Double jeopardy of renal insufficiency and anemia in patients undergoing percutaneous coronary interventions

Anemia and renal insufficiency impart an increased risk of mortality in patients with congestive heart failure. There is a paucity of data on the mortality hazard associated with anemia and renal insufficiency in patients undergoing percutaneous coronary intervention in the setting of contemporary practice. We analyzed the short- and long-term outcomes among patients enrolled in EPIC, EPILOG and EPISTENT trials according to degree of kidney dysfunction (glomerular filtration rate [GFR] <60, 60 to 75, and >75 ml/min/1.73 m2) and by hematocrit (<35, 35 to 39 and 40). GFR was calculated as GFR = 186 x (serum creatinine-1.154) x (age-0.203) x 1.212 (if black) or x 0.742 (if female). There were 20 deaths (3.2%) among 638 patients with a hematocrit of <35, 41 deaths among 2,066 patients (2.0%) with a hematocrit of 35 to 39, and 43 deaths in 3,618 patients (1.2%) with a hematocrit >40 at 6 months (p <0.001). Similarly, a significant increase in mortality was seen with lower GFR [33 of 1,168 (2.9%) at GFR <60, 33 of 1,766 (1.9%) at GFR 60 to 75 and 37 of 3,317 (1.1%) at GFR >75, p <0.001)]. Further, GFR and anemia independently and in combination predicted mortality at 3 years. Thus, renal insufficiency and anemia are significant independent and additive predictors of short- and long-term complications in patients undergoing percutaneous coronary intervention.     

Identification of mycobacterium species in contaminated cultures by polymerase chain reaction

STUDY OBJECTIVES: The detection of Mycobacterium sp on a culture remains the "gold standard" technique for the diagnosis of mycobacterial infections. A small percentage of these cultures, however, may be contaminated by other nonfastidious microorganisms, making accurate diagnosis difficult. We evaluated the use of a polymerase chain reaction (PCR) protocol that was specific for the genus Mycobacterium, and specifically for Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare, for the identification of Mycobacterium sp growing on contaminated cultures. DESIGN: This prospective study was designed to identify Mycobacterium sp growing on mycobacterial cultures contaminated with other microorganisms.Samples and patients: Twenty-six samples, taken from 23 patients with probable mycobacterial disease, that resulted in Mycobacterium growth but were contaminated during their processing were evaluated in this study. Clinical data and the clinical status of each patient were used to ascertain the final diagnosis. RESULTS: All samples studied here exhibited Mycobacterium growth on solid media but were contaminated by nonfastidious bacteria, compromising the biochemical identification of the Mycobacterium sp. PCR correctly identified the genus Mycobacterium in all samples. M tuberculosis was identified in 14 samples, and M avium in 10 samples. No amplification of M intracellulare was obtained, and in two samples there was amplification only for the genus Mycobacterium. In the cultures of those patients in whom a mycobacterial infection was evident, PCR identified M avium and M tuberculosis in samples from 6 and 12 patients, respectively. However, PCR identified M avium (two patients) and M tuberculosis (two patients) in the cultures of four patients for whom a mycobacterial disease could not be confirmed by our case definition. Finally, in two samples from one patient only the genus Mycobacterium was amplified by PCR. CONCLUSION: PCR, with its advantages of greater speed and effectiveness than conventional detection methods, was successfully used to identify the Mycobacterium sp growing on contaminated cultures.     

Clinical and Immunoinflammatory Evaluation of One‐Stage Full‐Mouth Ultrasonic Debridement as a Therapeutic Approach for Smokers With Generalized Aggressive Periodontitis: A Short‐Term Follow‐Up Study

Background: This study aims to evaluate the effect of one‐stage full‐mouth ultrasonic debridement (OSFMUD) on clinical and immunoinflammatory parameters in smokers with generalized aggressive periodontitis (GAgP). Methods: Fourteen smoking and 14 non‐smoking patients with GAgP were selected. After initial supragingival therapy, patients were treated by OSFMUD. Full‐mouth parameters evaluated were: 1) plaque index (PI); 2) bleeding scores (BS); 3) probing depth (PD); and 4) clinical attachment level (CAL). Clinical evaluation was performed, and gingival crevicular fluid (GCF) was collected for selected sites (ss) at baseline and 1, 3, and 6 months. GCF was analyzed via enzyme‐linked immunosorbent assay for: 1) receptor activator of nuclear factor‐κ B ligand (RANKL); 2) osteoprotegerin (OPG); 3) interleukin (IL)‐6; and 4) tumor necrosis factor (TNF)‐α, whereas secreted osteoclastogenic factor of activated T‐cells (SOFAT) was evaluated by Western blotting. Results: Significant reduction (P <0.05) was observed between baseline and 6 months for: 1) PI; 2) BS; and 3) PD, with no difference between smoking and non‐smoking patients (P >0.05). Regarding CAL, only non‐smoking patients showed a significant decrease (P <0.05). Significant reduction (P <0.05) was observed in both groups for: 1) PIss; 2) PDss; 3) bleeding on probing; and 4) relative CAL. Smoking and non‐smoking patients presented significantly decreased levels of IL‐6 and TNF‐α over time (P <0.05); however, no difference was observed between groups (P >0.05). RANKL was significantly different (P <0.05) only for non‐smokers at 6 months, whereas OPG was not significant (P >0.05). SOFAT expression was significantly lower (P <0.05) after OSFMUD for non‐smokers only. Conclusion: Considering the clinical and immunoinflammatory parameters evaluated in this short‐term follow‐up study, it can be concluded that OSFMUD can be used as an alternative treatment for smokers with GAgP.     

Knockout of renin-angiotensin system genes: Effects on vascular development

Pharmacologic inhibition of the renin-angiotensin system (RAS) is a widely accepted and effective treatment for hypertension. However, in the past several years, much attention has been focused on additional roles of the RAS including the possibility that its end-product, angiotensin II, could elicit end-organ pathologies independent of its effect on blood pressure. The ability to selectively delete genes in mice (by homologous recombination or gene knockouts) has led to new — and sometimes surprising — insights into the roles of the RAS in the developmental modeling and pathologic remodeling of the heart and blood vessels.     

Risk factors for congenital hypothyroidism: results of a population case-control study (1997-2003)

OBJECTIVE: To identify risk factors for permanent and transient congenital hypothyroidism (CH). DESIGN: A population-based case-control study was carried out by using the network created in Italy for the National Register of Infants with CH. METHODS: Four controls were enrolled for each new CH infant; 173 cases and 690 controls were enrolled in 4 years. In order to distinguish among risk factors for permanent and transient CH, diagnosis was re-evaluated 3 years after enrollment when there was a suspicion of transient CH being present. Familial, maternal, neonatal and environmental influences were investigated. RESULTS: An increased risk for permanent CH was detected in twins by a multivariate analysis (odds ratio (OR) = 12.2, 95% confidence interval (CI): 2.4-62.3). A statistically significant association with additional birth defects, female gender and gestational age >40 weeks was also confirmed. Although not significant, an increased risk of CH was observed among infants with a family history of thyroid diseases among parents (OR = 1.9, 95% CI: 0.7-5.2). Maternal diabetes was also found to be slightly associated with permanent CH (OR = 15.7, 95% CI: 0.9-523) in infants who were large for gestational age. With regard to transient CH, intrauterine growth retardation and preterm delivery were independent risk factors for this form of CH. CONCLUSION: This study showed that many risk factors contribute to the aetiology of CH. In particular, our results suggested a multifactorial origin of CH in which genetic and environmental factors play a role in the development of the disease.     

Standardization and comparison of endogenous erythroid colony assays performed with bone marrow or blood progenitors for the diagnosis of polycythemia vera

The reliability of the assay of endogenous erythroid colony (EEC) formation in serum-free, cytokine-free collagen-based media was investigated in a multicentric study including 140 patients with polyglobuly (80 polycythemia vera (PV), 54 secondary erythrocytosis (SE), six idiopathic erythrocytosis (IE)) and 10 healthy donors. In each center, EEC assays were performed in parallel with progenitor cells from bone marrow (BM) and peripheral blood (PB); two commercialized media and 'low' and 'high' cell plating densities were tested. Negativity of EEC assays was considered certain only when sufficient BFU-E growth was obtained in control cultures with cytokines. In the two media, EEC formation was specific - never observed in cultures of healthy donors or SE patients - and comparable. BM EEC assays were positive (presence of eythroid colonies) for 75% ('low' plating) to 100% ('high' plating) of PV patients; PB EEC assays were positive for 83.3% ('low' plating) to 93.7% ('high' plating) of PV patients (differences not significant). Depending on the medium, 86.2-93.7% of patients with a positive BM EEC assay had a positive PB EEC assay. Hence, a standardized collagen-based EEC assay can be performed with either BM or PB progenitors; the EEC assay described here is positive for at least 75% of PV patients when a single EEC assay is performed, and for at least 94% of PV patients when both BM and PB EEC assays are performed.     

Intrahepatic hepatitis C virus RNA quantification in microdissected hepatocytes

BACKGROUND/AIMS: Debate continues on whether serum and intrahepatic HCV viral loads are correlated and if HCV viral load correlates with the severity of liver disease. These difficulties may at least in part be linked to liver cell heterogeneity, when total liver extracts from HCV-infected individuals are tested for HCV RNA quantification. We have therefore investigated the feasibility of quantifying HCV replication using a laser-based microdissection technique. METHODS: We compared the results with those obtained for serum HCV RNA quantification and immunochemistry in the case of HCV antigen detection in the liver. Twenty-one HCV-positive patients with chronic active hepatitis (n=10) or cirrhosis (n=11) were analyzed. RESULTS: A positive correlation (P=0.0019) was observed between HCV RNA quantifications in sera and microdissected cells. Immunohistochemistry demonstrated that HCV antigen hepatocytes were randomly distributed within liver lobules. Their percentage varied in different patients (0-40%), but did not correlate with the HCV viral load. CONCLUSIONS: We have designed a sensitive methodology to evaluate the intrahepatic HCV viral load by combining a standardized RNA quantification method with microdissected hepatocytes from frozen liver needle biopsies. Our results directly demonstrate a positive correlation between serum and intrahepatic viral loads, which therefore provides a reliable reflection of intrahepatic HCV replication.     

Overexpression of dominant-negative Ikaros 6 protein is restricted to a subset of B common adult acute lymphoblastic leukemias that express high levels of the CD34 antigen

Ikaros is a critical regulator of hematopoiesis. Its effects result in part from the balance between the isoforms that are produced by differential splicing of the pre-mRNA. Short isoforms that lack the DNA-binding domain act as dominant negatives by binding long isoforms through the C-terminal zinc-finger domain, which allows for the homo- or heterodimerization of the proteins. There are a number of evidences that different subsets of murine hematopoietic progenitors - as defined by phenotype - have different patterns of Ikaros expression. Forced expression of short isoforms (Ik5, Ik6 or Ik7) in murine or human hematopoietic progenitors alters the differentiation capacities of these cells. Human leukemias provide additional information: because of the blockade in differentiation, leukemias represent an equivalent of a particular stage of human hematopoietic hierarchy. We and others have shown that human acute leukemias are heterogeneous for the pattern of Ikaros isoform expression. The present study focused on adult de novo B ALLs and the Ikaros 6 isoform. ProB (BI, n=3), common B (BII, n=15) and preB (BIII, n=3) ALL were identified by their phenotype. RT-PCR and Western blot analyses of blast cell protein lysates suggest that approximately 50% of BII leukemias overexpress Ikaros 6 RNA and protein. Comparison of BII cells with high or normal levels of Ik6 shows a higher level of expression for the membrane stem cell antigen CD34 in the former, as detected with flow cytometry and confirmed with DNA arrays.     

From the Cover: Radiation-induced neoantigens broaden the immunotherapeutic window of cancers with low mutational loads

Immunotherapies are a promising advance in cancer treatment. However, because only a subset of cancer patients benefits from these treatments it is important to find mechanisms that will broaden the responding patient population. Generally, tumors with high mutational burdens have the potential to express greater numbers of mutant neoantigens. As neoantigens can be targets of protective adaptive immunity, highly mutated tumors are more responsive to immunotherapy. Given that external beam radiation 1) is a standard-of-care cancer therapy, 2) induces expression of mutant proteins and potentially mutant neoantigens in treated cells, and 3) has been shown to synergize clinically with immune checkpoint therapy (ICT), we hypothesized that at least one mechanism of this synergy was the generation of de novo mutant neoantigen targets in irradiated cells. Herein, we use Kras^G12D x p53^−/− sarcoma cell lines (KP sarcomas) that we and others have shown to be nearly devoid of mutations, are poorly antigenic, are not controlled by ICT, and do not induce a protective antitumor memory response. However, following one in vitro dose of 4- or 9-Gy irradiation, KP sarcoma cells acquire mutational neoantigens and become sensitive to ICT in vivo in a T cell-dependent manner. We further demonstrate that some of the radiation-induced mutations generate cytotoxic CD8⁺ T cell responses, are protective in a vaccine model, and are sufficient to make the parental KP sarcoma line susceptible to ICT. These results provide a proof of concept that induction of new antigenic targets in irradiated tumor cells represents an additional mechanism explaining the clinical findings of the synergy between radiation and immunotherapy.

Immune checkpoint therapy (ICT) can lead to durable responses in subsets of cancer patients (1–8). On the basis of computational analyses, the patients who most benefit from ICT are those with cancers that have high mutational burden (9–18). For example, patients bearing tumors with high mutational burden caused by environmental exposure (such as ultraviolet-induced melanoma) or deficiencies in DNA repair (such as microsatellite instability-high colorectal cancers) tend to respond well to immunotherapy (18–26). Presumably the sensitivity of such cancers reflects the increased likelihood of formation of immunogenic, tumor-specific mutant neoantigens (27). We and others previously showed that certain tumor-specific neoantigens are major targets of natural and therapeutically induced antitumor responses in both mice and humans (28–41). Therefore, the presence of immunogenic tumor neoantigens is currently thought to contribute to tumor sensitivity to immunotherapy.However, many cancer patients do not respond to ICT, suggesting that their neoantigen burden is either of insufficient magnitude or immunogenicity to function as targets for T cell-dependent antitumor mechanisms. Indeed, there are many tumor types, such as acute myeloid leukemia, estrogen receptor-positive breast, and prostate cancers, that have limited mutational burdens and display low response rates to ICT (9, 13, 42, 43). Additionally, tumor cell clones expressing immunogenic neoantigens that develop during tumor evolution may be eliminated from tumors with high mutational burden by the process of cancer immunoediting, resulting in outgrowth of tumor cell clones with reduced immunogenicity that can then grow progressively in the presence of the unmanipulated immune system (33, 44, 45). Therefore, a process by which tumors with low neoantigen burden can acquire immunogenic mutations has the potential to expand the number of patients able to benefit from ICT.Ionizing radiation has been shown to elicit DNA damage in tumor cells, leading to an increase in overall mutational load (46–52). This damage is thought to occur primarily through generation of reactive oxygen species which induce base pair substitutions by mechanisms involving transitions, transversions, and/or faulty DNA repair (53). Multiple preclinical studies have demonstrated antitumor responses when focal radiation is combined with ICT in tumors that do not respond to ICT alone (54–60) and several clinical studies have demonstrated that human tumor patients have improved responsiveness to ICT following focal radiation (e.g., {"type":"clinical-trial","attrs":{"text":"NCT02303990","term_id":"NCT02303990"}}NCT02303990, {"type":"clinical-trial","attrs":{"text":"NCT02298946","term_id":"NCT02298946"}}NCT02298946, {"type":"clinical-trial","attrs":{"text":"NCT02383212","term_id":"NCT02383212"}}NCT02383212) (61–67). Radiation has been demonstrated to function as an in vivo tumor vaccine by inducing damage-associated molecular patterns (DAMP)-dependent immunogenic cell death (68), inducing DNA damage sensed by pattern recognition receptors (69, 70), enhancing access of immune effector cells to their cognate targets through tumor cell debulking and vasculature changes (71, 72), up-regulating major histocompatibility complex class I (MHC-I) receptors (73), up-regulating cell-surface molecules such as Fas (74), and augmenting tumor antigen cross-presentation by specific subsets of dendritic cells through up-regulation of type I interferon (IFN), which results in increased numbers and action of tumor-specific CD8⁺ T cells (75–77). However, none of these explanations take into account that following irradiation, tumor cells acquire novel mutations that may function as effective tumor neoantigens. In fact, two groups have demonstrated broadening of the T cell repertoire following radiation treatment of mouse 4T1 mammary tumors and B16F10 melanoma tumors (56, 78). Radiation-induced neoantigens may partially explain the broadening of the T cell repertoire reported during noncurative doses of irradiation.Given the above observations, we specifically explored whether one dose of in vitro irradiation could increase the immunogenicity of poorly immunogenic tumor cell lines through mechanisms involving the de novo generation of tumor-specific mutant neoantigens. For this purpose, we used a mouse Kras^G12D x p53^−/− sarcoma cell line as a model system since the R.D.S. and T.J. laboratories have previously shown that these tumor cells express a very limited number of somatic mutations, are essentially devoid of mutational neoantigens, and are nonimmunogenic and grow progressively in syngeneic wild-type (WT) mice either following treatment with control antibody or the combination of anti–PD-1/anti–CTLA-4 (34, 41). We find that treating these cell lines with noncurative doses of irradiation induces expression of somatic mutations, some of which function as neoantigens and render the sarcoma cells susceptible to ICT in vivo. These data support the concept that an additional mechanism underlying the synergy between radiation therapy and immunotherapy is that the former induces immunogenic mutations in tumors that now function as targets for the latter.