Introduction

In the next 20 years, medicine will change more than ithas in the past 2000. There are two main drivers of thischange: molecular biology and information technology[1]. However, despite the increasing sophistication ofthe basic science community to applying molecular biol-ogy to resolve biomedical issues, a parallel momentumfor exploiting these technologies has been relativelyslow to develop in the field of intensive care medicine.Thus, despite the enormous literature pertaining to the

molecular biology of the cell that has evolved over thepast decade, many intensivists have little understandingof the profound impact of molecular biology in the prac-tice of their own specialty, and in this sense the gap be-tween basic research and the clinical arena is widening[2]. To bridge the gap, a new breed of physicians willneed to develop basic scientific laboratory training aswell as critical care skills, not only to provide the transi-tion of molecular biological advances to clinical applica-tion, but also to keep the basic science research on track.We cannot predict the future direction and potential

J. VillarK.A. Siminovitch

Molecular intensive care medicine

Accepted: 6 April 1999

Supported by Fondo de InvestigacionSanitaria of Spain (#95/1769) and MedicalResearch Council of Canada

J. Villar ())Research Institute, Hospital de laCandelaria, Tenerife, Canary Islands,Spain; Critical Care, Mount Sinai Hospital,Toronto, Canada; Critical Care Medicine,Mercer University, Macon, Georgia, USA

K.A. SiminovitchDepartments of Medicine, Immunologyand Molecular & Medical Genetics,Samuel Lunenfeld Research Institute atthe Mount Sinai Hospital of Toronto,University of Toronto, Toronto, Canada

Mailing address:Research Institute, Hospital de laCandelaria, 38010 Santa Cruz de Tenerife,Canary Islands, SpainTel.: + 34 (9 22) 602389Fax: + 34 (9 22) 6005 62email: jvillar@cistia.es

Abstract The development ofmethods for the analysis of genestructure and function, referred to asrecombinant DNA (deoxyribonu-cleic acid) technology, has createdunprecedented opportunities forsignificantly improving the preven-tion or treatment of human diseases.Both practitioners working in thisfield and interested observers can-not fail to recognize that the re-markable progress in understandingdisease pathogenesis has placed uson the threshold of a new, revolu-tionary era of clinical practice. Inthis context, molecular medicine ±that is, the application of molecularbiology to elucidating the causes andpotential cures of disease, has be-come a major thrust of research atvirtually all medical schools. Incor-porating the techniques of molecu-lar biology into the research arsenalof the physician should provide newopportunities to dissect out and de-

fine the reversible and irreversibleintracellular processes giving rise toacute respiratory distress syndrome,sepsis, septic shock, or multiple sys-tem organ failure, the major causesof mortality in most intensive careunits.

Intensive Care Med (1999) 25: 652±661Ó Springer-Verlag 1999 REVIEW

successes of molecular medicine, but clearly the linksbetween bench and bedside must be strengthened suchthat the potential of recombinant DNA technologymay be fully realized in intensive care medicine.

Recombinant DNA technology has made possiblethe investigation of the molecular factors modulatingcellular responses to various metabolic and environ-mental stresses. For example, the molecular structureof a spectrum of physiological substances has been de-fined in this fashion and the molecular interactions re-sponsible for their behavior in vivo elucidated [3]. Themain purpose of this review is to introduce molecular bi-ology to those unfamiliar with the language of recombi-nant DNA technology. The ultimate goal is to enablephysicians to follow the exciting developments in thisfield and to begin to participate in bringing the remark-able advances of biomedical research to clinical prac-tice. After outlining the basic concepts and terminology,we discuss some specific examples of gene expressionand transfer that have been studied over the last10 years and their relevance to most common causes ofcritical illnesses.

Basic concepts: from DNA to molecular physiology

Anatomy of a gene

A gene is a sequence of DNA that encodes a functionalproduct. At the structural level, DNA, the fundamentalchemical component of every gene, is a double-strand-ed, helical chain comprised of a series of nucleotideslinked to a sugar (deoxyribose)-phosphate backbone.Each nucleotide contains a nitrogenous base, a pentoseand a phosphate group. The four bases found in DNAare cytosine (C), thymine (T), adenine (A), and guanine(G). It is the arrangement of these four nucleotides thatdistinguishes one gene from another. DNA is organizedinto two strands, with paired nucleotides on each, suchthat adenine binds thymine and guanine binds cytosine.This so-called ªcomplementaryº base pairing allows forduplication of the information content on each of theDNA strands, a structural feature which is key to theiraccurate replication during cell division. The DNA ineach cell is distributed among 23 chromosome pairsand includes about 3 ´ 109 base pairs [4].

The sequences of DNA within the genes are orga-nized into segments called exons which are separatedby introns. The exons are the DNA regions that codefor proteins while the intronic DNA sequence does notcontain protein coding information. In the human ge-nome, less than 10% of the DNA represents exon se-quences. In bacteria, the gene sequences are continuouswithout introns. A gene structure also includes sequenc-es of DNA that regulate the rate of gene transcriptionand ensure that it is initiated at the correct site within

the gene. The regulatory sequences that control the ex-pression of a gene are referred to as promoter sequencesand are often very similar (i. e., conserved) among dif-ferent genes. For example, one promoter element,known as the TATA box, is comprised of alternatingthymine and adenine nucleotides. TATA boxes are reg-ulators of transcription initiation; including directingRNA polymerase II to start mRNA synthesis at the cor-rect initiation site. Many other regulatory DNA se-quences have been identified in recent years and havebeen shown to play a variety of roles in directing therate at which given genes are transcribed as well as theirtemporal and spatial expression. The physical organiza-tion of the genome within each chromosome is verycomplex and is as yet not entirely understood. Structuralstudies to date indicate that the linear double-helixDNA molecule is wrapped for two turns around globu-lar proteins called histones. These histones are in turngrouped into a helical secondary structure of their own,and the secondary structure packed into loops whichgather together during mitosis to form the arms of thechromosomes. It is still unclear how this structure is un-ravelled so as to permit transcription of selected genesat the appropriate time.

In recent years, much attention has focused on theHuman Genome Project, an international effort de-signed to delineate the entire DNA sequence of Homosapiens [5]. Once available, the results of this labor-in-tensive technological enterprise should provide theframework for the more rapid identification of genes re-sponsible for disease susceptibility and genes encodingthe spectrum of structural and functional proteins thatmakes us what we are. Almost certainly, the informationderived from knowledge of the human genome se-quence, when combined with the increasingly sophisti-cated tools of molecular biology, will radically alterboth scientific enterprise and medical practice.

The mission of a gene: from DNA to protein

Protein production requires the induction of gene ex-pression, which for most depends upon activation oftranscription (Fig.1). Thus, the factors regulating genetranscription play a major role in modulating cell devel-opment and function. A primary mechanism for activat-ing transcription is the binding of an inducer protein tothe promoter region near the initial coding sequencesof a gene [6]. Such DNA-protein binding interactionsmight be induced by the interaction of a surface cell re-ceptor with some external cognate ligand (e.g., hor-mone, cytokine) and the consequent transduction ofthis stimuli via enzymatic signaling cascades from thecytosol to nucleus.

The synthesis of ribonucleic acid (RNA) from DNAis referred to as ªtranscriptionº (Fig. 2). The DNA mol-

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ecule serves as a blueprint or template for the synthesisof RNA, a single-stranded molecule in which the pen-tose is ribose and the base thymidine is replaced by ura-cil. Transcription of genes is catalyzed by the cellular en-zyme RNA polymerase II and involves unraveling theDNA double helix such that the noncoding or anti-senseDNA strand can be used as a template for the genera-tion of an RNA strand that is carrying a nucleotide se-quence essentially the same as the sense strand ofDNA. This RNA will undergo further processing beforeexport to the cytoplasm as messenger RNA (mRNA).These processing steps include the addition of a 7-methylguanosine group (the mRNA cap) at the begin-ning of the RNA molecule and the addition of longruns of adenosine [poly(A) tails] at the end of the mole-

cule. These modifications protect mRNA from rapiddegradation by intracellular ribonucleases and are be-lieved to facilitate mRNA transport to the cytosol. Inaddition, the initial RNA copy transcribed from thegene sequence also undergoes ªsplicingº, a processwhereby the intron sequences are excised and exon se-quences ligated, to generate a mature mRNA contain-ing only coding sequences (Fig.2).

ªTranslationº of the information encoded in themRNA transcript occurs in the cytoplasm on the ribo-some. Translation refers to the conversion of the RNAnucleotide sequence into an amino acid sequence. Setsof three nucleotides termed codons contain the informa-tion for one amino acid. Triplet combinations of the fournucleotides generate 64 possible distinc codons, but ourproteins are composed of only 20 amino acids. This re-sults in significant redundancy in the genetic code, withseveral different codons specifying the same amino acid(Fig. 3). For mRNA, uracil (U) replaces thymidine. Theredundancy of the genetic code offers some protectionagainst mutation. Translation involves the attachmentof the mRNA to a ribosomal unit and the subsequentpassage of the mRNA through this unit as translation en-sues. The mRNA is copied into protein by virtue of theassociation of each codon with a specific transfer RNAmolecule (tRNA) carrying an anticodon sequence atone end and the appropriate amino acid at the other.The tRNA acts as an adapter that adds amino acids tothe growing end of the polypeptide chain. In general,the first codon of many genes is an AUG sequence whichencodes for methionine, while the last codons of mostgenes are so-called ªstopº signals which do not encodean amino acid but instead provide a signal for termina-tion of translation. At this stage, mRNA dissociatesfrom the ribosomes and the newly made protein is re-

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Fig.1 The central dogma: from DNA to protein. A genetic mes-sage begins as a DNA molecule which serves as the template formessenger RNA. The mRNA directs the order of amino acids inprotein

Fig.2 Levels of gene expression: from DNA to protein. Schematicdepicting the basic structure of a gene and the flow from gene acti-vation to protein synthesis

Fig.3 The genetic code for translation. The mRNA is translatedinto protein, one codon at a time, by tRNA molecules which recog-nize the codon and transport the appropriate amino acid to thegrowing peptide chain

leased into the cytoplasm. Translation involves the gen-eration of a linear sequence of amino acids assembledon the ribosomes. Once the newly synthesized protein isreleased into the cytoplasm undergoes spontaneous fold-ing so as to acquire secondary and tertiary structure. This3-D structure together with other post-translationalmodifications, such as glycosylation and phosphoryla-tion, play a key role in protein function and/or stability.

Examining gene expression in critical illness

The cellular events involved in mediating organ inflam-mation, tissue damage, and repair are ultimately con-trolled at the molecular level and cannot be fully under-stood without consideration of the functions of the rele-vant genes and their products. It is now widely recog-nized that various cellular stimuli mediate their physio-logical effects by the induction of complex intracellularsignaling cascades which culminate in the activation orinduction of a particular gene or subset of genes. By ex-tension, activation leads to the synthesis of particularsets of proteins and a consequent change in cellular be-havior. Depending on the nature of the physiological orpathological perturbation, these steps, which couplecell stimulation to response, represent potential targetsfor interventive maneuvers and potential therapeuticstrategies. Accordingly, the investigation and definitionof the regulatory events controlling such cellular signal-

ing cascades represent a promising avenue toward thedesign of improved approaches to the treatment ofacute and chronic inflammatory conditions.

Tools of the game

As illustrated in Fig. 2, the functional capacity of a givengene can be influenced at several levels, including, mostnotably, the gene structure and the regulation of genetranscription. These properties can be assessed by anumber of well-defined recombinant DNA technologies[6, 7]. These include, for example, Southern analysis,which allows examination of a gene's structural integrityand Northern and Western analyses, which allow for as-sessment of gene expression at the mRNA and proteinlevel, respectively. These techniques are describedbriefly below (Table 1).

Southern blot analysis is used to assess DNA config-uration in a cell or cell population and takes advantageof the capacity of specialized so-called restriction en-zymes to cut and fragment DNA molecules. These enzi-mes are produced normally in bacteria and their effectson DNA are realized by the recognition of a specificset of nucleotide sequences. Thus, for example, an en-zyme which recognizes a particular six base pair se-quence will cut DNA isolated from a given source (bac-teria, mammalian cell, etc.) wherever this six base pairsequence occurs and thereby digest the DNA into thou-sands of fragments ranging from 100 to 20000 base pairsin length. These ªrestriction fragmentsº can be separat-ed based on their size and electrical charge by agarosegel electrophoresis and viewed under ultraviolet lightfollowing staining with ethidium bromide. For Southerntransfer, the DNA fragments spread across the agarosegel are exposed to salt conditions which cause the dou-ble strands to separate (denature). The fragments arethen transferred from the gel to a nitrocellulose filter,an electrostatic charged paper that binds the DNA.The filter is then exposed (hybridized) to a single-strandDNA probe, most often a radiolabeled cloned fragmentof DNA which has a complementary sequence to theDNA sequence under investigation. Finally, the DNA-containing filter is subjected to autoradiography to visu-alize restriction fragments associated with the radiola-beled probe. By this means, gross changes in gene struc-ture, such as deletion or truncation and even more sub-tle changes in the gene sequence which alter restrictionenzyme digestion, can be evaluated.

The profile of gene expression can be studied bymeasuring its immediate product, the specific mRNA.Northern analysis represents the most commonly usedtechnique for measuring gene expression at the level oftranscription and can be used to determine which tissuesexpress the gene of interest as well as changes in the lev-els of gene expression, for example, following cell stimu-

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Table 1 Tools of recombinant DNA technology

Definition Purpose

Restrictionenzymes

Bacterial proteinsthat cut the DNAinto fragments

To cut DNA wherevera specific nucleotidesequence occurs

Southern blot A technique of cut-ting DNA with arestriction enzyme

To detect variations inDNA sequences

Northern blot A technique foranalyzing mRNA

To study gene expres-sion at transcriptionallevel

Western blot A technique foranalyzing protein

To study gene expres-sion at translationallevel

In situhybridization

Hybridization of alabeled nucleic acidprobe directly tocells or tissues

To visualize DNAor RNA in tissue sec-tions or cells

Polymerase chainreaction

A technique ofrepetitive cyclesof DNA synthesis

To obtain about 106

copies of a singlefragment of DNA

Gene cloning Technique of isolat-ing a gene of parti-cular interest andmaking copies of it

To search for newgenes

lation or during development. For Northern analysis,RNA is isolated by chromatographic separation andthen electrophoresed over a denaturating gel, such asformaldehyde agarose. As for Southern analysis, theRNA is then transferred to nitrocellulose and hybrid-ized with a radiolabeled probe representing the gene ofinterest. Expression may also be assessed at the proteinlevel by Western blotting, a technique whereby proteinsare separated by electrophoresis through a polyacryla-mide gel, again transferred to nitrocellulose filters anddetected by the use of antibody probes. Gene expres-sion can also be studied in tissue sections and cell smearsdirectly either by in situ hybridization, wherein DNA orRNA probes are used to assess mRNA expression, or byimmunohistochemistry, by using antibody probes to as-sess protein expression in the target tissue or cells.

Another widely used approach for evaluating geneexpression involves the use of polymerase chain reaction(PCR) amplification, a technique which can be appliedto the assessment of gene expression in small quantitiesof tissue. PCR involves the exponential amplification ofa selected DNA segment by more than 1 million-foldby the induction of repeated cycles (about 30) of DNAsynthesis from a given DNA template [8]. The strategyhere is similar to the process of DNA synthesis utilizedby all living organisms, involving the use of DNA poly-merase and a ªprimerº fragment to generate a comple-mentary DNA (cDNA) strand from a selected DNAtemplate. However, in contrast to normal DNA synthe-sis in the cell, PCR is designed for the synthesis of many(millions) copies of the template DNA. This is madepossible by the use of Taq polymerase, an enzyme whichwithstands very high temperatures and which can thuswithstand the repeated cycles of DNA denaturing at95�C required for PCR amplification. PCR also requiresthe use of two oligonucleotide fragments which are com-plementary to the sequences that flank the DNA seg-ment to be amplified and which serve as primers forDNA amplification. To use PCR in analysis of gene ex-pression, this procedure includes an initial step whereinmRNA is isolated from the tissue/cells of interest and re-verse transcribed into a cDNA, which represents a DNAcopy of the mRNA and thus lacks the regulatory and in-tron sequences found in the genomic copy of the gene.This cDNA is then subjected to PCR amplification andthe product of this so-called reverse transcription-PCR(RT-PCR) provides an index of the level of gene expres-sion in the sample under study.

Tissue and disease-specific gene expression

Using standard biochemical techniques [9], DNA,mRNA or protein can be extracted from virtually anytissue or cell population and used in conjunction withthe techniques described above to assess the relevance

of altered gene transcription and translation to givenphysiological and pathological states [10, 11]. In consid-ering the pathogenesis of organ inflammation and dam-age, neutrophils, monocytes, macrophages, and plate-lets represent cell populations which are likely to be eti-ologically relevant and subjected to altered gene expres-sion during organ failure. Similarly, epithelial and en-dothelial cells respond to injury with acute alterationsin mediator generation and surface molecule expression[12, 13] and appear to act in concert with inflammatorycells to influence the tissue response to injury and in-flammatory stimuli. These interactions in turn areknown to induce the expression of various genes encod-ing proteins central to coagulation, fibrinolysis, and re-pair. Among these proteins, a critical set of moleculesinvolved in directing the inflammatory response is thefamily of cytokine proteins. Cytokines, a word derivedfrom the Greek terms for ªcellº and ªmoverº, are lowmolecular weight glycoproteins which may act locallyin a paracrine or autocrine fashion to influence cell be-havior, or more generally to induce substantial systemiceffects. The locally active cytokines are often producedby T-lymphocytes, whereas cytokines produced by mac-rophages are often active systemically. Advances inDNA technology have had a major impact on the identi-fication of specific cytokines and the definition of theirroles in tissue injury. Molecular studies of cytokine ex-pression have revealed that the induction of marked in-creases in the expression of certain cytokines followingtissue injury often correlates with the magnitude of tis-sue damage [14]. Among the cytokines particularlywell-studied in relation to tissue damage [12±15] are tu-mor necrosis factor alpha (TNFa), interleukin 1 (IL-1),IL-2, IL-6, IL-8, interferon gamma (IFN-g), transform-ing growth factor beta (TGFb), and platelet-derivedgrowth factor (PDGF). The influence of these and othercytokines on a particular cell or cell population may beinfluenced greatly by interactions with other cytokinesand other types of regulatory factors (Fig. 4). For exam-ple, the combined effects of IL-1, TNFa, and lipo-polysaccharide are believed to be responsible for the ac-tivation of epithelial and endothelial cells during endo-toxemia [16]. The regulatory mechanisms that controlthe acute-phase response during inflammation and or-gan injury are highly complex and include the releaseof cytokines, the transduction of signals from the cellmembrane to the nucleus where gene transcription isup- or downregulated, the processing of mRNA, and ul-timately the change in protein synthesis. These cyto-kine±cell interactions produce acute changes in cellfunction and may be associated with normal growthand repair, or alternatively with the pathogenesis of dis-ease if cytokine production becomes uncontrolled.Thus, inappropriate cytokine production and/or cell re-sponse to cytokine stimuli may lead to ongoing inflam-mation and chronic disease [17].

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Sepsis represents one of the most challenging prob-lems in critical care medicine. Despite the use of power-ful antimicrobial agents, sepsis continues to representthe commonest cause of respiratory failure, multiplesystem organ dysfunction, and death in the critically ill.Cumulative experimental and clinical evidence indicatea major role for cytokine production and systemic re-lease in sepsis-induced inflammatory responses. Thus,blocking cytokine activation or pharmacological effectswith specific cytokine-receptor antagonists represents alogical strategy for the treatment or attenuation of sep-sis-related inflammation [12, 18]. Strategies whichmake it possible selectively to downregulate the effectsof specific cytokines would be particularly attractivefrom the therapeutic perspective. However, achievingthis objective is far from straightforward, since the ef-fects of specific cytokine inhibition in vivo are extreme-ly unpredictable. For example, despite data from animalstudies showing a dramatic efficacy of antibodiesagainst endotoxin and TNF, and IL-1 receptor antago-nist in the treatment of sepsis, corresponding beneficialeffects have not been observed in human trials [19±21].It appears that information concerning cytokine biologywill need to be considerably enhanced before we cancontemplate the development of a ªmagic bulletº thatattenuates inflammatory responses during organ injuryand prevents organ dysfunction.

The gene as a drug

Recombinant DNA technology has also provided themeans to alter and improve the production of variousprotein products in vitro and in vivo, and thereby to ma-

nipulate cell function and behavior [22]. The ability tointroduce heterologous genes and regulatory elementsinto mammalian cells to induce gene expression in thetarget cells allows for the specific alteration of biochem-ical pathways in both normal and diseased cells. Thistechnology has thus paved the way for the insertion oftherapeutic genes into a recipient's cells, a methodologywith enormous promise for ameliorating many untreat-able diseases [23]. This requires efficient methods fordelivering active new genes into specific types of cellssuch that the inserted gene does not disrupt other cellfunctions and can be expressed for an appropriate peri-od of time. To this end, a great deal of effort has been di-rected at the design of vectors for the introduction ofgenes into human cells [24]. In particular, this work hasinvolved the use of recombinant adenoviruses and ret-roviruses which are highly efficient for cell infectionand that can be engineered to contain only the genes re-quired for infecting mammalian cells as well as the ther-apeutic genetic material. Vectors based on human ade-noviruses have shown more promise than retrovirusesfor in vivo gene delivery because they can be used totransfer recombinant genes efficiently into a wide varie-ty of dividing and nondividing cells. Nonviral methodsof gene therapy are also being evaluated. Liposomesare lipid vesicles which can encapsulate therapeuticagents and which can be ingested by macrophages andthus targeted to sites of infection and inflammation.Liposomes can be administered orally, transdermally,subcutaneously, intraocularly, intramuscularly, intra-peritoneally, and intratracheally [25] and are currentlybeing tested for use in conjunction with viruses to en-hance localized gene delivery. In addition to liposomes,plasmids are also being examined as vehicles for genedelivery. Plasmids are like viruses living parasitically inbacteria. Direct injection of human plasmid DNA con-structs have already been shown to engender goodgene delivery and expression in several animal modelsof human diseases [26]. However, the field of gene ther-apy is still in its infancy. There is a considerable need todesign improved vectors, possibly with viral and plasmidcomponents, the goal being to minimize vector immu-nogenicity, more selectively target the construct to aparticular cell type, and allow for the expression of largegenes and facilitate long-term gene expression whichmight be modulated in response to appropriate signals.Once such vectors are designed, gene therapy might in-deed be as simple as a shot in the arm.

Molecular intensive care medicine: part of our destiny

The molecular changes seen in critically ill tissues and or-gans are either exaggerations of normal physiology or in-appropriate expression of repair patterns. As our knowl-edge of molecular biology continues to expand, the po-

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Fig.4 Systematic effects of cytokine secretion. TNF, IL-1, and IL-6produced by monocytes in response to a given stimulus may influ-ence and modulate the functions of a broad spectrum of cell typessuch as B-lymphocytes, in the peripheral lymphoid organs, he-mopoietic precursor cells developing in the marrow, and hepatic,neuronal, and endothelial cells

tential for using this knowledge to alter the morbidityand mortality of human diseases increases. Recent ad-vances have facilitated a continuing need for the devel-opment of new diagnostic tests and treatments in allbranches of medicine, including infectious diseases, ge-netic disorders, inflammatory processes, and cancer.

Microbiologists now have highly sensitive, rapid, andspecific molecular methods of identifying infectiousagents by the direct detection of DNA or RNA se-quences that are unique to a particular organism. Anyclinical sample may now be analyzed it for the presenceof foreign, nonhuman DNA. The potential for molecu-lar diagnostics in the identification of microorganims isvirtually unlimited. The diagnostic utility of the PCRwas examined in 156 patients suspected of extrapulmo-nary tuberculosis by comparing the PCR results from11 different sites with the results of microscopy and cul-ture [27]. PCR had a much higher sensitivity than mi-croscopy and facilitated therapeutic decisions. In a sec-ond study, Tambic et al. [28] were able to identify anoutbreak of methicillin-resistant Staphylococcus aureusamong patients on an ICU using a DNA assay andpulsed field gel electrophoresis. Antibiotic-resistant mi-croorganisms have become a significant problem inmany medical centers. Most reported outbreaks haveoccurred in ICUs, dialysis units, and pediatric wards.Staff-to-patient transmission as well as contaminationof enviromental surfaces play an important part in noso-comial acquisition. The study by Tambic et al. showedthat several patients on the ICU and a further 5 patientson one ward that had received colonized patients trans-ferred from the ICU were affected by a highly virulent,methicillin-resistant Staphylococcus aureus strain withthe same antibiotic susceptibility patterns. Isolates withthe same characteristics were found on the hands ofone member of the ICU staff and 15 of 17 outbreakstrains had genetically identical amplified polymorphicDNA and gel electrophoresis profiles [28].

Airway epithelium is subjected to injury during in-flammation and exposure to a variety of inhaled and in-fectious agents. In addition, research over the past de-cade has shown that mechanical ventilation can by itselfproduce or worsen lung injury [29]. However, little isknown about the upregulation of gene products duringmechanical ventilation and repair of lung injury. Threerecent reports [30±32] are instrumental in understand-ing the inflammatory aspects of the pathogenesis of ven-tilator-induced lung injury and the influence of differentventilatory patterns on such pulmonary inflammatoryresponse. In all three, the authors investigated whethercyclic motion produced by certain modes of mechanicalventilation leads to induction, synthesis, and release ofcytokines from lung tissue. Intraalveolar gene expres-sion of TNFa was studied in surfactant-depleted rabbitsafter 1 h of conventional mechanical ventilation or highfrequency ventilation [30]. Lavage fluid samples were

analyzed for mRNA quantification by RT-PCR tech-nique. Although there were no differences in blood gas-es or lung lavage cytology between conventional andhigh frequency ventilation, the former produced largeincreases in TNFa mRNA in the alveolar cells, presum-ably by alveolar macrophages. High frequency ventila-tion produced minimal increases in TNFa gene tran-scripts. Tremblay et al. [31] examined the effects offour different ventilation strategies on lung inflammato-ry mediators in the presence and absence of a preexist-ing inflammatory stimulus. The production of cytokines(TNFa, IL-1b in the serum; TNFa, IL-1b, IL-6, IL-10,IFN-g, MIP-2 in the lavage fluid; TNFa in lung tissue)and c-fos (an immediate early response gene) was evalu-ated in an isolated rat lung model with and without sep-sis. TNFa mRNA was measured by Northern analysisupon completion of 2 h of ex vivo ventilation. In endot-oxin-induced lung injury animals, TNFa mRNA was sig-nificantly greater for the group ventilated using moder-ate tidal volume (15 ml/kg) and a high positive end-ex-piratory pressure (PEEP) (10 cmH2O) or zero PEEP ascompared to the control group or those ventilated withhigh volume (40 ml/kg) and zero PEEP. A similar patternwas found for induction of c-fos mRNA. von Bethmannet al. [32] investigated whether mechanical ventilationleads to induction, synthesis, and release of cytokinesfrom the lung. They explored in an isolated perfusedand ventilated mouse lung the effects of hyperventilationat a transpulmonary pressure of 25 cmH2O over 150 min.At the end of the experiments, they measured TNFa andIL-6 gene expression in lung extracts by RT-PCR, andTNFa, IL-4, IL-6, and IFN-g in the perfusate by en-zmye-linked immunosorbent assay (ELISA). Theyfound that hyperventilation resulted in a 1.75-fold in-creased expression of TNFa and IL-6 mRNA and a 12-fold and more than 3-fold increased production ofTNFa and IL-6, respectively. Although there are vari-ous differences between these experimental modelsand the clinical setting, these reports support the hy-pothesis that mechanical ventilation might contributeto the development of a systemic inflammatory re-sponse and multiple system organ failure.

Modern molecular methods have been developedthat allow the stable transfer of foreign DNA sequencesinto human and other mammalian somatic cells. Untilthe retroviral vectors ± the first truly efficient genetransfer tools ± became available, gene therapy re-mained an entirely conjectural and theoretical concept.The ability of humans to read and reconstruct theirown genetic makeup is unprecedented. In 1986, Kantoffet al. undertook one of the first studies involving a retro-virally transferred foreign gene in human cells [33]. Thefirst human gene transplant was carried out at the Na-tional Institutes of Health on September 1990 by FrenchAnderson and Steve Rosenberg and their colleagues[34]. The study involved the introduction of the neomy-

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cin resistance gene into tumor-infiltrating lymphocytes.Today, more than a 100 clinical gene therapy studieshave actually been approved (Table 2). Currently, threeforms of gene therapy are underway: gene insertion,gene repair, and gene surgery [35]. Anti-sense gene ther-apy, a modification of the gene insertion approach, in-volves the insertion of a DNA segment, which producesan anti-sense mRNA, preventing the mRNA from beingtranslated into proteins on the ribosomes [36].

It is an exciting time for respiratory and critical carescience. There is now an impressive range of potentialtreatments including gene therapy, anticytokine, andantiadhesion molecule approaches, and targeting of in-tracellular signal transduction pathways [37]. Severalstudies have demonstrated that it is feasible to transferthe normal human cystic fibrosis transmembrane con-ductance regulator complementary DNA to the respira-tory epithelium and that this will lead to production ofcystic fibrosis transmembrane conductance regulatorprotein and in some cases to a physiological response[38]. Other diseases that may be helped by gene therapyinclude gastrointestinal malignancies, viral hepatitis, he-mophilias, alpha-1antitrypsin deficiency, metabolic dis-eases of the liver, and bone marrow reconstitution aftertransplantation.

Recombinant retrovirus constructs containing a cyto-kine-cDNA can be used to infect cells in vitro and ob-

tain information about the pathogenesis of several pro-cesses that are relevant in clinical diseases. Xing et al.[39] constructed a replication-deficient adenoviral vec-tor expressing mouse IL-10 to investigate the therapeu-tic potential of IL-10 in an experimental model of en-dotoxemia. Intramuscular injection of this vector inmice resulted in active release of IL-10 protein into thebloodstream and inhibition of entotoxin-induced TNFaand IL-6 mRNA in different organs with a maximal re-duction seen in the lung.

Conclusion

All areas of medical research are being affected by theexplosion of knowledge and technology in the fields ofcellular and molecular biology [40, 41]. The rapid evolu-tion of biomedical research during the last two decades,due in large measure to recombinant DNA technology,has changed fundamentally the way medicine approach-es the pathogenesis and treatment of disease. In the fu-ture, a routine physical examination may include DNAanalysis from blood or tissue specimens to uncover ge-netic interactions between molecules. Today, the tech-niques of cellular and molecular biology can be appliedto the study of particular aspects of disease mechanismsalready identified, in finer and finer detail. As molecular

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Disease Gene defect ± gene insertion Target cells

Brain tumors Insertion of a replication-defectiveretrovirus vector bearing the herpessimplex thymidine kinase gene

Tumor cells become sensitiveto ganciclovir

Cystic fibrosis Human cystic fibrosis transmembraneconductance regulator

Lung epithelial cells

Duchenne's musculardystrophy

Dystrophin Muscle cells

Familial hyperchole-sterolemia

Liver receptor for low densitylipoprotein

Liver cells

Gaucher disease Glucocerebrosidase Bone marrow cells

Growth hormonedeficiency

Human growth hormone Myoblasts

Hemophilia A Factor VIII Liver cells or fibroblasts

Hemophilia B Factor IX Liver cells or fibroblasts

Inherited emphysema Alpha-1 antitrypsin Lung or liver cells

Melanoma Insertion of genes to increaseexpression of IL-2, IL-4, TNF, IFN-g

Lymphocytes

Metastatic colorectalcarcinoma

Insertion of a ªsuicideº gene(Escherichia coli cytosine deaminase)

Tumor cells

Renal carcinoma Transfection with IL-2 and IFN-agenes

Tumor cells

Severe combinedimmune deficiency

Adenosine deaminase Bone marrow stem cellsor T cells

Thalassemias b -globin Bone marrow cells

Table 2 Trials of gene therapy

medicine matures with increased clinical experience, itwill become part of the mainstream. However, thisshould not be regarded as distancing physiology fromthe use of molecular biology. The new intellectual chal-lenge is to reintegrate this information into an under-standing of whole tissue, organ, and organism function[42]. As the tools in molecular biology continue to pro-liferate, the concept of ªsystemic diseaseº will becomea waste/basket since patients could be screened for inju-ry susceptibility and evaluated for a particular, locally

given treatment based on molecular analysis. Certainly,DNA is part of our destiny. For this to happen, physi-cians taking care of patients with critical problemsmust understand the basic science and technology ofmolecular biology. We predict that active clinical scien-tists in the field of intensive care medicine will be in-volved in addressing this challenge, both for reasons ofaccess to clinical material and for reasons of access toclinical knowledge to improve our patients' outcome.

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