Small Amount Of Peripheral Blood Research Articles

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a). CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (>10(9) cells per patient) from healthy donors as well as cancer patients (Fig.1c,d). In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3). To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function. CD40-B cells efficiently take up, process and present antigens to T cells. They do not only prime naïve, but also expand memory T cells. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors.

CD40-activated B cells can be generated in high number and purity in cancer patients: analysis of immunogenicity and homing potential

Cellular adjuvants such as dendritic cells (DC) are in the focus of tumour immunotherapy. In DC-vaccine trials, induction of tumour antigen-specific immunity is observed frequently and well-documented clinical responses have been reported. However, the overall response rate is less than 3%, therefore alternative strategies are being investigated. CD40-activated B cells (CD40-B) have been characterized previously as an interesting alternative because they present antigen efficiently and can be expanded by several logs from small amounts of peripheral blood. To determine the central technical challenges of cell-based vaccines we performed a single-patient analysis of 502 patients from DC-based tumour vaccine trials and identified at least three factors contributing to their limited efficiency: (1) lack of cell numbers; (2) lack of documented purity thus high contamination of bystander cells; and (3) lack of quality control and thus heterogeneous or unknown expression of important surface molecules such as major histocompatibility complex (MHC) and chemokine receptors. Based on these findings we re-evaluated the CD40-B approach in cancer patients. Here, we show that proliferation of B cells from cancer patients is equivalent to that observed in healthy donors. Purity is always > 90% after 2 weeks and remains stable for several weeks. They have comparable antigen-presenting capability determined phenotypically and by allogeneic mixed lymphocyte reaction. Expression of CCR7 and CD62L was detected in all samples and B cells migrated towards the relevant homing chemokines. Taken together, CD40-B cells from cancer patients can be expanded in virtually unlimited numbers at high purity and full function concerning antigen-presentation and migratory properties.

Human plasma accelerates immortalization of B lymphocytes by Epstein–Barr virus

Serum and plasma contain species-specific factors that modulate cell population growth and function, and that are required for proliferation of most cell cultures. Foetal calf serum (FCS) is the most common source of these growth factors. We studied the effect of human plasma (HP) on the immortalization process of B lymphocytes by Epstein-Barr virus (EBV) was studied. The effect of HP as compared to FCS was done through assessment of cell proliferation. It was found that HP (autologous and non-autologous plasma) is more effective than FCS in generating lymphoblastoid cell lines, regardless of EBV status of the donors: 65% of HP-supplemented cultures developed into lymphoblastoid cell lines by 7-14 culture days, as compared to 16% of cultures with FCS. In addition, 6% of HP-supplemented cultures did not achieve becoming lymphoblastoid cell lines by day 35 in comparison to 94% of cultures with FCS. The higher proliferative effect of HP was not altered by heat inactivation or filtration. HP maintained its proliferative activity at 4 degrees C over 8 months, thus indicating that HP contains a stable growth factor(s), which accelerates B-lymphocyte immortalization. The results support other studies that recommend the use of autologous plasma for tissue culture, mainly in the case of autologous transplantation. Furthermore, the use of HP allows preparation of lymphoblastoid cell lines from a small amount of peripheral blood in a shorter period of time and with a higher rate of success.

Erythroid differentiation and maturation from peripheral CD34 + cells in liquid culture: Cellular and molecular characterization

In vitro models of human erythropoiesis are useful in studying the mechanisms of erythroid differentiation from BFU-E to mature erythrocytes both in normal and pathological conditions. Most of the available in vitro liquid cultures are from cell lines or are limited by the production of few erythroid cells mixed with myeloid cells. Here we describe an erythroid liquid culture system starting from CD34 +-enriched cells obtained from peripheral blood. CD34 + cells were cultured for 21 days in different conditions. Precisely stem cell factor (SCF, 20 ng/mL) and IL-3 (10 ng/mL) were added at starting point plus Epo (3 U/mL) at day 0 or 7 of culture with or without cyclosporine A (Cy; 1 mg/mL). In all the conditions, the highest recovery was obtained at day 14 of culture. Epo and Cy added at day 0 produced the highest cell expansion (170-fold mean amplification of the initial cell input by day 14) and recovery of erythroid cell. Sixty seven percent of the cells were GP + at day 7 and 97% by day 14 respectively. Most of the cells were proerythroblasts at day 7 and mature erythroblasts at day 14 (> 90% were benzidine pos). The presence of Cy favoured erythroid differentiation and maturation and reduced the percentage of non-erythroid CD45 + cells (2% with Cy versus 5% without Cy). Cells cultured with Epo and Cy reproduced erythropoiesis also at the molecular level. The results suggest that in 14 days different steps of human erythropoiesis from peripheral CD34 + cells could be reproduced, with high recovery of highly purified erythroid cells. The high number and purity of erythroid cells produced from a small amount of peripheral blood make this method useful for studying either normal or pathological erythropoiesis.

CD40-activated B cells express full lymph node homing triad and induce T-cell chemotaxis: potential as cellular adjuvants

AbstractCD40-activated B cells (CD40-B cells) have previously been introduced as an alternative source of antigen-presenting cells for immunotherapy. CD40-B cells can prime naive and expand memory T cells, and they can be generated in large numbers from very small amounts of peripheral blood derived from healthy individuals or cancer patients alike. Administration of CD40-B cells as a cellular adjuvant would require these cells to migrate toward secondary lymphoid organs and attract T cells in situ, processes guided by specific chemokines and chemokine receptors. Here, we demonstrate that primary, human CD40-B cells express a pattern of adhesion molecules and chemokine receptors necessary for homing to secondary lymphoid organs and have the capacity to migrate to cognate ligands. Furthermore, we show that CD40-B cells express important T-cell attractants and induce strong T-cell chemotaxis. These findings further support the use of CD40-B cells as cellular adjuvant for cancer immunotherapy.

A new method for histamine release from purified peripheral blood basophils using monoclonal antibody-coated magnetic beads

A new method for evaluating histamine release from purified basophils was developed. Basophil-containing leukocytes were directly purified from a small amount of peripheral blood using monoclonal antibody BA312-coated magnetic beads. The purified basophils still rosetted to magnetic beads maintained a normal response to anti-IgE and to dust mite allergen in comparison with the conventional method using washed leukocytes. This methodology facilitates the purification of basophils, anti-IgE- and allergen-induced histamine release, and subsequent histamine determination within only 3 h. The released histamine was analyzed by an enzyme-linked immunosorbent assay (ELISA) with a characteristic detection profile. Since all steps were performed in 96-well microplates, many clinical samples could be analyzed at the same time, permitting easy applications in routine laboratories.

CONTRIBUTION OF THE MOLECULAR GENETICS LABORATORY TO THE EVALUATION OF THE PERSISTENTLY HYPOTONIC INFANT. † 872

Pediatricians may be requested to elucidate the cause of nonspecific persistent generalized infantile hypotonia. Among the many causes in the differential diagnosis of a floppy baby are several familiar genetic diseases that can present with generalized hypotonia in the newborn period. Testing for these disorders, spinal muscular atrophy (SMA), myotonic dystrophy (DM) and Prader-Willi Syndrome (PWS), now requires only a small amount of peripheral blood, and positive results are diagnostic for that disease. The tests for SMA, DM, and recently PWS are done by DNA analysis to look for gene deletions(SMA), expansions (DM), or the absence of the paternal copy of chromosome 15(PWS). Previous analysis for PWS, including this study, has been done by chromosome fluorescence in-situ hybridization to look for deletions in the PWS region of chromosome 15. This technique can detect approximately 70% of those with PWS. Screening of 35 persistently hypotonic children for one or more of these three disorders revealed 7 positive results, 2 with DM, 1 with SMA and 4 with PWS, for a detection rate of 20%. In addition, in 8 of 8 cases in which a clinical diagnosis of SMA had been made, DNA analysis was used to confirm the diagnosis, aid in interpretation of prenatal results in subsequent pregnancies, or provide a conclusive diagnosis, without the need for a muscle biopsy. Positive findings for one of these genetic diseases can provide valuable information regarding clinical management and prognosis of the child, allow specific prenatal testing in future pregnancies and could lead to the identification of other at-risk relatives. Twenty per cent of hypotonic infants were found with one of these three genetic disorders, illustrating the significance of considering this panel of diseases in an infant presenting for genetic evaluation with nonspecific hypotonia.

The concomitant detection of gene mutation and micronucleus induction by mitomycin C in vivo using lacZ transgenic mice

A new assay system that can simultaneously provide gene mutagenicity and clastogenicity data in vivo is described. Transgenic mice (Muta TM Mouse) harboring the lacZ gene as a target for mutation analysis were injected intraperitoneally with mitomycin C (MMC), either once or on 5 successive days. Micronucleuc assays were performed with small amounts of peripheral blood collected from a tail vessel. The spontaneous frequency of micronucleated reticulocytes was 0.42%. For the mutation analysis, DNA was extracted from bone marrow and liver cells at several harvest times. The lacZ gene was rescued by lambda packaging and infection of E. coli C ( lac −), followed by plating on agarose plates containing X-gal. The spontaneous lacZ mutant frequencies were 37 and 29×10 −6 in bone marrow and liver, respectively. In the micronucleus assay, single treatments with 1.0 and 2.0 mg/kg of MMC induced micronuclei in 3.6 and 5.8% of reticulocytes, respectively, peaking 48 h after treatment. Muta TM Mouse sensivitiy to micronucleus induction was similar to nontransgenic strains used routinely for the micronucleus test. On the other hand, single treatments with MMC at 1.0 and 2.0 mg/kg did not induce any significant increases in the frequency of lacZ − mutants in bone marrow or liver. N-Ethyl- N-nitrosourea, used at 100 mg/kg as a positive control, yielded a 5-fold increase in mutant frequency above untreated animals in bone marrow only. After 5-day treatments, MMC induced approximately a 2-fold increase in mutant frequency in bone marrow only for the sublethal dose of 2 mg/kg. Therefore, this study indicated that the strong clastogenic activity of MMC in bone marrow was not accompanied by significant gene mutagenic activity.

Construction of human gene libraries from small amounts of peripheral blood: analysis of beta-like globin genes.

We describe a rapid procedure for constructing cloned human genomic libraries from small amounts of peripheral blood. High molecular weight DNA is isolated from 5-20 ml peripheral blood, partially cleaved with Eco R1, and 8-22 kb fragments are cloned using bacteriophage Charon 4A and suitable E. coli host. Using the approach we have isolated and characterized several non-alpha globin clones from a Kurdish Jew with homozygous beta thalassemia. The ability to isolate suitable amounts of high molecular weight DNA from peripheral blood provides a relatively simple means of constructing human gene libraries representing a variety of hemoglobin disorders.