In vitro assessment of the effects of platelet-rich plasma on the extracellular matrix metabolism of porcine intervertebral disc cells.
To determine whether platelet-rich plasma is effective in ...stimulating cell proliferation and extracellular matrix metabolism by porcine disc cells cultured in alginate beads.
Platelet-rich plasma is used to accelerate wound healing and tissue regeneration. Activated platelets release multiple growth factors that regulate cell proliferation, differentiation, and morphogenesis. Individual growth factors present in platelet-rich plasma have been demonstrated to affect the metabolism of intervertebral disc cells.
Platelet-poor and platelet-rich plasma was isolated from fresh porcine blood using a commercially available platelet concentration system. After preculture for 7 days and serum starvation for 24 hours, the beads containing nucleus pulposus and anulus fibrosus cells were then cultured for another 72 hours in serum-free medium, 10% fetal bovine serum, 10% platelet-poor plasma, or 10% platelet-rich plasma. The synthesis of proteoglycans and collagen, the accumulation of proteoglycans, and the DNA content were biochemically assessed.
Platelet-rich plasma had a mild stimulatory effect on cell proliferation of intervertebral disc cells. Platelet-rich plasma treatment significantly upregulated proteoglycan and collagen synthesis and proteoglycan accumulation when compared with platelet-poor plasma.
Platelet-rich plasma was effective in stimulating cell proliferation and extracellular matrix metabolism. The response to platelet-rich plasma was greater in the case of anulus fibrosus cells than of nucleuspulposus cells. The local administration of platelet-rich plasma might stimulate intervertebral disc repair. In addition, given the risks of using animal serum for tissue engineering, autologous blood may gain favor as a source of growth factors and serum supplements needed for stimulating cells to engineer intervertebral disc tissues.
The insect allatostatins obtained their names because they block the biosynthesis of juvenile hormone (a terpenoid) in the corpora allata (two endocrine organs near the insect brain). Chemically, the ...allatostatins can be subdivided into three different peptide groups: the A-type allatostatins, first discovered in cockroaches, which have the C-terminal sequence Y/FXFGLamide in common; the B-type allatostatins, first discovered in crickets, which all have the C-terminal sequence W(X)6Wamide; and the C-type allatostatins, first discovered in the moth Manduca sexta, which have an unrelated and nonamidated C terminus. We have previously reported the structure of an A-type allatostatin preprohormone from the fruitfly Drosophila melanogaster. Here we describe the molecular cloning of a B-type prepro-allatostatin from Drosophila (DAP-B). DAP-B is 211 amino acid residues long and contains one copy each of the following putative allatostatins: AWQSLQSSWamide (drostatin-B1), AWKSMNVAWamide (drostatin-B2), <EAQGWNKFRGAWamide (drostatin-B3), EPTWNNLKGMWamide (drostatin-B4), and DQWQKLHGGWamide (drostatin-B5). All five drostatins are novel peptide structures. The DAP-B gene has one intron and two exons and is located at position 74B1 on the left arm of the third chromosome. The gene is expressed in all developmental stages, but weakly in embryos and strongly in larvae. In situ hybridizations of larvae showed that neurons in the brain and abdominal ganglia and endocrine cells in the gut expressed DAP-B. This is the first published report of a B-type allatostatin preprohormone in insects, and the first paper describing the presence of B-type allatostatins in a representative of the insect order Diptera (flies).
The identification and quantitation of the metabolites of Statil™ in rat bile and urine were investigated by
1H- and
19F-NMR spectroscopy and liquid scintillation counting. Male Wistar rats received ...a single oral dose of 100 mg/kg of radiolabelled Statil™. Statil™ is known to produce glucuronide conjugates which are predominantly excreted into the bile in male rats. The complex multiphasic matrix of bile has been shown to make identification of the resonances by
1H-NMR spectroscopy very difficult as Statil™ appeared to be micellar bound giving rise to very broad signals. This not only impaired unambiguous signal characterisation but also quantification. The partial separation by SPEC-
1H-NMR spectroscopy enabled the disruption of the micellar matrices and hence enabled the identification of Statil™ predominantly as aglycone, and to a lesser extent as glucuronide conjugate. In addition, minor acyl migration products of Statil™ glucuronide could also be detected as they were separated during the SPEC-process.
19F-NMR spectroscopic measurements on whole bile confirmed their presence as a number of overlapped signals could be observed. The selectivity, simplicity and signal dispersion characteristic of
19F-NMR spectroscopy also enabled the calculation of dose related recoveries of Statil™ related material in the bile and urine samples without the need for a radiolabel. The aim of this work was to investigate the usefulness and limitations of NMR spectroscopy of intact bile and urine as a means of quantifying levels of drug metabolites. The results obtained from NMR spectroscopy are compared with those obtained using scintillation techniques. Scintillation counting yields unequivocal quantification results, provided the label is preserved in metabolites as has been the case here. In general, quantification by
19F-NMR results similar to those obtained by scintillation counting (in agreement within about 20%). However, discrepancies have been observed with very small and broad
19F-NMR signals in bile. Nevertheless,
19F-NMR spectroscopy of bile is a rapid and facile method for assessing metabolite levels of fluorinated drugs.
The application of HPLC-MS combined with principal components analysis (PCA) to the metabonomic analysis of mouse urine is demonstrated. Urine samples from three strains of mouse were analysed by ...gradient HPLC-MS combined with positive and negative electrospray time-of-flight mass spectrometry. Analysis of the resulting data using PCA enabled the samples to be discriminated between on the basis of gender, strain and diurnal variation. These preliminary results suggest that HPLC-MS-based approaches may have a useful role in metabonomic analysis that complements existing approaches.
Computer-assisted sperm analysis of fresh and frozen-thawed bovine sperm requires proper handling and preparation, and the type of slide used in the assessment is critical if the resultant data are ...to be useful quality control measurements. In the present study, 4 different slide viewing chambers, a Makler chamber, a clean slide-coverslip, or a 2- or 4-cell chamber Leja slide, were compared with assess their utility in providing reliable measurements of sperm motility variables. A Hamilton-Thorne IVOS Computer-Assisted Semen Analyzer (CASA) was the instrument used to determine sperm measurements utilizing the 4 different chambers. Fifty-eight different freeze batches of bovine semen that had been collected from 47 bulls at 7 sites that sex-sort sperm using Sexing Technologies sorting criteria were incorporated into the trial. Neither the percentage of motile sperm nor the percentage of progressively motile sperm differed for the Makler chamber vs. slide-coverslip comparisons. Similarly, total and progressively motile sperm did not differ between the 2- and 4-cell chambered Leja slides. However, total and progressive motility of sperm determined with the Makler chamber and slide-coverslip were greater (P < 0.0001) than motilities recorded by the 2- or 4-cell chambered Leja slides. Based on the results, the type of viewing chamber can affect the range of sperm motility values when CASA is used for quality control evaluations of thawed, cryopreserved sex-sorted sperm samples. PUBLICATION ABSTRACT
In the phase II multicohort CheckMate 142 study, nivolumab plus low-dose (1 mg/kg) ipilimumab provided robust and durable clinical benefit with a manageable safety profile in previously treated ...patients with microsatellite instability-high/mismatch repair-deficient (MSI-H/dMMR) metastatic colorectal cancer (mCRC) at 13.4- and 25.4-month median follow-up (Overman MJ, Lonardi S, Wong KYM et al. Durable clinical benefit with nivolumab plus ipilimumab in DNA mismatch repair-deficient/microsatellite instability-high metastatic colorectal cancer. J Clin Oncol. 2018;36:773-779. Overman MJ, Lonardi S, Wong KYM, et al. Nivolumab plus low-dose ipilimumab in previously treated patients with microsatellite instability-high/mismatch repair deficient metastatic colorectal cancer: long-term follow-up. J Clin Oncol. 2019;37:635). Here, we present results from the 4-year follow-up of these patients.
Patients received nivolumab (3 mg/kg) plus low-dose (1 mg/kg) ipilimumab every 3 weeks (four doses) followed by nivolumab (3 mg/kg) every 2 weeks until disease progression. Primary endpoint was investigator-assessed objective response rate (ORR; as per RECIST version 1.1).
A total of 119 patients were treated; 76% had ≥2 prior lines of therapy. Median follow-up was 50.9 months (range 46.9-62.7 months). Median duration of therapy was 24.9 months 95% confidence interval (CI) 15.8-33.2 months. Investigator-assessed ORR increased from 55% (95% CI 45% to 64%) at 13.4 months to 65% (95% CI 55% to 73%) at 50.9 months with a disease control rate of 81% (95% CI 72% to 87%). The complete response rate increased from 3% at 13.4 months to 13% at 50.9 months. Partial responses were observed in 52% of patients; 21% had stable disease, and 12% had progressive disease. Median time to response was 2.8 months (range 1.1-37.1 months), and median duration of response was not reached (range 1.4+ to 58.0+ months). At data cut-off, 37 (48%) patients had ongoing responses. Median progression-free survival was not reached 95% CI 38.4 months-not estimable (NE), and median overall survival was not reached (95% CI NE). Grade 3-4 treatment-related adverse events (TRAEs) were observed in 32% of patients; 13% of patients had any-grade TRAEs leading to discontinuation.
The results confirm long-term benefit of nivolumab plus low-dose ipilimumab for previously treated patients with MSI-H/dMMR mCRC. The safety profile was manageable with no new safety signals.
•In previously treated MSI-H/dMMR mCRC, nivolumab plus ipilimumab showed durable benefit with 4 years of follow-up.•ORR was 65%. The 48-month rates of progression-free survival and overall survival were 53% and 71%, respectively.•Significant and clinically meaningful improvements in quality of life were largely maintained with continuous treatment.•No new safety signals were identified, with a low rate (13% of patients) of any-grade TRAEs leading to discontinuation.•These results show the long-term benefit of nivolumab plus low-dose ipilimumab in previously treated MSI-H/dMMR mCRC.
This study examined DNA damage and postthaw motility of white-tailed deer sperm (n = 28) before and after sex selection and conventional sorting using MoFlo XDP SX flow cytometry. Semen samples from ...the same individuals were treated in 4 different ways: 1) chilled-extended sperm samples (without glycerol); 2) cryopreserved conventional samples, samples directly cryopreserved after the addition of extenders; 3) cryopreserved conventionally sorted samples, sorted samples to remove the dead sperm subpopulation; and 4) cryopreserved sex-sorted samples; sorted samples to remove the dead sperm subpopulation and separation of X- and Y-chromosome-bearing sperm. In all the cases (n = 6), conventional samples showed decreased postthaw motilities (43 ± 26%) when compared with X-sorted samples (59 ± 20%; P < 0.05) and Y-sorted samples (54 ± 20%; P > 0.05). The DNA fragmentation baseline was <5% for frozen-thawed conventional samples, but even less after sex sorting and conventional sorting: 2.4 and 1.7%, respectively. On the other hand, conventional samples showed greater (P < 0.05) DNA fragmentation than the sex-sorted sperm (n = 6) at 96 h (average of 4.8 ± 4.5% and 5.3 ± 4%, respectively). Conventionally sorted samples (n = 8) did not have greater (P > 0.05) DNA fragmentation when compared with the sex-sorted samples. Fragmentation of DNA on X-chromosome and Y-chromosome-bearing sorted sperm were not significantly different (n = 10, P > 0.05) after 96 h (2.6 ± 3.6% and 2.2 ± 0.5%, respectively). Future research should be implemented for examining the fertilizing potential of sex-sorted white-tailed deer sperm (e.g., AI fertility trials). PUBLICATION ABSTRACT
The first dedicated local electrode atom probes (LEAP a
trademark of Imago Scientific Instruments Corporation) have been
built and tested as commercial prototypes. Several key performance
parameters ...have been markedly improved relative to conventional
three-dimensional atom probe (3DAP) designs. The Imago LEAP can operate
at a sustained data collection rate of 1 million atoms/minute. This
is some 600 times faster than the next fastest atom probe and large
images can be collected in less than 1 h that otherwise would take many
days. The field of view of the Imago LEAP is about 40 times larger than
conventional 3DAPs. This makes it possible to analyze regions that are
about 100 nm diameter by 100 nm deep containing on the order of 50 to
100 million atoms with this instrument. Several example applications
that illustrate the advantages of the LEAP for materials analysis are
presented.
Immune checkpoint inhibitors (CPIs) activate antitumoral immune responses and are used to treat multiple types of primary and metastatic malignancies. Thyroid dysfunction is a known immune-related ...adverse event of CPI therapy. There are few data on the effect of CPI and CPI-induced thyroiditis on primary papillary thyroid carcinoma (PTC). We present a patient who developed CPI-induced thyroiditis during treatment for a nonthyroid malignancy and subsequent regression of a coexisting untreated primary PTC.
A 49-year-old man with metastatic colon adenocarcinoma was found to have a large right thyroid nodule with biopsy confirmation of PTC. He did not have compressive symptoms or evidence of metastatic PTC. Resection was not performed because of colon cancer therapy. Treatment with CPI (ezabenlimab, an anti–programmed cell death protein 1 antibody) was initiated for the treatment of colon cancer. Four months after the initiation of CPI therapy, testing showed thyroid–stimulating hormone and free thyroxine levels of 174.9 (0.3-4.0 mIU/L) and 0.67 (0.93-1.70 ng/dL), respectively, consistent with CPI-induced hypothyroidism. Levothyroxine therapy was initiated. Repeat imaging 3 months later demonstrated a decrease in the tumor size to 4.1 × 4.9 × 4.2 cm (calculated volume change, −8.3% from baseline). At the last imaging, 1 year after the onset of CPI-induced thyroiditis, the PTC continued to decrease in size and measured 2.9 × 3.9 × 3.2 cm (volume change, −60.7% from baseline).
CPI-induced thyroiditis suggests the development of an immune response against thyroid tissue and may reflect a similar increased immune response against PTC cells leading to tumor regression in this case.
Further research to assess the immunologic mechanism underlying this association is warranted to potentially develop improved immunotherapy for PTC.
Gene chip array (Affymetrix) data from liver tissue and high resolution
1
H
NMR spectra from intact liver tissue, tissue extracts and plasma have been analyzed to identify biochemical changes arising ...from hepatotoxicity in mice dosed with acetaminophen. These data sets have been co-interpreted in terms of common metabolic pathways. The principal metabolic changes comprised a decrease in hepatic glucose and glycogen in intact tissue, coupled with an increase in lipid content, with increases in the levels of glucose, pyruvate, acetate and lactate in plasma, and increases in alanine and lactate in the aqueous tissue extracts. Collectively these data provide evidence for an increased rate of hepatic glycolysis. The metabolic observations were consistent with the altered levels of gene expression relating to lipid and energy metabolism in liver which both preceded and were concurrent with the metabolic perturbations. The results show that these two technology platforms together offer a complementary view into cellular responses to toxic processes, providing new insight into the toxic consequences, even for well-studied therapeutic agents such as acetaminophen.
