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.     

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.     

Phenotypic dynamics of tumor progression in human malignant melanoma

The phenotypic changes in human melanoma cells during the course of tumor progression were studied with monoclonal antibodies (MAbs) against the melanoma-associated antigens (MAA) M.2.2.4, H.2.8.10, K.1.2, A.1.43, and A.10.33, and HLA-(A,B,C and D). Cryostat sections of 172 primary melanomas of the skin, 157 melanoma metastases and 56 nevi were investigated with an indirect immunoperoxidase method. Phenotypic heterogeneity was observed within lesions at all stages, and also within different tumors of the same patients. Despite this heterogeneity, principles of antigen expression were found. From the reaction pattern of MAbs, the following classifications of antigens were derived: "constitutive" markers of nevomelanocytic cells (M.2.2.4 and H.2.8.10) were found expressed over a wide range of local and systemic tumors. One MAA, K.1.2 (Suter et al., 1985), that declines with progression of melanoma, was classified as an "early" antigen, whereas MAA that appear in primary melanoma in proportion to invasiveness, and which are expressed in metastases of lymph nodes and visceral organs (A.1.43, and A.10.33), were classified as "late" markers of tumor progression. HLA-antigens were classified as "intermediate" markers, HLA-A,B,C, as an "early-intermediate", and HLA-DR as a "late-intermediate" marker. The occurrence of class II HLA, A.1.43-, and A.10.33-positive tumor cells in primary melanoma indicates a high metastatic potential of tumors, independent of tumor thickness. The data show that local and systemic progression of melanoma is associated with qualitative changes in tumor cells which can be recognized by MAbs.     

Pharmacological characterization of A1 adenosine receptors in isolated rat ventricular myocytes

Summary The aim of the present study was the characterization of adenosine receptors in isolated rat ventricular myocytes. The CAMP-levels of rat ventricular myocytes in the presence of 1 mol/l isoprenaline were reduced by up to 48% by adenosine analogues; the rank order of potency was: R-N⁶-phenylisopropyladenosine (IC₅₀ 60 nmol/1), 5-N-ethylcarboxamidoadenosine (IC₅₀ 360 nmol/l) and S-N⁶-phenylisopropyladenosine (IC₅₀ 16 ol/l). The adenosine receptor antagonist XAC (xanthine amine congener) antagonized the effect of R-N⁶-phenylisopropyladenosine in a concentration-dependent manner with a K_i-value of 20 nmol/l. The A₁ receptor-selective radioligand R-N⁶-¹²⁵I-p-hydroxyphenylisopropyladenosine bound to membranes prepared from rat ventricular myocytes in a saturable manner with a B _max of 17.7 fmol/mg protein and a K _D-value of 1.1 nmol/l. Adenosine analogues competed for the binding with the same rank order of potency as for the inhibition of the isoprenaline-induced cAMP-increase. GTP inhibited radioligand binding with an IC₅₀-value of 73 ol/l. These results suggest the presence of A₁ adenosine receptors on rat ventricular myocytes, which mediate an inhibition of adenylate cyclase. The receptors may be responsible for the effects of adenosine and its analogues on the heart.Abbreviations ¹²⁵I-HPIA R-N⁶-¹²⁵I-p-hydroxyphenylisopropyladenosine - PIA N⁶-phenylisopropyladenosine - NECA 5-N-ethyl-carboxamidoadenosine - XAC 8-4-[([(2-aminoethyl)aminocarbonyl]methyl)oxy]phenyl-1,3-dipropylxanthine (xanthine amine congener) - Ro 20-1724 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone - ScAMPTME 2-O-monosuccinyladenosine-3,5-cyclic monophosphate tyrosyl methyl ester - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - GTP guanosine-5-tri-phosphate Send offprint requests to D. Martens     

Ectopic Calcification and Hypophosphatemic Rickets: Natural History of ENPP1 and ABCC6 Deficiencies

Generalized arterial calcification of infancy (GACI) is a rare disorder caused by ENPP1 or ABCC6 variants. GACI is characterized by low pyrophosphate, arterial calcification, and high mortality during the first year of life, but the natural course and possible differences between the causative genes remain unknown. In all, 247 individual records for patients with GACI (from birth to 58.3 years of age) across 19 countries were reviewed. Overall mortality was 54.7% (13.4% in utero or stillborn), with a 50.4% probability of death before the age of 6 months (critical period). Contrary to previous publications, we found that bisphosphonate treatment had no survival benefit based on a start-time matched analysis and inconclusive results when initiated within 2 weeks of birth. Despite a similar prevalence of GACI phenotypes between ENPP1 and ABCC6 deficiencies, including arterial calcification (77.2% and 89.5%, respectively), organ calcification (65.8% and 84.2%, respectively), and cardiovascular complications (58.4% and 78.9%, respectively), mortality was higher for ENPP1 versus ABCC6 variants (40.5% versus 10.5%, respectively; p = 0.0157). Higher prevalence of rickets was reported in 70.8% of surviving affected individuals with ENPP1 compared with that of ABCC6 (11.8%; p = 0.0001). Eleven affected individuals presenting with rickets and without a GACI diagnosis, termed autosomal recessive hypophosphatemic rickets type 2 (ARHR2), all had confirmed ENPP1 variants. Approximately 70% of these patients demonstrated evidence of ectopic calcification or complications similar to those seen in individuals with GACI, which shows that ARHR2 is not a distinct condition from GACI but represents part of the spectrum of ENPP1 deficiency. Overall, this study identified an early mortality risk in GACI patients despite attempts to treat with bisphosphonates, high prevalence of rickets almost exclusive to ENPP1 deficiency, and a spectrum of heterogenous calcification and multiple organ complications with both ENPP1 and ABCC6 variants, which suggests an overlapping pathology. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR). This article has been contributed to by US Government employees and their work is in the public domain in the USA.     

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.     

Bases anatomiques de la mémoire

Résumé Les données cliniques et anatomo-pathologiques ont permis de suggérer le rôle du circuit hippocampo-mamillo-thalamique dans certains processus mnésiques. Récemment des critiques ont été apportées mettant en cause le rôle même de ce circuit et impliquant d'autres formations: hile du lobe temporal; noyau médio-dorsal du thalamus, amygdale. L'étude des relations anatomiques précises du circuit hippocampo-mamillo-thalamique permet de montrer l'importance dans le circuit lui-même de certaines formations comme le subiculum et l'aire entorhinale. Par ailleurs, ce circuit s'intègre totalement au sein du système limbique au sens large, tel que l'a défini Nauta (1961) et par là à des structures comme l'amygdale, le noyau médio-dorsal du thalamus, le cortex orbito-frontal, le septum et la réticulée mésencéphalique.Mais au sein même de ce système limbique, il semble exister une spécificité de relation des éléments en rapport avec le circuit hippocampo-mamillo-thalamique.Enfin, le circuit HMT ne peut pas se concevoir comme restreint au système limbique. Il est en relation étroite avec l'ensemble du cortex et particulièrement avec le cortex frontal, ce que rappellent les désordres mnésiques entraînés par les lésions de cette région.Le circuit HMT n'est donc pas la mémoire mais reste un modèle et un point de départ utile sinon indispensable dans son étude.