Effective prognostic and clinical risk stratification in COVID-19 using multimodality biomarkers.

In acute coronavirus disease 19 (COVID-19) patients, effective clinical risk stratification has important implications on treatment and therapeutic resource distribution. This article reviews the evidence behind a wide range of biomarkers with prognostic value in COVID-19. Patient characteristics and co-morbidities, such as cardiovascular and respiratory diseases, are associated with increased mortality risk. Peripheral oxygen saturation and arterial oxygenation are predictive of severe respiratory compromise, whereas risk scores such as the 4C-score enable multi-factorial prognostic risk estimation. Blood tests such as markers of inflammation, cardiac injury and d-dimer and abnormalities on electrocardiogram are linked to inpatient prognosis. Of the imaging modalities, lung ultrasound and echocardiography enable the bedside assessment of prognostic abnormalities in COVID-19. Chest radiograph (CXR) and computed tomography (CT) can inform about prognostic pulmonary pathologies, whereas cardiovascular CT detects high-risk features such as coronary artery and aortic calcification. Dynamic changes in biomarkers, such as blood tests, CXR, CT and electrocardiogram findings, can further inform about disease severity and prognosis. Despite the vast volumes of existing evidence, several gaps exist in our understanding of COVID-19 biomarkers. First, the pathophysiological basis on which these markers can foretell prognosis in COVID-19 remains poorly understood. Second, certain under-explored tests such as thoracic impedance assessment and cardiovascular magnetic resonance imaging deserve further investigation. Lastly, the prognostic values of most biomarkers in COVID-19 are derived from retrospective analyses. Prospective studies are required to validate these markers for guiding clinical decision-making and to facilitate their translation into clinical management pathways.

Modulation of allergic inflammation in the lung by a peptide derived from Mycobacteria tuberculosis chaperonin 60.1.

BACKGROUND: We have previously demonstrated that Mycobacteria tuberculosis chaperonin 60.1 inhibits leucocyte diapedesis and bronchial hyperresponsiveness in a murine model of allergic lung inflammation. METHODS: In the present study, we have investigated the effect of a shorter peptide sequence derived from Cpn 60.1, named IRL201104, on allergic lung inflammation induced by ovalbumin (OVA) in mice and by house dust mite (HDM) in guinea pigs, as well as investigating the action of IRL201104 on human cells in vitro. RESULTS: Pre-treatment of mice or guinea pigs with IRL201104 inhibits the infiltration of eosinophils to the lung, cytokine release, and in guinea pig skin, inhibits allergen-induced vascular permeability. The protective effect of intranasal IRL201104 against OVA-induced eosinophilia persisted for up to 20 days post-treatment. Moreover, OVA-sensitized mice treated intranasally with 20 ng/kg of IRL201104 show a significant increase in the expression of the anti-inflammatory molecule ubiquitin A20 and significant inhibition of the activation of NF-κB in lung tissue. Our results also show that A20 expression was significantly reduced in blood leucocytes and ASM obtained from patients with asthma compared to cells obtained from healthy subjects which were restored after incubation with IRL201104 in vitro, when added alone, or in combination with LPS or TNF-α in ASM. CONCLUSIONS: Our results suggest that a peptide derived from mycobacterial Cpn60.1 has a long-lasting anti-inflammatory and immunomodulatory activity which may help explain some of the protective effects of TB against allergic diseases.

A model-based analysis identifies differences in phenotypic resistance between in vitro and in vivo: implications for translational medicine within tuberculosis.

Proper characterization of drug effects on Mycobacterium tuberculosis relies on the characterization of phenotypically resistant bacteria to correctly establish exposure-response relationships. The aim of this work was to evaluate the potential difference in phenotypic resistance in in vitro compared to murine in vivo models using CFU data alone or CFU together with most probable number (MPN) data following resuscitation with culture supernatant. Predictions of in vitro and in vivo phenotypic resistance i.e. persisters, using the Multistate Tuberculosis Pharmacometric (MTP) model framework was evaluated based on bacterial cultures grown with and without drug exposure using CFU alone or CFU plus MPN data. Phenotypic resistance and total bacterial number in in vitro natural growth observations, i.e. without drug, was well predicted by the MTP model using only CFU data. Capturing the murine in vivo total bacterial number and persisters during natural growth did however require re-estimation of model parameter using both the CFU and MPN observations implying that the ratio of persisters to total bacterial burden is different in vitro compared to murine in vivo. The evaluation of the in vitro rifampicin drug effect revealed that higher resolution in the persister drug effect was seen using CFU and MPN compared to CFU alone although drug effects on the other bacterial populations were well predicted using only CFU data. The ratio of persistent bacteria to total bacteria was predicted to be different between in vitro and murine in vivo. This difference could have implications for subsequent translational efforts in tuberculosis drug development.

Mechanism of Action of a Membrane-Active Quinoline-Based Antimicrobial on Natural and Model Bacterial Membranes.

HT61 is a quinoline-derived antimicrobial, which exhibits bactericidal potency against both multiplying and quiescent methicillin resistant and sensitive Staphylococcus aureus, and has been proposed as an adjunct for other antimicrobials to extend their usefulness in the face of increasing antimicrobial resistance. In this study, we have examined HT61's effect on the permeability of S. aureus membranes and whether this putative activity can be attributed to an interaction with lipid bilayers. Using membrane potential and ATP release assays, we have shown that HT61 disrupts the membrane enough to result in depolarization of the membrane and release of intercellular constituents at concentrations above and below the minimum inhibitory concentration of the drug. Utilizing both monolayer subphase injection and neutron reflectometry, we have shown that increasing the anionic lipid content of the membrane leads to a more marked effect of the drug. In bilayers containing 25 mol % phosphatidylglycerol, neutron reflectometry data suggest that exposure to HT61 increases the level of solvent in the hydrophobic region of the membrane, which is indicative of gross structural damage. Increasing the proportion of PG elicits a concomitant level of membrane damage, resulting in almost total destruction when 75 mol % phosphatidylglycerol is present. We therefore propose that HT61's primary action is directed toward the cytoplasmic membrane of Gram-positive bacteria.

A multistate tuberculosis pharmacometric model: a framework for studying anti-tubercular drug effects in vitro.

OBJECTIVES: Mycobacterium tuberculosis can exist in different states in vitro, which can be denoted as fast multiplying, slow multiplying and non-multiplying. Characterizing the natural growth of M. tuberculosis could provide a framework for accurate characterization of drug effects on the different bacterial states. METHODS: The natural growth data of M. tuberculosis H37Rv used in this study consisted of viability defined as cfu versus time based on data from an in vitro hypoxia system. External validation of the natural growth model was conducted using data representing the rate of incorporation of radiolabelled methionine into proteins by the bacteria. Rifampicin time-kill curves from log-phase (0.25-16 mg/L) and stationary-phase (0.5-64 mg/L) cultures were used to assess the model's ability to describe drug effects by evaluating different linear and non-linear exposure-response relationships. RESULTS: The final pharmacometric model consisted of a three-compartment differential equation system representing fast-, slow- and non-multiplying bacteria. Model predictions correlated well with the external data (R(2) = 0.98). The rifampicin effects on log-phase and stationary-phase cultures were separately and simultaneously described by including the drug effect on the different bacterial states. The predicted reduction in log10 cfu after 14 days and at 0.5 mg/L was 2.2 and 0.8 in the log-phase and stationary-phase systems, respectively. CONCLUSIONS: The model provides predictions of the change in bacterial numbers for the different bacterial states with and without drug effect and could thus be used as a framework for studying anti-tubercular drug effects in vitro.

Enhancement by novel anti-methicillin-resistant Staphylococcus aureus compound HT61 of the activity of neomycin, gentamicin, mupirocin and chlorhexidine: in vitro and in vivo studies.

OBJECTIVES: Previously, we described a small quinoline-derived compound that exhibited selective bactericidal activity against methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA). It depolarizes the bacterial cell membrane. In this study, we investigated if HT61 was able to enhance the potency of other antibiotics, namely neomycin, gentamicin and mupirocin, and an antiseptic, namely chlorhexidine, against clinical isolates of MSSA and MRSA in vitro and in vivo. METHODS: The MICs were determined by the broth microdilution method. The effect of combinations was examined using the chequerboard method and time-kill curves. A murine skin infection model was used to evaluate the enhancement by HT61 of other antimicrobials. RESULTS: Using the fractional inhibitory concentration index, no interaction was seen in both MSSA and MRSA for the pair HT61 and gentamicin or the pair HT61 and neomycin. Synergism was seen for 65% of both MSSA and MRSA when HT61 was combined with chlorhexidine. There was also no interaction between HT61 and mupirocin. Time-kill analysis demonstrated significant synergistic activities when a low level of HT61 was combined with neomycin, gentamicin or chlorhexidine. The effect was more dramatic against non-multiplying bacteria against which the antimicrobials used were inactive on their own. Significant synergistic effects were also seen on mouse infected skin. CONCLUSIONS: We demonstrate that HT61, developed as a topical agent, acts as an enhancer that accelerates the activities of other antimicrobial agents against both MSSA and MRSA.

Comparison of Lymphocyte-CRP Ratio to Conventional Inflammatory Markers for Predicting Clinical Outcomes in COVID-19.

Background: In COVID-19 patients, lymphocyte-CRP ratio (LCR) is a promising biomarker for predicting adverse clinical outcomes. How well LCR performs compared to conventional inflammatory markers for prognosticating COVID-19 patients remains unclear, which hinders the clinical translation of this novel biomarker. Methods: In a cohort of COVID-19 inpatients, we characterised the clinical applicability of LCR by comparing its prognostic value against conventional inflammatory markers for predicting inpatient mortality and a composite of mortality, invasive/non-invasive ventilation and intensive care unit admissions. Results: Of the 413 COVID-19 patients, 100 (24%) patients suffered inpatient mortality. On Receiver Operating Characteristics analysis, LCR performed similarly to CRP for predicting mortality (AUC 0.74 vs. 0.71, p = 0.049) and the composite endpoint (AUC 0.76 vs. 0.76, p = 0.812). LCR outperformed lymphocyte counts (AUC 0.74 vs. 0.66, p = 0.002), platelet counts (AUC 0.74 vs. 0.61, p = 0.003) and white cell counts (AUC 0.74 vs. 0.54, p < 0.001) for predicting mortality. On Kaplan-Meier analysis, patients with a low LCR (below a 58 cut-off) had worse inpatient survival than patients with other LCR values (p < 0.001). Conclusion: LCR appears comparable to CRP, but outperformed other inflammatory markers, for prognosticating COVID-19 patients. Further studies are required to improve the diagnostic value of LCR to facilitate clinical translation.

Normal high-sensitivity cardiac troponin for ruling-out inpatient mortality in acute COVID-19.

INTRODUCTION: Assessment of inpatient mortality risk in COVID-19 patients is important for guiding clinical decision-making. High sensitivity cardiac troponin T (hs-cTnT) is a biomarker of cardiac injury associated with a worse prognosis in COVID-19. We explored how hs-cTnT could potentially be used in clinical practice for ruling in and ruling out mortality in COVID-19. METHOD: We tested the diagnostic value of hs-cTnT in laboratory-confirmed COVID-19 patients (≥18 years old) admitted to the Royal Berkshire Hospital (UK) between 1st March and 10th May 2020. A normal hs-cTnT was defined as a value within the 99th percentile of healthy individuals (≤14 ng/L), and an elevated hs-cTnT was defined as >14 ng/L. Adverse clinical outcome was defined as inpatient mortality related to COVID-19. RESULTS: A total of 191 COVID-19 patients (62% male; age 66±16 years) had hs-cTnT measured on admission. Of these patients, 124 (65%) had elevated hs-cTnT and 67 (35%) had normal hs-cTnT. On a group level, patients with elevated hs-cTnT had worse inpatient survival (p = 0.0014; Kaplan-Meier analysis) and higher risk of inpatient mortality (HR 5.84 [95% CI 1.29-26.4]; p = 0.02; Cox multivariate regression) compared to patients with normal hs-cTnT. On a per-patient level, a normal hs-cTnT had a negative predictive value of 94% (95% CI: 85-98%) for ruling out mortality, whilst an elevated hs-cTnT had a low positive predictive value of 38% (95% CI: 39-47%) for ruling in mortality. CONCLUSIONS: In this study cohort of COVID-19 patients, the potential clinical utility of hs-cTnT appears to rest in ruling out inpatient mortality. This finding, if prospectively validated in a larger study, may allow hs-cTnT to become an important biomarker to facilitate admission-avoidance and early safe discharge.

Low CRB-65 Scores Effectively Rule out Adverse Clinical Outcomes in COVID-19 Irrespective of Chest Radiographic Abnormalities.

Background: CRB-65 (Confusion; Respiratory rate ≥ 30/min; Blood pressure ≤ 90/60 mmHg; age ≥ 65 years) is a risk score for prognosticating patients with COVID-19 pneumonia. However, a significant proportion of COVID-19 patients have normal chest X-rays (CXRs). The influence of CXR abnormalities on the prognostic value of CRB-65 is unknown, limiting its wider applicability. Methods: We assessed the influence of CXR abnormalities on the prognostic value of CRB-65 in COVID-19. Results: In 589 study patients (71 years (IQR: 57-83); 57% males), 186 (32%) had normal CXRs. On ROC analysis, CRB-65 performed similarly in patients with normal vs. abnormal CXRs for predicting inpatient mortality (AUC 0.67 ± 0.05 vs. 0.69 ± 0.03). In patients with normal CXRs, a CRB-65 of 0 ruled out mortality, NIV requirement and critical illness (intubation and/or ICU admission) with negative predictive values (NPVs) of 94%, 98% and 99%, respectively. In patients with abnormal CXRs, a CRB-65 of 0 ruled out the same endpoints with NPVs of 91%, 83% and 86%, respectively. Patients with low CRB-65 scores had better inpatient survival than patients with high CRB-65 scores, irrespective of CXR abnormalities (all p < 0.05). Conclusions: CRB-65, CXR and CRP are independent predictors of mortality in COVID-19. Adding CXR findings (dichotomised to either normal or abnormal) to CRB-65 does not improve its prognostic accuracy. A low CRB-65 score of 0 may be a good rule-out test for adverse clinical outcomes in COVID-19 patients with normal or abnormal CXRs, which deserves prospective validation.

Characterisation of Ferritin-Lymphocyte Ratio in COVID-19.

Introduction: The ferritin-lymphocyte ratio (FLR) is a novel inflammatory biomarker for the assessment of acute COVID-19 patients. However, the prognostic value of FLR for predicting adverse clinical outcomes in COVID-19 remains unclear, which hinders its clinical translation. Methods: We characterised the prognostic value of FLR in COVID-19 patients, as compared to established inflammatory markers. Results: In 217 study patients (69 years [IQR: 55-82]; 60% males), FLR was weakly correlated with CRP (R = 0.108, p = 0.115) and white cell count (R = -0.144; p = 0.034). On ROC analysis, an FLR cut-off of 286 achieved a sensitivity of 86% and a specificity of 30% for predicting inpatient mortality (AUC 0.60, 95% CI: 0.53-0.67). The negative predictive values of FLR for ruling out mortality, non-invasive ventilation requirement and critical illness (intubation and/or ICU admission) were 86%, 85% and 93%, respectively. FLR performed similarly to CRP (AUC 0.60 vs. 0.64; p = 0.375) for predicting mortality, but worse than CRP for predicting non-fatal outcomes (all p < 0.05). On Kaplan-Meier analysis, COVID-19 patients with FLR values > 286 had worse inpatient survival than patients with FLR ≤ 286, p = 0.041. Conclusions: FLR has prognostic value in COVID-19 patients, and appears unrelated to other inflammatory markers such as CRP and WCC. FLR exhibits high sensitivity and negative predictive values for adverse clinical outcomes in COVID-19, and may be a good "rule-out" test. Further work is needed to improve the sensitivity of FLR and validate its role in prospective studies for guiding clinical management.

Mycobacterium tuberculosis chaperonin 60.1 inhibits leukocyte diapedesis in a murine model of allergic lung inflammation.

Chaperonin 60.1 from Mycobacterium tuberculosis suppressed allergic lung inflammation and bronchial hyperresponsiveness in mice by a mechanism that is yet to be clarified. To investigate the possible antiinflammatory mechanism(s) of action of Cpn60.1 in a model of allergic lung inflammation, ovalbumin (OVA)-allergic mice were pretreated with Cpn60.1 intranasally 20 minutes before each OVA aerosol challenge in a total of three treatments. Readouts were performed 24 hours after last challenge. Pretreatment with Cpn60.1 (1.0-0.001 µg) significantly inhibited the number of eosinophils in bronchoalveolar lavage fluid (OVA, 49.2 ± 12.3 versus Cpn60.1 [1 µg dose], 90.4 ± 2.3 × 10(4) cells/ml) and IL-5 release (OVA, 43 ± 8.5 versus Cpn60.1 [1 µg dose], 3 ± 11 pg/ml) but increased IL-12 levels (OVA, 50 ± 24 versus Cpn60.1 [1 µg dose], 103 ± 13 pg/ml). The effect of Cpn60.1 on cell recruitment and IL-5, but not IL-12, release was abolished in TLR-4 knockout mice. Intravital microscopy demonstrated that Cpn60.1 reduced chemokine-mediated leukocyte rolling and transmigration across the vessel wall (rolling cells: eotaxin, 11.7 ± 1.1 versus Cpn60.1 [1 µg dose], 2.8 ± 1 cells in 30 s). Similarly, Cpn60.1 reduced eotaxin-induced leukocyte migration in vitro (eotaxin, 17.3 ± 3.3 versus Cpn60.1 [0.1 µg dose], 3.3 ± 0.4 cells × 10(4)/ml). Immunostaining demonstrated that Cpn60.1 inhibits VCAM-1 and increases vascular endothelial-cadherin expression in lung vascular tissue, suggesting that the antiinflammatory effect of Cpn60.1 is partly mediated by altering the expression of adhesion molecules. This study shows that Cpn60.1 inhibits leukocyte diapedesis by a TLR-4 and an adhesion molecule-dependent mechanism in allergic inflammation in mice.

Antibiotics in phase II and III clinical trials.

There are 19 compounds in late-stage clinical trials, of which ten may be suitable for Gram-positive infections. However, there are only five compounds in development for Gram-negative infections, in addition to four broad-spectrum ones. There are two new classes in late-stage clinical development. This chapter discusses in some detail each of the antibiotics in Phase II and Phase III clinical trials. Only those that appear in the literature are covered. The shortage of compounds in development for Gram-negatives and the small number of new classes in the pipeline is of serious concern; this matter needs to be addressed by governments, the regulatory authorities, the pharmaceutical industry and academia urgently.

HPLC/MS/MS methodology for sensitive quantitation of monic acid A, the metabolic product of the antibiotic mupirocin.

Patients who are treated by self-medication with intranasal mupiricin (Bactroban™) for controlling meticillin-resistant Staphylococcus aureus may, or may not, adhere to their regimen. Herein, we describe a potential methodology for assessing adherence by measuring the gastric degradation product, monic acid A (MA), as a biomarker in urine. MA was isolated (~80% recovery) through a Waters Oasis HLB cartridge and detected (e.g. 25 pg on the column) by HPLC/MS/MS (API4000). Within a calculated 10(6)-fold margin, this analytical sensitivity should facilitate urinary MA quantitation if, for example, 1% of intranasal mupirocin is swallowed and degraded characteristically to MA by gastric acidity.

Comparison of the moonlighting actions of the two highly homologous chaperonin 60 proteins of Mycobacterium tuberculosis.

Evidence is emerging that the two chaperonin (Cpn) 60 proteins of Mycobacterium tuberculosis, Cpn60.1 and Cpn60.2, have moonlighting actions that may contribute to the pathology of tuberculosis. We studied the release of Cpn60.1 from M. tuberculosis and infected macrophage like cells and compared recombinant Cpn60.1 and Cpn60.2 in a range of cell-based assays to determine how similar the actions of these highly homologous proteins are. We now establish that Cpns are similar as follows: (i) Cpn60.1, as it has been shown for Cpn60.2, is released by M. tuberculosis in culture, and Cpn60.1 is furthermore released when the bacterium is in quiescent, but not activated, macrophage like cells, and (ii) both proteins only showed a partial requirement for MyD88 for the induction of proinflammatory cytokine production compared to lipopolysaccharide. However, we also found major differences in the cellular action of Cpns. (i) Cpn60.2 proved to be a more potent stimulator of whole blood leukocytes than Cpn60.1 and was the only one to induce tumor necrosis factor alpha synthesis. (ii) Cpn60.1 bound to ca. 90% of circulating monocytes compared to Cpn60.2, which bound <50% of these cells. Both chaperonins bound to different cell surface receptors, while monocyte activation by both proteins was completely abrogated in TLR4-/- mice, although Cpn60.2 also showed significant requirement for TLR2. Finally, an isogenic mutant lacking cpn60.1, but containing intact cpn60.2, was severely inhibited in generating multinucleate giant cells in an in vitro human granuloma assay. These results clearly show that, despite significant sequence homology, M. tuberculosis Cpn60 proteins interact in distinct ways with human or murine macrophages.

Antibiotic combination therapy against resistant bacterial infections: synergy, rejuvenation and resistance reduction.

Introduction: Anti-Microbial Resistance (AMR) is a pandemic which threatens modern medicine. There is a lack of effective drug treatment due to the slow pace, high cost and low achievable sales prices of new antibiotic monotherapies. New hope comes in the shape of antibiotic combination therapy, which although used by mother nature, is under-explored and could provide the solution to AMR.Areas covered: We performed a search of Pubmed and Medline using the keywords 'combination therapy', 'antimicrobial resistance' for articles between 1930 and 2019, as supplemented with other relevant references to our knowledge. We have reviewed the theoretical considerations for combination development and examine the existing and future clinical indications of combination therapies. We have discussed the potential of antibiotic combinations to provide therapeutic synergy, rejuvenating the effectiveness of old antibiotics to which the bacteria had developed resistance previously. We have examined the current thinking and evidence on resistance reduction using combination therapies, with a review on toxicity and drug-drug antagonism.Expert opinion: Antibiotic combination therapy, exploiting synergies, old-drug rejuvenation and resistance reduction could provide the solution to AMR. The number of pharmaceutical companies in this area is likely to expand, bringing promising combinations to the bedside, to save millions of lives worldwide.

Translational Model-Informed Approach for Selection of Tuberculosis Drug Combination Regimens in Early Clinical Development.

The development of optimal treatment regimens in tuberculosis (TB) remains challenging due to the need of combination therapy and possibility of pharmacodynamic (PD) interactions. Preclinical information about PD interactions needs to be used more optimally when designing early bactericidal activity (EBA) studies. In this work, we developed a translational approach which can allow for forward translation to predict efficacy of drug combination in EBA studies using the Multistate Tuberculosis Pharmacometric (MTP) and the General Pharmacodynamic Interaction (GPDI) models informed by in vitro static time-kill data. These models were linked with translational factors to account for differences between the in vitro system and humans. Our translational MTP-GPDI model approach was able to predict the EBA0-2 days , EBA0-5 days , and EBA0-14 days from different EBA studies of rifampicin and isoniazid in monotherapy and combination. Our translational model approach can contribute to an optimal dose selection of drug combinations in early TB clinical trials.

Targeting non-multiplying organisms as a way to develop novel antimicrobials.

Increasing resistance and decreasing numbers of antibiotics reaching the market point to a growing need for novel antibacterial drugs. Most antibiotics are very inefficient at killing non-multiplying bacteria, which live side by side with multiplying ones of the same strain in a clinical infection. Although non-multiplying bacteria do not usually cause disease, they can revert to the multiplying state that leads to overt disease, at which time resistance can emerge. Here we discuss the concept of developing antibacterial drugs by targeting non-multiplying organisms. We define non-multiplying bacteria, discuss the efficacy of existing antibiotics, and assess whether targeting these bacteria might lead to new antibiotics that will decrease the rate of emergence of resistance. Lastly, we review the potential of new molecular targets and live non-multiplying bacteria as possible routes for the development of novel antimicrobial drugs.

The two homologous chaperonin 60 proteins of Mycobacterium tuberculosis have distinct effects on monocyte differentiation into osteoclasts.

Mycobacterium tuberculosis produces two homologous chaperonin (Cpn)60 proteins, Cpn60.1 and Cpn60.2 (Hsp65). Both proteins stimulate human and murine monocyte cytokine synthesis but, unlike Cpn60 proteins from other microbial species, fail to stimulate the breakdown of cultured murine bone. Here, we have examined the mechanism of action of these proteins on bone remodelling and osteoclastogenesis, induced in vitro in murine calvarial explants and the murine monocyte cell line RAW264.7. Additionally, we have determined their effect on bone remodelling in vivo in an animal model of arthritis. Recombinant Cpn60.1 but not Cpn60.2 inhibited bone breakdown both in vitro, in murine calvaria and in vivo, in experimental arthritis. Analysis of the mechanism of action of Cpn60.1 suggests that this protein works by directly blocking the synthesis of the key osteoclast transcription factor, nuclear factor of activated T cells c1. The detection of circulating immunoreactive intact Cpn60.1 in a small number of patients with tuberculosis but not in healthy controls further suggests that the skeleton may be affected in patients with tuberculosis. Taken together, these findings reveal that M. tuberculosis Cpn60.1 is a potent and novel inhibitor of osteoclastogenesis both in vitro and in vivo and a potential cure for bone-resorptive diseases like osteoporosis.

A Mycobacterium tuberculosis mutant lacking the groEL homologue cpn60.1 is viable but fails to induce an inflammatory response in animal models of infection.

The causative agent of tuberculosis, Mycobacterium tuberculosis, has two chaperonin (Cpn60) proteins and one cochaperonin (Cpn10) protein. We show here that cpn60.2 and cpn10, but not cpn60.1, are essential for cell survival. A mutant lacking Cpn60.1 was indistinguishable from the wild-type organism in plate and broth culture and within murine macrophages, although it showed increased sensitivity to high temperature (55 degrees C). However, infection of mice with the Deltacpn60.1 mutant revealed a major difference from the wild-type organism. In spite of having equal numbers of bacteria in infected sites, the Deltacpn60.1 mutant failed to produce granulomatous inflammation in either mice or guinea pigs. This was associated with reduced cytokine expression in infected animals and macrophages. Cell wall lipid acid composition was not altered in the mutant strain. Thus, it appears that Cpn60.1 is an important agent in the regulation of the cytokine-dependent granulomatous response in M. tuberculosis infection.

A biphasic response from bladder epithelial cells induced by catheter material and bacteria: an in vitro study of the pathophysiology of catheter related urinary tract infection.

PURPOSE: Catheter associated urinary tract infection is the most common type of hospital acquired infection. An understanding of catheter associated urinary tract infection pathogenesis is needed to improve the control and treatment of these infections. We investigated the relationship among catheter material, bacteria and bladder epithelial cell reaction. MATERIALS AND METHODS: Urinary catheter sections and a clinical isolate of Escherichia coli were added to human bladder carcinoma epithelial cells in vitro in combination or independently. The catheters were rotated for 30 seconds over the cells, followed by incubation. The cytokines interleukin-6 and 8 were measured by enzyme-linked immunosorbent assay as indicators of inflammation and cell membrane disruption was assessed using a lactate dehydrogenase assay. RESULTS: The levels of lactate dehydrogenase release and cytokine production depended on the number of bacteria added. Bacteria grown for 3 days caused greater secretion of cytokines than bacteria grown overnight. Silicone catheter material alone caused immediate damage to cells with increased lactate dehydrogenase in the supernatant but little interleukin-6 or 8 production. Silicone catheters caused significantly less cytokine secretion from bladder cells than latex catheters. Conversely bacteria caused little immediate damage to cells but stimulated cytokine production after 12 hours. CONCLUSIONS: Disruption of bladder epithelial cell membranes in vitro occurred immediately as a result of physical abrasion caused by catheters but delayed inflammation occurred in response to bacterial infection.