Simple and fast one-step FRET assay of therapeutic mAb bevacizumab using anti-idiotype DNA aptamer for process analytical technology.

We developed an aptamer-based fluorescence resonance energy transfer (FRET) assay capable of recognizing therapeutic monoclonal antibody bevacizumab and rapidly quantifying its concentration with just one mixing step. In this assay, two fluorescent dyes (fluorescein and tetramethylrhodamine) labeled aptamers bind to two Fab regions on bevacizumab, and FRET fluorescence is observed when both dyes come into close proximity. We optimized this assay in three different formats, catering to a wide range of analytical needs. When applied to hybridoma culture samples in practical settings, this assay exhibited a signal response that was concentration-dependent, falling within the range of 50-2000 µg/mL. The coefficients of determination (r2) ranged from 0.998 to 0.999, and bias and precision results were within ±24.0 % and 20.3 %, respectively. Additionally, during thermal and UV stress testing, this assay demonstrated the ability to detect denatured samples in a manner comparable to conventional Size Exclusion Chromatography. Notably, it offers the added advantage of detecting decreases in binding activity without changes in molecular weight. In contrast to many existing process analytical technology tools, this assay not only identifies bevacizumab but also directly measures the quality attributes related to mAb efficacy, such as the binding activity. As a result, this assay holds great potential as a valuable platform for providing highly reliable quality attribute information in real-time. We consider this will make a significant contribution to the worldwide distribution of high-quality therapeutic mAbs in various aspects of antibody manufacturing, including production monitoring, quality control, commercial lot release, and stability testing.

Foreign body granulomas mimic peritoneal dissemination caused by incarcerated femoral hernia perforation: A case report.

BACKGROUND: Foreign body granuloma (FBG) is a well-known type of granulomatous formation, and intraabdominal FBG (IFBG) is primarily caused by surgical residues. Multifocal IFBGs caused by gastrointestinal perforation is an extremely rare and interesting clinicopathological condition that resembles peritoneal dissemination. Here, we present a case of IFBGs mimicking peritoneal dissemination caused by bowel perforation and describe the value of intraoperative pathological examinations for rapid IFBG diagnosis. CASE SUMMARY: An 86-year-old woman with an incarcerated femoral hernia was admitted to the hospital and underwent operation. During the operation, the incarcerated ileum was perforated during repair due to hemorrhage necrosis, and a small volume of enteric fluid leaked from the perforation. The incarcerated ileum was resected, and the femoral hernia was repaired without mesh. Four months later, a second operation was performed for an umbilical incisional hernia. During the second operation, multiple small, white nodules were observed throughout the abdominal cavity, resembling peritoneal dissemination. The results of peritoneal washing cytology in Douglas' pouch and the examination of frozen nodule sections were compatible with IFBG diagnosis, and incisional hernia repair was performed. CONCLUSION: IFBGs can mimic malignancy. Intraoperative pathological examinations and operation history are valuable for the rapid diagnosis to avoid excessive treatments.

Small-cell Lung Cancer in a Young Adult Nonsmoking Patient with Ectopic Adrenocorticotropin (ACTH) Production.

Cushing's syndrome due to young small-cell lung cancer (SCLC) is recognized as being extremely rare. We herein present the case of a 35-year-old nonsmoking man who presented with thirst and polyuria. Laboratory examinations showed hyperglycemia, hypokalemia and liver enzyme elevation. Imaging examinations revealed the presence of multiple liver tumors and lymph node swelling. The levels of serum neuroendocrine tumor markers were elevated. The patient was diagnosed with SCLC based on the pathological examination of a biopsy specimen from the right supraclavicular lymph node. The physical findings, including proximal myopathy, truncal obesity and pigmentation suggested high levels of glucocorticoids. An immunohistochemical examination of the tumor showed that it was positive for adrenocorticotropin (ACTH). An endocrinological investigation allowed for the definitive diagnosis of SCLC with ectopic ACTH production.

Grafting of poly(ethylene glycol) to poly-lysine augments its lifetime in blood circulation and accumulation in tumors without loss of the ability to associate with siRNA.

Poly-lysine has been studied as a carrier for the delivery of drugs and nucleic acids for at least a decade. It is an especially attractive carrier for DNA and RNA, because of its condensed cationic charges. In our previous study, we showed that poly(ethylene glycol) (PEG) grafted to poly-L-lysine (PLL) remarkably increased the life time of a small interfering RNA (siRNA) in blood circulation. In this study, we prepared a new series of PEG-grafted PLL (PLL-g-PEG) with various lengths (PEG 2kDa, 5kDa, and 10kDa and PLL 28kDa and 40kDa), to evaluate masking effects of PEG on cationic charges of PLL in vivo and the structural implications for biodistribution and tumoral accumulation. The best in the series, 40K10P37 (40kDa of PLL, 10kDa of PEG, 37mol% grafting) with molecular weight of 10(6) as determined by Multi-Angle Laser Light Scattering (MALLS), accumulated in tumors at about 8% of the injected dose per gram of tissue. Interestingly, a PLL-g-PEG conjugate pre-mixed with murine sera prevented degradation of siRNA, suggesting that PLL-g-PEG preferentially associates with siRNA in sera. Our results indicate grafting of PEG to the side chains of PLL augments its lifetime in blood circulation and tumoral accumulation without loss of the ability to associate with siRNA and support further evaluation of these cationic delivery carriers.

Polymer brush-stabilized polyplex for a siRNA carrier with long circulatory half-life.

Delivery systems of small interfering RNA (siRNA) are the key to siRNA therapeutic application. In this study, we prepared and evaluated a series of cationic comb-type copolymers (CCCs) possessing a polycationic backbone (less than 30 weight (wt) %) and abundant water-soluble side chains (more than 70 wt.%) as a siRNA carrier with prolonged blood circulation time. Markedly, the CCC with the higher side chain content (10 wt.% PLL and 90 wt.% PEG) showed stronger interaction with siRNA than that with the lower content (30 wt.% PLL and 70 wt.% PEG), suggesting that highly dense PEG brush reinforces interpolyelectrolyte complex between the PLL backbone and siRNA. The siRNA complexed with the CCC was resistant to nucleases in 90% plasma for 24 h in vitro. The CCC having the higher side chain content increased circulation time of siRNA in mouse bloodstream by 100-fold. Surprisingly, even when the CCC and siRNA were separately injected into mouse at 20 min interval, blood circulation of post-injected siRNA was significantly increased. These results imply that the CCC has higher selectivity in its ionic interaction with siRNA than other anionic substances in blood stream. To our knowledge, this is the first example of a polyplex carrier that prolongs blood circulation time of unmodified siRNA without resource-consuming preparation process.

Secondary torsion of vermiform appendix with mucinous cystadenoma.

Torsion of the vermiform appendix is a rare disorder, which causes abdominal symptoms indistinguishable from acute appendicitis. We report a case (a 34-year-old male) of secondary torsion of the vermiform appendix with mucinous cystadenoma. This case was characterized by mild inflammatory responses, pentazocine-resistant abdominal pain, and appendiceal tumor, which was not enhanced by the contrast medium on computed tomography presumably because of reduced blood flow by the torsion. These findings may be helpful for the preoperative diagnosis of secondary appendiceal torsion.

Successful treatment of multi-agent chemotherapy with rituximab for IgM plasma cell leukemia.

A 67-year-old woman presented with impaired general performance, suffering from fatigue, dyspnea on exertion, and paresthesia of the finger tips. The laboratory findings showed increased white blood cells at 11.37 x 10(3)cells/microl with 26.5% abnormal cells, low haemoglobin and, elevated creatinine, although serum lactate dehydrogenase and calcium levels were normal. Serum immunofixation was positive for monoclonal IgM-kappa paraprotein. Total serum protein and the IgM component were elevated. X-ray examination of the skeleton was normal. Bone marrow aspiration showed 59.5% infiltration of abnormal cells that were characterized by typical mature plasmacytoid morphology. Abnormal cells expressed surface CD20, surface CD138, and cytoplasmic IgM, but not surface CD56 nor surface IgM by flow cytometric immunophenotyping with CD38 gating. Immunohistochemistry showed surface CD38, surface CD20, and cytoplasmic IgM. The clinical findings led to the diagnosis of the IgM Plasma cell leukemia (PCL). The patient received multi-agent chemotherapy (VAD and EDAP with rituximab). The clinical symptoms disappeared, leading to the tumor load reduction. To the best of our knowledge, this is the first report of successful treatment of multi-agent chemotherapy with rituximab for IgM PCL.

[Effective measurement of coronary flow velocity reserve (CFVR) with transthoracic Doppler echocardiography (TTDE) for plasma cell leukemia with hyperviscosity syndrome].

A 67-year-old woman was admitted with impaired general performance, suffering from fatigue, chest oppression on exertion, and paresthesia of the finger trips. The laboratory findings showed increased white blood cells with abnormal cells, and serum immunofixation test showed monoclonal IgM kappa paraprotein. On flow cytometric immunophenotyping with CD38 gating, most of the abnormal cells expressed surface CD20, CD138, cytoplasmic IgM, but neither surface CD56 nor surface IgM. Immunohistochemical staining of abnormal cells was positive for surface CD38, surface CD20 and cytoplasmic IgM. The final diagnosis was plasma cell leukemia IgM kappa type. Electrocardiography (ECG) on admission showed ST depression in II, III, aV(F), V4, V5, and V6. Coronary angiography (CAG) is invasive and difficult for patients with renal failure, therefore the patient underwent transthoracic Doppler echocardiography (TTDE), which revealed reduced coronary flow velocity reserve (CFVR). Two courses of VAD therapy were administered, then the condition improved, the serum IgM level decreased, abnormal cells were decreased in peripheral blood and bone marrow aspirates, and the creatinine levels improved. With the return of normal ECG findings and improved CFVR, the abnormal ECG and reduction in CFVR was thought to be associated with the hyperviscosity syndrome in PCL. Noninvasive assessment of CFVR by TTDE is significantly useful for the patients who have renal failure and need chemotherapy.

[Acute pancreatitis during the treatment of relapsed acute promyelocytic leukemia with As2O3].

A 77-year-old man suffered from acute pancreatitis during the treatment of relapsed acute promyelocytic leukemia with As2O3. He presented with epigastralgia on day 25 during the treatment with As2O3. Pancreatic enzyme levels were elevated and the computed tomography scan of the abdomen showed swelling of the pancreas. As acute pancreatitis due to As2O3 was suspected, As2O3 was discontinued. Intravenous gabexate mesilate was administered, and the pancreatitis improved. Acute pancreatitis should be considered as a possible complication during treatment with As2O3.

Flow cytometric immunophenotyping of adult T-cell leukemia/lymphoma using CD3 gating.

Adult T-cell leukemia/lymphoma (ATLL) is a lymphoproliferative neoplasm of helper T lymphocytes caused by human T-cell leukemia virus type-1 (HTLV-1). The disease was first described in Kyushu, in southwestern Japan, and most frequently occurs in endemic areas, such as Japan, the Caribbean basin, West Africa, Brazil, and northern Iran. ATLL is essentially a disease of adults, characterized clinically by generalized lymphadenopathy, hepatosplenomegaly, skin lesions, and hypercalcemia. The prognosis of most patients is quite poor, with a median survival time of only 13 months, even if multiagent combination chemotherapy is given. In the present study, flow cytometric immunophenotyping with CD3 gating was performed on 30 samples from 26 patients who had been given a diagnosis of ATLL. The records of these patients also were reviewed retrospectively. In 14 of the 30 samples, an abnormal CD3(low) T-cell population was distinguishable from the normal T-cell populations by flow cytometric analysis. Herein we report a novel strategy for flow cytometric immunophenotyping of ATLL facilitated by CD3(low) gating.

[Intestinal bleeding during the treatment of chronic myeloid leukemia with imatinib mesylate].

A 35-year-old woman attended our hospital with chronic myeloid leukemia and was prescribed imatinib mesylate. She was admitted with lower abdominal pain, stomatitis, and hyposthenia after an increase in her dose of imatinib mesylate. When the treatment was changed to interferon-alpha and Ara-C, the lower abdominal pain, stomatitis, and hyposthenia improved, but bone marrow aspiration showed 36.4% blasts. After the treatment was changed back to an increased dose of imatinib mesylate (800 mg), the stomatitis deteriorated and intestinal bleeding reoccurred. Endoscopy demonstrated the presence of multiple ulcers in the ascending colon and 99mTc RBC scintigraphy demonstrated lesions of the large and small intestine. The patient declined any treatment except for transfusion and died suddenly after ten days. The present case suggests that we should carefully consider the possibility of intestinal bleeding when prescribing imatinib mesylate.

[Successful treatment of refractory mantle cell lymphoma with irinotecan].

A 59-year-old man presented in January 2003 with generalized lymphadenopathy. An inguinal lymph node biopsy showed mantle cell lymphoma (MCL). After four courses of Rituximab-CHOP therapy were administered, complete response (CR) was achieved. However, in August 2003, he presented with neck lymphadenopathy and was found to have relapsed. Several salvage therapies (ESHAP, Hyper-CVAD/MTX-ara-C) were administered, but CR was not achieved. After two courses of single-agent chemotherapy with CPT-11 (40 mg/m2) were administered on days 1, 2, 3, 8, 9, and 10, CR was achieved. Several studies reveal that the long-term prognosis for MCL with conventional therapy is poor. This report describes CPT-11 therapy for MCL and provides evidence that CPT-11 is another therapeutic option in refractory cases of MCL.

Pulmonary parenchymal infiltrates in a patient with CD20-positive multiple myeloma.

A 53-yr-old woman developed a dry cough after the completion of multi-agent chemotherapy. The biopsy specimens showed diffuse infiltrates with multiple myeloma (MM) cells. Immunohistochemistry revealed positive staining in MM cells with surface CD20, surface CD38, and cytoplasmic IgG. This report represents the first reported case of pulmonary parenchymal infiltrates in a patient with CD20-positive MM.

[Multiple organ relapse in primary de novo CD5+ diffuse large B cell lymphoma of the bone after a complete response].

A 57-year-old woman was admitted with swelling of the femur. MRI showed that an intramedullary lesion had expanded from the trunk to the distal portion where it had formed an extramedullary tumor mass. An open biopsy showed diffuse proliferation of abnormal lymphoid cells. Immunohistochemical staining and flow cytometry demonstrated LCA+, CD3-, CD23-, CD79a+, CD5+, IgM+, IgD- and kappa + and cyclin D1-. FISH analysis did not detect t(11;14)(q13;q32). The final diagnosis was de novo CD5+ diffuse large B-cell lymphoma (DLBL) of the bone at clinical stage IEA. The patient suffered a pathological fracture in the femur after two courses of CHOP. The therapy was changed to ESHAP and irradiation. The result was assessed as a complete remission (CR). One month later, the patient presented with epigastric pain. MRI showed the tumor at the spleen and kidney and hydronephrosis due to pelvic lymphadenopathy, but did not show a tumor in the femur. An open biopsy of the pelvic lymph node showed relapse. The tumor and hydronephrosis disappeared and necrosis in the kidney was observed on MRI after ESHAP. De novo CD5+ DLBL appears to constitute a unique subset of DLBL with an aggressive clinical course and requires established therapeutic strategies.