Multiplex polymerase chain reaction for the detection of mycobacterial DNA in cases of tuberculosis and sarcoidosis.

The aims of the present study were: (1) to design a sensitive and specific polymerase chain reaction-based method that would allow detection of most common human typical and atypical mycobacterial strains and (2) to apply the method on formalin-fixed paraffin-embedded (FFPE) tissue and sputum samples from patients with clinicopathological evidence of tuberculosis and sarcoidosis. Three sets of primers were selected. The first detects specifically members of the Mycobacterium tuberculosis (M. tuberculosis) complex, amplifying a 243 bp fragment of the gene encoding the immunogenic protein MPB 64, whereas the second traces members of the Mycobacterium avium (M. avium) complex producing a 91 bp fragment of the IS1110 element. The third pair of primers is specific for slow-growing mycobacteria, amplifying a 383 bp region of the 65 kDa mycobacterial antigen gene. Our multiplex polymerase chain reaction assay identified mycobacterial DNA of 10(-3) colony-forming units (CFU)/mL from sputum samples, 10(-5) CFU/mL from FFPE tissue samples and 10(-6) CFU/mL from pure broth cultures. By performing the method on 75 FFPE tissue samples with histological and clinical evidence of tuberculosis and 300 sputum specimens from patients suspected of tuberculosis, we found 38 M. tuberculosis complex, 7 M. avium complex, and 14 slow-growing mycobacteria positive samples in the first case and in the second we found 95 M. tuberculosis complex, 21 M. avium complex, and 35 slow-growing mycobacteria positive samples. The sensitivity of the assay was significantly higher than that of Ziehl-Neelsen and in some cases higher than culture, especially when applied on atypical mycobacteria. In addition, 25 cases histologically and clinically characterized as sarcoidosis were investigated for mycobacterial DNA sequences and in nine of these, DNA corresponding to M. tuberculosis complex was detected. The method described can be applied directly on FFPE and sputum samples and allows not only the detection of mycobacterial DNA, but also an assessment concerning the species involved.

Modulation of wild-type p53 activity by mutant p53 R273H depends on the p53 responsive element (p53RE). A comparative study between the p53REs of the MDM2, WAFI/Cip1 and Bax genes in the lung cancer environment. WAFI/Cip1 = WAF1/Cip1.

Wild-type (wt) p53 is a tumor-suppressor protein which acts via transcriptional and transcriptional-independent mechanisms. The transcriptional function of p53 is mediated by specific responsive elements (REs). The MDM2, WAF1/Cip1 and Bax genes possess p53REs and their activation by wt p53 induces cell cycle progression, arrest and programmed cell death (apoptosis), respectively. Mutations of the p53 gene are detected in more than 50% of the human malignant tumors. p53 mutants seem to have a more stable conformation and are suggested to exert dominant-negative inhibition of wt p53 in cells containing both wt and mutant (mt) alleles. However, recent studies show that certain mt p53 proteins posses a "gain of function" phenotype. In the present study, we examined the effects of the second most frequent p53 mutant R273H on the p53REs of the MDM2, WAF1/Cip1 and Bax genes in the H1299 non-small cell lung carcinoma cell line. Although mt p53 R273H alone was unable to bind and transactivate the corresponding p53REs, it enhanced the MDM2-p53RE mediated gene transcription of wt p53 (positive-dominant effect) and prevented the wt p53 transactivation of the p53REs of WAF1/Cip1 and Bax genes (negative-dominant effect). Our data suggest that in the appropriate environment, differential transcription of critical p53 target genes by certain p53 mutant proteins may illustrate another mechanism implicated in tumor development.

Human papilloma virus (HPV) is possibly involved in laryngeal but not in lung carcinogenesis.

Data on human papilloma virus (HPV) involvement in preneoplastic and neoplastic lesions of the larynx and lung are limited and conflicting. The presence of HPV was investigated in a series of laryngeal specimens and non-small cell lung carcinomas (NSCLCs). The laryngeal samples (154) comprised 14 cases with hyperplasia without dysplasia, 49 with dysplasia, and 91 squamous cell carcinomas (SqCCs). The NSCLCs included 31 SqCCs, 32 adenocarcinomas, and 5 undifferentiated large cell carcinomas. Furthermore, we examined, for HPV DNA sequences, 14 bronchial metaplastic squamous lesions located next to cancerous areas. We used a sensitive nested polymerase chain reaction assay (NPCR), dot blotting, and in situ hybridization. The findings were correlated with clinicopathologic features of the patients. In the laryngeal specimens, NPCR analysis showed HPV DNA in 20 (13%) of the 154 specimens. Notably, 19 of 20 HPV-positive cases were carcinomas and only one was a mild dysplastic lesion. Typing of the carcinomas showed single HPV 6, 16, 18, and 33 infection in 1 (1.1%), 12 (13.2%), 2 (2.2%), and 1 (1.1%) samples, respectively, and HPV 6/33, 16/33, and 6/18 coinfection in three carcinomas. In situ hybridization findings were in agreement with PCR results, with the exception of two cases in which HPV 18 DNA was detected only by PCR. HPV was more frequently observed in heavy smokers than in patients with low daily cigarette consumption and nonsmokers (P = .03). There was no correlation between virus infection and gender, grade, and lymph node status of the carcinomas. None of the NSCLCs or adjacent metaplastic squamous epithelium contained HPV DNA sequences. The presented data suggest a contributory role of HPV in late stages of laryngeal carcinogenesis, because all premalignant lesions were negative but one. This study does not support a potential role of HPV in the development of NSCLCs.

Alterations of the p16-pRb pathway and the chromosome locus 9p21-22 in non-small-cell lung carcinomas: relationship with p53 and MDM2 protein expression.

The p16-pRb and p53-MDM2 pathways represent vital cell cycle checkpoints. Recent studies provide evidence that these pathways are directly linked via MDM2-pRb interaction and p53 suppression of the RB1 gene. In the present study we investigated the alterations of this G1 phase protein network using immunohistochemical and molecular methods in a series of 68 non-small-cell lung carcinomas (NSCLCs) and correlated the findings with clinicopathological features and prognosis of the patients. Aberrant expression (Ab) of p16 and pRb was observed in 33 (49%) and 27 (40%) of the carcinomas, respectively. Analysis of the region that encodes for p16 by deletion mapping, a polymerase chain reaction (PCR)-based methylation assay and PCR single-strand conformation polymorphism (SSCP) analysis revealed that deletions and transcriptional silencing by methylation might represent the main mechanisms of CDKN2/p16ink4a inactivation in NSCLCs. The results of deletion mapping also suggest that other tumor suppressor genes may reside at the 9p21-22 region, which encodes for CDKN2/MTS1/p16ink4a, p14ARF, and MTS2/p15ink4b. In addition, microsatellite instability was observed with a frequency of 16% in the 9p21-22 chromosome area. Overexpression (P) of p53 and MDM2 proteins was found in 39 (58%) and 47 (70%) of the cases, respectively. A highly significant association was observed between p53 overexpression and p53 mutations (P = 0.006). Statistical analysis of the expression patterns of the biologically relevant molecules (p16/pRb, p53/MDM2, MDM2/pRb, and p53/pRb) showed coincident overexpression of p53 and MDM2 (P = 0.04) and that abnormal pRb was correlated with elevated levels of MDM2 (P = 0.013) and p53 (P = 0.01), respectively. We suggest that deregulated expression of these molecules may act synergistically. An important finding of the study was that multiple impairments (three and four molecules affected) of the p16/pRb/p53/MDM2 network occurred in a large proportion (43%) of the carcinomas. This finding in addition to the absence of correlation with clinical stage of the tumors suggests that multiple hits of this network may be a relatively early event in the development of a subset of NSCLCs. The relationship between the factors examined in the present study, clinicopathological features, and survival of the patients did not reveal any significant correlations with the exception of smoking, which was associated with microsatellite alterations (loss of heterozygosity and microsatellite instability) at the 9p21-22 locus (P = 0.04) and the immunophenotypes p53(P)/MDM2(P) (P = 0.04) and p16(Ab)/pRb(Ab)/p53(P)/MDM2(P) (P = 0.03), respectively. We suggest that in a subset of NSCLCs, simultaneous deregulation of the members of this network may represent one way of initiating the oncogenic procedure whereas in other NSCLC subgroups alternative pathways may play this role.

Effects of p53 mutants derived from lung carcinomas on the p53-responsive element (p53RE) of the MDM2 gene.

The present study represents a continuation of previous works in which we observed that lung carcinomas co-expressing MDM2 protein and p53 mutants (mt p53) exhibited more aggressive behaviour. In the above studies, we suggested a 'gain of function' mechanism of mt p53 proteins based on the fact that the MDM2 gene possesses a p53-responsive element (MDM2-p53RE). In this study, to prove our hypothesis, we selected 12 cases from a series of 51 bronchogenic carcinomas. In these 12 cases, we examined the ability of the expressed mt p53 to bind the MDM2-p53RE and correlated the findings with MDM2 expression. Furthermore, we constructed four of these p53 mutants and studied their transactivation properties by co-transfecting them with a reporter plasmid carrying MDM2-p53RE in the p53 null non-small-cell lung carcinoma cell line (NSCLC) H1299. We observed mutant p53 protein DNA-binding activity, which depended on the nature and the position of the amino acid substitution. The fact that the cases with DNA-binding activity were accompanied with MDM2 protein isoforms' overexpression is indicative of a 'gain of function' phenotype. This hypothesis was enforced by the findings of the transfection experiments, which revealed that certain p53 mutants enhanced the expression of the luciferase reporter gene either directly or indirectly via a dominant positive effect on the wild-type p53. In conclusion, this work is one first attempt to examine if the deregulation of the p53/MDM2 autoregulatory feedback loop is due to novel properties of certain p53 mutants in the specific environment of a subset of bronchogenic carcinomas.

Dependence of forced vital capacity manoeuvre on time course of preceding inspiration in patients with restrictive lung disease.

In normal subjects and patients with airway obstruction, flows during a forced vital capacity (FVC) manoeuvre are higher after a fast inspiration without an end-inspiratory pause (manoeuvre 1) as compared to a slow inspiration with an end-expiratory pause of approximately 5 s (manoeuvre 2). In this study, we investigated the influence of these manoeuvres on maximal expiratory volume-time and flow-volume curves in patients with restrictive lung disease. Eleven patients with restrictive lung disease were studied. Their average (+/-SD) lung function test results were: FVC=55+/-12% predicted value, forced expiratory volume in one second (FEV1) 52+/-20% pred, FEV1/FVC 85+/-6%, total lung capacity 55+/-8% pred, and carbon monoxide transfer factor 47+/-18% pred. The patients performed the two FVC manoeuvres in random order. We compared the ensuing spirograms and maximal expiratory flow-volume curves from which peak expiratory flow, FEV1, FEV1/FVC, maximal mid-expiratory flow, and maximal flows were computed. All spirometric indices were significantly higher with manoeuvre 1 than 2. Maximal expiratory flows at the same lung volume were also significantly higher with manoeuvre 1 than 2, in all patients. Routine spirometric indices, obtained during a forced vital capacity manoeuvre depend on the time course of the preceding inspiration in patients with restrictive lung disease. Therefore, the forced vital capacity manoeuvre should be standardized if used in clinical, epidemiological and research studies.

Somatostatin receptor scintigraphy of non-neuroendocrine malignancies with 111In-pentetreotide.

In-111 pentetreotide is a new radiolabelled [OctreoScan 111, Mallinckrodt Medical BV, Petten] somatostatin analog with high affinity to somatostatin receptors (SR). introduced for the in vivo imaging of SR positive tissues. In an attempt to evaluate its clinical usefulness for tissue characterization in malignancies without neuroendocrine expression in parallel with histological and radiological examinations, specific scintigraphy was performed on brain (6 cases), thyroid (6 cases) and breast (9 cases) tumors, and in lymphomas (9 cases) and melanomas (6 cases). A dose of 111MBq of In-111 pentetreotide was injected i.v. to each patient and scintimages at 6 and 22 hours (for comparison) p.i. were obtained. The primary lesion of the breast cancer population was imaged in all 9 cases as well as all the palpable axillary nodes in 4 cases. Three women with impalpable axillary lymph nodes scanned negative but had a positive biopsy. Both meningiomas were positive for SR scans: positive results were also obtained for the high grade astrocytoma and the craniopharyngioma: Two out of 6 patients with papillary thyroid cancer showed a marked radiotracer accumulation. Scintigraphy in all 5 lymphomas was positive for SR but did not detect the Tc-99m sulphur microcolloid [Lymphoscint, Solco, Basel, Suitzerland] imaged lymph nodes in 5 melanomectomized patients. When judging the imaging results of these non-neuroendocrine malignancies definite conclusions should not be drawn since the number of studied cases polymorph, was small for every cancer histotype; nevertheless SR scintigraphy does not seem to be reliable for tumor staging in non-neuroendocrine malignancies, but is more suitable for a tissue characterization and monitoring changes of SR expression during and after therapy.

Receptor scintigraphy of non-neuroendocrine cancers with In-111 pentetreotide.

Somatostatin receptors (SR) are surface markers characterizing not only APUDomas associated with neuroendocrine identities but also malignancies without neuroendocrine expression. Recently, the somatostatin analog pentetreotide was labeled with In-111 (OctreoScan 111, Mallinckrodt Medical BV, Petten, Holland) and introduced for the in vivo visualization in man of SR-positive tissues. In the present report, SR-specific scintigraphy is evaluated as a clinical tool for tissue characterization in correlation with histological and radiological examinations. Scintigraphy was focused and performed in cancer types without neuroendocrine tissue expression such as brain (n = 6) and breast tumors (n = 9) and lymphomas (n = 5). Scintigraphy was performed for comparison at 6 and 22 h after i.v. application of 111 MBq (3 mCi) of In-111-Pentetreotide. In the breast cancer group, the primary tumor was visualized in all 9 women as well as in all 4 cases with palpable axillary lymph nodes. Three women with a negative axillary node scan and impalpable nodes had positive biopsy. In two cases, mediastinal lymph node involvement was observed. So far the role of SR-positive breast cancer (BC) scans remains unknown. It is tempting to speculate that in resected women who are histologically and scintigraphically SR positive, it might be of value in the early detection of symptom-free recurrences. High densities of SR were present within both meningiomas, the high-grade astrocytoma and the craniopharyngioma. Differentiation of low- and high-grade astrocytomas could not be successfully achieved because both grades showed intense radioactivity uptake, even though high-grade tumors lack SR. The latter might be due to the damaged blood-brain barrier and the poor radioactivity washout observed in high-grade astrocytomas. All five lymphomas could be detected due to the presence of activated lymphocytes and macrophages that express SR at a sufficient density. In conclusion, SR scintigraphy in non-neuroendocrine malignancies does not seem to be reliable for an initial tumor staging but rather more suitable for a tissue characterization and extremely useful for monitoring changes of SR expression after treatment.

A molecular and immunohistochemical study of the MDM2 protein isoforms and p53 gene product in bronchogenic carcinoma.

Forty-one bronchogenic carcinomas were investigated for expression of MDM2 protein isoforms and their relationship to p53 protein levels and p53 gene alterations using molecular and immunohistochemical techniques. The findings were correlated with the pathological features of the carcinomas. MDM2 protein was overexpressed in 26 cases (63 percent). Western blot analysis with two monoclonal antibodies, 1B10 and IF2, revealed three MDM2 protein isoforms, p90, p57 and p76/74. p90 and p57 are capable of interacting with p53 protein, while p76/74 is not. Various patterns of MDM2 isoforms were seen. Although no correlation between the patterns and pathological features was observed, lymph node metastases were more frequent in the cases with MDM2 overexpression (P < 0.005). In 3 out of 17 specimens of normal lung tissue examined, there was a low level of expression of p90. Molecular analysis revealed that MDM2 overexpression was a consequence of increased transcription rather than MDM2 gene amplification. p53 protein was overexpressed in 21 cases (51 percent) and p53 gene alterations (mutations + allelic deletions) were detected in 23 patients (56 percent). A high degree of concordance (76 percent) between p53 mutations and p53 staining was noticed (P < 10(-5)). p53 gene alterations were significantly associated with lymph node disease (P < 0.01). MDM2 and p53 proteins were simultaneously detected in 21 cases (51 percent), of which 17 (42 percent) showed p53 and MDM2 overexpression. The latter group was positively correlated with p53 mutations (P < 0.05). A strong correlation between MDM2/p53 co-expression and lymph node metastases was observed (P < 0.001). The findings suggest that MDM2 overexpression is a common event in bronchogenic carcinoma. The selective expression of some MDM2 isoforms in neoplastic tissue and not in the surrounding normal areas underscores the pathological role of the various MDM2 products. Finally, the coexistence of MDM2 protein(s) and p53 aberrations (mutations and/or overexpression) in a subset of lung carcinomas may be indicative of a 'gain of function' phenotype, with more aggressive characteristics.

Immunohistochemical and molecular evaluation of the mdm-2 gene product in bronchogenic carcinoma.

In this study, we investigated immunohistochemically the expression of mdm-2 protein in 93 surgically resected bronchogenic carcinomas. The findings were correlated with p53 protein detection status and clinicopathologic data (histologic type, differentiation grade of the lesions, lymph node metastases, and smoking history of the patients). Thirty of the 93 immunohistochemically examined specimens were subjected to Northern blot and differential polymerase chain reaction analysis to look into the mechanism of mdm-2 overexpression. Finally, we studied the concordance between p53 immunohistochemical positivity and p53 gene alterations as assessed by the single-strand conformation polymorphism technique. Seventy-three (78%) and 67 (72%) of 93 carcinomas showed nuclear immunoreactivity for mdm-2 and p53 proteins, respectively. We observed a high degree of concordance (75%) between p53 mutations and p53 immunolabelling, which was even higher in the specimens with p53 positively in more than 50% of the cells (90%). Despite the high percentage of mdm-2 and p53 expression, the two molecules were simultaneously detected in 50 (54%) of 93 cases. Forty-two (84%) of the 50 cases were accompanied by p53 mobility shifts, which indicated mutations. Interestingly, statistical analysis revealed an almost significant correlation between the carcinomas with mdm-2/p53 coexpression and lymph node disease (P = 0.058), which indicated a possible "gain of function" phenotype. In addition, absence of reactivity for both proteins was statistically more frequent in the patients without lymph node disease (P = 0.006). The mdm-2-positive/p53-negative immunohistochemical profile was more often seen in adenocarcinomas (P = 0.003), especially in well-differentiated ones (P = 0.02), than in other histologic types of lung cancer, which suggested a p53-independent pathway of mdm-2 overexpression. Molecular analysis showed that mdm-2 overexpression was a consequence of increased transcription rather than of mdm-2 gene amplification. The smoking history of the patients was strongly related to p53 (P = 10(-4)) even in the group of adenocarcinomas (P = 0.012). No correlation was observed between cigarette consumption and mdm-2 immunoreactivity.

Small airways dysfunction in systemic sclerosis. A controlled study.

A debate exists regarding the importance of small airways disease in systemic sclerosis, while smoking seems to have a major effect on the exact prevalence. In order to evaluate small airways dysfunction (SAD) in a pure systemic sclerosis population, we performed pulmonary function studies in 31 nonsmoking patients and 31 age- and sex-matched nonsmoking control subjects. Patients' FVC, TLC, and Dco mean values were significantly lower compared with the corresponding values of the controls (p less than 0.05), while there was no difference in MEF25, RV, and RV/TLC. Seven (22.6 percent) of 31 patients and four controls (a nonsignificant difference) had evidence of SAD, namely a maximum expiratory flow at 25 percent of vital capacity (MEF25) less than 60 percent of predicted. Positive correlation (p less than 0.001) was found between MEF25 and FEV1/FVC in the patients. Moreover, no differences were found in abnormal lung function patients with and those without SAD in demographic, clinical, roentgenologic, and serologic features and results of pulmonary function tests. These findings suggest that SAD in our patients is not a characteristic and early manifestation of systemic sclerosis and that, when present, it is not correlated with the severity of the pulmonary involvement in scleroderma.

Organic brain syndrome with psychosis as an initial manifestation of systemic lupus erythematosus in an elderly woman.

This paper describes a rare case of organic brain syndrome with psychosis and clinically transverse myelopathy, as initial manifestations of systemic lupus erythematosus in an elderly woman. The identification and evaluation of antibodies to ribosome P in the serum and cerebrospinal fluid may be of help in such cases for differential diagnosis. The patient was treated successfully with 30 mg prednisone daily.

Cardiac performance in collagen diseases estimated by non-invasive methods.

The myocardial performance was studied in 3 collagen diseases, i.e. rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and progressive systemic sclerosis (PSS). Sixteen patients with SLE, 12 with RA and 11 with PSS were examined, measuring the systolic time intervals from the first derivative of the carotid pulse in all cases. The ejection fraction (EF), was evaluated in 33 patients using radionuclide left ventricular angiography. The systolic time intervals were compared to those of 103 normal persons and the EF to that of 22 normal controls. In the SLE group the pre-ejection period was significantly shorter, while the ejection period was longer than normally expected. In the same group, the EF was significantly higher that the EF of the control group. These differences could not be related to age, blood pressure, disease duration, coronary risk factors, heart rate or blood values. Most findings in the RA group tended to be opposite to those of SLE. It is concluded that in SLE before any direct involvement of the heart the systolic time intervals and the EF present features similar to those of increased cardiac performance.

Paget's bone disease, anaemia and vitiligo: a case report.

The coexistence of Paget's bone disease, pernicious anaemia and vitiligo is very rare. The pathogenesis of Paget's bone disease remains still unknown. We report here a patient having these three entities simultaneously and we suggest that some cases of Paget's bone disease be due to autoimmunity.

Regional blood flow distribution in dog during induced hypotension and low cardiac output. Spontaneous breathing versus artificial ventilation.

Respiratory muscle blood flow and organ blood flow was studied in two groups of dogs with radioactively labeled microspheres to assess the influence of the working respiratory muscles on the regional distribution of blood flow when arterial pressure and cardiac output were lowered by pericardial tamponade. In one group (n = 6), the dogs were paralyzed and mechanically ventilated (Mv), while in the other (n = 6), they were left to breathe spontaneously (Sb). Cardiac output fell to 30% of control values during tamponade in both groups and was maintained constant. None of the dogs was hypoxic. Ventilation in the Sb group peaked after 50 min of hypotension, but remained unchanged in the Mv group. Duplicate measurements of blood flow were made during a control period and after 50 min of tamponade (corresponding to the peak ventilation in Sb). Blood flow to the respiratory muscles increased significantly (P less than 0.001) during tamponade in Sb (diaphragmatic flow increased to 361% of control values), while it decreased in Mv. Although the arterial blood pressure and cardiac output were comparable in the two groups, blood flow distribution during tamponade was different. In Sb, the respiratory muscles received 21% of the cardiac output, compared with only 3% in the Mv group. Thus, by muscle paralysis and Mv, a large fraction of the cardiac output used by the working respiratory muscles can be made available for perfusion of other organs during low cardiac output state: blood flows to the liver, brain, and quadriceps muscles were significantly higher during tamponade in the Mv group compared with the Sb group. Similarly, blood lactate at all times after the induction of low cardiac output and hypotension was significantly lower in the Mv animals (P less than 0.005).