Longterm efficacy of interferon-alpha for extrahepatic disease associated with hepatitis C virus infection.

OBJECTIVE: To investigate longterm responsiveness to interferon-alpha (lFN-alpha) of patients with extrahepatic manifestations of hepatitis C virus (HCV) in a nonendemic area. METHODS: We prospectively evaluated 11 patients with extrahepatic manifestations of HCV infection, including 10 with Type II cryoglobulins, treated with IFN-alpha--9 had cutaneous vasculitis, 6 arthralgias, 7 neuropathy, and 4 glomerulonephritis. Liver biopsies were performed on all patients, although 6/11 had normal liver function tests. All received 3 M units IFN-alpha tiw, with total length of treatment ranging from 3 mo to 5 yrs. Periodic assessments were made of clinical activity, biochemical variables, cryoglobulin quantitation, and HCV copy number. RESULTS: Three patients were withdrawn because of toxicity. Three were nonresponders at 6, 16, and 17 mo of therapy, based on persistence of HCV RNA in blood, cryoprecipitates, and peripheral blood mononuclear cells. One patient was a partial responder at 3 yrs, with 2 major flares of cutaneous vasculitis occurring on separate attempts to withdraw IFN-alpha. Three patients (27.2%) were complete responders based on resolution of symptoms (purpura, neuropathy) and disappearance of cryoprecipitates and HCV RNA, but only one successfully tapered IFN-alpha after 3 yrs of treatment, with sustained resolution at followup 15 mo later. CONCLUSION: IFN-alpha is safely tolerated for prolonged periods in patients with extrahepatic HCV infection, and is particularly effective for treatment of cutaneous vasculitis. Careful monitoring is needed for evolution of liver pathology to cirrhosis, or for progression of renal or neurologic disease.

Isolated amyloidosis of anterior uvea and trabecular meshwork.

Amyloid deposition in the iris is rare, scanty and associated with systemic disease. We present a case of isolated localized heavy amyloid infiltration of the iris and describe a new hillock-pattern feature visualized using scanning electron microscopy.

Hepatitis C virus, autoimmunity and lymphoproliferation.

We summarize clinical, laboratory and pathologic details regarding a patient who presented with extrahepatic disease manifestations of hepatitis C virus (HCV) infection, including cryoglobulinemic leg ulcers due to cutaneous vasculitis, peripheral sensorimotor neuropathy, and recurrent pulmonary infiltrates. The patient had evidence for B-cell lymphoproliferation, diagnosed as extranodal lymphoma on initial (though not subsequent) bone marrow examination, retroperitoneal lymphadenopathy, and the presence of a Type II IgM6 monoclonal rheumatoid factor which became cryoprecipitable on complexing to IgG. Chronic hepatitis was mild on liver biopsy, though fibrotic changes developed over a three-year period of follow-up. She had consistently normal liver function tests, except for a brief rebound effect on discontinuing interferon-alpha, and preterminally. Symptoms were only partially responsive to trials of corticosteroids, cytotoxic agents, plasmapheresis and interferon, and the patient ultimately died at The Mount Sinai Hospital of sepsis. We review current information regarding the spectrum of extrahepatic HCV infection, including pathogenic factors relevant to its overlapping autoimmune, rheumatic and lymphoproliferative disease manifestations. The exact prevalence of these HCV-related syndromes among the 1% of the world population estimated to be infected by this virus remains to be delineated. Chronicity of infection, and lack of efficacy of currently available therapy in effecting sustained clearance of the virus from the host, have made this an important public health problem that is likely to increase in significance. Possible relationships to non-Hodgkin's lymphoma may present novel opportunities to delineate the basis for oncogenesis in HCV infection.

Guidelines for clinical and laboratory evaluation patients with monoclonal gammopathies.

This guideline provides the recommendations of an expert panel for the clinical and laboratory evaluation of patients suspected of having a clinical condition that produces a monoclonal protein in serum or urine. The recommendations describe the clinical conditions in which a monoclonal protein should be sought, the optimal sequence of testing to diagnose and monitor these patients, and the most effective laboratory procedures.

Evaluation of cryoglobulins.

Cryoglobulins are immunoglobulins that precipitate as serum is cooled below core body temperatures. A cryoglobulin screen is the observation of a serum specimen collected and separated while warm for cryoprecipitation over a period of up to 7 days. Values of the screening may be reported as a cryocrit, which is the volume percent of the precipitate compared with the total volume of serum. Further proof that the precipitate is indeed a cryoglobulin can be obtained by demonstrating resolubilization with warming and immunochemical analysis by immunofixation. Detailed characterization of cryoglobulins may also require rigorous washing of the precipitate, quantitation of total protein and immunoglobulins, and evaluation of serum for monoclonal gammopathy, rheumatoid factor activity, evidence of complement activation, and presence of hepatitis C virus seroreactivity or hepatitis C virus RNA. The single most important variable confounding standardization of cryoglobulin testing is the frequently improper separation of warm serum from other blood elements prior to screening and characterization.

Cryoglobulinemia.

Cryoglobulinemia may be found in a spectrum of disorders spanning clear-cut-B-cell neoplastic states, in which cryoprecipitation manifests as ischemic or occlusive vasculopathy, to a variety of immune complex diseases, in which vasculitis or glomerulonephritis may occur. Symptomatic cryoglobulinemia is many diseases, driven by and driving antibody-antigen responses, hepatic dysfunction, lymphoproliferation, and immune complexes. Distinguishing features that cause only some cryoglobulins to be symptomatic, elucidating the pathogenic mechanisms of HCV in cryoglobulin formation, and devising better therapies and more systematic evaluation of existing therapies are among the challenges for the future. Prognostication and classification will continue to rely on Brouet's classification (types I, II, and III), but additional features will probably include the presence or absence of HCV, HCV factors (genotype, titer), coexisting infections, B-cell clone burden, host factors, and immune system interactions (B- and T-cell idiotype networks, cytokines). Although antiviral therapy is a reasonable option for HCV-associated cryoglobulinemia, not all patients are HCV-positive, and only 60% to 80% of HCV-positive patients respond to IFN. In addition, not all patients tolerate IFN, and in those who do, the response is often short-lived once the treatment is discontinued. Only creative strategies, systematically studied, will provide long-awaited solutions.

Transthyretin Ile 122 and cardiac amyloidosis in African-Americans. 2 case reports.

Two cases of cardiac amyloidosis resulting from deposition of the Ile 122 variant of transthyretin in African-Americans are presented. These cases illustrate several typical features of this disorder, including electrocardiographic abnormalities and digoxin toxicity. Transthyretin Ile 122 is a common amyloidogenic variant in African-Americans (present as a heterozygous variant in 4% of this population); therefore, the diagnosis of transthyretin Ile 122 cardiac amyloidosis should be considered in African-Americans with unexplained restrictive cardiomyopathy or arrhythmias.

Fate of cerebrospinal fluid-borne amyloid beta-peptide: rapid clearance into blood and appreciable accumulation by cerebral arteries.

In Alzheimer's disease, the neuritic or senile amyloid plaques in hippocampus and association cortex, the diffuse plaques in brain areas such as the cerebellum and sensorimotor cortex, and the amyloid deposits in the walls of pial and parenchymal blood vessels are mainly composed of amyloid beta-peptides. In the present study, either soluble 40-residue amyloid beta-peptide radiolabeled with 125I (I-sAbeta) or [14C]polyethylene glycol ([14C]PEG, a reference material) was briefly infused into one lateral ventricle of normal rats. By 3.5 min, 30% of the I-sAbeta was cleared from ventricular CSF into blood; another 30% was removed over the next 6.5 min. No [14C]PEG was lost from the CSF-brain system during the first 5 min, and only 20% was cleared by 10 min. Much of the I-sAbeta that reached the subarachnoid space was retained by pial arteries and arterioles. Virtually no I-sAbeta was found in brain. The clearance of amyloid beta-peptides from the CSF-brain system, reported herein for normal rats, may be reduced in Alzheimer's disease, thus contributing to amyloid deposition in cerebral tissue and blood vessels.

Purification of Borrelia burgdorferi outer surface protein A (OspA) and analysis of antibody binding domains.

The major outer surface protein, OspA, of Borrelia burgdorferi is a lipoprotein which is a particular interest because of its potential as a vaccine candidate. However, serotypic and genetic analysis of OspA from both European and North American strains have demonstrated antigenic and structural heterogeneities. We purified OspA to homogeneity by exploiting its resistance to trypsin digestion. By treating spirochetes with trypsin and then using Triton X-114 extraction and ion-exchange chromatography, we obtained a yield of 2 mg of pure OspA protein per liter of culture. INtrinsic labeling with [14C]palmitic acid confirmed that OspA was lipidated, and partial digestion established lipidation at the amino-terminal end of the molecule. The reactivity of five anti-OspA murine monoclonal antibodies to nine different isolates of B. burgdorferi was ascertained by Western blot (immunoblot) analysis. Purified OspA was fragmented by enzymatic or chemical cleavage, and the monoclonal antibodies were able to define four distinct immunogenic domains. Further resolution of the epitope specificity to determine humoral and cellular immune responses to OspA has implications for vaccine development and for the utility of this protein as a reagent in diagnostic testing for Lyme borreliosis.

Characterization of a novel processing pathway for Alzheimer's amyloid beta precursor protein.

Amyloid beta (A beta) is a normal proteolytic fragment of a large precursor protein (beta PP) which undergoes altered conformation, leading to fibril formation. Two main beta PP processing pathways have been described, and we are now reporting the characterization of a third beta PP pathway. A membrane-associated 16 kDa component identified in human platelets isolated from normal donors. Based on size, immunoreactivity and amino acid sequence analysis, the fragment is a C-terminal beta PP component which starts at position 642 (APP770 numbering) and contains the intact A beta sequence. The presence of this novel pathway of beta PP processing in resting platelets suggest that it occurs as a normal event.

Three autosomal dominant corneal dystrophies map to chromosome 5q.

The two most common autosomal dominant dystrophies of the corneal stroma are lattice corneal dystrophy type I and granular dystrophy. A third autosomal dominant stromal dystrophy (Avellino) has also been recognized. Chromosome linkage analysis of four families with Avellino dystrophy mapped the disease-causing gene to chromosome 5q. Subsequent linkage analysis of two families with typical lattice dystrophy and two with typical granular dystrophy also revealed significant linkage with the same markers. Thus, each of three clinically and histopathologically distinct phenotypes is independently linked to 5q. The maximum combined lod score using all 114 affected patients was 28.6 with marker D5S393. None of the 14 known human amyloid-associated genes map to chromosome 5.

High-level expression and in vitro mutagenesis of a fibrillogenic 109-amino-acid C-terminal fragment of Alzheimer's-disease amyloid precursor protein.

We amplified DNA encoding the 3' 109 codons of Alzheimer's-disease amyloid precursor protein (APP) inclusive of the beta protein (A beta) and cytoplasmic domains from cDNA using oligonucleotide primers designed to facilitate cloning into the T7 expression vector pT7Ad23K13. We also modified this construct to generate recombinant molecules incorporating two recently described APP mutants by site-directed mutagenesis. Both native C109 (deletion construct inclusive of the C-terminal 109 residues of APP) and constructs with a single mutation at codon 642 (T-->G, resulting in a substitution of glycine for valine) or a double mutation at codons 595 (G-->T, substituting asparagine for lysine) and 596 (A-->C, substituting leucine for methionine) were expressed in Escherichia coli to levels of 5-20% of total bacterial protein after induction. The major constituent of expressed C109 protein had an apparent molecular mass of 16-18 kDa by SDS/PAGE and appeared to be the full-length construct by size and N-terminal microsequencing. Also present was a 4-5 kDa species that co-purified with C109, constituting only approximately 1% of expressed protein, which was revealed by Western-blot analysis with antibodies specific for A beta epitopes and after biotinylation of purified recombinant C109. This fragment shared N-terminal sequence with, and appeared to arise by proteolysis of, full-length C109 in biosynthetic labelling experiments. C109 spontaneously precipitated after dialysis against NaCl or water, and with prolonged (> 20 weeks) standing was found by electron microscopy to contain a minor (< 5%) fibrillar component that was reactive with antibodies to a C-terminal epitope of APP. Recombinant C109 appears to duplicate some of the biochemical and physicochemical properties of C-terminal A beta-inclusive fragments of APP that have been found in transfected cells, brain cortex and cerebral microvessels.

Bronchiolitis obliterans organizing pneumonia and rheumatoid arthritis.

The case of a 68-year-old woman who presented with dyspnea and upper lobe pulmonary infiltrates and shortly thereafter developed seropositive, erosive polyarticular rheumatoid arthritis (RA) is presented. An open-lung biopsy to evaluate progression of infiltrates showed bronchiolitis obliterans-organizing pneumonia (BOOP). Both lung and articular disease responded rapidly to corticosteroid therapy. Interrelationships between BOOP, bronchiolitis obliterans, and interstitial fibrosis with connective tissue disease are discussed.

Reactive arthropathy and autoimmunity in non-HIV-associated immunodeficiency.

The spectrum of arthropathy in non-HIV immune deficiency states includes arthritis due to prevalent infectious pathogens and autoimmunity that may in some instances be triggered by microorganisms. Joint symptoms may be clinical manifestations of disease, or they may develop later during therapy for immunodeficiency. Pathogenesis can be related to occult infection, loss of mucosal barrier function, defective clearance of immune complexes, or aberrant immune responses. Proper treatment includes an appreciation of likely pathogens, an understanding of the nature of immunologic deficits, and rigorous exclusion of immune dysfunction that may be secondary to treatment.

Cross-reactive antigenic domains of the flagellin protein of Borrelia burgdorferi.

The p41 flagellin of Borrelia burgdorferi is the most common antigen recognized by serum of patients with Lyme borreliosis. This antigen shares amino acid homology, particularly in the amino and carboxy termini, with periflagellar antigens found in other microorganisms including Treponema pallidum. We cloned and expressed the p41 open reading frame in Escherichia coli and expressed it both as TrpE fusion and full-length unfused proteins. Also, we generated deletion constructs of various portions of the gene. Sera from patients with late Lyme borreliosis and secondary syphilis were used to identify the recombinant proteins by immunoblot analysis. Sera from 26 patients with Lyme borreliosis, 20 with secondary syphilis and 10 controls were used to identify cross-reactive domains of the B. burgdorferi flagellin. The variable region (amino acids 131-234) of the protein was recognized by 59% (15/26) of patients with late Lyme borreliosis compared to 30% (6/20) of patients with secondary syphilis and no (0/10) control patients. It appears that cross-reactive epitopes between B. burgdorferi and T. pallidum extend to the variable region of the flagellin.

Gelsolin immunoreactivity in corneal amyloid, wound healing, and macular and granular dystrophies.

Immunohistologic studies of tissue sections obtained from patients with type 1 or type 2 lattice corneal dystrophy, polymorphic amyloid degeneration, or gelatinous amyloid degeneration were performed by using a monoclonal antibody raised to a chymotryptic fragment inclusive of the carboxy-terminal half of plasma gelsolin, and also with a series of polyclonal antibodies specific for synthetic peptides corresponding to immunogenic epitopes of gelsolin. These epitopes are parts of sequences at the amino- and carboxy-terminal ends of gelsolin, as well as adjacent to and inclusive of the codon 187 mutant 7-11 kD fragment that has been shown to be the subunit protein of amyloid fibrils occurring systemically in patients affected by Finnish type familial amyloidosis. These antibodies were also tested on tissue sections obtained from patients with granular and macular corneal dystrophy, corneal wounds, and normal control corneas. Specificity of staining was established by absorption with gelsolin purified from plasma, or the appropriate synthetic peptide. Gelsolin immunoreactivity was detected in the conjunctival and skin amyloid in familial amyloidosis by using familial amyloid (Finnish type) antibody. In other types of corneal amyloid, including lattice dystrophy type 1, immunoreactivity with gelsolin and synthetic peptides was observed adjacent to the deposits, but rarely within them. In macular dystrophy, variable staining of the deposits could result from the association of subunit proteins with glycosaminoglycans.

A 109-amino-acid C-terminal fragment of Alzheimer's-disease amyloid precursor protein contains a sequence, -RHDS-, that promotes cell adhesion.

Amyloid beta (A beta), the major constituent of the fibrils composing senile plaques and vascular amyloid deposits in Alzheimer's disease (AD) and related disorders, is a 39-42-residue self-aggregating degradation peptide of a larger multidomain membrane glycoprotein designated amyloid precursor protein (APP). An array of biological functions has been assigned to different APP domains, including growth regulation, neurotoxicity, inhibitory activity of serine proteinases and promotion of cell-cell and cell-matrix interactions. A beta is generated through an as-yet-unknown catabolic pathway that by-passes or inhibits the cleavage of APP within the A beta sequence. We have identified a 16 kDa intermediate APP C-terminal fragment containing A beta in leptomeningeal vessels of aged normal individuals and AD patients by means of its immunoreactivity with a panel of four different anti-(APP C-terminal) antibodies, indicating a different pathway of APP processing. Previous studies have indicated that the APP C-terminal domain is the most likely to be involved in cell-matrix interactions. A 109-amino-acid construct C109 with a sequence analogous to the C-terminal of APP (positions 587-695 of APP695), similar in length and immunoreactivity to the 16 kDa fragment, was found to promote cell adhesion. By use of synthetic peptides, this activity was initially located to the extracellular 28 residues of A beta. Inhibition studies demonstrated that the sequence RHDS (amino acids 5-8 of A beta, corresponding to residues 601-604 of APP695 was responsible for the adhesion-promoting activity. The interaction is dependent on bivalent cations and can be blocked either by the tetrapeptides RHDS and RGDS or by an anti-(beta 1 integrin) antibody. Thus, through integrin-like surface receptors, APP or its derivative proteolytic fragments containing the sequence RHDS may modulate cell-cell or cell-matrix interactions.

In situ localization of PCR-amplified human and viral cDNAs.

We describe a technique, called reverse transcriptase (RT) in situ PCR, whereby RNA may be nonisotopically detected in fixed cells when amplified by PCR after cDNA synthesis by RT. RT in situ PCR using primers specific for the measles virus generated an intense signal in most measles-infected HeLa cells, as compared to the weak signal generated in few cells using standard in situ hybridization analysis. The viral RNA that localized to the nucleus spared the nucleoli, was most evident when the RT step used the primer complementary to the negative genomic strand, and was demonstrated in all multinucleated cells and the majority of uninucleate cells. A hybridization signal was evident with standard RNA in situ hybridization using the human megakaryocyte cell line Dami and a probe for glycoprotein IIB (GIIB) mRNA but not a probe for amyloid precursor protein (APP) or gelsolin (GEL) mRNA. After RT in situ PCR, signals were evident for each target localizing to the nucleolus for APP and to perinucleolar and cytoplasmic locations for GEL and GIIB. The latter findings suggest that mRNAs may follow different geographic pathways as they progress from premessage to transcriptionally active message.

The 93-kilodalton protein of Borrelia burgdorferi: an immunodominant protoplasmic cylinder antigen.

Using immunoblots, we identified proteins of Borrelia burgdorferi recognized by sera from 62 patients with either acute or chronic Lyme disease. In all groups studied, the 41-kDa flagellar protein and a relatively minor 93-kDa protein (p93) were the most commonly recognized antigens in patients with acute and chronic disease due to B. burgdorferi. A murine monoclonal antibody (MAb 181.1) was developed against p93, and the antigen was detected by immunoblot analysis in four European and American strains of B. burgdorferi. On two-dimensional gel electrophoresis, p93 had an apparent pI of 6.8. Immunoelectronmicroscopy with MAb 181.1 demonstrated that p93 is located within the protoplasmic cylinder compartment of the organism. The gene encoding p93 was retrieved from a phage expression library. The derived amino acid sequence of p93 confirmed chemical characterization of the antigen, including its amino-terminal peptide sequence. The derived amino acid sequence predicted it to be predominantly alpha helical. A prominent antigenic domain located at the carboxy portion of the protein was recognized by human and rabbit polyclonal antisera and human (MAb D4) and mouse (MAb 181.1) MAbs.