Background: Despite research into the mechanisms of injury in blast-induced thoracic trauma using animal or clinical models, some questions regarding the exact nature of such inju-ries have not been fully addressed. The present study aims to investigate the temporal altera-tions in the quantity and functions of Alveolar Macrophages (AMs) associated with intrapulmo-nary haemorrhage in a prospective rodent model of primary blast lung injury. Methods: Female Wister rats were randomly divided into two experimental groups: 1. the blast group, which re-ceived a single standardised blast wave; 2. the control group, which received no trauma but un-derwent the same procedure as the experimental group. AMs were harvested, cultured, and stimulated. The number of AMs was determined, and their capacity for phagocytosis, respiratory burst, and release of cytokines was determined, with or without the contribution of blood com-ponents. Results: The number of AMs exhibited a time-dependent pattern of changes. Phagocy-tosis of dead Escherichia coli (E. coli) and superoxide production by AMs decreased 10 min and 24 h after trauma, but both parameters increased significantly 96 h after the insult. These results sug-gest a transiently impaired response of AMs to a bacterial stimulus in the initial period after thoracic trauma. However, the inclusion of blood components in the experiment further reduced the phagocytic capacity and respiratory burst of AMs. Conclusions: Blunt thoracic trauma in the present form and intensity impaired two important antibacterial functions of innate immunity, including the phagocytosis and superoxide generation capacity of AM and their ability to pro-duce TNF. These changes are followed by a time-dependent recovery. Alveolar haemorrhage has been shown to be at least partially involved in the impairment of the phagocytosis capacity of AMs.

BACKGROUND AND OBJECTIVES:Blast-induced lung injury is associated with inflammatory, which are characterised by disruption of the alveolar-capillary barrier, haemorrhage, pulmonary infiltrateration causing oedema formation, pro-inflammatory cytokine and chemokine release, and anti-inflammatory counter-regulation. The objective of the current study was to define sequence of such alterations in with establishing blast-induced lung injury in rats using an advanced blast generator.METHODS:Rats underwent a standardized blast wave trauma and were euthanised at defined time points. Non-traumatised animals served as sham controls. Obtained samples from bronchoalveolar lavage fluid (BALF) at each time-point were assessed for histology, leukocyte infiltration and cytokine/chemokine profile.RESULTS:After blast lung injury, significant haemorrhage and neutrophil infiltration were observed. Similarly, protein accumulation, lactate dehydrogenase activity (LDH), alveolar eicosanoid release, matrix metalloproteinase (MMP)-2 and -9, pro-Inflammatory cytokines, including tumour necrosis factor (TNF) and interleukin (IL) -6 raised up. While declining in the level of anti-inflammatory cytokine IL-10 occurred. Ultimately, pulmonary oedema developed that increased to its maximum level within the first 1.5 h, then recovered within 24 h.CONCLUSION:Using a stablished model, can facilitate the study of inflammatory response to blast lung injury. Following the blast injury, alteration in cytokine/chemokine profile and activity of cells in the alveolar space occurs, which eventuates in alveolar epithelial barrier dysfunction and oedema formation. Most of these parameters exhibit time-dependent return to their basal status that is an indication to resilience of lungs to blast-induced lung injury.

Objective: Current treatments for blast-induced lung injury are limited to supportive procedures including mechanical ventilation. The study aimed to investigate the role of post-trauma-induced oedema generation in the function of time and trauma intensity and the probable role of beta 2-adrenergic receptors (β2-ARs) agonists on pulmonary oedema. The study is conducted using an ex vivo model after an experimental in vivo blast-induced thorax trauma in rats. Methods: Rats were randomised and divided into two groups, blast and sham. The blast group were anaesthetised and exposed to the blast wave (3.16 ± 0.43 bar) at a distance of 3.5 cm from the thorax level. The rats were sacrificed 10 min after the blast, the lungs explanted and treated with terbutaline, formoterol, propranolol or amiloride to assess the involvement of sodium transport. Other groups of rats were exposed to distances of 5 and 7 cm from the thorax to reduce the intensity of the injury. Further, one group of rats was studied after 180 min and one after 360 min after a 3.5 cm blast injury. Sham controls were exposed to identical procedures except for receiving blast overpressure. Results: Lung injury and oedema generation depended on time after injury and injury intensity. Perfusion with amiloride resulted in a further increase in oedema formation as indicated by weight gain (p < 0.001), diminished tidal volume (Tv) (p < 0.001), and increased airway resistance (p < 0.001). Formoterol caused a significant increase in the Tv (p < 0.001) and a significant decrease in the airway resistance (p < 0.01), while the lung weight was not influenced. Trauma-related oedema was significantly reduced by terbutaline in terms of lung weight gain (p < 0.01), Tv (p < 0.001), and airway resistance (p < 0.01) compared to control blast-injured lungs. Terbutaline-induced effects were completely blocked by the β-receptor antagonist propranolol (p < 0.05). Similarly, amiloride, which was added to terbutaline perfusion, reversed terbutaline-induced weight gain reduction (p < 0.05). Conclusions: β2-adrenoceptor stimulation had a beneficial impact by amiloride-dependent sodium and therefore, fluid transport mechanisms on the short-term ex vivo oedema generation in a trauma-induced in vivo lung injury of rats.

Rationale: Alveolar liquid clearance is regulated by Na+ uptake through the apically expressed epithelial sodium channel (ENaC) and basolaterally localized Na+-K+-ATPase in type II alveolar epithelial cells. Dysfunction of these Na+ transporters during pulmonary inflammation can contribute to pulmonary edema.Objectives: In this study, we sought to determine the precise mechanism by which the TIP peptide, mimicking the lectin-like domain of tumor necrosis factor (TNF), stimulates Na+ uptake in a homologous cell system in the presence or absence of the bacterial toxin pneumolysin (PLY).Methods: We used a combined biochemical, electrophysiological, and molecular biological in vitro approach and assessed the physiological relevance of the lectin-like domain of TNF in alveolar liquid clearance in vivo by generating triple-mutant TNF knock-in mice that express a mutant TNF with deficient Na+ uptake stimulatory activity.Measurements and Main Results: TIP peptide directly activates ENaC, but not the Na+-K+-ATPase, upon binding to the carboxy-terminal domain of the α subunit of the channel. In the presence of PLY, a mediator of pneumococcal-induced pulmonary edema, this binding stabilizes the ENaC-PIP2-MARCKS complex, which is necessary for the open probability conformation of the channel and preserves ENaC-α protein expression, by means of blunting the protein kinase C-α pathway. Triple-mutant TNF knock-in mice are more prone than wild-type mice to develop edema with low-dose intratracheal PLY, correlating with reduced pulmonary ENaC-α subunit expression.Conclusions: These results demonstrate a novel TNF-mediated mechanism of direct ENaC activation and indicate a physiological role for the lectin-like domain of TNF in the resolution of alveolar edema during inflammation.

Background Tumor necrosis factor alpha (TNF) is able to kill cancer cells via receptor-mediated cell death requiring adenosine triphosphate (ATP). Clinical usage of TNF so far is largely limited by its profound hepatotoxicity. Recently, it was found in the murine system that specific protection of hepatocytes against TNF's detrimental effects can be achieved by fructose-mediated ATP depletion therein. Before employing this quite attractive selection principle in a first clinical trial, we here comprehensively investigated the interdependence between ATP depletion and TNF hepatotoxicity in both in vitro and ex vivo experiments based on usage of primary patient tissue materials. Methods Primary human hepatocytes, and both non-tumorous and tumorous patient-derived primary liver tissue slices were used to elucidate fructose-induced ATP depletion and TNF-induced cytotoxicity. Results PHH as well as tissue slices prepared from non-malignant human liver specimen undergoing a fructose-mediated ATP depletion were both demonstrated to be protected against TNF-induced cell death. In contrast, due to tumor-specific overexpression of hexokinase II, which imposes a profound bypass on hepatocytic-specific fructose catabolism, this was not the case for human tumorous liver tissues. Conclusion Normal human liver tissues can be protected transiently against TNF-induced cell death by systemic pretreatment with fructose used in non-toxic/physiologic concentrations. Selective TNF-targeting of primary and secondary tumors of the liver by transient and specific depletion of hepatocytic ATP opens up a new clinical avenue for the TNF-based treatment of liver cancers.

Fructose-induced hepatic ATP depletion prevents TNF-induced apoptosis, whereas it contrarily enhances CD95-induced hepatocyte apoptosis in vitro and in vivo. By contrast, transformed liver cells are not protected against TNF due to metabolic alterations, allowing selective tumor targeting. We analyzed the molecular mechanisms by which fructose modulates cytokine-induced apoptosis. A release of adenosine after fructose-induced ATP depletion, followed by a cAMP response, was demonstrated. Likewise, cAMP and adenosine mimicked per se the modulation by fructose of CD95- and TNF-induced apoptosis. The effects of fructose on cytokine-induced apoptosis were sensitive to inhibition of protein kinase A. Fructose prevented the pro-apoptotic, sustained phase of TNF-induced JNK signaling and thereby blocked bid-mediated activation of the intrinsic mitochondrial apoptosis pathway in a PKA-dependent manner. We explain the dichotomal effects of fructose on CD95- and TNF-induced cell death by the selective requirement of JNK signaling for the latter. These findings provide a mechanistic rationale for the protection of hepatocytes from TNF-induced cell death by pharmacological doses of fructose.

Background & aims: Despite its high potency, TNF's current use in medical oncology is restricted since effective doses leading to profound hepatotoxicity. Interestingly, TNF-induced cytotoxicity is known to be a highly ATP-dependent process. In primary murine hepatocytes it was recognized that fructose transiently depletes ATP which is due to a distinct enzyme configuration being specific for hepatocytes leading to a TNF resistance (Speicher et al., 2010). In light of the murine results, the goal of our current study was to find out whether the therapeutic benefits of fructose also apply to the human situation.

Histone deacetylases (HD) represent a novel target in cancer treatment, particularly for scattered small tumours such as the hepatocellular carcinoma (HCC). However, only few studies address the toxicological impact of HD Inhibitors (HDIs) on malignantly transformed cells versus primary hepatocytes. We examined whether and how different classes of HDIs sensitise the human HCC cell line HepG2, primary healthy murine and human liver cells towards the death receptor agonists TNFα and CD95L. Apicidin, M344 (N-hydroxy-7-(-4-dimethylaminobenzol)aminoheptanamide), CBHA (m-carboxycinnamic acid bis-hydroxamide) and VPA (valproic acid) sensitised liver cell cultures towards CD95-triggered apoptosis with the following potency: apicidin > M344 ≈ CBHA ≫ VPA. Apicidin sensitised towards CD95 also in the intact organ, i.e. in the isolated perfused mouse liver. No significant sensitisation towards TNFα was found in vitro. Western blot analysis showed that all HDIs studied downregulated the anti-apoptotic protein cFLIP, but only VPA additionally affected the expression level of XIAP. Furthermore, in models of the intrinsic apoptosis pathway, i.e. in HepG2 cells treated with Melphalan and in primary hepatocytes irradiated with UV light, only VPA exhibited significant sensitisation. These findings extend the biochemical, pharmacological and toxicological basis for HDI therapy and provide a caveat for clinical use in patients with an accompanying critical inflammatory state in which the CD95 system might be pre-activated.

We report here an affinity-proteomics approach that combines 2D-gel electrophoresis and immunoblotting with high performance mass spectrometry to the identification of both full length protein antigens and antigenic fragments of Chlamydophila pneumoniae (C. pneumoniae). The present affinity-mass spectrometry approach effectively utilized high resolution FTICR mass spectrometry and LC-tandem-MS for protein identification, and enabled the identification of several new highly antigenic C. pneumoniae proteins that were not hitherto reported or previously detected only in other Chlamydia species, such as Chlamydia trachomatis. Moreover, high resolution affinity-MS provided the identification of several neo-antigenic protein fragments containing N- and C-terminal, and central domains such as fragments of the membrane protein Pmp21 and the secreted chlamydial proteasome-like factor (Cpaf), representing specific biomarker candidates.

Objectives Pulmonary edema, which involves a reduced alveolar Na+ uptake capacity, can be a major complication of listeriosis. We have investigated the effect of the main virulence factor of L. monocytogenes, i.e. listeriolysin (LLO), on the expression of the crucial alpha subunit of ENaC and on Protein Kinase C (PKC)-alpha, a negative regulator of ENaC expression, in the human H441 cell line. We moreover assessed LLO effects on amiloride-sensitive Na+ currents in vitro and on edema formation in vivo. Results ENaC-alpha expression and amiloride-sensitive sodium uptake in H441 cells are significantly decreased upon LLO-treatment, an effect completely blunted by the PKC-alpha inhibitor Ro-32-0432. The TNF-derived TIP peptide blocks LLO-mediated PKC-alpha activation, significantly restores ENaC-alpha expression and increases amiloride-sensitive Na+ uptake. In vivo, LLO, upon intratracheal instillation, significantly increases lung wet-to-dry ratio, which can be blunted by the TIP peptide. Conclusion These results demonstrate that LLO contributes to edema formation by reducing ENaC function in a PKC-alpha-dependent manner. The TIP peptide restores impaired ENaC expression and activates Na+ uptake in LLO-treated airway epithelial cells, thus indicating its therapeutic potential for the treatment of listeriosis-related permeability edema. (Supported by grant RO1HL094609 from the NHBLI)

BACKGROUND & AIMS:TNF was the first cytokine employed for cancer therapy, but its use was limited due to its insufficient selectivity towards malignant cells. Fructose induces transient hepatic ATP depletion in humans and rodents due to the liver-specific fructose metabolism via fructokinase, while other cells e.g. Muscle cells metabolize fructose via hexokinase. Under ATP depleted conditions hepatocytes are protected against TNF-induced apoptosis. Our aim was to identify metabolic differences between normal and malignant liver cells that can be exploited for selective immunotherapy. METHODS:We analyzed the expression and activities of enzymes involved in fructose metabolism in primary hepatocytes and hepatoma cell lines. Furthermore, we studied the influence of hexokinase II (HKII) on fructose-mediated ATP depletion and cytoprotection in murine hepatocytes. RESULTS:Primary mouse, rat and human hepatocytes depleted of ATP by fructose were fully protected against TNF-induced cytotoxicity. By contrast, hepatic tumor cell lines showed increased HKII expression, lack of fructose-mediated ATP depletion and, therefore, remained susceptible to TNF/ActD-induced apoptosis. Inhibition of hexokinases restored fructose-induced ATP depletion in hepg2 cells. Finally, hypoxia-inducible factor1 (HIF1)-mediated up-regulation of HKII prevented fructose-induced ATP depletion and overexpression of HKII inhibited fructose-mediated cytoprotection against TNF-induced apoptosis in primary murine hepatocytes. CONCLUSION:Increased expression of HKII in malignant cells of hepatic origin shifts the fructose metabolism from liver- to muscle-type, thereby preventing ATP depletion and subsequent cytoprotection of the target cells. Therefore, healthy liver cells are transiently protected from TNF-mediated cell death by fructose-induced ATP depletion, while malignant cells can be selectively eliminated through TNF-induced apoptosis.

Listeriosis can lead to potentially lethal pulmonary complications in newborns and immune compromised patients, characterized by extensive permeability edema. Listeriolysin (LLO), the main virulence factor of Listeria monocytogenes, induces a dose-dependent hyperpermeability in monolayers of human lung microvascular endothelial cells in vitro. The permeability increasing activity of LLO, which is accompanied by an increased reactive oxygen species (ROS) generation, RhoA activation and myosin light chain (MLC) phosphorylation, can be completely inhibited by the protein kinase C (PKC) alpha/beta inhibitor GO6976, indicating a crucial role for PKC in the induction of barrier dysfunction. The TNF-derived TIP peptide, which mimics the lectin-like domain of the cytokine, blunts LLO-induced hyperpermeability in vitro, upon inhibiting LLO-induced protein kinase C-alpha activation, ROS generation and MLC phosphorylation and upon restoring the RhoA/Rac 1 balance. These results indicate that the lectin-like domain of TNF has a potential therapeutic value in protecting from LLO-induced pulmonary endothelial hyperpermeability.

β2-Agonists have been shown to increase alveolar fluid reabsorption, and at least part of their effect depends on active sodium transport from the alveolus into the epithelial cell by the amiloride-sensitive epithelial sodium channel (ENaC). Few data exist on their effect in the injured lung. The authors therefore investigated the effect of intrabronchially administered terbutaline pretransplantation by measuring outcome 1 day after experimental donor lung transplantation with severe injury due to prolonged ischemia. Orthotopic single left-sided lung allotransplantation was performed in female rats (Wistar to Wistar) after a total ischemic time of 20 hours. Graft PaO2/FiO2 in 6 recipients treated with 10−4 M terbutaline in 500 μ L NaCl 0.9% was superior 24 hours after transplantation, with a PaO2 of 329 (111 [SD]) mm Hg versus 5 vehicle controls with 44 (15) mm Hg (P =. 002). The beneficial effect of 10−4 M terbutaline was abrogated by 10−4 M of the sodium channel blocker amiloride to 71 (34) mm Hg in 3 recipients (P =. 028 versus terbutaline 10−4 M). Ten recipients receiving 10−5 M terbutaline in 500 μ L NaCl 0.9% showed inconsistent improvements of gas exchange, with a PaO2 of 158 (± 153) mm Hg (P =. 058). Terbutaline at a high dose significantly improved the transplanted rat lung function at 24 hours after transplantation. Part of it may be via activating epithelial sodium transport, thus suggesting an important role of alveolar fluid transport in such a model of acute lung injury.

Listeriosis can result in lethal pulmonary permeability edema, with reduced sodium uptake capacity and epithelial/endothelial hyperpermeability. This study investigated the effect of L. monocytogenes' main virulence factor, Listeriolysin O (LLO), on the serum and glucocorticoid dependent kinase 1 (SGK1) and on prostasin, both positive regulators of epithelial sodium channel (ENaC) expression. Moreover, the effects of the TNF‐derived TIP peptide, mimicking its lectin‐like domain, on SGK1 and prostasin expression and permeability in LLO‐treated human H441 cells were investigated. This peptide reduces edema formation in murine models. LLO significantly reduced T256 phosphorylation, required for full activation of SGK1, blunted prostasin expression and reduced expression of the crucial ENaC alpha subunit (Western Blotting). The toxin dose‐dependently induced hyperpermeability in H441 monolayers, as assessed in Electrical Cell Substrate Impedance Sensing. The TIP peptide significantly inhibited hyperpermeability and restored SGK1 activity at 24h in LLO‐treated H441 cells. LLO is thus implicated in permeability edema formation by reducing ENaC expression and epithelial barrier integrity. The TNF‐derived TIP peptide can counteract these effects, indicating its therapeutic potential for the treatment of listeriosis‐related permeability edema.

5-Azacytidine (5-aza-CR) is a DNA-hypomethylating antineoplastic agent used because of its inhibitory activity on DNA methyltransferases. Today, it is approved as an epigenetically active drug therapy for treatment of myelodysplastic disorders, with a contraindication as to pre-existing liver diseases. Because the mechanism of its hepatotoxicity is still unknown, we investigated the pharmacodynamic properties of 5-aza-CR with regard to death receptor/ligand-induced apoptosis and the mode of execution of cell death. In a time- and concentration-dependent manner, primary murine, human hepatocytes and HepG2 cells exposed to 5-aza-CR became highly sensitive toward cell death induced by CD95L, tumor necrosis factor (TNF)-related apoptosis-inducing ligand, or TNF. Cell death was characterized as apoptotic by membrane blebbing, chromatin condensation, and exposure of phosphatidylserine on the outer membrane. Neither 5-aza-2'-deoxycytidine nor the common DNA methyltransferase inhibitors S-(5'-adenosyl)-L-homocysteine or RG 108 showed any significant effects under these conditions. Despite the complete protection of HepG2 by high concentrations of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp( O-Me) fluoromethyl ketone (z-VAD-fmk), effector caspase-3/7 activity was completely abolished at approximately a 20-fold lower concentration of z-VAD-fmk. Under these conditions, the serine protease inhibitors N, alpha-tosyl-L-phenylalanine chloromethyl ketone, N, p-tosyl-L-lysine chloromethyl ketone, and 4-(2-aminoethyl)-benzenesulfonyl fluoride, respectively, conferred protection against death receptor ligands. We conclude that this caspase-independent apoptosis is executed by a yet-unidentified serine protease.

The heterocyclic organic compound ebselen (2-phenyl-1,2-benizsoselenazol-3(2H)-one) is a glutathione peroxidase mimick with protective properties against oxidative injury. Ebselen also has anti-inflammatory activity, including attenuation of tumor necrosis factor release and increase of interleukin-10, as shown in vivo, in inflammatory and ischemia-reperfusion injuries, including those of the lung. This study was designed to assess its effect on severe ischemia-reperfusion injury in a model of left-sided rat lung isotransplantation. Orthotopic single left-sided lung allotransplantation (Wistar to Wistar) was performed in female rats after a total ischemic time of 18 h. In nine recipients given 500 mg/kg oral ebselen 1 h before transplantation, graft PaO2/FiO2 was improved 24 h after transplantation, as evidenced with a mean (standard deviation) PaO2 of 139 (61) mmHg vs. eight controls with 65 (33) mmHg (p = 0.009). Bronchoalveolar PMN count was reduced to approximately 50% in the ebselen group compared with controls, whereas no difference in the tumor necrosis factor content was found. We conclude that the improvement of lung function in ebselen-treated transplanted rats is mainly the result of the anti-inflammatory activity of the drug during reperfusion.

The remarkable ability of TNF, especially in combination with Interferon-gamma or melphalan, to inhibit the growth of malignant tumor cells is so far unmatched. Unfortunately, its high systemic toxicity and hepatotoxicity prevent its systemic use in cancer patients. An elegant manner to circumvent this problem is the isolated limb and liver perfusion for the treatment of melanoma, soft tissue sarcoma and liver tumors, respectively, although the latter method can lead to a reversible hepatotoxicity. In order to allow also the treatment of other cancers with TNF, new strategies have to be developed that aim at sensitizing tumor cells to TNF and at reducing its systemic and liver toxicity, without losing its antitumor efficiency. Moreover, the lectin-like domain of TNF, which is spatially distinct from the receptor binding sites, could be useful in reducing cancer treatment-related pulmonary edema formation. This review will discuss some recent developments in these areas, which can lead to a renewed interest in TNF for the systemic treatment of cancer.

The skin sensitization potential of eight unsaturated and one saturated lipid (bio)chemicals was tested in both the LLNA and the GPMT to address the hypothesis that chemicals with unsaturated carbon–carbon double bonds may result in a higher number of unspecific (false positive) results in the LLNA compared to the GPMT. Seven substances (oleic acid, linoleic acid, linolenic acid, undecylenic acid, maleic acid, squalene and octinol) gave clear positive results in the LLNA (stimulation index (SI) ⩾ 3) and thus would require labelling as skin sensitizer. Fumaric acid and succinic acid gave clearly negative results. In the GPMT, besides some sporadic skin reactions, reproducible skin reactions indicating an allergic response were found in a few animals for four test substances. Based on the GPMT results, only undecylenic acid would have to be classified and labelled as a skin sensitizer according to the European Dangerous Substance Directive (67/548/EEC) (results for linoleic acid were inconclusive), while the other seven test substances would not require labelling. Possible mechanisms for unspecific skin cell stimulation and lymph node responses are discussed. In conclusion, the suitability of the LLNA for unsaturated compounds bearing structural similarity to the tested substances should be carefully considered and the GPMT should remain available as an accepted test method for skin sensitization hazard identification.

The controversial discussion about the role of Chlamydia pneumoniae in atherosclerosis cannot be solved without a reliable diagnosis that allows discrimination between past and persistent infections. Using a proteomic approach and immunoblotting with human sera, we identified 31 major C. pneumoniae Ags originating from 27 different C. pneumoniae proteins. More than half of the proteins represent Chlamydia Ags not described previously. Using a comparative analysis of spot reactivity Pmp6, OMP2, GroEL, DnaK, RpoA, EF-Tu, as well as CpB0704 and CpB0837, were found to be immunodominant. The comparison of Ab-response patterns of sera from subjects with and without evidence for persisting C. pneumoniae, determined by multiple PCR analysis of PBMC and vasculatory samples, resulted in differential reactivity for 12 proteins, which is not reflected by reactivity of the sera in the microimmunofluorescence test, the current gold standard for serodiagnosis. Although reactivity of sera from PCR-positive donors was increased toward RpoA, MOMP, YscC, Pmp10, PorB, Pmp21, GroEL, and Cpaf, the reactivity toward YscL, Rho, LCrE, and CpB0837 was decreased, reflecting the altered protein expression of persisting C. pneumoniae in vitro. Our data provide the first evidence of a unique Ab-response pattern associated with persistent C. pneumoniae infections, which is a prerequisite for the serological determination of persistently infected patients.