Resistance patterns of selected respiratory tract pathogens in Poland.

This study presents the results of a survey of the in-vitro susceptibility to antimicrobial agents of major pathogens responsible for community-acquired respiratory tract infections in Poland during 2002-2004. The collection of 1184 bacterial isolates comprised 398 Streptococcus pneumoniae, 344 Haemophilus influenzae, 302 Streptococcus pyogenes and 140 Moraxella catarrhalis. Among the pneumococcal isolates, 16.8% were penicillin-non-susceptible (PNSP), of which 80.6% were identified as multidrug-resistant. Overall, 9.0% of H. influenzae isolates were beta-lactamase-positive, although this percentage increased noticeably in the third year of the study. Based on PCR results, 12.8% of H. influenzae isolates were identified as low-level beta-lactamase-negative, ampicillin-resistant (BLNAR), and one isolate as low-level beta-lactamase-positive, amoxycillin-clavulanic acid-resistant (BLPACR). Pulsed-field gel electrophoresis (PFGE) classified 45 H. influenzae isolates with altered penicillin-binding proteins into 15 PFGE types, including two predominant types (with four and six sub-types) containing 15 and ten isolates, respectively. Resistance to tetracycline, erythromycin and clindamycin was found in 20.9%, 8.9% and 4.6% of S. pyogenes isolates, respectively. The production of beta-lactamase characterised 91.4% of M. catarrhalis isolates. In summary, the overall occurrence of PNSP in Poland remains stable, although there was a noticeable increase in the proportion of fully-resistant isolates. A rising trend in the prevalence of beta-lactamase producers and low-level BLNAR isolates was observed among Polish isolates of H. influenzae.

Characterisation of Neisseria meningitidis C:2b:P1.2,5 isolates in Poland.

This study aimed to characterise Neisseria meningitidis C:2b:P1.2,5 isolates from Poland, which have now become predominant among serogroup C isolates in this country. Overall, 44 isolates (25 invasive and 19 from contact carriers) were typed by whole-cell ELISA and pulsed-field gel electrophoresis. Additionally, the invasive isolates were analysed by multilocus sequence typing, which revealed that they all belonged to the ST-8 complex/cluster A4. The emergence of this clone in other countries has resulted in mass immunisation campaigns and has been associated with a higher level of decreased susceptibility to penicillin; however the present study detected only one isolate that was penicillin-non-susceptible.

Lack of specificity for antibodies to double stranded DNA found in four commercial kits.

Four commercially available kits (three enzyme linked immunosorbent assays and one modified Farr radioimmunoassay) were compared for their ability to detect specifically autoantibodies to double-stranded DNA (dsDNA) using 66 patient sera. This was assessed by comparing the results of the kits with those from an ELISA specifically measuring antibodies against highly purified dsDNA, single-stranded DNA (ssDNA), native DNA and histones. The RIA and two of the ELISAs seemed equally efficient at detecting antibodies to dsDNA, but all three also detected anti-ssDNA (the RIA being particularly bad for this). The need for highly purified dsDNA was clearly shown. The results obtained with one ELISA did not correlate with any variable investigated in this study. A total of 220 sera were assayed with the IDS RIA, of which 130 were recorded as positive. Of these, 50 sera seemed to contain no identifiable autoantibodies. This very high false positive rate may be due, at least in part, to precipitation of nonspecifically bound labelled DNA.

Anti-muscle antibodies in Graves' ophthalmopathy.

The nature of the humoral immune response in patients with Graves' ophthalmopathy has been investigated using a solid phase 125I Protein A binding assay. Retro-orbital muscle (R.O.M.), skeletal muscle (Sk.M.), R.O.M.-membranes, thyroid, kidney, liver, harderian gland, acetylcholine receptors (AchR), actin and myosin were used as target antigens. No significant difference in antibody binding profile to R.O.M. and Sk.M. was found indicating that the ophthalmic immunoglobulins (OIgs) were not recognising a R.O.M. specific antigen(s). Comparison between R.O.M. and R.O.M.-membranes, however, revealed that these antigens were detecting very different antibody populations. Using the former, it appeared that the predominant antibody population being measured was anti-myosin whereas the latter appeared to be detecting primarily anti-AchR antibodies. Anti-actin antibodies were also present in some of the sera. Thus a spectrum of anti-R.O.M. antibodies appears to be present in Graves' ophthalmopathy but the cross-reactivity of these with non-R.O.M. skeletal muscle and their similarity to those found in myasthenia gravis prevent them as yet being used to explain the specific immunopathology observed in this disease.

Immunity in Graves' disease at diagnosis: correlation between activated T cells and humoral immune factors.

Activated T cells, T-cell subsets, thyrotropin receptor antibodies and immune complexes were evaluated in 31 patients with newly diagnosed Graves' disease. Activated T cells were assayed by monoclonal antibodies against early (4F2) and late activation surface lymphocyte antigens (different epitopes of class II antigens). In comparison with the normal population, Graves' patients showed a significant decrease in the suppressor cytotoxic T-cell subset. Significant increases of 4F2-positive cells (70% of patients studied), class II antigen-positive cells (65%), thyrotropin receptor antibodies (93%), Clq-immune complexeses (44%) and conglutinin-immune complexes (37%) were observed. A significant inverse correlation between the increase in 4F2-positive cells and thyrotropin receptor antibody values was also observed. Lymphocytes from Graves' patients were cultured in the presence of thyrotropin receptor antibody-positive or -negative sera, with or without mitogen stimulation. Thyrotropin receptor antibodies were shown not to interfere with the expression of activation antigens in cultured cells. The different patterns of humoral and cellular immune phenomena may indicate the existence of either different stages of Graves' disease or a heterogeneity of the immunopathogenesis in different patients.

The lack of specificity of ophthalmic immunoglobulins in Graves' disease.

Immunoglobulins (Igs) binding to retro-orbital muscle (ROM) antigens, known as ophthalmic Igs (OIg), were measured using a 100,000 X g sediment of porcine ROM as antigen in a solid phase [125I]protein A binding assay. Serum samples from 50 control subjects bound from 0.60-2.42 times the amount of [125I]protein A as did the normal reference serum samples, defined as the OIg ratio. Serum from 95 patients with hyperthyroid Graves' disease had OIg ratios from 0.64-9.99, with 24 (25%) being positive [OIg ratio greater than 2.05 (mean + 2 SD of the normal group)]. Ten patients with euthyroid Graves' ophthalmopathy had OIg ratios from 1.01-6.33, with 6 (60%) being positive. Among those Graves' disease patients with ophthalmopathy (n = 19) and the euthyroid Graves' ophthalmopathy patients there was a good correlation between the severity of eye signs and the OIg ratio. The OIg-positive serum samples cross-reacted with skeletal muscle and thyroid as well as with ROM antigen. This lack of specificity contradicts previous reports, but does not rule out a role for these antibodies in the etiology of Graves' ophthalmopathy.

Immune responses to myelin antigens in Guillain-Barré syndrome.

Antibodies to nerve antigens were sought in the sera of 17 patients with acute Guillain-Barré syndrome (GBS), 11 with chronic relapsing demyelinating poly-radiculoneuropathy (CRP), 20 with other neuropathies (ON), 15 with other neurological diseases (OND) and 19 normal subjects. Complement-fixing antibodies to a suspension of human peripheral nerve tissue were identified in only 2 patients with GBS and 1 with chronic progressive neuropathy. Five GBS sera gave complement fixation reactions with rabbit sciatic nerve. The sera were also tested for galactocerebroside (Gal-C) binding activity using a solid phase assay. The range of values in all groups was the same, although the mean values for patients with GBS, ON and OND were higher than those of normal subjects. In a radioimmunoassay for antibodies to bovine P2 slightly more radiolabelled antigen was precipitated by the GBS group of sera than by sera from the other groups, but only one serum from the GBS and another from the CRP patients precipitated more than 10% of the label. Addition of bovine P2 to cultures of peripheral blood mononuclear cells from 11 patients with GBS did not cause significant stimulation. Immunoassay for antibody to myelin basic protein (MBP) showed an increased proportion of sera with low binding activity in the GBS and CRP groups. The results suggest that humoral immune responses to potentially neuritogenic antigens are found with marginally increased frequency in patients with GBS and CRP.

Spontaneous and experimental neuritis and the distribution of the myelin protein P2 in the nervous system.

The P2 contents of nervous tissues from the human, rabbit, guinea pig, and Lewis rat were measured by radioimmunoassay. The ventral spinal roots contained more P2 than any other tissue. Human dorsal roots and peripheral nerves contained 41-65% of the amount in human ventral roots. Human olfactory and optic nerves and brain contained 1.1-2.7%, spinal cord, 2.8%, cranial nerve VIII, 11%, and cerebral grey matter, 0%. The relative amounts in the rabbit nervous system were similar except that the spinal cord contained 20% of the amount in the ventral roots. Qualitative estimates in the guinea pig showed that the spinal roots and peripheral nerves contained more P2 than the spinal cord, and that none was present in the brain. In the Lewis rat, P2 could be detected in the spinal roots and peripheral nerves but not in the CNS. The distribution of P2 in the human nervous system parallels the incidence and severity of lesions in acute polyradiculoneuritis. It also explains the absence of any lesions in the CNS when experimental allergic neuritis is induced in the Lewis rat.

Immunocytochemical study of the appearance of P2 in developing rat peripheral nerve: comparison with other myelin components.

Using indirect immunofluorescence on both dissociated cell cultures and frozen sections from rat sciatic nerve, dorsal root ganglion and superior cervical ganglion, we have examined the development and distribution of the peripheral myelin protein P2. It first appears in development in sciatic nerve and dorsal root ganglion on the first day of birth, at about the same time as P0 and P1. Like galactocerebroside, P0 and P1, P2 disappears gradually from dissociated Schwann cells in culture. In adult sciatic nerve, and dorsal and ventral roots, it shows an uneven distribution and is absent from some myelinated axons. In electron micrographs the onset of myelination in the sciatic nerve occurs between the day of birth (day 0) and the first day after birth (day 1). Immunofluorescence studies on freshly dissociated cell suspensions, frozen sections and dissociated cell cultures at these early time points indicate that the myelin glycolipids galactocerebroside and sulphatide are present on the surface of many schwann cells at least one day before myelination starts while the myelin proteins P0, P1 and P2 are not detected until myelination begins. This suggests that the early appearance of galactocerebroside and sulphatide is an important step preceding the formation of compact myelin.

Immune responses in experimental allergic neuritis.

The antibody and cell mediated immune responses were investigated in inbred Lewis rats with experimental allergic neuritis (EAN) induced by either P2, a protein purified from the bovine cauda equina nerve roots, or whole bovine nerve root myelin. In the P2 immunised animals both antibodies to P2 detected by radioimmunoassay and cell-mediated immunity to P2 assayed by skin testing appeared before the onset of EAN and persisted during and after the disease. In the myelin immunised animals the antibody titres were lower and somewhat delayed and the skin tests became negative at the height of the disease. Complement-fixing antibodies to galactocerebroside, which have been implicated in the production of demyelination under some circumstances, could not be detected in the serum after immunisation with either P2 or myelin. EAN was transferred passively with lymph node cells from rats immunised with either P2 or myelin although anti-P2 antibodies could not be detected in the serum of recipients with EAN. The results favour a cell-mediated immune response to P2 as the most important pathogenetic mechanism in EAN induced wtih whole myelin in the rat.

Treatment of acute inflammatory polyneuropathy.

Most patients with acute inflammatory polyneuropathy (AIP) recover spontaneously, but the time course of the illness is unpredictable so that the results of treatment are difficult to assess. Three decades of retrospective reports of steroid treatment fail to demonstrate any striking beneficial effect. In a randomized trial of prednisolone, starting dose 60 mg daily, 21 treated patients improved more slowly than 19 untreated patients. By contrast, in rats immunized with bovine nerve root myelin, prednisolone at 10 mg/kg reduced the severity and duration of experimental allergic neuritis (EAN), the putative animal model for AIP. This discrepancy might reflect the greater difficulty of clinical as opposed to animal therapeutic trials or indicate that EAN is not the appropriate model for the human disease. Immunosuppressive drugs, plasmapheresis and other agents have also been employed, but their efficacy cannot be decided from the available case report. The role of similar agents in chronic progressive and relapsing inflammatory neuropathy cannot yet be resolved, but in some patients steroids do appear to be valuable.

The neuritogenicity and encephalitogenicity of P2 in the rat, guinea-pig and rabbit.

In inbred Lewis rats, P2 basic protein from bovine peripheral nervous system (PNS) myelin produced experimental allergic neuritis (EAN) without involvement of the brain or spinal cord. In guinea-pigs, bovine P2 did not produce EAN but large doses produced mild experimental allergic encephalomyelitis (EAE). In rabbits, bovine P2 produced both mild EAE and EAN. Human P2 produced severe EAN in the Lewis rat, but only mild EAN with quite marked EAE in the guinea-pig. Material cross-reacting with bovine P on immunodiffusion was identified in the extracts from the nerves of all three species but only in the spinal cord of the guinea-pig and rabbit, not in the rat spinal cord. The species differences in response to immunisation with P2 cannot be simply explained by the presence or absence of P2 in their PNS or CNS, but may reflect differences in the immune response.

Experimental allergic neuritis in the Lewis rat: characterization of the activity of peripheral myelin and its major basic protein,P2.

Experimental allergic neuritis has been produced in the inbred Lewis rat in the absence of experimental allergic encephalomyelitis (EAE) using bovine intradural root myelin. The lack of EAE is probably because P1 is only weakly encephalitogenic in the rat. One of the basic proteins of bovine peripheral myelin, P2, was isolated and demonstrated to be pure by amino acid analysis and SDS PAGE. It was found to have a molecular weight of 15,400 and contained 4 mol 1/2-cystine/mol. This P2 was found to be highly neuritogenic and is probably the sole neuritogenic antigen in this system. The successful demonstration of its neuritogenicity must be due in large part to the use of the inbred Lewis rat and bovine P2, but an explanation could also involve the omission of denaturing organic solvents, the prevention of oxidative denaturation and presumably the fact that any changes which may occur are not sufficient to prevent recognition of the active site by the immune system of the inbred Lewis rat. P2 was neuritogenic down to 5 micrograms/animal. Its activity was enhanced by but not dependent on the presence of Mycobacterium in the adjuvant. This suggested that release of P2 could possibly break tolerance and produce an auto-immune disease such as the Guillain--Barre syndrome.