[Methods for mathematical data processing in enzyme immunoassay. I. Theoretical bases].

The paper deals with the mathematical processing of enzyme immunoassay (EIA) data. The paper comprises two parts. Part 1 contains a brief comparative account of the basic ways of presenting experimental data and constructing a standard curve, as well as practical guidelines for choosing the mathematical methods for calculation of concentrations. The recommendations proposed in Part II are based on the comparative study of photometric equipment and software that is of common use in EIA diagnosis at the Russian clinical laboratories. The experiment imitates competitive analysis illustrates a relationship of the obtained clinical result to the choice of either mode of constructing the standard graph on the basis of the optic densities of calibration samples. The findings indicate that an incorrectly chosen method for data processing may make both a false rejection of the whole analysis on the basis of the erroneous calculation of the concentration of a test agent in the control serum and to underdiagnosis on the basis of the erroneous calculation of the concentration of a test agent in a patient's sample near the point of taking a medical decision.

[Cell response of rat liver parenchyma to the infection by the intestinal protozoan pathogen Cryptosporidium parvum (Sporozoa, Coccidia)]. / Reaktsiia kletok parenkhimy pecheni krys na zarazheniie kishechnym protozoinym patogenom Cryptosporidium parvum (Sporozoa, Coccidia).

In the present work, the authors' previous studies of a "distant action", exerted by an intestinal pathogen (Cryptosporidium parvum) on the liver of experimentally infected baby rats, were extended to include shifts in the quantity of glycogen, protein and nuclear DNA in the host liver at different degrees of infection. One of the outcomes of this work is the discovery of a very quick response of hepatocytes and a high sensitivity of rat liver to parasitic invasion even at a weak intensity of infection. 85-90 h after oocyst feeding to rats, glycogen quantity in their livers was 2.5 times lower that in the control. This suggests that the infected host liver worked under energetic starvation conditions. The proposed coefficients of general infection (I) and infection with intracellular stages (F) made it possible to distinguish between the total abundance of parasites in the host intestine during the whole period of infection, and the number of feeding intracellular stages available by the moment of autopsy. The glycogen amount in rat hepatocytes does not depend on I, and negatively correlates with F. Unlike, the protein content in hepatocytes positively correlates with I, being independent of F. Despite the obvious deficiency of amino acids in the infected rats, as a consequence of cryptosporidiosis-induced malabsorption, the protein synthesis in their hepatocytes was not at all inhibited but, on the contrary, much activated. This is a most characteristic feature of the distant action of C. parvum on the liver of parasitized host. With C. parvum infection, the share of polyploid hepatocytes does not correlate with either I, or F. However, compared to the control, the mean values of relative numbers of polyploid cells in weakly, moderately, and heavily infected animals (according to I values) were higher by 20, 100 and 100%, respectively. In hepatocyte nuclei of C. parvum infected rats, the total area of nucleoli increases almost by 30%. The above changes are discussed in terms of both the liver compensatory response to the existing pathology (diarrhea), and the host-parasite relationships. Studies into the distant action of an intestinal pathogen (C. parvum) on non-intestinal organs (liver) of the infected host may be qualified as a new and original approach to pathogenesis of protozoan infections (coccidioses sensu lato), to which young host specimens are known to be most susceptible.

[Formation and diversity of parasitophorous vacuoles in parasitic protozoa. The Coccidia (Sporozoa, Apicomplexa)]. / Puti formirovaniia parazitofornykh vakuolei i ikh raznoobrazie u paraziticheskikh prosteishikh. Koktsidii (Sporozoa, Apicomplexa).

Data on parasitophorous vacuole (PV) formation in host cells (HC) harbouring different intracellular protozoan parasites have been reviewed and critically analysed, with special reference to the main representatives of the Coccidia. The vacuole membrane (PVM) is the interface between host and parasite, playing a role in nutrient acquisition by the parasite from the HC. The PV phenomenon is regarded as a generalized HC response to the introduction of alien bodies (microorganisms), which eventually reflects the evolutionary established host-parasite relationships at cellular, subcellular and molecular levels. Special attention has been paid to the existing morpho-functional diversity of the PVs within the same genera and species of parasites, and even at different stages of the parasite life cycle. The PVM is generally considered to derive from the HC plasmalemma, whose biochemical composition undergoes significant changes as the intravacuolar parasite grows. The original HC proteins are selectively excluded from the PVM, while those of the parasite are incorporated. As the result, the changed PVM becomes not fusigenic for HC lysosomes. For Toxoplasma gondii and other cyst-forming coccidia (Isospora, Sarcocystis), a definite correlation has been noticed between the extent of rhoptry and dense granule secrets released by a zoite during HC internalization, on the one hand, and the pattern of the PV that forms, on the other one. In T. gondii, tachyzoites, known to discharge abundant secrets, commonly force the development of PVs limited with a single unit membrane and equipped with a tubulovesicular network in the lumen. Unlike, bradyzoites known to be deficient in secretory materials trigger the formation of PVs with a three-membrane lining composed of the changed invaginated plasmalemma in addition to two membranes of endoplasmic reticulum. The two different types of PV harbour, respectively, exoenteric and enteric stages of T. gondii, the latter being confined to the cat intestine only. Unlike, all endogenous stages of the classic intestinal coccidia (Eimeria spp.) develop within PVs limited with a single membrane, with some invaginations extending into the PV lumen. Unusual PV patterns are characteristic of the extracytoplasmic eimerian coccidia (Cryptosporidium, Epieimeria) and adeleid haemogreagarines (Karyolysus). In cyst-forming coccidia, the PVM is actively involved in tissue cyst wall formation, thus protecting the encysted parasites from recognition by the host immune system. All this strongly suggests that the PV is far from being an indifferent membraneous vesicle containing a parasite, but represents a metabolically active compartment in infected cells. Since all the coccidia are obligate intracellular parasites, the mode of their intimate interaction with the HC, largely accomplished via the PV and its membrane, is vital for their survival as biological species.

[Morphofunctional changes in hepatocytes during the early postnatal development of rats experimentally infected with the intestinal protozoan pathogen, Cryptosporidium parvum (Coccidia, Sporozoa)]. / Morfofunktsional'nye izmeneniia pecheni v rannem postnatal'nom razvitii u krys, éksperimental'no zarazhennykh kishechnym protozoinym patogenom Cryptosporidium parvum (Coccidia, Sporozoa).

Morphofunctional changes in hepatocytes of 10-14-day old rats were followed in norm and after experimental infection with different doses of oocysts of Cryptosporidium parvum. The liver index (ratio between the liver and body masses) varied with the intensity of invasion on the background of slowing down up to the total cessation of animal growth rates, and all this obviously pointed to severe pathology. In the infected rats, some cytological indices were shifted compared to the norm: protein amount and the average number of genomes per hepatocyte were seen to increase, the normal ratio between cells with different ploidy levels being violated. The particular correlation analysis was employed to distinguish between the ontogenetic (animal growth related) and pathologic (related to the infection intensity) polyploidization and hypertrophy in hepatocytes. In 10-14-day old rats, the former is affected primarily by the increase in the share of multinuclear hepatocytes, whereas the latter is accomplished by the increase in the number of cells with polyploid nuclei (4c and 4c x 2 cells). In the heavily infected rats, the ontogenetic polyploidy was almost totally suppressed due, presumably, to their growth rate inhibition, the rise in hepatocyte ploidy resulting form the obvious pathological changes in the liver. In the infected rats, the ontogenetic hypertrophy of hepatic parenchymatous cells was not manifested, and the observed protein accumulation in hepatocytes also resulted from the pathological changes in the liver. It is obvious that changes in cell hypertrophy (protein content) may serve as a more susceptible tool that readily perceives the host's stress experienced due to the parasitic infection (cryptosporidiosis), than cell ploidy: the levels of the respective responses of these two parameters differing by 4 times. However, due to the known reversible nature of hypertrophy, it cannot be used for the aims of a long-term prediction about the future mode of liver functioning in the animal that survived cryptosporidiosis. Unlike, such a parameter as frequencies of hepatocytes with different ploidy levels is much more useful in this respect.

[In vitro interaction between persisting oocysts of Cryptosporidium parvum and murine resident peritoneal macrophages. I. Effect of parasite on macrophage capability for oxidative burst]. / Vzaimootnosheniia persistiruiushchikh ootsist paraziticheskogo prosteishego kriptosporidiia Cryptosporidium parvum s rezidentnymi peritoneal'nymi makrofagami myshi in vitro. I. Vliianie parazita na sposobnost' zarazhennykh makrofagov k okislitel'nomu vzryvu.

Cryptosporidiosis in an opportunistic infection which poses a significant threat to the immunodeficient patients (including people with AIDS). The aim of the present work was to study whether oxidative burst, known as a nonspecific immune response of macrophages, may be modulated in vitro by persisting oocysts of a coccidian pathogen Cryptosporidium parvum. Live oocysts of C. parvum engulfed by murine perithoneal macrophages may persist within the phagosomes and retain their initial morphology for about 7 days following oocyst injection into macrophage monolayer. A short-term interaction of adherent macrophages with C. parvum oocyst suspension for 5-15 min caused oxidative burst in these macrophages. After a while, the intensity of this oxidative burst smoothly decreased to vanish within the following 2-6 h oocyst-macrophage cultivation. Dead oocysts caused no oxidative burst in macrophages. Co-cultivation of macrophages with oocysts for 1.0-1.5 days led to the appearance of macrophages, which had oocysts both contacting the cell surface and existing inside the phagosomes. In these macrophages the oxidative burst in response to the addition of a chemotactic peptide (fMLP) was considerably higher than in uninfected control cells. During a 1-4 day co-cultivation, the degree of oxidative burst caused by fMLP in macrophages containing only phagocytosed oocysts did not differ from that in the non-infected (oocyst-free) control. The data obtained enable us to propose that the products of oxidative burst in macrophages, formed in response to the interaction with C. parvum oocysts, do not kill the oocysts. These can survive for a long time in the phagosomes of macrophages. Such a long persistence of oocysts in phagosomes does not affect the capability of macrophages for oxidative burst in response to the action of fMLP.

[Oocyst structure and problem of coccidian taxonomy]. / Struktura ootsist i voprosy sistematiki koktsidii.

A comparative ultrastructural study was made of both thin- and thick-walled oocysts of Cryptosporidium parvum. According to the authors' findings, all the oocysts in C. parvum should be considered as thin-walled, since their walls have been composed of a single membrane or of two, closely apposed membranes without any additional substance in between. Despite the presence of two types of wall-forming bodies (WFB) in the maturing macrogamete or zygote, there is no evidence of their involvement in oocyst wall formation. In this concern, the function and destiny of WFB in C. parvum oocysts still remain obscure. Similar structure of the oocysts wall was reported elsewhere for thin-walled oocysts of fish coccidia of the genera Goussia and Eimeria. In C. parvum, the "thick-walled" oocysts differ from oocysts with thin walls in the availability in the former of a single sporocyst. The sporocyst wall consists of two unequal layers: a thin outer layer and a thicker inner one, in which a characteristic suture line is occasionally seen. By this feature the thick-walled oocysts of C. parvum bear similarities with oocysts of the cyst-forming coccidia (Cystoisospora, Toxoplasma, Sarcocystis) and of the genus Goussia: in all these the valves making up the sporocyst wall are joint just along the suture line. The literary and the authors' own data make it possible to suppose that the suture detected in C. parvum oocysts is located in the sporocyst wall, joining its valves, rather than in the oocyst wall proper, known to be composed of one or two, closely apposed unit membranes. Again, the availability of a suture (or sutures) in the sporocyst hardly provides enough reason to relate C. parvum with either cyst-forming, or fish coccidia, since this structure itself may be of a convergency character, rather than of systematic value. This may be substantiated, at least in part, by the authors' previous findings (Beyer, Sidorenko, 1984) of a similar structure, originally referred to as a "slit channel", in the intraerythrocytic capsule around gamont stage of haemogregarines--the adeleid coccidia of the genus Karyolysus. The suture-like structure could have originated in the evolution independently in different groups of parasitic protozoa to serve eventually as a suitable mechanism for immediate separation of elements involved in protective formation harbouring different developmental stages, including, for example, sporozoites in the eimeriid coccidia, or gamonts in the adeleid coccidia.

[The interrelationships of the coccidian Cryptosporidium parvum (Apicomplexa: Sporozoa) with the cells of the immune system in the mammalian host]. / Vzaimootnosheniia koktsidii Cryptosporidium parvum (Apicomplexa: Sporozoa) s kletkami immunnoi sistemy khoziaina-mlekopitaiushchego.

By means of an electron microscopic study of the intestine in young rats infected with Cryptosporidium parvum we observed a mass migration of immunocompetent cells of the host (eosinophils, neutrophils and macrophages) into the lumen of intestine. Some lymphocytes were also observed. Immunocompetent cells (except lymphocytes) included inside phagosomes with different endogenic states of C. parvum. Macrophages with typical extracytoplasmic parasitophorous vacuoles formed by C. parvum were also observed in the intestine lumen. Almost all stages of C. parvum could be observed on a surface of such macrophages. However, we did not find in lamina propria of intestine villi any macrophages with parasites. The place of macrophages infection is unknown. We suggest that surviving of C. parvum in macrophages is principally possible.

[Cellular interactions in the intracellular parasitism of cryptosporidia. I. The effect of Cryptosporidium parvum on the phosphatase activity in the small intestine enterocytes of experimentally infected newborn rat pups]. / Kletochnye vzaimodeistviia pri vnutrikletochnom parazitirovanii kriptosporidii. I. Vliianie kriptosporidii Cryptosporidium parvum na fosfataznuiu aktivnost' v énterotsitakh tonkoi kishki éksperimental'no zarazhennykh novorozhdennykh krysiat.

Cytochemical methods for detection of non-specific phosphatases were employed at the light microscope level for identification of enzymatic activity in the small intestine of new-born rats (6--11 days old), both infected and non-infected with the intestinal coccidium Cryptosporidium parvum. In the new-born rats, the level of alkaline and especially acid phosphatase is originally very low, suggesting their insignificant involvement in digestion processes in suckling animals compared to rats of older age (3 month old). However, a heavy colonization of the brush border of the intestinal villi of the new-born rats with cryptosporidia results in obvious inactivation of phosphatases in the infected enterocytes, in contrast to the neighbouring parasite-free host cells. The general picture of metabolic interaction between cells of a unicellular parasite (C. parvum) and those of its metazoan host (rat) much resembles that observed in the course of Elmeria spp. infection, but differs from that induced by Toxoplasma gondii endogenous stages in the cat intestine. Details of cell interaction with intracellular parasitism need additional studies at the ultrastructural level.