Treatment consistent with idiopathic multicentric Castleman disease guidelines is associated with improved outcomes.

Idiopathic multicentric Castleman disease (iMCD) is a rare hematologic disorder with an unknown etiology. Clinical presentation is heterogeneous, ranging from mild constitutional symptoms with lymphadenopathy to life-threatening multiorgan dysfunction. International, consensus treatment guidelines developed in 2018 relied upon a limited number of clinical trials and small case series; however, to our knowledge, real-world performance of these recommendations has not been subsequently studied. Siltuximab, a monoclonal antibody against interleukin 6 (IL6), is approved for the treatment of iMCD and recommended first-line, and tocilizumab, a monoclonal antibody directed against the IL6 receptor, is recommended when siltuximab is unavailable. Chemotherapy, rituximab, and immunomodulators are recommended as second- and third-line treatments based on limited evidence. Corticosteroid monotherapy is used by clinicians, although not recommended. Here, we draw upon the ACCELERATE Natural History Registry to inventory regimens and evaluate regimen response for 102 expert-confirmed iMCD cases. Siltuximab with/without (w/wo) corticosteroids was associated with a 52% response, whereas corticosteroid monotherapy was associated with a 3% response. Anti-IL6-directed therapy with siltuximab or tocilizumab demonstrated better response and more durability than was observed with rituximab w/wo corticosteroids. Cytotoxic chemotherapy was associated with a 52% response and was predominantly administered in patients characterized by thrombocytopenia, anasarca, fever, renal failure/reticulin fibrosis, and organomegaly. Our results provide evidence in support of current recommendations to administer anti-IL6 as first-line treatment, to administer cytotoxic chemotherapy in patients with severe refractory disease, and to limit corticosteroid monotherapy. Evidence remains limited for effective agents for patients who are refractory to anti-IL6-directed therapy. This trial was registered at www.clinicaltrials.gov as #NCT02817997.

A novel cryopreservation and biobanking strategy to study lymphoid tissue stromal cells in human disease.

Nonhematopoietic lymph node stromal cells (LNSCs) regulate lymphocyte trafficking, survival, and function for key roles in host defense, autoimmunity, alloimmunity, and lymphoproliferative disorders. However, the study of LNSCs in human diseases is complicated by a dependence on viable lymphoid tissues, which are most often excised prior to establishment of a specific diagnosis. Here, we demonstrate that cryopreservation can be used to bank lymphoid tissue for the study of LNSCs in human disease. Using human tonsils and lymph nodes (LN), lymphoid tissue fragments were cryopreserved for subsequent enzymatic digestion and recovery of viable nonhematopoietic cells. Flow cytometry and single-cell transcriptomics identified comparable proportions of LN stromal cell types in fresh and cryopreserved tissue. Moreover, cryopreservation had little effect on transcriptional profiles, which showed significant overlap between tonsils and LN. The presence and spatial distribution of transcriptionally defined cell types were confirmed by in situ analyses. Our broadly applicable approach promises to greatly enable research into the roles of LNSCs in human disease.

A novel cryopreservation and biobanking strategy to study lymphoid tissue stromal cells in human disease.

Non-hematopoietic lymph node stromal cells (LNSCs) regulate lymphocyte trafficking, survival, and function for key roles in host defense, autoimmunity, alloimmunity, and lymphoproliferative disorders. However, study of LNSCs in human diseases is complicated by a dependence on viable lymphoid tissues, which are most often excised prior to establishment of a specific diagnosis. Here, we demonstrate that cryopreservation can be used to bank lymphoid tissue for the study of LNSCs in human disease. Using human tonsils, lymphoid tissue fragments were cryopreserved for subsequent enzymatic digestion and recovery of viable non-hematopoietic cells. Flow cytometry and single-cell transcriptomics identified comparable proportions of LNSC cell types in fresh and cryopreserved tissue. Moreover, cryopreservation had little effect on transcriptional profiles, which showed significant overlap between tonsils and lymph nodes. The presence and spatial distribution of transcriptionally defined cell types was confirmed by in situ analyses. Our broadly applicable approach promises to greatly enable research into the roles of LNSC in human disease.

Characterization of the Serpentine Adeno-Associated Virus (SAAV) Capsid Structure: Receptor Interactions and Antigenicity.

Adeno-associated viruses (AAVs) are being developed as clinical gene therapy vectors. One issue undermining their broad use in the clinical setting is the high prevalence of circulating antibodies in the general population capable of neutralizing AAV vectors. Hence, there is a need for AAV vectors that can evade the preexisting immune response. One possible source of human naive vectors are AAVs that do not disseminate in the primate population, and one such example is serpentine AAV (SAAV). This study characterizes the structural and biophysical properties of the SAAV capsid and its receptor interactions and antigenicity. Single particle cryo-electron microscopy (cryo-EM) and thermal stability studies were conducted to characterize the SAAV capsid structure at pH 7.4, 6.0, 5.5, and 4.0, conditions experienced during cellular trafficking. Cell binding assays using Chinese hamster ovary (CHO) cell lines identified terminal sialic acid as the primary attachment receptor for SAAV similar to AAV1, 4, 5, and 6. The binding site of sialic acid to the SAAV capsid was mapped near the 2-fold axis toward the 2/5-fold wall, in a different location than AAV1, 4, 5, and 6. Towards determining the SAAV capsid antigenicity native immunodot blots showed that SAAV evades AAV serotype-specific mouse monoclonal antibodies. However, despite its reptilian origin, it was recognized by ~25% of 50 human sera tested, likely due to the presence of cross-reactive antibodies. These findings will inform future gene delivery applications using SAAV-based vectors and further aid the structural characterization and annotation of the repertoire of available AAV capsids. IMPORTANCE AAVs are widely studied therapeutic gene delivery vectors. However, preexisting antibodies and their detrimental effect on therapeutic efficacy are a primary challenge encountered during clinical trials. In order to circumvent preexisting neutralizing antibodies targeting mammalian AAV capsids, serpentine AAV (SAAV) was evaluated as a potential alternative to existing mammalian therapeutic vectors. The SAAV capsid was found to be thermostable at a wide range of environmental pH conditions, and its structure showed conservation of the core capsid topology but displays high structural variability on the surface. At the same time, it binds to a common receptor, sialic acid, that is also utilized by other AAVs already being utilized in gene therapy trials. Contrary to the initial hypothesis, SAAV capsids were recognized by one in four human sera tested, pointing to conserved amino acids around the 5-fold region as epitopes for cross-reacting antibodies.

Adeno-Associated Virus (AAV) Capsid Stability and Liposome Remodeling During Endo/Lysosomal pH Trafficking.

Adeno-associated viruses (AAVs) are small, non-pathogenic ssDNA viruses being used as therapeutic gene delivery vectors for the treatment of a variety of monogenic diseases. An obstacle to successful gene delivery is inefficient capsid trafficking through the endo/lysosomal pathway. This study aimed to characterize the AAV capsid stability and dynamics associated with this process for a select number of AAV serotypes, AAV1, AAV2, AAV5, and AAV8, at pHs representative of the early and late endosome, and the lysosome (6.0, 5.5, and 4.0, respectively). All AAV serotypes displayed thermal melt temperatures that varied with pH. The stability of AAV1, AAV2, and AAV8 increased in response to acidic conditions and then decreased at pH 4.0. In contrast, AAV5 demonstrated a consistent decrease in thermostability in response to acidification. Negative-stain EM visualization of liposomes in the presence of capsids at pH 5.5 or when heat shocked showed induced remodeling consistent with the externalization of the PLA2 domain of VP1u. These observations provide clues to the AAV capsid dynamics that facilitate successful infection. Finally, transduction assays revealed a pH and temperature dependence with low acidity and temperatures > 4 °C as detrimental factors.

The influence of sex and estrous cyclicity on cocaine and remifentanil demand in rats.

The application of behavioral economic demand theory in addiction science has proved useful for evaluating individual characteristics underlying abuse liability. Two factors that have received comparably little attention within this literature are sex and gonadal hormones. We determined cocaine and remifentanil demand in male and female rats using a within-session procedure. Cocaine and remifentanil demand were evaluated for 15 consecutive days using a balanced, crossover design that randomized drug order. This design allowed for the evaluation of temporal and exposure effects on two independent dimensions of demand, unconstrained demand (Q0 ) and demand elasticity (α). Estrous cyclicity was tracked to determine the contribution of phase to demand. No overall sex differences were observed. Increased unconstrained demand for cocaine and remifentanil was observed in females during periods in which estrogen was high (eg, estrus phase). Unconstrained remifentanil demand escalated over the 15-day testing period, but escalation was not observed for cocaine or for demand elasticity. A significant exposure effect was also observed in which greater prior remifentanil intake increased unconstrained cocaine demand and reduced cocaine demand elasticity. These effects were directionally specific as no significant effects of prior cocaine exposure were observed on remifentanil demand measures. These data suggest that unconstrained demand and demand elasticity do not differ between male and female subjects; however, that unconstrained demand is associated with estrous cyclicity. These findings also suggest that opioid exposure enhances subsequent demand for psychomotor stimulants, which may be important when considering recent increases in nonmedical prescription opioid use in the United States.

Adolescent d-amphetamine exposure enhances the acquisition of cocaine self-administration in male and female rats.

Diagnosis and treatment of attention-deficit/hyperactivity disorder (ADHD) has risen drastically over the past 20 years in the United States and abroad. Amphetamine-based prescription stimulants are the most prescribed treatment for ADHD and the diversion of these drugs has also increased. Reports indicate 61% of individuals with an ADHD medication prescription have sold or shared their medication. Exposure to prescription stimulants, especially for those without an ADHD diagnosis, may increase susceptibility to drugs of abuse. The present study aimed to model ADHD medication misuse during adolescence in male and female rats. The primary dependent measure was the acquisition of intravenous cocaine self-administration. Male and female, Long-Evans rats were exposed to d-amphetamine (0.7 mg/kg, i.p.) or saline in adolescence (35-41 days old), during which locomotor activity was measured. At approximately 75 days old, animals were implanted with jugular catheters. All animals then entered a 15-day acquisition procedure with no prior operant training. Finally, following acquisition all animals responded on a progressive-ratio (PR) schedule to obtain 0, 0.1, 0.3, and 1.0 mg/kg/infusion cocaine. Animals exposed to amphetamine acquired cocaine self-administration faster than saline-exposed controls when the acquisition criterion was operationally defined as two consecutive days with 12 infusions or greater. Discrete-time hazard modeling also found amphetamine exposure to increase the likelihood of acquiring cocaine self-administration. There were no differences detected during PR testing. These data suggest that individuals with histories of prescription stimulant misuse may be at increased risk to use other drugs of abuse. (PsycINFO Database Record

Thermal Stability as a Determinant of AAV Serotype Identity.

Currently, there are over 150 ongoing clinical trials utilizing adeno-associated viruses (AAVs) to target various genetic diseases, including hemophilia (AAV2 and AAV8), congenital heart failure (AAV1 and AAV6), cystic fibrosis (AAV2), rheumatoid arthritis (AAV2), and Batten disease (AAVrh.10). Prior to patient administration, AAV vectors must have their serotype, concentration, purity, and stability confirmed. Here, we report the application of differential scanning fluorimetry (DSF) as a good manufacturing practice (GMP) capable of determining the melting temperature (Tm) for AAV serotype identification. This is a simple, rapid, cost effective, and robust method utilizing small amounts of purified AAV capsids (∼25 µL of ∼1011 particles). AAV1-9 and AAVrh.10 exhibit specific Tms in buffer formulations commonly used in clinical trials. Notably, AAV2 and AAV3, which are the least stable, have varied Tms, whereas AAV5, the most stable, has a narrow Tm range in the different buffers, respectively. Vector stability was dictated by VP3 only, specifically, the ratio of basic/acidic amino acids, and was independent of VP1 and VP2 content or the genome packaged. Furthermore, stability of recombinant AAVs differing by a single basic or acidic amino acid residue are distinguishable. Hence, AAV DSF profiles can serve as a robust method for serotype identification of clinical vectors.