Association Between Use of Lung-Protective Ventilation With Lower Tidal Volumes and Clinical Outcomes Among Patients Without Acute Respiratory Distress Syndrome A Meta-analysis

Context Lung-protective mechanical ventilation with the use of lower tidal volumes has been found to improve outcomes of patients with acute respiratory distress syndrome (ARDS). It has been suggested that use of lower tidal volumes also benefits patients who do not have ARDS. Objective To determine whether use of lower tidal volumes is associated with improved outcomes of patients receiving ventilation who do not have ARDS. Data Sources MEDLINE, CINAHL, Web of Science, and Cochrane Central Register of Controlled Trials up to August 2012. Study Selection Eligible studies evaluated use of lower vs higher tidal volumes in patients without ARDS at onset of mechanical ventilation and reported lung injury development, overall mortality, pulmonary infection, atelectasis, and biochemical alterations. Data Extraction Three reviewers extracted data on study characteristics, methods, and outcomes. Disagreement was resolved by consensus. Data Synthesis Twenty articles (2822 participants) were included. Meta-analysis using a fixed-effects model showed a decrease in lung injury development (risk ratio [RR], 0.33; 95% CI, 0.23 to 0.47; I-2, 0%; number needed to treat [NNT], 11), and mortality (RR, 0.64; 95% CI, 0.46 to 0.89; I-2, 0%; NNT, 23) in patients receiving ventilation with lower tidal volumes. The results of lung injury development were similar when stratified by the type of study (randomized vs nonrandomized) and were significant only in randomized trials for pulmonary infection and only in nonrandomized trials for mortality. Meta-analysis using a random-effects model showed, in protective ventilation groups, a lower incidence of pulmonary infection (RR, 0.45; 95% CI, 0.22 to 0.92; I-2, 32%; NNT, 26), lower mean (SD) hospital length of stay (6.91 [2.36] vs 8.87 [2.93] days, respectively; standardized mean difference [SMD], 0.51; 95% CI, 0.20 to 0.82; I-2, 75%), higher mean (SD) PaCO2 levels (41.05 [3.79] vs 37.90 [4.19] mm Hg, respectively; SMD, -0.51; 95% CI, -0.70 to -0.32; I-2, 54%), and lower mean (SD) pH values (7.37 [0.03] vs 7.40 [0.04], respectively; SMD, 1.16; 95% CI, 0.31 to 2.02; I-2, 96%) but similar mean (SD) ratios of PaO2 to fraction of inspired oxygen (304.40 [65.7] vs 312.97 [68.13], respectively; SMD, 0.11; 95% CI, -0.06 to 0.27; I-2, 60%). Tidal volume gradients between the 2 groups did not influence significantly the final results. Conclusions Among patients without ARDS, protective ventilation with lower tidal volumes was associated with better clinical outcomes. Some of the limitations of the meta-analysis were the mixed setting of mechanical ventilation (intensive care unit or operating room) and the duration of mechanical ventilation. JAMA. 2012;308(16):1651-1659 www.jama.com

Whither lung EIT: Where are we, where do we want to go and what do we need to get there?

Breathing moves volumes of electrically insulating air into and out of the lungs, producing conductivity changes which can be seen by electrical impedance tomography (EIT). It has thus been apparent, since the early days of EIT research, that imaging of ventilation could become a key clinical application of EIT. In this paper, we review the current state and future prospects for lung EIT, by a synthesis of the presentations of the authors at the 'special lung sessions' of the annual biomedical EIT conferences in 2009-2011. We argue that lung EIT research has arrived at an important transition. It is now clear that valid and reproducible physiological information is available from EIT lung images. We must now ask the question: How can these data be used to help improve patient outcomes? To answer this question, we develop a classification of possible clinical scenarios in which EIT could play an important role, and we identify clinical and experimental research programmes and engineering developments required to turn EIT into a clinically useful tool for lung monitoring.

Effects of different tidal volumes in pulmonary and extrapulmonary lung injury with or without intraabdominal hypertension

We hypothesized that: (1) intraabdominal hypertension increases pulmonary inflammatory and fibrogenic responses in acute lung injury (ALI); (2) in the presence of intraabdominal hypertension, higher tidal volume reduces lung damage in extrapulmonary ALI, but not in pulmonary ALI. Wistar rats were randomly allocated to receive Escherichia coli lipopolysaccharide intratracheally (pulmonary ALI) or intraperitoneally (extrapulmonary ALI). After 24 h, animals were randomized into subgroups without or with intraabdominal hypertension (15 mmHg) and ventilated with positive end expiratory pressure = 5 cmH(2)O and tidal volume of 6 or 10 ml/kg during 1 h. Lung and chest wall mechanics, arterial blood gases, lung and distal organ histology, and interleukin (IL)-1 beta, IL-6, caspase-3 and type III procollagen (PCIII) mRNA expressions in lung tissue were analyzed. With intraabdominal hypertension, (1) chest-wall static elastance increased, and PCIII, IL-1 beta, IL-6, and caspase-3 expressions were more pronounced than in animals with normal intraabdominal pressure in both ALI groups; (2) in extrapulmonary ALI, higher tidal volume was associated with decreased atelectasis, and lower IL-6 and caspase-3 expressions; (3) in pulmonary ALI, higher tidal volume led to higher IL-6 expression; and (4) in pulmonary ALI, liver, kidney, and villi cell apoptosis was increased, but not affected by tidal volume. Intraabdominal hypertension increased inflammation and fibrogenesis in the lung independent of ALI etiology. In extrapulmonary ALI associated with intraabdominal hypertension, higher tidal volume improved lung morphometry with lower inflammation in lung tissue. Conversely, in pulmonary ALI associated with intraabdominal hypertension, higher tidal volume increased IL-6 expression.

How large is the lung recruitability in early acute respiratory distress syndrome: a prospective case series of patients monitored by computed tomography

Introduction: The benefits of higher positive end expiratory pressure (PEEP) in patients with acute respiratory distress syndrome (ARDS) have been modest, but few studies have fully tested the "open-lung hypothesis". This hypothesis states that most of the collapsed lung tissue observed in ARDS can be reversed at an acceptable clinical cost, potentially resulting in better lung protection, but requiring more intensive maneuvers. The short-/middle-term efficacy of a maximum recruitment strategy (MRS) was recently described in a small physiological study. The present study extends those results, describing a case-series of non-selected patients with early, severe ARDS submitted to MRS and followed until hospital discharge or death. Methods: MRS guided by thoracic computed tomography (CT) included two parts: a recruitment phase to calculate opening pressures (incremental steps under pressure-controlled ventilation up to maximum inspiratory pressures of 60 cmH(2)O, at constant driving-pressures of 15 cmH(2)O); and a PEEP titration phase (decremental PEEP steps from 25 to 10 cmH2O) used to estimate the minimum PEEP to keep lungs open. During all steps, we calculated the size of the non-aerated (-100 to +100 HU) compartment and the recruitability of the lungs (the percent mass of collapsed tissue re-aerated from baseline to maximum PEEP). Results: A total of 51 severe ARDS patients, with a mean age of 50.7 years (84% primary ARDS) was studied. The opening plateau-pressure was 59.6 (+/- 5.9 cmH(2)O), and the mean PEEP titrated after MRS was 24.6 (+/- 2.9 cmH(2)O). Mean PaO2/FiO(2) ratio increased from 125 (+/- 43) to 300 (+/- 103; P < 0.0001) after MRS and was sustained above 300 throughout seven days. Non-aerated parenchyma decreased significantly from 53.6% (interquartile range (IQR): 42.5 to 62.4) to 12.7% (IQR: 4.9 to 24.2) (P < 0.0001) after MRS. The potentially recruitable lung was estimated at 45% (IQR: 25 to 53). We did not observe major barotrauma or significant clinical complications associated with the maneuver. Conclusions: MRS could efficiently reverse hypoxemia and most of the collapsed lung tissue during the course of ARDS, compatible with a high lung recruitability in non-selected patients with early, severe ARDS. This strategy should be tested in a prospective randomized clinical trial.

Pentraxin 3 accelerates lung injury in high tidal volume ventilation in mice

Mechanical ventilation is the major cause of iatrogenic lung damage in intensive care units. Although inflammation is known to be involved in ventilator-induced lung injury (VILI), several aspects of this process are still unknown. Pentraxin 3 (PTX3) is an acute phase protein with important regulatory functions in inflammation which has been found elevated in patients with acute respiratory distress syndrome. This study aimed at investigating the direct effect of PTX3 production in the pathogenesis of VILI. Genetically modified mice deficient and that over express murine Ptx3 gene were subjected to high tidal volume ventilation (V-T = 45 mL/kg, PEEPzero). Morphological changes and time required for 50% increase in respiratory system elastance were evaluated. Gene expression profile in the lungs was also investigated in earlier times in Ptx3-overexpressing mice. Ptx3 knockout and wild-type mice developed same lung injury degree in similar times (156 +/- 42 min and 148 +/- 41 min, respectively: p = 0.8173). However, Ptx3 overexpression led to a faster development of VILI in Ptx3-overexpressing mice (77 +/- 29 min vs 118 +/- 41 min, p = 0.0225) which also displayed a faster kinetics of Il1b expression and elevated Ptx3, Cxcl1 and Ccl2 transcripts levels in comparison with wild-type mice assessed by quantitative real-time polymerase chain reaction. Ptx3 deficiency did not impacted the time for VILI induced by high tidal volume ventilation but Ptx3-overexpression increased inflammatory response and reflected in a faster VILI development. (C) 2012 Elsevier Ltd. All rights reserved.

Protective effects of bone marrow mononuclear cell therapy on lung and heart in an elastase-induced emphysema model

We hypothesized that bone marrow-derived mononuclear cell (BMDMC) therapy protects the lung and consequently the heart in experimental elastase-induced emphysema. Twenty-four female C57BL/6 mice were intratracheally instilled with saline (C group) or porcine pancreatic elastase (E group) once a week during 4 weeks. C and E groups were randomized into subgroups receiving saline (SAL) or male BMDMCs (2 x 10(6), CELL) intravenously 3 h after the first saline or elastase instillation. Compared to E-SAL group, E-CELL mice showed, at 5 weeks: lower mean linear intercept, neutrophil infiltration, elastolysis, collagen fiber deposition in alveolar septa and pulmonary vessel wall, lung cell apoptosis, right ventricle wall thickness and area, higher endothelial growth factor and insulin-like growth factor mRNA expressions in lung tissue, and reduced platelet-derived growth factor, transforming growth factor-beta, and caspase-3 expressions. In conclusion, BMDMC therapy was effective at modulating the inflammatory and remodeling processes in the present model of elastase-induced emphysema. (c) 2012 Elsevier B.V. All rights reserved.

N-acetylcysteine prevents pulmonary edema and acute kidney injury in rats with sepsis submitted to mechanical ventilation

Campos R, Shimizu MH, Volpini RA, de Bragan a AC, Andrade L, Lopes FD, Olivo C, Canale D, Seguro AC. N-acetylcysteine prevents pulmonary edema and acute kidney injury in rats with sepsis submitted to mechanical ventilation. Am J Physiol Lung Cell Mol Physiol 302: L640-L650, 2012. First published January 20, 2012; doi: 10.1152/ajplung.00097.2011.-Sepsis is a common cause of acute kidney injury (AKI) and acute lung injury. Oxidative stress plays as important role in such injury. The aim of this study was to evaluate the effects that the potent antioxidant N-acetylcysteine (NAC) has on renal and pulmonary function in rats with sepsis. Rats, treated or not with NAC (4.8 g/l in drinking water), underwent cecal ligation and puncture (CLP) 2 days after the initiation of NAC treatment, which was maintained throughout the study. At 24 h post-CLP, renal and pulmonary function were studied in four groups: control, control + NAC, CLP, and CLP + NAC. All animals were submitted to low-tidal-volume mechanical ventilation. We evaluated respiratory mechanics, the sodium cotransporters Na-K-2Cl (NKCC1) and the alpha-subunit of the epithelial sodium channel (alpha-ENaC), polymorphonuclear neutrophils, the edema index, oxidative stress (plasma thiobarbituric acid reactive substances and lung tissue 8-isoprostane), and glomerular filtration rate. The CLP rats developed AKI, which was ameliorated in the CLP + NAC rats. Sepsis-induced alterations in respiratory mechanics were also ameliorated by NAC. Edema indexes were lower in the CLP + NAC group, as was the wet-to-dry lung weight ratio. In CLP + NAC rats, alpha-ENaC expression was upregulated, whereas that of NKCC1 was downregulated, although the difference was not significant. In the CLP + NAC group, oxidative stress was significantly lower and survival rates were significantly higher than in the CLP group. The protective effects of NAC (against kidney and lung injury) are likely attributable to the decrease in oxidative stress, suggesting that NAC can be useful in the treatment of sepsis.

Protective effects of aerobic exercise on acute lung injury induced by LPS in mice

Abstract Introduction The regular practice of physical exercise has been associated with beneficial effects on various pulmonary conditions. We investigated the mechanisms involved in the protective effect of exercise in a model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Methods Mice were divided into four groups: Control (CTR), Exercise (Exe), LPS, and Exercise + LPS (Exe + LPS). Exercised mice were trained using low intensity daily exercise for five weeks. LPS and Exe + LPS mice received 200 µg of LPS intratracheally 48 hours after the last physical test. We measured exhaled nitric oxide (eNO); respiratory mechanics; neutrophil density in lung tissue; protein leakage; bronchoalveolar lavage fluid (BALF) cell counts; cytokine levels in BALF, plasma and lung tissue; antioxidant activity in lung tissue; and tissue expression of glucocorticoid receptors (Gre). Results LPS instillation resulted in increased eNO, neutrophils in BALF and tissue, pulmonary resistance and elastance, protein leakage, TNF-alpha in lung tissue, plasma levels of IL-6 and IL-10, and IL-1beta, IL-6 and KC levels in BALF compared to CTR (P ≤0.02). Aerobic exercise resulted in decreases in eNO levels, neutrophil density and TNF-alpha expression in lung tissue, pulmonary resistance and elastance, and increased the levels of IL-6, IL-10, superoxide dismutase (SOD-2) and Gre in lung tissue and IL-1beta in BALF compared to the LPS group (P ≤0.04). Conclusions Aerobic exercise plays important roles in protecting the lungs from the inflammatory effects of LPS-induced ALI. The effects of exercise are mainly mediated by the expression of anti-inflammatory cytokines and antioxidants, suggesting that exercise can modulate the inflammatory-anti-inflammatory and the oxidative-antioxidative balance in the early phase of ALI.

Losartan exerts no protective effects against acute pulmonary embolism-induced hemodynamic changes

The acute obstruction of pulmonary vessels by venous thrombi is a critical condition named acute pulmonary embolism (APE). During massive APE, severe pulmonary hypertension may lead to death secondary to right heart failure and circulatory shock. APE-induced pulmonary hypertension is aggravated by active pulmonary vasoconstriction. While blocking the effects of some vasoconstrictors exerts beneficial effects, no previous study has examined whether angiotensin II receptor blockers protect against the hemodynamic changes associated with APE. We examined the effects exerted by losartan on APE-induced hemodynamic changes. Hemodynamic evaluations were performed in non-embolized lambs treated with saline (n = 4) and in lambs that were embolized with silicon microspheres and treated with losartan (30 mg/kg followed by 1 mg/kg/h, n = 5) or saline (n = 7) infusions. The plasma and lung angiotensin-converting enzyme (ACE) activity were assessed using a fluorometric method. APE increased mean pulmonary arterial pressure (MPAP) and pulmonary vascular resistance index (PVRI) by 21 +/- 2 mmHg and 375 +/- 20 dyn s cm(-5) m(-2), respectively (P < 0.05). Losartan decreased MPAP significantly (by approximately 15%), without significant changes in PVRI and tended to decrease cardiac index (P > 0.05). Lung and plasma ACE activity were similar in both embolized and non-embolized animals. Our findings show evidence of lack of activation of the renin-angiotensin system during APE. The lack of significant effects of losartan on the pulmonary vascular resistance suggests that losartan does not protect against the hemodynamic changes found during APE.

Effects of Positive End-expiratory Pressure Titration and Recruitment Maneuver on Lung Inflammation and Hyperinflation in Experimental Acid Aspiration-induced Lung Injury

Background: In acute lung injury positive end-expiratory pressure (PEEP) and recruitment maneuver are proposed to optimize arterial oxygenation. The aim of the study was to evaluate the impact of such a strategy on lung histological inflammation and hyperinflation in pigs with acid aspiration-induced lung injury. Methods: Forty-seven pigs were randomly allocated in seven groups: (1) controls spontaneously breathing; (2) without lung injury, PEEP 5 cm H2O; (3) without lung injury, PEEP titration; (4) without lung injury, PEEP titration + recruitment maneuver; (5) with lung injury, PEEP 5 cm H2O; (6) with lung injury, PEEP titration; and (7) with lung injury, PEEP titration + recruitment maneuver. Acute lung injury was induced by intratracheal instillation of hydrochloric acid. PEEP titration was performed by incremental and decremental PEEP from 5 to 20 cm H2O for optimizing arterial oxygenation. Three recruitment maneuvers (pressure of 40 cm H2O maintained for 20 s) were applied to the assigned groups at each PEEP level. Proportion of lung inflammation, hemorrhage, edema, and alveolar wall disruption were recorded on each histological field. Mean alveolar area was measured in the aerated lung regions. Results: Acid aspiration increased mean alveolar area and produced alveolar wall disruption, lung edema, alveolar hemorrhage, and lung inflammation. PEEP titration significantly improved arterial oxygenation but simultaneously increased lung inflammation in juxta-diaphragmatic lung regions. Recruitment maneuver during PEEP titration did not induce additional increase in lung inflammation and alveolar hyperinflation. Conclusion: In a porcine model of acid aspiration-induced lung injury, PEEP titration aimed at optimizing arterial oxygenation, substantially increased lung inflammation. Recruitment maneuvers further improved arterial oxygenation without additional effects on inflammation and hyperinflation.

ROLE OF FOCAL ADHESION KINASE IN LUNG REMODELING OF ENDOTOXEMIC RATS

Despite significant advances in the care of critically ill patients, acute lung injury continues to be a complex problem with high mortality. The present study was designed to characterize early lipopolysaccharide (LPS)-induced pulmonary injury and small interfering RNA targeting focal adhesion kinase (FAK) as a possible therapeutic tool in the septic lung remodeling process. Male Wistar rats were assigned into endotoxemic group and control group. Total collagen deposition was performed 8, 16, and 24 h after LPS injection. Focal adhesion kinase expression, interstitial and vascular collagen deposition, and pulmonary mechanics were analyzed at 24 h. Intravenous injection of small interfering RNA targeting FAK was used to silence expression of the kinase in pulmonary tissue. Focal adhesion kinase, total collagen deposition, and pulmonary mechanics showed increased in LPS group. Types I, III, and V collagen showed increase in pulmonary parenchyma, but only type V increased in vessels 24 h after LPS injection. Focal adhesion kinase silencing prevented lung remodeling in pulmonary parenchyma at 24 h. In conclusion, LPS induced a precocious and important lung remodeling. There was fibrotic response in the lung characterized by increased amount in total and specific-type collagen. These data may explain the frequent clinical presentation during sepsis of reduced lung compliance, oxygen diffusion, and pulmonary hypertension. The fact that FAK silencing was protective against lung collagen deposition underscores the therapeutic potential of FAK targeting by small interfering RNA.

Regional lung perfusion estimated by electrical impedance tomography in a piglet model of lung collapse

Borges JB, Suarez-Sipmann F, Bohm SH, Tusman G, Melo A, Maripuu E, Sandstrom M, Park M, Costa EL, Hedenstierna G, Amato M. Regional lung perfusion estimated by electrical impedance tomography in a piglet model of lung collapse. J Appl Physiol 112: 225-236, 2012. First published September 29, 2011; doi: 10.1152/japplphysiol.01090.2010.-The assessment of the regional match between alveolar ventilation and perfusion in critically ill patients requires simultaneous measurements of both parameters. Ideally, assessment of lung perfusion should be performed in real-time with an imaging technology that provides, through fast acquisition of sequential images, information about the regional dynamics or regional kinetics of an appropriate tracer. We present a novel electrical impedance tomography (EIT)-based method that quantitatively estimates regional lung perfusion based on first-pass kinetics of a bolus of hypertonic saline contrast. Pulmonary blood flow was measured in six piglets during control and unilateral or bilateral lung collapse conditions. The first-pass kinetics method showed good agreement with the estimates obtained by single-photon-emission computerized tomography (SPECT). The mean difference (SPECT minus EIT) between fractional blood flow to lung areas suffering atelectasis was -0.6%, with a SD of 2.9%. This method outperformed the estimates of lung perfusion based on impedance pulsatility. In conclusion, we describe a novel method based on EIT for estimating regional lung perfusion at the bedside. In both healthy and injured lung conditions, the distribution of pulmonary blood flow as assessed by EIT agreed well with the one obtained by SPECT. The method proposed in this study has the potential to contribute to a better understanding of the behavior of regional perfusion under different lung and therapeutic conditions.

Comparison of 3 modes of automated weaning from mechanical ventilation: A bench study

Purpose: Automated weaning modes are available in some mechanical ventilators, but no studies compared them hitherto. We compared the performance of 3 automated modes under standard and challenging situations. Methods: We used a lung simulator to compare 3 automated modes, adaptive support ventilation (ASV), mandatory rate ventilation (MRV), and Smartcare, in 6 situations, weaning success, weaning failure, weaning success with extreme anxiety, weaning success with Cheyne-Stokes, weaning success with irregular breathing, and weaning failure with ineffective efforts. Results: The 3 modes correctly recognized the situations of weaning success and failure, even when anxiety or irregular breathing were present but incorrectly recognized weaning success with Cheyne-Stokes. MRV incorrectly recognized weaning failure with ineffective efforts. Time to pressure support (PS) stabilization was shorter for ASV (1-2 minutes for all situations) and MRV (1-7 minutes) than for Smartcare (8-78 minutes). ASV had higher rates of PS oscillations per 5 minutes (4-15), compared with Smartcare (0-1) and MRV (0-12), except when extreme anxiety was present. Conclusions: Smartcare, ASV, and MRV were equally able to recognize weaning success and failure, despite the presence of anxiety or irregular breathing but performed incorrectly in the presence of Cheyne-Stokes. PS behavior over the time differs among modes, with ASV showing larger and more frequent PS oscillations over the time. Clinical studies are needed to confirm our results. (C) 2012 Elsevier Inc. All rights reserved.

Risk Factors and Survival Impact of Primary Graft Dysfunction After Lung Transplantation in a Single Institution

Background. Lung transplantation has become a standard procedure for some end-stage lung diseases, but primary graft dysfunction (PGD) is an inherent problem that impacts early and late outcomes. The aim of this study was to define the incidence, risk factors, and impact of mechanical ventilation time on mortality rates among a retrospective cohort of lung transplantations performed in a single institution. Methods. We performed a retrospective study of 118 lung transplantations performed between January 2003 and July 2010. The most severe form of PGD (grade III) as defined at 48 and 72 hours was examined for risk factors by multivariable logistic regression models using donor, recipient, and transplant variables. Results. The overall incidence of PGD at 48 hours was 19.8%, and 15.4% at 72 hours. According multivariate analysis, risk factors associated with PGD were donor smoking history for 48 hours (adjusted odds ratio [OR], 4.83; 95% confidence interval [CI], 1.236-18.896; P = .022) and older donors for 72 hours (adjusted OR, 1.046; 95% CI, 0.997-1.098; P = .022). The operative mortality was 52.9% among patients with PGD versus 20.3% at 48 hours (P = .012). At 72 hours, the mortality rate was 58.3% versus 21.2% (P = .013). The 90-days mortality was also higher among patients with PGD. The mechanical ventilation time was longer in patients with PGD III at 48 hours namely, a mean time of 72 versus 24 hours (P = .001). When PGD was defined at 72 hours, the mean ventilation time was even longer, namely 151 versus 24 hours (P < .001). The mean overall survival for patients who developed PGD at 48 hours was 490.9 versus 1665.5 days for subjects without PGD (P = .001). Considering PGD only at 72 hours, the mean survival was 177.7 days for the PGD group and 1628.9 days for the other patients (P < .001). Conclusion. PGD showed an important impacts on operative and 90-day mortality rates, mechanical ventilation time, and overall survival among lung transplant patients. PGD at 72 hours was a better predictor of lung transplant outcomes than at 48 hours. The use of donors with a smoking history or of advanced age were risk factors for the development of PGD.

Improve protective efficacy of a TB DNA-HSP65 vaccine by BCG priming

Vaccines are considered by many to be one of the most successful medical interventions against infectious diseases. But many significant obstacles remain, such as optimizing DNA vaccines for use in humans or large animals. The amount of doses, route and easiness of administration are also important points to consider in the design of new DNA vaccines. Heterologous prime-boost regimens probably represent the best hope for an improved DNA vaccine strategy. In this study, we have shown that heterologous prime-boost vaccination against tuberculosis (TB) using intranasal BCG priming/DNA-HSP65 boosting (BCGin/DNA) provided significantly greater protection than that afforded by a single subcutaneous or intranasal dose of BCG. In addition, BCGin/DNA immunization was also more efficient in controlling bacterial loads than were the other prime-boost schedules evaluated or three doses of DNA-HSP65 as a naked DNA. The single dose of DNA-HSP65 booster enhanced the immunogenicity of a single subcutaneous BCG vaccination, as evidenced by the significantly higher serum levels of anti-Hsp65 IgG2a Th1-induced antibodies, as well as by the significantly greater production of IFN-γ by antigen-specific spleen cells. The BCG prime/DNA-HSP65 booster was also associated with better preservation of lung parenchyma.