Induction of tumor necrosis factor alpha in murine macrophages with various strains of Coxiella burnetii and their lipopolysaccharides.

The ability of various strains of Coxiella burnetii (C.b.) and their phase I and II lipopolysaccharides (LPSs) to induce tumor necrosis factor alpha (TNF-alpha) in peritoneal Balb/c mouse macrophages in vitro was investigated. Considerable differences in the induction ability were observed in dependence on the strain applied. In a TNF-alpha bioassay, the most effective inducers were both corpuscles and LPSs of the strains Priscilla and Scurry, followed by Nine Mile, Luga, and Henzerling I. In contrast, in ELISA, the most effective inducers were LPSs of the strains Luga and Henzerling, followed by Nine Mile, Priscilla, and Scurry. The role of toll-like receptor 4 (TLR4) in the induction was confirmed by the use of C3H/HeJ mouse macrophages. Thus, the induction of TNF-alpha was much higher in Balb/c mouse macrophages than that in TLR4-deficient C3H/HeJ mouse macrophages. Differences in the results of the bioassay and those of ELISA suggest a role of another secreted factor(s) induced with C.b. in murine macrophages that could act synergically with TNF-alpha in L929 cells in the bioassay. The observed differences in TNF-alpha induction might play a role in the pathobiology of Q fever.

Management of inflammatory corneal melt leading to central perforation in children: a retrospective study and review of literature.

PURPOSE: To assess the outcome of early therapeutic penetrating keratoplasty (PKP) for corneal melt leading to perforation in children. METHODS: Case notes of all the consecutive patients presenting with acute corneal perforation that underwent urgent therapeutic PKP between 2000 and 2010 to the practice of one of the authors, both NHS at Great Ormond Street Hospital for Children and private, were retrospectively reviewed. Onset of perforation, underlying cause, medical and surgical treatment, pre- and post-operative visual acuity, graft clarity, length of follow-up, and post-operative complications were recorded. RESULTS: Four eyes of four consecutive patients (mean age of 9.5 years and median 8.5 years, range 4-17 years) were treated for acquired acute onset corneal perforations. There were three females and one male. Etiologies included herpes simplex keratitis secondary to immune recovery disease post bone marrow transplantation, acanthamoeba keratitis, recessive dystrophic epidermolysis bullosa, and blepharokeratoconjunctivitis with acne rosacea. Pre-operative visual acuity ranged from hand movements to 6/150. All the patients had severe anterior chamber inflammation. All eyes improved in visual acuity ranging from 6/9 to 6/18 with clear grafts at last follow-up. There was no recurrence of melt or perforation. Mean follow-up was 67 months (median 44 months). CONCLUSION: PKP during the acute phase together with aggressive medical therapy and close follow-up may achieve good visual outcomes in children with corneal melt with perforation and should be considered. Waiting may sometimes allow the marked inflammatory response seen in children to cause irreversible structural and/or functional damage.

Endocrine disorders among long-term survivors of childhood head and neck rhabdomyosarcoma.

PURPOSE: Head and neck rhabdomyosarcoma (HNRMS) survivors are at increased risk of developing pituitary dysfunction as an adverse event of radiotherapy. Our aim was to investigate the frequency and risk factors for pituitary dysfunction in these survivors. Secondly, we aimed to compare the prevalence of pituitary dysfunction between survivors treated with external beam radiation therapy (EBRT) and survivors treated with the ablative surgery, moulage technique after loading brachytherapy, and surgical reconstruction (AMORE) procedure. METHODS: Eighty HNRMS survivors treated in London (EBRT based) and Amsterdam (AMORE based: AMORE if feasible, otherwise EBRT) in the period 1990-2010 and alive ≥ 2 years post-treatment were evaluated. Survivors were evaluated in multidisciplinary late-effects clinics, with measurement of linear growth, determination of thyroid function, and growth hormone parameters. Additional data, such as baseline characteristics, anthropometrics, pubertal stage, and the results of additional laboratory investigations, were retrieved from patient charts. RESULTS: Pituitary dysfunction was diagnosed in 24 in 80 (30%) survivors, after a median follow-up time of 11 years. Median time to develop pituitary dysfunction after HNRMS diagnosis was 3.0 years. Risk factors were EBRT-based therapy (odds ratio [OR] 2.06; 95% confidence interval [CI] 1.79-2.46), parameningeal tumour site (OR 1.83; 95% CI 1.60-2.17) and embryonal RMS histology (OR 1.49; 95% CI 1.19-1.90). CONCLUSIONS: Radiotherapy used for the treatment of HNRMS confers a significant risk of the development of pituitary dysfunction. AMORE-based treatment in children with HNRMS resulted in less pituitary dysfunction than treatment with conventional EBRT. Our findings underscore the importance of routine early endocrine follow-up in this specific population.

Phase variation of lipopolysaccharide of Coxiella burnetii, strain Priscilla during chick embryo yolk sac passaging.

Changes of lipopolysaccharide (LPS) of Coxiella burnetii strain Priscilla during chick embryo yolk sac passaging were observed by SDS-PAGE and immunoblot analysis. The course of LPS phase variation was similar to that found in other C. burnetti strains, i.e. a conversion of the phase I to the intermediate phase II after 10 passages. The intermediate phase II LPS of Priscilla strain was also detectable by immunoblot analysis using immune serum against Priscilla strain in the 30th passage.

Cell-mediated immune response to Coxiella burnetii antigens in Q fever convalescents and vaccinees.

Cellular immunity against phase I and II Coxiella burnetii antigens was evaluated by the lymphocyte blast transformation test in a) Q fever convalescents; b) persons immunized with a chemovaccine against Q fever; and c) control persons. The first two groups consisted of workers in a ricketsiology department. Of the 9 convalescents, all (100%) were positive against phase I antigen and 4 (44.4%) against phase II antigen. Of the 22 vaccinees, 77.3% were positive against phase I antigen and all were negative against phase II antigen. All 10 controls were negative against both antigens.

Long-term persistence of cell-mediated and humoral immune response in specifically resistant mice given live attenuated langat (E5 "14") virus.

In a long-term experiment, the features of humoral and cell-mediated immunity (CMI) were investigated in mice given intravenously (i.v.) a single dose of live attenuated Langat (E5 "14") virus of the tick-borne encephalitis (TBE) complex. The inapparent infection induced in mice specific resistance against challenge with virulent TBE virus, CMI was assayed by the capillary method of murine splenic lymphocyte migrarion inhibition (LMI) in the presence of partially purified TBE vurus. Significant LMI values were observed as early as 4 days after inoculation (p.i.), persisting for at least 15 months. Specific virus neutralizing (VN) antibodies were detected in the pooled serum of mice on the 4th day p.i. by a VN test potentiated with monospecific anti-mouse IgG serum. For the whole experimental period, significant antibody levels were found in the immunized mice which also displayed a complete resistance against 1000 s.c. LD50 of the virulent virus. Organ cultures of brain and spleen from these mice did not yield infections virus.

Coxiella burnetii phase I and II proteins studied by SDS-page.

Coxiella burnetii cells in both phase I and II reveal in sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) similar protein profiles with only small differences. C. burnetii protein profile in SDS-PAGE depended on the method of purification of C. burnetii cells from chick embryo yolk sacs. Immune mouse sera against C. burnetii phase I cells recognized in phase I and II cell protein profiles mainly the 61 K and 29 K proteins by the immunoblot method. Hyperimmune mouse and rabbit sera against phase I and II cells reacted in different way with phase I and II cells. Sera against phase I cells recognized in both phase I and II profiles more protein bands than sera against phase II cells. Thus phase I LPS present in phase I cells exerted adjuvant effect on the antibody response in animals immunized with phase I cells.

Transfer with dialysable transfer factor of T-lymphocyte cytolytic response to tick-bone encephalitis virus antigen in naive mice.

Transfer factor activity was demonstrated in the dialysable extract from lymphocytes from outbred donor mice, in which immunization with live attenuated Langat (E5 "14") virus induced a stage of high resistance against challenge with virulent tick-borne encephalitis virus. Administered to naive inbred recipient mice, the extract conveyed in them specific cytolytic activity, exerted by their T lymphocytes, as demonstrated by the 51Cr release assay on tick-borne encephalitis virus-infected syngeneic L-929 cells.

Cross-reactive killer T lymphocytes in a flavivirus infection.

Splenocytes from mice immunized intravenously with one dose of Langat or Yellow fever viruses were cytotoxic for target cells infected with virulent tick-borne encephalitis (TBE) virus; cytotoxicity was measured by 51Cr release from target L929 cells. No significant lysis of the TBE virus-infected taget cells with splenocytes from Sindbis virus-infected mice was noted.

In vitro studies on cell-mediated immune response to tick-borne encephalitis virus: findings in convalescents and human subclinical infections.

Peripheral blood leukocytes from tick-borne encephalitis (TBE) convelescents (manifest and inapparent forms) working with TBE virus-containing material and from individuals without specific serum virus neutralizing antibodies (VNA) were studied in capillary tube leukocyte migration experiments. Partially purified TBE virus preparations were used as antigen. Under standardized conditions a strong inhibitory reaction was observed in convalescents with significantly higher values in persons recovered from an abortive form of infection only. These results differed markedly from values recorded in persons without specific VNA. Significance of correlation between humoral VNA titres and the intensity of the cell-mediated component of the immune response (CM IR), as indicated by leukocyte migration inhibition (LMI) values, was less than P equals 0.25.

Cell-mediated immunity in flavivirus infections. I. Induction of cytotoxic T lymphocytes in mice by an attenuated virus from the tick-borne encephalitis complex and its group-reactive character.

The lytic activity of splenocytes from C3H mice immunized with a highly attenuated line of Langat virus [tick-borne encephalitis (TBE) complex] was determined by the in vitro 51Cr release assay on TBE virus (western subtype)-infected target L929 cells. After the spleen cell suspensions were depleted of T cells with anti-mouse theta serum, or of B cells with anti-mouse immunoglobulin, the cytotoxic effect was found dependent on the presence of T lymphocytes. During immunization with Langat virus, cytotoxic T lymphocytes were generated, specific for both the foreign virus-specified antigen and the self component of the host system studied, coded by the K allele of the H-2 complex. The peak of T lymphocyte cytotoxic response was attained on the 6th day after administration of the live virus. T lymphocytes from mice, single-shot immunized with a flavivirus, displayed distinct cross-reactive lysis when studied on target cells infected with other flaviviruses but not on cells infected with an alphavirus.

Immunogenicity and protective ability of corpuscular and soluble vaccines prepared from different Coxiella burnetii phase I strains.

BALB/c mice immunized intraperitoneally (ip) with killed purified Coxiella burnetii phase I corpuscular vaccines or trichloroacetic acid (TCA) extracts from phase I corpuscles (soluble vaccines) were protected against ip challenge with both homologous and heterologous C. burnetii phase I strains. Though the degree of protection, namely the inhibition of C. burnetii multiplication in the mouse spleen slightly varied, in general, corpuscular vaccines provided better protection than soluble ones. Cross-protection was accompanied by comparable levels of cell-mediated immune response as evaluated by lymphocyte transformation test (LTT). However, higher stimulation indices of LTT were obtained with homologous than with heterologous strains. The values of antibody response as determined by enzyme-linked immunosorbent assay (ELISA) were higher with homologous strains too. On average, both antibody-inducing and antibody-binding capabilities of the strains Priscilla and S were lower than those of the Nine Mile and Luga strains, except for values obtained with the antigens from homologous strains.

Antigen-specific transfer factor from mice immunized with an attenuated flavivirus: augmentation of inducing activity in semipurified splenocytic dialyzates.

Three large batches were prepared of lyzed splenocytic leukocyte dialyzate from SPF outbred mice, immunized with a live attenuated virus from the tick-borne encephalitis (TBE) complex. Total mass of freeze-dried dialyzates was 1.73 g. One mg of respective batches contained 2 X 10(5), 2 X 10(4) and 2 X 10(3) units of the transfer factor, specific for the flavivirus group-antigen, as estimated according to the capacity to induce specifically cytotoxic T-cells in the recipient C3H mice. The amount of protein and orcinol-reactive material (purine-bound ribose), the presumed components of the inducer's substrate, ranged in individual dialyzates from 9.9-12.4 and 0.72-0.80% of their dry mass. Materials from each batch obtained after double precipitation by ethanol were subjected to permeation chromatography on Sephadex G-25 columns and subsequent lyophilization of the peak with specific inducing activity. The final product represented on average 3.7 per cent of dry mass of the starting material. In comparison to the crude material, in one mg of the final product the protein and the orcinol-reactive material were reduced by 80 and 37 per cent, respectively, but an increment in the antigen-specific inducing capacity comprising 2-3 log10 units was observed. These findings add to the concept that a) macromolecules carrying the inducing activity can be separated from other constituents of the crude dialyzate and b) an increase in antigen-specific inducing activity titre was, besides partial concentration, mainly due to removal of suppressor or inhibitory factor(s) present in the crude dialysates and probably acting in vivo.

Concentration of murine antigen-specific transfer factor of defined potency.

Dialysates containing transfer factor (TF) activity were prepared from lyzed splenic cells of SPF mice immunized with live, peripherally avirulent Langat virus (TP21 E5 "14" clone) from the tick-borne encephalitis (TBE) complex. The amount of TF was estimated by its capacity to generate in recipient inbred C3H mice cytotoxic T lymphocytes inducing lysis of TBE virus-infected target cells as demonstrated by 51Cr--release assay. A 100 to 1000-fold concentration of TF activity was achieved by combination of the two-step ethanol precipitation of crude dialysates with subsequent fractionation on Sephadex G-25 by exclusion chromatography. Materials from individual concentration steps showed reduced amounts of admixtures, as revealed by absorbance profiles of their chromatograms. In the final product the protein content was most decreased.

Dialysable specific transfer factor in mice immunized with attenuated Langat virus from the tick-borne encephalitis complex: generation, action and quantitative assay.

Cytolytic T lymphocyte assay was developed in order to measure the response of inbred C3H mice to dialysable specific transfer factor (STF), induced in subadult outbred mice by one shot immunization with the attenuated Langat virus. The first STF activity in mice splenic leukocytes was detected between 48-72 hr after virus administration. The conversion of splenic T-cell cytotoxic response in C3H mice in vivo occurred between 15-21 hr after STF administration. The killing activity of T-cells, induced by STF, showed cross-reactive traits within the genus Flavivirus. STF, given prior to the live virus, augmented the specific cytolytic T-cell response. In the live virus-primed mice the booster effect was markedly enhanced when administration of STF preceded the second immunization dose. In the serum of STF recipients, interferon was irregularly detected attaining low levels for short time periods. Temperature of 56 degrees C for 60 min abolished the activity of least 10(4) murine STF units, temperature of 37 degrees C lowered after 24 hr this activity by 3 log10units. Chromatography of the dialyzed leukocyte lysate on Sephadex G-25 column yielded usually five peaks. The second peak showed an increased content of ribose-bound and protein materials and, as a rule, a relatively concentrated STF activity.

Immunogenicity of Coxiella burnetii whole cells and their outer membrane components.

The immunogenicity and protective efficacy of the phase I and phase II Coxiella burnetii whole cells (Cb I and Cb II) and their outer membrane components (OMC), i.e. phase I trichloroacetic acid extract (TCAE), phase I 29 K protein (PRO), phase I and II lipopolysaccharides (LPS I, LPS II), polysaccharides (PS I, PS II), and lipid A (LA I, LA II), were compared. The highest immune response was observed in BALB/c mice by Cb I in both humoral immunity and lymphocyte transformation assays, and in the protective effect as well. The immune response was also significant by Cb II, but their protective capacity was low. The OMC reacted variously. Only TCAE and PRO gave a high value of humoral immunity evaluated by the serological methods. All OMC reacted in the haemolytic plaque assay giving different responses. Lymphoproliferation of splenocytes was positive with all OMC using both Cb I and Cb II antigens with the exception of PS I and PS II in the case of Cb II antigen. The induction of protection against infectious Cb I was demonstrated after immunization with TCAE, PRO, and LPS I. Other OMC did not induce protection against this agent.

Experimental live tick-borne encephalitis vaccine (Labgat E5 "14" virus clone): volunteers 1 and 2 years after single-dose immunization.

Groups of volunteers given intramuscularly 5 or 6.5 dex or perorally 6.5 dex newborn mouse icLD50 of the plaquesegregated "14" clone of the Langat E5 virus strain (tick-borne encephalitis comples) were followed for periods of 12 and 24-27 months. Circulating specific virus neutralizing antibodies persisted in them in the absence of apparent reaction as evidenced by clinical, electroencephalographic and cerebrospinal fluid findings.

[The immunogenicity of a vaccine against enzootic abortion in sheep]. / Imunogénnost vakcíny proti enzootickému potratu oviec.

Immunogenicity of vaccine against enzootic abortion of ewes (EPO) was evaluated in sheep and laboratory white mice. The vaccine contained purified and formalin-inactivated corpuscules of Chlamydia psittaci. Experiment was performed on ten sheep, Slovak Merino breed, which were negative before vaccination in serological assays and blastic transformation of lymphocytes (BTL) tests. The animals were immunized subcutaneously with 2 ml of vaccine which contained 100 micrograms (sheep No. 1-4) and 20 micrograms (sheep No. 5-8) corpuscules of C. psittaci. Control group (No. 9-10) received the same volume of physiological saline. The second dose of vaccine was given one month after the first dose (No. 1-2--100 micrograms each, and No. 5-6--20 micrograms each). Blood for serological evaluation and BTL test was taken before vaccination and 1, 3, and 6 months after vaccination. The sheep which were given the second dose of vaccine were also evaluated two weeks thereafter. Antibody response in complement fixation reaction (CFR) and enzyme-linked immunosorbent assay (ELISA) was compared with the results of BTL. While the antibody response evaluated by CFR was sporadically positive after administration of the higher dose of vaccine only, in ELISA all sera were positive except one lower dose of vaccine (Tab. I). In all post-vaccination intervals positivity was confirmed by BTL test irrespective of the size and number of vaccine dose (Tab. II). Immunization of mice with one dose (100 micrograms) of vaccine significantly but not completely reduced multiplication of C. psittaci in the lungs of mice (Tab. III).

[Suggested low vision care for visually impaired children in Slovakia]. / Návrh systému starostlivosti o zrakovo postihnuté deti v SR.

INTRODUCTION: There is currently no system of registration for visually impaired children in Slovakia and the current prevalence of visual impairment (VI), low vision and blindness is unknown for this population. We propose a template for a process of registration of visually impaired children in Slovakia as well as a system for the Low Vision Health Core for this population. MATERIAL AND METHODS: Based on a literature search, we report our data of the estimated prevalence of VI in children in Slovakia and the number of registered students with VI. We have created a registration form and suggested a template of registration for VI children as well as the Health Care System for this population. RESULTS: In industrialized countries, the prevalence of VI, including blindness is 10 -22/10,000 in children aged less than 16 years. Extrapolating these figures to the Slovak population, we estimate that there will be between 1500 to 3200 VI children under the age of 19 years. Only 752 students with VI of this age were recorded in Slovakia in 2009/2010. We suggest that three Low Vision Centres for VI children should be adequate to cater for the VI population, each of which should provide all levels of care and that ophthalmologists should register patients with VI by filling the proposed registration form. CONCLUSION: The number of VI children in Slovakia appears to be very low. The only way of accurately assessing the prevalence is to introduce a VI registration system into the country, to be carried out by the ophthalmologists. We suggest that the Low vision service provided by the health authority needs to be divided into primary, secondary and tertiary care (including visual rehabilitation by low vision aids). Only if the Health Insurance will adequately remunerate the Ophthalmologists for the individual procedures will they be motivated enough to provide this level of health care to VI patients.

[Improvement in the outcome of visual impairment using low vision aids in children]. / Kompenzácia zrakového postihnutia u detí prostredníctvom optických pomôcok.

INTRODUCTION: Low visual aids (LVAs) are the simplest and the most economic method of reducing the consequences of visual impairment (VI). We evaluate prescribing of LVAs in order to improve the quality of life of visually impaired children. MATERIAL AND METHODS: We retrospectively reviewed the notes of 113 patients (pts.) with VI (visual acuity (VA) 6/18 or worse or with visual field defects) who attended the Low Vision Clinic, F.D. Roosevelt Hospital in Banska Bystrica, between Jan 2000 - Dec 2005. There were 61 boys and 52 girls with ages ranging from < 1 year to < 19 years (mean age 9.4 years, median 9 years). RESULTS: Of the 113 notes reviewed, only 109 of patients were cooperative in having their VA formally tested. Blindness (VA worse than 3/60) occurred in 31 pts. (28.4%) and Low vision (VA worse than 6/18 or equal to 3/60) in 67 (61.5%). Of the remaining 11 patients, 4 had gross field defects (and good VA), (indicating that the total number with Low vision was 71 (65%). The other 7 patients had either developmental delay or were too young to perform a formal visual field test. Blindness was caused by perinatal factors in 52% of cases, prenatal factors in 45% and postnatal factors in 3%. Low vision was caused in 74.6% of cases by prenatal factors. The causes of blindness according to the affected anatomical site were: optic nerve disease in 12 (39%), cerebral visual impairment (CVI) in 8 (26%), and retinal disease in 6 (20%). Low vision was caused by retinal disease in 23 (34.3%), lens pathology in 11 (16.5%) and CVI in 10(14.9%). 171 LVAs were prescribed to 74 patients (65%): 76 (44.4%) were magnifiers, 21 (12.3%) were hyperoculars or high adds, 65 (38%) were telescopes and 9 (5.5%) telescope extensions. An average of 1.5 devices was prescribed for each patient. Every patient who was prescribed an LVA had subjective and objective improvement of their visual function and functional vision. CONCLUSION: Blindness occurred in almost one third of our patients which agrees with the worldwide accepted estimate. The LVA are improving the quality of life of visually impaired children. The prescribing of LVA depends on the individual: it is not possible to anticipate the optical power of the LVA that a patient will need simply from looking at their diagnosis and measuring their VA because there are so many different requirements depending on their age and visual demands.