Massachusetts General Hospital

Palindromic rheumatism as a feature of alpha-gal syndrome: Initial observation on 18 patients from the capital of the Cherokee Nation.

Pathogenesis, newly recognized etiologies, and management of idiopathic anaphylaxis.

Idiopathic anaphylaxis (IA) is a life-threatening allergic disease and the most common diagnosis given to patients following an anaphylactic event. The inability of the healthcare provider and the patient to identify the trigger for anaphylaxis makes standard allergen avoidance measures ineffectual. IA is diagnosed after other causes of anaphylaxis have been excluded. Mast cell activation syndromes (MCAS), mastocytosis, IgE to galactose-alpha-1,3-galactose (α-gal), and certain medications have recently been recognized as causes of anaphylaxis that were previously labeled idiopathic. This review will describe the epidemiology and proposed theories of pathogenesis for IA, its diagnostic approach, its clinical management, and examine newly recognized disorders that were previously labeled as idiopathic anaphylaxis.

Determinants of food allergy.

Food allergy is an emerging epidemic in the United States and the Western world. The determination of factors that make certain foods allergenic is still not clearly understood. Only a tiny fraction of thousands of proteins and other molecules is responsible for inducing food allergy. In this review, the authors present 3 examples of food allergies with disparate clinical presentations: peanut, soy, and mammalian meat. The potential relationships between allergen structure and function, emphasizing the importance of cross-reactive determinants, immunoglobulin E antibodies to the oligosaccharides, and the immune responses induced in humans are discussed.

Panitumumab in patients with KRAS wild-type colorectal cancer after progression on cetuximab.

Cetuximab and panitumumab are monoclonal antibodies that target the epidermal growth factor receptor (EGFR) and are approved for the treatment of patients with KRAS wild-type meta-static colorectal cancer. There are no data that describe the activity of panitumumab in patients with progressive disease on cetuximab. We performed a single-arm phase II trial of panitumumab in patients with KRAS wild-type metastatic colorectal cancer that had progressed on prior cetuximab. We used a two-stage study design to treat patients with panitumumab at 6 mg/kg every 14 days (cycle length = 28 days). Treatment was continued until disease progression, death, inability to tolerate panitumumab, or study withdrawal. The primary endpoint was response rate; secondary endpoints included progression-free survival and overall survival. Twenty patients were treated in the first stage, with plans to treat an additional twelve patients if there was at least one objective response. We collected blood samples at baseline and prior to cycles 2 and 3 to evaluate for the presence of anti-cetuximab and anti-panitumumab antibodies. We treated twenty patients for a median of two cycles (range 1-4). No patients responded, and 45% had a best response of stable disease (no progression for at least two cycles). Median progression-free survival was 1.7 months and median overall survival was 5.2 months. Panitumumab was well tolerated. Thirteen patients (65%) had grade 1-2 dry skin or rash, and three patients had treatment-related grade 3 toxicities (one each with hyperglycemia, hyperbilirubinemia, and hypokalemia). No patients had detectable anti-cetuximab antibodies at any time point; one patient developed anti-panitumumab antibodies. Panitumumab has minimal benefit in patients with KRAS wild-type metastatic colorectal cancer that has progressed on prior cetuximab. Discussion Both cetuximab and panitumumab competitively inhibit ligand binding to EGFR, thereby promoting receptor internalization and blocking receptor-mediated signaling. Although the two agents have never been compared directly in a randomized clinical trial, they produce similar response rates when used alone as well as in combination with cytotoxic agents. Cetuximab is a chimeric antibody with approximately 30% murine protein, while panitumumab is a fully human monoclonal antibody. Correspondingly, rates of severe hypersensitivity reactions are somewhat increased with cetuximab (3%) compared to panitumumab (1%). However, the potential efficacy of panitumumab in patients who have developed disease progression on cetuximab has been an open question. Metges et al. (PANERB trial) prospectively treated 32 KRAS wild-type metastatic colorectal cancer patients with cetuximab and irinotecan followed by panitumumab monotherapy after progression. Remarkably, the authors reported an objective response rate of 22% to panitumumab, including a disease control rate (objective response plus stable disease) of 73% in 11 patients who had previously responded to cetuximab and irinotecan. In contrast, we found no responders and a stable disease rate of 45% with a median duration of only 1.7 months in our trial of 20 patients. Moreover, no patients had detectable anti-cetuximab antibodies at baseline. It is not clear to what extent the PANERB trial included patients without objective disease progression on cetuximab or for whom cetuximab-containing regimens may have been ceased due to toxicity in the absence of disease progression. In both circumstances, retreatment with panitumumab may be expected to demonstrate some degree of clinical activity. In our study, disease progression after at least 4 weeks of cetuximab documented radiographically or by increased carcinoembryonic antigen (CEA) levels was required for inclusion in order to ensure that the study population demonstrated unequivocal evidence of progression on cetuximab. While it remains possible that a small subset of patients may benefit from panitumumab after progression on cetuximab, our results suggest that this approach should not be adopted until predictive biomarkers for panitumumab response in this setting have been discovered and validated. Until then, patients who develop progression on cetuximab should be enrolled in trials of novel agents.

Phase II study of cisplatin plus cetuximab in advanced, recurrent, and previously treated cancers of the cervix and evaluation of epidermal growth factor receptor immunohistochemical expression: a Gynecologic Oncology Group study.

The purpose of this study was to evaluate the safety and efficacy of cetuximab (C225), an antibody that inhibits epidermal growth factor receptor (EGFR) activity, with cisplatin and to explore associations between EGFR protein expression with patient demographics or clinical outcome. Women with advanced, persistent, or recurrent carcinoma of the cervix were eligible. The women received cisplatin at 30mg/m(2) on days 1 and 8 with a loading dose of cetuximab at 400mg/m(2) followed by 250mg/m(2) on days 1, 8, and 15 in a 21day cycle. Adverse events were assessed with CTCAE v 3.0. Primary measure of efficacy was tumor response by RECIST. The study was stratified by prior chemotherapy (CT). EGFR protein expression in pre-treatment tumor was analyzed by immunohistochemistry. Between September 2004 and March 2008, 76 patients were enrolled. Of these, 69 were eligible and evaluable; 44 (64%) received prior chemotherapy. There were 4 responses in each group, prior chemotherapy and no chemotherapy, 9% and 16%, respectively. Grade 4 toxicities included anemia (1), allergy (1), metabolic (1), and vascular (1). The most common grade 3 toxicities were metabolic (15), dermatologic (8), fatigue (6), and gastrointestinal (6). EGFR protein was expressed in 47/48 (98%) of tumors analyzed with a median cellular expression of 81%. Exploratory analyses revealed a trend between the percentage of cells expressing EGFR protein and PFS (hazard ratio=1.76, 95% confidence interval=0.96-3.21). The combination of cetuximab with cisplatin was adequately tolerated but did not indicate additional benefit beyond cisplatin therapy.

Reduction of anti-Galalpha1,3Gal antibodies by infusion of types 2 and 6 gal trisaccharides conjugated to poly-L-lysine.

To investigate the specificity of anti-Galalpha1,3Gal (Gal) antibodies (Abs) with respect to Gal oligosaccharides of types 2 and 6, eight baboons received an intravenous infusion of either a poly-l-lysine conjugate of Gal type 2 (n = 5) or type 6 (n = 3), followed 48 h later by the alternative Gal type 6 or 2 conjugate, respectively. IgM Abs reactive to Gal type 2 were depleted by 80 to 89% by either Gal conjugate. IgM reactive to Gal type 6 was less efficiently depleted by the Gal type 2 conjugate (57% depletion) than the Gal type 6 (82% depletion). Gal-reactive IgG was depleted more slowly and less efficiently by either glycoconjugate (initially by only 28 to 54%). Our results indicate that the Gal type 6 conjugate depletes most anti-Gal IgM, but the Gal type 2 conjugate is less efficient in depleting anti-Gal IgM reactive with type 6. There remain small fractions of antibody that are unadsorbed, particularly of IgG, probably due to their low affinity and distribution in both the intra- and extra-vascular compartments.

Peritoneal cavity B cells are precursors of splenic IgM natural antibody-producing cells.

Peritoneal cavity B-1 cells are believed to produce IgM natural Abs. We have used alpha1,3-galactosyltransferase-deficient (GalT(-/-)) mice, which, like humans, produce IgM natural Abs against the carbohydrate epitope Galalpha1,3Gal (Gal), to demonstrate that peritoneal cavity B-1b cells with anti-Gal receptors produce anti-Gal IgM Abs only after LPS stimulation. Likewise, peritoneal cavity cells of GalT(-/-) and wild-type mice do not produce IgM Abs of other specificities without LPS stimulation. Development of Ab-secreting capacity is associated with loss of CD11b/CD18 (Mac-1) expression. In contrast, there are large numbers of cells producing anti-Gal and other IgM Abs in fresh splenocyte preparations from GalT(-/-) and (for non-Gal specificities) wild-type mice. These cells are Mac-1(-) but otherwise B-1b-like in their phenotype. We therefore hypothesized a pathway wherein peritoneal cavity B cells migrate into the spleen after activation in vivo and lose Mac-1 expression to become IgM Ab-producing cells. Consistent with this possibility, splenectomy reduced anti-Gal Ab production after immunization of GalT(-/-) mice with Gal-positive rabbit RBC. Furthermore, splenectomized B6 GalT(-/-), Ig micro -chain mutant ( micro (-/-)) (both Gal- and B cell-deficient) mice produced less anti-Gal IgM than nonsplenectomized controls after adoptive transfer of peritoneal cavity cells from B6 GalT(-/-) mice. When sorted GalT(-/-) Mac-1(+) peritoneal cavity B cells were adoptively transferred to B6 GalT(-/-), micro (-/-) mice, IgM Abs including anti-Gal appeared, and IgM-producing and Mac1(-) B cells were present in the spleen 5 wk after transfer. These findings demonstrate that peritoneal cavity Mac-1(+) B-1 cells are precursors of Mac-1(-) splenic IgM Ab-secreting cells.

Pig hematopoietic cell chimerism in baboons conditioned with a nonmyeloablative regimen and CD154 blockade.

In an attempt to induce mixed hematopoietic chimerism and transplantation tolerance in the pig-to-primate model, we have infused high-dose porcine peripheral blood progenitor cells (PBPC) into baboons pretreated with a nonmyeloablative regimen and anti-CD154 monoclonal antibody (mAb). Group 1 baboons (n=2) received a nonmyeloablative regimen including whole body irradiation, pharmacological immunosuppression, porcine hematopoietic growth factors, and immunoadsorption of anti-Galalpha1,3Gal (Gal) antibody before infusion of high doses of PBPC (2.7-4.6x10(10) cells/kg). In group 2 (n=5), cyclosporine was replaced by anti-CD154 mAb. Group 3 (n=3) received the group 1 regimen plus anti-CD154 mAb. In group 1, pig chimerism was detected in the blood by flow cytometry (FACS) for 5 days (with a maximum of 14%), and continuously up to 13 days by polymerase chain reaction (PCR). In group 2, pig chimerism was detectable for 5 days by FACS (maximum 33%) and continuously up to 28 days by PCR. In group 3, initial pig chimerism was detectable for 5 days by FACS (maximum 73%). Two of three baboons showed reappearance of pig cells on days 11 and 16, respectively. In one, in which no anti-Gal IgG could be detected for 30 days, pig cells were documented in the blood by FACS on days 16-22 (maximum 6% on day 19) and pig colony-forming cells were present in the blood on days 19-33, which we interpreted as evidence of engraftment. Microchimerism was continuous by PCR up to 33 days. These results suggest that there is no absolute barrier to pig hematopoietic cell engraftment in primates, and that this may be facilitated if the return of anti-Gal IgG can be prevented.

Adult porcine islet transplantation in baboons treated with conventional immunosuppression or a non-myeloablative regimen and CD154 blockade.

The aim of the present study was to assess the survival of adult porcine islets transplanted into baboons receiving either (I) conventional triple drug immunosuppressive therapy or (2) a non-myeloablative regimen and an anti-CD154 monoclonal antibody (mAb) aimed at tolerance-induction. Group 1 baboons (n = 3) were pancreatectomized prior to intraportal injection of 10,000 porcine islet equivalents (IE)/kg and immunosuppressed with anti-thymocyte globulin (ATG), cyclosporine and azathioprine. In Group 2 (n = 2), non-pancreatectomized baboons underwent induction therapy with whole body and thymic irradiation, and ATG. Extracorporeal immunoadsorption (EIA) of anti-Galalpha1,3Gal (Gal) antibody was carried out. Maintenance therapy was with cobra venom factor, cyclosporine. mycophenolate mofetil, methylprednisolone and anti-CD154 mAb. Porcine islets were injected intraportally (14,000 and 32,000 IE/kg, respectively) and high-dose pig mobilized peripheral blood progenitor cells (3 x 10(10) cells/kg) were infused into a systemic vein. Porcine islets were also implanted in the sternomastoid muscle to facilitate subsequent biopsies. In both groups. porcine C-peptide was measured, and histological examination of liver or sternomastoid muscle biopsies was performed at regular intervals. In Group 1, total pancreatectomy reduccd human C-peptide to < 0.1 ng/ml and induced insulin-requiring diabetes. The transplantation of porcine islets was followed by normalization of glycemia for 15-24 h. Porcine C-peptide was detected only transiently immediately after porcine islet injection (maximum 0.12 ng/ml). Histological examination of liver biopsies taken between days 2 and 19 did not reveal viable islets, but necrotic cell structures with mononuclear cell infiltrates were identified in portal venules. In Group 2, injection of porcine islets into non-pancreatectomized recipients induced a transient hypoglycemia (2-4 h) requiring concentrated intravenous dextrose administration. Porcine C-peptide was detectable for 5 and 3 days (maximum 2.8 and 1.0 ng/ml), respectively. Baboon #4 died on day 12 from small bowel intussusception. Liver and sternomastoid muscle biopsies showed well-preserved porcine islets, staining positive for insulin and glucacon, without signs of rejection. In baboon #5, viable islets were detected in the sternomastoid muscle biopsy on day 14, but not on day 28 or thereafter. A progressive mononuclear cell and macrophage infiltration was seen in the biopsies. In conclusion, conventional immunosuppression allowed survival of porcine islets in baboons for < 24 h. The non-myeloablative regimen prolonged survival of porcine islets for > 14 days. However, despite depletion of T cells, anti-Gal antibody and complement, and CD154-hlockade, porcine islets were rejected by day 28. These results suggest that powerful innate immune responses are involved in rejection of discordant xenogencic islets.

Engraftment of genetically modified bone marrow cells in sensitized hosts.

Expression of retrovirally transduced genes in bone marrow-derived cells can be used to establish stable long-term B- and T-cell tolerance. To determine whether preexisting antibodies may prohibit the use of gene therapy to establish tolerance, we examined the extent to which preexisting antibodies specific for the carbohydrate antigen Gal alpha1-3Gal beta1-4GlcNAc-R (alpha Gal) could affect engraftment and development of bone marrow progenitors expressing the enzyme UDPgalactose:beta-D-galactosyl-1,4-N-acetyl-D-glucosaminide alpha(1-3)galactosyltransferase (E.C. 2.4.1.151), or simply alpha GT, which synthesizes the alpha Gal epitope. Groups of alpha GT knockout mice (GT(0) mice) lacking alpha Gal were presensitized to alpha Gal by immunization and then lethally irradiated and reconstituted with varying numbers of alpha GT-transduced syngeneic bone marrow cells. Whereas unimmunized controls were reconstituted with as few as 2 x 10(6) transduced cells, a significant fraction of immunized mice reconstituted with 2 x 10(6) or 4 x 10(6) alpha GT-transduced cells failed to undergo bone marrow engraftment and died. Immunized mice in which radiation protection was achieved failed to express alpha Gal. However, radiation protection and expression of alpha Gal on bone marrow-derived cells, resulting in tolerance, could be achieved by increasing the number of transduced cells used to reconstitute immunized mice. Thus, although high levels of preexisting antibodies can be a significant barrier to engraftment, this barrier can be overcome by increasing the number of transduced cells used for reconstitution.

In vivo T-cell depletion enhances production of anti-GALalpha1,3GAL natural antibodies in alpha1,3-galactosyltransferase-deficient mice.

It has been reported that T-cell depletion by in vivo treatment with monoclonal antibodies results in polyclonal B-cell activation. However, its effects on B cells responding to Galalpha1,3Gal (Gal) epitopes remain unknown. alpha1,3-Galactosyltransferase-deficient (GalT-/-) mice were treated with depleting anti-CD4 and CD8 monoclonal antibodies. The kinetics of anti-Gal natural antibodies (NAb) and total immunoglobulin levels in their sera were evaluated. The frequencies of anti-Gal NAb-producing cells were determined in the various tissues of GalT-/- mice by enzyme-linked immunospot assay. In vivo T-cell depletion led to significant increases in both anti-Gal IgM and total IgM levels in sera of GalT-/- mice, but did not influence either anti-Gal IgG or total IgG levels. An increased frequency of anti-Gal and total IgM-producing cells was observed in the spleens and bone marrow of T-cell-depleted GalT-/-mice but not in peritoneal cavity cells. In vivo T-cell depletion facilitates anti-Gal IgM production, suggesting that T cells deliver inhibitory signals to B cells responding to Gal.

Plasma perfusion by apheresis through a Gal immunoaffinity column successfully depletes anti-Gal antibody: experience with 320 aphereses in baboons.

Anti-Galalpha1-3Gal (Gal) antibodies (Gal Ab) contribute to the rejection of porcine organs transplanted into primates. Extracorporeal immunoadsorption (EIA) has been developed to eliminate Gal Ab from the circulation. Between 1995 and 1999 we performed 320 EIAs in baboons using a COBE-Spectra apheresis unit incorporating a synthetic Gal immunoaffinity column. Three plasma volumes were immunoadsorbed on each occasion. The 221 consecutive EIAs performed in 41 immunosuppressed baboons between January 1997 and April 1999 form the basis of this review. Of these 41 baboons, 29 underwent a series of three or four EIAs at daily intervals, seven had multiple series of three EIAs, and the remainder underwent single or double EIAs. Serum Gal Ab levels were monitored by ELISA before and at intervals after the course of EIA. There were two fatal complications, one from a respiratory mishap (unrelated to the EIA) and one from persistent hypotension unresponsive to therapeutic interventions. Seven procedures (3%) were terminated early owing to technical difficulties and/or persistent hypotension. Mean pre-EIA Gal Ab levels in naive baboons were 33.1 microg/ml (IgM) and 14.5 microg/ml (IgG). Immediately after three consecutive EIAs, IgM was depleted by a mean of 97.3% and IgG by 99.4%. By 18 to 24 h later, Gal Ab was returning but depletion remained at 80.1% (IgM) and 84.7% (IgG). The subsequent rate of return of Gal Ab depended on the immunomodulatory protocol used. (1) With appropriate monitoring, EIA is an acceptably safe procedure, even in small (<10 kg) baboons. (2) Three consecutive EIAs are effective in removing >97% of Gal Ab. (3) In the majority of cases, return of Gal Ab begins within 24 h, irrespective of the immunomodulatory protocol.

Apheresis and column absorption for specific removal of Gal-alpha-1,3 Gal natural antibodies in a pig-to-baboon model.