Dierich Mp

HIV and HIV-infected cells differentially activate the human complement system independent of antibody

The human retroviruses HTLV-I and HIV-I have previously been shown not to be lysed by human serum. An interaction between HIV and the complement system, however, has not been investigated in any detail. In this report we show that purified HIV as well as HIV-infected cells activate the complement system. In the case of virus-infected cells this activation is mediated by the alternative pathway of complement, whereas the classical pathway seems to be in operation for the triggering of the complement system by purified virus and recombinant envelope glycoprotein (gp 160). We demonstrate that this leads to the deposition of C3b and/or C3bi on the surface of infected cells. But the HIV-infected cells are not lysed by human complement. C3 fragments deposited on the surface of HIV-infected cells are capable of mediating immune adherence to complement receptor-bearing cells, such as human erythrocytes and phagocytes. Whether this might have an influence on infectivity of HIV for certain cells bearing complement receptors has yet to be shown.

Structural and functional relationships among receptors and regulators of the complement system

The classical and alternative pathway of complement activation are regulated by a series of fluid phase and cell-bound factors, some of which at the same time serve as receptors for fragments of C3 and C4. These molecules are factor H, CR1 (C3b/C4b receptor), CR2 (C3d/EBV receptor), C4BP (C4b binding protein), DAF (decay accelerating factor), MCP (membrane cofactor protein; earlier designated p45/70), CR3 (iC3b receptor or Mac-1) and CR4 (protein 150/95). Due to structural, genetic and functional features these factors are members of one or several newly recognized large families of proteins: (1) molecules with 60 amino acids long repeats (H, CR1, CR2, C4BP, DAF); (2) proteins with 1,2-diacylglycerol membrane anchoring (DAF); (3) proteins with a heterodimer structure and preference for ligands containing the tripeptide arginine-glycine-asparagine (CR3, CR4). Recognizing the above mentioned regulators and receptors of the complement system as belonging to these protein families opens new perspectives for further genetic and functional research of mutual interest to complement and noncomplement scientists.

Neopterin and (beta)2-microglobulin as prognostic indices in human immunodeficiency virus type 1 infection

SummaryThe great majority of individuals with human immunodeficiency virus type 1 (HIV-1) infection presents with no signs or symptoms, or only lymphadenopathy. To initiate prophylactic measures in time it is necessary to establish risk criteria. CD4+ cell counts are significant predictors. Supplementary methods to improve the predictive information of CD4+ cell counts are still required. In addition, CD4+ cell counting is laborious, expensive, and restricted to specialized laboratories. Thus, there is also a place for more easily performed laboratory tests with similar predictive value as CD4+ cell counts. Neopterin and β2-microglobulin levels proved to be significant predictors of AIDS risk in HIV-1 seropositives. The predictive value of both parameters is equal to CD4+ cell counts and both markers are significant joint predictors in addition to CD4+ cell counts. Measurement of the parameters is done in serum (neopterin and β2-microglobulin) or urine (neopterin) specimens which reduces the risk of HIV-1 transmission compared to handling of whole-blood samples as it is required for cell counting. Although more studies are needed, especially in developing countries and in persons receiving zidovudine, it can be recommended to use neopterin and β2-microglobulin as additional marker to estimate AIDS risk in HIV-1 seropositive individuals. Moreover, both markers may be useful for this purpose without CD4+ cell counts if cell counting is not available.ZusammenfassungDie Mehrheit der mit dem humanen Immundefizienzvirus Typ 1 (HIV-1) infizierten Personen ist frei von Symptomen oder weist nur persistierende Lymphadenopathie auf. Es ist notwendig, Risikokriterien für das Fortschreiten der Erkrankung zu erstellen, um zeitgerecht prophylaktische Maßnahmen einleiten zu können. Die Zahl der CD4-positiven Zellen ist ein geeigneter Prognoseparameter. Die Zellzählung ist jedoch aufwendig, teuer und Speziallabors vorbehalten, sodaß zusätzliche einfache Untersuchungen erwünscht sind, um die Risikoabschätzung weiter zu verbessern. Wissenschaftliche Untersuchungen ergaben, daß Neopterin und ß2- Mikroglobulin bei der HIV-1 Infektion gleich gute Prognoseparameter sind wie die Zahl der CD4-positiven Zellen; beide Parameter liefern darüber hinaus zusätzliche Infromation. Die Bestimmung der Meßgrößen erfolgt aus Serum (Neopterin und ß2-Mikroglobulin) oder Urin (Neopterin), wodurch das Risiko einer eventuellen HIV-1 Übertragung bei der Testdurchführung im Vergleich zur Bearbeitung von Vollblut, wie es zur Zellzählung benötigt wird, verringert wird. Auch wenn noch weitere Untersuchungen vor allem in Entwicklungsländern und in mit Zidovudin behandelten HIV-1 Infizierten notwendig sind, können Neopterin und β2-Mikroglobulin als zusätzliche Parameter zur Abschätzung des AIDS-Risikos bei HIV-1 Infizierten empfohlen werden. Beide Meßgrößen sind auch dazu geeignet, die Bestimmung von CD4- positiven Zellen zu ersetzen, wenn die Möglichkeiten der Zellquantifizierung nicht gegeben sind.

Role of C3b receptors in the enhancement of interleukin-2-dependent T-cell proliferation

The mechanism by which the complement system influences immune responses to T-cell-dependent antigens has not yet been clarified. That is why we studied the effect of the third complement component (C3) on different T-cell-dependent processes using well-defined mouse T-cell lines. While C3 did not influence the interleukin-2 (IL-2) production of the ST2/K-9 helper T-cells, the IL-2-dependent proliferation of the ST1 line was shown to be dose-dependently enhanced by C3. It is proved that neither the haemolytic activity of C3 nor the C3a fragment had any role in the process. The effect of C3 on the IL-2-dependent T-cell growth is even more enhanced (up to five-fold) when using polymerised C3. When the ST1 cell line is cultured in the presence of the cross-linked ligand, T-cells formed 80% less rosettes with red blood cells coated with antibody and mouse or human C3b. It is strongly suggested that C3--particularly when aggregated--exerts its enhancing effect on the growth of IL-2-dependent cell lines by binding to C3b receptors present on such T-cells.

Correlation of hepatitis C virus antibodies with HIV-1 seropositivity in intravenous drug addicts

Since Choo, Kuo, and co-workers [1, 2] used a yeast expression system to synthesize a recombinant polypeptide of the hepatitis C virus (HCV) (a major etiologic virus of blood-borne non-A, non-B hepatitis (NANBH) many investigators have studied the prevalence of antibodies to HCV. High percentages of antibodies were detected in patients with post-transfusion NANBH [2, 3], in patients with hepatocellular carcinoma, hepatic cirrhosis [4, 5] and in haemophiliacs [3, 6]. In healthy blood donors the percentage of antibodies detected against HCV varied from 0.42% [7], 0.5% [2], 0.68% [8], to 7.3% [4]. We studied HCV-antibody prevalence in anti-H/V-1 positive (referred to as HIV +) and negative (HIV-) intravenous drug addicts (IVDA) in Western Austria. Antibodies to HCV were determined by ELISA purchased from Ortho Diagnostic Systems Inc., Raritan, N.Y., USA; code 933440, lot# hcvl03. All tests for detection of Hepatitis B markers were obtained from ABBOTT LAB., North Chicago, I1, USA: HBs-antigen Auszyme monoclehal, # 32281 M201), anti-HBc (~orzyme # 32565 M301), and anti-HBs (Ausab-eia, #32591 M202). In the sere of 30 HIV-positive and 36 H/V-negative IVDA we found 20 (66.6%) and 13 (36.1%), respectively, positive for anti-HCV in an ELISA system obtained from ORTHO whereas only three (4.1%) out of 73 healthy, young volunteers reacted to this test (Table 1 ). In addition, the prevalence of hepatitis B in these persons Was tested by the determination of hepatitis virus B surface antigen, of antiHBc, and anti-HBs antibodies. HBs-antigen and/or anti-HBs could be detected in 17 (56.6%) of HIV +, in 19 (52.7%) of H/VIVDA and in two (2.7%) of the healthy volunteers. Anti-HBc could be detected in 22 (73.3%) of HIV + and in 14 (38.8%) of the HIVIVDA, but in only one (1.37%) of the healthy volunteers. Our findings indicate that: a high prevalence of anti-HCV exists in IVDA, which is in agreement with data previously reported [3]; a higher prevalence of anti-HCV in H/V + IVDA is detectable when compared to HIVIVDA. A similar observation was made by Mortimer et al. [9] studying HCV seropositivity in H/V + and HIVhomosexual men;

HIGH FREQUENCY OF HTLV-III ANTIBODIES AMONG HETEROSEXUAL INTRAVENOUS DRUG ABUSERS IN THE AUSTRIAN TYROL

34 intravenous drug abuse prisoners (5 females 2 of whom were prostitutes and 29 heterosexual males; aged 20-34) have been tested for antibodies to human T-lymphotropic virus type III (HTLV-III) both in Innsbruck (by enzyme-linked immunosorbent assay (ELISA) and immunoperoxidase straining. No one had had a recent infection at the time of the study. All these drug abusers had used intravenous opioids for 1-12 years and 18 had also taken cocaine in the 2-12 years before the study. However they had been free of drugs since their imprisonment 1 week to 18 months ealier. Immunological studies were done by fluorescence-activated cell sorting with antibodies Leu 3a Leu 2a. Neopterin a marker of the activation status of the cellular immune system was measured in urine and serum by high performance liquid chromatography and radioimmunoassay respectively. Raised neopterin levels have been found in AIDS patients and in risk groups for AIDS. Serum antibodies to HTLV-III were found in 15 (44%) of the drug addicts 14 seropositive individuals being heterosexual males and 1 a female prostitute. 15/29 (52%) had reversed helper-suppressor lymphocyte ratios. Neopterin levels were raised in 16/33 (48%) urine and 15/34 (44%) serum samples. The HTLV-III seropositive and seronegative groups were similar in respect of previous hepatitis infections hepatitis A and B virus antigen-antibody status duration and type of drug abuse and detoxification time. HTLV-III seropositive drug abusers were more likely to have raised neopterin levels and reversed T-helper suppressor ratios (0.9 or less). The significance of the raised neopterin levels in 4 subjects without HTLV-III antibodies is not clear. A prospective study is needed to show whether HTLV-III antibodies develop primarily among individuals with increased neopterin levels or independently of neopterin levels. The frequency of HTLV-III antibodies in drug abusers in the Austrian Tyrol is high compared with figures from the United States Britain and West Germany especially since the drug abusers we studied although in prison at the time do not live in an area of high risk for AIDS. The high frequency of HTLV-III in drug abusers has implications for the spread of the virus into the community at large since the heterosexual group especially after rehabilitation will not be so limited in their social contacts as homosexuals are. Futhermore some drug addicts are non-registered prostitutes. (full text)

Neopterin screening and acute cytomegalovirus infections in blood donors

Since October 1986 the blood bank of the University Hospital in Innsbruck, Austria, has been testing all blood donations for increased neopterin concentrations in order to further improve the safety of blood transfusions [1]. Donations with neopterin levels above 10 nmol/1 are excluded from transfusion. Increased neopterin levels indicate activation of the cellular immune system [2]. Beginning in January 1988 we tested donations with increased neopterin concentrations for acute eytomegalovirus (CMV) infection by CMV IgM ELISA. Of 93,789 donations, 1,767 (1.88%) showed raised neopterin levels. These donors were routinely informed, thoroughly examined, and asked for a control blood sample four to five weeks after the primary donation. In addition, a control group of 1,092 randomly selected donors with neopterin concentrations below the cut-off limit of 10 nmol/1 were examined following the same protocol. Neopterin was determined by radioimmunoassay (Henning, Berlin, Germany). CMV IgM/IgG was tested by ELISA (Abbott Laboratories, North Chicago, Ill., USA). CMV IgM/IgG testing was confirmed by a second ELISA (Behring, Marburg, FRG). In addition to neopterin and CMV testing, all samples were screened for hepatitis B surface antigen and human immunodeficiency virus types 1 and/or 2 antibodies (Abbott), syphilis (Fuji, Vienna, Austria), and ALT levels (Boehringer, Vienna, Austria). Samples positive for CMV IgM antibodies were also tested for rheumatoid factors, heterophil antibodies (Bio-Merieux, Carbonniere les Bains, France), and Epstein-Barr virus IgG/IgM serostatus (Epignost, Linz, Austria). Rheumatoid factor testing was performed twice by latex agglutination (Serotherapeutisches Institut, Vienna and Gamma Biologicals, Houston, Tex., USA). Of 1,767 donations with increased neopterin levels, 93 (5.26%) were found to be positive for CMV IgM antibodies. Eleven of the 93 CMV IgM seropositives also had IgG antibodies. At the control visit mean neopterin levels decreased in donors who developed CMV IgG antibodies (Table 1). In 34 samples CMV IgM antibodies without CMV IgG were detected. Ten of 1,674 donors with increased neopterin who were negative for CMV IgM/IgG antibodies at the time of donation and negative for all the other tests had CMV IgM antibodies at the time of the control; neopterin was still elevated. Three of these donors were followed up for a second time four weeks later; they presented with CMV IgM and IgG antibodies, but with decreased neopterin levels (Table 1). Acute CMV infection may explain elevated neopterin at the time of donation, the increase of neopterin preceding the appearance of CMV IgM and/or IgG antibodies by 2-4 weeks. In the group with low neopterin at donation, only three (0.3%) were CMV IgM seropositive. Thus, acute CMV infection is significantly more prevalent Table 1. Association between CMV serologic findings and neopterin concentrations in blood donors

Involvement of complement in B-cell, T-cell and monocyte/macrophage activation

In the early 70s it had been shown, that for the immune response against T-dependent antigens C3 was necessary, while T-independent antigens, although activating the alternative pathway of complement, triggered antibody formation also in C-deficient mice. During recent years functional and biochemical knowledge about complement binding structures on B-cells and monocytes/macrophages continuously increased and, also, on T-cells C3 binding entities have been detected. In the case of B-cells and, at least in special experimental conditions, in the case of T-cells C3 can exert a proliferative response as long as the cells are prestimulated (excited) by anti-Ig or IL-2, respectively. Monocytes can bind C3b- or iC3b-carrying particles, but only when progressed to macrophages can they phagocytose such particles. Thus the concept evolves that B-cells, T-cells and monocytes can acquire competence for a C3-driven response when excited properly. The involvement of molecules such as CR1, CR2, factor H, IL-2-receptor and others with a basic structure of repeating units of 61 amino acids in the triggering processes is a surprising finding and certainly suggests their functional importance. In the case of T-independent antigens the structures triggering the alternative pathway of complement are the structures triggering monocytes directly. Whether these two functions have a causal relationship has to be shown.

C3 Binding Proteins of Foreign Origin

The generation of C3b from C3 releases the internal thioester and allows C3 to bind covalently to OH and NH2 groups, using the carbonyl group and leaving the SH group unoccupied. The molecules to which C3b binds in this way are commonly referred to as C3 acceptors and range from H2O to large fluid-phase and membrane molecules. C3b and its derivatives—iC3b, C3d,g, and C3d, covalently bound to C3 acceptors—have the potential to interact noncovalently with a number of fluid-phase and membrane-associated molecules (LAMBRIS 1988). Such molecules on pathogens are the subject of this chapter, particularly whether the presence of such C3 binding molecules constitute advantages for the pathogen in coping with the control mechanisms of the host, i.e., whether they can be considered as pathogenicity factors.

Use of a high-efficiency expression vector to isolate cDNA clones for factor H and map their positions within the molecule

A human liver cDNA library, cloned in a novel high-efficiency bacterial expression vector (PEX), was screened with an affinity-purified antibody to human factor H. Four distinct cDNA clones, H-2, H-40, H-46 and H-49, were identified. Of these, H-2 also reacted with two monoclonal antibodies to H, MAH-4 and OX-24, which were previously shown to recognize the 38,000 N-terminal tryptic fragment of H, carrying the binding site for C3b. By using polyclonal antibodies specific for the domains in H coded for by these cDNA-clones, it could be established that H-2 codes only for the 38,000 N-terminal tryptic fragment of H, whereas H-40, H-46 and H-49 are derived from the 142,000 C-terminal fragment of H. By subcloning H-2 the epitope for OX-24 could be localized as being coded near the central Sma-site of H-2.