Comparison of the Effect of Holder Pasteurization and High-Pressure Processing on Human Milk Bacterial Load and Bioactive Factors Preservation.

OBJECTIVES: This project aims at comparing the impact of Holder pasteurization (HoP) and high-pressure processing (HPP) on bacterial load and retention of immunological components in human milk. METHODS: Human milk samples discarded by the Public Mothers' milk bank (Montreal, Canada) for bacterial purpose were pooled (n = 6) and pasteurized either by heating in a water bath (62.5°C, 30 minutes) or by HPP treatment (425 MPa, four cycles of 6 minutes, initial milk temperature of 4°C or 37°C). Bacterial load, lysozyme activity, and levels of immunoglobulins, lactoferrin, lipase, and 26 cytokines were analyzed. Untreated milk samples from same pools served as control. RESULTS: HPP treatment of milk allows a similar elimination of bacteria than HoP; bacterial counts were under the detection limit [<3 colony-forming units (CFU)/mL] in 50% of milk pools after HPP treatment, compared to 17% for HoP. With initial heating of samples to 37°C before HPP treatment, inactivation to an extent under the detection limit was reached in 67% of pools. There is no significant difference in IgA, lysozyme, and cytokines concentrations between untreated milk and all treatment methods. While no significant difference was observed in the amount of lipase (P > 0.07) and IgG (P > 0.11) between untreated milk and HPP-treated milk samples, HoP seems to be damaging for these factors (P < 0.04). IgM is well preserved in HPP-4°C samples compared to untreated milk (P = 0.07) whereas a decrease is observed for this immunoglobulin levels in HPP-37°C and HoP samples (P < 0.01). Lactoferrin activity, is well maintained in HPP-37°C milk samples in comparison to untreated milk samples (P = 0.52). A decrease in activity of this molecule is noted for samples treated with HPP at 4°C (P = 0.02) and this decrease is even more pronounced for HoP samples (P = 0.004). CONCLUSIONS: HPP is a promising alternative to HoP for treatment of human milk intended to preterm babies. Our results demonstrate that HPP treatment of human milk provides safe milk with less detrimental effects on the biochemically and immunologically active milk components than HoP.

Rhesus D Antigenic Determinants on Residual Red Blood Cells in Apheresis and Buffy Coat Platelet Concentrates.

BACKGROUND: The level of residual red blood cells (RBCs) in platelet concentrates (PCs) is of interest because of clinical concerns related to alloimmunization to RBC antigens in transfused patients. This work aims at characterizing and quantifying the levels of intact and fragmented RBCs in apheresis (AP-PCs) and buffy coat PCs (BC-PCs) to assess their potential risk for RhD antigen alloimmunization. METHODS: After staining with anti-CD41 (platelets) and anti-CD235a (RBCs) antibodies, the size and density of RhD antigen on intact and fragmented RBCs were analyzed by flow cytometry. RESULTS: Residual RBC counts were 29 ± 22 × 106/unit in AP-PCs and 121 ± 54 × 106/unit in BC-PCs, which correspond to about 3 and 11 µL of RBCs by product, respectively. RhD expression was about 4 times higher on RBC particles in AP-PCs, and these particles contribute to 66 and 75% of the total antigenic load in BC-PCs and AP-PCs, respectively. CONCLUSIONS: Processing methods influence the quantity and nature of contaminating residual RBCs and RBC-derived particles in PCs. The estimation of residual RBCs in these blood products is generally based on measurements of intact RBCs, which might underestimate the risk for alloim-munization in transfused patients. The question of whether these RBC-derived particles can produce an immune response and, thus, should then be taken into consideration for Rh immune prophylactic treatments, remains to be clarified.

Major role of IgM in the neutralizing activity of convalescent plasma against SARS-CoV-2.

Characterization of the humoral response to SARS-CoV-2, the etiological agent of COVID-19, is essential to help control the infection. The neutralization activity of plasma from patients with COVID-19 decreases rapidly during the first weeks after recovery. However, the specific role of each immunoglobulin isotype in the overall neutralizing capacity is still not well understood. In this study, we select plasma from a cohort of convalescent patients with COVID-19 and selectively deplete immunoglobulin A, M, or G before testing the remaining neutralizing capacity of the depleted plasma. We find that depletion of immunoglobulin M is associated with the most substantial loss of virus neutralization, followed by immunoglobulin G. This observation may help design efficient antibody-based COVID-19 therapies and may also explain the increased susceptibility to SARS-CoV-2 of autoimmune patients receiving therapies that impair the production of immunoglobulin M (IgM).

Evaluation of the Tempo® System: Improving the Microbiological Quality Monitoring of Human Milk.

Background: Bacteriological testing of donor human milk is mostly done both before and after pasteurization to control contamination in the end-product and meet the microbiological standards. Although the plate count method represents a reliable and sensitive technique and is considered the gold standard for bacteriological testing, it is recognized for being time-consuming and requiring qualified personnel. Recently, faster testing technologies, mostly geared toward the food industry, have been developed. Among these, the bioMérieux TEMPO® system uses the most probable number method to assess microbiological content in a semi-automated fashion. Objective: The performances of the TEMPO® system in enumerating bacterial quality indicators in human milk were assessed and compared to the reference plate count method. Methods: Naturally and artificially contaminated human milk samples were used to compare the analytical performances of the TEMPO® system to the plate count technique. More specifically, bacteria belonging to the genera Bacillus, Enterobacteriaceae, Staphylococcus aureus, and total aerobic flora were screened using both methods. Bacteria isolated on agar plates containing selective media were identified by supplemental testing. Bacterial testing results and method parameters were compared using linear regression analyses and Bland-Altman approaches. Results: Naturally contaminated milk samples (n = 55) tested for total aerobic flora showed < 1 log (CFU/ml) discrepancy between the two methods in the output results for 98% of the samples. Comparative linear regression analyses demonstrate good correlations between the two methods (R 2 > 0.9). At lower levels of bacterial contamination, the TEMPO® method precision (C.V. < 8%) and accuracy (> 83%) were comparable to plate counts. Conclusions: The analytical performances of the TEMPO® system for human milk bacteriological testing are equivalent to the reference plate count method. Results from the TEMPO® system are available within a 24-h turnaround time from sample inoculation without the need for further supplemental testing, suggesting that this semi-automated method could be implemented within milk bank operations as an in-process monitoring technology to optimize end-product quality and safety.

Immunomodulation of human B cells following treatment with intravenous immunoglobulins involves increased phosphorylation of extracellular signal-regulated kinases 1 and 2.

In the treatment of autoimmune diseases, intravenous Igs (IVIg) are assumed to modulate immune cells through the binding of surface receptors. IVIg act upon definite human B cell populations to modulate Ig repertoire, and such modulation might proceed through intracellular signaling. However, the heterogeneity of human B cell populations complicates investigations of the intracellular pathways involved in IVIg-induced B cell modulation. The aim of this study was to establish a model allowing the screening of IVIg signal transduction in human B cell lines and to attempt transposing observations made in cell lines to normal human B lymphocytes. Nine human B cell lines were treated with IVIg with the goal of selecting the most suitable model for human B lymphocytes. The IgG(+) DB cell line, whose response was similar to that of human B lymphocytes, showed reduced IVIg modulation following addition of PD98059, an inhibitor of extracellular signal-regulated protein kinase 1/2 (ERK1/2). The IVIg-induced ERK1/2 phosphorylation was indeed proportional to the dosage of monomeric IVIg used when tested on DB cells as well as Pfeiffer cells, another IgG(+) cell line. In addition, two other intermediates, Grb2-associated binder 1 (Gab1) and Akt, showed increased phosphorylation in IVIg-treated DB cells. IVIg induction of ERK1/2 phosphorylation was finally observed in peripheral human B lymphocytes, specifically within the IgG(+) B cell population. In conclusion, IVIg immunomodulation of human B cells can thus be linked to intracellular transduction pathways involving the phosphorylation of ERK1/2, which in combination with Gab1 and Akt, may be related to B cell antigen receptor signaling.

Modulation of CD40-activated B lymphocytes by N-acetylcysteine involves decreased phosphorylation of STAT3.

B lymphocyte activation, maturation and reshaping require the interaction of its receptor CD40 with its ligand CD154, which is expressed on activated T lymphocytes. Metabolism in activated B lymphocytes is also characterized with several REDOX changes including fluctuation of Reactive Oxygen Species (ROS). Herein, we first confirm that stimulation of human peripheral blood B lymphocyte with CD154 increases intracellular ROS level. Then, by treatments with two well-known antioxidants, N-acetylcysteine (NAC) and Trolox, we further investigate the influence of REDOX fluctuation in CD40-activated B lymphocyte homeostasis in long term culture (13 days). Treatments with NAC increase viability, decrease proliferation and Ig secretion and enhance homoaggregation of B lymphocytes while Trolox only induces a marginal increase of their Ig secretion. The NAC-induced homoaggregation phenotype is paralleled with increased expressions of CD54, CD11a, CD27 and CD38. Mechanistically, a 24h exposure of B lymphocytes with NAC is sufficient to show strong inhibition of STAT3 phosphorylation. Besides, the treatment of B lymphocytes with the STAT3 inhibitor VI increases viability and decreases proliferation and secretion as in NAC-treated cells thus showing a role for STAT3 in these NAC-induced phenotypes. This study done in a human-based model provides new findings on how REDOX fluctuations may modulate CD40-activated B lymphocytes during immune response and provide additional hints on NAC its immunomodulatory functions.

Effective in vitro expansion of CD40-activated human B lymphocytes in a defined bovine protein-free medium.

CD40-CD154 interaction is used to culture human B lymphocytes, which are now viewed as effectors to potentially promote T lymphocyte response against malignant cells in cell-based therapy. Currently, the media used, based on bovine serum, are raising concerns for patient safety in such therapy. In this study, we have investigated whether human B lymphocytes could be cultured in the absence of bovine serum. Blood CD19(+) B lymphocytes were activated using interaction through CD40 in medium containing defined animals or human proteins and lipids, and were monitored during short-term periods (≤15 days). Conventional stem-cell medium and a medium containing human albumin instead of bovine albumin were tested. We observed that the response of B lymphocytes appeared influenced by lot-to-lot variability in low density lipoproteins (LDL). Nevertheless, B lymphocyte proliferation and secretion in serum-free and bovine protein-free media were quite similar to that of cells cultured in medium containing FBS. Our results show that CD40-activated B lymphocytes can be cultured for up to 15 days in a serum-free medium containing human albumin, LDL, α-tocopherol and chemically-defined lipids.

Estimation of the number of CD154 molecules in membrane extracts used as a source of CD40 stimulation of human B lymphocytes.

The CD40-CD154 interaction is better exemplified by a rheostat than by an on-off switch, and variations in its intensity can play a role in the regulation of B lymphocyte activation following primary and/or secondary humoral immune response. The CD40-CD154 interaction is often studied in co-culture models using CD154+ adherent cells, which can be problematic when performing protein or gene analyses. The use of membrane extracts prepared from CD154+-transfected cells can eliminate possible interferences caused by the presence of contaminating feeder cells. Given the dose-response effect of CD154 on target B cells, it is important to measure the amount of CD154 when using soluble membranes. We hereby report a simple method, based on cytometry analysis, to estimate the relative number of CD154 molecules in membrane extracts, allowing reproducibility in human B-cell activation level.

CD40-activated B cells from patients with systemic lupus erythematosus can be modulated by therapeutic immunoglobulins in vitro.

INTRODUCTION: Aberrant signaling within and between B and T cells, considered to be central in systemic lupus erythematosus (SLE), could depend on enhanced CD40-CD154 activation. As a result, autoreactive B cells, normally anergic, differentiate and secrete antibodies attacking several normal tissues. Thus restorating B cell homeostasis might help control this disease. In this study, two facets of SLE B cells were investigated, namely their in vitro response to CD40-CD154 and the effect of treatment with human immunoglobulins for intravenous use (IVIg). MATERIALS AND METHODS: Blood samples from SLE patients and healthy volunteers were obtained and used to isolate B cells, which were activated through CD40 in the presence or absence of IVIg. The phenotype, proliferation, and differentiation of the SLE B cells were determined and compared with those of control B cells using flow cytometry and standard ELISA. RESULTS: In this model, CD40-activated SLE B cells, as control B cells, proliferated and differentiated and were characterized by the emergence of CD19(lo)CD38(++)CD138(+)CD27(++) cells. IVIg treatment of the CD40-activated SLE B cells resulted in higher differentiation, characterized by increased secretion rates of IgG and IgM, as reported previously for control B cells. CONCLUSIONS: Taken as a whole, such accelerated differentiation of CD40-activated B cells suggests that IVIg may participate in re-equilibration of the antibody repertoire by replacing pathological antibodies by de novo harmless antibodies.

Human B lymphocytes and non-Hodgkin's lymphoma cells become polyploid in response to the protein kinase inhibitor SU6656.

We show that prolonged exposure of non-Hodgkin's lymphoma (NHL) cell lines to low doses of the Src family protein tyrosine kinases (SFKs) inhibitor SU6656 caused proliferation abrogation as a result of the formation of cells with single multilobed nuclei and several mitotic spindle poles, features similar to polyploid megakaryocytes. The propensity of the NHL B cells tested to undergo polyploid was unrelated to the presence of p53 mutations in these cells since comparable outcomes were observed in SU6656-exposed cultures of blood B lymphocytes derived from healthy individuals. Thus, in addition to its utility for the study of megakaryocyte polyploidization, our results show that SU6656 can also induce polyploidy in cells of lymphoid origin, revealing a chemotherapeutic potential for this inhibitor to limit tumor propagation of malignant B cell lymphomas, although not without affecting normal B cells as well.

Characterization of mononuclear cells remaining in the leukoreduction system chambers of apheresis instruments after routine platelet collection: a new source of viable human blood cells.

BACKGROUND: The yield of white blood cells (WBCs) extracted from whole-blood leukoreduction filters can be affected by the storage conditions and delay before filtration. Platelets (PLTs) collected with apheresis instruments (Trima Accel, Gambro BCT) are leukoreduced during the procedure on a fluidized particle bed in a leukoreduction chamber (LRS chamber). In this report, the residual cell content of these LRS chambers was characterized to determine whether it would be a valuable source of viable human blood cells. STUDY DESIGN AND METHODS: The content of LRS chambers was eluted by gravity, and peripheral blood mononuclear cells (PBMNCs) were purified on a Ficoll-Paque gradient. Analyses were performed before and after freezing. Proportions of CD3+, CD14+, CD16+, CD19+, CD34+, and CD45+ cells were determined by flow cytometry. The frequency of T cells expressing CD4, CD8, and CD27 and of B cells expressing immunoglobulin G (IgG), IgM, and CD27 was also determined. RESULTS: LRS chambers held approximately 10(9) CD45+ cells representing the normal proportions of CD3+, CD14+, CD16+, and CD19+ cell populations of PBMNCs. A small fraction of these CD45+ cells were CD34+CD38+ cells (0.3 +/- 0.2%). The viability of these cells, measured before and after freezing, was more than 95 percent. CONCLUSION: The residual cell content of Trima Accel LRS chambers recovered after PLT collection is a good source of viable monocytes and lymphocytes. These PBMNCs, containing CD3+, CD14+, CD16+, CD19+, and CD34+ cells can be frozen to prepare cell banks, which opens new avenues for utilization in several physiologic studies or even in cellular therapy applications.

Whole-blood leukoreduction filters are a source for cryopreserved cells for phenotypic and functional investigations on peripheral blood lymphocytes.

BACKGROUND: Leukoreduction of blood is now widely performed by blood banks, and the possibility of recovering 10(8) to 10(9) white blood cells (WBCs) from leukoreduction filters, which are usually discarded, represents a promising source for normal human cells. Previous studies with these filters to prepare WBCs have performed their experimentation with fresh cells only. Whether these filter-derived cells could also be used to prepare frozen cell banks to facilitate work organization and open new avenues for their utilization as references in physiological studies and clinical investigations was investigated. STUDY DESIGN AND METHODS: Blood samples or whole-blood leukoreduction filters were obtained, after informed consent, from volunteers or blood donors, respectively. The proportions of CD3+, CD14+, CD16+, CD19+, and CD45+ cells within peripheral blood mononuclear cells (PBMNCs) were determined by flow cytometry from all samples. B cells were isolated and their functional responses were evaluated in vitro. RESULTS: The yield of PBMNCs recovered from whole-blood leukoreduction filters was lower (50%) than the one with fresh blood samples but still provided 2 x 10(8) to 4 x 10(8) PBMNCs per unit. After one cycle of freezing-thawing, the proportions of B- and T-cell populations were similar to normal blood values. Purified B cells issued from whole-blood leukoreduction filters displayed normal phenotypes and functions. CONCLUSION: Leukoreduction filters represent a valuable source of PBMNCs. These cells could be easily recovered to prepare frozen cell banks useful in basic phenotypic and functional analyses involving the main subsets of B cells and the global T-cell population.

B cell proliferation following CD40 stimulation results in the expression and activation of Src protein tyrosine kinase.

Resting normal human B cells express negligible c-src mRNA or Src protein tyrosine kinase; however, upon induction of proliferation, these cells express high levels of both mRNA and protein and show a concomitant increase in tyrosine kinase activity of immunoprecipitated Src. Src expression was most pronounced upon stimulation with CD154, and to a lesser extent CD70, Staphylococcus aureus, Cowan strain I and phorbol ester, and correlated with the activation of the cells. Transfection of cDNA for human wild-type or kinase-dead Src into Raji B cells resulted in an increase and decrease, respectively, of the cell numbers in culture, showing a direct correlation of proliferation to the expression of Src that was corroborated using anti-sense oligodeoxynucleotides and chemical inhibitors. Furthermore, the human B cell lines, Namalwa, Daudi and Raji express low levels of Src but express very high levels of Src after stimulation with CD154 that showed a correlation with increased activation. This is the first report of Src detectable in normal B cells. The finding that Src expression is inducible and correlates with stimulation by CD154 and the proliferation of the B cells suggests that Src may play a specific role in normal and transformed B cell activation/proliferation pathways mediated primarily through CD40 stimulation.