Theories on the pathogenesis of endometriosis.

Endometriosis is a common, chronic inflammatory disease defined by the presence of extrauterine endometrial tissue. The aetiology of endometriosis is complex and multifactorial, where several not fully confirmed theories describe its pathogenesis. This review examines existing theories on the initiation and propagation of different types of endometriotic lesions, as well as critically appraises the myriad of biologically relevant evidence that support or oppose each of the proposed theories. The current literature suggests that stem cells, dysfunctional immune response, genetic predisposition, and aberrant peritoneal environment may all be involved in the establishment and propagation of endometriotic lesions. An orchestrated scientific and clinical effort is needed to consider all factors involved in the pathogenesis of this multifaceted disease and to propose novel therapeutic targets to reach effective treatments for this distressing condition.

Application of Dolichos biflorus in immunoassay detection of kidney collecting duct biomarkers.

Currently there are no biomarkers for detecting collecting duct damage in man. Antibodies to several collecting duct-specific antigens exist but sandwich assays have been difficult to establish due to the need for two different antibodies to the same protein. We hypothesized that a collecting duct-specific lectin could be used in combination with a collecting duct-specific antibody to negate the need for two different antibodies. The collecting duct specificity of selected antibodies (NiCa II 13C2, Pap XI 3C7, HuPaP VII 2B11 and aquaporin 2), was verified by immunohistochemistry. Aquaporin 2 and Pap XI 3C7 were used successfully in setting up assays with the lectin Dolichos biflorus, using the Meso Scale Discovery (MSD) platform. Antigen expression was highest in the papillae of rat and human kidney (corresponding to the greatest density of collecting ducts) and was also present in normal urine. We propose that further qualification and validation would lead to an assay for detecting collecting duct damage in man.

Meso scale discovery and luminex comparative analysis of calbindin D28K.

The sensitivity of different renal regions to xenobiotics requires the development of a multiplex immunoassay for the simultaneous analysis of kidney biomarkers. Calbindin D28K is a distal tubule-specific protein that can be detected in urine under pathological conditions. In this study, a pair of anti-calbindin D28K antibodies was used in an immunoassay for the detection of calbindin D28K expression in rat and human kidney and urine. Comparative analysis of the immunoassay was performed on the Meso Scale Development (MSD) and Luminex platforms. Analysis on both platforms detected calbindin D28K concentrations between 100 ng/mL and 100 pg/mL. Luminex detected 10-fold the amount of calbindin D28K in samples analyzed as compared to MSD, whereas calbindin D28K level in rat and human urine was below detection limit in both platforms. The application of the immunoassays described herein may be useful in toxicological and pathological studies of distal tubular damage in rats and human.

Production, purification, crystallization and preliminary X-ray diffraction analysis of the HIV-2-neutralizing V3 loop-specific Fab fragment 7C8.

7C8 is a mouse monoclonal antibody that is specific for the third hypervariable loop (V3 loop) of the human immunodeficiency virus type 2 (HIV-2) associated protein gp125. Fab fragments of 7C8 effectively neutralize HIV-2. 7C8 was expressed and purified from a hybridoma cell line in order to establish the molecular basis underlying the specificity of the 7C8 antibody for the V3 loop as well as the specific role of the elongated third complementarity-determining region of the heavy chain (CDRH3). The antibody was digested with papain and Fab fragments were purified using size-exclusion chromatography. Hanging-drop vapour-diffusion crystallization techniques were employed and the protein was crystallized in 50 mM ammonium sulfate, 100 mM Tris-HCl pH 8.5, 25%(w/v) PEG 8000 and 2.5%(w/v) PEG 400 at 275 K. The analysed crystals belonged to the rhombohedral space group P3(2)21, with unit-cell parameters a = b = 100.1, c = 196.8 A, and diffracted to 2.7 A resolution.

HIV-2 neutralization by intact V3-specific Fab fragments.

The V3 region of both HIV-1 gp120 and HIV-2 gp125 surface glycoprotein has been described as a target for neutralizing antibodies. In this study a conformation-sensitive (3C4) and a linear site-specific (7C8) anti-HIV-2 V3 monoclonal antibody (mAb) were characterized. The neutralization capacity of the purified mAbs and their respective papain-generated Fab fragments was analyzed. The Fabs were further characterized by sequence analysis. Our results demonstrate that neither purified mAbs were capable of neutralizing HIV-2, while intact Fab fragments from both mAbs blocked in vitro infection of HIV-2 isolates. Moreover, the conformation sensitive 3C4 Fab neutralized both subtype A and B HIV-2 isolates and SIVsm. Sequence analysis of the hypervariable regions of 3C4 Fab and 7C8 Fab revealed that the third CDR of the heavy chain (CDRH3) of the antibodies was not as long as many of the previously characterized neutralizing antibodies. Our findings suggest that whole 7C8 and 3C4 mAbs are sterically hindered from neutralizing HIV-2, whereas the smaller size of Fab fragments enables access to the V3 region on the virion surface.

Deletion of the V1/V2 region does not increase the accessibility of the V3 region of recombinant gp125.

Previous analyses of HIV-1 surface glycoprotein indicate that both the V1/V2 region and the interaction of gp120 with CD4 influence the accessibility of the V3 region on gp120. In this study we investigated the accessibility of the V3 region of HIV-2 recombinant gp125 proteins using V3-specific mAbs (7C8 and 3C4) and analyzed the binding kinetics of soluble CD4 (sCD4) to recombinant HIV-1 gp120 and HIV-2 gp125 proteins by surface plasmon resonance (SPR) analysis. Our results indicated that 7C8 recognized monomers of gp125 and gp125Delta v1v2, (lacking the V1/V2 region) while 3C4 was sensitive to the conformation of gp125, recognizing only oligomers of gp125Delta v1v2. Furthermore, SPR analysis of 7C8 binding to gp125 demonstrated that the deletion of the V1/V2 region did not increase the accessibility of the V3 region in gp125Delta v1v2. Comparative SPR analyses of sCD4 binding HIV recombinant surface glycoproteins revealed a lower affinity of sCD4 to gp125 as compared to gp120. Moreover, the analyses suggest that conformational changes only occur in HIV-1 gp120 upon interaction with CD4. We hypothesize that the V3 region is accessible in HIV-2 gp125 and thus may not require interaction with CD4 to induce conformational reorientation of the V1/V2 region.

Cloning, expression, and purification of HIV-2 gp125: A target for HIV vaccination.

The envelope of the human immunodeficiency virus (HIV) is the main target for neutralizing antibodies. We report the cloning, purification, and characterization of two recombinant forms of the envelope glycoprotein gp125 from a primary HIV-2SBL-6669 isolate. Both constructs were truncated at the N- and C-termini, and in the gp125deltav1v2 construct the variable V1 and V2 loops were deleted. The recombinant glycoproteins were stably expressed in Chinese hamster ovarian cells, producing soluble gp125 and gp125deltav1v2 at molecular weights of 74.2 and 56.9 kDa, respectively, and were purified from cell culture supernatants in a single step using Galanthus nivalis lectin chromatography. Circular dichroism analysis indicated a similar secondary structure for gp125 and gp125deltav1v2, and both proteins were recognized by HIV-2 serum antibodies in surface plasmon resonance assays. The high yield and purity of these constructs makes them suitable for structural and functional analyses, as well as vaccine studies.

Potent neutralizing serum immunoglobulin A (IgA) in human immunodeficiency virus type 2-exposed IgG-seronegative individuals.

The mechanisms behind the resistance to human immunodeficiency virus type 2 (HIV-2) infection are still not fully understood. In the present study, we explored the HIV-2-specific humoral serum immunoglobulin A (IgA) immune response in HIV-2-exposed IgG-seronegative (EGSN) individuals. Serum samples from heterosexual EGSN individuals and their known HIV-2-infected partners, as well as controls originating from Guinea-Bissau in Africa, were studied. Antibody reactivity to native and recombinant envelope glycoproteins was investigated, and the capacity of purified serum IgA to neutralize HIV-2(SBL6669) was tested. Our results showed that 16 of 25 EGSN samples exhibited reactivity against whole HIV-2 antigen, 6 of 25 samples reacted with recombinant gp36 (rgp36), and 3 of 25 samples were positive against HIV-2 rgp105; no reactivity to native HIV-2 gp125 was detected. Purified serum IgA antibodies from both EGSN and HIV-2-positive individuals, but not that from the negative controls, exhibited neutralization of HIV-2(SBL6669). The most potent neutralization activity was exhibited by IgA purified from EGSN compared to infected individuals' IgA. The antigenic pattern of the HIV-2-positive partners showed that all serum IgA samples were reactive to whole HIV-2 antigen, and 14 of 15 reacted with rgp36. For rgp105 and gp125, 5 of 15 and 4 of 15 samples exhibited binding, respectively. The serum of the EGSN group had a higher mean IgA concentration than that of the negative controls (P < 0.05). Thus, we describe HIV-2-specific serum IgA antigen reactivity and show a more potent serum IgA-mediated HIV-2-neutralizing activity in EGSN individuals than in HIV-2-infected patients.

Serum immunoglobulin A (IgA)-mediated immunity in human immunodeficiency virus type 2 (HIV-2) infection.

In the present study, we sought to define the importance of serum IgA (sIgA)-mediated immunity in HIV-2 infection. Serum samples from a total of 29 HIV-2-infected patients from Guinea-Bissau (n = 20) and Portugal (n = 9) were studied. Samples from seronegative individuals were used as controls. Antibody reactivity to native and recombinant envelope glycoproteins as well as peptides representing various regions of the envelope glycoproteins was investigated. Furthermore, the capacity of purified IgA to neutralize the HIV-2(SBL6669) strain was tested. All serum samples showed IgA reactivity against whole HIV-2 antigen. Twenty-eight out of 29 IgA samples (96%) reacted with native HIV-2 gp125, 26/29 (90%) with recombinant gp105, and 29/29 (100%) with recombinant gp36. When using peptides, the most prominent IgA reactivity was seen against the peptide representing aa 644-658 of the transmembranous protein gp36, to which 72% of the sera reacted. Purified sIgA antibodies showed neutralizing effects against HIV-2(SBL6669) in 17/29 cases (59%). This work describes the HIV-2-specific sIgA antigenic response. Moreover, our findings show, for the first time, that sIgA may play a role in the in vitro neutralization of HIV-2.