Martin K. Oaks

Differentiation of the expression of aldosterone synthase and 11β-hydroxylase mRNA in the rat adrenal cortex by reverse transcriptase-polymerase chain reaction

The adrenocortical enzymes of the steroidogenic late pathway in the rat are aldosterone synthase (P450aldo), which catalyzes the production of aldosterone, and 11 beta-hydroxylase (P45011 beta), which catalyzes the production of corticosterone throughout the cortex. These two enzymes are highly homologous and are encoded by the genes CYP11B2 and CYP11B1, respectively. The purpose of the present study is to describe the development of two sets of primers and the reverse transcription-polymerase chain reaction (RT-PCR) conditions that are capable of discriminating between rat P450aldo and P45011 beta mRNAs. The P450aldo primer set did not amplify full length cDNA P45011 beta plasmid and the P45011 beta primer set did not amplify full length cDNA P450aldo plasmid indicating minimal crosstalk. The fidelity of the PCR primers and method was further established by sequencing the PCR products and demonstration of virtual identity with the published sequences of P450aldo and P45011 beta. RT-PCR of mRNA from adrenal capsules (zona glomerulosa) and subcapsules (zona reticularis/fasciculata) from rats demonstrated no effect of sodium diet on the expression of P45011 beta mRNA but an approximately 8-fold greater expresison in P450aldo mRNA on low vs high sodium intake. Similar results were found when single hemicapsules were subjected to RT-PCR, demonstrating the sensitivity of the method. We conclude that the two sets of PCR primers and the RT-PCR method described are capable of evaluating the expression of the highly homologous mRNAs for P450aldo and P45011 beta with great precision and sensitivity.

Many de novo donor-specific antibodies recognize β2 -microglobulin-free, but not intact HLA heterodimers.

Solid‐phase single antigen bead (SAB) assays are standard of care for detection and identification of donor‐specific antibody (DSA) in patients who receive solid organ transplantation (SOT). While several studies have documented the reproducibility and sensitivity of SAB testing for DSA, there are little data available concerning its specificity. This study describes the identification of antibodies to β2‐microglobulin‐free human leukocyte antigen (β2‐m‐fHLA) heavy chains on SAB arrays and provides a reassessment of the clinical relevance of DSA testing by this platform. Post‐transplant sera from 55 patients who were positive for de novo donor‐specific antibodies on a SAB solid‐phase immunoassay were tested under denaturing conditions in order to identify antibodies reactive with β2‐m‐fHLA or native HLA (nHLA). Antibodies to β2‐m‐fHLA were present in nearly half of patients being monitored in the post‐transplant period. The frequency of antibodies to β2‐m‐fHLA was similar among DSA and HLA antigens that were irrelevant to the transplant (non‐DSA). Among the seven patients with clinical or pathologic antibody‐mediated rejection (AMR), none had antibodies to β2‐m‐fHLA exclusively; thus, the clinical relevance of β2‐m‐fHLA is unclear. Our data suggests that SAB testing produces false positive reactions due to the presence of β2‐m‐fHLA and these can lead to inappropriate assignment of unacceptable antigens during transplant listing and possibly inaccurate identification of DSA in the post‐transplant period.

Growth Hormone Therapy During Neonatal Hypoxia in Rats Body Composition, Bone Mineral Density, and Insulin-like Growth Factor-1 Expression

Hypoxia from birth results in a decrease in body weight gain, body size, and bone mineral density (BMD). The purpose of the present study was to determine whether short-term administration of growth hormone (GH) (rat GH; 100 µg/d) could attenuate some of these effects of neonatal hypoxia. Rat pups (with their lactating dams) were exposed to hypoxia (vs normoxic control) from birth. Hypoxia was continued until 14 d of age, with rat GH (vs vehicle control) administered daily. Hypoxia significantly inhibited body weight gain; GH therapy did not reverse this effect. GH therapy did reverse the inhibitory effect of hypoxia on tail length but not on body length. Hypoxia decreased BMD analyzed by dual X-ray absorptiometry (DXA); this effect was not reversed by GH therapy. Both GH therapy and hypoxia decreased the percentage of body fat analyzed by DXA, the effects of which were additive when combined. There were minimal effects of hypoxia and GH therapy on plasma insulin-like growth factor-1 (IGF-1), IGF-binding protein-3, and hepatic IGF-1 mRNA expression. We conclude that some of the effects of hypoxia on body habitus are reversed by GH therapy, but that short-term GH therapy did not prevent a loss of BMD. GH therapy for more than 14 days may be necessary to appreciate fully its potential in the treatment of the sequelae of neonatal hypoxia.

Xenoreactive antibodies and latent fibrin formation in VAD and cardiac transplant recipients can confound the detection and measurement of anti-AT1R antibodies

Autoantibodies to the angiotensin II type 1 receptor (AT1R) are thought to be important in antibody‐mediated rejection (AMR), especially in the absence of anti‐HLA antibodies. We used a variety of methods to examine the specificity of a commercially available kit designed to quantitate anti‐AT1R antibodies. We found that fibrin formation in serum samples from patients awaiting cardiac transplantation with ventricular assist devices (VADs) can produce falsely elevated anti‐AT1R values. In addition, absorption studies with a variety of cell lines with or without expression of human AT1R, and those that express xenoantigens, suggest that many of the antibodies detected in the AT1R test system are heterophilic and have reactivity to xenoantigens. Furthermore, we provide data that show that reactivity to the sialic acid Neu5Gc is a common finding among samples that are highest in anti‐AT1R levels. We conclude that a common laboratory method for quantitation of anti‐AT1R antibodies is nonspecific and overestimates the frequency of true positives. A reevaluation of the role that anti‐AT1R antibodies play in allograft function and patient outcomes is warranted.

Multiple Myeloma Vaccination Patterns in a Large Health System: A Pilot Study

Purpose Common reasons for hospitalization and death in patients with multiple myeloma (MM) are infections. As patients with MM are living longer and are treated with immunomodulatory drugs, there is a need to immunize against vaccine-preventable diseases and ultimately determine the efficacy of these vaccines. We evaluated vaccination practice patterns in MM patients at our health system using electronic medical records and data analytics. Methods This institutional review board-approved study retrospectively reviewed patients with MM who visited the health system from May 2012 to May 2014. Data collected included demographics, influenza vaccination (FV) and pneumonia vaccination (PV) history, hospitalization episodes and associated costs, and duration of survival. Patients were considered PV-positive if vaccinated within 5 years prior to study. FV was defined as optimal (two FV in 2012-2014), suboptimal (one FV in 2012-2014) or none (in 2012-2014). Results Of 411 MM patients, 55% were male and 85% Caucasian. Nearly 58% received PV in the past 5 years. FV was 15% optimal, 52% suboptimal and 33% none. A total of 444 hospitalizations involving 204 patients were observed over 2-year follow-up. More than

Multiple Myeloma Baseline Immunoglobulin G Level and Pneumococcal Vaccination Antibody Response

23 million was incurred from hospitalizations in the 2-year study period. There was no statistically significant difference in all-cause hospitalization and overall survival by FV and PV status. Conclusions Despite recommendations of vaccination in multiple myeloma, our cohort had low rates of influenza and pneumonia vaccination. FV and PV status did not show any significant association with additional hospitalization or overall survival in this pilot study. Future prospective studies are needed to ascertain the immunological and clinical efficacy and effectiveness of these vaccines in immunosuppressed patients.

Adrenocortical responses to ACTH in neonatal rats: effect of hypoxia from birth on corticosterone, StAR, and PBR

Infections are a major cause of morbidity and mortality in multiple myeloma (MM), a cancer of the immune system. Vaccination clinical efficacy endpoints have not been demonstrated, and there are limited data on surrogate markers of efficacy. This pilot study evaluated sequential immunologic markers after standard pneumococcal vaccination (PV) in patients with MM and non-MM controls. Vaccination was standard for PV (PCV13 or PPV23), with laboratory testing at baseline and at 2, 4, 12 and 24 weeks after vaccination. Immunoglobulin G (IgG) antibodies to pneumococcal antigens were detected by ELISA. Prevaccination total IgG levels and IgG subclass levels were also measured by ELISA. Four of 6 controls responded with at least a 2-fold increase in antibody concentration; only 2 controls had a sustained increase in concentration. Six of 8 patients with MM had at least a 2-fold antibody increase; however, only 2 of these patients showed a sustained increase of antipneumococcal antibody. Response rate differences were not statistically significant in this small pilot, and there was no relationship between responsiveness to PV and initial serum total IgG levels or IgG subclasses at study entry. Future prospective studies are needed to ascertain the immunological and clinical efficacy and effectiveness of various vaccines and vaccination strategies in MM.