Biomedical subjects
R D Karp
Publications and source records attributed to R D Karp.
Evolution of adaptive immunity: inducible responses in the American cockroach.
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A diphasic immune response against bacteria in the American cockroach.
The adult cockroach generates an adaptive humoral immune response exhibiting specificity and immunological memory when immunized with soluble proteins. In contrast, the response induced by bacteria in holometabolous insects is non-specific and short term, generally losing activity after 72 hr. We have found that the roach generates a diphasic response when injected with bacteria, displaying an acute non-specific phase initially, which is then superseded by a second response that is relatively long term and specific. Animals were immunized with either killed Pseudomonas aeruginosa or pyrogen-free Burns-Tracey saline (BTS) and challenged at various times post-immunization (p.i.) with a lethal dose of viable P. aeruginosa. The induction of immunity was measured by monitoring the percentage of survivors after challenge. Immunization with killed P. aeruginosa induced significant (P less than 0.05) protection against challenge as compared with BTS controls. The response was elicited by Day 1 p.i. and did not begin to decline until after Day 14 p.i. The specificity of the response was tested by immunizing animals with either killed Serratia marcescens, Enterobacter cloacae, Streptococcus lactis or Micrococcus lysodeikticus, and then challenging them with live P. aeruginosa. Significant protection against P. aeruginosa challenge was induced by any of the bacteria within the first 3 days p.i. However, by Day 4 the response began to show specificity. Immunization with P. aeruginosa induced significantly more protection than immunization with any one of the other organisms, and only the Gram-negative organisms (P. aeruginosa, E. cloacae and S. marcescens) induced any protection relative to controls.(ABSTRACT TRUNCATED AT 250 WORDS)
Short-term immunologic memory in the allograft response of the American cockroach, Periplaneta americana.
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Primary integumentary allograft reactivity in the American cockroach, Periplaneta americana.
Previous reports have failed to demonstrate integumentary allograft rejection in insects. We realized however, that these studies may not have fully appreciated the structure of the insect exoskeleton. Since the subcuticlar epidermal layer constitutes the only living tissue associated with insect integument, its destruction would indicate that the animal recognized and responded to the foreign tissue. Thus, we investigated allograft reactivity in the American cockroach, Periplaneta americana, by observing the fate of the epidermal portion of the integument. Each animal in a pair received a 3 X 4-mm integumentary allograft from its partner, as well as a 3 X 4-mm control autograft. The transplants were then examined histologically for signs of epidermal destruction at 0, 1, 3, 5, 7, and 10-70 days (in 10-day increments) posttransplantation. The results indicated that significant rejection of the allografts began by day 3, with peak reactivity occurring by day 7 when 92% of the grafts were scored as rejected. At later periods (greater than 20 days), the graft sites showed signs of repopulation by host epidermal cells. The allograft reaction was found to lag behind the xenograft reaction, which showed peak activity after only 1 day posttransplantation. Even so, allograft rejection in this insect occurred quite rapidly (as compared with some other invertebrates), and would appear to be due to a cytotoxic reaction against the epidermal layer.
Demonstration of cell-mediated cytotoxicity to allogeneic and xenogeneic tissue in the American cockroach, Periplaneta americana, using a combination in vivo/in vitro assay.
Previous reports have claimed that insects lack the ability to reject integumentary allografts. However, when we followed the fate of the epidermal layer underneath the inert cuticle, we observed that the American cockroach not only rejected xenografts, but also demonstrated impressive reactivity against allografts. We have now developed a combination in vivo/in vitro assay that can quantitate the extent of allograft reactivity in the roach. Pieces of filter paper (3 X 4-mm) were implanted under the second and fourth tergites of donor animals. After 7 days, implants seeded with host hemocytes were removed, washed, and incubated in culture medium containing 3H-thymidine for 24 hr. Labeled grafts were reimplanted into paired animals to detect cytotoxicity as follows: grafts removed from under the second tergite were placed back into their original positions to serve as autograft controls; grafts removed from under the fourth tergite were reciprocally transferred between paired animals; and grafts were recovered after various time intervals and processed for scintillation counting. There was no significant difference in counts between allografts and autografts at day 1. Autografts sampled on days 3, 5, 7, and 10 had significantly higher counts than allografts, with peak reactivity occurring between days 3 and 5. This indicates that allogeneic cells were selectively destroyed, and confirms our recent data from conventional grafting studies that insects have the ability to react to allografts.
Alteration of the protein composition in the haemolymph of American cockroaches immunized with soluble proteins.
Previous in vivo experiments demonstrating the existence of humoral immunity in cockroaches prompted us to investigate the composition and number of proteins that are newly induced or increased in quantity in the haemolymph of immune cockroaches compared to saline-injected control animals. SDS-PAGE analysis showed that six different haemolymph proteins of molecular weights 220,000, 162,000, 115,000, 102,000, 95,000 and 45,000 were induced in response to immunization by honeybee toxoid. However, only the 115,000 and 102,000 MW proteins consistently increased in quantity in the haemolymph of females immunized with various other soluble protein antigens, such as phospholipase A2 toxoid and inactivated creatine phosphokinase. Other experiments showed that the 115,000 MW band was not induced in immunized male cockroaches. Therefore, only the 102,000 MW protein was consistently enhanced in all samples of immune haemolymph from animals injected with various antigens. In addition, this protein was found in precipitates recovered from assays where immune haemolymph proteins were precipitated by the antigen. Gel filtration chromatography showed that this induced haemolymph protein is large, and bears an apparent native molecular weight of 600,000 +/- 80,000. This suggests that the 600,000 MW protein may consist of multiple subunits of 102,000.
Recent progress in studies on cell-mediated immune responsiveness in insects.
Comparative immunologists have been engaged in a search for the origins of cell-mediated immunity for a long time. One group of invertebrates that have been receiving increasing attention has been the insects. These animals not only serve as good experimental models, but are of great interest because of their evolutionary success. Several studies have shown that insects generally respond to particular foreign material by mounting an encapsulation reaction. On a more specific level, insects have also been found to be capable of quickly rejecting integumentary xenografts. A basic enigma however, has been the reported inability of insects to respond to integumentary allografts. Recent work from our lab indicates that if one concentrates on the fate of the soft tissue component of the integument, one can readily demonstrate allograft reactivity in these animals. We have now confirmed this by yet another system which monitors the loss of 3H-thymidine from labeled allo- and autografts. Thus, future studies promise to unravel some long sought after questions concerning the evolution of cellular defense mechanisms.
Ontogeny of the invertebrate humoral immune response: studies on various developmental stages of the American cockroach (Periplaneta americana).
Earlier studies revealed that a specific adaptive humoral immune response can be induced in the American cockroach (Periplaneta americana) to the soluble protein complex, Honeybee venom (HBT). We have undertaken a series of ontogenetic studies to determine if there are differences between the protective responses of roaches representing different developmental stages. Our results indicated that the response to HBT in the immature cockroach (nymphs weighing 200-500 mg) was characterized by a significant lag period before immune protection began to develop. However, by the second week of the response, reactivity was comparable to that of the adult. Old adult male roaches (animals 5 months into adulthood) displayed a significant decline in reactivity during the early phases of the response in comparison to younger adults, and in general, appeared to be less vigorous in generating protection. Both the nymph and old adult roaches demonstrated good secondary responsiveness. The results from these experiments indicated that the developmental stage of the roach could be directly correlated to the degree of immunocompetency possessed by the animal. This is similar to the ontogenetic sequence typically found for immune reactivity in vertebrates.
Preliminary characterization of the inducible humoral factor in the American cockroach (Periplaneta americana).
Previous studies have defined an inducible humoral immune response in the American cockroach that demonstrates specificity and memory. In addition, the response is affected by the age and gender of the animal. Preliminary studies have been initiated to determine which of the protein components found in immune hemolymph might be responsible for mediating this activity. Comparisons between hemolymphs from immune and control animals, using SDS-PAGE gel analysis, revealed that there was a unique band generated in immune animals with an approximate MW of 162K. In addition, several other bands, with MWs of 220K, 115K, 102K, 95K, 80K and 77K, appear to be enhanced in immune hemolymph. The 240K, 220K and 115K bands were found only in females, and so have been temporarily excluded from further study because they were gender-dependent. When hemolymph samples were absorbed with HBT toxoid, the 162K band was no longer present in immune hemolymph, and the 115K, 102K, 95K and the 80/77K doublet were all greatly reduced as compared to controls. Analysis of supernatants from 24 hour cultures of cells from immune and BTS-injected animals, revealed that immune cells secreted the 162K, 102K, 95K and 80K bands. The 115K band was barely detectable, and the 77K band appeared to be missing. Thus, these studies have provided some valuable initial information that reduces the number of proteins that should be considered for further study as mediators of the roach inducible humoral response.
Effect of gender on the inducible humoral immune response to honeybee venom in the American cockroach (Periplaneta americana).
It is well known that in higher animals, the female of the species usually possesses a superior immune response to that of the male. We investigated the possibility that this rule of nature might also be true amongst the invertebrates. Adult female American cockroaches (Periplaneta americana) were immunized with Honeybee venom, and their responses were compared to that of the adult male. The female primary response was found not only to be enhanced, but prolonged as compared to the male. The response was also specific and demonstrated long-term immunological memory. Thus, the humoral immune response of this advanced invertebrate shares yet another characteristic common to higher vertebrates, since the female of the species demonstrated much better immune responsiveness than the male.
The humoral immune response in the American cockroach, Periplaneta americana: reactivity to a defined antigen from honeybee venom, phospholipase A2.
Our previous experiments demonstrated that honeybee venom could induce a specific, adaptive humoral immune response in the American cockroach. Since honeybee venom is a complex substance made up of several proteins, a more defined antigen is needed for future characterization studies. One of the components of bee venom, phospholipase A2 (PA2) was found to be highly lethal and immunogenic in the roach. Roaches injected with PA2 generated a specific primary response that developed over a period of time, peaking within 10 days, and then gradually subsiding by the fifth week. Specificity of this response was demonstrated by the fact that immunized animals were protected against the original immunizing PA2, but not to PA2 from a heterologous source. In addition, a secondary response could be induced with PA2, demonstrating the existence of immunologic memory. Thus, we established that PA2 could induce as good, if not better, humoral responsiveness as whole bee venom, and therefore could be utilized as a more defined antigen in studies designed to characterize the inducible humoral factor in the roach.
Primary integumentary xenograft reactivity in the American cockroach, Periplaneta americana.
The nature of cell-mediated immunity in the American cockroach (Periplaneta americana) was studied by observing the events involved in graft reactivity. Adult roaches received integumentary xenografts from Blaberus giganteus, as well as control autografts. Grafts were scored histologically by counting the number of nuclei in a 100-mu-wide area under the center of the graft. The data showed that the reaction to the xenografts was characterized by quick recognition of foreignness by day 3 posttransplantation, followed by a slow decline in the cellular response, which lasted for more than 70 days. Control autografts did not appear to stimulate a reaction. Histological examination of xenografts revealed a disruption of the normal cytoarchitecture by a heavy cellular infiltrate. Our findings indicate that the outcome of the response to foreign integument in the roach is more characteristic of an encapsulation reaction than a classic rejection reaction.
Histological versus morphological assessment of graft rejection in invertebrates.
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Cryoultramicrotomy: evidence against melting and the use of a low temperature cement for specimen orientation.
A technique is presented showing the use of toluene as a low temperature cement to attach portions of frozen tissues in the desired orientation on to the cryoultramicrotome chuck. Examples of transverse sections of skeletal and vascular smooth muscle are illustrated. Electron probe analysis of these sections indicates that the distribution of elements is maintained. Evidence is presented against through section melting based on sectioning toluene at a temperature just below its melting point (178K). Toluene was sectioned (about 100 nm thickness) successfully at three block temperatures (177, 163 and 113 K) when the knife temperature was by 1 degree or mor colder than the m.p. of toluene. In all cases the toluene sections melted when the knife temperature reached 178K. We conclude that sufficient heat is not generated and/or transferred to the toluene sections during cryosectioning under our conditions to raise the temperature of a 100 nm section by 1 K or more.
Induction of specific humoral immunity to soluble proteins in the American cockroach (Periplaneta americana). I. Nature of the primary response.
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Induction of specific humoral immunity to soluble proteins in the American cockroach (Periplaneta americana). II. Nature of the secondary response.
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Micro mixed hemadsorption assay--a sensitive method for detecting antibodies against tumor-associated antigens.
Antibodies against methylcholanthrene (MCA)-induced sarcomas in Fischer (F344) rats were detected by a modified micro mixed hemadsorption (MHA)-assay. The assays detected anti-tumor antibodies as titers up to 1 : 320 in sera from hyperimmunized rats and at titers up to 1 : 160 in sera from rats bearing a 5-8 cm3 progressively growing tumor. MHA-titers decreased when sera were absorbed with sarcoma cells prior to MHA-assays. IgG antibodies in sera from tumor bearing rats showed titers of 1:20 and 1:5. These anti-tumor sera formed rosettes on the corresponding sarcoma cells as well as other sarcomes induced by MCA in F344 and Lewis strain rats tested. The assay was modified for a micro technique using a microtest plate (No. 3034, Falcon, CA). This modification yielded as assay requiring only 10 microliter of test sera. The test is quantitative and highly sensitive and results are reproducible. Several critical factors which influence test results in this assay were examined.