Contract development and manufacturing organization selection: critical considerations that can make or break your cell and gene therapy development.

BACKGROUND AIMS: With the increase in cell and gene therapy (CGT) clinical trials in recent years has come a subsequent increase in the number of contract development and manufacturing organizations (CDMOs). Successful transition from development and early-phase clinical trials to commercialization of a CGT product often depends on selecting the best-suited CDMO. However, many CGT companies are small biotech companies that lack expertise in the field or do not have experience selecting and transferring a process to a CDMO. METHODS: Given the interest in this topic, a roundtable with CGT developers and CDMO members at the 2023 annual meeting of the International Society of Cell and Gene Therapy Paris discussed these critical aspects of product development, including technical expertise, risk sharing and timing of partnerships. RESULTS AND CONCLUSIONS: Here, we'll analyze the considerations discussed by the panel and elaborate on other factors crucial for CGT development.

How Well Are Nurse Practitioners Prepared to Treat Common Musculoskeletal Conditions?

BACKGROUND: Musculoskeletal disorders affect a third of the U.S. population and are among the most prevalent, costly, and debilitating medical conditions. Basic familiarity with musculoskeletal conditions is essential for all primary care providers, including nurse practitioners (NPs). PURPOSE: The purpose of this study was: (1) to estimate the quantity of musculoskeletal education provided in NP programs; (2) to evaluate NPs' perceptions of their own confidence in providing musculoskeletal care versus other areas of primary care; and (3) to determine how well NPs score on a test of basic musculoskeletal knowledge. METHODS: An online self-reporting survey was conducted to evaluate NPs' knowledge, competence, and confidence in treating common musculoskeletal disorders. RESULTS: Most NPs receive fewer than 10 hours of musculoskeletal education, are subjectively less confident about their musculoskeletal skill set compared to other areas of primary care, and lack the basic knowledge to competently manage musculoskeletal problems in primary care. CONCLUSION: The findings of this study confirm earlier conclusions that, like our physician colleagues, the vast majority of nurse practitioners lack adequate preparation to manage common nonsurgical musculoskeletal problems.

GnRH immunization alters the expression and distribution of protein disulfide isomerases in the epididymis.

Hypogonadism is defined as the inadequate gonadal production of testosterone. Low serum testosterone leads to infertility by impairing spermatogenesis and reducing sperm count, however, the impact of hypogonadism in epididymal sperm maturation is poorly understood. From the testis, spermatozoa are transported into the epididymis where they find a specific microenvironment composed of a complex mixture of proteins that facilitate sperm storage and maturation. Inside the epididymal ductule, spermatozoa undergo several changes, resulting in their becoming capable of fertilizing eggs. Protein disulfide isomerases (PDIs) are known to participate in the folding and assembly of secreted proteins in the endoplasmic reticulum. However, little is known about the control and function of PDIs in the testis and epididymis, particularly during male development. The aim of this work was to compare the expression and distribution of PDI and PDIA3 (ERp57) in the testis and epididymis of healthy and GnRH-immunized boars. We detected higher amounts of PDIA3 and PDI in sperm preparations and fluid from the proximal regions of the epididymis of healthy boars. However, we observed an increase in PDIA3 expression in the testis and cauda epididymis in the immunocastrated group. GnRH-immunized boars showed a marked increase in PDI content in cauda spermatozoa and fluid, indicating a possible endocrine dysregulation of PDI. The results of our study suggest that PDIs are associated with epididymal sperm maturation and may be attractive candidates for monitoring male fertility.

Anisotropic strain transfer through the aortic valve and its relevance to the cellular mechanical environment.

Aortic valve interstitial cells are responsible for maintaining the valve in response to their local mechanical environment. However, the complex organization of the extracellular matrix means cell strains cannot be directly derived from gross strains, and knowledge of tissue structure-function correlations is fundamental towards understanding mechanotransduction. This study investigates strain transfer through the valve, hypothesizing that organization of the valve matrix leads to non-homogenous local strains. Radial and circumferential samples were cut from aortic valve leaflets and subjected to quasi-static mechanical characterization. Further samples were imaged using confocal microscopy, to determine local strains in the matrix. Mechanical data demonstrated that the valve was significantly stronger and stiffer when loaded circumferentially, comparable with previous studies. Micromechanical studies demonstrated that strain transfer through the matrix is anisotropic and indirect, with local strains consistently smaller than applied strains in both orientations. Under radial loading, strains were transferred linearly to cells. However, under circumferential loading, strains were only one-third of applied values, with a less direct relationship between applied and local strains. This may result from matrix reorganization, and be important for preventing cellular damage during normal valve function. These findings should be taken into account when investigating interstitial cell behaviours, such as cell metabolism and mechanotransduction.

Defining the protein-protein interactions of the mammalian endoplasmic reticulum oxidoreductases (EROs).

The ER (endoplasmic reticulum) is the site of protein folding for all eukaryotic secreted and plasma membrane proteins. Disulphide bonds are formed in many of these proteins through a dithiol-disulphide exchange chain comprising two types of protein catalysts: PDI (protein disulphide-isomerase) and ERO (ER oxidoreductase) proteins. This review will examine what we know about ERO function, and will then consider ERO interactions and their implications for mammalian oxidative protein folding.

Manipulation of oxidative protein folding and PDI redox state in mammalian cells.

In the endoplasmic reticulum (ER), disulfide bonds are simultaneously formed in nascent proteins and removed from incorrectly folded or assembled molecules. In this compartment, the redox state must be, therefore, precisely regulated. Here we show that both human Ero1-Lalpha and Ero1-Lbeta (hEROs) facilitate disulfide bond formation in immunoglobulin subunits by selectively oxidizing PDI. Disulfide bond formation is controlled by hEROs, which stand at a crucial point of an electron-flow starting from nascent secretory proteins and passing through PDI. The redox state of ERp57, another ER-resident oxidoreductase, is not affected by over-expression of Ero1-Lalpha, suggesting that parallel and specific pathways control oxidative protein folding in the ER. Mutants in the Ero1-Lalpha CXXCXXC motif act as dominant negatives by limiting immunoglobulin oxidation. PDI-dependent oxidative folding in living cells can thus be manipulated by using hERO variants.

The CXXCXXC motif determines the folding, structure and stability of human Ero1-Lalpha.

The presence of correctly formed disulfide bonds is crucial to the structure and function of proteins that are synthesized in the endoplasmic reticulum (ER). Disulfide bond formation occurs in the ER owing to the presence of several specialized catalysts and a suitable redox potential. Work in yeast has indicated that the ER resident glycoprotein Ero1p provides oxidizing equivalents to newly synthesized proteins via protein disulfide isomerase (PDI). Here we show that Ero1-Lalpha, the human homolog of Ero1p, exists as a collection of oxidized and reduced forms and covalently binds PDI. We analyzed Ero1-Lalpha cysteine mutants in the presumed active site C(391)VGCFKC(397). Our results demonstrate that this motif is important for protein folding, structural integrity, protein half-life and the stability of the Ero1-Lalpha-PDI complex.

Glycoprotein folding in the endoplasmic reticulum.

Our understanding of eukaryotic protein folding in the endoplasmic reticulum has increased enormously over the last 5 years. In this review, we summarize some of the major research themes that have captivated researchers in this field during the last years of the 20th century. We follow the path of a typical protein as it emerges from the ribosome and enters the reticular environment. While many of these events are shared between different polypeptide chains, we highlight some of the numerous differences between proteins, between cell types, and between the chaperones utilized by different ER glycoproteins. Finally, we consider the likely advances in this field as the new century unfolds and we address the prospect of a unified understanding of how protein folding, degradation, and translation are coordinated within a cell.

Managed care and critical pathway development: the joint replacement experience.

This article examines the economic, social, ethical, and political issues affecting total joint replacement patients in a managed care environment. Using general systems theory as a framework, it examines the interrelated historical events that have shaped the development of both joint replacement procedures and managed care, and discusses the extent to which these two phenomena have been mutually influential. Specifically, the article examines the initial development, implementation, and continuing evolution of clinical pathways as an easily identified and relatively discrete manifestation of managed care for the joint replacement population. While the overall impact of managed care is beyond the scope of this presentation, it is hoped that a focus on the practical application of clinical pathways to joint replacement will allow some general principles to emerge that may be useful for both patients and practitioners operating in other aspects of the managed care environment.

Allelic differences in the relationship between proteasome activity and MHC class I peptide loading.

MHC class I molecules are cell surface glycoproteins that play a pivotal role in the response to intracellular pathogens. The loading of MHC class I molecules with antigenic substrates takes place in the endoplasmic reticulum. This requires a functional TAP transporter, which translocates peptides into the endoplasmic reticulum from the cytosol. The generation of antigenic peptides from polypeptide precursors is thought to be mediated in the cytosol by the proteasome. Previously, we have demonstrated that inhibiting the proteasome with the specific covalent inhibitor lactacystin results in a direct reduction of peptide-loaded MHC class I molecules. This indicates that the proteasome is the limiting step in the MHC class I pathway. In this study we use isoelectric focusing to demonstrate that two related MHC class I alleles, HLA-A3 and HLA-A11, as well as HLA-B35 do not follow this behavior. In contrast to other class I alleles expressed by the same cells, these alleles are loaded with peptides and mature normally when proteasome activity is severely inhibited. Our observations highlight a new level of diversity in the MHC class I system and indicate that there are allele-specific differences in the linkage between proteasome activity and MHC class I peptide loading.

High performance density gradient electrophoresis of subcellular organelles, protein complexes and proteins.

A density gradient electrophoresis (DGE) apparatus (2.2 x, 14 cm) was constructed for the rapid separation of milligram quantities of proteins. By using binary buffers according to Bier (Electrophoresis 1993, 14, 1011-1018) proteins were rate-zonally separated in less than 60 min. Acidic proteins were separated in a pH 8.6, 56 microS/cm buffer, and basic proteins in a pH 5.4, 76 microS/cm buffer. Thus the A (pI 5.15) and B (pI 5.30) forms of beta-lactoglobulin as well as the sialylated glycoforms of apotransferrin were well separated at pH 8.6. The isoforms of myoglobin (pI 6.9 and 7.35, respectively), RNAse A (pI 9.45) and cytochrome c (pI 10.0) and lysozyme (pI 11) were separated at pH 5.4 within 80 min. On a 7 cm DGE column, subcellular organelles derived from HeLa cells were separated in standard electrophoresis buffer (655 microS/cm) for 90 min at 10 mA. Using a new low conductivity buffer (193 microS/cm) 20 min was sufficient to separate late endosomes, lysosomes, endoplasmic reticulum, early endosomes, plasma membrane, clathrin-coated pits, proteasomes, and clathrin-coated vesicles within a single run directly from a postnuclear supernatant.

Dynamics of proteasome distribution in living cells.

Proteasomes are proteolytic complexes involved in non-lysosomal degradation which are localized in both the cytoplasm and the nucleus. The dynamics of proteasomes in living cells is unclear, as is their targeting to proteins destined for degradation. To investigate the intracellular distribution and mobility of proteasomes in vivo, we generated a fusion protein of the proteasome subunit LMP2 and the green fluorescent protein (GFP). The LMP2-GFP chimera was quantitatively incorporated into catalytically active proteasomes. The GFP-tagged proteasomes were located within both the cytoplasm and the nucleus. Within these two compartments, proteasomes diffused rapidly, and bleaching experiments demonstrated that proteasomes were transported slowly and unidirectionally from the cytoplasm into the nucleus. During mitosis, when the nuclear envelope has disintegrated, proteasomes diffused rapidly throughout the dividing cell without encountering a selective barrier. Immediately after cell division, the restored nuclear envelope formed a new barrier for the diffusing proteasomes. Thus, proteasomes can be transported unidirectionally over the nuclear membrane, but can also enter the nucleus upon reassembly during cell division. Since proteasomes diffuse rapidly in the cytoplasm and nucleus, they may perform quality control by continuous collision with intracellular proteins, and degrading those proteins that are properly tagged or misfolded.

Practice parameters for the psychiatric assessment of infants and toddlers (0-36 months). American Academy of Child and Adolescent Psychiatry.

These practice parameters describe the psychiatric assessment of infants and toddlers (0-36 months) and support the growth of infant and toddler psychiatry, a rapidly developing field. Infants and toddlers are brought to clinical attention because of concerns about emotional, behavioral, relational, or developmental difficulties. It is axiomatic that the infant or toddler must be understood, evaluated, and treated within the context of the family. A perspective that is developmental, relational, and multidimensional and that borrows from the knowledge of multiple disciplines is essential. Collaborative efforts support the urgent need and incomparable opportunity to understand and to intervene early and preventively with young children and their families.

The proteasome-specific inhibitor lactacystin blocks presentation of cytotoxic T lymphocyte epitopes in human and murine cells.

We describe the effect of the proteasome specific inhibitor lactacystin on the metabolic stability of influenza nucleoprotein (NP) and on the generation of antigens presented by human and murine class I molecules of the major histocompatibility complex to cytotoxic T lymphocytes (CTL). We show that cells treated with lactacystin fail to present influenza antigens to influenza-specific CTL, but retain the capacity to present defined epitopes expressed as peptides intracellularly by recombinant vaccinia viruses. This block in antigen presentation can be overcome by expressing the viral protein within the lumen of the endoplasmic reticulum, confirming the specificity of lactacystin for cytosolic proteases. We also show that the effect of lactacystin on antigen presentation correlates with the block of breakdown of a rapidly degraded form of the influenza NP linked to ubiquitin. These results demonstrate that proteasome-dependent degradation plays an important role in the cytosolic generation of CTL epitopes.

Proteasome activity limits the assembly of MHC class I molecules after IFN-gamma stimulation.

For an effective CD8+ cytotoxic T cell response to occur during infection, MHC class I molecules must be loaded with antigenic peptides in the endoplasmic reticulum. The cytosolic factor responsible for peptide generation is believed to be the proteasome, with the TAP heterodimer mediating peptide transport into the endoplasmic reticulum. However, the rate-determining step(s) in this intracellular pathway of Ag presentation is currently unresolved. The availability of a specific and irreversible proteasome inhibitor called lactacystin has enabled us to determine the amount of proteasomes required for the peptide loading of MHC class I molecules in four cell types. In the absence of the IFN-gamma-inducible proteasome subunits LMP2 and LMP7, the trypsin-like (but not the chymotrypsin-like) activity of the proteasome is directly related to MHC class I peptide loading. However, IFN-gamma stimulation or assimilation of catalytic LMP2 and LMP7 subunits into proteasomes causes both chymotrypsin- and trypsin-like activities of the proteasome to become limiting for the loading of class I molecules. Our data suggest that upon full IFN-gamma stimulation, peptide supply by the proteasome is the limiting step in the assembly of MHC class I polypeptides. This mechanism may enable the cell to prevent competition between novel Ags and the pool of endogenous proteins for binding to MHC class I molecules.

High-resolution density gradient electrophoresis of proteins and subcellular organelles.

Following a concept developed by Bier et al. (Electrophoresis 1993, 14, 1011-1018), binary mixtures of amphoteric buffers with low conductivity and a good buffering capacity permit rapid rate zonal separation of proteins on a density gradient electrophoresis apparatus (7 cm, x 2.2 cm). At pH 8.66 and 250 V, beta-lactoglobulin (Mr 36600) was separated into the A and B isoforms within 44 min; human transferrin (Mr 76000-81000) was separated into its sialylated glycoforms and carbonic anhydrase (Mr 30000) separated into its isoenzymes. From these results we arrive at the term high-performance density gradient electrophoresis. Compartments belonging to the endosomal system were separated by density gradient electrophoresis. Early endosomes, recycling vesicles, intermediate endosomes, late endosomes and lysomes became well-separated after 80 min at 10 mA using [125I]transferrin and horseradish peroxidase as reporter molecules in pulse-chase regimes. Mixtures of Bier buffers and standard electrophoresis media permitted very short separation times (19 min at 10 mA) for the endosomal compartments. Concommittantly, endoplasmic reticulum and proteasomes were well resolved.

T and B cell responsiveness to donor class I MHC molecules and peptides in long survivors with kidney allografts.

LEW rats with long-surviving (> 100 days) (DA x LEW)F1 kidney allografts were generated by treating the recipients with cyclosporine for 14 days after grafting. All rats were monitored after transplantation for the development of antibodies to intact donor class I MHC molecules. Cyclosporine completely suppressed the early antibody response to intact DA class I MHC molecules in all 19 LEW rats. However, 17 of the 19 rats developed antibodies between four and six weeks after grafting-i.e., between two and four weeks after the cessation of cyclosporine therapy, and maintained high levels of antibody to the donor class I molecules in spite of the long-term presence of the allograft. The 2 rats that did not produce antibodies to donor class I MHC molecules, along with one of the 17 that did produce antibodies, were immunized with a synthetic peptide corresponding to a region of the DA class I MHC molecule known to be recognized by LEW CD4+ T cells via the indirect recognition pathway. All 3 long survivors developed self APC-dependent CD4+ T cell proliferation to the immunizing donor peptides, and strong antibody responses to these peptides. However, none of these long survivors suffered rejection episodes as a consequence of the peptide immunization. In one of the two long-surviving rats without antibodies to intact donor class I MHC molecules at the time of peptide priming, the peptide priming resulted in the prompt development of strong antibodies to intact donor class I molecules. However, the other of these 2 rats did not produce such antibodies after peptide priming. Thus in this model of kidney allograft tolerance, with long-term exposure of the recipient's immune system to donor antigens without evidence of rejection, none of the animals develops tolerance for the indirect T cell recognition of donor class I MHC antigens. In occasional animals, B cells potentially reactive to intact donor class I molecules are present and are adequately exposed to antigen but are quiescent because of the absence of T cell help, perhaps as a consequence of reversible T cell suppression or anergy. In other occasional animals, B cell nonreactivity (anergy or tolerance) to intact donor class I molecules appears to develop.

A three-cell cluster hypothesis for noncognate T-B collaboration via direct T cell recognition of allogeneic dendritic cells.

In this article, we propose that T cell help for B cells can occur via an unusual three-cell cluster, with recipient CD4+ T helper cells interacting via direct allorecognition with donor dendritic cell class II MHC antigens, recipient B cells interacting with MHC class I (or any other) antigen on the donor dendritic cell surface, and noncognate (i.e., antigen nonspecific) T-B collaboration. In this noncognate pathway, antigen processing by B cells is not required and T cell help is potent because of the high precursor T cell frequency for direct recognition of allogeneic class II MHC molecules. The data supporting this hypothesis are: 1. LEW rat strain recipients of interstitial dendritic cell-free (DAxLEW)F1 kidney allografts were shown to have no detectable antibody to donor class I MHC antigens at day 7 after grafting. By contrast, LEW recipients of normal (DAxLEW)F1 kidneys had strong antibody responses. 2. Consistent wih important role for donor dendritic cells in the early antibody response to donor class I MHC antigens was the finding that it was dependent on donor class II MHC antigens. PVG recipients, previously immunized with pure DA RT1.B class II MHC antigens, had virtually no antibody response to the class I MHC antigens of DA kidney allografts. 3. We confirmed the low and high responder status of PVG and LEW rats, respectively, to DA class I antigens by studying antibody responses to pure DA class I antigens. However, PVG and LEW recipients of DA kidney allografts did not differ in their antibody response to the donor DA class I MHC antigens. This is consistent with this response not requiring the processing and presentation of DA class I antigen by PVG recipients. 4. LEW recipients of interstitial dendritic cell-free (DAxLEW)F1 kidney allografts did eventually develop a strong antibody response to DA class I antigens, but this was delayed by several weeks. That this delayed antibody response was probably mediated by conventional T-B collaboration and that T help was rate limiting in this situation, was demonstrated by immunizing LEW recipients with a DA class I peptide. This markedly accelerated the kinetics of the antibody response to the dendritic cell-free (DAxLEW)F1 kidneys.