy containing cisplatin, were stained immunohistochemically for MT. In untreated tumors, 23% (15/64) of all tumors and 27% (15/56) of non-small-cell carcinoma (NSCLC) stained positive, while all eight small-cell carcinoma (SCLC) were negative. In treated tumors, 52% (17/33) of all tumors, 80% (12/15) of NSCLC and 28% (5/18) of SCLC stained positive. The proportion of positively-stained tumors was significantly higher in treated NSCLC compared with untreated NSCLC (P = 0.0005) and treated SCLC (P < 0.005). Our results indicate that MT expression increases following chemotherapy and that such expression may confer during resistance in lung cancer, especially NSCLC.

The multidrug resistance-associated protein gene confers drug resistance in human gastric and colon cancers.

To determine the expression of multidrug resistance-associated protein (MRP) gene and its role in gastric and colon cancers, we analyzed 10 gastric and 10 colon non-drug-selected cell lines and a similar number of tissue samples of these cancers. We compared the expression of MRP and mdrl mRNA in cell lines and tissues using reverse-transcriptase polymerase chain reaction. In mdrl-negative cells, the relationship between the level of MRP gene expression and sensitivity to anticancer drugs was examined. The effect of verapamil, an MRP-modulating agent, was also examined in these cells. The expression of MRP gene in gastric cancer cell lines varied from a low to a high level, but mdrl was not detected in any of these cell lines. Colon cancer cell lines expressed low to intermediate levels of MRP gene, and half of the cells co-expressed low to high levels of mdrl. In tissue samples, the expression pattern of the two multidrug resistance (MDR) genes was broadly similar to that described for the cell lines, except that most of the gastric cancer tissue samples did express low levels of mdrl. No significant correlation was observed between the level of MRP gene expression and sensitivity to anticancer drugs in gastric and colon cell lines. However, verapamil significantly increased the sensitivity to etoposide, doxorubicin and vincristine in cells highly expressing MRP gene. Our results indicate that MRP gene may be important in conferring MDR in gastric and colon cancer cells.

A prospective study of bronchoscopy for endotracheobronchial tuberculosis.

To clarify the role of bronchoscopy for endotracheobronchial tuberculosis (EBTB), we performed a prospective study in 45 patients with active pulmonary tuberculosis, in 15 (33%) of whom bronchoscopic findings related to EBTB were seen. The findings were classified into six types:edematous hyperemic, submucosal nodule, ulcerative (shallow and deep), polypoid, cicatrical, and compression type. Each of these types was related to the healing process and outcome after antituberculosis chemotherapy. Serial bronchoscopy revealed that lesions less advanced than the deep ulcerative type responded to chemotherapy; the deep ulcerative and polypoid types progressed to cicatrical bronchostenosis despite chemotherapy. In the follow-up of patients with lesions of the deep ulcerative and polypoid types, serial bronchoscopy after chemotherapy is recommended; these patients also require adjunctive therapy to prevent cicatrical bronchostenosis.

Levels of recombinant human granulocyte colony-stimulating factor in serum are inversely correlated with circulating neutrophil counts.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is effective in countering chemotherapy-induced neutropenia. However, serum rhG-CSF levels cannot be maintained throughout the course of rhG-CSF therapy. The drop in serum rhG-CSF levels may vary with the duration of rhG-CSF administration or with the circulating neutrophil counts. We investigated the relationship between serum G-CSF levels and circulating neutrophil counts and the pharmacokinetics of rhG-CSF for patients with lung cancer who had been treated with myelosuppressive chemotherapy and then with subcutaneous rhG-CSF (lenograstim, 2 micrograms per kg of body weight per day). Twelve patients were randomly assigned to four groups with different rhG-CSF therapy schedules. Serum G-CSF levels were measured by an enzyme immunoassay method. Serum G-CSF levels during the rhG-CSF therapy greatly exceeded endogenous G-CSF levels and were mainly due to the presence of exogenous rhG-CSF rather than increased levels of endogenous G-CSF. Despite the duration of rhG-CSF administration, serum G-CSF levels during rhG-CSF therapy were inversely correlated with circulating neutrophil counts (r2 = 0.73, P < 0.0001). The value for the area under the concentration-time curve of rhG-CSF on the day of neutrophilia was lower than that on the day of neutropenia (P < 0.05). Our results suggest that the fall in serum G-CSF levels during rhG-CSF therapy may result from increased clearance and/or decreased absorption of rhG-CSF, two processes related to circulating neutrophil counts.

Chronic bronchial foreign body mimicking peripheral lung tumor.

We describe a case with chronic bronchial foreign body presenting with recurrent hemoptysis mimicking a peripheral lung tumor, and the outcome of surgical resection. A 61-year-old female with recurrent hemoptysis had a peripheral nodule in the right lower lobe. A right bronchial arteriogram showed dilatation and hypervascularity in the nodule. Surgical removal of the nodule contained a small branch of a white cedar. Chronic bronchial foreign body, while rare in adults, should be considered in the differential diagnosis of peripheral lung tumors associated with recurrent hemoptysis.

Arteriovenous malformation of the bronchial artery showing endobronchial protrusion.

The bronchoscopic findings of arteriovenous malformation of the bronchial artery are compared with findings on angiography and pathology. A non-pulsatile protrusion with engorged vessels on its glossy and uneven surface was seen at the distal portion of the right anterior segmental bronchus. A bronchial angiogram demonstrated that the protrusion corresponded to the hypervascular area of convoluted vessels. Histologically, a glossy, uneven surface and engorged vessels represented normal mucosa overlaying an extramuscular tortuous artery and proliferation of submucosal tiny vessels, respectively. These bronchoscopic findings were important clues for diagnosis.