Ischemia but no obstructive coronary artery disease: more than meets the eye.

Symptomatic women with angina are more likely to have ischemia with no obstructive coronary arteries (INOCA) compared to men. In both men and women, the finding of INOCA is not benign and is associated with adverse cardiovascular events, including myocardial infarction, heart failure and angina hospitalizations. Women with INOCA have more angina and a lower quality of life compared to men, but they are often falsely reassured because of a lack of obstructive coronary artery disease (CAD) and a perception of low risk. Coronary microvascular dysfunction (CMD) is a key pathophysiologic contributor to INOCA, and non-invasive imaging methods are used to detect impaired microvascular flow. Coronary vasospasm is another mechanism of INOCA, and can co-exist with CMD, but usually requires invasive coronary function testing (CFT) with provocation testing for a definitive diagnosis. In addition to traditional heart disease risk factors, inflammatory, hormonal and psychological risk factors that impact microvascular tone are implicated in INOCA. Treatment of risk factors and use of anti-atherosclerotic and anti-anginal medications offer benefit. Increasing awareness and early referral to specialized centers that focus on INOCA management can improve patient-oriented outcomes. However, large, randomized treatment trials to investigate the impact on major adverse cardiovascular events (MACE) are needed. In this focused review, we discuss the prevalence, pathophysiology, presentation, diagnosis and treatment of INOCA.

Cold Pressor Testing and Sympathetic Nervous System Contribution to Ischemia with No Obstructive Coronary Artery Disease: Results from the Women's Ischemia Syndrome Evaluation-Coronary Vascular Dysfunction Project.

Study Objective: Cold Pressor Testing (CPT) is a known stimulus of the sympathetic nervous system (SNS). To better understand sympathetic contribution to coronary blood flow regulation in women with suspected ischemia and no obstructive coronary arteries (INOCA), we compared myocardial perfusion reserve during CPT stress cardiac magnetic resonance (CMR) imaging between women with suspected INOCA and reference subjects. Design: Prospective cohort. Setting: Academic hospital. Participants: 107 women with suspected INOCA and 21-age-matched reference women. Interventions: CPT stress CMR was performed with measurement of myocardial perfusion reserve index (MPRI), adjusted for rate pressure product (MPRIRPP). Invasive coronary function testing in a subset of INOCA women (n=42) evaluated for endothelial dysfunction in response to acetylcholine, including impaired coronary diameter response ≤0% and coronary blood flow response (ΔCBF) <50%. Main Outcome Measure: MPRIRPP. Results: Compared to reference women, the INOCA group demonstrated higher resting RPP (p=0.005) and CPT MPRIRPP (1.09±0.36 vs 0.83±0.18, p=0.002). Furthermore, INOCA women with impaired ΔCBF (n=23) had higher CPT MPRIRPP (p=0.044) compared to reference women despite lower left ventricular ejection fraction (64±7 % vs 69±2 %, p=0.005) and mass-to-volume ratio (0.79±0.15 vs 0.62±0.09, p<0.0001). These differences in CPT MPRIRPP did not persist after adjusting for age, body mass index, and history of hypertension. CPT MPRIRPP among INOCA women did not differ based on defined acetylcholine responses. Conclusions: Myocardial perfusion reserve to CPT stress is greater among women with INOCA compared to reference subjects. CPT induced a higher MPRIRPP also in women with coronary endothelial dysfunction, suggesting a greater contribution of the SNS to coronary flow than endothelial dysfunction. Further investigation in a larger cohort is needed.

Diagnosis of urogenital tuberculosis by multiplex-nested PCR targeting mpt64 (Rv1980c) and IS6110: comparison with multiplex PCR and GeneXpert® MTB/RIF.

A multiplex-nested PCR (M-nested PCR) targeting mpt64 (Rv1980c) + IS6110 was designed to detect Mycobacterium tuberculosis (Mtb) DNA within urine (n = 35), endometrial biopsies (n = 22) and menstrual blood (n = 3) of male/female UGTB patients, and results were compared with M-PCR using the same targets. Detection limit of the purified Mtb DNA was found to be 1 fg by M-nested PCR, which was 106 -fold lower than M-PCR. Moreover, sensitivities of 100% and 81·8% were obtained in confirmed (n = 5) and clinically suspected UGTB (n = 55) cases, respectively, by M-nested PCR, with a specificity of 97·1% (n = 70). Sensitivities attained by M-nested PCR were significantly higher (p < 0·05) than M-PCR in both clinically suspected and total UGTB (n = 60) cases. To confirm the true PCR-negative results, an internal amplification control, that is, human ß-globin gene (hbb) was incorporated in the M-nested PCR/M-PCR assays, wherein all the clinical specimens (positive/negative for mpt64/IS6110) were found to be positive for hbb. Some UGTB specimens (n = 35) were also subjected to GeneXpert® MTB/RIF assay that revealed a significantly lower (p < 0·001) sensitivity (17·1 vs 88·6%) than M-nested PCR, although high specificity (100%) was attained with GeneXpert. After validating the results in a higher number of UGTB specimens, our M-nested PCR may be translated into an attractive diagnostic kit.

Diagnosis of osteoarticular tuberculosis by immuno-PCR assay based on mycobacterial antigen 85 complex detection.

Diagnosis of osteoarticular tuberculosis (OATB) exhibits serious challenges owing to paucibacillary nature of specimens and localization of disease at sites that are difficult to access. We recently developed indirect immuno-PCR (I-PCR) and real-time I-PCR (RT-I-PCR) assays for the detection of mycobacterial antigen 85 complex (Ag85) in OATB patients. Detection limits for the purified Ag85 protein were found to be 1 and 41 fg ml-1 by I-PCR and RT-I-PCR, respectively, which were at least 105 -fold lower than respective ELISA. While spiking synovial fluids of non-TB control subjects with the purified Ag85 protein, LODs of 100 and 120 fg ml-1 were obtained by I-PCR and RT-I-PCR, respectively, thus demonstrating the sample matrix effect. Sensitivities of 87·5 and 70·5% were observed in bodily fluids of confirmed (n = 8) and clinically suspected (n = 51) OATB cases, respectively, by I-PCR, with a specificity of 93·9% (n = 33). Markedly, the sensitivities obtained by I-PCR/RT-I-PCR were significantly higher (P < 0·05-0·01) than ELISA and GeneXpert assay (n = 30). However, no substantial difference in sensitivity was observed between the I-PCR and RT-I-PCR assays. After further improving the accuracy of I-PCR, this test may lead to development of an attractive diagnostic kit.

Learning from UJAMBO: Perspectives on Gynecologic Care in African Immigrant and Refugee Women in Boston, Massachusetts.

African-born immigrant women, and particularly refugees and asylum seekers, are at risk for reproductive health disparities but inadequately use relevant gynecologic services. We sought to elucidate perspectives on gynecologic care in a population of Congolese and Somali immigrants. We conducted a secondary qualitative analysis of focus group data using a grounded theory approach and the Integrated Behavioral Model as our theoretical framework. Thirty one women participated in six focus groups. Participant beliefs included the states of pregnancy and/or pain as triggers for care, preferences included having female providers and those with familiarity with female genital cutting. Barriers included stigma, lack of partner support, and lack of resources to access care. Experiential attitudes, normative beliefs, and environmental constraints significantly mediated care preferences for/barriers to gynecologic health service utilization in this population. Centering of patient perspectives to adapt delivery of gynecologic care to immigrants and refugees may improve utilization and reduce disparities.

Purification and characterization of arylacetonitrile-specific nitrilase of Alcaligenes sp. MTCC 10675.

Arylacetonitrile-hydrolyzing nitrilase (E.C. 3.5.5.5) of Alcaligenes sp. MTCC 10675 has been purified by up to 46-fold to homogeneity and 32% yield using ammonium sulfate fractionation, Sephacryl S-300 gel permeation, and anion exchange chromatography. The molecular weight of the native enzyme was estimated to be 520 ± 60 kDa. The subunit has a molecular weight of 60 ± 14 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature of the purified enzyme were 6.5 and 50 °C, respectively. The purified arylacetonitrilase has a half-life of 3 H 20 Min at its optimum temperature. The value for Vmax, Km , kcat , and ki of enzyme for mandelonitrile as a substrate was 50 ± 05 µmol/Min/mg, 13 ± 02 mM, 26 ± 03 Sec(-) , and 32.4 ± 03 mM, respectively. Alcaligenes sp. MTCC 10675 arylacetonitrilase amino acid sequence has variations from other reported arylacetonitrilase, namely, A11G, N21H, D149N, S170T, P171R, S179A, Q180N, and S191A, and it has a high thermal stability and catalytic rate as compared with the already purified arylacetonitrilase.

Simultaneous purification of nitrile hydratase and amidase of Alcaligenes sp. MTCC 10674.

Alcaligenes sp. MTCC 10674 has a bienzymatic system for the hydrolysis of nitriles. The nitrile hydratase and amidase have been purified simultaneously to homogeneity using a combination of (NH)4SO4 precipitation, ion exchange chromatography and gel permeation chromatography. Nitrile hydratase and amidase have molecular weight of 47 and 114 kDa, respectively and exist as heterodimer. Optimum temperatures for maximum activity of nitrile hydratase and amidase were 15 °C (2.4 U/mg protein) and 45 °C (2.3 U/mg protein), respectively. Nitrile hydratase showed maximum 7.8 U/mg protein at 50 mM acrylonitrile and amidase has 9.2 U/mg protein at 25 mM propionamide. Nitrile hydratase has Vmax 10 µmol/min/mg and Km 40 mM, while amidase has Vmax 12.5 µmol/min/mg and Km 45.5 mM, respectively. Heavy metal ions Hg2+, Ag+, Pb2+ and Cu2+ were strong inhibitors of nitrile hydratase and amidase activity.

Optimization of arylacetonitrilase production from Alcaligenes sp. MTCC 10675 and its application in mandelic acid synthesis.

Alcaligenes sp. MTCC 10675 has been isolated from soil sample using enrichment method and has nitrilase catalytic system which is highly specific for the hydrolysis of arylaliphatic nitriles. Optimization of culture conditions using response surface methodology and inducer-mediated approach enhanced arylacetonitrilase production significantly (2.4-fold). Isobutyronitrile acted as an effective inducer for the induction of arylacetonitrilase, and it is highly specific for arylacetonitriles (phenyl acetonitrile and mandelonitrile). Arylacetonitrilase has no effect on its relative velocity (V r) up to 20 mM substrate (mandelonitrile) concentration and at 30 mM mandelonitrile, 23.4 % degree of inhibition (I d) was recorded. Half life of arylacetonitrilase of Alcaligenes sp. MTCC 10675 was 27.5 h at 25 °C. Hg(2+), Ag(+), Pb(3+), and Co(2+) were strong inhibitor of arylacetonitrilase activity which resulted into 100 %, 91 %, 84 %, and 83 % inhibition, respectively. Polar protic solvent (dichloromethane, dimethylsulphooxide, and n-butanol) reduce arylacetonitrilase activity up to 80-94 % at 10 % concentration. Alcaligenes sp. MTCC 10675 has higher biocatalytic activity, i.e., 3.9 gg(-1) dcw, which is highest in comparison to till reported organism. Arylacetonitrilase-mediated hydrolysis of racemic mandelonitrile resulted into R-(-) mandelic acid with 99.0 % enantiomeric excess (e.e.).

An isobutyronitrile-induced bienzymatic system of Alcaligenes sp. MTCC 10674 and its application in the synthesis of α-hydroxyisobutyric acid.

Alcaligenes sp. MTCC 10674 was isolated as acetone cyanohydrin hydrolyzing bacterium from soil of orchid gardens of Himachal Pradesh. Acetone cyanohydrin hydrolyzing activity of this organism comprised nitrile hydratase and amidase activities. It exhibited higher substrate specificity towards aliphatic hydroxynitrile (acetone cyanohydrin) in comparison to arylaliphatic hydroxynitrile. Isobutyronitrile (40 mM) acted as a carbon source as well as inducer for growth of Alcaligenes sp. MTCC 10674 and expression of acetone cyanohydrin hydrolyzing activity. Optimization of culture condition using response surface methodology increased acetone cyanohydrin hydrolyzing activity by 1.3-fold, while inducer mediation approach increased the activity by 1.2-fold. The half life of this enzyme was 25 h at 15 °C. V max and K m value for acetone cyanohydrin hydrolyzing enzyme was 0.71 µmol mg(-1) min(-1) and 14.3 mM, when acetone cyanohydrin was used as substrate. Acetone cyanohydrin hydrolyzing enzyme encountered product inhibition and IC50 and K i value were calculated to be 28 and 10.2 mM, respectively, when product α-hydroxyisobutyric acid was added in the reaction. Under optimized reaction conditions at 40 ml fed batch scale, 3 mg dcw ml (-) resting cells of Alcaligenes sp. MTCC 10674 fully converted 0.33 M acetone cyanohydrin into α-hydroxyisobutyric acid (1.02 g) in 6 h 40 min. The characterization of acetone cyanohydrins hydrolyzing activity revealed that it comprises bienzymatic nitrile hydrolyzing system, i.e. nitrile hydratase and amidase for the production of α-hydroxyisobutyric acid from acetone cyanohydrin and maximum 70 % yield is being reported for the first time.

Acute coronary syndrome as a first presentation of systemic lupus erythematosus in a teenager: revascularization by hybrid coronary artery bypass graft surgery and percutaneous coronary intervention: case report.

Patients with systemic lupus erythematosus (SLE) may present with acute coronary syndrome (ACS) due to coronary vasculitis or premature atherosclerosis. There is a paucity of data on invasive management strategies for young adults who present with an ACS secondary to active vasculitis. This article describes the case of a teenager who presented with an ACS secondary to lupus vasculitis as his first presentation of active SLE. Coronary angiography showed a left main equivalent lesion involving a proximal very large left anterior descending artery (LAD) and diagonal stenosis (with a diminutive left circumflex artery). The boy underwent a successful endoscopic coronary bypass surgery to his LAD followed by percutaneous coronary intervention to his diagonal artery. This case demonstrates the feasibility and safety of a hybrid coronary revascularization in a teenager with acute coronary syndrome due to coronary vasculitis.

Coronary heart disease in women: battle is won, but the war remains.

According to the most recent report of the US National Heart, Lung, and Blood Institute, mortality from coronary heart disease has declined in women from one in three to one in four. Due to massive campaigning efforts in educating the medical community and the general public, coronary heart disease has become increasingly recognized as a woman's disease. Indeed, it is the number one killer in women, exceeding cancer and infectious diseases. Numerous observational studies, clinical trials, and reports have indicated that there are gender-specific differences in the presentation, diagnosis, treatment, and outcomes of coronary heart disease. One common theme, not only in United States, but world-wide is the underutilization of known and validated medical and interventional therapies in women compared to men. Even though previously conducted large, randomized controlled trials had limited numbers of women, recent large scale cardiac trials in women have enabled the development of evidence-based guidelines for coronary heart disease diagnosis and management in women. Importantly, menopausal hormone therapy and antioxidant vitamin therapy do not protect post-menopausal women from coronary heart disease. Aggressive life-style and pharmacologic management of known coronary risk factors in women should be a top priority to improve coronary heart disease morbidity and mortality. Research data continue to emerge to fill the gaps of how gender affects atherosclerosis; in the meantime, continued patient and physician education to increase awareness of coronary heart disease may help to eliminate some of the gender-based disparities in the delivery of coronary care to women.

Molecular emergence of acute myeloid leukemia during treatment for acute lymphoblastic leukemia.

Therapy-related acute myeloid leukemias (t-AML) with translocations of the MLL gene are associated with the use of topoisomerase II inhibitors. We established the emergence of the malignant clone in a child who developed t-AML with a t(11;19) (q23;p13.3) during treatment for acute lymphoblastic leukemia (ALL). The MLL-ENL and the reciprocal ENL-MLL genomic fusions and their chimeric transcripts were characterized from samples collected at the time of t-AML diagnosis. We used PCR with patient-specific genomic primers to establish the emergence of the MLL-ENL fusion in serially obtained DNA samples. The MLL-ENL fusion was not detectable in bone marrow at the time of ALL diagnosis or after 2 months of chemotherapy (frequency <8.3 x 10(-7) cells(-1)). The genomic fusion was first detected in bone marrow after 6 months of treatment at a frequency of one in 4,000 mononuclear bone marrow cells; the frequency was one in 70 cells after 20 months of therapy. At the first detection of MLL-ENL, the only topoisomerase II inhibitors the patient had received were one dose of daunorubicin and two doses of etoposide. The MLL-ENL fusion was not detectable in blood at the time of ALL diagnosis or after 0.7, 2, 8, 10, and 12 months of therapy but was detectable in blood at 16 months (one in 2.3 x 10(4) cells). Recombinogenic Alu sequences bracketed the breakpoints in both fusions. These data indicate that the malignant clone was not present before therapy, arose early during chemotherapy, and was able to proliferate even during exposure to antileukemic therapy.

Fusion of two novel genes, RBM15 and MKL1, in the t(1;22)(p13;q13) of acute megakaryoblastic leukemia.

t(1;22) is the principal translocation of acute megakaryoblastic leukemias. Here we show this chromosomal rearrangement to result in the fusion of two novel genes, RNA-binding motif protein-15 (RBM15), an RNA recognition motif-encoding gene with homology to Drosophila spen, and Megakaryoblastic Leukemia-1 (MKL1), a gene encoding an SAP (SAF-A/B, Acinus and PIAS) DNA-binding domain.

From cofactor to enzymes. The molecular evolution of pyridoxal-5'-phosphate-dependent enzymes.

The pyridoxal-5'-phosphate (vitamin B(6))-dependent enzymes that act on amino acid substrates have multiple evolutionary origins. Thus, the common mechanistic features of B(6) enzymes are not accidental historical traits but reflect evolutionary or chemical necessities. The B(6) enzymes belong to four independent evolutionary lineages of paralogous proteins, of which the alpha family (with aspartate aminotransferase as the prototype enzyme) is by far the largest and most diverse. The considerably smaller beta family (tryptophan synthase beta as the prototype enzyme) is structurally and functionally more homogenous. Both the D-alanine aminotransferase family and the alanine racemase family consist of only a few enzymes. The primordial pyridoxal-5'-phosphate-dependent protein catalysts apparently first diverged into reaction-specific protoenzymes, which then diverged further by specializing for substrate specificity. Aminotransferases as well as amino acid decarboxylases are found in two different evolutionary lineages, providing examples of convergent enzyme evolution. The functional specialization of most B(6) enzymes seems to have already occurred in the universal ancestor cell before the divergence of eukaryotes, archebacteria, and eubacteria 1500 million years ago. Pyridoxal-5'-phosphate must have emerged very early in biological evolution; conceivably, metal ions and organic cofactors were the first biological catalysts. To simulate particular steps of molecular evolution, both the substrate and reaction specificity of existent B(6) enzymes were changed by substitution of active-site residues, and monoclonal pyridoxal-5'-phosphate-dependent catalytic antibodies were produced with selection criteria that might have been operative in the evolution of protein-assisted pyridoxal catalysis.

The molecular evolution of pyridoxal-5'-phosphate-dependent enzymes.

The pyridoxal-5-phosphate-dependent enzymes (B6 enzymes) that act on amino acid substrates are of multiple evolutionary origin. The numerous common mechanistic features of B6 enzymes thus are not historical traits passed on from a common ancestor enzyme but rather reflect evolutionary or chemical necessities. Family profile analysis of amino acid sequences supported by comparison of the available three-dimensional (3-D) crystal structures indicates that the B6 enzymes known to date belong to four independent evolutionary lineages of homologous (or more precisely paralogous) proteins, of which the alpha family is by far the largest. The alpha family (with aspartate aminotransferase as the prototype enzyme) includes enzymes that catalyze, with several exceptions, transformations of amino acids in which the covalency changes are limited to the same carbon atom that carries the amino group forming the imine linkage with the coenzyme (i.e., Calpha in most cases). Enzymes of the beta family (tryptophan synthase beta as the prototype enzyme) mainly catalyze replacement and elimination reactions at Cbeta. The D-alanine aminotransferase family and the alanine racemase family are the two other independent lineages, both with relatively few member enzymes. The primordial pyridoxal-5-phosphate-dependent enzymes apparently were regio-specific catalysts that first diverged into reaction-specific enzymes and then specialized for substrate specificity. Aminotransferases as well as amino acid decarboxylases are found in two different evolutionary lineages. Comparison of sequences from eukaryotic, archebacterial, and eubacterial species indicates that the functional specialization of most B6 enzymes has occurred already in the universal ancestor cell. The cofactor pyridoxal-5-phosphate must have emerged very early in biological evolution; conceivably, organic cofactors and metal ions were the first biological catalysts. In attempts to stimulate particular steps of molecular evolution, oligonucleotide-directed mutagenesis of active-site residues and directed molecular evolution have been applied to change both the substrate and reaction specificity of existent B6 enzymes. Pyridoxal-5-phosphate-dependent catalytic antibodies were elicited with a screening protocol that applied functional selection criteria as they might have been operative in the evolution of protein-assisted pyridoxal catalysis.

Rates of evolution of pyridoxal-5'-phosphate-dependent enzymes.

The pyridoxal-5'-phosphate-dependent enzymes (B(6) enzymes), that operate in the metabolism of amino acids, are of multiple evolutionary origin. To estimate their rates of evolution, a total of 180 sequences of 21 B(6) enzymes from distantly related eukaryotic species were compared. The enzymes belong to all four evolutionarily independent families of B(6) enzymes with different folds, i.e., the large alpha family, the beta family, the d-alanine aminotransferase family, and the alanine racemase family. Their unit evolutionary periods, i.e., the time for a 1% sequence difference to accumulate between branches, ranged from 4.6 to 45.1 million years. Both, fastest changing serine pyruvate aminotransferase and most slowly changing glutamate decarboxylase are members of the alpha family. The evolutionary rates of the few enzymes belonging to the other three families were interspersed among those of the alpha family members. Enzymes that catalyze the same reaction, e.g., transamination or amino acid decarboxylation, with different substrates show widely varying rates. The absence of correlations of the rate of evolution with either protein fold or type of catalyzed reaction suggests that individual functional constraints have determined the differential rates of evolution of B(6) enzymes.

Recognizing very distant sequence relationships among proteins by family profile analysis.

Family profile analysis (FPA), described in this paper, compares all available homologous amino acid sequences of a target family with the profile of a probe family while conventional sequence profile analysis (Gribskov M, Lüthy R, Eisenberg D. Meth Enzymol 1990;183:146-159) considers only a single target sequence in comparison with the probe family. The increased input of sequence information in FPA expands the range for sequence-based recognition of structural relationships. In the FPA algorithm, Zscores of each of the target sequences, obtained from a probe profile search over all known amino acid sequences, are averaged and then compared with the scores for sequences of 100 reference families in the same probe family search. The resulting F-Zscore of the target family, expressed in "effective standard deviations" of the mean Zscores of the reference families, with value above a threshold of 3.5 indicates a statistically significant evolutionary relationship between the target and probe families. The sensitivity of FPA to sequence information was tested with several protein families where distant relationships have been verified from known tertiary protein architectures, which included vitamin B6-dependent enzymes, (beta/alpha)8-barrel proteins, beta-trefoil proteins, and globins. In comparison to other methods, FPA proved to be significantly more sensitive, finding numerous new homologies. The FPA technique is not only useful to test a suspected relationship between probe and target families but also identifies possible target families in profile searches over all known primary structures.

Bacterial selenocysteine synthase--structural and functional properties.

Selenocysteine synthase from Escherichia coli is a pyridoxal-5'-phosphate-containing enzyme which catalyses the conversion of seryl-tRNA(Sec) into selenocysteyl-tRNA(Sec). Analysis of amino acid sequences indicated that selenocysteine synthase belongs to the alpha/gamma superfamily of pyridoxal-5'-phosphate-dependent enzymes. To identify the lysine residue carrying the prosthetic group, the genes coding for the selenocysteine synthases from Moorella thermoacetica and Desulfomicrobium baculatum were cloned and sequenced and their derived amino acid sequences were aligned with those from E. coli and Haemophilus influenzae. Three lysine residues were found to be conserved; they were mutated into asparagine and one of them, Lys295, was found to be essential for activity. Proteolytic fragmentation of the E. coli enzyme reduced with borohydride, and mass-spectrometric and sequence analysis of the chromophoric peptide proved that Lys295 was modified. Kinetic analysis of the enzyme showed that thiophosphate served as a substrate leading to cysteyl-tRNA(Sec) synthesis, albeit with a 330-fold lower catalytic efficiency. Selenide and, to a much lesser degree, sulfide could also be used by the enzyme but only at much higher concentrations. These data together with the finding that selenophosphate synthetase is highly specific for selenide indicate that the phosphate moiety of selenophosphate provides selenocysteine synthase with the discrimination specificity against sulfur.

Comparison of in vitro models for the study of Mycobacterium tuberculosis invasion and intracellular replication.

We recently evaluated several tissue culture model systems for the study of invasion and intracellular multiplication of Mycobacterium tuberculosis. These model systems include a human alveolar pneumocyte epithelial cell line, a murine macrophage cell line (J774), and fresh human peripheral blood-derived macrophages. Our data indicated that the initial level of association of M. tuberculosis with human alveolar pneumocyte cells (2%) was less than that observed with fresh human peripheral blood macrophages (9%) or J774 murine macrophages (13%) within 6 h of the addition of the bacteria. M. tuberculosis replicated in association with the pneumocyte cells by more than 55-fold by day 7 postinfection. In contrast, total bacteria] growth in the J774 cells and human macrophages was considerably less, with increases of only fourfold and threefold, respectively, over the same 7-day period. Amikacin, an aminoglycoside antimicrobial agent, was added to inhibit the growth of extracellular bacteria after the initial 6-h infection period. Decreases in viable counts were observed in all three cell cultures within the first 3 days after infection. However, unlike the case with either macrophage culture, intracellular bacterial CFU obtained from the infected pneumocytes increased by fourfold by day 7 after the addition of amikacin. These data indicate that M. tuberculosis infects and multiplies intracellularly in human lung epithelial cells and that these cells may be an alternative in vitro model for the study of intracellular multiplication of M. tuberculosis in the human lung.