Protection from chemotherapy-induced alopecia in a rat model.

Alopecia (hair loss) is among the most distressing side effects of cancer chemotherapy. Little progress has been made, however, in its prevention or treatment, partly because of the lack of suitable experimental model. In recent work on the treatment of myelogenous leukemia in the rat, the following observations were made: (i) treatment of 8-day-old rats with cytosine arabinoside consistently produced alopecia, and (ii) ImuVert, a biologic response modifier derived from the bacterium Serratia marcescens, uniformly produced complete protection against the alopecia. In subsequent experiments, both cyclophosphamide and doxorubicin also produced alopecia in this model, and the doxorubicin-induced alopecia was prevented by treatment with ImuVert. The potential relevance of these observations to chemotherapy-induced alopecia in the clinical setting should be examined.

Programmed cell death in terminally differentiating keratinocytes: role of endogenous endonuclease.

Mammalian epithelium is a tissue with a very high turnover rate. It consists of a rapidly proliferating compartment comprising basal and suprabasal keratinocytes, from which cells move upwards while differentiating into granular keratinocytes. The end product is shed as an enucleate corneocyte, which has a mechanically rigid, chemically resistant cross-linked keratinous envelope. The loss of the nucleus occurs specifically in the granular keratinocyte layer; here, cells with the classical apoptotic morphology of clumped and marginated condensed chromatin may be observed. This morphology is characteristic of "programmed" cell death in other systems, of which the lymphocyte has been most extensively studied, and is associated with the cleavage of nuclear DNA into nucleosome-sized fragments. In the present investigation we separated newborn mouse skin into basal and granular keratinocyte fractions and examined the state of the DNA in each fraction. Our results indicate that cells in the basal layer, while their DNA is perfectly intact, are preparing to die. DNA fragmentation is initiated in the granular keratinocyte layer and is identical in pattern to that seen in other examples of programmed cell death.

The myelopoietic effects of a Serratia marcescens-derived biologic response modifier in a mouse model of thermal injury.

The proliferative defects observed in phagocytic stem cells after major thermal injuries may be caused by an inadequate production of colony-stimulating factors (CSFs), a family of hemopoietic cytokines necessary for the production and function of granulocytes and monocytes. In this study a biologic response modifier (S-BRM) consisting of sized vesicles derived from the cell membrane and ribosomes of Serratia marcescens was investigated in a mouse model of thermal injury to determine its ability to augment postburn myelopoiesis. Treatment of burned mice with S-BRM was well tolerated and was associated with statistically significant increases in absolute numbers of circulating granulocytes and monocytes compared with burned mice receiving saline solution. In addition, the size of the splenic myeloid stem cell compartment, as measured by granulocyte-macrophage stem cell colony formation in soft agar, was markedly expanded. Finally, plasma levels of CSF were increased significantly in burned mice receiving S-BRM but were not elevated in burned littermates treated with saline solution. These data suggest that production of CSF is suboptimal after thermal injury and S-BRM is capable of up-regulating postburn myelopoiesis by causing the release of CSF into the systemic circulation.

Pilot study to assess the effects of early flea exposure on the development of flea hypersensitivity in cats.

This pilot study was to determine if early oral flea exposure reduces the incidence of flea allergy dermatitis (FAD) in cats. Eighteen kittens, assigned to three groups, received no flea exposure, oral flea exposure or flea infestation for 12 weeks. Then all the kittens were exposed continually to fleas for 31 weeks. Sensitization was monitored using intradermal testing (IDT), in vitro measurement of anti-flea saliva immunoglobulin E (IgE) and development of FAD. There was no statistically significant difference between groups in IDT reactions, in vitro data or clinical scores. The development of FAD was not associated with the presence of anti-flea saliva IgE. However, the development of a delayed reaction to flea bite was associated with symptoms after flea exposure. Although not statistically significant, the FAD scores in the oral group were lower than in the controls. Further studies are required to determine the role of oral flea exposure in the development of FAD in cats.

Augmentation of natural killer cell activity by ImuVert: a biological response modifier derived from Serratia marcescens.

The natural killer (NK) cell activity of peripheral blood mononuclear cells (PBMC) from healthy human volunteers was studied following in vitro incubation with ImuVert, a biological response modifier derived from the bacterium Serratia marcescens. Exposure of these cells to ImuVert for as little as 10 minutes followed by an additional incubation in vitro of at least 12 hours and optimally 18 hours resulted in a substantial, consistent, and dose-dependent augmentation of NK cell activity against K562 tumor cells. Additional studies indicate that the augmented cell expressed the leu 11 cell surface marker and that peripheral blood monocytes were essential in the induction of augmented NK cell activity but were not the effector cell of NK activity.

Augmentation of ADCC and cytotoxic T-cell activity with ImuVert.

We previously reported that natural killer cell activity of peripheral blood mononuclear cells (PBMC) from healthy human subjects was augmented following in vitro incubation in ImuVert, a biologic response modifier derived from the bacterium Serratia marcescens. In the current investigation, we found that exposure of PBMC to ImuVert, 3-40 micrograms/ml, for 18 hr, resulted in significant and dose-dependent augmentation of three other types of cell-mediated cytotoxicity: K cell-mediated antibody-dependent cellular cytotoxicity (ADCC), monocyte-mediated ADCC, and cytotoxic T-cell activity against allogeneic PBMC. These and previous findings suggest that ImuVert may have a broad range of stimulatory effects on immune function.

Treatment with Imuvert aborts development of chloroleukemia in newborn rats.

We have previously demonstrated that the successful transfer of rat chloroleukemia (Mia C51) cells to newborn rats is related to the host's inability to generate adequate levels of differentiation factor (DF). Thus, when the appropriate amount of DF was injected into rats bearing MIA C51 cells, the development of chloroleukemia was aborted. In the present study, we provide evidence that stimulation of endogenous differentiation activity (DA) production by the administration of a biologic response modifier (Imuvert) will like-wise abort the development of chloroleukemia. Imuvert at 50 micrograms/ml had no direct effect on growth, viability, or differentiation of MIA C51 cells. However, when monocytes from young rats or adult rats were stimulated with Imuvert in vivo or in vitro, there was significant increase in DA production. Treatment of young rats with Imuvert aborted the development of chloroleukemia from transplanted MIA C51 cells. It is concluded that stimulation of endogenous DA production may provide a potentially useful approach in the treatment of leukemia.

A recombinant group 1 house dust mite allergen, rDer f 1, with biological activities similar to those of the native allergen.

Serum IgE directed against Der f 1, a protease found in the feces of Dermatophagoides farinae, correlates well with allergic sensitization to house dust mite in humans and is a risk factor for developing asthma. Native Der f 1 (nDer f 1) is produced as a pre-pro form and processed to an approximately 25-kDa mature form. We have expressed recombinant forms of Der f 1 (rDer f 1) in Pichia pastoris using AOX1-promoter expression vectors. Fusion of either the pro-enzyme form or the mature form to the Saccharomyces cerevisiae alpha factor pre-pro sequence resulted in secretion of the mature form of the protein from P. pastoris. The secreted protein was heterogeneously glycosylated at a single N-glycosylation site and had an apparent molecular mass of 35-50 kDa. Both the alpha factor signal peptide and the pro-enzyme region were efficiently processed during secretion. A version of the pro-enzyme with a mutated consensus N-linked glycosylation site was secreted from P. pastoris as a mature, unglycosylated, approximately 25-kDa protein. The IgE binding activity of this unglycosylated rDer f 1 was similar to that of glycosylated forms produced by P. pastoris and to nDer f 1 obtained from mites. Thus, oligosaccharides are not required for secretion from P. pastoris or for IgE binding in vitro. Recombinant and native versions of Der f 1 displayed protease activity on casein zymogram gels. The availability of a highly purified recombinant Der f 1 will facilitate experimental and clinical studies of mite allergy.