Comparison of electron microscopic techniques for enumeration of endogenous retrovirus in mouse and Chinese hamster cell lines used for production of biologics.

Murine myeloma and Chinese hamster ovary cells are used widely in the manufacture of recombinant proteins for biopharmaceuticals. However, rodent cell lines express endogenous retrovirus, which necessitates appropriate design of purification processes to remove virus in excess of the calculated maximum retroviral load. Currently, electron microscopy is the method of choice for determination of retroviral titre in bulk harvest. In this study we compared three electron microscopy techniques to determine retroviral titre in bulk harvest. These were direct negative stain, negative stain after sucrose-density purification and thin section electron microscopy of pelleted supernatant. The study demonstrated that the level of C-type retrovirus associated with cells was predictive of the viral load in cell culture supernatants. The most accurate method for quantifying viral load was direct counting, followed by thin section of pelleted supernatant and negative stain after sucrose concentration. The most practical method was thin section of resuspended pelleted supernatant, which gave improved detection limits.

Degenerated tubules in the guinea pig testis after long-term vasectomy or sham operation.

The testes of eight unilaterally vasectomized and six sham-operated Dunkin Hartley guinea pigs were examined 3 years after operation by wax and resin histology and transmission electron microscopy. Degenerated tubules are reported that were common on the side of vasectomy but also found in the contralateral testes and in the controls. A central accumulation of macrophages, rich in phagocytosed debris including spermatozoal fragments, was surrounded by attenuated Sertoli cells, a markedly thickened basement membrane and myoid cells. At some sites macrophages impinged directly on the basement membrane. They probably represented highly degenerated seminiferous tubules. The study suggests that the response to injury of seminiferous tubules may show species variations. Macrophages did not feature in the degenerated seminiferous tubules we reported following vasectomy in the rat. However, the rat showed striking changes in the morphology of the basal laminae and myoid cells which did not occur in the guinea pig. Pathological changes have been reported in the human testis following vasectomy but their etiology is unclear. Studies in the guinea pig are enhancing understanding of the mechanisms and features of testicular damage.

Macrophages of the sperm granuloma 3 months after vasectomy in the Albino Swiss rat.

This study documents the appearance and arrangement of the macrophages of the sperm granuloma 3 months after vasectomy in the Albino Swiss rat. We found, as have others, that the macrophages form a distinct layer in the granuloma wall, external to the central mass of extravasated spermatozoa. Those closest to the spermatozoa showed much phagocytosis. Many had two, three, or more nuclei. The largest macrophages, giant cells of the foreign body and Langhans types, were generally farther from the sperm mass, but retained evidence of phagocytic activity in the form of sperm fragments. Macrophages, either mono- or multinucleate, frequently showed numerous surface microridges that interdigitated with those of neighboring cells. The microridges varied in length, but were of uniform thickness and cytoplasmic content and were rarely branched. Monocytes were frequently observed, indicating continuing significant recruitment of cells of the macrophage series to the granuloma. We also noted mononucleate cells in the connective tissue external to the macrophage layer. Their cytology suggested they were macrophages, but they showed much less phagocytic activity than those of the main macrophage layer and had distinctive aggregations of moderately electron-dense droplets.

Behavior of radial forearm skin flaps transplanted into the oral cavity.

Radial forearm skin flaps transplanted into the oral cavity in patients undergoing treatment for intra-oral tumors frequently acquire a gross appearance resembling that of buccal mucosa. This article reports studies of biopsies of the flaps in 53 patients, of which 21 showed changes at some stage in the follow-up period. The project aims to determine whether 1) the change in the epithelium to a mucosal form is genuine or reactive, 2) a creeping replacement of epidermis results from the surrounding buccal epithelium, 3) alterations in the epidermis could be associated with changes in the dermis, 4) the change is caused by radiotherapy. Examination of the biopsies by resin histology and transmission electron microscopy shows that the changes may be reversible: it suggests that they are reactive in nature, and related to the degree of dermal inflammation, rather than to the effect of radiotherapy. The dermis persists as such and is not replaced by buccal lamina propria.