An HPV-E6/E7 immunotherapy plus PD-1 checkpoint inhibition results in tumor regression and reduction in PD-L1 expression.

We have investigated if immunotherapy against human papilloma virus (HPV) using a viral gene delivery platform to immunize against HPV 16 genes E6 and E7 (Ad5 [E1-, E2b-]-E6/E7) combined with programmed death-ligand 1 (PD-1) blockade could increase therapeutic effect as compared to the vaccine alone. Ad5 [E1-, E2b-]-E6/E7 as a single agent induced HPV-E6/E7 cell-mediated immunity. Immunotherapy using Ad5 [E1-, E2b-]-E6/E7 resulted in clearance of small tumors and an overall survival benefit in mice with larger established tumors. When immunotherapy was combined with immune checkpoint blockade, an increased level of anti-tumor activity against large tumors was observed. Analysis of the tumor microenvironment in Ad5 [E1-, E2b-]-E6/E7 treated mice revealed elevated CD8(+) tumor infiltrating lymphocytes (TILs); however, we observed induction of suppressive mechanisms such as programmed death-ligand 1 (PD-L1) expression on tumor cells and an increase in PD-1(+) TILs. When Ad5 [E1-, E2b-]-E6/E7 immunotherapy was combined with anti-PD-1 antibody, we observed CD8(+) TILs at the same level but a reduction in tumor PD-L1 expression on tumor cells and reduced PD-1(+) TILs providing a mechanism by which combination therapy favors a tumor clearance state and a rationale for pairing antigen-specific vaccines with checkpoint inhibitors in future clinical trials.

A non-oncogenic HPV 16 E6/E7 vaccine enhances treatment of HPV expressing tumors.

Human papillomaviruses (HPVs) are the causative factor for >90% of cervical cancers and 25% of head and neck cancers. The incidence of HPV positive (+) head and neck squamous cell carcinomas has greatly increased in the last 30 years. E6 and E7 are the two key viral oncoproteins that induce and propagate cellular transformation. An immune response generated during cisplatin/radiation therapy improves tumor clearance of HPV(+) cancers. Augmenting this induced response during therapy with an adenoviral HPV16 E6/E7 vaccine improves long-term survival in pre-clinical models. Here, we describe the generation of an HPV16 E6/E7 construct, which contains mutations that render E6/E7 non-oncogenic, while preserving antigenicity. These mutations do not allow E6/E7 to degrade p53, pRb, PTPN13, or activate telomerase. Non-oncogenic E6/E7 (E6(Δ)/E7(Δ)) expressed as a stable integrant, or in the [E1-, E2b-] adenovirus, lacks the ability to transform human cells while retaining the ability to induce an HPV-specific immune response. Moreover, E6(Δ)/E7(Δ) plus chemotherapy/radiation statistically enhances clearance of established HPV(+) cancer in vivo.

An Ad5[E1-, E2b-]-HER2/neu vector induces immune responses and inhibits HER2/neu expressing tumor progression in Ad5 immune mice.

Immunotherapy is a promising approach for the treatment of cancers. Modified adenovirus 5 (Ad5) vectors have been used as a platform to deliver genes encoding tumor associated antigens (TAA). A major obstacle to Ad5 vector immunotherapy has been the induction of vector immunity following administration or the presence of pre-existing Ad5 immunity, which results in vector mitigation. It has been reported by us that the Ad5[E1-, E2b-] platform with unique deletions in the E1, E2b and E3 regions can induce potent cell mediated immunity (CMI) against delivered transgene products in the presence of pre-existing Ad5 immunity. Here we report the use of an Ad5[E1-, E2b-] vector platform expressing the TAA HER2/neu as a breast cancer immunotherapeutic agent. Ad5[E1-, E2b-]-HER2/neu induced potent CMI against HER2/neu in Ad5 naïve and Ad5 immune mice. Humoral responses were also induced and antibodies could lyse HER2/neu expressing tumor cells in the presence of complement in vitro. Ad5[E1-, E2b-]-HER2/neu prevented establishment of HER2/neu-expressing tumors and significantly inhibited progression of established tumors in Ad5 naïve and Ad5 immune murine models. These data demonstrate that in vivo delivery of Ad5[E1-, E2b-]-HER2/neu can induce anti-TAA immunity and inhibit progression of HER2/neu expressing cancers.

Oral tacrolimus treatment of severe colitis in children.

OBJECTIVE: To evaluate the efficacy of oral tacrolimus as an induction agent in steroid-refractory severe colitis. STUDY DESIGN: Open-label, multicenter trial of oral tacrolimus in patients with severe colitis. Patients not responding to conventional therapy received tacrolimus, 0.1 mg/kg/dose given twice a day, and the dosage was adjusted to achieve blood levels between 10 and 15 ng/mL. Response was defined as improvement in a number of clinical parameters (including abdominal pain, diarrhea, rectal bleeding, and cessation of transfusions). Patients who responded by 14 days continued to receive tacrolimus, and 6-mercaptopurine or azathioprine was added as a steroid-sparing agent 4 to 6 weeks after the tacrolimus was instituted. RESULTS: Fourteen patients were enrolled in the study. One patient elected to withdraw after 48 hours. Of the 13 remaining, 9 (69%) responded and were discharged. Tacrolimus was continued for 2 to 3 months in the responders, except for 1 patient who was given tacrolimus for 11 months. After 1 year of follow-up, only 5 (38%) patients were receiving maintenance therapy; the other 4 responders had undergone colectomy. CONCLUSION: Although tacrolimus is effective induction therapy for severe ulcerative or Crohn's colitis, fewer than 50% of patients treated will successfully achieve a long-term remission.

Cyclic vomiting syndrome: evolution in our understanding of a brain-gut disorder.

Cyclic vomiting syndrome (CVS) remains a mysterious disorder despite our increasing knowledge since its classic description by Gee in 1882. Its hallmark feature of recurrent, explosive bouts of vomiting punctuating periods of normal health causes substantial medical morbidity (50% of patients require intravenous therapy), as well as significant time lost from school (20 school absences per year) and work. Limited epidemiologic data indicate that CVS may occur more commonly than previously thought, affecting as many as 1.9% of school-aged children. Besides the relentless vomiting, the child usually has pallor (87%), lethargy (91%), anorexia (74%), nausea (72%), and abdominal pain (80%). There is evidence of clinical and physiologic overlap among CVS, abdominal migraine, and migraine headaches. We propose revised criteria for abdominal migraine that include pain as the predominant and consistent symptom, lack of abnormal screening tests, and in retrospect, either subsequent development of migraines or positive response to antimigraine medication. Besides migraines, other etiologic possibilities include mitochondrial DNA mutations, ion channelopathies, excessive hypothalamic-pituitary-adrenal axis activation, and heightened autonomic reactivity. The differential diagnosis includes idiopathic CVS (88%); gastrointestinal disorders (7%), including serious surgical disorders (e.g., malrotation); and extraintestinal disorders (5%), including serious surgical (brain stem neoplasm) and metabolic disorders (e.g., fatty acid oxidation disorder). Within the idiopathic group, there may be migraine, Sato's neuroendocrine, mitochondrial, and other subgroups. Treatment includes avoidance of triggers, prophylactic medication, supportive care, abortive medication, and family support. In the future, investigation into mitochondrial DNA mutations, ion channel defects, corticotropin-releasing factor, and serotonin and tachykinin receptor physiology and pharmacology may help discover the etiology and pathogenesis of this disorder.

Presenting symptoms and diagnostic lag in children with inflammatory bowel disease.

Presenting symptoms and their duration may affect the time that elapses prior to definitive diagnosis of inflammatory bowel disease (IBD). This study was undertaken to determine the mean duration of presenting symptoms and diagnostic lag in children with IBD. The medical records of all patients less than 19 years of age diagnosed with IBD at the pediatric gastroenterology clinic of Children's Hospital of Wisconsin between 1990-1995 were reviewed. The age at diagnosis, gender, presenting symptoms and duration, disease location, and diagnostic lag were analyzed. There were 91 children (49 male) diagnosed with IBD. Crohn's disease (CD) was diagnosed in 58, ulcerative colitis (UC) in 24, and indeterminate colitis in 9. The mean ages at diagnosis were 11.4 years for CD, 9.7 years for UC, and 7.8 years for indeterminate colitis. The most frequent presenting symptoms were abdominal pain, diarrhea, hematochezia, and weight loss. The average lag in diagnosis of CD was 7.1 months, which varied by disease location: small intestine 10.5 months, ileocolonic 7.5 months, and colonic 6.4 months. The average lag in diagnosis was 6.7 months for UC and 14 months for indeterminate colitis. Children presenting with growth failure had the longest diagnostic lag. (a) The elapsed time between symptom onset and the diagnosis of CD has decreased. (b) The diagnostic lag in CD decreases with distal colonic involvement. (c) Following onset of symptoms UC was diagnosed only slightly more rapidly than CD.

The heat-stable enterotoxin-guanylin receptor is expressed in rat hepatocytes and in a rat hepatoma (H-35) cell line.

BACKGROUND/AIMS: Guanylyl cyclase C (GC-C) is an intestinal transmembrane receptor which binds both guanylin, an endogenous ligand, and Escherichia coli heat-stable enterotoxin (STa) resulting in 5'-cyclic guanosine monophosphate (cGMP) accumulation and chloride secretion. In the adult rat, there is a high basal level of GC-C expression in the intestine, but not in the liver. Increased expression of GC-C in the rat liver has been demonstrated during the perinatal period as well as with liver regeneration and during an acute phase response. The aim of this study was to identify and utilize cell culture models to further characterize the expression of GC-C in the liver. METHODS: STa binding, STa-stimulated cGMP accumulation, and GC-C RNA expression by Northern analysis were determined in primary cultures of rat hepatocytes and H-35 cells, a rat hepatoma cell line, following treatment with dexamethasone and/or interleukin-6 (IL-6). RESULTS: In rat hepatocytes treated with the combination of dexamethasone and IL-6, there was an increase in STa binding, STa-stimulated cGMP accumulation, and GC-C RNA expression as compared to untreated cells. In H-35 cells treated with dexamethasone alone, there was an increase in STa binding, STa-stimulated cGMP accumulation, and GC-C RNA expression as compared to untreated cells. CONCLUSION: Primary cultures of rat hepatocytes and H-35 cells can be utilized to further study upregulation of GC-C in the hepatocyte. The expression of this receptor in hepatocytes, combined with the recent demonstration of circulating guanylin, is consistent with a functional role for GC-C in the liver.

Modulation of idiotypic and antiidiotypic immunoglobulin G responses in an alloimmune thrombocytopenic patient associated with extracorporeal protein A immunoadsorption.

In the present case study, a patient with Non-Hodgkin. Lymphoma underwent combination chemotherapy resulting in severe pancytopenia requiring transfusion support with blood products. The patient became refractory to random donor platelet transfusions and subsequently received five immunoadsorption treatments. The patient's clinical response to immunoadsorption therapy was assessed by monitoring platelet transfusion recovery and survival. In addition, changes in antibody responses were assessed. Early during the course of immunoadsorption therapy, antiplatelet immunoglobulin G (IgG) alloantibody was detected. There was a decline in antiplatelet IgG alloantibody levels by the last immunoadsorption treatment associated with increases to platelet correct count increments after completion of immunoadsorption therapy. In addition, elevated levels of antiidiotypic IgG antibody detected early during the course of therapy were significantly reduced by the last immunoadsorption treatment. This case study suggests that specific alloimmune idiotypic IgG antibody and corresponding antiidiotypic IgG antibody responses may be modulated in association with extracorporeal immunoadsorption employing protein A/silica columns.

Evidence for proteolytic cleavage of covalently bound protein A from a silica based extracorporeal immunoadsorbent and lack of relationship to treatment effects.

Studies were conducted to evaluate the potential cause for release of covalently bound Staphylococcal protein A (SpA) from a silica based extracorporeal immunoadsorbent matrix. In vitro tests revealed that SpA could be detected in human plasma, human serum, and chicken serum upon exposure to the immunoadsorbent matrix which had been treated to remove non-covalently bound SpA. In contrast, only minute quantities of SpA were detected after exposure of a physiologic mixture of purified albumin and immunoglobulin G (IgG) to the immunoadsorbent matrix. Additional tests, employing a cocktail of protease inhibitors and formalin as a general stabilizer and protease inhibitor, revealed significant inhibition of endogenous proteolytic activity present in plasma and serum. Prevention of this proteolytic activity also significantly inhibited the release of covalently bound SpA from the immunoadsorbent matrix upon contact with plasma or serum samples. Further analyses of serum samples from patients with immune thrombocytopenia, chemotherapy associated thrombotic thrombocytopenic purpura-hemolytic uremic syndrome, and breast cancer revealed a lack of association between the quantity of SpA proteolytically released and observed clinical responses or adverse effects experienced during immunoadsorption treatments. These studies indicate that SpA detected in plasma or serum after exposure to the immunoadsorbent is due to inherent endogenous proteolytic activity which cleaves protein fragments from the matrix and that these cleaved SpA fragments do not appear to contribute to the observed clinical responses or adverse effects in treated patients.

Specificity of antibody responses affected by extracorporeal immunoadsorption of plasma over columns of protein A silica.

A relationship is described between the interaction of circulating immune complexes (CIC) from plasma with staphylococcal protein A immunoadsorption treatment columns and modulation of antibody responses related to the specific CIC. Eluates from the initial immunoadsorption columns used to treat a series of patients with breast adenocarcinoma, cancer chemotherapy-associated thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (C-TTP/HUS), or immune thrombocytopenic purpura (ITP) were evaluated for disease-specific CIC containing Lex glycosphingolipid (Lex gl) adenocarcinoma-associated antigens or platelet autoantibody (anti-GPIIb/IIIa), together with the corresponding neutralizing antibody [anti-F(ab')2], and for nonspecific CIC containing cytomegalovirus (CMV) or herpes simplex virus type 1 (HSV-1) antigens. In addition, the levels of antibodies directed against CMV, HSV-1, Lex gl, and GPIIb/IIIa antigens, as well as anti-F(ab')2 antibodies, were compared in pretreatment and posttreatment serum samples. Columns used to treat breast adenocarcinoma patients contained only Lex gl CIC, and the only immunologic change observed after treatment was significant increases in anti-Lex gl antibodies in some patients. Columns used to treat C-TTP/HUS patients contained anti-GPIIb/IIIa-anti-F(ab')2 CIC, in addition to Lex gl CIC. After treatment, significant increases in anti-Lex gl and anti-F(ab')2 antibodies and significant decreases in anti-GPIIb/IIIa antibodies were observed in some patients. Columns used to treat ITP patients only exhibited anti-GPIIb/IIIa-anti-F(ab')2 CIC, and after treatment only decreases in anti-GPIIb/IIIa and increases in anti-F(ab')2 antibodies were observed in some patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Minimal toxicity during protein A immunoadsorption treatment of malignant disease: an outpatient therapy.

Extracorporeal removal or modulation of circulating immune complexes (CIC) from plasma of animals and humans with malignant disease may be associated with induction of immune-mediated anti-tumor responses. Immunoadsorption columns containing heat-killed and formalin-fixed Staphylococcus aureus or staphylococcal protein A have been used for this purpose but treatments have often been associated with cardiopulmonary toxicity. Recently, an immunoadsorption device containing highly purified protein A covalently attached to a silica matrix (PROSORBA column) was used to treat 142 patients with refractory malignancies and 22 of 104 patients evaluated for anti-tumor response had objectively measurable reduction in tumor burden. In contrast to earlier experience with other devices, the procedures used in this trial were well tolerated and could be performed on an outpatient basis. The most common side effects observed among 1,306 treatments were chills (28% of treatments), low grade fever (28%), and musculoskeletal pain (16%). Side effects were mild to moderate and required no treatment or only symptomatic treatment. Treatment schedules were interrupted due to side effects for only six patients and there were no treatment-related deaths. Of 64 patients available for long-term follow-up evaluation (mean of 11 months), none exhibited evidence of long-term treatment-related side effects. None of the patient deaths in that period were associated with short or long-term treatment-related side effects. Protein A-silica (PROSORBA columns) can be used safely for development of further experimental treatments of malignant disease.

Modulation of immunity in patients with autoimmune disease and cancer treated by extracorporeal immunoadsorption with PROSORBA columns.

Extensive animal studies and clinical observations support an immunosuppressive role for certain antibodies and circulating immune complexes (CIC) in malignant and autoimmune diseases. Investigators have attempted to correct or modulate dysfunction by removal of antibodies or CIC from plasma. Extra-corporeal immunoadsorption of plasma over columns containing a silica matrix and covalently attached highly purified staphylococcal protein A (PROSORBA column) is a procedure that specifically removes those plasma components by the interaction of protein A with the Fc region of IgG. The interaction of CIC with the Fc receptor on protein A has three specific results. First, there is direct removal of immunosuppressive CIC from the circulation. Studies of CIC-mediated immunosuppression in experimental systems have shown dose-response relationships over wide ranges of CIC concentrations. Thus, removal of CIC relative to the IgG antibody may be expected to exert some stimulation of the immune system. Second, the complement system is activated. Elevated levels of C3a, C4a, and C5a are observed in patients' circulating plasma after PROSORBA treatment. These levels peak one to three hours post-perfusion and are near normal levels by six hours post-perfusion. These complement components are stimulators of growth and activity of immune cells. In addition, by binding to CIC they stimulate clearance of CIC by the reticuloendothelial system. Thus, treatments may induce removal of more CIC than could be anticipated by the binding capacity of treatment columns. Third, antibody is released from CIC. Interaction of CIC with bound protein A with or without the aid of activated complement components leads to liberation of free antibody. Depending upon other factors, eg, amount of circulating antigen and/or unbound IgG, either free antibody or CIC containing more antibody relative to antigen (or both) may be infused into patients with the posttreatment plasma. Such CIC function as immune stimulators rather than suppressors of immune cell activity. The consequences of the treatments are summarized as follows. Stimulation of immune cellular activity is seen one to three hours posttreatment. During the first one to three treatments, cells of the granulocyte/macrophage series show the greatest increase. During and after treatments 2 to 4, lymphocytes show the greatest increase. At this point, increased blastogenic response to mitogens is observed along with an increase in the T helper/suppressor cell ratio.(ABSTRACT TRUNCATED AT 400 WORDS)

Presence of fucolipid antigens with mono- and dimeric X determinant (Lex) in the circulating immune complexes of patients with adenocarcinoma.

A series of fucosylated glycosphingolipids with the Lewisx (Lex) determinant (Gal beta 1----4[Fuc alpha 1----3]GlcNAc) have been shown to accumulate in human adenocarcinomas. Lex glycolipids were eluted from Protein A-silica columns over which plasma from patients with adenocarcinoma had previously been perfused. The fact that Protein A has strong affinity for IgG and IgG-immune complexes suggested that the Lex antigens isolated from Protein A eluates were complexed with IgG. Lewisx antigen eluted from Protein A columns banded in the immune complex-enriched region (below IgG) of neutral sucrose density gradients. A modified Raji cell assay and an anticomplement C1q enzyme-linked immunosorbent assay were also used for measurement of Lex antigen associated with C3- and C1q-CIC, respectively. Following affinity purification of Lex-IgG complexes and subsequent dissociation of these immune complexes, human antibodies were isolated which reacted with purified glycosphingolipids containing Lex. Levels of Lex-IgG complexes were found to be 2- to 5-fold higher in eluates of Protein A-silica columns perfused with plasma from adenocarcinoma patients compared to eluates from columns perfused with plasma from healthy individuals and patients with other cancers. These assays may prove to be of diagnostic and/or prognostic significance in adenocarcinoma.

Selective removal of antigen-complexed IgG from cat plasma by adsorption onto a protein A-silica matrix.

The binding of normal cat IgG, heat-aggregated cat IgG and specific immune complexes (IC) containing cat IgG to a silica matrix containing covalently bound Staphylococcus aureus protein A was evaluated. The amounts of serum relative to protein A-silica, the flow rates and the perfusion times were representative of those existing when protein A-silica columns are used for therapeutic extracorporeal immunoadsorption of IgG and IC from humans and animals. When cat IgG was present in a large excess, approximately one molecule was bound to the matrix per molecule of solid-phase protein A with a KA of 1.5 X 10(6) 1/mol. Aggregated and immune complexed IgG bound to the matrix with relatively higher affinity. IC prepared in vitro between the purified envelope glycoprotein of the feline leukemia virus (FeLV gp70) and affinity-purified cat antibodies bound to the matrix even though normal IgG was present in greater than 10,000-fold excess. Once bound, IC were not eluted from columns upon further perfusion with normal serum. However, bound IgG was eluted from columns by further perfusion of normal serum or IC. IC were at least five-fold more efficient than normal IgG in exerting this effect. The results suggest that protein A-silica columns can be used for preferential removal of IC from plasma in a clinical or experimental setting.

Extracorporeal perfusion of plasma over immobilized protein A in a patient with Kaposi's sarcoma and acquired immunodeficiency.

Plasma of a homosexual man with Kaposi's sarcoma and acquired immunodeficiency was perfused over a protein A column using a continuous-flow plasma-cell separator. Three liters of plasma was perfused during each procedure and returned to the patient at a plasma flow rate of 10-20 ml/min. Three treatments were performed over a 1-week period. Transient removal of IgG, immune complexes, antilymphocyte antibodies, and serum blocking factors to mitogenic lymphocyte stimulation was observed during each treatment. Although no striking regression occurred, microscopic examination of sarcomatous lesions revealed decrease of tumor cell density and collagen proliferation within the tumor. Immunohistochemical deposition of C3 was observed in the tumor lesions after treatment. Side effects from the treatment included a mild drop of systolic blood pressure, sinus tachycardia, and a decrease in platelet count.

Circulating immune complexes in rats with metastasizing or nonmetastasizing mammary tumor.

The dual rat metastatic and nonmetastatic mammary tumor model was characterized by the level of immune complexes in the sera of rats bearing these tumors. Circulating immune complexes (CIC) were measured at various intervals posttumor injection in Wistar-Furth (W/Fu) rats inoculated at 2 months of age with either 1 X 10(6) viable metastasizing (TMT-081) or nonmetastasizing (MT-100) tumor cells into the mammary fat pads. The Raji cell assay was used to measure CIC. No correlation between tumor size and CIC levels in the tumors were observed. While none of the sera from the rats bearing the nonmetastasizing tumors had CIC levels higher than 30 micrograms/ml, a small percentage of the animals bearing the metastasizing tumors had serum CIC levels higher than 30 micrograms/ml. This study suggests no clear difference between the amount of CIC in the sera of animals bearing either metastasizing or nonmetastasizing tumors.

Isolation of human and canine IgM utilizing protein A affinity chromatography.

Human and canine high molecular weight IgM's were isolated employing a system of G-200 column chromatography and passage through a protein A Sepharose 4B column. Polyacrylamide Gel Electrophoresis (PAGE) analysis of the isolated immunoglobulin revealed polypeptides corresponding to mu and light immunoglobulin chains of IgM which were identified immunochemically as IgM. Ultracentrifugation studies revealed that the isolated immunoglobulin co-migrated with 19S IgM markers. This simple and efficient procedure may serve as an alternative to classical isolation procedures of IgM from certain mammalian species.

Suppressive effect of 3-methylcholanthrene on phagocytic activity of mouse peritoneal macrophages for Torulopsis glabrata.

The effect of 3-methylcholanthrene (MCA) on the phagocytic activity of mouse peritoneal macrophages for Torulopsis glabrata was investigated. Macrophages were maintained in glass scintillation vials or on cover slips in Leighton tubes with the use of Hanks' balanced salt solution plus 30% horse serum. Graded amounts of MCA were incorporated into the medium and the macrophages were parasitized with viable cells of T. glabrata. Macrophages from C3H mice, a strain highly susceptible to MCA carcinogenesis, were more prone to the suppressive effect of MCA than were the macrophages from CFW mice, a relatively resistant strain. Significant suppressive effect on phagocytosis of macrophages from C3H mice was observed with 5 micrograms MCA/ml, whereas up to 50 micrograms MCA/ml did not alter the phagocytic activity of CFW macrophages. However, 100 micrograms MCA/ml also suppressed the phagocytosis of CFW macrophages. Suppression in phagocytosis of C3H macrophages was observed after 6 hours' exposure to MCA, whereas a similar effect on CFW macrophages was seen after 12 hours. Treatment with 100 micrograms MCA/ml imparied the fungicidal activity of both C3H and CFW macrophages. These results indicate a correlation between the suppressive effect of MCA on macrophage activity and the strain susceptibility of mice to chemical carcinogenesis.