Sheep Red Cells Research Articles

Does Plasma Inhibit the Activity of KCl Cotransport in Red Cells From LK Sheep?

Red cells from LK sheep represent an important paradigm for control of KCl cotransport activity, as well as being important to sheep erythroid function. A previous report (Godart et al., 1997) suggested that autologous plasma markedly inhibits red cell KCC activity and identified the presence of the bicarbonate/CO2 buffer system as the probable cause. Findings were restricted, however, to red cells from patients with sickle cell disease (SCD) swollen anisotonically and carried out at a very high O2 tension (c.700 mmHg). It was therefore important to investigate the generality of the effect described and whether it was also relevant to the two main stimuli for KCC activity encountered most often by circulating red cells in vivo - low pH in active muscle beds during exercise and high urea concentrations in the renal medulla during antidiuresis. Results confirm that inhibition was significant in response to anisotonic swelling with KCC activity in MOPS-buffered saline (MBS) vs. bicarbonate-buffered saline (BBS) and in MBS vs. plasma both reduced (by about 25 and 50%, respectively). By contrast, however, inhibition was absent at low pH and in high concentrations of urea. These findings suggest therefore that red cell KCC activity represents an important membrane permeability in vivo in red cells suspended in plasma. They are relevant, in particular, to sheep red cells, and may also be important by extension to those of other species and to the abnormal red cells found in human patients with SCD.

In Silico Prediction and Design of Uropathogenic Escherichia coli Alpha-Hemolysin Generate a Soluble and Hemolytic Recombinant Toxin

The secretion of α-hemolysin by uropathogenic Escherichia coli (UPEC) is commonly associated with the severity of urinary tract infections, which makes it a predictor of poor prognosis among patients. Accordingly, this toxin has become a target for diagnostic tests and therapeutic interventions. However, there are several obstacles associated with the process of α-hemolysin purification, therefore limiting its utilization in scientific investigations. In order to overcome the problems associated with α-hemolysin expression, after in silico prediction, a 20.48 kDa soluble α-hemolysin recombinant denoted rHlyA was constructed. This recombinant is composed by a 182 amino acid sequence localized in the aa542–723 region of the toxin molecule. The antigenic determinants of the rHlyA were estimated by bioinformatics analysis taking into consideration the tertiary form of the toxin, epitope analysis tools, and solubility inference. The results indicated that rHlyA has three antigenic domains localized in the aa555–565, aa600–610, and aa674–717 regions. Functional investigation of rHlyA demonstrated that it has hemolytic activity against sheep red cells, but no cytotoxic effect against epithelial bladder cells. In summary, the results obtained in this study indicate that rHlyA is a soluble recombinant protein that can be used as a tool in studies that aim to understand the mechanisms involved in the hemolytic and cytotoxic activities of α-hemolysin produced by UPEC. In addition, rHlyA can be applied to generate monoclonal and/or polyclonal antibodies that can be utilized in the development of diagnostic tests and therapeutic interventions.

Single-cell atlas of splenocytes reveals a critical role of a novel plasma cell‒specific marker Hspa13 in antibody class-switching recombination and somatic hypermutation

Our previous study had shown that member 13 (Hspa13) of heat shock protein family A (Hsp70) promotes plasma cell (PC) production and antibody secretion. To further explore Hspa13 expression and function, we combined single-cell RNA-sequencing and antigen receptor lineage (BCR) analysis to characterize sheep red cell‒primed splenocytes. The single-cell transcriptional profiles revealed that Hspa13 is specifically and highly expressed in PCs. These results suggest that Hspa13 is a novel PC-specific marker. In terms of its function, we found that the CD19cre-mediated conditional knock-out (cKO) of Hspa13 reduced the expression of Ebi3 and IL-10 in PCs. Ebi3 and IL-10 are important factors in IL-4‒secreting type 2 helper T cell (Th2) activation and differentiation. As expected, we found that the Hspa13 cKO reduced IL‒4-expressing follicular helper T (Tfh2) cells. Finally, the single-cell antigen receptor analysis demonstrated that the Hspa13 cKO reduced the Aicda-mediated antibody class-switching recombination (CSR) and somatic hypermutation (SHM) in germinal centers (GCs) B cells. Altogether, the single-cell atlas of splenocytes revealed a critical indirect role for the novel PC-specific marker Hspa13 in CSR and SHM in GC B cells by promoting Ebi3 and IL-10 expression in PCs to induce IL-4-expressing Tfh2 cells. Further exploration of Hspa13 expression and function will provide valuable clues for how to use Hspa13 in the treatment of autoimmune diseases.

Evaluation of virulence factors of Candida albicans isolated from HIV-positive individuals using HAART

The colonization by Candida species is one of the most important factors related to the development of oral candidiasis in HIV-infected individuals. The aim of the study was to evaluate and discuss the phospholipase, proteinase, DNAse and haemolytic activities of Candida albicans isolated from the oral cavity of HIV individuals with high efficiency antiretroviral therapy. Seventy-five isolates of C. albicans obtained from saliva samples of patients with HIV and 41 isolates from HIV-negative individuals were studied. Haemolytic activity was determined in Sabouraud dextrose agar plates containing 3% glucose and 7% sheep red cells. Culture medium containing DNA base-agar, egg yolk, and bovine albumin were used to determine DNase, phospholipase and proteinase activities, respectively. All isolates from the HIV patients group had haemolytic activity, 98% showed phospholipase activity, 92% were positive for proteinase and 32% DNAse activity. Regarding the group of indivídios HIV negative, all 41 isolates presented hemolytic activity, 90.2% showed phospholipase and proteinase activity and 12.2% were positive for DNAse. The phospholipase activity was more intense for the group of HIV positive individuals. DNase production was more frequently observed in the group of HIV-positive individuals. The percentage of isolates having DNAse activity was also significantly different between the groups of patients not using any antiretroviral therapy, those using transcriptase inhibitors and those using transcriptase inhibitor and protease inhibitor in combination.

Prevalence and antibiotic susceptibility pattern of methicillin resistant <i>Staphylococcus aureus</i> in burns and pressure ulcer patients

Methicillin resistant Staphylococcus aureus (MRSA) is a multidrug resistant bacterium that threatens the continued effectiveness of antibiotics worldwide. The objective of this study was to investigate the prevalence of MRSA and its antibiotic susceptibility pattern in patients with burns and bedsore. This was a cross- sectional study that was carried out at National Orthopaedic Hospital, Enugu, Nigeria. A structured questionnaire was used to obtain information on demographic and source of wounds. Pus from the wound was collected with swab sticks or 2ml syringe and analyzed bacteriologically, using mannitol salt agar sheep red cell blood agar. Isolates of Staphyloccus aureus were subjected to oxacillin and cefoxitin disc-diffusion assay and confirmed by chromogenic Brilliance MRSA 2 Agar; for identification of MRSA and MSSA. The MRSA and MSSA strains were tested for antimicrobial susceptibility patterns and multiple antibiotic index calculated. Of 104 wound swabs analyzed, 52 (50%) were Staphylococcus aureus isolates, while 21 (20.2%) were MRSA and 31 (29.8%) were MSSA. No significant differences were observed in the prevalence of MRSA among gender, duration of wounds, wound dressing interval and source of wound. There was an association between age, prolonged hospital admission MRSA infection. Methicillin-resistant Staphylococcus aureus isolates showed high resistance to ampicillin 90.5% followed by erytromycin 81% and ciprofloxacillin 71.4%.All the MRSA isolates were susceptible to vancomycin. All isolates of MRSA were resistant to β-lactams, aminoglycosides and quinolones group of antibiotic used. Minimum Inhibitory Concentration of vancomycin showed that the break point was between 0.5-2kg/ml and that of ampicillin was ranges from 4 kg/ml-128 kg/ml. MAR Index was >0.2 which indicates the resistance emanates from hospital. The high prevalence of MRSA and antibiotics resistance may increase the disease burden amongst these patients. It is necessary to establish an antimicrobial susceptibility surveillance system and to improve current infection control programs in the hospitals and community settings, to prevent the spread of MRSA.Keywords: MRSA, Brillience ChromAgar, ampicillin, vancomycin, multiple antibioticindex

Enzymatic and hemolytic activity in different Candida species

BackgroundCandida species, in conditions of microbiota imbalance or decreased immune defenses, may be one of the main human fungal pathogens. Virulence factors constitute the mechanisms used by the fungus to avoid host defenses. AimsThis study aimed to investigate the in vitro production of virulence factors, such as hemolytic activity, and deoxyribonuclease (DNase), proteinase, and phospholipase activities in Candida spp. MethodsFifty clinical isolates were analyzed for virulence factors: Candida albicans (15), Candida tropicalis (15), Candida parapsilosis (10), Candida glabrata (5), and Candida krusei (5). Hemolytic activity was determined in Sabouraud dextrose agar plates containing 3% glucose and 7% sheep red cells. Culture media containing, respectively, agar-base DNA, egg yolk, and bovine albumin were used to determine DNase, phospholipase and proteinase activities, respectively. ResultsForty-eight (96%) of 50 isolates showed hemolytic activity, with 10 (20%) positive for DNase, 19 (38%) for proteinase, and 16 (32%) for phospholipase. Statistically significant differences were observed between species for phospholipase (p<0.0001) and proteinase (p<0.05) production. ConclusionsIt is concluded that all species had hemolytic activity. DNase activity was detected in all species except in C. glabrata; proteinase activity was detected in C. albicans, C. tropicalis, and C. parapsilosis; and phospholipase activity was observed in C. albicans and C. tropicalis.

COMPLEMENT FACTOR H FUNCTIONAL ASSAY MAY HELP TO MONITOR ATYPICAL HAEMOLYTIC URAEMIC SYNDROME: A PILOT STUDY

Background: Atypical haemolytic uraemic syndrome (aHUS) results from uncontrolled complement system activation. Complement factor H gene mutations are common causes of aHUS. Plasmatherapy, including plasma infusions and/or plasma exchanges, has been tried in this setting with various successes. At present, we lack a specific marker to monitor functional factor H deficiency-related aHUS.Methods: We report the use of factor H functional assay in three patients with atypical haemolytic uraemic syndrome. This assay is based on the requirement of soluble complement regulators that bind sheep red cells to prevent haemolysis. As factor H is highly abundant in the plasma, its defect results in haemolysis. Factor H activity was also measured among plasma donors.Results: One patient suffered from a plasma-dependent form of atypical haemolytic uraemic syndrome. Plasma exchanges restored higher factor H activity and were associated with a 15-months disease-free period. In the two other patients, one with a failing renal graft and the other on chronic dialysis, a bout of thrombotic microangiopathy was preceded by a drop of haemolytic activity below normal values. Plasma from healthy donors (N = 65) showed only minimal variations of Factor H activity (mean activity: 98.3%, SD = 4.0).Conclusion: These preliminary data suggest that factor H activity could be of interest in both the diagnosis and the treatment by plasmatherapy of factor H-related aHUS

Infectious mononucleosis or glandular fever revisited

Infectious mononucleosis is also known as kissing disease or Pfeiffer’s disease. It is caused by the Epstein-Barr virus, a type of Herpes virus. More than 95% of adults worldwide have been exposed to this virus1 and may be at risk of reactivation in case of immune depression, advanced HIV infection,2 treatment of inflammatory diseases with anti-TNF (tumor necrosis factor)3 or immunosuppressive agents. In children, the disease is asymptomatic, or it can be characterized by a flu-like syndrome, and it is thought to be more prevalent in developing rather than developed countries.1 The symptomatic form of disease is commonly seen in teenagers who present with a sore throat, tonsillitis, lymphadenopathy, fever and fatigue. Classic symptoms also include petechiae, pharyngitis, splenomegaly and hepatitis. Thrombocytopenia and even pancytopenia may be present, as well as a cold agglutinin disease characterized by an autoimmune hemolytic anemia. Elevated hepatic transaminases can be found in roughly half of the patients. Ampicillin or amoxicillin may trigger a characteristic rash. The Epstein-Barr virus is spread via saliva, with an incubation period of 4 to 6 weeks and symptoms lasting for 2 to 3 weeks. The virus replicates in the endothelial cells of the pharynx and in the B cells (CD 21).1 This results in atypical lymphocytes or Downey cells. The diagnosis relies on atypical lymphocytes which resemble monocytes or on heterophile antibody tests which assess agglutination with sheep, guinea pig and horse red cells. A more sensitive test is the detection of IgG (reflecting a past infection) and IgM antibodies (suggesting a recent or current infection). Differential diagnoses can include Toxoplasma gondii or cytomegalovirus infection, bacterial tonsillitis, other causes of hepatitis, leukemia and influenza. Treatment is symptomatic and most cases resolve spontaneously but splenic rupture can occur, although rarely. For this reason, contact sports should be avoided. The mainstay of treatment includes non-steroidal anti-inflammatory drugs, especially ibuprofen. Antibiotics should not be used, as the etiological agent is a virus, or unless there is a concomitant streptococcal sore throat (in this case a penicillin derivative can be used). In the event of an upper airways obstruction, steroids may be used, but opioid analgesics are relatively contraindicated due to the risk of respiratory depression.

Resistance to Complement Dependent Cytotoxicity in CLL Cells From Patients Treated with Ofatumumab

Abstract 2836Monoclonal antibodies (mAb) are an effective but non-curative treatment of CLL. Although the mechanisms of action of mAb in vivo are not fully defined, complement dependent cytotoxicity (CDC) appears to play an important role. We have previously shown that addition of ofatumumab (OFA) significantly increases alemtuzumab (ALM) CDC in vitro and identified a subpopulation of CLL cells that are intrinsically resistant to activated complement. We propose that resistance to CDC could be an important cause of mAb treatment failure in CLL patients. To test the hypothesis that CLL cells treated in vivo with OFA would be resistant to subsequent OFA retreatment in vitro, we studied CLL cells and serum from 10 previously untreated patients with progressive CLL treated with pentostatin, cyclophosphamide, and ofatumumab (PCO). Methods.Patients received 300 mg of OFA followed by 2 mg/m2 of pentostatin and 600 mg/m2 of cyclophosphamide on day 1 of therapy. Samples were taken prior to treatment (S1), immediately after the OFA infusion and prior to chemotherapy (S2), and post-chemotherapy before the second dose of OFA on day 2 (S3). Mononuclear blood cells isolated from EDTA anticoagulated blood by Ficoll-Paque centrifugation were purified to achieve a concentration of > 90% CLL. CDC was assayed at a concentration of 2 × 106/ml CLL cells with 10 mg/ml of ALM (Genzyme), OFA (G.S.K.) or rituximab (RTX)(Genentech) in AIM V medium (Invitrogen, CA) and 10% normal human serum (10%NHS)(Sigma, MO) as a source of complement for 1 hour at 37°C. Absolute viable cell counts were measured by flow cytometry using counting beads (Trucount, BD, CA) and propidium iodide staining (Sigma, MO) with a FACSCalibur (BD, CA) and CellQuest Pro software (BD, CA). Percent CDC was calculated relative to counts for CLL cells treated with only 10%NHS. Binding of mAb and C3b to CLL cells was measured by flow cytometry with mouse anti-human Fc antibody FITC-HB43 and anti-C3b antibody FITC-7C12. C5 deficient serum (C5-serum)(Sigma, MO) was used to examine mAb and C3b binding without CDC. Results were expressed as delta mean fluorescent intensity (dMFI) relative to cells treated without mAb in 10%NHS or C5-serum as appropriate. Complement (CH50) was measured based on lysis of opsonized sheep red cells using standard methods. OFA concentrations in serum samples were determined by measuring the level of binding to Daudi cells compared to standards as previously described. Results:S1 CLL cells were significantly more susceptible to in vitro OFA CDC (median 30%) than S2 and S3 cells (both 0%, p=0.002)(Fig. 1). In contrast, high levels of CDC (median 86 – 88%) were induced in vitro by ALM in all these samples and prior in vivo exposure to OFA did not affect susceptibility to ALM CDC in vitro (p>0.1). CLL cells from S2&S3 had low levels of OFA binding both prior to and post in vitro exposure to OFA (median dMFI 2 and 3 respectively) which was significantly less than OFA binding for S1 (median dMFI 38 p=0.002) likely reflecting in vivo trogocytosis of bound OFA and CD20, as previously described for RTX. In vitro ALM binding was higher than OFA (median dMFI>150) in all specimens. C3b binding was low in S2&S3 both before and after in vitro OFA exposure (median dMFI 3 and 4 respectively) with significantly higher levels in S1 (median dMFI 94, p=0.002). C3b binding was higher in all CLL cells treated with ALM (median dMFI>358). OFA treatment resulted in marked decreases in serum complement levels in S2 (median 86%, range 65–98%) and S3 (median 78% range 61–88%) compared to S1. The median serum OFA concentration was 18.5 μg/ml (range 7.3–50.4) in S2 and 19.8 μg/ml (range 0–32.3) in S3. Conclusions:Circulating CLL cells from patients treated with OFA are resistant to in vitro OFA CDC primarily because of low levels of expression of CD20. The reduction in complement titers could limit in vivo CDC but we found no evidence that low levels of OFA were important. Cells surviving OFA in vivo do retain sensitivity to ALM CDC. Our data support the previous description of trogocytosis in CLL patients treated with RTX and suggest that lower doses of these mAb, that promote far lower levels of trogocytosis, could be more effective in sustaining CDC. In addition, our study provides pre-clinical data to support a clinical trial of combination therapy with OFA and ALM for CLL. Acknowledgment:This study was supported by funding from GlaxoSmithKline and the University of Iowa/Mayo Clinic Lymphoma SPORE (CA097274). [Display omitted] Disclosures:Taylor:Genmab: Consultancy; Glaxo Smith Kline: Research Funding. Zent:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Genzyme: Research Funding.

Modulation of in vivo immunoglobulin production by endogenous histamine and H1R and H2R agonists and antagonists

The present study was designed to delineate the immunomodulatory role of histamine receptors (H1R and H2R) and their antibody generation in a rabbit model. Six groups containing 18 rabbits each received either vehicle (sterile distilled water, 1ml/kg×b.i.d), histamine (100µg/kg×b.i.d.), H1R agonist (HTMT, 10µg/kg×b.i.d.), H2R agonist (amthamine, 10µg/kg×b.i.d.), H1R antagonist (pheniramine, 10mg/kg×b.i.d.) or H2R antagonist (ranitidine, 10mg/kg×b.i.d.). All animals were subsequently immunized with an intravenous injection of sheep red bood cells (SRBC). Estimations of total serum immunoglobulins (Igs), immunoglobulin M (IgM) and immunoglobulin G (IgG) were performed by ELISA and hemagglutination assay (HA) at days 0 (pre-immunization), 7, 14, 21, 28 and 58 (post-immunization). Both the ELISA and the HA showed similar production of Igs, IgM and IgG but the results were found comparatively more significant by ELISA as opposed to HA. Results showed that histamine could influence a detectable antibody response to SRBC early (i.e., at day 7), which lasted until day 58. Immunomodulatory processes showed suppression of an Ig generation in the H1R-antagonist group with enhancement in the H2R-antagonist group. The H1R-agonist group showed an increased Ig production in comparison to the H2R-agonist group. The IgM production was inhibited in the H1R-antagonist group as compared to the H2R-antagonist group, and it was also suppressed in H1R-agonist group as compared to H2R-agonist group. IgG production was inhibited in the H1R-antagonist group as opposed to the H2R-antagonist group. In contrast, the H1R-agonist group increased IgG production as compared to the H2R-agonist group. All the results were found to be statistically significant (p<0.05 or p<0.01). In conclusion, histamine and its receptor (H1R and H2R) agonists enhance antibody production by triggering the histamine receptors (H1R and H2R), and both the H1R antagonist and the H2R antagonist positively or negatively regulate the antibody production. The findings of this study may have clinical significance.

English

This study determined the haemolytic, haemagglutinating and antibiotic susceptibility activities of Plesiomonas shigelloides isolated from stool samples of patients attending different health centers in the Venda region of South Africa. P.shigelloides was isolated and identified using the API 20E, API 20NE systems. Antibiotic susceptibility profiles of the isolates were determined using the disc diffusion method and analyzed according to NCCLS standards. The hemolytic and hemagglutination activities of the isolates on human, sheep, pig and chicken red blood cells were determined using the plate and slide methods. A total of 89 (13%) P. shigelloide were isolated from 660 samples. The hemolytic activities of the isolates were variable with no heamolysis on sheep red cells. 33 (37%) of isolates were beta lactamase producers. There was a high level of resistance to the penicilllins with 100% resistance to Penicillin G, Amoxicillin and Ampicillin. This study has demonstrated multiple resistance to different antibiotics and production of beta lactamase. Most of the isolates showed evidence of pathogenicity as demonstrated by hemolytic and haemagglutinating activities.

Sheep red cell survival (RCS) can be followed using 4 different densities of biotin label

Anemia is a serious problem in critically ill neonates. To investigate anemia, to assess responses to red blood cell (RBC) transfusions and erythropoietin therapy, and to compare sources of RBCs and storage methods, simultaneous measurement of RCS would be useful. Here we report the validation in sheep (a common model for perinatal studies of physiology and erythropoiesis) of a method for independent measurement of RCS using 4 populations of RBCs labeled at distinct biotin densities.MethodsBy varying the biotinylating reagent from 12 to 96 ?g/mL RBC, the density of biotin on sheep RBCs was incrementally increased to produce 4 discrete populations. Biotinylated RBCs (BioRBCs) were reinfused, and blood was sampled over the next 4 months. RCS was determined using flow cytometric enumeration and by scintillation counting.ResultsBioRBCs produced parallel disappearance curves with life spans from the linear phase that were similar to the reference 14C‐cyanate method (mean potential lifespan = 66±2 vs 62 d).ConclusionRCS can be measured accurately and simultaneously in sheep using BioRBCs without radiolabeling. NIH P01 HL046925; Thrasher Research Foundation 02825‐3.

SEROLOGICAL DETECTION OF THYMOMA IN MYASTHENIA GRAVIS

A purified citric acid extract (CAE) was prepared from skeletal muscle and coated onto sheep red cells for use in an indirect hemagglutination test. Out of 64 MG sera examined, only 5 contained antibodies to the CAE. Radiological evidence of a thymoma was found in 1, but thymectomy revealed a lymphoepithelioma in all 5 patients. In 4 of the patients, the tumor was very small or medium-sized. None of the CAE-negative patients had clinical, radiological or other evidence of a thymoma. The test in therefore a supplementary diagnostic method for the detection of a thymoma at an early stage.

Lysophosphatidic Acid Protects Cancer Cells from Histone Deacetylase (HDAC) Inhibitor-induced Apoptosis through Activation of HDAC

Histone deacetylases (HDACs) catalyze the removal of acetyl groups from histones and contribute to transcriptional repression. In addition, the HDAC inhibitors induce apoptosis in cancer cells through alterations in histone acetylation and activation of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) apoptotic pathway. Lysophosphatidic acid (LPA) is a growth factor that promotes survival of cancer cells through activation of G protein-coupled receptors. Here we show that HDAC inhibitors can induce apoptosis through activation of the TRAIL apoptotic pathway, and LPA prevented HDAC inhibitor-induced apoptosis and increased TRAIL receptor DR4 (death receptor 4) protein expression. This was associated with increased HDAC1 recruitment to the DR4 promoter following LPA treatment and a reduction in HDAC inhibitor-induced histone acetylation in the DR4 promoter. In addition, LPA induces HDAC enzyme activity in a dose- and time-dependent manner, and this is associated with HDAC1 activation and increased binding of HDAC1 to HDAC2. Reducing the expression of HDAC1 significantly lowered LPA-induced HDAC activity and increased histone acetylation. LPA induction of HDAC activity was blocked by the LPA receptor antagonist, Ki16425, or by inhibiting receptor activation with pertussis toxin. Reducing the expression of the LPA receptor LPA(1) also blocked LPA-induced HDAC activation. In addition, LPA reduced histone acetyltransferase enzymatic activity. Finally, LPA attenuated the ability of the HDAC inhibitor to reduce HDAC activity. Thus, LPA enhances survival of cancer cells by increasing HDAC activity and reducing histone acetylation.

“B27 Disease” Is a New Autoimmune Disease That Affects Millions of People

"B27 disease" is a new autoimmune disease that afflicts millions of people throughout the world. "B27 disease" occurs in individuals who have ankylosing spondylitis (AS) or preankylosing spondylitis and/or uveitis and are also positive for HLA-B27. Molecular mimicry between the bowel microbe Klebsiella and the HLA-B27 molecule, as well as the spinal collagens types I, III, and IV, indicates a pathological mechanism involving autoimmunity. Antibodies to Klebsiella microbes have been reported in AS patients from 18 different countries. Sera from patients with AS show complement-dependent cytopathic activity against sheep red cells coated with HLA-B27 peptide antigens. Diagnosis of B27 disease can lead to early treatment, involving low-starch diet, sulfasalazine, and immunosuppressive and biological agents so as to prevent the irreversible bony changes of established classical AS. The concept of B27 disease provides a new approach to the study and treatment of these disorders and needs to be evaluated in prospective studies by the world rheumatological community.

Streptococcus agalactiae CAMP factor/protein B does not bind to human IgG

CAMP factor is an extracellular cytolytic protein produced by Streptococcus agalactiae. CAMP factor has been reported to bind the Fc fragments of immunoglobulin G (IgG) and has therefore also been called protein B, in analogy to protein A of Staphylococcus aureus. We attempted to characterize the interaction of protein B with IgG in more detail. In contrast to protein A, CAMP factor does not inhibit the activation of complement by hemolysin antibodies bound to sheep red cell surfaces. IgG also failed to inhibit the co-hemolytic activity of CAMP factor, which is in disagreement with previous findings. After co-incubation, CAMP factor and IgG were cleanly separated by gel filtration, indicating that no binding had occurred.

Virulence properties of Escherichia coli O111:H12 strains.

Seventeen Escherichia coli O111:H12 strains isolated from the feces of children with acute diarrhea were studied with regard to their adherence properties and other virulence characteristics. All strains showed an aggregative adherence pattern to HEp-2 cells and agglutinated bovine and sheep red cells in the presence of mannose. These strains did not have gene sequences homologous to the aggregative adherence fimbria I gene and did not react with any of the DNA probes used to detect other virulence genes in enteropathogens. With one exception, the O111:H12 strains did not induce fluid accumulation in the rabbit ileal loop assay, although 16 of the strains had the enteroaggregative E. coli heat-stable enterotoxin 1 (EAST) gene sequences. A 60-70 MDa plasmid was present in 16 of the strains studied. We conclude that the O111:H12 serotype, one of the first E. coli identified in infantile diarrhea, belongs to the enteroaggregative E. coli category but the genes encoding its adherence phenotype are distinct from those previously described.

Lysophosphatidic Acid (LPA) Protects Primary Chronic Lymphocytic Leukemia Cells from Apoptosis through LPA Receptor Activation of the Anti-apoptotic Protein AKT/PKB

Lysophosphatidic acid (LPA) protects epithelial and fibroblast cell lines from apoptosis. In B-cells, LPA acts as a growth factor promoting cell proliferation. Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD19+/CD5+ B-lymphocytes primarily through a block in apoptosis. The mechanisms underlying this defect are not fully understood. We investigated whether LPA could be a survival factor in CLL cells. Herein, we demonstrate that LPA protects B-cell lines BJAB and I-83 and primary CLL cells but not normal B-cells from fludarabine- and etoposide-induced apoptosis. Furthermore, LPA prevented spontaneous apoptosis in primary CLL cells. The LPA1 expression was found to be increased in primary CLL cells compared with normal B-cells correlating with LPA prevention of apoptosis. Treatment of primary CLL cells with the LPA receptor antagonist, diacylglycerol pyrophosphate, reverses the protective effect of LPA against apoptosis, and down-regulation of the LPA1 by siRNA blocked LPA-mediated protection against spontaneous apoptosis in primary CLL cells. The protective effect of LPA was inhibited by blocking activation of the phosphatidylinositol 3-kinase/AKT signaling pathway. These results indicate that LPA is a survival factor in B-cell lines and primary CLL cells but not normal B-cells. Thus, drugs targeting the LPA receptors might be an effective therapy against B-cell-derived malignancies such as CLL.

In vivo anti-complement effect of bilirubin-IXα

The effect of the IXα isomer of unconjugated bilirubin (UB) on complement-mediated intravascular hemolysis was evaluated in rats carrying naturally occurring heteroantibodies against sheep erythrocytes. Several doses of UB were administered i.v. to these animals in order to induce different levels of hyperbilirubinemia. Intravascular hemolysis was promoted by transfusion with a sheep red cell suspension. Hemoglobin in urine was assessed as a marker of intravascular hemolysis. The urinary excretion of hemoglobin was attenuated by UB in a dose-dependent manner. To establish whether complement was involved in the hemolytic reaction, we evaluated the hemolytic activity of complement in these same animals, before and after sheep erythrocyte transfusion. The significant consumption of complement, which was partially prevented by UB, corroborated its participation in the intravascular hemolytic reaction in the current experimental conditions. The data suggest an inhibitory action of UB on complement-mediated hemolysis in vivo.

Effects of protein kinase and phosphatase inhibitors and anti-L antisera on K + transport in LK sheep red cells

We investigated the role of protein phosphorylation in the action of anti-L on low potassium (LK) sheep red cells. Anti-L stimulated the Na/K pump by four- to fivefold, but Na/K pump activity in anti-L-stimulated or in control cells was unaffected by protein kinase/protein phosphatase (PK/PP) inhibitors. KCl co-transport activity was inhibited by anti-L (about 50%). Co-transport was stimulated by staurosporine; and inhibited by calyculin A, okadaic acid, tyrphostin B46 and genistein; with a similar pattern in both control and anti-L-treated cells. O 2 sensitivity of KCl co-transport was similar in control and anti-L-treated cells. Neither control nor anti-L-stimulated Na/K pump activities were O 2 sensitive. Incubation with urea stimulated KCl co-transport in both control and anti-L-treated cells. Inhibition of co-transport by anti-L was unaffected by low concentrations of urea but was reduced at higher urea concentrations. Na/K pump activity of control cells was unaffected by incubation with urea, but that in cells stimulated by anti-L was reduced, though not significantly. Under high hydrostatic pressure, KCl co-transport was stimulated, and the inhibitory effects of PP inhibition (okadaic acid), anti-L or combinations of the two were reduced. Results suggest that anti-L does not affect K + transport in LK sheep red cells via protein phosphorylation.