Anti-IgM induces transforming growth factor-beta sensitivity in a human B-lymphoma cell line: inhibition of growth is associated with a downregulation of mutant p53

We wished to examine the role of transforming growth factor-beta (TGF- beta) in the regulation of human lymphoma cell growth. The RL cell line is an immunoglobulin M (IgM)+, IgD+ B lymphoma cell line, which does not constitutively express receptors for TGF-beta, and thus has lost the ability to respond to the inhibitory effects of TGF-beta. We demonstrate here that anti-Ig antibodies can efficiently upregulate the expression of TGF-beta receptors and promote sensitivity to growth inhibition by TGF-beta. Furthermore, because TGF-beta has been shown to function in late G1 of the cell cycle, we examined the ability of TGF- beta to modulate two tumor suppressor proteins known to be critical regulators of the G1/S transition, Rb and p53. Rb is a 105- to 110-kD phosphoprotein, which has been shown to maintain its growth suppressive function when it is found in the hypophosphorylated state. Wild-type p53 is a 53-kD phosphoprotein that appears to be important in preventing cell-cycle progression and promoting apoptosis in cells with DNA damage, whereas mutant p53 can overcome those functions. We show here that TGF-beta treatment of phorbol myristate acetate (PMA) or anti- Ig-activated RL cells results in growth inhibition through a dual effect on Rb and mutant p53. After TGF-beta treatment, we observe a predominance of Rb in the hypophosphorylated, growth suppressive form. In addition, we show a decrease in levels of mRNA and protein for mutant p53. We also show that, although these changes are sufficient to halt progression through the cell cycle, the cells do not appear to undergo extensive programmed cell death following 72 hours of TGF-beta treatment. Thus, although these lymphoma cells maintain the capacity to be negatively growth regulated by TGF-beta, the ability of TGF-beta to induce apoptosis must be independently controlled.     

Early human thymocyte proliferation is regulated by an externally controlled autocrine transforming growth factor-beta 1 mechanism

Early thymocytes undergo extensive proliferation after their entry into the thymus, but cellular interactions and cytokines regulating this intrathymic step remain to be determined. We analyzed the effects of various T-cell growth factors and cellular interactions on in vitro proliferation of early CD2+CD3/TCR-CD4-CD8- (triple negative [TN]) human thymocytes. Freshly isolated TN cells were then assayed for their growth capacity after incubation with CD2I+III-monoclonal antibody (MoAb), recombinant human interleukin-2 (IL-2), IL-7, and/or IL-4. These cells displayed significant proliferative responses with IL-4, IL- 7, or CD2-MoAb+IL-2. The addition of recombinant transforming growth factor beta (TGF beta) or autologous irradiated CD3+CD8+CD4- cells to TN cell cultures dramatically decreased their growth responses to IL-2 and IL-7, whereas IL-4-induced proliferation was less sensitive to growth inhibition. We thus asked whether the CD8+ cell-derived inhibitory effect was due to TGF beta. The addition of neutralizing anti-TGF beta MoAb completely abolished CD8+ cell-derived inhibition of TN cell growth. Analysis of CD8+ cell-derived supernatants indicated that these cells had low TGF beta 1 production capacity, whereas TN cells secrete significantly high levels of TGF beta 1. Cell fixation studies showed that TN cells were the source of the TGF beta. TGF beta 1 released from TN cells was in the latent form that became the active inhibitory form through interaction of TN cells with CD8+ cells. Together, these data suggest a role for TGF beta 1 as an externally controlled, autocrine inhibitory factor for human early thymocytes, with a regulatory role in thymic T-cell output.     

Impact of interferon‐free regimens on the glomerular filtration rate during treatment of chronic hepatitis C in a real‐life cohort

Little data are available on renal toxicity exerted by direct‐acting antivirals (DAAs) in real life. The aim of this study was to assess the impact of direct‐acting antivirals against hepatitis C virus infection currently used in Spain and Portugal on the estimated glomerular filtration rate (eGFR) in clinical practise. From an international, prospective multicohort study, patients treated with DAAs for at least 12 weeks and with eGFR ≥30 mL/min per 1.73 m² at baseline were selected. eGFR was determined using the CKD‐EPI formula. A total of 1131 patients were included; 658 (58%) were HIV/HCV‐coinfected patients. Among the 901 patients treated for 12 weeks, median (interquartile range) eGFR was 100 (87‐107) at baseline vs 97 (85‐105) mL/min per 1.73 m² at week 12 of follow‐up (FU12) post‐treatment (P < .001). For HIV‐coinfected subjects who received tenofovir plus a ritonavir‐boosted HIV protease inhibitor (PI/r), baseline vs FU12 eGFR were 104 (86‐109) vs 104 (91‐110) mL/min per 1.73 m² (P = .913). Among subjects receiving ombitasvir/paritaprevir with or without dasabuvir, eGFR did not show any significant change. Of 1100 subjects with eGFR >60 mL/min per 1.73 m² at baseline, 22 (2%) had eGFR <60 mL/min per 1.73 m² at FU12, but none presented with eGFR <30 mL/min per 1.73 m². In conclusion, eGFR slightly declines during therapy with all‐oral DAAs and this effect persists up to 12 weeks after stopping treatment in subjects with normal to moderately impaired renal function, regardless of HIV status. Concomitant use of tenofovir plus PI/r does not seem to have an impact on eGFR.     

Anatomic site evaluation of the zygomatic bone for dental implant placement

Thirty human zygomatic bone specimens (15 females mean age 81.60 +/- 11.38 years, 15 males, mean age 78.47 +/- 6.58 years) were examined by quantitative computed tomography and histomorphometry. The aim of the study was to assess the bone mineral density, the trabecular bone volume and the trabecular bone pattern factor. Moreover, the anterior-posterior and the medio-lateral dimensions and the estimated implant length within the zygomatic bone were determined. For quantitative computed tomography the specimens were scanned together with a bone mimicking anthropomorphic reference phantom. The bone mineral density was calculated for the specimens in the plane of the intended direction of the implant placement. Subsequently, with the sawing and grinding technique, the specimens were prepared in the same plane for histomorphometry. The trabecular bone mineral density was 369.95 +/- 188.80 mg/cm3 for the female and 398.94 +/- 99.11 mg/cm3 for the male specimens (P = 0.23). The male trabecular bone volume showed a value of 27.32 +/- 9.49%, while the female group reached a value of 19.99 +/- 7.60% (P = 0.23). The trabecular bone pattern factor was 1.2 x 10-2 +/- 1.28 mm-1 for the male and 1.02 +/- 0.96 mm-1 for the female specimens (P = 0.045). The study reveals that the zygomatic bone consists of trabecular bone with parameters that are unfavourable for implant placement. However, the success of implants placed in the zygomatic bone is secured by the employment of at least four cortical portions.     

Lack of beneficial effects of platelet-rich plasma on sinus augmentation using a fluorohydroxyapatite or autogenous bone: an explorative study

BACKGROUND: Maxillary sinus augmentation is frequently necessary before placement of dental implants in the posterior maxilla. Besides autogenous bone graft, various bone substitutes have been used, with favourable results. Although platelet-rich plasma (PRP) has been used in the field of oral and maxillofacial surgery for years, its beneficial effects on osseous regeneration still remain unclear. The aim of this study was to evaluate the short and long time effects of PRP on single-stage sinus augmentation using autogenous bone or a fluorohydroxyapatite (Algipore) in a randomized prospective animal study. METHODS: After extraction of maxillary premolars of sixteen minipigs, the wounds were allowed to heal for 2 months. Then, sinus augmentations were performed bilaterally using one of the following grafting materials: autogenous bone and Algipore with or without PRP. Three dental implants (Ankylos) were installed in each sinus simultaneously. Four animals were euthanized at each period of observation (1, 2, 8 and 12 months). Implant-bearing specimens were sectioned bucco-lingually along the long axis of implants and undecalcified ground specimens were prepared. The bone-implant-contact (BIC) was measured by means of microradiographic examination. For histological evaluation, the specimens were stained with toluidin blue, and the percentage of the newly formed bone and the remaining bone substitute were evaluated. RESULTS: The grafting materials chosen showed increasing levels of BIC and newly formed bone throughout the period of observation in both PRP and non-PRP groups. Adding PRP resulted in lower BIC and newly formed bone compared with autogenous bone grafts or Algipore alone. However, a statistical significance was not found. The percentages of the remaining bone substitute in both the PRP and non-PRP groups were closely comparable in all observation periods. CONCLUSIONS: The application of PRP could not reveal significant beneficial effects on the BIC, the percentage of the newly formed bone and the remaining bone substitute in this study.     

Restoration of the lateral sinus wall using a collagen type I membrane for guided tissue regeneration.

A collagen type I membrane prepared from bovine pericard was used to restore and thicken the lateral sinus wall after resection in 6 G?ttingen minipigs. One rehydrated collagen type I membrane was placed on the intact sinus mucosa. A hydroxyapatite block was placed on this membrane and covered with a second membrane. After 6 months there was complete, bony regeneration in the space between the 2 membranes and thickening of the new bone plate compared to the untreated control side. This study shows that a collagen membrane can be applied for guided tissue regeneration.     

Influence of root development and root anatomy on the occurrence of periodontal damage during removal of third molars for transplantation: a histometric study

The purpose of this study was to determine the influence of root development and root anatomy on surgically induced periodontal damage to third molars resulting from removal prior to transplantation. Thirty impacted third molars were removed by a careful technique, whereas another 30 were removed by the usual vestibular osteotomy and served as controls. Periodontal damage was histometrically evaluated on serial cross sections of the whole root and classified according to five categories describing the extent of damage to periodontal fibers, cementoblasts, and the cementum layer. The results showed more extensive damage to the periodontal membrane in the more mature stages of root development. A statistically significant influence of root anatomy on the occurrence of periodontal damage was not found.     

Presence of mixed colony-forming cells in long-term hamster bone marrow suspension cultures: response to pokeweed spleen conditioned medium

In contrast to the murine system, long-term hamster bone marrow suspension cultures maintain proliferation of both pluripotent and committed stem cells in the absence of an adherent layer and without addition of exogenous factors, such as hydrocortisone. Addition of pokeweed-mitogen-stimulated hamster spleen conditioned medium (SCM) to these long-term suspension cultures produces an increase in the number of mixed colonies assayed in soft-agar, These mixed colonies, which contained four cell lineages--granulocytic, erythroid, megakaryocytic, and macrophage--could be generated from cells grown in suspension for over 6 mo. Addition of SCM also induces an initial rapid expansion of the myeloid compartment, and this expansion results in 70% of the cells being terminally differentiated granulocytes. In contrast, addition of SCM to hamster bone marrow cultures containing both adherent cells and hematopoietic stem cells produced no change in the number of mixed colonies generated in the culture. This system allows the in vitro study of the process of stem cell proliferation and differentiation and also provides a means to examine the relationship of adherent and supernatant bone marrow populations.     

Nonhomogeneous distribution of leukemia in the bone marrow during minimal residual disease

In a rat model (BNML) for human acute myelocytic leukemia the distribution of leukemic cells in bone marrow samples from various sites was investigated, using monoclonal antibodies (MoAbs) and flow cytometry. Rats were studied before chemotherapy as well as thereafter, ie, in the "minimal residual disease" (MRD) phase. Bone marrow from different types of bones was analyzed from each animal. Before treatment, the ratio of the measured extreme values (ie, highest/lowest value) for leukemic cell frequencies in bones from individual rats ranged from 3.7 to 11.7. During the MRD phase the ratios of the extremes ranged from a factor of 36 to more than 13,000 from one rat to another. The variability between bones of comparable size was estimated by studying the ribs from each individual animal. Within individuals the extremes differed by a factor of 1.2 to 4.0 before chemotherapy and from 2.4 to greater than 320 after chemotherapy. The variability within the marrow cavity of a single bone was determined by analyzing multiple samples from femoral bones cut into slices. The leukemic cell frequency appeared to vary considerably, ie, before treatment from 1.7 to 7.3 and during MRD from 4 to 28,000. The presented data may contribute to understanding the sometimes conflicting observations in leukemic patients. Improvement of methods for detecting MRD will not automatically lead to a more accurate estimation of the total tumor burden. The reliability of diagnoses based on the analysis of single bone marrow aspirates appears to be highly questionable.     

High-level expression and purification of a recombinant human erythropoietin produced using a baculovirus vector

Conditions presently have been established for the high-level expression and simplified purification of recombinant human erythropoietin produced in Spodoptera frugiperda cells. Expression, as mediated by infection with a recombinant baculovirus, was accomplished in suspension culture using reduced levels of serum and media supplements experimentally determined to provide optimum levels of factor production (500,000 U/L). Purification of this recombinant human erythropoietin to virtual homogeneity (greater than or equal to 99%) was accomplished via a simple three-step procedure involving isocratic elution from DEAE-Sephacel, reverse-phase high performance liquid chromatography (HPLC) on a C4 medium, and the single-step elution of purified hormone from concanavalin A agarose. Overall, an 890-fold purification was accomplished with a recovery of 80% as assayed in vitro. Biologically, this purified erythropoietin is highly active, possessing a specific activity in vitro of 200,000 U/mg protein. Chemically, this erythropoietin (molecular weight [mol wt] 26,200) appears exceptionally uniform in its oligosaccharide constitution (30%) as contrasted with heterogeneously glycosylated erythropoietins derived from mammalian cells (mol wt 30,000 to 38,000; 40% to 50% complex-type oligosaccharide). Thus, human erythropoietin as presently produced in an insect cell line comprises not only an abundant source of highly active, readily purified hormone for studies of its mechanism of action and cell surface receptor, but also represents a uniquely homogeneous form that should prove advantageous for direct structural analyses.