19 research outputs found
Differences in the distribution, phenotype and gene expression of subretinal microglia/macrophages in C57BL/6N (Crb1rd8/rd8) versus C57BL6/J (Crb1wt/wt) mice
Parafoveal acute middle maculopathy (PAMM) in sickle cell disease after discontinuation of hydroxyurea
Purpose: Paracentral acute middle maculopathy (PAMM) is a rare ophthalmologic emergency involving the intermediate and deep retinal capillary plexus that supply the retina's middle layers. This case report describes an episode of PAMM in a patient with sickle cell disease (SCD) to demonstrate the importance of early diagnosis, review potential pathophysiologic mechanisms, and finally discuss appropriate management in this patient population. Observations: A 33-year-old black female with SCD, who had recently discontinued disease-modifying therapy with hydroxyurea, presented with a central scotoma of the left eye. Examination showed superficial opacification and whitening of the temporal perifoveal macula. After an initial diagnosis of central retinal artery occlusion she was admitted for a stroke workup. MRI was negative for stroke, and the patient was discharged after undergoing a red blood cell exchange (RBCX). Follow-up exam and optical coherence tomography (OCT) findings were more consistent with PAMM. Conclusions and Importance: To our knowledge, this is the first report of PAMM after discontinuation of hydroxyurea in preparation for pregnancy. It highlights the importance of a multidisciplinary approach when treating peripartum patients with SCD and the need for further research regarding vaso-occlusive prophylactic agents and their effects in pregnancy to minimize morbidity during family planning
Factor H binds to the hypervariable region of many <em>Streptococcus pyogenes</em> M proteins but does not promote phagocytosis resistance or acute virulence
Phagocytosis tests with M1 and M3 strains.
<p>(<b>A</b>) Suspensions of the bacterial strains indicated were analyzed for ability to bind human FH. After incubation of a bacterial suspension with pure FH (50 µg/ml), the bacteria were washed twice and bound protein was eluted and analyzed by western blot, employing anti-FH for detection. The analysis included wild type M5, M1 and M3 strains and their M-negative mutants (ΔM5, ΔM1 and ΔM3, respectively). Pure FH was included as a control in the blot (right). (<b>B</b>) Phagocytosis assay in whole human blood with M1 and M3 strains, and M-negative mutants, as indicated. MF, multiplication factor.</p
Isolated HVRs derived from M5, M6 and M18 specifically bind FH.
<p>(<b>A</b>) Analysis by non-reducing SDS-PAGE of HVRs dimerized via a C-terminal cysteine residue. As previously observed <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003323#ppat.1003323-Morfeldt1" target="_blank">[26]</a>, dimerized HVRs move more slowly than expected in gels. (<b>B</b>) Binding of FH to isolated HVRs immobilized in microtiter wells. The wells were coated with 0.1 µg of the HVRs, as indicated, and tested for ability to bind added FH. (<b>C</b>) Immobilized HVRs, derived from FH-binding M proteins, specifically bind FH among all proteins in human serum. Whole human serum was applied to columns in which the HVRs indicated had been immobilized. After washings, bound protein was eluted and analyzed by SDS-PAGE. A column without HVR was used as control. Pure FH was included as a reference in the gel analysis (right). (<b>D</b>) Sequence alignment of the three FH-binding HVRs that were studied in isolated form. This alignment does not include a C-terminal Cys residue included to allow dimerization. The lengths of these HVR vary slightly from those previously reported <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003323#ppat.1003323-Lannergrd1" target="_blank">[79]</a>, because the position of the C-terminal end was chosen to allow optimal dimerization and FH-binding. Asterisks indicate residues identical in all three sequences. Pair-wise identities (based on regions present in both sequences) are indicated to the right.</p
Streptococcal M proteins vary in ability to bind human FH and C4BP.
<p>(<b>A</b>) Schematic representation of the human complement regulators FH and C4BP and of an M protein. For FH and C4BP, each circle represents an SCR domain. While FH is a single chain with 20 SCR domains, C4BP typically contains 7 α-chains with 8 SCR domains and one β-chain with 3 SCRs. An M protein has an N-terminal HVR and a more conserved C-terminal region that includes C repeats and the wall-anchoring region. The location of M protein binding sites in FH and C4BP are indicated. (<b>B</b>) SDS-PAGE analysis of the purified recombinant M proteins studied, five of which were of class I, and two of class II. The presence of doublet or triplet bands is typical for M proteins expressed in <i>E. coli</i> or <i>S. pyogenes </i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003323#ppat.1003323-Stenberg1" target="_blank">[32]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003323#ppat.1003323-Scott1" target="_blank">[82]</a>. (<b>C</b>) Binding of human FH, C4BP or fibrinogen (Fg) to pure M proteins immobilized in microtiter wells. The wells were coated with 0.1 µg M protein and human ligands (50 µl) were added at the following concentrations: FH 2 µg/ml, C4BP 1 µg/ml, Fg 0.28 µg/ml. Bound ligands were detected with specific antibodies. Binding is given in percent of maximal binding for each ligand. (<b>D</b>) Binding of human FH analyzed for the M-positive M5, M6 and M18 <i>S. pyogenes</i> strains and their M-negative mutants (ΔM5, ΔM6, and ΔM18, respectively). Bacterial suspensions were incubated with pure FH (50 µg/ml). After two washes bound protein was eluted and analyzed by western blot, employing anti-FH for detection. Pure FH was included as a control in the blot (right).</p
Characterization of a chimeric FH expressed by Tg mice, and infection experiments with Tg and normal mice.
<p>(<b>A</b>) SDS-PAGE and western blot analysis of purified chimeric FH (chim. FH), derived from mouse and human FH. This FH was compared with pure human Y402 FH (hFH) and pure mouse FH (mFH). Streptococcal protein Rib <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003323#ppat.1003323-StlhammarCarlemalm1" target="_blank">[81]</a> was included as negative control. The blot was probed with antiserum to human FH. Bound antibodies were detected with radiolabeled protein G followed by autoradiography. (<b>B</b>) Binding of the M5 protein to immobilized FH proteins. The proteins used were those shown in panel A. The wells of microtiter plates were coated with the protein indicated, using 0.1 µg protein per well, with protein Rib as negative control. The immobilized proteins were analyzed for ability to bind biotinylated pure M5 protein, added at the concentrations indicated, and bound M protein was detected by the addition of radiolabeled streptavidin. (<b>C</b>) Comparison of bacterial growth in wild type mice and Tg mice. The mice (n = 8 for wt and n = 8 for Tg) were subjected to invasive infection with a sublethal dose of the <i>S. pyogenes</i> M5 strain and sacrificed after 18 h, followed by determination of bacterial counts in the spleen. (<b>D</b>) Survival of wild type (wt) and Tg mice after infection with M5 bacteria. The mice (n = 7 for wt and n = 10 for Tg) were subjected to invasive infection with an ∼LD<sub>90</sub> dose of the <i>S. pyogenes</i> M5 strain and survival was recorded regularly, as indicated. (<b>E</b>) Survival of wild type (wt) and Tg mice after infection with M5 bacteria. The mice (n = 12 for wt and n = 12 for Tg) were subjected to invasive infection, as in D, but with a lower bacterial dose. (<b>F</b>) Growth of the <i>S. pyogenes</i> M5 strain in wild type mice injected with pure human Y402 FH (2×100 µg) or with PBS. The mice (n = 6 for FH and n = 6 for PBS) received FH or PBS 4 h before the infection and mixed with the infecting bacteria. Bacterial counts in spleens were analyzed, as in (C). (<b>G</b>) Growth of the <i>S. pyogenes</i> M5 strain in wild type mice injected with normal human serum (NHS) or normal mouse serum (NMS). The mice (n = 8 for NHS and n = 7 for NMS) were injected with two 200 µl doses of serum, one dose given 4 h before the infection and one mixed with the infecting bacteria. Analysis as in (C).</p
Antigenic properties of FH-binding HVRs.
<p>(<b>A</b>) Isolated HVRs show limited or no antigenic cross-reactivity. Antiserum to one HVR (specified at the top of a panel) was analyzed for reactivity with four immobilized HVRs, as indicated. The HVRs of M5 and M18 exhibited limited cross-reactivity, while the HVR of M6 did not cross-react with the other two HVRs. The non-FH-binding M1-HVR was included as a control. Pre-immune sera were used to obtain background values, which were subtracted from the values obtained with immune sera. (<b>B</b>) Binding of FH to the HVR of an M protein is inhibited by antiserum to the corresponding HVR. The biotinylated M protein indicated above each panel was used to detect FH immobilized in microtiter wells. The binding was inhibited with rabbit anti-HVR serum, diluted as indicated. Preimmune serum was used as a control.</p
FH-binding M proteins show species specificity and bind to the same region in FH.
<p>(<b>A</b>) The M5-HVR binds FH present in normal human serum (NHS) but not FH in normal mouse serum (NMS) or normal rabbit serum (NRS). In three separate tests, the sera were applied to a column containing the M5-HVR. After washes, bound protein was eluted and analyzed by SDS-PAGE. (<b>B</b>) Binding of a chimeric FH, expressed by Tg mice, to M proteins. The chimeric FH includes the SCR6-8 region of human FH (top). Serum from the Tg mice, or from wild type (wt) C57Bl/6 mice, was analyzed for presence of FH able to bind to the M protein indicated, immobilized in microtiter wells. (<b>C</b>) Different FH-binding HVRs bind to the same or overlapping site(s) in FH. A biotinylated form of the M5, M6 or M18 protein was used to detect FH immobilized in microtiter wells, and binding was inhibited with the four HVRs indicated, added at a concentration of 10 µM.</p