Marianna Kuropatwa

Retinal Antigens Are Recognized by Antibodies Present in Sera of Patients with Multiple Sclerosis

Multiple sclerosis (MS) is frequently accompanied by visual symptoms including those related to retinal disorders. Since they may be a consequence of an autoimmune reaction, we examined whether sera of patients with diagnosed MS and changes in visual-evoked potentials contain antibodies against retinal antigens (retAgs). Immunoblot analysis revealed that MS sera recognized mainly a 46-kD antigen, a 41-kD antigen, retinal arrestin, to a smaller extent also 70-, 56-, 43-, and 36-kD proteins. Patients whose sera showed the highest reactivity with 41- and 46-kD antigens had deficiencies in visual acuity, visual fields, ophthalmoscopy, and electroretinograms. Our observation suggests that antibodies to these retAgs may play a role in the origin of ophthalmologic impairment in MS.

Serum Antibodies to Retinal Antigens in Lung Cancer and Sarcoidosis

Objective: Autoantibodies to various neuronal proteins frequently accompany lung cancer and their appearance may precede cancer symptoms. In this study we examined which retinal antigens (RAs) are recognized by sera of patients with lung cancer and whether the occurrence of serum antibodies to particular RAs is characteristic for cancer in comparison with a noncancer lung disease. Methods: Sera of 72 patients with non-small-cell lung cancer (NSCLC), 29 with small-cell lung cancer (SCLC), 27 with sarcoidosis (S), and sera of 32 healthy donors were examined in immunoblotting using retinal extracts and purified RAs as antigens. Results: 69.0% of SCLC, 45.8% of NSCLC, and 44.4% of S sera displayed anti-RAs reactivity. Significantly less (p < 0.05; χ2 test) percent of healthy control sera reacted with RAs. Lung cancer sera recognized mainly 46-, 56-, and 36-kD and to a smaller extent also 96-, 72-, 43-, and 26-kD proteins. Most of them were recognized with about 2-fold lower frequencies by S and control sera. Only lung cancer sera contained very high-titer antibodies to 46- and 26-kD RAs, identified as α-enolase and recoverin, respectively. Conclusion: Antibodies to RAs occur more frequently and in higher titers in lung cancer (especially SCLC) than in sarcoidosis or control sera. Although antibodies to retinal α-enolase, recoverin and other RAs are present mainly or exclusively in lung cancer sera, none of them seems to be a specific marker of a particular disease.

Antibodies to 46-kDa retinal antigen in a patient with breast carcinoma and cancer-associated retinopathy

Cancer-associated retinopathy (CAR) is a rare paraneoplastic syndrome usually associated with small-cell lung carcinoma and serum autoantibodies against recoverin. We report the breast cancer woman with visual impairments and electrophysiological abnormalities characteristic of CAR. Her serum contained high-titer antibodies against α-enolase but not against other retinal proteins. This suggests that anti-enolase antibodies could be responsible for the development of CAR symptoms.

Different effects of soluble and particulate guanylyl cyclases on expression of inflammatory cytokines in rat peripheral blood mononuclear cells.

Inflammation involves the cooperation of various cells and biologically active molecules. An important intracellular messenger molecule participating in the regulation of the process is cyclic GMP (cGMP), which is synthesized by guanylyl cyclases (GCs). The GC family comprises cytosolic (soluble) and membrane-bound (particulate) enzymes. The aim of this study was to determine whether and how the synthesis of cGMP by various forms of GC affects the expression of inflammatory cytokines depending on the activity of the transcription factors NF-κB (nuclear factor-κB) and AP-1 (activator protein-1). We established that in rat peripheral blood mononuclear cells (PBMCs), synthesis of cGMP was elevated by sodium nitroprusside (SNP), the activator of soluble GC, and by atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP), the activators of particulate GC-A and GC-B, respectively. Stimulation of various GCs differently affected the expressions of the cytokines IL-1β, IL-6, and TNF-α in control cells and in cells activated by bacterial endotoxin (LPS). In control PBMCs their expression was elevated by stimulation of soluble, but not particulate, GC. SNP caused an increase in NF-κB activity, but had no influence on the activity of AP-1. The cells treated with LPS decreased the expressions of IL-1β, IL-6, and TNF-α in response to stimulation of particulate GC-A, but not other guanylyl cyclases. This inhibitory effect was a result of suppression of the activities of NF-κB and AP-1. Both effects that of SNP and of ANP, were cGMP dependent, as shown using its membrane-permeable analog 8-Br-cGMP. The implementation of specific inhibitors showed that the stimulatory effect of SNP was mediated by soluble GC and cGMP-dependent protein kinase (PKG-I). However, PKG-I was not involved in the inhibition of NF-κB and AP-1 activities by ANP in LPS-activated cells. Taken together, these results for the first time indicate that various GCs and various cGMP-dependent signaling pathways can modulate the activity of AP-1 and/or NF-κB and thus affect the expressions of IL-1β, IL-6, and TNF-α, which play important roles in the development of inflammation.

Colorimetric method for assay of serum γ-glutamyltransferase activity with some L-γ-glutamyl-carboxyanilides

Abstract A simple method for the assay of serum γ-glutamyltransferase activity with soluble l -γ-glutamyl-carboxyanilides is presented. It is based on transformation of aminobenzoic acid liberated by the enzyme into colour complex with 4-dimethyl-aminobenzaldehyde or with 4-dimethylaminocinnamaldehyde in acid medium. Among three carboxyanilides studied the highest enzyme activity was noted with l -γ-glutamyl-3-carboxyanilide and no activity with l -γ-glutamyl-2-carboxyanilide. A good correlation was demonstrated for serum γ-glutamyltransferase activity determined by the new colorimetric method and by the standard one with l -γ-glutamyl-3-carboxy-4-nitroanilide.

Enzyme-linked immunosorbent assay (ELISA) of acylase in serum of patients with viral hepatitis

Abstract Enzyme-linked immunosorbent assay of cobalt-activated acylase (form 2) from human liver has been described. A good correlation (r = 0.95) was noted between the acylase activities assayed by this immunological method and those measured colorimetrically in serum of patients with viral hepatitis. Stability of the serum acylase was also studied by both methods.

Sp1 mediates phorbol ester (PMA)-induced expression of membrane-bound guanylyl cyclase GC-A in human monocytic cells

Cyclic guanosine monophosphate (cGMP) is synthesized by two types of enzymes: particulate (membrane-bound) guanylyl cyclases (pGCs) and soluble (cytosolic) guanylyl cyclases (sGCs). sGCs are primarily activated by binding of nitric oxide to their prosthetic heme group while pGCs are activated by binding of peptide ligands to their extracellular domains. One of them, pGC type A (GC-A) is activated by atrial and brain natriuretic peptides (ANP and BNP, respectively). Human monocytes isolated from peripheral blood mononuclear cells have been found to display sGC expression without concomitant expression of GC-A. However, GC-A activity appears in monocytes under certain conditions but a molecular mechanism of GC-A expression is still poorly understood. In this report we show that phorbol ester (PMA) induces transcription of a gene encoding GC-A in human monocytic THP-1 cells. Moreover, we find that PMA-treated THP-1 cells raise cGMP content following treatment with ANP. Studies using pharmacological inhibitors of protein kinases suggest involvement of protein kinase C (PKC), mitogen extracellular kinases (MEK1/2), and extracellular signal-regulated kinases (ERK1/2) in PMA-induced expression of the GC-A encoding gene in THP-1 cells. Finally, we show that PMA stimulates binding of Sp1 transcription factor to GC-rich DNA sequences and mithramycin A (a selective Sp1 inhibitor) inhibits expression of the GC-A mRNA in PMA-treated THP-1 cells. Taken together, our findings suggest that the PMA-stimulated PKC and MEK/ERK signaling pathways induce Sp1-mediated transcription of the GC-A encoding gene in human monocytic THP-1 cells.

Phosphodiesterase 2 negatively regulates adenosine-induced transcription of the tyrosine hydroxylase gene in PC12 rat pheochromocytoma cells.

Adenosine induces expression of the tyrosine hydroxylase (TH) gene in PC12 cells. However, it is suggested that atrial natriuretic peptide (ANP) inhibits expression of this gene. Using real-time PCR and luciferase reporter assays we found that ANP significantly decreases the adenosine-induced transcription of the TH gene. Results of measurements of cyclic nucleotide concentrations indicated that ANP-induced accumulation of cGMP inhibits the adenosine-induced increase in cAMP level. Using selective phosphodiesterase 2 (PDE2) inhibitors and a synthetic cGMP analog activating PDE2, we found that PDE2 is involved in coupling the ANP-triggered signal to the cAMP metabolism. We have established that ANP-induced elevated levels of cGMP as well as cGMP analog stimulate hydrolytic activity of PDE2, leading to inhibition of adenosine-induced transcription of the TH gene. We conclude that ANP mediates negative regulation of TH gene expression via stimulation of PDE2-dependent cAMP breakdown in PC12 cells.