242 Siehe oben C VI Seite 309 ff.243 Schönke/Schröder/Cramer/Sternberg-Lieben § 15 Rn 60.244 Siehe unten D II Seite 368; vgl. MK/Ambos Vor §§ 3-7 Rn 3.245 Siehe unten D IV Seite 374 ff.246 Siehe unten D I 5 b Seite 365 f. zum Verbotsirrtum.247 EGMR v. 22.03.2001 - 34044/96 u.a., NJW 2001, 3035 (3039), Tz.79 ff.; EGMR v. 22.03.2011 - 37201/97, NJW 2011, 3042 (3044), Tz.75; BVerfG v. 24.10.1996 (Fn 223) Seite 130; BGH v. 03.11.1992 - 5 StR 370/92, BGHSt 39, 1 (26 f.); BGH v. 26.07.1994 - 5 StR 167/94, NJW 1994, 2708 (2709); Schmidt-Bleibtreu/Hofmann/Hupfauf/Schmähl Art. 103 Rn 40; Leibholz/Rinck/Burghart Art. 103 Rn 1260; Maunz/Dürig/Schmidt-Assmann Art. 103 Rn 255a; NK/Hassemer/Kargl § 1 Rn 48.248 Überblick hierzu Schmidt-Bleibtreu/Hofmann/Hupfauf/Schmähl Art. 103 Rn 40; Maunz/Dürig/Schmidt-Assmann Art. 103 Rn 255c; v.Mangoldt/Klein/Starck/Nolte Art. 103 Rn 125; Polakiewicz, Verfassungs- und völkerrechtliche Aspekte der strafrechtlichen Ahndung des Schusswaffeneinsatzes an der innerdeutschen Grenze, EuGRZ 1992, 177 (188).249 Sic!.
They include non-conventional international agreements, like the Helsinki Final Act (see for example, Freedom and Democracy Party (ÖZDEP) v. Turkey, no. 23885/94, § 40, 8 December 1999); treaties not ratified by the respondent State (see for example, Marckx, cited above); declarations of international organisations, like the Universal Declaration on Human Rights and other General Assembly Declarations (see for example, K.-H. W. v. Germany [GC], no. 37201/97, § 95, ECHR 2001-II); resolutions and recommendations of international organisations, like those of the Parliamentary Assembly and the Committee of Ministers of the Council of Europe (see for example, Mosley v. United Kingdom, no. 48009/08,,§§ 87, 119 and 124, 10 May 2011); General Comments of international organisations, like those adopted by the United Nations treaties bodies (see for example, Bayatyan v. Armenia [GC], no. 23459/03, § 105, ECHR 2007); and Codes of Conduct and Guidelines of international organisations, like those of the World Health Organisation (see for example, Oluic v. Croatia, no. 61260/08, § 60, 20 May 2010); commentaries and studies by ONGs, like the International Committee of the Red Cross study on customary international humanitarian law and commentaries on the Geneva conventions (see for example, Korbely v. Hungary, no. 9174/02, §§ 50, 51 and 90, ECHR 2008), and reports of individuals, like those of the United Nations Secretary-General (Korbely, cited above, § 90).
Certes, cette notion s'applique en principe à l'évolution progressive de la jurisprudence dans un même Etat de droit et sous un régime démocratique, mais elle garde toute sa valeur lorsqu'il y a eu, par exemple, succession de deux Etats (voir Streletz, Kessler et Krenz, précité, §§ 49 et 81-82 ; et K.-H.W. c. Allemagne [GC], no 37201/97, §§ 44 et 84-85, CEDH 2001-II (extraits)).
En l'espèce, cette pratique a vidé de sa substance la constitution et la législation de l'époque sur laquelle elle était censée se fonder, et ne saurait donc être qualifiée de «droit» au sens de l'article 7 de la Convention (voir, mutatis mutandis, Streletz, Kessler et Krenz précité, §§ 85 et 87 ; K.-H.W. c. Allemagne [GC], no 37201/97, §§ 88 et 90, CEDH 2001-II (extraits)).
In the performance of that task it is, notably, free to attribute to the facts of the case, as found to be established on the evidence before it, a characterisation in law different from that given by the applicant or, if need be, to view the facts in a different manner; furthermore, it has to take account not only of the original application but also of the additional documents intended to complete the latter by eliminating initial omissions or obscurities (see K.-H.W. v. Germany [GC], no. 37201/97, § 107, ECHR 2001-II (extracts), Camenzind v. Switzerland, judgment of 16 December 1997, Reports of Judgments and Decisions 1997-VIII, § 50 and Foti and Others v. Italy, judgment of 10 December 1982, Series A no. 56, pp.
Secondly, the Court observes that the interpretation in question does not appear to be the result of a gradual clarification of the rules of criminal liability through judicial interpretation from case to case (compare K.-H. W. v. Germany [GC], no. 37201/97, § 45, ECHR 2001-II).
The Court notes that it is free to attribute to the facts of the case a characterisation in law different from that given by the parties (see, among many other authorities, Foti and Others v. Italy, judgment of 10 December 1982, Series A no. 56, pp. 15-16, § 44; Camenzind v. Switzerland, judgment of 16 December 1997, Reports of Judgments and Decisions 1997-VIII, pp. 2895-96, § 50; and K.-H.W v. Germany [GC], no. 37201/97, § 107, ECHR 2001-II (extracts)).
37201/97, entstammt der Zeitschrift "ÖIMR-Newsletter" (NL 2001, 59) bzw. der entsprechenden Datenbank des Österreichischen Institutes für Menschenrechte, Salzburg, und wurde von diesem dem OGH zur Aufnahme in die Entscheidungsdokumentation Justiz im RIS zur Verfügung gestellt.


For a deed to be recorded, Georgia law requires that it be signed by the maker (grantor), attested by an officer as provided in O.C.G.A. 44-2-15, and attested by one other witness. O.C.G.A. Section 44-5-30. A deed is attested when an officer as provided in O.C.G.A. 44-2-15 and one or more natural persons see the maker sign the deed and they "attest" to the fact by signing the deed as officer and witness.
As of the July 1, 2015 revision to O.C.G.A. 44-5-30, an acknowledged deed will not be entitled to be recorded. "Acknowledging" a deed is not the same as "attesting" a deed. To be recorded, a deed must be "attested" by an officer as provided in O.C.G.A. 44-2-15, and attested by one other witness ". If there is only one (1) "attesting" witness, the deed is not entitled to be recorded, even if the deed is subsequently "acknowledged" and even if the one "attesting" witness is an authorized officer. O.C.G.A. Section 44-2-14.
The above requirements are the same for all deeds, regardless of their nature. Therefore, when attestation is not proper, it must be reported as a title defect.
For a deed to be recorded, Georgia law requires that it be signed by the maker (grantor) and attested by at least two (2) witnesses. O.C.G.A. Section 44-5-30. A deed is attested when two or more natural persons see the maker sign the deed and they "attest" to the fact by signing the deed as witnesses.
Also, the deed must be attested or acknowledged by an officer authorized by Georgia statutes. O.C.G.A. Section 44-2-14, 44-2-15. The official witness can be one of the two (2) required attesting witnesses. If the deed is not attested by an official witness, it may be acknowledged after execution and the acknowledgment certified on the deed by the official witness entitling the deed to be recorded. O.C.G.A. Section 44-2-16.
Acknowledgment involves the grantor's declaration before a notary public or other officer that, in fact, he is the maker named in the deed, that he signed the deed, and that the signature on the deed is in fact his signature; these facts provide the substance of the officer's certification of acknowledgment. White v. Margarahan, 87 Ga. 217, 13 S.E. 509 (1891) (overruled on other ground by Leeds Building Product, Inc. v. Sears Mortgage Corp. 267 Ga. 300, 477 S.E. 2d 565 (1996) which allows a deed that was properly attested or acknowledged on its face, but contained a latent defect, to provide constructive notice. Although customary in some jurisdictions, the Georgia Code does not require the maker's signature on the acknowledgment. O.C.G.A. Section 44-2-15.
An acknowledgment does not eliminate the requirement for at least two (2) attesting witnesses. "Acknowledging" a deed is not the same as "attesting" a deed. To be recorded, a deed must be "attested" by two (2) witnesses; if one of the "attesting witnesses is an authorized officer, there is no need for an "acknowledgment". If there are two (2) unofficial "attesting" witnesses, the deed is still not entitled to be recorded until "acknowledged" by an authorized officer. If there is only one (1) "attesting" witness, the deed is not entitled to be recorded, even if the deed is subsequently "acknowledged" and even if the one "attesting" witness is an authorized officer. O.C.G.A. Section 44-2-14.
The above requirements are the same for all deeds, regardless of their nature. Therefore, when attestation or acknowledgment is not proper, it must be reported as a title defect.
To admit deeds to secure debts or mortgages to record, they must be attested by an officer as provided in Code Section 44-2-15 and attested by one other witness. O.C.G.A. Section 44-14-61 and O.C.G.A. Section 44-14-33. A deed to secure debt or mortgage is attested when an officer as provided in O.C.G.A. 44-2-15 and one or more natural persons see the maker sign the deed to secure debt or mortgage and they "attest" to the fact by signing the deed to secure debt or mortgage as officer and witness.
When a security deed is defectively attested, it may be corrected by a new corrective deed or a modification agreement. See Revised State Bar of Georgia Title Standards §14.4. O.C.G.A. Section 14-2-18 provides that if a mortgage or security deed is not properly attested, the security deed may be recorded upon the affidavit of a subscribing witness, and the officer must testify to the execution of the deed and its attestation according to law. Also, O.C.G.A. Section 44-2-19 outlines procedures for recording a deed by affidavit when the subscribing witness or witnesses are dead, insane or have moved outside the state or otherwise incapacitated to make an affidavit.
O.C.G.A. §44-14-64 sets out that all transfers of deeds to secure debt….”shall be witnessed as required for deeds”.
When a quit claim deed is executed to release property from a Security Deed it should be attested in the same manner required for deeds, see Chapter 4.5. When a cancellation is executed to cancel the security deed of record, OCGA 44-14-67 provides a form and requires that any cancellation shall be substantially similar to the form provided, see Chapter 14.7. The form provides for a witness and notary, therefore good title practice requires that the cancellation should be executed in the same manner as deeds.
To admit deeds to secure debts to record, they must be attested or proved in the same manner prescribed by law for mortgages. O.C.G.A. Section 44-14-61. To admit a mortgage to record, it must be attested by or acknowledged before an officer as prescribed for the attestation or the acknowledgment of deeds of bargain and sale. In the case of real property, a mortgage must also be attested or acknowledged by one additional witness. O.C.G.A. Section 44-14-33.
When a security deed is defectively attested or acknowledged, it may be corrected by a new corrective deed or a modification agreement. See Revised State Bar of Georgia Title Standards §14.4. O.C.G.A. Section 14-2-18 provides that if a mortgage, or security deed, is neither attested by nor acknowledged before one of the officers set out in O.C.G.A. Section 44-2-15, the security deed may be recorded upon the affidavit of a subscribing witness, and the officer must testify to the execution of the deed and its attestation according to law. Also, O.C.G.A. Section 44-2-19 outlines procedures for recording a deed by affidavit when the subscribing witness or witnesses are dead, insane or have moved outside the state or otherwise incapacitated to make an affidavit.
A deed that is not properly attested or acknowledge is ineligible for recording, and may be rejected by the clerk of the court, or, even if recorded, will not serve as constructive notice. Coniff v. Hunnicutt, 157 Ga. 823, 122 S.E. 694 (1924). However, that actual notice from record, if seen in the record by the purchaser or his attorney, will be effective even if the deed is ineligible for recording, and thus will trigger inquiry notice. Gardner v. Granniss, 57 Ga. 539 (1876). This conforms with Georgia's race/notice standard which provides that a purchaser who has actual notice of a prior instrument does not have priority over a subsequent purchaser even if that prior instrument is unrecorded or defectively recorded. O.C.G.A. Section 44-2-3.
A deed that is not properly attested or acknowledged is ineligible for recording, and may be rejected by the clerk of the court, or, even if recorded, will not serve as constructive notice. Coniff v. Hunnicutt, 157 Ga. 823, 122 S.E. 694 (1924). However, that actual notice from record, if seen in the record by the purchaser or his attorney, will be effective even if the deed is ineligible for recording, and thus will trigger inquiry notice. Gardner v. Granniss, 57 Ga. 539 (1876). This conforms with Georgia's race/notice standard which provides that a purchaser who has actual notice of a prior instrument does not have priority over a subsequent purchaser even if that prior instrument is unrecorded or defectively recorded. O.C.G.A. Section 44-2-3.
Taken together, U.S. Bank National Association v. Gordon, 709 S.E. 2d 258 Ga (2011) and Wells Fargo Bank v. Gordon, 292 Ga. 474 (2013) establish that for a security deed to be "duly filed, recorded and indexed," in satisfaction of O.C.G.A. Section 44-14-33 and the 1995 Amendment, the security deed must not be facially defective and thus cannot be missing any required signatures or attestations and acknowledgments. The Supreme Court in the Gordon cases explicitly voiced concern that the relaxation of execution formalities, for equitable reasons, would increase the risk of fraud and would undermine the efficacy and purpose of such requirements for the recording system. Note that under §544(a) of the Bankruptcy Code, the Trustee enjoys a heightened hypothetical bona fide purchaser status, regardless of actual notice, that has not yet been applied outside of the bankruptcy context by the Georgia Supreme Court.
If the execution of any document in the chain of title is made by an attorney-in-fact, it is better practice that the title examiner be able to verify that: the power of attorney is dated, properly executed, witnessed and recorded; empowers the attorney to execute the document; refers to the real property as specifically as possible; and at the time the attorney executed the document, the power of attorney had not been terminated of record by the principal, nor was there any evidence of record that the principal was deceased or mentally incompetent at that time, if the power of attorney provides that it terminates in the event of mental incompetency. A power of attorney is not terminated and it remains durable if the principal becomes mentally incompetent, unless the power of attorney provides to the contrary. If the examiner is dissatisfied with any or these facts surrounding the power of attorney, then the examiner should raise his/her objections to the client to permit the closing attorney to resolve the matter.
Comment: See the Comment above to Standard 8.4.
When a corporate instrument is either (i) executed by a corporate officer, that person’s signature is attested by the secretary or assistant secretary (or other officer authorized to authenticate corporate records), and the corporate seal or a facsimile thereof is affixed, or (ii) executed by the president or vice president and that person’s signature is attested by the secretary or assistant secretary (or other officer authorized to authenticate corporate records), a title examiner may assume that the officer executing the document in fact holds the position indicated, that such person is authorized to execute the document, and that the officer’s signature is genuine.
Comment: Source — O.C.G.A. Section 14-2-151. See also O.C.G.A. Section 14-5-7, Clause (ii) above became effective July 1, 1992.
The Examiner should bear in mind that the evidentiary presumption afforded by O.C.G.A. Sections 14-2-151 and 14-5-7 do not establish recording requirements. Recording is governed by O.C.G.A. Section 44-2-14, which requires, with respect to execution, a signature, two witnesses, and an attestation or acknowledgment.attested by an officer as provided in O.C.G.A. 44-2-15, and attested by one other witness. Neither the presence of a corporate seal, nor the signature of an officer specified in O.C.G.A. Section 14-5-7, is required for recording.
Foreclosures in Georgia may be conducted in equity; pursuant to statutory procedures; by execution, levy and sheriff’s sale; and by non-judicial power of sale. The non-judicil power of sale is the most common method of foreclosure in Georgia. The most common security instrument in Georgia is the deed to secure debt also referred to as a security deed.
When a foreclosure appears in the chain of title, an examination of the security instrument which was foreclosed is necessary. The security instrument and succession of transfers to the current holder authorized to conduct the sale should be recorded. A foreclosure sale conducted after the statute of limitations has run on the security instrument is void. Failure to pay intangible recording tax applicable to the security instrument constitutes a bar to foreclosure. Such bar to foreclosure may be removed by payment of the tax plus penalty and interest. Since non-judicial power of sale is a contractual remedy, the terms and procedures for conducting the foreclosure sale must be set forth in the security instrument to be foreclosed. The instrument must also contain a valid power of attorney empowering the grantee to conduct the foreclosure sale and execute the deed under power of sale as the attorney in fact for the grantor.
While there is no statutory procedure for conducting non-judicial foreclosure sales, it is required that a non-judicial foreclosure sale be advertised and conducted in the same manner as sheriffs’ sales. If all or a part of the property to be foreclosed was to be used as a dwelling place of the debtor at the time the security instrument is entered into, Written notice of the sale must be given to the debtor by registered or certified mail sent no later than 30 days prior to the foreclosure sale date.
Comment: For a comparison of security deeds with other security instruments, see Pindar and Pindar, Ga. Real Est. Law, Section 21-4 (4th ed.).
Security deeds and transfers recorded after July 1, 1989, should include the mailing address of the grantee or transferee. However, failure to include the mailing address is not a defense to foreclosure (O.C.G.A. Sections 44-14-63 and 64). A foreclosure sale by a holder of a security instrument pursuant to an unrecorded transfer is valid. [Burgess v. Simmons, 207 Ga. 291, 61 S.E.2d 410 (1950)]. However, failure to record the transfer would require future examiners to inquire as to the authority of the party executing the deed under power of sale. [Pindar and Pindar, Ga. Real Est. Law, Section 21-46 (4th ed.)].O.C.G.A. §44-14-162(b) requires that the assignment into the foreclosing lender must be recorded prior to the time of the foreclosure sale.
O.C.G.A. Section 44-14-80 establishes a 7-year reverter following maturity of the debt or recording or delivery if no maturity is stated and a 20-year reverter from the date of the conveyance if no maturity is stated and the conveyance contains an affirmative statement that the parties intend to establish a perpetual or indefinite security interest in the property conveyed to secure a debt or debts. (See Standard 14.6). Prior to the 1994 amendment, this Code Section established a 20-year reverter following maturity of the debt or recording or delivery if no maturity was stated. The amended Code Section should not be relied upon as to security deeds executed prior to the effective date, April 19, 1994, of the 1994 amendment. O.C.G.A. Section 44-14-81 bars a foreclosure after the reversion period. The expiration of the statute of limitations on a note secured by a security deed does not bar foreclosure (O.C.G.A. Section 44-14-43).
O.C.G.A. Section 48-6-61 imposes a tax of $1.50 for each $500.00 or fraction thereof of the face amount of a long-term note secured by real estate as defined at O.C.G.A. Section 48-6-60 (2). O.C.G.A. Section 48-6-77(a) bars foreclosure unless applicable intangible recording tax is paid. This same Code Section imposes a penalty of 50% of the amount of the tax plus interest if such tax is not paid within 90 days of the date of the instrument.
For standards regarding powers of attorney, see Standard 8.5.
See O.C.G.A. Section 23-2-114 for power of sale in deed to secure debt and O.C.G.A. Section 44-14-162 for conducting as a sheriff’s sale.
See O.C.G.A. Sections 44-14-162.1 through 162.4 for notice requirements to debtor and deed recitals.


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Impaired APC functions may play important roles in chronicity of hepatitis C virus (HCV) and HIV infections. To investigate the separate and combined effects of HCV and HIV infection on immature dendritic cells (DCs), we evaluated myeloid-derived DC (MDC) and plasmacytoid-derived DC (PDC) frequencies and functions, measured by Toll-like receptor ligand-induced IFN-α and IL-12, in healthy controls and subjects with chronic HCV, HIV, and HCV-HIV infection. To evaluate the relation between innate and adaptive immunity, we measured HCV-specific IFN-γ-producing T cell frequency. MDC frequencies tended to be reduced in HIV infection (1.8-fold), while PDC frequencies were minimally reduced in HCV infection (1.4-fold). In contrast, a striking reduction in non-PDC-associated IFN-α production was observed in HIV-infected subjects (17-fold), while PDC-associated IFN-α production was markedly reduced in HCV-infected subjects (20-fold). Both non-PDC and PDC functions were impaired in HCV-HIV coinfection. MDC-associated IL-12 production was markedly reduced in both HCV and HIV-infected subjects (over 10-fold). Functional defects were attenuated with slowly progressive HIV infection. The proportion of subjects with HCV-specific T cell responses, and the number of Ags recognized were reduced in HCV-HIV subjects as compared with HCV singly infected subjects. A positive association was observed between MDC-associated IL-12 production and HCV-specific T cell frequency in HCV-infected subjects. These results indicate that immature DC function is dysregulated in HIV and HCV infections, but differentially, and that these defects are attenuated in slowly progressive HIV infection. These selectively different impairments may contribute to the reduced adaptive immune response to HCV in HCV-HIV coinfection.
There are over 3 million Americans chronically infected with hepatitis C virus (HCV).4 These individuals are at increased risk of developing cirrhosis, liver failure, and hepatocellular carcinoma (1, 2). HIV infection appears to alter the course of HCV-related disease, accelerating rates of progression to cirrhosis (3, 4, 5). Additionally, HCV infection has emerged as a cause of increased morbidity and mortality in HIV-infected individuals (6, 7). Despite a high prevalence of HCV-HIV coinfection, the interrelationships between these viruses are not completely understood.
The T cell response directed at HCV-derived Ags in the infected host is thought to be important in the pathogenesis of HCV-mediated disease, associated both with favorable clinical outcome as well as organ damage (8, 9). Additionally, effector dysfunction of HCV-reactive T cells has been proposed to contribute to the frequent persistence of HCV infection (10, 11). It appears that in chronically infected individuals, this dysfunction may be specific for HCV (10, 12, 13, 14). Moreover, we have previously found that CD4 cell responses to HCV are reduced in magnitude in individuals coinfected with HIV when compared with HCV singly infected individuals (13). The mechanism for this additional impairment, though potentially due to reduced CD4 cell numbers in HIV infection, is not clear. In contrast to HCV singly infected and HCV-HIV coinfected subjects with progressive HIV disease, we have also previously found that HCV-HIV coinfected subjects with slowly progressive HIV disease have higher frequencies of HCV-specific CD8 cells than observed in HCV singly infected individuals (14). HCV-HIV slow progressors also tended to have lower HCV plasma levels than did HCV-HIV-infected subjects with progressive HIV disease. One possible mechanism for T cell effector dysfunction in HCV infection is abnormal priming and stimulation of HCV-specific T cell populations by dysfunctional APC populations.
Dendritic cells (DC) are a distinct population of bone marrow-derived leukocytes that are capable of directing the magnitude, polarity, and effector function of the T cell response (15). This is possible because DCs possess pathogen recognition receptors, Ag capturing and processing machinery, and costimulatory molecules which allow them to act as professional APC. Immature DC are efficient at Ag uptake (16). Once activated, they are capable of efficiently migrating to lymphoid tissue and priming T cell responses. Mature DC, in addition to expressing necessary costimulatory signals, also secrete IFN-α and IL-12, which facilitate the effective priming of human type 1 cytokine-producing T cells (15, 17, 18, 19) and resistance to viral infection (20). DC precursors have been characterized by cell surface phenotype, and have been divided into two subsets (myeloid-derived DC (MDC) and plasmacytoid-derived DC (PDC) or immature MDCs and PDCs) (21). MDC are myeloid derived and in general are characterized by expression of CD11c, HLA DR, GM-CSFR, CD13, and CD33, and a lack of expression of IL-3R (CD123) or lineage marker expression. PDC are plasmacytoid derived and are characterized by the expression of CD123, and a lack of expression of CD11c or lineage marker. This latter cell type had initially been shown to be a dominant IFN-α-producing cell type upon viral challenge (22). However, other DC subtypes are also capable of dominant IFN-α secretion during viral infection (23). Initially, MDC and PDC were thought to prime primarily type 1 and type 2 T cell responses, respectively (21). However, subsequent data suggest that function (priming of type 1 vs type 2 responses) may vary with time between Ag exposure and presentation (24), and that PDC are capable of priming type 1 responses in an IL-12 and IFN-α-dependent fashion (18). Thus, circulating peripheral MDC and PDC populations can mature to DC populations capable of IFN-α and IL-12 secretion, and efficient priming of type 1 responses that may be important for control of chronic viral infections such as HCV and HIV.
Reduced numbers of PDC populations have been observed in HIV-infected individuals (25, 26, 27). These numbers are more markedly reduced in subjects with AIDS, and PDC number has been found to inversely correlate with HIV viral level and CD4 cell count, suggesting that these cells may be important in the control of HIV, or that they are reduced as a result of more advanced HIV disease (27). Furthermore, HSV and influenza-induced PDC IFN-α secretion has been found to be reduced in HIV-infected subjects (25, 26, 27). This IFN-α-producing function has also been found to inversely correlate with plasma HIV levels, suggesting the possible importance of this function in the control of HIV (25, 27). Additionally, MDC numbers have been found to be reduced in HIV-infected subjects (25, 26). In one study, MDC number was reduced in HIV-infected subjects with higher plasma viral levels (>5000 copies/ml), and MDC number was inversely associated with plasma HIV levels, again suggesting the potential importance of MDC number in the control of HIV (26). In addition to an IFN-α-producing deficit, APC from HIV-infected subjects are reduced in IL-12-producing function (28, 29). Therefore, it appears that MDC and PDC numbers and function are affected in HIV infection, and are either impaired by HIV replication or may be important in the control of HIV replication.
In HCV-infected subjects, cultured APC populations have been shown to have impaired function in allostimulatory mixed lymphocyte reactions (30, 31, 32). In one study, this reduced allostimulatory capacity was associated with reduced IFN-γ secretion in mixed lymphocyte reaction supernatants and reduced LPS-induced IL-12-secreting capacity (32). Consistent with this impairment in allostimulatory function are the findings that HCV-infected individuals have an impaired response to new antigenic stimulation with hepatitis A, hepatitis B, and influenza vaccinations (33, 34, 35), while responses to recall Ags are unimpaired (10, 13, 14, 36). Therefore, it appears that there is an APC-based impairment in development of T cell responses in HCV infection.
To investigate the mechanism underlying our previous finding that HIV infection impacts on HCV-specific T cell responses in individuals coinfected with both viruses, we explored the effect of HIV infection on APC dysfunction present in HCV infection. We conducted an analysis of circulating DC precursor frequency and function in HCV, HCV-HIV, and HIV-infected subjects. HIV-infected subjects with slow progressor phenotype were analyzed separately. Our data reveal a predominant deficit of non-PDC-associated IFN-α-secreting function in HIV-infected subjects, whereas we observed a predominant deficit of PDC-associated IFN-α-secreting function in HCV-infected subjects. Both functions are dramatically impaired in HCV-HIV-infected subjects. Furthermore, we observed defects in MDC-associated IL-12-producing function in both HIV and HCV-infected subjects. Moreover, HCV-HIV coinfected subjects were observed to have reduced HCV-specific IFN-γ-producing T cell responses in comparison to HCV singly infected subjects. Finally, there appears to be a relation between immature DC function and the presence of HCV-specific type 1 cytokine-producing T cell immunity. These data indicate that impairment of immature DC function differs between HCV and HIV infection, and offer one mechanism by which HIV infection may impact on the adaptive immune response to HCV.
Chronic HCV-infected subjects (n = 18) had detectable serum HCV Ab and RNA. HCV-HIV coinfected subjects (HCV-HIV progressors, n = 13; HCV-HIV slow progressors, n = 7) had, in addition, detectable HIV Abs. HIV singly infected subjects (HIV progressors, n = 15; HIV slow progressors, n = 4) had HIV Ab, but no detectable HCV Ab. Slow progressor subjects were HIV infected for at least 7 years, without clinical manifestations of HIV infection, without previous HIV therapy, and had CD4 cell counts >400/μl. No subject had previously received therapy for HCV. Eleven healthy controls had no HCV or HIV Abs. All studies were performed with approval of the institutional review boards for human studies at the Cleveland Veterans Affairs Medical Center and University Hospitals of Cleveland (Cleveland, OH).
Recombinant HCV core, NS3, NS4, and NS5 proteins were supplied by Chiron (Emeryville, CA). HCV core 15-mer peptides with 11-aa overlap (n = 45) were synthesized by the multipin technique (Chiron Technologies, Raleigh NC), and pooled together as a set. A set of 73 MHC class I-restricted HCV peptides representing portions of the HCV genotype 1 protein sequence, based upon previously described CD8 determinants (8, 37, 38, 39), were synthesized by the multipin technique (Chiron Technologies) and pooled together as a set. Where published sequences differed from those published by Choo et al. (40), additional peptides were designed to match the latter sequence. Sixty one of these 73 peptides have been previously described as a set (41). The remaining 12 peptides in the set of 73 were also based upon previously described CD8 determinants (10, 42, 43, 44) as follows: core 111-119 DPRRRSRNL, NS5 2594-2602 ALYDVVTKL, E2 530-539 GENDTDVFVL, E2 654-662 LEDRDRSEL, NS4 1941-1948 AARVTAIL, NS5 2152-2160 HEYPVGSQL, NS5 2161-2171 PCEPEPDVAVL, NS5 2221-2231 SPDAELIEANL, NS3 1359-1367 HPNIEEVAL, NS4B 1666-1675 VLAALAAYCL, NS5A 1987-1995 VLSDFKTWL, and NS5A 2140-2149 LLREEVSFRV.
Four-color FACS analysis was performed on freshly Ficoll-prepared PBMC populations (1 × 106 PBMC analyzed per sample). PBMC that lack lineage marker expression (CD3, CD14, CD16, CD19, CD20, and CD56) (Dendritic Cell Bundle kit; BD Biosciences, Mountain View, CA), express HLA DR, and express CD11c were identified as MDC; while PBMC that lack lineage marker expression, express HLA DR, and express CD123 were identified as PDC (15, 21, 22). Frequencies of these populations were determined as the proportion of live PBMC that stained negative or positive for the appropriate markers (Fig. 1⇓). Four-color analysis was performed on a FACSCalibur (BD Biosciences) flow cytometer using CellQuest Software (BD Biosciences). Notably, there are likely multiple myeloid-derived APC subsets (45, 46), and a small subset of CD16-expressing MDC that may not be as efficient as other MDC subsets in accessory function has previously been described (46, 47). Based upon our lineage marker gating, we have excluded this latter subset expressing CD16 in our analysis of immature MDC.
FACS analysis of PBMC for MDC and PDC frequency. Frequencies of MDC and PDC populations were determined as the proportion of live PBMC (initial gate based upon forward and side scatter) that lack lineage marker expression (CD3, CD14, CD16, CD19, CD20, and CD56) shown in second gate, express HLA DR, and express CD11c (MDC) or CD123 (PDC).
In some experiments, MDC and PDC subset analysis was combined with intracellular cytokine staining for IL-12 or IFN-α using mouse anti-human IL-12 (p40/p70) mAb PE (clone: C11.5; BD PharMingen, Mountain View, CA) or mouse monoclonal anti-human α2-IFN PE (clone: 225.2C; Chromaprobe, Aptos, CA).
Freshly prepared PBMC populations (600,000 cells/well) were stimulated in 96-well plates with medium alone, 50 μg/ml poly(I:C) (Amersham Biosciences, Piscataway, NJ), 25 μM CpG (ACCGATAACGTTGCCGGTGACGGCACCACG), or 25 μM CpG control (ACCGATACCGGTGCCGGTGACGGCACCACG) (48) for 20 h at 37°C in the presence of penicillin, streptomycin, l-glutamine, and 5% human AB serum (Gemini Bio-Products, Woodland, CA). Culture supernatants were quantitated for IFN-α by standard ELISA which measured human IFN-αA, α2, αA/D, αD, αK, and α4b (BioSource International, Camarillo, CA). Poly(I:C) and CpG concentration curves revealed that the above concentrations were within the upper linear range for stimulation of IFN-α production (not shown). Time curves performed revealed that a 20-h incubation was optimal for quantitating IFN-α production. IL-12 (p40/p70) was also quantitated from culture supernatants using standard ELISA (R&D Systems, Minneapolis MN). No detectable LPS contaminant was observed in poly(I:C) preparations using E-TOXATE assay (Sigma-Aldrich, St. Louis, MO). Depletions of CD11c or CD123-bearing cell populations were performed using RosetteSep depletion mixtures (StemCell Technologies, Vancouver, British Columbia, Canada) resulting in depletion of over 75% of MDC and 60% of PDC populations, respectively.
PBMC were plated (3 × 105 cells/well), in the presence (in duplicate) or absence (in triplicate) of pooled peptide (HCV core 15-mer pool at 2 μM each peptide and 0.5% DMSO or pool of 73 previously defined CD8 determinants, at 3.4 μM for each peptide and 0.5% DMSO) or protein Ag (10 μg/ml). Ninety-six-well ELISPOT cell cultures were incubated for 20 h at 37°C, developed, and analyzed as previously described (41, 49, 50, 51). A response was defined here as a peptide-specific IFN-γ-producing frequency ≥20/million PBMC, and 3 SDs greater than the mean frequency in the absence of peptide.
Continuous data are displayed as medians. Differences among groups were compared using ANOVA when samples were normally distributed, or using Kruskal Wallis when samples were not normally distributed. When differences existed, Tukey’s test was used to compare values between groups when normally distributed, and the Mann-Whitney U test was used when samples were not normally distributed. The nonparametric Spearman’s test was used to explore correlations. χ2 analysis was used to compare the proportion of subjects in each group with an HCV-specific T cell response. All analyses were done using SPSS version 11.0 (Chicago, IL).
Study subject characteristics are shown in Table I⇓. The HCV plasma level was lower in HCV-HIV slow progressors as compared with HCV-HIV progressors (480,000 vs 1,680,000 IU/ml; p = 0.04), extending upon our previously published finding where a nonsignificant trend existed (14). Plasma HIV levels and CD4 cell counts did not significantly differ between HCV-HIV progressors, HCV-HIV slow progressors, HIV slow progressors, and HIV-infected progressor subjects, though there was a trend toward higher CD4 cell counts in HCV-HIV slow progressors and HIV slow progressors vs HIV and HCV-HIV-infected subjects (median 719 and 727 vs 503 and 532 cells/μl, p > 0.05). Ten of 13 HCV-HIV progressors and 11 of 15 HIV-infected subjects were receiving antiretroviral therapy.
To assess MDC and PDC frequencies as a function of HCV vs HIV infection, we performed a cross-sectional analysis of PBMC populations freshly prepared from our groups of HCV-infected, HCV-HIV coinfected with progressive HIV disease, HCV-HIV-infected with slow progressor status, HIV slow progressors, HIV-infected progressors, and healthy control subjects. Because cell surface phenotype did not vary between HCV-HIV slow progressors and HIV slow progressors, these two groups are represented as the same group (HIV slow progressors). As shown in Fig. 2⇓, MDC frequencies tended to be reduced in HIV and HCV-HIV-infected subjects with progressive HIV disease as compared with HCV-infected subjects (median 0.11 ± 0.08%, 25/75th percentile 0.03/0.15% and 0.08 ± 0.05% 25/75th percentile 0.03/0.13% vs 0.2 ± 0.18%, 25/75th percentile 0.11/0.29%, p = 0.05 and p = 0.01; Fig. 2⇓A). In contrast, PDC frequency was minimally reduced in HCV and HCV-HIV-infected progressors as compared with healthy control subjects (median 0.08 ± 0.05%, 25/75th percentile 0.06/0.14% and 0.07 ± 0.05%, 25/75th percentile 0.03/0.13% vs 0.11 ± 0.13%, 25/75th percentile 0.09/0.36%, p = 0.04 and p = 0.04; Fig. 2⇓B). Overall, these data indicate that in HIV infection there tend to be modest (1.8-fold) reductions in frequencies of MDC populations, while in HCV infection there are minimal (1.4-fold) reductions in frequencies of PDC populations.
Frequencies of MDC and PDC in HCV, HCV-HIV, HIV slow progressor, HIV, and healthy control subject PBMC populations. Four-color FACS analysis was performed on freshly Ficoll-prepared PBMC populations obtained from HCV-infected (n = 16), HCV-HIV-infected (n = 13), HIV-infected with slow progressor status (n = 10; HCV-HIV slow progressors n = 3, and HIV slow progressors n = 4; combined together and referred to as HIV sp), HIV-infected (n = 12), and healthy control (n = 10) subjects. A, Frequencies of PBMC that lack lineage marker expression (CD3, CD14, CD16, CD19, CD20, and CD56), express HLA DR, and express CD11c (MDC). B, The frequencies of PBMC that lack lineage marker expression, express HLA DR, and express CD123 (PDC). Median values for each group are marked.
Because CD4 cell count often reflects the stage of HIV-related disease, and to investigate whether MDC and PDC frequency are correlated, we compared MDC and PDC frequencies, and CD4 cell numbers, in our subjects. We observed direct correlations between MDC and PDC frequencies in all groups separately and combined. Fig. 3⇓A and Table II⇓ show this relation for HIV and HCV-HIV coinfected groups combined (r = 0.75, p = <0.001). Additionally, CD4 cell count directly correlated with MDC frequency in HIV-infected groups combined (r = 0.65, p < 0.001, Fig. 3⇓B). There was also a weak direct correlation between CD4 cell count and PDC frequency in HIV and HCV-HIV coinfected subjects when combined (r = 0.32, p = 0.04, Fig. 3⇓C). Therefore, MDC and PDC frequencies are closely related to each other in both the presence and absence of viral infection with HCV or HIV, and each population is associated with CD4 cell number in HIV-infected subjects.
Correlations between MDC frequency, PDC frequency, and CD4 count. Correlation plots are shown for HIV and HCV-HIV coinfected subjects combined (n = 36). A, MDC vs PDC frequencies; B, CD4 cell count vs MDC frequency; C, CD4 cell count vs PDC frequency.
We next investigated MDC and PDC function in freshly prepared nonexpanded PBMC subpopulations by stimulation with the Toll-like receptor (TLR) ligands poly(I:C) and CpG oligodeoxynucleotides. Both poly(I:C) and CpG have been previously described to stimulate DC populations via TLR3 and TLR9, respectively (15, 52, 53). Poly(I:C) stimulates IFN-α secretion from non-PDC or MDC populations (48) (23, 53), while CpG stimulates IFN-α secretion from PDC populations (52, 54). To minimize in vitro artifact that purification or long-term cell culture can introduce, we used these two reagents to directly stimulate freshly prepared PBMC populations. Stimulation with control oligonucleotides lacking the CpG motif resulted in no IFN-α production (not shown). As shown in Fig. 4⇓, poly(I:C) induced IFN-α secretion, and this activity was inhibited by selective depletion of cells bearing the MDC marker CD11c. CpG also induced IFN-α secretion, and this activity was inhibited by selective depletion of cells bearing the PDC marker CD123. Intracellular staining for IL-12 followed by FACS analysis confirmed that MDC cells (Lin−/HLA DR+/CD11C+) produced IL-12 in response to poly(I:C) (not shown). Therefore, stimulation of freshly prepared PBMC populations with these two reagents allows for assessment of non-PDC-associated IFN-α, MDC-associated IL-12, and PDC-associated IFN-α-producing capacity.
Poly(I:C) and CpG-induced IFN-α is dependent on CD11c and CD123-expressing cell compartments, respectively. Depletion of CD11c or CD123-bearing cell populations was performed with three separate healthy control subjects, resulting in selective depletion of 75–90% of MDC and 60–90% of PDC populations, respectively. Additionally, evaluation of the lin−DR+CD11c− and CD123− populations revealed that CD11c depletion resulted in at least 75% reduction in the lin−DR+CD123− population, while CD123 depletion resulted in at least 39% reduction in the lin−DR+CD11c− population, indicating that depletion reagent did not just remove the surface molecule, but the cell population expressing the appropriate surface molecule (not shown). These same freshly prepared PBMC, CD11c-depleted PBMC, or CD123-depleted PBMC populations (600,000 cells) from these three healthy control subjects were stimulated with medium alone, poly(I:C) (50 μg/ml), or CpG (25 μM) 20 h at 37°C. Culture supernatants were quantitated for IFN-α by standard ELISA. Poly(I:C)-induced IFN-α ranged from 400 to 452 pg/ml and CpG-induced IFN-α ranged from 480 to 1500 pg/ml in nondepleted PBMC populations, while <10 pg/ml was observed in medium-containing wells (not shown). The proportion (percent) of nondepleted PBMC poly(I:C) or CpG-induced IFN-α-induced activity is shown for CD11c and CD123-depleted populations. Depletion of CD11c-bearing cells reduced poly(I:C)-induced IFN-α production by 90% (activity reduction corresponding closely with MDC depletion efficiency), while depletion of CD123-bearing cells reduced CpG-induced IFN-α production by 60% (again activity reduction corresponding closely with PDC depletion efficiency). This experiment was performed two additional times with one of the three healthy control subjects.
Because IFN-α plays a role in priming type 1 T cells, we next examined poly(I:C)-induced IFN-α (non-PDC associated) and CpG-induced IFN-α (PDC associated)-producing capacity in freshly prepared PBMC from our subjects. As shown in Fig. 5⇓A, poly(I:C)-induced IFN-α-producing capacity was reduced 17-fold in HIV and HCV-HIV subjects as compared with HCV-infected subjects (median 30 ± 323 pg/ml, 25/75th percentile 11.5/130 pg/ml and 40 ± 73 pg/ml, 25/75th percentile 14/120 pg/ml vs 510 ± 314 pg/ml, 25/75th percentile 290/762 pg/ml, p = 0.005 and p < 0.001), and reduced 12-fold in HCV-HIV-infected subjects when compared with healthy control subjects (median 40 ± 73 pg/ml, 25/75th percentile 14/120 pg/ml vs 380 ± 212 pg/ml, 25/75th percentile 195/627 pg/ml p = 0.03). Poly(I:C)-induced IFN-α production did not significantly differ between HCV-infected subjects and healthy control or HIV slow progressor (with or without HCV coinfection) subjects (510 ± 314 pg/ml, 25/75th percentile 290/762 pg/ml vs 380 ± 212 pg/ml, 25/75th percentile 195/627 pg/ml and 110 ± 238 pg/ml, 25/75th percentile 33/412 pg/ml, p > 0.05). In contrast, CpG-induced IFN-α-producing capacity was reduced 20- and 40-fold in HCV and HCV-HIV-infected subjects as compared with healthy control subjects (40 ± 239 pg/ml, 25/75th percentile 10/235 pg/ml and 20 ± 134 pg/ml, 25/75th percentile 10/100 pg/ml vs 800 ± 531 pg/ml, 25/75th percentile 35/1155 pg/ml, p = 0.02 and p = 0.008), while there was only a 6-fold nonsignificant reduction in activity in HIV slow progressors (with or without HCV coinfection) and HIV-infected progressor subjects as compared with healthy controls (135 ± 302 pg/ml, 25/75th percentile 33/412 pg/ml and 130 ± 563 pg/ml, 25/75th percentile 12/585 pg/ml vs 800 ± 531 pg/ml, 25/75th percentile 35/1155 pg/ml, p > 0.05 Fig. 5⇓B). In contrast to the 1.8- and 1.4-fold reductions in MDC and PDC frequencies observed in Fig. 2⇑, over 10-fold differences in functional activity are observed in most cases here. These results indicate that non-PDC and PDC populations are selectively and differently functionally impaired in HCV and HIV infections.
Poly(I:C)-induced IFN-α-producing activity (non-PDC-associated activity) is reduced in HIV infection, while CpG-induced IFN-α-producing activity (PDC-associated activity) is reduced in HCV infection. Freshly prepared PBMC populations obtained from subjects described in Fig. 2⇑ were stimulated with poly(I:C) (A) or CpG (B) as described in Fig. 4⇑, and IFN-α was quantitated from culture supernatants. C, IL-12 was quantitated from a subset of poly(I:C)-stimulated PBMC culture supernatants (10 HCV, 7 HCV-HIV, 8 HIV SP (6 with HCV coinfection), 13 HIV, and 8 healthy control subject samples analyzed for this graph).
Because IL-12 is also known to be important in priming type 1 T cells, we measured MDC-associated IL-12 production in response to poly(I:C). CpG failed to induce IL-12 in PBMC cultures in all subjects (not shown). In contrast, poly(I:C)-induced IL-12 was readily observed in healthy control subjects, while this capacity was dramatically (over 10-fold) reduced in HCV, HIV, and HCV-HIV-infected subjects as compared with healthy controls (8 ±27 pg/ml, 25/75th percentile 4.7/26.3 pg/ml, 6 ± 8.3 pg/ml, 25/75th percentile 3.5/9 pg/ml, and 5 ± 4 pg/ml, 25/75th percentile 2/6 pg/ml vs 83 ± 38 pg/ml, 25/75th percentile 25/90 pg/ml, p = 0.004, p < 0.001, p < 0.001, respectively; Fig. 5⇑C). IL-12 production was not significantly impaired in HIV slow progressors when compared with healthy controls (32 ± 35 pg/ml, 25/75th percentile 4/69 pg/ml vs 83 ± 38 pg/ml, 25/75th percentile 25/90 pg/ml p > 0.05). Therefore, poly(I:C)-induced MDC-associated IL-12 production is dramatically reduced in both HIV and HCV infections.
When we analyzed the relationships among non-PDC, MDC, and PDC activities, cell frequencies, and viral levels, a number of correlations were observed. Table II⇑ summarizes all nominally significant (p < 0.05) correlations. A positive correlation was observed between poly(I:C)-induced non-PDC IFN-α and MDC IL-12 production in all HCV-infected groups combined and all HIV-infected groups combined (r = 0.46 and r = 0.57, p = 0.03 and p = 0.001), while an inverse correlation between non-PDC IFN-α and MDC IL-12 production was observed in healthy control and HCV singly infected subjects (r = −0.68 and r = −0.75, p = 0.04 and p = 0.04), suggesting different relationships between these functions in the presence vs the absence of different chronic viral infections. Notably, inverse correlations were observed between HIV and HCV plasma levels and poly(I:C)-induced IFN-α-producing capacity in both HCV and HIV-infected subjects (r = −0.41 and r = −0.25, p = 0.03 and p = 0.02), while in HIV-infected subjects there was also an inverse relationship between poly(I:C)-induced IL-12 production and HIV levels (r = −0.53, p = 0.004). Finally, while there appears to be concordant regulation of non-PDC and PDC IFN-α-producing functions in healthy control subjects (r = 0.79, p = 0.002), this regulation appears to be affected in HCV and HIV infections (p > 0.05). Therefore, immature DC activity may play a role in control of viral level. Alternatively, viral level may determine immature DC activity. Again, there appear to be distinctly different relationships between virus and immature DC activity comparing chronic HCV and HIV infections.
HCV-specific IFN-γ-producing T cell responses were not observed in healthy control subjects (not shown). Consistent with our previous observation that HCV recombinant protein-specific proliferative responses are reduced in HCV-HIV subjects as compared with HCV-infected subjects (13), we found here that the proportion of subjects with an HCV Ag-specific IFN-γ-producing T cell response was reduced in HCV-HIV-infected groups as compared with HCV singly infected groups using a more complete set of HCV Ags (HCV core, NS3, NS4, and NS5 protein Ags; HCV core overlapping 15-mer peptides; and previously defined HCV CD8 determinants) (80 vs 11%, p = 0.04; Fig. 6⇓). Additionally, the number of protein Ags, or protein Ags and peptide Ags combined, that were recognized was reduced in HCV-HIV-infected subjects as compared with HCV singly infected subjects (p = 0.03 and p = 0.005, not shown). We did not distinguish between the CD4 and CD8 compartment for response here. Moreover, a positive correlation was observed between poly(I:C)-induced PBMC IL-12 production and HCV-specific T cell frequency in HCV singly infected subjects (r = 0.9, p = 0.02; Table II⇑). Therefore, in those subjects where the most dramatic reductions in MDC and PDC function are observed (HCV-HIV-infected subjects), fewer HCV-specific T cell responses were observed, while HCV-specific IFN-γ-producing T cell frequency appears to be directly related to MDC function.
HCV-HIV-infected subjects have fewer HCV-specific IFN-γ-producing T cells than do HCV singly infected subjects. IFN-γ ELISPOT assays were performed with PBMC from HCV, HCV-HIV, and healthy control subjects evaluated in Fig. 5⇑, using both peptide and protein antigenic stimuli as described in Materials and Methods. Proportion (percent) of subjects in HCV and HCV-HIV-infected groups that had HCV-specific IFN-γ-producing T cell responses are shown.
Impairment in APC function has been proposed to play a role in the persistence of HCV infection (30, 31, 32) and in the immune deficiencies of HIV infection (25, 26, 27). We describe here deficiencies in function out of proportion to deficiencies in frequency of immature DC populations circulating in the periphery of HCV and HIV-infected subjects. These deficiencies distinctly differ between HCV and HIV infection, and dually infected subjects have the most severe and combined deficiency of these functions. Specifically, non-PDC IFN-α-producing capacity was dramatically reduced in HIV-infected subject groups, with MDC frequencies only tending to be reduced. In contrast, PDC IFN-α-producing capacity was markedly reduced in HCV-infected subject groups, while PDC frequencies were only minimally reduced. PDC IFN-α production also tended to be reduced in the HIV singly infected subjects, though this did not reach statistical significance. Furthermore, MDC IL-12 production was markedly reduced in both HCV and HIV-infected subjects. HCV-HIV coinfected subjects displayed the most dramatic impairment in all functions. The observed inverse relationship between HIV and HCV viral levels and non-PDC IFN-α-producing capacity, and between MDC IL-12-producing activity and HIV levels suggests that these functional properties may play an important role in mounting or maintaining a successful immune response against chronic HCV and HIV infections. Finally, HIV-infected subjects with slow progressor phenotype did not differ from healthy controls with regard to MDC and PDC frequency or function, suggesting that dysfunction induced by both HCV and HIV infections was attenuated in these subjects. These results indicate that immature DC functions in HIV and HCV disease are dysregulated, but differentially, and implicate a mechanism by which APC function may contribute to the immune deficiencies of these two chronic viral infections.
To investigate the effect that these dysfunctional immature DC populations may have on the adaptive arm of the immune response in chronic HCV and HIV infection, we investigated HCV-specific T cell responses in these same subjects. We found a relation between MDC IL-12-producing activity and HCV-specific IFN-γ-producing T cell frequency in HCV singly infected subjects, indicating that this activity may be important for formation or maintenance of HCV-specific T cell responses. Moreover, HCV-specific IFN-γ-producing T cell frequency was reduced in HCV-HIV coinfected subjects with progressive HIV disease as compared with HCV singly infected subjects. These latter data extend upon our previous finding comparing CD4 proliferative responses in HCV vs HCV-HIV-infected individuals (14), including here a larger number of HCV Ags, though not distinguishing between the CD4 and CD8 compartment. Although the decreased frequency of HCV-specific IFN-γ-producing cells present in HCV-HIV-infected individuals may be related to reduced CD4 cell numbers or function that occur in HIV infection, this reduced activity may also be due in part to the decreased immature DC functional activities observed in the HCV-HIV coinfected subjects as compared with HCV or HIV singly infected subjects. Therefore, it appears that MDC and PDC dysfunction in HCV and HIV infections could directly impact upon HCV-specific T cells immunity.
Previous investigations have found reduced MDC and PDC numbers in HIV infection (25, 26, 27). Our data are in agreement with these previous reports (Table III⇓). PDC frequencies were found here to be similar in HCV and HIV-infected subjects, and lower than those in healthy control subjects. Likely because of high subject-to-subject variability in cell frequency, however, only the difference between HCV and healthy control subjects was observed to be significant. Additionally, reduced MDC and PDC cell frequencies were found here to be more pronounced in HCV-HIV coinfected subjects. Moreover, MDC and PDC frequencies in HIV or HCV-HIV-infected subjects with slow progressor status did not significantly differ from those in healthy control subjects, suggesting that slow progressor HIV phenotype is associated with a preservation of these cells. We further found a direct relation between MDC and PDC frequency in all groups (including healthy control subjects), suggesting that both populations are regulated in similar fashion. Our observation that CD4 cell count is related to immature DC frequency in HIV-infected subjects may reflect disease stage, or may reflect a particular dependent relationship between CD4 cell and immature DC cell homeostasis. Perhaps most importantly, the overall frequencies of MDC and PDC do not differ by >2-fold in any of the disease states we have examined. This modest quantitative difference contrasts dramatically with the more dramatic differences in immature DC activity that we observed.
HSV and influenza-induced PDC IFN-α-secreting capacity has previously been found to be reduced in peripheral blood-derived populations of HIV-infected subjects (25, 26, 27). Additionally, IFN-α-producing capacity has been previously found to correlate inversely with HIV plasma levels, consistent with an importance of this function in the control of HIV (25, 27). Our data in HIV-infected subjects are in agreement with these previous findings in that HIV-infected subjects tended to have reduced PDC-associated function. Our data extend upon these previous findings by revealing reduced non-PDC-associated function in HIV-infected subjects. We additionally find here a more pronounced reduction in PDC-associated IFN-α-producing activity in HCV-infected subjects. Previous reports have described a reduction in IL-12-secreting capacity in both HIV and HCV-infected subjects (28, 29, 32, 55). Our data are in agreement with these previous reports, and also indicate a relationship between IL-12-producing function and virus-specific T cell immunity in the case of HCV, and viral replication in the case of HIV. Together, these data support the notion that immature DC IFN-α and IL-12-producing functions play important roles in host defenses against HCV and HIV.
Because we have performed these functional assays with freshly prepared and minimally manipulated cell fractions (PBMC), these immature DC activities are less likely to be affected by in vitro artifact that may exist in more commonly studied systems where immature DC cell fractions are cultured for a period of days in the presence of cytokine before activity assay. Also, in comparison to activity studies performed with purified or enriched MDC or PDC populations, the activities measured here are more likely to reflect the complex interactions that occur in vivo. For example, in a recently published study by Lore et al. (54), purified uncultured MDC populations were unable to produce IFN-α in response to poly(I:C), while in the study by Kadowaki et al. (48) cultured MDC were capable of IFN-α secretion in response to poly(I:C). Our data with unpurified cell populations in short-term culture (<24 h) indicate poly(I:C)-induced IFN-α production is dependent on non-PDC, CD11c-bearing cell populations. CD11c-bearing cell populations other than MDC include monocytes, NK cells, and granulocytes. As an independent means of localizing activity to the cell compartment, preliminary data using intracellular staining and FACS analysis confirmed that PDC cells (Lin−/HLA DR+/CD123+) produced IFN-α in response to CpG, and not in response to CpG control or to poly(I:C) (not shown). Therefore, our data with poly(I:C)-induced IFN-α activity are consistent with previous literature (23, 48) in that it is dependent on non-PDC populations. If this activity is MDC derived, it may also require the presence of cell types other than MDC.
The mechanism by which immature DC cells become dysfunctional in HCV and HIV infections is currently unclear. The presence of HCV RNA within APC has been proposed as a possible cause of impaired allostimulatory function of these cells (31). In support of this is the observation that when DC from normal donors are transduced with the HCV gene containing adenovirus constructs, the ability to stimulate CD4 cells is impaired, resulting in incomplete activation (56). Similarly, direct infection of DC by HIV may cause a similar impairment (57). Such findings have led to the hypothesis that APC containing HCV are selectively impaired in T cell priming activity, while “uninfected” APC are capable of priming normal CD4 responses. Other potential causes of impaired peripheral immature DC function include a reduced formation of functional immature DC populations, an increased rate of destruction of these populations, trafficking of these populations from the periphery to the site of organ damage, or TLR desensitization in HCV and HIV infections. Evidence for the latter possibility has been shown in other systems, with operative mechanisms involving heterologous Toll receptor desensitization, enhanced expression of inhibitors of Toll-mediated signaling, and specific pathogen-encoded products that inhibit Toll-mediated signaling (58, 59, 60). Clearly, an improved understanding of immature DC function in the HCV and HIV-infected host should allow for important insight into both innate and adaptive immunity in humans with chronic viral infection.
In conclusion, these data implicate distinctly different immature DC functional impairments in HCV and HIV infections, and a dual and more pronounced impairment in immature DC function in HCV-HIV coinfected individuals. Interestingly, the HIV slow progressor phenotype appears to ameliorate both HCV and HIV dysfunctional immature DC phenotypes, indicating that these individuals preserve a number of cellular immune functions not only during HIV infection, but also during HCV infection. Because an association between immature DC function and adaptive immunity was observed, these immature DC dysfunctional phenotypes may play an important role in defining the reduced HCV-specific adaptive T cell response observed in HCV-HIV-infected individuals as compared with HCV singly infected individuals.
We thank Dr. Michael Houghton (Chiron) for recombinant hepatitis C proteins.
↵1 This work was supported by a Veterans Affairs Career Development Award and National Institutes of Health Grants RO1AI47839, PO1AI055793, UO1AI38858, and P30AI36219 (a Case Western Reserve University Center for AIDS Research Core Facility Development Award).
↵3 Current address: Boehringer Ingelheim Pharmaceuticals, Ridgebury, CT.
↵4 Abbreviations used in this paper: HCV, hepatitis C virus; DC, dendritic cell; MDC, myeloid-derived DC; PDC, plasmacytoid-derived DC; TLR, Toll-like receptor.
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Daniel DUNIGAN and John Berry, Defendants-Appellants.
James R. Ferguson, Asst. U.S. Atty., Criminal Receiving, Appellate Div., Chicago, Ill., for plaintiff-appellee.
Carl P. Clavelli, Schiller Park, Ill., for Daniel Dunigan.
Luis M. Galvan, Office of the Federal Defender Program, Chicago, Ill., for John Berry.
John G. McKenzie, Asst. U.S. Atty., Rockford, Ill., for petitioner-appellee.
Before BAUER, Chief Judge, and EASTERBROOK and MANION, Circuit Judges.
The appellants in this case are two civilians who were found by a jury to have participated in a surgical strike upon a supply building at the Glenview Naval Air Station: the objective of the mission was to steal factory sealed air conditioners being stored there. After a three day trial, the jury found Dunigan and Berry guilty of conspiring to steal the air conditioners, entering a United States building with intent to commit a theft, and knowingly converting United States property for their own use. We affirm.
In the early morning hours of November 22, 1983, a supply specialist at Glenview Navel Air base in Glenview, Illinois, called the sentry's station to report that the Public Works Center had been broken into. A subsequent investigation revealed that ten factory sealed air conditioners had been stolen from the building. Michael Seaman, a petty officer who was on duty as one of the sentries that evening, recalled seeing Don Allen, a jet mechanic shop supervisor, hastily leave the base in a Ford containing a large box in its trunk. Seaman recalled stating to the other sentry on duty, "if that T.V. falls out its mine." Allen was arrested the next day and agreed to cooperate in the investigation and prosecution of his cohorts.
At the appellants' trial, Allen testified that on the evening of November 21, 1983, Dunigan and Berry were waiting outside of his apartment in Dunigan's van. Dunigan asked Allen if he could provide them with an additional vehicle so that they could steal some air conditioners from the base. Dunigan sweetened the proposition by offering to cancel the $300 gambling debt Allen owed him if he participated. Allen agreed, but since his car was having mechanical problems, he borrowed his friend's Ford Torino. He followed Dunigan and Berry in the van to the Public Works Center. Allen testified that when they arrived, the door of the Center had already been broken into and two air conditioners were sitting outside the structure. Allen forced the door open further, and all three men loaded four air conditioners into the van and three into the Torino and took them to Dunigan's house. They then returned to the Center in the Torino and loaded up three more air conditioners, again taking them to Dunigan's house.
At that point, the three discussed "distribution or sale" of the goods. They agreed to load up the van with four air conditioners and travel to Chicago to Anthony Haywood's house in an attempt to hash out a sale. Upon arriving at Haywood's house, Dunigan and Berry waited in the van parked at the corner of 68th and Blackstone while Allen went upstairs to discuss the transaction. Soon thereafter, at approximately 3:30 a.m., Chicago Police Officers Thompson and Jones responded to a radio dispatch concerning a van with two male occupants parked at the intersection. Officer Thompson testified that they pulled up to the corner facing the van and parked approximately ten feet in front of it. Dunigan and Berry then exited the van and met the officers halfway between vehicles. The officers requested that the two produce their driver's licenses and asked what they were doing in the area. Berry responded that they had given a friend from Kankakee a ride to Chicago to get some money, and that he was in one of the apartment buildings, but they were unsure which building it was. Nor were they able to tell the officers the last name of their friend.
While Officer Jones called in to check the status of Berry's and Dunigan's driver's licenses, Officer Thompson shined his flashlight through the front windshield of the van. He observed large cardboard boxes stacked behind the seats. At that point, Thompson walked over to the vehicle, opened the passenger door, and shined his flashlight into the back of the van. He saw that the boxes were labeled "Air Conditioner" and that they were still factory sealed. He then reached inside the van and pushed one of the boxes to test whether it was full. Thompson walked back over to Dunigan and Berry and inquired about the air conditioners. Dunigan told Thompson that he and Berry were subcontractors and that they had bought the air conditioners that day at an auction; they were en route to Kankakee to install them in a building they were working on. When asked if he had a receipt of purchase, Dunigan said he did not. When asked for proof that they were subcontractors, Dunigan said they had none.
Officer Jones responded by using his radio to inquire whether there had been any burglaries in the area. The dispatcher stated that although no burglaries had been reported, Dunigan's license was suspended. At that point, the Officers took Dunigan and Berry to the station in a paddy wagon for questioning. Meanwhile, Allen and Haywood watched from the safe harbor of Haywood's apartment as the police drove off with their compatriots.
Dunigan and Berry were eventually released by the police without incident, and Allen and Haywood caught up with the two later that night at Dunigan's apartment in Glenview. Having regrouped, all four men proceeded to load four more air conditioners into the van and take them to Thomas Moses' apartment in Chicago. Moses was a friend of Haywood's who was willing to store the air conditioners in the basement of his apartment: Haywood had agreed to buy four of the air conditioners for $200 and offered to pay Moses $50 for storing them. Moses testified that after Dunigan and Berry haggled with Haywood over price, they helped to unload the air conditioners and put them in Moses' basement. Haywood then put a lock on the basement door. Moses recalled telling Haywood the next day that he had gotten "a steal of a deal," causing Haywood to smile in agreement.
The benefit of the bargain was fleeting, however, for Allen was arrested that very day. He confessed to his role in the theft and agreed to cooperate with both the police and Naval Investigative Service. Allen led navy investigators to Moses' basement, at which point four of the air conditioners were recovered. Police did not recover any air conditioners from Dunigan's apartment.
Before trial, Dunigan and Berry moved to suppress the statements and possible testimony of Officers Thompson and Jones. Defendants alleged that the officers unlawfully searched and seized their van. The trial court denied the motion after holding an evidentiary hearing and accepting the government's agreement not to present any evidence or testimony at or after the point that defendants were taken to the police station. At the conclusion of the trial that ensued, the jury found Dunigan and Berry guilty of all three counts.2 The defendants timely filed a notice of appeal from the order of judgment.
Appellants emphasize that the crux of the government's case consisted of the testimony of Allen, their coconspirator, and Officer Thompson, who provided circumstantial evidence concerning the appellants' possession of the air conditioners and their placement at Haywood's apartment. That being said, they argue that the evidence was insufficient for the jury to find the defendants guilty (although appellants do not say so, we presume that they are raising this claim relative to each of the three counts). The gravamen of this claim is the appellants' contention that Allen's testimony must be discounted because it was incredible as a matter of law. In making this argument, appellants virtually ignore the veritable Maginot Line that stands between the credibility of witnesses at trial and an appellate court's role on review. Appellants seek to charge that line at its highest point: they do not clear the peak.
When assessing a general claim that the evidence at trial was insufficient to allow the jury to reach its verdict, we are constrained to review the evidence in the light most favorable to the prosecution. Our inquiry is limited to a determination of whether any rational trier of fact could have found the elements of the offense charged beyond a reasonable doubt. United States v. Whaley, 830 F.2d 1469, 1472 (7th Cir.1987) (quoting Jackson v. Virginia, 443 U.S. 307, 319, 99 S.Ct. 2781, 2789, 61 L.Ed.2d 560 (1979)). The jury's verdict must stand unless "the record contains no evidence, regardless of how it is weighed, from which the jury could find guilt beyond a reasonable doubt." United States v. Bruun, 809 F.2d 397, 408 (7th Cir.1987). Our role is further limited when the credibility of a witness is at issue. This is a matter inherently within the province of the jury, and "absent extraordinary circumstances," this court will not reevaluate the testimony of a witness to determine his or her motives or other possible measures of reliability. United States v. Garner, 837 F.2d 1404, 1423 (7th Cir.1987).
When a conviction rests solely upon the uncorroborated testimony of an accomplice, we will uphold the verdict unless the accomplice's testimony is incredible as a matter of law. United States v. Cravero, 530 F.2d 666, 670 (5th Cir.1976). Appellants seem to be arguing that their conviction falls within this principle; however, it is clear that Allen's testimony is corroborated by the circumstantial evidence provided by both Officer Thompson and Moses. In any event, appellants are unable to show that Allen's testimony is legally "incredible."
To be incredible as a matter of law, a witness' testimony must be unbelievable on its face. In other words, it must have been either physically impossible for the witness to observe that which he or she claims occurred, or impossible under the laws of nature for the occurrence to have taken place at all. United States v. Garner, 581 F.2d 481, 485 (5th Cir.1978). Mere inconsistencies in the witness' testimony do not render it legally incredible. Id. See also United States v. Byrd, 771 F.2d 215, 224 (7th Cir.1985). Notwithstanding this demanding standard, appellants argue that Allen's testimony is incredible because it is improbable that they would have committed their acts without apparent concern about their repeated ferries in and out of the naval base, or without attempting to conceal their unauthorized entry into the Public Works Center. They also offer what they claim is "an entirely more probable" scenario in which they are depicted as two unsuspecting civilians whom Allen was able to persuade to drive to Chicago with a van full of air conditioners. Our first response is that these propositions are internally inconsistent. At one level, they urge us to presume that appellants would have conducted their criminal acts in an intelligent manner. But if "military intelligence" is an oxymoron, surely "rational criminal conduct" must also be one which we need not accept on principle. Going the other way, appellants ask us to presume that they were a couple of easy marks who could be persuaded to drive their own van full of air conditioners to Chicago and back twice in one evening without any culpable knowledge or participation in the theft or subsequent fence. These arguments are possible theories of defense which should have been presented to the jury. They do not constitute grounds for finding that Allen's testimony was incredible as a matter of law.
Appellants' second claim on appeal is that the trial court erred when it denied their motion to suppress the testimony of Officer Thompson. They reassert their contention that because the officers conducted an unlawful search and seizure of the van, Officer Thompson's entire testimony should have been suppressed.
I believe the officer did not violate any constitutional rights by inspecting further what he had seen in plain view, in that he walked to the passenger side of the van, saw the four large boxes, opened the door, and basically stepped into the van to push the boxes to see if they were full. And when he, I guess, determined that they were full, and also observed that they were factory sealed, it was appropriate to make inquiries of the defendants regarding the boxes.
At that point in time, again, there was no arrest. The statement in response to the question, the statement made by defendant Dunigan, that the air conditioners ... had been purchased at an auction, and that the air conditioners were being taken to Kankakee, that statement was not coerced in any way or obtained in any way by the police officers in violation of the defendants' constitutional rights.
We must rely on these findings of fact by the district court unless they are clearly erroneous. Similarly, "[a] district court's denial of a motion to suppress evidence will be affirmed on appeal unless it is clearly erroneous." United States v. Grier, 866 F.2d 908, 935 (7th Cir.1989) (quoting United States v. D'Antoni, 856 F.2d 975, 978-79 (7th Cir.1988)). Cf. United States v. Fuesting, 845 F.2d 664, 671-72 (7th Cir.1988) (abuse of discretion applied). We have also recognized that a district court's determination whether a police-citizen encounter constituted a "seizure" is one that will generally rest upon a fact specific inquiry directly related to the credibility of the witnesses in each particular case. United States v. Boden, 854 F.2d 983, 991 (7th Cir.1988) (citing cases). Thus we owe this determination particular deference and will not disturb it unless the conclusion is clearly erroneous. United States v. Espinosa-Alvarez, 839 F.2d 1201, 1205 (7th Cir.1987); United States v. Black, 675 F.2d 129, 135 (7th Cir.1982), cert. denied, 460 U.S. 1068, 103 S.Ct. 1520, 75 L.Ed.2d 945 (1983).
In the present case, the court found that up until the time that the officers took appellants to the police station in a paddy wagon, their encounter on the street was a consensual one. A consensual encounter exists when "no restraint of the liberty of the citizen is implicated, [rather] the voluntary cooperation of the citizen is elicited through non-coercive questioning; this type of contact does not rise to a level of a seizure." Black, 675 F.2d at 133. Stated in other terms, a police-citizen encounter rises to the level of a seizure under the Fourth Amendment when a reasonable person under the surrounding circumstances would have believed that he or she was not free to leave. Michigan v. Chesternut, 486 U.S. 567, 108 S.Ct. 1975, 1979, 100 L.Ed.2d 565 (1988) (reaffirming test articulated in United States v. Mendenhall, 446 U.S. 544, 554, 100 S.Ct. 1870, 1877, 64 L.Ed.2d 497 (1980) (opinion of Stewart, J.)).
In light of these principles, we conclude that the district court did not commit clear error in finding that Dunigan and Berry consented to the initial questioning of the officers. The officers did nothing that could objectively lead Dunigan and Berry to believe that they were not free to leave, at least up until the point that the police discovered that Dunigan's license was suspended. If the appellants felt constrained prior to that point, it was because they had been sitting in a van filled with stolen air conditioners and they did not want to raise the suspicions or ire of the officers. As the appellants stood in front of the van and talked to the officers, Officer Thompson was able to see the air conditioner boxes in plain view with the use of his flash light.
Whether Officer Thompson subsequently had probable cause to enter the van and shake the boxes to test their contents is a closer question. To establish probable cause, a "police officer must be able to point to specific and articulable facts which, taken together with rational inferences from those facts, reasonably warrant the intrusion." Terry v. Ohio, 392 U.S. 1, 21, 88 S.Ct. 1868, 1880, 20 L.Ed.2d 889 (1968). The totality of the circumstances in this instance include the fact that it was 3:30 a.m. when the officers received a radio dispatch that there were two suspicious men sitting in a van on the corner. It was a neighborhood with a high crime rate. When the police pulled up, the appellants proceeded to get out of their van and walk away from it toward the patrol car. When asked about their presence in the area, appellants offered a vague purpose for waiting there. They could not tell the police the last name of the friend they were waiting for, or which apartment he had gone into. The officers then saw the boxes in the back of the van. On balance, we believe that these facts gave Officer Thompson probable cause for believing that an offense had been or was being committed. Accordingly, we conclude that the district court properly rejected the motion to suppress.
The third issue which appellants raise on appeal is that their convictions for both burglary under Illinois law and theft of government property under 18 U.S.C. Sec. 641 merged into one offense and could not be properly charged as separate counts. This argument has absolutely no merit. It is well settled that a single transaction can give rise to more than one conviction under differing statutes. United States v. Allen, 798 F.2d 985, 1000 (7th Cir.1986). When two distinct statutes are involved, the question is whether the statutes were "directed at separate evils." Albernaz v. United States, 450 U.S. 333, 334, 101 S.Ct. 1137, 1140, 67 L.Ed.2d 275 (1981). It is apparent from the face of the provisions that the Illinois burglary statute is directed at an evil quite apart from that which Congress sought to address through 18 U.S.C. Sec. 641. See generally United States v. Langdon-Bey, 739 F.2d 1285, 1286-87 (7th Cir.1984). Moreover, the convictions are proper when analyzed under the test enumerated in Blockburger v. United States, 284 U.S. 299, 52 S.Ct. 180, 76 L.Ed. 306 (1932). There, the Court held that the appropriate inquiry is whether each statute involved "requires proof of a fact that the other does not." Id. at 304, 52 S.Ct. at 182. To prove burglary under the Illinois statute, the government was required to show that appellants entered a building without authority and with the intent to commit a theft. Theft of government property, on the other hand, requires proof that the appellants knowingly and intentionally stole or converted property belonging to the United States government. Under either approach, the appellants' convictions of the two counts under separate statutes was proper.

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