Mechanisms of Intracellular Communication in Cancer and Pathogen Spreading.

Cell-to-cell interactions are essential for proper development, homeostasis, and complex syncytia/organ formation and function. Intercellular communication are mediated by multiple mechanisms including soluble mediators, adhesion molecules and specific mechanisms of cell to cell communication such as Gap junctions (GJ), tunneling nanotubes (TNT), and exosomes. Only recently, has been discovered that TNTs and exosomes enable the exchange of large signaling molecules, RNA, viral products, antigens, and organelles opening new avenues of research and therapeutic approaches. The focus of this review is to summarize these recent findings in physiologic and pathologic conditions.

The hypoxia-regulated ectonucleotidase CD73 is a host determinant of HIV latency.

Deciphering the mechanisms underlying viral persistence is critical to achieving a cure for human immunodeficiency virus (HIV) infection. Here, we implement a systems approach to discover molecular signatures of HIV latently infected CD4+ T cells, identifying the immunosuppressive, adenosine-producing ectonucleotidase CD73 as a key surface marker of latent cells. Hypoxic conditioning, reflecting the lymphoid tissue microenvironment, increases the frequency of CD73+ CD4+ T cells and promotes HIV latency. Transcriptomic profiles of CD73+ CD4+ T cells favor viral quiescence, immune evasion, and cell survival. CD73+ CD4+ T cells are capable of harboring a functional HIV reservoir and reinitiating productive infection ex vivo. CD73 or adenosine receptor blockade facilitates latent HIV reactivation in vitro, mechanistically linking adenosine signaling to viral quiescence. Finally, tissue imaging of lymph nodes from HIV-infected individuals on antiretroviral therapy reveals spatial association between CD73 expression and HIV persistence in vivo. Our findings warrant development of HIV-cure strategies targeting the hypoxia-CD73-adenosine axis.

The role of Pannexin-1 channels, ATP, and purinergic receptors in the pathogenesis of HIV and SARS-CoV-2.

Infectious agents such as human immune deficiency virus-1 (HIV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) use host proteins to infect, replicate, and induce inflammation within the host. A critical component of these diseases is the axis between pannexin-1 channels, extracellular ATP, and purinergic receptors. Here, we describe the potential therapeutic role of Pannexin-1/purinergic approaches to prevent or reduce the devastating consequences of these pathogens.

The role of tunneling nanotubes during early stages of HIV infection and reactivation: implications in HIV cure.

Tunneling nanotubes (TNTs), also called cytonemes or tumor microtubes, correspond to cellular processes that enable long-range communication. TNTs are plasma membrane extensions that form tubular processes that connect the cytoplasm of two or more cells. TNTs are mostly expressed during the early stages of development and poorly expressed in adulthood. However, in disease conditions such as stroke, cancer, and viral infections such as HIV, TNTs proliferate, but their role is poorly understood. TNTs function has been associated with signaling coordination, organelle sharing, and the transfer of infectious agents such as HIV. Here, we describe the critical role and function of TNTs during HIV infection and reactivation, as well as the use of TNTs for cure strategies.

Glucuronoxylomannan intranasal challenge prior to Cryptococcus neoformans pulmonary infection enhances cerebral cryptococcosis in rodents.

The encapsulated fungus Cryptococcus neoformans is the most common cause of fungal meningitis, with the highest rate of disease in patients with AIDS or immunosuppression. This microbe enters the human body via inhalation of infectious particles. C. neoformans capsular polysaccharide, in which the major component is glucuronoxylomannan (GXM), extensively accumulates in tissues and compromises host immune responses. C. neoformans travels from the lungs to the bloodstream and crosses to the brain via transcytosis, paracytosis, or inside of phagocytes using a "Trojan horse" mechanism. The fungus causes life-threatening meningoencephalitis with high mortality rates. Hence, we investigated the impact of intranasal exogenous GXM administration on C. neoformans infection in C57BL/6 mice. GXM enhances cryptococcal pulmonary infection and facilitates fungal systemic dissemination and brain invasion. Pre-challenge of GXM results in detection of the polysaccharide in lungs, serum, and surprisingly brain, the latter likely reached through the nasal cavity. GXM significantly alters endothelial cell tight junction protein expression in vivo, suggesting significant implications for the C. neoformans mechanisms of brain invasion. Using a microtiter transwell system, we showed that GXM disrupts the trans-endothelial electrical resistance, weakening human brain endothelial cell monolayers co-cultured with pericytes, supportive cells of blood vessels/capillaries found in the blood-brain barrier (BBB) to promote C. neoformans BBB penetration. Our findings should be considered in the development of therapeutics to combat the devastating complications of cryptococcosis that results in an estimated ~200,000 deaths worldwide each year.

COVID-19 Lung Pathogenesis in SARS-CoV-2 Autopsy Cases.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major public health issue. COVID-19 is considered an airway/multi-systemic disease, and demise has been associated with an uncontrolled immune response and a cytokine storm in response to the virus. However, the lung pathology, immune response, and tissue damage associated with COVID-19 demise are poorly described and understood due to safety concerns. Using post-mortem lung tissues from uninfected and COVID-19 deadly cases as well as an unbiased combined analysis of histology, multi-viral and host markers staining, correlative microscopy, confocal, and image analysis, we identified three distinct phenotypes of COVID-19-induced lung damage. First, a COVID-19-induced hemorrhage characterized by minimal immune infiltration and large thrombus; Second, a COVID-19-induced immune infiltration with excessive immune cell infiltration but no hemorrhagic events. The third phenotype correspond to the combination of the two previous ones. We observed the loss of alveolar wall integrity, detachment of lung tissue pieces, fibroblast proliferation, and extensive fibrosis in all three phenotypes. Although lung tissues studied were from lethal COVID-19, a strong immune response was observed in all cases analyzed with significant B cell and poor T cell infiltrations, suggesting an exhausted or compromised immune cellular response in these patients. Overall, our data show that SARS-CoV-2-induced lung damage is highly heterogeneous. These individual differences need to be considered to understand the acute and long-term COVID-19 consequences.

Tunneling nanotubes, TNT, communicate glioblastoma with surrounding non-tumor astrocytes to adapt them to hypoxic and metabolic tumor conditions.

Cell-to-cell communication is essential for the development and proper function of multicellular systems. We and others demonstrated that tunneling nanotubes (TNT) proliferate in several pathological conditions such as HIV, cancer, and neurodegenerative diseases. However, the nature, function, and contribution of TNT to cancer pathogenesis are poorly understood. Our analyses demonstrate that TNT structures are induced between glioblastoma (GBM) cells and surrounding non-tumor astrocytes to transfer tumor-derived mitochondria. The mitochondrial transfer mediated by TNT resulted in the adaptation of non-tumor astrocytes to tumor-like metabolism and hypoxia conditions. In conclusion, TNT are an efficient cell-to-cell communication system used by cancer cells to adapt the microenvironment to the invasive nature of the tumor.

Central Nervous System (CNS) Viral Seeding by Mature Monocytes and Potential Therapies To Reduce CNS Viral Reservoirs in the cART Era.

The human immunodeficiency virus (HIV) enters the central nervous system (CNS) within a few days after primary infection, establishing viral reservoirs that persist even with combined antiretroviral therapy (cART). We show that monocytes from people living with HIV (PLWH) on suppressive cART harboring integrated HIV, viral mRNA, and/or viral proteins preferentially transmigrate across the blood-brain barrier (BBB) to CCL2 and are significantly enriched post-transmigration, and even more highly enriched posttransmigration than T cells with similar properties. Using HIV-infected ART-treated mature monocytes cultured in vitro, we recapitulate these findings and demonstrate that HIV+ CD14+ CD16+ ART-treated monocytes also preferentially transmigrate. Cenicriviroc and anti-JAM-A and anti-ALCAM antibodies significantly and preferentially reduce/block transmigration of HIV+ CD14+ CD16+ ART-treated monocytes. These findings highlight the importance of monocytes in CNS HIV reservoirs and suggest targets to eliminate their formation and reseeding.IMPORTANCE We characterized mechanisms of CNS viral reservoir establishment/replenishment using peripheral blood mononuclear cells (PBMC) of PLWH on cART and propose therapeutic targets to reduce/block selective entry of cells harboring HIV (HIV+) into the CNS. Using DNA/RNAscope, we show that CD14+ CD16+ monocytes with integrated HIV, transcriptionally active, and/or with active viral replication from PBMC of PLWH prescribed cART and virally suppressed, selectively transmigrate across a human BBB model. This is the first study to our knowledge demonstrating that monocytes from PLWH with HIV disease for approximately 22 years and with long-term documented suppression can still carry virus into the CNS that has potential to be reactivated and infectious. This selective entry into the CNS-and likely other tissues-indicates a mechanism of reservoir formation/reseeding in the cART era. Using blocking studies, we propose CCR2, JAM-A, and ALCAM as targets on HIV+ CD14+ CD16+ monocytes to reduce and/or prevent CNS reservoir replenishment and to treat HAND and other HIV-associated comorbidities.

Astrocytes are HIV reservoirs in the brain: A cell type with poor HIV infectivity and replication but efficient cell-to-cell viral transfer.

The major barrier to eradicating Human immunodeficiency virus-1 (HIV) infection is the generation of tissue-associated quiescent long-lasting viral reservoirs refractory to therapy. Upon interruption of anti-retroviral therapy (ART), HIV replication can be reactivated. Within the brain, microglia/macrophages and a small population of astrocytes are infected with HIV. However, the role of astrocytes as a potential viral reservoir is becoming more recognized because of the improved detection and quantification of HIV viral reservoirs. In this report, we examined the infectivity of human primary astrocytes in vivo and in vitro, and their capacity to maintain HIV infection, become latently infected, be reactivated, and transfer new HIV virions into neighboring cells. Analysis of human brain tissue sections obtained from HIV-infected individuals under effective and prolonged ART indicates that a small population of astrocytes has integrated HIV-DNA. In vitro experiments using HIV-infected human primary astrocyte cultures confirmed a low percentage of astrocytes had integrated HIV-DNA, with poor to undetectable replication. Even in the absence of ART, long-term culture results in latency that could be transiently reactivated with histone deacetylase inhibitor, tumor necrosis factor-alpha (TNF-α), or methamphetamine. Reactivation resulted in poor viral production but efficient cell-to-cell viral transfer into cells that support high viral replication. Together, our data provide a new understanding of astrocytes' role as viral reservoirs within the central nervous system (CNS).

Role of Tunneling Nanotube-like Structures during the Early Events of HIV Infection: Novel Features of Tissue Compartmentalization and Mechanism of HIV Spread.

HIV has become a chronic disease despite the effective use of antiretroviral therapy (ART). However, the mechanisms of tissue colonization, viral evolution, generation of viral reservoirs, and compartmentalization are still a matter of debate due to the challenges involved in examining early events of infection at the cellular and molecular level. Thus, there is still an urgent need to explore these areas to develop effective HIV cure strategies. In this study, we describe the early events of tissue colonization and compartmentalization as well as the role of tunneling nanotube-like structures during viral spread in the presence and absence of effective antiretroviral treatment. To examine these mechanisms, NOD/SCID IL-2 RG-/- humanized mice were either directly infected with HIVADA or with low numbers of HIVADA-infected leukocytes to limit tissue colonization in the presence and absence of TAK779, an effective CCR5 blocker of HIV entry. We identify that viral seeding in tissues occurs early in a tissue- and cell type-specific manner (24-72 h). Reduction in systemic HIV replication by TAK779 treatment did not affect tissue seeding or spreading, despite reduced systemic viral replication. Tissue-associated HIV-infected cells had different properties than cells in the circulation because the virus continues to spread in tissues in a tunneling nanotube-like structure-dependent manner, despite ART. Thus, understanding these mechanisms can provide new approaches to enhance the efficacy of existing ART and HIV infection cure strategies.

Tunneling Nanotubes Mediate Adaptation of Glioblastoma Cells to Temozolomide and Ionizing Radiation Treatment.

Glioblastoma (GBM) is the most prevalent and aggressive tumor in the central nervous system. Surgical resection followed by concurrent radiotherapy (ionizing radiation [IR]) and temozolomide (TMZ) is the standard of care for GBM. However, a large subset of patients offer resistance or become adapted to TMZ due mainly to the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). Thus, alternative mechanisms of MGMT deregulation have been proposed but are heretofore unproven. We show that heterogeneous GBM cells express tunneling nanotubes (TNTs) upon oxidative stress and TMZ/IR treatment. We identified that MGMT protein diffused from resistant to sensitive cells upon exposure to TMZ/IR, resulting in protection against cytotoxic therapy in a TNT-dependent manner. In vivo analysis of resected GBM tumors support our hypothesis that the MGMT protein, but not its mRNA, was associated with TNT biomarkers. We propose that targeting TNT formation could be an innovative strategy to overcome treatment resistance in GBM.

HIV infects astrocytes in vivo and egresses from the brain to the periphery.

HIV invades the brain during acute infection. Yet, it is unknown whether long-lived infected brain cells release productive virus that can egress from the brain to re-seed peripheral organs. This understanding has significant implication for the brain as a reservoir for HIV and most importantly HIV interplay between the brain and peripheral organs. Given the sheer number of astrocytes in the human brain and their controversial role in HIV infection, we evaluated their infection in vivo and whether HIV infected astrocytes can support HIV egress to peripheral organs. We developed two novel models of chimeric human astrocyte/human peripheral blood mononuclear cells: NOD/scid-IL-2Rgc null (NSG) mice (huAstro/HuPBMCs) whereby we transplanted HIV (non-pseudotyped or VSVg-pseudotyped) infected or uninfected primary human fetal astrocytes (NHAs) or an astrocytoma cell line (U138MG) into the brain of neonate or adult NSG mice and reconstituted the animals with human peripheral blood mononuclear cells (PBMCs). We also transplanted uninfected astrocytes into the brain of NSG mice and reconstituted with infected PBMCs to mimic a biological infection course. As expected, the xenotransplanted astrocytes did not escape/migrate out of the brain and the blood brain barrier (BBB) was intact in this model. We demonstrate that astrocytes support HIV infection in vivo and egress to peripheral organs, at least in part, through trafficking of infected CD4+ T cells out of the brain. Astrocyte-derived HIV egress persists, albeit at low levels, under combination antiretroviral therapy (cART). Egressed HIV evolved with a pattern and rate typical of acute peripheral infection. Lastly, analysis of human cortical or hippocampal brain regions of donors under cART revealed that astrocytes harbor between 0.4-5.2% integrated HIV gag DNA and 2-7% are HIV gag mRNA positive. These studies establish a paradigm shift in the dynamic interaction between the brain and peripheral organs which can inform eradication of HIV reservoirs.

HIV gp120 Protein Increases the Function of Connexin 43 Hemichannels and Pannexin-1 Channels in Astrocytes: Repercussions on Astroglial Function.

At least half of human immunodeficiency virus (HIV)-infected individuals suffer from a wide range of cognitive, behavioral and motor deficits, collectively known as HIV-associated neurocognitive disorders (HAND). The molecular mechanisms that amplify damage within the brain of HIV-infected individuals are unknown. Recently, we described that HIV augments the opening of connexin-43 (Cx43) hemichannels in cultured human astrocytes, which result in the collapse of neuronal processes. Whether HIV soluble viral proteins such as gp120, can regulate hemichannel opening in astrocytes is still ignored. These channels communicate the cytosol with the extracellular space during pathological conditions. We found that gp120 enhances the function of both Cx43 hemichannels and pannexin-1 channels in mouse cortical astrocytes. These effects depended on the activation of IL-1ß/TNF-α, p38 MAP kinase, iNOS, cytoplasmic Ca2+ and purinergic signaling. The gp120-induced channel opening resulted in alterations in Ca2+ dynamics, nitric oxide production and ATP release. Although the channel opening evoked by gp120 in astrocytes was reproduced in ex vivo brain preparations, these responses were heterogeneous depending on the CA1 region analyzed. We speculate that soluble gp120-induced activation of astroglial Cx43 hemichannels and pannexin-1 channels could be crucial for the pathogenesis of HAND.

Circulating levels of ATP is a biomarker of HIV cognitive impairment.

BACKGROUND: In developed countries, Human Immunodeficiency Virus type-1 (HIV-1) infection has become a chronic disease despite the positive effects of anti-retroviral therapies (ART), but still at least half of the HIV infected population shown signs of cognitive impairment. Therefore, biomarkers of HIV cognitive decline are urgently needed. METHODS: We analyze the opening of one of the larger channels expressed by humans, pannexin-1 (Panx-1) channels, in the uninfected and HIV infected population (n = 175). We determined channel opening and secretion of intracellular second messengers released through the channel such as PGE2 and ATP. Also, we correlated the opening of Panx-1 channels with the circulating levels of PGE2 and ATP as well as cogntive status of the individuals analyzed. FINDINGS: Here, we demonstrate that Panx-1 channels on fresh PBMCs obtained from uninfected individuals are closed and no significant amounts of PGE2 and ATP are detected in the circulation. In contrast, in all HIV-infected individuals analyzed, even the ones under effective ART, a spontaneous opening of Panx-1 channels and increased circulating levels of PGE2 and ATP were detected. Circulating levels of ATP were correlated with cognitive decline in the HIV-infected population supporting that ATP is a biomarker of cognitive disease in the HIV-infected population. INTERPRETATION: We propose that circulating levels of ATP could predict CNS compromise and lead to the breakthroughs necessary to detect and prevent brain compromise in the HIV-infected population.

HIV infection and latency induce a unique metabolic signature in human macrophages.

Currently, a major barrier to curing HIV infection is the generation of tissue-associated, non-replicating, long-lasting viral reservoirs that are refractory to therapy and can be reactivated upon anti-retroviral therapy interruption. One of these reservoirs are latently HIV-infected macrophages. Here, we show that HIV infection of macrophages results in survival of a small population of infected cells that are metabolically altered and characterized by mitochondrial fusion, lipid accumulation, and reduced mitochondrial ATP production. No changes in glycolysis were detected. Metabolic analysis indicated an essential role of succinate and other TCA metabolites in the tricarboxylic acid (TCA) cycle in mediating lipid accumulation and oxidative phosphorylation (OXPHOS) in the mitochondria. Furthermore, we show that while uninfected and HIV infected macrophages use fatty acids and glucose as primary sources of energy, surviving HIV infected macrophages also use glutamine/glutamate as a major energy source, and blocking these new sources of energy resulted in the killing of latent HIV infected macrophages. Together, our data provide a new understanding of the formation, properties, and potential novel ways to eliminate macrophage viral reservoirs.

HIV-1 reservoirs in urethral macrophages of patients under suppressive antiretroviral therapy.

Human immunodeficiency virus type 1 (HIV-1) eradication is prevented by the establishment on infection of cellular HIV-1 reservoirs that are not fully characterized, especially in genital mucosal tissues (the main HIV-1 entry portal on sexual transmission). Here, we show, using penile tissues from HIV-1-infected individuals under suppressive combination antiretroviral therapy, that urethral macrophages contain integrated HIV-1 DNA, RNA, proteins and intact virions in virus-containing compartment-like structures, whereas viral components remain undetectable in urethral T cells. Moreover, urethral cells specifically release replication-competent infectious HIV-1 following reactivation with the macrophage activator lipopolysaccharide, while the T-cell activator phytohaemagglutinin is ineffective. HIV-1 urethral reservoirs localize preferentially in a subset of polarized macrophages that highly expresses the interleukin-1 receptor, CD206 and interleukin-4 receptor, but not CD163. To our knowledge, these results are the first evidence that human urethral tissue macrophages constitute a principal HIV-1 reservoir. Such findings are determinant for therapeutic strategies aimed at HIV-1 eradication.

Role of cell-to-cell communication in cancer: New features, insights, and directions.

The current special issue entitled "Role of tunneling nanotubes (TNTs) in carcinogenesis" was designed to discuss the role of cell-to-cell communication, especially TNTs, in cancer pathogenesis. In addition, we discuss the exploitation of TNTs as a potential therapeutic target to prevent and reduce cancer incidence. It is accepted that cell-to-cell communication is essential for the development of multicellular systems, and it is coordinated by soluble factors, associated membrane proteins, exosomes, gap junction channels, and TNTs. An old belief in the cancer field is that cancer cells are "disconnected" from healthy cells, resulting in loss of cell-to-cell communication and neighbor control. However, recent data obtained from different kind of tumors indicate that TNTs and others forms of communication (exosomes and localized cell-to-cell communication) are highly expressed and functional during tumor development . In physiological conditions, TNTs are expressed by few cells, and their main function is to coordinate long-distance signaling. However, upon carcinogenesis, TNTs proliferate and provide an alternative route of communication to enable the transfer of several signaling molecules and organelles to spread disease and toxicity. We propose that TNTs and their cargo are an attractive therapeutic target to reduce or prevent cancer development. All these unique aspects of cell-to-cell diffusion and organelle sharing will be discussed in this special issue.

Identification, Localization, and Quantification of HIV Reservoirs Using Microscopy.

The major barrier to eradicating human immunodeficiency virus-1 (HIV) infection is the generation and extended survival of HIV reservoirs. In order to eradicate HIV infection, it is essential to detect, quantify, and characterize circulating and tissue-associated viral reservoirs in infected individuals. Currently, PCR-based technologies and Quantitative Viral Outgrowth Assays (Q-VOA) are the gold standards to detect viral reservoirs. However, these methods are limited to detecting circulating viral reservoirs, and it has been shown that they misrepresent the size of the reservoirs, largely because they detect only one component of the HIV life cycle and are unable to detect viral reservoirs in tissues. Here, we described the use of multiple detection systems to identify integrated HIV DNA or viral mRNA and several HIV proteins in circulating and tissue reservoirs using improved staining and microscopy techniques. We believe that this imaging-based approach for detecting HIV reservoirs will lead to breakthroughs necessary to eradicate these reservoirs. © 2018 by John Wiley & Sons, Inc.

Inflammatory mediators reduce surface PrPc on human BMVEC resulting in decreased barrier integrity.

The cellular prion protein (PrPc) is a surface adhesion molecule expressed at junctions of various cell types including brain microvascular endothelial cells (BMVEC) that are important components of the blood-brain barrier (BBB). PrPc is involved in several physiological processes including regulation of epithelial cell barrier function and monocyte migration across BMVEC. BBB dysfunction and disruption are significant events in central nervous system (CNS) inflammatory processes including HIV neuropathogenesis. Tumor necrosis factor (TNF)-α and vascular endothelial growth factor (VEGF) are two inflammatory factors that have been implicated in the processes that affect BBB integrity. To examine the effect of inflammation on PrPc expression in BMVEC, we used these mediators and found that TNF-α and VEGF decrease surface PrPc on primary human BMVEC. We also showed that these factors decrease total PrPc protein as well as mRNA, indicating that they regulate expression of this protein by de novo synthesis. To determine the effect of PrPc loss from the surface of BMVEC on barrier integrity, we used small hairpin RNAs to knockdown PrPc. We found that the absence of PrPc from BMVEC causes increased permeability as determined by a fluorescein isothiocyanate (FITC)-dextran permeability assay. This suggests that cell surface PrPc is essential for endothelial monolayer integrity. To determine the mechanism by which PrPc downregulation leads to increased permeability of an endothelial monolayer, we examined changes in expression and localization of tight junction proteins, occludin and claudin-5, and found that decreased PrPc leads to decreased total and membrane-associated occludin and claudin-5. We propose that an additional mechanism by which inflammatory factors affect endothelial monolayer permeability is by decreasing cell-associated PrPc. This increase in permeability may have subsequent consequences that lead to CNS damage.

Extreme Drug Tolerance of Mycobacterium tuberculosis in Caseum.

Tuberculosis (TB) recently became the leading infectious cause of death in adults, while attempts to shorten therapy have largely failed. Dormancy, persistence, and drug tolerance are among the factors driving the long therapy duration. Assays to measure in situ drug susceptibility of Mycobacterium tuberculosis bacteria in pulmonary lesions are needed if we are to discover new fast-acting regimens and address the global TB threat. Here we take a first step toward this goal and describe an ex vivo assay developed to measure the cidal activity of anti-TB drugs against M. tuberculosis bacilli present in cavity caseum obtained from rabbits with active TB. We show that caseum M. tuberculosis bacilli are largely nonreplicating, maintain viability over the course of the assay, and exhibit extreme tolerance to many first- and second-line TB drugs. Among the drugs tested, only the rifamycins fully sterilized caseum. A similar trend of phenotypic drug resistance was observed in the hypoxia- and starvation-induced nonreplicating models, but with notable qualitative and quantitative differences: (i) caseum M. tuberculosis exhibits higher drug tolerance than nonreplicating M. tuberculosis in the Wayne and Loebel models, and (ii) pyrazinamide is cidal in caseum but has no detectable activity in these classic nonreplicating assays. Thus, ex vivo caseum constitutes a unique tool to evaluate drug potency against slowly replicating or nonreplicating bacilli in their native caseous environment. Intracaseum cidal concentrations can now be related to the concentrations achieved in the necrotic foci of granulomas and cavities to establish correlations between clinical outcome and lesion-centered pharmacokinetics-pharmacodynamics (PK-PD) parameters.