Defective T suppressor-inducer cell function in human immune deficiency virus-seropositive hemophilia patients

In human immune deficiency virus (HIV)-seropositive hemophilia patients, a low number of CD4 + lymphocytes is found, as well as a low CD4+/CD8+ ratio. In previous studies, it has been shown that antigen- specific T-helper cell (CD4+) function was present and no excessive antigen-specific T-suppressor cell (CD8+) function could be demonstrated. In this report, we studied another activity of CD4+ cells, namely the capacity to induce T-suppressor cell activity. The results clearly show a selective dysfunction of CD4+ suppressor-inducer (Tsi) cell function. Since these HIV-seropositive hemophilia patients showed the presence of activated B cells in the peripheral circulation refractory to antigen-specific T-helper cell signals and secreting specific antibodies spontaneously, we raised the hypothesis that the activated B cells in the patients activate the Tsi cells in vivo. This constant activation leads to a functional exhaustion of the Tsi cell pool.     

G970R‐CFTR Mutation (c.2908G>C) Results Predominantly in a Splicing Defect

In previous work, participants with a G970R mutation in cystic fibrosis transmembrane conductance regulator (CFTR) (c.2908G>C) had numerically lower sweat chloride responses during ivacaftor treatment than participants with other CFTR gating mutations. The objective of this substudy was to characterize the molecular defect of the G970R mutation in vitro and assess the benefit of ivacaftor in participants with this mutation. This substudy assessed sweat chloride, spirometry findings, and nasal potential difference on and off ivacaftor treatment in three participants with a G970R/F508del genotype. Intestinal organoids derived from rectal biopsy specimens were used to assess ivacaftor response ex vivo and conduct messenger RNA splice and protein analyses. No consistent or meaningful trends were observed between on‐treatment and off‐treatment clinical assessments. Organoids did not respond to ivacaftor in forskolin‐induced swelling assays; no mature CFTR protein was detected in Western blots. Organoid RNA analysis demonstrated that 3 novel splice variants were created by G970R‐CFTR: exon 17 truncation, exons 13–15 and 17 skipping, and intron 17 retention. Functional and molecular analyses indicated that the c.2908G>C mutation caused a cryptic splicing defect. Organoids lacked an ex vivo response with ivacaftor and supported identification of the mechanism underlying the CFTR defect caused by c.2908G>C. Analysis of CFTR mutations indicated that cryptic splicing was a rare cause of mutation misclassification in engineered cell lines. This substudy used organoids as an alternative in vitro model for mutations, such as cryptic splice mutations that cannot be fully assessed using cDNA expressed in recombinant cell systems.

Study Highlights

• WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?

☑ Participants with the G970R mutation in the CFTR gene have lower sweat chloride responses to ivacaftor treatment than do participants with G551D or other non–G551D‐CFTR gating mutations.

• WHAT QUESTION DID THIS STUDY ADDRESS?

☑ This substudy characterized the molecular defect of the G970R‐CFTR mutation in vitro and assessed the benefit of ivacaftor in participants with this mutation.

• WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

☑ The functional and molecular analyses revealed that the G970R‐CFTR mutation is not responsive to ivacaftor treatment and identified the mechanism of the CFTR defect as a cryptic splicing defect.

• HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

☑ For mutations that cannot be fully assessed using recombinant cell systems, such as cryptic splicing defects, organoid in vitro assessments and RNA analyses can be used as alternatives to characterize mutations.

Cystic fibrosis (CF) is an autosomal recessive hereditary disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that result in decreased quantity and/or function of the CFTR protein. ¹ , ² The CFTR protein is an epithelial chloride channel aiding in the regulation of salt and fluid absorption and secretion that is located in the epithelia of multiple organs, including the lungs, pancreas, intestinal tract, liver, and vas deferens. ³ , ⁴ , ⁵ , ⁶ CF is a multisystemic disease with clinical manifestations, including lung function decline, chronic airway infections, pancreatic insufficiency, and malnutrition. ⁷ Ivacaftor (Kalydeco^(R); Vertex Pharmaceuticals, Boston, MA) is a CFTR modulator that increases chloride transport by potentiating the channel open probability (P₀ (or gating)) of the CFTR protein at the epithelial cell surface. ⁸ Currently, ivacaftor is indicated in the United States for people aged ≥ 4 months with CF with ≥ 1 CFTR mutation that is responsive to ivacaftor based on clinical and/or in vitro assay data ⁹ ; the approved genotypes and ages vary in other regions. ¹⁰ , ¹¹ , ¹² In clinical studies of people with CF with eligible genotypes, ivacaftor treatment has been shown to achieve improvements in CFTR function, lung function, risk of pulmonary exacerbations, pancreatic function, and nutritional status. ¹³ , ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ Among > 340 known CF‐causing CFTR mutations identified, 10 have historically been classified as class III mutations that result in severe defects in CFTR channel gating: G551D, G178R, S549N, S549R, G551S, G970R, G1244E, S1251N, S1255P, and G1349D. ² , ¹⁸ , ¹⁹ , ²⁰ , ²¹ In Fischer rat thyroid (FRT) cells engineered to individually express complementary DNAs (cDNAs) that express CFTR proteins coding each of these mutations, chloride transport as determined by electrophysiological studies was < 5% of normal CFTR, despite the presence of mature CFTR protein at levels similar to those of normal CFTR. Ivacaftor increased the channel P₀ of these 10 CFTR forms and caused an increase in CFTR‐mediated chloride transport to levels equivalent to > 10% above baseline. These in vitro data supported investigation of the potential clinical benefit with ivacaftor in people with CF with severe gating mutations. ⁸ In a clinical study of ivacaftor in participants with CF who had a non‐G551D severe gating mutation, participants with a G970R‐CFTR mutation had a numerically lower treatment response than participants with the other gating mutations. ¹⁴ Given this result, the molecular defect of the G970R mutation was further evaluated as part of the completed open‐label extension study KONTINUE. ¹⁵ Organoids, a cell culture technology platform, ²² enabled generation of an in vitro model from each participant that expresses the CFTR protein based on the genomic DNA rather than on a cDNA construct. These organoids were used for in vitro functional CFTR experiments to evaluate the response with ivacaftor and for further mechanistic studies to help clarify the range of functional defects caused by the G970R mutation.     

Bile salt metabolism is not the only factor contributing to Clostridioides (Clostridium) difficile disease severity in the murine model of disease

ABSTRACT

Susceptibility of patients to antibiotic-associated C. difficile disease is intimately associated with specific changes to gut microbiome composition. In particular, loss of microbes that modify bile salt acids (BSA) play a central role; primary bile acids stimulate spore germination whilst secondary bile acids limit C. difficile vegetative growth. To determine the relative contribution of bile salt (BS) metabolism on C. difficile disease severity, we treated mice with three combinations of antibiotics prior to infection. Mice given clindamycin alone became colonized but displayed no tissue pathology while severe disease, exemplified by weight loss and inflammatory tissue damage occurred in animals given a combination of five antibiotics and clindamycin. Animals given only the five antibiotic cocktails showed only transient colonization and no disease. C. difficile colonization was associated with a reduction in bacterial diversity, an inability to amplify bile salt hydrolase (BSH) sequences from fecal DNA and a relative increase in primary bile acids (pBA) in cecal lavages from infected mice. Further, the link between BSA modification and the microbiome was confirmed by the isolation of strains of Lactobacillus murinus that modified primary bile acids in vitro, thus preventing C. difficile germination. Interestingly, BSH activity did not correlate with disease severity which appeared linked to alternations in mucin, which may indirectly lead to increased exposure of the epithelial surface to inflammatory signals. These data confirm the role of microbial metabolic activity in protection of the gut and highlights the need for greater understanding the function of bacterial communities in disease prevention.     

Tamoxifen. A reappraisal of its pharmacodynamic and pharmacokinetic properties, and therapeutic use

Tamoxifen, a non-steroidal antioestrogen, represents a significant advance in treatment of female breast cancer. In trials of tamoxifen as postsurgical adjuvant treatment of early breast cancer, disease-free survival is consistently prolonged, representing an enhanced quality of life in association with tamoxifen's favourable adverse effect profile. Moreover, overview analysis indicates a survival benefit of approximately 20% at 5 years for all women, most clearly evident in women over 50 years, while a survival benefit independent of menopausal, nodal or oestrogen receptor status has been demonstrated in some individual trials. Thus, for postmenopausal women, tamoxifen is clearly optimal adjuvant treatment, although the relative benefit of adjuvant chemotherapy in node-negative patients requires clarification. A survival benefit for women under 50 has not been clearly demonstrated in overview analysis, but is not precluded by these rather limited data, and adjuvant treatment of premenopausal women with tamoxifen may also warrant serious consideration. Response rates to tamoxifen in advanced breast cancer are around 30 to 35%, increasing with patient selection for oestrogen receptor positivity. Tamoxifen must be regarded as first-line endocrine treatment in postmenopausal women, and may represent an alternative to first-line ovarian ablation in premenopausal women. An emergent role in primary therapy of elderly and frail patients with operable disease is apparent. Tamoxifen is also of benefit following surgery in male breast cancer, and may have a role as first-line endocrine treatment. Tamoxifen also has a potential role in other hormone-sensitive malignancies such as pancreatic carcinoma, and in treatment of benign breast disease. Finally, tamoxifen has a place in treatment of male and female infertility. because of adverse effects is rarely necessary. The most frequent adverse effects are related to the drug's anti-oestrogenic activity, and include hot flushes, nausea and/or vomiting, vaginal bleeding or discharge, and menstrual disturbances in premenopausal patients. Thus, tamoxifen continues to play a major role in management of female breast cancer in both early and advanced stages of disease, with a place also in treatment of male breast cancer and of infertility.     

Plasma ionized calcium monitoring during liver transplantation

Plasma ionized calcium and citrate concentrations were measured in 11 patients undergoing liver transplantation. During the anhepatic phase of the procedure, ionized calcium concentrations fell to as low as 40% of normal, in spite of calcium supplementation. Simultaneously, citrate concentrations rose to between 20 and 100 times preoperative levels. In two patients low plasma ionized calcium concentrations were associated with hypotension that responded to calcium infusion. Intraoperative monitoring of plasma ionized calcium during liver transplantation is helpful in the rational control of the patient's calcium status.     

Interleukin-5 is at 5q31 and is deleted in the 5q- syndrome

Human interleukin-5 (IL-5) is a selective eosinophilopoietic and eosinophil-activating growth hormone. By in situ hybridization this gene is mapped to chromosome 5q23.3 to 5q32. It is shown to be deleted in two patients with the 5q-syndrome and in one patient previously diagnosed with myelodysplasia whose condition had progressed to acute myeloblastic leukemia. The clustering of other genes involved in hematopoiesis (IL-3, granulocyte-macrophage colony-stimulating factor, feline sarcoma viral oncogene homolog, colony-stimulating factor 1) to the same region as IL-5 suggests a nonrandom localization and raises interesting questions concerning the evolution and regulation of these genes.     

Intraosseous lipoma of the ilium. A case report

Intraosseous lipoma is the rarest of the benign primary tumors of bone. It occurs most often in the metaphysis of long bones and is usually identified as an incidental roentgenographic finding. A 53-year-old man demonstrated a mass in his buttock and a lytic lesion of his ilium. Preoperative computed axial tomography (CAT) scanning was helpful in establishing its anatomic boundaries and the cellular composition of the mass. The CAT scan demonstrated the tumor extending anteriorly and posteriorly through the ilium and having a uniform soft tissue density with the same attenuation as adipose tissue. The patient was successfully treated with a marginal excision of the tumor. An intraosseous lipoma seems not to have been previously reported to involve the ilium or be associated with significant extension into soft tissue.