Specific activation of B-cell clones within the central nervous system in course of herpes simplex encephalitis

Total and virus-specific immunoglobulin (Ig) G oligoclonal bands were studied in paired serum and cerebrospinal fluid (CSF) of four patients with herpes simplex virus type 1 (HSV-1) encephalitis. We used the isoelectric focusing in agarose gel, a sensitive technique for protein separation, followed by passive transfer of proteins on nitrocellulose paper and specific immunostaining. Oligoclonal bands were observed in serum and CSF of all patients. HSV-1-specific oligoclonal IgG bands were present in the CSF only during a limited period of the disease, having their counterpart in serum during the remaining periods. Our findings contribute to tackle the issue of B-cell activation within central nervous system and peripheral blood compartments in course of HSV-1 encephalitis.     

Analysis of cerebrospinal fluid proteins by electrophoresis

The cerebrospinal fluid (CSF) is a specific ultrafiltrate of plasma, which surrounds the brain and spinal cord. The study of its proteins and their alteration may yield useful information on several neurological diseases. By using various electrophoretic separation techniques, several CSF proteins have been identified derived from plasma or from brain. Different one-dimensional methods, such as agarose gel electrophoresis and isoelectric focusing, are of similar value in identifying the non-specific oligoclonal bands, which are mainly helpful in the diagnosis of multiple sclerosis and other inflammatory diseases. Isoelectric focusing has a greater resolution than other one-dimensional methods, and it yields additional data about disease-associated proteins occurring in Alzheimer's disease, Huntington's chorea and amyotrophic lateral sclerosis. Silver-stained two-dimensional gels provide more information about the complex protein composition of CSF, particularly about proteins produced in the brain, such as apolipoprotein E and neuron-specific enolase. For the detection of oligoclonal antibodies, the investigation of protein changes revealed by Parkinson's disease, schizophrenia and Creutzfeldt—Jakob disease, and the analysis of CSF immune complexes, two-dimensional electrophoresis has a greater sensitivity.     

Specificity of oligoclonal IgG bands against myelin proteins in chronic relapsing EAE in guinea pigs

We showed previously by using imprint electroimmunofixation that the oligoclonal IgG in sera and CSF from chronic relapsing EAE in guinea pigs were specific to spinal cord and Mycobacterium tuberculosis. We now show that most oligoclonal IgG bands are directed predominantly against isolated myelin basic protein (MBP). Activity to the latter could be removed from sera or CSF by absorption with MBP but not with histone or lysozyme. The oligoclonal IgG reacted weakly with isolated proteolipid apoprotein, and lacked reactivity to myelin-associated glycoprotein. When the oligoclonal IgG activity to myelin proteins was removed from the sera by absorption with a preparation of delipidated myelin before imprint electroimmunofixation, a few bands in some sera still reacted with whole spinal cord homogenate. These results indicate that, in some sera, a part of the oligoclonal IgG was directed against non-myelin proteins or lipids. In contrast to chronic relapsing EAE, CSF oligoclonal IgG from patients with multiple sclerosis showed no reactivity against human brain homogenate, whole myelin, delipidated myelin, and MBP in imprint electroimmunofixation.     

Restricted heterogeneity of antibody to gp120 and p24 in AIDS

Neurologic complications and cerebrospinal fluid (CSF) abnormalities are common in AIDS. We found that a substantial number of AIDS patients with neurologic involvement had oligoclonal IgG bands in CSF and sera by IEF. Using an IEF-Ag overlay technique, anti-gp120 antibody activity was demonstrated more frequently than anti-p24 antibody activity. These antibody activities exhibited restricted heterogeneity of their IEF pattern; this restriction may contribute to the relatively low titers of neutralizing antibody found in AIDS sera. None of the CSF and serum oligoclonal bands showed anti-HIV antibody activity, suggesting that they are directed against opportunistic agents or result from immunodysregulation.     

Cerebrospinal-fluid profile in neuroborreliosis and its diagnostic significance

Selected cerebrospinal-fluid (CSF) parameters (intrathecal synthesis ofBorrelia-specific antibodies, oligoclonal IgG bands, CSF-to-serum quotient of albumin as a marker of blood-CSF barrier function and cytology) and typical CSF profile in neuroborreliosis were evaluated with the aim of elucidating possible clinical and laboratory similarities of neuroborreliosis (NB) and other neurological diseases (OND). From the cohort of 58 patients (38 diagnosed for NB, 20 with OND) NB patients had positiveBorrelia-specific IgG antibodies in 97 % and positiveBorrelia-specific IgM antibodies in 55 %; oligoclonal IgG bands were detected in 55 %. The blood-CSF barrier was impaired in 89 %, positive cytology was detected in 97 % of the NB patients. Evaluation of specific intrathecal synthesis improves CSF diagnosis of NB, therefore, a combined CSF analysis has to be considered along with the clinical picture and medical history when formulating the diagnosis of NB.     

Combined immunoabsorption and isoelectric focusing of barley and malt amylases in polyacrylamine gel.

A technique combining immunoabsorption, isoelectric focusing, and enzymatic characterizationin the same polyacrylamide gel is described. The a- and 0-amylases from barley seeds, an immune serum induced in rabbits by barley malt α-amylase, the immunoglobulin G (IgG) of the immune serum, and the IgG purified from a nonimmunized animal were used. The application of this technique in physiological and genetical studies to the identification of amylolytic enzymes which cannot be distinguished by existing chromogenic reactions and which have similar isoelectric points is discussed.     

Diagnostic usefulness of cerebrospinal fluid IgG in multiple sclerosis and other neurological disorders

MS is one of the most common neurologic disorders encountered in the United States. An increase in the CSF IgG index or IgG synthesis rate within CNS, and the presence of CSF oligoclonal bands, now serve as important tools for the diagnosis of MS. Although IEF shows better identification of a number of distinct oligoclonal bands compared to AGE, the latter appears to be a more convenient system for the average hospital's clinical laboratory. These findings are not specific to MS, and similar CSF abnormalities occur in other, more rarely neurologic diseases. It is generally easy to distinguish MS from these other diseases when a thorough clinical and laboratory evaluation is carried out. However, the detection of these CSF IgG abnormalities in non-MS patients may offer an important clue to the presence of a previously unsuspected chronic infection or inflammatory process involving the CNS.     

Oligoclonal IgG bands with and without measles antibody activity in sera of patients with subacute sclerosing panencephalitis (SSPE)

Oligoclonal IgG bands from SSPE sera were isolated by combination of Protein A-Sepharose 4B column and preparative isoelectric focusing gel procedures. Each eluted fraction, when examined in analytic IEF, showed two or three individual bands with isoelectric points close to one another, compared to approximately fifteen IgG bands seen in whole serum. When the bands were tested for measles antibody activity in immunofixation with measles virus followed by peroxidase staining, the bands eluted in pH region 8.5 to 9.3 were found to be measles specific, whereas those in pH 7.0 to 8.4 lacked significant measles activity. When eluted fractions containing groups of bands were absorbed with measles virus, the bands in pH region 8.5 to 9.3 were removed, whereas those in pH 7.0 to 8.4 region remained unchanged; this indicated that a number of oligoclonal IgG bands without measles virus activities are present in SSPE. The bands lacking measles-specific activity may be synthesized against other infectious agents or they may represent nonspecific activation of B cell clones.     

Oligoclonal IgG in rabbits with experimental allergic encephalomyelitis: Non-reactivity of the bands with sensitizing neural antigens

Oligoclonal IgG bands have recently been reported to occur in cerebrospinal fluid (CSF) and serum of rabbits with experimental allergic encephalomyelitis (EAE). To examine the specificity of these bands, a) individual bands eluted from rabbit CSF and sera were tested by radioimmunoassay (RIA) for anti-MBP activity and b) rabbit sera were absorbed with the neuroantigens used for sensitization. RIA of eluates from sequential agar gel slices of the entire IgG region of serum or CSF from MBP sensitized rabbits showed that anti-MBP activity occurred throughout the IgG region and did not localize to specific band-containing fractions. Furthermore, there was no change in banding patterns following absorption of EAE rabbit sera with washed brain homogenates, soluble MBP or MBP conjugated to Sepharose beads. Therefore, our results indicate that the oligoclonal IgG response in EAE is not preferentially directed against the sensitizing neuroantigen, and we raise the possibility of nonspecific B cell activation.     

Polyacrylamide gel electrophoresis: recovery of non-stained and stained proteins from gel slices

Following electrophoresis or isoelectric focusing in gels of polyacrylamide the protein band of interest is cut out and placed above a sucrose gradient column, containing carrier ampholytes (Pharmalyte). By electrophoresis, isoelectric focusing or displacement electrophoresis the proteins migrate out of the gel slice and into the isoelectric focusing column for concentration and further purification. From this column, the proteins can be withdrawn and their isoelectric points determined. Even after staining with Coomassie Brilliant Blue at least some proteins can be recovered by this technique and used for further analyses, for instance amino acid determinations. The focusing in a pH gradient by carrier ampholytes can be replaced by an electrophoresis in a conductivity gradient column. However, in comparison with isoelectric focusing, this concentration technique has the drawback of not permitting further purification of the eluted protein.     

Complete sharing of light chain spectrotypes by murine IgM and IgG anti-streptococcal antibodies.

In order to examine the diversity of antibody light chains, we have developed an analytic isoelectric focusing procedure which permits the routine analysis of L chains from antibodies raised in individual mice. We have used this technique to demonstrate that the light chains of IgM and IgG anti-group A streptococcal antibodies raised in SWR mice are probably shared. Interestingly, numerous light chain spectrotypes are shared between individual mice whose 7S antibody focusing patterns differ.     

Chlamydia pneumoniae and multiple sclerosis: no significant association

The cause of multiple sclerosis (MS) is unknown. Despite indications from epidemiological and identical-twin studies that MS is infectious, no virus or other infectious agent has been tightly linked to disease. The isolation of Chlamydia pneumoniae from the cerebrospinal fluid (CSF) of MS patients and the detection of both Chlamydia-specific DNA and antibody in MS CSF have been reported. Other analyses of brain and CSF have shown no significant difference in C. pneumoniae-specific DNA or antibody between MS and control subjects. Recent work has revealed intrathecal production of C. pneumoniae-specific IgG in only 24% of MS patients compared with 5% of control patients. More importantly, the major CSF oligoclonal bands from MS patients did not react to C. pneumoniae.     

Comparative analysis of the CD19+ and CD138+ cell antibody repertoires in the cerebrospinal fluid of patients with multiple sclerosis

Increased amounts of intrathecally synthesized IgG and oligoclonal bands have long been recognized as a hallmark of multiple sclerosis (MS). B cells and plasma cells are components of the inflammatory infiltrates in both active and chronic MS lesions, and increased numbers of these cells are present in MS cerebrospinal fluid (CSF). Single-cell RT-PCR was used to analyze both the CD19+ B cell and CD138+ plasma cell populations in CSF of two patients with clinically definite MS and of one MS patient whose CSF was obtained after a clinically isolated syndrome, but before the second episode. Sequence analysis of amplified IgG V region sequences identified the rearranged germline segments, extent of somatic mutation, and clonal relationships within and between the two cell populations in the three MS patients. Expanded B cell and plasma cell clones were detected in each MS CSF and in all three patients the CD138+ IgG repertoire was more restricted. However, little if any significant sequence overlap was observed between the CD19+ and CD138+ repertoires of each donor. Detection of plasma cell clones by single-cell PCR will facilitate the in vitro production of recombinant Abs useful in identifying disease-relevant Ags.     

Isolation of colony stimulating factor from human milk

Human milk contains colony stimulating factor (CSF), a polypeptide growth factor, which stimulates in in vitro bone marrow culture proliferation and differentiation of colony forming granulocytic macrophage progenitor cells (CFU-GM) to form colonies. This activity was not found in either bovine milk or colostrum when assayed in human or mouse bone marrow cells. The human milk CSF activity is destroyed by treatment with proteases. However, neither 6M urea, 4M guanidine hydrochloride, 5 mM dithiothreitol, nor exposure to pH 2 will inactivate the milk derived CSF. Gel filtration and isoelectric focusing indicate that human milk CSF differs biochemically from the other CSFs isolated from various sources and has a molecular weight between 250,000 and 240,000 and an isoelectric point between 4.4 and 4.9.     

Chronic meningitis and Lyme disease in Sweden.

We studied 35 patients with chronic meningitis. The neurological abnormalities included aseptic meningitis, cranial neuropathy (mostly facial palsy), motor and sensory peripheral radiculoneuropathy, and myelitis. Neurological symptoms were sometimes preceded by erythema chronicum migrans or an insect bite and were often accompanied by fever, malaise, profound fatigue, and weight loss. The cerebrospinal fluid (CSF) abnormalities consisted of a predominantly mononuclear pleocytosis, an elevated CSF protein (mean 2.3 g/l), intrathecal synthesis of oligoclonal immunoglobulin G, and, in half of the patients, a fall in the CSF/blood glucose ratio. High antibody titers to the Lyme spirochete and the Swedish Ixodes ricinus spirochete were demonstrated by immunofluorescence in 26 of the 35 patients. By imprint immunofixation of electrofocused samples of serum and CSF, intrathecal production of oligoclonal Lyme-spirochete-specific IgG was demonstrated in one patient with chronic meningitis. Four sequential paired samples of serum and CSF from this patient showed local synthesis of spirochete-specific antibodies in CSF. The 35 patients improved or recovered, sometimes dramatically, during a two-week course of intravenous penicillin G.     

Demonstration of a factor in the serum of homozygotes and heterozygotes for cystic fibrosis by a non-biological technique.

A factor has been isolated from serum of homozygotes and obligate heterozgotes for cystic fibrosis using isoelectric focusing and disc electrophoresis as analytical methods. The factor is focused within an IgG-fraction with an isoelectric point of pH 8 to 9 but differs from IgG in its lower molecular weight. It is thus similar to, if not identical with, the ciliary dyskinesia factor.     

Permeation of endogenous IgG with an anionic subpopulation into glomerular basement membrane in rat

Conflicting results of previous electron microscopy studies and concerns about the validity of immunoperoxidase technique employed in those studies to accurately localize endogenous IgG in rat glomerular basement membrane (GBM) prompted us to use other techniques to answer the following question: Does endogenous IgG permeate the matrix of GBM? Immunofluorescence, radioimmunoassay (RIA), isoelectric focusing, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunodetection on Western blots were used to detect endogenous IgG in GBM. Direct immunofluorescence of normal frozen rat kidney sections prepared from in vivo perfused kidney showed endogenous IgG in a linear pattern of staining in the GBM. RIA for rat IgG found the IgG content of collagenase-solubilized GBM to be 0.48% of the dry weight. Immunodetection for rat IgG on Western blots of SDS-PAGE-separated GBM demonstrated endogenous IgG in purified collagenase-solubilized GBM. IgG was detected as an intact molecule with covalently linked light and heavy chains and not as small immunoreactive catabolic fragments. Isoelectric focusing followed by immunodetection on Western blot showed that part of the endogenous IgG in GBM was anionic. The results clearly show that under normal conditions, endogenous IgG can permeate into the collagen matrix of GBM in rat and that some of this IgG is more anionic than the IgG in serum. These findings may assist in understanding the transit of autoantibodies to subepithelial glomerular antigens located beneath the matrix of GBM in membranous glomerulonephropathy.     

Molecular heterogeneity of secretory IgA anti-DNP antibodies

Evidence is presented that demonstrates that the microscale sucrose isoelectric focusing technique can be used to assess the molecular heterogeneity of secretory IgA anti-DNP antibodies. The data indicate that substitution of the DNP group on the bacterial carrier, pneumococcus, can limit the structural complexity of secretory IgA anti-DNP antibodies. The data support the concept that factors governing secretory IgA antibody heterogeneity are analogous to those influencing the heterogeneity of serum IgG antibodies.     

A monoclonal IgG4 (lambda) with factor V inhibitory activity.

A monoclonal IgG4 (lambda) with inhibitory activity to human coagulation factor V was isolated from the serum of a patient with a fatal hemorrhagic diathesis by using a combination of DE-52 ion exchange chromatography and isoelectric focusing techniques. Using the criteria for defining a monoclonal immunoglobulin of restricted mobility on protein electrophoresis, immunoelectrophoresis, and isoelectric focusing, as well as neutralization with class, subclass, and light chain type antisera, we are the first to demonstrate a factor V inhibitor as a monoclonal IgG4 (lambda) detectable in serum or plasma.     

Isoelectric focusing and isoelectric points of bovine liver histones

A technique for isoelectric focusing of total histones in very narrow pH gradients is described. The isoelectric focusing was performed in 5% acrylamide gels at the pH range 9–11 in long quartz tubes (24 cm) in a nitrogen atmosphere. The total bovine liver histones separated into five main fractions which were identified as H1, H3, H2B, H2A, and H4 histones, and their apparent isoelectric points were determined. The main fractions were further divided into several subfractions, the maximal number of bands being 12. The isoelectric point for H1 histone in 6.25 m urea solution in the presence of a nitrogen atmosphere was 8.90, and the corresponding values for H3, H2B, H2A, and H4 histones were 9.80, 9.90, 10.10, and 10.25, respectively. The focusing technique described here has a high resolution, reproducibility, and sensitivity. The technique can be used for preparative and quantitative analysis and for studies on specificity and developmental changes of histones.