Development of tetravalent, bispecific CCR5 antibodies with antiviral activity against CCR5 monoclonal antibody-resistant HIV-1 strains

In this study, we describe novel tetravalent, bispecific antibody derivatives that bind two different epitopes on the HIV coreceptor CCR5. The basic protein formats that we applied were derived from Morrison-type bispecific antibodies: whole IgGs to which we connected single-chain antibodies (scFvs) via (Gly4Ser)n sequences at either the C or N terminus of the light chain or heavy chain. By design optimization, including disulfide stabilization of scFvs or introduction of 30-amino-acid linkers, stable molecules could be obtained in amounts that were within the same range as or no less than 4-fold lower than those observed with monoclonal antibodies in transient expression assays. In contrast to monospecific CCR5 antibodies, bispecific antibody derivatives block two alternative docking sites of CCR5-tropic HIV strains on the CCR5 coreceptor. Consequently, these molecules showed 18- to 57-fold increased antiviral activities compared to the parent antibodies. Most importantly, one prototypic tetravalent CCR5 antibody had antiviral activity against virus strains resistant to the single parental antibodies. In summary, physical linkage of two CCR5 antibodies targeting different epitopes on the HIV coreceptor CCR5 resulted in tetravalent, bispecific antibodies with enhanced antiviral potency against wild-type and CCR5 antibody-resistant HIV-1 strains.     

Preferences for rehabilitation services among women with major limb amputations

Purpose

We present five cases of adult females with major limb amputations, their concerns and preferences for services across the life span.

Design

A convenience sample of five veteran and nonveteran women aged 19–58 with major limb amputations participating in a regional VA Prosthetics Conference in 2010 took part in a panel interview.

Findings

The concerns identified by these women as high priorities included independence and participation in a full range of life activities, limitations in access, patient decision‐making and body image concerns, and preferences for selected services. Maximizing function and quality of life for women amputees requires identifying patient preferences for rehabilitation and prosthetic services. Lessons learned could inform development of clinic‐based rehabilitation care, prosthetic services, and studies of women with major limb amputations.

Conclusions

As the current conflicts in Iraq and Afghanistan wind down, the number of women veterans seeking rehabilitation and prosthetic services will increase. With this information, rehabilitation and prosthetic service providers and organizations will be uniquely positioned to provide prevention and treatment of amputations for this growing population of women veterans in national care delivery systems and in communities.

Clinical Relevance

An open‐ended facilitated discussion among a panel of women with major limb amputations provided insights for providers and organizations with respect to needs, concerns, and preferences for rehabilitation and prosthetic services.     

A human antibody binds to alpha-galactose receptors and mimics the effects of Clostridium difficile toxin A in rat colon

BACKGROUND & AIMS: Nearly all human sera contain an immunoglobulin G antibody (antigalactose) that binds the trisaccharide Gal alpha 1-3Gal beta 1-4GlcNAc expressed on cells from most mammals but not humans. Because the Clostridium difficile toxin A receptor in rodents contains this trisaccharide, the aim of this study was to examine whether antigalactose could mimic the enterotoxic effects of toxin A and bind to receptors containing this trisaccharide. METHODS: Fluid secretion, [3H]-mannitol permeability, and release of rat mast cell protease II and prostaglandin E2 were measured after luminal exposure of rat colon to either purified human anti-galactose, control immunoglobulin G, toxin A, or buffer. RESULTS: Toxin A (5 micrograms) and antigalactose (250 micrograms) but not control immunoglobulin (250 micrograms) stimulated colonic fluid secretion and caused increased mannitol permeability and rat mast cell protease II release. Antigalactose and toxin A and, to a lesser degree, control immunoglobulin G also stimulated release of prostaglandin E2, but only toxin A produced acute inflammation of rat colonic mucosa. Antigalactose and toxin A bound specifically to a single class of colonic brush border receptors with dissociation constants of 10(-6) mol/L and 5.4 x 10(-8) mol/L, respectively. CONCLUSIONS: Fluid secretion, increased permeability, and mast cell activation occur in rat colon when toxin A or human antigalactose immunoglobulin G bind to receptors bearing the trisaccharide Gal alpha 1-3Gal beta 1-4GlcNAc. (Gastroenterology 1996 Jun;110(6):1704-12)     

Specificity of autoantibodies in autoimmune thrombocytopenia

In 42 patients with autoimmune thrombocytopenia (AITP) and a positive direct platelet suspension immunofluorescence test (PSIFT), the antigenic specificity of the autoantibodies was studied. Because the autoantibodies were often not detectable in the serum and additional HLA antibodies may disturb the reaction pattern with the platelet panel, we used eluates prepared from the patients' platelets for this study. Thirty-five patients had antibodies equally reactive with normal platelets, irrespective of their antigenic make-up, but not with the platelets from two Glanzmann's disease patients. Absorption and elution experiments in two patients showed that his was probably not due to the presence of a combination of anti-Zwa and anti-Zwb antibodies. Thus, the majority of autoantibodies against platelets seems to be directed against antigenic determinants not present on Glanzmann's disease platelets, but perhaps located on the platelet-membrane glycoproteins IIb and/or IIIa. In ten patients, antibodies of no, or still unknown, specificity were detected. Three of these had additional antibodies not reactive with the platelets of the two Glanzmann patients.     

Sera from patients with heparin-induced thrombocytopenia generate platelet-derived microparticles with procoagulant activity: an explanation for the thrombotic complications of heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia is characterized by moderate thrombocytopenia and thrombotic complications, whereas quinine/quinidine-induced thrombocytopenia usually presents with severe thrombocytopenia and bleeding. Using flow cytometry and assays of procoagulant activity, we investigated whether sera from patients with these immune drug reactions could stimulate normal platelets to generate platelet-derived microparticles with procoagulant activity. Sera or purified IgG from patients with heparin-induced thrombocytopenia stimulated the formation of platelet-derived microparticles in a heparin-dependent fashion. Further studies showed that heparin-induced thrombocytopenia sera also produced a marked increase in procoagulant activity. In contrast, sera from patients with quinine- or quinidine-induced thrombocytopenia did not generate platelet-derived microparticles nor generate increased procoagulant activity. However, quinine/quinidine-induced thrombocytopenia sera produced a significant increase in the binding of IgG to platelets in a drug-dependent fashion, whereas sera from patients with heparin-induced thrombocytopenia demonstrated no drug-dependent binding of IgG to platelets. We also observed increased levels of circulating microparticles in patients with acute heparin-induced thrombocytopenia compared with control patients. Our observations indicate that the generation of procoagulant platelet-derived microparticles in vivo is a plausible explanation for the thrombotic complications observed in some patients with heparin-induced thrombocytopenia.     

Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis

Apoptosis, or programmed cell death, is a general mechanism for removal of unwanted cells from the immune system. It is characterized by chromatin condensation, a reduction in cell volume, and endonuclease cleavage of DNA into oligonucleosomal length fragments. Apoptosis is also accompanied by a loss of membrane phospholipid asymmetry, resulting in the exposure of phosphatidylserine at the surface of the cell. Expression of phosphatidylserine at the cell surface plays an important role in the recognition and removal of apoptotic cells by macrophages. Here we describe a new method for the detection of apoptotic cells by flow cytometry, using the binding of fluorescein isothiocyanate-labeled annexin V to phosphatidylserine. When Burkitt lymphoma cell lines and freshly isolated germinal center B cells are cultured under apoptosis inducing conditions, all cells showing chromatin condensation strongly stain with annexin V, whereas normal cells are annexin V negative. Moreover, DNA fragmentation is only found in the annexin V-positive cells. The nonvital dye ethidium bromide was found to stain a subpopulation of the annexin V-positive apoptotic cells, increasing with time. Our results indicate that the phase in apoptosis that is characterized by chromatin condensation coincides with phosphatidylserine exposure. Importantly, it precedes membrane damage that might lead to release from the cells of enzymes that are harmful to the surrounding tissues. Annexin V may prove important in further unravelling the regulation of apoptosis.     

Immunoglobulin G from patients with heparin-induced thrombocytopenia binds to a complex of heparin and platelet factor 4

Heparin-induced thrombocytopenia (HIT) is an important complication of heparin therapy. Although there is general agreement that platelet activation in vitro by the HIT IgG is mediated by the platelet Fc receptor, the interaction among the antibody, heparin, and platelet membrane components is uncertain and debated. In this report, we describe studies designed to address these interactions. We found, as others have noted, that a variety of other sulfated polysaccharides could substitute for heparin in the reaction. Using polysaccharides selected for both size and charge, we found that reactivity depended on two independent factors: a certain minimum degree of sulfation per saccharide unit and a certain minimum size. Hence, highly sulfated but small (< 1,000 daltons) polysaccharides were not reactive nor were large but poorly sulfated polysaccharides. The ability of HIT IgG to recognize heparin by itself was tested by Ouchterlony gel diffusion, ammonium sulfate and polyethylene glycol precipitation, and equilibrium dialysis. No technique demonstrated reactivity. However, when platelet releasate was added to heparin and HIT IgG, a 50-fold increase in binding of radio-labeled heparin to HIT IgG was observed. The releasate was then depleted of proteins capable of binding to heparin by immunoaffinity chromatography. Only platelet factor 4-immunodepleted releasate lost its reactivity with HIT IgG and heparin. Finally, to determine whether the reaction occurred on the surface of platelets or in the fluid phase, washed platelets were incubated with HIT IgG or heparin and after a wash step, heparin or HIT IgG was added, respectively. Reactivity was only noted when platelets were preincubated with heparin. Consistent with these observations was the demonstration of the presence of PF4 on platelets using flow cytometry. These studies indicate that heparin and other large, highly sulfated polysaccharides bind to PF4 to form a reactive antigen on the platelet surface. HIT IgG then binds to this complex with activation of platelets through the platelet Fc receptors.     

Inferior Vena Caval and Right Atrial Thrombosis Complicating an Amebic Hepatic Abscess

We describe a 50-yr-old black laborer who presented with right lower chest pain, weight loss, and pedal edema. Ultrasonography and computed tomograms showed a large abscess cavity in the right lobe of the liver which extended very close to the inferior vena cava. The lumen of the adjacent inferior vena cava was partially occluded by thrombus, which could be traced up into the cavity of the right atrium. The hepatic veins were normally patent. Sterile blood-stained pus was aspirated from the abscess. Antibodies against Entamoeba histolytica were present in high titer in the patient's serum. Although propagation of hepatocellular carcinoma into the inferior vena cava and even up into the right atrium is well recognized, inferior vena caval thrombosis extending up into the right atrium has not hitherto been reported as a complication of amebic hepatic abscess.     

Bone-marrow microinvolvement in non-small cell lung cancer is not a reliable indicator of tumour recurrence and prognosis.

AIMS: This study aimed to examine the incidence of bone-marrow microinvolvement in non-small cell lung cancer (NSCLC) patients and its correlation with tumour recurrence and prognosis. METHODS: Between March 1997 and August 1998, we analysed 96 bone-marrow specimens (from the posterior iliac crest) of NSCLC patients before surgery. Tumour differentiation showed well differentiated carcinoma in six, moderately differentiated carcinoma in 69, and poorly differentiated carcinoma in 21. p-TNM staging showed stage Ia in five, stage Ib in 33, stage IIb in 19, stage IIIa in 26, stage IIIb in eight, and stage IV in five. The specimens were examined by immunohistochemical staining with anti-human cytokeratin AE1/AE3 and clone MNF116 mixed solution (Ab1, n=96) and/or Ber-EP4 (Ab2, n=80) to detect the presence of malignant epithelial cells in the bone marrow. RESULTS: Positive results were observed in 21 patients (21. 9%). The occurrence of bone-marrow microinvolvement was not related to patient age, sex, cell type, or TNM status. The 30-month disease-free survival rates were 50.2% and 53.9% in bone-marrow negative and bone-marrow positive patients, respectively (P=0.5670); the 30-month cumulative survival rates were 66.7% and 67.6% in bone-marrow negative and bone-marrow positive patients, respectively (P=0.9351). Multivariate analysis failed to demonstrate bone-marrow microinvolvement as an independent prognostic factor. CONCLUSIONS: Our results show that bone-marrow microinvolvement is not unusual, and its occurrence cannot be translated into early tumour recurrence or poor outcome during an intermediate-term follow-up, which means bone-marrow microinvolvement may be an epiphenomenon rather than true metastasis in NSCLC.