Volume 45 (1999) No. 2

Binding of Anti-CD4 Monoclonal Antibody to D1 Domain (CR1, CR2 epitopes) of CD4 Molecule
K. KOUBEK.........................67
Institute of Hematology and Blood Transfusion, Prague, Czech Republic
Corresponding author: Kristián Koubek, Institute of Hematology and Blood Transfusion, U Nemocnice 1, 128 20 Prague 2, Czech Republic. Fax: 420 2 299821; e-mail: Koubek@uhkt.cz.
Background.

To elucidate the mechanism of cytopathicity of the transformation-defective avian retrovirus tdPH2010, we examined the function of two point mutations we had previously found in the long terminal repeat U3 region of this virus. Our previous studies showed that the U3 region was responsible for the cytopathic effects. These mutations were a G-to-T mutation at position -126 from the transcription start site and a G-to-A mutation at -23. Site-directed mutagenesis was performed on a noncytopathic, wild-type virus BSU to alter the nucleotides at these two positions, one at a time, to those of tdPH2010. Cell growth assay using the altered viruses revealed that host cell growth was retarded only when both of these mutations were present. The two additional mutations previously found in the direct repeat 1-polypurine tract (dr1-PPT) region of tdPH2010 were present also in the noncytopathic strain tdPH2013. Site-directed mutagenesis confirmed that these two mutations had indeed no role in the cytopathic effect of tdPH2010. None of these mutations influenced virus production from the infected cells. We conclude that the cytopathic effects by tdPH2010 are ascribed to the two point mutations in the U3 region.
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Ovulated oocytes can be fertilized in vivo almost exclusively with spermatozoa of its own species only, while the species specificity of zona pellucida of in vitro matured oocytes is less restrictive. Our present experiments were undertaken to determine whether estrous oviductal fluid modifies the interaction between gametes of unrelated species. After incubation of in vitro matured sheep oocytes with bull spermatozoa, the penetration rate was 75.0%, whereas when the oocytes were matured in medium supplemented with 15% sheep oviductal fluid collected using the permanent indwelling oviductal cannulae, the penetration rate decreased to 4.8% (4/84). In reverse combination, 70.4% (38/54) cattle oocytes matured in vitro were penetrated with ram spermatozoa. The addition of oviductal fluid caused a drop in penetration by ram sperm to 38% (19/5O). In parallel experiments, no penetration was recorded when in vivo matured sheep oocytes were incubated with bull spermatozoa; high fertilization rates (79.4% - 27/34) were recorded when such eggs were incubated with ram spermatozoa, irrespective to the presence or to the absence of oviductal fluid in the medium. The results suggest that the properties of zonae pellucidae of ovulated and in vitro matured oocytes are not identical and may be modified by contact with estrous oviductal fluid.
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The presence of ZP autoantibodies in serum and Ffl samples of 109 women attending the ART Center in Kiev was investigated using IIF and ELISA. Positive serum and Ffl samples examined by both methods were found in 20 (18.34%) and in 19 (17.43%) patients respectively; 31 (28.44%) serum samples and 33 (30.27%) Ffl samples analyzed by IIF were positive; of the samples analyzed by ELISA 21 (19.26%) and 20 (18.34%), respectively, were positive. No relationship was found between ZP autoantibody incidence and the type and cause of infertility. A significant prevalence of ZP autoantibodies detected by ELISA in Ffl was found in patients with fertilization failure (39.13%) and with low fertilization rate (42.85%) when compared to patients with middle fertilization rate (5.71%) and high fertilization rate (8.1%). Clinical significance of ZP autoantibodies in Ffls for in vitro fertilization outcome was suggested.
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 Mast cells play a role in inflammation and immunological reactions. Cardiac mast cells, shown by sodium sulfate--alcian blue staining, were evaluated in endomyocardial biopsy specimens in patients with unexplained congestive heart failure. The results of histopathological analysis were consistent with active myocarditis according to the Dallas criteria in 10 patients (15%), borderline myocarditis in 9 (13.8%), and dilated cardiomyopathy in 25 patients (38.5%); these results were compared with a control group of 10 traffic accident victims. The highest numerical areal density of mast cells was found in active myocarditis (3.92 counts/mm2, SD = 1.84), followed by borderline myocarditis (2.76 counts/mm2, SD = 1.66), dilated cardiomyopathy (1.56 counts/mm2, SD = 0.45) and control group (0.77 counts/mm2, SD = 0.19). Degranulation involved 27% (SD = 3.6) of mast cells in active myocarditis, 18% (SD = 4.5) of mast cells in borderline myocarditis, 10.8% (SD = 3.12) of mast cells in dilated cardiomyopathy and 4% (SD = 2.0) of mast cells from autopsy tissue. The differences among the four groups were statistically significant (P < 0.001).
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CD4 molecule is a cell surface glycoprotein (55 kD) with four extracellular Ig-like domains and one cytoplasm domain associated with p56lck, a Src-like protein tyrosine kinase (Veillette et al., 1988).
CD4 is known to contact nonpolymorphic regions of MHC class II proteins and acts as a coreceptor in antigen recognition recognition (König et al., 1992). Human CD4 glycoprotein also serves as primary cellular receptor for human immunodeficiency virus (HIV) (Sattentau and Weiss, 1988). MHC II and gp120 molecules as well as human seminal plasma glycoprotein gp17 have been proposed to bind to overlapping sites involving the CDR2-like region in CD4 domain 1 (Autiero et al., 1995). In addition, interleukin 16 (IL-16) is a chemoattractant factor for CD4-positive cells and seems to bind to CD4 domain 3 (Center et al., 1996). Crystallographic studies of the four extracellular Ig-like domains of recombinant soluble CD4 have suggested that the full extracellular segment adopts an extended structure about 125Ő long and 25--50Ő wide (Maddon et al., 1985; Kwong et al., 1990; Ryu et al., 1990).
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