Long-term maintenance of hematopoietic stem cells does not require contact with embryo-derived stromal cells in cocultures

We recently established that two midgestation-derived stromal clones--UG26-1B6, urogenital ridge-derived, and EL08-1D2, embryonic liver-derived--support the maintenance of murine adult bone marrow and human cord blood hematopoietic repopulating stem cells (HSCs). In this study, we investigate whether direct HSC-stroma contact is required for this stem cell maintenance. Adult bone marrow ckit+ Ly-6C- side population (K6-SP) cells and stromal cells were cocultured under contact or noncontact conditions. These experiments showed that HSCs were maintained for at least 4 weeks in culture and that direct contact between HSCs and stromal cells was not required. To find out which factors might be involved in HSC maintenance, we compared the gene expression profile of EL08-1D2 and UG26-1B6 with four HSC-nonsupportive clones. We found that EL08-1D2 and UG26-1B6 both expressed 21 genes at a higher level, including the putative secreted factors fibroblast growth factor-7, insulin-like growth factor-binding proteins 3 and 4, pleiotrophin, pentaxin-related, and thrombospondin 2, whereas 11 genes, including GPX-3 and HSP27, were expressed at a lower level. In summary, we show for the first time long-term maintenance of adult bone marrow HSCs in stroma noncontact cultures and identify some secreted molecules that may be involved in this support.     

C-reactive protein concentration is not related to islet autoimmunity status in offspring of parents with type 1 diabetes

Autoimmunity may be associated with acute or chronic inflammation. In order to determine whether the inflammatory marker C-reactive protein (CRP) was an indicator of inflammatory events that precede, predict, or associate with islet autoimmunity or type 1 diabetes, CRP was measured in sequential antibody-negative, seroconversion, and follow-up-positive samples from 65 prospectively studied islet autoantibody-positive children. Although changes in CRP concentrations were observed in some children, overall CRP concentrations were similar in antibody-negative samples (median, 0.21 mg/L), antibody-positive samples (median, 0.26 mg/L), and samples at seroconversion (median, 0.26 mg/L). CRP concentrations at diabetes onset (median, 0.59 mg/L) were not significantly increased over antibody-negative samples (P = 0.07). CRP concentrations did not predict diabetes development. CRP concentrations were related to age (r = 0.26; P < 0.001) and were increased in samples obtained from October to January (P < 0.001). These findings suggest that CRP concentrations are not a valuable marker of progression to type 1 diabetes and highlight the importance of correcting analyses for seasonal variations.     

Cross-reactive trinitrophenylated peptides as antigens for class II major histocompatibility complex-restricted T cells and inducers of contact sensitivity in mice. Limited T cell receptor repertoire

The induction of contact sensitivity in mice by hapten reagents such as trinitrochlorobenzene (TNCB) involves the activation of class II major histocompatibility complex (MHC)-restricted, hapten-specific, CD4⁺ T cells. Reports from different laboratories have indicated that the relevant antigenic epitopes in such reactions might include hapten-conjugated, MHC class II-associated peptides. This study for the first time directly demonstrates that hapten-peptides account for the majority of determinants recognized by trinitrophenyl (TNP)-specific CD4⁺ T lymphocytes. The sequences of those TNP carrier peptides do not have to be related to mouse proteins. Thus, we show that TNP-modified peptides derived from mouse IgG, pigeon cytochrome c or staphylococcal nuclease known to bind to I-A^b or from λ represser with specificity to I-A^d as well as TNP-proteins such as bovine serum albumin, ovalbumin or keyhole limpet hemocyanin all create class II-restricted hapten determinants for a number of TNP-specific T cell clones and hybridomas. All of these cells were induced with cells modified by trinitrobenzene sulfonic acid (TNBS). In addition, we present arguments indicating that individual TNP-specific helper T cells may cross-react with different TNP-peptides bound to identical class II molecules. Chemical treatment of antigen-presenting cells with TNCB or TNBS may thus result in a limited number of particularly repetitive immunodominant hapten epitopes. Immunodominant epitopes were also indicated by an overrepresentation of the TCR elements Vβ2 and Vα10 in I-A^b/TNP-specific T cells. Most importantly, however, we demonstrate that TNP attached to lysine 97 in the staphylococcal nuclease peptide 93–105 (i.e. a clearly “non-self” sequence) is able to prime mice for subsequent elicitation of contact sensitivity by TNCB in the absence of foreign protein. We take this to indicate that those TNP-peptide determinants defined by us as immuno-dominant are responsible for the induction of contact sensitivity to haptens.     

Epidemiologische Ansätze zur Klärung wichtiger Forschungsfragen zu COVID-19 – eine Übersicht

Bundesgesundheitsblatt - Gesundheitsforschung - Gesundheitsschutz - Die Epidemiologie als wissenschaftliche Disziplin ist prädestiniert dafür, Kernfragen der COVID-19-Pandemie zu...     

Pedobarographic outcome after subtalar screw arthroereisis in flexible juvenile flatfoot

BackgroundFlatfoot is a frequent skeletal deformity in childhood that can be minimally invasively treated by arthroereisis. Question: Does the motion of juvenile flexible flatfoot normalize after arthroereisis?MethodPedographic measurements were obtained from 39 patients preoperatively, six months postoperatively and compared to a healthy group. The footprints were divided into 8 areas. The selected parameters were: contact area and force-time-integral.ResultsAfter surgery, a load shift from the medial to the lateral areas was detected under the midfoot and forefoot. The force-time-integral under the hallux normalized. However, under the lateral midfoot, the postoperative force-time-integral was significantly higher than in the control group.SignificanceThe study shows that arthroereisis is able to correct the medially displaced load distribution of juvenile flexible flatfoot. However, further investigations are required to find out if the higher punctual loading under the lateral midfoot may cause problems in the long term.     

Protroca: A Noninterventional Study on Prophylactic Lipegfilgrastim against Chemotherapy-Induced Neutropenia in Nonselected Breast Cancer Patients

BackgroundProtroca evaluated the efficacy and safety of primary and secondary prophylaxis of neutropenia with lipegfilgrastim (Lonquex®) in breast cancer patients receiving neoadjuvant or adjuvant chemotherapy (CT).Patients and MethodsOf the 255 patients enrolled, 248 patients were evaluable for the intent-to-treat (ITT) and 194 patients for the per-protocol set. Primary and secondary end points after lipegfilgrastim treatment were assessed.ResultsNine patients of the ITT set receiving lipegfilgrastim as primary prophylaxis (n = 222) had febrile neutropenia of grade 3–4 (5 patients) or infection of grade 3–4 (4 patients); 1/26 of those receiving secondary prophylaxis had an event. Dose reductions were performed in 9.5% of the patients. Postponement of cancer CT cycles for >3 days occurred in <15% of patients; 10.8% (92/851 AEs) and 8% (2/25 SAEs) of documented adverse events and serious adverse events, respectively, were related to lipegfilgrastim.ConclusionsApplication of lipegfilgrastim was effective as primary and secondary prophylaxis in the prevention of CT-induced neutropenia in breast cancer.     

Short-term and long-term results after early treatment with the castillo morales stimulating plate

The present study is divided into 2 parts: The first explains the concept of stimulating plate therapy at the Muenster Clinic and the second presents a longitudinal study of mouth and tongue posture in trisomy 21 children treated with this concept.47 children underwent a pre-treatment examination (Rec1), and 38 attended a first follow-up examination (Rec2) after an average of 4 months of stimulating plate treatment. The second follow-up examination (Rec3) was on average 53 months after the end of treatment and covered 18 patients. Data acquisition was based on standardized parent questionnaires and standardized clinical examination records. Rec2 showed in part a highly significant improvement in orofacial appearance. These results were confirmed by the parent interview, according to which the mouth and tongue posture improved in 76.3% of the children during the time they were wearing the plate. Even when the plate was not in place, the result remained stable in 65.8% of the patients. Up to Rec3, further improvement was recorded in some results. The symptoms: mouth mostly wide open and tongue mostly protruding way over lips were reduced. The number of children whose tongue was mostly in the oral cavity increased correspondingly.At Rec3, the parents also rated the mouth posture in 88.9% and the tongue posture in 77.7% of the children as stable or further improved.It can be concluded from the results of this study that stimulating plate treatment in combination with physiotherapy provides a long-term improvement in the mouth and tongue posture of children with Down's syndrome.     

Optimization and Standardization of the “Comet Assay” for Analyzing the Repair of DNA Damage in Cells

BACKGROUND: The "comet assay" has become an interesting and a very useful tool for the analysis of the induction and amount of DNA damage in single cells thus offering the opportunity to measure the effectiveness of DNA repair. On the basis of the Ostling and Johanson protocol we have developed a modified method with increased sensitivity and high reproducibility. MATERIAL AND METHODS: Human tumor cells or isolated human peripheral blood lymphocytes were analyzed in the experiments. The amount of DNA damage and the effectiveness of DNA repair was measured after X-irradiation using the "comet assay" technique. RESULTS: In this presentation the influences of different methodological factors like agarose concentration, buffer pH, electrophoresis time, electric field strength on the applicability of the "comet assay" are described in detail and optimum conditions for "comet assay" experiments have been evaluated. Additionally the authors will show a comparison of different fluorescent DNA dyes pointing out their advantages or disadvantages for "comet" analysis. The usefulness of this technique and its capabilities are exemplified by showing DNA repair kinetics of human lymphocytes of different healthy or radiosensitive donors after in-vitro irradiation with 2 Gy X-rays. CONCLUSIONS: This paper presents data on the optimization and standardization of the original "comet assay" leading to an extremely fast and practicable protocol in the field of single cell gel electrophoresis. After irradiation with 0.1 Gy an increase in the amount of DNA damage can be measured with high statistical significance and the DNA repair capacity of individual cells after X-ray doses of 2 Gy can be analyzed with high reproducibility. The results comparing DNA repair capacities of different donors point out that the "comet assay" may have the potential for the estimation of individual radiosensitivity.     

Penetration of ciprofloxacin, norfloxacin and ofloxacin into the aqueous humor using different topical application modes

 · Background: A prospective study was undertaken to determine the transcorneal penetration of three topically applied fluoroquinolones into aqueous humor. · Methods: Cataract patients (n=224) preoperatively received topically applied gyrase inhibitors (0.3% ciprofloxacin, 0.3% norfloxacin, 0.3% ofloxacin) in two different application modes. In application mode I, patients received on the day before operation 3×1 eye drop at 2-h intervals, and on the day of operation 3 drops at 1-h intervals. In application mode II, patients received 9 drops at 15-min intervals on the day of operation only. Just before cataract surgery 50–100 μl aqueous humor was aspirated and stored at –80 °C. The HPLC method was used for measuring the concentration. · Results: The highest concentrations of all tested antibiotics were measured after the mode of application in which one drop was given every 15 min between 06:00 and 08:00 hours before operation. In this mode, ciprofloxacin achieved a mean aqueous level of 379.8±327.8 μg/l (range 33–1388 μg/l), norfloxacin 182.1±118.1 μg/l (range 38–480 μg/l) and ofloxacin 563.9±372.1 μg/l (range 64–1455 μg/l). These mean concentration are all above the MIC₉₀ of gram-negative bacteria like Proteus mirabilis and Escherichia coli. In some cases the concentrations of ciprofloxacin and ofloxacin, but never norfloxacin, reached therapeutic values above the MIC₉₀ of Staphylococcus aureus and Staphylococcus epidermidis · Conclusions: The mean concentration value of 0.3% ciprofloxacin and of 0.3% ofloxacin in the aqueous humor reached the MIC₉₀ values of the frequently occurring gram-positive and gram-negative bacteria. Of the currently available topical fluoroquinolones, ofloxacin achieved the highest aqueous humor concentration. Considering the higher antimicrobial activity of ciprofloxacin, both ciprofloxacin and ofloxacin may be useful ophthalmic agents in antibacterial management, but they are not efficient against Streptococcus pneumoniae and Pseudomonas aeruginosa. Received: 8 September 1997 Revised version received: 19 March 1998 Accepted: 8 April 1998     

Intraocular silicone lenses in silicone oil: an experimental study

Background: To evaluate a potential effect of silicone oil on flexible silicone intraocular lenses, four lenses (STAAR AA-4203) were stored in silicone oil under sterile conditions for periods between 1 month and 3 years. Method: The edge and surface of the lenses were examined by scanning electron micrography and the findings compared with a lens of the same model which had been stored in Ringer's solution for 2 years. Results: After 1 year of silicone oil exposure, droplets of different sizes adherent to the surface of the lens were found. These changes proceeded to a wave-like appearance of the surface after 2 and 3 years of storage, so that a continuous layer of silicone oil polymers is probably covering the intraocular lens. Conclusion: Optical interference has to be considered a possibility if it turns out that the droplets cannot be removed during silicone oil evacuation. Consequently silicone intraocular lenses without hydrophilic preparation of the surface should not be implanted in eyes undergoing combined anterior and posterior segment surgery with silicone oil tamponade or in eyes with high risk for vitreoretinal complications.The authors state that they have no proprietary interest in the marketing of the products mentioned herein or competing products