Primary effusion lymphoma in an elderly patient effectively treated by lenalidomide: case report and review of literature.

Primary effusion lymphoma (PEL) is a rare aggressive subset of non-Hodgkin B-cell lymphoma. It is caused by Kaposi sarcoma-associated herpesvirus/human herpesvirus type 8 (KSHV/HHV8). It occurs mainly, but not exclusively, in HIV-positive patients. PEL predominantly develops in serous cavities and occasionally in extracavitary regions. PEL carries a very poor prognosis with a median survival time of <6 months. Indeed, currently used treatment modalities such as CHOP chemotherapy are far from achieving complete and sustainable remission. Therefore, there is no clear standard of care established in the treatment of PEL patients, stressing the need for novel-targeted approaches. Here, we have attempted a comprehensive assessment of the treatment of PEL, discussed avant-garde therapies and updated the state of preclinical research with promising clinical applications in the field. These include inhibitors of viral replication, modulators of cell signaling and inflammation, nuclear factor kappa B (NF-κB) and histone deacetylase inhibitors, and recently the combination of arsenic trioxide and interferon-alpha. Some of these targeted therapies have not yet reached clinical studies, although others were used in a few individual case reports with low numbers of patients. We also describe the first case of a 77-year-old, HIV-negative, HHV8-positive patient diagnosed with PEL limited to the pleural and peritoneal cavities. He received lenalidomide 25 mg/day for 21 days every 28 days. Treatment was well tolerated with no side effects. He rapidly improved after 1 month of treatment and progressively achieved complete remission persistent after 18 months of therapy. We believe that this review will bridge an important gap between classical chemotherapy and modern approaches of targeted therapy. Finally, our findings warrant further evaluation of lenalidomide in future prospective clinical studies.

The inflammatory response in acyl-CoA oxidase 1 deficiency (pseudoneonatal adrenoleukodystrophy).

Among several peroxisomal neurodegenerative disorders, the pseudoneonatal adrenoleukodystrophy (P-NALD) is characterized by the acyl-coenzyme A oxidase 1 (ACOX1) deficiency, which leads to the accumulation of very-long-chain fatty acids (VLCFA) and inflammatory demyelination. However, the components of this inflammatory process in P-NALD remain elusive. In this study, we used transcriptomic profiling and PCR array analyses to explore inflammatory gene expression in patient fibroblasts. Our results show the activation of IL-1 inflammatory pathway accompanied by the increased secretion of two IL-1 target genes, IL-6 and IL-8 cytokines. Human fibroblasts exposed to very-long-chain fatty acids exhibited increased mRNA expression of IL-1α and IL-1ß cytokines. Furthermore, expression of IL-6 and IL-8 cytokines in patient fibroblasts was down-regulated by MAPK, p38MAPK, and Jun N-terminal kinase inhibitors. Thus, the absence of acyl-coenzyme A oxidase 1 activity in P-NALD fibroblasts triggers an inflammatory process, in which the IL-1 pathway seems to be central. The use of specific kinase inhibitors may permit the modulation of the enhanced inflammatory status.

KSHV-transformed primary effusion lymphoma cells induce a VEGF-dependent angiogenesis and establish functional gap junctions with endothelial cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of primary effusion lymphoma (PEL) and of Kaposi's sarcoma. PEL is an aggressive proliferation of B cells with poor prognosis. We evaluated both in vitro and in vivo the potential role of angiogenic factors secreted by PEL cells, that is, their interaction with endothelial cells and their implication in the invasive behavior of tumoral cells. In vitro, PEL-induced angiogenesis is dependent on vascular endothelial growth factor (VEGF) and VEGF receptors. However, although PEL cells produce VEGF and basic fibroblast growth factor (b-FGF) transcripts, they only secrete VEGF in vitro. In vivo, very high levels of both VEGF and b-FGF were found in the ascitic fluid of NOD/SCID mice injected with PEL cells. We then show evidence of cell adhesion and gap junction-mediated heterocellular communication between PEL cells and endothelial cells. Finally, we show that PEL cells extravasate through the endothelial barrier and that the specific tyrosine kinase inhibitor of VEGF receptors, PTK-787/ZK-222584, the anti-VEGF antibody, bevacizumab or the gap junction inhibitor 18-alpha-glycyrrhetinic acid, partially attenuate PEL cell extravasation. Angiogenesis, cell adhesion and communication likely contribute to the development of PEL and represent potential therapeutic targets.

Unlocking diamond's potential as an electronic material.

In this paper, we review the suitability of diamond as a semiconductor material for high-performance electronic applications. The current status of the manufacture of synthetic diamond is reviewed and assessed. In particular, we consider the quality of intrinsic material now available and the challenges in making doped structures suitable for practical devices. Two practical applications are considered in detail. First, the development of high-voltage switches capable of switching voltages in excess of 10 kV. Second, the development of diamond MESFETs for high-frequency and high-power applications. Here device data are reported showing a current density of more than 30 mA mm(-1) along with small-signal RF measurements demonstrating gigahertz operation. We conclude by considering the remaining challenges which will need to be overcome if commercially attractive diamond electronic devices are to be manufactured.

PS-341 or a combination of arsenic trioxide and interferon-alpha inhibit growth and induce caspase-dependent apoptosis in KSHV/HHV-8-infected primary effusion lymphoma cells.

Kaposi's sarcoma (KS)-associated herpes virus (KSHV) is the causative agent of primary effusion lymphoma and of KS. Primary effusion lymphoma (PEL) is an aggressive proliferation of B cells. Conventional chemotherapy has limited benefits in PEL patients, and the prognosis is very poor. We previously reported that treatment of human T-cell leukemia virus type 1 (HTLV-1)-associated adult T-cell leukemia/lymphoma cells either with arsenic trioxide (As) combined to interferon-alpha (IFN-alpha) or with the bortezomib (PS-341) proteasome inhibitor induces cell cycle arrest and apoptosis, partly due to the reversal of the constitutive nuclear factor-kappaB (NF-kappaB) activation. PEL cells also display an activated NF-kappaB pathway that is necessary for their survival. This prompted us to investigate the effects of PS-341, or of the As/IFN-alpha combination on PEL cells. A dramatic inhibition of cell proliferation and induction of apoptosis was observed in PS-341 and in As/IFN-alpha treated cells. This was associated with the dissipation of the mitochondrial membrane potential, cytosolic release of cytochrome c, caspase activation and was reversed by the z-VAD caspase inhibitor. PS-341 and As/IFN-alpha treatment abrogated NF-kappaB translocation to the nucleus and decreased the levels of the anti-apoptotic protein Bcl-X(L). Altogether, these results provide a rational basis for a future therapeutic use of PS-341 or combined As and IFN-alpha in PEL patients.

Protection against cephalosporin-induced lipid peroxidation and nephrotoxicity by (+)-cyanidanol-3 and vitamin E.

The ability of the clinically used cephalosporins: cephalothin, cefotaxime and cefotiam to induce lipid peroxidation (LPO) and renal damage was compared to that of nephrotoxic cephaloridine under in vivo conditions. Glutathione was measured in rat liver or in renal cortex as non-protein sulfhydryls. LPO was measured in plasma, renal cortex and liver by the generation of malondialdehyde or as the increase in renal cortical concentration of conjugated dienes. Impairment of renal function was measured as the decrease in renal cortical accumulation of the organic anion p-aminohippurate (PAH). Administration of cephalosporins to rats as a single dose (2000 mg/kg, ip) induced a significant glutathione-depletion in the renal cortex with cephaloridine, and in the liver with cephaloridine, cephalothin and cefotiam. Treatment of rats with cephaloridine, cephalothin and cefotiam (200, 500, or 1000 mg kg-1 day-1, ip) for 5 days resulted in a dose-dependent increase of LPO in the renal cortex. While cephaloridine induced the highest concentration of conjugated diene, cefotaxime had no effect. Measurements of PAH accumulation in renal cortical slices from cephalosporin-treated rats showed a dose-dependent decrease in the renal cortical accumulation of PAH. Pretreatment with the antioxidants vitamin E or cyanidanol (400 mg kg-1 day-1, ip) 1 h before treatment with cephaloridine, cephalothin or cefotiam (1000 mg kg-1 day-1, ip) for 3 days inhibited cephalosporin-induced LPO and significantly reduced the impairment of renal cortical accumulation of PAH. The potential of different cephalosporins for inducing LPO and reducing PAH accumulation was ranked as follows: cephaloridine > cephalothin > cefotiam > cefotaxime.

Protection against cephalosporin-induced lipid peroxidation and nephrotoxicity by (+)-cyanidanol-3 and vitamin E

The ability of the clinically used cephalosporins: cephalothin, cefotaxime and cefotiam to induce lipid peroxidation (LPO) and renal damage was compared to that of nephrotoxic cephaloridine under in vivo conditions. Glutathione was measured in rat liver or in renal cortex as non-protein sulfhydryls. LPO was measured in plasma, renal cortex and liver by the generation of malondialdehyde or as the increase in renal cortical concentration of conjugated dienes. Impairment of renal function was measured as the decrease in renal cortical accumulation of the organic anion p-aminohippurate (PAH). Administration of cephalosporins to rats as a single dose (2000 mg/kg, ip) induced a significant glutathione-depletion in the renal cortex with cephaloridine, and in the liver with cephaloridine, cephalothin and cefotiam. Treatment of rats with cephaloridine, cephalothin and cefotiam (200, 500, or 1000 mg kg-1 day-1, ip) for 5 days resulted in a dose-dependent increase of LPO in the renal cortex. While cephaloridine induced the highest concentration of conjugated diene, cefotaxime had no effect. Measurements of PAH accumulation in renal cortical slices from cephalosporin-treated rats showed a dose-dependent decrease in the renal cortical accumulation of PAH. Pretreatment with the antioxidants vitamin E or cyanidanol (400 mg kg-1 day-1, ip) 1 h before treatment with cephaloridine, cephalothin or cefotiam (1000 mg kg-1 day-1, ip) for 3 days inhibited cephalosporin-induced LPO and significantly reduced the impairment of renal cortical accumulation of PAH. The potential of different cephalosporins for inducing LPO and reducing PAH accumulation was ranked as follows: cephaloridine > cephalothin > cefotiam > cefotaxime.

Effect of chronic exposure to cold on isoprenaline-induced cAMP accumulation and relaxation in the rat aorta.

Rats chronically exposed to cold (5 degrees C for 5 weeks) develop hypertension. Isoprenaline-induced vascular smooth muscle relaxation is increased in these animals. Our main objective was to compare isoprenaline-induced relaxation of aortae isolated from control and cold-acclimated rats and attempt to relate the differences to changes in receptor parameters (affinity and reserve) and signaling mechanisms. Isoprenaline (10(-9)-10(-5) M)-induced relaxation was enhanced significantly (p < 0.05) in aorta segments from cold-acclimated rats. There was a significant (p < 0.05) increase in the potency of isoprenaline but with no change in affinity. Isoprenaline produced 50% of the maximum response while occupying about 50% and about 15% of the receptors in isolated rat aorta segments from control and cold-treated rats, respectively. Forskolin and db-cAMP also concentration-dependently relaxed aorta segments from control and cold-acclimated rats. There was no difference in potency or maximum response to forskolin (which directly activates adenylyl cyclase) and db-cAMP. cAMP concentrations in the presence of isoprenaline were significantly (p < 0.05) higher in aorta segments from rats chronically exposed to cold when compared with aorta segments from control rats. These findings suggested that altered mechanisms upstream of activation of adenylyl cyclase are involved in the increased beta-adrenoceptor-induced relaxation.

[Verrucous carcinoma of the tongue occurring on lesions of lichen planus]. / Carcinome verruqueux de la langue survenu sur des lésions de lichen plan.

BACKGROUND: Verrucous carcinoma of the oral cavity is a rare entity that was formerly controversial. Etiopathogenesis remains unclear, notably as for its possible association with lichen planus. We report a case of verrucous carcinoma occurring in lesions of lichen planus of the tongue. CASE REPORT: A 78-year old, non smoking patient, with past history of cutaneous lichen planus presented for lesions of oral lichen planus affecting both the tongue and the palate. A treatment by topical tretinoin improved him in a spectacular way and brought about a remission which lasted 5 years. A recurrence occurred when the treatment was stopped; new whitish, warty cauliflower-like lesions appeared on the tongue. A biopsy confirmed the clinical suspicion of verrucous carcinoma. A laser resection was performed. Three months later, another recurrence was observed. A chemotherapy associating isotretinoin and methotrexate eliminated all lesions. The patient's condition is considered stable, under treatment, one year later. DISCUSSION: Verrucous carcinoma is a rare slow-growing oral tumor that is chiefly exophytic and does not metastasize, but it can invade and destroy oral tissues. Its clinical presentation contrasts with benign histologic features: papillomatosis, acanthosis, dysplasia in variable degrees. The occurrence on lesions of lichen planus, although "classic", is very rarely found in the literature. The treatment is not well codified. An additional chemotherapy seems necessary to prevent recurrences.

Detection of rifampin resistance in Mycobacterium tuberculosis in a single tube with molecular beacons.

Current clinical assays for determining antibiotic susceptibility in Mycobacterium tuberculosis require many weeks to complete due to the slow growth of the bacilli. Here we demonstrate an extremely sensitive single-tube PCR assay that takes less than 3 h and reliably identifies rifampin-resistant M. tuberculosis in DNA extracted directly from sputum. Ninety-five percent of mutations associated with rifampin resistance occur in an 81-bp core region of the bacterial RNA polymerase gene, rpoB. All mutations that occur within this region result in rifampin resistance. The assay uses novel nucleic acid hybridization probes called molecular beacons. Five different probes are used in the same reaction, each perfectly complementary to a different target sequence within the rpoB gene of rifampin-susceptible bacilli and each labeled with a differently colored fluorophore. Together, their target sequences encompass the entire core region. The generation of all five fluorescent colors during PCR amplification indicates that rifampin-susceptible M. tuberculosis is present. The presence of any mutation in the core region prevents the binding of one of the molecular beacons, resulting in the absence of one of the five fluorescent colors. When 148 M. tuberculosis clinical isolates of known susceptibility to rifampin were tested, mutations associated with rifampin resistance were detected in 63 of the 65 rifampin-resistant isolates, and no mutations were found in any of the 83 rifampin-susceptible isolates. When DNA extracted directly from the sputum of 11 patients infected with rifampin-resistant tuberculosis was tested, mutations were detected in all of the samples. The use of this rapid assay should enable early detection and treatment of drug-resistant tuberculosis in clinical settings.

Right ventricular form and function after percutaneous atrial septal defect device closure.

OBJECTIVES: We sought to assess the right heart's response to percutaneous device closure of moderate sized atrial septal defects (ASDs) in adults over a one-year follow-up period. BACKGROUND: Percutaneous ASD device closure is a safe and effective means of reducing or eliminating interatrial shunting. The response of the adult's right heart to device closure is incompletely understood. METHODS: Forty consecutive patients had 40 device implantations (32 with the CardioSeal implant and 8 with the Amplatzer device). The patients were assessed with echocardiography, chest radiography and electrocardiography before the procedure and at 1, 6 and 12 months. RESULTS: The mean ASD size was 13+/-4 mm, and the device size ranged from 33 to 40 mm for CardioSeal and 12 to 36 mm for Amplatzer. At one month, heart size (49% vs. 46%), four-chamber right ventricular (RV) size (45 vs. 41 mm), paradoxical septal motion (60% vs. 5%), QRS duration (125 vs. 119 ms), PR interval (181 vs. 155 ms) and echocardiographically determined pulmonary artery systolic pressure decreased significantly and was maintained at 12-month follow-up. At six months, right atrial length decreased from 50 to 47 mm. At one year, 29% of patients had persistent RV enlargement. CONCLUSIONS: Right heart morphology undergoes rapid improvement within one month of defect closure, with associated mechanoelectrical benefit. A small number of patients had persistent RV enlargement or pulmonary hypertension, or both, at one year. Our data support the application of transcatheter methods in achieving excellent hemodynamic and anatomic outcomes.

Genotypic analysis of Mycobacterium tuberculosis in two distinct populations using molecular beacons: implications for rapid susceptibility testing.

Past genotypic studies of Mycobacterium tuberculosis may have incorrectly estimated the importance of specific drug resistance mutations due to a number of sampling biases including an overrepresentation of multidrug-resistant (MDR) isolates. An accurate assessment of resistance mutations is crucial for understanding basic resistance mechanisms and designing genotypic drug resistance assays. We developed a rapid closed-tube PCR assay using fluorogenic reporter molecules called molecular beacons to detect reportedly common M. tuberculosis mutations associated with resistance to isoniazid and rifampin. The assay was used in a comparative genotypic investigation of two different study populations to determine whether these known mutations account for most cases of clinical drug resistance. We analyzed samples from a reference laboratory in Madrid, Spain, which receives an overrepresentation of MDR isolates similar to prior studies and from a community medical center in New York where almost all of the resistant isolates and an equal number of susceptible controls were available. The ability of the molecular beacon assay to predict resistance to isoniazid and rifampin was also assessed. The overall sensitivity and specificity of the assay for isoniazid resistance were 85 and 100%, respectively, and those for rifampin resistance were 98 and 100%, respectively. Rifampin resistance mutations were detected equally well in isolates from both study populations; however, isoniazid resistance mutations were detected in 94% of the isolates from Madrid but in only 76% of the isolates from New York (P = 0.02). In New York, isoniazid resistance mutations were significantly more common in the MDR isolates (94%) than in single-drug-resistant isolates (44%; P < 0.001). No association between previously described mutations in the kasA gene and isoniazid resistance was found. The first mutations that cause isoniazid resistance may often occur in sequences that have not been commonly associated with isoniazid resistance, possibly in other as yet uncharacterized genes. The molecular beacon assay was simple, rapid, and highly sensitive for the detection of rifampin-resistant M. tuberculosis isolates and for the detection of isoniazid resistance in MDR isolates.

Dissemination of Mycobacterium tuberculosis across the San Francisco Bay Area.

The propensity of Mycobacterium tuberculosis genotypes to spread across geographic boundaries was investigated by comparing the IS6110 and polymorphic GC-rich sequence patterns of M. tuberculosis isolates from San Francisco and the East Bay, two distinct regions separated by San Francisco Bay. Of 724 isolates from incident tuberculosis patients during 1992 and 1993, only 53 (7.3%) had patterns matching > or = 1 isolates from the other region. In the multivariable analysis of patient risk factors, an AIDS diagnosis (odds ratio [OR], 1.89; 95% confidence interval [CI], 1.00-3.57) and non-Asian race (OR, 3.43; 95% CI, 1.59-7.42) were associated with having an isolate with a matching pattern. Of 375 unique IS6110 patterns among San Francisco isolates, only 9 (2.4%) matched patterns of East Bay isolates. These population-based data suggest that in the San Francisco Bay Area, M. tuberculosis does not rapidly spread across geographic boundaries, and tuberculosis control efforts should focus on transmission within defined areas.

DNA fingerprinting with two probes decreases clustering of Mycobacterium tuberculosis.

DNA fingerprinting of Mycobacterium tuberculosis is used to study the epidemiology of tuberculosis, but the specificity of the widely used IS6110 technique has not been validated. Isolates from Denver, Colorado from December 1988 through June 1994 were fingerprinted with the IS6110 technique. Available records were reviewed for patients whose isolates were within IS6110-defined clusters, and these isolates were fingerprinted with an independent technique (pTBN12). Of 189 isolates, 86 (46%) were in IS6110-defined clusters. Clustering was inversely related to the number of copies of IS6110, ranging from 12 of 12 (100%) to 37 of 48 (77%) and 37 of 129 (29%) for isolates having one, two to five, and more than five copies (p < 0.001). Of the 86 isolates clustered with the IS6110 technique, 35 (41%) had unique pTBN12 fingerprints. Discordant results with the two fingerprinting techniques were more common among isolates having five or fewer copies of IS6110. Epidemiologic links were identified among four of 35 (11%) patients whose isolates had discordant fingerprinting results, as compared with 40 of 51 (78%) of those whose isolates matched by both IS6110 and pTBN12. DNA fingerprinting with the IS6110 technique was not a specific marker of DNA clonality, particularly among isolates having fewer than five copies of IS6110. The use of a supplemental DNA fingerprinting technique decreased clustering and improved the correlation between the transmission links predicted by molecular techniques and epidemiologic investigation.

Expression profile of MDM-2 proteins in chronic lymphocytic leukemia and their clinical relevance.

The MDM-2 oncoprotein exists in an autoregulatory feedback loop with the tumor suppressor protein p53. Therefore, intracellular levels of these two proteins may play important roles in cell proliferation and tumorigenesis. Several MDM-2 proteins (Mr 35-100 Kd) have been demonstrated in human cell lines. We report here the expression profile of MDM-2 and p53 proteins in 87 cases of chronic lymphocytic leukemia (CLL) as detected by immunoblot analysis. The MDM-2 proteins (p57, p59, p67, and p90) were found to be overexpressed in different combinations in 56/87 (64%) of cases of CLL when compared with normal volunteers. The MDM-2 protein p57 was predominantly overexpressed 46/87 (53%) in CLL. In 22/87 (25%) cases of CLL p57 was overexpressed alone, and in 24/87 (28%) cases it was co-overexpressed with other MDM-2 proteins p59/p67/p90. Six of the 87 cases of CLL showed overexpression of the tumor suppressor protein p53 by immunoblot analysis, and five of those cases also co-overexpress MDM-2 protein p57. No statistically significant correlation of MDM-2 protein overexpression to clinical disease stage and history of previous chemotherapy of CLL patients has been found. However, considering the oncogenic potential of overexpressed MDM-2 proteins, a possible role of MDM-2 proteins in the promotion of CLL disease remains to be evaluated.

The incidence of false-positive cultures for Mycobacterium tuberculosis.

The frequency of false-positive cultures for Mycobacterium tuberculosis due to cross-contamination has been difficult to determine because of the lack of specific strain markers. Isolates collected prospectively over 5 yr from a municipal health department laboratory underwent DNA fingerprinting using the IS6110 and pTBN12 sequences. We reviewed the clinical and laboratory records of all isolates that had matching DNA fingerprints and were processed within 42 d of each other; 8 isolates were classified as probable or definite false-positives, representing 4.0% (8/199) of the culture-positive patients. A convenience sample of 42 isolates from three other mycobacterial laboratories also underwent DNA fingerprinting, and five (12%) were found to be definite or probable false-positives. Cross-contamination during initial processing of specimens was the most common source of false-positive cultures. The source of cross-contamination for three false-positive cultures was a laboratory proficiency survey specimen containing strain H37Ra. Ten of the 13 patients were misdiagnosed as having tuberculosis, and seven received unnecessary multidrug treatment. Clinicians should be aware of the potential for false-positive cultures for M. tuberculosis, and mycobacteriology laboratories need to carefully review procedures to minimize this occurrence. DNA fingerprinting provides a valuable tool for the study of false-positive cultures.

Differences in the prevalence of IS6110 insertion sites in Mycobacterium tuberculosis strains: low and high copy number of IS6110.

SETTING: Mycobacterium tuberculosis (M. tuberculosis) isolates from various parts of the USA which have few copies of the insertion sequence IS6110. OBJECTIVES: To characterize the sites of insertion of IS6110 among M. tuberculosis isolates that have one to six copies of the insertion sequence. DESIGN: The mixed-linker polymerase chain reaction (ML-PCR) procedure was used to amplify the terminal repeats on the ends of IS6110 and adjacent flanking sequences. From the ML-PCR products, sequences flanking 14 copies of IS6110 in strains containing less than seven copies of the insertion were determined. Sequence information from the flanking deoxyribonucleic acid was used to construct flanking primers that can be used to indicate the presence of IS6110 at a particular site when paired with outbound IS6110 primers in a PCR. Over 200 strains of diverse origin were screened for the insertion of IS6110 at several distinct sites using this procedure. RESULTS: The direct repeat (DR) locus has been described as a highly preferred site for insertion of IS6110 in strains of M. tuberculosis. Another highly preferred site of insertion of IS6100, DK1, is herein described. Insertions at DK1 are highly prevalent in M. tuberculosis strains harboring two to six copies of IS6110. The prevalence of insertions at this site decreases in strains with more than six copies of IS6110, even though the sequence itself is present in strains lacking a copy of IS6110 at this site. CONCLUSION: In addition to the DR locus there are other conserved sites of insertion among M. tuberculosis strains. The data further suggest a separate lineage for the high copy and the low copy strains, and a possible sequential insertion of IS6110 in strains of M. tuberculosis with less than seven copies.

Usefulness of the secondary probe pTBN12 in DNA fingerprinting of Mycobacterium tuberculosis.

A comparison was made between DNA fingerprints of Mycobacterium tuberculosis produced with the insertion sequence IS6110 and those produced with the polymorphic GC-rich repetitive sequence contained in the plasmid pTBN12. A total of 302 M. tuberculosis isolates from the prison system in Madrid, Spain, and the Denver Public Health Department (Denver, Colo.) were analyzed with the two probes. Both probes identified the same isolates in the same clusters when the fingerprints had six or more copies of IS6110. Analysis of isolates with unique IS6110 fingerprints demonstrated that they were unique with pTBN12. The pTBN12 probe had greater discriminating power in isolates having five or fewer copies of IS6110. Forty-seven isolates from Denver having fewer than five copies of IS6110 which were grouped in 11 clusters with identical fingerprint patterns were subdivided into 35 different patterns by pTBN12. Isolates with IS6110 fingerprints with more than six copies of IS6110 that differed from one another by only one or two hybridizing bands were analyzed with pTBN12. Most of these sets of isolates demonstrated identical patterns with pTBN12. However, some exceptions were observed, suggesting that those having nearly identical IS6110 patterns should not necessarily be included in the same cluster. Since IS6110 provides more polymorphism in the fingerprint, it is most useful in identifying isolates with unique fingerprint patterns and those in clusters in which the isolates contain six or more copies of the insertion. However, it is necessary to employ a secondary probe, such as pTBN12, to discriminate isolates with five or fewer copies of IS6110 and those with similar but not identical IS6110 patterns.