R. M. Minchinton

Impact of Mannose-Binding Lectin on Susceptibility to Infectious Diseases

When the adaptive immune response is either immature or compromised, the innate immune system constitutes the principle defense against infection. Mannose-binding lectin (MBL) is a C-type serum lectin that plays a central role in the innate immune response. MBL binds microbial surface carbohydrates and mediates opsonophagocytosis directly and by activation of the lectin complement pathway. A wide variety of clinical isolates of bacteria, fungi, viruses, and parasites are bound by MBL. Three polymorphisms in the structural gene MBL2) and 2 promoter gene polymorphisms are commonly found that result in production of low serum levels of MBL. Clinical studies have shown that MBL insufficiency is associated with bacterial infection in patients with neutropenia and meningococcal sepsis. Low MBL levels appear to predispose persons to HIV infection. Numerous other potential infectious disease associations have been described. Therapy to supplement low MBL levels is being explored using either plasma-derived or recombinant material.

Analysis of the relationship between mannose-binding lectin (MBL) genotype, MBL levels and function in an Australian blood donor population.

The mannose‐binding lectin (MBL) pathway of complement activation is an important component of innate host defence. Numerous studies have described associations between the MBL genotype, MBL levels and disease susceptibility. However, genotyping and quantitative assays used in these studies have frequently been limited, and comprehensive data examining the interaction between structural and coding MBL genetic variants, MBL antigenic levels and MBL functional activity are lacking. Such data may be important for accurate planning and interpretation of studies of MBL and disease. This study has examined MBL in a cohort of 236 Australian blood donors. Five MBL promoter and coding single nucleotide polymorphisms were genotyped using polymerase chain reaction–sequence‐specific priming (PCR–SSP). Plasma levels of MBL antigen were quantified using a double‐antibody enzyme‐linked immunosorbent assay (ELISA), and functional MBL levels were quantified using a mannan‐binding assay. Activation of the complement pathway by MBL was measured in a C4‐deposition assay. Significant associations were found between both coding and promoter polymorphisms and MBL antigenic and functional levels. There was significant correlation between the results of MBL double‐antibody, mannan‐binding and C4‐deposition assays. Comprehensive MBL genotyping and functional MBL quantitation using mannan‐binding and C4‐deposition assays have the potential to be highly informative in MBL disease association studies.

Mannose binding lectin acute phase activity in patients with severe infection.

Mannose Binding Lectin (MBL) is a liver derived, circulating plasma protein that plays a pivotal role in innate immunity. MBL functions as a pathogen recognition molecule, opsonising organisms and initiating the complement cascade. MBL deficiency arising from mutations and promoter polymorphisms in the MBL2 gene is common and has been associated with risk, severity, and frequency of infection in a number of clinical settings. With MBL therapy on the horizon, the usefulness of replacement MBL therapy has been challenged by the notion, that as an acute phase protein, MBL levels may rise under stress to sufficient levels, in individuals who are usually deficient. This report demonstrates that in patients with sepsis and septic shock, the majority of patients do not display an MBL acute phase response: 41.4% of individuals maintained consistent MBL levels throughout hospital stay, 31.3% of individuals demonstrated a positive acute phase response, and a negative acute phase response was observed in 27.3% of individuals studied. Importantly, a positive acute phase response was generally observed in individuals with wild-type MBL2 genes. When a positive acute phase response was observed in individuals with coding mutation, these individuals demonstrated a normal MBL level on admission to hospital. Furthermore, no individual, regardless of genotype who was MBL deficient at admission was able to demonstrate a positive acute phase response into the normal MBL range. These findings indicate MBL demonstrates a variable acute phase response in the clinical setting of sepsis and septic shock.

Alloimmune neonatal neutropenia linked to anti‐HNA‐4a

Summary. This is a novel case report of alloimmune neonatal neutropenia (ANN) linked to the neutrophil antibody anti‐HNA‐4a (MART). Since its discovery, the HNA‐4a antigen has never been associated with any clinical neutropenia. A first‐born neonate with respiratory distress was found to be severely neutropenic, because of ANN. The broad reactivity of the antibody together with its capture by CD11b and CD18 in monoclonal antibody immobilization of granulocyte antigen test suggested HNA‐4a specificity. DNA sequencing confirmed that the father is HNA‐4a‐positive and that the mother is HNA‐4a‐negative, supporting the diagnosis of ANN linked to MART.

Investigating transfusion-related acute lung injury (TRALI)

Abstract

Human neutrophil antigen‐4a gene frequencies in an Australian population, determined by a new polymerase chain reaction method using sequence‐specific primers

Summary. Human neutrophil antigen‐4a (HNA‐4a) is a high‐frequency (99% in the USA) neutrophil antigen, which has recently been linked to a case of alloimmune neonatal neutropenia. We have devised a new polymerase chain reaction sequence‐specific primer (PCR‐SSP) method to assess HNA‐4a genotype, and used it to determine the HNA‐4a gene frequencies in an Australian population. The gene frequency was found to be 0·906, which is the same as in the American population. The PCR‐SSP genotyping method perfectly correlates with serological phenotyping and is efficient for screening large numbers of samples.

Clinical scale ex vivo manufacture of neutrophils from hematopoietic progenitor cells

Dose‐intensive chemotherapy results in an obligatory period of severe neutropenia during which patients are at high risk of infection. While patient support with donor neutrophils is possible, this option is restricted due to donor availability and logistic complications. To overcome these problems, we explored the possibility of large scale ex vivo manufacture of neutrophils from hematopoietic progenitor cells (HPC). CD34+ HPC isolated from umbilical cord blood (UCB) and mobilized peripheral blood (mPB) were expanded in serum‐free medium supplemented with stem cell factor, granulocyte colony stimulating factor, and a thrombopoietin peptide mimetic. After 15 days of cultivation a 5,800‐fold expansion in cell number was achieved for UCB, and up to 4,000‐fold for mPB, comprising 40% and 60% mature neutrophils respectively. Ex vivo expanded neutrophils exhibited respiratory burst activity similar to that for donor neutrophils, and were capable of killing Candida albicans in vitro. These yields correspond to a more than 10‐fold improvement over current methods, and are sufficient for the production of multiple neutrophil transfusion doses per HPC donation. To enable clinical scale manufacture, we adapted our protocol for use in a wave‐type bioreactor at a volume of 10 L. This is the first demonstration of a large scale bioprocess suitable for routine manufacture of a mature blood cell product from HPC, and could enable prophylactic neutrophil support for chemotherapy patients. Biotechnol. Bioeng. 2009; 104: 832–840

The systemic inflammatory response syndrome induces functional changes and relative hyporesponsiveness in neutrophils

PURPOSE To study the effects of systemic inflammatory response syndrome (SIRS) on polymorhonuclear neutrophil (PMN) function and phenotype by comparing neutrophils from critically ill patients with SIRS against those from healthy blood donors. MATERIAL AND METHODS Intensive care unit patients (n = 110) who met at least one SIRS criterion were recruited to the study. One hundred healthy blood donors were recruited as normal controls. RESULTS Polymorphonuclear cells from critically ill patients with SIRS were more resistant to activation than PMNs from healthy donors, but when stimulated had an exaggerated microbicidal response. Buffer-treated PMNs from patients with SIRS had significantly higher CD43 surface expression that may inhibit heterotypic cellular contact or ligand stimulation of membrane receptors, had significantly lower expression of IgG receptor CD16, demonstrated resistance to shedding of L-selectin when primed by platelet-activating factor which could be pro-inflammatory, and had reduced respiratory burst when primed by platelet-activating factor than activated by formyl-Met-Leu-Phe. CONCLUSION The phenotypic and functional changes observed in neutrophils in the critically ill indicate that they require a higher level of stimulus to become activated. This may represent an auto-protective mechanism where the neutrophils in the already inflamed host may, by this mechanism, avoid excessive inflammation reducing the risk of further host cell injury and death.

Neutrophil antibody diagnostics and screening: review of the classical versus the emerging.

Background and Objectives  Severe transfusion‐related acute lung injury (TRALI) events have been linked to donor‐derived neutrophil antibodies. The journey to developing mass donor neutrophil antibody screening platforms is challenged by the fragility of neutrophils and their unique‐specific antigenic characteristics.

Cardiopulmonary bypass induces enduring alterations to host neutrophil physiology: a single-center longitudinal observational study.

Studies during and immediately post-cardiopulmonary bypass (CPB) surgery have revealed that neutrophils (PMNs) are pivotal to post-CPB inflammation and innate immunity. The aim of this study was to investigate the effects of CPB on the PMN phenotype and respiratory burst function over a longer post-CPB period (up to day 5). Blood samples were collected pre-CPB and on days 1, 3, and 5 post-CPB from 20 patients. Changes to PMN surface expression of CD16, CD62L, CD11b, CD18, and CD43, and PMN respiratory burst activity were measured, together with the white blood cell count and absolute PMN count. Cardiopulmonary bypass induced neutrophilia on days 1 and 3. One day post-CPB, CD16 expression reached a nadir (P = 0.001), and platelet-activating factor-induced CD18 increase was depressed (P < 0.05). Three days post-CPB, CD43 expression peaked (P < 0.05), with a concomitant resistance to N-formyl-Met-Leu-Phe-induced CD11b upregulation (P < 0.05). The PMN respiratory burst activity declined continuously post-CPB until day 5. Neutrophilia on days 1 and 3 was associated with changes to surface molecules expression that may reduce PMN activation response. This study demonstrated that CPB depresses the respiratory burst activity of host PMNs for an extraordinarily longer period of at least 5 days even after neutrophilia had resolved. Collectively, the changes portray an autoprotective yet responsive homeostatic balance.