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PubMed2002

Immunological approach in the evaluation of regional lymph nodes of patients with squamous cell carcinoma of the head and neck.

In cancer, regional lymph node (LN) cells are one of the first components of the immune system to have contact with tumor cells or their products. Therefore, the phenotype and functional properties of hematopoietic cells present within the tumor-draining LN are important to understanding their role in the control of malignant cells. Based on the locoregional metastatic behavior of squamous cell carcinoma of the head and neck (SCCH&N) region, we analyzed tumor-draining lymph nodes from SCCH&N patients to obtain insights into regional tumor immunity. Using a three-color fluorescent labeling technique, surface antigen expression was visualized in mononuclear cells of lymph nodes that were obtained from head and neck cancer patients and compared to mononuclear cells of normal lymph nodes. Cell cycle analyses were performed using propidium iodide. Proliferation after phytohemagglutinin stimulation was measured by a sodium tetrazolium-based assay. LN histology was correlated with flow cytometric findings. Regional lymph nodes of head and neck cancer patients undergo morphologic and functional changes. Flow cytometry revealed a decrease in CD8(+) T cells and in some lymph nodes the presence of second or third populations of larger cells with distinct size and granularity that expressed both T (gammadelta/alphabeta) and different natural killer cell markers. Moreover, cell cycle analyses and proliferation assays showed a diminished response to mitogenic stimuli. These changes were found in both metastatic and hyperplastic lymph nodes from head and neck cancer patients; however, no alterations were found in control lymph nodes or peripheral blood mononuclear cells from noncancer patients. The immune alterations detected in lymphocytes present within the draining lymph nodes of head and neck cancer patients may improve our understanding of how tumor cells escape host immunosurveillance. However, this dysfunction in local draining lymph nodes may not be detected systemically.

Adult↗
PubMed2007

Monitoring the genotoxic effects of radiosynovectomy with Re-186 in paediatric age group undergoing therapy for haemophilic synovitis.

PURPOSE: The aim of this study was to investigate the genotoxic effect on the peripheral blood lymphocytes potentially induced by Re-186 in paediatric age group undergoing radiosynovectomy for haemophilic synovitis, by using chromosomal aberration analysis (CA) and the micronuclei (MN) assay for detecting chromosomal aberrations, as well as the sister chromatid exchanges (SCE) technique for assessing DNA damage. METHODS: Cytogenetic analyses were evaluated in 20 boys (mean age: 13.8 +/- 2.7 years) before, and 2 and 90 days after radiosynovectomy from the peripheral lymphocytes of the patients. Joint retention and extra-articular spread of the radionuclides were evaluated by using quantitative gamma camera imaging. RESULTS: Imaging after radiosynovectomy revealed local lymph node visualization in 8 (40%) patients and hepatosplenic visualization in 3 (15%) patients due to extra-articular leakage of radioactive material. The mean frequency of chromosome aberrations (0.2 +/- 0.4/1000 cells) determined prior to the onset of therapy was not significantly increased in comparison with control values obtained 2 days (0.4 +/- 0.5/1000 cells) and 90 days (0.2 +/- 0.4/1000 cells) after therapy (P = 0.754 and P = 1.0). In the analysis of MN and SCE, when we compare the baseline levels, the mean MN and SCE frequencies were slightly higher in the control analyses performed 2 and 90 days after radiosynovectomy but there were no significant differences between baseline and control levels (chi(2) = 2.621, P = 0.270 and F = 0.573, P = 0.569, respectively). CONCLUSION: The major finding of this study with relatively small sample is that, radiosynovectomy with Re-186 does not seem to induce early genotoxic effects on the peripheral blood lymphocytes in paediatric age group.

Adolescent↗
PubMed1992

T-cell-dependent popliteal lymph node reactions to platinum compounds in mice.

The requirements for sensitization to complex salts of platinum were investigated in a mouse model by means of the popliteal lymph node (PLN) assay. A single subcutaneous injection of dissolved hexachloroplatinates without adjuvant induced a vigorous primary immune reaction in the draining PLN. Dose-dependent lymph node activation was determined by an increase in both PLN weight and cellularity. In C57BL/6 mice, peak reactions were obtained around day 6 after administration of 90-180 nmol Na2[PtCl6] or (NH4)2[PtCl6] per animal. Mice primed to [PtCl6]2- mounted an enhanced response upon local restimulation with suboptimal doses of the same but not unrelated compounds, indicating a specific secondary response. T cells were required to elicit PLN reactions to [PtCl6]2-, because athymic nude mice completely failed to respond, in contrast to their +/nu littermates. Differences between various inbred strains of mice revealed that Pt-induced PLN responses are genetically controlled. Moreover, the immunogenicity of Pt salts in mice is not confined to hexachloroplatinates, but other compounds, such as the antineoplastic agent cis-dichlorodiamine platinum, are able to induce comparable PLN reactions.

Animals↗
PubMed1991

Lymphokine-activated killer traffic assay and our preliminary clinical results of regional arterial infusion of lymphokine-activated killer cells for renal cell carcinoma.

In vivo distribution of radiolabeled lymphokine-activated killer (LAK) cells that were administered either through the caudal vein or the abdominal aorta of Lewis rats was studied. The LAK traffic study showed remarkable increase in radioactivity in several organs peripheral to the arterial injection site. We began treating the patients with metastatic renal cancer by means of regional arterial administration of LAK cells. This treatment was supported by the results of the traffic assay. Our regional LAK therapy, which involves a weekly leukapheresis in combination with daily systemic low dose interleukin-2 administration, has been in practice for over 2.5 years. Seven out of 12 metastatic lesions in the 7 patients treated showed regression. Two responding cases have been alive for over 2 years. Responding lesions were bone, muscle, para-aortic lymph node, and/or retroperitoneum. The regional arterial administration of LAK cells is local therapy. However, this mode of therapy could be useful for those patients with extrapulmonary nonresectable metastasis and at least one injectable feeding artery. This study shows our laboratory data concerning the localization of LAK cells transferred via arterial route and our clinical cases treated with regional arterial administration of LAK cells.

Animals↗
PubMed1989

Molecular mechanisms of lymphocyte extravasation. III. The loss of lymphocyte extravasation potential induced by pertussis toxin is not mediated via the activation of protein kinase C.

The present study evaluated whether protein kinase C (PKC) activation was involved in the lymphocytosis promoting properties of pertussis toxin (Ptx). The exposure of mouse lymphocytes to phorbol esters (as a means to selectively activate PKC) caused a depression in their subsequent capacity to localize into lymph nodes and Peyer's patches in vivo. This pattern of inhibition was quite similar to that observed with lymphocytes treated with Ptx. The mechanisms responsible for the observed decreases in localization to lymphoid organs caused by these two agents, however, appeared to be distinct. Exposure of lymphocytes to PMA was followed by a time and dosage-dependent decrease in the surface density of MEL-14 defined homing receptors. Ptx-treated lymphocytes retained normal density of this homing receptor. Consequently, PMA-treated lymphocytes lost their capacity to bind to high-endothelial venules in in vitro lymph node binding assays while Ptx-treated cells retained normal high-endothelial venule binding potential. We conclude from this study that: 1) the activation of PKC in lymphocytes by PMA can alter their recirculation properties via mechanisms that diminish their expression of surface receptors which support extravasation into lymph node and mucosal lymphoid tissues, and 2) even though Ptx has been reported to elevate the rate of inositol phosphate turnover in lymphocytes, the loss of extravasation potential of Ptx-treated lymphocytes is not mediated via the modification of surface homing receptors as observed in cells exposed to the known PKC activator, PMA.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMedDate not supplied

Impact of epidermal growth factor (EGF) radioreceptor analysis on long-term survival of gastric cancer patients.

BACKGROUND: The EGF receptor/ligand system seems to be involved in the regulation of gastric mucosa proliferation and progression of gastric carcinomas. PATIENTS AND METHODS: EGF receptor levels were quantitatively determined in 47 gastric carcinomas by 125J [EGF] radioreceptor assays in membrane preparations of tumor samples or corresponding adjacent mucosa. Specific receptor binding was determined by the analysis of displacement curves by non-linear least-square regression analysis using an estimated model of 'goodness of fit'. RESULTS: Increased EGF receptor binding was observed in gastric carcinomas (mean +/- SEM: 11.87 +/- 1.9 fmol/mg protein) in comparison to adjacent normal gastric mucosa ( 5.28 +/- 1.0 fmol/mg protein, p = 0.003). Elevated EGF receptor levels were especially found in more invasive T3/4 carcinomas, tumors with positive lymph nodes, advanced UICC III carcinomas, undifferentiated tumors, carcinomas of the diffuse-type according to Lauren's classification and gastric carcinomas localized distal from the cardia. In histopathologically normal appearing gastric mucosa, EGF-receptor levels were significantly decreased relative to corresponding tumor samples from advanced UICC stages (UICC I vs UICC I/II: p = 0.008) or tumors with low levels of differentiation (G2 vs G3: p = 0.028). Overall survival was significantly reduced in patients with advanced gastric carcinomas according to UICC classification (UICC III vs UICC I/II: 18.8 vs 45.5 months, p = 0.016). A subgroup analysis of gastric carcinomas localized distal from the cardia indicated, that increased EGF-receptor levels were an independent indicator of poor prognosis as determined by univariate (p = 0.020) and multivariate analysis (p = 0.042). CONCLUSION: Gastric carcinomas with increased EGF receptors might be a possible target for anticancer strategies blocking the EGF receptor/ligand pathway.

Epidermal Growth Factor↗
PubMed1987

Lymphogenous and hematogenous metastasis of Lewis lung carcinoma in the mouse.

Quantitative and temporal comparisons were made between lymphogenous and hematogenous metastasis in the non-immunogenic model system of mice bearing transplanted 3LL tumors in their hind feet. The experimental evidence indicates that cancer cells disseminate from clinically detectable primary cancers by non-exclusive routes in the blood-stream and in the lymphatics. Following a delay of approximately 2 weeks after injection of 3LL cancer cells into the foot, local lymph-node micrometastases occur, together with the first appearance of overt hematogenous metastases in the lungs. The anatomic extent of lymph-node involvement, determined by bioassays of orthotopic grafts, of ipsilateral popliteal, inguinal and lumbar nodes, provides an accurate indicator of hematogenous metastasis, even though lymphogenous and hematogenous metastasis are operationally independent in this tumor/host system.

Animals↗
PubMed1992

Locally increased metastatic efficiency as a reason for preferential metastasis of solid tumors to lymph nodes.

Metastases from solid tumors to lymph nodes do not portend as poor a prognosis as metastases to other sites. The authors wished to determine whether specific subpopulations of cells metastasized to lymph nodes and whether they have different properties than cells metastatic to visceral sites. Repetitive selection for "spontaneous" metastases of a B16 melanoma to either lung or lymph node increased the incidence of lymph node metastases. Cells derived from pulmonary and lymph node metastases were assayed for their ability to adhere to cryostat sections of lung and lymph node and respond to target organ-conditioned media in serum-free conditions. Both cell types were four times more adherent to lymph node than lung, and consistently attached to the hilar and subcapsular sinuses. Attachment of cells derived from pulmonary metastases to either tissue was threefold greater than that of cells derived from nodal metastases. Lung-conditioned media stimulated proliferation of both cell types, and transiently induced differentiated morphology in cells derived from lymph node metastases, but not in cells from pulmonary metastases. Neither effect was found in lymph-node-conditioned medium. These results suggest that cells metastasize to lymph nodes preferentially not because of a specific predilection for lymph node, but because it is an easy site to colonize. Adhesive interactions in the lymph node rather than trophic ones appear to account for this effect. Cells metastatic to lymph node may be less "malignant" than cells metastatic to visceral sites because less has been required for them to succeed as a metastatic focus.

Animals↗
PubMed1989

Prognostic factors for recurrence and cosmesis in 393 patients after radiation therapy for early mammary carcinoma.

Between 1978 and 1985, 393 of 2,765 (14%) patients with operable cancer of the breast (clinical stage T0-3N0-2M0) were irradiated after excisional biopsy and staging axillary dissection. Of 77 patients with microscopic axillary metastases, 68 received systemic adjuvant therapy. Treatment failed locally in 26 cases, and there were seven patients with distant metastasis. The three major factors for increased local treatment failure were (a) age below 40 years (P = .003), (b) negative estrogen receptor assay result (P = .03), and (c) failure to deliver a radiation boost dose when tumor was present at the margin of the specimen (P = .002). The size of the tumor, the nodal status, the progesterone receptor assay result, and the presence of ductal carcinoma in situ mixed with infiltrating carcinoma did not show a significant influence on local recurrence. In 274 of 393 (70%) patients, cosmesis was evaluated. The four major factors affecting cosmesis favorably were (a) utilization of a wedge (P less than .0001); (b) treatment of two fields a day (P less than .0001); (c) failure to use a separate treatment port to the regional lymph nodes, so as to avoid field junctions (P = .0003); and (d) small size of specimen (less than 50 cm2) (P = .0171). A second or third cancer was found in 39 of the 393 (10%) patients; contralateral breast cancer was the most common form (n = 23), followed by genitourinary cancer (n = 5). The most frequent complication was arm edema (6%).

Adult↗
PubMed1986

Immunological relationships between murine surrogate mothers and transplanted embryos, as studied by local GVH reactivity.

C57BL/Ks (H-2d) female mice were transplanted with early (stage 2) embryos of the A/J (H-2a) strain. Spleens from mice exhibiting successful pregnancies were tested at days 16 to 19 of gestation in a local graft versus host (LGVH) assay using (C57BL/Ks X A/J)F1 recipients and proved to be significantly more reactive than virgin controls or mice carrying transplanted syngeneic fetuses. This increased reactivity was specific for the transplanted embryo's strain. Other controls included donors with semi-allogeneic (F1) transplanted fetuses and females naturally pregnant by allogeneic males which did not give reactions significantly different from virgin control spleen cells. Para-aortic lymph node cells (PALN) obtained from the same A/J embryo-transplanted females showed a strong T suppressive activity both on their own spleen cell (SC) reaction as well as on the reaction obtained with virgin SC. This suppressive activity also appeared to be embryo-strain specific. Serological tests revealed the presence of mast cell-degranulating (anaphylactic) antibodies but not of hemagglutinating or complement-fixing cytotoxic activities. The A/J offspring obtained after embryo transfer to C57BL/Ks females presented at the age of two months significantly lower LGVH reactivity against the surrogate mother's strain. The differences in the responsiveness of the mice transplanted with allogeneic embryos compared with those with conventional pregnancies are discussed.

Animals↗
PubMed1995

Virus-specific antigen presentation by different subsets of cells from lung and mediastinal lymph node tissues of influenza virus-infected mice.

Immune responses at mucosal sites are thought to be initiated in the draining lymph nodes, where dendritic cells present viral antigens and induce naive T cells to proliferate and to become effectors. Formal proof that antigen-presenting cells (APC) do indeed localize to the regional lymph nodes has been lacking for viral infections of the respiratory tract. Influenza virus was detected in the draining mediastinal lymph nodes (MLN) early after intranasal inoculation, with peak virus titers in this tissue measured at 2 days postinfection. Virus-specific cytotoxic T-lymphocyte responses were first detected in the MLN 1 day later. Macrophages, dendritic cells, and B lymphocytes were isolated from influenza virus-infected mice and assayed for the capacity to stimulate a major histocompatibility complex class I-restricted virus-specific T-cell hybridoma. All APC populations from lungs and MLN contained virus and thus had the potential to present antigen to CD8+ T cells. The APC recovered from the lungs of influenza virus-infected mice and dendritic cells from the MLN were able to stimulate virus-specific responses. The lack of a virus-specific T-cell response to B cells corresponds to the small number of virus-positive B lymphocytes in the MLN. These results indicate that dendritic cells and macrophages are antigen positive in mice acutely infected with an influenza A virus and that dendritic cells are probably responsible for initiating the cytotoxic T-lymphocyte response to influenza virus in the draining lymph nodes.

Animals↗
PubMed1983

A guinea pig model to study effects of persistent intrabronchial antigenic stimulation and inflammation.

Chronic antigenic stimulation and inflammation of the bronchial tree occurs in several diseases involving microbial airway colonization. We developed a guinea pig model to study the effects of persistent intrabronchial antigenic stimulation with keyhole limpet hemocyanin (KLH). Sepharose beads 200 to 300 micron in diameter conjugated with 300 micrograms KLH were injected via a tracheostomy catheter, causing them to lodge in the bronchial tree. When lung sections from these animals and animals given uncoupled sepharose beads were examined histologically, it became apparent that in this model the persistent antigenic stimulus is accompanied by local inflammation caused by an irritant effect of the beads. To determine how the intrabronchial antigen was distributed locally and systemically, 125I labeled KLH-coupled beads were administered. Radioactivity remained predominantly in the lung, where 13 +/- 5% of the administered dose was still detectable by Day 30. Autoradiographs of Day 30 lung sections demonstrated that the radioactivity was concentrated on the individual beads, indicating that it largely represented KLH persisting in the bronchial tree. Minute amounts of radioactivity were detected systemically and in the regional lymph nodes (LN). A radioimmune assay of serum collected on Day 1 demonstrated that some of the circulating radioactivity represented antigenic KLH. When anti-KLH antibody-forming cells (AFC) were measured in lung homogenate, regional LN, blood, and spleen cell preparations after KLH-coupled bead administration, they were initially detected only in the spleen. Significant concentrations appeared in the lung homogenate, regional LN, and spleen by Day 10 and thereafter through Day 20.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Monoclonal antibody identification and characterization of a Mr 43,000 membrane glycoprotein associated with human breast cancer.

A monoclonal antibody (323/A3) with a high degree of selectivity for binding to breast cancer cells was produced by immunization of mice with MCF-7 human breast cancer cells. The antigen recognized by 323/A3 on MCF-7 appears to be surface localized, and by enzyme-linked immunosorbent assay, the antibody was found to bind strongly with four of six breast cancer cell lines examined while no binding was detectable with nonbreast cancer cell lines. In vivo distribution of the 323/A3 antigen was screened by immunoperoxidase staining of formalin-fixed paraffin sections of normal human tissues and tumors. Among breast tissues, positive staining was detected with 75% (6 of 8) of metastatic lymph nodes, 59% (76 of 128) of primary breast tumors, 20% (13 of 63) of benign breast lesions, and 0% (0 of 10) of normal breast. No immunostaining was detected with a large variety and number of other normal human tissues with the exception of staining observed with epithelium of normal colon. Antigen distribution appears not to be disease specific, since positive staining was also observed with adenocarcinomas other than breast. The antigen recognized by the 323/A3 antibody was identified by Western blot analysis as a Mr 43,000 protein. The glycoprotein nature of the antigen was demonstrated by its binding to concanavalin A, specific elution with sugar, and immunoprecipitation of a Mr 43,000 radiolabeled protein from extracts of MCF-7 cells after pulse labeling with [3H]glucosamine. The 323/A3 antigen appears to be the same Mr 43,000 protein in cell lines as in breast tumors in vivo. Based on a comparison with the molecular weights of other known tumor-associated antigens and with their immunocytochemical tissue distribution, the Mr 43,000 glycoprotein described here represents a tumor-associated antigen previously undescribed in breast cancer or in other tumors. Since the Mr 43,000 glycoprotein is present on the surface of most breast cancer cells and is either absent or expressed at very low levels in most normal tissues including normal breast, the monoclonal antibody described here may have potential applications in diagnosis and management of breast cancer.

Antibodies, Monoclonal↗
PubMed1985

The shedding of viable cells into the local lymph by tumours growing in the gut of rats.

Suspensions of syngeneic sarcoma cells were injected into the Peyer's patches of rats from which the mesenteric nodes had been removed. By later cannulating the thoracic duct of such rats it was possible to collect peripheral intestinal lymph that had come directly from the tumour bearing area without being filtered through a regional node. The number of viable tumour cells in the lymph coming from the tumours was monitored by culturing the whole lymph cells in a limiting dilution assay. The tumours grew to a diameter of approximately 1 cm in 25 days and during this time tumour cells were present in the lymph at a ratio of approximately 1 tumour cell per 10(5) lymph cells. In euthymic rats this number declined as the immune response developed. In athymic rats the number increased by approximately 10 fold during the experiments. It was concluded that the shedding of viable cells parallels the linear, not the volumetric dimensions of the tumour.

Animals↗
PubMed1995

Analysis of the expression and secretion of isotypes of sheep B cell immunoglobulins with a panel of isotype-specific monoclonal antibodies.

Monoclonal antibodies to sheep light chain, IgM and IgG were produced and used to assess total immunoglobulin (Ig) synthesis by sheep B cells in culture and antibodies to specific antigens. By using these antibodies in a dual fluorescence-activated cell sorting analysis of sheep efferent lymph B lymphocytes the percentage change in surface Ig isotype of B lymphoblasts from IgM to IgG after the antigenic stimulation of the local lymph node was measured. An extension of this analysis to paired blood and afferent or efferent lymph B cells made it possible to investigate the recirculation characteristics of B cells expressing different Ig isotypes.

Animals↗
PubMedDate not supplied

Antifertility effects of neem (Azadirachta indica) oil in male rats by single intra-vas administration: an alternate approach to vasectomy.

An alternate approach to vasectomy for long-term male contraception following a single intra-vas application of a traditional plant (Azadirachta indica) product having immunomodulatory properties is described. Male Wistar rats of proven fertility were given a single dose (50 microliters) of neem oil in the lumen of the vas deferens on each side; control animals received the same volume of peanut oil. Animals were put on continuous mating 4 weeks after the treatment, with females of proven fertility. While the control animals impregnated the female partners, all males treated with neem oil remained infertile throughout the 8 months of observation period. Epididymal and vas histology were normal without any inflammatory changes or obstruction. The intra-vas administration of neem oil resulted in a block of spermatogenesis without affecting testosterone production; the seminiferous tubules, although reduced in diameter, appeared normal and contained mostly early spermatogenic cells. No anti-sperm antibody could be detected in the serum. Unilateral administration of neem oil in the vas resulted in a significant reduction of testicular size and spermatogenic block only on the side of application; the draining lymph node cells of the treated side also showed enhanced proliferative response to in vitro mitogen challenge. These results indicate that the testicular effects following intra-vas application of neem oil may possibly be mediated by a local immune mechanism.

Animals↗
PubMed1998

Time course of the porcine cellular and humoral immune responses in vivo against pseudorabies virus after inoculation and challenge: significance of in vitro antigenic restimulation.

We investigated the time course of porcine cellular and humoral immune responses against pseudorabies virus (PRV) after pigs were inoculated with PRV gE(-) mutant strain M141 and challenged with wild-type virus NIA-3. Peripheral blood mononuclear cells (PBMC) were isolated from blood samples; half were used directly and half were restimulated with PRV in vitro before use in a cytolytic assay. We determined time course and extent of PRV-specific lymphoproliferative and cytolytic response. In addition, serum samples were examined for neutralizing antibodies. After inoculation, the frequency of various lymphocyte subsets in peripheral blood was determined by FACScan. One week after inoculation, T-lymphocytes proliferated abundantly and a B-lymphocyte response was observed. When PBMC were used directly without restimulation, only 15% of the PRV-infected target cells were lysed, and about 15-20% of uninfected target cells were lysed. In contrast, when PBMC were restimulated with PRV, up to 50% of the PRV-infected target cells were lysed while only 30% of the uninfected target cells were lysed. The frequency of various T-lymphocyte subsets in the circulation did not change significantly after inoculation, which indicates that the number of PRV-specific lymphocytes in circulation was very small. After challenge, the T-lymphocyte response was enhanced, but the B-lymphocyte response was not. When PBMC were used directly, only 20% of the PRV-infected and uninfected target cells were lysed after challenge. In contrast, when PBMC were restimulated with PRV, they again lysed more PRV-infected target cells than uninfected target cells. Cytolytic cells were detected for a longer period after challenge than after inoculation. Since it was only possible to clearly detect cytolysis after lymphocytes were restimulated with PRV, it may be that they do not preferentially localize in blood or that they are too few in blood to be detected without further antigenic restimulation in vitro. These lymphocytes may instead localize in other tissues, such as mucosal tissues, tonsils and draining lymph nodes. Whether such a reservoir of PRV-specific cytolytic cells is important in clearing the virus is still unknown. In this study we demonstrated PRV-specific lymphocytes in circulation after they were restimulated in vitro with PRV.

Animals↗
PubMed1999

Tumor necrosis factor-alpha and virus expression in the central nervous system of cats infected with feline immunodeficiency virus.

Cytokine disregulation has been implicated in the pathogenesis of lentivirus-induced diseases. In the present study, 18 specific pathogen free (SPF) cats were inoculated with feline immunodeficiency virus (FIV) Petaluma strain and sacrificed at different times post-infection. Five additional SPF cats were used as controls. The cell localization of the cytokine tumor necrosis factor alpha (TNF-alpha) in the central nervous system (CNS) was determined by immunohistochemical and morphometric analyses with a polyclonal rabbit anti-human TNF-alpha antibody. TNF-alpha and FIV RNA were measured using competitive reverse transcriptase polymerase chain reaction (PCR) assays and the number of proviral genomes was estimated by competitive PCR. Portions of frontal cortex were collected from each animal and both formalin-fixed and snap-frozen and stored at -80 degrees C until used. The results showed that TNF-alpha is present mainly in astrocytes and microglial cells. Morphometric analysis showed that areas of TNF-alpha production increased in the early phases of infection. Molecular analyses demonstrated that the kinetics of proviral loads in the CNS were comparable to what observed in lymph nodes and peripheral blood mononuclear cells, with the peaks in the early and late stages of infection. A positive correlation was found between viral parameters and TNF-alpha transcription, the strongest relationship was found between the transcription of the cytokine and viral RNA load. These results confirm that invasion of CNS by FIV occurs soon after virus exposure and that during this phase there is an increase of local viral loads with concomitant up-regulation of TNF-alpha expression. During the asymptomatic phase viral replication remains low in spite of the progression of CNS alterations. The dissociation between the viral load and the lesions observed suggests the importance of an indirect mechanism for the progression of these lesions, even if TNF-alpha seems to play a role particularly in the early phase of infection.

Animals↗