Exploring the feasibility of selective depletion of T lymphocyte subsets by whole blood immunoadsorption cytapheresis.

Normal turnover of T lymphocytes is slow relative to other blood cells. Consequently, the physical removal of circulating leucocytes by thoracic duct drainage, repeated leukapheresis or blood filtration results in T cell depletion and immunosuppression. However, clinical use of such procedures is impractical compared with immunosuppressive drugs or radiation. None the less, immunosuppression by physical depletion of T cells, avoiding the systemic toxicities of drugs and radiation, might have clinical advantages if immunophenotypically distinct T cell subsets could be depleted selectively. Recent advances in targeted plasma protein apheresis using adsorbent macrobead columns prompted us to determine whether analogous techniques might permit CD4+ T lymphocytes to be removed selectively from whole blood. To explore this possibility, we linked murine anti-human-CD4 and isotype-identical control monoclonal antibodies (mAbs) to agarose, polyacrylamide and polystyrene macrobeads (150-350 microm) and then evaluated the selectivity, specificity and efficiency of macrobead columns to remove CD4+ T cells from anti-coagulated whole blood at varying mAb densities and flow rates. We also examined saturation kinetics and Fc-oriention versus random coupling of mAbs to macrobeads. Sepharose 6MB macrobead (250-350 microm) columns proved to be most effective, selectively removing up to 98% of CD4+ T cells from whole blood. Moreover, depletion efficiency and selectivity were retained when these columns were reused after elution of adherent CD4+ cells. These studies indicate that selective depletion of T lymphocyte subsets by whole blood immunoadsorption apheresis using mAb-linked macrobead columns may be feasible on a clinical scale. It is possible that such apheresis techniques could achieve targeted forms of immunosuppression not possible with drugs or radiation.

Treatment of autoimmune disease by immunomodulation through extracorporeal elimination and intravenous immunoglobulin.

If customary drug mediated immunosuppressive therapy leads to intolerable side effects or is inefficient, extracorporeal elimination and untargeted or targeted immunoglobin therapy modulating the immune response are taken into consideration for the treatment of patients with autoimmune diseases. Both elimination and immunoglobulin therapy, are not alternatives but appear to act complementary, if sequentially applied. Selective immunoadsorption is increasingly applied for extracorporeal elimination and is able to replace plasma exchange therapy. Both, hydrophobic interaction chromatography and affinity chromatography are effective in given clinical conditions. Therapeutic affinity chromatography appears to be superior to hydrophobic interaction chromatography, if an effective, rapid elimination of the disease promoting agent is desired. Experience with therapeutic chromatography collected in the past, indicates that a rapid elimination of immunoglobulins and the subsequent intravenous infusion of 7S immunoglobulin is superior to elimination or untargeted immunomodulation alone. These experimental approaches lead to an extension of the available treatment modalities. However, controlled trials rather than anecdotal reports are needed, to provide substantial information.

Technical and clinical experience with protein A immunoadsorption columns.

Protein A adsorption (Immunosorba, Excorim, Sweden) is a potent tool for the rapid removal of antibodies from the circulation. In comparison to conventional plasma exchange therapy, Protein A adsorption offers the advantage of processing large amounts of plasma, removing up to 87% of the initial level of IgG in one session without a clinically significant loss of fibrinogen. So far 61 treatments have been carried out in our department without serious side effects.

[Elution of bacteria adhering to the epithelium in the rumen in sheep and their detection using electron microscopy]. / Elúcia baktérií adherovaných na epitel bachora oviec a ich elektrónovomikroskopický dôkaz.

A method of preparing the pure concentrate of epithelium-adherent bacteria in sheep rumen was worked out and it was tested in 24 samples of the rumen wall, obtained from 12 slaughtered sheep. The purity of the bacterial eluate was checked by transmission electron microscopy and negative staining, followed by electron-microscopic evaluation. Besides bacterial cells no residues of feed, epithelium and other undesirable impurities were found out in the eluate. The percent yield of this method was determined by scanning electron microscopy and it made 93.3%. An isolated pure concentrate of the mixture of bacterial cultures, previously adhered to the rumen epithelium, was prepared which can be used for morphological and biochemical studies of this interesting group of rumen bacteria.

[The effect of a protein-free diet on the submicroscopic structure of striated muscles in Japanese quail]. / Odraz bezbielkovinovej diéty na submikroskopickú struktúru priecne-pruhovanej svaloviny japonskej prepelice.

The effect of protein-free diet on the striated muscle m. gastrocnemius of Japanese quail was studied in three test groups and the data were compared with a control group. Each test group included three birds. The first group was fed protein-free diet for three days, the second group for six days and the third for nine days. Egg albumin was absent in the protein-free diet. The remaining components (maize starch, germ oil, cellulose, mineral and vitamin premix) were represented by the same percentage in both diets. In the second group of birds given protein-free diet for six days, doubled Z-lines were found. No such alterations were found in the remaining groups in comparison with the controls. Since doubled Z-lines occurred only in the group given the protein-free diet for six days, further study is needed to provide evidence indicating that the doubling of Z-line is due to this factor.

[Electron microscopy studies of surface cells and cells in the area of the glandular neck of normal stomach mucosa in humans]. / Elektronenmikroskopische Untersuchungen an Oberflächenzellen und Zellen im Drüsenhalsbereich der normalen Magenschleimhaut des Menschen.

The authors describe the submicroscopical structure of surface epithelial cells of the mucosa and cells of the stomach gland cervix in men. On the basis of electronmicroscopic studies of bioptic samples of the stomach mucosa the surface epithelium of the stomach mucosa in men was found to be covered with high cylindrical cells showing typical small, relatively low microvilli at the luminal side with numerous mucous granules in the supranuclear zone of the cells. The basal part of the cell is extremely dense containing numerous ribosomes, a Golgi apparatus and mitochondriae. Isthmic cells were found to differ from surface cells by the location of the nucleus and the amount of mucous granules, which are larger and granular in the rule. The cells of this zone are mutually connected by desmosomes. The granular endoplasmic reticulum is present at a relatively lesser amount as compared to the former group of cells. These cervical cells are of importance from the viewpoint of regeneration and may differentiate into the other cell types of the stomach mucosa.

[Abnormalities of the sperm neck of poultry]. / Deformácie stavby krcka spermie hydiny.

Defects in the structure of the middle piece of poultry spermatozoon were studied by the electron-microscopic method. The defects included loose jointing of the head and falgellum owing to the shallow invagination fossa of the nucleus, and structural and mainly shape alterations of the proximal centriole. These defects existed in spermatozoa with a normal shape of nucleus as well as in those with rounded or otherwise irregularly shaped nuclei.

[Electron microscopic studies of striated muscles of the Japanese quail (Coturnix coturnix japonica) obtained from eggs centrifuged during incubation]. / Elektronenmikroskopisches Studium der quergestreiften Muskulatur der japanischen Wachtel (Coturnix coturnix japonica) aus während der Inkubation zentrifugierten Eiern.

Reported in this paper are results obtained from investigations into the effect of hypergravitation on embryogenesis of striped muscles in Japanese quail. Specimens of striped muscles were excised from the birds after hatching and used in the study. The eggs had been centrifuged on zero day as well as on the first and 15th days of incubation. The muscle fibrils were found to proliferate in all groups, that is the control group, the group with centrifugation on zero day, and that with centrifugation on the first and 15th days. The process thus is characterised as physiological, known to occur prenatally and postnatally in developing individuals.

[The relationship between fermentation and the morphological structure of rumen epithelium in sheep fed pelleted feeds]. / Vztah medzi fermentáciou a morfologickou struktúrou epitelu bachora oviec krmených tvarovaným krmivom.

Wethers were fed complete granular feed rations including 41.81% of grass hay, 25.28% of barley, 15.37% of sawdust, 14.98% of molasses, 1.32% of urea, and 1.24% of mineral supplement in dry matter for 24 weeks. Samples of the dorsal rumen sac of these wethers were subjected to patho-anatomical, histological, histo-chemical, and electron-microscopical examination. Volatile fatty acids were also determined in the rumen fluid of slaughtered animals. The control group was given the same diet in the classical form with long hay. The rumen contents of the slaughtered animals of the experimental group had an increased level of total volatile fatty acids (125.93 mM) and butyric acid (17.8 M%). The acetate:propionate ratio was 3.66. No substantial differences were observed in enzymatic activity. Electronograms recorded an increase in the number of T cells and keratinizing cells -- this suggests an increased intensity of the process of keratinization.

[Electron microscopic and histochemical studies of the transverse striated musculature of birds after long-term hypokinesia]. / Elektronenmikroskopische und histochemische Studien an der quergestreiften Muskulatur der Vögel nach langfristiger Hypokinese.

Electromicroscopical and histochemical studies of the gastrocnemius muscle has been carried out in 4 months old cockerels of the laying hybrid after hypokinesis lasting 15 and 30 days. It was found that restricted movement resulted in dystrophic changes of myofibrils, enlargement of the sarcoplasmic reticulum and oedem of interfibrillar spaces. Histochemical studies revealed focuses of increased activity of non-specific esterase decreased activity of dehydrogenase of lactic acid and a positive reaction of acid phosphatase.

[Ultrastructural study of the hypothalamic region after administration of aurothioglucose in mice]. / Ultrastruktúrne pozorovanie hypotalamickej oblasti po aplikácii aurothioglukózy u mysí.

Aurothioglucose dose of 1 mg g-1 1. w. was applied intraperitoneally to experimental female mice. After 24 hours the animals were decapitated and samples for ultrastructural examination were immediately taken from the region of the hypothalamic ventromedial nucleus. The following findings were obtained from the study of 243 electronograms of 8 experimental animals: 1. dense round particles 0.034 to 0.016 mu in size, representing the gold of aurothioglucose; 2. parenchymatous dystrophy of the mitochondria; 3. peeling off of the individual layers of myelin sheath and the granularity of these layers corresponding to the disintegration of nerve fibres.

[Submicroscopic structure of small intestine cells of lambs during a course of experimental inflammation]. / Submikroskopická struktúra buniek sliznice tenkého creva jahniat v priebehu experimentálneho zápalu.

The changes observed in the submicroscopic structure of the enterocytes of the jejunum of lambs in which inflammation has been chemically evoked justify the assumption that 12 hours after application of the chemical agent some differences exist, particularly in cellular reaction in the submucosa, in comparison with inflammations evoked by the infective agent. The submicroscopic changes in the enterocytes correspond to acute inflammation and indicate alterations on cell components which considerably reduce the absorptive area of the intestinal epithelium, and on organellae essential for the function of the enterocyte. In this way the natural barriers in the intestinal wall are damaged, leading to derangements in the absorption and passage of the nutrients taken in.

[Ultrastructural studies of mitochondria in striated muscles of birds with reference to experimental hypokinesis]. / Studium der Ultrastruktur von Mitochondrien in der quergestreiften Muskulatur der Vögel unter Berücksichtigung der experimentell hervorgerufenen Hypokinese.

Electron microscopic studies have been carried out on the mitochondria of the transversely striated muscles with regard to experimental hypokinasis. As compared to the control group the mitochondria of m. pectoralis thoracicus and the m. iliotibialis posterior in hypokinetic birds reveal marked changes. In filamentous and ovoid mitochondria vacuoles can be observed which in some cases produced larger light formations with following disappearance of the cristae and destruction of mitochondria. Fat particles located at the poles of the altered mitochondria, sporadically occurring also laterally, presented another finding. The Z-lines of the sarcomer did not form a continuous line, but were somewhat shifted.