Real-time PCR detection of bacteria belonging to the Firmicutes Phylum.

Members of the bacterial Phylum Firmicutes occupy a wide range of habitats and can be either beneficial or detrimental in diverse settings, including food- and beverage-related industries. Firmicutes are responsible for the vast majority of beer-spoilage incidents and, as such, they have a substantial financial impact in the brewing industry. Rapid detection and identification of a bacterium as a Firmicutes is difficult due to widespread genetic transfer and genome reduction resulting in phenotypic diversity in these bacteria. Here we describe a real-time multiplex PCR to detect and differentiate Firmicutes associated with beer-spoilage from non-Firmicutes bacteria that may be present as benign environmental contaminants. A region of the 16S rRNA gene was identified and predicted to be highly conserved amongst, and essentially specific for, Firmicutes. A real-time PCR assay using a hydrolysis probe targeting this region of the 16S rRNA gene was experimentally shown to detect ten genera of Firmicutes known to be beer spoilers, but does not cross-react with eleven of twelve non-Firmicutes genera which can periodically appear in beer. Only one non-Firmicutes species, Zymomonas mobilis, weakly reacted with the Firmicutes probe. This rPCR assay has a standard curve that is linear over six orders of magnitude of DNA, with a quantitation limit of DNA from <10 bacteria. When used to detect bacteria present in beer, the assay was able to detect 50-100 colony forming units (CFU) of Firmicutes directly from 2.5 cm membranes used to filter 100 ml of contaminated beer. Through incorporation of a 4.7 cm filter and an overnight pre-enrichment incubation, the sensitivity was increased to 2.5-10 CFU per package of beer (341 ml). When multiplexed with a second hydrolysis probe targeting a universal region of the 16S rRNA gene, the assay reliably differentiates between Firmicutes and non-Firmicutes bacteria found in breweries.

Circulating immune complexes in rats bearing 6-thioguanine-resistant variants of the 13762 mammary adenocarcinoma.

The relationship between immune complex (IC) formation and tumor cell metastatic potential was investigated in rats inoculated in the footpad with parental 13762 mammary adenocarcinoma cells or 6-thioguanine-resistant (TGR) variant cells. These cell lines are either highly metastatic (13762), nonmetastatic (TGR), or occasionally metastatic ( TGRrev , TGRrevM ). The 13762 TGR rat tumor model thus provides the opportunity to examine host immune responses to tumor cells of different phenotypes, but derived from the same parent tumor line. IC levels were low in 13762 tumor-bearing rats. In contrast, animals with TGR tumors had high levels of ICs in their sera, while animals bearing TGRrev and TGRrevM tumors had intermediate levels of ICs. In this rat tumor model system, IC formation is inversely related to the metastatic potential of the tumor lines.

Circulating immune complexes and immunoglobulin M-class rheumatoid factor in rats bearing mammary adenocarcinomas which vary in ability to metastasize.

In order to explore whether immune complex (IC) formation and immunoglobulin M-class rheumatoid factor (RF) synthesis are related to tumor progression, solid-phase enzyme immunoassays were used to test for ICs and RF in rats bearing three different syngeneic mammary adenocarcinomas. The mammary adenocarcinoma cell lines used produced either extensive metastasis (13762), metastasis in only a proportion of the animals given injections (R3230AC), or no metastasis (DMBA8). DMBA8 and 13762 tumor-bearing rats developed only low levels of circulating ICs. Of 18 animals bearing R3230AC tumors, four developed palpable lymph node metastasis (macrometastasis), while another five showed evidence of metastasis only upon histological examination (micrometastasis). R3230AC tumor-bearing animals which did not develop metastasis were found to have significantly higher IC levels than those rats with metastasis. Several sera from rats bearing R3230AC tumors were fractionated by molecular sieve chromatography. Most of the ICs in these sera were 7S to 19S in size. Significant RF synthesis occurred only in rats bearing R3230AC tumors and only during terminal tumor growth. These results show that IC formation and RF synthesis varies in animals bearing different mammary adenocarcinomas.

One-way humoral immune cross-reactivity between bovine spinal cord protein and bovine myelin basic protein.

Whether bovine myelin basic protein (BP) and bovine spinal cord protein (SCP) cross-react at the humoral immune level was assessed with a sensitive solid-phase enzyme immunoassay. We found that a hyperimmune anti-SCP serum reacted strongly with SCP and cross-reacted nearly as well with BP. A hyperimmune anti-BP serum reacted only with BP. Antigenic competition analysis revealed that SCP and BP both inhibited binding of the hyperimmune anti-SCP serum to solid-phase adsorbed SCP and BP, while only BP inhibited binding of the hyperimmune anti-BP serum to solid-phase adsorbed BP. Finally, BP cross-reactivity antibodies were present in early bleedings from rabbits immunized with SCP that had been passed through an anti-BP immunosorbent column. These results clearly show there is a one-way humoral immune cross-reactivity between SCP and BP which goes in the direction of SCP to BP.

Herpes simplex virus subunit antibodies in patients with Parkinson's disease.

Serum IgG antibodies against herpes simplex virus (HSV) type 1 capsid, envelope, and excreted antigens in 52 patients with idiopathic Parkinson's disease, and in their age- and sex-matched controls, were assayed with a solid-phase radioimmunoassay. When compared with the controls, patients with Parkinson's disease were found to have a substantially increased antibody response against each of the HSV subunit antigens tested. The increased antibody response in patients with Parkinson's disease was not associated with the occurrence of recurrent HSV infections, since the difference in antibody levels was most evident when comparing patients without recurrent HSV infections with their respective control group. Consequently, the increased HSV antibody response in patient with Parkinson's disease might depend on some antigenic stimulation other than ordinary recurrent HSV infections, or alternatively, on the generally enhanced immunological reaction of the patients against HSV.

Immunotherapy of the rat 13762SC mammary adenocarcinoma by vaccinia virus augmentation of tumor immunity.

We studied whether vaccinia virus (VV) functioned as an immunogenic carrier in augmenting anti-tumor immunity in rats bearing a syngeneic metastatic tumor. The primary tumor was induced by injecting 10(6) 13762SC mammary adenocarcinoma cells subcutaneously into the right hind footpad of Fischer 344 rats. A concomitant anti-tumor response is induced by the tumor as demonstrated by the inhibited growth of a second tumor challenge given in the contralateral footpad 3-15 days later. Attempts were made to increase the concomitant immunity by injecting tumor-bearing animals intramuscularly with irradiated, VV-infected or uninfected 13762SC cells without adjuvant. Provided the immunotherapy was done within 5 days of the tumor challenge, administration of 10(6)-10(7) irradiated, VV-infected 13762SC cells resulted in significantly slower tumor growth, or led to complete tumor regression, compared to control animals given no treatment. In contrast, tumor growth in animals given only VV or given irradiated, uninfected 13762SC cells, alone or mixed with VV, was the same as that in control animals. Kinetics of early primary tumor growth were predictive of a longer-term anti-tumor effect. Rechallenge of 13762SC tumor-cured animals with either the homologous or with a heterologous syngeneic mammary adenocarcinoma showed the animals to be specifically 13762SC tumor-resistant, since only rats challenged with the heterologous mammary adenocarcinoma developed progressive tumors. We interpret these results to mean that early immunotherapy with irradiated, VV-infected 13762SC cells enhances an on-going anti-tumor immune response sufficiently to cause rejection of the primary tumor and any metastases that have occurred. We also believe that later immunotherapy with irradiated, VV-infected cells has no effect due to tumor-induced immunosuppression becoming paramount.

Antibodies to coronaviruses OC43 and 229E in multiple sclerosis patients.

Multiple sclerosis (MS) and matched control sera had similar antibody titers to coronaviruses OC43 and 229E when tested by a radioimmunoassay method. In contrast, cerebrospinal fluid from MS patients contained coronavirus antibodies more frequently and in higher titers than matched controls. Intrathecal antibody synthesis to OC43 and 229E viruses was detected in 41% (9/22) and 26% (7/27) of MS patients, respectively, but was not found in any of the neurologic control patients. This intrathecal antibody synthesis may mean that coronaviruses play an etiologic or pathogenic role in MS. Alternatively, intrathecal synthesis of coronavirus antibodies may be but part of a generalized and variable intrathecal antibody synthesis that is typical for MS patients.

Porcine C1q and the solid-phase immunoassay of human immune complexes.

Experiments were undertaken to determine if porcine C1q could replace human C1q in the solid-phase immunoassay of human immune complexes (ICs). Porcine C1q was obtained by a two-cycle precipitation method involving dialysis against chelating agents in low ionic strength buffer. C1q was adsorbed to polystyrene beads and in vivo- or in vitro-formed ICs binding to the solid-phase C1q were detected with 125I-labeled or horseradish peroxidase-conjugated anti-human gamma antibodies. Unfractionated, heat-aggregated human gamma globulin (delta IgG) could be detected at 20 ng/ml when diluted in buffer only. The detection threshold changed to 40-80 ng delta IgG/ml when the assay was run with buffer containing normal human serum diluted 1: 1000 (the serum dilution used for detecting natural ICs). Analysis of systemic lupus erythematosus sera revealed that 60% contained highly significant levels of ICs (binding greater than or equal to 3 S.D. above the mean of controls). Comparison with platelet aggregation test results revealed a highly significant correlation between the two methods (P less than 0.0001), even though each assay detected ICs in several serum specimens negative in the other test. These results demonstrate that porcine C1q can functionally replace human C1q in the solid-phase immunoassay of human ICs. Since porcine blood is normally a waste product of the meat-processing industry, it is an obvious source of easily isolated C1q for use in such an assay.

In vitro proliferation of lymphocytes from cyclophosphamide-pretreated mice immunized with antigen mixed with dimethyl dioctadecyl ammonium bromide.

A blastogenesis assay employing lymphocytes from cyclophosphamide-pretreated mice immunized with antigen mixed with the immunopotentiating compound dimethyl dioctadecyl ammonium bromide is described. The model antigen used for determining the assay parameters was inactivated purified measles virus. The optimal time for removal of immunologically primed T cells was 7 days after immunization of mice pretreated 2 days previously with 200 mg of cyclophosphamide/kg. The peak lymphoproliferative response was found to occur after 3-5 days in culture, depending on the concentration of antigen used. Although fetal bovine serum and syngeneic mouse serum each worked well as a medium supplement, significantly higher specific and lower non-specific lymphoproliferation were obtained when the mouse serum was used. Most of the lymphocytes responding to antigen were of the Ly 1.2 phenotype. Specificity of the blastogenic response was shown by a lack of cross-reactivity among measles virus, herpes simplex virus type 1 and vesicular stomatitis virus antigens. This approach to a mouse blastogenesis assay involves an easy way to induce strong T cell priming in mice, while still providing an assay which has an ideal combination of low non-specific and high antigen-specific responses.

Polystyrene balls as the solid-phase of a double-antibody radioimmunoassay for human serum albumin.

Polystyrene balls have been incorporated as the solid-phase of a model double-antibody radioimmunoassay for human serum albumin. Purified IgG from the secondary antiserum is adsorbed on the 6.4 mm diameter balls. The solid-phase secondary antibody is then used to separate primary antibody bound iodinated antigen from unbound antigen. The secondary antibody coated polystyrene balls are easily prepared and manipulated; several hundred sample dilutions can readily be processed in a single assay. Assay background values of 1.5% or less are consistently obtained without extensive or special washing procedures.

Solid-phase radioimmunoassay of herpes simplex virus IgG and IgM antibodies.

A solid-phase radioimmunoassay for detection of herpes simplex virus-specific IgG and IgM antibodies in human serum specimens is presented. Virus antigen is adsorbed on polystyrene balls and antibodies which attach to the antigen are detected by 125I-labeled antihuman-gamma or antihuman-mu immunoglobulins. A total of 76 specimens have been tested. The appearance of virus-specific IgG and IgM antibodies in primary herpetic infections was readily demonstrated. When serum samples from patients with past exposure to herpes simplex virus were tested, endpoint titers of virus-specific IgG antibodies were found to be 8 to 2048 times higher than titers determined by a complement fixation test. Apparent cross reactivity with varicella-zoster virus was observed in the present radioimmunoassay.

Adherent cells suppress measles and herpes simplex I virus-induced blastogenesis of multiple sclerosis lymphocytes.

Viral antigen-induced blastogenesis of lymphocytes from multiple sclerosis (MS) patients was investigated to determine if the responses were actively suppressed. We found that depletion of adherent cells increased measles and herpes simplex I virus antigen-induced transformation of MS lymphocytes. Addition of indomethacin to cultures of unfractionated MS lymphocytes also caused an increase in viral antigen-induced responses. These two facts, plus finding that the cell type mediating the immunosuppression did not rosette with 2-aminoethylisothiouronium bromide hydrobromide-treated sheep red blood cells, indicate that the suppressed T-cell responsiveness of MS patients is caused by macrophages rather than T-cells. These results have a major implication for the divergent published data on blastogenesis induced in MS patient lymphocytes by specific antigens, viral or otherwise. We feel the inconsistencies may simply have arisen from the different lymphocyte isolation and washing procedures used giving variable levels of macrophages and, hence, variable levels of immune suppression. This clearly suggests that induction of blastogenesis in MS patient lymphocytes by a wider array of infectious agent antigens and by various neural antigens should now be undertaken using adherent cell-depleted lymphocytes.

Prior infection by respiratory syncytial virus or parainfluenza viruses augments virus-specific IgG responses induced by the measles/mumps/rubella vaccine.

We found previously that immunizing cyclophosphamide-treated mice with one Paramyxoviridae virus mixed with dimethyl dioctadecyl ammonium bromide induces T cells which apparently also recognize other Paramyxoviridae viruses. This finding and the fact that respiratory syncytial virus (RSV) and parainfluenza viruses (PIVs) infect children early in life led us to ask if prior RSV or PIV infections influence the antibody response to measles and mumps vaccine viruses. Detection of virus-specific IgG in serum specimens collected randomly or at defined times after measles/mumps/rubella (MMR) vaccination was done with solid-phase enzyme immunoassays. The antibody-binding data obtained were converted to serum antibody titers by an immunoassay curve-fitting computer program. Prior infection by RSV and PIVs correlated with an augmented IgG response not only to measles and mumps virus, but also to rubella virus. Furthermore, the augmentation was greater for responders below the median response. These data show that common early childhood viral infections can influence immunity induced by the MMR vaccine.

T cell cross-reactivity among viruses of the paramyxoviridae.

Blastogenesis and delayed-type hypersensitivity assays were used to examine mouse T cell responses to five viruses representing the three genera of the Paramyxoviridae. Cross-reactive T cell responses were observed in a lympho-proliferative assay for measles, mumps, respiratory syncytial, canine distemper and parainfluenza type 3 virus. Confirmation of T cell cross-reactivity among measles, mumps and respiratory syncytial virus was obtained with a delayed-type hypersensitivity test. These results show that T cell cross-reactivity is common for Paramyxoviridae viruses, even though these viruses show virtually no inter-genus antibody cross-reactivity. The cross-reactivity among respiratory syncytial, measles and mumps virus at the T cell level may have implications for usage of the attenuated measles/mumps/rubella (MMR) vaccine. Respiratory syncytial virus is contacted by many children before they receive the MMR vaccine and T cells induced by respiratory syncytial virus may influence subsequent development of immunity to measles and/or mumps virus.

IgM-class rheumatoid factor interference in the solid-phase radioimmunoassay of rubella-specific IgM antibodies.

The interference of IgM-class rheumatoid factor (RF) in the solid-phase radioimmunoassay (RIA) of rubella virus IgM antibodies was studied. Acute rubella infections did not significantly activate RF. False-positive rubella antibody results were obtained, however, when patients with raised RF levels were tested. If a low rubella IgG antibody titre was present, a high level of RF was required to cause a false-positive IgM result; conversely, in sera with high IgG titres, only a low level of RF was required for interference. Although the false-positive IgM titres obtained were generally low, thet did show a positive correlation to both RF levels and rubella IgG titres. False-positive results were successfully avoided by removing the RF by absorption with heat-aggregated human gamma globulin. The absorption procedure did not affect true rubella IgM antibody titres.

Release of Candida albicans yeast antigens upon interaction with human neutrophils in vitro.

Candida albicans is the leading cause of invasive candidosis. As conventional tests do not reliably detect invasive infection, attention has turned to the detection of C. albicans antigens circulating in blood. As antigen tests for invasive candidosis could be improved if C. albicans antigens released upon phagocytosis were defined, this study was undertaken to characterise antigens released during the interaction of yeasts and human neutrophils in vitro. An enzyme immunoassay developed previously to detect what were believed to be predominantly C. albicans cytoplasmic antigens in patients with invasive candidosis was used to follow the neutrophil-mediated release of yeast antigens. Serum opsonisation enhanced antigen release, which was rapid and essentially complete by 1 h. When fresh C. albicans yeasts were added to medium from cultures of neutrophils plus yeasts or neutrophils plus latex beads, additional yeast antigens were released. Medium from neutrophils plus yeasts or from yeasts alone had similar immunoblot patterns with rabbit antibodies to a C. albicans cytoplasmic antigen preparation, with the reactive antigens generally being of higher mol. wt than the reactive antigens in the antigen mixture used for preparation of the antiserum. The two supernates also had similar immunoblot patterns with rabbit anti-C. albicans cell-wall mannan antibodies. These results suggest that yeast surface antigens are released quickly during phagocytosis by neutrophils. Detection of such yeast surface antigens, possibly together with selected yeast cytoplasmic antigens, should improve the sensitivity of C. albicans antigen assays.

The use of bacteriophages to differentiate serologically cross-reactive isolates of Klebsiella pneumoniae.

In serological typing of Klebsiella pneumoniae strains from human, equine and environmental sources, the capsular identity of many isolates could not be determined because of serological cross-reactivity. A panel of 91 bacteriophages able to lyse each of the 77 capsular serotypes of K. pneumoniae was isolated and tested for the ability to distinguish between strains in a collection of 17 clinical isolates of K. pneumoniae which exhibited cross-reactivity with two or more capsular type sera. Most isolates could be assigned a capsular type by performing a simple streak test with bacteriophage, although some required the application of an efficiency of plating analysis to discern capsular type. Bacteriophage typing was found to be an effective, inexpensive and clinically practical adjunct to serotyping in distinguishing serologically cross-reactive K. pneumoniae isolates, irrespective of their origin.

Radioimmunoassay of herpes-simplex and measles virus antibodies in serum and cerebrospinal fluid of patients without infectious or demyelinating diseases of the central nervous system.

A solid-phase radioimmunoassay was used to detect IgG antibodies against herpes-simplex virus antigens (capsid, envelope and excreted) and against measles virus antigen in serum and cerebrospinal fluid (CSF) specimens of 61 patients with no evidence of infectious or demyelinating disease of the central nervous system. Quantitative determinations of IgG and albumin in serum and CSF were also performed. Of the 61 serum and 61 CSF samples tested, 57 and 56 respectively contained antibodies against subunit antigens of herpes simplex virus. Antibody against measles virus was found in 59 serum and 47 CSF specimens. A positive correlation (P less than 0-001) was found between each of the four serum to CSF antibody ratios and the serum to CSF total IgG ratios. This indicated that the distribution of antiviral IgG antibodies in serum and CSF normally follows the distribution of total IgG. The ratios between viral antibody in serum and CSF were also correlated with albumin ratios (P less than 0-05). An inverse relation (P less than 0-001) was found between the age of the patients and their serum to CSF albumin ratios, but not their IgG ratios, suggesting that the albumin ratio is a useful indicator of a blood brain barrier lesion and that the IgG ratio should be used in evaluating disturbed antibody ratios.

Replication of measles virus in the human plasma cell leukemia-derived LICR-LON-HMy2 cell line.

Measles virus is usually grown in human or monkey fibroblast cells. We now show that LICR-LON-HMy2 (LL2) cells, a human plasma cell leukemia-derived line which grows in suspension culture, will permissively support replication of measles virus to an extent achievable with Vero cells. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of measles virions produced by LL2 cells showed a polypeptide pattern typical of measles virus. As well, measles virus-infected LL2 cells, like infected Vero cells, were found to secrete large amounts of virus hemagglutinin, but not other virus proteins. We thus conclude that LL2 cells can be effectively used to produce milligram amounts of measles virus and that virus-clarified culture medium from measles virus-infected LL2 cells is a potential source for purifying virus hemagglutinin.

Coincidental fluctuations of humoral immunity and clinical progression in a patient with subacute sclerosing panencephalitis.

Serum and cerebrospinal fluid were obtained from a 6-year-old male with subacute sclerosing panencephalitis (SSPE). Specimens were collected over a 9-month period beginning in the unusually acute phase and ending in a more quiescent phase of the disease. Immune complexes, auto-antibodies and viral antibodies were measured by radio-immunoassays. Fluctuations in these humoral immune parameters coincided with cessation of the acute phase of this disease. The results show that neurological changes in SSPE patients can be reflected in immune responses within both the peripheral circulation and the central nervous system.