Engraftment and Humoral Immunity in SCID and RAG-2-Deficient Mice Transplanted with Human Peripheral Blood Lymphocytes

SCID and RAG-2 deficient mice were transplanted intraperitoneally with human peripheral blood lymphocytes (hu-PBL-SCID and hu-PBL-RAG mice). Seven days after transplantation tbe mice were immunized with a pneumococcal polysaccharide vaccine. Flow cytometry analysis of cells from the peritoneal cavity and the spleen after 8–10 weeks revealed that human cells had more limited engraftment in RAG than in SCID recipient mice, and that more human cells were found in the spleen than in the peritoneal cavity. Functionality of the human cells recovered from these two locations was explored by the counting of human immunoglobulin secreting cells (hu-ISC). A total of 83% of the hu-PBL-SCID mice and 29% of the hu-PBL-RAG mice had detectable hu-ISC in the peritoneal cavity and/or the spleen. The kinetic profiles of human immunoglobulins in the mouse sera during the experiment showed donor dependency. More than 90% of the hu-PBL-SCID mice had detectable levels of human IgG, IgM and IgA, while 78% had detectable levels of IgE, whereas detectable levels of IgG, IgM, IgA and IgE were measured in 37%, 64%, 68% and 23% of the hu-PBL-RAG mice, respectively. Forty-seven per cent of immunized hu-PBL-SCID mice showed a human antipneumococcal IgG level that was significantly above the background level in non-immunized mice, while none of the hu-PBL-RAG mice produced any detectable levels of human antipneumococcal IgG. In short, human PBL showed a better engraftment and a better antibody response when transplanted into SCID mice than into RAG mice.     

T-Cell-Dependent Production of IgG by Human Cord Blood B Cells in Reconstituted SCID Mice

Reconstitution of severe combined immunodeficient (SCID) mice with human lymphocytes has recently allowed the elucidation of abnormalities of immune responses in various immunological disorders. In the present study, mononuclear cells (MNC) from neonatal cord blood and adult peripheral blood were intraperitoneally injected into SCID mice to examine induction of human Ig in respective mice recipients. Human IgG was consistently detected in the serum of SCID transferred with adult MNC, but only a few SCID recipients of cord blood MNC showed detectable but low levels of IgG in the serum. The combination experiments of isolated B and T cells disclosed that some interactions between B and T cells might be necessary for IgG production in transferred SCID mice. Notably, transfer of cord blood B cells with adult but not cord blood T cells resulted in efficient induction of IgG, associated with a change in subclass distribution. The results suggest that inability of neonatal B cells to produce IgG can be overcome by transfer with adult mature T cells into SCID mice.     

A Peptide Domain on Gingipain R Which Confers Immunity against Porphyromonas gingivalis Infection in Mice

The cysteine proteinases referred to as gingipains R (gingipain R1 and gingipain R2) and gingipain K produced by Porphyromonas gingivalis are virulence factors of this periodontal pathogen which likely act by interrupting host defense mechanisms and by participating in the penetration and destruction of host connective tissue. To examine the effect of immunization with gingipains R on the ability of P. gingivalis to colonize and invade in the mouse chamber model, BALB/c mice were immunized intraperitoneally with the 95-kDa gingipain R1, the 50-kDa gingipain R2, or multiple antigenic peptide (MAP)-conjugated gingipain R-derived peptides and then challenged with P. gingivalis. Immunization of mice with the 95-kDa gingipain R1, the 50-kDa gingipain R2, or a peptide derived from the N-terminal sequence of the catalytic domain of gingipains R (peptide A) followed by challenge with P. gingivalis A7436 resulted in protection from P. gingivalis invasion. In contrast, immunization with peptides corresponding to either a sequence encompassing the catalytic cysteine residue of gingipains R (peptide B) or an identical sequence within the catalytic domains of gingipain R1 and gingipain K (peptide C), followed by challenge with P. gingivalis, did not protect animals, nor did immunization with a peptide corresponding to sequences within the adhesion/hemagglutinin domain of gingipain R1 (peptide D) which have been shown to be directly involved in the hemagglutinin activity of gingipain R1. However, the immunoglobulin G (IgG) titer obtained following immunization with peptide D was comparable to that obtained following immunization with the N-terminal peptide (peptide A). Competitive enzyme-linked immunosorbent assays, using either the 95-kDa gingipain R1 or gingipain K as the competing soluble antigen, indicated that 42 and 53% of the antibodies induced by immunization with heat-killed bacteria recognize gingipain R1 and gingipain K, respectively; however, even at very high concentrations, the 50-kDa gingipain R2 did not hinder IgG binding to P. gingivalis. These results indicate that antibodies directed to the amino-terminal region of the catalytic domain of gingipains R are capable of inducing a protective immune response against P. gingivalis infection in the mouse chamber model.     

C-Reactive Protein Protects Mice against Pneumococcal Infection via Both Phosphocholine-Dependent and Phosphocholine-Independent Mechanisms

The mechanism of action of C-reactive protein (CRP) in protecting mice against lethal Streptococcus pneumoniae infection is unknown. The involvement of the phosphocholine (PCh)-binding property of CRP in its antipneumococcal function previously has been explored twice, with conflicting results. In this study, using three different intravenous sepsis mouse models, we investigated the role of the PCh-binding property of CRP by employing a CRP mutant incapable of binding to PCh. The ability of wild-type CRP to protect mice against infection was found to differ in the three models; the protective ability of wild-type CRP decreased when the severity of infection was increased, as determined by measuring mortality and bacteremia. In the first animal model, in which we used 25 μg of CRP and 10⁷ CFU of pneumococci, both wild-type and mutant CRP protected mice against infection, suggesting that the protection was independent of the PCh-binding activity of CRP. In the second model, in which we used 25 μg of CRP and 5 × 10⁷ CFU of pneumococci, mutant CRP was not protective while wild-type CRP was, suggesting that the protection was dependent on the PCh-binding activity of CRP. In the third model, in which we used 150 μg of CRP and 10⁷ CFU of pneumococci, mutant CRP was as protective as wild-type CRP, again indicating that the protection was independent of the PCh-binding activity of CRP. We conclude that both PCh-dependent and PCh-independent mechanisms are involved in the CRP-mediated decrease in bacteremia and the resulting protection of mice against pneumococcal infection.     

Intranasal Immunization with Pneumococcal Polysaccharide Conjugate Vaccines Protects Mice against Invasive Pneumococcal Infections

Host defenses against Streptococcus pneumoniae depend largely on opsonophagocytosis mediated by antibodies and complement. Since pneumococcus is a respiratory pathogen, mucosal immune responses may play a significant role in the defense against pneumococcal infections. Thus, mucosal vaccination may be an alternative approach to current immunization strategies, but effective adjuvants are required. Protein antigens induce significant mucosal immunoglobulin A (IgA) and systemic IgG responses when administered intranasally (i. n.) with the glyceride-polysorbate based adjuvant RhinoVax (RV) both in experimental animals and humans. The immunogenicity and efficacy of pneumococcal polysaccharide conjugate vaccines (PNC) of serotypes 1 and 3 was studied in mice after i.n. immunization with RV. Antibodies were measured in serum (IgM, IgG, and IgA) and saliva (IgA) and compared to antibody titers induced by parenteral immunization. The PNCs induced significant systemic IgG and IgA antibodies after i.n. immunization only when given with RV and, for serotype 1, serum IgG titers were comparable to titers induced by subcutaneous immunization. In addition, i.n. immunization with PNC-1 in RV elicited detectable mucosal IgA. These results demonstrate that RV is a potent mucosal adjuvant for polysaccharides conjugated to proteins. A majority of the PNC-1-immunized mice were protected against both bacteremia and pneumonia after i.n. challenge with a lethal dose of serotype 1 pneumococci, and protection correlated significantly with the serum IgG titers. Similarly, the survival of mice immunized i.n. with PNC-3 in RV was significantly prolonged. These results indicate that mucosal vaccination with PNC and adjuvants may be an alternative strategy for prevention against pneumococcal infections.     

Reactivity of Antiserum to Human Brain with Peripheral Blood Lymphocytes

Antisera to human brain (AHBS) and human thymocytes (AHTS) were produced in rabbits and selectively absorbed to render them specific for T cells. After absorption AHBS, but not AHTS, lost most of its cytotoxic activity against T cells. Absorbed AHBS bound up to 95% of peripheral blood T lymphocytes as detected by indirect immunofluorescence and inhibited up to 46% of the lytic activity of AHTS; however, it was incapable of inhibiting the E-rosette formation of T lymphocytes. All 10 samples of human peripheral blood lymphocytes, pretreated with AHBS, were significantly suppressed in their response to antigens, but fewer samples were affected in their response to mitogens and to allogeneic stimulation, indicating diversity in the nature of the receptors involved in the cellular responses.     

Carbohydrate Metabolism in PHA Stimulated Human Peripheral Blood Lymphocytes

Human peripheral blood lymphocytes (PBL) stimulated in vitro by phytohemoagglutinin (PHA) manifest augmented glycolysis and oxidation of glucose-1-¹⁴C, indicating an increased utilization of the pentose pathway. Lactic acid production, as index of increased glycolysis, follows the same kinetic of thymidine incorporation and can he easily quantitated by an enzymatic assay.     

Topical Resiquimod Protects against Visceral Infection with Leishmania infantum chagasi in Mice

New prevention and treatment strategies are needed for visceral leishmaniasis, particularly ones that can be deployed simply and inexpensively in areas where leishmaniasis is endemic. Synthetic molecules that activate Toll-like receptor 7 and 8 (TLR7/8) pathways have previously been demonstrated to enhance protection against cutaneous leishmaniasis. We initially sought to determine whether the TLR7/8-activating molecule resiquimod might serve as an effective vaccine adjuvant targeting visceral leishmaniasis caused by infection with Leishmania infantum chagasi. Resiquimod was topically applied to the skin of mice either prior to or after systemic infection with L. infantum chagasi, and parasite burdens were assessed. Surprisingly, topical resiquimod application alone, in the absence of vaccination, conferred robust resistance to mice against future intravenous challenge with virulent L. infantum chagasi. This protection against L. infantum chagasi infection persisted as long as 8 weeks after the final topical resiquimod treatment. In addition, in mice with existing infections, therapeutic treatment with topical resiquimod led to significantly lower visceral parasite loads. Resiquimod increased trafficking of leukocytes, including B cells, CD4⁺ and CD8⁺ T cells, dendritic cells, macrophages, and granulocytes, in livers and spleens, which are the key target organs of visceralizing infection. We conclude that topical resiquimod leads to systemic immune modulation and confers durable protection against visceralizing L. infantum chagasi infection, in both prophylactic and therapeutic settings. These studies support continued studies of TLR-modulating agents to determine mechanisms of protection and also provide a rationale for translational development of a critically needed, novel class of topical, preventative, and therapeutic agents for these lethal infections.     

A Complex Adenovirus-Vectored Vaccine against Rift Valley Fever Virus Protects Mice against Lethal Infection in the Presence of Preexisting Vector Immunity

Rift Valley fever virus (RVFV) has been cited as a potential biological-weapon threat due to the serious and fatal disease it causes in humans and animals and the fact that this mosquito-borne virus can be lethal in an aerosolized form. Current human and veterinary vaccines against RVFV, however, are outdated, inefficient, and unsafe. We have incorporated the RVFV glycoprotein genes into a nonreplicating complex adenovirus (CAdVax) vector platform to develop a novel RVFV vaccine. Mice vaccinated with the CAdVax-based vaccine produced potent humoral immune responses and were protected against lethal RVFV infection. Additionally, protection was elicited in mice despite preexisting immunity to the adenovirus vector.Rift Valley fever virus (RVFV) is an arthropod-borne bunyavirus that can cause severe disease in humans. Disease symptoms range from benign flulike symptoms to more severe disease involving retinal lesions, hemorrhagic fever, or encephalitis, and disease may be fatal (1, 4, 37). Outbreaks among livestock, often signaled by mass spontaneous abortions, can have a significant economic impact. RVFV outbreaks historically have been limited to sub-Saharan Africa but are highly transmissible and have since spread into other regions, including Saudi Arabia and Yemen (2, 34, 36, 47, 48). Additionally, the severity of human RVFV outbreaks may be increasing; among identified cases in an outbreak in Sudan in late 2007, the mortality rate reached 35% (49). Human infection with RVFV occurs most commonly through contact with infected animals but can also result from mosquito bites during periods when the virus is circulating at high densities in these hosts. Accidental infection of scientists in a laboratory setting, however, indicates that the virus is fully capable of aerosol transmission (15, 39), and this has also been demonstrated with animal models (5, 7).Control of the arthropod host and immunization of livestock might be effective approaches to limit natural outbreaks of the disease, but no effective and safe vaccine is yet available. More importantly, these approaches do not address the threat that RVFV poses as an agent for bioterrorism. The virus is widespread in diverse parts of Africa and can be propagated easily and efficiently in vitro (14). Because of its disease potential, aerosolized RVFV could be used as a weapon to threaten human life and to devastate livestock and the economy (11, 39). Release of aerosolized RVFV in confined spaces, such as public buildings and subways, may enhance spread of the virus among civilians. With such potential in mind, RVFV has been placed on the CDC''s list of select agents and is an NIAID category A priority pathogen. Therefore, developing a vaccine protective against RVFV infection for human use is a critical strategy to address this threat.It is widely agreed that an effective RVFV vaccine should elicit potent neutralizing antibodies and provide complete sterilizing immunity. Prior studies have shown that passive transfer of immune sera completely protects naïve mice from lethal challenge with RVFV (35). Yet several RVFV vaccines fail to elicit a potent neutralizing antibody response or are deemed inappropriate for human vaccination due to safety concerns. While live-attenuated virus vaccines have been used against RVFV for livestock, these vaccines are unacceptable for human use, since they are known to cause abortions in cattle (6) and are teratogenic in sheep (23). A formalin-inactivated RVFV vaccine was developed in the 1980s for use with military personnel (25), but it is very weakly immunogenic, requiring a series of booster immunizations (16, 31). Other attenuated virus vaccines have been developed and tested with various animal species (9, 28, 29, 43), but these vaccines are considered unsafe for human use given their potential to revert to the pathogenic virus.In an attempt to develop safer and more potent RVFV vaccines, several subunit and recombinant vaccine approaches have been explored. RVFV is an enveloped phlebovirus of the Bunyaviridae family that carries two glycoproteins on its surface, and these viral components are the likely targets for a protective immune response. These N-terminal and C-terminal glycoproteins, named Gn and Gc, respectively, are encoded on the M segment of the genome and are synthesized as part of polyprotein precursors, which can also include an additional 14-kDa N-terminal component depending on selection of alternative translation initiation codons (17, 24). Antibodies against Gn and Gc effectively neutralize virus by blocking virus-receptor interaction and cell entry events and may also play a role in complement-mediated clearance of virus. It was shown previously that lysates of insect cells infected with a baculovirus expressing the M segment elicited a protective immune response to RVFV in mice (35). Importantly, protection against challenge could be provided to naïve mice via passive transfer of immune sera, thus demonstrating the key role of neutralizing antibodies in providing protection against RVFV infection and the importance of Gn and Gc as antigens in eliciting a potent humoral immune response.Recently, Wallace et al. compared three different vaccination approaches for RVFV: a DNA vaccine, a recombinant-protein vaccine, and a recombinant lumpy skin disease virus (rLSDV) vector vaccine expressing Gn and Gc (44). Both a recombinant form of the Gc protein and the rLSDV-RVFV vector protected mice from challenge with RVFV after a single immunization. While the use of rLSDV-RVFV is feasible only as a potential veterinary vaccine against RVFV and sheeppox virus, the result supports the use of RVFV glycoproteins for a vaccine. Expression of the Gc protein alone using a Venezuelan equine encephalitis virus (VEEV) replicon provided protection against RVFV challenge in mice following a single injection (19). Mice were also protected from challenge following immunization with a VEEV expressing a 318-amino-acid fragment of Gc fused to the E2 glycoprotein of the vector. However, the authors reported difficulties in propagation of the Gn-expressing VEEV replicons and suggested that Gn vectors would be problematic for large-scale manufacturing. Another promising vaccine candidate is RVFV virus-like particles, assembled from envelope and nucleocapsid proteins of the virus but lacking an intact viral genome (20). These replication-deficient virus-like particles have recently been shown to protect against RVFV challenge in mice that received a series of three immunizations, but their production on a large scale remains a challenge (30).To address the lack of a safe, effective, and practical RVFV vaccine, we have utilized a proven vaccine platform based on our complex adenovirus (CAdVax) vector system. Adenovirus (Ad) vectors are widely studied viral vectors for vaccines and gene therapy applications and have been tested in hundreds of clinical trials worldwide, with a favorable safety profile (18). Ad vectors are capable of expressing high levels of transgenes, are efficiently produced in large quantities, and are highly immunogenic as vaccines. An additional capability of the CAdVax platform is the expression of multiple antigen inserts independently. RVFV glycoproteins have shown promise as targets of neutralizing immunity and form the primary surface components of RVFV. Since neutralizing antibodies against the RVFV envelope glycoproteins are the key component of protective immunity against RVFV infection, we developed and tested a CAdVax vaccine to direct simultaneous, de novo expression of the Gn and Gc glycoproteins. We found this vaccine to be highly immunogenic in mice, eliciting strong immune responses and providing complete protection against lethal challenge with RVFV. The CAdVax-RVF vaccine also showed efficacy in animals that were previously immunized with a heterologous CAdVax vector, suggesting that preexisting immunity to the Ad type 5 (Ad5) vector has little effect on limiting induction of a potent immune response to CAdVax vector antigens in vaccinated mice. Here, we present the results of these experiments and discuss their implications for the development and large-scale production of a safe and effective RVFV vaccine.     

Intranasal Immunization with Heat-Inactivated Streptococcus pneumoniae Protects Mice against Systemic Pneumococcal Infection

In order to study the mucosal and serum antibody response to polysaccharide-encapsulated bacteria in mice, a preparation of heat-inactivated Streptococcus pneumoniae type 4 was administered, with and without cholera toxin, at various mucosal sites. It appeared that intranasal immunization of nonanesthesized animals was superior to either oral, gastric, or colonic-rectal antigen delivery with regard to the induction of serum immunoglobulin G (IgG) and IgA, as well as saliva IgA antibodies specific for pneumococci. The marked IgA antibody response in feces after intranasal, but not after oral or gastric, immunization is suggestive of a cellular link between the nasal induction site and the distant mucosal effector sites. Intranasal immunization also induced antibodies in serum and in mucosal secretions against type-specific capsular polysaccharide. IgA and IgG antibody levels in pulmonary lavage fluids correlated well with saliva IgA and serum IgG antibodies, respectively. Antibody determinations in pulmonary secretions may therefore be redundant in some cases, and the number of experimental animals may be reduced accordingly. After intraperitoneal challenge with type 4 pneumococci, mice immunized intranasally were protected against both systemic infection and death, even without the use of cholera toxin as a mucosal adjuvant. Thus, an efficient intranasal vaccine against invasive pneumococcal disease may be based on a very simple formulation with whole killed pneumococci.     

Theoylline Induced Non Specific Suppressor Activity in Human Peripheral Blood Lymphocytes

It is wellknown that theophylline yields phenotypic changes on suppressor cells. In the present study we investigated the possibility that theophylline could directly induce a suppressor activity on a lymphocyte subpopulation. We observed that a short preincubation (120 min at 37°C) with theophylline (1mM) activates human peripheral blood lymphocytes to suppress mitogenic response of autologous cells. This activity was not evident on a T cell subpopulation depleted of theophylline-sensitive (T-sens) lymphocytes.

Theophylline mediated suppressor activity is only present in the Concanavalin A stimulated cultures, thus suggesting a synergism between Concanavalin A and theophylline in the expression of non specific suppression. Moreover we observed that after a 24 hrs preincubation of lymphocytes in complete culture medium there was a complete loss of theophylline-induced suppression. Such a preincubation time also produced a decrease in the theophylline-mediated enhancement of intracellular 3',5' cyclic adenosine monophosphate levels and the impairment of E-rosette formation, suggesting that theophylline acts mainly on a “short-lived” suppressor lymphocyte subset.     

Synthesis and Fragmentation of DNA in Phytohaemagglutinin-Stimulated Human Peripheral Blood Lymphocytes

In phytohaemagglutinin-stimulated lymphocytes, pulse labelled with tritiated thymidine for one minute, the acid-precipitable radioactivity was in the form of fragments that banded at the top of an alkaline sucrose gradient. When the radioactivity was chased with unlabelled thymidine for 2 hrs, most of the acid-precipitable radioactivity banded with the bulk of the DNA in the lower half of the gradient. On further chasing from 5 to 24 hrs, the radioactive DNA was fragmented and could be located on the lighter side of the gradients. This later fragmentation of radioactive DNA was associated with a loss of acid-precipi table radioactivity from the cells and appearance of DNA in the medium. After 24 hrs, approximately 67% of the acid-precipitable radioactivity was present in the medium. 60 to 70% of the radioactivity put out into the medium was acid precipitable, alkali resistant and DNase sensitive.     

Characterization of NKT Cells in Human Peripheral Blood and Decidual Lymphocytes

PROBLEM: To examine whether natural killer (NKT) cells are present in human pregnancy decidua. METHOD OF STUDY: We calculated the percentage of CD3+CD161+Valpha 24+-NKT cells in peripheral blood and early pregnancy decidua, and analyzed intracellular cytokines, interleukin (IL)-4 and interferon (IFN)gamma in NKT cells using flow cytometry. RESULTS: A distinct subset of CD3+ CD161+ lymphocytes expressing an invariant antigen receptor encoded by the Valpha24 and Vbeta11 segment was accumulated in the decidua. In pregnant subjects the percentages of NKT cells were significantly increased in the decidua compared with peripheral blood. Both NKT cells in the decidua and the peripheral blood had an ability to rapidly produce cytokine associated with Th1 (IFNgamma) and Th2 (IL-4). Interestingly, the percentages of IL-4 and IFNgamma producing NKT cells were significantly higher in the decidua compared with the peripheral blood. CONCLUSIONS: These findings suggest that NKT cells might control the Th1/Th2 balance by producing IL-4 and IFNgamma at the feto-maternal interface.     

Complement Regulatory Protein CD46 Protects against Choroidal Neovascularization in Mice

Dysregulation of the complement system is increasingly recognized as a contributing factor in age-related macular degeneration. Although the complement regulator CD46 is expressed ubiquitously in humans, in mouse it was previously thought to be expressed only on spermatozoa. We detected CD46 mRNA and protein in the posterior ocular segment (neuronal retina, retinal pigment epithelium, and choroid) of wild-type (WT) C57BL/6J mice. Cd46^−/− knockout mice exhibited increased levels of the membrane attack complex and of vascular endothelial growth factor (VEGF) in the retina and choroid. The Cd46^−/− mice were also more susceptible to laser-induced choroidal neovascularization (CNV). In Cd46^−/− mice, 19% of laser spots were positive for CNV at day 2 after treatment, but no positive spots were detected in WT mice. At day 3, 42% of laser spots were positive in Cd46^−/− mice, but only 11% in WT mice. A fully developed CNV complex was noted in both Cd46^−/− and WT mice at day 7; however, lesion size was significantly (P < 0.05) increased in Cd46^−/− mice. Our findings provide evidence for expression of CD46 in the mouse eye and a role for CD46 in protection against laser-induced CNV. We propose that the Cd46^−/− mouse has a greater susceptibility to experimental CNV because of insufficient complement inhibition, which leads to increased membrane attack complex deposition and VEGF expression.Age-related macular degeneration (AMD) is a leading worldwide cause of central vision loss in individuals over the age of 50.^1–5 The prevalence of AMD is growing because of increased longevity. The disease brings negative changes in life style. AMD patients often cannot perform daily tasks of living, such as reading or driving. Current estimates are that it requires 575 to 733 million dollars annually to treat AMD in the United States.⁵ Thus, AMD profoundly affects the quality of life, creating a serious social and public health problem.^3,4Two major clinical phenotypes of AMD are recognized: nonexudative (dry type) and exudative (wet type). Wet AMD is frequently associated with central blindness, and choroidal neovascularization (CNV) is the hallmark of this type in humans. Agents available for treating exudative AMD reduce the rate of vision loss, but they do not reverse damage; furthermore, they are associated with a variety of ocular complications, require repetitive administration, and are expensive.^1–4AMD is a disease with numerous risk factors, and multiple pathological mechanisms have been reported.^6–10 Evidence accumulated during the past decade, primarily through genetic studies, strongly indicates that the alternative pathway of the complement system plays an important role in AMD pathogenesis in humans.^3,11–15 Reports from multiple investigators have established that the membrane attack complex (MAC) C5b-9, formed as a result of alternative pathway complement engagement, participates in mediating animal models of CNV.^{11,12,16–23} Complement regulatory proteins control the complement cascades,^24,25 and their deficiency has been reported to predispose to the development of experimental CNV.^18,20CD46 [alias membrane cofactor protein (MCP)] is a widely expressed transmembrane glycoprotein in primates that serves as a complement regulatory protein^26,27 and is present in the normal human eye.^28–31 It binds C3b and C4b and then serves as a cofactor protein for the cleavage of these two substrates by serine protease factor I. In rodents, however, CD46 expression has been firmly established only for the inner acrosomal membrane of spermatozoa.^32–34 Our goal in the present study was to investigate whether CD46 is expressed in the mouse eye. After this was unequivocally shown, we explored its role in a murine model of laser-induced CNV.^11,12,35,36     

霍乱毒素佐剂联合弓形虫ESA鼻内免疫小鼠抗弓形虫感染作用

目的 观察霍乱毒素（cholera toxin,CT）佐剂和弓形虫排泄-分泌抗原（ESA）鼻内免疫小鼠诱导的抗弓形虫感染作用。方法 6周龄BALB/c小鼠60只,随机分为3组,每组20只。分别用PBS 20μl、ESA 20μg或CT 1.0μg＋ESA 20μg每只滴鼻免疫2次,间隔2周。末次免疫后14 d,用4×104个弓形虫速殖子每只灌胃攻击所有小鼠,观察小鼠健康及死亡情况。速殖子攻击后30 d,计数肝、脑组织内弓形虫速殖子。结果 CT作为佐剂联合弓形虫ESA滴鼻免疫小鼠的健康状况明显好于PBS组和ESA组,存活率（95%）也显著高于PBS组（55%）。与PBS组相比,CT＋ESA组肝和脑组织内速殖子数分别减少了80.19%（P〈0.001）和78.24%（P〈0.005）。CT作为佐剂联合ESA滴鼻免疫小鼠诱导了高水平的黏膜免疫应答和系统免疫应答。结论 CT作为佐剂联合弓形虫ESA滴鼻免疫小鼠诱导的免疫应答可有效抵抗弓形虫速殖子攻击。     

PHA-Induced Activation of Suppressor Cells in Normal Human Peripheral Blood Lymphocytes

Normal human peripheral blood and tonsil lymphocytes can be stimulated to proliferate by phytohemagglutinin (PHA). When cells cultured with this mitogen for 3 days were transferred fo fresh autologous lymphocytes in fresh medium with PHA, the mitogen response of the fresh lymphocytes was suppressed. The suppression required the presence of viable cells, in that culture supernatants alone were not inhibitory and cell extracts showed only marginal inhibition. Approximately equivalent numbers of previously stimulated cells were required to produce optimal suppression of the PHA response of fresh cells. Cells irradiated after PHA stimulation were as effective as nonirradiated cells in causing suppression. PHA-stimulated cells also inhibited concanavalin A-induced proliferation and a mixed lymphocyte reaction. However, PHA-stimulated cells only partially inhibited the response to pokeweed mitogen. The suppressive effects were fully retained by a nylon-wool-enriched T-cell fraction but not by a B-cell-enriched fraction.     

Studying the Proliferation of Human Peripheral Blood T Lymphocytes in Serum-Free Medium

We compared the cultivation of human peripheral blood lymphocytes in serum-free medium Hybris-2 and RPMI 1640 medium with 10% fetal bovine serum in the presence of phytohemagglutinin and interleukin-2. The optimal concentration of phytohemagglutinin significantly differed in serum-free and serum-containing media (0.5 and 5 mg/ml, respectively). Both mitogens were more potent in stimulating the proliferation of lymphocytes in serum-free medium than in serum-containing medium. Strong proliferation of CD3⁺ and CD4⁺ T lymphocytes was observed in both media. The dynamics of other markers was similar in serum-free and serum-containing media. However, significant differences were revealed between individual donors. Our results indicate that the developed serum-free medium may be used in lymphocyte cultivation for scientific, diagnostic, and therapeutic purposes. Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 10-15, 2009 An erratum to this article can be found at     

Perforin-Expressing Lymphocytes in Peripheral Blood and Decidua of Human First-Trimester Pathological Pregnancies

PROBLEM: We have shown previously that the decidua of first-trimester human pregnancy is heavily infiltrated with perforin-positive cells. The aim was to detect expression of perforin in both decidual lymphocytes (DL) and peripheral blood lymphocytes (PBL) in the first trimester of pathological pregnancies: Anembryonic pregnancy and missed abortion. METHOD: Decidual tissue from a normal pregnancy group and from pathological pregnancies was obtained by vaginal curettage. Perforin (an intracellular antigen) and the cell surface antigens CD3, CD4, CD8, CD16, CD56, CD11c, and CD45RA were quantified simultaneously by flow cytometric analysis. RESULTS: In the missed abortion group, we found: 1) a relative decrease in the frequency of both CD4⁺P⁺ cells and CD56⁺P⁺ cells as well as the mean fluorescence intensity for perforin; 2) a relative increase of CD16⁺P⁺ PBL cells; and 3) a relative increase of CD4⁺ cells in PBL compared with anembryonic pregnancy and normal pregnancy. There was also a significant relative decrease in the proportion of CD4⁺ and CD8⁺ cells among perforin-positive PBL in both anembryonic pregnancy and missed abortion. CONCLUSION: Our results show that significant decreases in the prevalence of perforin-positive lymphoid cells, their subpopulations, and mean fluorescence intensity for perforin are associated with pregnancy failure.