Bacterial safety of cell-based therapeutic preparations, focusing on haematopoietic progenitor cells.

Bacterial safety of cellular preparations, especially haematopoietic progenitor cells (HPCs), as well as advanced therapy medicinal products (ATMPs) derived from stem cells of various origins, present a challenge for physicians, manufacturers and regulators. The article describes the background and practical issues in this area and illustrates why sterility of these products cannot currently be guaranteed. Advantages and limitations of approaches both for classical sterility testing and for microbiological control using automated culture systems are discussed. The review considers novel approaches for growth-based rapid microbiological control with high sensitivity and faster availability of results, as well as new methods for rapid bacterial detection in cellular preparations enabling meaningful information about product contamination within one to two hours. Generally, however, these direct rapid methods are less sensitive and have greater sampling error compared with the growth-based methods. Opportunities for pyrogen testing of cell therapeutics are also discussed. There is an urgent need for development of novel principles and methods applicable to bacterial safety of cellular therapeutics. We also need a major shift in approach from the traditional view of sterility evaluation (identify anything and everything) to a new thinking about how to find what is clinically relevant within the time frame available for the special clinical circumstances in which these products are used. The review concludes with recommendations for optimization of microbiological control of cellular preparations, focusing on HPCs.

Establishment of the first international repository for transfusion-relevant bacteria reference strains: ISBT working party transfusion-transmitted infectious diseases (WP-TTID), subgroup on bacteria.

BACKGROUND: Bacterial contamination of platelet concentrates (PCs) still remains a significant problem in transfusion with potential important clinical consequences, including death. The International Society of Blood Transfusion Working Party on Transfusion-Transmitted Infectious Diseases, Subgroup on Bacteria, organised an international study on Transfusion-Relevant Bacteria References to be used as a tool for development, validation and comparison of both bacterial screening and pathogen reduction methods. MATERIAL AND METHODS: Four Bacteria References (Staphylococcus epidermidis PEI-B-06, Streptococcus pyogenes PEI-B-20, Klebsiella pneumoniae PEI-B-08 and Escherichia coli PEI-B-19) were selected regarding their ability to proliferate to high counts in PCs and distributed anonymised to 14 laboratories in 10 countries for identification, enumeration and bacterial proliferation in PCs after low spiking (0·3 and 0·03 CFU/ml), to simulate contamination occurring during blood donation. RESULTS: Bacteria References were correctly identified in 98% of all 52 identifications. S. pyogenes and E. coli grew in PCs in 11 out of 12 laboratories, and K. pneumoniae and S. epidermidis replicated in all participating laboratories. The results of bacterial counts were very consistent between laboratories: the 95% confidence intervals were for S. epidermidis: 1·19-1·32 × 10(7) CFU/ml, S. pyogenes: 0·58-0·69 × 10(7) CFU/ml, K. pneumoniae: 18·71-20·26 × 10(7) CFU/ml and E. coli: 1·78-2·10 × 10(7) CFU/ml. CONCLUSION: The study was undertaken as a proof of principle with the aim to demonstrate (i) the quality, stability and suitability of the bacterial strains for low-titre spiking of blood components, (ii) the property of donor-independent proliferation in PCs, and (iii) their suitability for worldwide shipping of deep frozen, blinded pathogenic bacteria. These aims were successfully fulfilled. The WHO Expert Committee Biological Standardisation has approved the adoption of these four bacteria strains as the first Repository for Transfusion-Relevant Bacteria Reference Strains and, additionally, endorsed as a project the addition of six further bacteria strain preparations suitable for control of platelet contamination as the next step of enlargement of the repository.

FACS technology used in a new rapid bacterial detection method.

Culture methods for bacterial detection (BacT/ALERT or Pall eBDS) are currently implemented in blood donor screening procedures in many countries. Experience in the first years after implementation of these detection assays showed that although the analytical sensitivity was extremely high (about 1 CFU mL(-1)), the majority of these platelets were still transfused before a positive screening result was attained. Rapid technologies were developed to more effectively prevent transfusion-transmitted bacterial infection. In this study, a new rapid bacterial detection method based on fluorescence-activated cell sorting (FACS) technology was developed. Bacteria were stained with thiazole orange dye for 5 min and measurement was taken immediately after staining. The entire process took only 30 min. Six transfusion-relevant bacteria strains were tested in a spiking study. Without pre-incubation in a special bacteria growth medium, analytical sensitivity ranged between 10(5) CFU mL(-1) (Klebsiella oxytoca and Serratia marcesens) and 10(3) CFU mL(-1) (Escherichia coli). Sensitivity could be improved to 10(1) CFU mL(-1) for all tested bacteria by adding a pre-incubation step (6 h at 37 degrees C). Although preliminary in nature, results of our study suggest that bacterial detection by FACS technology in conjunction with a pre-incubation step offers a sensitive alternative technology to culture methods. Additionally, it provides the benefit of a rapid test time and the opportunity of preventing bacterial transmitted infections more effectively.

Fluorescence quencher improves SCANSYSTEM for rapid bacterial detection.

BACKGROUND AND OBJECTIVES: The optimized scansystem could detect contaminated platelet products within 24 h. However, the system's sensitivity was reduced by a high fluorescence background even in sterile samples, which led to the necessity of a well-trained staff for confirmation of microscope results. MATERIALS AND METHODS: A new protocol of the optimized scansystem with the addition of a fluorescence quencher was evaluated. Pool platelet concentrates contaminated with five transfusion-relevant bacterial strains were tested in a blind study. RESULTS: In conjunction with new analysis software, the new quenching dye was able to reduce significantly unspecific background fluorescence. Sensitivity was best for Bacillus cereus and Escherichia coli (3 CFU/ml). DISCUSSION: The application of a fluorescence quencher enables automated discrimination of positive and negative test results in 60% of all analysed samples.

Analysis of leukocyte binding to depletion filters: role of passive binding, interaction with platelets, and plasma components.

Since limited knowledge exists on the mechanisms which regulate cell binding to leukocyte removal filter surfaces, we investigated the binding patterns of leukocytes to individual layers of leukocyte depletion filters. After passage of 1 unit of whole blood, blotting of isolated filter layers on glass slides or elution of cells from filter layers revealed that most leukocytes were located within the first 10 of a total of 28 filter layers, peaking at layers 6 to 8, with granulocytes binding on average to earlier filter layers than lymphocytes. Leukocytes preincubated with inhibitors of actin activation showed unchanged distribution between filter layers, suggesting that cytoskeletal activation does not significantly contribute to their binding. When leukocytes were directly incubated with single filter layers, binding of up to 30% of input cells was recorded in the absence of Ca(2+). Immunohistological analyses showed colocalization of platelets and leukocytes, with co-clustering of platelets and leukocytes. Monocytes and to some degree lymphocytes but not granulocytes competed with platelets for filter binding. Precoating of filter layers with individual plasma components showed that hyaluronic acid, plasma type fibronectin, and fibrinogen all increased the binding of leukocytes compared with albumin coating. In conclusion, leukocytes can bind passively to filters in a process which does not require Ca(2+), which is independent of cytoskeletal activation and which may depend on individual plasma components. These results are of importance when new selective cell enrichment or depletion strategies through specific filters are envisaged.

Characterization and crystallization of a novel Sarcocystis muris lectin, SML-2.

A novel lectin (SML-2) consisting of 138 amino acids was isolated from cyst merozoites of Sarcocystis muris and sequenced by Edman degradation and mass spectrometry. All 12 cysteinyl residues are involved in disulfide bridges, four of which are attributed to a characteristic pattern of cysteines as found in the so-called PAN-module superfamily. Crystals of SML-2 diffracting to 2.1 A resolution at a synchrotron were grown by the hanging-drop vapour-diffusion technique. They belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 53.6, b = 128.8, c = 158.2 A and eight molecules in the asymmetric unit. SML-2 cocrystallized with Au galactose results in two different crystal forms. The first form is isomorphous with the native crystals and the second form adopts space group C222(1), with unit-cell parameters a = 74.7, b = 82.0, c = 131.0 A, and diffracts to 2.4 A at a rotating-anode X-ray generator.

[Effect of the preservative thiomersal on the release of interleukin-1 beta from human peripheral blood cells]. / Einfluss des Konservierungsmittels Thiomersal auf die Interleukin-1 Beta Ausschüttung humaner peripherer Blutzellen.

Several vaccines have been tested in the human whole blood pyrogen test as an alternative to the rabbit pyrogen test. As reported previously by our group, the alternative test system is basically applicable to vaccines. The widely used conservative thiomersal is influencing the test system. Surprisingly, the induction of fever inducing cytokines by endotoxin contaminations and other pyrogens can be suppressed by thiomersal.

Growth hormone treatment of breeding bulls used for artificial insemination improves fertilization rates.

To evaluate new therapeutical concepts for male subfertility, we tested the effects of exogenous recombinant bovine growth hormone (rbGH) on various endocrine and metabolic parameters both in blood and in seminal plasma of bulls. Sperm quality was assessed morphometrically and by monitoring the number of successful artificial inseminations (AIs) defined as non-return rates (NRR). Aliquots of 450 semen samples were used from each bull and each experimental period (4 wk before, 14 weeks during and 6 wk after treatment). Six out of ten sires (average age 8.4 years) were treated every two weeks with 640-mg depot formulated rbGH (Eli Lilly). Four bulls received vehicle only. Blood plasma bGH, IGF-I, insulin and glucose concentrations were increased with rbGH treatment. In seminal plasma there was no effect of rbGH treatment on fructose and citrate or on testosterone concentrations. With one exception, rbGH-treated bulls had greater IGFBP-3 concentrations in seminal plasma. Motility of spermatozoa after freezing and thawing was increased compared with pretreatment rates. Most interestingly, the number of successful AIs was increased by an average of 6.0% NRR when ejaculates from rbGH-treated bulls were used.

Expression, purification, and biochemical characterization of a recombinant lectin of Sarcocystis muris (Apicomplexa) cyst merozoites.

The mature major microneme protein of Sarcocystis muris cyst merozoites, which is known as a dimeric lectin with high affinity to galactose and some of its derivatives, was expressed in Escherichia colias a histidine-tagged fusion protein. The recombinant polypeptide, which was recognized by a monoclonal antibody directed against the native lectin, was purified from inclusion bodies after solubilization and refolding, using a combination of metal chelate and lactose affinity chromatography. The apparent molecular mass of the refolded polypeptide as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoreses was 16 kDa, whereas gel filtration chromatography clearly demonstrated that the recombinant protein, like its native counterpart, exists as a homodimer of two non-covalently associated subunits. Inhibition of haemagglutination suggests that the combining site of the recombinant lectin recognizes N-acetyl-galactosamine as the dominant sugar, thus confirming the correct folding of the monosaccharide combining site in the renatured lectin. To the best of our knowledge, this work represents the first reported detailed characterization of a recombinant lectin from apicomplexan parasites, and may contribute to a better understanding of the process of host cell recognition and invasion by these obligate intracellular protozoa.

Characterization of indole-negative Bacteroides fragilis group species with use of polymerase chain reaction fingerprinting and resistance profiles.

Biochemical tests alone do not adequately differentiate the various Bacteroides species, groups, and antimicrobial-resistant variants. Consequently, we used a polymerase chain reaction (PCR) fingerprinting technique, with either a single nonspecific primer derived from the t-DNA intergenic spacer region (T3B) or a single primer that anneals to minisatellite DNA sequences (M13 core), to identify and characterize 58 clinical isolates of Bacteroides fragilis group species (B. fragilis, B. distasonis, and B. caccae). In addition to species- and subspecies-specific differences, 4 strains of B. fragilis, 1 of B. distasonis, and 3 of B. caccae that showed increased resistance to imipenem, ampicillin, and ampicillin/sulbactam also produced unique PCR fingerprint profiles. Analysis by the clinical source of isolation (i.e. blood or intraabdominal, skin, or soft-tissue infection) indicated that no particular PCR fingerprint type was associated with greater pathogenicity of any individual clinical source. The PCR fingerprinting technique proves to be a useful tool for species identification and taxonomic studies, as well as for epidemiological studies of Bacteroides species.

[Comparison of standard methods for the preparation of egg yolk antibodies]. / Vergleich von Standardmethoden zur Präparation von Dotterantikörpern.

Chicken egg yolk antibodies (lgY) play an increasing role as alternative to mammalian polyclonal antibodies. They are widely used in biomedical research, for diagnostics, prophylaxis, and therapy of diseases. The extraction steps of IgY from egg yolk must be simple, with high output of purified antibodies. The aim of the present study was a comparison of different purification methods of egg yolk antibodies. The results of eight extraction methods of IgY and method combinations were investigated by PAGE and densitometric analysis. It has been demonstrated, that the IgY preparation with dextran sulfate is very effective, quick and simple to perform. It is well-suited in combination with other methods, e.g. ammonium sulfate precipitation.

Identification and strain differentiation of 'Bacteroides fragilis group' species and Prevotella bivia by PCR fingerprinting.

Using single consensus primers of genomic nucleotide sequences, PCR-generated fingerprints were used for identification and differentiation of the Bacteroides fragilis group (B. fragilis, B. thetaiotaomicron, B. ovatus, B. distasonis, B. vulgatus) and Prevotella bivia (B. bivius) by comparing the DNA profiles with those of reference strains from the American Type Culture Collection and German Culture Collection. When primed by a single primer phage M13 core sequence, intra-species specific differences and species-specific bands were detected. Using primers derived from the evolutionarily conserved tRNA gene sequence, species-specific patterns were produced. A computer program, GelManager, was used to analyze the profiles and generate dendrograms. The correlation coefficients determined from the DNA fingerprint profiles of the clinical isolates (using the M13 core primer) fell within a narrow range, reflecting a high level of homology within the species. Based on the dendrograms, strains of one species were clearly differentiated from strains of other species. For comparison, SDS-PAGE analysis of whole cell extracts was also performed to obtain protein band patterns of various strains. Because of the simplicity of the PCR fingerprinting method and the ease of performance of computerized evaluation of data, this technique is a useful method for both species and strain differentiation, as well as for characterization of Bacteroides species and Prevotella bivia.

[Cross-reacting HLA antigens as a factor increasing the risk of childlessness in a family]. / Perekrestnoreagiruiushchie antigeny sistemy HLA kak fator, povyshaiushchii risk bezdetnosti v sem'e.

A comparative analysis of HLA phenotypes of fertile and infertile spouses revealed that among the infertile the sum of spouses showed compatibility in two or three HLA antigens. A detailed analysis also showed that 10 infertile couples exhibited identical and 2 antigens with a similar structure and function with so-called cross-reactions increasing the histocompatibility index to 75%. The significance of these facts for infertility risk factors is discussed.

The impact of subspecialty training on the management of advanced ovarian cancer.

A retrospective study was conducted to determine the influence of subspecialty training in gynecologic oncology as well as several other covariates on the feasibility, operative mortality, and survival benefits of cytoreductive surgery for 263 patients with stages IIIC and IVA epithelial ovarian cancer. Covariates most predictive of an optimal (< or = 1 cm) cytoreductive outcome were the diameter of the largest metastases before cytoreduction (< or = 10 cm vs > 10 cm, P < 0.001) and the specialty training of the physicians present at surgery (gynecologic oncologists vs other, P < 0.001). Age influenced operative mortality most (< 60 vs > or = 60, P < 0.001). Covariates found to most significantly influence survival time include the specialty training of the physicians present at surgery (gynecologic oncologists vs other, P < 0.0001), cytoreductive outcome (complete vs optimal, P = 0.001, optimal vs suboptimal, P < 0.0001), grade of tumor (grade 1 vs grades 2 and 3, P = 0.01), and pelvic disease status (frozen pelvis vs mobile primary tumor, P = 0.03). We conclude that patients with advanced epithelial ovarian cancer should undergo aggressive cytoreductive surgery by gynecologic oncologists, with the objective to remove all macroscopic disease. Subsequent treatment with platinum-based chemotherapy offers the best chance for long-term survival or cure.

Sarcoid-like lesions associated with epithelial ovarian adenocarcinoma.

A woman with poorly differentiated endometrioid adenocarcinoma of the ovary had sarcoid-like lesions in the bone marrow. Sarcoid-like lesions may be misinterpreted as metastatic disease, resulting in inappropriate modification of therapy.

Activation of human CD4+ and CD8+ cells by Sarcocystis gigantea lectin.

As recently reported, Sarcocystis gigantea lectin (SGL) is a powerful mitogen and a polyclonal activator (Syn. S. ovifelis) of human peripheral B-cells. In the present study we investigated the reactivity of human T-helper (CD4) and T-suppressor (CD8) cells to SGL. Mononuclear cells (MNCs) from five newborns and six adults were examined cytofluorometrically for the expression of cell-surface differentiation and activation antigens using a set of seven monoclonal antibodies. In all, 96% of cord-blood and 81% of adult CD4 cells expressed receptors for interleukin-2 (Tac+) after 64 and 164 h microgram/ml). The percentages of neonatal and adult Tac+ CD8 cells amounted to 67% and 59%, respectively. Major histocompatibility complex (MHC) class II antigens identified using the monoclonal anti-human leukocyte antigen (HLA)-DR antibody L243 were expressed on 30% (CD4) and 44% (CD8) of adult T-cells. Neonatal HLA-DR+ T-lymphocytes were not detectable. In parallel, functional tests were performed to examine cell proliferation and MNC antibody production.