Best Practices for Microbial Challenge In-use Studies to Evaluate the Microbial Growth Potential of Parenteral Biological Products; Industry and Regulatory Considerations.

Microbial challenge in-use studies are performed to evaluate the potential for microbial proliferation in preservative-free single dose biological products after first puncture and potential accidental contamination during dose preparation (e.g. reconstitution, dilution) and storage. These studies, in addition to physicochemical in-use stability assessments, are used as part of product registration to define in-use hold times in Prescribing Information and in the pharmacy manual in the case of clinical products. There are no formal guidance documents describing regulator expectations on how to conduct microbial challenge in-use studies and interpret microbial data to assign in-use storage hold-times. In lieu of guidance, US Food and Drug Administration (FDA) regulators have authored publications and presentations describing regulator expectations. Insufficient or unavailable microbial challenge data can result in shortened in-use hold times, thus microbial challenge data enables flexibility for health care providers (HCPs) and patients, while ensuring patient safety. A cross-industry/FDA in-use microbial working group was formed through the Innovation & Quality (IQ) Consortium to gain alignment among industry practice and regulator expectations. The working group assessed regulatory guidance, current industry practice via a blinded survey of IQ Consortium member companies, and scientific rationale to align on recommendations for experimental design, execution of microbial challenge in-use studies, and a decision tree for microbial data interpretation to assign in-use hold times. Besides the study execution and data interpretation, additional considerations are discussed including use of platform data for clinical stage products, closed system transfer devices (CSTDs), transport of dose solutions, long infusion times, and the use of USP <797> by HCPs for preparing sterile drugs for administration. The recommendations provided in this manuscript will help streamline biological product development, ensure consistency on assignment of in-use hold times in biological product labels across industry, and provide maximum allowable flexibility to HCPs and patients, while ensuring patient safety.

The impact of the Covid-19 pandemic and government intervention on active mobility.

With data from automated counting stations and controlling for weather and calendar effects, we estimate the isolated impacts of the "first wave" of Covid-19 pandemic and subsequent government intervention (contact restrictions and closures of public spaces) on walking and cycling in 10 German cities. Pedestrian traffic in pedestrian zones decreases with higher local incidence values, and with stricter government intervention. There are ambiguous effects for cycling, which decreases in cities with a higher modal share of cycling, and increases in others. Moreover, we find impact heterogeneity with respect to different weekdays and hours of the day, both for cycling and walking. Additionally, we use data on overall mobility changes, which were derived from mobile phone data, in order to estimate the modal share changes of cycling. In almost all cities, the modal share of cycling increases during the pandemic, with higher increases in non-bicycle cities and during stronger lockdown interventions.

ICH: Strengths, Weaknesses, and Future Tasks.

The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) started in 1990 with the mission to harmonize technical guidelines and obligations for the development and authorization of human pharmaceuticals through the cooperation of drug regulatory authorities and the pharmaceutical industry of the main drug markets in the world. To evaluate the achievements of ICH over the last 23 years, 30 European experts were interviewed. The interviewee panel was selected to encompass representatives of both the pharmaceutical industry (20 interview partners) and the health authorities (10 interview partners). Many of the interview partners have actively contributed to ICH by leading or participating in working groups. Based on the interviews, this article highlights the strengths and weaknesses of ICH with a focus on ideas and visions to improve the status quo.

Hsp42 is required for sequestration of protein aggregates into deposition sites in Saccharomyces cerevisiae.

The aggregation of proteins inside cells is an organized process with cytoprotective function. In Saccharomyces cerevisiae, aggregating proteins are spatially sequestered to either juxtanuclear or peripheral sites, which target distinct quality control pathways for refolding and degradation. The cellular machinery driving the sequestration of misfolded proteins to these sites is unknown. In this paper, we show that one of the two small heat shock proteins of yeast, Hsp42, is essential for the formation of peripheral aggregates during physiological heat stress. Hsp42 preferentially localizes to peripheral aggregates but is largely absent from juxtanuclear aggregates, which still form in hsp42Δ cells. Transferring the amino-terminal domain of Hsp42 to Hsp26, which does not participate in aggregate sorting, enables Hsp26 to replace Hsp42 function. Our data suggest that Hsp42 acts via its amino-terminal domain to coaggregate with misfolded proteins and perhaps link such complexes to further sorting factors.

Chronic pancreatitis is associated with disease-specific regulatory T-cell responses.

BACKGROUND & AIMS: Chronic pancreatitis is characterized by alternating phases of acute inflammation and quiescent disease. Involvement of T-cell responses has been suggested, but pancreatitis-specific T cells have not been described. METHODS: We characterized T-cell responses against pancreatitis, pancreatic carcinoma-associated antigens, and tetanus toxoid in the bone marrow, blood, and/or pancreatitis lesions of patients with pancreatitis, pancreatic cancer, and healthy individuals. T cells were functionally characterized by antigen-dependent secretion of interferon (IFN)-gamma, interleukin (Il)-4, and IL-10, which indicate type 1, type 2, or regulatory T-cell responses, respectively. Regulatory T cells were characterized by multicolor flow cytometry. Isolated regulatory T cells were tested for their capacity to recognize pancreatitis-associated antigens and to suppress conventional T cells in an antigen-dependent manner. T cell-derived cytokines in tissue lesions were quantified by enzyme-linked immunosorbent assay. RESULTS: Chronic pancreatitis patients showed similar to pancreatic cancer patients and healthy individuals type 1 T-cell responses against tetanus toxoid; however, they exhibited strong IL-10-based T-cell responses against pancreatitis-associated but not pancreatic carcinoma-associated antigens. T cells from pancreatic cancer patients responded to pancreatic cancer-associated but not pancreatitis-associated antigens with IFN-gamma secretion. Pancreatitis-specific IL-10 responses were mediated by IL-10(+)IFN-gamma(-)FoxP3(+) regulatory T cells, which were expanded in the blood, bone marrow, and pancreatitis lesions and possessed the potential to suppress the proliferation of autologous conventional T cells in an antigen-specific manner. Pancreatitis lesions, in comparison with pancreatic carcinomas, contained increased concentrations of IL-10 and reduced levels of IFN-gamma, suggesting pancreatitis-specific activity of regulatory T cells in situ. CONCLUSIONS: Chronic pancreatitis is associated with disease-specific regulatory T-cell responses.

Tumor infiltrating T lymphocytes in colorectal cancer: Tumor-selective activation and cytotoxic activity in situ.

OBJECTIVE: To examine whether tumor-selective infiltration, activation, and cytotoxic activity of tumor infiltrating T lymphocytes (TIL) can be demonstrated in situ in colorectal cancer samples. SUMMARY BACKGROUND DATA: Recent studies indicated a correlation between the presence of TIL and an improved prognosis in colorectal cancer. However, tumor-selective activation and cytotoxic activity of CD8 TIL in situ in colorectal cancer patients have not yet been examined. METHODS: Tumor samples from 49 patients and corresponding normal mucosa samples from 23 patients with colorectal cancer (UICC stages II-IV) were examined for TIL. Two-color fluorescence immunohistochemistry and multicolor flowcytometric (FACS) analysis were used for quantification of CD8 T cells and measurement of their activation status (CD69-expression) and cytotoxic activity (CD107a-expression) in situ. Presence of tumor antigen-reactive T cells in tumor, blood, and bone marrow was evaluated by IFN-gamma Elispot analysis. RESULTS: While absolute numbers of CD8 T cells were similar, CD4 T helper cells were significantly increased in tumor tissue compared with normal mucosa. There was a significantly higher proportion of activated and cytotoxically active CD8 TIL in colorectal cancer compared with normal mucosa. Increased activation, cytotoxic activity, and functional reactivity of TIL were correlated with the presence of functional tumor antigen-reactive T cells in the blood and bone marrow. The proportion of activated TIL decreased significantly with higher tumor stage. CONCLUSIONS: Tumor-selective activation and cytotoxic activity of CD8 TIL and tumor-selective migration of CD4 T helper cells were demonstrated in colorectal cancer for the first time. Our data support the immunogenicity of colorectal cancer and suggest clinical significance of tumor-specific immune responses.