Nonimmune Host Research Articles

ALLOGENEIC RED BLOOD CELLS FAIL TO INDUCE HAEMAGGLUTINATING ANTIBODIES OR CELLULAR ALLOIMMUNITY IN RATS AND ARE IMMUNOSUPPRESSIVE

Purified rat red blood cells, which express the A region antigens of the major histocompatibility complex (MHC) with which anti-RT-1 (H-1; Ag-B) haemagglutinins combine, fail to stimulate the production of either haemagglutinating antibodies or cellular immunity in allogeneic hosts. Allogeneic red blood cells may in appropriate circumstances be mildly immunosuppressive. Intraperitoneally injected allogeneic red blood cells persist in the host circulation for several weeks. The immune response to skin allografts does not accelerate the destruction of circulating donor strain red blood cells, although the administration of alloantiserum with a high haemagglutinin titre can do so. The manner in which allogeneic red blood cells effect immunosuppression is unknown. Approximately 75% of the red blood cells administered i.v. to nonimmune allogeneic hosts are destroyed, by mechanisms unknown, during the first 6 days after injection. By contrast, about one-half of this number of red blood cells are destroyed by syngeneic hosts during the same period. Red blood cells and platelets in rodents are probably similar in expressing A region (rats) or K-D region (mice) antigens of the MHC, in being nonimmunogenic by themselves and in their capacity for effecting limited immunosuppression. If human platelets have similar attributes, the administration of platelets bearing the appropriate HLA-A, B, and C antigens might be a useful adjunct to conventional immunosuppression in the management of allografts in man. Likewise, the platelet component of blood transfusions might account for their beneficial effect in some renal allograft recipients.

Immune Response in Bladder Cancer Patients

Patients with various stages of bladder cancer underwent skin testing with dinitrochlorobenzene, recall antigens and Croton oil. In vitro lymphocyte evaluation was performed with T and B cell quantitation, T cell cytotoxicity and non-T cell cytotoxicity. Increasing disease stage was reflected by a greater percentage of negative responses to all skin tests. No response to dinitrochlorobenzene was found in 52 per cent of the patients with superficial tumor and 83 per cent of the patients with metastatic disease failed to respond (p < 0.025). After surgical removal of the bladder cancer in patients with superficial disease there was a significant decrease in negative response (p < 0.05). Skin test response in patients with invasive disease who were tested before and after cystectomy demonstrated no significant differences in responses.Assessment of dinitrochlorobenzene skin test response indicates that 1) dinitrochlorobenzene response is related to stage of disease, 2) survival of patients with metastatic tumor was significantly better if the dinitrochlorobenzene test was positive and 3) bladder cancer alters the non-immune host inflammatory system in addition to the delayed hypersensitivity immune system. Inflammatory (non-immune) function as measured by Croton oil response correlated with stage of disease (p < 0.001) but not with prognosis. None of the recall antigen responses correlated with prognosis. Streptokinase-streptodornase was the only recall antigen that related to tumor stage (p < 0.001). Lymphocyte evaluation indicated that bladder cancer patients, regardless of stage of disease, have significant variability, above and below the norm in T cell percentages and antibody-dependent cytotoxicity when compared to age-matched controls. Phytohemagglutinin-dependent cytotoxicity showed a significantly decreased variability after tumor removal (p < 0.001). Lymphocyte cytotoxicity in bladder cancer is greater than in the non-cancer population. This is particularly true in those with metastatic tumor. After removal of the tumor phytohemagglutinin-dependent cytotoxicity returned toward normal

Macrophage Immunity to Influenza Virus: &lt;i&gt;in vitro &lt;/i&gt;and &lt;i&gt;in vivo &lt;/i&gt;Studies

Using M-TUR, a macrophage-adapted avian influenza A virus (Hav1, Nav3), antiviral resistance of peritoneal macrophages obtained from specifically or nonspecifically immunized mice towards in vitro infection was assessed. M-TUR grew to high titers in macrophages from nonimmune mice thereby causing a marked cytopathic effect. In contrast, peritoneal macrophages from mice specifically immunized with TUR virus were not affected by infection with M-TUR in vitro. This antiviral immunity was specific: mice immunized with antigenetically unrelated influenza strains such as influenza A/Hong Kong/1/68 (H3, N2) or influenza B/Lee yielded susceptible macrophages. Specific macrophage immunity could be abrogated by trypsin treatment in vitro. Susceptible macrophages from nonimmune hosts became resistant following in vitro exposure to homologous anti-TUR sera. Peritoneal exudate cells from BCG-infected animals were less susceptible to in vitro challenge with M-TUR than control macrophages. In vivo treatment of mice with the unspecific immunostimulants BCG or Corynebacterium parvum did not protect the animals against lethal infection with a hepatotropic variant of TUR.

Membrane sialic acid on target particles modulates their phagocytosis by a trypsin-sensitive mechanism on human monocytes.

Monolayers of human peripheral blood monocytes in the absence of exogenous proteins ingest a variety of natural particulate activators of the human alternative complement pathway. Sheep erythrocytes, which do not ordinarily activate the human alternative complement pathway or initiate a direct monocyte phagocytic response, can be modified to exhibit both functions by the deletion or alteration of membrane sialic acid residues. Enzymatic removal of the sialic acid residues with sialidase or their conversion to heptulosonic acid derivatives by limited oxidation with NaIO4 and reduction with BH4- have equivalent dose-response effects on the capacity of the altered sheep erythrocytes to initiate the phagocytic response by human monocytes or to activate the alternative pathway in human serum. The deposition of C3b on native sheep erythrocytes had little effect on their ingestion by human monocytes, whereas the fixation of C3b on desialated sheep erythrocytes had a synergistic effect on the percentage of monocytes ingesting such a particle. The monocyte receptor essential for ingestion of desialated sheep erythrocytes or desialated sheep erythrocytes bearing C3b was inactivated by concentrations of trypsin that also prevented the monocytes from ingesting natural activators of the human alternative complement pathway, but did not alter the receptors for C3b or the Fc portion of IgG. The capacity of the nonimmune host to respond to desialated particles by initiating the monocyte ingestive process and by activating the alternative complement pathway to provide the synergy afforded by C3b deposition on that particle represents a primitive biochemical basis for differentiation of nonself from self.

Tumor-Growth Suppression in Vivo: Cooperation between Immune Lymphoid Cells Sensitized in Vitro and Nonimmune Bone Marrow Cells

Abstract Spleen cells from rats immunized with syngeneic (C58NT)D Gross virus-induced lymphoma cells restimulated in vitro with mitomycin C-treated (C58NT)D cells become cytotoxic to tumor targets and demonstrate an increased ability to adoptively confer antitumor immunity to nonimmune recipients. The purpose of the present studies was to determine whether nonimmune host cells participated in the local inhibition of tumor growth mediated by in vitro-activated cytotoxic cell populations. Adoptive transfer of antitumor immunity to irradiated syngeneic rats was more efficient when the recipients were reconstituted with normal bone marrow. A quantitative relationship was observed between the number of bone marrow cells administered and tumor-growth inhibition. Bone marrow cells from thymectomized, thoracic duct drained rats similarly were able to reconstitute lethally irradiated thymectomized recipients. Lymph node, thymus, or spleen cells were inactive when tested under the same conditions. Peritoneal-exudate (PE) cells contributed to decreased tumor growth in the presence or absence of sensitized lymphocytes. The results show that nonimmune bone marrow cells, although not required for tumor-growth suppression, interact synergistically with immune lymphocytes. These results suggest that in vitro-sensitized cells appear to inhibit tumor growth in vivo by at least two independent mechanisms: a direct, presumably cytotoxic effect on tumortarget cells and by indirect recruitment of a nonimmune radiosensitive bone marrow-derived host cell.

Splenectomy and infection

In the last two decades, physicians have become more aware of the spleen's role in natural immunity. The spleen is composed of two distinct but interrelated compartments. Fixed phagocytes of the reticuloendothelial system (RES) reside in the red pulp while lymphoid tissue is found in the follicles and periarteriolar sheaths of the white pulp. These two anatomic divisions are reflected in the spleen's two separate immunologic functions. On the other hand, the spleen serves as an important organ of clearance of hematogenously borne bacteria in the nonimmune host.¹On the other hand, it plays an important role in the production of early antibody in response to particulate antigens circulating in the bloodstream.²It is the strategic location of these two separate functions "midstream" in the circulation that accounts for its unique role in the nonimmune host's defense against blood-borne bacteria. A number of studies in animals have documented

Splenectomy and Infection

In the last two decades, physicians have become more aware of the spleen's role in natural immunity. The spleen is composed of two distinct but interrelated compartments. Fixed phagocytes of the reticuloendothelial system (RES) reside in the red pulp while lymphoid tissue is found in the follicles and periarteriolar sheaths of the white pulp. These two anatomic divisions are reflected in the spleen's two separate immunologic functions. On the other hand, the spleen serves as an important organ of clearance of hematogenously borne bacteria in the nonimmune host.¹On the other hand, it plays an important role in the production of early antibody in response to particulate antigens circulating in the bloodstream.²It is the strategic location of these two separate functions "midstream" in the circulation that accounts for its unique role in the nonimmune host's defense against blood-borne bacteria. A number of studies in animals have documented

Chemical Attraction between Adults of Nippostrongylus brasiliensis: Description of the Phenomenon and Effects of Host Immunity

Attraction between adults of Nippostrongylus brasiliensis was studied both in vivo and in vitro particularly with regard to the effects of host immunity on the behavior of the parasite. Most worms were found in clusters in the intestines of larval-infected rats but the number of isolated worms, particularly females, was greater in 14-day (immune) than in 7-day infected (nonimmune) hosts. Intubation of small numbers of normal adults into uninfected rats resulted in recovery of mostly aggregated worms unless infections consisted only of males. In contrast, immune-damaged worms exhibited little aggregation regardless of the sexes of worms instilled. Analysis of pairing between worms in vitro indicated that attraction occurred in the absence of host factors between all permutations of normal worms except male-male combinations. Pairing between damaged worms occurred only between males and females and not between worms of the same sex. Attraction to worm excretory and secretory products (ES) indicated that chemical factors mediated pairing. Normal female ES was attractive to both sexes whereas damaged female ES failed to attract either sex. ES from both normal and damaged males attracted females but not males thereby confirming the results of other experiments.

Participation of complement in the nonimmune host defense against experimental Haemophilus influenzae type b septicemia and meningitis.

This study was undertaken to determine whether the terminal complement components (C3-9) are involved in the nonimmune host defense against Haemophilus influenzae type b septicemia and meningitis. Using cobra venom factor, infant rats were depleted of C3 and C5. After intranasal challenge with H. influenzae type b, the complement-depleted rats developed a greater incidence and magnitude of bacteremia and a higher mortality rate. In contrast to the effects on bacteremia, complement depletion did not directly influence either the occurrence of meningitis or bacterial multiplication within the cerebrospinal fluid. These experiments provide evidence that the complement system may be an important mechanism of natural immunity to H. influenzae type b.

Dissemination and Causes of Infection

Specific immunization or vaccination, like antibiotics, may be of limited value because of the narrowness of their spectrum. On the other hand, if feasible, enhancement of some portion of the nonspecific, nonimmune host defense mechanism holds the most promise for the surgeon and his patient.

The Role of the Capsular Polysaccharide in the Activation of the Alternative Pathway by the Pneumococcus

Abstract The alternative pathway plays a significant role in the non-immune host's defense against the pneumococcus. The following studies concern the role of the capsular polysaccharide in the activation of the alternative pathway by the pneumococcus. Some pneumococcal capsular polysaccharides, but not all, are able to activate the alternative pathway. Soluble purified capsular polysaccharide types 1, 4, and 25 activate C3 in C4-deficient guinea pig serum (C4D GPS), where types 2, 3, 14, and 19 do not. Since capsular polysaccharides exist in their native form attached to the pneumococcal surface, selected capsular polysaccharides were also tested when coupled by CrCl3 to a particulate carrier, sheep erythrocytes. When attached to sheep erythrocytes, capsular polysaccharide types 2 and 3 failed to consume C3 in C4D GPS, paralleling the results obtained when they were in solution. In contrast, type 25 capsular polysaccharide coupled to erythrocytes not only consumed C3 in C4D GPS, as it had when in solution, but also initiated alternative pathway lysis of the erythrocytes.

The Role of the Capsular Polysaccharide in the Activation of the Alternative Pathway by the Pneumococcus

Previous studies have shown that when pneumococci are incubated in normal, nonimmune serum, they activate the alternative pathway and opsonically active C3b is fixed to the surface of the organism. Other studies have demonstrated that C3-dependent opsonization via the alternative pathway plays a significant role in the nonimmune host's defense against the pneumococcus. The present studies concern the role of the capsular polysaccharide in initiating the activation of the alternative pathway by the pneumococcus. Some pneumococcal capsular polysaccharide types, but not all, are able to activate the alternative pathway. Soluble purified capsular polysaccharide types 1, 4 and 25 activate the alternative pathway, whereas types 2, 3, 14, and 19 do not. Since the capsular polysaccharides exist in their native form attached to the pneumococcal surface, selected capsular polysaccharides were also tested for their ability to activate the alternative pathway when attached to a particulate carrier, sheep erythrocytes. Capsular polysaccharide types 2 and 3 failed to activate the alternative pathway when attached to sheep erythrocytes, paralleling the results obtained when these capsular polysaccharides were in solution. In contrast, the type 25 capsular polysaccharide not only activated the alternative pathway when attached to sheep erythrocytes, as it had when in solution, but it also initiated alternative pathway-mediated lysis of the erythrocytes. The capsular polysaccharide is not required for the activation of the alternative pathway by the pneumococcus. Although all types of encapsulated pneumococci are able to activate the alternative pathway, not all the purified capsular polysaccharide types are able to do so. In addition, a nonencapsulated pneumococcus, derived originally from a type 2 organism, activates the alternative pathway as well as a fully encapsulated type 2 pneumococcus.

Phagocytosis of Francisella tularensis by Rhesus monkey peripheral leukocytes

Phagocytosis of Francisella tularensis by rhesus monkey peripheral neutrophils (PMN, was assessed by autoradiography, electron microscopy, and biochemical techniques. PMN, in contrast to mononuclear phagocytes, were unable to phagocytize F. tularensis in vitro in the absence of immune serum. It is postulated that in the nonimmune host the innate inability of PMN to phagocytize F. tularensis deletes one of the possible mechanisms of host defense to bacterial infection and thus permits the early dissemination of F. tularensis to intracellular sites of specific target tissues.

THE BIOLOGIC SIGNIFICANCE OF THE THIRD COMPONENT OF COMPLEMENT (C3) IN NONIMMUNE HOST DEFENSES

Studies performed in vitro have demonstrated that C3 acts as an opsonin when activated to C3b and fixed to bacteria. In order to investigate the significance of C3 as an opsonin in vivo the following experiments were performed in normal, nonimmune mice. Mice were totally depleted of serum C3, as measured in a hemolytic assay, by the injection of cobra venom factor(CoVF). Concurrent with the C3 depletion their serum opsonizing activity decreased to a level less than 20% of normal. When mice were challenged with an intraperitoneal injection of pneumococci(Pn) immediately after the CoVF treatment, the LD50 was 1.8 × 106 as compared to 6.8 × 107 in saline treated control animals (P<0.05). When the CoVF was given only 6 hours after the Pn challenge the LD50 was the same as in the control mice. If Pn were preopsonized in vitro with nonimmune mouse serum and then injected into CoVF treated animals, the LD50 was the same as that in control animals. These studies demonstrate that there is a biologically significant in vivo role for C3 as an opsonin in the nonimmune host's defense against bacterial infection and that that role is most significant in the early stages of bacterial invasion.

Immunity to Hymenolepis citelli by Peromyscus maniculatus: Genetic Control and Ecological Implications

Acquired resistance to Hymenolepis citelli in Peromyscus maniculatus, the white-footed deer mouse, is shown to be controlled by a single autosomal dominant gene R (recessive gene designated r). This resistance could be transferred to uninfected hosts with lymphoid cells but not serum from infected resistant animals. Treatment with heterologous antilymphocyte serum depressed the ability of competent hosts to resist infections. A comparison of the response elicited against 2 strains of H. citelli (Utah and California) illustrated that chance-bred P. m. sonoriensis respond more rapidly and in greater incidence to the California strain than to the Utah strain. Homozygous recessive (r/r) hosts, susceptible to H. citelli (Utah), respond in significant numbers to the California strain of the tapeworm. Natural infections of H. citelli in Peromyscus do not occur uniformly throughout the environment but occur in small foci favoring existence of both intermediate and definitive hosts. Infections rarely occur outside such foci. We postulate that H. citelli is propagated in such foci by genetically nonimmune (r/r) hosts comprising about 25% of the host population. A previous study (Wassom et al., 1973), utilizing the tapeworm Hymenolepis citelli and its natural host Peromyscus maniculatus, illustrated that a tapeworm living exclusively in the gut lumen could elicit a protective resistance in some individuals. Such hosts, after initial acceptance of infection, responded by eliminating the worms and resisting reinfection. Other individuals were incapable of developing resistance. Primary infections were maintained and secondary infections could be imposed upon initial ones. The present study was conducted to evaluate the genetic aspects of this host resistance, and to determine whether the response is mediated by specific immune mechanisms or by nonspecific factors. MATERIALS AND METHODS All experimental animals were laboratory-reared Peromyscus maniculatus sonoriensis fed Purina Laboratory Chow. The isolation of H. citelli from wild-caught P. maniculatus and its maintenance in the laboratory as well as procedures for infection have been previously reported (Wassom et al., 1973). Immune serum was pooled from 2 groups of chance-bred P. maniculatus infected on day 0 with 10 cysticercoids of H. citelli, challenged with reinfection on day 14 or 21, and bled 7 days later. Received for publication 16 July 1973. * Department of Biology, University of Utah, Salt Lake City, Utah 84112. t Department of Pathology, University of Utah, Salt Lake City, Utah 84112. 47 Lymphoid-cell suspensions from the thymus, spleen, and lymph nodes of infected animals were prepared by mincing the tissues in Minimal Essential Medium for Suspension-Earle's BSS (MEMMicrobiological Associates), filtering through nylon gauze, and centrifuging at 60 g for 10 min. Red blood cells were lysed with Tris-buffered hypotonic ammonium chloride (Boyle, 1968), and the cells washed 4 times in MEM. Viability of cells was determined by trypan blue exclusion (Boyse et al., 1962). Cell concentrations were determined with standard microscopic counting chambers. Antilymphocyte serum (ALS) was prepared in rabbits by injection of Mus musculus lymphoid cell membranes in Freund's complete adjuvant. Injection of 0.5 ml of this ALS intraperitoneally on days 0, 2, and 4 was shown to delay median rejection times of Mus musculus allogeneic skin grafts (H2' to H2k) from 10.5 to 13.7 days. Skin grafts were not attempted in P. maniculatus. However, injection of 0.4 ml ALS intraperitoneally to P. maniculatus on days 0, 2, and 4 was shown to reduce hemagglutinin titers to SRBC by 4 doubling dilutions. This ALS is thus an effective immunosuppressive agent in P. maniculatus.

Studies of delayed hypersensitivity to L. Monocytogenes in mice: Nature of cells involved in passive transfers

C3Hf/Umc mice were immunized by an intravenous injection of a sublethal dose of live Listeria monocytogenes. The animals developed delayed-type hypersensitivity (DH) concomitant with infectious immunity to this organism. Delayed hypersensitivity could be transferred to normal lethally irradiated mice with spleen cells from immune animals. The immune cells cells responsible for transfer of adoptive immunity were susceptible to in vitro cytolytic action of anti-theta iso-antibody and complement, since such treatment rendered these cells incapable of further passive transfer of specific immunity to Listeria. The acquired DH to Listeria persisted in mice after 900 R lethal irradiation, provided normal syngeneic bone marrow cells were also administered, thus indicating the persistance of a cell population in the immune irradiated mice, resistant to effects of radiation. The radio resistant nature of this immune cell population was further demonstrated by passive transfer with spleen cells, derived from preimmunized lethally irradiated mice to normal syngeneic mice or to lethally irradiated nonimmunized hosts reconstituted with normal bone marrow which then responded to antigenic challenge with DH. Treatment of the immune radio resistant spleen cells in vitro with anti-theta and complement eliminated passive transfers of DH by these cells; however, this effect was less obvious than similar treatment of the immune, nonirradiated, spleen cells.

Indirect visualization of Staphylococcus aureus protein A.

Specific but nonimmunologic reaction between staphylococcal protein A and the Fc portion of gamma globulin provided the basis for ultrastructural studies to determine the localization of protein A, using intact staphylococci and labeled myeloma gamma G-globulin. Protein A appeared to be part of the outermost layer of the staphylococcal cell wall. Strains with protein A demonstrated a coating of myeloma globulin over the entire bacterial surface. There was no coating of strains without protein A. Identification of protein A on the surface of the staphylococcal cell wall provides evidence that this may be the first material in contact with host environment. It probably accounts for apparent cross-reactions of staphylococci with antibodies to many antigens. More importantly, even in the nonimmune host protein A immunoglobulin reactivity may initiate complement activation and inflammatory reactions including chemotaxis and pus formation.

Potentiation of experimental arbovirus encephalitis by immunosuppressive doses of cyclophosphamide.

THE role of the immune response in recovery from primary viral infection has been debated for many years, in part because of the inconclusive nature of the relevant evidence1,2. The ability of children with defective immunoglobulin synthesis to recover uneventfully from certain viral infections3, and the recognition of non-immune host defences such as interferon4, have suggested that the immune response plays a small part. The recent development of an increasing variety of techniques for the production of immunosuppression permits a re-examination of the question. Using the potent alkylating agent, cyclophosphamide5,6, we have found that administration of immunosuppressive doses of the drug converts a silent abortive arbovirus infection of the rodent central nervous system (CNS) into a lethal encephalitis.

Integration of lambda and lambda-b2 genomes in nonimmune host bacteria carrying a lambda cryptic prophage.

Abstract Bacteria that carry a λ cryptic (λ cry) prophage are not λ-immune and can be further lysogenized with suitably marked λ phages. The genetic structure of these double lysogens, which are obtained at high frequency, has been analyzed by P1 transduction. When the superinfecting phage is λb2+, it enters with equal frequency either between gal and λ cry or between λ cry and bio. In contrast, λb2 enters efficiently only near bio. No case insertion of the second phage genome within the λ cry prophage has been found out of 40 strains studied. These results fit the idea that in nonimmune bacteria lysogenization is due to an effective, phage-directed integrating mechanism, presumably an enzyme specific for the host and phage attachment sites. The results also imply that λb2 still possesses both the ability to produce such an enzyme and at least a part of the substrate on which the integrating enzyme acts. Singly lysogenic bacteria that appear to carry only a λb2 genome as an integrated prophage have been obtained; these prove to be low-phage yielders. Multiple lysogens in which one or more prophages are b2 upon UV induction are normal yielders. The phage they produce consists mainly of phage genomes that contain neighboring segments of adjacent prophages (internal excision).

Resistance of Galleria mellonella (Linnaeus) to the Tipula iridescent virus at high temperatures

At 23° to 25°C, the larvae of the greater wax moth, Galleria mellonella (Linnaeus), which had been inoculated intrahemocoelically with the noninclusion virus of Tipula paludosa Meigen died of infection with the virus. At temperatures above 30°C, the virus-inoculated larvae survived to adults. Inasmuch as the virus was inoculated directly into the hemocoel, the resistance of the larvae at high temperatures was due to the destruction of the virus or the prevention of virus multiplication by immune and nonimmune host reactions, and not to the failure of the virus to penetrate into the hemocoel.