S Lombardi

Renal involvement in Feline Immunodeficiency Virus infection: p24 antigen detection, virus isolation and PCR analysis

Renal alterations characterized morphologically by glomerular and tubulo-interstitial lesions and clinically by a heavy proteinuria and sometimes by renal failure are frequent in feline immunodeficiency virus (FIV) infected cats. To investigate the possible role of local FIV replication in the genesis of this renal damage, renal tissues of 15 consecutive naturally infected and five non-infected cats were examined for traces of the virus by immunohistochemistry, using a monoclonal anti-p24 antibody in a streptavidin-biotin peroxidase labeled system, cultivation and polymerase chain reaction (PCR). Tubular epithelial cells as well as scattered interstitial inflammatory and glomerular cells were positive for p24 antigen in 13 cats. Viral isolation was successful in seven cats, and FIV gag DNA and RNA sequences were detected in 14 and five cats, respectively. Control cats were constantly negative. Although not conclusive, these results suggest that a direct role of FIV in the induction of the renal damage observed in infected animals is possible.

DETECTION OF FELINE IMMUNODEFICIENCY VIRUS P24 ANTIGEN AND P24-SPECIFIC ANTIBODIES BY MONOCLONAL ANTIBODY-BASED ASSAYS

A panel of monoclonal antibodies (mAbs) detecting distinct B-cell epitopes on p24 core viral protein of feline immunodeficiency virus (FIV) were employed to develop immunoassays to measure p24 concentration in culture and serum samples, to localize p24 in FIV-infected cells and tissues, and to detect anti-p24 antibodies in cat sera. In its optimized configuration the p24 capture assay detected as little as 0.25 ng/ml of protein. The assay was found at least as sensitive as the reverse transcriptase activity assay in FIV-infected lymphocyte cultures and proved capable of detecting p24 antigen in acid pretreated sera from a high proportion of FIV-infected cats. The mAbs were also successfully used to detect the p24 antigen in permeated FIV-infected cells by flow cytometry and in tissue sections from FIV-infected cats by immunohistochemical staining. Anti-p24 antibodies in FIV-infected cat sera were assayed by a competitive capture ELISA which readily identified occasional false positive results provided by a standard ELISA using purified whole FIV-coated wells.

Epitope mapping of the V3 domain of feline immunodeficiency virus envelope glycoprotein by monoclonal antibodies

A panel of six IgG monoclonal antibodies (MAbs) was produced by immunizing mice with a 22 amino acid synthetic peptide, designated V3.3, of the third variable region of feline immunodeficiency virus (FIV) envelope glycoprotein. This peptide is known to induce neutralizing antibodies in cats. In ELISA all MAbs reacted with purified SDS-disrupted FIV and in flow cytometry all MAbs stained permeated, persistently infected FL4 cells but not unfixed FL4 cells; this indicated that the MAbs recognize essentially cryptic epitopes of the gp100 V3 loop. By direct ELISA using partially overlapping synthetic peptides and by competition binding studies, the anti-V3.3 MAbs were shown to detect at least four distinct epitopes, two located in the amino-terminal half and two in the carboxy-terminal half of the sequence. When tested for neutralizing activity by the syncytium inhibition assay in Crandell feline kidney cells, all anti-V3.3 MAbs neutralized FIV at high dilution. However, at low dilution two MAbs exhibited much less neutralizing activity. These results indicate that the V3 region of FIV contains multiple epitopes involved in neutralization.

Circulating immune complexes and analysis of renal immune deposits in feline immunodeficiency virus‐infected cats

Total immunoglobulin content and concentration of immune complexes (IC) were determined in the sera of 51 cats infected with feline immunodeficiency virus (FIV) and of 40 controls. IgG and IgM were quantified by radial immunodiffusion and circulating IC (CIC) by the CIC‐conglutinin assay. IgG fractions were obtained by acid elution from kidney tissues of 15 FIV‐infected and five negative control cats to investigate the possible role of IC in the genesis of renal damage observed in infected animals. Mean concentrations of IgG and circulating IC were higher in FIV‐infected cats than in controls (29·6±6·7 versus 23·0±1·9 mg/dl (mean±s.d.) P < 0001; and 66·5 ± 17·0 versus 27·4 ± 19·9% I, P < 0·001, respectively), while IgM levels were only slightly increased (0·9 ± 0·05 versus 0·87 ± 0·04 mg/dl, P < 0·02). Immunoglobulin fractions were eluted from 10 of the 15 renal tissue samples from FIV‐infected cats and were found to be polyclonal and at least partly specific for FIV antigens. These findings confirm the presence of a B cell activation in FIV‐infected cats and demonstrate the presence of high levels of CIC in their sera. The presence of immune deposits in renal tissues suggests that IC might play a role in the pathogenesis of the renal damage observed in FIV‐infected cats.

Regulation of the Development of Plaque-Forming Cells to Bromelain-Treated Syngeneic Mouse Erythrocytes in Bone Marrow Cell Cultures

The development of plaque-forming cells (PFC) to bromelain-treated syngeneic mouse red blood cells (Br-MRBC) was studied in bone marrow cell (BMC) cultures. It was found that the number of marrow PFC to Br-MRBC does not show the typical spontaneous increase observed in spleen cell (SPC), or peritoneal cell (PC) cultures. The number of anti-Br-MRBC PFC was markedly increased by lipopolysaccharide (LPS), even in conditions in which cell proliferation was blocked by mitomycin C, suggesting the presence of high numbers of Br-MRBC-specific precursor cells, potentially capable of differentiating into autoantibody-producing cells, in the marrow. Moreover, the low levels of anti-Br-MRBC PFC were further reduced in the presence of concanavalin A (Con A). The addition of Con A-activated BMC to BMC, SPC, or PC cultures actively suppressed the development of anti-Br-MRBC PFC. Con A-activated BM suppressor cells were found to be Thy 1.2-negative, Ig-negative, nonadherent cells. A possible role for the BM suppressor cell in tolerance to self antigens is discussed.

Cell-mediated immunity and delayed-type hypersensitivity in Pseudomonas aeruginosa-infected mice.

In mice repeated systemic injections ofPseudomonas aeruginosa viable cells were able to induce a specific delayed-type hypersensitivity, which was evaluated as increase both in footpad swelling and in the weight of popliteal lymph nodes, after a challenge in the footpad. Unfractionated spleen cells or T lymphocyte-enriched spleen cells from sensitized donors were able to specifically transfer the delayed-type hypersensitivity to syngeneic recipients but failed to protect them against a lethal challenge withP. aeruginosa. In contrast, serum or B lymphocyte and macrophage-enriched spleen cells from the same donors were capable of transferring protective immunity but failed to induce any delayed-type hypersensitivity reaction in the recipients.These results clearly show that in systemicP. aeruginosa infections a dissociation between delayed-type hypersensitivity and acquired cellular resistance occurs.