Single-center randomized trial of T-reg graft alone vs T-reg graft plus tacrolimus for the prevention of acute GVHD.

ABSTRACT: Allogeneic hematopoietic cell transplantation (HCT) is a curative therapy for hematological malignancies for which graft-versus-host disease (GVHD) remains a major complication. The use of donor T-regulatory cells (Tregs) to prevent GVHD appears promising, including in our previous evaluation of an engineered graft product (T-reg graft) consisting of the timed, sequential infusion of CD34+ hematopoietic stem cells and high-purity Tregs followed by conventional T cells. However, whether immunosuppressive prophylaxis can be removed from this protocol remains unclear. We report the results of the first stage of an open-label single-center phase 2 study (NCT01660607) investigating T-reg graft in myeloablative HCT of HLA-matched and 9/10-matched recipients. Twenty-four patients were randomized to receive T-reg graft alone (n = 12) or T-reg graft plus single-agent GVHD prophylaxis (n = 12) to determine whether T-reg graft alone was noninferior in preventing acute GVHD. All patients developed full-donor myeloid chimerism. Patients with T-reg graft alone vs with prophylaxis had incidences of grade 3 to 4 acute GVHD of 58% vs 8% (P = .005) and grade 3 to 4 of 17% vs 0% (P = .149), respectively. The incidence of moderate-to-severe chronic GVHD was 28% in the T-reg graft alone arm vs 0% with prophylaxis (P = .056). Among patients with T-reg graft and prophylaxis, CD4+ T-cell-to-Treg ratios were reduced after transplantation, gene expression profiles showed reduced CD4+ proliferation, and the achievement of full-donor T-cell chimerism was delayed. This study indicates that T-reg graft with single-agent tacrolimus is preferred over T-reg graft alone for the prevention of acute GVHD. This trial was registered at www.clinicaltrials.gov as #NCT01660607.

Surveillance for multidrug resistant Escherichia coli carriage in cattle, dogs and humans reveals predominance of CMY-2, CTX-M-15 and CTX-M-9 groups of ß-lactamases.

Global spread of antimicrobial multidrug resistance (MDR) in human and veterinary medicine relies upon diagnostics, surveillance and stewardship to guide mitigation. Utilizing surveillance of fecal samples from our service area for detecting MDR Escherichia coli carriage in humans (2143), dogs (627), and cattle (130), we found isolates resistant to third/fourth generation cephems present in 3.7 %, 13.1 %, and 51.5 %, respectively. CMY-2, CTX-M-15-like and CTX-M9 group genes in descending order were predominant in all hosts and accounted for 83.3 % of non-wild-type gene targets. MDR carriage mirrored cephem non-susceptibility rates as published in annual antibiograms for humans and dogs; notably, no carbapenem-resistant carriage isolates were detected. Given the scale of MDR E. coli carriage in cattle (14X) and dogs (3.5X) compared to humans, bench-marking of the resistance gene pool by host species utilizing regional One Health surveillance may aid in assessing occupational and geographic risks for acquiring resistance and for monitoring of mitigation strategies.

The voltage sensing phosphatase (VSP) localizes to the apical membrane of kidney tubule epithelial cells.

Voltage-sensing phosphatases (VSPs) are transmembrane proteins that couple changes in membrane potential to hydrolysis of inositol signaling lipids. VSPs catalyze the dephosphorylation of phosphatidylinositol phosphates (PIPs) that regulate diverse aspects of cell membrane physiology including cell division, growth and migration. VSPs are highly conserved among chordates, and their RNA transcripts have been detected in the adult and embryonic stages of frogs, fish, chickens, mice and humans. However, the subcellular localization and biological function of VSP remains unknown. Using reverse transcriptase-PCR (RT-PCR), we show that both Xenopus laevis VSPs (Xl-VSP1 and Xl-VSP2) mRNAs are expressed in early embryos, suggesting that both Xl-VSPs are involved in early tadpole development. To understand which embryonic tissues express Xl-VSP mRNA, we used in situ hybridization (ISH) and found Xl-VSP mRNA in both the brain and kidney of NF stage 32-36 embryos. By Western blot analysis with a VSP antibody, we show increasing levels of Xl-VSP protein in the developing embryo, and by immunohistochemistry (IHC), we demonstrate that Xl-VSP protein is specifically localized to the apical membrane of both embryonic and adult kidney tubules. We further characterized the catalytic activity of both Xl-VSP homologs and found that while Xl-VSP1 catalyzes 3- and 5-phosphate removal, Xl-VSP2 is a less efficient 3-phosphatase with different substrate specificity. Our results suggest that Xl-VSP1 and Xl-VSP2 serve different functional roles and that VSPs are an integral component of voltage-dependent PIP signaling pathways during vertebrate kidney tubule development and function.

Multisite reproducibility of the broth microdilution method for susceptibility testing of Nocardia species.

Antimicrobial susceptibility testing (AST) of clinical isolates of Nocardia is recommended to detect resistance to commonly used antimicrobial agents; such testing is complicated by difficulties in inoculum preparation and test interpretation. In this study, six laboratories performed repetitive broth microdilution testing on single strains of Nocardia brasiliensis, Nocardia cyriacigeorgica, Nocardia farcinica, Nocardia nova, and Nocardia wallacei. For each isolate, a total of 30 microdilution panels from three different lots were tested at most sites. The goal of the study was to determine the inter- and intralaboratory reproducibility of susceptibility testing of this group of isolates. Acceptable agreement (>90% agreement at ±1 dilution of the MIC mode) was found for amikacin, ciprofloxacin, clarithromycin, and moxifloxacin. After eliminating MIC values from single laboratories whose results showed the greatest deviation from those of the remaining laboratories, acceptable agreement was also found for amoxicillin-clavulanic acid, linezolid, minocycline, and tobramycin. Results showed unsatisfactory reproducibility of broth microdilution testing of ceftriaxone with N. cyriacigeorgica and N. wallacei, tigecycline with N. brasiliensis and N. cyriacigeorgica, and sulfonamides with N. farcinica and N. wallacei. N. nova ATCC BAA-2227 is proposed as a quality control organism for AST of Nocardia sp., and the use of a disk diffusion test for sulfisoxazole is proposed as a check of the adequacy of the inoculum and to confirm sulfonamide MIC results.

Multilaboratory testing of antifungal combinations against a quality control isolate of Candida krusei.

Candida krusei ATCC 6258 was tested by eight laboratories using 96-well plates containing checkerboard pairwise combinations of amphotericin B (AMB), posaconazole (PSC), caspofungin (CSP), and voriconazole (VRC). The methodology led to reproducible results across the laboratories. All drug combinations yielded MICs lower than the MICs of any two drugs tested singly, and combinations of AMB, PSC, CSP, and VRC were indifferent (no antagonism) by summations of fractional inhibitory concentration.

Factors influencing broth microdilution antimicrobial susceptibility test results for dalbavancin, a new glycopeptide agent.

Performance of antimicrobial susceptibility tests with new agents requires careful consideration of the properties of the antimicrobial to ensure that the tests are standardized, reproducible, and reflect the true potency of the drug. Dalbavancin is a new glycopeptide with potent activity against gram-positive bacterial species. The investigations described here demonstrated that methodologic modifications of procedures are necessary to ensure consistent test results, both for quality control and for routine testing of clinical isolates. Dimethyl sulfoxide is the preferred primary solvent. The addition of 0.002% polysorbate-80 (a surfactant) to dalbavancin-containing wells in the reference broth microdilution assay resulted in consistent and reproducible MIC results for three quality control strains: Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, and Streptococcus pneumoniae ATCC 49619. The same degree of consistency was observed among clinical isolates of gram-positive bacterial species tested in several clinical laboratories. These results indicate that the addition of 0.002% (final concentration) of the surfactant in broth microdilution tests produces optimal dalbavancin MICs required for accurate and reproducible clinical laboratory tests, without untoward influences of substrate binding or media constituents.

Epitope escape mutation and decay of human immunodeficiency virus type 1-specific CTL responses.

To investigate possible mechanisms behind HIV-1 escape from CTL, we performed detailed longitudinal analysis of Gag (SLYNTVATL)- and RT (ILKEPVHGV)-specific CTL responses and plasma epitope sequences in five individuals. Among those with CTL against consensus epitope sequences, epitope mutations developed over several years, invariably followed by decay of the CTL targeting the consensus epitopes. The maturation state of the CTL varied among individuals and appeared to affect the rate of epitope mutation and CTL decay, despite similar IFN-gamma production. Escape mutations were oligoclonal, suggesting fitness constraints. The timing of escape indicated that the net selective advantage of escape mutants was slight, further underscoring the importance of understanding factors determining selective pressure and viral fitness in vivo. Our data show surprisingly consistent decay of CTL responses after epitope escape mutation and provide insight into potential mechanisms for both immune failure and shifting CTL specificities.