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PubMed2000

Value of reverse transcription polymerase chain reaction assay in pathological stage T3N0 prostate cancer.

BACKGROUND: We tested the ability of the nested reverse transcription polymerase chain reaction (RT-PCR) assay to detect signs of biochemical recurrence of prostate cancer in the lymph nodes and peripheral blood of patients with pT3N0 prostate cancer. METHODS: Using lymph nodes and pre- and postoperative peripheral blood dissected from 30 patients with pT3N0 prostate cancer treated by radical prostatectomy, we used RT-PCR for prostate-specific membrane antigen (PSM) and serum prostate-specific antigen (PSA) to determine the presence of prostate cancer. Results of the nested RT-PCR assay were compared with pathological stages and biochemical recurrence. RESULTS: Two of 13 patients with capsular penetration (15%), 6 of 10 patients with invasion of seminal vesicles (60%), and 3 of 7 patients with a positive surgical margin (43%) were RT-PCR-positive for PSM and/or PSA in the lymph nodes. Results of preoperative RT-PCRs of peripheral blood for PSM and for PSA significantly differed between positive and negative results of RT-PCR in lymph nodes (P < 0.001 and P < 0.001, respectively). Results of postoperative RT-PCRs of peripheral blood for PSM and for PSA also significantly different between positive and negative results of RT-PCR in lymph nodes (P = 0.011 and P = 0.001, respectively). Nine of 11 patients with positive nested RT-PCR for PSM and/or PSA in the lymph nodes (82%) experienced biochemical recurrence. Significant difference in Kaplan-Meier recurrence-free actuarial curves was noted between patients who nested positive and negative on RT-PCR in the lymph nodes, pre- and postoperative peripheral blood, biopsy and prostatectomy Gleason score, and preoperative PSA values. In multivariate analysis, biopsy and prostatectomy Gleason score (P = 0.026, P = 0.020, respectively), pre- and postoperative RT-PCR for PSM in peripheral blood (P = 0.030 and P = 0.040, respectively), and RT-PCR for PSM in lymph nodes (P = 0.035) were independent prognostic factors. CONCLUSIONS: Nested RT-PCR assay of the lymph nodes or peripheral blood significantly predicted biochemical recurrence after surgery. It may help identify patients at risk for recurrence and progression of prostate cancer.

Aged↗
PubMed2001

Tumor necrosis factor-dependent segmental control of MIG expression by high endothelial venules in inflamed lymph nodes regulates monocyte recruitment.

Monocytes recruited from the blood are key contributors to the nature of an immune response. While monocyte recruitment in a subset of immunopathologies has been well studied and largely attributed to the chemokine monocyte chemoattractant protein (MCP)-1, mechanisms mediating such recruitment to other sites of inflammation remain elusive. Here, we showed that localized inflammation resulted in an increased binding of monocytes to perifollicular high endothelial venules (HEVs) of lymph nodes draining a local inflammatory site. Quantitative PCR analyses revealed the upregulation of many chemokines in the inflamed lymph node, including MCP-1 and MIG. HEVs did not express detectable levels of MCP-1; however, a subset of HEVs in inflamed lymph nodes in wild-type (but not tumor necrosis factor [TNF] null mice) expressed MIG and this subset of HEVs preferentially supported monocyte binding. Expression of CXCR3, the receptor for MIG, was detected on a small subset of peripheral blood monocytes and on a significant percentage of recruited monocytes. Most importantly, in both ex vivo and in vivo assays, neutralizing anti-MIG antibodies blocked monocyte binding to inflamed lymph node HEVs. Together, these results suggest that the lymph node microenvironment can dictate the nature of molecules expressed on HEV subsets in a TNF-dependent fashion and that inflammation-induced MIG expression by HEVs can mediate monocyte recruitment.

Animals↗
PubMed1997

Measurement of interleukin-6, interleukin-10, and tumor necrosis factor-alpha levels in tissues and plasma after thermal injury in mice.

BACKGROUND: Cytokines are important modulators of physiologic alterations after thermal injury. Indeed, an increase in the level of circulating cytokines has been documented after thermal injury. However, the mechanism of the increase has not been clarified. We determined cytokine levels in local tissue after thermal injury to identify the tissues responsible for the increase. METHODS: Female C57BL/6 mice each received a 20% full-thickness burn injury. Blood, burned skin, unburned skin, muscle underlying the burn, and muscle of the thigh, liver, spleen, and mesenteric lymph node were sampled at 1, 2, 4, 8, and 24 hours after injury. Uninjured control mice were treated similarly. The samples were cultured, and concentrations of tumor necrosis factor-alpha, interleukin-6 (IL-6), and IL-10 in the culture media were measured by using an enzyme-linked immunosorbent assay. RESULTS: IL-6 levels in unburned skin were significantly increased at 1 hour and decreased at 24 hours, compared with the control. IL-6 levels in muscle underlying the burn were significantly decreased at 8 hours. No elevation of plasma IL-6 levels was observed after injury. Neither tumor necrosis factor-alpha IL-10 was detected in any tissue. CONCLUSIONS: Results indicate that unburned skin may be a major source of IL-6 production after thermal injury and may contribute to the physiologic alterations occurring after such injury.

Animals↗
PubMed1981

Spontaneous (H)-thymidine uptake in histological subgroups of human B-cell lymphomas.

Spontaneous (3H)-TdR incorporation was studied in cell suspensions from 64 patients with monoclonal B-cell neoplasias. Among various incubation periods a long-term (20 h) assay with (3H)-TdR had the greatest discriminatory power versus non-neoplastic lymph node cell suspensions. The (3H)-TdR uptake correlated positively with cell volume, nuclear volume, and cells in S + G + M phase of the cell cycle. A high degree of heterogeneity with regard to (3H)-TdR incorporation was found within several histological groups of the Kiel classification, especially in low-grade malignant lymphomas of centroblastic/centrocytic origin and in the lymphoplasmacytoid groups. In highly malignant lymphomas (3H)-TdR uptake was statistically, significantly higher than in lymphomas of low-grade malignancy. Patients with localized disease (stages I and II) and those with "B'-symptoms showed increased incorporation of radioactive thymidine as compared to patients with disseminated mined by histopathology and spontaneous uptake of (3H)-TdR.

Adult↗
PubMedDate not supplied

Canine experimental infection: intradermal inoculation of Leishmania infantum promastigotes.

Five mixed breed dogs were inoculated intradermally (ID) with cultured virulent stationary phase promastigotes of Leishmania infantum Nicole, 1908 stocks recently isolated. Parasite transformations in the skin of ID infected dogs were monitored from the moment of inoculation and for 48 h, by skin biopsies. Anti-Leishmania antibody levels were measured by indirect immunofluorescence assay, counterimmunoelectrophoresis and direct agglutination test, and clinical conditions were examined. Thirty minutes after ID inoculation the first amastigotes were visualised and 3 to 4 h after inoculation the promastigotes were phagocytized by neutrophils and by a few macrophages. These cells parasitised by amastigotes progressively disappeared from the skin and 24 h after inoculation parasites were no longer observed. Local granulomes were not observed, however, serological conversion for antibodies anti-Leishmania was achieved in all dogs. Direct agglutination test was the only technique positive in all inoculated dogs. Amastigotes were found in the popliteal lymph node in one dog three months after inoculation. This work demonstrates that, with this inoculum, the promastigotes were transformed into amastigotes and were up taken by neutrophils and macrophages. The surviving parasites may have been disseminated in the canine organism, eliciting a humoral response in all cases.

Agglutination Tests↗
PubMed1985

The effect of intraperitoneal and intramammary immunization of sheep on the numbers of antibody-containing cells in the mammary gland, and antibody titres in blood serum and mammary secretions.

The contribution of gut-associated lymphoid tissue (GALT) to the local response in the mammary gland is well documented in laboratory animals and has been evaluated in this study in ruminants. Ewes were immunized intraperitoneally (IP) with antigen in Freund's complete adjuvant (FCA), a procedure which stimulates the production of antibodies of the IgA class in the intestine, and challenged intramammarily (IMam) either during colostrum formation or mammary gland involution. Despite a substantial IgA antibody-containing cell (ACC) response in the intestine in IP immunized sheep, there was no evidence to suggest a relocation of IgA-specific ACC to the mammary gland. There was, however, an IgA antibody response in mammary secretion of IP immunized animals, regardless of whether the mammary gland was locally immunized, but the origin of this antibody is unclear. IP/IMam immunized sheep did have an enhanced antigen-specific ACC response of the IgG1 isotype in locally immunized glands, but whether these cells were of GALT or systemic origin is also unclear.

Animals↗
PubMed1991

Enzymological evidence for the indispensability of small intestine in the synthesis of arginine from glutamate. I. Pyrroline-5-carboxylate synthase.

The in vivo synthesis of arginine from glutamate in mammals requires seven enzymes to cooperate. Pyrroline-5-carboxylate synthase (PCS) is the first enzyme required. In order to establish the interorgan dependency of arginine synthesis, we quantitated PCS activity in as many as 32 rat tissues and found that the activity was concentrated only in the upper small intestine. Minor activity was found in pancreas, thymus, lymph node, and some other tissues: this was confirmed by the dependency on specific substrates, the loss of activity in the presence of an inhibitor, and identifying the reduced product as proline. No difference in activity was found between male and female rats on a milligram protein basis. The strict tissue localization of PCS and the localization of other enzymes of arginine synthesis previously reported clearly indicate that the upper small intestine is an indispensable tissue for the arginine synthesis from glutamate. Many of the tissues examined showed an activity to form an unknown product from glutamate. When assayed by the previously reported radiometric assay procedure using an AG1-X8 column (acetate), the product was not separated from PC and caused false-positive activities of PCS. An improved procedure was developed to overcome this technical difficulty. The new procedure enabled us to detect even 20 pmol PC without contamination by the adjoining unknown product. A preliminary characterization of the unknown product was achieved.

Animals↗
PubMed2004

Identification of sialoadhesin as a dominant lymph node counter-receptor for mouse macrophage galactose-type C-type lectin 1.

In the sensitization phase of contact hypersensitivity in mice, dermal macrophages (MOs) expressing MO galactose-type C-type lectin1 (MGL1) are known to migrate from the dermis to lymph nodes (LNs) where they accumulate in the subcapsular sinus, interfollicular regions, and areas surrounding high endothelial venules. We hypothesize that the interactions between MGL1 and its ligands determine the localizations of MGL1-positive cells within the LNs. In the present study, our major aim was to isolate MGL1 counter-receptor(s) from lysates of LNs using affinity chromatography with immobilized recombinant MGL1. Fractions bound and eluted with EDTA were analyzed by SDS-PAGE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. One of the predominant components was sialoadhesin (Sn, Siglec-1). Sn from lysates of LNs was immobilized on microtiter plates precoated with anti-Sn monoclonal antibody, and binding of recombinant MGL1 and adhesion of cells expressing MGL1 were tested. The binding of recombinant MGL1 to Sn was shown to be dependent on Ca2+ and N-glycans on Sn. MGL1-transfected Chinese hamster ovary cells adhered to the Sn-coated plates, whereas mock transfectants did not. Immunohistochemical localization of anti-Sn monoclonal antibody in LN coincided with the subcapsular sinus area to which recombinant MGL1 was bound. Furthermore, the distribution of MGL1+ cells after sensitization with FITC was demonstrated to overlap with that of Sn within the subcapsular sinus of draining LNs. These results suggest that Sn acts as an endogenous counter-receptor for MGL1.

Animals↗
PubMed1996

Comparison of analysis of the different prostate-specific antigen forms in serum for detection of clinically localized prostate cancer.

OBJECTIVES: To compare different forms and ratios of serum prostate-specific antigen (PSA) to determine which form or ratio provides optimal diagnostic specificity and sensitivity in distinguishing between benign prostatic hyperplasia (BPH) and clinically localized prostate cancer. METHODS: Serum samples were obtained from 47 patients with BPH and 39 with clinically localized prostate cancer. Patients with BPH underwent either transurethral resection of the prostate or transurethral microwave thermotherapy. Patients with prostate cancer, all of whom had no metastases on radionucleotide bone scans and no pelvic lymph node involvement, underwent either radical external beam radiation therapy or radical retropubic prostatectomy. All patients had pretreatment serum PSA levels between 1 and 20 ng/mL. The different forms of serum PSA (free PSA [PSA-F], PSA complexed to alpha 1-antichymotrypsin [PSA-ACT], and total PSA [PSA-T]) were measured using different monoclonal antibodies against PSA and ACT and immunofluorometric assay techniques. Furthermore, three ratios (PSA-F/PSA-T, PSA-ACT/PSA-T, and PSA-F/PSA-ACT) were calculated. RESULTS: By receiver operating characteristic curve analysis, the performance of the different forms and ratios were compared. The PSA-F/PSA-T ratio had the greatest area under the curve (AUC, 0.776), significantly larger than that for PSA-T (0.612; P = 0.024). For PSA-ACT/PSA-T, the AUC was 0.695 (P = 0.283 versus PSA-T) and 0.773 for PSA-F/PSA-ACT (P = 0.051 versus PSA-T). At a cutoff level < 0.17, PSA-F/PSA-T had a sensitivity of 79%, a specificity of 66%, and a positive predictive value of 66% compared with 74%, 38%, and 50%, respectively, for PSA-T at a cutoff level > 4.0 ng/mL. CONCLUSIONS: The PSA-F/PSA-T ratio gives the best diagnostic performance compared with that for other forms and ratios of PSA and will reduce the number of prostatic biopsies in patients with BPH.

Adult↗
PubMed1995

Repeated exposure to silicone gel can induce delayed hypersensitivity.

The possible immunologic reactivity of silicone gel remains speculative and controversial. In this laboratory, a quantitative lymphocyte localization assay has been developed and well studied using pure lymphocytes collected by the technique of lymph vessel cannulation in sheep. The kinetics of antigen-specific immune responses (e.g., tuberculin reaction) in this model are well described and accepted. Using the known parameters regarding the response to purified protein derivative and the classic adjuvant Freund's Complete Adjuvant, this study was designed to identify the possible antigen-specific immunologic response, in the form of delayed-type hypersensitivity, after repeated exposure to silicone gel. Pure lymphocytes were collected by cannulating the efferent vessel of a subcutaneous lymph node in four groups of primed sheep which, 30 days previously, had received intradermal injections of 0.9% saline (negative controls; n = 6), Freund's Complete Adjuvant only (positive controls; n = 6), silicone gel (n = 7), or Freund's Complete Adjuvant homogenized with silicone gel (n = 7) in an attempt to induce sensitization. Multiple (1040) intradermal skin tests were performed using silicone gel, purified protein derivative, and 0.9% saline. After the skin lesions had developed for 48 hours, 5 x 10(8) lymphocytes were labeled in vitro with indium-111, returned intravenously, and allowed to circulate for 3 hours. Sheep were euthanized, the skin lesions were removed, and the radioactivity was counted in a gamma spectrometer. The radioactivity in each skin lesion is considered a measure of lymphocyte accumulation. The occurrence of augmented accumulation after reexposure to an antigen is a hallmark of delayed-type hypersensitivity. The purified protein derivative and saline lesions functioned as positive and negative controls, with counts per minute (cpm +/- standard error) of 2404 +/- 478 (Freund's Complete Adjuvant group) and 149 +/- 21 (saline group), respectively. Significantly greater (p = 0.0021) radioactivity was found in the silicone gel sites (310 +/- 35) in the silicone gel-primed group and the Freund's Complete Adjuvant plus silicone gel group (453 +/- 44; p = 0.0004) than in normal skin in each group. These data suggest that it may be possible to induce an antigen-specific lymphocyte-mediated response to silicone gel.

Animals↗
PubMed1999

HMGIC expression in human adult and fetal tissues and in uterine leiomyomata.

The high-mobility-group (HMG) protein gene, HMGIC, is localized to chromosome 12, band q15, a region often rearranged in benign mesenchymal tumors, including uterine leiomyomata. Although some evidence suggests a role in regulation of cell proliferation, the precise function of HMGIC in the development or progression of these tumors remains unclear. We investigated HMGIC expression in 17 fetal tissues (adrenal, aorta, bone, brain, heart, intestine, kidney, liver, lung, muscle, ovary, placenta, skin, spleen, stomach, testis, and uterus) and 10 adult tissues (aorta, brain, cerebellum, fat, kidney, liver, lung, lymph node, myometrium, and spinal cord) by Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) assays. Comparisons between HMGIC gene expression in tumor samples from 11 uterine leiomyomata and 7 normal matched myometrium or in vitro cell cultures (chorionic villi, placenta, myometrium, leiomyoma, and skin) were also performed. The gene was expressed in all fetal tissues tested but only in adult lung and kidney. HMGIC was also expressed in leiomyoma tumor samples containing t(12;14) and in all in vitro cell cultures. The pattern of HMGIC expression suggests that this gene is important in rapidly proliferating human fetal tissues. Restoration of expression in leiomyomata required dysregulation of HMGIC. Transcripts of HMGIC can also be detected after in vitro cell culture, suggesting that HMGIC expression may be affected by factors present in culture media and serum. Genes Chromosomes Cancer 25:316-322, 1999.

Adult↗
PubMed1992

Role of adhesion molecules in the immune reaction to M-MSV-induced tumors.

We have investigated the in vivo role of 2 different adhesion molecules, LFA-1 and LECAM-1, in the immune reaction to Moloney-murine-sarcoma-virus(M-MSV)-induced tumors, which undergo a peculiar spontaneous regression due to generation of a strong virus-specific cytotoxic-T-lymphocyte(CTL) response. Repeated administration of anti-LFA-1 monoclonal antibody (FD441.8 MAb), i.p. or at the site of virus inoculation, enhanced tumor growth and delayed regression, while i.p. administration of anti-LECAM-1 MEL-14 MAb gave rise to tumors that grew progressively and caused host death. Evaluation of the immunological response in MAb-treated mice showed reduced generation of virus-specific CTL precursors (p) in the spleen of animals given FD441.8 MAb i.p.; CTLp frequency in locally treated mice overlapped with that of control mice injected with virus only. FD441.8 MAb treatment did not interfere with CTL homing in the tumor, since the frequency of M-MSV-specific CTLps in sarcomas was similar in treated and control mice. Cytofluorimetric analysis indicated that the majority of tumor-infiltrating lymphocytes (TIL) from MAb-treated mice were covered by anti-LFA-1 MAb, and lacked cytotoxic activity when assayed against target cells bearing relevant tumor antigens. Instead, in mice injected i.p. with MEL-14 MAb, a very low frequency of CTLps was detected in lymph nodes draining the tumor area, and within the tumor. Our results indicate that enhanced tumor growth, depending on the MAb used, is the resultant of an inhibitory effect on different T-lymphocyte functions. Tumor progression in anti-LFA-1 MAb-injected mice is explained mostly by blockage of CTL lytic activity at the tumor site; in mice receiving i.p. MEL-14 MAb treatment, by the failure of naive T lymphocytes to enter peripheral lymph nodes and subsequently by the lack of generation of tumor-specific CTLs.

Animals↗
PubMed2002

Serum cytokeratin fragment 21.1 (CYFRA 21.1) as tumour marker for breast cancer: comparison with carbohydrate antigen 15.3 (CA 15.3) and carcinoembryonic antigen (CEA).

Serum carbohydrate antigen 15.3 (CA 15.3) and carcinoembryonic antigen (CEA) are currently employed in clinical practice as markers for breast cancer, particularly in the follow-up and therapy monitoring. However, the American Society for Clinical Oncology (ASCO) stated in its clinical practice guidelines for the use of tumour markers in breast carcinoma that neither CA 15.3 nor CEA are recommended for routine use in screening, diagnosis and surveillance after primary treatment, or in monitoring response to treatment, because current literature data are insufficient. Cytokeratin fragment 21.1 (CYFRA 21.1) assay detects a serum fragment of cytokeratin 19 (CK19) and is employed in the diagnosis and management of lung cancer, particularly of squamous cell histotype. Breast carcinoma has been demonstrated to express CK19 fragments in the primary and metastatic lesions and CK19 mRNA is detectable in peripheral blood from patients affected by breast cancer. We measured serum markers CYFRA 21.1, CEA and CA 15.3 in the sera from 212 females affected by histologically proven breast carcinoma. Patients comprised 96 individuals with untreated primary disease (54 stage I-II, 18 stage III and 24 stage IV), 30 regional (chest-wall and/or lymph-nodes) relapsing disease and 68 metastatic (haematogenous metastases) relapsing disease. Forty-eight patients previously treated by surgery and without any evidence of disease were enrolled to evaluate the role of serum markers in the monitoring for recurrence of the disease. One hundred healthy age-matched females and 65 patients affected by benign mammary gland disease (including 38 patients with mastopathy and 27 with fibroadenoma) were enrolled as controls. Serum levels of all markers increased from controls to patients affected by breast cancer, from stage I-II to stage IV of the breast cancer and from local to advanced recurrence. The comparison of diagnostic accuracy in the detection of primary and relapsing breast cancer showed no significant differences between markers. Univariate and multivariate survival analysis showed a significant statistically prognostic value for CA 15.3 and CYFRA 21.1 but not for CEA. However, the factors N and M were confirmed to be very strong predictors of the patients' survival. Finally, CEA and CYFRA 21.1 detected less recurrences than CA 15.3. In conclusion, our data show no significant improvement in the diagnosis, prognostic evaluationand follow-up of breast cancer by CYFRA 21.1 and CEA assays compared to CA 15.3 assay. Considering the ASCO statement on tumour markers in breast cancer, the CYFRA 21.1 assay should not be employed in clinical practice.

Adult↗
PubMed1999

The changing face of AIDS-related opportunism: cryptococcosis in the highly active antiretroviral therapy (HAART) era. Case reports and literature review.

Only nine cases of AIDS-related cryptococcosis have been reported until now in patients receiving highly active antiretroviral therapy (HAART), all of them with abnormal clinical features. Two HIV-infected patients who experienced an atypical relapse of cryptococcosis shortly after the start of HAART and despite maintenance antifungal treatment, are described. Six different relapses of cryptococcal meningitis were observed in a 28-month period in a patient who obtained a poor immune recovery after HAART (as shown by a CD4+ lymphocyte count ranging from 78 to 149 cells/microL, opposed to a baseline level of 98 cells/microL). On the other hand, a patient with favorable immunological response to HAART (as expressed by a CD4+ count growing from 7 to 186 cells/microL), experienced isolated multiple indolent cryptococcal abscesses involving head, neck, the anterior thoracic wall, and regional lymph nodes, with repeatedly negative cultures, and diagnosis obtained by both histopathologic study and positive serum antigen assay. Both our case reports are representative of novel correlations between opportunistic pathogens and immune reactivity, descending from the introduction of HAART. The first episode describes an exceedingly elevated number of disease relapses despite HAART and antifungal maintenance treatment, which may descend from an incomplete immune response to antiretroviral therapy, possibly responsible for failure in obtaining eradication of yeasts, but also for lack of disease dissemination (usually leading to a lethal multivisceral involvement in the pre-HAART era). The abnormal disease course and localization of second reported patient well depicts an "immune reconstitution syndrome" probably representing a flare-up of a latent fungal infection, caused by a rapidly effective HAART. In patients treated with HAART, AIDS-related cryptococcosis cannot therefore be ruled out by the absence of neurological involvement, and by persistingly negative cultures.

AIDS-Related Opportunistic Infections↗
PubMed1994

Activation of high endothelial venules in peripheral lymph nodes. The involvement of interferon-gamma.

The introduction of Freund's complete adjuvant into footpads of mice leads to functional changes of the high endothelial venules (HEV) in the draining lymph nodes. Using an in vitro adhesion assay, increased binding of monocytes and dendritic cells could be seen. These effects are also seen after injection of high doses of INF-gamma. No changes in the expression of vascular addressins, VCAM-1, E selection or ICAM-1 could be observed but the binding of monocytes could be blocked with an antibody against Mac-1. Depletion studies have made it clear that macrophages in the lymph node are not directly involved in the activation of the HEV. The induction of additional adhesion mechanisms on HEV could be an efficient way to recruit inflammatory cells into the lymph node, thereby regulating the local immune response.

Animals↗
PubMed2005

Immunomodulation by topical particle-mediated administration of cytokine plasmid DNA suppresses herpetic stromal keratitis without impairment of antiviral defense.

BACKGROUND: We investigated whether the course of herpetic stromal keratitis (HSK) in BALB/c mice could be altered by topical gene-gun-mediated administration of interleukin (IL)-4 or IL-10 plasmid DNA. METHODS: Corneas of BALB/c mice were transfected with plasmids expressing beta-galactosidase (beta-gal), IL-4, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), and pCR3.1 (control) 2 days before Herpes simplex virus-1 (HSV-1; KOS) infection. Development of keratitis and cell infiltration were studied. HSV-1 replication was monitored by plaque assay. Expression of cytokines was detected by enzyme-linked immunosorbent assay. HSV-specific proliferation in the regional lymph nodes and spleens was measured. HSV-1 neutralizing antibody titers and IgG2A/IgG1 ratios were determined. RESULTS: Expression of beta-gal was found in the treated corneas, but not in other tissues. IL-4 or IL-10 plasmid administration induced cytokine production in the corneas. After treatment with 300 psi, the severity of HSK was attenuated (each P<0.05), and the numbers of infiltrating inflammatory cells were lower than in the pCR3.1-treated controls (P<0.001). IL-6, but not IL-1alpha, expression in the cornea was reduced after treatment with IL-4 or IL-10 plasmid DNA. The HSV-1-specific DTH response, corneal Th1 cytokine profile, IgG/IgG2a/IgG1 ratio, neutralizing antibody titers, and virus clearance did not differ between the groups. CONCLUSIONS: Thus, topically administered IL-4 and IL-10 plasmid DNA can lead to a milder course of HSK without impeding viral clearance. The gene gun technique for corneal delivery of plasmid cytokine DNA may be useful for modulating local immune responses without affecting antiviral defense.

Administration, Topical↗
PubMed1990

Elimination of spleen and of lymph node macrophages and its difference in the effect on the immune response to particulate antigens.

To study the role of macrophages in the in situ immune response to particulate antigens in spleen and popliteal lymph nodes (PLN), mice were injected with dichloromethylene diphosphonate (Cl2MDP)-containing liposomes to eliminate macrophages, followed by immunization with trinitrophenylated sheep red blood cells (TNP-SRBC). Depletion of macrophages in the spleen caused a strong decrease in the number of antibody-forming cells (AFC), which develop after intravenous (i.v.) injection of the antigen. These results strongly suggested the involvement of splenic macrophages in the processing of TNP-SRBC. In particular, the populations of marginal zone macrophages may be involved in the inductive phase of an antibody response to particulate antigens. These macrophages are strategically positioned at the end of the white pulp capillaries in the marginal zone of the spleen and they have their cell processes between the marginal zone-B cells. Elimination of macrophages in PLN had no effect on the number of AFC, which develop after subcutaneous (s.c.) injection of the antigen in the hind footpads. This indicates that the macrophages are not essential for the induction of a local immune response to the particulate antigen TNP-SRBC. After depletion of lymph node macrophages, the number of AFC developing in the spleen after s.c. footpad injection of the antigen increased and the anti-TNP serum titers were elevated. This may well be caused by the fact that more of the antigen reaches the circulation and subsequently stimulates the spleen.

Animals↗
PubMed1981

Studies with a monoclonal antibody on the distribution of Thy-1 in the lymphoid and extracellular connective tissues of the dog.

BALB/c mice were immunized with canine T lymphocytes from lymph node and used in cell fusion experiments to derive monoclonal antibodies to the T lymphocyte surface. The cloned hybrid line F3-20-7 described in this paper was shown to be secreting antibodies directed at canine Thy-1 since (1) the tissue distribution of the antigen corresponded precisely to the tissue distribution expected of canine Thy-1 from previous studies and (2) the antigen purified from F3-20-7 monoclonal antibody affinity columns (a) had the same mobility on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as pure rat Thy-1 and (b) could inhibit and assay shown previously to be directed at canine Thy-1 on the basis of cross-reactivity with rat Thy-1. Studies with the fluorescence-activated cell sorter showed that all thymocytes and lymph node T lymphocytes of the dog were Thy-1 positive, establishing that Thy-1 is a T cell marker in the dog, as in the mouse, although the presence of a small percentage of Thy-1-positive B cells could not be entirely excluded. In addition, 15% of the nucleated bone marrow cells were Thy-1 positive, which is similar to the finding in rat bone marrow. Localization studies on frozen sections of skin demonstrated large amounts of Thy-1 organized in a highly structured manner around the basal parts of the hair follicles. This covering of Thy-1 was lost as the follicles approached the surface. Epidermal cells were Thy-1 negative. In the kidney, Thy-1 was shown to be strongly associated with all of the basement membranes of the medulla. The kidney cortex, including glomeruli was essentially negative, except for the occasional staining of basement membranes, possibly of collecting ducts.

Animals↗