Forty years on in general practice.
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Biomedical subjects
Publications and source records attributed to R Ross.
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Two pools of rabbit anti-guinea pig neutrophil serum (ANS) were prepared using an intravenous (ANS I) or subcutaneous (ANS II) route of immunization with proteose peptone-stimulated peritoneal exudate neutrophils (PMNs) from albino guinea pigs. In vitro, both pools of ANS contained high titers of agglutinating antibodies to neutrophils and lower titers against lymphocytes and red cells. Agglutinins against all three cell types could be selectively removed by absorption. The in vivo hematologic effects of both the absorbed and unabsorbed antisera were examined after intraperitoneal administration, and the effects of ANS on neutrophils in blood, bone marrow, and peritoneal cavity were examined by light and electron microscopy of spleen, liver, lung, lymph node, buffy coat, bone marrow and pellets of peritoneal cells removed at various time intervals within 24 hours. Injection of either antisera caused a rapid decrease in circulating neutrophils and lymphocytes, which reached their lowest levels within 12 hours. Neutrophils that disappeared from the circulation were sequestered primarily in liver and spleen where they were phagocytized, as morphologically intact cells, by macrophages and then rapidly digested. Immature bone marrow neutrophils as young as early myelocytes were ingested by macrophages in the marrow at 6 hours or later after ANS. Neutrophils that were phagocytized were apparently opsonized by ANS since there was no ultrastructural evidence of neutrophil lysis in blood or bone marrow after ANS treatment. However, both lysed and ingested neutrophils were observed in the peritoneal cavity. Absorption of ANS with neutrophils removed the ability of the serum to produce neutropenia. However, absorption of ANS with lymphocytes did not alter the lymphopenia produced by the antiserum. The fate of lymphocytes leaving the peripheral circulation was not apparent. Lymphocytes did not accumulate in liver or spleen sinusoids and were not ingested by macrophages in these organs, as were the neutrophils. There was no evidence of paracortical depletion or extensive phagocytosis of lymphocytes in lymph nodes after ANS, as other investigators have reported after administration of antilymphocyte serum.
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Healing skin wounds were studied in a series of parabiotic rats. The femurs of one parabiont of each pair were shielded whilst both animals were given 800 r from a Co(60) source. The animals were wounded 3 days after irradiation. Each animal with partially shielded marrow was then given tritiated thymidine intraperitoneally daily while the cross-circulation was arrested by clamping. After the thymidine-(3)H had cleared the blood, the clamp was released. Animals were sacrificed, and wounds were prepared for radioautography 1, 2, and 6 days after wounding. In the wounds of the shielded animals thymidine-(3)H was observed in epidermis, endothelium, leukocytes, fibroblasts, and mast cells. Only neutrophilic leukocytes, monocytes, and lymphocytes were labeled, as determined by light and electron microscope radioautography, in the wounds of each nonshielded parabiont. None of the many fibroblasts present were found to contain label in the wounds of the nonshielded parabionts through the 6 day period. These observations provide further evidence that wound fibroblasts do not arise from hematogenous precursors and, therefore, must arise from adjacent connective tissue cells.
The two morphologically different constituents of the mature elastic fiber, the central amorphous and the peripheral microfibrillar components, have been separated and partially characterized. A pure preparation of elastic fibers was obtained from fetal bovine ligamentum nuchae by extraction of the homogenized ligament with 5 M guanidine followed by digestion with collagenase. The resultant preparation consisted of elastic fibers which were morphologically identical with those seen in vivo. The microfibrillar components of these elastic fibers were removed either by proteolytic enzymes or by reduction of disulfide bonds with dithioerythritol in 5 M guanidine. The microfibrils solubilized by both methods were rich in polar, hydroxy, and sulfur-containing amino acids and contained less glycine, valine, and proline than the amorphous component of the elastic fiber. In contrast, the amino acid composition of the amorphous component was identical with that previously described for elastin. This component demonstrated selective susceptibility to elastase digestion, but was relatively resistant to the action of other proteolytic enzymes and to reduction. These observations establish that the microfibrils consist of a different connective tissue protein (or proteins) that is neither collagen nor elastin. During embryologic development the microfibrils form an aggregate structure before the amorphous component is secreted. These microfibrils may therefore play a primary role in the morphogenesis of the elastic fiber.
The three species of the genus Bordetella-B. pertussis, B. parapertussis, and B. bronchiseptica-have many antigens in common. Studies on representative strains of these species have shown that there are only a few specific antigens in each species. Whole-cell vaccines and extracts from B. pertussis contained specific mouse-protective antigen and a histamine-sensitizing factor. In addition, whole-cell vaccines and some saline extracts protected mice against intracranial challenge with B. bronchiseptica. Cells and a saline extract of B. parapertussis also protected against B. bronchiseptica but not against B. pertussis. Whole cells of B. bronchiseptica protected against B. bronchiseptica, but only one of three saline extracts protected against this challenge. Neither whole cells nor saline extracts from B. bronchiseptica protected against B. pertussis. The antigen in B. pertussis responsible for cross-protection against B. bronchiseptica was less resistant to heat than the protective antigen in B. bronchiseptica. Since histamine-sensitizing factor was not detected in B. bronchiseptica or B. parapertussis cells or extracts, this factor is not required to protect mice against B. bronchiseptica challenge. Whether B. pertussis vaccines protected against B. bronchiseptica by a nonspecific mechanism was not established, but it is clear that the specific antigen responsible for protection against B. pertussis was found only in B. pertussis and not in B. bronchiseptica or B. parapertussis.
A series of linearly incised superficial skin wounds was made on the forearms of young adult male volunteers. Wounds were sampled at several intervals between 3 hr and 21 days after wounding, for study by light and electron microscopy. The light microscopic observations show that regeneration of epidermis in human wounds conforms chronologically to that reported for the epidermis in superficial wound repair in laboratory animals. It is further shown that "ruffling" of cell membranes characterizes the cells of the migrating epidermis in early wound healing. This study reveals that the basement lamina and hemidesmosomes are established by epidermis in contact with the fibrin net at the base of early wounds. Epidermal cells in the wound environment are shown to be phagocytic. Analysis of the submicroscopic cytology of differentiating and maturing regenerated epidermis reveals that, in the sequence of events, the formation of filaments, basal lamina, and desmosomes is followed chronologically by evolution of keratohyalin granules and, subsequently, by keratinization of the surface epidermal elements. The entire sequence of migration, differentiation, and ultimate keratinization in the superficial wounds studied requires 3-5 days for completion.
Connective tissue repair was studied in a series of skin wounds in young adult males. The tissues were examined at 3, 12, and 24 hr, and at 2, 3, 5, 7, 14, and 21 days after wounding. The neutrophilic leukocytes contain within membrane-bounded vacuoles some fibrin and serum protein from the wound; however, most of the granulocytes lyse and release their cytoplasmic contents into the extracellular space. The mononuclear cells undergo a series of morphologic alterations during which they develop a modest amount of relatively poorly developed rough endoplasmic reticulum and an extensive system of smooth-surfaced membranes prior to active phagocytosis. They could be clearly distinguished from immature fibroblasts by the differences in the development of their organelles, particularly the rough endoplasmic reticulum. The perivascular connective tissue adjacent to the wound contains cells which appear like poorly developed or immature fibroblasts. The development of these cells into mature fibroblasts can be followed during the different stages of wound repair. Intimate contact was observed between basal cells of the regenerated epidermis and monocytes in the wound below: cytoplasmic projections of the basal cells extended beneath the basement lamina to the surface of the monocytes. Such contacts were seen only on the 4th-7th day after wounding. Their possible significance is discussed.
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The fine structure of the estrogen-primed uterus was examined in two series of rats, with emphasis upon the alterations in smooth muscle cells and fibroblasts. The first series of animals were mature animals that were sacrificed at diestrus or estrus. The second series consisted of prepubertal rats (57-70 g) that received subcutaneous injections of estradiol-17 beta in 20% alcohol. Four groups of animals received the hormone twice daily for 3 days for a total dose of 0.06, 0.6, 6.0, or 60.0 microg, respectively. An estrogenic response was observed in all groups as indicated by an increase in uterine weight. Control groups consisted of either untreated animals or animals receiving 20% alcohol. All animals were sacrificed on the 4th day. The fibroblasts and smooth muscle cells in the controls were similar to their counterparts in the mature animal in diestrus. They were small, contained relatively little rough endoplasmic reticulum, and the connective tissue cells appeared like fibrocytes. All of the estrogen-treated animals were similar in appearance and were comparable to their counterparts in the mature animal in estrus. Both the smooth muscle cells and the fibroblasts were increased in size, demonstrated a marked enlargement and dilation of ergastoplasmic cisternae, and contained increased numbers of attached and free cytoplasmic ribosomes. The presence of an extensive rough endoplasmic reticulum in the smooth muscle cells of the stimulated uterus is in marked contrast to the appearance of these cells in other tissues. These observations correlate with previous biochemical studies by other workers, in which estrogens have been shown to promote the synthesis of uterine RNA, collagen, and noncollagenous protein, and suggest that smooth muscle cells may participate in the synthesis of connective tissue proteins.
This study has presented the fine structure changes in the eosinophilic leukocyte in the rat uterus during the estrous cycle. Eosinophils were seen to emigrate into the uterine connective tissues from the blood stream. Just prior to, during estrus, and 1 day postestrus, eosinophilic leukocytes underwent lysis releasing their contents into the extracellular spaces and both whole eosinophils and individual granules from lysed cells were ingested by resident macrophages. No phagocytic activity by eosinophils was observed. The possible relationship of the turnover of eosinophils to the profound morphologic and chemical changes in the uterus during the estrous cycle was discussed.
Regions of attachment have been observed between connective tissue cells from four different structures: fibroblasts in embryonic and fetal tendons, fibroblasts in fetal ligamentum nuchae, odontoblasts, and osteoblasts. Morpho logically these sites appear to be focal and to consist of an approximation of the plasma membranes of adjacent cells to within approximately 200 A. In the region of approximation both the extracellular space and the cytoplasm adjacent to the plasmalemma are increased in density. We have postulated a role for these sites in the maintenance of structural integrity.
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The fine structure of developing elastic fibers in bovine ligamentum nuchae and rat flexor digital tendon was examined. Elastic fibers were found to contain two distinct morphologic components in sections stained with uranyl acetate and lead. These components are 100 A fibrils and a central, almost amorphous nonstaining area. During development, the first identifiable elastic fibers are composed of aggregates of fine fibrils approximately 100 A in diameter. With advancing age, somewhat amorphous regions appear surrounded by these fibrils. These regions increase in prominence until in mature elastic fibers they are the predominant structure surrounded by a mantle of 100 A fibrils. Specific staining characteristics for each of the two components of the elastic fiber as well as for the collagen fibrils in these tissues can be demonstrated after staining with lead, uranyl acetate, or phosphotungstic acid. The 100 A fibrils stain with both uranyl acetate and lead, whereas the central regions of the elastic fibers stain only with phosphotungstic acid. Collagen fibrils stain with uranyl acetate or phosphotungstic acid, but not with lead. These staining reactions imply either a chemical or an organizational difference in these structures. The significance and possible nature of the two morphologic components of the elastic fiber remain to be elucidated.
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The uptake, intracellular transport, and secretion of protein by guinea pig wound fibroblasts was studied by electron microscope radioautography using L-proline-3,4-H(3) as a tracer. Experiments were performed to determine the curve of concentration of free amino acid in the blood after intraperitoneal administration of the labeled proline. Radioautographs were quantitatively analyzed and the concentration of isotope, in grains per unit area, was determined for the following cellular and extracellular compartments: ergastoplasm, Golgi complex, peripheral cytoplasmic structures, and collagen. The concentration of label, expressed as number of grains per unit area of each subcellular system, reveals the period during which each cellular compartment is maximally labeled, and presents a clearer picture of the passage of the label through each of these compartments. The data demonstrate appearance of the label at maximum concentration in the ergastoplasm 15 minutes after injection, and this compartment remains maximally labeled for 2 hours. In the Golgi complex, concentration is not maximal until 60 minutes after injection of isotope, and appears to decrease before or at about the same rate as that of the ergastoplasm. The present experiment is consistent with previous light microscope radioautographic studies, and no storage phase was found in the fibroblasts. The findings are not simply consistent with a direct precursor-product relationship between the contents of the ergastoplasm and those of the Golgi complex. Morphologic observations of regions in the fibroblast interpretable as possible sites of communication between the ergastoplasm and the extracellular space, together with the kinetic studies, permit the suggestion of an alternate pathway of passage of at least some of the synthesized protein directly from the ergastoplasmic cisternae to the cell exterior.