Erythropoietin-specific cell cycle progression in erythroid subclones of the interleukin-3-dependent cell line 32D [published erratum appears in Blood 1993 Jun 1;81(11):3168]

Recently, a variety of growth factor-dependent subclones of the murine interleukin-3 (IL-3)-dependent cell line 32D have been isolated. These subclones include those dependent for growth on erythropoietin (Epo) (32D Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF) (32D GM), or granulocyte colony-stimulating factor (G-CSF) (32D G). 32D Epo1.1 is a revertant of 32D Epo and is capable of growing in IL-3. These cell lines express the differentiation program appropriate to the specific growth factor and depend on the growth factors not only for proliferation but also for survival. To determine how the signal for proliferation is triggered by various growth factors, we examined the DNA histograms and the expression of cell cycle-specific genes in the different cell lines. The cell cycle-specific genes analyzed were myc (early G1), myb (late G1), and the structural genes for the calcium- binding protein 2A9 (middle G1) and histone H3 (G1-S boundary). The DNA histogram analysis of cells in the logarithmic phase of growth showed that approximately 40% of 32D, 32D GM, 32D G, and 32D Epo1.1 (growing in IL-3) were cells with a 2N DNA content (and therefore in G0/G1), and another 40% have a DNA content intermediate between 2N and 4N (in S phase). In contrast, 32D Epo and 32D Epo1.1 (growing in Epo) had fewer cells in the G0/G1 phase of the cell cycle compared with the number of cells that were in the S phase (19% to 31% v 69% to 78%, respectively). Because all the cell lines have comparable doubling times (15 to 18 hours), the cell distribution among the phases of the cell cycle is proportional to the length of the phase. Therefore, cells growing in IL- 3 (32D and 32D Epo1.1), GM-CSF (32D GM), or G-CSF (32D G) progress along the cycle in a manner typical of previously reported nontransformed cell lines. In contrast, cells growing in Epo (32D Epo or 32D Epo1.1) spend relatively less time in G0/G1 and correspondingly more time in S. These data were confirmed by the analysis of the tritiated thymidine (3H-TdR) suicide rate and of the expression of cell cycle-specific genes. The 32D and 32D Epo1.1 cells growing in IL-3 had a suicide rate of congruent to 50%, whereas the suicide rate of 32D Epo and 32D Epo1.1 growing in Epo was higher than 75%.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hyperbaric oxygen therapy improves angiogenesis and bone formation in critical sized diaphyseal defects

Besides the use of autologous bone grafting several osteoconductive and osteoinductive methods have been reported to improve bone healing. However, persistent non‐union occurs in a considerable number of cases and compromised angiogenesis is suspected to impede bone regeneration. Hyperbaric oxygen therapy (HBO) improves angiogenesis. This study evaluates the effects of HBO on bone defects treated with autologous bone grafting in a bone defect model in rabbits. Twenty‐four New‐Zealand White Rabbits were subjected to a unilateral critical sized diaphyseal radius bone defect and treated with autologous cancellous bone transplantation. The study groups were exposed to an additional HBO treatment regimen. Bone regeneration was evaluated radiologically and histologically at 3 and 6 weeks, angiogenesis was assessed by immunohistochemistry at three and six weeks. The additional administration of HBO resulted in a significantly increased new bone formation and angiogenesis compared to the sole treatment with autologous bone grafting. These results were apparent after three and six weeks of treatment. The addition of HBO therapy to autologous bone grafts leads to significantly improved bone regeneration. The increase in angiogenesis observed could play a crucial role for the results observed. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:513–520, 2015.     

Proline at the P2 position in protein C is important for calcium- mediated regulation of protein C activation and secretion

Protein C is a vitamin K-dependent plasma serine protease zymogen, which upon activation, functions as an anticoagulant. Protein C activation is catalyzed by a complex of thrombin (T) with thrombomodulin (TM). This activation is Ca(2+)-dependent, but Ca2+ inhibits protein C activation by thrombin alone. In most proteases, specificity is determined primarily by the residues that lie near the scissile bond. In protein C, the P2 position is Pro, whereas in the fibrinogen A chain, P2 is Val. We have expressed a Pro-->Val mutant of protein C (P168V) in mammalian cells. At saturating Ca2+, the P168V and wild-type proteins were activated by the T-TM complex equivalently, but half maximal rates of activation were obtained at 50 mumol/L Ca2+ for wild type and approximately 5 mmol/L Ca2+ for the P168V mutant. In the absence of TM, Ca2+ no longer inhibited the activation of the P168V mutant. These results indicate that Pro168 influences the Ca(2+)- dependent conformational changes in protein C that control activation. Recently, a patient with thrombotic complications has been identified with a Pro168-->Leu substitution. Both the P168V and the P168L mutation lead to impaired secretion caused by retention within the cell.     

Existence of a pool of T-lymphocyte colony-forming cells (T-CFC) in human bone marrow and their place in the differentiation of the T- lymphocyte lineage

The existence and characteristics of bone marrow T-cell progenitors have not yet been established in man. Several pieces of evidence such as the reconstitution of certain immunodeficiencies by bone marrow graft suggest that T-cell precursors are present in the bone marrow. We report the growth of T-cell colonies from bone marrow populations using PHA-stimulated lymphocyte-conditioned medium containing T-cell growth factor (TCGF). Rosetting experiments and complement-dependent cytotoxicity assays with monoclonal antibodies indicate that the bone marrow T colony-forming cells (T-CFC) are E- OKT 3- and la+, i.e., immature progenitors. The colonies derived from these cells have the phenotype of mature T cells: E + OKT 3 + la- with either helper (OKT 4+) and suppressor (OKT 8 +) antigens. These results suggest that a thymic microenvironment may not be necessary for the in vitro proliferation and differentiation of the T-cell lineage in adult humans. These methodologies may permit direct investigation of early phenomena concerning the T-cell lineage, such as the acquisition of self-tolerance, the formation of a repertoire of specificities, and the HLA restriction phenomena that we believe takes place before the thymic maturation.     

Severity of alcohol-induced painful peripheral neuropathy in female rats: role of estrogen and protein kinase (A and Cepsilon)

Small-fiber painful peripheral neuropathy, a complication of chronic ethanol ingestion, is more severe in women. In the present study, we have replicated this clinical finding in the rat and evaluated for a role of estrogen and second messenger signaling pathways. The alcohol diet (6.5% ethanol volume:volume in Lieber-DeCarli formula) induced hyperalgesia with more rapid onset and severity in females. Following ovariectomy, alcohol failed to induce hyperalgesia in female rats, well past its time to onset in gonad intact males and females. Estrogen replacement reinstated alcohol neuropathy in the female rat. The protein kinase A (PKA) inhibitor (Walsh inhibitor peptide, WIPTIDE) only attenuated alcohol-induced hyperalgesia in female rats. Inhibitors of protein kinase Cepsilon (PKCepsilon-I) and extracellular-signal related kinase (ERK) 1/2 (2'-amino-3'-methoxyflavone (PD98059) and 1,4-diamino-2, 3-dicyano-1, 4-bis (2-aminophenylthio) butadiene (U0126)) attenuated hyperalgesia in males and females, however the degree of attenuation produced by PKCepsilon-I was much greater in females. In conclusion, estrogen plays an important role in the expression of pain associated with alcohol neuropathy in the female rat. In contrast to inflammatory hyperalgesia, in which only the contribution of PKCepsilon signaling is sexually dimorphic, in alcohol neuropathy PKA as well as PKCepsilon signaling is highly sexually dimorphic.     

Facial nerve injury during temporal artery biopsy

Temporal artery biopsy is considered the gold standard investigation of giant cell arteritis and is recommended in suspected cases despite a sensitivity of 81–91%. This review highlights the potential risk of facial nerve injury during temporal artery biopsy and introduces recent advances in the emerging role of imaging modalities. When these non-invasive techniques are used in conjunction with American College of Rheumatology scoring, which includes clinical features and biochemical test results, temporal artery biopsy may be avoided in selected cases.