Acute renal failure in dense deposit disease: complete recovery after combination therapy with immunosuppressant and plasma exchange.

We describe the clinical course of a female adolescent who was followed because of isolated microhematuria and hypocomplementemia before admission to hospital with a sudden onset of acute renal failure. At presentation, she exhibited complement consumption through the complement alternative pathway (AP) while other serologic tests were negative. Renal biopsy revealed dense deposit disease (DDD) with a crescentic pattern. Intravenous methylprednisolone, followed by plasma exchange (PE), and intravenous cyclophosphamide pulses were started shortly after admission. C3NeF and anti-factor H antibody tests were negative. Serum factor H and I levels were normal as well as factor H activity. Screening for mutation in the factor H gene revealed the H402 allele variant. Clinical remission, defined as normalization in renal function and in the activity levels of the complement AP, was noted at one month post-presentation and throughout the follow-up. A repeat renal biopsy showed the disappearance of crescent formation, whereas electron microscopy revealed no regression in dense transformation of the lamina densa. In summary, our patient was successfully treated with immunosuppressant and PE. The absence of known factors associated with DDD suggests that, in this particular case, other regulatory mechanisms of complement AP might have been involved in the disease process.

Epoetin beta in the treatment of anemia in patients with advanced gastrointestinal cancer.

PURPOSE: The possibility that epoetin beta (EPO) could increase hemoglobin (B-Hb) levels and improve quality of life (QoL) in patients with advanced gastrointestinal cancers was investigated. PATIENTS AND METHODS: One hundred patients with gastric, pancreatic, biliary, or colorectal cancers and subnormal B-Hb levels were included in a randomized study to test low-dose EPO (2,000 U subcutaneously thrice weekly [2,000 group]) against a higher dose (10,000 U times three [10,000 group]). Eighty-four patients were treated with chemotherapy. QoL was evaluated using the European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30 instrument. RESULTS: At baseline, mean B-Hb was 108 g/L with no difference between the groups. In the 10,000 group, an increase in B-Hb (>10 g/L) was seen in 30 (73%) patients treated with chemotherapy, after a median of 4 weeks, whereas a corresponding increase in the 2,000 group was seen in 15 (30%) patients after a median of 10 weeks (P < .001). A difference in the proportion of responders (five of eight v one of eight) was also seen in the group of patients not treated with chemotherapy. The proportion of responders was independent of baseline endogenous serum EPO level or observed/predicted log10 serum (S)-EPO levels. Patients who demonstrated improved B-Hb levels also showed improvements in QoL parameters. Tumor response was usually also associated with QoL improvements. CONCLUSION: Treatment with EPO at a dose of 10,000 U thrice weekly can rapidly and safely increase B-Hb levels in a high proportion of patients with advanced gastrointestinal cancers. QoL is influenced by the B-Hb increase, but also by the course of the underlying malignancy. It is therefore difficult to define clearly the clinical relevance of the B-Hb increase as such.

Quantitation of C-reactive protein in cerebrospinal fluid and serum by zone immunoelectrophoresis assay (ZIA).

The zone immunoelectrophoresis assay (ZIA) for C-reactive protein (CRP) determinations is easy to perform and requires only small amount of antiserum, e.g., 25-100 and 0.5-1.0 microliter anti-CRP antibody/20 serum and CSF samples, respectively. For quantitating CSF-CRP the immunoprecipitates formed were stained using alkaline phosphatase-conjugated secondary antibodies and the lowest standard concentration used was 30 micrograms/l. The immunoprecipitates formed when measuring CRP in serum were stained by Coomasie brilliant blue R250 with a detection limit of about 300 micrograms/l. CRP was determined in cerebrospinal fluid in 27 patients with bacterial meningitis (range less than 0.03-23.0 mg/l) and in 25 patients with viral meningitis (range less than 0.03-0.23 mg/l). CRP was quantitated in 52 sera by both the CRP ZIA method (y) and by electroimmunoassay (x). The correlation coefficient was r = 0.992 with the regression line y = 1.024 x + 0.855.

Differences in the E3 regions of the canine adenovirus type 1 and type 2.

Sequence analysis of a region extending between the pVIII and the fiber gene of Canine adenovirus type 1 (Cav-1, Utrecht) and type 2 (Cav-2, Manhattan) was performed. The results revealed a high level of identity between the two viruses when the pVIII gene and the N-terminal part of the fiber gene were compared. The open reading frames of region E3 in of Cav-1 and Cav-2 encoded a 22 kDa and 40.7 kDa polypeptide, respectively. The Cav-1 and Cav-2 E3 region polypeptides shared conserved amino and carboxyl domains. In Cav-2 an extra sequence of about 500 nucleotides was present, appearing like an in frame insertion of foreign DNA. It can be speculated that this insertion in the E3 region contributes to the observed biological differences between Cav-1 and Cav-2.

Analysis of the large (L) protein gene of the porcine rubulavirus LPMV: identification of possible functional domains.

The complete nucleotide sequence of the porcine rubulavirus LPMV (La Piedad Michoacan virus) large (L) protein gene was determined and analysed. The L mRNA was found to span 6,786 nucleotides, containing one single large open reading frame (ORF), putatively encoding a polypeptide of 2,251 amino acids. By aligning the amino acid sequence of the LPMV L-protein with L-protein of a number of viruses belonging to the order mononegavirale, a high degree of similarity between the LPMV L-protein and other rubula virus L-proteins was demonstrated, extending through almost the whole protein. Additionally we could identify several regions as being highly conserved among all studied viruses of the order mononegavirale. The significance of these regions are discussed.

Evolution of H3N8 equine influenza virus from 1963 to 1991.

The antigenic properties of H3N8 influenza viruses isolated from outbreaks of equine influenza in Sweden between 1979 and 1991 have been studied in hemagglutination inhibition tests with polyclonal and monoclonal antisera, and antigenic drift of the virus has been demonstrated. To clarify the basis of the antigenic drift, amino acid sequences of the globular head regions (HA1) of the hemagglutinin membrane glycoproteins of virus strains from 1979, 1984, 1988 and 1990 have been deduced from the nucleotide sequences of the hemagglutinin genes, and the sequence information has been used to construct a phylogenetic tree of H3N8 equine influenza strains. Several strains from previous studies have been included to give a clearer picture of viral evolution in an international context.

Equine influenza virus from the 1991 Swedish epizootic shows major genetic and antigenic divergence from the prototype virus.

The antigenic properties of H3N8 equine influenza virus from the Swedish epizootic of 1991 differ from those of A/eq 2/Fontainebleau/79 (representative of the Swedish vaccine strain) in hemagglutination inhibition tests. The amino acid sequence of the hemagglutinin (HA) of an isolate from the 1991 outbreak was deduced from the nucleotide sequence and comparison was made to the A/eq 2/Fontainebleau/79 strain. Twenty-three amino acid substitutions were found, 10 mapping onto areas of the HA known to bind antibodies in human H3 influenza viruses. The amino acid changes together with the serological data suggest that a major antigenic drift has taken place in equine H3N8 viruses in Sweden and we conclude that recent strains of the virus must be incorporated into vaccines on a regular basis if epizootics of equine influenza are to be controlled in the future.

Plasmid profile analysis and restriction enzyme fingerprinting of Salmonella DO-group strains.

In Sweden Salmonella dublin is the most common serotype within the DO-group isolated from animals. In recent years also salmonella strains belonging to the DO-group but lacking H-antigen have been isolated from cattle in different areas. It was not possible to further differentiate the latter strains by serological methods. However, all Salmonella dublin strains and those strains lacking H-antigen carried a 50 Mdal plasmid exhibiting the same EcoRI and Hind III restriction enzyme digestion pattern. Two of the 26 strains contained an additional 5 Mdal plasmid. Other serotypes within the DO-group investigated, namely Salmonella enteritidis and Salmonella panama, carried plasmids smaller than 50 Mdal. The plasmid profiles and restriction enzyme digestion patterns indicated that the salmonella strains lacking H-antigen were variants of Salmonella dublin. Thus, analysis of plasmid profile and restriction enzyme fingerprinting are useful complements to serological methods in the differentiation of salmonella DO-group strains.

Single-dose etoposide in advanced pancreatic and biliary cancer, a phase II study.

Palliative chemotherapy can add to the duration and quality of life in patients with advanced pancreatic and biliary cancer, albeit in a limited way. Between March 1995 and October 1997, 31 symptomatic patients were treated with etoposide in a phase II trial. Measurements of objective and subjective responses were performed, the latter by the treating physician and with the method of clinical benefit response (CBR). Quality of life was evaluated with the EORTC QLQ-C30 questionnaire. A partial response was seen in 2 (6%) patients. Subjective responses/quality of life gains were seen in 6 (19%), 7 (23%) and 9 (29%) patients, respectively, with the different methods. Median survival was 4.5 months. WHO grade 3 and 4 toxicity, alopecia excluded, was seen in 20% of the patients. The clinical activity of etoposide is limited, and in the same low range as other drugs in these diseases.

Genetic drift of equine 2 influenza A virus (H3N8), 1963-1988: analysis by oligonucleotide mapping.

Comparative analysis by RNA oligonucleotide fingerprints of total genomic RNA as well as the individual RNA segments of equine 2 influenza A virus strains from 1963, 1968, 1979, 1984, 1987 and 1988 revealed genetic diversity. Strains from the epizootic outbreak during 1978-1979 showed minor differences among their genomes. The Swedish isolates from 1979 up to 1988 showed increasing genomic heterogeneity indicating genetic drift.

The molecular biology of the porcine paramyxovirus LPMV.

Protein and genomic studies of a previously uncharacterized porcine paramyxovirus (designated LPMV) confirmed that it was a member of the paramyxovirus genus. The nucleotide sequences and deduced amino acids of the complete P-gene, M-gene, F-gene and HN-gene as well as the intergenic sequences have been determined. Comparative sequence analysis of the M-gene of LPMV revealed the closest relationship of LPMV was to human mumps virus with a homology of 46% and 55% at the amino acid and nucleotide levels respectively. The P-gene of LPMV is transcribed to V protein mRNA and by editing of the gene to the P protein mRNA. The LPMV P-gene has the coding capacity for an additional protein of 126 amino acids, a C protein.

Detection of aerolysin gene in Aeromonas strains isolated from drinking water, fish and foods by the polymerase chain reaction.

A polymerase chain reaction (PCR) technique was used to assay the presence of the aerolysin gene in a total of 89 Aeromonas hydrophila and A. sobria strains isolated from drinking water, fish and foods. These strains were also characterized for the production of virulence factors such as haemolysin, protease and cytotoxin. The primers used in the PCR targeted a 209-bp fragment of the aer gene coding for the beta-haemolysin and detected template DNA only in haemolytic A. hydrophila strains. The cell-free culture supernatants of these aerolysin-positive A. hydrophila strains were also cytotoxic to the HeLa and McCoy cells. The haemolytic A. sobria and non-haemolytic A. hydrophila were consistently negative in the PCR assay. Primer specificity was determined in the PCR by using a control haemolytic Escherichia coli, Streptococcus pyogenes and a restriction endonuclease assay. The PCR clearly identified the aerolysin-producing strains of A. hydrophila and may have application as a rapid species-specific virulence test.

Cerebral abnormalities in Wilson's disease as evaluated by ultra-low-field magnetic resonance imaging and computerized image processing.

The cerebral involvement of a 13-yr-old boy with Wilson's disease was serially evaluated during the first 18 mo of D-penicillamine treatment. An ultra-low-field magnetic resonance imaging (ULF MRI) system, operating at 0.02 T, with computerized image processing was used. The half-yr period prior to the clinical diagnosis was set, the patient had showed poor school performance, emotional lability, deteriorating handwriting, progressively slow, gross, and fine motor functions, and a fixed rigid smile. No overt signs of liver disease were found. With D-penicillamine treatment (1-1.5 g/d) a continuous improvement was seen. The pretreatment MRI investigation showed pronounced pathological transformation in the basal ganglia. However, changes were seen also in most other parts of the brain indicating diffuse involvement. During treatment the computerized MR images became gradually more normal. The current magnetic resonance imaging system with computerized image processing is a sensitive and simple method for evaluation of subtle parenchymal changes of the brain.

Lymphocyte subpopulations and immunoglobulin production in IgA nephropathy.

Lymphocyte subpopulations were identified and Ig production in vitro was studied in patients with IgA nephropathy and in age-matched controls. The investigations were performed in an infection-free interval. The proportions of T-lymphocytes with Fc receptors for IgG and IgM, respectively, did not differ between patients and controls. T-suppressor (Leu 2A) and T-helper (Leu 3A) cells, as defined by monoclonal antibodies, were within normal range. The PWM-stimulated in vitro synthesis of IgA, IgG and IgM was somewhat higher in the patients than in the controls. However, the differences were not statistically significant. Thus, in the infection-free interval no evidence for a uniform disturbance in the lymphocyte subsets could be found.

Simplified polyacrylamide gel electrophoresis and silver staining for analysis and preparation of influenza virus RNA segments.

The influenza virus has a genome consisting of eight RNA segments. A simplified technique to study the RNA segmental pattern by silver staining after gel electrophoresis has been developed. In addition, individual RNA segments could be isolated by a combination of polyacrylamide gel electrophoresis and isotachophoresis.

A comparative study on the use of virus and antibody detection techniques for the diagnosis of La Piedad Michoacan paramyxovirus (LPMV) infection in pigs.

La Piedad Michoacan paramyxovirus (LPMV) is newly recognized paramyxovirus that has been associated with neurologic and reproductive disorders in pigs in Mexico. To date, no comparative study of methods for the diagnosis of infection with this virus has been published. In this study, we identified tissues containing maximum virus load to optimize virus isolation procedures, and we compared this method to a rapid diagnostic test employing immunostaining of impression smears for LPMV antigens. In addition, several of the available tests for detecting LPMV antibodies were compared for their sensitivity in detecting seroconversion. Pigs used for the study of virus load in tissues and serologic studies were inoculated at 17 days of age with 10(7.00) TCID50 of LPMV. Serial blood samples were collected from selected pigs, and selected pigs were necropsied over a 14-day period. Pigs used in the investigation comparing standard virus isolation techniques to immunostaining of impression smears were inoculated at 3 days of age as described above and necropsied over an 8-day period. The results demonstrate that in the 17-day-old pigs maximum virus titers were detected in olfactory bulb at 5 days postinoculation (PI) and in midbrain at 9 days PI. In addition, the most consistent recovery of high titer virus was from tonsil (3-9 days PI) and olfactory bulb (4-9 days PI). Immunostaining of impression smears was as sensitive as virus isolation when selected tissues (lung, midbrain, olfactory bulb) were compared, with virus detected by both methods in 11/13 samples and in 1 sample each by immunostaining and virus isolation, respectively. All of the serology tests investigated detected seroconversion in pigs by 8 days PI. The identification of target organs where highest virus titers are found combined with immunofluorescent methods for the detection of LPMV antigens and a comparative study of the available serologic tests should facilitate the selection of techniques suitable for any laboratory to diagnose LPMV infection in pigs.

A sequential study of experimental porcine paramyxovirus (LPMV) infection of pigs: immunostaining of cryostat sections and virus isolation.

La Piedad Michoacan Paramyxovirus (LPMV) is a recently recognized paramyxovirus infecting pigs throughout Mexico. Disease syndromes observed in field cases associated with LPMV infection include neurologic, respiratory, and reproductive disorders. Clinical signs and the distribution of LPMV virus and antigen in tissue samples from pigs experimentally infected with LPMV by natural routes were studied. Severe neurologic disease and death occurred following experimental inoculation of 3- and 17-day-old pigs. All of the pigs inoculated at 3 days of age were either dead or moribund by 8 days after inoculation, whereas 30% of the pigs inoculated at 17 days of age were affected. Virus was consistently recovered from or demonstrated in tissues from the respiratory tract of both groups of pigs. LPMV and antigen were also demonstrated in central nervous system (CNS) tissues from these pigs; however, differences in virus distribution within the CNS were demonstrated in the 2 groups. In the pigs inoculated at 17 days of age, isolation of LPMV was restricted to the olfactory bulb and midbrain. In contrast, in the pigs inoculated at 3 days of age, isolation of LPMV was more widespread throughout the CNS tissue examined. Virus excretion studies indicated that nasal spread of LPMV was more important than fecal spread. Comparatively large quantities of infectious LPMV were consistently recovered from urine samples of experimentally infected pigs.

Diagnosis of pseudorabies virus infection in pigs with specific DNA probes.

Two DNA probes directed against pseudorabies virus DNA were developed for use in a dot hybridisation to detect the presence of pseudorabies virus in infected tissues. The specificity of the probes and their sensitivity were analysed. There was a strong correlation between the results obtained by dot hybridisation and those obtained by conventional methods of virus isolation from the same tissue specimens.

Rapid detection of Aujeszky's disease (pseudorabies) virus infection of pigs by direct filter hybridisation of nasal and tonsillar specimens.

A direct filter hybridisation method has been developed to diagnose acute Aujeszky's disease in live pigs. The advantages of the method are easy, fast sample processing; no DNA-purification is needed, and the hybridisation itself is simplified. The direct filter hybridisation method has been tested on pseudorabies virus infected cultured cells, experimentally infected pigs and on specimens from an outbreak of Aujeszky's disease. Virus isolation and filter hybridisation gave comparable results, indicating that the direct filter hybridisation method is a good tool for rapid diagnosis. It is independent of cell culture facilities and the disease can be diagnosed in live animals within 15 hours.

Establishment and characterisation of a porcine rubulavirus (LPMV) persistent infection in porcine kidney cells.

Porcine rubulavirus (LPMV) can establish persistent infections in porcine kidney cells. Cell cultures characterised at passages 25 and 65 demonstrated haemadsorption, formation of syncytia, and a slower growth rate. The nucleoprotein (NP) and haemagglutinin-neuraminidase (HN) protein were present in all cells, although not to the same extent as in wild type infected cells. Incubation of the cell cultures with virus neutralising antibodies could not cure them from the infection. The cells were resistant to LPMV high multiplicity superinfection, but lysed rapidly upon infection with VSV. These cells thus fulfilled the criteria of a true persistent infection. Viral particles were released into the medium from the persistently infected cells as measured by HA and infection of PK-15 cells with medium from the persistently infected cells. The infectious titer of the virus released from the persistently infected cells was 3 logs lower compared to wild type virus, the HN titer still being comparable. Virus released from the persistently infected cells was unable to cause a lytic infection in PK-15 cells, and showed a reduced ability to spread when compared to a LPMV lytic infection.