Lymphocyte reconstitution following autologous stem cell transplantation for progressive MS.

BACKGROUND: Autologous stem cell transplantation (ASCT) for progressive multiple sclerosis (MS) may reset the immune repertoire. OBJECTIVE: The objective of this paper is to analyse lymphocyte recovery in patients with progressive MS treated with ASCT. METHODS: Patients with progressive MS not responding to conventional treatment underwent ASCT following conditioning with high-dose cyclophosphamide and antithymocyte globulin. Lymphocyte subset analysis was performed before ASCT and for two years following ASCT. Neurological function was assessed by the EDSS before ASCT and for three years post-ASCT. RESULTS: CD4+ T-cells fell significantly post-transplant and did not return to baseline levels. Recent thymic emigrants and naïve T-cells fell sharply post-transplant but returned to baseline by nine months and twelve months, respectively. T-regulatory cells declined post-transplant and did not return to baseline levels. Th1 and Th2 cells did not change significantly while Th17 cells fell post-transplant but recovered to baseline by six months. Neurological function remained stable in the majority of patients. Progression-free survival was 69% at three years. CONCLUSION: This study demonstrates major changes in the composition of lymphocyte subsets following ASCT for progressive MS. In particular, ablation and subsequent recovery of thymic output is consistent with the concept that ASCT can reset the immune repertoire in MS patients.

Fibroblast movements during contraction of collagen lattices--a quantitative study using a new three-dimensional time-lapse technique with phase-contrast laser scanning microscopy.

In this study we assessed the behavior of fibroblasts during contraction of collagen lattices. We applied a new technique for three-dimensional time-lapse studies of movements of living cells using phase-contrast laser scanning microscopy. Five anchored and five floating collagen lattices were studied regarding the activity of cells during a 7-h period of active contraction. Three-dimensional reconstructions of the fibroblasts and their extensions were made from datasets of 16-26 "optical sections" 5 microm apart recorded hourly during the period of measurements. The distance between fibroblast nuclei in the floating lattices decreased by a mean of 6.8 microm, but remained constant in the anchored group. Only minor variations were found in the angle between a line connecting any two nuclei and the tangent of the lattice margin. The lengths of the cellular extensions continuously changed by shortening and extending, and an increasing number of intercellular contacts were established with time. The angle between the extensions and the periphery of the lattice varied continually, and no distinct pattern of arrangement of the extensions was seen. In conclusion, we have shown in living cells in vitro that fibroblasts do not appear to move around within lattices during contraction but rather send out and withdraw cellular extensions continuously. This speaks against cellular locomotion or movement as a main feature of contraction. Time-lapse scanning laser microscopy has also been shown to be a suitable method to study cellular behavior quantitatively in three dimensions during lattice contraction.

The effect of diclofenac (Voltaren) on the contraction of collagen lattices.

The inhibition of prostaglandin synthesis by diclofenac was studied with regard to effects on connective-tissue contraction and on the chemotaxis of fibroblasts stimulated by platelet-derived growth factor type BB (PDGF-BB). Collagen lattices populated with human fibroblasts responded to diclofenac with significantly increased contraction; the peak effect occurred at a dose of 5 micrograms/ml. Using a two-chamber system, PDGF-BB significantly increased the chemotactic activity of fibroblasts, and addition of diclofenac further increased this activity. Higher doses of diclofenac resulted in cell death, which was also reflected in lessened contraction of lattices with 50 micrograms/ml diclofenac, half of the cells were dead. The study showed that inhibition of prostaglandin synthesis by diclofenac increases the contraction of collagen lattices populated with human fibroblasts and increases the chemotactic activity of fibroblasts stimulated with PDGF-BB.

Capsular contracture with textured versus smooth saline-filled implants for breast augmentation: a prospective clinical study.

Texturization of silicone-filled breast implants has been shown to reduce the incidence of capsular contracture. A double-blind clinical study was undertaken to compare this incidence in saline-filled implants with textured or with smooth surfaces. Twenty-one women underwent mammary augmentation with a textured implant in one breast and a smooth implant in the other. The implants were placed subglandularly. All operations were performed by the same surgeon and all follow-up examinations by another. Breast hardness was evaluated 6 months postoperatively with applanation tonometry, using Baker's grading, and after 12 months, now also with a questionnaire concerning the patient's evaluation. Capsular contracture (Baker 3) had occurred in 33 percent of the breasts at the end of the study, and was bilateral in five cases. The incidence of contracture and the patients' views on the results did not differ between textured and smooth prostheses or between right and left breasts. Five patients requested reoperation, two of them because of breast hardness. Texturization of saline-filled implants thus did not reduce the incidence of capsular contracture.

Lysosomal heterogeneity between and within cells with respect to resistance against oxidative stress.

The prevailing opinion on lysosomal endurance is that, as long as the cells are still alive, these organelles are generally quite stable and, thus, do not induce cell damage by leaking their numerous powerful hydrolytic enzymes to the cytosol. We suggest that this opinion is basically wrong and consider that many lysosomes are quite vulnerable, especially to oxidative stress. Moreover, we suggest that cellular degeneration, including apoptosis as well as necrosis, follows upon lysosomal disruption. We have found differing stability of lysosomal membranes to oxidative stress, not only among different cell types, but also between cells of the same type and between lysosomes of individual cells. We suggest that cellular resistance to oxidative stress is mainly a function of three parameters: (i) the capacity to degrade hydrogen peroxide before it reaches, and may diffuse into, the acidic vacuolar compartment; (ii) the resistance to reactive oxygen species of lysosomal membranes; and (iii) the intralysosomal amounts of redox-active, low molecular weight iron. Iron-catalysed intralysosomal reactions, if pronounced enough, result in peroxidation and destabilization of the lysosomal membrane. Owing to differences in the cellular synthesis of hydrogen peroxide-degrading enzymes, degree of autophagocytotic degradation of iron-containing metalloproteins, lysosomal localization within the cytoplasm and intralysosomal iron chelation, the above three parameters may vary between both different and similar cells and between lysosomes of individual cells as well, explaining their observed variability with respect to resistance against oxidative stress.

Alpha-smooth muscle actin expression in rat and mouse mesenteric wounds after transforming growth factor-beta1 treatment.

Rat mesenteric perforations heal by contraction within 5 to 7 days, whereas mouse mesenteric perforations seldom close within 3 weeks unless stimulated by transforming growth factor-beta1. In this article, we quantified the expression of alpha-smooth muscle actin by quantitative-reverse transcription-polymerase chain reaction and the orientation of actin filaments at the wound margin by Fourier transformation image analysis after treatment with transforming growth factor-beta1. The expression of transforming growth factor-beta1 and its type II receptor was also assessed. Actin filaments were shown to increase with time at the wound margin in both species and the expression of alpha-smooth muscle actin mRNA increased simultaneously. Transforming growth factor-beta1 enhanced the alpha-smooth muscle actin expression four to five times in rats and three to four times in mice on day 5, but the number of copies expressed per cell was 15-fold higher in rats than in mice. Transforming growth factor-beta1 was down-regulated after wounding in free peritoneal cells of rats, but maintained until day 5 in transforming growth factor-beta1-treated mice. The main finding of this study was that untreated, normal rats expressed substantially more alpha-smooth muscle actin than mice. After treatment with transforming growth factor-beta1, this expression increased similarly in both species. It can be hypothesized that normal closure of mesenteric perforations requires a minimum level of actin expression. This level is not reached in normal mice, but is exceeded after stimulation. Perforations in the rat always close, because the alpha-smooth muscle actin expression is always above this level.

Specific binding of EGF in connective tissue repair.

Epidermal growth factor (EGF) is known to stimulate wound repair, including connective tissue repair as observed in the perforated rat mesentery. In the present study we assessed changes in expression of EGF receptors during healing of connective tissue by measuring the binding of [125I]EGF to perforated and unperforated mesenteric membranes. Autoradiographic grain density was measured on flat mounted or sectioned mesenteric tissue. Laparotomy alone caused an inflammatory reaction in the abdominal cavity and significantly (p < 0.04) increased the binding of [125I]EGF to unperforated membranes by 70% on days 1 and 3 postoperatively. In perforated mesenteric membranes, the binding of EGF in a 1 mm wide zone around the incision was significantly higher (p < 0.05) than EGF binding in the adjacent tissue on days 3 through 7. Furthermore, the average grain density in a 100 microns wide segment around the incision was approximately twice as high (p < 0.008) as the grain density in the mesentery 100 to 500 microns around the incision or in adjacent tissue (p < 0.004). These results indicate that expression of EGF receptor increases in the region of regenerating connective tissue and supports the hypothesis that EGF receptor plays a key role in mesenteric wound healing.

A double-embedding technique for thin tissue membranes.

A new double-embedding technique for thin tissue membranes is presented. This technique is useful for thin membranes such as mesenteric membranes from rodents, which usually measure only 10 microns in thickness. Several membranes are fixed and mounted on four needles located at the bottom of a plastic box. The box is filled with agarose at 50 C and then allowed to solidify. The agarose block is then removed, dehydrated in alcohol, cleared with HistoPetrol (isoparaffin hydrocarbons), permeated with paraffin and sectioned. The morphology is comparable to that obtained with methacrylate plastic embedding but is less time-consuming, less hazardous since no plastic hardener and activator are used and makes immunohistochemical studies easier.

Experimental mast cell activation improves connective tissue repair in the perforated rat mesentery.

The participation of mast cells in connective tissue repair was studied using the perforated-rat-mesentery model. Perforation of mesenteric membranes was performed during laparotomy of anesthetized Sprague-Dawley rats. Laparotomy significantly reduced the histamine content of the mesenteric membranes on day 1 postoperatively and perforation as such reduced the histamine content even more on days 1-10. Mast cell activation induced by a single intraperitoneal injection of Compound 48/80 two days prior to operation, significantly improved healing on days 5-7 postoperatively. Daily injections of Compound 48/80 for 5 days prior to operation showed significantly better healing compared to such injections for five days postoperatively. Administration of lupitidine, a long acting histamine H2-receptor antagonist, two times daily starting on the day of 48/80 injection to day 4 after operation did not apparently affect healing. The results indicate that mast cells may be activated during normal wound healing and that a preoperative, pharmacological activation improves healing. Furthermore, histamine does not seem to be of major importance for the beneficial effect of such mast cell activation on connective tissue repair.