Fatal systemic candidiasis of gastrointestinal origin: an experimental model in mice compromised by anti-cancer treatment.

An experimental model of fatal systemic candidiasis originating from the gastrointestinal (GI) tract of compromised mice is presented. ICR female mice were compromised by a single anti-cancer treatment: irradiation (4 or 6 Greys i.e. 400-600 rads), methotrexate (MTX) (3 mg per mouse, intraperitoneally) or 5-fluorouracil (5FU) (200 mg kg-1, intravenously). Three days later, compromised and non-treated control mice were exposed to Candida albicans administered orally. Morbidity and mortality due to candidiasis were monitored for 30 days post-candidal inoculation. Increased and longer GI colonization was noted among the MTX and 5FU treated mice, or 6 Greys irradiated mice (up to 92.3% for over 30 days in anti-cancer treated mice). The stomach was found to be the major part of the GI tract involved in fungal colonization. A significant number (53.8-83.3%) of the anti-cancer treated mice developed systemic candidiasis originating from the GI tract, which was fatal in 30-80% of the infected animals. In systemically infected animals, candidal antigen was demonstrated in the serum, and fungal abscesses containing C. albicans were observed in the liver, kidneys and spleen. C. albicans was isolated from the infected organs. The severity of the infection, as reflected by the number of fungi in visceral organs, and by mortality during the 30 days post-candidal inoculation, indicated differences in the course and nature of the infection among the three treatment groups (i.e. MTX, 5FU, 6 Greys).(ABSTRACT TRUNCATED AT 250 WORDS)

Spinal cord response to laser treatment of injured peripheral nerve.

The authors describe the changes occurring in the spinal cord of rats subjected to crush injury of the sciatic nerve followed by low-power laser irradiation of the injured nerve. Such laser treatment of the crushed peripheral nerve has been found to mitigate the degenerative changes in the corresponding neurons of the spinal cord and induce proliferation of neuroglia both in astrocytes and oligodendrocytes. This suggests a higher metabolism in neurons and a better ability for myelin production under the influence of laser treatment.

Systemic effects of low-power laser irradiation on the peripheral and central nervous system, cutaneous wounds, and burns.

In this paper, we direct attention to the systemic effect of low-power helium-neon (HeNe) laser irradiation on the recovery of the injured peripheral and central nervous system, as well as healing of cutaneous wounds and burns. Laser irradiation on only the right side in bilaterally inflicted cutaneous wounds enhanced recovery in both sides compared to the nonirradiated control group (P less than .01). Similar results were obtained in bilateral burns: irradiating one of the burned sites also caused accelerated healing in the nonirradiated site (P less than .01). However, in the nonirradiated control group, all rats suffered advanced necrosis of the feet and bilateral gangrene. Low-power HeNe laser irradiation applied to a crushed injured sciatic nerve in the right leg in a bilaterally inflicted crush injury, significantly increased the compound action potential in the left nonirradiated leg as well. The statistical analysis shows a highly significant difference between the laser-treated group and the control nonirradiated group (P less than .001). Finally, the systemic effect was found in the spinal cord segments corresponding to the crushed sciatic nerves. The bilateral retrograde degeneration of the motor neurons of the spinal cord expected after the bilateral crush injury of the peripheral nerves was greatly reduced in the laser treated group. The systemic effects reported here are relevant in terms of the clinical application of low-power laser irradiation as well as for basic research into the possible mechanisms involved.

New methods of treatment of severely injured sciatic nerve and spinal cord. An experimental study.

It has been demonstrated that low-energy laser irradiation (LELI) applied simultaneously to the injured sciatic nerve and the corresponding segment of the spinal cord, accelerates the process of regeneration of the injured peripheral nerve. A beneficial influence of LELI was also observed when it was applied to the spinal cord following transection and implantation of a segment of an autologous sciatic nerve, but further studies are necessary to evaluate if real regeneration or only earlier distal cord automatism occurred. Both methods are proposed for treatment of peripheral nerve lesions (PNS) and spinal cord injuries.

Induction of gamma-glutamyl transferase by dexamethasone in cultured fetal rat hepatocytes.

Hepatocytes isolated as a relatively pure population from normal fetal rats were maintained in primary monolayer culture for 4-10 days. Hepatocytes exhibited a small increase in basal gamma-glutamyl transferase (GGT) activity over time. Exposure to dexamethasone (10(-6) mol/l) elicited a rise in GGT activity after a lag of 24 h. The presence of the steroid was necessary to maintain induction, and its removal resulted in reversal of induction. The maximal response was 2- to 3-fold, 72 h after exposure to the steroid. After this maximal response, a gradual decay in enzyme activity occurred, despite the continuous presence of the hormone. Actinomycin D or cycloheximide given prior to/or simultaneously with the steroid prevented the induction, thus suggesting that both RNA and protein biosynthesis are necessary for induction to occur.

Induction of protection against candidiasis in tumor-bearing mice by vaccination with Candida albicans ribosomes.

The aim of this study was to determine whether vaccination of tumor-bearing mice with ribosomes of Candida albicans would result in the induction of protective immunity. Therefore, (BB)F1 mice were inoculated intraperitoneally with the thymoma, EL-4, then immunized with ribosomes purified from C. albicans, and challenged intravenously with C. albicans to assess the protective response. Unimmunized mice, both normal and tumor-bearing, treated in the same manner served as controls. Both normal and tumor-bearing mice developed protective immunity in response to immunization with ribosomes. The protected mice had fewer colony-forming units in their kidneys 3 days after intravenous challenge than the unimmunized control animals, regardless of tumor status. Moreover, all unimmunized mice, tumor-bearing and normal, developed delayed hypersensitivity to the ribosomes, as well as ribosome-specific antibody, both of which were of the same order of magnitude. Therefore, the EL-4 thymoma does not suppress the immune system of mice, and they are capable of responding immunologically in a normal manner to C. albicans.

Liver damage induced by Oriental hornet venom sac extract at the level of subcellular fractions.

The hepatotoxic effect of venom sac extract (VSE) of the Oriental hornet has already been demonstrated using the well-known models of experimental toxicology: in vivo, isolated in situ and in vitro. The present work deals with a series of 48 rats treated daily with 5 mg VSE/kg body weight for 1-14 days. Serum activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and gamma-glutamyl transferase (GGT) were measured. Liver tissue fractionation was performed. Detailed information on the topographical and functional aspects of some enzyme changes was obtained in respect to the number of envenomations. The biochemical alterations are partially reversible. The biochemically proven liver damage induced by VSE correlated well with previous electron microscopic observations of damage to mitochondria and cell membranes.

Response of peripheral nerve to He-Ne laser: experimental studies.

Low-energy He-Ne laser irradiation (LELI) was found to affect the electric activity and morphology in both intact and severely injured peripheral nerves in rats. Action potential (AP) in the healthy nerve increased by 33% following a single transcutaneous irradiation. Similar irradiation in crushed nerves caused AP to increase significantly over the AP of nonirradiated crushed nerve. Morphological observations revealed that a laser-irradiated injured nerve had diminished scar tissue as compared to an injured but not an irradiated nerve.

Gamma-glutamyltranspeptidase in murine lymphomas.

Five murine lymphoma cell lines were assayed for the content and activity of gamma-glutamyltranspeptidase (GTT). All lines [S49; L-12; 230-23-8 (C57 Black); 2M3 and RA3-2C2] contained detectable amounts of GGT. The specific activities of GGT were low and ranged between 1.2 and 2.3 mU/mg protein in cells growth in vitro. A highly malignant variant of the S49 line was also grown in vivo in BALB/c mice. This subline invariably produces both solid and ascitic tumors with infiltrations into the pancreas, liver and spleen. GGT levels in the tumor cells were low and independent of tumor type (solid, ascitic), location, passage number or inoculum size. Infiltrations of S49 tumor cells in liver and spleen were invariably GGT--negative as judged by histochemical examination. GGT activities in suspension cultures prepared from solid, as well ascitic tumors were low. Occasional high GGT activity of solid tumors was due to the presence of pancreas cells in them. The only host tissue significantly responding to the presence of tumors by elevated GGT levels was the liver. Compilation of data from this study and those of others clearly indicates that low GGT level is a typical property of tumors originating in the immune system.

Histopathological changes in rat liver following repeated vespine envenomation.

The histopathological picture of rat liver was studied after repeated envenomation with hornet (Vespa orientalis) venom sac extract (VSE). Light microscopical and histochemical investigation showed foci of necrosis of the hepatocytes, a decrease in glycogen content and in succinic dehydrogenase activity, some fat infiltration and an increase in alkaline phosphatase activity. Electron microscopy showed a decrease in the number of mitochondria per cell as well as a decrease in their cristae, enlargement of bile canaliculi, and destruction of the endothelium of the sinusoids bringing the cytoplasm of the necrotic hepatocytes in contact with the lumen of the sinusoid. The rat liver is proposed as a model for the study of pathological effects of animal toxins.

Protection against systemic infections with various Candida species elicited by vaccination with Candida albicans ribosomes.

This study investigated whether subcutaneous vaccination of mice with ribosomes from Candida albicans strain CBS 562 would also provide protection against infections by other isolates of Candida. Experiments with a total of 628 mice demonstrated that vaccination induced significant protection against heterologous C. albicans (serotypes A and B) and C. tropicalis isolates in terms of their 30 day survival rates. In all instances, however, protection was lower than that obtained against the homologous strain. In addition, a significant decrease in fungal colonization of the kidneys was found in immunized animals as compared to the non immunized controls. Cell-mediated immune responses against cytoplasmic extracts of the various fungi, as detected in vivo by the foot pad swelling test and in vitro by the lymphocyte transformation assay, were induced by the C. albicans ribosomal vaccination. The results show it is possible to induce cross protection to various Candida species by immunization with C. albicans ribosomes.

Systemic candidiasis in mice immunized with Candida albicans ribosomes.

In view of our previous findings that vaccination of mice with Candida albicans ribosomes protects them against experimental systemic candidiasis, the aim of this study was to investigate the effect of this vaccination on the course of infection in immunized animals. Since the kidney is the major target in systemic candidal infection, we concentrated in this research on studying the histopathology and determining quantitatively the candidal colonization of this organ. The experiments were carried out at various time intervals after intravenous inoculation with live C. albicans. The colonization of kidneys in immunized mice was markedly lower than that in controls. The maximal difference in renal colonization between immunized and non immunized animals was observed when relatively low challenge doses were used. The inhibition of candidal multiplication in immunized mice seemed to be correlated to their increased resistance against lethal challenge, as expressed by a significantly higher survival rate. Histopathological changes and fungal elements were found in kidneys of control mice as early as 20 h post infection, while the kidneys of immunized mice did not seem affected by the disease. Moreover, even 3 days post infection, the kidneys of vaccinated animals still seemed normal. In conclusion, apparently the ribosomal vaccination leads to diminished colonization of the major site of infection in candidiasis, thus affording protection to the immunized animals against these infections.

Culture of thymic epithelial cells from mice and age-related studies on the growing cells.

Culture of epithelial cells from the thymus of mice was achieved in a medium modified to favor epithelial growth while inhibiting proliferation of fibroblasts. Epithelial cells were identified by the presence of desmosomes in electron microscopic preparations and by antibody to intermediate filaments containing keratin. Morphologically, the cells thus positively identified displayed two main patterns: carpets of large flat cells resembling paving stones which are confluent along the length of their membranes, and networks of cells interconnected by long cytoplasmic processes. These two types of cells were dominant in cultures derived from mice of all ages tested (newborn to nine months) but the relative proportion of each type appeared to change with the age of the donor mice and also with the concentrations of cortisone in the culture medium. Autoradiography revealed that the cultured cells were dividing, and that (in the presence of cortisone) the rate of DNA synthesis was decreased in a portion of the epithelial cells derived from mice in which thymic involution was already underway.

Cell-mediated immunity following experimental vaccinations with Candida albicans ribosomes.

The aim of the present study was to ascertain whether cell-mediated immunity (CMI) is induced in animals by vaccination with Candida albicans ribosomes. Delayed type hypersensitivity (DTH) was detected in vivo in ribosome-vaccinated mice and guinea pigs by the footpad swelling and skin tests, respectively. The observed DTH was similar to that induced by live C. albicans organisms. A lymphocyte transformation assay was used for in vitro detection of CMI. The tritiated thymidine incorporation assays revealed that spleen lymphocytes from mice immunized with C. albicans ribosomes were stimulated by the ribosomal antigen. The findings establish that C. albicans ribosomes are able to induce CMI in experimental animals.

In vitro and in vivo adherence of Candida albicans to mucosal surfaces.

In continuation of a previous study, chitin soluble extract (CSE) and anti-Candida albicans antibodies were found to inhibit the in vitro adherence of C. albicans to human vaginal epithelial cells by 60-70%. Pretreatment of epithelial cells with chitin, CSE or N-acetyl-glucosamine (NAG) decreased the percentage of adherence, while pretreatment of yeasts had no such effect, indicating that chitin or its derivatives may be involved in mediating the adherence of C. albicans. In vivo attachment of C. albicans to epithelial cells was studied in an experimental murine vaginitis created by inoculating mice intravaginally with yeasts at the oestrus stage. Attachment of the yeasts to exfoliated vaginal epithelial cells was followed microscopically, and the course of infection was assessed histopathologically in tissue sections at various times post-yeast inoculation. The infection rate at 24-h post-yeast inoculation was approximately 50%. Attempts were made to block in vivo attachment and prevent infection by pretreating 208 mice with either CSE, NAG, mannan or phosphate-buffered saline (PBS) prior to inoculation with yeasts. The infection rate among mice pretreated with mannan or PBS was 41.6% and 43.8%, respectively. Among CSE- or NAG-pretreated animals, the rate ranged from 0-9%. Treatment of mice with CSE or NAG after inoculation of the yeasts did not prevent infection. The data from the in vivo experiments reveal that pretreatment of animals with chitin derivatives blocks attachment of yeasts to the vaginal mucosal surfaces and leads to the prevention of vaginal infection in an experimental model.

Oriental hornet venom enhances wound healing and repair in rat skin, possibly through its collagenolytic activity.

Oriental hornet (Vespa orientalis) venom sac extract (VSE) was topically applied to experimental wounds produced on the back skin of rats in order to observe histologically its effect on the healing process. In other studies the collagenolytic effect of VSE was investigated. The results clearly indicated that application of VSE enhanced the process of wound healing. Two weeks after wound induction a thick epidermis and maturation of the dermal connective tissue were seen in the experimental animals, whereas in the controls the dermis was thin and still showed granulation tissue, and the epidermis was thin. Incubation of VSE with purified neutral salt-soluble collagen (NSSC) labeled by reduction with [3H]NaBH4 brought about degradation of the substrate. It is assumed that VSE enhances the process of wound healing in the skin, probably due to its collagenolytic effect.

The effect of chloroquine upon the developing eye of the chick embryo.

The effect of chloroquine on the eyes of 8 (8DE) and 15 (15DE) day old chick embryos has been studied. Microphthalmus was observed in 30% of the 8DE and 80% of the 15DE. The most conspicuous changes were seen in the retina. In the 8DE, chloroquine mostly prevented the development of photo-receptors with subsequent adhesions between the pigment epithelium and external granular layer, or caused a destruction of the pigment epithelium with subsequent chorio-retinal adhesions. In the 15DE, chloroquine caused destruction of the whole retina. The choroid showed a hyperproliferation of the melanocytes in the 8DE, and edema in the 15DE. The lens showed moderate vacuolisation in the 8DE and severe cataract formation in the 15DE.

The effect of chloroquine upon the developing lens.

Applied to the developing lens of the 14-day-old chick embryo, in organ culture conditions, chloroquine prevented the elongation of the primary lens fibres, destroyed the equatorial ones and provoked vacuolisation and/or destruction in the epithelial cells.