FMRFamide-related peptides inHymenolepis diminuta: Immunohistochemistry and radioimmunoassay

The localization of FMRFamide-related peptide (FaRP) immunoreactivity was determined during different stages of development of the rat tapewormHymenolepis diminuta. In the adult worm (14 days old), FaRP immunostaining was most intense in the scolex and concentrated in the central nervous system (cerebral ganglia and transverse commissures) and around the lips of the suckers. In the strobila, medial and lateral longitudinal nerve cords (LNCs) and ladder-like connecting commissures were the only tissue stained. Immunoreactivity in the medial LNCs of the adult tapeworms extended only to and included proglottides containing developing testis and seminal receptacle but disappeared in proglottides in which primordial ovaries were first detected. Radioimmunoassay confirmed that FaRPs were concentrated in the scolex/neck region of the adult worm (3.9±1.5 pmol mg protein^–1), whereas the lowest concentrations (0.2±0.19 pmol mg protein^–1) were recovered from the regions of the strobila containing shelled eggs. The pattern of FaRP immunoreactivity observed in 5- and 7-day-old worms was similar to that seen in adult worms, but in 2- and 3-day-old worms the pattern of immunoreactivity observed in the cerebral ganglia, transverse commissures, and LNCs differed significantly as compared with that seen in older worms. These results indicate differential utilization and/or roles for FaRPs during development and suggest both central and sensory roles in this tapeworm.     

A morphological and cytochemical study of sperm development inHymenolepis diminuta

Summary Sperm development inHymenolepis diminuta was studied by light and electron microscopy and cytochemical techniques. Spermatogenesis involves a complex series of events in which syncytia are formed. At the light-microscope level, spermatozoa formation was first observed as a threading out of the peripheral cytoplasm of the 64-nuclear syncytium. Electronmicroscopic studies confirmed and expanded the light-microscopic observations. Cytochemical and morphological observations demonstrated that mature spermatozoa ofHymenolepis diminuta lack mitochondria. Glycogen was present only in mature spermatozoa and only in the form of -particles. Acid phosphatase was demonstrated in the axoneme of mature spermatozoa.     

Ca2+ inhibition of brush border alkaline phosphatase activity inHymenolepis diminuta

The brush border membrane ofHymenolepis diminuta contains several Ca²⁺-dependent enzymes. Following our isolation of a Ca²⁺-dependent modulator protein we examined the kinetic properties of the brush border marker alkaline phosphatase from fractionated and crude tegument. We show that this enzyme is inhibited by Ca²⁺ concentrations approaching those in the calcareous corpuscles ofH. diminuta.Abbreviations CT crude tegument - FT fractionated tegument - EGTA ethylene glycol bis (amino-ethyl-ether) tetra acetic acid     

Immune response to Acinetobacter calcoaceticus infection in man

After growth in an iron-depleted chemically-defined medium Acinetobacter calcoaceticus expressed four high mol. wt outer-membrane proteins (OMPs) which were repressed under iron supplementation or in a complex laboratory medium. Immunoblotting with serum from a septicaemic patient infected with A. calcoaceticus revealed antibody binding to these iron-repressible OMPs, indicating that they were expressed in vivo, and also to the 42- and 18-Kda OMPs. Although the antibody response to the OMPs did not vary significantly during convalescence, the response to the O-polysaccharide component of lipopolysaccharide decreased significantly. However, antibodies in serum from patients with A. calcoaceticus wound infections reacted with the iron repressible OMPs and a 54-Kda antigen suggesting a difference in immune recognition between local and systemic infection.     

Immune response to infection with Mycobacterium ulcerans

Mycobacterium ulcerans is a slow-growing, acid-fast bacillus that causes chronic necrotizing skin ulcers known as Buruli ulcers. Previously reported information on immunity to this mycobacterium is limited. We examined immune responses to M. ulcerans and M. bovis BCG in patients with M. ulcerans disease and in 20 healthy control subjects (10 tuberculin test positive and 10 tuberculin test negative). Cell-mediated immunity was assessed by stimulating peripheral blood mononuclear cells (PBMC) with whole mycobacteria and then measuring PBMC proliferation and the production of gamma interferon (IFN-gamma). Humoral immunity was assessed by immunoblotting. PBMC from all subjects showed significantly greater proliferation and IFN-gamma production in response to stimulation with living mycobacteria compared with killed cells. However, PBMC from subjects with past or current M. ulcerans disease showed significantly reduced proliferation and production of IFN-gamma in response to stimulation with live M. ulcerans or M. bovis than PBMC from healthy, tuberculin test-positive subjects (P < 0.001) and showed results in these assays comparable to those of tuberculin test-negative subjects (P > 0.2). Serum from 9 of 11 patients with M. ulcerans disease, but no control subject, contained antibodies to M. ulcerans. The results indicate that patients with M. ulcerans infection mount an immune response to M. ulcerans as evidenced by antibody production, but they demonstrate profound systemic T-cell anergy to mycobacterial antigens. These findings may explain some of the distinct clinical and pathological features of M. ulcerans-induced disease.     

Stage conversion ofToxoplasma gondii in mouse brain during infection and immunodepression

The kinetics and pattern of expression of bra-dyzoite-specific proteins were studied in mouse brain during infection withToxoplasma gondii. Parasites found in the brain 6 days after ingestion of cysts were expressing only tachyzoite-specific proteins (anti-SAGl antibodies being used as a marker). Bradyzoite-specific protein (Pb36) expression was first found after 9 days in vacuoles containing mixed parasites simultaneously expressing SAG1 and Pb36 or cysts containing parasites expressing only the bradyzoite marker. Reactivation of toxoplasmosis was studied in mouse brain using cortico-steroids for immunosuppression. Parasites expressing SAGI were first found 6 days after the beginning of treatment, but a very heterogeneous pattern was found throughout the study. We simultaneously found vacuoles containing parasites expressing only SAGI or containing intermediate stages or cysts containing parasites expressing only bradyzoite proteins. A striking observation was the multiplication of cysts in foci, suggesting that the immune suppression triggered the release of parasites from preexisting cysts but that the factors inducing bradyzoite development remained fully effective in driving parasites into this pathway.     

Immune response to infection with Salmonella typhimurium in mice

Infection of mice with Salmonella typhimurium results in systemic infection and a disease similar to that seen in humans after infection with S. typhi. The innate immune system can restrict replication of S. typhimurium to a certain degree, but for effective control and eradication of bacteria, acquired immunity is essential. Salmonella infection induces the generation of specific CD4+ and CD8+ T cells, and both T cell populations are important for protection during primary and secondary responses, although the mechanisms underlying T cell-mediated protection are not yet completely understood. Infection with S. typhimurium also results in a strong antibody response to Salmonella antigens and, in contrast to most other intracellular bacteria, this antibody response participates in protection. In summary, the response to S. typhimurium involves both T and B cell-mediated immunity, and mechanisms mediated by both lymphocyte populations are important for control of primary infection and protection against secondary infection.     

Immune response to persistent mycobacterial infection in mice.

Mycobacterium marinum has been recommended as a possible model of M. leprae for use in laboratory studies of antileprosy immunity. M. marinum introduced into the footpads of normal mice underwent a steady decline in viability, with less than 1% survival after a 30-day period. Small numbers of viable bacilli were recovered from the footpads of these mice up to 12 months later. Similarly, mice infected with M. simiae exhibited bacterial populations that persisted for up to 18 months with little change in viability. Injection of M. simiae into the footpads was followed by an extensive redistribution of the organisms in the tissues. Eventally, bacterial counts for footpads and draining lymph nodes stabilized, with small numbers of bacilli still present in the footpads 18 months later. Persistent growth, with little sign of any immune response, was also observed in mice infected with several strains of M. avium, as well as with one strain of M. intracellulare. Other strains of M. intracellulare, as well as M. vaccae and M. nonchromogenicum, failed to establish persistent infections in normal mice, regardless of whether they were introduced by an intravenous or subcutaneous (footpad) route. The relevance of these findings is discussed in relation to antileprosy immunity in experimental animals and in humans.     

Immune response to extracellular and somatic antigens in streptococcal infection and sequelae

The aim of the present study is to define the temporal relationships of the IgM and IgG responses to Streptococcal group A carbohydrate (CHO) in rabbits and in man. Rabbits were immunized with group A streptococci and the development of anti-group A carbohydrate (ACHO) was studied. ACHO which appeared one week after the beginning of immunization belonged to the 19S class of immunoglobulins (IgM). A two- to four-fold rise in ACHO titers and immunoglobulins of the 7S class (IgG) were observed after two weeks. Three weeks after the beginning of immunization, the ACHO titer was at a maximum. In the following months no further rises in titer were seen, and the antibodies belonged mostly to the IgG class. IgM and IgG responses to streptococcal CHO and to extracellular antigens in patients with pharyngitis, acute rheumatic fever (ARF), and acute glomerulonephritis (AGN) were studied. Higher values of IgM were found in pharyngitis and AGN sera than in ARF sera, probably reflecting the interval between streprococcal infection and time of bleeding. ACHO antibodies persisted in patients' sera for long periods and belonged to IgG and IgM. This suggests a continuous, rather than a persistent, production of ACHO.     

Immune complexes in serum of rats during infection withPlasmodium berghei

Large amounts of immune complexes were present in the serum of infected rats early in infection when parasitemias were low. As the infection progressed and parasitemia increased and then decreased, the amounts of immune complexes in the serum also fell. This result suggests that increased efficiency of complex clearance was an important factor in determining the levels of immune complexes in the serum. In high performance liquid chromatography (HPLC), the complexes in the serum migrated as a peak with material of 350 kDa and greater in mass. They sedimented in a sucrose gradient as a band with a sedimentation coefficient of 22s, which was calculated to yield a mass of approximately 1100 kDa. Immunoelectrophoresis and radial immunodiffusion showed that IgG was the major immunoglobulin in the complexes. As the IgG content of the complexes increased, the levels of complexes in the serum generally decreased. HPLC analysis of precipitated complexes suggested that they contained loosely bound albumin. Serum proteins were affected by the infection. A depletion of free immunoglobulin was observed during the initial period of immune complex formation.     

Immune responsiveness associated with experimental Encephalitozoon intestinalis infection in immunocompetent rats

PURPOSE: Microsporidial infections have been recognized as an increasingly important infection in immunocompromized patients, particularly those infected with HIV/AIDS. This study was designed to study immune responses associated with experimental Encephalitozoon intestinalis infection in immunecompetent rats. MATERIALS AND METHODS: Thirty-four rats in 3 groups, A (Control), B (Intraperitoneal) and C (Oral) were given injections of 0.5 ml of 2 x 10(6) of purified spores of Encephalitotozoon intestinalis spores and were observed for serum specific IgG for 21 days using both Direct and Indirect ELISA. RESULTS: In indirect ELISA, specific lgG were detected on days 7, 14 and 21 for the group B rats and on day 21 for group C and in direct ELISA method, specific lgG were detected in-group B rats on days 7 and 21, for group C rats on day 21 only, while in the control rats, specific lgG were not detected. There was no significant difference between the direct and indirect methods (df=1, X(2), P>0.05). E. intestinalis was observed in stool samples of rats in 1/12 (08.33%) on days 14 and 21 in group B and in 4/10 (33.33%), 3/10 (25.00%) and 2/10 (16.67%) on days 7, 14 and 21 respectively in group C. In-group, A which is the control rats, no microsporidia were observed on days 0, 7, 14 and 21. CONCLUSIONS: There were no changes in the T-lymphocyte counts of rats prior to and after inoculation with spores. Extensive lesions were observed along the intestinal walls especially on the middle and lower sections of group C rats only.     

Immune response and latent infection after topical treatment of herpes simplex virus infection in hairless mice.

Treatment of herpes simplex virus (HSV)-infected hairless mice with a 2% phosphonoacetic acid (PAA) ointment prevented the appearance of virus-induced skin lesions and subsequent central nervous system (CNS) involvement. Treatment started 24 h after infection significantly reduced the intensity of the skin lesions and also prevented CNS involvement. After four to six applications of PAA ointment, a moderate skin erythemia developed, followed by scaling and complete healing 7 days after cessation of the treatment. Mice treated early after HSV infection had low or undetectable levels of virus-specific antibodies but were completely resistant to reinfection. Early treatment prevented the development of a latent ganglionic infection, but treatment initiated 24 h after infection could not prevent the establishment of the latent infection. PAA-treated and HSV-infected mice with nondetectable levels of antibodies did not develop, with a single exception, a latent ganglionic infection unpon reinfection. The cell-mediated immune response determined by levels of [14C]thymidine incorporation in Ficoll-Hypaque-purified spleen lymphocytes cultures was low in PAA-treated mice; it increased slightly after challenge infection but was strong in mice that proved to harbor a latent HSV infection in the ganglia.     

Influenza virus infection of the guinea pig: Immune response and resistance

Guinea pigs were inoculated by intranasal inoculation with unadapted, influenza virus A/England/42/72, and virus was recovered from nasal washings between 3 and 10 days post-inoculation. Infected animals did not exhibit a febrile response to infection, did not produce local antibody and produced only relatively low levels of serum antibody. However, they developed delayed-type hypersensitivity to influenza virus, demonstrable by both skin tests and macrophage migration inhibition tests, which was similar to that of man. The relevance of the influenza virus specific delayed hypersensitivity in immunity to infection was examined in this model. Guinea pigs previously infected with virus or passively immunized with hyperimmune serum were relatively resistant to reinfection with influenza virus A/England/42/72. Inoculation of guinea pigs with spleen cells from immune donor animals, together with or without immune serum, did not give or enhance resistance to challenge virus infection. The results do not suggest a role for delayed hypersensitivity response in immunity to influenza virus infection.     

Immune response in Wistar rats with high and low level of situational anxiety

A large sample of Wistar rats was divided into 2 groups of high-anxiety and low-anxiety animals by the time spent in the open arms of the elevated plus-maze. This selection was based on the criterion of time (low-anxiety animals, not less than 10 sec; high-anxiety animals, not more than 2 sec). Immunization with T-dependent antigen was performed on the day of behavioral testing. The number of rosette-forming cells in high-anxiety rats significantly decreased on day 5 after immunization. A genetically determined relationship probably exists between low activity of the immune response and high level of reactive anxiety. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 11, pp. 549–551, November, 2007     

Coproantigens in gut tapeworm infections:Hymenolepis diminuta in rats

A capture enzyme-linked immunosorbent assay (ELISA) for the detection ofHymenolepis diminuta coproantigen in detergent-solubilised faecal supernatants was developed. The assay was sensitive to 400 ngH. diminuta protein/ml faecal supernatant and to 30 ng/ml in PBS 0.3% Tween. Faecal antigen levels started to rise on day 7 post infection and peaked on day 20. Following drug treatment of infected rats with 10 mg/kg praziquantel, there was an initial steep rise in faecal antigen levels, followed by a decline to background levels for uninfected animals by day 7 post treatment. When assayed against faecal supernatants prepared fromH. microstoma, H. citelli, Diphyllobothrium ditrenum, Necator americanus, Strongyloides ratti andNematospiroides dubius infections as well as uninfected animals, all results from these groups were 2 SD below those for animals infected withH. diminuta. The application of such a test to important intestinal cestodiases in man is discussed.     

Does normal lymphocyte DNA synthesis in response to PHA exclude cell-mediated immunodepression?

PHA stimulation assay was the first in vitro method for evaluating the T-cell function, and this T-cell proliferative response has been routinely used to discriminate between normal subjects and patients with deficiency in cell-mediated immunity. However, [3H]thymidine incorporation into lymphocyte DNA can be studied by using additional in vitro assay methods since they measure different lymphocyte activation pathways. In the present study we selected three different tests to investigate the reliability of this single approach: PHA induced lymphocyte DNA synthesis; T lymphocyte DNA synthesis to anti-T3 monoclonal antibody (OKT3); autologous mixed lymphocyte reaction (AMLR). In addition, IL-2 receptor expression on the membrane of T-cell stimulated in AMLR both with PHA and anti-T3 was evaluated. This study was performed in various groups of subjects: normal young controls, aged healthy individuals, and patients with Alzheimer's disease (AD), Retinitis Pigmentosa (RP), and with cell-mediated immunodeficiency and clinical evidence of recurrent viral infections (ID). The data reported herein show heterogeneity of results in each group studied and demonstrate the necessity of employing more than one laboratory test for the routine evaluation of T-cell-mediated immunity.     

Immune response after experimental allergic encephalomyelitis in rats subjected to calorie restriction

Male Lewis rats (6 weeks-old) were submitted to a calorie restriction equivalent to 33% or 66% of food restriction. Fifteen days later, groups of 7 animals were injected with complete Freund's adjuvant plus spinal cord homogenate (SCH) to induce experimental allergic encephalomyelitis (EAE) or with complete Freund's adjuvant alone. EAE was defined solely on clinical grounds. Rats were assessed daily for clinical signs of EAE and were killed on day 15 after immunization. Both diet and SCH injection diminished body weight significantly. In contrast to rats receiving a normal diet or a 33% calorie-restricted diet, rats subjected to severe calorie restriction did not exhibit clinical signs of EAE. Concomitantly with the lack of disease manifestation, 66% of calorie-restricted rats injected with SCH showed significantly less splenic and lymph node mitogenic response to concanavalin A (Con A) and a higher splenic response to lipopolysaccharide. Fewer splenic, lymph node and thymic CD4⁺ cells, greater numbers of splenic and lymph node CD8⁺ and CD4⁺- CD8⁺ cells, and fewer splenic T, B and T-B cells, and lymph node and thymic B and T-B cells were observed. There was impaired interferon (IFN)-γ production occurred in the three examined tissues. The results are compatible with the view that the acute phase of EAE can be curtailed by severe calorie restriction, presumably through impaired IFN-γ production.     

Immune response of rats to subcellular fractions of isologous brain and liver

The titres of complement fixing antibodies in the sera of rats injected with the soluble fraction of rat brain emulsified in Freunds' complete adjuvant (FCA) were usually below 10.

In contrast, injections of the nuclear, mitochondrial or microsomal fractions of rat brain in FCA were followed by the appearances of heat stable complement fixing and haemagglutinating antibodies within a few days, the highest antibody levels being attained about 6 days after the first injection of a particulate fraction. The microsomal fraction was the most efficient particulate antigen. After 2 weeks of immunization about 60 per cent of the complement fixing activity of the antisera was due to 19S antibody and the remainder to 7S antibody.

The response to injections of the nuclear and microsomal fractions of rat liver followed a similar time course but produced levels of complement fixing antibodies that were consistently lower than those engendered by the corresponding brain antigens.

     

Immune response to hepatitis A virus capsid proteins after infection.

This study was undertaken to determine the immune response of humans to viral capsid polypeptides of hepatitis A virus (HAV) after natural infection, which is very important for vaccine development. Antiviral capsids in 73 serum samples from patients with acute and chronic HAV infections were analyzed by immunoblotting against individual HAV capsid polypeptides (VP1, VP2, VP3, and VP4) by using a cell culture-based HAV antigen. For reference, total anti-HAV immunoglobulin G (IgG) and anti-HAV IgM were also determined by radioimmunoassay. As a result, a dominant immune response against VP1 (98% IgG, 94% IgM) was found in the acute phase. However, many other sera also reacted with VP0 (88% IgG; 35% IgM) and VP3 (81% IgG and 29% IgM). In contrast to the acute phase, anti-VP1, anti-VP0, and anti-VP3, IgG antibodies against all three viral proteins (29, 29, and 73% respectively), especially those against VP3, were found years after onset of HAV disease and over long periods in the sera of hepatitis patients. These results suggest that antibodies for capsid polypeptides are present over an extended period in the sera of HAV-infected patients. They are likely of importance in maintaining long-term immunity.     

Pathogenesis and antibody response to a cytomegalovirus infection in newborn rats

The present study described the kinetics of Rat cytomegalovirus (RCMV) infection in newborn rats by monitoring infectious virus and viral antigens in various organs, viral DNA in the blood (DNAemia) and antibody response. These parameters were evaluated quantitatively using double-antibody sandwich ELISA (DAS-ELISA), real-time PCR, indirect ELISA and virus infectivity assay. For the first time DAS-ELISA was used for detection of RCMV antigen directly from organ samples. The relationships between the presence of viral antigens in the infected organs and antibody levels were established by the Spearman's rank test. It was found that the virus was present in the blood, spleen, liver, lungs, and kidneys earlier than in the salivary glands. Furthermore, the early immunity of the newborn rats led to a delayed seroconversion. We suggested that the prolonged presence of the virus in salivary glands could augment the antibody response that conversely might be responsible for a reduction of viremia. This study expanded our understanding of RCMV pathogenesis leading to improved therapeutic and preventive treatment regimens particularly for the neonatal Human cytomegalovirus (HCMV) infections. Additionally, the detection procedures developed in this study such as DAS-ELISA and real-time PCR could serve as alternative techniques for rapid screening of large number of samples.