Collaborative Study for Analysis of Subvisible Particles Using Flow Imaging and Light Obscuration: Experiences in Japanese Biopharmaceutical Consortium

The evaluation of subvisible particles, including protein aggregates, in therapeutic protein products has been of great interest for both pharmaceutical manufacturers and regulatory agencies. To date, the flow imaging (FI) method has emerged as a powerful tool instead of light obscuration (LO) due to the fact that (1) protein aggregates contain highly transparent particles and thereby escape detection by LO and (2) FI provides detailed morphological characteristics of subvisible particles. However, the FI method has not yet been standardized nor listed in any compendium. In an attempt to assess the applicability of the standardization of the FI method, we conducted a collaborative study using FI and LO instruments in a Japanese biopharmaceutical consortium. Three types of subvisible particle preparations were shared across 12 laboratories and analyzed for their sizes and counts. The results were compared between the methods (FI and LO), inter-laboratories, and inter-instruments (Micro Flow Imaging and FlowCam). We clarified the marked difference between the detectability of FI and LO when counting highly transparent protein aggregates in the preparations. Although FlowCam provided a relatively higher number of particles compared with MFI, consistent results were obtained using the instrument from the same manufacturer in all 3 samples.     

Improvement of region of interest extraction and scanning method of computer-aided diagnosis system for osteoporosis using panoramic radiographs

Objectives

Patients undergoing osteoporosis treatment benefit greatly from early detection. We previously developed a computer-aided diagnosis (CAD) system to identify osteoporosis using panoramic radiographs. However, the region of interest (ROI) was relatively small, and the method to select suitable ROIs was labor-intensive. This study aimed to expand the ROI and perform semi-automatized extraction of ROIs. The diagnostic performance and operating time were also assessed.

Methods

We used panoramic radiographs and skeletal bone mineral density data of 200 postmenopausal women. Using the reference point that we defined by averaging 100 panoramic images as the lower mandibular border under the mental foramen, a 400?×?100-pixel ROI was automatically extracted and divided into four 100?×?100-pixel blocks. Valid blocks were analyzed using program 1, which examined each block separately, and program 2, which divided the blocks into smaller segments and performed scans/analyses across blocks. Diagnostic performance was evaluated using another set of 100 panoramic images.

Results

Most ROIs (97.0%) were correctly extracted. The operation time decreased to 51.4% for program 1 and to 69.3% for program 2. The sensitivity, specificity, and accuracy for identifying osteoporosis were 84.0, 68.0, and 72.0% for program 1 and 92.0, 62.7, and 70.0% for program 2, respectively. Compared with the previous conventional system, program 2 recorded a slightly higher sensitivity, although it occasionally also elicited false positives.

Conclusions

Patients at risk for osteoporosis can be identified more rapidly using this new CAD system, which may contribute to earlier detection and intervention and improved medical care.

     

Creation of Straight-Chain Cationic Polysaccharide-Based Bile Salt Sequestrants Made from Euglenoid β-1,3-Glucan as Potential Antidiabetic Agents

Purpose

The objective of this work was to evaluate the potential of polymeric spherical and aspherical invasive nanocarriers, loaded with antibiotic, to access and treat intracellular bacterial infections.

Methods

Aspherical nanocarriers were prepared by stretching of spherical precursors, and both aspherical and spherical nanocarriers were surface-functionalized with the invasive protein InvA497. The relative uptake of nanocarriers into HEp-2 epithelial cells was then assessed. Nanocarriers were subsequently loaded with a preparation of the non-permeable antibiotic gentamicin, and tested for their ability to treat HEp-2 cells infected with the enteroinvasive bacterium Shigella flexneri.

Results

InvA497-functionalized nanocarriers of both spherical and aspherical shape showed a significantly improved rate and extent of uptake into HEp-2 cells in comparison to non-functionalized nanocarriers. Functionalized and antibiotic-loaded nanocarriers demonstrated a dose dependent killing of intracellular S. flexneri. A slight but significant enhancement of intracellular bacterial killing was also observed with aspherical as compared to spherical functionalized nanocarriers at the highest tested concentration.

Conclusions

InvA497-functionalized, polymer-based nanocarriers were able to efficiently deliver a non-permeable antibiotic across host cell membranes to affect killing of intracellular bacteria. Functionalized nanocarriers with an aspherical shape showed an interesting future potential for intracellular infection therapy.

     

Allogeneic hematopoietic stem cell transplantation for aplastic anemia with pre-transplant conditioning using fludarabine,reduced-dose cyclophosphamide,and low-dose thymoglobulin: A KSGCT prospective study

The optimal pre-transplant conditioning for aplastic anemia (AA) remains unclear. We performed a prospective study on allogeneic transplantation from a related or unrelated donor for adult patients with AA. We assessed whether reduced-dose cyclophosphamide (CY) could decrease toxicity while maintaining engraftment, and low-dose thymoglobulin could safely prevent graft-vs-host disease (GVHD). The pre-transplant conditioning regimen consisted of fludarabine 120 mg/m², CY 100 mg/kg, and thymoglobulin 2.5 mg/kg with or without 2 Gy of total body irradiation. Twenty-seven patients with a median age of 36 years were analyzed. Sixteen patients received graft from related donors. The stem cell source was bone marrow in 26 patients. All of the patients but one, who died early, achieved neutrophil engraftment at a median of 19 days. Mixed chimerism was observed in six and five patients at days 30 and 90, respectively. Only one patient experienced secondary engraftment failure with complete donor-type chimerism. None of the patients developed severe acute GVHD. The cumulative incidence of chronic GVHD was 37.7% at 1 year. The overall survival rate was 96.3% at 1 year and 3 years. A high EB virus-DNA load was detected in one patient at days 60. No one developed EBV-lymphoproliferative disorder within a year. The results suggest that the conditioning regimen in this study was safe and effective. However, relatively high incidence of chronic GVHD needs further improvement.     

Indigo naturalis is effective even in treatment-refractory patients with ulcerative colitis: a post hoc analysis from the INDIGO study

Background

We recently reported the efficacy of indigo naturalis (IN) in patients with active ulcerative colitis (UC) in a randomized controlled trial (INDIGO study). However, few studies have been conducted to investigate whether IN is effective even in treatment-refractory cases, such as in those with steroid dependency and anti-TNF refractoriness.

Methods

In the INDIGO study, 86 patients with active UC were randomly assigned to an IN group (0.5–2.0 g daily) or placebo group. The rate of clinical response (CR), mucosal healing (MH), and change in fecal calprotectin (FCP) levels was compared between refractory [patients with steroid-dependent disease, previous use of anti-TNF-α, and concomitant use of immunomodulators (IM)] and non-refractory patients. We also analyzed factors predicting CR and MH at week 8.

Results

The rates of CR of IN group were significantly higher than placebo group, even in patients with steroid-dependent disease (p < 0.001), previous use of anti-TNF-α (p = 0.002), and concomitant use of IM (p = 0.013). The rates of MH in IN group were significantly higher than in placebo group in patients with steroid-dependent disease (p = 0.009). In the IN group, median FCP levels, at week 8, were significantly lower than baseline in patients with steroid-dependent disease and patients with the previous use of anti-TNF-α (p < 0.001, respectively). Multivariate analysis indicated that the previous use of anti-TNF-α was not a predictive factor for CR and MH at week 8.

Conclusions

In a sub-analysis of data from a randomized placebo-controlled trial, we found that IN may be useful even in patients with steroid-dependent disease and patients with the previous use of anti-TNF-α.

     

Cryptococcal meningitis accompanying lymphocytic inflammation predominantly in cerebral deep white matter: A possible manifestation of immune reconstitution inflammatory syndrome

Cryptococcal meningitis is rarely complicated by immune‐mediated leukoencephalopathy, but the precise pathomechanism is uncertain. A 72‐year‐old Japanese man treated with prednisolone for Sweet disease developed a subacute progression of meningitis, which was considered as neuro‐Sweet disease. A treatment by methylprednisolone rapidly improved CSF findings with a remarkable decrease in lymphocyte numbers in the blood, but the patient's consciousness still worsened after the cessation of the treatment. The patient developed cryptococcal meningitis and MRI showed abnormal intensities predominantly in the cerebral deep white matter along with the recovery of lymphocyte numbers in the blood, which resulted in death. A postmortem examination of the brain revealed degenerative lesions, especially at the cerebral white matter and cortex adjacent to the leptomeninges abundantly infiltrated by Cryptococcus neoformans. In the affected cerebral deep white matter, perivascular infiltration of lymphocytes was prominent in coexistence with reactive astrocytes and vascular proliferation, but these findings were not observed in the subcortical and cortical lesions. Cryptococcus neoformans was not present within the brain parenchyma. This is the first report of a case suggesting that cryptococcal meningitis can accompany lymphocytic inflammation predominantly in cerebral deep white matter as a possible manifestation of immune reconstitution inflammatory syndrome.     

Psychological distress in an earthquake-devastated area with pre-existing high rate of suicide

On 12 March 2011 an earthquake devastated the Matsunoyama and Matsudai districts of Tōkamachi City, Niigata, Japan. These areas had high pre-existing suicide rates, especially among the elderly. We investigated whether mental health status became worse among the sufferers 5 months after the earthquake, and what kind of factors were implicated in any changes. A 15-item questionnaire that tapped earthquake-related variables and the Kessler 10 Psychological Distress Scale to measure psychological distress were distributed to 1923 residents aged over 40 years. The mean age (S.D.) of the total 1731 respondents (male, 805; female, 926) was 68.2 (13.1) years. Of these, we assessed K10 scores from 1346 respondents. The mean scores (S.D.) for K10 and K6 (six selected items from the K10) were 5.8 (6.3) and 3.4 (3.9), respectively. Among the respondents, 9.1% and 3.2% obtained a score of K10 ≥15 and K6 ≥13, respectively. These scores showed slightly higher psychological distress, especially among the elderly, in comparison with existing community-based data. Categorical regression analysis revealed significant and relatively strong effects of initial psychological impact, decrease in sleep hours, advanced age, and decrease in interpersonal relationships within the community on the K10 score. The last item suggests the importance of socio-environmental factors in post-disaster mental health.