Long-term anti-arthritic and anti-osteoporotic effects of raloxifene in established experimental postmenopausal polyarthritis.

Both oestrogen deficiency and the inflammatory disease contribute to the generalized bone loss seen in postmenopausal rheumatoid arthritis (RA). Oestradiol and the selective oestrogen receptor modulator raloxifene have been shown to ameliorate the disease in collagen-induced arthritis (CIA), a well-established animal model for human RA. The aim of this study was to investigate whether raloxifene-treatment would be beneficial in long-term treatment of established CIA, both regarding anti-arthritic and anti-osteoporotic properties. Female dilute brown agouti mice were ovariectomized and CIA was induced. Raloxifene or vehicle treatment was administered 5 days per week, and the clinical arthritis score was evaluated continuously. At termination, bone mineral density was analysed, paws were collected for histological examination and sera were analysed for markers of bone and cartilage turnover, as well as antibodies to type II collagen and levels of interleukin (IL)-6. Treatment with raloxifene is beneficial in long-term treatment of established CIA. It hampers the disease severity and frequency, protects the joints from destruction and protects against the development of osteoporosis. The proinflammatory cytokine IL-6 was down-regulated in raloxifene-treated mice compared with controls. The serum levels of antibodies to collagen were not affected by raloxifene-treatment. Long-term treatment with raloxifene has both anti-arthritic and anti-osteoporotic effects in established experimental postmenopausal polyarthritis.

Identification of target cells for the genomic effects of estrogens in bone.

Estrogen has bone protective effects, but the exact mechanism behind these effects remains unclear. The aim of the present study was to identify the primary target cells in bone for the classical genomic effects of estrogens in vivo. For this purpose we have used reporter mice with a luciferase gene under the control of three estrogen-responsive elements (EREs), enabling detection of in vivo activation of gene transcription. Three-month-old ovariectomized mice were treated with a single dose (50 mug/kg) 17beta-estradiol (E2). Luciferase activity was analyzed in several tissues and in different bone marrow-derived lymphocyte enriched/depleted preparations using MacsMouse CD19 (for B lymphocytes) or CD90 (for T lymphocytes) MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Histological characterization of cells with high luciferase content was performed using immunohistochemistry. Both cortical bone and bone marrow displayed a rapid (within 1 h) and pronounced E2-induced increase in luciferase activity. The luciferase activity in total bone marrow and in bone marrow depleted of lymphocytes was increased six to eight times more than in either B-lymphocyte or T-lymphocyte enriched cell fractions 4 h after the E2 injection, demonstrating that mature lymphocytes are not major direct targets for the genomic effect of estrogens in bone. Immunohistochemistry identified clear luciferase staining in hypertrophic growth plate chondrocytes, megakaryocytes, osteoblasts, and lining cells, whereas no staining was seen in proliferative chondrocyte. Although most of the osteocytes did not display any detectable luciferase staining, a subpopulation of osteocytes both in cortical and trabecular bone stained positive for luciferase. In conclusion, hypertrophic growth plate chondrocytes, megakaryocytes, osteoblasts, lining cells, and a subpopulation of osteocytes were identified to respond to estrogen via the classical ERE-mediated genomic pathway in bone. Furthermore, our findings indicate that possible direct estrogenic effects on the majority of osteocytes, not staining positive for luciferase, on proliferative chondrocytes and on mature lymphocytes are mediated by non-ERE actions.

HIV isolation and antigen detection in infected individuals and their seronegative sexual partners.

We have examined 39 couples, each consisting of one HIV-seropositive index case and one seronegative sexual partner. HIV isolations, HIV antigen (HIV-Ag) tests and HIV antibody tests were performed on samples from these 78 individuals. Results were compared with those of 68 unselected individuals. Neither HIV, nor HIV-Ag was detected in any of the seronegative individuals. HIV-Ag tests, but not HIV isolations showed positive results with a significantly lower frequency in symptomatic index cases than in unselected patients with symptoms. This indicates that the absence of HIV-Ag in the serum may be correlated with a low level of contagiousness.

Increasing intrathecal lymphocytosis and immunoglobulin G production in neurologically asymptomatic HIV-1 infection.

Cerebrospinal fluid from 34 human immunodeficiency virus (HIV-1) seropositive patients, only four of whom had HIV-related neurological symptoms, was examined by cytology, protein quantification, isoelectric focusing and specific serological tests. CSF lymphocytosis and evidence of intrathecal IgG production, found in 21 and 20 respectively of the 34 patients, correlated significantly with the duration of the infection. Increasing IgG index was found in two patients with repeated CSF examinations during greater than 7 years. Intrathecal HIV antibodies were detected on Western blot in 32/34 patients. HIV antigen test positive in 5/34 sera was negative in all 34 CSF samples. Intrathecal B cell activation seems to increase during the early HIV infection.

Immunotherapy in liver tumors: II. Intratumoral injection with activated tumor-infiltrating lymphocytes, intrasplenic administration of recombinant interleukin-2 and interferon alpha causes tumor regression and lysis.

This study tested the effect of intratumoral injection with activated tumor-infiltrating lymphocytes (TIL), and simultaneous administration of recombinant interleukin-2 (rIL-2) and interferon alpha (IFN-alpha) (LII protocol), on mouse liver tumor. Group I (n = 10) served as the controls. Group II (n = 17) received rIL-2 + IFN-alpha schedule. Group III (n = 20) received the LII protocol. A total of 5 x 10(6) of TIL were injected into 4 sites of a tumor in a single treatment. rIL-2 (1 x 10(6) IU) on the first day and IFN-alpha (1 x 10(5) IU) on the second day were alternately given with a total of 10 treatment doses that were completed in 20 days. Tumor remission or regression rates of 29% and 40% were obtained in groups II and III, respectively, but no remission was obtained in the controls. A large number of TIL were also observed in the tumors treated with the LII protocol. Making comparisons between the control group and IL-2 + IFN-alpha schedule, and the control group and LII protocol, the ratios of cytolytic activity of TIL in vitro were 0:32 and 0:57, respectively. We conclude that the LII protocol appears to be more effective in the treatment of mouse liver tumor than the IL-2 + IFN-alpha schedule, and that it may be a new promise for the treatment of patients with liver malignancies.

Tumour vessel damage resulting from laser-induced hyperthermia alone and in combination with photodynamic therapy.

This study examined tumour vessel injury resulting from laser-induced hyperthermia alone and in combination with photodynamic therapy (PDT) in the treatment of rat liver tumours by means of scanning electron microscopy. A total of 18 Wistar rats were divided into three groups. Group I (six animals) underwent hyperthermia for 15 min (15-min hyperthermia). Group II (six animals) underwent hyperthermia for 30 min (30-min hyperthermia). Group III (six animals) received the combined treatment of PDT and 30-min hyperthermia. For PDT, delta-amino laevulinic acid at a dose of 60 mg/kg of body weight was intravenously administered 60 min before irradiation at 635 nm. The morphological results indicated that 15-min hyperthermia gave rise to an increase in permeability of the vessels in the treated tumour. Thirty-min hyperthermia caused extreme oedema of vascular endothelial cells and restrictive openings of tumour branch vessels. The combined therapy of PDT and hyperthermia destroyed tumour vasculature. Large breaks of the inner wall of the treated tumour vessels were deeply involved in the basement membrane of the vessel. The results indicate that there may be a close link between inhibition of tumour growth and degree of damage to tumour vessels.

Reaction of the vascular system in the trachea of the rabbit exposed to fractionated irradiation with and without the addition of misonidazole.

As the radiation field used in the radiation therapy of malignancies in the thoracic cavity often exposes the trachea to ionizing irradiation, it is important to ascertain the effects of radiation on this tissue either as a single therapy or in combination with radiosensitizers. In the study reported here the vascular area in the subepithelial layer of the trachea has been calculated in 160 rabbits treated in four ways: (1) 10 rabbits received no treatment and served as controls; (2) 50 rabbits were given 100 mg misonidazole daily on consecutive days, with the individual total dose ranging from 100 to 1000 mg; (3) 50 rabbits were treated with misonidazole in the same way, but were also exposed to radiation (2 Gy/F) at 15-30 min later; (4) 50 rabbits received only fractionated radiation (2 Gy/F). The total radiation dose in the irradiated animals ranged from 2 to 20 Gy. In the treated groups, an oedema was observed in both the ciliary cell layer and in the subepithelial area. In the group given only irradiation, this oedema was dose-dependent, but no such dose-dependency was observed in the two groups treated with misonidazole. The vascular area in the groups treated with misonidazole was significantly increased as compared with the group given only irradiation and the control group; this was valid both with and without correction for the oedema. There was a significant correlation between the oedema and the vascular area in the groups treated with misonidazole, which was not found in the group irradiated without the drug.

Scanning electron microscopy and transmission electron microscopy of the ciliated cells of the trachea of the rabbit treated with misonidazole alone and in combination with ionizing radiation.

The trachea is often located in the treatment volume when irradiating malignant tumours in the thorax. In order to evaluate possible synergism between misonidazole and irradiation on this tissue, the following studies were made. Fifty rabbits were treated with daily injections of 100 mg misonidazole given i.p. on consecutive days from 1 to 10 days. Morphological investigations of the trachea were made with scanning electron microscopy (SEM), transmission electron microscopy (TEM) and light microscopy (LM). Physiological examinations were performed with recording of the ciliary beat frequency. The results were compared with those from a group of 100 rabbits given misonidazole in a similar manner and exposed to irradiation (2 Gy) 15-30 min after each injection. Ten rabbits were used as controls. The results are compared to the effect of fractionated irradiation alone with 2 Gy/day. Fractionated irradiation of the ciliary epithelium in the trachea of the rabbit has shown dose-dependent physiological and morphological effects. Misonidazole potentiates these effects of radiation with a more pronounced change of the ciliary beat frequency and an increased metabolic activity as could be visualized on TEM. The combination of drug and irradiation also induced a hyperplasia of the ciliary epithelium. Misonidazole itself had no effect on the ciliary beat frequency, but caused a hypoplasia of the ciliary epithelium.

Multiple-system organ damage resulting from prolonged hepatic inflow interruption.

BACKGROUND: It has been reported that patients undergoing major hepatectomy tolerated 90 and 127 minutes of continuous hepatic inflow interruption with no evidence of permanent damage to the liver. We questioned the safety and feasibility of the interruption beyond 90 minutes in normothermic human beings. We also postulated that, besides injury to the liver per se, extended continuous hepatic inflow interruption would cause extrahepatic multiple-system organ damage in subjects exposed to continuous hepatic inflow interruption for 90 or 120 minutes. DESIGN: Fifty Sprague-Dawley rats were divided into three groups. Group 1 served as controls that had only laparotomy. Group 2 underwent continuous hepatic inflow interruption for 90 minutes, and group 3 was subjected to continuous hepatic inflow interruption for 120 minutes. Scanning electron microscopy and transmission electron microscopy were used to evaluate ultrastructural alterations in the liver, lung, heart, and intestine. SETTING: Lund (Sweden) University Hospital and Top Cancer Institute, Lund. INTERVENTIONS: Intraoperative and postoperative infusion and blood transfusion were given in all experimental animals. MAIN OUTCOME MEASURES: Animal survival and manifestations of multiple-system organ failure. RESULTS: In rats with continuous hepatic inflow interruption for 90 or 120 minutes, scanning electron microscopy showed a necrotic surface of the liver cells together with fibrin exudation. Hepatic sinusoids and intrahepatic nerves also had severe injury. Destruction of pulmonary structures and breakdown of microcirculation in the lung were characterized by thinned and ruptured walls of alveoli and a greatly decreased number of capillaries in the damaged alveolar wall. Transmission electron microscopy showed four types of ultrastructural changes, ie, necrosis of epithelial cells, extremely swollen mitochondria in intestinal cells, death of mucosal cells, and increased permeability of vessels in the injured intestine. The affected heart manifested highly enlarged mitochondria in myocardial cells, thickened vascular walls, and scattered necrotic lesions in myocardial tissue. CONCLUSIONS: Multiple-system organ failure resulting from ischemia-reperfusion injury and obstacle of portal hemodynamics in a subject subjected to an extended continuous hepatic inflow interruption is an unrecognized new disorder that may cause a high mortality rate. Our preliminary results indicated that animals subjected to continuous hepatic inflow interruption for 90 or 120 minutes developed various injuries to the liver, lung, heart, and gut. Therefore, we believe that continuous hepatic inflow interruption exceeding 90 minutes could also be hazardous in human beings.

Studies on ciliated epithelia of the human genital tract. II. The mucociliary wave pattern of fallopian tube epithelium.

The ciliary activity of human fallopian tube epithelium was studied in vitro by using a technique which included registration of the light reflections from the mucociliary waves on the epithelial surface. In all registrations, the mucociliary waves moved toward the uterine end of the specimen. The frequencies of the reflections of the mucociliary waves ranged between 960 and 1368 cpm, with a mean of 1191 cpm (2 SD, +/- 176) and a median of 1210 cpm. Significant differences in the mucociliary wave frequencies were not observed between different parts of the fallopian tube nor between registrations performed during the follicular and luteal phases of the menstrual cycle. Scanning electron microscopy of the specimens revealed no morphologic changes of the cilia during the menstrual cycle.

Intra-operative laser-induced photodynamic therapy in the treatment of experimental hepatic tumours.

OBJECTIVE: To examine the effect of photodynamic therapy (PDT) on experimental liver tumours in rats. DESIGN: An experimental liver tumour model was used. Each of a group of rats had two tumours simultaneously inoculated into its liver. The tumour located in the left hepatic lobe was used for PDT, and the other one, in the median lobe, as a control. The haem precursor delta-amino laevulinic acid (ALA), at a dose of 30 mg/kg body weight, was injected 60 min before laser irradiation. Rats in group I received ALA through a femoral vein. Those in group II received ALA through the portal vein. Group III had an injection of ALA solution through the portal vein plus hepatic inflow occlusion. Three and 6 days after the treatment, the rats were killed, and the tumours were measured, and ultrastructural changes were examined using scanning electron microscopy. SETTING: Lund University Medical Laser Centre, Lund, Sweden. RESULTS: The mean tumour volume of the treated tumours increased by factors of 1.9, 1.5 and 1.7 in groups I, II and III, respectively, compared with the pretreatment baseline value. However, the mean tumour volume in the control tumours increased by factors of 9.5, 4.3 and 4.8 in the respective groups. Under the light microscope, marked necrosis of the treated tumour and the surrounding liver tissue was observed. Scanning electron microscopy revealed heavy damage to the cells and vessels in the treated tumour. CONCLUSION: PDT with ALA is an effective treatment modality for rat liver tumours.

The mucociliary activity of the upper respiratory tract. I. A method for use in experimental studies on human material.

A method for standardized recordings of the mucociliary activity of the mucosa in the human respiratory tract is described. Nasal polyps, adenoid vegetations and biopsy material from maxillary sinuses have been used for preliminary in vitro experiments. The dependence of the mucociliary activity on oxygen supply from the surrounding air is emphasized. The influence of varying gas mixtures on the mucociliary activity can be thoroughly studied. The method is also applicable for in vivo recordings in the operating theatre during Luc Caldwell operations. A combination of in vivo and in vitro studies is under way in order to elucidate the aetiology, treatment, and prognosis of diseases of the upper respiratory tract.

Lymphocyte locomotion. I. The initiation, velocity, pattern, and path of locomotion in vitro.

The locomotive behaviour of human lymphocytes in coverslip preparations of clotted autoplasma was studied at +37 degrees C. Lymphocytes isolated from peripheral blood or from the tonsils did not move prior to membrane activation by means of incubation with phytohemagglutinin (PHA). After PHA stimulation the locomotion of 19 lymphocytes was analysed by time-lapse fliming. The locomotion was random, as evidenced by a median locomotive index of 0.64 (Q1-Q3 0.04-0.75) and comparatively slow, median velocity 15 mum/min (Q1-Q3 12-18). The locomotion of 5 other lymphocytes was studied at high magnification. It is suggested that the characteristic polarity of wandering lymphocytes, indicating the direction of movement, can be utilized in the analysis of the lymphocyte traffic in tissue sections of post-capillary high-endothelium venules.

Lymphocyte locomotion. II. The lymphocyte traffic over the post-capillary venules analysed by phase contrast microscopy of thin sections of rat lymph nodes.

Thin sections of lymph nodes from 14 rats were examined by phase contrast microscopy as regards direction of lymphoctes with amoeboid movement configuration (AMC) relative to the basement membrane of post-capillary high-endothelium venules (HE-cenules). Out of 118 lymphocytes with AMC, 82 appeared to be on theyr way into the venule from the lymph node parenchyma. This observation suggests that the lymphocyte traffic over the HE-venules is bi-directional, with the main migratory stream of lymphocytes from the lymph node parenchyma into the post-capillary venules.

Intra- and extracellular activities of ciliated cells. A model for functional studies.

The mucociliary activity of the mucosa of the respiratory tract is used as an experimental model for exposure studies regarding air pollution and pharmaca. It is possible to record the intracellular electrical activity simultaneously with the extracellular mechanical movements of cilia.

Mucociliary wave pattern. A comparative analysis of extracellular and intracellular activities.

Experimental in vitro studies have been made on the intracellular electrical activity and the extracellular wave movements of ciliary cells in rabbit trachea. The following results were obtained: 1. Surface light reflections from the mucous layer and from the ends of moving cilia showed about the same frequency and amplitude pattern as the intracellular action potential oscillations at 40 degrees C and 50 degrees C. At lower temperatures, however, there was a considerable discrepancy between them. 2. Surface light reflections from mucociliary wave movements had the same frequency in two areas 20 mum apart, but amplitude variations were out of phase. 3. Intracellular oscillations with a frequency of 18-21 per sec and a maximum amplitude of 1 mV have been recorded. Amplitude variations indicating rhythmical frequency variations have also been recorded. 4. The mean frequency of the intracellular action potential oscillations showed no remarkable differences at 20 degrees C, 30 degrees C, 40 degrees C and 50 degrees C.

Lymphocyte locomotion III. ultrastructural studies of the lymphocyte traffic over the postcapillary venules of rat lymph nodes.

The direction of lymphocytes with amoeboid movement configuration (AMC) was evaluated by means of electron microscopy of ultrathin sections of post-capillary high-endothelium venules (HE-venules) from rat lymph nodes. Out of 104 lymphocytes, 68 lymphocytes appeared to be on their way towards the lumen of the HE-venule and 36 lymphocytes appeared to be moving away from the venular lumen at the moment of fixation. This difference, which was statistically significant (p=0.0024), is thought to reflect the relative size of the migration stream of lymphocytes at the moment of fixation.

A scanning microscope study of vocal cord carcinoma.

A scanning electron microscope study of cell surface topography in squamous cell carcinoma of the vocal cord has demonstrated characteristic changes. The malignant cells showed an irregular surface pattern with an abundance of microvilli-like structures of different sizes and shapes. In addition, the contact between the malignant cells was reduced. Normal vocal cord cells were closely associated and had a smooth surface with microridges.

[Controlled, clinical trial of isoprinosine administration to HIV-infected patients. Results of a Danish/Swedish multicenter study. The Scandinavian Isoprinosine Study Group]. / Kontrolleret, klinisk afprøvning af isoprinosin til HIV-smittede. Resultater af en dansk/svensk multicenterundersøgelse. The Scandinavian Isoprinosine Study Group.

The safety and efficacy of isoprinosine in HIV-infected individuals were assessed in a multicentre, randomized, double-blind, 24-week study phase, followed by an optional 24-week open treatment phase. The results of the double-blind phase have been reported separately. Of 866 HIV-seropositive individuals randomized, 832 were eligible for efficacy analysis. On completion of the double-blind phase, 596 patients started open treatment. All patients were evaluated with regard to progression to AIDS. Within 48 weeks, 10/412 patients (2.4%) assigned isoprinosine and 27/420 (6.4%) assigned placebo progressed to AIDS (p = 0.005; odds ratio: 2.8, 95% CI: 1.3-6.2). Intention-to-treat analysis showed identical results. No severe adverse reactions or toxicities were observed. We conclude that HIV-infected individuals without AIDS may be safely and effectively treated with isoprinosine.