Autoimmune Limbic Encephalitis in a Patient with Acute Encephalopathy and Hyponatremia.

Acute encephalopathy is a common clinical presentation for hospital admissions. Autoimmune encephalitis is a rare cause of encephalopathy which has increasingly been recognized over the last decade. The detection of various neuronal antibodies has helped diagnose these syndromes, but they have limited availability, mostly in the developed countries. We present a case of a middle-aged female presenting with memory impairment, gait disturbances, and hyponatremia. A clinical diagnosis of autoimmune limbic encephalitis was made based on faciobrachial dystonic seizures, SIADH, and MRI changes 10 days prior to autoantibody titer returned. Prompt treatment with steroids and intravenous immunoglobulin was started with improvement in her neurological symptoms. This case highlights the importance of considering autoimmune encephalitis syndromes in the differential diagnosis of patients with classical neurological presentations and prompt diagnosis and immunotherapy to improve neurological outcomes.

Evaluation of single- and dual antigen delayed fluorescence immunoassay in comparison to an ELISA and the in vivo toxin neutralisation test for detection of diphtheria toxin antibodies.

An evaluation of the delayed fluorescence immunoassay (Delfia) against an ELISA method for determination of diphtheria antitoxin levels in serum was performed. The Delfia was also validated in the in vivo toxin neutralisation test (Txn) in rabbits. Two variants of the Delfia were studied, a single-antigen Delfia (sDelfia) with only the diphtheria toxin included and a dual-antigen Delfia (dDelfia) with tetanus toxoid included for simultaneous detection of antibodies against two antigens. The diphtheria antitoxin cut-off levels in the sDelfia and the dDelfia were 0.004 and 0.002 AU/ml, respectively, which is lower than the internationally accepted level showing any protection against diphtheria (0.01 IU/ml). Both Delfia variants showed good correlation with the ELISA procedure above the ELISA cut-off level of 0.02 AU/ml. Results from samples assayed in the in vivo Txn assay indicated that the low antitoxin levels detected by the Delfia were valid. These results show that the Delfia could be considered as an in vitro reference method for detection of diphtheria antitoxin in seroepidemiological surveys and vaccine studies.

Mortality and morbidity from invasive bacterial infections during a clinical trial of acellular pertussis vaccines in Sweden.

A double blind placebo-controlled efficacy trial of two acellular pertussis vaccines was conducted in 3801 6- to 11-month-old children. Four vaccinated children died during 7 to 9 months follow-up as a result of Haemophilus influenzae type b meningitis, heroin intoxication with concomitant pneumonia, suspected septicemia, and Neisseria meningitidis Group B septicemia. From the actual death rate in children belonging to the same birth cohort in Sweden that could have been eligible for the trial, one death was expected among vaccinated children. Several investigations were carried out to examine the possibility that the deaths could be causally related to the vaccination. The relative risk for hospitalization due to systemic or respiratory infections was 1.07 (95% confidence interval, 0.95 to 1.20) and 0.83 (95% confidence interval, 0.64 to 1.08) in the vaccine groups as compared with the placebo group. Subsets of the population were studied for signs of immunosuppression. There was no indication of immunoglobulin deficiency or any sign of clinically significant leukopenia or lymphocytosis in vaccine recipients. The results of this analysis provide no evidence for a causal relation between vaccination with the studied acellular pertussis vaccines and altered resistance to invasive disease caused by encapsulated bacteria. The hypothesis that the two variables are related, however, cannot be refuted from these data.

Neutralization of tetanus toxin by human monoclonal antibodies directed against tetanus toxin fragment C.

Two hybridomas (designated 143 and 147) producing human monoclonal antibodies (IgG1) directed against tetanus toxin were established by fusion of Epstein-Barr virus transformed human peripheral B lymphocytes with the heteromyeloma SPAM-8. The hybridomas produced antibodies in concentrations of approx. 3.5 micrograms/ml (hybridoma 143) and 6.4 micrograms/ml (hybridoma 147) using conventional flask cultures and 33.9 micrograms/ml and 36.2 micrograms/ml, respectively, in dialysis cultures. The antibodies were shown to react with tetanus toxin, toxoid and fragment C in ELISA, and reactivity with tetanus toxin and fragment C was confirmed in SDS-polyacrylamide gel electrophoresis followed by Western blots. The antibody binding sites were located to two different epitopes of fragment C as shown in a competition assay using biotinylated antibodies. Furthermore, binding of both antibodies to fragment C was inhibited by the addition of the receptor-associated ganglioside GT1b. Neutralization of tetanus toxin in concentrations equivalent to 100-120 IU per mg of antibody was observed for both antibodies in a mouse protection assay.

The importance of storage temperature to the quality of freeze-dried biological products.

Several important practical steps are recognized in the process of freeze-drying of biologic material. The first of these is the selection of conditions optimal for the stability of the substance at the normal storage temperature, usually +5 degrees C. The second step involves the collection of material, storage at some choice freezing temperature (preferably around -20 degrees C) until use. The third step consists of the freeze-drying process itself, with preliminary freezing at another temperature, usually between -60 degrees and -80 degrees C. How these steps affect the quality of biologic products, proteins and polysaccharides has been the subject of study in our laboratories. The results have led us to the conclusion that molecular rearrangements occur at certain critical frozen temperatures which, in combination with time, leads to inactivation in some cases, stability in others. Such critical temperatures can be measured for other products and systems.

An enzymatic time/temperature device for monitoring the handling of perishable commodities.

Kockums Chemical AB, Malmö, Sweden, has developed a biochemical indicator, the i-point TTM (Time-Temperature Monitor). The indicator responds to temperature in much the same way as perishable commodities in which loss of quality is directly related to the combined effects of the degree and the duration of storage temperature. Primary application of such indicators are expected for frozen, refrigerated and fresh food products. The device can be useful in monitoring the handling of various temperature sensitive noon-food products as well. Reacting at a relatively low rate under optimum temperture conditions and at accelerated rates as temperature rises, the indicator chemically integrates and accumulates both the length and degree of all temperature experiences. When pre-selected time-temperature limits have been exceeded a distinct irreversible color change will occur, indicating that action is required--the particular depending upon the use being made of the commodity.

Optimization of immunization schedule to standardize antibody response in mice to pertussis vaccines.

The intracerebral challenge test for determining the protective potency of pertussis vaccines has long been in use. In an effort to elucidate what antibodies are responsible for such protection, a study was undertaken to analyse serum antibodies against filamentous hemagglutinin, lymphocytosis promoting factor and agglutinogens. Conventional pertussis vaccines induced antibodies to FHA and agglutinogens in mice whereas no demonstrable antibodies to LPF were stimulated. By toxoiding LPF on cells with 0.4 per cent formaldehyde, preparations were obtained which induced anti-LPF antibodies in mice. This treatment however, resulted in considerable reduction in potency as judged by the intracerebral challenge test. Apparently the protective potency of pertussis vaccines in mice is based on the adjuvant properties of LPF and not its antigenic properties. An optimized schedule is outlined for the laboratory standardization of pertussis vaccines by measuring antibody responses and especially anti-LPF in mice. Highest titers were obtained in mice when the interval between immunization and booster injections was 28 days.

Bordetella pertussis serotype of clinical isolates in Sweden during 1970-1995 and influence of vaccine efficacy studies.

During the years 1970 to 1995, the serotypes of collected clinical isolates from the different regions of Sweden were examined. Vaccination using whole-cell pertussis vaccine decreased during the year preceding cessation of general vaccination of children in 1979. Although the total number of clinical isolates examined was limited up to 1978 compared to later years, serotype 1, 2, 3 clinical isolates generally predominated. During periodical pertussis outbreaks, serotype 1, 3 strains were isolated, the proportion of which was about equal to that of serotype 1, 2, 3 isolates. Following cessation of general vaccination in 1979, the proportion of both serotypes 1, 2, 3 and 1, 3 isolates gradually diminished, together contributing about 20 percent of the total number of isolates in 1995. Clinical isolates of serotype 1, 2 were identified in the year 1977, just before cessation of general vaccination in Sweden using whole-cell vaccine. Since then, this serotype has gradually increased to over 80 percent of total isolates in 1995, possibly indicating that the use of whole-cell pertussis vaccine was effective against this serotype or that the vaccine influenced serotype expression. Vaccine efficacy studies conducted in Sweden during 1986-87 using one and two-component vaccines (BIKEN) and in 1994-95 using two-component (SmithKline Beecham Pharmaceuticals) and five-component (Connaught Laboratories, Ltd.) vaccines are examined with the object of studying whether the vaccines showed any influence on the different serotypes of B. pertussis causing disease.

The standardization of an assay for pertussis toxin and antitoxin in microplate culture of Chinese hamster ovary cells.

A microplate assay, based on the clustering effect induced by pertussis toxin (PT) in Chinese hamster ovary (CHO) cells, has been developed and standardized. Toxin titration is done directly in the culture microplate by twofold dilutions of 25 microliters of test material to which are added 10 000 freshly trypsinized cells in 200 microliters of culture medium per well. The dilution causing the clustering effect is determined by direct microscopic observation after 48 h of incubation. The method allows detection of 50-100 pg toxin per millilitre. For determination of neutralizing antibodies (antitoxin), twofold dilutions of 25 microliters of antiserum are first made directly in the culture microplate. Thereafter 25 microliters of toxin, containing four times the minimal clustering concentration, is added to each well. After three hours for neutralization at +37 degrees C, cells are added, incubated and examined as above. The assay has been found to be simple and reproducible for measuring the antibody response to PT in human and different animal sera. For titration of bacteria associated toxin, the CHO cells are seeded and incubated for 24 h before the addition of bacteria. Incubation and examination are done as described for toxin titration.

Potency assay and characterization of lymphocytosis promoting factor in whole cell and acellular pertussis vaccines.

Potency assay in mice to evaluate the immunogenic properties of a number of candidate Lymphocytosis Promoting Factor (LPF) antigen in whole cell and acellular pertussis vaccines is described. Potency was estimated both by the conventional WHO intracerebral challenge test and an LPF-toxin challenge method. The latter method involved intraperitoneal injection of dilutions of test and reference preparations followed by challenging the animals 28 days later by LPF combined with AlPO4 adjuvant and estimating the total white blood cells three days after challenge. The method has been in routine use for the past four years and has given consistent results with reproducible specific activity and ED50 values. LPF antigen prepared by different chemical treatment or by genetic alteration of the molecule showed differences in specific activity, although different batches made by the same procedure appeared to give consistent values. Only two preparations, did not give specific activity close to what was given by LPF in whole cell vaccine by the LPF challenge method. Some preparations did not give any activity. In the intracerebral challenge method, many preparations failed to protect, while some gave a low degree of protection. The content of residual ADP ribosylating activity also varied. These three tests can be used for characterizing the LPF antigen of acellular pertussis vaccines.

Diphtheria antitoxin response to DTP vaccines used in Swedish pertussis vaccine trials, persistence and projection for timing of booster.

Data from two Swedish pertussis vaccine trials with various combination vaccines were used to compare anti-diphtheria antitoxin concentrations over time between different vaccines, vaccine lots and vaccine schedules. The immune responses were measured with a validated ELISA method.Results are given for 1326 children, born 1992, that were recruited to the placebo (DT)-controlled Trial I which used a 2, 4, 6 month schedule. Two DTP acellular and one DTP whole cell vaccine were used. No DT boosters were given until 5 years of age. Trial II recruited children born 1993-94 and compared three DTP acellular vaccines with one DTP whole cell vaccine. Results are given for 306 children in a 2, 4, 6 month schedule and for 531 children in a 3, 5, 12 month schedule. The latter schedule gave significantly higher diphtheria antitoxin concentrations post third dose. The various DTP acellular vaccines and an inefficacious DTP whole cell vaccine gave lower antitoxin concentrations than both an efficacious DTP whole cell vaccine and the DT vaccine. The larger differences in antigen response between vaccines was reduced in the course of time. Generally, an initial rapid decline of antitoxin concentration was followed by a slower decline; the change typically occurring when the antitoxin concentration reached 0.13-0.16 EU/ml. The time needed to reach this level was between 6 and 10 months based on the initial vaccine response.A "best-fit" combined exponential regression model was used to predict the optimal timing for booster vaccinations against diphtheria.Our data support a 3, 5, 12 month schedule followed by a fourth dose 4-5 years after the third dose, depending upon the vaccine used.

Acellular pertussis vaccine in adults: adverse reactions and immune response.

An acellular pertussis vaccine JNIH-6 containing pertussis toxin and filamentous hemagglutinin was evaluated in adult volunteers with regard to adverse reactions and antibody response. Adverse reactions were few and mild. A late onset local reaction was seen in 22 of the 47 vaccinees (47%) as compared to none of the 20 subjects receiving a placebo, the carrier solution of aluminium phosphate of the vaccine. The reaction, which manifested itself on the 6th to 8th day after vaccination, consisted in all cases of an induration and/or swelling considered insignificant by the majority of the subjects. The reaction was only found in vaccinees receiving a first dose of vaccine and was independent of the prevaccination antitoxin level. The vaccine induced a highly satisfactory antibody response to both filamentous hemagglutinin and pertussis toxin.

A clinical trial of a monocomponent pertussis toxoid vaccine.

We compared a monocomponent pertussis toxoid vaccine preparation (JNIH-7) in a double-blind, randomized study with another component vaccine (JNIH-6) containing pertussis toxoid and filamentous hemagglutinin in equal proportions. Monocomponent pertussis toxoid vaccines have not been tested previously in children. This trial comprised 255 previously nonimmunized children 6 to 10 months old who were given two 0.5-mL subcutaneous injections of either vaccine four to five weeks apart. Prevaccination and postvaccination serum samples from 235 infants showed an antitoxin seroconversion rate of 100% in both groups. Adverse reactions were few and mild, with local reactions occurring more often after administration of the second dose and among recipients of the two-component vaccine (P less than .05).

Preliminary data from a clinical trial (phase 2) of an acellular pertussis vaccine, J-NIH-6.

In the search for a new and better pertussis vaccine for general vaccination in Sweden, a clinical trial of an adsorbed acellular vaccine from Japan has been performed. The vaccine used was produced by Biken and contained only F-HA and the toxoid of pertussis toxin (LPF). The aims were: To demonstrate the serological response to primary vaccination of this adsorbed acellular vaccine in comparison to a plain whole cell vaccine. To compare the incidence of side reactions between the two types of vaccines and the adjuvant (aluminium phosphate) of the acellular vaccine used as placebo.