Are the Opsonophagocytic Activities of Antibodies in Infant Sera Measured by Different Pneumococcal Phagocytosis Assays Comparable?

Host protection against Streptococcus pneumoniae is mainly mediated by opsonin-dependent phagocytosis. Several techniques for measuring opsonophagocytic activity (OPA) of antibodies to S. pneumoniae have been standardized and used. These include the viable cell-assay, flow-cytometric assays, and an assay utilizing radiolabeled bacteria. Using these different methods, we measured the OPA of antibodies to S. pneumoniae types 6B and 19F from the sera of infants immunized with a pneumococcal conjugate vaccine, PncCRM. Generally, the results obtained by the various techniques correlated well, although serotype-specific differences were found (6B, r = 0.78 to 0.95, P < 0.001; 19F, r = 0.50 to 0.84, P < 0.001). The same serotype-specific differences were observed for the relationship between the concentrations of specific immunoglobulin G antibodies measured by enzyme immunoassay and the OPA. Since the sensitivities of the OPA assays differed, the most prominent discrepancies between the techniques were found at low antibody concentrations.     

What is the Practical Significance of Antibodies to Interferons?

Interferons are generally recognised as the treatment of choice in some infectious diseases, such as chronic hepatitis B and C. Since the early clinical trials it was documented that the therapeutic use of interferons could be complicated by the development of antibodies able to neutralise or to bind to the interferon molecule. This finding is not surprising if one considers that natural or therapy-induced antibodies to interleukin (IL)-1, IL-2, IL-6, IL-10, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, insulin and recombinant factor VIII have been reported in humans. Since hormones, cytokines, biological response modifiers and homeostatic agents are being used for the therapeutic management of many infectious, autoimmune and neoplastic diseases, the possibility that therapy-induced antibodies can develop in humans should be considered. In this article we summarise what is currently known about the clinical significance of antibodies to interferons in interferon-treated patients. The presence of circulating antibodies may affect the response to interferon. Antibody development may be clinically significant, depending on the titre and the time of appearance. In particular, the response to interferon therapy may be affected by antibodies when they appear early in therapy and at high titre.     

Pneumococcus with the “6E” cps Locus Produces Serotype 6B Capsular Polysaccharide

Genetic studies of serogroup 6 isolates of Streptococcus pneumoniae identified putative serotype 6E. Although its capsular polysaccharide structure has not been elucidated, putative serotype 6E is described in an increasing number of studies as a potentially new serotype. We show here that SPEC6B, which is widely used as a target strain for serotype 6B opsonophagocytosis assays, has the genetic features of the putative serotype 6E but produces capsular polysaccharide identical to 6B capsular polysaccharide as determined by one-dimensional (1D) and 2D nuclear magnetic resonance (NMR). Thus, putative serotype 6E is a mere genetic variant of serotype 6B. Also, SPEC6B is appropriate as a target strain for serotype 6B opsonophagocytosis assays. This example illustrates the difficulties of assigning new bacterial serotypes based on genetic findings alone.     

Are the enzyme immunoassays for antibodies to pneumococcal capsular polysaccharides serotype specific?

The specificity of antibody binding to pneumococcal capsular polysaccharides (Pnc PSs) measured by enzyme immunoassay (EIA) was studied by inhibition of antibody binding by homologous and heterologous PSs. We found extensive cross-reactivity of antibody binding to type 6B, 19F, and 23F PSs but not to type 14 PS, even after treatment with cell wall PS (CPS). The cross-reactive antibody was highly prevalent in sera of infants and adults with naturally acquired antibody, but not in sera of infants and adults immunized with pneumococcal vaccines. However, a type 11A antibody response was seen after vaccination with heterologous PSs. Monoclonal antibodies prepared against a type 6B PS-tetanus toxoid conjugate recognized also other than the specific type of PS in the EIA, implying the possible existence of a cross-reactive epitope. Remarkable differences in specificity among type 6B PS preparations from different manufacturers were found. Moreover, different lots of type 11A PS from the same manufacturer showed differences in specificity. The results suggest that some Pnc PS preparations may contain cross-reactive epitopes or impurities, other than CPS, that are common to many types of Pnc PS. The specificity of antibodies, especially in sera from nonimmunized subjects, measured by EIA can be questioned.     

Are we poised to target ACE2 for the next generation of antihypertensives?

Antihypertensive drugs based on the blockade of the renin–angiotensin system (RAS) target classical components of this system, i.e., angiotensin-converting enzyme (ACE) and angiotensin (Ang) II type 1 receptor. These antihypertensives are well-recognized and successful, if prescribed properly, in reducing high blood pressure, but much less effective in preventing and reverting end-organ damage induced by cardiovascular disease (CVD) and hypertension. Thus, new strategies and new drug targets that are more effective must be discovered. Recent identification of a counterregulatory axis of the RAS [ACE2, Ang-(1–7), and Mas receptor] that is potentially important in promoting vasoprotective effects offers a novel target for CVD therapeutics. In this brief review, we will highlight the functional characteristics of this axis with special emphasis on ACE2 and its possible involvement in the pathophysiology of the CVD. In addition, we will present our views on the potential of ACE2 as a new target for the development of innovative antihypertensives by highlighting the development and functional findings obtained with small molecules ACE2 activators.     

Are we closer to developing threshold limit values for allergens in the workplace?

The use of immunoassays has facilitated the measurement of high molecular weight sensitizers, usually protein molecules, in the picogram and nanogram per cubic meter range. This facilitated the evaluation of exposure response relationships for bakery workers, exposed to wheat allergens and fungal alpha-amylase and other groups exposed to other allergens such as laboratory animal workers. The application for the standard setting is still limited and requires rigorous standardization, but can be expected in the near future.     

Are we closer to selective immunotherapy for autoimmune diseases?

The discipline of immunotherapy has promised much in the restricted confines of the laboratory but, so far, has delivered little in the harsh reality of the clinic. At a recent conference to celebrate the opening of Roche Milano Ricerche¹, the hopes, successes and failures in this complex field were presented for examination.     

Pneumococcal Vaccination in High-Risk Individuals: Are We Doing It Right?

Controversy exists regarding the optimal use of the 23-valent pneumococcal conjugate vaccine for the protection of high-risk individuals, such as children and adults with immunocompromising conditions and the elderly. The effectiveness and immunogenicity of 23-valent pneumococcal polysaccharide vaccine (PPV23) are limited in such high-risk populations compared to the healthy, with meta-analyses failing to provide robust evidence on vaccine efficacy against invasive pneumococcal disease (IPD) or pneumonia. Moreover, several studies have demonstrated a PPV23-induced state of immune tolerance or hyporesponsiveness to subsequent vaccination, where the response to revaccination does not reach the levels achieved with primary vaccination. The clinical significance of hyporesponsiveness is not yet clarified, but attenuated humoral and cellular response could lead to reduced levels of protection and increased susceptibility to pneumococcal disease. As disease epidemiology among high-risk groups shows that we are still in need of maximum serotype coverage, the optimal use of PPV23 in the context of combined conjugate/polysaccharide vaccine schedules is an important priority. In this minireview, we discuss PPV23-induced hyporesponsiveness and its implications in designing highly effective vaccination schedules for the optimal protection for high-risk individuals.     

High Prevalence of Human Anti‐bovine IgG Antibodies as the Major Cause of False Positive Reactions in Two‐Site Immunoassays Based on Monoclonal Antibodies

Abstract

A sandwich ELISA for quantification of the endometrial protein PP14 revealed false positive reactions in 81% of male sera (n?=?54). The PP14 ELISA was based on two monoclonal antibodies (Mabs) with different epitope specificities—a catcher and a biotinylated indicator. The monoclonal antibodies were purified by protein G affinity chromatography from culture supernatant containing 10% (v/v) fetal calf serum (FCS). Human anti‐animal IgG (bovine, mouse, horse, and swine) antibodies and human anti‐bovine serum albumin antibodies were measured using an ELISA design, with direct bridging of the solid phase and biotinylated antigens. The false positive reactions were abolished by addition of 1% (v/v) bovine serum to the dilution buffer (DB). Human anti‐bovine IgG antibodies (HABIA) were detected in 99 out of 104 sera from blood donors (50 females; 54 males). HABIA levels in male sera (n?=?54) were positively correlated to the false positive signals in the PP14 ELISA (r?=?0.923; p?<?0.0001). Antibodies to IgG from other mammalian species (mouse, horse, and swine) were also detected in the donor sera, but levels and frequencies were lower compared to that of HABIA. Furthermore, HABIA were positively correlated to human anti‐bovine serum albumin antibodies in the donor sera (r?=?0.639; p?<?0.0001; n?=?103). HABIA (prevalence 95%) cause false positive reactions due to crossbinding of contaminating bovine IgG and/or crossreaction with mouse IgG in two‐site immunoassays. The apparent presence of human anti‐mouse IgG antibodies (HAMA), described to create false positive results, may be due to a crossreacting fraction of the polyclonal circulating antibodies against bovine IgG.     

Are prions related to the emergence of early life?

DNA and RNA are the modern cellular molecules related to the storage and processing of the genetic information. However, in the Earth primeval environment conditions, these two molecules are far from being the best option for this function due to their great complexity and sensibility to heat. Experiments have been showing that proteins are very stable and reliable molecules even in very extreme conditions and, under certain circumstances, could be related to the transmission of certain phenotypes that are inherited in a non-Mendelian manner. Prions, infective proteins that are associated to several neurological diseases among mammals by replacing their dominant native state of prion protein by a misfolded one, are remarkably resistant to even the most extreme environments. Furthermore, prions are also associated to the transmission of certain fungal traits in an epigenetical model. These two characteristics support the hypothesis that prions are a possible relic of early stage peptide evolution and may represent the reminiscence of a very ancient analogical code of biological transmission of information rather than the digital one represented by modern nucleic acids.     

Are we prepared to deliver gene-targeted therapies for rare diseases?

The cost and time needed to conduct whole-genome sequencing (WGS) have decreased significantly in the last 20 years. At the same time, the number of conditions with a known molecular basis has steadily increased, as has the number of investigational new drug applications for novel gene-based therapeutics. The prospect of precision gene-targeted therapy for all seems in reach… or is it? Here we consider practical and strategic considerations that need to be addressed to establish a foundation for the early, effective, and equitable delivery of these treatments.     

Are inflammatory phagocytes responsible for resistance to facultative intracellular bacteria?

At least 38 deaths from listeriosis in the United States in recent months have drawn attention to how little we know about resistance to facultative intracellular bacteria. Much more is known about resistance to obligate intracellular parasites. Macrophages, activated by T cell-derived macrophage activating factors (MAF), are able to kill obligate intracellular parasites and tumor cells^1–10 including Leishmania, certain trypanosomes, Toxoplasma, the obligate intracellular bacterium Rickettsia, and perhaps bacteria such as mycobacteria and Legionella. However, macrophages, stimulated by MAF, may not be the only host cells which can defend against infection by facultative intracellular bacteria such as Salmonella typhimurium or Listeria monocytogenes. Six different observations made by Priscilla Campbell and colleagues, and by others, suggest that it is not the so-called ‘activated’ macrophage which is primarily responsible for resistance against facultative intracellular bacteria. Rather, she proposes that an early inflammatory cell recently recruited in response to an inflammatory stimulus — a cell whose presence seems to be under the control of immunologically-specific T cells - plays a critical role in resistance to infection by these organisms.     

Are GnRH antagonists comparable to agonists for use in IVF?

We believe that appropriate comparison of optimal GnRH agonist and antagonist regimens has not been performed yet. Currently available meta-analyses included all comparative studies between GnRH agonists and antagonists performed so far, including less than optimal GnRH antagonist regimens. After critical appraisal of the various studied GnRH antagonist regimens in terms of follicular development and IVF outcome, we postulate that early suppression of endogenous FSH results in optimal follicular development. Additionally, stable and early suppression of LH and progesterone levels during the entire period of stimulation may be an advantage for implantation and pregnancy outcome. In this respect, single dose and particularly flexible protocols seem to be less advantageous. Early FSH and LH suppression can be achieved by early GnRH antagonist administration (stimulation day 1) or by oral contraceptive (OC) pretreatment. More studies comparing long GnRH agonist protocols with 'long' GnRH antagonist protocols, with enough power to identify differences in pregnancy rates, are required before appropriate comparison can be made.