Cytotoxic effects of pamidronate on monolayers of human intestinal epithelial (Caco-2) cells and its epithelial transport.

Pamidronate (APD) is a new drug in the treatment of osteolytic bone diseases. Caco-2 cells were used to study the cytotoxic effects of APD on intestinal epithelium and also the transport (mechanism) of APD across the intestinal epithelium. We investigated the cytotoxic effect of APD by combining two spectrophotometric assays [neutral red (NR) uptake and lactate dehydrogenase (LDH) release] with a morphological assay (electron microscopy). The amount of APD transported across the Caco-2 monolayer was measured by HPLC. The permeability of the monolayer was studied by determining the transepithelial electrical resistance (TEER). The results show that after exposing the Caco-2 cells to increasing concentrations of APD [dose range calculated on the basis of relevance to the oral dose administered to patients] the NR uptake decreased while LDH loss increased, which is indicative of a cytotoxic effect of APD. Ultrastructural alterations, including a widening in intercellular spaces and, at higher doses, complete cell death, were observed. The transport percentage of nontoxic doses of APD was low, while the TEER decreased with increasing doses of APD. In conclusion, APD is cytotoxic for Caco-2 cells. As the transport percentage of nontoxic doses of APD is low and APD reduces the TEER, it is hypothesized that APD is transported paracellularly.

On the relationship between X-bodies and symptom development in plants infected with different tobamoviruses.

The relationship between systemic mosaic symptoms and the occurrence of viral 126-kDa protein in X-bodies was studied in tobacco infected with the tobacco mild green mosaic virus (TMGMV) strains U2, U5, and ribgrass mosaic virus (RMV) strain HR, and in other plant species infected with tobacco mosaic virus (TMV) strain W U 1. Strains U2, U5, and HR coded for proteins of 126, 126, and 130 kDa, respectively, but these were not recognized by antisera against the corresponding protein from W U 1. Only the HR 130-kDa protein reacted with an antiserum raised against a peptide of amino acids 849-863 from the sequence of W U 1. Electron microscopic analysis established the presence of virus clusters in the cytoplasm, as well as in chloroplasts, in leaf tissue infected with U 2 or U 5, and adjacent to nuclei and chloroplasts in scattered cells infected with HR. X-bodies were not detected after infection with any of these strains, but were large and adjacent to nuclei in W U 1-infected tomato displaying severe mosaic symptoms. Large X-bodies were detected near nuclei in W U 1-infected tomato displaying severe mosaic symptoms, but none were detected after infection of tobacco with any of the other tobamoviruses. The induction of X-bodies appears to be characteristic of some tobamovirus only and, at best, can only be associated with, rather than causative of, the severity of symptoms induced by those viruses.

Accumulation of the 126 kDa protein of tobacco mosaic virus during systemic infection analysed by immunocytochemistry and ELISA.

Systemic infection of tobacco with tobacco mosaic virus (TMV) strain WU1, is accompanied by massive accumulation of the virus-coded non-structural 126 kDa protein in X-bodies. The development of X-bodies and the time course of the increase in 126 kDa protein in systemically infected leaves were analyzed by immunocytochemistry and ELISA, respectively, using an antiserum raised against a fusion protein of beta-galactosidase and part of the 126 kDa protein. The ELISA assay developed enabled routine detection of viral 126 kDa (as well as 183 kDa) protein in samples of less than 5 mg of systemically infected leaves. Plants were inoculated by differential temperature treatment, whereafter the accumulation of 126 kDa protein was related to viral multiplication, the development of X-bodies and the formation of symptoms. Both 126 kDa protein and coat protein became detectable between 40 and 66 h after transfer of the plants and increased in parallel up to 200 h. Vein clearing was visible at 66 h, followed by mosaic in the newly developed leaves at 112 h. By electron microscopical analysis small X-bodies, weakly labelled with antibodies against the 126 kDa protein, were detected as early as 24 h after transfer. At this stage they were not associated with nuclei. Thereafter, however, X-bodies increased in size and 126 kDa labelling density, and were increasingly often observed attached to nuclei. In emerging leaves that developed mosaic symptoms, X-bodies were associated with nuclei already at an early stage. These observations are consistent with the hypothesis that association of X-bodies with nuclei may lead to symptom induction, when the leaf is invaded by the virus early in its development.

A histological study of tissue response to simulated cleft palate surgery at different ages in beagle dogs.

The response of maxillary tissues to mucoperiosteal manipulation at different ages was studied microscopically early after operation and at maturity. Thirty-two Beagles were divided into three experimental groups, a sham-operated group and a control group. At the age of 6, 16 or 25 weeks in the experimental groups an elliptical mucoperiosteal flap was excised in the midsagittal region of the palate, relaxation incisions were made and the mucoperiosteum was elevated and closed in the midline leaving two areas of denuded bone adjacent to the teeth. The animals were killed 1, 2 or 3 weeks after the operation or at the age of 37 weeks and serial sections of the jaw tissues were examined. Two weeks after the operation, scar had formed, its lack of elastic fibres indicating a more rigid type of tissue. The palatal bone showed rapid trabecular deposition. Cervico-palatal and apico-buccal bone resorption in the alveolar socket indicated medial tipping of the teeth. The different soft tissue composition persisted until maturity. In the areas where scar tissue had developed, attachment by means of Sharpey's fibres from the mucoperiosteum to the palatal bone was found at the age of 37 weeks. Thus the different mechanical properties of scar tissue adjacent to the teeth and its attachment to the palatal bone may influence the growth and development of dento-alveolar structures.

Maxillary arch dimensions after palatal surgery at different ages on beagle dogs.

Changes of maxillary arch dimensions were studied longitudinally until maturity, after palatal surgery was performed on 37 beagle dogs at different ages. The dogs were divided into three experimental groups, a sham group, and a control group. At the age of 6, 16, or 25 weeks in the experimental groups, we created a soft-tissue cleft in the medial region of the palate, made relaxation incisions, and elevated and closed the mucoperiosteum in the midline, thus leaving two areas of denuded bone adjacent to the dentition. We made dental casts regularly until the age of 37 weeks, and studied maxillary arch dimensions. Surgery performed before or during the transition of teeth did not change maxillary arch dimensions in the deciduous dentition, but after the transition, the increase in arch width in the pre-molar region was reduced. Surgery performed after completion of the permanent dentition did not change maxillary arch dimensions. Delay of palatal surgery until the transition of teeth was completed favored normal development of the maxillary dental arch.

Association of viral 126 kDa protein-containing X-bodies with nuclei in mosaic-diseased tobacco leaves.

During the development of systemic mosaic symptoms in tobacco mosaic virus (TMV)-infected tobacco, the viral non-structural 126-kDa-protein was present among the chromatin-associated proteins in fractionated leaf homogenates [Van Telgen HJ et al. (1984) Virology 143: 612-616]. Using an antiserum raised against a fusion protein of beta-galactosidase and part of the 126-kDa-protein of TMV, this viral protein was detected by immunoelectron microscopy in X-bodies in infected tissue. No labelling of nuclei was apparent. However, in embedded purified nuclear preparations from systemically infected leaves amorphous structures, most likely X-bodies, were present and specifically labelled. In contrast, using antibodies against tobacco histones, only nuclei were labelled. Antibodies against viral coat protein labelled crystalline virus inclusions in the cytoplasm and did not react with nuclei. Light microscopic analysis indicated that X-bodies were almost always associated with nuclei. Thus, the presence of X-bodies in nuclear preparations appeared to result from adherence of the X-bodies to the nuclei.

Growth of the maxilla after soft tissue palatal surgery at different ages in beagle dogs: a longitudinal radiographic study.

This study investigated maxillary growth after palatal surgery at different ages in beagle dogs. Soft tissue clefts were created in the median region of the palate at the age of 6 (group 1), 16 (group 2), and 25 weeks (group 3). Relaxation incisions were made, and the mucoperiosteum was elevated and closed in the midline, leaving two areas of denuded bone adjacent to the dentition. Metallic implants were placed in the palate, and dorso-ventral and lateral radiographs were made until the age of 37 weeks to study sutural growth and overall growth of the maxilla. In groups 1 and 2, growth in the midpalatal suture was less, compared to the control, from 16 to 25 weeks and from 16 to 37 weeks of age. In group 2, less increase was also found in the transverse palatal sutures. Group 3, the sham group, and the control group did not show significant differences. None of the dimensions measured on the lateral radiographs showed any significant differences for any group. It was concluded that palatal surgery as performed in this study has no influence on vertical and antero-posterior maxillary growth; only transverse palatal growth is influenced to a minor degree, especially when surgery is performed before or during the eruption of the permanent posterior teeth.

Mucoperiosteal migration after palatal surgery in beagle dogs. A longitudinal radiographic study.

In this study, the effect of palatal surgery on the mucoperiosteum was investigated radiographically. 33 beagle dogs were used, divided into 3 experimental groups, a control group and a sham operated group. At the age of 6, 16 or 25 weeks in the experimental groups, a soft tissue defect was created in the median region of the palate by excising a standardized elliptical mucoperiosteal flap. This defect was closed according to the Von Langenbeck technique. Metallic implants were placed in the mucoperiosteum. Dorso-ventral radiographs were taken until the age of 37 weeks. Increments of distances between these mucoperiosteum markers were calculated. Wound contraction in the denuded areas, recorded as the approaching of the opposite metallic implants followed the same pattern for all experimental groups and was restricted to the first 2 experimental weeks. In all experimental groups, the anterior and posterior overall palatal width diminished during the early wound healing compared to the control group. The largest initial effect was found in the anterior region of the palate. The margins of the mucoperiosteal tissue in the median region of the palate moved towards the denuded areas during the first postoperative week. Palatal surgery as performed did not influence antero-posterior distances on the palatal mucoperiosteum.

Wound healing of the palatal mucoperiosteum in beagle dogs after surgery at different ages.

The aim of this study was to investigate macroscopic wound healing after palatal surgery at three different ages. A total of 37 beagle dogs was used, divided into three experimental groups, a control group and a sham-operated group. Palatal surgery was performed at the age of 6, 16 or 25 weeks respectively. The animals of the sham group and the control group were studied from the age of 6 weeks on. All animals were studied longitudinally over a period of six weeks. The three experimental groups were compared mutually and the youngest experimental group was compared with the sham group and the control group. Clinically the wound healing in the median region was complete after two weeks in all animals. In the denuded areas the wound healing continued for the youngest experimental group for 2 to 3 weeks postoperatively and for the two older age groups for 4 to 5 weeks postoperatively. The wound contraction in the denuded areas, recorded as the increasing approximation of the opposite tattoo points was larger in the two older age groups than in the youngest one and was restricted mainly to the first postoperative week. This effect seemed to be permanent because no compensating increase in distance was found later on. It was concluded that shortly after operation wound contraction was mainly responsible for the reduction of the surface area of the denuded bone, but later on, epithelial cell proliferation was the predominant factor.

A study of maternal ECG characteristics before and during intravenous tocolysis with beta-sympathicomimetics. Effects of i.v. tocolysis on maternal ECG characteristics.

Severe maternal complications during beta-mimetic therapy have been reported. In a study of maternal complications related to intravenous tocolysis, which covering a period of 2 years, we paid special attention to the maternal ECG before and during treatment. There was a high prevalence of pretreatment ECG changes: tachycardia (23.6%), disorders of impulse conduction (43.6%), ST-depression (14.5%) and disorders of repolarization (43.6%). During intravenous beta-mimetic therapy there was an increase in the prevalence of tachycardia, prolonged QT-time and disorders of repolarization. We also studied the course in time of the different ECG characteristics during treatment. With regard to the ST-depression, a possible physiological adaptation to the beta-mimetic drug is described. We could not find this possible adaptation with regard to the other ECG characteristics. None of the women in the studied group showed clinical signs of myocardial ischemia, notwithstanding the high prevalence of ECG changes. We conclude that the ECG criteria for discontinuation of tocolytic therapy need re-evaluation.