Injection Of Keyhole Limpet Hemocyanin Research Articles

Isolation and partial characterization of dogfish (Scyliorhinus canicula) antibody

A humoral immune response was elicited in adult dogfish by intramuscular injection of keyhole limpet haemocyanin (KLH). The antibody raised to this antigen was measured by an agglutination technique. The antibody-containing fraction of the serum was isolated in a two-step procedure utilizing gel nitration on Sepharose 6B and slab gel electrophoresis. A rabbit antiserum was then raised to the purified immunoglobulin. Further gel filtration studies revealed that the anti-KLH agglutinating activity was restricted to a highmolecular-weight serum fraction (approximately 800000) in fish immunized for up to 18 months. No anti-KLH precipitating activity was detected in fish at any stage of immunization; however immunodiffusion studies with the rabbit antiserum raised against dogfish antibody confirmed the existence of an 800000 molecular weight form and also a lighter species weighing 160000 daltons in both normal and immunized fish. The antigenic specificity of this latter molecule was not demonstrated. Immunoelectrophoretic analysis of the antibody revealed a neutral/slight anodic mobility, and after reduction and alkylation the presence of two polypeptide chains weighing 18200 and 75 860 daltons was demonstrated by thin-layer gel chromatography. These physicochemical characteristics make dogfish antibody analogous to mammalian immunoglobulin M (IgM).

Passive transfer of immune-mediated middle ear inflammation and effusion.

Antigenic challenge in the middle ear of animals previously sensitized intradermally results in an immunologically mediated inflammatory response, including the generation of serous effusion in the middle ear cavity. The mechanisms which contribute to this immune-mediated otitis media with effusion (OME) were investigated by passive transfer with either purified IgG or with sensitized lymphocytes. A peritoneal exudate was raised in strain 13 guinea pigs by repeated injection of keyhole limpet hemocyanin (KLH) with complete Freund's adjuvant. IgG and lymphocytes were isolated from exudates and used for passive sensitization. Middle ear challenge following passive transfer of IgG specific to KLH produced effusion and inflammation which was equivalent to that observed in actively immunized animals, but resulted in significantly greater numbers of leukocytes infiltrating the middle ear. Unlike in the active response, few of the cells infiltrating the middle ear mucosa and middle ear cavity were eosinophils. Middle ear challenge with KLH following passive transfer with sensitized lymphocytes produced no middle ear effusion, relatively little mucosal inflammation, and sharply reduced exocytosis of leukocytes into the middle ear when compared with the active immune response or even with unimmunized controls. It is concluded that middle ear effusion, inflammation, and leukocytic exocytosis are mediated primarily by the IgG fraction of humoral immunity in our model of immune OME.

Humoral and Cell-Mediated Immunity in Peripheral Blood following Introduction of Antigen into the Middle Ear

Serum levels of specific IgG and the sensitization of peripheral blood T-lymphocytes were measured in guinea pigs after single-dose antigenic sensitization by two routes: intratympanic and intradermal injection. Keyhole limpet hemocyanin (KLH) served as the antigen. Intratympanic injection of antigen resulted in much lower levels of circulating anti-KLH IgG than intradermal injection. When KLH was conjugated with alum to produce nonspecific inflammation and serve as adjuvant, the intratympanic route was considerably enhanced, but remained much less effective than the intradermal route. Development of an IgG response was also somewhat less rapid following intratympanic than following intradermal administration. Marked sensitization of circulating T-lymphocytes was seen after intradermal injection of alum-precipitated KLH. A much weaker, though still positive, response was seen after intradermal injection of KLH alone and with the intratympanic injection of alum-precipitated KLH. No T-lymphocyte sensitization could be detected after intratympanic injection of KLH alone. It was concluded that the afferent limb of both humoral (IgG) and cell-mediated immunity was operative in the middle ear. Therefore, the middle ear does not represent an immunologically "privileged" site. On the other hand, the afferent limb from the middle ear appears to operate less effectively and rapidly than that from the dermis. This observation is consistent with observations in other mucosal systems.

Experimentally induced alterations in periapical tissues of the cat.

Ten cats were immunized with subcutaneous injections of keyhole limpet hemocyanin (KLH). After it was determined that the experimental animals had developed circulating antibodies to KLH, challenge doses of KLH were administered via the root canal system. The radiographic and histologic findings suggest that antigen-antibody complex reactions can occur in periapical tissues of teeth, and that they can play a role in the pathogenesis of periapical lesions. In contrast, no radiographic and histologic changes were noted in non-immunized cats.

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.

A Special Report: Four-year Study of a Boy with Combined Immune Deficiency Maintained in Strict Reverse Isolation from Birth

A 4-year study of a boy with combined immune deficiency is presented, and the impact of this disease on various aspects of his growth and development is examined. There is no evidence of immune deficiency in either parent or in the genetic background on the maternal side. Three children of a brother of the mother's father may have had immune deficiencies but two have grown to be teenagers with no problems. Another died. At autopsy, however, lymph nodes appeared normal. The deceased older brother had severe combined immune deficiency (SCID). The autopsy findings showed Pneumocystis carinii pneumonia to be the direct cause of death and these findings contributed to the diagnosis of SCID. After a successful germ-free birth, the male infant (DV) was placed in the isolator. Laboratory tests were normal except that the x-rays showed no thymic shadow, his absolute lymphocyte count ranged from 399-440/mm and the lymphocytes showed no proliferative response to phytohemagglutinin (PHA). Specific tests showed the antibody-producing immune system and the cell-mediated immune system to be severely defective. The patient's lymphocytes elicited positive responses by lymphocytes from father, mother, and sister. Subsequent search in national and international tissue-typing laboratories has shown four HLA matches but none has been nonreactive in mixed lymphocyte culture (MLC). therefore, this patient has remained in isolation to the present; now he is 4 years old. Approximately 35 species of microorganisms, mostly transient contaminants, have been isolated, taking into account that the same organism may have been identified under different names in different laboratories. Those isolated frequently and in sufficiently high concentration to indicate colonization have been speciated as follows: anaerobes-Propionibacterium acnes, Lactobacillus catenaforme (disappeared spontaneously), Bacteroides oralis ss. elongatus, Clostridium (perenne, hastiforme, bifermentans), Bacteroides clostridiiformis ss. clostridiiformis; aerobes-Alcaligenes faecalis (eradicated by antibiotics), Staphylococcus epidermidis, Enterobacter agglomerans, Microcossu sp. subgroup 1, Bacillus pulvifaciens (disappeared spontaneously); yeasts-Candida (tropicalis, parapsilosis). Seven are considered to be probable components of the current autoflora: P. acnes, C. bifermentans, B. clostridiiformis ss. clostridiiformis, S. epidermidis, Micrococcus sp. subgroup 1, E. agglomerans, C. parapsilosis. No viruses or protozoa have been isolated. At age 3 years, the mean quantitation of anaerobic cells was 7.9 X 10(7) viable cells/g feces; this falls short of the mean anaerobic load from normal children. The mean aerobic concentration was 1.2 X 10(8) viable cells/g feces, not unlike normal children. Qualitatively his flora has abnormally few species and lacks those most common in normal subjects. This child has had no evidence of infection and has always been in excellent health even though some organisms which could be pathogenic under some circumstances have been isolated...

Basophils in tuberculin and "Jones-Mote" delayed reactions of humans.

Jones-Mote reactions are delayed, erythematous, and mildly indurated cutaneous reactions originally described in humans sensitized by skin injection of heterologous proteins. Similar reactions in guinea pigs contain many basophils and are called cutaneous basophil hypersensitivity. In contrast, guinea pigs immunized with mycobacterial adjuvants have classical tuberculin-type delayed hypersensitivity reactions, which contain few basophils. This has led to a new classification of delayed responses, based largely on the presence or absence of basophils. We induced sensitization for Jones-Mote reactions in 20 normal humans by intradermal injections of keyhole limpet hemocyanin. Skin tests with KLH 1 wk later showed erythematous and indurated delyaed reactions in all subjects. Rebuck skin windows showed specific accumulations of basophils with a delayed time-course in 18 of 20 subjects. In 12 normals sensitized with oxazolone-keyhole limpet hemocyanin conjugates, skin reactions and in vitro lymphocyte stimulation showed carrier and not hapten specificity, suggesting that cutaneous responses were probably mediated by T cells. A comparative study of strongly positive PPD skin tests in patients with tuberculosis showed significant basophil accumulations in five of nine subjects. Thus, basophils occurred in human tuberculin and Jones-Mote reactions and were not a distinguishing feature of Jones-Mote reactions. We suggest that the occurrence of basophils at delayed reactions is under complex regulation and that basophil accumulations are an aspect of delayed hypersensitivity, rather than an indication of a distinctive and separate response.

Differential Effects of Cyclophosphamide on the B and T Cell Compartments of Adult Mice

Abstract Adult mice were given a single sublethal dose of cyclophosphamide. Effects of the drug on the bone marrow-derived (B) and thymus-derived (T) cell compartments were measured by studies on lymphocyte transformation, lymphoid tissue morphology and responsiveness to a primary injection of keyhole limpet hemocyanin. These studies indicated that the effect of cyclophosphamide on the B cell compartment was more severe and longer-lasting than the effect on the T cell compartment.

In vitro studies of “antigenic competition” : I. The comparative responses of normal and “immune” lymphoid cell populations

A model has been presented for the mechanism of nonspecific antigen-induced suppression (“antigenic competition”) under in vitro conditions. The antigens used were Keyhole limpet hemocyanin (KLH) and horse erythrocytes, the former being injected into mice intravenously, and the latter being used as the antigen with which the spleen cells, derived from KLH-immune mice 2 days later, were cultured in vitro. It was observed that there was a specific and brief time interval after KLH injection during which suppression of the response to horse RBC in vitro occurred. The degree of suppression was proportional to the dosage of the first antigen, but independent of the dosage of the second. Suppression lasted for the duration of a 7-day period of culture, and was attributed to a decrease in antigen-reactive units specific for the horse RBC. These observations were discussed in the light of recent theories of antigen-induced suppression and antibody formation.

Distinctions between rabbit lymph node cell populations responding to primary and secondary injections of keyhole limpet hemocyanin.

AbstractRabbits were injected once or twice into the hind foot pads with alum‐precipitated keyhole limpet hemocyanin. At the height of the primary or secondary responses individual rabbits were sacrificed for the preparation of lymph node cell suspensions from the regional lymph nodes. These cells were employed for the in vitro study of antibody synthesis by the incorporation of 14C‐leucine and the secretion of antibody by the time of appearance of radioactive antibody in the medium. The primary response cells rapidly synthesized and secreted IgM and IgG antibodies. The secondary response cells rapidly synthesized IgM and IgG antibodies, secreted IgG antibody promptly, but secreted the IgM antibody with a lag of six to ten hours. Microsomal fractions could not be prepared from the primary response cells, but were readily produced from the cells of the secondary response. The primary response cells contained mainly free ribosomes, those of the secondary response predominantly membrane‐bound ribosomes. It was postulated that IgM antibody was not secreted until it was glyco‐sylated in the Golgi apparatus and that the lag in secretion entailed the time for this rate‐limiting step to occur.

Hemagglutinin Antibody Response of Normal and Aleutian Disease-Affected Mink to Keyhole Limpet Hemocyanin

Abstract After injection of Aleutian disease (AD) virus into mink homozygous for the Aleutian gene, the amount of serum γ globulin steadily increased and the amount of serum albumin decreased early in the disease and remained relatively stationary thereafter. Alterations in the α and β globulin concentrations were inconsistent. AD virus suppressed the primary antibody response of normal mink inoculated with keyhole limpet hemocyanin (KLH). This suppression began at about the time the serum γ globulin levels of mink inoculated with AD virus started to increase and was consistent, whether KLH was injected before or concomitantly with AD virus. When the primary injection of KLH was given before or concomitantly with AD virus, an excellent anamnestic response occurred in AD-affected mink. Antibody responses to KLH in mink affected with AD at the time of primary stimulation were extremely poor. The mink most severely affected at the time that they were inoculated with KLH gave the poorest primary and anamnestic responses.