C.Max Lang

Histologic Development of the Sheath That Forms Around Long-Term Implanted Central Venous Catheters

BACKGROUND Chronically implanted catheters often become covered with a thin, white adherent covering of tissue that has been referred to as a fibrin sheath. This tissue often interferes with catheter function. METHODS To chronicle the development of this sheath, rats were implanted with silicone rubber central venous catheters. Five rats were euthanized at 3,7, and 60 days postimplantation so that gross necropsy and histology could be performed on the catheterized vessels. RESULTS The coating that developed around the external portion of the catheter started as a dark red thrombus containing fibrin and progressed into vascularized, fibrous connective tissue. CONCLUSIONS The translucent to white sheath that forms around chronically implanted catheters is not composed of fibrin and is therefore not likely to be dissolved by fibrinolytic agents such as urokinase, streptokinase, or tissue plasminogen activator.

Human melanoma metastasis is inhibited following ex vivo treatment with an antisense oligonucleotide to protein kinase C-α

To determine whether alteration of PKC alpha expression would affect the metastatic potential of human melanoma cells, replicate cultures of C8161 cells were treated in vitro with a phosphorothioate antisense oligodeoxynucleotide (ODN) that specifically inhibits PKC alpha expression (ISIS-3521). Control C8161 cultures were treated with a scrambled sequence ODN, cationic liposomes or were left untreated. Northern blots demonstrated 70% inhibition of PKC alpha mRNA in ISIS-3521-treated cells compared to controls. Metastasis was suppressed by 75% when ISIS-3521-treated cells were injected intravenously into athymic mice. These results show that PKC alpha expression is important in the regulation of human melanoma metastasis.

Opioid receptor blockade during prenatal life modifies postnatal behavioral development.

The ontogeny of physical characteristics, spontaneous motor, and sensorimotor behaviors of preweaning rats, as well as ambulation and emotionality at weaning (day 21) were studied in rats exposed to 50 mg/kg naltrexone (NTX) or saline (controls) daily throughout gestation by maternal administration; all animals were cross-fostered to untreated mothers at birth. Morphine challenge tests and nociceptive measures revealed that this dosage of opioid antagonist blocked opioid receptors for 24 h. At birth and weaning, animals in the NTX group weighed 12 and 20%, respectively, more than control offspring. The age at which a specific physical characteristic, spontaneous motor behavior, or reflex initially appeared and the age at which 100% of the animals demonstrated a particular characteristic/behavior often were accelerated in animals prenatally exposed to NTX. The frequency of ambulation was subnormal in the NTX group, and the frequency and/or incidence of rearing, grooming, wet-dog shakes, and defecation were reduced from normal levels in these opioid antagonist-exposed rats. These results imply that interactions of endogenous opioid systems during embryogenesis are determinants of somatic, physical, and behavioral development in postnatal life.

In vivo effects of chronic treatment with [MET5]-enkephalin on hematological values and natural killer cell activity in athymic mice.

The role of endogenous opioids in immunological mechanisms was examined by subjecting athymic (nu/nu) mice to chronic injections of the opioid agonist [Met5]-enkephalin (MET) or continuous opioid receptor blockade with naltrexone (NTX). After 8 days of treatment, neither excess peptide nor deprivation of opioids from receptors had any effect on body weight, spleen index (spleen to body weight ratio), total and differential white blood cell counts, and natural killer (NK) cell activity in peripheral blood or splenic lymphocytes. At 28 days, chronic treatment with MET or NTX had no effect on any of these parameters with the exception of an elevation from controls in NK cell activity in peripheral blood in mice receiving NTX, and subnormal NK cell activity related to splenic lymphocytes in the MET group. These results suggest that chronic exposure to an opioid agonist, or persistent opioid receptor blockade, have little influence on a variety of immunological properties in athymic mice, suggesting that native opioids such as MET do not play a marked role in defense mechanisms in the athymic mouse.

Inhibition of human colon cancer by intermittent opioid receptor blockade with naltrexone

Nude mice inoculated with human colon cancer (HT-29) and receiving 0.1 mg/kg naltrexone (NTX) beginning immediately after tumor cell injection exhibited a marked retardation in tumorigenicity. This dosage of NTX, which blocked opioid receptors for 6-8 h/day, resulted in a delay of 2.4-fold in tumor appearance compared to control subjects. At the time (10 days) when all control mice had tumors, 80% of the mice in the 0.1 mg/kg NTX group had no signs of neoplasia. Binding capacity, but not affinity, of [3H][Met5]-enkephalin was reduced 85% of control levels in tumor tissue from mice of the 0.1 NTX group. Plasma, but not tumor tissue levels of [Met5]-enkephalin were elevated (2.5-fold) in contrast to control values. These results suggest that daily intermittent opioid receptor blockade with NTX provokes the interaction of opioids and receptors in the interval following drug availability, with opioids serving to inhibit tumorigenicity of human colon cancer.

Cardiovascular and respiratory effects of tiletamine-zolazepam

The combination of tiletamine and zolazepam is an important dissociative anesthetic-tranquilizer. However, little is known about the effects of this combination on the heart and respiration in rats. Adult, male rats anesthetized with tiletamine-zolazepam alone or tiletamine-zolazepam combined with xylazine or butorphanol were evaluated for changes in heart rate, mean arterial blood pressure, arterial blood pH, and blood gases during a 75-min period of anesthesia. Rats anesthetized with tiletamine-zolazepam had increased mean arterial blood pressure and less respiratory depression than did rats anesthetized with sodium pentobarbital. Tiletamine-zolazepam combined with xylazine at either dose produced bradycardia and a marked hypotension that persisted throughout the 75-min period. This combination produced respiratory depression comparable to tiletamine-zolazepam alone. The addition of butorphanol to tiletamine-zolazepam caused a transient hypotension and bradycardia. Tiletamine-zolazepam plus butorphanol produced a mild to severe respiratory depression that was dose and time dependent. These results demonstrate that: a) Tiletamine-zolazepam is cardiostimulatory, a property consistent with the known cardiovascular effects of other dissociative anesthetics; b) xylazine plus tiletamine-zolazepam is a potent cardiovascular depressant combination; and c) tiletamine-zolazepam plus butorphanol at specific doses is an anesthetic-analgesic combination with minimal effects on cardiovascular and respiratory function.

Antinociceptive properties of tiletamine-zolazepam improved by addition of xylazine or butorphanol

A combination of tiletamine HCl and zolazepam HCl is frequently used as an anesthetic, but little is known about the antinociceptive properties of tiletamine-zolazepam. The antinociceptive properties of tiletamine-zolazepam alone or combined with xylazine or butorphanol were determined in the adult male rate using the tail-flick test. Changes in tail-flick latency were determined at 15, 45, and 75 min after IP drug administration of sterile water, sodium pentobarbital, morphine, tiletamine-zolazepam, xylazine, butorphanol, and tiletamine-zolazepam plus xylazine or butorphanol. Tail-flick latency approximated 100% maximum possible effect (MPE) at 15-75 min postinjection in morphine-treated rats. Tiletamine-zolazepam, xylazine, and butorphanol alone, at any dose utilized, produced less than 50% MPE. However, the combination of tiletamine-zolazepam with butorphanol or xylazine increased tail-flick latency approximately three times greater than tiletamine-zolazepam alone. These results demonstrate that: a) consonant with earlier findings, analgesia and anesthesia are independent states; b) tiletamine-zolazepam is not an effective combination with respect to analgesia; but c) in concert with appropriate drugs, it can exhibit potent antinociceptive properties.

Development of Keratoacanthomas and Squamous Cell Carcinomas in Transgenic Rabbits with Targeted Expression of EJras. Oncogene in Epidermis

Activated ras genes have been frequently identified in both benign and malignant human tumors, including keratoacanthoma and squamous cell carcinoma. In this study, we developed two lines of transgenic rabbits in which the expression of EJras has been specifically targeted to the rabbit epidermal keratinocytes, using the upstream regulatory region of cottontail rabbit papillomavirus. All of the F1 transgenic progenies developed multiple keratoacanthomas at about 3 days after birth. The rabbits developed an average of 20 tumors, which usually reached the size of approximately 1 cm in diameter and then spontaneously regressed in about 2 months, similar to keratoacanthoma regression in humans. In addition, up to 18% of the rabbits then developed squamous cell carcinoma at about 5 months of age. The expression of EJras was detectable in all of the keratoacanthomas and squamous cell carcinomas. These results strongly support the involvement of the ras oncogene in both the initiation and regression of keratoacanthoma, and in the development of squamous cell carcinomas. These novel transgenic rabbits, with their consistent tumorigenic phenotype at an early age, high similarity to the human lesions, and easy accessibility for examination, manipulation, biopsy, and treatment, should provide a unique model system for studying ras activation-related tumor initiation, regression, and progression, and for evaluating antitumor therapies.

Methylation of cottontail rabbit papillomavirus DNA and tissue-specific expression in transgenic rabbits

Transgenic rabbits carrying multiple copies of CRPV genomic DNA were described previously (Peng et al. (1993) J. Virol. 67, 1698-1701). CRPV DNA was detectable in all tissues of the transgenic rabbits, however, the transcripts of CRPV genes only were found in skin and skin tumors. Tumor development was also restricted to skin. To study the mechanism involving tissue-specific expression of CRPV genes in rabbits, cellular DNAs, isolated from different normal tissues and skin tumors, were digested with the two isoschizomeric restriction endonucleases MspI (methylation resistant) and HpaII (methylation sensitive), respectively, and analyzed by Southern blot. CRPV DNA, especially its upstream regulatory region (URR), was extensively methylated in all normal tissues, but methylation was remarkably reduced in skin tumors. These data suggest that extensive methylation of CRPV genome, especially in the URR, might be a factor in controlling its tissue-specific expression.

Reinitiated expression of EJras transgene in targeted epidermal cells of transgenic rabbits by cottontail rabbit papillomavirus infection

Transgenic rabbits carrying the EJras oncogene have been established in our laboratory (Am. J. Pathol. 155 (1999) 315). The expression of the ras gene is targeted to the epidermal keratinocytes using the upstream regulatory region (URR) of the cottontail rabbit papillomavirus (CRPV). All of the transgenic rabbits develop keratoacanthomas at multiple sites in the skin at 2-3 days after birth, and the tumors spontaneously regress in 1.5-2 months. With regression of the keratoacanthomas, the rabbits appear normal and EJras expression is undetectable in their skin. To determine if CRPV infection would reinitiate the expression of the EJras transgene and make the rabbits more sensitive to tumorigenesis, the rabbits were infected with CRPV at 2 months of age when the keratoacanthomas had regressed. This study shows that CRPV infection of the transgenic rabbit skin could shorten the latency required for CRPV papilloma initiation, and significantly increase the tumor growth and persistence rate compared with non-transgenic rabbits. Furthermore, EJras expression became detectable in the CRPV induced papillomas in transgenic rabbits, but not in the papillomas of non-transgenic rabbits. These results indicate that CRPV infection is able to reinitiate the expression of the CRPV URR controlled EJras oncogene carried by the transgenic rabbits and that the expression of EJras can enhance the tumorigenesis of CRPV infection.