Surveillance during an era of rapidly changing poliovirus epidemiology in India: the role of one vs. two stool specimens in poliovirus detection, 2000-2010.

Since 2004, efforts to improve poliovirus detection have significantly increased the volume of specimen testing from acute flaccid paralysis (AFP) patients in India. One option to decrease collection and testing burden would be collecting only a single stool specimen instead of two. We investigated stool specimen sensitivity for poliovirus detection in India to estimate the contribution of the second specimen. We reviewed poliovirus isolation data for 303984 children aged <15 years with AFP during 2000-2010. Using maximum-likelihood estimation, we determined specimen sensitivity of each stool specimen, combined sensitivity of both specimens, and sensitivity added by the second specimen. Of 5184 AFP patients with poliovirus isolates, 382 (7.4%) were identified only by the second specimen. Sensitivity was 91.4% for the first specimen and 84.5% for the second specimen; the second specimen added 7.3% sensitivity, giving a combined sensitivity of 98.7%. Combined sensitivity declined, and added sensitivity increased, as the time from paralysis onset to stool collection increased (P = 0.032). The sensitivity added by the second specimen is important to detect the last chains of poliovirus transmission and to achieve certification of polio eradication. For sensitive surveillance, two stool specimens should continue to be collected from each AFP patient in India.

Functional outcome of patients 12 and 48 weeks after acute traumatic tetraplegia and paraplegia: data analysis from 2004-2009.

OBJECTIVES: During the first rehabilitation of patients with traumatic spinal cord injuries (SCIs), professional skills are learned, which can be objectified in an independent measurement score. The aims of this study were to record the skills of patients 12 and 48 weeks after acute trauma and perform an analysis of the data to identify provisions of importance. METHODS: All patients from 2004 to 2009 who experienced traumatic SCI were included in this investigation. Data recording were accomplished by the European Multi-Centre Study about Spinal Cord Injury (EMSCI) databank. Patients were divided into tetraplegia and paraplegia groups. Parameters were age at injury, the American Spinal Injury Association-Score, level of lesion and spinal cord independence measure (SCIM-Score) 12 and 48 weeks after traumatic spinal cord lesion. A questionnaire was also added to help clarify where deficiencies were prevalent. RESULTS: Data analysis of 103 tetraplegic and 110 paraplegic patients showed no correlation between the ASIA score, level of lesion, age and SCIM score. On average, tetraplegic patients had a SCIM score of 43 points 12 weeks after treatment, with 81% showing an increase to 58 points after 48 weeks. Paraplegic patients showed an average SCIM score of 60 points after 12 weeks, with 71% experiencing an increase to 71 points after 48 weeks. In all, 9% of tetraplegic patients and 19% of paraplegic patients experienced a decrease of SCIM points after 48 weeks, which occurred mainly in the bladder and intestinal control subgroups. Results of the questionnaire were not helpful for clarifying the location of the deficiencies. CONCLUSION: Most of the patients experienced an increase of SCIM points 48 weeks after traumatic SCI. However, data also showed that, especially in paraplegic patients, special attention must be given to bladder and intestinal management to avoid negative late-term consequences.

Evolving epidemiology of poliovirus serotype 2 following withdrawal of the serotype 2 oral poliovirus vaccine.

Although there have been no cases of serotype 2 wild poliovirus for more than 20 years, transmission of serotype 2 vaccine-derived poliovirus (VDPV2) and associated paralytic cases in several continents represent a threat to eradication. The withdrawal of the serotype 2 component of oral poliovirus vaccine (OPV2) was implemented in April 2016 to stop VDPV2 emergence and secure eradication of all serotype 2 poliovirus. Globally, children born after this date have limited immunity to prevent transmission. Using a statistical model, we estimated the emergence date and source of VDPV2s detected between May 2016 and November 2019. Outbreak response campaigns with monovalent OPV2 are the only available method to induce immunity to prevent transmission. Yet our analysis shows that using monovalent OPV2 is generating more paralytic VDPV2 outbreaks with the potential for establishing endemic transmission. A novel OPV2, for which two candidates are currently in clinical trials, is urgently required, together with a contingency strategy if this vaccine does not materialize or perform as anticipated.

Establishment and characterization of permanent cell lines from patients with acute and hairy cell leukemia.

Four cell lines were established from peripheral blood of patients with leukemia. All lines express the Epstein--Barr virus nuclear antigen (EBNA) and therefore should be classified as lymphoblastoid cell lines. However, one of the lines UHKT-5 established from a patient with acute myelomonocytic leukemia has some features not typical for lymphoblastoid cell lines. The cells resemble to macrophages with the phagocytic ability for yeasts, strong alpha-naphthylacetate esterase positivity in phagocytizing cells and unusually long villi. Another line UHKT-7 established from a patient with hairy cell leukemia expresses the same isotype of membrane immunoglobulins as the original hairy cells.

Human urinary bladder carcinoma cell line (T24): immunological studies and search for oncornavirus in T24 cell population and derived clones.

Cytotoxicity of mononuclear cell (MNC) preparations isolated from peripheral blood of patients with urinary bladder carcinomas of a transitional (BTCC) or squamous (BSCC) type and from blood of control subjects was examined on the target cells of T24 cell line. In pilot experiments the percentage of cytotoxic MNC preparations was found to be similar in patients with tumors of BTCC (European, 56%;African, 60%) and BSCC (54%) type. Cytotoxicity of MNC prepared from control subjects was detected in 10% of cases or less. These data suggest an antigenic cross-reactivity between urinary bladder carcinomas of transitional and squamous cell type. The cytotoxic MNC from peripheral blood of patients with BTCC and control subjects were fractionated by adherence in nylon wool columns, and the cytotoxicity of adherent and nonadherent MNC subpopulations was examined. In the majority of cytotoxic MNC preparations from both donors with and without BTCC, the cytotoxic activity was associated with nonadherent MNC. Comparison of immunosensitivity of T24 cell population with the cloned T24 subpopulations indicated that clones with various digrees of immunosensitivity are present in the T24 cell population. However, immunosensitivity of the clones was never higher than the immunosensitivity of the T24 cell population. A search for oncornavirus particles produced spontaneously or after treatment with virus production activators performed with regard to the possible association of oncornaviruses and antigens responsible for cell-mediated cytotoxicity gave thus far negative results.

T-cell hybrids. IV. One of parental cell lines is dominant in determining membrane characteristics of hybrid cells.

Membrane characteristics of parental thymic lymphoma cells (BW5147, EL-4R) and their hybrids (BH2) derived by PEG-promoted cell fusion were compared. Isoelectric focusing of the cell populations, titration of membrane proton binding groups and quantitative examination of cell-substrate adhesiveness indicated that the BW5147 cells behaved as dominant in determining cell membrane characteristics of the BH2 hybrids.

Long-term preservation of transfecting activity of DNA isolated from rat virogenic XC cells transformed by Prague strain of Rous sarcoma virus.

The DNA isolated from rat virogenic XC cells transformed by Rous sarcoma virus retains standard transfecting activity for 7 months when the DNA solution in 0.1 X PBS, containing 10% glycerol is stored at -70 degrees C. The value of the sedimentation constant S20w does not significantly change during storage.

Large-scale production, concentration and purification of Rous Sarcoma Virus in tissue culture.

A method is described for long-term standard production of purified virus from cultures of PR-RSV-C-transformed chicken cells. The mean yields were 4.91 mg of purified virus per liter of culture medium. Cells were grown in long-term cultures for 26 weeks. A combination of polyethylene glycol 6 000 precipitation and zonal centrifugation has been found satisfactory for virus concentration and purification.

Fractionation of proteins from Rous sarcoma virus and avian myeloblastosis virus by isoelectric focusing.

The isoelectric focusing technique in the pH gradients was used for a preparative isolation of proteins from Rous sarcoma virus and avian myeloblastosis virus. The purified major gs protein, p27 (pI = 9.1) and the gP86 (pI = 5.3) were obtained after disruption of virus with 1% non-ionogenic detergent in the presence of 6M urea. The p10, p15, and p19 were present in the same range of pH (pI = 6.8). A strongly basic protein, immunologically active, presumably the p12, was found in the alkaline region of the pH gradient 10.8. These proteins fully retained their immunological activity. On the other hand, in the acidic region of the pH gradient between pH 4 and 5, strong precipitates were regularly found. These precipitates were complexes which were formed by interaction of the acid components of ampholines with the viral proteins during isoelectric focusing. Almost all viral proteins were present, differing only in quantity. The complexes were stabile in 1% non-inogenic detergent and 6M urea. They were dissociated with 1% SDS and 5M urea, and had no immunological activity. The methods of virus disruption and possibilities of formation of the ampholine-viral protein complexes are discussed.

The expression of avian gs antigen in mammalian Rous sarcoma virus-transformed cells after treatment with 5-iododeoxyuridine and dexamethasone.

The content of avian gs antigen in RSV-transformed rat cells LW13-RsK1 and LW13-RsK4 showed a transient increase after treatment with 5-iododeoxyuridine. Higher levels of gs antigen were found when treated cells were cultivated in the presence of dexamethasone. In no case was the production of RSV found in treated RSV-transformed rat cell lines when, in addition, XC cells were tested.

The presence of retroviral particles in hybridoma cell lines.

The presence of very high numbers of type C and type A retroviral particles was repeatedly confirmed not only in myeloma cells NSI, Ag 8 and in thymoma cells BW5147, EL 4 but also in B and T cell hybridomas constructed from the myeloma and thymoma cells used. Retroviral particles were demonstrated by current electron-microscopic and physicochemical methods. Biological tests for the induction of possible malignant or any pathological changes in the artificially infected sensitive cells during long-term cultivations in vitro or in vivo gave negative results. Nevertheless, on account of the hitherto unknown action of retroviruses one can suppose that monoclonal products from B and T cell hybridomas, particularly when used for practical purposes, should be purified because of their infectious nature.

An enzyme-linked immunosorbent assay for detecting the chicken B cells.

Two modifications of an indirect enzyme immunoassay described here allow the discernment of chicken B and T cells when a polyclonal rabbit anti-chicken Ig antibody or a monoclonal antibody reactive with chicken IgM and IgG heavy chains is used. Comparison of both types of antibody in ELISA and in indirect immunofluorescence suggests that they can readily be exploited for detection of lymphoid cells with surface immunoglobulin markers.

Attempts ot activate endogenous virus expression with Marek's disease virus DNA.

Several DNAs derived from Marek's disease virus-infected cells and tissues were tested for in vitro infectivity and for the ability to activate avian endogenous type C virus. The DNA isolated from tumour tissue, peripheral blood buffy coat cells, MDV-infected tissue cultures, lymphoblastoid cell lines and feather follicle epithelium cells from MDV-infected birds elicited a negative response in transfection assays. The MDV DNAs isolated did not activate the endogenous type C virus from cell cultures derived from the C, I and M chicken lines. Activation was observed only in one experiment in the early period after transfection with MDV DNA. The treatment with DNase destroyed this MDV DNA activity, and lambda phage DNA and cell DNAs did not activate the endogenous viruses. Repeated experiments failed to confirm the early activation of endogenous viruses.

Detection of antibodies to hepatitis B surface antigen (HBsAg) using monoclonal antibody and the avidin-biotin system.

A direct ELISA using biotinylated HBsAg and a competitive ELISA using biotinylated monoclonal antibody were developed for the detection of antibodies to HBsAg. Both tests are capable of detecting 0.1 I.U. of anti-HBsAg antibody/ml. The direct ELISA was compared with a SPRIA test for anti-HBsAg antibody in human sera.

Virus-specific nucleotide sequences in duck cells transformed by chicken and duck-adapted Rous sarcoma virus.

Adaptation of PR-RSV-C on duck cells results in successful and efficient replication of the adapted virus in duck cells. The adapted variant, daPR-RSV-C, was compared with the parental chicken-cell derived PR-RSV-C. No differences in the efficiency of integration and in the number of integrated proviral copies in duck cells were found. However, the structure of proviral DNA of the adapted virus was different. Whereas EcoRI and HindIII digestion showed no differences between chicken-cell derived PR-RSV-C and the daPR-RSV-C, a new restriction site was found for BamHI endonuclease, which is probably located at the 3' end of the env gene.

Evidence for animal origin of cells and rescued tumour virus from a putative human osteosarcoma cell line.

Human osteosarcoma cell lines T 1 and ZT 1 were analyzed for host origin. The results indicated that these lines must have been contaminated with rat x mouse hybrid cells. The isolated virus was identified as an avian sarcoma virus belonging to the C subgroup.

Immunoregulatory effect of a synthetic peptide corresponding to a region of protein p24 of HIV.

The effect of a synthetic peptide, corresponding to a sequence of HIV-1 p24 protein (amino acids 218-237), on in vitro immune responses was studied. The peptide inhibited in a dose-dependent manner the induction of an anti-SRC antibody response and of a PPD-specific proliferative response of human PBL. On the other hand, PHA-induced proliferation of human PBL and PPD-induced proliferation of a PPD-specific human T-cell line were not modified by comparable amounts of the peptide. These results suggest that structures from a protein (p24), present in the serum throughout the course of HIV infection, are able to interfere with the inductive stages of specific immune responses. These findings may help to unravel some of the pathogenic mechanisms of AIDS and may contribute to the development of vaccine strategies.

Solid phase enzyme immunoassay of human interleukin 2 utilizing antibodies against synthetic IL-2 peptides.

In the present study, solid phase enzyme immunoassay utilizing antibodies against synthetic IL-2 peptides was used for quantitative measurements of human recombinant and lymphoid IL-2 preparations. The 27-peptide MCF-III-6 (Leu-Glu-His-Leu-Leu-Leu-Asp-Leu-Gln-Met-Ile-Leu-Asn-Gly-Ile-Asn-Asn-- Tyr-Lys-Asn-Pro-Lys-Leu-Thr-Arg-Met-Leu) that comprises the region 14-40 from the IL-2 amino acid sequence was synthesized and used for immunization of rabbits. Resulting anti-MCF-III-6 polyvalent rabbit antibodies reacted specifically in EIA up to dilution of 10(-7) with the MCF-III-6 peptide used for immunization as well as with 16-peptide I-16 (Cys-Nle-Gly-Ile-Asn-Asn-Tyr-Lys-Asn-Pro-Lys-Leu-Thr-Arg-Met-Leu) that comprises the region 27-40 from the IL-2 amino acid sequence. The anti-MCF-III-6 antibody reacted also with human recombinant IL-2 preparations obtained from three producers (Cetus, Riga and Amersham), to the concentration of 0.1 ng/ml, and with various human lymphoid IL-2 preparations. Direct correlation was observed between quantitative measurements of human lymphoid IL-2 by EIA and by CTLL bioassay. It can be concluded that utilization of synthetic IL-2 peptides provides a suitable and comparatively unexpensive immunogen for the production of IL-2 antibodies and that the solid phase EIA using such antibodies can be employed as a rapid, reproducible, and sensitive method for quantitative examination of both recombinant and lymphoid IL-2 preparations.

Antiviral and anticellular effects of synthetic (2'-5')-oligoadenylate (A 2' p 5' A 2' p 5' A) in Rauscher murine leukaemia.

Antiviral and antileukaemic effects of the synthetic (2'-5')-oligoadenylate trimer [(2'-5')-ApApA] were demonstrated in BALB/c mice infected with Rauscher murine leukaemia virus (RMLV) by intraperitoneal (i.p.) treatment for 5-20 days (100 micrograms--1 mg daily doses) as evidenced by 72% suppression of viraemia and by decreased activity of serum reverse transcriptase levels. Electron microscopy revealed more than 95% inhibition of RMLV replication as compared to controls in transformed spleen cells from mice treated 5 times with 1 mg dose of (2'-5') ApApA. A significant and dose-dependent reduction of spleen weights of the RMLV-infected mice treated with (2'-5') ApApA was also observed. The antileukaemic effect of (2'-5-') ApApA was enhanced by simultaneous i.p. injection of amphotericin B (20 micrograms/mouse). In comparison to the effect of interferon (IFN) on RNA tumour viruses, our results suggest a higher antiviral activity of the synthetic (2'-5') ApApA oligonucleotide in suppressing RMLV replication in vivo.

[Our approach to the care of chronic instability of the knee joint]. / Nás prístup k osetrování chronických nestabilit kolenního kloubu.

Chronic instability of the knee joint is a serious problem for the affected patient and the attending physician. Diagnosis must reveal which structures of the soft knee are insufficient. We use as a basis in particular a detailed clinical examination which frequently must be made under short-term general anaesthesia. As to auxiliary examination methods, we recommend X-rays in forced positions, possibly arthroscopy. Defects of the cruciate ligaments can be evaluated well also by means of computed tomography. Operation should be indicated carefully. We consider the extent of the patient's complaints, the ability of dynamic compensation of the instability, the presence of exudates, haematomas, signs of arthritis, locomotor restrictions and, last not least, also the assumed ability of the patient to cooperate in rehabilitation. In surgical treatment of instabilities it is necessary to respect carefully biomechanical principles of the function of the knee joint. In prostheses of the cruciate ligaments we prefer direct reconstruction with an autologous graft to extraarticular operations. It seems that in older patients also reconstruction with synthetic ligaments will be feasible.