Injection Of Keyhole Limpet Hemocyanin Research Articles

501 The effect of dietary camelina, flaxseed, and canola oil supplementation on skin fatty acid profile, inflammation and immune responses in healthy adult horses

Abstract As n-6 and n-3 fatty acids (FA) have pro- and anti-inflammatory effects, respectively, supplemental oils have the potential to impact inflammation, the immune response, and skin and coat health. This study aimed to evaluate the effects of camelina (CAM), in comparison to canola (OLA) and flaxseed (FLX) oil on the skin FA profile, inflammation, and the immune response in horses. Horses [n = 24; 14.2 ± 4.8 yr; 518 ± 63 kg body weight (BW)] were acclimated to sunflower oil over 4 wk. Groups were balanced by location, age, weight, sex, and breed and randomly assigned one of the three treatments (CAM, OLA, FLX). Horses were fed their assigned oil at a dose of 0.37 g/kg BW for 16 wk. Skin samples were collected on wk 16, and the skin FA composition was analyzed by gas chromatography. On wk 10 and 12, keyhole limpet hemocyanin (KLH) was administered intramuscularly to each horse to stimulate a systemic immune response. Blood was collected on wk 14 and 16 for the evaluation of plasma KLH immunoglobulin G (IgG) concentrations using a modified ELISA protocol and results were presented as optical density units. On wk 12, after the first KLH injection, a delayed-type hypersensitivity (DTH) test was performed by intradermally injecting KLH and the area of swelling was measured using the software ImageJ at 0.5-, 1-, 8-, 24-, 48-, 72-, and 96-h post-injection. Data were analyzed using the GLIMMIX procedure of SAS considering the effects of treatment, time, and their interaction as fixed, and the effects of horse and location as random. Repeated measures on horses were modelled using a spatial power covariance structure. Significance was declared at P ≤ 0.05 and when fixed effects were significant, means were separated using Tukey-Kramer adjustments. Immune and inflammatory responses were produced by the KLH injections; however, there were no differences among treatment groups regarding KLH antibody and DTH response (P > 0.44). The percentages of palmitic (P = 0.02) and stearic acid (P = 0.03) in the skin at wk 16 were greater in FLX as compared with CAM but neither differed from the OLA. The percentage of linoleic acid (P = 0.01) was greater in CAM than OLA but neither differed from FLX at wk 16. The percentage of all other FA was similar among treatment groups (P > 0.06). These results suggest that CAM is comparable to FLX and OLA regarding support of an immune and inflammatory response, and the oil FA composition was not reflected in the skin FA profile after feeding for 16 wk as hypothesized. In conclusion, at the dose provided, CAM supports a similar immune and inflammatory response to KLH as FLX and OLA; however, the lack of incorporation of FA from the diet into the skin should be further explored.

Enhancing immune responses in Japanese flounder (Paralichthys olivaceus) to Keyhole Limpet Hemocyanin by intramuscularly conjugating a new type of chemokine, JfCXCL8_L1b

CXCL8 is a high-profile chemokine in vertebrates. In our previous study, CXCL8_L1b was first identified in Japanese flounder and triggered varying immune responses in infected fish compared with Japanese flounder CXCL8_L1a (JfCXCL8_L1a). To further understand the immune functions of Japanese flounder CXCL8_L1b (JfCXCL8_L1b), in vitro and in vivo experiments were performed using the recombinant protein rCXCL8_L1b. Our results showed that rCXCL8_L1b significantly induced all the detected immune genes except IgM and IgD in peripheral blood leukocytes (PBLs) of Japanese flounder. Intramuscular injection of Keyhole Limpet Hemocyanin (KLH) did not cause inflammation responses in fish muscle. In the spleen of injected fish, IgM was the only detected gene expression. In contrast, all detected genes in the KLH group were significantly up-regulated in the head kidney, indicating the primary immune organ response to KLH was the head kidney instead of the spleen. At the injection site, we observed significantly induced receptor gene CXCR1 in KLH + rCXCL8_L1a, KLH + rCXCL8_L1b, and KLH + rCXCL8_L1a + rCXCL8_L1b groups on day 1, while significantly induced receptor gene CXCR2 was only observed in the KLH + rCXCL8_L1b group on day 3. Conversely, gene expression of JfCXCL8_L1a and CXCR2 were down-regulated in the head kidney of the KLH + rCXCL8_L1b group compared with the KLH + rCXCL8_L1a group. Intramuscular injection of KLH significantly induced an antibody titer response to KLH at weeks 2 and 4. Compared with rCXCL8_L1a, rCXCL8_L1b, as the immunologic adjuvant of KLH, significantly enhanced the antibody titer response to KLH at weeks 2 and 4. Collectively, this study reveals that JfCXCL8_L1a and JfCXCL8_L1b have different immune pathways, and JfCXCL8_L1b plays a significant role in enhancing the adaptive immunity of T cell-dependent antigen.

Exposure of low-temperature plasma after vaccination in tongue promotes systemic IgM induction against spike protein of SARS-CoV-2

COVID-19 has been pandemic since 2020 with persistent generation of new variants. Cellular receptor for SARS-CoV-2 is angiotensin-converting enzyme 2 (ACE2), where transmembrane serine protease-2 (TMPRSS2) is essential for viral internalization. We recently reported abundant expression of ACE2 and TMPRSS2 in the oral cavity of humans and mice. Therefore, oral cavity may work for COVID-19 infection gates. Here we undertook to evaluate whether vaccination in the tongue harbors any merit in comparison to subcutaneous injection. Low-temperature plasma (LTP) is the fourth physical state of matters with ionization above gas but at body temperature. LTP provides complex chemistry, eventually supplying oxidative and/or nitrosative stress on the interface. LTP-associated cellular death has been reported to cause apoptosis and/or ferroptosis. However, there is few data available on immunogenicity retention after LTP exposure. We therefore studied the effect of LTP exposure after the injection of keyhole limpet hemocyanin (KLH) or spike 2 protein of SARS-CoV-2 to the tongue of six-week-old male BALB/c mice, compared to subcutaneous vaccination. Whereas LTP did not change the expression of ACE2 and TMPRSS2 in the tongue, repeated LTP exposure after tongue vaccination significantly promoted systemic and specific IgM production at day 11. In contrast, repeated LTP exposure after subcutaneous vaccination of KLH decreased systemic IgM production. Of note, tongue injection produced significantly higher titer of IgM and IgG in the case of KLH. In conclusion, LTP significantly reinforced humoral immunity by IgM after tongue injection. Vaccination to the tongue can be a novel strategy to acquire immediate immunity.

Keyhole Limpet Hemocyanin-Conjugated Peptides from Hepatitis C Virus Glycoproteins Elicit Neutralizing Antibodies in BALB/c Mice.

Currently, no vaccine to prevent hepatitis C virus (HCV) infection is available. A major challenge in developing an HCV vaccine is the high diversity of HCV sequences. The purpose of immunization with viral glycoproteins is to induce a potent and long-lasting cellular and humoral immune response. However, this strategy only achieves limited protection, and antigen selection plays a crucial role in vaccine design. In this study, we investigated the humoral immune responses induced by intraperitoneal injection of keyhole limpet hemocyanin conjugated with 4 highly conserved peptides, including amino acids [aa]317-325 from E1 and aa418-429, aa502-518, and aa685-693 from E2, or 3 peptides from hypervariable region 1 (HVR1) of E2, including the N terminus of HVR1 (N-HVR1, aa384-396), C terminus of HVR1 (C-HVR1, aa397-410), and HVR1 in BALB/c mice. The neutralizing activity against HCV genotypes 1-6 was assessed using the cell culture HCV (HCVcc) system. The results showed that the 4 conserved peptides efficiently induced antibodies with potent neutralizing activity against 3 or 4 genotypes. Antibodies induced by aa685-693 conferred potent protection (>50%) against genotypes 2, 4, and 5. Peptide N-HVR1 elicited antibodies with the most potent neutralization activities against 3 HCV genotypes: TNcc(1a), S52(3a), and ED43(4a). These findings suggested that peptides within HCV glycoproteins could serve as potent immunogens for vaccine design and development.

Rindopepimut with Bevacizumab for Patients with Relapsed EGFRvIII-Expressing Glioblastoma (ReACT): Results of a Double-Blind Randomized Phase II Trial.

Rindopepimut is a vaccine targeting the tumor-specific EGF driver mutation, EGFRvIII. The ReACT study investigated whether the addition of rindopepimut to standard bevacizumab improved outcome for patients with relapsed, EGFRvIII-positive glioblastoma. In this double-blind, randomized, phase II study (NCT01498328) conducted at 26 hospitals in the United States, bevacizumab-naïve patients with recurrent EGFRvIII-positive glioblastoma were randomized to receive rindopepimut or a control injection of keyhole limpet hemocyanin, each concurrent with bevacizumab. The primary endpoint was 6-month progression-free survival (PFS6) by central review with a one-sided significance of 0.2. Between May 2012 and 2014, 73 patients were randomized (36 rindopepimut, 37 control). Rindopepimut toxicity included transient, low-grade local reactions. As primary endpoint, PFS6 was 28% (10/36) for rindopepimut compared with 16% (6/37) for control (P = 0.12, one-sided). Secondary and exploratory endpoints also favored the rindopepimut group including a statistically significant survival advantage [HR, 0.53; 95% confidence interval (CI), 0.32-0.88; two-sided log-rank P = 0.01], a higher ORR [30% (9/30) vs. 18% (6/34; P = 0.38)], median duration of response [7.8 months (95% CI, 3.5-22.2) vs. 5.6 (95% CI, 3.7-7.4)], and ability to discontinue steroids for ≥6 months [33% (6/18) vs. 0% (0/19)]. Eighty percent of rindopepimut-treated patients achieved robust anti-EGFRvIII titers (≥1:12,800), which were associated with prolonged survival (HR = 0.17; 95% CI, 0.07-0.45; P < 0.0001). Our randomized trial supports the potential for targeted immunotherapy among patients with GBM, but the therapeutic benefit requires validation due to the small sample size and potential heterogeneity of bevacizumab response among recurrent patients with GBM.See related commentary by Wick and Wagener, p. 1535.

Immunological characteristics of Interleukin-2 receptor subunit beta (IL-2Rβ) in flounder (Paralichtlys olivaceus): Implication for IL-2R function

Interleukin-2 receptor subunit beta of flounder (Paralichthys olivace, fIL-2Rβ) was annotated on the NCBI, its gene was cloned and characterized functionally in this study. And then the amino acids sequences and tertiary structure of fIL-2Rβ were analyzed, respectively. RT-PCR and ImageJ analyzed showed that fIL-2Rβ mRNA were expressed in the gill, spleen, kidney, intestines, liver, blood, muscle and skin, which showed high signals in spleen and blood. And then the recombinant protein of fIL-2Rβ extracellular region and its polyclonal antibodies were produced, native fIL-2Rβ molecules in flounder peripheral blood leukocytes (PBLs) were identified at 60.7 kDa by Mass spectrometry, which were in accordance with the molecular mass of full fIL-2Rβ protein calculated on the predicted protein sequence. Then the IL-2Rβ+ cell in T/B lymphocytes were characterized by Flow cytometry and indirect immunofluorescence assay, respectively. The results showed that the percentages of IL-2Rβ+ leukocytes, IL-2Rβ+/CD4+, IL-2Rβ+/IgM+ lymphocytes were 18.4 ± 2.7%, 4.5 ± 0.8%, 4.3% ± 0.5 in PBLs, and were 13.6 ± 0.9%, 4.6 ± 1.1%, 6.1% ± 0.4 in spleen, similarly, the percentages of IL-2Rβ+ leukocytes, IL-2Rβ+/CD4+, IL-2Rβ+/IgM+ lymphocytes were 9.4 ± 0.3%, 4.0 ± 0.5%, 5.7 ± 0.1% in head kidney, respectively. After KLH injection, compared with control group, the gene expression of IL-2, IL-2Rβ, CD3, TCR, CD79b and IgM in spleen of flounder were up-regulated, respectively (p < 0.05). And the FCM results showed that the percentages of IL-2Rβ+ leukocytes in PBLs were significantly increased post Keyhole limpet hemocyanin (KLH) injection, which peaked 23.9 ± 0.9% at 9th day (p < 0.05). To our knowledge, those results first reported that the characteristics of IL-2R and IL-2R + molecules were expressed on both B and T lymphocytes in fish. At the same time, this study lays a foundation for further exploring the interaction between IL-2 and IL-2R to promote cell proliferation and carrying out biological functions.

Paraquat Preferentially Induces Apoptosis of Late Stage Effector Lymphocyte and Impairs Memory Immune Response in Mice.

Paraquat (PQ) is a toxic non-selective herbicide. To date, the effect of PQ on memory immune response is still unknown. We investigated the impact of PQ on memory immune response. Adult C57BL/6 mice were subcutaneously injected with 2 mg/kg PQ, 20 mg/kg PQ or vehicle control every three days for two weeks. A single injection of keyhole limpet hemocyanin (KLH) at day four after the initial PQ treatment was used to induce a primary immune response; a second KLH challenge was performed at three months post the first KLH immunization to induce a secondary immune response. In steady state, treatment with 20 mg/kg PQ reduced the level of serum total IgG, but not that of IgM; treatment with 20 mg/kg PQ decreased the number of effector and memory lymphocytes, but not naïve or inactivated lymphocytes. During the primary immune response to KLH, treatment with 20 mg/kg PQ did not influence the proliferation of lymphocytes or expression of co-stimulatory molecules. Instead, treatment with 20 mg/kg PQ increased the apoptosis of lymphocytes at late stage, but not early stage of the primary immune response. During the secondary immune response to KLH, treatment with 20 mg/kg PQ reduced the serum anti-KLH IgG and KLH-responsive CD4 T cells and B cells. Moreover, effector or activated lymphocytes were more sensitive to PQ-induced apoptosis in vitro. Treatment with 2 mg/kg PQ did not impact memory immune response to KLH. Thus, treatment with 20 mg/kg PQ increased apoptosis of late stage effector cells to yield less memory cells and thereafter impair memory immune response, providing a novel understanding of the immunotoxicity of PQ.

Augmented primary humoral immune response and decreased cell-mediated immunity by Murraya koenigii in rats

Murraya koenigii (Rutaceae) (curry patta: Hindi) of the family Rutaceae is used in the traditional Indian system of medicine for its immunomodulatory properties. The essential oil of the leaves of M. koenigii possesses antimicrobial, antifungal, and pesticidal activities and is used for the treatment of amebiasis, diabetes, and hepatitis. The present study was performed to evaluate the effect of M. koenigii on humoral and cell-mediated immune responses in rats. Aqueous extract of M. koenigii leaves was administered orally in a dose of 350 mg/kg. Cell-mediated immunity was assessed by measuring foot pad thickness following sensitization by injection of keyhole limpet hemocyanin and subsequent challenge by the same. Humoral immunity was assessed by measurement of hemagglutination titer to sheep red blood cells (SRBCs). In the humoral immune response, the administration of M. koenigii [350 mg/kg per os (p.o.)] from day 1 to day 7 after sensitization with SRBC on day 0 caused a significant increase in the primary anti-SRBC titer. However, the secondary immune response was decreased significantly (p<0.05) as shown by a decrease in secondary anti-SRBC titer measured on day 11 following a booster dose of antigen on day 8. In the delayed-type hypersensitivity test, M. koenigii (350 mg/kg, p.o.), when administered for 14 days, produced a significant (p<0.05) decrease in foot pad thickness when compared with the control group. Thus, these results suggest that oral administration of M. koenigii augments primary humoral immune response and decreases cell-mediated immunity.

Evaluation of canine T-cell dependent antibody response to the primary and secondary immunization with keyhole limpet hemocyanin

T-cell dependent antibody response (TDAR) incorporating both primary and secondary responses to keyhole limpet hemocyanin (KLH) in canine models have not yet been fully understood. To develop a practical dog TDAR model, we characterized primary and secondary antibody responses by intravenous or intramuscular immunization of KLH twice at intervals of 8 days during a 28-day course of study. Primary immunization with KLH by both routes induced a maximum IgM response on 6 to 8 days after the treatment, whereas the IgG response started 6 to 8 days after the treatment with relatively low levels. Remarkable increases in anti-KLH IgG levels (about 10-times compared with the primary response) were produced 5 to 7 days after the secondary KLH immunization by both routes. These results indicate that IgM-predominant and IgG-predominant responses were respectively induced by the primary and secondary immunization. Furthermore, the intravenous route showed higher baseline titers of primary and secondary anti-KLH IgM responses, suggesting that intravenous immunization of KLH might be a more suitable method for immunotoxicity evaluation. No remarkable inter-individual variability was noted in our canine models. Treatment with cyclophosphamide at 2 mg/kg/day for a consecutive 28 days significantly suppressed primary and secondary anti-KLH IgM and IgG responses induced by KLH injection on Days 15 and 23 of CPA treatment. These results demonstrate that these experimental designs could provide valuable information about the influence on both the primary and secondary humoral immune responses in dogs when exposed to potential immunomodulatory drugs.

Reproduction of PMWS in immunostimulated SPF piglets transfected with infectious cloned genomic DNA of type 2 porcine circovirus

Postweaning multisystemic wasting syndrome (PMWS) is a recently emerged disease affecting pigs. Type 2 porcine circovirus (PCV2) has been associated with this syndrome although other factors are required in association with this virus for PMWS expression. The aim of this study was to investigate whether general immunostimulation (injections of keyhole limpet hemocyanin emulsified in incomplete Freund adjuvant and of thioglycollate medium) could strengthen the severity of PMWS in six-week-old specific-pathogen-free (SPF) piglets transfected with pure tandem-cloned PCV2 DNA by the intramuscular route. Non-immunostimulated piglets transfected with the viral clone did not present clinical signs but only mild pathological microlesions characteristic of PMWS. These piglets seroconverted and high viral genome loads and infectious titers were detected in the lymphoid organs at the end of the trial. Mild-to-moderate forms of PMWS were generally observed in the immunostimulated transfected piglets, as well as one severe form for a piglet (8003) which died. These piglets with mild-to-moderate forms had higher DNA loads than the transfected-only animals. Thus, viral replication was enhanced by immunostimulation. This is the first time that clinical PMWS has been reported in an SPF immunostimulated piglet infected with a pure inoculum consisting of tandem-cloned PCV2 DNA. This result confirms that PCV2 is the agent of PMWS and that immunostimulation could enhance PMWS in SPF piglets transfected with a PCV2 DNA clone.

T-Cell-Dependent Antibody Response: Assay Development in Cynomolgus Monkeys

The current study was designed to develop and test a T-cell dependent antibody response to keyhole limpet hemocyanin (KLH) in cynomolgus monkeys. In an optimization experiment, monkeys (3/sex) were given a single intramuscular injection of KLH at 10 mg/animal to evaluate the kinetics of the antibody response. Serum samples were collected pretest, and on Days 4, 6, 8, 11, 15 and 22 for measurement of anti-KLH IgM and IgG endpoint titers. In a subsequent experiment, female monkeys (3/group) were treated once daily by gavage with the immunosuppressive agent cyclosporine (Neoral) at 0, 10 and 50 mg/kg for 21 days, and the effects of drug treatment on anti-KLH IgM and IgG responses were determined. The effects of cyclosporine on hematology, biochemistry, bone marrow, organ weights, gross and histopathology, and peripheral lymphocyte subsets also were evaluated. Robust anti-KLH IgM and IgG responses were seen in monkeys given a single intramuscular injection of KLH at 10 mg/animal, with peak antibody responses at approximately 10–14 days post-immunization for anti-KLH IgM, and 14–21 days for anti-KLH IgG. Decreases in anti-KLH IgG endpoint titers were seen in 1 monkey given cyclosporine at 10 mg/kg, and 1 monkey dosed at 50 mg/kg. Relative to vehicle control animals, mild lymphoid depletion was evident in lymph nodes and tonsil of monkeys with suppressed anti-KLH IgG titers. Collectively, these findings in individual animals provided evidence of cyclosporine-induced immunosuppression. Cyclosporine at 10 and 50 mg/kg did not alter anti-KLH IgM production, hematology, biochemistry, bone marrow, organ weights, or peripheral lymphocyte subsets. Lastly, the results of this study demonstrated that KLH immunization at 10 mg/animal did not alter the standard toxicity endpoints evaluated in control animals.

Defective Neo-Antigen Response to Keyhole Limpet Hemocyanin (KLH) Vaccines in Patients after Hematopoietic Cell Transplantation.

Relapse is the most common cause of treatment failure for advanced cancer, even those treated with autologous hematopoietic cell transplantation (HCT). Effective tumor-specific immunotherapy may decrease relapse, however, this will fail if the immune system is not able to respond appropriately. To test the integrity of the immune response, we vaccinated 27 normal volunteers, 14 patients after HCT, and 19 patients with advanced cancer with a single injection of KLH, a neo-antigen that humans have not been exposed to previously. KLH (Intracel, Rockville, MD or Biosyn Corp, Carlsbad, CA) was given as a single 1 mg subcutaneous injection. Immune response was measured by ELISA, proliferation or ELISPOT (validated for cryopreserved cells) assays on blood 1 month after vaccination compared to preimmunization. Intracel KLH (containing the intact KLH molecule, 6000–8000 kDA) induced a potent IgM, IgG1 and IgG2 response as well as a cellular response measured by proliferation assay in normal volunteers (n=17). Because of sporadic availability, Biosyn KLH, produced as 350/390 kDA subunits of KLH, was then tested. Biosyn KLH alone failed to induce either a humoral or cellular response. We hypothesized that lack of a response to a single Biosyn KLH immunization was due to the product purity and decreased complexity of size resulting in loss of adjuvant properties compared to the intact KLH product. Biosyn KLH was then mixed with the oil/surfactant Montanide ISA-51, which completely restored immunogenicity to a single 1 mg KLH test vaccine in normal subjects (n=10) similar to the Intracel intact KLH product. In marked contrast to the normal response, humoral responses were significantly impaired in 11 patients 58–144 [median 103] days after autologous HCT and in 1 as late as 16 months after HCT. This latter patient was the only one who mounted a cellular response to KLH after HCT. Patients with metastatic melanoma and renal cell carcinoma exhibited an intermediate response (Table). The decreased KLH-specific IgG2 response after HCT was partially explained by the decreased total IgG2 levels after transplant as compared to normal volunteers and solid tumor patients. Total IgG1 and IgM immunoglubulin levels were not significantly different between groups. The defect in isotype switching correlated with decreases in absolute CD4 counts between the normal (891±58/mm3), solid tumor (501±73/mm3) and HCT (290±119/mm3) cohorts. We conclude that patients with cancer and especially those patients after autologous HCT have immune defects significantly impairing neo-antigen responses. This has important implications for tumor vaccine strategies in these settings. KLH vaccines are safe, and 100% of normal volunteers require only a single injection to test cellular and humoral neo-antigen responses including isotype switching, an optimal platform for definitive testing of strategies to promote immune reconstitution after HCT.Response to KLH 1 month after vaccination.ReadoutNormal (n=27)Solid Tumor (n=19)HCT (n=14)IgM*7.7 ± 1.37.6 ± 2.11.4 ± 0.28IgG1*234 ± 5037.3 ± 13.27.9 ± 4.5IgG2*34 ± 5.112.7 ± 4.10.67 ± 0.23ELISPOT#311 ± 61 (n=10)Not done14 ± 9 (n=7)* values are mean ± SEM in mcg/ml; # reported as spots/million cells

Photoperiod controls the induction, retention, and retrieval of antigen-specific immunological memory.

Changes in day length affect several measures of immunity in seasonally breeding mammals. In Siberian hamsters (Phodopus sungorus), short day lengths suppress specific secondary antibody responses to the keyhole limpet hemocyanin (KLH) antigen and enhance cutaneous delayed-type hypersensitivity (DTH) responses to dinitrofluorobenzene (DNFB). These experiments tested whether day length affects secondary antibody and DTH responses by altering immune function solely during the interval after the initial exposure to each antigen, solely during the interval after the second exposure, or during both stages of the respective immune responses. Adult male Siberian hamsters were exposed to either a long (16 h light/day; LD) or a short (8 h light/day; SD) photoperiod for 7.5 wk before receiving an initial exposure to each antigen (KLH injection, cutaneous DNFB treatment; separate groups of animals for each antigen). A subset of LD hamsters was transferred to the SD photo-period, and a subset of SD hamsters was transferred to the LD photoperiod. Other hamsters remained in LD or SD. Eight weeks later, all hamsters were challenged with a second subcutaneous injection of KLH or a second application of DNFB to the ear, and immune responses were measured. Exposure to SD during the primary antibody response did not affect secondary IgG responses, but SD exposure during the secondary response significantly suppressed IgG production independent of day length during the initial KLH treatment. In contrast, exposure to SD during the DNFB challenge enhanced the ensuing DTH response, but this enhancement depended on the photoperiod prevailing during the initial exposure. Exposure to SD during the sensitization stage did not enhance DTH in hamsters subsequently exposed to LD. The data suggest that short photoperiods have enduring effects on immune responsiveness and on the establishment and retention of immunological memory.

Blockage of immune-mediated inner ear damage by etanercept.

Etanercept will be able to reduce the inflammation and hearing loss associated with experimentally induced labyrinthitis. Inner ear immune responses cause hearing loss that may be reversible with pharmacologic treatment. Etanercept, tumor necrosis factor receptor blocker, was investigated in a guinea pig model of immune-mediated hearing loss. Sterile labyrinthitis was created by injection of keyhole limpet hemocyanin into the inner ear after systemic sensitization to keyhole limpet hemocyanin with adjuvant. Labyrinthitis involves infiltration of inflammatory cells and hearing loss detectable 3 to 5 days after challenge with keyhole limpet hemocyanin. Etanercept was administered either systemically (2.5 mg) 30 minutes before intracochlear challenge with keyhole limpet hemocyanin, with a second intraperitoneal dose (2.5 mg) 3 days later or locally by long-term infusion into the scala tympani with an osmotic pump (5.0 microg/h for 7 days). Auditory evoked brainstem response thresholds were measured before and after treatment to determine hearing loss. Cochleas were evaluated for the amount of inflammation. Hearing loss in the untreated systemic group averaged 71 +/- 21 dB versus 37 +/- 32 dB in the etanercept-treated animals (t test, P < 0.001). There was also less inflammation in the cochleas from etanercept-treated animals (t test, P < 0.01). Hearing loss with local administration of etanercept was 59 +/- 31 dB in the nontreated ears and 18 +/- 8 dB in the treated ears (t test, P < 0.02). Inflammation was also less (t test, P < 0.01). Etanercept was not ototoxic. Prompt intervention with the anti-inflammatory drug etanercept significantly reduces inflammation sufficient for substantive hearing preservation.

Proinflammatory cytokine expression in the endolymphatic sac during inner ear inflammation.

The inner ear is capable of rapidly mounting an immune response that can ultimately lead to cochlear degeneration and permanent hearing loss. The role of the endolymphatic sac in this immune process is not clear. In order to investigate the cytokine expression of cells within the endolymphatic sac, a secondary inner ear immune response to keyhole limpet hemocyanin (KLH) was created in mice. The animals were sacrificed 3-48 h and 7 days following initiation of the immune response. The cochleas and endolymphatic sacs were assayed by immunocytochemistry for IL-1beta, TNFalpha, and IL-6. Three hours after KLH challenge of the scala tympani, the perisaccular tissue of the endolymphatic sac contained more inflammatory cells than the scala tympani or endolymphatic sac lumen. Only a few of these cells, however, expressed the proinflammatory cytokines IL-1beta and TNFalpha between 3 and 12 h after KLH injection. On the other hand, TNFalpha, which plays an important role in the cochlear secondary immune response, was expressed in cells in the endolymphatic sac lumen. The maximum percentage of cells expressing TNFalpha was seen later than in the scala tympani. Animals treated with systemic injection of the TNF blocker, etanercept, showed a reduction in the number of cells in the endolymphatic sac lumen. It is concluded that the cells in the endolymphatic sac lumen contribute to the amplification of the adaptive immune response by expressing TNFalpha, while the infiltration of cells into the perisaccular connective tissue is part of the nonspecific, innate, cochlear immune response.

Tumor Necrosis Factor‐α, an Initiator, and Etanercept, an Inhibitor of Cochlear Inflammation

The inner ear rapidly mounts an immune response that can lead to cochlear degeneration and permanent hearing loss. Identification of proinflammatory cytokine expression during the initiation of the response should lead to rational therapeutic strategies that block the response, reducing damaging sequelae. A cochlear immune response to keyhole limpet hemocyanin (KLH) injected into the inner ear or subarachnoid space of sensitized animals was created. Etanercept was administered to a group of animals to blunt the immune response. Cochleae were immunoassayed for expression of interleukin-1beta, tumor necrosis factor-alpha, and interleukin-6 and evaluated for the amount of cochlear-infiltrated cells. Tumor necrosis factor-alpha and interleukin-1beta were expressed by infiltrated cells shortly after KLH injection. Tumor necrosis factor-alpha was expressed whether the antigen was introduced with or without surgical trauma. Interleukin-1beta was also expressed by the cochlear fibrocytes during the immune response and in surgical control animals, but not after intrathecal injection of antigen. Interleukin-6 expression was minimal during the response. Based on this observation, animals were treated with systemic injection of Etanercept, which reduced cochlear infiltrating cell number and cochlear fibrosis. Interleukin-1beta expression is a general cochlear response to trauma, whereas tumor necrosis factor-alpha expression in the infiltrated immunocompetent cells is the cytokine that induces amplification of the response that leads to cochlear disease.

CXCR5-transduced bone marrow-derived dendritic cells traffic to B cell zones of lymph nodes and modify antigen-specific immune responses.

Skin-derived migratory dendritic cells (DC), in contrast to bone marrow-derived DC (BMDC), express CXCR5, respond to the chemokine CXC ligand 13 (CXCL13) in vitro, and are capable of migrating to B cell zones (BCZ) in lymph nodes (LN) in vivo. Herein, we analyzed the surface phenotype of skin-derived migratory DC and found that 15-35% of MHC class II(high) cells showed high levels of expression of CXCR5 but expressed low levels of DEC205, a suggested characteristic of dermal-type DC in mice. To study the effects of CXCR5 on the trafficking dynamics of DC, we stably expressed CXCR5 in BMDC by retroviral gene transduction. CXCR5 was detected by flow cytometry on transduced cells, which responded to CXCL13 in vitro in chemotaxis assays (3-fold over nontransduced BMDC, p < 0.01). When injected into the footpads of mice, approximately 40% of injected CXCR5-BMDC were observed in BCZ of draining LN. Mice were vaccinated with CXCR5- and vector-BMDC that were pulsed with keyhole limpet hemocyanin (KLH) to induce Ag-specific cellular and humoral immune responses. Mice injected with CXCR5-BMDC (vs vector-BMDC) demonstrated marginally less footpad swelling in response to intradermal injection of KLH. Interestingly, significantly higher levels of KLH-specific IgG (p < 0.05) and IgM (p < 0.01) were found in the serum of mice injected with CXCR5-BMDC compared with mice immunized with vector-transduced BMDC. Thus, CXCR5 is predominantly expressed by dermal-type DC. Moreover, CXCR5 directs BMDC to BCZ of LN in vivo and modifies Ag-specific immune responses induced by BMDC vaccination.

Expression of caspase-activated deoxyribonuclease (CAD) and caspase 3 (CPP32) in the hydropic cochlea of guinea pigs – second report

Apoptosis was induced in the cochlea by the injection of keyhole limpet hemocyanin (KLH) into the endolymphatic sac of guinea pigs and immunohistochemically examined. Keyhole limpet hemocyanin was injected into the right endolymphatic sac. The temporal bones were fixed via cardiac infusion of fixative and immunohistochemically stained for caspase-activated deoxyribonuclease or caspase 3. Endolymphatic hydrops became evident in the cochlea 1 day after the injection of keyhole limpet hemocyanin (n=6). The temporal bones in the control group did not show any caspase-activated deoxyribonuclease or caspase 3 immunoreactivity (n=6). Immunoreactivity for caspase 3 was detected in the supporting cells of the organ of Corti, the stria vascularis and the spiral ganglion cells. Caspase-activated deoxyribonuclease was also detected in the same areas. These findings suggest that apoptosis is involved in the pathogenesis of endolymphatic hydrops. This phenomenon could lead to cochlear dysfunction, as seen in endolymphatic hydrops.

Altered immune responses to a heterologous protein in ponies with heavy gastrointestinal parasite burdens.

This study was performed to test the hypothesis that immunity to heterologous vaccination would improve when the parasites were removed. It was also expected that parasitised ponies would exhibit a biased Th2 cytokine response to KLH immunisation. Helminth parasites are common in horses even in the era of highly effective broad-spectrum antiparasiticides. These parasites have been shown to alter the outcome to heterologous immunisation in a number of host species. The effect of gastrointestinal parasites on heterologous vaccination has not been addressed in equids. In the current study, humoral, lymphoproliferative, and cytokine responses to a single i.m. injection of keyhole limpet haemocyanin (KLH) were compared between groups of ponies with high, medium or low gastrointestinal parasite burdens. Antibody levels determined by ELISA showed that animals with low levels of parasites had a trend toward increased KLH specific total immunoglobulin, IgG(T) and IgA compared to heavily parasitised ponies. Medium and heavily parasitised ponies demonstrated a trend toward reduced lymphoproliferative response to KLH that was not restored after the addition of interleukin-2 (Il-2). Cells from these ponies also produced significantly lower levels of IL-4 compared to lightly parasitised ponies. These data indicate that heavily parasitised ponies have uniformly decreased cellular and humoral immune responses to soluble protein immunisation. The mechanisms involved may have potential deleterious effects on standard vaccine protocols of parasitised equines.

Laparoscopic Nissen fundoplication with carbon dioxide pneumoperitoneum preserves cell-mediated immunity in an immature animal model

Purpose: The aim of this study is to elucidate the effects of laparoscopic Nissen fundoplication (LNF) with carbon dioxide (CO2) or helium (He) on the cell-mediated immune response in a pediatric animal model compared with open Nissen fundoplication (ONF). Methods: Cell immune response was evaluated in 45 1-week-old Sprague Dawley rats using the delayed type hypersensitivity (DTH) skin test. Animals were sensitized against keyhole limpet hemocyanin (KLH) by subcutaneous injection (0.5 mg) in complete Freund's adjuvant. Animals were challenged 2 weeks later by an intradermal injection of KLH (0.3 mg) in sterile saline (challenge 1, baseline). Rats with positive DTH skin reaction at 24 and 48 hours after challenge 1 were put randomly into 4 groups (n = 10 each): I, only anesthesia (control); II, LNF with CO2, III, LNF with He; IV, ONF. Animals were injected intradermally with KLH (0.3 mg) immediately before the procedures (challenge 2) and 3 and 6 days postoperatively (challenges 3 and 4). Results: DTH skin reactions were measured 24 and 48 hours after each challenge. There were no significant changes in cell-mediated immunosuppression after LNF with CO2. However, a transient cell-mediated immunosuppression was observed after LNF with He and ONF. All fundoplications were intact at the time of necropsy. Conclusions: These data suggest a transient suppression of cell-mediated immunity in open procedures when compared with laparoscopic interventions using CO2 in a pediatric animal model. In addition, the type of gas used during laparoscopy also may modulate this transient immunosuppression. J Pediatr Surg 36:1564-1568. Copyright © 2001 by W.B. Saunders Company.