Slow human immunodeficiency virus (HIV) infectivity correlated with low HIV coreceptor levels.

The absolute number of CD4+ lymphocytes in blood is prognostic for disease progression, yet the cell surface density of CD4 receptors or chemokine receptors on a single cell has not previously been found to be predictive of human immunodeficiency virus (HIV) infectivity outcome. It has recently been shown that human leukocyte elastase (HLE) and its ligand alpha(1) proteinase inhibitor (alpha(1)PI; alpha(1)antitrypsin) act as HIV fusion cofactors. The present study shows that decreased HIV infectivity is significantly correlated with decreased cell surface density of HLE but not with decreased CD4 nor chemokine receptors. In vitro HIV infectivity outcome in this study was predicted by the surface density of HLE on mononuclear phagocytes but not on lymphocytes. The set point HLE surface density was in part determined by alpha(1)PI. Decreased circulating alpha(1)PI was correlated with increased cell surface HLE and with increased HIV infectivity. The correlation of HIV infectivity outcome with surface HLE and circulating alpha(1)PI supports the utility of these HIV cofactors in diagnostic analysis and therapeutic intervention.

Self antigen prognostic for human immunodeficiency virus disease progression.

We have recently found that an extracellular protein, alpha(1) proteinase inhibitor (alpha(1)PI; alpha(1) antitrypsin), is required for in vitro human immunodeficiency virus (HIV) infectivity outcome. We show here in a study of HIV-seropositive patients that decreased viral load is significantly correlated with decreased circulating alpha(1)PI. In the asymptomatic category of HIV disease, 100% of patients manifest deficient levels of active alpha(1)PI, a condition known to lead to degenerative lung diseases and a dramatically reduced life span. Further, HIV-associated alpha(1)PI deficiency is correlated with circulating anti-alpha(1)PI immunoglobulin G. These results suggest that preventing HIV-associated alpha(1)PI deficiency may provide a strategic target for preventing HIV-associated pathophysiology.

Specific activity of alpha1proteinase inhibitor and alpha2macroglobulin in human serum: application to insulin-dependent diabetes mellitus.

The shifting balance between proteinases and proteinase inhibitors in blood, a function of their relative affinities and concentrations, has long been hypothesized to influence immune competency. The identification of proteinase-activated receptor responses in cells of the mononuclear phagocyte system suggests a potential explanation. The major serum proteinase inhibitor, alpha1proteinase inhibitor (alpha1PI, alpha1-antitrypsin), has been reported to increase in concentration during inflammation. Quantitative determination of serum alpha1PI has traditionally been performed nephelometrically; however, antigenically quantitated levels may not be representative of functional capacity. It has previously been observed that alpha1PI in serum exhibits bimodal behavior as the result of various concentrations of proteinase inhibitors, specifically alpha2macroglobulin (alpha2M) and inter-alpha-trypsin inhibitor, which compete in binding to a panel of serine proteinases. Consequently, it has not previously been possible to assign a numerical value for the specific activity of these competing proteinase inhibitors in serum. By applying known constants representing the association of proteinase inhibitors with porcine pancreatic elastase (PPE), the theoretical relationship between the functional and antigenic values for alpha1PI and alpha2M has been empirically derived allowing, for the first time, the calculation of their specific activities in serum. As predicted, the serum concentration of alpha1PI was found to be highly correlated with residual uninhibited PPE catalytic activity in healthy individuals, but not in individuals exhibiting fragmented or complexed alpha1PI. Using these techniques, both the antigenic and functional levels of alpha1PI were determined in sera from subjects with insulin-dependent diabetes mellitus (IDDM) who had been clinically diagnosed as having either periodontal disease or gingival health. Determination of quantitative levels by antigen-capture suggests that the IDDM subjects with periodontitis manifest dramatically increased levels of fragmented serum alpha1PI compared with their orally healthy counterparts or normal controls. In contrast, functional analysis of serum alpha1PI revealed no differences between the three subject populations. The elevated levels of antigenically determined serum alpha1PI reflect the inflammatory status of periodontal disease. These results support the importance of and provide methodology for determining the functionally active levels of alpha1PI allowing reexamination of changes detected during the acute phase of inflammation, replacement therapy, and longitudinal studies in relevant disease processes including malignancy and diabetes.

Alpha 1-antitrypsin deficiency and pregnancy.

Alpha1-antitrypsin deficiency is an inherited pulmonary disorder which results from a deficiency of a major plasma protease inhibitor. The onset and severity of symptoms vary widely and depend on the genotype and whether the patient smokes cigarettes. Alpha1-antitrypsin in pregnancy has only been previously reported twice. Our patient had a functional serum alpha1-antitrypsin level which was 15% of normal but was clinically asymptomatic and she did not smoke. Her genotype revealed a non-ZZ pattern. Her obstetric history was complicated by preterm labor in each of her five ongoing pregnancies. Alpha1-antitrypsin deficiency is inherited via two codominant autosomal genes. Although there is great variability in severity of disease, seriously affected patients may have emphysema and hepatic abnormalities. Patients with non-ZZ genotypes or who are heterozygotes may have favorable pregnancy outcomes.

Inhibition of HIV-1 by modification of a host membrane protease.

While it is clear that CD4 is the receptor for the gp120 envelope protein of HIV-1, substantial evidence suggests that other host cell proteins are required for successful membrane fusion. Studies were initiated to examine the potential for a protein receptor which has an elastase-like character to participate in fusion of HIV-1 with permissive host cells. A synthetic elastase inhibitor was shown to significantly reduce HIV-1 infectivity when present during, but not after, the initial contact between virus and cells. A human T cell elastase-like membrane component was purified and shown to be lipid-associated. By competitive inhibition, the purified protein was shown to bind gp160 within the HIV-1 fusion domain. The binding parameters of whole T cell membrane extract, with a hydrophobic pentapeptide representative of the fusion domain, suggested an elastase-like protein is the single, secondary T cell receptor for HIV-1 (K = 1 x 10(3) M-1). The pentapeptide interacted with porcine and human (epithelial and polymorphonuclear leukocyte), but not murine, elastase isoforms, suggesting its participation in the permissiveness of host cells to infection.

T cell antigen receptor immune complexes demonstrating biologic and proteolytic activity.

An elastase-like protease, recently recognized as a specific product of murine T cells, may functionally associate with the classical TCR. T cell elastase, found in combination with the natural elastase inhibitor alpha 1-antitrypsin (alpha 1-protease inhibitor, alpha 1-PI), is produced by both CD4+ and CD8+ T lymphocytes. T cell elastase and alpha 1-PI are found chemically associated with the TCR in an antigen-specific complex reminiscent of the Ig-complement 'immune complexes'.

Elastase is a constituent product of T cells.

Proteases produced by immune cells have been found to be important components of the immune response to antigen. A protease previously unrecognized as a specific T cell product has been identified which has the gene sequence, serologic crossreactivity, and enzymatic specificity of elastase. T cell elastase, found in combination with the natural elastase inhibitor alpha 1-antitrypsin (alpha 1-protease inhibitor, alpha 1-PI), is produced by both CD4+ and CD8+ T lymphocytes, and is found both in a membrane-bound and in a soluble form in murine T cell lines.

Solid-phase antigen binding by purified immunoproteins from antigen-specific monoclonal T cell hybridomas.

We have developed two distinct solid-phase immunoassays for the detection of antigen binding activity by products of antigen binding T cell hybridomas in the absence of MHC. Two suppressor T cell hybridomas studied (34s-18 and 34s-704) are specific for keyhole limpet hemocyanin, a protein antigen, and the other suppressor T cell hybridoma (51H7D) binds specifically to the arsonate hapten. We have adapted these hybridomas to growth in serum-free medium and have isolated molecules with antigen binding activity both from the cell membranes and from the culture fluid in which the cells had been grown. The antigen binding molecules (ABM) produced by the KLH-specific hybridomas bound best to native hemocyanin; binding was decreased when KLH was denatured by reduction and alkylation and no binding was found to an arthropod (Limulus) hemocyanin. The arsonate binding hybridoma, on the other hand, produced molecules specific for this hapten; they showed no capacity to bind KLH. The antigen binding molecules affinity-purified from all three T hybridomas have intact masses of either 145,000, 67,000 or 48,000 when run in SDS-PAGE under non-reducing conditions. Following reduction, ABM resolve in SDS-PAGE into a complex of polypeptide chains having apparent masses of 65,000, 56,000 and 49,000, with either a pair of bands at 26,000 and 22,000, or with a single band at 32,000, which is consistent with the size of translation products of mRNA previously isolated from these hybridomas. Two of the hybridomas, 34s-18 and 34s-704, used for isolation of antigen binding products in this study, were previously reported to lack detectable rearranged gamma or beta genes and therefore to lack expression of the alpha/beta or gamma/delta heterodimers. The antigen binding molecules react in solid-phase immunoassay with some antibodies specific for variable (first framework) region and joining (J) region peptide sequences predicted from T cell receptor gene sequence. Furthermore, the affinity-purified antigen binding molecules from mouse T cell hybridomas cross-react in ELISA with goat anti-rabbit IgG and not with protein G, thus allowing the use of these commercially available reagents in standard laboratory assays. Interestingly, ABM anchored in intact cell membranes, which could be shown to specifically bind antigen, did not cross-react with goat anti-rabbit IgG, indicating that the cross-reactive moiety is not detectable when the ABM are in this situation.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for the binding of human serum amyloid P component to Clq and Fab gamma.

In order to clarify the mechanism of interaction of serum amyloid P component (SAP) with complement, the interaction of SAP with C1q and with IgG was studied. It is known that SAP binds Sepharose in the presence of calcium. When purified 125I-C1q was incubated with SAP prior to Sepharose affinity chromatography, 125I-C1q was retained. However, in the absence of SAP, the 125I-C1q was not retained. To further examine the interaction of SAP with C1q, isolated SAP was incubated at varying ratios with C1q in the presence of 1.5 mM Ca2+. These mixtures were subsequently examined via crossed immunoelectrophoresis against goat anti-SAP. A change in the electrophoretic behavior of SAP was observed in the presence of C1q. In other studies, it was observed that SAP might interact with the collagen-like stem of C1q. In these latter studies, 125I-SAP was incubated with pepsin digests of C1q in a microtitre solid-phase binding assay. In addition, a microtitre solid-phase binding assay was utilized in order to investigate the possible binding of isolated 125I-SAP with IgG. Interestingly in the presence of Ca2+, human IgG and Fab gamma, but not Fc gamma, were found to bind 125I-SAP.