Proof of concept of a new plasma complement Factor H from waste plasma fraction.

Introduction: Complement factor H (FH) is a major regulator of the complement alternative pathway, its mutations predispose to an uncontrolled activation in the kidney and on blood cells and to secondary C3 deficiency. Plasma exchange has been used to correct for FH deficiency and although the therapeutic potential of purified FH has been suggested by in vivo experiments in animal models, a clinical approved FH concentrate is not yet available. We aimed to develop a purification process of FH from a waste fraction rather than whole plasma allowing a more efficient and ethical use of blood and plasma donations. Methods: Waste fractions from industrial plasma fractionation (pooled human plasma) were analyzed for FH content by ELISA. FH was purified from unused fraction III and its decay acceleration, cofactor, and C3 binding capacity were characterized in vitro. Biodistribution was assessed by high-resolution dynamic PET imaging. Finally, the efficacy of the purified FH preparation was tested in the mouse model of C3 glomerulopathy (Cfh-/- mice). Results: Our purification method resulted in a high yield of highly purified (92,07%), pathogen-safe FH. FH concentrate is intact and fully functional as demonstrated by in vitro functional assays. The biodistribution revealed lower renal and liver clearance of human FH in Cfh-/- mice than in wt mice. Treatment of Cfh-/- mice documented its efficacy in limiting C3 activation and promoting the clearance of C3 glomerular deposits. Conclusion: We developed an efficient and economical system for purifying intact and functional FH, starting from waste material of industrial plasma fractionation. The FH concentrate could therefore constitute possible treatments options of patients with C3 glomerulopathy, particularly for those with FH deficiency, but also for patients with other diseases associated with alternative pathway activation.

Acquired Complement Regulatory Gene Mutations and Hematopoietic Stem Cell Transplant-Related Thrombotic Microangiopathy.

Hematopoietic stem cell transplant-related thrombotic microangiopathy (HSCT-TMA) is a severe complication whose pathophysiology is unknown. We describe 6 patients in which the disease was associated with complement regulatory gene abnormalities received from their respective donors. It is suggested that mutated and transplanted monocyte-derived cells are responsible for production of abnormal proteins, complement dysregulation, and, ultimately, for the disease. This observation might have important drawbacks as far as HSCT-TMA pathophysiology and treatment are concerned.

Rapid isolation of pure Complement Factor H from serum for functional studies by the use of a monoclonal antibody that discriminates FH from all the other isoforms.

Several mutations have been identified in the gene coding for Complement Factor H (FH) from patients with atypical Hemolytic Uraemic Syndrome (aHUS), Age-related Macular Degeneration (AMD) and Membranoproliferative Glomerulonephritis (MPGN). These data allow for a precise description of the structural changes affecting FH, but a simple test for specifically assessing FH function routinely is not yet of common use. We have produced and characterised a monoclonal antibody (5H5) which discriminates between FH and the smaller FH-like 1 and FH-related proteins and show here that it specifically binds to FH without detecting the smaller isoforms. We therefore used this mAb for a quick, one-step micro-purification of FH directly from control sera and showed that this affinity chromatography procedure is not disruptive of its cofactor function. We also developed a modified sheep erythrocytes haemolysis test using our antibody and affinity-purified FH. These tests can be used in conjunction for assessing the function of FH purified from patients affected by FH-related diseases. Moreover we used this mAb to develop a FH-specific ELISA test.

CCL28 induces mucosal homing of HIV-1-specific IgA-secreting plasma cells in mice immunized with HIV-1 virus-like particles.

Mucosae-associated epithelial chemokine (MEC or CCL28) binds to CCR3 and CCR10 and recruits IgA-secreting plasma cells (IgA-ASCs) in the mucosal lamina propria. The ability of this chemokine to enhance migration of IgA-ASCs to mucosal sites was assessed in a mouse immunization model using HIV-1(IIIB) Virus-like particles (VLPs). Mice receiving either HIV-1(IIIB) VLPs alone, CCL28 alone, or the irrelevant CCL19 chemokine were used as controls. Results showed a significantly increased CCR3 and CCR10 expression on CD19(+) splenocytes of HIV-1(IIIB) VPL-CCL28-treated mice. HIV-1 Env-specific IFN-γ, IL-4 and IL-5 production, total IgA, anti-Env IgA as well as gastro-intestinal mucosal IgA-secreting plasma cells were also significantly augmented in these mice. Notably, sera and vaginal secretions from HIV-1(IIIB) VLP-CCL28-treated mice exhibited an enhanced neutralizing activity against both a HIV-1/B-subtype laboratory strain and a heterologous HIV-1/C-subtype primary isolate. These data suggest that CCL28 could be useful in enhancing the IgA immune response that will likely play a pivotal role in prophylactic HIV vaccines.

Häävikko's method to assess dental age in Italian children.

The aim of this study was to determine if Häävikko's maturation standards are applicable to Italian children. The sample included 500 healthy Caucasian children 3.9-15.4 years of age: 267 girls [mean age 9.6 years, standard deviation (SD) 2.1] and 233 boys (mean age 9.9 years, SD 2.1), living in Italy. All dental ages were assessed from panoramic films by one examiner using Häävikko's method. A second examiner independently scored 48 panoramic films to evaluate the reproducibility of the dental age measurements. A good correlation (0.95) was found, as shown by Cohen's kappa. To evaluate the relationship between dental age estimated by Häävikko's standards and the chronological age of the Italian sample, Bland and Altman's graphical method was employed. Moreover, centiles of dental age were constructed both for girls and boys using the LMS (L=skewness, M=median, S=coefficient of variation) method of Cole and Green. It was found that Häävikko's standards tended to underestimate chronological age in this Italian sample. Dental maturation standards as described by Häävikko do not appear suitable for Italian children; instead, centile curves constructed for girls and boys using the LMS method could be used for the estimation of dental age in the Italian population.

Two amino acid substitutions within the first external loop of CCR5 induce human immunodeficiency virus-blocking antibodies in mice and chickens.

Antibodies to the first loop (ECL1) of CCR5 have been identified in human immunodeficiency virus (HIV)-exposed uninfected individuals (ESN) and in HIV-positive nonprogressing subjects. Thus, these antibodies may confer resistance against HIV infection. To define which amino acids are involved in antibody binding to CCR5, we performed a peptide-scanning assay and studied the immunogenicity of peptides in animal models. A panel of synthetic peptides spanning the CCR5-ECL1 region and displaying glycine or alanine substitutions was assayed for antibody binding with a pool of natural anti-CCR5 antibodies. We used mice and chickens to study the immunogenicity of mutagenized peptide. Structural characterization by nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations were performed to better understand the structural and conformational features of the mutagenized peptide. Amino acid substitutions in positions Ala95 and Ala96 (A(95)-A(96)) increased antibody-peptide binding compared to that of the wild-type peptide (Asp(95)-Phe(96)). The Ala95-96 peptide was shown to induce, in mice and chickens, antibodies displaying biological activity at very low concentrations. Strikingly, chicken antibodies to the Ala95-96 peptide specifically recognize human CCR5 molecules, downregulate receptors from lymphocytes, inhibit CCR5-dependent chemotaxis, and prevent infection by several R5 viruses, displaying 50% inhibitory concentrations of less than 3 ng/ml. NMR spectroscopy and molecular dynamics simulations proved the high flexibility of isolated epitopes and suggested that A(95)-A(96) substitutions determine a slightly higher tendency to generate helical conformations combined with a lower steric hindrance of the side chains in the peptides. These findings may be relevant to the induction of strong and efficient HIV-blocking antibodies.

The mucosae-associated epithelial chemokine (MEC/CCL28) modulates immunity in HIV infection.

BACKGROUND: CCL28 (MEC) binds to CCR3 and CCR10 and recruits IgA-secreting plasma cells (IgA-ASC) in the mucosal lamina propria (MLP). Mucosal HIV-specific IgA are detected in HIV-infection and exposure. The CCL28 circuit was analyzed in HIV-infected and-exposed individuals and in HIV-unexposed controls; the effect of CCL28 administration on gastrointestinal MLP IgA-ASC was verified in a mouse model. METHODOLOGY/FINDINGS: CCL28 was augmented in breast milk (BM) plasma and saliva of HIV-infected and -exposed individuals; CCR3+ and CCR10+ B lymphocytes were increased in these same individuals. Additionally: 1) CCL28 concentration in BM was associated with longer survival in HIV vertically-infected children; and 2) gastro-intestinal mucosal IgA-ASC were significantly increased in VSV-immunized mice receiving CCL28. CONCLUSIONS: CCL28 mediates mucosal immunity in HIV exposure and infection. CCL28-including constructs should be considered in mucosal vaccines to prevent HIV infection of the gastro-intestinal MLP via modulation of IgA-ASC.

Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G: a possible role in the resistance to HIV of HIV-exposed seronegative individuals.

Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G), a human cytidine deaminase, is a potent inhibitor of HIV replication. To explore a possible role of this protein in modulating in vivo susceptibility to HIV infection, we analyzed APOBEC3G expression in HIV-exposed seronegative individuals, HIV-seropositive patients, and healthy control subjects. The results showed that the expression of APOBEC3G is significantly increased in peripheral blood mononuclear cells (PBMCs)--mainly CD14(+) cells--and in cervical tissues of HIV-exposed seronegative individuals. Higher APOBEC3G expression correlated with a reduced susceptibility of PBMCs to in vitro infection with the HIV-1(Ba-L) R5 strain. APOBEC3G could be important in modulating in vivo susceptibility to sexually transmitted HIV infection.

Immunization with gp120-depleted whole killed HIV immunogen and a second-generation CpG DNA elicits strong HIV-specific responses in mice.

HIV-1 Immunogen is a gp120-depleted whole killed virus vaccine candidate formulated with Incomplete Freund's Adjuvant (HIV-IFA). We evaluated in a mouse model the immunogenicity of HIV-IFA by itself and when combined with HYB2055, an immunomodulatory oligonucleotide consisting of a novel DNA structure and synthetic CpR immunostimulatory motif, as an adjuvant. C57/BL6 mice were immunized with HIV-IFA alone or combined with HYB2055. Mice treated with HYB2055 or with PBS were used as controls. Compared to HIV-IFA alone, immunization with HIV-IFA and HYB2055 combination elicited strong production of HIV- and p24-specific IFNgamma, RANTES, MIP 1alpha, and MIP 1beta, as well as high titers of HIV- and p24-specific antibodies. Inclusion of HYB2055 also reduced levels of IL-5 produced by HIV-IFA alone. HYB2055 enhances the immunogenicity of HIV-IFA and shifts responses towards a type 1 cytokine profile. The immune enhancing effects of HYB2055 adjuvant were dose-dependent. These findings warrant clinical evaluation of the HIV-1 immunogen/HYB2055 candidate as a therapeutic vaccine for HIV-1 infected patients.

Cardiac precursors in human bone marrow and cord blood: in vitro cell cardiogenesis.

BACKGROUND: Cell transplantation has come of age but numerous questions still remain. Which type of cell should be used? Cardiac precursors are present in mouse bone marrow and used to repair the infarcted myocardium in mice. We searched for these precursors in human bone marrow and analyzed gene expression patterns in cells induced to differentiate in vitro. METHODS: Cells from human bone marrow were isolated and cultured in medium supplemented with autologous serum and 5% CO2. Cell characterization was performed by immunocytochemical analysis. mRNA was isolated and retrotranscribed. The active genes were detected with polymerase chain reaction by using specific oligonucleotides. RESULTS: Some inducers pushed the cell through different stages of cardiogenesis, with expression of cardiac transcriptional activators and structural proteins. Some combinations of stimuli were able to drive cells to advanced stages of cardiogenesis. CONCLUSIONS: These studies lead to an exact description of in vitro cardiogenesis in humans. Our aim was also to assess the residual proliferative capacity of cells and to enhance the differentiation efficiency, thus maximizing their repair capacity and the likelihood that they functionally integrate with the surrounding cardiac tissue.