Virulent Tuberculosis Research Articles

Studying the tumor microenvironment under pressure

Meenal Datta is an assistant professor in the Department of Aerospace and Mechanical Engineering at the University of Notre Dame. Prof Datta received her PhD in Chemical and Biological Engineering from Tufts University in 2018, after which she completed a postdoctoral fellowship at Harvard Medical School and Massachusetts General Hospital. Her research focuses on deciphering the atypical tumor microenvironment that drives disease progression and treatment resistance in incurable cancers. By understanding and overcoming the biological, chemical, electrical, and mechanical abnormalities found in solid tumors, new therapeutic approaches can be discovered. Prof. Datta specializes in multidisciplinary and mechanism-based preclinical research that has the potential to be rapidly translated to improve treatment approaches in the clinic. She has spent her time as a researcher deciphering and reprogramming abnormal tissue microenvironments that present in a variety of diseases ranging from virulent tuberculosis to benign schwannoma to deadly glioblastoma that, surprisingly, share unifying features: abnormal blood vessels, abundant extracellular matrix, immunosuppression, and mechanopathologies. During her PhD, Dr Datta normalized the aberrant blood vasculature found in pulmonary tuberculosis granulomas to improve drug delivery. In her postdoctoral training, Dr Datta re-engineered the immunosuppressive brain tumor microenvironment to improve glioblastoma response to immunotherapy. As the director of the Tumor Immune Microenvironment & Mechanics Lab at Notre Dame (the TIME lab). Prof Datta’s research group is applying engineering fundamentals and problem-solving approaches to explore mechano-immunological phenomena in the tumor microenvironment and discover novel biophysical targets of interest.

EVALUATION OF THE ANTI-MYCOBACTERIAL, ANTI-OXIDATIVE AND ANTI-PROLIFERATIVE ACTIVITIES OF RUBIA CORDIFOLIA ETHANOLIC LEAF EXTRACT SUB-FRACTIONS IN HUMAN LYMPHOCYTES AND NEUTROPHILS

Background. Rubia cordifolia has been used to treat various diseases and exhibits antimicrobial, anti-inflammatory,
 analgesic and antidiarrheal activities. The plant has not been investigated for its anti-mycobacterial activity against
 virulent tuberculosis strains as well as cytotoxic and anti-oxidant activities in mononuclear cells and neutrophils.
 Methods. Ethanolic and dichloromethane leaf extract fractions of Rubia cordifolia (0.2- 125μg/ml) were screened for
 anti-mycobacterial activity using a fluorescent microplate assay. The anti-oxidant activity of the fractions was tested
 using the radical scavenging assay, while their cytotoxicity to lymphocytes was measured using the WST-8 assay.
 Reactive oxygen species production by neutrophils exposed to fractions was tested using Cell Meter™ Fluorimetric
 ROS Assay kit.
 Results. The ethanolic leaf fractions exhibited overall superior anti-mycobacterial activity than the dichloromethane
 group and were further screened for anti-oxidant and cytotoxic activities. Ethanolic sub-fractions K2F-3.2, K2F-3.3 and
 K2F-3.4 showed significant antioxidant activities at concentration of 50 μg/ml to 200 μg/ml and higher in both the cell
 free-based radical scavenging activity and total reactive oxygen species production assays. For lymphocytes, the test
 agents showed anti-proliferative activity at 25 μg/ml to 200 μg/ml for sub-fraction K2F-3.3B and 50 μg/ml to 200
 μg/ml for agents K2F-3.2 and K2F-3.4.
 Conclusion. The current study is the first to record the in vitro anti-mycobacterial, anti-oxidant and cytotoxic activities
 of Rubia cordifolia plant leaf extract sub-fractions using the given experimental setups and further research activities to
 identify the bioactive components are to be pursued.

Regulation of matrix metalloproteinase‐1, ‐3, and ‐9 inMycobacterium tuberculosis‐dependentrespiratory networks by the rapamycin‐sensitive PI3K/p70S6Kcascade

This study was designed to investigate the role of the phosphatidyl inositol 3-kinase (PI3K)/AKT/p70(S6K) signaling path on regulation of primary normal human bronchial epithelial cell-derived matrix metalloproteinase (MMP)-1, -3, and -9 expression in tuberculosis (TB). These MMPs are key in pathological extracellular matrix degradation in TB. Normal human bronchial epithelials were stimulated with conditioned medium from monocytes infected with virulent TB (CoMTb) and components of the PI3K/AKT signaling pathway blocked using specific chemical inhibitors and siRNA. MMP gene expression was measured by RT-PCR and secretion by ELISA, luminex, or zymography. Phospho-p70 S6K was detected by Western blot analysis and activity blocked by rapamycin. Chemical blockade of the proximal catalytic PI3K p110 subunit augmented MMP-1 and MMP-9 in a dose-dependent manner (all P<0.001) but suppressed MMP-3 (P<0.01). Targeted siRNA studies identified the p110α isoform as key causing 5-fold increase in TB network-dependent MMP-1 secretion to 4900 ± 1100 pg/ml. Specific inhibition of the AKT node suppressed all 3 MMPs. Phospho-p70(S6K) was identified in the cellular model, and rapamycin, a p70(S6K) inhibitor, inhibited MMP-1 (P<0.001) and MMP-3 (P<0.01) but not MMP-9. Controls were epithelial cells that were unstimulated or exposed to conditioned medium from monocytes not exposed to TB. In summary, blockade of the proximal PI3K catalytic subunit increases MMP-1 and MMP-9, whereas rapamycin decreased both MMP-1 and MMP-3. The regulation of the PI3K path in TB is complex, MMP specific, and a potential immunotherapeutic target in diseases characterized by tissue destruction.

Inhibition of TNF‐α‐induced Cyclooxygenase‐2 Expression by Mycobacterium bovis BCG in Human Alveolar Epithelial A549 Cells

Cyclooxygenase-2 (COX-2) is implicated in pathophysiological processes associated with the initiation or maintenance of host inflammatory responses to infection. Our results demonstrates that Mycobacterium bovis BCG (M. bovis BCG) downregulates tumour necrosis factor-alpha (TNF-alpha)-induced COX-2 gene expression in alveolar epithelial cells by inhibiting the phosphorylations of Raf-1 and p38 kinases. Further, M. bovis BCG-mediated inhibition of COX-2 or p38 mitogen-activated protein kinase could be reversed by Calyculin A, a selective inhibitor of Ser/Thr phosphatases. Moreover, M. bovis BCG inhibited the TNF-alpha-triggered NF-kappaB activation following IkappaB degradation. Taken together, these results suggest that the attenuation of COX-2 expression by vaccine strain, M. bovis BCG, represents a novel strategy to maintain robust host proinflammatory responses to subsequent challenges with virulent tuberculosis bacilli.

Reagentless Detection of Mycobacteria tuberculosis H37Ra in Respiratory Effluents in Minutes

Two similar mycobacteria, Mycobacteria tuberculosis H37Ra and Mycobacteria smegmatis are rapidly detected and identified within samples containing a complex background of respiratory effluents using single-particle aerosol mass spectrometry (SPAMS). M. tuberculosis H37Ra (TBa), an avirulent strain, is used as a surrogate for virulent tuberculosis; M. smegmatis (MSm) is utilized as a near-neighbor confounder for TBa. Bovine lung surfactant and human exhaled breath condensate are used as first-order surrogates for infected human lung expirations from patients with pulmonary tuberculosis. This simulated background sputum is mixed with TBa or MSm and nebulized to produce conglomerate aerosol particles, single particles that contain a bacterium embedded within a background respiratory matrix. Mass spectra of single conglomerate particles exhibit ions associated with both respiratory effluents and mycobacteria. Spectral features distinguishing TBa from MSm in pure and conglomerate particles are shown. SPAMS pattern matching alarm algorithms are able to distinguish TBa-containing particles from background matrix and MSm for >50% of the test particles, which is sufficient to enable a high probability of detection and a low false alarm rate if an adequate number of such particles are present. These results indicate the potential usefulness of SPAMS for rapid, reagentless tuberculosis screening.

Effect of mycobacteria on sensitivity to the cytotoxic effects of tumor necrosis factor

Unlike Mycobacterium leprae, Mycobacterium tuberculosis is not found inside cells other than macrophages and polymorphonuclear cells in vivo, yet previous work has revealed that in vitro it readily enters all cell lines tested. Moreover, these cells are not killed by the intracellular mycobacteria. We report here that when fibroblasts take up live (but not killed) M. tuberculosis H37Rv, they develop greatly increased sensitivity to the toxic effects of tumor necrosis factor (TNF) whether the cell line is inherently sensitive to TNF or not. Ultrasonically disrupted M. tuberculosis also has this property. The increased sensitivity is seen in the absence of metabolic inhibitors, although addition of emetine, an inhibitor of protein synthesis, causes the effect to manifest itself earlier and at a lower concentration of TNF. In contrast, infection with Mycobacterium bovis bacillus Calmette-Guérin induces little or no increased sensitivity to TNF, whereas Mycobacterium avium and M. tuberculosis H37Ra have intermediate sensitivities. We discuss the possibility that virulent tuberculosis strains produce a factor which distorts the normal protective function of TNF, rendering it toxic to host tissues and leading to the classical immunopathology of tuberculous lesions.

Induction of nonspecific acquired resistance and delayed-type hypersensitivity, but not specific acquired resistance in mice inoculated with killed mycobacterial vaccines.

A number of nonliving mycobacterial preparations were tested in vivo for their capacity to generate various relevant parameters of cellular immunity. All preparations tested had some detectable activity in raising resistance to challenge with Mycobacterium tuberculosis or with Listeria monocytogenes and in conferring the ability to mount a delayed-type hypersensitivity response to tuberculin. This report presents the first evidence, however, that none of these preparations were able to generate protective T cells capable of adoptive immunization against virulent tuberculosis. These data are discussed in terms of the use of these preparations in generating M. tuberculosis-reactive T-cell lines and the application of these lines in the continuing search for an improved vaccine against tuberculosis.

Ammonia inhibits phagosome-lysosome fusion in macrophages.

When foreign bodies, including many microorganisms, are ingested by cultured macrophages, they become enclosed in phagosomes, with which lysosomes usually fuse and then discharge their enzymes and other contents into the resulting phagolysosomes. Such fusion is, however, diminished or absent after the phagocytosis of some pathogens, notably Mycobacterium tuberculosis and Toxoplasma gondii. Assuming that the nonfusion is due to active inhibition by the intrapoagosomal microbe, identification of an inhibitor should clarify the lysosomal control mechanism. It has been suggested that strongly acidic sulphatides present in virulent tuberculosis, which, like other substances with polyanionic structural features, can themselves block phagosome-lysosome fusion (P-LF), may contribute to the negative lysosome response to ingested tubercle bacilli. We report here another possibility, based on inhibition of fusion of yeast-containing phagosomes by filtrates from cultures of tubercle bacilli on traditional-type defined media; we show that the ammonia content of such filtrates is sufficient to account for their effect. This inhibition of fusion seems to be an hitherto unrecognized intracellular consequence of added ammonia, in striking contrast to the enhancement produced by some lipophilic amines.

The possible role of ribonucleic acid in the mechanism of resistance to tuberculosis

The ribonucleic acid level in the lungs and liver of albino mice and guinea pigs proved to increase considerably under the effect of immunization with the BCG vaccine. Infection with a virulent tuberculosis strain provoked a considerable rise of the ribonucleic acid concentration in the lungs of albino mice relatively resistant to tuberculosis, whereas the organs of guinea pigs sensitive to tuberculosis exhibited a normal range of variations in the ribonucleic acid level. It is suggested that the rise of the ribonucleic acid level in the organs of the immunized animals and in the lungs of the infected mice is connected with the mechanism of artificial immunity and natural resistance to tuberculosis.

THE BRAZILIAN EXPERIENCE IN PREVENTION OF TUBERCULOSIS WITH A CONCURRENT METHOD OF BCG VACCINATION

The BCG-Moreau Strain, which has been experimentally studied and used in the vaccination against tuberculosis in Brazil for 30 years, is tolerated so well by human beings that it can be given orally in repeated high doses to individuals in any allergic condition, inclusive of tuberculosis, without any inconvenience and without the glandular enlargements produced by administration of the other BCG strains by mouth. When the BCG-Moreau vaccine is prepared from 10-day cultures and its vitality is properly preserved, a single dose of 100 mg to newborn infants and 200 mg to other individuals with negative tuberculin tests confers cutaneous tuberculin allergy in more than 97% of the cases; so, the oral dose is equivalent to the parenteral methods of BCG vaccination as to the postvaccinal allergization. The protection so conferred against active tuberculosis reduces mortality and morbidity from tuberculosis to levels six and eight times lower. The fortnightly or monthly repetition of such doses until reaching a total of 600 mg (newborns) or 1200 mg (other individuals), i.e., the concurrent BCG vaccination, does not produce lymphadenitis, and generally makes the post vaccinal tuberculin allergy disappear; but the specific protection conferred by the single dose against the forms of the virulent tuberculosis is brought to a maximum by it. The inhibitory effects of the concurrent BCG vaccination can be also observed in patients who continue to be exposed to contagion; these present very low morbidity and mortality from tuberculosis also. The newborn infants who received concurrent BCG vaccination, even those who live with tubercubous people, have presented low morbidity and mortality from tuberculosis since 11 years of age when they were vaccinated; they have not received any additional dose of vaccine. In the individuals vaccinated with concurrent BCG, virulent super-infections frequently cause exacerbation of the tuberculin allergy, but rarely provoke roentgenographic pulmonary signs coinciding with hyperergy, and exceptionally determine progredient tuberculosis on guinea pigs inoculated with gastric contents. Few deaths of children vaccinated with concurrent BCG have been observed; all of them occurred in very infective places ("favelas"). When presenting positive tuberculin tests, children, adolescents and adults tolerate, without any inconvenience, repeated BCG-Moreau doses of 200 to 400 mg, at weekly, fortnightly or monthly intervals, even a total of several 10 gm doses of the vaccinegerm; these vaccinations determine a variable frequency of deallergization coinciding with a manifest increase in the specific resistance against tuberculosis. Children who contracted primary infections of tuberculosis tolerate oral BCG-Moreau vaccination perfectly, even if high doses are repeated; the vaccine gives them variable deallergization, and their specific resistance against the disease is always increased; it is no matter whether they are treated with antibiotics or not. The same facts are being observed regarding the tuberculous reinfection of adults. These data indicate that the original idea of prevention "e sensu strictiori" by the BCG has yielded ground to the concept of true immunization with live bacilli. In the course of this immunization there is both exaltation of the specific resistance (which can reach complete immunity) and attenuation, sometimes extinction, of the allergy which is created at first.