Enzymatic digestion of adult human articular cartilage yields a small fraction of the total available cells

We investigated whether different protocols for the digestion of adult human articular cartilage influence the cell yield and capacity to attach and proliferate in culture dishes. Chondrocyte yields were expressed as a percentage of the total number of cells in the tissue, determined both histologically (using the dissector method) and biochemically (measuring the DNA content of tissue digests). Human cartilage specimens (n = 79) were digested using different protocols based on combinations of collagenase II (CGN), trypsin/EDTA, hyaluronidase, and tosyllysylchloromethane (TLCM). Yields of viable chondrocytes were the highest within a specific range of CGN concentrations and digestion times, but always < 22% of the total available cells. The combination of CGN with trypsin/EDTA or TLCM accelerated the digestion process but did not significantly increase cell yields. The percentage of viable cells that attached to culture dishes ranged 75-85% (< 19% of the total) and was reduced by TLCM. Doubling times of attached cells were comparable in all experimental groups. Our results indicate that chondrocyte yields and capacity to attach and proliferate are not highly sensitive to the specific isolation protocol used. However, typically used cartilage digestion protocols yield only a small fraction of the total available cells, possibly introducing an uncontrolled selection of certain chondrocyte subpopulations.     

Relations between chronic stimulation-induced changes in contractile properties and the Ca2+-sequestering system of rat and rabbit fast-twitch muscles

This study compares changes in contractile properties, Parvalbumin content, and Ca²⁺-uptake by the sarcoplasmic reticulum (SR) of low-frequency stimulated rat and rabbit tibialis anterior (TA) muscles. Time to peak tension increased 1.8-fold in 35-day stimulated rabbit TA, while no change occurred in rat TA. Isometric twitch tension increased 2-fold in rabbit TA, but was unaltered in rat TA. Parvalbumin (PA) content was more than 90% reduced in rabbit TA, but only 60% in rat TA after 35 days. At this time, PA content of the stimulated rat TA was still higher than that of normal rabbit TA. Taking into account the suggested role of PA as a cytosolic Ca²⁺ buffer, its decrease could lead to an impaired free Ca²⁺-decay with a prolonged active state and a higher tension output during a single twitch. This would explain why chronic stimulation led to an increase in isometric twitch tension in rabbit TA, but not in rat TA. The 1.6-fold rise in half-relaxation time of 35-day stimulated rat and rabbit TA most likely resulted from a 50% reduced Ca²⁺-uptake by the SR, due to a still unknown modification of the Ca²⁺-transport ATPase.     

(Co)polymers of L-lactide, 1. Synthesis,thermal properties and hydrolytic degradation

Copolymers of L -lactide with D -lactide

1 According to IUPAC the name dilactide is preferred to lactide.

, glycolide

2 IUPAC name: diglycolide.

, ε-caprolactone and trimethylene carbonate, and networks with spiro-bis-dimethylene-carbonate (2,4,7,9-tetraoxaspiro[5.5]undecane-3,8-dione) were prepared in bulk at standardized polymerization conditions. The properties of the nascent copolymers were evaluated with respect to the nature of the comonomer. Copolymerization with comonomers entailing low glass transition temperature simultaneously reduces the crystallinity. 300 MHz ¹H nuclear magnetic resonance is shown to be a useful technique for the determination of the average monomer sequence lengths in ε-caprolactone and trimethylene carbonate copolymers. The presence of crystallizable L -lactide sequences, due to differences in monomer reactivity, has a large effect on the thermal properties of the copolymer as well as on the long-term degradation characteristics.     

Differences in maintenance of CD8+ and CD4+ bacteria-specific effector-memory T cell populations

Our knowledge about the kinetics and dynamics of complex pathogen-specific CD8(+) T cell responses and the in vivo development of CD8(+) memory T cells has increased substantially over the past years; in comparison, relatively little is known about the CD4(+) T cell compartment. We monitored and directly compared the phenotypical changes of pathogen (Listeria monocytogenes)-specific CD8(+) and CD4(+) T cell responses under conditions leading to effective and long-lasting protective immunity. We found that the general kinetics of bacteria-specific CD8(+) and CD4(+) T cells during the effector and post-effector phases are synchronized. However, later during the memory phase, CD8(+) and CD4(+) T cell populations differ substantially. Whereas CD8(+) memory T cell populations with immediate effector function are readily detectable in lymphoid and non-lymphoid tissues and remain remarkably stable in size, antigen-specific CD4(+) effector-memory T cells decline continuously in frequency over time. These findings have important implications for the better understanding of the in vivo development of protective immunity towards intracellular pathogens.     

Long-chain acyl-CoA esters and phosphatidylinositol phosphates modulate ATP inhibition of Katp channels by the same mechanism

Phosphatidylinositol phosphates (PIPs, e.g. PIP₂) and long-chain acyl-CoA esters (e.g. oleoyl-CoA) are potent activators of K _atp channels that are thought to link K _atp channel activity to the cellular metabolism of PIPs and fatty acids. Here we show that the two types of lipid act by the same mechanism: oleoyl-CoA potently reduced the ATP sensitivity of cardiac (Kir6.2/SUR2A) and pancreatic (Kir6.2/SUR1) K _atp channels in a way very similar to PIP₂. Mutations (R54Q, R176A) in the C- and N-terminus of Kir6.2 that greatly reduced the PIP₂ modulation of ATP sensitivity likewise reduced the modulation by oleoyl-CoA, indicating that the two lipids interact with the same site. Polyvalent cations reduced the effect of oleoyl-CoA and PIP₂ on the ATP sensitivity with similar potency suggesting that electrostatic interactions are of similar importance. However, experiments with differently charged inhibitory adenosine phosphates (ATP^4-, ADP^3- and 2'(3')- O -(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP^2-)) and diadenosine tetraphosphate (Ap₄A^5-) ruled out a mechanism where oleoyl-CoA or PIP₂ attenuate ATP inhibition by reducing ATP binding through electrostatic repulsion. Surprisingly, CoA (the head group of oleoyl-CoA) did not activate but inhibited K _atp channels (IC₅₀= 265 ± 33 μM). We provide evidence that CoA and diadenosine polyphosphates (e.g. Ap₄A) are ligands of the inhibitory ATP-binding site on Kir6.2.     

Oxidative stress and sleep apnoea in clinically healthy infants in the first year of life

Summary Question of the study   Respiratory instability as well as tissue damage by free radicals (oxidative stress) have been hypothesized to play a role in cases of sudden and unexpected infant death in the first year of life. The ratio of the oxidized/reduced form of redox compounds in the circulation could be used as a marker of oxidative stress. Therefore, the sleep apnoea rate and redox status of coenzyme Q10 (CoQ10) (percentage of the oxidized form in total CoQ10) were measured in a population of clinically healthy infants in their first year of life in order to study whether a physiological parameter of respiratory instability is related to a biochemical parameter of oxidative stress. Patients and methods   Between May and December 1999, 323 infants in the first year of life were referred to a paediatric sleep laboratory. Sleep apnoea rate, periodic breathing and parameters of oxygenation (SaO₂ and TcPO₂) were calculated based on polysomnographic recordings. The CoQ10 redox status was calculated based on high-pressure liquid chromatographic (HPLC) analysis. Results   Statistical analysis showed an age-dependent decrease in apnoea rate ( r = – 0.38) and CoQ10 redox status ( r = – 0.40). An increased CoQ10 redox status (median: 16.6 %; range: 7.3 – 29.7 %) was found in infants with high apnoea rates above the 90^th percentile of a reference group in comparison with infants with apnoea rates below the 90^th percentile of a reference group (median: 10.4 %; range: 5.1 – 20.4 %; P = 0.031). Conclusions   These findings may indicate that high apnoea rates are accompanied by increased formation of free radicals in clinically healthy infants in the first year of life.     

Emergence of translocation t(9;11)-positive leukemia during treatment of childhood acute lymphoblastic leukemia

Therapy-related acute myeloid leukemia (t-AML) characterized by the t(9;11)(p22;q23) translocation is one of the most frequent secondary malignancies. The timing of the initiation of translocation and of development of the malignant t(9;11) clone during chemotherapy is presently unknown. In the present study, we backtracked bone marrow samples from three children during treatment for acute lymphoblastic leukemia (ALL). Two patients developed a t(9;11)-positive t-AML 19 and 30 months after therapy start, whereas the third patient, diagnosed with a rare t(9;11)-positive ALL, suffered from an ALL relapse 23 months after initial diagnosis. The genomic MLL-MLLT3 (MLL-AF9) fusion site was amplified by a multiplex, nested long-range PCR and used as a clonal marker for quantification of the MLL-MLLT3-positive cells during chemotherapy. The t(9;11)-positive clone was detectable 13 and 18 months after therapy start in both t-AML cases, which was 6-12 months before clinical diagnosis of the secondary malignancy. In the t(9;11)-positive ALL patient, the identical leukemic clone reoccurred during maintenance therapy after a short molecular remission, 8 months before clinically overt ALL relapse. The time course and characteristics of the genomic breakpoints in the present t-AML cases support the hypothesis of translocation formation as a result of defective breakage repair after topoisomerase II cleavage.     

Heat shock protein 60 from Chlamydia pneumoniae elicits an unusual set of inflammatory responses via Toll-like receptor 2 and 4 in vivo

Heat shock protein 60 (HSP60) from Chlamydia pneumoniae was described to trigger in vitro inflammatory and cytokine responses including TNF and IL-12p40. Although it can be found in atherosclerotic plaques of patients, the stimulatory potential of chlamydial and other HSP60 in vivo is unclear. We now report that chlamydial HSP60 fails to induce TNF expression in vivo, and significant serum levels of IL-12p40 are only found upon intraperitoneal injection of high doses of HSP60 or after intravenous application. Upon purification of chlamydial HSP60 with polymyxin B-agarose columns, its ability to induce TNF secretion in vitro is much reduced. However, purified chlamydial HSP60 causes increased serum levels of the CXC chemokines KC and MIP2 in vivo, as well as a strong accumulation of polymorphonuclear neutrophils (PMN) in the peritoneal cavity upon intraperitoneal challenge. With respect to PMN accumulation, chlamydial HSP60 is more potent than endotoxin or the CpG oligonucleotide 1668. The responses observed are completely abolished in Toll-like receptor (TLR)2/4-double-deficient mice, while single-deficient mice respond almost normally. Furthermore, KC induction and PMN accumulation are largely dependent on MyD88. In conclusion, HSP60 from C. pneumoniae triggers inflammatory responses in vivo that differ from responses induced by endotoxin or CpG oligonucleotides and are dependent on TLR2 and 4.