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The prototypical genetic autoimmune disease is immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome, a severe pediatric disease with limited treatment options. IPEX syndrome is caused by mutations in the forkhead box protein 3 (FOXP3) gene, which plays a critical role in immune regulation. As a monogenic disease, IPEX is an ideal candidate for a therapeutic approach in which autologous hematopoietic stem and progenitor (HSPC) cells or T cells are gene edited ex vivo and reinfused. Here, we describe a CRISPR-based gene correction permitting regulated expression of FOXP3 protein. We demonstrate that gene editing preserves HSPC differentiation potential, and that edited regulatory and effector T cells maintain their in vitro phenotype and function. Additionally, we show that this strategy is suitable for IPEX patient cells with diverse mutations. These results demonstrate the feasibility of gene correction, which will be instrumental for the development of therapeutic approaches for other genetic autoimmune diseases.

Q fever is a disease caused by Coxiella burnetii , which can cause serious illness in humans and abortions in goats. A Q fever outbreak among an unvaccinated goat herd led to a 65% loss of the kid crop in spring 2018. To assess the impact of the outbreak on the herd and environment, longitudinal surveillance of the ranch was conducted across three samplings in September 2018, April 2019, and May 2022. Antibodies against C. burnetii were monitored by an indirect immunofluorescence assay. Shedding was monitored through analysis of vaginal/fecal swabs and milk. Environmental swabs and bulk soil were collected from various locations around the ranch. Animal and environmental samples were analyzed for C. burnetii DNA by PCR. Herd-level seroprevalence decreased from 89% in 2018 to 84.3% in 2019, and 64.5% in 2022. Overall herd shedding was 14.4% in 2018, 7.4% in 2019, and 6.7% in 2022. The percentage of C. burnetii-positive environmental samples was 83.7% in 2018, 51.7% in 2019, and 28.6% in 2022. Serological evidence suggests that new infections were occurring in the herd 4 years post-abortion storm. This study demonstrates the presence of C. burnetii shedding and environmental contamination in a goat operation at least four kidding seasons after an outbreak. A better understanding of management practices that can improve outcomes for infected herds, particularly in areas without access to vaccines against C. burnetii , is needed to better protect operators and the public., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Miller, Priestley, Smith, Cherry and Kersh.)

Since 1999, doxycycline and hydroxychloroquine have been the recommended treatment for chronic Q fever, a life-threatening disease caused by the bacterial pathogen, Coxiella burnetii . Despite the duration of its use, the treatment is not ideal due to the lengthy treatment time, high mortality rate, resistant strains, and the potential for contraindicated usage. A literature search was conducted to identify studies that screened large panels of drugs against C. burnetii to identify novel targets with potential efficacy against C. burnetii . Twelve candidate antimicrobials approved for use in humans by the US Food and Drug Administration were selected and minimum inhibitory concentrations (MICs) were determined against the low virulence strain Nine Mile phase II. Rifabutin and rifaximin were the best performing antibiotics tested with MICs of ≤0.01 µg mL -1 . Further screening of these top candidates was conducted alongside two drugs from the same class, rifampin, well-characterized , and rifapentine, not previously reported against C. burnetii . These were screened against virulent strains of C. burnetii representing three clinically relevant genotypes. Rifapentine was the most effective in the human monocytic leukemia cell line, THP-1, with a MIC ≤0.01 µg mL -1 . In the human kidney epithelial cell line, A-498, efficacy of rifapentine, rifampin, and rifabutin varied across C. burnetii strains with MICs between ≤0.001 and 0.01 µg mL -1 . Rifampin, rifabutin, and rifapentine were all bactericidal against C. burnetii ; however, rifabutin and rifapentine demonstrated impressive bactericidal activity as low as 0.1 µg mL -1 and should be further explored as alternative Q fever treatments given their efficacy in vitro ., Importance: This work will help inform investigators and physicians about potential alternative antimicrobial therapies targeting the causative agent of Q fever, Coxiella burnetii . Chronic Q fever is difficult to treat, and alternative antimicrobials are needed. This manuscript explores the efficacy of rifamycin antibiotics against virulent strains of C. burnetii representing three clinically relevant genotypes in vitro . Importantly, this study determines the susceptibility of C. burnetii to rifapentine, which has not been previously reported. Evaluation of the bactericidal activity of the rifamycins reveals that rifabutin and rifapentine are bactericidal at low concentrations, which is unusual for antibiotics against C. burnetii ., Competing Interests: The authors declare no conflict of interest.

Coxiella burnetii is a bacterial pathogen capable of causing serious disease in humans and abortions in goats. Infected goats can shed C . burnetii through urine, feces, and parturient byproducts, which can lead to infections in humans when the bacteria are inhaled. Goats are important C . burnetii reservoirs as evidenced by goat-related outbreaks across the world. To better understand the current landscape of C . burnetii infection in the domestic goat population, 4,121 vaginal swabs from 388 operations across the United States were analyzed for the presence of C . burnetii by IS1111 PCR as part of the United States Department of Agriculture, Animal Plant Health Inspection Service, Veterinary Services' National Animal Health Monitoring System Goats 2019 Study. In total, 1.5% (61/4121) of swabs representing 10.3% (40/388) (weighted estimate of 7.8, 95% CI 4.4-13.5) of operations were positive for C . burnetii DNA. The quantity of C . burnetii on positive swabs was low with an average Ct of 37.9. Factors associated with greater odds of testing positive included suspected Q fever in the herd in the previous 3 years, the presence of wild deer or elk on the operation, and the utilization of hormones for estrus synchronization. Factors associated with reduced odds of testing positive include the presence of kittens and treatment of herds with high tannin concentrate plants, diatomaceous earth, and tetrahydropyrimidines. In vitro analysis demonstrated an inhibitory effect of the tetrahydropyrimidine, pyrantel pamoate, on the growth of C . burnetii in axenic media as low as 1 μg per mL. The final multivariable logistic regression modeling identified the presence of wild predators on the operation or adjacent property (OR = 9.0, 95% CI 1.3-61.6, p value = 0.0248) as a risk factor for C . burnetii infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Miller, Branan, Priestley, Álvarez-Alonso, Cherry, Smith, Urie, Wiedenheft, Bliss, Marshall and Kersh.)

Treatment of severe eczema in patients with primary immunodeficiencies can be particularly challenging as there are no guidelines with regards to these conditions. Dupilumab is an interleukin (IL)-4Rα antagonist that inhibits both IL-4 and IL-13 and is approved for the treatment of atopic dermatitis in pediatric patients. In this report, we describe a patient with a case of severe eczema in the context of Wiskott-Aldrich syndrome-related disorder, who was successfully treated with dupilumab., (© 2023 Wiley Periodicals LLC.)

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by life-threatening infections and inflammatory conditions. Hematopoietic cell transplantation (HCT) is the definitive treatment for CGD, but questions remain regarding patient selection and impact of active disease on transplant outcomes. We performed a multi-institutional retrospective and prospective study of 391 patients with CGD treated either conventionally (non-HCT) enrolled from 2004 to 2018 or with HCT from 1996 to 2018. Median follow-up after HCT was 3.7 years with a 3-year overall survival of 82% and event-free survival of 69%. In a multivariate analysis, a Lansky/Karnofsky score <90 and use of HLA-mismatched donors negatively affected survival. Age, genotype, and oxidase status did not affect outcomes. Before HCT, patients had higher infection density, higher frequency of noninfectious lung and liver diseases, and more steroid use than conventionally treated patients; however, these issues did not adversely affect HCT survival. Presence of pre-HCT inflammatory conditions was associated with chronic graft-versus-host disease. Graft failure or receipt of a second HCT occurred in 17.6% of the patients and was associated with melphalan-based conditioning and/or early mixed chimerism. At 3 to 5 years after HCT, patients had improved growth and nutrition, resolved infections and inflammatory disease, and lower rates of antimicrobial prophylaxis or corticosteroid use compared with both their baseline and those of conventionally treated patients. HCT leads to durable resolution of CGD symptoms and lowers the burden of the disease. Patients with active infection or inflammation are candidates for transplants; HCT should be considered before the development of comorbidities that could affect performance status. This trial was registered at www.clinicaltrials.gov as #NCT02082353.

Background: Shearer et al in 2014 articulated well-defined criteria for the diagnosis and classification of severe combined immunodeficiency (SCID) as part of the Primary Immune Deficiency Treatment Consortium's (PIDTC's) prospective and retrospective studies of SCID., Objective: Because of the advent of newborn screening for SCID and expanded availability of genetic sequencing, revision of the PIDTC 2014 Criteria was needed., Methods: We developed and tested updated PIDTC 2022 SCID Definitions by analyzing 379 patients proposed for prospective enrollment into Protocol 6901, focusing on the ability to distinguish patients with various SCID subtypes., Results: According to PIDTC 2022 Definitions, 18 of 353 patients eligible per 2014 Criteria were considered not to have SCID, whereas 11 of 26 patients ineligible per 2014 Criteria were determined to have SCID. Of note, very low numbers of autologous T cells (<0.05 × 10 9 /L) characterized typical SCID under the 2022 Definitions. Pathogenic variant(s) in SCID-associated genes was identified in 93% of patients, with 7 genes (IL2RG, RAG1, ADA, IL7R, DCLRE1C, JAK3, and RAG2) accounting for 89% of typical SCID. Three genotypes (RAG1, ADA, and RMRP) accounted for 57% of cases of leaky/atypical SCID; there were 13 other rare genotypes. Patients with leaky/atypical SCID were more likely to be diagnosed at more than age 1 year than those with typical SCID lacking maternal T cells: 20% versus 1% (P < .001). Although repeat testing proved important, an initial CD3 T-cell count of less than 0.05 × 10 9 /L differentiated cases of typical SCID lacking maternal cells from leaky/atypical SCID: 97% versus 7% (P < .001)., Conclusions: The PIDTC 2022 Definitions describe SCID and its subtypes more precisely than before, facilitating analyses of SCID characteristics and outcomes., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The DNA-repair enzyme Artemis is essential for rearrangement of T- and B-cell receptors. Mutations in DCLRE1C , which encodes Artemis, cause Artemis-deficient severe combined immunodeficiency (ART-SCID), which is poorly responsive to allogeneic hematopoietic-cell transplantation., Methods: We carried out a phase 1-2 clinical study of the transfusion of autologous CD34+ cells, transfected with a lentiviral vector containing DCLRE1C , in 10 infants with newly diagnosed ART-SCID. We followed them for a median of 31.2 months., Results: Marrow harvest, busulfan conditioning, and lentiviral-transduced CD34+ cell infusion produced the expected grade 3 or 4 adverse events. All the procedures met prespecified criteria for feasibility at 42 days after infusion. Gene-marked T cells were detected at 6 to 16 weeks after infusion in all the patients. Five of 6 patients who were followed for at least 24 months had T-cell immune reconstitution at a median of 12 months. The diversity of T-cell receptor β chains normalized by 6 to 12 months. Four patients who were followed for at least 24 months had sufficient B-cell numbers, IgM concentration, or IgM isohemagglutinin titers to permit discontinuation of IgG infusions. Three of these 4 patients had normal immunization responses, and the fourth has started immunizations. Vector insertion sites showed no evidence of clonal expansion. One patient who presented with cytomegalovirus infection received a second infusion of gene-corrected cells to achieve T-cell immunity sufficient for viral clearance. Autoimmune hemolytic anemia developed in 4 patients 4 to 11 months after infusion; this condition resolved after reconstitution of T-cell immunity. All 10 patients were healthy at the time of this report., Conclusions: Infusion of lentiviral gene-corrected autologous CD34+ cells, preceded by pharmacologically targeted low-exposure busulfan, in infants with newly diagnosed ART-SCID resulted in genetically corrected and functional T and B cells. (Funded by the California Institute for Regenerative Medicine and the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT03538899.)., (Copyright © 2022 Massachusetts Medical Society.)

Background: Robotic-assisted pancreatic surgery is limited to specialized high-volume centers and selected patient cohorts. Especially for patients with a history of previous abdominal surgeries, the standard procedure remains open surgery due to the fear of complications caused by abdominal adhesions., Methods: Clinical data of all consecutive patients undergoing robotic-assisted pancreatic surgery using the daVinci Xi system (Intuitive Surgical) at our center (Department of Surgery, Universitätsmedizin Berlin, Germany) were collected prospectively and further analyzed from October 2017 to October 2020. Prior abdominal surgeries were specified according to the surgical approach and localization. In univariate and multivariate analysis, baseline and perioperative parameters of patients with a history of prior abdominal surgeries (PS) were compared to those of patients with no history of prior abdominal surgeries (NPS)., Results: Out of 131 patients undergoing robotic-assisted pancreatic surgery, 62 (47%) had a history of abdominal surgery. Previous procedures included most often appendectomy (32%) followed by gynecological surgery (29%) and cholecystectomy (27%). 24% of PS had received multiple surgeries prior to the robotic-assisted pancreatic resections. Baseline characteristics and comorbidities were comparable between the groups. We did not detect differences in the duration of surgery (262 min), conversion rates (10%), and postoperative complications between NPS and PS. Postoperative pancreatic fistula (POPF), postpancreatectomy hemorrhage (PPH), and in-house mortality showed no significant differences between the two groups. Multivariate analysis revealed male sex and high BMI as a potential predictive factor for severe postoperative complications. Other characteristics like the type of pancreatic resection, ASA, and underlying malignancy showed no difference in the multivariable analysis., Conclusions: We propose robotic-assisted pancreatic surgery to be safe and feasible for patients with a history of minor prior abdominal surgery. Hence, each patient should individually be evaluated for a minimally invasive approach regardless of a history of previous operations., (© 2022. The Author(s).)

Chronic active Epstein-Barr virus (EBV) disease (CAEBV) is characterized by high levels of EBV predominantly in T and/or natural killer cells with lymphoproliferation, organ failure due to infiltration of tissues with virus-infected cells, hemophagocytic lymphohistiocytosis, and/or lymphoma. The disease is more common in Asia than in the United States and Europe. Although allogeneic hematopoietic stem cell transplantation (HSCT) is considered the only curative therapy for CAEBV, its efficacy and the best treatment modality to reduce disease severity prior to HSCT is unknown. Here, we retrospectively assessed an international cohort of 57 patients outside of Asia. Treatment of the disease varied widely, although most patients ultimately proceeded to HSCT. Though patients undergoing HSCT had better survival than those who did not (55% vs 25%, P < .01), there was still a high rate of death in both groups. Mortality was largely not affected by age, ethnicity, cell-type involvement, or disease complications, but development of lymphoma showed a trend with increased mortality (56% vs 35%, P = .1). The overwhelming majority (75%) of patients who died after HSCT succumbed to relapsed disease. CAEBV remains challenging to treat when advanced disease is present. Outcomes would likely improve with better disease control strategies, earlier referral for HSCT, and close follow-up after HSCT including aggressive management of rising EBV DNA levels in the blood., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Evidence suggests that Coxiella burnetii , which is shed in the milk, urine, feces, and birth products of infected domestic ruminants, can lead to Q fever disease following consumption of unpasteurized dairy products; however, C. burnetii is not believed to be a major gastrointestinal pathogen. Most infections are associated with inhalation of aerosols generated from the excreta of domestic ruminants. We recently demonstrated that C. burnetii delivered by oral gavage (OG) resulted in dissemination and an immune response; however, it is unclear how infection via the oral route compares to other well-established routes. Therefore, we delivered three strains of C. burnetii (representing three pertinent sequence types in the United States, such as ST16, ST20, and ST8) to immunocompetent mice in four doses via aerosol challenge (AC), intraperitoneal injection (IP), or OG. Low dose (10^5) of ST16 by OG was insufficient to cause infection, yet doses 1,000- or 100-fold lower by IP or AC, respectively, induced a robust immune response and dissemination. Despite being able to induce an immune response in a dose-dependent manner, administration of C. burnetii via OG is the least efficient route tested. Not only were the immune responses and bacterial loads diminished in mice exposed by OG relative to AC or IP, the efficiency of transmission was also inferior. High doses (10^8) were not sufficient to ensure transmission to 100% of the ST20 or ST8 cohorts. These results may provide some basis for why ingestion of C. burnetii as a mode of Q fever transmission is not often reported.

Coxiella burnetii is an obligate intracellular bacterium that causes the human disease Q fever, which can manifest as an acute flu-like illness or a long-term chronic illness, such as endocarditis. Three genotypes (ST8, ST16, and ST20) of Coxiella burnetii are commonly found in the contemporary US and are associated with specific animal hosts. Although all three genotypes have been isolated from humans with Q fever, studies comparing virulence between C. burnetii sequence types have been rare. Here, groups of mice were infected via aerosol inoculation with isolates derived from cow's milk, environmental, animal, and human samples. Mice were monitored for weight loss and blood samples were takenweekly. Animals were euthanized at 2- and 12-weeks post-infection, and bacterial burden was determined for tissues by real-time PCR. The levels of anti-Coxiella antibodies and selected inflammatory cytokines were determined for serum samples. Weight loss and splenomegaly were observed in mice infected with ST20 and ST16 isolates but were absent in the mice infected with ST8 isolates. Bacterial concentrations in the tissues were lower in the ST8 isolates at 2 weeks post-infection relative to all other isolates. ST16 and ST20 isolates induced robust antibody and cytokine responses, while ST8 isolates produced significantly lower anti- C. burnetii titers early in the infection but saw increased titers in some animals several weeks post-infection. The data suggest that the ST8 isolates are less virulent in this mouse model, as they produce less robust antibody responses that are slow to develop, relative to the ST16 and ST20 isolates.

Q fever is a disease caused by the bacterial pathogen Coxiella burnetii . This hardy organism can easily spread long distances in the wind, and only a few infectious aerosolized particles are necessary to cause serious illness. Presentations of Q fever disease can be wide-ranging, allowing it to masquerade as other illnesses and highlight the importance of laboratory testing for diagnosis and treatment. This review summarizes Q fever's epidemiology and clinical presentations and presents classical laboratory diagnostic assays and novel approaches to detecting this troubling disease.

Questionnaire data have linked contact with ruminants to the risk of esophageal squamous cell carcinoma (ESCC) in high-risk Asian populations. To better understand this observed association, we investigated exposure to two major zoonotic ruminant pathogens relative to ESCC risk. Using enzyme-linked immunosorbent assay, immunofluorescence assay, and Brucella microagglutination test assays, we measured immunoglobulin G anti- Coxiella burnetii and anti- Brucella spp. antibodies in patients with ESCC ( n  = 177) and population-based controls ( n  = 177) matched by age, gender, and residence area from the Golestan case-control study in Iran. We found a similarly high seroprevalence of C. burnetii in ESCC cases and controls (75% and 80%, respectively), and a similarly low seroprevalence of Brucella spp. (0% and 0.6%, respectively). While documenting a high exposure to one of two zoonotic ruminant infections, this exposure failed to explain the observed association of ruminant contact and ESCC risk in this high-risk population.

We evaluated the performance of self-collected anterior nasal swab (ANS) and saliva samples compared with healthcare worker-collected nasopharyngeal swab specimens used to test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We used the same PCR diagnostic panel to test all self-collected and healthcare worker-collected samples from participants at a public hospital in Atlanta, Georgia, USA. Among 1,076 participants, 51.9% were men, 57.1% were >50 years of age, 81.2% were Black (non-Hispanic), and 74.9% reported >1 chronic medical condition. In total, 8.0% tested positive for SARS-CoV-2. Compared with nasopharyngeal swab samples, ANS samples had a sensitivity of 59% and saliva samples a sensitivity of 68%. Among participants tested 3-7 days after symptom onset, ANS samples had a sensitivity of 80% and saliva samples a sensitivity of 85%. Sensitivity varied by specimen type and patient characteristics. These findings can help physicians interpret PCR results for SARS-CoV-2.

Serology is essential for Q fever diagnostics, a disease caused by the bacterial pathogen Coxiella burnetii. The gold standard test is an immunofluorescence assay utilizing whole cell antigens, which are both dangerous and laborious to produce. Complexities of the antigen coupled with the subjective nature of the assay lead to decreased uniformity of test results and underscore the need for improved methodologies. Thirty-three C. burnetii proteins, previously identified as immunoreactive, were screened for reactivity to naturally infected goat serum. Based on reactivity, 10 proteins were analyzed in a secondary screen against human serum from healthy donors. Assay sensitivity and specificity ranged from 21 to 71% and 90 to 100%, respectively. Three promising antigens were identified based on receiver operating characteristic curve analysis (CBU&#95;1718, CBU&#95;0307, and CBU&#95;1398). Five multiplex assays failed to outperform the individual proteins, with sensitivities and specificities ranging from 29 to 57% and 90 to 100%, respectively. Truncating the top antigen, CBU&#95;1718, had no effect on specificity (90%); yet sensitivity decreased dramatically (71% to 21%). Through this study, we have expanded the subset of C. burnetii immunoreactive proteins validated by enzyme-linked immunosorbent assay and demonstrate the effect of novel antigen combinations and protein truncations on assay performance.

Wiskott-Aldrich syndrome (WAS) is an X-linked disease caused by mutations in the WAS gene, leading to thrombocytopenia, eczema, recurrent infections, autoimmune disease, and malignancy. Hematopoietic cell transplantation (HCT) is the primary curative approach, with the goal of correcting the underlying immunodeficiency and thrombocytopenia. HCT outcomes have improved over time, particularly for patients with HLA-matched sibling and unrelated donors. We report the outcomes of 129 patients with WAS who underwent HCT at 29 Primary Immune Deficiency Treatment Consortium centers from 2005 through 2015. Median age at HCT was 1.2 years. Most patients (65%) received myeloablative busulfan-based conditioning. With a median follow-up of 4.5 years, the 5-year overall survival (OS) was 91%. Superior 5-year OS was observed in patients <5 vs ≥5 years of age at the time of HCT (94% vs 66%; overall P = .0008). OS was excellent regardless of donor type, even in cord blood recipients (90%). Conditioning intensity did not affect OS, but was associated with donor T-cell and myeloid engraftment after HCT. Specifically, patients who received fludarabine/melphalan-based reduced-intensity regimens were more likely to have donor myeloid chimerism <50% early after HCT. In addition, higher platelet counts were observed among recipients who achieved full (>95%) vs low-level (5%-49%) donor myeloid engraftment. In summary, HCT outcomes for WAS have improved since 2005, compared with prior reports. HCT at a younger age continues to be associated with superior outcomes supporting the recommendation for early HCT. High-level donor myeloid engraftment is important for platelet reconstitution after either myeloablative or busulfan-containing reduced intensity conditioning. (This trial was registered at www.clinicaltrials.gov as #NCT02064933.).

Primary Immune Regulatory Disorders (PIRD) are an expanding group of diseases caused by gene defects in several different immune pathways, such as regulatory T cell function. Patients with PIRD develop clinical manifestations associated with diminished and exaggerated immune responses. Management of these patients is complicated; oftentimes immunosuppressive therapies are insufficient, and patients may require hematopoietic cell transplant (HCT) for treatment. Analysis of HCT data in PIRD patients have previously focused on a single gene defect. This study surveyed transplanted patients with a phenotypic clinical picture consistent with PIRD treated in 33 Primary Immune Deficiency Treatment Consortium centers and European centers. Our data showed that PIRD patients often had immunodeficient and autoimmune features affecting multiple organ systems. Transplantation resulted in resolution of disease manifestations in more than half of the patients with an overall 5-years survival of 67%. This study, the first to encompass disorders across the PIRD spectrum, highlights the need for further research in PIRD management., (Copyright © 2020 Chan, Leiding, Liu, Logan, Burroughs, Allenspach, Skoda-Smith, Uzel, Notarangelo, Slatter, Gennery, Smith, Pai, Jordan, Marsh, Cowan, Dvorak, Craddock, Prockop, Chandrakasan, Kapoor, Buckley, Parikh, Chellapandian, Oshrine, Bednarski, Cooper, Shenoy, Davila Saldana, Forbes, Martinez, Haddad, Shyr, Chen, Sullivan, Heimall, Wright, Bhatia, Cuvelier, Goldman, Meyts, Miller, Seidel, Vander Lugt, Bacchetta, Weinacht, Andolina, Caywood, Chong, de la Morena, Aquino, Shereck, Walter, Dorsey, Seroogy, Griffith, Kohn, Puck, Pulsipher and Torgerson.)

Coxiella burnetii, the causative agent of Q fever, is widely present in dairy products around the world. It has been isolated from unpasteurised milk and cheese and can survive for extended periods of time under typical storage conditions for these products. Although consumption of contaminated dairy products has been suggested as a potential route for transmission, it remains controversial. Given the high prevalence of C. burnetii in dairy products, we sought to examine the feasibility of transmitting the major sequence types (ST16, ST8 and ST20) of C. burnetii circulating in the United States. We delivered three strains of C. burnetii, comprising each sequence type, directly into the stomachs of immunocompetent BALB/c mice via oral gavage (OG) and assessed them for clinical symptoms, serological response and bacterial dissemination. We found that mice receiving C. burnetii by OG had notable splenomegaly only after infection with ST16. A robust immune response and persistence in the stomach and mesenteric lymph nodes were observed in mice receiving ST16 and ST20 by OG, and dissemination of C. burnetii to peripheral tissues was observed in all OG infected mice. These findings support the oral route as a mode of transmission for C. burnetii.

Introduction: The effects of RIC for HSCT on male fertility remain unknown. We investigated spermatogenesis and gonadal hormonal status among adolescent male patients who received RIC HSCT for non-malignant diseases., Patients and Methods: Patients with non-malignant disease who had undergone a RIC HSCT were recruited and evaluated for spermatogenesis via semen analysis and gonadal hormonal function via serum hormone levels. Those who had received prior chemotherapy or radiation were excluded from the study. We reviewed the charts to record demographic factors, conditioning regimen and complications during and after transplant., Results: Five patients were enrolled. The median age at the time of transplant was 15 years (range, 11-19 years), and the median time between bone marrow transplant and semen analysis was 5 years (range, 3-11 years). Median age of patients was 20 years (range, 18-25 years) at the time of the study. Serum FSH and LH levels were elevated in four patients, and inhibin B levels were low for age in three patients. Semen analysis showed two patients had azoospermia, and the remaining three patients showed severe oligozoospermia. Normal morphology and motility were seen in only one patient., Conclusion: This case series suggests that RIC transplants may be associated with impaired spermatogenesis and sequential follow-up is necessary given the potential for either permanent impairment or delayed recovery. Further larger studies are needed to confirm these findings., (© 2019 Wiley Periodicals, Inc.)

Rationale: "Noninfectious" pulmonary complications are significant causes of morbidity and mortality after allogeneic hematopoietic cell transplant. Early-onset viral reactivations or infections are common after transplant. Whether the first-onset viral infection causes noninfectious pulmonary complications is unknown. Objectives: To determine whether the first-onset viral infection within 100 days after transplant predisposes to development of noninfectious pulmonary complications. Methods: We performed a retrospective review of 738 allogeneic hematopoietic cell transplant patients enrolled from 2005 to 2011. We also established a novel bone marrow transplantation mouse model to test whether herpesviral reactivation after transplant causes organ injury. Measurements and Main Results: First-onset viral infections with human herpesvirus 6 or Epstein-Barr virus within 100 days after transplant increase the risk of developing idiopathic pneumonia syndrome (adjusted hazard ratio [aHR], 5.52; 95% confidence interval [CI], 1.61-18.96; P  = 0.007; and aHR, 9.21; 95% CI, 2.63-32.18; P  = 0.001, respectively). First infection with human cytomegalovirus increases risk of bronchiolitis obliterans syndrome (aHR, 2.88; 95% CI, 1.50-5.55; P  = 0.002) and grade II-IV acute graft-versus-host disease (aHR, 1.59; 95% CI, 1.06-2.39; P  = 0.02). Murine roseolovirus, a homolog of human herpesvirus 6, can also be reactivated in the lung and other organs after bone marrow transplantation. Reactivation of murine roseolovirus induced an idiopathic pneumonia syndrome-like phenotype and aggravated acute graft-versus-host disease. Conclusions: First-onset herpesviral infection within 100 days after allogeneic hematopoietic cell transplant increases risk of pulmonary complications. Experimentally reactivating murine roseolovirus causes organ injury similar to phenotypes seen in human transplant recipients.

Laboratory testing for Q fever (Coxiella burnetii) is essential for a differential diagnosis, yet little is known about Q fever diagnostic testing practices in the United States. We retrospectively analyzed Q fever immunoglobulin G (IgG) indirect immunofluorescence assay (IFA) testing data between 1/1/2012-10/31/2016 from ARUP, LabCorp, Mayo Medical Laboratories, and Quest Diagnostics. Data included IgG phase I and phase II titers, patient age and sex, and state and date of specimen collection. On average, 12,821 specimens were tested for Q fever annually by the participating laboratories. Of 64,106 total specimens, 84.1% tested negative for C. burnetii-specific antibodies. Positive titers ranged from 16 to 262,144 against both phase I and phase II antigens. Submission of specimens peaked during the summer months, and more specimens were submitted from the West North Central division. Testing occurred more frequently in males (53%) and increased with age. In conclusion, few U.S. Q fever cases are reported, despite large volumes of diagnostic specimens tested. Review of commercial laboratory data revealed a lack of paired serology samples and patterns of serology titers that differ from case reporting diagnostic criteria.

Viral infections can be life threatening in patients with severe combined immunodeficiency (SCID) and other forms of profound primary immunodeficiency disorders both before and after hematopoietic stem cell transplantation (HSCT). Adoptive immunotherapy with virus-specific T cells (VSTs) has been utilized in many patients in the setting of HSCT, but has very rarely been attempted for treatment of viral infections before HSCT. Here we describe the use of VSTs in an infant with RAG1 SCID who had developed disseminated adenovirus which failed to improve on cidofovir. Adenovirus cleared following 2 doses of VSTs and marrow infusion from a matched unrelated donor, without incidence of graft versus host disease. T cell receptor-b sequencing demonstrated expansion of adenovirus-specific T cell fraction of the VSTs, suggesting that infusion facilitated viral clearance. This report suggests that VSTs are likely safe in the pre-HSCT period, and may be a useful bridge therapy for infants with SCID and persistent viral infections., (Copyright © 2018 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Background: After hematopoietic stem cell transplantation (HSCT) autoimmune hemolytic anemia (AIHA) is a known and fairly common complication. It is often refractory to conventional therapies including corticosteroids, intravenous immunoglobulin, splenectomy, and the more recently described use of monoclonal antibodies. The high morbidity associated with these severe persistent cases elucidates the gaps in alternative therapies available for treatment., Study Design and Methods: We described the successful use of abatacept for severe refractory AIHA after HSCT in three patients., Results: Three pediatric patients with refractory AIHA after allogeneic stem cell transplantation were observed to be unresponsive to multitude immunosuppressive therapies, resulting in persistent transfusion dependency. Treatment with abatacept, a fusion protein that inhibits T-cell activation by binding to CD80/CD86 on antigen-presenting cells (APCs), thus blocking the required CD28 interaction between APCs and T cells, resulted in the resolution of hemolysis., Conclusion: Abatacept may provide significant clinical benefit in the management of AIHA after HSCT., (© 2018 AABB.)

Staphylococcus aureus possess three alternative σ factors, including a lone extracytoplasmic function σ factor, σS. Our group previously identified and characterized this element, mapping three sigS promoters, demonstrating its inducibility during stress and virulence inducing conditions and demonstrating a role for this factor in disease causation. In the present study, we identify a fourth promoter of the sigS operon, termed P4, located in a unique position internal to the sigS coding region. Transcriptional profiling revealed that expression from P4 is dominant to the three upstream promoters, particularly upon exposure to chemical stressors that elicit DNA damage and disrupt cell wall stability; each of which have previously been shown to stimulate sigS expression. Importantly, expression of this fourth promoter, followed by at least one or more of the upstream promoters, is induced during growth in serum and upon phagocytosis by RAW 264.7 murine macrophage-like cells. Finally, we demonstrate that a downstream gene, SACOL1829, bears a large 3΄ UTR that spans the sigS-SACOL1828 coding region, and may serve to compete with the P4 transcript to inhibit σS production. Collectively, these findings reveal a unique operon architecture for the sigS locus that indicates the potential for novel regulatory mechanisms governing its expression.

Despite the mammalian host actively sequestering iron to limit pathogenicity, heme (or hemin when oxidized) and hemoproteins serve as important sources of iron for many bloodborne pathogens. The HmuRSTUV hemin uptake system allows Yersinia species to uptake and utilize hemin and hemoproteins as iron sources. HmuR is a TonB-dependent outer membrane receptor for hemin and hemoproteins. HmuTUV comprise a inner membrane ABC transporter that transports hemin and hemoproteins from the periplasmic space into the bacterial cytoplasm, where it is degraded by HmuS. Here we show that hmuSTUV but not hmuR are expressed under iron replete conditions, whereas hmuR as well as hmuSTUV are expressed under iron limiting conditions, suggesting complex transcriptional control. Indeed, expression of hmuSTUV in the presence of inorganic iron, but not in the presence of hemin, requires the global regulator IscR acting from a promoter in the intergenic region between hmuR and hmuS . This effect of IscR appears to be direct by binding a site mapped by DNaseI footprinting. In contrast, expression of hmuR under iron limiting conditions requires derepression of the ferric uptake regulator Fur acting from the hmuR promoter, as Fur binding upstream of hmuR was demonstrated biochemically. Differential expression by both Fur and IscR would facilitate maximal hemin uptake and utilization when iron and heme availability is low while maintaining the capacity for periplasmic removal and cytosolic detoxification of heme under a wider variety of conditions. We also demonstrate that a Y. pseudotuberculosis Δ iscR mutant has a survival defect when incubated in whole blood, in which iron is sequestered by heme-containing proteins. Surprisingly, this phenotype was independent of the Hmu system, the type III secretion system, complement, and the ability of Yersinia to replicate intracellularly. These results suggest that IscR regulates multiple virulence factors important for Yersinia survival and growth in mammalian tissues and reveal a surprising complexity of heme uptake expression and function under differing conditions of iron.

The occurrence of infections after allogeneic hematopoietic stem cell transplantation (HCT) is nearly universal. However, the relationship between infections and graft-versus-host disease (GVHD) is complex and attribution of infectious-related mortality is highly inconsistent, making comparison of infectious complication rates across allogeneic HCT clinical studies difficult. We categorized infectious complications from diagnosis or 1 year before HCT (whichever occurred later) through 2 years after HCT according to timing, frequency, causative organism, severity, and contribution to mortality for 431 consecutive patients who underwent allogeneic HCT from 2008 to 2011. We then assessed the contribution of risk factors, such as the frequency of pre-HCT infections and post-HCT GVHD, on post-HCT infection frequency and severity. We found that each pre-HCT bacterial infection/year leads to an additional 2.15 post-HCT bacterial infection/year (P = .004). Pre-HCT viral and fungal infections were not predictors for post-HCT infections. Acute GVHD (aGVHD) significantly increased the risk of developing life-threatening (hazard ratio [HR], 1.97; 95% confidence interval [CI], 1.33 to 2.90) and fatal (HR, 2.8; 95% CI, 1.10 to 7.08) infections. Furthermore, patients who develop aGVHD experienced ~60% more infections than patients who never develop aGVHD. Quantification of infection frequency and severity for patients with and without GVHD may facilitate comparison of infectious outcomes across allogeneic HCT trials., (Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

The iron overload disorder hereditary hemochromatosis (HH) predisposes humans to serious disseminated infection with pathogenic Yersinia as well as several other pathogens. Recently, we showed that the iron-sulfur cluster coordinating transcription factor IscR is required for type III secretion in Y. pseudotuberculosis by direct control of the T3SS master regulator LcrF. In E. coli and Yersinia, IscR levels are predicted to be regulated by iron bioavailability, oxygen tension, and oxidative stress, such that iron depletion should lead to increased IscR levels. To investigate how host iron overload influences Y. pseudotuberculosis virulence and the requirement for the Ysc type III secretion system (T3SS), we utilized two distinct murine models of HH: hemojuvelin knockout mice that mimic severe, early-onset HH as well as mice with the Hfe (C282Y∕C282Y) mutation carried by 10% of people of Northern European descent, associated with adult-onset HH. Hjv (-∕-) and Hfe (C282Y∕C282Y) transgenic mice displayed enhanced colonization of deep tissues by Y. pseudotuberculosis following oral inoculation, recapitulating enhanced susceptibility of humans with HH to disseminated infection with enteropathogenic Yersinia. Importantly, HH mice orally infected with Y. pseudotuberculosis lacking the T3SS-encoding virulence plasmid, pYV, displayed increased deep tissue colonization relative to wildtype mice. Consistent with previous reports using monocytes from HH vs. healthy donors, macrophages isolated from Hfe (C282Y∕C282Y) mice were defective in Yersinia uptake compared to wildtype macrophages, indicating that the anti-phagocytic property of the Yersinia T3SS plays a less important role in HH animals. These data suggest that Yersinia may rely on distinct virulence factors to cause disease in healthy vs. HH hosts.

Iron-sulfur clusters act as important cofactors for a number of transcriptional regulators in bacteria, including many mammalian pathogens. The sensitivity of iron-sulfur clusters to iron availability, oxygen tension, and reactive oxygen and nitrogen species enables bacteria to use such regulators to adapt their gene expression profiles rapidly in response to changing environmental conditions. In this review, we discuss how the [4Fe-4S] or [2Fe-2S] cluster-containing regulators FNR, Wbl, aconitase, IscR, NsrR, SoxR, and AirSR contribute to bacterial pathogenesis through control of both metabolism and classical virulence factors. In addition, we briefly review mammalian iron homeostasis as well as oxidative/nitrosative stress to provide context for understanding the function of bacterial iron-sulfur cluster sensors in different niches within the host.

Staphylococcus aureus possesses a lone extracytoplasmic function (ECF) sigma factor, σ(S). In Bacillus subtilis, the ECF sigma factor, σ(W), is activated through a proteolytic cascade that begins with cleavage of the RsiW anti-sigma factor by a site-1 protease (S1P), PrsW. We have identified a PrsW homologue in S. aureus (termed PrsS) and explored its role in σ(S) regulation. Herein, we demonstrate that although a cognate σ(S) anti-sigma factor currently remains elusive, prsS phenocopies sigS in a wealth of regards. Specifically, prsS expression mimics the upregulation observed for sigS in response to DNA-damaging agents, cell wall-targeting antibiotics and during ex vivo growth in human serum and murine macrophages. prsS mutants also display the same sensitivities of sigS mutants to the DNA-damaging agents methyl methane sulfonate (MMS) and hydrogen peroxide, and the cell wall-targeting antibiotics ampicillin, bacitracin and penicillin-G. These phenotypes appear to be explained by alterations in abundance of proteins involved in drug resistance (Pbp2a, FemB, HmrA) and the response to DNA damage (BmrA, Hpt, Tag). Our findings seem to be mediated by putative proteolytic activity of PrsS, as site-directed mutagenesis of predicted catalytic residues fails to rescue the sensitivity of the mutant to H2O2 and MMS. Finally, a role for PrsS in S. aureus virulence was identified using human and murine models of infection. Collectively, our data indicate that PrsS and σ(S) function in a similar manner, and perhaps mediate virulence and resistance to DNA damage and cell wall-targeting antibiotics, via a common pathway., (© 2015 The Authors.)

Background: We previously identified an ECF sigma factor, σS, that is important in the stress and virulence response of Staphylococcus aureus. Transcriptional profiling of sigS revealed that it is differentially expressed in many laboratory and clinical isolates, suggesting the existence of regulatory networks that modulates its expression., Results: To identify regulators of sigS, we performed a pull down assay using S. aureus lysates and the sigS promoter. Through this we identified CymR as a negative effector of sigS expression. Electrophoretic mobility shift assays (EMSAs) revealed that CymR directly binds to the sigS promoter and negatively effects transcription. To more globally explore genetic regulation of sigS, a Tn551 transposon screen was performed, and identified insertions in genes that are involved in amino acid biosynthesis, DNA replication, recombination and repair pathways, and transcriptional regulators. In efforts to identify gain of function mutations, methyl nitro-nitrosoguanidine mutagenesis was performed on a sigS-lacZ reporter fusion strain. From this a number of clones displaying sigS upregulation were subject to whole genome sequencing, leading to the identification of the lactose phosphotransferase repressor, lacR, and the membrane histidine kinase, kdpD, as central regulators of sigS expression. Again using EMSAs we determined that LacR is an indirect regulator of sigS expression, while the response regulator, KdpE, directly binds to the promoter region of sigS., Conclusions: Collectively, our work suggests a complex regulatory network exists in S. aureus that modulates expression of the ECF sigma factor, σS.

Type III secretion systems (T3SS) are essential for virulence in dozens of pathogens, but are not required for growth outside the host. Therefore, the T3SS of many bacterial species are under tight regulatory control. To increase our understanding of the molecular mechanisms behind T3SS regulation, we performed a transposon screen to identify genes important for T3SS function in the food-borne pathogen Yersinia pseudotuberculosis. We identified two unique transposon insertions in YPTB2860, a gene that displays 79% identity with the E. coli iron-sulfur cluster regulator, IscR. A Y. pseudotuberculosis iscR in-frame deletion mutant (ΔiscR) was deficient in secretion of Ysc T3SS effector proteins and in targeting macrophages through the T3SS. To determine the mechanism behind IscR control of the Ysc T3SS, we carried out transcriptome and bioinformatic analysis to identify Y. pseudotuberculosis genes regulated by IscR. We discovered a putative IscR binding motif upstream of the Y. pseudotuberculosis yscW-lcrF operon. As LcrF controls transcription of a number of critical T3SS genes in Yersinia, we hypothesized that Yersinia IscR may control the Ysc T3SS through LcrF. Indeed, purified IscR bound to the identified yscW-lcrF promoter motif and mRNA levels of lcrF and 24 other T3SS genes were reduced in Y. pseudotuberculosis in the absence of IscR. Importantly, mice orally infected with the Y. pseudotuberculosis ΔiscR mutant displayed decreased bacterial burden in Peyer's patches, mesenteric lymph nodes, spleens, and livers, indicating an essential role for IscR in Y. pseudotuberculosis virulence. This study presents the first characterization of Yersinia IscR and provides evidence that IscR is critical for virulence and type III secretion through direct regulation of the T3SS master regulator, LcrF.

Staphylococcus aureus is an opportunistic pathogen that presents severe health care concerns due to the prevalence of multiple antibiotic-resistant strains. New treatment strategies are urgently needed, which requires an understanding of disease causation mechanisms. Complement is one of the first lines of defense against bacterial pathogens, and S. aureus expresses several specific complement inhibitors. The effect of extracellular proteases from this bacterium on complement, however, has been the subject of limited investigation, except for a recent report regarding cleavage of the C3 component by aureolysin (Aur). We demonstrate here that four major extracellular proteases of S. aureus are potent complement inhibitors. Incubation of human serum with the cysteine proteases staphopain A and staphopain B, the serine protease V8 and the metalloproteinase Aur resulted in a drastic decrease in the hemolytic activity of serum, whereas two staphylococcal serine proteases D and E, had no effect. These four proteases were found to inhibit all pathways of complement due to the efficient degradation of several crucial components. Furthermore, S. aureus mutants lacking proteolytic enzymes were found to be more efficiently killed in human blood. Taken together, the major proteases of S. aureus appear to be important for pathogen-mediated evasion of the human complement system., (© 2013 S. Karger AG, Basel.)

Previously we identified a novel component of the Staphylococcus aureus regulatory network, an extracytoplasmic function σ-factor, σ(S), involved in stress response and disease causation. Here we present additional characterization of σ(S), demonstrating a role for it in protection against DNA damage, cell wall disruption, and interaction with components of the innate immune system. Promoter mapping reveals the existence of three unique sigS start sites, one of which appears to be subject to autoregulation. Transcriptional profiling revealed that sigS expression remains low in a number of S. aureus wild types but is upregulated in the highly mutated strain RN4220. Further analysis demonstrates that sigS expression is inducible upon exposure to a variety of chemical stressors that elicit DNA damage, including methyl methanesulfonate and ciprofloxacin, as well as those that disrupt cell wall stability, such as ampicillin and oxacillin. Significantly, expression of sigS is highly induced during growth in serum and upon phagocytosis by RAW 264.7 murine macrophage-like cells. Phenotypically, σ(S) mutants display sensitivity to a broad range of DNA-damaging agents and cell wall-targeting antibiotics. Furthermore, the survivability of σ(S) mutants is strongly impacted during challenge by components of the innate immune system. Collectively, our data suggest that σ(S) likely serves dual functions within the S. aureus cell, protecting against both cytoplasmic and extracytoplasmic stresses. This further argues for its important, and perhaps novel, role in the S. aureus stress and virulence responses.

Staphylococcus aureus is a leading human pathogen of both hospital and community-associated diseases worldwide. This organism causes a wealth of infections within the human host as a result of the vast arsenal of toxins encoded within its genome. Previous transcriptomic studies have shown that toxin production in S. aureus can be strongly impacted by the negative regulator CodY. CodY acts by directly, and indirectly (via Agr), repressing toxin production during times of plentiful nutrition. In this study, we use iTRAQ-based proteomics for the first time to study virulence determinant production in S. aureus, so as to correlate transcriptional observations with actual changes in protein synthesis. Using a codY mutant in the epidemic CA-MRSA clone USA300 we demonstrate that deletion of this transcription factor results in a major upregulation of toxin synthesis in both post-exponential and stationary growth. Specifically, we observe hyper-production of secreted proteases, leukocidins and hemolysins in both growth phases in the USA300 codY mutant. Our findings demonstrate the power of mass spectrometry-based quantitative proteomics for studying toxin production in S. aureus, and the importance of CodY to this central process in disease causation and infection., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Staphylococcus aureus possesses 16 two-component systems (TCSs), two of which (GraRS and NsaRS) belong to the intramembrane-sensing histidine kinase (IM-HK) family, which is conserved within the firmicutes. NsaRS has recently been documented as being important for nisin resistance in S. aureus. In this study, we present a characterization of NsaRS and reveal that, as with other IM-HK TCSs, it responds to disruptions in the cell envelope. Analysis using a lacZ reporter-gene fusion demonstrated that nsaRS expression is upregulated by a variety of cell-envelope-damaging antibiotics, including phosphomycin, ampicillin, nisin, gramicidin, carbonyl cyanide m-chlorophenylhydrazone and penicillin G. Additionally, we reveal that NsaRS regulates a downstream transporter NsaAB during nisin-induced stress. NsaS mutants also display a 200-fold decreased ability to develop resistance to the cell-wall-targeting antibiotic bacitracin. Microarray analysis reveals that the transcription of 245 genes is altered in an nsaS mutant, with the vast majority being downregulated. Included within this list are genes involved in transport, drug resistance, cell envelope synthesis, transcriptional regulation, amino acid metabolism and virulence. Using inductively coupled plasma-MS we observed a decrease in intracellular divalent metal ions in an nsaS mutant when grown under low abundance conditions. Characterization of cells using electron microscopy reveals that nsaS mutants have alterations in cell envelope structure. Finally, a variety of virulence-related phenotypes are impaired in nsaS mutants, including biofilm formation, resistance to killing by human macrophages and survival in whole human blood. Thus, NsaRS is important in sensing cell damage in S. aureus and functions to reprogram gene expression to modify cell envelope architecture, facilitating adaptation and survival.

S. aureus is a highly successful pathogen that is speculated to be the most common cause of human disease. The progression of disease in S. aureus is subject to multi-factorial regulation, in response to the environments encountered during growth. This adaptive nature is thought to be central to pathogenesis, and is the result of multiple regulatory mechanisms employed in gene regulation. In this work we describe the existence of a novel S. aureus regulator, an as yet uncharacterized ECF-sigma factor (sigma(S)), that appears to be an important component of the stress and pathogenic responses of this organism. Using biochemical approaches we have shown that sigma(S) is able to associates with core-RNAP, and initiate transcription from its own coding region. Using a mutant strain we determined that sigma(S) is important for S. aureus survival during starvation, extended exposure to elevated growth temperatures, and Triton X-100 induced lysis. Coculture studies reveal that a sigma(S) mutant is significantly outcompeted by its parental strain, which is only exacerbated during prolonged growth (7 days), or in the presence of stressor compounds. Interestingly, transcriptional analysis determined that under standard conditions, S. aureus SH1000 does not initiate expression of sigS. Assays performed hourly for 72 h revealed expression in typically background ranges. Analysis of a potential anti-sigma factor, encoded downstream of sigS, revealed it to have no obvious role in the upregulation of sigS expression. Using a murine model of septic arthritis, sigS-mutant infected animals lost significantly less weight, developed septic arthritis at significantly lower levels, and had increased survival rates. Studies of mounted immune responses reveal that sigS-mutant infected animals had significantly lower levels of IL-6, indicating only a weak immunological response. Finally, strains of S. aureus lacking sigS were far less able to undergo systemic dissemination, as determined by bacterial loads in the kidneys of infected animals. These results establish that sigma(S) is an important component in S. aureus fitness, and in its adaptation to stress. Additionally it appears to have a significant role in its pathogenic nature, and likely represents a key component in the S. aureus regulatory network.

Nineteen cases of a distinctive type of malignant small-cell tumor are presented. The main features of the entity are as follows: a predilection for adolescent males (mean age: 18.6 years); predominant or exclusive intra-abdominal location, with only inconstant and secondary organ involvement; nesting pattern of growth; focal rhabdoid features; intense desmoplastic reaction; immunohistochemical reactivity for epithelial [keratin, epithelial membrane antigen (EMA)], neural [neuron-specific enolase (NSE)], and muscle (desmin) markers; and highly aggressive behavior. It is proposed that this represents yet another member of the continuously enlarging and evolving family of small round (blue) cell tumors of infancy and childhood that features, more than any other member of this group, the capacity for simultaneous multidirectional phenotypical expression.

Histone methylation by extracts of rat brain or liver was inhibited and tRNA methylation stimulated by the addition of a number of naturally occurring polyamines. The effect was age independent although the methylase activities are highly age-related. Spermine and/or histamine stimulated methylation of cytosine and adenine to a far grease activity, were more sensitive to inhibition by adenosine than were liver extracts. Adenosine inhibited the methylation of guanine to a greater extent than of cytosine or adenine. Methylation of both tRNA and histone by liver enzyme was inhibited by L-dopa, dopamine and epinephrine. Methylation by brain enzyme was also blocked, but less extensively. The response of liver extracts to these catecholamines was highly age-related. The phenolic amines, octopamine, synephrine, serotonin and tyramine, stimulated tRNA methylation slightly while inhibiting histone methylation by both liver and brain extracts and these effects showed no age dependency. Analysis of the data suggests that most of these compounds do not act by competing for the available S-adenosylmethionine.

A Lineweaver-Burk analysis of a kinetic study of tRNA methylation by a 30-50% (NH4)2SO4 fraction from a weanling rat liver extract showed competitive inhibition with a Km for S-adenosylmethionine = 0.66 - 10(-6) M and a Ki for 3,4-dihydroxyphenylethylamine (dopamine) = 4 - 10(-5) M. The dopamine-inhibited methylation of tRNA appears to be linear with time. Rapid-flow dialysis studies indicated a S-adenosylmethionine binding constant of 0.65 - 10(-6) M. Dopamine appeared to interfere with the binding of S-adenosylmethionine to the weanling rat liver protein preparation but did not affect the binding of S-adenosylmethionine to protein in several systems in which dopamine did not inhibit tRNA methylase activity.

Association of koilocytic changes with cervical squamous cell carcinoma is well documented. The studies of such concurrent association may miss those cases of papillomavirus infection where cytomorphologic expression of virus has disappeared by the time carcinoma appears. The authors studied cytologic material before the diagnosis was made of cervical squamous cell carcinoma in situ in 25 patients. Twenty-two (88%) of the 25 patients showed koilocytosis compared with only 6 out of 57 (10.5%) in the control group. The findings of this study support a possible predisposing role of papillomavirus in squamous cell carcinoma of the cervix.