Electromyographic activity of knee stabilizer muscles during six different balance board stimuli after anterior cruciate ligament surgery.

The purpose of this study was to compare the electrical activity of the knee stabilizers, in patients with ACL (anterior cruciate ligament) reconstructed and uninjured individuals during different balance board stimuli. Eleven post-surgery individuals and eleven uninjured controls participated in the study. The muscular activity of the vastus medialis obliquus, vastus lateralis, semitendinosus, biceps femoris and gastrocnemius medial were analyzed by surface electromyography during the execution of six different balance board activities. All electromyographic data were reported as percentage of RMS mean values obtained in maximal voluntary isometric contractions (MVIC) for each muscle. When comparing the individuals with ACL reconstructed and uninjured controls, minor electromyographic activity was observed (MVIC %) for all the muscles in the surgery group (P < 0.05), however, when comparing each exercise between the groups, a statistically significant difference for vastus lateralis was demonstrated in the floor exercise (P = 0.02) and for gastrocnemius on the round board (P = 0.04). Individuals ACL reconstructed presented a decrease in muscular activity during different balance board stimuli, which suggests that compensatory alterations after ACL may still exist even after a surgery to repair an ACL rupture.

Antisperm antibodies detected by mixed agglutination reaction and immunobead test are not associated with chronic inflammation and infection of the seminal tract.

The association between chronic inflammatory/infectious diseases of the male reproductive tract and the presence of antisperm antibodies (ASA) in semen is still controversial. We compared the results of the mixed agglutinin reaction (MAR) test and immunobead test for detecting ASA type IgG and IgA in 133 patients attending our special outpatient department for andrological infections and evaluated the differences in the detection rate of ASA. Patients were divided into three groups: a study group that included 79 patients with symptomatic nonacute inflammatory/infectious diseases of the seminal tract, a control group (n = 44) and a third group of men with a history of successful vasectomy reversal (n = 10). The two tests correlated in a statistically significant manner for the detection of IgG and IgA in all groups. The overall positive detection rate of clinical significant levels of IgG and IgA was 2.5% and 1.3% (respectively) in the patients with inflammation/infection of the seminal tract. No statistical significant difference in the detection rate of ASA levels between the inflammatory/infectious group and the controls was detected. The results of the MAR test and immunobead test have a statistical significant correlation and their results provide evidence that there is no association between inflammatory/infectious diseases of the male reproductive tract and the presence of ASA in semen.

Biochemical and pharmacological investigations of selected cyanobacteria.

Cyanobacteria are a very old group of prokaryotic organisms that produce a variety of secondary metabolites with antibiotic, algicide, cytotoxic, immunosuppressive and enzyme inhibiting activities. In the last decades structures of pure compounds have been determined as phenols, peptides, alkaloids or terpenoids (Falch, 1996). Screening of lipophilic and hydrophilic extracts from cultured cyanobacteria or waterbloom material, isolated from German lakes and the Baltic sea for antiviral, antibiotic, immunomodulating and enzyme inhibiting activity in different in vitro systems revealed strains with interesting effects. These strains were cultivated in 45 litre photobioreactors to produce enough biomass for bioassay-guided isolation of the active substances. First results characterising active substances are reported.

Potentiation of HIV envelope glycoprotein and other immunogens by endotoxin (ET) and its molecular fragments.

The structural requirements for the immunopotentiating (adjuvant) effect of endotoxin (ET) were investigated. Mild hydrolysis (0.2 N acetic acid at 95 degrees C) was applied to various ET preparations and the lipid rich (Lipid A) and polysaccharide-rich (PS) preparations obtained were tested as adjuvants on three immunogens: sheep red blood cells (SRBC), L-glutamine: L-lysine: L-alanine containing random synthetic polypeptide (GLA-40), and recombinant HIV viral envelope polypeptide (CBre3). It was found that not only the Lipid A precipitates, but under certain hydrolytic conditions the non-toxic PS preparations were also potent adjuvants. The exact conditions of hydrolysis which led to the isolation of immune adjuvant bacterial products were established. These materials were also tested for endotoxicity (Limulus lysate clotting, chick embryo lethality and local Shwartzman skin reactivity), as well as for TNF generating activities. It was found that TNF generation runs parallel with toxicity of the samples, but it does not follow the adjuvant activity of the isolates. Chemical analysis of the preparations indicated that they did not contain residual ET or Lipid A, however, they did not exclude that deacylated and dephosphorylated skeletal remains of ET are among those components in these preparations which have immunomodulatory activity.

Molecular requirements of endotoxin (ET) actions: changes in the immune adjuvant, TNF liberating and toxic properties of endotoxin during alkaline hydrolysis.

The effect of alkaline pH and alkaline hydrolysis on the physico-chemical and biological properties of endotoxin (ET) isolated from Serratia marcescens ATCC 13477 by the Biovin procedure was studied. Major emphasis was put on the ion exchange column chromatography and immune adjuvant activity (ADA) of the alkali treated samples. To measure changes in some endotoxicity parameters, Limulus lysate clotting (LAL), chick embryo lethality, Shwartzman skin reactivity and in vitro TNF release were measured. The toxic properties of ET, with the unique exception of the Shwartzman skin reactivity, rapidly diminished during alkaline treatment. As immunogen CBre3, a recombinant HIV glycoprotein which spans the C terminus of gp 120 and the N terminus of gp 41, was used in CD-1 mice, alkali treated and immediately neutralized ET samples (zero time) were inactive as adjuvants, in some cases immunosuppression could be clearly seen. But if the alkaline hydrolysis was continued for 6 h, the ADA became higher than it had been for the starting ET sample. Further alkaline hydrolysis eliminated the ADA of the samples. Both NaOH and propylamine acted similarly on the ET preparation. Reaction kinetic studies of the NaOH detoxification indicated the cleavage of ester bound acyl groups with low binding energy. Chemical analyses of the samples revealed that changes occurred in the fatty acid composition, characterized by a loss of approximately half of the 3-OH myristic acid content.

Incidence of periodontitis recurrence in treated patients with and without cultivable Actinobacillus actinomycetemcomitans, Prevotella intermedia, and Porphyromonas gingivalis: a prospective study.

A total of 98 adults previously treated for moderate to advanced periodontitis and on a trimonthly recall schedule were screened for the presence of critical levels of Actinobacillus actinomycetemcomitans, Prevotella (Bacteroides) intermedia, and Porphyromonas (Bacteroides) gingivalis. Patients with at least 2 positive sites were placed in a positive group and patients without or with low levels of these bacteria in a negative group. During the 30-month study the incidence of disease recurrence was greater in the positive group, but did not reach statistical significance. Positive patients with deeper pockets tended to be at greater risk of developing recurrent disease than those with shallower pockets. In the positive group only, both A. actinomycetemcomitans recovery and antibody levels to A. actinomycetemcomitans strain NCTC 9710 (serotype c) were inversely correlated with disease recurrence. The presence of A. actinomycetemcomitans and P. intermedia above critical levels did not reliably predict future episodes of disease recurrence in this population. The sparse recovery of P. gingivalis did not permit us to assess its diagnostic value. With the exception of P. gingivalis, for which insufficient data were available, the results indicate that the presence or absence of the above bacterial species cannot of itself serve as a reliable predictor of future episodes of recurrent disease in a population of treated patients on a regular trimonthly recall schedule.

Column liquid chromatography of endotoxins.

A new, fast and highly reproducible column liquid chromatographic method was elaborated for the analysis and small-scale preparative isolation of endotoxin from Serratia marcescens Bizio (ATCC No. 264). This procedure detects contaminants of such preparations with high sensitivity and it is capable of separating them from endotoxic components. Extensive heterogeneity of both 5% trichloroacetic acid and phenol-water-extracted endotoxin preparations was recorded. Heterogeneity among the endotoxic components of purified preparations could also be detected by this method. Measurements of biological activities, such as Limulus amoebocyte lysate activation, lymphoblastogenesis (mitogenicity) and tumor necrosis factor (TNF) liberation were carried out on the chromatographically separated fractions. During these studies, non-toxic but in vitro TNF-generating components of crude endotoxin extracts were also detected.

Immunoadjuvanticity of endotoxins and nontoxic derivatives for normal and leukemic immunocytes.

Studies with FLV infected mice, a model for retrovirus induced acquired immunodeficiency, showed that intact lipopolysaccharide rich extract from Serratia marcescens as well as the nontoxic polysaccharide derivative free of lipid A were equally adjuvantic in enhancing antibody formation to sheep erythrocytes, both in vivo and in vitro. The PS-rich endotoxin derivative had little or no toxic activity in leukemic animals as occurred with intact endotoxin. The adjuvanticity of both the nontoxic polysaccharide derivative as well as the intact endotoxin in enhancing antibody formation in FLV infected mice was evident also in vitro when spleen cells from infected animals were immunized with sheep erythrocytes simultaneously with the polysaccharide in comparison with the LPS. Supernatants from normal spleen cells treated in vitro either with the polysaccharide or the intact endotoxin showed immunoenhancing helper activity for both normal and FLV infected spleen cells and this enhancing activity was due to IL-1 induced by either bacterial product. Thus the immunoenhancing soluble mediator, i.e., IL-1, is induced equally by PS or LPS and has immunorestorative activity for FLV infected animals. The potential value of the nontoxic PS as an immunoadjuvant in retrovirus immunosuppressed lymphoid cells is evident. The results of these studies suggest that further investigations concerning the nature and mechanism involved in such adjuvancticity is warranted.

Isolation of a nonendotoxic antitumor preparation from Serratia marcescens.

White-type polysaccharide preparation (WPS) obtained from Serratia marcescens bacteria by hot 0.2 N acetic acid extraction was shown to have antitumor effects. These were manifested by enhanced resistance to the take of TA3 transplantable murine adenocarcinoma and by the induction of regression of Meth A sarcoma in mice. Optimal conditions for the liberation and isolation of these substances were sought to achieve the highest antitumor activity and the lowest endotoxin (ET) content. Simultaneously, the activities of the WPS preparations were tested in various tests which are frequently used as in vitro correlates of in vivo antitumor effects, such as the activation of macrophage cytotoxicity, activation of natural killer (NK) cells, and tumor necrosis factor (TNF) generation. We found that the enhanced resistance to the take of TA3 tumor correlated with ET content of the WPS preparations. Preparations with reduced or no ET content showed diminishing activity in this assay or were without any measurable effect. The induction of TNF production and NK activation did not show such close relationship with the ET content. This was particularly evident if testing WPS samples obtained after 60 or 120 min hydrolysis at 90 degrees C. The greatest discrepancy was found between ET content and the Meth A regression induction. Samples with no detectable ET content and no activity in the macrophage, NK, or TNF tests were potent inducers of Meth A regression. Partial purification of such WPS samples could be achieved and a preparation was obtained with high Meth A regression capacity. Preliminary chemical analysis of this preparation showed 25.5% amino acid, 53.7% neutral carbohydrate, less than 0.4% KDO, 0.8% hexosamine, less than 0.1% phosphorous, and less 1.0% long-chain carboxylic acid content. The above chemical analytical data are not consistent with designating such preparations as ET or ET derivatives, such as Lipid A or its split products. This conclusion was confirmed by the lack of endotoxic properties as determined by biological assays on this preparation.

Thin-layer chromatography of endotoxins, their derivatives and contaminants.

Thin-layer chromatographic (TLC) separation techniques were used to analyze the heterogeneity of various preparations which included smooth and rough endotoxins (ET), Lipid A precipitates and synthetic Lipid A samples and a novel cytotoxic bacterial lipid. Furthermore, carbohydrate-rich split products (PS) of ET were also separated on commercial silica-coated plates. Satisfactory results were obtained by two-dimensional TLC or by the combination of chromatography followed by high-voltage electrophoresis in the separation of PS of ET cleaved by mild acetic hydrolysis. Several spray reagents were found which were eminently suitable to detect carbohydrate containing compounds. Less specific but generally useful spray reagents were also developed which gave strong color reactions with lipids, proteinaceous and carbohydrate containing split products of the ET preparations. Improved chromatographic resolution has also revealed substantial heterogeneity in both rough and smooth ET samples. Three biological activities of the separated components could be determined. These were antigenicity detected by reactivity with monoclonal antibodies on the TLC plates, endotoxicity, determined by the Limulus amoebocyte lysate (LAL) test and direct cytotoxicity of P815 cells in vitro. Considerable amounts of non-endotoxic and non-antigenic contaminants could be detected in all preparations tested. Significant amounts of free Lipid A were also found in smooth ETs. Thus a new level of complexity is recognized by TLC within these preparations.

Non-toxic endotoxin polysaccharide induces soluble mediators which potentiate antibody production by murine retrovirus-suppressed splenocytes.

Mice infected with Friend leukemia virus show marked acquired immunodeficiency characterized by the impairment of immune function of spleen cells to various antigens, both in vivo and in vitro. The large mol. wt. endotoxin derived from Serratia marcescens, as well as a smaller non-toxic polysaccharide derivative, were found to augment the antibody responsiveness of spleen cells from normal as well as FLV-infected mice. In addition, serum from normal donor mice pretreated with BCG and injected either with endotoxin or the polysaccharide derivative potentiated the antibody response of spleen cells from both normal and FLV-infected mice. Similar enhancement was induced by "antibody response helper factor(s)" present in 3-5 day spleen culture supernatants from endotoxin or polysaccharide-treated spleen cells from normal mice. Enhancement of the antibody response of spleen cells from FLV-infected mice by the antibody helper activity was due to stimulation of B-lymphocytes and reversal of a defect in antibody helper factor(s) formation by macrophages. Similar antibody response enhancing activity was induced by both endotoxin and the non-toxic polysaccharide derivative in cultures of normal spleen cells, adherent spleen cell populations, peritoneal cells and the P388D1 macrophage cell line.

Cytotoxicity of a novel lipid-like bacterial product.

A highly cytotoxic, lipid-like compound was isolated from a Serratia marcescens strain currently under identification. We have named the compound DCX for its direct cytotoxic activity on various cell types in culture. DCX was purified by preparative thin layer chromatography from chloroform: methanol = 4:1 extracts of whole bacteria, and is chromatographically homogeneous. The effect of DCX on cells is dose, time, and temperature dependent. DCX is particularly toxic to the mastocytoma cell line P815 (TD50 = 75 pg/ml). Three other malignant or transformed murine cell lines were sensitive to the cytotoxic action of DCX. The effect of DCX was also tested on normal cells (human gingival fibroblasts), which showed greater resistance to DCX than the other cells tested.

Distinctive immunomodulatory effects of endotoxin and nontoxic lipopolysaccharide derivatives in lymphoid cell cultures.

Endotoxin derived from Serratia marcescens, as well as a hydrolyzed polysaccharide-rich derivative from the bacteria similar to the White-type polysaccharide (WPS), and hydrolyzed split products of endotoxin, including Lipid A and the nontoxic polysaccharide (PS), were studied in terms of their ability to induce mitogenic proliferation of murine spleen cells in vitro and enhancement of antibody formation to sheep erythrocytes. The intact endotoxin and the Lipid A component induced marked proliferative activity of normal lymphoid cells, but the PS components obtained after a 30 min or longer mild hydrolysis of the endotoxin had much less mitogenic activity. The WPS preparation retained mitogenic activity after 30 min but lost it during continued hydrolysis. Nevertheless, all of the components were marked adjuvants for murine spleen cells in vitro in terms of enhancing the antibody response to sheep red cells. Both the intact endotoxin as well as the Lipid A component induced interleukin-1 and interferons, including alpha/beta and gamma, following stimulation of spleen cell cultures for 24 h in vitro. Although only the endotoxin and Lipid A, but not the PS components, were mitogenic for murine splenocytes, all of these preparations had immune adjuvant effects and induced spleen cells to produce or release interferons. However, the PS-rich derivative did not induce tumor necrosis factor. These distinct properties of components of endotoxin indicate that the nontoxic PS-rich derivative, as compared to the toxic endotoxin or Lipid A, has some immunostimulatory activities but lacks others.

Enhanced antibody response in retrovirus-infected mice treated with endotoxin or nontoxic polysaccharide derivative.

Friend leukemia virus (FLV) is a retrovirus which causes marked suppression of the immune response of genetically susceptible mice. In the present study the depressed antibody response to sheep erythrocytes by spleen cells from FLV-infected mice was partially reversed by injection of either a bacterial endotoxin or a nontoxic polysaccharide derivative directly into infected mice or by addition to spleen cell cultures from these mice immunized in vitro with sheep red blood cells (SRBC). The endotoxin and PS in a dose-related manner markedly increased the antibody responsiveness of the spleen cells to SRBC. Thus these results indicate that the nontoxic polysaccharide derivative has properties equivalent to the toxic endotoxin in enhancing the antibody responsiveness of FLV-suppressed spleen cells to a T-cell-dependent antigen like SRBC.

Fetal alcohol syndrome. Eye malformations in a mouse model.

Acute maternal ethanol administration on gestational day 7 (gastrulation stage) in C57Bl/6J mice results in a spectrum of ocular malformations. A deficiency in the anterior neural plate observable within 24 hours of exposure results in corresponding defects in the optic sulcus and subsequent optic vesicle. Deficiency in the size of the lens vesicle induced by a small optic vesicle is demonstrable as microphakia in older embryos. Delayed detachment of the lens vesicle from the surface ectoderm manifests in the live offspring as progressive corneal opacification and vascularization related to defects in corneal endothelium and Descemet's membrane. Anterior segment dysgenesis results in persistent iridocorneal adhesions, dyscoria, and abnormal formation of the anterior chamber. In contrast, ethanol exposure on day 8 of gestation did not result in eye malformations. Thus, it appears that many of the ocular abnormalities associated with fetal alcohol syndrome may result from an acute insult to the optic primordia during a very specific period that corresponds to the third week after fertilization in the human.

Review of the molecular requirements of endotoxic actions.

Old and new data are compiled to confirm earlier claims and to substantiate new concepts regarding the structural requirements of endotoxic reactions. The major points can be summarized as follows. (1) Recent results obtained by the use of synthetic analogues of lipid A structures proved unequivocally that the lipid moiety of the endotoxin is the carrier of toxic properties. Incomplete or attenuated structures are inactive in some or all toxic reactions, depending on the extent of deviations from the structure of the native product. (2) On the other hand, some beneficial reactions can be initiated not only by the complete structures but by their structural remains, which are no longer toxic. (3) Some of the split products in the lipid-free and polysaccharide-rich preparations can induce beneficial reactions. (4) Gram-negative bacteria can produce endotoxin-unrelated and beneficial compounds. Conventional endotoxin preparations are heterogeneous and often contain some of these unrelated substances. (5) Elucidation of the exact structural requirements of endotoxic reactions has just begun. It appears to be safe to predict that individual reactions will require highly specific structures or physicochemical properties. The potential rewards of such research might be quite significant by facilitating our understanding of the molecular events of the beneficial effects of endotoxins and by directing the search for preparations with therapeutic applications.

Interferon induction by endotoxin-derived nontoxic polysaccharides.

Polysaccharide-rich preparations from Serratia marcescens-derived endotoxin and components thereof, including Lipid A, were studied in terms of their ability to induce interferon (IFN) activity in murine spleen cell cultures in vitro. Although the polysaccharide-rich derivatives, similar to intact endotoxin, were only weak stimulators for IFN induction, pretreatment of splenocyte cultures with recombinant interleukin-2 (IL-2) significantly increased IFN-inducing activity. Both an endotoxin-derived polysaccharide and a "White type" polysaccharide prepared from intact Serratia had similar ability to induce IFN in vitro, but only when spleen cells were first treated with IL-2. The polysaccharide preparations were nontoxic as compared with the high degree of toxicity of the intact endotoxin, yet induced similar IFN levels as whole endotoxin. Much of the IFN induced by these preparations was of the gamma type, since activity was either not neutralized or only incompletely neutralized by treatment with anti-alpha/beta interferon antibody.

Time dependency of endotoxin-induced resistance to transplantable tumors in mice.

A single injection of bacterial endotoxin induced cyclic changes of enhanced or reduced host reactivities, as determined in some measurements of the beneficial effects of endotoxin, such as immune potentiation or protection against lethal irradiation. The results described here show that endotoxin-induced resistance to subsequent challenge with transplantable tumors such as Lewis lung carcinoma or L1210 murine leukemia is similarly time dependent. While certain time intervals between endotoxin treatment and tumor challenge will provide significant protection, others will render the recipients highly susceptible to the same tumor. It was also observed that L1210-bearing mice have a lower resistance to endotoxin injections than tumor-free mice of the same strain.

Metachromatic assay for the quantitative determination of bacterial endotoxins.

A metachromatic dye, 1,9-dimethylmethylene blue (DMB) reacts with bacterial endotoxins. This interaction results in a shift of the absorption maximum of DMB to shorter wavelengths. The findings indicate that the negatively charged lipid moiety of the endotoxic lipopolysaccharide reacts with DMB. The lowest amount of endotoxin detectable by the procedure described here is approximately one microgram. The dye could not be used in the presence of serum components. DMB mixed to column chromatographic effluent showed good resolution in continuous monitoring of endotoxin components leaving the column.