The Changes in the T helper 1 (Th1) and T helper 2 (Th2) Cytokine BalanceDuring HIV-1 Infection are Indicative of an Allergic Response to ViralProteins that may be Reversed by Th2 Cytokine Inhibitors and Immune

Response Modifiers – a Review and Hypothesis

YECHIEL BECKER*

Department of Molecular Virology, Faculty of Medicine, The Hebrew University of Jerusalem, P.O.B 12272,

Jerusalem 91120, Israel

Received September 20, 2003; Accepted October 1, 2003

Abstract. The HIV-1 infection in humans induces an early cellular immune response to react to the viralproteins with a cytotoxic T cell (CTL) response that fails to inhibit virus replication and the spread of thevirus. It became evident that the progression of the disease causes chronic changes to the immune system ofwhich a gradual increase in IgE antibodies is one of its features. When the HIV-1 epidemic began, therelation between the gradual increase in IgE content and AIDS was not understood, but later it became amarker for disease prognosis. The advances in the knowledge on T helper 1 (Th1) and T helper 2 (Th2) cellsrevealed that Th1 cells produce cytokines that stimulate the proliferation of CTLs. Th2 cells producecytokines that are responsible for the activation of the humoral immune response in healthy people. Studieson both Th1 and Th2 cytokine synthesis revealed an aberration in HIV-1 infected people. Clerici andShearer presented a hypothesis (1993) whereby Th1 cell activity declines and Th2 activity increases (theTh1 fi Th2 switch hypothesis) in HIV-1 infected people. In fact, experiments concerning this hypothesisultimately supported the premise that the switch involves a critical change in the cytokine balance, whichleads to the contraction of AIDS. However, the research community must still discern why such aTh1 fi Th2 switch takes place in infected people and how it can be reversed. The present review points tothe fact that a similar Th1 fi Th2 switch constitutes the response of allergic people to environmentalallergens. HIV-1 patients and allergic people that are exposed to allergens respond with an increasedsynthesis of Th2 cytokines and IgE, together with a decrease in Th1 cytokines. The studies on allergen-induced Th2 cells revealed that the Th2 cytokine IL-4 induces B cells to synthesize IgE, and cytokine IL-5 isthe inducer of eosinophilia, just as in HIV-1 infection. The difference between the HIV-1 infection andallergies is the ability of IL-4 to induce the synthesis in T cells of the HIV-1 coreceptor CXCR4 that selectsfrom the replicating virus a syncytium-inducing (SI) virus, a variant virus that replicates rapidly. Thepresent hypothesis implicates the viral proteins in the induction of Th2 cytokine synthesis. This suggeststhat in viral proteins, allergen-like domains may be responsible for the activation of Th2 cytokine synthesis.Based on the analogy of the responses of humans to allergens and HIV-1, the following hypotheses issuggested: (a) Removal of allergen-like domains from viral genes by genetic engineering may provide viralproteins for vaccine development. (b) Attempts to treat allergic patients with IL-4 receptor inhibitorssuggests that the ‘‘Th2 fi Th1 Reversion’’ constitutes a possible approach to inhibiting the Th2 cytokinesand inducing a revival of the anti-viral Th1 response.

Key words: HIV-1 infection, allergic response to HIV-1, Th1 to Th2 switch, Th2 to Th1 reversion, Inhi-bition of JL-4, immune response modifiers, Modifications in gp120, implications for vaccines

*E-mail: becker@md.huji.ac.il

Virus Genes 28:1, 5–18, 2004� 2004 Kluwer Academic Publishers. Manufactured in The Netherlands.

Introduction

Recently, 24 leading experts in the fields of HIV-1and AIDS research published a manifest entitled,‘‘The Need for Global HIV Vaccine Enterprise’’[1] stating that since the 1983 discovery of HIV-1,the cause of human AIDS, the pandemic proceedsat a speed of 45 million new infections by 2010 and70 millions will die by 2020. Due to the unsuc-cessful anti-HIV-1 vaccine trials, the authors pro-pose that HIV-1 vaccine enterprise be designed asa ‘‘high quality collaborative research system.’’There is no doubt that an international scientificcooperation on the development of effective andprotective anti-HIV-1 vaccines to immunize unin-fected people worldwide will help to stop theAIDS pandemic, similar to the effort to eliminatesmallpox disease by vaccination with vaccinia vi-rus that was achieved in 1977. However, the 45million of HIV-1 infected people and all the othersthat are predicted to die by the year 2020 need tobe saved from AIDS as early as possible.

With the current advances in the research on themolecular-immunological basis of allergy, it be-came evident that aeroallergens in susceptible in-dividuals induce T helper type 2 (Th2) cells toproduce the cytokines IL-4, IL-5, and IL-13. IL-4directs B cells to produce anti-allergen IgE mole-cules. Mast cells, dendritic cells (DCs), and Lan-gerhans cells (LCs) are induced to synthesize IgEreceptors (FceRI). Anti-allergen IgE antibodiesbind to IgE receptors (FceRI) and the bound IgEmolecules interact with the allergens and furtherinduce IgE synthesis by B cells. IL-4 also inhibits Thelper 1 cells (Th1) and prevents the production ofIL-2, IL-12, and INF-c that are necessary for thedevelopment of cytotoxic T cells (CTLs). The al-lergen-induced immune response in patients withatopic dermatitis affects the humoral and cellularimmune responses. It is recognized that allergicindividuals who were infected or immunized withvaccinia virus lack effective anti-viral immune re-sponses developed a marked infection [2]. Thereby,allergic people are prohibited from smallpox vac-cination.

With this in mind, I have reviewed the studies onthe enhanced production of IgE in HIV-1 infectedpeople and the reports that increased IgE in in-fected individuals is indicative of a poor prognosis.According to the reported evidence, cytokines IL-

4, IL-5, and IL-13 are increased during infectionwith deleterious effects on the humoral and cellularimmunity, which are similar to the immune defi-ciency of allergic patients. Reports on the in-creased levels of IgE synthesis in HIV-1 infectedpeople have appeared since 1983 [3, 4], 2 yearsafter the discovery of HIV-1. A review of the lite-rature during the last 20-years reveals that thecomplex phenomena that are associated with theHIV-1 infection and AIDS are dependent on ge-netic factors that protect or enhance virus repli-cation. It was also noted that the early anti-viralCTL response against HIV-1 failed, while IgEsynthesis prevented the production of effectiveanti-viral immuno-globulins. The possibility thatthe HIV-1/AIDS infection induces an allergic re-sponse in infected people was not studied. Thepresent hypothesis suggests that defining the HIV-1/AIDS disease as a form of allergy may lead tothe development of viral proteins from which al-lergen-like domains will be deleted. The use ofnon-allergen viral proteins for the development ofanti-HIV-1 vaccines may lead to effective anti-viralcellular and humoral inducing vaccines. Further-more, this hypothesis contends that inhibition ofTh2 cell activity and revival of Th1 cytokine syn-thesis (Th2 fi Th1 reversion hypothesis) in HIV-1/AIDS patients may be achieved by treatmentwith immune response modifiers or drugs that areused to treat allergies.

Observations on Increased IgE Levels in HIV-1

Infected Patients

Serum IgE in HIV-1 Infected Adults

Wright et al. [3] reported that, ‘‘HIV-1 infection ischaracterized by a progressive depletion of helperT lymphocytes and, like allergic diseases is asso-ciated with altered T cell regulation.’’ The authorsquoted five previous studies from 1983 to 1989that measured serum IgE levels in HIV infectedpeople. The studies revealed high IgE levels inHIV-1 patients with allergic symptoms. Conse-quently, the authors suggested that the increase inIgE in these individuals may be attributed to adifference in the amount of Th2 cytokine produc-tion and IgE synthesis. The results of this study [3]showed that 59% of HlV-infected male subjects

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with CD4 number £ 200/mm3 had a serum level of>100 Iu/ml. The mean level of this group was132 Iu/ml, significantly greater than the levels inHIV-negative heterosexuals and homosexuals andinfected subjects with CD4 number > 200/mm3.The authors concluded that advanced HIV infec-tion (CD4 numbers £ 200/mm3) is associated withincreased IgE levels that indicate an abnormal Tcell regulation of IgE synthesis by B cells. Thedifferences in T cell induction of IgE synthesis byactivated B cells may be related to differences inlymphokines in HIV-infected people that havebeen found to impair the production of INF-c [4].

Israel-Biet et al. [5] performed an evaluation ofserum IgE, IgA, IgG, and IgM in a cohort of 315HIV infected individuals with or without AIDSand compared the results to those of 100 HIV-seronegative subjects. It was found that IgE levelswere significantly higher in HIV-infected than inseronegative individuals. A strong correlation ap-peared between IgE and the immune status asassessed by CD4 cell counts in patients withCD4 £ 300/mm3 or CD4 > 300/mm3. The IgElevels appear to be a strong indicator that distin-guishes between patients in late stages of HIV in-fection. The authors reported that IgA, IgG, andIgM also significantly increased during HIV in-fection, but the slope of their increase was lesssteep than that of IgE.

In 1983, Ammann et al. [6] reported that theHIV epidemic is characterized by the frequent in-crease of IgE in patients with AIDS. Israel-Bietet al. [5] suggested that in HIV infection, IgEoverproduction could be related to CD4 cell de-pletion associated with a decreased ability of CD4cells to produce interferon (INF). A markedlyhigher amount of activated CD4 T cells clones thatspontaneously synthesize IL-4 have been reportedto be found in HIV-1 patients [7].

Miguez-Burbano et al. [8] reported that in earlystages of HIV-1 infection, the elevation of plasmaIgE levels preceded the decline of CD4 cells. Toexamine the possible impact of HIV-1 on IgElevels, comparisons between cohorts of HIV-1seropositive and seronegative drug users andhomosexual men were conducted. All HIV-1seropositives had significantly higher IgE levelsthan HIV-1 seronegative subjects. These resultssuggested that IgE might serve as a sensitivemarker to the evolution of HIV-1 disease. Shor-

Posner et al. [9] reported that increases in IgElevels are prevalent during the early stages of HIVdisease, independent of CD4 cell count, and isparticularly evident in persons with low plasmalevels of vitamin E.

Mazza et al. [10] studied 29 HIV-infected pa-tients to determine whether IgE dysregulation isaccompanied by significant changes in T and Bmarkers. An analysis of the results from the firstyear revealed a subset of patients with marked IgEelevations in serum. This increased IgE did notreact with any of eight atopic allergens (fromDermatophagoides fariane, oak, grass, Cladospo-rium, Alternaria, dog, cat, and short ragweed).

Elevated IgE Levels in HIV-1 Infected Children

In order to understand the possible cause of theallergic nasal symptoms in HIV-1 infected people,Koutsonikolis et al. [11] applied an enzyme im-munoassay in the sera of 29 HIV-1 infected chil-dren with chronic nasal symptoms, sinusitis, andotitis media. It was found that 9 of 29 children(31%) had initial IgE levels greater than 2 standarddeviations higher than age matched controls. Nopatients had detectable specific IgE to any of sixallergens (Dermatopagoides pteronyssinus, Ameri-can cockroach, Bahia grass, short ragweed, cat,and oak). It was concluded that the elevated IgEmay reflect and independent polyclonal activationof B lymphocytes. Similar results were reported byVigano et al. [12].

IgE Against HIV Proteins in Clinically HealthyChildren with HIV Disease

Secord et al. [13] reported that elevated serum IgEwas detected in 7/30 (26%) of children with HIVinfection (intrapartum or transplacenta infections)between the ages of 8–12 years old (long-termsurvivors). Four of seven children with elevatedIgE were of the Puerto Rican ethnic group, com-pared to only 9% (2/23) in the normal or low IgEgroup. None of the children had opportunisticinfections and none failed to thrive. In these HIV-1infected children the serum IgE antibodies wereagainst HIV proteins. Two of the seven PuertoRican children had IgE anti-p24 and IgE anti-gp160 and one child had only IgE anti-p24. IgE anti-p24 and IgE anti-p17 were detected in the serum of

The Changes in the T helper 1 7

one African-American child who did not haveelevated serum IgE.

Khalife et al. [14] reported on the presence ofIgE anti-HIV proteins in the serum of adults withHIV infection and hemophilia.

The role of Nitric Oxide in Promoting the Effect ofHIV-1 Infection and gp120 on IL-4 Induced IgEProduction by Human Mononuclear Cells

The above studies [13, 14] revealed that in HIV-1infected children, IgE molecules in the blood wereinduced by the viral proteins gpl60, p24, and p17.Duglas et al. [15] demonstrated that in vitro in-fection of human peripheral blood mononuclearcells by HIV-1 monocytotropic (Ba-L) or lympho-cytotropic (LAI) strains promoted IL-4 that in-duced IgE production. The viral membraneglycoproteins gp120 specifically potentiated, in adose response manner, IL-4 induced IgE produc-tion and did not affect IL-4 induced IgA, IgG, andIgM production. Viral gp160 also upregulated IL-4 induced IgE, but gp41 was ineffective. It wasfound that inhibition of nitric oxide (NO) in IgEproduction using a specific inhibitor of NO syn-thase pathways abolished IgE production that wasinduced by HIV-1 gp120.

A Th1 to Th2 Switch During HIV-1 Infection

The Th1 fi Th2 Switch Hypothesis by Clerici andShearer [16, 17]

The studies on the elevated IgE production againstviral proteins without affecting the synthesis ofIgA, IgG, and IgM may suggest that the viralgp120 glycoprotein and possibly p24 and p17 haveamino acid domains that have a quality of allergicproteins. The increase in IgE production prior tothe stage of AIDS may indicate that at this stagethe Th2 CD4þ cells are induced by viral proteinsto produce the cytokines IL-4, IL-5, and IL-13 atincreased levels. IL-4 inhibits Th1 cells and thesynthesis of the Th1 cytokines and the anti-viralCTL activity.

Changes in IL-2 and IL-4 contents in HIV-1seropositive individuals were reported by Clericiet al. [16] who have tested whether the loss of IL-2production that is characteristic of HIV infection

is associated with augmented production of IL-4.The authors reported that earlier stages of HIVinfection are dominated by IL-4 and a concomi-tant inhibition of IL-2 production (IL-4 > IL-2).It was reported that �50% of HIV-exposed sero-negative individuals showed evidence of Th1 cellspriming to HIV envelope protein and exhibited anIL-2 > IL-4 cytokine profile.

Clerici and Shearer [17] presented a model,based on their findings that a ‘‘Th1 fi Th2switch’’ is a critical step in the etiology of HIVinfection. The model suggests that an imbalance inthe Th1-type and Th2-type responses to HIV-1infection contributes to the immune dysregulationduring infection. Resistance to HIV infection and/or progression to AIDS is dependent on a Th1 >Th2 dominance. The authors’ hypothesis is basedon these findings: (1) many seronegative HIV ex-posed individuals generate strong Th1-type re-sponse to HIV antigens; (2) progression to AIDSis characterized by loss of IL-2 and INF-c pro-duction with concomitant increase in IL-4 and IL-10 [17].

The Clerici-Shearer ‘‘Th1 fi Th2 Switch’’Hypothesis in HIV Infection is Criticized

Romagnini et al. [18] reviewed the above studiesand reported that CD4þ Th2 and most Th0 clonesefficiently supported HIV replication whereas Th1clones did not. These results were taken to suggestpreferential HIV replication in T cells producingTh2-type cytokines [19] rather than a ‘‘Th1 fi Th2Switch’’ during HIV replication, as suggested byClerici and Shearer [17].

Granziosi et al. [20] reported on the constitutivecytokine expression in unfractionated and sorted Tcell populations isolated from peripheral bloodand lymph nodes of HIV infected individuals atdifferent stages of the disease. It was found thatexpression of IL-2 and IL-4 was barely detectableregardless of the particular stage of the disease.CD8þ cells expressed large amounts of IFN-c andIL-10 and the levels of these cytokines remainedstable throughout the course of infection. Theseresults were taken to indicate that a switch fromTh1 to Th2 cytokines does not occur during theprogression of HIV disease. In a study on thekinetics of cytokine expression during primaryHIV-1 infection, Graziosi et al. [21] determined

8 Becker

the constitutive expression of IL-2, IL-4, IL-6, IL-10, INF-c, and TNF-a in sequential peripheralblood mononuclear cell samples taken from nineindividuals with primary HIV-1 infection. It wasreported that very little expression of IL-2 and IL-4 in circulating CD4þ T lymphocytes was found.However, a lymph node biopsy in one patient, atday 28 after onset of symptoms, revealed sub-stantial levels of IL-2 and to a lesser extent of IL-4.In addition, the level of expression of IL-6, IL-10,IFN-c were three fold higher in the lymph nodecompared to peripheral blood. The conclusion wasthat the lack of detection of IL-2 and IL-4 ex-pression in peripheral blood cells does not meanthat a similar situation exists in other lymphoidcompartments.

Tanaka et al. [22] used a method that alloweddirect measurements of intracellular cytokines inCD4þ CD8) T cells in 48 HIV infected and 16uninfected individuals. The percentages of CD4þ

T cells that produced IL-2 were 23.6% in infectedindividuals and 34.9% in uninfected; IFN-c was13.7% versus 13.2%; IL-4 1.3% versus 1.0%; andIL-5 1.2% versus 0.9%. The population of IL-2producing cells decreased in proportion to the re-duction in CD4 counts. The conclusion was thatthe Th1 to Th2 switch did not occur in the mode-rate and advanced stages of HIV-1 infection.

Additional Studies on T helper type 1 and type 2Cytokines in HIV-1 Infections

Maggi et al. [23] analyzed circulating or skin in-filtrating T lymphocytes at the clonal level of twoHIV-1 infected individuals who suffered fromJob’s-like syndrome (HIV-1 infection with ecze-matous skin dermatitis (recurrent) and sinopulmo-nary infection and hyper-IgE without circulatingCD4þ T cells). Most of the CD3þ T cell clonesfrom both patients were CD4) CD8þ cells with Tcell receptor (TCR) abþ. Other clones were CD4),CD8), TCRaþ that exhibited no mRNA for eitherCD4 or CD8 molecules. The vast majority of bothcell types did not produce IFN-c, exhibited re-duced cytolytic activity, produced large amountsof IL-4 and IL-5, and provided B cells with Th2functions for IgE synthesis. The authors concludedthat a switch of cytolytic CD8þ T cells, whichexhibit a Th1-like cytokine secretion profile to cells

that synthesize Th2 cytokines occurs in the courseof HIV-1 infection. These cells may contribute tothe weakened defenses against virus infection andaccount for elevated IgE, eosinophilia and allergic-like clinical manifestations that are found in aproportion of HIV-1 infected people.

Autran et al. [24] reported that CD4þ CD7þ Tcells release significant amounts of IL-2 into theculture supernatant. CD4þ CD7) T cells releaseinto the culture medium of the cytokines IL-4 andIL-10. In HIV-1 infected patients, the expansion ofCD4þ CD7) T cells in relationship with diseaseprogression was associated to an in vivo activationof the CD4þ CD7� T cells as assessed by cellsurface expression of HLA-DR, CD25, CD71, andCD57 markers. The CD4þ CD7) T cells mightplay a role in T helper cell dysfunctions duringHIV infection.

Meroni et al. (25) reported that the early phaseof HIV infection is characterized by a decrease inmitogen-stimulated IL-2 and IFN-c productionand increased mitogen stimulated IL-4 and IL-10production. The authors concluded that HIV in-fection is a complex, dynamic immunologicalprocess even in its earliest phases.

Klein et al. [26] characterized the changes inTh1/Th2 cytokine production by peripheral bloodmononuclear cells during the course of HIV in-fection by single cell staining. The authors noted acontinuous decrease in IFN-c producing CD4þ Tcells in the course of HIV infection and a dramaticreduction of IL-2 expressing cells among the CD4þ

T cells in patients with AIDS. In contrast to Th1cytokines, the frequency of Th2 cytokine express-ing cells among CD4þ T cells increased in HIVinfected people. It was concluded that the resultsdemonstrate a shift of Th1 to Th2-type cytokineexpression by CD4þ T cells in the course of HIVinfection.

Wasik et al. [27] examined the relationship be-tween HIV specific CTL activity and the profile ofcytokine production that is induced by monoclo-nal antibodies (mAb) to CD3 and by HIV enve-lope peptides P18 and Tl in peripheral bloodmonocyte cells that were taken from children be-tween 6 months and three and a half years oldwith prenatal HIV infection. It was found thatanti-HIV cellular immunity was demonstratedonly during early stages of disease and undetectedin children with rapidly progressing disease and in

The Changes in the T helper 1 9

uninfected children of HIVþ and HIV) mothers.Levels of IL-2 mRNA in anti-CD3 mAb and HIV-1 envelope (env) induced T cells varied and washigher in the infected children with high frequen-cies of HIV-specific CTL precursors. IFN-c andIL-4 production by CD4þT cell clones that werestimulated with anti-CD3 mAb and env peptidesexhibited an expanded proportion of Th2 and Th0clones in HIV infected children with lower HIVspecific CTL activity. Children with high CTLactivity had increased Th1 clones. The authorsconcluded that decreased CTLs with anti-HIVactivity might be associated with the induction of aTh2 cytokine response. They concluded that therole of cytokines in the generation of HIV specificCTL responses might be a key to the develop-ment of immunomodulatory and vaccine strate-gies to interrupt the vertical transmission ofHIV.

Summary of the Findings Regarding the TimeCourse of the Changes in the Immune Response inHIV-1 Infected Children and Adults

The studies on the presence of IgE in the serum ofHIV-1 infected people led Clerici and Shearer [17]to hypothesize that at a later stage of infectioncytokine synthesis by Th1 cells declines and Th2cells are activated to synthesize IL-4, IL-5, and IL-13. The increased production of IL-4 causes B cellsto synthesize IgE antibodies that increase in theserum in the presence of IgA, IgG, and IgM. IL-4inhibits cytokine synthesis by Th1 cells and CTLactivities. The results of studies [23–27] revealedthat a marked decrease in Th1 cytokines IL-12 andIFN-c and marked increase of Th2 cytokines IL-4,IL-5 during HIV-1 infection. This lead to an in-crease in IgE level an indicator of a poor prognosisand the patients’ entering to the stage of AIDS.

Synthesis of IgE in HIV-1 Patients Resembles IgESynthesis in People with Allergies

Allergic responses in humans are characterized bythe synthesis of allergen specific IgE antibodies[28]. The allergic hypersensitivity reaction is trig-gered by allergens that aggregate on preformedIgE antibodies, which bind to the a-chain of thehigh affinity Fc receptor (FceRI) on the surface of

mast cells. Allergens must have at least two dis-tinct and spatially separated antibody bindingepitopes to aggregate onto FceRI complexes andfunction as allergens. Research on the solutionstructure of a mite allergen from Dermatophago-ides fariane (Der f 2) revealed that the Der f 2allergen is a single domain protein with an im-munoglobulin fold [29]. Aalberse [30] analyzed 40allergenic proteins (or parts of these proteins) ofwhich the protein folds are either known or can bepredicted on basis of homology and classified theminto four structural families: (1) anti-parallel bstrands: the immunoglobulin fold family, serineproteases, and soybean-type trypsin inhibitor; (2)anti-parallel b sheets that are immediately associ-ated with one or more a-helices; (3) a + b struc-tures in which the a-and b-structural elements arenot immediately associated and (4) a-helicalstructures. The author concluded that allergenshave no characteristic structural features otherthan that they need to reach and stimulate immunecells and mast cells. The discovery of IgE anti-bodies with anti-gp120, p24, and p17 in the serumof HIV-1 infected children [13, 14] opens the pos-sibility that HIV-1 gp120 peptides that are pre-sented by HLA class II molecules may haveallergenic properties. If this hypothesis is con-firmed, there may be an explanation to the un-successful attempts to vaccinate people withvaccines containing gp120 [1].

Additional Implications of the Th1 fi Th2 Switch

Hypothesis

Th2 Cytokines Cause the Selection of Syncytium-Inducing (SI) HIV-1 Variants

Trumpfheller et al. [31] analyzed cytokine andchemokine mRNAs production in lymph nodeswith follicular hyperplasia from HIV-infected pa-tients by in situ hybridization. It was discoveredthat in HIVþ patients a large number of cells in theT cells-dependent areas of the lymph nodes syn-thesized IFN-c, RANTES, MIP-la, and MP-1bmRNA, but only a few cells were found to produceIL-12p35. Lymph nodes from healthy individualscontained T cells that produced cytokines andchemokines. IL-4 producing cells were not found

10 Becker

in the infected and control lymph nodes. The samefinding was made in a limited number of samplesfrom patients with advanced disease.

In contrast to these studies, Torres et al. [32]investigated the relationship between serum cyto-kine pattern of T helper (Th) cell response and theacquisition of SI HIV-1 variants in HIV-1 sero-positive patients that were treated with anti-re-troviral drugs. It was reported that patients inwhom HIV-1 was converted to a SI virus showedat baseline lower levels of IL-2 and higher con-centrations of IL-4 than those who remained per-sistently negative for the SI variant of HIV-1.Shortly after the phenotype conversion, SI-virusconverting patients were characterized by signifi-cantly high IL-4 concentrations in the serum andby low levels of IFN-c. The authors concludedthat HIV-1 patients with non-syncytium (NSI)virus had serum cytokine pattern of Th1/Th2 re-sponse and differed from patients with SI virusphenotype. The Th2 cytokine pattern is the reasonfor the acquisition of the SI virus phenotype. HIV-1 variants are classified as non-syncytium inducing(NSI) virus, mostly low-replicating strains, andhigh replicating SI variants. The appearance of SIstrains during the chronic phase of HIV-1 infec-tion is associated with an increased rate of CD4þ

cell decline and more aggressive course of thedisease (32).

The Dual Effect of IL-4 on the HIV-1 PhenotypicCytokine Switch and Disease Progression

Valentine et al. [33] reported that IL-4 inhibits thepropagation of NSI HIV-1 variant by two mech-anisms: (1) IL-4 regulates the two major HIV co-receptors CCR5 and CXCR4, in human peripheralblood mononuclear cells. In the presence of IL-4,the synthesis of the CXCR4 receptor increases andthe synthesis of CCR5 expression in primaryCD4þ T lymphocytes declines; (2) IL-4 stimulatesthe expression of all HIV-1 isolates via a tran-scription activation mechanism. Similar findingswere reported by Wang et al. [34]. Biasin et al. [35]analyzed HIV-infected and uninfected individualsfrom Maharashtra–Mumbai, India. They reportedthat the synthesis of CXCR4 mRNA, surface ex-pression of the receptor, IL-4 mRNA synthesis,and T allele frequency at nucleotide)589 in the IL-

4 gene and of high IL-4 production – were in-creased in Indian patients relative to Italians. Theauthors indicated that due to the fact that IL-4stimulates CXCR4 expression, this observationcould be explained by the higher frequency of the)589T IL-4 polymorphism.

Kwa et al. [36] reported that in HIV-1 patientsbelonging to the Amsterdam cohort of homosex-ual men the )589T polymorphism in the promoterof the IL-4 gene was associated with a delayedacquisition of CXCR4 HIV-1 variants and a re-duced number of CCR5 memory CD4 T cells.Nakayama et al. [37] reported that the )589Tpolymorphism in the IL-4 gene protects againstHIV-1 disease progression.

Anti-Viral a-Defensins 1, 2, and 3 Synthesis byCD8þ T Cells, Neutrophils and other Cell Typesis Inhibited by IL-4

Mackewicz et al. [38] and Levy et al. [39] reportedthat the conditioned medium of CD8þ T cellsfrom HIV-1 infected individuals produced anunknown factor (CAF) that inhibited HIV repli-cation in cultured CD4þ T cells by a nonlytic, non-MHC restricted mechanism. Recently, Zhanget al. [40] identified a cluster of proteins that wassecreted when CD8þ T cells from long-term non-progressors with HIV-1 infection were stimulatedin vitro. These proteins were identified as a-de-fensin 1, 2, and 3. Synthetic and purified prepa-rations of a-defensins also inhibited the replicationof HIV-1 isolates in vitro. Neutrophils and CD8þ

T cells from normal blood donors were foundto have a-defensins in the cell cytoplasm withinsmall granules. Upon stimulation, CD8þ cells lostthe a-defensin-positive granules. The principlesource for a-defensin 1, 2, and 3 are the neutro-phils, but additional producers are NK cells, cdTcells, B cells, monocyte/macrophages, and epithe-lial cells.

Ong et al. [41] studied the effect of the Th2 cyto-kines IL-4 and IL-13 on the synthesis of the anti-microbial peptide b-defensin in skin keratinocytes.They reported that IL-4 and IL-13 inhibited theproduction of defensins synthesis by the kerati-nocytes. The defensins and cathelicidins are part ofthe human innate immune system. They functionas chemokines to attract immature DCs and

The Changes in the T helper 1 11

lymphocytes to site of infection. Moreover, theyserve as anti-bacterial and anti-viral substances.

Mode of Action of IL-4 and IL-5 Receptors (IL-

4Ra and IL-5R) in T Cells

The Role of IL-4 and IL-4Ra in the Th2 ImmuneResponse Against Allergens

Herrick et al. [42] investigated the role of IL-4 inthe Th2 response to an allergen by exposing miceto soluble ovalbumin (OVA), which was appliedby epicutaneous application to mouse skin underocclusive skin patch using IL- 4�/� mice. Theauthors reported that the Th2 response in thesemice remained intact after exposure to OVA.However, when STAT6 �/� mice (deficient in theSignal Traducer And Transcription activator #6)were exposed to OVA, the mice mounted a Th1-type response. Thus, mice lacking the IL-4 genewere able to induce a Th2 response, but the signalactivator for STAT6 was required for the activa-tion of the Th2 response. The mode of action ofIL-4 was summarized by Kelly-Welch et al. [43]:IL-4 signaling requires two receptors, type I andtype II, while only the type II receptor mediatesIL-13 signaling. IL-4 receptors are composed oftwo transmembrane proteins: the IL-4Ra chainthat binds IL-4 with a high affinity, leading to thedimerization with another protein to form eithertype I or type II receptor. In cells of the hemato-poietic lineage, the type I receptor arises by re-cruitment of a common gamma chain (cC). Innon-hematopoietic cells, the type II receptor isformed by the interaction of the IL-4Ra chain withthe IL-13Ral chain instead of the cC chain.

The cytoplasmic tail of IL-4 and IL-13 receptorsassociate with tyrosine kinases of the Janus family(Jakl-3, Tyk2). IL-4Ra associates with Jakl and cCwith Jak3 and the dimerization of the receptorsubunits enhances Jak activity, leading to thephosphorylation of tyrosine residues in the cyto-plasmic domain of IL-4Ra chain. These phos-photyrosines, then, act as docking sites forsignaling molecules that contain tyrosine-bindingdomains. One critical signaling pathway activa-ted by the IL-4 receptor leads to tyrosine phos-phorylation of STAT6, a latent cytoplasmictranscription factor. The phosphorylated STAT6

subsequently disengages from IL-4Ra chain, di-merizes, migrates to the nucleus and binds to con-sensus sequences found in the promoters of IL-4and IL-13 regulated genes and to the IL-4 respon-sive genes IL-4Ra, I-epsilon (Ie) gene, MHC classII, and CD23 (FceRI). The transcription generatesan immature RNA from the CE region (Ie) of theIg gene, an early step in IgE class switching. Thetranscript is termed germline e(G e) [44].

Mutated IL-4 antagonist peptides [45] orSTAT6-binding peptides [46] were reported to in-hibit the activity of IL-4 receptor. Such IL-4 an-tagonists inhibit the IL-4 induced STAT6phosphorylation and prevent IgE production byactivated B cells.

IL-5 and IL-5 Receptor (IL-5R) and theirBiological Activities

The third cytokine produced by the Th2 cells is IL-5, a hematopoietic growth and differentiation fac-tor for eosinophils and basophils. The human IL-5receptor (IL-5R) consists of an a chain that canbind IL-5 by itself with intermediate affinity, and a bsubunit that forms a high affinity receptor togetherwith the a chain. Cornelis et al. [47] transfectedmouse cells that expressed the common b chain ofIL-5R with a human IL-5Ra cDNA and inducedcell proliferation in response to picomolar concen-trations of hIL-5, suggesting that a functional hIL-5R was reconstituted. It was found that HL-5Rinduced tyrosine phosphorylation of the b chain,Jak2 kinase and cytosolic DNA binding activity.Similar results were reported by Chen et al. [48].

The mature hIL-5 cytokine consists of 115amino acids containing two cysteine residues andtwo potential N-glycosylation sites. Ingley et al.[49] reported on the purification of hIL-5 fromyeast and baculovirus systems that were trans-fected with hIL-5 cDNA and produced many hIL-5 recombinants that had a high specific activity inhuman peripheral eosinophil maintenance assay.Van der Westhuizen et al. [50] reported that dele-tion of the region 5¢ to the gene increased threefoldIL-5 production, while the deletion of the region 3¢to the gene decreased fourfold the IL-5 produc-tion. This indicated that there are control elementsin a 928 bp region 3¢ to the gene.

Mackewicz et al. [38] reported that IL-5 en-hances the replication of HIV in CD4þ lympho-

12 Becker

cytes. Kozyrev et al. [51] have constructed SIV/HIV-1 (sHIVs) chimeric viruses with an insertedhIL-5 gene in the viral vpr gene and examined theeffect of the inserted IL-5 gene expression on viralreplication. The SIV/HIV-1 chimeric virus ge-nomes were deleted for the vpr and nef genes.SHIVs were found to be infectious and the repli-cation competent viruses were used to infect hu-man T cell lines and monkey peripheral bloodmononuclear cells. The replication of SHIVs thatexpressed hIL-5 was faster than the parental vi-ruses. In a subsequent paper, Kozyrev et al. [52]reported that infection of rhesus monkeys withSHIVs that expressed hIL-5 was faster and re-sulted in greater virus replication and antibodyproduction than the nef-deleted parental SHIVinfected monkeys.

Tiffani et al. [53] studied the expression ofchemokine receptor 1 (CCR1) and 3 (CCR3) ineosinophilic HL-60 cells and reported that thesereceptors are differentially regulated by IL-5.CCR3 was expressed later and at lower levels thanCCR1 and could be further induced by IL-5,whereas IL-5 had little or no effect on CCR1 ex-pression. These results suggest that CCR3 couldalso function as an HIV-1 coreceptor in maturingeosinophils, but RANTES and MCP-3 ligands ofCCR3 did not inhibit HIV-1 env-dependent fu-sion. IL-5 is an important eosinophiloprotein thatregulates the hypereosinophilia, which is associa-ted with helminth infections. The infectionby HIV-1 of helminth-infected people may accel-erate the deterioration of the immune system andthe progression to AIDS.

The regulatory mechanisms of IL-5 productionwere studied by Heijink et al. [54] who demon-strated that IL-2 counteracts the CAMP-mediatedinhibitory effects on IL-5 secretion by modulatingphosphatidylinositide 3-kinase (P3-K)-dependentsignaling. The authors suggested that in asthma,increased IL-2 signaling may lead to enhanced IL-5 secretion.

Hypothesis, Conclusions and Suggestions

Implications of the Hypothesis and Conclusions

(i) The hypothesis suggests that a ‘‘Th2 fi Th1Reversion’’ is needed to enable infected individuals

to mount an active anti-HIV-1 CTL response anda concomitant inhibition of IgE synthesis, whichmay allow the synthesis of anti-viral antibodies.The reversion to Th1 cytokine synthesis will res-urrect the innate, cellular, and humoral immuneresponses. As a result, HIV-1 patients will be ableto clear the virus infection and recover.

(ii) The synthesis of IgE in HIV-1 infectedchildren and adults indicates the that ‘‘allergen-like domains’’ may be present in the HIV-1structural proteins gp120, p24, and p17, since anti-HIV-1 IgE antibodies were found in HIV-1 in-fected children. This suggests that ‘‘allergen-likepeptides’’ from the viral proteins triggered IgEsynthesis.

(iii) The similarities between IgE responses inHIV-1 infected people and IgE responses to envi-ronmental allergens in patients with atopic der-matitis and other allergies suggest that HIV-1/AIDS disease in humans is a form of virus-inducedallergy.

(iv) The hypothesis suggests the need to ap-proach HIV-1 infection and AIDS from the per-spective that the HIV-1 induced allergy may betreated with drugs that inhibit Th2 cytokine pro-duction or the receptor activity of IL-4R and IL-5R, as well as the activation of Th1 cytokineproduction.

(v) The hypothesis suggests that the identifica-tion and removal by genetic engineering of ‘‘al-lergen-like domains’’ from the viral proteinsgp120, p24, p17, and other proteins may provideviral proteins for the development of protectiveanti-HIV-1 vaccines.

Hypothesis: to Prevent HIV-1 Infection and theProgression to AIDS, it is Necessary to Reverse theCytokine Situation in the Patients from Th2 to Th1:‘‘Th2 fi Th1 Reversion’’ hypothesis

HIV-1 infections initially induce an anti-viral CTLresponse, but with enhanced virus replication it ishypothesized that viral ‘‘allergen-like domains’’ inthe viral structural proteins that are presented byHLA class II molecules on dendritic cells to Th2cells induce the synthesis of the cytokine IL-4, IL-5, and IL-13. Presentation of allergens to B cells inthe presence of IL-4 induce the synthesis of anti-allergen IgE antibodies that bind to FceRI (IgEreceptors) on the surface of DCs and mast cells.

The Changes in the T helper 1 13

The receptor bound IgE molecules interact withthe allergen-like domains of viral proteins andperpetuate the synthesis of anti-viral IgE. The in-crease in Th2 cytokine IL-4 inhibits the ability ofTh1 cells to synthesize IL-2, IL-12, and IFN-c, aswell as the anti-viral CTL activity and CTL pre-cursors. The progression of HIV-1 infection leadsto AIDS and to a complete failure of the immunesystem resembling an untreated allergy. Conse-quently, the hypothesis makes the following pre-dictions:(a) It is important to identify the ‘‘allergen-like

domains’’ in the viral proteins using animalmodels of allergy. If allergenic domains areidentified, genetic engineering of the viralgenes for structural and non-structural pro-teins will produce viral proteins that are free ofallergen-like domains for the development ofanti-HIV vaccines.

(b) In addition to the treatment of HIV-1 infectedpeople with anti-virals, there is a need to useanti-allergenic substances to achieve the re-version from Th2 cytokine expression to theactivation of Th1 cytokine production(Th2 fi Th1 reversion hypothesis). Inhibitionof IL-4R activity may be obtained with an-tagonists that are used for treatment of allergicpeople, IL-4 antagonists that are being deve-loped, and immune response modifiers thatinhibit Th2 cells and stimulate Th1 cells.

Ways to Achieve the ‘‘Th2 fi Th1 Reversion’’ in theTreatment of HIV-1 Infections

Antagonists of the IL-4 Signaling PathwayAllergic patients are treated with anti-inflamma-tory and immunosuppressive corticosteroid drugs.So et al. [55] reported that corticosteroids at con-centrations of 10)4–10)6 M inhibited the IL-4signaling pathway in human primary cells by downregulating the IL-4 induced IL-4R expression andSTAT6 activation at the level of tyrosine phos-phorylation.

Another approach to down regulate IL-4 acti-vity is the use of mutated IL-4 polypeptide anta-gonists that bind to IL-4Ra and abrogatethe signal transduction by the IL-4Ra. Stolzenberget al. [46] used a murine IL-4 antagonist that wasproduced by deleting the C-terminal amino acidthat follows the amino acid Q119 in the IL-4

amino acid sequence. The antagonist was studiedin an asthma model in mice and was found to in-hibit IL-4/IL-13 induced STAT6 phosphorylationand IgE production in splenocyte cultures in vitro.

Attempts to prevent the synthesis of anti-aller-gen IgE in allergic people does not require theinduction of an active CTL response since the al-lergenic proteins are the inducers of the allergicconditions. In contrast, a dual approach is neededto achieve the inhibition of IgE synthesis and in-duction of Th1 cytokine synthesis in HIV-1 pa-tients: drugs that both inhibit Th2 cell activity andactivate Th1-induced CTL anti-viral activity toclear HIV-1 from infected tissues.

The Possibility of Achieving the ‘‘Th2 fi Th1Reversion’’ in the HIV-1 Infection via Treatmentswith Immune Response Modifiers(a) The immune response modifier imiquimod (S-26308, R-837, AldaraTM, manufactured by 3MPharmaceuticals, St Paul, MN 55144, USA) wasreported to have completed phase III clinical trialsand has been shown to be safe and effective in thetreatment of patients with genital warts [56]. Imi-quimod and its derivatives are compounds fromthe imidazoquinoline family and have demon-strated potent anti-viral and anti-tumor propertiesin animal models. Imiquimod’s anti-viral activitywas studied in guinea pigs infected with HSV bytopical or intraviginal treatments. The animalswere protected from primary HSV infection. Theanti-viral activity of imiquimod was demonstratedin cytomegalovirus infected guinea pigs andarbovirus infected mice [57]. Upon oral adminis-tration to cynomolgus monkeys, one of the imi-quimod derivatives (S-28463) stimulated a dose-dependent increase in serum concentrations ofINFa, TNFa, IL-1 receptor antagonist (IL-lRa)and IL-6 while imiquimod induced increases inIFN and IL-1Ra. Oral applications of imiquimodinduced high IFN-a in rodents and primates with aminimal effective dose of 2–3 mg/kg [57].

Stanley [58] summarized the mechanism of ac-tion and therapeutic potential of imiquimod, amodulator of innate immune response of DCs andmacrophages in the induction and regulation ofTh1 responses. Imiquimod modulates an innateimmune response by activating plasmacytoid DCsand macrophages via binding to Toll-like receptor7 (TLR7), which induces the synthesis of IFN-a

14 Becker

and proinflammatory Th1 cytokines, and the ac-tivation of cell-mediated immunity. Wagner et al.[59] reported that imiquimod and its derivative R-848 (resiquimod) modulate Th1 and Th2 cytokineproduction. Furthermore, imiquimod and R-848induced human cell cultures of CD4+ and CD8þ

lymphocytes to synthesize and release IL-12 andIFN-c. R-848 (resiquimod) and imiquimod inhi-bited IL-4 and IL-5 production in human cell cul-ture systems. The inhibition of IL-5 in response toR-848 in cultures of CD4 lymphocytes and mac-rophages is mediated by IFN-a. The authors sug-gested that, ‘‘Imiquimod and R-848 may haveclinical utility in diseases where cell-mediated im-mune responses are important and in diseases as-sociated with over expression of IL-4 or IL-5 suchas atopic disease’’ [59].

(b) The immune response modifier berberine, abenzodioxoloquinolizine that has been isolatedfrom medicinal plants, was reported by Kim et al.[60] to induce interleukin-12 (IL-12) production inmacrophages and DCs in vitro. The authors re-ported that IL-12 is an inducer of IFN-c and aninhibitor of IL-4 in CD4þ T cells. Treatment ofmacrophages with anti-IL-12 mAb to cultures ofmannose-binding lectin-treated macrophages re-stored the induction of IL-4 and inhibited the in-duction of IFN-c by T cells. Kim et al. concludedthat berberine-mediated induction of IL-12 pro-duction in antigen presenting cells ‘‘may be usefulin the treatment of Th2-mediated immunologicaldisorders.’’

The HIV-1 Enigma and the Current Knowledge on

the Immunology of HIV/AIDS

The AIDS Pandemic and the Progress in theResearch of HIV-1 Pathogenicity andImmupathogenesis

After 20 years of active HIV-1 research and over40,000 publications on the subject, the scientificcommunity seems no closer to gaining an under-standing of why the vast majority of HIV-1 in-fected individuals, even those who were treatedwith anti-virals, will eventually develop AIDS.During the past 20, over 20 million people havelost their lives. Additionally, 42 million others areinfected and 15,000 people are infected every day.

An editorial in Nature Medicine entitled‘‘20 Years of HIV-1 Science’’ [61] summarized thestate of HIV-1/AIDS: ‘‘The stark reality of theAIDS pandemic twenty years after its causativeagent was first recognized is chilling. Even as thesestatistics become familiar they still hold the powerof shock, and we should never lose sight of thestaggering burden that HIV places on many popu-lations of the world.’’ In some countries, HIV-1/AIDS has drastically reduced the average lifespanfrom 63 to 36 years and has left 14 million or-phaned children in its wake [61].

The Current Understanding of theImmunopathology of HIV-1/AIDS

On the twentieth anniversary of the discovery ofHIV-1 – the causative virus of AIDS – NatureMedicine, published a series of excellent reviewson key issues of HIV-1 pathogenesis, immuno-pathogenesis, vaccines, and anti-virals. Moreover,the authors carefully analyzed the state of devel-opments in the fields of molecular biology andimmunology of HIV-1/AIDS as well as the plansfor future research. Two of the reviews expertlypresent the current understanding of the immuno-pathology of HIV-1/AIDS disease [62] and thedifficulties encountered in the efforts to developanti-HIV-1 vaccines [63].

In a review, Letvin and Walker [62] discuss thesignificance of anti-HIV-1 responses in infectedpeople. The authors indicated that HIV-1 neu-tralizing antibodies are apparently irrelevant to theearly control of HIV-1 replication, while virusspecific CD8þ cytotoxic T cells (CTLs) are activein the early weeks after HIV-1 infection [64].However, they fail to stop the virus infection. Theauthors stated that, ‘‘There have been numerousproposed mechanisms for the immune dysfunc-tion, but based on prior animal studies of immunefailure in chronic viral infections, it is likely thatlack of sufficient HIV-specific CD4þ T-helper cellproliferation and expansion is a crucial to thisimpairment.’’ Thereafter, the study by McKayet al. [63] on CD4 T-lymphocyte cytokine expres-sion in HIV/SIV chimeric virus infected monkeysis discussed. It was reported that HIV-1 infectionresults in a dysfunction of CD4 lymphocytes.However, the intracellular events that contribute

The Changes in the T helper 1 15

to the CD4 T lymphocyte dysfunction continue topuzzle researchers.

Regarding the subject of immunotherapy, theauthors discussed adoptive therapy, infusion ofanti-viral monoclonal antibodies or viral antigenspecific CTLs. After years of research, the authorsconcluded that it is not known if these treatmentsaffect disease progression. Augmentation of CD8þ

T cell responses through antigen-specific enhance-ment by therapeutic immunization did not succeed.

The finding by McKay et al. [63] may be ex-plained by Clerici and Shearer’s hypothesis that a‘‘Th1 fi Th2 Switch’’ [17] occurred in humansand animals after HIV-1 or SIV infection. Thepresent hypothesis suggests that a reversion of thevirus related inhibition of Th2 cells and the acti-vation of Th1 activity (Th2 fi Th1 Reversion)may be achieved by treating HIV-1/AIDS patientswith inhibitors such as IL-4 and IL-4Ra antago-nists, anti-allergy drugs, or immune responsemodifiers.

Current Attempts to Develop HTV-1 Vaccines

McMichael and Hanke [65] reviewed the designand experimental approach to the developmentof anti-HIV vaccines that will be able to induceantiviral antibodies and/or CTLs. The authorsdiscussed the three main challenges to thedevelopment of an effective HIV vaccine: (1) avaccine that is capable of stimulating the equiva-lent of the five known monoclonal neutralizingantibodies in high titers; (2) optimization of CD8þ

T cell-inducing vaccines; and increased capacity tocarry out phase three trials in developing countriesto immunize tens of millions of people.

The present hypothesis contends that in order toachieve these goals, a search for the presence of‘‘allergen-like domains’’ in the viral structural andnon-structural proteins is needed. It is imperativeto determine whether the immunization of healthvolunteers that were immunized with HIV-1 gp120vaccines responded to the vaccine with the syn-thesis of IgE. Since the response of HIV-1 infectedpeople to HIV-1 lead to IgE synthesis and immunedysfunction, the characterization of the IgE anti-bodies specificity needs to be further studied. Theidentification of allergen-like domains in the viralprotein may enable their deletion from the viral

proteins. This, then, may contribute to the deve-lopment of effective anti-HIV-1 vaccines [66].

Albert Sabin’s Challenge to kill HIV-1 Infected TCells is Met

In 1992, Albert Sabin [67] wrote on the improba-bility of effective vaccination against HIV due toits intracellular transmission: ‘‘The main challengeis to find a way to kill cells with chromosomallyintegrated HIV cDNA without harming normalcells’’ [66]. The present hypothesis – ‘‘Th2 fi Th1Reversion’’ – may be the answer to Sabin’s chal-lenge, as the Th2 cells are infected by HIV-1 andhave integrated viral DNA genomes. As suggestedabove, the inhibition of Th2 cells may prevent theexpression of integrated viral DNA and may thuscontrol the infection. The ‘‘Th2 fi Th1 Rever-sion’’ is essential to the efforts to prevent the HIV-1 infection from skewing the immune response.This approach may open the way for an effectivemeans with which to combat HIV-1/AIDS.

Acknowledgements

The author thanks Professor G. Darai, the Insti-tute for Medical Virology, Karl-Ruprecht Uni-versity, Heidelberg, Germany, for his advice, Dr.C.D. Enk, the Department of Dermatology,Hadassah University Hospital, Jerusalem, for theinformation on imiquimod, and Mrs. R. Bendheimfor her financial support. Thanks are due to Mr.A. Levin and Mr. A. Aronsky for their technicalhelp. The study was supported by the Foundationfor the Study of Molecular Virology and Cell Bio-logy, Paradise Valley, Arizona, Mrs. R. Bendheim,President.

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18 Becker

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