Case report: Molecular profiling facilitates the diagnosis of a challenging case of lung cancer with choriocarcinoma features.

Accurate diagnoses are crucial in determining the most effective treatment across different cancers. In challenging cases, morphology-based traditional pathology methods have important limitations, while molecular profiling can provide valuable information to guide clinical decisions. We present a 35-year female with lung cancer with choriocarcinoma features. Her disease involved the right lower lung, brain, and thoracic lymph nodes. The pathology from brain metastasis was reported as "metastatic choriocarcinoma" (a germ cell tumor) by local pathologists. She initiated carboplatin and etoposide, a regimen for choriocarcinoma. Subsequently, her case was assessed by pathologists from an academic cancer center, who gave the diagnosis of "adenocarcinoma with aberrant expression of ß-hCG" and finally pathologists at our hospital, who gave the diagnosis of "poorly differentiated carcinoma with choriocarcinoma features". Genomic profiling detected a KRAS G13R mutation and transcriptomics profiling was suggestive of lung origin. The patient was treated with carboplatin/paclitaxel/ipilimumab/nivolumab followed by consolidation radiation therapy. She had no evidence of progression to date, 16 months after the initial presentation. The molecular profiling could facilitate diagnosing of challenging cancer cases. In addition, chemoimmunotherapy and local consolidation radiation therapy may provide promising therapeutic options for patients with lung cancer exhibiting choriocarcinoma features.

Cytopathology assistance for optimizing interventional diagnostic procedures.

Herein, we review the components of Rapid On-Site Evaluation (ROSE) and the mechanics of Fine Needle Aspiration (FNA) to prepare cytopathologists to assist radiologists in optimizing their diagnostic procedures. The performance of FNA differs among proceduralists (interventional radiologists, general radiologists, bronchoscopists, endoscopists, surgeons, and clinicians), organ systems, diseases, and cancer types. The discussion is necessarily broad. Although practiced, professional aspects of ROSE interaction are not typically discussed in the literature. The target audience is primarily trainees and pathologists in an early stage of their career, but we hope that some ideas may be of general benefit. The information presented in this article is partially derived from experience in a busy tertiary care center with active ROSE services.

Primary Pancreatic Lymphoma Evaluated by Fine-Needle Aspiration.

OBJECTIVES: Primary pancreatic lymphoma (PPL) is rare, mimicking pancreatic ductal adenocarcinoma (PDAC) clinically and radiologically. The aim of this study is to evaluate the clinical, radiologic, and pathological characteristics of PPL diagnosed by fine-needle aspiration (FNA) in our institution. METHODS: Patient clinical, radiologic, and pathological information was collected from the electronic health record system. RESULTS: In total, 11 of 4,353 pancreatic FNAs met the criteria. The most common clinical symptom was jaundice, followed by abdominal pain, weight loss, and diarrhea. Abnormal laboratory findings included elevated alkaline phosphatase, total bilirubin, lactate dehydrogenase, and cancer antigen 19-9. Abnormal radiologic findings included pancreatic mass, biliary dilatation, vessel encasement, and common bile duct encasement and thickening. Five patients underwent more than 1 tissue sampling procedure before the final diagnosis of lymphoma. Final pathologic diagnosis included 7 large B-cell lymphomas and 4 follicular lymphomas. Flow cytometric analysis was performed on 9 specimens, and all demonstrated an aberrant monoclonal B-cell population. CONCLUSIONS: PPL mimics PDAC clinically and radiologically and could be a challenge for pathologic diagnosis if lymphoma is not included in the differential diagnosis during immediate evaluation. If lymphoma is suspected during immediate evaluation, PPL could be reliably diagnosed by FNA with the aid of ancillary studies.

Adequacy evaluation and use of pancreatic adenocarcinoma specimens for next-generation sequencing acquired by endoscopic ultrasound-guided FNA and FNB.

BACKGROUND: Endoscopic ultrasound-guided tissue acquisition (EUS-TA), especially endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA), is the mainstay of tissue acquisition for the diagnosis of pancreatic ductal adenocarcinoma (PDAC). Recently, endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) using flexible biopsy needles has been used for patients with PDAC in an effort to increase diagnostic yields and biomarker testing. However, the role of EUS-TA in biomarker testing for personalized therapy or precise chemotherapy for PDAC is not well established. METHODS: PDAC cases with specimens acquired through concurrent EUS-FNA and EUS-FNB were identified retrospectively. Smears were prepared from EUS-FNA sampling, and cell blocks (CBs) were prepared from EUS-FNB sampling. Rapid onsite evaluation was conducted for all cases for diagnostic adequacy. The adequacy for biomarker testing, including next-generation sequencing (NGS) and immunohistochemistry (IHC) assays, was evaluated, and cases with smears and CBs adequate for NGS were processed for targeted NGS. RESULTS: There were 26 PDAC cases concurrently sampled by EUS-FNA and EUS-FNB. EUS-FNA smears for all 26 cases and EUS-FNB CBs for 20 cases (77%) were diagnostic for PDAC. Twenty-one smears (81%) and 11 CBs (42%) were adequate for NGS. Nine cases with both smears and CBs adequate for NGS underwent NGS, which identified clinically significant gene mutation variants, including KRAS, TP53, and SMAD4 mutations. CONCLUSIONS: Both EUS-FNA and EUS-FNB can provide optimal material for targeted NGS for PDACs. In PDAC cases subjected to concurrent EUS-FNA and EUS-FNB, EUS-FNA specimens had greater diagnostic yields and more adequate material for NGS than EUS-FNB specimens, whereas EUS-FNB was more suitable for IHC-based biomarker testing.

Pancreatic paraganglioma diagnosed by endoscopic ultrasound-guided fine needle aspiration: A case report and review of literature.

BACKGROUND: Pancreatic paragangliomas (PPGL) are rare benign neuroendocrine neoplasms but malignancy can occur. PPGL are often misdiagnosed as pancreatic neuroendocrine tumor or pancreatic adenocarcinoma. CASE SUMMARY: We reviewed 47 case reports of PPGL published in PubMed to date. Fifteen patients (15/47) with PPGL underwent endoscopic ultrasound-guided fine needle aspiration (EUS-FNA). Only six (6/15) were correctly diagnosed as PPGL. All patients with PPGL underwent surgical resection except three (one patient surgery was aborted because of hypertensive crisis, two patients had metastasis or involvement of major vessels). Our patient remained on close surveillance as she was asymptomatic. CONCLUSION: Accurate preoperative diagnosis of PPGL can be safely achieved by EUS-FNA with immunohistochemistry. Multidisciplinary team approach should be considered to bring the optimal results in the management of PPGL.

Solid Tumor Metastases to the Pancreas Diagnosed Using Fine-Needle Aspiration.

OBJECTIVES: Solid tumor metastases to the pancreas are rare, and only limited literature on the topic is available. In this retrospective study, we reviewed 107 cases of solid tumor metastases to the pancreas diagnosed by fine-needle aspiration (FNA) from 2005 to 2019. METHODS: Information including the patients' clinical histories, radiologic and pathologic findings, treatments, and follow-up were collected. RESULTS: The mean age of the patients was 62.4 years. The mean tumor size was 2.64 cm with even distribution throughout the pancreas (head, neck and body, and tail). The most common primary site was the kidney, followed by the lung, skin, and breast and the gynecologic, gastrointestinal, and genitourinary tracts. The most common tumor type was carcinoma, followed by melanoma and sarcoma. In comparison to patients with nonkidney primary cancers, those with primary renal cell carcinoma had a longer median interval between primary diagnosis and metastasis (8.5 vs 4.0 years; P < .01), less often had metastasis outside the pancreas (38% vs 74%; P < .01), and had a significantly longer 5-year survival rate (65.7% vs 24.8%; P < .01). CONCLUSIONS: FNA plays a crucial role in diagnosing metastases to the pancreas. Metastases originating from kidney and nonkidney primary tumors have distinct clinicopathologic features and prognoses.

TRPV6 as A Target for Cancer Therapy.

Two decades ago a class of ion channels, hitherto unsuspected, was discovered. In mammals these Transient Receptor Potential channels (TRPs) have not only expanded in number (to 26 functional channels) but also expanded the view of our interface with the physical and chemical environment. Some are heat and cold sensors while others monitor endogenous and/or exogenous chemical signals. Some TRP channels monitor osmotic potential, and others measure cell movement, stretching, and fluid flow. Many TRP channels are major players in nociception and integration of pain signals. One member of the vanilloid sub-family of channels is TRPV6. This channel is highly selective for divalent cations, particularly calcium, and plays a part in general whole-body calcium homeostasis, capturing calcium in the gut from the diet. TRPV6 can be greatly elevated in a number of cancers deriving from epithelia and considerable study has been made of its role in the cancer phenotype where calcium control is dysfunctional. This review compiles and updates recent published work on TRPV6 as a promising drug target in a number of cancers including those afflicting breast, ovarian, prostate and pancreatic tissues.

A proposal for pathologic processing of breast implant capsules in patients with suspected breast implant anaplastic large cell lymphoma.

Breast implant anaplastic large cell lymphoma is an entity recently recognized by the World Health Organization. The tumor arises around textured-surface breast implants and is usually confined to the surrounding fibrous capsule. Currently, there are no recommendations for handling and sampling of capsules from patients with suspected breast implant anaplastic large cell lymphoma without a grossly identifiable tumor. We analyzed complete capsulectomies without distinct gross lesions from patients with breast implant anaplastic large cell lymphoma. The gross appearance of the capsules as well as the presence, extent and depth of tumor cells on the luminal side and number of sections involved by lymphoma were determined by review of routine stains and CD30 immunohistochemistry. We then used a mathematical model that included the extent of tumor cells and number of positive sections to calculate the minimum number of sections required to identify 95% of randomly distributed lesions. We identified 50 patients with breast implant anaplastic large cell lymphoma who had complete capsulectomies. The implants were textured in all 32 (100%) cases with available information. Anaplastic large cell lymphoma was found in 44/50 (88%) capsules; no tumor was found in six (12%) patients who had lymphoma cells only in the effusion. The median number of sections reviewed was 20 (range, 2-240), the median percentage of sections involved by tumor was 6% (range, 0-90%), and the median percentage of sections involved by lymphoma was 10% (range, 0-90%). Invasion deep into or through the capsule was identified in 18/50 (36%) patients. In patients with breast implant anaplastic large cell lymphoma without a grossly identifiable tumor we identified a spectrum of involvement and we propose a protocol for handling, sampling and reporting these cases. The number of sections to exclude the presence of lymphoma with more than 95% certainty was supported by a mathematic rationale.

Cytologic Evaluation of Positron Emission Tomography-Computed Tomography-Positive Lymph Nodes Sampled by Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration: Experience at a Large Cancer Center.

CONTEXT.­: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is routinely used to evaluate mediastinal lymph nodes (LNs), especially for cancer staging. There are limited large studies evaluating the cytologic, radiologic, and clinical features of 18F-fluorodeoxy glucose positron emission tomography-computed tomography-positive (PET-CT+) LNs. OBJECTIVE.­: To compare cytologic, radiologic, and clinical features of PET-CT+, cytology-malignant (PET-CT+/Cyto+) and PET-CT+, cytology-benign (PET-CT+/Cyto-) LNs. DESIGN.­: The pathology database was searched for cases of mediastinal LNs obtained by EBUS-TBNA from January 1, 2015 to December 31, 2015. The cytologic, radiologic, and clinical features were collected for all PET-CT+ LNs. RESULTS.­: Of 2267 mediastinal LNs obtained by EBUS-TBNA during this period, 577 LNs met the criteria. Of the latter, 263 (46%) were PET-CT+/Cyto+ and 314 (54%) were PET-CT+/Cyto-. All of the patients with PET-CT+/Cyto+ results had a prior or concurrent diagnosis of malignancy as compared to 89% of patients with PET-CT+/Cyto- results. Of the 224 patients with PET-CT+/Cyto+ LNs, 177 (79%) had metastases from lung primary, 43 (19%) had metastases from nonlung primaries, and 7 (3%) had lymphoma. Average LN size was larger in the PET-CT+/Cyto+ group than in the PET-CT+/Cyto- group (14.6 mm versus 9.58 mm), and mean standardized uptake value in PET-CT+/Cyto+ LNs was higher than that of PET-CT+/Cyto- LNs (10.05 versus 5.99). Significant cytologic findings in PET-CT+/Cyto- cases were necrosis and granulomatous inflammation, including 3 cases with fungal organisms. CONCLUSIONS.­: PET-CT positivity alone was nonspecific for malignancy and insufficient to guide management of patients with mediastinal adenopathy, but specificity could be improved when combined with LN size and standardized uptake value.

Inhibition of Transient Receptor Potential Vanilloid 6 channel, elevated in human ovarian cancers, reduces tumour growth in a xenograft model.

Background: Transient Receptor Potential Vanilloid 6 (TRPV6), a non-voltage gated calcium channel, is implicated in malignancies and correlates with Gleason scores in prostate cancer and with poor prognosis in breast cancer. Data on the TRPV6 status of ovarian malignancies has not received significant attention. The effect of inhibiting TRPV6 activity on ovarian tumour growth has never been reported. Methods: We quantified TRPV6 mRNA and protein in biopsies of five types of ovarian cancer at different stages and grades by quantitative PCR and immunohistochemistry respectively. We verified the presence of TRPV6 in SKOV-3 cells and xenografts by Western Blotting. NOD/SCID mice bearing xenografted ovarian tumours derived from SKOV-3 were treated daily with TRPV6-antagonistic peptides (SOR-C13 and SOR-C27) at 400, 600 and 800 mg/kg delivered intraperitoneally (i.p.) over 12 days. Data from qPCR and tumour growth experiments were compared with a Student's t-test. Immunohistochemical ranking of staining were compared with Kruskall-Wallace one-way ANOVA and Dunn's Multiple Comparison post-test. Results: TRPV6 mRNA and protein are significantly elevated at all stages and grades of 5 ovarian cancer types over normal tissue. Overall qPCR log2 values (n, mean, ± SEM) for mRNA in tumour (n = 165, 5.06 ± 0.16) were greater (p < 0.05) than normal tissues (n = 26, 0.45 ± 0.41). All stages and grades included in the biopsy arrays were significantly greater than normal tissues. Immunohistochemical staining of TRPV6 was ranked >2 (faint in most cells) in 80.5% of tumours (123) while 92% of normal tissues (23) ranked ≤ 2. Daily i.p. injection with SOR-C13 (400, 600 and 800 mg/kg) over 12 days inhibits tumour growth (59%) at the highest dose compared to non-treated controls. SOR-C27 at 800 mg/kg SOR-C27 inhibited tumour growth 55% after 12 days. Results of daily and intermittent dosing (Days 1, 2, 3 and 8, 9, 10) with SOR-C13 were indistinguishable. Conclusion: TRPV6 mRNA and protein are elevated in biopsies of ovarian cancers compared to normal tissue. Inhibition of TRPV6 activity significantly reduces ovarian tumour growth providing evidence that TRPV6 is a feasible oncology target in ovarian cancers.

Sensitivity of fine-needle aspiration biopsy for diagnosing and grading follicular lymphomas using a multiparameter approach in a cancer center.

OBJECTIVES: The diagnosis and grading of follicular lymphomas (FLs) by fine-needle aspiration biopsy (FNAB) has not been systematically compared with core needle biopsy (CNB). We evaluated the sensitivity of FNAB in diagnosing and grading FLs using a multiparameter approach in a large cancer center. METHODS: We retrospectively identified CNBs of lymph nodes diagnosed as FL that also had a concurrently acquired FNAB on the same site. The majority of cases had flow cytometric analysis and these results were available for interpretation of both the FNAB and CNB. RESULTS: Out of 342 patients, CNB diagnoses included 291 (85%) low-grade (LG) FLs, 30 (9%) high-grade (HG) FLs, and 21 (6%) non-graded FLs/other. FNAB diagnoses included 194 (57%) LG FLs, 19 (6%) HG FLs, 93 (27%) non-graded FLs, 9 (3%) large B-cell lymphomas (LBCL) of follicle center origin, and 27 (7%) insufficient for diagnosis/other. Review of non-graded FLs showed 45% LG, 35% indeterminate due to polymorphous lymphoid cells with increased numbers of large cells, and 20% scant cellularity. Sensitivity of FNAB for diagnosing FL was 89%, and 66% for LG FL. The latter increased (94%), however, when grading was performed. CONCLUSION: FNAB is highly sensitive for diagnosing FLs when cellular material for cytomorphology and flow cytometric analysis is obtained, and grading is feasible for most LG FLs. A subset of FLs composed of a polymorphous lymphoid population with increased numbers of large cells may be more difficult to grade, and HG FLs can be difficult to distinguish from CD10-positive diffuse LBCLs.

Effect of Freezing on Lyophilization Process Performance and Drug Product Cake Appearance.

This study highlights the significance of the freezing step and the critical role it can play in modulating process performance and product quality during freeze-drying. For the model protein formulation evaluated, the mechanism of freezing had a significant impact on cake appearance, a potential critical product quality attribute for a lyophilized drug product. Contrary to common knowledge, a freezing step with annealing resulted in 20% increase in primary drying time compared to without annealing. In addition, annealing resulted in poor cake appearance with shrinkage, cracks, and formation of a distinct skin at the top surface of the cake. Finally, higher product resistance (7.5 cm(2).Torr.hr/g) was observed in the case of annealing compared to when annealing was not included (5 cm(2).Torr.hr/g), which explains the longer primary drying time due to reduced sublimation rates. An alternative freezing option using controlled ice nucleation resulted in reduced primary drying time (i.e., 30% reduction compared to annealing) and a more homogenous batch with elegant uniform (i.e., significantly improved) cake appearance. Here, a mechanistic understanding of the distinct differences in cake appearance as a function of freezing mechanism is proposed within the context of ice nucleation temperature, ice crystal growth, and presumed solute distribution within the frozen matrix.

Clonidine inhibits anti-non-Gal IgM xenoantibody elicited in multiple pig-to-primate models.

BACKGROUND: Survival of vascularized xenografts is dependent on pre-emptive inhibition of the xenoantibody response against galactosyltransferase knockout (GTKO) porcine organs. Our analysis in multiple GTKO pig-to-primate models of xenotransplantation has demonstrated that the anti-non-gal-α-1,3-gal (anti-non-Gal) xenoantibody response displays limited structural diversity. This allowed our group to identify an experimental compound which selectively inhibited induced anti-non-Gal IgM xenoantibodies. However, because this compound had an unknown safety profile, we extended this line of research to include screening small molecules with known safety profiles allowing rapid advancement to large animal models. METHODS: The NIH clinical collections of small molecules were screened by ELISA for their ability to inhibit xenoantibody binding to GTKO pig endothelial cells. Serum collected from non-immunosuppressed rhesus monkeys at day 14 post-injection with GTKO pig endothelial cells was utilized as a source of elicited xenoantibody for initial screening. Virtual small molecule screening based on xenoantibody structure was used to assess the likelihood that the identified small molecules bound xenoantibody directly. As a proxy for selectivity, ELISAs against tetanus toxoid and the natural antigens laminin, thyroglobulin, and single-stranded DNA (ssDNA) were utilized to assess the ability of the identified reagents to inhibit additional antibody responses. The identified inhibitory small molecules were further tested for their ability to inhibit xenoantibody elicited in multiple settings, including rhesus monkeys pre-treated with an anti-non-Gal selective anti-idiotypic antibody, non-immunosuppressed rhesus monkeys immunized with wild-type fetal pig isletlike cell clusters, and non-immunosuppressed baboons transplanted with GTKO multiple transgenic pig kidneys. RESULTS: Four clinically relevant small molecules inhibited anti-non-Gal IgM binding to GTKO pig endothelial cells in vitro. Three of these drugs displayed a limited region of structural similarity suggesting they may inhibit xenoantibody by a similar mechanism. One of these, the anti-hypertensive agent clonidine, displayed only minimal inhibition of antibodies elicited by vaccination against tetanus toxoid or pre-existing natural antibodies against laminin, thyroglobulin, or ssDNA. Furthermore, clonidine inhibited elicited anti-non-Gal IgM from all animals that demonstrated a xenoantibody response in each experimental setting. CONCLUSIONS: Clinically relevant small molecule drugs with known safety profiles can inhibit xenoantibody elicited against non-Gal antigens in diverse experimental xenotransplantation settings. These molecules are ready to be tested in large animal models. However, it will first be necessary to optimize the timing and dosing required to inhibit xenoantibodies in vivo.

Folding and binding energy of a calmodulin-binding cell antiproliferative peptide.

We carry out a computational study of a calmodulin-binding peptide shown to be effective in reducing cell proliferation. We find several folded states for two short variants of different length of the peptide and determine the location of the binding site on calmodulin, the binding free energy for the different conformers and structural details that play a role in optimal binding. Binding to a hydrophobic pocket in calmodulin occurs via an anchoring phenylalanine residue of the natively disordered peptide, and is enhanced when a neighbouring hydrophobic residue acts as a co-anchor. The shorter sequence possesses better binding to calmodulin, which is encouraging in terms of the development of non-peptide analogues as therapeutic agents.

Rhesus monkeys and baboons develop clotting factor VIII inhibitors in response to porcine endothelial cells or islets.

BACKGROUND: Xenotransplantation of porcine organs holds promise of solving the human organ donor shortage. The use of α-1,3-galactosyltransferase knockout (GTKO) pig donors mitigates hyperacute rejection, while delayed rejection is currently precipitated by potent immune and hemostatic complications. Previous analysis by our laboratory suggests that clotting factor VIII (FVIII) inhibitors might be elicited by the structurally restricted xenoantibody response which occurs after transplantation of either pig GTKO/hCD55/hCD59/hHT transgenic neonatal islet cell clusters or GTKO endothelial cells. METHODS: A recombinant xenoantibody was generated using sequences from baboons demonstrating an active xenoantibody response at day 28 after GTKO/hCD55/hCD59/hHT transgenic pig neonatal islet cell cluster transplantation. Rhesus monkeys were immunized with GTKO pig endothelial cells to stimulate an anti-non-Gal xenoantibody response. Serum was collected at days 0 and 7 after immunization. A two-stage chromogenic assay was used to measure FVIII cofactor activity and identify antibodies which inhibit FVIII function. Molecular modeling and molecular dynamics simulations were used to predict antibody structure and the residues which contribute to antibody-FVIII interactions. Competition ELISA was used to verify predictions at the domain structural level. RESULTS: Antibodies that inhibit recombinant human FVIII function are elicited after non-human primates are transplanted with either GTKO pig neonatal islet cell clusters or endothelial cells. There is an apparent increase in inhibitor titer by 15 Bethesda units (Bu) after transplant, where an increase greater than 5 Bu can indicate pathology in humans. Furthermore, competition ELISA verifies the computer modeled prediction that the recombinant xenoantibody, H66K12, binds the C1 domain of FVIII. CONCLUSIONS: The development of FVIII inhibitors is a novel illustration of the potential impact the humoral immune response can have on coagulative dysfunction in xenotransplantation. However, the contribution of these antibodies to rejection pathology requires further evaluation because "normal" coagulation parameters after successful xenotransplantation are not fully understood.

Anti-non-Gal-specific combination treatment with an anti-idiotypic Ab and an inhibitory small molecule mitigates the xenoantibody response.

BACKGROUND: B-cell depletion significantly extends survival of α-1,3-galactosyltranferase knockout (GTKO) porcine organs in pig-to-primate models. Our previous work demonstrated that the anti-non-Gal xenoantibody response is structurally restricted. Selective inhibition of xenoantigen/xenoantibody interactions could prolong xenograft survival while preserving B-cell-mediated immune surveillance. METHODS: The anti-idiotypic antibody, B4N190, was selected from a synthetic human phage display library after enrichment against a recombinant anti-non-Gal xenoantibody followed by functional testing in vitro. The inhibitory small molecule, JMS022, was selected from the NCI diversity set III using virtual screening based on predicted xenoantibody structure. Three rhesus monkeys were pre-treated with anti-non-Gal-specific single-chain anti-idiotypic antibody, B4N190. A total of five monkeys, including two untreated controls, were then immunized with GTKO porcine endothelial cells to initiate an anti-non-α-1,3-Gal (non-Gal) xenoantibody response. The efficacy of the inhibitory small molecule specific for anti-non-Gal xenoantibody, JMS022, was tested in vitro. RESULTS: After the combination of in vivo anti-id and in vitro small molecule treatments, IgM xenoantibody binding to GTKO cells was reduced to pre-immunization levels in two-thirds of animals; however, some xenoantibodies remained in the third animal. Furthermore, when treated with anti-id alone, all three experimental animals displayed a lower anti-non-Gal IgG xenoantibody response compared with controls. Treatment with anti-idiotypic antibody alone reduced IgM xenoantibody response intensity in only one of three monkeys injected with GTKO pig endothelial cells. In the one experimental animal, which displayed reduced IgM and IgG responses, select B-cell subsets were also reduced by anti-id therapy alone. Furthermore, natural antibody responses, including anti-laminin, anti-ssDNA, and anti-thyroglobulin antibodies were intact despite targeted depletion of anti-non-Gal xenoantibodies in vivo indicating that selective reduction of xenoantibodies can be accomplished without total B-cell depletion. CONCLUSIONS: This preliminary study demonstrates the strength of approaches designed to selectively inhibit anti-non-Gal xenoantibody. Both anti-non-Gal-specific anti-idiotypic antibody and small molecules can be used to selectively limit xenoantibody responses.

Xenoantibody response to porcine islet cell transplantation using GTKO, CD55, CD59, and fucosyltransferase multiple transgenic donors.

BACKGROUND: Promising developments in porcine islet xenotransplantation could resolve the donor pancreas shortage for patients with type 1 diabetes. Using α1,3-galactosyltransferase gene knockout (GTKO) donor pigs with multiple transgenes should extend xenoislet survival via reducing complement activation, thrombus formation, and the requirement for exogenous immune suppression. Studying the xenoantibody response to GTKO/hCD55/hCD59/hHT islets in the pig-to-baboon model, and comparing it with previously analyzed responses, would allow the development of inhibitory reagents capable of targeting conserved idiotypic regions. METHODS: We generated IgM heavy and light chain gene libraries from 10 untreated baboons and three baboons at 28 days following transplantation of GTKO/hCD55/hCD59/hHT pig neonatal islet cell clusters with immunosuppression. Flow cytometry was used to confirm the induction of a xenoantibody response. IgM germline gene usage was compared pre- and post-transplant. Homology modeling was used to compare the structure of xenoantibodies elicited after transplantation of GTKO/hCD55/hCD59/hHT pig islets with those induced by GTKO and wild-type pig endothelial cells without further genetic modification. RESULTS: IgM xenoantibodies that bind to GTKO pig cells and wild-type pig cells were induced after transplantation. These anti-non-Gal antibodies were encoded by the IGHV3-66*02 (Δ28%) and IGKV1-12*02 (Δ25%) alleles, for the immunoglobulin heavy and light chains, respectively. IGHV3-66 is 86.7% similar to IGHV3-21 which was elicited by rhesus monkeys in response to GTKO endothelial cells. Heavy chain genes most similar to IGHV3-66 were found to utilize the IGHJ4 gene in 85% of V-D regions analyzed. However, unlike the wild-type response, a consensus complementary determining region 3 was not identified. CONCLUSIONS: Additional genetic modifications in transgenic GTKO pigs do not substantially modify the structure of the restricted group of anti-non-Gal xenoantibodies that mediate induced xenoantibody responses with or without immunosuppression. The use of this information to develop new therapeutic agents to target this restricted response will likely be beneficial for long-term islet cell survival and for developing targeted immunosuppressive regimens with less toxicity.

In vivo detection of human TRPV6-rich tumors with anti-cancer peptides derived from soricidin.

Soricidin is a 54-amino acid peptide found in the paralytic venom of the northern short-tailed shrew (Blarina brevicauda) and has been found to inhibit the transient receptor potential of vallinoid type 6 (TRPV6) calcium channels. We report that two shorter peptides, SOR-C13 and SOR-C27, derived from the C-terminus of soricidin, are high-affinity antagonists of human TRPV6 channels that are up-regulated in a number of cancers. Herein, we report molecular imaging methods that demonstrate the in vivo diagnostic potential of SOR-C13 and SOR-C27 to target tumor sites in mice bearing ovarian or prostate tumors. Our results suggest that these novel peptides may provide an avenue to deliver diagnostic and therapeutic reagents directly to TRPV6-rich tumors and, as such, have potential applications for a range of carcinomas including ovarian, breast, thyroid, prostate and colon, as well as certain leukemia's and lymphomas.

Role of BCL2-associated athanogene 1 in differential sensitivity of human endothelial cells to glucocorticoids.

OBJECTIVE: Chronic therapy with synthetic glucocorticoids has been associated with cardiovascular side effects, although differential interindividual susceptibility to glucocorticoids has been observed. The objective of this study was to identify the molecular mechanisms leading to differential glucocorticoid responses in endothelial cells. APPROACH AND RESULTS: We tested the sensitivity of 42 human umbilical vein endothelial cells (HUVECs) to dexamethasone as determined by changes in gene expression, promoter transactivation, and procoagulant activity. We identified that 16 HUVECs were sensitive in every test, 14 HUVECs were sensitive in at least 1 test and 12 HUVECs were resistant in every test to dexamethasone. Nuclear translocation assays revealed that Dex-sensitive HUVECs have higher basal and Dex-stimulated levels of nuclear glucocorticoid receptor compared with Dex-resistant HUVECs. Cycloheximide assays revealed that Dex-resistant HUVECs have significantly shorter glucocorticoid receptor protein half-lives than Dex-sensitive HUVECs. Dex-resistant HUVECs have a stronger interaction of glucocorticoid receptor with the proteasomal recruiting protein, BCL2-associated athanogene 1 (BAG1), as shown by immunoprecipitation assays. Silencing BAG1 expression increased Dex-sensitivity in resistant HUVECs, whereas BAG1 overexpression decreased Dex-sensitivity in sensitive HUVECs. Finally, Dex-resistant HUVECs presented higher BAG1 expression than Dex-sensitive HUVECs. CONCLUSIONS: In vitro endothelial sensitivity to Dex varies within individuals and is inversely proportional to BAG1 protein expression and glucocorticoid receptor protein turnover.

Vaccinelike and prophylactic treatments of EAE with novel I-domain antigen conjugates (IDAC): targeting multiple antigenic peptides to APC.

The objective of this work is to utilize novel I-domain antigenic-peptide conjugates (IDAC) for targeting antigenic peptides to antigen-presenting cells (APC) to simulate tolerance in experimental autoimmune encephalomyelitis (EAE). IDAC-1 and IDAC-3 molecules are conjugates between the I-domain protein and PLP-Cys and Ac-PLP-Cys-NH(2) peptides, respectively, tethered to N-terminus and Lys residues on the I-domain. The hypothesis is that the I-domain protein binds to ICAM-1 and PLP peptide binds to MHC-II on the surface of APC; this binding event inhibits the formation of the immunological synapse at the APC-T-cell interface to alter T-cell differentiation from inflammatory to regulatory phenotypes. Conjugation of peptides to the I-domain did not change the secondary structure of IDAC molecules as determined by circular dichroism spectroscopy. The efficacies of IDAC-1 and -3 were evaluated in EAE mice by administering iv or sc injections of IDAC in a prophylactic or a vaccinelike dosing schedule. IDAC-3 was better than IDAC-1 in suppressing and delaying the onset of EAE when delivered in prophylactic and vaccinelike manners. IDAC-3 also suppressed subsequent relapse of the disease. The production of IL-17 was lowered in the IDAC-3-treated mice compared to those treated with PBS. In contrast, the production of IL-10 was increased, suggesting that there is a shift from inflammatory to regulatory T-cell populations in IDAC-3-treated mice. In conclusion, the I-domain can effectively deliver antigenic peptides in a vaccinelike or prophylactic manner for inducing immunotolerance in the EAE mouse model.