Improved treatment of systemic blood infections using antibiotics with extracorporeal opsonin hemoadsorption.

Here we describe development of an extracorporeal hemoadsorption device for sepsis therapy that employs commercially available polysulfone or polyethersulfone hollow fiber filters similar to those used clinically for hemodialysis, covalently coated with a genetically engineered form of the human opsonin Mannose Binding Lectin linked to an Fc domain (FcMBL) that can cleanse a broad range of pathogens and endotoxin from flowing blood without having to first determine their identity. When tested with human whole blood in vitro, the FcMBL hemoadsorption filter (FcMBL-HF) produced efficient (90-99%) removal of Gram negative (Escherichia coli) and positive (Staphylococcus aureus) bacteria, fungi (Candida albicans) and lipopolysaccharide (LPS)-endotoxin. When tested in rats, extracorporeal therapy with the FcMBL-HF device reduced circulating pathogen and endotoxin levels by more than 99%, and prevented pathogen engraftment and inflammatory cell recruitment in the spleen, lung, liver and kidney when compared to controls. Studies in rats revealed that treatment with bacteriocidal antibiotics resulted in a major increase in the release of microbial fragments or 'pathogen-associated molecular patterns' (PAMPs) in vivo, and that these PAMPs were efficiently removed from blood within 2 h using the FcMBL-HF; in contrast, they remained at high levels in animals treated with antibiotics alone. Importantly, cleansing of PAMPs from the blood of antibiotic-treated animals with the FcMBL-hemoadsorbent device resulted in reduced organ pathogen and endotoxin loads, suppressed inflammatory responses, and resulted in more stable vital signs compared to treatment with antibiotics alone. As PAMPs trigger the cytokine cascades that lead to development of systemic inflammatory response syndrome and contribute to septic shock and death, co-administration of FcMBL-hemoadsorption with antibiotics could offer a more effective approach to sepsis therapy.

Antigenic modulation and rituximab resistance.

Several types of B-cell lymphoma have been successfully treated with rituximab, and approval by the US Food and Drug Administration for use of rituximab in the treatment of rheumatoid arthritis has increased interest in targeting CD20 on B cells for other indications. Although large amounts of rituximab can be infused into humans with no apparent dose-limiting toxicity, recent evidence suggests that the body's effector mechanisms, including complement-mediated cytotoxicity and natural killer (NK) cell-mediated killing, can be saturated or exhausted at high burdens of rituximab-opsonized B cells. One of the consequences of this saturation phenomenon is that the opsonized B cells are instead processed by a different pathway mediated by FcgammaR on effector cells. In this alternative pathway, both rituximab and CD20 are removed ("shaved") from the B cells and are taken up by monocytes/macrophages. This process, formerly called antigenic modulation, appears to occur in several compartments in the body and may play a key role in the development of resistance to rituximab therapy.

Formulation of multifunctional oil-in-water nanosized emulsions for active and passive targeting of drugs to otherwise inaccessible internal organs of the human body.

Oil-in-water (o/w) type nanosized emulsions (NE) have been widely investigated as vehicles/carrier for the formulation and delivery of drugs with a broad range of applications. A comprehensive summary is presented on how to formulate the multifunctional o/w NE for active and passive targeting of drugs to otherwise inaccessible internal organs of the human body. The NE is classified into three generations based on its development over the last couple of decades to make ultimately a better colloidal carrier for a target site within the internal and external organs/parts of the body, thus allowing site-specific drug delivery and/or enhanced drug absorption. The third generation NE has tremendous application for drug absorption enhancement and for 'ferrying' compounds across cell membranes in comparison to its first and second generation counterparts. Furthermore, the third generation NE provides an interesting opportunity for use as drug delivery vehicles for numerous therapeutics that can range in size from small molecules to macromolecules.

Prophylactic and therapeutic efficacy of a fully human immunoglobulin G1 monoclonal antibody to Pseudomonas aeruginosa alginate in murine keratitis infection.

Treatment of ulcerative keratitis due to Pseudomonas aeruginosa is difficult, time-consuming, and uncomfortable owing to the need for the frequent application of antibiotic drops to the infected corneal surface. We examined here whether a fully human immunoglobulin G1 monoclonal antibody (MAb) specific to the conserved alginate surface polysaccharide of P. aeruginosa could mediate protective immunity against typically nonmucoid strains isolated from human cases of keratitis. MAb F429 effectively opsonized alginate-positive, but not alginate-negative, nonmucoid strains in conjunction with phagocytes and complement. Prophylactic administration of MAb F429 18 h prior to infection with two clinical isolates significantly reduced bacterial levels in the eye and the associated corneal pathology. Along similar lines, systemic intraperitoneal injection, as well as topical application of the MAb onto the infected eye, starting 8 h postinfection in both experimental protocols resulted in significant reductions in bacteria in the eye, as well as minimizing pathological damage to the cornea. These findings indicate that MAb F429 could be useful as an additional therapeutic component for the treatment of P. aeruginosa keratitis.

A single-institution, 20-year prospective experience with an affordable Fc-receptor blockade method to treat patients with chronic, refractory autoimmune thrombocytopenic purpura.

In a 20-year period in a single institution, 34 patients with chronic, refractory autoimmune thrombocytopenic purpura were prospectively treated with ex vivo anti-D opsonized autologous red blood cells. All patients had received previous treatment with steroids and/or immunosuppressive agents, and 11 had been splenectomized. Twenty one patients had an increase in the platelet count; in five cases, the increase was more than 50 x 10(9)/L platelets and in 16 the increase was more than 100 x 10(9)/L platelets. Early responses were observed in 20 patients and late responses in seven, whereas seven patients (20%) did not respond at all. Nine of the 20 individuals who achieved an ER had a subsequent drop in the platelet count; however, only three had a drop below 50 x 10(9)/L. When last censored, of the 34 patients, 24 (70%) had a platelet count above 50 x 10(9)/L. The 84-month thrombocytopenia-free (over 50 x 10(9)/L platelets) status of the whole group is 70%, whereas the 84-month complete remission (over 100 x 10(9)/L platelets) status of the whole group is 50%. It is concluded that the use of ex vivo anti-D opsonized red blood cells may represent another, substantially cheaper treatment of patients with chronic, refractory, autoimmune thrombocytopenic purpura.

Antitumor antibodies in the treatment of cancer: Fc receptors link opsonic antibody with cellular immunity.

Engineered antibody therapeutics have provided new treatment options in cancer. Genetic evidence in man and in the mouse suggests that Fc receptor (FcR) engagement contributes mechanistically to the therapeutic activity of naked antibodies. Preferential activation of activating FcRs and limited engagement of inhibitory FcRs enhance tumor responses in mouse models. Thus, engineered Fc domains with favorable affinities for specific FcR types may prove to be clinically superior.

Antibody to capsular polysaccharide of Streptococcus pneumoniae at the time of hospital admission for Pneumococcal pneumonia.

IgG to capsular polysaccharide (CPS) of Streptococcus pneumoniae is thought to provide the greatest degree of protection against pneumococcal disease. Serum obtained at hospital admission from 14 (27%) of 51 patients with bacteremic pneumococcal pneumonia and 11 (37%) of 30 with nonbacteremic pneumococcal pneumonia contained IgG to CPS of the infecting serotype; these percentages are similar to the prevalence of IgG to CPS in a control population. However, when compared with antibody from healthy adults, this IgG had far less capacity to opsonize the infecting pneumococcal serotype for phagocytosis in vitro by normal human polymorphonuclear leukocytes or to protect mice against experimental challenge. Failure to opsonize correlated closely with failure to protect mice, and each of these parameters correlated well with poor avidity for CPS. Future vaccine studies may need to examine the functional capacity of antibodies as a surrogate for infection, in addition to measuring their concentration in serum.

Complementary immunotherapy in the treatment of chronic osteomyelitis.

The authors present a retrospective study of 492 cases of chronic osteomyelitis treated with active immunotherapy to complement surgical and antibiotic treatment. The systematic application of this treatment, which is based on the use of antistaphylococcal vaccines and/or auto-vaccines resulted in the osteomyelitic process being cured in 80 per cent of cases, with complete closure of the fistulae maintained for at least three years. The authors also report the preliminary results obtained with passive immunotherapy in twenty-six patients with chronic osteomyelitis that had proved resistant to all other forms of treatment. Sero-immunological investigations in these patients revealed a deficiency in the opsonifying capacity of the serum. Treatment with opsonin precursors produced complete recession of symptoms and clinical signs, maintained for at least a year, in 50 per cent of these patients.

Functional characteristics of a modified immunoglobulin preparation for intravenous administration: summary of studies of opsonic and protective activity against group B streptococci.

Group B streptococci (GBS) remains a major cause of morbidity and mortality in newborn babies, despite antibiotic therapy, Recent studies suggest that phagocytosis and killing of GBS is ineffective due to a deficiency in anti-GBS antibody. Using an opsonophagocytic bacterial assay and a suckling rat model of GBS sepsis, we analyzed a modified human immunoglobulin for opsonic and protective antibody. Immune globulin (IGIV) prepared for intravenous use (Gamimune, Cutter Laboratories, Inc.) was highly protective in this experimental GBS model. Using the opsonophagocytic assay, antibody activity to several strains of types Ia, II, and III GBS were also demonstrated with IGIV. To ensure that the IGIV activity was in fact antibody, globulin from IGIV was isolated and analyzed. Purified IgG retained opsonic activity in vitro and also provided protection in experimental GBS disease. Variation in the quantity of IgG necessary for protection was observed in different GBS strains. Since IGIV has opsonic and protective activity against several strains and serotypes of GBS, its intravenous administration may provide a valuable adjunct to standard antibiotic therapy for neonatal GBS infections.

Increased creatinine clearance following cryoprecipitate infusion in trauma and surgical patients with decreased renal function.

Deficiency of opsonic alpha 2 surface binding (SB) glycoprotein (cold-insoluble globulin, plasma fibrinectin) is related to depressed reticulendothelial function as well as to multiple organ failure after tissue injury and sepsis. Cryoprecipitate (250 ml), extracted from 10 units of human plasma, was infused over 60 minutes into 11 hypo-opsonemic patients with decreased renal function. Cardiac output, mean arterial pressure, creatinine clearance, and limb blood flow were measured before and at intervals of 14 to 20, 35 to 44, and 60 to 66 hours following cryoprecipitate infusion. Before infusion, the mean creatinine clearance was 30 +/- 4 ml/min/M2 body surface area (BSA) and increased to 40 +/- 6 ml/min/M2 BSA at 14 to 20 hrs (p < 0.05); to 40 +/- 4 ml/min/M2 BSA at 35 to 44 hrs (p < 0.05); and to 40 +/- 5 ml/min/M2 BSA at 60 to 66 hrs (p < 0.05). In contrast, mean arterial pressure and cardiac index at each time interval showed no significant changes from the pretreatment values of 81 +/- 6 mm Hg and 3.4 +/- .2 L/min/M2 BSA, respectively. Limb blood flow increased significantly at 4 hours and returned to control values by 35 to 44 hours. Thus cryoprecipitate infusion to critically ill trauma and surgical patients with depressed renal function may improve glomerular filtration rate independently of mean arterial pressure or cardiac output. This improved renal function may be related to increased reticuloendothelial clearance of blood-borne particulates and/or improved microcirculatory function and lends support to the concept that RES failure may be involved in the etiology of multiple organ failure secondary to combined tissue injury and sepsis.

Cardiovascular hemodynamics after opsonic alpha-2-surface binding glycoprotein therapy in injured patients.

Depression of reticuloendothelial (RE) phagocytic function has been clearly documented following trauma and operation. This phagocytic failure is mediated in part by depletion of an opsonic glycoprotein. Depletion of this opsonic protein may result in prolonged blood retention of potentially harmful particulates that may interfere with the microcirculation and may possibly result in altered organ function. Isolation and identification of this opsonic protein has led to the finding of the identity between opsonic glycoprotein and cold insoluble globulin (CIg) or so-called plasma fibronectin. Since CIg is concentrated in cryoprecipitate, this blood component was used as a readily available source of opsonic protein for replacement studies. Nine patients were studied following a 1-hour infusion of cryoprecipitate obtained from 10 units of plasma and suspended in a volume of 250 ml. Both the pulmonary shunt fraction and the fraction of dead space ventilation decreased significantly (P = 0.02) after cryoprecipitate administration. Limb blood flow (P = 0.001), limb oxygen consumption (P = 0.001), and reactive hyperemia of the limb (P = 0.05) increased significantly following cryoprecipitate infusion. Cardiac output, total oxygen consumption did not change consistently. The data demonstrate that the infusion of cryoprecipitate resulted in improved pulmonary and microcirculatory function--possibly due to opsonic glycoprotein replacement.

Opsonic alpha2 surface binding glycoprotein therapy during sepsis.

A pronounced depletion of an opsonic protein for hepatic reticuloendothelial (RE) phagocytosis has been demonstrated in critically ill trauma patients. This opsonic alpha(2) surface binding (SB) glycoprotein has immunologic identity and a similar amino acid composition to cold insoluble globulin (CIg). Since CIg can be concentrated in cryoprecipitate, it was utilized as a readily available source of opsonic alpha(2)SB glycoprotein for replacement therapy after injury with documented hypoopsonemia. Six septic patients (2 multiple trauma, 2 thermal burn, and 2 intra-abdominal abscess) were studied to test whether cryoprecipitate infusion would restore this humoral component. Pre- and posttherapy opsonin levels were determined by bioassay and electroimmunoassay. In all patients, severe opsonin depletion was reversed following cryoprecipitate infusion. All patients had a rapid improvement in febrile state, normalization of leukocyte levels, and improvement in pulmonary function as evidenced by decreasing requirements for end expiratory pressure at lowered levels of inspired oxygen. One patient was studied more extensively and demonstrated an increase in cardiac output, limb blood flow, total body and limb oxygen delivery, total body and limb oxygen consumption and a progressive decrease in pulmonary shunt fraction. Thus, opsonic alpha(2)SB glycoprotein deficiency can be reversed by cryoprecipitate infusion in critically ill septic injured patients. Replacement of this humoral factor may be an important therapeutic modality in prevention of multiple organ failure, but it should be administered only after documentation of hypoopsonemia in traumatized patients.

Macrophage-mediated destruction of human malignant cells in vivo.

Macrophages require a plasma component, designated "recognition factor" (RF), for the expression of optimal function. The RF activity was profoundly depleted in plasma from patients with malignant disease, and the degree of depletion and the severity of the malignant state seemed to be related. Since experiments demonstrated that an active RF significantly inhibited tumor growth, clinical studies were initiated to investigate the influence of intratumor administration of an active RF fraction. Glucan, a potent macrophage activator, was also employed alone or combined with RF. These studies were undertaken to enhance the recognition of malignant cells by macrophages and to mobilize and activate macrophages intralesionally. The initial 9 patients studied had malignant melanoma, adenosquamous carcinoma of the lung, or carcinoma of the breast. Control and experimental lesions were injected; subsequently biopsies were performed at varying intervals for histologic evaluation. Always when glucan or glucan and RF fraction were administered intralesionally, the size of the lesion was strikingly reduced in as short a period as 5 days. This reduction was associated with necrosis of the tumor and a monocytic infiltrate. In small lesions, resolution was complete, whereas in large lesions, resolution was partial. The amount of glucan injected and the quantity of residual tumor appeared to be related. The induced necrosis of the tumor nodule was associated with an increase in plasma levels of circulating RF activity.