Effects Of Keyhole Limpet Hemocyanin Research Articles

Keyhole limpet hemocyanin: an effective adjunct against melanoma in vivo

Background We have previously demonstrated the potent in vitro antiproliferative effects of keyhole limpet hemocyanin (KLH) against melanoma. Our prior studies directed us to hypothesize that KLH would be effective in vivo against melanoma, alone and in combination with conventional immunotherapy. Methods Mice were inoculated with 2 × 10 7 HTB68 cells and randomized to 6 groups. Treatment groups consisted of control, KLH 200 μg, alpha interferon (AIFN) 1000 IU, interleukin-2 (IL-2) 5000 IU, KLH + AIFN, and KLH + IL-2. Results KLH + IL-2 exhibited the greatest reduction in tumor volume (30%) as compared to control ( P = .014), followed by KLH + AIFN (28%, P = .031). Singly treated animals had less tumor inhibition: IL-2 (30%, P = .022), KLH (18%, not significant), and AIFN (16%, not significant). Conclusions KLH augments the effects of AIFN, one of the standard immunotherapeutic agents against melanoma in vivo. Further in vivo and early clinical studies into the effects of KLH as both a single and combined agent are warranted.

Keyhole limpet hemocyanin induces the activation and maturation of human dendritic cells through the involvement of mannose receptor

Keyhole limpet hemocyanin (KLH) is a xenoantigen largely used in vitro as an immunogen to study primary antigen-specific T cell responses and in vivo as a vaccine component with optimal carrier qualities. So far, the mechanisms by which KLH exerts its immunostimulatory properties are still largely unknown. In particular, although dendritic cells (DCs) play a central role in the initiation and activation of immune responses, the effects of KLH on these cells have been poorly explored. In the present study we investigated the effects of KLH on DCs differentiated in vitro from human monocytes. We observed that KLH promotes the activation and maturation of DCs, as assessed by up-regulation of the surface expression of CD80, CD86, CD40, HLA-DR and CD83. Moreover, even if KLH stimulated the production of IL-12 and IL-10 by DCs, the final balance was clearly in favour of IL-12. According to these stimulatory effects, KLH significantly increased the allostimulatory activity of DCs. To verify whether these effects of KLH may be related to the binding of this highly glycosilated molecule to mannose receptor (MR), we performed inhibition experiments with anti-MR antibody. Results showed that the stimulatory activity of KLH is indeed partially mediated by its interaction with MR. Taken together, our results seem to indicate that KLH does promote the maturation of DCs endowed with the ability to stimulate cell-mediated immune responses. We suggest that this property of KLH may represent a novel further mechanism by which this molecule may exert its efficacy when co-administered with others antigens in immunotherapeutic protocols.

In vivoMRI visualization of endolymphatic hydrops induced by keyhole limpet hemocyanin round window immunization

Endolymphatic hydrops is considered one of the principal pathological manifestations of Meniere's disease and abnormal immunoreaction may be one of the aetiologies of the disease. To investigate the effect of keyhole limpet hemocyanin (KLH) immunization on cochlear morphology and function in vivo, guinea pigs were inoculated with KLH systemically and received round window membrane delivery (group I). In the control group (group II), KLH was replaced with phosphate buffered saline. In vivo gadolinium diethylenetriaminepentaacetic acidbismethylamide MRI was obtained and compound action potentials were measured. In MRI, evident endolymphatic hydrops was detected in 2 of the 4 animals in group I and blood-endolymph barrier permeability increased in 3 of the 4 animals. The membranous lateral wall and the spiral lamina, in continuation with the organ of Corti, were also demonstrated in the MRI. More than 10 dB hearing loss was found in 2 out of the 4 animals in group I. Neither endolymphatic hydrops nor blood-e...

Inhibition of melanoma growth by hemocyanin occurs via early apoptotic pathways

Background We hypothesized that keyhole limpet hemocyanin (KLH) would reduce cellular proliferation and effect apoptosis of melanoma cell lines in vitro. Methods Two human melanoma cell lines (HTB68 and HTB72) were subjected to a dose-response treatment regimen of KLH (0.4 μg to 100 μg/well). Cell viability was tested by MTT assay (SIGMA, St Louis, MO) at 72 hours. Apoptosis and necrosis were measured by the Annexin V FITC assay (Biovision Inc, Mountain View, CA). Results Melanoma cell proliferation was significantly reduced in the HTB68 cell line treated with 6.3 μg or higher doses of KLH. A significant reduction in cell growth was also observed in the HTB72 cells at 50 and 100 μg of KLH. KLH increased early apoptotic activity, whereas both late apoptosis and necrosis were decreased by the addition of KLH. Conclusions KLH significantly reduces cellular proliferation in vitro in melanoma, via early apoptotic pathways. The results warrant in vivo studies into the effects of KLH in melanoma.

In vitro effects of keyhole limpet hemocyanin in breast and pancreatic cancer in regards to cell growth, cytokine production, and apoptosis

Background We have previously shown the inhibitory effects of keyhole limpet hemocyanin (KLH) against breast and pancreatic cancer in vitro. We hypothesize that its actions in breast and pancreas cancer cells are via apoptotic or cytokine pathways. Methods Two breast cancer cell lines, ZR75-1 and MCF-7, and one pancreas cancer cell line, PANC-1, were treated with KLH at 500 μg, 250 μg, and 250 ng/mL. Cell viability, cytokine production, and apoptosis were measured. Results Significant growth inhibition was observed in all cell lines at all KLH concentrations tested. Significant changes in cytokine production were observed in all cell lines. An increase in early and late apoptotic activity was observed in the MCF-7, whereas a reduction in late apoptotic activity was observed in the ZR75-1 cells. Conclusions KLH directly inhibits the growth of human breast and pancreas cancer in vitro by apoptotic and nonapoptotic mechanisms.

Vaccination alone or in combination with pyridostigmine promotes and prolongs activation of stress‐activated kinases induced by stress in the mouse brain

Gulf war illnesses (GWI) are currently affecting thousands of veterans. To date, the molecular mechanisms underlying the pathogenesis of these illnesses remain unknown. During Gulf war I, military personnel were exposed to multiple stressors, one or more vaccines, pyridostigmine (PY), and other chemicals. In our previous studies, we found that stress induces activation of mitogen activated protein-kinase kinase 4 (MKK4) and c-Jun-N-terminal kinase (JNK) in the mouse brain (Liu et al. 2004). Our working hypothesis is that stress, vaccination, and PY may synergistically induce activation of MKK4 and JNK in the brain, leading to over-activation of these kinases and neurological injuries. To test our hypothesis, we examined the effect of keyhole limpet hemocyanin (KLH) immunization alone or in combination with PY on activation of MKK4 and JNK induced by stress. We found that KLH immunization alone had a small effect on MKK4 or JNK activity but it significantly enhanced and prolonged activation of these kinases induced by stress, from a few hours to several days. Additionally, KLH immunization caused activation of p38MAPK. PY treatment further enhanced and prolonged activation of these kinases induced by stress in combination with KLH immunization and triggered activation of caspase-3. Our current studies suggest that stress, vaccination, and PY may synergistically act on multiple stress-activated kinases in the brain to cause neurological impairments in GWI.

Central CRH Suppresses Specific Antibody Responses: Effects of β-Adrenoceptor Antagonism and Adrenalectomy

Central corticotropin releasing hormone (CRH)-induced activation of the sympathetic nervous system and/or the pituitary–adrenal axis is hypothesized to mediate suppression of in vivo specific antibody responses. To test whether β-adrenergic receptor activation is involved in the immunosuppressive effects of central CRH, rats were pretreated with propranolol or saline before intracerebroventricular infusion of CRH and immunization with keyhole limpet hemocyanin (KLH). KLH (3 μg/kg) immunization induced significant increases in circulating levels of antigen-specific IgM and IgG. Central infusion of CRH (200 pmol) suppressed both IgM and IgG responses. Pretreatment with propranolol (20 mg/kg IP) reversed CRH-induced suppression of IgG responses but had no effect on IgM levels. To test whether adrenal activation also plays a role in the effects of KLH on specific antibody responses, a separate group of animals underwent adrenalectomy prior to CRH infusion and immunization with KLH. As compared to nonadrenalectomized control rats, adrenalectomized rats showed a reduction of antibody responses, and CRH failed to induce a further suppression of IgM or IgG responses in adrenalectomized animals. Collectively, these data suggest that β-adrenoceptors mediate the suppression of primary antibody responses induced by central CRH. Moreover, the adrenals may promote optimal primary antibody responses after exposure to physiological levels of antigen.

Keyhole Limpet Hemocyanine Can Enhance the Natural Killer Activity of Patients with Transitional Cell Carcinoma of the Bladder

It has been shown that natural killer (NK) cells are involved in the immunosurveillance of tumors. In patients with transitional cell carcinoma (TCC) of the bladder, there is a negative correlation between the levels of NK activity in peripheral blood mononuclear cells (PBMNCs) and both the clinical evolution and the pathological stages of the disease. We have investigated the immunoregulatory effect of keyhole limpet hemocyanin (KLH) on the NK activity of patients with TCC of the bladder. We found that KLH enhances the cytotoxic activity of PBMNC from patients with superficial TCC against NK-sensitive target cells in a time-dependent manner. This KLH-enhanced cytotoxicity is not directed against NK-resistant target cells. However, KLH fails to normalize the depressed NK activity of PBMNCs from patients with infiltrative TCC. The present in vitro studies suggest that this immunoregulatory effect of KLH on NK cytotoxicity may be implicated in its therapeutic effect on superficial TCC of the bladder.

Immunotherapy in Bladder Cancer with Keyhole-Limpet Hemocyanin: A Randomized Study

A randomized, controlled study was begun in 1982 on the effect of keyhole-limpet hemocyanin and mitomycin C in the prevention of recurrent superficial bladder cancer (stages pTa to pTl, grades 0 to 3) in 44 patients. All tumors were resected and all patients were presumed to be free of tumor at initiation of the prophylactic instillations. Before the bladder instillation program was begun all patients in the keyhole-limpet hemocyanin group Ia were immunized with 1mg. keyholelimpet hemocyanin intracutaneously and then monthly bladder instillations of 10mg. were given. The control group lb received 20mg. mitomycin C monthly.Of the 21 patients in the keyhole-limpet hemocyanin group la (mean followup 20.7 months) 3 (14.2 per cent) had recurrences, compared to 9 of 23 (39.1 per cent) in the mitomycin C group lb (mean followup 18.3 months). The over-all preventive effect was significantly better (p less than 0.05, chi-square) in keyhole-limpet hemocyanin-treated patients than in those given mitomycin C.In 1984 a new single drug study (group II) was started with keyhole-limpet hemocyanin alone, administered as in group la. Of 81 patients in group II (nonrandomized, mean followup 22.8 months) 17 (20.9 per cent) had recurrences. Of the patients given keyhole-limpet hemocyanin 20 of 21 (95.2 per cent) in group la and 70 of 81 (86.4 per cent) in group II had complete and partial prevention (downgrading), compared to 16 of 23 (69.5 per cent) in group lb. Our study was established to analyze the effect of a new method of immunotherapy; no adverse local or systemic side effects were noted.

Regulation of the in vitro anamnestic antibody response by cyclic AMP : I. Antigen-induced uptake of nucleotides and nucleosides by lymphocytes

Addition of N 6, O 2′-dibutyryl cAMP (DbcAMP) to keyhole limpet hemocyanin (KLH)-primed rabbit lymph node cells for 1 hr, followed by its removal and the addition of KLH, had no effect on the subsequent antibody response, whereas addition of KLH for 1 hr followed by DbcAMP resulted in a 100% enhancement of antibody synthesis. Addition of cholera enterotoxin (CT), which rapidly and irreversibly binds to lymphocytes and activates adenylate cyclase, either before or after the addition of antigen, elevated the antibody response by 100%. These results suggested that some antigen-induced event(s) may be required for DbcAMP to exert its enhancing effects on the antibody response. The effect of KLH on the uptake of DbcAMP by KLH-primed lymph node cells was investigated. One and one hundred micrograms of KLH, which induce optimal and supraoptimal antibody synthesis, respectively, promoted maximal uptake of DbcAMP. This induced uptake was first detectable about 12 hr after addition of KLH, and it peaked during 24–48 hr of culture. DbcAMP uptake induced by a brief exposure of KLH (0–1 hr) was equivalent to that observed with long-term KLH addition (0–24 hr). KLH-induced DbcAMP uptake required KLH-reactive lymphocytes and represented active transport. Antibody to rabbit T lymphocytes inhibited this antigen-induced uptake. The mitogens concanavalin A (Con A) (T cells) and goat anti-rabbit Fab' (anti-Fab') (B cells) also stimulated DbcAMP uptake, as did human serum albumin (HSA) and myoglobulin (Mb) when added to homologously primed cells, indicating the generality of the phenomenon. [ 3H]DbcAMP entered the cells as di- or monobutyryl cAMP with about 40% metabolized to 5′AMP. This uptake could be competitively inhibited by other adenine or guanine nucleotides and nucleosides.

The effect of Keyhole Limpet Hemocyanin (KLH) on the rat bladder

Immunostimulation with agents such as BCG or KLH may represent an adjuvant therapy in treating patients with bladder cancer. To investigate the effects of direct instillation and injection of KLH into the bladder of sensitized and nonsensitized rats, KLH was injected submucosally into the bladder. Histologically, a marked inflammatory reaction was seen without ulcerations.

Cell Fractionation on Affinity Columns

Nonimmune C57BL/6f mouse spleen lymphocytes were fractionated on glass-bead columns coated with keyhole limpet hemocyanin (KLH). Adherent cells conferred enhanced humoral anti-KLH responses on irradiated syngeneic recipients when compared with nonadherent or unfractionated cells. This effect was unaltered by simultaneous transfer of unfractionated nonimmune thymocytes, but it was not observed when cells were fractionated on a column coated with control antigen, when nonviable cells were transferred, and when cells were fractionated in the presence of free KLH. No adjuvant effect of KLH transferred with cells was demonstrable. By autoradiography with <sup>125</sup>I-KLH, adherent lymphocytes were enriched in antigen-binding cells (0.24%), relative to nonadherent cells (0.03%,) with an estimated incidence of 0.06% for KLH-binding lymphocytes in nonimmune C57BL/6f mouse spleen.

Keyhole limpet hemocyanin (KLH)-lipid bilayer membrane (BLM) interaction

The effect of keyhole limpet hemocyanin (KLH) on bimolecular lipid membranes formed in different buffers and at varying pH values was studied. The KLH was added after forming the black film. Upon its addition, the electrical resistance of the film decreased by as much as 6 orders of magnitude. The maximum change was observed in membranes bathed in “C” media at pH 7.1.